Patent Publication Number: US-2019192636-A1

Title: Compositions and methods for treatment of type viii collagen deficiencies

Description:
SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 9, 2017, is named 0100-0016PR1_SL.txt and is 92,924 bytes in size. 
     TECHNICAL FIELD OF THE INVENTION 
     The present invention relates to compositions and methods for the treatment of Type VII collagen deficiencies such as dystrophic epidermolysis bullosa comprising the administration of autologous genetically modified cells comprising a nucleotide sequence encoding Type VII collagen (C7) or a functional variant thereof. 
     BACKGROUND OF THE INVENTION 
     Type VII collagen (C7) is a protein important for anchoring fibril formation at the dermal-epidermal junction (DEJ), which holds together the layers of skin (Burgeson 1993; Leigh et al. 1988). Dystrophic epidermolysis bullosa (DEB) is an inherited disease characterized by a deficiency in C7 resulting in an impairment of anchoring fibrils to adhere between the epidermis and the underlying dermis affecting the skin and organs. Mutations of the COL7A1 gene encoding C7 causes the C7 deficiency. Patients suffering from DEB are highly susceptible to severe blistering. Dominant dystrophic epidermolysis bullosa (DDEB) is characterized by generalized blistering. Transient bullous dermatosis is a form of DDEB, which corrects itself during infancy. Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating skin-blistering disorder. RDEB inversa is another form of RDEB, where blisters form on areas where skin rubs on skin. Without these fibrils, skin layers separate causing severe blistering, open wounds and scarring in response to any kind of friction, including normal daily activities like rubbing or scratching. 
     There are currently no curative treatments and patients largely rely on palliative wound care. Preclinical studies employing a number of strategies show encouraging results suggesting that the effects of the disease can be corrected by restoring C7 function in RDEB skin. Initial approaches relied on grafting fragile engineered epidermal tissue and require removal of the skin tissue to be replaced, resulting in scarring (Mavilio et al. 2006; Chen, Nat. Genet. 2002; Siprashvili 2010). Other methods have included delivery of the corrective C7 by direct virus-based expression and systemic delivery of recombinant protein (Remington 2009; Woodley et al. 2003). All of these potential treatments have a number of drawbacks including cost and biosafety concerns. 
     The use of replication-defective, self-inactivating (SIN) lentiviral vectors (LV) offers several advantages for gene therapy: 1) ability to transduce both dividing and non-dividing cells, 2) high transduction efficiency, 3) long-term sustained transgene expression, 4) the ability to dispense sequences encoding viral proteins that might trigger an immune response, 5) an increased safety profile due to transcriptionally inactive SIN long terminal repeat (LTR), and 6) an ability to package large transgenes. Safety studies from clinical trials using LV vectors for gene therapy have showed no preferential integration in or near proto-oncogenes or tumor suppressor genes. Patients who have been dosed at 10 billion LV vector-transduced T cells per subject and followed for a median of 4 years, showed no leukemia or other adverse events. Trials with the indications of metachromatic leukodystrophy, Wiskott-Aldrich Syndrome (WAS) and X-adrenoleukodystrophy (ALD), revealed no aberrant clonal expansion seen up to 21 months, 32 months, and 36 months of follow up, respectively (Biffi et al. 2013; Aiuti et al. 2013). 
     Treatment with autologous genetically-modified human dermal fibroblasts (GM-HDF) offers a promising alternative based on correcting the hereditary defect ex vivo in the patients&#39; own fibroblasts (Ortiz-Urda et al. 2003; Woodley et al. 2003). It has been reported that hCOL7A1 gene-corrected fibroblasts are better than keratinocytes at supplying C7 to the basement membrane zone (BMZ) in mice with RDEB skin grafts (Goto et al. 2006). In addition, injection of fibroblasts can be more easily completed than grafting of keratinocytes. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a method of treating a patient suffering from a Type VII collagen (C7) deficiency comprising obtaining cells from the dermis or epidermis, preferably fibroblasts or keratinocytes, of a C7-deficient patient, contacting the cells with a transducing lentiviral vector particle comprising the COL7A1 gene or a functional variant thereof to form an autologous genetically modified cell comprising the COL7A1 gene or functional variant thereof having a vector copy number wherein said lentiviral vector particle has a transducing vector copy number in the range of 0.1 to 5.0 copies per cell, culturing said autologous genetically modified cell, and administering the genetically modified cells to the C7-deficient patient. The administration may be done by injection, preferably intradermal injection. In one aspect of the invention, the C7 deficiency is dystrophic epidermolysis bullosa (DEB), such as recessive dystrophic epidermolysis bullosa (RDEB) or dominant dystrophic epidermolysis bullosa (DDEB). Subtypes of RDEB are also envisioned to be treated according to the present invention including Hallopeau-Siemens, non-Hallopeau-Siemens RDEB, RDEB inversa, pretibial RDEB, acral RDEB, and RDEB centripetalis. 
     In one embodiment of the invention, the transducing lentiviral vector particle is constructed from a transfer lentiviral vector comprising (a) a modified 5′ long terminal repeat in LTR, wherein the promoter of the modified 5′ LTR is a cytomegalovirus promoter, (b) the COL7A1 gene or a functional variant thereof, (c) at least one lentiviral central polypurine tract, (d) a hepatitis B virus post-transcriptional regulatory element (PRE), and (e) a modified 3′ LTR, wherein the modified 3′ LTR comprises a deletion relative to the wild-type 3′ LTR, wherein the COL7A1 gene or functional variant is incorporated into the cells to form genetically modified cells having a functional COL7A1 gene or functional variant thereof. 
     In an embodiment, there are at least two lentiviral central polypurine tract elements. In another embodiment, the PRE is woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Preferably, the transfer lentiviral vector used to construct the transducing vector is selected from the group consisting of pSMPUW and pFUGW. Preferably, the transducing lentiviral vector particle is INXN-2004 or INXN-2002. 
     Administration of the genetically modified fibroblasts autologous to the C7-deficient patient may be done in any suitable manner, including by injection, topically, orally, or embedded in a biocompatible matrix. 
     Another aspect of the invention is directed to the autologous genetically modified fibroblasts from a C7-deficient patient, such as RDEB or DDEB patient, transduced with a lentiviral vector particle comprising a functional COL7A1 gene and expressing type VII collagen, wherein the lentiviral vector particle has a transducing vector copy number in the range of 0.1 to 5.0 copies per cell. 
     Another embodiment is directed to a self-inactivating lentiviral vector formed from a transfer vector comprising (a) a modified 5′ long terminal in LTR, wherein the promoter of the modified 5′ LTR is a cytomegalovirus promoter, (b) the COL7A1 gene or a functional variant thereof, (c) at least one lentiviral central polypurine tract element, (d) a hepatitis B virus post-transcriptional regulatory element (PRE), and (e) a modified 3′ LTR, wherein the modified 3′ LTR comprises a deletion relative to the wild-type 3′ LTR. In one embodiment, the vector comprises at least two lentiviral central polypurine tract elements and the PRE is woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The vector of the invention includes the vector designated INXN-2004 and comprising the sequence of IGE308 plasmid; or the vector designated INXN-2002. 
     Another embodiment of the invention is directed to pharmaceutical compositions comprising a fibroblast obtained from a patient suffering from RDEB or DDEB transduced with a lentiviral vector designated INXN-2004 comprising the sequence of IGE308 plasmid; or transduced with a lentiviral vector designated INXN-2002. 
     In one embodiment, the present invention is directed to cells transduced in vitro or ex vivo using the vectors, such as INXN-2002 and INXN-2004. 
     In one embodiment, the present invention is directed to transduced autologous fibroblasts using vectors INXN-2002 or INXN-2004, which have been obtained and propagated according to methods described in U.S. Pat. No. 8,728,819 and International Patent Application. 
     WO2008/027984 entitled “Methods for culturing minimally-passaged fibroblasts and uses thereof”, each of which is hereby incorporated by reference herein. 
     In one embodiment, the present invention is directed to use of concentrated lentivirus for transduction of human autologous dermal cells, such as fibroblasts and/or keratinocytes. 
     In another embodiment, the present invention is directed to transduction of human autologous dermal cells, such as fibroblasts and/or keratinocytes via centrifugation with lentivirus. 
     In one embodiment, the present invention is directed to use of both concentrated lentivirus and centrifugation for transduction of human autologous dermal cells, such as fibroblasts and/or keratinocytes. 
     In an embodiment, the present invention is directed to use of human autologous dermal cells, such as fibroblasts and/or keratinocytes, which have been contacted with lentivirus in two or more separate transduction processes. 
     In one embodiment, the present invention is directed to use human autologous dermal cells, such as fibroblasts and/or keratinocytes, which have been contacted with lentivirus in two or more separate transduction processes, wherein the cells are cultured and passaged one, two, or three times, before the second or any subsequent transduction process. 
     The present invention also relates to the treatment of patients suffering from pseudosyndactyly comprising administering to said patient an autologous population of cells obtained from said patient transduced with a lentiviral vector particle comprising a functional COL7A1 gene or its functional variant thereof, and expressing type VII collagen. 
     The invention further relates to a method of treating, inhibiting or reducing the blistering of a dystrophic epidermolysis bullosa (DEB) or methods of treating lesions in patients suffering from RDEB comprising administering the autologous genetically modified cells of the present invention. 
     Another aspect of the invention relates to isolated population of genetically modified fibroblasts autologous to a C7-deficient patient, such as DDEB or RDEB patient, transduced with a vector particle comprising a functional COL7A1 gene or functional variant thereof and expressing type VII collagen, as well as methods of making these isolated populations. 
     The invention also relates to a method of increasing the integrated transgene copy number per cell in genetically modified human dermal fibroblasts or keratinocytes comprising contacting a transducing lentiviral vector comprising a nucleotide sequence encoding a COL7A1 gene or a functional variant thereof with a human dermal fibroblast or keratinocyte obtained from a C7-deficient patient to form a transduction composition. The transduction composition is subjected to spinoculation to form transduced human dermal fibroblast or keratinocyte, wherein the integrated transgene copy number of the transduced human dermal fibroblasts or keratinocytes is higher relative to a transduction composition not subjected to spinoculation. The invention further includes a super-transduction step, wherein the transduced cells are further contacted with a second transducing lentiviral vector to form a second trandusction composition, which is optionally subjected to spinoculation. In one aspect, the transducing lentiviral vector is INXN-2002 or INXN-2004, preferably INXN-2004. It has been found that the the transduced human dermal fibroblast or keratinocytes have at least or about 1, 2, 5, 10, 20, 25, 27, 28, 29, 30, 35, 40, 45, or 50-fold greater integrated transgene copy number per cell relative to a transduction composition not subjected to spinoculation or super-transduction. In another embodiment, the invention relates to having an integrated transgene copy number per cell of at least 0.05, 0.09, 0.41, or 0.74 or a range of between about 0.1 to about 5, 0.1 to about 1, 0.4 to about 1, or 0.4 to about 0.75. Expression has been found to be increased using the transduction methods described herein. For example, it has been found that expression of C7 has increased by 10, 25, 50, 100, 150, or 200-fold relative to transduced human dermal fibroblasts or keratinocytes not subjected to spinoculation or a second transduction. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A more complete understanding of the present invention may be obtained by reference to the accompanying drawings, when considered in conjunction with the subsequent detailed description. The embodiments illustrated in the drawings are intended only to exemplify the invention and should not be construed as limiting the invention to the illustrated embodiments. 
         FIG. 1A  depicts type VII collagen (C7) trimers, which form anchoring fibrils. The COL7A1 gene encodes a 290-kDa alpha chain and three of the chains form a triple helix (trimer). Image from Bruckner-Tuderman L.  Molecular Therapy  (2008) 17:6-7. 
         FIG. 1B  depicts the general structure of normal and RDEB skin. C7 anchoring fibrils bind to other collagens, extracellular matrix proteins, and Lam332 to mediate attachment of the dermis to the epidermis. Absence of anchoring fibrils can lead to blister formation. 
         FIG. 2  describes a cGMP-scale GM-HDF production process according to one aspect of the present invention. A C7 expression cassette was cloned into a self-inactivating lentivirus backbone. A pilot-scale production of lentiviral vector (INXN-2002) comprising COL7A1 gene (LV-COLT) with a titer of ˜9×10 6  IU/mL was generated for use in the cGMP-scale production process. Fibroblasts were isolated from RDEB patient biopsies, grown, then split into three arms for mock, high-, and low-dose LV-COLT transduction. Each arm of fibroblasts was grown to 2×CS10 scale then cryopreserved (Drug Substance). For patient treatment, Drug Substance vials are thawed and formulated (Drug Product), then shipped back to the clinic for wound-site injection of the originating patient. 
         FIG. 3A  provides the LV-COLT (for INXN-2002) copy number per cell. Drug Substance vials were thawed and assayed for LV-COLT DNA copies per cell using qPCR. Primers were specific for the LV shuttle vector. Results demonstrate dose-dependent levels of copies per cell with an average of 0.38 and 0.18 copies from the High and Low Dose arms, respectively. 
         FIG. 3B  provides the C7 expression levels produced by RDEB patient fibroblasts transduced with LV-COLT: Drug Substance vials were thawed and cultured for 3 days. Conditioned cell culture supernatants were collected and assayed for C7 levels by ELISA. Results show virus dose-dependent protein expression that ranges from 60 to 120 ng/mL C7 in LV-COLT-transduced cells. 
         FIG. 3C  displays the trimeric form of C7 produced by RDEB patient fibroblasts transduced with LV-COL7: Conditioned cell culture supernatants were also used in an immunoprecipitation assay. Immunoprecipitated C7 was separated on non-denaturing SDS-PAGE and visualized by western blot. The C7 produced by RDEB fibroblasts was predominantly trimeric with LV-COL7-transduced cells expressing more COL7 than mock-transduced cells (Arm C). Some lower molecular weight species showing immunoreactivity, were also observed. 
         FIG. 4A  depicts binding of purified COL7 to Lam332. COL7 was expressed in CHO-DG44 cells, was purified by size exclusion chromatography, and was assayed for preferential binding to Lam332 as compared to BSA to establish the assay (increase in OD450 corresponds to increase in COL7 binding to Laminin332). 
         FIG. 4B  depicts Lam332 binding of COL7 in LV-COL7-transduced fibroblasts (as transduced by the INXN-2002 vector) culture supernatants. Drug Substance vials were thawed and cultured for 3 days. Conditioned cell culture supernatants were collected and assayed for binding to Lam332 compared with BSA control. Results show virus dose-dependent binding to Lam332. 
         FIG. 5  depicts composite RDEB skin grafts on the dorsum of SCID mice that were injected intradermally with 1×10 6  GM-HDF (as transduced by the INXN-2002 vector) and analyzed by immunofluorescent staining with human COL7 specific antibodies. Representative images are shown. Localization of COL7 was observed in composite grafts (n=4) generated with RDEB keratinocytes at Day 10-post intradermal injection of 1×10 6  GM-HDF. Positive control grafts generated from normal keratinocytes and fibroblasts showed intense COL7 staining and negative control grafts did not show COL7 staining at baseline measurements (arrows at DEJ for negative baseline comparison). 
         FIG. 6  provides a schematic of the pSMPUW lentiviral expression plasmid vector. 
         FIG. 7  compares the features of the pSMPUW lentiviral expression vector relative to 3rd generation lentivirus expression vectors. 
         FIG. 8  provides a schematic of the pFUGW lentiviral expression plasmid vector. 
         FIG. 9  depicts an electron micrograph of HIV-1 virus particles. 
         FIG. 10  provides the immunoprecipitation results for TR8 GM-HDFs detecting the formation of C7 trimers. 
         FIG. 11A  provides the immunoprecipitation results for TR10 and ER1 GM-HDFs detecting the formation of C7 trimers. 
         FIG. 11B  provides the immunoprecipitation results for TR9 detecting the formation of C7 trimers. 
         FIG. 12  shows the results of Lam332 binding results by C7 as expressed by GM-HDFs. 
         FIG. 13  provides a schematic of the lentiviral vector plasmid construct, which encodes a COL7A1 gene. 
         FIG. 14  presents a schematic of the proviral RNA genome structure of the INXN-2002 lentiviral vector. 
         FIG. 15  shows a schematic depicting the details of the mature INXN-2002 virus particle. 
         FIG. 16  provides a schematic of the in vitro immortalization assay to assess the potential for insertional genotoxicity of INXN-2002. 
         FIG. 17  represents typical cell morphology and structures of FXC-007 in culture. 
         FIG. 18  provides a graph of the C7 expression levels as determined by ELISA for particular dosages of the TR8 GM-HDFs. 
         FIG. 19  provides the western blot showing the immunoprecipitation of C7 trimers against NC1-specific antibodies as produced by FCX-007 GM-HDFs. 
         FIG. 20  shows the assay readout (optical density at 450 nm (OD450)) for a binding assay to detect the interaction of C7 with laminin 332 or bovine serum albumin (BSA)-coated wells. Bound C7 was detecting using a C7 NC1-specific antibody and an HRP-conjugated secondary antibody. 
         FIG. 21A  graphically represents the amount of cell migration of normal and RDEB patient fibroblasts transduced with the LV-COLT over time. 
         FIG. 21B  provides images of the cell migration of normal and RDEB patient fibroblasts transduced with the LV-COLT over time. 
         FIG. 22  provides the schematic for the cloning of human COL7A1 gene. 
         FIG. 23  provides the schematic of the cloning of the COL7A1 gene into the pSMPUW expression vector to produce the INXN-2002 lentiviral vector transfer plasmid, IGE230. 
         FIG. 24  provides the schematic representation of the construction of the INXN-2004 lentiviral vector transfer plasmid (IGE308). 
         FIG. 25A  schematically depict the gene elements of the IGE308 plasmid. 
         FIG. 25B  schematically depict the gene elements of the IGE308 plasmid. 
         FIG. 26  provides the immunofluorescence analysis of GM-HDF injected composite grafts. 
         FIG. 27  provides a graph representing the LV-COLT transcript levels as measured by its fold change value over control cells. 
         FIG. 28  provides representative images of C7 staining for INXN-2002 and LV-HA-COL7-transduced RDEB fibroblasts. 
         FIG. 29  provides a graphical representation of the C7 protein levels as measured in the cell supernatants of GM-HDFs transduced by TR8, TR9, T10 and ER1. 
         FIG. 30  graphically represents the correction of hypermotility of GM-HDFs over time for TR8, TR9, TR10 and ER1. 
         FIG. 31  depicts representative indirect immunofluorescence (IF) images to examine C7 protein expression by GM-HDF cells using an antibody specific for C7 at two magnifications, 20× and 5×. (Only Arm A was tested for TR12.1 and TR13. Only Arm A of TR8 is shown for comparison of previous processes.) 
         FIG. 32  provides the percentage of C7-positive (C7+) cells of GM-HDFs for each training run arm of TR11, TR12.1 and TR13. 
         FIG. 33  provides a graph representation of the amount of C7 protein levels expressed for TR8, TR11, TR12.1 and 13. Error bars represent standard deviation; n=3. Only Arm A results are shown for TR8, TR12.1, and TR13. 
         FIG. 34  provides the SDS-PAGE/immunoblots for the detection of C7 trimers (a) TR11, (b) TR12.1 and TR13 GM-HDFs. 
         FIG. 35  depicts a graph of C7 expressed by GM-HDF that bind to Laminin 332 (Lam332) for TR11, TR12.1 and TR13. 
         FIG. 36  provides the results of cell migration assays depicting the correction of hypermotility (% migration) over time for GM-HDFs for TR11, TR12.1 and TR13. The hypermotility of normal human dermal fibroblasts (NHDF) and a control fibroblasts from recessive dystrophic epidermolysis bullosa cells are provided. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of this invention. Although any compositions, methods, kits, and means for communicating information similar or equivalent to those described herein can be used to practice this invention, the preferred compositions, methods, kits, and means for communicating information are described herein. 
     All references cited herein are incorporated herein by reference to the full extent allowed by law. The discussion of those references is intended merely to summarize the assertions made by their authors. No admission is made that any reference (or a portion of any reference) is relevant prior art. Applicants reserve the right to challenge the accuracy and pertinence of any cited reference. 
     In order that the present invention may be more readily understood, certain terms are herein defined. Additional definitions are set forth throughout the detailed description. 
     The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” 
     Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. Typically, the term is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% variability depending on the situation. 
     The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” 
     As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention. 
     Nucleic acids and/or nucleic acid sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are homologous when their encoding DNAs are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. The homologous molecules can be termed homologs. For example, any naturally occurring proteins, as described herein, can be modified by any available mutagenesis method. When expressed, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establish homology. Methods for determining sequence identity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available. 
     The terms “identical” or “sequence identity” in the context of two nucleic acid sequences or amino acid sequences of polypeptides refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. A “comparison window”, as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned optimally. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482; by the alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443; by the search for similarity method of Pearson and Lipman (1988) Proc. Nat. Acad. Sci U.S.A. 85:2444; by computerized implementations of these algorithms (including, but not limited to CLUSTAL in the PC/Gene program by Intelligentics, Mountain View Calif, GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.); the CLUSTAL program is well described by Higgins and Sharp (1988) Gene 73:237-244 and Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-10890; Huang et al (1992) Computer Applications in the Biosciences 8:155-165; and Pearson et al. (1994) Methods in Molecular Biology 24:307-331. Alignment is also often performed by inspection and manual alignment. 
     In one class of embodiments, the polypeptides herein are at least 70%, generally at least 75%, optionally at least 80%, 85%, 90%, 98% or 99% or more identical to a reference polypeptide, or a fragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or more identical to a reference nucleic acid or a fragment thereof, e.g., as measured by BLASTN (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned. 
     The term “substantially identical” as applied to nucleic acid or amino acid sequences means that a nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, preferably at least 95%, more preferably at least 98% and most preferably at least 99%, compared to a reference sequence using the programs described above (preferably BLAST) using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantially identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. 
     A “functional variant” of a protein disclosed herein can, for example, comprise the amino acid sequence of the reference protein with at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid substitutions. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and Schirmer, R. H., Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and Schirmer, R. H., supra). Examples of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, for example, lysine for arginine and vice versa such that a positive charge may be maintained; glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained; serine for threonine such that a free —OH can be maintained; and glutamine for asparagine such that a free —NH 2  can be maintained. In one embodiment of the present invention, a COL7A1 gene encodes for the C7 protein or a functional variant thereof, provided that the variant is capable of anchoring the fibrils between the epidermis and dermis. 
     Alternatively or additionally, the functional variants can comprise the amino acid sequence of the reference protein with at least one non-conservative amino acid substitution. “Non-conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc. In this case, it is preferable for the non-conservative amino acid substitution to not interfere with, or inhibit the biological activity of, the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the reference sequence. 
     Proteins disclosed herein (including functional portions and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and include, for example, aminocyclohexane carboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptane carboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine. 
     The invention includes the vectors INXN-2002; and IGE308 also referred to herein as INXN-2004, as well as vectors substantially identical and/or homologous thereto as well as functional variants thereof. 
     The term “patient” or “subject” refers to mammals, including humans and animals. 
     The term “treating” refers to reducing or alleviating the symptoms, and/or preventing relapses and/or the progression of DEB, including RDEB and/or DDEB. 
     The term “autologous cells” refers to cells obtained and then returned to the same individual. In particular, cells may be removed from the body of a DEB patient, which may subsequently be genetically modified, cultivated to proliferate, and then returned to the body of the patient from which they were originally removed. 
     The term “transduction” refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection. 
     The term “transducing lentiviral vector” or “transducing lentiviral vector particle” refers to the infectious lentiviral vector particles formed from the co-transfection of a packaging cell line with the lentiviral expression/transfer plasmid vector comprising the COL7A1 gene or functional variant thereof, a packaging vector(s), and an envelope vector. The transducing lentiviral vector is harvested from the supernatant of the producer cell culture after transfection. Suitable packaging cell lines are known in the art and include, for example, the 293T cell line. 
     The term “lentiviral vector” refers to a vector containing structural and functional genetic elements outside the LTRs that are primarily derived from a lentivirus. 
     The term “self-inactivating vector” (SIN) refers to vectors in which the 3′ LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Consequently, the vectors are capable of infecting and then integrating into the host genome only once, and cannot be passed further. SIN vectors greatly reduce risk of creating unwanted replication-competent virus since the 3′ LTR U3 region has been modified to prevent viral transcription beyond the first round of replication, eliminating the ability of the virus to be passed. 
     The term “pharmaceutically acceptable carrier” is employed herein to refer to liquid solutions which are, within the scope of sound medical judgment, suitable for use in contact with the autologous, genetically-modified cells without affecting their activity, and without being toxic to the tissues of human beings and animals or causing irritation, allergic response, or other complications, commensurate with a reasonable benefit/risk ratio. A useful pharmaceutically acceptable carrier may be an injectable solution which is biocompatible with the autologous, genetically-modified cells and does not reduce their activity or cause their death. 
     The present invention relates to an autologous, genetically-modified cell therapy for C7-deficient patients. “C7-deficient patients” either lack or have substantially reduced production of type VII collagen (C7), which is important for anchoring fibril formation at the dermal-epidermal junction (DEJ), resulting in dystrophic epidermolysis bullosa (DEB). The present invention relates to harvesting DEB patient cells, such as fibroblasts or keratinocytes, genetically modifying the harvested cells to insert a functional COL7A1 gene or functional variant thereof, expanding the genetically modified cells, and administering a population of the genetically-modified cells back into the DEB patient. 
     Cells are harvested from a patient suffering from DEB. Fibroblasts or keratinocytes are preferred. The cells may be obtained through known methods, including from patient skin samples, scrapings, and biopsies, for example. In one embodiment, the cells are obtained from the non-blistering skin of a DEB patient. In another embodiment, the cells are obtained from the blistering skin of a DEB patient. The cells harvested from a DEB patient have either a mutated, non-functional or missing COL7A1 gene. These cells are cultured using standard cell culture techniques. The harvested patient&#39;s cells are subsequently treated ex vivo with genetic material encoding a functional type VII collagen gene (C7) or functional variant thereof. 
     The COL7A1 gene has the nucleic acid, SEQ ID NO:1, which encodes for a 290 kDa alpha chain, wherein three chains form a triple helix (trimer), as depicted in  FIG. 1A . C7 has the amino acid sequence of SEQ ID NO: 2 and is the protein important for anchoring fibril formation at the dermal-epidermal junction (DEJ). Mutations in the C7 gene cause an absence or reduction of C7, which make up anchoring fibrils that maintain binding of the epidermis to the dermis. C7 anchoring fibrils bind to other collagens, extracellular matrix proteins, and Lam332 that mediate the attachment of the dermis to the epidermis, as shown in  FIG. 1B . The absence of anchoring fibrils leads to blistering of the skin. The invention also includes using vectors comprising nucleic acids and polypeptides substantially identical to COL7A1 and C7 respectively. In another embodiment, the present invention includes vectors that comprise nucleic acids that encode a functional variant of the COL7A1 or C7 protein, provided that the variant is capable of anchoring the fibrils between the epidermis and dermis. Another aspect of the invention includes the use of only the open reading frame of the COL7A1 gene, which encodes the C7 protein or a functional variant thereof. In some embodiments, the COL7A1 nucleic acid is codon-optimized for increased expression of C7 in the transduced cell, as is known in the art. 
     In accordance with the present invention, the COL7A1 can be transduced into the harvested DEB cells using a transducing lentiviral vector, which is replication defective and self-inactivating. Self-inactivating lentiviral vectors are derived from the human immunodeficiency virus (HIV-1), which are pseudotyped with a heterologous VSV-G envelope protein instead of the HIV-1 envelope protein. As previously reported, a 400 bp deletion is introduced to the U3 region of the LTR resulting in a self-inactivating (SIN) vector (Zuffery 1998). The vectors of the present invention are distinguished from 2nd and 3rd generation SIN vectors. In particular, the vectors of the present invention have been modified to enhance the safety features and increase the cloning capacity as further described herein. 
     Vectors used to construct the transducing lentiviral vectors of the present invention are introduced via transfection or infection into a packaging cell line. The packaging cell line produces transducing lentiviral vector particles that contain the vector genome. After cotransfection of the packaging vectors, transfer vector, and an envelope vector to the packaging cell line, the recombinant virus is recovered from the culture media and titered by standard methods used by those of skill in the art. Thus, the packaging constructs can be introduced into human cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as kanamycin, neomycin, DHFR, or Glutamine synthetase, followed by selection in the presence of the appropriate drug and isolation of clones. The selectable marker gene can be linked physically to the packaging genes in the construct. 
     Stable cell lines wherein the packaging functions are configured to be expressed by a suitable packaging cell are known. For example, see U.S. Pat. No. 5,686,279; and Ory et al., (1996), which describe packaging cells. The packaging cells with a lentiviral vector incorporated in them form producer cells. Producer cells are thus cells or cell-lines that can produce or release packaged infectious viral particles carrying the therapeutic gene of interest. An example of a suitable lentiviral vector packaging cell lines includes 293 cells. 
     In one embodiment of the invention, the transducing lentiviral vector is constructed from a lentiviral expression plasmid vector comprising (a) a modified 5′ long terminal repeat (LTR), wherein the promoter of the modified 5′ LTR is a cytomegalovirus promoter, (b) the COL7A1 gene, (c) at least one lentiviral central polypurine tract element, and (d) a modified 3′ LTR, wherein the modified 3′ LTR comprises a deletion relative to the wild-type 3′ LTR and comprises a deletion of a hepatitis B virus post-transcriptional regulatory element (PRE). 
     In another aspect of the invention, the deleted hepatitis B virus post-transcriptional regulatory element (PRE) is a woodchuck post-transcriptional regulatory element PRE (WPRE). 
     In another aspect of the invention, the lentiviral expression plasmid vector comprises two or more lentiviral central polypurine tract (cPPT) elements. Incorporation of cPPT element has been found to increase gene expression levels. 
     In another aspect of the invention, the 3′ LTR may be modified to delete a 400 bp fragment (as disclosed by Zuffery 1998), instead of the commonly used 133 bp deletion of standard 3rd generation SIN LV vectors. The 400 bp deletion is believed to increase safety by preventing read-through transcription as well increase viral titer because the vector transcript is more stable in packaging cells. 
     In yet another aspect of the invention, the lentiviral expression plasmid may incorporate a hepatitis B virus post-transcriptional regulatory element (PRE), which is preferably woodchuck post-transcriptional regulatory element PRE (WPRE). WPRE has been found to increase the viral titer of the vector. 
     The addition of the cPPT, and modified 3′LTR deletion has been shown to improve the function of the lentiviral vector comprising the COL7A1 gene of the present invention to increase gene expression levels, viral titer, and safety. 
     The present invention further includes the use of lentiviral transfer vectors in which certain elements common for 3rd generation lentiviral expression vectors are deleted. In particular, safety concerns for use of lentiviral vectors in humans hinge on the fear of generating a replication-competent lentivirus, which may arise from recombination between split genomes of 3rd generation lentiviral vectors. (Tareen et al., 2013.). The gag sequence and/or the rev-responsive elements (RRE) may be deleted from the viral vector, in order to reduce sequence homology with other helper plasmids and thereby increase safety to humans. Accordingly, in one embodiment of the invention, the gag sequence is absent in the vector. In another embodiment of the invention, both the gag sequence and RRE is absent in the vector. 
     The starting materials to generate the lentiviral vectors of the present invention is preferably a lentiviral expression plasmid vector that comprises a cPPT and PRE that can accommodate the insertion of the large COL7A1 gene (8.89 kbp) or a functional variant thereof. Preferably, the starting material to construct the transducing lentiviral vector of the present invention is selected from the lentiviral expression plasmid vectors, pSMPUW (Cell Biolabs, Inc., San Diego, Calif.) and pFUGW (Addgene, Cambridge, Mass.). 
     In one aspect of the invention, the pSMPUW lentiviral expression plasmid vector is selected for construction of the transducing lentiviral vector.  FIG. 6  shows a schematic of the genetic elements in the pSMPUW lentiviral expression plasmid vector. Features of the pSMPUW lentiviral expression vector have been modified from 3rd generation lentivirus expression vectors in order to enhance gene expression levels and safety features, as depicted in  FIG. 7 . In particular, the pSMPUW lentiviral expression vector encodes for a multicloning site (MCS) followed by the Woodchuck Hepatitis Virus Post-transcriptional regulatory element (WPRE). The residual gag (Agag) and the RRE element were removed from the pSMPUW vector construct. Additionally, the pSMPUW vector construct utilized a larger 400 bp deletion in the 3′LTR U3 region instead of the commonly used 133 bp deletion in a standard 3rd generation SIN LV vector. Wild type COL7A1 gene was incorporated into the pSMPUW vector. 
     In another aspect of the invention, the pFUGW lentiviral expression plasmid is selected for construction of the transducing lentiviral vector.  FIG. 8  shows a schematic of the genetic elements in this plasmid vector. Features of the pFUGW vector include a RRE, two cPPT elements, and a WPRE element, which may improve lentiviral production and transgene expression. 
     In another aspect of the invention, the WPRE element has been deleted from the lentiviral vector. 
     In an embodiment of the present invention, transducing lentiviral vector particles of the present invention are designated INXN-2002 (vector transfer plasmid—IGE230) or INXN-2004 (vector transfer plasmid—IGE308), or a substantially identical vector comprising functional variants thereof. 
     The cells harvested from the DEB patient are transformed with a functional COL7A1 gene. In one aspect of the invention, the cells are transduced with a lentiviral vector having the COL7A1 gene. In another aspect of the invention, the lentiviral vector is a self-inactivating vector. The copy number of the integrated transgene can be assessed using any known methods. For example, copy number may be determined through quantitative PCR, multiplex ligation-dependent probe amplification, fluorescent in situ hybridization (FISH), microarray-based copy number screening, and conventional karyotyping. The number of copies of the transgene integrated into each cell may be modulated by the virus dose given to the cells during production. The integrated transgene copy number per cell in the RDEB harvested cells transduced with a COL7A1-containing vector is dose dependent. 
     Cells harvested from the DEB patient and transformed with the functional COL7A1 gene will have a functional COL7A1 gene and exhibit normal cell morphologies. These cells may be caused to proliferate or expanded in culture using standard cell culture techniques. 
     Normal fibroblast morphological characteristics are observed among harvested fibroblasts transduced with COL7A1-transducing vectors within the scope of the present invention. For example, normal fibroblast morphologies include cells displaying elongated, fusiform or spindle appearance with slender extensions. Normal morphologies further include cells appearing larger, flattened stellate cells which may have cytoplasmic leading edges.  FIG. 9  displays an example of normal cell morphology for fibroblasts transduced with a COL7A1-transducing vector. 
     The production of C7 produced by harvested DEB patient cells transduced with a COL7A1-transducing vector has also been observed. In particular, the formation of C7 trimers is important in the assembly of anchoring fibrils. It has been found that DEB patient cells transduced with a COL7A1-transducing vector are capable of forming the C7 trimers with the correct structure, size, and function. 
     In one aspect of the invention, it is possible to verify that the C7 expressed by fibroblasts transduced with a COL7A1-transducing vector will be capable of forming anchoring fibrils using immunoprecipitation with an anti-C7 specific antibody. For example, the anti-C7 specific antibody, fNC1, may be used for selective capture and the concentration of C7 from culture supernatants for detection by SDS-PAGE/immunoblot. For example,  FIGS. 9 and 10  demonstrate that C7 produced by fibroblasts transduced with a COL7A1-transducing vector were predominantly trimeric. 
     In another aspect of the invention, the function of C7 may be assessed using known methods, such as using a laminin binding assay or cell migration assay. C7 has been shown to bind immobilized extracellular matrix (ECM) components, including fibronectin, Laminin 332 (Lam332), COL1, and COL4 (Chen, et al., 2002a). The interaction between C7 and Lam332 occurs through the NH2-terminal NC1 domain of C7 and is dependent upon the native conformation of both Lam332 and C7 NC1 (Rousselle, et al., 1997). The association between C7 and Lam332 is important for establishing correct Lam332 architecture at the dermal-epidermal junction. Such organization is important for interactions with extracellular ligands and cell surface receptors, and for cell signaling (Waterman, et al., 2007). In one aspect of the invention, an ELISA using an antibody against the C7 NC1 domain to detect binding of C7 to purified Lam332 has been developed. Although a C7/Lam332 binding ELISA has already been described in the literature (Chen, et al., 2002a), to our knowledge it has never been used to test C7 present in the supernatants of transduced cells. Results in  FIG. 12  show dose-dependent binding to Lam332 by C7 expressed by GM-HDFs from the Training and Engineering runs. 
     In addition, a cell migration assay can be used to assess functional C7 activity. Previous studies have shown that RDEB fibroblasts and keratinocytes show an increase in motility relative to their normal counterparts, and that normal motility can be restored by expression of C7 (Chen, et al., 2000; Chen, et al., 2002b; Cogan, et al. 2014; Baldeschi et al., 2003). Suitable assay includes the colloidal gold salt migration assay to measure the migration of fibroblasts and keratinocytes, or a wound healing assay to measure the migration of keratinocytes. Cells having functional C7 activity will exhibit a reduced motility relative to RDEB cells in such assays. 
     The present invention is directed, in one aspect, to pharmaceutical formulations comprising the autologous, genetically-modified cells from DEB patients. These cells may be present in any amount suitable for the delivery to the patient in which the cells were originally harvested. For example, that cells may be present in a cell concentration of 1.0-5.0×10 7  cells/mL, 1.0-4.0×10 7  cells/mL, 1.0-3.0×10 7  cells/mL, or 1.0-2.0×10 7  cells/mL. The cells are present in present in a suspension suitable to sustain the viability of the cells, such as in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). In particular, the viability of the cells in the formulation are present in an amount of ≥60%, 70%, 75% or 80%. Suitable excipients may also be present in the formulation, such as phosphate buffered saline which may be used to wash the cells from thawed vials containing the autologous, genetically-modified. Preferably, no phenol red is present in the final formulation. 
     The autologous genetically modified cells of the present invention have a transducing vector copy number of at least or about 0.05, 0.09, 0.41, or 0.74 or in the range of about 0.1 to about 6.0, about 0.1 to about 5.5, about 0.1 to about 5.0, about 0.1 to about 4.5, about 0.1 to about 4.0, about 0.1 to about 3.5, about 0.1 to about 3.0, about 0.1 to about 1, 0.4 to about 1, or 0.4 to about 0.75. Preferably, the transducing vector copy number is in the range of about 0.1 about 5.0. It has been found that the the transduced human dermal fibroblast or keratinocytes have at least or about 1, 2, 5, 10, 20, 25, 27, 28, 29, 30, 35, 40, 45, or 50-fold greater integrated transgene copy number per cell relative to a transduction composition not subjected to spinoculation or super-transduction. Moreover, the C7 protein expression values of the FCX-007 cells of the present invention are ≥300, 350, 400, 450, 500, 550, or 600 ng/day/E6 cell. Preferably, the C7 protein expression values of the autologous genetically-modified cells of the present invention are ≥500 ng/day/E6 cell. The present invention has also found that expression of C7 has increased by 10, 25, 50, 100, 150, or 200-fold relative to transduced human dermal fibroblasts or keratinocytes not subjected to spinoculation or a second transduction. 
     The multiplicity of infection (MOI) refers to the number of vector particles per cell used in transduction. In accordance with the present invention, the desired transducing vector copy number has been achieved even as the MOIs decreases, as shown in the examples below. For example, the invention relates to MOIs of ≤15, ≤14, ≤13, ≤12, ≤11, or ≤10. Preferably, the MOI is between about 1 to 10. 
     The formulations of the present invention may be used to treat various maladies of patients suffering from Type VII deficiency. In particular, the formulations of the present invention are suitable to treat DEB, including DDEB or RDEB. In an embodiment of the invention, subtype of RDEB may also be treated, including but not limited to Hallopeau-Seiemens, non-Hallopeau-Siemens RDEB, RDEB inversa, pretibial RDEB, acral RDEB, and RDEB centripetalis. 
     The invention further relates to the treatment, reduction, prevention, and/or inhibition of various symptoms attributed to C7 deficiencies. For example, DEB is known to cause scarring after blisters, which may cause contracture deformities, swallowing difficulty if the mouth and esophagus are affected, fusion of fingers and toes, and limited mobility. Therefore, in one aspect of the invention envisions the treatment, reduction, inhibition, and/or prevention of pseudosyndactyly, also known as mitten hand syndrome. In another aspect, the invention relates to the treatment, reduction, inhibition, and/or prevention of deep fibrosis and/or scarring associated with RDEB and/or DDEB, which may result in milia, joint contractures, generalized soft tissue fibrosis, organ fibrosis, corneal lesions, scarring plaques, scarring alopecia, nail dystrophy, ankyloglosia, and increased frequency of dental caries, for example. In another aspect, the invention relates to the treatment, reduction, prevention, and/or inhibition of oral mucosa lesions and gastrointestinal lesions associated with RDEB. Another embodiment relates to the treatment, prevention, reduction and/or inhibition of blisters associated with DEB patients. 
     Administration of the autologous genetically modified cells may be done at any appropriate time as one skilled in the art would be capable of determining based on the needs of the patient. For example, administration may be done once, once a day, once a month, once a quarter, or 1-2 times per year. 
     The formulation of the present invention containing the autologous genetically modified cells may be administered to the patient by any known method, including but not limited to injection, topically, orally, or embedded in a biocompatible matrix. The injection may be, e.g., parenteral, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraarterial, ocular and intraperitoneal or direct injection into a specific organ/tissue, e.g. prostate or liver. Topically, the formulation may be administered directly to an affected site, such as at the site of a lesion. Debridement of the affected tissue can precede the direct application of the formulation to the site. Alternatively, the formulation may be encapsulated in a suitable delivery system, such as in a polymer capsule, or embedded in a biocompatible matrix or graft, e.g., collagen matrix, in a hydrogel, skin graft, or a mesh. 
     The autologous genetically modified cells of the present invention may be administered solely or in combination with other treatments for a patient suffering from DEB. Examples of therapeutic agents used for treating DEB include topical care, such as Zorblisa, skin grafts, anti-inflammatories, antibodies, other potential gene or cell-based therapies. In another embodiment, the modified cells of the present invention may be administered for for the skin or muoscal areas where bone marrow transplant therapies (or other system cellular therapies such as mesenchymal stem cells) did not provide sufficient therapy. The C7-expressing ability of the autologous genetically modified cells is preferably not impaired by a combination with other therapeutic agents. 
     The present invention further relates to a method of enhancing or increasing the integrated transgene copy number per cell in the genetically modified cells obtained from C7-deficient patients and transduced according to the present invention. In particular, it has been found that certain steps during transduction substantially enhanced the copy number of the transgene in these cells. In one aspect of the invention, the cells obtained from the C7-deficient patient are contacted with the lentiviral vector of the present invention and that composition is subjected to spinoculation, also known as spin transduction. In this manner, the lentiviral vectors are spun onto the targeted cells. The addition of spinoculation has unexpectedly increased the copy number per cells in the genetically modified human dermal fibroblasts by a level of ≥2-fold relative to a transduction without spinoculation or transduction. However, in order to identify additional methods to increase copy number, it has unexpectedly been found that a second transduction step (or super-transduction) in which the targeted cells are passaged through the first transduction with spinoculation, and then subsequently passaged through a second transduction optionally with spinoculation. In this manner, it has been found that this super-transduction with spinoculation increases the copy number by an additional 2-fold increase relative to the original traditional transduction method that did not include spinoculation or super-transduction. 
     In another embodiment, it has been found that changing the lentiviral vector from INXN-2002 to INXN-2004 increased the transgene copy number in the genetically modified human dermal fibroblasts by 4-fold relative to traditional transduction without spinoculation or super-transduction. 
     Through extensively evaluating integrated transgene copy numbers, it has been found the cumulative change of adding spinoculation, adding super-transduction with spinoculation, and changing from INXN-2002 to INXN-2004, relative to the original transduction process that did not include spinoculation or super-transduction, unexpectedly increased the copy number&gt;27-fold (see Example 9: comparing TR12.1 and TR3 to TR8 in original studies (which did not include spinoculation or super-transduction). Accordingly, the present invention relates to increased the copy number of the transduced cells from the C7-deficient patient. 
     To further illustrate the invention, the following non-limiting examples are provided. 
     EXAMPLES 
     FCX-007 is a suspension of live, autologous human dermal fibroblast cells genetically modified using either a lentiviral vector (INXN-2002) (as shown in Example 1) or a lentiviral vector (INXN-2004) (as shown in Example 2) to express the human collagen type 7 protein. 
     Example 1 
     A. Elucidation of Structure and Characteristics of FCX-007 Transduced with Lentiviral Vector INXN-2002 
     The FCX-007 cells derived from the lentiviral vector INXN-2002 cells are suspended in a cryopreservation medium consisting of Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) without fetal bovine serum (50.0%), Profreeze-CDM™ (42.5%) and dimethyl sulfoxide (DMSO) (7.5%). The structural features for FCX-007 Drug Substance (DS) include the primary structure of the autologous human dermal fibroblast (HDF) cells and the structure of the lentiviral vector used to transduce and gene modify the HDF cells. 
     B. Lentiviral Vector (INXN-2002) 
     INXN-2002 Lentiviral Vector (LV), which is used to transduce and to introduce the human collagen 7A1 gene into the HDF cells, is a recombinant lentiviral vector encoding the human collagen 7A1 gene. INXN-2002 LV is a self-inactivating (SIN) lentiviral vector that is constructed based on the human immunodeficiency virus type 1 (HIV-1) pseudotyped with a heterologous VSV-G envelope protein. The virus particle for INXN-2002 lentiviral vector is approximately 120 nm in diameter and is comprised of numerous proteins with two copies of a single-stranded RNA genome. A specific molecular formula, molecular weight, or stereochemistry is not available. 
     Structural features for INXN-2002 LV include the primary structure of the RNA viral genome and the structure of the viral particle. The primary structure of the RNA genome of INXN-2002 is determined by the full nucleotide sequencing of the viral genome. The viral particles structure is deduced from the particle structure of HIV-1 with added VSV-G protein pseudotyping. An overview of the nucleic acid structure and the structure of the viral particle are provided below. 
     C. INXN-2002 RNA Viral Genome Structure 
     INXN-2002 LV is a self-inactivating (SIN) lentiviral vector that is constructed based on the human immunodeficiency virus type 1 (HIV-1) pseudotyped with a heterologous VSV-G envelope protein instead of the HIV-1 envelope protein. A 400 bp deletion is introduced to the U3 region of the LTR resulting in a self-inactivating (SIN) vector (Zuffery 1998). The pSMPUW lentiviral expression plasmid vector (Cell Biolabs, Inc., San Diego, Calif.) was selected for construction of the INXN-2002 LV.  FIG. 6  shows a schematic of the genetic elements in the pSMPUW lentiviral expression plasmid vector. 
     The coding elements between the 5′ and 3′ LTRs of the HIV-1 virus are fully gutted. The vector encodes for a multicloning site followed by the Woodchuck Hepatitis Virus Posttransscriptional Regulatory Element (WPRE). In order to maximize the cloning capacity of the vector, these elements were removed by digesting the vector with BamHI from the multicloning site and KpnI at the 5′ end of the 3′ left terminal repeat. The COL7A1 gene expression cassette with a CMV promoter is cloned into the digested vector by single strand annealing to generate the lentiviral vector plasmid construct encoding the COL7A1 gene as shown in  FIG. 13 . 
     The lentiviral vector plasmid construct is co-transfected into HEK293T cells with three helper plasmids (pCMV-G, pCMV-Rev2 and pCgp) to produce the INXN-2002 lentiviral vector particles. pCMV-G plasmid provides the VSV-G pseudotyping surface protein, pCMV-Rev2 provides the HIV-1 Rev protein for efficient RNA transport and packaging, and pCgp provides the structural and viral enzyme proteins for production of the INXN-2002 lentiviral vector particles.  FIG. 14  shows a schematic of the proviral RNA genome structure of the INXN-2002 lentiviral vector. 
     D. INXN-2002 Lentiviral Vector Particle Structure 
     INXN-2002 lentiviral vector is constructed based on a HIV-1 derived vector backbone and has a similar viral particle structure to the HIV-1 virus.  FIG. 9  shows an electron micrograph of HIV-1 particles. 
     The HIV-1 particle has a spherical shape of approximately 120 nm in diameter, with an estimated molecular weight of 277MDa (Carlson 2008). The particle has a lipid bilayer membrane stubbed with envelope protein. The envelope protein interacts with the receptors on the target cells for infection and delivery of the RNA viral genome to the target cells. A cone shaped nucleo-core, where two strands of viral RNA genome is housed, can be observed inside the virus particle. The cone shaped nucleo-core is formed by the viral capsid protein. 
     For INXN-2002, VSV-G (glycoprotein of the vesicular stomatitis virus (VSV-G)) is used as a substitute for the HIV-1 envelope proteins resulting in improved vector stability, target cell tropism, and transduction efficiency (Cronin 2005). 
     During production, INXN-2002 viral particles assemble and bud out from the surface of transfected HEK293T cells. The VSV-G protein is provided by the pCMV-G helper plasmid, the vector core and enzyme proteins are provided by the pCgp helper plasmid, and the Rev protein, which is needed for efficient RNA genome transport and packaging into the viral particle, is provided by the pCMV-Rev2 plasmid. It is noted that all the other HIV-1 accessory proteins including Vpu, Vif, Vpr, Nef, and Tat, are deleted from the INXN-2002 vector. 
     After budding from the producer cell surface, the protease enzyme packaged inside the virus particle cleaves the Gag precursor protein into its constituent proteins (MA, CA, NC), to convert the immature virion into a mature infectious INXN-2002 vector particle.  FIG. 15  shows a schematic depicting the details of the mature INXN-2002 virus particle. 
     Two strands of the INXN-2002 RNA genome are packaged inside the cone shaped core formed by the Capsid protein (CA). The nucleocapsid (NC) protein forms a stable complex with the RNA genome inside the capsid core. The matrix protein forms a coat on the inner surface of the membrane. The virus buds through the cell plasma membrane spiked with the VSV-G envelope protein and forms the lipid envelope. Based on the recent three dimensional analysis of HIV-1 virus particle structure (Carlson 2008), Table 1 shows the component proteins that make up the INXN-2002 lentiviral vector and a brief description of the function of each of the components. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 INXN-2002 Lentiviral Vector Major Component Proteins 
               
            
           
           
               
               
               
            
               
                   
                 Protein 
                   
               
               
                 Component 
                 MW 
               
               
                 Proteins 
                 (kD) 
                 Protein functions 
               
               
                   
               
               
                 Capsid (CA) 
                 24 
                 Forms the nucleo-core 
               
               
                 Matrix (MA) 
                 17 
                 Forms the protein coat on the inner surface of 
               
               
                   
                   
                 the lipid membrane 
               
               
                 Nucleocapsid 
                  7 
                 Forms a stable complex with the RNA genome 
               
               
                 (NC) 
               
               
                 VSV-G 
                 69 
                 Pseudotyped envelope protein interacts and 
               
               
                   
                   
                 binds to receptors on target cells for trans- 
               
               
                   
                   
                 duction to deliver the vector RNA genome to 
               
               
                   
                   
                 the cells 
               
               
                 Protease 
                 11 
                 Plays an important role in the maturation of 
               
               
                   
                   
                 the INXN-2002 vector by cleaving the Gag 
               
               
                   
                   
                 proteins to its functional constituents, CA, 
               
               
                   
                   
                 MA, and NC 
               
               
                 Reverse 
                 66/51 
                 Builds a DNA copy of the viral RNA genome 
               
               
                 transcriptase 
               
               
                 Integrase 
                 31 
                 Inserts the DNA copy of the viral RNA genome 
               
               
                   
                   
                 into the infected cell genome 
               
               
                   
               
            
           
         
       
     
     E. INXN-2002 Characterization 
     Table 2 provides a list of the characterization assays and specifications for INXN-2002 manufactured by City of Hope. Characterization assay results are provided in the attached CoA for INXN-2002 lot number 0786-240-0002-1, the lot intended for use in manufacture of the FCX-007 clinical product. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Characterization of INXN-2002 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Target 
               
               
                 Cate- 
                   
                 Test 
                   
                   
                 Specifica- 
               
               
                 gory 
                 Assay 
                 Method 
                 Lab 
                 SOP 
                 tion 
               
               
                   
               
               
                 Identity 
                 Vector 
                 RT-PCR 
                 CoH 
                 QC-SOP- 
                 Band of 
               
               
                   
                 identity 
                   
                   
                 0843 
                 correct 
               
               
                   
                   
                   
                   
                   
                 size 
               
               
                   
                   
                   
                   
                   
                 detected 
               
               
                   
                 Vector 
                 Southern 
                 Indiana 
                 VP-10-17.16 
                 Report result 
               
               
                   
                 insert 
                 blot analy- 
                 Univer- 
               
               
                   
                 stability 
                 sis 
                 sity 
               
               
                 Potency 
                 Viral 
                 ELISA 
                 Indiana 
                 VP-10-05.06 
                 Report result 
               
               
                   
                 antigen 
                   
                 Univer- 
               
               
                   
                 Physical 
                   
                 sity 
               
               
                   
                 Titer (p24) 
               
               
                   
                 TU titer 
                 H1299 
                 BioRe- 
                 016135.BSV 
                 Report result 
               
               
                   
                   
                 trans- 
                 liance 
               
               
                   
                   
                 duction 
               
               
                   
                   
                 with qPCR 
               
               
                   
                   
                 readout 
               
               
                   
                 C7 
                 ELISA 
                 BioRe- 
                 016136.BSV 
                 Report result 
               
               
                   
                 expression 
                   
                 liance 
               
               
                   
                 (Potency) 
               
               
                 Appear- 
                 Appear- 
                 Visual 
                 CoH 
                 QC-SOP- 
                 Opaque 
               
               
                   
                 ance 
                 inspection 
                   
                 0734 
                 solution 
               
               
                 ance 
                 pH 
                 pH meter 
                 CoH 
                 QC-SOP- 
                 6.9-7.8 
               
               
                 and pH 
                   
                   
                   
                 0675 
               
               
                   
               
            
           
         
       
     
     F. Characterization of INXN-2002 by an In Vitro Immortalization Assay 
     The potential for insertional genotoxicity of INXN-2002 was evaluated using an in vitro immortalization (IVIM) assay. The test was conducted at Cincinnati Children&#39;s Hospital Medical Center, Division of Experimental Hematology &amp; Cancer Biology (CCHMC). 
     The principle of the IVIM test is based on the understanding that normal Lineage negative (Lin−) bone marrow (BM) cells will stop proliferating after 3-4 weeks in vitro, but in the presence of certain vector integrations which cause upregulation of proto-oncogenes, some clones will continue to proliferate after 5 or more weeks. The number of such immortalized clones is representative of the oncogenic potential of the vector. The immortalized clones are expanded and further analyzed for stem cell markers by FACS, vector copy number by qPCR or the site of integration by LAM-PCR. Insertion in common integration sites (cis) such as Evil or Prdm 16 is frequently observed in immortalized clones generated in IVIM assays (Calmels et al., 2005; Modlich et al., 2008). 
     Lineage-negative (Lin−) bone marrow (BM) cells from C57BL/6 mice were isolated from complete BM by magnetic sorting using lineage-specific antibodies. The Lin− BM cells were then cultured and stimulated in complete growth medium for 2 days. On Day 4, cells were transduced with INXN-2002 vector in a 48-well plate coated with RetroNectin (10 μg/cm 2 , Takara).  FIG. 16  shows a schematic setup of the IVIM assay. 
     To enhance the INXN-2002 transduction efficiency on Lin− BM cells, INXN-2002 vector was concentrated approximately 14-fold using a spin concentrator. For transduction, 80 μL of the concentrated INXN-2002 was added to each well containing 1.5×10 5  cells with 150 μL of transduction medium. The plate was centrifuged for 1000 g at 32° C. for 70 minutes (spinocculation) to further enhance the transduction efficiency. Cells were incubated at 37° C., 5% CO 2  incubator 0/N. 
     In order to achieve a high INXN-2002 vector copy number in the transduced Lin− BM cells, INXN-2002 transduction was repeated on Day 5, Day 6, and Day 7 for a total of 4 consecutive transductions. 
     The transduced Lin− BM cells were expanded for 10 days and cell concentration was adjusted to 2-4×10 5  cells/ml every two days, and fresh medium was provided as needed. On Day 10 post transduction, cells were harvested and counted. A portion of the cells was then submitted for DNA isolation and qPCR to determine the vector copy number (VCN). The remaining cells were placed back into culture for potential plating of the IVIM assay between days 18-21. 
     Table 3 shows the pilot test run result of INXN-2002 transduced Lin− BM cells harvested on Day 10. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Day 10 Post-Transduction Lin- BM Cells Analysis 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Vector Copy 
               
               
                 Test Article 
                 Total Cells 
                 Cell Viability 
                 per Cell 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 INXN-2002 
                 1.5 × 10 6   
                 89% 
                 0.09 
               
               
                 Control vector-1 
                 2.7 × 10 6   
                 95% 
                 0.81 
               
               
                 Control vector-2 
                 2.1 × 10 6   
                 91% 
                 3.58 
               
               
                 Mock 
                 3.0 × 10 6   
                 88% 
                 0.0 
               
               
                   
               
            
           
         
       
     
     Despite the use of concentrated INXN-2002 vector and 4× consecutive spinocculation transduction, the vector copy number reached in the Lin− BM cells was significantly lower than the targeted copy number of 1-3 expected for the IVIM test. The pilot test was terminated. 
     Because of the low vector copy numbers experienced in the IVIM assays as well as the low vector copy numbers achieved in all the RDEB fibroblast cell transduction runs, it is believed that this lot of INXN-2002 poses minimal risk of insertional genotoxicity when used to transduce RDEB fibroblast cells. No further analysis of this lot of INXN-2002 by the IVIM assay was pursued. 
     G. FCS-007 Transduced with INXN-2002 
     The human dermal fibroblast cells used for the manufacture of FCX-007 are derived from a live skin biopsy. The biopsy is digested using Liberase® (Roche) to release the dermal fibroblast cells and then the cells are expanded in culture using standard cell culture techniques. At the completion of culture expansion after INXN-2002 transduction, the cells are harvested and washed, then formulated to contain 1.0-3.0×10 7  cells/mL. The DS is tested for purity and confirmed to contain ≥98% fibroblasts by CD90 staining with cell viability of ≥85%. 
     FCX-007 cells transduced with the INXN-2002 LV in the DS formulation display typical fibroblast morphologies when cultured on tissue culture surfaces. Specifically, cells may display an elongated, fusiform or spindle appearance with slender extensions, or cells may appear as larger, flattened stellate cells which may have cytoplasmic leading edges. A mixture of these morphologies may also be observed.  FIG. 17  shows a typical cell morphology and structure of FCX-007 DS in culture. 
     The cells express proteins characteristic of normal fibroblasts including the fibroblast specific marker, CD90 (Thy-1), a 35 kDa cell-surface glycoprotein, and the extracellular matrix proteins, such as various types of collagen. 
     H. FCX-007 Derived from Lentiviral Vector INXN-2002: Drug Substance Release Characterization 
     FCX-007 Drug Substance is characterized for release at three stages during manufacturing: in-process, bulk harvest, and after cryopreservation (DS). Table 4 provides a list of the characterization assays and specifications for release of FCX-007 DS manufactured by PCT. Characterization assay results for the finished Drug Substance are provided in the attached CoTs for Training Run 8 Arm A, Arm B and Arm C (Non-Transduced Control). 
                     TABLE 4                  FCX-007 Drug Substance Release Characterization                                 Stage   Assay   Test Method   SOP   Specification               In-Process   Morphology   Microscopic   PCT SOP-0334   Pass               evaluation           Cell confluence   Microscopic   PCT SOP-0334   Pass               evaluation       Bulk   Cell count   Hemacytometer   PCT SOP-0329   ≥1E9       Harvest   Cell viability   Hemacytometer   PCT SOP-0329   ≥85%       Drug   Cell Count   Hemacytometer   PCT SOP-0329   1.0-2.7E7 cells/mL       Substance-   Cell Viability   Hemacytometer   PCT SOP-0329   &gt;/=85%       Cryovial   Purity   FACS   PCT SOP WI-1067   &gt;/=98% CD-90+           COL7A1 genecopy #   qPCR   BioReliance109011.BSV   Report result           C7 expression   ELISA   PCT SOP-0338   Report result                    
I. Additional FCX-007 DS Derived from INXN-2002 Characterization
 
     Additional characterization of FCX-007 has been performed on the training production runs to confirm expression of functional C7. The representative assessments shown below are from Training Run 8 (TR8) where cells were transduced with either a high dose (3.4 IU/cell) or low dose (1.7 IU/cell) of LV-COLT, or were mock-transduced. These transduction arms are also referred to as Arm A, B, and C, respectively. 
     J. FCX-007 DS Transduced with INXN-2002: C7 Expression Level 
     An enzyme-linked immunofluorescence assay (ELISA) was developed for the purpose of quantifying C7 expression by FCX-007. For this assay, TR8 Drug Substance vials (high dose, low dose, and mock-transduced) were thawed and cultured for 3 days and conditioned cell culture supernatants were collected and assayed for C7. Results in  FIG. 18  show virus dose-dependent protein expression that ranges from 60 to 120 ng/mL C7 in LV-COLT-transduced cells. 
     K. FCX-007 DS Transduced with INXN-2002: C7 Trimer Formation 
     Anchoring fibrils are formed from the assembly of C7 trimers. The proper expression and formation of C7 trimers by FCX-007 can be detected by immunoprecipitation of C7 followed by non-denaturing SDS-PAGE/immunoblot analyses. For  FIG. 19 , C7 was immunoprecipitated from FCX-007 cell culture supernatants at passage 1 and passage 2 post-thaw using a NC1-specific antibody and was separated on non-denaturing SDS-PAGE then visualized by western blot. The C7 produced by RDEB fibroblasts was predominantly trimeric (red arrow; ˜870 kDa) with LV-COLT-transduced cells (Transduction Arms A and B) expressing more C7 than mock-transduced cells (Arm C, starred). Monomeric (290 kDa) and dimeric (580 kDa) forms were also observed. Assay controls included immunopreciptation of purified C7 (Pur COLT) and immunoprecipitation without antibody or test sample (IP Controls). 
     L. FCX-007 Transduced with INXN-2002: C7 Binding to Lam332 
     C7 interacts with Laminin332 at the dermal/epidermal junction (DEJ). The interaction between C7 and Lammin332 is important for anchoring fibril functionality (Chen 2002, Rousselle 1997, Waterman 2007). A binding assay for detection of this interaction was developed at Intrexon. Drug Substance vials (high dose, low dose, and mock-transduced) were thawed and cultured for 2 days. Conditioned cell culture supernatants were collected and were incubated with Lam332 or bovine serum albumin (BSA)-coated wells and bound C7 was detected using a C7 NC1—specific antibody and an HRP-conjugated secondary antibody. The assay readout is optical density at 450 nm (OD450). Results in  FIG. 20  show virus dose-dependent binding to Lam332 compared with a BSA control. 
     M. FCX-007 Derived from INXN-2002 Migration Assessment 
     A migration assay was developed to assess function of C7. Chen 2000 has demonstrated that RDEB patient skin cells migrate faster than normal skin cells into an artificial wound margin created on tissue culture vessels, and that application of C7 can restore the migration rate. Normal human dermal fibroblasts (NHDFs; Lonza) and cells from FCX-007 Drug Substance vials were thawed and cultured. Cells were seeded into culture dishes with an insert to prevent cell adherence in a small strip of the culture dish. The strip was then removed and the rate at which the cells migrated into the open area was monitored by microscopy and quantified using the irregular shape delineation plugin for ImageJ software.  FIGS. 21A-21B  show both the percent migration (A) and the images of migration (B) for this assay. The results show that the mock-transduced RDEB patient fibroblasts migrate into the open area faster than NHDFs, and transduction with LV-COLT reverts the patient cells to a rate of migration similar to NHDFs. These results are consistent with those described by Chen 2000. 
     Example 2: FCX-007 Transduced with INXN-2004 Lentiviral Vector 
     FCX-007 is an autologous fibroblast cell product genetically modified by INXN-2004 lentiviral vector (LV-COLT) to express the human collagen 7 protein (C7). The materials used to manufacture FCX-007 DS/INXN-2004 are described below are similar to that set forth above in Example 1, except that the IGE-230 LV-COLT vector plasmid was used for the production of INXN-2002. IGE-308 LV-COLT vector plasmid was used to make the INXN-2004 vector. The same helper plasmids, pCMV-G, pCMV-Rev2, and pCgp were used to co-transfect a 293 WCB cell line. 
     INXN-2004 Lentiviral Vector 
     IGE308 LV-COLT Vector Transfer Plasmid 
     The IGE308 plasmid was constructed using standard molecular cloning methods. The construction process involved cloning of the human COL7A1 gene and introduction of the cloned COL7A1 gene into a lentiviral vector (SIN) backbone, pFUGW, to produce the IGE308 LV-COL7 vector transfer plasmid. 
     Cloning of the Human COL7A1 Gene 
     The COL7A1 gene cloned into the IGE308 LV-COL7 vector transfer plasmid is the same COL7A1 gene cloned into INXN-2002 (IGE230 vector transfer plasmid). 
     Primers were designed and produced with the Takara PrimeScript reverse transcriptase to amplify the COL7A1 gene from human genomic cDNA. Four primer pairs, as shown in Table 5 were designed, each to amplify approximately 2 kb of the COL7A1 gene. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                  Primer Sets used to Amplify the Human COL7A1 Gene 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Expected 
               
               
                 Forward 
                   
                 Reverse 
                   
                 Amplicon 
               
               
                 Primer 
                 Primer Sequence 
                 Primer 
                 Primer Sequence 
                 Size (bp) 
               
               
                   
               
               
                 Col1F1 
                 CGACTTGTGTTGGGACTGG 
                 Col1R3 
                 CCCGCACAGTGTAGCTAA 
                 1753 
               
               
                 (SEQ ID 
                 CTAGCGCCACCATGACGCT 
                 (SEQ ID 
                 GCCC 
                   
               
               
                 NO: 3) 
                 GCGGCTTCTGGTGGC 
                 NO: 7) 
                   
                   
               
               
                   
               
               
                 Col1F3 
                 GGGCTTAGCTACACTGTGC 
                 Col1R2 
                 CCTTTGGACAATACACTG 
                 2059 
               
               
                 (SEQ ID 
                 GGG 
                 (SEQ ID 
                 GGCAGG 
                   
               
               
                 NO: 4) 
                   
                 NO: 8) 
                   
                   
               
               
                   
               
               
                 Col1F2 
                 CCTGCCCAGTGTATTGTCC 
                 Col1R4 
                 CAGATGCCTTGATGCCAG 
                 2127 
               
               
                 (SEQ ID 
                 AAAGG 
                 (SEQ ID 
                 CAG 
                   
               
               
                 NO: 5) 
                   
                 NO: 9) 
                   
                   
               
               
                   
               
               
                 Col1F4 
                 CTGCTGGCATCAAGGCATC 
                 Col1R1 
                 CGTGATTTCATTTGCTAC 
                 2993 
               
               
                 (SEQ ID 
                 TG 
                 (SEQ ID 
                 ACGTAATCGATTCAGTCC 
                   
               
               
                 NO: 6) 
                   
                 NO: 10) 
                 TGGGCAGTACCTGTCCC 
               
               
                   
               
            
           
         
       
     
     Products were amplified with two primer pairs (CollF2/CollR4 and CollF4/CollR1) designed to amplify the 5′ end 3790 base pairs (bp) of COL7A1 from human genomic cDNA along with a 5′ overhang for cloning into the expression vector. The two 5′ PCR products (2127 bp and 2993 bp) were joined by overlap extension PCR to generate a 3790 bp final product. 
     In order to clone the remainder of the gene, a fragment of DNA (341 bp, Col1A1-3, Table 6 was synthesized encompassing the final 322 bp of the COL7A1 gene with overlaps to the PCR amplified 5′ 3790 bp gene fragment and the cloning vector. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Col1A1-3 Fragment Sequence 
               
            
           
           
               
               
            
               
                 Fragment 
                 Sequence 
               
               
                   
               
               
                 Col1A1-3 
                 CGCTCCCAGAACATCACCTACCACTGCAAGAACAGCG 
               
               
                 (SEQ ID 
                 TGGCCTACATGGACCAGCAGACTGGCAACCTCAAGAA 
               
               
                 NO: 11) 
                 CGGCCCTGCTCCTCCAGGGCTCAACGAGATCGAGATC 
               
               
                   
                 CGCGCCGAGGGCAACAGCCGCTTCACCTACAGCGTCA 
               
               
                   
                 CTGTCGATGGCTGCACGAGTCACACCGGAGCCTGGGG 
               
               
                   
                 CAAGACAGTGATTGAATACAAAACCACCAAGACCTCC 
               
               
                   
                 CGCCTGCGCATCATCGATGTGGCCCCCTTGGACGTTG 
               
               
                   
                 GTGCCCCAGACCAGGAATTCGGCTTCGACGTTGGCCA 
               
               
                   
                 TGTCTGCTTCCTGTAAATCGATTACGTGTAGCAAATG 
               
               
                   
                 AAATCACG 
               
               
                   
               
            
           
         
       
     
     The two COL7A1 gene fragments (3790 bp and Col1A1-3) were assembled into an inducible expression vector (VVN-257673) that had previously been digested with NheI and ClaI using the In-Fusion HD Cloning Kit. Bacterial clones were screened by PCR with primers specific to the backbone vector and COL7A1 sequence to identify candidates for sequencing. Positive clones were confirmed by digestion and COL7A1 fragment DNA sequencing. The resulting plasmid was named VVN-4311835. 
     VVN-4311835 and a COL7A1 expression plasmid (SC300011) purchased from Origene were digested with BstZ17I and SapI. A 6276 bp fragment of the COL7A1 gene was cloned from SC300011 into VVN-4311835. The resulting plasmid, VVN-4311835 (used the same plasmid number), was completely sequenced and the COL7A1 sequence was confirmed to be complete and without mutations. 
     VVN-4311835 plasmid was digested with NheI and ClaI, two restriction enzymes with sites just outside of the COL7A1 coding sequence. The excised COL7A1 gene was cloned into a constitutive expression vector, VVN-257231 which was also digested with NheI and ClaI, to produce VVN-4319958. VVN-4319958 expresses the full length human COL7A1 under the control of the constitutive CMV promoter. 
       FIG. 22  provides a schematic for the cloning of the human COL7A1 gene. 
     Introduction of the Cloned COL7A1 Gene into a Lentiviral Vector (SIN) 
     The pSMPUW lentiviral expression vector (VPK-211, Cell Biolabs, Inc., San Diego, Calif.) was initially selected for construction of INXN-2002 lentiviral vector encoding the COL7A1 gene based on its enhanced safety features and large cloning capacity. 
       FIG. 7  compares the pSMPUW vector to a standard 3rd generation SIN LV vector. The pSMPUW vector encodes for a multicloning site (MCS) followed by the Woodchuck Hepatitis Virus Post-transcriptional regulatory element (WPRE). The residual gag (Agag) and the RRE element were removed from the pSMPUW vector construct. Additionally, the pSMPUW vector construct utilized a larger 400 bp deletion in the 3′LTR U3 region instead of the commonly used 133 bp deletion in a standard 3rd generation SIN LV vector. 
     Details of the cloning of the COL7A1 gene into the pSMPUW lentiviral expression vector for construction of INXN-2002 lentiviral vector were described above in Example 1, and are further described below. 
     An insert was generated containing a CMV promoter followed by a Kozak sequence and a truncated version of the COL7A1 gene with BclI and SapI restriction sites to use for introduction of the entire coding sequence. This insert was synthesized in two fragments, CColG and ColG2 (IDT) (Table 8). These two fragments were assembled with the digested pSMPUW vector using the In-Fusion HD Cloning Kit. Positive clones were identified using colony PCR with a primer specific to the plasmid backbone (63968-3R), and a primer specific to the COL7A1 gene (ColS14). Plasmids from positive clones were purified and sequence confirmed to generate IGE228. IGE228 was digested with FspI and BamHI. 
     Initial attempts to clone the COL7A1 gene using the BclI and SapI sites were unsuccessful. An alternate strategy was devised using a PCR product as a linker to bypass the Bell restriction site. Primers EPF5 and Col1R3 were used to generate a PCR product from the plasmid template VVN-4319958. This product was digested with BamHI, which cuts 48 bp upstream of the 5′ end of COL7A1, and FspI, which cuts 1630 bp into the 5′ end of COL7A1 to generate the 5′ linker. 
     The wild type COL7A1 gene was cut with FspI and SapI from VVN-4319958. This fragment was ligated to the digested IGE228 and the 5′ linker to generate a lentivirus construct for the expression of COL7A1, VVN-4580853 (also named IGE230). Positive clones were identified using the primers ColS13, specific to COL7A1 and 63968-3R, specific to the lentiviral backbone. The plasmid was sequenced from the CMV promoter to the 3′ end of COL7A1. 
     Table 7 below shows the synthetic gene sequences and primers used for introduction of the COL7A1 gene into the pSMPUW lentiviral expression vector. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Synthetic Gene Elements and Primers 
               
            
           
           
               
               
            
               
                 Gene Element/ 
                   
               
               
                 Primer Name 
                 Sequence 
               
               
                   
               
               
                 CColG 
                 TTCAAATTTTCGGGGGATCGCATTAGTTATTAATAGTAATCAATTACGGGG 
               
               
                 (SEQ ID NO: 
                 TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAA 
               
               
                 12) 
                 ATGGCCCGCCTGGTTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAA 
               
               
                   
                 TGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT 
               
               
                   
                 GGGTGGAGTATTTACGGTAAACTGCCCACTTGGTAGTACATCAAGTGTATC 
               
               
                   
                 ATACGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT 
               
               
                   
                 GGTATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACAT 
               
               
                   
                 CTGCGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATC 
               
               
                   
                 AATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCC 
               
               
                   
                 ATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCA 
               
               
                   
                 AAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTA 
               
               
                   
                 CGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGC 
               
               
                   
                 TAGCATTGTGTGCTTTTCGGGCCACCATGACGCTGCGGCTTCTGGTGGCCGC 
               
               
                   
                 GCTCTGCGCCGGGATCCTGGCAGAGGCGCCCCGAGTGCGAGCCCAGCACA 
               
               
                   
                 GGGAGAGAGTGAC 
               
               
                   
               
               
                 ColG2 
                 AGTGCGAGCCCAGCACAGGGAGAGAGTGACCTGCACGCGCCTTTACGCCG 
               
               
                 (SEQ ID NO: 
                 CTGACATTGTGTTCTTACTGGATGGCTCCTCATCCATTGGCCGCAGCAATTT 
               
               
                 13) 
                 CCGCGAGGTCCGCAGCTTTCTCGAAGGGCTGGTGCTGCCTTTCTCTGGAGC 
               
               
                   
                 AGCCAGTGCACAGGGTGTGCGCTTTGCCACAGTGCAGTACAGCGATGACCC 
               
               
                   
                 ACGGACAGAGTTCGGCCTGGATGCACTTGGCTCTGGGGGTGATGTGATCCG 
               
               
                   
                 CGCCATCCGTGAGCTTAGCTACAAGGGGGGCAACACTCGCACAGGGGCTG 
               
               
                   
                 CAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGG 
               
               
                   
                 TGTCCCCAAGGTCTGCATCCTGATCACGTCTCTCATGCAGAGGAGGAAGAG 
               
               
                   
                 CGGGTACCCCCTGAGGATGATGAGTACTCTGAATACTCCGAGTATTCTGTG 
               
               
                   
                 GAGGAGTACCAGGACCCTGAAGCTCCTTGGGATAGTGATGACCCCTGTTCC 
               
               
                   
                 CTGCCACTGGATGAGGGCTCCTGCACTGCCTACACCCTGCGCTGGTACCAT 
               
               
                   
                 CGGGCTGTGACAGGCAGCACAGAGGCCTGTCACCCTTTTGTCTATGGTGGC 
               
               
                   
                 TGTGGAGGGAATGCCAACCGTTTTGGGACCCGTGAGGCCTGCGAGCGCCGC 
               
               
                   
                 TGCCCACCCCGGGTGGTCCAGAGCCAGGGGACAGGTACTGCCCAGGACTG 
               
               
                   
                 AGTACCTTTAAGACCAATGACTTACAA 
               
               
                   
               
               
                 63968-3R 
                 ATGGAAAAACGCCAGCAACG 
               
               
                 (SEQ ID NO: 
                   
               
               
                 14) 
                   
               
               
                   
               
               
                 ColS14 (SEQ 
                 CTGTCACCCTTTTGTCTATG 
               
               
                 ID NO: 15) 
                   
               
               
                   
               
               
                 EPF5 (SEQ ID 
                 GTTGCCGGACACTTCTTGTCCTCT 
               
               
                 NO: 16) 
                   
               
               
                   
               
               
                 Col1R3 (SEQ 
                 CCCGCACAGTGTAGCTAAGCCC 
               
               
                 ID NO: 17) 
                   
               
               
                   
               
               
                 ColS13 (SEQ 
                 AGAGGCCCCGAAGGACTTCA 
               
               
                 ID NO: 18) 
               
               
                   
               
            
           
         
       
     
       FIG. 23  provides a schematic of the cloning of the COL7A1 gene into the pSMPUW expression vector to produce the INXN-2002 lentiviral vector transfer plasmid, IGE230. 
     Results from continuing development indicated that deletion of the RRE element and the use of a large 400 bp deletion in the 3′LTR U3 region in the pSMPUW vector construct, combined with the requirement to package a large COL7A1 gene (8.8 kbp), resulted in a negative impact on LV-COLT vector production (infectious titer) and subsequent transduction and C7 protein expression in RDEB fibroblast cells. 
     A 3 rd  GEN SIN vector, pFUGW (FUGW, plasmid #14883, www.addgene.org) was selected to construct a second generation LV-COLT vector, INXN-2004. INXN-2004 was constructed to improve the vector copy number.  FIG. 8  provides a schematic of the genetic elements in the pFUGW lentiviral expression plasmid vector. 
     The pFUGW vector contains a RRE, two cPPT elements, as well as a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) element for achieving improved lentiviral vector production and transgene expression. The IGE308 lentiviral vector transfer plasmid was constructed to maximize the transgene cloning capacity to accommodate the insertion of the large COL7A1 gene (8.8 kbp). 
     The WPRE element, the hUBC promoter and the GFP reporter gene were removed through digestion of the vector with PacI and XhoI. An insert was generated containing the 5′ end of the CMV promoter followed by a small fragment of the 3′ end of COL7A1 gene. This insert (ColCN) was synthesized at IDT as a G-Block fragment (sequence in Table 8). The fragment was assembled with the digested pFUGW vector using a Gibson cloning derivative method. Positive clones were identified using colony PCR with primers specific to the plasmid backbone (FugwQCF and FugwQCR). Plasmids from positive clones were purified and sequence confirmed to generate IGE301. 
     IGE301 was then digested with BaeI. IGE230 was digested with AseI and SapI to create a fragment containing the 3′ end of the CMV promoter, the 5′ UTR, and the majority of the COL7A1 gene. The fragment was transferred to the BaeI-digested IGE301 vector using a Gibson cloning derivative method. Positive clones were identified using the primers ColS13, specific to COL7A1 and FugwQCR, specific to the lentiviral backbone. Plasmids from positive clones were purified and sequence-confirmed to generate IGE308. 
     Table 8 below provides the synthetic gene sequences and primers used for introduction of the COL7A1 gene into the pFUGW lentiviral expression vector. 
     Plasmids were Maxi-prepped with the Qiagen MaxiPrep Kit per the manufacturer&#39;s protocol. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Synthetic Gene Elements and Primers used in the 
               
               
                 Final Steps of IGE308 Creation 
               
            
           
           
               
               
            
               
                 Gene Element/ 
                   
               
               
                 Primer Name 
                 Sequence 
               
               
                   
               
               
                 ColCN 
                 Acagcagagatccagtttggttaattaagcatta 
               
               
                 (g-block) 
                 gttattaatagtaatcaattacggggtcattaag 
               
               
                 (SEQ ID NO: 
                 actcggccttagaaccccagtatcagcagaaccg 
               
               
                 19) 
                 tcgtctctcatgcagaggaggaagagcgggtacc 
               
               
                   
                 ccctgaggatgatgagactctgaatactccgagt 
               
               
                   
                 attctgtggaggagtaccaggaccctgaagctcc 
               
               
                   
                 ttgggatagtgatgacccctgttccctgccactg 
               
               
                   
                 gatgagggctcctgcactgcctacaccctgcgct 
               
               
                   
                 ggtaccatcgggctgtgacaggcagcacagaggc 
               
               
                   
                 ctgtcacccttttgtctatggtggctgtggaggg 
               
               
                   
                 aatgccaaccgttttgggacccgtgaggcctgcg 
               
               
                   
                 agcgccgctgcccaccccgggtggtccagagcca 
               
               
                   
                 ggggacaggtactgcccaggactgatcgataccg 
               
               
                   
                 tcgacctcgagacctagaaaaacat 
               
               
                   
               
               
                 FugwQCF 
                 GTAGACATAATAGCAACAGAC 
               
               
                 (SEQ ID NO: 
                   
               
               
                 20) 
                   
               
               
                   
               
               
                 FugwQCR 
                 TATTGCTACTTGTGATTGCTC 
               
               
                 (SEQ ID NO: 
                   
               
               
                 21) 
                   
               
               
                   
               
               
                 ColS13 (SEQ 
                 AGAGGCCCCGAAGGACTTCA 
               
               
                 ID NO: 22) 
               
               
                   
               
            
           
         
       
     
       FIG. 24  provides a schematic representation of the construction of the INXN-2004 lentiviral vector transfer plasmid (IGE308). 
     Production of IGE-308 Plasmid 
     The IGE308 plasmid was produced using a process consisting of five steps:
         transformation, glycerol stock production, scale up, capture, diafiltration and formulation.       

     Transformation: a seed stock of the IGE308 plasmid was provided to Aldevron for transformation into the competent  E. coli  (DH10B). Transformation plates were stored at 34° C. which is the standard for lentiviral constructs. 
     Glycerol stock production—Seed cultures were created by picking isolated colonies from the transformation plate. Each culture was mini prepped and the best seed culture, as determined via agarose gel, was used to make the working glycerol stocks. Scale Up—One 500 mL culture was grown in each of the following media types: Rapid Growth media and Maximum Yield media (for a total of two 500 mL cultures) in order to compare media. The cultures were inoculated using the glycerol stocks created in step two and a full panel of QC assays was run on each prep. This also allowed Aldevron to test the stability of the plasmid in each media type. 
     Based on the initial QC results Rapid Growth media was chosen for growth of the transformed  E. coli  in shaker flasks at 34° C. A total of 8 L of culture was prepared. 
     Capture—The 8 L of  E. coli  culture was lysed and initially purified using DMAE anion exchange chromatography which produced a total of 833.8 mg plasmid. An additional purification step was run using HIC (Hydrophobic Interaction Chromatography) chromatography over OS resin. This step yielded 663 mg of purified DNA. 
     Concentration and Formulation—The purified plasmid was then adjusted to the final buffer and concentration via serial diafiltration. The final buffer used was TE. 
     A total of 200 mg of IGE308 plasmid was shipped at a concentration of 1.4 mg/mL. The IGE308 plasmid was analyzed prior to further manufacture of the INXN-2004. 
     Analysis of IGE308 Plasmid Sequence 
     The IGE308 plasmid used for INXN-2004 manufacture was fully sequenced at SeqWright under GLP conditions using primer walking and oligonucleotide synthesis to yield 4-fold, bi-directional sequence coverage. The IGE308 plasmid has a size of 16,777 bp. The sequence showed 100% match to the IGE308 construction reference sequence provided to SeqWright. Gene elements in the IGE308 plasmid are illustrated in  FIGS. 25A-25B . Table 9 provides a list of gene elements with their corresponding functions. 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Size and Functions of Gene Elements in IGE308 Plasmid 
               
            
           
           
               
               
               
               
            
               
                 Location 
                 Size (bp) 
                 Element 
                 Function 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                  1-238 
                 238 
                 pFUGW backbone 
                 Backbone element 
               
               
                 239-815 
                 577 
                 CMV promoter 
                 Promoter to drive expression of the proviral genome 
               
               
                  239-1015 
                 777 
                 5′ LTR 
                 Proviral genome packaging and integration 
               
               
                 835-894 
                 60 
                 R region of 5′ LTR 
                 Repeat region in 5′ and 3′ LTRs, transcription 
               
               
                   
                   
                   
                 initiation site 
               
               
                  895-1015 
                 121 
                 U5 region of 5′ 
                 Unique 5′ sequence contains the Tat binding site 
               
               
                   
                   
                 LTR 
                 and packaging sequences of HIV 
               
               
                 1016-1125 
                 110 
                 pFUGW backbone 
                 Backbone element 
               
               
                 1126-1170 
                 45 
                 Psi packaging 
                 RNA target site for packaging the viral RNA 
               
               
                   
                   
                 signal 
                 genome into viral capsid during replication 
               
               
                 1171-1679 
                 509 
                 pFUGW backbone 
                 Backbone element 
               
               
                 1680-1913 
                 234 
                 RRE 
                 Facilitates mRNA transcript nucleus export when 
               
               
                   
                   
                   
                 bound to Rev protein 
               
               
                 1914-2443 
                 529 
                 pFUGW backbone 
                 Backbone element 
               
               
                 2444-2459 
                 16 
                 cPPT 
                 Recognition site for proviral DNA synthesis, 
               
               
                   
                   
                   
                 increases transduction efficiency and transgene 
               
               
                   
                   
                   
                 expression 
               
               
                 2460-2601 
                 142 
                 pFUGW backbone 
                 Backbone element 
               
               
                 2602-3199 
                 598 
                 CMV promoter 
                 Promoter to drive the expression of COL7A1 gene 
               
               
                   
                   
                   
                 inside the LV-COL7 vector 
               
               
                 3200-3217 
                 18 
                 5UTR 
                 Synthetic 5′UTR fragment to improve the 
               
               
                   
                   
                   
                 translation of the COL7A1 (derived from  Rahnella   
               
               
                   
                   
                   
                   aquatilis , ATCC 33071) 
               
               
                 3218-3223 
                 6 
                 Kozak 
                 Translation enhancement 
               
               
                  3224-12058 
                 8835 
                 Collagen 7A1 ORF 
                 Collagen 7A1 ORF encoding the human wild type 
               
               
                   
                   
                 with TGA stop 
                 collagen 7 protein 
               
               
                   
                   
                 codon 
               
               
                 12059-12070 
                 12 
                 Stuffer 
                 Remnant sequence resulted from DNA cloning 
               
               
                 12071-12762 
                 692 
                 3′LTR 
                 Polyadenylation signal and proviral genome 
               
               
                   
                   
                   
                 packaging and integration 
               
               
                 12071-12581 
                 511 
                 U3 region of 3′ 
                 SIN deletion (133 bp) located in this region 
               
               
                   
                   
                 LTR 
               
               
                 12245-12260 
                 16 
                 cPPT 
                 Recognition site for proviral DNA synthesis, 
               
               
                   
                   
                   
                 increases transduction efficiency and transgene 
               
               
                   
                   
                   
                 expression 
               
               
                 12582-12641 
                 60 
                 R region of 3′ LTR 
                 Repeat region in 5′ and 3′ LTRs, transcription 
               
               
                   
                   
                   
                 initiation site 
               
               
                 12642-12762 
                 121 
                 U5 region of 3′ 
                 Unique 5′ sequence contains the Tat binding site 
               
               
                   
                   
                 LTR 
                 and packaging sequences of HIV 
               
               
                 12763-12790 
                 28 
                 pFUGW backbone 
                 Backbone element 
               
               
                 12791-13018 
                 228 
                 bGHpA terminator 
                 Sequence to stop transcription of DNA 
               
               
                 13019-13080 
                 62 
                 pFUGW backbone 
                 Backbone element 
               
               
                 13081-13387 
                 307 
                 F1 origin 
                 F1 phage origin of replication 
               
               
                 13388-13948 
                 561 
                 pFUGW backbone 
                 Backbone element 
               
               
                 13949-14323 
                 375 
                 Zeocin resistance 
                 Mammalian selection marker 
               
               
                   
                   
                 gene 
               
               
                 14324-14455 
                 132 
                 pFUGW backbone 
                 Backbone element 
               
               
                 14456-14575 
                 120 
                 SV40 pA 
                 Sequence to stop transcription of DNA 
               
               
                   
                   
                 terminator 
               
               
                 14576-15009 
                 434 
                 pFUGW backbone 
                 Backbone element 
               
               
                 15010-15629 
                 620 
                 pUC origin 
                 Bacterial DNA replication start 
               
               
                 15630-15783 
                 154 
                 pFUGW backbone 
                 Backbone element 
               
               
                 15784-16644 
                 861 
                 Ampicillin 
                 Plasmid selection marker 
               
               
                   
                   
                 resistance gene 
               
               
                 16645-16685 
                 41 
                 pFUGW backbone 
                 Backbone element 
               
               
                 16686-16714 
                 29 
                 Ampicillin 
                 Promoter to drive expression of the ampicillin 
               
               
                   
                   
                 resistance promoter 
                 resistance gene 
               
               
                 16715-16777 
                 63 
                 pFUGW backbone 
                 Backbone element 
               
               
                   
               
            
           
         
       
     
     The COL7A1 gene sequence in the IGE308 plasmid (without the stop codon TGA) was compared to the GenBank  Homo sapiens  collagen, type VII, alpha 1 (COL7A1) sequence (NM_000094.3). Sequence alignment is provided in Appendix 4. Three silent point mutations were identified as listed in Table 10 below. No sequence gap was identified. The three silent point mutations have no impact on the encoded C7 protein. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Sequence Comparison of COL7A1 
               
               
                 in IGE308 and GenBank Consensus 
               
            
           
           
               
               
               
               
            
               
                 Nucleotide 
                 COL7A1 in 
                 GenBank Consensus 
                   
               
               
                 Position 
                 IGE308 
                 (NM_000094.3) 
                 Impact 
               
               
                   
               
               
                 6040 
                 G 
                 A 
                 Silent point mutation 
               
               
                 8674 
                 A 
                 G 
                 Silent point mutation 
               
               
                 8731 
                 G 
                 C 
                 Silent point mutation 
               
               
                   
               
            
           
         
       
     
     Helper Plasmids, pCMV-G, pCMV-Rev2 and pCgp 
     The three helper plasmids used for INXN-2004 LV vector production, pCMV-G, pCMV-Rev2 and pCgp, were provided by CoH. 
     293T Working Cell Bank (WCB) 
     The 293T WCB used for INXN-2004 LV vector production was provided by CoH. 
     Other Starting Materials for INXN-2004 Manufacture 
     Other starting materials used for INXN-2004 LV vector production were also provided by CoH. 
     Biopsy Tissue 
     Three 3-4 mm punch skin biopsies (dermis and epidermis layers) are collected from an un-blistered area of the RDEB subject&#39;s body using standard aseptic practices. The biopsies are collected by the treating physician, placed into a vial containing cold, sterile phosphate buffered saline (PBS), and shipped via next day delivery in a refrigerated Infectious Shipper container, which is appropriate for shipping potentially biohazardous tissue and is designed to maintain a temperature of 2-8° C. 
     a. Reagents, Solvents, and Auxiliary Materials 
     i. Reagents and Solvents 
     Two animal-sourced reagents are used in the FCX-007 Drug Substance manufacturing process: Fetal Bovine Serum (FBS) and Trypsin-EDTA solution. Complete Growth Medium 
     Complete Growth Medium (CGM) is used in FCX-007 manufacturing during all the cell culture expansion steps. CGM is prepared by mixing 18 L of IMDM in a 20 L bag with 2 L of FBS through aseptic transfer. The formulated CGM bag is stored in a 5±3° C. refrigerator. 
     a. Initiation Growth Medium 
     Initiation Growth Medium (IGM) is used in FCX-007 manufacturing for the initial seeding of fibroblasts after biopsy digestion into a T-75 cell culture flask in the presence of antibiotics. Initiation Growth Medium is prepared fresh by adding 0.6 mL of the 100-fold concentrated antibiotic solution (GA) aseptically inside an ISO5 BSC to 58.2 mL of CGM in a 500 mL square media bottle. Final concentrations are 0.4 mg/mL gentamicin, 0.295 μg/mL amphotericin B. GSH solution (1.2 mL of 50× solution) is also added to the 500 mL square media bottle. The bottles are closed and inverted 3-5 times to mix. The IGM is warmed inside a 37° C. incubator for at least 60 minutes before immediate use. 
     The Initiation Growth Medium is prepared fresh immediately prior to use and therefore no expiration date is applied and no Quality Control release tests are performed. 
     b. Transduction Medium 
     Transduction Medium™ is used during the INXN-2004 transduction of fibroblast cells. Transduction Medium is prepared fresh inside an ISO5 BSC by adding 40 mL IMDM to a 250 mL centrifuge tube containing 10 mL CGM to dilute the FBS concentration to 2% in the medium. The 250 mL tube is mixed and placed inside a 37.0° C. Incubator to warm until needed for INXN-2004 transduction. 
     The Transduction Medium is prepared fresh immediately prior to use and therefore no expiration date is applied and no Quality Control release tests are performed. 
     c. GSH Solution 
     GSH is supplemented to the Initiation Growth Medium (IGM) and CGM used in the early stage T-75 and T-175 fibroblast cell cultures to enhance cell growth. A 50×GSH stock solution is prepared inside an ISO5 BSC by dissolving 51.1 g of GSH into 1 L of IMDM. The solution is 0.22 μm filtered. The filtered solution is aliquoted into 50 mL centrifuge tubes at 20 mL per tube and stored frozen at −80° C. for further use. 
     d. RetroNectin™ Solution 
     RetroNectin™ is used in the FCX-007 production process to enhance the INXN-2004 transduction efficiency on fibroblast cells. Inside an ISO5 BSC, using a 5 mL syringe and a 18 g needle, 2.5 mL WFI is aseptically transferred into a vial of clinical grade RetroNectin® to reconstitute RetroNectin® at 1.0 mg/mL. RetroNectin® is dissolved thoroughly by swirling gently. The entire contents of the RetroNectin vial are withdrawn using the attached 5 mL syringe. The 18 g needle is aseptically replaced with a 0.22 μm syringe filter onto the syringe, and the reconstituted RetroNectin® is sterile filtered into a 250 mL centrifuge tube. Using an appropriately sized pipette, 122.5 mL PBS is transferred to the reconstituted RetroNectin® in the 250 mL centrifuge tube to dilute the RetroNectin® to 20 μg/mL, which is then mixed well using a pipette. Using an appropriately sized pipette, 6.3 mL of the diluted RetroNectin® solution is added to each of T-25 flasks to coat the surface with RetroNectin® at 5 μg/cm 2 . For the first round of INXN-2004 transduction, 5× to 10×T-25 flasks are prepared. For INXN-2004 super transduction, 18×T-25 flasks are prepared. 
     The RetroNectin™ solution is prepared fresh immediately prior to use and therefore no expiration date is applied and no Quality Control release tests are performed. 
     Cryopreservation Medium 
     Cryopreservation Medium is a two-fold concentrated solution that is added to harvested and washed fibroblasts during the FCX-007 manufacturing process to formulate the cell seed stock and FCX-007 Drug Substance for storage in liquid nitrogen. Cryopreservation Medium is prepared by mixing 0.85 volume ProFreeze™-CDM (2×) with 0.15 volume DMSO to obtain one volume Cryopreservation Medium. 
     The Cryopreservation Medium is prepared fresh immediately prior to use and therefore no expiration date is applied and no Quality Control release tests are performed. 
     Example 3: Training Run 8, 9 and 10 and Enhanced Biopsy Enzymatic Digestion 
     Training Run 8 and Enhanced Biopsy Enzymatic Digestion 
     In the above TRs, the biopsy digestion method was suspected to not be able to achieve effective digestion of the 3-4 mm sized biopsy tissue. In TR8, the application of additional shear force during the digestion process was incorporated into the process to improve the overall digestion efficiency. The digestion process was modified to the following: pulse vortex the centrifuge tube at maximum setting for 5 seconds, every 15±2 minutes during the digestion, returning the centrifuge tube to the orbital shaker after each vortex; at the end of the 60 minutes of incubation, pulse vortex the centrifuge tube at maximum setting for 10 seconds. 
     A biopsy from an RDEB donor was processed and digested using the enhanced digestion method. Cells from the digestion were seeded into a T-75 flask using culture medium supplemented with GSH. Cells showed good growth and reached 90% confluence on day 14 for passaging. To evaluate the INXN-2002 transduction conditions, cells harvested from the T-75 flask were seeded into 3×T-175 flasks, as Control, Arm A, and Arm B. Cells in all three arms were expanded, transduced with the 10 L pilot LV-COLT vector in 1-CS (the control arm was mock transduced), and then further expanded into 2×10-CS before being harvested for cryopreservation. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Cell Growth and Cell Yields in Training Run 8 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Days in 
                 Viable 
                 Cell 
                   
               
               
                 Activity 
                 Culture 
                 Cells 
                 Viability 
                 Note 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Biopsy digestion, seed into 
                 0 
                 3.4 × 10 5   
                 99% 
                 Good cell growth was 
               
               
                 one T-75 flask, with 35 mL 
                   
                   
                   
                 observed in the T-75 flask; 
               
               
                 culture medium, 37° C. and 
                   
                   
                   
                 approximately 90% 
               
               
                 5% CO 2   
                   
                   
                   
                 confluence on Day 14. 
               
               
                 Passage 1: to three T-175 
                 14 
                 6.5 × 10 6   
                 97% 
                 Flask 1: mock transduction 
               
               
                 flasks with 50 mL culture 
                   
                   
                   
                 control 
               
               
                 medium, 37° C. and 10% 
                   
                   
                   
                 Flask 2: LV transduction 
               
               
                 CO 2   
                   
                   
                   
                 (Arm A) 
               
               
                   
                   
                   
                   
                 Flask 3: LV transduction 
               
               
                   
                   
                   
                   
                 (Arm B) 
               
               
                 Passage 2: to 1-CS and LV 
               
               
                 transduction 
               
               
                 Control 
                 20 
                 1.4 × 10 7   
                 98% 
                 Cells were harvested from 
               
               
                 Arm A 
                 20 
                 1.0 × 10 7   
                 97% 
                 each of the T-175 flask 
               
               
                 Arm B 
                 20 
                 1.0 × 10 7   
                 98% 
                 and expanded into 
               
               
                   
                   
                   
                   
                 corresponding 1-CS for 
               
               
                   
                   
                   
                   
                 LV-COL7 transduction 
               
               
                   
                   
                   
                   
                 (CoH Pilot LV-COL7). 
               
               
                 Passage 3: to 10-CS 
               
               
                 Control 
                 26 
                 6.1 × 10 7   
                 99% 
                 Cells from each arm were 
               
               
                 Arm A 
                 27 
                 6.6 × 10 7   
                 99% 
                 harvested and expanded to 
               
               
                 Arm B 
                 26 
                 6.4 × 10 7   
                 99% 
                 one corresponding 10-CS. 
               
               
                 Passage 4: to 2x 10-CS 
               
               
                 Control 
                 35 
                 4.0 × 10 8   
                 99% 
                 Cells from each arm were 
               
               
                 Arm A 
                 39 
                 3.3 × 10 8   
                 98% 
                 harvested and expanded to 
               
               
                 Arm B 
                 38 
                 3.8 × 10 8   
                 98% 
                 two corresponding 10-CS. 
               
               
                 Harvest: 2x10-CS 
               
               
                 Control 
                 45 
                 6.9 × 10 8   
                 95% 
                 Cells were harvested from 
               
               
                 Arm A 
                 47 
                 4.0 × 10 8   
                 98% 
                 each of the 2x10-CS for 
               
               
                 Arm B 
                 46 
                 5.3 × 10 8   
                 97% 
                 cryopreservation and testing. 
               
               
                   
               
            
           
         
       
     
     The total number of cells harvested from the 10-CS was considered adequate for further production of FCX-007 drug product for injection. The harvested cells were tested for COL7A1 gene copy number (transduction efficiency), C7 protein expression, cell viability, and cell purity. Table 12 provides the analysis results for the harvested cells from all three arms. 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Analysis of Cells Harvested from Training Run 8 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 INXN-2002 
                 COL7 
                 C7 protein 
                   
                 Cell 
                   
               
               
                   
                 Transduction 
                 A1gene 
                 expression 2   
                 Purity 2   
                 Viability 
                   
               
               
                 Condition 
                 MOI (IU/cell) 1   
                 copy/cell 2   
                 (ng/mL) 
                 (% CD90 + ) 
                 (%) 
                 Endotoxin 
               
               
                   
               
               
                 Arm A 
                 3.4 
                 0.021 
                 59.4 
                 100% 
                 95% 
                 Pass 
               
               
                 Arm B 
                 1.7 
                 0.017 
                 46.6 
                 100% 
                 93% 
                 Pass 
               
               
                 Control 
                 Mock 
                 BLQ 3   
                 BLQ 3   
                 100% 
                 96% 
                 Pass 
               
               
                   
               
               
                   1 INXN-2002 IU titer: 9.2 × 10 6  IU/mL which was determined at Intrexon using an early development stage H1299 infectious titer assay. 
               
               
                   2 Details of the assays are provided in Section 3.2.S.4. 
               
               
                   3 BLQ: below limit of quantitation 
               
            
           
         
       
     
     A Multiplicity of Infection (MOI) dose dependent increase in COL7A1 gene copy number and C7 protein expression was observed. Details of the INXN-2002 transduction development and optimization are described below. Although low INXN-2002 IU titer limited the transduction MOI, and the subsequent COL7A1 gene copy number, C7 protein expression was observed to be biologically relevant in the preclinical setting as evidenced by the in vitro and in vivo results. Overall, the results show that RDEB fibroblast cells can be transduced with INXN-2002 and express functional C7 protein. 
     The Arm A cell product from TR8 was used for Proof of Concept studies and toxicology and biodistribution studies. 
     Training Run 9 
     A biopsy from an RDEB donor was processed and digested using the enhanced digestion method. Cells from the digestion were seeded into a T-75 flask using culture medium supplemented with GSH. Similar to TR8, cells showed good growth and reached 90% confluence on Day 19 for passaging. To evaluate the LV-COLT transduction conditions, cells harvested from the T-75 flask were seeded into 3×T-175 flasks, as Control, Arm A, and Arm B. Cells in all three arms were expanded, transduced with LV-HA-COLT vector (containing an HA tag in the construct) in 1-CS (the control arm was mock transduced), then further expanded into 2×10-CS before being harvested. The harvested cells were tested for COL7A1 gene copy number (transduction efficiency), C7 protein expression, and cell purity. Table 13 provides the cell growth in TR9. 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 Cell Growth and Cell Yields in Training Run 9 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Days in 
                 Viable 
                 Cell 
                   
               
               
                 Activity 
                 Culture 
                 Cells 
                 Viability 
                 Note 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Biopsy digestion, seed into 
                 0 
                 NA 
                 NA 
                 Good cell growth was 
               
               
                 one T-75 flask, with 35 mL 
                   
                   
                   
                 observed in the T-75 flask; 
               
               
                 culture medium, 37° C. and 
                   
                   
                   
                 approximately 90% 
               
               
                 5% CO 2   
                   
                   
                   
                 confluence on Day 19. 
               
               
                 Passage 1: to three T-175 
                 19 
                 4.5 × 10 6   
                 99% 
                 Flask 1: mock transduction 
               
               
                 flasks with 50 mL culture 
                   
                   
                   
                 control 
               
               
                 medium, 37° C. and 10% 
                   
                   
                   
                 Flask 2: LV transduction 
               
               
                 CO 2   
                   
                   
                   
                 (Arm A) 
               
               
                   
                   
                   
                   
                 Flask 3: LV transduction 
               
               
                   
                   
                   
                   
                 (Arm B) 
               
               
                 Passage 2: to 1-CS and LV 
               
               
                 transduction 
               
               
                 Control 
                 26 
                 1.0 × 10 7   
                 99% 
                 Cells were harvested from 
               
               
                 Arm A 
                 25 
                 7.1 × 10 6   
                 97% 
                 each of the T-175 flask and 
               
               
                 Arm B 
                 25 
                 7.8 × 10 6   
                 98% 
                 expanded into corresponding 
               
               
                   
                   
                   
                   
                 1-CS for LV-COL7 
               
               
                   
                   
                   
                   
                 transduction (CoH Pilot LV- 
               
               
                   
                   
                   
                   
                 HA-COL7, a research vector)  1.   
               
               
                 Passage 3: to 10-CS 
               
               
                 Control 
                 33 
                 3.8 × 10 7   
                 99% 
                 Cells from each arm were 
               
               
                 Arm A 
                 32 
                 5.3 × 10 7   
                 98% 
                 harvested and expanded to 
               
               
                 Arm B 
                 31 
                 2.5 × 10 7   
                 97% 
                 one corresponding 10-CS. 
               
               
                 Passage 4: to 2x 10-CS 
               
               
                 Control 
                 46 
                 2.6 × 10 8   
                 97% 
                 Cells from each arm were 
               
               
                 Arm A 
                 42 
                 3.6 × 10 8   
                 98% 
                 harvested and expanded to 
               
               
                 Arm B 
                 45 
                 2.8 × 10 8   
                 97% 
                 two corresponding 10-CS. 2   
               
               
                 Harvest: 2x10-CS 
               
               
                 Control 
                 61 
                 7.6 × 10 8   
                 100%  
                 Cells were harvested from 
               
               
                 Arm A 
                 53 
                 5.3 × 10 8   
                 97% 
                 each of the 2x10-CS for 
               
               
                 Arm B 
                 60 
                 5.5 × 10 8   
                 98% 
                 cryopreservation and testing. 
               
               
                   
               
               
                   1  LV-HA-COL7 vector is a research vector which incorporated an HA tag to the COL7A1 sequence in the INXN-2002 LV vector. 
               
               
                   2 The total number of cells harvested from the 10-CS is considered adequate for further production of FCX-007 drug product for injection. 
               
            
           
         
       
     
     Table 14 provides the analysis results for the harvested cells from all three arms. 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 Analysis of Cells Harvested from Training Run 9 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 LV-HA-Col7 
                 Col7 
                 Col7 
                   
                   
                   
               
               
                   
                 Transduction 
                 Gene 
                 Protein 
                 Purity 2   
                 Cell 
                   
               
               
                 Condi- 
                 MOI 
                 Copy/ 
                 Expression 
                 (% 
                 Viability 
                 Endo- 
               
               
                 tion 
                 (IU/cell) 1   
                 Cell 2   
                 (ng/mL) 
                 CD90 + ) 
                 (%) 
                 toxin 
               
               
                   
               
               
                 Arm A 
                 1.0 
                 0.009 
                 71.4 
                 100% 
                 92% 
                 Pass 
               
               
                 Arm B 
                 1.9 
                 0.021 
                 91.6 
                 99% 
                 95% 
                 Pass 
               
               
                 Control 
                 Mock 
                 BLQ 3   
                 BLQ 3   
                 100% 
                 92% 
                 Pass 
               
               
                   
               
               
                   1 LV-HA-Col7 vector titer: 2.0 × 10 6  IU/mL which was determined at Intrexon using an early development stage H1299 infectious titer assay. 
               
               
                   2 Details of the assays are provided in Section 3.2.S.4. 
               
               
                   3 BLQ: below limit of quantitation 
               
            
           
         
       
     
     An MOI dose dependent increase in COL7A1 gene copy number and C7 protein expression was again observed. Again, the results show that RDEB fibroblast cells can be successfully grown, transduced with LV-HA-COLT vector, and express C7 protein. 
     Training Run 10 and Scale-Up to Six 10-CS 
     According to the proposed clinical protocol (See Module 5), 4×10 8  FCX-007 drug product cells are needed for a single treatment dose, with a possibility of repeat dosing. To meet the projected FCX-007 product needs, it is necessary to scale-up the final cell expansion step to six 10-CSs based on the cell yields attained in TR8 and TR9. 
     A biopsy from an RDEB donor was processed and digested using the enhanced digestion method. Cells from the digestion were seeded into a T-75 flask using culture medium supplemented with GSH. Cells showed good growth and reached 90% confluence on Day 19 for passaging. Cells harvested from the T-75 flask were seeded into 2×T-175 flasks, one for INXN-2002 transduction, and one for cell substrate control. Cells from one flask were expanded, transduced with INXN-2002 in 1-CS, and then further expanded into 6×10-CS before being harvested. Table 15 provides the cell growth in TR10. 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 Cell Growth and Cell Yields in Training Run 10 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Days in 
                 Viable 
                 Cell 
                   
               
               
                 Activity 
                 Culture 
                 Cells 
                 Viability 
                 Note 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Biopsy digestion, seed into 
                 0 
                 NA 
                 NA 
                 Good cell growth was 
               
               
                 one T-75 flask, with 35 mL 
                   
                   
                   
                 observed in the T-75 flask; 
               
               
                 culture medium, 37° C. and 
                   
                   
                   
                 approximately 90% 
               
               
                 5% CO 2   
                   
                   
                   
                 confluence on Day 19. 
               
               
                 Passage 1: to two T-175 
                 19 
                 3.5 × 10 6   
                 98% 
                 Flask 1: LV transduction 
               
               
                 flasks with 50 mL culture 
                   
                   
                   
                 Flask 2: control cells for analysis 
               
               
                 medium, 37° C. and 10% 
               
               
                 CO 2   
               
               
                 Passage 2: to 1-CS and LV 
                 30 
                 4.6 × 10 6   
                 96% 
                 Cells from Flask 1 were harvested 
               
               
                 transduction 
                   
                   
                   
                 and expanded into 1- 
               
               
                   
                   
                   
                   
                 CS for LV-COL7 transduction 
               
               
                   
                   
                   
                   
                 (GMP grade INXN-2002). 
               
               
                 Passage 3: to 10-CS 
                 38 
                 2.1 × 10 7   
                 97% 
                 Cells were harvested and 
               
               
                   
                   
                   
                   
                 expanded to one 10-CS. 
               
               
                 Passage 4: to 6x 10-CS 
                 60 
                 1.9 × 10 8   
                 95% 
                 Cells were harvested and 
               
               
                   
                   
                   
                   
                 expanded to six 10-CS. 
               
               
                 Harvest: 6x 10-CS 
                 75 
                 5.8 × 10 8   
                 99% 
                 Cells were harvested from 
               
               
                   
                   
                   
                   
                 the 6x10-CS for 
               
               
                   
                   
                   
                   
                 cryopreservation and testing. 
               
               
                   
               
            
           
         
       
     
     Relative to cells in TR8 and TR9, cells in TR10 grew slower and yielded fewer cells at each passage step, indicating potential variability among different RDEB donors. 
     The total number of cells harvested from the 6×10-CS is considered adequate for production of one dosage of FCX-007 drug product for injection. 
     Table 16 provides the analysis results for the harvested cells. 
     
       
         
           
               
             
               
                 TABLE 16 
               
             
            
               
                   
               
               
                 Analysis of Cells Harvested from TR10 
               
            
           
           
               
               
               
               
               
               
            
               
                 INXN-2002 
                   
                   
                   
                 Cell 
                   
               
               
                 Trans- 
                 COL7 
                 C7 Protein 
                 Purity 2   
                 Via- 
               
               
                 duction MOI 
                 Gene 
                 Expression 2   
                 (% 
                 bility 
                 Endo- 
               
               
                 (IU/cell) 1   
                 Copy/Cell 2   
                 (ng/mL) 
                 CD90 + ) 
                 (%) 
                 toxin 
               
               
                   
               
               
                 2.0 
                 0.013 
                 60.09 
                 100% 
                 92% 
                 Pass 
               
               
                   
               
               
                   1 INXN-2002 IU titer: 9.2 × 10 6  IU/mL which was determined at Intrexon using an early development stage H1299 infectious titer assay. 
               
               
                   2 Details of the assays are provided in Section 3.2.S.4. 
               
            
           
         
       
     
     Example 4 
     INXN-2002 Transduction Development and Optimization 
     Lentiviral transduction of dermal fibroblast cells was initially developed and optimized using normal human dermal fibroblast cells (NHDF; Lonza, CC-2511) cultured in 96-well plates with a model lentiviral GFP (GeneCopoeia, LP-EGFP-LV105-0205). 
     Using GeneCopoeia&#39;s LV transduction protocol as a starting point, initial optimization evaluated conditions including cell density at time of transduction, transduction culture volume, use of RetroNectin™, super-infection (re-transducing cells with virus on two consecutive days), time of cell plating (at time of transduction versus one day prior to transduction), and serum content in transduction media. The optimal transduction procedure was then implemented in a GMP production setting. Details of the studies are described below. 
     A. Effect of Cell Seeding Conditions and RetroNectin™ Coating for LV Transduction 
     NHDFs in exponential growth phase were harvested and seeded into 96-well plates on two different dates at different seeding densities. One set of non tissue culture treated 96-well plates were coated with RetroNectin™ per the manufacturer&#39;s recommendation (the RetroNectin™ coating density was 20 μg/cm 2 ). For cells seeded one day before LV transduction (Day −1), the spent medium is removed and fresh medium (100 μL) with the LV vector is added to the wells on Day 0 for transduction. For the day of transduction condition (Day 0), cells and LV vector in 100 μL of fresh medium is added simultaneously to the wells. The MOI used was 2000 vp/cell for all conditions. After an overnight incubation, the medium was removed and the cells were fed with 100 μL fresh medium for further culture. At ninety-six hours post-transduction, the cells were harvested for transduction efficiency analysis by FACS analysis of GFP signal on a BD LSRII and analyzed using FlowJo software (v.10). Table 17 provides the LV transduction efficiencies under the different conditions. 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 Effect of Cell Seeding Condition and RetroNectin ™ Coating 
               
               
                 on LV Transduction of Fibroblast Cells (% GFP Positive Cells) 
               
            
           
           
               
               
               
            
               
                   
                 Without RetroNectin ™ 
                 With RetroNectin ™ 
               
               
                 Cell Seeding 
                 Coating 
                 Coating 
               
            
           
           
               
               
               
               
               
            
               
                 Density (Cells/Well; 
                 Day −1 Cell 
                 Day 0 Cell 
                 Day −1 Cell 
                 Day 0 Cell 
               
               
                 Cells/Cm 2 ) 1   
                 Seeding 
                 Seeding 
                 Seeding 
                 Seeding 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 3000; 9375 
                 6.8% 
                 11.2% 
                 NC 
                 22.6% 
               
               
                 1500; 4688 
                 3.3% 
                 7.2% 
                 NC 
                 18.2% 
               
               
                   
               
               
                   1 Surface area for a 96-well plate well: 0.32 cm 2   
               
               
                 NC = Not conducted 
               
            
           
         
       
     
     The results demonstrate that it is not necessary to pre-seed the cells on the day before LV transduction. Cells and LV vector can be added simultaneously at the time of transduction, which is convenient for GMP manufacturing operations. Coating the culture surface with RetroNectin™ prior to LV transduction significantly increased the LV transduction efficiency. The higher cell seeding density of approximately 1×10 4  cells/cm 2  is desired during LV transduction, as the lower cell seeding density resulted in lower LV transduction efficiency. 
     B. Effect of MOI and Super-Transduction on LV Transduction Efficiency 
     In this study, the effects of MOI and super-transduction on LV transduction of fibroblast cells were examined. 
     A set of non tissue culture treated 96-well plates was pre-coated with RetroNectin™ (20 μg/cm 2 ) before being used for transduction. At the time of transduction, 3000 cells with LV vector in a volume of 100 μL were added to a well. After an overnight incubation, the medium was removed and the cells were fed with 100 μL fresh medium for further culture. For the super-transduction, the culture medium was removed from the wells one day after the initial transduction (approximately 24 hours), and the same amount of LV vector in 100 μL fresh medium was added to the wells. After 3 hours of transduction, the medium was removed and the cells were fed with 100 μL fresh medium for further culture. Ninety-six hours post-transduction, the cells were harvested for transduction efficiency analysis by FACS analysis of GFP signal on a BD LSRII and analyzed using FlowJo software (v.10). Table 18 provides the LV transduction efficiencies under the different conditions. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 Effect of MOI and Super-Transduction on LV Transduction 
               
               
                 of Fibroblast Cells (% GFP Positive Cells) 
               
            
           
           
               
               
               
            
               
                 MOI (vp/cell) 
                 Without Super-Transduction 
                 With Super-Transduction 
               
               
                   
               
            
           
           
               
               
               
            
               
                 0 
                 1.6% 
                 2.0% 
               
               
                 200 
                 4.3% 
                 5.8% 
               
               
                 500 
                 7.7% 
                 10.0% 
               
               
                 1000 
                 15.3% 
                 16.3% 
               
               
                 2000 
                 22.6% 
                 27.5% 
               
               
                   
               
            
           
         
       
     
     An increasingly higher MOI resulted in higher transduction efficiencies with and without super-transduction. Super-transduction resulted in an incremental increase in LV transduction efficiency. However, the increase was not considered significant, and was not implemented in the GMP production setting. 
     C. LV Transduction Culture Volume and FBS Concentration 
     Lentiviral vector particles first need to make contact with fibroblast cells in culture in order to achieve transduction. Like other viral particles, LV particles in solution follow Brownian motion, and a productive transduction is generally a random event. Reduction of culture medium depth to a minimum volume or the use of spino-transduction has been shown to improve viral vector transduction on target cells (Nyberg-Hoffman 1997). The effect of transduction volume/culture depth and medium FBS concentration on LV transduction of fibroblast cells were evaluated. 
     A set of non tissue culture treated 96-well plates was pre-coated with RetroNectin™ (20 μg/cm 2 ) before being used for transduction. At the time of transduction, 3000 cells with LV vector in a volume of 100 μL or 50 μL of medium with 10% FBS were added to the wells. The same transduction conditions were repeated in medium with 2% FBS. After an overnight incubation, the medium was removed and the cells were all fed with 100 μL fresh medium with 10% FBS for further culture. The MOI used was 2000 vp/cell for all conditions. Ninety-six hours post-transduction, the cells were harvested for transduction efficiency analysis by FACS analysis of GFP signal on a BD LSRII and analyzed using FlowJo software. Table 19 provides the LV transduction efficiencies under the different conditions. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 Effect of Culture Volume (Depth) and FBS 
               
               
                 on LV Transduction (% GFP Positive Cells) 
               
            
           
           
               
               
               
            
               
                 Transduction 
                   
                   
               
               
                 Medium Volume 
                 Transduction 
               
               
                 in 96-well plate 
                 Medium Depth 
                 Transduction Medium FBS % 
               
            
           
           
               
               
               
               
            
               
                 (μL) 
                 (mm) 
                 2% 
                 10% 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 50 
                 1.6 
                 46.7% 
                 46.2% 
               
               
                 100 
                 3.1 
                 31.8% 
                     27% 
               
               
                   
               
            
           
         
       
     
     As expected, reducing the transduction medium volume (depth) resulted in a noticeable increase in LV transduction efficiency. Additionally, results indicate that using a 2% FBS transduction medium is beneficial for LV transduction. Based on these results, a 2% FBS transduction medium was incorporated into the GMP manufacturing process, while keeping the transduction medium volume to a minimum, approximately 60 mL in 1-CS. In the GMP manufacturing process for FCX-007, a 1-layer CellSTACK® with a surface area of 636 cm 2  will be used for LV transduction of fibroblast cells. It was determined that it is feasible to use a transduction volume of 60 mL (depth 0.9 mm) without causing detrimental effect (e.g. drying) on the cells during the 3 hours transduction period. 
     D. Evaluations of RetroNectin™ Coating and the Type of Culture Surface 
     RetroNectin™ coating of the culture surface significantly increased the LV transduction of fibroblast cells. The RetroNectin™ manufacturer recommends a RetroNectin™ coating amount in the range of 4 μg/cm 2  to 20 μg/cm 2 . To minimize the amount of RetroNectin™ used without negatively impacting the LV transduction of fibroblast cells, the amount of RetroNectin™ used for coating was evaluated. 
     Furthermore, the RetroNectin™ manufacturer recommends use of a non tissue culture treated plastic surface for RetroNectin™ coating. However, culture flasks and CellSTACKs® used for fibroblast cell culture are all tissue culture-treated. Both tissue culture treated and non tissue culture treated 96-well plates were coated with different amounts of RetroNectin™ and evaluated for LV transduction of fibroblast cells. 
     Both non tissue culture treated and tissue culture treated 96-well plates were pre-coated with different amounts of RetroNectin™ (20 μg/cm 2 , 10 μg/cm 2 , and 5 μg/cm 2 ) before being used for transduction. At the time of transduction, 3000 cells with LV vector in a volume of 50 μL of transduction medium with 2% FBS were added to each well. After 3 hours transduction, the medium was removed and the cells were all fed with 100 μL fresh medium with 10% FBS for further culture. The MOI used was 2000 vp/cell for all conditions. Ninety-six hours post-transduction, the cells were harvested for transduction efficiency analysis by FACS analysis of GFP signal on a BD LSRII and analyzed using FlowJo software. Table 20 provides the LV transduction efficiencies under the different conditions. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 Effect of RetroNectin ™ Amount and Culture Surface Type on 
               
               
                 LV Transduction of Fibroblast Cells (% GFP Positive Cells) 
               
            
           
           
               
               
            
               
                   
                 Amount of RetroNectin ™ Used for Coating 
               
               
                   
                 (μg/cm 2 ) 
               
            
           
           
               
               
               
               
            
               
                 Type of Culture Surface 
                 5 
                 10 
                 20 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Tissue culture treated 
                 27.8% 
                 25.8% 
                 28.1% 
               
               
                 Non tissue culture treated 
                 38.2% 
                 38.8% 
                 38.5% 
               
               
                   
               
            
           
         
       
     
     Comparable efficiencies of LV transduction of fibroblast were observed for all three doses of RetroNectin™ used for coating. As a result, a RetroNectin™ coating density of 5 μg/cm 2  was selected for use in the FCX-007 production process. Although lower LV transduction efficiency was observed in the tissue culture treated surface, the difference is not considered significant enough to prevent the use of tissue culture treated flasks in the INXN-2002 transduction step of the FCX-007 manufacturing process. 
     Summary of the Development and Optimization of Fibroblast LV Transduction 
     Based on the study results described above using the LV-GFP model vector for fibroblast cell transduction, the following LV transduction protocol was selected for INXN-2002 transduction of fibroblast cells derived from RDEB donor: 
     Pre-coat the culture surface with RetroNectin™ at 5 μg/cm 2 . Tissue culture treated flasks can be used for RetroNectin™ coating. 
     Add cells and LV vector simultaneously at the time of transduction, with a cell seeding density of approximately 1×10 4  cells/cm 2 . 
     Use a high MOI which does not cause toxic effect on cells to achieve high transduction efficiency (10 mL of INXN-2002 LV-Col7 vector per transduction). 
     Transduce for 3 hours at 37° C. in a transduction medium with 2% FBS, and then change or feed cells with a medium which contains 10% FBS. 
     Use a low transduction medium volume, 50 μL/well for a 96-well plate or 60 mL for a 1-layer CellSTACK®, for high transduction efficiency. 
     Super-transduction is not required. 
     F. INXN-2002 L V Transduction of Fibroblast Cells 
     Based on the LV transduction protocol developed above, INXN-2002 was used to transduce normal human fibroblast cells (NHDF, Lonza, CC-2511) in a 96-well plate. Considering the relative low titer of the INXN-2002, three doses of INXN-2002 were used for transduction, 12.5 μL (1:4 dilution), 3.1 μL (1:16 dilution), and 0.8 μL (1:64 dilution). The transduced cells were passaged three times to ensure stable integration of the LV-COLT vector into the transduced cell genome. At passage 3, genomic DNA was extracted from the cells and analyzed by qPCR using a primer specific to the LV-COLT vector sequence. The transduction efficiency was quantified as gene copy numbers per cell. Table 21 provides the transduction efficiency as measured by gene copy number per cell. 
     
       
         
           
               
             
               
                 TABLE 21 
               
             
            
               
                   
               
               
                 INXN-2002 Transduction of Fibroblast Cells 
               
            
           
           
               
               
            
               
                 INXN-2002 Vector Dosing 1   
                 Gene 
               
            
           
           
               
               
               
               
            
               
                 Dilution 
                 Vector volume per well 
                 Calculated MOI 
                 Copy Number 
               
               
                 factor 
                 of 96-well plate (μL) 
                 (IU/cell) 
                 per Cell 2   
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1/4  
                 12.5 
                 40 
                 0.08 
               
               
                 1/16 
                 3.1 
                 10 
                 0.86 
               
               
                 1/64 
                 0.8 
                 2.5 
                 0.11 
               
               
                   
               
               
                   1 INXN-2002 IU titer: 9.2 × 10 6  IU/mL which was determined at Intrexon using an early development stage H1299 infectious titer assay. 
               
               
                   2 Gene copy number per cell was determined at Intrexon using an early development stage qPCR assay, which resulted in an approximately 10-fold higher gene copy number compared to the fully developed assay that was transferred to BioReliance for FCX-007 product analysis. The difference was due to the use of a different qPCR primer set and circular plasmid standards in the early development stage assay. 
               
            
           
         
       
     
     At the highest dose of INXN-2002 vector, ¼ dilution (MOI=40), a significant toxic effect on cells were observed: most of the cells did not recover from the transduction step, resulting in a minimal gene copy number per cell at the end of the cell expansion. The optimal INXN-2002 MOI was approximately 10 IU/cell, resulting in a gene copy number of 0.86. 
     INXN-2002 LV Transduction in Training Runs 
     Following the LV transduction protocol (Section [0260]) and the INXN-2002 transduction vector dosing/MOI results from the 96-well studies, INXN-2002 transduction of RDEB fibroblast cells was conducted at scale in TR8, TR9, and TR10. The 1-layer CellSTACK® used for INXN-2002 transduction was pre-coated with RetroNectin™ at 5 μg/cm 2 . At the time of transduction, cells and INXN-2002 were added simultaneously in 60 mL of transduction medium with 2% FBS to the 1-layer CellSTACK®. After 3 hours in the incubator at 37° C., the cells were fed with 130 mL of complete growth medium with 10% FBS. The transduced cells were passaged twice, first to one 10-layer CellSTACK®, and then to two or six 10-layer CellSTACKs® for cell harvest. Gene copy numbers in the harvested cells were analyzed by qPCR. Table 22 provides the INXN-2002 transduction results from the executed Training Runs. 
     
       
         
           
               
             
               
                 TABLE 22 
               
             
            
               
                   
               
               
                 Summary of LV-COL7 Transduction in Training Runs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                 LV-COL7 
                   
                 Amount of Vector 
                   
                   
                 Gene 
               
               
                   
                 LV- 
                 Vector 
                   
                 Used for 
                 Number of 
                   
                 Copy 
               
               
                 Training 
                 COL7 
                 Titer 
                   
                 Transduction (mL) 
                 Cells for 
                 MOI 1   
                 per 
               
               
                 Run 
                 Vector 
                 (IU/mL) 
                 Arm 
                 (dilution factor) 
                 Transduction 
                 (IU/cell) 
                 Cell 2   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 8 
                 INXN- 
                 9.2 × 10 6   
                 A 
                 3.75 (1/16) 
                 1.0 × 10 7   
                 3.4 
                 0.021 
               
               
                   
                 2002 LV- 
                   
                 B 
                 1.88(1/32) 
                 1.0 × 10 7   
                 1.7 
                 0.017 
               
               
                   
                 COL7 
                   
                 Control 
                 0 
                 1.4 × 10 7   
                 0 
                 BLQ 1   
               
               
                   
                 (Pilot) 
                   
                   
                   
                   
                   
                   
               
               
                 9 
                 LV-HA- 
                 2.0 × 10 6   
                 A 
                 3.75 (1/16) 
                 7.1 × 10 6   
                 1.0 
                 0.009 
               
               
                   
                 COL7 
                   
                 B 
                 7.5 (1/8) 
                 7.4 × 10 6   
                 1.9 
                 0.021 
               
               
                   
                 (Pilot) 
                   
                 Control 
                 0 
                 1.0 × 10 7   
                 0 
                 BLQ 3   
               
               
                 10 
                 INXN- 
                 2.3 × 10 6   
                 TR10 
                 4 (1/15) 
                 4.6 × 10 6   
                 2.0 
                 0.013 
               
               
                   
                 2002 LV- 
                   
                   
                   
                   
                   
                   
               
               
                   
                 COL7 
                   
                   
                   
                   
                   
                   
               
               
                   
                 (GMP) 
               
               
                   
               
               
                   1 MOI was determined based on the INXN-2002 LV-Col7 vector IU titer determined at Intrexon using an early development stage H1299 infectious titer assay, which resulted in approximately 10-fold higher titer value compared to the fully developed assay that was transferred to BioReliance for FCX-007 product analysis. The difference was due to the use of a different qPCR primer set and circular plasmid standards in the early development stage assay. 
               
               
                   2 Gene copy per cell was quantified using the fully developed qPCR assay, which was transferred to BioReliance for FCX-007 product analysis 
               
               
                   3 BLQ: Below the limit of quantitation 
               
            
           
         
       
     
     Following the LV transduction protocol developed using the small scale 96-well plates, successful INXN-2002 transduction of RDEB fibroblast cells was achieved at scale for FCX-007 manufacture. Gene copy numbers in the transduced cells increased with an increased MOI of transduction. Because of the low titer of INXN-2002, gene copy numbers in the transduced, harvested cell product were relatively low. In order to produce FCX-007 cell product with a feasibly high gene copy number, 10 mL of INXN-2002 was selected for GMP production transduction, as it caused minimal toxic effects on cells during transduction. 
     Engineering Run 
     In preparation for GMP manufacturing of FCX-007, and finalization of the production Master Batch Records (MBR), an engineering run was executed using a biopsy from an RDEB donor with approved production batch records. The GMP grade INXN-2002 LV vector was used for transduction. Table 23 provides cell growth figures from the Engineering Run. 
     
       
         
           
               
             
               
                 TABLE 23 
               
             
            
               
                   
               
               
                 Cell Growth and Cell Yields in Engineering Run 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Days in 
                 Viable 
                 Cell 
                   
               
               
                 Activity 
                 Culture 
                 Cells 
                 Viability 
                 Note 
               
               
                   
               
               
                 Biopsy digestion, seed into one T-75 
                 0 
                 9.6 × 
                 92% 
                 Good cell growth in the T-75 flask, 
               
               
                 flask, with 35 mL culture medium, 
                   
                 10 5   
                   
                 reached approximately 90% 
               
               
                 37° C. and 5% CO 2   
                   
                   
                   
                 confluence on Day 19 
               
               
                 Passage 1: to one T-175 flask and one 
                 20 
                 3.5 × 
                 99% 
                 T-175 Flask: LV transduction 
               
               
                 T-25 flask, 37° C. and 10% CO 2   
                   
                 10 6   
                   
                 T-25 Flask: control cells for analysis 
               
               
                 Passage 2: to 1-CS and LV 
                 29 
                 9.9 × 
                 98% 
                 Cells from T-175 Flask were 
               
               
                 transduction 
                   
                 10 6   
                   
                 harvested and expanded into 1-CS 
               
               
                   
                   
                   
                   
                 for LV-COL7 transduction (GMP 
               
               
                   
                   
                   
                   
                 grade INXN-2002) 
               
               
                 Passage 3: to 10-CS 
                 38 
                 3.3 × 
                 98% 
                 Cells were harvested and expanded 
               
               
                   
                   
                 10 7   
                   
                 to one 10-CS. 
               
               
                 Passage 4: to 6 × 10-CS 
                 49 
                 3.1 × 
                 96% 
                 Cells were harvested and expanded 
               
               
                   
                   
                 10 8   
                   
                 to six 10-CS. 
               
               
                 Harvest: 6 × 10-CS 
                 60 
                 9.6 × 
                 97% 
                 Cells were harvested from the 6 × 10- 
               
               
                   
                   
                 10 8   
                   
                 CS for cryopreservation and testing 
               
               
                   
               
            
           
         
       
     
     Cell growth and yields from the ER are comparable to those in TR8 and TR9, and are noticeably faster and higher relative to TR10, indicating cell growth variability among different RDEB donors. The harvested cells were filled and cryopreserved as shown in Table 24. 
     
       
         
           
               
             
               
                 TABLE 24 
               
             
            
               
                   
               
               
                 FCX-007 Drug Substance Vials Filled in Engineering Run 
               
            
           
           
               
               
               
               
            
               
                   
                 Fill 
                 Number of 
                   
               
               
                 Vials 
                 Volume (mL) 
                 Vials Filled 
                 Purpose 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 2 mL cryovial 
                 1.2 
                 10 
                 Bulk Drug Substance 
               
               
                 5 mL cryovial 
                 4.5 
                 6 
                 Bulk Drug Substance 
               
               
                 2 mL cryovial 
                 1.2 
                 2 
                 Sterility test 
               
               
                 2 mL cryovial 
                 0.6 
                 4 
                 QC test 
               
               
                 2 mL cryovial 
                 0.6 
                 6 
                 Bulk Drug Substance 
               
               
                   
                   
                   
                 Stability Test 
               
               
                   
               
            
           
         
       
     
     The total number of cells harvested from the 6×10-CS is considered adequate for production of two doses of FCX-007 drug product for injection. 
     The harvested cells were tested according to the proposed FCX-007 Drug Substance Specifications. Table 25 provides the analysis results for the harvested cells. 
     
       
         
           
               
             
               
                 TABLE 25 
               
             
            
               
                   
               
               
                 FCX-007 Drug Substance Engineering Run Analysis 
               
            
           
           
               
               
               
               
               
               
            
               
                 Test 1   
                 Test Method 
                 Lab 
                 SOP 
                 Specifications 
                 Test Result 
               
               
                   
               
               
                 
                   Mycoplasma 
                 
                 USP&lt;63&gt; 
                 BioReliance 
                 102063GMP.BSV 
                 Negative 
                 Negative 
               
               
                 Replication Competent 
                 C8166 cell 
                 BioReliance 
                 009130GMP.BUK 
                 No RCL detected 
                 ND 4   
               
               
                 Lentivirus 
               
               
                 Sterility 
                 USP&lt;71&gt; 
                 BioReliance 
                 510120GMP.BSV 
                 No Growth 
                 No growth 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Endotoxin 
                 Endo safe 
                 PCT 
                 SOP-0249 
                 ≤5.0 EU/mL 
                 &lt;2.00 
                 EU/mL 
               
               
                 Cell Count 
                 Hemacytometer 
                 PCT 
                 SOP-0329 
                 1.0-3.0 × 10 7   
                 1.6 × 10 7   
                 cells/mL 
               
               
                   
                   
                   
                   
                 cells/mL 
               
            
           
           
               
               
               
               
               
               
            
               
                 Cell Viability 
                 Hemacytometer 
                 PCT 
                 SOP-0329 
                 ≥85% viability 
                  94% 
               
               
                 Purity 
                 FACS Calibur 
                 PCT 
                 SOP-1067 
                 ≥98% CD 90 +   
                 100% 
               
               
                 Residual VSV-G Protein 
                 VSV-G ELISA 
                 Intrexon 2   
                 VSV-G ELISA 
                 Report Result 
                 BLQ 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 COL7A Gene Copy Number 
                 qPCR 
                 Intrexon 2   
                 COL7A Gene Copy 
                 Report Result 
                 0.025 
                 copies/cell 
               
               
                   
                   
                   
                 Number 
               
               
                 COL7A Gene Expression 
                 Col 7 ELISA 
                 Intrexon 3   
                 COL7A Gene 
                 Report Results 
                 66.4 
                 ng/mL 
               
               
                   
                   
                   
                 Expression 
               
               
                   
               
               
                   1 Details of the assays are provided in Section 3.2.S.4.2. 
               
               
                   2 Intrexon in house assay. Assays are being transferred to BioReliance for GMP release test 
               
               
                   3 Intrexon in house assay. Assays are being transferred to PCT for GMP release test 
               
               
                   4 RCL testing was not performed on the cell product material as the material was not used for further studies 
               
            
           
         
       
     
     Example 5—Description of Manufacturing Process Development to Improve LV-Col7 Transduction Efficiency 
     FCX-007 cell products manufactured using the process described above had low levels of gene modification efficiency as indicated by the low gene copy number and C7 expression levels when INXN-2002 vector was used for cell transduction. Two approaches were pursued to increase the LV-Col7 transduction efficiency on RDEB fibroblast cells: 1) development of a new LV-Col7 lentiviral vector (INXN-2004) which offers better vector titer and improved stable transgene (collagen VII) expression in the transduced fibroblast cells, and 2) further optimization of the LV-Col7 transduction process on fibroblast cells. Details of the development of the new INXN-2004 vector are described in Section 3.2.S.2.3. Studies in further optimization of the LV-Col7 transduction process, as well as cell expansion procedures are described below. 
     Spin Transduction (Spinoculation) 
     Spin transduction involves centrifugation of viral vectors onto the target cells under a g force environment. Spin transduction (spinoculation) has been reported in the literature to increase retroviral vector transduction efficiency on target cells (J Virol Methods. 1995 August; 54(2-3):131-43. Centrifugal enhancement of retroviral mediated gene transfer. Bahnson AB1, Dunigan J T, Baysal B E, Mohney T, Atchison R W, Nimgaonkar M T, Ball E D, Barranger J A. and Hum Gene Ther. 1994 January; 5(1):19-28. Improved methods of retroviral vector transduction and production for gene therapy. Kotani H1, Newton P B 3rd, Zhang S, Chiang Y L, Otto E, Weaver L, Blaese R M, Anderson W F, McGarrity G J.). 
     RDEB fibroblast cells from the previous TR8 were cultured. Cells in exponential growth phase were harvested and used for this study. The previous INXN-2002 vector lots were used for transduction. For spin transduction, 1×10 3  cells with different MOIs of INXN-2002 in 504, of transduction media (IMDM+2% FBS) were added into each well of a 96-well tissue culture coated plates which were pre-coated with RetroNectin (5 μg/cm 2 ). The plates containing cells plus INXN-2002 vector were centrifuged at 1300 g, 4° C. for 1.5 hours. After centrifugation, the plates were placed into a 37° C. incubator to allow the cells to recover and grow for 3 hours. After which, 100 μl of complete culture media (IMDM+10FBS+GSH) was added to each well. The plates were returned to the incubator for further culture. The standard non-spin transduction described above was used as a control. After transduction, cells were passaged three times, 3-5 days culture between each cell passage. At the last passage culture supernatants were harvested to evaluate C7 protein expression levels, while the cells were harvested for gene copy number per cell analysis. Table 26 shows the Col7 expression levels for the different transduction conditions. 
     
       
         
           
               
             
               
                 TABLE 26 
               
             
            
               
                   
               
               
                 C7 Expression Level 
               
            
           
           
               
               
               
            
               
                   
                 Vector Dilution 
                 Transduction Method 
               
            
           
           
               
               
               
               
               
            
               
                 INXN- 
                 MOI 
                 used for 
                 Spin 
                 Non-spin 
               
               
                 2002 
                 (IU/cell) 
                 Transduction 
                 Transduction 
                 transduction 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 GMP lot 
                 6.31 
                   
                  257.4 ± 62.17 
                 311.75 ± 70.89 
               
               
                   
                 1.60 
                   
                 539.50 ± 70.49 
                 251.27 ± 22.41 
               
               
                   
                 0.4 
                   
                 243.94 ± 3.40  
                 207.51 ± 25.26 
               
               
                 Pilot lot 
                 30 
                   
                 327.76 ± 21.69 
                 238.56 ± 27.93 
               
               
                   
                 15 
                   
                 391.59 ± 42.82 
                 208.39 ± 16.80 
               
               
                   
                 7.5 
                   
                 269.07 ± 25.51 
                 214.23 ± 16.83 
               
               
                   
               
            
           
         
       
     
     Table 27 provides the vector copy number per cells of the harvested cells. 
     
       
         
           
               
             
               
                 TABLE 27 
               
             
            
               
                   
               
               
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
            
               
                   
                 Vector dilution 
                 Transduction Method 
               
            
           
           
               
               
               
               
               
            
               
                 INXN- 
                 MOI 
                 used for 
                 Spin 
                 Non-spin 
               
               
                 2002 
                 (IU/cell) 
                 transduction 
                 Transduction 
                 transduction 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 GMP lot 
                 6.31 
                   
                 0.11 ± 0.02 
                 0.10 ± 0.02 
               
               
                   
                 1.60 
                   
                 0.11 ± 0.01 
                 0.06 ± 0.00 
               
               
                   
                 0.4 
                   
                 0.10 ± 0.03 
                 0.04 ± 0.01 
               
               
                 Pilot lot 
                 30 
                   
                 0.09 ± 0.01 
                 0.06 ± 0.02 
               
               
                   
                 15 
                   
                 0.09 ± 0.02 
                 0.05 ± 0.01 
               
               
                   
                 7.5 
                   
                 0.05 ± 0.03 
                 0.07 ± 0.02 
               
               
                   
               
               
                 INXN-2002, IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
               
                 INXN-2002, IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     The results show a modest increase in both Col7 production levels and vector copy number per cell using the spin transduction method, especially at the lower MOIs and at the INXN-2002 vector dilutions tested. 
     Optimization of Spin Transduction Parameters 
     The spin transduction parameters, including centrifugation time, speed and temperature, were evaluated with an aim to further increase the INXN-2002 transduction efficiency. The same cell culture condition as described above was used in this study. In the initial experiment, centrifugation temperature was examined. 96-well plates containing cells plus INXN-2002 vector were centrifuged at either 4° C. or 25° C. (RT) for 1.5 hours at 1300 g. This was followed by another experiment where 96-well plates containing cells plus INXN-2002 vector were centrifuged at 4° C. or 25° C. (RT) for 1 or 2 hours, at either a high speed of 1300 g, or a low speed of 300 g, to examine the effect of centrifugation speed on INXN-2002 transduction efficiency. In both experiments, cells were returned to a 37° C. incubator for incubation post centrifugation. Three hours later, 100 μl of complete culture media was added to each well. Cells were passaged three times before being harvested for Col7 expression analysis in the culture supernatant and vector copy number per cell analysis in the harvested cells. 
     Table 28 provides the effect of spin transduction temperature on Col7 expression levels and vector copy number per cell. The standard non-spin transduction was included as a control. 
     Again, increased INXN-2002 transduction, as indicated by higher C7 protein expression and vector copy number per cell, was demonstrated using spin transduction compared to the standard no spin transduction. Spin transduction temperature did not demonstrate impact on the overall transduction efficiency. 
     Table 29 below provides the effect of centrifugation speed as well as temperature and time on INXN-2002 transduction efficiency. 
     
       
         
           
               
             
               
                 TABLE 28 
               
             
            
               
                   
               
               
                 The Effect of Spin Transduction Temperature on INXN-2002 Transduction 
               
               
                 Efficiency 
               
            
           
           
               
               
               
            
               
                 INXN- 
                 C7 Expression (ng C7/day/E6 cells) 
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
               
               
            
               
                 2002 
                   
                 Spin Transduction 
                   
                 Spin Transduction 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 MOI 
                 No spin 
                 4° C. 
                 RT 
                 No Spin 
                 4° C. 
                 RT 
               
               
                   
               
               
                 18 
                 105.84 ± 11.69 
                 177.36 ± 24.98 
                 313.59 ± 39.13 
                 0.07 ± 0.04 
                 0.07 ± 0.02 
                 0.08 ± 0.03 
               
               
                  6 
                 117.82 ± 14.79 
                 196.26 ± 23.75 
                 143.61 ± 19.01 
                 0.02 ± 0.01 
                 0.08 ± 0.03 
                 0.05 ± 0.00 
               
               
                  2 
                  50.05 ± 13.60 
                 136.55 ± 6.82  
                 105.57 ± 17.90 
                 0.01 ± 0.00 
                 0.02 ± 0.00 
                 0.03 ± 0.02 
               
               
                   
               
               
                 INXN-2002 IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 29 
               
             
            
               
                   
               
               
                 Effect of Spin Transduction Temperature, Time, and Speed on INXN-2002 
               
               
                 Transduction Efficiency 
               
            
           
           
               
               
               
            
               
                 INXN- 
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 2002 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
            
           
           
               
               
               
            
               
                 MOI 
                 C7 Expression (ng C7/day/E6 cells) 
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
               
               
            
               
                 Temp 
                 4° C. 
                 RT 
                 4° C. 
                 RT 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Speed (xg)  
                 1300 
                 300 
                 1300 
                 300 
                 1300 
                 300 
                 1300 
                 300 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 time (hr) 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
                 1 
                 2 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 3.8 
                 208.88 ± 
                 247.03 ± 
                 221.49 ± 
                 310.93 ± 
                 359.47 ± 
                 201.06 ± 
                 168.98 ± 
                 201.82 ± 
                 0.08 ± 
                 0.07 ± 
                 0.06 ± 
                 0.09 ± 
                 0.11 ± 
                 0.06 ± 
                 0.07 ± 
                 0.07 ± 
               
               
                   
                 13.61 
                 10.09 
                 4.08 
                 0.41 
                 3.85 
                 5.37 
                 3.34 
                 9.41 
                 0.01 
                 0.01 
                 0.01 
                 0.03 
                 0.01 
                 0.01 
                 0.01 
                 0.02 
               
               
                 1.9 
                 180.34 ± 
                 265.4 ± 
                 213.83 ± 
                 301.82 ± 
                 240.61 ± 
                 180.36 ± 
                 212.76 ± 
                 177.16 ± 
                 0.06 ± 
                 0.10 ± 
                 0.05 ± 
                 0.06 ± 
                 0.10 ± 
                 0.07 ± 
                 0.06 ± 
                 0.05 ± 
               
               
                   
                 12.33 
                 1.33 
                 0.58 
                 11.54 
                 2.68 
                 3.10 
                 3.19 
                 5.92 
                 0.02 
                 0.02 
                 0.00 
                 0.01 
                 0.02 
                 0.02 
                 0.01 
                 0.01 
               
               
                   
               
               
                 INXN-2002 IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     The results indicate a modest improvement in INXN-2002 transduction efficiency, as indicated by C7 protein expression and vector copy number, when spin transduction was conduced at RT, 1300 g, for 1 hour. The significance of the improvement is not known and the initially developed spin transduction parameters (centrifugation at 1300 g for 1.5 hours at 4° C.) were selected for future study use. 
     Super Transduction to Further Increase INXN-2002 Transduction Efficiency 
     Super transduction (a second transduction) was evaluated in the early development of INXN-2002 transduction, which is described above. Super transduction was evaluated again in combination with spin transduction to further increase INXN-2002 transduction efficiency on fibroblast cells. Unlike the previous method, in the current study&#39;s super transduction was performed on cells one passage after the initial spin transduction. The inclusion of the one cell passage is expected to allow the cells to recover from the initial spin transduction and become more receptive to the 2 nd  super transduction. 
     Similarly, RDEB fibroblast cells from the previous TR8 in exponential growth phase were harvested and used for this study. The optimized spin transduction method described above, centrifugation at 1300 g for 1.5 hours at 4° C., was used for INXN-2002 transduction. The transduced cells were passaged once at 72-96 hours post the initial spin transduction. After 72-96 hours of culture in the second passage, cells were harvested and re-transduced using the same spin transduction conditions. After the super spin transduction, cells were passaged an additional three times before being harvested for Col7 expression analysis in the culture supernatant, and vector copy number per cell analysis in the harvested cells. The INXN-2002 vector was used for the study. Table 30 provides the effect of super spin transduction on INXN-2002 transduction efficiency. 
     
       
         
           
               
             
               
                 TABLE 30 
               
             
            
               
                   
               
               
                 Effect of Super Spin Transduction 
               
            
           
           
               
               
               
            
               
                   
                 C7 Protein Expression 
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
               
               
            
               
                 INXN- 
                 One Round 
                   
                 One Round 
                   
               
               
                 2002 
                 of Spin 
                 Super Spin 
                 of Spin 
                 Super Spin 
               
               
                 MOI 
                 Transduction 
                 Transduction 
                 Transduction 
                 Transduction 
               
               
                   
               
               
                 1.9 
                 257.8 ± 3.37 
                 431.9 ± 95.5 
                 0.07 ± 0.03 
                 0.22 ± 0.07 
               
               
                   
               
               
                 INXN-2002 IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     Super spin transduction resulted in a significant increase in INXN-2002 transduction efficiency on RDEB fibroblast cells. C7 protein expression increased approximately 1.7-fold and the vector copy number per cell increased approximately 3-fold. 
     The data demonstrate a 40% increase in Col7 expression and a 60% increase in copy number per cell with the additional transduction. Due to this increase, super-infection was added to the production protocol. 
     Transduction with the INXN-2004 Vector 
     Concurrent with the development of the improved transduction method, a new LV-Col7 vector (referenced as “INXN-2004”) based on a different lentiviral vector backbone (that is, pFUGW) was also developed. 
     INXN-2004 Single Spin Transduction 
     RDEB fibroblast cells from the previous TR8 in exponential growth phase were harvested and used for this study. In the first study, single round of spin transduction as described above was carried out using a pilot lot of INXN-2004 vector. The standard no-spin transduction was included in the study as a control. Cells were passaged three times before being harvested for Col7 expression analysis in the culture supernatant, and vector copy number per cell analysis in the harvested cells. Table 31 provides the Col7 expression levels and vector copy number per cell using the INXN-2004 vector transduction. 
     
       
         
           
               
             
               
                 TABLE 31 
               
             
            
               
                   
               
               
                 C7 Protein Expression and Vector Copy Number 
               
               
                 per Cell using INXN-2004 Transduction 
               
            
           
           
               
               
               
            
               
                 INXN- 
                 C7 Protein Expression 
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
               
               
            
               
                 2004 
                 No Spin 
                 Spin 
                 No Spin 
                 Spin 
               
               
                 MOI 
                 Transduction 
                 Transduction 
                 Transduction 
                 Transduction 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 17 
                 2312.46 ± 0.03 
                 3960.58 ± 0.02 
                 0.16 ± 0.03 
                 0.05 ± 0.02 
               
               
                 4.25 
                 1668.46 ± 0.06 
                 3602.10 ± 0.06 
                 0.21 ± 0.06 
                 0.27 ± 0.06 
               
               
                 1.06 
                  724.12 ± 0.03 
                 2566.88 ± 0.10 
                 0.06 ± 0.03 
                 0.21 ± 0.10 
               
               
                 0.27 
                  168.61 ± 0.00 
                 1936.72 ± 0.00 
                 0.02 ± 0.00 
                 0.13 ± 0.00 
               
               
                   
               
               
                 INXN-2004 IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     Relative to the INXN-2002 vector, a significant improvement in transduction efficiency was achieved with use of INXN-2004 in combination with the spin transduction method. Additionally, the INXN-2004 vector appears to be significantly more active in transducing RDEB fibroblast cells even under a no-spin transduction condition, as indicated by the approximately 10-fold higher C7 protein expression and vector copy number per cell. The results support the use of the pFUGW base for construction of the new INXN-2004 vector. 
     Consistent to what is reported above, spin transduction appears to exacerbate the INXN-2004 vector toxic effect on fibroblast cells at higher MOI conditions, as indicated by the lower C7 expression levels and vector copy numbers at MOI17. This does not impact FCX-007 manufacturing as the use of a lower MOI is demonstrated to be as effective. 
     INXN-2004 Super Spin Transduction 
     In the second study, super spin transduction (as described above) was used for INXN-2004 transduction. The scale of transduction was increased from a 96-well plate to a T-25 flask. The transduction parameters remained unchanged, including the same RetroNectin™ coating density of 5 μg/cm2, and the same cell density at transduction of 1×10 4  cells/cm 2 . Transduction volume in the T-25 flask was 4.2 mL. For transduction, the T-25 flask containing the cells and virus was centrifuged at 4° C., for 1.5 hours at 1300 g. Cells were returned to a 37° C. incubator and incubated for three hours. After the initial incubation, 4.2 mL of complete culture media was added to the flask for further incubation. Cells were passaged three times before being harvested for Col7 expression analysis in the culture supernatant, and vector copy number per cell analysis in the harvested cells. Table 32 provides the effect of super spin transduction on INXN-2004 vector transduction efficiency. A pilot lot of INXN-2004 vector was used for the study. 
     
       
         
           
               
             
               
                 TABLE 32 
               
             
            
               
                   
               
               
                 Effect of Super Spin Transduction for the INXN-2004 Vector 
               
            
           
           
               
               
               
            
               
                 INXN- 
                 C7 Protein Expression 
                 Vector Copy Number per Cell 
               
            
           
           
               
               
               
               
               
            
               
                 2004 
                 Single Spin 
                 Super Spin 
                 Single Spin 
                 Super Spin 
               
               
                 MOI 
                 Transduction 
                 Transduction 
                 Transduction 
                 Transduction 
               
               
                   
               
               
                 0.11 
                 3178 ± 20.53 
                 4029 ± 59.67 
                 0.12 ± 0.007 
                 0.57 ± 0.134 
               
               
                   
               
               
                 INXN-2004 IU titer: 2.36 × 10 6  IU/mL (determined at Intrexon) 
               
            
           
         
       
     
     As observed for the previous INXN-2002 vector, super spin transduction resulted in a significant increase, approximately 5-fold, in vector copy number per cell in the transduction cells, despite a relatively modest increase in Col7 protein expression level, under a low MOI transduction condition. 
     Summary of Transduction Development and Optimization 
     Significant improvements in LV-Col7 transduction efficiency on RDEB fibroblast cells was achieved in continued process development, both with the previous INXN-2002 and the new INXN-2004 vector. The improvements are to enable the manufacturing of a biologically potent FCX-007 cell product. Based on the manufacturing process development study results, the following transduction procedure was chosen for manufacturing of FCX-007 using INXN-2004 vector for transduction. 
     Pre-coat culture surface with RetroNectin at 5 μg/cm 2 . Tissue culture-treated flasks are used for RetroNectin™ coating. 
     Add cells and INXN-2004 vector simultaneously at the time of transduction. Cell seeding density is approximately 1×10 4  cells/cm 2 . 
     Centrifuge cells and INXN-2004 vector transduction mixture in the transduction vessel at 4° C., for 1.5 hours at 1300 g. 
     Incubate cells for 1.5-2.0 hours at 37° C. post transduction in transduction medium with 2% FBS, and then change or feed cells with complete culture medium with 10% FBS. 
     Use a low transduction medium volume (50 μL/well for a 96-well plate, or 0.35 mL/cm 2 ). 
     Perform a super-transduction on cells after one passage post the initial transduction. 
     Example 6—Analysis of RDEB Genetically Modified Human Dermal Fibroblasts Transduced by INXN-2002 
     RDEB Genetically Modified Human Dermal Fibroblasts (GM-HDF) derived from INXN-2002 lentiviral vector was analyzed in order to: 
     Evaluate localization of COLT expression in established composite human skin grafts (prepared on porcine dermis) comprised of RDEB keratinocytes by ID injection. 
     Assess potential carcinogenicity/tumorgenicity in composite RDEB human skin grafted (prepared on porcine dermis) on SCID mice treated with RDEB GM-HDF 
     An initial attempt to evaluate GM-HDF (derived from INXN-2002) function by seeding fibroblasts on the reticular side of composite graft cultures was unsuccessful, possibly due to inadequate migration of GM-HDF into the devitalized porcine dermis or insufficient time for adequate diffusion and deposition of rC7 at Day 19. An alternative approach was used to evaluate function by direct ID injection of existing grafts in six back-up animals on study. RDEB composite grafts prepared on porcine dermis appear to be more robust than anticipated, making direct injection into established RDEB skin grafts possible. This approach more closely mimics the disease and clinical route of administration. 
     This study report presents data from an initial evaluation of ID injection of GM-HDF into established RDEB grafts. Additional studies to achieve long term objectives for in vivo pharmacology are planned. 
     A. Study Materials 
     A.1 Test Article(s) 
     Test article was prepared and tested for Drug Substance release specifications prior to shipment. The test article for skin graft injection was prepared one day prior to each scheduled day of treatment. GM-HDF was thawed, washed, resuspended in DMEM, and evaluated against release specifications. The GM-HDF was shipped for overnight delivery and administered within 48 hours. Non-corrected RDEB HDF (mock transduced) was isolated and expanded from the identical patient as the GM-HDF by the same methods. 
     The test article and cells to be used for preparation of composite skin grafts was shipped frozen. Cells used for skin graft preparation will be thawed and cultured for testing. 
     Lot number TR8 (Non-Transduced Control) was used as the RDEB-HDF negative control. Lot number TR8 Arm A (GM-HDF-LV-COLT) was used as the GM-HDF test article. 
     Test article analysis was performed at the manufacturing facility prior to shipment. Remaining test article will be stored at −70° C. for 1 year. The test article will be stored in Dulbecco&#39;s Modified Eagle Medium (DMEM). 
     A.2. Other Chemicals and Materials 
     A list of other materials is provided below in Table 33. 
     
       
         
           
               
             
               
                 TABLE 33 
               
             
            
               
                   
               
               
                 Other Study Materials 
               
            
           
           
               
               
               
            
               
                   
                 Description 
                 Source 
               
               
                   
                   
               
               
                   
                 Normal Porcine Skin 
                 Porcine cadaver 
               
               
                   
                 RDEB keratinocytes 
                 Human skin biopsy 
               
               
                   
                 RDEB fibroblasts 
                 Human skin biopsy 
               
               
                   
                 Normal keratinocytes 
                 Human skin biopsy 
               
               
                   
                 Normal fibroblasts 
                 Human skin biopsy 
               
               
                   
                   
               
            
           
         
       
     
     Group 1 (Negative Control) Cells: Untransduced RDEB cells from the donor from training run 8 were thawed, and passaged once prior to transfection. Two days after passaging the cells, the cells were collected and 1.2×10 6  cells were transfected in a 50 ml conical tube in low serum growth (ATCC PCS-201-030 and PCS-201-041) media using the lipid based transfection agent Transfex (ATCC) with COLT plasmid VVN 4317513. After a 10 minutes incubation with complexed Transfex, DNA and Optimem (LifeTech), 1×10 6  transfected cells were transferred to a T150 flask, and 2×10 5  cells were placed in a T25 and placed in a 37° C. 5% CO2 incubator for 16 hours, at which time the media was removed and replace with full growth media (IMDM (Sigma), 920 mg/L of L-Glutathione Reduced (Sigma) 10% heat inactivated FBS (Atlantic Biologicals) and 1×Glutamax (LifeTech)). Cells were then returned to the 37° C. 5% CO2 incubator until time of shipment. At time of shipment all cell flask was filled to capacity, such that all parts of the flask were in contact with media regardless of the flask orientation. 
     B. Study Design 
     B.1. Randomization 
     Due to high failure rates of RDEB human skin grafts, mice in these groups of animals are not randomized. 
     B.2. Justification for Species and Number on Study 
     The NOD. CB.17-PRKDC scid/scid mouse is the standard species for use in proof of concept preclinical efficacy/toxicology studies and is a rodent species of choice for such evaluations using the human xenograft models. This study was designed to minimize the number of animals on study that would provide sufficient data to evaluate the test articles in. 
     B.3. Route of Administration 
     Fibroblasts will be administered by intradermal injection for normal skin grafts. Composite grafts will be administered cells by seeding prior to grafting. 
     B.4. Dose Administration 
     Intradermal cell injections of lentivirus transduced (COL7A1-corrected) RDEB GM-HDF (using INXN-2002) and negative controls (vehicle or RDEB-HDF) will be done with a 30-gauge needle. The injection will be performed by first piercing the skin, then directing the needle as superficially as possible back upward toward the surface. 
     Composite grafts will be administered cells by seeding prior to grafting according to protocol outlined in the in vitro study with the distinction that the composite culture system will not be lifted to air-fluid interface prior to grafting. 
     B.4.1. Preparation of Fibroblasts for Injection (Composite Grafts) 
     Fibroblasts were removed from the liquid nitrogen storage at one week prior to injection, thawed in 37° C. water bath until a small ice crystal remains, then disinfected and transferred to BSC. Vials were resuspended in 10 ml of PBS wash volume and spun for 10 minutes at 1000 RPM. The supernatant was discarded and the cells were washed in DMEM+10% FBS 1× Antibiotic media and re-spun for 10 minutes at 1000 RPM. The pellet was resuspended in appropriate volume and plated on 15 cm TC dishes. Media was changed every other day and passaged at 80% confluence until grafts are ready to inject. On the day of injection, fibroblasts were trypsinized, neutralized in DMEM+10% FBS media and spun 10 min at 10000 RPM, the pellet of cells was resuspended and an aliquot of cells was taken, cells were counted with trypan blue stain to determine viability. The cells were resuspended such that 1.0×10 6  cells in 50 uL volume is drawn up into syringe. A 30 g needle was attached and placed at 4° C. until mouse was ready tobe injected. 
     
       
         
           
               
             
               
                 TABLE 34 
               
             
            
               
                   
               
               
                 Composite Skin Grafts (Fibroblast Seeding) 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 Collec- 
               
               
                   
                   
                   
                   
                   
                   
                   
                 tion 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Number of Cells 
                 of 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                 Composite Skin Graft 
                 Keratino- 
                   
                 Speci- 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Grp 
                 N 
                 Sex 
                 Keratinocytes 
                 Fibroblasts 
                 cytes 
                 Fibroblasts 
                 mens 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 1 
                 3 
                 M 
                 WT 
                 WT 
                 1 × 10 6   
                    1 × 10 6   
                 Day 15 
               
               
                 2 
                 3 
                 M 
                 RDEB 
                 RDEB 
                 1 × 10 6   
                    1 × 10 6   
                 Day 15 
               
               
                   
                   
                   
                 Keratinocytes 
                 HDF 
                   
                   
                   
               
               
                 3 
                 3 
                 M 
                 RDEB 
                 RDEB- 
                 1 × 10 6   
                    1 × 10 6   
                 Day 15 
               
               
                   
                   
                   
                 Keratinocytes 
                 GM-HDF 
                   
                   
                   
               
               
                 4 
                 3 
                 M 
                 RDEB 
                 RDEB- 
                 1 × 10 6   
                    5 × 10 5   
                 Day 15 
               
               
                   
                   
                   
                 Keratinocytes 
                 GM-HDF 
                   
                   
                   
               
               
                 5 
                 3 
                 M 
                 RDEB 
                 RDEB- 
                 1 × 10 6   
                  2.5 × 10 5   
                 Day 15 
               
               
                   
                   
                   
                 Keratinocytes 
                 GM-HDF 
                   
                   
                   
               
               
                 6 
                 3 
                 M 
                 RDEB 
                 RDEB- 
                 1 × 10 6   
                 1.25 × 10 5   
                 Day 15 
               
               
                   
                   
                   
                 Keratinocytes 
                 GM-HDF 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 35 
               
             
            
               
                   
               
               
                 Composite Skin Grafts (Fibroblast Intradermal Injections) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Grp 
                 N 
                 Sex 
                 Test Article 
                 Dose 
                 Injection 
                 Graft Description 
               
               
                   
               
               
                 1 
                 2 
                 M 
                 RDEB-HDF 
                 1 × 10 6   
                 Composite 
                 1 million RDEB GM- 
               
               
                   
                   
                   
                   
                   
                 Graft 
                 HDF, 1 million RDEB 
               
               
                   
                   
                   
                   
                   
                   
                 KC 
               
               
                 2 
                 4 
                 M 
                 RDEB-GM- 
                 1 × 10 6   
                 Composite 
                 0.125 million RDEB 
               
               
                   
                   
                   
                 HDF 
                   
                 Graft 
                 GM-HDF, 1 million 
               
               
                   
                   
                   
                   
                   
                   
                 RDEB KC 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 36 
               
             
            
               
                   
               
               
                 Study Groups for the Evaluation of ID-Injected Xenografts 
               
            
           
           
               
               
            
               
                   
                 Condition 
               
            
           
           
               
               
               
               
            
               
                   
                 Graft Pre-Seeding Prep 
                   
                   
               
               
                   
                 Conditions 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Keratin- 
                   
                 Injected 
                 Date of 
               
               
                   
                 ocytes 
                 Fibroblasts 
                 Fibroblasts 
                 Harvest 
               
               
                   
                   
               
            
           
           
               
            
               
                 Control Groups 
               
            
           
           
               
               
               
               
               
            
               
                 Normal 
                 Normal 
                 Normal FB 
                 N/A 
                 Day 19 post- 
               
               
                   
                 KC 
                   
                   
                 grafting 
               
               
                 FDEB 
                 RDEB 
                 RDEB 
                 N/A 
                 Day 19 post- 
               
               
                   
                   
                   
                   
                 grafting 
               
            
           
           
               
            
               
                 Experimental Groups 
               
            
           
           
               
               
               
               
               
            
               
                 1A 
                 RDEB 
                 TR8/A 1 million 
                 TR8 
                 10 days post- 
               
               
                   
                   
                   
                 nontransduced 
                 injection 
               
               
                 1B 
                 RDEB 
                 TR8/A 1 million 
                 TR8 
                 10 days post- 
               
               
                   
                   
                   
                 nontransduced 
                 injection 
               
               
                 2A 
                 RDEB 
                 TR8/A 0.125 mil 
                 TR8/ArmA 
                 10 days post- 
               
               
                   
                   
                   
                   
                 injection 
               
               
                 2B 
                 RDEB 
                 TR8/A 0.125 mil 
                 TR8/ArmA 
                 10 days post- 
               
               
                   
                   
                   
                   
                 injection 
               
               
                 2C 
                 RDEB 
                 TR8/A 0.125 mil 
                 TR8/ArmA 
                 10 days post- 
               
               
                   
                   
                   
                   
                 injection 
               
               
                 2D 
                 RDEB 
                 TR8/A 0.125 mil 
                 TR8/ArmA 
                 10 days post- 
               
               
                   
                   
                   
                   
                 injection 
               
               
                   
               
            
           
         
       
     
     C. Skin Grafts 
     C.1 RDEB Human Skin Grafts 
     C.1.1 Fibroblasts 
     Genetically engineered human fibroblasts (RDEB GM-HDF) produced by lentiviral gene transfer, including the INXN-2002 lentiviral vector, along with normal fibroblasts and non-corrected RDEB fibroblasts as controls, are the cell types used for initially infusing dermis skin. Cells are cultured onto devitalized dermis to form skin equivalents approximately 2 cm square. Regenerated skin composites consist of human keratinocytes cultured atop fibroblast-infused human devitalized dermis. 
     Isolation and Culture of Primary Keratinocytes 
     Wild-type keratinocyte cells are isolated from neonatal foreskin, whereas primary RDEB keratinocytes are isolated from patient skin samples. The latter is obtained as a 6 mm punch biopsy submerged in 15 mL of keratinocyte growth medium 50/50V in a 15 mL centrifuge tube shipped on wet ice. No bubbles can be present in shipped samples. Medium 50/50V contains 50% medium 154 (Invitrogen) supplemented with HKGS (0.2% bovine pituitary extract [BPE], bovine insulin [5 mg/mL], hydrocortisone [0.18 mg/mL], bovine transferrin [5 mg/mL], human epidermal growth factor [0.2 ng/mL]) and 50% keratinocyte SFM (KSFM; Invitrogen) supplemented with recombinant human EGF1-53 BPE. Antibiotics AV100 (amikacin/vancomycin) are added to reduce bacterial contamination. 
     Separate dermis and epidermis by treating with 25 U/mL dispase (Becton Dickinson) overnight at 4° C. Epidermis is then peeled from dermis and placed in a 15 mL centrifuge tube with 5 mL TrypLE 10×. Epidermal “peel” is incubated in a 37° C. water bath for 15-30 min with gentle agitation every 5 min. TrypLE is neutralized with equal volume DTI (defined trypsin inhibitor) or DMEM+10% FBS. Cells are pelleted at 1200 rpm and plated in 12 mL 50/50V onto T75 Corning® PurecoatTMcollagen 1 mimetic flasks. In the event of PureCoat™ flask unavailability from manufacturer, 1 ml of collagen 1 coating solution per 10 cm plastic dish for 15 minutes at 37° C. can be used instead. Collagen 1 coating is a 0.2 uM filtered solution of Vitrogen 100 Collagen (1 ml), HEPES (2 ml), BSA (100 uL of 100 mg/ml) and 1×HBSS (100 ml). The excess collagen coating is aspirated prior to cell seeding. Flasks are swirled to ensure an even distribution of cells and placed in a 37° C. 5% CO 2  humidified incubator for at least one night before any manipulation or observation. Cells are allowed to adapt to tissue culture conditions for 3 days. After adaptation, medium is changed once every 2 days or as needed. Keratinocytes are passaged onto normal tissue culture plastic when they are approximately 70% confluent. 
     In the 50/50V media keratinocytes grow well for five passages. Keratinocytes are utilized within the first four passages. 
     Preparation of Porcine Devitalized Dermis 
     Obtain sheets of split thickness porcine skin. This may be prepared in similar fashion to devitalized human dermis (as above) from porcine skin harvested by dermatome. Wash the porcine dermis 3× in sterile PBS containing 1× penicillin/streptomycin/Amphotericin/gentamicin and incubate in PBS with 1M NaCl and 2× antibiotics at 37° C. for 72 hours. Separate epidermis and discard, wash 3× with 1×PBS containing antibiotics to remove excess salt and gentamicin in solution. 
     Confirm sterility by culturing a piece of porcine dermis in 50/50V or KGM for 4 days. Dermis can be stored at 4° C. in antibiotic solution, changing the PBS weekly. Dermis should not be stored for more than a few months before use. 
     Organotypic Culture Seeding Fibroblasts 
     Cut devitalized dermis into 2 cm square pieces. Place dermis basement membrane side down into 6 well culture plates and allow dermis to dry slightly. 
     Seed fibroblasts into each well, centrifuge 1200 rpm for 5 minutes and return to 37° C. with 5% humidified CO 2  for 3-4 days, changing media every day. During this time, fibroblasts attach to the dermis and begin to migrate into the tissue. 
     Seeding Keratinocytes 
     After the fibroblasts have migrated through the dermis, carefully detach dermis and transfer to annular dermal support (ADS). Add a thin layer of Matrigel to seal and secure dermis in device. Add 5 mL KGM to stromal compartment of ADS. KGM is a 3:1 mixture of DMEM:Ham&#39;s F12 supplemented with FBS (10%), adenine (1.8×10-4 M), hydrocortisone (0.4 μg/mL), insulin (5 μg/mL), cholera toxin (1×10-10 M), EGF (10 ng/mL), transferrin (5 μg/mL), and triiodo-L-thyronine (1.36 ng/mL). Trypsinize, count, and seed keratinocytes in a total volume of 100 μL of KGM per ADS. Return ADS assembly to incubator. Do not disturb ADS for at least 24 hrs. The keratinocytes at the top of the dermis must remain at the air liquid interface. Change the KGM in the lower chamber every day. Organotypic culture will continue for 1-2 weeks. 
     Grafting Organotypic Culture 
     Organotypic cultures will be ready to graft after 7-10 days. Grafting will be conducted as described above for normal human skin grafts. Bandages and sutures will be removed after 7-10 days and animal will be rewrapped with fresh bandaging for another week. Thereafter graft will be exposed to air to mature. 
     Harvesting Grafts and Sectioning 
     Humanely sacrifice mice and carefully harvest graft. Bisect graft and cut an additional 1 mm slice. Trim slice down to a 1 mm square and send for immunoelectron microscopy (IEM). Freeze remaining halves in OCT on dry ice while noting the central edge. Sectioning will start at this edge. One half will be sectioned for immediate analysis and remaining half will be stored for later analysis. Cut ten 8 μm sections starting from the central edge and sequentially label. Discard the next forty sections. Repeat until end of graft is reached. Air dry and fix with ice cold 50/50 acetone/methanol. 
     Injection of Established RDEB Composite Grafts 
     Fibroblasts will be intradermally injected directly into the skin following of the established graft. Injections will be no greater than 50 μL in volume. 
     Injections were performed on mice previously grafted with composite grafts as described herein: Devitalized porcine dermis was seeded by centrifugation with variable dosage of RDEB GM-HDF and grown in tissue culture for 1 week. Subsequently, the dermis was flipped over and 1 million RDEB keratinocytes were seeded and allowed to grow in culture for one more week. After this two week period in tissue culture as described above, the grafts were placed on SCID mice and sutured in place, bandaged for 13 days. The dressings were removed at that time, and at 38-41 days post grafting, grafts were injected with 50 uL volume of fibroblasts. 
     D. Experimental Procedures 
     D.1. Localization of COLT 
     Immunofluorescent microscopy was used to evaluate expression of type VII collagen. For immunofluorescence (IF) analyses of skin cultures with anti-human C7 specific antibody NP185. 
     D.1.2. Methods for NP 185 Antibody Immunofluorescence Microscopy 
     Composite grafts were harvested from SCID mice immediately after CO 2  euthanasia was performed. The grafts were bisected and placed in OCT blocks. Frozen blocks of tissue were sectioned on a Leica CM1850 cryostat at 8 μM thickness at −21° C. and fixed in ice cold 50% methanol/50% acetone. Slides were rehydrated and washed in 1×PBS three times, incubated in primary antibody (NP185 10 ug/ml; mouse anti-Human Collagen VII) for 1 hour at room temperature. Slides were washed and incubated in Alexa 488 tagged goat anti-mouse IgG antibody (1:400, 1 hour, Room Temperature, life technologies) and a nuclear Hoescht 33342 counterstain (Life technologies). Samples were washed three times in 1×PBS, mounted using fluoromount prior to imaging. Images were taken on a Zeiss Observer.Z1 fluorescence microscope at 20× magnification. 
     Grafts harvested at Day 19 were included as positive and negative control arms. Experimental arms were injected Day 38, 39, or 41 and harvested Day 48, 49, or 51 (Table 37). 
     
       
         
           
               
             
               
                 TABLE 37 
               
             
            
               
                   
               
               
                 Graft, Injection, and Harvest Days 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Day of 
                 Day of 
                 Day of 
               
               
                 Study Arm 
                 N 
                 Graft 
                 Injection 
                 Harvest 
               
               
                   
               
            
           
           
               
            
               
                 Control Arms 
               
            
           
           
               
               
               
               
               
            
               
                 Positive (Normal 
                 1 
                 0 
                 N/A 
                 19 
               
               
                 keratinocytes, 
               
               
                 normal fibroblasts) 
               
               
                 Negative (RDEB 
                 1 
                 0 
                 N/A 
                 19 
               
               
                 keratinocytes, 
               
               
                 RDEB fibroblasts) 
               
            
           
           
               
            
               
                 Experiment Arms 
               
            
           
           
               
               
               
               
               
            
               
                 Non-transduced 
                 2 
                 0 
                 41 
                 51 
               
               
                 RDEB fibroblasts 
               
               
                 GM-HDF 
                 4 
                 0 
                 38 (n = 3) or 
                 48 (n = 3) or 
               
               
                   
                   
                   
                 39 (n = 1) 
                 49 (n = 1) 
               
               
                   
               
            
           
         
       
     
     E. Data Analysis 
     IF images were prepared and provided to the PI in a blinded fashion. Data was analyzed and interpreted by the PI prior to unblinding. 
     F. Results 
     Representative results from the IF analysis of the harvested graft tissue are presented in  FIG. 26 . 
     C7 staining at DEJ was visualized in all NP185 staining conditions excluding the negative control (image “RDEB uncorrected”—see arrows at DEJ for negative baseline comparison). With regard to the positive controls, there was intense DEJ staining seen in the DEJ of normal keratinocytes/fibroblasts seeded grafts. There was a small focus of positive staining seen in the group 1 injected with uncorrected fibroblasts (Image 1b) in a small section that could represent previously seeded corrected fibroblasts, however the remainder of the uncorrected fibroblast injected grafts in both mice appear to be less intense (as represented in image 1a). There was C7 staining seen at the DEJ in the representative images of four mouse grafts injected with corrected fibroblasts (4a-d) were observed even after only 10-days post-injection. 
     No tumors were observed in any grafts during the course of the study. 
     G. Conclusions 
     RDEB GM-HDF TR8 intradermally injected at the approximate proposed clinical dose produced C7 that localized to the DEJ in vivo in composite grafts of RDEB keratinocytes on devitalized pig dermis. Additionally results from seeding of GM-HDF in composite grafts prior to grafting indicate that GM-HDF could persist in expression C7 that localizes to the DEJ. This model can be used to characterize the localization, durability, persistence and phenotype correction approximating the effect of GM-HDF predictive of the clinical dose. 
     Results of these studies show intradermal injections of autologous GM-HDF as an effective treatment of RDEB in patients. 
     Example 7—Non-GLP In Vitro GM-HDF Cell Characterization and Proof-of-Concept Assessments 
     The objectives of this study are the following: (a) characterize the GM-HDFs produced using the anticipated at-scale GMP production method to be used for the treatment of RDEB patients; (b) assess copy number of integrated LV and expression levels of C7; and (c) confirm the functionality of C7 expressed by the GM-HDFs 
     A. Study Materials 
     A.1. Test Article(s) 
     GM-HDFs from Training runs 8, 9, and 10, and the Engineering run (as described above) were characterized. 
                     TABLE 38                  General production information for TR8, TR9, TR10, and ER1                                             LV-COL7           Production       MOI   and TU   Production       Run   Arm   (IU/cell)   titer   Scale                                         TR8   A   3.4   INXN-2002   2 x 10-layer           B   1.7   (Pilot)   CellSTACKs ®           Control 1     0   9.2 × 10 6                     IU/mL       TR9   A   1.0   LV-HA-COL7   2 x 10-layer           B   1.9   (Pilot)   CellSTACKs ®           Control 1     0   2.0 × 10 6                     IU/mL       TR10 2     A   2.0   INXN-2002   6 x 10-layer           Control 1     0   (GMP)   CellSTACKs ®                   2.3 × 10 6                     IU/mL       ER1 2     A   2.6   INXN-2002   6 x 10-layer                   (GMP)   CellSTACKs ®                   2.3 × 10 6                     IU/mL                 1 Control arm cells were mock-transduced         2 Only one LV-COL7 transduction arm was generated for TR10 and for ER1            
B. Primer and Probe Sequences Used in qPCR Analyses
 
     
       
         
           
               
             
               
                 TABLE 39 
               
             
            
               
                   
               
               
                 Primer and Probe Sequences used for qPCR Assays 
               
            
           
           
               
               
               
            
               
                 Name 
                 Sequence 1   
                 Assay 
               
               
                   
               
               
                 PR13843 
                 ACCTGAAAGCGAAAGGGAAAC (SEQ 
                 LV-COL7 copy number 
               
               
                 forward 2   
                 ID NO: 25) 
                   
               
               
                   
               
               
                 PR13843 
                 CACCCATCTCTCTCCTTCTAGCC (SEQ 
                 LV-COL7 copy number 
               
               
                 reverse 2   
                 ID NO: 26) 
                   
               
               
                   
               
               
                 PR13843 probe 2   
                 6-FAM- 
                 LV-COL7 copy number 
               
               
                   
                 AGCTCTCTCGACGCAGGACTCGGC- 
                   
               
               
                   
                 3′IB FQ (SEQ ID NO: 27) 
                   
               
               
                   
               
               
                 PR13653 forward 
                 CACTCCCAACGAAGACAAGAT (SEQ ID 
                 COL7A1 mRNA expression 
               
               
                   
                 NO: 28) 
                   
               
               
                   
               
               
                 PR13653 reverse 
                 GTCTAACCAGAGAGACCCAGTA (SEQ 
                 COL7A1 mRNA expression 
               
               
                   
                 ID NO: 29) 
                   
               
               
                   
               
               
                 PR13653 probe 
                 6-FAM 
                 COL7A1 mRNA expression 
               
               
                   
                 TTTGTAAACCGGTGCAGCTGCTTT 3′IB 
                   
               
               
                   
                 FQ (SEQ ID NO: 30) 
               
               
                   
               
               
                   1 Primers/probes purchased from IDT. 
               
               
                   2 LV-specific primer/probe sequences derived from Greenberg et al. (2006). 
               
            
           
         
       
     
     C. Experimental Procedures 
     C.1 LV-COLT Copy Number 
     Nucleic acid isolation was performed using Qiagen&#39;s AllPrep kit according to the manufacturer&#39;s instructions. gDNA isolated from 3×10 5  GM-HDF cells was normalized to 12.5 ng/μl. 8 μl of the normalized gDNA was used in a 20 μL assay (10 μL Taqman® Gene Express, 1.8 μL nuclease-free water, 0.06 μL of 100 μM forward primer, 0.06 μL of 100 μM reverse primer, 0.04 μL of 100 μM Taqman® probe). A standard curve of serially diluted linearized C7 lentiviral shuttle vector (1e6 copies/reaction to 5 copies/reaction), plus 4 μL of human gDNA (1.5e4 cells/reaction) were also assayed in the 20 μL assay mentioned above. The Taqman® assay used was PR13843. 8 μL of an additional standard curve of commercial human gDNA (Clontech) (1.5e4 cells/reaction to 2e2 cells/reaction) in a 20 μL assay (10 μL Taqman® Gene Express, 1.0 L nuclease-free water, 1 μL of 20×ACTB primer/probe set) was also performed. All samples were run on an ABI7900 using the following cycling parameters: 2 minutes at 50° C., 10 minutes at 95° C., and 40 cycles of 15 sec at 95° C. and 1 minute at 60° C. 
     C.2 COL7A1 RT-qPCR 
     Vials of GM-HDF cells were thawed and gDNA and RNA were isolated using Qiagen&#39;s AllPrep™ kit. RNA (800 μg) was used to generate cDNA using Quanta&#39;s gScript™ per manufacturer&#39;s instructions. The cDNA and gDNA were then tested using Taqman® assay PR13653 as described in above. Data were normalized to housekeeping gene ACTB (dCT), and then compared to Control data (ddCT). The resulting ddCT is converted to fold change using the formula 2{circumflex over ( )}−(ddCT). 
     C.3 C7 Immunofluorescence 
     For immunofluorescence analyses, 1.2×10 4  GM-HDFs were allowed to attach to PDL/Lamin—coated coverslips in 24-well plates overnight and then fixed and permeabilized with a 50%/50% mix of methanol/acetone. The coverslips were washed 3 times with 1×PBS and then blocked with 10% goat serum in PBS for 30 minutes at room temperature. After three additional washes with PBS, the coverslips were incubated with 1.25 μg/mL fNC1 antibody in 1% goat serum/PBS, followed by 3 additional washes with PBS and incubation with 5 μg/mL Alexa Fluor® 555—conjugated goat anti-rabbit IgG in 1% goat serum/PBS for 1 hour at room temperature. Coverslips were stained with NucBlue® Live Cell Stain Ready Probes Reagent before being mounted onto slides. Images were acquired on a Zeiss Axio Observer microscope at 20× magnification using an exposure time of 290 ms. NHDFs or GM-HDFs were fixed, permeabilized, and stained with NucBlueLive® Cell Stain to visualize nuclei (blue) or with the fNC1 antibody and Alexa Fluor® 555-congugated goat anti-rabbit IgG (5 μg/mL) to visualize C7 expression (red). Images were acquired at 20× magnification using an exposure time of 290 ms. 
     C.4 C7 Protein ELISA 
     Briefly, a standard curve of purified His-NC1 fragment (9.8-625 ng/mL) or collected supernatants containing C7 protein (from GM-HDF in culture for three to five days) were immobilized to a Nunc MaxiSorp® 96-well plate overnight at 4° C. Standards and samples were tested in the same sample matrix (20% RDEB fibroblast conditioned media). Coated wells were washed with PBST and blocked with 3% BSA/PBS for 1 hour at 37° C. Detection was accomplished using a polyclonal anti-NC1 Ab (fNC1, 0.5 μg/mL) followed by incubation with secondary antibody donkey anti-rabbit IgG HRP (Jackson ImmunoResearch, 0.08 μg/mL). Bound antibodies were detected via colorimetric development with TMB substrate solution. Following quenching of the reaction, absorbance was measured at 450 nm on the SpectraMax® Plus 384 (Molecular Devices). 
     C.5 Immunoprecipitation of C7 Trimers 
     First, magnetic Protein G beads were washed with 1×PBS-T. Beads were bound to the magnet for a minimum of 2 minutes prior to removal of supernatant and in all subsequent steps. Following the washes, the beads were coated with 5 μg of anti-C7 fNC1 and incubated for 10 minutes with rotation (Glas-Col, setting 30˜14 rpm at room temperature). Beads were bound to the magnet to remove the supernatant and washed with Ab binding/Wash buffer. Supernatants were collected from GM-HDFs in culture for three to five days. C7 containing supernatant was added to the bead/C7 supernatant mix and was incubated overnight at 4° C. with rotation (setting 30˜14 rpm). The next day the beads were bound to the magnet and washed three times using Wash buffer. Target antigen was eluted in 20 μL of elution buffer (50 mM glycine, pH 2.8, and 10 μl of 4× Loading Dye). The samples were denatured at 70° C. for 10 minutes. 12 μl of a total of 30 μl (remaining sample was stored at 4° C.) were then loaded (per well) into a 12 well 3-8% Tris Acetate gel and run for 4 hours at 150 volts. The gel was removed from the cassette and soaked in Transfer Buffer containing 10% methanol for 20 minutes. Overnight wet transfer of the gel to a nitrocellulose membrane was performed at 15 volts at 4° C. The next day, the voltage was increased to 50 volts for 30 min on ice. Upon completion of transfer, the blot was blocked using 5% milk in TBS-T with agitation (Labnet ProBlot™Rocker 25, setting 60 rpm) for 2 hours at room temperature and incubated with commercially available anti-C7 LH7.2 (0.25 μg/mL) with agitation for 2 hours at room temperature. The blot was washed 3 times with 1×TBS-T (5 min each) with agitation before being probed with HRP-conjugated goat anti-mouse IgG (0.1 μg/mL) with agitation for 1 hour at room temperature. The blot was developed using Lumiglo UltraTMChemiluminescent substrate and Fujifilm LAS 3000 Imaging System. 
     C.6 Lam332 Binding 
     96-well MaxiSorp™ plates were coated with 1 μg Lam332 or BSA in 100 mM carbonate buffer, pH 9.3 overnight at 4° C. The plates were rinsed five times with PBS-T, and then blocked with 1% BSA in PBS-T at room temperature for 1 hour. Coated wells were rinsed five times with PBS-T and incubated with supernatants from transduced cells overnight at 4° C. Following three more washes with PBS-T, bound C7 was incubated with the fNC1 antibody (0.5 μg/mL, PBS-T) at room temperature for 3 hours, followed by incubation with HRP-conjugated donkey anti-rabbit IgG (0.8 μg/mL final concentration in PBS-T) at room temperature for 2 hrs. Detection of the C7-bound antibodies was via a colorimetric reaction using TMB and measured by reading the absorbance of the product at 450 nm on a SpectraMax® Plus 384 plate reader (Molecular Devices). All incubations were carried out on an orbital shaker (GeneMate) at a speed setting of 2. 
     C.7. Cell Migration Assay 
     CytoSelect™ 24-well wound-healing assay kit from Cell Biolabs, Inc. was used for the cell migration assay. GM-HDFs (0.8×10 5 ) were seeded in the presence of a wound insert in 24-well plates and grown for 48 hours. The wound insert was then removed and the cells were further cultured in reduced (1%) serum media containing 15 μg/mL type I collagen (COL1) for 8 hours. Brightfield images of the wounds were acquired 0, 2, 4, 6, and 8 hours after removal of the wound insert using the Celigo® Imaging Cytometry System (Cyntellect). The surface area of the wound at each time point was measured using ImageJ (National Institutes of Health). The data is reported as the percentage of migration (% Migration) covering the wound area. 
     C.7. Data Analysis 
     qPCR was performed using an ABI 7900 instrument utilizing SDS2.3 software. The experimental data were viewed and exported in SDS 2.4 software. ImagJ software was used to quantitate percent cell migration for the assay described above. ImageJ is an open source image processing program developed by the Nation Institute of Health and designed for scientific multidimensional images. 
     D. Results 
     D.1. Characterization of C7 Expression 
     D.1.1. LV-COLT Copy Number and Transcript Levels 
     Taqman® assays were designed against various regions of the viral shuttle vector. Of the Taqman® assays tested, designs targeting the C7 coding sequence demonstrated unacceptable levels of background amplification. The Taqman® assay selected, PR13843, was targeted to a sequence found at the 5′ end of the LV backbone as described in Greenberg et al. (2006). 
     GM-HDF training run cells were obtained, washed with dPBS, and resuspended in RLT buffer. Qiagen&#39;s AllPrep kit was used to isolate the DNA (for copy number) and RNA (for mRNA transcript level) from the drug substance vials from each arm of each training run. The isolated DNA and RNA were then subjected to qPCR and RT-qPCR assays as described above in Section C.1 and C.2, respectively. Table 40 shows the copy number/cell from Training Runs 8, 9, and 10 (TR8, TR9 and TR10) and the Engineering Run (ER1). The data demonstrate that the number of copies of transgene integrated into each cell can be modulated by the virus dose given to the cells during production, and is less than 1 copy/cell. Similarly, the LV-COLT transcript levels, shown in  FIG. 40 , also correlate with the virus dose and copy number, with Arms A and B from the training runs expressing higher levels (3 to 5 LOG) of the lentiviral C7 sequence compared to Control cells. 
     
       
         
           
               
             
               
                 TABLE 40 
               
             
            
               
                   
               
               
                 LV-COL7 Copy Number in GM-HDF Production Run Cells 
               
            
           
           
               
               
               
               
               
            
               
                 Production 
                 LV-COL7 
                   
                 MOI 
                 Gene Copy per 
               
               
                 Run 
                 Used 
                 Arm 
                 (IU/cell) 
                 Cell 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 TR8 
                 INXN-2002 
                 A 
                 3.4 
                 0.021 ± 0.007 
               
               
                   
                 (Pilot) 
                 B 
                 1.7 
                 0.017 ± 0.007 
               
               
                   
                   
                 Control 1   
                 0 
                 BLQ 2   
               
               
                 TR9 
                 LV-HA-COL7 
                 A 
                 1.0 
                 0.009 ± 0.002 
               
               
                   
                 (Pilot) 
                 B 
                 1.9 
                 0.021 ± 0.002 
               
               
                   
                   
                 Control 1   
                 0 
                 BLQ 2   
               
               
                 TR10 3   
                 INXN-2002 
                 A (3) 
                 2.0 
                 0.013 ± 0.002 
               
               
                   
                 (GMP) 
                 Control 1   
                 0 
                 BLQ 2   
               
               
                 ER1 3   
                 INXN-2002 
                 A (3) 
                 2.6 
                 0.025 ± 0.005 
               
               
                   
                 (GMP) 
                 Control 1   
                 0 
                 BLQ 2   
               
               
                   
               
               
                   1 Control arms were mock-transduced and were not exposed to LV-COL7 
               
               
                   2 BLQ: below limit of quantitation 
               
               
                   3 Only a single transduction arm was generated for TR10 and for ER1 
               
            
           
         
       
     
     D.1.2. C7 Protein Expression 
     Indirect immunofluorescence (IF) was used to examine C7 protein expression by GM-HDF cells using an antibody specific for the C7. Normal human dermal fibroblasts (NHDFs) were used as a positive control for this assay and the control arms of the training runs were used as negative controls. In the representative images shown in  FIG. 28  below, a small percentage (&lt;10%) of INXN-2002 and LV-HA-COL7-transduced RDEB fibroblasts show C7 staining, which is consistent with the copy number of integrated LV (Table 18). The signal from the GM-HDFs that are C7-positive is brighter than the signal from NHDFs, indicating greater amounts of C7 produced by GM-HDFs. 
     A direct ELISA was developed by Intrexon to measure C7 secreted by GM-HDFs into the cell culture media. This assay, as described above in Section C.4, utilized an NC1 region-specific polyclonal antibody. ELISA assay results of the GM-HDFs from the three training runs show that C7 expression is LV dose dependent with expression levels ranging from 45-90 ng/mL of cell supernatants ( FIG. 29 ). 
     D.2. Functional Assessment of C7 Expressed by GM-HDFs 9.2.1. Formation of C7 Trimers 
     The formation of C7 trimers is important in the assembly of anchoring fibrils. To verify that the C7 expressed by GM-HDFs will be capable of forming anchoring fibrils, we assessed whether or not the expressed C7 formed trimers using immunoprecipitation (IP) with an anti-C7 specific antibody (fNC1) for selective capture, then concentration of C7 from culture supernatants for detection by SDS-PAGE/immunoblot. 
     The optimal conditions for immunoprecipitation were established by testing a commercial C7 antibody and the fNC1 C7 antibody. The coating of protein G beads with fNC1 resulted in higher levels of C7 binding compared to coating with the commercially available antibody. Controls used in this assay include a no antibody control to rule out non-specific binding of C7 to the beads, as well as antibody coated beads incubated with and without purified C7 spiked into conditioned media as controls for specificity of C7 isolated from GM-HDF culture supernatants. 
     Immunoprecipitation results for TR8 and for TR10 and ER1 showed that the C7 produced by GM-HDF was predominantly trimeric ( FIG. 10 , and  FIG. 11 , respectively). Some lower molecular weight species showing immunoreactivity were also observed, and included the dimeric and 290 kDa monomeric forms. IP of C7 from GM-HDFs of TR9 was detected on the immunoblot at low levels by both an anti-C7 antibody (left panel,  FIG. 11B ) as well as an antibody specific for the HA tag incorporated into the C7 protein (right panel,  FIG. 11B ). 
     D.2.2. Lam332 Binding by C7 Expressed from GM-HDFs 
     C7 has been shown to bind immobilized extracellular matrix (ECM) components, including fibronectin, Laminin 332 (Lam332), COL1, and COL4 (Chen, et al., 2002a). The interaction between C7 and Lam332 occurs through the NH2-terminal NC1 domain of C7 and is dependent upon the native conformation of both Lam332 and C7 NC1 (Rousselle, et al., 1997). The association between C7 and Lam332 is important for establishing correct Lam332 architecture at the dermal-epidermal junction. Such organization is important for interactions with extracellular ligands and cell surface receptors, and for cell signaling (Waterman, et al., 2007). Intrexon developed an ELISA using an antibody against the C7 NC1 domain to detect binding of C7 to purified Lam332. Although a C7/Lam332 binding ELISA has already been described in the literature (Chen, et al., 2002a), to our knowledge it has never been used to test C7 present in the supernatants of transduced cells. Results in  FIG. 12  show dose-dependent binding to Lam332 by C7 expressed by GM-HDFs from the Training and Engineering runs. 
     D.2.3. Cell Migration Assay 
     Previous studies have shown that RDEB fibroblasts and keratinocytes show an increase in motility relative to their normal counterparts, and that normal motility can be restored by expression of C7 (Chen, et al., 2000; Chen, et al., 2002b; Cogan, et al. 2014; Baldeschi et al., 2003). Most of these studies employed either the colloidal gold salt migration assay to measure the migration of fibroblasts and keratinocytes, or a wound-healing assay to measure the migration of keratinocytes. Here we used the CytoSelect™ 24-well wound-healing assay kit (Cell Biolabs, Inc.) to measure the motility of NHDFs and GM-HDFs. 
     Immunofluorescent staining for C7 revealed that only a small percentage (&lt;10%) of transduced cells expressed high levels of the protein, raising the question as to whether a small number of C7-producing cells could have a global effect on cell migration. To determine if the presence of C7 in the culture media is sufficient to reverse the RDEB hyper-motility phenotype, the wound healing assay was carried out on GM-HDFs from the three training runs, with NHDFs as a positive control and the mock-transduced Control (RDEB) arms as the negative control. 
     Control fibroblasts from TR8, TR10, and ER1 exhibit a hyper-motility phenotype that is reversed by the expression of C7 ( FIG. 30 ). However, the Control fibroblasts from TR9 do not exhibit a significant hyper-motility phenotype, making it difficult to investigate the functionality of HA-tagged C7 in this assay. 
     Conclusions 
     The integrated transgene copy number per cell in GM-HDFs was dependent on the virus dose ranging from 0.009 to 0.03 transgene copies per cell. 
     Dose-dependent C7 expression from the GM-HDF cells was confirmed by qRT-PCR, immunofluorescence staining, and ELISA. 
     The structure of the C7 expressed by the GM-HDF cells was confirmed to be predominantly trimeric by immunoprecipitation/SDS-PAGE/immunoblot analysis for TR8, TR10, and ER1. TR9 showed minimal trimer formation. 
     The C7 produced from the TR8 GM-HDF cells was demonstrated to be functional by binding to Laminin332 in an in vitro binding assay and by correction of the hypermotility phenotype of Control RDEB cells in an in vitro migration assay. TR9 showed binding to Laminin332, however, migration assay results were equivocal. TR9 HA-GM-HDFs generated from transduction with LV-HA-COLT showed low levels of trimer, less binding to Lam332, and lack of clear hypermotility correction. 
     Due to lack of confirmation of in vitro functionality of HA-C7 expressed by TR9 HA-GM-HDFs, TR8 cells were chosen for in vitro and in vivo hybrid pharmacology/toxicology evaluations. This eliminated the ability to assess expression, localization and persistence in normal human skin or RDEB composite grafts prepared with devitalized human skin. Ultimately, composite grafts prepared with devitalized porcine dermis were used for these studies. Immunostaining with human specific anti-C7 antibody was used to distinguish rC7 form native C7 in this approach. 
     Example 8—Drug Product 
     FCX-007 Drug Product consists of a sterile suspension of each patient&#39;s own living autologous fibroblast cells, which are gene modified using INXN-2004 LV-COLT to encode the human wild type COL7A1 gene, formulated in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) without phenol red. 
     Formulation Development 
     Development of FCX-007 formulation and cell dosage was based on Fibrocell&#39;s manufacturing experience with azficel-T (LAVIV®); a live, autologous fibroblast cell product for injection. Fibroblast cells have been successfully formulated in DMEM at a cell concentration range of 0.5-3.0×10 7  cells/mL without viscosity concerns (azficel-T BLA #125348). Data suggest viscosity becomes a concern for product injection when cell concentration reaches 5.8×10 7  cells/mL. As a result, DMEM at a cell concentration of 1.0-3.0×10 7  cells/mL was selected as the formulation for FCX-007 DP. 
     Excess 
     Up to ten FCX-007 DP vials are shipped to the clinic as one dose for treatment injection. At the time of administration, the DP vial is gently inverted three times, and then 1 mL from each vial is drawn aseptically into a sterile 1 mL syringe with a 21 gauge needle. The 21 gauge needle is then replaced with a 30 gauge needle for intradermal administration. The process is repeated for the remaining 9 product vials. 
     FCX-007 DP vials are filled with 1.2 mL of the fibroblast cell suspension into a 2.0 mL capacity vial for a recoverable volume of 1.0 mL. The 0.2 mL excess is intended to ensure that 1.0 mL can be obtained from the vial at the time of administration in the clinic. 
     a. Physicochemical and Biological Properties 
     i. Physicochemical Properties 
     FCX-007 DP is presented as a gene modified autologous fibroblast cell suspension in DMEM without phenol red with a 1.2 mL fill in a 2 mL cryovial. The cells are demonstrated by assays to be a living culture. Table 41 shows the physicochemical properties of FCX-007 DP. 
     
       
         
           
               
             
               
                 TABLE 41 
               
             
            
               
                   
               
               
                 Physicochemical Properties of FCX-007 DP 
               
            
           
           
               
               
               
            
               
                   
                 Physicochemical Property 
                 Value 
               
               
                   
                   
               
               
                   
                 Cell Count 
                 1.0-3.0 × 10 7  cells 
               
               
                   
                 Cell Viability 
                 ≥70% 
               
               
                   
                   
               
            
           
         
       
     
     Biological Properties 
     FCX-007 is a sterile autologous fibroblast cell product that is genetically modified to express the human collagen 7 protein (C7). INXN-2004 vector copy number per cell and expression of the C7 protein are unique biological properties of FCX-007 DP. FCX-007 DP is a live cell suspension product that is produced from FCX-007 DS by washing the thawed FCX-007 DS cells in PBS and DMEM buffers. INXN-2004 vector copy number per cell and C7 protein expression are analyzed on the FCX-007 DS prior to release to manufacture FCX-007 DP Table 42 shows the biological properties of FCX-007 DP. 
     
       
         
           
               
             
               
                 TABLE 42 
               
             
            
               
                   
               
               
                 Biological Properties of FCX-007 DP 
               
            
           
           
               
               
               
            
               
                 Biological Property 
                 Value 
                 Stage Measured 
               
               
                   
               
               
                 Cell Type Purity/Identity 
                 ≥98% CD 90+ 
                 Drug Substance 
               
               
                 (fibroblast) 
               
               
                 INXN-2002 Vector Copy 
                 0.1-5.0 
                 Drug Substance 
               
               
                 Number 
               
               
                 C7 Protein Expression 
                 ≥500 ng/day/E6 cells 
                 Drug Substance 
               
               
                 Sterility 
                 No Growth (Sterile) 
                 Drug Product 
               
               
                   
               
            
           
         
       
     
     Batch Formula 
     To prepare the FCX-007 Drug Product, FCX-007 Drug Substance vials are removed from the vapor phase of liquid nitrogen frozen storage, thawed, and pooled inside an ISO 5 BSC. The cells are washed with phosphate buffered saline (PBS), followed by a wash with Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) without phenol red before being suspended in DMEM without phenol red to a target concentration of 1.0-3.0×10 7  cells/mL. The formulated cells are then filled into 2 mL cryovials at 1.2 mL/vial. Drug Product material prepared for each injection treatment is defined as a batch. The proposed treatment batch size is up to ten (10) 2 mL vials containing 1.2 mL of cell suspension. 
     Table 43 provides a list of all components used in the Drug Product manufacturing process. 
     
       
         
           
               
             
               
                 TABLE 43 
               
             
            
               
                   
               
               
                 Composition and Quantity of FCX-007 Drug Product Batch 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Quality 
                 Quantity per 
                 Quantity per Batch 
               
               
                 Component 
                 Function 
                 Standard 
                 DP Vial 1   
                 (10 vials per batch) 
               
               
                   
               
               
                 FCX-007 DS 
                 Drug 
                 Drug Product 
                 1.2-3.6 × 10 7   
                 1.2-3.6 × 10 8   
               
               
                   
                 Substance 
                 Certificate of 
                 cells 
                 cells 
               
               
                   
                   
                 Analysis 
               
               
                 PBS 
                 Diluent used 
                 Manufacturer&#39;s 
                 Minimal 
                 Minimal 
               
               
                   
                 for washing 
                 Certificate 
                 residual trace 2   
                 residual trace 2   
               
               
                   
                 Drug 
                 of Analysis 
               
               
                   
                 Substance 
               
               
                   
                 cells 
               
               
                 DMEM 
                 Buffer for 
                 Manufacturer&#39;s 
                 1.2 mL 
                 12 mL 
               
               
                 without 
                 cell 
                 Certificate 
               
               
                 phenol red 
                 suspension 
                 of Analysis 
               
               
                   
               
               
                   1 Vial contains 1.2 mL of FCX-007 cell suspension 
               
               
                   2 PBS wash residual is diluted with DMEM without phenol red such that trace amounts of PBS is possible but not quantifiable 
               
            
           
         
       
     
     Description of Manufacturing Process and Process Controls 
     b. Manufacturing Process Flow Diagram 
     Manufacturing of the FCX-007 Drug Product commences when FCX-007 Drug 
     Substance vials are removed from liquid nitrogen storage and thawed using a ThermoMixer®. In an ISO5 BSC the thawed FCX-007 DS cells are pooled into a 250 mL centrifuge tube containing &gt;5× bulk volume of phosphate buffered saline (PBS) wash buffer for re-suspension. The cells are centrifuged at 1000 rpm for 10 minutes at 5±3° C. and the PBS wash buffer is removed. This is followed by a wash with &gt;5× bulk volume of Dulbecco&#39;s Modified Eagle Medium (DMEM) by re-suspension and centrifugation at 1000 rpm for 10 minutes at 5±3° C. The DMEM wash buffer is removed. The washed cells are re-suspended in DMEM without phenol red to a target concentration of 2.0×10 7  cells/mL. The cell suspension is manually filled into sterile 2.0 mL cryovials in an ISO5 BSC to a volume of 1.2 mL per vial to produce the FCX-007 DP vials. The Drug Product vials are stored at 2-8° C. until released by QA for shipment in a Credo Cube™ shipper to the clinical site. 
     Manufacturing Process Description 
     FCX-007 Drug Product is manufactured as indicated above. INXN-2002 is a synonymous name for FCX-007. 
     Step 1: Removal of FCX-007 Drug Substance Vials from Liquid Nitrogen Storage and Lot Verification 
     The FCX-007 Drug Substance, which is stored in a liquid nitrogen freezer, is requested from Materials Management. FCX-007 Drug Substance vials are then pulled for this batch of FCX-007 Drug Product production before being transferred to the cleanroom suite. 
     Step 2: Thaw Cryovials 
     A Line Clearance of the cleanroom suite is performed by QA per SOP-0099 GMP Production Line Clearance/GTP Line Cleaning in order to release the room for processing use. Operators initiate room, personnel, and BSC environmental monitoring (EM) per SOP-0129 (In Process Environmental and Personnel Monitoring) prior to processing and non-viable EM sampling in the BSC during processing. 
     The FCX-007 DS cyrovials are disinfected and thawed in a ThermoMixer® equilibrated to 37° C. until cell suspension is almost completely thawed with a sliver of ice crystals remaining. The thawed vials are transferred to a BSC. 
     Step 3: PBS Wash 
     Inside the BSC, using a pipette, transfer approximately 150 mL of PBS (&gt;5 volumes of the FCX-007 DS cell suspension) into a 250 mL centrifuge tube. The contents of the thawed cryovials are transferred into the 250 mL centrifuge tube. Use the diluted cell suspension to rinse the pipette. Use a pipette to transfer 3.0 mL of fresh PBS to each cryovial as a rinse. Transfer the rinse to a 250 mL centrifuge tube. Swirl the tube gently to mix the cell suspension. Transfer the centrifuge tube to a centrifuge and centrifuge the cells at 1000 RPM for 10 minutes at 5±3° C. After centrifugation, remove the PBS wash supernatant from the tube. 
     i. Step 4: DMEM Wash 
     Using a pipette, add approximately 200 mL of DMEM without phenol red (&gt;5 volumes of the FCX-007 DS cell suspension) to the tube to re-suspend the cell pellet. Swirl the tube gently to mix the cell suspension. Transfer the centrifuge tube to a centrifuge and centrifuge the cells at 1000 RPM for 10 minutes at 5±3° C. After centrifugation, using a pipette, transfer 0.9 mL of the supernatant from the centrifuge bottle into each of 2 separate 2 mL cryovials (1.8 mL total) labeled “Lot, QC-Sterility”. Set the cryovials labeled for sterility aside in the BSC. Aspirate the remaining DMEM wash supernatant from the centrifuge tube. 
     Step 5: Cell Pellet Re-Suspension in DMEM 
     The volume of DMEM used to re-suspend the cell pellet is calculated as: 
     Volume of thawed FCX-007 DS cell suspension divided by 1.5 
     The 1/1.5 volume factor compensates for possible cell loss during the wash steps, in order to result in a cell concentration in the specified range of 1.0-3.0×10 7  cells/mL after re-suspension. 
     Use a pipette to transfer the calculated amount of DMEM to re-suspend the cell pellet in the centrifuge tube. Use a pipette to transfer 0.1 mL of the cell suspension into a 2 mL cryovial labeled “Lot, QC-Count and Viability” and submit the cryovial to QC for cell counting. Use a pipette to measure the remaining total cell suspension volume and place the cell suspension into a 2-8° C. refrigerator for later final fill use. 
     Step 5a: Re-dilution (if needed) 
     If the QC cell concentration is above 3.0×10 7  cells/mL, perform an additional dilution using fresh DMEM to target a cell concentration of 2.0×10 7  cells/mL. Re-submit a cell suspension sample to QC for cell count to confirm final cell concentration. 
     Step 6: Final Fill 
     Retrieve the cells from the refrigerator and transfer the cells to the BSC for final fill. Use a pipette to fill the well-mixed cell suspension manually into 2 mL cryovials in the following order: 
     Fill 0.1 mL of cell suspension into the first sterility sample vial (added to retained sample from Step 4) 
     Fill the product vials at 1.2 mL per vial 
     Fill 0.1 mL of cell suspension into the second sterility sample vial (added to retained sample from Step 4) 
     Fill the QC vial at ≤1.8 mL per vial 
     Submit the sterility and QC vials for testing. 
     Step 7: Store DP Vials for Release and Shipment 
     Hold the DP vials at 5±3° C. for release and shipment to the clinical site. 
     Description of the In-Process Controls 
     Environmental Controls 
     All cell culture manipulations are carried out in a certified ISO 5 Biological Safety Cabinets (BSCs) within an ISO7 environmental background using aseptic techniques. The final Drug Product 2 mL cryovial containers are purchased pre-sterilized from Corning. The operators performing the final fill are qualified for aseptic fill operations. 
     In-Process Controls and Testing 
     The process controls and testing performed for release of FCX-007 Drug Product are described below. 
     Step 1 
     The correct FCX-007 Drug Substance lot is verified by QA before use in FCX-007 DP manufacture. 
     Step 2 
     The temperature of the ThermoMixer® used to thaw FCX-007 Drug Substance vials is controlled at 37° C. The content of each vial is thawed until a sliver of ice still remains in the vial to prevent overheating. 
     Step 3 
     The PBS wash volume is controlled at ≥5 volumes of the FCX-007 DS cell pool to ensure adequate washing of the cells. The centrifugation conditions are controlled at 1000 rpm, 10 minutes and 5±3° C. to ensure sufficient cell pelleting. 
     Step 4 
     Similarly to the PBS wash step, the DMEM wash volume is controlled at ≥5 volumes of the FCX-007 DS cell pool to ensure adequate washing of the cells. The centrifugation conditions are controlled at 1000 rpm, 10 minutes and 5±3° C. to ensure sufficient cell pelleting. Two 900 μL aliquots of DMEM wash supernatant are taken for use in sterility testing of the final product. 
     Step 5 and 5(a) (if needed) 
     The re-suspended cell concentration is controlled at 1.0-3.0×10 7  cells/mL, and viability≥70%. 
     Step 6 
     Fill volume of the FCX-007 DP vials is controlled at 1.2 mL fill per vial. A final cell count and viability measurement, as well as Gram stain is performed on the filled QC vial. The two sterility vials are submitted for sterility testing. The results of this sterility test are not received until after Drug Product release, so release of DP is done on the basis of a negative Gram Stain. The use of a rapid microbial test in this situation is indicated in Guidance for FDA Reviewers and Sponsors “Content and Review of Chemistry, Manufacturing, and Control (CMC) Information for Human Somatic Cell Therapy Investigation New Drug Applications (INDs)” of April 2008. 
     ii. Control of Materials 
     The FCX-007 Drug Product manufacturing process utilizes single use, disposable products that will come in contact with raw materials (e.g. pipets, centrifuge tubes, and cryovials) to remove cleaning and carryover issues from consideration. Raw materials selected for use in the manufacturing process are acceptable for pharmaceutical processing as being USP VI compliant. All disposable items are purchased pre-sterilized by gamma irradiation. The PBS and DMEM wash buffers are purchased sterile from commercial suppliers. Steps in the process have the appropriate process parameters identified, such as time and temperature. 
     Example 9—In Vitro Characterization of Optimized GM-HDF 
     This example describes characterization of GM-HDFs generated using an enhanced LV transduction procedure the LV-COLT construct INXN-2004. Three training runs (TR11, TR12.1, and TR13) were executed to generate cells for the analyses at GMP-scale. TR11 was transduced with the LV-COLT vector INXN-2002 and was used to evaluate a centrifugation-enhanced (spinoculation) LV transduction procedure with and without a second transduction step (super-transduction). TR12.1 and TR13 were transduced with the LV-COLT vector INXN-2004 utilizing enhanced transduction procedures as determined through evaluation of TR11. 
     GM-HDFs from each training run were characterized using assays to assess results such as copy number of integrated LV-COLT, protein expression of C7, and functionality of the C7 expressed by GM-HDFs. 
     LV integration and C7 expression levels increased first with the addition of the super-transduction step and increased further with use of the LV-COLT INXN-2004. C7 expressed from GM-HDFs from all three training runs assembled into the trimeric form and bound to Laminin332, which is important in the formation of anchoring fibrils. Expression of C7 by the GM-HDFs was able to reverse hypermotility observed in RDEB fibroblasts. GM-HDFs from TR12.1 and 13 were selected to be used in in vivo GLP toxicology studies. 
     Objectives in this study were to (a) demonstrate improvement in copy number of integrated LV and expression of C7 resulting from 1) an enhanced transduction procedure incorporated into the at-scale GMP production method and 2) adoption of the INXN-2004 LV-COLT construct and (b) to confirm the functionality of C7 expressed by the GM-HDFs 
     1. Study Materials 
     1.1. Test Article(s) 
     GM-HDFs from Training Runs (TR) 11, 12.1, and 13 were characterized. 
     
       
         
           
               
             
               
                 TABLE 44 
               
             
            
               
                   
               
               
                 General production information for TR11, TR12.1, and TR13 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 LV-COL7 
                   
                 MOI 
                 Super- 
                   
               
               
                 Produc- 
                 and TU 
                   
                 (IU/ 
                 trans- 
                 Production 
               
               
                 tion Run 
                 titer 
                 Arm 
                 cell) 
                 duction 
                 Scale 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 TR11 
                 INXN-2002 
                 A 
                 0.77 
                 Yes 
                 2 x 10-layer 
               
               
                   
                 (GMP) 
                 B 
                 0.77 
                 No 
                 CellSTACKs ® 
               
               
                   
                 1.2 × 10 5   
                 Control 1   
                 0 
                 n/a 
               
               
                   
                 IU/mL 
               
               
                 TR12.1 
                 INXN-2004 
                 A 
                 0.12 
                 Yes 
                 2 x 10-layer 
               
               
                   
                 (GMP) 
                   
                   
                   
                 CellSTACKs ® 
               
               
                   
                 1.4 × 10 5   
                 B 
                 0.12 
                 No 
                 T-175 2   
               
               
                   
                 IU/mL 
               
               
                 TR13 
                 INXN-2004 
                 A 
                 0.12 
                 Yes 
                 2 x 10-layer 
               
               
                   
                 (GMP) 
                   
                   
                   
                 CellSTACKs ® 
               
               
                   
                 1.4 × 10 5   
                 B 
                 0.12 
                 No 
                 T-175 2   
               
               
                   
                 IU/mL 
               
               
                   
               
               
                   1 Control arm cells were mock-transduced 
               
               
                   2 Arm B of TR12.1 and TR13 were for research only. Passaging of these arms terminated early and, thus, did not complete the full-scale production process. 
               
            
           
         
       
     
     1.2. Primer and Probe Sequences 
       
     
       
         
           
               
             
               
                 TABLE 45 
               
             
            
               
                   
               
               
                 Primer and Probe Sequences used for qPCR Assays 
               
            
           
           
               
               
               
            
               
                 Name 
                 Sequence 1   
                 Assay 
               
               
                   
               
               
                 PR13843 forward 2   
                 ACCTGAAAGCGAAAGGGAAAC (SEQ ID 
                 LV-COL7 copy number 
               
               
                   
                 NO: 31) 
                   
               
               
                   
               
               
                 PR13843 reverse 2   
                 CACCCATCTCTCTCCTTCTAGCC (SEQ ID 
                 LV-COL7 copy number 
               
               
                   
                 NO: 32) 
                   
               
               
                   
               
               
                 PR13843 probe 2   
                 6-FAM- 
                 LV-COL7 copy number 
               
               
                   
                 AGCTCTCTCGACGCAGGACTCGGC-3′IB 
                   
               
               
                   
                 FQ (SEQ ID NO: 33) 
               
               
                   
               
               
                   1 Primers/probes purchased from IDT. 
               
               
                   2 LV-specific primer/probe sequences derived from Greenberg et al. (2006). 
               
            
           
         
       
     
     2. Experimental Procedures 
     2.1. LV-COL7 Copy Number 
     Nucleic acid isolation was performed using Qiagen&#39;s AllPrep kit according to the manufacturer&#39;s instructions. gDNA was isolated from 6.7×10 5  GM-HDF cells, and 8 μL of gDNA was used in a 20 μL assay (10 μL Taqman® Gene Express, 1.8 μL nuclease-free water, 0.06 μL of 100 μM forward primer, 0.06 μL of 100 μM reverse primer, 0.04 μL of 100 μM Taqman® probe). A standard curve of serially diluted linearized LV-COL7 lentiviral shuttle vector (1e6 copies/reaction to 5 copies/reaction), plus 4 μL of human gDNA (1.5e4 cells/reaction) were also assayed in the 20 μL assay mentioned above. The Taqman® assay used was PR13843. 8 μL of an additional standard curve of commercial human gDNA (Clontech) (1.5e4 cells/reaction to 2e2 cells/reaction) in a 20 μL assay (10 μL Taqman® Gene Express, 1.0 L nuclease-free water, 1 μL of 20×ACTB primer/probe set) was also performed. All samples were run in triplicate on an ABI7900HT using the following cycling parameters: 2 minutes at 50° C., 10 minutes at 95° C., and 40 cycles of 15 sec at 95° C. and 1 minute at 60° C. 
     2.2. C7 Immunofluorescence 
     For immunofluorescence analyses, 1.2×10 4  GM-HDFs were allowed to attach to PDL/Lamin-coated coverslips in 24-well plates overnight and then fixed and permeabilized with a 50%/50% mix of methanol/acetone. The coverslips were washed 3 times with 1×PBS and then blocked with 10% goat serum in PBS for 30 minutes at room temperature. After three additional washes with PBS, the coverslips were incubated with 1.25 μg/mL of a polyclonal fNC1 antibody (α-fNC1) in 1% goat serum/PBS, followed by 3 additional washes with PBS and incubation with 5 μg/mL Alexa Fluor® 555-conjugated goat anti-rabbit IgG in 1% goat serum/PBS for 1 hour at room temperature. Coverslips were stained with NucBlue® Live Cell Stain Ready Probes Reagent before being mounted onto slides. Images were acquired on a Zeiss Axio Observer microscope at 20× magnification using an exposure time of 290 ms. NHDFs or GM-HDFs were fixed, permeabilized, and stained with NucBlue® Live Cell Stain to visualize nuclei (blue) or with the fNC1 antibody and Alexa Fluor® 555-congugated goat anti-rabbit IgG (5 μg/mL) to visualize C7 expression (red). Images were acquired at 20× and at 5× magnification using an exposure time of 290 ms. 
     2.3. C7 Flow Cytometry 
     GM-HDFs (˜500,000 cells) were collected into each well of a 96-well plate V-bottom plate, concentrated by centrifugation (1500 rpm, 5 min, room temp.), and the culture media supernatants removed. The cells were then resuspended in 200 μL of CytoFix, transferred to a 96-well V-bottom plate, and incubated for 30 min at 4° C. Following fixation, the fixative is then removed by centrifugation (1500 rpm, 5 min, 4° C.) and the cells permeabilized with 200 μL ice-cold 100% methanol for 30 min on ice. Cells were then held at −20° C. prior to staining procedure. For staining, permeabilized cells were washed twice with 200 μL incubation buffer (0.5 g BSA in 100 mL PBS), with centrifugation (300 rpm, 5 min, room temp.) in between washes to remove the buffer. Washed cells were resuspended in 100 μL of incubation buffer containing α-fNC1 diluted 1:400 and incubated for 1 hour at room temp. Cells were then washed twice as described above and resuspended in 100 μL of incubation buffer containing AlexaFluor 555-Rabbit IgG secondary antibody diluted 1:2000 and incubated for 30 min at room temp. Cells were then washed twice as described above and resuspended in 100 μL of PBS. C7-expressing cells were enumerated by flow cytometry using BD LSRII and BD FACSDiva Software (V6.2) with the following parameters: FSC Voltage=484, SSC Voltage=209, PE=500. 
     2.4. C7 Protein ELISA 
     Briefly, a standard curve of purified His-NC1 fragment (9.8-625 ng/mL) or collected supernatants containing C7 protein (from GM-HDF in culture for three to five days) were immobilized to a Nunc MaxiSorp® 96-well plate overnight at 4° C. Standards and samples were tested in the same sample matrix (10% RDEB fibroblast conditioned media). Coated wells were washed with PBST and blocked with 3% BSA/PBS for 1 hour at 37° C. Detection was accomplished using α-fNC1 Ab (0.5 μg/mL) followed by incubation with secondary antibody donkey anti-rabbit IgG HRP (Jackson ImmunoResearch, 0.08 μg/mL). Bound antibodies were detected via colorimetric development with TMB substrate solution. Following quenching of the reaction, absorbance was measured at 450 nm on the SpectraMax® Plus 384 (Molecular Devices). 
     2.5. Immunoprecipitation of C7 Trimers 
     First, magnetic Protein G beads were washed with 1×PBS-T. Beads were bound to the magnet for a minimum of 2 minutes prior to removal of supernatant and in all subsequent steps. Following the washes, the beads were coated with 5 μg of α-fNC1 and incubated for 10 minutes with rotation (Glas-Col, setting 30-14 rpm at room temperature). Beads were bound to the magnet to remove the supernatant and washed with Ab binding/Wash buffer. Supernatants were collected from GM-HDFs in culture for three to five days. C7 containing supernatant was added to the bead/C7 supernatant mix and was incubated overnight at 4° C. with rotation (setting 30-14 rpm). The next day the beads were bound to the magnet and washed three times using Wash buffer. Target antigen was eluted in 20 μL of elution buffer (50 mM glycine, pH 2.8, and 10 μL of 4× Loading Dye). The samples were denatured at 70° C. for 10 minutes. 12 μL of a total of 30 μL (remaining sample was stored at 4° C.) were then loaded (per well) into a 12 well 3-8% Tris Acetate gel and run for 4 hours at 150 volts. The gel was removed from the cassette and soaked in Transfer Buffer containing 10% methanol for 20 minutes. Overnight wet transfer of the gel to a nitrocellulose membrane was performed at 15 volts at 4° C. The next day, the voltage was increased to 50 volts for 30 min on ice. Upon completion of transfer, the blot was blocked using 5% milk in TBS-T with agitation (Labnet ProBlot™ Rocker 25, setting 60 rpm) for 2 hours at room temperature and incubated with commercially available anti-C7 LH7.2 (0.25 μg/mL) with agitation for 2 hours at room temperature. The blot was washed 3 times with 1×TBS-T (5 min each) with agitation before being probed with HRP-conjugated goat anti-mouse IgG (0.1 μg/mL) with agitation for 1 hour at room temperature. The blot was developed using Lumiglo Ultra™ Chemiluminescent substrate and Fujifilm LAS 3000 Imaging System. 
     2.6. Lam332 Binding 
     96-well MaxiSorp™ plates were coated with 1 μg Lam332 or BSA in 100 mM carbonate buffer, pH 9.3 overnight at 4° C. The plates were rinsed five times with PBS-T, and then blocked with 1% BSA in PBS-T at room temperature for 1 hour. Coated wells were rinsed five times with PBS-T and incubated with supernatants from transduced cells overnight at 4° C. Following three more washes with PBS-T, bound C7 was incubated with the α-fNC1 (0.5 μg/mL, PBS-T) at room temperature for 3 hours, followed by incubation with HRP-conjugated donkey anti-rabbit IgG (0.8 μg/mL final concentration in PBS-T) at room temperature for 2 hrs. Detection of the C7-bound antibodies was via a colorimetric reaction using TMB and measured by reading the absorbance of the product at 450 nm on a SpectraMax® Plus 384 plate reader (Molecular Devices). All incubations were carried out on an orbital shaker (GeneMate) at a speed setting of 2. 
     2.7. Cell Migration Assay 
     CytoSelect™ 24-well wound-healing assay kit from Cell Biolabs, Inc. was used for the cell migration assay. GM-HDFs (0.8×10 5 ) were seeded in the presence of a wound insert in 24-well plates and grown for 48 hours. The wound insert was then removed and the cells were further cultured in reduced (1%) serum media containing 15 μg/mL type I collagen (COL1) for 8 hours. Brightfield images of the wounds were acquired 0, 2, 4, 6, and 8 hours after removal of the wound insert using the Celigo® Imaging Cytometry System (Cyntellect). The surface area of the wound at each time point was measured using ImageJ (National Institutes of Health). The data is reported as the percentage of migration (% Migration) covering the wound area. 
     3. Data Analysis 
     qPCR was performed using an ABI 7900 instrument utilizing SDS2.3 software. The experimental data were viewed and exported in SDS 2.4 software. FlowJo V10 software was used to analyze Flow Cytometry data. ImagJ software was used to quantitate percent cell migration for the assay described in Section 2.7. ImageJ is an open source image processing program developed by the Nation Institute of Health and designed for scientific multidimensional images. 
     4. Results 
     4.1. Characterization of C7 Expression 
     The overall objectives of this evaluation were to increase copy number and protein expression through a comparison of transduction methods and LV vectors. Specific objectives were to:
         1. Evaluate the effect of super-transduction on integrated copy number   2. Evaluate the effect of spinoculation on integrated copy number   3. Compare copy number values between INXN-2002 and INXN-2004 under super-transduced and spinoculated conditions.       

     Two GMP lots of LV-COLT constructs were used for these studies: the INXN-2002 and the construct INXN-2004 as described in this Example. 
     4.1.1. LV-COLT Copy Number 
     GM-HDF training run cells were obtained and copy number was determined as described above in Section 2.1. Table 46 below provides the copy number/cell from Training Runs utilizing the new transduction methods. Initial optimization implemented the addition of centrifugation during the transduction step (“spinoculation”). Standard transduction had been used previously with INXN-2002 and resulted in integrated copy numbers ranging from 0.013 to 0.025. Addition of only the spinoculation step for transduction with INXN-2002 resulted in a ≥2-fold increase in the integrated copy number to 0.05 per cell (TR11 Arm B, Table 46) compared to TR8 results from previous studies. 
     To further enhance the integrated copy number, a second transduction (“super-transduction”) was added to the GM-HDF production process. Addition of super-transduction, also utilizing spinoculation, further increased integrated copy numbers of INXN-2002 by approximately 2-fold (TR11 Arm A, Table 46). 
     Small-scale research studies (not shown) demonstrated a second-generation INXN-2004 LV-COLT construct, which utilizes an alternative 3 rd -generation self-inactivating LV backbone, had the potential to achieve higher integrated copy numbers, resulting in higher expression levels, while utilizing a lower multiplicity of infection (MOI) than used for INXN-2002. When used at GMP-scale with the optimized transduction procedure (spinoculations with super-transduction), INXN-2004 had further improved integrated LV copy numbers of approximately 4- to 8-fold over the INXN-2002 construct to achieve average copy numbers of 0.41 to 0.74 (TR12.1 and TR13, respectively, Table 46). 
     Of note, Arms B of TR12.1 and TR13 were not tested for copy numbers as they were not passaged through the entire production process (Table 44). Accurate copy number assessments require cells that have been passaged several times following transduction to ensure that episomal copies of LV, which can artificially inflate measured copy numbers, no longer remain. 
     
       
         
           
               
             
               
                 TABLE 46 
               
             
            
               
                   
               
               
                 LV-COL7 Copy Number in GM-HDF Production Run Cells 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 MOI 
                 Super- 
                   
               
               
                 Production 
                 LV-COL7 
                   
                 (IU/ 
                 trans- 
                 Gene Copy 
               
               
                 Run 
                 Used 
                 Arm 
                 cell) 
                 duction 1   
                 per Cell 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 TR8 2   
                 INXN-2002 
                 A 
                 3.4 
                 No 
                 0.021 ± 0.005 
               
               
                   
                 (pilot) 
               
               
                 TR11 
                 INXN-2002 
                 A 
                 0.77 
                 Yes 
                 0.09 ± 0.03 
               
               
                   
                 (GMP) 
                 B 
                 0.77 
                 No 
                 0.05 ± 0.01 
               
               
                   
                   
                 Control 3   
                 n/a 
                 No 
                 BLQ 4   
               
               
                 TR12.1 5   
                 INXN-2004 
                 A 
                 0.12 
                 Yes 
                 0.41 ± 0.19 
               
               
                   
                 (GMP) 
               
               
                 TR13 5   
                 INXN-2004 
                 A 
                 0.12 
                 Yes 
                 0.74 ± 0.10 
               
               
                   
                 (GMP) 
               
               
                   
               
               
                   1 Only indicated arms were super-transduced; all TR11, TR12.1, and TR13 arms were spinoculated 
               
               
                   2 TR8 Arm A data is presented as a comparator for previous results presented in RDEB-ADSO-2 
               
               
                   3 Control arm was mock-transduced and was not exposed to LV-COL7 
               
               
                   4 BLQ: below lower limit of quantitation 
               
               
                   5 Arm B for both TR12.1 and TR13 was not carried through the full production process and not tested for LV-COL7 copy numbers 
               
            
           
         
       
     
     4.1.2. C7 Protein Expression 
     Indirect immunofluorescence (IF) was used to examine C7 protein expression by GM-HDF cells using an antibody specific for the C7. TR11 Arms A &amp; B, and only Arm A of TR12.1 and TR13 were analyzed by IF. Normal human dermal fibroblasts (NHDFs) were used as a positive control and the control arm of TR11 was used as a negative control. Representative images at two magnifications, 20× and 5× are shown below in  FIG. 31 . Also included as a comparator for the original process are IF images of cells from TR8 Arm A which had integrated LV-COLT copy numbers of 0.015 per cell. Consistent with the copy number of integrated LV presented in Table 46 above, only a small number of TR11 GM-HDFs were visualized with C7 antibodies, though still greater than the number of C7-positive cells from TR8. Furthermore, the higher copy numbers measured from TR12.1 and TR13 GM-HDFs correlate to higher numbers of cells positive for C7 detection. Of note, the signal from the GM-HDFs that are C7-positive is brighter than the signal from NHDFs, suggesting greater amounts of C7 produced by GM-HDFs per cell. 
     The specific number of cells expressing C7 was then quantified using a flow cytometry assay. As described in Section 2.3, this assay utilized an NC1 region-specific polyclonal antibody. For this experiment, Arm B cells from TR12.1 and TR13 were included to compare the single transduction procedure (Arm B) with the super-transduction procedure (Arm A) using second-generation LV-COLT construct (INXN-2004). As shown in  FIG. 32  below, the percentage of C7-positive (C7+) cells for each training run arm is consistent with the qualitative number of cells expressing C7 as detected by IF ( FIG. 32 ), and correlates to the measured copy number (Table 46). Comparison of C7+ cells between Arm A and Arm B for TR12.1 and TR13 confirms the near doubling of expression levels that would be predicted by the addition of a second transduction. 
     A direct ELISA was used to measure C7 secreted by GM-HDFs into the cell culture media. This assay, as described in Section 2.4, also utilized the NC1 region-specific polyclonal antibody. ELISA assay results of the GM-HDFs from the three training runs show that C7 expression is dependent on the level of integrated LV copy numbers with expression levels reaching approximately 2300 ng/day/1 e6 cells ( FIG. 33 ). Compared to TR8 Arm A as a representative result from previous studies (RDEB-ADSO-2), this represents a ≥200-fold increase in C7 expression ( FIG. 33 ). 
     4.2. Functional Assessment of C7 Expressed by GM-HDFs 
     4.2.1. Formation of C7 Trimers 
     The formation of C7 trimers is essential in the assembly of anchoring fibrils (Bruckner-Tuderman, 1999). To verify that the C7 expressed by GM-HDFs will be capable of forming anchoring fibrils, GM-HDFs were assessed for the formation of C7 trimers using immunoprecipitation (IP) with an anti-C7 specific antibody (fNC1) for selective capture, then concentration of C7 from culture supernatants for detection by SDS-PAGE/immunoblot. Purified recombinant C7 was used as a positive control and the capture step without C7-specific antibody was used as a negative control. IP results for the three training runs showed that the C7 produced by the GM-HDFs was predominantly trimeric ( FIG. 34 ) with some lower molecular weight species showing immunoreactivity present, likely the dimeric and monomeric forms of C7. 
     4.2.2. Lam332 Binding by C7 Expressed from GM-HDFs 
     C7 has been shown to bind immobilized extracellular matrix (ECM) components, including fibronectin, Laminin 332 (Lam332), COL1, and COL4 (Chen, et al., 2002a). The interaction between C7 and Lam332 occurs through the NH2-terminal NC1 domain of C7 and is dependent upon the native conformation of both Lam332 and C7 NC1 (Rousselle, et al., 1997). The association between C7 and Lam332 is important for establishing correct Lam332 architecture at the dermal-epidermal junction. Such organization is critical for interactions with extracellular ligands and cell surface receptors, and for cell signaling (Waterman, et al., 2007). Intrexon developed an ELISA using an antibody against the C7 NC1 domain to detect binding of C7 to purified Lam332. Results from two experiments, presented in  FIG. 35 , show copy number-dependent binding to Lam332 by C7 expressed by GM-HDFs from the three Training runs. TR11 Control cells were used as a negative control in both experiments. 
     4.2.3. Cell Migration Assay 
     Previous studies have shown that RDEB fibroblasts and keratinocytes show an increase in motility relative to their normal counterparts, and that normal motility can be restored by expression of C7 (Chen, et al., 2000; Chen, et al., 2002b; Cogan, et al. 2014; Baldeschi et al., 2003). Most of these studies employed either the colloidal gold salt migration assay to measure the migration of fibroblasts and keratinocytes, or a wound-healing assay to measure the migration of keratinocytes. In this Example a CytoSelect™ 24-well wound-healing assay kit (Cell Biolabs, Inc.) was used to measure the motility of NHDFs and GM-HDFs. 
     To determine whether the presence of C7 in the culture media is sufficient to reverse the RDEB hyper-motility phenotype, the wound healing assay was carried out on GM-HDFs from the three training runs, with NHDFs as a positive control and the mock-transduced Control (RDEB) arms as the negative control. Control fibroblasts from TR11 Arm C (RDEB) exhibited a hyper-motility phenotype that is reversed by the expression of C7 ( FIG. 36 ). Interestingly, the levels of phenotype reversal was not dependent upon the integrated copy number/C7 expression levels, suggesting that even low levels of C7 expression are enough to reverse the hyper-motility phenotype. 
     5. Conclusions 
     The integrated transgene copy number per cell in GM-HDFs was improved by both the addition of enhanced transduction methods (spinoculation and super-transduction) and a change to the LV-COLT construct INXN-2004, with levels as high as 0.74 copies per cell achieved. The fold-improvements achieved with each process change are as follows:
         The addition of spinoculation: ≥2-fold   The addition of super-transduction (with spinoculation): ˜2-fold   The change from INXN-2002 to INXN-2004: &gt;4-fold   The total cumulative change from original process: average of &gt;27-fold       

     (TR12.1 and TR13 compared to TR8 used in original studies) 
     Integrated copy number-dependent C7 expression from the GM-HDF cells was confirmed by immunofluorescence staining, flow cytometry, and ELISA. A &gt;200-fold improvement in C7 expression was achieved relative to TR8 results presented in IND submission 16582 Serial 0000. 
     The structure of the C7 expressed by the GM-HDF cells was confirmed to be predominantly trimeric by immunoprecipitation/SDS-PAGE/immunoblot analysis. 
     In addition, the C7 produced from the GM-HDF cells was confirmed to be functional with binding to Laminin332 in vitro and by correction of the hypermotility phenotype of control RDEB cells in an in vitro migration assay. 
                                 APPENDIX 4                  COL7A1   ATGACGCTGCGGCTTCTGGTGGCCGCGCTCTGCGCCGGGATCCTGGCAGAGGCGCCCCGA   60           Genbank   ATGACGCTGCGGCTTCTGGTGGCCGCGCTCTGCGCCGGGATCCTGGCAGAGGCGCCCCGA   60               ************************************************************                       COL7A1   GTGCGAGCCCAGCACAGGGAGAGAGTGACCTGCACGCGCCTTTACGCCGCTGACATTGTG   120           Genbank   GTGCGAGCCCAGCACAGGGAGAGAGTGACCTGCACGCGCCTTTACGCCGCTGACATTGTG   120               ************************************************************                       COL7A1   TTCTTACTGGATGGCTCCTCATCCATTGGCCGCAGCAATTTCCGCGAGGTCCGCAGCTTT   180           Genbank   TTCTTACTGGATGGCTCCTCATCCATTGGCCGCAGCAATTTCCGCGAGGTCCGCAGCTTT   180               ************************************************************                       COL7A1   CTCGAAGGGCTGGTGCTGCCTTTCTCTGGAGCAGCCAGTGCACAGGGTGTGCGCTTTGCC   240           Genbank   CTCGAAGGGCTGGTGCTGCCTTTCTCTGGAGCAGCCAGTGCACAGGGTGTGCGCTTTGCC   240               ************************************************************                       COL7A1   ACAGTGCAGTACAGCGATGACCCACGGACAGAGTTCGGCCTGGATGCACTTGGCTCTGGG   300           Genbank   ACAGTGCAGTACAGCGATGACCCACGGACAGAGTTCGGCCTGGATGCACTTGGCTCTGGG   300               ************************************************************                       COL7A1   GGTGATGTGATCCGCGCCATCCGTGAGCTTAGCTACAAGGGGGGCAACACTCGCACAGGG   360           Genbank   GGTGATGTGATCCGCGCCATCCGTGAGCTTAGCTACAAGGGGGGCAACACTCGCACAGGG   360               ************************************************************                       COL7A1   GCTGCAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGGTGTC   420           Genbank   GCTGCAATTCTCCATGTGGCTGACCATGTCTTCCTGCCCCAGCTGGCCCGACCTGGTGTC   420               ************************************************************                       COL7A1   CCCAAGGTCTGCATCCTGATCACAGACGGGAAGTCCCAGGACCTGGTGGACACAGCTGCC   480           Genbank   CCCAAGGTCTGCATCCTGATCACAGACGGGAAGTCCCAGGACCTGGTGGACACAGCTGCC   480               ************************************************************                       COL7A1   CAAAGGCTGAAGGGGCAGGGGGTCAAGCTATTTGCTGTGGGGATCAAGAATGCTGACCCT   540           Genbank   CAAAGGCTGAAGGGGCAGGGGGTCAAGCTATTTGCTGTGGGGATCAAGAATGCTGACCCT   540               ************************************************************                       COL7A1   GAGGAGCTGAAGCGAGTTGCCTCACAGCCCACCAGTGACTTCTTCTTCTTCGTCAATGAC   600           Genbank   GAGGAGCTGAAGCGAGTTGCCTCACAGCCCACCAGTGACTTCTTCTTCTTCGTCAATGAC   600               ************************************************************                       COL7A1   TTCAGCATCTTGAGGACACTACTGCCCCTCGTTTCCCGGAGAGTGTGCACGACTGCTGGT   660           Genbank   TTCAGCATCTTGAGGACACTACTGCCCCTCGTTTCCCGGAGAGTGTGCACGACTGCTGGT   660               ************************************************************                       COL7A1   GGCGTGCCTGTGACCCGACCTCCGGATGACTCGACCTCTGCTCCACGAGACCTGGTGCTG   720           Genbank   GGCGTGCCTGTGACCCGACCTCCGGATGACTCGACCTCTGCTCCACGAGACCTGGTGCTG   720               ************************************************************                       COL7A1   TCTGAGCCAAGCAGCCAATCCTTGAGAGTACAGTGGACAGCGGCCAGTGGCCCTGTGACT   780           Genbank   TCTGAGCCAAGCAGCCAATCCTTGAGAGTACAGTGGACAGCGGCCAGTGGCCCTGTGACT   780               ************************************************************                       COL7A1   GGCTACAAGGTCCAGTACACTCCTCTGACGGGGCTGGGACAGCCACTGCCGAGTGAGCGG   840           Genbank   GGCTACAAGGTCCAGTACACTCCTCTGACGGGGCTGGGACAGCCACTGCCGAGTGAGCGG   840               ************************************************************                       COL7A1   CAGGAGGTGAACGTCCCAGCTGGTGAGACCAGTGTGCGGCTGCGGGGTCTCCGGCCACTG   900           Genbank   CAGGAGGTGAACGTCCCAGCTGGTGAGACCAGTGTGCGGCTGCGGGGTCTCCGGCCACTG   900               ************************************************************                       COL7A1   ACCGAGTACCAAGTGACTGTGATTGCCCTCTACGCCAACAGCATCGGGGAGGCTGTGAGC   960           Genbank   ACCGAGTACCAAGTGACTGTGATTGCCCTCTACGCCAACAGCATCGGGGAGGCTGTGAGC   960               ************************************************************                       COL7A1   GGGACAGCTCGGACCACTGCCCTAGAAGGGCCGGAACTGACCATCCAGAATACCACAGCC   1020           Genbank   GGGACAGCTCGGACCACTGCCCTAGAAGGGCCGGAACTGACCATCCAGAATACCACAGCC   1020               ************************************************************                       COL7A1   CACAGCCTCCTGGTGGCCTGGCGGAGTGTGCCAGGTGCCACTGGCTACCGTGTGACATGG   1080           Genbank   CACAGCCTCCTGGTGGCCTGGCGGAGTGTGCCAGGTGCCACTGGCTACCGTGTGACATGG   1080               ************************************************************                       COL7A1   CGGGTCCTCAGTGGTGGGCCCACACAGCAGCAGGAGCTGGGCCCTGGGCAGGGTTCAGTG   1140           Genbank   CGGGTCCTCAGTGGTGGGCCCACACAGCAGCAGGAGCTGGGCCCTGGGCAGGGTTCAGTG   1140               ************************************************************                       COL7A1   TTGCTGCGTGACTTGGAGCCTGGCACGGACTATGAGGTGACCGTGAGCACCCTATTTGGC   1200           Genbank   TTGCTGCGTGACTTGGAGCCTGGCACGGACTATGAGGTGACCGTGAGCACCCTATTTGGC   1200               ************************************************************                       COL7A1   CGCAGTGTGGGGCCCGCCACTTCCCTGATGGCTCGCACTGACGCTTCTGTTGAGCAGACC   1260           Genbank   CGCAGTGTGGGGCCCGCCACTTCCCTGATGGCTCGCACTGACGCTTCTGTTGAGCAGACC   1260               ************************************************************                       COL7A1   CTGCGCCCGGTCATCCTGGGCCCCACATCCATCCTCCTTTCCTGGAACTTGGTGCCTGAG   1320           Genbank   CTGCGCCCGGTCATCCTGGGCCCCACATCCATCCTCCTTTCCTGGAACTTGGTGCCTGAG   1320               ************************************************************                       COL7A1   GCCCGTGGCTACCGGTTGGAATGGCGGCGTGAGACTGGCTTGGAGCCACCGCAGAAGGTG   1380           Genbank   GCCCGTGGCTACCGGTTGGAATGGCGGCGTGAGACTGGCTTGGAGCCACCGCAGAAGGTG   1380               ************************************************************                       COL7A1   GTACTGCCCTCTGATGTGACCCGCTACCAGTTGGATGGGCTGCAGCCGGGCACTGAGTAC   1440           Genbank   GTACTGCCCTCTGATGTGACCCGCTACCAGTTGGATGGGCTGCAGCCGGGCACTGAGTAC   1440               ************************************************************                       COL7A1   CGCCTCACACTCTACACTCTGCTGGAGGGCCACGAGGTGGCCACCCCTGCAACCGTGGTT   1500           Genbank   CGCCTCACACTCTACACTCTGCTGGAGGGCCACGAGGTGGCCACCCCTGCAACCGTGGTT   1500               ************************************************************                       COL7A1   CCCACTGGACCAGAGCTGCCTGTGAGCCCTGTAACAGACCTGCAAGCCACCGAGCTGCCC   1560           Genbank   CCCACTGGACCAGAGCTGCCTGTGAGCCCTGTAACAGACCTGCAAGCCACCGAGCTGCCC   1560               ************************************************************                       COL7A1   GGGCAGCGGGTGCGAGTGTCCTGGAGCCCAGTCCCTGGTGCCACCCAGTACCGCATCATT   1620           Genbank   GGGCAGCGGGTGCGAGTGTCCTGGAGCCCAGTCCCTGGTGCCACCCAGTACCGCATCATT   1620               ************************************************************                       COL7A1   GTGCGCAGCACCCAGGGGGTTGAGCGGACCCTGGTGCTTCCTGGGAGTCAGACAGCATTC   1680           Genbank   GTGCGCAGCACCCAGGGGGTTGAGCGGACCCTGGTGCTTCCTGGGAGTCAGACAGCATTC   1680               ************************************************************                       COL7A1   GACTTGGATGACGTTCAGGCTGGGCTTAGCTACACTGTGCGGGTGTCTGCTCGAGTGGGT   1740           Genbank   GACTTGGATGACGTTCAGGCTGGGCTTAGCTACACTGTGCGGGTGTCTGCTCGAGTGGGT   1740               ************************************************************                       COL7A1   CCCCGTGAGGGCAGTGCCAGTGTCCTCACTGTCCGCCGGGAGCCGGAAACTCCACTTGCT   1800           Genbank   CCCCGTGAGGGCAGTGCCAGTGTCCTCACTGTCCGCCGGGAGCCGGAAACTCCACTTGCT   1800               ************************************************************                       COL7A1   GTTCCAGGGCTGCGGGTTGTGGTGTCAGATGCAACGCGAGTGAGGGTGGCCTGGGGACCC   1860           Genbank   GTTCCAGGGCTGCGGGTTGTGGTGTCAGATGCAACGCGAGTGAGGGTGGCCTGGGGACCC   1860               ************************************************************                       COL7A1   GTCCCTGGAGCCAGTGGATTTCGGATTAGCTGGAGCACAGGCAGTGGTCCGGAGTCCAGC   1920           Genbank   GTCCCTGGAGCCAGTGGATTTCGGATTAGCTGGAGCACAGGCAGTGGTCCGGAGTCCAGC   1920               ************************************************************                       COL7A1   CAGACACTGCCCCCAGACTCTACTGCCACAGACATCACAGGGCTGCAGCCTGGAACCACC   1980           Genbank   CAGACACTGCCCCCAGACTCTACTGCCACAGACATCACAGGGCTGCAGCCTGGAACCACC   1980               ************************************************************                       COL7A1   TACCAGGTGGCTGTGTCGGTACTGCGAGGCAGAGAGGAGGGCCCTGCTGCAGTCATCGTG   2040           Genbank   TACCAGGTGGCTGTGTCGGTACTGCGAGGCAGAGAGGAGGGCCCTGCTGCAGTCATCGTG   2040               ************************************************************                       COL7A1   GCTCGAACGGACCCACTGGGCCCAGTGAGGACGGTCCATGTGACTCAGGCCAGCAGCTCA   2100           Genbank   GCTCGAACGGACCCACTGGGCCCAGTGAGGACGGTCCATGTGACTCAGGCCAGCAGCTCA   2100               ************************************************************                       COL7A1   TCTGTCACCATTACCTGGACCAGGGTTCCTGGCGCCACAGGATACAGGGTTTCCTGGCAC   2160           Genbank   TCTGTCACCATTACCTGGACCAGGGTTCCTGGCGCCACAGGATACAGGGTTTCCTGGCAC   2160               ************************************************************                       COL7A1   TCAGCCCACGGCCCAGAGAAATCCCAGTTGGTTTCTGGGGAGGCCACGGTGGCTGAGCTG   2220           Genbank   TCAGCCCACGGCCCAGAGAAATCCCAGTTGGTTTCTGGGGAGGCCACGGTGGCTGAGCTG   2220               ************************************************************                       COL7A1   GATGGACTGGAGCCAGATACTGAGTATACGGTGCATGTGAGGGCCCATGTGGCTGGCGTG   2280           Genbank   GATGGACTGGAGCCAGATACTGAGTATACGGTGCATGTGAGGGCCCATGTGGCTGGCGTG   2280               ************************************************************                       COL7A1   GATGGGCCCCCTGCCTCTGTGGTTGTGAGGACTGCCCCTGAGCCTGTGGGTCGTGTGTCG   2340           Genbank   GATGGGCCCCCTGCCTCTGTGGTTGTGAGGACTGCCCCTGAGCCTGTGGGTCGTGTGTCG   2340               ************************************************************                       COL7A1   AGGCTGCAGATCCTCAATGCTTCCAGCGACGTTCTACGGATCACCTGGGTAGGGGTCACT   2400           Genbank   AGGCTGCAGATCCTCAATGCTTCCAGCGACGTTCTACGGATCACCTGGGTAGGGGTCACT   2400               ************************************************************                       COL7A1   GGAGCCACAGCTTACAGACTGGCCTGGGGCCGGAGTGAAGGCGGCCCCATGAGGCACCAG   2460           Genbank   GGAGCCACAGCTTACAGACTGGCCTGGGGCCGGAGTGAAGGCGGCCCCATGAGGCACCAG   2460               ************************************************************                       COL7A1   ATACTCCCAGGAAACACAGACTCTGCAGAGATCCGGGGTCTCGPAGGTGGAGTCAGCTAC   2520           Genbank   ATACTCCCAGGAAACACAGACTCTGCAGAGATCCGGGGTCTCGAAGGTGGAGTCAGCTAC   2520               ************************************************************                       COL7A1   TCAGTGCGAGTGACTGCACTTGTCGGGGACCGCGAGGGCACACCTGTCTCCATTGTTGTC   2580           Genbank   TCAGTGCGAGTGACTGCACTTGTCGGGGACCGCGAGGGCACACCTGTCTCCATTGTTGTC   2580               ************************************************************                       COL7A1   ACTACGCCGCCTGAGGCTCCGCCAGCCCTGGGGACGCTTCACGTGGTGCAGCGCGGGGAG   2640           Genbank   ACTACGCCGCCTGAGGCTCCGCCAGCCCTGGGGACGCTTCACGTGGTGCAGCGCGGGGAG   2640               ************************************************************                       COL7A1   CACTCGCTGAGGCTGCGCTGGGAGCCGGTGCCCAGAGCGCAGGGCTTCCTTCTGCACTGG   2700           Genbank   CACTCGCTGAGGCTGCGCTGGGAGCCGGTGCCCAGAGCGCAGGGCTTCCTTCTGCACTGG   2700               ************************************************************                       COL7A1   CAACCTGAGGGTGGCCAGGAACAGTCCCGGGTCCTGGGGCCCGAGCTCAGCAGCTATCAC   2760           Genbank   CAACCTGAGGGTGGCCAGGAACAGTCCCGGGTCCTGGGGCCCGAGCTCAGCAGCTATCAC   2760               ************************************************************                       COL7A1 Genbank                         2820 2820           COL7A1   GGAGAAGGGCCCTCTGCAGAGGTGACTGCGCGCACTGAGTCACCTCGTGTTCCAAGCATT   2880           Genbank   GGAGAAGGGCCCTCTGCAGAGGTGACTGCGCGCACTGAGTCACCTCGTGTTCCAAGCATT   2880               ************************************************************                       COL7A1   GAACTACGTGTGGTGGACACCTCGATCGACTCGGTGACTTTGGCCTGGACTCCAGTGTCC   2940           Genbank   GAACTACGTGTGGTGGACACCTCGATCGACTCGGTGACTTTGGCCTGGACTCCAGTGTCC   2940               ************************************************************                       COL7A1   AGGGGATCCAGCTACATCCTATCCTGGCGGCGACTCAGAGGCCCTGGCCAGGAAGTGCCT   3000           Genbank   AGGGCATCCAGCTACATCCTATCCTGGCGGCCACTCAGAGGCCCTGGCCAGGAAGTGCCT   3000               ************************************************************                       COL7A1   GGGTCCCCGCAGACACTTCCAGGGATCTCAAGCTCCCAGCGGGTGACAGGGCTAGAGCCT   3060           Genbank   GGGTCCCCGCAGACACTTCCAGGGATCTCAAGCTCCCAGCGGGTGACAGGGCTAGAGCCT   3060               ************************************************************                       COL7A1   GGCGTCTCTTACATCTTCTCCCTGACGCCTGTCCTGGATGGTGTGCGGGGTCCTGAGGCA   3120           Genbank   GGCGTCTCTTACATCTTCTCCCTGACGCCTGTCCTGGATGGTGTGCGGGGTCCTGAGGCA   3120               ************************************************************                       COL7A1   TCTGTCACACAGACGCCAGTGTGCCCCCGTGGCCTGGCGGATGTGGTGTTCCTACCACAT   3180           Genbank   TCTGTCACACAGACGCCAGTGTGCCCCCGTGGCCTGGCGGATGTGGTGTTCCTACCACAT   3180               ************************************************************                       COL7A1   GCCACTCAAGACAATGCTCACCGTGCGGAGGCTACGAGGAGGGTCCTGGAGCGTCTGGTG   3240           Genbank   GCCACTCAAGACAATGCTCACCGTGCGGAGGCTACGAGGAGGGTCCTGGAGCGTCTGGTG   3240               ************************************************************                       COL7A1   TTGGGACTTGGGCCTCTTGGGCCACAGGCAGTTCAGGTTGGCCTGCTGTCTTAGAGTCAT   3300           Genbank   TTGGCACTTGGGCCTCTTGGGCCACAGGGAGTTCAGGTTGGCCTGCTGTCTTACAGTCAT   3300               ************************************************************                       COL7A1   CGGCCCTCCCCACTGTTCCCACTGAATGGCTCCCATGACCTTGGCATTATCTTGCAAAGG   3360           Genbank   CGGCCCTCCCCACTGTTCCCACTGAATGGCTCCCATGACCTTGGCATTATCTTGCAAAGG   3360               ************************************************************                       COL7A1   ATCCGTGACATGGCCTACATGGACCCAAGTGGGAACAACCTGGGCACAGCCGTGGTCACA   3420           Genbank   ATCCGTGACATGCCCTACATGGACCCAAGTGGGAACAACCTGGGCACAGCCGTGGTCACA   3420               ************************************************************                       COL7A1   GCTCACAGATACATGTTGGCACCAGATGCTCCTGGGCGCCGCCAGCACGTACCAGGGGTG   3480           Genbank   GCTCACAGATACATGTTGGCACCAGATGCTCCTGGGCGCCGCCAGCACGTACCAGGGGTG   3480               ************************************************************                       COL7A1   ATGGTTCTGCTAGTGGATGAACCCTTGAGAGGTGAGATATTCAGCCCCATCCGTGAGGCC   3540           Genbank   ATGGTTCTGCTAGTGGATGAACCCTTGAGAGGTGACATATTCAGCCCCATCCGTGAGGCC   3540               ************************************************************                       COL7A1   CAGGCTTCTGGGCTTAATGTGGTGATGTTGGGAATGGCTGGAGCGGACCCAGAGCAGCTG   3600           Genbank   CAGGCTTCTGGGCTTAATGTGGTGATGTTGGGAATGGCTGGAGCGGACCCAGAGCAGCTG   3600               ************************************************************                       COL7A1   CGTCGCTTGGCGCCGGGTATGGACTCTGTCCAGACCTTCTTCGCCGTGGATGATGGGCCA   3660           Genbank   CGTCGCTTGGCGCCGGGTATGGACTCTGTCCAGACCTTCTTCGCCGTGGATGATGGGCCA   3660               ************************************************************                       COL7A1   AGCCTGGACCAGGCAGTCAGTGGTCTGGCCACAGCCCTGTGTCAGGCATCCTTCACTACT   3720           Genbank   AGCCTGGACCAGGCAGTCAGTGGTCTGGCCACAGCCCTGTGTCAGGCATCCTTCACTACT   3720               ************************************************************                       COL7A1   CAGCCCCGGCCAGAGCCCTGCCCAGTGTATTGTCCAAAGGGCCAGAAGGGGGAACCTGGA   3780           Genbank   CAGCCCCGGCCAGAGCCCTGCCCAGTGTATTGTCCAAAGGGCCAGAAGGGGGAACCTGGA   3780               ************************************************************                       COL7A1   GAGATGGGCCTGAGAGGACAAGTTGGGCCTCCTGGCGACCCTGGCCTCCCGGGCAGGACC   3840           Genbank   GAGATGGGCCTGAGAGGACAAGTTGGGCCTCCTGGCGACCCTGGCCTCCCGGGCAGGACC   3840               ************************************************************                       COL7A1   GGTGCTCCCGGCCCCCAGGGGCCCCCTGGAAGTGCCACTGCCAAGGGCGAGAGGGGCTTC   3900           Genbank   GGTGCTCCCGGCCCCCAGGGGCCCCCTGGAAGTGCCACTGCCAAGGGCGAGAGGGGCTTC   3900               ************************************************************                       COL7A1   CCTGGAGCAGATGGGCGTCCAGGCAGCCCTGGCCGCGCCGGGAATCCTGGGACCCCTGGA   3960           Genbank   CCTGGAGCAGATGGGCGTCCAGGCAGCCCTGGCCGCGCCGGGAATCCTGGGACCCCTGGA   3960               ************************************************************                       COL7A1   GCCCCTGGCCTAAAGGGCTCTCCAGGGTTGCCTGGCCCTCGTGGGGACCCGGGAGAGCGA   4020           Genbank   GCCCCTGGCCTAAAGGGCTCTCCAGGGTTGCCTGGCCCTCGTGGGGACCCGGGAGAGCGA   4020               ************************************************************                       COL7A1   GGACCTCGAGGCCCAAAGGGGGAGCCGGGGGCTCCCGGACAAGTCATCGGAGGTGAAGGA   4080           Genbank   GGACCTCGAGGCCCAAAGGGGGAGCCGGGGGCTCCCGGACAAGTCATCGGAGGTGAAGGA   4080               ************************************************************                       COL7A1   CCTGGGCTTCCTGGGCGGAAAGGGGACCCTGGACCATCGGGCCCCCCTGGACCTCGTGGA   4140           Genbank   CCTGGGCTTCCTGGGCGGAAAGGGGACCCTGGACCATCGGGCCCCCCTGGACCTCGTGGA   4140               ************************************************************                       COL7A1   CCACTGGGGGACCCAGGACCCCGTGGCCCCCCAGGGCTTCCTGGAACAGCCATGAAGGGT   4200           Genbank   CCACTGGGGGACCCAGGACCCCGTGGCCCCCCAGGGCTTCCTGGAACAGCCATGAAGGGT   4200               ************************************************************                       COL7A1   GACAAAGGCGATCGTGGGGAGCGGGGTCCCCCTGGACCAGGTGAAGGTGGCATTGCTCCT   4260           Genbank   GACAAAGGCGATCGTGGGGAGCGGGGTCCCCCTGGACCAGGTGAAGGTGGCATTGCTCCT   4260               ************************************************************                       COL7A1   GGGGAGCCTGGGCTGCCGGGTCTTCCCGGAAGCCCTGGACCCCAAGGCCCCGTTGGCCCC   4320           Genbank   GGGGAGCCTGGGCTGCCGGGTCTTCCCGGAAGCCCTGGACCCCAAGGCCCCGTTGGCCCC   4320               ************************************************************                       COL7A1   CCTGGAAAGAAAGGAGAAAAAGGTGACTCTGAGGATGGAGCTCCAGGCCTCCCAGGACAA   4380           Genbank   CCTGGAAAGAAAGGAGAAAAAGGTGACTCTGAGGATGGAGCTCCAGGCCTCCCAGGACAA   4380               ************************************************************                       COL7A1   CCTGGGTCTCCGGGTGAGCAGGGCCCACGGGGACCTCCTGGAGCTATTGGCCCCAAAGGT   4440           Genbank   CCTGGGTCTCCGGGTGAGCAGGGCCCACGGGGACCTCCTGGAGCTATTGGCCCCAAAGGT   4440               ************************************************************                       COL7A1   GACCGGGGCTTTCCAGGGCCCCTGGGTGAGGCTGGAGAGAAGGGCGAACGTGGACCCCCA   4500           Genbank   GACCGGGGCTTTCCAGGGCCCCTGGGTGAGGCTGGAGAGAAGGGCGAACGTGGACCCCCA   4500               ************************************************************                       COL7A1   GGCCCAGCGGGATCCCGGGGGCTGCCAGGGGTTGCTGGACGTCCTGGAGCCAAGGGTCCT   4560           Genbank   GGCCCAGCGGGATCCCGGGGGCTGCCAGGGGTTGCTGGACGTCCTGGAGCCAAGGGTCCT   4560               ************************************************************                       COL7A1   GAAGGGCCACCAGGACCCACTGGCCGCCAAGGAGAGAAGGGGGAGCCTGGTCGCCCTGGG   4620           Genbank   GAAGGGCCACCAGGACCCACTGGCCGCCAAGGAGAGAAGGGGGAGCCTGGTCGCCCTGGG   4620               ************************************************************                       COL7A1   GACCCTGCAGTGGTGGGACCTGCTGTTGCTGGACCCAAAGGAGAAAAGGGAGATGTGGGG   4680           Genbank   GACCCTGCAGTGGTGGGACCTGCTGTTGCTGGACCCAAAGGAGAAAAGGGAGATGTGGGG   4680               ************************************************************                       COL7A1   CCCGCTGGGCCCAGAGGAGCTACCGGAGTCCAAGGGGAACGGGGCCCACCCGGCTTGGTT   4740           Genbank   CCCGCTGGGCCCAGAGGAGCTACCGGAGTCCAAGGGGAACGGGGCCCACCCGGCTTGGTT   4740               ************************************************************                       COL7A1   CTTCCTGGAGACCCTGGCCCCAAGGGAGACCCTGGAGACCGGGGTCCCATTGGCCTTACT   4800           Genbank   CTTCCTGGAGACCCTGGCCCCAAGGGAGACCCTGGAGACCGGGGTCCCATTGGCCTTACT   4800               ************************************************************                       COL7A1   GGCAGAGCAGGACCCCCAGGTGACTCAGGGCCTCCTGGAGAGAAGGGAGACCCTGGGCGG   4860           Genbank   GGCAGAGCAGGACCCCCAGGTGACTCAGGGCCTCCTGGAGAGAAGGGAGACCCTGGGCGG   4860               ************************************************************                       COL7A1   CCTGGCCCCCCAGGACCTGTTGGCCCCCGAGGACGAGATGGTGAAGTTGGAGAGAAAGGT   4920           Genbank   CCTGGCCCCCCAGGACCTGTTGGCCCCCGAGGACGAGATGGTGAAGTTGGAGAGAAAGGT   4920               ************************************************************                       COL7A1   GACGAGGGTCCTCCGGGTGACCCGGGTTTGCCTGGAAAAGCAGGCGAGCGTGGCCTTCGG   4980           Genbank   GACGAGGGTCCTCCGGGTGACCCGGGTTTGCCTGGAAAAGCAGGCGAGCGTGGCCTTCGG   4980               ************************************************************                       COL7A1   GGGGCACCTGGAGTTCGGGGGCCTGTGGGTGAAAAGGGAGACCAGGGAGATCCTGGAGAG   5040           Genbank   GGGGCACCTGGAGTTCGGGGGCCTGTGGGTGAAAAGGGAGACCAGGGAGATCCTGGAGAG   5040               ************************************************************                       COL7A1   GATGGACGAAATGGCAGCCCTGGATCATCTGGACCCAAGGGTGACCGTGGGGAGCCGGGT   5100           Genbank   GATGGACGAAATGGCAGCCCTGGATCATCTGGACCCAAGGGTGACCGTGGGGAGCCGGGT   5100               ************************************************************                       COL7A1   CCCCCAGGACCCCCGGGACGGCTGGTAGACACAGGACCTGGAGCCAGAGAGAAGGGAGAG   5160           Genbank   CCCCCAGGACCCCCGGGACGGCTGGTAGACACAGGACCTGGAGCCAGAGAGAAGGGAGAG   5160               ************************************************************                       COL7A1   CCTGGGGACCGCGGACAAGAGGGTCCTCGAGGGCCCAAGGGTGATCCTGGCCTCCCTGGA   5220           Genbank   CCTGGGGACCGCGGACAAGAGGGTCCTCGAGGGCCCAAGGGTGATCCTGGCCTCCCTGGA   5220               ************************************************************                       COL7A1   GCCCCTGGGGAAAGGGGCATTGAAGGGTTTCGGGGACCCCCAGGCCCACAGGGGGACCCA   5280           Genbank   GCCCCTGGGGAAAGGGGCATTGAAGGGTTTCGGGGACCCCCAGGCCCACAGGGGGACCCA   5280               ************************************************************                       COL7A1   GGTGTCCGAGGCCCAGCAGGAGAAAAGGGTGACCGGGGTCCCCCTGGGCTGGATGGCCGG   5340           Genbank   GGTGTCCGAGGCCCAGCAGGAGAAAAGGGTGACCGGGGTCCCCCTGGGCTGGATGGCCGG   5340               ************************************************************                       COL7A1   AGCGGACTGGATGGGAAACCAGGAGCCGCTGGGCCCTCTGGGCCGAATGGTGCTGCAGGC   5400           Genbank   AGCGGACTGGATGGGAAACCAGGAGCCGCTGGGCCCTCTGGGCCGAATGGTGCTGCAGGC   5400               ************************************************************                       COL7A1 Genbank                         5460 5460           COL7A1 Genbank                         5520 5520           COL7A1   AATGGAAAAAACGGAGAACCTGGGGACCCTGGAGAAGACGGGAGGAAGGGAGAGAAAGGA   5580           Genbank   AATGGAAAAAACGGAGAACCTGGGGACCCTGGAGAAGACGGGAGGAAGGGAGAGAAAGGA   5580               ************************************************************                       COL7A1   GATTCAGGCGCCTCTGGGAGAGAAGGTCGTGATGGCCCCAAGGGTGAGCGTGGAGCTCCT   5640           Genbank   GATTCAGGCGCCTCTGGGAGAGAAGGTCGTGATGGCCCCAAGGGTGAGCGTGGAGCTCCT   5640               ************************************************************                       COL7A1   GGTATCCTTGGACCCCAGGGGCCTCCAGGCCTCCCAGGGCCAGTGGGCCCTCCTGGCCAG   5700           Genbank   GGTATCCTTGGACCCCAGGGGCCTCCAGGCCTCCCAGGGCCAGTGGGCCCTCCTGGCCAG   5700               ************************************************************                       COL7A1   GGTTTTCCTGGTGTCCCAGGAGGCACGGGCCCCAAGGGTGACCGTGGGGAGACTGGATCC   5760           Genbank   GGTTTTCCTGGTGTCCCAGGAGGCACGGGCCCCAAGGGTGACCGTGGGGAGACTGGATCC   5760               ************************************************************                       COL7A1   AAAGGGGAGCAGGGCCTCCCTGGAGAGCGTGGCCTGCGAGGAGAGCCTGGAAGTGTGCCG   5820           Genbank   AAAGGGGAGCAGGGCCTCCCTGGAGAGCGTGGCCTGCGAGGAGAGCCTGGAAGTGTGCCG   5820               ************************************************************                       COL7A1   AATGTGGATCGGTTGCTGGAAACTGCTGGCATCAAGGCATCTGCCCTGCGGGAGATCGTG   5880           Genbank   AATGTGGATCGGTTGCTGGAAACTGCTGGCATCAAGGCATCTGCCCTGCGGGAGATCGTG   5880               ************************************************************                       COL7A1   GAGACCTGGGATGAGAGCTCTGGTAGCTTCCTGCCTGTGCCCGAACGGCGTCGAGGCCCC   5940           Genbank   GAGACCTGGGATGAGAGCTCTGGTAGCTTCCTGCCTGTGCCCGAACGGCGTCGAGGCCCC   5940               ************************************************************                       COL7A1   AAGGGGGACTCAGGCGAACAGGGCCCCCCAGGCAAGGAGGGCCCCATCGGCTTTCCTGGA   6000           Genbank   AAGGGGGACTCAGGCGAACAGGGCCCCCCAGGCAAGGAGGGCCCCATCGGCTTTCCTGGA   6000               ************************************************************                       COL7A1   GAACGCGGGCTGAAGGGCGACCGTGGAGACCCTGGCCCTCAGGGGCCACCTGGTCTGGCC   6060           Genbank   GAACGCGGGCTGAAGGGCGACCGTGGAGACCCTGGCCCTCAGGGGCCACCTGGTCTGGCC   6060               ************************************************************                       COL7A1   CTTGGGGAGAGGGGCCCCCCCGGGCCTTCCGGCCTTGCCGGGGAGCCTGGAAAGCCTGGT   6120           Genbank   CTTGGGGAGAGGGGCCCCCCCGGGCCTTCCGGCCTTGCCGGGGAGCCTGGAAAGCCTGGT   6120               ************************************************************                       COL7A1   ATTCCCGGGCTCCCAGGCAGGGCTGGGGGTGTGGGAGAGGCAGGAAGGCCAGGAGAGAGG   6180           Genbank   ATTCCCGGGCTCCCAGGCAGGGCTGGGGGTGTGGGAGAGGCAGGAAGGCCAGGAGAGAGG   6180               ************************************************************                       COL7A1   GGAGAACGGGGAGAGAAAGGAGAACGTGGAGAACAGGGCAGAGATGGCCCTCCTGGACTC   6240           Genbank   GGAGAACGGGGAGAGAAAGGAGAACGTGGAGAACAGGGCAGAGATGGCCCTCCTGGACTC   6240               ************************************************************                       COL7A1   CCTGGAACCCCTGGGCCCCCCGGACCCCCTGGCCCCAAGGTGTCTGTGGATGAGCCAGGT   6300           Genbank   CCTGGAACCCCTGGGCCCCCCGGACCCCCTGGCCCCAAGGTGTCTGTGGATGAGCCAGGT   6300               ************************************************************                       COL7A1   CCTGGACTCTCTGGAGAACAGGGACCCCCTGGACTCAAGGGTGCTAAGGGGGAGCCGGGC   6360           Genbank   CCTGGACTCTCTGGAGAACAGGGACCCCCTGGACTCAAGGGTGCTAAGGGGGAGCCGGGC   6360               ************************************************************                       COL7A1   AGCAATGGTGACCAAGGTCCCAAAGGAGACAGGGGTGTGCCAGGCATCAAAGGAGACCGG   6420           Genbank   AGCAATGGTGACCAAGGTCCCAAAGGAGACAGGGGTGTGCCAGGCATCAAAGGAGACCGG   6420               ************************************************************                       COL7A1   GGAGAGCCTGGACCGAGGGGTCAGGACGGCAACCCGGGTCTACCAGGAGAGCGTGGTATG   6480           Genbank   GGAGAGCCTGGACCGAGGGGTCAGGACGGCAACCCGGGTCTACCAGGAGAGCGTGGTATG   6480               ************************************************************                       COL7A1   GCTGGGCCTGAAGGGAAGCCGGGTCTGCAGGGTCCAAGAGGCCCCCCTGGCCCAGTGGGT   6540           Genbank   GCTGGGCCTGAAGGGAAGCCGGGTCTGCAGGGTCCAAGAGGCCCCCCTGGCCCAGTGGGT   6540               ************************************************************                       COL7A1   GGTCATGGAGACCCTGGACCACCTGGTGCCCCGGGTCTTGCTGGCCCTGCAGGACCCCAA   6600           Genbank   GGTCATGGAGACCCTGGACCACCTGGTGCCCCGGGTCTTGCTGGCCCTGCAGGACCCCAA   6600               ************************************************************                       COL7A1   GGACCTTCTGGCCTGAAGGGGGAGCCTGGAGAGACAGGACCTCCAGGACGGGGCCTGACT   6660           Genbank   GGACCTTCTGGCCTGAAGGGGGAGCCTGGAGAGACAGGACCTCCAGGACGGGGCCTGACT   6660               ************************************************************                       COL7A1   GGACCTACTGGAGCTGTGGGACTTCCTGGACCCCCCGGCCCTTCAGGCCTTGTGGGTCCA   6720           Genbank   GGACCTACTGGAGCTGTGGGACTTCCTGGACCCCCCGGCCCTTCAGGCCTTGTGGGTCCA   6720               ************************************************************                       COL7A1   CAGGGGTCTCCAGGTTTGCCTGGACAAGTGGGGGAGACAGGGAAGCCGGGAGCCCCAGGT   6780           Genbank   CAGGGGTCTCCAGGTTTGCCTGGACAAGTGGGGGAGACAGGGAAGCCGGGAGCCCCAGGT   6780               ************************************************************                       COL7A1   CGAGATGGTGCCAGTGGAAAAGATGGAGACAGAGGGAGCCCTGGTGTGCCAGGGTCACCA   6840           Genbank   CGAGATGGTGCCAGTGGAAAAGATGGAGACAGAGGGAGCCCTGGTGTGCCAGGGTCACCA   6840               ************************************************************                       COL7A1   GGTCTGCCTGGCCCTGTCGGACCTAAAGGAGAACCTGGCCCCACGGGGGCCCCTGGACAG   6900           Genbank   GGTCTGCCTGGCCCTGTCGGACCTAAAGGAGAACCTGGCCCCACGGGGGCCCCTGGACAG   6900               ************************************************************                       COL7A1   GCTGTGGTCGGGCTCCCTGGAGCAAAGGGAGAGAAGGGAGCCCCTGGAGGCCTTGCTGGA   6960           Genbank   GCTGTGGTCGGGCTCCCTGGAGCAAAGGGAGAGAAGGGAGCCCCTGGAGGCCTTGCTGGA   6960               ************************************************************                       COL7A1   GACCTGGTGGGTGAGCCGGGAGCCAAAGGTGACCGAGGACTGCCAGGGCCGCGAGGCGAG   7020           Genbank   GACCTGGTGGGTGAGCCGGGAGCCAAAGGTGACCGAGGACTGCCAGGGCCGCGAGGCGAG   7020               ************************************************************                       COL7A1   AAGGGTGAAGCTGGCCGTGCAGGGGAGCCCGGAGACCCTGGGGAAGATGGTCAGAAAGGG   7080           Genbank   AAGGGTGAAGCTGGCCGTGCAGGGGAGCCCGGAGACCCTGGGGAAGATGGTCAGAAAGGG   7080               ************************************************************                       COL7A1   GCTCCAGGACCCAAAGGTTTCAAGGGTGACCCAGGAGTCGGGGTCCCGGGCTCCCCTGGG   7140           Genbank   GCTCCAGGACCCAAAGGTTTCAAGGGTGACCCAGGAGTCGGGGTCCCGGGCTCCCCTGGG   7140               ************************************************************                       COL7A1   CCTCCTGGCCCTCCAGGTGTGAAGGGAGATCTGGGCCTCCCTGGCCTGCCCGGTGCTCCT   7200           Genbank   CCTCCTGGCCCTCCAGGTGTGAAGGGAGATCTGGGCCTCCCTGGCCTGCCCGGTGCTCCT   7200               ************************************************************                       COL7A1   GGTGTTGTTGGGTTCCCGGGTCAGACAGGCCCTCGAGGAGAGATGGGTCAGCCAGGCCCT   7260           Genbank   GGTGTTGTTGGGTTCCCGGGTCAGACAGGCCCTCGAGGAGAGATGGGTCAGCCAGGCCCT   7260               ************************************************************                       COL7A1   AGTGGAGAGCGGGGTCTGGCAGGCCCCCCAGGGAGAGAAGGAATCCCAGGACCCCTGGGG   7320           Genbank   AGTGGAGAGCGGGGTCTGGCAGGCCCCCCAGGGAGAGAAGGAATCCCAGGACCCCTGGGG   7320               ************************************************************                       COL7A1   CCACCTGGACCACCGGGGTCAGTGGGACCACCTGGGGCCTCTGGACTCAAAGGGAGACAAG   7380           Genbank   CCACCTGGACCACCGGGGTCAGTGGGACCACCTGGGGCCTCTGGACTCAAAGGGAGACAAG   7380               ************************************************************                       COL7A1   GGAGACCCTGGAGTAGGGCTGCCTGGGCCCCGAGGCGAGCGTGGGGAGCCAGGCATCCGG   7440           Genbank   GGAGACCCTGGAGTAGGGCTGCCTGGGCCCCGAGGCGAGCGTGGGGAGCCAGGCATCCGG   7440               ************************************************************                       COL7A1   GGTGAAGATGGCCGCCCCGGCCAGGAGGGACCCCGAGGACTCACGGGGCCCCCTGGCAGC   7500           Genbank   GGTGAAGATGGCCGCCCCGGCCAGGAGGGACCCCGAGGACTCACGGGGCCCCCTGGCAGC   7500               ************************************************************                       COL7A1   AGGGGAGAGCGTGGGGAGAAGGGTGATGTTGGGAGTGCAGGACTAAAGGGTGACAAGGGA   7560           Genbank   AGGGGAGAGCGTGGGGAGAAGGGTGATGTTGGGAGTGCAGGACTAAAGGGTGACAAGGGA   7560               ************************************************************                       COL7A1   GACTCAGCTGTGATCCTGGGGCCTCCACGCCCACGGGGTGCCAAGGGGGACATGGGTGAA   7620           Genbank   GACTCAGCTGTGATCCTGGGGCCTCCAGGCCCACGGGGTGCCAAGGGGGACATGGGTGAA   7620               ************************************************************                       COL7A1   CGAGGGCCTCGGGGCTTGGATGGTGACAAAGGACCTCGGGGAGACAATGGGGACCCTGGT   7680           Genbank   CGAGGGCCTCGGGGCTTGGATGGTGACAAAGGACCTCGGGGAGACAATGGGCACCCTGGT   7680               ************************************************************                       COL7A1   GACAAGGGCAGCAAGGGAGAGCCTGGTGACAAGGGCTCAGCCGGGTTGCCAGGACTGCGT   7740           Genbank   GACAAGGGCAGCAAGGGAGAGCCTGGTGACAAGGGCTCAGCCGGGTTGCCAGGACTGCGT   7740               ************************************************************                       COL7A1   GGACTCCTGGGACCCCAGGGTCAACCTGGTGCAGCAGGGATCCCTGGTGACCCCGGATCC   7800           Genbank   GGACTCCTGGGACCCCAGGGTCAACCTGGTGCAGCAGGGATCCCTGGTGACCCCGGATCC   7800               ************************************************************                       COL7A1   CCAGGAAAGGATGGAGTGCCTGGTATCCGAGGAGAAAAAGGAGATGTTGGCTTCATGGGT   7860           Genbank   CCAGGAAAGGATGGAGTGCCTGGTATCCGAGGAGAAAAAGGAGATGTTGGCTTCATGGGT   7860               ************************************************************                       COL7A1   CCCCGGGCCCTCAAGGGTGAACGCGGAGTGAAGGGAGCCTGTGGCCTTGATGGAGAGAAG   7920           Genbank   CCCCGGGGCCTCAAGGGTGAACGGGGAGTGAAGGGAGCCTGTGGCCTTGATGGAGAGAAG   7920               ************************************************************                       COL7A1   GGAGACAAGGGAGAAGCTGGTCCCCCAGGCCGCCCCGGGCTGGCAGGACACAAAGGAGAG   7980           Genbank   GGAGACAAGGGAGAAGCTGGTCCCCCAGGCCGCCCCGGGCTGGCAGGACACAAAGGAGAG   7980               ************************************************************                       COL7A1   ATGGGGGAGCCTGGTGTGCCGGGCCACTCGGGGGCCCCTGGCAAGGAGGGCCTGATCGGT   8040           Genbank   ATGGGGGAGCCTGGTGTGCCGGGCCAGTCGGGGGCCCCTGGCAAGGAGGGCCTGATCGGT   8040               ************************************************************                       COL7A1   CCCAAGGGTGACCGAGGCTTTGACGGGCAGCCAGGCCCCAAGGGTGACCAGGGCGAGAAA   8100           Genbank   CCCAAGGGTGACCGAGGCTTTGACGGGCAGCCAGGCCCCAAGGGTGACCAGGGCGAGAAA   8100               ************************************************************                       COL7A1   GGGGAGCGGGGAACCCCAGGAATTGGGGGCTTCCCAGGCCCCAGTGGAAATGATGGCTCT   $160           Genbank   GGGGAGCGGGGAACCCCAGGAATTGGGGGCTTCCCAGGCCCCAGTGGAAATGATGGCTCT   8160               ************************************************************                       COL7A1   GCTGGTCCCCCAGGGCCACCTGGCAGTGTTGGTCCCAGAGGCCCCGAAGGACTTCAGGGC   8220           Genbank   GCTGGTCCCCCAGGGCCACCTGGCAGTGTTGGTCCCAGAGGCCCCGAAGGACTTCAGGGC   8220               ************************************************************                       COL7A1   CAGAAGGGTGAGCGAGGTCCCCCCGGAGAGAGAGTGGTGGGGGCTCCTGGGGTCCCTGGA   8280           Genbank   CAGAAGGGTGAGCGAGGCCCCCCCGGAGAGAGAGTGGTGGGGGCTCCTGGGGTCCCTGGA   8280               ************************************************************                       COL7A1   GCTCCTGGCGAGAGAGGGGAGCAGGGGCGGCCAGGGCCTGCCGGTCCTCGAGGCGAGAAG   8340           Genbank   GCTCCTGGCGAGAGAGGGGAGCAGGGGCGGCCAGGGCCTGCCGGTCCTCGAGGCGAGAAG   8340               ************************************************************                       COL7A1   GGAGAAGCTGCACTGACGGAGGATGACATCCGGGGCTTTGTGCGCCAAGAGATGAGTCAG   8400           Genbank   GGAGAAGCTGCACTGACGGAGGATGACATCCGGGGCTTTGTGCGCCAAGAGATGAGTCAG   8400               ************************************************************                       COL7A1   CACTGTGCCTGCCAGGGCCAGTTCATCGCATCTGGATCACGACCCCTCCCTAGTTATGCT   8460           Genbank   CACTGTGCCTGCCAGGGCCAGTTCATCGCATCTGGATCACGACCCCTCCCTAGTTATGCT   8460               ************************************************************                       COL7A1   GCAGACACTGCCGGCTCCCAGCTCCATGCTGTGCCTGTGCTCCGCGTCTCTCATGCAGAG   8520           Genbank   GCAGACACTGCCGGCTCCCAGCTCCATGCTGTGCCTGTGCTCCGCGTCTCTCATGCAGAG   8520               ************************************************************                       COL7A1   GAGGAAGAGCGGGTACCCCCTGAGGATGATGAGTACTCTGAATACTCCGAGTATTCTGTG   8580           Genbank   GAGGAAGAGCGGGTACCCCCTGAGGATGATGAGTACTCTGAATACTCCGAGTATTCTGTG   8580               ************************************************************                       COL7A1   GAGGAGTACCAGGACCCTGAAGCTCCTTGGGATAGTGATGACCCCTGTTCCCTGCCACTG   8640           Genbank   GAGGAGTACCAGGACCCTGAAGCTCCTTGGGATAGTGATGACCCCTGTTCCCTGCCACTG   8640               ************************************************************                       COL7A1   GATGAGGGCTCCTGCACTGCCTACACCCTGCGCTGGTACCATCGGGCTGTGACAGGCAGC   8700           Genbank   GATGAGGGCTCCTGCACTGCCTACACCCTGCGCTGGTACCATCGGGCTGTGACAGGCAGC   8700               ************************************************************                       COL7A1   ACAGAGGCCTGTCACCCTTTTGTCTATGGTGGCTGTGGAGGGAATGCCAACCGTTTTGGG   8760           Genbank   ACAGAGGCCTGTCACCCTTTTGTCTATGGTGGCTGTGGAGGGAATGCCAACCGTTTTGGG   8760               ************************************************************                       COL7A1   ACCCGTGAGGCCTGCGAGCGCCGCTGCCCACCCCGGGTGGTCCAGAGCCAGGGGACAGGT   8820           Genbank   ACCCGTGAGGCCTGCGAGCGCCGCTGCCCACCCCGGGTGGTCCAGAGCCAGGGGACAGGT   8820               ************************************************************                       COL7A1   ACTGCCCAGGAC   8832   (SEQ ID NO: 23)       Genbank   ACTGCCCAGGAC   8832   (SEQ ID NO: 24)           ************                    
The following is a comparative analysis of 60/010,743-ITX-00001_CONTIG SEQUENCE (SEQ ID NO:34) relative to IGE308_REFSEQUENCE (SEQ ID NO: 34).
 
     
       
         
           
               
               
            
               
                 60010743-ITX-00001_Comparative_Analysis 
                   
               
               
                 60010743-ITX_Final.SPF 
               
            
           
           
               
               
               
               
            
               
                     IGE308_RefSequence 
                     #1 
                 GTCGACGGAT CGGGAGATCT CCCGATCCCC TATGGTGCAC TCTCAGTACA ATCTGCTCTG ATGCCGCATA GTTAAGCCAG 
                   
               
               
                     60010743-ITX-00001_Contig 
                     #1 
                 GTCGACGGAT CGGGAGATCT CCCGATCCCC TATGGTGCAC TCTCAGTACA ATCTGCTCTG ATGCCGCATA GTTAAGCCAG 
               
               
                   
                     #1 
                 GTCGACGGAT CGGGAGATCT CCCGATCCCC TATGGTGCAC TCTCAGTACA ATCTGCTCTG ATGCCGCATA GTTAAGCCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                    #81 
                 TATCTGCTCC CTGCTTGTGT GTTGGAGGTC GCTGAGTAGT GCGCGAGCAA AATTTAAGCT ACAACAAGGC AAGGCTTGAC 
               
               
                     60010743-ITX-00001_Contig 
                    #81 
                 TATCTGCTCC CTGCTTGTGT GTTGGAGGTC GCTGAGTAGT GCGCGAGCAA AATTTAAGCT ACAACAAGGC AAGGCTTGAC 
               
               
                   
                    #81 
                 TATCTGCTCC CTGCTTGTGT GTTGGAGGTC GCTGAGTAGT GCGCGAGCAA AATTTAAGCT ACAACAAGGC AAGGCTTGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #161 
                 CGACAATTGC ATGAAGAATC TGCTTAGGGT TAGGCGTTTT GCGCTGCTTC GCGATGTACG GGCCAGATAT ACGCGTTGAC 
               
               
                     60010743-ITX-00001_Contig 
                   #161 
                 CGACAATTGC ATGAAGAATC TGCTTAGGGT TAGGCGTTTT GCGCTGCTTC GCGATGTACG GGCCAGATAT ACGCGTTGAC 
               
               
                   
                   #161 
                 CGACAATTGC ATGAAGAATC TGCTTAGGGT TAGGCGTTTT GCGCTGCTTC GCGATGTACG GGCCAGATAT ACGCGTTGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #241 
                 ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA 
               
               
                     60010743-ITX-00001_Contig 
                   #241 
                 ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA 
               
               
                   
                   #241 
                 ATTGATTATT GACTAGTTAT TAATAGTAAT CAATTACGGG GTCATTAGTT CATAGCCCAT ATATGGAGTT CCGCGTTACA 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #321 
                 TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
               
               
                     60010743-ITX-00001_Contig 
                   #321 
                 TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
               
               
                   
                   #321 
                 TAACTTACGG TAAATGGCCC GCCTGGCTGA CCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT ATGTTCCCAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #401 
                 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA GTACATCAAG 
               
               
                     60010743-ITX-00001_Contig 
                   #401 
                 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA GTACATCAAG 
               
               
                   
                   #401 
                 AGTAACGCCA ATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC GGTAAACTGC CCACTTGGCA GTACATCAAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #481 
                 TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC 
               
               
                     60010743-ITX-00001_Contig 
                   #481 
                 TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC 
               
               
                   
                   #481 
                 TGTATCATAT GCCAAGTACG CCCCCTATTG ACGTCAATGA CGGTAAATGG CCCGCCTGGC ATTATGCCCA GTACATGACC 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #561 
                 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT 
               
               
                     60010743-ITX-00001_Contig 
                   #561 
                 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT 
               
               
                   
                   #561 
                 TTATGGGACT TTCCTACTTG GCAGTACATC TACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT GGCAGTACAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #641 
                 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC 
               
               
                     60010743-ITX-00001_Contig 
                   #641 
                 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC 
               
               
                   
                   #641 
                 CAATGGGCGT GGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC CCATTGACGT CAATGGGAGT TTGTTTTGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #721 
                 ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 
               
               
                     60010743-ITX-00001_Contig 
                   #721 
                 ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 
               
               
                   
                   #721 
                 ACCAAAATCA ACGGGACTTT CCAAAATGTC GTAACAACTC CGCCCCATTG ACGCAAATGG GCGGTAGGCG TGTACGGTGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #801 
                 GAGGTCTATA TAAGCAGCGC GTTTTGCCTG TACTGGGTCT CTCTGGTTAG ACCAGATCTG AGCCTGGGAG CTCTCTGGCT 
               
               
                     60010743-ITX-00001_Contig 
                   #801 
                 GAGGTCTATA TAAGCAGCGC GTTTTGCCTG TACTGGGTCT CTCTGGTTAG ACCAGATCTG AGCCTGGGAG CTCTCTGGCT 
               
               
                   
                   #801 
                 GAGGTCTATA TAAGCAGCGC GTTTTGCCTG TACTGGGTCT CTCTGGTTAG ACCAGATCTG AGCCTGGGAG CTCTCTGGCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #881 
                 AACTAGGGAA CCCACTGCTT AAGCCTCAAT AAAGCTTGCC TTGAGTGCTT CAAGTAGTGT GTGCCCGTCT GTTGTGTGAC 
               
               
                     60010743-ITX-00001_Contig 
                   #881 
                 AACTAGGGAA CCCACTGCTT AAGCCTCAAT AAAGCTTGCC TTGAGTGCTT CAAGTAGTGT GTGCCCGTCT GTTGTGTGAC 
               
               
                   
                   #881 
                 AACTAGGGAA CCCACTGCTT AAGCCTCAAT AAAGCTTGCC TTGAGTGCTT CAAGTAGTGT GTGCCCGTCT GTTGTGTGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                   #961 
                 TCTGGTAACT AGAGATCCCT CAGACCCTTT TAGTCAGTGT GGAAAATCTC TAGCAGTGGC GCCCGAACAG GGACTTGAAA 
               
               
                     60010743-ITX-00001_Contig 
                   #961 
                 TCTGGTAACT AGAGATCCCT CAGACCCTTT TAGTCAGTGT GGAAAATCTC TAGCAGTGGC GCCCGAACAG GGACTTGAAA 
               
               
                   
                   #961 
                 TCTGGTAACT AGAGATCCCT CAGACCCTTT TAGTCAGTGT GGAAAATCTC TAGCAGTGGC GCCCGAACAG GGACTTGAAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1041 
                 GCGAAAGGGA AACCAGAGGA GCTCTCTCGA CGCAGGACTC GGCTTGCTGA AGCGCGCACG GCAAGAGGCG AGGGGCGGCG 
               
               
                     60010743-ITX-00001_Contig 
                  #1041 
                 GCGAAAGGGA AACCAGAGGA GCTCTCTCGA CGCAGGACTC GGCTTGCTGA AGCGCGCACG GCAAGAGGCG AGGGGCGGCG 
               
               
                   
                  #1041 
                 GCGAAAGGGA AACCAGAGGA GCTCTCTCGA CGCAGGACTC GGCTTGCTGA AGCGCGCACG GCAAGAGGCG AGGGGCGGCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1121 
                 ACTGGTGAGT ACGCCAAAAA TTTTGACTAG CGGAGGCTAG AAGGAGAGAG ATGGGTGCGA GAGCGTCAGT ATTAAGCGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #1121 
                 ACTGGTGAGT ACGCCAAAAA TTTTGACTAG CGGAGGCTAG AAGGAGAGAG ATGGGTGCGA GAGCGTCAGT ATTAAGCGGG 
               
               
                   
                  #1121 
                 ACTGGTGAGT ACGCCAAAAA TTTTGACTAG CGGAGGCTAG AAGGAGAGAG ATGGGTGCGA GAGCGTCAGT ATTAAGCGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1201 
                 GGAGAATTAG ATCGCGATGG GAAAAAATTC GGTTAAGGCC AGGGGGAAAG AAAAAATATA AATTAAAACA TATAGTATGG 
               
               
                     60010743-ITX-00001_Contig 
                  #1201 
                 GGAGAATTAG ATCGCGATGG GAAAAAATTC GGTTAAGGCC AGGGGGAAAG AAAAAATATA AATTAAAACA TATAGTATGG 
               
               
                   
                  #1201 
                 GGAGAATTAG ATCGCGATGG GAAAAAATTC GGTTAAGGCC AGGGGGAAAG AAAAAATATA AATTAAAACA TATAGTATGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1281 
                 GCAAGCAGGG AGCTAGAACG ATTCGCAGTT AATCCTGGCC TGTTAGAAAC ATCAGAAGGC TGTAGACAAA TACTGGGACA 
               
               
                     60010743-ITX-00001_Contig 
                  #1281 
                 GCAAGCAGGG AGCTAGAACG ATTCGCAGTT AATCCTGGCC TGTTAGAAAC ATCAGAAGGC TGTAGACAAA TACTGGGACA 
               
               
                   
                  #1281 
                 GCAAGCAGGG AGCTAGAACG ATTCGCAGTT AATCCTGGCC TGTTAGAAAC ATCAGAAGGC TGTAGACAAA TACTGGGACA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1361 
                 GCTACAACCA TCCCTTCAGA CAGGATCAGA AGAACTTAGA TCATTATATA ATACAGTAGC AACCCTCTAT TGTGTGCATC 
               
               
                     60010743-ITX-00001_Contig 
                  #1361 
                 GCTACAACCA TCCCTTCAGA CAGGATCAGA AGAACTTAGA TCATTATATA ATACAGTAGC AACCCTCTAT TGTGTGCATC 
               
               
                   
                  #1361 
                 GCTACAACCA TCCCTTCAGA CAGGATCAGA AGAACTTAGA TCATTATATA ATACAGTAGC AACCCTCTAT TGTGTGCATC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1441 
                 AAAGGATAGA GATAAAAGAC ACCAAGGAAG CTTTAGACAA GATAGAGGAA GAGCAAAACA AAAGTAAGAC CACCGCACAG 
               
               
                     60010743-ITX-00001_Contig 
                  #1441 
                 AAAGGATAGA GATAAAAGAC ACCAAGGAAG CTTTAGACAA GATAGAGGAA GAGCAAAACA AAAGTAAGAC CACCGCACAG 
               
               
                   
                  #1441 
                 AAAGGATAGA GATAAAAGAC ACCAAGGAAG CTTTAGACAA GATAGAGGAA GAGCAAAACA AAAGTAAGAC CACCGCACAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1521 
                 CAAGCGGCCG CTGATCTTCA GACCTGGAGG AGGAGATATG AGGGACAATT GGAGAAGTGA ATTATATAAA TATAAAGTAG 
               
               
                     60010743-ITX-00001_Contig 
                  #1521 
                 CAAGCGGCCG CTGATCTTCA GACCTGGAGG AGGAGATATG AGGGACAATT GGAGAAGTGA ATTATATAAA TATAAAGTAG 
               
               
                   
                  #1521 
                 CAAGCGGCCG CTGATCTTCA GACCTGGAGG AGGAGATATG AGGGACAATT GGAGAAGTGA ATTATATAAA TATAAAGTAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1601 
                 TAAAAATTGA ACCATTAGGA GTAGCACCCA CCAAGGCAAA GAGAAGAGTG GTGCAGAGAG AAAAAAGAGC AGTGGGAATA 
               
               
                     60010743-ITX-00001_Contig 
                  #1601 
                 TAAAAATTGA ACCATTAGGA GTAGCACCCA CCAAGGCAAA GAGAAGAGTG GTGCAGAGAG AAAAAAGAGC AGTGGGAATA 
               
               
                   
                  #1601 
                 TAAAAATTGA ACCATTAGGA GTAGCACCCA CCAAGGCAAA GAGAAGAGTG GTGCAGAGAG AAAAAAGAGC AGTGGGAATA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1681 
                 GGAGCTTTGT TCCTTGGGTT CTTGGGAGCA GCAGGAAGCA CTATGGGCGC AGCGTCAATG ACGCTGACGG TACAGGCCAG 
               
               
                     60010743-ITX-00001_Contig 
                  #1681 
                 GGAGCTTTGT TCCTTGGGTT CTTGGGAGCA GCAGGAAGCA CTATGGGCGC AGCGTCAATG ACGCTGACGG TACAGGCCAG 
               
               
                   
                  #1681 
                 GGAGCTTTGT TCCTTGGGTT CTTGGGAGCA GCAGGAAGCA CTATGGGCGC AGCGTCAATG ACGCTGACGG TACAGGCCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1761 
                 ACAATTATTG TCTGGTATAG TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA ACAGCATCTG TTGCAACTCA 
               
               
                     60010743-ITX-00001_Contig 
                  #1761 
                 ACAATTATTG TCTGGTATAG TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA ACAGCATCTG TTGCAACTCA 
               
               
                   
                  #1761 
                 ACAATTATTG TCTGGTATAG TGCAGCAGCA GAACAATTTG CTGAGGGCTA TTGAGGCGCA ACAGCATCTG TTGCAACTCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1841 
                 CAGTCTGGGG CATCAAGCAG CTCCAGGCAA GAATCCTGGC TGTGGAAAGA TACCTAAAGG ATCAACAGCT CCTGGGGATT 
               
               
                     60010743-ITX-00001_Contig 
                  #1841 
                 CAGTCTGGGG CATCAAGCAG CTCCAGGCAA GAATCCTGGC TGTGGAAAGA TACCTAAAGG ATCAACAGCT CCTGGGGATT 
               
               
                   
                  #1841 
                 CAGTCTGGGG CATCAAGCAG CTCCAGGCAA GAATCCTGGC TGTGGAAAGA TACCTAAAGG ATCAACAGCT CCTGGGGATT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #1921 
                 TGGGGTTGCT CTGGAAAACT CATTTGCACC ACTGCTGTGC CTTGGAATGC TAGTTGGAGT AATAAATCTC TGGAACAGAT 
               
               
                     60010743-ITX-00001_Contig 
                  #1921 
                 TGGGGTTGCT CTGGAAAACT CATTTGCACC ACTGCTGTGC CTTGGAATGC TAGTTGGAGT AATAAATCTC TGGAACAGAT 
               
               
                   
                  #1921 
                 TGGGGTTGCT CTGGAAAACT CATTTGCACC ACTGCTGTGC CTTGGAATGC TAGTTGGAGT AATAAATCTC TGGAACAGAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2001 
                 TTGGAATCAC ACGACCTGGA TGGAGTGGGA CAGAGAAATT AACAATTACA CAAGCTTAAT ACACTCCTTA ATTGAAGAAT 
               
               
                     60010743-ITX-00001_Contig 
                  #2001 
                 TTGGAATCAC ACGACCTGGA TGGAGTGGGA CAGAGAAATT AACAATTACA CAAGCTTAAT ACACTCCTTA ATTGAAGAAT 
               
               
                   
                  #2001 
                 TTGGAATCAC ACGACCTGGA TGGAGTGGGA CAGAGAAATT AACAATTACA CAAGCTTAAT ACACTCCTTA ATTGAAGAAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2081 
                 CGCAAAACCA GCAAGAAAAG AATGAACAAG AATTATTGGA ATTAGATAAA TGGGCAAGTT TGTGGAATTG GTTTAACATA 
               
               
                     60010743-ITX-00001_Contig 
                  #2081 
                 CGCAAAACCA GCAAGAAAAG AATGAACAAG AATTATTGGA ATTAGATAAA TGGGCAAGTT TGTGGAATTG GTTTAACATA 
               
               
                   
                  #2081 
                 CGCAAAACCA GCAAGAAAAG AATGAACAAG AATTATTGGA ATTAGATAAA TGGGCAAGTT TGTGGAATTG GTTTAACATA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2161 
                 ACAAATTGGC TGTGGTATAT AAAATTATTC ATAATGATAG TAGGAGGCTT GGTAGGTTTA AGAATAGTTT TTGCTGTACT 
               
               
                     60010743-ITX-00001_Contig 
                  #2161 
                 ACAAATTGGC TGTGGTATAT AAAATTATTC ATAATGATAG TAGGAGGCTT GGTAGGTTTA AGAATAGTTT TTGCTGTACT 
               
               
                   
                  #2161 
                 ACAAATTGGC TGTGGTATAT AAAATTATTC ATAATGATAG TAGGAGGCTT GGTAGGTTTA AGAATAGTTT TTGCTGTACT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2241 
                 TTCTATAGTG AATAGAGTTA GGCAGGGATA TTCACCATTA TCGTTTCAGA CCCACCTCCC AACCCCGAGG GGACCCGACA 
               
               
                     60010743-ITX-00001_Contig 
                  #2241 
                 TTCTATAGTG AATAGAGTTA GGCAGGGATA TTCACCATTA TCGTTTCAGA CCCACCTCCC AACCCCGAGG GGACCCGACA 
               
               
                   
                  #2241 
                 TTCTATAGTG AATAGAGTTA GGCAGGGATA TTCACCATTA TCGTTTCAGA CCCACCTCCC AACCCCGAGG GGACCCGACA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2321 
                 GGCCCGAAGG AATAGAAGAA GAAGGTGGAG AGAGAGACAG AGACAGATCC ATTCGATTAG TGAACGGATC GGCACTGCGT 
               
               
                     60010743-ITX-00001_Contig 
                  #2321 
                 GGCCCGAAGG AATAGAAGAA GAAGGTGGAG AGAGAGACAG AGACAGATCC ATTCGATTAG TGAACGGATC GGCACTGCGT 
               
               
                   
                  #2321 
                 GGCCCGAAGG AATAGAAGAA GAAGGTGGAG AGAGAGACAG AGACAGATCC ATTCGATTAG TGAACGGATC GGCACTGCGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2401 
                 GCGCCAATTC TGCAGACAAA TGGCAGTATT CATCCACAAT TTTAAAAGAA AAGGGGGGAT TGGGGGGTAC AGTGCAGGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #2401 
                 GCGCCAATTC TGCAGACAAA TGGCAGTATT CATCCACAAT TTTAAAAGAA AAGGGGGGAT TGGGGGGTAC AGTGCAGGGG 
               
               
                   
                  #2401 
                 GCGCCAATTC TGCAGACAAA TGGCAGTATT CATCCACAAT TTTAAAAGAA AAGGGGGGAT TGGGGGGTAC AGTGCAGGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2481 
                 AAAGAATAGT AGACATAATA GCAACAGACA TACAAACTAA AGAATTACAA AAACAAATTA CAAAAATTCA AAATTTTCGG 
               
               
                     60010743-ITX-00001_Contig 
                  #2481 
                 AAAGAATAGT AGACATAATA GCAACAGACA TACAAACTAA AGAATTACAA AAACAAATTA CAAAAATTCA AAATTTTCGG 
               
               
                   
                  #2481 
                 AAAGAATAGT AGACATAATA GCAACAGACA TACAAACTAA AGAATTACAA AAACAAATTA CAAAAATTCA AAATTTTCGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2561 
                 GTTTATTACA GGGACAGCAG AGATCCAGTT TGGTTAATTA AGCATTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG 
               
               
                     60010743-ITX-00001_Contig 
                  #2561 
                 GTTTATTACA GGGACAGCAG AGATCCAGTT TGGTTAATTA AGCATTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG 
               
               
                   
                  #2561 
                 GTTTATTACA GGGACAGCAG AGATCCAGTT TGGTTAATTA AGCATTAGTT ATTAATAGTA ATCAATTACG GGGTCATTAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2641 
                 TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGTT GACCGCCCAA CGACCCCCGC 
               
               
                     60010743-ITX-00001_Contig 
                  #2641 
                 TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGTT GACCGCCCAA CGACCCCCGC 
               
               
                   
                  #2641 
                 TTCATAGCCC ATATATGGAG TTCCGCGTTA CATAACTTAC GGTAAATGGC CCGCCTGGTT GACCGCCCAA CGACCCCCGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2721 
                 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT 
               
               
                     60010743-ITX-00001_Contig 
                  #2721 
                 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT 
               
               
                   
                  #2721 
                 CCATTGACGT CAATAATGAC GTATGTTCCC ATAGTAACGC CAATAGGGAC TTTCCATTGA CGTCAATGGG TGGAGTATTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2801 
                 ACGGTAAACT GCCCACTTGG TAGTACATCA AGTGTATCAT ACGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT 
               
               
                     60010743-ITX-00001_Contig 
                  #2801 
                 ACGGTAAACT GCCCACTTGG TAGTACATCA AGTGTATCAT ACGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT 
               
               
                   
                  #2801 
                 ACGGTAAACT GCCCACTTGG TAGTACATCA AGTGTATCAT ACGCCAAGTA CGCCCCCTAT TGACGTCAAT GACGGTAAAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2881 
                 GGCCCGCCTG GTATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTGCGTATT AGTCATCGCT 
               
               
                     60010743-ITX-00001_Contig 
                  #2881 
                 GGCCCGCCTG GTATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTGCGTATT AGTCATCGCT 
               
               
                   
                  #2881 
                 GGCCCGCCTG GTATTATGCC CAGTACATGA CCTTATGGGA CTTTCCTACT TGGCAGTACA TCTGCGTATT AGTCATCGCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #2961 
                 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #2961 
                 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA 
               
               
                   
                  #2961 
                 ATTACCATGG TGATGCGGTT TTGGCAGTAC ATCAATGGGC GTGGATAGCG GTTTGACTCA CGGGGATTTC CAAGTCTCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3041 
                 CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT 
               
               
                     60010743-ITX-00001_Contig 
                  #3041 
                 CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT 
               
               
                   
                  #3041 
                 CCCCATTGAC GTCAATGGGA GTTTGTTTTG GCACCAAAAT CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3121 
                 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTGGTTTAG TGAACCGTCA GATCCGCTAG 
               
               
                     60010743-ITX-00001_Contig 
                  #3121 
                 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTGGTTTAG TGAACCGTCA GATCCGCTAG 
               
               
                   
                  #3121 
                 TGACGCAAAT GGGCGGTAGG CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTGGTTTAG TGAACCGTCA GATCCGCTAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3201 
                 CATTGTGTGC TTTTCGGGCC ACCATGACGC TGCGGCTTCT GGTGGCCGCG CTCTGCGCCG GGATCCTGGC AGAGGCGCCC 
               
               
                     60010743-ITX-00001_Contig 
                  #3201 
                 CATTGTGTGC TTTTCGGGCC ACCATGACGC TGCGGCTTCT GGTGGCCGCG CTCTGCGCCG GGATCCTGGC AGAGGCGCCC 
               
               
                   
                  #3201 
                 CATTGTGTGC TTTTCGGGCC ACCATGACGC TGCGGCTTCT GGTGGCCGCG CTCTGCGCCG GGATCCTGGC AGAGGCGCCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3281 
                 CGAGTGCGAG CCCAGCACAG GGAGAGAGTG ACCTGCACGC GCCTTTACGC CGCTGACATT GTGTTCTTAC TGGATGGCTC 
               
               
                     60010743-ITX-00001_Contig 
                  #3281 
                 CGAGTGCGAG CCCAGCACAG GGAGAGAGTG ACCTGCACGC GCCTTTACGC CGCTGACATT GTGTTCTTAC TGGATGGCTC 
               
               
                   
                  #3281 
                 CGAGTGCGAG CCCAGCACAG GGAGAGAGTG ACCTGCACGC GCCTTTACGC CGCTGACATT GTGTTCTTAC TGGATGGCTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3361 
                 CTCATCCATT GGCCGCAGCA ATTTCCGCGA GGTCCGCAGC TTTCTCGAAG GGCTGGTGCT GCCTTTCTCT GGAGCAGCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #3361 
                 CTCATCCATT GGCCGCAGCA ATTTCCGCGA GGTCCGCAGC TTTCTCGAAG GGCTGGTGCT GCCTTTCTCT GGAGCAGCCA 
               
               
                   
                  #3361 
                 CTCATCCATT GGCCGCAGCA ATTTCCGCGA GGTCCGCAGC TTTCTCGAAG GGCTGGTGCT GCCTTTCTCT GGAGCAGCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3441 
                 GTGCACAGGG TGTGCGCTTT GCCACAGTGC AGTACAGCGA TGACCCACGG ACAGAGTTCG GCCTGGATGC ACTTGGCTCT 
               
               
                     60010743-ITX-00001_Contig 
                  #3441 
                 GTGCACAGGG TGTGCGCTTT GCCACAGTGC AGTACAGCGA TGACCCACGG ACAGAGTTCG GCCTGGATGC ACTTGGCTCT 
               
               
                   
                  #3441 
                 GTGCACAGGG TGTGCGCTTT GCCACAGTGC AGTACAGCGA TGACCCACGG ACAGAGTTCG GCCTGGATGC ACTTGGCTCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3521 
                 GGGGGTGATG TGATCCGCGC CATCCGTGAG CTTAGCTACA AGGGGGGCAA CACTCGCACA GGGGCTGCAA TTCTCCATGT 
               
               
                     60010743-ITX-00001_Contig 
                  #3521 
                 GGGGGTGATG TGATCCGCGC CATCCGTGAG CTTAGCTACA AGGGGGGCAA CACTCGCACA GGGGCTGCAA TTCTCCATGT 
               
               
                   
                  #3521 
                 GGGGGTGATG TGATCCGCGC CATCCGTGAG CTTAGCTACA AGGGGGGCAA CACTCGCACA GGGGCTGCAA TTCTCCATGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3601 
                 GGCTGACCAT GTCTTCCTGC CCCAGCTGGC CCGACCTGGT GTCCCCAAGG TCTGCATCCT GATCACAGAC GGGAAGTCCC 
               
               
                     60010743-ITX-00001_Contig 
                  #3601 
                 GGCTGACCAT GTCTTCCTGC CCCAGCTGGC CCGACCTGGT GTCCCCAAGG TCTGCATCCT GATCACAGAC GGGAAGTCCC 
               
               
                   
                  #3601 
                 GGCTGACCAT GTCTTCCTGC CCCAGCTGGC CCGACCTGGT GTCCCCAAGG TCTGCATCCT GATCACAGAC GGGAAGTCCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3681 
                 AGGACCTGGT GGACACAGCT GCCCAAAGGC TGAAGGGGCA GGGGGTCAAG CTATTTGCTG TGGGGATCAA GAATGCTGAC 
               
               
                     60010743-ITX-00001_Contig 
                  #3681 
                 AGGACCTGGT GGACACAGCT GCCCAAAGGC TGAAGGGGCA GGGGGTCAAG CTATTTGCTG TGGGGATCAA GAATGCTGAC 
               
               
                   
                  #3681 
                 AGGACCTGGT GGACACAGCT GCCCAAAGGC TGAAGGGGCA GGGGGTCAAG CTATTTGCTG TGGGGATCAA GAATGCTGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3761 
                 CCTGAGGAGC TGAAGCGAGT TGCCTCACAG CCCACCAGTG ACTTCTTCTT CTTCGTCAAT GACTTCAGCA TCTTGAGGAC 
               
               
                     60010743-ITX-00001_Contig 
                  #3761 
                 CCTGAGGAGC TGAAGCGAGT TGCCTCACAG CCCACCAGTG ACTTCTTCTT CTTCGTCAAT GACTTCAGCA TCTTGAGGAC 
               
               
                   
                  #3761 
                 CCTGAGGAGC TGAAGCGAGT TGCCTCACAG CCCACCAGTG ACTTCTTCTT CTTCGTCAAT GACTTCAGCA TCTTGAGGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3841 
                 ACTACTGCCC CTCGTTTCCC GGAGAGTGTG CACGACTGCT GGTGGCGTGC CTGTGACCCG ACCTCCGGAT GACTCGACCT 
               
               
                     60010743-ITX-00001_Contig 
                  #3841 
                 ACTACTGCCC CTCGTTTCCC GGAGAGTGTG CACGACTGCT GGTGGCGTGC CTGTGACCCG ACCTCCGGAT GACTCGACCT 
               
               
                   
                  #3841 
                 ACTACTGCCC CTCGTTTCCC GGAGAGTGTG CACGACTGCT GGTGGCGTGC CTGTGACCCG ACCTCCGGAT GACTCGACCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #3921 
                 CTGCTCCACG AGACCTGGTG CTGTCTGAGC CAAGCAGCCA ATCCTTGAGA GTACAGTGGA CAGCGGCCAG TGGCCCTGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #3921 
                 CTGCTCCACG AGACCTGGTG CTGTCTGAGC CAAGCAGCCA ATCCTTGAGA GTACAGTGGA CAGCGGCCAG TGGCCCTGTG 
               
               
                   
                  #3921 
                 CTGCTCCACG AGACCTGGTG CTGTCTGAGC CAAGCAGCCA ATCCTTGAGA GTACAGTGGA CAGCGGCCAG TGGCCCTGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4001 
                 ACTGGCTACA AGGTCCAGTA CACTCCTCTG ACGGGGCTGG GACAGCCACT GCCGAGTGAG CGGCAGGAGG TGAACGTCCC 
               
               
                     60010743-ITX-00001_Contig 
                  #4001 
                 ACTGGCTACA AGGTCCAGTA CACTCCTCTG ACGGGGCTGG GACAGCCACT GCCGAGTGAG CGGCAGGAGG TGAACGTCCC 
               
               
                   
                  #4001 
                 ACTGGCTACA AGGTCCAGTA CACTCCTCTG ACGGGGCTGG GACAGCCACT GCCGAGTGAG CGGCAGGAGG TGAACGTCCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4081 
                 AGCTGGTGAG ACCAGTGTGC GGCTGCGGGG TCTCCGGCCA CTGACCGAGT ACCAAGTGAC TGTGATTGCC CTCTACGCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #4081 
                 AGCTGGTGAG ACCAGTGTGC GGCTGCGGGG TCTCCGGCCA CTGACCGAGT ACCAAGTGAC TGTGATTGCC CTCTACGCCA 
               
               
                   
                  #4081 
                 AGCTGGTGAG ACCAGTGTGC GGCTGCGGGG TCTCCGGCCA CTGACCGAGT ACCAAGTGAC TGTGATTGCC CTCTACGCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4161 
                 ACAGCATCGG GGAGGCTGTG AGCGGGACAG CTCGGACCAC TGCCCTAGAA GGGCCGGAAC TGACCATCCA GAATACCACA 
               
               
                     60010743-ITX-00001_Contig 
                  #4161 
                 ACAGCATCGG GGAGGCTGTG AGCGGGACAG CTCGGACCAC TGCCCTAGAA GGGCCGGAAC TGACCATCCA GAATACCACA 
               
               
                   
                  #4161 
                 ACAGCATCGG GGAGGCTGTG AGCGGGACAG CTCGGACCAC TGCCCTAGAA GGGCCGGAAC TGACCATCCA GAATACCACA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4241 
                 GCCCACAGCC TCCTGGTGGC CTGGCGGAGT GTGCCAGGTG CCACTGGCTA CCGTGTGACA TGGCGGGTCC TCAGTGGTGG 
               
               
                     60010743-ITX-00001_Contig 
                  #4241 
                 GCCCACAGCC TCCTGGTGGC CTGGCGGAGT GTGCCAGGTG CCACTGGCTA CCGTGTGACA TGGCGGGTCC TCAGTGGTGG 
               
               
                   
                  #4241 
                 GCCCACAGCC TCCTGGTGGC CTGGCGGAGT GTGCCAGGTG CCACTGGCTA CCGTGTGACA TGGCGGGTCC TCAGTGGTGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4321 
                 GCCCACACAG CAGCAGGAGC TGGGCCCTGG GCAGGGTTCA GTGTTGCTGC GTGACTTGGA GCCTGGCACG GACTATGAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #4321 
                 GCCCACACAG CAGCAGGAGC TGGGCCCTGG GCAGGGTTCA GTGTTGCTGC GTGACTTGGA GCCTGGCACG GACTATGAGG 
               
               
                   
                  #4321 
                 GCCCACACAG CAGCAGGAGC TGGGCCCTGG GCAGGGTTCA GTGTTGCTGC GTGACTTGGA GCCTGGCACG GACTATGAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4401 
                 TGACCGTGAG CACCCTATTT GGCCGCAGTG TGGGGCCCGC CACTTCCCTG ATGGCTCGCA CTGACGCTTC TGTTGAGCAG 
               
               
                     60010743-ITX-00001_Contig 
                  #4401 
                 TGACCGTGAG CACCCTATTT GGCCGCAGTG TGGGGCCCGC CACTTCCCTG ATGGCTCGCA CTGACGCTTC TGTTGAGCAG 
               
               
                   
                  #4401 
                 TGACCGTGAG CACCCTATTT GGCCGCAGTG TGGGGCCCGC CACTTCCCTG ATGGCTCGCA CTGACGCTTC TGTTGAGCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4481 
                 ACCCTGCGCC CGGTCATCCT GGGCCCCACA TCCATCCTCC TTTCCTGGAA CTTGGTGCCT GAGGCCCGTG GCTACCGGTT 
               
               
                     60010743-ITX-00001_Contig 
                  #4481 
                 ACCCTGCGCC CGGTCATCCT GGGCCCCACA TCCATCCTCC TTTCCTGGAA CTTGGTGCCT GAGGCCCGTG GCTACCGGTT 
               
               
                   
                  #4481 
                 ACCCTGCGCC CGGTCATCCT GGGCCCCACA TCCATCCTCC TTTCCTGGAA CTTGGTGCCT GAGGCCCGTG GCTACCGGTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4561 
                 GGAATGGCGG CGTGAGACTG GCTTGGAGCC ACCGCAGAAG GTGGTACTGC CCTCTGATGT GACCCGCTAC CAGTTGGATG 
               
               
                     60010743-ITX-00001_Contig 
                  #4561 
                 GGAATGGCGG CGTGAGACTG GCTTGGAGCC ACCGCAGAAG GTGGTACTGC CCTCTGATGT GACCCGCTAC CAGTTGGATG 
               
               
                   
                  #4561 
                 GGAATGGCGG CGTGAGACTG GCTTGGAGCC ACCGCAGAAG GTGGTACTGC CCTCTGATGT GACCCGCTAC CAGTTGGATG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4641 
                 GGCTGCAGCC GGGCACTGAG TACCGCCTCA CACTCTACAC TCTGCTGGAG GGCCACGAGG TGGCCACCCC TGCAACCGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #4641 
                 GGCTGCAGCC GGGCACTGAG TACCGCCTCA CACTCTACAC TCTGCTGGAG GGCCACGAGG TGGCCACCCC TGCAACCGTG 
               
               
                   
                  #4641 
                 GGCTGCAGCC GGGCACTGAG TACCGCCTCA CACTCTACAC TCTGCTGGAG GGCCACGAGG TGGCCACCCC TGCAACCGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4721 
                 GTTCCCACTG GACCAGAGCT GCCTGTGAGC CCTGTAACAG ACCTGCAAGC CACCGAGCTG CCCGGGCAGC GGGTGCGAGT 
               
               
                     60010743-ITX-00001_Contig 
                  #4721 
                 GTTCCCACTG GACCAGAGCT GCCTGTGAGC CCTGTAACAG ACCTGCAAGC CACCGAGCTG CCCGGGCAGC GGGTGCGAGT 
               
               
                   
                  #4721 
                 GTTCCCACTG GACCAGAGCT GCCTGTGAGC CCTGTAACAG ACCTGCAAGC CACCGAGCTG CCCGGGCAGC GGGTGCGAGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4801 
                 GTCCTGGAGC CCAGTCCCTG GTGCCACCCA GTACCGCATC ATTGTGCGCA GCACCCAGGG GGTTGAGCGG ACCCTGGTGC 
               
               
                     60010743-ITX-00001_Contig 
                  #4801 
                 GTCCTGGAGC CCAGTCCCTG GTGCCACCCA GTACCGCATC ATTGTGCGCA GCACCCAGGG GGTTGAGCGG ACCCTGGTGC 
               
               
                   
                  #4801 
                 GTCCTGGAGC CCAGTCCCTG GTGCCACCCA GTACCGCATC ATTGTGCGCA GCACCCAGGG GGTTGAGCGG ACCCTGGTGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4881 
                 TTCCTGGGAG TCAGACAGCA TTCGACTTGG ATGACGTTCA GGCTGGGCTT AGCTACACTG TGCGGGTGTC TGCTCGAGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #4881 
                 TTCCTGGGAG TCAGACAGCA TTCGACTTGG ATGACGTTCA GGCTGGGCTT AGCTACACTG TGCGGGTGTC TGCTCGAGTG 
               
               
                   
                  #4881 
                 TTCCTGGGAG TCAGACAGCA TTCGACTTGG ATGACGTTCA GGCTGGGCTT AGCTACACTG TGCGGGTGTC TGCTCGAGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #4961 
                 GGTCCCCGTG AGGGCAGTGC CAGTGTCCTC ACTGTCCGCC GGGAGCCGGA AACTCCACTT GCTGTTCCAG GGCTGCGGGT 
               
               
                     60010743-ITX-00001_Contig 
                  #4961 
                 GGTCCCCGTG AGGGCAGTGC CAGTGTCCTC ACTGTCCGCC GGGAGCCGGA AACTCCACTT GCTGTTCCAG GGCTGCGGGT 
               
               
                   
                  #4961 
                 GGTCCCCGTG AGGGCAGTGC CAGTGTCCTC ACTGTCCGCC GGGAGCCGGA AACTCCACTT GCTGTTCCAG GGCTGCGGGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5041 
                 TGTGGTGTCA GATGCAACGC GAGTGAGGGT GGCCTGGGGA CCCGTCCCTG GAGCCAGTGG ATTTCGGATT AGCTGGAGCA 
               
               
                     60010743-ITX-00001_Contig 
                  #5041 
                 TGTGGTGTCA GATGCAACGC GAGTGAGGGT GGCCTGGGGA CCCGTCCCTG GAGCCAGTGG ATTTCGGATT AGCTGGAGCA 
               
               
                   
                  #5041 
                 TGTGGTGTCA GATGCAACGC GAGTGAGGGT GGCCTGGGGA CCCGTCCCTG GAGCCAGTGG ATTTCGGATT AGCTGGAGCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5121 
                 CAGGCAGTGG TCCGGAGTCC AGCCAGACAC TGCCCCCAGA CTCTACTGCC ACAGACATCA CAGGGCTGCA GCCTGGAACC 
               
               
                     60010743-ITX-00001_Contig 
                  #5121 
                 CAGGCAGTGG TCCGGAGTCC AGCCAGACAC TGCCCCCAGA CTCTACTGCC ACAGACATCA CAGGGCTGCA GCCTGGAACC 
               
               
                   
                  #5121 
                 CAGGCAGTGG TCCGGAGTCC AGCCAGACAC TGCCCCCAGA CTCTACTGCC ACAGACATCA CAGGGCTGCA GCCTGGAACC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5201 
                 ACCTACCAGG TGGCTGTGTC GGTACTGCGA GGCAGAGAGG AGGGCCCTGC TGCAGTCATC GTGGCTCGAA CGGACCCACT 
               
               
                     60010743-ITX-00001_Contig 
                  #5201 
                 ACCTACCAGG TGGCTGTGTC GGTACTGCGA GGCAGAGAGG AGGGCCCTGC TGCAGTCATC GTGGCTCGAA CGGACCCACT 
               
               
                   
                  #5201 
                 ACCTACCAGG TGGCTGTGTC GGTACTGCGA GGCAGAGAGG AGGGCCCTGC TGCAGTCATC GTGGCTCGAA CGGACCCACT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5281 
                 GGGCCCAGTG AGGACGGTCC ATGTGACTCA GGCCAGCAGC TCATCTGTCA CCATTACCTG GACCAGGGTT CCTGGCGCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #5281 
                 GGGCCCAGTG AGGACGGTCC ATGTGACTCA GGCCAGCAGC TCATCTGTCA CCATTACCTG GACCAGGGTT CCTGGCGCCA 
               
               
                   
                  #5281 
                 GGGCCCAGTG AGGACGGTCC ATGTGACTCA GGCCAGCAGC TCATCTGTCA CCATTACCTG GACCAGGGTT CCTGGCGCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5361 
                 CAGGATACAG GGTTTCCTGG CACTCAGCCC ACGGCCCAGA GAAATCCCAG TTGGTTTCTG GGGAGGCCAC GGTGGCTGAG 
               
               
                     60010743-ITX-00001_Contig 
                  #5361 
                 CAGGATACAG GGTTTCCTGG CACTCAGCCC ACGGCCCAGA GAAATCCCAG TTGGTTTCTG GGGAGGCCAC GGTGGCTGAG 
               
               
                   
                  #5361 
                 CAGGATACAG GGTTTCCTGG CACTCAGCCC ACGGCCCAGA GAAATCCCAG TTGGTTTCTG GGGAGGCCAC GGTGGCTGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5441 
                 CTGGATGGAC TGGAGCCAGA TACTGAGTAT ACGGTGCATG TGAGGGCCCA TGTGGCTGGC GTGGATGGGC CCCCTGCCTC 
               
               
                     60010743-ITX-00001_Contig 
                  #5441 
                 CTGGATGGAC TGGAGCCAGA TACTGAGTAT ACGGTGCATG TGAGGGCCCA TGTGGCTGGC GTGGATGGGC CCCCTGCCTC 
               
               
                   
                  #5441 
                 CTGGATGGAC TGGAGCCAGA TACTGAGTAT ACGGTGCATG TGAGGGCCCA TGTGGCTGGC GTGGATGGGC CCCCTGCCTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5521 
                 TGTGGTTGTG AGGACTGCCC CTGAGCCTGT GGGTCGTGTG TCGAGGCTGC AGATCCTCAA TGCTTCCAGC GACGTTCTAC 
               
               
                     60010743-ITX-00001_Contig 
                  #5521 
                 TGTGGTTGTG AGGACTGCCC CTGAGCCTGT GGGTCGTGTG TCGAGGCTGC AGATCCTCAA TGCTTCCAGC GACGTTCTAC 
               
               
                   
                  #5521 
                 TGTGGTTGTG AGGACTGCCC CTGAGCCTGT GGGTCGTGTG TCGAGGCTGC AGATCCTCAA TGCTTCCAGC GACGTTCTAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5601 
                 GGATCACCTG GGTAGGGGTC ACTGGAGCCA CAGCTTACAG ACTGGCCTGG GGCCGGAGTG AAGGCGGCCC CATGAGGCAC 
               
               
                     60010743-ITX-00001_Contig 
                  #5601 
                 GGATCACCTG GGTAGGGGTC ACTGGAGCCA CAGCTTACAG ACTGGCCTGG GGCCGGAGTG AAGGCGGCCC CATGAGGCAC 
               
               
                   
                  #5601 
                 GGATCACCTG GGTAGGGGTC ACTGGAGCCA CAGCTTACAG ACTGGCCTGG GGCCGGAGTG AAGGCGGCCC CATGAGGCAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5681 
                 CAGATACTCC CAGGAAACAC AGACTCTGCA GAGATCCGGG GTCTCGAAGG TGGAGTCAGC TACTCAGTGC GAGTGACTGC 
               
               
                     60010743-ITX-00001_Contig 
                  #5681 
                 CAGATACTCC CAGGAAACAC AGACTCTGCA GAGATCCGGG GTCTCGAAGG TGGAGTCAGC TACTCAGTGC GAGTGACTGC 
               
               
                   
                  #5681 
                 CAGATACTCC CAGGAAACAC AGACTCTGCA GAGATCCGGG GTCTCGAAGG TGGAGTCAGC TACTCAGTGC GAGTGACTGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5761 
                 ACTTGTCGGG GACCGCGAGG GCACACCTGT CTCCATTGTT GTCACTACGC CGCCTGAGGC TCCGCCAGCC CTGGGGACGC 
               
               
                     60010743-ITX-00001_Contig 
                  #5761 
                 ACTTGTCGGG GACCGCGAGG GCACACCTGT CTCCATTGTT GTCACTACGC CGCCTGAGGC TCCGCCAGCC CTGGGGACGC 
               
               
                   
                  #5761 
                 ACTTGTCGGG GACCGCGAGG GCACACCTGT CTCCATTGTT GTCACTACGC CGCCTGAGGC TCCGCCAGCC CTGGGGACGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5841 
                 TTCACGTGGT GCAGCGCGGG GAGCACTCGC TGAGGCTGCG CTGGGAGCCG GTGCCCAGAG CGCAGGGCTT CCTTCTGCAC 
               
               
                     60010743-ITX-00001_Contig 
                  #5841 
                 TTCACGTGGT GCAGCGCGGG GAGCACTCGC TGAGGCTGCG CTGGGAGCCG GTGCCCAGAG CGCAGGGCTT CCTTCTGCAC 
               
               
                   
                  #5841 
                 TTCACGTGGT GCAGCGCGGG GAGCACTCGC TGAGGCTGCG CTGGGAGCCG GTGCCCAGAG CGCAGGGCTT CCTTCTGCAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #5921 
                 TGGCAACCTG AGGGTGGCCA GGAACAGTCC CGGGTCCTGG GGCCCGAGCT CAGCAGCTAT CACCTGGACG GGCTGGAGCC 
               
               
                     60010743-ITX-00001_Contig 
                  #5921 
                 TGGCAACCTG AGGGTGGCCA GGAACAGTCC CGGGTCCTGG GGCCCGAGCT CAGCAGCTAT CACCTGGACG GGCTGGAGCC 
               
               
                   
                  #5921 
                 TGGCAACCTG AGGGTGGCCA GGAACAGTCC CGGGTCCTGG GGCCCGAGCT CAGCAGCTAT CACCTGGACG GGCTGGAGCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6001 
                 AGCGACACAG TACCGCGTGA GGCTGAGTGT CCTAGGGCCG GCTGGAGAAG GGCCCTCTGC AGAGGTGACT GCGCGCACTG 
               
               
                     60010743-ITX-00001_Contig 
                  #6001 
                 AGCGACACAG TACCGCGTGA GGCTGAGTGT CCTAGGGCCG GCTGGAGAAG GGCCCTCTGC AGAGGTGACT GCGCGCACTG 
               
               
                   
                  #6001 
                 AGCGACACAG TACCGCGTGA GGCTGAGTGT CCTAGGGCCG GCTGGAGAAG GGCCCTCTGC AGAGGTGACT GCGCGCACTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6081 
                 AGTCACCTCG TGTTCCAAGC ATTGAACTAC GTGTGGTGGA CACCTCGATC GACTCGGTGA CTTTGGCCTG GACTCCAGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #6081 
                 AGTCACCTCG TGTTCCAAGC ATTGAACTAC GTGTGGTGGA CACCTCGATC GACTCGGTGA CTTTGGCCTG GACTCCAGTG 
               
               
                   
                  #6081 
                 AGTCACCTCG TGTTCCAAGC ATTGAACTAC GTGTGGTGGA CACCTCGATC GACTCGGTGA CTTTGGCCTG GACTCCAGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6161 
                 TCCAGGGCAT CCAGCTACAT CCTATCCTGG CGGCCACTCA GAGGCCCTGG CCAGGAAGTG CCTGGGTCCC CGCAGACACT 
               
               
                     60010743-ITX-00001_Contig 
                  #6161 
                 TCCAGGGCAT CCAGCTACAT CCTATCCTGG CGGCCACTCA GAGGCCCTGG CCAGGAAGTG CCTGGGTCCC CGCAGACACT 
               
               
                   
                  #6161 
                 TCCAGGGCAT CCAGCTACAT CCTATCCTGG CGGCCACTCA GAGGCCCTGG CCAGGAAGTG CCTGGGTCCC CGCAGACACT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6241 
                 TCCAGGGATC TCAAGCTCCC AGCGGGTGAC AGGGCTAGAG CCTGGCGTCT CTTACATCTT CTCCCTGACG CCTGTCCTGG 
               
               
                     60010743-ITX-00001_Contig 
                  #6241 
                 TCCAGGGATC TCAAGCTCCC AGCGGGTGAC AGGGCTAGAG CCTGGCGTCT CTTACATCTT CTCCCTGACG CCTGTCCTGG 
               
               
                   
                  #6241 
                 TCCAGGGATC TCAAGCTCCC AGCGGGTGAC AGGGCTAGAG CCTGGCGTCT CTTACATCTT CTCCCTGACG CCTGTCCTGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6321 
                 ATGGTGTGCG GGGTCCTGAG GCATCTGTCA CACAGACGCC AGTGTGCCCC CGTGGCCTGG CGGATGTGGT GTTCCTACCA 
               
               
                     60010743-ITX-00001_Contig 
                  #6321 
                 ATGGTGTGCG GGGTCCTGAG GCATCTGTCA CACAGACGCC AGTGTGCCCC CGTGGCCTGG CGGATGTGGT GTTCCTACCA 
               
               
                   
                  #6321 
                 ATGGTGTGCG GGGTCCTGAG GCATCTGTCA CACAGACGCC AGTGTGCCCC CGTGGCCTGG CGGATGTGGT GTTCCTACCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6401 
                 CATGCCACTC AAGACAATGC TCACCGTGCG GAGGCTACGA GGAGGGTCCT GGAGCGTCTG GTGTTGGCAC TTGGGCCTCT 
               
               
                     60010743-ITX-00001_Contig 
                  #6401 
                 CATGCCACTC AAGACAATGC TCACCGTGCG GAGGCTACGA GGAGGGTCCT GGAGCGTCTG GTGTTGGCAC TTGGGCCTCT 
               
               
                   
                  #6401 
                 CATGCCACTC AAGACAATGC TCACCGTGCG GAGGCTACGA GGAGGGTCCT GGAGCGTCTG GTGTTGGCAC TTGGGCCTCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6481 
                 TGGGCCACAG GCAGTTCAGG TTGGCCTGCT GTCTTACAGT CATCGGCCCT CCCCACTGTT CCCACTGAAT GGCTCCCATG 
               
               
                     60010743-ITX-00001_Contig 
                  #6481 
                 TGGGCCACAG GCAGTTCAGG TTGGCCTGCT GTCTTACAGT CATCGGCCCT CCCCACTGTT CCCACTGAAT GGCTCCCATG 
               
               
                   
                  #6481 
                 TGGGCCACAG GCAGTTCAGG TTGGCCTGCT GTCTTACAGT CATCGGCCCT CCCCACTGTT CCCACTGAAT GGCTCCCATG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6561 
                 ACCTTGGCAT TATCTTGCAA AGGATCCGTG ACATGCCCTA CATGGACCCA AGTGGGAACA ACCTGGGCAC AGCCGTGGTC 
               
               
                     60010743-ITX-00001_Contig 
                  #6561 
                 ACCTTGGCAT TATCTTGCAA AGGATCCGTG ACATGCCCTA CATGGACCCA AGTGGGAACA ACCTGGGCAC AGCCGTGGTC 
               
               
                   
                  #6561 
                 ACCTTGGCAT TATCTTGCAA AGGATCCGTG ACATGCCCTA CATGGACCCA AGTGGGAACA ACCTGGGCAC AGCCGTGGTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6641 
                 ACAGCTCACA GATACATGTT GGCACCAGAT GCTCCTGGGC GCCGCCAGCA CGTACCAGGG GTGATGGTTC TGCTAGTGGA 
               
               
                     60010743-ITX-00001_Contig 
                  #6641 
                 ACAGCTCACA GATACATGTT GGCACCAGAT GCTCCTGGGC GCCGCCAGCA CGTACCAGGG GTGATGGTTC TGCTAGTGGA 
               
               
                   
                  #6641 
                 ACAGCTCACA GATACATGTT GGCACCAGAT GCTCCTGGGC GCCGCCAGCA CGTACCAGGG GTGATGGTTC TGCTAGTGGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6721 
                 TGAACCCTTG AGAGGTGACA TATTCAGCCC CATCCGTGAG GCCCAGGCTT CTGGGCTTAA TGTGGTGATG TTGGGAATGG 
               
               
                     60010743-ITX-00001_Contig 
                  #6721 
                 TGAACCCTTG AGAGGTGACA TATTCAGCCC CATCCGTGAG GCCCAGGCTT CTGGGCTTAA TGTGGTGATG TTGGGAATGG 
               
               
                   
                  #6721 
                 TGAACCCTTG AGAGGTGACA TATTCAGCCC CATCCGTGAG GCCCAGGCTT CTGGGCTTAA TGTGGTGATG TTGGGAATGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6801 
                 CTGGAGCGGA CCCAGAGCAG CTGCGTCGCT TGGCGCCGGG TATGGACTCT GTCCAGACCT TCTTCGCCGT GGATGATGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #6801 
                 CTGGAGCGGA CCCAGAGCAG CTGCGTCGCT TGGCGCCGGG TATGGACTCT GTCCAGACCT TCTTCGCCGT GGATGATGGG 
               
               
                   
                  #6801 
                 CTGGAGCGGA CCCAGAGCAG CTGCGTCGCT TGGCGCCGGG TATGGACTCT GTCCAGACCT TCTTCGCCGT GGATGATGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6881 
                 CCAAGCCTGG ACCAGGCAGT CAGTGGTCTG GCCACAGCCC TGTGTCAGGC ATCCTTCACT ACTCAGCCCC GGCCAGAGCC 
               
               
                     60010743-ITX-00001_Contig 
                  #6881 
                 CCAAGCCTGG ACCAGGCAGT CAGTGGTCTG GCCACAGCCC TGTGTCAGGC ATCCTTCACT ACTCAGCCCC GGCCAGAGCC 
               
               
                   
                  #6881 
                 CCAAGCCTGG ACCAGGCAGT CAGTGGTCTG GCCACAGCCC TGTGTCAGGC ATCCTTCACT ACTCAGCCCC GGCCAGAGCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #6961 
                 CTGCCCAGTG TATTGTCCAA AGGGCCAGAA GGGGGAACCT GGAGAGATGG GCCTGAGAGG ACAAGTTGGG CCTCCTGGCG 
               
               
                     60010743-ITX-00001_Contig 
                  #6961 
                 CTGCCCAGTG TATTGTCCAA AGGGCCAGAA GGGGGAACCT GGAGAGATGG GCCTGAGAGG ACAAGTTGGG CCTCCTGGCG 
               
               
                   
                  #6961 
                 CTGCCCAGTG TATTGTCCAA AGGGCCAGAA GGGGGAACCT GGAGAGATGG GCCTGAGAGG ACAAGTTGGG CCTCCTGGCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7041 
                 ACCCTGGCCT CCCGGGCAGG ACCGGTGCTC CCGGCCCCCA GGGGCCCCCT GGAAGTGCCA CTGCCAAGGG CGAGAGGGGC 
               
               
                     60010743-ITX-00001_Contig 
                  #7041 
                 ACCCTGGCCT CCCGGGCAGG ACCGGTGCTC CCGGCCCCCA GGGGCCCCCT GGAAGTGCCA CTGCCAAGGG CGAGAGGGGC 
               
               
                   
                  #7041 
                 ACCCTGGCCT CCCGGGCAGG ACCGGTGCTC CCGGCCCCCA GGGGCCCCCT GGAAGTGCCA CTGCCAAGGG CGAGAGGGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7121 
                 TTCCCTGGAG CAGATGGGCG TCCAGGCAGC CCTGGCCGCG CCGGGAATCC TGGGACCCCT GGAGCCCCTG GCCTAAAGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #7121 
                 TTCCCTGGAG CAGATGGGCG TCCAGGCAGC CCTGGCCGCG CCGGGAATCC TGGGACCCCT GGAGCCCCTG GCCTAAAGGG 
               
               
                   
                  #7121 
                 TTCCCTGGAG CAGATGGGCG TCCAGGCAGC CCTGGCCGCG CCGGGAATCC TGGGACCCCT GGAGCCCCTG GCCTAAAGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7201 
                 CTCTCCAGGG TTGCCTGGCC CTCGTGGGGA CCCGGGAGAG CGAGGACCTC GAGGCCCAAA GGGGGAGCCG GGGGCTCCCG 
               
               
                     60010743-ITX-00001_Contig 
                  #7201 
                 CTCTCCAGGG TTGCCTGGCC CTCGTGGGGA CCCGGGAGAG CGAGGACCTC GAGGCCCAAA GGGGGAGCCG GGGGCTCCCG 
               
               
                   
                  #7201 
                 CTCTCCAGGG TTGCCTGGCC CTCGTGGGGA CCCGGGAGAG CGAGGACCTC GAGGCCCAAA GGGGGAGCCG GGGGCTCCCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7281 
                 GACAAGTCAT CGGAGGTGAA GGACCTGGGC TTCCTGGGCG GAAAGGGGAC CCTGGACCAT CGGGCCCCCC TGGACCTCGT 
               
               
                     60010743-ITX-00001_Contig 
                  #7281 
                 GACAAGTCAT CGGAGGTGAA GGACCTGGGC TTCCTGGGCG GAAAGGGGAC CCTGGACCAT CGGGCCCCCC TGGACCTCGT 
               
               
                   
                  #7281 
                 GACAAGTCAT CGGAGGTGAA GGACCTGGGC TTCCTGGGCG GAAAGGGGAC CCTGGACCAT CGGGCCCCCC TGGACCTCGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7361 
                 GGACCACTGG GGGACCCAGG ACCCCGTGGC CCCCCAGGGC TTCCTGGAAC AGCCATGAAG GGTGACAAAG GCGATCGTGG 
               
               
                     60010743-ITX-00001_Contig 
                  #7361 
                 GGACCACTGG GGGACCCAGG ACCCCGTGGC CCCCCAGGGC TTCCTGGAAC AGCCATGAAG GGTGACAAAG GCGATCGTGG 
               
               
                   
                  #7361 
                 GGACCACTGG GGGACCCAGG ACCCCGTGGC CCCCCAGGGC TTCCTGGAAC AGCCATGAAG GGTGACAAAG GCGATCGTGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7441 
                 GGAGCGGGGT CCCCCTGGAC CAGGTGAAGG TGGCATTGCT CCTGGGGAGC CTGGGCTGCC GGGTCTTCCC GGAAGCCCTG 
               
               
                     60010743-ITX-00001_Contig 
                  #7441 
                 GGAGCGGGGT CCCCCTGGAC CAGGTGAAGG TGGCATTGCT CCTGGGGAGC CTGGGCTGCC GGGTCTTCCC GGAAGCCCTG 
               
               
                   
                  #7441 
                 GGAGCGGGGT CCCCCTGGAC CAGGTGAAGG TGGCATTGCT CCTGGGGAGC CTGGGCTGCC GGGTCTTCCC GGAAGCCCTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7521 
                 GACCCCAAGG CCCCGTTGGC CCCCCTGGAA AGAAAGGAGA AAAAGGTGAC TCTGAGGATG GAGCTCCAGG CCTCCCAGGA 
               
               
                     60010743-ITX-00001_Contig 
                  #7521 
                 GACCCCAAGG CCCCGTTGGC CCCCCTGGAA AGAAAGGAGA AAAAGGTGAC TCTGAGGATG GAGCTCCAGG CCTCCCAGGA 
               
               
                   
                  #7521 
                 GACCCCAAGG CCCCGTTGGC CCCCCTGGAA AGAAAGGAGA AAAAGGTGAC TCTGAGGATG GAGCTCCAGG CCTCCCAGGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7601 
                 CAACCTGGGT CTCCGGGTGA GCAGGGCCCA CGGGGACCTC CTGGAGCTAT TGGCCCCAAA GGTGACCGGG GCTTTCCAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #7601 
                 CAACCTGGGT CTCCGGGTGA GCAGGGCCCA CGGGGACCTC CTGGAGCTAT TGGCCCCAAA GGTGACCGGG GCTTTCCAGG 
               
               
                   
                  #7601 
                 CAACCTGGGT CTCCGGGTGA GCAGGGCCCA CGGGGACCTC CTGGAGCTAT TGGCCCCAAA GGTGACCGGG GCTTTCCAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7681 
                 GCCCCTGGGT GAGGCTGGAG AGAAGGGCGA ACGTGGACCC CCAGGCCCAG CGGGATCCCG GGGGCTGCCA GGGGTTGCTG 
               
               
                     60010743-ITX-00001_Contig 
                  #7681 
                 GCCCCTGGGT GAGGCTGGAG AGAAGGGCGA ACGTGGACCC CCAGGCCCAG CGGGATCCCG GGGGCTGCCA GGGGTTGCTG 
               
               
                   
                  #7681 
                 GCCCCTGGGT GAGGCTGGAG AGAAGGGCGA ACGTGGACCC CCAGGCCCAG CGGGATCCCG GGGGCTGCCA GGGGTTGCTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7761 
                 GACGTCCTGG AGCCAAGGGT CCTGAAGGGC CACCAGGACC CACTGGCCGC CAAGGAGAGA AGGGGGAGCC TGGTCGCCCT 
               
               
                     60010743-ITX-00001_Contig 
                  #7761 
                 GACGTCCTGG AGCCAAGGGT CCTGAAGGGC CACCAGGACC CACTGGCCGC CAAGGAGAGA AGGGGGAGCC TGGTCGCCCT 
               
               
                   
                  #7761 
                 GACGTCCTGG AGCCAAGGGT CCTGAAGGGC CACCAGGACC CACTGGCCGC CAAGGAGAGA AGGGGGAGCC TGGTCGCCCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7841 
                 GGGGACCCTG CAGTGGTGGG ACCTGCTGTT GCTGGACCCA AAGGAGAAAA GGGAGATGTG GGGCCCGCTG GGCCCAGAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #7841 
                 GGGGACCCTG CAGTGGTGGG ACCTGCTGTT GCTGGACCCA AAGGAGAAAA GGGAGATGTG GGGCCCGCTG GGCCCAGAGG 
               
               
                   
                  #7841 
                 GGGGACCCTG CAGTGGTGGG ACCTGCTGTT GCTGGACCCA AAGGAGAAAA GGGAGATGTG GGGCCCGCTG GGCCCAGAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #7921 
                 AGCTACCGGA GTCCAAGGGG AACGGGGCCC ACCCGGCTTG GTTCTTCCTG GAGACCCTGG CCCCAAGGGA GACCCTGGAG 
               
               
                     60010743-ITX-00001_Contig 
                  #7921 
                 AGCTACCGGA GTCCAAGGGG AACGGGGCCC ACCCGGCTTG GTTCTTCCTG GAGACCCTGG CCCCAAGGGA GACCCTGGAG 
               
               
                   
                  #7921 
                 AGCTACCGGA GTCCAAGGGG AACGGGGCCC ACCCGGCTTG GTTCTTCCTG GAGACCCTGG CCCCAAGGGA GACCCTGGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8001 
                 ACCGGGGTCC CATTGGCCTT ACTGGCAGAG CAGGACCCCC AGGTGACTCA GGGCCTCCTG GAGAGAAGGG AGACCCTGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #8001 
                 ACCGGGGTCC CATTGGCCTT ACTGGCAGAG CAGGACCCCC AGGTGACTCA GGGCCTCCTG GAGAGAAGGG AGACCCTGGG 
               
               
                   
                  #8001 
                 ACCGGGGTCC CATTGGCCTT ACTGGCAGAG CAGGACCCCC AGGTGACTCA GGGCCTCCTG GAGAGAAGGG AGACCCTGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8081 
                 CGGCCTGGCC CCCCAGGACC TGTTGGCCCC CGAGGACGAG ATGGTGAAGT TGGAGAGAAA GGTGACGAGG GTCCTCCGGG 
               
               
                     60010743-ITX-00001_Contig 
                  #8081 
                 CGGCCTGGCC CCCCAGGACC TGTTGGCCCC CGAGGACGAG ATGGTGAAGT TGGAGAGAAA GGTGACGAGG GTCCTCCGGG 
               
               
                   
                  #8081 
                 CGGCCTGGCC CCCCAGGACC TGTTGGCCCC CGAGGACGAG ATGGTGAAGT TGGAGAGAAA GGTGACGAGG GTCCTCCGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8161 
                 TGACCCGGGT TTGCCTGGAA AAGCAGGCGA GCGTGGCCTT CGGGGGGCAC CTGGAGTTCG GGGGCCTGTG GGTGAAAAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #8161 
                 TGACCCGGGT TTGCCTGGAA AAGCAGGCGA GCGTGGCCTT CGGGGGGCAC CTGGAGTTCG GGGGCCTGTG GGTGAAAAGG 
               
               
                   
                  #8161 
                 TGACCCGGGT TTGCCTGGAA AAGCAGGCGA GCGTGGCCTT CGGGGGGCAC CTGGAGTTCG GGGGCCTGTG GGTGAAAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8241 
                 GAGACCAGGG AGATCCTGGA GAGGATGGAC GAAATGGCAG CCCTGGATCA TCTGGACCCA AGGGTGACCG TGGGGAGCCG 
               
               
                     60010743-ITX-00001_Contig 
                  #8241 
                 GAGACCAGGG AGATCCTGGA GAGGATGGAC GAAATGGCAG CCCTGGATCA TCTGGACCCA AGGGTGACCG TGGGGAGCCG 
               
               
                   
                  #8241 
                 GAGACCAGGG AGATCCTGGA GAGGATGGAC GAAATGGCAG CCCTGGATCA TCTGGACCCA AGGGTGACCG TGGGGAGCCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8321 
                 GGTCCCCCAG GACCCCCGGG ACGGCTGGTA GACACAGGAC CTGGAGCCAG AGAGAAGGGA GAGCCTGGGG ACCGCGGACA 
               
               
                     60010743-ITX-00001_Contig 
                  #8321 
                 GGTCCCCCAG GACCCCCGGG ACGGCTGGTA GACACAGGAC CTGGAGCCAG AGAGAAGGGA GAGCCTGGGG ACCGCGGACA 
               
               
                   
                  #8321 
                 GGTCCCCCAG GACCCCCGGG ACGGCTGGTA GACACAGGAC CTGGAGCCAG AGAGAAGGGA GAGCCTGGGG ACCGCGGACA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8401 
                 AGAGGGTCCT CGAGGGCCCA AGGGTGATCC TGGCCTCCCT GGAGCCCCTG GGGAAAGGGG CATTGAAGGG TTTCGGGGAC 
               
               
                     60010743-ITX-00001_Contig 
                  #8401 
                 AGAGGGTCCT CGAGGGCCCA AGGGTGATCC TGGCCTCCCT GGAGCCCCTG GGGAAAGGGG CATTGAAGGG TTTCGGGGAC 
               
               
                   
                  #8401 
                 AGAGGGTCCT CGAGGGCCCA AGGGTGATCC TGGCCTCCCT GGAGCCCCTG GGGAAAGGGG CATTGAAGGG TTTCGGGGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8481 
                 CCCCAGGCCC ACAGGGGGAC CCAGGTGTCC GAGGCCCAGC AGGAGAAAAG GGTGACCGGG GTCCCCCTGG GCTGGATGGC 
               
               
                     60010743-ITX-00001_Contig 
                  #8481 
                 CCCCAGGCCC ACAGGGGGAC CCAGGTGTCC GAGGCCCAGC AGGAGAAAAG GGTGACCGGG GTCCCCCTGG GCTGGATGGC 
               
               
                   
                  #8481 
                 CCCCAGGCCC ACAGGGGGAC CCAGGTGTCC GAGGCCCAGC AGGAGAAAAG GGTGACCGGG GTCCCCCTGG GCTGGATGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8561 
                 CGGAGCGGAC TGGATGGGAA ACCAGGAGCC GCTGGGCCCT CTGGGCCGAA TGGTGCTGCA GGCAAAGCTG GGGACCCAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #8561 
                 CGGAGCGGAC TGGATGGGAA ACCAGGAGCC GCTGGGCCCT CTGGGCCGAA TGGTGCTGCA GGCAAAGCTG GGGACCCAGG 
               
               
                   
                  #8561 
                 CGGAGCGGAC TGGATGGGAA ACCAGGAGCC GCTGGGCCCT CTGGGCCGAA TGGTGCTGCA GGCAAAGCTG GGGACCCAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8641 
                 GAGAGACGGG CTTCCAGGCC TCCGTGGAGA ACAAGGCCTC CCTGGCCCCT CTGGTCCCCC TGGATTACCG GGAAAGCCAG 
               
               
                     60010743-ITX-00001_Contig 
                  #8641 
                 GAGAGACGGG CTTCCAGGCC TCCGTGGAGA ACAAGGCCTC CCTGGCCCCT CTGGTCCCCC TGGATTACCG GGAAAGCCAG 
               
               
                   
                  #8641 
                 GAGAGACGGG CTTCCAGGCC TCCGTGGAGA ACAAGGCCTC CCTGGCCCCT CTGGTCCCCC TGGATTACCG GGAAAGCCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8721 
                 GCGAGGATGG GAAACCTGGC CTGAATGGAA AAAACGGAGA ACCTGGGGAC CCTGGAGAAG ACGGGAGGAA GGGAGAGAAA 
               
               
                     60010743-ITX-00001_Contig 
                  #8721 
                 GCGAGGATGG GAAACCTGGC CTGAATGGAA AAAACGGAGA ACCTGGGGAC CCTGGAGAAG ACGGGAGGAA GGGAGAGAAA 
               
               
                   
                  #8721 
                 GCGAGGATGG GAAACCTGGC CTGAATGGAA AAAACGGAGA ACCTGGGGAC CCTGGAGAAG ACGGGAGGAA GGGAGAGAAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8801 
                 GGAGATTCAG GCGCCTCTGG GAGAGAAGGT CGTGATGGCC CCAAGGGTGA GCGTGGAGCT CCTGGTATCC TTGGACCCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #8801 
                 GGAGATTCAG GCGCCTCTGG GAGAGAAGGT CGTGATGGCC CCAAGGGTGA GCGTGGAGCT CCTGGTATCC TTGGACCCCA 
               
               
                   
                  #8801 
                 GGAGATTCAG GCGCCTCTGG GAGAGAAGGT CGTGATGGCC CCAAGGGTGA GCGTGGAGCT CCTGGTATCC TTGGACCCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8881 
                 GGGGCCTCCA GGCCTCCCAG GGCCAGTGGG CCCTCCTGGC CAGGGTTTTC CTGGTGTCCC AGGAGGCACG GGCCCCAAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #8881 
                 GGGGCCTCCA GGCCTCCCAG GGCCAGTGGG CCCTCCTGGC CAGGGTTTTC CTGGTGTCCC AGGAGGCACG GGCCCCAAGG 
               
               
                   
                  #8881 
                 GGGGCCTCCA GGCCTCCCAG GGCCAGTGGG CCCTCCTGGC CAGGGTTTTC CTGGTGTCCC AGGAGGCACG GGCCCCAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #8961 
                 GTGACCGTGG GGAGACTGGA TCCAAAGGGG AGCAGGGCCT CCCTGGAGAG CGTGGCCTGC GAGGAGAGCC TGGAAGTGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #8961 
                 GTGACCGTGG GGAGACTGGA TCCAAAGGGG AGCAGGGCCT CCCTGGAGAG CGTGGCCTGC GAGGAGAGCC TGGAAGTGTG 
               
               
                   
                  #8961 
                 GTGACCGTGG GGAGACTGGA TCCAAAGGGG AGCAGGGCCT CCCTGGAGAG CGTGGCCTGC GAGGAGAGCC TGGAAGTGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9041 
                 CCGAATGTGG ATCGGTTGCT GGAAACTGCT GGCATCAAGG CATCTGCCCT GCGGGAGATC GTGGAGACCT GGGATGAGAG 
               
               
                     60010743-ITX-00001_Contig 
                  #9041 
                 CCGAATGTGG ATCGGTTGCT GGAAACTGCT GGCATCAAGG CATCTGCCCT GCGGGAGATC GTGGAGACCT GGGATGAGAG 
               
               
                   
                  #9041 
                 CCGAATGTGG ATCGGTTGCT GGAAACTGCT GGCATCAAGG CATCTGCCCT GCGGGAGATC GTGGAGACCT GGGATGAGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9121 
                 CTCTGGTAGC TTCCTGCCTG TGCCCGAACG GCGTCGAGGC CCCAAGGGGG ACTCAGGCGA ACAGGGCCCC CCAGGCAAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #9121 
                 CTCTGGTAGC TTCCTGCCTG TGCCCGAACG GCGTCGAGGC CCCAAGGGGG ACTCAGGCGA ACAGGGCCCC CCAGGCAAGG 
               
               
                   
                  #9121 
                 CTCTGGTAGC TTCCTGCCTG TGCCCGAACG GCGTCGAGGC CCCAAGGGGG ACTCAGGCGA ACAGGGCCCC CCAGGCAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9201 
                 AGGGCCCCAT CGGCTTTCCT GGAGAACGCG GGCTGAAGGG CGACCGTGGA GACCCTGGCC CTCAGGGGCC ACCTGGTCTG 
               
               
                     60010743-ITX-00001_Contig 
                  #9201 
                 AGGGCCCCAT CGGCTTTCCT GGAGAACGCG GGCTGAAGGG CGACCGTGGA GACCCTGGCC CTCAGGGGCC ACCTGGTCTG 
               
               
                   
                  #9201 
                 AGGGCCCCAT CGGCTTTCCT GGAGAACGCG GGCTGAAGGG CGACCGTGGA GACCCTGGCC CTCAGGGGCC ACCTGGTCTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9281 
                 GCCCTTGGGG AGAGGGGCCC CCCCGGGCCT TCCGGCCTTG CCGGGGAGCC TGGAAAGCCT GGTATTCCCG GGCTCCCAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #9281 
                 GCCCTTGGGG AGAGGGGCCC CCCCGGGCCT TCCGGCCTTG CCGGGGAGCC TGGAAAGCCT GGTATTCCCG GGCTCCCAGG 
               
               
                   
                  #9281 
                 GCCCTTGGGG AGAGGGGCCC CCCCGGGCCT TCCGGCCTTG CCGGGGAGCC TGGAAAGCCT GGTATTCCCG GGCTCCCAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9361 
                 CAGGGCTGGG GGTGTGGGAG AGGCAGGAAG GCCAGGAGAG AGGGGAGAAC GGGGAGAGAA AGGAGAACGT GGAGAACAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #9361 
                 CAGGGCTGGG GGTGTGGGAG AGGCAGGAAG GCCAGGAGAG AGGGGAGAAC GGGGAGAGAA AGGAGAACGT GGAGAACAGG 
               
               
                   
                  #9361 
                 CAGGGCTGGG GGTGTGGGAG AGGCAGGAAG GCCAGGAGAG AGGGGAGAAC GGGGAGAGAA AGGAGAACGT GGAGAACAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9441 
                 GCAGAGATGG CCCTCCTGGA CTCCCTGGAA CCCCTGGGCC CCCCGGACCC CCTGGCCCCA AGGTGTCTGT GGATGAGCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #9441 
                 GCAGAGATGG CCCTCCTGGA CTCCCTGGAA CCCCTGGGCC CCCCGGACCC CCTGGCCCCA AGGTGTCTGT GGATGAGCCA 
               
               
                   
                  #9441 
                 GCAGAGATGG CCCTCCTGGA CTCCCTGGAA CCCCTGGGCC CCCCGGACCC CCTGGCCCCA AGGTGTCTGT GGATGAGCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9521 
                 GGTCCTGGAC TCTCTGGAGA ACAGGGACCC CCTGGACTCA AGGGTGCTAA GGGGGAGCCG GGCAGCAATG GTGACCAAGG 
               
               
                     60010743-ITX-00001_Contig 
                  #9521 
                 GGTCCTGGAC TCTCTGGAGA ACAGGGACCC CCTGGACTCA AGGGTGCTAA GGGGGAGCCG GGCAGCAATG GTGACCAAGG 
               
               
                   
                  #9521 
                 GGTCCTGGAC TCTCTGGAGA ACAGGGACCC CCTGGACTCA AGGGTGCTAA GGGGGAGCCG GGCAGCAATG GTGACCAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9601 
                 TCCCAAAGGA GACAGGGGTG TGCCAGGCAT CAAAGGAGAC CGGGGAGAGC CTGGACCGAG GGGTCAGGAC GGCAACCCGG 
               
               
                     60010743-ITX-00001_Contig 
                  #9601 
                 TCCCAAAGGA GACAGGGGTG TGCCAGGCAT CAAAGGAGAC CGGGGAGAGC CTGGACCGAG GGGTCAGGAC GGCAACCCGG 
               
               
                   
                  #9601 
                 TCCCAAAGGA GACAGGGGTG TGCCAGGCAT CAAAGGAGAC CGGGGAGAGC CTGGACCGAG GGGTCAGGAC GGCAACCCGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9681 
                 GTCTACCAGG AGAGCGTGGT ATGGCTGGGC CTGAAGGGAA GCCGGGTCTG CAGGGTCCAA GAGGCCCCCC TGGCCCAGTG 
               
               
                     60010743-ITX-00001_Contig 
                  #9681 
                 GTCTACCAGG AGAGCGTGGT ATGGCTGGGC CTGAAGGGAA GCCGGGTCTG CAGGGTCCAA GAGGCCCCCC TGGCCCAGTG 
               
               
                   
                  #9681 
                 GTCTACCAGG AGAGCGTGGT ATGGCTGGGC CTGAAGGGAA GCCGGGTCTG CAGGGTCCAA GAGGCCCCCC TGGCCCAGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9761 
                 GGTGGTCATG GAGACCCTGG ACCACCTGGT GCCCCGGGTC TTGCTGGCCC TGCAGGACCC CAAGGACCTT CTGGCCTGAA 
               
               
                     60010743-ITX-00001_Contig 
                  #9761 
                 GGTGGTCATG GAGACCCTGG ACCACCTGGT GCCCCGGGTC TTGCTGGCCC TGCAGGACCC CAAGGACCTT CTGGCCTGAA 
               
               
                   
                  #9761 
                 GGTGGTCATG GAGACCCTGG ACCACCTGGT GCCCCGGGTC TTGCTGGCCC TGCAGGACCC CAAGGACCTT CTGGCCTGAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9841 
                 GGGGGAGCCT GGAGAGACAG GACCTCCAGG ACGGGGCCTG ACTGGACCTA CTGGAGCTGT GGGACTTCCT GGACCCCCCG 
               
               
                     60010743-ITX-00001_Contig 
                  #9841 
                 GGGGGAGCCT GGAGAGACAG GACCTCCAGG ACGGGGCCTG ACTGGACCTA CTGGAGCTGT GGGACTTCCT GGACCCCCCG 
               
               
                   
                  #9841 
                 GGGGGAGCCT GGAGAGACAG GACCTCCAGG ACGGGGCCTG ACTGGACCTA CTGGAGCTGT GGGACTTCCT GGACCCCCCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                  #9921 
                 GCCCTTCAGG CCTTGTGGGT CCACAGGGGT CTCCAGGTTT GCCTGGACAA GTGGGGGAGA CAGGGAAGCC GGGAGCCCCA 
               
               
                     60010743-ITX-00001_Contig 
                  #9921 
                 GCCCTTCAGG CCTTGTGGGT CCACAGGGGT CTCCAGGTTT GCCTGGACAA GTGGGGGAGA CAGGGAAGCC GGGAGCCCCA 
               
               
                   
                  #9921 
                 GCCCTTCAGG CCTTGTGGGT CCACAGGGGT CTCCAGGTTT GCCTGGACAA GTGGGGGAGA CAGGGAAGCC GGGAGCCCCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10001 
                 GGTCGAGATG GTGCCAGTGG AAAAGATGGA GACAGAGGGA GCCCTGGTGT GCCAGGGTCA CCAGGTCTGC CTGGCCCTGT 
               
               
                     60010743-ITX-00001_Contig 
                 #10001 
                 GGTCGAGATG GTGCCAGTGG AAAAGATGGA GACAGAGGGA GCCCTGGTGT GCCAGGGTCA CCAGGTCTGC CTGGCCCTGT 
               
               
                   
                 #10001 
                 GGTCGAGATG GTGCCAGTGG AAAAGATGGA GACAGAGGGA GCCCTGGTGT GCCAGGGTCA CCAGGTCTGC CTGGCCCTGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10081 
                 CGGACCTAAA GGAGAACCTG GCCCCACGGG GGCCCCTGGA CAGGCTGTGG TCGGGCTCCC TGGAGCAAAG GGAGAGAAGG 
               
               
                     60010743-ITX-00001_Contig 
                 #10081 
                 CGGACCTAAA GGAGAACCTG GCCCCACGGG GGCCCCTGGA CAGGCTGTGG TCGGGCTCCC TGGAGCAAAG GGAGAGAAGG 
               
               
                   
                 #10081 
                 CGGACCTAAA GGAGAACCTG GCCCCACGGG GGCCCCTGGA CAGGCTGTGG TCGGGCTCCC TGGAGCAAAG GGAGAGAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10161 
                 GAGCCCCTGG AGGCCTTGCT GGAGACCTGG TGGGTGAGCC GGGAGCCAAA GGTGACCGAG GACTGCCAGG GCCGCGAGGC 
               
               
                     60010743-ITX-00001_Contig 
                 #10161 
                 GAGCCCCTGG AGGCCTTGCT GGAGACCTGG TGGGTGAGCC GGGAGCCAAA GGTGACCGAG GACTGCCAGG GCCGCGAGGC 
               
               
                   
                 #10161 
                 GAGCCCCTGG AGGCCTTGCT GGAGACCTGG TGGGTGAGCC GGGAGCCAAA GGTGACCGAG GACTGCCAGG GCCGCGAGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10241 
                 GAGAAGGGTG AAGCTGGCCG TGCAGGGGAG CCCGGAGACC CTGGGGAAGA TGGTCAGAAA GGGGCTCCAG GACCCAAAGG 
               
               
                     60010743-ITX-00001_Contig 
                 #10241 
                 GAGAAGGGTG AAGCTGGCCG TGCAGGGGAG CCCGGAGACC CTGGGGAAGA TGGTCAGAAA GGGGCTCCAG GACCCAAAGG 
               
               
                   
                 #10241 
                 GAGAAGGGTG AAGCTGGCCG TGCAGGGGAG CCCGGAGACC CTGGGGAAGA TGGTCAGAAA GGGGCTCCAG GACCCAAAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10321 
                 TTTCAAGGGT GACCCAGGAG TCGGGGTCCC GGGCTCCCCT GGGCCTCCTG GCCCTCCAGG TGTGAAGGGA GATCTGGGCC 
               
               
                     60010743-ITX-00001_Contig 
                 #10321 
                 TTTCAAGGGT GACCCAGGAG TCGGGGTCCC GGGCTCCCCT GGGCCTCCTG GCCCTCCAGG TGTGAAGGGA GATCTGGGCC 
               
               
                   
                 #10321 
                 TTTCAAGGGT GACCCAGGAG TCGGGGTCCC GGGCTCCCCT GGGCCTCCTG GCCCTCCAGG TGTGAAGGGA GATCTGGGCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10401 
                 TCCCTGGCCT GCCCGGTGCT CCTGGTGTTG TTGGGTTCCC GGGTCAGACA GGCCCTCGAG GAGAGATGGG TCAGCCAGGC 
               
               
                     60010743-ITX-00001_Contig 
                 #10401 
                 TCCCTGGCCT GCCCGGTGCT CCTGGTGTTG TTGGGTTCCC GGGTCAGACA GGCCCTCGAG GAGAGATGGG TCAGCCAGGC 
               
               
                   
                 #10401 
                 TCCCTGGCCT GCCCGGTGCT CCTGGTGTTG TTGGGTTCCC GGGTCAGACA GGCCCTCGAG GAGAGATGGG TCAGCCAGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10481 
                 CCTAGTGGAG AGCGGGGTCT GGCAGGCCCC CCAGGGAGAG AAGGAATCCC AGGACCCCTG GGGCCACCTG GACCACCGGG 
               
               
                     60010743-ITX-00001_Contig 
                 #10481 
                 CCTAGTGGAG AGCGGGGTCT GGCAGGCCCC CCAGGGAGAG AAGGAATCCC AGGACCCCTG GGGCCACCTG GACCACCGGG 
               
               
                   
                 #10481 
                 CCTAGTGGAG AGCGGGGTCT GGCAGGCCCC CCAGGGAGAG AAGGAATCCC AGGACCCCTG GGGCCACCTG GACCACCGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10561 
                 GTCAGTGGGA CCACCTGGGG CCTCTGGACT CAAAGGAGAC AAGGGAGACC CTGGAGTAGG GCTGCCTGGG CCCCGAGGCG 
               
               
                     60010743-ITX-00001_Contig 
                 #10561 
                 GTCAGTGGGA CCACCTGGGG CCTCTGGACT CAAAGGAGAC AAGGGAGACC CTGGAGTAGG GCTGCCTGGG CCCCGAGGCG 
               
               
                   
                 #10561 
                 GTCAGTGGGA CCACCTGGGG CCTCTGGACT CAAAGGAGAC AAGGGAGACC CTGGAGTAGG GCTGCCTGGG CCCCGAGGCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10641 
                 AGCGTGGGGA GCCAGGCATC CGGGGTGAAG ATGGCCGCCC CGGCCAGGAG GGACCCCGAG GACTCACGGG GCCCCCTGGC 
               
               
                     60010743-ITX-00001_Contig 
                 #10641 
                 AGCGTGGGGA GCCAGGCATC CGGGGTGAAG ATGGCCGCCC CGGCCAGGAG GGACCCCGAG GACTCACGGG GCCCCCTGGC 
               
               
                   
                 #10641 
                 AGCGTGGGGA GCCAGGCATC CGGGGTGAAG ATGGCCGCCC CGGCCAGGAG GGACCCCGAG GACTCACGGG GCCCCCTGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10721 
                 AGCAGGGGAG AGCGTGGGGA GAAGGGTGAT GTTGGGAGTG CAGGACTAAA GGGTGACAAG GGAGACTCAG CTGTGATCCT 
               
               
                     60010743-ITX-00001_Contig 
                 #10721 
                 AGCAGGGGAG AGCGTGGGGA GAAGGGTGAT GTTGGGAGTG CAGGACTAAA GGGTGACAAG GGAGACTCAG CTGTGATCCT 
               
               
                   
                 #10721 
                 AGCAGGGGAG AGCGTGGGGA GAAGGGTGAT GTTGGGAGTG CAGGACTAAA GGGTGACAAG GGAGACTCAG CTGTGATCCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10801 
                 GGGGCCTCCA GGCCCACGGG GTGCCAAGGG GGACATGGGT GAACGAGGGC CTCGGGGCTT GGATGGTGAC AAAGGACCTC 
               
               
                     60010743-ITX-00001_Contig 
                 #10801 
                 GGGGCCTCCA GGCCCACGGG GTGCCAAGGG GGACATGGGT GAACGAGGGC CTCGGGGCTT GGATGGTGAC AAAGGACCTC 
               
               
                   
                 #10801 
                 GGGGCCTCCA GGCCCACGGG GTGCCAAGGG GGACATGGGT GAACGAGGGC CTCGGGGCTT GGATGGTGAC AAAGGACCTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10881 
                 GGGGAGACAA TGGGGACCCT GGTGACAAGG GCAGCAAGGG AGAGCCTGGT GACAAGGGCT CAGCCGGGTT GCCAGGACTG 
               
               
                     60010743-ITX-00001_Contig 
                 #10881 
                 GGGGAGACAA TGGGGACCCT GGTGACAAGG GCAGCAAGGG AGAGCCTGGT GACAAGGGCT CAGCCGGGTT GCCAGGACTG 
               
               
                   
                 #10881 
                 GGGGAGACAA TGGGGACCCT GGTGACAAGG GCAGCAAGGG AGAGCCTGGT GACAAGGGCT CAGCCGGGTT GCCAGGACTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #10961 
                 CGTGGACTCC TGGGACCCCA GGGTCAACCT GGTGCAGCAG GGATCCCTGG TGACCCGGGA TCCCCAGGAA AGGATGGAGT 
               
               
                     60010743-ITX-00001_Contig 
                 #10961 
                 CGTGGACTCC TGGGACCCCA GGGTCAACCT GGTGCAGCAG GGATCCCTGG TGACCCGGGA TCCCCAGGAA AGGATGGAGT 
               
               
                   
                 #10961 
                 CGTGGACTCC TGGGACCCCA GGGTCAACCT GGTGCAGCAG GGATCCCTGG TGACCCGGGA TCCCCAGGAA AGGATGGAGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11041 
                 GCCTGGTATC CGAGGAGAAA AAGGAGATGT TGGCTTCATG GGTCCCCGGG GCCTCAAGGG TGAACGGGGA GTGAAGGGAG 
               
               
                     60010743-ITX-00001_Contig 
                 #11041 
                 GCCTGGTATC CGAGGAGAAA AAGGAGATGT TGGCTTCATG GGTCCCCGGG GCCTCAAGGG TGAACGGGGA GTGAAGGGAG 
               
               
                   
                 #11041 
                 GCCTGGTATC CGAGGAGAAA AAGGAGATGT TGGCTTCATG GGTCCCCGGG GCCTCAAGGG TGAACGGGGA GTGAAGGGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11121 
                 CCTGTGGCCT TGATGGAGAG AAGGGAGACA AGGGAGAAGC TGGTCCCCCA GGCCGCCCCG GGCTGGCAGG ACACAAAGGA 
               
               
                     60010743-ITX-00001_Contig 
                 #11121 
                 CCTGTGGCCT TGATGGAGAG AAGGGAGACA AGGGAGAAGC TGGTCCCCCA GGCCGCCCCG GGCTGGCAGG ACACAAAGGA 
               
               
                   
                 #11121 
                 CCTGTGGCCT TGATGGAGAG AAGGGAGACA AGGGAGAAGC TGGTCCCCCA GGCCGCCCCG GGCTGGCAGG ACACAAAGGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11201 
                 GAGATGGGGG AGCCTGGTGT GCCGGGCCAG TCGGGGGCCC CTGGCAAGGA GGGCCTGATC GGTCCCAAGG GTGACCGAGG 
               
               
                     60010743-ITX-00001_Contig 
                 #11201 
                 GAGATGGGGG AGCCTGGTGT GCCGGGCCAG TCGGGGGCCC CTGGCAAGGA GGGCCTGATC GGTCCCAAGG GTGACCGAGG 
               
               
                   
                 #11201 
                 GAGATGGGGG AGCCTGGTGT GCCGGGCCAG TCGGGGGCCC CTGGCAAGGA GGGCCTGATC GGTCCCAAGG GTGACCGAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11281 
                 CTTTGACGGG CAGCCAGGCC CCAAGGGTGA CCAGGGCGAG AAAGGGGAGC GGGGAACCCC AGGAATTGGG GGCTTCCCAG 
               
               
                     60010743-ITX-00001_Contig 
                 #11281 
                 CTTTGACGGG CAGCCAGGCC CCAAGGGTGA CCAGGGCGAG AAAGGGGAGC GGGGAACCCC AGGAATTGGG GGCTTCCCAG 
               
               
                   
                 #11281 
                 CTTTGACGGG CAGCCAGGCC CCAAGGGTGA CCAGGGCGAG AAAGGGGAGC GGGGAACCCC AGGAATTGGG GGCTTCCCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11361 
                 GCCCCAGTGG AAATGATGGC TCTGCTGGTC CCCCAGGGCC ACCTGGCAGT GTTGGTCCCA GAGGCCCCGA AGGACTTCAG 
               
               
                     60010743-ITX-00001_Contig 
                 #11361 
                 GCCCCAGTGG AAATGATGGC TCTGCTGGTC CCCCAGGGCC ACCTGGCAGT GTTGGTCCCA GAGGCCCCGA AGGACTTCAG 
               
               
                   
                 #11361 
                 GCCCCAGTGG AAATGATGGC TCTGCTGGTC CCCCAGGGCC ACCTGGCAGT GTTGGTCCCA GAGGCCCCGA AGGACTTCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11441 
                 GGCCAGAAGG GTGAGCGAGG TCCCCCCGGA GAGAGAGTGG TGGGGGCTCC TGGGGTCCCT GGAGCTCCTG GCGAGAGAGG 
               
               
                     60010743-ITX-00001_Contig 
                 #11441 
                 GGCCAGAAGG GTGAGCGAGG TCCCCCCGGA GAGAGAGTGG TGGGGGCTCC TGGGGTCCCT GGAGCTCCTG GCGAGAGAGG 
               
               
                   
                 #11441 
                 GGCCAGAAGG GTGAGCGAGG TCCCCCCGGA GAGAGAGTGG TGGGGGCTCC TGGGGTCCCT GGAGCTCCTG GCGAGAGAGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11521 
                 GGAGCAGGGG CGGCCAGGGC CTGCCGGTCC TCGAGGCGAG AAGGGAGAAG CTGCACTGAC GGAGGATGAC ATCCGGGGCT 
               
               
                     60010743-ITX-00001_Contig 
                 #11521 
                 GGAGCAGGGG CGGCCAGGGC CTGCCGGTCC TCGAGGCGAG AAGGGAGAAG CTGCACTGAC GGAGGATGAC ATCCGGGGCT 
               
               
                   
                 #11521 
                 GGAGCAGGGG CGGCCAGGGC CTGCCGGTCC TCGAGGCGAG AAGGGAGAAG CTGCACTGAC GGAGGATGAC ATCCGGGGCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11601 
                 TTGTGCGCCA AGAGATGAGT CAGCACTGTG CCTGCCAGGG CCAGTTCATC GCATCTGGAT CACGACCCCT CCCTAGTTAT 
               
               
                     60010743-ITX-00001_Contig 
                 #11601 
                 TTGTGCGCCA AGAGATGAGT CAGCACTGTG CCTGCCAGGG CCAGTTCATC GCATCTGGAT CACGACCCCT CCCTAGTTAT 
               
               
                   
                 #11601 
                 TTGTGCGCCA AGAGATGAGT CAGCACTGTG CCTGCCAGGG CCAGTTCATC GCATCTGGAT CACGACCCCT CCCTAGTTAT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11681 
                 GCTGCAGACA CTGCCGGCTC CCAGCTCCAT GCTGTGCCTG TGCTCCGCGT CTCTCATGCA GAGGAGGAAG AGCGGGTACC 
               
               
                     60010743-ITX-00001_Contig 
                 #11681 
                 GCTGCAGACA CTGCCGGCTC CCAGCTCCAT GCTGTGCCTG TGCTCCGCGT CTCTCATGCA GAGGAGGAAG AGCGGGTACC 
               
               
                   
                 #11681 
                 GCTGCAGACA CTGCCGGCTC CCAGCTCCAT GCTGTGCCTG TGCTCCGCGT CTCTCATGCA GAGGAGGAAG AGCGGGTACC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11761 
                 CCCTGAGGAT GATGAGTACT CTGAATACTC CGAGTATTCT GTGGAGGAGT ACCAGGACCC TGAAGCTCCT TGGGATAGTG 
               
               
                     60010743-ITX-00001_Contig 
                 #11761 
                 CCCTGAGGAT GATGAGTACT CTGAATACTC CGAGTATTCT GTGGAGGAGT ACCAGGACCC TGAAGCTCCT TGGGATAGTG 
               
               
                   
                 #11761 
                 CCCTGAGGAT GATGAGTACT CTGAATACTC CGAGTATTCT GTGGAGGAGT ACCAGGACCC TGAAGCTCCT TGGGATAGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11841 
                 ATGACCCCTG TTCCCTGCCA CTGGATGAGG GCTCCTGCAC TGCCTACACC CTGCGCTGGT ACCATCGGGC TGTGACAGGC 
               
               
                     60010743-ITX-00001_Contig 
                 #11841 
                 ATGACCCCTG TTCCCTGCCA CTGGATGAGG GCTCCTGCAC TGCCTACACC CTGCGCTGGT ACCATCGGGC TGTGACAGGC 
               
               
                   
                 #11841 
                 ATGACCCCTG TTCCCTGCCA CTGGATGAGG GCTCCTGCAC TGCCTACACC CTGCGCTGGT ACCATCGGGC TGTGACAGGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #11921 
                 AGCACAGAGG CCTGTCACCC TTTTGTCTAT GGTGGCTGTG GAGGGAATGC CAACCGTTTT GGGACCCGTG AGGCCTGCGA 
               
               
                     60010743-ITX-00001_Contig 
                 #11921 
                 AGCACAGAGG CCTGTCACCC TTTTGTCTAT GGTGGCTGTG GAGGGAATGC CAACCGTTTT GGGACCCGTG AGGCCTGCGA 
               
               
                   
                 #11921 
                 AGCACAGAGG CCTGTCACCC TTTTGTCTAT GGTGGCTGTG GAGGGAATGC CAACCGTTTT GGGACCCGTG AGGCCTGCGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12001 
                 GCGCCGCTGC CCACCCCGGG TGGTCCAGAG CCAGGGGACA GGTACTGCCC AGGACTGATC GATACCGTCG ACCTCGAGAC 
               
               
                     60010743-ITX-00001_Contig 
                 #12001 
                 GCGCCGCTGC CCACCCCGGG TGGTCCAGAG CCAGGGGACA GGTACTGCCC AGGACTGATC GATACCGTCG ACCTCGAGAC 
               
               
                   
                 #12001 
                 GCGCCGCTGC CCACCCCGGG TGGTCCAGAG CCAGGGGACA GGTACTGCCC AGGACTGATC GATACCGTCG ACCTCGAGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12081 
                 CTAGAAAAAC ATGGAGCAAT CACAAGTAGC AATACAGCAG CTACCAATGC TGATTGTGCC TGGCTAGAAG CACAAGAGGA 
               
               
                     60010743-ITX-00001_Contig 
                 #12081 
                 CTAGAAAAAC ATGGAGCAAT CACAAGTAGC AATACAGCAG CTACCAATGC TGATTGTGCC TGGCTAGAAG CACAAGAGGA 
               
               
                   
                 #12081 
                 CTAGAAAAAC ATGGAGCAAT CACAAGTAGC AATACAGCAG CTACCAATGC TGATTGTGCC TGGCTAGAAG CACAAGAGGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12161 
                 GGAGGAGGTG GGTTTTCCAG TCACACCTCA GGTACCTTTA AGACCAATGA CTTACAAGGC AGCTGTAGAT CTTAGCCACT 
               
               
                     60010743-ITX-00001_Contig 
                 #12161 
                 GGAGGAGGTG GGTTTTCCAG TCACACCTCA GGTACCTTTA AGACCAATGA CTTACAAGGC AGCTGTAGAT CTTAGCCACT 
               
               
                   
                 #12161 
                 GGAGGAGGTG GGTTTTCCAG TCACACCTCA GGTACCTTTA AGACCAATGA CTTACAAGGC AGCTGTAGAT CTTAGCCACT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12241 
                 TTTTAAAAGA AAAGGGGGGA CTGGAAGGGC TAATTCACTC CCAACGAAGA CAAGATATCC TTGATCTGTG GATCTACCAC 
               
               
                     60010743-ITX-00001_Contig 
                 #12241 
                 TTTTAAAAGA AAAGGGGGGA CTGGAAGGGC TAATTCACTC CCAACGAAGA CAAGATATCC TTGATCTGTG GATCTACCAC 
               
               
                   
                 #12241 
                 TTTTAAAAGA AAAGGGGGGA CTGGAAGGGC TAATTCACTC CCAACGAAGA CAAGATATCC TTGATCTGTG GATCTACCAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12321 
                 ACACAAGGCT ACTTCCCTGA TTGGCAGAAC TACACACCAG GGCCAGGGAT CAGATATCCA CTGACCTTTG GATGGTGCTA 
               
               
                     60010743-ITX-00001_Contig 
                 #12321 
                 ACACAAGGCT ACTTCCCTGA TTGGCAGAAC TACACACCAG GGCCAGGGAT CAGATATCCA CTGACCTTTG GATGGTGCTA 
               
               
                   
                 #12321 
                 ACACAAGGCT ACTTCCCTGA TTGGCAGAAC TACACACCAG GGCCAGGGAT CAGATATCCA CTGACCTTTG GATGGTGCTA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12401 
                 CAAGCTAGTA CCAGTTGAGC AAGAGAAGGT AGAAGAAGCC AATGAAGGAG AGAACACCCG CTTGTTACAC CCTGTGAGCC 
               
               
                     60010743-ITX-00001_Contig 
                 #12401 
                 CAAGCTAGTA CCAGTTGAGC AAGAGAAGGT AGAAGAAGCC AATGAAGGAG AGAACACCCG CTTGTTACAC CCTGTGAGCC 
               
               
                   
                 #12401 
                 CAAGCTAGTA CCAGTTGAGC AAGAGAAGGT AGAAGAAGCC AATGAAGGAG AGAACACCCG CTTGTTACAC CCTGTGAGCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12481 
                 TGCATGGGAT GGATGACCCG GAGAGAGAAG TATTAGAGTG GAGGTTTGAC AGCCGCCTAG CATTTCATCA CATGGCCCGA 
               
               
                     60010743-ITX-00001_Contig 
                 #12481 
                 TGCATGGGAT GGATGACCCG GAGAGAGAAG TATTAGAGTG GAGGTTTGAC AGCCGCCTAG CATTTCATCA CATGGCCCGA 
               
               
                   
                 #12481 
                 TGCATGGGAT GGATGACCCG GAGAGAGAAG TATTAGAGTG GAGGTTTGAC AGCCGCCTAG CATTTCATCA CATGGCCCGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12561 
                 GAGCTGCATC CGGACTGTAC TGGGTCTCTC TGGTTAGACC AGATCTGAGC CTGGGAGCTC TCTGGCTAAC TAGGGAACCC 
               
               
                     60010743-ITX-00001_Contig 
                 #12561 
                 GAGCTGCATC CGGACTGTAC TGGGTCTCTC TGGTTAGACC AGATCTGAGC CTGGGAGCTC TCTGGCTAAC TAGGGAACCC 
               
               
                   
                 #12561 
                 GAGCTGCATC CGGACTGTAC TGGGTCTCTC TGGTTAGACC AGATCTGAGC CTGGGAGCTC TCTGGCTAAC TAGGGAACCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12641 
                 ACTGCTTAAG CCTCAATAAA GCTTGCCTTG AGTGCTTCAA GTAGTGTGTG CCCGTCTGTT GTGTGACTCT GGTAACTAGA 
               
               
                     60010743-ITX-00001_Contig 
                 #12641 
                 ACTGCTTAAG CCTCAATAAA GCTTGCCTTG AGTGCTTCAA GTAGTGTGTG CCCGTCTGTT GTGTGACTCT GGTAACTAGA 
               
               
                   
                 #12641 
                 ACTGCTTAAG CCTCAATAAA GCTTGCCTTG AGTGCTTCAA GTAGTGTGTG CCCGTCTGTT GTGTGACTCT GGTAACTAGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12721 
                 GATCCCTCAG ACCCTTTTAG TCAGTGTGGA AAATCTCTAG CAGGGCCCGT TTAAACCCGC TGATCAGCCT CGACTGTGCC 
               
               
                     60010743-ITX-00001_Contig 
                 #12721 
                 GATCCCTCAG ACCCTTTTAG TCAGTGTGGA AAATCTCTAG CAGGGCCCGT TTAAACCCGC TGATCAGCCT CGACTGTGCC 
               
               
                   
                 #12721 
                 GATCCCTCAG ACCCTTTTAG TCAGTGTGGA AAATCTCTAG CAGGGCCCGT TTAAACCCGC TGATCAGCCT CGACTGTGCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12801 
                 TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT 
               
               
                     60010743-ITX-00001_Contig 
                 #12801 
                 TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT 
               
               
                   
                 #12801 
                 TTCTAGTTGC CAGCCATCTG TTGTTTGCCC CTCCCCCGTG CCTTCCTTGA CCCTGGAAGG TGCCACTCCC ACTGTCCTTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12881 
                 CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC 
               
               
                     60010743-ITX-00001_Contig 
                 #12881 
                 CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC 
               
               
                   
                 #12881 
                 CCTAATAAAA TGAGGAAATT GCATCGCATT GTCTGAGTAG GTGTCATTCT ATTCTGGGGG GTGGGGTGGG GCAGGACAGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #12961 
                 AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG AAAGAACCAG 
               
               
                     60010743-ITX-00001_Contig 
                 #12961 
                 AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG AAAGAACCAG 
               
               
                   
                 #12961 
                 AAGGGGGAGG ATTGGGAAGA CAATAGCAGG CATGCTGGGG ATGCGGTGGG CTCTATGGCT TCTGAGGCGG AAAGAACCAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13041 
                 CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG CGCAGCGTGA 
               
               
                     60010743-ITX-00001_Contig 
                 #13041 
                 CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG CGCAGCGTGA 
               
               
                   
                 #13041 
                 CTGGGGCTCT AGGGGGTATC CCCACGCGCC CTGTAGCGGC GCATTAAGCG CGGCGGGTGT GGTGGTTACG CGCAGCGTGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13121 
                 CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 
               
               
                     60010743-ITX-00001_Contig 
                 #13121 
                 CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 
               
               
                   
                 #13121 
                 CCGCTACACT TGCCAGCGCC CTAGCGCCCG CTCCTTTCGC TTTCTTCCCT TCCTTTCTCG CCACGTTCGC CGGCTTTCCC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13201 
                 CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA 
               
               
                     60010743-ITX-00001_Contig 
                 #13201 
                 CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA 
               
               
                   
                 #13201 
                 CGTCAAGCTC TAAATCGGGG GCTCCCTTTA GGGTTCCGAT TTAGTGCTTT ACGGCACCTC GACCCCAAAA AACTTGATTA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13281 
                 GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA 
               
               
                     60010743-ITX-00001_Contig 
                 #13281 
                 GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA 
               
               
                   
                 #13281 
                 GGGTGATGGT TCACGTAGTG GGCCATCGCC CTGATAGACG GTTTTTCGCC CTTTGACGTT GGAGTCCACG TTCTTTAATA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13361 
                 GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 
               
               
                     60010743-ITX-00001_Contig 
                 #13361 
                 GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 
               
               
                   
                 #13361 
                 GTGGACTCTT GTTCCAAACT GGAACAACAC TCAACCCTAT CTCGGTCTAT TCTTTTGATT TATAAGGGAT TTTGCCGATT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13441 
                 TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG TCAGTTAGGG 
               
               
                     60010743-ITX-00001_Contig 
                 #13441 
                 TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG TCAGTTAGGG 
               
               
                   
                 #13441 
                 TCGGCCTATT GGTTAAAAAA TGAGCTGATT TAACAAAAAT TTAACGCGAA TTAATTCTGT GGAATGTGTG TCAGTTAGGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13521 
                 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA 
               
               
                     60010743-ITX-00001_Contig 
                 #13521 
                 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA 
               
               
                   
                 #13521 
                 TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA TCTCAATTAG TCAGCAACCA GGTGTGGAAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13601 
                 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC 
               
               
                     60010743-ITX-00001_Contig 
                 #13601 
                 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC 
               
               
                   
                 #13601 
                 GTCCCCAGGC TCCCCAGCAG GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG CCCCTAACTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13681 
                 CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG 
               
               
                     60010743-ITX-00001_Contig 
                 #13681 
                 CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG 
               
               
                   
                 #13681 
                 CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13761 
                 GCCGAGGCCG CCTCTGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT 
               
               
                     60010743-ITX-00001_Contig 
                 #13761 
                 GCCGAGGCCG CCTCTGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT 
               
               
                   
                 #13761 
                 GCCGAGGCCG CCTCTGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC CTAGGCTTTT GCAAAAAGCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13841 
                 CCCGGGAGCT TGTATATCCA TTTTCGGATC TGATCAGCAC GTGTTGACAA TTAATCATCG GCATAGTATA TCGGCATAGT 
               
               
                     60010743-ITX-00001_Contig 
                 #13841 
                 CCCGGGAGCT TGTATATCCA TTTTCGGATC TGATCAGCAC GTGTTGACAA TTAATCATCG GCATAGTATA TCGGCATAGT 
               
               
                   
                 #13841 
                 CCCGGGAGCT TGTATATCCA TTTTCGGATC TGATCAGCAC GTGTTGACAA TTAATCATCG GCATAGTATA TCGGCATAGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #13921 
                 ATAATACGAC AAGGTGAGGA ACTAAACCAT GGCCAAGTTG ACCAGTGCCG TTCCGGTGCT CACCGCGCGC GACGTCGCCG 
               
               
                     60010743-ITX-00001_Contig 
                 #13921 
                 ATAATACGAC AAGGTGAGGA ACTAAACCAT GGCCAAGTTG ACCAGTGCCG TTCCGGTGCT CACCGCGCGC GACGTCGCCG 
               
               
                   
                 #13921 
                 ATAATACGAC AAGGTGAGGA ACTAAACCAT GGCCAAGTTG ACCAGTGCCG TTCCGGTGCT CACCGCGCGC GACGTCGCCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14001 
                 GAGCGGTCGA GTTCTGGACC GACCGGCTCG GGTTCTCCCG GGACTTCGTG GAGGACGACT TCGCCGGTGT GGTCCGGGAC 
               
               
                     60010743-ITX-00001_Contig 
                 #14001 
                 GAGCGGTCGA GTTCTGGACC GACCGGCTCG GGTTCTCCCG GGACTTCGTG GAGGACGACT TCGCCGGTGT GGTCCGGGAC 
               
               
                   
                 #14001 
                 GAGCGGTCGA GTTCTGGACC GACCGGCTCG GGTTCTCCCG GGACTTCGTG GAGGACGACT TCGCCGGTGT GGTCCGGGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14081 
                 GACGTGACCC TGTTCATCAG CGCGGTCCAG GACCAGGTGG TGCCGGACAA CACCCTGGCC TGGGTGTGGG TGCGCGGCCT 
               
               
                     60010743-ITX-00001_Contig 
                 #14081 
                 GACGTGACCC TGTTCATCAG CGCGGTCCAG GACCAGGTGG TGCCGGACAA CACCCTGGCC TGGGTGTGGG TGCGCGGCCT 
               
               
                   
                 #14081 
                 GACGTGACCC TGTTCATCAG CGCGGTCCAG GACCAGGTGG TGCCGGACAA CACCCTGGCC TGGGTGTGGG TGCGCGGCCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14161 
                 GGACGAGCTG TACGCCGAGT GGTCGGAGGT CGTGTCCACG AACTTCCGGG ACGCCTCCGG GCCGGCCATG ACCGAGATCG 
               
               
                     60010743-ITX-00001_Contig 
                 #14161 
                 GGACGAGCTG TACGCCGAGT GGTCGGAGGT CGTGTCCACG AACTTCCGGG ACGCCTCCGG GCCGGCCATG ACCGAGATCG 
               
               
                   
                 #14161 
                 GGACGAGCTG TACGCCGAGT GGTCGGAGGT CGTGTCCACG AACTTCCGGG ACGCCTCCGG GCCGGCCATG ACCGAGATCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14241 
                 GCGAGCAGCC GTGGGGGCGG GAGTTCGCCC TGCGCGACCC GGCCGGCAAC TGCGTGCACT TCGTGGCCGA GGAGCAGGAC 
               
               
                     60010743-ITX-00001_Contig 
                 #14241 
                 GCGAGCAGCC GTGGGGGCGG GAGTTCGCCC TGCGCGACCC GGCCGGCAAC TGCGTGCACT TCGTGGCCGA GGAGCAGGAC 
               
               
                   
                 #14241 
                 GCGAGCAGCC GTGGGGGCGG GAGTTCGCCC TGCGCGACCC GGCCGGCAAC TGCGTGCACT TCGTGGCCGA GGAGCAGGAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14321 
                 TGACACGTGC TACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG 
               
               
                     60010743-ITX-00001_Contig 
                 #14321 
                 TGACACGTGC TACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG 
               
               
                   
                 #14321 
                 TGACACGTGC TACGAGATTT CGATTCCACC GCCGCCTTCT ATGAAAGGTT GGGCTTCGGA ATCGTTTTCC GGGACGCCGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14401 
                 CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC CCAACTTGTT TATTGCAGCT TATAATGGTT 
               
               
                     60010743-ITX-00001_Contig 
                 #14401 
                 CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC CCAACTTGTT TATTGCAGCT TATAATGGTT 
               
               
                   
                 #14401 
                 CTGGATGATC CTCCAGCGCG GGGATCTCAT GCTGGAGTTC TTCGCCCACC CCAACTTGTT TATTGCAGCT TATAATGGTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14481 
                 ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 
               
               
                     60010743-ITX-00001_Contig 
                 #14481 
                 ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 
               
               
                   
                 #14481 
                 ACAAATAAAG CAATAGCATC ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14561 
                 ATCAATGTAT CTTATCATGT CTGTATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT 
               
               
                     60010743-ITX-00001_Contig 
                 #14561 
                 ATCAATGTAT CTTATCATGT CTGTATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT 
               
               
                   
                 #14561 
                 ATCAATGTAT CTTATCATGT CTGTATACCG TCGACCTCTA GCTAGAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14641 
                 GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG 
               
               
                     60010743-ITX-00001_Contig 
                 #14641 
                 GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG 
               
               
                   
                 #14641 
                 GTGAAATTGT TATCCGCTCA CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14721 
                 TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA 
               
               
                     60010743-ITX-00001_Contig 
                 #14721 
                 TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA 
               
               
                   
                 #14721 
                 TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT CGTGCCAGCT GCATTAATGA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14801 
                 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 
               
               
                     60010743-ITX-00001_Contig 
                 #14801 
                 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 
               
               
                   
                 #14801 
                 ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14881 
                 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 
               
               
                     60010743-ITX-00001_Contig 
                 #14881 
                 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 
               
               
                   
                 #14881 
                 CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #14961 
                 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 
               
               
                     60010743-ITX-00001_Contig 
                 #14961 
                 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 
               
               
                   
                 #14961 
                 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15041 
                 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 
               
               
                     60010743-ITX-00001_Contig 
                 #15041 
                 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 
               
               
                   
                 #15041 
                 CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15121 
                 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG 
               
               
                     60010743-ITX-00001_Contig 
                 #15121 
                 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG 
               
               
                   
                 #15121 
                 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15201 
                 GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 
               
               
                     60010743-ITX-00001_Contig 
                 #15201 
                 GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 
               
               
                   
                 #15201 
                 GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15281 
                 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 
               
               
                     60010743-ITX-00001_Contig 
                 #15281 
                 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 
               
               
                   
                 #15281 
                 CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15361 
                 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT 
               
               
                     60010743-ITX-00001_Contig 
                 #15361 
                 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT 
               
               
                   
                 #15361 
                 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15441 
                 GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 
               
               
                     60010743-ITX-00001_Contig 
                 #15441 
                 GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 
               
               
                   
                 #15441 
                 GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15521 
                 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 
               
               
                     60010743-ITX-00001_Contig 
                 #15521 
                 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 
               
               
                   
                 #15521 
                 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15601 
                 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 
               
               
                     60010743-ITX-00001_Contig 
                 #15601 
                 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 
               
               
                   
                 #15601 
                 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15681 
                 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 
               
               
                     60010743-ITX-00001_Contig 
                 #15681 
                 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 
               
               
                   
                 #15681 
                 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15761 
                 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 
               
               
                     60010743-ITX-00001_Contig 
                 #15761 
                 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 
               
               
                   
                 #15761 
                 TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15841 
                 CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC 
               
               
                     60010743-ITX-00001_Contig 
                 #15841 
                 CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC 
               
               
                   
                 #15841 
                 CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #15921 
                 CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 
               
               
                     60010743-ITX-00001_Contig 
                 #15921 
                 CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 
               
               
                   
                 #15921 
                 CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16001 
                 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 
               
               
                     60010743-ITX-00001_Contig 
                 #16001 
                 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 
               
               
                   
                 #16001 
                 GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16081 
                 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 
               
               
                     60010743-ITX-00001_Contig 
                 #16081 
                 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 
               
               
                   
                 #16081 
                 CAACGTTGTT GCCATTGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16161 
                 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 
               
               
                     60010743-ITX-00001_Contig 
                 #16161 
                 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 
               
               
                   
                 #16161 
                 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16241 
                 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT 
               
               
                     60010743-ITX-00001_Contig 
                 #16241 
                 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT 
               
               
                   
                 #16241 
                 AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16321 
                 TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA 
               
               
                     60010743-ITX-00001_Contig 
                 #16321 
                 TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA 
               
               
                   
                 #16321 
                 TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16401 
                 TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 
               
               
                     60010743-ITX-00001_Contig 
                 #16401 
                 TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 
               
               
                   
                 #16401 
                 TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16481 
                 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 
               
               
                     60010743-ITX-00001_Contig 
                 #16481 
                 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 
               
               
                   
                 #16481 
                 AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC 
               
               
                   
               
               
                     IGE308_RefSequence 
                 #16561 
                 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC 
               
               
                     60010743-ITX-00001_Contig 
                 #16561 
                 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC 
               
               
                   
                 #16561 
                 CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC 
               
            
           
         
       
     
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