# Patent

## Title
Tumor therapy with replication competent sindbis viral vectors

## Abstract

Disclosed herein are methods for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising systematically administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector, wherein said vector encodes a suicide gene.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application Ser. No. 61/030,367, filed Feb. 21, 2008, the contents of which are hereby incorporated by reference in their entirety.


The United States government has certain rights to this invention, by virtue of the funding received from grant CA1100687 from the National Institutes of Health.


FIELD OF THE INVENTION
The present invention is directed to methods to treat mammals suffering from tumors and to monitor anticancer therapy using Sindbis viral vectors and pharmaceutical formulations for use in the methods. In particular, the vectors are replication competent Sindbis Viral Vectors and the tumors are solid tumors expressing increased levels of High Affinity Laminin Receptors (LAMR) compared to normal cells of the same lineage.
BACKGROUND OF THE INVENTION
One type of gene therapy of tumors, gene-directed enzyme-prodrug therapy (GDEPT), holds considerable promise, although practical considerations limit its clinical applicability. These include the lack of acceptable noninvasive methods that are adaptable to humans for selective tumor targeting of the therapeutic genetic material. Sindbis virus is an oncolytic, alphavirus that selectively targets tumors through the 67-kDa laminin receptor (LAMR).
Gene therapy targets the genome of tumor cells as a basis for a highly selective and nontoxic anticancer therapy. To enhance selectively and specificity to the killing of cancer cells, several enzyme/prodrug systems—such as carboxylesterase/CPT-11 (1), cytosine deaminase/5-fluoro-cytosine (2), and herpes simplex virus thymidine kinase type 1 (HSVtk)/ganciclovir (GCV) (3,4)—have been developed for gene-directed enzyme-prodrug therapy (GDEPT). In this strategy, tumor cells are transduced with therapeutic genes that encode enzymes for specific conversion/activation of prodrugs, which are toxicologically inert at relatively high doses, into highly toxic metabolites for tumor killing.
In addition to a proper vector system, cancer GDEPT therapy would greatly benefit from a means to noninvasively monitor the GDEPT enzyme activity after vector treatments in vivo. Such capability could improve the Sindbis-based HSVtk/GCV GDEPT in clinical settings by providing important information to address 2 critical questions: (i) Do the vectors systemically target tumor cells and spare normal tissues? (i) Do the tumors have sufficient expression levels of the enzyme for tumor eradication by subsequent prodrug activation? In addition, monitoring during the therapy could facilitate optimizing the dose and dosing schedule of the prodrug to reduce unwanted side affects.
U.S. Pat. No. 7,306,712 discloses that vectors based on Sindbis virus, a blood-borne alphavirus transmitted through mosquito bites, infect tumor cells specifically and systemically throughout the body. The tumor specificity of Sindbis vectors may be mediated by the 67-kDa high-affinity laminin receptor (LAMR), which is over expressed in several types of human tumors. Another advantageous property of Sindbis vectors for cancer therapy is that, without carrying cytotoxic genes, they have been shown to induce apoptosis in mammalian cells. Furthermore, as Sindbis vectors are capable of expressing very high levels of their transduced suicide genes in infected tumor cells, the efficient production of the enzymes for sufficient prodrug conversion is ensured.
U.S. patent application Ser. No. 10/920,030 discloses methods and compositions for detecting cancer cells and monitoring cancer therapy using replication defective Sindbis virus vectors.
U.S. Pat. No. 7,303,798 discloses novel defective Sindbis virus vectors and their use in treating tumors in mammals.
SUMMARY OF THE INVENTION
The present inventors have unexpectedly discovered that replication competent (RC) Sindbis viral vectors have enhanced anti-tumor and cancer therapy monitoring activities when used with tumors which express higher levels of LAMR than normal cells of the same lineage. The RC Sindbis virus vectors are based on the mut-4 replication defective Sindbis virus vector disclosed in the '798 patent.
In one aspect, the present invention provides a method for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising systematically administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector, wherein said vector encodes a suicide gene.
In another aspect, the present invention provides a method for monitoring anti-cancer therapy in a mammal harboring a solid tumor which expresses higher levels of LAMR than normal cells of the same lineage comprising administering to a mammal in need of such treatment a diagnostically effective amount of a Replication Competent (RC) Sindbis virus vector comprising a gene encoding a detectable label, and determining the amount of cancer cells in the body of said mammal, wherein the amount of cancer cells is proportional to the amount of label produced by said cancer cells and said vector encodes a suicide gene.
In a further aspect, the present invention provides a method for identifying cancer cells which expresses higher levels of LAMR than normal cells of the same lineage in the body of a mammal comprising administering to a mammal in need of such treatment a diagnostically effective amount of a mut-4 RC Sindbis virus vector comprising a gene encoding a detectable label, and assaying for said label, wherein said cell is a cancer cell if it expresses said label and said vector encodes a suicide gene.
In a still further aspect, the present invention provides a method for determining the amount of cancer cells which expresses higher levels of LAMR than normal cells of the same lineage in the body of a mammal comprising the steps of (a) administering to a mammal in need of such treatment a diagnostically effective amount of a mut-4 RC Sindbis virus vector comprising a gene encoding a detectable label, and (b) determining the amount of said detectable label, wherein the amount of cancer cells in the body of said mammal is proportional to the amount of said label and said vector encodes a suicide gene.
In yet another aspect, the present invention provides a Replication Competent mut-4 Sindbis virus vector, wherein said vector encodes a suicide gene.
In a still further aspect, the present invention provides a pharmaceutical formulation or dosage form for administration to a mammal suffering from a solid tumor which expresses higher levels of LAMR than normal cells of the same lineage comprising a mut-4 RC Sindbis virus vector and a pharmaceutically acceptable carrier or diluent, wherein said vector further comprises a suicide gene.
These and other aspects of the present invention will be apparent to those of ordinary skill in the art in light of the present description and drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1(A-D). Various Sindbis vector systems. (A) The wild-type Sindbis virus RNA genome has two major groups of genes: non-structural genes on the 5′ side and structural genes on the 3′ side. (B) The conventional two-component replication defective (RD) system contains a replicon RNA for therapeutic gene expression, and a helper RNA to provide structural genes for vector production. (C) A replication-capable (RC) vector system with integrated structural genes. (D) a RC vector system with a suicide gene fused in-frame with the ns3 gene to achieve “controlled” vector propagation and replication in tumor cells. The ns3 gene encodes a protein which is critical for viral replication and survival.
FIG. 2 (A-E). Replication-capable (RC) Sindbis virus vectors show superior tumor targeting and killing over conventional replication-defective (RD) systems. (A and B) Design of conventional RD-Sindbis/Fluc and a prototype RC-Sindbis/Fluc both carrying the firefly luciferase gene as reporter. (C) Bioluminescent imaging of tumor-bearing mice that received two consecutive daily treatments of RD- or RC-Sindbis/Fluc. The first dose was on day 0 and the last dose was on day 1. Subcutaneous BHK tumors were implanted on the right hind limb. (D) Quantitative representation of tumor signals after two consecutive treatments on day 2. (E) Tumor volume measured on day 10.
FIG. 3. (A-D). A suicide gene further enhances the therapeutic efficacy of Sindbis virus vectors. (A) Design of a conventional RD vector capable of expressing HSVtk for prodrug activation. (B) Bioluminescent imaging of ES2/Fluc ovarian cancer cells that express firefly luciferase for monitoring disease progression. SCID mice were inoculated with ES2/Fluc cells on day 0. Daily treatments with RD-Sindbis/HSVtk and GCV started on day 3. The combination of Sindbis virus vectors and the prodrug ganciclovir (GCV) significantly improved anti-tumor efficacy. (C and D) Quantitative representations of the imaging data.
FIG. 4 is a map of the pSP6-R/NS3-HSVtk/Fluc-Mut4 plasmid. pSP6-R/NS3-HSVtk/Fluc-Mut4 is a Sindbis RC vector construction based on pSP6-R and Mut-4, which provides the replicase genes and structural genes respectively.



DETAILED DESCRIPTION OF THE INVENTION
The instant invention takes advantage of the natural affinity of an alphavirus, particularly Sindbis virus, for tumor cells, in particular, for solid tumors that express higher levels of high affinity laminin receptors (alternatively referred to herein as LAMR or HALR), as compared to normal cells of the same lineage. The term “high affinity laminin receptor” or “LAMR” has its ordinary meaning in the art, i.e., the Mr 67,000 laminin receptor that can function as the receptor for Sindbis virus entry into cells (Wang et al., J. Virol. 1992, 66:4992-5001; Strauss et al., Arch. Virol. Suppl. 1994, 9:473-84).
Accordingly, the present invention provides a method for treating a mammal (e.g. human) suffering from a tumor that expresses greater levels of high affinity laminin receptor (LAMR) compared to normal cells of the same lineage. The method comprises administering to a mammal harboring such a tumor an amount of a vector effective to treat the tumor, wherein the vector has a preferential affinity for LAMR and the vector genome comprises a single component.
While not bound by any particular theory, three sets of observations may account for the remarkable anti-tumor efficiency of Sindbis vector-based therapy of the present invention. First, the LAMR can function as the receptor for Sindbis virus entry into cells of most species (Wang et al., J. Virol., 1992, 66:4992-5001; and Strauss et al., Arch. Virol. Suppl., 1994, 9:473-484). Second, it is widely recognized that expression of the LAMR is markedly elevated in many types of cancers (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145). In fact, a significant correlation has been established between the increased expression of Mr 67,000 LAMR and cancers of the breast (Menard et al., 1998, supra; Paolo Viacava et al., J. Pathol., 1997, 182:36-44; Martignone et al., J. Natl. Cancer Inst., 1993, 85:398-402), thyroid (Basolo et al., Clin. Cancer Res., 1996, 2:1777-1780), colon (San Juan et al., J Pathol., 1996, 179:376-380), prostate (Menard S et al., Breast Cancer Res. Treat, 1998, 52:137-145), stomach (de Manzoni et al., Jpn J Clin. Oncol., 1998, 28:534-537), pancreas (Pelosi et al., J Pathol., 1997, 183:62-69), ovary (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145; and van den Brule et al., Eur J Cancer, 1996, 32A: 1598-1602.), melanocytes (Taraboletti et al., J Natl. Cancer Inst., 1993, 85:235-240), lung (Menard et al., Breast Cancer Res. Treat, 1998, 52:137-145), liver (Ozaki et al., Gut, 1998, 43:837-842), endometrium, and uterus (van den Brule et al., Hum Pathol, 1996, 27:1185-1191). Indeed, data on more than 4000 cases of different tumors from diverse organs studied by immunohistochemistry are all concordant with a role for HALR in invasiveness, metastasis, and tumor growth (Menard et al., Breast Cancer Res. Treat., 1998, 52:137-145). Sindbis vectors, which are naturally blood-borne, can easily travel through the circulation and specifically home to and target growing and metastatic tumors expressing increased levels of LAMR. Finally, Sindbis virus is well known to be highly apoptotic for mammalian cells (Levine et al., Nature 1993, 739-742; Jan et al. J Virol., 1999: 10296-10302; Jan et al. J Virol 2000 6425-6432). Cell death begins within a few hours of infection and by 48-96 hours virtually all infected cells are dead (Sawai et al., Mol Genet Metab. 1999, 67:36-42; Griffin et al., Ann. Rev., 1997, Microbiol. 51:565-592).
The Sindbis vectors of the present invention, do not infect normal cells to the same extent in vivo compared to tumor cells. This allows for a differential effect in vector therapy, e.g., infection by Sindbis vectors results in the death of tumor cells leading to tumor elimination without apparent deleterious effects to other tissues and organs of the treated subjects. This phenomenon may be explained by the observation that an increased number of LAMR in tumors versus normal cells leads to a high number of exposed or unoccupied receptors on tumor cells (Liotta, L. A. Cancer Research, 1986, 46:1-7; Aznavoorian et al., 1992, Molecular Aspects of Tumor Cell Invasion and Metastasis, pp. 1368-1383). For example, it has been demonstrated that breast carcinoma and colon carcinoma tissues contain a higher number of exposed (unoccupied) LAMR compared to benign lesions (Liotta et al., 1985, Exp. Cell Res., 156:117-26; Barsky et al., Breast Cancer Res. Treat., 1984, 4:181-188; Terranova et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 444-448). These excess unoccupied LAMR receptors on tumor cells, which are not found in normal cells, may be available for Sindbis virus binding, infection, and induction of cell death.
The invention advantageously provides a method for treating a mammal suffering from a tumor, in which the cells of the tumor express greater levels of LAMR compared to normal cells of the same lineage. The different levels of LAMRs result in target-mediated delivery, i.e., preferential binding of vectors of the invention to tumor cells. “Greater levels” of expression generally refer herein to levels that are expressed by tumor cells (as compared to non-tumor cells) and result in such preferential binding, e.g., at least a 3-fold greater binding, preferably at least a 30-fold greater binding and, most preferably at least a 300-fold greater binding. The increased level of expression in tumor cells can be evaluated on an absolute scale, i.e., relative to any other LAMR expressing non-tumor cells described, or on a relative scale, i.e., relative to the level expressed by untransformed cells in the same lineage as the transformed cancer cells (e.g., melanocytes in the case of melanoma; hepatocytes in the case of hepatic carcinoma; ovarian endothelial cells in the case of ovarian adenocarcinoma, renal endothelial or epithelial cells in the case of renal carcinoma).
As used herein, the term “infectious”, “replication competent” or “replication capable”, when used to describe a Sindbis virus vector RNA molecule, means an RNA molecule which is self-replicating and provides for transcription in a host cell. The term “replication”, when used in conjunction with a Sindbis virus genomic RNA vector RNA molecule means production of full-length equivalents of (+)-strand RNA using (−)-strand RNA as a template.
As used herein, the term “tumor” refers to a malignant tissue comprising transformed cells that grow uncontrollably. Tumors include leukemias, lymphomas, myelomas, plasmacytomas, and the like; and solid tumors. Examples of solid tumors that can be treated according to the invention include sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, epidermoid carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, neuroglioma, and retinoblastoma. As noted above, the method of the invention depends on expression of LAMRs by cells of the tumor targeted for treatment.
The term “about” or “approximately” usually means within an acceptable error range for the type of value and method of measurement. For example, it can mean within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
The phrase “pharmaceutically acceptable”, as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.
The term “therapeutically effective” when applied to a dose or an amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a mammal in need thereof As used herein with respect to viral vectors of the invention, the term “therapeutically effective amount/dose” refers to the amount/dose of a vector or pharmaceutical composition containing the vector that is sufficient to produce an effective anti-tumor response upon administration to a mammal.
As disclosed in Ser. No. 10/920,030 the present inventors previously discovered and devised methods for detecting tumor cells and monitoring cancer therapy. The methods are based on the natural preference of Sindbis virus to infect human cancer cells that express higher level of 37/67-KDa laminin receptor (LAMR) than normal, non-cancerous cells.
Various Sindbis viral vectors for cancer gene therapy have been designed based on the RNA genome of wild-type virus (FIG. 1A). The wild-type genome contains two major parts. The first part, located on the 5′ side of the genome, carries all of the non-structural genes (ns1, ns2, ns3, and ns4) and a packaging signal in the ns1 region. The expression of the ns1-4 genes leads to synthesis of the non-structural proteins nsp 1-4 that form the viral replicase. The replicase is necessary for expression of the structural genes in the second part of the genome at 3′ side for virus formation. In order to do so, the replicase specifically recognizes a short stretch of sequences between the non-structural and structural genes, called the sub-genomic promoter (PSG). One structural protein, the capsid protein, specifically recognizes the packaging signal at the ns1 region and picks it up to form viral particles with the rest of the structural proteins.
FIG. 1B depicts the conventional design of a replication defective Sindbis viral vector that is capable of efficient delivery of therapeutic genes while being unable to propagate and generate more vector particles. This particular system has two components. Replicon RNA contains all of the non-structural genes, while the structural genes are replaced with the therapeutic gene to be delivered. In order to generate vector particles, the structural genes are provided in trans using a helper RNA, the second component of this system. Unlike replicon RNA, the helper RNA contains the non-structural genes and lacks the packaging signal. Therefore, the produced vector particles only carry replicon RNA with the gene of interest.
One major advantage of such conventional dual-component vector systems is that the produced vector particle is replication-defective (RD) and is safer for clinical use. However, this advantage may become a significant drawback for cancer genes therapy. The goal of cancer gene therapy is to infect the majority of tumor cells and deliver the therapeutic genes for tumor detection or eradication. To achieve this goal using a replication-defective system may require a repetitive treatment regime and high doses of vectors. In some cases, such high doses may not be easily obtained using a replication defective system.
On the other hand, a Sindbis vector system that is capable of “controlled” replication and propagation is of great interest for cancer gene therapy. Such a replication-capable (RC) system should comprise a single-component to ensure efficient propagation of the vector in the tumor. That is, the system does not require a helper component for replication. One major benefit of such a system is that fewer treatments would be required and a lower dose should be sufficient to achieve successful therapeutic outcomes while retaining the same tumor targeting capability as RD vectors. Preferably, a safety mechanism is incorporated in the vector to ensure that the vector is eliminated in order to prevent unwanted toxicity, if any, associated with the propagation of the vector.
The present invention provides a replication-capable (RC) Sindbis viral vector (FIG. 1C) which was tested to see if a single-component system performed better than the conventional dual-component system. Instead of using a separate helper RNA vector to carry the structural genes, the structural genes are directly inserted into a conventional replicon vector, along with a dedicated sub-genomic promoter following the therapeutic gene. The dedicated second promoter guarantees that the efficient expression of the structural genes for high-level vector production occurs. A simple dual-promoter RC system was first developed in order to study the function of the sub-genomic promoter in mammalian cells (5,6). This type of RC system was later used to deliver antigen encoding genes for vaccination purposes (7-9). Since Sindbis virus infects mosquito cells, a simple RC system was also used to study Sindbis virus spreading in mosquitoes (10). In light of the present inventors' discovery that RD Sindbis vectors can target tumors in living animals, a single-component RC Sindbis vector has been used to detect/treat tumors in mouse tumor models (11). However, instead of using a dedicated sub-genomic promoter, the prior art RC system used a cleavable component to release the reporter protein from the structural proteins and, therefore, significantly reduced the titer of vector production (12).
FIG. 2 shows the results of an experiment comparing the anti-tumor activity of replication defective (RD, FIG. 2A) and replication competent (RC, FIG. 2B) Sindbis virus vectors. In this set of experiments, a subcutaneously induced BHK tumor model (on the right hind limb of SCID mice) was used to test and compare these two vector systems. In order to evaluate specific tumor targeting and elimination in this model, both vector systems carry a firefly luciferase gene as a reporter. The bioluminescent signals generated in the tumors can be easily detected and analyzed using the IVISTM imaging system. Mice received only two consecutive treatments of RD or RC vectors via systemic (intravenous) injections on a day 0 and day 1. No further treatment was administrated. FIG. 2C depicts the imaging result on day 2 and FIG. 2D quantitatively shows the level of bioluminescent signals in tumors that directly reflects vector infection level. The data indicate that the RC vector system has about a 30 fold increase in infectivity compared to the conventional RD vector system. Higher infectivity is also reflected in enhanced tumor killing as evidenced by tumor size reduction on day 10 (FIG. 2E).
These data provide proof-of-concept results in support of the use of a RC Sindbis vector system for cancer gene therapy. The capability of the RC vector to propagate and spread to the tumor dramatically enhances the ability of Sindbis vectors to target and kill cancer cells. The data also show that the same level of tumor detection can be achieved using a lower range of effective doses of the RC vector compared to conventional RD vectors. In the example depicted in FIG. 2, 106 RD or RC vector particles were intravenously administered into mice. The signals from tumors treated with the RC vectors were about 30 fold higher than the RD signals. Therefore, the same imaging intensity in tumors was achieved using a lower dose (104 to 105) of RC vectors. Interestingly, no toxicity was associated with this prototype RC vector administered at 106 level and no bioluminescent signals, except in residual tumors, were observed in animals up to 20 days after vector injections, suggesting that using the same level of RC vector, as high as 106 per dose, should not cause side effects. The higher dose is useful not only for detecting small lesions but also for better tumor killing and therapeutic effects. Since humans are about 1000 times the body weight of mice, the expected range of effective amounts for humans will range between about 107 and about 1010 particles per dose. The lower range (107-108) is sufficient to detect tumor masses and higher range (108-1010) are better for tumor eradication.
For use in the present invention, the RC Sindbis virus vectors can be produced as described in U.S. Pat. Nos. 7,306,712 and 7,303,798 and in the example below. This involves the described in vitro transcription/electroporation method.
However, as mentioned above, a safety mechanism significantly reduces the risk, if any, of toxicity by controlling the propagation of the RC vector system. In a preferred embodiment, a “suicide gene” is incorporated into one of the Sindbis virus non-structural genes that are essential for viral propagation and survival. FIG. 1C depicts the concept of such a design. A suicide gene, which encodes an enzyme capable of activating inert prodrugs into cytotoxic metabolites, is fused in frame with the ns3 gene to ensure co-expression with non-structural protein 3, an essential component of viral replicase. A particular region of the ns3 gene has been shown to be suitable for fusion without compromising the function of nsp3. Therefore, by such design the RC vector is genetically tagged with this safety mechanism which can shut-off vector propagation by killing the vector producing cell during or after the treatment regime.
In addition to serving as a safety feature, the fused suicide gene provides another advantage. The tumor cells that are selectively infected by the vector are more susceptible and sensitive to the prodrug treatment, since they would not only face the killing imposed by Sindbis infection, but also are exposed to toxic metabolites as a result of prodrug activation. In this regard, it has been discovered that activated toxic metabolites can passively diffuse to neighboring uninfected tumor cells to further enhance tumor killing. This is called a “bystander effect”. The bystander effect plays an important role in the eradication of surrounding untransduced (uninfected) tumor cells. This is caused by transmission of the activated prodrug from the transduced tumor cells (which may be only a small fraction of total tumor mass) to uninfected tumor cells. In the HSVtk/GCV system, the activated GCV is not membrane permeable because of its highly charged phosphate groups. However, it can be transferred to uninfected cells via the gap junctions or through the exchange of apoptotic vesicles that kill the surrounding untransduced tumor cells (14).
Several suicide genes and their appropriate prodrugs are available and suitable for use with the Sindbis virus vector in this embodiment. For example, as disclosed herein, a conventional Sindbis virus vector carrying a thymidine kinase gene isolated from herpes simplex virus (HSVtk) significantly enhanced tumor killing (FIG. 3A) (Horsburgh B C et al, Recurrent acyclovir-resistant herpes simplex in an immunocompromised patient; can strain differences compensate for loss of thymidine kinase in pathogenesis? J. Infect. Dis., 178 (3), 618-625, 1998). A specific prodrug, ganciclovir (GCV), has been developed to target HSVtk, and has been clinically approved for treatment of cytomegalovirus and herpes simplex virus infection in humans. (Ganciclovir, GCV, is marketed under the trade name CYTOVENETM by Roche Laboratories Inc.)
Additional examples of suicide genes are thymidine kinase of Varicella Zoster virus (VZV-tk) (disclosed in Lacey S F et al, Analysis of mutations in the thymidine kinase genes of drug-resistant varicella-zoster virus populations using the polymerase chain reaction, J. Gen. Virol. 72 (PT 3), 623-630, 1991) and the bacterial gene cytosine deaminase (Perna N T et al, Genome sequence of enterohaemorrhagic Escherichia coli O157:H7, Nature 409 (6819), 529-533, 2001).
The prodrugs useful in the methods of the present invention are any that can be converted to a toxic product, i.e., toxic to tumor cells. A preferred prodrug is ganciclovir, which is converted in vivo to a toxic compound by HSV-tk (Chen et al., Cancer Res. 1996, 56: 3758-3762). Other representative examples of prodrugs include acyclovir, FIAU [1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil] (FIALURIDINETM, Moravek Biochemicals and Radiochemicals), 6-methoxypurine arabinoside (converted by VZV-tk), and 5-fluorocytosine (converted by cytosine deaminise) (5-fluorocytosine, Roche).
Prodrugs, may be readily administered to patients by physicians having ordinary skill in the art. Using methods known in the field, such physicians would also be able to determine the most appropriate dose and route for the administration of the prodrug. For example, ganciclovir is preferably administered systemically (e.g. orally or parenterally) in a dose of about 1-20 mg/day/kg body weight; acyclovir is administered in a dose of about 1-100 mg/day/kg body weight, and FIAU is administered in a dose of about 1-50 mg/day/kg body weight.
In the example below, SCID mice were intraperitoneally implanted with ES2 human ovarian cancer cells. In order to track and monitor disease progression, the ES2 cells were genetically engineered to express a firefly luciferase gene for bioluminescent imaging. Therefore, the bioluminescent signal intensity is proportional to the tumor load in these animals (FIG. 3B). Tumor-bearing mice were either mock treated or received daily treatments with a conventional RD-Sindbis/HSVtk vector. Some mice also received daily GCV treatments to determine if the prodrug enhanced tumor killing in conjunction with Sindbis vector treatment. Quantitative analysis indicated that, without HSVtk expression, unactivated GCV confers no therapeutic effect on tumor loading (FIG. 3C). On the contrary, GCV dramatically enhanced the Sindbis HSVtk vector treatments and suppressed tumor growth (FIG. 3D).
In an alternate embodiment, the vectors of the present invention can be used to detect cancer cells and monitor anti-cancer therapy. Previously, the present inventors used an optical bioluminescence imaging system and RD Sindbis virus vectors to detect tumor-specific targeting of Sindbis virus vectors in small animals (15, 16 and Ser. No. 10/920,030). The advantages of bioluminescent imaging include short imaging time, low costs, and ease of use. However, optical imaging methods suffer from very substantial attenuation of the light signal and, thus, are not amenable to applications in large animals and in patients. Recent advances in optical and radionuclide imaging technology provide several methods for non-invasive monitoring of marker gene expression in living animals. On the other hand, radionuclide imaging methods such as g-camera, SPECT, and PET have excellent depth sensitivity and can detect accumulation of gene expression within the transfected tumors anywhere in the body and on the basis of gene expression imaging (17,18,19). A major advantage of PET is the ability to generate quantitative high spatial resolution, 3-dimensional images. When combined with other forms of tomographic imaging, such as CT or MRI, fusion images of functional and anatomic data provides more detailed in situ information of marker genes' expression and localization.
As disclosed in copending Ser. No. 10/920,030, the present inventors have discovered that imaging can be translated into photon counts produced by the detectable label delivered to cancer cells and that these are proportional to the amount of tumor cells that remain alive. Therefore, the present invention can be used to monitor anti-cancer therapy as follows. Patients can be administered a diagnostically-effective amount of the RC Sindbis vector of the present invention comprising a detectable label before the onset of treatment, and this value can be compared to one obtained upon administration of a diagnostically effective amount of a Sindbis virus comprising a detectable label after therapy has been completed. In this way, it is possible to determine the extent of tumor kill. Since only living tumor cells would contain the label, therapy would continue only until a minimal amount of label is detected.
Since Sindbis virus vectors are gene transfer vectors, the cancer cells are labeled using genetic markers incorporated into the RC Sindbis virus vectors. In this embodiment, the genes useful for live tumor monitoring or labeling include but are not limited to the Green Fluorescence Protein (GFP) gene, [Cormack, B. P. et al. (1966) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173:33-38] the Firefly luciferase (Fluc) gene, [de Wet, J. R., et al. (1987) Firefly luciferase gene: structure and expression in mammalian cells Mol. Cell Biol. 7 (2), 725-737], the Renilla luciferase (Rluc) gene [Lorenz, W. W. et al. (1991) Isolation and expression of a cDNA encoding Renilla reinformis luciferase, Proc. Natl. Acad. Sci. U.S.A. 88 (10), 4438-4442] and the dopamine-2 receptor (D2R) gene. The use of the D2R gene as a reporter gene in living animals is disclosed in MacLaren et al. (Gene Therapy 6:785-791 (1999)) and Yaghoubi et al. (Gene Therapy 8:1072-1080 (2001)) These genes can be incorporated into the Sindbis virus vectors of the present invention using techniques well known to those of ordinary skill in the art, as described in Bredenbeek P. J. et al. (1993) (Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs, J. Virol.; 67(11):6439-46.)
Cells expressing the genetic markers of the present invention can be identified as follows: for the HSV-tk gene, the subject can be administered radiolabeled 9-[(4[18F]fluro-3-hydroxymethylbutyl)guanine (FHBG), administered intravenously, about 6000 μCi/Kg body weight of the recipient, (commercially available from PET Imaging Science Center, U. of South California). Expression of HSV-tk activity in tumor cells results in the accumulation of radiolabeled FHBG and can be monitored by Positron Emission Tomography (PET). In vivo GFP expressing tumor cells can be monitored by fluoresence microscopic examination of tissue sections. Tissue sections of Fluc or Rluc expressing tumor cells can be monitored by Cooled Charge-Coupled Device (CCD) cameras in vivo (commercially available from Xenogen Corp., Alamenda, Calif.). D2R activity can be identified by administering 3-(2-[18F]fluoroethyl)spiperone ([18F]FESP) and monitored by PET.
A subject to whom the diagnostic compound of the present invention has been administered as an effective diagnostic monitor for a disease or disorder is preferably a human, but can be any animal, including a laboratory animal in the context of a clinical trial or screening or activity experiment. Thus, as can be readily appreciated by those of ordinary skill in the art, the methods and compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals, such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., avian species, such as chickens, turkeys, songbirds, etc., ire., for veterinary medical use.
In summary, the single-component RC Sindbis vector system of the present invention dramatically enhances the tumor targeting, monitoring and killing capability of replication-capable Sindbis vectors, and incorporation of suicide genes provides an additional layer of protection to achieve “controlled” propagation in tumors and enhances tumor cell killing by RC Sindbis virus vectors.
In a preferred embodiment, the RC vectors are derived from the RD mut-4 vector disclosed in U.S. Pat. No. 7,303,798. The mut-4 vector is similar to the SinRep5 system (Invitrogen Corp.), except for changes in the amino acid sequences in the E2 protein. Since this protein is directly involved in vector binding and targeting to tumor cells, it is expected that RC vectors derived from the mut-4 vector will have the same improved binding capability as the RD vectors.
The construction of the RC mut-4 vector containing the HSV-tk gene is shown in Paper Example 1 below.
The present invention also provides pharmaceutical formulations or dosage forms for administration to mammals.
When formulated in a pharmaceutical composition, the vectors of the present invention can be admixed with a pharmaceutically acceptable carrier or excipient. The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that are “generally regarded as safe”, e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicles with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including but not limited to one or more of a binder (for compressed pills), an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
The preferred route of administration of the vectors of the present invention, for treatment and monitoring, is parenteral and most preferably systemic. This includes, but is not limited to intravenous, intraperitoneal, intra-arteriole, intramuscular, intradermal, subcutaneous, intranasal and oral. These routes of administration will permit homing of the vector to tumor cells wherein, only Sindbis virus is a blood-borne virus. Therefore, gene therapy vectors based on this virus have an advantage over other viral vectors that are not adapted to travel in the bloodstream. This property is largely responsible for the observation that systemic administration of Sindbis viral vectors by i.p. or i.v. injections, target and infect only tumors expressing greater amounts of LAMR than normal cells of the same lineage growing s.c., i.p., intrapancreatically, or in the lungs. Thus, the blood-borne nature of Sindbis viral vectors provides them with the capacity to treat malignancies and monitor cancer therapy.
The present invention is described below in examples which are intended to further describe the invention without limiting the scope therapy.
Materials and Methods
Vector Construction
The prototype RC-Sindbis/Fluc vector was constructed using the pSinRep5/Fluc plasmid as a backbone. In order to make the vector replication competent, a DNA segment containing a sub-genomic promoter and the Sindbis viral structural genes was excised from the ptRNA-DHBB plasmid (Invitrogen Corp., Carlsbad, Calif.) using NsiI and BamHI enzymes and then inserted into pSinRep5/Fluc at the StuI site. Therefore the constructed pSinRep5/Fluc-tBB plasmid has two independent sub-genomic promoters to drive expression of firefly luciferase and Sindbis viral structural proteins.
For construction of a RC vector with a suicide gene in the HSVtk gene was fused in-frame with the Ns3 gene at the SpeI site in pSinRep5/Fluc-tBB. The HSVtk gene (from the pORF-HSVtk plasmid, Invivogen, San Diego, Calif.) was inserted at this site to generate the pSinRep5-nsp3-HSVtk/Fluc-tBB plasmid.
Vector Preparation
The RC-Sindbis/Fluc vector was prepared using an in vitro transcription/electroporation method as described U.S. Pat. Nos. 7,306,712 and 7,303,798. The plasmid pSinRep5/Fluc-tBB was linearized using the NotI restriction enzyme. The linearized plasmid DNA was then used as template for in vitro transcription. The in vitro transcription was done in a total volume of 20 μL using a commercially available SP6 in vitro transcription kit (Ambion Inc., Austin, Tex.). Transcribed RNA (20 μL) was then electroporated into 6×106 BHK cells and cultured at 37° C. in a 10 cm dish containing 10 mL of αMEM (Invitrogen Corp.) with 10% FCS. The next day, the culture media was replaced with 9 mL of OptiMEM (Invitrogen). The OptiMEM was then harvested and stored at −80° C.
In Vivo Imaging
BHK tumors were induced in female SCID mice (Taconic, Germantown, N.Y.) by subcutaneous injection of 2 million BHK cells. Ten days later, on day 0, mice were split into two groups. One group of five mice received the first intravenous injection of 106 particles of RD-Sindbis/Fluc, and the other group received the first i.v. injection of 106 particles of RC-Sindbis/Fluc. The next day (day 1), both groups received a second dose (106) of i.v. treatments. Twenty-four hours later (day 2), tumor luminescence signals were measured using the IVIS® spectrum imaging system (Caliper LifeSciences, Hopkinton, Mass.) and tumor specific signals were analyzed using Living Image 3.0 software. Five minutes before imaging, 0.3 mL of 15 mg/mL D-luciferin (Promega, Madison, Wis.) was i.p. injected in order to generate bioluminescent signals. Tumor sizes were measured using calipers and volumes were calculated using the formula: 4π/3×length×width×height.
An ES-2/Fluc ovarian cancer model was used to test if the HSVtk suicide gene enhanced the therapeutic effects of Sindbis vectors. The prodrug GCV (CYTOVENE-IV®, The Roche Laboratories Inc.) enhanced the killing of Sindbis/tk-infected ES-2/Fluc cells in vivo, as determined by the IVIS® system, which is capable of non-invasive detection of bioluminescent signal generated by ES-2/Fluc tumors. SCID mice were inoculated with ES-2/Fluc on day 0. Daily GDEPT treatments involving i.p. injections of RD-Sindbis/tk and GCV (25 mg/Kg of body weight) were started on day 3. The Sindbis/tk −GCV group (n=5) received Sindbis/tk treatments but no GCV. The Sindbis/tk +GCV group (n=5) received both Sindbis/tk and GCV treatments. The Control group (n=5) was neither treated with Sindbis/tk nor GCV. The Control +GCV group (n=5) received no Sindbis/tk but was treated with GCV. Disease progression was monitored and the whole body photon counts were determined using the IVIS® system on days 1, 3, 6, 8, 12 and 14. Representative images of each treatment group are shown in FIG. 2.
In FIG. 2, tumor luciferase signals were higher and tumor volumes were lower using the RC vector compared to the RD vector. The average tumor luciferase signal in RC-treated mice was 43,970,000 photons, which was much higher than the average of 1,776,000 photons in RD-treated animals. The average tumor size of RC-treated tumors was 410 mm3 and for RD-treated tumor the average size was 1609 mm3.
PAPER EXAMPLE 1
The plasmid pSP6-R/NS3-HSVtk/Fluc-Mut4 enclodes a Sindbis virus RC vector construction based on pSP6-R and Mut-4, which provide replicase genes and structural genes respectively. Its sequence is set forth in Appendix A and a map of the plasmid is shown in FIG. 4. In an alternate embodiment, the promoter is T7 (Ambun, Austin, Tex. ??) Its construction is described as follows.
A DNA segment containing a sub-genomic promoter and the Sindbis viral structural genes is excised from the pSP6-Mut4 plasmid (disclosed in U.S. Pat. No. 7,303,798) using NsiI and BamHI enzymes and then inserted into pSP6-R/Fluc at the PmlI site. In addition, the HSVtk gene fragment (from the pORF-HSVtk plasmid (Invivogen, San Diego, Calif.) is inserted at the SpeI site in the ns3 region on pSP6-R/Fluc-tBB to generate the pSP6-R/nsp3-HSVtk/Fluc-tBB plasmid. A map showing the pSP6-R/nsp3-HSVtk/Fluc-tBB plasmid is shown in FIG. 4 and its sequence is set forth in Appendix A below.
In FIG. 4, the location of the genes is set forth below:
Sindbis non-structural genes locations (bp):
















NS1
60-1979 (SEQ. ID NO.: 2)



NS2
1980-4100 (SEQ. ID NO.: 3)



NS3
4101-5261 and 6399-6878 (SEQ. ID NO.: 4)



NS4
6879-8729 (SEQ. ID NO.: 5)



First Psg
8722-8734 (SEQ. ID NO.: 6)



Second Psg
10766-10789 (SEQ. ID NO. 7)



Structural Genes:
10833-14567 (SEQ. ID NO.: 8)










In addition, the vector may also comprise a suicide gene, such as the thymidine kinase (TK) gene located within the NS3 gene (Nucleotide 5262-6398).
REFERENCES


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    2. Austin E A, Huber B E. A first step in the development of gene therapy for colorectal carcinoma: cloning, sequencing, and expression of Escherichia coli cytosine deaminase. Mol Pharmacol. 1993:43:380-387.
    3. Caruso M. Panis Y. Gagandeep S, Houssin D, Salzmann J L, Klatzmann D. Regression of established macroscopic liver metastases after in situ transduction of a suicide gene. Proc Natl Acad Sci U.S.A. 1993; 90:7024-7028.
    4. Sterman D H, Treat J, Litzky L A, et al. Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma. Hum Gene Ther. 1998; 9:1083-1092.
    5. Levis, R., Schlesinger, S. & Huang, H. V. Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription. J Virol 64, 1726-33 (1990).
    6. Raju, R. & Huang, H. V. Analysis of Sindbis virus promoter recognition in vivo, using novel vectors with two subgenomic mRNA promoters. J Virol 65, 2501-10 (1991).
    7. Hahn, C. S., Hahn, Y. S., Braciale, T. J. & Rice, C. M. Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation. Proc Natl Acad Sci U.S.A. 89, 2679-83 (1992).
    8. Pugachev, K. V., Mason, P. W., Shope, R. E. & Frey, T. K. Double-subgenomic Sindbis virus recombinants expressing immunogenic proteins of Japanese encephalitis virus induce significant protection in mice against lethal JEV infection. Virology 212, 587-94 (1995).
    9. Tsuji, M. et al. Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice. J Virol 72, 6907-10 (1998).
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APPENDIX A


    
    
        pS6-R/nsp3-HSVtk/Fluc-Mut4 (SEQ ID NO. I
    
    











    1
ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATTGCACT




TAACTGCCGC ATCATGTGTG ATAACTTAGT TTGTCGGCTG GTTAACGTGA


 


   51
ACCATCACAA TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG



TGGTAGTGTT ACCTCTTCGG TCATCATTTG CATCTGCATC TGGGGGTCTC


 


  101
TCCGTTTGTC GTGCAACTGC AAAAAAGCTT CCCGCAATTT GAGGTAGTAG



AGGCAAACAG CACGTTGACG TTTTTTCGAA GGGCGTTAAA CTCCATCATC


 


  151
CACAGCAGGT CACTCCAAAT GACCATGCTA ATGCCAGAGC ATTTTCGCAT



GTGTCGTCCA GTGAGGTTTA CTGGTACGAT TACGGTCTCG TAAAAGCGTA


 


  201
CTGGCCAGTA AACTAATCGA GCTGGAGGTT CCTACCACAG CGACGATCTT



GACCGGTCAT TTGATTAGCT CGACCTCCAA GGATGGTGTC GCTGCTAGAA


 


  251
GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAG CACCAGTATC



CCTGTATCCG TCGCGTGGCC GAGCATCTTA CAAAAGGCTC GTGGTCATAG


 


  301
ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAA



TAACACAGAC GGGGTACGCA TCAGGTCTTC TGGGCCTGGC GTACTACTTT


 


  351
TATGCCAGTA AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT



ATACGGTCAT TTGACCGCCT TTTTCGCACG TTCTAATGTT TGTTCTTGAA


 


  401
GCATGAGAAG ATTAAGGATC TCCGGACCGT ACTTGATACG CCGGATGCTG



CGTACTCTTC TAATTCCTAG AGGCCTGGCA TGAACTATGC GGCCTACGAC


 


  451
AAACACCATC GCTCTGCTTT CACAACGATG TTACCTGCAA CATGCGTGCC



TTTGTGGTAG CGAGACGAAA GTGTTGCTAC AATGGACGTT GTACGCACGG


 


  501
GAATATTCCG TCATGCAGGA CGTGTATATC AACGCTCCCG GAACTATCTA



CTTATAAGGC AGTACGTCCT GCACATATAG TTGCGAGGGC CTTGATAGAT


 


  551
TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATT GGCTTCGACA



AGTAGTCCGA TACTTTCCGC ACGCCTGGGA CATGACCTAA CCGAAGCTGT


 


  601
CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAAC



GGTGGGTCAA GTACAAGAGC CGATACCGTC CAAGCATGGG ACGCATGTTG


 


  651
ACCAACTGGG CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG



TGGTTGACCC GGCTGCTCTT TCAGGAACTT CGCGCATTGT AGCCTGAAAC


 


  701
CAGCACAAAG CTGAGTGAAG GTAGGACAGG AAAATTGTCG ATAATGAGGA



GTCGTGTTTC GACTCACTTC CATCCTGTCC TTTTAACAGC TATTACTCCT


 


  751
AGAAGGAGTT GAAGCCCGGG TCGCGGGTTT ATTTCTCCGT AGGATCGACA



TCTTCCTCAA CTTCGGGCCC AGCGCCCAAA TAAAGAGGCA TCCTAGCTGT


 


  801
CTTTATCCAG AACACAGAGC CAGCTTGCAG AGCTGGCATC TTCCATCGGT



GAAATAGGTC TTGTGTCTCG GTCGAACGTC TCGACCGTAG AAGGTAGCCA


 


  851
CTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGT GATACAGTGG



CAAGGTGAAC TTACCTTTCG TCAGCATGTG AACGGCGACA CTATGTCACC


 


  901
TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATC



ACTCAACGCT TCCGATGCAT CACTTCTTTT AGTGGTAGTC AGGGCCCTAG


 


  951
ACGGGAGAAA CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT



TGCCCTCTTT GGCACCCTAT GCGCCAATGT GTGTTATCGC TCCCGAAGAA


 


 1001
GCTATGCAAA GTTACTGACA CAGTAAAAGG AGAACGGGTA TCGTTCCCTG



CGATACGTTT CAATGACTGT GTCATTTTCC TCTTGCCCAT AGCAAGGGAC


 


 1051
TGTGCACGTA CATCCCGGCC ACCATATGCG ATCAGATGAC TGGTATAATG



ACACGTGCAT GTAGGGCCGG TGGTATACGC TAGTCTACTG ACCATATTAC


 


 1101
GCCACGGATA TATCACCTGA CGATGCACAA AAACTTCTGG TTGGGCTCAA



CGGTGCCTAT ATAGTGGACT GCTACGTGTT TTTGAAGACC AACCCGAGTT


 


 1151
CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACC AACACCATGC



GGTCGCTTAA CAGTAATTGC CATCCTGATT GTCCTTGTGG TTGTGGTACG


 


 1201
AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAG



TTTTAATGGA AGACGGCTAG TATCGTGTTC CCAAGTCGTT TACCCGATTC


 


 1251
GAGCGCAAGG ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG



CTCGCGTTCC TACTAGAACT ATTGCTCTTT TACGACCCAT GATCTCTTGC


 


 1301
CAAGCTTACG TATGGCTGCT TGTGGGCGTT TCGCACTAAG AAAGTACATT



GTTCGAATGC ATACCGACGA ACACCCGCAA AGCGTGATTC TTTCATGTAA


 


 1351
CGTTTTATCG CCCACCTGGA ACGCAGACCA TCGTAAAAGT CCCAGCCTCT



GCAAAATAGC GGGTGGACCT TGCGTCTGGT AGCATTTTCA GGGTCGGAGA


 


 1401
TTTAGCGCTT TTCCCATGTC GTCCGTATGG ACGACCTCTT TGCCCATGTC



AAATCGCGAA AAGGGTACAG CAGGCATACC TGCTGGAGAA ACGGGTACAG


 


 1451
GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAG GAGGAAAAAC



CGACTCCGTC TTTAACTTTG ACCGTAACGT TGGTTTCTTC CTCCTTTTTG


 


 1501
TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAG



ACGACGTCCA GAGCCTCCTT AATCAGTACC TCCGGTTCCG ACGAAAACTC


 


 1551
GATGCTCAGG AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC



CTACGAGTCC TCCTTCGGTC TCGCCTCTTC GAGGCTCTTC GTGAAGGTGG


 


 1601
ATTAGTGGCA GACAAAGGCA TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG



TAATCACCGT CTGTTTCCGT AGCTCCGTCG GCGTCTTCAA CAGACGCTTC


 


 1651
TGGAGGGGCT CCAGGCGGAC ATCGGAGCAG CATTAGTTGA AACCCCGCGC



ACCTCCCCGA GGTCCGCCTG TAGCCTCGTC GTAATCAACT TTGGGGCGCG


 


 1701
GGTCACGTAA GGATAATACC TCAAGCAAAT GACCGTATGA TCGGACAGTA



CCAGTGCATT CCTATTATGG AGTTCGTTTA CTGGCATACT AGCCTGTCAT


 


 1751
TATCGTTGTC TCGCCAAACT CTGTGCTGAA GAATGCCAAA CTCGCACCAG



ATAGCAACAG AGCGGTTTGA GACACGACTT CTTACGGTTT GAGCGTGGTC


 


 1801
CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCA



GCGTGGGCGA TCGTCTAGTC CAATTCTAGT ATTGTGTGAG GCCTTCTAGT


 


 1851
GGAAGGTACG CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG



CCTTCCATGC GCCAGCTTGG TATGCTGCGA TTTCATGACT ACGGTCGTCC


 


 1901
AGGTGCCGTA CCATGGCCAG AATTCCTAGC ACTGAGTGAG AGCGCCACGT



TCCACGGCAT GGTACCGGTC TTAAGGATCG TGACTCACTC TCGCGGTGCA


 


 1951
TAGTGTACAA CGAAAGAGAG TTTGTGAACC GCAAACTATA CCACATTGCC



ATCACATGTT GCTTTCTCTC AAACACTTGG CGTTTGATAT GGTGTAACGG


 


 2001
ATGCATGGCC CCGCCAAGAA TACAGAAGAG GAGCAGTACA AGGTTACAAA



TACGTACCGG GGCGGTTCTT ATGTCTTCTC CTCGTCATGT TCCAATGTTT


 


 2051
GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGAC AAGAAGCGTT



CCGTCTCGAA CGTCTTTGTC TCATGCACAA ACTGCACCTG TTCTTCGCAA


 


 2101
GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACC



CGCAATTCTT CCTTCTTCGG AGTCCAGACC AGGAGAGCCC TCTTGACTGG


 


 2151
AACCCTCCCT ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC



TTGGGAGGGA TAGTACTCGA TCGAGACCTC CCTGACTTCT GGGCTGGACG


 


 2201
GGTCCCGTAC AAGGTCGAAA CAATAGGAGT GATAGGCACA CCGGGGTCGG



CCAGGGCATG TTCCAGCTTT GTTATCCTCA CTATCCGTGT GGCCCCAGCC


 


 2251
GCAAGTCAGC TATTATCAAG TCAACTGTCA CGGCACGAGA TCTTGTTACC



CGTTCAGTCG ATAATAGTTC AGTTGACAGT GCCGTGCTCT AGAACAATGG


 


 2301
AGCGGAAAGA AAGAAAATTG TCGCGAAATT GAGGCCGACG TGCTAAGACT



TCGCCTTTCT TTCTTTTAAC AGCGCTTTAA CTCCGGCTGC ACGATTCTGA


 


 2351
GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTT ATGCTCAACG



CTCCCCATAC GTCTAATGCA GCTTCTGTCA TCTAAGCCAA TACGAGTTGC


 


 2401
GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGC



CTACGGTGTT TCGGCATCTT CACGACATGC AACTGCTTCG CAAGCGCACG


 


 2451
CACGCAGGAG CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA



GTGCGTCCTC GTGATGAACG GAACTAACGA TAGCAGTCCG GGGCGTTCTT


 


 2501
GGTAGTACTA TGCGGAGACC CCATGCAATG CGGATTCTTC AACATGATGC



CCATCATGAT ACGCCTCTGG GGTACGTTAC GCCTAAGAAG TTGTACTACG


 


 2551
AACTAAAGGT ACATTTCAAT CACCCTGAAA AAGACATATG CACCAAGACA



TTGATTTCCA TGTAAAGTTA GTGGGACTTT TTCTGTATAC GTGGTTCTGT


 


 2601
TTCTACAAGT ATATCTCCCG GCGTTGCACA CAGCCAGTTA CAGCTATTGT



AAGATGTTCA TATAGAGGGC CGCAACGTGT GTCGGTCAAT GTCGATAACA


 


 2651
ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAAC CCGTGCAAGA



TAGCTGTGAC GTAATGCTAC CTTTCTACTT TTGGTGCTTG GGCACGTTCT


 


 2701
AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGAT



TCTTGTAACT TTAGCTATAA TGTCCCCGGT GTTTCGGCTT CGGTCCCCTA


 


 2751
ATCATCCTGA CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA



TAGTAGGACT GTACAAAGGC GCCCACCCAA TTCGTTAACG TTTAGCTGAT


 


 2801
TCCCGGACAT GAAGTAATGA CAGCCGCGGC CTCACAAGGG CTAACCAGAA



AGGGCCTGTA CTTCATTACT GTCGGCGCCG GAGTGTTCCC GATTGGTCTT


 


 2851
AAGGAGTGTA TGCCGTCCGG CAAAAAGTCA ATGAAAACCC ACTGTACGCG



TTCCTCACAT ACGGCAGGCC GTTTTTCAGT TACTTTTGGG TGACATGCGC


 


 2901
ATcACATCAG AGCATGTGAA CGTGTTGCTC ACCCGCACTG AGGACAGGCT



TAGTGTAGTC TCGTACACTT GCACAACGAG TGGGCGTGAC TCCTGTCCGA


 


 2951
AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAG CTCACTAACA



TCACACCTTT TGGAACGTCC CGCTGGGTAC CTAATTCGTC GAGTGATTGT


 


 3001
TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACAC



ATGGATTTCC TTTGAAAGTC CGATGATATC TCCTGACCCT TCGACTTGTG


 


 3051
AAGGGAATAA TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT



TTCCCTTATT AACGACGTTA TTTGTCGGGG TGAGGGGCAC GGTTAGGCAA


 


 3101
CAGCTGCAAG ACCAACGTTT GCTGGGCGAA AGCATTGGAA CCGATACTAG



GTCGACGTTC TGGTTGCAAA CGACCCGCTT TCGTAACCTT GGCTATGATC


 


 3151
CCACGGCCGG TATCGTACTT ACCGGTTGCC AGTGGAGCGA ACTGTTCCCA



GGTGCCGGCC ATAGCATGAA TGGCCAACGG TCACCTCGCT TGACAAGGGT


 


 3201
CAGTTTGCGG ATGACAAACC ACATTCGGCC ATTTACGCCT TAGACGTAAT



GTCAAACGCC TACTGTTTGG TGTAAGCCGG TAAATGCGGA ATCTGCATTA


 


 3251
TTGCATTAAG TTTTTCGGCA TGGACTTGAC AAGCGGACTG TTTTCTAAAC



AACGTAATTC AAAAAGCCGT ACCTGAACTG TTCGCCTGAC AAAAGATTTG


 


 3301
AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCT



TCTCGTAGGG TGATTGCATG GTAGGGCGGC TAAGTCGCTC CGGCCATCGA


 


 3351
CATTGGGACA ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT



GTAACCCTGT TGTCGGGTCC TTGGGCGTTC ATACCCATGC TAGTGCGGTA


 


 3401
TGCCGCCGAA CTCTCCCGTA GATTTCCGGT GTTCCAGCTA GCTGGGAAGG



ACGGCGGCTT GAGAGGGCAT CTAAAGGCCA CAAGGTCGAT CGACCCTTCC


 


 3451
GCACACAACT TGATTTGCAG ACGGGGAGAA CCAGAGTTAT CTCTGCACAG



CGTGTGTTGA ACTAAACGTC TGCCCCTCTT GGTCTCAATA GAGACGTGTC


 


 3501
CATAACCTGG TCCCGGTGAA CCGCAATCTT CCTCACGCCT TAGTCCCCGA



GTATTGGACC AGGGCCACTT GGCGTTAGAA GGAGTGCGGA ATCAGGGGCT


 


 3551
GTACAAGGAG AAGCAACCCG GCCCGGTCGA AAAATTCTTG AACCAGTTCA



CATGTTCCTC TTCGTTGGGC CGGGCCAGCT TTTTAAGAAC TTGGTCAAGT


 


 3601
AACACCACTC AGTACTTGTG GTATCAGAGG AAAAAATTGA AGCTCCCCGT



TTGTGGTGAG TCATGAACAC CATAGTCTCC TTTTTTAACT TCGAGGGGCA


 


 3651
AAGAGAATCG AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA



TTCTCTTAGC TTACCTAGCG GGGCTAACCG TATCGGCCAC GTCTATTCTT


 


 3701
CTACAACCTG GCTTTCGGGT TTCCGCCGCA GGCACGGTAC GACCTGGTGT



GATGTTGGAC CGAAAGCCCA AAGGCGGCGT CCGTGCCATG CTGGACCACA


 


 3751
TCATCAACAT TGGAACTAAA TACAGAAACC ACCACTTTCA GCAGTGCGAA



AGTAGTTGTA ACCTTGATTT ATGTCTTTGG TGGTGAAAGT CGTCACGCTT


 


 3801
GACCATGCGG CGACCTTAAA AACCCTTTCG CGTTCGGCCC TGAATTGCCT



CTGGTACGCC GCTGGAATTT TTGGGAAAGC GCAAGCCGGG ACTTAACGGA


 


 3851
TAACCCAGOA GGCACCCTCG TGGTGAAGTC CTATGGCTAC GCCGACCGCA



ATTGGGTCCT CCGTGGGAGC ACCACTTCAG GATACCGATG CGGCTGGCGT


 


 3901
ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCT



TGTCACTCCT GCATCAGTGG CGAGAACGGT CTTTCAAACA GTCCCACAGA


 


 3951
GCAGCGAGAC CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT



CGTCGCTCTG GTCTAACACA GAGTTCGTTA TGTCTTTACA TGGACTAAAA


 


 4001
CCGACAACTA GACAACAGCC GTACACGGCA ATTCACCCCG CACCATCTGA



GGCTGTTGAT CTGTTGTCGG CATGTGCCGT TAAGTGGGGC GTGGTAGACT


 


 4051
ATTGCGTGAT TTCGTCCGTG TATGAGGGTA CAAGAGATGG AGTTGGAGCC



TAACGCACTA AAGCAGGCAC ATACTCCCAT GTTCTCTACC TCAACCTCGG


 


 4101
GCGCCGTCAT ACCGCACCAA AAGGGAGAAT ATTGCTGACT GTCAAGAGGA



CGCGGCAGTA TGGCGTGGTT TTCCCTCTTA TAACGACTGA CAGTTCTCCT


 


 4151
AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGC GAAGGAGTCT



TCGTCAACAG TTGCGTCGGT TAGGCGACCC ATCTGGTCCG CTTCCTCAGA


 


 4201
GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACG



CGGCACGGTA GATATTTGCA ACCGGCTGGT CAAAATGGCT AAGTCGGTGC


 


 4251
GAGACAGGCA CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA



CTCTGTCCGT GGCGTTCTTA CTGACACACG GATCCTTTCT TTCACTAGGT


 


 4301
CGCGGTCGGC CCTGATTTCC GGAAGCACCC AGAAGCAGAA GCCTTGAAAT



GCGCCAGCCG GGACTAAAGG CCTTCGTGGG TCTTCGTCTT CGGAACTTTA


 


 4351
TGCTACAAAA CGCCTACCAT GCAGTGGCAG ACTTAGTAAA TGAACATAAC



ACGATGTTTT GCGGATGGTA CGTCACCGTC TGAATCATTT ACTTGTATTG


 


 4401
ATCAAGTCTG TCGCCATTCC ACTGCTATCT ACAGGCATTT ACGCAGCCGG



TAGTTCAGAC AGCGGTAAGG TGACGATAGA TGTCCGTAAA TGCGTCGGCC


 


 4451
AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACC GCGCTAGACA



TTTTCTGGCG GAACTTCATA GTGAATTGAC GAACTGTTGG CGCGATCTGT


 


 4501
GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAA



CTTGACTGCG CCTGCATTGG TAGATAACGG ACCTATTCTT CACCTTCCTT


 


 4551
AGAATCGACG CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA



TCTTAGCTGC GCCGTGAGGT TGAATTCCTC AGACATTGTC TCGACTTCCT


 


 4601
TGAAGATATG GAGATCGACG ATGAGTTAGT ATGGATCCAT CCAGACAGTT



ACTTCTATAC CTCTAGCTGC TACTCAATCA TACCTAGGTA GGTCTGTCAA


 


 4651
GCTTGAAGGG AAGAAAGGGA TTCAGTACTA CAAAAGGAAA ATTGTATTCG



CGAACTTCCC TTCTTTCCCT AAGTCATGAT GTTTTCCTTT TAACATAAGC


 


 4701
TACTTCGAAG GCACCAAATT CCATCAAGCA GCAAAAGACA TGGCGGAGAT



ATGAAGCTTC CGTGGTTTAA GGTAGTTCGT CGTTTTCTGT ACCGCCTCTA


 


 4751
AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAA CTGTGTGCCT



TTTCCAGGAC AAGGGATTAC TGGTCCTTTC ATTACTTGTT GACACACGGA


 


 4801
ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGAC



TGTATAACCC ACTCTGGTAC CTTCGTTAGG CGCTTTTCAC GGGCCAGCTG


 


 4851
CATAACCCGT CGTCTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA



GTATTGGGCA GCAGATCGGG CGGGTTTTGC AACGGCACGG AAACGTACAT


 


 4901
TGCCATGACG CCAGAAAGGG TCCACAGACT TAGAAGCAAT AACGTCAAAG



ACGGTACTGC GGTCTTTCCC AGGTGTCTGA ATCTTCGTTA TTGCAGTTTC


 


 4951
AAGTTACAGT ATGCTCCTCC ACCCCCCTTC CTAAGCACAA AATTAAGAAT



TTCAATGTCA TACGAGGAGG TGGGGGGAAG GATTCGTGTT TTAATTCTTA


 


 5001
GTTCAGAAGG TTCAGTGCAC GAAAGTAGTC CTGTTTAATC CGCACACTCC



CAAGTCTTCC AAGTCACGTG CTTTCATCAG GACAAATTAG GCGTGTGAGG


 


 5051
CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAA CAGCCTACCG



GCGTAAGCAA GGGCGGGCAT TCATGTATCT TCACGGTCTT GTCGGATGGC


 


 5101
CTCCTCCTGC ACAGGCCGAG GAGGCCCCCC AAGTTGTAGC GACACCGTCA



GAGGAGGACG TGTCCGGCTC CTCCGGGGGC TTCAACATCG CTGTGGCAGT


 


 5151
CCATCTACAG CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA



GGTAGATGTC GACTATTGTG GAGCGAACTA CAGTGTCTGT AGAGTGACCT


 


 5201
TATGGATGAC AGTAGCGAAG GCTCACTTTT TTCGAGCTTT AGCGGATCGG



ATACCTACTG TCATCGCTTC CGAGTGAAAA AAGCTCGAAA TCGCCTAGCC


 


 5251
ACAACTCTAT TACTAGTGCC TCGTACCCCG GCCATCAACA CGCGTCTGCG



TGTTGAGATA ATGATCACGG AGCATGGGGC CGGTAGTTGT GCGCAGACGC


 


 5301
TTCGACCAGG CTGCGCGTTC TCGCGGCCAT AGCAACCGAC GTACGGCGTT



AAGCTGGTCC GACGCGCAAG AGCGCCGGTA TCGTTGGCTG CATGCCGCAA


 


 5351
GCGCCCTCGC CGGCAGCAAG AAGCCACGGA AGTCCGCCCG GAGCAGAAAA



CGCGGGAGCG GCCGTCGTTC TTCGGTGCCT TCAGGCGGGC CTCGTCTTTT


 


 5401
TGCCCACGCT ACTGCGGGTT TATATAGACG GTCCCCACGG GATGGGGAAA



ACGGGTGCGA TGACGCCCAA ATATATCTGC CAGGGGTGCC CTACCCCTTT


 


 5451
ACCACCACCA CGCAACTGCT GGTGGCCCTG GGTTCGCGCG ACGATATCGT



TGGTGGTGGT GCGTTGACGA CCACCGGGAC CCAAGCGCGC TGCTATAGCA


 


 5501
CTACGTACCC GAGCCGATGA CTTACTGGCG GGTGCTGGGG GCTTCCGAGA



GATGCATGGG CTCGGCTACT GAATGACCGC CCACGACCCC CGAAGGCTCT


 


 5551
CAATCGCGAA CATCTACACC ACACAACACC GCCTCGACCA GGGTGAGATA



GTTAGCGCTT GTAGATGTGG TGTGTTGTGG CGGAGCTGGT CCCACTCTAT


 


 5601
TCGGCCGGGG ACGCGGCGGT GGTAATGACA AGCGCCCAGA TAACAATGGG



AGCCGGCCCC TGCGCCGCCA CCATTACTGT TCGCGGGTCT ATTGTTACCC


 


 5651
CATGCCTTAT GCCGTGACCG ACGCCGTTCT GGCTCCTCAT ATCGGGGGGG



GTACGGAATA CGGCACTGGC TGCGGCAAGA CCGAGGAGTA TAGCCCCCCC


 


 5701
AGGCTGGGAG CTCACATGCC CCGCCCCCGG CCCTCACCCT CATCTTCGAC



TCCGACCCTC GAGTGTACGG GGCGGGGGCC GGGAGTGGGA GTAGAAGCTG


 


 5751
CGCCATCCCA TCGCCGCCCT CCTGTGCTAC CCGGCCGCGC GGTACCTTAT



GCGGTAGGGT AGCGGCGGGA GGACACGATG GGCCGGCGCG CCATGGAATA


 


 5801
GGGCAGCATG ACCCCCCAGG CCGTGCTGGC GTTCGTGGCC CTCATCCCGC



CCCGTCGTAC TGGGGGGTCC GGCACGACCG CAAGCACCGG GAGTAGGGCG


 


 5851
CGACCTTGCC CGGCACCAACATCGTGCTTG GGGCCCTTCC GGAGGACAGA



GCTGGAACGG GCCGTGGTTG TAGCACGAAC CCCGGGAAGG CCTCCTGTCT


 


 5901
CACATCGACC GCCTGGCCAA ACGCCAGCGC CCCGGCGAGC GGCTGGACCT



GTGTAGCTGG CGGACCGGTT TGCGGTCGCG GGGCCGCTCG CCGACCTGGA


 


 5951
GGCTATGCTG GCTGCGATTC GCCGCGTTTA CGGGCTACTT GCCAATACGG



CCGATACGAC CGACGCTAAG CGGCGCAAAT GCCCGATGAA CGGTTATGCC


 


 6001
TGCGGTATCT GCAGTGCGGC GGGTCGTGGC GGGAGGACTG GGGACAGCTT



ACGCCATAGA CGTCACGCCG CCCAGCACCG CCCTCCTGAC CCCTGTCGAA


 


 6051
TCGGGGACGG CCGTGCCGCC CCAGGGTGCC GAGCCCCAGA GCAACGCGGG



AGCCCCTGCC GGCACGGCGG GGTCCCACGG CTCGGGGTCT CGTTGCGCCC


 


 6101
CCCACGACCC CATATCGGGG ACACGTTATT TACCCTGTTT CGGGCCCCCG



GGGTGCTGGG GTATAGCCCC TGTGCAATAA ATGGGACAAA GCCCGGGGGC


 


 6151
AGTTGCTGGC CCCCAACGGC GACCTGTATA ACGTGTTTGC CTGGGCCTTG



TCAACGACCG GGGGTTGCCG CTGGACATAT TGCACAAACG GACCCGGAAC


 


 6201
GACGTCTTGG CCAAACGCCT CCGTTCCATG CACGTCTTTA TCCTGGATTA



CTGCAGAACC GGTTTGCGGA GGCAAGGTAC GTGCAGAAAT AGGACCTAAT


 


 6251
CGACCAATCG CCCGCCGGCT GCCGGGACGC CCTGCTGCAA CTTACCTCCG



GCTGGTTAGC GGGCGGCCGA CGGCCCTGCG GGACGACGTT GAATGGAGGC


 


 6301
GGATGGTCCA GACCCACGTC ACCACCCCCG GCTCCATACC GACGATATGC



CCTACCAGGT CTGGGTGCAG TGGTGGGGGC CGAGGTATGG CTGCTATACG


 


 6351
GACCTGGCGC GCACGTTTGC CCGGGAGATG GGGGAGGCTA ACACTAGTAT



CTGGACCGCG CGTGCAAACG GGCCCTCTAC CCCCTCCGAT TGTGATCATA


 


 6401
GGACAGTTGG TCGTCAGGAC CTAGTTCACT AGAGATAGTA GACCGAAGGC



CCTGTCAACC AGCAGTCCTG GATCAAGTGA TCTCTATCAT CTGGCTTCCG


 


 6451
AGGTGGTGGT GGCTGACGTT CATGCCGTCC AAGAGCCTGC CCCTATTCCA



TCCACCACCA CCGACTGCAA GTACGGCAGG TTCTCGGACG GGGATAAGGT


 


 6501
CCGCCAAGGC TAAAGAAGAT GGCCCGCCTG GCAGCGGCAA GAAAAGAGCC



GGCGGTTCCG ATTTCTTCTA CCGGGCGGAC CGTCGCCGTT CTTTTCTCGG


 


 6551
CACTCCACCG GCAAGCAATA GCTCTGAGTC CCTCCACCTC TCTTTTGGTG



GTGAGGTGGC CGTTCGTTAT CGAGACTCAG GGAGGTGGAG AGAAAACCAC


 


 6601
GGGTATCCAT GTCCCTCGGA TCAATTTTCG ACGGAGAGAC GGCCCGCCAG



CCCATAGGTA CAGGGAGCCT AGTTAAAAGC TGCCTCTCTG CCGGGCGGTC


 


 6651
GCAGCGGTAC AACCCCTGGC AACAGGCCCC ACGGATGTGC CTATGTCTTT



CGTCGCCATG TTGGGGACCG TTGTCCGGGG TGCCTACACG GATACAGAAA


 


 6701
CGGATCGTTT TCCGACGGAG AGATTGATGA GCTGAGCCGC AGAGTAACTG



GCCTAGCAAA AGGCTGCCTC TCTAACTACT CGACTCGGCG TCTCATTGAC


 


 6751
AGTCCGAACC CGTCCTGTTT GGATCATTTG AACCGGGCGA AGTGAACTCA



TCAGGCTTGG GCAGGACAAA CCTAGTAAAC TTGGCCCGCT TCACTTGAGT


 


 6801
ATTATATCGT CCCGATCAGC CGTATCTTTT CCACTACGCA AGCAGAGACG



TAATATAGCA GGGCTAGTCG GCATAGAAAA GGTGATGCGT TCGTCTCTGC


 


 6851
TAGACGCAGG AGCAGGAGGA CTGAATACTG ACTAACCGGG GTAGGTGGGT



ATCTGCGTCC TCGTCCTCCT GACTTATGAC TGATTGGCCC CATCCACCCA


 


 6901
ACATATTTTC GACGGACACA GGCCCTGGGC ACTTGCAAAA GAAGTCCGTT



TGTATAAAAG CTGCCTGTGT CCGGGACCCG TGAACGTTTT CTTCAGGCAA


 


 6951
CTGCAGAACC AGCTTACAGA ACCGACCTTG GAGCGCAATG TCCTGGAAAG



GACGTCTTGG TCGAATGTCT TGGCTGGAAC CTCGCGTTAC AGGACCTTTC


 


 7001
AATTCATGCC CCGGTGCTCG ACACGTCGAA AGAGGAACAA CTCAAACTCA



TTAAGTACGG GGCCACGAGC TGTGCAGCTT TCTCCTTGTT GAGTTTGAGT


 


 7051
GGTACCAGAT GATGCCCACC GAAGCCAACA AAAGTAGGTA CCAGTCTCGT



CCATGGTCTA CTACGGGTGG CTTCGGTTGT TTTCATCCAT GGTCAGAGCA


 


 7101
AAAGTAGAAA ATCAGAAAGC CATAACCACT GAGCGACTAC TGTCAGGACT



TTTCATCTTT TAGTCTTTCG GTATTGGTGA CTCGCTGATG ACAGTCCTGA


 


 7151
ACGACTGTAT AACTCTGCCA CAGATCAGCC AGAATGCTAT AAGATCACCT



TGCTGACATA TTGAGACGGT GTCTAGTCGG TCTTACGATA TTCTAGTGGA


 


 7201
ATCCGAAACC ATTGTACTCC AGTAGCGTAC CGGCGAACTA CTCCGATCCA



TAGGCTTTGG TAACATGAGG TCATCGCATG GCCGCTTGAT GAGGCTAGGT


 


 7251
CAGTTCGCTG TAGCTGTCTG TAACAACTAT CTGCATGAGA ACTATCCGAC



GTCAAGCGAC ATCGACAGAC ATTGTTGATA GACGTACTCT TGATAGGCTG


 


 7301
AGTAGCATCT TATCAGATTA CTGACGAGTA CCATGCTTAC TTGGATATGG



TCATCGTAGA ATAGTCTAAT GACTGCTCAT GCTACGAATG AACCTATACC


 


 7351
TAGACGGGAC AGTCGCCTGC CTGGATACTG CAACCTTCTG CCCCGCTAAG



ATCTGCCCTG TCAGCGGACG GACCTATGAC GTTGGAAGAC GGGGCGATTC


 


 7401
CTTAGAAGTT ACCCGAAAAA ACATGAGTAT AGAGCCCCGA ATATCCGCAG



GAATCTTCAA TGGGCTTTTT TGTACTCATA TCTCGGGGCT TATAGGCGTC


 


 7451
TGCGGTTCCA TCAGCGATGC AGAACACGCT ACAAAATGTG CTCATTGCCG



ACGCCAAGGT AGTCGCTACG TCTTGTGCGA TGTTTTACAC GAGTAACGGC


 


 7501
CAACTAAAAG AAATTGCAAC GTCACGCAGA TGCGTGAACT GCCAACACTG



GTTGATTTTC TTTAACGTTG CAGTGCGTCT ACGCACTTGA CGGTTGTGAC


 


 7551
GACTCAGCGA CATTCAATGT CGAATGCTTT CGAAAATATG CATGTAATGA



CTGAGTCGCT GTAAGTTACA GCTTACGAAA GCTTTTATAC GTACATTACT


 


 7601
CGAGTATTGG GAGGAGTTCG CTCGGAAGCC AATTAGGATT ACCACTGAGT



GCTCATAACC CTCCTCAAGC GAGCCTTCGG TTAATCCTAA TGGTGACTCA


 


 7651
TTGTCACCGC ATATGTAGCT AGACTGAAAG GCCCTAAGGC CGCCGCACTA



AACAGTGGCG TATACATCGA TCTGACTTTC CGGGATTCCG GCGGCGTGAT


 


 7701
TTTGCAAAGA CGTATAATTT GGTCCCATTG CAAGAAGTGC CTATGGATAG



AAACGTTTCT GCATATTAAA CCAGGGTAAC GTTCTTCACG GATACCTATC


 


 7751
ATTCGTCATG GACATGAAAA GAGACGTGAA AGTTACACCA GGCACGAAAC



TAAGCAGTAC CTGTACTTTT CTCTGCACTT TCAATGTGGT CCGTGCTTTG


 


 7801
ACACAGAAGA AAGACCGAAA GTACAAGTGA TACAAGCCGC AGAACCCCTG



TGTGTCTTCT TTCTGGCTTT CATGTTCACT ATGTTCGGCG TCTTGGGGAC


 


 7851
GCGACTGCTT ACTTATGCGG GATTCACCGG GAATTAGTGC GTAGGCTTAC



CGCTGACGAA TGAATACGCC CTAAGTGGCC CTTAATCACG CATCCGAATG


 


 7901
GGCCGTCTTG CTTCCAAACA TTCACACGCT TTTTGACATG TCGGCGGAGG



CCGGCAGAAC GAAGGTTTGT AAGTGTGCGA AAAACTGTAC AGCCGCCTCC


 


 7951
ATTTTGATGC AATCATAGCA GAACACTTCA AGCAAGGCGA CCCGGTACTG



TAAAACTACG TTAGTATCGT CTTGTGAAGT TCGTTCCGCT GGGCCATGAC


 


 8001
GAGACGGATA TCGCATGATT CGACAAAAGC CAAGACGACG CTATGGCGTT



CTCTGCCTAT AGCGTAGTAA GCTGTTTTCG GTTCTGCTGC GATACCGCAA


 


 8051
AACCGGTCTG ATGATCTTGG AGGACCTGGG TGTGGATCAA CCACTACTCG



TTGGCCAGAC TACTAGAACC TCCTGGACCC ACACCTAGTT GGTGATGAGC


 


 8101
ACTTGATCGA GTGCGCCTTT GGAGAAATAT CATCCACCCA TCTACCTACG



TGAACTAGCT CACGCGGAAA CCTCTTTATA GTAGGTGGGT AGATGGATGC


 


 8151
GGTACTCGTT TTAAATTCGG GGCGATGATG AAATCCGGAA TGTTCCTCAC



CCATGAGCAA AATTTAAGCC CCGCTACTAC TTTAGGCCTT ACAAGGAGTG


 


 8201
ACTTTTTGTC AACACAGTTT TGAATGTCGT TATCGCCAGC AGAGTACTAG



TGAAAAACAG TTGTGTCAAA ACTTACAGCA ATAGCGGTCG TCTCATGATC


 


 8251
AAGAGCGGCT TAAAACGTCC AGATGTGCAG CGTTCATTGG CGACGACAAC



TTCTCGCCGA ATTTTGCAGG TCTACACGTC GCAAGTAACC GCTGCTGTTG


 


 8301
ATCATACATG GAGTAGTATC TGACAAAGAA ATGGCTGAGA GGTGCGCCAC



TAGTATGTAC CTCATCATAG ACTGTTTCTT TACCGACTCT CCACGCGGTG


 


 8351
CTGGCTCAAC ATGGAGGTTA AGATCATCGA CGCAGTCATC GGTGAGAGAC



GACCGAGTTG TACCTCCAAT TCTAGTAGCT GCGTCAGTAG CCACTCTCTG


 


 8401
CACCTTACTT CTGCGGCGGA TTTATCTTGC AAGATTCGGT TACTTCCACA



GTGGAATGAA GACGCCGCCT AAATAGAACG TTCTAAGCCA ATGAAGGTGT


 


 8451
GCGTGCCGCG TGGCGGACCC CCTGAAAAGG CTGTTTAAGT TGGGTAAACC



CGCACGGCGC ACCGCCTGGG GGACTTTTCC GACAAATTCA ACCCATTTGG


 


 8501
GCTCCCAGCC GACGACGAGC AAGACGAAGA CAGAAGACGC GCTCTGCTAG



CGAGGGTCGG CTGCTGCTCG TTCTGCTTCT GTCTTCTGCG CGAGACGATC


 


 8551
ATGAAACAAA GGCGTGGTTT AGAGTAGGTA TAACAGGCAC TTTAGCAGTG



TACTTTGTTT CCGCACCAAA TCTCATCCAT ATTGTCCGTG AAATCGTCAC


 


 8601
GCCGTGACGA CCCGGTATGA GGTAGACAAT ATTACACCTG TCCTACTGGC



CGGCACTGCT GGGCCATACT CCATCTGTTA TAATGTGGAC AGGATGACCG


 


 8651
ATTGAGAACT TTTGCCCAGA GCAAAAGAGC ATTCCAAGCC ATCAGAGGGG



TAACTCTTGA AAACGGGTCT CGTTTTCTCG TAAGGTTCGG TAGTCTCCCC


 


 8701
AAATAAAGCA TCTCTACGGT GGTCCTAAAT AGTCAGCATA GTACATTTCA



TTTATTTCGT AGAGATGCCA CCAGGATTTA TCAGTCGTAT CATGTAAAGT


 


 8751
TCTGACTAAT ACTACAACAC CACCACCTCT AGCCCGGGCT CGAGATCTGC



AGACTGATTA TGATGTTGTG GTGGTGGAGA TCGGGCCCGA GCTCTAGACG


 


 8801
GATCTAAGTA AGCTTGGCAT TCCGGTACTG TTGGTAAAGC CACCATGGAA



CTAGATTCAT TCGAACCGTA AGGCCATGAC AACCATTTCG GTGGTACCTT


 


 8851
GACGCCAAAA ACATAAAGAA AGGCCCGGCG CCATTCTATC CGCTGGAAGA



CTGCGGTTTT TGTATTTCTT TCCGGGCCGC GGTAAGATAG GCGACCTTCT


 


 8901
TGGAACCGCT GGAGAGCAAC TGCATAAGGC TATGAAGAGA TACGCCCTGG



ACCTTGGCGA CCTCTCGTTG ACGTATTCCG ATACTTCTCT ATGCGGGACC


 


 8951
TTCCTGGAAC AATTGCTTTT ACAGATGCAC ATATCGAGGT GGACATCACT



AAGGACCTTG TTAACGAAAA TGTCTACGTG TATAGCTCCA CCTGTAGTGA


 


 9001
TACGCTGAGT ACTTCGAAAT GTCCGTTCGG TTGGCAGAAG CTATGAAACG



ATGCGACTCA TGAAGCTTTA CAGGCAAGCC AACCGTCTTC GATACTTTGC


 


 9051
ATATGGGCTG AATACAAATC ACAGAATCGT CGTATGCAGT GAAAACTCTC



TATACCCGAC TTATGTTTAG TGTCTTAGCA GCATACGTCA CTTTTGAGAG


 


 9101
TTCAATTCTT TATGCCGGTG TTGGGCGCGT TATTTATCGG AGTTGCAGTT



AAGTTAAGAA ATACGGCCAC AACCCGCGCA ATAAATAGCC TCAACGTCAA


 


 9151
GCGCCCGCGA ACGACATTTA TAATGAACGT GAATTGCTCA ACAGTATGGG



CGCGGGCGCT TGCTGTAAAT ATTACTTGCA CTTAACGAGT TGTCATACCC


 


 9201
CATTTCGCAG CCTACCGTGG TGTTCGTTTC CAAAAAGGGG TTGCAAAAAA



GTAAAGCGTC GGATGGCACC ACAAGCAAAG GTTTTTCCCC AACGTTTTTT


 


 9251
TTTTGAACGT GCAAAAAAAG CTCCCAATCA TCCAAAAAAT TATTATCATG



AAAACTTGCA CGTTTTTTTC GAGGGTTAGT AGGTTTTTTA ATAATAGTAC


 


 9301
GATTCTAAAA CGGATTACCA GCGATTTCAG TCGATGTACA CGTTCGTCAC



CTAAGATTTT GCCTAATGGT CCCTAAAGTC AGCTACATGT GCAAGCAGTG


 


 9351
ATCTCATCTA CCTCCCGGTT TTAATGAATA CGATTTTGTG CCAGAGTCCT



TAGAGTAGAT GGAGGGCCAA AATTACTTAT GCTAAAACAC GGTCTCAGGA


 


 9401
TCGATAGGGA CAAGACAATT GCACTGATCA TGAACTCCTC TGGATCTACT



AGCTATCCCT GTTCTGTTAA CGTGACTAGT ACTTGAGGAG ACCTAGATGA


 


 9451
GGTCTGCCTA AAGGTGTCGC TCTGCCTCAT AGAACTGCCT GCGTGAGATT



CCAGACGGAT TTCCACAGCG AGACGGAGTA TCTTGACGGA CGCACTCTAA


 


 9501
CTCGCATGCC AGAGATCCTA TTTTTGGCAA TCAAATCATT CCGGATACTG



GAGCGTACGG TCTCTAGGAT AAAAACCGTT AGTTTAGTAA GGCCTATGAC


 


 9551
CGATTTTAAG TGTTGTTCCA TTCCATCACG GTTTTGGAAT GTTTACTACA



GCTAAAATTC ACAACAAGGT AAGGTAGTGC CAAAACCTTA CAAATGATGT


 


 9601
CTCGGATATT TGATATGTGG ATTTCGAGTC GTCTTAATGT ATAGATTTGA



GAGCCTATAA ACTATACACC TAAAGCTCAG CAGAATTACA TATCTAAACT


 


 9651
AGAAGAGCTG TTTCTGAGGA GCCTTCAGGA TTACAAGATT CAAAGTGCGC



TCTTCTCGAC AAAGACTCCT CGGAAGTCCT AATGTTCTAA GTTTCACGCG


 


 9701
TGCTGGTGCC AACCCTATTC TCCTTCTTCG CCAAAAGCAC TCTGATTGAC



ACGACCACGG TTGGGATAAG AGGAAGAAGC GGTTTTCGTG AGACTAACTG


 


 9751
AAATACGATT TATCTAATTT ACACGAAATT GCTTCTGGTG GCGCTCCCCT



TTTATGCTAA ATAGATTAAA TGTGCTTTAA CGAAGACCAC CGCGAGGGGA


 


 9801
CTCTAAGGAA GTCGGGGAAG CGGTTGCCAA GAGGTTCCAT CTGCCAGGTA



GAGATTCCTT CAGCCCCTTC GCCAACGGTT CTCCAAGGTA GACGGTCCAT


 


 9851
TCAGGCAAGG ATATGGGCTC ACTGAGACTA CATCAGCTAT TCTGATTACA



AGTCCGTTCC TATACCCGAG TGACTCTGAT GTAGTCGATA AGACTAATGT


 


 9901
CCCGAGGGGG ATGATAAACC GGGCGCGGTC GGTAAAGTTG TTCCATTTTT



GGGCTCCCCC TACTATTTGG CCCGCGCCAG CCATTTCAAC AAGGTAAAAA


 


 9951
TGAAGCGAAG GTTGTCGATC TGGATACCGC GAAAACGCTG GGCGTTAATC



ACTTCGCTTC CAACACCTAG ACCTATGGCC CTTTTGCGAC CCGCAATTAG


 


10001
AAAGAGGCGA ACTGTGTGTG AGAGGTCCTA TGATTATGTC CGGTTATGTA



TTTCTCCGCT TGACACACAC TCTCCAGGAT ACTAATACAG GCCAATACAT


 


10051
AACAATCCGG AAGCGACCAA CGCCTTGATT GACAAGGATG GATGGCTACA



TTGTTAGGCC TTCGCTGGTT GCGGAACTAA CTGTTCCTAC CTACCGATGT


 


10101
TTCTGGAGAC ATAGCTTACT GGGACGAAGA CGAACACTTC TTCATCGTTG



AAGACCTCTG TATCGAATGA CCCTGCTTCT GCTTGTGAAG AAGTAGCAAC


 


10151
ACCGCCTGAA GTCTCTGATT AAGTACAAAG GCTATCAGGT GGCTCCCGCT



TGGCGGACTT CAGAGACTAA TTCATGTTTC CGATAGTCCA CCGAGGGCGA


 


10201
GAATTGGAAT CCATCTTGCT CCAACACCCC AACATCTTCG ACGCAGGTGT



CTTAACCTTA GGTAGAACGA GGTTGTGGGG TTGTAGAAGC TGCGTCCACA


 


10251
CGCAGGTCTT CCCGACGATG ACGCCGGTGA ACTTCCCGCC GCCGTTGTTG



GCGTCCAGAA GGGCTGCTAC TGCGGCCACT TGAAGGGCGG CGGCAACAAC


 


10301
TTTTGGAGCA CGGAAAGACG ATGACGGAAA AAGAGATCGT GGATTACGTC



AAAACCTCGT GCCTTTCTGC TACTGCCTTT TTCTCTAGCA CCTAATGCAG


 


10351
GCCAGTCAAG TAACAACCGC GAAAAAGTTG CGCGGAGGAG TTGTGTTTGT



CGGTCAGTTC ATTGTTGGCG CTTTTTCAAC GCGCCTCCTC AACACAAACA


 


10401
GGACGAAGTA CCGAAAGGTC TTACCGGAAA ACTCGACGCA AGAAAAATCA



CCTGCTTCAT GGCTTTCCAG AATGGCCTTT TGAGCTGCGT TCTTTTTAGT


 


10451
GAGAGATCCT CATAAAGGCC AAGAAGGGCG GAAAGATCGC CGTGTAATTC



CTCTCTAGGA GTATTTCCGG TTCTTCCCGC CTTTCTAGCG GCACATTAAG


 


10501
TAGAGGCGCG CCGATCTCAC GATCCCCTGA AAAGGCTGTT TAAGTTGGGT



ATCTCCGCGC GGCTAGAGTG CTAGGGGACT TTTCCGACAA ATTCAACCCA


 


10551
AAACCGCTCC CAGCCGACGA CGAGCAAGAC GAAGACAGAA GACGCGCTCT



TTTGGCGAGG GTCGGCTGCT GCTCGTTCTG CTTCTGTCTT CTGCGCGAGA


 


10601
GCTAGATGAA ACAAAGGCGT GGTTTAGAGT AGGTATAACA GGCACTTTAG



CGATCTACTT TGTTTCCGCA CCAAATCTCA TCCATATTGT CCGTGAAATC


 


10651
CAGTGGCCGT GACGACCCGG TATGAGGTAG ACAATATTAC ACCTGTCCTA



GTCACCGGCA CTGCTGGGCC ATACTCCATC TGTTATAATG TGGACAGGAT


 


10701
CTGGCATTGA GAACTTTTGC CCAGAGCAAA AGAGCATTCC AAGCCATCAG



GACCGTAACT CTTGAAAACG GGTCTCGTTT TCTCGTAAGG TTCGGTAGTC


 


10751
AGGGGAAATA AAGCATCTCT ACGGTGGTCC TAAATAGTCA GCATAGTACA



TCCCCTTTAT TTCGTAGAGA TGCCACCAGG ATTTATCAGT CGTATCATGT


 


10801
TTTCATCTGA CTAATACTAC AACACCACCA CCATGAATAG AGGATTCTTT



AAAGTAGACT GATTATGATG TTGTGGTGGT GGTACTTATC TCCTAAGAAA


 


10851
AACATGCTCG GCCGCCGCCC CTTCCCGGCC CCCACTGCCA TGTGGAGGCC



TTGTACGAGC CGGCGGCGGG GAAGGGCCGG GGGTGACGGT ACACCTCCGG


 


10901
GCGGAGAAGG AGGCAGGCGG CCCCGATGCC TGCCCGCAAC GGGCTGGCTT



CGCCTCTTCC TCCGTCCGCC GGGGCTACGG ACGGGCGTTG CCCGACCGAA


 


10951
CTCAAATCCA GCAACTGACC ACAGCCGTCA GTGCCCTAGT CATTGGACAG



GAGTTTAGGT CGTTGACTGG TGTCGGCAGT CACGGGATCA GTAACCTGTC


 


11001
GCAACTAGAC CTCAACCCCC ACGTCCACGC CAGCCACCGC GCCAGAAGAA



CGTTGATCTG GAGTTGGGGG TGCAGGTGCG GTCGGTGGCG CGGTCTTCTT


 


11051
GCAGGCGCCC AAGCAACCAC CGAAGCCGAA GAAACCAAAA ACGCAGGAGA



CGTCCGCGGG TTCGTTGGTG GCTTCGGCTT CTTTGGTTTT TGCGTCCTCT


 


11101
AGAAGAAGAA GCAACCTGCA AAACCCAAAC CCGGAAAGAG ACAGCGCATG



TCTTCTTCTT CGTTGGACGT TTTGGGTTTG GGCCTTTCTC TGTCGCGTAC


 


11151
GCACTTAAGT TGGAGGCCGA CAGATTGTTC GACGTCAAGA ACGAGGACGG



CGTGAATTCA ACCTCCGGCT GTCTAACAAG CTGCAGTTCT TGCTCCTGCC


 


11201
AGATGTCATC GGGCACGCAC TGGCCATGGA AGGAAAGGTA ATGAAACCTC



TCTACAGTAG CCCGTGCGTG ACCGGTACCT TCCTTTCCAT TACTTTGGAG


 


11251
TGCACGTGAA AGGAACCATC GACCACCCTG TGCTATCAAA GCTCAAATTT



ACGTGCACTT TCCTTGGTAG CTGGTGGGAC ACGATAGTTT CGAGTTTAAA


 


11301
ACCAAGTCGT CAGCATACGA CATGGAGTTC GCACAGTTGC CAGTCAACAT



TGGTTCAGcA GTCGTATGCT GTACCTCAAG CGTGTCAACG GTCAGTTGTA


 


11351
GAGAAGTGAG GCATTCACCT ACACCAGTGA ACACCCCGAA GOATTCTATA



CTCTTCACTC CGTAAGTGGA TGTGGTCACT TGTGGGGCTT CCTAAGATAT


 


11401
ACTGGCACCA CGGAGCGGTG CAGTATAGTG GAGGTAGATT TACCATCCCT



TGACCGTGGT GCCTCGCCAC GTCATATCAC CTCCATCTAA ATGGTAGGGA


 


11451
CGCGGAGTAG GAGGCAGAGG AGACAGCGGT CGTCCGATCA TGGATAACTC



GCGCCTCATC CTCCGTCTCC TCTGTCGCCA GCAGGCTAGT ACCTATTGAG


 


11501
CGGTCGGGTT GTCGCGATAG TCCTCGGTGG AGCTGATCAA GGAACACGAA



GCCAGCCCAA CAGCGCTATC AGGAGCCACC TCGACTACTT CCTTGTGCTT


 


11551
CTGCCCTTTC GGTCGTCACC TGGAATAGTA AAGGGAAGAC AATTAAGACG



GACGGGAAAG CCAGCAGTGG ACCTTATCAT TTCCCTTCTG TTAATTCTGC


 


11601
ACCCCGGAAG GGACAGAAGA GTGGTCCGCA GCACCACTGG TCACGGCAAT



TGGGGCCTTC CCTGTCTTCT CACCAGGCGT CGTGGTGACC AGTGCCGTTA


 


11651
GTGTTTGCTC GGAAATGTGA GCTTCCCATG CGACCGCCCG CCCACATGCT



CACAAACGAG CCTTTACACT CGAAGGGTAC GCTGGCGGGC GGGTGTACGA


 


11701
ATACCCGCGA ACCTTCCAGA GCCCTCGACA TCCTTGAAGA GAACGTGAAC



TATGGGCGCT TGGAAGGTCT CGGGAGCTGT AGGAACTTCT CTTGCACTTG


 


11751
CATGAGGCCT ACGATACCCT GCTCAATGCC ATATTGCGGT GCGGATCGTC



GTACTCCGGA TGCTATGGGA CGAGTTACGG TATAACGCCA CGCCTAGCAG


 


11801
TGGCAGAAGC AAAAGAAGCG TCATCGATGA CTTTACCCTG ACCAGCCCCT



ACCGTCTTCG TTTTCTTCGC AGTAGCTACT GAAATGGGAC TGGTCGGGGA


 


11851
ACTTGGGCAC ATGCTCGTAC TGCCACCATA CTGAACCGTG CTTCAGCCCT



TGAACCCGTG TACGAGCATG ACGGTGGTAT GACTTGGCAC GAAGTCGGGA


 


11901
GTTAAGATCG AGCAGGTCTG GGACGAAGCG GACGATAACA CCATACGCAT



CAATTCTAGC TCGTCCAGAC CCTGCTTCGC CTGCTATTGT GGTATGCGTA


 


11951
ACAGACTTCC GCCCAGTTTG GATACGACCA AAGCGGAGCA GCAAGCGCAA



TGTCTGAAGG CGGGTCAAAC CTATGCTGGT TTCGCCTCGT CGTTCGCGTT


 


12001
ACAAGTACCG CTACATGTCG CTTAAGCAGG ATCACACCGT TAAAGAAGGC



TGTTCATGGC CATGTACAGC GAATTCGTCC TAGTGTGGCA ATTTCTTCCG


 


12051
ACCATGGATG ACATCAAGAT TAGGACCTCA GGACCGTGTA GAAGGCTTAG



TGGTACCTAC TGTAGTTCTA ATCGTGGAGT CCTGGCACAT CTTCCGAATC


 


12101
CTACAAAGGA TACTTTCTCC TCGCAAAATG CCCTCCAGGG GACAGCGTAA



GATGTTTCCT ATGAAAGAGG AGCGTTTTAC GGGAGGTCCC CTGTCGCATT


 


12151
CGGTTAGCAT AGTGAGTAGC AACTCAGCAA CGTCATGTAC ACTGGCCCGC



GCCAATCGTA TCACTCATCG TTGAGTCGTT GCAGTACATG TGACCGGGCG


 


12201
AAGATAAAAC CAAAATTCGT GGGACGGGAA AAATATGATC TACCTCCCGT



TTCTATTTTG GTTTTAAGCA CCCTGCCCTT TTTATACTAG ATGGAGGGCA


 


12251
TCACGGTAAA AAAATTCCTT GCACAGTGTA CGACCGTCTG AAAGAAACAA



AGTGCCATTT TTTTAAGGAA CGTGTCACAT GCTGGCAGAC TTTCTTTGTT


 


12301
CTGCAGGCTA CATCACTATG CACAGGCCGG GCCCGCACGC TTATACATCC



GACGTCCGAT GTAGTGATAC GTGTCCGGCC CGGGCGTGCG AATATGTAGG


 


12351
TACCTGGAAG AATCATCAGG GAAAGTTTAC GCAAAGCCGC CATCTGGGAA



ATGGACCTTC TTAGTAGTCC CTTTCAAATG CGTTTCGGCG GTAGACCCTT


 


12401
GAACATTACG TATGAGTGCA AGTGCGGCGA CTACAAGACC GGAACCGTTT



CTTGTAATGC ATACTCACGT TCACGCCGCT GATGTTCTGG CCTTGGCAAA


 


12451
CGACCCGCAC CGAAATCACT GGTTGCACCG CCATCAAGCA GTGCGTCGCC



GCTGGGCGTG GCTTTAGTGA CCAACGTGGC GGTAGTTCGT CACGCAGCGG


 


12 501
TATAAGAGCG ACCAAACGAA GTGGGTCTTC AACTCACCGG ACTTGATCCG



ATATTCTCGC TGGTTTGCTT CACCCAGAAG TTGAGTGGCC TGAACTAGGC


 


12551
ACATGACGAC CACACGGTCC AAGGGAAATT GCATTTGCCT TTCAAGTTGA



TGTACTGCTG GTGTGCCAGG TTCCCTTTAA CGTAAACGGA AAGTTCAACT


 


12601
TCCCGAGTAC CTGCATGGTC CCTGTTGCCC ACGCGCCGAA TGTAATACAT



AGGGCTCATG GACGTACCAG GGACAACGGG TGCGCGGCTT ACATTATGTA


 


12651
GGCTTTAAAC ACATCAGCCT CCAATTAGAT ACAGACCACT TGACATTGCT



CCGAAATTTG TGTAGTCGGA GGTTAATCTA TGTCTGGTGA ACTGTAACGA


 


12701
CACCACCAGO AGACTAGGGG CAAACCCGGA ACCAACCACT GAATGGATCG



GTGGTGGTCC TCTGATCCCC GTTTGGGCCT TGGTTGGTGA CTTACCTAGC


 


12751
TCGGAAAGAC GGTCAGAAAC TTCACCGTCG ACCGAGATGG CCTGGAATAC



AGCCTTTCTG CCAGTCTTTG AAGTGGCAGC TGGCTCTACC GGACCTTATG


 


12801
ATATGGGGAA ATCATGAGCC AGTGAGGGTC TATGCCCAAG AGTCAGCACC



TATACCCCTT TAGTACTCGG TCACTCCCAG ATACGGGTTC TCAGTCGTGG


 


12851
AGGAGACCCT CACGGATGGC CACACGAAAT AGTACAGCAT TACTACCATC



TCCTCTGGGA GTGCCTACCG GTGTGCTTTA TCATGTCGTA ATGATGGTAG


 


12901
GCCATCCTGT GTACACCATC TTAGCCGTCG CATCAGCTAC CGTGGCGATG



CGGTAGGACA CATGTGGTAG AATCGGCAGC GTAGTCGATG GCACCGCTAC


 


12951
ATGATTGGCG TAACTGTTGC AGTGTTATGT GCCTGTAAAG CGCGCCGTGA



TACTAACCGC ATTGACAACG TCACAATACA CGGACATTTC GCGCGGCACT


 


13001
GTGCCTGACG CCATACGCCC TGGCCCCAAA CGCCGTAATC CCAACTTCGC



CACGGACTGC GGTATGCGGG ACCGGGGTTT GCGGCATTAG GGTTGAAGCG


 


13051
TGGCACTCTT GTGCTGCGTT AGGTCGGCCA ATGCTGAAAC GTTCACCGAG



ACCGTGAGAA CACGACGCAA TCCAGCCGGT TACGACTTTG CAAGTGGCTC


 


13101
ACCATGAGTT ACTTGTGGTC GAACAGTCAG CCGTTCTTCT GGGTCCAGTT



TGGTACTCAA TGAACACCAG CTTGTCAGTC GGCAAGAAGA CCCAGGTCAA


 


13151
GTGCATACCT TTGGCCGCTT TCATCGTTCT AATGCGCTGC TGCTCCTGCT



CACGTATGGA AACCGGCGAA AGTAGCAAGA TTACGCGACG ACGAGGACGA


 


13201
GCCTGCCTTT TTTAGTGGTT GCCGGCGCCT ACCTGGCGAA GGTAGACGCC



CGGACGGAAA AAATCACCAA CGGCCGCGGA TGGACCGCTT CCATCTGCGG


 


13251
TACGAACATG CGACCACTGT TCCAAATGTG CCACAGATAC CGTATAAGGC



ATGCTTGTAC GCTGGTGACA AGGTTTACAC GGTGTCTATG GCATATTCCG


 


13301
ACTTGTTGAA AGGGCAGGGT ATGCCCCGCT CAATTTGGAG ATCACTGTCA



TGAACAACTT TCCCGTCCCA TACGGGGCGA GTTAAACCTC TAGTGACAGT


 


13351
TGTCCTCGGA GGTTTTGCCT TCCACCAACC AAGAGTACAT TACCTGCAAA



ACAGGAGCCT CCAAAACGGA AGGTGGTTGG TTCTCATGTA ATGGACGTTT


 


13401
TTCACCACTG TGGTCCCCTC CCCAAAAATC AAATGCTGCG GCTCCTTGGA



AAGTGGTGAC ACCAGGGGAG GGGTTTTTAG TTTACGACGC CGAGGAACCT


 


13451
ATGTCAGCCG GCCGTTCATG CAGACTATAC CTGCAAGGTC TTCGGAGGGG



TACAGTCGGC CGGCAAGTAC GTCTGATATG GACGTTCCAG AAGCCTCCCC


 


13501
TCTACCCCTT TATGTGGGGA GGAGCGCAAT GTTTTTGCGA CAGTGAGAAC



AGATGGGGAA ATACACCCCT CCTCGCGTTA CAAAAACGCT GTCACTCTTG


 


13551
AGCCAGATGA GTGAGGCGTA CGTCGAACTG TCAGCAGATT GCGCGTCTGA



TCGGTCTACT CACTCCGCAT GCAGCTTGAC AGTCGTCTAA CGCGCAGACT


 


13601
CCACGCGCAG GCGATTAAGG TGCACACTGC CGCGATGAAA GTAGGACTGC



GGTGCGCGTC CGCTAATTCC ACGTGTGACG GCGCTACTTT CATCCTGACG


 


13651
GTATAGTGTA CGGGAACACT ACCAGTTTCC TAGATGTGTA CGTGAACGGA



CATATCACAT GCCCTTGTGA TGGTCAAAGG ATCTACACAT GCACTTGCCT


 


13701
GTCACACCAG GAACGTCTAA AGACTTGAAA GTCATAGCTG GACCAATTTC



CAGTGTGGTC CTTGCAGATT TCTGAACTTT CAGTATCGAC CTGGTTAAAG


 


13751
AGCATCGTTT ACGCCATTCG ATCATAAGGT CGTTATCCAT CGCGGCCTGG



TCGTAGCAAA TGCGGTAAGC TAGTATTCCA GCAATAGGTA GCGCCGGACC


 


13801
TGTACAACTA TGACTTCCCG GAATATGGAG CGATGAAACC AGGAGCGTTT



ACATGTTGAT ACTGAAGGGC CTTATACCTC GCTACTTTGG TCCTCGCAAA


 


13851
GGAGACATTC AAGCTACCTC CTTGACTAGC AAGGATCTCA TCGCCAGCAC



CCTCTGTAAG TTCGATGGAG GAACTGATCG TTCCTAGAGT AGCGGTCGTG


 


13901
AGACATTAGG CTACTCAAGC CTTCCGCCAA GAACGTGCAT GTCCCGTACA



TCTGTAATCC GATGAGTTCG GAAGGCGGTT CTTGCACGTA CAGGGCATGT


 


13951
CGCAGGCCGC ATCAGGATTT GAGATGTGGA AAAACAACTC AGGCCGCCCA



GCGTCCGGCG TAGTCCTAAA CTCTACACCT TTTTGTTGAG TCCGGCGGGT


 


14001
CTGCAGGAAA CCGCACCTTT CGGGTGTAAG ATTGCAGTAA ATCCGCTCCG



GACGTCCTTT GGCGTGGAAA GCCCACATTC TAACGTCATT TAGGCGAGGC


 


14051
AGCGGTGGAC TGTTCATACG GGAACATTCC CATTTCTATT GACATCCCGA



TCGCCACCTG ACAAGTATGC CCTTGTAAGG GTAAAGATAA CTGTAGGGCT


 


14101
ACGCTGCCTT TATCAGGACA TCAGATGCAC CACTGGTCTC AACAGTCAAA



TGCGACGGAA ATAGTCCTGT AGTCTACGTG GTGACCAGAG TTGTCAGTTT


 


14151
TGTGAAGTCA GTGAGTGCAC TTATTCAGCA GACTTCGGCG GGATGGCCAC



ACACTTCAGT CACTCACGTG AATAAGTCGT CTGAAGCCGC CCTACCGGTG


 


14201
CCTGCAGTAT GTATCCGACC GCGAAGGTCA ATGCCCCGTA CATTCGCATT



GGACGTCATA CATAGGCTGG CGCTTCCAGT TACGGGGCAT GTAAGCCTAA


 


14251
CGAGCACAGC AACTCTCCAA GAGTCGACAG TACATGTCCT GGAGAAAGGA



GCTCGTGTCG TTGAGAGGTT CTCAGCTGTC ATGTACAGGA CCTCTTTCCT


 


14301
GCGGTGACAG TACACTTTAG CACCGCGAGT CCACAGGCGA ACTTTATCGT



CGCCACTGTC ATGTGAAATC GTGGCGCTCA GGTGTCCGCT TGAAATAGCA


 


14351
ATCGCTGTGT GGGAAGAAGA CAACATGCAA TGCAGAATGT AAACCACCAG



TAGCGACACA CCCTTCTTCT GTTGTACGTT ACGTCTTACA TTTGGTGGTC


 


14401
CTGACCATAT CGTGAGCACC CCGCACAAAA ATGACCAAGA ATTTCAAGCC



GACTGGTATA GCACTCGTGG GGCGTGTTTT TACTGGTTCT TAAAGTTCGG


 


14451
GCCATCTCAA AAACATCATG GAGTTGGCTG TTTGCCCTTT TCGGCGGCGC



CGGTAGAGTT TTTGTAGTAC CTCAACCGAC AAACGGGAAA AGCCGCCGCG


 


14501
CTCGTCGCTA TTAATTATAG GACTTATGAT TTTTGCTTGC AGCATGATGC



GAGCAGCGAT AATTAATATC CTGAATACTA AAAACGAACG TCGTACTACG


 


14551
TGACTAGCAC ACGAAGATGA CCGCTACGCC CCAATGATCC GACCAGCAAA



ACTGATCGTG TGCTTCTACT GGCGATGCGG GGTTACTAGG CTGGTCGTTT


 


14601
ACTCGATGTA CTTCCGAGGA ACTGATGTGC ATAAGTGAGC ATGCGTTTAA



TGAGCTACAT GAAGGCTCCT TGACTACACG TATTCACTCG TACGCAAATT


 


14651
ACTGGGCCCA ATGTTCCCCA ATGATCCGAC CAGCAAAACT CGATGTACTT



TGACCCGGGT TACAAGGGGT TACTAGGCTG GTCGTTTTGA GCTACATGAA


 


14701
CCGAGGAACT GATGTGCATA ATGCATCAGG CTGGTACATT AGATCCCCGC



GGCTCCTTGA CTACACGTAT TACGTAGTCC GACCATGTAA TCTAGGGGCG


 


14751
TTACCGCGGG CAATATAGCA ACACTAAAAA CTCGATGTAC TTCCGAGGAA



AATGGCGCCC GTTATATCGT TGTGATTTTT GAGCTACATG AAGGCTCCTT


 


14801
GCGCAGTGCA TAATGCTGCG CAGTGTTGCC ACATAACCAC TATATTAACC



CGCGTCACGT ATTACGACGC GTCACAACGG TGTATTGGTG ATATAATTGG


 


14851
ATTTATCTAG CGGACGCCAA AAACTCAATG TATTTCTGAG GAAGCGTGGT



TAAATAGATC GCCTGCGGTT TTTGAGTTAC ATAAAGACTC CTTCGCACCA


 


14901
GCATAATGCC ACGCAGCGTC TGCATAACTT TTATTATTTC TTTTATTAAT



CGTATTACGG TGCGTCGCAG ACGTATTGAA AATAATAAAG AAAATAATTA


 


14951
CAACAAAATT TTGTTTTTAA CATTTCAAAA AAAAAAAAAA AAAAAAAAAA



GTTGTTTTAA AACAAAAATT GTAAAGTTTT TTTTTTTTTT TTTTTTTTTT


 


15001
AAAATTTAAA TTAATTAAGC GGCCGCCTCG AGGACGTCAG



TTTTTTTTTT TTTTAAATTT AATTAATTCG CCGGCGGAGC TCCTGCAGTC


 


15051
GTGGCACTTT TCGGGGAAAT GTGCGCGGAA CCCCTATTTG TTTATTTTTC



CACCGTGAAA AGCCCCTTTA CACGCGCCTT GGGGATAAAC AAATAAAAAG


 


15101
TAAATACATT CAAATATGTA TCCGCTCATG AGACAATAAC CCTGATAAAT



ATTTATGTAA GTTTATACAT AGGCGAGTAC TCTGTTATTG GGACTATTTA


 


15151
GCTTCAATAA TATTGAAAAA GGAAGAGTAT GAGTATTCAA CATTTCCGTG



CGAAGTTATT ATAACTTTTT CCTTCTCATA CTCATAAGTT GTAAAGGCAC


 


15201
TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC



AGCGGGAATA AGGGAAAAAA CGCCGTAAAA CGGAAGGACA AAAACGAGTG


 


15251
CCAGAAACGC TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCACG



GGTCTTTGCG ACCACTTTCA TTTTCTACGA CTTCTAGTCA ACCCACGTGC


 


15301
AGTGGGTTAC ATCGAACTGG ATCTCAACAG CGGTAAGATC CTTGAGAGTT



TCACCCAATG TAGCTTGACC TAGAGTTGTC GCCATTCTAG GAACTCTCAA


 


15351
TTCGCCCCGA AGAACGTTTT CCAATGATGA GCACTTTTAA AGTTCTGCTA



AAGCGGGGCT TCTTGCAAAA GGTTACTACT CGTGAAAATT TCAAGACGAT


 


15401
TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC AACTCGGTCG



ACACCGCGCC ATAATAGGGC ATAACTGCGG CCCGTTCTCG TTGAOCCAGC


 


15451
CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG



GGCGTATGTG ATAAGAGTCT TACTGAACCA ACTCATGAGT GGTCAGTGTC


 


15501
AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC



TTTTCGTAGA ATGCCTACCG TACTGTCATT CTCTTAATAC GTCACGACGG


 


15551
ATAACCATGA GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATCGG



TATTGGTACT CACTATTGTG ACGCCGGTTG AATGAAGACT GTTGCTAGCC


 


15601
AGGACCGAAG GAGCTAACCG CTTTTTTGCA CAACATGGGG GATCATGTAA



TCCTGGCTTC CTCGATTGGC GAAAAAACGT GTTGTACCCC CTAGTACATT


 


15651
CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA ATGAAGCCAT ACCAAACGAC



GAGCGGAACT AGCAACCCTT GGCCTCGACT TACTTCGGTA TGGTTTGCTG


 


15701
GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACGT TGCGCAAACT



CTCGCACTGT GGTGCTACGG ACATCGTTAC CGTTGTTGCA ACGCGTTTGA


 


15751
ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT



TAATTGACCG CTTGATGAAT GAGATCGAAG GGCCGTTGTT AATTATCTGA


 


15801
GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG



CCTACCTCCG CCTATTTCAA CGTCCTGGTG AAGACGCGAG CCGGGAAGGC


 


15851
GCTGGCTGGT TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG



CGACCGACCA AATAACGACT ATTTAGACCT CGGCCACTCG CACCCAGAGC


 


15901
CGGTATCATT GCAGCACTGG GGCCAGATGG TAAGCCCTCC CGTATCGTAG



GCCATAGTAA CGTCGTGACC CCGGTCTACC ATTCGGGAGG GCATAGCATC


 


15951
TTATCTACAC GACGGGGAGT CAGGCAACTA TGGATGAACG AAATAGACAG



AATAGATGTG CTGCCCCTCA GTCCGTTGAT ACCTACTTGC TTTATCTGTC


 


16001
ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC TGTCAGACCA



TAGCGACTCT ATCCACGGAG TGACTAATTC GTAACCATTG ACAGTCTGGT


 


16051
AGTTTACTCA TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA



TCAAATGAGT ATATATGAAA TCTAACTAAA TTTTGAAGTA AAAATTAAAT


 


16101
AAAGGATCTA GGTGAAGATC CTTTTTGATA ATCTCATGAC CAAAATCCCT



TTTCCTAGAT CCACTTCTAG GAAAAACTAT TAGAGTACTG GTTTTAGGGA


 


16151
TAACGTGAGT TTTCGTTCCA CTGAGCGTCA GACCCCGTAG AAAAGATCAA



ATTGCACTCA AAAGCAAGGT GACTCGCAGT CTGGGGCATC TTTTCTAGTT


 


16201
AGGATCTTCT TGAGATCCTT TTTTTCTGCG CGTAATCTGC TGCTTGCAAA



TCCTAGAAGA ACTCTAGGAA AAAAAGACGC GCATTAGACG ACGAACGTTT


 


16251
CAAAAAAACC ACCGCTACCA GCGGTGGTTT GTTTGCCGGA TCAAGAGCTA



GTTTTTTTGG TGGCGATGGT CGCCACCAAA CAAACGGCCT AGTTCTCGAT


 


16301
CCAACTCTTT TTCCGAAGGT AACTGGCTTC AGCAGAGCGC AGATACCAAA



GGTTGAGAAA AAGGCTTCCA TTGACCGAAG TCGTCTCGCG TCTATGGTTT


 


16351
TACTGGTCTT CTAGTGTAGC CGTAGTTAGG CCACCACTTC AAGAACTCTG



ATGACCAGAA GATCACATCG GCATCAATCC GGTGGTGAAG TTCTTGAGAC


 


16401
TAGCACCGCC TACATACCTC GCTCTGCTAA TCCTGTTACC AGTGGCTGCT



ATCGTGGCGG ATGTATGGAG CGAGACGATT AGGACAATGG TCACCGACGA


 


16451
GCCAGTGGCG ATAAGTCGTG TCTTACCGGG TTGGACTCAA GACGATAGTT



CGGTCACCGC TATTCAGCAC AGAATGGCCC AACCTGAGTT CTGCTATCAA


 


16501
ACCGGATAAG GCGCAGCGGT CGGGCTGAAC GGGGGGTTCG TGCACACAGC



TGGCCTATTC CGCGTCGCCA GCCCGACTTG CCCCCCAAGC ACGTGTGTCG


 


16551
CCAGCTTGGA GCGAACGACC TACACCGAAC TGAGATACCT ACAGCGTGAG



GGTCGAACCT CGCTTGCTGG ATGTGGCTTG ACTCTATGGA TGTCGCACTC


 


16601
CTATGAGAAA GCGCCACGCT TCCCGAAGGG AGAAAGGCGG ACAGGTATCC



GATACTCTTT CGCGGTGCGA AGGGCTTCCC TCTTTCCGCC TGTCCATAGG


 


16651
GGTAAGCGGC AGGGTCGGAA CAGGAGAGCG CACGAGGGAG CTTCCAGGGG



CCATTCGCCG TCCCAGCCTT GTCCTCTCGC GTGCTCCCTC GAAGGTCCCC


 


16701
GAAACGCCTG GTATCTTTAT AGTCCTGTCG GGTTTCGCCA CCTCTGACTT



CTTTGCGGAC CATAGAAATA TCAGGACACC CCAAAGCGGT GGAGACTGAA


 


16751
GAGCGTCGAT TTTTGTGATG CTCGTCAGGG GGGCGGAGCC TATGGAAAAA



CTCGCAGCTA AAAACACTAC GAGCAGTCCC CCCGCCTCGG ATACCTTTTT


 


16801
CGCCAGCAAC GCGGCCTTTT TACGGTTCCT GGCCTTTTGC TGGCCTTTTG



GCGGTCGTTG CGCCGGAAAA ATGCCAAGGA CCGGAAAACG ACCGGAAAAC


 


16851
CTCACATGTG GGAGGCTAGA GTACATTTAG GTGACACTAT AGAA



GAGTGTACAC CCTCCGATCT CATGTAAATC CACTGTGATA TCTT






The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
It is further to be understood that all values are approximate, and are provided for description.
Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

## Claims

1. A method for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising intraperitoneally administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector and a therapeutically effective amount of a prodrug, wherein said vector encodes a suicide gene, and wherein said RC Sindbis virus vector is a RC mut-4 vector comprising SEQ. ID NOS.: 2-8 and said administration causes reduction in size of said tumor.

2. The method of claim 1 wherein said suicide gene is the Herpes virus thymidine kinase gene.

3. The method of claim 1, wherein said suicide gene is the Vericella zoster Virus thymidine kinase gene.

4. The method of claim 1, wherein said suicide gene is the cytosine deaminase gene.

5. The method of claim 2 wherein said prodrug is ganciclovir.

6. The method of claim 2 wherein said prodrug is 5-fluorocytosine.

7. The method of claim 2 wherein said prodrug is acyclovir.

8. The method of claim 2 wherein said prodrug is FIAU.

9. A method for treating a mammal harboring a solid tumor which expresses higher levels of High Affinity Laminin Receptors (LAMR) than normal cells of the same lineage comprising parenterally administering to a mammal in need of such treatment a therapeutically effective amount of a Replication Competent (RC) Sindbis virus vector, wherein said vector encodes a suicide gene and, wherein said RC Sindbis virus vector is a RC mut-4 vector comprising SEQ. ID NOS.: 2-8, and said administration causes a reduction in tumor size.

10. The method of claim 9 further comprising administering an effective amount of a pro drug.

11. The method of claim 9 wherein said suicide gene is the Herpes virus thymidine kinase gene.

12. The method of claim 10 wherein said prodrug is ganciclovir.