Patent Publication Number: US-2023140346-A1

Title: Promoter Sequences for In Vitro and In Vivo Expression of Gene Therapy Products in CD3+ Cells

Description:
BACKGROUND 
     There is great interest in developing the ability to modify the activity of specific classes of immune cells by targeted expression of exogenous genes introduced into these cells. Different methods are available for introducing DNA encoding desired transgenes into specific cell types, such as CD3+ T-cells, including plasmids and viral vectors, which are often packaged into delivery vehicles such as targeted nanoparticles. However, even if a transgene can be delivered successfully and specifically into the desired cells, there remains a need to present the transgene in a context suitable for sufficient expression to occur in the target cells. To that end, there is a need to develop promoter sequences that can drive required levels of expression of transgenes in specific populations of cells, such as CD3+ cells, so that immunotherapeutic and other treatment paradigms can be carried out successfully. 
     SUMMARY 
     The present technology provides several promoter sequences suitable to drive expression of a transgene in CD3+ T-cells. The promoter sequences can be used in vectors, such as viral vectors, when placed 5′ to the open reading frame of a transgene intended for expression in CD3+ T-cells. Preferably, the expression supported by the promoters of the present technology is selective for CD3+ T-cells. For example, the promoters preferably support expression of the transgene at a level which is about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, or even 50-fold or 100-fold higher in CD3+ cells, such as CD3+ T-cells than in CD3− cells, such as CD19+, CD3− B-cells. 
     Promoters of the present technology include nucleic acid sequences, such as DNA or RNA oligonucleotide sequences comprising or consisting of the nucleotide sequence of any of SEQ ID NOS:2-16 or 19-29, or comprising or consisting of a sequence variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOS:2-16 or 19-29. 
     Other promoters of the present technology can comprise or consist of one or more fragments of any of SEQ ID NOS:2-16 or 19-29, or combinations of such fragments assembled in any order, optionally including linker sequences between the fragments. Such fragments can be any 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 or more, 300 or more, 350 or more, 400 or more, 500 or more, 600 or more, or 700 or more consecutive nucleotides of any of SEQ ID NOS:2-16 or 19-29. Fragments can be taken from the 5′ end or the 3′ end of any of SEQ ID NOS:2-16 or 19-29. Fragments can comprise or consist of the 100, 200, 300, 400, 500, 600, or 700 consecutive nucleotides selected starting at the 3′ end of any of SEQ ID NOS:1-16 and 19-29 and moving towards the 5′ end. Fragments also can comprise or consist of the 500, 1000, or 1500 consecutive nucleotides selected starting at the 3′ end of any of SEQ ID NOS:1-10, 12-16, and 19-29 and moving towards the 5′ end. Fragments also can comprise or consist of any binding site of a transcription factor, such as transcription factors NF-kappaB, AP-1, STAT, GATA-3, and NFAT, as identified in SEQ ID NOS:2-16 and 19-29, which can be combined with or inserted into any of SEQ ID NOS:2-16 or 19-29, or fragments or variants thereof as defined above. Preferably, promoters of the present technology contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, or 20 transcription factor binding sites selected from binding sites for one or more of NF-kappaB, AP-1, STAT, GATA-3, and NFAT, or variants thereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to any of the transcription factor binding sites shown in any of SEQ ID NOS:2-16 and 19-29. The transcription factor binding sites can be positioned within the promoter sequence at any desired position in the sequence and in any order. Binding sites for other transcription factors also can be included in promoters of the present technology. Preferred promoter sequences are ICOS (SEQ ID NO:13) and CTLA4 (SEQ ID NO:7). Other preferred promoter sequences are the CD3+ specific promoters LAIR2 (SEQ ID NO:2), TNFS8 (SEQ ID NO:3), TCR (SEQ ID NO:11) and LTK (SEQ ID NO:14); each of these promoters contains fewer than 2 NFκB sites, fewer than 8 NFAT boxes, and fewer than 8 NFκB+AP1 sites. 
     The technology can be further summarized in the following list of features. 
     1. A promoter sequence for use in expression of a transgene under control of the promoter sequence in a CD3+ cell, the promoter sequence comprising nucleotides 1501-2000 of any of SEQ ID NOS:2-10 or 12-16, or a variant thereof having at least 90% identity to said sequence.
 
2. The promoter sequence of feature 1, wherein the promoter sequence comprises nucleotides 1001-2000 of any one of SEQ ID NOS:2-10 or 12-16 or a variant thereof having at least 90% identity to said sequence.
 
3. The promoter sequence of feature 2, wherein the promoter sequence comprises nucleotides 501-2000 of any one of SEQ ID NOS:2-10 or 12-16 or a variant thereof having at least 90% identity to said sequence.
 
4. The promoter sequence of feature 3, wherein the promoter sequence comprises the nucleotide sequence of any one of SEQ ID NOS:2-16 or a variant thereof having at least 90% identity to said sequence.
 
5. The promoter sequence of any of features 1-4, wherein the promoter sequence comprises a binding sequence for one or more transcription factors selected from the group consisting of NF-kappaB, AP-1, STAT, GATA-3, and NFAT.
 
6. The promoter sequence of any of features 1-5, wherein the promoter is capable of expressing the transgene at a higher level in CD3+ cells compared to CD3− cells.
 
7. The promoter sequence of feature 6, wherein the ratio of expression in CD3+ cells to CD3-cells is at least 2:1.
 
8. A promoter sequence for use in expression of a transgene under control of the promoter sequence in a CD3+ cell, the promoter sequence comprising SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:13.
 
9. A vector, plasmid, or nucleic acid molecule comprising the promoter sequence of any of features 1-8.
 
10. The vector of feature 9 which is a viral vector.
 
11. The viral vector of feature 10 which is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a herpes simplex virus.
 
12. The viral vector of any of features 9-11 which is incorporated into a nanoparticle.
 
13. The viral vector of any of features 9-11 which is not incorporated into a nanoparticle.
 
14. The viral vector of any of features 9-11 whose envelope lacks a fusion protein.
 
15. The vector of any of features 9-14, wherein the vector comprises a transgene encoding a product selected from the group consisting of chimeric antigen receptors (CARs), checkpoint inhibitors, cytokines, chemokines, antibodies and antigen binding fragments and variants thereof, enzymes, structural proteins, and reporter genes.
 
16. The nucleic acid molecule of feature 9 which is an RNA molecule.
 
17. A cell comprising the vector, plasmid, or nucleic acid molecule of any of features 9-16.
 
18. The cell of feature 17, wherein the cell comprises a genome-integrated viral vector.
 
19. The cell of feature 17, wherein the cell comprises an episomal form of the vector.
 
20. A nanoparticle comprising the vector of any of features 9-15, wherein the nanoparticle is capable of delivery of the vector into a CD3+ cell.
 
21. The nanoparticle of feature 20, wherein the nanoparticle comprises a targeting moiety that promotes selective entry of the nanoparticle into CD3+ cells.
 
22. The nanoparticle of feature 20 or 21, wherein the nanoparticle is also capable of delivery of the vector into a CD3− cell.
 
23. The nanoparticle of any of features 20-22, wherein the nanoparticle comprises a polymer.
 
24. The nanoparticle of feature 23, wherein the polymer is a poly(beta-amino ester).
 
25. A method of expressing a transgene in a CD3+ cell, the method comprising the steps of:
 
     (a) providing the vector of any of features 9-15, or the nanoparticle of any of features 20-24, and a CD3+ cell, wherein the vector comprises said transgene; 
     (b) transducing or transfecting the cell with the vector; and 
     (c) allowing the transgene to be expressed in the transduced or transfected cell. 
     26. The method of feature 25, wherein the vector is a lentiviral vector.
 
27. The method of feature 25, wherein the CD3+ cell is CD4+, CD4−, CD8+, or CD8−.
 
28. The method of feature 25, wherein step (b) comprises contacting the vector with a mixture of CD3+ and CD3− cells, and wherein the CD3+ cells are selectively transduced.
 
29. The method of any of features 25-28, wherein step (b) is performed in vitro.
 
30. The method of any of features 25-28, wherein step (b) is performed in vivo and comprises administration of the vector by intravenous, intratumoral, intramedullary, or intraperitoneal injection.
 
31. A method of making the vector of any one of features 9-15, the method comprising adding the promoter sequence of any of features 1-8 to a vector for use in transducing a CD3+ cell.
 
32. The method of feature 31, wherein said promoter sequence does not support expression of the transgene in packaging cells or producer cells used to make the vector.
 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIGS.  1 A,  1 B and  1 C  show in vitro results obtained with lentiviral vectors (LV) encoding green fluorescent protein (GFP) under the control of different human T-cell specific promoters. The transduction efficiency is given for each tested promoter at 72 h post-transduction of HEK293T cells ( FIG.  1 A ), Ramos cells ( FIG.  1 B ), and Jurkat cells ( FIG.  1 C ) analyzed by flow cytometry. Control cells were not transduced (NT) but were kept in culture throughout the experiment and a positive control was obtained by cells transduced with LV carrying the ubiquitous CMV promoter to drive the expression of GFP. For each promoter, 3 different Multiple of Infection (MOI) were evaluated. 
         FIGS.  2 A,  2 B and  2 C  show in vitro results obtained after the transduction of human PBMCs with lentiviral vectors (LV) encoding GFP under the control of different human T-cell specific promoters. The transduction efficiency was measured for each tested promoter at 72 h post-transduction by flow cytometry either with total CD45+ cells ( FIG.  2 A ), CD19+ cells ( FIG.  2 B ) or CD3+ cells ( FIG.  2 C ). Control cells (NT) were not transduced, but were activated and kept in culture throughout the experiment. 
         FIG.  3 A  shows the expression level of CD19 CAR on LV293 producing cells 72 h post-transfection with the packaging plasmid (pARA-pack) and the proviral plasmid (pARA-hUBC-CAR-CD19). CD19 CAR expression was measured by flow cytometry with CAR CD19 detection reagent and anti-biotin secondary antibody (α-biotin). Control cells were not transfected with the 2 plasmids and background staining was assessed with the anti-biotin antibody alone. 
         FIG.  3 B  shows the expression level of different transgenes (GFP and CD19 CAR) after transduction of PBMCs with PBAE encapsulated VSV-G− (“Bald”) LV or non-encapsulated VSV-G− (“Bald”) LV encoding for GFP or CD19 CAR under the control of ubiquitous promoters CMV and hUBC respectively. 
     
    
    
     DETAILED DESCRIPTION 
     The present technology provides promoter sequences for use in expressing any desired transgene in CD3+ cells. Each of the promoter sequences can be inserted into a vector for expression of a transgene just upstream of the transgene sequence, in the 5′ untranslated region most proximal to the transgene. The promoters are specific for expression in CD3+ cells, which adds an increased level of safety and specificity when using the transgene for gene transfer or immunotherapy, especially for in vivo therapy in which the vector is introduced into a patient. 
     One aspect of the present technology is a promoter sequence for use in expression of a transgene in a CD3+ cell. The promoter sequence includes at least nucleotides 1501-2000 of any of SEQ ID NOS:2-10 or 12-16 or a variant thereof. Alternatively, the promoter sequence can include nucleotides 1001-2000 of any one of SEQ ID NOS:2-10 or 12-16, nucleotides 501-2000 of any one of SEQ ID NOS:2-10 or 12-16, or nucleotides 1-2000 of any one of SEQ ID NOS:2-10 or 12-16, or a sequence variant of any of these. Further promoter sequences can be derived from any of SEQ ID NOS:2-16, or a variant thereof, by including one or more blocks of nucleotides starting from the 3′ end of the sequence and extending in a 5′ direction; such blocks of nucleotides can contain 200, 300, 400, 500, 600, 700, or more nucleotides, and extending up to a maximum of the first nucleotide of the sequence at the 5′ end. See, for example, SEQ ID NOS:19-29, described in Example 8. Sequence variants as described above can have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the stated sequence or its complement. 
     Another aspect of a promoter sequence of the present technology is that it can contain a binding sequence for one or more transcription factors selected from the group consisting of NF-kappaB, AP-1, STAT, GATA-3, and NFAT, or another transcription factor. The transcription factor binding sites can occur in any combination, in any position, and in any order within the promoter sequence. An online tool for identifying transcription factor binding sites in promoter sequences or putative promoter sequences is available at http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3. 
     Yet another aspect of a promoter sequence of the present technology is that it is capable of selectively promoting the in vitro or in vivo expression of a transgene in CD3+ cells compared to CD3− cells. For example, the ratio of expression in CD3+ cells to CD3− cells can be at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, or even 50:1 or 100:1, or higher. The promoter sequence can promote the expression of a transgene in CD3+ cells that are either CD4+ or CD4−, in CD3+ cells that are CD8+ or CD8− or in CD3+ cells that are CD4+ CD8+ or CD4− CD8−. 
     Still another aspect of the technology is a vector containing any of the above-described promoter sequences. The vector can be, for example, a viral vector, such as a lentiviral vector (LV). The vector can also be a “bald” LV, or other viral vector, which lacks a virus fusion protein on the surface of the vector particles, such as described in WO 2019/145796A2, which is incorporated by reference in its entirety herein. Other viral vectors such as retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus used as gene-delivery vehicles can incorporate the above-described promoter sequences. The vector can be present in a cell, including cells used for immunotherapy or used to produce a recombinant protein. A promoter of the present technology also can be packaged into a plasmid or nucleic acid molecule, such as an RNA molecule, including an mRNA molecule, together with one or more transgenes under its control, for delivery to cells. The vector, plasmid, or nucleic acid molecule can be packaged in a nanoparticle, such as a polymer-containing nanoparticle, including nanoparticles comprising or consisting of polymer-encapsulated vector. Polymers of such nanoparticles can be poly(beta-amino ester)s (PBAEs), including PBAE polymers containing oligopeptide end caps. The vector contains a transgene for expression in a target cell. The transgene can be any gene, including genes intended for immunotherapy involving enhancing the activity of a T-cell or a sub-population of T-cells, including CD4+ cells, CD8+ cells, NK cells, Th cells, or Treg cells. 
     While the promoters of the present technology can be used to promote the expression of any transgene, the transgene can encode, for example, an anti-checkpoint protein or polypeptide, such as an inhibitor of CTLA-4, PD1, PDL1, LAG-3, TIM 3, B7-H3, ICOS, IDO, 4-1BB, or CD47. The transgene alternatively can encode a chimeric antigen receptor (CAR) having binding specificity for any desired antigen, such as a tumor antigen or an antigen on the surface of a pathogen such as a bacterium, virus, yeast, or parasite. The CAR can be a universal CAR, which binds to an adapter molecule having a domain for CAR binding as well as an antigen binding domain suitable for binding to an antigen such as a tumor antigen or an antigen on the surface of a pathogen such as a bacterium, virus, yeast, or parasite. The transgene can be a protein or a combination of proteins able to elicit an immune response and act as a vaccine. The elicited immune response can be prophylactic or therapeutic, and can stimulate an immune response against bacteria, viruses, other microbial pathogens, or cancer cells, or another undesired cell type found in the body of a subject. 
     Another aspect of the present technology is a method of expressing a transgene in a CD3+ cell. The method includes the steps of: (a) providing a vector or nanoparticle as described above and a CD3+ cell; (b) transducing the cell, either in vitro or in vivo, with the vector or nanoparticle; and (c) allowing the transgene to be expressed in the transduced cell. The vector or nanoparticles contains the transgene and a promoter as described above in the 5′ untranslated region proximal to the gene. 
     Even another aspect of the present technology is a method of making the vector described above. The method includes adding any of the above-described promoter sequences to a vector for use in transducing a CD3+ cell. 
     EXAMPLES 
     Example 1. Production of Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters 
     Different batches of lentiviral vectors (LVs) were prepared and tested in vitro to investigate transgene expression. The LVs were made using the following materials and methods. 
     Selection of T-Cell Promoters 
     T lymphocytes represent a diverse population of CD3-positive immune cells, the main types being cytotoxic T-cells (CD8 + , Tc cells), helper T-cells (CD4 + , Th cells) and regulatory T-cells (Tregs). The hematopoietic lineage expressing the CD3 receptor also includes immature populations at various stages of differentiation (CD4 + /CD8 − , CD4 − /CD8 + , CD4+/CD8+) and innate lymphoid cells from which originate natural killer cells (NK cells). 
     Promoters for specific expression in CD3-positive cells were designed using orthogonal methods. A set of 15 promoters was selected for overexpression in CD3+ cells (SEQ ID NOS:2-16); these promoters are listed in Table 1. Among this group, 3 promoters (SEQ ID NOS:8, 12, and 15) have been described in the literature as partially repressed in Treg and B cells. 
     Molecular Cloning of T-Cell Promoters 
     The promoters were ordered as synthetic genes flanked by Mlul and BamHI restriction sites and subcloned into the transfer vector plasmid pARA-CMV-GFP upstream of the open reading frame (ORF) encoding Green Fluorescent Protein (GFP). CMV promoter (SEQ ID NO:1) was chosen as a control as it drives expression of transgenes at a high level in a ubiquitous manner. For SEQ ID NOS:3, 4, and 5, Mlul and BglII sites were chosen as flanking sites due to the presence of BamHI sites within the promoter regions. 
     The same strategy was followed to subclone the promoters with a T cell specific activity demonstrated in cell assays in a second transfer vector plasmid pARA-hUBC-CAR-CD19 upstream of the ORF encoding a chimeric antigen receptor (CAR) specific for the human CD19 antigen. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Summary of 15 human promoters selected for expression in CD3-positive cells. SEQ ID NO:, gene function and predicted 
               
               
                 expression pattern in the CD3 +  lymphoid lineage are given. Unknown: data not available in browsed databases. 
               
            
           
           
               
               
            
               
                   
                 Predicted expression pattern within CD3 +  lymphoid lineage 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 SEQ 
                   
                   
                   
                   
                   
                   
                   
                   
                 Innate 
               
               
                 ID 
                   
                   
                   
                   
                   
                   
                   
                   
                 lymphoid 
               
               
                 NO: 
                 Promoter 
                 Gene function 
                 CD4 +   
                 CD8 +   
                 Treg 
                 CD4 + /CD8 −   
                 CD4 − /CD8 +   
                 CD4 + /CD8 +   
                 cells 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 2 
                 LAIR2 1   
                 Leucocyte-associated 
                 + 
                 + 
                 + 
                 Unknown 
                 Unknown 
                 Unknown 
                 + 
               
               
                   
                   
                 Ig-like receptor 2 
               
               
                 3 
                 TNFSF8 
                 Tumor necrosis factor 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 − 
               
               
                   
                   
                 ligand superfamily 
               
               
                   
                   
                 member 8 
               
               
                 4 
                 UBASH3A 
                 Ubiquitin- associated 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                   
                 and SH3 domain 
               
               
                   
                   
                 containing A 
               
               
                 5 
                 ZAP70 
                 Zeta chain- associated 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                   
                 protein kinase 70 
               
               
                 6 
                 BCL11B 
                 Zinc finger protein 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
               
               
                 7 
                 CTLA4 
                 cytotoxic T- 
                 + 
                 − 
                 + 
                 + 
                 − 
                 − 
                 − 
               
               
                   
                   
                 lymphocyte-associated 
               
               
                   
                   
                 protein 4 
               
               
                 8 
                 TCF 
                 T-cell specific 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 − 
               
               
                   
                   
                 transcription factor, 
               
               
                   
                   
                 HMG-box 
               
               
                 9 
                 TIGIT 
                 T cell immunoreceptor 
                 + 
                 + 
                 + 
                 − 
                 − 
                 − 
                 + 
               
               
                   
                   
                 with Ig and ITIM 
               
               
                 10 
                 GIMAP7 
                 GTPase, IMAP family 
                 + 
                 + 
                 + 
                 + 
                 + 
                 − 
                 + 
               
               
                   
                   
                 member 7 
               
               
                 11 
                 TCR 2   
                 T cell receptor Vβ 8.1 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
               
               
                 12 
                 EOME 3   
                 Eomesodermin 
                 + 
                 + 
                 − 
                 − 
                 − 
                 − 
                 + 
               
               
                 13 
                 ICOS 
                 Inducible T-cell co- 
                 + 
                 + 
                 + 
                 + 
                 + 
                 − 
                 + 
               
               
                   
                   
                 stimulator 
               
               
                 14 
                 LCK 
                 Lymphocyte specific 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
                 Unknown 
               
               
                   
                   
                 protein tyrosine kinase 
               
               
                 15 
                 ITK 4   
                 IL-2 inducible T-cell 
                 + 
                 + 
                 − 
                 + 
                 + 
                 + 
                 + 
               
               
                   
                   
                 kinase 
               
               
                 16 
                 IL2RA 
                 IL2 receptor alpha 
                 − 
                 − 
                 + 
                 − 
                 − 
                 − 
                 − 
               
               
                   
               
            
           
         
       
     
     Materials 
     The transfer vector plasmids were pARA-XXX-GFP and pARA-XXX-CAR-CD19. The kanamycin-resistant plasmids coding for the provirus (a non-pathogenic and non-replicative recombinant proviral DNA derived from HIV-1, strain NL4-3), in which expression cassettes encoding GFP or CAR CD19 were cloned. The insert contained the transgene, the promoter for transgene expression and sequences added to increase the transgene expression and to allow the lentiviral vector to transduce all cell types including non-mitotic ones. The promoters were the human T-cell promoters or the CMV promoter devoid of any enhancer sequence. The non-coding sequences and expression signals corresponded to Long Terminal Repeat sequences (LTR) with the whole cis-active elements for the 5′LTR (U3-R-U5) and the deleted one for the 3′LTR, hence lacking the promoter region (ΔU3-R-U5). For the transcription and integration experiments, encapsidation sequences (SD and 5′Gag), the central PolyPurine Tract/Central Termination Site for the nuclear translocation of the vectors, and the BGH polyadenylation site were added. 
     The packaging plasmid was pARA-Pack. The kanamycin resistant plasmid encoded for the structural lentiviral proteins (GAG, POL, TAT and REV) used in trans for the encapsidation of the lentiviral provirus. The coding sequences corresponded to a polycistronic gene gag-pol-tat-rev, coding for the structural (Matrix MA, Capsid CA and Nucleocapside NC), enzymatic (Protease PR, Integrase IN and Reverse Transcriptase RT) and regulatory (TAT and REV) proteins. The non-coding sequences and expression signals corresponded to a minimal promoter from CMV for transcription initiation, a polyadenylation signal from the insulin gene for transcription termination, and an HIV-1 Rev Responsive Element (RRE) participating for the nuclear export of the packaging RNA. 
     The envelope plasmid, when used, was pENV1. This kanamycin-resistant plasmid encoded glycoprotein G from the Vesicular Stomatitis Virus (VSV-G) Indiana strain, used for the pseudotyping of some of the lentiviral vectors. The VSV-G genes were codon optimized for expression in human cells, and the gene was cloned into pVAX1 plasmid (Invitrogen). The coding sequences corresponded to codon-optimized VSV-G gene, and the noncoding sequences and expression signals corresponded to a minimal promoter from CMV for transcription initiation, and the BGH polyadenylation site to stabilize the RNA. 
     Production of VSV-G −  (“Bald”) Lentiviral Vector Particles 
     LV293 cells were seeded at 5×10 5  cells/mL in 2×3000 mL Erlenmeyer flasks (Corning) in 1000 mL of LV-Max Production Medium (Gibco Invitrogen). The two Erlenmeyers were incubated at 37° C., 65 rpm under humidified 8% CO 2 . The day after seeding, the transient transfection was performed. PEIPro transfectant reagent (PolyPlus Transfection, Illkirch, France) was mixed with transfer vector plasmids (pARA-CMV-GFP or pARA-XXX-GFP or pARA-XXX-CAR-CD19) and packaging plasmid (pARA-Pack). After incubation at room temperature, the mix PEIPro/Plasmid was added dropwise to the cell line and incubated at 37° C., 65 rpm under humidified 8% CO 2 . At day 3, the lentivector production was stimulated by sodium butyrate at 5 mM final concentration. The bulk mixture was incubated at 37° C., 65 rpm under humidified 8% CO 2  for 24 hours. After clarification by deep filtration at 5 and 0.5 μm (Pall Corporation), the clarified bulk mixture was incubated 1 hour at room temperature for DNase treatment. 
     Lentivector purification was performed by chromatography on a Q mustang membrane (Pall Corporation) and eluted by NaCl gradient. Tangential flow filtration was performed on a 100 kDa HYDROSORT membrane (Sartorius), which allowed to reduce the volume and to formulate in specific buffer at pH 7, ensuring at least 2 years of stability. After sterile filtration at 0.22 μm (Millipore), the bulk drug product was filled in 2 mL glass vials with aliquots less than 1 ml, then labelled, frozen and stored at &lt;−70° C. 
     The bald LV number was evaluated by physical titer quantification with two methods: p24 ELISA and qRT-PCR. The p24 ELISA was performed by detection and quantitation of the lentivirus associated HIV-1 p24 core protein only (Cell Biolabs Inc.). A pre-treatment of the samples allows to distinguish the free p24 from destroyed Lentivectors. The qRT-PCR was performed by purification of lentiviral RNA with Nucleospin RNA virus kit (Macherey Nagel) and quantitation with Lenti-X qRT-PCR titration kit (Takara). For each LV batch, physical titer, particle size and size distribution were measured by Nanoparticle Tracking Analyzer and Dynamic Light Scattering (Viewsizer 3000 and NanoPartica SZ-100V2 instruments respectively, Horiba Instruments Inc., USA) Analyses were performed at room temperature after dilution of LV in formulation buffer (10-fold for DLS and 300-fold for NTA) but without filtration not to impact biophysical properties of the samples. The results were determined using the control Horiba softwares of the equipments. 
     Production of VSV-G +  (“Pseudotyped”) Lentiviral Vector Particles 
     The same above-described method was used except that PEIPro transfectant reagent (PolyPlus, 115-010) was mixed with transfer vector plasmid (pARA-CMV-GFP or pARA-XXX-GFP or pAra-XXX-CAR-CD19), packaging plasmid (pARA-Pack) and the envelope plasmid (pENV1). Titration for the pseudotyped Lentivectors was performed by quantitative PCR three days post-transduction of HEK293T cells (8×10 5  cells/well) with clarified bulk obtained during the bioproduction. 
     As shown in Table 2, production yields were generally as effective as those obtained with the CMV promoter despite the fact that these promoter regions were significantly longer. For all constructs, titers were at least in the 10 7  TU/mL range and were consistent among different production runs. The lowest titers were obtained with promoters based on SEQ ID 4 and 8. Yields of production are a criteria for the selection of the promoters, as a low yield implies future difficulties for industrialization. In any case, no issue that would impact industrial bioproduction was observed. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Summary of production yields obtained for pseudotyped LV coding 
               
               
                 for GFP under the control of 14 human promoters identified by 
               
               
                 a bioinformatics approach to be expressed in CD3-positive cells. 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Size of 
                   
                   
               
               
                   
                   
                 promoter 
                 Gene of 
                 Infectious titer 
               
               
                 SEQ ID 
                 Promoter 
                 region (bp) 
                 interest 
                 qPCR (TU/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 CMV 
                 776 
                 GFP 
                 2.38 × 10 7   
               
               
                 2 
                 LAIR2 
                 2000 
                 GFP 
                 2.61 × 10 7   
               
               
                 3 
                 TNFSF8 
                 2000 
                 GFP 
                 2.63 × 10 7   
               
               
                 4 
                 UBASH3A 
                 2000 
                 GFP 
                 1.05 × 10 7   
               
               
                 5 
                 ZAP70 
                 2000 
                 GFP 
                 3.93 × 10 7   
               
               
                 6 
                 BCL11B 
                 2000 
                 GFP 
                 2.99 × 10 7   
               
               
                 7 
                 CTLA4 
                 2000 
                 GFP 
                 1.73 × 10 7   
               
               
                 8 
                 TCF 
                 2000 
                 GFP 
                 1.18 × 10 7   
               
               
                 9 
                 TIGIT 
                 2000 
                 GFP 
                 4.44 × 10 7   
               
               
                 10 
                 GIMAP7 
                 2000 
                 GFP 
                 4.49 × 10 7   
               
               
                 11 
                 TCR 
                 781 
                 GFP 
                 3.54 × 10 7   
               
               
                 12 
                 EOME 
                 2000 
                 GFP 
                 1.56 × 10 7   
               
               
                 13 
                 ICOS 
                 2000 
                 GFP 
                 2.16 × 10 7   
               
               
                 14 
                 LCK 
                 2000 
                 GFP 
                 6.00 × 10 7   
               
               
                 15 
                 ITK 
                 2000 
                 GFP 
                 1.72 × 10 7   
               
               
                   
               
            
           
         
       
     
     Example 2. Transduction of HEK293T, Ramos, and Jurkat Cells by Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters 
     In order to investigate the CD3-specific activity of the 15 promoters identified by bioinformatic tools, we used pseudotyped Lentiviral Vectors carrying a green fluorescent protein (GFP) transgene under the control of a given promoter to transduce different human cell types and analyze GFP expression. These studies were performed on CD3-positive Jurkat (Acute T Cell Leukemia Human Cell Line—ATCC TIB-152), CD3-negative Ramos (Burkitt&#39;s Lymphoma Human Cell Line—ATCC CRL-1596) and non-lymphocyte HEK293T (Human Embryonic Kidney Cell Line—ATCC CRL-1573). 
     HEKT293 cells were seeded in 24-well plates at a density of 8×10 4  cells per well in DMEM medium (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen), 1 penicillin/streptomycin and incubated for 4 h to adhere. Cells were then transduced by replacing the medium with 300 μL of Lentiviral Vector (at a MOI of 1, 5 or 10) in culture medium or culture medium (NT controls), followed by incubation at 37° C., 5% CO 2  for 2 h. After adsorption, 1 mL of complete medium was added to each well. At 72 h post transduction, the cells were trypsinized and resuspended in 200 μL of Cellfix 1×, and the percentage of cells expressing GFP was determined with an Attune N×T flow cytometer (ThermoFisher) using the BL1 channel. 
     Jurkat and Ramos cells were seeded in 24-well plates at a density of 8×10 4  cells per well in RPMI-1640 medium (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen), 1% penicillin/streptomycin. Cells were then transduced by replacing the medium with 300 μL of Lentiviral Vector (at a Multiple of Infection (MOI) of 10, 30 or 50) in culture medium or culture medium alone (NT controls). After 2 h incubation at 37° C., 5% CO 2 , 500 μL of fresh complete medium was added to each well. The percentage of cells expressing GFP transgene was determined 72 h post-transduction with an Attune N×T flow cytometer using the BL1 channel. 
     The percentages of viable and GFP-positive cells were determined by gating on debris excluded/viable/single cells. Graphed data represent means of triplicates of a representative experiment. 
     The results presented in  FIG.  1 A  (HEK293T cells),  1 B (Ramos CD3− cells) and  1 C (Jurkat CD3+ cells) show that, for all cell types, differences in expression patterns were observed between the promoters. Although lower than levels observed with ubiquitous CMV promoter, significant GFP expression (above 10%) was measured in human CD3-negative lymphocytes (Ramos cells) and non-lymphocyte cells (HEK293T) at the 3 tested MOI with promoters ZAP70 (SEQ ID NO: 5), BCL11B (SEQ ID NO: 6), TCF7 (SEQ ID NO: 8), TIGIT (SEQ ID NO: 9), GIMAP7 (SEQ ID NO: 10) and EOMES (SEQ ID NO: 12) ruling out any specificity for CD3-positive cells. 
     Promoters TNFS8 (SEQ ID NO: 3), UBASH3 (SEQ ID NO: 4), CTLA4 (SEQ ID NO: 7), ICOS (SEQ ID NO: 13) and LCK (SEQ ID NO: 14) drove the highest GFP expression (above 40% at MOI 10) in CD3-positive Jurkat cells at levels comparable with CMV promoter. However, background GFP expression (5-10% GFP-positive cells) was detected in CD3-negative cells with these promoters. A dose-dependent and strictly restricted to CD3-positive Jurkat cells GFP expression was achieved with promoters LAIR2 (SEQ ID NO: 2), TCR (SEQ ID NO: 11) and ITK (SEQ ID NO: 15). The CD3-specific activity of these last three promoters resulted in weaker GFP expression with maximal values of 45% reached at MOI of 50. 
     Of note, none of the tested promoters had an impact on viability of transduced cells (data not shown). 
     Example 3. Transduction of Human PBMCs Using Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters 
     Purification of Human PBMCs 
     Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats obtained from healthy donors (Etablissement Francais du Sang, Division Rhones-Alpes). After diluting the blood with DPBS, the PBMCs were separated over a FICOLL density gradient (GE Healthcare) and washed twice with DPBS. Then, residual Red Blood Cells were lysed during a 5 min incubation of PBMCs in ACK lysis buffer (Gibco) and an additional DPBS washing. PBMCs were frozen at a density of 20×10 6  cells/mL in 10% DMSO (Sigma), 90% FBS (Gibco) and stored at −150° C. until use. 
     Activation and Transduction of PBMCs with VSV-G +  (“Pseudotyped”) Lentiviral Vector 
     The CD3-specific activity of the six promoters screened in Example 2 was evaluated in human quiescent cells with LV carrying a GFP transgene under the control of a given promoter to transduce previously frozen human PBMCs. Thawed human PBMCs were seeded in 24-well plates at a density of 1×10 6  cells per well in RPMI medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), activated in presence of CD3-CD28 Dynabeads (Gibco) and incubated for 72 h at 37° C. and 5% CO 2 . Activated PMBCs were then pooled and transduced with encapsulated pseudotyped LV (at a MOI of 20, 50 or 100) in culture medium or culture medium alone (NT controls) in 24-well plates at a density of 1×10 5  cells. After 2 h incubation at 37° C., 5% CO 2 , 500 μL of fresh culture medium (Gibco Invitrogen) was added to each well and incubated for 72h. The percentage of cells expressing GFP was determined 72 h post-transduction with an Attune N×T flow cytometer. The phenotype of transduced cells expressing GFP transgene was determined by flow cytometry staining with antibodies specific for the following cell types following manufacturer&#39;s instructions (Biolegend): CD3-AF700, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421 and Zombie NIR for live/dead discrimination. After 30 min incubation at 4° C., cells were centrifuged at 500×g for 2 min and fixed with CelIFix solution (BD Biosciences). Fluorescence-positive cells were counted by flow cytometry (AttuneNXT; Invitrogen, Inc.) on BL1 (GFP), RL2 (AF700 dye), RL3 (Zombie NIR), VL1 (BV421 dye), VL2 (BV510 dye), VL3 (BV605 dye), VL4 (BV711 dye) and YL4 (PE-Cy7 dye) channels. Cell phenotypes were defined for CD45+, viable and single cells as follows: T lymphocytes (CD3 pos -CD19 neg ), B lymphocytes (CD3 neg -CD19 pos ), NK cells (CD3 neg -CD19 neg -CD56 pos ), monocytes (CD14 pos ) and granulocytes (SSC high -CD16 pos ). 
     The results presented in  FIG.  2 A  (whole CD45+ PBMCs),  2 B (CD19+ cells among PBMCs) and  2 C (CD3+ cells among PBMCs) show the differences in the GFP expression patterns obtained with the tested promoters. The highest GFP expression (above 40%) was measured on total CD45+ PBMCs and among gated CD3+ T cells at the 2 tested MOI with promoters CTLA4 (SEQ ID NO: 7) and ICOS (SEQ ID NO: 13). However, GFP expression was weaker in CD3+ primary cells than on CD3+ cell lines for LAIR2 (SEQ ID NO: 2), LCK (SEQ ID NO: 14), TCR (SEQ ID NO: 11) and TNFS8 (SEQ ID NO: 3) (below 20%). In addition, only LCK (SEQ ID NO: 14) at MOI 100 was shown to induce GFP expression in CD19+ cells at the same level as for CMV. The other selected promoters resulted in weaker GFP expression than CMV, which confirms their CD3+ selectivity of both cell lines and primary samples. Finally, none of the tested promoters had an impact on viability of transduced primary cells (data not shown). 
     Example 4. Transduction of Primary Lymphocytes and PBMCs Using PBAE-Encapsulated VSV-G− (“Bald”) LV 
     The CD3-specific activity of the promoters screened in Examples 2 and 3 is evaluated in human quiescent cells with PBAE-encapsulated bald LV carrying a GFP transgene under the control of a given promoter to transduce human PBMCs. PBMCs are isolated from buffy coats obtained from healthy donors (Etablissement Francais du Sang, Division Rhones-Alpes) or from blood samples from lymphoma patients as purified and frozen cells available at Lonza and CALYM Network (Centre Hospitalier Lyon-Sud, France) Biobanks. 
     Purification of Human PBMCs 
     After diluting the fresh blood with DPBS, the PBMCs are separated over a FICOLL density gradient (GE Healthcare) and washed twice with DPBS. Then, residual red blood cells are lysed during a 5 min incubation of PBMCs in ACK lysis buffer (Gibco) and an additional DPBS washing. PBMCs are frozen at density of 20×10 6  cells/mL in 10% DMSO (Sigma), 90% FBS (Gibco) and stored at −150° C. until use. 
     Transduction of PBAE-Encapsulation of VSV-G− (“Bald”) Lentiviral Vector 
     Because non-dividing cells are generally difficult to transduce with LV (without cytokine and CD3-CD28 activation), oligopeptide-modified poly(beta-amino ester) (OM-PBAE) polymers are used as transfection agents instead. OM-PBAEs have already been described as transfection agents that form polymer-encapsulated vehicles able to deliver genetic material (plasmids or other nucleic acid molecules) to eukaryotic cells (US2016/0145348A1, Mangraviti et al. 2015, Anderson et al. 2004, WO2016/116887). OM-PBAEs have been successfully used to coat transduction-deficient lentiviral vectors and engineer human cells to stably express various transgenes including reporter genes (Green Fluorescent Protein -GFP and mCherry) and CARs (see WO2019/145796). The polymers used in the following encapsulation experiments are poly(beta-amino esters) (PBAEs) conjugated to charged peptides. Polymer PBAE-CR3 refers to PBAE conjugated to the peptide CRRR (SEQ ID NO:17 (same peptide at both ends). PBAE-CH3 polymer refers to PBAE conjugated to the peptide CHHH (SEQ ID NO:18). Mixtures of these OM-PBAEs are tested at a 60/40 molar ratio. 
     Human PBMCs are seeded in 24-well plates at a density of 1×10 5  cells per well in RPMI medium containing 10% FBS and 1% penicillin/streptomycin. Cells are then transduced by replacing the medium with 100 to 300 μL of encapsulated pseudotyped LV in culture medium or culture medium alone (NT controls). After 2 h incubation at 37° C., 5% CO 2 , 600 μL of fresh complete medium are added to each well. The percentage of cells expressing GFP is determined 72 h post-transduction with an Attune N×T flow cytometer using the BL1 channel. The phenotype of transduced cells expressing GFP transgene is determined by flow cytometry staining with antibodies specific for the following cell types following manufacturer&#39;s instructions (Biolegend): CD3-AF700, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421 and Zombie NIR for live/dead discrimination. After 30 min incubation at 4° C., cells are centrifuged at 500×g for 2 min and fixed with CelIFix solution (BD Biosciences). Fluorescence-positive cells are counted by flow cytometry (AttuneNXT; Invitrogen, Inc.) on BL1 (GFP), RL2 (AF700 dye), RL3 (Zombie NIR), VL1 (BV421 dye), VL2 (BV510 dye), VL3 (BV605 dye), VL4 (BV711 dye) and YL4 (PE-Cy7 dye) channels. Cell phenotypes are defined for CD45+, viable and single cells as follows: T lymphocytes (CD3pos-CD19neg), B lymphocytes (CD3 neg-CD19pos), NK cells (CD3neg-CD19neg-CD56pos), monocytes (CD14pos) and granulocytes (SSChigh-CD16pos). 
     Finally, this set of experiments is carried out with the promoters that showed the most stringent CD3-specific activity for the expression of intracellular GFP but in the context of transgenes expressed on the surface of lymphocytes, LV without the VSV-G envelope and carrying an anti-CD19 CAR under the control of a given promoter are used to transduce PBMCs from healthy donors and lymphoma patients. The percentage of cells expressing CAR CD19 is determined 72 h post-transduction by flow cytometry with a human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions using the BL1 channel. 
     Example 5. In Vivo Transduction of Peripheral Blood Mononuclear Cells with VSV-G −  (“Bald”) Lentiviral Vector Particles Encapsulated in OM-PBAEs 
     The CD3-specific activity of the 2 best promoters confirmed in Example 4 to trigger the expression of GFP in lymphocytes is evaluated in vivo in immunodeficient NSG mice engrafted with human PBMCs from healthy donors or PBMCs from lymphoma patients. Another mouse model which can be used is NSG mice engrafted with human CD34-positive hematopoeitic stem cells that exhibit multi-lineage engraftment of human immune cell populations. Nanoparticles described in Example 4 consisting in LV carrying a GFP transgene under the control of a given promoter and formulated with and without PBAEs covalently linked with CD3 targeting agents are repeatedly injected intravenously in mice that have previously been injected with PBMCs or CD34-positive hematopoietic stem cells. Control animals are injected with vehicle or transduction-deficient lentiviral vectors lacking the VSV-G envelope and not encapsulated in PBAE polymers. 
     In vivo biodistribution of expressed GFP transgene is evaluated weekly over 24 to 90 days on whole blood cells by flow cytometry staining with specific antibody panels purchased from Biolegend: general panel (CD3-AF700, CD11b-APC, CD11c-PE, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421, CD66b-PerCP-Cy5.5, HLA-DR PE-Dazzle594, and Zombie NIR) and T cell panel (CD3-AF700, CD4-PerCP-Cy5.5, CD8-BV605, CD25-PE, CD45-BV510, CD45RA-BV711, CD69-PE-Dazzle594, CD127-APC, TCRg/d-PE-Cy7, CCR7 (CD197)-BV421 and Zombie NIR). 
     At sacrifice, the phenotype of GFP-expressing cells is determined on blood cells and cell suspension prepared from collected spleens and bone marrows. Tissue biodistribution of genome-integrated lentiviral vector is analyzed by duplex quantitative PCR on genomic DNA extracted from blood and organs collected at sacrifice. Treatment toxicity is evaluated by determining blood cell counts (flow cytometry as already described in Example 2), ALT/AST hepatic enzymes (enzyme activity kits) and cytokine levels (Th1/Th2 Cytometry Bead Array) in blood samples collected pre-treatment and at weekly post-treatment intervals. Behavior of animals, body weight, water and food consumption are recorded 3 times a week as additional read-outs for treatment safety and tolerance. 
     Finally, this set of animal experiments is carried out with the promoters selected in Example 4 but in the context of an anti-CD19 CAR transgene expressed on the surface of lymphocytes. The only difference with the above-described animal procedures lies in the evaluation of the phenotype of CD19 CAR-expressing cells that is determined by flow cytometry with the human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions already described in Example 4. 
     Example 6. Efficacy of In Vivo Transduction of Peripheral Blood Mononuclear Cells with VSV-G −  (“Bald”) Lentiviral Vector Particles Encapsulated in OM-PBAEs 
     The CD3-specific activity of the 2 best promoters screened in Example 4 to trigger the expression of CAR CD19 in lymphocytes is evaluated in vivo in immunodeficient NSG mice bearing Ramos cancer cells modified to constitutively express the Luciferase reporter gene. Different NSG mice models are used: NSG mice engrafted with human PBMCs from healthy donors or PBMCs from lymphoma patients and finally NSG mice engrafted with human CD34-positive hematopoeitic stem cells that exhibit multi-lineage engraftment of human immune cell populations. 
     Nanoparticles described in Example 4 consisting of LV carrying a CAR CD19 transgene under the control of a given promoter and formulated with and without PBAEs covalently linked with CD3 targeting agents are repeatedly injected intravenously in mice that have previously been injected with Ramos-Luc cells. Control animals are injected with vehicle or transduction-deficient LV lacking the VSV-G envelope and not encapsulated in PBAE polymers. 
     In vivo efficacy is evaluated weekly over 24 to 90 days by whole animal bioluminescence imaging to measure the tumor growth and rate associated with circulating 
     Ramos-Luc cells and survival in the treated groups versus controls. Treatment toxicity is evaluated by determining blood cell counts (flow cytometry as already described in Example 5), ALT/AST hepatic enzymes (enzyme activity kits) and cytokine levels (Cytometry Bead Array) in blood samples collected pre-treatment and at weekly post-treatment intervals. Behavior of animals, body weight, water and food consumption are recorded 3 times a week as additional read-outs for treatment safety and tolerance. 
     Example 7. Design of T-Cell Specific Chimeric Promoters 
     The promoter sequences described above contain some transcription factor binding sites, including NFκB; AP1, Stats, GATA and NFAT. Table 3 summarizes the presence of those binding sequences inside each promoter sequence. Transcription factor binding sites are depicted in the sequences as well. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Mapping of the promoter sequences with T-cell specific transcription 
               
               
                 factor binding sites. Indicated in italics are the promoters 
               
               
                 driving an unspecific expression of the transgene (according 
               
               
                 to the experiments described in FIG. 1). 
               
            
           
           
               
               
               
               
               
               
            
               
                 Promoter 
                 NFκB 
                 AP-1 
                 STATS 
                 GATA-3 
                 NFAT 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 
                   EOMES 
                 
                 
                   3 
                 
                 
                   4 
                 
                 
                   13 
                 
                 
                   3 
                 
                 
                   13 
                 
               
               
                 LCK 
                 2 
                 6 
                 6 
                 2 
                 6 
               
               
                 
                   BCL11B 
                 
                 
                   3 
                 
                 
                   3 
                 
                 
                   6 
                 
                 
                   2 
                 
                 
                   4 
                 
               
               
                 
                   ZAP70 
                 
                 
                   3 
                 
                 
                   5 
                 
                 
                   6 
                 
                 
                   1 
                 
                 
                   7 
                 
               
               
                 ICOS 
                 5 
                 7 
                 8 
                 4 
                 10 
               
               
                 UBASH3A 
                 7 
                 4 
                 15 
                 7 
                 0 
               
               
                 
                   GIMAP7 
                 
                 
                   2 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   5 
                 
                 
                   12 
                 
               
               
                 LAIR2 
                 1 
                 7 
                 8 
                 1 
                 5 
               
               
                 CTLA4 
                 3 
                 4 
                 5 
                 2 
                 6 
               
               
                 
                   ICR2A 
                 
                 
                   8 
                 
                 
                   1 
                 
                 
                   7 
                 
                 
                   1 
                 
                 
                   5 
                 
               
               
                 TCR 
                 2 
                 1 
                 3 
                 1 
                 2 
               
               
                 
                   TIGIT 
                 
                 
                   5 
                 
                 
                   5 
                 
                 
                   6 
                 
                 
                   4 
                 
                 
                   6 
                 
               
               
                 
                   TCF7 
                 
                 
                   1 
                 
                 
                   6 
                 
                 
                   6 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 TNFS8 
                 2 
                 4 
                 9 
                 6 
                 7 
               
               
                 ITK 
                 2 
                 4 
                 12 
                 8 
                 8 
               
               
                   
               
               
                 The CD3+ specific promoters (LAIR2, TNFS8, TCR and ITK) present less than 2 NFκB sites, less than 8 NFAT boxes and less than 8 NFκB + AP1 sites. 
               
            
           
         
       
     
     Example 8: Optimization and Shortening of Promoter Sequences 
     To define the significant regions involved in inducible gene expression and increased transgene expression level 5,6,7 , 5′ deletion constructs were designed by shortening of 2 kb-promoters. 
     Shortening and Molecular Cloning of Tcell Promoters 
     Among the set of 15 promoters, CTLA4 (SEQ ID NO: 7) and ICOS (SEQ ID NO: 13) were selected for overexpression in CD3-expressing cell lines and primary cells, and low or no expression in non-CD3-expressing cells. The promoters were shortened from 5′ to 3′ by PCR using the synthetic gene as template and Phusion HF high fidelity DNA polymerase (Thermofisher). Forward primers, containing a Mul restriction site, were specific of 5′ regions of CTLA4 and ICOS and reverse primers, containing a BglII site (for SEQ ID NOS: 24-25) or a BamHI site (for SEQ ID NOS: 19-23, 26-29) were specific to the 3′ end of CTLA4 or ICOS. PCR fragments were then subcloned in the transfer vector plasmid pARA-CMV-GFP upstream of the open reading frame (ORF) encoding GFP and instead of the CMV promoter. 
     The same strategy was followed to subclone the truncated promoters in a second transfer vector plasmid pARA-hUBC-CAR-CD19 upstream of the ORF encoding a chimeric antigen receptor (CAR) specific for the human CD19 antigen. Sequence identity of truncated promoters and in frame insertion upstream of the transgene were verified by Sanger sequencing. Resulting optimized promoter sequences are described in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Shortened CTLA4 and ICOS Promoters. 
               
            
           
           
               
               
               
            
               
                   
                   
                 Size of promoter 
               
               
                 SEQ ID 
                 Promoter 
                 region (bp) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 19 
                 CTLA4_1 
                 1844 
               
               
                 20 
                 CTLA4_2 
                 1237 
               
               
                 21 
                 CTLA4_3 
                 1048 
               
               
                 22 
                 CTLA4_4 
                 745 
               
               
                 23 
                 CTLA4_5 
                 344 
               
               
                 24 
                 ICOS_1 
                 1703 
               
               
                 25 
                 ICOS_2 
                 1443 
               
               
                 26 
                 ICOS_3 
                 1219 
               
               
                 27 
                 ICOS_4 
                 1000 
               
               
                 28 
                 ICOS_5 
                 837 
               
               
                 29 
                 ICOS_6 
                 550 
               
               
                   
               
            
           
         
       
     
     LV Production and Transduction of Human Cell Lines and Primary Cells 
     Transfer plasmids encoding GFP and a CD19-targeted CAR were used to produce LV and transduced cell lines and primary cells as described in Examples 3 and 4. GFP or CD19 CAR transgene expression were measured by flow cytometry. 
     Example 9: “Pseudotyping” of Lentiviral Vectors with CARs 
     The experiments of Examples 3 and 4 showed that bald LV packaged into nanoparticles, such as by OM-PBAE encapsulation, have the ability to transduce PBMCs, including T lymphocytes, and direct the expression of a transgene. Since the goal of CAR T cell therapy is to introduce a CAR into T cells and express the CAR on the surface of such T cells, the question arises whether the presence of any expressed CAR on the surface of LV intended for use in producing CAR T cells can serve to pseudotype the LV, and possibly direct the LV to transduce cells that were intended for attack by CAR T cells. The experiments described below were designed to address this question. 
     Production of Lentiviral Vector Particles Coding for CD19 CAR 
     Production of bald LV particles was performed as described in Example 1. Briefly, LV293 cells were transiently transfected the day after seeding. PEIPro transfectant reagent (PolyPlus Transfection, Illkirch, France) was mixed with the transfer vector plasmid pARA-hUBC-CAR-CD19 and the packaging plasmid pARA-Pack. Bald LV particles encapsulated in OM-PBAEs were prepared as described in Example 3 and used to transduce LV293 cells or human PBMCs. 
     CD19 CAR Detection 
     After a DPBS washing step, cells (LV293 transfected and untransfected or LV-transduced PBMCs) were stained with human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions as already described in Example 4. 
     The results presented in  FIG.  3 A  show CD19 CAR expression on the surface of LV293 cells used for the production of LVs (transfected with pARA-hUBC-CAR-CD19 and pARA-Pack plasmids). Untransfected cells were used as control. A strong expression of the CD19 CAR was detected only on the surface of LV293-producing cells after transfection. 
     In addition, results presented in  FIG.  3 B  show expression obtained on human PBMCs transduced with VSV-G− (“Bald”) LV encoding CD19 CAR under the control of hUBC promoter or GFP under the control of CMV promoter. While no expression of GFP was observed in PBMCs exposed to bald LVs, their encapsulation in OM-PBAE polymers restored their transduction efficiency. When VSV-G− (“Bald”) LV encoding CD19 CAR were used to transduce PBMCs, similar expression levels of the chimeric receptor were observed in presence or absence of the MO-PBAE polymers. 
     Altogether, these results suggest that the use of a ubiquitous promoter to control the expression of CD19 CARs results in the expression of chimeric receptor on the surface of the LV293 cell line that produces LVs (bald or pseudotyped) decorated with membrane proteins. This pseudotyping with CD19 CARs must be sufficient to allow the bald vector to bind to cells and generate an artefactual CD19-CAR signal, because the bald LV are deficient for transduction by nature, even though the CD19-CAR is not an efficient pseudotyping protein, as it does not allow endosomal escape of the LV). This “pseudotyping” may lead to safety issues, as the regular LV (with VSV-G) will directly target CD19+B lymphocytes both in vitro and in vivo. The results obtained here indicate that use of a T-cell specific promoter as described herein will abrogate the expression of CD19 CAR at the surface of LV293, and no pseudotyping of LV will be observed. This will increase the safety margin of LV designed for producing CAR T cells. 
     Sequences 
     Transcription factor binding sites are depicted in each promoter sequence as follows: NFkB (bold), AP-1 (underlined), STATS (italics), GATA-3 (double-underlined) and NFAT 5 (boxed). Of note, some binding sites are overlapping. 
     
       
         
           
               
               
            
               
                 SEQ ID NO: 1 
                   
               
               
                 GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC 
                   
               
               
                   
               
               
                 GCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA 
               
               
                   
               
               
                 CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT 
               
               
                   
               
               
                 GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT 
               
               
                   
               
               
                 ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC 
               
               
                   
               
               
                 ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT 
               
               
                   
               
               
                 TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT 
               
               
                   
               
               
                 TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG 
               
               
                   
               
               
                 GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC 
               
               
                   
               
               
                 SEQ ID NO: 2 
                   
               
               
                 CCCACTTGCTCCCTGGGCTTGGAGCACAACCACTCAAACAGAACTGGCTTTTGGTCAGTAAG 
                   
               
               
                   
               
               
                 GAAGAAGTGAGCAACGGCTGCGGTGTAGACCCTCGTCAATGCCTGCGACGGTTACACCTGGA 
               
               
                   
               
               
                 GACAAGCTCCCCAGTGTCCTCAGGAGCAGCGGAGATGAGAATCCATGATAGGGTGGGCTCTG 
               
               
                   
               
               
                 TCCCCCTCAGCTCCGTGATGCCGAAATGCACTGCTGGTCCTGGTCCTGCTCCTCATTCCACA 
               
               
                   
               
               
                 CCCGGCTGAGTGCCCATCTGACCCCAGACCTCAACGCGAGGTTCTAAGCACTGTCTCCTGAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GTGTGTGTGAATGTGTGTGTGTATTATACATAATATATATAATTACAACATTGTTAATGGGG 
               
               
                   
               
               
                 CCGGGCGCGGTGGCTCACACCTGTAATCACAGCACTTTGGGAGGCTGAGATGGGCAGATCAC 
               
               
                   
               
               
                 ATGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAATA 
               
               
                   
               
               
                 CAAAAATTAGCCGGGCATGGTGGCGTGCGCCTGTAGTCCCGGCTACTCAGCAGGC TGAGGCA   
               
               
                   
               
               
                 AGAGAATTGCTTGAGCCTGGGAGGCGGAGGTTGCAG TGAGCCA AGATTGCACCACTGCACTC 
               
               
                   
               
               
                 CAGCCTGGGCAACAGAGTACGACTCCATCTCCACACACACATACACACACACACACACACAC 
               
               
                   
               
               
                 ACAAATTGTTAATTGTATATGTATAATTATAATAGTTTATATATATTAACAATTATATATAA 
               
               
                   
               
               
                 ACTTGCATATATATAGTATTTGCTGTATTATTATATATAAACAATTATATATGTAATGACTG 
               
               
                   
               
               
                 TATAAAAT AGATAAAC AATTTTAACTAATAATATTATATTAATTATATTATTATTAGATGTA 
               
               
                   
               
               
                 ATAATTATAATTATTTATATATAATATTTATTATTATGTATCATTGTTAGAACACTTAACGT 
               
               
                   
               
               
                 GAGCTCTGTCCTCTTAACAAATTTCAAGTGAACAAGACGTTATTGCTGACGATGGGTCGTAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GTGACATCATGCAGTACTTGGTCTCTGCCTCTGCGTCGCTTGGCGTGATGTCCTCAGGTTTC 
               
               
                   
               
               
                 GTCCGTGTTGTCGCCCATGGCAGAATTT TCTTCC TTGTTTAAGGCTGAATAG TATTCC CCTG 
               
               
                   
               
               
                 TGTGTGCACCACATTTTCTATATCAATTCTTCTATCAATGGACATTTAGATGATTTTCACGT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTAGTTTTTTG AGGAACCTCCCC ACTGTTCTCTATAGTGAGTGCACAATTTTTCAGCCTCCC 
               
               
                   
               
               
                 AAAGTGCTGGGATCACAGGCG TGAGCCA CCGCGCCCGGCGCACTGTAGGATCTTTTTTAATG 
               
               
                   
               
               
                 CATTATATACCTTGCTGAGATTTTAGCAGAGATCACAATATTAAAAA CTTGGGGAAGGATT T 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAGCGGTGGTCGTTAATGTGTGGAGTTGGGATGACAT CCAAGTCA GTTGGTTGCACGACCTT 
               
               
                   
               
               
                 TATTCTGTCTTGTCCCATAGATTTAGAAAGAGGC TGACACA TCGGGTAACTAGTTTAAGGTC 
               
               
                   
               
               
                 ATCTGATCATGCGGGTAAGCGACAT TTTTCAGAAACCA AGGCCCTCCCTCTCATCTCACTAG 
               
               
                   
               
               
                 TGGGAAGGGTGGAAAG 
               
               
                   
               
               
                 SEQ ID NO: 3 
                   
               
               
                 TTTTCCACTGCCCTTCTCAGCCCCACAGGCCTTCAGGAAGACCCGAACTTCAAGCAAAGCCT 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGCTGAACAAGAGGGCCAAGGGAGCAGCTGGTTCTCAGCAACTCTGGGCAGACCACAGAGC 
               
               
                   
               
               
                 CCTTGCTACCCACTCACTGTCCGTGCCCACCAGAGGACACAGCCTTCTCCCCAAATCAGCCA 
               
               
                   
               
               
                 GGTACATGCCCCAGAAAACACTGGCTTGCCTCGTTCCCACCTTAATTACCGGACCAAACGAA 
               
               
                   
               
               
                 CGTGAACACACTGTTTTCAAAACCAAACTCAATTGGGATCACGGGGGCCTCGG TTTCCTTGA   
               
               
                   
               
               
                 TTGTAAAATGGGTATATCGCCCCCCACTCTATTTAGCCCAAGAAAACA ATTTATCT CTTGAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATCCTAAAGCCTGATCTAATTTGGGAGATGCTCAGAAGTTTTGGTTCTATGCAAGAACAGCA 
               
               
                   
               
               
                 GTGGTAATAATCCAAGCTTGGCTTTAGACAC AGGACGTTTCC TTAGGGGCATCTGGGATCTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGAATGAGCT TCTTCC ACGGTGATACCAAATGGAGCTTTCAAAGGCCCACATCTGGAGGCAG 
               
               
                   
               
               
                 CAGCTATGCAGTGATTAACATTTTAAACGGTATTTTGAAATGGAGATCATTAGTAACCACAG 
               
               
                   
               
               
                 ATGTGATC TGACTCTG TCCCCCAGGTAATCTGTCTA TTGTATCT AAATTCCAGACTTAGCCC 
               
               
                   
               
               
                 AGTAGACAGCTTGGGATGTTTAACAGGAATTGTCCAACACCATCCCCAAATCTATTTTTATT 
               
               
                   
               
               
                 CATGGAGTACTCTGACATCATCTCGCTTGG TCTTCC TGA TGACTGTA ATGCAGATTGGGAAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACTTCAGGTTTTAGCTTTCAGGTGCAGTGAAGATTGAA TGACTTAG CACGAGCTTTCAGCCA 
               
               
                   
               
               
                 GGCAGGCTGCAAAGTGCACCCAAGTTCTTTCAGTGATCGACACTTGCGACTTAGGTTG AGAT   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAGGGTGGAGGTGGAAGTGGAATGAAGCCACATGTAACTCCTAGCGCTGTGCCTGAGACAGT 
               
               
                   
               
               
                 AGAGGTTCAATTA TAGTAGTCA CATACACACACAACACATACATACACAAGACACAACACAC 
               
               
                   
               
               
                 CACACACAACACATACATACACACCACACACAACACTTACATACGCACCACACTCGGCATGC 
               
               
                   
               
               
                 ATTGCAGATCACAAATGCACACCACACACACACTGCATACATATACCATAGAGAACACACAA 
               
               
                   
               
               
                 CACCTACACATAACCCCATACCACAAATACACACACACTACATAGTACACCACGCAGAACAC 
               
               
                   
               
               
                 ACACAGCCCCCATATTCCACACCACACCATCTCACTGCCAATT CCTTCC CCTCTTCATGAGT 
               
               
                   
               
               
                 TTTACGGCAGGTCCAGGTTCATCTGCCAGTTTAACAGATCCCAAAACTTCTGCAGGAGTGTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATTACACTGCAGTCCGAGGGATCCGGCCTCCCTGAGACCCAGCAAGGACT CATTATCT GGGG 
               
               
                   
               
               
                 AGGTCTTCTGAGCCACAGGCCTCGCTGAAAGAAGGTGCAGCTTCTTGAACAGGAAGGCGTTT 
               
               
                   
               
               
                 TGTGGCAGAGTCTAAA 
               
               
                   
               
               
                 SEQ ID NO: 4 
                   
               
               
                 CAGGATAGCCCTTTCAATGCGGAAGTAGATGAAAGTTTACCCCTACCTCACACCATACACAG 
                   
               
               
                   
               
               
                 AATGAATTCAAATGGATCATAGAGCTAAATATGAAGAGCAAAAGTAGCAAAACTTCTAGAAC 
               
               
                   
               
               
                 AAAACATAGATGATCTTCATCGATGTTGGATTAACAACAATCTCTTGAACAGGATGCAGAAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CTACACACCACCCCCACCACAGAGTGGCTAAAATTAGGGACTGATAATATTAAGTGTGCATG 
               
               
                   
               
               
                 AGGATATAGAGCTTTGTACAACTGCTTGGCAGTTCCTAATGGAGTTTCACGTGCTCTGTGAT 
               
               
                   
               
               
                 CCAACAACCCCACTCCTAGATGCACAC CTAACAGAAATGC ATGCACAAGGCACTCAAAGACA 
               
               
                   
               
               
                 CATACAAGAACGTCCTCAGCAACATTAGTTACAATAGCAAAACAACCGAAAACTTCCTGAAT 
               
               
                   
               
               
                 GCATATCAACAGAGAAATGGATAAATGAATTATGTTATATTTATTTTATTTATTTATTTTTG 
               
               
                   
               
               
                 AGCTGGAGTCTCACTCTGTCCCAGGCTGGAGTGCAGTGATGCAATCTTTGCTCACTGTAACC 
               
               
                   
               
               
                 TCCACCTCCTGGGTTCAAATGATTCTCCTGCTTCACCCTCCTGAGTAGCTGGGATTACAGGC 
               
               
                   
               
               
                 ATGCACCACCATGCCCAACTAATTTTTGTATTTTTTAGTAGAGACAGGGTTTCAACATGTTG 
               
               
                   
               
               
                 GCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCCCCCCCTTGGCCTCCCAAAGTC 
               
               
                   
               
               
                 CCAAAGTGCTGGGATTACAGGCG TGAGCCA CCACGCCTGGCTGGTATATTTATTAATCAAAT 
               
               
                   
               
               
                 ACTACACAGCAATAACAGTGAACTAACTGTGGCTACAAGCAATAATGTGGATAAATTTTACA 
               
               
                   
               
               
                 GGCATAAT TTGAGGAAAA AGAAATGAGATATGATAGAGAATATGCTATATGGTTCTATTTAC 
               
               
                   
               
               
                 ATAAGGCTCAACCACAGGCACAATTCATATACAAGTGATACAGCAGTTTACTTTCTGGGGGA 
               
               
                   
               
               
                 GGGAGGAGATAACTGGAGGAGGCTGAGCTGGGGGCAGAGGTGCATCTGAGGTTCTGAGAATG 
               
               
                   
               
               
                 TTCCAGAGCAGGCTATCTCCACCTTGGCACTAGTGAAATTTTGTTGGGGAGACTGTCCTGGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACCACCCCCGCCTCA TTTTGGAAAC AAAAATGTCTCCAGACA TTTCCAAAT GTCCCCTTGGG 
               
               
                   
               
               
                 TGGAGGAGGTA CAAAATTCCC CTGGTTGCCCTGTTTGATTTTGGGTCGTGGGGGAATTAGGG 
               
               
                   
               
               
                 GTGTGGGCTCAT TTATGGAAAT TCCTTGAGCTATACA CATATGATTCA TCAACTTTTCTGTA 
               
               
                   
               
               
                 CACATATAAAGCTTTTAAAGTTTTTAAAATAAAAAAACAAGTCAATGAA TGACTGA AAGAAC 
               
               
                   
               
               
                 CAGTAAG TTTTCCAGCA ATCCGAGACAATAGATAGAGTGGGGTTTAATGTCTTGTTCACAGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TAGCCAAGTCCCTACAGAGGCTGATCCCGTGCAGGGGCTCGGTTGGACCTTTAGCCTCAAAC 
               
               
                   
               
               
                 TCCCG TGGTGGAAAC AGTTAGGATTGGTGGACGCTGCACCCAGCATCCTGCAAACAGAATTC 
               
               
                   
               
               
                 TGAAAACTGATATCTCATGCAAACAGGATATCAACTT TTGTCAGAAAGAT CAAAAAGAGCTT 
               
               
                   
               
               
                 GAGGTTTTTCTTCACA 
               
               
                   
               
               
                 SEQ ID NO: 5 
                   
               
               
                 CATGTGCGTACAGCAATGTCTGGCACATAATCAGTGCGACTCTCATTATTGTAGTATGAATG 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AAGAAATGAGCGTCCAGCATGGATGTGAAGGGATGCTTGCTGCACCTGGAGTTTCAAGATGC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGTAGCCTAGAGCCGGAACTCAGATTTTCTAAAGGGGATTCAGGCGGCCAGAGGATAGTAGA 
               
               
                   
               
               
                 GGGCCAGAAAGGAGGTCCCCAAGGAGGCTTCGGGGTAGGGTGTGAGGGGATCCTCAGGCAGA 
               
               
                   
               
               
                 CTGGGGAGCTGAGCCTGTTGTAACCGAGCGAGTTACAGAGAAACGCCGCACTTTGAGACGAA 
               
               
                   
               
               
                 TTCAGGGGTCCTTTATTAGCTGGCGACTGAGAGACAGCTAGTGCTCAAAATTCTCTCAGCCC 
               
               
                   
               
               
                 CAAAGAAGGGGCTTGATTTTCTTTTATACTTTGGTTTAGAAAGGACAGGTGGGGGTCTAAAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGTTCCAGGTGCAGGGGCTTAAAATCTATCACAAGGTGATAGACACGGGGCTTTGGGCGTTA 
               
               
                   
               
               
                 TCAACCGGACACAAACGCCGGGGCTCTGGGTGCTATTAACCGGGCGAATTCCTGGGAACTGC 
               
               
                   
               
               
                 GGATATAGCTTGCCA CAGTATCT TATCAGTTAATTGCATTCTTGGATGTGCTGGGAGTCAGC 
               
               
                   
               
               
                 TTGCACAAATTAAGTCCTTGAGGAAGCGGGGTGGGTAAGGGGCTGCAAATGAAAGAGCCAAG 
               
               
                   
               
               
                 ATGGAGTCTGTCTGGCTCTCTTAGCTAAGG GAGAGTCA ATTCAGGTTAAAACAAGGTAGGGT 
               
               
                   
               
               
                 ATCACAAGCCCACTTAACAAGGGCAGCAGGACCCCAAGAAGAAAAGCTTTAGGAGTCTCCAC 
               
               
                   
               
               
                 AGTGGGCCCAGGGCAGTTCCACACAGGTCTCTGAGGCCCCACAGACAGGAGAGCTGTGACGA 
               
               
                   
               
               
                 CTCCCTTAGTGCCCAAGAAAGCAAGGAGGTGGTGGGCAAGGGGCTCCCAGAGGCCTTGGGGC 
               
               
                   
               
               
                 ACTAGAGGGGAGATGGAGCCGAGGGAGTGGCTCTGCAGGCCCCTCTCTGAGAGAGTGCATGA 
               
               
                   
               
               
                 GGTGTGGCCCCAGGCCCAAGGGTAGGGGGTGCACAGGGTGAGGGAAGGGAGGAGAGGAAGAG 
               
               
                   
               
               
                 GAGGAGGAGGTGGTGGCCACAGCGGGATGGCACTCAGCCAGGTTCAGCTT CGTG   GGAAAGGT   
               
               
                   
               
               
                   CCC AGGTGGGCCGGGCTAGCACTGGGGATGCCCTGGCTCTGTGTCTCGGTTGGGTGGC TGAC   
               
               
                   
               
               
                   TCC TCCTGGAACGCATCCCTGACATCCTCCAGGCTGGCTTCCCCATACTCTGGGGACACACA 
               
               
                   
               
               
                 GTGCCCAGGCTTCCGGCCTCCCAGCCCTGGCTCCCTGT GGACTCA GCTGCCCCCAACAAGCC 
               
               
                   
               
               
                 TGCGAGCCACTGGAGAGCAAGGGCAGGCTGTCCTTGCCATCCAGATGCCTGGCACAGAAGGG 
               
               
                   
               
               
                 GTTCTCACCTTCGGGGTATGGGC TGACTCAG TGGGTTTCTCCTCCCTCCAGCTCTAACCTGC 
               
               
                   
               
               
                 CTGTGGAATAAACAAATCACTCCTCTGGGTTGTCTAGCTGGTGCAGGCCCCAGGGCGACGCC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CCACTGCTGCCTACCCTCCGGTTCCAGGTATGCAGGCTTCCTCCCTTCTGACGGTTCCTGCT 
               
               
                   
               
               
                 GCTGGAGTCGTCCTTCCTGAAACCCTGCCTTTGCTTAGCCTCATTCCCATCTCTCAGTCCCA 
               
               
                   
               
               
                 TCCTGCAGCTGGGCAGGCAGTGCTGGG CCCC   GGAAATGCCCT CTGCCTCCCTGGAGCACGTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SEQ ID NO: 6 
                   
               
               
                 GCTTCACATCGCTAGTTCTCAGAAAGAGAGCGCGAGTTGATCTTTCAAATTTAGAGTCCGGG 
                   
               
               
                   
               
               
                 CGAGTAGGGATGGCCAGTTTATGAATGTCTTAAAGAAGTTTATTAAAGGTGCGTGTGGGCAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCCCTACTAGACTTTGGACCACGTGGAGGTGGGGTGTTGGGGGTGGGCAGCCGGGTGCAGCC 
               
               
                   
               
               
                 TGCCCGGCCAGGGCGAGGAGGTGCAAGTCCGCGCCCCGCCCCCGGTCTCCATCCGCTCGGCC 
               
               
                   
               
               
                 TCGCGTCCGCCCACCGTGGAGCCCACAGTTTACAGAACTGCAAGCCAGGGCACATTTTATTG 
               
               
                   
               
               
                 TTATTATTTTTTAGGGTATGGGACTGGAAGGGATCTCTTTCTGTTCTCGCTCACGGACTCTG 
               
               
                   
               
               
                 GGCGCTGTTAAATTGTTGGTGCCATCGCCCCACCCAAGACCTTCGGGACGAAAACAAAAACC 
               
               
                   
               
               
                 CCCGTGTGCCCC CCGA   GGAAAAGGCCC CGAGGGAGGCGGCGCTGAGGCGTCACGGCCTCGCC 
               
               
                   
               
               
                 ATGCGCCCGGGTGAAGCCGCCGGAGCCAGGCCTGGGGGCGGAGTGGGGTGGGTGCAGCTGGA 
               
               
                   
               
               
                 CTCGCCCGCGAAGCCTAGCCGGAGCCAGGCCCCGACCCGCCCTGGCCGCCTCCAGAGAAACC 
               
               
                   
               
               
                 CTGAGAAGGGAGAGGAGGCCAGCCGCGTGGCGCGCCCAGCCCTGGAGGCCAGGTCATCAAAT 
               
               
                   
               
               
                 TGACAAAGTTGGCCACAGCCTGGGGAATGGGGGGTGTCCACGGGGGCCGTGGTCTGGCCTTG 
               
               
                   
               
               
                 TCCCCGGGCCACGGAGAGGCCCCACAACTCCTCTTGTCCTCGCCGGTTGGAGATCAAACGGC 
               
               
                   
               
               
                 TGACAAGGCAGCTGCGTCCTTCCCAGTCTGA CAGGCATTTCC   AAGTC CTACCCTGGGCCGAG 
               
               
                   
               
               
                 GAGCGCGGGGGGCGGGGTG TGACTTCC AGGTTGCTGTGATTGTCAGGGGCGCCTCC TGGAGT   
               
               
                   
               
               
                   CA CCCACCC GAGTATCT GCGGAGATTTGGCCACACCTACTCCTGGGCTGGCAAGTCCAAGTC 
               
               
                   
               
               
                 CCTCTCCCACGTGGAGCGCCCCGGTCCCTTTACCCTCTCGCTTGCCATAGACCCAGTTTAGG 
               
               
                   
               
               
                 AGGTGGGGTCGTGTTTGACCCCAGGAGTTAATCGGGGTAAGGTGAGGGGAGGGGACAAAGAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACGTGCTCTTGATTCTGTTGTCTTAGTGTCGTTTATTTTGTCATCCTCACCCTCCCAGAGTC 
               
               
                   
               
               
                 TGTAATACCAGGATTTATATGGAGACACTTTTTGGAAGTTGAACTCCAACATCTACTCCCCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCCCCCACCCCCAAAGTCCAAGTCCGAAAGAGCTTTTGGGTGGGTGGAGACTTGCAT TGATT   
               
               
                   
               
               
                   CA ATTTAGTTCACTCACACAGCACCCCGCCCCCCTTCTCAGGGGGTCCTGCTCCCAGGATGG 
               
               
                   
               
               
                 AGG AGATATAA GGACGATTTTTCTTTTATTTTAAAATAAGCTGCCCAGTGGCCCCCCCCAAC 
               
               
                   
               
               
                 CCCCTCCCGCTGTTGCGCAGCCGGGGCTCGGGGGAGATGAGCGCACAAAAACGCGGTTTGCA 
               
               
                   
               
               
                 CGTGTGTCCGGCTTGGGCTGCGGGTGTGCGCAACTGGCGACTGTGTGTGTGTGTGTGTGAGT 
               
               
                   
               
               
                 GTGCGCGCGCGCGCGAGTGTGTCTCTGTGTGTGCTTTCTTGTTCTCTTACAGGGTACAATGT 
               
               
                   
               
               
                 TAAAAAGCCACCGCTAGTCGCCCCCAGTGCTCCGACTCTCTGGGTCTTTTTGTCTCTAGTGC 
               
               
                   
               
               
                 AGATTAAACGTCACGTCCGCACTTGAACTTGAATTTTATCCCATTGTACAGAGGCAGCCCCA 
               
               
                   
               
               
                 GCCATAGAGAGACCGA 
               
               
                   
               
               
                 SEQ ID NO: 7 
                   
               
               
                 TAGAAAGCTTTCCAAATCCTGGTGCCTGGCGTATTCCAATAGTCTTCTTCCCAGTCTTCCTG 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   TCA TACCAGTTTGCTCCTTATGTTTCATTGGCCCTTGCTGCTAAGAGCATCCGCTTGCACCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CTTTAGCCCATGTTATTCTTCTTGTCTGAATATCCACCCTTTTCTCTGTTCTCAATAATAAG 
               
               
                   
               
               
                 TTCAGGCTTTTCGTCTTCTGAGAAGCCCTTTC TGACTTCC ACAGGCTGAACCACTGGCTTCT 
               
               
                   
               
               
                 GCTCCTCTACATAATACTTCAATTCCAGCATTGATCTCACTCTATCATGATCATGGGTTTAG 
               
               
                   
               
               
                 CTGTCTGTCCCTGCCACTGCTGTGTGTTCCTCTTGAGGGCAGGAACATTTGTTTTTCACTTT 
               
               
                   
               
               
                 TTAAAAAACCTCTGTTGCCCAGTCTGGCATTAGGAAGTGCCCATTAGGTTGTTATTGCTTGT 
               
               
                   
               
               
                 TGGCGCTTGAGCTGGGGCTTGAAGGTTTCTATAATGTGTAGCAGTGTATAGAAAACAGGCAG 
               
               
                   
               
               
                 GTCAGAAAAGGCTTCTGTGCATCACACCAACATGGCACATGTATACATATGTAACAAATCTG 
               
               
                   
               
               
                 CATGTTGTGCACATGTACCCTAAAACTTAAAGTATAATAATAATAAAATTTTAAAAAAAAAA 
               
               
                   
               
               
                 AGAAGAGGCTTCCTGGAGGAGATGACAGCTGAGCTAAGTCCTGGAGGATGAGAAGGAGTATA 
               
               
                   
               
               
                 AAATA AGATAATA GGAGAAAAAAGGCAGTAGGAACAGCATGGGTAAAGGTGATGAGGCCTGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGCCAAAGAGGGAGGCATTTGGTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGT 
               
               
                   
               
               
                 GGACAAT GGGAAACCAT GGACGGACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGT 
               
               
                   
               
               
                 CTGTTTGCATGTCAGCCTTCTAGAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCAT 
               
               
                   
               
               
                 CATTCAATCCTAAGTGCACAGA ATTCCGGGCATA TTACAGGTTCCCCATGAATGTTTCTTTC 
               
               
                   
               
               
                 TTTATTAAAATGTATGAAAACTCTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTT 
               
               
                   
               
               
                 TTGTTAGATCATTGGTCCTGTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAAT 
               
               
                   
               
               
                 CTTCACTTTTGAATTTTCTGCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATG 
               
               
                   
               
               
                 GACGGCTCTAAT CTCTTGAATCA TTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AAATGCTAAATGGAT TTAGGAGAAATAA ACTTATTTGTAAAGCTGTCAAGGGACCATTAGAA 
               
               
                   
               
               
                 GGATGGTGCTTCAC AGATAGAA TACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATT 
               
               
                   
               
               
                 AGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGG 
               
               
                   
               
               
                 ACTGGATGGTTAAGGATGCCCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGTTGTCTTTTCGACGTAACAGCTAAACCCACGGCT TCCTTTCTCGTAA AACCAAAACAAA 
               
               
                   
               
               
                 AAGGCTTTCTATTCAA 
               
               
                   
               
               
                 SEQ ID NO: 8 
                   
               
               
                 TGAGGCGTGAGCGTCTACAGTGAACCCAGCACAGAAACCTGCTAGGGGAGCTGCTGTTGACT 
                   
               
               
                   
               
               
                 GCATCGCGATCCAAAGGACCGGCGTCTTTTGTAGATGCAGGGGCTGAGCCAGGCCAAGCGCG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGCCAGGGTCAACTTCAGAGTCATGGGCTCCCTAAATGCGACTTCTAGGGTTGAGTTGCTG 
               
               
                   
               
               
                 TGGACGAGCGACCCATGTCGGAATCCCGCGCCCACGTGGCTGCCCAAAGTTCCGAGTCTCCG 
               
               
                   
               
               
                 GGCTGCAGGT TCTAGTCA CGGAACCGAGTTGGGAGAGTCATAGGGGCTGGGACTTGGAGGAT 
               
               
                   
               
               
                 CGGCTGAGGTCCGGTGCTCTTGGCTGTGTTCGCGGCTCGGAGCCGTCGCCTGACTGAGGGGC 
               
               
                   
               
               
                 CCGTCACAGATGTGTGATGTATAAGCTCTGCACGCAACAGGAGCTCAATAAATGTGCGAAGG 
               
               
                   
               
               
                 GGGGTATACTTATGTTCGCACTGTATGCAGGCGGCCTAGAAGGAAGTCCCTGATTGGCACAG 
               
               
                   
               
               
                 GGATGGAGGATGGGGCAAGAGCCGCAACAGCGCCGCGGAGTTCCAACGCTGCCGGTTCCCTG 
               
               
                   
               
               
                 GGGTACGAGCACAGCCTCAAGCAGCCTCAAGCCCTAGGAAGCCCCCAGTTCAAAGCACAGGG 
               
               
                   
               
               
                 CGCATTGGAGCCTGGGCACGATACAGTTCACACCACGGCTGCGATGGTAAGCCACGCCCAAG 
               
               
                   
               
               
                 TCCCAAGGGCCTAGGGGACCCCCGCCCTCCACAGCCGGAGGAGAAACCTGGGCGCAGAAAGC 
               
               
                   
               
               
                 A GGGG     GAATATCT   GGTTGTAGGTGAGTAAGCGGGGTCAGGAGTTCCCGTTAGAGTCTCTGCG 
               
               
                   
               
               
                 TTTCGGGAGAAGGGTGATC ATTCCCAG GCTTGTCCGACGTCTCTCTCAGGGTGCGCTCCGGA 
               
               
                   
               
               
                 AGAGCGAGCCCTTTAAGGCTATGCCGAGTGGGCGCGTCCCGGCCTCTCCCGGGAGAGGAGAG 
               
               
                   
               
               
                 GCGGGGCGGACCTGTGTCCCGCCCCCGGCCCGGCCCGCCCCCAGTGCCCGCCCCGCCCCCGG 
               
               
                   
               
               
                 CACTCGGCCGGCGGCGCCTTTGATGTTCCGACCCGCCAGCTCGCGGAGCCGCTCTGCCCCGC 
               
               
                   
               
               
                 GCCCTAGCCCGCGCCTGCAGCCCGCCCAGGCGGAGTCAGCCCGCGCTCCGCCCGCCGCGATC 
               
               
                   
               
               
                 CGAGCTCGGAGGTTCGGACTCCGGGCTCGCCGCCCCCCGGGCCGGCTCCGCGCCCCGCACTC 
               
               
                   
               
               
                 CCGGCGCCCAGCGCCCCGCGCCCCGGCGGGCGGAGCGCACCATGCCGCAGCTGGACTCCGGC 
               
               
                   
               
               
                 GGGGGCG GCGCGGGCGGCGGCGA CGACCTCGGCGCGCCGGACGAGCTGCTGG CCTTCC AGGA 
               
               
                   
               
               
                 TGAAGGCGAGGAGCAGGACGACAAGAGCCGCGACAGCGCCGCCGGTCCCGAGCGCGACCTGG 
               
               
                   
               
               
                 CCGAGCTCAAGTCGTCGCTCGTGAACGAGT CCGAGGGCGC GGCCGGCGGCGCAGGGATCCCG 
               
               
                   
               
               
                 GGGGTC CCGGGGGCCG GCGCCGGGGCCCGCGGCGAGGCCGAGGT GAGCCCCCGC CGGCGCCG 
               
               
                   
               
               
                 GCT CCTCCCCCGC GGTCGCCGCGCCGCGCCGCCCCAGTTGCGCGCGGCCCTCGGGGTCTCCA 
               
               
                   
               
               
                 GCGCGCAGAGCGTCCCTGCCCCGGCGTCGGCCCCGACCCCCGCGGTCCCACCGCCCCTCACT 
               
               
                   
               
               
                 CCCCTCCGGTTCTCCCTCCAGGCTCTCGGGCG GGAACA CGCTGCGCAGAGACTCTTCCCGGA 
               
               
                   
               
               
                 CAA ACTTCC AGAGCCCCTGGAGGACGGTGAGTTTCTGCCCGGCCCG GCTTCC CTTCGTCGCG 
               
               
                   
               
               
                 CTCAGGCCCTGGCCTCGGTGGGACGGGGACGCCAAGGACCGCGGGGAGCCGGGTGCCTCCCC 
               
               
                   
               
               
                 CACCGCAGCTCAGGAGGCGGCAGAACCCAGGGGTGGAGAGTGGGGGGCGGGCTTCCCGGGCG 
               
               
                   
               
               
                 CCGCCGGGTCGAGTCA 
               
               
                   
               
               
                 SEQ ID NO: 9 
                   
               
               
                 GACCAATATGGTGAAACCCCATCTCTACTAAACACAAAAAATTAGCTGGGCGTGGTGGTGCA 
                   
               
               
                   
               
               
                 TGCCTGTACTTGGGAGGC TGAGGCAGGAG AATCGCTTGAACCCGGGAGGTGGGAGTTGCAG T   
               
               
                   
               
               
                   GAGCCA AGACTGTGCCATTGTATTCCAGCCTGGGCAACAAGAGAGAAACTCCATCAAATAAA 
               
               
                   
               
               
                 TAAATAAATAAATAGGTCATGGGGATTGATTTCATCAGATTGCTTATCAGAAT CAAAGGAAA   
               
               
                   
               
               
                   T GATTATAGGCTTTGAAAAATATAGACCTTATATTTATTATTAGTGGATTTTGCAATATTGA 
               
               
                   
               
               
                 ATCATTCTTGCTACCCTGGAATAAACCCTACCTGGTGATTATGTATTTTGTTTAATATATTA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TGGTGTTCTTTAATTGTGTGCAATCTTAATCACTTTAATATCAGTTTTGAATTCATATCATA 
               
               
                   
               
               
                 AAACTAATGAAGAAGGCTTTATGTTCTATGTCTGTATATTTTAAGTAACATTGAAATTATCT 
               
               
                   
               
               
                 GCTCTTCAGAAGTTTGTTGTAGTCTCTTTGTGAAATCTTCTAGGTATGGCATTCTTTTTGCT 
               
               
                   
               
               
                 GGGGACAGGAGGAGCTCTTGATGCTTGAGGGAGTCTTGGAATGATATGTTCACGTGTACTTC 
               
               
                   
               
               
                 TATCCCAGTCCTAGTTGTCTTGGA TGACTGAG GGCTCTAGGTAAGAGGA AGAAGGAAAGA AG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGACTGAGAGTAACAATGATTCTTTGCTTGACCAAACTTTGTCAGCAAAGTTTGTCCTGAA 
               
               
                   
               
               
                 CCTTCTCCTAGGCCCATCCATGCACTTCCTTGTAAAATCCATTTTTTAGCCAAGAACAAGGA 
               
               
                   
               
               
                 TAAATCAGTTTTGCAAGAACCCCTATCCCC CTGTATCT GATCACCCT TGATATCT TATCAGG 
               
               
                   
               
               
                 TTTCTCATCCTCCACCATTTCTCAGTTCATGTCTGATCTACCCTGGCCTGTCTTTAGCAAGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AATTGAGCCCAATCTCTCCCCCTCTGCAAAATCCCATTGCCTTGGTCTCTATACCTATTGTG 
               
               
                   
               
               
                 ATGGTCCTGAATAAAGGCTGCCTTACCTTTAAGAAGTGTCATTGCCTAAGTTGTCTAATTAT 
               
               
                   
               
               
                 ACGATAGTTTAATCCCATCTAGAATAGAGGAGGAGAAGGAGGGAAGCAATGTGGGTGTGTAG 
               
               
                   
               
               
                 GAGGGTTGTGAAGAGAGGCGGCAGGAGAGGCCTAAGCTGAGAGGATCAGCC GCCTCTTCCC A 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GG AGATAGAT CCTCAGAGCCACCTCACGGGAGGGAGTTTTGACAAAC AGAAGTCA GCGGTCC 
               
               
                   
               
               
                 CACTCCCAAGTTCTCCTCATTTGTATTTGCCAGTGT CAGCCAAGTCCC ATGGCTTGTTAATA 
               
               
                   
               
               
                 AGCCTCAGATCACGGGCAGTCCTTTCGCGTGGAGTCTGATTTTCTGAGGCAGCTCTCTCCAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAGACAGCTGGGGGTGATGGGACACAGGGCGGGCCCTTCCTGGTGCCTGGGGCGGCCAGGTC 
               
               
                   
               
               
                 CCCTTGTCCCCCCTGTGTCAGGGCTCCCGTGCTTCCTCGCCCTTCTCTCCTAACATCTAAGG 
               
               
                   
               
               
                 CAGGAGGCAGGGCCTTCTCGCCCCCCGCCATAATATAACCTGCCACCAGGAGGCGCTGTTCT 
               
               
                   
               
               
                 ACAGGATGGAGAGTGG 
               
               
                   
               
               
                 SEQ ID NO: 10 
                   
               
               
                 TGGTCACTATTTACTGTGTGAGTTCCACTGGAGACAAGCACACTTGCTCCAATCCCAGAGTC 
                   
               
               
                   
               
               
                 TACACCTGACCGGGAATTTTTCATTTTGGATTCTGATTGAG GGAAAATACTG ATATCCAATG 
               
               
                   
               
               
                 GGCTATAAGGGGGACCTTT CAGTTTCTGTTAA CAGCATCTGAGTTCTATGCACGTCCTGGAA 
               
               
                   
               
               
                 AGGCACCTGCCCACTGGCTAATTCACATGTTGTAATCCCAACACTGT AAATATCTGACTTCA   
               
               
                   
               
               
                 CATTATACCCATGTAA ATCCTGTCTCA GTAGTTCTGGACTCAGCCATGGCTGCACTAATGTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGAGTATGCCGTGCTAGATGGAC AGAAGTCA ACTTGTCAAATCCTCTTCCTATTTCTACTT 
               
               
                   
               
               
                 GGAGCTCGTAGTGAGAAACGTTGGCGGAACATACTACTTCACTGGCTCTAGTGGATGGAGGC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CATGTTTGAGTGATTCAACTTGTGTTCTTGAACTAAAG CTCACAGAAAGTGA CAAAAATCCA 
               
               
                   
               
               
                 AATATTCCAAAGAAGGCTTTTG TTTTATCT TCCTTTGAAGATGC AGATATAT CCTACTTAGC 
               
               
                   
               
               
                 GGCGACGACGGCCAGCGACTCCCTACAGGCTGCCAGTGGTGCGGACCTGGGCGGGTGCCGGA 
               
               
                   
               
               
                 GCCCGGATCACAGAGGGGCAAGGCGAGCGCTCTAGGCCCACTCCTGGCGTGGGGCGCTGGAG 
               
               
                   
               
               
                 GGTGGCAAGGCCGCGACAGGGGCTGTGGGCTTGGAACTCTAAACATAGTATTCTTACGTAGC 
               
               
                   
               
               
                 CAACTG GAATATCT GGTCAACCGTGAGAGCTTTGGGAACCTGCCTCTCACCTCAAGAAACCT 
               
               
                   
               
               
                 CCAGAGCAGATTGGAACCATCCACTGGGTAAATTCT GGAAAATCTGT CTCTGCCTGAGACTG 
               
               
                   
               
               
                 GGGTAGGAAGATGTGGGGTAGAGAGGAAGGTGGGGAAGGGATGTAGGATCTATTCCTTGGGT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GGTTCCTAATGATTTAGACTCTGCCTTTGAGCCTCAGTGCTGTTAACTTTTGAATGAGTAAC 
               
               
                   
               
               
                 TTGAGGCCTTCCTGAAAGACTATAATAAAAAACCCATATTATACAATCACTAAGAACCCCCA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCCTCCCACTCCACCCTGGGAAGACAATCAGGTCCTGTGCCTTGTTTTCTAGGCAATATTTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACCAGATTATGGAGCCGTATTT CTGTTTCTTTAAA GACAAACATTTTTGTGTGTGTGGCTCC 
               
               
                   
               
               
                 ACCCAGCCTGAGCTTC 
               
               
                   
               
               
                 SEQ ID NO: 11 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 G GGAAAAAGCCA GGTGTTAATGATGAAAAAACATTCAACTTTTCTACCGCTACTAATAACAT 
               
               
                   
               
               
                 TTAATTCAAGTACTGAGAACATTTACCTCAAATCTTCAAGAATAAGACAATATTATCCCCTT 
               
               
                   
               
               
                 TCTCTTTTATTGTCGGACTAGAGAATGTGAGAGAGGTTACATTCCATGGG CTTTGGGA ATTT 
               
               
                   
               
               
                 AATATGGTTCAAGGATAAACACACCCAGGTTTTTTCACTCCAGAGAAGAGCTTCAAATATAAT 
               
               
                   
               
               
                 CCAGTTTTCAGGTCATCAGCTCAGCTCTTGTATCCCTAACAATGCGGTTGACATACCGTCTT 
               
               
                   
               
               
                 CTCACATAGTCTAAACTCCTAAACTCACTAAGCCATACTTTAAAGTACATATAAAGGACTAG 
               
               
                   
               
               
                 AAGCACCAAGCTACCAGTGAGACGAAGA GGAGAGTTTCC ACAGAAGCTGGCTTCAAATAAGA 
               
               
                   
               
               
                 CAATGAGTTCATCTTTAAATACTTGCCATTTGAGGTGCAGATGGATATAGTTGGCAGGC TCC   
               
               
                   
               
               
                   TATCT AACGCATGTTATGCACAAGCTACCGTGAATTGATAATATCAAAACAAATATCCAGCG 
               
               
                   
               
               
                 AGCCTCTGCAAGTGTGCATCTCTATTTCACACCAATTATAGTT GACTTAA TTCCTCCCTCAT 
               
               
                   
               
               
                 TCATCTCCCAGAGATGCAGCCTCCTCTTAAAGAAGTTGCGGCTGGTGGCCCATTCAGTGATG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SEQ ID NO: 12 
                   
               
               
                 TAGCTCCAAATAACGTGAGATTAGGTCTCACATTAGGGAGATTTCTCATCCCTACTTTTGCC 
                   
               
               
                   
               
               
                 TT AGATATCT TGCAACTCAGGTAAAGGATAATGTGGAGGATCCTACGAATAGTAATCAGGAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAAGGCATGGACAATGTAGTGATCAAAACAGCCGTGCCAAACACTAACTATACTGATGACAC 
               
               
                   
               
               
                 AAAACGCAGGAAGCGCAGGGCAGAACGCGCTCAGTACCAGCTTTCTGGCGATCTGCTCCTGG 
               
               
                   
               
               
                 CCACTCTGTCCCAGGACCTACAAGGCCACCGCCCCAGCCCACTAGTCTCAACTTGCCCCCTC 
               
               
                   
               
               
                 CCCTGTGGCTGTGCCTTGACCCTGAAAATCTTCGCGCACTCCTGGGTTTCACCACCACACAC 
               
               
                   
               
               
                 ACGCGAGCGCGCGAGCGCGCGAACACACACATCAAAAAAGAAAAAAAAAACCGTGATAAATG 
               
               
                   
               
               
                 CCCTGGAGAGTCTAAGTGCATTAAGGACCTCGTTCTCGGAGACCCCAGACATGCCGATGTTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCGGCGTCTGTAATTGCTTATTAACAGCGAATATTCAGGCTTCTCCTTATCCGCAACGAAAC 
               
               
                   
               
               
                 GTGCCCCCCGCTTCCGTAATAATGAAACGATAAAATATGACGGCCCCGCTCTTGA ATCTATC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACTAATTATTCTACCTTCTGTATTTGCCGCAGAGCAGAGG CGCAGGGAA TCCTAACTGTGGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   C TCCCCGAAAGCAGTTGTGTTGGCGTGAGTATGAAGTTGGTGTTAGCTCGCTTAGTTTCACT 
               
               
                   
               
               
                 CTTGTCCTGTCCCCAGTACCCTGTCCAAGCTTCTGATTTAGAAGCTGAGAGCTTAGGTCCTC 
               
               
                   
               
               
                 CAGGATGGCAATGTGCTCTGGCCGTCCCCGGAATCCCAAAAGCTTAG CGCGAGTTTCC TCTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 C TTCCCTGCAAGCT ATCAGCTTGAAGAGTCTTTCTCTCTCTGGCCTCTAAAATCTGCTCCAG 
               
               
                   
               
               
                 CCATCTCATTCCTCCAGCGGAGGGCTGAGA TGACTAAT GCGCCAAGGGTGGACACCGCTATG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAGGCGACTTGATCCAATTAGTAAATGCCTAATTGAATTAGTGTCACCTCCACCAGCCAATA 
               
               
                   
               
               
                 GGAGGGTCTCCGGGCTGTGA GGCCGGGAA GCCCCTCCCTGGCCGCAAATATATAAAGCAGCT 
               
               
                   
               
               
                 AGTGACAGCCCATAAG 
               
               
                   
               
               
                 SEQ ID NO: 13 
                   
               
               
                 ATAATTTCTAGCCAAATAAATGAGGATAAGTGTGTGAGTGTGGTGGGGTAGTTAAGAGTTTG 
                   
               
               
                   
               
               
                 GAATCCAGAGTTTCACTGTCTGGATTTGAACAGGCTCCGTTGCTCATCAGCTCTG TGACTCC   
               
               
                   
               
               
                 GGGCAAA TGACTGA ATCTCTCTAACTCTCAGTTTTCTCATCTATGAAATGAGAATAAGTAAC 
               
               
                   
               
               
                 AGACTCTCCCTCGCTGAGTTGTTTGAGAACGGGATCAGACAATGCACGTGGAGCCCCGTGTC 
               
               
                   
               
               
                 TGGCACACACTATGTCCTCATTGCATATTTATTATTAGGTTTCTGCTTTGTATTTTTCTCTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   TA CTCAGTTCCAGAGCTAGGCCTCACCTAGGTTGGAAGAAGTCAATTGTTCTCCACTGCCTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCTACGGGGAGTACAGTGAAAAGGGAGGAGATTGGGGTAGCAAGGTCCCAGGGGTCCTGGCA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCATTCCTAGTGGCTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGCCAGACTCTCTAAA 
               
               
                   
               
               
                 TCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTG TGCCTCA G TT   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATATT 
               
               
                   
               
               
                 TTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTA AGATATAT TATTTCATA 
               
               
                   
               
               
                 TAAAACATTTAAGTACATATACATAT AATTATCT ATGTAATTATTATTGTTATTGTTGTTAT 
               
               
                   
               
               
                 TGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGTC 
               
               
                   
               
               
                 TCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CATAAATA TCATTTCTCAGAT TCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTAACCCCCATAACTAT TGATTCA GAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AATAATCCATGCATTGTT TAACTCA TGTCATAAGCAATAATGCCTTTCATATAGCCATTGGC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   
               
               
                 SEQ ID NO: 14 
                   
               
               
                 AAAAATACAAAAGTTAGCCAAGCGTGATGGTGCATGCCTGTAATCCCAACTACTCAGGAGGC 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACTGGGTTCGGTGGTGCATGCCTATAATCCTAGCATTTGGGGAGGCCAAGGCAGGAGGATTG 
               
               
                   
               
               
                 CTTGAGCCCAGGAGTTCGAGATCAGACTGGGCAACATAGTGAGACCTCATCTCTACAAAAAA 
               
               
                   
               
               
                 ATAATCAGCTGGGCATGATGGTGTGCACCTTTAGTCTCAGCTACTGGGGAGGTTGAGGTGGG 
               
               
                   
               
               
                 AAGATTGCTTGAGCCCAGGAGGTCTAAGGGGATTGCGCCACTGCACTTCAGCCTGGGAGAGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGCGAGAGAGCGCACGCATTTCAAAGCAGGGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TAGTTCAGAACACAGGCTGTGTGTGTGGGGGTGTGTGGGGGTGCCTGTGTGTGGGTGTTTGT 
               
               
                   
               
               
                 GTGTGTGTGAGTGTGGGTGTGTGG GGGTATCT ATGGAGGGGATGCCTGTGTGTGTGTGTAAG 
               
               
                   
               
               
                 GTGAGTGTGTGGATGTGTGCATGGGTGTGGGTGAGTGTGTGGAGGTGCCTGTGTGTGTGTAT 
               
               
                   
               
               
                 GAATGTGTGTTGGGGAGGGGAGAATGTGTGTGTGTGGGGTGAGTGCGTGTGTGTGGGGGTGA 
               
               
                   
               
               
                 ATGCGTGTGTGTGTGTATGTGGGTGTGACTGTGTGGGTGTGTGGACGAGTGTGTGTGTTCCT 
               
               
                   
               
               
                 GGGTGTGGGAGCCTGTGTGTGTAGGGGGAGTATGTATAGGGTGTGTGCATGAATGTGTGTGT 
               
               
                   
               
               
                 GGGTACGTGTGTGAGAGTGGGTGCCTGTGTGTGGGGGGTGAGTGTGTGTGTGGGGGGGCACT 
               
               
                   
               
               
                 TGTGGAGGGTGAGTGTATGTGTTTACTGAGTGTGAGTGTGGGTGCCTGTGTGTGGGAGGG TG   
               
               
                   
               
               
                   AGTCT GTGTGTGAGTGTGTG GGGGAGTACCTG TGAGGGGTGAGTGTGTGTGTTTATGTGAAA 
               
               
                   
               
               
                 GTGTGTGTGTGTGGATGCCTCTGTGGAGGTGGGATAGGGGGTGCCTCTGTGTGTGTGTGTGA 
               
               
                   
               
               
                 GAGTGTGTGTGTGTAGGGTGTGTATATGTATAGGGTGTGTGTGAGTGTGTGTGTGTGAGAGA 
               
               
                   
               
               
                 GTGTGTGTGTGGCAGAATAGACTGCGGAGGTGGATTTCATCTTGATATGAAAGGTCTGGAAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AGGAGGAGCTGCAGGGACTCCGGGGGCTTCAAAGTGAGGGCCCCACTCTGCTTCAGGCAAAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   CACTG CTAAAACAGGCAAATAAACAAAAAAAAAGTTATGGCCAAC AGAGTCA CTGGAGGGTT 
               
               
                   
               
               
                 TTCTGCTGGGGAGAAGCAAGCCCGTGTTTGAAGGAACCCTGTGAGATGACTGTGGGCTGTG T   
               
               
                   
               
               
                   GAGGGGAA CAGCGGGGGCTTGATGGTGGACTTCGGGAGCAGAAGCCTCTTTCTCAGCCTCCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CCAACACAGGGTACTGGCAGAGGGAGAGGGAGGGGGCAGAGGCAGGAAGTGGGTAACTAGAC 
               
               
                   
               
               
                 TAACAAAGGTGCCTGTGGCGGTTTGCCCATCCCAGGTGGGAGGGTGGGGCTAGGGCTCAGGG 
               
               
                   
               
               
                 GCCGTGTGTGAATTTA 
               
               
                   
               
               
                 SEQ ID NO: 15 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AAAGATGAGTCTCAGTTAGTGGGTTTGATGGCTCTATGAGAAAGAGAATAAAATTACTTACG 
               
               
                   
               
               
                 ATGACACTCCATGTAGACTGAGCAAACGAAAGACTCCTAAGTATTGCAACAGCTACTGATGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAGGTGTAAA ATGTACTTTCC   TCAG AGGAGTTTGTAGTCTAGTTTGAGAAGATAGCACTG TA   
               
               
                   
               
               
                   TTATCT AGGTTTGCTGGTAGTGACAGAAACCTCAAAATAACTGAGACTTAGAAAAT AGATAT   
               
               
                   
               
               
                   TA AAAAATGTAATCCAGGGCTGGCATGGAAGATCCATGGTCACCAGGGTCCCAGGCTCT TTC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TGACCAGAA GACAGTCA TACAACTACACCTAGCTACAAGGGAGGCTAAAATTTGTAGTCTTT 
               
               
                   
               
               
                 ATTCTTGCTGGCCAGATGCCC AGATAACT GTCAGGGATTCTGTTACTCTAGTAAGAAGGAGA 
               
               
                   
               
               
                 GAGTAGCTATTTG GGAATA GTAGCAATCCCTACCTCAAGCCCATACTCAGAAAAAAAAAA TA   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AATTTAAAATATACCAGTACCGTACCCTCCTGGGTGTCTTGATTATGCTGGGGCATCCAGTA 
               
               
                   
               
               
                 TATGAAATAAAATTAGGCTGTTGTCTGGTAATTTACATTTAAAAATAAACATTGGAGAATTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   ATCT GCTTACACTAAAATCTCTTGATTATTAACATAGTAGAGGTAACTGTCACACATTATAC 
               
               
                   
               
               
                 TTCAACTATGTACTAACTCTAAGGTGACATGTA TGACTAGC ACCTACCATTCATAAGATGTA 
               
               
                   
               
               
                 TGGGATTCAGTCCTCCAAAGGACCCTCACAA GGAAGC TAAGGATCAG AGAAGTCA TGGCTTG 
               
               
                   
               
               
                 CTCAAAGAGAAACAGCATAGTAAGTGACAGAGCTG GGAATC CAACACAAGTTTGTCTGTTTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   CC ACAAAACGTCAGGACAGAAA GGCAGTCA TCTATATGTAGCATTGAGAAATTTCTATGGGC 
               
               
                   
               
               
                 TTTAATTTAAATCTTAAAAACTAATAAACTTCAGCAACATATTTCTTCACTTTAATGTGTGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AATTTTTCTTGAACATCTGTTTTATGTGTCAGGTACTGTGATGGTTGCTGAGTATATCATGG 
               
               
                   
               
               
                 TAAGCAAAACCACACATTGTCCTTACCCCTTGTGCTACATGATTTAACTGCTGGATGGATTA 
               
               
                   
               
               
                 AACCA GGAACT ATAAAGATTAAACTGTAATCCTTACCACAATTTTGAGTTTACAGTTTTTCA 
               
               
                   
               
               
                 AATATCATTTCAAGCCATTAGCAAAGGCTGTATGTATTTTTTACATATGCCTCCTCGTTTTG 
               
               
                   
               
               
                 TGAATTTTGAAAGGATGTGGTTTCGGCCTTTGACATCAGAGGAGAAGCTCAGCTATGTTGGC 
               
               
                   
               
               
                 TGAACGTTGATAGAA AGATAACG TTGAAGGCAAGTTGCCCTTGAGCAGCTCTCTGAAGATCA 
               
               
                   
               
               
                 ACTGCCTCCACATTGC 
               
               
                   
               
               
                 SEQ ID NO: 16 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                 CATCAGCAAACGACGACAGAGCGTTCATCCGTAAGGTGAACCAGAAAAGCCAGTTCAATGAC 
               
               
                   
               
               
                 TTGTTTAACCATGGTCCATCTCAGAACCAAGAGTTGGGCCTCTTAT TTACCAGAAAAAT TGT 
               
               
                   
               
               
                 GGGGGCTTTGTGATATGGCTTTAAAAAAATCTTGTAATTGCCAGGCGTGGTGGCTCACACCT 
               
               
                   
               
               
                 GTAATCCCAGCACTTTGGGAGGCCGAGGTGGGTGAATCGCCTAAGGTCAGGAGTTCGAGACC 
               
               
                   
               
               
                 AGCCTGACCAACATGGTGAAACTCCGTCTCTACTAAAAATACAAAAACTAGCTGGATGTGGT 
               
               
                   
               
               
                 GACGCGTGCCTGTAATCCTAGCTACTCAGGAGGCTGACGCAGGAGAATCACTTGAACCTGGG 
               
               
                   
               
               
                 AGGCAGAGGTTGCAG TGAGCCAAGAT TGTGCCATTGCGCTCCAAAAAAAAAAAAAAAAAGAC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GATGGAAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAG 
               
               
                   
               
               
                 CCTGCACCTCCTCGGTTCCAGCTATT CTCTTGTCTCA GCCTCC TGAGTAA CTGGGATTACAG 
               
               
                   
               
               
                 GCGCCCGCCACTACGCCTGGCTAATTTTTGTATTT TTAGTAGAAATGG GGTTTTACCATGTT 
               
               
                   
               
               
                 GGCCAGACTGGTCTCAAACTCCCGACCTCAGGTGATCTGCC TGCCTCA GCCTCCCAAAGTGC 
               
               
                   
               
               
                 TGGAATTACAGGCGTGTGCCACTGCGCCTGGCTAATTTTTTTTTTTTTTTTTTTTTTTTTTT 
               
               
                   
               
               
                 TTTTTTAGTAGAGACGGTGGTTTCACCATGTCATCCAGGCTGGTCTCAAACTCCTGACCTCA 
               
               
                   
               
               
                 GGTGATCCACCCACCTTGGTCTACCAAAGTGCTCGGATTACAGGCATGAGCCACCAGGCCCA 
               
               
                   
               
               
                 GTCAACGTGATGTGTTTTGGAACCCTGAATTCCTTGGCTTGCCCGGAGGGTTTTCTTTTTGT 
               
               
                   
               
               
                   TAATATCT TTGCTTGCTTTCTAGTATTTAAAAAATTGTGTTTTGCTCTAACTATGCAATGGC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAGAGCTCTGATCAATAAATAAGCAAGACTGAATTTTACAAAATAATCCAAAGTTTAAAACC 
               
               
                   
               
               
                 AAAGCCCACTTTTTGCATGATCCT TTAAGAGAAAGAA ATCTGGAAGCAAAACACCTTATAAA 
               
               
                   
               
               
                 ATGACAATGCACTTTCAGGAGCCCAGGGCACTGTGGTGAAATGATGATGGCTAGTACAGGTT 
               
               
                   
               
               
                 ATAAGC CTTGGGGAA TTATTTATGAATTCTCAGGATCCTTCAGTTCGCCGCATCCTTCTCCA 
               
               
                   
               
               
                 TTATTTGAATATTGGAGGCTGCCTGACCAGAATCTTGTCAGGACTTTGCTCCTTCATCCCAG 
               
               
                   
               
               
                 GTGGTCCCGGC TGACTCCT GAGGACGTTACAGCCCTGAGGGGA GGACTCA GCTTATGAAGTG 
               
               
                   
               
               
                 CTGGGTGAGACCACTGCCAAGAAGTGCTTGCTCACCCTACCTTCAACGGCA GGGGAATCTCC   
               
               
                   
               
               
                 CTCTCCTTTTATGGGCGTAGCTGAAGAAAGGATTCATAAATGAAGTTCAATCCTTCTCATCA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAAACTATATTGTCATCAAAAAAAAAAAAAAAAAAAACACTTCCTATATTTGAGATGAGAG 
               
               
                   
               
               
                 AAGAGAGTGCTAGGCA 
               
               
                   
               
               
                 SEQ ID NO: 17 
                   
               
               
                 Cys-Arg-Arg-Arg 
                   
               
               
                   
               
               
                 SEQ ID NO: 18 
                   
               
               
                 Cys-His-His-His 
                   
               
               
                   
               
               
                 SEQ ID NO: 19 
                   
               
               
                 CCTTGCTGCTAAGAGCATCCGCTTGCACCTTCTGCTCATCCCCAGACAAGCTTTGTCCTGTG 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TCCACCCTTTTCTCTGTTCTCAATAATAAGTTCAGGCTTTTCGTCTTCTGAGAAGCCCTTTC 
               
               
                   
               
               
                   TGACTTCC ACAGGCTGAACCACTGGCTTCTGCTCCTCTACATAATACTTCAATTCCAGCATT 
               
               
                   
               
               
                 GATCTCACTCTATCATGATCATGGGTTTAGCTGTCTGTCCCTGCCACTGCTGTGTGTTCCTC 
               
               
                   
               
               
                 TTGAGGGCAGGAACATTTGTTTTTCACTTTTTAAAAAACCTCTGTTGCCCAGTCTGGCATTA 
               
               
                   
               
               
                 GGAAGTGCCCATTAGGTTGTTATTGCTTGTTGGCGCTTGAGCTGGGGCTTGAAGGTTTCTAT 
               
               
                   
               
               
                 AATGTGTAGCAGTGTATAGAAAACAGGCAGGTCAGAAAAGGCTTCTGTGCATCACACCAACA 
               
               
                   
               
               
                 TGGCACATGTATACATATGTAACAAATCTGCATGTTGTGCACATGTACCCTAAAACTTAAAG 
               
               
                   
               
               
                 TATAATAATAATAAAATTTTAAAAAAAAAAAGAAGAGGCTTCCTGGAGGAGATGACAGCTGA 
               
               
                   
               
               
                 GCTAAGTCCTGGAGGATGAGAAGGAGTATAAAATA AGATAATA GGAGAAAAAAGGCAGTAGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 AAGGGGGAATGGGAGGGAATGCTGGGGTACAGGCCAAAGAGGGAGGCATTTGGTGAGTATTC 
               
               
                   
               
               
                 TGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAAT GGGAAACCAT GGACGGACTGGAGTA 
               
               
                   
               
               
                 GGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTTCTAGAAGCCCCTT 
               
               
                   
               
               
                 AAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCACAGA ATTCCGGGCA   
               
               
                   
               
               
                   TA TTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAAACTCTCCAGATTT 
               
               
                   
               
               
                 AAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCTGTCTACAGCTGTC 
               
               
                   
               
               
                 ACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCTGCTTGAAAAATTT 
               
               
                   
               
               
                 GTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAAT CTCTTGAATCA TTTGGGTTG 
               
               
                   
               
               
                 GCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGT TGAGTTA AGGCTTTTAAGAACA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TCTTGTGATTTTAGT TTTTTTCTCTTAA CCAAATGCTAAATGGAT TTAGGAGAAATAA ACTT 
               
               
                   
               
               
                 ATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCAC AGATAGAA TACAGTTTTT 
               
               
                   
               
               
                 ATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGT 
               
               
                   
               
               
                 AGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCAGAAGATTGAA 
               
               
                   
               
               
                 TAAAATTGGGATTTAGGAGGACCCTTGTACTCCA GGAAATTCTCC AAGTCTCCACTTAGTTA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATTGGCTTGTTTTGTTCAGTTGAGTGCTTGAGGTTGTCTTTTCGACGTAACAGCTAAACCCA 
               
               
                   
               
               
                 CGGCT TCCTTTCTCGTAA AACCAAAACAAAAAGGCTTTCTATTCAA 
               
               
                   
               
               
                 SEQ ID NO: 20 
                   
               
               
                 AGATGACAGCTGAGCTAAGTCCTGGAGGATGAGAAGGAGTATAAAATA AGATAATA GGAGAA 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTGGTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAAT GGGAAACCAT GG 
               
               
                   
               
               
                 ACGGACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTT 
               
               
                   
               
               
                 CTAGAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCAC 
               
               
                   
               
               
                 AGA ATTCCGGGCATA TTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAA 
               
               
                   
               
               
                 ACTCTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCT 
               
               
                   
               
               
                 GTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCT 
               
               
                   
               
               
                 GCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAAT CTCTTGA   
               
               
                   
               
               
                   ATCA TTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGT TGAGTTA AG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTTTCAGTTTATTTCTTGTGATTTTAGT TTTTTTCTCTTAA CCAAATGCTAAATGGAT TTAG   
               
               
                   
               
               
                   GAGAAATAA ACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCAC AGATA   
               
               
                   
               
               
                   GAA TACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAG 
               
               
                   
               
               
                 TTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGC 
               
               
                   
               
               
                 CCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCA GGAAATTCTCC AAGT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACAGCTAAACCCACGGCT TCCTTTCTCGTAA AACCAAAACAAAAAGGCTTTCTATTCAA 
               
               
                   
               
               
                 SEQ ID NO: 21 
                   
               
               
                 GTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAAT GGGAAACCAT GGACG 
                   
               
               
                   
               
               
                 GACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTTCTA 
               
               
                   
               
               
                 GAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCACAGA 
               
               
                   
               
               
                   ATTCCGGGCATA TTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAAACT 
               
               
                   
               
               
                 CTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCTGTC 
               
               
                   
               
               
                 TACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCTGCT 
               
               
                   
               
               
                 TGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAAT CTCTTGAATC   
               
               
                   
               
               
                   A TTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGT TGAGTTA AGGCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TCAGTTTATTTCTTGTGATTTTAGT TTTTTTCTCTTAA CCAAATGCTAAATGGAT TTAGGAG   
               
               
                   
               
               
                   AAATAA ACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCAC AGATAGAA   
               
               
                   
               
               
                 TACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTA 
               
               
                   
               
               
                 GCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCA 
               
               
                   
               
               
                 GAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCA GGAAATTCTCC AAGTCTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GCTAAACCCACGGCT TCCTTTCTCGTAA AACCAAAACAAAAAGGCTTTCTATTCAA 
               
               
                   
               
               
                 SEQ ID NO: 22 
                   
               
               
                 TCCTGTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATT 
                   
               
               
                   
               
               
                 TTCTGCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAAT CTC   
               
               
                   
               
               
                   TTGAATCA TTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGT TGAGT   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CCTATTTTCAGTTTATTTCTTGTGATTTTAGT TTTTTTCTCTTAA CCAAATGCTAAATGGAT 
               
               
                   
               
               
                   TTAGGAGAAATAA ACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCAC A   
               
               
                   
               
               
                   GATAGAA TACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAG 
               
               
                   
               
               
                 AAAGTTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGG 
               
               
                   
               
               
                 ATGCCCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCA GGAAATTCTCC   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CGTAACAGCTAAACCCACGGCT TCCTTTCTCGTAA AACCAAAACAAAAAGGCTTTCTATTCA 
               
               
                   
               
               
                 A 
               
               
                   
               
               
                 SEQ ID NO: 23 
                   
               
               
                 TAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGTAGTTTTGGAGTT 
                   
               
               
                   
               
               
                 GTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCAGAAGATTGAATAAAATTGGGAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TGTTCAGTTGAGTGCTTGAGGTTGTCTTTTCGACGTAACAGCTAAACCCACGGCT TCCTTTC   
               
               
                   
               
               
                   TCGTAA AACCAAAACAAAAAGGCTTTCTATTCAA 
               
               
                   
               
               
                 SEQ ID NO: 24 
                   
               
               
                 GTATTTTTCTCTGTGCAACAGCCCTGAGCTCTAGAACTTATGACAGCTTTTCTCAAGTGAAG 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   CTTTCCTCAT GATGCTACGGGGAGTACAGTGAAAAGGGAGGAGATTGGGGTAGCAAGGTCCC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GGGGGATCCCTGCAGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCA 
               
               
                   
               
               
                 TTCCTAGTGGCTGGCCTAAAGCCCCAAACCTGGAATCTGTTTCAGAGCCTGGACCTTTTGGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GATCAGCAGGCCTTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGC 
               
               
                   
               
               
                 CAGACTCTCTAAATCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGT 
               
               
                   
               
               
                 CTG TGCCTCA G TTTTCCTCAT CTGTAAAATGGGAGATTAGTGGTCACCATTGGGATGATTAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAA 
               
               
                   
               
               
                 AAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTA AGAT   
               
               
                   
               
               
                   ATAT TATTTCATATAAAACATTTAAGTACATATACATAT AATTATCT ATGTAATTATTATTG 
               
               
                   
               
               
                 TTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTA 
               
               
                   
               
               
                 AACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACAATGAATGCCACATAAATA TCATTTCTCAGAT TCCTATGATGCTCTTCTTTCAGATCTTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTTTTAGGTCCTGTTAACCCCCATAACTAT TGATTCA GAGAAGTAGGGTGGTTCTGAAAAAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 SEQ ID NO: 25 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCATTCCTAGTGGCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGCCAGACTCTCTAA 
               
               
                   
               
               
                 ATCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTG TGCCTCA G T   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATAT 
               
               
                   
               
               
                 TTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTA AGATATAT TATTTCAT 
               
               
                   
               
               
                 ATAAAACATTTAAGTACATATACATAT AATTATCT ATGTAATTATTATTGTTATTGTTGTTA 
               
               
                   
               
               
                 TTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGT 
               
               
                   
               
               
                 CTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACATAAATA TCATTTCTCAGAT TCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GTTAACCCCCATAACTAT TGATTCA GAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAATAATCCATGCATTGTT TAACTCA TGTCATAAGCAATAATGCCTTTCATATAGCCATTGG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 SEQ ID NO: 26 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TCTGGATGGAGGGATCAGCAGGCCTTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGA 
               
               
                   
               
               
                 GGACTCTGGAGCCAGACTCTCTAAATCATAGCACTGACACTTACTATATGACTTGGGCCAGG 
               
               
                   
               
               
                 TTCGTTACCTGTCTG TGCCTCA G TTTTCCTCAT CTGTAAAATGGGAGATTAGTGGTCACCAT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TATATACAAACAGTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTAT 
               
               
                   
               
               
                 ATTCATATATAAAAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATT 
               
               
                   
               
               
                 TTGCATTA AGATATAT TATTTCATATAAAACATTTAAGTACATATACATAT AATTATCT ATG 
               
               
                   
               
               
                 TAATTATTATTGTTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCAC 
               
               
                   
               
               
                 GGCAGGCTTTTAAACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ACATTGCAATCTACAATGAATGCCACATAAATA TCATTTCTCAGAT TCCTATGATGCTCTTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATCATGTGAGTATTTTTAGGTCCTGTTAACCCCCATAACTAT TGATTCA GAGAAGTAGGGTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 SEQ ID NO: 27 
                   
               
               
                 CTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTG TGCCTCA G TTTTCCTCAT   
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATGTATAAACTATAGTTATAATGTAAATATATATACAAACAGTAACCATTTACATGCATATA 
               
               
                   
               
               
                 AAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATATTTTATATAT 
               
               
                   
               
               
                 AAAATATATTACATTTAATTTTTTACATTTTGCATTA AGATATAT TATTTCATATAAAACAT 
               
               
                   
               
               
                 TTAAGTACATATACATAT AATTATCT ATGTAATTATTATTGTTATTGTTGTTATTGTCTACT 
               
               
                   
               
               
                 TTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGTCTCACTGAG 
               
               
                   
               
               
                 CCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAGGGTTTAAG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                   TCATTTCTCAGAT TCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTCTATAATAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CATAACTAT TGATTCA GAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCTCTTTAACT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 ATGCATTGTT TAACTCA TGTCATAAGCAATAATGCCTTTCATATAGCCATTGGCATCAAAGA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 SEQ ID NO: 28 
                   
               
               
                 CAGTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATAT 
                   
               
               
                   
               
               
                 AAAAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTA AG   
               
               
                   
               
               
                   ATATAT TATTTCATATAAAACATTTAAGTACATATACATAT AATTATCT ATGTAATTATTAT 
               
               
                   
               
               
                 TGTTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTT 
               
               
                   
               
               
                 TAAACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CTACAATGAATGCCACATAAATA TCATTTCTCAGAT TCCTATGATGCTCTTCTTTCAGATCT 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TATTTTTAGGTCCTGTTAACCCCCATAACTAT TGATTCA GAGAAGTAGGGTGGTTCTGAAAA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 SEQ ID NO: 29 
                   
               
               
                 CCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAGGGTTTAAGTTAGTTTAAAACTGAC 
                   
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 TATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTCTATAATAATTTTGTTTGTTTCTTG 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 GAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCTCTTTAACTTGTTTTATAGGAACCA 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
     As used herein, “consisting essentially of” allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with the alternative expressions “consisting essentially of” or “consisting of”. 
     While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein. 
     REFERENCES 
     
         
         1. Blood. 2014 Apr. 24; 123(17):e68-78 
         2. Molecular and cellular biology 1997, p. 4220-4229 
         3. Science 7 Nov. 2003: Vol. 302, Issue 5647, pp. 1041-1043 
         4. J Immunol. 2015 Oct. 1; 195(7):3058-70 
         5. J Immunol. 1996 156:4154-4159 
         6. J Biol. Chem. 2006 Sep. 29; Vol. 281, No 39, pp. 28666-28678 
         7. European J Immunol. 2012. 42:1850-1862