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RECOMBINANT VECTORS SUITABLE FOR THE TREATMENT OF IPEX SYNDROME

20220136006 · 2022-05-05

    Inventors

    Cpc classification

    International classification

    Abstract

    IPEX (Immune dysregulation Polyendocinopathy X linked) syndrome is a primary immunodeficience caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. Preclinical and clinical studies suggest that T cell gene therapy approaches designed to selectively restore the repertoire of Treg cells by transfer of wild type FOXP3 gene is a promising potential cure for IPEX. However, there is still a need for a vector that can be used efficiently for the preparation of said Treg cells. The inventors thus compared 6 different lentiviral constructs according to 4 criteria (vector titers, level of transduction of human CD4+ T cells, level of expression of FOXP3 and ΔLNGFR genes, degree of correlation between both expression) and selected one construct comprising a bidirectional EFS-PGK promoter that showed remarkable efficiency.

    Claims

    1. A recombinant nucleic acid molecule comprising a bidirectional EFS-PGK promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction, wherein the bidirectional EFS-PGK promoter comprises an EFS portion derived from the EFS promoter and a PGK portion derived from the PGK promoter, wherein the first transgene is under the control of the EFS portion of the bidirectional promoter and encodes a protein that is not constitutively expressed by a T cell and the second transgene is under the control of the PGK portion of the bidirectional promoter and encodes a transcription factor.

    2. The recombinant nucleic acid molecule of claim 1 wherein the EFS portion comprises a nucleic sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:3.

    3. The recombinant nucleic acid molecule of claim 1 wherein the PGK portion comprises a nucleic sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:2.

    4. The recombinant nucleic acid molecule of claim 1 wherein the EFS portion and the PGK portion are separated by a spacer sequence.

    5. The recombinant nucleic acid molecule of claim 4 wherein the spacer sequence comprises a nucleic sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:4.

    6. The recombinant nucleic acid molecule of claim 1 wherein the bidirectional promoter comprises a nucleic acid sequence having at least 80% of identity with the sequence as set forth in SEQ ID NO:5.

    7. The recombinant nucleic acid molecule of claim 1 wherein the sequences of the transgenes are codon-optimized.

    8. The recombinant nucleic acid molecule of claim 1 wherein the first transgene that is under the control of the EFS portion of the bidirectional promoter encodes for a low-affinity nerve growth factor receptor (LNGFR).

    9. The recombinant nucleic acid molecule of claim 1 wherein the second transgene that is under the control of the PGK portion of the bidirectional promoter encodes for FoxP3.

    10. The recombinant nucleic acid molecule of claim 1 which comprises: i) a first nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID: 8 ii) a second nucleic acid sequence having at least 80% of identity with the nucleic acid sequence acid sequence as set forth in SEQ ID NO:5 and iii) a third nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:7.

    11. The recombinant acid molecule of claim 1 which comprises a nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:11.

    12. A lentiviral vector which comprises the recombinant acid molecule of claim 1.

    13. (canceled)

    14. A method of producing a population of Treg cells, comprising, transfecting or transducing a population of T cells in vitro or ex vivo with the lentiviral vector of claim 12.

    15. A population of Treg cells obtainable by the method of claim 14.

    16. A method of treating an autoimmune disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of Treg cells of claim 15.

    17. The method of claim 16 wherein the autoimmune disease is IPEX syndrome.

    18. A nucleic acid sequence as set forth in SEQ ID NO:7.

    19. The method of claim 14, wherein the population of Treg cells that express LNGFR at their cell surface.

    20. The recombinant nucleic acid molecule of claim 8, wherein the LNGFR is a low-affinity nerve growth factor receptor truncated of its intracytoplasmic part (ΔLNGFR).

    Description

    FIGURES

    [0063] FIG. 1 depicts the different constructions tested by the inventors.

    [0064] FIG. 2 depicts the flow cytometry analysis at Day 5.

    EXAMPLE

    [0065] We compared 6 different lentiviral constructs according to 4 criteria (vector titers, level of transduction of human CD4+ T cells, level of expression of FOXP3 and ΔLNGFR genes, degree of correlation between both expression) (FIG. 1): [0066] #91: unidirectional, EFS-FOXP3, PGK-ΔLNGFR [0067] #95: unidirectional, PGK-FOXP3, EFS-ΔLNGFR [0068] #101: bidirectional, ΔLNGFR-EFS,PGK-FOXP3 [0069] #103: bidirectional, ΔLNGFR-mCMV, EF1a-FOXP3 [0070] #151: bicistronic, EF1a-ΔLNGFR-T2A-FOXP3 [0071] #155: bicistronic, EF1a-FOXP3-T2A-ΔLNGFR

    [0072] Table 1 below illustrates vector titer, transduction efficiency measured in vector copy number (VCN) per cell at day 12 of culture, and co-expression of FOXP3 and ΔLNGFR measured by flow cytometry indicated as % of CD4+ T cells at day 5. In some cases, ΔLNGFR+ cells were sorted at day 5, further cultured for 12 days and analyzed by flow cytometry at D12 (FIG. 2).

    TABLE-US-00011 TABLE 1 % LNGFR + % LNGFR + VCN FOXP3 + FOXP3 + (D12) Vector Titer (D12) (D5) after sorting at D5 #91  1.49 × 10e9 4   13.2  ND #95  ND ND 9.1 ND #101  4.1 × 10e9 4   69   88 #103  1.3 × 10e9  0.45 11.8  ND #151 2.35 × 10e8  0.61 25.9  ND #155 6.36 × 10e7  0.31 20.6  ND

    [0073] Constructs #151 and #155 were excluded because of low titers. #95 and #103 were excluded because of low levels of co-expression of FOXP3 and ΔLNGFR genes and low VCN for #103. #91 was excluded because of the low level of expression of FOXP3. To note, the bidirectional construct tested by Passerini and coll (Passerini et al., 2013) that we reproduce herein with the codon optimized version (#103) was not efficient in terms of correlation of expression of FOXP3 and ΔLNGFR genes. The only constructs that fulfilled the 4 criteria defined above is the bidirectional designs including forward hFOXP3co under the control of the PGK promoter and reverse ΔLNGFRco under the control of EFS promoter (#101, pCCL.ΔLNGFRco.EFS.PGK.hFOXP3co).

    REFERENCES

    [0074] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0075] Aarts-Riemens, T., M. E. Emmelot, L. F. Verdonck, and T. Mutis. 2008. Forced overexpression of either of the two common human Foxp3 isoforms can induce regulatory T cells from CD4(+)CD25(−) cells. Eur J Immunol 38:1381-1390. [0076] Aiuti, A., R. Bacchetta, R. Seger, A. Villa, and M. Cavazzana-Calvo. 2012. Gene therapy for primary immunodeficiencies: Part 2. Curr Opin Immunol 24:585-591. [0077] Aiuti, A., S. Vai, A. Mortellaro, G. Casorati, F. Ficara, G. Andolfi, G. Ferrari, A. Tabucchi, F. Carlucci, H. D. Ochs, L. D. Notarangelo, M. G. Roncarolo, and C. Bordignon. 2002. Immune reconstitution in ADA-SCID after PBL gene therapy and discontinuation of enzyme replacement. Nat Med 8:423-425. [0078] Allan, S. E., A. N. Alstad, N. Merindol, N. K. Crellin, M. Amendola, R. Bacchetta, L. Naldini, M. G. Roncarolo, H. Soudeyns, and M. K. Levings. 2008. Generation of potent and stable human CD4+T regulatory cells by activation-independent expression of FOXP3. Mol Ther 16:194-202. [0079] Barzaghi, F., L. Passerini, and R. Bacchetta. 2012. Immune dysregulation, polyendocrinopathy, enteropathy, x-linked syndrome: a paradigm of immunodeficiency with autoimmunity. Front Immunol 3:211. [0080] Bennett, C. L., J. Christie, F. Ramsdell, M. E. Brunkow, P. J. Ferguson, L. Whitesell, T. E. Kelly, F. T. Saulsbury, P. F. Chance, and H. D. Ochs. 2001. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 27:20-21. [0081] Blaese, R. M., K. W. Culver, A. D. Miller, C. S. Carter, T. Fleisher, M. Clerici, G. Shearer, L. Chang, Y. Chiang, P. Tolstoshev, J. J. Greenblatt, S. A. Rosenberg, H. Klein, M. Berger, C. A. Mullen, W. J. Ramsey, L. Muul, R. A. Morgan, and W. F. Anderson. 1995. T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 270:475-480. [0082] Bonino, C., G. Ferrari, S. Verzeletti, P. Servida, E. Zappone, L. Ruggieri, M. Ponzoni, S. Rossini, F. Mavilio, C. Traversari, C. Bordignon. 1997. HSV-TK Gene Transfer into Donor Lymphocytes for Control of Allogeneic Graft-Versus-Leukemia. Science 276: 1717-1724. [0083] Bonini, C., M. K. Brenner, H. E. Heslop, and R. A. Morgan. 2011. Genetic modification of T cells. Biol Blood Marrow Transplant 17:S15-20. [0084] Brunkow, M. E., E. W. Jeffery, K. A. Hjerrild, B. Paeper, L. B. Clark, S. A. Yasayko, J. E. Wilkinson, D. Galas, S. F. Ziegler, and F. Ramsdell. 2001. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet 27:68-73. [0085] Ferraro, A., A. M. D′Alise, T. Raj, N. Asinovski, R. Phillips, A. Ergun, J. M. Replogle, A. Bernier, L. Laffel, B. E. Stranger, P. L. De Jager, D. Mathis, and C. Benoist. 2014. Interindividual variation in human T regulatory cells. Proc Natl Acad Sci USA 111:E1111-1120. [0086] Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 4:330-336. [0087] Fu, W., A. Ergun, T. Lu, J. A. Hill, S. Haxhinasto, M. S. Fassett, R. Gazit, S. Adoro, L. Glimcher, S. Chan, P. Kastner, D. Rossi, J. J. Collins, D. Mathis, and C. Benoist. 2012. A multiply redundant genetic switch ‘locks in’ the transcriptional signature of regulatory T cells. Nat Immunol 13:972-980. [0088] Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057-1061. [0089] Horino, S., Y. Sasahara, M. Sato, H. Niizuma, S. Kumaki, D. Abukawa, A. Sato, M. Imaizumi, H. Kanegane, Y. Kamachi, S. Sasaki, K. Terui, E. Ito, I. Kobayashi, T. Ariga, S. Tsuchiya, and S. Kure. 2014. Selective expansion of donor-derived regulatory T cells after allogeneic bone marrow transplantation in a patient with IPEX syndrome. Pediatr Transplant 18:E25-30. [0090] Kalos, M., and C. H. June. 2013. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39:49-60. [0091] Kasow, K. A., V. M. Morales-Tirado, D. Wichlan, S. A. Shurtleff, A. Abraham, D. A. Persons, and J. M. Riberdy. 2011. Therapeutic in vivo selection of thymic-derived natural T regulatory cells following non-myeloablative hematopoietic stem cell transplant for IPEX. Clin Immunol 141:169-176. [0092] Khattri, R., T. Cox, S. A. Yasayko, and F. Ramsdell. 2003. An essential role for Scurfin in CD4+CD25+T regulatory cells. Nat Immunol 4:337-342. [0093] Li, W., L. Wang, H. Katoh, R. Liu, P. Zheng, and Y. Liu. 2011. Identification of a tumor suppressor relay between the FOXP3 and the Hippo pathways in breast and prostate cancers. Cancer Res 71:2162-2171. [0094] Mottet, C., H. H. Uhlig, and F. Powrie. 2003. Cutting edge: cure of colitis by CD4+CD25+ regulatory T cells. J Immunol 170:3939-3943. [0095] Muul, L. M., L. M. Tuschong, S. L. Soenen, G. J. Jagadeesh, W. J. Ramsey, Z. Long, C. S. Carter, E. K. Garabedian, M. Alleyne, M. Brown, W. Bernstein, S. H. Schurman, T. A. Fleisher, S. F. Leitman, C. E. Dunbar, R. M. Blaese, and F. Candotti. 2003. Persistence and expression of the adenosine deaminase gene for 12 years and immune reaction to gene transfer components: long-term results of the first clinical gene therapy trial. Blood 101:2563-2569. [0096] Passerini, L., E. Rossi Mel, C. Sartirana, G. Fousteri, A. Bondanza, L. Naldini, M. G. Roncarolo, and R. Bacchetta. 2013. CD4(+) T cells from IPEX patients convert into functional and stable regulatory T cells by FOXP3 gene transfer. Sci Transl Med 5:215ra174. [0097] Provasi, E., P. Genovese, A. Lombardo, Z. Magnani, P. Q. Liu, A. Reik, V. Chu, D. E. Paschon, L. Zhang, J. Kuball, B. Camisa, A. Bondanza, G. Casorati, M. Ponzoni, F. Ciceri, C. Bordignon, P. D. Greenberg, M. C. Holmes, P. D. Gregory, L. Naldini, and C. Bonini. 2012. Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer. Nat Med 18:807-815. [0098] Sawant, D. V., and D. A. Vignali. 2014. Once a Treg, always a Treg? Immunol Rev 259:173-191. [0099] Seidel, M. G., G. Fritsch, T. Lion, B. Jurgens, A. Heitger, R. Bacchetta, A. Lawitschka, C. Peters, H. Gadner, and S. Matthes-Martin. 2009. Selective engraftment of donor CD4+25high FOXP3-positive T cells in IPEX syndrome after nonmyeloablative hematopoietic stem cell transplantation. Blood 113:5689-5691. [0100] Tang, Q., K. J. Henriksen, M. Bi, E. B. Finger, G. Szot, J. Ye, E. L. Masteller, H. McDevitt, M. Bonyhadi, and J. A. Bluestone. 2004. In vitro-expanded antigen-specific regulatory T cells suppress autoimmune diabetes. J Exp Med 199:1455-1465. [0101] Thornton, A. M., and E. M. Shevach. 1998. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 188:287-296. [0102] Trzonkowski, P., M. Bieniaszewska, J. Juscinska, A. Dobyszuk, A. Krzystyniak, N. Marek, J. Mysliwska, and A. Hellmann. 2009. First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127-T regulatory cells. Clin Immunol 133:22-26. [0103] Wieckiewicz, J., R. Goto, and K. J. Wood. 2010. T regulatory cells and the control of alloimmunity: from characterisation to clinical application. Curr Opin Immunol 22:662-668. [0104] Wildin, R. S., F. Ramsdell, J. Peake, F. Faravelli, J. L. Casanova, N. Buist, E. Levy-Lahad, M. Mazzella, O. Goulet, L. Perroni, F. D. Bricarelli, G. Byrne, M. McEuen, S. Proll, M. Appleby, and M. E. Brunkow. 2001. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet 27:18-20. [0105] Yagi, H., T. Nomura, K. Nakamura, S. Yamazaki, T. Kitawaki, S. Hori, M. Maeda, M. Onodera, T. Uchiyama, S. Fujii, and S. Sakaguchi. 2004. Crucial role of FOXP3 in the development and function of human CD25+CD4+ regulatory T cells. Int Immunol 16:1643-1656.