RECOMBINANT VECTORS SUITABLE FOR THE TREATMENT OF IPEX SYNDROME

Abstract

IPEX (Immune dysregulation Polyendocrinopathy X linked) syndrome is a primary immunodeficiency 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 PGK-EF1a promoter that showed remarkable efficiency.

Claims

1. A recombinant nucleic acid molecule comprising a bidirectional PGK-EF1a promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction, wherein the bidirectional PGK-EF1a promoter comprises a first PGK portion and a second EF1a portion, and wherein the first transgene is under control of the first PGK portion of the bidirectional PGK-EF1a promoter and encodes a protein that is not constitutively expressed by a T cell, and the second transgene is under control of the second EF1a portion of the bidirectional PGK-EF1a promoter and encodes a transcription factor.

2. The recombinant nucleic acid molecule of claim 1 wherein the first PGK 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 second EF1a 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 first PGK portion and the second EF1a 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 first transgene and the second transgene are codon-optimized.

8. The recombinant nucleic acid molecule of claim 1 wherein the first transgene that is under the control of the first PGK 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 second EF1a 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:6.

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 the step of 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 recombinant nucleic acid molecule of claim 8 wherein an intracytoplasmic part (ΔLNGFR) is truncated from the LNGFR.

Description

FIGURES

[0062] FIG. 1 depicts the different constructions tested by the inventors.

[0063] FIG. 2 depicts the flow cytometry analysis at Day 5.

[0064] FIG. 3 depicts the transduction efficacy quantified by the percentage of ΔLNGFR at day 5 after transduction in WT and Scurfy CD4+ T cells. Transduction efficiency was significantly increased with LNGFRp-eFOXP3 vector compared to LNGFR.t2a.FOXP3 both for WT and Scurfy CD4+ T cells (with a p-value=0.002 and =0.007 respectively, Mann-Whitney test). On the opposite, transduction efficacy with the mock vectors was higher with the T2A construct with p-value=0.02 and =0.04 respectively in WT and Scurfy CD4+ T cells. (n=3 independent experiment for WT CD4+ T cells and 2 independent experiments for Scurfy CD4+Tc ells).

EXAMPLE

[0065] Material and Methods

[0066] Mice

[0067] Scurfy phenotype was obtained by backcrossing on B6.129S7-Rag1tmlMom/J background, allowing generation of homozygous XSf/XSf.Rag1−/− female. Crossing of these female with WT C57BL/6J mice result in the birth only of diseased XSf/Y.Rag1−/+ male.

[0068] WT CD4 T Cells

[0069] Splenocytes were harvested from C57BL/6J by aseptic removal. After gentle crushing of spleens through a 70 μM mesh filter, CD4+ T cells were isolated by negative selection using EasySep Mouse CD4+ T cell Isolation Kit (StemCell Technologies, Grenoble, France). Purity exceeded 90%.

[0070] Scurfy CD4 T Cells

[0071] From XSf/Y.Rag1−/+ mice of 10 days, lymph nodes were collected and CD4+ T cells were separated using Murine CD4+ T cell Isolation kit (Miltenyi Biotec, Paris, France). Briefly, CD4+ collected from lymph nodes were labeled with a cocktail of biotinylated antibodies targeting CD4− cells, followed by labeling with anti-biotin magnetic beads. Cells were separated on an LS column (Miltenyi Biotec) and CD4+ cells were collected in the flow through. Purity exceeded 90%.

[0072] WT Tregs CD4+CD25+

[0073] Splenocytes and lymph nodes were harvested from B6LY5.1 CD45.1 (8-12 weeks) and CD4+ T cells were isolated using EasySep Mouse CD4+ T cell Isolation Kit. A staining of CD25+ cells was performed with an anti-CD25 PE antibody (clone PC61, BD Biosciences, Le Pont de Claix, France), and then CD4+CD25+ cells were sorted on SH800 (Sony Biotechnology, Weybridge, UK) or ARIA II (BD Biosciences) cells sorters with a nozzle of 100 μm. For Treg suppression assay, CD4+CD25− cells were also sorted.

[0074] Lentiviral Vector

[0075] The cDNA for a truncated codon-optimized human ΔLNGFR and/or a codon optimized human FOXP3 was cloned in a pCCL backbone with different designs. Bidirectional vector contains ΔLNGFR in a reverse position under the control of respectively PGK or mCMV promoters, FOXP3 is under the control of EF1a promoter. A unidirectional polyA sequence was added to terminate transduction of the reverse gene.

[0076] In T2A designs, expression is under the control of EF1a. Two constructs were built: ΔLNGFR followed by the T2A sequence and FOXP3 or FOXP3 followed by the T2A and ΔLNGFR.

[0077] Lentiviral vectors were packaged with a VSV-G pseudotype as previously described. Production of bidirectional constructs was increased thanks to co-transfection with NovB2 plasmid26.

[0078] T Cell Transduction

[0079] Freshly isolated CD4+ T cells were plated at 4.10.sup.6 cells/mL in round bottom plate in RPMI 1640 medium+GlutaMax (GIBCO, Thermo Fisher Scientific, Montigny-Le-Bretonneux, France) supplemented with 10% fetal bovine serum (GIBCO), 1% Penicillin-Streptomycin (GIBCO), 0.1% 2-mercaptoethanol (GIBCO). Medium was supplemented with recombinant murine IL-2 (Peprotech, Rocky Hill, USA) at a concentration of 100 UI/ml for WT CD4 T cells or 300 UI/ml for Scurfy CD4 T cells. Cells were activated and expanded with anti-CD3/CD28 Dynabeads (GIBCO) at a 1:1 bead:cell ratio. Transduction was performed according the protocol previously described.sup.43 (ref article LB). Briefly transduction medium (RPMI supplemented with 0.25 mg/ml Lentiboost (Sirion Biotech, FlashTherapeutics, Toulouse, France)) was added to cells with lentiviral vector at a MOI 10 concomitantly with activation and incubated overnight. Transduced cells were stained at day 5 after transduction by ΔLNGFR PE antibodies (clone ME20.4-1.H4, Miltenyi Biotec) and sorted on SH800 (Sony Biotechnology).

[0080] Determination of Vector Copies Number

[0081] Genomic DNA was extracted from samples 10 days after transduction using a Genomic DNA Purification kit (Qiagen, Cergy-Pontoise, France). VCN were quantified using qPCR or ddPCR. qPCR were performed following the protocol previously described.sup.43. For ddPCR, gDNA were first digested by Hind III HF (New England Biolabs, Evry, France) then mixed with ddPCR Mastermix (Bio-rad, Marnes-la-Coquette, France), primers and probes specific to the HIV Psi region (Bio-Rad) and a sequence in the murine genome (Titin) or human genome (Albumin) for normalization. Droplet generation was performed using the QX100 Droplet. The concentration of specific amplified portions was quantified using the QX200 Droplet Reader/Quantasoft VI.7 (Bio-Rad).

[0082] Treg Suppression Assay

[0083] Bonafide WT Treg cells (CD4+CD25+) or indicated engineered CD4+ Tcells from Scurfy mice were co-cultured with Tconv (WT CD4+CD25-, 1.104 cells/well) and stimulated with anti-CD3 (1 μg/ml) in the presence of mitomycin C (50 μg/ml) (Merck KGaA) treated splenocytes, 1.104/well) with complete RPMI medium in round-bottom 96-well plates. Treg cells or engineered CD4+ Tcells were labeled with 5 μM Cell trace violet proliferation dye (Thermo Fisher Scientific) whereas Tconv cells were labeled with 5 μM CFSE to differentiate the two populations.

[0084] Suppressive cells were co-cultured with Tconv at degressive ratio Treg:Tconv (1:1, 2:1, 4:1, 8:1, 16:1, 32:1, 64:1) for 3 days followed by FACS analysis (MACSquant, Miltenyi Biotec). 7AAD staining was added to remove dead cells. Proliferation index was calculated with FlowJo (BD Biosciences) modelisation.

[0085] Flow Cytometry

[0086] Single cell suspensions from spleen and lymph nodes were obtained by gentle crushing of spleens through a 70 μM mesh filter. Samples from the lung and the liver were prepared after digestion with Collagenase IV (Thermo Fischer Scientific) followed by gentle crushing of spleens through a 100 μM mesh filter.

[0087] Samples were prepared for flow cytometry using the following method: Cells were resus-pended in 100 uL of FACS buffer (phosphate buffered saline (PBS, Corning)/2% Fetal Bovine Serum [GIBCO]) and incubated with 2 uL of each antibody 7AAD (Miltenyi Biotec) for 20-30 min at 4 C.

[0088] Cells were washed once in FACS buffer prior to analysis. For intracellular FoxP3 staining, cells were first stained with cell surface markers and fixable viability dye eF780 (eBioscience, Thermo Fischer Scientific) as described above. After washing, cells were fixed and permeabilized using the FoxP3 staining buffer set eBioscience, Thermo Fischer Scientific) according to manufacturers' directions. Human FoxP3-APC (eBioscience, Thermo Fischer Scientific) was added for 30-60 min at RT. Samples were acquired on a MACSquant flow cytometer (Miltenyi Biotec), BD LSR Fortessa cytometer (BD Biosciences) or a Sony Spectral SH6800 (Sony Biotechnology). Data were analyzed using FlowJo V10 (TreeStar). The following antibodies were used: anti-mouse CD62L APC-Cy7 clone MEL-14, CD44APC clone IM7 (BD Biotechnology), CD45.1 APC-Cy7 clone A20, CD45.2 PeCy7 clone 104, CD134 clone OX-40 Brilliant Violet 421, CD279 (PD-1) clone 29F.1A12 Brilliant Violet 605, CD25 clone PC61 Brilliant Violet 711, TIGIT clone Vstm3 1G9 PE, CD357 (GITR) clone DTA-1 PerCP/Cy5.5, CD39 clone Duha59 PE/Cy7 and CD152 clone UC10-4B9 PE/Dazzle (Sony Biotechnology) and human ΔLNGFR PE clone ME20.4-1.H4 (Miltenyi Biotec), Helios clone 22F6 eF450 and human FOXP3 APC Clone PCH101 (eBioscience, Thermo Fischer Scientific)

[0089] Histology

[0090] Lung, liver and ear was collected after mice euthanasia and fixed in PFA 4% (Sigma). Tissues section was stained with HE and inflammation was analyzed as described by Workman and al. 44.

[0091] Statistical Analysis

[0092] Values are represented as means±SD, unless stated otherwise. GraphPad Prism 6.0 was used for all statistical analyses. P value was calculated with a confidence interval of 95% to indicate the statistical significance between groups. Statistical test included non-parametric Mann-Whitney test, Fischer exact test or two ways ANOVA depending on the dataset. A P value <0.05 was considered statistically significant. Statistically significant differences between groups are noted in figures with asterisks (*p<0.05, **p<0.01, ***p<0.001, ****p <0.0001). Correlations were performed with a non-parametric Spearman correlation. Survival was analyzed with Log-rank test (Mantel-Cox).

[0093] Ethics

[0094] Animal procedure received our institution ethics committee agreement and Ministère de l'Agriculture agreement according to European directive 2010/63/UE.

Results

Example 1

[0095] 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 zLNGFR genes, degree of correlation between both expression) (FIG. 1):

[0096] #91: unidirectional, EFS-FOXP3, PGK-ΔLNGFR

[0097] #95: unidirectional, PGK-FOXP3, EFS-ΔLNGFR

[0098] #99: bidirectional, ΔLNGFR-PGK, EF1a-FOXP3

[0099] #103: bidirectional, ΔLNGFR-mCMV, EF1a-FOXP3

[0100] #151: bicistronic, EF1a-ΔLNGFR-T2A-FOXP3

[0101] #155: bicistronic, EF1a-FOXP3-T2A-ΔLNGFR

[0102] Table 1 below illustrates vector titer, transduction efficiency measured in vector copy number (VCN) per cell at day 12 of culture, and coexpression 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 analysed by flow cytometry at D12 (FIG. 2).

TABLE-US-00012 TABLE 1 % % LNGFR+FOXP3+ VCN LNGFR+FOXP3+ (D 12) Vector Titer (D 12) (D 5) after sorting at D 5 #91 1.49 × 10e9 4 13.2 ND #95 ND ND 9.1 ND #99 1.36 × 10e9 1.18 32.7 83.6 #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

[0103] Constructs #151 and #155 were excluded because of low titers. #95 and #103 were excluded because of low levels of coexpression of FOXP3 and ZLNGFR 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 ZLNGFR genes. The only constructs that fulfilled the 4 criteria defined above is the bidirectional designs including forward hFOXP3co under the control of the EF1 promoter and reverse ΔLNGFRco under the control of PGK promoter (#99, pCCL.ΔLNGFRco.PGK.EF1a.hFOXP3co).

Example 2

[0104] Concomitant expression of two genes, here FOXP3 and ΔLNGFR reporter, can be obtained by either bicistronic construct or bidirectional promoters. We generated vectors (i) one with the bidirectional promoters architecture, one allowing FOXP3 expression under the control of the ubiquitous elongation factor 1 alpha (EF1a) and ΔLNGFR under the control of phosphoglycerate kinase (PGK) human promoter and their mock counterpart containing only the ΔLNGFR reporter (LNGFRp-eFOXP3 and LNGFRp-e, also known as #99) and (ii) two bicistronic using 2A self-cleaving peptide system with their mock counterpart (named eLNGFR.t2a.FOXP3 and eLNGFR.t2a, also known as #151 vs. eFOXP3.t2a.LNGFR and e.t2a.LNGFR, also known #155) (FIG. 1).

[0105] With the exception of LNGFRp-e, bidirectional vectors' titers quantified by titration assay were more than 10 fold higher as compared to bicistronic T2A vectors (Table 2).

TABLE-US-00013 TABLE 2 Vectors Titer (Ig/mL) LNGFRp-e 1.4 × 10.sup.8 LNGFRp-eFOXP3 (#99) 1.5 × 10.sup.9 eLNGFR.t2a 5.9 × 10.sup.7 eLNGFR.t2a.FOXP3 (#151) 1.1 × 10.sup.8 e.t2a.LNGFR 2.3 × 10.sup.7 eFOXP3.t2a.LNGFR (#155) 6.4 × 10.sup.7

[0106] Despite a bidirectional design the titer was sufficient thanks to the use for production of NovB2 which inhibits the RNA interference mechanism induced by the reverse transcript 26. CD4+ T cell isolated from WT mice were activated with anti-CD3/CD28, IL-2 and simultaneously transduced at a MOI 10 with the 4 constructs and their empty control counterparts. Surface ΔLNGFR and intracellular FOXP3 expressions were evaluated by flow cytometry 5 days after transduction (Data not shown). The level of transduction ranged from 5.2 to 25.2% ΔLNGFR+ cells. Correlation between FOXP3 and ΔLNGFR was respectively quantified with Spearman correlation at expression r.sup.2=0.51, r.sup.2=0.54, r.sup.2=0.66 and r.sup.2=0.61 respectively for LNGFRp-eFOXP3, eLNGFR.t2a.FOXP3 and eFOXP3.t2a.LNGFR vectors. eFOXP3.t2a.LNGFR vector was excluded for further evaluation because transduction efficiency was low. Both LNGFRp-eFOXP3 and eLNGFR.t2a.FOXP3 constructs were further tested in Scurfy CD4+ T cells. Scurfy CD4+ T cells are highly sensitive to cell sorting and culture, probably as a consequence of chronically activated environment. Therefore, we first optimized Scurfy CD4+ T cell sorting starting from lymph nodes to limit the contamination by B cells, granulocytes and monocytes and to reach purity above 95%. Viability of Scurfy CD4+T in culture was improved by the selection of donor mice of an age beyond 12 days to limit inflammation and maintained above 80% up to 12 days using 300 UI/mL IL-2 (compared to 100 UI/ml for WT CD4 T cells). As shown in FIG. 3, the level of transduction after 5 days was significantly higher with the bidirectional LNGFRp-eFOXP3 construct as compared to the bicistronic eLNGFR.t2a.FOXP3 construct not only for WT CD4+T lymphocytes, but also for Scurfy CD4+T lymphocytes (p-value=0.002 and 0.007 respectively). Transduced cells were sorted five days after transduction and expanded for 7 supplemental days for VCN quantification. VCN in WT CD4+T lymphocytes ranged between 0.9 and 1.9 for bidirectional design. VCN range was similar in Scurfy mice with bidirectional design ranging between 0.9 and 1.4. The poor viability of Scurfy CD4 cells transduced with T2A vectors did not allow quantification of VNC in those conditions. After 5 days of culture, mean fluorescent intensity of FOXP3 expression was similar between CD4.sup.LNGFRp-eFOXP3 and CD.sup.4eLNGFR.t2a.FOXP3 in WT cells but was significantly increased with the bidirectional LNGFRp-eFOXP3 construct in Scurfy CD4+T lymphocytes (p-value=0.04) (Data not shown). Since the three required criteria were achieved (efficient transduction, suitable correlation between FOXP3 and ΔLNGFR expression and stability in vitro), LNGFRp-eFOXP3 and its mock counterpart LNGFRp-e were selected for functional evaluation. They will be named LNGFR.FOXP3 and LNGFR vector thereafter.

[0107] Thymic Tregs are defined by a specific combination of surface molecules, which includes particularly CTLA-4 and CD25. At day 7 post transduction, CTLA-4 and CD25 expression was higher in CD4.sup.LNGFR.FOXP3 transduced cells as compared to CD4.sup.LNGFR transduced cells (Data not shown). Functional evaluation of transduced cells was performed using an in vitro suppression assay. ΔLNGFR+ cells were sorted at day 5 post-transduction with either LNGFR.FOXP3 or LNGFR vector. The capacity of sorted cells (CD4.sup.LNGFR.FOXP3 and CD4.sup.LNGFR) to suppress proliferation of CFSE labelled CD4+CD25− Tconv was measured at a 1:1 up to 1:64 suppressor-to-effector ratio and compared to that of WT CD4+CD25.sup.high Tregs as positive controls, and untransduced Scurfy CD4+ T cells (CD4.sup.UT) as negative controls (Data not shown). CD4.sup.LNGFR and CD4.sup.UT resulted in a small level of proliferation inhibition up to ratio 1:4 whereas CD4.sup.LNGFR.FOXP3 transduced CD4+ T cells were able to suppress Tconv proliferation as well as WT Tregs (up to a suppressor-to-effector ratio of 1:32).

[0108] Discussion:

[0109] In the present work, we developed a bidirectional lentiviral vector allowing the coexpression of hFOXP3 together with a ΔLNGFR surface reporter. In this study, murine HSPC were collected from Scurfy mice rescued by WT splenocytes injection and transduced. After engraftment in WT mice, corrected CD4 T cells were collected and demonstrated their ability to prevent the onset of Scurfy phenotype. The total number of corrected Scurfy CD4+ T cells injected in Scurfy neonates was ranging between 1.8×10.sup.7 and 2.5×10.sup.7 corresponding to a putative Tregs dose ranging between 9.8×10.sup.5 and 1.4×10.sup.6 cells. Moreover these results were obtained with high VCN ranging between 3.3 and 5.8. These results demonstrated the feasibility of a HSPC gene therapy but could suggest that the expression of FOXP3 driven by the endogenous promoter might require higher vector copy number and higher cell dose as compared to FOXP3 expression in CD4+ T cells driven by our vector. Our work demonstrated the advantage of a curative strategy based on genetic engineering of CD4+ T cells. The vector we designed allowed inducing a suppressive function in CD4 T cell at lower VCN between 1 and 2. Gene therapy of CD4 T cells with the expression of FOXP3 under the control of an ubiquitous promoter or gene therapy of HSCT with vector allowing the expression of FOXP3 under the control of its own regulated promoter are additive strategies.

REFERENCES

[0110] 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. [0111] 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. [0112] 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. [0113] 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. [0114] 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. [0115] 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. [0116] 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. [0117] 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. [0118] 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. [0119] 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. [0120] 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. [0121] 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. [0122] 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. [0123] 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. [0124] Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science 299:1057-1061. [0125] 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. [0126] Kalos, M., and C. H. June. 2013. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39:49-60. [0127] 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. [0128] 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. [0129] 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. [0130] Masiuk K E, Laborada J, Roncarolo M G, Hollis R P, Kohn D B. Lentiviral Gene Therapy in HSCs Restores Lineage-Specific Foxp3 Expression and Suppresses Autoimmunity in a Mouse Model of IPEX Syndrome. Cell Stem Cell. 2019; 24(2):309-317.e7. doi:10.1016/j.stem.2018.12.003 [0131] 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. [0132] 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. [0133] 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. [0134] 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. [0135] Sawant, D. V., and D. A. Vignali. 2014. Once a Treg, always a Treg? Immunol Rev 259:173-191. [0136] 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. [0137] 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. [0138] 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. [0139] 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. [0140] 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. [0141] 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. [0142] 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.