Optimized RAG1-deficient gene therapy

Abstract

The present invention provides novel expression cassettes, retroviral plasmids, vectors, virions, compositions, and recombinant cells comprising a promoter operably linked to a codon-optimized recombination-activated (RAG1) transgene. These novel expression cassettes, retroviral plasmids, vectors, virions, compositions, and recombinant cells are useful for treating diseases caused by complete or partial loss of function of the protein encoded by the rag-1 gene, such as RAG-deficient severe combined immunodeficiency disease (RAG1-SCID), Omenn's syndrome (OS), atypical SCID, or combined immunodeficiency disease (CID). Corresponding methods of treatment are also provided.

Description

[Technical Field]

[0001] The present invention provides novel expression cassettes, retroviral plasmids, vectors, virions, compositions, and recombinant cells comprising a promoter operably linked to a codon-optimized recombination-activated (RAG1) transgene. These novel expression cassettes, retroviral plasmids, vectors, virions, compositions, and recombinant cells are useful for treating diseases caused by complete or partial loss of function of the protein encoded by the rag-1 gene, such as RAG-deficient severe combined immunodeficiency (RAG1-SCID), Omenn's syndrome (OS), atypical SCID, or combined immunodeficiency (CID). Corresponding methods of treatment are also provided. [Background technology]

[0002] background Gene therapy for rare inherited immune disorders has recently become a clinical reality, particularly for severe combined immunodeficiency (SCID). For example, two major types of SCID (ADA-SCID and X-SCID) have been successfully treated with autologous stem cell-based gene therapy. However, there is no treatment for the most common type of SCID, SCID with recombination deficiency (e.g., RAG-deficient SCID; also known as RAG-SCID), due to the high genetic complexity involved.

[0003] Patients with RAG-deficient SCID have mutations in either RAG1 or RAG2, which are required for genetic recombination of the T cell receptor (TCR) and B cell receptor (BCR). Affected children typically experience a variety of severe, life-threatening infections, including pneumonia, meningitis, and sepsis. The only current treatment for RAG-SCID is replacement of diseased bone marrow with healthy, unmodified allogeneic stem cells via allogeneic stem cell transplantation (allo-SCT). While overall survival rates are satisfactory for matched SCT recipients, outcomes are significantly worse for mismatched SCT recipients, who account for the majority of cases.

[0004] Furthermore, approximately 25% of transplant recipients develop graft-versus-host disease, significantly reducing outcomes in terms of morbidity, immune reconstitution, and transplant-related mortality (Gennery, 2010). Thus, transplant outcomes in RAG-SCID (and other recombination-deficient T-SCID and B-SCID) are significantly worse than in B-cell-containing SCID (i.e., TB+SCID). Collectively, these data suggest that allogeneic SCT, the only currently available treatment option, has significant limitations in terms of both cure potential and survival potential, demonstrating the urgent need for new and improved strategies based on gene correction of autologous stem cells.

[0005] Successful clinical trials using autologous stem cell-based gene therapy have been conducted for the treatment of X-linked SCID and ADA-SCID, but these trials have revealed severe adverse effects: the development of lymphoproliferative disorders / leukemia. In both cases, T-cell acute lymphoblastic leukemia (T-ALL) occurred as a direct result of insertional gene mutagenesis caused by the retroviral vector used to deliver the therapeutic gene. Following this severe failure of gene therapy, recent studies have shown that next-generation vectors, particularly those with inactivated viral promoter / enhancer sequences (self-inactivating vectors or SIN vectors), significantly reduce the incidence of insertional mutagenesis.

[0006] Recent clinical trials targeting X-linked SCID and ADA-SCID have demonstrated the safety and efficacy of SIN lentiviral vectors, facilitating the clinical development of genetically modified hematopoietic stem cells. However, unlike X-linked SCID and ADA-SCID, gene therapy for the treatment of RAG-SCID has proven extremely challenging. Previous attempts (Lagrésle-Peyrou, 2006) used gammaretroviral vectors in preclinical Rag1- / - models, but this carried a high risk of insertional mutagenesis. While RAG1 gammaretroviral vectors were more easily able to correct the defect, SIN lentiviral vectors initially failed to express the therapeutic RAG1 gene, resulting in a "leaky" SCID or omen-like phenotype. Due to differences in the expression levels and transduction efficiencies achieved for therapeutic genes, conflicting results have been observed in this field (van Til., 2014).

[0007] New and improved strategies for treating RAG1-deficient severe combined immunodeficiency (SCID) and Omenn's syndrome (OS) are needed. Summary of the Invention

[0008] Disclosure summary Using clinically tolerated lentiviral gene therapy and a codon-optimized RAG1 transgene sequence, the inventors surprisingly discovered a minimal threshold of RAG1 expression that results in therapeutic benefit in a preclinical model of RAG-deficient SCID.

[0009] We designed clinically relevant lentiviral SIN plasmids with different internal promoters driving the expression of a codon-optimized RAG1 gene. We evaluated the efficacy of various low-copy-number plasmids using Rag1- / - mice as a preclinical model of RAG1-SCID and confirmed that B and T cell reconstitution was directly correlated with RAG1 expression. Mice with low RAG1 expression exhibited insufficient immune reconstitution, whereas high RAG1 expression resulted in phenotypically and functionally equivalent lymphoid reconstitution compared with mice administered wild-type stem cells. Remarkably, transplantation of CD34+ cells from a RAG1-SCID patient transfected with a clinical RAG1 plasmid into NOD SCID gamma (NSG) mice completely restored human B and T cell development. Together with favorable safety data, our results provide a solid foundation for human clinical trials of RAG1-deficient SCID.

[0010] The present inventors have provided a new system for inducing and maintaining therapeutic threshold levels of RAG1 expression in RAG-deficient cells using a novel codon-optimized RAG1 transgene sequence. The inventors have shown that a therapeutic effect (in terms of in vivo B cell and T cell reconstitution) is observed when RAG1 expression levels are at least three-fold higher for B cell recovery (10-fold higher for T cell recovery) than a specific housekeeping gene, such as ABL1. Thus, a minimum threshold of three-fold higher expression is shown herein to have a beneficial therapeutic effect. The inventors have shown for the first time that such levels of RAG1 expression can be achieved using a low-copy-number retroviral plasmid encoding a codon-optimized RAG1 transgene (i.e., when using a codon-optimized RAG1 transgene sequence, RAG1 expression levels at least three-fold higher than ABL1 can be achieved in cells even when the copy number of the RAG1 transgene (in the context of the expression cassette) integrated into the cell's genome is five or fewer). As used herein, as is well known in the art, "low copy number" refers to a plasmid that integrates into the genome of a target cell at a frequency of 5 copies per cell or less (i.e., 5 copies or less, 4 copies or less, 3 copies or less, 2 copies or less, 1 copy or less, 0.5 copies or less, 0.4 copies or less, 0.3 copies or less, 0.2 copies or less, etc., per cell). The use of low copy number plasmids is advantageous because it significantly reduces the incidence of insertional mutagenesis during gene therapy. Advantageously, the inventors have shown that beneficial effects can be obtained even at copy numbers as low as 0.2 per cell.

[0011] The present invention is exemplified using low copy number plasmids, specifically self-inactivating (SIN) lentiviral (LV) plasmids containing the pCCL backbone. This plasmid is particularly advantageous because it can be produced at higher titers than other LV backbones. However, other low copy number plasmids are also useful in the present invention, as they may similarly offer the advantage of significantly reducing the incidence of insertional mutagenesis. Alternative low copy number plasmids are described in detail elsewhere herein.

[0012] The present inventors have demonstrated the threshold level required for RAG1 expression using the MND promoter. Surprisingly, when the MND promoter is operably linked to a codon-optimized RAG1 transgene, the RAG1 expression level achieved in vivo from a low-copy-number plasmid is sufficient to induce B cell and T cell reconstitution. Thus, the present inventors have confirmed that the combination of a low-copy-number plasmid, a codon-optimized RAG1 transgene sequence, and a strong promoter such as MND is sufficient to induce therapeutic levels of RAG1 expression in vivo. While the present invention uses the MND promoter for example, other strong promoters that induce comparable (or higher) levels of RAG1 expression can also be used. For example, in other systems, the CMV, RSV, and CAG promoters are known to drive high levels of expression of linked transgenes. The threshold level of RAG1 expression required for therapeutic effect is now known (as provided herein for the first time), and other promoters known to be equivalent to MND (such as the CMV, RSV, and cag promoters) can also be similarly applied in the present invention to achieve the desired effect. The present invention therefore encompasses the use of such promoters as alternatives to MND.

[0013] The data provided herein utilize a codon-optimized sequence of RAG1 as a RAG1 transgene operably linked to a required promoter (e.g., MND; although others, such as CMV, RSV, or CAG promoters, can also be used). As described in detail elsewhere herein, the use of a codon-optimized RAG1 sequence is advantageous because it can result in higher viral titers and increased RAG1 protein stability. Thus, the use of a codon-optimized transgene sequence helps achieve the minimum threshold of RAG expression required for therapeutic efficacy (i.e., at levels at least three-fold higher in cells than certain housekeeping genes, such as ABL1, even when five or fewer copies of the RAG1 transgene are integrated into the cell's genome).

[0014] In one aspect, an expression cassette is provided comprising a promoter operably linked to a RAG1 transgene comprising the nucleic acid sequence of SEQ ID NO:2, which expression cassette, when expressed in a human CD34+ hematopoietic stem cell having five or fewer copies of the expression cassette integrated into its genome, produces an expression product at a level at least three times higher than the expression level of ABL1 in the cell.

[0015] Suitably, the promoter may be selected from MND, CMV, RSV and CAG.

[0016] Thus, there is provided an expression cassette comprising a promoter operably linked to a RAG1 transgene comprising the nucleic acid sequence of SEQ ID NO: 2, wherein the promoter is selected from MND, CMV, RSV, and cag. In one example, the RAG1 transgene comprises the nucleic acid sequence of SEQ ID NO: 4. Suitably, the expression cassette, when expressed in human CD34+ hematopoietic stem cells having five or fewer copies of the expression cassette integrated into their genome, produces an expression product at a level at least three-fold higher than the expression level of ABL1 in the cells.

[0017] Preferably, the RAG1 transgene encodes a polypeptide comprising the sequence of SEQ ID NO:1.

[0018] Suitably, the RAG1 transgene may comprise the nucleic acid sequence of SEQ ID NO:4.

[0019] Suitably, the promoter may be MND.

[0020] Preferably, the expression cassette may further comprise a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0021] In one aspect, a retroviral plasmid comprising an expression set of the present invention is provided.

[0022] Suitably, the plasmid may be a self-inactivating (SIN) lentiviral plasmid.

[0023] Suitably, the plasmid may comprise a pCCL backbone.

[0024] Suitably, the plasmid may comprise a pCCL backbone, a nucleotide sequence encoding WPRE, an MND promoter and a transgene comprising the nucleic acid sequence of SEQ ID NO:4.

[0025] Preferably, the plasmid may contain the sequence of FIG.

[0026] In one aspect, a virion is provided that comprises an expression cassette of the invention.

[0027] In one aspect, a composition is provided comprising an expression cassette of the invention or a plasmid of the invention, or a virion of the invention, and a pharmaceutically acceptable adjuvant, carrier, excipient, or diluent.

[0028] In one aspect, a recombinant CD34+ hematopoietic stem cell comprising an expression cassette of the invention is provided.

[0029] In one aspect, the present invention provides an ex vivo method for producing recombinant CD34+ hematopoietic stem cells, the method comprising contacting a cell with a plasmid of the invention or a virion of the invention under conditions in which the expression cassette is incorporated into and expressed in the cell, thereby producing recombinant CD34+ hematopoietic stem cells.

[0030] In one aspect, an expression cassette, plasmid, composition, virion or recombinant cell of the invention is provided for use in therapy.

[0031] Suitably, the expression cassette, vector, composition, virion, or recombinant cell may be for use in treating RAG1-deficient SCID, Omenn's syndrome (OS), atypical SCID, or combined immunodeficiency (CID). For example, the expression cassette, vector, composition, virion, or recombinant cell may be for use in treating RAG1-deficient SCID or Omenn's syndrome (OS).

[0032] In one aspect, there is provided a method of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an expression cassette, plasmid, composition, virion particle, or recombinant cell of the invention.

[0033] Preferably, the subject may have RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID, or combined immunodeficiency (CID). For example, the subject may have RAG1-deficient SCID or Omenn syndrome (OS).

[0034]

[0010] In one aspect, there is provided a method of treating RAG1 deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID) in a subject in need thereof, the method comprising: (i) extracting CD34+ hematopoietic stem cells from the subject; (ii) contacting the cell of (i) with a virion of the invention or a plasmid of the invention; (iii) incubating the cells of (ii) for a period of time; and (iv) introducing the cells of (iii) into the subject. Includes.

[0035] Suitably, the method may further comprise the step of administering chemotherapy or other conditioning regimen to said subject prior to step (iv). [Brief explanation of the drawings]

[0036] [Figure 1]Figure 1: Identification of the optimal SIN LV plasmid for restoring immune reconstitution in Rag1 deficiency. A) Four different SIN LV plasmids in a CCL backbone carrying different promoters (Cbx3-MND, MND, PGK, and UCOE promoters) were tested to drive expression of a codon-optimized version of RAG1. B) Representative FACS plots showing the recovery of B220hi+ B cells in the BM. C) The total number of B220hi+ B cells in the PB (upper panel) and the total number of different B cell subsets in the BM (bone marrow) 16 weeks after SC transplantation. Graphs show the mean and standard deviation of a pilot experiment using 2-3 mice per group (Mann-Withney test, one-tailed, *p ≤ 0.05). D) Representative FACS plots of thymic reconstitution (CD4 vs. CD8) using different constructs. E) Total numbers of T cells (CD3+TCRαβ+) in the PB (upper panel) and total numbers of different T cell subsets in the thymus (lower panel) 16 weeks after transplantation. Graphs show the mean and standard deviation of a pilot experiment with 2-3 mice per group (Mann-Withney test, one-tailed, *p≦0.05). [Figure 2]Figure 2: Correlation of immune reconstitution with RAG1 expression and safety of each vector. A) Correlation of the total number of B220+ cells (right panel) and the total number of B220+IgM+ cells (center panel) with coRAG1 expression in the BM. Correlation of VCN (vector / plasmid copy number) and coRAG1 expression levels in the BM of immune-reconstituted mice (left panel, red = achieved immune reconstitution). B) Correlation of total thymocytes (right panel) and DP cells (center panel) with coRAG1 expression levels in the thymus. Correlation of VCN and coRAG1 expression levels in the thymus of immune-reconstituted mice (left panel, red = achieved immune reconstitution). Immune reconstitution was achieved in all but one mouse. Only extremely high RAG1 levels resulted in low / minimal reconstitution. Data shown are representative of three independent in vivo experiments (circles, hexagons, and squares: filled = MND promoter; open = other promoters). Each dot represents one mouse; all but one mouse achieved immune reconstitution. C) To assess safety, IVIM assays were performed on the different constructs (mock cells served as a negative control, and the RSF91 gammaretroviral vector served as a positive control). Data represent the results of three complete IVIM assays. D) GeneScan analysis of TCR Vβ repertoire. A total of 24 Vβ families were analyzed using splenocytes from three mice per group. A combined score across all families was calculated for the different constructs. E) A representative sample of GeneScan plots of four different families is shown (x-axis indicates CDR3 length; y-axis indicates fluorescence intensity of the runoff product).

[0037] [Figure 3]Figure 3: Extensive immune reconstitution in mice receiving gene therapy SC with clinical-grade MND-coRag1 vector. A) Representative plots of B cell reconstitution in the blood (B220+IgM / IgD cells, upper panel) and B cell development in the BM (B220+CD19+ cells, lower panel) 24 weeks after transplantation. B) Total number of B cells (B220+CD11b / CD43- cells) in the PB. Mann-Whitney test (KO control vs. MND-coRAG1, one-tailed, *p<0.05; **p<0.01). C) Subset distribution of immature B cells (B220+CD93+ cells; left panel) and mature B cells (B220+CD93- cells; right panel) in the spleen. Two-way ANOVA test; ***p<0.001; ****p<0.0001. D) Representative plots of T cell reconstitution in blood (CD3+TCRab+ cells; upper panel) and T cell development in the thymus (CD4 cells vs. CD8 cells; lower panel) 24 weeks after transplantation. E) Total number of T cells (CD3+TCRab+ cells) in PB at the end of the experiment (week 24). Mann-Whitney test (KO control vs. MND-coRAG1, one-tailed; *p<0.05; **p<0.01). F) Distribution of naive, effector, and central memory T cell subsets within the CD4 (CD3+TCRab+CD4+; left panel) and CD8 (CD3+TCRab+CD8+; right panel) T cell subsets in the spleen: Distribution of naive (CD44-CD62L+), effector (CD44+CD62L-), and central memory (CD44+CD62L+) cells in PB 24 weeks after transplantation. G) Left panel: Hematoxylin and eosin staining of mesenteric lymph nodes (LN) (scale bar = 200 μm) and spleen (scale bar = 100 μm). R. Representative FoxP3 staining in spleen tissue (scale = 100 μm). Representative images from one WT control, one KO control, and one MND-coRAG1 GT mouse. Arrows indicate FoxP3 positivity in germinal centers. Right panel: Histological analysis of thymic reconstitution by hematoxylin and eosin staining (scale bar = 50 μm) and cytokeratin staining (scale bar represents 50 μm).Representative images of WT control and MND-coRAG1 mice. [Figure 4] Figure 4: Functional Ig and TCR reconstitution and Ig class switching after Rag1 gene therapy. A) GeneScan analysis of the TCR Vβ repertoire. Spleen cells from three WT control mice, one KO control mouse, and eight MND-coRAG1 mice (immunized and non-immunized) were used to analyze a total of 24 Vβ families. A combined score for all families was calculated. A representative sample of GeneScan plots for three different families is shown (x-axis indicates CDR3 length, y-axis indicates fluorescence intensity of efflux products). B) Quantification of total IgG and IgM in serum by ELISA. C) Quantification of TNP-specific IgG in serum from immunized mice. Each dot represents the value obtained from one mouse. One-way ANOVA test. *p<0.05. [Figure 5]Figure 5: Preclinical safety testing of the clinical-grade MND-co.oRag1 vector. A) Biodistribution of the vector in immune and non-immune organs was assessed by qPCR using DNA samples from a total of 16 organs. Each dot represents a value from one mouse. B) Analysis of the LV insertion site by nrLAM-PCR was performed on DNA isolated from BM obtained from a Rag1- / - untransduced control mouse (Mock) and four MND-coRAG1 mice (male non-immunized / immunized mice, female non-immunized / immunized mice). The gel shows the results of linear amplification from the 3' LTR and 5' LTR, respectively (L = 1 kb + marker). C) The replication frequency (RF) of the control samples Mock or RSF91 and the test vector MND-coRAG1 was compared with data from a meta-analysis of control samples (Mock-MA, RSF91-MA, lv-SF-MA [lentiviral vector with an SFFV promoter]). Data points below the limit of detection (LOD; plates with no wells above the MTT threshold) were manually inserted into the graph (due to the logarithmic y-axis scale). Above the graph, the percentage of positive (left numbers) and negative (right numbers) plates by MTT assay is shown. Differences in the incidence of positive and negative assays compared to Mock-MA or RSF91-MA were analyzed by Fisher's exact test with Benjamini-Hochberg correction (*P<0.05; **P<0.01; ***P<0.001; NS=not significant). Above the LOD, the bar indicates the mean RF.

[0038] [Figure 6]Figure 6: Restoration of B and T cell development in Rag1 SCID patient cells. A) Mice were transplanted with CD34+ purified mock-transduced cells (65,000) or MND-CoRAG1-transduced cells (65,000). Representative FACS plots of human B cells (CD13 / 33-CD19+CD20+ cells; upper panel) and total B cell numbers (CD13 / 33-CD19+CD20+IgD / IgM cells; lower panel) in the spleen are shown. B) Representative FACS plots of human T cells (CD3+TCRαβ+; upper panel) and total T cell, CD4 and CD8 T cell numbers (lower panel) in the PB. C) Human T cell development in the thymus. Representative FACS plots (CD4 vs. CD8) and distribution of different T cell subsets within the thymus. D) Quantification of total human IgM by ELISA using serum from NSG mice transplanted with SCID control CD34+ cells, SCID patient CD34+ cells, and SCID MND-coRAG1 CD34+ cells. E) Human TCR Vβ and Vγ repertoire analysis of DNA isolated from NSG thymus (SCID patient and SCID MND-coRAG1) using the TCRB + TCRG T cell clonality assay (x-axis represents fragment size; y-axis represents fluorescence intensity of efflux products). F) Analysis of LV insertion sites by nrLAM-PCR of DNA isolated from BM obtained from untransfected cells (Mock) of an NSG SCID patient and an NSG SCID MND-coRAG1 mouse. The gel shows the linear amplification from the 5' LTR (L = 1 kb + marker). Data from n = 1 independent experiment per condition. [Figure 7]Figure 7 shows immune development after gene therapy in the Rag1- / - mouse model. A) Temporal distribution of B cells (CD11b / CD43-B220+ cells; left panel) and T cells (CD3+TCRαβ+ cells; right panel) in the PB after SC transplantation with different constructs (Cbx3-coRAG1, MND-coRAG1, PGK-coRAG1, and UCOE-coRAG1). B) Temporal distribution of B cells (CD11b / CD43-B220+ cells; left panel) and T cells (CD3+TCRαβ+ cells; right panel) in the PB after SC transplantation with the clinical MND-coRAG1 batch. C) Distribution of B cell subsets in the BM (left panel) and T cell populations in the thymus (right panel) 20 weeks after SC transplantation. Graphs show the mean and standard deviation for three control and eight gene therapy mice. D) Histological analysis of liver (scale bar = 100 μm), kidney (scale bar = 200 μm), lung (scale bar = 100 μm), and BM (scale bar = 100 μm) stained with hematoxylin and eosin. Representative images of WT control, KO control, and MND-coRAG1 mice. E) Quantification of total IgE in serum by ELISA. Each dot represents the value obtained for one mouse. One-way ANOVA test. *p<0.05.

[0039] [Figure 8]Figure 8 shows human immune reconstitution after CD34+ MND-coRAG1 transplantation. A) Percentage of human chimerism (hCD45+ / (hCD45+mCD45+)) in immune organs of NSG mice transplanted with CD34+ SCID patient cells and CD34+ SCID patient cells transduced with MND-coRAG1 24 weeks after transplantation (one NSG mouse per condition). B) Percentage of human B cells in peripheral blood over time during transplantation (CD19+ cells per total hCD45+ cells). C) Percentage of human T cell development over time in PB during transplantation (CD3+ cells / total hCD45+ cells). D) Flow cytometry analysis of thymocytes 24 weeks after transplantation showing different stages of T cell development. E) Human IgH and IgK repertoires of DNA isolated from NSG BM (SCID patient and SCID MND-coRAG1) were analyzed using an IgH+IgK B cell clonality assay (x-axis indicates fragment size, y-axis indicates fluorescence intensity of efflux products). [Figure 9] FIG. 9 shows the complete plasmid sequence of LV-MND-coRAG1. [Figure 10] Figure 10 shows histological analysis of the skin and intestine after MND-coRAG1 gene therapy in the Rag1- / - mouse model. Representative images were obtained from WT control, KO control, and MND-coRAG1 mice (non-immunized and immunized). For skin, scale bar = 100 μm; for intestine, scale bar = 50 μm. Samples were stained with hematoxylin and eosin. DETAILED DESCRIPTION OF THE INVENTION

[0040] Detailed Description We designed clinically useful lentiviral SIN plasmids harboring various internal promoters operably linked to a codon-optimized RAG1 transgene to identify the minimum threshold of RAG1 expression required for therapeutic efficacy in vivo.

[0041] Rag1 as a preclinical model for RAG1-SCID - / -We evaluated the efficacy of various low-copy-number plasmids carrying a codon-optimized RAG1 transgene in mice and confirmed that B and T cell reconstitution was directly correlated with RAG1 expression. Mice with low RAG1 expression exhibited poor immune reconstitution, whereas high RAG1 expression resulted in phenotypic and functional lymphoid reconstitution comparable to that of mice administered wild-type stem cells. Surprisingly, CD34 expression in RAG1-SCID patients transfected with clinical RAG1 plasmids was significantly higher than that in control mice. + When the cells were transplanted into NSG mice, human B- and T-cell development was fully restored.

[0042] To facilitate the understanding of this invention, several terms are defined below.

[0043] Expression cassette An expression cassette is provided that includes a codon-optimized RAG1 transgene operably linked to a promoter. The RAG1 transgene may encode the amino acid sequence set forth in SEQ ID NO: 1 (human RAG1), a homolog thereof, or a functional variant thereof (e.g., a conservative amino acid sequence variant thereof).

[0044] The term "expression cassette" refers to a nucleic acid molecule containing one or more transcriptional regulatory elements (including, but not limited to, promoters, enhancers and / or regulatory elements, polyadenylation sequences, introns, etc.) that drive expression of a transgene in one or more desired cell types, tissues, or organs. The expression cassettes of the present invention are synthetic nucleic acid molecules.

[0045] As used herein, the term "nucleic acid" generally refers to an oligomer or polymer (preferably a linear polymer) of any length composed essentially of nucleotides. A nucleotide unit typically comprises a heterocyclic base, a sugar residue, and at least one, e.g., one, two, or three, phosphate groups (including modified or substituted phosphate groups). Heterocyclic bases may include, inter alia, purine and pyrimidine bases commonly present in natural nucleic acids, such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as well as other natural bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated) non-natural or derivatized bases. Sugar groups may include, inter alia, pentose (pentofuranosyl) groups, such as ribose and / or 2-deoxyribose, which are common in natural nucleic acids, or arabinose, 2-deoxyarabinose, threose, or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as contemplated herein may contain natural nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may contain modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications to the phosphate group or sugar may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" further preferably encompasses DNA, RNA, and DNA-RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g., chemically synthesized) DNA, RNA, or DNA-RNA hybrids. Nucleic acids may be natural, e.g., naturally occurring or isolated from nature, or non-natural, e.g., recombinant, i.e., produced by recombinant DNA technology, and / or partially or entirely chemically or biochemically synthesized. "Nucleic acids" may be double-stranded, partially double-stranded, or single-stranded. If single-stranded, the nucleic acid may be the sense or antisense strand. Furthermore, nucleic acids may be circular or linear.

[0046] The expression cassette may comprise DNA or RNA.

[0047] The term "synthetic nucleic acid" as used herein relates to a nucleic acid molecule that does not occur in nature.

[0048] As used herein, the term "transgene" refers to an exogenous nucleic acid sequence, i.e., a non-naturally occurring sequence that includes other elements (e.g., transcriptional regulatory elements such as a promoter) found in an expression cassette. In one example, a transgene is a gene that encodes an industrially or pharmaceutically useful compound or a gene that encodes a desirable trait. In this specification, a transgene of interest is a RAG1 transgene.

[0049] RAG1 transgene: human RAG1 and its homologues Thus, the present invention provides an expression cassette comprising a codon-optimized RAG1 transgene operably linked to a promoter.

[0050] A RAG1 transgene is a nucleic acid sequence that encodes a RAG1 protein. For the avoidance of doubt, a transgene does not necessarily contain all of the native elements of endogenous RAG1; for example, a transgene may be the corresponding cDNA of RAG1 (i.e., without endogenous introns, etc.). As used herein, the term "recombinase activating gene-1 (RAG1)" refers to the protein encoded by the RAG1 gene.

[0051] RAG1 and RAG2 combine to form the RAG complex, a multiprotein complex that mediates the DNA cleavage step during VDJ recombination. This complex can create double-strand breaks by cleaving DNA at conserved recombination signal sequences (RSSs). The RAG complex recognizes the RSSs flanking the V, D, and J regions of the genes encoding the constant regions of both the heavy and light chains of antibodies. The complex binds to the RSSs and nicks the DNA, which removes the RSSs and ultimately joins the V, D, and J sequences.

[0052] RAG1 is thought to possess most of the catalytic activity of the RAG complex. The RAG1 protein is the component that binds and cleaves DNA, and in this way, RAG1 is involved in activating immunoglobulin VDJ recombination. RAG2 does not appear to have endonuclease activity or DNA binding ability, but serves as a cofactor. Its main function is to interact with RAG1 and activate its endonuclease function.

[0053] Defects in the genes encoding RAG1 and RAG2 cause several diseases. In this context, deletion of RAG1 and RAG2 in mouse models impairs T and B cell maturation and functionally deletes mature T and B cells from the immune system.

[0054] In one example, the RAG1 transgene comprises a nucleotide sequence encoding the human RAG1 protein (SEQ ID NO: 1). Alternatively, the RAG1 sequence may be from a different species, such as pig, mouse, rat, non-human primate, etc.

[0055] The sequences of the human RAG1 gene and protein are known (see, e.g., unique identifiers: HGNC:HGNC:9831 HUGO Human Gene Nomenclature Committee related to Ensembl:ENSG00000166349 MIM:179615). For ease of reference, the human RAG1 protein sequence is provided in SEQ ID NO:1.

[0056] The sequences of the mouse RAG1 gene and protein are known (e.g., unique identifiers for mouse RAG1 include: ENSMUST00000078494; ENSMUSP00000077584; ENSMUSG00000061311).

[0057] The sequences of the rat RAG1 gene and protein are known (see, for example, unique identifiers: Ensembl: ENSRNOG00000004630; ENSRNOT00000006115; ENSRNOP00000006115; ENSRNOG00000004630).

[0058] RAG1 protein: functional variants The RAG1 transgene may comprise a nucleotide sequence encoding a native human, mouse, rat, etc. RAG1 protein, or a functional variant thereof (e.g., a human, mouse, or rat RAG1 functional variant). Examples of functional variant RAG1 proteins are conservative amino acid substitution variants of native RAG1 (i.e., sequences that vary from the native sequence of the human, mouse, or rat RAG1 sequence by only one or more conservative amino acid substitutions).

[0059] A "functional mutant" retains the functional ability of the RAG1 protein. In other words, a functional RAG1 mutant can generate a double-strand break by cleaving DNA at a conserved recombination signal sequence (RSS). Those skilled in the art can readily know how to identify polypeptides with this activity using routine tests known in the art. Suitable experiments for identifying functional RAG1 polypeptides are summarized below.

[0060] Functional RAG1 protein sequences can be identified using a functional complementation test. This test can use the lentivirus described in the Examples section below, which contains a RAG1 transgene encoding the RAG1 mutant to be tested. Lin-bone marrow cells are used as a source of hematopoietic stem cells and transduced with a recombinant lentivirus encoding the RAG1 sequence to be tested. These cells are then transduced with Rag1. - / - The cells are transplanted into mice and T cell development is monitored. After 8-12 weeks, CD3+ TCRαβ+ T cells are generated, and if the number of T cells is at least 50% of that of wild-type stem cells, the sequence is considered successful.

[0061] A suitable test for RAG1 activity that we have performed and that can be routinely performed by a person skilled in the art is outlined below: C57BL / 6 wild-type mice and C57BL / 6 Rag1 - / -Mouse bone marrow (BM) cells were obtained from mouse femurs and tibiae. The bones were flushed or crushed, and the cells were passed through a 0.7 μm cell strainer (Falcon), washed, and viably frozen. After thawing, lineage-negative cells were isolated using a mouse lineage depletion kit and an AUTOMacs cell sorter (Miltenyi Biotech). Lineage-negative cells were stimulated overnight in StemSpam-SFEM containing penicillin / steptamycin (5,000 units / 5,000 μg / ml; Gibco) and supplemented with 50 ng / mL recombinant mouse FMS-related tyrosine kinase 3 ligand (rmFLT3L; R&D Systems), 100 ng / mL recombinant mouse Stem-Cell Factor (rmSCF; R&D Systems), and 10 ng / mL recombinant mouse thrombopoietin (rmTPO; R&D Systems). Subsequently, Rag1 - / - Cells were transduced with different lentiviruses using 4 μg / ml proteamine sulfate (Sigma-Aldrich) by spinoculation at 800 × g and 32°C for 1 hour. Cells were cultured in cytokine-supplemented medium at 37°C and 5% CO for 24 hours.

[0062] Control mock-transduced cells (C57BL / 6 wild-type cells as WT control, Rag1 - / - cells are referred to as KO control) and transduced Rag1 - / - Mouse cells (up to 5.10 5 Cells / mouse) were cultured in phenol red-free Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) containing supportive Rag1 - / - Spleen cells (3.10 6 cells / mouse) and pretreated Rag1 - / -Recipient mice were transplanted via tail vein injection. Recipient mice (8–12 weeks old) were conditioned by a single dose of whole-body orthovoltage X-rays (8.08 Gy) 24 h before transplantation or by two consecutive doses of 25 mg / kg busulfan (Sigma-Aldrich) administered 48 and 24 h before transplantation.

[0063] Mice used for transplantation were housed in a specific pathogen-free environment. For the first 4 weeks after transplantation, mice were supplemented with DietGel recovery food (Clear H2O) and antibiotic water containing 0.07 mg / mL polymyxin B (Bupha Uitgeest), 0.0875 mg / mL ciprofloxacin (Bayer bv), and 0.1 mg / mL amphotericin B (Bristol-Myers Squibb). The mice were observed daily. Peripheral blood (PB) was collected from mice via tail vein incision every 4 weeks until the end of the study. PB, thymus, spleen, and BM were obtained from mice euthanized with CO2.

[0064] Single-cell suspensions were prepared from spleens by squeezing the organs through a 70 μM cell strainer (BD Falcon), and single-cell suspensions from BM were prepared as described above. Erythrocytes from spleens were lysed using NH₄Cl (8.4 g / L) / KHCO₃ (1 g / L) solution. The single-cell suspensions were counted and stained with the antibodies listed in Table 1. Cells were incubated for 30 min at 4°C in the dark with an antibody mix solution containing antibodies directly conjugated to their optimal working solution in FACS buffer (PBS pH 7.4, 0.1% azide, 0.2% BSA). After washing with FACS buffer, a second 30-min incubation step was performed with streptavidin-conjugated antibody solution at 4°C. Cells were measured using a FACS-Canto II and an LSR Fortessa X-20 (BD Biosciences), and data were analyzed using FlowJO software (Tree Star).

[0065] The antibodies used in the optimal panel are listed below. At a minimum, CD3, CD4, CD8, and TCRβ were included for staining. [Table 1]

[0066] Thus, a RAG1 polypeptide may comprise the amino acid sequence set forth in SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), or may be a functional variant (or functional fragment) thereof. Such variants may be naturally occurring (e.g., allelic), synthetic, or synthetically improved functional variants of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence).

[0067] A functional variant generally contains only conservative substitutions of one or more amino acids of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), or may contain substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein. Thus, a functional variant of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence) may be a conservative amino acid sequence variant of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence).

[0068] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence) that does not have RAG1 activity. Non-functional variants generally may include non-conservative substitutions, deletions, insertions, or premature truncations of the amino acid sequence of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants (e.g., functional and non-functional allelic variants) are well known to those skilled in the art.

[0069] A summary of the critical and non-critical amino acids of RAG1 is provided in Luigi D. Notarangelo, Min-Sung Kim, Jolan E. Walter & Yu Nee Lee Nature Reviews Immunology volume 16, pages 234-246 (2016). Thus, one skilled in the art can readily identify amino acids that can be substituted to provide functional variants (or functional fragments), such as conservative amino acid sequence variants of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence).

[0070] A functional variant may comprise an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), or a portion or fragment thereof. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 1), or a portion or fragment thereof.

[0071] In one example, the RAG1 transgene encodes a polypeptide comprising the sequence of SEQ ID NO: 1, or a conservative amino acid sequence variant thereof.

[0072] As used herein, a "native" polypeptide refers to an amino acid sequence that occurs in nature.

[0073] A "non-essential" (or "non-critical") amino acid residue is one that can be altered from a wild-type sequence (e.g., the sequence of SEQ ID NO: 1) without abolishing, or more preferably, substantially altering, biological activity, whereas an "essential" (or "critical") amino acid residue is one that is subject to such an alteration. For example, conserved amino acid residues are predicted to remain unchanged, except that amino acid residues within the hydrophobic core of a domain can generally be replaced with other residues of approximately equivalent hydrophobicity without significantly altering activity.

[0074] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential (or non-critical) amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly and the resulting mutants screened for biological activity to identify mutants that retain activity.

[0075] Conservative amino acid substitution variants of RAG1 may have at least one (e.g., two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, ten or less, etc.) conservative amino acid substitution compared to native human, mouse or rat RAG1 (identified above using unique identifiers).

[0076] Sequence of the RAG1 transgene; variations at the nucleic acid sequence level By "RAG1 transgene" is meant any nucleic acid sequence that encodes a functional RAG1 protein (eg, human, mouse, or rat RAG1, or a functional variant thereof, such as an amino acid substitution variant).

[0077] The RAG1 nucleotide sequences described herein are codon-optimized. As used herein, "codon-optimized" (or "co") refers to a polynucleotide sequence encoding a RAG1 protein that has been altered relative to the native polynucleotide sequence without altering the encoded amino acid sequence. This term is widely known in the art. Codon optimization of a polynucleotide sequence can have several effects that increase the overall translation efficiency / expression level of the RAG1 protein in cells.

[0078] for example, 1.Effects on RNA secondary structure Because secondary structures at the 5' end of mRNA affect translation efficiency, synonymous changes in this region of mRNA can have a significant impact on gene expression. Therefore, codon usage in non-coding DNA regions plays a major role in RNA secondary structure and downstream protein expression, and may be subject to further selective pressure. In particular, strong secondary structures at ribosome binding sites and initiation codons can inhibit translation, and mRNA folding at the 5' end can result in large fluctuations in protein levels.

[0079] Herein, the RAG1 nucleotide sequence may be codon optimized to increase the GC content of the coding sequence.

[0080] 2. Effects on transcription / gene expression Heterologous gene expression is used in many biotechnology fields, including protein production and metabolic engineering. Because tRNA pools vary among organisms, placing a specific coding sequence in a non-native environment can reduce transcription and translation efficiency. In overexpressed transgenes, the corresponding mRNA accounts for a large proportion of the total RNA in the cell. Rare codons in the transcript can lead to reduced ribosome utilization and depletion, ultimately resulting in reduced heterologous protein production levels. However, overexpressing heterologous genes using codons optimized for a specific host's tRNA pool can also lead to amino acid depletion or alter the tRNA pool equilibrium. This method of tailoring codons to the host's tRNA abundance has traditionally been used for heterologous gene expression. However, new strategies for optimizing heterologous gene expression take into account global nucleotide content, such as local mRNA folding, codon pair bias, codon ramp, or codon correlation.

[0081] Specific codon biases are also observed in some endogenous genes, such as those involved in amino acid depletion. For example, amino acid biosynthetic enzymes are poorly adapted to the normal tRNA abundance, but preferentially use codons that are well adapted to the tRNA pool under depletion conditions. Thus, codon usage can introduce an additional level of transcriptional regulation for proper gene expression under specific cellular conditions.

[0082] In this regard, the nucleotide sequence of RAG1 can be codon optimized to include removal of alternative splice sites and cryptic splice sites, and optimized codon usage for human tRNAs.

[0083] 3. Effect on translation elongation rate Generally, for highly expressed genes, translation elongation rates are faster for transcripts with high codon compatibility with the tRNA pool and slower for transcripts with rare codons. This correlation between codon translation rate and cognate tRNA concentration further regulates translation elongation rates, providing several benefits to organisms. Specifically, codon usage can globally regulate these rates, and rare codons may contribute to translation accuracy at the expense of speed.

[0084] In this regard, the RAG1 nucleotide sequence can be codon-optimized to include codon usage optimized for human tRNAs.

[0085] 4. Effect on protein folding In vivo, protein folding is vectorial, with the N-terminus of a protein emerging from the translating ribosome and exposed to solvent before its C-terminal region. As a result, cotranslational protein folding imposes spatial and temporal constraints on the process by which a polypeptide chain folds. Because mRNA translation rate is linked to protein folding and codon adaptation is linked to translation elongation, manipulation at the sequence level has been shown to be effective for modulating and improving protein folding. Several studies have shown that for certain proteins, translational pauses may occur as a result of local mRNA structure and be necessary for proper folding. Furthermore, synonymous mutations have been shown to have significant effects on the nascent protein folding process and can even alter the substrate specificity of enzymes. These studies suggest that codon usage influences the rate at which polypeptides emerge vectorially from the ribosome, which in turn may influence the protein folding pathway across the available structural space.

[0086] Codon-optimized RAG1 polynucleotide sequences are encompassed herein, regardless of the means of codon optimization.

[0087] Our analysis of the human RAG1 cDNA sequence revealed several possibilities for improving the DNA sequence without affecting the amino acid sequence, due to the presence of many rare codons in the native RAG1 gene. Most of these codons were replaced with more frequently used codons from Homo sapiens genes. The GC content was also increased to enhance mRNA stability. Finally, 21 cis-acting motifs (prokaryotic inhibitory motifs, splice donor sites, poly(A) sites, and RNA instability motifs) that could adversely affect expression were removed. The amino acid sequence was left unchanged, allowing regulatory mechanisms at the protein level to function normally.

[0088] In one non-limiting example, a RAG1 codon-optimized transgene may encode the amino acid sequence of SEQ ID NO: 1 and include the nucleic acid sequence of SEQ ID NO: 2. In other words, a RAG1 transgene may encode the human RAG1 protein (SEQ ID NO: 1) while having a nucleic acid sequence that differs from the native RAG1 nucleic acid sequence (SEQ ID NO: 3) due to (at least) codon optimization of the RAG1 catalytic domain. The nucleic acid sequence shown in SEQ ID NO: 2 is the core catalytic domain sequence of human RAG1, indicating which nucleic acids were altered during codon optimization. The inventors have demonstrated that codon optimization of RAG1 is beneficial for optimal expression of the RAG1 transgene. Advantageously, the codon-optimized sequence described herein for the RAG1 catalytic domain (SEQ ID NO: 2) does not adversely affect RAG1 catalytic domain function, which is important for RAG1 activity. Therefore, suitable base sequences are provided for codon-optimized variants of the RAG1 transgene. Thus, a codon-optimized variant of the RAG1 transgene comprises the codon-optimized catalytic domain shown in SEQ ID NO: 2, and any additional codon optimizations can be performed in other regions of the RAG1 transgene.

[0089] Thus, for the avoidance of doubt, a RAG1 nucleic acid sequence may differ from the native RAG1 sequence of SEQ ID NO: 3 in at least the catalytic domain (optional codon optimization may be performed in other regions of the RAG1 transgene sequence), whilst still encoding a functional RAG1 polypeptide as set forth in SEQ ID NO: 1.

[0090] The sequence of a codon-optimized human RAG1 transgene that has been successfully used by the present inventors is shown in SEQ ID NO: 4. Thus, in one example, an expression cassette is provided that comprises the RAG1 transgene of SEQ ID NO: 4 operably linked to a promoter. Suitable promoters are described below.

[0091] As described herein, the RAG1 transgene is operably linked to a promoter within an expression cassette. As used herein, the terms "operably linked," "operably associated," or equivalent expressions refer to the positioning of various nucleic acid elements relative to one another so that they are functionally linked and can interact with one another in their intended manner. Such elements include, but are not limited to, promoters, enhancers and / or regulatory elements, polyadenylation sequences, one or more introns and / or exons, and the coding sequence of the gene of interest to be expressed. When properly oriented or operably linked, these nucleic acid sequence elements can interact with one another to modulate each other's activity, ultimately affecting the expression level of the expression product. Modulation refers to increasing, decreasing, or maintaining the activity level of a particular element. The relative position of each element with respect to other elements is expressed in terms of the 5' and 3' ends of each element, and the distance between any particular elements can be referred to by the number of intervening nucleotides (i.e., spacer sequences) or base pairs between the elements. As will be understood by those skilled in the art, operably linked refers to functional activity and does not necessarily relate to linkage in natural spatial relationship.

[0092] As used herein, a "spacer sequence" or "spacer" is a nucleic acid sequence that separates two functional nucleic acid sequences. It can have essentially any sequence as long as it does not prevent the functional nucleic acid sequences from functioning as desired. Generally, it is a non-functional sequence that exists solely to separate adjacent functional nucleic acid sequences from one another.

[0093] As used herein, the term "promoter" generally refers to a nucleic acid sequence located upstream of a nucleic acid sequence to be transcribed. A promoter is generally required for transcription, i.e., it initiates transcription. A promoter allows for the appropriate activation or repression of transcription of the coding sequence under its control. A promoter usually contains specific sequences that are recognized and bound by multiple transcription factors (TFs). Binding of the TF to the promoter sequence recruits RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene. Numerous promoters are known in the art.

[0094] The promoters described herein may be described as "strong promoters" because they drive high-level expression of an operably linked transgene in a cell. Generally, the promoter drives expression of an operably linked RAG1 transgene in a cell (e.g., recombinant human CD34+ hematopoietic stem cells) such that the expression product of the RAG1 transgene in the cell is at least x-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cell (e.g., recombinant human CD34+ hematopoietic stem cells). As used herein, "x-fold higher" includes at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and at least 10-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cell (e.g., recombinant human CD34+ hematopoietic stem cells). As will be apparent to those skilled in the art, "expression product" encompasses all products produced during expression of a transgene, and thus encompasses not only proteins but also mRNA (transcripts) of the transgene. Methods for measuring the level of an expression product in a cell are well known in the art. For example, the expression product of the transgene can be measured at the transcript (mRNA) level or at the protein level.

[0095] To detect the level of mRNA in a sample, any known mRNA detection method can be used. For example, the level of a specific mRNA in a sample can be measured using Southern blot analysis or Northern blot analysis, polymerase chain reaction, or probe array. In one embodiment, the sample can be contacted with a nucleic acid molecule (i.e., a probe such as a labeled probe) that can specifically hybridize to a specific mRNA.

[0096] Alternatively, the level of a particular mRNA in a sample can be assessed by nucleic acid amplification, for example, by rtPCR, ligase chain reaction, self-sustained sequence replication, transcription amplification, or any other nucleic acid amplification method, followed by detection of the amplified molecules, using techniques known in the art.

[0097] Any known protein detection method can be used to detect the level of a protein in a sample. Generally, protein detection methods involve contacting a sample with an agent or antibody that selectively binds to the protein and measuring the level of a particular protein in the sample. Preferably, the agent or antibody is labeled, for example, with a detectable label. Suitable antibodies are polyclonal or monoclonal antibodies. Antibody fragments such as Fab or F(ab')2 can also be used. As used herein, the term "labeled" refers to both direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of a probe or antibody by reactivity with a detectable substance.

[0098] The level of a specific protein biomarker in a sample can be determined by techniques known in the art, such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), Western blot analysis, and lateral flow devices (LFDs) using membrane-bound antibodies specific for the protein biomarker. Alternatively, mass spectrometry can be used to detect and quantitate the level of a specific biomarker protein in a sample. Such methods are routinely used in the art.

[0099] The level of the expression product can be normalized by comparing it to the level of a housekeeping gene (e.g., a constitutively expressed mRNA or protein) in the sample. A suitable housekeeping gene is ABL1, but other genes may be used. Normalization in this manner allows the expression level of one sample to be compared with another sample, or between samples from different sources.

[0100] Advantageously, the promoters described herein drive the required level of expression of a transgene that is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell) when the copy number of the expression cassette integrated into the genome of the cell is 5 or less.

[0101] In other words, the promoters described herein are capable of driving the required level of expression of the RAG1 transgene even when the promoter is in a plasmid that is a low copy number plasmid. The term low copy number plasmid is well known in the art (and is used to describe vectors that integrate into the genome at a frequency of 5 or fewer copies per cell (i.e., 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer copies per cell, 0.5 or fewer, 0.4 or fewer, 0.3 or fewer, 0.2 or fewer, etc.). See, e.g., 1. Poletti V, Charrier S, Corre G, Gjata B, Vignaud A, Zhang F, Rothe M,Schambach A, Gaspar HB, Thrasher AJ, Mavilio F. “Preclinical Development of a Lentiviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency.” Mol Ther Methods Clin Dev. 2018 Mar 10;9:257-269. doi:10.1016 / j.omtm.2018.03.002. eCollection 2018 Jun 15. PubMed PMID: 29707600; PubMed Central PMCID: PMC5918176. 2. Siler U, Paruzynski A, Holtgreve-Grez H, Kuzmenko E, Koehl U, Renner ED, Alhan C, de Loosdrecht AA, Schwaeble J, Pfluger T, Tchinda J, Schmugge M, Jauch A, Naundorf S, Kuehlcke K, Notheis G, Guengor T, Kalle CV, Schmidt M, Grez M, Seger R, Reichenbach J. “Successful Combination of Sequential Gene Therapy and Rescue Allo-HSCT in Two Children with X-CGD - Importance of Timing.” Curr Gene Ther. 2015;15(4):416-27. PubMed PMID: 25981636. 3. Greene MR, Lockey T, Mehta PK, Kim YS, Eldridge PW, Gray JT, Sorrentino BP. “Transduction of human CD34+ repopulating cells with a self-inactivating lentiviral vector for SCID-X1 produced at clinical scale by a stable cell line.” Hum Gene Ther Methods. 2012 Oct;23(5):297-308. doi: 10.1089 / hgtb.2012.150. Epub 2012 Nov 7. PubMed PMID: 23075105; PubMed Central PMCID: PMC373213.

[0102] Those skilled in the art can easily identify suitable promoters using routine methods. For example, a potential promoter of interest may be operably linked to the codon-optimized RAG1 nucleic acid sequence (SEQ ID NO: 4) described herein within the plasmid backbone (pCCL) described herein, and the resulting plasmid may be introduced into the Rag1- / - mouse preclinical model of RAG-SCID described herein. The level of RAG1 expression product may then be measured as described in the Examples section below and compared with the ABL1 level described herein. If the RAG1 expression level is at least three-fold (e.g., 10-fold) higher than the ABL1 level, the promoter being tested is considered suitable for the present invention and therefore falls within the scope of the claimed invention. Detailed descriptions of methods that can be used to test potential promoters of interest are provided in the Examples section below. Alternative / supplementary methods are also known to those skilled in the art.

[0103] The strength of the promoter can be most easily tested by examining the expression of the therapeutic RAG1 gene by Q-PCR in CD34+ cells. As a reference, a housekeeping gene such as ABL1 is used in the same assay. The ratio of the expression levels of the two is a direct indicator of promoter strength.

[0104] qPCR was used to quantitatively analyze mRNA expression using WPRE, coRAG1, and ABL1 as targets. Total RNA from single-cell suspensions was purified using the RNeasy Mini kit (Qiagen) and reverse-transcribed into cDNA using the Superscript III kit (Invitrogen). Genomic DNA was extracted from single-cell suspensions using the GeneElute Mammalian Genomic DNA kit (Sigma-Aldrich). Genomic DNA was extracted from mouse organs and tissues using the Dneasy Blood and Tissue Kit (Qiagen). qPCR was performed using TaqMan Universal Master Mix II (Thermofisher) in combination with specific probes for the indicated genes from the Universal Probe Library (Roche). The primers and probes used are listed in Table 2. PCR reactions were performed using a StepOnePlus Real-Time PCR system (Thermofisher). All samples should be performed in triplicate. Exemplary primers that can be used are as follows:

[0105] [Table 2]

[0106] For example, suitable promoters include MND, CMV, RSV, and CAG promoters. These promoters are well known; see, for example, Daniela Zychlinski, Axel Schambach, Ute Modlich, Tobias Maetzig, Johann Meyer, Elke Grassman, Anjali Mishra, Christopher Baum, "Physiological Promoters Reduce the Genotoxic Risk of Integrating Gene Vectors," Molecular Therapy, Volume 16, Issue 4, 2008, Pages 718-725, ISSN 1525-0016, https: / / doi.org / 10.1038 / mt.2008.5; Astrakhan A, Sather BD, Ryu BY, Khim S, Singh S, Humblet-Baron S, Ochs HD, Miao CH, Rawlings DJ. “Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome.” Blood. 2012 May 10;119(19):4395-407. doi: 10.1182 / blood-2011-03-340711, Yaguchi M, Ohashi Y, Tsubota T, Sato A, Koyano KW, Wang N, Miyashita Y. “Characterization of the properties of seven promoters in the motor cortex of rats and monkeys after lentiviral vector-mediated gene transfer.” Hum Gene Ther Methods. 2013 Dec;24(6):333-44. doi: See 10.1089 / hgtb.2012.238.

[0107] The MND promoter can be universally identified by the unique identifier GenBank: LZ103461.1. Its sequence is also set forth herein as SEQ ID NO: 5. Similarly, the CMV promoter can be universally identified by the unique identifier GenBank: AB902850.1 (ncl 1114-1493). The RSV promoter can be universally identified by the unique identifier GenBank: GM964660.1. Furthermore, the CAG CMV early enhancer / chicken β-actin [CAG] promoter can be universally identified by the unique identifier Pubmed / 1144964.

[0108] Thus, in one example, an expression cassette is provided that includes a RAG1 transgene operably linked to an MND promoter. In this example, when the promoter is MND, the RAG1 transgene is a codon-optimized version of the human RAG1 transgene as set forth in SEQ ID NO:2 or SEQ ID NO:4, wherein the transgene encodes the protein of SEQ ID NO:1 but does not have the native RAG1 nucleic acid sequence of SEQ ID NO:3. Advantageously, the expression product of the RAG1 transgene (when operably linked to the MND promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) is at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell. This is particularly advantageous when the expression cassette is present in a low copy number in the cell, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and when the expression product of the RAG1 transgene (when operably linked to the MND promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell).

[0109] In another example, an expression cassette is provided that includes a RAG1 transgene operably linked to a CMV promoter. In this example, when the promoter is CMV, the RAG1 transgene can be a human RAG1 transgene, or a codon-optimized version thereof (as shown in SEQ ID NO:2 or SEQ ID NO:4, where the transgene encodes the protein of SEQ ID NO:1 but does not have the native RAG1 nucleic acid sequence of SEQ ID NO:3). Advantageously, the expression product of the RAG1 transgene (when operably linked to a CMV promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell. This is particularly advantageous when the expression cassette is present in the cell at low copy number, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and when the expression product of the RAG1 transgene (when operably linked to a CMV promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell).

[0110] Also provided is an expression cassette comprising a RAG1 transgene operably linked to an RSV promoter. In this example, when the promoter is RSV, the RAG1 transgene may be a codon-optimized version of the human RAG1 transgene (as shown in SEQ ID NO:2 or SEQ ID NO:4, where the transgene encodes the protein of SEQ ID NO:1 but does not have the native RAG1 nucleic acid sequence of SEQ ID NO:3). Advantageously, the expression product of the RAG1 transgene (when operably linked to an RSV promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells) is at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell. This is particularly advantageous when the expression cassette is present in a low copy number in the cell, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and when the expression product of the RAG1 transgene (when operably linked to an RSV promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell).

[0111] Also provided is an expression cassette comprising a RAG1 transgene operably linked to a CAG promoter. In this example, when the promoter is CAG, the RAG1 transgene may be a codon-optimized version of the human RAG1 transgene (shown in SEQ ID NO:2 or SEQ ID NO:4, where the transgene encodes the protein of SEQ ID NO:1 but does not have the native RAG1 nucleic acid sequence of SEQ ID NO:3). Advantageously, the expression product of the RAG1 transgene (operably linked to a CAG promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) is at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell. This is particularly advantageous when the expression cassette is present in a low copy number in the cell, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and when the expression product of the RAG1 transgene (operably linked to a CAG promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell).

[0112] As described herein, the expression product of the RAG1 transgene (when operably linked to a promoter and expressed in a cell) advantageously is at least three-fold higher than the level of a housekeeping gene (such as ABL1) in the cell. This is particularly advantageous when the expression cassette is present in the cell at low copy number, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and the expression product of the RAG1 transgene (when operably linked to a promoter and expressed in the cell) remains at a level at least three-fold higher than the level of a housekeeping gene (such as ABL1) in the cell). Exemplary cells are described herein as recombinant human CD34+ hematopoietic stem cells. However, any cell in which an expression cassette is integrated into its genome is equally relevant, including, but not limited to, HSCs (e.g., CD34+ HSCs), leukocytes, patient-specific induced pluripotent stem cells (iPSCs), or hematopoietic progenitor cells, including mesenchymal stem cells.

[0113] To normalize or compare transgene expression levels between cells, samples, or studies, the Abelson murine leukemia viral oncogene homolog 1 (ABL1) gene is commonly used as a control gene because ABL1 gene transcription levels do not vary significantly between normal and leukemia samples (Beillard et al., 2003). Therefore, ABL1 can be used to normalize or compare expression levels obtained for the expression product of the RAG1 transgene (e.g., RAG1 transcript or protein levels in cells). Methods for measuring and comparing ABL1 to an expression product of interest are well known in the art and are described elsewhere herein.

[0114] Additionally, additional elements may be included in the expression cassette to optimize expression of the desired transgene.

[0115] For example, an expression cassette can include any combination, or indeed all, of the following elements, in any order well known in the art:

[0116] [Table 3]

[0117] In one example, an expression cassette is provided that includes a RAG1 transgene operably linked to a promoter, wherein the expression cassette further includes a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). WPRE sequences are well known in the art; see, e.g., Zanta-Boussif MA, Charrier S, Brice-Ouzet A, Martin S, Opolon P, Thrasher AJ, Hope TJ, Galy A. "Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS." Gene Ther. 2009 May;16(5):605-19. doi: 10.1038 / gt.2009.3

[0118] In other words, the expression cassette may comprise a RAG1 transgene operably linked to an MND promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In one example, the expression cassette may comprise a (human) RAG1 transgene (or a codon-optimized sequence thereof) operably linked to an MND promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence for human RAG1 is shown in SEQ ID NO:2 or SEQ ID NO:4.

[0119] In another example, the expression cassette may comprise a RAG1 transgene operably linked to a CMV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In one example, the expression cassette may comprise a (human) RAG1 transgene (or a codon-optimized sequence thereof) operably linked to a CMV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence for human RAG1 is set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0120] In another example, the expression cassette may comprise a RAG1 transgene operably linked to an RSV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In one example, the expression cassette may comprise a (human) RAG1 transgene (or a codon-optimized sequence thereof) operably linked to an RSV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence for human RAG1 is set forth in SEQ ID NO:2 or SEQ ID NO:4.

[0121] In another example, the expression cassette may comprise a RAG1 transgene operably linked to a CAG promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In one example, the expression cassette may comprise a (human) RAG1 transgene (or a codon-optimized sequence thereof) operably linked to a CAG promoter, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence for human RAG1 is shown in SEQ ID NO:2 or SEQ ID NO:4.

[0122] Advantageously, the expression product of the RAG1 transgene (when operably linked to a suitable promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell. This is particularly advantageous when the expression cassette is present in the cell at low copy number, e.g., when the copy number of the expression cassette integrated into the genome of the cell is 5 or less (and the expression product of the RAG1 transgene (when operably linked to a suitable promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at a level at least three-fold higher than that of a housekeeping gene (e.g., ABL1) in the cell).

[0123] Retroviral Plasmids Provided herein are retroviral plasmids, also called transfer plasmids.

[0124] The retroviral plasmid contains an expression cassette comprising a RAG1 transgene operably linked to a promoter. Suitable RAG1 transgenes are described elsewhere herein. For example, the RAG1 transgene can be a human RAG1 transgene. The human RAG1 transgene is codon-optimized as described elsewhere herein (see, for example, SEQ ID NO: 2 or SEQ ID NO: 4).

[0125] Suitable promoters are provided herein. As described elsewhere herein, the promoters described herein drive high-level expression of an operably linked transgene in a cell. Advantageously, these promoters can drive the required level of expression of a RAG1 transgene even when the expression cassette is part of a low-copy-number retroviral plasmid. For example, the promoters described herein can drive expression of an operably linked RAG1 transgene in a cell such that the expression product of the RAG1 transgene in the cell is at a level at least x-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cell (e.g., recombinant human CD34+ hematopoietic stem cells). As used herein, "x-fold higher" includes at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cell (e.g., recombinant human CD34+ hematopoietic stem cells).

[0126] The retroviral plasmid may contain any of the expression cassettes described herein. For example, the retroviral plasmid may contain an expression cassette comprising a RAG1 transgene operably linked to an MND promoter. Such expression cassettes are described in detail elsewhere herein.

[0127] In another example, the retroviral plasmid may contain an expression cassette comprising a RAG1 transgene operably linked to a CMV promoter. In a further example, the retroviral plasmid may contain an expression cassette comprising a RAG1 transgene operably linked to a CAG promoter. In another example, the retroviral plasmid may contain an expression cassette comprising a RAG1 transgene operably linked to an RSV promoter. Such expression cassettes are described in detail elsewhere herein.

[0128] In this specification, the terms "plasmid" and "vector" are used interchangeably unless otherwise specified.

[0129] The term "vector" is well known in the art and refers to a nucleic acid molecule, e.g., DNA or RNA, into which an expression cassette described herein can be inserted. A vector is used to transport an inserted nucleic acid molecule (here, an expression cassette comprising a promoter operably linked to the RAG1 transgene) into a suitable host cell. A vector generally contains all necessary elements that allow the inserted nucleic acid molecule to be transcribed and, preferably, the transcript to be translated into a polypeptide, so that once the vector is inside a host cell, it can replicate independently of or co-representatively with the host's chromosomal DNA. Multiple copies of the vector and its inserted nucleic acid molecule can be generated. A vector can be an episomal vector (i.e., one that does not integrate into the host cell's genome) or a vector that integrates into the host cell's genome. A vector can be a non-viral or viral vector. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS), and pBR322 or their derivatives lacking bacterial sequences (minicircles)), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. To accommodate larger inserts, larger vectors such as artificial chromosomes (bacterial (BAC), yeast (YAC), or human (HAC)) may be used. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpes viruses, and hepatitis virus vectors. Generally, although not necessarily, viral vectors are replication-deficient because viral genes essential for replication have been removed from the viral vector, thereby losing the ability to propagate in a given cell. However, some viral vectors can be adapted to replicate specifically in a given cell, such as cancer cells, and are commonly used to induce cell-specific oncolysis.Virosomes are a non-limiting example of vectors that contain both viral and non-viral elements, particularly liposomes combined with inactivated HIV or influenza viruses. Another example is a viral plasmid mixed with a cationic lipid.

[0130] The term "retroviral plasmid" is also well known in the art and, as used herein, refers to a plasmid derived from an RNA virus known as a retrovirus. Retroviruses have the ability to insert one or more copies of their genome into the genome of a host cell. Gammaretroviral and lentiviral plasmids are attractive for gene therapy. These plasmids have been refined and developed to mediate stable genetic modification of treated cells by chromosomal integration of the transferred plasmid genome. This technology is useful not only for research purposes but also for clinical gene therapy aimed at long-term correction of genetic defects in stem and progenitor cells. Retroviral plasmid particles have been engineered with tropisms for various target cells. Gammaretroviral and lentiviral plasmids have been used in over 300 clinical trials to date, representing treatment options for a variety of diseases.

[0131] In one example, the retroviral plasmids described herein are lentiviral plasmids. Alternative retroviral plasmids that can be used include MFG and MSCV.

[0132] The retroviral plasmid may be a self-inactivating (SIN) lentiviral plasmid, which is useful because the viral promoter / enhancer sequences are inactivated, significantly reducing the incidence of insertional mutagenesis.

[0133] In one example, a SIN lentiviral plasmid contains the pCCL backbone. The pCCL backbone is advantageous because it is a third-generation LV plasmid well known in the art, allowing for the production of virion particles at high titers and the concentration of virion supernatants to even higher titers required for clinical applications. Alternative SIN lentiviral plasmids include pRRL, pRLL, and pCLL. These are all lentiviral transfer plasmids containing a plasmid backbone containing the simian virus 40 polyadenylation and (enhancer-less) replication origin sequences downstream of the chimeric Rous sarcoma virus (RSV)-HIV or CMV-HIV 5' LTR and the HIV 3' LTR, replacing most of the human sequences remaining at the HIV integration site. In pRRL, the enhancer and promoter of the RSV U3 region (nucleotides -233 to -1 relative to the transcription start site, GenBank Accession No. J02342) are linked to the R region of the HIV-1 LTR. In pRLL, the RSV enhancer sequence (nucleotides -233 to -50) was linked to the HIV-1 promoter region (position -78 relative to the transcription start site). In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start site; GenBank Accession No. K03104) were linked to the HIV-1 R region. In pCLL, the CMV enhancer (nucleotides -673 to -220) was linked to the HIV-1 promoter region (position -78).

[0134] Thus, by way of example, a retroviral plasmid may include: 1) an expression cassette comprising a RAG1 transgene (e.g., human RAG1, which may be codon-optimized as described herein; see SEQ ID NO:2 or SEQ ID NO:4) operably linked to an MND promoter, and 2) a SIN lentiviral backbone, e.g., with a pCCL backbone.

[0135] In another example, the retroviral plasmid may include 1) an expression cassette including a RAG1 transgene (e.g., human RAG1, which may be codon-optimized as described herein; see SEQ ID NO:2 or SEQ ID NO:4) operably linked to a CMV promoter, and 2) a SIN lentiviral backbone, e.g., with a pCCL backbone.

[0136] In another example, the retroviral plasmid may include 1) an expression cassette containing a RAG1 transgene (e.g., human RAG1, which may be codon-optimized as described herein; see SEQ ID NO:2 or SEQ ID NO:4) operably linked to an RSV promoter, and 2) a SIN lentiviral backbone, e.g., with a pCCL backbone.

[0137] In another example, the retroviral plasmid may include 1) an expression cassette including a RAG1 transgene (e.g., human RAG1, which may be codon-optimized as described herein; see SEQ ID NO:2 or SEQ ID NO:4) operably linked to a cag promoter, and 2) a SIN lentiviral backbone, e.g., with a pCCL backbone.

[0138] As described elsewhere herein, the expression cassettes provided herein may have additional elements, such as, for example, a nucleotide sequence encoding the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0139] For example, the retroviral plasmid may contain the sequence of FIG.

[0140] composition Also provided are compositions comprising the expression cassettes, plasmids, or virions described herein together with pharmaceutically acceptable excipients, adjuvants, diluents, and / or carriers. Compositions may typically include pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, supplemental immune enhancing agents such as adjuvants and cytokines, and any other therapeutic agents or compounds.

[0141] As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual together with the selected binding protein without causing undesired biological effects or interacting adversely with any of the other components of the pharmaceutical composition in which it is included.

[0142] An excipient is a natural or synthetic substance formulated with an active substance (e.g., an expression cassette, a plasmid, or a virion) to increase the bulk of the formulation, such as by promoting drug absorption or dissolution, or to enhance the therapeutic effect of the active ingredient in the final dosage form. Excipients are also useful in the manufacturing process, facilitating the handling of the active substance, such as by increasing the flowability or non-stickiness of the powder, as well as enhancing in vitro stability, such as preventing degradation during the expected storage period. Pharmaceutically acceptable excipients are well known in the art. Therefore, suitable excipients can be easily identified by those skilled in the art. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, etc.

[0143] Adjuvants are pharmacological and immunological agents that adjust the effect of other agents in the formulation.Pharmaceutically acceptable adjuvants are well known in the art.Therefore, those skilled in the art can easily identify suitable adjuvants.

[0144] A diluent is a drug that is diluted. Pharmaceutically acceptable diluents are well known in the art. Therefore, a person skilled in the art can easily identify a suitable diluent.

[0145] The carrier is non-toxic to recipients at the dosage and concentration used, and is compatible with other ingredients of the formulation. The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Therefore, suitable carriers can be easily identified by those skilled in the art.

[0146] Virus particle production The retroviral plasmids described herein, e.g., lentiviral plasmids, can be used to produce virions. To increase the safety of the virions, the components required for virion production are separated into multiple plasmids (three for the second-generation system and four for the third-generation system). The components of both systems are as follows: A lentiviral transfer plasmid encoding an insert of interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate integration of the transfer plasmid sequence into the host genome. Generally, sequences between and including the LTRs are integrated into the host genome upon viral transduction. Most lentiviral transfer plasmids are based on the HIV-1 virus. For safety reasons, all transfer plasmids are replication-incompetent and may also contain a deletion in the 3' LTR, resulting in self-inactivating (SIN) virus after integration. Packaging plasmids (may be one or two plasmids) · Envelope plasmids. As an example, SIN lentiviral plasmids are used herein as they are considered safer for gene therapy applications.

[0147] The most important factor to consider and optimize is the transfer plasmid containing the expression cassette. Second-generation lentiviral plasmids utilize viral LTR promoters to drive gene expression, while third-generation transfer plasmids utilize hybrid LTR promoters. Additional or specialized promoters can also be included in the transfer plasmid; for example, the pSico plasmid contains a U6 promoter to drive shRNA expression. Other features that can be included in the transfer plasmid include Tet- or Cre-based control and fluorescent fusions or reporters.

[0148] Third-generation systems further improve upon the safety of second-generation systems in several key ways. First, the packaging system is split into two halves: one for the Rev-encoding plasmid and one for the Gag- and Pol-encoding plasmids. Second, third-generation systems eliminate Tat by adding a chimeric 5' LTR fused to a heterologous promoter to the transfer plasmid. Transgene expression from this promoter is no longer dependent on Tat transactivation. Third-generation transfer plasmids can be packaged by either second- or third-generation packaging systems.

[0149] Methods for producing transgenic retroviral (e.g., lentiviral) virions are widely known in the art (e.g., protocols such as Pike-Overzet, Leukemia, 2011). Briefly, 3-4 plasmids are transfected into A293T cells; after a medium change and a short incubation period, the supernatant containing the virions is removed and saved or centrifuged to concentrate the virions. Crude or concentrated virions can then be used to transfect cells of interest. The viral titer can then be measured.

[0150] Thus, also provided herein are virions comprising an expression cassette comprising a RAG1 transgene operably linked to a promoter. Components of suitable expression cassettes are described elsewhere herein.

[0151] For the avoidance of doubt, the expression cassette present in the virion may comprise an RNA nucleic acid sequence, for example, the expression cassette present in the virion may comprise an RNA sequence corresponding to any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.

[0152] As used herein, "transfection" broadly refers to any process of intentionally introducing nucleic acid into a cell, including the introduction of viral and non-viral vectors, and encompasses terms and processes such as transformation and transduction. Examples include transfection with viral vectors, transformation with plasmid vectors, electroporation (Fromm et al. (1986) Nature 319:791-3), lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7), microinjection (Mueller et al. (1978) Cell 15:579-85), Agrobacterium-mediated transcription (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7), direct uptake of DNA, whisker-mediated transformation, and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0153] treatment method Provided herein is a method for treating patients who do not have a functional Rag1 gene or RAG1 protein. For example, provided herein is a method for treating patients with RAG1-deficient severe combined immunodeficiency (RAG1-SCID) or Omenn's syndrome. The complete loss of RAG1 function in humans leads to severe immunodeficiency in humans. Therefore, patients who do not have a functional Rag1 gene of RAG1 protein are generally identified in infancy.

[0154] The method provided herein is also for treating patients with at least one mutation in the RAG1 protein. In other words, this method is for treating diseases caused by at least one mutation in the RAG1 protein. These diseases are characterized by partial loss of functional RAG1 in patients, i.e., the patient may have a mild (hypomorphic) RAG1 mutation. Diseases caused by mild RAG1 mutations worsen at a slower rate than diseases caused by complete loss of RAG1 function, because RAG1 mutants can maintain partial recombination activity. Therefore, diseases with mild RAG1 mutations may develop life-threatening complications several years later. Although this disease is often underdiagnosed, it may be far more common than RAG1-SCID or OS. Next-generation sequencing of patients with primary immunodeficiency has revealed many mild RAG1 mutations, but there is currently no curative treatment for them. In fact, functional evaluation of 71 RAG1 mutants has been performed to date. The phenotype associated with mild RAG1 mutations is combined immunodeficiency with granulomatous and / or autoimmune diseases (CID-G / A). RAG1 deficiency can be measured by quantifying recombination activity in vitro. An example of a disease caused by mild RAG1 mutations that can be treated by the methods described herein is atypical SCID or combined immunodeficiency (CID). CID is a group of diseases characterized by mild RAG1 mutations that lead to a reduced immune repertoire.

[0155] The methods provided herein are particularly useful in treating patients with RAG1-deficient severe combined immunodeficiency (RAG1-SCID) or Omenn's syndrome. However, as noted above, they are also useful in treating patients with atypical SCID or combined immunodeficiency (CID). Thus, although the present invention is described primarily in the context of RAG1-SCID or Omenn's syndrome, all such aspects of the invention apply equally to atypical SCID or combined immunodeficiency (CID).

[0156] The method may involve ex vivo cell-based therapy. Suitable methodology for use in such methods is well known in the art; see, for example, “Improving Lentiviral Transduction of CD34+ Hematopoietic Stem and Progenitor Cells”April 2018 Human Gene Therapy Methods 29(2) DOI: 10.1089 / hgtb.2017.085; or PLoS One. 2009 Jul 30;4(7):e6461. doi: 10.1371 / journal.pone.0006461. “Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem / progenitor cells.” Millington M1, Arndt A, Boyd M, Applegate T, Shen S.

[0157] For example, hematopoietic progenitor cells, such as HSCs (e.g., CD34+ HSCs), can be isolated from a patient. Methods for doing so are described elsewhere herein. The genomes of these cells can be modified using expression cassettes, plasmids, virions, or compositions and methods described herein. The recombinant cells can then be transplanted back into the patient.

[0158] The terms "hematopoietic progenitor cell" and "hematopoietic stem cell (HSC)" refer to cells of the stem cell lineage that give rise to all blood cell types, including erythroid cells (erythrocytes or red blood cells (RBCs)), myeloid cells (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, dendritic cells), and lymphoid cells (T cells, B cells, NK cells).

[0159] Preferably, the hematopoietic progenitor cells, e.g., HSCs, express at least one of the following cell surface markers characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thyl / CD90+, CD381o / -, and C-kit / CDI17+. Most preferably, the hematopoietic progenitor cells are CD34+ HSCs.

[0160] HSCs are an important target for gene therapy because they provide a long-term source of corrected cells. HSCs give rise to both myeloid and lymphoid blood cells. Mature blood cells have a limited lifespan and must be continuously replaced throughout life. Blood cells are continuously produced by the proliferation and differentiation of a population of multipotent hematopoietic stem cells, which can be replenished through self-renewal. The bone marrow (BM) is the primary site of hematopoiesis in humans and a good source of hematopoietic stem and progenitor cells (HSPCs). HSPCs are also present in small amounts in peripheral blood (PB). Depending on the indication or treatment, their numbers may increase. HSC progeny mature through stages to generate multipotent and lineage-committed progenitors, including lymphoid progenitors that give rise to RAG1-expressing cells. Because B- and T-cell progenitors are two cell populations that require RAG1 activity, they can be transfected at a pre-repopulating stage, but correcting progenitor cells has the advantage of providing a continuous source of corrected cells.

[0161] Thus, the present method can include an ex vivo method for generating recombinant CD34+ hematopoietic stem cells, comprising contacting CD34+ hematopoietic stem cells with virions described herein under conditions in which the expression cassette is incorporated into the cells and expressed, thereby generating recombinant CD34+ hematopoietic stem cells. As used herein, "conditions in which the expression cassette is incorporated into the cells and expressed, thereby generating recombinant CD34+ hematopoietic stem cells" can include culturing cells in the presence of appropriate medium and growth factors, followed by incubation with lentiviral virions described herein. Optionally, retronectin, proteamine sulfate, or other compounds that promote viral transduction (transduction enhancers) can be included.

[0162] In one example, CD34+ cells are isolated from a patient's blood or bone marrow and cultured ex vivo under GMP-grade conditions using media and growth factors, followed by an additional incubation with lentiviral virions containing retronectin, proteamine sulfate, or other compounds that facilitate viral transduction (transduction enhancers). Culture continues, and in some cases, a second viral "hit" is administered. At the end of the culture period, the cells are harvested and collected in an intravenous bag for administration to the patient (or frozen in liquid nitrogen until needed, then thawed and administered IV).

[0163] The term "recombinant" cell means a cell that contains at least one integrated expression cassette.

[0164] Thus, also provided herein are recombinant CD34+ hematopoietic stem cells comprising an expression cassette that includes a RAG1 transgene operably linked to a promoter (details of which are described elsewhere herein). Advantageously, when the promoters described herein are used in combination with the transgenes described herein, the required level of transgene expression is achieved even when low-copy retroviral plasmids are used. In other words, when the expression cassettes, plasmids, and virions described herein are used, the expression product of the RAG1 transgene in the resulting recombinant CD34+ hematopoietic stem cells is at a level at least three-fold higher than that of intracellular ABL1 when the copy number of the expression cassette integrated into the genome of the recombinant human CD34+ hematopoietic stem cells is five or less.

[0165] Advantageously, the combination of the promoter and RAG1 transgene in the expression cassette drives expression of RAG1 in each of the above cell types to a minimum therapeutic threshold level (due to the nature of the promoter used; i.e., due to its ability to drive expression of the transgene so that its expression product is at a level at least three times higher in the cell than the expression level of a housekeeping gene such as ABL1 (even when the copy number of the expression cassette integrated into the cell's genome is 5 or less, i.e., when a low-copy-number plasmid is used)).

[0166] Thus, in one example, a method of treating RAG1 deficient SCID or OS in a subject is provided, the method comprising the steps of: (i) extracting CD34+ hematopoietic stem cells from a subject; (ii) contacting the cells of (i) with a virion described herein; (iii) incubating the cells of (ii) for a period of time, preferably 12 to 84 hours, more preferably 12 to 72 hours; and (iv) introducing the cells of (iii) into a subject in need of treatment.

[0167] To extract CD34+ hematopoietic stem cells, tissue or fluid can be biopsied or aspirated from the bone marrow of the subject.Biopsy or aspirated can be carried out according to any method known in the art.For example, bone marrow aspiration (aspiration) uses a thick needle to be inserted into the pelvic bone to collect bone marrow.

[0168] Hematopoietic progenitor cells may be extracted from biopsies or aspirates by any method known in the art. For example, CD34+ cells may be enriched using the CliniMACS® Cell Selection System (Miltenyi Biotec). CD34+ cells may also be weakly stimulated with cytokines (e.g., SCF, rhTPO, rhFLT3) in serum-free medium (e.g., CellGrow SCGM medium, CellGenix).

[0169] The cells may then be contacted with the virions and incubated together for a suitable period of time using methods well known in the art.

[0170] Before transplanting the recombinant cells into the patient, clearance of the bone marrow niche may be required. Current methods rely on radiotherapy and / or chemotherapy. Therefore, the method of the present invention may include a step of administering chemotherapy to the subject before step (iv). Suitable chemotherapy regimens are well known to those skilled in the art.

[0171] However, due to the limitations and side effects of radiation and / or chemotherapy, safer conditioning regimens have been and are currently being developed, such as immunodepletion of bone marrow cells with antibodies or antibody-toxin conjugates against hematopoietic cell surface markers such as CD17, c-kit, etc. Such methods can also form part of the methods described herein.

[0172] The method of the present invention then includes the step of returning the cells to the subject in need of treatment. As used herein, this also refers to transplanting the recombinant cells back into the patient. This transplantation step can be accomplished using any transplantation method known in the art. For example, the recombinant cells may be administered to the patient by injecting them directly into the patient's bloodstream.

[0173] By introducing the expression cassette into autologous cells that are derived from the patient in need and are therefore already a perfect immunological match, it is possible to generate cells that can be safely reintroduced into the patient, effectively generating a population of cells that are effective in ameliorating one or more clinical conditions associated with the patient's disease.

[0174] The above examples refer to hematopoietic stem cells, however, white blood cells isolated from the patient can alternatively be used in the above treatments.

[0175] Patient-specific induced pluripotent stem cells (iPSCs) may be generated. The genomes of these iPS cells may then be modified using expression cassettes, plasmids, virions, or compositions and methods described herein. The iPSCs may then be differentiated into hematopoietic progenitor cells or leukocytes. Finally, the hematopoietic progenitor cells or leukocytes may be transplanted into the patient.

[0176] Alternatively, mesenchymal stem cells can be isolated from a patient and used in the above treatments.

[0177] One advantage of ex vivo cell therapy is the ability to perform comprehensive analysis of therapeutic agents prior to administration. Additionally, specific cell populations, including clonal populations, can be isolated or enriched prior to transplantation.

[0178] Also described are in vivo-based therapies, which utilize the materials and methods described herein to correct the chromosomal DNA of patient cells. Preferably, the cells are leukocytes, bone marrow cells, hematopoietic progenitor cells, HSCs, or HSC CD34+ cells.

[0179] Although blood cells present attractive targets for ex vivo treatment and therapy, increased efficiency in delivery may enable direct in vivo delivery to other B and T cell precursors, such as HSCs and / or CD34+ cells. Ideally, targeting and integration of expression cassettes could be directed to relevant cells.

[0180] The advantage of in vivo gene therapy is that the therapeutic agent is easy to manufacture and administer.The same therapeutic approach and treatment method can be used to treat multiple patients, for example, multiple patients who share the same or similar genotype or allele.In contrast, ex vivo cell therapy usually requires the use of the patient's own cells, which are isolated, manipulated, and then returned to the same patient.

[0181] Pharmaceutically acceptable carriers for recombinant cells The ex vivo methods of administering recombinant cells to a subject contemplated herein involve the use of a therapeutic composition comprising the recombinant cells.

[0182] Therapeutic compositions comprise a physiologically acceptable carrier together with the recombinant cell composition, and optionally, at least one additional bioactive agent described herein dissolved or dispersed therein as an active ingredient. Preferably, the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless desired.

[0183] Generally, the recombinant cells described herein are administered as a suspension containing a pharmaceutically acceptable carrier. Those skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition does not contain buffers, compounds, cryopreservatives, preservatives, or other agents in an amount that substantially prevents the viability of the cells delivered to a subject. The formulation containing the recombinant cells may contain, for example, an osmotic buffer that allows the integrity of the cell membrane to be maintained, and, if necessary, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with progenitor cells as described herein using routine experimentation.

[0184] The recombinant cell composition may also be emulsified or presented as a liposomal composition, provided that the emulsification procedure does not adversely affect the viability of the cells. The recombinant cells and any other active ingredient(s) may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient(s) in amounts suitable for use in the therapeutic methods described herein.

[0185] Additional agents contained in the recombinant cell composition can include pharmaceutically acceptable salts of the components contained therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, etc. Salts can also be formed with free carboxyl groups using inorganic bases such as sodium, potassium, ammonium, calcium, or iron, or organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0186] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers include sterile aqueous solutions containing no substances in addition to the active ingredient and water, or phosphate-buffered saline solutions containing buffers such as sodium phosphate, saline, or both at physiological pH values. Additionally, aqueous carriers can contain multiple buffer salts, as well as salts such as sodium chloride and potassium chloride, glucose, polyethylene glycol, and other solutes. Liquid compositions can contain liquid phases in addition to and in addition to water. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in a recombinant cell composition that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.

[0187] Administration and efficacy of recombinant cells The terms "administer," "introduce," and "implant" are used interchangeably to refer to placing recombinant cells, e.g., HPSC cells, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect is achieved. Recombinant cells, e.g., HPSC cells, can be administered by any suitable route that delivers at least a portion of the transplanted cells or cellular components to a desired location in a subject in a viable state. The survival period of the cells after administration to a subject can be a few hours, e.g., 24 hours, several days, several years, or even the lifetime of the patient, i.e., long-term engraftment. For example, in some embodiments described herein, an effective amount of myogenic progenitor cells is administered via a systemic route, such as intraperitoneally or intravenously (IV).

[0188] The terms "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any subject for whom diagnosis, treatment, or therapy is desired. For purposes of the present invention, a subject may be a primate, preferably a human, or other mammal, such as a dog, cat, horse, pig, goat, or cow.

[0189] When provided prophylactically, the recombinant cells described herein can be administered to a subject prior to any symptoms of SCID and / or Omenn's syndrome, for example, prior to the onset of alpha / beta T-cell lymphopenia with gamma / delta T-cell proliferation, severe cytomegalovirus (CMV) infection, autoimmunity, chronic inflammation of the skin, eosinophilia, reproductive problems, lymphadenopathy, splenomegaly, diarrhea, and hepatomegaly. Thus, prophylactic administration of a population of hematopoietic progenitor cells helps to prevent SCID and / or Omenn's syndrome.

[0190] When provided therapeutically, HPSCs are provided at (or after) the onset of symptoms or signs of SCID and / or Omenn's syndrome, eg, at the onset of disease.

[0191] Preferably, the HPSC population administered in accordance with the methods described herein comprises allogeneic HPSCs obtained from one or more donors. "Allogeneic" refers to HPSCs, or a biological sample containing HPSCs, obtained from one or more different donors of the same species who are not genetically identical at one or more loci. For example, the HPSC population administered to a subject may be derived from one or more unrelated donor subjects, or from one or more non-identical siblings. Preferably, a syngeneic hematopoietic progenitor cell population can be used, such as one obtained from genetically identical animals or from identical twins. Alternatively, the HPSCs can be autologous, i.e., the HPSCs are obtained or isolated from the subject and administered to the same subject, i.e., the donor and recipient are the same.

[0192] The term "effective amount" refers to the amount of a population of recombinant cells or their progeny required to prevent or alleviate at least one sign or symptom of SCID and / or Omenn syndrome, and refers to a composition in an amount sufficient to achieve the desired effect, e.g., treating a subject with SCID and / or Omenn syndrome. Accordingly, the term "therapeutically effective amount" refers to a sufficient amount of recombinant cells or a composition comprising recombinant cells to promote a particular effect when administered to a typical subject with or at risk of SCID and / or Omenn syndrome. Effective amounts also include amounts sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., including, but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It is understood that an appropriate "effective amount" in any given case can be determined by one of ordinary skill in the art using routine experimentation.

[0193] Preferably, the effective amount of HPSCs is at least 10 2 At least 5 x 10 hPSCs 2 At least 10 hPSCs 3 At least 5 x 10 hPSCs 3 At least 10 hPSCs 4 At least 5 x 10 hPSCs 4 At least 10 hPSCs 5 At least 2 x 10 hPSCs 5 At least 3 x 10 hPSCs 5 At least 4 x 10 hPSCs 5 At least 5 x 10 hPSCs 5 At least 6 x 10 hPSCs 5 Contains at least 7 x 10 hPSCs 5 At least 8 x 10 hPSCs 5 At least 9 x 10 hPSCs 5 At least 1 x 10 hPSCs 6 At least 2 x 10 hPSCs 6 At least 3 x 10 hPSCs 6 At least 4 x 10 hPSCs 6At least 5 x 10 hPSCs 6 At least 6 x 10 hPSCs 6 At least 7 x 10 hPSCs 6 At least 8 x 10 hPSCs 6 At least 9 x 10 hPSCs 6 HPSCs, or multiples thereof. The HPSCs may be derived from one or more donors or obtained from an autologous source. Preferably, the HPSCs described herein are expanded in culture before administration to a subject in need thereof.

[0194] "Administration" means delivering the HPSC composition to a subject by a method or route that results in at least partial localization of the cell composition at the desired site. The cell composition can be administered by any suitable route that results in effective treatment in the subject, i.e., administration delivers the cell composition to the desired site in the subject, and at least a portion of the delivered composition, i.e., at least 1 x 10 4 The cells are delivered to the desired site for a certain period of time. Methods of administration include injection, infusion, drip infusion, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial spinal injection, and drip infusion. In some embodiments, the route is intravenous. Cell delivery can be by injection or infusion.

[0195] Preferably, the cells are administered systemically. The terms "systemic administration," "administered systemically," "peripheral administration," and "peripherally administered" refer to administering a population of progenitor cells other than directly to a target site, tissue, or organ so that they instead enter the subject's circulatory system and are therefore subject to metabolic and other similar processes.

[0196] The effectiveness of a composition for treating SCID and / or Omenn's syndrome can be determined by one of skill in the art. Treatment is considered "effective" if any one or more of the signs or symptoms of the disease are altered in a beneficial manner. By way of example, treatment is considered effective when the level of a functional RAG1 protein of interest is at least three times higher in CD34+ cells than the level of an appropriate housekeeping gene (e.g., ABL1). Efficacy can also be measured by a lack of worsening of an individual's symptoms (e.g., halting or at least slowing disease progression) as assessed by the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including: (1) inhibiting the disease, e.g., preventing or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing symptomatic remission; and (3) preventing or reducing the likelihood of symptom onset.

[0197] The treatment methods of the present invention improve one or more symptoms associated with SCID and / or Omenn's syndrome by increasing the amount of functional RAG1 in an individual. Early symptoms commonly associated with SCID and / or Omenn's syndrome include, for example, the development of alpha / beta T-cell lymphopenia with gamma / delta T-cell proliferation, severe cytomegalovirus (CMV) infection, autoimmunity, chronic inflammation of the skin, eosinophilia, growth failure, lymphadenopathy, splenomegaly, diarrhea, and hepatomegaly.

[0198] kit Also provided herein are kits for carrying out the methods of the present invention. The kits may include one or more of the expression cassettes of the present invention, the plasmids of the present invention, or the virions of the present invention, and / or any nucleic acid or protein molecules or any combination thereof necessary to carry out aspects of the methods of the present invention. Suitably, the kits may include reagents and / or reagents for reconstitution and / or dilution of the plasmid(s). Suitably, the components of the kit may be in separate containers or may be mixed in a single container.

[0199] Preferably, the above kit further comprises one or more additional reagents, such additional reagents being selected from buffers, buffers for introducing polypeptides or polynucleotides into cells, washing buffers, control reagents, etc. Buffers include stabilization buffers, reconstitution buffers, dilution buffers, etc.

[0200] In addition to the above-mentioned components, the kit may further include instructions for using the kit components to practice the method. The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be included in the kit as a package insert or may be on the label of the container (i.e., associated with the packaging or subpackaging) of the kit or its components. The instructions may be present as an electronic storage data file residing on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not included in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment includes a kit that includes a web address where the instructions can be viewed or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions may be recorded on a suitable substrate.

[0201] General definition As used herein, "complementary" or "complementarity" refers to the Watson-Crick base pairing of two nucleic acid sequences. For example, the sequence 5'-AGT-3' will bind to the complementary sequence 3'-TCA-5'. Complementarity between two nucleic acid sequences can be "partial," where only some bases bind with their complement, or "complete," where all bases in the sequence bind with their complementary bases. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0202] The term "hybridizing" refers to the annealing of two at least partially complementary nucleotide sequences in a hybridization process. To enable hybridization, complementary nucleic acid molecules are typically thermally or chemically denatured to melt the duplex into two single strands and / or remove secondary structures, such as hairpins, from single-stranded nucleic acids. Hybridization stringency is influenced by conditions such as temperature, salt concentration, and the composition of the hybridization buffer. Conventional hybridization conditions are described, for example, in Sambrook (2001) Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York. However, those skilled in the art will appreciate that numerous different hybridization conditions can be designed depending on the known or predicted homology and / or length of the nucleic acid sequences. High stringency conditions for hybridization include high temperature and / or low sodium / salt concentration (salts include, for example, sodium chloride, NaCl, and sodium citrate) and / or inclusion of formamide in the hybridization buffer and / or reducing the concentration of compounds such as SDS (sodium dodecyl sulfate detergent) in the hybridization buffer and / or eliminating compounds such as dextran sulfate or polyethylene glycol (which promote molecular crowding) from the hybridization buffer. As a non-limiting example, typical salt and temperature conditions for stringent hybridization are 1x SSC, 0.5% SDS, and 65°C. The abbreviation SSC refers to the buffer used in nucleic acid hybridization solutions. One liter of 20x (20-fold concentrated) stock SSC buffer (pH 7.0) contains 175.3 g of sodium chloride and 88.2 g of sodium citrate. A typical time to achieve hybridization is 12 hours.

[0203] The terms "identity" and "identical" refer to the sequence similarity between two nucleic acid molecules, such as two polymer molecules, e.g., two DNA molecules. Sequence alignment and sequence identity determination can be performed, for example, using the Basic Local Alignment Search Tool (BLAST), first described in Altschul et al. 1990 (J Mol Biol 215:403-10), e.g., the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250).

[0204] Methods for aligning sequences for comparison are well known in the art. Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed discussion of sequence alignment methods and homology calculations is provided, for example, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0205] National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST (商標) ; Altschul et al. (1990)) is available in conjunction with several sequence analysis programs from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the Internet. A description of how to determine sequence identity using this program is available at BLAST on the Internet. (商標) For nucleic acid sequence comparisons, use BLAST. (商標) The "Blast 2 sequences" function of the (Blastn) program can be used with default parameters. Nucleic acid sequences that are more similar to a reference sequence will have an increased percent identity when evaluated in this manner. Typically, the percent sequence identity is calculated over the entire length of the sequence.

[0206] For example, the Needleman-Wunsch algorithm suitably finds a global optimal alignment using the following scoring parameters: match score: +2, mismatch score: -3, gap penalty: gap open 5, gap extension 2. The percent identity of the resulting optimal global alignment is suitably calculated by multiplying the ratio of the number of aligned bases to the total length of the alignment (the length of the alignment includes both matches and mismatches) by 100.

[0207] While the specification provides detailed descriptions of making and using various embodiments of the invention, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not limit the scope of the invention.

[0208] The practice of the present invention may employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology which are within the skill of those in the art, and such techniques are fully explained in the literature. For example, Current Protocols in Molecular Biology (Ausubel, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gait ed., 1984); US Patent No. 4,683,195; Nucleic Acid Hybridization (Harries and Higgins eds. 1984); Transcription and Translation (Hames and Higgins eds. 1984); Culture of Animal Cells (Freshney, Alan R. Liss, Inc, 1987), Immobilized Cells and Enzymes (IRL Press, 1986), Perbal, A Practical Guide to Molecular Cloning (1984), Methods in Enzymology series (Abelson and Simon, eds. -in-chief, Academic Press, Inc., New York), especially volumes 154 and 155 (Wu et al. eds.), volume 185 "Gene Expression Technology" (Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (Miller and Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); and Handbook of Experimental Immunology, Vols. Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0209] Terms defined herein have meanings commonly understood by one of ordinary skill in the art relevant to the present invention. Terms such as "a," "an," and "the" are not intended to refer to only a single entity, but include general classes for which specific examples may be used for illustration. While terms herein are used to describe particular aspects of the present invention, their use does not limit the present invention except as outlined in the claims. The present disclosure relates, for example, to the following: [Section 1] 1. An expression cassette comprising a promoter operably linked to a RAG1 transgene comprising the nucleic acid sequence of SEQ ID NO:2, wherein the promoter is selected from MND, CMV, RSV and CAG. [Section 2] The expression cassette of item 1, wherein the RAG1 transgene comprises the nucleic acid sequence of SEQ ID NO:4. [Section 3] Item 1. The expression cassette according to Item 1, wherein when the expression cassette is expressed in human CD34+ hematopoietic stem cells having a genome copy number of 5 or less, an expression product is produced at a level at least 3 times higher than the expression level of ABL1 in the cells. [Section 4] Item 4. The expression cassette of any one of items 1 to 3, wherein the promoter is MND. [Section 5] 5. The expression cassette of any one of paragraphs 1 to 4, wherein the expression cassette further comprises a nucleotide sequence encoding a Woodchuck Hepatitis Virus (WHP) post-transcriptional regulator (WPRE). [Section 6] A retroviral plasmid comprising the expression cassette of any one of items 1 to 5. [Section 7] The plasmid of paragraph 6, wherein the plasmid is a self-inactivating (SIN) lentiviral plasmid. [Section 8] The plasmid of paragraph 7, wherein the plasmid comprises a pCCL backbone. [Section 9] 9. The plasmid of any one of paragraphs 6 to 8, wherein the plasmid comprises a pCCL backbone, a nucleotide sequence encoding WPRE, an MND promoter, and a transgene comprising the nucleic acid sequence of SEQ ID NO:4. [Section 10] A virion comprising the expression cassette of any one of paragraphs 1 to 5. [Section 11] A composition comprising the expression cassette of any one of paragraphs 1 to 5 or the plasmid of any one of paragraphs 6 to 9, or the virion of paragraph 10, and a pharmaceutically acceptable adjuvant, carrier, excipient, or diluent. [Section 12] A recombinant CD34+ hematopoietic stem cell comprising the expression cassette of any one of items 1 to 5. [Section 13] An ex vivo method for generating recombinant CD34+ hematopoietic stem cells, comprising contacting cells with the plasmid of any one of paragraphs 6 to 9 or the virion of paragraph 10 under conditions in which the expression cassette is taken up by the cells and expressed, thereby producing recombinant CD34+ hematopoietic stem cells. [Section 14] 13. The expression cassette, plasmid, composition, virion or recombinant cell of any one of paragraphs 1 to 12 for use in therapy. [Section 15] 15. The expression cassette, vector, composition, virion, or recombinant cell for use according to paragraph 14, wherein the expression cassette, vector, composition, virion, or recombinant cell is for use in treating RAG1-deficient severe combined immunodeficiency (SCID), Omenn's syndrome (OS), atypical SCID, or combined immunodeficiency (CID). [Section 16] A method of treating a subject comprising administering to a subject in need thereof a therapeutically effective amount of an expression cassette, plasmid, composition, virion particle or recombinant cell described in any one of paragraphs 1 to 12. [Section 17] The method of paragraph 16, wherein the subject has RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID, or combined immunodeficiency (CID). [Section 18] The method of claim 17, wherein the SCID is a RAG1-deficient SCID. [Section 19] (i) extracting CD34+ hematopoietic stem cells from a subject; (ii) contacting the cell of (i) with the virion of paragraph 10 or the plasmid of paragraphs 6 to 9; (iii) incubating the cells of (ii) for a period of time; and (iv) introducing the cells of (iii) into the subject. 10. A method for treating RAG1 deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID) in a subject in need thereof, comprising: [Section 20] 20. The method of paragraph 19, further comprising administering chemotherapy or other conditioning regimen to the subject prior to step (iv). [Example]

[0210] Example result MND promoter as the optimal vector for correcting Rag1 deficiency. At the start of this project, we constructed four SIN LV plasmids in a CCL scaffold and tested four promoters previously used in other clinical trials. The four promoters, PGK (phosphoglycerate kinase), MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted), UCOE (chromatin-remodeling element), and a combination of UCOE and MND (Cbx-MND), were used to drive expression of codon-optimized versions of RAG1 (Figure 1A). These transfer vectors were combined with GAG-Pol, REV, and envelope (VSV-G) plasmids to generate recombinant lentiviruses, which were then used to transduce lineage-negative BM cells from Rag1-deficient mice. Rag1 knockout (KO) mice were transplanted with wild-type (WT) stem cells, mock-transduced Rag1KO stem cells, or stem cells treated with gene therapy using the four promoters. Mice were bled every 4 weeks and sacrificed after 16 weeks. Subsequently, we performed broad-spectrum analysis of viral copy number (VCN), Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), and therapeutic gene RAG1 expression using flow cytometry and Q-PCR (Figure 7A). Reflecting the known promoter strength of these four vectors, initial testing yielded broad-spectrum RAG1 expression. Mice were sacrificed after 4 months or earlier if they showed signs of disease, and immune organs were analyzed by flow cytometry. Restoration of IgM+B220+ B cells in the BM (Figure 1B) was observed in mice treated with wild-type (wt) stem cells and gene therapy mice treated with MND-coRAG1, occasionally in mice carrying the Cbx3-MND element, but not in mice treated with the PGK or UCOE promoters (Figure 1B,C).Mock transduction of Rag1 KO stem cells did not restore B cell development, as expected, and cells were arrested at the pre-B cell stage.

[0211] We next analyzed the expression of thymic T cells using CD4 and CD8 markers. Normal T cell development, encompassing the full spectrum of DP and SP developmental stages, was observed in wt and MND-coRAG1 cells, but not in the other promoters used (Figure 1D, E).

[0212] We observed that many mice (n = 4 of 9) in the low coRAG1 expression group developed skin rashes, whereas animals in the high coRAG1 expression group and those administered wild-type or unmodified Rag1 knockout cells did not develop any health problems.

[0213] To better understand the effects of the various promoters used, we analyzed the relationship between RAG1 expression in the BM and the number of B cells generated in the BM (Figure 2A) and the number of T cells generated in the thymus (Figure 2B), two major lymphoid organs in which RAG genes are activated. For B cell development, a clear linear correlation was observed between RAG1 expression and B220+ cells in the BM up to 10-fold above housekeeping gene levels. For T cells, we identified a threshold at which RAG1 expression was minimal, approximately 10-fold above housekeeping control levels. In mice reconstituted with stem cells expressing coRAG1 below this threshold, almost no thymic T cells developed.

[0214] In addition to efficacy, safety is also an important aspect for the clinical use of gene therapy vectors. We used the IVIM assay, a currently accepted standard for viral vector safety, as an additional selection criterion. All four vectors showed at least 50-fold lower frequencies of insertional mutagenic events than the classic RSF91 gammaretroviral vector (Figure 2C), and only the UCOE vector showed significantly lower replication efficiency than the other three promoters.

[0215] Finally, we confirmed the diversity and clonality of the TCRβ repertoire generated in the gene-treated mice (Figure 2D). We performed GeneScan analysis on 24 Vb genes and calculated a cumulative complexity score. Again, the MND promoter performed similarly to WT mice, as shown in the representative plot and the highest score.

[0216] Therefore, the present inventors determined that the pCCL-MND-coRAG1 LV vector was the optimal vector and proceeded with the production of a GMP-grade vector. Using this clinical-grade vector, the following tests were performed, and further preclinical studies are currently underway.

[0217] Extensive preclinical testing of the pCCL-MND-coRAG1 LV vector in Rag1 − / − mice. Initial analysis of eight Rag1 - / - mice treated with the MND vector, positive control (wt stem cells), and negative control (mock-transduced Rag1 - / - stem cells) confirmed favorable B cell reconstitution in the periphery (PB) and BM (Figure 3A), although their numbers remained lower than those of mice treated with wt stem cells (Figure 3B and Figure 7B). This may be due to a partial arrest in development from the pre-B to immature B cell stage, derived from cells transduced with coRAG1 levels insufficient to support complete Ig rearrangement (Figure 7C). Alternatively, residual pro-B and pre-B cells may inhibit B cell development by occupying critical developmental niches. However, in the spleens of gene-treated mice, immature and mature B cell subsets were found at similar rates (Figure 3C). Regarding T cells, most GT mice showed nearly normal thymic T cell development and thymocyte numbers (Figure 3D and Figure 7C). However, peripheral T cell numbers recovered to approximately 30% of normal (Figure 3E). The proportion of naive CD4 and CD8 T cells was somewhat lower, and effector memory subsets were increased (Figure 3F). This is likely due to the constitutive proliferation of early T cells shed from the thymus. In addition to flow cytometric analysis of primary and secondary immune organs, we also confirmed immune system recovery by histological analysis. The spleen, lymph nodes, and thymus exhibited remarkably normal architecture after GT (Figure 3G), comparable to that of mice treated with wt stem cells and distinct from that of negative control mice treated with mock-transduced Rag1- / - cells. Importantly, FoxP3 expression, which directs T cells toward the CD4+ regulatory T cell lineage (Treg), was restored in mice treated with MND-coRAG1 gene therapy (Fig. 3G).

[0218] Functional reconstitution of immunity after Rag1 gene therapy Next, we examined whether the generated T and B cells possessed diverse repertoires and could mount immune responses against T cell-dependent neoantigens. GeneScan analysis revealed a diverse TCR Vb repertoire, which was somewhat less complex than that of mice reconstituted with wt stem cells before immunization (Figure 4A), but no statistically significant differences in immune repertoire were observed after immunization. Total IgM, IgG, and IgE levels were also measured (Figure 4B and Figure 7E), and reached near-normal levels in GT-treated mice. We investigated whether the generated T and B cells cooperated to mount a vigorous immune response by measuring the production of TNP-specific IgG antibodies using TNP-KLH as a T cell-specific antigen. Serum TNP-specific IgG levels were similar in wt stem cell-treated and GT-treated mice (Figure 4C).

[0219] Individual TCR Vb family analysis demonstrated that the MND-coRAG1 construct provided a rearrangement pattern comparable to that of WT controls with polyclonal TCR Vb families, without the disruption of TCR Vb usage or oligoclonal expansion observed with other constructs. Importantly, for the clinical MND-coRAG1 batch, the immune diversity of treated mice was comparable to that of WT control mice both before and after immunization. Importantly, while the CID mouse model is deficient in responses to B cell-dependent T cell antigens, our TNP-KLH-immunized MND-co RAG1 gene-treated mice were able to successfully mount immune responses to B cell-dependent T cell antigens at levels comparable to control mice, suggesting that gene therapy-treated mice do not exhibit a CID phenotype but rather are able to overcome this immunodeficient phenotype.

[0220] Preclinical safety testing of vectors As required by regulatory authorities, clinical-grade vectors were tested for the presence of replication-competent virus (RCL) by an external laboratory. The vectors were negative in two independent tests (data not shown). Other safety tests typically require vector biodistribution in vivo, confirmation of vector insertion sites (especially for potential clonal development), and insertional mutagenesis tests such as IVIM.

[0221] We confirmed vector distribution in multiple perfused organs of all GT-treated mice (Figure 5A). Perfusion was performed to remove most of the blood cells that would otherwise carry the vector. As expected, due to positive selection of coRAG1-transduced cells, high VCN was observed in the thymus, followed by other immune system organs, such as the spleen, bone marrow, lymph nodes, and peripheral blood. All other organs showed significantly lower signals, with the exception of occasional positivity in the stomach and lungs. This may be due to incomplete perfusion or infrequent progression of infection in individual mice (Figure 7D, Table 4).

[0222] [Table 4]

[0223] Importantly, pathological examination of histological slides from 29 different organs per mouse revealed that MNDCoR No abnormalities were observed in mice treated with AG1 gene therapy. The most characteristic pathology of Omenn syndrome (OS) and atypical SCID models is a severe phenotype accompanied by erythroderma, skin infiltration, and eosinophilia. We performed extensive pathology examination of mice treated with the MND-CoRAG1 gene therapy vector, but did not detect any features characteristic of OS / atypical SCID. Indeed, Figure 7D shows lung and liver pathology, which demonstrates a normal phenotype similar to that of WT-treated mice, without abnormal T cell infiltration. We also examined the skin and small intestine to confirm that mice treated with the MND-coRAG1 vector did not exhibit the OS or atypical SCID phenotype. Clinical symptoms of skin disease were absent in all groups (no ulcers, crusts, redness, or hair loss) (Figure 10). Furthermore, histological analysis of the skin from all groups confirmed that the hallmarks of Omenn-like syndrome, such as severe alopecia, erythroderma, and dense dermal inflammation composed of lymphocytes and eosinophils, were absent in MND-coRAG1-treated mice.We extensively sampled the small intestine (duodenum, jejunum, and ileum) and large intestine (cecum, colon, and rectum), but did not find severe inflammatory infiltrates resembling Omenn-like syndrome.

[0224] Next, we confirmed the viral insertion site using nrLAM-PCR (Figure 5B). This is a highly sensitive technique that can detect clonal insertions as distinct bands (which can be sequenced if necessary) (Gabriel et al., 2014). We observed only bands indicative of polyclonal hematopoiesis, with little or no oligoclonality, except for a few minor bands. We conclude that there was no evidence of vector-induced clonal selection. This is consistent with the findings of others using SIN LV vectors in hematopoietic stem cells (HSCs).

[0225] The safety of clinical MND-coRAG1 was also tested using an IVIM assay. In separate independent experiments, the clinical vector did not result in clonal expansion, similar to the results of mock-transduced cells (Figure 5C). This is likely due to its higher purity compared to the research vector, resulting in improved functional titers and fewer side effects.

[0226] Restoration of B- and T-cell development in RAG1 SCID patient cells We have previously demonstrated that transplantation of BM CD34+ cells from SCID patients into NSG mice can confirm the stage at which T cell development ceases in human SCID. This model should also be suitable as a preclinical efficacy model using patient cells. Therefore, we purified CD34+ cells from cryopreserved BM cells of a RAG1-SCID patient. This patient was hypomorphic, with some residual B cells but no T cells. We transplanted mock-transduced or MND-coRAG1-transduced CD34+ cells into busulfan-treated mice and monitored T and B cell development over time. Human cell engraftment was similar between mice transplanted with gene therapy-treated cells and mice transplanted with mock-transduced cells, indicating that gene therapy does not affect human cell engraftment. As expected, B cells were observed in mock-transduced humanized mice, but significantly more B cells were found in the spleens of GT-treated CD34+ cells (Figures 6A and 8B). Furthermore, the B cells present displayed polyclonal Ig rearrangement (Figure 8E) and produced immunoglobulins, as evidenced by the detection of human IgM in the serum of mice (Figure 6D), although there was a trend toward a more polyclonal repertoire after GT.

[0227] Surprisingly, mice transplanted with mock-transduced RAG1-SCID cells failed to develop T cells, whereas gene-treated mice clearly developed detectable T cells (Figures 6B and 8C). We also examined the thymus after sacrificing the mice. Because the patient was hypomorphic, we confirmed the presence of several stages of T cell development, including all DN, ISP, and early CD3-DP stages (Figure 6C). However, none of the cells were CD3+, and there were no late CD3+ DP or SP thymocytes, suggesting that TCRα rearrangement in particular was affected by this RAG1 mutation. Finally, we confirmed rearrangements in TCRB and TCRG by GeneScan analysis. Due to very limited material, we were unable to analyze all possible Vg and Vb genes. However, selected gene segments showed more in-frame rearrangements for TCRG in the gene-therapy group, whereas rearrangements for TCRB were only detected in the GT group (Figure 6E). nRLAM_PCR on splenocytes revealed a polyclonal pattern with no signs of clonal dominance ( Figure 6F ).

[0228] Consideration RAG1-SCID patients have impaired TCR and BCR genetic assembly. Affected children typically suffer from a variety of severe, life-threatening infections. Currently, the only treatment for RAG1-SCID is replacement of the diseased bone marrow with healthy, unmodified allogeneic stem cells. While the overall survival rate after matched-donor SCT is satisfactory, outcomes are significantly worse after mismatched-donor SCT, which accounts for the majority of cases. Furthermore, approximately 25% of patients treated with allogeneic SCT develop graft-versus-host disease, which significantly impairs outcomes in terms of morbidity, immune reconstitution, and transplant-related mortality (Gennery et al.). Furthermore, transplant outcomes in RAG-SCID (and other recombination-deficient TB-SCID) are significantly worse than those in B-cell-containing SCID (i.e., T-B+ SCID) (Gennery et al.).

[0229] Transplantation of genetically corrected autologous HSCs eliminates the risks associated with allogeneic stem cell transplantation (GvHD and rejection), making it a valuable alternative, especially for patients without a matched donor. Gene therapy using LV or RV SIN vectors for X-SCID has been successful and has been shown to be free of the genotoxicity problems previously associated with gamma-retroviral vectors. (Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman MH, Pike-Overzet K, Chatters SJ, de Ridder D, Gilmour KC, Adams S, Thornhill SI, Parsley KL, Staal FJ, Gale RE, Linch DC, Bayford J, Brown L, Quaye M, Kinnon C, Ancliff P, Webb DK, Schmidt M, von Kalle C, Gaspar HB, Thrasher AJ. J Clin Invest. 2008) Sep;118(9):3143-50). For ADA-SCID, both the RV vector (now sold as an approved treatment under the name Strimvelis) and the LV vector have shown excellent clinical results comparable to matched donor hematopoietic stem cell transplantation. See Morgan, RA, Gray, D., Lomova, A., and Kohn, DB (2017). Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned. Cell stem cell 21, 574-590.

[0230] Unlike X-linked SCID and ADA-SCID, developing gene therapy for RAG-SCID has been challenging. Previous attempts (Lagrésle-Peyrou et al., 2008) used gammaretroviral vectors in preclinical Rag1- / - models, but this carried a high risk of insertional mutagenesis. While RAG1 gammaretroviral vectors could more easily correct the defect, SIN lentiviral vectors initially failed to express the therapeutic RAG1 gene, resulting in a 'leaky' SCID or omen-like phenotype. Here, we demonstrate that durable, functional immune reconstitution can be achieved even with low VCN. We also demonstrate that human RAG1 deficiency can be functionally restored in patient cells, providing important additional efficacy data necessary for successful clinical implementation.

[0231] In this study, we selected a SIN LV vector using the MND promoter because this relatively strong promoter has been shown to be the most effective in preclinical models. The MND promoter has previously been used in gene therapy trials for ADA-SCID and adrenoleukodystrophy (ALD), with no reported insertional mutagenesis. Furthermore, preclinical safety data have demonstrated that the MND-coRAG1 vector is relatively safe. We found that the MND-coRAG1 SIN LV vector can restore immunity without gross abnormalities or histopathology, suggesting that this vector has the potential to treat a wide range of RAG1-mediated diseases.

[0232] Clinical trials have shown that gene therapy for ADA-SCID and X-linked SCID provides significant clinical benefits while significantly reducing healthcare costs. We anticipate similar benefits from our approach to treating RAG1-SCID patients, as it may reduce suboptimal outcomes in (mismatched) allogeneic transplants, which often require immunoglobulin administration and treatment for infections and GvHD-related complications.

[0233] material and method mouse C57BL / 6 Rag1- / - mice were obtained from The Jackson Laboratory (USA). C57BL / 6 wild-type mice and NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ(NSG) mice were purchased from Charles River (France). Mice were bred and cared for in the animal facility of the Leiden University Medical Center (LUMC). All animal experiments were approved by the Dutch Central Committee for Animal Experimentation (Centrale Commissie Dierproeven, CCD).

[0234] Lentiviral vectors and vector production The RAG1 gene sequence was optimized as described by Pike-Overzet et al. (2011), resulting in 90% of codons conforming to the codon bias of Homo sapiens genes. Furthermore, the GC content was increased from 48% to 61%, and the number of cis-acting motifs was reduced from 21 to 0. The optimized RAG1 sequence was synthesized by GeneArt (Regensburg, Germany). The codon-optimized RAG1 (coRAG1) was cloned into the self-inactivating lentiviral pCCL plasmid to obtain Cbx3.MND.coRAG1 (hereafter referred to as Cbx3-coRAG1), pCCL-MND-coRAG1 (hereafter referred to as MND-coRAG1), pCCL-PGK-coRAG1 (hereafter referred to as PGK-coRAG1), and pCCL-UCOE-coRAG1 (hereafter referred to as UCOE-coRAG1). DNA sequencing of the transgene was performed to verify the transgene constructs. The helper plasmids pMDLg / pRRE, pRSV-Rev, and pMD2.VSVG for lentivirus production were kindly provided by L. Naldini (San Raffaele Telethon Institute for Gene Therapy, Milan, Italy) (Dull et al., 1998). Large-scale helper plasmid preparations were obtained through PlasmidFactory (Bielefeld, Germany).

[0235] 293T cells were transiently transfected with the transfer and helper plasmids using X-tremeGene HP DNA transfection reagent (Sigma-Aldrich). Lentivirus was harvested 24, 30, and 48 hours after transfection, filtered through a 0.22 μm pore filter (Whatmann), and stored at -80°C. Pooled lentiviral supernatants were purified by ultracentrifugation (Beckman Optima). (商標)The vector was concentrated under vacuum for 16 hours at 10,000 rpm and 4°C using a LE-80K rotor SW32Ti. The pellet was resuspended in StemSpan Serum-Free expansion medium (SFEM; Stemcell Technologies Inc.) and aliquoted to avoid repeated freeze / thaw cycles. Because a suitable anti-RAG1 antibody was unavailable, we determined the viral titer using qPCR as described below. Clinical GMP-grade vectors were produced by Batavia Biosciences (Leiden, The Netherlands), tested and validated in mouse Rag1-deficient bone marrow cells and human CD34+ cells, dispensed into 200 ml vials, and stored at -80°C until use.

[0236] Transduction of mouse lineage-negative bone marrow cells and human CD34+ cells Mouse bone marrow (BM) cells were obtained from the femurs and tibias of C57BL / 6 wild-type and C57BL / 6 Rag1- / - mice. The bones were washed or crushed, and the cells were passed through a 0.7 μm cell strainer (Falco), washed, and then frozen viably. After thawing, lineage-negative cells were isolated using a mouse lineage depletion kit and an AUTOMacs cell sorter (Miltenyi Biotech). Lineage-negative cells were stimulated overnight in StemSpam-SFEM containing penicillin / steptamycin (5,000 units / 5,000 μg / 00; Gibco) and supplemented with 50 ng / mL recombinant mouse mutant-related tyrosine kinase 3 ligand (rmFLT3L; R&D systems), 100 ng / mL recombinant mouse Stem-Cell Factor (rmSCF; R&D systems), and 10 ng / mL recombinant mouse thrombopoietin (rmTPO; R&D systems). - / -Cells were transduced with different lentiviruses by spinoculation with 4 μg / ml proteamine sulfate (Sigma-Aldrich) at 800×g for 1 hour at 32° C. Cells were cultured in cytokine-supplemented medium at 37° C. in 5% CO for 24 hours.

[0237] Human bone marrow from a child diagnosed with SCID was obtained in accordance with the guidelines of the Medical Ethics Committee and IRB of the Leiden University Medical Center. The patient was a compound heterozygote with the following confirmed mutations: RAG1 allele 1 C 256-257 deletion AA, allele 2 C 1677 G>T. Mononuclear cells were isolated by Ficoll gradient centrifugation, frozen in fetal calf serum (Grenier Bio-one) / 10% DMSO (Sigma-Aldrich), and stored in liquid nitrogen. After thawing, human CD34 was purified using a CD34 MicroBead UltraPure Kit (Milteny Biotec). + Cells were isolated and enriched for CD34 + Cells were stimulated overnight in X-VIVO15 medium without gentamicin and phenol red (Lonza) and 1% human albumin (200 g / L; Sanquin) supplemented with 300 ng / ml huSCF (Milteny Biotec), 100 ng / ml huTPO (Milteny Biotec), 300 ng / ml huFlt3L (Milteny Biotec), and 10 ng / ml huIL3 (Milteny Biotec) in Pen / Strep medium. Cells were transduced as described above in complete X-VIVO-15 medium containing 4 μg / ml proteamine sulfate and cultured for 24 hours.

[0238] Rag1 - / - Transplantation of mice and NSG mice Control mock-transduced cells (C57BL / 6 wild-type cells as WT control and Rag1 cells as Rag1 control) were cultured in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) without phenol red. - / -cells referred to as KO control) and transduced Rag1 - / - Mouse cells (up to 5.10 5 cells / mouse) supporting Rag1 - / - Spleen cells (3.10 6 cells / mouse) and pre-adjusted Rag1 - / - Transplantation was performed via tail vein injection into recipient mice. Recipient mice (8-12 weeks old) were conditioned by a single dose of orthovoltage X-rays (8.08 Gy) or two consecutive doses of 25 mg / kg busulfan (Sigma-Aldrich) (48 and 24 hours before transplantation). After overnight culture, 60,000-70,000 human CD34 cells were cultured. + Cells were resuspended in phenol red (Gibco)-free IMDM and transplanted intravenously into busulfan-preconditioned NSG recipient mice (5-week-old mice, busulfan conditioning as described above). Mice used for transplantation were kept in a specific pathogen-free environment. For the first 4 weeks after transplantation, mice were supplemented with DietGel recovery food (Clear H2O) and antibiotic water containing 0.07 mg / mL polymyxin B (Bupha Uitgeest), 0.0875 mg / mL ciprofloxacin (Bayer bv), and 0.1 mg / mL amphotericin B (Bristol-Myers Squibb). Mice were observed daily. Peripheral blood (PB) was collected from mice by tail vein incision every 4 weeks until the end of the experiment. PB, thymus, spleen, and BM were collected from mice euthanized with CO2.

[0239] immunization Four weeks before the end of the experiment, mice were immunized with synthetic TNP-KLH antigen. 100 μg of TNP-KLH (Biosearch Technologies Inc.) dissolved in 50% Imject Alum (Thermo Scientific) was injected intraperitoneally (ip). Three weeks later, mice were boosted ip with 100 μg of TNP-KLH in PBS. Serum was collected before the booster injection and 1 week after the injection.

[0240] Flow cytometry Single-cell suspensions from the thymus and spleen were prepared by squeezing the organs with a 70 μM cell strainer (BD Falcon), and single-cell suspensions from the BM were prepared as described above. Red blood cells from the PB and spleen were lysed using a NH4Cl (8.4 g / L) / KHCO3 (1 g / L) solution. The single-cell suspensions were counted and stained with the antibodies listed in Table 1.

[0241] Briefly, cells were incubated in the dark at 4°C for 30 min with an antibody mixture containing antibodies directly conjugated to an optimal working solution in FACS buffer (PBS pH 7.4, 0.1% azide, 0.2% BSA). After washing with FACS buffer, a second incubation step was performed with streptavidin-conjugated antibody solution for 30 min at 4°C. Where necessary, 7AAD (BD Biosciences) was used as a viability dye. Cells were measured using a FACS-Canto II and an LSR Fortessa X-20 (BD Biosciences), and data were analyzed using FlowJO software (Tree Star).

[0242] Determination of vector copy number (VCN) and coRag1 expression by RT-qPCR Genomic lentiviral RNA, proviral DNA copies, and transgene mRNA expression were quantitatively analyzed by qPCR using WPRE, coRAG1, ABL1, and PTBP2 as targets. Total RNA from single-cell suspensions was purified using the RNeasy Mini kit (Qiagen) and reverse-transcribed to cDNA using the Superscript III kit (Invitrogen). Genomic DNA was extracted from single-cell suspensions using the GeneElute Mammalian Genomic DNA kit (Sigma-Aldrich). Genomic DNA was extracted from mouse organs and tissues using the Dneasy Blood and Tissue Kit (Qiagen). VCN was determined in DNA samples by detection of WPRE and PTBP2. qPCR was performed using TaqMan Universal Master Mix II (Thermofisher) with specific probes for the indicated genes from the Universal Probe Library (Roche). The primers and probes used are listed in Tables 5 and 6. PCR reactions were performed using a StepOnePlus Real-Time PCR system (Thermofisher). All samples were run in triplicate.

[0243] [Table 5]

[0244] [Table 6]

[0245] Serum immunoglobulin quantification Mouse IgG, IgM, IgE, TNP-specific IgG, and human IgM were measured by sandwich enzyme-linked immunosorbent assay (ELISA). NUNC Maxisop plates (Thermo Scientific) were coated with unlabeled anti-mouse IgG, IgM (11E10), and IgE antibodies (SouthernBiotech) or unlabeled anti-human IgM antibodies (kindly provided by Dr. Karahan, Jackson Immuno Research Laboratories, LUMC). For TNP-specific IgG detection, plates were coated with synthetic TNP-KLH (Biosearch Technologies Inc.). Blocking was performed with 1% BSA / PBS (mouse) or 2% BSA / 0.025 Tween / PBS (human) for 1 hour at room temperature (RT), followed by incubation with serial dilutions of the resulting sera for 3 hours at RT. After washing, plates were incubated with biotin-labeled anti-mouse IgG, IgM, and IgE (SouthernBiotech) or anti-human IgM (Novex Life Technologies, kindly provided by Dr. Karahan, LUMC) for 30 minutes at room temperature. For detection, plates were incubated with streptavidin-horseradish peroxidase (Jackson Immuno Research Laboratories) for 30 minutes at room temperature, followed by azino-bis-ethylbenzthiazoline sulfonic acid (ABTS, Sigma-Aldrich) as substrate. Data were acquired at 415 nm using a Bio-Rad iMark microplate reader and MPM 6 software (Bio-Rad). Antibody concentrations were calculated based on purified IgG, IgM, and IgE proteins (SouthernBiotech) and human reference serum (Bethyl Laboratories, kindly provided by Dr. Karahan, LUMC).

[0246] Repertoire analysis Total RNA was purified from mouse spleen cells and reverse transcribed into cDNA as described above. The GeneScan procedure for mouse T cell repertoire analysis was adapted from (Pannetier et al., 1993). cDNA was amplified using FAM-labeled C gene segment-specific primers and 24 TCR Vβ-specific primers (see Table 6). GeneScan (商標) 500 ROX (商標) (ThermoFisher) was used as an internal size standard. The labeled PCR products were run on an ABI Prism® Genetic Analyzer (Applied Biosystems) for fragment analysis. The raw spectratyping data were analyzed, visualized, and scored using ScoreSpec, a novel spectratyping algorithm for estimating immunological diversity (Cordes et al., manuscript in preparation). ScoreSpec identifies and scores individual spectratyping peak patterns, including overall peak distribution (Gaussian distribution), individual peak shapes, and correction for out-of-frame TCR transcripts. Scores range from 0, indicating no peaks were detected, to 100, indicating a diverse TCR repertoire.

[0247] Human immunoglobulin and T cell receptor repertoires generated in NSG mice were analyzed using DNA samples collected from the BM and thymus (DNA was extracted as described above). Rearrangements were analyzed using the EuroClonality / BOMED-2 multiplex PCR protocol (van Dongen et al., 2003). Amplification of IgH, IgK, TCRβ, and TCRγ rearrangements was performed according to the instructions for the IGH + IGK B Cell Clonality Assay (InvivoScribe) and the TCRB + TCRG T Cell Clonality Assay (InvivoScribe), respectively. PCR products were analyzed by differential fluorescence detection using an ABI-3730 instrument (Applied Biosystems) for fragment analysis. Output files were visualized and analyzed using ScoreSpec.

[0248] Non-limiting linear amplification-mediated PCR (nrLAM-PCR) Lentiviral insertion sites were analyzed by nrLAM-PCR of mouse bone marrow DNA samples as described (Gabriel et al., 2014); Schmidt M. et al (2014) J. Vis. Exp. (88), e51543.

[0249] In vitro immortalization assay (IVIM) The genotoxicity of viral vectors (Cbx3-coRAG1, MND-coRAG1, PGK-coRAG1, UCOE-coRAG1) was quantified as described (Modlich et al., 2006) Baum et al. (2006) Blood 108:2545-2553.

[0250] Gross and histopathology Dissections were performed and organs were collected for gross and microscopic examination (List of organs collected X). The selection of organs for gross pathology and histopathological analysis followed applicable European and international guidelines (EMEA 1995, WHO 2005) (WHO, 2005). Gross pathology included examination of the external body surface, orifices, thoracoabdominal area, and body cavities (organs analyzed are listed in Table 4).

[0251] For histopathological examination, organs were fixed in 4% neutral buffered formalin for 24 hours, embedded in paraffin, and then sectioned at 5 μm for hematoxylin and eosin (HE) staining and immunohistochemistry according to standard procedures (Bancroft and Gamble, 2008). All slides were examined blindly by a European Commission-certified pathologist (ECVP).

[0252] statistics Statistics were calculated and graphs were prepared using GraphPad Prism 6 (GraphPad Software). Statistical significance was determined by standard one-tailed Mann-Whitney U test or ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0253] array SEQ ID NO: 1: RAG1 human protein sequence (1043 aa) MAASFPPTLGLSAPDEIQHPHIKFSEWKFKLFRVRSFEKTPEAQKEKKDSFEGKPSLEQSPAVLDKAD GQKPVPTQPLLKAHPKFSKKFHDNEKARGKAIHQANLRHLCRICGNSFRADEHNRRYPVHGPVDGKTLGL LRKKEKRATSWPDLIAKVFRIDVKADVDSIHPTEFCHNCWSIMHRKFSSAPCEVYFPRNVTMEWHPHTPS CDICNTARRGLKRKSLQPNLQLSKKLKTVLDQARQARQHKRRAQARISSKDVMKKIANCSKIHLSTKLLA VDFPEHFVKSISCQICEHILADPVETNCKHVFCRVCILRCLKVMGSYCPSCRYPCFPTDLESPVKSFLSV LNSLMVKCPAKECNEEVSLEKYNHISSHKESKEIFVHINKGGRPRQHLLSLTRRAQKHRLRELKLQVKA FADKEEGGDVKSVCMTLFLLALRARNEHRQADELEAIMQGKGSGLQPAVCLAIRVNTFLSCSQYHKMYRT VKAITGRQIFQPLHALRNAEKVLLPGYHHFEWQPPLKNVSSSTDVGIIDGLSGLSSSSVDDYPVDTIAKRF RYDSALVSALMDMEEDILEGMRSQDLDDYLNGPFTVVVKESCDGMGDVSEKHGSGPVVPEKAVRFSFTIM KITIAHSSQNVKVFEEAKPNSELCCKPLCLMLADESDHETLTAILSPLIAEREAMKSSELMLELGGILRT FKFIFRGTGYDEKLVREVEGLEASGSVYICTLCDATRLEASQNLVFHSITRSHAENLERYEVWRSNPYHE SVEELRDRVKGVSAKPFIETVPSIDALHCDIGNAAEFYKIFQLEIGEVYKNPNASKEERKRWQATLDKHL RKKMNLKPIMRMNGNFARKLMTKETVDAVCELIPSEERHEALRELMDLYLKMKPVWRSSCPAKECPESLC QYSFNSQRFAELLSTKFKYRYEGKITNYFHKTLAHVPEIIERDGSIGAWASEGNESGNKLFRRFRKMNAR QSKCYEMEDVLKHHWLYTSKYLQKFMNAHNALKTSGFTMNPQASLGDPLGIEDSLESQDSMEF SEQ ID NO: 2: Codon-optimized nucleic acid sequence encoding the human RAG1 catalytic domain SEQ ID NO: 3: RAG1 cDNA sequence SEQ ID NO: 4: Codon-optimized RAG1 DNA sequence SEQ ID NO: 5: MND promoter sequence tttatttagt ctccagaaaa aggggggaat gaaagacccc acctgtaggt ttggcaagct aggatcaagg ttaggaacag agagacagca gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagttggaa cagcagaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtccc gccctcagca gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagccca SEQ ID NO: 6: Primer 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' SEQ ID NO: 7: Primer 5'-GATGTAGTTGCTTGGGACCCA-3' SEQ ID NO: 8: Probe 5'FAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA 3' SEQ ID NO: 9: Primer 5' CAACTGCAAGCACGTGTTCTG 3' SEQ ID NO: 10: Primer 5' GCAGTAGCTGCCCATCACTTT 3' SEQ ID NO: 11: Probe 5'FAM AGAGTGTGCATCCTGCGGTGCCT TAMRA 3' For SEQ ID NOs: 12 to 43, see Tables 5 and 6 and Figure 9.

[0254] Reference list Beillard et al., Evaluation of candidate control genes for diagnosis and residual disease detection in leukemic patients using 'real-time' quantitative reverse-transcriptase polymerase chain reaction (RQ-PCR) - a Europe against cancer program - Leukemia volume 17, pages 2474-2486 (2003) Bancroft, JD, and Gamble, M. (2008). Theory and Practice of Histological Techniques (Churchill Livingstone). Baum, C., Kustikova, O., Modlich, U., Li, Z., and Fehse, B. (2006). Mutagenesis and oncogenesis by chromosomal insertion of gene transfer vectors. Hum Gene Ther 17, 253-263. Dull, T., Zufferey, R., Kelly, M., Mandel, RJ, Nguyen, M., Trono, D., and Naldini, L. (1998). A third-generation lentivirus vector with a conditional packaging system. J Virol 72, 8463-8471. Gabriel, R., Kutschera, I., Bartholomae, C.C., von Kalle, C., and Schmidt, M. (2014). Linear amplification mediated PCR--localization of genetic elements and characterization of unknown flanking DNA. J Vis Exp, e51543. Gennery, A.R., Slatter, M.A., Grandin, L., Taupin, P., Cant, A.J., Veys, P., Amrolia, P.J., Gaspar, H.B., Davies, E.G., Friedrich, W., et al. Transplantation of hematopoietic stem cells and long-term survival for primary immunodeficiencies in Europe: entering a new century, do we do better? The Journal of allergy and clinical immunology 126, 602-610 e601-611. Howe, S.J., Mansour, M.R., Schwarzwaelder, K., Bartholomae, C., Hubank, M., Kempski, H., Brugman, M.H., Pike-Overzet, K., Chatters, S.J., de Ridder, D., et al. (2008). Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. The Journal of clinical investigation 118, 3143-3150. Lagresle-Peyrou, C., Benjelloun, F., Hue, C., Andre-Schmutz, I., Bonhomme, D., Forveille, M., Beldjord, K., Hacein-Bey-Abina, S., De Villartay, J.P., Charneau, P., et al. (2008). Restoration of human B-cell differentiation into NOD-SCID mice engrafted with gene-corrected CD34+ cells isolated from Artemis or RAG1-deficient patients. Molecular therapy : the journal of the American Society of Gene Therapy 16, 396-403. Lagresle-Peyrou, C., Yates, F., Malassis-Seris, M., Hue, C., Morillon, E., Garrigue, A., Liu, A., Hajdari, P., Stockholm, D., Danos, O., et al. (2006). Long-term immune reconstitution in RAG-1-deficient mice treated by retroviral gene therapy: a balance between efficiency and toxicity. Blood 107, 63-72. Modlich, U., Bohne, J., Schmidt, M., von Kalle, C., Knoess, S., Schambach, A., and Baum, C. (2006). Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood 108, 2545-2553. Pannetier, C., Cochet, M., Darche, S., Casrouge, A., Zoeller, M., and Kourilsky, P. (1993). The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments. Proceedings of the National Academy of Sciences of the United States of America 90, 4319-4323. Pike-Overzet, K., Baum, C., Bredius, R.G., Cavazzana, M., Driessen, G.J., Fibbe, W.E., Gaspar, H.B., Hoeben, R.C., Lagresle-Peyrou, C., Lankester, A., et al. (2014). Successful RAG1-SCID gene therapy depends on the level of RAG1 expression. The Journal of allergy and clinical immunology 134, 242-243. Pike-Overzet, K., de Ridder, D., Weerkamp, F., Baert, M.R., Verstegen, M.M., Brugman, M.H., Howe, S.J., Reinders, M.J., Thrasher, A.J., Wagemaker, G., et al. (2006). Gene therapy: is IL2RG oncogenic in T-cell development? Nature 443, E5; discussion E6-7. Pike-Overzet, K., Rodijk, M., Ng, Y.Y., Baert, M.R., Lagresle-Peyrou, C., Schambach, A., Zhang, F., Hoeben, R.C., Hacein-Bey-Abina, S., Lankester, A.C., et al. (2011). Correction of murine Rag1 deficiency by self-inactivating lentiviral vector-mediated gene transfer. Leukemia 25, 1471-1483. Pike-Overzet, K., van der Burg, M., Wagemaker, G., van Dongen, J.J., and Staal, F.J. (2007). New insights and unresolved issues regarding insertional mutagenesis in X-linked SCID gene therapy. Molecular therapy : the journal of the American Society of Gene Therapy 15, 1910-1916. van Dongen, J.J.M., Langerak, A.W., Brueggemann, M., Evans, P.A.S., Hummel, M., Lavender, F.L., Delabesse, E., Davi, F., Schuuring, E., Garcia-Sanz, R., et al. (2003). Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17, 2257. van Til, N.P., Sarwari, R., Visser, T.P., Hauer, J., Lagresle-Peyrou, C., van der Velden, G., Malshetty, V., Cortes, P., Jollet, A., Danos, O., et al. (2014). Recombination-activating gene 1 (Rag1)-deficient mice with severe combined immunodeficiency treated with lentiviral gene therapy demonstrate autoimmune Omenn-like syndrome. Journal of Allergy and Clinical Immunology 133, 1116-1123. WHO (2005). WHO guidelines on nonclinical evaluation of vaccines, W.H. Organization, ed. (Tech Rep Ser), pp. 31-63.

Claims

1. A self-inactivating (SIN) lentiviral plasmid comprising an expression cassette comprising the MND promoter operably linked to a RAG1 transgene comprising the nucleic acid sequence of SEQ ID NO:

4.

2. 2. The SIN lentiviral plasmid of claim 1, wherein the expression cassette further comprises a nucleotide sequence encoding a woodchuck hepatitis virus (WHP) post-transcriptional regulator (WPRE).

3. 3. The SIN lentiviral plasmid of claim 1 or 2, wherein the plasmid comprises a pCCL backbone.

4. 4. A SIN lentiviral plasmid according to any one of claims 1 to 3, wherein the plasmid comprises a pCCL backbone, a nucleotide sequence encoding WPRE, an MND promoter and a transgene comprising the nucleic acid sequence of SEQ ID NO:

4.

5. A lentiviral virion comprising an expression cassette comprising an MND promoter operably linked to a RAG1 transgene comprising the nucleic acid sequence of SEQ ID NO:

4.

6. A composition comprising a SIN lentiviral plasmid described in any one of claims 1 to 4 or a lentiviral virion described in claim 5, and a pharmaceutically acceptable adjuvant, carrier, excipient or diluent.

7. A recombinant CD34+ hematopoietic stem cell comprising a SIN lentiviral plasmid according to any one of claims 1 to 4 or a lentiviral virion according to claim 5.

8. An ex vivo method for generating recombinant CD34+ hematopoietic stem cells, comprising contacting cells with a SIN lentiviral plasmid described in any one of claims 1 to 4 or a lentiviral virion described in claim 5 under conditions in which the expression cassette is taken up by the cells and expressed, thereby producing recombinant CD34+ hematopoietic stem cells.

9. 8. A SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell according to any one of claims 1 to 7 for use in therapy.

10. A SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell as described in claim 9, wherein the SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell is for use in treating RAG1-deficient severe combined immunodeficiency disease (SCID), Omenn's syndrome (OS), atypical SCID or combined immunodeficiency disease (CID).

11. A SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell described in any one of claims 1 to 7 for use in a method of treating a subject comprising administering a therapeutically effective amount of a SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell described in any one of claims 1 to 7 to a subject in need thereof.

12. The SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell of claim 11, wherein the subject has RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID).

13. The SIN lentiviral plasmid, composition, lentiviral virion or recombinant cell of claim 12, wherein the SCID is a RAG1-deficient SCID.

14. (i) extracting CD34+ hematopoietic stem cells from a subject; (ii) contacting the cells of (i) with the lentiviral virion of claim 5 or the SIN lentiviral plasmid of any one of claims 1 to 4; (iii) incubating the cells of (ii) for a period of time; and (iv) introducing the cells of (iii) into the subject. For use in a method for treating RAG1 deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID) in a subject in need thereof, comprising: A lentiviral virion according to claim 5 or a SIN lentiviral plasmid according to any one of claims 1 to 4.

15. 15. The lentiviral virion or SIN lentiviral plasmid of claim 14, further comprising the step of administering chemotherapy or other conditioning regimen to the subject prior to step (iv).

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