Optimized rag1 deficient gene therapy

Abstract

To provide a novel and improved method for treating RAG1-deficient severe combined immunodeficiency (RAG1-SCID) and Omenn Syndrome (OS).SOLUTION: Provided is an expression cassette comprising a promoter operably linked to a RAG1 transgene containing a specific nucleic acid sequence, the expression cassette being useful for the treatment of diseases caused by complete or partial loss-of-function of the protein encoded by the rag-1 gene, such as RAG1-deficient severe combined immunodeficiency (RAG1-SCID), Omenn Syndrome (OS), atypical SCID, or combined immunodeficiency (CID), wherein the promoter is selected from MND, CMV, RSV, and CAG.SELECTED DRAWING: Figure 1-1

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 recombinant activation (RAG1) transgene. These novel expression cassettes, retroviral plasmids, vectors, virions, compositions and recombinant cells are useful for the treatment of 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 syndrome (OS), atypical SCID or combined immunodeficiency (CID). Also provided is a corresponding treatment method.

Background Art

[0002] Background Gene therapy for rare hereditary immune diseases has become clinically realistic in recent years, especially for severe combined immunodeficiency (SCID). For example, two major types of SCID (ADA-SCID, X-SCID) have been successfully treated by autologous stem cell-based gene therapy. However, for SCID with recombination deficiency, the most common form of SCID (e.g., RAG-deficient SCID; also known as RAG-SCID), there is no treatment method due to the high complexity of the genes involved.

[0003] Patients with RAG-deficient SCID have mutations in either RAG1 or RAG2, which are required for gene recombination of the T cell receptor (TCR) and B cell receptor (BCR). Affected children usually experience various life-threatening severe infections, including pneumonia, meningitis and sepsis. At present, the only treatment method for RAG-SCID is to replace the affected bone marrow with healthy unmodified allogeneic stem cells by allogeneic stem cell transplantation (allo-SCT). The overall survival rate of matched SCT recipients is satisfactory, but the results of unmatched SCT recipients, which account for the majority of cases, are significantly worse.

[0004] Furthermore, approximately 25% of transplant patients develop graft-versus-host disease, significantly reducing outcomes in terms of morbidity, immune reconstitution, and transplant-related mortality (Gennery, 2010). Therefore, transplant outcomes in RAG-SCID (and other recombinant-deficient T-SCID and B-SCID) are significantly worse than those in SCID with B cells (i.e., T-B+ SCID). Overall, these data suggest that allo-SCT, the only available treatment option currently, has significant limitations with respect to both the potential for treatment and the potential for survival, indicating the urgent need for new and improved strategies based on genetic modification 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 serious 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 mutations by the retroviral vectors used for the introduction of the therapeutic gene. After this serious failure of gene therapy, recent studies have shown that next-generation vectors, particularly vectors in which viral promoter / enhancer sequences are inactivated (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 shown that SIN lentiviral vectors are safe and highly effective, indicating that the clinical development of genetically modified hematopoietic stem cells is promoted. However, unlike X-linked SCID and ADA-SCID, it has been very difficult to use gene therapy for the treatment of RAG-SCID. In previous attempts (Lagresle-Peyrou, 2006), gamma-retroviral vectors were used in a preclinical Rag1- / - model, but this had a high risk of insertional mutagenesis. Although the RAG1 gamma-retroviral vector was able to correct the deficiency more easily, initially in the SIN lentiviral vector, the expression of the therapeutic RAG1 gene was insufficient, resulting in a "leaky" SCID or Omenn-like phenotype. Conflicting results have been observed in this field due to differences in the expression levels and transduction efficiencies obtained for the therapeutic genes (van Til., 2014).

[0007] New and improved strategies are needed to treat RAG1-deficient severe combined immunodeficiency (SCID) and Omenn syndrome (OS). SUMMARY OF THE INVENTION

[0008] Summary of the Disclosure The inventors have surprisingly found the minimum threshold of RAG1 expression that provides a therapeutic effect in a preclinical model of RAG-deficient SCID using clinically acceptable lentiviral gene therapy and a codon-optimized RAG1 transgene sequence.

[0009] The inventors designed clinically relevant lentiviral SIN plasmids with different internal promoters that promote the expression of codon-optimized RAG1 gene. Using Rag1− / − mice as a preclinical model of RAG1-SCID, the inventors evaluated the efficacy of various plasmids at low copy numbers and confirmed that the reconstitution of B cells and T cells was directly correlated with RAG1 expression. Mice with low RAG1 expression had insufficient immune reconstitution, but high RAG1 expression resulted in phenotypic and functional lymphocyte reconstitution equivalent to that of mice administered wild-type stem cells. Surprisingly, transplantation of CD34+ cells from RAG1-SCID patients transfected with the clinical RAG1 plasmid into NOD SCID gamma (NSG) mice completely restored human B cell and T cell development. Together with favorable safety data, the inventors' results provide a strong basis for human clinical trials of RAG1-deficient SCID.

[0010] The inventors 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 showed that a therapeutic effect is observed when the RAG1 expression level is at least three-fold higher for B cell reconstitution (ten-fold for T cell reconstitution) compared to certain housekeeping genes such as ABL1 (from the perspective of in vivo B and T cell reconstitution). Thus, a minimum threshold of three-fold higher expression is shown herein to have a beneficial therapeutic effect. The inventors first showed 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 the codon-optimized RAG1 transgene sequence, even when the copy number of the RAG1 transgene integrated into the cell's genome (in the context of the expression cassette) is 5 or less, an RAG1 expression level at least three-fold higher than ABL1 in the cell can be achieved). As is well known in the art herein, "low copy number" means a plasmid that integrates into the genome of a target cell at a frequency of 5 copies or less per cell (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 a copy number of about 0.2 per cell.

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

[0012] The inventors have demonstrated the threshold levels required for RAG1 expression using the MND promoter. Surprisingly, when the MND promoter is operably linked to a codon-optimized RAG1 transgene, the level of RAG1 expression achieved from a low-copy plasmid in vivo is sufficient to induce B cell and T cell reconstitution. Thus, the inventors have confirmed that a combination of a low-copy plasmid, a codon-optimized RAG1 transgene sequence, and a strong promoter such as MND is sufficient to induce RAG1 expression to therapeutic levels in vivo. In the present invention, the MND promoter is exemplified, but other strong promoters that induce RAG1 expression at equivalent (or higher) levels can also be used. For example, in other systems, the CMV, RSV, and CAG promoters are known to promote high-level expression of the linked transgene. Currently, the threshold of RAG1 expression required for a therapeutic effect is 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 obtain the desired effect. Thus, the present invention encompasses the use of such promoters as an alternative to MND.

[0013] The data provided herein utilize the codon-optimized sequence of RAG1 as a RAG1 transgene operably linked to the required promoter (e.g., MND; other ones such as the CMV, RSV, or CAG promoter can also be used). As described in detail in other parts of this specification, the use of the codon-optimized RAG1 sequence is advantageous because it can obtain a higher viral titer and enhance the stability of the RAG1 protein. Thus, the use of the codon-optimized transgene sequence helps to achieve the minimum threshold of RAG expression required to obtain a therapeutic effect (i.e., at least 3-fold higher level in cells than certain housekeeping genes such as ABL1 even if the copy of the RAG1 transgene integrated into the cell genome is 5 or less).

[0014] On the one hand, 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 when the expression cassette is expressed in human CD34+ hematopoietic stem cells in which the copy number of the expression cassette integrated into its genome is 5 or less, it produces an expression product at a level at least 3-fold higher than the expression level of ABL1 in the cells.

[0015] Preferably, the promoter can be selected from MND, CMV, RSV, and CAG.

[0016] Accordingly, 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. Preferably, when the expression cassette is expressed in human CD34+ hematopoietic stem cells having 5 or fewer copies of the expression cassette integrated into their genome, it produces an expression product at a level at least 3-fold higher than the expression level of ABL1 in those cells.

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

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

[0019] Preferably, the promoter may be MND.

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

[0021] On the one hand, there is provided a retroviral plasmid comprising the expression set of the present invention.

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

[0023] Preferably, the plasmid may contain a pCCL backbone.

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

[0025] Preferably, the plasmid may contain the sequence of FIG. 9.

[0026] On the one hand, there is provided a virion containing the expression cassette of the present invention.

[0027] On the one hand, there is provided a composition containing the expression cassette of the present invention, or the plasmid of the present invention, or the virion of the present invention, and a pharmaceutically acceptable adjuvant, carrier, excipient or diluent.

[0028] On the one hand, there is provided a recombinant CD34+ hematopoietic stem cell containing the expression cassette of the present invention.

[0029] On the one hand, the present invention provides an ex vivo method for generating recombinant CD34+ hematopoietic stem cells, the method comprising contacting the cells with the plasmid of the present invention or the virion of the present invention under conditions such that the expression cassette is integrated into the cells and expressed, to generate recombinant CD34+ hematopoietic stem cells.

[0030] On the one hand, the expression cassette, plasmid, composition, virion or recombinant cell of the present invention is provided for use in therapy.

[0031] Suitably, the expression cassette, vector, composition, virion or recombinant cell may be for use in the treatment of RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID). For example, the expression cassette, vector, composition, virion or recombinant cell may be for use in the treatment of RAG1-deficient SCID or Omenn 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 the expression cassette, plasmid, composition, virion particle or recombinant cell of the present invention.

[0033] Suitably, 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] 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 cells of (i) with the virion of the present invention or the plasmid of the present invention; (iii) incubating the cells of (ii) for a certain period of time; and (iv) introducing the cells of (iii) into the subject comprising.

[0035] Suitably, the method may further comprise administering to the subject a chemotherapy or other conditioning regimen prior to step (iv). BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

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Mode for Carrying Out the Invention

[0040] Detailed Description The inventors designed a clinically useful lentiviral SIN plasmid having various internal promoters linked to the codon-optimized Rag1 transgene operably to identify the minimum threshold of Rag1 expression required to obtain a therapeutic effect in vivo.

[0041] Using Rag1 - / - mice as a preclinical model of RAG1-SCID, the inventors evaluated the efficacy of various low-copy number plasmids having a codon-optimized Rag1 transgene and confirmed that the reconstitution of B cells and T cells was directly correlated with Rag1 expression. Mice with low Rag1 expression had insufficient immune reconstitution, but high Rag1 expression resulted in reconstitution of phenotypic and functional lymphocytes equivalent to that of mice administered wild-type stem cells. Surprisingly, when CD34 + cells of RAG1-SCID patients transfected with a clinical Rag1 plasmid were transplanted into NSG mice, the generation of human B cells and T cells was completely restored.

[0042] To facilitate understanding of the present invention, some terms are defined below.

[0043] Expression Cassette Provided is an expression cassette comprising a codon-optimized RAG1 transgene operably linked to a promoter. The RAG1 transgene may encode the amino acid sequence shown 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” means a nucleic acid molecule comprising one or more transcriptional regulatory elements (including, but not limited to, promoters, enhancers and / or regulatory elements, polyadenylation sequences, and introns) that promote the expression of a transgene in one or more desired cell types, tissues, or organs. The expression cassette of the present invention is a synthetic nucleic acid molecule.

[0045] As used herein, the term "nucleic acid" generally refers to any length of oligomer or polymer (preferably a linear polymer) essentially composed of nucleotides. A nucleotide unit generally includes a heterocyclic base, a sugar residue, and at least one, for example one, two, or three phosphate groups (including modified or substituted phosphate groups). Heterocyclic bases include, in particular, purine bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), pyrimidine bases widely present in natural nucleic acids, other natural bases (e.g., xanthine, inosine, hypoxanthine), and chemically or biochemically modified (e.g., methylated) unnatural or derivatized bases. Sugar groups include, inter alia, pentose (pentofuranose) groups such as ribose and / or 2-deoxyribose, which are preferably 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 intended herein may include natural nucleotides, modified nucleotides, or mixtures thereof. Modified nucleotides may include modified heterocyclic bases, modified sugar moieties, modified phosphate groups, or combinations thereof. Modifications of the phosphate group or sugar may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term "nucleic acid" more 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., those existing in nature or isolated from nature, or unnatural, e.g., those produced by recombinant, i.e., recombinant DNA technology, and / or partially or wholly chemically or biochemically synthesized. A "nucleic acid" may be double-stranded, partially double-stranded, or single-stranded. In the case of single-stranded, the nucleic acid may be a sense strand or an antisense strand. Further, the nucleic acid may be circular or linear.

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

[0047] The term "synthetic nucleic acid" as used herein refers to nucleic acid molecules that do not occur naturally.

[0048] The term "transgene" as used herein refers to an exogenous nucleic acid sequence, i.e., a sequence that does not occur naturally and includes other elements (e.g., transcriptional regulatory elements such as promoters) found within the expression cassette. In one example, the transgene is a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable trait. Throughout this specification, the transgene of interest is the RAG1 transgene.

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

[0050] The RAG1 transgene is a nucleic acid sequence encoding the RAG1 protein. To avoid misunderstanding, the transgene does not necessarily contain all of the native elements of endogenous RAG1. For example, the transgene may be the corresponding cDNA of RAG1 (i.e., without endogenous introns, etc.). The term "recombinase activating gene-1 (RAG1)" as used herein refers to the protein encoded by the RAG1 gene.

[0051] RAG1 and RAG2 together form the RAG complex. The RAG complex is a multi - protein complex that mediates the DNA cleavage step during VDJ recombination. This complex can cleave double - strands by cleaving DNA at conserved recombination signal sequences (RSS). The RAG complex recognizes RSS adjacent to the V, D, and J regions of genes encoding the constant regions of both the heavy and light chains of antibodies. This complex binds to the RSS and nicks the DNA. As a result, the RSS is removed and ultimately the V, D, and J sequences are joined together.

[0052] RAG1 is thought to have most of the catalytic activity of the RAG complex. The RAG1 protein is the component that binds to and cleaves DNA, and in this way RAG1 is involved in the activation of immunoglobulin VDJ recombination. RAG2 does not seem to have endonuclease activity or DNA - binding ability, but serves as a co - factor. 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 a mouse model impairs the maturation of T cells and B cells and functionally deletes mature T cells and B cells from the immune system.

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

[0055] The sequences of the human RAG1 gene and protein are known (for example, see the 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 (for example, the unique identifiers for mouse RAG1 include: ENSMUST00000078494; ENSMUSP00000077584; ENSMUSG00000061311).

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

[0058] RAG1 Protein: Functional Variants The RAG1 transgene may contain a nucleotide sequence encoding a RAG1 protein from a natural human, mouse or rat, 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 natural RAG1 (i.e., sequences that vary from the native sequence of human, mouse or rat RAG1 sequences by only one or more conservative amino acid substitutions).

[0059] "Functional variants" retain the functional capabilities of the RAG1 protein. In other words, functional RAG1 variants can cleave double-stranded DNA by cleaving DNA at conserved recombination signal sequences (RSSs). One of ordinary skill in the art can readily know how to identify polypeptides having this activity using routine assays known in the art. Suitable experiments for identifying functional RAG1 polypeptides are summarized below.

[0060] Functional RAG1 protein sequences can be identified using functional complementation assays. In this assay, the lentiviruses described in the following Examples section containing the RAG1 transgene encoding the RAG1 variant to be tested can be used. 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 - / - transplanted into Rag1 mice and the development of T cells is followed. If CD3+ TCRαβ+ T cells develop after 8 - 12 weeks and the number of T cells is at least 50% of that of wild-type stem cells, this sequence is considered successful.

[0061] The outline of a preferred assay for RAG1 activity that the inventors have conducted and that one of ordinary skill in the art can routinely perform is as follows: C57BL / 6 wild-type mice and C57BL / 6 Rag1 - / -Mouse bone marrow (BM) cells were obtained from the femurs and tibias of mice. The obtained bones were flushed or ground, and the cells were passed through a 0.7-μm cell strainer (Falcon), washed, and frozen in a viable state. After thawing, lineage-negative cells were isolated using a mouse lineage depletion kit and an AUTOMacs cell sorter (Miltenyi Biotech). The lineage-negative cells were stimulated overnight with StemSpam-SFEM supplemented with penicillin / streptomycin (5,000 units / 5,000 μg / ml; Gibco), 50 ng / mL of recombinant mouse FMS-related tyrosine kinase 3 ligand (rmFLT3L; R&D systems), 100 ng / mL of recombinant mouse Stem-Cell Factor (rmSCF; R&D systems), and 10 ng / mL of recombinant mouse thrombopoietin (rmTPO; R&D systems). Subsequently, Rag1 - / - Cells were transduced with different lentiviruses by performing a 1-hour spin-oculation at 800 x g and 32°C using 4 μg / ml of proteamine sulfate (Sigma-Aldrich). The cells were cultured for 24 hours at 37°C and 5% CO2 using cytokine-supplemented medium.

[0062] Control mock-transduced cells (C57BL / 6 wild-type cells as WT controls, Rag1 - / - cells are referred to as KO controls) and transduced Rag1 - / - mouse cells (up to 5.10 5 cells / mouse) were mixed in Iscove's Modified Dulbecco's Medium (IMDM) (Gibco) without phenol red with supportive Rag1 - / - spleen cells (3.10 6 cells / mouse) and pre-treated Rag1 - / -The recipient mice were transplanted by tail vein injection. Recipient mice (8 - 12 weeks old) were conditioned by a single whole-body irradiation with orthovoltage X-rays (8.08 Gy) 24 hours before transplantation, or by two consecutive administrations of 25 mg / kg busulfan (Sigma-Aldrich) (48 hours and 24 hours before transplantation).

[0063] The mice used for transplantation were housed in a specific pathogen-free facility. During the first 4 weeks after transplantation, the mice were additionally given DietGel Recovery Food (Clear H2O) and antibiotic water containing 0.07 mg / mL polymyxin B (Bupha Uitgeest), 0.0875 mg / mL ciprofloxacin (Bayer b.v.) and 0.1 mg / mL amphotericin B (Bristol-Myers Squibb), and the condition of the mice was observed daily. Peripheral blood (PB) of the mice was collected by tail vein incision every 4 weeks until the end of the experiment. PB, thymus, spleen and BM were obtained from the mice euthanized with CO2.

[0064] A single cell suspension from the spleen was prepared by squeezing the organ through a 70 μM cell strainer (BD Falcon), and a single cell suspension from the BM was prepared by the above method. Erythrocytes from the spleen were lysed using an NH4Cl (8.4 g / L) / KHCO3 (1 g / L) solution. The single cell suspension was counted and stained with the antibodies shown in Table 1. That is, the cells were incubated in the dark at 4°C for 30 minutes using an antibody mix solution containing an antibody directly conjugated to the optimal working solution in FACS buffer (PBS pH 7.4, 0.1% azide, 0.2% BSA). After washing with FACS buffer, a second 30-minute incubation step was performed at 4°C using a streptavidin-conjugated antibody solution. The cells were measured with a FACS-CantoII and an LSR Fortessa X-20 (BD Biosciences), and the data were analyzed using FlowJO software (Tree Star).

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

Table 1

[0066] Accordingly, the RAG1 polypeptide may contain the amino acid sequence shown 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., alleles) of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), synthetic, or synthetically improved functional variants.

[0067] Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence), or may include substitutions, deletions, or insertions of non-essential amino acids in non-essential regions of the protein. Accordingly, functional variants of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence) may be conservative amino acid sequence variants of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence).

[0068] Non-functional variants are amino acid sequence variants of SEQ ID NO: 1 (or an equivalent mouse or rat RAG1 sequence) that do 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 important amino acids or important 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 important and unimportant amino acids of RAG1 is described in Luigi D. Notarangelo, Min-Sung Kim, Jolan E. Walter & Yu Nee Lee Nature Reviews Immunology volume 16, pages 234-246 (2016). Thus, a person skilled in the art can easily identify the 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 equivalent mouse or rat RAG1 sequences).

[0070] Functional variants can include amino acid sequences 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 equivalent mouse or rat RAG1 sequences), or a portion or fragment thereof. Preferably, the percentage of identity can be calculated as the percentage of identity to the full length of a control 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, "native" polypeptide means an amino acid sequence that exists in nature.

[0073] "Non-essential" (or "non-critical") amino acid residues are residues that can be changed from the wild-type sequence (e.g., the sequence of SEQ ID NO:1) without abolishing biological activity, or more preferably, without substantially changing biological activity, while "essential" (or "critical") amino acid residues are those that result in such a change. For example, amino acid residues within the hydrophobic core of a domain are generally predicted not to be altered without particular change, except that they can be replaced with other residues having substantially equivalent hydrophobicity without significantly changing activity.

[0074] "Conservative amino acid substitutions" are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), among others. Thus, non-essential (or non-critical) amino acid residues in a protein are preferably replaced with another amino acid residue of 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 mutants of RAG1 may have at least one (e.g., 2 or less, 3 or less, 4 or less, 5 or less, 6 or less, 7 or less, 8 or less, 9 or less, 10 or less, etc.) conservative amino acid substitutions compared to native human, mouse or rat RAG1 (identified above using their respective identifiers).

[0076] Sequence of the introduced RAG1 gene; mutations at the nucleic acid sequence level The "introduced RAG1 gene" means any nucleic acid sequence encoding a functional RAG1 protein (for example, human, mouse or rat RAG1, or a functional variant such as an amino acid substitution variant thereof).

[0077] The RAG1 nucleotide sequences described herein are codon-optimized. As used herein, "codon-optimized" (or "c.o.") means a polynucleotide sequence encoding a RAG1 protein that has been modified relative to the native polynucleotide sequence without changing 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 Since the secondary structure at the 5'-end of mRNA affects 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 selection pressure. In particular, strong secondary structures at the ribosome binding site or start codon inhibit translation, and folding of mRNA at the 5'-end results in large variations at the protein level.

[0079] In the present specification, the RAG1 nucleotide sequence can be codon-optimized such that the GC content of the coding sequence is increased.

[0080] 2. Effects on transcription / gene expression Heterologous gene expression is used in many fields of biotechnology, such as protein production and metabolic engineering. Since tRNA pools vary among organisms, placing a specific coding sequence in a non-native environment may reduce the efficiency of transcription and translation. In overexpressed transgenes, the corresponding mRNA occupies most of the total RNA in the cell. If the transcript contains rare codons, the utilization efficiency of ribosomes decreases and becomes depleted, ultimately potentially reducing the level of heterologous protein production. However, overexpressing a heterologous gene using codons optimized for a specific host's tRNA pool may also deplete amino acids or change the equilibrium state of the tRNA pool. The method of adjusting codons according to the tRNA amount of this host has conventionally been used for heterologous gene expression. However, new strategies for optimizing heterologous gene expression consider global nucleotide content, such as local mRNA folding, codon pair bias, codon ramp, or codon correlation.

[0081] Bias in special codons is also seen in some endogenous genes that are involved in amino acid depletion. For example, amino acid biosynthetic enzymes are not very adapted to the normal abundance of tRNA, but in the depleted state, they preferentially use codons that are adapted to the tRNA pool. Therefore, codon usage can introduce an additional level of transcriptional regulation for appropriate gene expression under specific cellular conditions.

[0082] From such a perspective, the nucleotide sequence of RAG1 can be codon-optimized to include removal of alternative splicing sites and potential splicing sites, and optimized codon usage for human tRNA.

[0083] 3. Influence on translation elongation rate Generally, in the case of highly expressed genes, the translation elongation rate is faster for transcripts with high codon adaptability to the tRNA pool and slower for transcripts with rare codons. This correlation between codon translation rate and cognate tRNA concentration will further regulate the translation elongation rate and bring several advantages to organisms. Specifically, codon usage can globally regulate these rates, and rare codons may contribute to translation accuracy at the expense of speed.

[0084] From this perspective, the RAG1 nucleotide sequence can be codon-optimized to include codon usage optimized for human tRNA.

[0085] 4. Effects on Protein Folding Protein folding in vivo occurs vectorially, with the N-terminus of the protein emerging from the translating ribosome and being exposed to the solvent earlier than its more C-terminal region. As a result, in the cotranslational folding of proteins, spatial and temporal constraints occur during the process of polypeptide chain folding. Since the translation rate of mRNA is related to protein folding and codon adaptation is related to translation elongation, it has been considered that sequence-level manipulation is effective for regulating or improving protein folding. Some studies have shown that for certain proteins, translational pauses occur as a result of local mRNA structure, which may be necessary for proper folding. Furthermore, it has been shown that synonymous mutations can have a significant impact on the folding process of nascent proteins and even change the substrate specificity of enzymes. From these studies, it is suggested that codon usage affects the rate at which polypeptides emerge vectorially from ribosomes, which in turn may affect the protein folding pathway throughout the available structural space.

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

[0087] Analysis of the human RAG1 cDNA sequence by the inventors revealed several possibilities for improving the DNA sequence without affecting the amino acid sequence, since many rare codons exist in the native RAG1 gene. Most of these codons were replaced with more frequently used codons of the Homo sapiens gene. Also, the GC content was increased to enhance mRNA stability. Finally, 21 cis-acting motifs (prokaryotic inhibitory motifs, splice donor sites, polyA sites, RNA instability motifs) that might have an adverse effect on expression were removed. Since no changes were made to the amino acid sequence, it allows the regulatory mechanisms occurring at the protein level to function properly.

[0088] In a non-limiting example, the RAG1 codon-optimized transgene may encode the amino acid sequence of SEQ ID NO: 1 and may contain the nucleic acid sequence of SEQ ID NO: 2. In other words, the RAG1 transgene may encode the human RAG1 protein (SEQ ID NO: 1), while having a nucleic acid sequence different from the native RAG1 nucleic acid sequence (SEQ ID NO: 3) (at least) due to the 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 changed during codon optimization. The inventors have shown that codon optimization of RAG1 is beneficial for optimal expression of the RAG1 transgene. Advantageously, the codon-optimized sequence (SEQ ID NO: 2) described herein for the RAG1 catalytic domain does not adversely affect the RAG1 catalytic domain function important for RAG1 activity. Thus, a base sequence suitable for the codon-optimized variant of the RAG1 transgene is provided. Accordingly, the codon-optimized variant of the RAG1 transgene contains the codon-optimized catalytic domain shown in SEQ ID NO: 2, and any additional codon optimization can be performed in other regions of the RAG1 transgene.

[0089] Therefore, to avoid misunderstanding, the RAG1 nucleic acid sequence may differ from the native RAG1 sequence of SEQ ID NO: 3, at least in the catalytic domain (any codon optimization may be performed in other regions of the RAG1 transgene sequence), while on the other hand, it may encode a functional RAG1 polypeptide as shown in SEQ ID NO: 1.

[0090] The sequence of the codon-optimized human RAG1 transgene successfully used by the inventors is shown in SEQ ID NO: 4. Accordingly, 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 term "operably linked" or "operably joined" or equivalent expressions mean that the nucleic acid elements are arranged in relation to each other such that they are functionally linked and can interact with each other in the intended manner. Such elements include, but are not limited to, a promoter, an enhancer and / or regulatory element, a polyadenylation sequence, one or more introns and / or exons, and the coding sequence of the gene of interest to be expressed. These nucleic acid sequence elements, when appropriately oriented or operably linked, act on each other to regulate each other's activities and ultimately can affect the expression level of the expression product. Regulation means 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 nucleotides (i.e., spacer sequence) or base pairs intervening between the elements. As will be understood by those skilled in the art, being operably linked means functional activity and is not necessarily related to a linkage in a natural positional relationship.

[0092] As used herein, the "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 sequence from functioning as desired. Generally, it is a non-functional sequence that exists only to separate adjacent functional nucleic acid sequences from each other.

[0093] As used herein, the term "promoter" generally refers to a nucleic acid sequence located upstream of the nucleic acid sequence to be transcribed. A promoter is generally necessary for transcription, i.e., it initiates transcription. A promoter allows for proper 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). When a TF binds to the promoter sequence, RNA polymerase, an enzyme that synthesizes RNA from the coding region of a gene, is recruited. A very large number of promoters are known in the art.

[0094] The promoter described in this specification can be described as a "strong promoter" because it drives high-level expression of an operably linked transgene intracellularly. Generally, the promoter drives the expression in cells of an operably linked RAG1 transgene such that the expression product of the RAG1 transgene in the cells (e.g., recombinant human CD34+ hematopoietic stem cells) is at least x-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cells (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 cells (e.g., recombinant human CD34+ hematopoietic stem cells). As will be apparent to those skilled in the art, the "expression product" encompasses all products generated during the expression of the transgene, and thus includes not only proteins but also the mRNA (transcript) of the transgene. Methods for measuring the level of the expression product in cells are well known in the art. For example, the expression product of the transgene can be measured at the transcript (mRNA) level or 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 a probe array, etc. In one aspect, the sample may be contacted with a nucleic acid molecule (i.e., a probe such as a labeled probe) that can specifically hybridize to the specific mRNA.

[0096] Alternatively, the level of a specific mRNA in a sample can be evaluated by a nucleic acid amplification method, such as 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] To detect the level of protein in a sample, any known protein detection method can be used. Generally, protein detection methods involve contacting a sample with an agent or antibody that selectively binds to the protein to measure the level of a specific protein in the sample. Preferably, the agent or antibody is labeled with a detectable label, for example. 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” means direct labeling of a probe or antibody by coupling (i.e., physically binding) 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 immunoassay (EIA), radioimmunoassay (RIA), Western blot analysis, and lateral flow devices (LFDs) utilizing membrane-bound antibodies specific for the protein biomarker. Alternatively, mass spectrometry can be used to detect and quantify the level of a specific biomarker protein in a sample. Such methods are routinely performed in the art.

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

[0100] Advantageously, the promoter described herein drives expression of the transgene at a level 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 promoter described herein can drive expression of the RAG1 transgene at the required level 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 a vector that integrates into the genome at a frequency of 5 or less copies per cell (i.e., 5 or less, 4 or less, 3 or less, 2 or less, 1 or less copies per cell, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less, etc.)). See, for example: 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] Suitable promoters can be readily identified by one of ordinary skill in the art using conventional methods. For example, a potential promoter of interest may be operably linked to the codon-optimized RAG1 nucleic acid sequence (SEQ ID NO: 4) within the plasmid backbone (pCCL) described herein, and the resulting plasmid may be introduced into the preclinical Rag1− / − mouse model of RAG-SCID described herein. The level of the RAG1 expression product can then be measured as described in the Examples section below and compared to the ABL1 level described herein. If the RAG1 expression level is at least 3-fold (e.g., 10-fold) higher than the ABL1 level, the promoter being tested is considered suitable for the present invention and is thus within the scope of the claimed invention. A detailed description of the methods that can be used to test a potential promoter of interest is provided in the Examples section below. Alternative / supplemental methods are also known to those of ordinary skill in the art.

[0103] The strength of a promoter can be most readily tested by assaying 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 these two expression levels is a direct indicator of promoter strength.

[0104] For the quantitative analysis of mRNA expression using WPRE, c.o.RAG1, and ABL1 as targets, qPCR was used. 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 designated genes from the Universal Probe Library (Roche). The primers and probes used are shown in Table 2. The PCR reaction was carried out on a StepOnePlus Real-Time PCR system (Thermofisher). All samples need to be performed in triplicate. Exemplary primers that can be used are as follows.

[0105]

Table 2

[0106] As an example, suitable promoters include MND, CMV, RSV, and CAG. These promoters are well-known; 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、 See 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: 10.1089 / hgtb.2012.238.

[0107] The MND promoter can be universally identified by the unique identifier: GenBank: LZ103461.1. Its sequence is also shown 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] Accordingly, 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 the codon-optimized version of the human RAG1 transgene shown in SEQ ID NO: 2 or SEQ ID NO: 4. Here, the transgene encodes the protein of SEQ ID NO: 1 and does not have the native RAG1 nucleic acid sequence of SEQ ID NO: 3. Advantageously, the expression product of the RAG1 transgene is at least 3-fold higher than a housekeeping gene (such as ABL1) in the cell when operably linked to the MND promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell). This is particularly advantageous when the expression cassette is present at a low copy number in the cell, e.g., when the copy number of the expression cassette integrated into the cell's genome is 5 or less (and when the expression product of the RAG1 transgene remains at least 3-fold higher than a housekeeping gene (such as ABL1) in the cell when operably linked to the MND promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell)).

[0109] In another example, an expression cassette is provided that comprises a RAG1 transgene operably linked to a CMV promoter. In this example, when the promoter is CMV, the RAG1 transgene may be a human RAG1 transgene, or the transgene may be its codon-optimized version (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 is at least 3-fold higher in level than a housekeeping gene (such as ABL1) in the cell when operably linked to the CMV promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell). This is particularly advantageous when the expression cassette is present at 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 CMV promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell)) remains at least 3-fold higher in level than a housekeeping gene (such as ABL1) in the cell).

[0110] An expression cassette is also provided that contains 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, 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 is at least 3-fold higher than the level of a housekeeping gene (such as ABL1) in the cell when operably linked to the RSV promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell). This is particularly advantageous when the expression cassette is present at a low copy number in the cell, e.g., when the copy number of the expression cassette integrated into the cell's genome is 5 or less (and when the expression product of the RAG1 transgene (operably linked to the RSV promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell)) remains at least 3-fold higher than the level of a housekeeping gene (such as 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. Here, the transgene encodes the protein of SEQ ID NO: 1 and does not have the native RAG1 nucleic acid sequence of SEQ ID NO: 3). Advantageously, the expression product of the RAG1 transgene is at least 3-fold higher in level than a housekeeping gene (such as ABL1) in the cell when operably linked to the CAG promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells). This is particularly advantageous when the expression cassette is present in the cell at a low copy number, e.g., when the copy number of the expression cassette integrated into the cell's genome is 5 or less (and when the expression product of the RAG1 transgene (operably linked to the CAG promoter and expressed in a cell (e.g., recombinant human CD34+ hematopoietic stem cells)) remains at least 3-fold higher in level than a housekeeping gene (such as 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) is advantageously at least 3-fold higher in level than a housekeeping gene (such as ABL1) in the cell. This is particularly advantageous when the expression cassette is present in the cell at a low copy number, e.g., when the copy number of the expression cassette integrated into the cell's genome is 5 or less (and when the expression product of the RAG1 transgene (operably linked to a promoter and expressed in a cell) remains at least 3-fold higher in level than 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 the expression cassette is integrated into its genome is similarly relevant and includes, for example, without limitation, hematopoietic progenitor cells such as HSCs (e.g., CD34+ HSCs), leukocytes, patient-specific induced pluripotent stem cells (iPSCs), or mesenchymal stem cells.

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

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

[0115] For example, the expression cassette can include any combination, or indeed all, of the following elements, and the order is 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, where the expression cassette further includes a nucleotide sequence encoding the post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus (WHP). The sequence of WPRE is well known in the art; see, for example, 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] Alternatively, the expression cassette may include a RAG1 transgene operably linked to an MND promoter, where the expression cassette further includes a nucleotide sequence encoding the post-transcriptional regulatory element (WPRE) of woodchuck hepatitis virus (WHP). In one example, the expression cassette may include a (human) RAG1 transgene (or a codon-optimized sequence thereof) operably linked to an MND promoter, where the expression cassette further includes a nucleotide sequence encoding the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence of 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 the 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 the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence of human RAG1 is shown 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 a RSV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding the 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 RSV promoter, wherein the expression cassette further comprises a nucleotide sequence encoding the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence of human RAG1 is shown 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 the 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 the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). An example of a codon-optimized sequence of human RAG1 is shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0122] Advantageously, the expression product of the RAG1 transgene is at least 3-fold higher than a housekeeping gene (e.g., ABL1) in the cell when (operatively linked to a suitable promoter and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell)). This is particularly advantageous when the expression cassette is present in the cell at a 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 remains at least 3-fold higher than a housekeeping gene (such as ABL1) in the cell when (operatively linked to a suitable one and expressed in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell))).

[0123] Retroviral Plasmid Provided herein is a retroviral plasmid. The retroviral plasmid is also referred to as a transfer plasmid.

[0124] The retroviral plasmid contains an expression cassette comprising a RAG1 transgene operatively 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 (e.g., see SEQ ID NO: 2 or SEQ ID NO: 4).

[0125] In this specification, suitable promoters are provided. As described elsewhere in this specification, the promoters described herein drive high-level expression of an operably linked transgene intracellularly. Advantageously, these promoters can drive the required level of expression of the RAG1 transgene even when the expression cassette is part of a low-copy-number retroviral plasmid. For example, the promoters described herein can drive the expression of an operably linked RAG1 transgene such that the expression product of the RAG1 transgene in the cell (e.g., a recombinant human CD34+ hematopoietic stem cell) is at least x-fold higher than the corresponding expression product of a housekeeping gene (e.g., ABL1) in the cell. 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, at least 10-fold higher compared to the corresponding expression product of a housekeeping gene (e.g., ABL1) in a cell (e.g., a recombinant human CD34+ hematopoietic stem cell).

[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 in this specification.

[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 a RSV promoter. Such expression cassettes are described in detail elsewhere in this specification.

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

[0129] The term "vector" is well known in the art and refers to a nucleic acid molecule, such as DNA or RNA, into which the expression cassette described herein can be inserted. A vector is used to transport the inserted nucleic acid molecule (here, an expression cassette containing a promoter operably linked to the RAG1 transgene) into a suitable host cell. A vector generally contains all the elements necessary to enable transcription of the inserted nucleic acid molecule and preferably translation of the transcript into a polypeptide, so that once inside the host cell, the vector can replicate independently of or simultaneously with the host's chromosomal DNA. Multiple copies of the vector and its inserted nucleic acid molecule can be generated. The vector can be an episomal vector (i.e., not integrated into the genome of the host cell) or a vector integrated into the host cell genome. The vector can be a non-viral vector or a viral vector. Non-viral vectors include, but are not limited to, plasmid vectors (e.g., pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 lacking bacterial sequences (minicircles) or their derivatives), transposon-based vectors (e.g., PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors, such as artificial chromosomes (bacterial (BAC), yeast (YAC) or human (HAC)), may be used to accommodate larger inserts. Viral vectors are derived from viruses and include, but are not limited to, retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, herpesviruses, hepatitis virus vectors, etc. Generally, but not always, viral vectors are replication-defective because essential viral genes for replication have been removed from the viral vector, resulting in a loss of the ability to grow in a given cell. However, some viral vectors can be adapted to specifically replicate in a given cell, such as a cancer cell, and are generally used to cause (cancer) cell-specific lysis (oncolysis).A virosome is a non-limiting example of a vector containing both viral and non-viral elements, in particular a combination of a liposome and inactivated HIV or influenza virus. Another example is a mixture of a viral plasmid and a cationic lipid.

[0130] The term "retroviral plasmid" is also well known in the art and herein means 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. Gamma-retroviral plasmids and lentiviral plasmids are attractive for gene therapy purposes. These plasmids have been improved and developed to mediate stable genetic modification of treated cells by integrating the transplanted plasmid genome onto the chromosome. This technology is useful not only for research purposes but also for clinical gene therapy aimed at long-term correction of genetic defects such as in stem cells and progenitor cells. Retroviral plasmid particles with tropism for various target cells have been designed. Gamma-retroviral plasmids and lentiviral plasmids have been used in more than 300 clinical trials to date and cover treatment options for various diseases.

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

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

[0133] In one example, the SIN lentiviral plasmid contains the pCCL backbone. The pCCL backbone is well-known in the art and is advantageous because it enables the production of virion particles at high titers and allows for the concentration of the virion supernatant to even higher titers required for clinical applications. Alternative SIN lentiviral plasmids include pRRL, pRLL, and pCLL. These are all lentiviral transfer plasmids that contain the sequence of simian virus 40 polyadenylation and the origin of replication (without enhancer) downstream of the 5' LTR of chimeric Rous sarcoma virus (RSV)-HIV or CMV-HIV and the 3' LTR of HIV, and contain a plasmid backbone in which most of the human sequences remaining at the integration site of HIV have been replaced. In pRRL, the enhancer and promoter of the U3 region of RSV (nucleotides -233 to -1 relative to the transcription start site, GenBank Accession No. J02342) are bound to the R region of the HIV-1 LTR. In pRLL, the sequence of the enhancer of RSV (nucleotides -233 to -50) is bound to the promoter region of HIV-1 (position -78 relative to the transcription start site). In pCCL, the enhancer and promoter of CMV (nucleotides -673 to -1 relative to the transcription start site, GenBank Accession No. K03104) were bound to the R region of HIV-1. In pCLL, the enhancer of CMV (nucleotides -673 to -220) was bound to the promoter region of HIV-1 (position -78).

[0134] Thus, as an example, the retroviral plasmid may include 1) an expression cassette containing a RAG1 transgene operably linked to an MND promoter (e.g., human RAG1 which may be codon-optimized as described herein; see SEQ ID NO: 2 or SEQ ID NO: 4), and 2) a SIN lentiviral backbone having, for example, 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 undesirable biological effects or interacting harmfully with any of the other components of the pharmaceutical composition in which it is contained.

[0142] An excipient is a natural or synthetic substance formulated with an active substance (e.g., an expression cassette, plasmid or virion), and is formulated for the purpose of increasing the formulation, such as promoting the absorption or dissolution of a drug, or for the purpose of enhancing the therapeutic effect on the active ingredient of the final dosage form. Excipients are also useful in the manufacturing process, and in addition to facilitating the handling of the active substance, such as enhancing the fluidity or non-stickiness of the powder, they can enhance the in vitro stability, such as preventing degradation during the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Thus, suitable excipients can be readily identified by those skilled in the art. For example, suitable pharmaceutically acceptable excipients include water, physiological saline, aqueous dextrose solution, glycerol, ethanol and the like.

[0143] An adjuvant is a pharmacological and immunological agent that modulates the effects of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. Thus, suitable adjuvants can be readily identified by those skilled in the art.

[0144] A diluent is an agent that dilutes. Pharmaceutically acceptable diluents are well known in the art. Thus, suitable diluents can be readily identified by those skilled in the art.

[0145] The carrier is non-toxic to the recipient at the administered dosages and concentrations used and is compatible with the other components of the formulation. The term "carrier" refers to natural or synthetic organic or inorganic components with which the active ingredient is combined to facilitate its application. Pharmaceutically acceptable carriers are well known in the art. Accordingly, suitable carriers can be readily identified by those skilled in the art.

[0146] Virus Particle Production The retroviral plasmids described herein, such as lentiviral plasmids, can be used to produce virions. To enhance the safety of virions, the components necessary for virion production are divided among multiple plasmids (three in the second-generation system and four in the third-generation system). The components of both systems are as follows. · A lentiviral transfer plasmid encoding the insert of interest. The transgene sequence is flanked by long terminal repeat (LTR) sequences, which facilitate the integration of the transfer plasmid sequence into the host genome. Generally, the sequence between the LTRs and the sequence containing the LTRs are integrated into the host genome upon viral transduction. Many lentiviral transfer plasmids are based on the HIV-1 virus. For safety reasons, all transfer plasmids are replication-incompetent and may further contain a deletion in the 3' LTR, rendering the integrated virus self-inactivating (SIN). · A packaging plasmid (in some cases one or two plasmids) · An envelope plasmid. As an example, SIN lentiviral plasmids are used herein because they are considered to be safer for gene therapy applications.

[0147] The most important elements and optimizations to consider are the transfer plasmids containing the expression cassettes. Second-generation lentiviral plasmids utilize the viral LTR promoter to express genes, 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 the U6 promoter to facilitate shRNA expression. Other functions that can be included in the transfer plasmid include Tet- or Cre-based controls and fluorescent fusions or reporters.

[0148] The third-generation system further improves the safety of the second-generation system in several important aspects. First, the packaging system is split into two plasmids, one encoding Rev and the other encoding Gag and Pol. Second, in the third generation, Tat is eliminated by adding a chimeric 5’LTR fused to a heterologous promoter to the transfer plasmid. Expression of the transgene from this promoter is no longer dependent on Tat transactivation. Third-generation transfer plasmids can be packaged by either second-generation or third-generation packaging systems.

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

[0150] Accordingly, provided herein are virions comprising an expression cassette comprising a RAG1 transgene operably linked to a promoter. Suitable components of the expression cassette are described elsewhere herein.

[0151] To avoid misunderstanding, the expression cassette present within the virion may comprise an RNA nucleic acid sequence. For example, the expression cassette present within 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 for the intentional introduction of nucleic acid into a cell, including the introduction of viral vectors and non-viral vectors, and includes terms and processes such as transformation, transduction, etc. Examples include transfection by viral vectors, transformation by 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 transfer (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] Therapy Provided herein are methods of treating patients who do not have a functional Rag1 gene or RAG1 protein. For example, provided herein are methods of treating patients with Rag1-deficient severe combined immunodeficiency (RAG1-SCID) or Omenn syndrome. Complete loss of function of RAG1 in humans results in severe immunodeficiency in humans. Thus, patients who do not have a functional Rag1 gene for the RAG1 protein are generally identified in infancy.

[0154] The methods provided herein are also for treating patients having at least one mutation in the RAG1 protein. In other words, this method is for treating diseases caused by at least one mutation of the RAG1 protein. These diseases are characterized in that functional RAG1 is partially lost in the patient, that is, the patient may have a hypomorphic RAG1 mutation. Diseases caused by hypomorphic RAG1 mutations progress at a slower rate than diseases caused by complete loss of RAG1 function because the RAG1 mutant can maintain partial recombination activity. Therefore, in diseases associated with hypomorphic RAG1 mutations, life-threatening complications may appear several years later. The disease is often underdiagnosed but may be much more common than RAG1-SCID or OS. As a result of next-generation sequencing of primary immunodeficiency patients, many hypomorphic RAG1 mutations have been revealed, but there is currently no curative treatment for them. In fact, functional evaluations of 71 RAG1 mutants have been performed so far. Phenotypes associated with hypomorphic RAG1 mutations are combined immunodeficiency with granulomas and / or autoimmunity (CID-G / A). RAG1 deficiency can be measured by quantification of recombination activity in vitro. Examples of diseases caused by hypomorphic RAG1 mutations that can be treated by the methods described herein are atypical SCID or combined immunodeficiency (CID). CID is a series of diseases characterized by hypomorphic RAG1 mutations that lead to a decrease in the immune repertoire.

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

[0156] The method may include an ex vivo cell-based therapy. Suitable methodologies for use in such methods are well known in the art; see, for example, the following. “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 therefor are described elsewhere herein. The genomes of these cells can be altered by using the 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 cells" and "hematopoietic stem cells (HSCs)" refer to cells of a 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, hematopoietic progenitor cells, such as HSCs, express at least one of the following cell surface markers characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thyl / CD90+, CD38lo / -, and C-kit / CD117+. Most preferably, the hematopoietic progenitor cells are CD34+ HSCs.

[0160] HSCs are an important target for gene therapy because they serve as a long-term source of modified cells. HSCs give rise to both myeloid and lymphoid blood cells. The lifespan of mature blood cells is limited and they need to be continuously replaced throughout life. Blood cells are continuously produced by the proliferation and differentiation of a population of pluripotent hematopoietic stem cells that can be replenished by self-renewal. Bone marrow (BM) is the major site of human hematopoiesis and a good source of hematopoietic stem and progenitor cells (HSPCs). HSPCs are also present in small numbers in peripheral blood (PB). Depending on the indication or treatment, their number may increase. The progeny of HSCs generate pluripotent and lineage-committed progenitor cells, including lymphoid progenitor cells that give rise to cells expressing RAG1. Since the progenitors of B cells and T cells are two cell populations that require the activity of RAG1, they can be transfected at a pre-rearrangement stage, but modifying the progenitors has the advantage of continuing to be a source of modified cells.

[0161] Accordingly, the method may include an ex vivo method for generating recombinant CD34+ hematopoietic stem cells, which includes contacting CD34+ hematopoietic stem cells with the virions described herein under conditions where the expression cassette is taken up by the cells and expressed to generate recombinant CD34+ hematopoietic stem cells. The “conditions under which the expression cassette is taken up by the cells and expressed to generate recombinant CD34+ hematopoietic stem cells” as used herein may include culturing the cells in the presence of an appropriate medium and growth factors and then incubating with the lentiviral virions described herein. Optionally, it may include Retronectin, protamine sulfate or other compounds that facilitate the introduction of the virus (transduction enhancer).

[0162] In one example, CD34+ cells are isolated from a patient's blood or bone marrow, cultured ex vivo under GMP-grade conditions using a medium and growth factors, and then further incubated with lentiviral virions containing Retronectin, protamine sulfate or other compounds that facilitate the introduction of the virus (transduction enhancer). Further culturing is continued, and in some cases, a second viral “hit” may be performed. At the end of the culture period, the cells can be harvested, collected in a bag for intravenous administration, and administered to the patient (or frozen in liquid nitrogen until needed and then thawed for IV (intravenous) administration).

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

[0164] Accordingly, the present specification also provides recombinant CD34+ hematopoietic stem cells comprising an expression cassette comprising a RAG1 transgene operably linked to a promoter (the details of which are described elsewhere in the present specification). Advantageously, when the promoters described in the present specification are used in combination with the transgenes described in the present specification, the required level of transgene expression is achieved even when using a retroviral plasmid with a low copy number. In other words, when using the expression cassettes, plasmids and virions described in the present specification, the expression product of the RAG1 transgene in the resulting recombinant CD34+ hematopoietic stem cells will be at least 3-fold higher than that of ABL1 in the cells when the copy number of the expression cassette integrated into the genome of the recombinant human CD34+ hematopoietic stem cells is 5 or less.

[0165] Advantageously, the combination of the promoter and the RAG1 transgene in the expression cassette drives the expression of RAG1 in each of the above cell types to a therapeutic minimum threshold level (due to the nature of the promoter used; i.e., the ability of the transgene expression product to drive the expression of the transgene such that it is at least 3-fold higher than the expression level of a housekeeping gene such as ABL1 in the cells even when the copy number of the expression cassette integrated into the cell's genome is 5 or less, i.e., when using a plasmid with a low copy number).

[0166] Accordingly, in one example, a method for treating RAG1-deficient SCID or OS in a subject is provided, the method comprising the following steps: (i) extracting CD34+ hematopoietic stem cells from the subject; (ii) contacting the cells of (i) with the virions described in the present specification; (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 the subject in need of treatment.

[0167] To extract CD34+ hematopoietic stem cells, a biopsy or aspiration of tissue or body fluid may be performed from the subject's bone marrow. The biopsy or aspiration can be carried out according to any of the methods known in the art. For example, in bone marrow puncture (aspiration), a thick needle is inserted into the pelvic bone to collect bone marrow.

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

[0169] Thereafter, the cells may be contacted with the virions and incubated together for an appropriate period using methods well known in the art.

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

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

[0172] Next, the method of the present invention includes the step of returning cells to a subject in need of treatment. As used herein, it also means returning and transplanting recombinant cells to a patient. This transplantation step can be achieved using any of the methods of transplantation known in the art. For example, the recombinant cells may be administered to the patient, such as by directly injecting them into the patient's blood.

[0173] By introducing the expression cassette into autologous cells derived from the patient in need and thus already fully immunologically matched, cells can be generated that can be safely reintroduced into the patient, effectively yielding a population of cells effective in improving one or more clinical conditions associated with the patient's disease.

[0174] The above example refers to hematopoietic stem cells. However, alternatively, leukocytes isolated from the patient can be used in the above-described treatment method.

[0175] Patient-specific induced pluripotent stem cells (iPSCs) may be created. Next, the genomes of these iPS cells may be modified using the expression cassettes, plasmids, virions or compositions and methods described herein. Next, these iPSCs may 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 the patient and used in the above-described treatment.

[0177] One advantage of ex vivo cell therapy is that a comprehensive analysis of the therapeutic agent can be performed prior to administration. Furthermore, specific cell populations, including clonal populations, can be isolated or enriched prior to transplantation.

[0178] Also described is an in vivo-based treatment method. In this method, the chromosomal DNA of the patient's cells is corrected using the materials and methods described herein. Preferably, the cells are leukocytes, bone marrow cells, hematopoietic progenitor cells, HSCs or HSC CD34+ cells.

[0179] Blood cells present attractive targets for ex vivo treatment and therapy, but increased effectiveness in delivery could enable direct in vivo delivery to other B cell and T cell precursors such as HSCs and / or CD34+ cells. Ideally, targeting and integration of the expression cassette can be directed to the relevant cells.

[0180] The advantages of in vivo gene therapy are that the manufacture and administration of therapeutic agents are easy. The same therapeutic approach and treatment can potentially be used to treat multiple patients, e.g., a number of patients sharing the same or similar genotypes or alleles. In contrast, for ex vivo cell therapy, it is usually necessary to use the patient's own cells, which are isolated, manipulated, and returned to the same patient.

[0181] Pharmaceutically Acceptable Carrier for Recombinant Cells The ex vivo method of administering to a subject a recombinant cell contemplated herein involves the use of a therapeutic composition comprising the recombinant cell.

[0182] The therapeutic composition comprises a physiologically acceptable carrier together with the recombinant cell composition and, if desired, at least one additional bioactive agent described herein dissolved or dispersed therein as an active ingredient. Preferably, the therapeutic composition is substantially non-immunogenic when administered to a mammalian or human patient for therapeutic purposes, unless desired.

[0183] <  In general, the recombinant cells described herein are administered as a suspension containing a pharmaceutically acceptable carrier. One of ordinary skill in the art would recognize that the pharmaceutically acceptable carrier used in the cell composition does not contain an amount of buffer, compound, cryopreservative, preservative, or other agent that would substantially interfere with the viability of the cells being delivered to the subject. Formulations containing recombinant cells may contain, for example, an osmotic buffer that allows maintenance of the integrity of the cell membrane, and, if desired, nutrients to maintain the viability of the cells or enhance engraftment upon administration. Such formulations and suspensions are known to those of ordinary skill in the art and / or can be adapted for use with progenitor cells as described herein using routine experimentation.

[0184] The recombinant cell composition can 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 can be mixed in an amount suitable for use in the therapeutic methods described herein with a pharmaceutically acceptable excipient that is compatible with the active ingredient.

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

[0186] Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers include sterile aqueous solutions that contain no substances other than the active ingredient and water, or buffered solutions such as phosphate buffered saline that contain a buffer such as sodium phosphate at physiological pH values, physiological saline, or both. Additionally, aqueous carriers can contain multiple buffer salts, as well as salts such as sodium chloride and potassium chloride, and solutes such as glucose and polyethylene glycol. The liquid composition can contain, in addition to water and excluding water, a liquid phase. Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of the active compound used in the recombinant cell composition effective for treating a particular disorder or condition depends 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 "transplant" are used interchangeably with respect to the placement of recombinant cells, such as HPSC cells, by a method or route that results in at least partial localization of the introduced cells to a desired site, such as a site of injury or repair, such that the desired effect is obtained. Recombinant cells, such as HPSC cells, can be administered by any suitable route by which at least a portion of the transplanted cells or cell components are delivered to the desired location in the subject in a viable state. The survival period of the cells after administration to the subject can be several hours, such as 24 hours, several days, several years, or the lifetime of the patient, i.e., a long-term engraftment period. For example, in some embodiments described herein, an effective amount of myogenic progenitor cells is administered via a systemic administration route such as the intraperitoneal route or the intravenous route (IV).

[0188] The terms "individual", "subject", "host" and "patient" are used interchangeably herein and mean any subject for which diagnosis, treatment or therapy is desired. For the purposes of the present invention, the subject can 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 the onset of any symptoms of SCID and / or Omenn 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 disorders, lymph node swelling, spleen swelling, diarrhea, and liver enlargement. Thus, prophylactic administration of a hematopoietic progenitor cell population serves to prevent SCID and / or Omenn syndrome.

[0190] When provided therapeutically, HPSCs are provided at the onset (or thereafter) of symptoms or signs of SCID and / or Omenn syndrome, for example, at the onset of the disease.

[0191] Preferably, the HPSC population administered according to the methods described herein comprises allogeneic HPSCs obtained from one or more donors. "Allogeneic" means HPSCs or biological samples containing HPSCs obtained from different donors of the same species, where the genes at one or more loci are not identical. For example, the HPSC population administered to a subject may be derived from one or more unrelated donor subjects, or one or more non-identical siblings. Preferably, syngeneic hematopoietic progenitor cell populations, such as those obtained from genetically identical animals or from monozygotic twins, can be used. Alternatively, the HPSCs are autologous cells, 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" means the amount of a population of recombinant cells or a population of their progeny necessary to prevent or alleviate at least one or more signs or symptoms of SCID and / or Omenn syndrome, and relates to a composition in an amount sufficient only to treat a subject having the desired effect, e.g., SCID and / or Omenn syndrome. Thus, the term "therapeutically effective amount" means an amount of recombinant cells or a composition comprising recombinant cells sufficient to promote a particular effect when administered to a typical subject having or at risk of having SCID and / or Omenn syndrome. Also included in the effective amount is an amount sufficient to prevent or delay the onset of symptoms of the disease, to change the course of symptoms of the disease (e.g., but not limited to, slowing the progression of symptoms of the disease), or to reverse the symptoms of the disease. It is understood that in any given case, the appropriate "effective amount" can be determined by one of ordinary skill in the art using routine experimentation.

[0193] Preferably, the effective amount of HPSC is at least 10 2 HPSCs, at least 5×10 2 HPSCs, at least 10 3 HPSCs, at least 5×10 3 HPSCs, at least 10 4 HPSCs, at least 5×10 4 HPSCs, at least 10 5 HPSCs, at least 2×10 5 HPSCs, at least 3×10 5 HPSCs, at least 4×10 5 HPSCs, at least 5×10 5 HPSCs, at least 6×10 5 HPSCs, at least 7×10 5 HPSCs, at least 8×10 5 HPSCs, at least 9×10 5 HPSCs, at least 1×10 6 HPSCs, at least 2×10 6 HPSCs, at least 3×10 6 HPSCs, at least 4×10 6cells of HPSC, at least 5×10 6 cells of HPSC, at least 6×10 6 cells of HPSC, at least 7×10 6 cells of HPSC, at least 8×10 6 cells of HPSC, at least 9×10 6 cells of HPSC, or multiples thereof. The HPSC are derived from one or more donors or obtained from autologous sources. Preferably, the HPSC described herein are expanded in culture prior to administration to a subject in need thereof.

[0194] "Administration" means delivering an HPSC composition to a subject by a method or route that results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in the subject, i.e., delivered to a desired site in the subject and at least a portion of the delivered composition, i.e., at least 1×10 4 cells are delivered to the desired site for a period of time. Methods of administration include injection, infusion, drip, 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, intracerebrospinal injection, and drip. In some embodiments, the route is intravenous. For delivery of cells, administration by injection or infusion is possible.

[0195] Preferably, the cells are administered systemically. The terms "systemic administration", "administered systemically", "peripheral administration", and "administered peripherally" mean administering a population of progenitor cells other than by direct administration to a target site, tissue, or organ, instead into the subject's circulatory system and thus subjecting them to metabolism and other similar processes.

[0196] The effectiveness of a composition for the treatment of SCID and / or Omenn syndrome can be determined by those skilled in the art. Treatment is considered "effective" if any one or more of the signs or symptoms of the disease change in a beneficial way. As an example, when the level of the functional RAG1 protein of interest is at least three-fold higher in CD34+ cells than the level of an appropriate housekeeping gene (e.g., ABL1), the treatment is considered effective. Also, effectiveness can be measured by the non-worsening of the symptoms of an individual evaluated by the need for hospitalization or medical intervention (e.g., the progression of the disease stops or at least slows down). Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals) and includes the following: (1) suppressing the disease, e.g., preventing or slowing the progression of symptoms; or (2) alleviating the disease, e.g., bringing about remission of symptoms; and (3) preventing or reducing the likelihood of the onset of symptoms.

[0197] The treatment method of the present invention improves one or more symptoms associated with SCID and / or Omenn syndrome by increasing the amount of functional RAG1 in an individual. Initial symptoms commonly associated with SCID and / or Omenn syndrome include, for example, the onset of alpha / beta T cell lymphopenia with gamma / delta T cell proliferation, severe cytomegalovirus (CMV) infection, autoimmunity, chronic skin inflammation, eosinophilia, growth retardation, lymph node swelling, spleen swelling, diarrhea, and liver enlargement.

[0198] Kit Also, this specification provides a kit for implementing the method of the present invention. The kit may include one or more of the expression cassette of the present invention, the plasmid of the present invention, or the virion of the present invention, and / or any nucleic acid or protein molecule or any combination thereof necessary for implementing the aspects of the method of the present invention. Preferably, the kit may include reagents for the reconstruction and / or dilution of the plasmid(s). Preferably, the components of the kit may be in separate containers or may be mixed in a single container.

[0199] Preferably, the above kit further includes one or more additional reagents, and such additional reagents are selected from buffers, polypeptides or buffers for introducing polynucleotides into cells, washing buffers, control reagents, etc. Examples of the buffer solution include a stabilization buffer, a reconstruction buffer, a dilution buffer, etc.

[0200] In addition to the above components, the kit may further include instructions for using the components of the kit to implement the method. The instructions for implementing 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 described on the label of the container of the kit or its components (i.e., related to the packaging or sub-packaging). The instructions can exist as an electronic storage data file on a suitable computer-readable storage medium such as a CD-ROM, a diskette, a flash drive, etc. In some examples, the actual instructions are not included in the kit, but means for obtaining the instructions from a remote source (e.g., via the Internet) can be provided. Examples of this aspect include a kit including a web address where the instructions can be viewed or downloaded. Similar to the instructions, the means for obtaining the instructions can be recorded on a suitable substrate.

[0201] General Definitions As used herein, "complementary" or "complementarity" means Watson-Crick base pairing of two nucleic acid sequences. For example, for the sequence 5'-AGT-3', it binds to the complementary sequence 3'-TCA-5'. Complementarity between two nucleic acid sequences can be "partial", where only some bases bind to their complements, or "complete", where all bases in the sequence bind to their complementary bases. The degree of complementarity between nucleic acid strands greatly affects the efficiency and strength of hybridization between nucleic acid strands.

[0202] The term "hybridizing" means annealing to two nucleotide sequences that are at least partially complementary in a hybridization process. To enable hybridization, the complementary nucleic acid molecules are generally heat-denatured or chemically denatured to melt the double-strand into two single-strands and / or to remove secondary structures such as hairpins from single-stranded nucleic acids. The stringency of hybridization is affected 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. One of ordinary skill in the art will understand that numerous different hybridization conditions can be designed depending on the known or expected homology and / or length of the nucleic acid sequences. High stringency conditions for hybridization include high temperature and / or low sodium / salt concentration (the salt contains sodium such as NaCl or Na citrate, for example) and / or reducing the concentration of compounds such as formamide in the hybridization buffer and / or SDS (sodium dodecyl sulfate detergent) in the hybridization buffer and / or excluding compounds such as dextran sulfate or polyethylene glycol (which promotes molecular crowding) from the hybridization buffer. By way of non-limiting example, representative salt and temperature conditions for stringent hybridization are 1×SSC, 0.5% SDS, 65°C. The abbreviation SSC means 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 representative time to achieve hybridization is 12 hours.

[0203] The terms "identity" and "identical" mean sequence similarity between two polymer molecules, such as between two nucleic acid molecules such as two DNA molecules. Determination of sequence alignment and sequence identity 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), such as 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. U.S.A. 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. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed discussion of sequence alignment methods and homology calculations is described, 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 connection with several sequence analysis programs from several information sources, including the National Center for Biotechnology Information (Bethesda, MD), and from the Internet. A description of how to determine sequence identity using this program is available from the BLAST (商標) "Help" section on the Internet. For comparison of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST (商標) (Blastn) program can be used with default parameters. Nucleic acid sequences with even higher similarity to a control sequence will have increased percent identity when evaluated by this method. Usually, the percentage of sequence identity is calculated over the entire length of the sequence.

[0206] For example, using the Needleman-Wunsch algorithm, a global optimal alignment is preferably found 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 preferably 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] Although the various aspects of the invention are described in detail herein, it should be understood that the invention provides many applicable inventive concepts that can be embodied in a variety of specific situations. The specific aspects discussed herein are merely illustrative of specific methods for making and using the invention and do not limit the scope of the invention.

[0208] In the practice of the present invention, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art, may be employed. Such techniques are well 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 (M. J. Gait ed., 1984); U.S. 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), the "Methods in Enzymology" series (Abelson and Simon, eds.-in-chief, Academic Press, Inc, New York), particularly 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, N.Y., 1986), etc.

[0209] The terms defined herein have meanings commonly understood by those skilled in the art related to the present invention. Terms such as "a", "an", "the", etc. are not intended to mean only a single entity, but include general classes for which specific examples can be used for illustration. The terms herein are used to describe specific aspects of the present invention, but their use does not limit the present invention, except as outlined in the claims.

Examples

[0210] Examples Results The MND promoter as an optimal vector for correcting Rag1 deficiency. At the start of this project, the inventors constructed four SIN LV plasmids with a CCL backbone and tested four promoters previously used in other clinical trials. Four promoters, namely PGK (Phospho Glycerate 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 promote the expression of the codon-optimized version of RAG1 (Figure 1A). These transfer vectors were combined with GAG-Pol, REV, and envelope (VSV-G) plasmids to generate recombinant lentiviruses, which were used for introduction into lineage-negative BM cells of Rag1-deficient mice. Rag1 knockout (KO) mice were transplanted with wild-type (WT) stem cells, mock-transduced Rag1 KO stem cells, or stem cells that had undergone gene therapy using the four promoters. Mice were bled every four weeks and sacrificed after 16 weeks. Subsequently, flow cytometry and Q-PCR were used to extensively analyze the viral copy number (VCN), WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element), and the expression of the therapeutic gene RAG1 (Figure 7A). Reflecting the known promoter strengths of these four vectors, a wide range of RAG1 expression was obtained in the initial test. Mice were sacrificed four months later or earlier if signs of illness were observed, and the immune organs were analyzed by flow cytometry. Recovery of IgM+B220+B cells in BM (Figure 1B) was seen in mice treated with wild-type (wt) stem cells and gene therapy mice treated with MND-c.o.RAG1, and occasionally in mice with the Cbx3-MND element, but not in mice using the PGK or UCOE promoter (Figure 1B, C).Even when mock-introduced with Rag1 KO stem cells, B cell development did not recover as expected, and the cells were blocked at the pre B cell stage.

[0211] Next, the expression of T cells in the thymus was analyzed using CD4 and CD8 markers. Normal development of T cells with the full spectrum of DP and SP developmental stages was observed in wt cells and MND-c.o.RAG1 cells, but not in the other promoters used (Fig. 1D, E).

[0212] The inventors confirmed that skin rash was observed in many mice (4 out of 9) in the low c.o.RAG1 expression group, but no health problems were seen in animals in the high c.o.RAG1 expression group and animals administered with wild-type cells or unmodified Rag1 knockout cells.

[0213] To more deeply understand the effects of the various promoters used, the inventors analyzed the relationship between the expression of RAG1 in the BM, the number of B cells generated in the BM (Fig. 2A), and the number of T cells generated in the thymus (Fig. 2B), which are two major lymphoid organs where the RAG gene is activated. In B cell development, a clear linear correlation up to 10-fold of the housekeeping gene level was seen between RAG1 expression and B220+ cells in the BM. For T cells, the inventors confirmed that there was a threshold at which RAG1 expression was minimized, which was approximately 10-fold of the housekeeping control level. In mice reconstituted with stem cells having c.o.RAG1 expression lower than this threshold, thymic T cell development hardly occurred.

[0214] For the clinical use of gene therapy vectors, in addition to efficacy, safety is also an important aspect. The present inventors used the IVIM assay, which is the currently accepted standard for the safety of viral vectors, as an additional selection criterion. All four vectors were shown to have a frequency of insertional mutagenic events at least 50-fold lower than that of the classical RSF91 gamma-retroviral vector (Figure 2C), and only the UCOE vector showed significantly lower replication efficiency than the other three promoters.

[0215] Finally, the present inventors confirmed the diversity and clonality of the TCRβ repertoire generated in mice that received gene therapy (Figure 2D). The inventors performed GeneScan analysis on 24 Vb genes and calculated the cumulative complexity score. Again, as shown by the representative plots and the highest scores, the MND promoter showed performance similar to that of WT mice.

[0216] Therefore, the present inventors determined that the pCCL-MND-c.o.RAG1 LV vector was the optimal vector and proceeded with the production of GMP-grade vectors. Using this clinical-grade vector, the following tests were conducted and further preclinical trials are underway.

[0217] Extensive preclinical trials of the pCCL-MND-coRAG1 LV vector in Rag1- / - mice. Initial analysis of eight Rag1− / − mice treated with the MND vector, positive controls (wt stem cells), and negative controls (mock-transduced Rag1− / − stem cells) confirmed good B cell reconstitution in the periphery (PB) and BM (Figure 3A), although the numbers remained lower than those in mice administered wt stem cells (Figures 3B and 7B). This is thought to be because development from pre B to immature B cell stage is partially arrested in cells transduced with c.o.RAG1 levels that are insufficient to support complete Ig rearrangement (Figure 7C). Alternatively, it is possible that residual pro B and pre B cells are inhibiting B cell development by occupying developmentally important niches. However, in the spleens of gene therapy mice, subsets of immature and mature B cells were seen at the same proportions (Figure 3C). For T cells, most GT mice had near-normal T cell development in the thymus and near-normal numbers of thymocytes (Figures 3D and 7C), although peripheral T cell numbers recovered to only about 30% of normal (Figure 3E), with slightly lower proportions of naive CD4 and CD8 T cells and increased effector memory subsets (Figure 3F), which is thought to be due to continuous proliferation of early T cells that have emigrated from the thymus. The inventors confirmed immune system recovery by histological analysis in addition to flow cytometry analysis of primary and secondary immune organs. The spleen, lymph nodes, and thymus showed surprisingly normal structure after GT (Figure 3G), equivalent to mice treated with wt stem cells and quite different from negative control mice treated with mock-transduced Rag1− / − cells. Importantly, in mice receiving MND-coRAG1 gene therapy, expression of FoxP3, which induces T cells into the CD4+ regulatory T cell line (T reg), was restored (Figure 3G).

[0218] Functional Reconstitution of Immunity after Rag1 Gene Therapy Next, the inventors verified whether the generated T cells and B cells have diverse repertoires and can mount an immune response against T cell-dependent neoantigens. In GeneScan analysis, diverse TCR Vβ repertoires were shown, which were slightly less complex than those in mice reconstituted with wt stem cells before immunization (Figure 4A), but there were no statistical differences in the immune repertoires after immunization. Total IgM, IgG, and IgE levels were also confirmed (Figure 4B and Figure 7E), and reached values close to normal levels in GT-treated mice. The inventors used TNP-KLH as a T cell-specific antigen and measured the production of TNP-specific IgG antibodies to examine whether the generated T cells and B cells cooperate to mount an active immune response. The levels of TNP-specific IgG in the serum were comparable between mice treated with wt stem cells and GT-treated mice (Figure 4C).

[0219] When examining each TCR Vβ family individually, the MNDCoRAG1 construct provided a rearrangement pattern equivalent to that of the WT control with a polyclonal TCR Vβ family, and showed no perturbation of TCR Vβ usage or oligoclonal expansion (as observed with other constructs). Importantly, in the clinical-grade MND-c.o.RAG1 batch, the immune diversity of the treated mice was equivalent to that of WT control mice before and after immunization. Importantly, in the CID mouse model, the response to B cell-dependent T cell antigens is defective, but the MND-c.o.RAG1 gene therapy mice immunized with our TNP-KLH were able to initiate an immune response at a level equivalent to that of control mice against B cell-dependent T cell antigens, and it is thought that the mice treated with gene therapy did not exhibit the CID phenotype, but rather were able to overcome this immunodeficiency phenotype.

[0220] Preclinical safety testing of the vector In accordance with the requirements of the regulatory authorities, the clinical-grade vector was tested by an external institution for the presence of replication-competent virus (RCL). As a result, the vector was negative in two independent tests (data not shown). Other safety tests commonly required include in vivo biodistribution of the vector, confirmation of vector insertion sites (especially regarding the possibility of clonal occurrence), and insertional mutagenesis tests such as IVIM.

[0221] The inventors confirmed the distribution of the vector in a large number of perfused organs of all GT-treated mice (Figure 5A). Perfusion was performed to remove most of the blood cells that should carry the vector by leukocytes. As expected, high VCN was observed in the thymus, followed by VCN in other immune system organs, spleen, bone marrow, lymph nodes, and peripheral blood, since c.o.RAG1-transduced cells were positively selected. In all other organs, the signal was significantly low except for rare positive signals in the stomach and lungs, which is thought to be due to incomplete perfusion or rare progression of infection in individual mice (Figure 7D, Table 4).

[0222]

Table 4

[0223] Importantly, histological slides of 29 different organs were pathologically examined for each mouse, and MNDCoR No abnormalities were observed in the mice treated with AG1 gene therapy. The most characteristic pathologies of the Omenn syndrome (OS) and the atypical SCID model are a severe phenotype accompanied by erythroderma, skin infiltration, and eosinophilia. The inventors conducted extensive pathological examinations of mice treated with the MNDCoRAG1 gene therapy vector, but the characteristics of OS / atypical SCID were not detected. In fact, Figure 7D shows the pathology of the lung and liver, showing a normal phenotype like that of WT-treated mice without abnormal T cell infiltration. In addition, the inventors confirmed the skin and small intestine to confirm that the mice treated with the MND-c.o.RAG1 vector did not exhibit the phenotype of OS or atypical SCID. As a result, no clinical symptoms of skin disease were observed in all groups (ulcers, crusts, erythema, hair loss were not seen) (Figure 10). Furthermore, histological analysis of the skin in all groups confirmed that the characteristics of Omenn-like syndrome, such as severe hair loss, skin erythroderma, and dense dermal inflammation consisting of lymphocytes and eosinophils in the skin, were absent in MND-c.o.RAG1-treated mice. The inventors collected extensive samples from the small intestine (duodenum, jejunum, and ileum) as well as the large intestine (cecum, colon, and rectum), but no severe inflammatory infiltration similar to Omenn-like syndrome was observed.

[0224] Next, the inventors confirmed the viral insertion sites using nrLAM-PCR (Figure 5B). This is a highly sensitive technique that can detect cloned insertions as individual bands (which can be sequenced if necessary) (Gabriel et al., 2014). The inventors observed only bands showing polyclonal hematopoiesis with little oligoclonality, except for some minor bands. The inventors concluded that there was no evidence of clone selection induced by the vector. This is consistent with the findings of other researchers when using SIN LV vectors for hematopoietic stem cells (HSCs).

[0225] The safety of clinical MND-c.o.RAG1 was also tested using the IVIM assay. In different independent experiments, no clone growth was observed for the clinical vector, and the results were close to those of mock-transduced cells (Figure 5C). This is presumably because the purity is higher than that of the research vector, resulting in an improved functional titer and fewer side effects.

[0226] Recovery of B cell and T cell development in RAG1 SCID patient cells The inventors have previously shown that by transplanting BM CD34+ cells from SCID patients into NSG mice, it is possible to confirm where T cell development stops in human SCID. This model should also be suitable as a preclinical efficacy model using patient cells. Therefore, the inventors purified CD34+ cells from cryopreserved BM cells of RAG1-SCID patients. This patient was hypomorphic and had some remaining B cells but no T cells. The inventors transplanted mock-transduced CD34+ cells or MND-c.o.RAG1-transduced CD34+ cells into busulfan-treated mice and observed the development of T cells and B cells over time. Since human cell engraftment was similar between mice transplanted with gene therapy-treated cells and mice transplanted with mock-transduced cells, it was found that gene therapy does not affect human cell engraftment. As expected, B cells were observed in mock-transduced humanized mice, but many more B cells were seen in the spleens of GT-treated CD34+ cells (Figures 6A and 8B). In addition, the existing B cells showed polyclonal Ig rearrangement (Figure 8E) and produced immunoglobulins such that human IgM was detected in the sera of the mice (Figure 6D), but there was a tendency for the polyclonal repertoire to increase after GT.

[0227] Surprisingly, while T cells did not develop in mice transplanted with mock-transduced RAG1-SCID cells, clearly detectable T cell development was observed in gene therapy mice (Figure 6B and Figure 8C). In addition, the inventors examined the thymus after sacrificing the mice. Because the patient was hypomorphic, the inventors confirmed that there were several stages of T cell development, including all DN, ISP, and early CD3-DP stages (Figure 6C). However, there were no CD3+ cells, and no late CD3+ DP thymocytes or SP thymocytes, suggesting that in particular, TCRα rearrangement was affected by this RAG1 mutation. Finally, the inventors confirmed TCRB and TCRG rearrangement by Gene Scan analysis. Due to the very limited material, not all possible Vγ genes and Vβ genes could be analyzed, but the selected gene segments showed more in-frame rearrangements in the gene therapy-treated group for TCRG, while rearrangements were detected only in the GT group for TCRB (Figure 6E). Polyclonal patterns without signs of clonal dominance were revealed by nRLAM-PCR in spleen cells (Figure 6F).

[0228] Discussion Patients with RAG1-SCID have impaired genetic assembly of the TCR and BCR. Affected children typically develop a variety of severe life-threatening infections. Currently, the only treatment for RAG1-SCID is to replace the affected bone marrow with healthy, unmodified allogeneic stem cells. The overall survival rate of matched donor SCT is satisfactory, but the results are significantly worse in unmatched donor SCT, which accounts for the majority of cases. Furthermore, approximately 25% of patients who have received allogeneic SCT develop graft-versus-host disease, which significantly impairs the outcome in terms of morbidity, immune reconstitution, and transplant-related mortality (Gennery et al.). Additionally, transplant outcomes in RAG-SCID (and other recombination-deficient T-B-SCID) are significantly worse than those in SCID with B cells (i.e., T-B+ SCID) (Gennery et al.).

[0229] Transplanting genetically modified 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 shown no genotoxicity issues seen when using γ-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 RV vectors (currently sold as a therapy approved under the name Strimvelis) and LV vectors have shown excellent clinical outcomes comparable to hematopoietic stem cell transplantation by a matched donor. See Morgan, R.A., Gray, D., Lomova, A., and Kohn, D.B. (2017). Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned. Cell stem cell 21, 574-590.

[0230] Unlike X-linked SCID and ADA-SCID, the development of gene therapy for RAG-SCID has been extremely difficult. In previous attempts (Lagresle-Peyrou et al., 2008), gammaretroviral vectors were used in preclinical Rag1- / - models, but this carried a high risk of insertional mutagenesis. Although the RAG1 gammaretroviral vector was able to correct the deficiency more easily, the SIN lentiviral vector initially had insufficient expression of the therapeutic RAG1 gene, causing a 'leaky' SCID or Omenn-like phenotype. Here, the inventors show that durable functional immune reconstitution can be obtained even at low VCN. The inventors also show 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, a SIN LV vector using the MND promoter was selected because this rather strong promoter is the most effective in preclinical models. The MND promoter has been used in previous gene therapy trials for ADA-SCID and Adrenoleukodystrophy (ALD), and there have been no reports of insertional mutagenesis. Also, from preclinical safety data, the MND-coRAG1 vector has been found to be relatively safe. The inventors have found that the SIN LV vector using MND-coRAG1 can restore immunity without overall abnormalities or histological pathology, and thus this vector has the ability to treat a wide range of diseases mediated by RAG1.

[0232] In clinical trials, gene therapy for ADA-SCID and X-linked SCID has been shown to provide significant clinical benefits and also to greatly reduce healthcare-related costs. The inventors expect similar benefits since our approach for treating RAG1-SCID patients can reduce suboptimal outcomes in (mismatched) allogeneic transplantation, which often requires administration of immunoglobulins and treatment of infections and GvHD-related complications.

[0233] Materials and Methods 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). The mice were housed and maintained in the animal facility of the Leiden University Medical Center (LUMC). All animal experiments were approved by the Central Committee for Animal Experiments (Centrale Commissie Dierproeven, CCD) of the Netherlands.

[0234] Lentiviral Vector and Vector Production As a result of optimizing the RAG1 gene sequence according to the description of Pike-Overzet et al. (2011), 90% of the codons were compatible with the codon bias of the Homo sapiens gene. Furthermore, the GC content increased from 48% to 61%, and the number of cis-acting motifs decreased from 21 to 0. The optimized RAG1 sequence was synthesized by GeneArt (Regensburg, Germany). Codon-optimized RAG1 (c.o.RAG1) was cloned into the self-inactivating lentiviral pCCL plasmid to obtain Cbx3.MND.coRAG1 (hereinafter referred to as Cbx3-c.o.RAG1), pCCL-MND-c.o.RAG1 (hereinafter referred to as MND-c.o.RAG1), pCCL-PGK-c.o.RAG1 (hereinafter referred to as PGK-c.o.RAG1) and (hereinafter referred to as UCOE-c.o.RAG1). DNA sequencing of the transgene was performed to verify the gene transfer construct. 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 preparation of the helper plasmid was obtained through PlasmidFactory (Bielefeld, Germany).

[0235] 293T cells were transiently transfected with the transfer plasmid and the helper plasmid using the X-tremeGene HP DNA transfection reagent (Sigma-Aldrich). Lentivirus was harvested 24 hours, 30 hours and 48 hours after transfection, filtered through a 0.22 μm pore filter (Whatmann), and stored at -80°C. The pooled lentiviral supernatant was ultracentrifuged (Beckman Optima (商標)Using an LE-80K rotor (SW32Ti), it was concentrated under vacuum at 10,000 rpm and 4 °C for 16 hours. The pellet was resuspended in StemSpan Serum-Free expansion medium (SFEM; Stemcell Technologies Inc) and aliquoted to avoid repeated freeze / thaw cycles. Since a suitable anti-RAG1 antibody was not available, the inventors determined the virus titer using qPCR as described below. Clinical-grade GMP vectors were generated by Batavia Biosciences (Leiden, The Netherlands), tested and verified in mouse Rag1-deficient bone marrow cells and human CD34+ cells, aliquoted into 200 ml vials, and stored at -80 °C until use.

[0236] Transduction of Mouse Strained-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 mice and C57BL / 6 Rag1 - / - mice. The obtained bones were washed or crushed, and the cells were passed through a 0.7 μm cell strainer (Falco), washed, and then frozen as viable cells. After thawing, lineage-negative cells were isolated using a mouse lineage depletion kit and an AUTOMacs cell sorter (Miltenyi Biotech). The lineage-negative cells were stimulated overnight in StemSpam-SFEM containing penicillin / streptomycin (5,000 units / 5,000 μg / 00; Gibco), 50 ng / mL of recombinant mouse FMS-like tyrosine kinase 3 ligand (rmFLT3L; R&D systems), 100 ng / mL of recombinant mouse Stem-Cell Factor (rmSCF; R&D systems), and 10 ng / mL of recombinant mouse thrombopoietin (rmTPO; R&D systems). Subsequently, Rag1 - / -Cells were transduced with different lentiviruses by spin - incubation at 800 x g, 32 °C for 1 hour using 4 μg / ml of protamine sulfate (Sigma - Aldrich). Cells were cultured at 37 °C in 5% CO2 for 24 hours using cytokine - supplemented medium.

[0237] Human bone marrow from children diagnosed with SCID was obtained according to the guidelines of the Medical Ethics Committee and IRB of the Leiden University Medical Center. This patient was a compound heterozygote with the following identified 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 + cells were isolated. The enriched CD34 + cells were stimulated overnight in Pen / Strep medium containing X - VIVO15 medium without gentamicin and phenol red (Lonza) - 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). Cells were transduced with X - VIVO - 15 complete medium containing 4 μg / mL of protamine sulfate as described above and cultured for 24 hours.

[0238] Rag1 - / - Transplantation of Mice and NSG Mice In Iscove's Modified Dulbecco's Medium (IMDM) without phenol red (Gibco), control mock - transduced cells (C57BL / 6 wild - type cells as WT control and Rag1 - / -cells (referred to as KO controls) and transduced Rag1 - / - mouse cells (up to 5.10 5 cells / mouse) were mixed with supporting Rag1 - / - spleen cells (3.10 6 cells / mouse) and injected into the tail vein of pre-conditioned Rag1 - / - recipient mice for transplantation. Recipient mice (8 - 12 weeks old) were conditioned by a single whole-body irradiation with a conventional voltage X-ray (8.08 Gy) 24 hours before transplantation, or by two consecutive administrations of 25 mg / kg of busulfan (Sigma-Aldrich) (48 hours and 24 hours before transplantation). After overnight culture, 60,000 - 70,000 human CD34 + cells were resuspended in phenol red-free (IMDM) and transplanted intravenously into NSG recipient mice (5-week-old mice, busulfan conditioning as described above). The mice used for transplantation were kept in a specific pathogen-free location. During the first 4 weeks after transplantation, DietGel Recovery Food (Clear H2O) and antibiotic water containing 0.07 mg / mL polymyxin B (Bupha Uitgeest), 0.0875 mg / mL ciprofloxacin (Bayer b.v.) and 0.1 mg / mL amphotericin B (Bristol-Myers Squibb) were additionally given, and the condition of the mice was observed daily. Peripheral blood (PB) of the mice was collected 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, the mice were immunized with a synthetic TNP-KLH antigen. A solution of 100 μg of TNP-KLH (Biosearch Technologies Inc.) dissolved in 50% Imject Alum (Thermo Scientific) was injected intraperitoneally (i.p.). Three weeks later, the mice were given a booster injection of 100 μg TNP-KLH in PBS i.p. 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 compressing the organs using a 70 μM cell strainer (BD Falcon), and the single cell suspension from the BM was prepared by the above method. Red blood cells from the PB and spleen were lysed using an NH4Cl (8.4 g / L) / KHCO3 (1 g / L) solution. The single cell suspensions were counted and stained with the antibodies shown in Table 1.

[0241] In summary, cells were incubated for 30 minutes at 4 °C in the dark using an antibody mixture solution containing an antibody directly conjugated to the 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 for 30 minutes at 4 °C using an antibody solution conjugated with streptavidin. If necessary, 7AAD (BD Biosciences) was used as a viability dye. Cells were measured using a FACS-CantoII and an LSR Fortessa X-20 (BD Biosciences), and the data were analyzed using FlowJO software (Tree Star).

[0242] Determination of Vector Copy Number (VCN) and c.o.Rag1 Expression by RT-qPCR Targeting WPRE, c.o.RAG1, ABL1, and PTBP2, genomic lentiviral RNA, proviral DNA copies, and transgene mRNA expression were quantitatively analyzed by qPCR. 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). VCN was determined on DNA samples by detection of WPRE and PTBP2. qPCR was performed using TaqMan Universal Master Mix II (Thermofisher) with specific probes for the designated genes of the Universal Probe Library (Roche). The primers and probes used are shown in Tables 5 and 6. PCR reactions were performed on a StepOnePlus Real-Time PCR system (Thermofisher). All samples were run in triplicate.

[0243]

Table 5

[0244]

Table 6

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

[0246] Repertoire Analysis Total RNA was purified from mouse spleen cells and reverse-transcribed into cDNA by the above method. The GeneScan analysis procedure for the mouse T cell repertoire was referred to (Pannetier et al., 1993). The cDNA was amplified using a FAM-labeled C gene segment-specific primer and 24 types of 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 (registered trademark) Genetic Analyzer (Applied Biosystems) for fragment analysis. The raw data of the spectral types were analyzed, visualized, and scored by ScoreSpec, a new spectral type analysis algorithm for estimating immunological diversity (Cordes et al, manuscript in preparation). ScoreSpec discriminates and scores the peak patterns of individual spectral types, such as the overall peak distribution (Gaussian distribution), the shape of individual peaks, and the correction of TCR transcripts outside the frame. The scores range from 0 when no peaks are detected to 100 when diverse TCR repertoires are detected.

[0247] The human immunoglobulin and T cell receptor repertoires generated in NSG mice were analyzed with DNA samples collected from the BM and thymus (DNA was extracted by the above method). 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 of the IGH + IGK B cell Clonality Assay (InvivoScribe) and the TCRB+TCRG T cell Clonality Assay (InvivoScribe), respectively. The PCR products were analyzed by differential fluorescence detection using an ABI-3730 instrument (Applied Biosystems) for fragment analysis. The output files were visualized and analyzed using ScoreSpec.

[0248] Non-Restrictive Linear Amplification-Mediated PCR (nrLAM-PCR) The insertion site of the lentivirus was analyzed by nrLAM-PCR of mouse bone marrow DNA samples as described in (Gabriel et al., 2014); Schmidt M. et al (2014) J. Vis. Exp. (88), e51543.

[0249] In Vitro Immortalization Assay (IVIM) The genotoxicity of the viral vectors (Cbx3-c.o.RAG1, MND-c.o.RAG1, PGK-c.o.RAG1, UCOE-c.o.RAG1) was quantified as described in (Modlich et al., 2006) Baum et al. (2006) Blood 108:2545-2553.

[0250] Gross Pathology and Histopathology Dissection was performed, organs were collected, and gross and microscopic examinations were carried out (list X of the collected organs). The selection of organs for gross pathological examination and histopathological analysis followed the applicable European and international guidelines (EMEA 1995, WHO 2005) (WHO, 2005). For gross pathological examination, the outer surface of the body, openings, thorax, abdomen, and body cavities were examined (the analyzed organs are shown in Table 4).

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

[0252] Statistics Statistical calculations and graph creation were performed using GraphPad Prism6 (GraphPad Software). Statistical significance was determined by standard one-sided Mann-Whitney U test or ANOVA test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0253] Sequence SEQ ID NO: 1: RAG1 human protein sequence (1043 aa) MAASFPPTLGLSSAPDEIQHPHIKFSEWKFKLFRVRSFEKTPEEAQKEKKDSFEGKPSLEQSPAVLDKAD GQKPVPTQPLLKAHPKFSKKFHDNEKARGKAIHQANLRHLCRICGNSFRADEHNRRYPVHGPVDGKTLGL LRKKEKRATSWPDLIAKVFRIDVKADVDSIHPTEFCHNCWSIMHRKFSSAPCEVYFPRNVTMEWHPHTPS CDICNTARRGLKRKSLQPNLQLSKKLKTVLDQARQARQHKRRAQARISSKDVMKKIANCSKIHLSTKLLA VDFPEHFVKSISCQICEHILADPVETNCKHVFCRVCILRCLKVMGSYCPSCRYPCFPTDLESPVKSFLSV LNSLMVKCPAKECNEEVSLEKYNHHISSHKESKEIFVHINKGGRPRQHLLSLTRRAQKHRLRELKLQVKA FADKEEGGDVKSVCMTLFLLALRARNEHRQADELEAIMQGKGSGLQPAVCLAIRVNTFLSCSQYHKMYRT VKAITGRQIFQPLHALRNAEKVLLPGYHHFEWQPPLKNVSSSTDVGIIDGLSGLSSSVDDYPVDTIAKRF RYDSALVSALMDMEEDILEGMRSQDLDDYLNGPFTVVVKESCDGMGDVSEKHGSGPVVPEKAVRFSFTIM KITIAHSSQNVKVFEEAKPNSELCCKPLCLMLADESDHETLTAILSPLIAEREAMKSSELMLELGGILRT FKFIFRGTGYDEKLVREVEGLEASGSVYICTLCDATRLEASQNLVFHSITRSHAENLERYEVWRSNPYHE SVEELRDRVKGVSAKPFIETVPSIDALHCDIGNAAEFYKIFQLEIGEVYKNPNASKEERKRWQATLDKHL RKKMNLKPIMRMNGNFARKLMTKETVDAVCELIPSEERHEALRELMDLYLKMKPVWRSSCPAKECPESLC QYSFNSQRFAELLSTKFKYRYEGKITNYFHKTLAHVPEIIERDGSIGAWASEGNESGNKLFRRFRKMNAR QSKCYEMEDVLKHHWLYTSKYLQKFMNAHNALKTSGFTMNPQASLGDPLGIEDSLESQDSMEF Sequence number 2: Codon-optimized nucleic acid sequence encoding the human RAG1 catalytic domain Accession No. 3: RAG1 cDNA sequence Accession number 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.[[ID=⑦]] [[ID=⑧]]

[0254] [[ID=⑨]] [[ID=⑩]] List of References [[ID=⑪]] [[ID=⑫]]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)[[ID=⑬]] [[ID=⑭]]Bancroft, J.D., and Gamble, M. (2008). Theory and Practice of Histological Techniques (Churchill Livingstone).[[ID=⑮]] [[ID=⑯]]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.[[ID=⑰]] [[ID=⑱]]Dull, T., Zufferey, R., Kelly, M., Mandel, R.J., 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

**Claim 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. **Claim 2** The expression cassette according to claim 1, wherein the RAG1 transgene comprises the nucleic acid sequence of SEQ ID NO:

4. **Claim 3** The expression cassette according to claim 1, wherein when the expression cassette is expressed in human CD34+ hematopoietic stem cells in which the copy number of the expression cassette integrated into the genome is 5 or less, the expression product is produced at a level at least 3 times higher than the expression level of ABL1 in the cells. **Claim 4** The expression cassette according to any one of claims 1 to 3, wherein the promoter is MND. **Claim 5** The expression cassette according to any one of claims 1 to 4, wherein the expression cassette further comprises a nucleotide sequence encoding the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). **Claim 6** A retroviral plasmid comprising the expression cassette according to any one of claims 1 to 5. **Claim 7** The plasmid according to claim 6, wherein the plasmid is a self-inactivating (SIN) lentiviral plasmid. **Claim 8** The plasmid according to claim 7, wherein the plasmid comprises a pCCL backbone. **Claim 9** The plasmid according to any one of claims 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. **Claim 10** A virion comprising the expression cassette according to any one of claims 1 to 5. **Claim 11** A composition comprising the expression cassette according to any one of claims 1 to 5, the plasmid according to any one of claims 6 to 9, or the virion according to claim 10, and a pharmaceutically acceptable adjuvant, carrier, excipient, or diluent. **Claim 12** A recombinant CD34+ hematopoietic stem cell comprising the expression cassette according to any one of claims 1 to 5. **Claim 13** An ex vivo method for generating recombinant CD34+ hematopoietic stem cells, the method comprising contacting the cells with the plasmid according to any one of claims 6 to 9 or the virion according to claim 10 under conditions such that the expression cassette is taken up and expressed in the cells to produce recombinant CD34+ hematopoietic stem cells. **Claim 14** The expression cassette, plasmid, composition, virion or recombinant cell according to any one of claims 1 to 12 for use in treatment.

15. The expression cassette, plasmid, composition, virion or recombinant cell for use according to claim 14, wherein the expression cassette, vector, composition, virion or recombinant cell is for use in the treatment of RAG1-deficient severe combined immunodeficiency (SCID), Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID).

16. A method of treating a subject comprising administering to the subject in need thereof a therapeutically effective amount of the expression cassette, plasmid, composition, virion particle or recombinant cell according to any one of claims 1 to 12.

17. The method according to claim 16, wherein the subject has RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID).

18. The method according to claim 17, wherein the SCID is RAG1-deficient SCID.

19. (i) Extracting CD34+ hematopoietic stem cells from a subject; (ii) Contacting the cells of (i) with the virion according to claim 10 or the plasmid according to claims 6 to 9; (iii) Incubating the cells of (ii) for a period of time; and (iv) Introducing the cells of (iii) into the subject A method of treating RAG1-deficient SCID, Omenn syndrome (OS), atypical SCID or combined immunodeficiency (CID) in a subject in need thereof, comprising:

20. The method according to claim 19, further comprising administering to the subject a chemotherapy or other pretreatment regimen prior to step (iv).

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