Gene therapy for dock8 deficiency

Abstract

Described herein are systems including nucleic acid constructs for DOCK8 protein expression in cells and uses thereof. Further described are Lentiviral vectors containing these expression systems, and methods of use thereof for treating a DOCK8 deficiency in a subject in need.

Description

GENE THERAPY FOR DOCK8 DEFICIENCYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 467,552, filed on May 18, 2023, and entitled GENE THERAPY FOR DOCKS DEFICIENCY, which is incorporated herein in its entirety by reference.SEQUENCE LISTING STATEMENT

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML format copy, created on May 14, 2024, is named P-636966- PC_05_14_2024 SL.xml and is 241.2 kilobytes in size.BACKGROUND

[0003] DOCKS deficiency results from mutations in DOCK8, a large gene on chromosome 9 comprised of 48 exons that encodes a protein about 190 kDa in size. The DOCKS protein C is constitutively expressed in cells of hematopoietic origin and is highly expressed by healthy lymphocytes. Affected patients present within the first months to early years of life and are severely affected by the disease. The median survival is 10-20 years and is complicated by multiple disease-related comorbidities, including recalcitrant infections, autoimmunity, and predisposition to the development of malignancies. Similar to many other inborn errors of immunity, allogeneic HSCT can be curative for DOCK8 deficiency but is frequently complicated by acute and chronic GvHD, sinusoidal obstruction syndrome, and worsening of pre-existing disease. Despite the associated risks of allogeneic bone marrow transplantation, the therapeutic benefits of allogeneic HSCT suggests that ex vivo gene modification of autologous HSPC may be an effective treatment for the disease with fewer side effects.

[0004] Autologous bone marrow transplantation would not require an HLA-matched donor and would allow affected individuals to be transplanted when they are younger, which is associated with improved overall survival.SUMMARY

[0005] Disclosed here are systems for expressing D0CK8 protein and uses thereof.

[0006] In certain aspects, a system for expressing human D0CK8 protein in a cell, comprises a first nucleic acid encoding a first fragment of a human DOCK8 protein fused at the C terminal to an N-intein; and a second nucleic acid encoding a second fragment of the human D0CK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the DOCK8 protein ligated to the second fragment of the D0CK8 protein forms a full-length human D0CK8 protein, wherein the first fragment of the D0CK8 protein and the second fragment of the DOCK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), and wherein said first nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the first fragment of DOCK8 and said second nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the C-intein, wherein optionally said promoter comprises EFS. In a related aspect, the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 25, or the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 37.

[0007] In another related aspect, the first nucleic acid encoding a first fragment of a human DOCK8 is codon-optimized and wherein said second nucleic acid encoding a second fragment of the human DOCK8 protein is codon-optimized.

[0008] In another related aspect, the second fragment of the DOCK8 protein comprise an extein linker where said linker comprises C-F or C-F-N. In a further related aspect, the N- intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence, wherein optionally said nucleotide sequence encoding said CfaN amino acid sequence and said nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell. In yet another further related aspect, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and theamino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23 or SEQ ID NO: 27; or the amino acid sequence of said first fragment of the human D0CK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42.

[0009] In another related aspect of the system, the first nucleic acid is comprised in a first lentiviral vector (first LV) and the second nucleic acid is comprised in a second lentiviral vector (second LV). In a further related aspect, the first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors. Still another further related aspect, the first nucleic acid further comprises a WPRE element located 3' of said nucleic acid encoding the N-intein and said second nucleic acid further comprises a WPRE element located 3 ' of said nucleic acid encoding the second fragment of DOCK8 protein.

[0010] In another related aspect of the system, the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30.

[0011] In yet another related aspect of the system, the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39.

[0012] Disclosed herein in one aspect is a pair of recombinant lentiviral vectors (LVs) comprising: a first LV comprising a first nucleic acid encoding a first fragment of a human DOCK8 protein fused at the C termina to an N-intein; and a second LV comprising a second nucleic acid encoding a second fragment of the human DOCK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the DOCK8 protein ligated to the second fragment of the DOCK8 protein forms a full-length DOCK8 protein, wherein the first fragment of the DOCK8 protein and the second fragment of the DOCK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), andwherein said first nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the first fragment of D0CK8 and said second nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the C-intein.

[0013] In a related aspect to the pair of LVs, the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 25, or the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 37. In another related aspect to the pair of LVs, the first nucleic acid encoding a first fragment of a human DOCK8 is codon-optimized and wherein said second nucleic acid encoding a second fragment of the human DOCK8 protein is codon-optimized.

[0014] In a related aspect to the pair of LVs, the second fragment of the DOCK8 protein comprise an extein linker where said linker comprises C-F or C-F-N. In a further related aspect, the N-intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence, wherein optionally said nucleotide sequence encoding said CfaN amino acid sequence and said nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell. In yet another further related aspect, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23 or SEQ ID NO: 27; or the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42.

[0015] In another related aspect of the pair of LVs, the first nucleic acid further comprises a WPRE element located 3' of said nucleic acid encoding the N-intein and said second nucleic acid further comprises a WPRE element located 3 ' of said nucleic acid encoding the second fragment of DOCK8 protein.

[0016] In still another related aspect of the pair of LVs, the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30. In yet another related aspectof the pair of LVs, the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39. In a further related aspect of the pair of LVs, the first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors.

[0017] In one aspect, disclosed herein is a host cell transduced with the pair of lentiviral vectors according to another of the aspects disclosed herein. In a related aspect, the cell is a stem cell derived from bone marrow, umbilical cord blood, or from peripheral blood, or any combination thereof, or is a hematopoietic progenitor cell. In another related aspect, the human hematopoietic progenitor cell is a CD34+ cell.

[0018] In one aspect, disclosed herein is method of treating a DOCK8 deficiency in a human subject, said method comprising: transducing a stem cell and / or progenitor cell from said subject with a pair of lentiviral vectors according to claim 12 to produce transduced cells expressing functional DOCK8 protein; and transplanting said transduced cell or cells derived therefrom into said subject, where said cells therefrom express functional DOCK8 protein. In a related aspect, the DOCK8 protein is constitutively expressed. In another related aspect, the cell is a stem cell or human hematopoietic progenitor cell or a combination thereof. In a further related aspect, the stem cell is derived from bone marrow. In yet another further related aspect, the human hematopoietic progenitor cell is a CD34+ cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The subject matter regarded as the system for expressing DOCK8 protein in a cell and methods of use thereof, is particularly pointed out and distinctly claimed in the concluding portion of the specification. The system for expressing DOCK8 protein in a cell and methods of use thereof, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0020] Figures 1A-1D. Figure 1A shows a schematic of the two half-DOCK8 cDNAs integrating into the genome of a peripheral blood stem cell (PBSC). P / P indicates Primer / Probe sites. E / P indicates an upstream enhancer / promotor. pA is a polyA sequence. Figure IB shows percent (%) integration as measured by the different probes. Figure 1Cshows viability of the transduced PBSC. Figure ID shows proliferation (Fold Expansion) of the transduced PBSC.

[0021] Figure 2. DOCK8 lentiviral vectors include an internal EFS promoter driving expression of two codon-optimized versions of the therapeutic gene cassette with and without WPRE. Codon optimization was performed using either GeneArt or GenScript methods. The nucleotide sequence of the vector comprising EFS-GeneArtDOCK8-WPRE is set forth in SEQ ID NO: 4.

[0022] Figures 3A-3B. DOCK8 lentiviral vectors were tested in Jurkat T cells. Figure 3A. GeneArt codon optimization of the DOCK8 cDNA resulted in restoration of DOCK8 expression. Y axis represents Count. X-axis represent Dock8. Figure 3B. Inclusion of the WPRE sequence improved DOCK8 expression at similar vector copy number (VCN) when measured by flow cytometry.

[0023] Figures 4A and 4B. The VCN of PBSC transduced with EFS-GeneArtDOCK8- WPRE at le7 TU / ml (Figure 4A) or the VCN of PBSC transduced with EFS- GeneArtDOCK8-WPRE at various TU / mL (Figure 4B), alone or in combination with various transduction enhancers. Cells are typically transduced after one day pre-stimulation, except samples labeled as day 2, which were transduced after 2 days pre-stimulation. 2hit samples refer to those which were transduced twice, after one and two days pre-stimulation. Results are from 5 different PBSC donors mixed together.

[0024] Figures 5A-5B. The beta-can shape of the GFP protein is shown in Figure 5A. The split site (red line) for GFP was chosen at one of the short helical segments at the ends of the beta-sheet cylinder. Figure 5B. Schematic of GFP-CfaN and CfaC-GFP (C-F) expression plasmids.

[0025] Figure 6. K562 cells electroporated with the CfaN or CfaC plasmids alone do not express GFP. (top row) Cells electroporated with both plasmids express high levels of GFP. Both CFN and CF adjacent extein sequences appear to allow equal levels of protein splicing.

[0026] Figure 7. DOCK8 protein structure (top) and two DHR domains important for protein function (bottom) from Biggs et al. (2017) DOCK8 deficiency: Insights into pathophysiology, clinical features and management. Clin Immunol. 181:75-82.

[0027] Figure 8. Two different split sites in the DOCK8 protein were tested at amino acid positions 939 and 1267. The split site is just before the 939thamino acid of DOCK8 (939 plasmid vectors) or split site for this cassette is just after the 1267th amino acid of DOCK8(1267 plasmid vectors).

[0028] Each plasmid vector of paired plasmid vectors {See for example Figures 12-20) comprises nucleic acid sequences encoding either the N-terminal or C-terminal half of DOCK8. A skilled artisan would recognize that the term “half’ is used to denote either a N- terminal (59 half of DOCK8 protein (nucleic acid) or a C-terminal (39 half of DOCK8 protein (nucleic acid). The term half is not meant to infer or define a quantity of the D0CK8 protein or nucleotide sequence as being exactly half (50%). While the two halves when spliced together form a full-length DOCK8, the term “half’ is employed to denote a portion of the protein or nucleotide sequence encoding the protein. The split sites in these 939 or 1267 vectors are as described here for Figure 8.

[0029] Figure 9. Immunoblots demonstrating restoration of DOCK8 protein expression in Jurkat T cells treated with a combination of the CfaN and CfaC intein / DOCK8 plasmids. Untreated Jurkat T cells do not express DOCK8 at baseline, and cells treated with only one of the intein plasmids does not express detectable DOCK8 protein. Positive controls include primary T cells from healthy individuals and K562 cell lines.

[0030] Figure 10. Map of AAV donor sequences for sequential editing approach left (59 side. pFb-e3g4-L-shortHA (8922 bp).

[0031] Figure 11. Map of AAV donor sequences for sequential editing approach right (39 side pAAV-e3g4-R-flag-shortHA (7440 bp).

[0032] Figure 12. Map of lentiviral plasmid sequence for gADOCK8-WPRE Lentiviral approach. mCCL-EFS-Dock8ga-WPRE (13207 bp).

[0033] Figure 13. Map of D0CK8 939 CfaN Intein plasmid sequence. 939 DOCK8- CfaN_2 (7253 bp).

[0034] Figure 14. Map of DOCK8 939 CfaN Intein Lentiviral plasmid sequence. mCCL-EFS-939Dock8-CfaN-WPRE (9959 bp).

[0035] Figure 15. Map of DOCK8 939 CfaC-CF Intein plasmid sequence. 939 CfaC- CF-DOCK8-bGHpA (7730 bp).

[0036] Figure 16. Map of DOCK8 939 CfaC-CF Intein Lentiviral plasmid sequence. mCCL-EFS-939Dock8-CfaC-CF-WPRE (10436 bp).

[0037] Figure 17. Map of DOCK8 939 CfaC-CFN Intein plasmid sequence. 939 CfaC- CFN-DOCK8-bGHpA (7733 bp).

[0038] Figure 18. Map of DOCK8 1267 CfaN Intein Lentiviral plasmid sequence.mCCL-EFS-1267Dock8-CfaN-WPRE (10946 bp).

[0039] Figure 19. Map of D0CK8 1267 CfaC-CFN plasmid sequence. 1267 CfaC- CFN-D0CK8-bGHpA (6743 bp).

[0040] Figure 20. Map of D0CK8 1267 CfaC-CFN Lentiviral plasmid sequence. mCCL-EFS-1267Dock8-CfaC-CFN-WPRE (9449 bp).

[0041] Figure 21. V ector Copy Number (VCN) of Jurkat cells 2 weeks post transduction with intein Lentiviral vectors.

[0042] Figures 22A and 22B. VCN and Expression of D0CK8 was analyzed in Jurkat cells transduced with different doses of intein Lentivirus 2 weeks post transduction.

[0043] Figure 23. Western Blot of transduced Jurkat cells demonstrates D0CK8 expression.

[0044] Figures 24A and 24B. Intein Lentiviral vectors in human Peripheral Blood Stem Cells (PBSC).

[0045] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.DETAILED DESCRIPTION

[0046] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the system for expressing DOCKS protein in a stem and or progenitor cell and methods of use thereof. However, it will be understood by those skilled in the art that the systems and vectors comprising the DOCK8 protein and the methods of use thereof may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the DOCK8 system and vectors.DOCK8 Protein and Systems for Expressing a human DOCK8 protein

[0047] In humans, the Dedicator Of Cytokinesis 8 (DOCK8) gene encodes a DOCK8 protein that is a guanine nucleotide releasing factor involved in intracellular signaling networks. DOCK8 protein (2099 AA; -190 kDa) is exceptionally large, which can lead tochallenges for heterologous expression as part of a gene therapy regime.

[0048] In some embodiments, disclosed herein is a system for expressing human DOCK8 protein in a cell, wherein the system comprises: a first nucleic acid encoding a first fragment of a human DOCK8 protein fused at the C terminal to an N-intein; and a second nucleic acid encoding a second fragment of the human DOCK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the DOCK8 protein ligated to the second fragment of the DOCK8 protein forms a full-length human DOCK8 protein, wherein the first fragment of the D0CK8 protein and the second fragment of the D0CK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), and wherein said first nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the first fragment of DOCK8 and said second nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the C-intein.

[0049] One skilled in the art would appreciate that it is important to choose split sites that do not appear to contribute to intra-protein interactions because the DOCK8 extein that is fused to the CfaC intein requires a CF or CFN linker that will be incorporated into the resulting DOCK8 protein. The split sites of 939 and 1267 meet this criteria. The 939 split site is just before the 939thamino acid of DOCK8 (939 plasmid vectors). The 1267 split site for cassette is just after the 1267thamino acid of DOCK8 (1267 plasmid vectors).

[0050] In some embodiments, the promoter comprises a constitutive promoter . In some embodiments, said constitutive promoter comprises an EFS promoter, a PGK (phosphoglycerate kinase 1) promoter, a CMV (cytomegalovirus immediate) promoter, a MND promoter (synthetic promoter containing U3 region of modified Maloney murine leukemia virus and enhancer from myeloproliferative sarcoma virus), an endogenous DOCK8 promoter, or a EF-1 alpha promoter (human eukaryotic translation elongation factor 1 alpha 1). In some embodiments, said constitutive promoter comprises an EFS promoter. While in some embodiments, the shortened EF-1 alpha promoter is used in the Examples below, a skilled artisan would recognize that the full length version of the EF-1 alphapromoter could also be used.

[0051] In some embodiments, a system comprises a first nucleic acid and a second nucleic acid, each encoding a fragment of human DOCK8 protein, wherein the two fragments when ligated together form a full-length human DOCK8 protein. In some embodiments of a system, each nucleic acid is comprised in a vector.

[0052] In some embodiments, the first fragment comprises the DOCK homology region 1 (DHR1) and the second fragment comprises the DOCK homology region 2 (DHR2).

[0053] In some embodiments of a system, the first and second nucleic acids may integrate into a host cell genome, and once integrated express the full-length DOCK8 protein. In some embodiments, expression is constitutive. In some embodiments, the expressed full- length DOCK8 protein promotes STAT3 phosphorylation and translocation to the nucleus.

[0054] The term “first fragment of DOCK8” and grammatical variations thereof, may be used interchangeably with the term “first half’ or “first half of DOCK8” and grammatical variations thereof, having all the same meanings and qualities. Similarly, the term “second fragment of DOCK8” and grammatical variations thereof, many be used interchangeably with the term “second half’ or “second half of DOCK8” and grammatical variations thereof, having all the same meanings and qualities.

[0055] A skilled artisan would recognize that the term “half’ as used herein, refers to a portion or a part of DOCK8, wherein a first and second half of DOCK8 together form a full- length DOCK8 protein or encode a full-length DOCK8 protein, although each half is not defined as being 50% of DOCK8. For example, in certain embodiments, a first half of a DOCK8 protein comprises the protein up until the amino acid before position 939. The split site is just before the 939thamino acid of DOCK8 so the DOCK8 cDNA for this sequence ends at the codon encoding amino acid 938. In that same embodiment, the second half includes amino acid 939 of DOCK8 until the end of the DOCK8 protein sequence. These halves are termed “939DOCK8” first half and second half. Similarly, in certain embodiments, a first half of a DOCK8 protein comprises the protein up until and including amino acid 1267. The split site for this cassette is just after the 1267th amino acid of DOCK8. In that same embodiment, the second half includes amino acid 1268 of DOCK8 through to the end of the DOCK8 protein sequence. These halves are termed “1267DOCK8” first half and second half.

[0056] In some embodiments, a first nucleic acid and a second nucleic acid each comprisea promoter to drive the expression of the encoded D0CK8 fragment. In certain embodiments, the promoter comprises a constitutive human promoter. In certain embodiments, the promoter comprises an elongation factor la (EF-l ) promoter. In some embodiments, the promoter comprises an EF-l Short (EFS) intron-less promoter.

[0057] In some embodiments, a first nucleic acid encodes a first fragment of a human DOCK8 protein fused at the C terminal to an N-intein. In some embodiments, the N-intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence, wherein optionally said nucleotide sequence encoding said CfaN amino acid sequence and said nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell. In some embodiments, the N-intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence. In some embodiments, the nucleotide sequence encoding the CfaN amino acid sequence and the nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell. In some embodiments, a codon-optimized CfaN nucleotide sequence is set forth in SEQ ID NO: 16. In some embodiments, a CfaN amino acid sequence is set forth in SEQ ID NO: 19. In some embodiments, a codon-optimized CfaC nucleotide sequence is set forth in SEQ ID NO: 21. In some embodiments, a CfaC amino acid sequence is set forth in SEQ ID NO: 24.

[0058] In some embodiments, a first nucleic acid encodes a first half of DOCK8 fused at the C terminal to an CfaN. In some embodiments, a first nucleic acid encodes a first half of 939DOCK8 fused at the C terminal to an CfaN. In some embodiments, a first nucleic acid encodes a first half of 1267DOCK8 fused at the C terminal to an CfaN.

[0059] In some embodiments, a first nucleic acid encodes a first fragment of a human DOCK8 protein fused at the C terminal to an N-intein. In some embodiments, a first nucleic acid encodes a first half of DOCK8 fused at the C terminal to an CfaN. In some embodiments, a first nucleic acid encodes a first half of 939DOCK8 fused at the C terminal to an CfaN. In some embodiments, a first nucleic acid encodes a first half of 1267DOCK8 fused at the C terminal to an CfaN.

[0060] In some embodiments, a second nucleic acid encodes a second fragment of the human DOCK8 protein fused at the N terminal with a C-intein. In some embodiments, a second nucleic acid encodes a second half of DOCK8 fused at the N terminal with CfaC. Insome embodiments, a second half of D0CK8 protein comprises an extein linker. In some embodiments, the linker comprises amino acids Cysteine-Phenylalanine (C-F) or Cysteine- Phenylalanine- Asparagine (C-F-N). In some embodiments, a second nucleic acid encodes a second half of 939DOCK8 fused at the N terminal to C-F-CfaC. In some embodiments, a second nucleic acid encodes a second half of 939DOCK8 fused at the N terminal to C-F-N- CfaC. In some embodiments, a second nucleic acid encodes a second half of 1267DOCK8 fused at the N terminal to C-F-N-CfaC, wherein the N residue is part of the 1267DOCK8 second half amino acid sequence.

[0061] In some embodiments, the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 25, or the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 37. In some embodiments, the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 25. In some embodiments, the amino acid sequence of said first fragment of DOCK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of DOCK8 is set forth in SEQ ID NO: 37.

[0062] In some embodiments, the nucleotide sequence encoding the DOCK8 protein is codon-optimized for translation in humans. In some embodiments, the nucleotide sequence encoding a first fragment of DOCK8 is codon-optimized. In some embodiments, the nucleotide sequence encoding a second fragment of DOCK8 is codon-optimized. In some embodiments, the first nucleic acid encoding a first fragment of a human DOCK8 is codon- optimized for translation in humans and the second nucleic acid encoding a second fragment of the human DOCK8 protein is codon-optimized for translation in humans.

[0063] In some embodiments, the optimized nucleic acid sequence encoding said first fragment of DOCK8 is set forth in SEQ ID NO: 15 and the optimized nucleic acid sequence encoding said second fragment of DOCK8 is set forth in SEQ ID NO: 22, or the optimized nucleic acid sequence encoding first fragment of DOCK8 is set forth in SEQ ID NO: 32 and the optimized nucleic acid sequence encoding said second fragment of DOCK8 is set forth in SEQ ID NO: 36. In some embodiments, the optimized nucleic acid sequence encoding said first fragment of DOCK8 is set forth in SEQ ID NO: 15 and the optimized nucleic acidsequence encoding second fragment of D0CK8 is set forth in SEQ ID NO: 22. In some embodiments, the optimized nucleic acid sequence encoding said first fragment of D0CK8 is set forth in SEQ ID NO: 32 and the optimized nucleic acid sequence encoding said second fragment of D0CK8 is set forth in SEQ ID NO: 36.

[0064] In some embodiments, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23 or SEQ ID NO: 27; or the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42. In some embodiments, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23. In some embodiments, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 27. In some embodiments, the amino acid sequence of said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42.

[0065] In some embodiments, the nucleic acid sequence encoding said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 14 and the nucleic acid sequence encoding said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 20 or SEQ ID NO: 26; or the nucleic acid sequence encoding said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 31 and the nucleic acid sequence encoding said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 35. In some embodiments, the nucleic acid sequence encoding said first fragment of the human DOCK8 protein fused at the Cterminal to an N-intein is set forth in SEQ ID NO: 14 and the nucleic acid sequence encoding said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 20. In some embodiments, the nucleic acid sequence encoding said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 14 and the nucleic acid sequence encoding said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO:26. In some embodiments, the nucleic acid sequence encoding said first fragment of the human DOCK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 31 and the nucleic acid sequence encoding said second fragment of the human DOCK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 35.

[0066] In some embodiments, the cell in which the system for expressing DOCK8 is a human host cell. In some embodiments, the human host cell comprises a stem cell. In some embodiments, a human stem cell is derived from bone marrow, umbilical cord, or peripheral blood, or any combination thereof. In some embodiments, a human stem cell is derived from bone marrow. In some embodiments, a human stem cell is derived from umbilical cord. In some embodiments, a human stem cell is derived from peripheral blood. In some embodiments, a human stem cell is derived from bone marrow, umbilical cord, and peripheral blood. In some embodiments, the human host cell comprises a human hematopoietic progenitor cell. In some embodiments, a human hematopoietic progenitor cell comprises a CD34+cell. In some embodiments, a human host cell comprises a combination of stem cells and hematopoietic progenitor cells, which may be referred to as hematopoietic stem and progenitor cells (HSPC).

[0067] In certain embodiments, a first nucleic acid is comprised in a first lend viral vector (first LV) and a second nucleic acid is comprised in a second lentiviral vector (second LV). In some embodiments, the first LV and the second LV are each TAT-independent and selfinactivating (SIN) lentiviral vectors. A skilled artisan would appreciate that TAT- independent and self-inactivating (SIN) lentiviral vectors and their use are well known in the art, for example but not limited to the disclosure present in Kohn et al., Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency. N Engl J Med. 2021 May 27;384(21):2002-2013; Cowan et al. Lentiviral Gene Therapy for Artemis-Deficient SCID. N Engl J Med. 2022 Dec 22;387(25):2344-2355; and Poletti and Mavilio Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases. Viruses. 2021 Aug 2; 13(8): 1526.Similarly, the skilled artisan would appreciate and know how to comprise nucleic acid sequences, for example a first nucleic acid sequence or a second nucleic acid sequence in a TAT-independent and self-inactivating (SIN) lenti viral vector based on knowledge in the art.

[0068] In some embodiments, a first nucleic acid comprised in a first LV further comprises a WPRE element located 3' of said nucleic acid encoding the N-intein and s second nucleic acid comprised in a second LV further comprises a WPRE element located 3 ' of said nucleic acid encoding the second fragment of DOCK8 protein.

[0069] In some embodiments of a system, the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30, or the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39. In some embodiments of a system, the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30. In some embodiments of a system, the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29. In some embodiments of a system, the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 30. In some embodiments of a system, the nucleic acid sequence of a first nucleic acid comprised in a first LV is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39.Lentiviral Vectors comprising a System for expressing human DOCK8

[0070] In some embodiments, disclosed herein is a pair of recombinant lentiviral vectors (LVs) comprising: a first LV comprising a first nucleic acid encoding a first fragment of a human DOCK8 protein fused at the C termina to an N-intein; and a second LV comprising a second nucleic acid encoding a second fragment of the human DOCK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the DOCK8 protein ligated to the second fragment of the DOCK8 protein forms a full-length DOCK8 protein,wherein the first fragment of the D0CK8 protein and the second fragment of the D0CK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), and wherein said first nucleic acid further comprises a promoter located 5 ' of said nucleic acid encoding the first fragment of D0CK8 and said second nucleic acid further comprises a promoter located 5 ' of said nucleic acid encoding the C-intein.

[0071] In certain embodiments, said first LV and said second LV are each TAT- independent and self-inactivating (SIN) lend viral vectors. The elements and amino acid and nucleic sequences have been described in detail above (DOCK8 Protein and Systems for Expressing a human DOCK8 protein).

[0072] In some embodiments of a pair of recombinant LVs, the nucleic acid sequence of a first LV is set forth in SEQ ID NO: 6 and the nucleic acid sequence of a second LV is set forth in SEQ ID NO: 8. In some embodiments of a pair of recombinant LVs, the nucleic acid sequence of a first LV is set forth in SEQ ID NO: 11 and the nucleic acid sequence of a second LV is set forth in SEQ ID NO: 13. One skilled in the art would appreciate that the LV backbones comprised in the LV vectors set forth in SEQ ID NO: 6, 8, 11, and 13, would be interchangeable with other TAT-independent and self-inactivating (SIN) LV vector backbones, and would further understand how to build such LV vectors based on knowledge in the art and the disclosure herein of at least the DOCK8 and Intein components.Host cells

[0073] In some embodiments, a host cell may be transduced with the pair of lenti viral vectors (LVs) as described herein. In some embodiments, a host cell is transduced using methods well known in the art.

[0074] In some embodiments, a host cell is a human host cell. In some embodiments, a host cell comprises any of the immune cells such as lymphocytes, neutrophils, dendritic cells, monocytes, etc. In some embodiments, the transduction is in vitro. In some embodiments, the transduced host cell expresses a full-length DOCK8 protein. In some embodiments, the transduced host cell expresses a full-length, functional DOCK8 protein. In some embodiments, the transduced host cell expresses a full-length, functional DOCK8 proteinconstitutively.

[0075] In some embodiments, the human host cell comprises a stem cell. In some embodiments, a human stem cell is derived from bone marrow, umbilical cord, or peripheral blood, or any combination thereof. In some embodiments, a human stem cell is derived from bone marrow. In some embodiments, a human stem cell is derived from umbilical cord. In some embodiments, a human stem cell is derived from peripheral blood. In some embodiments, a human stem cell is derived from bone marrow, umbilical cord, and peripheral blood. In some embodiments, the human host cell comprises a human hematopoietic progenitor cell. In some embodiments, a human hematopoietic progenitor cell comprises a CD34+cell. In some embodiments, a human host cell comprises a combination of stem cells and hematopoietic progenitor cells, which may be referred to as hematopoietic stem and progenitor cells (HSPC).

[0076] In some embodiments, the pair of LVs comprises a system for expressing human DOCK8 protein in a cell, wherein the system comprises: a first nucleic acid encoding a first fragment of a human DOCK8 protein fused at the C terminal to an N-intein; and a second nucleic acid encoding a second fragment of the human DOCK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the DOCK8 protein ligated to the second fragment of the DOCK8 protein forms a full-length human DOCK8 protein, as described in detail above (DOCK8 Protein ancl Systems for Expressing a human DOCK8 protein).Treating DOCK8 Deficiency

[0077] The are multiple known patient mutations in DOCK8 gene that lead to DOCK8 deficiency (See, Figure 1 in Ravendran el al. CRISPR / Cas-Based Gene Editing Strategies for DOCK8 Immunodeficiency Syndrome. Front Genome Ed. 2022 Mar 17;4:793010) and it is known in the art that DOCK8 protein is expressed primarily in hematopoietic tissues.

[0078] In some embodiments, disclosed herein is a method of treating a DOCK8 deficiency in a human subject, said method comprising: transducing a stem cell and / or progenitor cell from said subject with a pair of lentiviral vectors as described herein above (See, Sections Lentiviral Vectors comprising a System for expressing human DOCK8 & DOCK8 Protein and Systems for Expressing a human DOCK8 protein incorporated herein in their entirety) toproduce transduced cells expressing functional D0CK8 protein; and transplanting said transduced cell or cells derived therefrom into said subject, where said cells therefrom express functional D0CK8 protein.

[0079] In some embodiments, of a methods for treating a DOCK8 deficiency, wherein the DOCK8 protein is constitutively expressed in said transduced cell. In some embodiments, the constitutive expression of DOCK8 protein treats the DOCK8 deficiency. In some embodiments, the constitutive expression of DOCK8 protein improves the subject’s physical wellbeing.

[0080] In some embodiments, the stem cell and or progenitor cell is (are) obtained from said subject, and is therefore autologous with the subject. This is an advantage over known therapies in the art for DOCK8 deficiency, for example, allogeneic hematopoietic stem cell transplant (HSCT) has been used to treat DOCK8 deficiency but this method frequently results in acute and or chronic Graft-versus-host disease (GvHD). Gene therapy using autologous cells, including stem cells and hematopoietic progenitor cells that are corrected with normal DOCK8 gene expression, will have a beneficial effect on blood cell production or function without the immunologic complications of HSCT.

[0081] In some embodiments, the cell is a combination of stem cells and human hematopoietic progenitor cells. In some embodiments, the stem cell is derived from bone marrow. In some embodiments, the human hematopoietic progenitor cell is a CD34+ cell. In some embodiments, the cell is a stem cell derived from bone marrow. In some embodiments, the cell is a human hematopoietic progenitor cell, and said human hematopoietic progenitor cell is a CD34+ cell.Additional Embodiments

[0082] Embodiment 1 : A system for expressing DOCK8 protein in a cell said system comprising: a first nucleic acid encoding a first fragment of a DOCK8 protein attached at the C termina to an N-intein (In); and a second nucleic acid encoding a second fragment of a DOCK8 protein attached at the N terminal with a C-intein (Ic); where the first fragment of a DOCK8 protein ligated to the second fragment of a DOCK8 protein forms a full-length normal (wildtype) DOCK8 protein.

[0083] Embodiment 2: The system of embodiment 1, wherein the N-intein and C-inteincomprise a Cfa split intein where the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence.

[0084] Embodiment 3: The system according to any one of embodiments 1-2, wherein said the first nucleic acid encoding an N-intein and the second nucleic acid encoding said C- intein are codon optimized for expression in a human cell.

[0085] Embodiment 4: The system according to any one of embodiments 1-3, wherein the first fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions.

[0086] Embodiment 5: The system according to any one of embodiments 1-3, wherein the first fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide the split between said first fragment and said second fragment between DOCK homology region 1 (DHR1) and DOCK homology region 2 (DHR2).

[0087] Embodiment 6: The system of embodiment 5, wherein the first fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide the split between said first fragment and said second fragment before amino acid 939 or after amino acid 1267.

[0088] Embodiment 7: The system according to any one of embodiments 1-6, wherein said second fragment of a DOCK8 protein comprise an extein linker where said linker comprise C-F or C-F-N.

[0089] Embodiment 8: The system according to any one of embodiments 1-7, wherein said first nucleic acid is disposed in a first lenti viral vector (first LV) and said second nucleic acid is disposed in a second lentiviral vector (second LV).

[0090] Embodiment 9: The system of embodiment 8, wherein said first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors.

[0091] Embodiment 10: The system according to any one of embodiments 8-9, wherein said lentiviral vectors each contain a WPRE element.

[0092] Embodiment 11: A set of recombinant lentiviral vectors (LVs) for the treatment of DOCK8 deficiency, said set of vectors comprising: a first LV comprising a first nucleic acid encoding a first fragment of a DOCK8 protein attached at the C termina to an N-intein (In); and a second LV comprising a second nucleic acid encoding a second fragment of aDOCK8 protein attached at the N terminal with a C-intein (1c); where the first fragment of a D0CK8 protein ligated to the second fragment of a D0CK8 protein forms a full-length normal (wildtype) D0CK8 protein.

[0093] Embodiment 12: The set of lentiviral vectors of embodiment 11, wherein the N- intein and C-intein comprise a Cfa split intein where the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence.

[0094] Embodiment 13: The set of lentiviral vectors according to any one of embodiments 11-12, wherein said the first nucleic acid encoding an N-intein and the second nucleic acid encoding said C-intein are codon optimized for expression in a human cell.

[0095] Embodiment 14: The set of lentiviral vectors according to any one of embodiments 11-13, wherein the first fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions.

[0096] Embodiment 15: The set of lentiviral vectors according to any one of embodiments 11 -13, wherein thefirst fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide the split between said first fragment and said second fragment between DOCK homology region 1 (DHR1) and DOCK homology region 2 (DHR2).

[0097] Embodiment 16: The set of lentiviral vectors of embodiment 15, wherein the first fragment of a DOCK8 protein and the second fragment of a DOCK8 protein are selected to provide the split between said first fragment and said second fragment is before amino acid 939 or after amino acid 1267.

[0098] Embodiment 17: The set of lentiviral vectors according to any one of embodiments 11-16, wherein said second fragment of a DOCK8 protein comprise an extein linker where said linker comprise C-F or C-F-N.

[0099] Embodiment 18: The set of lentiviral vectors according to any one of embodiments 11-17, wherein said first nucleic acid is disposed in a first lentiviral vector (first LV) and said second nucleic acid is disposed in a second lentiviral vector (second LV).[000100] Embodiment 19: The set of lentiviral vectors of embodiment 18, wherein said first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors.[000101] Embodiment 20: The set of lentiviral vectors according to any one ofembodiments 18-19, wherein said lentiviral vectors each contain a WPRE element.[000102] Embodiment 21 : A host cell transduced with a set of lentiviral vectors according to any one of embodiments 11-20.[000103] Embodiment 22: The host cell of embodiment 21, wherein the cell is a stem cell. [000104] Embodiment 23: The host cell of embodiment 22, wherein said cell is a stem cell derived from bone marrow, and / or from umbilical cord blood, and / or from peripheral blood. [000105] Embodiment 24: The host cell of embodiment 21 , wherein the cell is a 293T cell. [000106] Embodiment 25: The host cell of embodiment 21, wherein the cell is a human hematopoietic progenitor cell.[000107] Embodiment 26: The host cell of embodiment 25, wherein the human hematopoietic progenitor cell is a CD34+ cell.[000108] Embodiment 27: A method of treating a DOCK8 deficiency in a subject, said method comprising: transducing a stem cell and / or progenitor cell from said subject with a system according to any one of embodiments 1 -7 to produce transduced cells; and transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express functional DOCK8 protein.[000109] Embodiment 28: The method of embodiment 27, wherein the expression of DOCK8 protein recapitulates in vivo the expression pattern of the wildtype DOCK8 protein. [000110] Embodiment 29: The method according to any one of embodiments 27-28, wherein said transducing comprises introducing a set of lentiviral vectors according to any one of embodiments 11 -20.[000111] Embodiment 30: The method according to any one of embodiments 27-29, wherein the cell is a stem cell.[000112] Embodiment 31 : The method of embodiment 30, wherein said cell is a stem cell derived from bone marrow.[000113] Embodiment 32: The method according to any one of embodiments 27-29, wherein the cell is a human hematopoietic progenitor cell.[000114] Embodiment 33: The method of embodiment 32, wherein the human hematopoietic progenitor cell is a CD34+ cell.[000115] Embodiment 34: A method of introducing a nucleic acid that encodes a normal (wildtype) full length DOC8 protein into a cell, said method comprising:- using a programmable nuclease to perform targeted genome editing to introduce a first corrective nucleic acid that encodes a first fragment of a normal (non-mutated) D0CK8 protein; and using a programmable nuclease to perform targeted genome editing to introduce a second corrective nucleic acid that encodes a second fragment of a normal (nonmutated) D0CK8 protein; wherein said second corrective nucleic acid is inserted adjacent to said first corrective nucleic acid and said first corrective nucleic acid combined with said second corrective nucleic acid encode a full length normal D0CK8 protein.[000116] Embodiment 35: The method of embodiment 34, wherein said programmable nuclease comprise a nuclease selected from the group consisting of a zinc finger nuclease (ZFN), a transcription activator like effector nuclease (TALEN), and a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 nuclease.[000117] Embodiment 36: The method of embodiment 35, wherein said programmable nuclease comprises a CRISPR / Cas9 nuclease.[000118] Embodiment 37: The method according to any one of embodiments 34-36, wherein introduction of said first corrective nucleic acid into said cell is by the use of a first AAV vector and introduction of said second corrective nucleic acid into said cell is by the use of a second AAV vector.[000119] Embodiment 38: The method of embodiment 37, wherein said first AAV vector and said second AAV vector are independently selected from the group consisting of AAV 1 , AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9.[000120] Embodiment 39: The method of embodiment 38 wherein said first AAV vector and said second AAV vector are AAV6 vectors.[000121] Embodiment 40: The method according to any one of embodiments 34-39, wherein said first corrective nucleic acid and said second corrective nucleic acid are codon optimized.[000122] Embodiment 41: The method according to any one of embodiments 34-40, wherein said cell is a cell derived from a subject that has a DOCK8 mutation.[000123] Embodiment 42: The method of embodiment 41, wherein said cell is a hematopoietic stem cell.[000124] Embodiment 43: The method of embodiment 41, wherein said cell is ahematopoietic cell precursor.[000125] Embodiment 44: The method according to any one of embodiments 34-43, wherein said first corrective nucleic acid and said second corrective nucleic acid are inserted downstream of the endogenous DOCK8 promoter in said cell.[000126] Embodiment 45: The method of embodiment 44, wherein said first corrective nucleic acid and said second corrective nucleic acid are inserted between the promoter and exonl of the endogenous DOCK8 gene in said cell.[000127] Embodiment 46: A method of treating a subject that has one or more DOCK8 mutations, said method comprising:! using a method according to any one of embodiments 34-45 transducing a stem cell and / or progenitor cell obtained from said subject with a nucleic acid that encodes a normal (wildtype) full length DOC8 protein to produce transduced cells; and transplanting said transduced cell or cells derived therefrom into said subject where said cells or derivatives therefrom express a normal (wildtype) full length DOC8 protein.[000128] Embodiment 47: The method of embodiment 46, wherein the expression of said full length DOC8 protein recapitulates in vivo the expression pattern of the wildtype DOCK8 gene.[000129] Embodiment 48: The method according to any one of embodiments 46-47, wherein the cell is a stem cell.[000130] Embodiment 49: The host cell of embodiment 48, wherein said cell is a stem cell derived from bone marrow.[000131] Embodiment 50: The method according to any one of embodiments 46-47, wherein the cell is a human hematopoietic progenitor cell.[000132] Embodiment 51: The method of embodiment 50, wherein the human hematopoietic progenitor cell is a CD34+ cell.EXAMPLESExample 1: Integration of DOCK8 cDNAs in Stem Cells Using CRISPR / Cas9 Editing[000133] Objective: To develop a human stem cell transfer method for the treatment of DOCK8 deficiency using CRISPR / Cas9 editing of human hematopoietic stem cells.Methods:[000134] sgRNA and AAV6 donor vector sequences[000135] sgRNA e3g4: uccacagggucauaaaacug (SEQ ID NO: 1)[000136] pFb-e3g4-L-shortHA (Figure 10; GeneART sequence optimized DOCK8 fragment is from nucleotides 4488-7967) : gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttt tcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttg atttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacg tttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatg agacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttac atcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtact caccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcg gccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttc tgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgg ggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgct gagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtag aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtag ccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtg gcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccga agggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgga aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaa gcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcagaccagccgcgtaacctggcaaaatcggttacggttgagtaataaatggatgccctgcgtaagcgggtgtgggcggacaataaagtcttaaactgaacaaaatagatctaaac tatgacaataaagtcttaaactagacagaatagttgtaaactgaaatcagtccagttatgctgtgaaaaagcatactggacttttgttatg gctaaagcaaactcttcattttctgaagtgcaaattgcccgtcgtattaaagaggggcgtggccaagggcatggtaaagactatattc gcggcgttgtgacaatttaccgaacaactccgcggccgggaagccgatctcggcttgaacgaattgttaggtggcggtacttgggt cgatatcaaagtgcatcacttcttcccgtatgcccaactttgtatagagagccactgcgggatcgtcaccgtaatctgcttgcacgtag atcacataagcaccaagcgcgttggcctcatgcttgaggagattgatgagcgcggtggcaatgccctgcctccggtgctcgccgg agactgcgagatcatagatatagatctcactacgcggctgctcaaacctgggcagaacgtaagccgcgagagcgccaacaaccg cttcttggtcgaaggcagcaagcgcgatgaatgtcttactacggagcaagttcccgaggtaatcggagtccggctgatgttgggag taggtggctacgtctccgaactcacgaccgaaaagatcaagagcagcccgcatggatttgacttggtcagggccgagcctacatgt gcgaatgatgcccatacttgagccacctaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctccataacatcaaac atcgacccacggcgtaacgcgcttgctgcttggatgcccgaggcatagactgtacaaaaaaacagtcataacaagccatgaaaac cgccactgcgccgttaccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagtttacga accgaacaggcttatgtcaactgggttcgtgccttcatccglttccacggtgtgcgtcacccggcaaccttgggcagcagcgaagtc gaggcatttctgtcctggctggcgaacgagcgcaaggtttcggtctccacgcatcgtcaggcattggcggccttgctgttcttctacg gcaaggtgctgtgcacggatctgccctggcttcaggagatcggaagacctcggccgtcgcggcgcttgccggtggtgctgaccc cggatgaagtggttcgcatcctcggttttctggaaggcgagcatcgtttgttcgcccaggactctagctatagttctagtggttggcta cagcttgcatgcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttg gtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcg tactagtTCTAGAggaaaattgtctccgttccctagttcactgtatttgggtaaacaaaacatggcctcggagagcctttgcggg agaaattataaaaattcagcaaagctttgtgatctcattttggatctcctcaggattcctcatcaagttgcaattaaggagagaaaaattc cgccttggggtgggattttaattactcatcatgttatgacttgactttctgtgctgcatttttttccatatcactacttctgtgtttgacagctcc tccaaagagtcgctccgttttatgccaggagcgaagtacttactaagtcaaaggaaagcaaggcaaacatctactattgcctttgtgc ttttaccagaaaactgggtgagaacctccttttccttttccctgcccctccagcctcaAttCtatgaccctgtggagccagtggacttt gaaggacttctgATGACCCACCTGAACAGCCTGGATGTGCAGCTGGCCCAAGAGCTG GGCGACTTCACCGACGATGATCTGGACGTGGTGTTCACCCCTAAAGAGTGCAGAACCCTGCAGCCTAGCCTGCCTGAAGAAGGCGTGGAACTGGATCCTCACGTG CGGGATTGTGTGCAGACCTACATCCGCGAGTGGCTGATCGTGAACCGGAAGA ATCAGGGCAGCCCAGAGATCTGCGGCTTCAAGAAAACCGGCAGCCGGAAGGA CTTCCACAAGACCCTGCCTAAGCAGACCTTCGAGAGCGAGACACTGGAATGC TCTGAGCCTGCTGCTCAGGCCGGACCTAGACACCTGAATGTGCTGTGTGACGT GTCCGGCAAGGGACCTGTGACCGCCTGCGATTTCGACCTGAGAAGTCTGCAGC CCGACAAGCGGCTGGAAAACCTGCTCCAGCAAGTGTCCGCCGAGGACTTCGAGAAGCAGAACGAGGAAGCCAGACGGACCAACAGACAGGCCGAGCTGTTTGCTCTGTACCCCAGCGTGGACGAGGAAGATGCCGTCGAGATTAGACCCGTGCCTGAGTGCCCAAAAGAGCACCTGGGCAACAGAATCCTGGTCAAGCTGCTGACCCTGAAGTTCGAGATCGAGATTGAGCCCCTGTTCGCCTCTATCGCCCTGTACGACGTGAAAGAGCGGAAGAAGATCAGCGAGAACTTCCACTGCGACCTGAACTCCGACCAGTTCAAGGGCTTCCTGAGAGCCCACACACCATCTGTGGCCGCTAGCTCTCAGGCCAGATCTGCCGTGTTCAGCGTGACATACCCCAGCAGCGACATCTACCTGGTGGTCAAGATCGAGAAGGTCCTGCAGCAGGGCGAGATCGGCGATTGTGCCGAGCCTTACACCGTGATCAAAGAGAGCGACGGCGGCAAGAGCAAAGAGAAGATTGAGAAGCTGAAGCTGCAGGCCGAGAGCTTCTGTCAGCGGCTGGGCAAGTACAGAATGCCCTTTGCCTGGGCTCCTATCAGCCTGTCCAGCTTCTTCAACGTGTCCACACTGGAACGGGAAGTGACCGACGTGGACTCTGTCGTGGGCAGATCTAGCGTGGGCGAGAGAAGGACACTGGCTCAGTCTAGACGGCTGAGCGAGAGAGCCCTGAGCCTGGAAGAAAATGGCGTGGGCAGCAACTTCAAGACCAGCACTCTGTCCGTGTCTAGCTTCTTTAAGCAAGAGGGCGACAGACTGAGCGACGAGGACCTGTTCAAGTTCCTGGCCGACTACAAGCGGAGCAGCTCCCTGCAGAGAAGAGTGAAGTCTATCCCTGGCCTGCTGCGCCTGGAAATCTCTACAGCCCCTGAGATCATCAACTGCTGTCTGACCCCTGAAATGCTGCCCGTGAAGCCCTTTCCAGAGAACCGGACCAGACCTCACAAAGAGATCCTGGAATTCCCCACCAGAGAAGTGTACGTGCCCCACACCGTGTACCGGAACCTGCTGTATGTGTACCCACAGCGGCTGAACTTCGTGAACAAGCTGGCCTCCGCCAGAAACATCACCATCAAGATCCAGTTTATGTGCGGCGAGGACGCCAGCAACGCCATGCCTGTGATCTTCGGCAAGTCTAGCGGCCCTGAGTTCCTGCAAGAGGTGTACACAGCCGTGACCTACCACAACAAGAGCCCCGACTTCTACGAGGAAGTGAAGATTAAGCTGCCCGCCAAGCTGACCGTGAATCACCATCTGCTGTTCACCTTCTACCACATCAGCTGCCAGCAGAAACAGGGCGCCTCTGTGGAAACACTGCTGGGCTATAGCTGGCTGCCCATCCTGCTGAACGAGAGACTGCAGACCGGCAGCTACTGTCTGCCTGTGGCTCTGGAAAAGCTGCCACCTAACTACAGCATGCACTCCGCCGAGAAGGTGCCCCTGCAGAATCCTCCTATTAAGTGGGCCGAGGGCCACAAGGGCGTGTTCAATATCGAGGTGCAGGCCGTGTCCTCCGTGCACACCCAGGATAACCACCTGGAAAAGTTCTTCACCCTGTGCCACAGCCTCGAGAGCCAAGTGACATTCCCCATCCGCGTGCTGGACCAGAAAATCTCCGAGATGGCCCTGGAACACGAGCTGAAACTGAGCATCATCTGCCTGAATAGCAGCAGACTGGAACCCCTGGTGCTGTTCCTGCATCTGGTGCTGGACAAGCTGTTCCAGCTGAGCGTGCAGCCCATGGTTATCGCCGGACAGACCGCCAACTTCAGCCAGTTCGCCTTTGAGAGCGTGGTGGCCATTGCCAACAGCCTGCACAACAGCAAGGACCTGAGCAAGGATCAGCACGGCAGAAACTGCCTGCTGGCCTCTTACGTGCACTACGTGTTCAGACTGCCTGAGGTGCAGAGGGACGTGCCAAAATCTGG CGCTCCTACCGCTCTGCTGGACCCCAGAAGCTATCACACCTACGGCAGAACAT CTGCCGCCGCTGTGTCCTCTAAACTCCTGCAGGCTAGAGTGATGTCCAGCAGC AACCCTGATCTGGCCGGAACACACAGCGCCGCTGATGAAGAAGTGAAAAACA TCATGAGCAGCAAGATCGCCGACCGGAACTGCAGCCGGATGAGCTACTACTG TAGCGGCAGCTCTGATGCCCCTAGCTCTCCAGCTGCTCCTAGACCTGCCAGCAAGAAGCACTTTCACGAGGAACTGGCCCTGCAGATGGTCGTGTCTACCGGCATGGTTCGAGAGACAGTGTTTAAGTACGCCTGGTTCTTCTTCGAACTGCTGGTCAAGAGCATGGCCCAGCACGTGCACAACATGGACAAGCGGGACAGCTTCCGGCGGACCAGATTCAGCGACAGATTCATGGACGACATCACCACCATCGTGAACGTGGTCACCAGCGAGATTGCTGCCCTGCTGGTTAAGCCCCAGAAAGAGAACGAGCAGGCCGAAAAGATGAATATCTCTCTGGCCTTCTTCCTCTACGACCTGCTGAGCCTGATGGACCGGGGCTTCGTGTTCAACCTGATCCGGCACTACTGCTCCCAGCTGTCTGCCAAGCTGTCCAACCTGCCTACACTGATCTCTATGAGACTCGAGTTCCTGAGGATCCTGTGCAGCCACGAGCACTACCTGAACCTGAATCTGTTCTTCATGAACGCCGACACAGCCCCTACAAGCCCCTGTCCTTCTATCAGCAGCCAGAACAGCAGCAGCTGTAGCAGCTTTCAGGATCAGAAAATCGCCAGCATGTTCGATCTGACCAGCGAGTACCGGCAGCAGCACTTTCTGACCGGCCTGCTGTTTACCGAACTGGCAGCCGCTCTTGATGCCGAAGGCGAGGGAATCAGCAAGGTGCAGCGGAAAGC CGTGTCTGCCATTCACAGCCTGCTGTCTAGCCACGACCTGGATCCTAGATGCG TGAAGCCTGAAGTGAAAGTGAAAATCGCTGCCCTGTACCTGCCTCTCGTGGGCATCATTCTGGATGCCCTGCCTCAGttttatgaccctgtggagccagtggactttgaaggacttctgatgacaca cctgaacagcctggatgtgcagcttgcccaggagctcggggacttcactgatgacgacttggacgtggtgttcacgccaaaggaat gtaggactttgcagccctctttgccggaggaagggtaaatagttttctaaaatgtagatgtgattgggattgtcatgattgttttcaataa gtgggtaggggagatgccttcaatctgaacttaaaaataaaataaaattactcaatccattcaaatgtgtgggacagctatatgatatc atcatgtaagtatagatagtttttaaattagtttggccagagattttgaaaagctatgtgaagactaaggGTAACCACGTGC GGACCGAGCGGCCGCaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggc cgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggtctgagacaataaccctgataaatgcttcaataatgtaagctctaggagatccgaaccagataagtgaaatctagttccaaactattttgt catttttaattttcgtattagcttacgacgctacacccagttcccatctattttgtcactcttccctaaataatccttaaaaactccatttccac ccctcccagttcccaactattttgtccgcccacagcggggcatttttcttcctgttatgtttttaatcaaacatcctgccaactccatgtga caaaccgtcatcttcggctactttttctctgtcacagaatgaaaatttttctgtcatctcttcgttattaatgtttgtaattgactgaatatcaac gcttatttgcagcctgaatggcgaatg (SEQ ID NO: 2).[000137] The pFb-3eg4-L-short HA plasmid includes a plasmid backbone (named pFB) is used for packaging the AAV. pFb-3eg4-L-short HA includes DOCK8 amino acids 1-1160. [000138] pFB-e3g4-Rflag-shortHA (Figure 11; GeneART sequence optimized DOCK8 fragment is from nucleotides 1207-4218): gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagc gcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttc cgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttt tcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttg atttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacg tttacaatttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatg agacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgc ggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttac atcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtact caccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcg gccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat cgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgc aaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttc tgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgg ggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgct gagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtag aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtag ccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtg gcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgga aaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaa gcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcagaccagccgcgtaacctggcaaaatc ggttacggttgagtaataaatggatgccctgcgtaagcgggtgtgggcggacaataaagtcttaaactgaacaaaatagatctaaac tatgacaataaagtcttaaactagacagaatagttgtaaactgaaatcagtccagttatgctgtgaaaaagcatactggacttttgttatg gctaaagcaaactcttcattttctgaagtgcaaattgcccgtcgtattaaagaggggcgtggccaagggcatggtaaagactatattc gcggcgttgtgacaatttaccgaacaactccgcggccgggaagccgatctcggcttgaacgaattgttaggtggcggtacttgggt cgatatcaaagtgcatcacttcttcccgtatgcccaactttgtatagagagccactgcgggatcgtcaccgtaatctgcttgcacgtag atcacataagcaccaagcgcgttggcctcatgcttgaggagattgatgagcgcggtggcaatgccctgcctccggtgctcgccgg agactgcgagatcatagatatagatctcactacgcggctgctcaaacctgggcagaacgtaagccgcgagagcgccaacaaccg cttcttggtcgaaggcagcaagcgcgatgaatgtcttactacggagcaagttcccgaggtaatcggagtccggctgatgttgggag taggtggctacgtctccgaactcacgaccgaaaagatcaagagcagcccgcatggatttgacttggtcagggccgagcctacatgt gcgaatgatgcccatacttgagccacctaactttgttttagggcgactgccctgctgcgtaacatcgttgctgctccataacatcaaac atcgacccacggcgtaacgcgcttgctgcttggatgcccgaggcatagactgtacaaaaaaacagtcataacaagccatgaaaac cgccactgcgccgttaccaccgctgcgttcggtcaaggttctggaccagttgcgtgagcgcatacgctacttgcattacagtttacga accgaacaggcttatgtcaactgggttcgtgccttcatccgtttccacggtgtgcgtcacccggcaaccttgggcagcagcgaagtc gaggcatttctgtcctggctggcgaacgagcgcaaggtttcggtctccacgcatcgtcaggcattggcggccttgctgttcttctacg gcaaggtgctgtgcacggatctgccctggcttcaggagatcggaagacctcggccgtcgcggcgcttgccggtggtgctgaccc cggatgaagtggttcgcatcctcggttttctggaaggcgagcatcgtttgttcgcccaggactctagctatagttctagtggttggcta cagcttgcatgcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttg gtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcctgcggccgcacgcg tactagtTCTAGAGTTCCTGAGGATCCTGTGCAGCCACGAGCACTACCTGAACCTG AATCTGTTCTTCATGAACGCCGACACAGCCCCTACAAGCCCCTGTCCTTCTATC AGCAGCCAGAACAGCAGCAGCTGTAGCAGCTTTCAGGATCAGAAAATCGCCA GCATGTTCGATCTGACCAGCGAGTACCGGCAGCAGCACTTTCTGACCGGCCTG CTGTTTACCGAACTGGCAGCCGCTCTTGATGCCGAAGGCGAGGGAATCAGCA AGGTGCAGCGGAAAGCCGTGTCTGCCATTCACAGCCTGCTGTCTAGCCACGAC CTGGATCCTAGATGCGTGAAGCCTGAAGTGAAAGTGAAAATCGCTGCCCTGT ACCTGCCTCTCGTGGGCATCATTCTGGATGCCCTGCCTCAGCTGTGTGATTTCA CCGTGGCCGACACCAGACGGTACAGAACCTCTGGCTCCGACGAGGAACAAGAAGGCGCTGGCGCCATCAACCAGAATGTGGCCCTTGCCATTGCCGGGAACAACTTCAATCTGAAAACCAGCGGCATCGTGCTGAGCAGCCTGCCTTACAAGCAGTACAACATGCTGAATGCCGACACTACCCGCAACCTGATGATCTGCTTCCTGTGGATTATGAAGAACGCCGATCAGAGCCTGATTCGGAAGTGGATTGCCGACCTGCCAAGCACTCAGCTGAACCGGATTCTGGACCTCCTGTTCATCTGCGTGCTGTGCTTCGAGTACAAGGGCAAGCAGTCCAGCGACAAGGTGTCCACTCAGGTGCTGCAGAAAAGCCGGGATGTGAAGGCCAGGCTGGAAGAGGCACTTCTGAGAGGCGAAGGCGCCAGAGGCGAGATGATGAGGCGTAGAGCCCCTGGCAACGATAGATTCCCCGGCCTGAATGAGAACCTGCGGTGGAAGAAAGAGCAGACCCATTGGAGACAGGCCAACGAGAAACTGGACAAGACCAAGGCCGAACTGGACCAAGAGGCCCTGATCTCTGGCAACCTGGCCACAGAAGCCCACCTGATTATCCTGGACATGCAAGAGAACATCATCCAGGCCAGTAGCGCCCTGGATTGCAAGGATTCTCTGCTCGGAGGCGTGCTGAGGGTGCTCGTGAATAGCCTGAATTGCGACCAGAGCACCACCTACCTGACACACTGCTTCGCCACACTGAGGGCCCTGATTGCCAAGTTTGGGGACCTGCTGTTCGAGGAAGAGGTGGAACAGTGCTTCGACCTGTGTCACCAAGTGCTGCACCACTGCAGCAGCTCCATGGACGTGACAAGATCCCAGGCCTGCGCCACTCTGTATCTGCTGATGCGGTTTAGCTTCGGCGCCACCAGCAATTTCGCCAGAGTGAAGATGCAAGTGACCATGAGCCTGGCCAGCCTCGTTGGAAGGGCCCCTGACTTCAATGAGGAACACCTCAGAAGAAGCCTGCGGACCATCCTGGCCTACAGCGAAGAGGATACCGCCATGCAGATGACCCCATTTCCAACACAAGTGGAAGAACTGCTGTGCAACCTGAATTCCATCCTGTACGATACCGTGAAGATGAGAGAGTTCCAAGAGGACCCCGAGATGCTGATGGATCTGATGTACCGGATCGCCAAGAGCTACCAGGCCTCTCCTGATCTGAGACTGACCTGGCTGCAGAACATGGCCGAGAAGCACACCAAGAAGAAGTGCTACACCGAGGCCGCCATGTGCCTGGTTCATGCTGCTGCACTCGTGGCCGAGTACCTGAGCATGCTGGAAGATCACTCCTACCTGCCAGTGGGCAGCGTGTCCTTCCAGAACATCAGCTCCAACGTCCTGGAAGAGTCCGTGGTGTCCGAGGACACACTGAGCCCAGATGAGGATGGCGTGTGTGCCGGACAGTACTTTACCGAGTCTGGCCTCGTGGGCCTTCTGGAACAGGCCGCCGAACTGTTTTCTACCGGCGGACTGTACGAGACAGTGAACGAAGTGTACAAGCTGGTCATCCCCATCCTGGAAGCCCACAGAGAGTTCAGAAAGCTGACACTGACCCACTCCAAGCTGCAGCGGGCCTTCGACAGCATTGTGAACAAGGACCACAAGCGGATGTTCGGCACCTACTTCAGAGTGGGCTTTTTCGGCAGCAAATTCGGCGACCTGGACGAGCAAGAGTTCGTGTACAAAGAGCCCGCCATCACCAAGCTGCCTGAGATCTCCCATCGGCTGGAAGCCTTCTACGGCCAGTGTTTTGGCGCCGAGTTCGTGGAAGTGATCAAGGACAGCACCCCTGTGGATAAGACCAAACTGGACCCTAACAAGGCCTACATCCAGATCACCTTCGTGGAACCCTACTTCGACGAGTACGAGATGAAGGATAGAGTGACCTACTTTGAGAAGAACTTCAACCTGCGGCGGTTCATGTACACCACACCTTTTACACTGGAAGGCAGACCCAGGGGCGAGCTGCACGAGCAGTATAGAAGAAACACCGTGCTGACCACCATGCACGCATTCCCTTACATCAAGACCCGGATCAGCGTGATCCAGAAAGAAGAGTTTGTCCTGACACCAATCGAGGTGGCCATCGAGGACATGAAGAAGAAAACCCTCCAGCTGGCCGTGGCCATCAATCAAGAACCTCCAGACGCCAAGATGCTCCAGATGGTGCTCCAGGGATCTGTGGGCGCCACAGTGAATCAGGGACCTCTGGAAGTGGCCCAAGTGTTCCTGGCTGAGATCCCCGCCGATCCTAAGCTGTACAGACACCATAACAAGCTGCGGCTCTGCTTCAAAGAATTCATCATGAGATGTGGCGAGGCCGTGGAAAAGAACAAGAGACTGATCACCGCCGACCAGAGAGAGTACCAGCAAGAGCTGAAGAAAAACTACAACAAGCTCAAAGAAAATCTGCGGCCCATGATCGAGAGAAAGATCCCCGAGCTGTACAAGCCCATCTTCCGCGTGGAAAGCCAGAAGCGGGACTCCTTTCACAGATCCAGCTTTAGGAAGTGCGAGACACAGCTGTCCCAGGGCTCTtcgagcgactacaaagaccatgacggtg attataaagatcatgacatcgattacaaggatgacgatgacaagTGAGAAAAGCCATCTTCATTCGTGGAGACTGTGGCCCTGCAACCCTGGAGAAGGACTTGCTGGTACTTAAAAAATGGGACATTTGCCACCCAGGACTGACTGTACACTCCCTGATCAGCCAGCACTCTGGAAGCTTTGGGATCCCAGGAACCATGGAATTATTCCCAAATGGACTCTGACCAGATTTTTGCCATACTGGGGGGTGGCGGGATGGAGGATGGGTACTCAGGCATGACTGCGTATTTATTAAAGTGTGTTTTTCCACAATGTACCAAACAAGGCATAAGCAGCTTCTCCTGCTGACTGGCCAATCACTGCCCATCTGAGAGATGATTTCCTCTGGCCCATATTTGAATTTATTGGAGTAACTCAAATTGCCTGAGGAAAAATGGAAAAATTATCCACCAGTCGATTCAAACTGAATTTCACTCTTTATAGGAAGGCAGGGCAAACTTGTAGGAGTACGAAACATTTTCAATAAATCTACAAAGGGAAGCCTTACTACAATTCCAAAAATCATCATGGTTGGAAATTTGGGAGGAGATTATTTGTGAACTTGTTACCCTTTTGGTAATGGTGGACTAATTGCTGTATAGTTATTTTTGTTTTATTATTACTGTTACATTAATTTAACATGCATTTATAGAAGAATACATTCAAAGCACTGATGTAGGAGATACACGGTACTTGGAGCAGTCAGCCAGAAATCACAGATACTGCTTTCACTTAAATGGAAACAATTCTCCGATAATGCTTTGCTTTTTTTCTTATGTCACTCTTGTGTACTATCTATTTTTCTCCTCTCTGGGACCAAGTTTCTTTTTA TAAAGCAATAATATCTCTGTTTTCATTTCAGAACATTGTGCTGTCTGTCAGCAT ATGTATATCAGCTACAAAATATATTCAACTTTGACTTCTTTTGACAAAGGACTT TAGGAAAAAGAGGAACAAAGACATTATTTGAGAATTAAATTATATATTTTTAA TATGACTGTGACCTTGACTGATAATAAAGATGTAATAAGAATTGCAAGCTAAA ttttatgaccctgtggagccagtggactttgaaggacttctgatgacacacctgaacagcctggatgtgcagcttgcccaggagctc ggggacttcactgatgacgacttggacgtggtgttcacgccaaaggaatgtaggactttgcagccctctttgccggaggaagggta aatagttttctaaaatgtagatgtgattgggattgtcatgattgttttcaataagtgggtaggggagatgccttcaatctgaacttaaaaat aaaataaaattactcaatccattcaaatgtgtgggacagctatatgatatcatcatgtaagtatagatagtttttaaattagtttggccaga gattttgaaaagctatgtgaagactaaggGTAACCACGTGCGGACCGAGCGGCCGCaggaacccctagt gatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgc ccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggtctgagacaataaccctgataaatgcttcaataatgtaagc tctaggagatccgaaccagataagtgaaatctagttccaaactattttgtcatttttaattttcgtattagcttacgacgctacacccagttc ccatctattttgtcactcttccctaaataatccttaaaaactccatttccacccctcccagttcccaactattttgtccgcccacagcgggg catttttcttcctgttatgtttttaatcaaacatcctgccaactccatgtgacaaaccgtcatcttcggctactttttctctgtcacagaatgaa aatttttctgtcatctcttcgttattaatgtttgtaattgactgaatatcaacgcttatttgcagcctgaatggcgaatg (SEQ ID NO: 3).[000139] The pFB-e3g4-Rflag-shortHA plasmid includes a plasmid backbone (named pFB) is used for packaging the AAV. pFB-e3g4-Rflag-shortHA includes DOCK8 amino acids 1161-2099.[000140] The pFb-e3g4-L-shortHA and pFB-e3g4-Rflag-shortHA comprising the 5 ' and 3 ' DOCK8 halves were designed based on the CRISPR editing approach.[000141] Healthy donor derived peripheral blood stem cells (PBSC) were pre-stimulated in X-VIVO15 Serum-free Hematopoietic Cell Medium (Lonza Bioscience) with 50 ng / mL Human Stem Cell Factor (hSCF; Peprotech), 50 ng / mL human fms like tyrosine kinase 3 (hFlt-3; Peprotech) ligand, and 50 ng / mL human thrombopoietin (hTPO; Peprotech). After two days in culture, DOCK8 sgRNA e3g4 (SEQ ID NO: 1) pre-complexed to Cas9 protein were delivered to PBSC as ribonucleoprotein complexes using the BTX square wave electroporator (250 V, 5 mS; Harvard Apparatus).[000142] PBSC cells used herein comprise a mix of stem and progenitor cells, also called HSPC - Hematopoietic Stem and Progenitor Cells.[000143] Immediately following electroporation, cells were transduced with the two AAV 6donors, each containing half of the D0CK8 cDNA with their respective homology arms (SEQ ID NO: 2 and SEQ ID NO: 3) for 16-24 hours to allow for site specific integration.[000144] Rates of site specific integration were quantified by droplet digital PCR with three different sets of probes, one binding in the 5’ end of the cDNA donor, one in the 3’ end, and one where the two halves of the DOCK8 cDNA come together (Figures 1A and IB).[000145] Viability and fold expansion were measured by trypan blue exclusion 24 hours after treatment (Figures 1C and ID).Results:[000146] Gene integration occurred in 12.5% of cells when measured with the Left Primer / Probe set, while 15.1% gene integration was quantified using the Right Primer / Probe set. The Center Primer / Probe set measured 5.6% gene integration (Figure IB). The center P / P is measuring integration of both halves of the DOCK8 cDNA by the two AAV donors, while the Left and Right P / P are also detecting events in which only the pFb-e3g4-L-shortHA (SEQ ID NO: 2) or pFB-e3g4-Rflag-shortHA (SEQ ID NO: 3) has integrated independent of the other. Use of the dual AAV system does not significantly affect cell viability and fold expansion as measured by trypan blue exclusion (Figures 1C and ID).[000147] These results demonstrated the sequential integration of DOCK8 cDNA (e3g4-L- short HA donor was integrated first followed by integration of the e3g4-Rflag-short HA) is enabled by two AAV6 donors in peripheral blood stem cell (PBSC).Example 2: Integration of Full-length DOCKS cDNAs in Stem Cells Using Lentiviral Vectors[000148] Objective: To develop a human stem cell transfer method for the treatment of DOCK8 deficiency using Lentiviral vector transduction of human hematopoietic stem cells approach.[000149] Materials[000150] Vector and component sequences[000151] Elongation factor 1 alpha short (EFS): ggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccg gtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaacc gtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtgtcgtga (SEQ ID NO: 40).[000152] Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE):ttccgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgc tttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtgg cccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctc ctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcgg ctgttgggcactgacaattccgtggtgttgtcggggaaATCATcgtcctttccTtggctgctcgcctgtgttgccacctggattct gcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctct tccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgc (SEQ ID NO: 41).[000153] mCCL-EFS-DOCK8ga-WPRE (Figure 12): acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgc ccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccg atttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttc gccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatt tataagggatttlgccgatttcggcctattggttaaaaaatgagctgattlaacaaaaatttaacgcgaattttaacaaaatattaacgctt acaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaga caataaccctgataaatgcttcaataatagcacctagatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgc acgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgt gttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggca gcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctg ctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcgg cggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaag ccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagca tgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcat cgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaa tgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctga attattaacgcttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatcaggtggcacttttcggggaa atgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaccaaaatcccttaacgtgagttttcgttcc actgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaa ccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagata ccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctg ttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtc gggctgaacgggggggtcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctat gagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctc gtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttc tttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagc gcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgc agctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccc aggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatt acgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttggccattgcatacgttgtatccatatcata atatgtacatttatattggctcatgtccaacattaccgccatgttgacattgattattgactagttattaatagtaatcaattacggggtcatt agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccatt gacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccc acttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgccca gtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatc aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccatlgacgtcaatgggagtttgttttggcaccaaaatc aacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagc agagctcgtttagtgaaccgGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTC TGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGC TTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTC AGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCagtggcgcccgaacagggacttgaaagcgaa agggaaaccagaggagctctcTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAG AGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGC TAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGaattagat cgcgatgggaaaaaattcggtaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctag aacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacag gatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaag ctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcGGCCGCTgatcttcagacctggag gaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaag gcaaagagaagagtggtgcagagagaaaaaagaGCAGTGGGAATaggagctttgttccttgggttcttgggagcagca ggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaattt gctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtg gaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgcta gttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaa tacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtac tttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccg aaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatCacaaatggcagtattcatcc acaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaacta aagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagaggctccggtgcccgtcagtgggcag agcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggt aaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtga acgttctttttcgcaacgggtttgccgccagaacacaggtgtcgtgaGACAGACGAGGTTTGCGCTTGGCTGG GCATGTTCCGCGGCTACTCTGCGGCGCGCCAGGCCCCCGCTTTCCGCACCCCGC GACCCTAGAAGCCACCGAACCGCCGGCGGGCCATGGCCACACTGCCTAGCGCTG AGAGAAGGGCCTTCGCTCTGAAGATCAACCGGTACAGCAGCGCCGAGATCCGGAA GCAGTTTACCCTGCCTCCTAACCTGGGCCAGTACCACAGACAGAGCATCAGCACCA GCGGCTTCCCATCTCTGCAGCTGCCCCAGTTCTACGACCCTGTGGAACCCGTGGAT TTCGAGGGCCTGCTGATGACCCACCTGAACAGCCTGGATGTGCAGCTGGCCCAAG A GCTGGGCGA CTTCA CCGA CGA TGA TCTGGA CGTGGTGTTCA CCCCTAAA GA GTGC AGAACCCTGCAGCCTAGCCTGCCTGAAGAAGGCGTGGAACTGGATCCTCACGTGC GGGATTGTGTGCAGACCTACATCCGCGAGTGGCTGATCGTGAACCGGAAGAATCA GGGCAGCCCAGAGATCTGCGGCTTCAAGAAAACCGGCAGCCGGAAGGACTTCCAC AAGACCCTGCCTAAGCAGACCTTCGAGAGCGAGACACTGGAATGCTCTGAGCCTG CTGCTCAGGCCGGACCTAGACACCTGAATGTGCTGTGTGACGTGTCCGGCAAGGG ACCTGTGACCGCCTGCGATTTCGACCTGAGAAGTCTGCAGCCCGACAAGCGGCTG GAAAACCTGCTCCAGCAAGTGTCCGCCGAGGACTTCGAGAAGCAGAACGAGGAAG CCAGACGGACCAACAGACAGGCCGAGCTGTTTGCTCTGTACCCCAGCGTGGACGA GGAAGATGCCGTCGAGATTAGACCCGTGCCTGAGTGCCCAAAAGAGCACCTGGGC AACAGAATCCTGGTCAAGCTGCTGACCCTGAAGTTCGAGATCGAGATTGAGCCCCT GTTCGCCTCTATCGCCCTGTACGACGTGAAAGAGCGGAAGAAGATCAGCGAGAACT TCCACTGCGACCTGAACTCCGACCAGTTCAAGGGCTTCCTGAGAGCCCACACACCA TCTGTGGCCGCTAGCTCTCAGGCCAGATCTGCCGTGTTCAGCGTGACATACCCCAG CAGCGACATCTACCTGGTGGTCAAGATCGAGAAGGTCCTGCAGCAGGGCGAGATC GGCGATTGTGCCGAGCCTTACACCGTGATCAAAGAGAGCGACGGCGGCAAGAGCA AAGAGAAGATTGAGAAGCTGAAGCTGCAGGCCGAGAGCTTCTGTCAGCGGCTGGG CAAGTACAGAATGCCCTTTGCCTGGGCTCCTATCAGCCTGTCCAGCTTCTTCAACGTGTCCACACTGGAACGGGAAGTGACCGACGTGGACTCTGTCGTGGGCAGATCTAGCGTGGGCGAGAGAAGGACACTGGCTCAGTCTAGACGGCTGAGCGAGAGAGCCCTGAGCCTGGAAGAAAATGGCGTGGGCAGCAACTTCAAGACCAGCACTCTGTCCGTGTCTAGCTTCTTTAAGCAAGAGGGCGACAGACTGAGCGACGAGGACCTGTTCAAGTTCCTGGCCGACTACAAGCGGAGCAGCTCCCTGCAGAGAAGAGTGAAGTCTATCCCTGGCCTGCTGCGCCTGGAAATCTCTACAGCCCCTGAGATCATCAACTGCTGTCTGACCCCTGAAATGCTGCCCGTGAAGCCCTTTCCAGAGAACCGGACCAGACCTCACAAAGAGATCCTGGAATTCCCCACCAGAGAAGTGTACGTGCCCCACACCGTGTACCGGAACCTGCTGTATGTGTACCCACAGCGGCTGAACTTCGTGAACAAGCTGGCCTCCGCCAGAAACATCACCATCAAGATCCAGTTTATGTGCGGCGAGGACGCCAGCAACGCCATGCCTGTGATCTTCGGCAAGTCTAGCGGCCCTGAGTTCCTGCAAGAGGTGTACACAGCCGTGACCTACCACAACAAGAGCCCCGACTTCTACGAGGAAGTGAAGATTAAGCTGCCCGCCAAGCTGACCGTGAATCACCATCTGCTGTTCACCTTCTACCACATCAGCTGCCAGCAGAAACAGGGCGCCTCTGTGGAAACACTGCTGGGCTATAGCTGGCTGCCCATCCTGCTGAACGAGAGACTGCAGACCGGCAGCTACTGTCTGCCTGTGGCTCTGGAAAAGCTGCCACCTAACTACAGCATGCACTCCGCCGAGAAGGTGCCCCTGCAGAATCCTCCTATTAAGTGGGCCGAGGGCCACAAGGGCGTGTTCAATATCGAGGTGCAGGCCGTGTCCTCCGTGCACACCCAGGATAACCACCTGGAAAAGTICTTCACCCTGTGCCACAGCCTCGAGAGCCAAGTGACATTCCCCATCCGCGTGCTGGACCAGAAAATCTCCGAGATGGCCCTGGAACACGAGCTGAAACTGAGCATCATCTGCCTGAATAGCAGCAGACTGGAACCCCTGGTGCTGTTCCTGCATCTGGTGCTGGACAAGCTGTICCAGCTGAGCGTGCAGCCCATGGTTATCGCCGGACAGACCGCCAACTTCAGCCAGTTCGCCTTTGAGAGCGTGGTGGCCATTGCCAACAGCCTGCACAACAGCAAGGACCTGAGCAAGGATCAGCACGGCAGAAACTGCCTGCTGGCCTCTTACGTGCACTACGTGTTCAGACTGCCTGAGGTGCAGAGGGACGTGCCAAAATCTGGCGCTCCTACCGCTCTGCTGGACCCCAGAAGCTATCACACCTACGGCAGAACATCTGCCGCCGCTGTGTCCTCTAAACTCCTGCAGGCTAGAGTGATGTCCAGCAGCAACCCTGATCTGGCCGGAACACACAGCGCCGCTGATGAAGAAGTGAAAAACATCATGAGCAGCAAGATCGCCGACCGGAACTGCAGCCGGATGAGCTACTACTGTAGCGGCAGCTCTGATGCCCCTAGCTCTCCAGCTGCTCCTAGACCTGCCAGCAAGAAGCACnTCACGAGGAACTGGCCCTGCAGATGGTCGTGTCTACCGGCATGG7TCGAGAGACAGTGTTTAAGTACGCCTGGTTCTTC7TCGAACTGCTGGTCAAGAGCATGGCCCAGCACGTGCACAACATGGACAAGCGGGACAGCTTCCGGCGGACCAGATTCAGCGACAGATTCATGGACGACATCACCACCATCGTGAACGT GGTCACCAGCGAGATTGCTGCCCTGCTGGTTAAGCCCCAGAAAGAGAACGAGCAG GCCGAAAAGATGAATATCTCTCTGGCCTTCTTCCTCTACGACCTGCTGAGCCTGATG GACCGGGGCTTCGTGTTCAACCTGATCCGGCACTACTGCTCCCAGCTGTCTGCCAA GCTGTCCAACCTGCCTACACTGATCTCTATGAGACTCGAGTTCCTGAGGATCCTGT GCAGCCACGAGCACTACCTGAACCTGAATCTGTTCTTCATGAACGCCGACACAGCC CCTACAAGCCCCTGTCCTTCTATCAGCAGCCAGAACAGCAGCAGCTGTAGCAGCTT TCAGGATCAGAAAATCGCCAGCATGTTCGATCTGACCAGCGAGTACCGGCAGCAGC ACTTTCTGACCGGCCTGCTGTTTACCGAACTGGCAGCCGCTCTTGATGCCGAAGGC GAGGGAATCAGCAAGGTGCAGCGGAAAGCCGTGTCTGCCATTCACAGCCTGCTGT CTAGCCACGACCTGGATCCTAGATGCGTGAAGCCTGAAGTGAAAGTGAAAATCGCT GCCCTGTACCTGCCTCTCGTGGGCATCATTCTGGATGCCCTGCCTCAGCTGTGTGA TTTCACCGTGGCCGACACCAGACGGTACAGAACCTCTGGCTCCGACGAGGAACAA GAAGGCGCTGGCGCCATCAACCAGAATGTGGCCCTTGCCATTGCCGGGAACAACT TCAA TCTGAAAA CCA GCGGCA TCGTGCTGA GCA GCCTGCCTTA CAA GCA GT A CAA C ATGCTGAATGCCGACACTACCCGCAACCTGATGATCTGCTTCCTGTGGATTATGAAG AACGCCGATCAGAGCCTGATTCGGAAGTGGATTGCCGACCTGCCAAGCACTCAGCT GAACCGGATTCTGGACCTCCTGTTCATCTGCGTGCTGTGCTTCGAGTACAAGGGCA AGCAGTCCAGCGACAAGGTGTCCACTCAGGTGCTGCAGAAAAGCCGGGATGTGAAGGCCAGGCTGGAAGAGGCACTTCTGAGAGGCGAAGGCGCCAGAGGCGAGATGAT GAGGCGTAGAGCCCCTGGCAACGATAGATTCCCCGGCCTGAATGAGAACCTGCGG TGGAAGAAAGAGCAGACCCATTGGAGACAGGCCAACGAGAAACTGGACAAGACCA AGGCCGAACTGGACCAAGAGGCCCTGATCTCTGGCAACCTGGCCACAGAAGCCCA CCTGATTATCCTGGACATGCAAGAGAACATCATCCAGGCCAGTAGCGCCCTGGATT GCAAGGATTCTCTGCTCGGAGGCGTGCTGAGGGTGCTCGTGAATAGCCTGAATTG CGACCAGAGCACCACCTACCTGACACACTGCTTCGCCACACTGAGGGCCCTGATTG CCAAGTTTGGGGACCTGCTGTTCGAGGAAGAGGTGGAACAGTGCTTCGACCTGTGT CACCAAGTGCTGCACCACTGCAGCAGCTCCATGGACGTGACAAGATCCCAGGCCT GCGCCACTCTGTATCTGCTGATGCGGTTTAGCTTCGGCGCCACCAGCAATTTCGCC AGAGTGAAGATGCAAGTGACCATGAGCCTGGCCAGCCTCGTTGGAAGGGCCCCTG ACTTCAATGAGGAACACCTCAGAAGAAGCCTGCGGACCATCCTGGCCTACAGCGAA GAGGATACCGCCATGCAGATGACCCCATTICCAACACAAGTGGAAGAACTGCTGTGCAACCTGAATTCCATCCTGTACGATACCGTGAAGATGAGAGAGTTCCAAGAGGACC CCGAGA TGCTGATGGA TCTGA TGTA CCGGATCGCCAA GAGCTA CCAGGCCTCTCCT GATCTGAGACTGACCTGGCTGCAGAACATGGCCGAGAAGCACACCAAGAAGAAGT GCTACACCGAGGCCGCCATGTGCCTGGTTCATGCTGCTGCACTCGTGGCCGAGTA CCTGAGCATGCTGGAAGATCACTCCTACCTGCCAGTGGGCAGCGTGTCCTTCCAGA ACATCAGCTCCAACGTCCTGGAAGAGTCCGTGGTGTCCGAGGACACACTGAGCCC AGATGAGGATGGCGTGTGTGCCGGACAGTACTTTACCGAGTCTGGCCTCGTGGGC CTTCTGGAACAGGCCGCCGAACTGTTTTCTACCGGCGGACTGTACGAGACAGTGAA CGAAGTGTACAAGCTGGTCATCCCCATCCTGGAAGCCCACAGAGAGTTCAGAAAGC TGACACTGACCCACTCCAAGCTGCAGCGGGCCTTCGACAGCATTGTGAACAAGGAC CACAAGCGGATGTTCGGCACCTACTTCAGAGTGGGCTTTTTCGGCAGCAAATTCGG CGACCTGGACGAGCAAGAGTTCGTGTACAAAGAGCCCGCCATCACCAAGCTGCCT GAGATCTCCCATCGGCTGGAAGCCTTCTACGGCCAGTGTTTTGGCGCCGAGTTCGT GGAAGTGATCAAGGACAGCACCCCTGTGGATAAGACCAAACTGGACCCTAACAAGG CCTA CA TCCA GA TCA CCTTCGTGGAA CCCTA CTTCGA CGA GT A CGA GA TGAA GGA T AGAGTGACCTACTTTGAGAAGAACTrCAACCTGCGGCGGlTCATGTACACCACACC TTTTACACTGGAAGGCAGACCCAGGGGCGAGCTGCACGAGCAGTATAGAAGAAAC ACCGTGCTGACCACCATGCACGCATTCCCTTACATCAAGACCCGGATCAGCGTGAT CCAGAAAGAAGAGTTTGTCCTGACACCAATCGAGGTGGCCATCGAGGACATGAAGAAGAAAACCCTCCAGCTGGCCGTGGCCATCAATCAAGAACCTCCAGACGCCAAGATG CTCCAGATGGTGCTCCAGGGATCTGTGGGCGCCACAGTGAATCAGGGACCTCTGG AAGTGGCCCAAGTGTTCCTGGCTGAGATCCCCGCCGATCCTAAGCTGTACAGACAC CATAACAAGCTGCGGCTCTGCTTCAAAGAATTCATCATGAGATGTGGCGAGGCCGT GGAAAAGAACAAGAGACTGATCACCGCCGACCAGAGAGAGTACCAGCAAGAGCTG AAGAAAAACTACAACAAGCTCAAAGAAAATCTGCGGCCCATGATCGAGAGAAAGATC CCCGAGCTGTACAAGCCCATCTTCCGCGTGGAAAGCCAGAAGCGGGACTCCTTTCA CAGATCCAGCITTAGGAAGTGCGAGACACAGCTGTCCCAGGGCTCTrGAttccgataatc aacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgccttt gtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtca ggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccggga ctttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggca ctgacaattccgtggtgttgtcggggaaATCATcgtcctttccTtggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttc gccttcgccctcagacgagtcggatctccctttgggccgcctccccgcTaaaagaaaaggggggactggaagggctaattcact cccaacgaagacaagatctgctttttgcttgtactGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGG GAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCC TTGAGTGCTTCAAGTAGTGTGTTGGTTTTTTGTGTGTGCATTGTCTGAGTAGGT GTCATTCTATTCTGGGGGGTGGGGTGGGGCAGCACAGCAAGGGGGAGGATTG GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGagtagtagttcatg tcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaata aagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtc tggctctagctatcccgcccctaactccgcccATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATT CTCCGCCCCATGGCTCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCC TCGGCCTCTGCATAAATAAAAAAAATTAGTCGAGGCTTTTTTGGAGGCCTAGG gacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggc gttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttccca acagttgcgcagcctgaatggcgaatggg (SEQ ID NO: 4). The nucleic acid sequence encoding the GeneArt optimized DOCK8 protein is Italicized and represents nucleotide positions 5430- 11729 of the mCCL-EFS-DOCK8ga-WPRE vector. A description of the lentiviral transfer vector construct containing the CMV-HIV 5’LTR for the mCCL vectors disclosed (including SEQ ID NOs: 4, 6, 8, 11, and 13) herein can be found in Dull et al., A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998 Nov;72(ll):8463-71. Methods and Results'.[000154] PBSC cells used herein comprise a mix of stem and progenitor cells, also called HSPC - Hematopoietic Stem and Progenitor Cells.[000155] Given the constitutive nature of DOCK8 expression in cells of hematopoietic lineage, as a first step, the feasibility of gene addition through lentiviral vectors was evaluated. HIV-1 based lentiviral vectors in which the LTR is deleted and the transgene is driven by an internal promoter were used in this approach as insertional mutagenesis is a concern with gamma-retroviral vectors. Self-inactivating lentiviral vectors not only possess a safer vector integration profile but an improved ability to transduce quiescent, engrafting HSC. (Zufferey et al., Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 1998 Dec;72(12):9873-80.)[000156] Two codon-optimization algorithms (GeneArt (ga) versus GenScript) were usedto generate the D0CK8 cDNA in forward orientation with expression driven by the elongation factor 1 alpha short (EFS; SEQ ID NO: 40) as the internal promoter. (Figure 2). There has been significant experience with the EFS promoter in current clinical trials of gene therapy given its ability to maintain high expression levels of therapeutic transgenes in hematopoietic cells while exhibiting low rates of transactivation of neighboring genomic loci. In addition, the Woodchuck Hepatitis Virus Post-transcriptional Regulatory Element (WPRE; SEQ ID NO: 41), which is widely used in lentiviral therapies to stabilize the viral RNA and mRNA of the expressed transgene, was included in two versions of the DOCK8 donors.[000157] DOCK8 lentiviral vectors were first tested in Jurkat cell lines, which do not express DOCK8 at baseline (any detectable DOCK8 expression after viral transduction is from the transgene.) DOCK8 expression was restored only in cells treated with the EFS- GeneArtDOCK8 vector, with minimal DOCK8 expression when using the GenScript codon optimization. (Figure 3A) Inclusion of the WPRE sequence improved DOCK8 expression at similar vector copy number (VCN) when measured by flow cytometry. (Figure 3B).[000158] Moving forward with the EFS-GeneArtDOCK8-WPRE lentiviral vector (Figure 2; SEQ ID NO: 4), peripheral blood stem cells (PBSC) from healthy donors were transduced at le7 TU / mL with a combination of various small molecules previously shown to enhance transduction efficiency, including Lentiboost (Sirion Biotech)., cyclosporin H, and protamine sulfate. VCN 12-14 days after transduction is shown in Figure 4. Increasing amounts of virus beyond le7 TU / mL did not appear to increase VCN. Addition of protamine sulfate in addition to LentiBoost increased transduction efficiencies, with improvement when transduction was performed after two day pre-stimulation in XVIV015 media (Lonza Biosciences) with 50 ng / mL of hSCF, hFlt3L, and hTPO. There was no additional benefit from transducing cells both on day 1 and day 2 of pre-stimulation.[000159] While VCNs of 0.4 (reflecting that 40% of cells contain the therapeutic transgene) are likely to significantly ameliorate the disease phenotype, improved methods to deliver large transgenes more efficiently were considered worth exploring.Example 3: Integration of GFP Split Inteins[000160] Objective’. To assess integration of GFP split inteins in human cells using protein splicing.Methods and Results’.[000161] Investigations using inteins were begun in an effort to identify improved methods for the delivery of large transgenes, such as D0CK8, into cells. Inteins are protein segments capable of ligating flanking exteins (external proteins) into a new protein in a process known as protein splicing. Specifically, split inteins divided into two fragments called N-intein and C-intein may be capable of restoring D0CK8 expression using two lentiviral vectors. An example of a split intein is the Cfa split intein (Stevens AJ, et al. (2016) Design of a Split Intein with Exceptional Protein Splicing Activity. J. Am. Chem. Soc. 138: 2162-2165).[000162] As the Cfa split intein has been evaluated largely in non-mammalian cells, the published sequences of CfaN and CfaC were codon-optimized using ThermoFisher’s GeneOptimizer software to account for human codon usage bias.[000163] Codon-Optimized CfaN nucleotide sequence:TGCCTGAGCTACGACACCGAGATCCTGACCGTGGAATACGGCTTCCTGCCTAT CGGCAAGATCGTGGAAGAACGGATCGAGTGCACCGTGTACACCGTGGACAAG AACGGCTTCGTGTACACACAGCCTATCGCTCAGTGGCACAACCGGGGAGAGC AAGAGGTGTTCGAGTACTGCCTGGAAGATGGCAGCATCATCCGGGCCACCAA GGACCACAAGTTCATGACCACCGACGGCCAGATGCTGCCCATCGACGAGATC TTTGAGAGAGGCCTGGACCTGAAACAGGTGGACGGACTTCCT (SEQ ID NO: 16).[000164] CfaN amino acid sequence:CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVF EYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP (SEQ ID NO: 19) [000165] Codon-Optimized CfaC nucleotide sequence:GTGAAGATCATCAGCAGAAAGAGCCTGGGCACCCAGAACGTGTACGACATCG GCGTGGAAAAGGACCACAACTTTCTGCTCAAGAACGGCCTGGTGGCCAGCAAC (SEQ ID NO: 21)[000166] CfaC amino acid sequence:VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN (SEQ ID NO: 24)[000167] First, the ability of the human codon-optimized Cfa split intein to splice together two halves of the green fluorescent protein (GFP) was evaluated.[000168] Two separate expression plasmids driven by the CMV promoter were cloned to contain either the CfaN sequence (SEQ ID NO: 16) fused to the first half of GFP or the second half of GFP fused to the CfaC sequence (SEQ ID NO: 21) (Figure 5B). Twoversions of the CfaC plasmids were created to test the required extein amino acids adjacent to the intein, which can be C-F-N (cysteine-phenylalanine-asparagine) or C-F (the plasmid with the C-F-N is not shown but is identical to the CfaC-GFP plasmid except at the site of the C-F). The split site for the GFP protein was chosen at one of the short helical segments on the ends of the beta-sheet cylinder to avoid disruption of the beta-can that is important forGFP expression. (Figure 5A).[000169] These sequences were then tested in human cell lines, K562 cells, derived from the bone marrow of a female with chronic myelogenous leukemia. Briefly, K562 cells were electroporated on the Lonza 4D platform (Lonza Biosciences) with a total of lug of plasmid. Cells that received the CfaN or CfaC plasmids alone did not express GFP, while those that received both expressed high levels of GFP as measured by flow cytometry. (Figure 6). The C-F linker for the CfaC intein resulted in similarly efficient protein splicing compared to the C-F-N linker.Example 4: Integration of D0CK8 cDNA Split Inteins[000170] Objectives: To assess if splicing of the DOCK8 protein would be feasible by inteins.[000171] Materials[000172] Vector and component sequences[000173] The plasmid vector constructs provided in Table 1 below were used in Example 4 and Example 5. In these experiments, the nucleotide sequences for both DOCK8 fragments(halves) and the intein halves were codon-optimized using Gene Art algorithms.[000174] Table 1. Plasmid Vector Constructs[000175] The nucleotide and amino acid sequences of the DOCK8 fragments alone or fusedto an intein component are provided in Table 2 below. In addition, the nucleotide sequences of expression cassettes are also provided in Table 2. To identify which portion of DOCK8 is represented in the below sequences, the information presented in the parentheses indicates either the 1stor 2ndfragment, also called the first or second half of DOCK8. One skilled in the art would appreciate that in certain instances a TGA stop codon is present at the 3' end of some of the nucleic acid sequences below, for example SEQ ID NO: 14.[000176] Table 2: Plasmid Vector ComponentsMethods and Results:[000177] To assess if splicing of the DOCK8 protein would be feasible by inteins, additional plasmids were cloned containing two halves of the DOCK8 protein with their respective inteins. (See Table 1 for the amino acid and nucleic acid sequences of DOCK8 plasmids and components thereof.) However, in contrast to GFP, the DOCK8 protein is large and complex and is known to have two domains (DHR1 and DHR2) critical for protein function. (Figure 7; Biggs et al (2017) DOCK8 deficiency: Insights into pathophysiology, clinical features and management. Clin Immunol. 181:75-82.) Therefore, intein split sites were identified in the region between the two Dock Homology Regions at amino acids 939 or 1267 in external loops that do not obviously contribute to intra-protein interactions (Figure 8).[000178] For the 939 CfaC intein, both the C-F-N and C-F extein linkers were tested; for the 1267 CfaC intein, only the C-F-N extein linker was tested since that particular split site naturally starts with an N (asparagine).[000179] The intein plasmids used were:939 DOCK8-CfaN (SEQ ID NO: 5): This is the N-terminal intein sequence containing the first fragment (half) of the human DOCK8 codon-optimized cDNAfollowed by the CfaN intein sequence. The split site is just before the 939thamino acid of DOCKS so the DOCK8 cDNA for this sequence ends at the codon encoding amino acid 938.• 939 CfaC-CFN-DOCK8 (SEQ ID NO: 7): This is the C terminal DOCK8 intein starting with the CfaC intein sequence followed by the second fragment (half) of the human DOCK8 codon-optimized cDNA sequence. Note, the CfaC intein has a required linker sequence between the intein and the extein that can be either -CF- (cysteine-phenylalanine) or -CFN- (cysteine-phenylalanine-asparagine). Since the efficiency of each of these linkers is unknown for intein splicing, both versions were designed and tested.• 939 CfaC-CF-DOCK8 (SEQ ID NO: 9): Same as 939 CfaC-CFN-DOCK8 except with the -CF- (cysteine-phenylalanine linker).• 1267 DOCK8-CfaN: (SEQ ID NO: 10) This is the N-terminal intein sequence containing the first fragment (half) of the human DOCK8 codon-optimized cDNA followed by the CfaN intein sequence. The split site for this cassette is just after the 1267th amino acid of DOCK8.• 1267 CfaC-CFN-DOCK8: (SEQ ID NO: 12) This is the C terminal DOCK8 intein starting with the CfaC intein sequence followed by the second fragment (half) of the human DOCK8 codon-optimized cDNA sequence. Note, we chose this split site because the first amino acid of the C terminal half of the protein naturally starts with an phenylalanine followed by an asparagine, so we only needed to add cysteine to create a -CFN- linker.[000180] Intein plasmids were tested in the Jurkat T cell line, which does not express DOCK8. DOCK8 inteins were introduced to Jurkat cells via electroporation using the Lonza 4D Nucleofector. 1 ug of each plasmid listed above were used in each experimental condition. One day after electroporation, protein lysates were collected, quantified, and analyzed for DOCK8 protein expression by immunoblot.[000181] For both the 939 and 1267 split sites, only samples that receive both the CfaN and CfaC components of the DOCK8 inteins form DOCK8 protein visible at 190 kDa. Control samples for the Western blot include T cells and K562 cell lines, both of which are positive for DOCK8 and untreated Jurkat cells, which do not express DOCK8 at baseline.[000182] As shown in Figure 9, untreated Jurkat cells are negative for DOCK8, while thepositive controls, which include primary human T cells and K562 cells express D0CK8. Jurkat cells that received only half of the D0CK8 protein are negative by immunoblot, while samples receiving both the CfaN and CfaC intein plasmids have restored D0CK8 expression. In addition, both the CfaC-C-F and CfaC-C-F-N plasmids appear to restore D0CK8 expression equally at the 939 D0CK8 split site. As both the 939 CfaC-CF-D0CK8- bGHpA (Seq ID No 7) and 939 CfaC-CFN-D0CK8-bGHpA (Seq ID No 9) perform similarly in these experiments, moving forward the CF linker (Seq ID No 7) was used, given this would result in fewer added amino acids to the D0CK8 protein.[000183] In all, these experiments demonstrate the ability of inteins to restore expression of the DOCK8 protein. Additional work using inteins delivered by lentiviral vectors was performed in primary hematopoietic stem cells.Example 5: Integration of DOCKS cDNA Split Inteins in Stem Cells Using Lentiviral Vectors[000184] Objectives: To develop a human stem cell transfer method for the treatment of DOCK8 deficiency using Lentiviral vector transduction of human hematopoietic stem cells approach.[000185] Materials: Lentiviral vectors and sequences thereof are provided in Table 1 (Example 4 above) and Figures 14, 16, 18, and 20. . In these experiments the nucleotide sequence for both DOCK8 and the intein halves were codon-optimized using GeneArt algorithms.[000186] PBSC cells used herein comprise a mix of stem and progenitor cells, also called HSPC - Hematopoietic Stem and Progenitor Cells.Methods and Results:[000187] Jurkat T cells were transduced with either mCCL-EFS-939DOCK8-CfaN-WPRE (SEQ ID NO: 6) with mCCL-EFS-939Dock8-CfaC-CF-WPRE (SEQ ID NO: 8), or with mCCL-EFS-1267Dock8-CfaN-WPRE (SEQ ID NO: 11) with mCCL-EFS-1267Dock8- CfaC-CFN-WPRE (SEQ ID NO: 13) at a range of concentrations from 2e5 TU / mL up to 6e6 TU / mL. Two weeks post-transduction, genomic DNA was isolated from cells and vector copy number (VCN) was quantified by droplet digital PCR (ddPCR).[000188] Figure 21 shows that VCNs increased in a dose dependent manner based on the amount of vector added and ranged from 1.56 up to 16.1.[000189] To gage if the concentration of Lentiviral vectors influenced expression level ofI llD0CK8, flow cytometric analysis of D0CK8 expression in Jurkat T cells transduced with either the 939 or 1267 intein lenti viral vector pairs at a range of concentrations from 2e5 to 6e6 TU / mL was performed.[000190] The results show that there is a dose dependent response based on the amount of lentiviral vector used in terms of both VCN quantified 2 weeks after transduction by ddPCR or DOCK8 expression measured by flow cytometry. DOCK8 positive cells range from 23% to 93% from the lowest to the highest concentrations of virus shown in Figures 22A-22B.[000191] Transduced Jurkat cells were also assessed for DOCK8 expression by Western blot (Figure 23). Mock treated Jurkat cells do not have detected DOCK8 protein (190kDa) while cells transduced with the 939 or 1267 lentiviral pairs have detectable DOCK8 expression. CD4 T cells are included as a positive control.[000192] Figure 24A shows healthy donor derived peripheral blood stem cells (PBSC) were pre-stimulated for either one or two days in XV1V015 with 50 ng / mL hSCF, 50 ng / mL hFlt31igand, and 50 ng / mL hTPO. After 1 or 2 days of pre-stimulation, PBSC were transduced with the mCCL-EFS-939DOCK8-CfaN-WPRE (SEQ ID NO: 6) and mCCL- EFS-939Dock8-CfaC-CF-WPRE (SEQ ID NO: 8) lentiviral vectors at a range of TU / mL, with or without LentiBoost (Sirion Biotech). Vector copy number was quantified by ddPCR and shown above. With the addition of LentiBoost, transduction efficiency is improved and VCNs reach >1.5.[000193] Figure 24B shows similar results in PBSC transduced with mCCL-EFS- 1267Dock8-CfaN-WPRE (SEQ ID NO: 11) and mCCL-EFS-1267Dock8-CfaC-CFN- WPRE (SEQ ID NO: 13) lentiviral vectors at a range of concentrations from 6e6 to 2e8 TU / mL with LentiBoost. The results show that increasing amounts of lentiviral vector improves VCN.[000194] DOCK8 promotes STAT3 phosphorylation and translocation to the nucleus. Therefore, gene modified cells that properly express DOCK8, as has been shown here, should also have normal phosphorylation of STAT3 upon immune stimulation as detected by Western blot and flow cytometry.[000195] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the systems forexpressing D0CK8 and uses thereof.

Claims

CLAIMSWhat is claimed is:

1. A system for expressing human D0CK8 protein in a cell, said system comprising: a first nucleic acid encoding a first fragment of a human D0CK8 protein fused at the C terminal to an N-intein; and a second nucleic acid encoding a second fragment of the human D0CK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the D0CK8 protein ligated to the second fragment of the D0CK8 protein forms a full-length human D0CK8 protein, wherein the first fragment of the D0CK8 protein and the second fragment of the D0CK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), and wherein said first nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the first fragment of D0CK8 and said second nucleic acid further comprises a promoter located 5 ' of said nucleic acid encoding the C-intein, wherein optionally said promoter comprises EFS.

2. The system according to claim 1 , wherein the amino acid sequence of said first fragment of D0CK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of D0CK8 is set forth in SEQ ID NO: 25, or the amino acid sequence of said first fragment of D0CK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of D0CK8 is set forth in SEQ ID NO: 37.

3. The system according to claim 1, wherein said first nucleic acid encoding a first fragment of a human D0CK8 is codon-optimized and wherein said second nucleic acid encoding a second fragment of the human D0CK8 protein is codon-optimized.

4. The system according to claim 1 , wherein said second fragment of the D0CK8 protein comprise an extein linker where said linker comprises C-F or C-F-N.

5. The system according to claim 1, wherein the N-intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence, wherein optionally said nucleotide sequence encoding said CfaN amino acid sequence and said nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell.

6. The system according to claim 5, wherein the amino acid sequence of said first fragment of the human D0CK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23 or SEQ ID NO: 27; or the amino acid sequence of said first fragment of the human D0CK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42.

7. The system according to claim 1, wherein the first nucleic acid is comprised in a first lentiviral vector (first LV) and the second nucleic acid is comprised in a second lenti viral vector (second LV).

8. The system according to claim 7, wherein said first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors.

9. The system according to claim 7, wherein said first nucleic acid further comprises a WPRE element located 3' of said nucleic acid encoding the N-intein and said second nucleic acid further comprises a WPRE element located 3 ' of said nucleic acid encoding the second fragment of D0CK8 protein.

10. The system according to claim 9, wherein the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30.

11. The system according to claim 9, wherein the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39.

12. A pair of recombinant lentiviral vectors (LVs) comprising: a first LV comprising a first nucleic acid encoding a first fragment of a human D0CK8 protein fused at the C termina to an N-intein; and a second LV comprising a second nucleic acid encoding a second fragment of the human D0CK8 protein fused at the N terminal with a C-intein; wherein the first fragment of the D0CK8 protein ligated to the second fragment of the D0CK8 protein forms a full-length D0CK8 protein, wherein the first fragment of the D0CK8 protein and the second fragment of the D0CK8 protein are selected to provide a split between said first fragment and said second fragment in an external loop that does not contribute to intra-protein interactions, wherein said first fragment comprises the DOCK homology region 1 (DHR1) and said second fragment comprises the DOCK homology region 2 (DHR2), and wherein said first nucleic acid further comprises a promoter located 5 ' of said nucleic acid encoding the first fragment of D0CK8 and said second nucleic acid further comprises a promoter located 5' of said nucleic acid encoding the C-intein.

13. The pair of lentiviral vectors of claim 12, wherein the amino acid sequence of said first fragment of D0CK8 is set forth in SEQ ID NO: 18 and the amino acid sequence of said second fragment of D0CK8 is set forth in SEQ ID NO: 25, or the amino acid sequence of said first fragment of D0CK8 is set forth in SEQ ID NO: 34 and the amino acid sequence of said second fragment of D0CK8 is set forth in SEQ ID NO: 37.

14. The pair of lentiviral vectors according to claim 12, wherein said first nucleic acid encoding a first fragment of a human D0CK8 is codon-optimized and wherein said second nucleic acid encoding a second fragment of the human D0CK8 protein is codon- optimized.

15. The pair of lentiviral vectors according to claim 12, wherein said second fragment of the D0CK8 protein comprise an extein linker where said linker comprises C-F or C- F-N.

16. The pair of lentiviral vectors according to claim 12, wherein the N-intein and C-intein comprise a Cfa split intein, wherein the N-intein comprises a CfaN sequence and the C-intein comprise a CfaC sequence, wherein optionally said nucleotide sequence encoding said CfaN amino acid sequence and said nucleotide sequence encoding said CfaC amino acid sequence are codon optimized for expression in a human cell.

17. The pair of lentiviral vectors according to claim 16, wherein the amino acid sequence of said first fragment of the human D0CK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 17 and the amino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 23 or SEQ ID NO: 27; or the amino acid sequence of said first fragment of the human D0CK8 protein fused at the C terminal to an N-intein is set forth in SEQ ID NO: 33 and the amino acid sequence of said second fragment of the human D0CK8 protein fused at the N terminal with a C-intein is set forth in SEQ ID NO: 42.

18. The pair of lentiviral vectors according to claim 12, wherein said first nucleic acid further comprises a WPRE element located 3' of said nucleic acid encoding the N-intein and said second nucleic acid further comprises a WPRE element located 3 ' of said nucleic acid encoding the second fragment of D0CK8 protein.

19. The pair of lentiviral vectors according to claim 18, wherein the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 28 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 29 or SEQ ID NO: 30.

20. The pair of lentiviral vectors according to claim 18, wherein the nucleic acid sequence of said first nucleic acid is set forth in SEQ ID NO: 38 and the nucleic acid sequence of said second nucleic acid is set forth in SEQ ID NO: 39.

21. The pair of lentiviral vectors of claim 12, wherein said first LV and said second LV are each TAT-independent and self-inactivating (SIN) lentiviral vectors.

22. A host cell transduced with the pair of lentiviral vectors according to claim 12.

23. The host cell according to claim 22, wherein the cell is a stem cell derived from bone marrow, umbilical cord blood, or from peripheral blood, or any combination thereof,or is a hematopoietic progenitor cell.

24. The host cell according to claim 23, wherein the human hematopoietic progenitor cell is a CD34+ cell.

25. A method of treating a D0CK8 deficiency in a human subject, said method comprising: transducing a stem cell and / or progenitor cell from said subject with a pair of lentiviral vectors according to claim 12 to produce transduced cells expressing functional D0CK8 protein; and transplanting said transduced cell or cells derived therefrom into said subject, where said cells therefrom express functional D0CK8 protein.

26. The method of claim 25, wherein the D0CK8 protein is constitutively expressed.

27. The method of claim 25, wherein said cell is a stem cell or human hematopoietic progenitor cell or a combination thereof.

28. The method of claim 27, wherein said stem cell is derived from bone marrow.

29. The method according to claim 27, wherein said human hematopoietic progenitor cell is a CD34+ cell.

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