JPWO2019210042A5

JPWO2019210042A5 -

Info

Publication number: JPWO2019210042A5
Application number: JP2020560400A
Authority: JP
Publication date: 2022-11-25

Claims

1. A method of making a genetically modified CD34 ⁺ cell comprising delivering a nuclease or a nucleic acid encoding a nuclease and a donor template to the CD34 ⁺ cell,
a first homology arm in which the donor template has homology to a sequence within (a) the FOXP3 gene, the AAVS1 locus or the TRA gene of a CD34 ⁺ cell;
(b) a second homologous arm having homology to a sequence within the same gene or locus as the gene or locus to which said first homologous arm has homology;
(c) a promoter; and (d) a sequence encoding FOXP3 or a functional derivative thereof,
A method, characterized in that said promoter and the sequence encoding said FOXP3 or a functional derivative thereof are located between said first homologous arm and said second homologous arm.

2. The method of claim 1, wherein said nuclease is an RNA-guided DNA endonuclease, a zinc finger nuclease, or a transcription activator-like effector nuclease (TALEN).

3. The method of claim 2, wherein said RNA-guided DNA endonuclease is a Cas endonuclease.

4. The method of claim 3, wherein said Cas endonuclease is Cas9 endonuclease.

3. The method of claim 2, further comprising delivering a guide RNA (gRNA) comprising a spacer sequence complementary to the FOXP3 gene, the AAVS1 locus or the TRA gene to said CD34 ⁺ cells.

The gRNA is
i) a spacer sequence set forth in any of SEQ ID NOs: 1-7, 15-20, and 27-29, or no more than 3 compared to any of SEQ ID NOs: 1-7, 15-20, and 27-29 variants of said spacer sequence with mismatches;
and/or
iii) a spacer sequence set forth in any of SEQ ID NOs: 2, 3 and 5, or a variant of said spacer sequence having no more than 3 mismatches compared to any of SEQ ID NOs: 2, 3 and 5. The method described in .

2. The method of claim 1, wherein said first homology arm has homology to a sequence within the FOXP3 locus and said second homology arm has homology to a sequence within the FOXP3 locus.

(a) said FOXP3 or functional derivative thereof is wild-type human FOXP3;
(b) said donor template is encoded by an adeno-associated virus (AAV) vector;
and/or (c) the promoter is the MND promoter, the PGK promoter, or the EF1α promoter.

2. The method of claim 1, wherein the sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in said cell.

10. The method of claim 9, wherein the sequence encoding FOXP3 or a functional derivative thereof is the FOXP3 cDNA sequence.

11. The method of claim 10, wherein said FOXP3 cDNA sequence comprises at least 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:110.

2. The method of claim 1, wherein said donor template further comprises a sequence encoding a selectable marker, further comprising separating cells expressing said selectable marker from cells not expressing said selectable marker.

Genetically modified CD34 ⁺ cells produced by the method of any one of claims 1-12.

14. The genetically modified CD34 ⁺ cell of claim 13, wherein the sequence encoding FOXP3 or a functional derivative thereof is FOXP3 cDNA and the promoter is the MND promoter.

A pharmaceutical composition comprising the genetically modified CD34 ⁺ cells according to claim 13 or 14 and a pharmaceutically acceptable additive.

A CD34 ⁺ cell comprising a nucleic acid sequence comprising a promoter operably linked to a FOXP3 cDNA sequence encoding FOXP3,
A CD34 ⁺ cell, wherein said promoter and FOXP3 cDNA sequence are located within the FOXP3 gene, the AAVS1 locus or the TRAC gene of said CD34 ⁺ cell.

17. The CD34 ⁺ cell of claim 13, 14 or 16, comprising a nucleic acid encoding a T cell receptor (TCR).

17. The CD34 ⁺ cell of claim 13, 14 or 16, comprising a nucleic acid encoding a chimeric antigen receptor (CAR).

19. The cell of any one of claims 13-14 and 16-18, which is for treatment or prevention of a disease or condition in a subject.

20. The cells of claim 19, characterized in that they differentiate into T cells in a subject.

20. The cells of claim 19, characterized in that they differentiate into FOXP3+ regulatory T cells in a subject.

20. The cell of claim 19, wherein said disease or condition is an inflammatory disease, an autoimmune disease, or a condition associated with solid organ transplantation.

The disease is IPEX syndrome, graft-versus-host disease (GVHD), systemic lupus erythematosus, scleroderma, fused anemia, vasculitis, type 1 diabetes, Graves' disease, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis , Goodpasture syndrome, muscle disease, severe combined immunodeficiency, Di George syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, have leukocyte adhesion deficiency, NF-κB essential modulator (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferation, and/or ataxia telangiectasia; 20. A cell according to claim 19.

(a) a first homology arm having homology to sequences within the FOXP3 gene, the AAVS1 locus, or the TRAC gene of CD34 ⁺ cells;
(b) a second arm of homology having homology to a sequence within the same locus as the locus to which said first arm of homology has homology;
(c) a promoter; and (d) a sequence encoding FOXP3 or a functional derivative thereof,
A nucleic acid, characterized in that said promoter and a sequence encoding said FOXP3 or a functional derivative thereof are located between said first homologous arm and said second homologous arm.

(a) said FOXP3 or functional derivative thereof is wild-type human FOXP3;
and/or (b) the promoter is the MND promoter, the PGK promoter, or the EF1α promoter.

26. The nucleic acid of claim 25, wherein the sequence encoding FOXP3 or a functional derivative thereof is codon-optimized for expression in human cells.

27. The nucleic acid of claim 26, wherein the sequence encoding FOXP3 or a functional derivative thereof is the FOXP3 cDNA sequence.

28. The nucleic acid of claim 27, wherein said FOXP3 cDNA sequence comprises at least 90% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:110.

An adeno-associated virus (AAV) vector comprising a nucleic acid according to any one of claims 24-28.

A lipid nanoparticle comprising the nucleic acid of any one of claims 24-28.

31. The AAV vector of claim 29, or the lipid nanoparticle of claim 30, for the treatment or prevention of an inflammatory disease, an autoimmune disease, or a condition associated with solid organ transplantation in a subject.

IPEX syndrome, graft-versus-host disease (GVHD), systemic lupus erythematosus, scleroderma, fused anemia, vasculitis, type 1 diabetes, Graves' disease, inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, Goodpasture's syndrome , muscle disease, severe combined immunodeficiency, Di George syndrome, hyper-IgE syndrome, unclassifiable immunodeficiency, chronic granulomatosis, Wiskott-Aldrich syndrome, autoimmune lymphoproliferative syndrome, hyper-IgM syndrome, leukocyte adhesion deficiency , NF-κB essential modulator (NEMO) mutation, selective immunoglobulin A deficiency, X-linked agammaglobulinemia, X-linked lymphoproliferation, and/or ataxia telangiectasia. 32. The AAV vector or lipid nanoparticle of claim 31.