A treatment method for autoimmune diseases using CD4 T cells in which the expression of the endogenous FOXP3 gene has been stabilized by genetic recombination.

Abstract

To provide a method for producing a recombinant cell expressing FOXP3, and to provide a method of treatment.SOLUTION: In some embodiments, the methods of the invention may comprise: providing a first nucleotide sequence comprising a coding strand comprising one or more regulatory elements and a FOXP3 gene or a portion thereof; providing a nuclease; and editing the one or more regulatory elements and optionally gene editing the first nucleotide sequence by editing the FOXP3 gene or a part thereof. Furthermore, a method for treating a subject having an autoimmune disease and a method for treating a subject who has caused side effects of organ transplantation are provided.SELECTED DRAWING: None

Claims

[Claim 1] A nucleic acid editing template for stabilizing FOXP3 expression in cells, (i) One or more heterogeneous regulatory factors, (ii) A heterologous constitutive promoter operably ligated to the FOXP3 gene, or to a portion of the FOXP3 gene that includes at least a portion of the first coding exon of the FOXP3 gene. Includes, including code chain, A nucleic acid editing template in which the heteroconstitutive promoter is located upstream of the first coding exon or a portion thereof, and a T reg-specific demethylation region (TSDR) does not exist between the heteroconstitutive promoter on the coding strand and the first coding exon or a portion thereof. [Claim 2] The nucleic acid editing template according to claim 1, wherein the heterogeneous promoter is an EF1α promoter, a PGK promoter, or a MND promoter. [Claim 3] The nucleic acid editing template according to claim 1 or 2, wherein the heterogeneous constituent promoter is an MND promoter. [Claim 4] The nucleic acid editing template according to any one of claims 1 to 3, wherein the first coding exon is a synthesized first coding exon. [Claim 5] The nucleic acid editing template according to any one of claims 1 to 3, wherein the first coding exon is an endogenous first coding exon. [Claim 6] The nucleic acid editing template according to any one of claims 1 to 5, further comprising a heterologous transcription enhancer region. [Claim 7] The nucleic acid editing template according to any one of claims 1 to 6, further comprising a heterologous transcription activation region. [Claim 8] The nucleic acid editing template according to any one of claims 1 to 7, further comprising a ubiquitous chromatin opening element (UCOE). [Claim 9] A method for producing nucleic acids for the stable expression of FOXP3 in cells in vitro or ex vivo, (i)T reg To provide a first nucleic acid containing a specific demethylation region (TSDR) and the FOXP3 gene; (ii) To provide a nuclease that can be cleaved at a target gene locus or a second nucleic acid encoding the nuclease; and (iii) Perform gene editing by inserting a heterologous constitutive promoter into the first nucleic acid. Includes, The FOXP3 gene includes a first coding exon, A method in which, after insertion of the heterogeneous promoter, (a) the heterogeneous promoter is located upstream of the first coding exon, and (b) the TSDR is not present between the heterogeneous promoter and the first coding exon. [Claim 10] The method according to claim 9, wherein the target gene locus is located in the first coding exon. [Claim 11] The method according to claim 9, wherein the target gene locus is located within 10 base pairs, 20 base pairs, 30 base pairs, 40 base pairs, 50 base pairs, 60 base pairs, 70 base pairs, 80 base pairs, 90 base pairs, 100 base pairs, or 110 base pairs upstream of the first coating exon, or within a number of base pairs defined by any two of these numbers. [Claim 12] The method according to any one of claims 9 to 11, wherein the gene editing generates a synthesized first coding exon. [Claim 13] The method according to any one of claims 9 to 12, wherein the nuclease is Cas9, zinc finger nuclease, or TALEN. [Claim 14] The method according to any one of claims 9 to 13, wherein the heterogeneous promoter is an EF1α promoter, a PGK promoter, or a MND promoter. [Claim 15] The method according to any one of claims 9 to 14, wherein the heterogeneous constituent promoter is an MND promoter. [Claim 16] The method according to any one of claims 9 to 15, further comprising inserting one or more heterologous regulatory factors into the first nucleic acid such that the gene editing is operably linked to the FOXP3 gene. [Claim 17] The method according to claim 16, wherein one or more heterologous regulatory factors include heterologous transcription enhancer regions. [Claim 18] The method according to claim 16 or 17, wherein the one or more heterologous regulatory factors include heterologous transcriptional activation regions. [Claim 19] The method according to any one of claims 16 to 18, wherein the one or more heterogeneous regulatory factors include a ubiquitous chromatin opening element (UCOE).

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