Method for treating autoimmune disease using cd4 t-cells with engineered stabilization of expression of endogenous foxp3 gene

Abstract

Disclosed are methods of making a genetically cell that expressed FOXP3 and methods of treatment. In some embodiments, the method comprises providing a first nucleotide sequence, wherein the first nucleotide sequence comprises a coding strand, the coding strand comprising one or more regulatory elements and a FOXP3 gene or portion thereof providing a nuclease and performing a gene editing process on the first nucleotide sequence, which edits said one or more regulatory elements, and optionally edits the FOXP3 gene or portion thereof. Methods of treating a subject suffering from an autoimmune disease and subjects suffering the effects of organ transplantation are also provided.

1 . (canceled) 
     
     
         2 . A genetically modified cell comprising a heterologous promoter operably linked to a FOXP3 gene on a nucleic acid in the cell genome, wherein the heterologous promoter is:
 (i) downstream from a Treg-specific demethylated region (TSDR) of the FOXP3 gene; and   (ii) upstream from a first coding exon of the FOXP3 gene.   
     
     
         3 . The genetically modified cell of  claim 2 , wherein the heterologous promoter is a constitutive promoter. 
     
     
         4 . The genetically modified cell of  claim 3 , wherein the constitutive promoter is an EF-1α promoter, MND promoter, or PGK promoter. 
     
     
         5 . The genetically modified cell of  claim 2 , wherein the heterologous promoter is an MND promoter. 
     
     
         6 . The genetically modified cell of  claim 2 , wherein the heterologous promoter is an inducible promoter. 
     
     
         7 . The genetically modified cell of  claim 2 , wherein the first coding exon of the FOXP3 gene is a natural first coding exon of an endogenous FOXP3 gene. 
     
     
         8 . The genetically modified cell of  claim 2 , wherein the first coding exon of the FOXP3 gene is a synthetic first coding exon. 
     
     
         9 . The genetically modified cell of  claim 2 , wherein the nucleic acid further comprises a heterologous transcriptional enhancer domain. 
     
     
         10 . The genetically modified cell of  claim 2 , wherein the nucleic acid further comprises a heterologous transcriptional activation domain. 
     
     
         11 . The genetically modified cell of  claim 2 , wherein the nucleic acid further comprises a ubiquitous chromatin opening element (UCOE). 
     
     
         12 . The genetically modified cell of  claim 2 , wherein the cell is a CD4 +  T cell. 
     
     
         13 . The genetically modified cell of  claim 2 , wherein the cell is a CD8 +  T cell. 
     
     
         14 . The genetically modified cell of  claim 2 , wherein the cell is a regulatory T (Treg) cell. 
     
     
         15 . The genetically modified cell of  claim 14 , wherein the cell is CD25 + , CD127 − , CTLA-4 + , and/or LAG3 + . 
     
     
         16 . The genetically modified cell of  claim 2 , wherein the cell expresses a T cell receptor (TCR). 
     
     
         17 . The genetically modified cell of  claim 2 , wherein the cell expresses a chimeric antigen receptor (CAR). 
     
     
         18 . The genetically modified cell of  claim 2 , wherein the cell is a CD4 +  regulatory T (Treg) cell expressing a T cell receptor (TCR), and wherein the heterologous promoter is an MND promoter. 
     
     
         19 . The genetically modified cell of  claim 2 , wherein the cell is a CD4 +  regulatory T (Treg) cell expressing a chimeric antigen receptor (CAR), and wherein the heterologous promoter is an MND promoter. 
     
     
         20 . The genetically modified cell of  claim 2 , wherein the cell is a CD8 +  regulatory T (Treg) cell expressing a chimeric antigen receptor (CAR), and wherein the heterologous promoter is an MND promoter. 
     
     
         21 . A pharmaceutical composition comprising the genetically modified cell of  claim 2 , and a pharmaceutically acceptable excipient.