Recombinant vectors suitable for the treatment of IPEX syndrome
IPEX (Immune dysregulation Polyendocrinopathy X linked) syndrome is a primary immunodeficiency caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. Preclinical and clinical studies suggest that T cell gene therapy approaches designed to selectively restore the repertoire of Treg cells by transfer of wild type FOXP3 gene is a promising potential cure for IPEX. However, there is still a need for a vector that can be used efficiently for the preparation of said Treg cells. The inventors thus compared 6 different lentiviral constructs according to 4 criteria (vector titers, level of transduction of human CD4+ T cells, level of expression of FOXP3 and ΔLNGFR genes, degree of correlation between both expression) and selected one construct comprising a bidirectional PGK-EF1a promoter that showed remarkable efficiency.
1 . A method of producing a population of Treg cells, comprising the step of transfecting or transducing a population of T cells in vitro or ex vivo with a lentiviral vector comprising a recombinant nucleic acid molecule, wherein the recombinant nucleic acid molecule comprises
a bidirectional PGK-EF1a promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction, wherein the bidirectional PGK-EF1a promoter comprises a nucleic acid sequence as set forth in SEQ ID NO:5, and wherein
the first transgene is under control of the first PGK portion of the bidirectional PGK-EF1a promoter and encodes for a truncated low-affinity nerve growth factor receptor (LNGFR) comprising a nucleic acid sequence as set forth in SEQ ID NO: 8, and
the second transgene is under control of the second EF1a portion of the bidirectional PGK-EF1a promoter and encodes for FoxP3.
2 . A population of Treg cells obtainable by the method of claim 1 .
3 . The method of claim 1 , wherein the sequences of the first transgene and the second transgene are codon-optimized.
4 . The method of claim 1 , wherein
the second transgene comprises a nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:7.
5 . The method of claim 1 , wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence having at least 80% of identity with the nucleic acid sequence as set forth in SEQ ID NO:11.
6 . The method of claim 1 , wherein the recombinant nucleic acid molecule comprises a nucleic acid sequence as set forth in SEQ ID NO:11.