Abstract
Recombinase-activating gene-1 (RAG1)-deficient severe combined immunodeficiency (SCID) patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. Gene therapy is an alternative for those RAG1-SCID patients who lack a suitable bone marrow donor. We designed lentiviral vectors with different internal promoters driving codon-optimized RAG1 to ensure optimal expression. We used Rag1−/− mice as a preclinical model for RAG1-SCID to assess the efficacy of the various vectors. We observed that B and T cell reconstitution directly correlated with RAG1 expression. Mice with low RAG1 expression showed poor immune reconstitution; however, higher expression resulted in phenotypic and functional lymphocyte reconstitution comparable to mice receiving wild-type stem cells. No signs of genotoxicity were found. Additionally, RAG1-SCID patient CD34+ cells transduced with our clinical RAG1 vector and transplanted into NSG mice led to improved human B and T cell development. Considering this efficacy outcome, together with favorable safety data, these results substantiate the need for a clinical trial for RAG1-SCID.
Highlights
Severe combined immunodeficiency (SCID) is a life-threatening disorder of the adaptive immune system.[1]
We found that UCOE-c.o.recombination-activating gene-1 (RAG1) reached a lower viral copy number (VCN) (Figure 1D) than did the other vectors, and PGK-c.o.RAG1 was the vector with the lowest promoter strength (Figure 1F)
Both PGK and UCOE-c.o.RAG1 only resulted in low levels of c.o.RAG1 expression (Figure 1E) whereas quite high levels are known to be required for immune reconstitution.[24,26,33]
Summary
Severe combined immunodeficiency (SCID) is a life-threatening disorder of the adaptive immune system.[1] In all forms of SCID, the development of T cells in the thymus is arrested due to genetic defects in genes essential for this complex process, while concomitant deficiencies in B lymphocytes and natural killer (NK) cells depend on the SCID genotype. Affected infants are born with a severe T lymphocyte deficiency and die within the first year of life unless effective treatment is given. The third major type of SCID is formed by recombination deficiencies. In these types of SCID the recombination machinery that is responsible for variable diversity joining (V(D)J) recombination of T cell receptor (TCR) and immunoglobulin (Ig) genes is affected.
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