Correction of Murine Rag2 Severe Combined Immunodeficiency by Lentiviral Gene Therapy Using a Codon-optimized RAG2 Therapeutic Transgene

Abstract

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2 −/− mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2 −/− mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.

RAG gene defects at the verge of immunodeficiency and immune dysregulation

“…When Rag2 −/− mice were transplanted with Rag2 −/− lineage-negative bone marrow cells transduced with a lentiviral vector expressing codon-optimized hRAG2 (coRAG2) under the control of ubiquitous chromatin-opening element (UCOE), good levels of both T-and B-cell reconstitution were observed at a VCN of 1. 146 In particular, gene therapy-treated mice showed normalization of thymic morphology with appearance of AIRE + mature mTECs. In the periphery, polyclonality of the T-cell repertoire, normalization of T-cell subsets and of immunoglobulin levels, and production of antibodies to T-dependent and Tindependent antigens were detected.…”

Human RAG mutations: biochemistry and clinical implications

“…More promising preclinical results have been obtained with gene therapy for RAG2 deficiency 95,96 . In particular, the use of vectors expressing codon-optimized human RAG2 and containing other modifications to reduce the risk of silencing of the RAG2 transgene resulted in significant improvement of peripheral T cell and B cell immunity in mice even at a relatively low vector copy number, in spite of a subnormal number of single positive T cells in the thymus and the persistence of a higher proportion of pro-B cells in the bone marrow 96 .…”

“…In particular, the use of vectors expressing codon-optimized human RAG2 and containing other modifications to reduce the risk of silencing of the RAG2 transgene resulted in significant improvement of peripheral T cell and B cell immunity in mice even at a relatively low vector copy number, in spite of a subnormal number of single positive T cells in the thymus and the persistence of a higher proportion of pro-B cells in the bone marrow 96 . These data offer hope for the development of gene therapy for human RAG2 deficiency in the near future, but it remains to be seen whether this strategy will be efficacious in patients with hypomorphic RAG2 mutations, for whom competition between endogenous, uncorrected, lymphoid progenitors and gene-transduced cells might affect the quality of immune reconstitution.…”

How I treat severe combined immunodeficiency

“…Current guidelines for the management of SCID-X1 were agreed upon by the Inborn Errors Working Party of the ESID/EBMT and are shown in Figure 4. 43 Preclinical studies in murine models have shown the success of gene therapy in correcting T-and B-cell development defects in the RAG1/2 60,61 and Artemis forms of SCID, 62 and it is likely that clinical trials for these forms will start in the next few years.…”