Gene therapy for Athabascan SCID

Abstract

Artemis is an endonuclease characterized as a key factor involved in both nonhomologous end joining (NHEJ) and variable (diversity) joining (V(D)J) recombination. Mutations in the gene encoding Artemis result in a radiationsensitive form of severe combined immunodeficiency (SCID) found at a high incidence in Athabascan-speaking Native Americans (SCID-A) and characterized by the absence of mature B and T lymphocytes. Early treatment is critical since otherwise the disease results in severe infections that ultimately lead to fatality at a young age. The current therapy for SCID-A is allogeneic hematopoeitic cell transplantation (HCT); however, HCT often results in incomplete reconstitution of B lymphocytes and may lead to complications such as graft versus host disease. Transplantation with genetically corrected autologous cells is an alternative approach that may provide improved treatment of SCID-A. Lentiviral vectors pseudotyped with VSV-G are compelling candidate vectors for gene transfer considering their high transduction efficiency and capability to mediate gene transfer in non-dividing cells populations, such as quiescent hematopoietic stem cells. Accordingly, I developed several lentiviral vectors for the transduction of human Artemis cDNA into hematopoeitic cells for the correction of a murine model of SCID-A. Upon characterization of these vectors I found that Artemis over-expression results in a decrease in cell survival due to genomic DNA fragmentation, cell cycle arrest, and ultimately apoptosis. These data emphasize the importance of transgene regulation and demonstrate the necessity of establishing conditions that provide Artemis expression at a level iv that is non-toxic yet sufficient to complement Artemis deficiency. To this end, I subsequently recovered and characterized the endogenous human Artemis promoter (APro) as a one-kilobase region located directly upstream of the human Artemis translational start site. APro conferred a moderate level of reporter gene expression in vitro and in vivo, including secondary mouse transplant recipients, thus demonstrating reliable expression after lentiviral gene transfer into hematopoeitic stem cells. Subsequently, I compared innate regulation of the human Artemis cDNA using its own endogenous promoter sequence to that of the strong EF1α and more moderate PGK promoter for the capacity to mediate correction of a murine model of Artemis deficiency presenting a B- T- phenotype and exhibiting no leakiness (mArt -/-). Transplantation with both APro-hArtemis and PGK-hArtemis transduced mArt -/- marrow led to complete reconstitution of the immune compartment in the recipient animals. Beginning at 8 weeks posttransplant, the recipient animals had wild-type levels of CD3+CD4+ and CD3+CD8+ T lymphocytes and B220+NK1.1- B lymphocytes, cell populations that are absent in mArt -/- immunodeficient mice. However, transplantation with EF1α-hArtemis transduced marrow did not support immune reconstitution, suggestive of cytotoxic effects caused by Artemis over-expression. AProhArtemis treated mice exhibited restored IgM and IgG responses against 4- hydroxyl-3-nitrophenylacetyl hapten conjugated-keyhole limpet hemocyanin as well as restored cellular immune function, as assessed by in vitro stimulation of isolated splenocytes with anti-CD3 or concanavalin A. These results demonstrate that the naturally regulated Artemis lentiviral vector effectively complemented murine SCID-A, contributing to the development and advancement of gene transfer as a clinically relevant and feasible approach for treatment of SCID-A in humans.