In vivo and ex vivo gene editing for Artemis-deficient SCID

Primary Immune Deficiencies (PID) are a heterogeneous class of diseases ascribed to individuals exhibiting deficiency or severe impairment of cellular and/or humoral immunity. This proposal focuses on a form of PID associated with radiation sensitivity and defects in V(D)J rearrangement caused by mutations in the DCLRE1C (Artemis) gene, “ART-SCID”. Artemis is a hairpin-opening enzyme that plays an essential role in V(D)J recombination, immunoglobulin and T cell receptor gene rearrangement in immunodevelopment. The only effective therapy for ART-SCID is allogeneic hematopoietic stem cell transplant (HSCT), for which there is a significant risk of morbidity and mortality. Our team demonstrated that ex vivo lentiviral correction of Artemis deficiency in a mouse model of SCID-A was most effectively accomplished using the endogenous Artemis promoter to regulate expression, forming the basis for our teams’ clinical trial initiated in 2018 (NCT03538899). However, results from clinical trials using viral vectors have highlighted some of the limitations of genomically dispersed vector-mediated integration, such as genotoxicity associated with insertional oncogene activation, genomic instability, transgene silencing, and disrupted proliferative control. What we envision in this proposal is the next generation of genetic therapies for Art-SCID by chromosomal site-directed correction of the endogenous Artemis gene in hematopoietic stem cells. The Specific Aims of this proposal address key questions in the development of a targeted genetic approach for therapy of ART-SCID and PID in general: Aim 1: What is the feasibility of targeted modification of the Artemis locus at a single genetic lesion for restoration of immune function? To address this question, we have developed an Adenosine Base Editing (ABE) approach that functionally restores a TAA nonsense mutation in the Artemis gene that is prevalent in Navajo and Apache Native Americans. We have also established a new mouse strain transgenic for the human TAA nonsense mutation (SCID-A mice). Using electroporation conditions that we have shown to be effective for introduction of CRISPR gene editing reagents, in Aim 1 we will deliver ABE along with guide RNA targeting the Artemis TAA stop codon in mouse hematopoietic stem cells transplanted into recipient SCID-A mice, evaluating the animals for restored immunity. Aim 2: How can targeted genetic modification be extended from ex vivo to in vivo delivery of CRISPR reagents targeting the Artemis gene? In Aim 2 we will test adenovirus vectors and AAV vectors targeted to hematopoietic stem cells for in vivo delivery of ABE and restoration of immunity in SCID-A mice. Results from these studies will have immediate implications for genetic therapy of ART-SCID and by extension for therapy of PID in general.