The Pre-Clinical Development of Gene Therapy for DOCK8 Deficiency

Abstract

Background: DOCK8 deficiency is a combined immunodeficiency leading to recurrent infections and severe allergic inflammation. If left untreated, survival rates into adulthood are low. While allogeneic hematopoietic stem cell transplant (HSCT) can cure this disease, suitable donors are not universally available, and morbidity such as graft-versus-host disease (GvHD) remains problematic. The development of novel curative therapies for these patients is therefore considered a high-priority. Objective: To develop a pre-clinical model of gene therapy (GT) for DOCK8 deficiency. Methods: To determine which viral vector would be best suited for in vivo transplantation experiments in Dock8-/- mice, we first generated and compared the expression of a codon-optimized cDNA of DOCK8 in a lentiviral (LV) versus alpharetroviral (aRV) backbone by transducing the human Jurkat T cell line (DOCK8 null), and comparing DOCK8 expression of both vectors by flow cytometry analysis. We next evaluated the efficacy of various ubiquitous promoters (SFFV, MND, EFS) at driving DOCK8 expression using the same methods. To test whether these vectors could reconstitute DOCK8 function, we stimulated transduced Jurkat T cells with IL- 21 and assessed restoration of STAT3 phosphorylation via flow cytometry. Vector copy numbers (VCN) were determined by qPCR. Results: The lentiviral construct consistently drove better DOCK8 expression (% DOCK8+ cells: 16.7% vs 2.0%, p<0.01; MFI: 256.7 vs 233.7, p=0.02) and better DOCK8 function (pSTAT3+ cells: 16.4% vs 3.1%). When compared to MND, the EFS promoter required a higher VCN for the same transduction efficiency (% DOCK8+ cells 35.6% vs 35.2%, p=0.9; MFI 275.3 vs 221.7, p=0.005, mean VCN 5.3 vs 3.4 copies/cell, p=0.02). Finally, we discovered significant toxicity from overexpression of DOCK8 after transduction of murine HSCs. Conclusion: Taken together, these data indicate the potential of lentiviral vectors at driving high DOCK8 expression with restoration of function in the context of strong nonselective promoters, highlighting the feasibility of their use in gene therapy for DOCK8-deficient patients. However, we also uncovered unexpected toxicity from DOCK8 overexpression in HSCs. A vector with a more tightly regulated pattern of DOCK8 expression is therefore required to pursue in vivo GT experiments in a Dock8-/- mouse model.