Abstract
Though successful clinical gene therapy using viral vectors to deliver corrective genes to hematopoietic stem cells (HSCs) has been achieved in a select group of individuals, gene transfer efficiency limits application from disorders in which higher gene transfer rates are necessary. Pools of evidence have revealed a distinguishable arm of the innate immune system that potentially limits the delivery of vector into target cell populations. To address the contribution of the innate immune system in limiting delivery to hematopoietic stem cell targets, we analyzed the endogenous expression of innate immune genes among mobilized peripheral blood (PB) CD34+ cells before and after two 36 hour exposures to VSV-G psuedotyped lentiviral vectors. Semi-quantitative analysis of endogenously expressed innate immune genes by real-time PCR revealed a relative 100 and 400 fold greater expression of APOBEC3G (A3G) and 2′–5′ Oligoadenylate synthetase 2a (OAS), respectively, in PB CD34+ cells after a 24 hour prestimulation in the presence of Flt-3 ligand, SCF, and TPO relative to 293T cells. PB mononuclear cell (PBMNC) exhibited a 140 and 100 fold greater expression of A3G and OAS, respectively, over 293T cultures. Following transduction with a VSV-G pseudotyped lentivirus carrying GFP (m.o.i 40), a 2–5 fold increase in PKR and a 4 fold increase of OAS, both at 36 hours post 1st and 2nd viral exposure, was observed. Interestingly, lentiviral transduction induced a 3 fold increase in A3G at 36 hours after the 1st lentiviral transduction only, with no further increase after the second. Furthermore, HeLa, 293T, and PBMNC cultures were tested for their responsiveness to interferon(s); established regulators, effectors, and activators of the innate and adaptive immune response. Addition of 103U/ml IFNa-2a in PBMC cultures induced a 2 fold increase in A3G, a 5 fold increase in OAS, and a 20 fold increase in PKR within 3hours after treatment, when normalized to b-actin expression. Likewise, IFNa-2a generated a 5 fold increase in OAS in 293T, PBMC, and PB CD34+ cells. In contrast, IFNa-2a did not generate A3G responsiveness in Hela or 293T cultures. To investigate the role double-stranded RNA (dsRNA) has on the gene activity of dsRNA-activated innate proteins, cultures where titrated with synthetic RNA, polyinosinic-cytidylic acid (pI:C). A3G mRNA expression increased by 1800 fold in PBMC cultures compared to transfection with DNA. In contrast, OAS expression was activated 5 fold relative to DNA. Although A3G gene activity was increased by 80 fold in PB CD34+ cultures, the gene was less responsive requiring 200 times more concentrated pI:C (10μg/ml). As expected, OAS gene activity was non responsive to synthetic dsRNA in CD34+ cultures. To further investigate these findings, siRNA targeted at A3G and OAS messages will be used to investigate their role in limiting transduction efficiency. These data argue the potential for improving transduction efficiency to clinical utility by abrogating the expression of A3G and OAS during in vitro PB CD34+ cell transduction. Innate immune gene responsiveness among PB CD34+ cells may confer the ability to induce and maintain a strong intrinsic antiviral response decreasing transduction efficiency.
Effects of Overexpression of the Egyptian Fruit Bat Innate Immune Genes on Filovirus Infections in the Host Cells
