Abstract
Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (IM) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2–3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the IM or the intravenous (IV) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.
Highlights
Recombinant Adeno-Associated Virus-mediated gene transfer can sustain long-term expression of a protein in large animal models after a single administration in skeletal muscle [1,2,3,4]
Similar to other non-integrating viruses [12,13], we recently demonstrated that the Recombinant Adeno-Associated Virus (rAAV) episomes assimilate, in nonhuman primates (NHP) skeletal muscle, into a chromatin-like structure where nucleosomes are regularly assembled along the viral genome in a pattern similar to cellular chromatin [4]
We have previously shown that the rAAV-mediated expression of the LEA29Y transgene under the Rous Sarcoma Virus promoter (RSVp) transcriptional control was stable for more than 2 years in IM injected primates (Mac 1, 2, and 9) [4]
Summary
Recombinant Adeno-Associated Virus (rAAV)-mediated gene transfer can sustain long-term expression of a protein in large animal models after a single administration in skeletal muscle [1,2,3,4]. After intramuscular (IM) injection of single-stranded AAV (ssAAV) vectors in nonhuman primates (NHP), a rise in the level of vector-expressed secreted proteins is seen in the serum during the first 2–3 months (Phase 1), followed by a progressive decrease (Phase 2) [1,2,3,4]. The protein concentration reaches a steady-state (Phase 3) 6–8 months post-injection (pi) at half the maximum level. Whereas rAAV trafficking and double-stranded conversion characterizing Phase 1 were extensively studied [8,9], the mechanism(s) underlying decreasing protein level in Phase 2 in NHP has not been elucidated
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