Abstract
Viral vectors are extensively purified for use in biomedical research, in order to separate biologically active virus particles and to eliminate production related impurities that are assumed to be detrimental to the host. For recombinant adeno-associated virus (rAAV) vectors this is typically accomplished using density gradient-based methods, which are tedious and require specialized ultracentrifugation equipment. In order to streamline the preparation of rAAV vectors for pilot and small animal studies, we recently devised a simple ultrafiltration approach that permits rapid virus concentration and partial removal of production-related impurities. Here we show that systemic administration of such rapidly prepared (RP) rAAV8 vectors in mice is safe and efficiently transduces the liver. Across a range of doses, delivery of RP rAAV8-CMV-eGFP vector induced enhanced green fluorescent protein (eGFP) expression in liver that was comparable to that obtained from a conventional iodixanol gradient-purified (IP) vector. Surprisingly, no liver inflammation or systemic cytokine induction was detected in RP rAAV injected animals, revealing that residual impurities in the viral vector preparation are not deleterious to the host. Together, these data demonstrate that partially purified rAAV vector can be safely and effectively administered in vivo. The speed and versatility of the RP method and lack of need for cumbersome density gradients or expensive ultracentrifuge equipment will enable more widespread use of RP prepared rAAV vectors, such as for pilot liver gene transfer studies.
Highlights
Recombinant adeno-associated virus vectors are versatile tools for gene therapy due to their ability to mediate transgene expression for an extended period, their low immunogenicity profile and their wide tissue tropism
The day before transfection 7.5 x 106 HEK293A cells per T-150 flask (Corning) were plated in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and antibiotics. 3.75 μg of endotoxin-free pAAV2/8, 7.5 μg of pAd-Helper, Ultrafiltered rAAV8 efficiently transduces liver and 3.75 μg of pAAV-cytomegalovirus immediate-early promoter (CMV)-eGFP expressing enhanced green fluorescent protein under the control of a Cytomegalovirus promoter were transfected per T-150 flask using PEI “Max” transfection reagent (Polysciences, Inc.) in DMEM containing 2% FBS. 72 hrs post-transfection cells and culture media were harvested by adding 300 μl of 0.5 M EDTA
iodixanol purified (IP) and rapidly prepared (RP)-based rAAV8-CMV-eGFP vectors were intraperitoneally injected in groups of mice (n = 6) with 7.50 x 1011 vg/mouse, a dose level results in efficient liver transduction with this serotype and injection route [11] and that we demonstrated to induce supra-physiological transgene expression in the liver [12]
Summary
Recombinant adeno-associated virus (rAAV) vectors are versatile tools for gene therapy due to their ability to mediate transgene expression for an extended period, their low immunogenicity profile and their wide tissue tropism. They are based on adeno-associated virus (AAV), which is a small, single-stranded DNA virus belonging to the Parvoviridae family that can infect both dividing and non-dividing cells, but which requires a helper virus to replicate and is not known. Ultrafiltered rAAV8 efficiently transduces liver funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript
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