Ultrafiltered recombinant AAV8 vector can be safely administered in vivo and efficiently transduces liver

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.

AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria

“…The OHSU Molecular Virology Support Core produced large-scale preparations of recombinant rAAV2/8 viruses, referenced as rAAV2/8_LSP_SpCas9 and AAV2/ 8_Ex7RepairTemp_U6G1, using standard triple plasmid transfection procedures into cultured HEK293 cells and purified by iodixanol gradient ultracentrifugation. 49 AAV titers were determined using ITR-based qPCR analysis.…”

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload

“…via tail vein with an AAV2/8 virus encoding the full-length murine Tfr2 ORF under the regulation of a hepatocyte-specific promoter (AAV2/8-Tfr2). PBS-injected mice were used as controls because previously published PBS-and AAV2/8-GFP-injected mice indicated no differences in responses to any of the parameters measured (37,38). RT-quantitative PCR and Western blot analysis confirmed liver-specific expression of TFR2 3 weeks after injection (Fig.…”

Approaches to purification and concentration of rAAV vectors for gene therapy

“…This method did not affect either the efficiency of transduction in mouse liver cells or the induction of inflammation after systemic delivery. [ 94 ]…”