Production of Recombinant Adeno‐Associated Viral Vectors for In Vitro and In Vivo Use

Abstract

Adeno-associated virus is a nonpathogenic human virus that has been developed into a gene-delivery vector due to its high efficiency of infection in many different cell types and its ability to persist and lead to long-term gene expression. The vector is also a valuable tool in molecular biology experiments. This unit describes efficient methods to generate high-titer, research-grade, adenovirus-free, recombinant single-stranded and self-complementary adeno-associated virus in various serotypes, along with methods to quantify the viral vectors.

Effects of adeno‐associated virus‐2‐mediated human BMP‐7 gene transfection on the phenotype of nucleus pulposus cells

“…Disadvantages of using AAV are the limited packaging constraints (<5 kb) and the laborious and technically difficult procedures required for viral AAV vector production. [14][15][16][17][18][19][20] Based on the empirical advantages of rAAV2 vectors and its clinical potential, rAAV2 vectors were selected for this study to deliver the human BMP-7 gene into canine NP cells. The recombinant AAV2-hBMP7 vector was constructed and able to successfully transfect the canine NP cells, as determined by PCR analysis, and to express hBMP7 protein shown by Western blot.…”

“…12 Compared with other vector systems, recombinant adeno-associated virus-2 (rAAV2) vectors have several properties making them attractive candidates for musculoskeletal gene therapy. 13 These properties include: (i) the absence of inflammatory, cytotoxic, or cell-mediated immune host responses that attack the transduced cells and threaten the host 14 ; (ii) a broad cellular tropism 14,15 ; (iii) the ability to infect nondividing cells 16,17 ; (iv) the ability to deliver lifetime gene expression in some cell types [18][19][20] ; and (v) the production of high titers (>10 13 vg/ml) using easy ultra-high grade purification techniques.…”

In vivo disruption of latent HSV by designer endonuclease therapy

“…AAV vectors of all serotypes were generated by transiently transfecting 293 cells using polyethylenimine (PEI) according to the method of Choi et al (53). Briefy, HEK293 cells were transfected with a scAAV or ssAAV vector plasmid, a plasmid that expresses the AAV rep and capsid proteins, and a helper plasmid that expresses adenovirus helper proteins (pHelper).…”

“…At 24 hours after transfection, media was changed to serum-free DMEM, and after 72 hours, cells were collected and resuspended in AAV lysis buffer (50mM Tris, 150 mM NaCl, pH 8.5) before freeze-thawing 4 times. AAV stocks were purified by iodixanol gradient separation (53, 54) followed by concentration into PBS using an Amicon Ultra-15 column (EMD Millipore) before storage at –80°C. All AAV vector stocks were quantified by qPCR using primers/probes against the AAV ITR, with linearized plasmid DNA as a standard, according to the method of Aurnhammer et al (55).…”

AAV-Mediated Gene Targeting Is Significantly Enhanced by Transient Inhibition of Nonhomologous End Joining or the ProteasomeIn Vivo

“…Virus was pelleted and precipitated with 1 M CaCl 2 , followed by 40% PEG-8000 treatment and resuspension in a sodium/HEPES buffer and rotated overnight at 4°C. After resuspension, purification by sequential cesium chloride density centrifugation was performed as described (Choi et al, 2007) with the following modifications. Peak fractions from both gradients were determined by optical refractometry.…”