High-Titer Adeno-Associated Viral Vectors from a Rep/Cap Cell Line and Hybrid Shuttle Virus

Abstract

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function

“…Viral vector was produced by Targeted Genetics (Seattle, WA, USA), with the use of a B50 packaging cell line. 35 The rAAV titers were determined by dot blot, generating vector concentrations of 1Â10 11 and 1Â10 12 vg ml À1 . Subretinal injections were performed as previously described.…”

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…37,38 Stable packaging cell lines were created from HeLa cells by integration of the native AAV genome including a Rep-Cap gene cassette, but without both ITR sequences. 34,39,40 Induction of Rep and Cap proteins expression from a naturally silent native AAV promoter requires Ad E1 protein provided in trans from a helper virus. 39 Alternatively, Rep-Cap protein expression can be driven by a heterologous inducible promoter.…”

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

“…The B50 cell line expressing AAV Reps and Cap proteins was provided by Dr J Wilson (Institute for Human Gene Therapy and Department of Molecular and Cellular Engineering, University of Pennsylvania and The Wistar Institute, Philadelphia, PA, USA) and maintained and used for production of rAAV as described. 10 Plasmid psub201 containing AAV rep and cap genes were provided by Dr J Samulski (Human Gene Therapy Center, University of North Carolina at Chapel Hill, NC, USA) 30 and used for construction of rAdAAVrep-cap and rAAVGFP. Padeasy1 and pShuttle plasmid DNAs were provided by Dr B Vogelstein (Baltimore, MD, USA) 31 and used for construction of recombinant adenoviruses.…”

“…7,23 The rAAVGFP produced was purified from cell lysates by two cycles of CsCl 2 gradients followed by heat inactivation of recombinant adenoviruses at 56°C for 30 min as described. 9,10 To obtain an accurate titer for the rAAV-GFP if was necessary to eliminate possible contamination with rAdAAV-GFP, which also would result in expression of the GFP protein in the test cells. A neutralizing adenoviral antibody 1D6.14 9 was first titrated by limited dilution with a constant amount of AdCMVLacZ on 293 cells.…”

“…[9][10][11][12] The necessary helper functions are then provided by infection with a helper adenovirus (Ad) or transfection with plasmid DNA encoding Ad helper functions. 9 Although several packaging cell lines expressing the rep and cap gene products have been successfully used to achieve relatively high-titer production of rAAV, large-scale production of rAAV is still limited by the requirement for transfection of multiple DNAs encoding Ad helper functions or AAV rep and cap gene products.…”