Helper virus-free, optically controllable, and two-plasmid-based production of adeno-associated virus vectors of serotypes 1 to 6

Abstract

We present a simple and safe strategy for producing high-titer adeno-associated virus (AAV) vectors derived from six different AAV serotypes (AAV-1 to AAV-6). The method, referred to as "HOT," is helper virus free, optically controllable, and based on transfection of only two plasmids, i.e., an AAV vector construct and one of six novel AAV helper plasmids. The latter were engineered to carry AAV serotype rep and cap genes together with adenoviral helper functions, as well as unique fluorescent protein expression cassettes, allowing confirmation of successful transfection and identification of the transfected plasmid. Cross-packaging of vector DNA derived from AAV-2, -3, or -6 was up to 10-fold more efficient using our novel plasmids, compared to a conservative adenovirus-dependent method. We also identified a variety of useful antibodies, allowing detection of Rep or VP proteins, or assembled capsids, of all six AAV serotypes. Finally, we describe unique cell tropisms and kinetics of transgene expression for AAV serotype vectors in primary or transformed cells from four different species. In sum, the HOT strategy and the antibodies presented here, together with the reported findings, should facilitate and support the further development of AAV serotype vectors as powerful new tools for human gene therapy.

Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection

“…Helper plasmids used for cross-packaging were generously provided by Dr Kleinsmidt (University of Heidelberg, Germany) and used as described earlier. 65 Briefly, pTRCGW (50 mg per roller bottle) and the helper plasmids (ratio 1:3) were co-transfected into 293T cells cultured in roller bottles (850 cm 2 surface, Greiner, Alphen aan Den Rijn, The Netherlands) using calcium phosphate. Each roller bottle contained 2.5 Â 10 7 cells.…”

“…Characterization of purified rAAV stocks included quantitative PCR analysis to determine the number of genomic copies (GCs), SDS-polyacrylamide gel electrophoresis analysis for visualization of the three capsid proteins 65 and an infection assay of 293T cells to verify the infectivity. The infection assay was performed using serial dilutions of the vector, by adding the viral vector to camptothecin-treated (4.8 mg ml À1 for 4 h) 293T cells.…”

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…AAV2 vectors carrying wt capsids were constructed by co-transfection of pUF2CMV-Luc with the helper plasmid pDP2. 53 For production of AAV2 vectors with detargeted capsids, pDG (R484E;R585E) containing double mutations of capsid protein amino acids (R484E; R585E) involved in heparin binding of AAV2 was co-transfected with pUF2CMV-Luc. 38 For generation of the targeted d-sarcoglycan vectors, we used pMT187VNSTRLPXX2, pDGDVP and pdsCMV-MLC0.26-SGCD, a derivate of pdsAAV-CMV-GFP 54 with the murine d-sarcoglycan cDNA introduced with BsrGI/NheI under the control of a MluI/HindIII fragment of the CMV-enhanced myosin light chain (CMVenhMLC) promoter.…”

Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6

“…Single-stranded rAAV2/6 expressing eGFP under control of a hybrid construct driven by the cytomegalovirus (CMV) promoter and the human beta-globin intron (glb), was produced in 293AAV cells as described previously. 24 Cell lysates were purified on a heparin affinity column (HiTrap Heparin HP, GE Healthcare, Upsala, Sweden) and the concentrated eluate titrated by (i) real-time PCR against the vector genome sequence (viral particles) and (ii) infection of 293T cells and flow cytometry analysis 48-h post infection (transducing units). Animals were injected into the gastrocnemius muscle of the right leg with 1.3 Â 10 12 viral particles (3.3 Â 10 9 transducing units) of rAAV2/6.…”