AAV VECTORS: GENOME AND CAPSID MODIFICATIONS 18. Incorporation of the Green Fluorescent Protein into the Adeno-Associated Virus Type 2 Capsid Kerstin Lux,1 Nico Goerlitz,1 Kristin Leike,1 Daniela Goldnau,1 Stefan Finke,2 Michael Hallek,1,3,4 Hildegard Buening.1 1 Gene Center, Ludwig Maximilian University of Munich, Munich, Germany; 2Max von Pettenkofer Institute, Ludwig Maximilian University of Munich, Munich, Germany; 3First Department of Internal Medicine, University Hospital Cologne, Cologne, Germany; 4GSF-National Center for Research and Environment, KKG Gene Therapy, Munich, Germany. Adeno-Associated Virus type 2 (AAV) is a small, nonenveloped, icosahedral virus of approximately 25 nm in diameter that packages a single-stranded DNA. Until now, no human disease caused by AAV has been detected. This and other features as e.g. its ability to transduce both dividing and non-dividing cells, its low immunogenicity and its broad tropism make of AAV a promising system for the development as gene therapy vector. However, many aspects of its infectious biology still remain to be elucidated. Green fluorescent protein (GFP) has been extensively used to study intracellular trafficking of proteins. Therefore, we incorporated GFP into the AAV capsid in order to allow a direct visualization of the infectious process. Based on earlier results, obtained by Yang et al. (1998), we generated an N-terminal fusion of the GFP protein with the second largest capsid protein, named VP2. We could show by transient transfection assays that this fusion protein is tranported into the nucleus like wild type capsid protein. Next, we generated viral particles containing the GFP-VP2 fusion protein. Viral progeny was obtained with titers comparable to wild type AAV and could be purified by iodixanol gradient centrifugation or heparin affinity chromatography. The GFP-VP2 fusion protein was detected together with the other wild type capsid proteins in Western Blot analysis of purified viral preparations. The particle to capsid ratio showed that the fusion protein does not interfere with viral genome packaging. Furthermore, HeLa cells infected with GFP-VP2 containing virions resulted in eGFP positive cells measurable by FACS analysis. Such infections could be inhibited by the addition of heparin. Finally, fluorescent viral particles could be visualized by live cell imaging and fluorescent microscopy. First results will be presented.
The Transcapsidation of AAV Serotypes
Joseph E. Rabinowitz,2 Dawn E. Bowles,2 Susan M. Faust,2 Julie G. Ledford,2 Scott E. Cunningham,2 R. Jude Samulski.1 1 Department of Pharmacology; 2Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC. The ability to pseudotype enveloped viruses enables the recombinant genome of one variety of virus to transduce cells susceptible to another kind of virus. Adeno-associated viruses (AAV) which lack an envelope cannot be pseudotyped according to this strict definition. Instead the capsid components (Vp1, Vp2, and Vp3) from the different serotypes should be able to be mixed during production and used to transcapsidate rAAV genomes. Whether the resulting mixed viruses exhibited altered or novel transduction profiles can be evaluated. Pair wise combinations of AAV serotypes 1-5 helper plasmids were used to produce transcapsidated rAAV in an otherwise standard transfection protocol. Five ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these helper plasmids were used in the production scheme. High titer rAAV was obtained with mixtures that included either serotypes 1, 2, or 3 while rAAV of intermediate titer was obtained from serotype 5 mixtures. Only mixtures containing the AAV4 capsid exhibited reduced packaging capacity. Binding profiles of these mixed virus preparations to either heparin sulfate (HS) or mucin agarose were tested. Transduction of five cell lines (HeLa, C2C12, CHO K1, S8
CHO pgsD, and CHO pgsE), was used to further evaluate the phenotypes of these transcapsidated virions. From these studies four transduction profiles were observed: i. small to no change regardless of ratio, ii. a gradual increase in transduction consistent with titration of a second capsid component, iii. an abrupt increase in transduction (threshold effect) dependent on specific ratios used, or iv. an unexpected increase (synergy) in transduction. The synergistic profile was observed only with mixtures of AAV1 helper combined with type 2 or type 3 recipient helpers. At least two components contributed to this observed synergy: i. heparin mediated binding from AAV2 and ii. an as of yet identified enhancement activity from AAV1 structural proteins. The in vivo transduction capabilities of these AAV1/AAV2 mixed virions were evaluated using a luciferase imaging system following direct injection into mouse muscle. The synergy observed in vitro was reproduced in mouse muscle. This straight forward approach of mixing different AAV helper plasmids to generate mixed AAV virions can be applied to all AAV serotypes and should allow for continued analyses of AAV capsid assembly, receptor-mediated binding, and virus trafficking. Exploitation of this approach in generating custom designed AAV vectors should be of significant value to the field of gene therapy
20. Directed Evolution of AAV2 Yields Novel Capsid Variants Narendra Maheshri,1 Brian Kaspar, David V. Schaffer.1 Chemical Engineering & Neuroscience, University of California at Berkeley, Berkeley, CA. 1
Adeno-associated virus (AAV) is a highly promising gene delivery vehicle, though gene transfer barriers can in cases limit its efficiency. These barriers arise at every step of delivery: the transit of the vector from injection to a cell surface, receptor binding and uptake, intracellular trafficking, and nuclear steps. The AAV gene transfer properties at each of these steps are determined by its capsid structure. Previous capsid modifications that alter AAV tropism, as well as the existence of multiple AAV serotypes, suggest that the structure-function relationships of the AAV capsid are reasonably plastic. We have taken advantage of this remarkable capsid plasticity to generate a large random mutant AAV library (1e6) and select for mutant AAV virions that can overcome several barriers to infection. Specifically, we have selected the AAV2 library for infectious particles with altered heparan sulfate (HS) affinity and for the ability to evade an AAV2 immune response. First, we have generated mutants with both lower and higher affinity to heparin, which could prove valuable in controlling the therapeutic zone of an AAV vector in tissues where ECM HS hinders AAV2 diffusion. Furthermore, we have generated vector variants that have greater than 100-fold improved resistance to human serum that neutralizes wild type AAV2, yet retain AAV2 gene delivery efficiency. These vectors may enable high gene delivery efficiency even in patients with preexisting immunity, and the locations of point mutations on the capsid surface suggest new regions of functional importance to the virus. These AAV libraries therefore both provide useful variants for gene therapy applications as well as offer new avenues to dissect basic AAV biology.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy