MUTAGENESIS OF AAV CAPSID PROTEINS FOR ENHANCED TRANSDUCTION OF CARDIAC CELL TYPES

Background: Adeno- associated virus serotype 9 (AAV9) has a high transduction efficiency for cardiac tissues, along with liver, skeletal muscle, pancreatic tissue, and the eye, versus other AAV serotypes. Unfortunately, nonspecific targeting to the intended tissue can result in the need for use of higher particle numbers to obtain intended transduction of the target tissue, which may also increase chances of initiating innate and adaptive immune responses. Such limitations require the need to bioengineer AAV9, and other serotypes including AAV6 and AAV8, in order to develop recombinant AAV vectors with cardiac tissue specific targeting and altered surface antigenic properties that may evade phosphorylation mediated degradation, neutralization, and clearance. Methods: Random mutagenesis PCR was used to mutate the external surface domains of AAV9 and chimeric AAV9-2 capsids. Mutations in the VP3 capsid sequences, cloned into helper plasmids pXR9 and chimeric pXR9-2 (substituting the 3-fold loop region of AAV9's VP3 with AAV2's), were used to generate a small library of rAAV9 or chimeric 9-2 vectors with mutant capsids. For accurate comparison, parent plasmids AAV9-2, AAV9 and AAV2 (with no mutations) were also used to generate rAAV vectors, each packaging the luciferase transgene. Mutant or parent vectors expressing luciferase were used to transduce HEK293 cells (HEK293s), neonatal cardiomyocytes (NCMs), and neonatal or adult rat cardiac fibroblasts (RCFs), in vitro (1,000 vp/cell). Relative luciferase activities and protein concentrations were measured (RLU/ug/uL), respectively. A second approach at mutagenesis of the pXR9-2 capsid was employed. Surface-exposed T and Y residues were substituted for Valine or Phenylalanine and an ischemic myocardium targeting peptide (ICTP) motif was inserted at site 588 of the VP1 capsid protein. Results: A small library of mutant rAAV9 or 9-2 vectors was screened in HEK293s, NCMs, and RCFs in vitro. Luciferase assay results led to the selection of several mutants, believed to have enhanced levels of transgene expression. After large scale production of mutant and parent vectors, in vitro transduction assays revealed mutant vectors did not transduce NCMs or RCFs cultures better than their respective parent vectors. Recombinant AAV9-2R mutants, containing all or some point mutations and/or ICTP, produced similar results. Some mutants exhibited changes in tropism properties that were different from the parent vectors. Still, parent vector AAV9-2 displayed robust transduction efficiency, much greater than the other parent or mutant vectors. These results encourage us to continue our investigation into its unique properties. Conclusions: This study reflects the difficulties of AAV capsid mutagenesis. We hope that this study can serve as a tool to further our understanding of AAV's molecular and structural biology properties.

Grantor dc:publisher

Temple University. Libraries

Year dc:date.issued

2015

Author dc:creator

• Bowman, Latricia

Advisor dc:contributor.advisor

• Rabinowitz, Joseph

Committee members dc:contributor.committeemember

• Eguchi, Satoru
• Recchia, Fabio

• Virology
• Biology, Molecular
• Genetics

Statement dc:rights

• IN COPYRIGHT- This Rights Statement can be used for an Item that is in copyright. Using this statement implies that the organization making this Item available has determined that the Item is in copyright and either is the rights-holder, has obtained permission from the rights-holder(s) to make their Work(s) available, or makes the Item available under an exception or limitation to copyright (including Fair Use) that entitles it to make the Item available.

Licence dc:rights.uri

• http://rightsstatements.org/vocab/InC/1.0/

Language dc:language.iso

eng

Dc Identifier Other

920555130

OAI identifier oai:identifier

oai:scholarshare.temple.edu:20.500.12613/2617