Loading...
MUTAGENESIS OF AAV CAPSID PROTEINS FOR ENHANCED TRANSDUCTION OF CARDIAC CELL TYPES
Bowman, Latricia
Bowman, Latricia
Citations
Altmetric:
Genre
Thesis/Dissertation
Date
2015
Advisor
Committee member
Group
Department
Biomedical Sciences
Subject
Permanent link to this record
Collections
Research Projects
Organizational Units
Journal Issue
DOI
http://dx.doi.org/10.34944/dspace/2599
Abstract
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.
Description
Citation
Citation to related work
Has part
ADA compliance
For Americans with Disabilities Act (ADA) accommodation, including help with reading this content, please contact scholarshare@temple.edu