Presentation Title

Point Mutations in Surface-Exposed Tyrosines on the Adeno-Associated Virus DJ Capsid to Improve Transduction Efficiency In Vivo

Faculty Mentor

Beatriz Ferran Perez, Michael Markus Bachschmid

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

173

Session

poster 1

Type of Presentation

Poster

Subject Area

health_nutrition_clinical_science

Abstract

Gene therapy using adeno-associated virus (AAV) is becoming a popular method of introducing healthy or modified genes into animals and patients. Adeno-associated viruses, from the parvovirus family are preferred vectors due to their high transduction efficiency, safety, negligible immune-response, and extended stable gene expression. Recent phase I/II clinical trials with AAV2 are promising but require high vector doses to achieve sufficient gene expression. A 2008 study by Zhong L. Li found that changing specific surface-exposed tyrosines to phenylalanine on the AAV2 capsid significantly increased viral stability and transduction efficiency. Our study aims to improve further the transduction efficiency of the AAV-DJ capsid, a vector engineered for liver gene therapy. Using PyMOL, we performed molecular modeling of the viral DJ capsid and identified surface-exposed tyrosines. We mutated potential tyrosine residues to phenylalanine with site-directed mutagenesis and confirmed the clones by sequencing. To produce the wild type and mutant AAVs, we transfected HEK293T cells with pHelper, a viral expression plasmid encoding green fluorescent protein (pAAV-IRES-EGFP), and the respective replication/capsid vectors (pRep/Cap). We purified the virus by PEG-precipitation and determined the titer with quantitative real-time PCR. We cloned and verified DJ capsid mutants with Y443F, Y445F, and Y732F. Unexpectedly, preliminary data has shown that mutant capsids generated higher titers than wild type AAV-DJ. Using these mutations, we transduced HepG2 cells, with three concentrations. After seven days of incubation, we quantified fluorescence using the Keyence BIOREVO 9000 microscope. Mutants Y443F and Y445F yielded a 3 to 4-fold increase in fluorescence compared to the parental DJ capsid. Current efforts to produce more of these mutant viruses are underway, with the next step being in vivo transduction of mice to quantify the efficiency of these capsids. If successful, this improved AAV vector may lower doses for gene therapy uses.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Point Mutations in Surface-Exposed Tyrosines on the Adeno-Associated Virus DJ Capsid to Improve Transduction Efficiency In Vivo

173

Gene therapy using adeno-associated virus (AAV) is becoming a popular method of introducing healthy or modified genes into animals and patients. Adeno-associated viruses, from the parvovirus family are preferred vectors due to their high transduction efficiency, safety, negligible immune-response, and extended stable gene expression. Recent phase I/II clinical trials with AAV2 are promising but require high vector doses to achieve sufficient gene expression. A 2008 study by Zhong L. Li found that changing specific surface-exposed tyrosines to phenylalanine on the AAV2 capsid significantly increased viral stability and transduction efficiency. Our study aims to improve further the transduction efficiency of the AAV-DJ capsid, a vector engineered for liver gene therapy. Using PyMOL, we performed molecular modeling of the viral DJ capsid and identified surface-exposed tyrosines. We mutated potential tyrosine residues to phenylalanine with site-directed mutagenesis and confirmed the clones by sequencing. To produce the wild type and mutant AAVs, we transfected HEK293T cells with pHelper, a viral expression plasmid encoding green fluorescent protein (pAAV-IRES-EGFP), and the respective replication/capsid vectors (pRep/Cap). We purified the virus by PEG-precipitation and determined the titer with quantitative real-time PCR. We cloned and verified DJ capsid mutants with Y443F, Y445F, and Y732F. Unexpectedly, preliminary data has shown that mutant capsids generated higher titers than wild type AAV-DJ. Using these mutations, we transduced HepG2 cells, with three concentrations. After seven days of incubation, we quantified fluorescence using the Keyence BIOREVO 9000 microscope. Mutants Y443F and Y445F yielded a 3 to 4-fold increase in fluorescence compared to the parental DJ capsid. Current efforts to produce more of these mutant viruses are underway, with the next step being in vivo transduction of mice to quantify the efficiency of these capsids. If successful, this improved AAV vector may lower doses for gene therapy uses.