Adeno-Associated Virus Serotype 6 Capsid Tyrosine-to-Phenylalanine Mutations Improve Gene Transfer to Skeletal Muscle

Abstract

Adeno-associated viral (AAV) vectors are the most efficient in vivo gene transfer tools for gene therapy applications. Efforts have been made to translate encouraging results in small animal models to human patients. However, the need for large quantities of vector for clinical application remains a great challenge. Developing novel AAV vectors with enhanced infectivity may reduce the high vector dose requirement for many applications such as gene therapy for muscular dystrophy. Selective mutation of AAV capsid surface-exposed tyrosine (Y) is a novel strategy to improve transduction efficiency. AAV6 has been considered one of the most robust muscle gene delivery vehicles. Here, we hypothesize that AAV6 transduction efficiency can be further enhanced by mutating surface Y to phenylalanine (F). We found that mutants AAV6-Y445F and AAV6-Y731F, especially the former, achieved more efficient gene transfer than the original AAV6 after intramuscular administration to mice. Expression of both firefly luciferase and alkaline phosphatase reporter genes increased up to 8-fold and DNA copy numbers in muscle increased up to 6-fold. Our results suggest that tyrosine-mutant AAV6 vectors may represent powerful tools for testing muscle gene therapy in animal models and potentially in humans.

A Simplified Immune Suppression Scheme Leads to Persistent Micro-dystrophin Expression in Duchenne Muscular Dystrophy Dogs

“…The expression of the canine microgene is controlled by the cytomegalovirus (CMV) promoter, an engineered Kozak sequence and the SV40 polyadenylation signal. The Y445F tyrosine modified AAV-6 was purified through three rounds of cesium chloride isopycnic ultracentrifugation using our published protocol (Zhong et al, 2008;Qiao et al, 2010). The resulting viruses were termed AV.AP and AV.lDys.…”

“…Nevertheless, post-AAV infection BUN was still within the normal range ( < 28 mg/dL). AAV vectors were packaged in a modified AAV-6 capsid in which the tyrosine residue at the position 445 was replaced by phenylalanine (Zhong et al, 2008;Qiao et al, 2010). Two different AAV vectors were tested.…”

“…We applied CSP and MMF for a total of 5 weeks (starting from 1 week before AAV injection). We used Y445F tyrosine mutant AAV-6 vectors that express either a foreign reporter gene (human placental alkaline phosphatase gene, AP) or a therapeutic canine micro-dystrophin gene (Zhong et al, 2008;Qiao et al, 2010). AAV was injected into the extensor carpi radialis (ECR) and extensor carpi ulnaris (ECU) muscles of young adult dystrophic dogs.…”

“…Thirdly, it is possible that the AAV serotype used in our study and that of Wang et al (2007b) may have contributed to the difference. Wang and colleagues used the original AAV-6, but in our study we used a newly described Y445F tyrosine mutant AAV-6 (Zhong et al, 2008;Qiao et al, 2010). We recently found that Y445F-mutated AAV-6 resulted in higher transduction in murine muscle than the original AAV-6 (Qiao et al, 2010).…”

“…Wang and colleagues used the original AAV-6, but in our study we used a newly described Y445F tyrosine mutant AAV-6 (Zhong et al, 2008;Qiao et al, 2010). We recently found that Y445F-mutated AAV-6 resulted in higher transduction in murine muscle than the original AAV-6 (Qiao et al, 2010). Although future studies are needed to determine whether Y445F tyrosine mutant AAV-6 is stronger and/or less immunogenic in dystrophic dog muscles, robust transduction we observed with both *This is different from the protocol used for human micro-dystrophin vector (depicted in Fig 1).…”