Bioengineered Viral Platform for Intramuscular Passive Vaccine Delivery to Human Skeletal Muscle

Abstract

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.

Using a barcoded AAV capsid library to select for novel clinically relevant gene therapy vectors

“…The cost and time of the screening process can be minimized by predeterming the appropriate MOI and number of selection rounds for a particular screen. For previous library screening studies in our lab that did not employ barcoded libraries, four or more rounds of selection were routinely performed (Grimm et al, 2008;Lisowski et al, 2014;Paulk et al, 2018a;Paulk et al, 2018b). However, in the current study, we found that three rounds of selection were sufficient to enrich and amplify variants with improved transduction of the target cell type to a level where they could be isolated reliably.…”

“…While the possibility of cross-packaging, which is characterized by sequence differences between the packaging capsid and the corresponding viral genome, cannot be excluded, several of the enriched capsids from our screen were found to exhibit the desired phenotype, suggesting that cross-packaging or mosaicism was not a major problem in our libraries. Moreover, when we had previously generated AAV libraries using high concentrations of AAV plasmid during transfection, we had been successful in generating variants with greatly improved phenotypes (Grimm et al, 2008;Lisowski et al, 2014;Paulk et al, 2018a;Paulk et al, 2018b), suggesting that for reasons unknown so far there seems to be a strong capsid genotype linkage in case of AAV packaging (Grimm & Zolotukhin, 2015). A recent report by the Grimm group laid to rest the concern that functionality of AAV capsid libraries generated for directed evolution studies might be severely compromised by inactivation of the assembly activating protein (AAP) in a large proportion of the chimeric variant pool (Herrmann et al, 2018b).…”

“…In 2008 our group was the first to report the development of an improved AAV variants by subjecting a capsid shuffled AAV library to multiple rounds of selection on the target cell type (Grimm et al, 2008). Since then, we and others have used this approach to identify AAV variants with improved properties (Asuri et al, 2012;Gray et al, 2010;Grimm et al, 2008;Li et al, 2008;Lisowski et al, 2014;Maguire et al, 2010;Paulk et al, 2018a;Paulk et al, 2018b;Siu et al, 2017;Tervo et al, 2016;Yang et al, 2009;Yang et al, 2011).…”

Methods Matter -- Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors

“…To determine whether these potency differences were restricted to IM delivery of rAAV1, we repeated the same experiment with rAAV8-a serotype currently being tested as an intramuscular HIV passive vaccine vector 33 , despite reports of poor transduction in human skeletal muscle 34 and other human tissues 35,36 , as well as high neutralizing antibody levels in HIV+ individuals 37 . Here again, twelve mice (6 male and 6 female) were injected IM with 5e10 vg of ssAAV8-CMV-FLuc vector and liveimaged weekly for FLuc expression (Figures 3f, S13c).…”

“…Administration of rAAV IM is typically only done when treating muscle disorders or when using muscle as a secretion depot for passive vaccines 34 . Many gene therapy trials administer vector intravenously (IV) to facilitate distribution to internal organs not easily accessible by direct injection.…”