Novel Chimeric Gene Therapy Vectors Based on Adeno-Associated Virus and Four Different Mammalian Bocaviruses

Abstract

Parvoviruses are highly attractive templates for the engineering of safe, efficient, and specific gene therapy vectors, as best exemplified by adeno-associated virus (AAV). Another candidate that currently garners increasing attention is human bocavirus 1 (HBoV1). Notably, HBoV1 capsids can cross-package recombinant (r)AAV2 genomes, yielding rAAV2/HBoV1 chimeras that specifically transduce polarized human airway epithelia (pHAEs). Here, we largely expanded the repertoire of rAAV/BoV chimeras, by assembling packaging plasmids encoding the capsid genes of four additional primate bocaviruses, HBoV2–4 and GBoV (Gorilla BoV). Capsid protein expression and efficient rAAV cross-packaging were validated by immunoblotting and qPCR, respectively. Interestingly, not only HBoV1 but also HBoV4 and GBoV transduced pHAEs as well as primary human lung organoids. Flow cytometry analysis of pHAEs revealed distinct cellular specificities between the BoV isolates, with HBoV1 targeting ciliated, club, and KRT5+ basal cells, whereas HBoV4 showed a preference for KRT5+ basal cells. Surprisingly, primary human hepatocytes, skeletal muscle cells, and T cells were also highly amenable to rAAV/BoV transduction. Finally, we adapted our pipeline for AAV capsid gene shuffling to all five BoV isolates. Collectively, our chimeric rAAV/BoV vectors and bocaviral capsid library represent valuable new resources to dissect BoV biology and to breed unique gene therapy vectors.

Twenty-Five Years of Structural Parvovirology

“…The development of Parvovirinae members as biologics is primarily focused on the engineering of capsids of members that can be used as viral vectors in gene delivery applications, such as the AAVs [121], or more recently also bocaviral vectors [122]. For such vectors, a transgene expression cassette is packaged into the capsids instead of the wt viral genome [123].…”

The Interplay between Adeno-Associated Virus and Its Helper Viruses

“…They combined a distinct set of AdV and bocavirus helper genes and found that rAAV titers were significantly increased when compared to rAAV vector titers that have been produced with either AdV or bocavirus helper factors alone. Lastly, other bocaviruses, such as HBoV2–4 and the gorilla bocavirus, were recently found to provide helper factors for the generation of AAV gene therapy vectors (GBoV) [ 227 ].…”

Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

“…An alternative strategy that is being investigated to deliver large genes is to leverage the capsid of the human bocavirus 1 (HBoV1), 46,47 an autonomous parvovirus relative of AAV, with a 5.5 kb genome. Indeed, it has been shown that an oversize AAV2 vector genome, including the CFTR expression cassette, can be effectively packaged in the HBoV1 capsid, resulting in efficient transduction of the human-polarized airway epithelia.…”