Efficacy of Codelivery of Dual AAV2/5 Vectors in the Murine Retina and Hippocampus

Abstract

Recombinant adeno-associated virus (AAV) represents an efficient system for neuronal transduction. However, a potential drawback of AAV is its restricted packaging capacity of approximately 5 kb. To bypass this limitation, a number of dual-and triple-vector strategies divide the transgene(s) between two or three AAVs. The success of these approaches relies directly on efficient cotransduction of the component AAVs. Although proof of concept for these stratagems has been demonstrated, the underlying cotransduction rate has not been analyzed quantitatively. In this study, cotransduction efficiencies in both retina and hippocampus have been investigated, using two reporter AAVs expressing either a green (GFP) or red (DsR) fluorescent protein.Transduction efficiencies were monitored via microscopy, flow cytometry, and quantitative PCR. After viral transduction with 1.5 · 10 9 viral particles of each of the reporter AAVs, approximately one-third of the retinal cells expressed one or both transgenes at levels detectable by native fluorescence. Notably, the majority of the remaining retinal cells were also transduced and expressed the reporters at lower levels, which were detectable only by immunolabeling. Flow cytometric analysis demonstrated cotransduction rates of up to 55% with the two reporter AAVs in retinal cells. Modifying the ratio of the two coadministered AAVs resulted in altered mRNA expression levels of the two reporter genes in cotransduced cell populations. The study suggests that codelivery of AAV is an efficient means of expanding the therapeutic application of AAV in neurons.

Copackaging of Multiple Adeno-Associated Viral Vectors in a Single Production Step

“…In other cases, it may prove necessary to coordinate and differentially control transgene expression to different target regions with tissue-restricted promoters, such as the central nervous system, eye, or systemically, while avoiding expression in antigen presenting cells and provoking a deleterious immune response Palfi et al, 2012;Bosch et al, 2014;Fagoe et al, 2014). In these lines of examples, a copackaging strategy was recently employed by Xiao's group to deliver a functional factor VIII in a model of hemophilia A (Wang et al, 2014).…”

“…Such as for indications where two or more subunits are needed (e.g., hexosaminidase A and B for TaySachs disease) or indications where the expression of the therapeutic gene needs to be elevated in specific tissues, which could be mediated by the use of different promoters upstream of the same therapeutic transgene (Pacak et al, 2009;Palfi et al, 2012;Fagoe et al, 2014). For instance, targeting gene expression to the liver for the purposes of immune tolerance induction while providing an additional vector to correct systemic pathology would allow for the simultaneous treatment of many congenital metabolic myopathies wherein immune responses have proven deleterious to the efficacy of gene therapy.…”

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids

“…27 On the other hand, approximately 6% of all human proteins have a coding sequence that exceeds 4 kb, 28 which makes it difficult, if not impossible, to fit expression cassettes for these cDNAs into a single AAV genome. This group of proteins includes several therapeutic targets for a number of diseases (e.g., see Table 1).…”

Retinal gene delivery by adeno-associated virus (AAV) vectors: Strategies and applications

“…In another study, codelivery of two separate AAV vectors expressing for the RHO shRNA and the silencing-resistant RHO transgenes was successfully applied to treat the P347S mouse model of autosomal dominant retinitis pigmentosa [120]. The success of this approach is based on the fact that multiple AAV particles can transduce the same cell [120][121][122].…”