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

Abstract

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration-at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only *5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of *3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then-through a variety of mechanismsresult in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction.

Adeno-Associated Virus Gene Therapy for Liver Disease

“…30,31 To overcome the size limitation challenge, dual vector systems have been proposed, where the larger genome would be recreated through recombination of the partial genomes. 32 In addition, the effort to develop cassettes for factor VIII (FVIII) expression serves as an object lesson in setting expectations for both efficacy and quality of large-genome rAAV. Bearing in mind the well-established packaging “cliff” of approximately 5.2 kb, success has only come after considerable effort from individual groups to minimize the size of the protein itself (deletion of the B-domain), establish the bare minimum regulatory sequences required for a reasonable level of tissue-specific expression, and test alternate serotypes for greater size tolerance.…”

Systemic delivery of adeno-associated viral vectors

“…This limits the use of AAV for a number of diseases including DMD and dysferlin-deficient myopathy. Various dual AAV strategies have been developed to overcome this hurdle (reviewed in [56-58]). Optimized dual AAV vectors have reach transduction efficiency of the single AAV vector [59,60].…”

Viral vectors for therapy of neurologic diseases

“…One approach to overcome this limitation is the dual AAV vector system, where an oversized transgene is split across two AAV vectors to reconstitute the full-length cDNA upon delivery (Yan et al, 2000). Full-length assembly can occur through recombination of homologous regions or/and trans-splicing (Chamberlain et al, 2016). Dual AAV vectors have shown therapeutic efficacy in mouse models of retinal disorders (Trapani et al, 2014).…”