Constructing and packaging scAAV vectors and detecting their transduction efficiency

Gene therapy is a form of molecular medicine that promises to provide new treatments for a large number of inherited and acquired diseases. One of the biggest stumbling blocks to successful widespread application of genetic treatments is the development of safe and effective vectors with which to ferry genetic material into cells. Adeno-associated virus （AAV）, a nonpathogenic human parvovirus, has gained attention as a potentially safe vector for gene transfer and gene therapy. The AAV genome is a single-stranded DNA, which is transcriptionally inactive. It has been reported that viral second-strand DNA synthesis is the rate-limiting step in AAV-mediated transgene expression. Self-complementary adeno-associated viral（scAAV） vectors bypass the requirement for viral second-strand DNA synthesis, and lead to a significant increase in transduction efficiency, scAAV vectors have been successfully constructed by Xiao and Smulski in the USA in 2003. Recently, we have developed and packaged scAAV, and reported as following.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency

Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.

Engineered AAV13 variants with enhanced transduction and confined spread

Precise targeting of specific regions within the central nervous system(CNS)is crucial for both scientific research and gene therapy in the context of brain diseases.Adeno-associated virus 13(AAV13)is known for its restricted diffusion range within the CNS,making it an ideal choice for precise labeling and administration within small brain regions.However,AAV13 mediates relatively low expression of target genes.Here,we introduced specifically engineered modifications to the AAV13 capsid protein to enhance its transduction efficiency.We first constructed AAV13-YF by mutating tyrosine to phenylalanine on the surface of the AAV13 capsid.We then inserted the 7m8 peptide,known to enhance cell transduction,into positions 587/588 and 585/586 of the AAV13 capsid,resulting in two distinct variants named AAV13-587-7m8 and AAV13-585-7m8,respectively.We found that AAV13-YF exhibited superior in vitro infectivity in HEK293T cells compared to AAV13,while AAV13-587-7m8 and AAV13-585-7m8 showed enhanced CNS infection capabilities in C57BL/6 mice,with AAV13-587-7m8 infection retaining a limited spread range.These modified AAV13 variants hold promising potential for applications in gene therapy and neuroscience research.

The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo

Objective: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus(rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based r AAV2 vector delivery strategy.Methods: The melittin peptide was inserted into the rAAV2 capsid either in the loop Ⅷ of all viral proteins(VPs) or at the N terminus of VP2. Various r AAV2-gfp or-fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.Results: rAAV2 vectors with melittin peptide inserted in the loop Ⅷ of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of r AAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.Conclusion: Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.