Project description:Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Project description:Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction.
Project description:[15:18] Toole, Estee Adeno-Associated Viruses (AAVs) comprise an area of rapidly growing interest due to their ability to act as a gene delivery vehicle in novel gene therapy strategies and vaccine development. Peptide mapping is a common technique in the biopharmaceutical industry to confirm the correct sequence, product purity, PTMs, and stability. However, conventional peptide mapping is time-consuming and has proven difficult to reproduce with viral capsids because of their high structural stability and the suboptimal localization of trypsin cleavage sites in the AAV protein sequences. In this study, we present an optimized workflow that provides thorough characterization within one day. This workflow is also highly reproducible due to its simplicity having very few steps, and easy to perform proteolytic digestion utilizing thermally-stable pepsin, active at 70°C in acidic conditions. The acidic conditions of the peptic digestions drive viral capsid denaturation and improve cleavage site accessibility. We characterized the efficiency and ease of digestion through peptide mapping of the AAV2 viral capsid protein. Using nanoflow liquid chromatography coupled with tandem mass spectrometry, we achieved 100% sequence coverage of the low abundance VP1 capsid protein with a digestion process taking only 10 minutes to prepare and 30 minutes to complete the digestion.