Nonviral vector strategies for ultrasound targeted microbubble destruction-mediated hepatic gene therapy

Ph.D. University of Hawaii at Manoa 2014.Includes bibliographical references.The goal of this research was to identify an improved delivery system and vectors for noninvasive gene therapy of Hemophilia B. Ultrasound Targeted Microbubble Destruction (UTMD) is a platform technology that can deliver geneexpression vectors bound to the shells of lipid microbubbles to organs accessible to ultrasound. In UTMD, the DNA-loaded microbubbles are injected intravenously and are disrupted at the target organ by acoustic cavitation at a resonant frequency of the bubbles, resulting in delivery of expression plasmids to the target organ. Our aim was to apply UTMD to the hepatic delivery of conventional (pcDNA3), piggyBac (pmGENIE) and Sleeping Beauty (SB100x) transposon-based, and minicircle (MC) DNA vectors. We measured reporter and human blood coagulation factor IX transgene expression in the mouse liver and plasma driven by constitutive and tissue-specific promoters. In vitro we demonstrated a ten-fold increase in the level of reporter expression in HEK293 cells after transfection with a piggyBac transposase system over three weeks. In C57Bl/6 mice, we first compared pmGENIE2-luciferase to pcDNA3-luciferase, and observed UTMD-mediated liver-specific expression of pmGENIE2 for an average of 24 days (n=12), compared to 4 days with the pcDNA3 (n=7) (p=0.037). Expression of the reporter constructs was initially predominately located proximal to blood vessels while expression past three days was more evenly distributed through the parenchyma of the liver. We also used nonrestrictive linear amplification mediated (nrLAM) PCR to evaluate the genomic integration sites of the pmGENIE reporter vectors in vitro and in vivo. Chromosomal integration sites were randomly distributed in genomic DNA samples isolated from mouse 3T3 cells transfected with pmGENIE3-eGFP in vitro but were predominately targeted to specific chromosomes in livers from C57BL/6 mice transfected with pmGENIE3-luc (n=13) in vivo. Thus, the UTMD delivery of transposon-based vectors has revealed an unexpected tropism for certain murine chromosomal sites in vivo that is not seen in vitro. We delivered various reporter constructs to the liver by UTMD and compared reporter expression levels and hepatic localization over two weeks. We initially observed robust transgene levels from all vectors, however, the intensity of expression remained 10 to 1000-fold stronger in mice from the pmGENIE3-luc and liver-specific pZY53-luc treatments compared to pcDNA3, SB100X, or minicircle constructs. The greatest hepatic specificity of transgene expression was observed for the alpha1 antitrypsin promoter-driven pZY53-luc, which supports the use of vectors with tissue-specific promoters to further enhance UTMD site-specificity. We also evaluated these vectors with liverspecific promoters and the human factor IX (FIX) gene in HepG2 cells and in C57Bl/6 mice. The optimal hFIX vectors (pZY53-hFIX and pmGENIE3-hFIX) were delivered to the livers of FIX deficient (-/-) mice to determine whether we could ameliorate the Hemophilia B coagulopathy. Immunofluorescence and bioluminescence imaging demonstrated successful UTMD-mediated hepatic transfection in the FIX (-/-) mice from co-deliveries of the hFIX vectors (pZY53-hFIX or pmGENIE3-hFIX) with the liver-specific reporter, pZY53-luc. Immunofluorescence assays performed on livers harvested one, four or five, and twelve days after UTMD revealed hFIX expression in hepatocytes, predominantly surrounding hepatic blood vessels. We analyzed plasma collected at the same time-points from FIX (-/-) mice after treatment with pZY53-hFIX and pmGENIE3 hFIX and detected human FIX expression by Western blotting indicating that the transfected mouse hepatocytes were secreting hFIX. Activated partial thromboplastin time (APTT) assays performed on treated plasma samples showed significantly reduced clotting times compared to untreated mutant control plasma four to five days (p=0.001 and p=0.012 for pZY53-hFIX and pmGENIE3-hFIX, respectively) and twelve days (p=0.02 and p=0.001 for pZY53-hFIX and pmGENIE3-hFIX, respectively) after UTMD. Together these data show that our method of UTMD-mediated hFIX delivery resulted in successful transfection and secretion of hFIX from the livers of FIX (-/-) mice. These results demonstrate potentially therapeutic levels of FIX expression and support consideration of UTMD as an approach for hepatic gene therapy. Overall, the combination of UTMD and transposon-based expression constructs provides a minimally invasive strategy for delivery of therapeutic genes to the liver. This may be useful for the treatment of many hepatic gene deficiency disorders, including Hemophilia B