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GLP-1, and its long-acting analogue exendin-4, may potentially induce islet regeneration in type I diabetes (T1D) and, in settings of type 2 diabetes, enhance insulin secretion leading to improved glycemic control. Previously, we delivered and expressed genes critical to pancreatic development and differentiation by direct injection into the pancreas for the purpose of reversing diabetes. Specifically, similar to the procedure of endoscopic retrograde cholengiopancreatography (ERCP), we successfully delivered the adeno-associated virus-8 (AAV-8) vector via the pancreatic ducts to the pancreas of mice. This vector was efficient in infecting pancreatic cells in vivo, and induced expression of human alpha anti-trypsin, (AAT) without inducing pancreatitis. We therefore constructed AAV-8 vectors encoding the human AAT signal peptide fused to GLP-1 containing a hydrophilic octapeptide epitope DYKDDDDK, named FLAG (sGLP1). Following in vitro transfection, this construct produced significant levels of active GLP-1 product in supernatants (>200nM/L). To determine the effects of intra-pancreatic expression of GLP-1 on diabetes reversal, we injected sGLP1 vectors (1.5 x 10[sup 11] vector genomes/mouse), or luciferase-enhanced yellow fluorescent protein gene (AAV-8-LucEYFP, 1.2 x 10[sup 10] vector genomes/mouse) (EYFP) into the pancreas of low-dose streptozotocin (STZ) induced diabetic C57BL/6 mice. sGLP1 significantly improved BG levels (194+/-15.4 mg/dl) and reversed diabetes in STZ mice 13 weeks post injection, while EYFP recipients maintained hyperglycemia (330+/-5.1 mg/dl; P=0.0001). All sGLP1 recipients had supraphysiologic levels of GLP-1 (>600pM/L in sera 4 to 13 weeks post injection (experiments ongoing). Control groups (EYFP, untreated, STZ-alone treated control groups) expressed <13pM/L. Glucagon and leptin were reduced in sGLP1 recipients (P=0.007), as was weight. Improved intraperitoneal glucose tolerance tests (IPGTT) were noted 8 weeks post injection (P=0.007). In conclusion, our studies clearly demonstrate the feasibility of direct pancreatic gene delivery of GLP-1 with long-term expression. Further studies will determine location of expression within the pancreas, islet cell mass, beta cell apoptosis, cell proliferation and the precise mechanisms of protection. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing α-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 × 1010 vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT (“long serpin”) indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency–related emphysema.Molecular Therapy (2008) 17 1, 81–87 doi:10.1038/mt.2008.217 [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated viral (rAAV) vectors have been widely used in pulmonary gene therapy research. In this study, we evaluated the transduction and expression efficiencies of several AAV serotypes and AAV2 capsid mutants with specific pulmonary targeting ligands in the mouse lung. The noninvasive intranasal delivery was compared with the traditional intratracheal lung delivery. The rAAV8 was the most efficient serotype at expressing alpha-1-antitrypsin (AAT) in the lung among all the tested serotypes and mutants. A dose of 1 x 10(10) vg of rAAV8-CB-AAT transduced a high percentage of cells in the lung when delivered intratrachealy. The serum and the broncho-alveolar lavage fluid (BALF) levels of human AAT (hAAT) were about 6- and 2.5-fold higher, respectively, than those of rAAV5 group. Among the rAAV2 capsid mutants, the rAAV2 capsid mutants that display a peptide sequence from hAAT ("long serpin") indicated a twofold increase in transgene expression. For most vectors, the serum hAAT levels achieved after intranasal delivery were 1/2 to 1/3 of those with the intratracheal method. Overall, rAAV8 was the most promising vector for the future application in gene therapy of pulmonary diseases such as AAT deficiency-related emphysema.