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Abstract: Gene transfer into pancreatic cells in vivo could be of immense therapeutic benefit in cases of type 1 diabetes (T1D) through the production of molecules capable of interrupting the progression of autoimmunity or promoting regeneration of insulin-secreting β cells. We adapted a clinically relevant surgical technique (endoscopic retrograde cholangiopancreatography) to deliver rAAV encoding human α1-antitrypsin (approved gene symbol SERPINA1) to the pancreas of 3-week-old Fisher 344 rats and C57BL/6 mice. We compared natural as well as bioengineered serotypes of rAAV (rAAV1, rAAV2/Apo (B. R. Burkhardt et al., 2003, Ann. N. Y. Acad. Sci. 1005, 237–241) [1], rAAV8) as well as different promoters (chicken β-actin, human insulin) for their expression in vivo. Rats injected with rAAV1 showed the highest hAAT expression (week 2, rAAV1/CB-AT, 579 ± 457 ng/ml). In mice, rAAV8 vector delivered the highest serum concentration of hAAT (week 2, rAAV8/CB-AT, 19 ± 6 μg/ml). The chicken β-actin promoter provided the highest expression in both rodent experiments. Immunohistochemical staining indicated transduction primarily of pancreatic acinar cells with either the rAAV1/CB-AT vector in the rat or the rAAV8/CB-AT vector in the mouse. This study demonstrates that rAAV vectors can be designed to deliver therapeutic genes efficiently to the pancreas and achieve high levels of gene expression and may be useful in treating pancreatic disorders, including T1D. [Copyright &y& Elsevier]

Abstract: Studies in animals and human clinical trials demonstrate the safety and persistence of recombinant adeno-associated viral (rAAV) serotype 2 vectors in a variety of tissues. rAAV vectors of other serotypes are also being developed for efficient gene transfer. To date, the literature describing these vectors has relied on physical or transducing titers to determine dose, but few, if any, infectious titers have been presented. This is due in large part to the lack of reagents and methods that would facilitate the infectious titering of vectors other than serotype 2. Here, we describe reagents and methods for infectious titering of AAV2 ITR-containing vectors pseudotyped with other AAV capsid serotypes and demonstrate their utility by titering pseudotyped rAAV1 or rAAV5 vectors. Cell lines are screened for optimal transduction using a vector of a particular serotype that expresses a marker transgene. Once a cell line and vector serotype are matched, a recombinant herpes simplex virus vector expressing AAV2 rep and cap genes provides helper functions that amplify the rAAV vector genome. The vector genomes are then detected and a titer is calculated. These methods generate reliable infectious titers for AAV vectors of different serotypes, thus enhancing product characterization and reducing risk in future clinical applications. [Copyright &y& Elsevier]

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]