785. A Novel Adeno Associated Virus Vector That Efficiently Transduces Airway Epithelium In Vivo and Can Be Re-Administered
Authors: Maria Limberis, Roberto Calcedo, Julio SanMiguel, Julie C. Johnston, Deirdre McMenamin, Peter L. Bell, James M. Wilson
Journal: Molecular Therapy (2006)

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Airway|[ndash]|directed gene transfer has emerged as a promising approach for the treatment of cystic fibrosis and |[alpha]|-1-antitrypsin deficiency (AAT). It will be important to re-administer the vector for treatment of these chronic disorders. We have shown previously that AAV2/9 can efficiently target and transduce the murine upper and lower airways in vivo. As such we studied the performance of AAV2/9 in the setting of pre-existing immunity to homologous vector.

C57Bl/6 mice were inoculated intratracheally with a single dose (1E+11 genome copies) of either AAV2/9 or AAV2/5 expressing AAT. We found that AAV2/9-mediated AAT expression was relatively stable for the duration of the experiment (9 months) and at the peak of gene expression was 60-fold higher than that of AAV2/5. Within two weeks of vector administration, high levels (>1:2560) of serum|[ndash]|circulating neutralizing antibodies (NAB) were observed in both AAV2/9 and AAV2/5 treated mice. Mice were divided into groups and designated groups were subjected to a second instillation of the same vector serotype expressing a different transgene (nLacZ) at specific time points after delivery of the AAV2/9- or AAV2/5|[ndash]| AAT vectors. Mice were sacrificed 21 days following the AAV|[ndash]| nLacZ vector instillation and the lung and nose processed for nLacZ expression. The serum NAB titers in the AAV2/9 treatment group remained high (average of 1:2560) up to day 112 and decreased to |[sim]|1:640 by day 217. Interestingly, we found that AAV2/9 was able to circumvent the AAV2/9-specific NABs, resulting in nLacZ gene transfer levels in nasal and lung airways similar to that of age-matched naive mice at all time points. In AAV2/5-treated mice, no transduced cells were seen in either the nasal or lung airways over a broad range of serum|[ndash]|circulating NAB titers (1:56000 to 1:1280). Some level of gene transfer was achieved with AAV2/5-nLacZ when administered 196 days after the AAT vector which was when serum NAB declined to 1:320|[ndash]|1:160. To explain why AAV2/9 can efficiently transduce the lung airway epithelium in the presence of high titer serum|[ndash]| circulating NAB, the bronchoalveolar lavage fluid (BALF) isolated from mice at the first time point (day 49) and at the end of the experiment (day 217) was analyzed for AAV-specific NABs. We did not observe any NAB titer, IgG or IgA responses to AAV2/9 vector. In contrast, when BALF from AAV2/5 treated mice was analyzed, NAB titers ranged from 1:160 to 1:1280, vector IgG responses from 1:100 to 1:12800 and vector IgA responses from 1:100|[ndash]|1:1600.

Our results suggest that AAV2/9 is an excellent candidate for clinical applications that require long term transgene expression. We show that AAV2/9 can be re-administered to lung with out diminution of gene transfer efficiency while AAV2/5 can not. This finding further supports the development of AAV2/9 for use in clinical applications for chronic diseases in which vector re-administration will be necessary.