(609) Immunosuppression Improves Transduction Following Repeat Administration of AAV2.5T to Ferret Lungs

Background: Gene replacement is a mutation agnostic approach to the treatment of cystic fibrosis (CF). Adeno-associated virus vectors (AAV) have a strong track record for clinical success. However, AAV vectors do not readily integrate and induce humoral and cellular responses that need to be overcome to achieve successful therapeutic efficacy upon repeat administration. Previous work demonstrated that repeated rAAV2.5T vector administered to ferret lungs leads to reduced transgene expression and higher levels of neutralizing antibodies (NAbs) compared to a single-dose rAAV2.5T administration. We hypothesized that immunosuppression (IS) could limit NAb production and improve transgene expression.

Methods: Twenty-six-day old ferrets were given three immunosuppressants (tri-IS; cyclosporine, methylprednisolone (MP), and azathioprine), MP alone, or no IS for a period of 1 month starting at 2 days prior to AAV administration. Ferrets were initially dosed with AAV2.5T-fCFTRdR (ferret CFTRdeltaR) via an atomizer at four-weeks old. Four weeks later, the ferrets were administered AAV2.5T-gLuc (secreted gaussia luciferase). Immunosuppression treatment ended on the day of AAV2.5T-gLuc administration. Transgene expression (gLuc) and NAb levels were monitored in the blood over the next 14-days and in the bronchiolar lavage fluid (BALF) at necropsy, 14 days post-dosing of the AAV2.5T-gLuc vector.

Results: Both immunosuppression strategies effectively improved transgene expression following a second intratracheal administration of rAAV2.5T to ferret lungs. The tri-IS and MP regimens led to gLuc levels in the plasma that were 79.6-fold (tri-IS) and 28.3-fold (MP) higher than repeat-dose animals with no IS. Similarly, gLuc levels in the BALF were 85.8-fold (tri-IS) and 19.1-fold (MP) higher than the control animals that were repeat dosed with AAV2.5T but had no IS. The IS strategies did not prevent the generation of capsid-specific humoral immunity elicited by the 1st dose of the AAV2.5T vector or activation of that response, as demonstrated by the increase of NAbs in the plasma after the 2nd dose of AAV2.5T vector. However, the levels of the NAbs in tri-IS or MP repeat-dose groups were lower in the plasma (2.6- and 2.3-fold, respectively) and in the BALF (4.2- and 4.4-fold, respectively) than animals without IS at 14-days following AAV2.5T redosing. In particular, plasma IgG levels were significantly inhibited by tri-IS or MP when compared to redosing without IS. Furthermore, no significant AAV2.5T-specific cellular immune response was detected in any group by ELISpot. Quantification of splenocyte derived cytokines, IL-2, IL-4, IL-6, IFNγ, IFNα, IFNβ and TNFα by RT-qPCR showed that mRNA levels for IL-4 and IFNα from the tri-IS group, but not the MP group, were significantly inhibited compared to the non-IS AAV2.5T redosing control group.

Conclusions: Our data suggest that suppressing IgG and pro-inflammatory immune responses may be important to avoid lower levels of transduction upon repeat administration of AAV2.5T. Collectively, our data suggest that tri-IS or MP treatment may improve transgene expression following AAV2.5T redosing. Future work will focus on identifying the durability of transgene expression and NAb responses after a single dose of AAV2.5T vector and delineating the molecular immune mechanisms that limit transduction following repeat-dosing to the ferret airway.

Acknowledgements: This work was supported by Spirovant Sciences, Inc., Philadelphia, PA.

References: