P13
Nanoparticle Based Enhancement and Retargeting of AAV-Mediated Gene Delivery
S A Shakouri(1,2), A Hoenig(1,2), S Hanusch(1,2), V Rajendran(1,2), I Luksch(1,2), A Dastmaltschi(1,2), A Bähr(1,2), N Raad(1,2), T Ziegler(1,2), T Bozoglu(1,2), C Kupatt(1,2)
1. Klinik und Poliklinik für Innere Medizin I, Klinikum rechts der Isar, Technical University Munich, Germany; 2. German Center for Cardiovascular Research (DZHK), Munich Heart Alliance, Munich, Germany.
Background:
Adeno-associated viruses (AAV) are currently the gold standard for gene transfer applications in vivo, due to low immunogenicity, and natural and engineered serotypes providing different tissue and cell tropism. However, several shortcomings, such as large doses required for effective transduction or limitations on transducable cell types in target organs, leave room to improve.
Methods:
Previously, we have demonstrated efficient polyamidoamine (PAMAM) mediated retargeting of AAV9 from cardiomyocytes to microvascular endothelial cells¹. Employing a similar methodology, comprised of mixed in vitro and in vivo biopanning of phage libraries, followed NGS analysis of sublibraries, peptides targeting other interstitial cell types, as well as cardiomyocytes, were identified. Transduction efficacies of modified AAV9 encoding cell type specific promoter driven reporter genes were determined via flow cytometry and immunohistochemisty. Furthermore, the effects of nanoparticle modification facilitating neutralizing antibody escape was elucidated.
Results:
In mTmG mouse model, pPDGFRa-Cre encoding AAV9 coated with fibroblast targeting peptides conjugated PAMAM resulted in transduction of up to 15% of PDGFRa⁺ cells. Similarly, pericyte retargeting with NG2 promoter and pericyte specific peptide transduced 13% of NG2⁺ cells. Moreover, TNNT2 promoter and myocytic peptides reduced the necessary dose for cardiomyocyte transduction by half a log. Finally, PAMAM coating reduced susceptibility of AAV9 to neutralizing antibodies from seropositive porcine serum in vitro.
Conclusion:
This gene delivery toolkit, comprised of promoter – PAMAM – peptide formulations, would allow spatial and temporal resolution required for dissection of a plethora of clinically relevant cardiovascular processes ranging from fibrosis to angiogenesis.