Nanoparticle Based Enhancement and Retargeting of AAV-Mediated Gene Delivery

Background:

Adeno-associated viruses (AAV) are currently the gold standard for gene transfer and gene editing applications in vivo, due to its low integration rate and low immunogenicity, as well as a variety of natural and engineered serotypes providing different tissue and cell type tropism. However, several shortcomings, such as large doses required for effective transduction, limitations on transducable cell types in target organs, and unfeasibility of repeat dosing due to neutralizing antibody buildup, leaves room to improve.

Methods:

Previously, we have demonstrated efficient polyamidoamine (PAMAM) mediated retargeting of AAV9 from cardiomyocytes to microvascular endothelial cells, a practically untransducable component of the myocardium¹. 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.  This finding led to a currently undergoing in vivo murine trial, probing feasibility of repeat dosing facilitated with PAMAM coating and NA-Pass, a compound capable of reducing neutralizing antibodies in hIVIg supplemented mice to wildtype levels.

Conclusion:

The gene delivery toolkit we are developing in this study, comprised of retargeting capabilities of promoter – PAMAM – peptide formulations, and AAV9 readministrability improvements via antibody escape with PAMAM coating and antibody elimination with NA-Pass, impoves the spatial and temporal resolution required for dissection of a plethora of clinically relevant cardiovascular processes ranging from fibrosis to angiogenesis.