Adeno-associated virus (AAV) is favorable choice 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. AAV serotype 9 is known to have strong tropism to cardiac and skeletal muscle. However, myocyte compartment of these organs are the principal targets of AAV9, whereas transduction is particularly inefficient in the cells of the microcirculatory network, such as endothelial cells and pericytes. This exclusive cell type specificity prevents a plethora of potential applications of AAV mediated gene transfer in vascular medicine and research.

Previously we have demonstrated efficient retargeting of AAV9 particles to microvascular endothelium (1). Briefly, we identified an endothelial-affine heptamer peptide via phage-display on endothelial culture cells. Second generation (G2) polyamidoamine (PAMAM) dendrimers were modified by conjugating this peptide via a polyethyleneglycol (PEG) linker. AAV9 particles were coated with the modified PAMAM immediately before application. We were able to show efficient transduction in mTmG reporter mouse and pig models, as well as functional relevance in a variety of settings including atherosclerosis and Cas9 mediated knockouts.

Purpose:

The aim of this study is to apply the above-described methodology to achieve retargeting of AAV9 to the other principal component cell type of the microvasculature, the pericyte.

Methods:

Bio-panning of a phage display library (Ph.D. CX7C) on pericytic culture cells (CCL-226) was carried out for 2 rounds. Resulting restricted library was applied to wild type C57BL/6 mice after chloroquine injections. Cardiac and peripheral muscle pericytes were isolated by Liberase digestion and PDFGRb Macs sorting. NGS-amplicon sequencing was carried out to obtain heptameric peptide sequences. After normalization of sequence counts for bacterial amplification bias, 20 peptides were selected for synthesis and conjugated to PAMAM G2 dendrimers via PEG linker. A prescreening was carried out by coating AAV9-eGFP with the modified PAMAMs and application via I.M. injections to wild type C57BL/6 mice. Relative efficacies in pericyte transduction were assessed via flow cytometric analysis of eGFP⁺/NG2⁺ cells. To further elucidate most efficacious of the peptides, an AAV9 encoding Cre under NG2 promoter control (AAV9-proNG2-Cre) was generated. 5 most efficient peptide-PAMAM conjugates were used to coat AAV9-proNG2-Cre and applied to mTmG reporter mice via tail vein injections. Efficacy of pericyte retargeting was assessed via flow cytometry and confocal microscopy.

Results:

Flow cytometric analysis of skeletal muscle cell suspensions from transduced mTmG-mice showed that up to 40% of NG2⁺ cells were eGFP positive. Confocal imaging of cardiac and skeletal muscle sections of revealed transduced NG2⁺ and PDGFRb⁺ cells with ratios in accordance with flow cytometry results (Fig. 1).

Conclusion:

Efficient gene transfer to pericytes are of utmost utility in clinical settings where microcirculatory network is compromised, such as atherosclerosis or sepsis. Therefore, pericyte retargeted AAV9 provides researchers with a novel tool to overcome this limitation in microvascular medicine and research.

References:

1.         T. Bozoglu, et al. Endothelial Retargeting of AAV9 In Vivo. Advanced Science. 9, 2103867 (2022).