Abstract 524 Specific targeting of cardiac fibroblasts by capsid engineered and in vivo selected adeno-associated viral vectors

Clin Res Cardiol (2023). https://doi.org/10.1007/s00392-023-02180-w
Specific targeting of cardiac fibroblasts by capsid engineered and in vivo selected adeno-associated viral vectors
S. Groß¹, L. Rode¹, S. Biß¹, H. Büning², C. Bär¹, T. Thum¹
¹Institut für Molekulare und Translationale Therapiestrategien, OE-8886, Medizinische Hochschule Hannover, Hannover; ²Experimentelle Hämatologie, Medizinische Hochschule Hannover, Hannover;
Adeno-associated viruses (AAV) present one of the most promising gene delivery systems for preclinical and clinical applications. Despite a grand selection of naturally occurring serotypes with different tissue tropisms and a suitable vector for heart-directed gene transfers (AAV6 or AAV9 depending on the species), transduction of cardiac fibroblasts (CF) is rather inefficient. Abnormal fibroblast phenotypes, in particular myofibroblasts and matrifibrocytes conversion, are a hallmark of pathological cardiac remodeling. Although cardiovascular diseases are quite complex and underlying mechanisms may differ, cardiac fibrosis is an often-underestimated key player in the development of heart failure. However, direct anti-fibrotic treatments that are scarce and current therapies lack efficiency. One major obstacle is the specific targeting of (activated) fibroblasts, which represents an unmet medical need beyond the heart. For this reason, a large AAV2 capsid-modified library was screened in mice with pressure overload-induced cardiac hypertrophy (after transverse aortic constriction, TAC) in three rounds of selection. Nineteen distinct variants were identified as cardiac fibroblast-enriched. To enable a characterization of all variants in parallel and simultaneously reducing animal numbers, a GFP-expressing barcode library was generated. Since capsid formation was either compromised or some variants formed unstable capsids, 14 AAV candidates were selected for the final CF library that was injected into 6-week TAC mice. Two weeks post injection, hearts were explanted and digested into cardiomyocytes, cardiac fibroblasts and endothelial cell fractions. Next-generation sequencing (NGS) identified three highly enriched variants in the CF fraction and two further variants characterized by an increased transduction of both cardiomyocytes and fibroblasts. Known AAV off target organs such as liver and spleen were examined together with organs that harbor noteworthy sources of fibroblasts. Subsequent in vitro investigations confirmed the improved transduction of mouse CFs with the three most enriched variants (Var1, 4 and 14). Next, human cardiac fibroblasts (HCFs), either purchased, or isolated from explanted hearts or from biopsies obtained prior to the implantation of a left ventricular assist device were used for subsequent experiments. These cells mainly consist of activated fibroblasts considering their origin from heart failure patients. Interestingly, the three AAV capsid variants indeed showed a strongly enhanced in vitro targeting of HCFs compared to the parental AAV2. Similar results were obtained in human lung, liver and kidney fibroblasts. Lowered in vitro neutralization by human IVIG revealed a partial immune escape of the novel variants, whereas a heparin competition assay revealed an altered cell-entry receptor usage. Both are important and desirable features towards a clinical development. In summary, these novel fibroblast-targeting AAV vectors represent a pioneering inventions of utmost importance for future clinical advances in the field of gene therapy for cardiovascular diseases.
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