Evolving Adeno-Associated Virus for Kidney Gene Transfer

Abstract

Chronic kidney disease (CKD) is estimated to affect 8-16% of the population worldwide and has continued to increase by 31.7% over the last ten years. Ultimately, CKD progresses to end-stage renal disease (ESRD), where dialysis or kidney transplantation are currently the only viable treatment options. Dialysis only facilitates a very small fraction of normal kidney function and requires multiple prolonged sessions per week and is characterized by poor patient survival, with the average life expectancy being 5-10 years. On the other hand, kidney transplantation has developed into a successful long-term therapy but has its own limitations –– due to donor organ scarcity, risk of organ rejection and the need for lifelong immunosuppression, transplantation remains a double-edged sword. Importantly, many kidney diseases such as cystinuria, polycystic kidney disease, and cystinosis amongst others can cause CKD and have underlying genetic etiologies that may be amenable to gene therapy or genome editing, yet there are currently no feasible options for targeting these disorders using gene therapy. Taken together, there is a critical unmet need for an effective and safe kidney-targeting gene delivery vehicle that can transcend the limitations of current treatments for kidney disease.Within the gene therapy framework, recombinant adeno-associated virus (AAV) vectors constitute a promising gene delivery platform for the treatment of a variety of human diseases. With the approval of five AAV-based products by the FDA for treating ocular, hematological and neuromuscular disorders, there are ~ 200 completed or active gene therapy clinical trials registered at ClinicalTrials.gov as of September 2024. However, little progress has been made to date in achieving effective therapeutic gene transfer to kidneys. Due to the intrinsic filtering function and complex physiology of kidneys, successful gene delivery has proven challenging. Moreover, naturally occurring AAV serotypes have either no kidney-tropism or broad, nonspecific tissue tropism, and can lack translatability across traditionally used biomedical research animals, impeding development of AAV vectors for preclinical and clinical applications.To overcome this barrier, we first characterized AAV.cc47, an AAV9-derived capsid from a previous cross-species evolution effort with enhanced transduction profiles across multiple tissues, in multiple relevant preclinical kidney models. mCherry reporter studies in mice demonstrated robust expression in proximal tubules. In human kidney organoids, AAV.cc47 demonstrated enhanced transduction efficiency. Increased potency of AAV.cc47 for transducing kidneys was further corroborated in ex vivo perfused pig kidneys and in situ delivered nonhuman primate kidneys. We demonstrated efficacy and efficient transduction in delivering a therapeutically relevant construct in nonhuman primate kidneys using different routes of administration to kidney for successful gene delivery.To further improve efficacy, we used insights from our AAV.cc47 study to inform our approach for evolving kidney-targeting capsids. We evolved new cross-species compatible, AAV-kidney (AAV.k) variants by cycling AAV capsid libraries across different kidney systems and using different routes of administration to target the kidney. Our robust approach utilized intravenous dosing in mice and pigs, ex vivo machine perfusion in isolated rhesus macaque kidneys using both ureteral and arterial delivery as well as human organoid cultures to cycle AAV capsid libraries. By employing a multi-model and multi-species evolution strategy in vivo and ex vivo, we discovered and characterized novel AAV.k variants, AAV.k20 and AAV.k13, promising vectors for therapeutic gene transfer applications in kidney diseases and transplantation.In conclusion, these findings underscore the potential of AAVs as a transformative tool in gene therapy for kidney diseases and lay the foundation for future preclinical and clinical development of AAV-based kidney gene therapies. These advancements not only provide promising solutions for treating genetic kidney diseases but also have the potential to transform transplantation strategies. Lastly, our approach provides a pipeline for discovering other AAV.k variants capable of transducing different segments of the nephron or other disease relevant kidney cell-types.