Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution

• Published in: Molecular therapy. Methods & clinical development Vol. 28; pp. 220 - 237
• Main Authors: Cabanes-Creus, Marti, Navarro, Renina Gale, Liao, Sophia H.Y., Scott, Suzanne, Carlessi, Rodrigo, Roca-Pinilla, Ramon, Knight, Maddison, Baltazar, Grober, Zhu, Erhua, Jones, Matthew, Denisenko, Elena, Forrest, Alistair R.R., Alexander, Ian E., Tirnitz-Parker, Janina E.E., Lisowski, Leszek
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 09.03.2023; American Society of Gene & Cell Therapy; Elsevier
• Subjects: adeno-associated vector; gene therapy; humanized FRG; liver lobule; liver zonation; lobular transduction; Original; single-nucleus RNA sequencing; liver lobule; liver zonation; single-nucleus RNA sequencing; gene therapy; lobular transduction; humanized FRG; adeno-associated vector
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2022.12.014

Recent clinical successes have intensified interest in using adeno-associated virus (AAV) vectors for therapeutic gene delivery. The liver is a key clinical target, given its critical physiological functions and involvement in a wide range of genetic diseases. In the present study, we first investigated the validity of a liver xenograft mouse model repopulated with primary hepatocytes using single-nucleus RNA sequencing (sn-RNA-seq) by studying the transcriptomic profile of human hepatocytes pre- and post-engraftment. Complementary immunofluorescence analyses performed in highly engrafted animals confirmed that the human hepatocytes organize and present appropriate patterns of zone-dependent enzyme expression in this model. Next, we tested a set of rationally designed HSPG de-targeted AAV-LK03 variants for relative transduction performance in human hepatocytes. We used immunofluorescence, next-generation sequencing, and single-nucleus transcriptomics data from highly engrafted FRG mice to demonstrate that the optimally HSPG de-targeted AAV-LK03 displayed a significantly improved lobular transduction profile in this model. [Display omitted] Using single-nucleus RNA sequencing and complementary immunofluorescence analyses, the authors confirmed that human hepatocytes engrafted in xenograft mice organize and present appropriate patterns of liver-zone-dependent enzyme expression. Having confirmed this, the authors successfully performed targeted bioengineering of a clinical AAV variant, AAV-LK03, to improve its lobular transduction profile.