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

Marti Cabanes-Creus; Renina Gale Navarro; Sophia H Y Liao; Suzanne Scott; Rodrigo Carlessi; Ramon Roca-Pinilla; Maddison Knight; Grober Baltazar; Erhua Zhu; Matthew Jones; Elena Denisenko; Alistair R R Forrest; Ian E Alexander; Janina E E Tirnitz-Parker; Leszek Lisowski

doi:10.1016/j.omtm.2022.12.014

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

Mol Ther Methods Clin Dev. 2023 Jan 2:28:220-237. doi: 10.1016/j.omtm.2022.12.014. eCollection 2023 Mar 9.

Authors

Marti Cabanes-Creus¹, Renina Gale Navarro¹, Sophia H Y Liao¹, Suzanne Scott¹, Rodrigo Carlessi^{2

3}, Ramon Roca-Pinilla¹, Maddison Knight¹, Grober Baltazar¹, Erhua Zhu⁴, Matthew Jones³, Elena Denisenko³, Alistair R R Forrest³, Ian E Alexander^{4

5}, Janina E E Tirnitz-Parker^{2

6}, Leszek Lisowski^{1

7}

Affiliations

¹ Translational Vectorology Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW 2145, Australia.
² Curtin Medical School, Curtin Health Innovation Research Institute, Curtin University, Bentley, WA 6102, Australia.
³ Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, The University of Western Australia, Nedlands, WA 6009, Australia.
⁴ Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Faculty of Medicine and Health, The University of Sydney, and Sydney Children's Hospitals Network, Westmead, NSW 2145, Australia.
⁵ Discipline of Child and Adolescent Health, The University of Sydney, Sydney Medical School, Faculty of Medicine and Health, Westmead, NSW 2145, Australia.
⁶ UWA Centre for Medical Research, The University of Western Australia, Nedlands, WA 6009, Australia.
⁷ Laboratory of Molecular Oncology and Innovative Therapies, Military Institute of Medicine, 04-141 Warsaw, Poland.

Abstract

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.

Keywords: adeno-associated vector; gene therapy; humanized FRG; liver lobule; liver zonation; lobular transduction; single-nucleus RNA sequencing.