TY - JOUR
T1 - Characterization of the humanized FRG mouse model and development of an AAV-LK03 variant with improved liver lobular biodistribution
AU - Cabanes-Creus, Marti
AU - Navarro, Renina Gale
AU - Liao, Sophia H.Y.
AU - Scott, Suzanne
AU - Carlessi, Rodrigo
AU - Roca-Pinilla, Ramon
AU - Knight, Maddison
AU - Baltazar, Grober
AU - Zhu, Erhua
AU - Jones, Matthew
AU - Denisenko, Elena
AU - Forrest, Alistair R.R.
AU - Alexander, Ian E.
AU - Tirnitz-Parker, Janina E.E.
AU - Lisowski, Leszek
N1 - Funding Information:
We thank CMRI Vector and Genome Engineering Facility (VGEF) for help in vector preparation. We also thank the Cytometry Facility of the Westmead Institute for Medical Research (WIMR) for help with sorting of murine and human hepatocytes. We also would like to thank all the members of the CMRI Bioresources, with special thanks to S. Dimech. Figures 2C, 3A, 3C, 5A, and 5B were generated with BioRender (https://biorender.com). This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC) to L.L. and I.E.A. (APP1108311, APP1156431 and APP1161583) and Paediatrio Paediatric Precision Medicine Program to L.L. (PPM1 K5116/RD274) as well as by funding from LogicBio Therapeutics. L.L. was also supported by research grants from the Department of Science and Higher Education of Ministry of National Defense, Republic of Poland, (“Kościuszko” k/10/8047/DNiSW/T – WIHE/3) and from the National Science Centre, Republic of Poland (OPUS 13) (UMO-2017/25/B/NZ1/02790). M.C-C. was also supported by a 2021 New South Wales (NSW) Ministry of Health, Office of Health and Medical Research (OHMR) Early-Mid Career Research Grant - Gene and Cell Therapy. Conceptualization, M.C.-C. and L.L.; methodology, M.C.-C. R.G.N. S.H.Y.L. R.C. and E.Z.; software, S.S. R.C. M.J. E.D. A.R.R.F. and J.E.E.T.-P.; investigation, M.C.-C. R.G.N. S.H.Y.L. R.C. M.K. and G.B.; writing – original draft, M.C.-C.; writing – review and editing, M.C.-C. S.S. and L.L.; funding acquisition, I.E.A. J.E.E.T.-P. and L.L.; visualization, M.C.-C. and R.R.-P.; supervision, M.C.-C. and L.L. L.L. is a cofounder of LogicBio Therapeutics, and L.L. and I.E.A. are co-founders of Exigen Biotherapeutics, companies that utilize similar technologies broadly discussed in this paper.
Funding Information:
We thank CMRI Vector and Genome Engineering Facility (VGEF) for help in vector preparation. We also thank the Cytometry Facility of the Westmead Institute for Medical Research (WIMR) for help with sorting of murine and human hepatocytes. We also would like to thank all the members of the CMRI Bioresources, with special thanks to S. Dimech. Figures 2 C, 3 A, 3C, 5 A, and 5B were generated with BioRender ( https://biorender.com ). This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC) to L.L. and I.E.A. ( APP1108311 , APP1156431 and APP1161583 ) and Paediatrio Paediatric Precision Medicine Program to L.L. (PPM1 K5116/RD274) as well as by funding from LogicBio Therapeutics. L.L. was also supported by research grants from the Department of Science and Higher Education of Ministry of National Defense , Republic of Poland, (“Kościuszko” k/10/8047/DNiSW/T – WIHE/3) and from the National Science Centre, Republic of Poland (OPUS 13) (UMO-2017/25/B/NZ1/02790). M.C-C. was also supported by a 2021 New South Wales (NSW) Ministry of Health, Office of Health and Medical Research (OHMR) Early-Mid Career Research Grant - Gene and Cell Therapy.
Publisher Copyright:
© 2023 The Authors
PY - 2023/3/9
Y1 - 2023/3/9
N2 - 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.
AB - 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.
KW - adeno-associated vector
KW - gene therapy
KW - humanized FRG
KW - liver lobule
KW - liver zonation
KW - lobular transduction
KW - single-nucleus RNA sequencing
UR - http://www.scopus.com/inward/record.url?scp=85146297629&partnerID=8YFLogxK
U2 - 10.1016/j.omtm.2022.12.014
DO - 10.1016/j.omtm.2022.12.014
M3 - Article
C2 - 36700121
AN - SCOPUS:85146297629
SN - 2329-0501
VL - 28
SP - 220
EP - 237
JO - Molecular Therapy - Methods and Clinical Development
JF - Molecular Therapy - Methods and Clinical Development
ER -
