TY - JOUR
T1 - Resolution of hepatic fibrosis after ZFN-mediated gene editing in the PiZ mouse model of human α1-antitrypsin deficiency
AU - Li, Yanfeng
AU - Guha, Chandan
AU - Asp, Patrik
AU - Wang, Xia
AU - Tchaikovskya, Tatyana L.
AU - Kim, Kenneth
AU - Mendel, Matthew
AU - Cost, Gregory J.
AU - Perlmutter, David H.
AU - Roy-Chowdhury, Namita
AU - Fox, Ira J.
AU - Conway, Anthony
AU - Roy-Chowdhury, Jayanta
N1 - Publisher Copyright:
Copyright © 2023 The Author(s).
PY - 2023/2
Y1 - 2023/2
N2 - Background: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver. Methods: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI). PiZ mice were injected i.v. with rAAV-TI alone or the rAAV-ZFNs at a low (7.5 × 1010vg/mouse, LD) or a high dose (1.5×1011vg/ mouse, HD), with or without rAAV-TI. Two weeks and 6 months after treatment, livers were harvested for molecular, histological, and biochemical analyses. Results: Two weeks after treatment, deep sequencing of the hepatic SA1-ATZ transgene pool showed 6% ± 3% or 15% ± 4% nonhomologous end joining in mice receiving LD or HD rAAV-ZFN, respectively, which increased to 36% ± 12% and 36% ± 12%, respectively, 6 months after treatment. Two weeks postinjection of rAAV-TI with LD or HD of rAAV-ZFN, repair by targeted insertion occurred in 0.10% ± 0.09% and 0.25% ± 0.14% of SA1-ATZ transgenes, respectively, which increased to 5.2% ± 5.0% and 33% ± 13%, respectively, 6 months after treatment. Six months after rAAV-ZFN administration, there was a marked clearance of ATZ globules from hepatocytes, and resolution of liver fibrosis, along with reduction of hepatic TAZ/WWTR1, hedgehog ligands, Gli2, a TIMP, and collagen content. Conclusions: ZFN-mediated SA1-ATZ transgene disruption provides a proliferative advantage to ATZ-depleted hepatocytes, enabling them to repopulate the liver and reverse hepatic fibrosis.
AB - Background: α1-antitrypsin deficiency is most commonly caused by a mutation in exon-7 of SERPINA1 (SA1-ATZ), resulting in hepatocellular accumulation of a misfolded variant (ATZ). Human SA1-ATZ-transgenic (PiZ) mice exhibit hepatocellular ATZ accumulation and liver fibrosis. We hypothesized that disrupting the SA1-ATZ transgene in PiZ mice by in vivo genome editing would confer a proliferative advantage to the genome-edited hepatocytes, enabling them to repopulate the liver. Methods: To create a targeted DNA break in exon-7 of the SA1-ATZ transgene, we generated 2 recombinant adeno-associated viruses (rAAV) expressing a zinc-finger nuclease pair (rAAV-ZFN), and another rAAV for gene correction by targeted insertion (rAAV-TI). PiZ mice were injected i.v. with rAAV-TI alone or the rAAV-ZFNs at a low (7.5 × 1010vg/mouse, LD) or a high dose (1.5×1011vg/ mouse, HD), with or without rAAV-TI. Two weeks and 6 months after treatment, livers were harvested for molecular, histological, and biochemical analyses. Results: Two weeks after treatment, deep sequencing of the hepatic SA1-ATZ transgene pool showed 6% ± 3% or 15% ± 4% nonhomologous end joining in mice receiving LD or HD rAAV-ZFN, respectively, which increased to 36% ± 12% and 36% ± 12%, respectively, 6 months after treatment. Two weeks postinjection of rAAV-TI with LD or HD of rAAV-ZFN, repair by targeted insertion occurred in 0.10% ± 0.09% and 0.25% ± 0.14% of SA1-ATZ transgenes, respectively, which increased to 5.2% ± 5.0% and 33% ± 13%, respectively, 6 months after treatment. Six months after rAAV-ZFN administration, there was a marked clearance of ATZ globules from hepatocytes, and resolution of liver fibrosis, along with reduction of hepatic TAZ/WWTR1, hedgehog ligands, Gli2, a TIMP, and collagen content. Conclusions: ZFN-mediated SA1-ATZ transgene disruption provides a proliferative advantage to ATZ-depleted hepatocytes, enabling them to repopulate the liver and reverse hepatic fibrosis.
UR - http://www.scopus.com/inward/record.url?scp=85204522166&partnerID=8YFLogxK
U2 - 10.1097/HC9.0000000000000070
DO - 10.1097/HC9.0000000000000070
M3 - Article
C2 - 36848094
AN - SCOPUS:85204522166
SN - 2471-254X
VL - 7
SP - e0070
JO - Hepatology Communications
JF - Hepatology Communications
IS - 3
M1 - e0070
ER -