Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of α-1 Antitrypsin Deficiency

Abstract

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…Such an effect is not readily observed in in-vivo lung or liver gene therapy, but examples do exist, and a proliferative advantage can be established artificially. For example, correcting the common PI*Z mutation of AAT deficiency or inhibiting its expression with a microRNA prevents the accumulation of PI*Z polymers in the hepatocytes and give those cells a survival advantage over non-corrected cells that have a tendency to undergo stress-induced apoptosis [87]. A more synthetic approach was taken by Nygaard and colleagues, who developed a vector that, in addition to expressing a therapeutic transgene, also expressed a short hairpin RNA (shRNA) that protects against a toxic drug [88].…”

Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

“…To date, there have been relatively few studies using genome editing for successful homology-directed repair (HDR) in vivo, none of which have used human-specific guide RNAs or donor templates. These studies have all employed murine models where either a selective growth advantage is conferred on the corrected cells, such as models of hereditary tyrosinemia type I, [11][12][13] or where the correction of only a small percentage of cells can result in phenotypic improvements, as is the case for haemophilia B. 14 Alternatively, studies targeting primary hepatocytes in vivo have exploited the more active non-homologous end joining (NHEJ) pathway for locus-specific disruption of the murine Pcsk9 15,16 or human PCSK9 17 genes.…”

Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

“…Third, to overcome the absence of selective pressure for corrected cells in other diseases, a clinically relevant protocol will need to be optimized for enhancing proliferation of transplanted cells to phenotypically relevant levels. Currently, a number of genetic and drug‐induced injury methods have demonstrated efficacy in preclinical models . However, to date, no clinically relevant methodology has been demonstrated.…”