Complete and long-term correction of CN1 in Gunn adults using lentiviral vectors
Previous attempts to treat adult Gunn rats with retroviral vectors failed because of an immune response directed against the transgene product. We constructed a tolerogenic lentiviral vectors by incorporing mir-142-3p target sequences in vector backbone and we achieved an unprecedent complete and definitive correction of adult Gunn rat, animal model of the Crigler-Najjar Type 1 liver disease, after a single systemic injection of lentiviral vectors.
Priming of hepatocytes enhances in vivo lentiviral transduction in adult animals
We identified that phenobarbital and cholic acid, 2 clinically relevant compounds that primed quiescent hepatocytes into G1 phase, increased hepatocyte in vivo transduction by about 10-fold and 5-fold, respectively .
Lentiviral vectors prevents the long-term onset of hepatic tumors in GSD1a mice
We reported for the first time that a partial correction of diseased hepatocytes prevented the natural onset of tumors in a hereditary liver disease, for instance glycogen storage disease type 1a (GSD1a). After partial restoration of glucose-6 phosphatase activity (25%) in mice modeling GSD1a using lentiviral vectors, mice presented normal hepatic triglyceride levels and were protected against the development of hepatic tumors at 9 months post-injection while most of untreated GSD1a mice developed millimetric tumors.
In vivo genome editing
ZFNs are composed of 2 monomers that dimerize to induce sequence-specific double-strand breaks. The 2 monomers are highly homologous (>90%) and thereby they recombine when packaged into recombinant retrovirus. By using a codon swapping strategy, we constructed a single integration-defective lentiviral vector encoding both ZFN monomer (in collaboration with IA) and reported for the first time targeted DSBs in primary cells (hepatocytes) and in vivo (liver).
We also worked at defining a genome editing strategy to repair the mutated ugt1a11 gene in the Gunn rat. We co-delivered ZFNs targeting ugt1a11 exon 4 and of an exogenous donor for gene repair using AAV vectors. In treated rats, we observed a significant decrease in bilirubinemia by up to 50% at 15 weeks post-injection. We detected expression of repaired UGT1A1 mRNA as well as presence of conjugated bilirubin in the bile at time of sacrifice (week 27 post-injection), showing unequivocal evidence of UGT1A1 activity and long-term persistence of corrected hepatocytes.
Ex vivo liver gene therapy
Having showed the feasibility of an ex vivo clinically-relevant approach, in which isolated hepatocytes were transduced in Suspension with lentiviral vector and Immediately Transplanted (SLIT) combined with a partial portal embolization to improve cell engraftment in the liver in non-human primate model, we showed now its feasibility in the Watanabe heritable hyperlipidemic rabbit, the most accurate animal model of familial hypercholesterolemia. By extrapolating all our results obtained in pre-clinical models to humans, we expect to obtain a 5-10% liver repopulation efficacy, which is sufficient to treat the majority of inherited metabolic liver diseases.
Regenerative medicine using IPS cells
Human induced pluripotent stem cells (hPSC) could be an ideal source for cell therapy because they represent an unlimited source of hepatocytes (pStemHep). Our overall objectives are: (i) to solve issues regarding clinical application of pluripotent stem cells with the aim of restoring defective hepatic function(s) in patients with severe metabolic liver disease and (ii) to develop immunomodulatory strategies to induce tolerance to hepatocyte transplants. We have defined a GMP compatible-method to differentiate pStemHep. We generated pStemHep until the stage of hepatic progenitors and showed that these pStemHep stably decreased bilirubinemia (30% decrease) throughout the study after transplantation in tacrolimus-immunosupressed Gunn rats without side effects. These results demonstrate the therapeutic potential of hPSC cell-based regenerative medicine for treating inherited metabolic liver diseases in absence of any selective growth advantage of transplanted cells over resident hepatocytes. Therapeutic efficacy in the Gunn rats was equivalent to that we observed after transplantation of hepatocytes isolated from human livers. We are evaluating some biomaterials to improve both cell implantation efficacy and protection against immune response.