*2.4. Prospects of Ex Vivo Gene Therapy for PFIC*

Applying gene therapy to isolated patient cells has major important advantages over in vivo approaches, like absence of interference of pre-existing immunity, the lack of an immune response towards the vector, no vector toxicity, exclusive targeting of the affected cell type, and the less invasive procedure [28]. Ex vivo gene therapy was the first form to show therapeutic efficacy in correcting inherited diseases of the hematopoietic stem cells (HSCs) [77]. This ex vivo correction benefited largely from the extensive experience with bone-marrow transplantations for tissue culture and pre-conditioning of the patient. Several HSC ex vivo gene therapies have shown long-term therapeutic efficacy and one has been registered, namely Strimvelis [78–80]. Transplantation of donor derived hepatocytes has been applied in patients suffering from different forms of inherited liver disorders [81]. Several studies report a partial correction of the disease indicating functionality of the transplanted hepatocytes. In all studies, this effect appeared to be transient, most likely due to immune responses towards the donor derived hepatocytes. Correcting patient derived hepatocytes would, by overcoming immune mediated loss of transplanted hepatocytes, provide long term correction. However, a small clinical study to treat familial hypercholesterolemia caused by low-density lipoprotein receptor (LDLR) deficiency did not provide therapeutic efficacy [82]. In part, this low efficacy could be explained by the use of a retroviral vector that, in contrast to a lentiviral vector, does not transduce non-dividing cells, such as mature hepatocytes. In addition, this trial also showed the complexity of the procedure including partial resection of the liver to isolate, culture and transduce the amount of hepatocytes needed for therapeutic efficacy. Further, in contrast to bone marrow transplantation, the experience with conditioning of the recipient is limited. All taken together, this approach only renders feasibility if corrected hepatocytes have a survival advantage resulting in a partial repopulation of the liver by the corrected cells. The liver damage observed in all types of PFIC does indicate that corrected hepatocytes may be able to repopulate the liver in these patients. Studies in relevant pre-clinical models for PFIC2 and 3 demonstrate that transplanted hepatocytes indeed repopulate the liver [52,67]. For the other types of PFIC a significant survival advantage of corrected cells has not been demonstrated, hence it is less clear if this approach will be suitable.

Correction of patient hepatocytes using ex vivo gene therapy and subsequent transplantation does require integration of the therapeutic construct in, or correction of, the host genome. The most effective vector for this type of gene therapy are the lentiviral vectors that transduce non-dividing cells such as hepatocytes. These vectors do integrate mostly in active genes which could increase the risk of tumorigenesis. For traditional retroviral vectors this risk indeed resulted in the development of acute leukemia, as shown in two trials [47,83]. In comparison to the vector used in these older studies, the safety of lentiviral vectors has been improved [84]. These third generation lentiviral vectors have been applied in several clinical trials and no tumor formation has been seen [85–87]. Ex vivo gene therapy using mature hepatocytes obtained from the patient liver will be a very challenging procedure especially in view of the large number of cells needed. To render this method applicable, cell proliferation will be essential to provide sufficient numbers for effective gene therapy. In this respect the use of stem cells, either induced pluripotent stem cells (iPSCs) or from the liver, remains necessary. In combination with a survival effect, these ex vivo corrected cells can be effective. A recent study for a severe skin disease resulted in complete recovery upon transplanting corrected skin stem cells [88]. For the liver this will be more complicated, but may be possible in future perspective.
