*2.5. Clinical Feasibility*

To summarize, in vivo gene therapy using AAV vectors shows safety and efficacy in the clinic for several inherited diseases, including disorders affecting the liver. Within these therapies, the non-integrating approach has been extensively explored. In view of the early onset of severe damage and the persistent proliferation in case of insufficient efficacy, for all PFIC, integrating approaches are necessary for improved clinical application. Ex vivo gene therapy in adult hepatocytes followed by transplantation seems a potential option due to the survival benefit of the corrected cells that will promote liver repopulation. In view of the complicated procedure and inefficient grafting, in vivo gene therapy currently looks more promising for all types of PFIC.

The predominant concern for integrating gene therapy is induction of tumorigenesis, fueled by the early trials in severe combined immunodeficiency (SCID) patients [77]. The current adequate focus on tumorigenesis within gene therapy research will accelerate the development of safe, genome integrating gene therapeutics. State of the art integrating in vivo gene therapy approaches, including CRISPR/Cas9 show great promise in preclinical research because integration is guided, and therefore controlled, by Cas9 [56]. Currently, a first AAV-directed Cas9 driven gene therapy clinical trial for Leber's congenital amaurosis 10 (LCA10) has been approved and is recruiting (NCT03872479). Particularly safety assessments will determine conclusive outcomes for future in vivo application of integrating gene therapies in other inherited diseases.

Easily accessible organs like the eye, cervix, and liver are good targets for integrating gene therapy and future perspectives likely include new therapeutics for disorders within these target organs [89]. Proliferation advantages of corrected hepatocytes in stressed and diseased livers represent an important argument to choose for integrating gene therapy in certain types of PFIC.

Feasibility of gene therapy as a curative treatment differs between the types of PFIC. Deficiency in ABCB11 (PFIC2) show a complicated clinical expectation as the disease is cell autonomous and patients can develop hepatocellular carcinomas (HCC) or cholangiocarcinoma's (CCC) [90–92]. Although integrating gene therapy results in liver repopulation, presence of non-corrected cells cannot be excluded and maintains a permanent risk. Therefore even upon effective treatment in these patients, the risk of developing HCC and subsequent OLT remains [93]. Current available gene therapy methods seem an effective option to prevent liver failure until a suitable donor liver is available, but cannot replace liver transplantation in PFIC2 patients.

In PFIC5, deficiency of FXR, the potential role of gene therapy is comparable to that in patients with PFIC2. FXR is essential for transcription of the *ABCB11* gene and the expression of BSEP in all hepatocytes. Like PFIC2 patients, PFIC5 patients are at risk to develop HCC at an early age. Even upon effective gene therapy, presence of non-corrected hepatocytes results in a persisting risk for HCC development. In addition, the broad range of processes regulated by FXR results in a complicated disease and therefore correction of the diseased liver will not cure the pathology in all affected tissues. PFIC6 is caused by mutations in specific parts of the MYO5B protein, affecting its translocational capacity of proteins to the apical membrane [75]. Most MYO5B-deficient patients dominantly suffer from Microvillus Inclusion Disease [7]. Only for a sub-group of patients suffering from PFIC6, in which the liver pathology is the main symptom, liver directed gene therapy deleting the expression of the mutated MYO5B seems a feasible option.

In view of currently available methods, gene therapy to treat PFIC1, 3, and 4 seems the most feasible for clinical translation. Deficiencies of ABCB4 is the best studied type where three different pre-clinical studies all showed efficacy and proof of concept for liver directed in vivo gene therapy. The liver pathology caused by ATP8B1 deficiency (PFIC1) is also an attractive target for integrating gene therapy since the survival benefit of the corrected cells will promote liver repopulation. However, this approach will not address the extra-hepatic symptoms and it is not clear if an effective treatment will result in the development of a fatty liver, as seen upon OLT in these PFIC patients. In patients suffering from PFIC4,

re-expression of TJP2 needed for the formation of functional tight junction formation in the liver, may also result in a survival benefit of the corrected cells [11]. Since pathology caused by TJP2 deficiency seems restricted to the liver, this appears a good target for in vivo gene therapy. Proof of concept studies require the development of a conditional liver specific TJP2 knock-out model for PFIC4 because the whole body knock-out is not viable.
