**3. Conclusions**

In this review, we discussed the most recent in vivo and ex vivo approaches for using gene therapy in patients with PFIC, and addressed future developments within this field. Further, we elaborated on the clinical feasibility for all types of PFIC. The safety and efficacy results from many clinical trials conducting gene therapy are sufficiently positive to push the application of gene therapy for PFIC patients closer to the clinic. We can conclude that state of the art in vivo non-integrating gene therapy approaches will most likely not provide lifelong correction, due to a loss of AAV vector genomes caused by hepatocyte proliferation. However, the transient correction can slow down the decrease in liver function. Only integrating strategies that provide survival benefit to the corrected cells seem suitable for sustained efficacy in PFIC.

The feasibility to translate state of the art in vivo integrating gene therapies, upon showing proof of concept in pre-clinical models, into the clinic depends on the type of PFIC. Although integrating gene therapy would cure the PFIC2 and 5 phenotype, these patients remain at risk to develop HCC or CCC. Gene therapy for PFIC6 will only be effective for the small group of patients with the mutations in the motor domain of MYO5B that dominantly suffer from liver symptoms. Although MYO5B is too large to be packaged into AAVs, treating PFIC6 by knocking out gene expression of the mutated protein in the liver using AAV-mediated gene therapy or another approach to deliver CRISPR/Cas9 specifically to the hepatocytes, in theory could be a promising option. In most of MYO5B deficient patients, the pathology in the intestine is much more severe and will not be mitigated by AAV-mediated in vivo liver-directed gene therapy. PFIC1, 3 and 4 do seem good candidates for integrating gene therapy strategies targeting the diseased hepatocytes and could provide life-long correction in patients suffering from these severe life-threatening disorders.

**Author Contributions:** Conceptualization, M.W. and P.J.B.; writing—original draft preparation, M.W. and P.J.B.; writing—review and editing, R.P.J.O.-E. All authors have read and agreed to the published version of the manuscript

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
