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Application of classic liver-directed gene replacement strategies is limited in genetic diseases characterized by liver injury due to hepatocyte proliferation, resulting in decline of therapeutic transgene expression and potential genotoxic risk. Wilson disease (WD) is a life-threatening autosomal disorder of copper homeostasis caused by pathogenic variants in copper transporter ATP7B and characterized by toxic copper accumulation, resulting in severe liver and brain diseases. Genome editing holds promise for the treatment of WD; nevertheless, to rescue copper homeostasis, ATP7B function must be restored in at least 25% of the hepatocytes, which surpasses by far genome-editing correction rates. We applied a liver-directed, nuclease-free genome editing approach, based on adeno-associated viral vector–mediated (AAV-mediated) targeted integration of a promoterless mini-ATP7B cDNA into the albumin (Alb) locus. Administration of AAV-Alb–mini-ATP7B in 2 WD mouse models resulted in extensive liver repopulation by genome-edited hepatocytes holding a proliferative advantage over nonedited ones, and ameliorated liver injury and copper metabolism. Furthermore, combination of genome editing with a copper chelator, currently used for WD treatment, achieved greater disease improvement compared with chelation therapy alone. Nuclease-free genome editing provided therapeutic efficacy and may represent a safer and longer-lasting alternative to classic gene replacement strategies for WD.

Abstract Birt‐Hogg‐Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss‐of‐function pathogenic variants in the gene encoding the tumor‐suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney‐specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient‐derived tumor samples revealed increased activation of TFEB/TFE3‐mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line‐derived xenografts (CDXs). Our findings demonstrate in disease‐relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.

Abstract Gyrate atrophy of choroid and retina (GACR) is a chorioretinal degeneration caused by pathogenic variants in the gene encoding ornithine aminotransferase (OAT), an enzyme mainly expressed in liver. Affected patients have increased ornithine concentrations in blood and other body fluids and develop progressive constriction of vision fields leading to blindness. Current therapies are unsatisfactory and better treatments are highly needed. In two mouse models of OAT deficiency that recapitulates biochemical and retinal changes of GACR, we investigated the efficacy of an intravenously injected serotype 8 adeno‐associated (AAV8) vector expressing OAT under the control of a hepatocyte‐specific promoter. Following injections, OAT‐deficient mice showed reductions of ornithine concentrations in blood and eye cups compared with control mice injected with a vector expressing green fluorescent protein. AAV‐injected mice showed improved electroretinogram response and partial restoration of retinal structure up to one‐year post‐injection. In summary, hepatic OAT expression by AAV8 vector was effective at correction of hyperornithinemia and improved function and structure of the retina. In conclusion, this study provides proof‐of‐concept of efficacy of liver‐directed AAV‐mediated gene therapy of GACR.

Wilson disease (WD) is a genetic disorder of copper homeostasis, caused by deficiency of the copper transporter ATP7B. Gene therapy with recombinant adeno-associated vectors (AAV) holds promises for WD treatment. However, the full-length human ATP7B gene exceeds the limited AAV cargo capacity, hampering the applicability of AAV in this disease context. To overcome this limitation, we designed a dual AAV vector approach using split intein technology. Split inteins catalyze seamless ligation of two separate polypeptides in a highly specific manner. We selected a DnaE intein from Nostoc punctiforme (Npu) that recognizes a specific tripeptide in the human ATP7B coding sequence. We generated two AAVs expressing either the 5′-half of a codon-optimized human ATP7B cDNA followed by the N-terminal Npu DnaE intein or the C-terminal Npu DnaE intein followed by the 3′-half of ATP7B cDNA, under the control of a liver-specific promoter. Intravenous co-injection of the two vectors in wild-type and Atp7b−/− mice resulted in efficient reconstitution of full-length ATP7B protein in the liver. Moreover, Atp7b−/− mice treated with intein-ATP7B vectors were protected from liver damage and showed improvements in copper homeostasis. Taken together, these data demonstrate the efficacy of split intein technology to drive the reconstitution of full-length human ATP7B and to rescue copper-mediated liver damage in Atp7b−/− mice, paving the way to the development of a new gene therapy approach for WD.

Hyperammonemia occurs in liver diseases affecting ureagenesis, and is life-threatening. Here, the authors show that liver UDP-GlcNAc is increased during hyperammonemia, leading to O-GlcNAcylation of the rate-limiting ureagenesis enzyme CPS1, that enhanced ureagenesis and ammonia detoxification. They also showed that pharmacological increase of protein O-GlcNAcylation reduces hyperammonemia in mouse models of liver disease.

Abstract Liver gene therapy with adeno‐associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X‐linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti‐F8 antibodies which neutralise F8 activity. Taking advantage of split‐intein‐mediated protein trans‐splicing, we divided the coding sequence of the large and highly secreted F8‐N6 variant in two separate AAV‐intein vectors whose co‐administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti‐F8 antibodies unlike animals treated with the single oversized AAV‐F8 vector under clinical development. Therefore, liver gene therapy with AAV‐F8‐N6 intein should be considered as a potential therapeutic strategy for HemA.

Limits of AAV-mediated gene therapy include targeting dominant mutations and inducing long-term transgene expression. Here, the authors show that AAV-HITI results in efficient allele-independent integration of a donor DNA in both retina and liver providing therapeutic benefit in mouse models of either a genetic form of blindness or a lysosomal storage disease, respectively.

Autophagy is a critical metabolic process that acts as a major self-digestion and recycling pathway contributing to maintain cellular homeostasis. An emerging field of research supports the therapeutic modulation of autophagy for treating human neurodegenerative disorders, in which toxic aggregates are accumulated in neurons. Our previous study identified Ezrin protein as an inhibitor of autophagy and lysosomal functions in the retina; thus, in turn, identifying it as a potential pharmacological target for increasing retinal cell clearance to treat inherited retinal dystrophies in which misfolded proteins have accumulated. This study aimed to verify the therapeutic inhibition of Ezrin to induce clearance of toxic aggregates in a mouse model for a dominant form of retinitis pigmentosa (i.e., RHOP23H/+). We found that daily inhibition of Ezrin significantly decreased the accumulation of misfolded RHOP23H aggregates. Remarkably, induction of autophagy, by a drug-mediated pulsatile inhibition of Ezrin, promoted the lysosomal clearance of disease-linked RHOP23H aggregates. This was accompanied with a reduction of endoplasmic reticulum (ER)-stress, robust decrease of photoreceptors' cell death, amelioration in both retinal morphology and function culminating in a better preservation of vision. Our study opens new perspectives for a pulsatile pharmacological induction of autophagy as a mutation-independent therapy paving the way toward a more effective therapeutic strategy to treat these devastating retinal disorders due to an accumulation of intracellular toxic aggregates.