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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.

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.

Artículo escrito por un elevado número de autores, solo se referencian el que aparece en primer lugar, el nombre del grupo de colaboración, si le hubiere, y los autores pertenecientes a la UAM, Life-threatening hyperammonemia occurs in both inherited and acquired liver diseases affecting ureagenesis, the main pathway for detoxification of neurotoxic ammonia in mammals. Protein O-GlcNAcylation is a reversible and nutrient-sensitive post-translational modification using as substrate UDP-GlcNAc, the end-product of hexosamine biosynthesis pathway. Here we show that increased liver UDP-GlcNAc during hyperammonemia increases protein O-GlcNAcylation and enhances ureagenesis. Mechanistically, O-GlcNAcylation on specific threonine residues increased the catalytic efficiency for ammonia of carbamoyl phosphate synthetase 1 (CPS1), the rate-limiting enzyme in ureagenesis. Pharmacological inhibition of O-GlcNAcase, the enzyme removing O-GlcNAc from proteins, resulted in clinically relevant reductions of systemic ammonia in both genetic (hypomorphic mouse model of propionic acidemia) and acquired (thioacetamide-induced acute liver failure) mouse models of liver diseases. In conclusion, by fine-tuned control of ammonia entry into ureagenesis, hepatic O-GlcNAcylation of CPS1 increases ammonia detoxification and is a novel target for therapy of hyperammonemia in both genetic and acquired diseases, This work was supported by grants of Fondazione Telethon Italy (to N.B.‐P.), MIUR (PRIN2017 to N.B.‐P.), of the Swiss National Science Foundation (grant 320030_176088 to J.H.), of the US National Institutes of Health grants (R21NS091654 and R01NS100979 both to G.S.L.), of the Spanish Ministry of Science and Innovation (PID2019-105344RB-I00/AEI/10.13039/501100011033 toL.R.D. and E.R.), and by a Wellcome Trust Investigator Award (110061to D.M.F.v.A.)

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

Adeno-associated viral (AAV) vectors have emerged as the preferred platform for in vivo gene transfer because of their combined efficacy and safety. However, insertional mutagenesis with the subsequent development of hepatocellular carcinomas (HCCs) has been recurrently noted in newborn mice treated with high doses of AAV, and more recently, the association of wild-type AAV integrations in a subset of human HCCs has been documented. Here, we address, in a comprehensive, prospective study, the long-term risk of tumorigenicity in young adult mice following delivery of single-stranded AAVs targeting liver. HCC incidence in mice treated with therapeutic and reporter AAVs was low, in contrast to what has been previously documented in mice treated as newborns with higher doses of AAV. Specifically, HCCs developed in 6 out 76 of AAV-treated mice, and a pathogenic integration of AAV was found in only one tumor. Also, no evidence of liver tumorigenesis was found in juvenile AAV-treated mucopolysaccharidosis type VI (MPS VI) cats followed as long as 8 years after vector administration. Together, our results support the low risk of tumorigenesis associated with AAV-mediated gene transfer targeting juvenile/young adult livers, although constant monitoring of subjects enrolled in AAV clinical trial is advisable., Graphical Abstract, Systemic delivery of high doses of adeno-associated viral (AAV) vectors causes insertional mutagenesis and hepatocellular carcinomas in newborn mice. The study shows that this risk is lower in young adult mice and juvenile cats using AAV doses similar to those used in many clinical applications.

α1-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop-/- mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop-/- mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter-based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1–3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB—unlike other substrates of mTORC1, such as S6K and 4E-BP1— is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt–Hogg–Dube syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt–Hogg–Dube syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer. Dysregulation of an mTORC1 substrate-specific mechanism leads to constitutive activation of TFEB, and promotes kidney cystogenesis and tumorigenesis in a mouse model of Birt–Hogg–Dube syndrome.

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., RHO

Challenges to the widespread application of gene therapy with adeno-associated viral (AAV) vectors include dominant conditions due to gain-of-function mutations which require allele-specific knockout, as well as long-term transgene expression from proliferating tissues, which is hampered by AAV DNA episomal status. To overcome these challenges, we used CRISPR/Cas9-mediated homology-independent targeted integration (HITI) in retina and liver as paradigmatic target tissues. We show that AAV-HITI targets photoreceptors of both mouse and pig retina, and this results in significant improvements to retinal morphology and function in mice with autosomal dominant retinitis pigmentosa. In addition, we show that neonatal systemic AAV-HITI delivery achieves stable liver transgene expression and phenotypic improvement in a mouse model of a severe lysosomal storage disease. We also show that HITI applications predominantly result in on-target editing. These results lay the groundwork for the application of AAV-HITI for the treatment of diseases affecting various organs.

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid β progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a “molecular tweezer” that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs., Graphical Abstract, Fraldi and colleagues demonstrated that multiple amyloid proteins progressively aggregate in neurons of a severe lysosomal storage disease, impairing autophagy degradation and triggering neurodegeneration. They also showed that inhibiting amyloid deposition protects against neurodegeneration, thus providing evidence that amyloid aggregation is a new attractive target for the treatment of LSDs.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.

PURPOSE: We studied microRNAs as potential biomarkers for Pompe disease. METHODS: We analyzed microRNA expression by small RNA-seq in tissues from the disease murine model at two different ages (3 and 9 months), and in plasma from Pompe patients. RESULTS: In the mouse model we found 211 microRNAs that were differentially expressed in gastrocnemii and 66 in heart, with a different pattern of expression at different ages. In a preliminary analysis in plasma from six patients 55 microRNAs were differentially expressed. Sixteen of these microRNAs were common to those dysregulated in mouse tissues. These microRNAs are known to modulate the expression of genes involved in relevant pathways for Pompe disease pathophysiology (autophagy, muscle regeneration, muscle atrophy). One of these microRNAs, miR-133a, was selected for further quantitative real-time polymerase chain reaction analysis in plasma samples from 52 patients, obtained from seven Italian and Dutch biobanks. miR-133a levels were significantly higher in Pompe disease patients than in controls and correlated with phenotype severity, with higher levels in infantile compared with late-onset patients. In three infantile patients miR-133a decreased after start of enzyme replacement therapy and evidence of clinical improvement. CONCLUSION: Circulating microRNAs may represent additional biomarkers of Pompe disease severity and of response to therapy.

Pompe disease is a metabolic myopathy due to acid alpha‐glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha‐glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients’ cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N‐acetylcysteine, idebenone, resveratrol, edaravone) improved alpha‐glucosidase activity in rhGAA‐treated cells, enhanced enzyme processing, and improved mannose‐6‐phosphate receptor localization. When co‐administered with rhGAA, antioxidants improved alpha‐glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder., Enzyme replacement therapy (ERT) with recombinant human alpha‐glucosidase (rhGAA) is currently the standard of care for the treatment of Pompe disease. However, this approach shows important limitations. We have tested whether modulation of oxidative stress may improve the efficacy of ERT.

α(1)-Antitrypsin (AAT) encoded by the SERPINA1 gene is an acute-phase protein synthesized in the liver and secreted into the circulation. Its primary role is to protect lung tissue by inhibiting neutrophil elastase. The Z allele of SERPINA1 encodes a mutant AAT, named ATZ, that changes the protein structure and leads to its misfolding and polymerization, which cause endoplasmic reticulum (ER) stress and liver disease through a gain-of-function toxic mechanism. Hepatic retention of ATZ results in deficiency of one of the most important circulating proteinase inhibitors and predisposes to early-onset emphysema through a loss-of-function mechanism. The pathogenetic mechanisms underlying the liver disease are not completely understood. C/EBP-homologous protein (CHOP), a transcription factor induced by ER stress, was found among the most up-regulated genes in livers of PiZ mice that express ATZ and in human livers of patients homozygous for the Z allele. Compared with controls, juvenile PiZ/Chop(−/−) mice showed reduced hepatic ATZ and a transcriptional response indicative of decreased ER stress by RNA-Seq analysis. Livers of PiZ/Chop(−/−) mice also showed reduced SERPINA1 mRNA levels. By chromatin immunoprecipitations and luciferase reporter–based transfection assays, CHOP was found to up-regulate SERPINA1 cooperating with c-JUN, which was previously shown to up-regulate SERPINA1, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele. In summary, CHOP and c-JUN up-regulate SERPINA1 transcription and play an important role in hepatic disease by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

Alpha-1 antitrypsin (AAT) deficiency is a common genetic disorder with lung and liver involvement. Most patients carry the Z allele in SERPINA1 that encodes a mutant AAT (ATZ) forming hepatotoxic polymers. We found CHOP upregulation and activation in both mouse (PiZ) and human livers expressing ATZ. Compared to controls, juvenile PiZ/Chop-/- mice showed reduction in hepatic ATZ and transcriptional response to endoplasmic reticulum stress, as consequence of CHOP-mediated increase of SERPINA1 transcription. CHOP was found to upregulate SERPINA1 though binding with c-JUN on SERPINA1 regulatory elements, thus aggravating hepatic accumulation of ATZ. Increased CHOP levels were detected in diseased livers of children homozygous for the Z allele.Compared to adults, AAT deficiency in infants has different severity and prognosis. Based on our findings, CHOP-c-JUN complex upregulates SERPINA1 transcription and play an important role in the hepatic disease pathogenesis by increasing the burden of proteotoxic ATZ, particularly in the pediatric population.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light‐responsive miR‐211 controls lysosomal biogenesis at the beginning of light–dark transitions in the RPE by targeting Ezrin, a cytoskeleton‐associated protein essential for the regulation of calcium homeostasis. miR‐211‐mediated down‐regulation of Ezrin leads to Ca(2+) influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light‐mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR‐211(−/−) mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR‐211(−/−) phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

Graphical abstract, Highlights • Pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) translocate to the nucleus in injured liver cells. • Increased nuclear PDHC and LDH induce increased nuclear acetyl-CoA and lactate, histone hyper-acetylation, and damage response gene expression. • Inhibition of histone acetylation or inhibition of PDHC and LDH reduce damage and inflammation in injured livers. • Galloflavin inhibits both PDH and LDH and protects from damage and mortality induced by hepatotoxins., Background & Aims Acute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression. Methods Levels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure. Results Levels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival. Conclusion PDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure. Lay summary Acute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.

Ammonia is a potent neurotoxin that is detoxified mainly by the urea cycle in the liver. Hyperammonemia is a common complication of a wide variety of both inherited and acquired liver diseases. If not treated early and thoroughly, it results in encephalopathy and death. Here, we found that hepatic autophagy is critically involved in systemic ammonia homeostasis by providing key urea-cycle intermediates and ATP. Hepatic autophagy is triggered in vivo by hyperammonemia through an α-ketoglutarate-dependent inhibition of the mammalian target of rapamycin complex 1, and deficiency of autophagy impairs ammonia detoxification. In contrast, autophagy enhancement by means of hepatic gene transfer of the master regulator of autophagy transcription factor EB or treatments with the autophagy enhancers rapamycin and Tat-Beclin-1 increased ureagenesis and protected against hyperammonemia in a variety of acute and chronic hyperammonemia animal models, including acute liver failure and ornithine transcarbamylase deficiency, the most frequent urea-cycle disorder. In conclusion, hepatic autophagy is an important mechanism for ammonia detoxification because of its support of urea synthesis, and its enhancement has potential for therapy of both primary and secondary causes of hyperammonemia.

Pompe disease (PD) is a metabolic myopathy due to acid alpha-glucosidase deficiency and characterized by extensive glycogen storage and impaired autophagy. We previously showed that modulation of autophagy and lysosomal exocytosis by overexpression of the transcription factor EB (TFEB) gene was effective in improving muscle pathology in PD mice injected intramuscularly with an AAV-TFEB vector. Here we have evaluated the effects of TFEB systemic delivery on muscle pathology and on functional performance, a primary measure of efficacy in a disorder like PD. We treated 1-month-old PD mice with an AAV2.9-MCK-TFEB vector. An animal cohort was analyzed at 3 months for muscle and heart pathology. A second cohort was followed at different timepoints for functional analysis. In muscles from TFEB-treated mice we observed reduced PAS staining and improved ultrastructure, with reduced number and increased translucency of lysosomes, while total glycogen content remained unchanged. We also observed statistically significant improvements in rotarod performance in treated animals compared to AAV2.9-MCK-eGFP-treated mice at 5 and 8 months. Cardiac echography showed significant reduction in left-ventricular diameters. These results show that TFEB overexpression and modulation of autophagy result in improvements of muscle pathology and of functional performance in the PD murine model, with delayed disease progression.

In vivo gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. We recently demonstrated that AAV8-mediated liver gene transfer is effective in animal models of mucopolysaccharidosis type VI (MPS VI), a rare lysosomal storage disease that is caused by arylsulfatase B (ARSB) deficiency. In preparing for a first-in-human trial, we performed non-clinical studies to assess the safety of intravenous administrations of AAV2/8.TBG.hARSB produced under good manufacturing practice-like conditions. No toxicity was observed in AAV-treated mice, except for a transient increase in alanine aminotransferase in females and thyroid epithelial hypertrophy. AAV2/8.TBG.hARSB biodistribution and expression confirmed the liver as the main site of both infection and transduction. Shedding and breeding studies suggest that the risk of both horizontal and germline transmission is minimal. An AAV dose-response study in MPS VI mice was performed to define the range of doses to be used in the clinical study. Overall, these data support the non-clinical safety and efficacy of AAV2/8.TBG.hARSB and pave the way for a phase I/II clinical trial based on intravascular infusions of AAV8 in patients with MPS VI.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors—master regulators of lysosomal and melanosomal biogenesis and autophagy—control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

Mucopolysaccharidosis type IIIA (MPS-IIIA, Sanfilippo A) is one of the most severe lysosomal storage disorder (LSD) caused by the inherited deficiency of sulfamidase, a lysosomal sulfatase enzyme involved in the stepwise degradation of heparan sulfates (HS). MPS-IIIA patients show multisystemic problems, including a strong impairment of central nervous system (CNS), mild somatic involvement, and ocular manifestations that result in significant visual impairment. Despite the CNS and somatic pathology have been well characterized, studies on visual system and function remain partially explored. Here, we characterized the retina morphology and functionality in MPS-IIIA mouse model and analyzed how the SGSH deficiency affects the autophagic flux. MPS-IIIA mice exhibited a progressive retinal dystrophy characterized by significant alterations in visual function. The photoreceptor degeneration was associated with HS accumulation and a block of autophagy pathway. These events caused a reactive microgliosis, and a development of apoptotic processes in MPS-IIIA mouse retina. Overall, this study provides the first phenotypic spectrum of retinal disorders in MPS-IIIA and significantly contributes for diagnosis, counseling, and potential therapies development.

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathwaysand suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is itstranslocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of severaldiseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bonegrowth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomalassociation and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associatedBeclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads toa blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens,the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling orstimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, andameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis ofskeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.

Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies.Molecular Therapy (2016); doi:10.1038/mt.2016.181.

Mucopolysaccharidosis type II (MPSII), or Hunter syndrome, is a devastating disorder associated with a shortened life expectancy. Patients affected by MPSII have a variety of symptoms that affect all organs of the body and may include progressive cognitive impairment. MPSII is due to inactivity of the enzyme iduronate-2-sulfatase (IDS), which results in the accumulation of storage material in the lysosomes, such as dermatan and heparan sulfates, with consequent cell degeneration in all tissues including, in the severe phenotype, neurodegeneration in the central nervous system (CNS). To date, the only treatment available is systemic infusion of IDS, which ameliorates exclusively certain visceral defects. Therefore, it is important to simultaneously treat the visceral and CNS defects of the MPSII patients. Here, we have developed enzyme replacement therapy (ERT) protocols in a mouse model that allow the IDS to reach the brain, with the substantial correction of the CNS phenotype and of the neurobehavioral features. Treatments were beneficial even in adult and old MPSII mice, using relatively low doses of infused IDS over long intervals. This study demonstrates that CNS defects of MPSII mice can be treated by systemic ERT, providing the potential for development of an effective treatment for MPSII patients.

Sulfatases are enzymes that hydrolyse a diverse range of sulfate esters. Deficiency of lysosomal sulfatases leads to human diseases characterized by the accumulation of either GAGs (glycosaminoglycans) or sulfolipids. The catalytic activity of sulfatases resides in a unique formylglycine residue in their active site generated by the post-translational modification of a highly conserved cysteine residue. This modification is performed by SUMF1 (sulfatase-modifying factor 1), which is an essential factor for sulfatase activities. Mutations in the SUMF1 gene cause MSD (multiple sulfatase deficiency), an autosomal recessive disease in which the activities of all sulfatases are profoundly reduced. In previous studies, we have shown that SUMF1 has an enhancing effect on sulfatase activity when co-expressed with sulfatase genes in COS-7 cells. In the present study, we demonstrate that SUMF1 displays an enhancing effect on sulfatases activity when co-delivered with a sulfatase cDNA via AAV (adeno-associated virus) and LV (lentivirus) vectors in cells from individuals affected by five different diseases owing to sulfatase deficiencies or from murine models of the same diseases [i.e. MLD (metachromatic leukodystrophy), CDPX (X-linked dominant chondrodysplasia punctata) and MPS (mucopolysaccharidosis) II, IIIA and VI]. The SUMF1-enhancing effect on sulfatase activity resulted in an improved clearance of the intracellular GAG or sulfolipid accumulation. Moreover, we demonstrate that the SUMF1-enhancing effect is also present in vivo after AAV-mediated delivery of the sulfamidase gene to the muscle of MPSIIIA mice, resulting in a more efficient rescue of the phenotype. These results indicate that co-delivery of SUMF1 may enhance the efficacy of gene therapy in several sulfatase deficiencies.

Enzyme replacement therapy (ERT) is the current standard of care for Mucopolysaccharidosis type VI (MPS VI) that is caused by deficiency of arylsulfatase B (ARSB), which results in widespread accumulation and excretion of toxic glycosaminoglycans (GAGs). However, ERT is associated with inconvenient multiple and costly administrations and fails to ameliorate cardiac, visual and bone abnormalities.To overcome ERT limitations, we developed a successful gene therapy approach based on a single administration of AAV2/8 that targets liver of MPS VI animal models. Importantly, we showed that a single systemic administration of AAV2/8 at high doses (2×10e12 gc/kg) is at least as effective as the current ERT therapeutic regimen based on weekly infusions of recombinant human ARSB (rhARSB). If this translates to humans, gene therapy could replace ERT for MPS VI. However, the administration of high doses of AAV2/8 requires a challenging and costly production process, and results in cell-mediated immune responses that eliminate transduced hepatocytes. Similarly, weekly ERT infusions are costly and require high patient compliance.We therefore evaluated in a mouse model of MPS VI whether the combination of low doses of AAV2/8 at 6×10e11 or 2×10e11 gc/kg with a rarified ERT schedule (1mg/kg once a month) may be as effective as the single treatments at high doses or frequent regimen.Significant increase of ARSB activity was found in liver of all treated mice. Detectable but low activity was variably observed in spleen and kidney and was associated with significant reduction of tissue GAGs, regardless of treatment and ARSB activity levels, similarly to what observed in mice treated with high doses of AAV2/8 or weekly ERT. This supports previous data indicating that low enzymatic levels improve MPS VI visceral phenotype. Evaluation of GAG storage in myocardium and heart valves is in progress.Urinary GAG, which are a sensitive biomarker of systemic clearance of lysosomal storage and, thus of therapeutic efficacy, are slightly reduced in mice treated with either monthly ERT or 2×10e11 gc/kg of AAV2/8. The reduction is more consistent at 6×10e11 gc/kg. Importantly, urinary GAG decreased more in mice receiving the combined therapy than in those receiving single treatments. In particular, urinary GAG reduction in mice treated with both 6×10e11 gc/kg of AAV2/8 and monthly ERT was comparable to that obtained following administration of either high doses of AAV2/8, i.e 2×10e12 gc/kg, or weekly ERT.The data collected so far show similar efficacy between low-dose gene therapy combined with rarified ERT and the corresponding single treatments at high doses or frequent regimen. This should increase the safety and reduce the risks and costs associated with both therapeutic approaches.

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

The transcription factor TBX1 is the major gene involved in 22q11.2 deletion syndrome (22q11.2DS). Using mouse models of these diseases, we have previously shown that TBX1 activates VEGFR3 in endothelial cells (EC), and that this interaction is critical for the development of the lymphatic vasculature. In this study, we show that TBX1 regulates brain angiogenesis. Using loss-of-function genetics and molecular approaches, we show that TBX1 regulates the VEGFR3 and DLL4 genes in brain ECs. In mice, loss of TBX1 causes global brain vascular defects, comprising brain vessel hyperplasia, enhanced angiogenic sprouting and vessel network disorganization. This phenotype is recapitulated in EC-specific Tbx1 conditional mutants and in an EC-only 3-dimensional cell culture system (matrigel), indicating that the brain vascular phenotype is cell autonomous. Furthermore, EC-specific conditional Tbx1 mutants have poorly perfused brain vessels and brain hypoxia, indicating that the expanded vascular network is functionally impaired. In EC-matrigel cultures, a Notch1 agonist is able to partially rescue microtubule hyperbranching induced by TBX1 knockdown. Thus, we have identified a novel transcriptional regulator of angiogenesis that exerts its effect in brain by negatively regulating angiogenesis through the DLL4/Notch1-VEGFR3 regulatory axis. Given the similarity of the phenotypic consequences of TBX1 mutation in humans and mice, this unexpected role of TBX1 in murine brain vascularization should stimulate clinicians to search for brain microvascular anomalies in 22q11.2DS patients and to evaluate whether some of the anatomical and functional brain anomalies in patients may have a microvascular origin.

Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1 alpha subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1 alpha in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1 alpha through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.

Mucopolysaccharidoses type IIIA (MPS-IIIA) is a neurodegenerative lysosomal storage disorder (LSD) caused by inherited defects of the sulphamidase gene. Here, we used a systemic gene transfer approach to demonstrate the therapeutic efficacy of a chimeric sulphamidase, which was engineered by adding the signal peptide (sp) from the highly secreted iduronate-2-sulphatase (IDS) and the blood-brain barrier (BBB)-binding domain (BD) from the Apolipoprotein B (ApoB-BD). A single intravascular administration of AAV2/8 carrying the modified sulphamidase was performed in adult MPS-IIIA mice in order to target the liver and convert it to a factory organ for sustained systemic release of the modified sulphamidase. We showed that while the IDS sp replacement results in increased enzyme secretion, the addition of the ApoB-BD allows efficient BBB transcytosis and restoration of sulphamidase activity in the brain of treated mice. This, in turn, resulted in an overall improvement of brain pathology and recovery of a normal behavioural phenotype. Our results provide a novel feasible strategy to develop minimally invasive therapies for the treatment of brain pathology in MPS-IIIA and other neurodegenerative LSDs. ->See accompanying article emmm.201302668 Gene transfer of a liver-targeted sulfamidase engineered for increased secretion and blood brain barrier permeability, effectively ameliorates overall brain pathology and behavioural phenotype in treated Mucopolysaccharidosis (MPS) type IIIA mice. © 2013.

Crigler-Najjar syndrome type I is caused by mutations of the uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) gene resulting in life-threatening increase of serum bilirubin. Life-long correction of hyperbilirubinemia was previously shown with intravenous injection of high doses of a helper-dependent adenoviral (HDAd) vector expressing UGT1A1 in the Gunn rat, the animal model of Crigler-Najjar syndrome. However, such high vector doses can activate an acute and potentially lethal inflammatory response with elevated serum interleukin-6 (IL-6). To overcome this obstacle, we investigated safety and efficacy of direct injections of low HDAd doses delivered directly into the liver parenchyma of Gunn rats. Direct hepatic injections performed by either laparotomy or ultrasound-guided percutaneous injections were compared with the same doses given by intravenous injections. A greater reduction of hyperbilirubinemia and increased conjugated bilirubin in bile were achieved with 1 × 10(11) vp/kg by direct liver injections compared with intravenous injections. In sharp contrast to intravenous injections, direct hepatic injections neither raised serum IL-6 nor resulted in thrombocytopenia. In conclusion, ultrasound-guided percutaneous injection of HDAd vectors into liver parenchyma resulted in improved hepatocyte transduction and reduced toxicity compared with systemic injections and is clinically attractive for liver-directed gene therapy of Crigler-Najjar syndrome.

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Crigler-Najjar syndrome is an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels, but liver transplantation is the only permanent treatment. Muscle-directed gene therapy has several advantages, including easy and safe access through simple intramuscular injections, and has been investigated in human clinical trials. In this study, we have investigated the efficacy of adeno-associated viral (AAV) vector-mediated muscle-directed gene therapy in the preclinical animal model of Crigler-Najjar syndrome, that is the Gunn rat. Serotype 1 AAV vector expressing rat UGT1A1 under the control of muscle-specific creatine kinase promoter was injected at a dose of 3×10(12) genome copies/kg into the muscles of Gunn rats and resulted in expression of UGT1A1 protein and functionally active enzyme in injected muscles. AAV-injected Gunn rats showed an approximately 50% reduction in serum bilirubin levels as compared with saline-treated controls, and this reduction was sustained for at least 1 year postinjection. Increased excretion of alkali-labile metabolites of bilirubin in bile and urine was detected in AAV-injected animals. High-performance liquid chromatography analysis of bile from AAV-injected Gunn rats showed a metabolite with retention time close to that of bilirubin diglucuronide. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type 1.

Dysfunctional mitochondria are a well-known disease hallmark. The accumulation of aberrant mitochondria can alter cell homeostasis, thus resulting in tissue degeneration. Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by the buildup of undegraded material inside the lysosomes that leads to autophagic-lysosomal dysfunction. In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction. However, the mechanisms underlying mitochondrial aberrations and how these are involved in tissue pathogenesis remain largely unexplored. In normal conditions, mitochondrial clearance occurs by mitophagy, a selective form of autophagy, which relies on a parkin-mediated mitochondrial priming and subsequent sequestration by autophagosomes. Here, we performed a detailed analysis of key steps of mitophagy in a mouse model of multiple sulfatase deficiency (MSD), a severe type of LSD characterized by both neurological and systemic involvement. We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death. Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration. Together, these data shed new light on the mechanisms underlying mitochondrial dysfunction in LSDs and on their tissue-specific differential contribution to the pathogenesis of this group of metabolic disorders.

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

Mucopolysacccharidosis (MPS) IIIB is an inherited lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase (NAGLU). The disease is characterized by mild somatic features and severe neurological involvement with high mortality. Although several therapeutic approaches have been applied to the murine model of the disease, no effective therapy is available for patients. In this study, we used the lentiviral-NAGLU vector to deliver the functional human NAGLU gene into the brain of young adult MPS IIIB mice. We report the restoration of active enzyme with a sustained expression throughout a large portion of the brain, and a significantly improved behavioral performance of treated animals. Moreover, we analyzed the effect of therapy on the expression profile of some genes related to neurotrophic signaling molecules and inflammatory cytokines previously found altered in MPS IIIB mice. At 1 month from treatment, the level of cerebellin 1 (Cbln1) was decreased while the brain-derived neurotrophic factor (Bdnf) expression was increased, both reaching normal values. At 6 months from treatment a significant reduction in the expression of all the inflammation- and oxidative stress-related genes was observed, as well as the maintenance of the correction of the Bdnf gene expression. These results indicate that NAGLU delivery from intracerebral sources has the capacity to alleviate most disease manifestations in MPS IIIB mice; furthermore, Bdnf might be a response-to-therapy biomarker for MPS IIIB.

Sulfatases are involved in several biological functions such as degradation of macromolecules in the lysosomes. In patients with multiple sulfatase deficiency, mutations in theSUMF1gene cause a reduction of sulfatase activities because of a posttranslational modification defect. We have generated a mouse line carrying a null mutation in theSumf1gene. Sulfatase activities are completely absent inSumf1−/−mice, indicating that Sumf1 is indispensable for sulfatase activation and that mammals, differently from bacteria, have a single sulfatase modification system. Similarly to multiple sulfatase deficiency patients,Sumf1−/−mice display frequent early mortality, congenital growth retardation, skeletal abnormalities, and neurological defects. All examined tissues showed progressive cell vacuolization and significant lysosomal storage of glycosaminoglycans.Sumf1−/−mice showed a generalized inflammatory process characterized by a massive presence of highly vacuolated macrophages, which are the main site of lysosomal storage. Activated microglia were detected in the cerebellum and brain cortex associated with remarkable astroglyosis and neuronal cell loss. Between 4 and 6 months of age, we detected a strong increase in the expression levels of inflammatory cytokines and of apoptotic markers in both the CNS and liver, demonstrating that inflammation and apoptosis occur at the late stage of disease and suggesting that they play an important role in both the systemic and CNS phenotypes observed in lysosomal disorders. This mouse model, in which the function of an entire protein family has been silenced, offers a unique opportunity to study sulfatase function and the mechanisms underlying lysosomal storage diseases.

Background The BIO14.6 hamster is an excellent animal model for inherited cardiomyopathy, because of its lethal and well-documented course, due to a spontaneous deletion of delta-sarcoglycan gene promoter and first exon. The muscle disease is progressive and average lifespan is 11 months, because heart slowly dilates towards heart failure. Methodology/Principal Findings Based on the ability of adeno-associated viral (AAV) vectors to transduce heart together with skeletal muscle following systemic administration, we delivered human delta-sarcoglycan cDNA into male BIO14.6 hamsters by testing different ages of injection, routes of administration and AAV serotypes. Body-wide restoration of delta-SG expression was associated with functional reconstitution of the sarcoglycan complex and with significant lowering of centralized nuclei and fibrosis in skeletal muscle. Motor ability and cardiac functions were completely rescued. However, BIO14.6 hamsters having less than 70% of fibers recovering sarcoglycan developed cardiomyopathy, even if the total rescued protein was normal. When we used serotype 2/8 in combination with serotype 2/1, lifespan was extended up to 22 months with sustained heart function improvement. Conclusions/Significance Our data support multiple systemic administrations of AAV as a general therapeutic strategy for clinical trials in cardiomyopathies and muscle disorders.