Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

Role of trafficking protein particle complex 2 in medaka development.

The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders.

Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin.

Mutant Z alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZ are needed. Transient receptor potential mucolipin-1 (TRPML1) is a lysosomal Ca2+ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small-molecule-mediated activation of TRPML1 reduces hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.

TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.

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.

New mouse models with hypomorphic SUMF1 variants mimic attenuated forms of multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultrarare lysosomal storage disorder due to deficiency of all known sulfatases. MSD is caused by mutations in the Sulfatase Modifying Factor 1 (SUMF1) gene encoding the enzyme responsible for the post-translational modification and activation of all sulfatases. Most MSD patients carry hypomorph SUMF1 variants resulting in variable degrees of residual sulfatase activities. In contrast, Sumf1 null mice with complete deficiency in all sulfatase enzyme activities, have very short lifespan with significant pre-wean lethality, owing to a challenging preclinical model. To overcome this limitation, we genetically engineered and characterized in mice two commonly identified patient-based SUMF1 pathogenic variants, namely p.Ser153Pro and p.Ala277Val. These pathogenic missense variants correspond to variants detected in patients with attenuated MSD presenting with partial-enzyme deficiency and relatively less severe disease. These novel MSD mouse models have a longer lifespan and show biochemical and pathological abnormalities observed in humans. In conclusion, mice harboring the p.Ser153Pro or the p.Ala277Val variant mimic the attenuated MSD and are attractive preclinical models for investigation of pathogenesis and treatments for MSD.

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

Partial Retraction of: The EMBO Journal (2010) 29: 3607-3620. DOI: 10.1038/emboj.2010.237 | Published online 24 September 2010 Journal statement The journal contacted the authors in February 2022 about potential image insertions and duplications in Fig 4A and 4E. In the absence of source data, the authors are retracting Fig 4A, the lower panel of Fig 4E (LAMP1 immunoblot), and the following statements in the text that rely on these data: "Quantitative analysis showed that the percentage of Flotillin-1 associated with DRMs was increased in LSD endolysosomal membranes (Figure 4A), indicating an increased amount of cholesterol-enriched regions in these membrane samples." "LAMP1 also displayed a similar distribution profile in WT and LSD cells (Figure 4E)". Author statement The authors could not verify the aberrations in panel A of Fig 4 and the lower immunoblot (LAMP1) of 4E because the original source data are no longer available (12 years after publication, which is beyond the institute's 10-year data retention policy). The authors wish to clarify that the main conclusions of the paper are not affected by the retraction of Figure panels 4A and 4E for the following reasons: Figure panel 4A supports the observation that there are increased cholesterol-enhanced regions in LSD samples. This finding is also supported by data provided in figs 4B, 4C and 4D. Figure panel 4E: The LAMP1 blot in Fig 4E shows that the distribution of protein normally excluded from DRMs is not altered between Wt and LSD samples. This result is also supported by the upper blot in this panel (Transferrin receptor). The authors apologize for these errors and agree with this corrigendum; no response could be obtained from AL.

Fluoxetine ameliorates mucopolysaccharidosis type IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.

Retinal Degeneration in MPS-IIIA Mouse Model.

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 Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease.

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 ß 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.

Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA.

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.

Effects of non-euphoric plant cannabinoids on muscle quality and performance of dystrophic mdx mice.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy greatly contributes to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV), to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo. EXPERIMENTAL APPROACH: Using quantitative PCR, western blots and [Ca2+ ]i measurements, we explored the effects of CBD and CBDV on the differentiation of both murine and human skeletal muscle cells as well as their potential interaction with TRP channels. Male dystrophic mdx mice were injected i.p. with CBD or CBDV at different stages of the disease. After treatment, locomotor tests and biochemical analyses were used to evaluate their effects on inflammation and autophagy. KEY RESULTS: CBD and CBDV promoted the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. In primary satellite cells and myoblasts isolated from healthy and/or DMD donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case, mostly via TRPA1 activation. In mdx mice, CBD (60 mg·kg-1 ) and CBDV (60 mg·kg-1 ) prevented the loss of locomotor activity, reduced inflammation and restored autophagy. CONCLUSION AND IMPLICATIONS: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Gene therapy for mucopolysaccharidoses: in vivo and ex vivo approaches.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by a deficiency in lysosomal enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs). The current therapeutic strategies of enzyme replacement therapy and allogeneic hematopoietic stem cell transplantation have been reported to reduce patient morbidity and to improve their quality of life, but they are associated with persistence of residual disease burden, in particular at the neurocognitive and musculoskeletal levels. This indicates the need for more efficacious treatments capable of effective and rapid enzyme delivery to the affected organs, especially the brain and the skeleton. Gene therapy (GT) strategies aimed at correcting the genetic defect in patient cells could represent a significant improvement for the treatment of MPS when compared with conventional approaches. While in-vivo GT strategies foresee the administration of viral vector particles directly to patients with the aim of providing normal complementary DNA to the affected cells, ex-vivo GT approaches are based on the ex-vivo transduction of patient cells that are subsequently infused back. This review provides insights into the state-of-art accomplishments made with in vivo and ex vivo GT-based approaches in MPS and provide a vision for the future in the medical community.

Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevents neurodegeneration in lysosomal storage diseases.

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs.

Brain Targeting in MPS-IIIA.

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a childhood metabolic neuropathology caused by the inherited deficiency of the lysosomal enzyme sulfamidase and is characterized by the accumulation of undegraded glycosaminoglycans in the lysosomes of cells and tissues of affected patients. MPS-IIIA represents one of the most common forms of lysosomal storage disorders (LSDs) and to date there is no cure. Since neurodegeneration is the most relevant pathological feature in MPS-IIIA patients, the treatment of the central nervous system (CNS) lesions represents the goal of any effective therapy for this devastating disorder. During the last years many advances have been made in developing and testing new therapies for brain involvement in MPS-IIIA. These studies have been possible because of the availability of mouse and dog models that recapitulate the MPS-IIIA neuropathological features. Some of these approaches are based on direct CNS administration routes through which the therapeutic molecules access the CNS via the parenchyma (intracerebral injections) or via the cerebrospinal fluid (intraventricular/intrathecal injections). These approaches are highly invasive and poorly suited for clinical use. Minimally invasive approaches are based on systemic injections into the blood stream of therapeutics capable of crossing the blood-brain barrier (BBB). This review will present the background of the clinic and pathology aspects of MPS-IIIA and will describe the current MPS-IIIA preclinical and clinical studies focusing on how a systemic therapeutic strategy based on crossing the BBB has been successfully used to treat CNS pathology and behavioral abnormalities in a mouse model of MPS-IIIA. Future clinical applications of this approach to MPS-IIIA patients will be also discussed together with the possibility of using similar strategies in other LSDs with neurological involvement.

A Comprehensive Map of CNS Transduction by Eight Recombinant Adeno-associated Virus Serotypes Upon Cerebrospinal Fluid Administration in Pigs.

Cerebrospinal fluid administration of recombinant adeno-associated viral (rAAV) vectors has been demonstrated to be effective in delivering therapeutic genes to the central nervous system (CNS) in different disease animal models. However, a quantitative and qualitative analysis of transduction patterns of the most promising rAAV serotypes for brain targeting in large animal models is missing. Here, we characterize distribution, transduction efficiency, and cellular targeting of rAAV serotypes 1, 2, 5, 7, 9, rh.10, rh.39, and rh.43 delivered into the cisterna magna of wild-type pigs. rAAV9 showed the highest transduction efficiency and the widest distribution capability among the vectors tested. Moreover, rAAV9 robustly transduced both glia and neurons, including the motor neurons of the spinal cord. Relevant cell transduction specificity of the glia was observed after rAAV1 and rAAV7 delivery. rAAV7 also displayed a specific tropism to Purkinje cells. Evaluation of biochemical and hematological markers suggested that all rAAV serotypes tested were well tolerated. This study provides a comprehensive CNS transduction map in a useful preclinical large animal model enabling the selection of potentially clinically transferable rAAV serotypes based on disease specificity. Therefore, our data are instrumental for the clinical evaluation of these rAAV vectors in human neurodegenerative diseases.

Transdermal spinal catheter placement in piglets: Description and validation of the technique.

BACKGROUND: The swine species represents a perfect model for translational medicine due to its physiological and anatomical resemblance to humans. The development of techniques for spinal catheter insertion in swine is significantly useful but, at the moment, the only technique described requires laminectomy as a surgical approach. NEW METHOD: The proposed techniques represent a transdermal approach for catheter placement in piglets. The study was divided into Phase I (anatomical study on 8 cadavers) and Phase II (in vivo application of the technique in 20 anaesthetised 30-day old piglets). A spinal needle was introduced between the L2 and L3 spinous processes with a ventro-cranial orientation until cerebro-spinal fluid leakage. It was then replaced with a Tuohy needle, used to introduce the catheter into the intrathecal space. Before inserting the catheter, the approximate length from the insertion point to the external projection of the Cisterna Magna was measured using the gradation markings on the device. RESULTS: The technique described allowed spinal catheter placement in all piglets. In Phase I, the correct placement was confirmed using fluoroscopy while, in Phase II, cerebrospinal fluid leakage from the needle was relied on. No clinical alterations were detected either during the procedure or during the following days. COMPARISON WITH EXISTING METHOD: This technique is easy and requires less skilled operators when compared to the other existing method which involves a surgical approach. Moreover, being less invasive, it potentially leads to fewer complications. CONCLUSIONS: In conclusion, the technique can be performed safely in piglets, and provides an easier and less invasive approach for spinal catheter insertion.

A highly secreted sulphamidase engineered to cross the blood-brain barrier corrects brain lesions of mice with mucopolysaccharidoses type IIIA.

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.

Efficacy of a combined intracerebral and systemic gene delivery approach for the treatment of a severe lysosomal storage disorder.

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.

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders.

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.