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GM1 gangliosidosis is a fatal neurodegenerative disease caused by a deficiency of lysosomal β-galactosidase. In its most severe form, GM1 gangliosidosis causes death by 4 years of age, and no effective treatments exist. Previous work has shown that injection of the brain parenchyma with an adeno-associated viral (AAV) vector provides pronounced therapeutic benefit in a feline GM1 model. To develop a less invasive treatment for the brain and increase systemic biodistribution, intravenous injection of AAV9 was evaluated. AAV9 expressing feline β-galactosidase was intravenously administered at 1.5×1013 vector genomes/kg body weight to six GM1 cats at ∼1 month of age. The animals were divided into two cohorts: (i) a long-term group, which was followed to humane end point; and (ii) a short-term group, which was analysed 16 weeks post-treatment. Clinical assessments included neurological exams, CSF and urine biomarkers, and 7 T MRI and magentic resonance spectroscopy (MRS). Post-mortem analysis included β-galactosidase and virus distribution, histological analysis and ganglioside content. Untreated GM1 animals survived 8.0 ± 0.6 months while intravenous treatment increased survival to an average of 3.5 years (n = 2) with substantial improvements in quality of life and neurological function. Neurological abnormalities, which in untreated animals progress to the inability to stand and debilitating neurological disease by 8 months of age, were mild in all treated animals. CSF biomarkers were normalized, indicating decreased CNS cell damage in the treated animals. Urinary glycosaminoglycans decreased to normal levels in the long-term cohort. MRI and MRS showed partial preservation of the brain in treated animals, which was supported by post-mortem histological evaluation. β-Galactosidase activity was increased throughout the CNS, reaching carrier levels in much of the cerebrum and normal levels in the cerebellum, spinal cord and CSF. Ganglioside accumulation was significantly reduced by treatment. Peripheral tissues such as heart, skeletal muscle, and sciatic nerve also had normal β-galactosidase activity in treated GM1 cats. GM1 histopathology was largely corrected with treatment. There was no evidence of tumorigenesis or toxicity. Restoration of β-galactosidase activity in the CNS and peripheral organs by intravenous gene therapy led to profound increases in lifespan and quality of life in GM1 cats. These data support the promise of intravenous gene therapy as a safe, effective treatment for GM1 gangliosidosis.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme β-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.

Global gene delivery to the CNS has therapeutic importance for the treatment of neurological disorders that affect the entire CNS. Due to direct contact with the CNS, cerebrospinal fluid (CSF) is an attractive route for CNS gene delivery. A safe and effective route to achieve global gene distribution in the CNS is needed, and administration of genes through the cisterna magna (CM) via a suboccipital puncture results in broad distribution in the brain and spinal cord. However, translation of this technique to clinical practice is challenging due to the risk of serious and potentially fatal complications in patients. Herein, we report development of a gene therapy delivery method to the CM through adaptation of an intravascular microcatheter, which can be safely navigated intrathecally under fluoroscopic guidance. We examined the safety, reproducibility, and distribution/transduction of this method in sheep using a self-complementary adeno-associated virus 9 (scAAV9)-GFP vector. This technique was used to treat two Tay-Sachs disease patients (30 months old and 7 months old) with AAV gene therapy. No adverse effects were observed during infusion or post-treatment. This delivery technique is a safe and minimally invasive alternative to direct infusion into the CM, achieving broad distribution of AAV gene transfer to the CNS.

Familial amyloidotic polyneuropathy (FAP) is a neurodegenerative disorder characterized by extracellular deposition of amyloid fibrils composed by mutated transthyretin (TTR) mainly in the peripheral nervous system. At present, liver transplantation is still the standard treatment to halt the progression of clinical symptoms in FAP, but new therapeutic strategies are emerging, including the use of TTR stabilizers. Here we propose to establish a new gene therapy approach using adeno-associated virus (AAV) vectors to deliver the trans-suppressor TTR T119M variant to the liver of transgenic TTR V30M mice at different ages. This TTR variant is known for its ability to stabilize the tetrameric protein. Analysis of the gastrointestinal tract of AAV-treated animals revealed a significant reduction in deposition of TTR non-fibrillar aggregates in as much as 34% in stomach and 30% in colon, as well as decreased levels of biomarkers associated with TTR deposition, namely the endoplasmic reticulum stress marker BiP and the extracellular matrix protein MMP-9. Moreover, we showed with different studies that our approach leads to an increase in tetrameric and more stable forms of TTR, in favor of destabilized monomers. Altogether our data suggest the possibility to use this gene therapy approach in a prophylactic manner to prevent FAP pathology.

Transthyretin (TTR) is a plasma and cerebrospinal fluid (CSF)-circulating homotetrameric protein. More than 100 point mutations have been identified in the TTR gene and several are related with amyloid diseases. Here we focused our attention in the TTR L12P variant associated with severe peripheral neuropathy and leptomeningeal amyloidosis. By using different cell lines derived from tissues specialized on TTR synthesis, such as the hepatocyte and the choroid plexus expressing WT, V30M, or L12P TTR variants we analyzed secretion, intracellular aggregation and degradation patterns. Also, we used liver-specific AAV gene transfer to assess expression of the L12P variant in vivo. We found the following: (i) decreased secretion with intracellular aggregation of TTR L12P in hepatoma cells relative to WT and V30M variant; this differential property of TTR L12P variant was also observed in mice injected with L12P AAV vector; (ii) differential N-glycosylation pattern of L12P variant in hepatoma cell lysates, conditioned media and mouse sera, which might represent an escape mechanism from ERAD degradation; (iii) intracellular L12P TTR aggregates mainly localized to lysosomes in cultured cells and liver; and (iv) none of the above findings were present in choroid plexus derived cells, suggesting particular secretion/quality control mechanisms that might contribute to leptomeningeal amyloidosis associated with the L12P variant. These observations open new avenues for the treatment of TTR associated leptomeningeal amyloidosis.

Carvedilol is a β-antagonist with strong anti-oxidant effect in lipids, proteins and superoxide production protecting biological membranes from oxidative stress. We hypothesised a possible therapeutical application of carvedilol in familial amyloidotic polyneuropathy (FAP), a neurodegenerative disease caused by deposition of transthyretin (TTR) amyloid fibrils. Oxidative stress, apoptosis and inflammation related to aggregation of non-fibrillar and fibrillar TTR have been detected both in pre-symptomatic and symptomatic stages of the disease, respectively. In this study we show by semi-quantitative immunohistochemistry and western blot analysis that administration of carvedilol to a transgenic mouse model of FAP influences the expression of oxidative and apoptotic biomarkers usually associated with TTR deposition, namely oxidation products such as HNE and 8-OHdG, and markers of ER stress. Most important, carvedilol treatment significantly reduced TTR aggregates in as much as 50%. We also observed that carvedilol rescues a mice neuroblastoma cell line from death induced by TTR oligomers, by decreasing activation of BiP and other ER stress biomarkers. Since carvedilol has no effect on TTR aggregation "in vitro", this finding points for the "in vivo" modulation of TTR aggregation by oxidative stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutical measures in the FAP population.

Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent anti-apoptotic and anti-oxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with transthyretin (TTR) deposition in Familial Amyloidotic Polyneuropathy (FAP), we investigated the possible TUDCA therapeutical application in this disease. We show by semi-quantitative immunohistochemistry and western blotting that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2α, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most important, TUDCA treatment significantly reduced TTR toxic aggregates in as much as 75%. Since TUDCA has no effect on TTR aggregation “in vitro”, this finding points for the “in vivo” modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutic measures in FAP.

The identification of specific biomarkers provides opportunities to develop early diagnostic parameters, monitor disease progression, and test drug efficiency in clinical trials. We previously demonstrated that in familial amyloidotic polyneuropathy (FAP) related to the abnormal extracellular tissue deposition of mutant transthyretin (TTR), inflammatory and apoptotic pathways are triggered in the presymptomatic stages of the disease, when nonfibrillar TTR deposits are present. In the present work, to better define biomarkers for future assessment of prophylactic and therapeutic drugs in the treatment of FAP, we extended the search for oxidative stress and apoptotic biomarkers to clinical samples and animal models presenting nonfibrillar and fibrillar TTR. We found that lipid peroxidation measured by hydroxynonenal, oxidative DNA damage measured by 8-hydroxy-2′-deoxyguanosine, and cellular redox homeostasis measured by glutaredoxin 1 were consistently increased in biopsy specimens from FAP patients and in tissues from transgenic mouse models presenting nonfibrillar TTR deposition. Death-receptor Fas, caspase-8, and Bax were also found to be increased, indicative of the involvement of death receptors in the observed apoptosis process. Removal of TTR deposition by an immunization protocol resulted in significant decreases of the selected markers we describe, corroborating the relationship between TTR deposition, oxidative stress, and apoptosis. Taken together, our results provide a robust biomarker profile for initial experimental animal studies and clinical trials to assess the application of the selected markers in therapies aimed at removal and/or inhibition of TTR polymerization.