Gene Therapy in Patients with the Crigler-Najjar Syndrome.

BACKGROUND: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 µmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 µmol per liter. The patients who received the higher dose had bilirubin levels below 300 µmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 µmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 µmol per liter). CONCLUSIONS: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).

Multiyear Factor VIII Expression after AAV Gene Transfer for Hemophilia A.

BACKGROUND: The goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose. METHODS: In this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII. RESULTS: The median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration). CONCLUSIONS: Sustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor.

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

Gene therapy access: Global challenges, opportunities, and views from Brazil, South Africa, and India.

Gene and cell therapies for a variety of life-limiting illnesses are under investigation, and a small number of commercial products have successfully obtained regulatory approval. The cost of treatment is high, and clinical studies evaluating safety and efficacy are performed predominately in high-income countries. We reviewed the current status of gene and cell therapies in low- and middle-income countries and highlighted the need and current barriers to access. The state of product development in Brazil, South Africa, and India is discussed, including lessons learned from American Society of Gene and Cell Therapy (ASGCT)-sponsored virtual symposia in each of these countries.

Analysis of Adeno-Associated Virus Serotype 8 (AAV8)-antibody complexes using epitope mapping by molecular imprinting leads to the identification of Fab peptides that potentially evade AAV8 neutralisation.

Gene therapy is a promising approach for treating genetic disorders by delivering therapeutic genes to replace or correct malfunctioning genes. However, the introduced gene therapy vector can trigger an immune response, leading to reduced efficacy and potential harm to the patient. To improve the efficiency and safety of gene therapy, preventing the immune response to the vector is crucial. This can be achieved through the use of immunosuppressive drugs, vector engineering to evade the immune system, or delivery methods that bypass the immune system altogether. By reducing the immune response, gene therapy can deliver therapeutic genes more effectively and potentially cure genetic diseases. In this study, a novel molecular imprinting technique, combined with mass-spectrometry and bioinformatics, was used to identify four antigen-binding fragments (Fab) sequences of Adeno-Associated Virus (AAV) - neutralising antibodies capable of binding to AAV. The identified Fab peptides were shown to prevent AAV8's binding to antibodies, demonstrating their potential to improve gene therapy efficiency by preventing the immune response.

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome.

A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer.

Gene therapy with adeno-associated virus (AAV) vectors has demonstrated safety and long-term efficacy in a number of trials across target organs, including eye, liver, skeletal muscle, and the central nervous system. Since the initial evidence that AAV vectors can elicit capsid T cell responses in humans, which can affect the duration of transgene expression, much progress has been made in understanding and modulating AAV vector immunogenicity. It is now well established that exposure to wild-type AAV results in priming of the immune system against the virus, with development of both humoral and T cell immunity. Aside from the neutralizing effect of antibodies, the impact of pre-existing immunity to AAV on gene transfer is still poorly understood. Herein, we review data emerging from clinical trials across a broad range of gene therapy applications. Common features of immune responses to AAV can be found, suggesting, for example, that vector immunogenicity is dose-dependent, and that innate immunity plays an important role in the outcome of gene transfer. A range of host-specific factors are also likely to be important, and a comprehensive understanding of the mechanisms driving AAV vector immunogenicity in humans will be key to unlocking the full potential of in vivo gene therapy.

The Effect of CpG Sequences on Capsid-Specific CD8+ T Cell Responses to AAV Vector Gene Transfer.

Transfer of genes by adeno-associated virus (AAV) vectors is benefiting patients with particular genetic defects. Challenges remain by rejection of AAV-transduced cells, which may be caused by CD8+ T lymphocytes directed to AAV capsid antigens. Reducing the number of CpG motifs from the genome of AAV vectors reduces expansion of naive T cells directed against an epitope within the capsid. In contrast, AAV capsid-specific memory CD8+ T cells respond more vigorously to AAV vectors lacking CpG motifs than to those with CpG motifs presumably reflecting dampening of T cell expansion by cytokines from the innate immune system. Depending on the purification method, AAV vector preparations can contain substantial amounts of empty AAV particles that failed to package the genome. Others have used empty particles as decoys to AAV-neutralizing antibodies. We tested if empty AAV vectors given alone or mixed with genome-containing AAV vectors induce proliferation of naive or memory CD8+ T cells directed to an antigen within an AAV capsid. Naive CD8+ T cells failed to respond to empty AAV vectors, which in contrast induced expansion of AAV-specific memory CD8+ T cells.

SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease.

Glial cell-derived neurotrophic factor (GDNF) has a potent action in promoting the survival of dopamine (DA) neurons. Several studies indicate that increasing GDNF levels may be beneficial for the treatment of Parkinson's disease (PD) by reducing neurodegeneration of DA neurons. Despite a plethora of preclinical studies showing GDNF efficacy in PD animal models, its application in humans remains questionable for its poor efficacy and side effects due to its uncontrolled, ectopic expression. Here we took advantage of SINEUPs, a new class of antisense long non-coding RNA, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. By synthesizing a SINEUP targeting Gdnf mRNA, we were able to increase endogenous GDNF protein levels by about 2-fold. Adeno-associated virus (AAV)9-mediated delivery in the striatum of wild-type (WT) mice led to an increase of endogenous GDNF protein for at least 6 months and the potentiation of the DA system's functions while showing no side effects. Furthermore, SINEUP-GDNF was able to ameliorate motor deficits and neurodegeneration of DA neurons in a PD neurochemical mouse model. Our data indicate that SINEUP-GDNF could represent a new strategy to increase endogenous GDNF protein levels in a more physiological manner for therapeutic treatments of PD.

Rescue of Advanced Pompe Disease in Mice with Hepatic Expression of Secretable Acid α-Glucosidase.

Pompe disease is a neuromuscular disorder caused by disease-associated variants in the gene encoding for the lysosomal enzyme acid α-glucosidase (GAA), which converts lysosomal glycogen to glucose. We previously reported full rescue of Pompe disease in symptomatic 4-month-old Gaa knockout (Gaa-/-) mice by adeno-associated virus (AAV) vector-mediated liver gene transfer of an engineered secretable form of GAA (secGAA). Here, we showed that hepatic expression of secGAA rescues the phenotype of 4-month-old Gaa-/- mice at vector doses at which the native form of GAA has little to no therapeutic effect. Based on these results, we then treated severely affected 9-month-old Gaa-/- mice with an AAV vector expressing secGAA and followed the animals for 9 months thereafter. AAV-treated Gaa-/- mice showed complete reversal of the Pompe phenotype, with rescue of glycogen accumulation in most tissues, including the central nervous system, and normalization of muscle strength. Transcriptomic profiling of skeletal muscle showed rescue of most altered pathways, including those involved in mitochondrial defects, a finding supported by structural and biochemical analyses, which also showed restoration of lysosomal function. Together, these results provide insight into the reversibility of advanced Pompe disease in the Gaa-/- mouse model via liver gene transfer of secGAA.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 1010 to 2 × 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12-15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.

Exon-specific U1 snRNAs improve ELP1 exon 20 definition and rescue ELP1 protein expression in a familial dysautonomia mouse model.

Familial dysautonomia (FD) is a rare genetic disease with no treatment, caused by an intronic point mutation (c.2204+6T>C) that negatively affects the definition of exon 20 in the elongator complex protein 1 gene (ELP1 also known as IKBKAP). This substitution modifies the 5' splice site and, in combination with regulatory splicing factors, induces different levels of exon 20 skipping, in various tissues. Here, we evaluated the therapeutic potential of a novel class of U1 snRNA molecules, exon-specific U1s (ExSpeU1s), in correcting ELP1 exon 20 recognition. Lentivirus-mediated expression of ELP1-ExSpeU1 in FD fibroblasts improved ELP1 splicing and protein levels. We next focused on a transgenic mouse model that recapitulates the same tissue-specific mis-splicing seen in FD patients. Intraperitoneal delivery of ELP1-ExSpeU1s-adeno-associated virus particles successfully increased the production of full-length human ELP1 transcript and protein. This splice-switching class of molecules is the first to specifically correct the ELP1 exon 20 splicing defect. Our data provide proof of principle of ExSpeU1s-adeno-associated virus particles as a novel therapeutic strategy for FD.

Prevalence and long-term monitoring of humoral immunity against adeno-associated virus in Duchenne Muscular Dystrophy patients.

Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.

Rescue of GSDIII Phenotype with Gene Transfer Requires Liver- and Muscle-Targeted GDE Expression.

Glycogen storage disease type III (GSDIII) is an autosomal recessive disorder caused by a deficiency of glycogen-debranching enzyme (GDE), which results in profound liver metabolism impairment and muscle weakness. To date, no cure is available for GSDIII and current treatments are mostly based on diet. Here we describe the development of a mouse model of GSDIII, which faithfully recapitulates the main features of the human condition. We used this model to develop and test novel therapies based on adeno-associated virus (AAV) vector-mediated gene transfer. First, we showed that overexpression of the lysosomal enzyme alpha-acid glucosidase (GAA) with an AAV vector led to a decrease in liver glycogen content but failed to reverse the disease phenotype. Using dual overlapping AAV vectors expressing the GDE transgene in muscle, we showed functional rescue with no impact on glucose metabolism. Liver expression of GDE, conversely, had a direct impact on blood glucose levels. These results provide proof of concept of correction of GSDIII with AAV vectors, and they indicate that restoration of the enzyme deficiency in muscle and liver is necessary to address both the metabolic and neuromuscular manifestations of the disease.

Bioengineered AAV Capsids with Combined High Human Liver Transduction In Vivo and Unique Humoral Seroreactivity.

Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.

Autophagy determines efficiency of liver-directed gene therapy with adeno-associated viral vectors.

Use of adeno-associated viral (AAV) vectors for liver-directed gene therapy has shown considerable success, particularly in patients with severe hemophilia B. However, the high vector doses required to reach therapeutic levels of transgene expression caused liver inflammation in some patients that selectively destroyed transduced hepatocytes. We hypothesized that such detrimental immune responses can be avoided by enhancing the efficacy of AAV vectors in hepatocytes. Because autophagy is a key liver response to environmental stresses, we characterized the impact of hepatic autophagy on AAV infection. We found that AAV induced mammalian target of rapamycin (mTOR)-dependent autophagy in human hepatocytes. This cell response was critically required for efficient transduction because under conditions of impaired autophagy (pharmacological inhibition, small interfering RNA knockdown of autophagic proteins, or suppression by food intake), recombinant AAV-mediated transgene expression was markedly reduced, both in vitro and in vivo. Taking advantage of this dependence, we employed pharmacological inducers of autophagy to increase the level of autophagy. This resulted in greatly improved transduction efficiency of AAV vectors in human and mouse hepatocytes independent of the transgene, driving promoter, or AAV serotype and was subsequently confirmed in vivo. Specifically, short-term treatment with a single dose of torin 1 significantly increased vector-mediated hepatic expression of erythropoietin in C57BL/6 mice. Similarly, coadministration of rapamycin with AAV vectors resulted in markedly enhanced expression of human acid-α-glucosidase in nonhuman primates. CONCLUSION: We identified autophagy as a pivotal cell response determining the efficiency of AAVs intracellular processing in hepatocytes and thus the outcome of liver-directed gene therapy using AAV vectors and showed in a proof-of-principle study how this virus-host interaction can be employed to enhance efficacy of this vector system. (Hepatology 2017;66:252-265).

Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM.

Biochemical, histological and functional correction of mucopolysaccharidosis type IIIB by intra-cerebrospinal fluid gene therapy.

Gene therapy is an attractive tool for the treatment of monogenic disorders, in particular for lysosomal storage diseases (LSD) caused by deficiencies in secretable lysosomal enzymes in which neither full restoration of normal enzymatic activity nor transduction of all affected cells are necessary. However, some LSD such as Mucopolysaccharidosis Type IIIB (MPSIIIB) are challenging because the disease's main target organ is the brain and enzymes do not efficiently cross the blood-brain barrier even if present at very high concentration in circulation. To overcome these limitations, we delivered AAV9 vectors encoding for α-N-acetylglucosaminidase (NAGLU) to the Cerebrospinal Fluid (CSF) of MPSIIIB mice with the disease already detectable at biochemical, histological and functional level. Restoration of enzymatic activity in Central Nervous System (CNS) resulted in normalization of glycosaminoglycan content and lysosomal physiology, resolved neuroinflammation and restored the pattern of gene expression in brain similar to that of healthy animals. Additionally, transduction of the liver due to passage of vectors to the circulation led to whole-body disease correction. Treated animals also showed reversal of behavioural deficits and extended lifespan. Importantly, when the levels of enzymatic activity were monitored in the CSF of dogs following administration of canine NAGLU-coding vectors to animals that were either naïve or had pre-existing immunity against AAV9, similar levels of activity were achieved, suggesting that CNS efficacy would not be compromised in patients seropositive for AAV9. Our studies provide a strong rationale for the clinical development of this novel therapeutic approach as the treatment for MPSIIIB.