Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy.

OBJECTIVE: Pompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. METHODS: PD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. RESULTS: PD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. CONCLUSIONS: This gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

Ferritin But Not Iron Increases in Retina Upon Systemic Iron Overload in Diabetic and Iron-Dextran Injected Mice.

Purpose: Iron overload causes oxidative damage in the retina, and it has been involved in the pathogeny of diabetic retinopathy, which is one of the leading causes of blindness in the adult population worldwide. However, how systemic iron enters the retina during diabetes and the role of blood retinal barrier (BRB) in this process remains unclear. Methods: The db/db mouse, a well-known model of type 2 diabetes, and a model of systemic iron overload induced by iron dextran intraperitoneal injection, were used. Perls staining and mass spectrophotometry were used to study iron content. Western blot and immunohistochemistry of iron handling proteins were performed to study systemic and retinal iron metabolism. BRB function was assessed by analyzing vascular leakage in fundus angiographies, whole retinas, and retinal sections and by studying the status of tight junctions using transmission electron microscopy and Western blot analysis. Results: Twenty-week-old db/db mice with systemic iron overload presented ferritin overexpression without iron increase in the retina and did not show any sign of BRB breakdown. These findings were also observed in iron dextran-injected mice. In those animals, after BRB breakdown induced by cryopexy, iron entered massively in the retina. Conclusions: Our results suggested that BRB protects the retina from excessive iron entry in early stages of diabetic retinopathy. Furthermore, ferritin overexpression before iron increase may prepare the retina for a potential BRB breakdown and iron entry from the systemic circulation.

Treatment of skeletal and non-skeletal alterations of Mucopolysaccharidosis type IVA by AAV-mediated gene therapy.

Mucopolysaccharidosis type IVA (MPSIVA) or Morquio A disease, a lysosomal storage disorder, is caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency, resulting in keratan sulfate (KS) and chondroitin-6-sulfate accumulation. Patients develop severe skeletal dysplasia, early cartilage deterioration and life-threatening heart and tracheal complications. There is no cure and enzyme replacement therapy cannot correct skeletal abnormalities. Here, using CRISPR/Cas9 technology, we generate the first MPSIVA rat model recapitulating all skeletal and non-skeletal alterations experienced by patients. Treatment of MPSIVA rats with adeno-associated viral vector serotype 9 encoding Galns (AAV9-Galns) results in widespread transduction of bones, cartilage and peripheral tissues. This led to long-term (1 year) increase of GALNS activity and whole-body correction of KS levels, thus preventing body size reduction and severe alterations of bones, teeth, joints, trachea and heart. This study demonstrates the potential of AAV9-Galns gene therapy to correct the disabling MPSIVA pathology, providing strong rationale for future clinical translation to MPSIVA patients.

TIM2 modulates retinal iron levels and is involved in blood-retinal barrier breakdown.

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.

Cerebellar Astrocyte Transduction as Gene Therapy for Megalencephalic Leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare genetic disorder belonging to the group of vacuolating leukodystrophies. It is characterized by megalencephaly, loss of motor functions, epilepsy, and mild mental decline. In brain biopsies of MLC patients, vacuoles were observed in myelin and in astrocytes surrounding blood vessels. There is no therapy for MLC patients, only supportive treatment. We show here a preclinical gene therapy approach for MLC using the Mlc1 knock-out mouse. An adeno-associated virus coding for human MLC1 under the control of the glial fibrillary acidic protein promoter was injected in the cerebellar subarachnoid space of Mlc1 knock-out and wild-type animals at 2 months of age, before the onset of the disease, as a preventive approach. We also tested a therapeutic strategy by injecting the animals at 5 months, once the histopathological abnormalities are starting, or at 15 months, when they have progressed to a more severe pathology. MLC1 expression in the cerebellum restored the adhesion molecule GlialCAM and the chloride channel ClC-2 localization in Bergmann glia, which both are mislocalized in Mlc1 knock-out model. More importantly, myelin vacuolation was extremely reduced in treated mice at all ages and correlated with the amount of expressed MLC1 in Bergmann glia, indicating not only the preventive potential of this strategy but also its therapeutic capacity. In summary, here we provide the first therapeutic approach for patients affected with MLC. This work may have also implications to treat other diseases affecting motor function such as ataxias.

FGF21 gene therapy as treatment for obesity and insulin resistance.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. Fibroblast growth factor 21 (FGF21) is considered a promising therapeutic agent for T2D/obesity. Native FGF21 has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF21. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for > 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF21. Furthermore, FGF21 overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF21 gene therapy to treat obesity, insulin resistance, and T2D.

Long-Term Efficacy and Safety of Insulin and Glucokinase Gene Therapy for Diabetes: 8-Year Follow-Up in Dogs.

Diabetes is a complex metabolic disease that exposes patients to the deleterious effects of hyperglycemia on various organs. Achievement of normoglycemia with exogenous insulin treatment requires the use of high doses of hormone, which increases the risk of life-threatening hypoglycemic episodes. We developed a gene therapy approach to control diabetic hyperglycemia based on co-expression of the insulin and glucokinase genes in skeletal muscle. Previous studies proved the feasibility of gene delivery to large diabetic animals with adeno-associated viral (AAV) vectors. Here, we report the long-term (∼8 years) follow-up after a single administration of therapeutic vectors to diabetic dogs. Successful, multi-year control of glycemia was achieved without the need of supplementation with exogenous insulin. Metabolic correction was demonstrated through normalization of serum levels of fructosamine, triglycerides, and cholesterol and remarkable improvement in the response to an oral glucose challenge. The persistence of vector genomes and therapeutic transgene expression years after vector delivery was documented in multiple samples from treated muscles, which showed normal morphology. Thus, this study demonstrates the long-term efficacy and safety of insulin and glucokinase gene transfer in large animals and especially the ability of the system to respond to the changes in metabolic needs as animals grow older.

Correction: L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy.

[This corrects the article DOI: 10.1371/journal.pone.0106974.].

Disease correction by AAV-mediated gene therapy in a new mouse model of mucopolysaccharidosis type IIID.

Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID.

CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome).

Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-Ids) to the cerebrospinal fluid of MPSII mice with already established disease. Treated mice showed a significant increase in IDS activity throughout the encephalon, with full resolution of lysosomal storage lesions, reversal of lysosomal dysfunction, normalization of brain transcriptomic signature, and disappearance of neuroinflammation. Moreover, our vector also transduced the liver, providing a peripheral source of therapeutic protein that corrected storage pathology in visceral organs, with evidence of cross-correction of nontransduced organs by circulating enzyme. Importantly, AAV9-Ids-treated MPSII mice showed normalization of behavioral deficits and considerably prolonged survival. These results provide a strong proof of concept for the clinical translation of our approach for the treatment of Hunter syndrome patients with cognitive impairment.

ALOX5AP Overexpression in Adipose Tissue Leads to LXA4 Production and Protection Against Diet-Induced Obesity and Insulin Resistance.

Eicosanoids, such as leukotriene B4 (LTB4) and lipoxin A4 (LXA4), may play a key role during obesity. While LTB4 is involved in adipose tissue inflammation and insulin resistance, LXA4 may exert anti-inflammatory effects and alleviate hepatic steatosis. Both lipid mediators derive from the same pathway, in which arachidonate 5-lipoxygenase (ALOX5) and its partner, arachidonate 5-lipoxygenase-activating protein (ALOX5AP), are involved. ALOX5 and ALOX5AP expression is increased in humans and rodents with obesity and insulin resistance. We found that transgenic mice overexpressing ALOX5AP in adipose tissue had higher LXA4 rather than higher LTB4 levels, were leaner, and showed increased energy expenditure, partly due to browning of white adipose tissue (WAT). Upregulation of hepatic LXR and Cyp7a1 led to higher bile acid synthesis, which may have contributed to increased thermogenesis. In addition, transgenic mice were protected against diet-induced obesity, insulin resistance, and inflammation. Finally, treatment of C57BL/6J mice with LXA4, which showed browning of WAT, strongly suggests that LXA4 is responsible for the transgenic mice phenotype. Thus, our data support that LXA4 may hold great potential for the future development of therapeutic strategies for obesity and related diseases.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies.

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD.

HMGA1 overexpression in adipose tissue impairs adipogenesis and prevents diet-induced obesity and insulin resistance.

High-Mobility-Group-A1 (HMGA1) proteins are non-histone proteins that regulate chromatin structure and gene expression during embryogenesis, tumourigenesis and immune responses. In vitro studies suggest that HMGA1 proteins may be required to regulate adipogenesis. To examine the role of HMGA1 in vivo, we generated transgenic mice overexpressing HMGA1 in adipose tissues. HMGA1 transgenic mice showed a marked reduction in white and brown adipose tissue mass that was associated with downregulation of genes involved in adipogenesis and concomitant upregulation of preadipocyte markers. Reduced adipogenesis and decreased fat mass were not associated with altered glucose homeostasis since HMGA1 transgenic mice fed a regular-chow diet exhibited normal glucose tolerance and insulin sensitivity. However, when fed a high-fat diet, overexpression of HMGA1 resulted in decreased body-weight gain, reduced fat mass, but improved insulin sensitivity and glucose tolerance. Although HMGA1 transgenic mice exhibited impaired glucose uptake in adipose tissue due to impaired adipogenesis, the increased glucose uptake observed in skeletal muscle may account for the improved glucose homeostasis. Our results indicate that HMGA1 plays an important function in the regulation of white and brown adipogenesis in vivo and suggests that impaired adipocyte differentiation and decreased fat mass is not always associated with impaired whole-body glucose homeostasis.

Insulin-like Growth Factor 2 Overexpression Induces ß-Cell Dysfunction and Increases Beta-cell Susceptibility to Damage.

The human insulin-like growth factor 2 (IGF2) and insulin genes are located within the same genomic region. Although human genomic studies have demonstrated associations between diabetes and the insulin/IGF2 locus or the IGF2 mRNA-binding protein 2 (IGF2BP2), the role of IGF2 in diabetes pathogenesis is not fully understood. We previously described that transgenic mice overexpressing IGF2 specifically in ß-cells (Tg-IGF2) develop a pre-diabetic state. Here, we characterized the effects of IGF2 on ß-cell functionality. Overexpression of IGF2 led to ß-cell dedifferentiation and endoplasmic reticulum stress causing islet dysfunction in vivo. Both adenovirus-mediated overexpression of IGF2 and treatment of adult wild-type islets with recombinant IGF2 in vitro further confirmed the direct implication of IGF2 on ß-cell dysfunction. Treatment of Tg-IGF2 mice with subdiabetogenic doses of streptozotocin or crossing these mice with a transgenic model of islet lymphocytic infiltration promoted the development of overt diabetes, suggesting that IGF2 makes islets more susceptible to ß-cell damage and immune attack. These results indicate that increased local levels of IGF2 in pancreatic islets may predispose to the onset of diabetes. This study unravels an unprecedented role of IGF2 on ß-cells function.

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.

L-ferritin binding to scara5: a new iron traffic pathway potentially implicated in retinopathy.

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.

Pancreatic transduction by helper-dependent adenoviral vectors via intraductal delivery.

Pancreatic gene transfer could be useful to treat several diseases, such as diabetes mellitus, cystic fibrosis, chronic pancreatitis, or pancreatic cancer. Helper-dependent adenoviral vectors (HDAds) are promising tools for gene therapy because of their large cloning capacity, high levels of transgene expression, and long-term persistence in immunocompetent animals. Nevertheless, the ability of HDAds to transduce the pancreas in vivo has not been investigated yet. Here, we have generated HDAds carrying pancreas-specific expression cassettes, that is, driven either by the elastase or insulin promoter, using a novel and convenient plasmid family and homologous recombination in bacteria. These HDAds were delivered to the pancreas of immunocompetent mice via intrapancreatic duct injection. HDAds, encoding a CMV-GFP reporter cassette, were able to transduce acinar and islet cells, but transgene expression was lost 15 days postinjection in correlation with severe lymphocytic infiltration. When HDAds encoding GFP under the control of the specific elastase promoter were used, expression was detected in acinar cells, but similarly, the expression almost disappeared 30 days postinjection and lymphocytic infiltration was also observed. In contrast, long-term transgene expression (>8 months) was achieved with HDAds carrying the insulin promoter and the secretable alkaline phosphatase as the reporter gene. Notably, transduction of the liver, the preferred target for adenovirus, was minimal by this route of delivery. These data indicate that HDAds could be used for pancreatic gene therapy but that selection of the expression cassette is of critical importance to achieve long-term expression of the transgene in this tissue.

Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer.

Interleukin 2 (IL2) is the key cytokine supporting survival and function of regulatory T cells (Tregs). We recently reported that low-dose IL2 safely expands/stimulates Tregs and improves autoimmune conditions in humans. Further development of IL2 in autoimmune diseases will require chronic IL2 administration, which could affect beneficial effector immune responses regulated by Tregs. We used recombinant adeno-associated viral vector (rAAV)-mediated gene transfer to continuously release IL2 in mice and assessed its long-term effects on immune responses. A single rAAV-IL2 injection enabled sustained stimulation and expansion of Tregs without inducing Teff activation and prevented diabetes in NOD mice. After several weeks of IL2 production, mice responded normally to a viral challenge and to vaccination, and had pregnancies with offspring that developed normally. They showed no change in the occurrence and growth of chemically-induced tumors. Altogether, chronic low-dose IL2 treatment does not affect beneficial effector immune responses at doses that prevent autoimmune diabetes.

Programmed cell senescence during mammalian embryonic development.

Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-ß/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.

Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy.

For most lysosomal storage diseases (LSDs) affecting the CNS, there is currently no cure. The BBB, which limits the bioavailability of drugs administered systemically, and the short half-life of lysosomal enzymes, hamper the development of effective therapies. Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomic recessive LSD caused by a deficiency in sulfamidase, a sulfatase involved in the stepwise degradation of glycosaminoglycan (GAG) heparan sulfate. Here, we demonstrate that intracerebrospinal fluid (intra-CSF) administration of serotype 9 adenoassociated viral vectors (AAV9s) encoding sulfamidase corrects both CNS and somatic pathology in MPS IIIA mice. Following vector administration, enzymatic activity increased throughout the brain and in serum, leading to whole body correction of GAG accumulation and lysosomal pathology, normalization of behavioral deficits, and prolonged survival. To test this strategy in a larger animal, we treated beagle dogs using intracisternal or intracerebroventricular delivery. Administration of sulfamidase-encoding AAV9 resulted in transgenic expression throughout the CNS and liver and increased sulfamidase activity in CSF. High-titer serum antibodies against AAV9 only partially blocked CSF-mediated gene transfer to the brains of dogs. Consistently, anti-AAV antibody titers were lower in CSF than in serum collected from healthy and MPS IIIA-affected children. These results support the clinical translation of this approach for the treatment of MPS IIIA and other LSDs with CNS involvement.