Combination HSCT and intravenous AAV-mediated gene therapy in a canine model proves pivotal for translation of Krabbe disease therapy.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

Transduction characteristics of alternative adeno-associated virus serotypes in the cat brain by intracisternal delivery.

Multiple studies have examined the transduction characteristics of different AAV serotypes in the mouse brain, where they can exhibit significantly different patterns of transduction. The pattern of transduction also varies with the route of administration. Much less information exists for the transduction characteristics in large-brained animals. Large animal models have brains that are closer in size and organization to the human brain, such as being gyrencephalic compared to the lissencephalic rodent brains, pathway organization, and certain electrophysiologic properties. Large animal models are used as translational intermediates to develop gene therapies to treat human diseases. Various AAV serotypes and routes of delivery have been used to study the correction of pathology in the brain in lysosomal storage diseases. In this study, we evaluated the ability of selected AAV serotypes to transduce cells in the cat brain when delivered into the cerebrospinal fluid via the cisterna magna. We previously showed that AAV1 transduced significantly greater numbers of cells than AAV9 in the cat brain by this route. In the present study, we evaluated serotypes closely related to AAVs 1 and 9 (AAVs 6, AS, hu32) that may mediate more extensive transduction, as well as AAVs 4 and 5, which primarily transduce choroid plexus epithelial (CPE) and ependymal lining cells in the rodent brain. The related serotypes tended to have similar patterns of transduction but were divergent in some specific brain structures.

Efficacy and Safety of a Krabbe Disease Gene Therapy.

Krabbe disease is a lysosomal storage disease caused by mutations in the gene that encodes galactosylceramidase, in which galactosylsphingosine (psychosine) accumulation drives demyelination in the central and peripheral nervous systems, ultimately progressing to death in early childhood. Gene therapy, alone or in combination with transplant, has been developed for almost two decades in mouse models, with increasing therapeutic benefit paralleling the improvement of next-generation adeno-associated virus (AAV) vectors. This effort has recently shown remarkable efficacy in the canine model of the disease by two different groups that used either systemic or cerebrospinal fluid (CSF) administration of AAVrh10 or AAV9. Building on our experience developing CSF-delivered, AAV-based drug products for a variety of neurodegenerative disorders, we conducted efficacy, pharmacology, and safety studies of AAVhu68 delivered to the CSF in two relevant natural Krabbe animal models, and in nonhuman primates. In newborn Twitcher mice, the highest dose (1 × 1011 genome copies [GC]) of AAVhu68.hGALC injected into the lateral ventricle led to a median survival of 130 days compared to 40.5 days in vehicle-treated mice. When this dose was administered intravenously, the median survival was 49 days. A single intracisterna magna injection of AAVhu68.cGALC at 3 × 1013 GC into presymptomatic Krabbe dogs increased survival for up to 85 weeks compared to 12 weeks in controls. It prevented psychosine accumulation in the CSF, preserved peripheral nerve myelination, ambulation, and decreased brain neuroinflammation and demyelination, although some regions remained abnormal. In a Good Laboratory Practice-compliant toxicology study, we administered the clinical candidate into the cisterna magna of 18 juvenile rhesus macaques at 3 doses that displayed efficacy in mice. We observed no dose-limiting toxicity and sporadic minimal degeneration of dorsal root ganglia (DRG) neurons. Our studies demonstrate the efficacy, scalability, and safety of a single cisterna magna AAVhu68 administration to treat Krabbe disease. ClinicalTrials.Gov ID: NCT04771416.

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.

Global CNS correction in a large brain model of human alpha-mannosidosis by intravascular gene therapy.

Intravascular injection of certain adeno-associated virus vector serotypes can cross the blood-brain barrier to deliver a gene into the CNS. However, gene distribution has been much more limited within the brains of large animals compared to rodents, rendering this approach suboptimal for treatment of the global brain lesions present in most human neurogenetic diseases. The most commonly used serotype in animal and human studies is 9, which also has the property of being transported via axonal pathways to distal neurons. A small number of other serotypes share this property, three of which were tested intravenously in mice compared to 9. Serotype hu.11 transduced fewer cells in the brain than 9, rh8 was similar to 9, but hu.32 mediated substantially greater transduction than the others throughout the mouse brain. To evaluate the potential for therapeutic application of the hu.32 serotype in a gyrencephalic brain of larger mammals, a hu.32 vector expressing the green fluorescent protein reporter gene was evaluated in the cat. Transduction was widely distributed in the cat brain, including in the cerebral cortex, an important target since mental retardation is an important component of many of the human neurogenetic diseases. The therapeutic potential of a hu.32 serotype vector was evaluated in the cat homologue of the human lysosomal storage disease alpha-mannosidosis, which has globally distributed lysosomal storage lesions in the brain. Treated alpha-mannosidosis cats had reduced severity of neurological signs and extended life spans compared to untreated cats. The extent of therapy was dose dependent and intra-arterial injection was more effective than intravenous delivery. Pre-mortem, non-invasive magnetic resonance spectroscopy and diffusion tensor imaging detected differences between the low and high doses, and showed normalization of grey and white matter imaging parameters at the higher dose. The imaging analysis was corroborated by post-mortem histological analysis, which showed reversal of histopathology throughout the brain with the high dose, intra-arterial treatment. The hu.32 serotype would appear to provide a significant advantage for effective treatment of the gyrencephalic brain by systemic adeno-associated virus delivery in human neurological diseases with widespread brain lesions.

Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.

Cerebrospinal fluid and serum glycosphingolipid biomarkers in canine globoid cell leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD, Krabbe disease, Krabbe's disease) is caused by genetic mutations in the gene encoding, galactosylceramidase (GALC). Deficiency of this enzyme results in central and peripheral nervous system pathology, and is characterized by loss of myelin and an infiltration of globoid cells. The canine model of GLD provides a translational model which faithfully recapitulates much of the human disease pathology. Targeted lipidomic analysis was conducted in serum and cerebrospinal fluid (CSF) over the lifetime of GLD affected and normal canines, and in brain tissue at humane endpoint to better understand disease progression and identify potential biomarkers of disease. Psychosine, a substrate of GALC and primary contributor to the pathology in GLD, was observed to be significantly elevated in the serum and CSF by 2 or 4 weeks of age, respectively, and steadily increased over the lifetime of affected animals. Importantly, psychosine concentration strongly correlated with disease severity. Galactosylceramide, glucosylceramide, and lactosylceramide were also found to be elevated in the CSF of affected animals and increased with age. Psychosine and galactosylceramide were found to be significantly increased in brain tissue at humane endpoint. This study identified several biomarkers which may be useful in the development of therapeutics for GLD.

Pharmacokinetics and distribution of 2-hydroxypropyl-ß-cyclodextrin following a single intrathecal dose to cats.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is an experimental therapy for Niemann-Pick disease type C (NPC) that reduced neuronal cholesterol and ganglioside storage, reduced Purkinje cell death, and increased lifespan in npc1-/- mice and NPC1 cats. In this study, tissue distribution was investigated in normal cats that received a single 120-mg dose of [14 C]-HP-ß-CD (approximately 200 µCi/cat) via the cerebellomedullary cistern (CBMC) and lumbar cistern. One cat was euthanized at each of various time points up to 24 hours postdose for subsequent processing and quantitative whole-body autoradiographic analysis. HP-ß-CD-derived radioactivity absorbed from the CBMC was widely distributed to cat tissues; most tissues were observed to have reached their highest concentration at 1 hour postdose. HP-ß-CD-derived radioactivity penetrated into the deeper parts of the central nervous system with the highest concentration at 4 hours (403 µg Eq/g or 0.28 mM) and remained high (49.7 µg Eq/g or 0.03 mM) at 24 hours. The relatively long half-life (11-30 hours) in cerebral ventricles and the subarachnoid space surrounding the brain and spinal cord might contribute to the efficacy of HP-ß-CD in NPC1 cats. Other tissues with high concentrations of radioactivity were nasal turbinates, pituitary gland, and urinary bladder, while relatively low concentrations were observed in blood and bile.

AAVrh10 Gene Therapy Ameliorates Central and Peripheral Nervous System Disease in Canine Globoid Cell Leukodystrophy (Krabbe Disease).

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

LC3 Immunostaining in the Inferior Olivary Nuclei of Cats With Niemann-Pick Disease Type C1 Is Associated With Patterned Purkinje Cell Loss.

The feline model of Niemann-Pick disease, type C1 (NPC1) recapitulates the clinical, neuropathological, and biochemical abnormalities present in children with NPC1. The hallmarks of disease are the lysosomal storage of unesterified cholesterol and multiple sphingolipids in neurons, and the spatial and temporal distribution of Purkinje cell death. In feline NPC1 brain, microtubule-associated protein 1 light chain 3 (LC3) accumulations, indicating autophagosomes, were found within axons and presynaptic terminals. High densities of accumulated LC3 were seen in subdivisions of the inferior olive, which project to cerebellar regions that show the most Purkinje cell loss, suggesting that autophagic abnormalities in specific climbing fibers may contribute to the spatial pattern of Purkinje cell loss seen. Biweekly intrathecal administration of 2-hydroxypropyl-beta cyclodextrin (HPßCD) ameliorated neurological dysfunction, reduced cholesterol and sphingolipid accumulation, and increased lifespan in NPC1 cats. LC3 pathology was reduced in treated animals suggesting that HPßCD administration also ameliorates autophagic abnormalities. This study is the first to (i) identify specific brain regions exhibiting autophagic abnormalities in any species with NPC1, (ii) provide evidence of differential vulnerability among discrete brain nuclei and pathways, and (iii) show the amelioration of these abnormalities in NPC1 cats treated with HPßCD.

Evaluation of Intrathecal Routes of Administration for Adeno-Associated Viral Vectors in Large Animals.

Delivery of adeno-associated viral (AAV) vectors into the cerebrospinal fluid (CSF) can achieve gene transfer to cells throughout the brain and spinal cord, potentially making many neurological diseases tractable gene therapy targets. Identifying the optimal route of CSF access for intrathecal AAV delivery will be a critical step in translating this approach to clinical practice. We previously demonstrated that vector injection into the cisterna magna is a safe and effective method for intrathecal AAV delivery in nonhuman primates; however, this procedure is not commonly used in clinical practice. More routine methods of administration into the CSF are now being explored, including intracerebroventricular (ICV) injection and injection through a lumbar puncture. In this study, we compared ICV and intracisternal (IC) AAV administration in dogs. We also evaluated vector administration via lumbar puncture in nonhuman primates, with some animals placed in the Trendelenburg position after injection, a maneuver that has been suggested to improve cranial distribution of vector. In the dog study, ICV and IC vector administration resulted in similarly efficient transduction throughout the brain and spinal cord. However, animals in the ICV cohort developed encephalitis associated with a T-cell response to the transgene product, a phenomenon that was not observed in the IC cohort. In the nonhuman primate study, transduction efficiency was not improved by placing animals in the Trendelenburg position after injection. These findings illustrate important limitations of commonly used methods for CSF access in the context of AAV delivery, and will be important for informing the selection of a route of administration for first-in-human studies.

Clinical, electrophysiological, and biochemical markers of peripheral and central nervous system disease in canine globoid cell leukodystrophy (Krabbe's disease).

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a debilitating and always fatal pediatric neurodegenerative disease caused by a mutation in the gene encoding the hydrolytic enzyme galactosylceramidase (GALC). In the absence of GALC, progressive loss of myelin and accumulation of a neurotoxic substrate lead to incapacitating loss of motor and cognitive function and death, typically by 2 years of age. Currently, there is no cure. Recent convincing evidence of the therapeutic potential of combining gene and cell therapies in the murine model of GLD has accelerated the requirement for validated markers of disease to evaluate therapeutic efficacy. Here we demonstrate clinically relevant and quantifiable measures of central (CNS) and peripheral (PNS) nervous system disease progression in the naturally occurring canine model of GLD. As measured by brainstem auditory-evoked response testing, GLD dogs demonstrated a significant increase in I-V interpeak latency and hearing threshold at all time points. Motor nerve conduction velocities (NCVs) in GLD dogs were significantly lower than normal by 12-16 weeks of age, and sensory NCV was significantly lower than normal by 8-12 weeks of age, serving as a sensitive indicator of peripheral nerve dysfunction. Post-mortem histological evaluations confirmed neuroimaging and electrodiagnostic assessments and detailed loss of myelin and accumulation of storage product in the CNS and the PNS. Additionally, cerebrospinal fluid psychosine concentrations were significantly elevated in GLD dogs, demonstrating potential as a biochemical marker of disease. These data demonstrate that CNS and PNS disease progression can be quantified over time in the canine model of GLD with tools identical to those used to assess human patients. © 2016 Wiley Periodicals, Inc.

Neonatal tolerance induction enables accurate evaluation of gene therapy for MPS I in a canine model.

High fidelity animal models of human disease are essential for preclinical evaluation of novel gene and protein therapeutics. However, these studies can be complicated by exaggerated immune responses against the human transgene. Here we demonstrate that dogs with a genetic deficiency of the enzyme α-l-iduronidase (IDUA), a model of the lysosomal storage disease mucopolysaccharidosis type I (MPS I), can be rendered immunologically tolerant to human IDUA through neonatal exposure to the enzyme. Using MPS I dogs tolerized to human IDUA as neonates, we evaluated intrathecal delivery of an adeno-associated virus serotype 9 vector expressing human IDUA as a therapy for the central nervous system manifestations of MPS I. These studies established the efficacy of the human vector in the canine model, and allowed for estimation of the minimum effective dose, providing key information for the design of first-in-human trials. This approach can facilitate evaluation of human therapeutics in relevant animal models, and may also have clinical applications for the prevention of immune responses to gene and protein replacement therapies.

Cerebrospinal Fluid Calbindin D Concentration as a Biomarker of Cerebellar Disease Progression in Niemann-Pick Type C1 Disease.

Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-ß-cyclodextrin (HPßCD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HPßCD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HPßCD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.

Clinical Improvement of Alpha-mannosidosis Cat Following a Single Cisterna Magna Infusion of AAV1.

Lysosomal storage diseases (LSDs) are debilitating neurometabolic disorders for most of which long-term effective therapies have not been developed. Gene therapy is a potential treatment but a critical barrier to treating the brain is the need for global correction. We tested the efficacy of cisterna magna infusion of adeno-associated virus type 1 (AAV1) expressing feline alpha-mannosidase gene in the postsymptomatic alpha-mannosidosis (AMD) cat, a homologue of the human disease. Lysosomal alpha-mannosidase (MANB) activity in the cerebrospinal fluid (CSF) and serum were increased above the control values in untreated AMD cats. Clinical neurological signs were delayed in onset and reduced in severity. The lifespan of the treated cats was significantly extended. Postmortem histopathology showed resolution of lysosomal storage lesions throughout the brain. MANB activity in brain tissue was significantly above the levels of untreated tissues. The results demonstrate that a single cisterna magna injection of AAV1 into the CSF can mediate widespread neuronal transduction of the brain and meaningful clinical improvement. Thus, cisterna magna gene delivery by AAV1 appears to be a viable strategy for treatment of the whole brain in AMD and should be applicable to many of the neurotropic LSDs as well as other neurogenetic disorders.

Diffusion Tensor Imaging for Assessing Brain Gray and White Matter Abnormalities in a Feline Model of α-Mannosidosis.

α-Mannosidosis (AMD) is an autosomal recessively inherited lysosomal storage disorder affecting brain function and structure. We performed ex vivo and in vivo diffusion tensor imaging (DTI) on the brains of AMD-affected cats to assess gray and white matter abnormalities. A multi-atlas approach was used to generate a brain template to process the ex vivo DTI data. The probabilistic label method was used to measure fractional anisotropy (FA), mean diffusivity, axial diffusivity, and radial diffusivity values from gray and white matter regions from ex vivo DTI. Regional analysis from various regions of the gray matter (frontal cortex, cingulate gyrus, caudate nucleus, hippocampus, thalamus, and occipital cortex), and white matter (corpus callosum, corticospinal tract, cerebral peduncle, external and internal capsule) was also performed on both ex vivo and in vivo DTI. Ex vivo DTI revealed significantly reduced FA from both gray and white matter regions in AMD-affected cats compared to controls. Significantly reduced FA was also observed from in vivo DTI of AMD-affected cats compared to controls, with lower FA values observed in all white matter regions. We also observed significantly increased axial and radial diffusivity values in various gray and white matter regions in AMD cats from both ex vivo and in vivo DTI data. Imaging findings were correlated with histopathologic analyses suggesting that DTI studies can further aid in the characterization of AMD by assessing the microstructural abnormalities in both white and gray matter.

Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in ß-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates.

The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.

Intracisternal cyclodextrin prevents cerebellar dysfunction and Purkinje cell death in feline Niemann-Pick type C1 disease.

Niemann-Pick type C1 (NPC) disease is a lysosomal storage disease caused by mutations in the NPC1 gene, leading to an increase in unesterified cholesterol and several sphingolipids, and resulting in hepatic disease and progressive neurological disease. We show that subcutaneous administration of the pharmaceutical excipient 2-hydroxypropyl-ß-cyclodextrin (HPßCD) to cats with NPC disease ameliorated hepatic disease, but doses sufficient to reduce neurological disease resulted in pulmonary toxicity. However, direct administration of HPßCD into the cisterna magna of presymptomatic cats with NPC disease prevented the onset of cerebellar dysfunction for greater than a year and resulted in a reduction in Purkinje cell loss and near-normal concentrations of cholesterol and sphingolipids. Moreover, administration of intracisternal HPßCD to NPC cats with ongoing cerebellar dysfunction slowed disease progression, increased survival time, and decreased the accumulation of brain gangliosides. An increase in hearing threshold was identified as a potential adverse effect. These studies in a feline animal model have provided critical data on efficacy and safety of drug administration directly into the central nervous system that will be important for advancing HPßCD into clinical trials.

Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I.

Patients with mucopolysaccharidosis type I (MPS I), a genetic deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), exhibit accumulation of glycosaminoglycans in tissues, with resulting diverse clinical manifestations including neurological, ocular, skeletal, and cardiac disease. MPS I is currently treated with hematopoietic stem cell transplantation or weekly enzyme infusions, but these therapies have significant drawbacks for patient safety and quality of life and do not effectively address some of the most critical clinical sequelae, such as life-threatening cardiac valve involvement. Using the naturally occurring feline model of MPS I, we tested liver-directed gene therapy as a means of achieving long-term systemic IDUA reconstitution. We treated four MPS I cats at 3-5 mo of age with an adeno-associated virus serotype 8 vector expressing feline IDUA from a liver-specific promoter. We observed sustained serum enzyme activity for 6 mo at ∼ 30% of normal levels in one animal, and in excess of normal levels in three animals. Remarkably, treated animals not only demonstrated reductions in glycosaminoglycan storage in most tissues, but most also exhibited complete resolution of aortic valve lesions, an effect that has not been previously observed in this animal model or in MPS I patients treated with current therapies. These data point to clinically meaningful benefits of the robust enzyme expression achieved with hepatic gene transfer that extend beyond the economic and quality of life advantages over lifelong enzyme infusions.