Adeno-associated viral (AAV) vectors have shown promise as a platform for gene therapy of neurological disorders. Achieving global gene delivery to the central nervous system (CNS) is key for ...development of effective therapies for many of these diseases. Here we report the isolation of a novel CNS tropic AAV capsid, AAV-B1, after a single round of in vivo selection from an AAV capsid library. Systemic injection of AAV-B1 vector in adult mice and cat resulted in widespread gene transfer throughout the CNS with transduction of multiple neuronal subpopulations. In addition, AAV-B1 transduces muscle, β-cells, pulmonary alveoli, and retinal vasculature at high efficiency. This vector is more efficient than AAV9 for gene delivery to mouse brain, spinal cord, muscle, pancreas, and lung. Together with reduced sensitivity to neutralization by antibodies in pooled human sera, the broad transduction profile of AAV-B1 represents an important improvement over AAV9 for CNS gene therapy.
Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an ...effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33–50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.
Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental ...mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex ...deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.
GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes the lysosomal hydrolase acid β-galactosidase (β-gal). β-gal deficiency leads to ...toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. LYS-GM101 is an AAVrh.10-based gene therapy vector carrying the human GLB1 cDNA. The efficacy of intra-cerebrospinal fluid injection of LYS-GM101 analogs was demonstrated in GM1 mouse and cat models with widespread diffusion of β-gal and correction of GM1 ganglioside accumulation in the CNS without observable adverse effects. Clinical dose selection was performed, based on a good-laboratory-practice study, in nonhuman primates (NHPs) using the clinical LYS-GM101 vector. A broadly distributed increase of β-gal activity was observed in NHP brain 3 months after intra-cisterna magna injection of LYS-GM101 at 1.0 × 1012 vg/mL CSF and 4.0 × 1012 vg/mL CSF, with 20% and 60% increases compared with vehicle-treated animals, respectively. Histopathologic examination revealed asymptomatic adverse changes in the sensory pathways of the spinal cord and dorsal root ganglia in both sexes and at both doses. Taken as a whole, these pre-clinical data support the initiation of a clinical study with LYS-GM101 for the treatment of GM1 gangliosidosis.
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GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by defects of the enzyme β-galactosidase. LYS-GM101 is a gene therapy vector supplying the missing enzyme to the central nervous system. We describe efficacy and safety in animal models, supporting initiation of a clinical study using LYS-GM101 in GM1 gangliosidosis patients.
Gene replacement therapy is a rational therapeutic strategy and clinical intervention for neurodegenerative disorders like Canavan disease, a leukodystrophy caused by biallelic mutations in the ...aspartoacylase (ASPA) gene. We aimed to investigate whether simultaneous intravenous (i.v.) and intracerebroventricular (i.c.v.) administration of rAAV9-CB6-ASPA provides a safe and effective therapeutic strategy in an open-label, individual-patient, expanded-access trial for Canavan disease. Immunomodulation was given prophylactically prior to adeno-associated virus (AAV) treatment to prevent an immune response to ASPA or the vector capsid. The patient served as his own control, and change from baseline was assessed by clinical pathology tests, vector genomes in the blood, antibodies against ASPA and AAV capsids, levels of cerebrospinal fluid (CSF) N-acetylaspartate (NAA), brain water content and morphology, clinical status, and motor function tests. Two years post treatment, the patient’s white matter myelination had increased, motor function was improved, and he remained free of typical severe epilepsy. NAA level was reduced at 3 months and remained stable up to 4 years post treatment. Immunomodulation prior to AAV exposure enables repeat dosing and has prevented an anti-transgene immune response. Dual-route administration of gene therapy may improve treatment outcomes.
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Byrne and colleagues demonstrated that simultaneous intravenous and intracerebroventricular administration of rAAV9-CB6-AspA in a single patient with Canavan disease is a safe approach to improve the patient’s white matter myelination and overall motor function. Prophylactic immunomodulation and the dual route of administration might have improved the outcome.
Oligonucleotide therapeutics offer great promise in the treatment of previously untreatable neurodegenerative disorders; however, there are some challenges to overcome in pre-clinical studies. (1) ...They carry a well-established dose-related acute neurotoxicity at the time of administration. (2) Repeated administration into the cerebrospinal fluid may be required for long-term therapeutic effect. Modifying oligonucleotide formulation has been postulated to prevent acute toxicity, but a sensitive and quantitative way to track seizure activity in pre-clinical studies is lacking. The use of intracerebroventricular (i.c.v.) catheters offers a solution for repeated dosing; however, fixation techniques in large animal models are not standardized and are not reliable. Here we describe a novel surgical technique in a sheep model for i.c.v. delivery of neurotherapeutics based on the fixation of the i.c.v. catheter with a 3D-printed anchorage system composed of plastic and ceramic parts, compatible with magnetic resonance imaging, computed tomography, and electroencephalography (EEG). Our technique allowed tracking electrical brain activity in awake animals via EEG and video recording during and for the 24-h period after administration of a novel oligonucleotide in sheep. Its anchoring efficiency was demonstrated for at least 2 months and will be tested for up to a year in ongoing studies.
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Benatti and colleagues describe novel technique troubleshooting challenges of administering new drugs into the brain of a large animal. The technique consists of single surgical intervention, allowing multiple new drugs infusions over at least 2 months. It allows drug safety assessment by electroencephalography since infusions are made with animals under sedation.
GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted ...consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats—either untreated or AAV treated for more than 5 years—and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.
GM1 gangliosidosis is a fatal, untreatable neurodegenerative disease of children and adults. Gray-Edwards and colleagues demonstrate >5-year survival in AAV-treated GM1 cats and describe novel minimally invasive biomarkers for use in clinical trials. Analyses of blood, cerebrospinal fluid, electroencephalography, MRI, and MRS are included from GM1 cats and human patients.
GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats ...treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline β-galactosidase (β-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where β-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and β-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.
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