rAAV-Mediated Gene Transfer For Study of Pathological Mechanisms and Therapeutic Intervention in Canavan\u27s Disease: A Dissertation

Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism

MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression

Recombinant adeno-associated viruses (rAAVs) that can cross the blood-brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5\u27 and 3\u27 mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications

Gene therapy for Canavan\u27s disease takes a step forward

Canavan\u27s disease (CD) is a rare but devastating pediatric leukodystrophy that causes progressive spongy neurodegeneration and is invariably fatal in congenital form.1 The disease is associated with \u3e54 loss-of-function mutations2,3,4 in the enzyme aspartoacylase (ASPA), leads to accumulation of the substrate N-acetyl aspartic acid (NAA) in the brain, and is diagnosed via the presence of NAA aciduria.1 CD is characterized by dysmyelination, intramyelinic edema (leading to hydrocephalus), and extensive vacuolation of the central nervous system (CNS) white matter.5 Currently there is no established therapy that affects progression of the disease, and survival is based primarily on improved general medical care. A previous gene therapy attempt using liposome-encapsulated plasmid DNA6 had shown encouraging although transient decreases in local NAA concentrations in the treated brains, which prompted a gene therapy clinical protocol using recombinant AAV serotype 2 (rAAV2) in the hope of better dissemination of the vector and more sustainable NAA reductions.7 In a recent issue of Science Translational Medicine, Leone et al.8 report long-term follow-up of 13 of the 28 patients enrolled in this trial, who received intracranial injections of first-generation rAAV vectors-based on serotype 2 nearly a decade ago

Making the White Matter Matters: Progress in Understanding Canavan\u27s Disease and Therapeutic Interventions Through Eight Decades

Canavan\u27s disease (CD) is a fatal autosomal recessive pediatric leukodystrophy in which patients show severe neurodegeneration and typically die by the age of 10, though life expectancy in patients can be highly variable. Currently, there is no effective treatment for CD; however, gene therapy seems to be a feasible approach to combat the disease. Being a monogenic defect, the disease provides an excellent model system to develop gene therapy approaches that can be extended to other monogenic leukodystrophies and neurodegenerative diseases. CD results from mutations in a single gene aspartoacylase which hydrolyses N-acetyl aspartic acid (NAA) which accumulates in its absences. Since CD is one of the few diseases that show high NAA levels, it can also be used to study the enigmatic biological role of NAA. The disease was first described in 1931, and this review traces the progress made in the past 8 decades to understand the disease by enumerating current hypotheses and ongoing palliative measures to alleviate patient symptoms in the context of the latest advances in the field

Gene transfer to the CNS using recombinant adeno-associated virus

Recombinant adeno-associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to target the adult CNS by intravascular injections. Curr. Protoc. Microbiol. 29:14D.5.1-14D.5.18. (c) 2013 by John Wiley and Sons, Inc

Gene transfer in the liver using recombinant adeno-associated virus

Liver-directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno-associated virus (rAAV) is a popular gene delivery vehicle for gene therapy, and intravenous delivery of some rAAV serotypes results in very efficient transduction in the liver. rAAV-mediated gene transfer to the liver can be used to create somatic transgenic animals or disease models for studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a biofactory for production of coagulation factors, insulin, growth hormones, and other non-hepatic proteins. Hence, efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long-standing interest to research scientists and clinicians alike. This unit describes methods for delivery of rAAV vectors by several injection routes, followed by a range of analytical methods for assessing the expression, activity, and effects of the transgene and its product. Curr. Protoc. Microbiol. 29:14D.6.1-14D.6.32. (c) 2013 by John Wiley and Sons, Inc

Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system

Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research

rAAV Gene Therapy in a Canavan\u27s Disease Mouse Model Reveals Immune Impairments and an Extended Pathology Beyond the Central Nervous System

Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease

Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10

Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets

A single intravenous rAAV injection as late as P20 achieves efficacious and sustained CNS Gene therapy in canavan mice

Canavan\u27s disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤ 1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20