Giridhar Murlidharan

Biology of adeno-associated viral vectors in the central nervous system

Gene therapy is a promising approach for treating a spectrum of neurological and neurodegenerative disorders by delivering corrective genes to the central nervous system (CNS). In particular, adeno-associated viruses (AAVs) have emerged as promising tools for clinical gene transfer in a broad range of genetic disorders with neurological manifestations. In the current review, we have attempted to bridge our understanding of the biology of different AAV strains with their transduction profiles, cellular tropisms, and transport mechanisms within the CNS. Continued efforts to dissect AAV-host interactions within the brain are likely to aid in the development of improved vectors for CNS-directed gene transfer applications in the clinic.

CNS-restricted Transduction and CRISPR/Cas9-mediated Gene Deletion with an Engineered AAV Vector

Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.Gene therapy using recombinant adeno-associated viral (AAV) vectors is emerging as a promising approach to treat central nervous system disorders such as Spinal muscular atrophy, Batten, Parkinson and Alzheimer disease amongst others. A critical remaining challenge for central nervous system-targeted gene therapy, silencing or gene editing is to limit potential vector dose-related toxicity in off-target cells and organs. Here, we characterize a lab-derived AAV chimeric (AAV2g9), which displays favorable central nervous system attributes derived from both parental counterparts, AAV2 and AAV9. This synthetic AAV strain displays preferential, robust, and widespread neuronal transduction within the brain and decreased glial tropism. Importantly, we observed minimal systemic leakage, decreased sequestration and gene transfer in off-target organs with AAV2g9, when administered into the cerebrospinal fluid. A single intracranial injection of AAV2g9 vectors encoding guide RNAs targeting the schizophrenia risk gene MIR137 (encoding MIR137) in CRISPR/Cas9 knockin mice resulted in brain-specific gene deletion with no detectable events in the liver. This engineered AAV vector is a promising platform for treating neurological disorders through gene therapy, silencing or editing modalities.

Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4-/- mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4-/- mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.

AAV Gene Therapy for MPS1-associated Corneal Blindness.

Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.

Unique Glycan Signatures Regulate Adeno-Associated Virus Tropism in the Developing Brain

ABSTRACT Adeno-associated viruses (AAV) are thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways. The role of host receptors that mediate these processes is not well understood. In the current study, we utilized AAV serotype 4 (AAV4) as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain. Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans in AAV4 transduction. Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low-affinity AAV4 mutant (AAV4.18) displayed a striking shift in cellular tropism from 2,3-linked SA+ ependymal lining to 2,8-linked PSA+ migrating progenitors in the rostral migratory stream and olfactory bulb. In addition, this gain-of-function phenotype correlated with robust CNS spread of AAV4.18 through paravascular transport pathways. Consistent with these observations, altering glycan dynamics within the brain by coadministering SA- and PSA-specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors. We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain. IMPORTANCE Viruses invade the CNS through various mechanisms. In the current study, we utilized AAV as a model to study the dynamics of virus-carbohydrate interactions in the developing brain and their impact on viral tropism. Our findings suggest that carbohydrate content can be exploited to regulate viral transport and tropism in the brain.

Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier

Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

Aquaporin-4 dependent glymphatic solute transport in rodent brain

The glymphatic system is a brain-wide metabolite clearance pathway, impairment of which in post-traumatic and ischemic brain or healthy aging is proposed to contribute to intracerebral accumulation of amyloid-β and tau proteins. Glymphatic perivascular influx of cerebrospinal fluid (CSF) depends upon the expression and perivascular localization of the astroglial water channel aquaporin-4 (AQP4). Prompted by a recent publication that failed to find an effect of Aqp4 knockout on perivascular CSF tracer influx and interstitial fluid (ISF) tracer dispersion, four independent research groups have herein re-examined the importance of Aqp4 in glymphatic fluid transport. We concur in finding that CSF tracer influx, as well as fluorescently-tagged amyloid-β efflux, are significantly faster in wild-type mice than in three different transgenic lines featuring disruption of the Aqp4 gene and one line in which AQP4 expression lacks the critical perivascular localization (Snta1 knockout). These data validate the role of AQP4 in supporting fluid and solute transport and efflux in brain in accordance with the glymphatic system model.

30. Modulation of Intracellular Calcium Enhances AAV Transduction in the CNS

Recent preclinical studies and clinical trials utilizing recombinant AAV vectors have highlighted vector dose-related toxicity as a potential safety concern. Therefore, strategies to enhance AAV transduction may mitigate this toxicity, as they may allow for administration of lower vector doses. While some strategies rely on modification of the capsid to achieve such, pharmacological modulation of the cellular environment often has the benefit of enhancing AAV transduction independent of capsid serotype. Intracellular calcium is important in the life cycles of several viruses, such as Ebola virus and rotavirus. Several small molecule drugs and biologics exist that can modify intracellular calcium levels in a specific manner. In the current study, we sought to determine if modulation of intracellular calcium levels affects AAV transduction. Increasing intracellular calcium levels using either thapsigargin or ionomycin decreased AAV transduction by an order of magnitude. Conversely, decreasing intracellular calcium levels using the cell permanent calcium chelator BAPTA-AM increased transduction by 10 to 100-fold in a cell independent fashion. In addition, the effects of modification of intracellular calcium are not dependent on AAV serotype. Furthermore, in vivo studies performed in mice demonstrate that BAPTA-AM augments transduction by different AAV serotypes, when administered intracranially. In summary, our results support the preclinical evaluation of drugs and biologics that modulate intracellular calcium in the CNS and evaluate their potential for affecting AAV transduction. Results from ongoing studies in mouse models of disease will be presented.

14. Next Generation AAV Vectors for Limiting Systemic Leakage and Improving Safety Following CNS Administration

Intracranial or intrathecal administration of certain AAV vectors for CNS gene transfer is accompanied with systemic leakage into off-target organs such as the liver and spleen. Both preclinical and clinical studies have highlighted potential concerns related to high vector dose-related immunotoxicity and more recently, hepatic genotoxicity in mouse models. In order to address these potential safety issues and reduce the effective dose required to achieve efficient transgene expression in the CNS, we have rationally engineered next generation AAV vectors that show robust CNS spread and efficient transduction, while demonstrating minimal leakage into the systemic circulation. Direct CNS administration or intrathecal infusion of AAV9 results in highly efficient gene expression in neuronal and glial cellular populations in neonatal and adult mice in vivo. However, AAV9 vectors are also disseminated into the blood circulation accompanied by broad vector biodistribution and reporter gene expression in the heart, liver, spleen and kidney. CNS-to-liver and CNS-to-spleen ratios of vector genome copy numbers ranging from 0.3 to 1 were observed. A prototype, engineered AAV strain demonstrated similar potential for spread and high transduction efficiency in neonatal and adult mice. However, transgene expression was primarily restricted to neurons and virtually no leakage into systemic organs was observed regardless of CNS injection route. Preliminary studies in rhesus macaques also confirm the ability of the engineered AAV strain to spread and globally transduce the primate brain. Additional biodistribution data from rodent and primate models is forthcoming. These studies provide a roadmap for addressing clinical gene therapy challenges through continued vector development and confirm that natural AAV isolates are excellent platforms for building next generation vectors with robust transduction efficiency and improved safety profiles.

Gene Therapy of CNS Disorders Using Recombinant AAV Vectors

Corrective intervention for CNS disorders typically requires replenishment of depleted biomolecules (e.g., catabolic enzymes), protection of neurons and glia from premature death, or utilization of CNS cells as bio-factories for production of neurotransmitters or their biological precursors/cofactors. Gene therapy offers the ability to treat disorders in various organs by delivering therapeutic transgenes for regaining lost functionality. Adeno-associated viruses (AAV) have emerged as the vector of choice for CNS gene therapy. This chapter summarizes key observations made during preclinical and clinical evaluations of AAV vectors toward gene therapy of two broad categories of CNS disorders, namely metabolic storage disorders and movement disorders.