Widespread correction of brain pathology in feline alpha-mannosidosis by dose escalation of intracisternal AAV vector injection.

Alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions in the brain and other tissues. Enzyme replacement therapy is available but is not approved for treating the CNS, since the enzyme does not penetrate the blood-brain barrier. However, intellectual disability is a major manifestation of the disease; thus, a complimentary treatment is needed. While enzyme replacement therapy into the brain is technically feasible, it requires ports and frequent administration over time that are difficult to manage medically. Infusion of adeno-associated viral vectors into the cerebrospinal fluid is an attractive route for broadly targeting brain cells. We demonstrate here the widespread post-symptomatic correction of the globally distributed storage lesions by infusion of a high dose of AAV1-feline alpha-mannosidase (fMANB) into the CSF via the cisterna magna in the gyrencephalic alpha-mannosidosis cat brain. Significant improvements in clinical parameters occurred, and widespread global correction was documented pre-mortem by non-invasive magnetic resonance imaging. Postmortem analysis demonstrated high levels of MANB activity and reversal of lysosomal storage lesions throughout the brain. Thus, CSF treatment by adeno-associated viral vector gene therapy appears to be a suitable complement to systemic enzyme replacement therapy to potentially treat the whole patient.

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.

Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration. Notably, while these xenografts were rejected in dogs that were not immunosuppressed, transplants in most dogs receiving systemic immunosuppression survived up to 3-5 months postinjection. Moreover, differentiation of donor PRPCs into photoreceptors with synaptic pedicle-like structures that established contact with second-order neurons was enhanced in rcd1/PDE6B mutant dogs. Together, our findings set the stage for evaluating functional vision restoration following photoreceptor replacement in canine models of inherited retinal degeneration.

Islet1 Precursors Contribute to Mature Interneuron Subtypes in Mouse Neocortex.

Cortical interneurons (GABAergic cells) arise during embryogenesis primarily from the medial and caudal ganglionic eminences (MGE and CGE, respectively) with a small population generated from the preoptic area (POA). Progenitors from the lateral ganglionic eminence (LGE) are thought to only generate GABAergic medium spiny neurons that populate the striatum and project to the globus pallidus. Here, we report evidence that neuronal precursors that express the LGE-specific transcription factor Islet1 (Isl1) can give rise to a small population of cortical interneurons. Lineage tracing and homozygous deletion of Nkx2.1 in Isl1 fate-mapped mice showed that neighboring MGE/POA-specific Nkx2.1 cells and LGE-specific Isl1 cells make both common and distinct lineal contributions towards cortical interneuron fate. Although the majority of cells had overlapping transcriptional domains between Nkx2.1 and Isl1, a population of Isl1-only derived cells also contributed to the adult cerebral cortex. The data indicate that Isl1-derived cells may originate from both the LGE and the adjacent LGE/MGE boundary regions to generate diverse neuronal progeny. Thus, a small population of neocortical interneurons appear to originate from Isl-1-positive precursors.

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.

In Situ Hybridization for Detection of AAV-Mediated Gene Expression.

Techniques to localize vector transgenes in cells and tissues are essential in order to fully characterize gene therapy outcomes. In situ hybridization (ISH) uses synthesized complementary RNA or DNA nucleotide probes to localize and detect sequences of interest in fixed cells, tissue sections, or whole tissue mounts. Variations in techniques include adding labels to probes, such as fluorophores, which can allow for the simultaneous visualization of multiple targets. Here we provide the steps necessary to: (1) label probes for colorimetric visualization and (2) perform ISH on OCT cryo-preserved fixed frozen tissues.

Malformation of the Posterior Cerebellar Vermis Is a Common Neuroanatomical Phenotype of Genetically Engineered Mice on the C57BL/6 Background.

C57BL/6 mice exhibit spontaneous cerebellar malformations consisting of heterotopic neurons and glia in the molecular layer of the posterior vermis, indicative of neuronal migration defect during cerebellar development. Recognizing that many genetically engineered (GE) mouse lines are produced from C57BL/6 ES cells or backcrossed to this strain, we performed histological analyses and found that cerebellar heterotopia were a common feature present in the majority of GE lines on this background. Furthermore, we identify GE mouse lines that will be valuable in the study of cerebellar malformations including diverse driver, reporter, and optogenetic lines. Finally, we discuss the implications that these data have on the use of C57BL/6 mice and GE mice on this background in studies of cerebellar development or as models of disease.

Dopamine induces soluble α-synuclein oligomers and nigrostriatal degeneration.

Parkinson's disease (PD) is defined by the loss of dopaminergic neurons in the substantia nigra and the formation of Lewy body inclusions containing aggregated α-synuclein. Efforts to explain dopamine neuron vulnerability are hindered by the lack of dopaminergic cell death in α-synuclein transgenic mice. To address this, we manipulated both dopamine levels and α-synuclein expression. Nigrally targeted expression of mutant tyrosine hydroxylase with enhanced catalytic activity increased dopamine levels without damaging neurons in non-transgenic mice. In contrast, raising dopamine levels in mice expressing human A53T mutant α-synuclein induced progressive nigrostriatal degeneration and reduced locomotion. Dopamine elevation in A53T mice increased levels of potentially toxic α-synuclein oligomers, resulting in conformationally and functionally modified species. Moreover, in genetically tractable Caenorhabditis elegans models, expression of α-synuclein mutated at the site of interaction with dopamine prevented dopamine-induced toxicity. These data suggest that a unique mechanism links two cardinal features of PD: dopaminergic cell death and α-synuclein aggregation.

Stem Cell Therapy for the Central Nervous System in Lysosomal Storage Diseases.

Neurological diseases with genetic etiologies result in the loss or dysfunction of neural cells throughout the CNS. At present, few treatment options exist for the majority of neurogenetic diseases. Stem cell transplantation (SCT) into the CNS has the potential to be an effective treatment modality because progenitor cells may replace lost cells in the diseased brain, provide multiple trophic factors, or deliver missing proteins. This review focuses on the use of SCT in lysosomal storage diseases (LSDs), a large group of monogenic disorders with prominent CNS disease. In most patients the CNS disease results in intellectual disability that is refractory to current standard-of-care treatment. A large amount of preclinical work on brain-directed SCT has been performed in rodent LSD models. Cell types that have been used for direct delivery into the CNS include neural stem cells, embryonic and induced pluripotent stem cells, and mesenchymal stem cells. Hematopoietic stem cells have been an effective therapy for the CNS in a few LSDs and may be augmented by overexpression of the missing gene. Current barriers and potential strategies to improve SCT for translation into effective patient therapies are discussed.

Integrated analysis of proteome and transcriptome changes in the mucopolysaccharidosis type VII mouse hippocampus.

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the deficiency of ß-glucuronidase. In this study, we compared the changes relative to normal littermates in the proteome and transcriptome of the hippocampus in the C57Bl/6 mouse model of MPS VII, which has well-documented histopathological and neurodegenerative changes. A completely different set of significant changes between normal and MPS VII littermates were found in each assay. Nevertheless, the functional annotation terms generated by the two methods showed agreement in many of the processes, which also corresponded to known pathology associated with the disease. Additionally, assay-specific changes were found, which in the proteomic analysis included mitochondria, energy generation, and cytoskeletal differences in the mutant, while the transcriptome differences included immune, vesicular, and extracellular matrix changes. In addition, the transcriptomic changes in the mutant hippocampus were concordant with those in a MPS VII mouse caused by the same mutation but on a different background inbred strain.

Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.

More than one hundred naturally occurring variants of adeno-associated virus (AAV) have been identified, and this library has been further expanded by an array of techniques for modification of the viral capsid. AAV capsid variants possess unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding. AAV capsids can be chemically modified to alter tropism, can be produced as hybrid vectors that combine the properties of multiple serotypes, and can carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency. This large number of AAV variants and engineered capsids provides a varied toolkit for gene delivery to the CNS and retina, with specialized vectors available for many applications, but selecting a capsid variant from the array of available vectors can be difficult. This chapter describes the unique properties of a range of AAV variants and engineered capsids, and provides a guide for selecting the appropriate vector for specific applications in the CNS and retina.

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.

Bilateral single-site intracerebral injection of a nonpathogenic herpes simplex virus-1 vector decreases anxiogenic behavior in MPS VII mice.

Genetic diseases of the brain usually have pathologic lesions distributed throughout, thus requiring global correction. Herpes simplex virus-1 (HSV-1) vectors may be especially useful for gene delivery in these disorders since they can spread trans-synaptically along neuronal pathways to distal sites from a localized injection. We have previously shown that a nonpathogenic HSV-1 (strain 1716), which is deleted in the ICP34.5 gene, and expressing the lysosomal enzyme ß-glucuronidase (GUSB) from the latency-associated transcript (LAT) promoter, spreads within the brains of GUSB-deficient mucopolysaccharidosis VII mice to reverse the pathognomonic storage lesions throughout the diseased brain. In this study, we tested the ability of the 1716 LAT-GUSB vector to improve behavioral deficits. The treatment significantly decreased anxiogenic behaviors associated with the mutation, as indicated by open-field behavior and decreased neophobia in a novel object-recognition task. The treated mice also exhibited an improvement in cognitive function associated with the cerebral cortex in a familiar object test. The results indicate the functional therapeutic potential of the 1716 LAT-GUSB vector.

Ex vivo gene therapy using patient iPSC-derived NSCs reverses pathology in the brain of a homologous mouse model.

Neural stem cell (NSC) transplantation is a promising strategy for delivering therapeutic proteins in the brain. We evaluated a complete process of ex vivo gene therapy using human induced pluripotent stem cell (iPSC)-derived NSC transplants in a well-characterized mouse model of a human lysosomal storage disease, Sly disease. Human Sly disease fibroblasts were reprogrammed into iPSCs, differentiated into a stable and expandable population of NSCs, genetically corrected with a transposon vector, and assessed for engraftment in NOD/SCID mice. Following neonatal intraventricular transplantation, the NSCs engraft along the rostrocaudal axis of the CNS primarily within white matter tracts and survive for at least 4 months. Genetically corrected iPSC-NSCs transplanted post-symptomatically into the striatum of adult Sly disease mice reversed neuropathology in a zone surrounding the grafts, while control mock-corrected grafts did not. The results demonstrate the potential for ex vivo gene therapy in the brain using human NSCs from autologous, non-neural tissues.

Adeno-associated virus serotypes 1, 8, and 9 share conserved mechanisms for anterograde and retrograde axonal transport.

Adeno-associated virus (AAV) vectors often undergo long-distance axonal transport after brain injection. This leads to transduction of brain regions distal to the injection site, although the extent of axonal transport and distal transduction varies widely among AAV serotypes. The mechanisms driving this variability are poorly understood. This is a critical problem for applications that require focal gene expression within a specific brain region, and also impedes the utilization of vector transport for applications requiring widespread delivery of transgene to the brain. Here, we compared AAV serotypes 1 and 9, which frequently demonstrate distal transduction, with serotype 8, which rarely spreads beyond the injection site. To examine directional AAV transport in vitro, we used a microfluidic chamber to apply dye-labeled AAV to the axon termini or to the cell bodies of primary rat embryonic cortical neurons. All three serotypes were actively transported along axons, with transport characterized by high velocities and prolonged runs in both the anterograde and retrograde directions. Coinfection with pairs of serotypes indicated that AAV1, 8, and 9 share the same intracellular compartments for axonal transport. In vivo, both AAV8 and 9 demonstrated anterograde and retrograde transport within a nonreciprocal circuit after injection into adult mouse brain, with highly similar distributions of distal transduction. However, in mass-cultured neurons, we found that AAV1 was more frequently transported than AAV8 or 9, and that the frequency of AAV9 transport could be enhanced by increasing receptor availability. Thus, while these serotypes share conserved mechanisms for axonal transport both in vitro and in vivo, the frequency of transport can vary among serotypes, and axonal transport can be markedly increased by enhancing vector uptake. This suggests that variability in distal transduction in vivo likely results from differential uptake at the plasma membrane, rather than fundamental differences in transport mechanisms among AAV serotypes.

Long-distance axonal transport of AAV9 is driven by dynein and kinesin-2 and is trafficked in a highly motile Rab7-positive compartment.

Adeno-associated virus (AAV) vectors can move along axonal pathways after brain injection, resulting in transduction of distal brain regions. This can enhance the spread of therapeutic gene transfer and improve treatment of neurogenetic disorders that require global correction. To better understand the underlying cellular mechanisms that drive AAV trafficking in neurons, we investigated the axonal transport of dye-conjugated AAV9, utilizing microfluidic primary neuron cultures that isolate cell bodies from axon termini and permit independent analysis of retrograde and anterograde axonal transport. After entry, AAV was trafficked into nonmotile early and recycling endosomes, exocytic vesicles, and a retrograde-directed late endosome/lysosome compartment. Rab7-positive late endosomes/lysosomes that contained AAV were highly motile, exhibiting faster retrograde velocities and less pausing than Rab7-positive endosomes without virus. Inhibitor experiments indicated that the retrograde transport of AAV within these endosomes is driven by cytoplasmic dynein and requires Rab7 function, whereas anterograde transport of AAV is driven by kinesin-2 and exhibits unusually rapid velocities. Furthermore, increasing AAV9 uptake by neuraminidase treatment significantly enhanced virus transport in both directions. These findings provide novel insights into AAV trafficking within neurons, which should enhance progress toward the utilization of AAV for improved distribution of transgene delivery within the brain.

High-resolution magnetic resonance microscopy and diffusion tensor imaging to assess brain structural abnormalities in the murine mucopolysaccharidosis VII model.

High-resolution microscopic magnetic resonance imaging (µMRI) and diffusion tensor imaging (DTI) were performed to characterize brain structural abnormalities in a mouse model of mucopolysaccharidosis type VII (MPS VII). Microscopic magnetic resonance imaging demonstrated a decrease in the volume of anterior commissure and corpus callosum and a slight increase in the volume of the hippocampus in MPS VII versus wild-type mice. Diffusion tensor imaging indices were analyzed in gray and white matter. In vivo and ex vivo DTI demonstrated significantly reduced fractional anisotropy in the anterior commissure, corpus callosum, external capsule, and hippocampus in MPS VII versus control brains. Significantly increased mean diffusivity was also found in the anterior commissure and corpus callosum from ex vivo DTI. Significantly reduced linear anisotropy was observed from the hippocampus from in vivo DTI, whereas significantly decreased planar anisotropy and spherical anisotropy were observed in the external capsule from only ex vivo DTI. There were corresponding morphologic differences in the brains of MPS VII mice by hematoxylin and eosin staining. Luxol fast blue staining demonstrated less intense staining of the corpus callosum and external capsule; myelin abnormalities in the corpus callosum were also demonstrated quantitatively in toluidine blue-stained sections and confirmed by electron microscopy. These results demonstrate the potential for µMRI and DTI for quantitative assessment of brain pathology in murine models of brain diseases.

Quantitative, noninvasive, in vivo longitudinal monitoring of gene expression in the brain by co-AAV transduction with a PET reporter gene.

In vivo imaging of vector transgene expression would be particularly valuable for repetitive monitoring of therapy in the brain, where invasive tissue sampling is contraindicated. We evaluated adeno-associated virus vector expression of a dopamine-2 receptor (D2R) mutant (D2R80A) by positron emission tomography in the brains of mice and cats. D2R80A is inactivated for intracellular signaling and binds subphysiologic amounts of the radioactive [(18)F]-fallypride analog of dopamine. The [(18)F]-fallypride signal bound to D2R80A in the injection site was normalized to the signal from endogenous D2R in the striatum and showed stable levels of expression within individual animals. A separate adeno-associated virus type 1 vector with identical gene expression control elements, expressing green fluorescent protein or a therapeutic gene, was coinjected with the D2R80A vector at equal doses into specific sites. Both transgenes had similar levels of gene expression by immunohistochemistry, in situ hybridization, and quantitative PCR assays, demonstrating that D2R80A is a faithful surrogate measure for expression of a gene of interest. This dual vector approach allows the D2R80A gene to be used with any therapeutic gene and to be injected into a single site for monitoring while the therapeutic gene can be distributed more widely as needed in each disease.

Engraftment of nonintegrating neural stem cells differentially perturbs cortical activity in a dose-dependent manner.

Neural stem cell (NSC) therapy represents a potentially powerful approach for gene transfer in the diseased central nervous system. However, transplanted primary, embryonic stem cell- and induced pluripotent stem cell-derived NSCs generate largely undifferentiated progeny. Understanding how physiologically immature cells influence host activity is critical to evaluating the therapeutic utility of NSCs. Earlier inquiries were limited to single-cell recordings and did not address the emergent properties of neuronal ensembles. To interrogate cortical networks post-transplant, we used voltage sensitive dye imaging in mouse neocortical brain slices, which permits high temporal resolution analysis of neural activity. Although moderate NSC engraftment largely preserved host physiology, subtle defects in the activation properties of synaptic inputs were induced. High-density engraftment severely dampened cortical excitability, markedly reducing the amplitude, spatial extent, and velocity of propagating synaptic potentials in layers 2-6. These global effects may be mediated by specific disruptions in excitatory network structure in deep layers. We propose that depletion of endogenous cells in engrafted neocortex contributes to circuit alterations. Our data provide the first evidence that nonintegrating cells cause differential host impairment as a function of engrafted load. Moreover, they emphasize the necessity for efficient differentiation methods and proper controls for engraftment effects that interfere with the benefits of NSC therapy.