The benefit of early diagnosis and treatment has been demonstrated in animal models of several lysosomal storage diseases. In a clinical setting, however, diagnoses are often not made until after patients become symptomatic. The lysosomal storage disease alpha-mannosidosis is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions throughout the brain and other tissues. In a feline model of alpha-mannosidosis, we previously demonstrated complete correction of the brain following delivery of AAVhu.32-fMANB via the carotid artery in the early symptomatic stage. Here, we investigate the efficacy of AAV gene therapy on globally distributed storage lesions in animals with advanced disease. Some improvements in clinical parameters were observed, however these improvements were less than in animals with less advanced disease. Although the treated animals were improved compared to untreated animals, increasing the vector dose did not further improve clinical outcomes. These results further demonstrate the importance of early detection and treatment of a lysosomal storage disease to successful outcomes. Despite this, partial correction extended the lifespan of diseased cats and may be medically beneficial to patients by slowing or stabilizing the progressive degenerative course of disease.

The choroid plexuses (CP) are highly vascularized structures that project into the ventricles of the vertebrate brain. The polarized epithelia of the CP produce cerebrospinal fluid by transporting water and ions into the ventricles from the blood and normally secrete a large number of proteins. We assessed the feasibility of selective CP transduction with recombinant adeno-associated virus (rAAV) gene therapy vectors for treatment of lysosomal storage disease (LSD), a broad category of neurometabolic illness associated with significant burdens to affected patients and their families. There are no ideal or complete therapeutic options currently available, especially for the central nervous system manifestations of LSDs. Alpha-mannosidosis (AMD) is an autosomal recessive prototypical LSD caused by deficiency of lysosomal alpha-mannosidase and characterized by cerebellar ataxia, neurocognitive disability, facial and skeletal abnormalities, hearing impairment, and mild immune deficiency. In a murine model of AMD, we compared the biochemical effects of CSF-directed rAAV serotypes 1, 4, 5, 6, and 9. Recombinant AAV1 and rAAV6, two closely related serotypes whose capsid sequences differ by only six amino acids, showed the most robust transduction of CP in mouse brain, consistent with their transduction of CPE in nonhuman primates and cats, as well as in other structures. We found restoration of LAMAN enzyme activity comparable to or higher than AMD heterozygote levels in the brain globally (olfactory bulb, cortex, cerebellum, brainstem). Further IND-generating preclinical experiments will advance rAAV6-LAMAN, which appears to be the most promising choroid plexus-targeting candidate serotype for future clinical translation to treat AMD.

Progressive photoreceptor loss in retinal degenerative diseases leads to irreversible vision impairment. Transplantation of human embryonic or induced pluripotent stem cell-derived photoreceptor precursor cells (PRPCs) offers potential for vision restoration. However, substantial early donor cell loss remains a major challenge. This study aims to elucidate the mechanisms underlying early PRPC loss and to evaluate host retinal responses to transplantation. PRPCs derived from human embryonic stem cells (hESC)-based retinal organoids were subretinally transplanted into both normal and degenerated canine retinas to investigate the impact of host retinal degeneration on transplant survival and integration. Single-cell RNA sequencing (scRNAseq) was performed on transplanted PRPCs 3 days post-transplantation into normal canine retinas, as well as on host retinal cells to identify molecular pathways associated with early donor cell loss. Non-invasive multimodal retinal imaging and immunohistochemical analyses were conducted to assess PRPC survival, integration, and host immune responses. Despite systemic immunosuppression, extensive early loss of human PRPCs occurred within the first week following xenotransplantation into both normal and degenerated canine retinas, suggesting that factors beyond immune activation contribute to donor cell loss. Transcriptomic analysis identified metabolic stress as a key driver of early donor cell death, characterized by dysregulation of mitochondrial function and oxidative phosphorylation pathways. Microglial infiltration into the donor cell mass was also observed in normal retinas, suggesting a response to donor cell stress and apoptosis. Beyond the initial phase of cell death, surviving donor cells integrated and persisted when transplanted into retinas with a partially preserved outer nuclear layer, whereas cell loss continued when intervention occurred at end-stage degeneration. Metabolic stress represents a critical barrier to PRPC survival following transplantation. Strategies aimed at enhancing metabolic resilience may improve transplantation outcomes. Furthermore, host retinal responses shape the transplant microenvironment, influencing donor cell survival and integration. These findings highlight the need for targeted interventions to mitigate early metabolic stress and optimize PRPC transplantation strategies for retinal degenerative diseases.

The lysosomal storage disease alpha-mannosidosis (AMD) is caused by a genetic deficiency of lysosomal alpha-mannosidase, leading to the widespread presence of storage lesions in the brain and other tissues. Animal models of lysosomal diseases have demonstrated the benefit of early treatment; however, many human diagnoses occur after patients are symptomatic. We demonstrate here partial correction of the globally distributed storage lesions by infusion of a high dose of adeno-associated virus 1-feline alpha-mannosidase into the cerebrospinal fluid via the cisterna magna in the gyrencephalic AMD cat brain at different ages, corresponding with different stages of disease progression. Significant improvements in clinical parameters were observed, and partial correction was documented pre mortem by non-invasive magnetic resonance spectroscopy and diffusion tensor imaging. Post mortem analysis demonstrated that higher levels of lysosomal alpha-mannosidase activity in animals treated at 12 weeks of age did not translate into increased correction of lysosomal storage lesions throughout the brain when compared with animals treated at earlier time points. These results further demonstrate the importance of early detection and treatment of a lysosomal storage disease to successful outcomes.

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.

Widespread distribution of transduced brain cells following delivery of AAV vectors into the cerebrospinal fluid (CSF) of the cisterna magna (CM) has been demonstrated in large animal brains. In humans, intraventricular injection is preferred to intracisternal injection for CSF delivery due to the risk of brain stem injury. One study in the dog reported adverse reactions to AAV vectors expressing GFP injected into the lateral ventricle but not when injected into the CM. In contrast, AAV expressing mammalian genes in diseased animals have not triggered adverse responses since many genetic diseases also have compromised immune systems. Differences in circulation of CSF from each site could potentially affect vector spread within the brain, but a direct comparison has not been made using both a mammalian gene and immunologically normal animals. In this study we evaluated the dopamine-2-receptor (D2R) variant D2R80A, which is inactivated for intracellular signaling and has been used as a reporter gene in large animal brains. No adverse reactions to the D2R80A gene were observed from either injection route in normal dogs and both routes resulted in comparable distribution of D2R80A within the brain.

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.

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. More-over, 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.

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.

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.

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.

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 has long been thought to contribute to neurodegeneration in Parkinson's disease. The authors show that dopamine-induced neuron death in the substantia nigra is dependent on α-synuclein and coincides with increased levels of α-synuclein oligomers. The results suggest a synergistic interaction between dopamine and α-synuclein that underlies neuronal vulnerability in disease. 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.

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.