Progress in developing transgenic monkey model for Huntington's disease.

Huntington's disease (HD) is a complex neurodegenerative disorder that has no cure. Although treatments can often be given to relieve symptoms, the neuropathology associated with HD cannot be stopped or reversed. HD is characterized by degeneration of the striatum and associated pathways that leads to impairment in motor and cognitive functions as well as psychiatric disturbances. Although cell and rodent models for HD exist, longitudinal study in a transgenic HD nonhuman primate (i.e., rhesus macaque; HD monkeys) shows high similarity in its progression with human patients. Progressive brain atrophy and changes in white matter integrity examined by magnetic resonance imaging are coherent with the decline in cognitive behaviors related to corticostriatal functions and neuropathology. HD monkeys also express higher anxiety and irritability/aggression similar to human HD patients that other model systems have not yet replicated. While a comparative model approach is critical for advancing our understanding of HD pathogenesis, HD monkeys could provide a unique platform for preclinical studies and long-term assessment of translatable outcome measures. This review summarizes the progress in the development of the transgenic HD monkey model and the opportunities for advancing HD preclinical research.

Characterization of a murine model of SMA.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease, which is the leading genetic cause of mortality in children. To date no effective treatment exists for SMA. The genetic basis for SMA has been well documented as a mutation in the gene for survival of motor neuron (SMN). Because there is an understanding of which gene needs to be replaced (SMN) and where it needs to be replaced (spinal motor systems), SMA is an ideal target for gene replacement via gene therapy. While a variety of animal models for SMA exist, they are either too fulminant to realistically test most gene delivery strategies, or too mild to provide a robust read out of the therapeutic effect. The field, therefore, requires a robust model with a slower symptomatic progression. A conditional knockout of SMN in neuronal cell types, giving a phenotype of functional motor defects, weight loss and reduced life expectancy partially satisfies this need (Frugier, Tiziano et al. 2000). This Cre/LoxP mediated neuron specific model presents an attractive alternative. In the present manuscript, we characterize the functional motor deficits of the model. We observed a decline in locomotor ability, as assessed by open field testing. The finer functions of motor skills such as righting reflex and grip strength were also observed to degenerate in the SMA mice. The decline in motor function that we observed here correlates with the anatomical decline in motor neurons and motor axons presented in the literature (Ferri, Melki et al. 2004). This work adds to our understanding and knowledge base of this Cre/LoxP model and provides a basis from which functional recovery, following interventions can be assessed.

Characterization of dental pulp stem/stromal cells of Huntington monkey tooth germs.

BACKGROUND: Dental pulp stem/stromal cells (DPSCs) are categorized as adult stem cells (ASCs) that retain multipotent differentiation capabilities. DPSCs can be isolated from individuals at any age and are considered to be true personal stem cells, making DPSCs one of the potential options for stem cell therapy. However, the properties of DPSCs from individuals with an inherited genetic disorder, such as Huntington's disease (HD), have not been fully investigated. RESULTS: To examine if mutant huntingtin (htt) protein impacts DPSC properties, we have established DPSCs from tooth germ of transgenic monkeys that expressed both mutant htt and green fluorescent protein (GFP) genes (rHD/G-DPSCs), and from a monkey that expressed only the GFP gene (rG-DPSCs), which served as a control. Although mutant htt and oligomeric htt aggregates were overtly present in rHD/G-DPSCs, all rHD/G-DPSCs and rG-DPSCs shared similar characteristics, including self-renewal, multipotent differentiation capabilities, expression of stemness and differentiation markers, and cell surface antigen profile. CONCLUSIONS: Our results suggest that DPSCs from Huntington monkeys retain ASC properties. Thus DPSCs derived from individuals with genetic disorders such as HD could be a potential source of personal stem cells for therapeutic purposes.

Intramuscular administration of a VEGF zinc finger transcription factor activator (VEGF-ZFP-TF) improves functional outcomes in SOD1 rats.

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron loss leading to paralysis and death. Vascular endothelial growth factor (VEGF) has angiogenic, neurotrophic, and neuroprotective properties, and has preserved neuromuscular function and protected motor neurons in rats engineered to overexpress the human gene coding the mutated G93A form of the superoxide dismutase-1 (SOD1). We assessed the effects of intramuscular administration of a plasmid that encodes a zinc finger protein transcription factor (ZFP-TF) engineered to induce VEGF expression in the SOD1 rat model of ALS. Weekly injections of the plasmid preserved ipsilateral hindlimb grip strength and markedly improved rotarod performance in SOD1 rats compared to the vehicle-treated group. The number of motor neurons and the proportion of innervated neuromuscular junctions were similar in both groups. In conclusion, our data suggest that administration of the VEGF-ZFP-TF may be neuroprotective and has potential as a safe and practical approach for the management of motor disability in ALS.

Assessment of hippocampal adeno-associated viral vector gene delivery via frameless stereotaxis in a nonhuman primate.

BACKGROUND/AIMS: Expression of the neuropeptide galanin in hippocampal neurons reduces seizures in the kainic acid rodent model of epilepsy. In order to translate these findings into a human clinical trial, the safety and feasibility of hippocampal adeno-associated viral (AAV) vector expression must be demonstrated in a nonhuman primate model. METHODS: The Stealth Frameless Stereotactic System and Navigus Biopsy Appliance (Medtronic) were used to inject self-complementary AAV2 carrying the gene for green fluorescent protein (GFP) into monkey hippocampi. Using a single occipital trajectory per side (n = 8 trajectories), multiple injections spaced by 5 mm were delivered to each hippocampus. RESULTS: GFP was expressed in both neuronal and glial cells. Injections led to nonhomogeneous gene expression, suggesting closer spacing of injections may lead to more gene expression. Increasing injection volumes entailed a general increase in volume of expression, but there was no overlap of expression within the 5-mm injection interval. Efforts to avoid the occipital horn failed to prevent leaking of vector into the ventricle, and resulted in deviation of the trajectory at proximal points from the hippocampus. CONCLUSION: Using the occipital approach, adequate cannulation of the monkey hippocampus will require transventricular trajectories.

Monkey hybrid stem cells develop cellular features of Huntington's disease.

BACKGROUND: Pluripotent stem cells that are capable of differentiating into different cell types and develop robust hallmark cellular features are useful tools for clarifying the impact of developmental events on neurodegenerative diseases such as Huntington's disease. Additionally, a Huntington's cell model that develops robust pathological features of Huntington's disease would be valuable for drug discovery research. RESULTS: To test this hypothesis, a pluripotent Huntington's disease monkey hybrid cell line (TrES1) was established from a tetraploid Huntington's disease monkey blastocyst generated by the fusion of transgenic Huntington's monkey skin fibroblast and a wild-type non-transgenic monkey oocyte. The TrES1 developed key Huntington's disease cellular pathological features that paralleled neural development. It expressed mutant huntingtin and stem cell markers, was capable of differentiating to neural cells, and developed teratoma in severely compromised immune deficient (SCID) mice. Interestingly, the expression of mutant htt, the accumulation of oligomeric mutant htt and the formation of intranuclear inclusions paralleled neural development in vitro , and even mutant htt was ubiquitously expressed. This suggests the development of Huntington's disease cellular features is influenced by neural developmental events. CONCLUSIONS: Huntington's disease cellular features is influenced by neural developmental events. These results are the first to demonstrate that a pluripotent stem cell line is able to mimic Huntington's disease progression that parallels neural development, which could be a useful cell model for investigating the developmental impact on Huntington's disease pathogenesis.

Putative dental pulp-derived stem/stromal cells promote proliferation and differentiation of endogenous neural cells in the hippocampus of mice.

Until now, interest in dental pulp stem/stromal cell (DPSC) research has centered on mineralization and tooth repair. Beginning a new paradigm in DPSC research, we grafted undifferentiated, untreated DPSCs into the hippocampus of immune-suppressed mice. The rhesus DPSC (rDPSC) line used was established from the dental pulp of rhesus macaques and found to be similar to human bone marrow/mesenchymal stem cells, which express Nanog, Rex-1, Oct-4, and various cell surface antigens, and have multipotent differentiation capability. Implantation of rDPSCs into the hippocampus of mice stimulated proliferation of endogenous neural cells and resulted in the recruitment of pre-existing Nestin(+) neural progenitor cells (NPCs) and beta-tubulin-III(+) mature neurons to the site of the graft. Additionally, many cells born during the first 7 days after implantation proliferated, forming NPCs and neurons, and, to a lesser extent, underwent astrogliosis, forming astrocytes and microglia, by 30 days after implantation. Although the DPSC graft itself was short term, it had long-term effects by promoting growth factor signaling. Implantation of DPSCs enhanced the expression of ciliary neurotrophic factor, vascular endothelial growth factor, and fibroblast growth factor for up to 30 days after implantation. In conclusion, grafting rDPSCs promotes proliferation, cell recruitment, and maturation of endogenous stem/progenitor cells by modulating the local microenvironment. Our results suggest that DPSCs have a valuable, unique therapeutic potential, specifically as a stimulator and modulator of the local repair response in the central nervous system. DPSCs would be a preferable cell source for therapy due to the possibility of a "personalized" stem cell, avoiding the problems associated with host immune rejection. Disclosure of potential conflicts of interest is found at the end of this article.

Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery.

Gene therapy for motor neuron diseases requires efficient gene delivery to motor neurons (MNs) throughout the spinal cord and brainstem. The present study compared adeno-associated viral (AAV) vector serotypes 1, 6, 8, and 9 for spinal cord delivery in adult mice, by the intraparenchymal or intrathecal route of administration. Whereas intraparenchymal injections resulted in local transduction of the lumbar segment of the spinal cord, intrathecal injections led to a broader distribution, transducing cells along the sacral, lumbar, and lower thoracic spinal cord. Overall, AAV6 and AAV9 performed better than the other serotypes. Dramatic differences in cell-specific expression patterns could be observed when constructs bearing the chicken ß-actin (Cba) versus cytomegalovirus (CMV) promoter were compared. In summary, intrathecal delivery of AAV6 or AAV9 vectors containing the CMV promoter yielded the strongest levels of biodistribution and MN transduction in the spinal cord.

Viral vector-mediated gene transfer for CNS disease.

IMPORTANCE OF THE FIELD: Gene therapy is a promising strategy for the treatment of many neurological disorders that currently lack effective treatment. Recent improvements in vectorology and vector engineering have improved overall safety and delivery of viral vectors. AREAS COVERED IN THIS REVIEW: This review discusses the current state of viral vector development and clinical use, as well as routes of delivery, and clinical trials for neurological disorders. WHAT THE READER WILL GAIN: Viral vectors may be delivered directly or remotely to the CNS, largely depending on the nature of the disease and the tropism of the vector. Nonetheless, delivery remains one of the major limitations of successful gene transfer to the CNS. TAKE HOME MESSAGE: Although the majority of clinical trials have centered on gene replacement and neuroprotection approaches, the field is advancing in the direction of neuromodulation, gene silencing and other newer strategies.

Human multipotent stromal cells (MSCs) increase neurogenesis and decrease atrophy of the striatum in a transgenic mouse model for Huntington's disease.

BACKGROUND: Implantation of human multipotent stromal cells from bone marrow (hMSCs) into the dentate gyrus of the hippocampus of mice was previously shown to stimulate proliferation, migration and neural differentiation of endogenous neural stem cells. We hypothesized that hMSCs would be beneficial in a mouse model of Huntington disease (HD) due to these neurogenic effects. RESULTS: We implanted hMSCs into the striatum of transgenic mice (N171-82Q) that are a model for HD. The implanted hMSCs rapidly disappeared over 3 to 15 days. However, they increased proliferation and neural differentiation of endogenous neural stem cells for up to 30 days. They also increased neurotrophic signaling and decreased atrophy of the striatum in 3-month old HD mice implanted with hMSCs one month earlier. CONCLUSIONS: The results therefore suggested that neural implantation of hMSCs may be of benefit in HD but a number of parameters of dose, treatment schedule, and route of administration need to be optimized.

A conditioning lesion provides selective protection in a rat model of Amyotrophic Lateral Sclerosis.

BACKGROUND: Amyotrophic Lateral Sclerosis (ALS) is neurodegenerative disease characterized by muscle weakness and atrophy due to progressive motoneuron loss. The death of motoneuron is preceded by the failure of neuromuscular junctions (NMJs) and axonal retraction. Thus, to develop an effective ALS therapy you must simultaneously preserve motoneuron somas, motor axons and NMJs. A conditioning lesion has the potential to accomplish this since it has been shown to enhance neuronal survival and recovery from trauma in a variety of contexts. METHODOLOGY/PRINCIPAL FINDINGS: To test the effects of a conditioning lesion in a model of familial ALS we administered a tibial nerve crush injury to presymptomatic fALS(G93A) rats. We examined its effects on motor function, motoneuron somas, motor axons, and NMJs. Our experiments revealed a novel paradigm for the conditioning lesion effect. Specifically we found that the motor functional decline in fALS(G93A) rats that received a conditioning lesion was delayed and less severe. These improvements in motor function corresponded to greater motoneuron survival, reduced motor axonopathy, and enhanced NMJ maintenance at disease end-stage. Furthermore, the increased NMJ maintenance was selective for muscle compartments innervated by the most resilient (slow) motoneuron subtypes, but was absent in muscle compartments innervated by the most vulnerable (fast fatigable) motoneuron subtypes. CONCLUSIONS/SIGNIFICANCE: These findings support the development of strategies aimed at mimicking the conditioning lesion effect to treat ALS as well as underlined the importance of considering the heterogeneity of motoneuron sub-types when evaluating prospective ALS therapeutics.