Marina E. Emborg - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To explore if oral administration of pioglitazone can be used as neuroprotective therapy in Parkinson's disease. The identification of therapies aimed to slow or stop the progressive dopaminergic neuronal loss has become a priority for Parkinson's disease. Pioglitazone is an FDA approved, antidiabetic therapy found to have anti-inflammatory and anti-oxidant properties. Our results suggest that pioglitazone have neuroprotective effects against MPTP-induced behavioral and anatomical damage. We are currently working with the M. J. Fox Foundation towards its clinical translation and follow up studies including comparisons with selctive PPARgamma inhibitors. This research used Animal Services, CPI, Assay Services, and Pathology Services. Publication pending.

Aging; Animals; Autopsy; Brain; CPI; CRISP; Clinical Trials; Clinical Trials, Unspecified; Computer Retrieval of Information on Scientific Projects Database; Data; Encephalon; Encephalons; Environmental Toxin; Funding; GDNF; GDNF gene; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genetic Intervention; Grant; Idiopathic Parkinson Disease; Image; Injection of therapeutic agent; Injections; Institution; Intervention, Genetic; Investigators; Lentiviral Vector; Lentivirus Vector; Lewy Body Parkinson Disease; Macaca mulatta; Mammals, Primates; Methods; Molecular Biology, Gene Therapy; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsonian; Parkinsonian Condition; Parkinsonian Diseases; Parkinsonian Disorders; Parkinsonian Syndrome; Parkinsonism; Parkinsons disease; Pathology; Patients; Primary Parkinsonism; Primates; Publications; Recovery of Function; Reporting; Research; Research Personnel; Research Resources; Researchers; Resources; Rhesus; Rhesus Macaque; Rhesus Monkey; Risk Factors; Safety; Scientific Publication; Senescence; Services; Source; Therapy, DNA; Toxic Environmental Agents; Toxic Environmental Substances; United States National Institutes of Health; Work; aged; base; behavior test; behavioral test; clinical investigation; cyclopropapyrroloindole; environmental toxicant; functional recovery; gene therapy; genetic therapy; imaging; male; necropsy; neurotoxic; postmortem; prevent; preventing; senescent

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To assess neuroprotective strategies for Parkinson's disease using gene therapy to deliver the trophic factor GDNF [unreadable] or the antiapoptotic molecule Bcl-2.[unreadable] [unreadable] Aging and environmental toxins have been identified as risk factors for Parkinson's disease. In order to assess the [unreadable] potential for functional recovery induced by GDNF of a diseased aged primate brain, old male rhesus monkeys [unreadable] presenting a parkinsonian syndrome received intracerebral injections of lentiviral vectors encoding for GDNF or a [unreadable] control treatment. Our results using behavioral tests, imaging and postmortem brain analysis indicate that the [unreadable] aged primate brain exposed to a neurotoxic insult is responsive to GDNF trophic stimulation locally delivered by [unreadable] lentiviral vectors. The value of this research is directly applied to clinical trials to prevent progression of [unreadable] Parkinsonism in patients and this work also provides data on the safety of the method of delivery using lentiviral vectors. [unreadable] This research used WNPRC Animal Services and Aging Resources.[unreadable] [unreadable] Funding note: NIH R01NS040578-01 funding ended during this reporting period, and publications are linked here based on [unreadable] project results.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To explore if oral administration of pioglitazone can be used as neuroprotective therapy in Parkinson's disease.[unreadable] [unreadable] The identification of therapies aimed to slow or stop the progressive dopaminergic neuronal loss has become a priority for [unreadable] Parkinson's disease. Pioglitazone is an FDA approved, antidiabetic therapy found to have anti-inflammatory and anti-[unreadable] oxidant properties. During the last year, we completed the behavioral data collection for this study. We are now performing [unreadable] histological processing to assess if behavioral improvements are associated to nigrostriatal dopaminergic preservation. This [unreadable] research use WNPRC Animal Services and Assay Services.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To explore the use of human neural progenitor stem cells (HNPSCs) genetically modified to deliver the trophic [unreadable] factor GDNF as a therapy for Parkinson's disease and compare its effects to in vivo delivery and unmodified HNPSCs.[unreadable] [unreadable] HNPSCs have been proposed as a source of cells for ex vivo gene therapy. We performed a pilot study using HNPSCs [unreadable] genetically modified to deliver the trophic factor GDNF to assess viability and safety in the monkey brain. Our results [unreadable] showed HNPSCs survival and production of GDNF in the monkey brain via genetically modified HNPSCs and restoration/[unreadable] protection of dopaminergic fibers in target areas. Based on this data, we started a second project with extensive behavioral [unreadable] testing comparing the effects of 1) lentiviral vectors delivering GDNF vs. 2) HNPSCs delivering GDNF vs. 3) HNPSCs alone [unreadable] vs. 4) control media. The impact of this study is that it assesses, side-by-side, in vivo vs. ex vivo, gene therapy for the [unreadable] delivery of trophic factors. It also assesses whether HNPSCs by themselves can affect neurodegeneration. These are key [unreadable] questions for the application of these new biological therapies for the treatment of Parkinson's disease and other [unreadable] neurodegenerative disorders. This Research used WNPRC Animal Services and Stem Cell Resources.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To design a successful convection-enhanced infusion protocol for human intra-putamenal infusions of solutions carrying drugs. Success means, in this context, covering the putamen and/or caudate nucleus, with minimal coverage of surrounding structures. The goal of this particular study is to develop such a protocol in rhesus monkeys, and to offer a prediction for the results of a similar protocol in humans. We will estimate the likelihood of achieving a given coverage using this protocol. We are also specifically comparing current proposed methods of brain delivery. We have established all the infusion methods and are performing routing infusions and gels in nonhuman primates using intraoperative imaging. This research used WNPRC Assay Services and CPI. PUBLICATION: J Kimmelman, A John London, B Ravina, T Ramsay, M Bernstein, A Fine, FW Stahnisch, ME Emborg, MD, Launching Invasive, First-in-Human Trials Against Parkinson's Disease: Ethical Considerations. Movement Disorders. Vol. 24, No. 13, 2009, pp. 1893[unreadable]1901. Another publication has been submitted.

CRISP; China; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Idiopathic Parkinson Disease; Institution; International; Investigators; Lewy Body Parkinson Disease; Mainland China; NIH; National Institutes of Health; National Institutes of Health (U.S.); Paralysis Agitans; Parkinson; Parkinson Disease; Parkinson's; Parkinson's disease; Parkinsons disease; Primary Parkinsonism; Programs (PT); Programs [Publication Type]; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Study models; United States National Institutes of Health; non-human primate; nonhuman primate; pre-clinical; preclinical; programs

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To develop tests to access autonomic dysfunction induced by systemic administration of the neurotoxin 6 hydroxydopamine. PD is associated with loss of autonomic innervation. The heart is one key organ affected. As such, PD patients present heart dysautonomia symptoms like autostatic hypotension or arrhythmias. Currently, there are not well characterized nonhuman primate models of these problems. Through an interdisciplinary team consisting of PD experts, cardiologists and imaging experts, we are developing a battery of tests to access autonomic dysfunction induced by systemic administration of the neurotoxin 6 hydroxydopamine. Preliminary results from this project suggest that intravenous administration of 6-OHDA induces neurodegeneration of catecholaminergic neurons innervating the heart and can be used to model heart autonomic dysfunction in PD. This research uses WNPRC Assay Services and CPI. A publication is in preparation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To derive dopamineric neurons from induced pluripotent stem cells (iPS cells) to treat Parkinson's Disease in macaque models of PD. This project has just begun and progress is forthcoming. This research used WNPRC Assay Services and CPI.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To assess neuroprotective properties of the PPAR GAMMA AGONIST LY554862 in treating Parkinson's disease. Our laboratory has recently shown that chronic oral administration of pioglitazone induces modest neuroprotection against MPTP intoxication in a primate model. Based on this information we continued to investigate PPAR-[unreadable] activation as a possible therapeutic target for PD. This research used WNPRC Assay Services and is precursor work to nonhuman primate studies. A publication is in preparation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: To assess penetration of pioglitazone into the cerebral spinal fluid (CSF). This project is completed: Pioglitazone, a thiazoledionedione currently FDA approved as an antidiabetic treatment, has been proposed as a neuroprotective therapy for neurological disorders. We previously demonstrated in nonhuman primates that daily oral pioglitazone dosing protects against the functional and anatomical effects of the parkinsogenic toxin MPTP. In this study we continued our clinical translation analysis of this compound by analyzing the ability of different doses of pioglitazone to penetrate the CSF. Five adult female rhesus monkeys (5-6 kg) received once daily oral administration of placebo, 2.5 mg/kg, 5.0 mg/kg mg or 7.5 mg /kg of pioglitazone for 7 days. There was a lapse of 1 week between a change in dose and plasma and CSF sampling to allow for steady state. Plasma and CSF samples were obtained at each baseline and 5 hours after the last dose of pioglitazone. Levels of pioglitazone were evaluated by HPLC methods. In plasma, comparison between pioglitazone levels showed, as expected, that administration of 5 mg/kg induced higher levels than 2.5 mg/kg. Yet, in four of the five monkeys 7.5 mg/kg dosing did not result in consistently higher increases of pioglitazone plasma levels compared to 5 mg/kg. These data suggest increased drug elimination with 7.5 mg/kg. CSF levels of pioglitazone compared to plasma showed more individual variability. A dose of 2.5 mg/kg induced non-detectable levels in two of the five animals. Both, 5 and 7.5 mg/kg had consistent measurable levels in CSF. Four of the five monkeys had higher CSF levels of pioglitazone after receiving 5 mg/kg compared to 7.5 mg/kg. The results confirmed the ability of pioglitazone to penetrate CSF and suggest that in rhesus monkeys 5 mg/kg was an efficient dose to consistently induce detectable higher levels of pioglitazone in plasma and CSF. This research used CPI and Assay Services. Publication is pending.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objective: This project will assess the feasibility of producing iPS derived Dopaminergic autologous for restoration of Parkinson's disease. This project, funded by a UW-ICTR Type I grant, has just begun and progress is forthcoming. This research used WNPRC Assay Services and CPI.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To improve the methods of intracerebral delivery of trophic factor gene therapy by comparing different viral vectors systems and using real time MRI imaging for intracerebral navigation and monitoring of infusions. This ICTR Type I project recently began and progress is forthcoming.

DESCRIPTION (provided by applicant): Nonmotor symptoms of Parkinson's disease (PD), such as cardiac autonomic dysfunction (dysautonomia), greatly affect patients' quality of life. They are frequently unrecognized as PD symptoms, many times undiagnosed and overall poorly managed, as they do not respond to typical anti-parkinsonian therapies. Progress towards improving treatments and biomarkers have been hampered by the lack of animal models. We have developed a nonhuman primate (NHP) model of cardiac dysautonomia by intravenous delivery of the neurotoxin 6-OHDA and developed a battery of tests to characterize the model. We have also demonstrated that oral dosing of the peroxisome proliferator activator receptor gamma (PPAR?) agonist pioglitazone modulates inflammation and oxidative stress, inducing neuroprotection in a NHP model of PD with typical nigrostriatal degeneration. Based on these studies we hypothesize that pioglitazone can be neuroprotective in the NHP model of cardiac dysautonomia and that the therapeutic effects are mediated via a reduction in inflammation and oxidative stress. To evaluate this hypothesis we propose: Specific Aim 1: To evaluate whether chronic oral dosing of the PPAR? agonist pioglitazone prevents 6-OHDA-induced peripheral catecholaminergic neurodegeneration and downregulates mechanisms of inflammation and oxidative stress in a NHP model of cardiac dysautonomia. We will use state-of-the-art PET imaging and radioligands to evaluate in vivo cardiac markers of catecholaminergic innervation ([C11]MHED), inflammation ([C11]PK11195) and oxidative stress ([61/64Cu]ATSM) before and after treatments. We will correlate the imaging data with clinical measures (ECG, blood pressure, activity), circulating metabolites (e.g.: catecholamines, cytokines and PGC?-1) and morphological data (e.g.: regional myocardial quantification of TH, HLA-DR, nitrotyrosine and alpha synuclein expression), to analyze how the different measures relate to catecholaminergic loss and preservation. These technologies will allow us to evaluate mechanisms of neurodegeneration and neuroprotection while validating biomarkers for clinical application.

DESCRIPTION (provided by applicant): Nonhuman primates (NHP) offer the most appropriate experimental model for many areas of human biomedical research, particularly in reproductive medicine, immunology and transplantation, and neurological disease. Furthermore, monkey induced pluripotent stem cells (iPSCs) are envisioned as a valuable renewable resource of cells for preclinical testing of personalized regenerative medicine approaches. Based on initial advances in common marmoset assisted reproductive technologies (ARTs) developed at the Wisconsin National Primate Research Center (WNPRC), the production of transgenic common marmosets expressing green fluorescent protein, including the demonstration of germline transmission, has been reported. Marmoset monkeys present several advantages for transgenic approaches, including the ability to routinely carry multiple offspring (rapidly increasing cohort size), facile reproductive management, and a shorter lifespan, which facilitates the study of age-related diseases such as diabetes, arthritis and Parkinson's disease (PD). In regards to PD, identification of specific alleles of the leucine rich repeat kinase 2 (LRRK2) in familial and sporadic cases of PD supports the development of a NHP model expressing these variants, as proof-of-principle for the contribution of human alleles to disease pathophysiology. We propose to advance the development of animal disease models for stem cell-based therapeutic applications with two Specific Aims: Specific Aim 1. To optimize reprogramming of iPSCs from common marmoset fibroblasts, to use transgene expression and genomic editing to define the effect of alleles associated with PD on in vitro neural differentiation in isogenic iPSC lines, and to use genomic editing tools to define the feasibility, efficiency and accuracy of genomic editing in IVF-derived common marmoset embryos. Specific Aim 2. To produce transgenic marmoset monkeys carrying wild-type and PD-associated human LRRK2 alleles and evaluate a proof-of-principle cohort for PD pathophysiology. The long-term goal of this work is to provide investigators with marmoset iPSC lines, and genetically modified common marmosets as platforms for translational research, particularly in the treatment of diseases for which primate species are the most suitable models. Animal experiments will be performed at WNPRC, which is one of the few facilities in North America housing an experimental common marmoset colony, and the only Center where marmosets, primate embryology, and cutting edge neurological translational models are actively being used to test therapies for human disease. The proposed generation and analysis of transgenic monkeys and associated modified iPSCs will provide a platform to create disease specific models and cells, and develop tests of therapeutic approaches, including personalized medicine using engraftment of iPSCs.

Project Summary: CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode- generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the brain/neuron represents a major hurdle due to its dense structure and the blood?brain barrier (BBB). The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein (RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our approach, in a demonstration, by targeting the amyloid precursor protein (APP) ? relevant to Alzheimer's disease (AD) in healthy mice and monkey models. During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%), versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus macaques. Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide range of human diseases, including debilitating neurodegenerative diseases for which there are no cures.

ABSTRACT Frontotemporal dementia (FTD) with or without parkinsonism, is an Alzheimer?s disease related dementia (ADRD) that has been linked to sporadic and familial mutations in the MAPT gene, including the autosomal dominant R406W missense point mutation. Patients with FTD MAPT R406W present progressive memory loss, and late-onset of parkinsonism, personality changes, and/or language deficits. Typical pathological findings include abnormal intraneuronal accumulation of phosphorylated tau filaments (tauopathy), frontotemporal atrophy, neuronal loss, and gliosis. At the Wisconsin National Primate Research Center (WNPRC) in collaboration with Baylor College of Medicine, a rhesus monkey was identified as a carrier of the MAPT R406W mutation, identical to human patients. Progress in the development of biomarkers and treatments for FTD and other dementias has been hampered by the lack of faithful animal models of the disorder. Nonhuman primates (NHPs), in particular rhesus macaques, are an ideal species to study dementias and related neurodegeneration, due to the complexity of their behavior and neuroanatomy. In the R61 phase of this application we propose to phenotype the MAPT R406 carriers of this family and to identify additional MAPT R406W mutation carriers. The rhesus will be evaluated with a battery of age-appropriate behavioral tests (cognitive, mood and motor), MRI (for neuroanatomical and volumetric analysis) and F18-MK6240 PET (to visualize tau accumulation) and compared to age- and sex- matched controls. These animals will be the founders of a breeding colony of MAPT R406W carriers. During the R33 phase we will aim to create a MAPT R406W rhesus colony resource, by breeding the mutated rhesus and generating induced pluripotent stem cells (iPSCs) from the carriers? fibroblasts. The offspring will be genotyped and neurobehaviorally evaluated. The iPSCs will be a platform to study the effects of ADRD alleles on in vitro neural differentiation. The overarching goal of this project is to generate an NHP resource for the ADRD research community. A well characterized NHP model of genetic FTD, supported by in vivo behavioral and imaging outcome measures and associated iPSC lines, will help understand the pathological mechanisms of the disease, which could lead to the identification of biomarkers and therapeutic targets. We have assembled a strong team of investigators across different disciplines focused on translational neuroscience and neurodegenerative diseases, human and nonhuman primate genetics, stem cells, and, most importantly, experts devoted to nonhuman primate breeding and care.