Ole Isacson - US grants

In the last decade, McLean Hospital, affiliated with the Harvard Medical School, has been one of the world's most productive sites for neuroscience research. For the most part, laboratory research at McLean is done at the Mailman Research Center (MRC). Constructed in 1945, the MRC currently provides laboratory support and space for 24 investigators, 26 post-doctoral students, 7 graduate students and additional undergraduate and graduate students during the summer. At present, neuroscience research is performed in the Biological Research Laboratory facility. Researchers engage in programs of related projects studying the cytostructural elements essential for normal neuronal growth and development and signal transduction mechanisms mediating neuronal plasticity in development and aging. However, due to its antiquated support facilities, coupled with significant building code modifications and requirements, the facility no longer provides adequate laboratory space. Infrastructure utilities, including HVAC, electrical, plumbing, water filtration, and acid neutralization systems are in desperate need of replacement. High technology instrumentation and techniques required to conduct many of the extremely complex analytical tasks cannot be sustained in the presently configured space. This award will provide partial funding for the replacement of the Biological Research Laboratory. Upon completion, modernized research space will be available to support investigations in neurodevelopmental, molecular and structural neuroscience. The research programs will benefit by an improved infrastructure that will be capable of supporting advanced techniques and by furnishing a clean environment required for operating the instrumentation utilizing these techniques. This renovation project will provide the neuroscience program with a laboratory environment designed to complement modern research procedures required to meet high technology research protocols.

technology /technique development; computed axial tomography; positron emission tomography; magnetic resonance imaging; drug screening /evaluation; Primates; Mammalia; biological products; nervous system; psychology; behavioral /social science research tag;

technology /technique development; computed axial tomography; positron emission tomography; magnetic resonance imaging; drug screening /evaluation; Primates; Mammalia; biological products; nervous system; psychology; behavioral /social science research tag;

Huntington's disease (HD) and Parkinson's disease (PD) are neurodegenerative diseases. The neuropathological, neurochemical and behavioral hallmarks of HD can be replicated in the non-human primate by stereotaxic excitotoxic lesions of the caudate-putamen (Isacson et al. 1989). In a MPTP chronic lesion model of PD, we are currently pharmacologically testing drugs for therapeutic effects in combination with transplantation of fetal neurons. Behavioral testing of the HD and PD models is therefore of utmost importance for developing a neurobiological basis for new treatments of neurodegenerative disease. The HD model determines the neurological basis for basal ganglia dependent dyskinesia. This year we will continue our lesion and transplantations studies in combination with PET and MRI studies to develop a therapy against the symptoms seen in HD and PD.

Huntington's disease (HD) and Parkinson's disease (PD) are neurodegenerative diseases. The neuropathological, neurochemical and behavioral hallmarks of HD can be replicated in the non-human primate by stereotaxic excitotoxic lesions of the caudate-putamen (Isacson et al. 1989). In a MPTP chronic lesion model of PD, we are currently pharmacologically testing drugs for therapeutic effects in combination with transplantation of fetal neurons. Behavioral testing of the HD and PD models is therefore of utmost importance for developing a neurobiological basis for new treatments of neurodegenerative disease. The HD model determines the neurological basis for basal ganglia dependent dyskinesia. This year we will continue our lesion and transplantations studies in combination with PET and MRI studies to develop a therapy against the symptoms seen in HD and PD.

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. This project focuses on the development of novel therapeutic approaches for treatment of Parkinson's disease.

xenotransplantation; Huntington's disease; Primates; animal colony; genetically modified animals;

Parkinson's disease; nervous system regeneration; Primates; disease /disorder model; animal colony; neurosurgery; positron emission tomography; magnetic resonance imaging;

embryonic stem cell; Parkinson's disease; disease /disorder model; Primates; animal colony;

CRISP; Computer Retrieval of Information on Scientific Projects Database; Disease; Disorder; Funding; Grant; Huntington Chorea; Huntington Disease; Huntington's; Huntington's Disease; Huntington's Disease Pathway; Huntingtons Disease; Institution; Investigators; NIH; National Institutes of Health; National Institutes of Health (U.S.); Progressive Chorea, Hereditary, Chronic (Huntington); Research; Research Personnel; Research Resources; Researchers; Resources; Source; United States National Institutes of Health; disease/disorder; new therapeutics; next generation therapeutics; novel therapeutics

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. The use of primate stem cells in Parkinson's disease models.

DESCRIPTION (provided by applicant): We have developed a plan for a NINDS research organization/consortium to develop, characterize and study iPS cell lines for Parkinson's disease (PD). The consortium is organized with several top research teams with both clinical and basic science expertise for PD. The overall goal of this consortium of multiple- Pis (see leadership plan) is to organize the rapidly expanding iPS stem cell field into useful scientific applications and evaluations for clinically oriented PD research. The organizational structure includes a centralized site to generate the iPS cell lines to distribute these to the research teams, and a secure website (www.PDiPS.org). This central facility (Core A) is available at McLean/Harvard and is led by Drs. Isacson, Feng (SUNY) and Jaenisch at MIT. This Core function is also linked with a clinical core (Core B, in close collaboration with Proj 1 - Dr Wszolek) for patient material and analysis, headed by Dr Karen Marder at Columbia University. These types of activities are recorded and coordinated with databases (pd.doc and dbGaP) for known PD mutations and clinical histories, with PD patient fibroblast and blood cell banking made at Coriell cell repository, which also deposits the PD iPS cell lines generated by Core A. With the stem cell iPS cell lines basic characterization fulfilled by Core A and B, these are distributed to the outstanding investigative teams. Project 1: Research team 1 involves both a genetic and genomic analysis team, which also provides insight into new mutations, both at the familial and genetic association level as risk factors. This team is led by Zbigniew Wszolek as a clinical director and Matt Farrer for genetic studies (as Pis). Their recent analysis and genetic technology will benefit the continued work. In Project 2 there is a molecular characterization of the various cell lines. This team is headed by Dr. Ted Dawson, who will be working with his collaborators to have iPS line analyses from relevant PD patient material. Dr. Dawson also has a current collaboration through Project 1 with Dr. Wszolek, and they are already moving towards setting up these cell lines for molecular characterization. In Project 3 a U. Penn research team led by Drs. Lee and Trojanowski will carry out investigation into PD protein processing, degradation and autophagy analysis of PD patients'iPS cells. Their outstanding expertise in this field will allow a state of the art analysis of changes relevant to such cell biological processes. Project 4 will be an essential in vitro physiological and toxicological analysis, and critical in vivo bioassays for long-term evaluation of PD iPS cell derived DA neurons. This project team is headed by Drs. Isacson and Surmeier, who by collaboration will perform both in vitro and in vivo assays for any altered electrophysiologic and neurotoxic response of the PD iPS derived cells;providing neurobiology of disease information for neuronal/neuritic growth, vulnerability and calcium buffering capacities. Finally, Project 5, headed by Dr Serge Przedborski, will examine the highly relevant PD molecular mechanisms and pathology of mitochondria in differentiated PD iPS neurons. Overall, this PD iPS consortium will have an exceptional research capacity, which is currently ready to deploy by the independent strengths and expertise of the individual research teams. The GO grant will enable a consortium that quickly can provide a national resource for iPS cell lines relevant to PD, to be characterized in a meaningful way by the assembled collaborators, and made available as useful reagents to the large community of present and future PD researchers. PUBLIC HEALTH RELEVANCE: We use stem cells generated from Parkinson's disease patients'skin to produce a specific population of purified neurons that retain the authentic genetic risks for the disease. We will determine if these neurons are more susceptible to Parkinson's disease-like degeneration than neurons generated from healthy people's skin. Our data will aim to establish these neurons as a powerful new approach to understanding the complex disease process and a future tool for drug discovery.

Dr. Isacson will continue the role of leader of the PD IPS consortium that he began in 2009, when the ARRA GO grants (RC2s) funded the PD, ALS and HD consortia. His experience and leadership will strengthen the ability of the PD IPS consortium to achieve the goals set out in the RFA for this U24 grant. This PD iPS Consortium will now be organized into three Project Cores, a) Clinical and Genetic Core (Z. Wszoiek, core PI), b) Differentiation and Reporter Core (L. Studer and D. Krainc, core Pis), c) Cell Function and Pathophysiology Core (T. Dawson, core PI). These three cores will be supported by the research resource cores: Administration (O. Isacson, core PI) and the Reprogramming contract research organizations (CROs) (Harvard Stem Cell Institute and New York Stem Cell Foundation) (see letters of collaboration). (For overview of organization, see Fig. 1, Introduction). The cores include research studies at eight separate institutions (McLean/Harvard, Columbia, Mayo-Jacksonville, Memorial Sloan Keftering, Massachusetts General Hospital/Harvard, Johns Hopkins University, University of Pennsylvania and Northwestern University. In order to foster the synergies and lines of communication necessary for the successful achievement of the scientific and technical challenges to achieving the consortium goals and timelines, the Administrative Core will provide the organization and support for the administration of this U24 grant. The Consortium Director, Prof Ole Isacson, is responsible for ensuring that coordination and core functions are appropriately managed. Dr. Isacson's expertise in stem cell use exceeds a decade, and he has productively directed NIH-funded Centers and Programs, including the recent RC2 GO grant that currently funds this PD IPS Consortium. Because the interactions between the members of this Consortium are essential to the accomplishment of the Consortium goals, Dr. Isacson will hold a conference call every 2 months between all of the Core Pis and the members of the Executive Science Committee (see Fig. 2, Introduction). He will promote the continuous cooperation between investigators, and the sending of cells for deposit to Coriell. On the administrative day-to-day detail, Ms. Sandra Pohlman has been the chief administrator of the Center for Neuroregeneration Research at McLean Hospital/Harvard Medical School for over 10 years. Her role is to manage resources and funds, and ensure proper accounting and spending according to the research plan and research cost categories. Her involvement as an Administrator is exemplified by the cooperative agreements involved. She has carried out such activities previously for large Center grants and the current PD IPS consortium. Ms. Pohlman serves as the coordinator for communication between the investigators on this proposal, their grants administrators and sponsored research offices for preparation and administration of budgets and research reports.

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DESCRIPTION (provided by applicant): As a continuation of the NIH-funded GO-grant work on PD IPS cell genetic lines our 9 research teams are now proposing a U24 grant consortium, according to the new RFA NS-11-011. The tool generation for public and scientific use of Parkinson's disease (PD) patient derived IPS cell lines will continue and new lines with mutations, isogenic genetically repaired iPS lines, and inserts of reporter systems for studying such lines will be established by the funds provided by the U grant. The PD iPS consortium director, Dr. Ole Isacson, and the Executive Science Committee consisting of Core leaders, the IPS resource representative and an NIH representative will carry out the work through the activity of: the (1) Clinical and Genetic Core led by Zbigniew Wszoiek that will provide necessary patient fibroblast lines, including GBA, FTD and additional LRRK2 and alpha synuclein mutations, along with a mRNA sequencing and expression laboratory. These lines will be reprogrammed according to the priorities set by the RFA at (2) contracting research organizations (CROs) at the Harvard Stem Cell Institute with their Directors Drs. Rossi and Cowan at the iPS Core facility, and the New York Stem Cell Foundation (NYSCF) with the Director Dr. Scott Noggle, providing the new lines in a timely manner. The (3) Reprogramming, Differentiation Reporter (and Isogenic Repair) Core is led by Lorenz Studer and Dimitri Krainc, who will provide a robust differentiation protocol for dopamine neurons and genetically repair PINK1 and LRRK2 G2019S into their isogenic forms and also add fluorescent reporter genes to these PD iPS cells. These tools and reagents will enable the assignments of the (4) Cell Function and Pathophysiology Core led by Ted Dawson, who will direct the teamwork around the phenotypes and etiobiology discovery of PD. The value provided by this U24 grant proposal is realized by the (a) new iPS lines provided, and (b) the engineered PD IPS lines as exceptionally useful human cellular tools for understanding PD, as well as (c) collaborative and shared use of cells lines for drug discovery.

DESCRIPTION (provided by applicant): High throughput sequencing of the genome-wide transcriptome will generate many new hypotheses regarding the genetic interactions underlying neurodegenerative mechanisms. We expect that marker genes of biologically relevant pathways are needed to efficiently interrogate the large sets of raw data produced by the experiments. The goal of this new R21 proposal is to help validate the neuronal transcriptome as a tool for the study of Parkinson's disease. Cells from well-defined familial cases of Parkinson's disease, although rare, provide an excellent starting point for such analyses. We and others have shown that cells from Parkinson's disease patients carrying LRRK2 or PINK1 mutations demonstrate mitochondrial deficits. Importantly, the molecular mechanisms that connect LRRK2 or PINK1 mutations to the mitochondrial deficits remain unclear. Using an innovative approach to interpreting the neuronal transcriptome, we aim to generate new hypotheses regarding the molecular mechanisms of LRRK2 or PINK1 associated mitochondrial deficits. We will link the functional mitochondrial deficits of human neurons carrying Parkinson's disease associated LRRK2 and PINK1 mutations that we have observed to the RNA sequences expressed by the neurons. By combining high- throughput transcriptome sequencing with new quantitative PCR arrays, we propose to determine the expression level and sequence signatures of mitochondrial DNA (SA1) and nuclear DNA (SA2) encoded genes that mark the mitochondrial deficits of patient-derived neurons. Multiple clones of age-matched, sibling and isogenic control iPSCs will be used to minimize the influence of genomic variation across neuronal samples. In a first step towards our goal, RNA molecules from induced pluripotent stem cell (iPSC)-derived neurons carrying LRRK2, PINK1 mutations and showing mitochondrial deficits or healthy subjects were sequenced. Preliminary analyses of the RNAseq data confirmed the presence of the LRRK2 and PINK1 mutations in the neuronal transcriptome. Furthermore, our analysis suggests that the functional mitochondrial deficits are associated with aberrant processing of mitochondrial DNA-encoded transcripts. These data from human neurons can be used to establish a reasonable and coherent framework of cell biological responses to interpret a patient's transcriptome. Our results will be instructive and critical to future attempts to effectively analyze human cell biological phenotypes with the multiple underlying genetic interactions of sporadic forms of Parkinson's disease.

Synapses and axons are targets of inflammation-induced neurodegeneration. Synaptic dysfunction and loss usually precedes degeneration of the neurons, and are early features of several neurodegenerative diseases including Lewy body disease dementia. Our preliminary data show that the complement system is activated after elevation of brain glycosphingolipids, which are involved in the pathophysiology of Lewy body dementia and related disorders. The in vivo experiments outlined in this proposal determine complement activation, the immune system and synaptic neurodegeneration in dementia of the Lewy body type. In addition, in these preclinical animal models, the cognitive effects of inhibiting the complement pathway will be determined. We specifically hypothesize that (1) C1q and the complement pathway is involved in synaptic dysfunction and loss in experimental Lewy body dementia models in which synaptic degeneration and inflammatory response are key pre-degenerative features, and (2) novel stabilized, long-acting antisense oligonucleotides can effectively reduce C1qA and C3 in experimental Lewy body dementia models. These experiments will address whether C1q and C3 are involved in the degeneration of cortical and hippocampal neurons using (a) global brain human Thy1 ?-synuclein overexpression, (b) regional AAV human ?-synuclein expression, and (c) systemic and brain glycosphingolipid-induced ?-synucleinopathy and inflammation using GbaD409V knockin mice. Cognitive behavioral assays include novel object recognition tasks and Y-maze. By peripheral and central knockout in experimental Lewy body dementia models these experiments are also designed to distinguish between the local role of C1q and C3 in eliminating synapses in the brain (by brain antisense oligonucleotide targeting) and the potentially needed phagocytic function and systemic influence (by transgenic knockout). Investigating synaptic loss mediated by modulators of the complement pathway is a paradigm for understanding cortical and hippocampal neuron degeneration in models of Lewy body and related dementias. The findings will impact the understanding and therapeutic options to potentially control complement-mediated elimination of synapses and degeneration of neurons in Lewy body dementia.

Abstract: This is a revised application, modified to fully respond to reviewers? comments and FDA interactions since the application (FDA pre-IND meeting held on Sept. 25, 2018). The proposed work will complete IND-enabling studies to progress cell replacement paradigms into the clinic using induced pluripotent stem cell (iPSC)- derived dopamine (DA) neurons, and a first-in-man clinical trial for autologous transplantation in Parkinson?s disease (PD). Cell replacement therapy with midbrain dopamine (mDA) neurons provides cellular and synaptic repair in the parkinsonian brain, and addresses both the motor symptoms of PD as well as levodopa-induced dyskinesias. Our previous fetal cell transplantation work shows that in PD patients transplanted mDA neurons remain healthy and can provide remarkable therapeutic benefit for decades. While fetal cell transplantations are not scalable for a larger patient population and require immunosuppression, iPSCs are a promising alternative cell source. iPSCs generated from PD patients can be differentiated into midbrain dopaminergic cells, frozen and used for autologous transplantation. The NINDS CREATE Bio Development Track U01 proposal over 5 years consists of milestones within four Specific Aims, that includes a Phase I clinical trial in human patients with PD. In Specific Aim 1 we will transfer the remaining mDA neuron product quality control assays for qualification in the cGMP facility, perform FDA- guided quality control of excipients for the clinical product, and produce mDA neurons to be used in IND- enabling studies. In Specific Aim 2, definitive IND-enabling studies will be performed to test the safety (tumorigenicity and biodistribution) and efficacy of human iPSC-derived frozen-thawed mDA neurons in rodents, as well as testing of the planned clinical delivery device in non-human primates. Specific Aim 3 will include IND package preparation and filing for an Investigator-initiated Phase I clinical trial, recruitment of patients with PD and generation of autologous cGMP iPSCs and mDA neurons as well as release criteria testing of the cryopreserved clinical product. Finally, Specific Aim 4 is a first-in-human clinical Phase I interventional, open-label clinical trial in 6 patients with sporadic PD, to test the safety and efficacy of autologous transplantation of frozen-thawed mDA neurons. This highly innovative autologous CMC iPS cell technology U01 proposal for cell replacement clinical trials in PD patients provides a necessary step and exploration for the development of successful cell therapy for PD and several neurological disorders.