Mark Tuszynski - US grants

Gene modification may benefit the therapy of human neurodegenerative conditions such as Alzheimer's disease (AD) and Parkinson's disease (PD). Over the last 10 years, we have demonstrated that ex vivo NGF gene transfer to the primate brain can prevent cholinergic neuronal death, and reverse spontaneous cholinergic neuronal decline with aging. These findings have led to the first clinical trial of gene therapy to treat an age-related neurodegenerative disorder. While our ex vivo clinical trial of NGF gene delivery will test the hypothesis that NGF delivery to cholinergic neurons may delay progression in AD, ex vivo gene delivery is generally a cumbersome and expensive approach for gene therapy in the CNS. In vivo gene therapy, wherein vectors capable of infecting non-dividing cells are directly injected into the brain, offers a simpler and more realistic approach to gene delivery in large numbers of people. Substantial advances in the field of in vivo vectorology have been made over the past 5 years, with recent reports of sustained and safe gene expression in both rodent and primate models of neurological disease. Thus, this project will determine whether in vivo gene therapy can delivery therapeutic molecules to the adult primate brain in a safe and effective manner to prevent neuronal degeneration and ameliorate cognitive decline. Further, we will characterize effects of aging on cognition to generate novel information regarding mechanisms of neural dysfunction with primate aging, and the sensitivity of this dysfunction to growth factor gene delivery. Finally, we aim to develop a primate in vivo brain model of amyloid over-expression. Specific Aim 1: Optimal in vivo vectors for gene therapy: Comparison of AAV and Lentivirus for amount, duration and safety of gene expression Specific Aim 2: Determine whether in vivo NGF transfer will prevent cholinergic neuronal degeneration and sustain gene expression for prolonged time periods. Specific Aim 3: Determine whether in vivo NGF transfer in aged rhesus monkeys will ameliorate cognitive deficits and atrophy of basal forebrain cholinergic neurons. Specific Aim 4: Develop a model for amyloid over-expression, and examine potential neural toxicity, in the young and aged primate brain.

musculoskeletal system; animal tissue; transplantation; skeletal system; nervous system; trauma; rehabilitation; gene therapy; biotechnology; Primates; Mammalia;

Significance Spinal cord injury is a common and debilitating human disease for which currenttreatments are unsatisfactory. In rodent studies, we have shown that grafts of cells genetically modified to produce nervous system growth factors can elicit robust growth of axons and, in some cases, functional recovery after spinal cord injury. In the present experiments, we are determining whether grafts of autologous primate cells genetically modified to produce neurotrophic factors will promote axonal growth and functional amelioration in the primate spinal cord. Positive findings from these experiments could lead to new treatments for human spinal cord injury. Objectives Examine whether grafts of autologous fibroblasts genetically modified to produce nerve growth factor (NGF) to the unlesioned primate spinal cord elicit axonal growth. It will be determined whether principles of neurotrophism observed in the rodent spinal cord are replicated in the primate spinal cord. Previous studies in rodents have demonstrated that grafts of NGF-producing genetically modified cells elicit robust growth of primary sensory axons and coerulospinal axons in the unlesioned rat spinal cord. Before beginning partial lesion experiments in primates, this aim will first determine whether NGF-producing cell grafts to the intact spinal cord also elicit growth, both to provide evidence that neurotrophic factor principles are valid in primates, and to justify proceeding to partial lesion experiments. Results Grafts of NGF-secreting cells elicited robust growth of sensory and coerulospinal axons in the primate spinal cord, thereby establishing the validity of neurotrophic factor principles in primates. Future Directions Determine whether grafts of fibroblasts genetically modified to produce neurotrophin-3 (NT-3) to the partially lesioned primate spinal cord elicit axonal growth and functional recovery. KEYWORDS gene therapy, spinal injury,

DESCRIPTION: This project will test the central hypothesis that cellular transplants of Schwann cells genetically modified to produce augmented amounts of neuro-trophins will promote CNS repair. Schwann cells are readily obtainable by peripheral nerve biopsy, sustainable as purified primary cultures, and possess intrinsic properties that favor their use in CNS repair, including the ability to myelinate CNS or PNS axons and the ability to secrete extracellular matrix molecules to support and guide new axonal growth. In extensive preliminary experiments, the investigators have transduced Schwann cells to produce and secrete supraphysiological levels of human nerve growth factor (hNGF) and human neurotrophin-3 (hNT3). In proposed experiments, two in vivo models will be used to evaluate the ability of genetically modified Schwann cells to promote neural repair. A spinal cord injury model will examine the ability of genetically modified Schwann cells to promote axonal regrowth. A model of basal forebrain cholineric neuronal degeneration will examine whether genetically modified Schwann cells rescue degenerating neurons. The choice of neurotrophic factors is based upon published data from the principal investigator's laboratory demonstrating in vivo axonal regeneration and neuronal rescue using neurotrophin infusions or grafts of genetically modified fibroblasts. Preliminary in vivo Schwann cell data indicates the practicality and feasibility of addressing the proposal's central hypothesis in these models. The following Specific Aims will be addressed: 1) Determine whether primary Schwann cells genetically modified to express and secrete augmented amounts of human nerve growth factor (hNGF) will promote axonal repair after spinal cord injury. 2) Determine whether primary Schwann cells genetically modified to express and secrete augmented amounts of human neurotrophin-3 (hNT3) will promote axonal repair after spinal cord injury. 3) Determine whether primary Schwann cells genetically modified to express and secrete augmented amounts of human NGF will rescue degenerating host cholinergic neurons in the brain.

Estrogen replacement therapy in elderly women has been reported to diminish the risk of developing Alzheimer's disease (AD) and to improve response to anti-cholinesterase therapy in women already diagnosed with AD. Further, recent evidence indicates that estrogen may modulate the expression of neuronal growth factors and their receptors in the brain. These findings suggest that neuronal plasticity in the brain may be modulated in part by estrogen-linked mechanisms. To investigate this possibility, our laboratory has explored interactions of estrogen-responsive neurons with growth factors and their receptors. We have found extensive co-expression of estrogen and neurotrophin systems in both cortical regions and forebrain projections to cortical regions in the brains of rodents and primates. Studies in this proposal will examine the effects of neuronal injury and estrogen loss on neural plasticity to test the hypothesis that estrogens modulate neural plasticity to test the hypothesis that estrogens modulate neural plasticity through interactions with neurotrophic factor systems in the brain. Mechanisms of such interactions will be elucidated. This work will build upon techniques and methods that are will-established in the PI's lab and that have led to useful insights into the nature of neurotrophin biology in the rodent and primate brains. The specific aims will be addressed: Aim 1: Do neuronal populations in the primate and rodent brains co-express estrogen receptors and neurotrophins or their receptors, reflecting co- modulation of these systems? Aim 2: What effects do natural fluctuations in estrogen levels exert on cortical synapse density and on expression of NGF, BDNF and neurotrophin receptors in the brain? Aims 3 and 4: What effect does estrogen loss exert on synapse density in cortical target regions, and on expression of neurotrophins and their receptors, in the brains of adult and aged rats (Aim 3) and in the brains of adult primates (Aim 4)? Aim 5: Do estrogens and growth factors act synergistically to enhance neural plasticity? These studies will provide insights regarding estrogen effects on going, cognition, and neurotrophic factors, leading to potential novel therapeutic approaches to AD.

nervous system; aging; Mammalia;

transplantation; nervous system; genetic manipulation; genetically modified animals; aging; gene therapy; animal tissue; Mammalia;

DESCRIPTION (provided by applicant): Progress in preclinical research over the last two decades has contributed substantially to our knowledge of factors that can promote axonal growth after spinal cord injury. However, there is a relative paucity of information regarding molecular mechanisms that might augment and guide axonal growth after injury in the adult. One important family of molecules that plays an important role in axon growth and guidance during development is the netrin family of diffusible molecules, and their associated receptors, DCC (deleted colon carcinoma) and UNC-5 family proteins. This project will characterize the natural expression of netrin and its receptors after adult spinal cord injury, then manipulate the potential ability of axons to respond to these molecules using ex vivo gene delivery of netrin to the injured spinal cord. Two specific aims will be examined: Specific Aim 1: Characterize expression of netrins and their receptors (members of the DCC and UNC-5 family) in the intact and lesioned adult rat spinal cord. Specific Aim 2: Determine whether netrin over-expression in the injured adult spinal cord influences the extent or direction of axonal growth.

[unreadable] DESCRIPTION (provided by applicant): Recent progress has been made in understanding mechanisms and consequences of injury to the CNS, and in augmenting CNS plasticity and regeneration after experimental spinal cord injury (SCI) in non-primates. The relevance of this work to injured primate systems remains to be established. Differences in neural anatomy and function, and inter-species differences in immune and inflammatory responses to injury, raise questions regarding the simple translation of findings from rodents to primates. In the last 4 years, our consortium has established a reliable and practical model of SCI in primates, and has tested potential treatments in this model. We have found both similarities and differences in the anatomy and functional properties of intact and lesioned rodent and primate spinal cords. Further, we have preliminary evidence that plasticity can be augmented after primate SCI, leading to improved functional outcomes. The renewal of this program will build upon these findings, aiming to contribute both mechanistic and empirical knowledge leading to the development of therapeutic strategies for promoting recovery from primate SCI. This project brings together research groups in a collaborative effort to advance the understanding and treatment of SCI in the following aims over the next 5 years: Specific Aim 1: Determine Mechanisms Underlying Spontaneous Recovery After Acute C5-6 Hemisection Lesions in Primates: Anatomical, Electrophysiological and Functional Studies. Specific Aim 2: Determine Whether BDNF Delivery Into and Below a C5-6 Hemisection Lesion Will Promote Axonal Sprouting or Regeneration, and Functional Recovery. Specific Aim 3: Determine Whether BDNF Delivery Into and Below a C5-6 Hemisection Lesion, + cAMP Augmentation, Will Promote Axonal Sprouting or Regeneration, and Functional Recovery. [unreadable] [unreadable] [unreadable]

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. Alzheimer's disease is the most common neurological degenerative disorder, affecting both critical nerve centers of the brain as well as cognition. Currently, Alzheimer's disease affects 4 million people in the U.S. Existing therapies for this disease are few in number and relatively ineffective. Novel therapies are needed. A promising class of natural biological proteins called nervous system growth factors offers the potential to reduce cell degeneration in the brain in such disorders as Alzheimer's disease. Gene therapy offers a uniquely effective means of delivering these growth factors to the brain.

DESCRIPTION (provided by applicant): Lesioned spinal cord long-tract axons can grow into a lesion/graft site if growth factors are locally provided. Separately, intraganglionic elevation of cAMP can promote dorsal column sensory sprouting after spinal cord lesions. However, achieving host axonal growth beyond cell grafts placed in spinal cord lesion sites has proven difficult, yet will likely be required for functionally meaningful recovery after SCI. Recently, we succeeded in promoting axonal regeneration into and beyond sites of cervical SCI using a combinatorial approach of: 1) cAMP stimulation of the neuronal soma, 2) autologous cell bridges in the lesion site, and 3) growth factor gradients provided as stimuli to axons in and beyond the lesion site. Controls lacking all three of these treatments did not exhibit bridging. This project will further develop this approach, identify its mechanistic underpinnings, and reduce it to potential clinical practicality. The following specific aims will be pursued: Specific Aim 1: Determine whether a combinatorial approach to regeneration will promote axonal regeneration and functional recovery after spinal cord lesions. Specific Aim 2: Determine whether a combinatorial approach to regeneration will promote tissue sparing, axonal regeneration and functional recovery after acute spinal cord contusions. Specific Aim 3: Determine whether a combinatorial approach to regeneration will promote axonal regeneration and functional improvement after chronic spinal cord contusions. The UCSD Center for Neural Repair also has in place primate SCI models for proof-of-concept testing of these findings, allowing potential reduction to practicality. We further have a track record of responsibly translating promising therapies from the bench to bedside.

DESCRIPTION (provided by applicant): The purpose of this Mentored Clinical Scientist Development Program is to support the career development of outstanding physician trainees in an aging-related neuroscience discipline, leading-to-research independence. Training will be conducted as a joint program taking advantage of the diverse and extensive resources of the local neuroscience community, including the University of California-San Diego, Scripps Research Foundation, the Salk Institute, and the Burnham Foundation. Outstanding applicants for the program will be recruited both locally from graduating residents of the clinical neurology program in the Department of Neurosciences at UCSD, and from neurology training programs nationally. 1 slot per year is requested for a 5 year period, totaling 5 training positions. The program will be administered by the Department of Neurosciences, a joint basic science-clinical department. The faculty possesses considerable strength in the genetic, molecular, cellular, systems, behavioral and modeling aspects of nervous system aging research. The faculty are also active in translational research, thereby directly impacting the treatment of human disease. The training program will provide appointees three types of academic experiences to lead to research independence: 1) Basic neuroscience training, accomplished by formal Neuroscience Graduate Program course work and laboratory research rotations, 2) a 3-4 year period of intensive mentored research, and 3) a continuous curriculum of symposia, coursework in aging, and lectures. Trainees who do not have a Ph.D. degree will have the opportunity to enroll in the UCSD Neuroscience Graduate Program and earn a Neuroscience doctoral degree The training program intends to expose the trainees to a breadth of neuroscience topics that are required to approach aging-related questions from a comprehensive and scholarly vantage point, ranging from the study of gene expression to whole systems/behavioral approaches. Individual trainees will then choose a discipline within which to focus and foster a career research topic.

American; CRISP; Computer Retrieval of Information on Scientific Projects Database; Funding; Gene Transfer Clinical; Gene Transfer Procedure; Gene-Tx; Genetic Intervention; Goals; Grant; Institution; Intervention, Genetic; Investigators; Mammals, Primates; Modeling; Molecular Biology, Gene Therapy; Myelopathy, Traumatic; NIH; National Institutes of Health; National Institutes of Health (U.S.); Natural regeneration; Primates; Recovery; Recovery of Function; Regeneration; Research; Research Personnel; Research Resources; Researchers; Resources; Source; Spinal Cord Trauma; Spinal Trauma; Spinal cord injured; Spinal cord injuries; Spinal cord injury; Therapy, DNA; United States National Institutes of Health; functional recovery; gene therapy; genetic therapy; regenerate

The aim of this Program Project is to explore influences of genes and the environment on neuronal vulnerability to degeneration in aging and Alzheimer's disease (AD), thereby identifying potential translational therapies for humans with AD. The resubmission of this program project will both build upon progress from the previous period of funding, and explore new evidence that physical activity influences cognitive and cellular features of neural function. Each project focuses on these central themes while examining individual mechanisms and specific models in detail. Project 1 focuses on effects of Neuregulin 1 gene expression on AB load, neuronal structure, neurogenesis and behavior on a background of aging and amyloid mutations in mice. Project 2 tests the hypothesis that genetic approaches to modifying amyloid processing, including overexpression of amyloid-degrading enzymes, can improve neuronal structural, electrophysiology, neurogenesis and behavior in amyloid mutant mice. Project 3 examines the extent to which glutamatergic and nicotinic mechanisms influence amyloid processing, caspace activation, neural structure and neurogenesis. Project 4 tests the hypothesis that BDNF ameliorates neuronal dysfunction and death in primate models of entorhinal/hippocampal degeneration using methods clinically practical in AD, and explores whether augmented physical activity in aged primates improves neuronal structure, cognitive ability and neurogenesis. Core A will coordinate activities and administration of all projects and Cores, and provide statistical support. Core B, the Vector Core, will support the a variety of recombinant vector needs that constitute a common theme in all projects. Core C will provide anatomical and electrophysiological support in evaluating effects of interventions from all projects on neuronal structure, synaptic plasticity, neurogenesis and behavior. The interdependency of resources and skills between Projects/Cores, as well as our past success with this PPG, support the argument that the Program Project is the most efficient and cost-effective way to address these issues.

PROJECT SUMMARY (See instructions): Lentiviral vectors have the unique ability to efficiently transduce non-dividing cells. Since the majority of the cells in the CNS are post mitotic, recombinant Lentiviral vectors can introduce genes in a variety of cell types including neurons and astrocytes. Lentiviral vectors can also be used to knock-down gene expression using RNA interference (RNAi) technology. The vectors can be generate shRNA to specifically compromise the expression of a target gene. Lentiviral vectors can also be designed to regulate gene expression. Adeno associated viral (AAV) vectors can also be used to introduce genes into the CNS. An advantage of AAV is the ability to generate very high titers of recombinant viruses which can allow easier genetic manipulation of higher animals. In some situations, to identify dividing neurons (neurogenesis), retroviral vectors which transduce only dividing cells will be used. Core B will provide higher titer recombinant viruses to all the investigators. Core B will also offer the possibility of training students and post doctoral fellows in the Pi's laboratory to generate viral vectors. Each PI has indicated their expected needs and we have listed them in Core B text.

PROJECT SUMMARY (See instructions): The goal of the proposed research is to contribute to the elucidation of the molecular mechanism of Alzheimer's disease with a focus on the deficits observed in synaptic transmission, synaptic plasticity and memory and learning in a mouse model of Alzheimer's disease. The mouse model that will be used in the experiments is a mouse engineered to over expressing the human APP protein carrying mutations which are known to cause Alzheimer's disease in humans. We have demonstrated that these mice exhibit deficits in synaptic transmission, synaptic plasticity, and spatial learning. We will also examine the role of neurogenesis and exercise in Alzheimer's disease. Aim 1 of this proposal is to examine the role of nicotinic receptors in Alzheimer's disease. Behavioral studies have linked the cholinergic system to learning and memory, which is intriguing given the observation that Alzheimer's patients have a deficiency in memory function and cortical nicotinic receptors. The first class of drugs approved for the treatment of Alzheimer's disease, which still today constitutes the best available treatment, are the cholinesterase inhibitors (Tacrine, Donepezil, Rivastigmine, Galantamine), which boost cholinergic function. Aim 1 of this proposal will be to test the hypothesis that binding of the beta-amyloid fragment (1-42) to the nicotinic receptor alpha 7 leads to the pathology seen in Alzheimer's disease. These experiments may either support or refute the idea that the cholinergic receptor system plays an important role in Alzheimer's disease. Either outcome will provide important information for developing strategies to prevent or reverse Alzheimer's disease. Aim 2 of this proposal is to test the role of the APP C-terminal caspase cleavage site and its proteolytic products in Alzheimer's disease. We have shown that mice over expressing the mutant human APP protein that causes human Alzheimer's disease exhibit deficits in synaptic transmission, synaptic plasticity and spatial learning. On the other hand, mice expressing the same mutant APP protein but with a deletion of a caspase cleavage site near the APP protein c-terminal are protected from the deficits. These observations predict that APP cterminal proteolytic fragments are causing synaptic damage and this hypothesis will be tested directly.

The objective of Core A is to organize and administrate the activities of the Program Project. The specific tasks include keeping track of personnel, ordering and monitoring purchase of supplies, equipment, and other expenses to include travel arrangements and review of the monthly financial reports on each project. Core A will also be responsible for arranging the group meetings, and writing up the meeting notes. Core A will also include a biostatistics group lead by Dr. Ronald Thomas that will provide expertise in biostatistics and manage a web-based database for the projects/core.

PROJECT SUMMARY (See instructions): This is the second re-submission of the competitive renewal. Accumulation of AB early in AD appears to play an important role in the mechanisms of synaptic damage and neurodegeneration leading to cognitive deficits. During the previous period of funding we focused at developing new models and treatments for AD using lentiviral vectors expressing or blocking AB degrading enzymes such s neprilysin (NEP). NEP might be important for AD also because it¿s possible involvement in neuroprotection and as an interface between environment and genetic regulation of synaptic plasticity responses. NEP cleaves bioactive transmitters such as Neuropeptide Y (NPY) resulting in trophic C-terminal fragments (CTFs). In this context, we hypothesize that among other AB-degrading enzymes, NEP might be involved in neuroprotection in APP transgenic (tg) mice by regulating synaptic remodeling and neurogenesis in the hippocampus. The main objective of this collaborative competitive renewal will be to investigate the molecular mechanisms through which interactions between environment and AB-degrading enzymes regulate synaptic regeneration and neurogenesis during aging and in AD. To this end we propose the following: Aim 1. To determine the AB-independent effects of NEP and other A^-degrading enzymes in synaptic regeneration during aging to murine models. Aim 2. To investigate the molecular mechanisms through which AB-degrading enzymes regulate synaptic regeneration during aging and in APP tg models of AD. Aim 3. To investigate the combined effects of physical activity and expression of AB-degrading enzymes on neurogenesis during aging and in APP tg models of AD. Aim 4. To evaluate the potention therapeutical and neuroprotective effects of NPY fragments in APP tg models of AD. In collaboration with the Cores with will perform studies of synaptic plasticity, neurogenesis and enhanced physical activity in APP tg, NEP, APP and NPY deficient mice, treated with lentiviral vectors expressing AB- degrading enzymes and NPY fragments. Together, these studies might also help in develop new treatments for AD that will have the dual role of reducing accumulation of neurotoxic Ab species and promoting neurogenesis.

DESCRIPTION (provided by applicant): Approximately 1.2 million Americans have sustained some form of spinal cord injury (SCI), with an estimated annual economic impact of $20 billion. Our overarching vision is to use a multimodality approach to promote axon regeneration and improve behavioral outcomes in the most challenging and clinically predictive SCI model, complete spinal transection. We will optimize and reduce to practicality bioengineered, template agarose guidance scaffolds for axonal regeneration based on substantial experience from a collaborative, multi-PI effort that pairs leaders in bioengineering at Michigan State University with a top national laboratory in the field of SCI research at UC San Diego. Specific Aim 1: Optimize Templated Agarose Scaffold Design Optimize scaffold design by adding: a)drug delivery capabilities using layer by layer (LBL) technology to control release of growth factors, and b) biodegradability properties by integration of hydrogels or agarose/polyelectrolyte composites. Specific Aim 2: UseTemplated Agarose Scaffolds to Promote Host Axonal Regeneration After T3 Complete Spinal Cord Injury Aim 2 will use optimized scaffolds from Aim 1 to enhance axonal growth into and beyond SCI lesions in vivo. Success in this model can lead to clinical translation. Specific Aim 3: Use Templated Agarose Scaffolds to Promote Formation of Neuronal Relay Circuits After T3 Complete Spinal Cord Injury Aim 3 will combine two cutting edge technologies - bioengineered scaffolds and stem cells - to formulate a new generation of therapies that could constitute the most promising approach yet for treating SCI. The natural progression of this work can lead to translation to our non-human primate model of SCI, and then to clinical trials. PUBLIC HEALTH RELEVANCE: Approximately 1.2 million Americans have sustained some form of spinal cord injury (SCI), with an estimated annual economic impact of $20 billion.SCI tragically disables its victims and extracts a psychological toll on patients and caregivers. This project incorporates material science, drug delivery, tissue engineering, stem cell biology and neurosciences to promote axon regeneration and improve behavioral outcomesafter SCI.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common neurodegenerative disorder, afflicting 5 million people in the U.S. alone. This U grant application will support studies leading to the filing of an Investigational New Drug (IND) application to the FDA for Brain-Derived Neurotrophic Factor (BDNF) gene delivery in AD. We have completed proof-of-concept studies in mice, rats and non-human primates, demonstrating that BDNF prevents entorhinal cortical neuronal cell loss, enhances synaptic markers, reverses molecular and biochemical features associated with AD, and improves learning and memory. These effects extend into the hippocampus, thereby treating key memory circuitry of the brain. Importantly, this approach provides a much-needed alternative to amyloid-modifying therapeutics currently under development, providing future possibilities for combined therapies if both prove to be partly effective. We propose gene delivery of BDNF because of the need to administer this protein directly into the brain and sustain its delivery over time. In the proposed work plan, we will manufacture adeno-associated virus serotype 2 (AAV2) vectors expressing human BDNF at a GMP facility, then use this clinical-grade material to perform IND-enabling studies in two species (rat and primates). In addition, we will generate expertise in accurately targeting and delivering AAV2-BDNF to the primate entorhinal cortex using real-time, MR-guided imaging. The following aims will be performed: Aim 1: Produce AAV2-BDNF for IND-enabling safety/toxicity/dosing studies. Aim 2: Optimize AAV2-BDNF gene delivery to the entorhinal cortex in non-human primates using convection-enhanced delivery and real-time MR guidance. Aim 3: Safety/toxicity/dosing/biodistribution studies in rodents and non-human primates. Aim 4: Draft and Submit an IND Application. Relevance: Successful completion of this work will lead to clinical translation of a new approach to prevent cell loss and stimulate neural function in a common, severe and disabling neurodegenerative disorder.

PROJECT SUMMARY/ABSTRACT This is a new multi-PI proposal from our California Spinal Cord Injury (SCI) consortium to continue to translate exciting results from the transplantation of neural stem cells (NSCs) from rodent to primate, and to evaluate efficacy and safety in our non-human primate (NHP) cervical contusion SCI model. Our multi-center consortium has examined recovery of function and its anatomical correlates in a series of studies using a cervical hemisection model. We have discovered spontaneous and extensive plasticity of the corticospinal tract (CST) system that had not been appreciated in previous rodent studies. We have developed the first large NHP model of cervical hemicontusion SCI together with an open-field scoring system and novel in-cage forelimb activity and hand function tests to evaluate functional outcomes. The wealth of new information and directions speak to the value of this shared approach to using the very valuable primate model. This project focuses on translation of our NHP stem cell work. We now report that neural stem cells (NSCs) derived from human spinal cord grafted early to hemisection sites in NHP SCI, extend very large numbers of axons over very long distances, and that these transplants appear to enhance long-term recovery of hand function, and support CST regeneration into the graft. NSCs derived from the approved human embryonic stem cell H9 (H9 hESCNSCs) also support CST regeneration into spinal cord grafts in the NHP after SCI. Further, we have advanced our cell therapy strategy to produce the first H9 hESCNSCs caudalized to move them towards a spinal cord fate, and have shown that transplants of these cells in rodents promote much more vigorous regeneration of CST axons7. Therefore, in this proposal in NHPs, we will transplant caudalized hESCNSCs into a contusion lesion at a more chronic and clinically relevant six week time point. We hypothesize that these grafts will support robust CST regeneration and enhance recovery of forelimb function, and provide a relay for CST axons to influence forelimb circuitry in the C8-T1 cord. We will use anterograde and retrograde tracing, IHC and transfection of graft cells and correlate the connectional data with recovery, and test the long-term survival, safety, and functional effects of these transplants.

This is a proposal to map the ?connectome? of the primate fine motor control system. This work has the potential to lead to a new and remarkably detailed understanding of anatomical mechanisms underlying corticospinal fine motor control in primates, as well as reorganization of motor systems after injury that are associated with functional loss and recovery. Aim 1: The Intact Primate Corticospinal Connectome Aim 1 will map the intact corticospinal connectome underlying hand control in the rhesus monkey using new viral tools. These viral tools will identify all projections and collateral branches arising from corticospinal neurons that project to C8 spinal cord segments to influence fine motor control of the hand. Unprecedented detail and specificity will be achieved using efficient retrograde transport of AAV9-Cre injected into spinal segment C8, and motor cortex injections of Cre-dependent AAV5-Flex-GFP and AAV5-Flex-TdTomato. All projections and collateral branches of this corticospinal hand control system throughout the neuraxis will be mapped, quantified, and rendered in 3D. The data will be uploaded to a free, publicly accessible website (https://neurodata.io/data/) for use by the research community. Aim 2: The Lesioned Primate Corticospinal Connectome How does injury affect the corticospinal connectome? How does the corticospinal system adapt to injury and alter its set of outputs, and how are these changes associated with functional recovery? We will use the elegant and novel tools of Aim 1 to map the injured and reorganized corticospinal connectome, and relate these changes to spontaneous, partial recovery of hand function after C7 hemisection lesions. This work will provide new understanding of primate anatomical and functional adaptation to injury at an unprecedented depth, and identify potential targets for enhancing functional repair. 1

Project Summary/Abstract Nearly 300,000 Americans have sustained some form of spinal cord injury (SCI), and effective therapies to promote recovery of neural function are lacking. Our overarching vision is to create an ex-vivo tissue that can replace the damaged spinal cord and enable formation of new relay circuits across sites of even severe injury. Our extensive rodent work with 3D printed biomimetic scaffolds shows that this approach can result in electrophysiological and functional recovery after complete spinal cord transection, the most severe model of spinal cord injury. This project aims to demonstrate the feasibility of scaling up a 3D printed biomimetic scaffold, loaded with human neural stem cells, to a clinically relevant, non-human primate model of spinal cord contusion. There are 3 objectives to this project: 1. Image the primate contusion-lesioned spinal cord by MRI scan. 2. Generate a 3D model and print individual scaffolds that conform to each subject's injury. 3. Implant 2 subjects with scaffolds: 1 Empty scaffold, 1 scaffold loaded with human neural stem cells. A demonstration of feasibility will lead to an R01 application in the non-human primate, with potential clinical translation.

Abstract Brain-derived neurotrophic factor (BDNF) is a nervous system growth factor that enhances synaptic plasticity and regulates neuronal function. BDNF gene therapy for Alzheimer?s disease (AD) is a promising alternative to amyloid- and tau-targeted therapies: BDNF reduces neuronal degeneration and stimulates neuronal activity in rodent and non-human primate models of AD. Direct injection of an Adeno- Associated Virus (AAV) vector into entorhinal cortex mediates safe and long-lasting BDNF expression, and will soon begin human clinical trials. Although promising, intraparenchymal AAV-BDNF injection is invasive and treats only a small percentage of the cerebral cortex. Intrathecal administration of AAV9-BDNF to the cerebrospinal fluid could solve these problems by broadly treating the entire cortex from a single minimally invasive infusion. We recently reported that two hours of Trendelenburg positioning, in which the body lies supine on a reclining table with the head 30° below the feet, dramatically increases the strength and consistency of gene transfer to cerebral cortex after intrathecal AAV9 infusion in rats. More than 95% of transduced cells in cortex are neurons, and gene expression in off-target brain regions and spinal cord is minimal. This novel delivery method has strong potential for clinical treatment of AD. We propose systematic preclinical testing of intrathecal AAV9-BDNF gene therapy for AD. Aim 1 will test therapeutic efficacy by directly comparing intrathecal and intraparenchymal AAV9-BDNF infusion in a transgenic mouse model of AD and analyzing behavioral and anatomical outcomes. Aim 2 will test the safety of intrathecal AAV9-BDNF infusion at escalating doses and over prolonged treatment periods in the non-human primate. Aim 3 will enhance the specificity of intrathecal AAV9-BDNF therapy by testing cell-specific promoters to reduce or eliminate off- target gene expression. These studies aim to simplify delivery, enhance efficacy, and increase clinical feasibility of BDNF gene therapy for AD, and will support both upcoming clinical trials and preclinical development of new gene therapies for AD.

Project Summary/Abstract Nearly 300,000 Americans have sustained some form of spinal cord injury (SCI), and effective therapies to promote recovery of neural function are lacking. Our overarching vision is to create an ex-vivo tissue that can replace the damaged spinal cord and enable formation of new relay circuits across sites of even severe injury. Our extensive rodent work with 3D printed biomimetic scaffolds shows that this approach can result in electrophysiological and functional recovery after complete spinal cord transection, the most severe model of spinal cord injury. This project aims to assess the efficacy of 3D printed biomimetic scaffolds, loaded with human neural progenitor cells and implanted acutely, in a long-term clinically relevant, non-human primate model of cervical spinal cord contusion. There are 2 objectives to this project: 1. Analysis of anatomical outcomes - Biocompatibility, integration, degradation, vascularization, host axon regeneration into the scaffold, and neural progenitor cell-derived axon outgrowth from the scaffold into the host. 2. Analysis of functional outcomes. Animals will be functionally assessed on tasks of right- hand function, including Brinkman board, home cage-based robotic manipulation, forelimb and hindlimb use in an open field/exercise enclosure, and sensation. Successful performance outcomes of the grant may lead to clinical trials, with resulting high impact.

Project Summary/Abstract: We propose to conduct a first-in-human clinical trial of BDNF gene therapy in Alzheimer?s Disease (AD) and Mild Cognitive Impairment (MCI), aiming to reduce neuronal loss and to activate neuronal function. BDNF (Brain-Derived Neurotrophic Factor) is actively produced and utilized in cortical circuits throughout life to sustain neuronal function and circuits. In animal models of AD, BDNF builds new synapses, prevents neuronal death and activates neurons; thus, BDNF offers the potential to slow or actually reverse cognitive decline in established AD and MCI. Proof-of-concept studies have been performed in mice, rats and rhesus monkeys. Because BDNF is a relatively large and polar protein that does not cross the blood brain barrier, we will use intraparenchymal gene therapy to deliver BDNF directly into the entorhinal cortex. BDNF will be neuronally trafficked into the hippocampus. BDNF will be delivered using adeno- associated serotype 2 vectors (AAV2), which have now been utilized in hundreds of patients in CNS gene therapy trials. We will utilize start-of-the-art methods for gene delivery, employing real-time MR guidance and convection-enhanced delivery (CED) in collaboration with the world leaders in this technology at Ohio State University (OSU). A total of 12 patients (6 AD and 6 MCI) will be recruited from two clinical sites: UCSD and Case Western. All patients will undergo gene delivery at OSU. The primary outcome measure will be safety, together with secondary cognitive outcome measures that reflect memory-specific and global cognitive measures. Serum, CSF and imaging biomarkers will be collected. If AAV2- BDNF gene delivery is safe and well-tolerated, and exhibit possible cognitive benefits, we will advance to Phase 2 trials. An IND for this program is under review by the FDA, and the trial will begin upon FDA clearance. Two dose groups will be studied: 3x1011 vg/ml and 1x1012 vg/ml. Relevance: Effective disease-modifying therapies for AD and MCI have not been identified. BDNF gene delivery offers the potential to slow or reverse cognitive decline in established AD by building new synapses, stimulating neuronal function and reducing neuronal death. Our approach also offers the potential for combination therapy with amyloid- and tau-modifying therapies.

Project Summary Efforts to promote recovery of function after human spinal cord injury (SCI) will likely require interventions targeting the corticospinal motor system, the most important pathway for voluntary motor control in humans. In a series of studies over the past 4 years we have found that corticospinal tract (CST) axons regenerate into spinal cord neural stem cell (NSC) grafts placed into sites of SCI in mice, rats and monkeys. These regenerating CST axons form synapses with the graft, and the graft in turn extends very large numbers of new axons from the injury site over long distances into the distal spinal cord. Neural relays across the injury are thereby formed, supporting functional improvement. This work is on a human translational path and IND-enabling work is in progress. This grant proposes two new directions that will be critically important in supporting human translation. First, we recently reported that injured adult mouse CST neurons revert to an embryonic transcriptional state that lasts for two weeks after SCI, a time during which CST axons can regenerate. This finding establishes a critical period for intervention after mouse SCI to support recovery. Does the same transcriptional reversion to a pro-growth embryonic state occur in the primate brain? If so, how long does it last? Work in Aim 1 will definitively answer this question, identifying for the first time what may be an optimal time window for therapeutic intervention of any type to support functional recovery in primates, including humans. We will perform RNA sequencing (RNAseq) specifically of CST neurons after SCI in rhesus monkeys using intersectional viral approaches, based on supportive preliminary data in monkeys. In Aim 2 we propose for the first time using novel viral vectors to anterogradely, trans-synaptically trace primate corticospinal projections to the spinal cord. Our preliminary studies demonstrate that rodent CST axons project nearly entirely to spinal cord interneurons, whereas in primates the vast preponderance of CST axons terminate directly on alpha motor neurons. Knowing the precise targets of CST projections to the spinal cord will both markedly extend our basic knowledge of motor system organization in primates, and will allow optimization of stem cell graft properties to enhance neural relay formation across sites of SCI. Unlike other neural stem cell programs for SCI, our work aims to directly re-form critical neural relays across a severe injury, rather than target spared axons through grafts of OPCs; knowledge gained from this aim could markedly improve relay formation across injury sites in the primate system.