Aileen J. Anderson - US grants

DESCRIPTION (provided by applicant): Previous studies have investigated the cellular inflammatory response and regulation of inflammatory cytokines following spinal cord injury (SCI). Infiltration of inflammatory cells and corresponding cytokine production has been predicted to contribute to secondary injury via several mechanisms, and studies suggest that inhibition of inflammation can be beneficial to recovery. Conversely, other studies have raised the tantalizing possibility that, at least under some conditions, stimulation of the cellular immune system may provide neuroprotective benefits or enhance recovery from CNS injury. Critically, however, the principal humoral immune component of these inflammatory events, the complement cascade, has not been investigated. While antibodies are a critical component of homologous (host) defense, complement is a principal effector of both the innate and adaptive immune system. Our preliminary data provide novel evidence for: 1) complement immunoreactivity in association with neurons, oligodendrocytes, and axons after SCI, 2) improved functional recovery and histological outcome following complement depletion in contusion-injured rats, and 3) improved functional recovery in mice deficient in the C5 component of complement. In this proposal, we investigate the mechanism of action of complement depletion in SCI, cellular source of complement after SCI, and predominant pathway(s) for complement-mediated impairments in functional recovery and tissue damage after SCI. We hypothesize that: 1) The functional improvements derived from complement depletion will be associated with inhibition of neutrophil, macrophage/monocyte, microgial, and T-cell recruitment, inhibition of neuron and oligodendrocyte cell loss, and reduction in glial scar formation; 2) In addition to serum-derived complement from Blood-Spinal Barrier (BSB) opening immediately after injury, local CNS cell synthesis is a component of complement deposition after SCI; and 3) Complement-mediated impairments in functional recovery and tissue damage activation after SCI are predominantly dependent upon the terminal pathway. These studies will provide an understanding of the specific pathological role of complement activation after SCI, and clarify appropriate potential targets for therapeutic complement inhibition in SCI, which will be increasingly important as new complement inhibitors are brought to clinical trials for CNS injury.

DESCRIPTION (provided by applicant): A multitude of events contribute to the ultimate outcome following SCI, including neuron, oligodendrocyte and axonal loss, demyelination, glial scar formation and inhibitory molecule deposition, and endogenous capacity for regeneration. These events define critical points for investigation of the mechanism of action of interventions that affect functional recovery. The development of cell-based therapeutic strategies, including cultured Schwann and olfactory ensheathing cells, fetal spinal cord tissues, and embryonic stem cell (ES)-derived progenitors, is of strong current interest for SCI. In particular, CNS Stem Cells (CNS-SC), which have the ability to migrate and differentiate into neurons, oligodendrocytes and astrocytes upon transplantation could benefit the injured spinal cord in a variety of ways. These include differentiation and functional engraftment of new neurons and oligodendrocytes, modifying the regenerative or remyelination potential of host cells, and decreasing host glial scaring or deposition of inhibitory matrix molecules (e.g. proteoglycans). We have found that cells from CNS-SC banks initiated from prospectively isolated human fetal brain using monoclonal antibody based fluorescence activated cell sorting (FACS) survive and engraft in contusion-injured immunodeficient NOD-scid mice. Contusion-injured mice transplanted with human CNSSC neurospheres 9 days post-SCI show improved recovery of open field locomotor function. These highly enriched human CNS-SC can be reproducibly isolated, are capable of long term growth in culture as neurospheres, and our preliminary data suggest that they maintain their capacity to differentiate into neurons and oligodendrocytes in the injured spinal cord. The objective of this proposal is to experimentally test the basis for the observed functional recovery, testing the hypothesis that human CNS-SC either differentiate and functionally engraft or modify the host response to injury as described above. Further, we will also test the hypothesis that exercise will act synergistically with cell transplantation to improve locomotor recovery, based on its known role in promoting neurogenesis and our data demonstrating enhancement of locomotor outcome in contusion-injured mice in a voluntary wheel running paradigm.

DESCRIPTION (provided by applicant): Injury to the spinal cord results in paralysis below the level of the injury, and there are no current therapies that are able to restore function. Limited regeneration occurs as result of the local environment, which is deficient in stimulatory factors and has an excess of inhibitory factors. Our long-term goal is to develop multi- functional biomaterials that bridge the injury site to control the microenvironment to promote and direct axonal growth into and through, and to re-enter the host tissue to form functional connections with intact circuitry. In the previous funding periods, we have developed multiple channel bridges that mechanically stabilize the injury that limits secondary damage, and using a transgenic mouse model with a GFP reporter construct expressed predominantly in the corticospinal tract (CST), we demonstrated that large numbers of CST axons grow through the bridge, re-enter the host tissue, and extend up to 3 mm down the cord by 10 weeks post- implantation. Additionally, we have an unparalleled ability to localize delivery of gene therapy vectors, with which expression of neurotrophic factors significantly enhanced the number of regenerating axons. This proposal builds on these results and focuses on enhancing the number of neural progenitors (either through recruitment or transplantation) and promoting their differentiation into mature oligodendrocytes that can myelinate axons and functionally reconnect a significant number of regenerating axons with the intact circuitry below the injury. Our development of bridges is targeted toward the 14% of spinal cord injuries that result from penetrating wounds that create a gap in the spinal cord, and may necessitate a different approach to restoring function than contusion/compression injuries. We propose that providing a bridge soon after a penetrating injury in order to stabilize the spinal cord and attenuate the host response. The bridges could be an off-the- shelf product that is readily available for implantation, and the bridge is initially designed to target survival, migration, and differentiatin of the endogenous progenitor cell population. Alternatively, we investigate delivery of neural stem cells rostral and caudal to the bridge a week or more after the bridge is implanted. While a bridge can be delivered soon after injury, stem cell transplants immediately after injury are contraindicated, as the cells are allogeneic and would require immunosuppression. The survival, recruitment, proliferation, and differentiation of endogenous or exogenous progenitor cells will be targeted through the immune response at the scaffold (Aim 1). We propose to use the bridges to modulate the macrophage phenotype towards M2 in order to promote secretion of pro-regenerative factors following injury. Alternatively, we propose to delivery trophic factors tht target the function of progenitor cells by complementary pathways. The bridge platform can support multiple aspects of the regenerative process, and the well-defined components, which have been used in the clinic, may facilitate the ultimate translation to the clinic.

DESCRIPTION (provided by applicant): We previously demonstrated pre-clinical efficacy of Human Central Nervous System Stem Cells (HuCNS-SC) in multiple rodent models of thoracic spinal cord injury (SCI). HuCNS-SC transplanted at 9, 30, and 60 days post-injury (dpi) engraft, survive, migrate, and exhibit predominant differentiation into oligodendrocytes and neurons. Critically, transplanted cells promoted recovery of locomotor function and showed no evidence of allodynia at each of these transplantation times. These data contributed to a Phase I/II SwissMedic trial targeting chronically injured individuals suffering from thoracic SCIs; the first patient received HuCNS-SC in September 2011 at the University of Zurich. Critically, 52% of clinical cases of SCIs occur at the cervical level. Targeting the chronic SCI population yields a much larger subject cohort and advantages for clinical safety and efficacy assessment (e.g. spontaneous recovery is a significant confound acutely). Discrimination of a therapy induced improvement for SCI is dependent, in part, on the spontaneous recovery rate. Spontaneous recovery is highest in acute thoracic subjects, and lowest in chronic cervical subjects. An acute thoracic SCI trial has been estimated to require as many as 225-250 ASIA A subjects and 1,100 ASIA B subjects to detect a moderate effect. In contrast, a chronic cervical SCI trial may require as few as 25 ASIA A subjects, and 50 ASIA B subjects. Further, a small gain of function in the cervical region affords a proportionally greater change in quality of life compared to a similar gain at the thoracic level. We have generated a unilateral cervical SCI model using immunodeficient Rag2g/c mice to optimize xeno-engraftment, and demonstrated that 9 dpi transplantation of HuCNS-SC promotes recovery of locomotor function with no adverse effects, establishing pre-clinical efficacy of HuCNS-SC. The UCI-StemCells Inc. team already has considerable experience in clinical translation using HuCNS-SC and has sought preliminary feedback from the FDA for a cervical SCI indication in the form of a pre-preIND consultation. This proposal is based on our FDA interactions and seeks to expand our current cervical SCI efficacy data to evaluate the intended clinical cell lot of HuCNS-SC for both efficacy and long-term safety/toxicology addressing the following Specific Aims: Aim 1: Establish efficacy and test cell delivery site for the HuCNS-SC CCL in a 9 dpi transplantation unilateral cervical SCI mouse model. Aim 2: Establish efficacy and test cell delivery site for the HuCNS-SC CCL in a 60 dpi transplantation unilateral cervical SCI mouse model. Aim 3: Assess the safety of the HuCNS-SC CCL in a rodent model of autonomic dysreflexia. Aim 4: Establish long-term safety/toxicology of the HuCNS-SC CCL in the established unilateral cervical SCI mouse model. Aim 5: If dictated by the FDA in a pre-IND meeting in Year 2, establish efficacy and safety/toxicology for the HuCNS-SC CCL in a second species (ATN rat), using a hybrid study design and cervical SCI rats receiving supplemental immunosuppression with anti-sialoGM1 antibody treatment. The aims of this project will enable IND application for the use of this HuCNS-SC clinical cell lot for cervical SCI as a therapeutic target.

Summary: Regeneration of tissues following injury can be limited due to the development of strong inflammatory responses that can lead to substantial cell death and inappropriate conditioning of the local environment, which becomes deficient in stimulatory factors and has an excess of inhibitory factors. Our long-term goal is to develop nanoparticles that reprogram the phenotype of monocytes and neutrophils in the blood after trauma, resulting in altered trafficking and anti-inflammatory phenotypes that reduce the extent of damage and may support an environment that leads to enhanced regeneration. The premise of the proposed research is based on our preliminary data indicating the ability to deliver nanoparticles in a minimally invasive manner that target inflammatory monocytes and neutrophils in the blood to reprogram their function, which leads to substantial functional recovery in a spinal cord model. The particles may enhance recovery by multiple mechanisms, including reducing immune cell accumulation at the injury, modulating the splenic microenvironment that is known to coordinate inflammatory responses, or directly inducing an anti-inflammatory or pro-regenerative environment at the injury. The following aims employ nanoparticles with differential binding to monocytes and neutrophils, which influences their phenotype such as trafficking and cytokine production. Importantly, the reprogramming is mediated solely by the physicochemical properties of the nanoparticles (e.g., size, charge, composition) and does not involve an active pharmaceutical ingredient (API), which have been discontinued from many applications due to the risk-benefit ratio. The focus herein is to identify the mechanism by which the particles are enhancing functional recovery, which may also identify design parameters that are more efficient. Aim 1 will investigate nanoparticle association with innate immune cells in circulation, and their subsequent trafficking and phenotype in the inflammatory response. Nanoparticle injection following SCI has led to substantial recovery gains we aim to identify the mechanisms by which the particles are promoting recovery. Particles that induce differential binding, phenotypic polarization, and trafficking of monocytes and neutrophils will be investigated. Aim 2 will investigate the impact of the reprogrammed immune cells on the microenvironment within the spleen and spinal cord. Stromal and immune cells are initially investigated throughout recovery, and we subsequently investigate the extent of axon growth, myelination, and functional recovery. Collectively, these studies will determine the relationship between nanoparticle properties, immune modulation, and the capacity of the environment to reduce damage and enhance functional recovery. We propose that the particles that reprogram based on their physicochemical properties have the potential to be a transformational therapy for trauma by providing a readily available, non-invasive means to reprogram inflammatory monocytes and neutrophils in order to limit damage and enhance regeneration.

Spinal Cord Injury (SCI) causes paralysis below the level of damage, which results from neuron and oligodendrocyte cell death, axonal loss, demyelination, and critically, the limited capacity of spinal cord neurons to regenerate. In contrast to patients with contusion injuries, individuals with penetrating SCI do not recover some function due to plasticity and are reliant on reconnection of spinal pathways, such as through biomaterial bridge that support true axonal regeneration. Although spinal cord neurons have the innate capacity to regenerate, they are limited by the environment, which contains an insufficient supply of factors to promote regeneration, and an abundant supply of factors that inhibit regeneration. Our long-term goal is to develop a combination therapy based on biomaterials that can 1) bridge, 2) modulate the injury microenvironment, 3) drive axon growth through an inhibitory milieu enabling the promotion and direction of axonal growth into, through, and re-entering spared host tissue to form functional connections with intact circuitry below the injury. We have shown that the bridge architecture leads to integration with the host tissue, reduces secondary injury, and prevents cyst formation. The channels of the bridge support robust axonal ingrowth into and through the bridge for corticospinal tract (CST) axons and extend >2 mm down the cord by 10 weeks post-implantation. Bridge implantation enhances functional recovery by itself, and modification of the bridge to express anti-inflammatory factors further enhances function recovery by decreasing the secondary damage and initiating a regenerative program that consists of genes associated with neural development and repair. This proposal builds on these results and focuses on regeneration at chronic time points by providing anti-inflammatory factors acutely after a penetrating injury combined with a biomaterial bridge at a chronic time points. We hypothesize that acute delivery of factors to reduce inflammation will minimize inhibitory molecules and spare regeneration competent axons adjacent to the injury, and that combination of this approach with delayed bridge implantation and pharmaceutical microtubule stabilization will drive directed axon regrowth through the channels to re-enter the caudal parenchyma and synapse onto intact circuitry in chronic SCI. Toward this goal, gene delivery will be used to modulate inflammation and reduce inhibitory molecule expression during the acute stage of injury (Aim 1). Regeneration at chronic times is investigated using bridges in combination with the microtubule stabilizer epothilone B (EpoB), which drives axon growth through the injury to connect with intact circuitry (Aims 2). The combination of acute and chronic therapies is investigated in Aim 3. The bridge platform can support multiple aspects of the regenerative process, and the well-defined components, which have been used in the clinic, may facilitate the ultimate translation to the clinic. These studies provide critical information on how early injury interventions can impact regeneration at later times.