Nicholas M. Boulis - US grants

DESCRIPTION (provided by applicant): The present proposal will continue the career development of Dr. Boulis as a clinician-scientist in the field of Functional Neurosurgery, focusing on an alternative to existing strategies for Neuromodulation. Neuromodulation, the manipulation of neural activity within anatomically discrete targets, is the principle tool of Functional Neurosurgery, finding application in the treatment of movement disorders, pain, spasticity, epilepsy and psychiatric disease. It has largely replaced the destruction of neural tissue as a means to treat refractory functional disorders. Nonetheless, the focused delivery of electric current is incapable of pharmacological specificity and requires electronic neural prostheses that carry a significant complication rate. Viral gene therapy has several advantages over implanted devices for the treatment of functional neural disorders. Neuronal gene expression can be achieved through minimally invasive stereotactic injection. Moreover, the tropism of viral vectors can be engineered through manipulation of the virus surface to target the vectors to individual cell types as well as limit and direct the spread of gene expression. Finally, viral gene expression can be achieved in a sustained fashion in neurons without disrupting their architecture or synaptic structure. Thus, gene transfer can be used to manipulate functioning neural structures in a fashion that current surgical procedures cannot achieve, providing the dual advantage of both pharmacologic and anatomic specificities. The following proposal explores the development of vectors to achieve controlled modulation of synaptic function using the best available inducible gene expression systems for regulated release of the clostridial tetanus toxin light (LC) gene and the inwardly rectifying potassium channel (Kir2.1) gene. Aims of the current proposal will test the following hypotheses: 1) AAV mediated LC synaptic inhibition is durable and less immunogenic than delivery mediated by Adenovirus, and that durable expression can be regulated by the Tet-on system. 2) Expression cassette modification, targeting expression to motor neurons and transgene delivery to axons, can improve the potency and specificity of LC gene-based neural inhibition. 3) The Rheoswitch(r) inducible expression system will improve controlled LC delivery. 4) Neuronal Kir2.1 gene expression can safely inhibit neuronal activity with potency exceeding that of LC mediated synaptic inhibition.

DESCRIPTION (provided by applicant): Motor neuron diseases (MND), such as Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA), are progressive neurodegenerative disorders that share the common characteristic of upper and/or lower motor neuron (MN) degeneration. Although the molecular mechanisms underlying MND are not entirely clear, all forms ultimately lead to apoptotic motor neuron death. Therapeutic strategies for MND, using trophic factors, or anti-apoptotic proteins can confer MN protection regardless of the specific mechanism of injury. Currently, gene therapy is one of the most promising candidates to deliver these treatments in MND, using lentiviral and adeno-associated viral (AAV) vectors. Because of the attractive safety profile of AAV vectors a variety of clinical trials are ongoing or planned for application to neurodegenerative diseases including ALS. Despite their appealing characteristics, AAV vectors have high affinity for skeletal muscle, as well as limited tropism for axon terminals, impeding MN gene delivery after IM injection. These limitations halted the aggressive development of a clinical trial for the treatment of ALS through intramuscular AAV.IGF-I injection. To overcome this barrier, we have modified the vector's capsid through the insertion of a novel peptide (Tet1) with high MN affinity and retrograde transport, increasing AAV mediated MN gene delivery. The present grant seeks support to identify the optimal targeted AAV vector for enhanced MN gene delivery, and demonstrate improved survival in the rat model of ALS compared to the earlier generation vector. Substantially improved retrograde delivery will prompt a return to the aggressive development of a clinical trial for ALS gene therapy. Our application will attempt to demonstrate that: 1. Capsid Mutation of the AAV Cap gene, incorporating novel neuronal binding peptides into the virus'coat, can increase the efficiency and specificity of MN gene delivery. 2. Targeted AAV-mediated IGF-I gene expression will protect MNs in SOD1 rats. 3. Peptide insertion may enhance neuronal delivery following alternative delivery routes, such as intra-arterial and intrathecal injections. PUBLIC HEALTH RELEVANCE: Death and disability in Motor neuron diseases (MND), such as Amyotrophic Lateral Sclerosis (ALS), result from death of cells in the nervous system called motor neurons (MN). In the present application, we will engineer viruses for safe and enhanced delivery of therapeutic genes to motor neurons. These viruses will be capable of delivering genes to the spinal cord after simple muscle injection, providing a safe approach to gene therapy for ALS.

DESCRIPTION (provided by applicant): This proposal outlines a plan for a Phase 1b clinical trial for the injection of human spinal cord derived stem cells (HSSCs) into the cervical spinal cord of patients with ALS. This trial is a follow up to a trial already underway at Emory University, where 12 ALS patients have been injected with the same HSSCs into the lumbar spinal cord. In order to move this therapeutic approach closer to a clinical trial to determine if t is effective in ameliorating disease, we are proposing to test the safety of HSSC injection into the cervical spinal cord. Motor neurons in the cervical spinal cord innervate the respiratory diaphragm, the loss of which is typically the cause of death in ALS patients. We propose that the protection of these neurons is likely to prolong life by preserving respiratory function. This safety trial will employ progressive dose escalation to determine the maximum tolerated dose that can be used for the long term goal of performing Phase 2 and Phase 3 efficacy trials. There are two specific aims. In Aim 1 we propose to sequentially escalate the dose of delivery as defined by 1) the number of cells/injection, 2) the number of injections into the cord, and 3) either unilateral or bilateral injections. This dose escalation scheme is designed to safely and efficiently test our ability to achieve a pre-defined target therapeutic dose, which can be used in the next phase of testing therapeutic efficacy. Aim 2 of this proposal is to examine several exploratory endpoints that may be used to test the efficacy of this therapy in future Phase 2 and Phase 3 trials. These include measures of respiratory function, diaphragm function, muscle strength, and electrical characteristics of muscle (Electrical Impedance Myography). Successful completion of this Phase 1b trial will allow for testing of this highly innovative approach to the treatment of ALS. The impact will extend beyond patients with ALS since this novel trial will provide data on surgical approach and safety, as well as trial design that will be highly relevant to cellular therapeutics for spinal cord injury, multiple sclerosis, spinal muscular atrophies, as well as other neurological diseases.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating neuromuscular disorder striking about 1 person in 40,000 each year. Individuals with ALS exhibit rapid loss of muscle control, muscle atrophy, and death due to respiratory failure. The cause of ALS is the progressive denervation of muscle by motor neurons. There is currently no cure for this disease, and the only approved therapy has a very modest effect on the disease progression. Clearly, there is a pressing need for more effective therapies. One possible route would be to use neuroprotective factors which, due to their general mode of action, may have utility in other neuromuscular disorders as well. Our long-term objective for this project is to develop gene therapy for ALS. Previous studies have investigated the use of neuroprotective factors. These molecules, such as insulin-like growth factor 1 (IGF-1) provide anti-apoptotic signals for motor neurons as well as promoting neurite outgrowth. These molecules seemed promising in animal studies. However, clinical trials demonstrated that scaling the dose to humans poses daunting challenges. A more effective approach might be to use gene therapy to allow the patients' own cells to produce the therapeutic factor. Several studies, including our own, have shown this approach has merit. However, these studies used techniques that have not scaled up well in larger animal models. Intraparenchymal injection into the spinal cord results in only localized transgene expression and thus would require an unreasonably large number of injections in humans. Retrograde transport in motor neurons of vector injected into muscle was also effective in a rodent model of ALS, but again would likely have limited clinical applicability due to the muscle mass that would need to be injected. In this proposal we will investigate efficacy of intrathecally administered gene therapy expressing IGF-1 in the SOD1-G93A rat model of ALS. In Specific Aim 1, we show that our gene therapy can promote motor neuron survival and protect the integrity of neuromuscular junctions. In addition we will show that this therapy attenuates the activation of astrocytes and microglia that helps contribute to th destruction of motor neurons. Furthermore, we will investigate the possibility that motor neurons can develop tolerance to elevated levels of IGF-1, a phenomenon that could limit the effectiveness of this therapy long-term. In Specific Aim 2, we will show that the improvements found in Aim 1 translate into improved motor function and increased life span. SOD1 rats will be evaluated using the grip strength, rotarod, and open field tests to evaluate several aspects of motor function. In addition, life span, age at disease onset, and the rate of disease progression will be measured to show efficacy. This study will provide the proof-of-principle data necessary to support future clinical trials of this approach.

? DESCRIPTION (provided by applicant): Our goal is to design, fabricate, and validate a novel class of nanofiber-based conduits for the surgical repair of transected peripheral nerves by restoring their continuity and functions. We will take a bioengineering approach to the repair of large defects in thick nerves. Based on knowledge of the anatomy of a peripheral nerve, as well as the biochemistry and cell biology involved in its injury and repair, we will design and fabricat multi-tubular nerve guidance conduits (mNGCs) with a honeycomb structure from a biodegradable polymer by electrospinning. The mNGC will be constructed by inserting a hexagonal array of seven small, single-tubular conduits into a large conduit. The wall of both small and large conduits has a tri-layer structure. A non-woven sheet of random nanofibers will be used as the outer layer to circumvent any possible tearing during surgery, together with an inner layer of uniaxially aligned nanofibers to provide longitudinal guidance for axonal extension. A phase-change material (PCM) sensitive to temperature change will be applied as a thin, porous layer to glue together these two layers of nanofibers. The PCM will be per-loaded with bioactive molecules for pulse release to digest the inhibitory chondroitin sulfate proteoglycan and promote neurite extension. The inner surface of each small conduit will also be seeded with Schwann cells derived from autologous bone mesenchymal stem cells (BMSCs) to support neurite outgrowth. We will evaluate the conduits using clinically relevant animal models capable of recreating the repair features in humans. The scope of this research includes: i) fabrication of the conduits using electrospun nanofibers; ii) in vitro evaluation of the differentiation of BMSCs on various types of nanofiber scaffolds under pulse release of bioactive molecules; iii) evaluation of the efficacy of single-tubular NGCs for sciatic nerve repair in a rat model; and iv) evaluation of the efficacy of multi-tubular NGCs for median nerve repair in an ovine model.

PROJECT SUMMARY High-grade Spinal Cord Glioma (SCG) is an orphan disease that results in significant morbidity and mortality, with no effective treatment options available. Despite significant advances in our knowledge of the disease process, there have unfortunately been limited changes to the clinical outcomes. In part, this represents the malignant nature of a disease that is refractory to the standard of care. On the other hand, this raises the question of the translational value of existing preclinical animal models, especially from a surgical standpoint ? where widely scalable large animal models of SCG were previously unavailable. To this end, the Boulis and Canoll laboratories partnered to begin addressing this gap in the field by developing a minipig SCG model. Through lentiviral targeting of the well implicated RTK/RAS/PI3K and p53 pathways, our preliminary data demonstrates the induction of high-grade astrocytoma with histopathologic, radiologic, and transcriptomic characterization in 100% of minipigs. Consequently, we posit that the next steps to advancement of this model system are to modulate tumor phenotype and to demonstrate its utility in a directly translatable surgical application. In the enclosed proposal, we will begin by evaluating the induction of SCG by targeting common genetic lesions implicated in the human disease including PDGFB, P53, CDKN2A, EGFR, and PTEN (AIM 1). This represents the opportunity to produce highly characterized SCG lesions for therapeutic testing in an immunocompetent, more anatomically relevant, large animal model. In parallel, we will apply our existing minipig SCG model (AIM 2) to perform the first intra-tumoral convection enhanced delivery (CED) study for SCG in a large animal. Rodent studies of chemotherapeutic CED for SCG have reported suppression of tumor growth and amelioration of neurologic deficits. However, these data cannot be readily scaled for translation due to anatomic limitations of rodent systems. Despite an ongoing Phase I human trial for CED in SCG, drug distribution and CED parameters are poorly understood. Indeed, failures of CED in human trials for intracranial glioma can be attributed to both ineffective drug distribution and single treatments. As such, our study will employ implanted pumps for prolonged intratumoral CED. We will investigate parameters (flow rate, volume of infusion) to evaluate optimal readouts (volume of distribution, reflux, safety, radiologic vs chemotherapeutic distribution). These data will have immediate translational impact on present and future trials.