Brian K. Kaspar - US grants

DESCRIPTION (provided by applicant): Gene therapy has significant promise for the treatment of neurodegeneration. However, current gene delivery technology must be engineered for improved delivery efficiency, targeting to affected cell types, and the ability to transduce large and often surgically inaccessible regions of tissue. The recent findings that AAV vectors undergo retrograde transport along axonal projections also raises the intriguing possibility for efficient targeting and delivery of therapeutic payloads to cells that are delicate and otherwise difficult to surgically access such as motor neurons residing in the spinal cord. However, we have found that the efficiency of AAV retrograde transport can be limiting in a mouse model. We therefore propose to employ a directed molecular evolution approach to develop novel AAV capsid variants with enhanced retrograde transport in vivo. We anticipate this work may significantly improve the therapeutic efficacy and cost effectiveness of retrogradely delivered biologics to target difficult to access areas of the nervous system. Improvements in the retrograde transport ability of AAV would therefore allow more efficient delivery to spinal cord motor neurons for numerous diseases including Amyotrophic Lateral Sclerosis (ALS) and spinal cord injury. Our Specific Aims are: 1. To determine whether directed evolution can yield AAV variants with enhanced retrograde transport properties in vivo. 2. To characterize the enhanced retrograde transport variants for transduction efficiency and analyze sequence-function relationships for in vivo gene delivery of reporter genes. 3. To determine whether retrograde transport variants harboring the neuroprotective gene IGF-1 can enhance therapeutic efficacy in a mouse model of ALS. We believe the development of our library of AAV mutants will provide for novel reagents to be provided to the research community.

[unreadable] DESCRIPTION (provided by applicant): In pursuit of improving gene delivery to all areas of the CNS in a safe and efficient manner, the objective of this proposal is to develop, optimize, and test this alternate delivery route to overcome the limitations of the blood brain barrier, which is a specific focus of this Request for Applications. Our strategy is centered on a specialized region of the brain termed the deep cerebellar nuclei (DCN). We will employ Adeno- Associated Viral Vector (AAV), a vector that has shown to be non-pathogenic with safety profiles being established in numerous human clinical trials as well as initial signs of clinical success in diseases such as Parkinson's disease and a viral vector that we have considerable experience with 1, 2 . We have recently examined this novel global gene delivery approach in a mouse model for Amyotrophic Lateral Sclerosis (ALS) with the approach appearing to be robust and therefore feasible. Several reports have shown the potential actions of neurotrophic factors to delay disease onset and progression 4-6 . However, challenges still remain to effectively translate these approaches to the clinic. We hypothesize that this one-time single injection delivery strategy to the DCN will result in global CNS transduction, allowing for widespread trophic support to the brainstem, spinal cord and motor cortex. We further hypothesize that this approach will greatly increase motor coordination, extend survival, and rescue neuropathological symptoms when the delivery approach is tested in ALS transgenic mice. The main goal of this proposal is to define a more effective therapeutic route for global gene delivery utilizing an AAV vector. This approach may be amenable for treating multiple neurological diseases and conditions, primarily associated either with the spinal cord such as ALS and spinal cord injury, or with the brain such as Alzheimer's and Parkinson's Disease. Our specific aims are shown below. 1.) To determine whether a single injection of AAV to the DCN targets the CNS and all regions of the spinal cord. 2.) To determine the efficacy of DCN gene delivery of AAV-IGF-1 in an animal model of ALS. Current gene therapy remains to be a challenge due to effective methods to target the CNS including the difficulty to access the spinal cord. Systemic delivery of gene therapy vectors to the CNS has been challenging due largely by their inability to cross the blood-brain barrier (BBB). Our strategy is centered on targeting a specialized region of the brain termed the deep cerebellar nuclei (DCN) with adeno-associated vectors (AAV) to deliver transgenes. We have decided to target the DCN because of its extensive connections with all regions of the spinal cord and brainstem, and would thus serve as an ideal gene delivery route. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is a genetic disorder characterized by the loss of lower motor neurons (LMNs) residing along the entire length of the spinal cord. SMA is caused by a reduction in the expression of the survival motor neuron (SMN) protein. The disease has an incidence of 1 in 6000, and in general, the earlier the symptoms, the shorter lifespan of patients. To date there are no effective therapies, particularly for the most severe forms of this lethal disease. Many lines of evidence support that increased amounts of SMN protein in motor neurons lessen disease severity. To date, therapeutic approaches have mainly focused on evaluating drugs for increasing SMN levels or enhance residual SMN function. Despite years of screening, no drugs have been identified to be fully effective for increasing SMN levels for a restorative therapy. Given the known genetic defect, in which SMN levels are decreased, gene replacement strategies may offer promise for SMA patients. New enhancements in viral vectors are demonstrating significant promise for a number of neurological disorders. This proposal seeks to develop novel gene delivery platforms based on a non-toxic, long-term expressing vector system;adeno-associated virus (AAV). While gene and drug delivery to the spinal cord has remained a challenge, we have recently discovered a unique ability for AAV serotype 9 to be highly efficient at delivering genes to spinal cord motor neurons of new born mice following vascular delivery. Indeed, in some areas of the spinal cord, over 90% of motor neurons were targeted by an AAV9-GFP marker gene, demonstrating the potential for AAV9 to be an effective gene delivery platform for SMA. Furthermore, we have demonstrated preliminary results that AAV9 is efficient at delivering genes to skeletal muscle across the entire body. These results are meaningful given that AAV9 targets both motor neurons and skeletal muscles, which are significantly affected in this disease. Our proposal seeks to further enhance this delivery platform in efforts to develop successful treatment strategies in SMA. This work will provide important information for translating an SMN gene replacement strategy to the clinic for SMA patients and provide needed information in efforts to develop an Investigational New Drug (IND) application. The aims of this proposal are specific goals for this NINDS developmental program to advance promising therapies to the clinic. Aim 1: Optimize vascular injection of AAV9-GFP to neonatal animals for maximal gene delivery to lower motor neurons. Aim 2: Evaluate optimal delivery of AAV9 expressing SMN for neonatal gene replacement in a mouse model of Type 2 SMA for improved function and survival. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is a genetic disorder characterized by the loss of lower motor neurons (LMNs) residing along the entire length of the spinal cord. To date, therapeutic approaches have mainly focused on evaluating drugs for increasing SMN levels or enhance residual SMN function. Despite years of screening, no drugs have been identified to be fully effective for increasing SMN levels for a restorative therapy. Given the known genetic defect, in which SMN levels are decreased, gene replacement strategies may offer promise for SMA patients. We have discovered a novel delivery platform for highly efficient targeting of motor neurons in neonatal animals. We will develop these findings for the potential to translate into human SMA clinical trials.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease, which results in muscle paralysis and ultimate respiratory failure and death. The underlying cause for ALS remains unknown with no cure. Numerous reports, including work from our laboratory have demonstrated the potential for neurotrophic factors to be highly therapeutic in rodent models of familial ALS (fALS). Indeed, Insulin-like growth factor-1 (IGF-1), glial derived neurotrophic factor (GDNF), and vascular endothelial growth factor (VEGF) delivered at disease onset in ALS rodent models have demonstrated profound effects in delaying disease progression. Recent studies have surprisingly demonstrated that astrocytes and microglia expressing a mutation in the enzyme superoxide dismutase can exacerbate motor neuron death, supporting earlier studies that ALS is a non-cell autonomous disease. Specifically, glial cells have been shown to develop aberrant activity, secreting toxic signals that lead to motor neuron demise. Based on these results, therapies designed to neutralize glial cell toxicity would be highly beneficial to ALS patients. Until genetic screening identifies new ALS inducing genes, therapies that could potentially prolong the lives of ALS patients should be developed. The mechanism by which neurotrophic factors prolong survival and motor function has remained elusive. Recent preliminary work by our laboratory has demonstrated that both IGF-1 and VEGF can act to suppress the mutant glial cell mediated toxicity. In this proposal, we will investigate the relative efficiency of IGF-1, VEGF and GDNF to suppress aberrant glial activity and delay motor neuron death. Specifically, these factors will be tested utilizing an invaluable in vitro model for ALS that was recently developed in our laboratory. We will also test these factors in an in vivo AAV (Adeno-Associated Virus) gene delivery paradigm that efficiently targets all regions of the spinal cord. We will analyze the mechanism by which these trophic factors mediate their effects on astrocytes and finally, we will test an optimal combination of these factors in familial fALS mice using our expertise in AAV vector gene delivery to the CNS. The specific aims of this proposal are: Specific Aim 1.) To determine the efficiency of neuroprotection and reduction of glial cell toxicity using potent neurotrophins in an in vitro based model of fALS. Specific Aim 2.) To determine whether IGF-1, VEGF, and GDNF act in combination to alter aberrant ALS glial activity and provide additive neuroprotection in an in vitro based model of fALS. Specific Aim 3.) To determine whether a combinatorial neurotrophic factor therapy is beneficial in a mouse model of fALS. PUBLIC HEALTH RELEVANCE Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease, which results in muscle paralysis and ultimate respiratory failure and death. The underlying cause for ALS remains unknown with no cure. We have shown the potential for neurotrophic factors to be highly therapeutic in rodent models of familial ALS including IGF-1, VEGF, and GDNF. Our proposal focuses on evaluating optimal trophic factors individually or in combination to delay motor neuron degeneration. We have developed an in vitro based model of ALS that utilizes stem cells directed to motor neurons and ALS containing astrocytes, which recapitulates the disease. We will subsequently test the optimal combination of these factors using gene delivery in a rodent model of this devastating disease in order to define an optimal therapy for this debilitating disorder.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is one of the most common neuromuscular diseases worldwide. It is a devastating motor neuron (MN) disease, resulting in muscle paralysis and ultimately respiratory failure leading to death. There is currently no effective therapy to significantly alter the disease course in humans. 90% of ALS cases are sporadic (SALS), in which no known genetic or environmental cause is known. The remaining 10% of patients have a familial variant of the disease (FALS). Approximately 20% of FALS have mutations within the gene encoding superoxide dismutase 1 (SOD1). For these FALS patients, mutant SOD1 (mtSOD1) is a clear pharmacological target, and it is well established that reduction of mtSOD1 prolongs lifespan in several ALS mouse models. The clinical and pathological presentation of both FALS and SALS is identical, in which striking gliosis, increase proliferation of astrocytes and microglia, occurs accompanied by selective MN cell death. Recent studies using mtSOD1 animal models have shown that non-neuronal cells contribute to disease onset and progression, and evidence for non-cell autonomous motor neuron death has mounted. Indeed, it has been shown that the reduction of mtSOD1 expression in astrocytes by breeding transgenic mice contacting the floxed mtSOD1 with mice expressing Cre from the glial fibrillary acidic protein (GFAP) promoter had significant effects in the disease These crosses showed that disease progression was significantly slowed and the effect on survival was profound. However, an important question remains from these studies: What is the timing for removing mtSOD1 from astrocytes that delays disease progression and extends survival? We believe that it is important to identify the latest time point during disease progression in which the removal of mtSOD1 is still efficacious, as many patients are diagnosed after disease onset. Therefore, it is crucial for therapy development to identify the 'point of no return' in ALS. We recently reported a very efficient method to target glia, predominantly astrocytes, in the adult Central Nervous System via systemic delivery of a vector based on Adeno-associated virus serotype 9 (AAV9).This discovery will enable our laboratory to efficiently suppress mtSOD1 in glia at various time points during disease (onset, early and late phase) in ALS mouse models allowing us to address this important question and forming the basis of this translational proposal. Specifically, we will: Determine the clinically relevant time frames when reduction of mtSOD1 in astrocytes provides therapeutic benefit. At the completion of this project, it is our expectation that we will have identified the optimal as well as the latest time points for decreasing mtSOD1 in glia that results in delayed disease progression and extends survival of SOD1G93A mice. This work will generate important insights to unravel the optimal therapeutic time window for attenuating aberrant glial cell toxicity in ALS providing the basis for a translational approach to remove mtSOD1 in patients.

DESCRIPTION (provided by applicant): The autosomal recessive disorder proximal spinal muscular atrophy (SMA) is the most common genetic cause of infant death and has an incidence of 1:6,000-10,000 live births1. The most common form is type 1 SMA or Werdnig-Hoffman Disease which results in hypotonia and progressive weakness and is typically recognized in the first few months of life2. Diagnosis of type 1 SMA occurs in the first 6 months and death usually occurs by age two. There is no treatment available to slow or halt disease progression, but our recent preclinical studies utilizing gene delivery in newborn rodent models of SMA suggest gene therapy may hold promise. SMA is an attractive disease for gene therapy because it is a single gene defect that results in low amounts of the survival motor neuron (SMN) protein (hypomorph) versus a total deficiency3. SMN is a ubiquitously expressed protein that is essential in all tissues and is not associated with toxicity when over expressed in mice4 or in humans5 with multiple copies of the gene. In addition, disease severity correlates with SMN protein levels emphasizing the potential therapeutic benefit for SMN as a treatment strategy.1 We have discovered that systemically delivered AAV96-SMN delivered early to a severe rodent model of SMA7 results in substantial rescue of the lifespan8 . This work was published in Nature Biotechnology in 20109,10 as well as similar reports 11-13. We have expanded this work in additional studies with now over 50 animals that have been treated with scAAV9-SMN and had remarkably consistent results with long survival of SMA mice. In the initial phases of treatment the evaluator is completely blinded. However, while blinding is maintained throughout the study, it is obviously difficult to maintain a true blind status with suc a dramatic effect on survival10. This sets the stage to advance to human clinical trials. However, systemic gene delivery, while it has certain advantages for the youngest patients, has limitations when advancing to the large and older patients. First, systemic gene delivery requires significant amounts of virus, and may be technically and economically challenging. Second, systemic gene delivery targets all organs, which may increase safety considerations, and in patients with neutralizing antibodies against AAV9 be less effective. We and others have recently discovered that AAV9, when delivered to the CSF results in efficient motor neuron targeting with a fraction of the virus utilized in our systemic gene delivery studies14-16. When virus is delivered to the CSF, via intraventricular injections, we and others13,17 have demonstrated that SMA mice survive significantly longer, greater than 100 days of age, which is again a remarkable finding in a mouse model that typically dies at 15 days of age (preliminary data). This formulates the basis for the proposed U01 application to advance to IND submission and human clinical trials. The specific Aims proposed for this U01 are: Aim 1: Develop pre-clinical data of CSF delivered AAV9-SMN in the 7 mouse model of SMA to support a pre-IND Aim 2: Optimize intrathecal dosing in nonhuman primates for most efficient targeting of spinal motor neurons using scAAV9.CB.GFP Aim 3: Assess toxicology and immune response following AAV9-SMN administration in mice and nonhuman primates (non-GLP) and submit a Pre-IND to the FDA. Aim 4: Formally assess toxicology, immune response, biodistribution and long-term expression in mice that will support an IND application to the FDA (GLP). Aim 5: Prepare and Submit an IND to the FDA.

DESCRIPTION (provided by applicant): Spinal muscular atrophy (SMA) is an autosomal recessive disorder which is the most common genetic cause of infant death. SMA is caused by loss or mutation of survival motor neuron 1 gene (SMN1) and retention of the SMN2 gene, which leads to insufficient levels of SMN for motor neurons. Introduction of scAAV9-SMN either through the vascular system or via intrathecal (CSF) delivery in SMA mice results in a marked correction of the phenotype. The advantage of intrathecal delivery is the lower titer of virus that can be used for correction, thus simplifying the production of scAAV9-SMN. While the efficiency of transduction of motor neurons has been investigated in pig and in monkeys, the ability to correct a SMA phenotype in a large animal model has not been investigated. This is due to fact that there is no large animal model of SMA currently available. A large animal model of SMA can be used to address the importance of high SMN levels in motor neurons, the efficiency of SMN transduction required to obtain a clinical benefit, and when SMN needs to be induced to obtain clinical benefit. We therefore propose to optimize scAAV9-SMN delivery to motor neurons in a clinically relevant large animal porcine model of SMA and develop gene therapy protocols for the treatment of SMA in this model. In particular, we will address transduction efficiency including repeat administration and when SMN must be introduced to correct a pig model of SMA. We will create the pig SMA model by reducing SMN in the required neurons using scAAV9- shRNA: SMN knockdown. The shRNA is specific for pig SMN and does not affect the levels of human SMN. The shRNA is delivered via intrathecal delivery of a scAAV9shRNA construct that expresses SMN and GFP. Injection is performed in 5-day-old piglets and EMG, clinical presentation, and a ramp test will be used to evaluate the affected pig. The GFP can be used to follow the efficiency of motor neuron transduction and EMG to follow the progression of functional motor neuron loss. We will then introduce scAAV9-SMN at various stages of the disease including pre-symptomatic, at the first signs of phenotype, and later stages when the hind limbs are severely weak. Furthermore, we will determine if restoration of SMN levels must occur prior to CMAP and MUNE reduction, which will in turn indicate when SMN needs to be restored in SMA patients to obtain the greatest clinical benefit. These experiments will set the parameters for all SMN induction therapies in SMA and allow determination of when to introduce a therapeutic agent.