Beverly L. Davidson, PhD, University of Michigan - US grants

Many inborn errors of metabolism cause devastating neurologic disease. Representative of such disorders are the mucopolysaccharidoses (MPS), and specifically, beta-glucuronidase deficiency or MPS VII. Systemic treatments for the MPS have no effect on CNS involvement. Thus direct methods to correct the deficiency in brain should be developed, evaluated, and optimized. In the initial two years of funding of this award we have identified key limitations to efficacious application of adenoviral vectors to brain. Importantly, our data suggest that the limitations can be overcome, and that a therapeutic response can be achieved. Specifically, we hypothesize that increased transduction efficiency will improve efficacy of adenoviral mediated gone transfer in the CNS of gusmps/gusmps mice. Preliminary data using adenoviruses containing knobs from other serotypes support this hypothesis. Also, our data show that E4 deletion in the viral backbone, or transient inhibition of antigen presenting cell activation and T cell stimulation at the time of adenovirus vector delivery, significantly prolongs transgene expression. Finally, we hypothesize that transduction of cells responsible for moderating the constituents of the CSF, or targeting the vector to the vascular endothelium will increase the distribution of corrected cells to deficient mice and improve efficacy.

DESCRIPTION (Abstract of the application) The goal of this program is to develop a gene therapy approach for stable, efficient gene transfer to liver using retroviral vectors. Our preliminary data show that we can achieve gene transfer to liver with Moloney murine virus based retroviral vectors without prior toxic injury or surgical removal of hepatocytes, and that gene expression with reporter viruses persists for at least 9 months. This is accomplished by prior exposure of the liver to growth factors, and then subsequent i.v. infusion of high titer (>10e8 colony forming units/ml) retrovirus at the peak of hepatocyte proliferation. Additional preliminary data suggests that the growth factors to be studied, recombinant keratinocyte growth factor (rKGF) and recombinant hepatocyte growth factor (rHGF) induce different populations of hepatocytes to proliferate. Also, hepatocytes are responsive to a second exposure of rKGF. In the first aim of this proposal we will take advantage of these properties to optimize the levels of gene transfer that can be achieved using reporter viruses. In the second aim of this proposal experiments will be done to determine the potential therapeutic application of this method to the treatment of hyperlipidemia in low density lipoprotein receptor (LDLr) and apoprotein E (apoE)/LDLr double knock out (KO) mice. We have previously established biochemical and morphometric methods to quantitate alterations in lipid metabolism and atherosclerosis in these murine models. We will test the effects of LDLr gene transfer on cholesterol profiles, rate of VLDL and LDL clearance, and progression and severity of atherosclerotic disease. We hypothesize that a sufficient number of hepatocytes can be induced to proliferate with growth factors, subsequently allowing efficient gene transfer with retroviral vectors. We further hypothesize that LDLr gene replacement in murine models of hyperlipidemia will restore the ability of their hepatocytes to maintain normal plasma lipid profiles in vivo, preventing atherosclerosis. At the completion of these studies we will understand the level of gene transfer required to see a therapeutic response. Understanding the correlation between the number of transduced cells and modulation of disease severity is important in translating this approach to larger animal models, and ultimately, to humans.

transfection /expression vector; technology /technique development; gene therapy; biotechnology; cystic fibrosis; biomedical facility; Adenoviridae; helper virus; fluorescent dye /probe; plasmids;

Vector Core The Vector Core is a service facility supported in part by the University of Iowa College of Medicine but also by RO1s, program projects (PPGs), Center Grants (P30s) and SCORs. The facility provides an integrative function in facilitating gene transfer studies in both applied and basic research. GOALS The specific aims of the Vector Core for this PPG proposal are i) to prepare working stocks of previously generated recombinant viruses for investigators to perform gene transfer experiments, ii) test all viruses for wild type contamination and pfu/particle ration, iii) generate new recombinant viruses as needed including cloning into shuttle vectors, iv) assist in the evaluation of initial gene transfer studies by helping set up molecular and/or enzymatic assays. By providing these services, the Vector Core substantially reduced the overall cost and time of setting up routine vector production in each investigators laboratory.

DESCRIPTION (provided by applicant): The cumulative incidence of lysosomal storage diseases has been estimated at 1 in 10,000 live births with 65% demonstrating significant CNS involvement. Systemic therapies can effectively alleviate visceral disease, but the relative impermeability of the human blood-brain barrier has prevented effective correction of CNS deficits. Thus, directed application of enzyme to the brain is required. We hypothesize that recombinant feline immunodeficiency virus (FIV) vectors can be developed and used to complement the enzyme deficiency and accompanying CNS disease. Because many lysosomal enzymes are secreted as well as traffic directly to the lysosome, and uptake of secreted enzyme occurs from transduced and non-transduced cells, delivery to all deficient cells is not required. However, enzyme must reach a significant proportion of the brain for benefit, but optimal methods to accomplish global correction for the LSDs are unknown. Novel envelopes for pseudotyping FIV, identified during the prior award, give us the opportunity to now test how best to achieve this. We will use the beta-glucuronidase deficient (MPS VII) mouse as a model of progressive CNS lysosomal storage disease to address our questions. Two hypotheses will be tested 1) delivery of FIV pseudotyped with envelopes that provide broad transduction profiles or delivery of enzyme from the CSF will reverse functional deficits in the MPS VII mouse brain and 2) an envelope that provides for transduction of resident neuroprogenitor cells will correct functional deficits in the MPS VII brain. Our data will reveal the levels of enzyme required for correction of functional deficits, and the optimal cell targets for achieving persistent global correction of neuropathology and functional restoration in MPS VII brain. The importance of these studies extends to other lysosomal storage diseases affecting the CNS, and many neuropathological conditions for which lentivirus-mediated therapy may be beneficial in future years.

The University of Iowa (UI) will increase the number of underrepresented minorities in biomedical research by retaining minority undergraduates in the biosciences and transitioning them into biomedical research graduate programs and ultimately to careers in biomedical research. 1. To increase retention of UI undergraduate minority students in the biosciences. 2. To facilitate the entry of undergraduate minority students into graduate and professional programs in the biosciences. 3. To encourage minority dental, medical, nursing, pharmacy and other health professional students to consider academic research careers-by providing them with research training and experience. Specific Aims: 1. To provide minority students with pre-college foundation courses, continuing academic support and laboratory research experiences. 2. To enable minority to develop mentor relationships with successful faculty biomedical researchers. Mentors will employ minority students in their biomedical research laboratories. 3. To provide minority students with continuous enrichment seminars in social, ethnic and regulatory aspects of biomedical science; these will convey the excitement and promise of biomedical research careers. 4. To prepare minority bioscience undergraduates to enter biomedical graduate training programs. 5. To engage minority students in MD, DDS and other health professional programs in biomedical research and careers. The project will provide minority students with academic, financial and social support. in the summer immediately after high school graduation, they will begin courses on, Interpreting Scientific and Medical Literature, and Library and WWW Research. In year 1, students will serve as library research assistants to their faculty mentors. During the freshman year, and for four years thereafter, study groups run by graduate assistants, tutoring services, and participation in weekly bioscience seminars and receptions, will help students to succeed in their required and elective courses (mathematics, biology and chemistry, etc.). The next summer will include an Introduction to Laboratory Sciences Course (cell biology, immunobiology, neurobiology, and genetics) that incorporates discussion of The Responsible Conduct of Research. After completing this course, minority students will be RAs in their mentors' laboratories. Throughout their undergraduate years, minority students will continue RAships in mentors' labs. In summer number 3, minority students will complete a course in biomedical graduate admissions preparation and career planning. We will transition project students directly into supportive UI biomedical research graduate programs laboratories. The project features on-going evaluation and revision of program efforts so as to best meet minority students needs.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] No treatment exists for any dominantly inherited neurodegenerative disease. In the dominant polyglutamine (polyQ) diseases expansion of a CAG triplet repeat results in a lengthened polyglutamine domain, conferring a dominant toxic property on the gene product. One promising strategy for therapy is therefore reduction of disease protein expression. Recent discoveries coupled with our own preliminary results suggest that small inhibitory RNA (siRNA) may be particularly effective in accomplishing this goal. In this proposal we will use spinocerebellar ataxia type I (SCAl), the best-characterized polyQ disorder, as our principal disease paradigm to test siRNA as a therapeutic approach. Neurodegeneration in SCAl is caused by CAG-repeat expansion in ataxin-1. Here, we will test the effect of siRNA in culture and animal models of SCA-1, which are perfectly suited to test the general therapeutic utility of this approach.

The Gene Transfer Core at the University of Iowa is integrated into multiple gene therapy projects directed at the study of diseases of the lung with particular application to cystic fibrosis. Gene Transfer Core staff are active participants in the development of gene transfer technologies. Interactions between Gene Transfer Core staff and colleagues, Zabner, Welsh, and McCray allows for cross-fertilization of ideas, technical advancements, and innovations in vector design. Additionally the Gene Transfer Core benefits from interactions with investigators in the Cardiovascular, Macular Degeneration and CF Centers. The Gene Transfer facility's overall objective is to support Drs. Zabner, Welsh and McCray in the use of gene transfer technologies. This includes consultation with them, development of novel vectors, collaborative testing of vectors generated for function and purity, and finally routine preparation. The Gene Transfer Core staff and investigators are in close contact through all phases of vector design and generation. Thus, the Core serves as both a research and development facility for gene transfer studies, and a service facility for routine vector preparations. As a part of the service the Gene Transfer Core will provide purified and concentrated preparations of recombinant adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus). This facility will also provide access to standard cell lines, expression plasmids, and stocks of recombinant reporter viruses. The main responsibilities of the Core will be: Prepare recombinant vectors; Quality control; Vector Dissemination; Maintain a database of vector stocks available for use; Catalogue plasmid database of expression vectors; develop new expression vectors as needed; Develop novel methods for virus production; Design and develop novel vectors.

This project is focused on the development of RNAi as therapy for HD. We previously demonstrated the ability of RNAi to silence a disease allele in an animal model of a related neurogenetic disease, Spinocerebellar ataxia type 1, resulting in improved behavior and neuropathology. We will now test several specific hypotheses regarding inhibition of mutant htt expression by RNAi: 1) RNAi can protect, and/or reverse, the neuropathology in mouse models of human Huntington's disease. Earlier studies in an HD mouse model with an inducible mutant allele demonstrate that if expression of the disease allele is abrogated, behavior and pathology improve. RNAi for silencing could yield a similar, promising benefit. 2) RNAi targeted to htt cDNA polymorphisms can be utilized for allele-specific silencing of mutant htt. One published disease-linked polymorphism and several novel ones will be tested for their utility to specifically silence the disease allele in cell culture studies. 3) Regulated RNAi can be achieved in vivo, and can be used to address duration of efficacy. HD usually takes decades to develop. If RNAi is beneficial it is unlikely that lifelong suppression of expression is required for sustained benefit. Thus we will take advantage of our recently developed regulated RNAi vectors to determine how long RNAi-induced improvements last and whether any problems from expressing RNAi in HD brain resolve once vector-expressed RNAi is reduced. 4) The off target effects of RNAi are minimal and can be resolved by cessation of RNAi. Earlier microarray analyses on HD mice brain from various models have shown that transcriptional changes induced by mutant htt are confined to a circumscribed set of genes. Regulatable vectors allow us to test if off-target effects resolve when shRNAs are no longer expressed.

Gene transfer is becoming increasingly important as a research tool and has clinical potential as well. The Gene Transfer Vector Core (GTVC) brings expertise to cancer from gene transfer experience in multiple other areas of biomedicine. The interaction with multiple investigators from various disciplines and experience with multiple different types of tissues allows for cross-fertilization of ideas, technical advancements, and innovations in vector technologies. The GTVC provides 1) Consultation with the Principal Investigator 2) Development of novel vectors 3) Purified and concentrated preparations of recombinant adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus) 4) Collaborative testing of vectors generated for function and purity, and finally routine preparation 5) Standard cell lines, expression plasmids, and stocks of recombinant reporter viruses GTVC staff and investigators are in close contact through all phases of vector design and generation. Thus, the GTVC serves as both a research and development facility for gene transfer studies, and a service facility for routine vector preparations

Description (provided by applicant): Despite recent progress that has defined their genetic basis, many inherited neurological diseases remain untreatable and ultimately fatal. In particular, disorders caused by dominant mutations, including Huntington's disease (HD), familial Alzheimer's disease (AD) and DYT1 dystonia (DYT1), progress inexorably due to the toxic or dominant-negative actions of the encoded disease proteins. RNA interference RNAi) has recently emerged as a powerful tool by which to suppress specific genes. In vitro work has now established that dominant disease genes (including those in HD, AD and DYT1) can be silenced by RNAi, in some cases in an allele-specific manner that suppresses only the disease allele. It is unknown, however, whether this technology can work in the mammalian brain to prevent or cure such diseases. The projects in this program address this important question, building on recent advances in RNAi technology and viral-mediated gene transfer to the brain. Project 1 explores the potential of RNAi to prevent or reverse neuropathology in mouse models of HD, one of at least nine neurodegenerative diseases caused by expanded polyglutamine. Project 2 tests whether two genes central to the pathogenesis of familial and sporadic AD can be suppressed by RNAi. Studies will compare the utility of silencing BACE, APP, or both genes, in preventing the development of pathological features in mouse models of AD. Project 3 takes advantage of new cellular and mouse models of DYT1 to test allele-specific silencing of DYT1, and to address new theories about the pathogenic mechanisms of this disease. The PPG benefits from outstanding support by the Vector Core, the Neuropathology Core and the Administrative Core. Together, these studies will provide answers to questions about the efficacy, specificity and longevity of RNAi in the mammalian brain. They will also take us closer to our long-term goal of developing RNAi as therapy for HD, AD, DYT1 and related neurological diseases.

Description (provided by applicant): Despite recent progress that has defined their genetic basis, many inherited neurological diseases remain untreatable and ultimately fatal. In particular, disorders caused by dominant mutations, including Huntington's disease (HD), familial Alzheimer's disease (AD) and DYT1 dystonia (DYT1), progress inexorably due to the toxic or dominant-negative actions of the encoded disease proteins. RNA interference RNAi) has recently emerged as a powerful tool by which to suppress specific genes. In vitro work has now established that dominant disease genes (including those in HD, AD and DYT1) can be silenced by RNAi, in some cases in an allele-specific manner that suppresses only the disease allele. It is unknown, however, whether this technology can work in the mammalian brain to prevent or cure such diseases. The projects in this program address this important question, building on recent advances in RNAi technology and viral-mediated gene transfer to the brain. Project 1 explores the potential of RNAi to prevent or reverse neuropathology in mouse models of HD, one of at least nine neurodegenerative diseases caused by expanded polyglutamine. Project 2 tests whether two genes central to the pathogenesis of familial and sporadic AD can be suppressed by RNAi. Studies will compare the utility of silencing BACE, APP, or both genes, in preventing the development of pathological features in mouse models of AD. Project 3 takes advantage of new cellular and mouse models of DYT1 to test allele-specific silencing of DYT1, and to address new theories about the pathogenic mechanisms of this disease. The PPG benefits from outstanding support by the Vector Core, the Neuropathology Core and the Administrative Core. Together, these studies will provide answers to questions about the efficacy, specificity and longevity of RNAi in the mammalian brain. They will also take us closer to our long-term goal of developing RNAi as therapy for HD, AD, DYT1 and related neurological diseases.

The Gene Transfer Core at the University of Iowa is integrated into multiple gene therapy projects directed at the study of diseases of the lung with particular application to cystic fibrosis. Gene Transfer Core staff are active participants in the development of gene transfer technologies. Interactions between Gene Transfer Core staff and colleagues, Zabner, Welsh, and McCray allows for cross-fertilization of ideas, technical advancements, and innovations in vector design. Additionally the Gene Transfer Core benefits from interactions with investigators in the Cardiovascular, Macular Degeneration and CF Centers. The Gene Transfer facility's overall objective is to support Drs. Zabner, Welsh and McCray in the use of gene transfer technologies. This includes consultation with them, development of novel vectors, collaborative testing of vectors generated for function and purity, and finally routine preparation. The Gene Transfer Core staff and investigators are in close contact through all phases of vector design and generation. Thus, the Core serves as both a research and development facility for gene transfer studies, and a service facility for routine vector preparations. As a part of the service the Gene Transfer Core will provide purified and concentrated preparations of recombinant adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus). This facility will also provide access to standard cell lines, expression plasmids, and stocks of recombinant reporter viruses. The main responsibilities of the Core will be: Prepare recombinant vectors; Quality control; Vector Dissemination; Maintain a database of vector stocks available for use; Catalogue plasmid database of expression vectors; develop new expression vectors as needed; Develop novel methods for virus production; Design and develop novel vectors.

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are noncoding RNAs expressed within the genomes of multiple species. Prior work has demonstrated that miRNAs are expressed in developing and adult tissues, and their expression can be constitutive, temporal, ubiquitous, or cell-specific. A new class of negative regulators of expression, miRNAs work by inhibiting translation of target messenger RNAs (mRNAs) via base pairing with mRNA 3'untranslated regions. This process is called RNA interference (RNAi). Since the discovery of RNAi in worms, which was awarded the Nobel prize in physiology or medicine in 2006, studies of naturally occurring RNAi in developing vertebrate organs and in adult animals have revealed their important contribution to cellular phenotypes. Recent work has shown that miRNAs participate in teratogen-mediated changes. We also know that teratogens impair proliferation of neural progenitors. What we do not know, however, is the interface between miRNAs and neural progenitor cell maintenance, proliferation and differentiation. In this proposal we will first use a focused approach to assess how a candidate miRNA, miR-34a, participates in cell fate decisions in neural progenitor cells in culture and in vivo. In additional studies we will ascertain how perturbing miR-34a levels impacts target protein expression. Two aims are proposed. In Aim 1 we will use anti-miRs and miRexpression vectors to test how miR-34a impacts neuronal differentiation in loss- and gain- of function experiments, respectively, in vitro and in vivo. In Aim 2 we will use anti-miRs and miR-expression vectors to test how miR-34a levels effects miR-34a target proteins.

[unreadable] DESCRIPTION (provided by applicant): The planned research addresses a problem of fundamental importance to the neuroscience community - brain delivery. While some studies suggest that bioengineered reagents can reach the brain when delivered into the systemic vasculature, the complex nature of these previously developed reagents makes implementation for science unwieldy, and adaptation as a therapeutic medicine unlikely. Only a handful of laboratories are capable of generating and using these complex reagents, and for most cases, there has been little progress towards translation to therapies despite more than 10 years of engineering and testing in animal models. We propose that new methods merging chemical simplicity with the power of in vivo selection can solve this monumental problem. In this proposal, we will test the feasibility of developing RNA aptamers capable of directing a payload to neurons, astrocytes, or brain endothelia cells. PUBLIC HEALTH RELEVANCE: This EUREKA proposal addresses a problem of fundamental importance to the scientific and medical community - getting material into the brain without directly injecting it into the brain. Some earlier work made a complex of materials that could deliver a gene of interest or a small peptide to the brain. But these studies were done more than a decade ago and yet this approach is still not used widely. This is probably because it is very labor intensive to make these complex materials. Also, because the systems currently used have many components, it is not likely to be developed into a drug. To get around this problem, we propose to use a very simple molecule, RNA. We can generate 10^12 different RNAs that naturally fold into various structures. Interestingly, these structures are capable of binding to a variety of cell surface proteins or other cell surface material. In our preliminary work, we found that delivery of some of these RNAs into the tail vein of the mouse could reach the brain. What we propose here is to develop more aptamers, and test their ability to reach different types of brain cells following delivery into the mouse tail vein. If we are successful, we would have made an enormous leap in the biosciences. Our data will be of exceptional importance and find broad use in the scientific community. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): 15-NS-103, 'Demonstration of "proof-of-concept" for a new therapeutic approach in a neurological disease.'Children with inherited lysosomal storage disorders (LSDs) affecting the central nervous system (CNS) succumb to disease because of lack of therapeutic options. Although these diseases result from deficiency of soluble lysosomal enzymes, and systemic administration is effective for correction of peripheral organs, methods for treating the brain are few. Presently, repetitive direct introduction of recombinant enzyme, introduction of stem cells, or multiple injections of recombinant viruses are being investigated as therapies. We have recently developed two complimentary approaches to achieve enzyme correction in brain, and shown efficacy in mouse models of LSD. In one method, we demonstrated that a single injection of adeno-associated virus (AAV) vector with tropism to the ventricular lining ependymal cells corrected the storage disease and reversed established behavioral deficits throughout the CNS. In a second series of the studies, we identified peptide epitopes that homed to the brain, and cloned these epitopes into the AAV capsid. When injected into the peripheral vasculature, the resultant peptide-modified AAVs were redirected from principally targeting hepatocytes, to target also the cerebrum, cerebellum, brain stem and spinal cord. As a result, established neuropathological and behavioral deficits were corrected. Both of these novel approaches show remarkable promise and their development into translational programs would benefit greatly from testing in large animal models. Thus, this proposal will combine the Davidson's laboratory expertise in vector development and delivery with the Katz and Coates laboratories expertise in canine models of human disease. In recent work Katz and Coates have characterized a canine model of late infantile ceroid lipofuscinosis (LINCL), resulting from mutations in the gene encoding tripeptidyl peptidase I (TPP1). This model shows progressive neurological disease and pathological hallmarks that closely mirror LINCL. As stated in the RFA, the challenge is to enter into the NINDS translational research program. Our published data, and evidence presented here demonstrating efficacy of the approaches in the LINCL mouse model supports the proposed research to establish proof-of-concept in the canine model of LINCL. In this work we will test the safety and efficacy of gene therapies designed to correct central nervous system manifestations in a dog model of childhood-onset neurological diseases. These studies are an important step in a translational research program encompassing established investigators and new trainees at the University of Iowa and the University of Missouri, which builds upon preliminary work in rodent models showing robust improvements in behavioral manifestations and neuropathology shortly introduction of therapeutic vectors.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15): 15-NS-102: Translation of Gene Silencing Therapeutics. The proposed challenge set forth in the RFA is to extend the current understanding of the feasibility and safety of RNA interference (RNAi) therapeutics for the treatment of chronic neurological disorders from rodent models of disease to a more clinically relevant species. The current proposal outlines a systematic approach to translate work we, and others, have undertaken to investigate RNAi as a potential therapy for the neurological disorder, Huntington's disease (HD) in cell culture and rodent models and apply these findings to the non-human primate (NHP). HD is a fatal, genetic disorder caused by mutations in HTT, which encodes huntingtin (HTT), and affects approximately 30,000 people in the United States alone. HD is characterized by hyperkinetic movements, loss of cognitive abilities and severe emotional disturbances. RNAi has emerged as a candidate therapy for HD because it can reduce disease gene expression. However two critical questions important to the general application of RNAi to brain disorders, and of specific importance to HD, have yet to be addressed. One, is long-term application of therapeutic RNAi to primate brain feasible for chronic neurodegenerative diseases like HD? Second, is the primate brain tolerant of knock down of both normal and mutant HTT alleles? To address the former question we, and others, have built the tools necessary for sustained expression of candidate therapeutic RNAi in brain using viral vectors. In this manner, questions of chronic application can be tested without re-delivery issues. In this proposal we will test the feasibility of long-term therapeutic RNAi using recombinant adeno-associated virus (AAV) vectors. To address the second question, we have built and tested therapeutic RNAi in rodent models of HD, and show improvements in disease-specific phenotypes, including survival. With these tools we can now answer the questions posed in a more relevant model, the normal NHP (rhesus macaque;Macaca mullata). The proposed studies are a collaboration between two laboratories: the Davidson Laboratory at the University of Iowa and the Ojeda Laboratory at the Oregon National Primate Research Center (ONPRC). The Davidson Laboratory has experience developing and testing RNAi therapeutics in rodents, while the Ojeda Laboratory has expertise in stereotaxic delivery of viral vectors to the NHP brain. Also, the ONPRC has methodologies, equipment and personnel in place that, along with Dr. Ojeda's group, can evaluate if application of HTT suppression, or RNAi in general, induces neuropathology or neurological symptoms after delivery of RNAi expression vectors to NHP brain. While these proof-of-principle studies will provide a platform to rigorously evaluate RNAi therapeutics for HD, information derived from our work can be applied to RNAi therapies for other chronic, neurological disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (Lou Gherig's disease), dystonia, spinocerebellar ataxias and Rett's Syndrome. Moreover, the results from our studies will provide the necessary tools for laboratories investigating RNAi therapy for other brain disorders in the NHP, such as neuropathic pain, encephalitis, various carcinomas, anxiety and depression. In this work we will test the safety and efficiency of gene therapies designed to treat the fatal neurogenetic disorder, Huntington's disease, in a clinical relevant model, the nonhuman primate. Experiments are designed to test questions relevant to moving therapeutic gene silencing strategies from efficacy studies in rodent models to the clinic, and to address important issues regarding this therapy for Huntington's disease. This work is a collaborative effort between laboratories at the University of Iowa and the Oregon National Primate Research Center at the Oregon Health Sciences University.

Vector Core The Vector Core at the University of Iowa is integrated into multiple gene therapy projects directed at the study of diseases of the lung, eye, brain, liver, heart and muscle, as well as at the study of the cardiovascular and neuromuscular systems. Also, Vector Core staff are active participants in the development of gene transfer technologies used by the Cardiovascular, Macular Degeneration, and Cystic Fibrosis Centers. The resulting interaction with multiple investigators from various disciplines allows for cross-fertilization of ideas, technical advancements, and innovations in vector designs. The overall objective of the Vector Core facility is to support investigators in the use of gene transfer technologies. This includes consultation with the Principle Investigator and staff, development of novel vectors, collaborative testing of the vectors generated for function and purity, and routine preparation of vectors (including quality control). The Vector Core staff and investigators are in close contact through all phases of vector design and generation. Thus, the Core serves both as a research and development facility for gene transfer studies and a service facility for routine vector preparation. As a part of its service, the Vector Core provides purified and concentrated preparations of recombinant adenovirus, adeno-associated virus (AAV), and retrovirus (including lentivirus). This facility will also provide access to standard cell lines, expression plasmids, and stocks of recombinant reporter viruses. The main responsibilities of the Core will be: ¿ Preparation of recombinant vectors ¿ Dissemination of vectors ¿ Maintainance of database of available vector stocks ¿ Maintainance of database of available expression vectors; development of new expression vectors ¿ Development of novel methods for virus production ¿ Consultation on and assistance in the design and development of novel vectors

Progressive and chronic airway inflammation and infection are hallmarks of cystic fibrosis (CF) lung disease. A reduction of the host inflammatory response could favorably modify the disease phenotype. Indeed this has been the goal of clinical trials with steroids and non-steroidal anti-inflammatory drugs. Another approach to reduce inflammation is to silence pro-inflammatory mediators by RNA interference (RNAi). Here, we propose to use several novel methods to accomplish RNAi in the airways. In one approach, we hypothesize that virally expressed inhibitory RNAs can effectively transduce airway epithelia and inhibit expression of our target genes. We will use methods developed in our laboratories for optimal inhibitory RNA expression. In a second approach, we will use peptide-siRNA complexes. In these experiments, we will take advantage of peptides that bind to airway epithelia to direct siRNA uptake and target gene silencing. For both viral and nonviral experiments, we will first test the effectiveness in vitro using well differentiated airway epithelia. Once optimized in vitro, the tools will be tested in vivo in wild type, and then CF pigs. Importantly, the methods we develop to deliver RNAi and reduce geneexpression in vitro and in vivo will have direct relevance to other pro-inflammatory targets, and newly identified participants in CF ainway disease.

No abstract provided

DESCRIPTION (provided by applicant): Abstract Cystic fibrosis (CF) is the most common lethal genetic disorder among Caucasians. As a consequence of mutations in the CFTR gene, most CF patients die from progressive lung disease for which there is no curative treatment. Expression of the CFTR cDNA in as few as 6- 10% of respiratory epithelia can correct the anion transport defect, and therefore gene therapy holds great promise for this autosomal recessive disease. Viral vectors such as adeno- associated virus (AAV) are among the safest tools available to deliver a corrective cargo to the airways, however, inefficient delivery continues to limit the field. We propose to overcome this limitation by using novel peptide epitopes that bind efficiently to the surface of well-differentiaed primary CF airway epithelia from humans and from a novel CF pig model. Our overall hypothesis is that peptide motifs with affinity to the apical surface of CF airway epithelia can be identified via phage panning, and incorporated into an AAV capsid to improve vector tropism for the airways. The newly engineered vectors will be used to correct the CF phenotype by gene addition in well-differentiated epithelia. This collaborative proposal combines expertise in CF and airway epithelial cell biology (Drs. McCray and Zabner) with expertise in phage panning and vector engineering (Drs. Davidson and Zabner). These PIs also have considerable expertise in gene therapy. This proposal encompasses two aims, In Aim 1, we will use panning with a phage display library to identify peptide motifs with affinity to the mucosal surface of well-differentiaed CF airway epithelia. We will test insert those motifs into new AAV capsids that emerged through capsid shuffling strategies and show improved transduction profiles, for improved targeting to the apical surface of CF airway epithelia. In aim 2, we will use the peptide ligand-modified AAVs to correct the CFTR anion transport and host defense defects in CF airway epithelia. PUBLIC HEALTH RELEVANCE: Project Narrative CF is one of the most common genetic diseases, yet new therapies for this recessively inherited disorder based on a molecular understanding of the disease have been slow to advance. One promising approach is gene replacement of the mutant CFTR to the major site of destruction, the lung. Unfortunately however, methods to deliver the corrected gene product to cell that line the airways in the lung are inefficient. In this work, we propose hih risk, yet high impact studies that will identify novel methods for achieving efficient delivery to he intact airway epithelia. Our innovative methods could also be applied to the broader spectrum of airway diseases, thus strengthening the overall impact of our findings.

DESCRIPTION (provided by applicant): Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) is a childhood-onset autosomal recessive neurodegenerative disease. This disorder, a lysosomal storage disease (LSD), is caused by mutations in CLN2, the gene that encodes the lysosomal hydrolase tripeptidyl peptidase I (TPP1). TPP1 deficiency causes epileptic seizures, vision impairment, cognitive and motor dysfunction, and is invariably fatal by late childhood. Currently, there is no treatment or cure for this devastating disease. The major barrier to treatment is the delivery of bioactive TPP1 enzyme to the central nervous system, a goal hindered by the selectivity of the blood-brain-barrier (BBB). In recent studies, our lab has engineered a brain-homing adeno-associated virus (AAV) gene delivery vector. Brain tropism of the virus is conferred by a peptide, GMNAFRA, which we identified by in vivo phage display panning in a mouse mode of LINCL. The GMN peptide has affinity for the luminal surface of brain vascular endothelial cells, which are the primary component of the BBB. When delivered peripherally, this peptide-modified virus (GMN-AAV) travels to, and transduces brain endothelia. Significantly, delivery of GMN-AAV encoding CLN2 in LINCL mice restored TPP1 expression in the brain and corrected neuropathological defects. Thus, this novel gene therapy approach bypasses the BBB by engineering endothelial cells to secrete recombinant enzyme directly into the underlying neuropil. In order to advance this exciting and promising gene therapy approach to LINCL patients, elucidation of the identity of the brain endothelial receptor molecule that interacts with the GMN peptide is required. The overall goal of this proposal is to perform experiments to discover this molecule, which mediates GMN-AAV brain targeting and transduction. Our hypothesis is that the receptor is a plasma membrane-associated protein or glycan moiety expressed on the surface of brain vascular endothelial cells in TPP1 deficient brain. In aim one, we will use affinity purification and quantitative mass spectrometry methods to isolate and identify the GMN-AAV receptor. In aim two, we will use mass spectrometry glycan profiling, and a mammalian glycan array developed by the NIH-operated Consortium for Functional Glycomics to determine whether GMN-AAV binds to specific glycans or classes of glycans that may be present on the surface of brain endothelial cells. Completion of these aims will enable us to advance the translation of this novel gene therapy to children with LINCL. In addition, this therapeutic strategy could be broadly applied to other LSDs that cause central nervous system dysfunction and neurodegeneration.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is one of several dominant neurodegenerative diseases caused by a similar toxic gain of function mutation in the disease protein: expansion of a polyglutamine (polyQ)-encoding tract. Currently, no therapy exists for HD. RNA interference (RNAi) has emerged as a leading method to reduce disease gene expression by targeting and degrading the encoding mRNA. Our preliminary work demonstrates that vector-mediated RNAi can reduce huntington (Htt) expression and improve disease phenotypes in mouse models of disease. We used minimal off-target silencing as a primary objective in vector design, taking advantage of bioinformatics and microarrays to identify transcriptional consequences of the inhibitory RNAs. These vectors, though minimized for off-sequence silencing, still reduce expression of both HTT alleles. Recent analysis of the HD population suggests that there are 4-5 predominant single nucleotide polymorphisms (SNPs) representing almost 90% of HD patients. This data gives us the opportunity to test if vectors for allele-directed silencing with minimized off-targeting are safe and effective in vivo. For this, we developed novel transgenic mice, with the mutant transgenes engineered to contain these relevant SNPs. We propose to now test if artificial miRNAs targeting disease-linked polymorphisms can preferentially silence mutant htt alleles in vivo. Finally, we have used data on transcriptional dysregulation in HD to identify, build and preliminarily test endogenously regulated promoters for expression control of inhibitory RNAs to further improve their safety profile. We will now test the capabilities of these promoters to drive RNAi in a disease responsive manner in vitro and in vivo.

ABSTRACT JNCL is a devastating childhood-onset neurodegenerative disease caused by deficiency in CLN3, a membrane-integral protein with unresolved function. Using a Cln3-reporter mouse, we previously discovered that in the postnatal mouse brain, expression is limited to a few subpopulations of neurons, but is widespread in endothelial cells (EC) that line the vasculature. Our continued studies show that brain EC from CLN3-deficient mice display impairments in drug efflux, regulatory volume response, and endocytosis. These functions are critical to normal operation of the blood-brain barrier (BBB), which governs selective passage of molecules between blood and brain, mediating import of nutrients and export of toxic substances away from proximal neurons. We postulate that brain EC dysfunction plays a dominant role in JNCL pathogenesis, and that restoring CLN3 to EC will alleviate disease progression. The current proposal seeks to test our hypothesis via two main Aims. For Aim 1 we plan to use a transgenic mouse approach to trigger EC-exclusive expression of CLN3, and assess whether this prevents the development of behavioral and pathological measures of JNCL. In Aim 2, we plan to generate an adeno-associated virus (AAV) vector with tropism for brain endothelium to test the efficacy of CLN3 gene transfer in the JNCL mouse model. Accomplishment of these aims will provide valuable information relevant to JNCL pathogenesis and potential treatment avenues. Should our hypothesis prove correct, this would distinguish central nervous system EC as a therapeutic target for JNCL. Moreover, therapeutic efficacy of our gene transfer approach, in which the vector is administered via intra-vascular injection, would be an exciting outcome with hope for translation to JNCL patients, and possibly more broadly to other neurodegenerative diseases.

DESCRIPTION (provided by applicant): A CAG repeat expansion in exon 1 of the HD gene product, huntingtin, causes Huntington's disease (HD), a fatal neurodegenerative disease for which there is no cure or neuroprotective treatment. Dysregulation of transcription is a major feature of HD pathogenesis, as indicated by a large body of work using RNA array techniques, and work on specific transcription factors and their targets. More recent studies have also suggested a role for huntingtin in RNA processing. Prior work on gene expression alterations in HD brain tissues used 3' biased gene expression arrays. Of increasing importance in many human diseases, particularly neurodegenerative diseases, is the occurrence of aberrant alternative pre-mRNA splicing. However, conventional gene expression techniques are not well suited to quantitative analysis of alternative splicing patterns, and do not sample rare transcript well. Several lines of evidence from our preliminary work suggest global splicing abnormalities in HD. For example, we reported earlier that microRNA miR-124 was significantly reduced in HD brains. Work by Maniatis and colleagues showed that miR-124 promotes neuronal-specific alternative splicing events by down- regulating an important tissue-specific splicing regulator, polypyrimidine tract-binding protein (PTBP1). Consistent with the decrease in miR-124, we have preliminary evidence for significantly increased PTBP1 mRNA levels in HD patient samples. Moreover, preliminary data suggest that several exons in genes regulated by PTBP1 show corresponding changes in exon inclusion/exclusion in HD brain. Inclusion or exclusion of non-constitutive exons can have dramatic effects on transcript stability and protein activity. Thus transcriptome alterations in HD may extend beyond up- and down-regulated genes to include changes in gene and protein isoforms. Assessing these events on a global scale for HD will aid efforts to unravel disease pathophysiology, and may identify new drug targets for therapy. In our work, which encompasses 3 aims, we will move from identification of the altered HD transcriptome, to validation, to in vitro and in vivo studies to test their relevance on HD phenotypes. These studies combine the genomics and bioinformatics expertise of the Xing lab and the HD expertise of the Ross and Davidson labs. The functional relevance of those changes will be elucidated using gain and loss of function studies in the Davidson and Ross labs, where both groups have substantial experience with HD models.

? DESCRIPTION (provided by applicant): Huntington's disease (HD) is an inherited neurodegenerative disease that strikes in the prime of life and has no disease-modifying treatment. HD is caused by CAG repeat expansion in the HD gene, causing complex and extensive cellular dysfunction. The identification of cellular targets that impact disease onset and progression and enlighten further mechanistic understanding of these targets are critical for development of new treatments. Mutant HTT (mHTT) and toxic fragments derived from the mutant protein are in a dynamic equilibrium poised to shift the homeostatic network from the appropriate balance of protein folding, misfolding, oligomerization and degradation to one in which that balance is disrupted. Upon network disruption, cellular proteins accumulate and degradation pathways become impaired. Our studies suggest that the E3 SUMO ligase, PIAS1, may be an important regulatory switch in this dynamic equilibrium. In published findings, we identified PIAS1 as a novel modulator of both SUMO-1 and SUMO-2 modification and accumulation of mHTT protein in cultured cells and that reduction of PIAS in Drosophila delays phenotypes caused by repeat expanded HTT. In recent preliminary data, we find that reduction of PIAS1 expression in R6/2 mice confers robust neuroprotection, suggesting PIAS may provide a selective therapeutic target. The communication and involvement between E3 SUMO ligases and protein clearance pathways are not well understood with respect to misfolded and accumulated proteins. In addition to functioning as a SUMO E3 ligase, PIAS is implicated in regulating transcription of proinflammatory cytokine signaling and innate immune response pathways. Therefore, clarifying the PIAS1 network in HD systems will provide a crucial understanding as to its role in HD pathology. We hypothesize that PIAS1 is a key regulator of HTT SUMOylation and accumulation, that it can modulate HD pathogenesis and that it may be a novel target for development of HD therapies. We propose to use cell based assays and in vivo studies to advance our mechanistic understanding of PIAS1-mediated networks, and validate PIAS1 as a molecular target for HD drug development. Specifically we will carry out the following proposed aims: Aim 1: PIAS1 modulation in HD mouse models. Aim 2: PIAS1 network in mHTT expressing neural cells. Aim 3: In vivo effects of mHTT expression in heterozygous PIAS1-null mice. Aim 4: Functional significance of PIAS1 domains in disease modifying pathways.

? DESCRIPTION (provided by applicant): This application requests funding for the 2015 Gordon Research Conference on CAG Triplet Repeat Disorders and the associated Graduate Research Seminar to be held at the Il Ciocco Resort in Barga, Italy from May 30 - June 5, 2015. This will be the eighth Gordon Research Conference on CAG Triplet Repeat Disorders. The previous five conferences have alternated between American (Mount Holyoke College, 2001, 2005; Waterville Valley NH, 2009 and 2013) and European (Il Ciocco, Italy 2003, 2011 and Aussois, France 2007) sites. This is the third year that there will be an associated Graduate Research Seminar. The CAG Triplet Repeat Disorders are a group of largely untreatable inherited neurological disorders which result from an expansion in a CAG trinucleotide repeat in the mutant genes. This group of diseases includes Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA, Kennedy's disease), spinocerebellar ataxias types 1, 2, 3, 6, 7, and 17, and dentatorubropallidoluysian atrophy (DRPLA). The disease-inducing CAG repeat expansion occurs in coding regions within the affected gene and can confer toxicity via RNA-dependent and/or protein-dependent mechanisms. Differences in the anatomical distribution of selective neuronal degeneration make it imperative to unravel their natural histories, cellular dysfunction and gene, RNA and protein pathways involved in the pathogenesis. To increase the pace of basic research discovery and set in place the contacts and clinical resources necessary to move the basic science forward, perhaps into the clinic, a multidisciplinary research effort is required. It is essential that collaborative projects between scientists from diverse disciplines ranging from organic chemistry and fruit fly genetics to neurology and human clinical trials be established. The conference on CAG Triplet Repeat Disorders will gather together young investigators and established senior scientists to deliver provoking lectures on the cutting-edge of science. In keeping with the Gordon Research Conference format, there will be generous time allocated for both structured discussions led by peers and for informal discussion and social interactions to facilitate collaboration. Strong emphasis is placed on training and mentoring of young scientists, and time will be devoted to career issues. All participants will be required to present posters. The 2015 GRC has new innovations including a devoted session within the GRC to GRS participants and more late-breaking talks to be pulled from the abstracts from prior years. Priority will be given to women, minorities and persons with disabilities when selecting participants for all sessions.

DESCRIPTION (provided by applicant): Spinocerebellar ataxia type 1 (SCA1) is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective treatment strategies for this disease. Previously, we showed that RNA interference (RNAi)-mediated silencing of ataxin-1 mRNA provides therapeutic benefit in mouse models of SCA1. Adeno-associated viral (AAV) delivery of an engineered microRNA (miRNA) targeting ataxin-1 to the cerebellum of well-established SCA1 transgenic mouse models improved motor phenotypes, neuropathology, and transcriptional changes. We now propose to move our lead vector forward by completing a series of milestones that will lead us to a Phase 1 clinical trial in human subjects. In the UH2 phase of this application we will 1) perform comparability testing of the proposed vector in nonhuman primates to confirm our proposed dosing regimen, and 2) hold a Pre-IND Type B meeting with the CBER of the FDA to receive input on the design of the planned GLP tox study and the proposed Phase 1 protocol. In the UH3 phase of this application we will 1) produce GMP- process comparable vector (GLP vector), 2) perform the planned GLP pharm/tox study in nonhuman primates and rodents, 3) produce GMP-grade vector, 4) prepare and file the IND with the FDA, and 5) perform a phase I/II clinical trial in SCA1 subjects.

Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the gradual appearance of progressive cognitive dysfunction with neuronal death in multiple areas of the cerebral cortex. Current therapies are symptomatic and there are no available treatments that alter the natural history of the disease. In previous proof-of-concept studies, and backed by strong human genetics data, we demonstrated that a single injection of an adeno-associated viral (AAV) vector into the lateral ventricle of AD mice leads to sustained APOE2 expression from ependymal cells and secretion into the cerebrospinal fluid (CSF). Once in the CSF, this protein distributes throughout the entire brain with a beneficial effect on many AD-related phenotypes. Moreover, therapeutic levels of expression were also achieved using the same approach in non-human primates. Here, we propose to move our therapeutic vector through a milestone-driven process that ends with an IND application for a Phase 1 clinical trial in human subjects. Specifically, we propose to 1) hold a Pre-IND Type B meeting with the CBER of the FDA to receive input on the design of the planned GLP tox study and the proposed Phase 1 protocol; 2) produce GMP-process comparable vector (GLP vector); 3) perform IND-enabling GLP pharm/tox studies in nonhuman primates (Rhesus macaques); 4) generate GMP-grade vector; and 5) prepare and file the IND with the FDA for a phase I/II clinical trial in subjects with early symptomatic AD.

Project Summary/Abstract Lysosomal diseases represent more than 70 disorders caused by inherited defects in a spectrum of organellar proteins; they affect many organ systems progressively to cause chronic illness and premature death. The Lysosomal Disease Gordon Research Conference (GRC) has played a crucial role in stimulating discoveries in this field. The 2019 Lysosomal Disease GRC/Gordon Research Seminar (GRS), held at the Hotel Galvez in Galveston, Texas, will offer a critical venue for addressing major topics in lysosomal biology, disease mechanisms, diagnosis and therapy, including (i) lysosomal systems biology, where `big data' is opening the door to genetic and cellular systems at play in the pathophysiology of disease: (ii) innovations in methods that reverse, rather than prevent or stabilize, pathogenesis, a critical advance as many individuals with these disorders come to light long after the pathology is established: (iii) emerging tools to study lysosomal diseases, at the cellular level and in the whole animal; (iv) mechanisms of pathogenesis in disorders that have recently been linked to lysosomal dysfunction; (v) progress in our understanding of lysosome-resident channels, a field that is emerging as critical to ascertaining how various deficiencies impair lysosome function; (vi) lysosome biology; and (vii) advances in clinical approaches focused on lysosomal disease. This conference is unique in the academic calendar and an outstanding complement to other existing lysosomal disease forums in USA and Europe. To address these topics we have invited, as speakers and discussants, 42 scientists and clinicians working in the lysosomal disease and related fields. Importantly, there is a mix of junior and senior investigators from many countries, and we made every effort to include women and minorities. Additionally, we have formatted this year's conference for an additional 12 speakers to be selected from the abstracts, giving us an excellent opportunity to balance the program among junior and senior investigators, women and men, and to encourage minority participants. Of the current program presented in the application, 95% have accepted our invitation. Based on historical data for this conference, we anticipate that approximately one-third of attendees (aside from invited speakers and discussants) will be junior investigators, postdoctoral fellows and graduate students, and all together there will be approximately 160 people focused on lysosome disease, from the science underlying the field to the translation of those findings to patients suffering from lysosomal disease. Finally, the associated GRS insures that the very best and the brightest of the next generation of lysosomal disease researchers will also be integrated within the GRC community. The discussion and cross fertilization of ideas and approaches occurring as part of the GRC and GRS meetings will accelerate our understanding of the role of the lysosomal system in health and in disease and continue to advance the development of effective therapies.