Paula R. Clemens - US grants

The goal of the research proposed for this award is to develop strategies for gene therapy in patients with Duchenne muscular dystrophy (DMD). The ideal gene transfer therapy for patients with DMD would be systemic, safe, efficient and targeted to tissues that normally express dystrophin. A promising candidate carrier for dystrophin gene delivery is a replication-deficient viral vector. To date, the retroviral and adenoviral vectors have proved safe and are therefore potentially useful for human gene transfer. However, these vectors can accept DNA inserts of approximately one-half the length of the recombinant dystrophin cDNA. We propose to accommodate the insert size limitation of these vectors by truncating the dystrophin cDNA driven by the muscle creatine kinase promoter. The construction of truncated recombinant dystrophin cDNA molecules will be achieved by engineering deletions of varying sizes in the spectrin-repeat region of dystrophin. The characterization of dystrophin deletions in patients with mild phenotypes has revealed deletions in this region. A polymerase chain reaction-based strategy will be used to precisely delete integral numbers of spectrin-like repeats. It may also be possible to truncate the promoter region and the 3' untranslated region without adversely affecting dystrophin function. After construction, these recombinant genes will be used to generate retroviral and adenoviral vectors that will first be tested for expression in cell culture. The primary aim of these studies will be the production of viral vectors with stable inserts, high viral titer, and expression of functional dystrophin. Dystrophin expression, which will be detected by immunocytochemistry and Western analysis, from truncated, recombinant dystrophin constructs with different deletions may yield important information about the relationship between specific regions of dystrophin and its expression. A second goal of the expression studies in cell culture is to develop a better understanding of the factors that affect gene uptake and stable expression in muscle cells using these viral vectors. Promising constructs for gene therapy will be used to generate transgenic mice on the mdx mouse background; the mdx mouse is a biochemical and genetic model for DMD. These studies will allow demonstration of the tissue-specific expression of dystrophin and the effect of the recombinant, truncated dystrophin gene on the levels of dystrophin-associated proteins in skeletal muscle in vivo. In vivo studies are also planned that will demonstrate the efficacy of dystrophin gene transfer into the mdx mouse using retroviral and adenoviral vectors.

DESCRIPTION (Abstract): Therapeutic gene transfer is a logical approach to the treatment of inherited genetic deficiency diseases, many of which have no other adequate treatment. However, persistent expression of the therapeutic gene in the target tissue and/or the capability of repeat administration will be required. To date, adenoviral (Ad) vector- mediated gene transfer trials have resulted in an immune response that eliminates the therapeutic protein. This immunity may be due either to the expression of the therapeutic protein itself, to Ad proteins or both. The future success of human gene therapy trials using the Ad vector as a vehicle will likely depend on a comprehensive understanding of this immune response and novel strategies to modulate it. Although the therapeutic protein to be provided by a gene therapy vector is a self-protein in healthy individuals, the patients who would be treated by gene transfer lack this self-protein due to a germ-line mutation (a null mutant). Examples include Factor IX deficiency, cystic fibrosis and Duchenne's muscular dystrophy (DMD). DMD provides an excellent model with which to study gene transfer treatment for an inherited protein deficiency because the mutant gene and defective protein are known, the target tissue is easily accessible and the mdx mouse strain, which models human DMD, is readily available. The investigators recently have described the development and use of a novel high-capacity Ad vector that has all viral genes removed and can accommodate 30 kb of insert DNA. This vector is the most promising to date for decreasing the immunity induced by therapeutic Ad vector- mediated gene delivery; no AD antigens are expressed from the vector, and the use of a muscle-specific promoter should reduce antigen presentation by professional antigen presenting cells. The five aims of this application will lead to the characterization and modulation of the immune response induced by therapeutic gene delivery to skeletal muscle. The first group (Aims 1, 2 and 3) will analyze the immune response to specific antigens. The second group (Aims 4 and 5) has as a common thread the modification of vector characteristics to improve high- capacity Ad vector-mediated gene delivery to muscle by modulating the immune response to the antigens studies in Aims 1-3.

The goal of the Mouse Breeding Core is to provide mice and hamsters to Program investigators in a cost-effective and dependable manner. Both control and dystrophic mice and hamsters will be bred to maintain a colony of sufficient size such that ample animals of specified age can be supplied to each of the research projects in this Program. This Core has been in operation for early two years, and has been run by Dr. Clemens during this time. Support has previously been derived from a parent-run foundation, as part of the DMDRC. Setting of priorities of the Core will be done by Dr. Clemens, with the Program Director(Dr. Hoffman) arbitrating any conflicts with regards mouse or hamster requirements. Day-to-day operation of the Core, including distribution of mice and hamsters, will be done by a technician, Ms. Christy Bruton, who has been responsible for the Core to date.

Adenoviral vectors have held a prominent position in efforts to deliver therapeutic genes to cells, including muscle. However, a major limitation to first generation adenoviral vectors has been the aggressive immune response that these vectors elicit in vivo. We have recently described the design and construction of a novel adenoviral vector that appears to circumvent many of the immunological problems encountered by previous vectors. The 'helper-dependent' system expresses no virally-encoded antigens, and has a very large carrying capacity for DNA. High-level, muscle-specific murine dystrophin expression in mdx mouse muscle was demonstrated using one such recombinant vector. However, the longevity of dystrophin expression was limited by immune responses to beta-galactosidase that was co-expressed by the vector. The next generation of dystrophin-expressing, high capacity adenoviral vectors eliminates the lacZ gene cassette and uses novel helper virus technology to further decrease helper virus in the purified vector preparation. In this grant proposal, critical aspects of gene delivery, persistence and therapeutic effect will be studied in order to accomplish long-term genetic complementation of dystrophin-deficient muscle. Effects on muscle function will be assayed biochemically, histologically and physiologically. In Aim 1 the immunological effects of gene delivery on homologous proteins will be explored by cross- species dystrophin gene delivery. In Aim 2 novel strategies of adenoviral vector delivery, including muscle-specific targeting, will be tested. Aim 3 investigates the physiological benefits of full-length dystrophin delivery using a high-capacity adenoviral vector. Mouse age and immunosuppressive treatment are variables that will be tested for their effects on indices on muscle function afforded by adenoviral vector-mediated dystrophin delivery. In Aim 4, the biochemical, functional and immunological benefits of adenoviral vector-mediated gene transfer to fetal muscle will be investigated.

[unreadable] DESCRIPTION (provided by applicant): Despite many successful examples of gene transfer to limited areas of postnatal skeletal muscle, regional or systemic delivery suitable to treat inherited muscle diseases such as Duchenne muscular dystrophy (DMD) has proved elusive. Gene or cell transfer during the fetal stage holds the potential of more efficient and widespread genetic complementation of dystrophin deficiency in DMD. An important rationale to pursue therapeutic strategies for the dystrophic fetus is that muscle stem cells are more plentiful in fetal muscle than in adult muscle. Furthermore, viral vector-mediated gene delivery and engraftment of genetically engineered muscle precursor cells to skeletal muscle may be enhanced during this fetal stage. Therefore, we will explore the feasibility of performing gene and cell transfer to muscle in utero exploiting the benefits of lower tissue mass and immune immaturity of the fetus as well as the earlier stage of muscle development. [unreadable] Specific Aims: [unreadable] 1. To evaluate in utero dystrophin gene delivery using high-capacity adenoviral (HC-Ad) vectors. [unreadable] 2. To evaluate in utero dystrophin gene delivery using adeno-associated viral (AAV) vectors. [unreadable] 3. To evaluate in utero gene delivery using early progenitor myogenic cells [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Despite many successful examples of gene transfer to limited areas of postnatal skeletal muscle, regional or systemic delivery suitable to treat inherited muscle diseases such as Duchenne muscular dystrophy (DMD) has proved elusive. Gene or cell transfer during the fetal stage holds the potential of more efficient and widespread genetic complementation of dystrophin deficiency in DMD. An important rationale to pursue therapeutic strategies for the dystrophic fetus is that muscle stem cells are more plentiful in fetal muscle than in adult muscle. Furthermore, viral vector-mediated gene delivery and engraftment of genetically engineered muscle precursor cells to skeletal muscle may be enhanced during this fetal stage. Therefore, we will explore the feasibility of performing gene and cell transfer to muscle in utero exploiting the benefits of lower tissue mass and immune immaturity of the fetus as well as the earlier stage of muscle development. [unreadable] Specific Aims: [unreadable] 1. To evaluate in utero dystrophin gene delivery using high-capacity adenoviral (HC-Ad) vectors. [unreadable] 2. To evaluate in utero dystrophin gene delivery using adeno-associated viral (AAV) vectors. [unreadable] 3. To evaluate in utero gene delivery using early progenitor myogenic cells [unreadable] [unreadable]

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by mutations in tiie dystrophiin gene. Patients have partial loss-of-function of the dystrophin protein due to in-frame gene deletions, or other hypomorphic alleles. BMD genotype/phenotype studies have been limited to date, and there have been no longitudinal natural history studies in a multi-center setting. The intent ofexon skipping is to produce BMD-like internally deleted, in-frame dystrophin proteins as a therapeutic intervention for Duchenne muscular dystrophy (DMD) patients. Many clinically mild BMD patients have been described, some with very large deletions, and some who show only high serum creatine kinase levels with little or no associated clinical symptoms. However, there are also many BMD patients, with in-frame deletions of the rod domain, who have a severe muscle dystrophic phenotype, often as severe as the classic phenotype of DMD. A better understanding ofthe BMD phenotype is critical to the design and evaluation of drug development programs based on exon skipping. In this project, we propose a natural history study of BMD participants with specific in-frame deletions that correspond to the mutations generated by exon skipping of exons 45, 51 or 53. These reflect the 'target'deletions of DMD exon skipping resulting from the three antisense oligonucleotide drugs studied in this CORT, hence linking Project 3 with Projects 1 and 2. We will use a collaborative network of clinical research centers, the Cooperative International Neuromuscular Research Group (CINRG) to recruit participants. The CINRG group has an ongoing federally-funded longitudinal history study of 348 DMD participants in addition to multiple completed and ongoing clinical trials. The current census comprising all CINRG sites is 472 BMD patients. We will characterize the BMD phenotype, and correlate specific abnormal dystrophin proteins with the range of clinical outcomes. As the first natural history study for BMD, the proposed project has high impact in the field of emerging molecular therapeutics for DMD and contributes to the translational CORT focus of furthering research toward exon skipping therapy for DMD.

DESCRIPTION (provided by applicant): To translate the rapid progress in neurological therapeutic developments to patient care, clinical trials must keep pace with preclinical discoveries. Hurdles encountered in neuroscience clinical trials include delays in regulatory approvals, slow recruitment, participants lost to follow-up and slow translation of findings to clinical practice. The University of Pittsburgh has unique and significant resources and a proven track record of excellent performance in clinical trials. The aims of the proposed NINDS Network of Excellence in Neuroscience Trials at the University of Pittsburgh (NEXT-UP) are: (1) to develop an administrative structure to rapidly implement NEXT trials across of wide range of neurological conditions;(2) to foster collaboration and outreach in NEXT trials among community physicians, patients and patient support groups;and (3) to promote careers in clinical trial research by integrating training with NEXT-UP activities. The NEXT-UP leadership plan reflects these goals with a Program Director (Lawrence R. Wechsler, MD) and co-Program Director;an Advisory Committee with an adult neurologist, pediatric neurologist, neurosurgeon and rotating member with trial-specific expertise;and a Program Coordinator. Collaboration will be established by meeting regularly and feedback sessions. Recruitment will occur through high-volume referral providers, active surveillance and community outreach. Outreach includes the NEXT-UP Academy, registry, website and Study Partner Program. Efficiency and monitoring will be accomplished through: a tier-1 federated IRB model;standardized trial agreements;establishing a timeline and benchmarks for each trial;monthly reports to the NEXT-UP Leadership from co-Investigators;and Corrective Action plans for challenges encountered. Promotion of careers in clinical trial research will include a training plan involving all career levels including: NEXT-UP workshops, a NEXT-UP elective, and hands on-experience implementing a NEXT trial with a more senior co-investigator. The combination of the University of Pittsburgh Medical Center's regional dominance in healthcare and this innovative proposal will allow NEXT-UP to become a model site in the NEXT network with efficiently run clinical trials integrating investigators with community providers, patients and trainees. PUBLIC HEALTH RELEVANCE: Potential treatments for neurological disorders are rapidly developing. However delays occur in translating this to care for patients due to the lack of efficiency of clinical trials. The proposed NINDS Network of Excellence in Neuroscience trials at the University of Pittsburgh will creatively leverage its extensive resources to rapidly implement clinical trials with academic investigators, community providers and patients.

DESCRIPTION (provided by applicant): The most effective drug identified to date for Duchenne muscular dystrophy (DMD) is daily high dose glucocorticoids (prednisone, deflazacort). However, the benefits of the anti-inflammatory pharmacological activities of glucocorticoids may not be realized for a patient with DMD, or indeed with one of a large number of diverse inflammatory conditions, due to unacceptable side effects of the therapy. The balance of efficacy and side effects leads to significant variation in glucocorticoid use for the clinical management of DMD due to side effects of short stature, osteopenia, mood changes, and altered muscle bulk. A novel dissociative steroid, VBP15, was developed with a more optimal risk-benefit ratio for the treatment of youth with DMD. Preclinical research with VBP15 showed that the anti-inflammatory mechanism, due largely to inhibition of NF-?B activation, is further augmented by beneficial modulation of leukocyte extravasation and increased plasma membrane stabilization. Furthermore, VBP15 has significantly less binding to the glucocorticoid response element (GRE) in target gene enhancers and repressors. Preclinical studies demonstrate that reduced GRE binding results in fewer adverse effects and suggests that there will be fewer glucocorticoid-induced side effects in humans treated with VBP15. Extensive preclinical efficacy and toxicology research support the development of VBP15 for first-in-human clinical use. In multiple pre-clinical models of inflammatory disease, including the mdx murine model of DMD, VBP15 reduces side effects, widens therapeutic windows, and retains or enhances efficacy relative to traditional glucocorticoids. In this application, we propose planning activities to support a Phase 2a (safety, tolerability, dose-finding, PK and PD) clinical trial of VBP15. An extension to this Phase 2a study will integrate pharmacodynamics biomarker panels and MRI outcomes to query time-dependent effects on inflammation and membrane stability in young (4-7 yr. old) DMD patients.

? DESCRIPTION (provided by applicant): ReveraGen BioPharma is a clinical stage drug development company that is developing VBP15, a novel dissociative steroidal class drug. The initial indication for development is Duchenne muscular dystrophy, where VBP15 holds promise for retaining or increasing efficacy of glucocorticoids, while reducing side effects (bone fragilit, stunting of growth). VBP15 is currently in Phase I clinical trials in adult volunteers. This proposed Phase II SBIR research is to carry out a Phase IIa clinical trial in Duchenne muscular dystrophy boys, steroid naïve, ages 4-7 yrs. Aim 1 is to test four dose levels of VBP15 in a multiple ascending dose (MAD) trial design, with two weeks on drug. Patients completing the Phase IIa clinical trial will be offered enrollment into a six month extension study. The goal is t test pediatric pharmacokinetics, tolerability and safety in the Phase IIa clinical trial, and assessments of efficacy (time to stand velocity) and safety (change in body mass index) in the six month extension study to aid dose selection in the future Phase IIb registration trial. Aim 2 i to develop a pharmacodynamics biomarker panel that can assess safety and efficacy markers in acute time frames from peripheral blood. We have completed a natural history of DMD serum biomarkers using both SomaScan and proteomics discovery methods, and present preliminary data on glucocorticoid- associated efficacy and safety biomarkers. The Phase II SBIR research will compare VBP15 biomarkers to glucocorticoid biomarkers as a means of assessing comparative safety and potential efficacy.

To translate the rapid progress in neurological therapeutic developments to patient care, clinical trials must keep pace with preclinical discoveries. Hurdles encountered in neuroscience clinical trials include delays in regulatory approvals, slow recruitment, participants lost to follow-up and slow translation of findings to clinical practice. The University of Pittsburgh has unique and significant resources and a proven track record of excellent performance in clinical trials, and in NeuroNEXT in particular. The aims of the proposed NINDS Network of Excellence in Neuroscience Trials at the University of Pittsburgh (NEXT-UP) are: (1) to develop an administrative structure to rapidly implement NeuroNEXT trials across of wide range of neurological conditions; (2) to foster collaboration and outreach in NeuroNEXT trials among academic physicians, community physicians, patients and patient support groups; and (3) to promote careers in clinical trial research by integrating clinical research training of early-stage investigators with NEXT-UP activities. The NEXT-UP leadership plan reflects these goals with a Program Director (Paula R. Clemens, MD) and co-Program Director (Lawrence R. Wechsler, MD); an Advisory Committee incorporating expertise and liaison to adult and pediatric neurology clinicians and patients; an early stage investigator; and a Program Coordinator. Collaboration will be established by meeting regularly and outreach to providers and the community. Recruitment will occur through high-volume referral providers and community outreach. Efficiency and monitoring will be accomplished through use of a central IRB and standardized trial agreements; establishing a timeline and benchmarks for each trial; monthly reports to the NEXT-UP Leadership from co-Investigators; and Corrective Action plans for challenges encountered. Promotion of careers in clinical trial research will include a training plan for early stage clinical neuroscience investigators. The combination of the University of Pittsburgh Medical Center's regional dominance in healthcare and this innovative proposal will allow NEXT-UP to continue to be a model site in the NeuroNEXT network with efficiently run clinical trials integrating investigators with community providers, patients and trainees.

Abstract ReveraGen BioPharma is a clinical stage drug development company that is developing vamorolone (VBP15), a first-in-class dissociative steroidal drug. The initial indication being tested is Duchenne muscular dystrophy (DMD), where vamorolone holds promise for retaining or increasing efficacy of glucocorticoids, while reducing adverse effects (bone fragility, stunting of growth, weight gain, insulin resistance). ReveraGen received a NINDS SBIR Phase II grant in support of the Phase 2a studies in 4 to <7 year old DMD boys in 2016 (protocols VBP15-002; VBP15-003) (R44NS095423). The original trial was planned for four dose groups, with 2-6 boys per dose group (up to 24 patients). After award of the grant, the size of the trial was increased to 48 DMD boys (12 per dose group; n=48). Despite the narrow age range in a rare disease, the trial was fully enrolled within 15 months, and the Phase 2a study completed in November 2017, within the time frame of the SBIR Phase II grant (15 February 2016-31 January 2018). All doses were well-tolerated (0.25 mg/k/gday ? 6.0 mg/kg/day; or to 10-times the typical prednisone dose in DMD boys). Pharmacokinetics showed a well- behaved drug, with PK similar to corticosteroids (short half-life of 2-3 hrs, and no drug accumulation between doses). There was no significant change in body mass index (primary clinical safety outcome relative to prednisone). Pharmacodynamic biomarker results showed improved safety compared to corticosteroids, with no evidence of insulin resistance at any dose, and a >10 fold improved safety margin for bone turnover and adrenal suppression. Here, we request partial support for the pivotal double blind Phase 2b study of 120 DMD boys, ages 4 to <7 years. The Phase 2b study (VBP15-004) includes four groups with six months treatment (placebo, prednisone, low dose vamorolone, high dose vamorolone), followed by an additional 6 months treatment with two dose levels of vamorolone. Co-funding for the Phase 2b study is from the European Commission ($7M Horizons 2020 grant awarded in 2016), venture philanthropy support by non-profit foundations, and an option agreement for future sales and marketing. Aim 1 is to test 6 months of treatment of two dose levels of vamorolone, with efficacy (time to stand) vs. placebo, and safety (change in body mass index) vs. prednisone. Aim 2 is to bridge pharmacodynamic biomarkers to clinical outcomes of both safety and efficacy. Anticipated new matching funding during the proposed award period includes a 2nd milestone payment on the option agreement ($15M August 2018). The completion of the proposed Phase IIB SBIR research will lead to discussions with the FDA regarding possible accelerated approval, with Phase III trials in the post-marketing period. The bridging of novel safety and efficacy biomarkers to clinical outcomes will enable the utilization of these biomarkers to expand labeling to younger DMD children (0-4 years), and efficient testing of vamorolone for treatment of new indications (eg. Becker muscular dystrophy, juvenile dermatomyositis), thus providing the potential to significantly augment the treatment options and quality of life for patients with genetic and autoimmune neuromuscular disorders.

Abstract (Administrative Supplement) In this supplemental project, we will study an innovative and cost-effective approach to returning individual and aggregate, non-genetic clinical trial data to families of boys with Duchenne muscular dystrophy (DMD) participating in vamorolone trials (n=168). The assessment of this approach will involve extensive community stakeholder input and return of individual and aggregate trial data to participants in VBP15-002, 003, and LTE. A centralized, sponsor- initiated design will be utilized for re-consent and connecting patients with their data, using a single coordinator to preserve privacy firewall between patients and industry study teams. Before and following return of data, surveys will be administered to the child?s parents and physicians regarding ethics and logistics. This information will then inform return of data to participants in VBP15-004. The Parent Grant is a SBIR Phase 2 and 2B NINDS award for support of three clinical trials of vamorolone in DMD (VBP15-002 and VBP15-003 [48 patients, 4-7 years; completed]; VBP15-004 [120 patients, 4-7 years, enrollment complete 2019).

Project Summary Novel treatments are desperately needed by patients with Becker muscular dystrophy (BMD), and anti- inflammatory therapies hold great promise for treating patients with this condition. However, the slow progressive nature of the disease, small numbers of patients, variable onset and clinical presentation, and lack of biomarkers presents a serious barrier to drug development in BMD. A first-in-class steroid, vamorolone, has shown promise for the treatment of young boys with Duchenne muscular dystrophy and is in a randomized, blinded and controlled, confirmatory study for this indication. In this project, we propose a 24-week pilot trial of vamorolone 6 mg/kg/day in 30 ambulatory patients with BMD at 2 sites, evaluating safety and tolerability. Biomarkers, creatine kinase, macrophage derived chemokine, and microRNA 146a, and the timed 10 meter run/walk test will also be evaluated throughout the trial, to assess the usefulness of these potential treatment responsive biomarkers to this anti-inflammatory drug in BMD patients. If vamorolone is demonstrated to be safe and tolerable in this study, a controlled study of vamorolone treatment for BMD patients will be planned. Evaluation of pharmacodynamic biomarkers in this short trial serves to provide objective evidence for drug mechanism of action; if there is expected change in this pilot trial, these biomarkers would be further studied in the next phase trial.