Rashmi Gopal-Srivastava

Clinical research for rare disease : Opportunities, challenges, and solutions

Over 7000 rare diseases, each <200,000 US residents, affect nearly 30 million people in the United States. Furthermore, for the 10% of people with a rare disease and for their families, these disorders no longer seem rare. Molecular genetics have characterized the cause of many rare diseases and provide unprecedented opportunities for identifying patients, determining phenotypes, and devising treatments to prevent, stabilize, or improve each disease. Rare disease research poses challenges to investigators requiring specific approaches to: (1) the design of clinical studies; (2) the funding of research programs; (3) the discovery, testing, and approval of new treatments, and (4) the training of clinical scientists. Rigorous, statistically-valid, natural history-controlled, cross-over, and n-of-1 trials can establish efficacy and support regulatory approval of new treatments for rare diseases. The U.S. Orphan Drug Act of the U.S. FDA has stimulated industry investment in clinical trials to develop treatments for rare diseases. For trainees interested in finding a treatment for a rare disease, a commitment to longitudinal care of patients provides a base for the characterization of phenotype and natural history, a stimulus for innovation, a target population for research and helps fund training and research. The scientific methodology, financial resources, and logistics of clinical research for rare diseases have changed dramatically in the past two decades resulting in increased understanding of the pathophysiology of these disorders and direct benefit to patients.

Involvement of Retinoic Acid/Retinoid Receptors in the Regulation of Murine αB-crystallin/Small Heat Shock Protein Gene Expression in the Lens

Crystallins are a diverse group of abundant soluble proteins that are responsible for the refractive properties of the transparent eye lens. We showed previously that Pax-6 can activate the αB-crystallin/small heat shock protein promoter via the lens-specific regulatory regions LSR1 (−147/−118) and LSR2 (−78/−46). Here we demonstrate that retinoic acid can induce the accumulation of αB-crystallin in N/N1003A lens cells and that retinoic acid receptor heterodimers (retinoic acid receptor/retinoid X receptor; RAR/RXR) can transactivate LSR1 and LSR2 in cotransfection experiments. DNase I footprinting experiments demonstrated that purified RAR/RXR heterodimers will occupy sequences resembling retinoic acid response elements within LSR1 and LSR2. Electrophoretic mobility shift assays using antibodies indicated that LSR1 and LSR2 can interact with endogenous RAR/RXR complexes in extracts of cultured lens cells. Pax-6 and RAR/RXR together had an additive effect on the activation of αB-promoter in the transfected lens cells. Thus, the αB-crystallin gene is activated by Pax-6 and retinoic acid receptors, making these transcription factors examples of proteins that have critical roles in early development as well as in the expression of proteins characterizing terminal differentiation.

Expression of the murine alpha B-crystallin gene in lens and skeletal muscle: identification of a muscle-preferred enhancer.

The alpha B-crystallin gene is expressed at high levels in lens and at lower levels in some other tissues, notably skeletal and cardiac muscle, kidney, lung, and brain. A promoter fragment of the murine alpha B-crystallin gene extending from positions -661 to +44 and linked to the bacterial chloramphenicol acetyltransferase (CAT) gene showed preferential expression in lens and skeletal muscle in transgenic mice. Transfection experiments revealed that a region between positions -426 and -257 is absolutely required for expression in C2C12 and G8 myotubes, while sequences downstream from position -115 appear to be determinants for lens expression. In association with a heterologous promoter, a -427 to -259 fragment functions as a strong enhancer in C2C12 myotubes and less efficiently in myoblasts and lens. Gel shift and methylation interference studies demonstrated that nuclear proteins from C2C12 myoblasts and myotubes specifically bind to the enhancer.

The murine alpha B-crystallin/small heat shock protein enhancer: identification of alpha BE-1, alpha BE-2, alpha BE-3, and MRF control elements.

The murine alpha B-crystallin gene (a member of the small heat shock protein family) is expressed constitutively at high levels in the lens and at lower levels in many other tissues, including skeletal muscle. We have previously used the herpes simplex virus thymidine kinase promoter fused to the human growth hormone gene to identify an alpha B-crystallin enhancer at positions -427 to -259 that has high activity in muscle and low activity in lens cell lines. In the study reported here, we performed DNase I footprinting, transfection, mutagenesis, and electrophoretic mobility shift experiments using the murine C2C12 muscle and alpha TN4-1 lens cell lines and the rabbit N/N1003A lens cell line to identify sequences responsible for activity of this enhancer. Enhancer activity in both the muscle and lens cells was dependent on novel elements called alpha BE-1 (-407 to -397), alpha BE-2 (-360 to -327), and alpha BE-3 (-317 to -306). These elements were also weakly occupied by nuclear proteins in L929 cells, which appear to express the alpha B-crystallin gene at a very low level (detectable only by the polymerase chain reaction). A fourth element containing a consensus muscle regulatory factor-binding site called MRF (-300 to -288) was occupied and used only by the C2C12 muscle cells. Cotransfection in NIH 3T3 cells and antibody-gel shift experiments using C2C12 nuclear extracts indicated that MyoD, myogen, or a similar member of this family can activate the alpha B-crystallin enhancer by interaction with the MRF site. Taken together, we conclude that the alpha BE-1, alpha BE-2, and alpha BE-3 elements are shared by both lens and muscle cells, but the MRF element is used only in muscle cells, providing the first example of a muscle-specific control element in a crystallin gene.

Recruitment of enzymes and stress proteins as lens crystallins

The major water-soluble proteins--or crystallins--of the eye lens are either identical to or derived from proteins with non-refractive functions in numerous tissues. In general, the recruitment of crystallins has come from metabolic enzymes (usually with detoxification functions) or stress proteins. Some crystallins have been recruited without duplication of the original gene (i.e., lactate dehydrogenase B and alpha-enolase), while others have incurred one (i.e., argininosuccinate lyase and a small heat shock protein) or several (i.e., glutathione S-transferase) gene duplications. Enzyme (or stress protein)-crystallins often maintain their non-refractive function in the lens and/or other tissues as well as their refractive role, a process we call gene sharing. alpha-Crystallin/small heat shock protein/molecular chaperone is of special interest since it is the major crystallin of humans. There are two alpha-crystallin genes (alpha A and alpha B), with alpha B retaining the full functions of a small heat shock protein. Here we describe recent evidence indicating that alpha A and alpha B have kinase activity, which would make them members of the enzyme-crystallins. We also describe various regulatory elements of the mouse alpha-crystallin genes responsible for their expression in the lens and, for alpha B, in skeletal muscle. Delineating the control elements for gene expression of these multifunctional protective proteins provides the foundations for their eventual use in gene therapy. Finally, comparison of the mouse and chicken alpha A-crystallin genes reveals similarities and differences in their functional cis-acting elements, indicative of evolution at the level of gene regulation.

Differential use of the regulatory elements of the αB-crystallin enhancer in cultured murine lung (MLg), lens (αTN4-1) and muscle (C2C12) cells

Abstract The mouse αB-crystallin-encoding gene (αB-cry) is highly expressed in the lens and expressed to lesser extents in other tissues. Here, we investigated αB-cry expression in mouse-lung-derived MLg cells. Two sizes of MLg αB-cry transcripts comigrated with αB-cry transcripts contained in total and poly(A)+RNA from mouse lung, with preference for the larger species in the MLg cells. Expression of both αB-cry promoter/cat reporter gene constructs and αB-cry enhancer (nt -427/-259)/herpes simplex virus (HSV) thymidine kinase promoter (ptk)/human growth hormone reporter gene (hGH) constructs was studied in transfected MLg cells and the results compared with those obtained from αTN4-1 lens and C2C12 muscle cells. The αB-cry enhancer increased activity of the endogenous and tk promoters approx. 2-fold in the MLg cells, in contrast to its 3-7-fold effect in αTN4-1 cells and 17-20-fold effect in C2C12 myotubes. Site-specific mutagenesis of the previously identified enhancer control elements, αBE-1 (nt -407 to -397), αBE-2 (-360 to -327) and MRF (-300 to -288), decreased enhancer strength in transfected MLg cells. DNase I footprinting showed that MLg nuclear proteins occupy only αBE-1 and αBE-2. Previous data have shown that lens cells use αBE-1, αBE-2 and αBE-3, while muscle cells use, in addition, the muscle regulatory factor-binding site (MRF). Thus, the present experiments correlate tissue-specific enhancer strength and the number of control elements utilized.

Gene Therapy for Rare Diseases: Summary of a National Institutes of Health Workshop, September 13, 2012

Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.

Gene Therapy: Charting a Future Course—Summary of a National Institutes of Health Workshop, April 12, 2013

Recently, the gene therapy field has begun to experience clinical successes in a number of different diseases using various approaches and vectors. The workshop Gene Therapy: Charting a Future Course, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities, brought together early and mid-career researchers to discuss the key scientific challenges and opportunities, ethical and communication issues, and NIH and foundation resources available to facilitate further clinical advances.

Creating a multi-center rare disease consortium – the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR)

Eosinophilic gastrointestinal disorders (EGIDs) affect various segments of the gastrointestinal tract. Since these disorders are rare, collaboration is essential to enroll subjects in clinical studies and study the broader population. The Rare Diseases Clinical Research Network (RDCRN), a program of the National Center for Advancing Translational Sciences (NCATS), funded the Consortium of Eosinophilic Gastrointestinal Disease Researchers (CEGIR) in 2014 to advance the field of EGIDs. CEGIR facilitates collaboration among various centers, subspecialties, patients, professional organizations and patient-advocacy groups and includes 14 clinical sites. It has successfully initiated two large multi-center clinical studies looking to refine EGID diagnoses and management. Several pilot studies are underway that focus on various aspects of EGIDs including novel therapeutic interventions, diagnostic and monitoring methods, and the role of the microbiome in pathogenesis. CEGIR currently nurtures five physician-scholars through a career training development program and has published more than 40 manuscripts since its inception. This review focuses on CEGIR’s operating model and progress and how it facilitates a framework for exchange of ideas and stimulates research and innovation. This consortium provides a model for progress on other potential clinical areas.

Female Sex and Gender in Lung/Sleep Health and Disease: Increased Understanding of Basic Biological, Pathophysiological and Behavioral Mechanisms Leading to Better Health for Female Patients with Lung Disease

Abstract Female sex/gender is an undercharacterized variable in studies related to lung development and disease. Notwithstanding, many aspects of lung and sleep biology and pathobiology are impacted by female sex and female reproductive transitions. These may manifest as differential gene expression or peculiar organ development. Some conditions are more prevalent in women, such as asthma and insomnia, or, in the case of lymphangioleiomyomatosis, are seen almost exclusively in women. In other diseases, presentation differs, such as the higher frequency of exacerbations experienced by women with chronic obstructive pulmonary disease or greater cardiac morbidity among women with sleep‐disordered breathing. Recent advances in ‐omics and behavioral science provide an opportunity to specifically address sex‐based differences and explore research needs and opportunities that will elucidate biochemical pathways, thus enabling more targeted/personalized therapies. To explore the status of and opportunities for research in this area, the NHLBI, in partnership with the NIH Office of Research on Womens Health and the Office of Rare Diseases Research, convened a workshop of investigators in Bethesda, Maryland on September 18 and 19, 2017. At the workshop, the participants reviewed the current understanding of the biological, behavioral, and clinical implications of female sex and gender on lung and sleep health and disease, and formulated recommendations that address research gaps, with a view to achieving better health outcomes through more precise management of female patients with nonneoplastic lung disease. This report summarizes those discussions.