Advances in genomic science, cellular and organismal biology, and structural biology, combined with a greater public appreciation of the value to the nations health of biomedical research, have set the stage for an explosive growth in knowledge and practical applications in the biosciences. Partnerships among industry, academia and government will be needed to bring these opportunities to fruition.
Facilitating Innovation in a New Era of Drug Discovery: An Industry Perspective
Gordon Binder, Chair and Chief Executive Officer, Amgen, Inc.
The FDA is striving to adapt to new technologies and attending ethical issues at a time when rising incomes, the aging of the population and other economic and demographic factors push up healthcare costs. In the face of these challenges, the agency sometimes responds with over-regulation. The speaker believes the system, while striking a good balance between opportunities and risks, would benefit from a streamlined, and strengthened, advisory board system. He advises that the system include more experts from outside the regulatory community. And while a small number of boards could be dispensed with, those remaining should also meet more often, and the agency should act more quickly upon their recommendations. In closing, the speaker suggests that the industry and the FDA should increase their efforts toward cooperation, especially in the area of implementing the aspects of FDAMA designed to make the development process move more surely and efficiently.
The first wave of products of agricultural biotechnology (including transgenic plants) that have reached, or will reach, the market are based on relatively simple technologies, and involved the transfer or modification of one or several genes. The next generation of products will involve single or multiple genes, some of which will derive from non-plant organisms, and others that will be developed de novo. This will include genes that are modified for functions different than the wild type gene; for example, genes that produce proteins with unique enzymatic functions, and proteins that act as dominant negative inhibitors of functions of wild type genes; chimeric proteins to inhibit multiple biochemical processes; novel proteins that regulate the expression of a single or multiple genes; novel peptides selected by display technologies that inhibit pathogens, insects, or key regulatory pathways; proteins and other products that increase the health and welfare of the consumer; etc. It is likely that some of these products will have high specificity and efficacy, yet will be substantially novel in composition and use. Some of these products will present new challenges for regulatory and marketing strategies: nevertheless, because of the potential of new products to improve the human condition, and contribute to the efficiency and sustainability of agriculture, it is important to encourage the research and development of such products.
Enhanced Output Traits from Agricultural Crops
John Pierce, Ph.D., Director, Discovery Research, DuPont Agricultural Products
The impact of modern biotechnology is beginning to be felt across the world of agriculture. The nutritional quality and value of important crops such as corn, soybean, wheat, and rice are being enhanced by modern genetics, using both conventional and transgenic approaches. Crops with improvements in both the quantity and quality of the major seed components--protein, oil, and carbohydrate--are already starting to be commercialized, and there are many products in the pipeline. Numerous additional opportunities exist to enhance the nutritional and functional value of crop plants through modification of other seed components. This talk will outline some of the current and near term prospects for crop improvement improvements which will have important effects on animal nutrition, human health, and agricultural productivity.
Improving Human Health Via Better Plants
Ganesh Kishore, Ph.D., Monsanto Company
The remarkable penetration of the first generation of plant biotechnology products into the farming arena in North America is an illustration of the power of this technology in creating beneficial and convenient products for agriculture. These products create significant value by increasing agricultural productivity and indirectly benefit the consumers. The tools of plant biotechnology are now being harnessed to directly benefit the consumers by increasing the nutrient density of crops. Two distinct types of opportunities are being explored. First is the increase in content of known nutrients and second is related to the discovery of role of phytonutrients in regulating human health and disease. In the former context, we have focused on increasing the pro-vitamin A content of vegetable oil. Vitamin A deficiency is a major global problem and could be addressed at least in part by growing major crops with a higher level of precursors of vitamin A such as Beta-carotene. Canola plants which substantially overproduce Beta-carotene in its seeds have been produced using a gene encoding, a rate-limiting step in this pathway. With respect to phytonutrients, we are developing compounds that decrease total and LDL cholesterol levels in humans and thus provide dietary intervention mechanisms for managing cholesterol, an important biomarker of cardiovascular health. Longer term, our goal is to leverage the tools of biotechnology to produce and deliver these compounds in a safe, efficacious and cost effective manner.
Transgenic Livestock: Biotechnology for the Production of Innovative Animals and Products
Robert J. Wall, Ph.D., United States Department of Agriculture, Agriculture Research Service, LPSI, Gene Evaluation and Mapping Laboratory
Since the first transgenic farm animals were produced in 1985, animal scientists have envisioned using genetic engineering to improve growth characteristics, modify the composition of milk and enhance disease resistance of livestock. These goals have been pursued in an effort to reduce animal production costs and thereby insuring an adequate, less expensive supply of healthful food to consumers. Though progress has been slow in comparison to the use of transgenic livestock as pharmaceutical production systems and organ donors, the insights gained over the last 15 years suggests that genetically engineered farm animals will become part of the human food supply, possibly within the next two decades. Several transgenic livestock projects will be discussed as examples of where we have been and some speculation will be offered in an attempt to predict where we are headed.
Vaccines from Edible Plants
Carol O. Tacket, Ph.D., Center for Vaccine Development, Baltimore, MD
Plant biotechnology techniques have been used to create potato plants which contain a gene derived from diarrheagenic E. coli. The cloned gene encodes the nontoxic B subunit of the heat labile enterotoxin of E. coli. Healthy adult volunteers who ingested 50 or 100 gm of these raw transgenic potatoes developed serum anti-toxin and specific antibody secreting cells. This clinical study supports the principle that vaccine antigens may be delivered by the oral route in edible plants. Further studies will evaluate other vaccine antigens.
Antimicrobial resistance is a serious problem in the U.S., involving bacteria, parasites, viruses, and fungi. Strategies to prevent the development and spread of antimicrobial resistance differ for various pathogens. Surveillance for resistance is a shared need across all strategies and pathogens for measuring the impact of resistance and the evaluation of prevention interventions. Effective monitoring of resistance and developing effective prevention strategies will require partnerships between CDC, other federal agencies, state health departments, health care delivery organizations, industry, and professional societies, among others, both domestically and internationally.
Biochemical and Genomic Approaches to the Identification of Compounds with Activity Against Mycobacterium tuberculosis
Clifton E. Barry III, Ph.D., Chief, Tuberculosis Research Section, NIAID, National Institutes of Health
The recently completed genome of Mycobacterium tuberculosis represents a wealth of biochemical information that could be used to guide and inform the development of new chemo- and immuno-therapies. This talk will address current research utilizing both genomic information and biochemical studies to establish the mechanism of action of commonly used chemotherapies. The mechanism of isoniazid action will be used to illustrate methods of exploitation of such information in the development of high throughput assays and novel chemotherapies. Comparative metabolic studies also suggest that pathogenic mycobacteria are dependent upon host membranes for intracellular survival. The abundance of lipid catabolic enzymes encoded in the genome substantiates such studies and suggests that inhibiting utilization of these molecules may represent a uniquely effective drug target.
Mutators Among Escherichia coli and Salmonella enterica: Adaptation and Emergence of Bacterial Pathogens
T.A. Cebula, B. Li, W.L. Payne, and J.E. LeClerc. Division of Molecular Biological Research and Evaluation, CFSAN, FDA
Genetic change (mutation) and exchange (recombination) provide the genetic diversity upon which selection works to establish a specific microbe in its particular niche. How successful a microbe is at surviving the diverse challenges of an ever-changing environment ultimately rests upon the relative diversity within the microbial population at large. Under adverse conditions, a high mutation rate might be anticipated since it would increase the chances of spawning the rare mutant needed to survive - be it to escape immune surveillance, to elude therapeutic intervention, or to evade the manifold barriers meant to keep microbial populations in check. In this vein, we discuss here the importance of particular mutators, those defective in methyl-directed mismatch repair (MMR), that we found relatively frequently among natural isolates of Escherichia coli and Salmonella enterica. We weigh the role of MMR mutators in microbial evolution, for such mutators are not only hypermutable but promiscuous as well.
A live attenuated strain of the simian immunodeficiency virus (SIV) with large deletions in nef, vpr, and in the negative regulatory element (NRE) was pathogenic in 100% of infant macaques vaccinated orally, intravenously or intra-amniotically. Long-term follow-up of 16 vaccinated adult macaques showed recurrence of viremia (4 animals), and signs of disease (2 animals). The molecularly cloned vaccine strain underwent a number of mutations during the years after vaccination. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines attenuated by similar mechanisms may also have retained the potential to cause AIDS and related diseases.
Production of Secretory IgA Plantibodies
Elliott Fineman, M.S., J.D., C.E.O., Planet Biotechnology, Inc.
Genetically engineered (transgenic) plants have several advantages for production of protein therapeutics and vaccines. Among these are cost and scale of production, lack of human pathogens in plant hosts, limited toxicity and facile construction. Our laboratory first demonstrated that plants can assemble large amounts of secretory IgA (SIgA) - the major mucosal antibody synthesized by the body. This opens the opportunity of novel treatments for many unaddressed medical problems using mucosal, enteric or topically applied plantibodies. The first therapeutic SIgA plantibody evaluated in clinical trials inhibits tooth adherence of S. mutans, the major pathogen causing dental caries. Results from and regulatory issues related to clinical studies of plantibodies and other plant produced proteins will be discussed.
FDA Efforts on Safety Assessment of Vectors Used for Gene Therapy
Carolyn A. Wilson, Ph.D., Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, FDA
The Center for Biologics Evaluation and Research is charged with ensuring the safety, efficacy, potency and purity of biological products. While general regulatory principles apply to the review of gene therapy clinical protocols, the rapidly evolving nature of these products requires flexibility and case-by-case assessment of the unique safety issues involved. As gene therapy clinical protocols proceed, we reassess safety concerns in an iterative manner, and the regulatory concerns of phase I clinical trials may differ from those of later phase testing. More gene therapy clinical trials are proceeding to post-phase I testing, and these issues will be discussed.
Gene Therapies: A Newer Model of Federal Regulation
Philip D. Noguchi, M.D., Director, Division of Cellular and Gene Therapies, Office of Therapeutics Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration
In 1986, FDA announced that gene therapy products would be considered biological products subject to licensure by FDA under section 351 of the Public Health Service Act. Prior to the revolution in biotechnology in the early 1980's, most biologic products were either viral or bacterial vaccines, blood or blood products or allergenic extracts. CBER, the lead center for biological products responded to the rapid growth in biotechnology by forming a number of new product divisions including, in 1993, the Division of Cellular and Gene Therapies (DCGT) which has the lead role in regulating gene therapy. Also in 1993, FDA issued a Federal Register Notice that discussed the regulatory program for gene therapies (58 Fed. Reg. 53,248 [14 October 1993]). Investigational clinical trials in humans with gene therapy products are subject to the general requirements for drugs and biologics in Title 21 of the Code of Federal Regulations (CFR), including 21 CFR Part 312, the Investigational New Drug Application (IND). As of November, 1998, over 220 INDs for human gene therapy have been submitted to the Agency, with an average of 30 to 40 per year for the past several years. As these gene therapy products move from phase 1 through phase II and phase III, other portions of the 21 series pertain, including the 200 series on Current Good Manufacturing Practices (GMPS), the 300 series on new drug applications, the 600 series on product license requirements and various aspects of government reinvention such as elimination of the establishment license, accelerated approvals, oncology initiative and the tissue initiative of March 1997. Given the rapid escalation of industry involvement, how would one find out about the latest events and progress in gene therapies? The first place to look would be on the World Wide Web, but not on the FDA home page, but rather at http://www.nih.gov/od/orda/, the home page of the Office of Recombinant DNA Activities (ORDA) of the National Institutes of Health (NIH). In addition to a listing of studies and investigators, one can find at this site a rich history of the public discussions in the area of human gene therapy through the minutes of the Recombinant DNA Advisory Committee (RAC). This unusually public access to an FDA regulated area has an equally unusual history. In 1975, scientists worldwide voluntarily halted laboratory research in the then novel area of recombinant DNA research, on the basis that the risks of such research were unknown and could have devastating effects on animal and human life. An ad hoc gathering of distinguished scientists met in Asilomar, California in 1975, and from those debates the RAC was constituted within the office of the NIH Director. The charge of the RAC was to review in public each experiment involving recombinant DNA research. Over time the RAC declared classes of experiments to be exempt from review and now currently only reviews in public those human gene therapy protocols considered to be novel enough to warrant public discussion. The evolution of RAC oversight to prospectively discuss emerging ethical and safety issues, such as in utero gene therapy and lentivirus vectors, will serve an increasingly important role in helping the FDA regulate and advance the area of gene therapies.
The biotechnology revolution has resulted in major advances in our understanding of the molecular biology of cell and tissue function, and has provided an impressive array of new technologies to monitor the molecules that control these functions. These advances have created major opportunities for improved approaches to nonclinical and clinical safety assessment. These include: 1) damage-inducible responses as indicators (biomarkers) of functional damage to cells and tissues, 2) genetic technology for individual genotyping and monitoring of mutations and genetic polymorphisms in vivo, 3) transgenic technologies that allow the construction of new animal models with engineered characteristics, such as rapid tumor formation, humanized receptors, and recoverable genetic targets, and 4) improved biomarkers of cell and tissue pathology. Examples of these new approaches and how they may lead to improved safety assessment will be discussed.
Use of Transgenics, P53 and Tg.AC, and the Carcinogenicity of Genotoxic and Nongenotoxic NCE's
R. E. Stoll, Ph.D., Director of Toxicology and Safety Assessment, Boehringer Ingelheim Pharmaceuticals, Inc.
The presentation will entail Boehringer Ingelheim Pharmaceuticals Inc.'s experience in evaluating and using the Tg.AC and/or P53 transgenic mouse in the carcinogenicity assessment of drugs. A case history, oxymetholone, tested in both the Tg.AC and P53 mouse will be presented. Historical and positive control data will also be presented, along with basic design methodology issues when planning on performing such 6 month transgenic studies.
Genotyping of Metabolic Polymorphisms for Prediction of Adverse Drug Reactions and Human Cancer Susceptibility
Fred F. Kadlubar1,2, Patricia A. Thompson1,2, Nicholas P. Lang2, and Michael E. Hogan3, 1N.C.T.R., FDA, Jefferson, AR 72079, 2Arkansas Cancer Research Center, Little Rock, AR 72205, and Genometrix Corp., The Woodlands, TX, 77381
Genetic polymorphisms have long been associated with inter-individual differences in susceptibility to adverse drug reactions, cancer, and other diseases in humans. Such polymorphisms often involve single base changes in coding or in non-coding regions of genes that can alter the biological function and/or the expression of proteins involved in drug or carcinogen activation or detoxification. Examples of individual susceptibility include drug-induced hepatotoxicities and cancers of the lung, urinary bladder, colon, breast, and prostate. Although genotyping has usually been based on PCR-RFLP analyses, the advent of high-throughput DNA microarray technology should allow molecular epidemiological studies for individual assessment of drug efficacy and cancer risk.
Surrogate Markers for Efficacy and Safety Testing
J. Carl Barrett, National Institute of Environmental Health Sciences, National Institutes of Health
With the discovery of new gene targets and the use of combinatorial chemistry to create large numbers of lead compounds, the rate limiting step in drug discovery and development becomes clinical studies of efficacy and safety. The potential of surrogate markers for efficacy is high in drug development to provide proof of principle and ultimately for drug registration. Surrogates of safety are also crucial throughout the drug discovery and development processes. New technological developments allow improvement of animal models by transgenic methodologicals, identification of susceptible individuals on the basis of genetic polymorphisms, and use of gene expression profiles for study of drug efficacy and safety. cDNA microarrays allow studies in animals and humans of drug mechanism of action and effectiveness as well as multiple toxic effects in target and nontarget tissues. Through development of a combination of these technologies, it is predicted that surrogate markers will be a key component of future pharmaceutical and regulatory decisions.
Tissue engineering has been defined as the application of the principles of life sciences and engineering to develop biological substitutes for the restoration, maintenance, modification, improvement, or replacement of tissue or organ function. In this context, Tissue Engineered Medical Products (TEMPs) include a broad range of products such as: human and animal tissues or organs for transplantation; processed, selected, or expanded mammalian cells in combination with or without biomaterials; and totally synthetic materials of biomimetic design. To date, this multi-disciplinary technology has given rise to a diverse array of products for many different medical conditions, affecting virtually every organ system in the body. Some of these products have been approved by the U.S. Food and Drug Administration (FDA) while others are under regulatory evaluation or experimental investigation. As with all medical products, TEMPs are subject to assessments of safety and effectiveness, the basic elements of premarket product review. These are evaluated in the manufacturers claim of intended use, manufacturing and quality control procedures, and the products performance. Where specific questions of product safety and effectiveness cannot reasonably be determined in the premarket studies, further product evaluation in a postmarket study may be appropriate. he FDA is committed to maintaining a productive dialogue with the tissue engineering industry and consumers and to develop the appropriate oversight for Tissue Engineered Medical Products (TEMPs) to contribute to establishing their proper place in clinical medicine. As part of this effort, the FDA has adopted multiple, cooperative, and multi-disciplinary approaches across the appropriate FDA centers to develop the necessary oversight for TEMPs. These approaches include initiatives of cross-cutting working groups such as the FDA InterCenter Tissue Engineering Working Group and the Tissue Reference Group, as well as the InterCenter Agreements, a Reinventing Government Initiative (the Proposed Approach to Regulation of Cellular- and Tissue-Based Products), and rulemaking. In addition, the FDA utilizes other vehicles such as guidance documents and standards to aid the product review process and TEMP development. As tissue engineering technology evolves leading to new and different products, the development of new and different product-specific issues will be expected. Therefore, the FDA, working with the industry and other interested parties, will continue to seek resolution of such issues through its continuing research, review, and assessment of TEMPs. In some cases, lessons learned from previous product classes, such as recombinant DNA technology, may be applicable. In other cases, the FDA will look to the best scientific minds and methods to determine innovative and appropriate resolution.
Tissue Engineering Solutions to Clinical Problems
Joseph P. Vacanti, M.D., John Homans Professor of Surgery, Harvard Medical School and Massachusetts General Hospital
The field of Tissue Engineering is now emerging internationally as a multidisciplinary scientific effort to generate new tissues for all fields of reconstructive surgery. Current therapies often fall short of achieving optimal results of patients lacking sufficient functioning tissue due to disease, trauma, or congenital absence. Replacing destroyed structures with new living tissue is the ultimate goal. Progress towards this goal will be discussed using many clinical examples.
Polymeric Based Systems for Tissue Engineering
Robert S. Langer, Sc.D., Massachusetts Institute of Technology
Tissue loss or organ failure is one of the most devastating problems facing patients today. We discuss here a new approach to address this problem involving the use of bioerodible polymers which can serve as implantable scaffolds for mammalian cells to create new organ transplants. By appropriately engineering the polymer scaffold, high concentrations of cells can be provided to aid in the treatment of metabolic or structural deficiencies. One critical issue is polymer synthesis in particular, it is important to be able to design polymers that are biocompatible and can interact with mammalian cells in such a way as to promote adhesion and growth of specific cell types. A second issue involves polymer manufacturing. The goal here is to design a polymer matrix that is mechanically strong yet has a very large surface to volume ratio so as to be able to accommodate a large number of cells. An additional important area of study involves reactor design which must be optimized so as to enable cells to grow in a desirable fashion. In vivo studies examining the effectiveness of the engineered tissue or organ are the final and ultimate test of performance. We and our collaborators have used these approaches to create cartilage, skin, liver, tendon, bone, intestine, urologic structures, and other tissues in animals or humans.
Cell Design Principles in Cell and Gene Therapy and for Bioartificial Organs
Lola M. Reid, Ph.D., Professor, Program in Molecular Biology and Biotechnology, UNC School of Medicine
The ability to do human liver cell or gene therapy or to develop human bioartificial livers is severely constrained by the limited amounts of normal human tissue available. Almost all healthy tissue samples, from individuals over 6 months of age, go automatically into liver transplantation programs. We are pursuing an alternative which is to use flow cytometry methodologies to isolate and purify hepatic progenitor populations that are capable of extensive expansion and the ability to mature into all of the cells of the adult tissues. The methodologies have been developed utilizing rodent model systems and are now being adapted to human tissue. The microenvironment (hormones, nutrients, extracellular matrix) for ex vivo expansion and differentiation of the progenitors has been defined and overlaps with that for mature liver cells but also has a number of distinctions. The purified rodent hepataic progenitors proved capable of reconstituting adult liver tissue giving hope to a similar phenomenon in humans. Over the past year, antigenic profiles for human hepatic progenitors have been defined enabling us to isolate essentially all human liver cells expressing alpha-fetoprotein. The subpopulation of cells enriched for AFP is being tested as a candidate for cell therapy and for the creation of bioartificial livers intended as liver assist devices for patients in liver failure.
The Biomaterials Component for Tissue Engineered Medical Products
Jeffrey A. Hubbell, Ph.D., Institute for Biomedical Engineering and Department for Materials, Swiss Federal Institute of Technology and University of Zurich, Switzerland
Tissue engineered products make more extensive use of biological principles and interactions than more typical biomedical implants, and much of this regulation of biological interaction proceeds through the biomaterial component of the product. Accordingly, materials may be designed specifically for the envisioned tissue engineered product. These materials may be biological in origin, derivatised biologicals, or biomimetic synthetics. The goals of the incorporation of such biological functionality may be to provide adhesion signals for cells, to induce cell migration, to promote the retention of a particular cell phenotype, to release an incorporated growth factor, to induce a desirable multi-cellular assembly and morphology, or to be remodeled under the biological influences of cells in the tissue response. Such materials are constructed for applications as diverse as matrices for transplantation of cells, matrices for immunoisolation of cells, matrices for the promotion and guidance of cell infiltration, matrices for guidance of tissue remodelling and morphogenesis, and matrices for the modulation of healing, regeneration and implant integration.
Codifying Tissue Engineering Technology and Products
Peter C. Johnson, M.D., President, Pittsburgh Tissue Engineering Initiative; Chair, American Society for Testing and Materials Committee F04, Division IV on Tissue Engineered Medical Products
Engineered tissues are complex products whose construction and use can be predicted to require substantial changes in the ways that researchers, industry and government regulators communicate with one another. The development of engineered tissues requires unprecedented interdisciplinary scientific cooperation. Similarly, the review and regulation of these products for human use has required an unusual degree of interagency cooperation. In order to facilitate these interactions, a standards development effort for tissue engineered medical products was proposed at the ASTM in Spring 1997. This process was designed to antedate the appearance of most products since it was realized that a preliminary mechanism was necessary to foster dialogue between regulators and technology developers during the actual process of technology development. In addition, it was deemed important to structure the codification of tissue engineered medical products in a consensus-based fashion. The structure of Division IV and the ways in which it is achieving these goals en route to the development of standards for tissue based products will be presented.
The genetically polymorphic human cytochrome P450s designated CYP2D6 and CYP2C19 have been shown to be involved in the metabolism of a large number of important drugs including codeine, antiarrhythmics such as flecainide, tricyclic antidepressants such as desipramine, antipsychotics such as haloperidol and risperidone and anxiolytics including diazepam and fluvoxamine. The genetically-mediated absence of these isoforms results in changes in drug exposure (area under the plasma-concentration time curve) that vary between 5 and 20-fold. Clinical consequences that include lack of effect (codeine), proarrhythmia (flecainide), seizures (desipramine), extrapyrramidal toxicity (haloperidol and risperidone) and prolonged sedation (diazepam) are of potentially great importance to patients and their health care providers. The determination of metabolic genotype for these enzymes using relatively small amounts of DNA obtained from whole blood, buccal epithelia or hair follicles has the potential to accurately predict genotype using a method that does not require careful ingestion of a probe drug and then a timed urine or blood collection, and that would make large scale testing of populations easier. We have tested over 4000 samples of DNA obtained from various ethnic groups and clinical trials over the past three years for their CYP2D6 and CYP2C19 genotype using mutation-specific primers with endonucleases for the *1, *3, *4, and *5 mutations of CYP2D6 and the *1, *2, and *3 mutations of CYP2C19. In the past year we have also tested the AffymetrixTM GeneChip® CYP450 system. Conventional PCR and mutation-specific endonuclease digestion requires separate amplifications and digestions be done for each mutation tested, and is relatively slow and work-intensive. On the other hand, it is more forgiving and technically less demanding than multiplex primer amplification, for the results of amplification are very sensitive to DNA quality. The AffymetrixTM GeneChip® CYP450 system is able to simultaneously detect 17 CYP2D6 and 3 CYP2C19 mutations. The fact that the amplification for this system requires seven pairs of primers mandates the need for a very controlled template free area, since the annealing temperature used must be very permissive to allow all amplicons to be amplified. Optimizing yield for one band may cause a decrease in the yield of another. The fragmentation and fluorescent labeling of amplicons before array hybridization is routine, but the hybridization is the rate-limiting step in an otherwise efficient process. Laser scanning of the fluorescently-labeled chip generated is carried out in a few minutes, but interpretation of the data requires software that does not "over-call" the data, or limit its interpretation. The ability to detect large numbers of CYP2D6 and CYP2C19 mutations in a more through manner is a technical advantage over conventional genotyping that is particularly important in situations where the determined genotype does not appear to match the phenotype.
Detection and Disinfection of Transmissible Spongiform Encephalopathy Agents
David M. Asher, M.D., Chief, Laboratory of Method Development, Division of Viral Products, Office of Vaccine Research and Review, Center for Biologics Evaluation and Research, FDA
FDA must reduce the likelihood that regulated products are contaminated with infectious agents causing transmissible spongiform encephalopathies (TSE agents or "prions") by assessing purity of animal and human source materials and evaluating manufacturing processes purported to eliminate TSE agents. Detection methods vary in sensitivity, specific TSE agents detected and time required. Transgenic mice expressing prion-proteins (PrP) of more susceptible species may improve detection. Immunoassays of protease-resistant PrP, uniquely associated with TSE, are less sensitive than infectivity assays but rapid. A method to evaluate virucidal disinfectants was adapted to TSE agents. Substantial infectivity remained detectable on surfaces after soaking in disinfectant, and some survived autoclaving. Combined treatments are under study.
Rapid Electrochemiluminescent Detection of E. coli O157:H7 Contamination and Electrochemiluminescent
PCR Confirmation
C.G. Crawford, Chandi Wijey, Pina Fratamico, USDA, ARS, Eastern Regional Research Center, Wyndmoor, PA; Paul Converse, IGEN International Inc., Gaithersburg, MD
We have developed an immunomagnetic-electrochemiluminescent (IM-ECL) screening assay for E. coli O157:H7. The assay utilizes anti-E. coli O157 coated paramagnetic beads and anti-E. coli O157 antibody labeled with a ruthenium compound. Meat samples are incubated in an enrichment medium for 5 hours at 42° C. Beads and labeled antibody are added to 1.0 ml of the enriched sample, gently mixed for 60 minutes, then analyzed by the ORIGEN instrument. Each analysis takes less than one minute, so 50 samples can be analyzed in under an hour. The test can detect 1 CFU E. coli O157:H7 in the original sample after the 5 hour enrichment period. We have also developed an IM-ECL PCR assay for the confirmation of the presence of the flagellar H7 gene, and a multiplex PCR assay for the simultaneous detection of the H7, and the attaching and effacing gene (eae).
A Sensitive PCR-Based Reverse Transcriptase Method for the Detection of Retroviruses
Keith Peden, Ph.D., Senior Staff Fellow/Microbiologist, Laboratory of Retroviruses, Division of Viral Products, Office of Vaccines Research & Review, Center for Biologics Evaluation and Research, FDA
The reverse transcriptase (RT) assay is a general method for the detection of any retrovirus, since all retroviruses carry in their viral particles (virions) multiple copies of this enzyme. Thus, the RT assay has the potential to detect all types of retroviruses regardless of the sequence of the RNA genome present in the virion. The problem with the conventional RT assay is that it is not sensitive enough to reveal the presence of the low levels of retroviruses that may be expected as contaminants of viral vaccines or other biological products or to measure the levels of retroviruses in clinical specimens during the course of disease, such as in AIDS. In the last few years, several highly-sensitive RT assays have been developed that are about one million-fold more sensitive than conventional RT assays. These are PCR-based assays and, while they have names such as PERT and Amp-RT assays, they can be generically referred to as PBRT assays for PCR-based RT assays. These assays have the potential to detect a single virion in a ten microliter sample and thus can detect retroviruses at a level of 100 particles per milliliter. Recent modifications to the PBRT assay have been incorporated such that the assay is now linear over at least six orders of magnitude.
The Role of Genetic Testing in the Practice of Medicine
Neil A. Holtzman, M.D., M.P.H., Professor, Genetics and Public Policy Studies, Johns Hopkins Medical Institutions
That genetic discovery will significantly transform the practice of medicine rests on several assumptions: (1) Knowledge of genotype will be a valid predictor of future disease; (2) Individualizing therapy on the basis of inherited risk will improve outcome; (3) The genetic approach is more efficient than others; (4) It will be acceptable to the public and to third-party payers. The assumptions are most likely to be met for highly penetrant Mendelian disorders, most of which are rare. Seldom will they be met for the vast majority of those with common disorders. Among this large group, finding healthy people with a family history of a disease may benefit some, but most disease will occur in those without a significant family history.
The FDA has the awesome responsibility for protecting the health of the American public by ensuring the safety, efficacy, purity and honest labeling of foods, human and veterinary drugs, biologics, medical devices and cosmetics - products that in aggregate constitute over 25% of the nation's Gross Domestic Product. Since its earliest inception in 1906, the agency has linked its regulatory responsibilities with the conduct of original research to enhance the quality of its regulatory performance and advance the underlying scientific disciplines, and has attempted to embed its commitment to "science-based regulation" deeply within the agency's fabric. Today, at a time when astonishing and rapid advances are transforming the foundational scientific and technological disciplines, and the translation of these advances into wholly new classes of medical products - drugs and devices - occurs with unprecedented rapidity, the need for scientifically astute regulation, informed and supported by an intramural base of mission-focussed, high quality, well-organized and well-managed scientific research has never been more compelling. Yet, that research base has perhaps never been so critically at risk.
Consumer Issues in Food Biotechnology Regulation
Marion Nestle, Ph.D., M.P.H., Department of Nutrition and Food Studies, New York University
The FDA's science-based regulatory policies for food biotechnology have elicited consumer demands for premarket notification, premarket testing, and labeling of food products derived from genetically modified agricultural products. Consumer concerns also focus on whether and how FDA plans to address the issue of health claims for "nutritional genomic" or "functional" foods foods bioengineered for a particular therapeutic purpose such as increased vitamin, phytochemical, or omega-3-fatty acid content.
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Last updated on 1998-NOV-28 by frf.