H U M A N G E N E T H E R A P Y 2:243-249 (1991) Mary A n n Liebert, Inc., Publishers
R e g u l a t o r y C o n c e r n s in H u m a n
Gene
Therapy
SUZANNE L. EPSTEIN
ABSTRACT Gene therapy in humans is now being undertaken in an investigational setting. Such therapy involves the administration of biological products to h u m a n patients. A document entitled, "Points to Consider in H u m a n Somatic Cell Therapy and G e n e Therapy" has been prepared by the Center for Biologies Evaluation and Research ( C B E R ) of the Food and D r u g Administration ( F D A ) and is published elsewhere in this issue. This paper provides explanatory material about the C B E R regulatory process and the scientific and regulatory basis for the requests for data in that document.
handled on a case-by-case basis but follows a common set of principles. In the case of gene therapy, the field is evolving so rapidly that any set of standards might be out of date before it The two institutions that have major regulatory responsibility over human gene therapy are the N I H and the F D A . The could be announced, and so the approach must be a case-by-case N I H has previously published its "Points to Consider" approach that is responsive to the specific proposals and the changing scientific information available. This paper does not document. The F D A publishes a draft of its proposed "Points to Consider" on pages 251-256 of this issue. In this constitute a formal policy statement, but rather is a summary of issues in this area and of FDA's approach to these issues. accompanying article, Epstein provides the background
OVERVIEW S U M M A R Y
information on which the F D A "Points" are based.
R E G U L A T O R Y RESPONSIBILITIES INTRODUCTION In many cases, trials of human gene therapy are subject not to F D A oversight but also to review by institutional review Many OF THE medical interventions that can beonly characterized as gene therapy involve the administration of boards and, at least in the case of federally funded studies, the biological products to humans. This article describes in general NIH Recombinant D N A Advisory Committee (RAC) and its terms some of the regulatory and scientific considerations Human Gene Therapy Subcommittee. The focus and philosophy pertinent to Food and Drug Administration (FDA) review of of these review processes may at times be different from that of the FDA. Table 1 summarizes some differences between the applications in this area. Details can be found in the F D A documents mentioned below and in the "Points to Consider in roles and responsibilities of the F D A and the R A C . Most Human Somatic Cell Therapy and Gene Therapy" published importantly, the F D A examines product development and manelsewhere in this issue. The present discussion is limited to ufacturing, quality control issues, and safety testing involving somatic cell gene therapy; regulation of gene therapy intended to primary data about manufactured batches of material to be used modify genetic material of recipient germ cells would involve clinically. F D A regulation and review applies to both the product and the process of its production. Another distinction is additional considerations. The regulation of biological products has not historically lent that many (not all) meetings of the R A C are public, while the itself to checklists or rigid sets of rules. The products are too F D A provides confidential review since some sponsors consider diverse, and the same sets of tests are not appropriate or even the information they submit to be proprietary. It should also be mentioned that the "Points to Consider in the possible for all products. Regulation of biologies is therefore
Molecular Immunology Laboratory, Division of Biochemistry and Biophysics, FDA, CBER, Bethesda, M D 20892.
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244 Table 1. Regulation of Gene Therapy: NIH Recombinant DNA Advisory Committee and Its Human Gene Therapy Subcommittee Compared to FDA NIH-RAC
FDA
Confidential (some meetings open) Staff available for interaction and consultation on day-to-day basis Main areas of expertise, and of information scrutinized: Basic sciences Basic sciences Clinical medicine Clinical medicine Regulatory issues Law Manufacturing processes Ethics Quality control Theology Preclinical testing Preclinical testing Open, public (some meetings closed) Six meetings per year
Design and Submission of H u m a n Somatic-Cell Gene Therapy Protocols" issued by the N I H R A C is a distinct document from the F D A "Points to Consider on H u m a n Somatic Cell Therapy and Gene Therapy." The F D A has issued "Points to Consider" documents in a variety of areas, and the term was not changed due to overlapping use by the R A C . The Center for Biologies Evaluation and Research ( C B E R ) is the F D A component that has reviewed existing gene therapy protocols. C B E R has several mechanisms for facilitating the review process. A variety of documents are available, including those called "Points to Consider," which are not regulations but rather present the current thinking of the C B E R staff about important issues in a given area of product development and testing. These documents can be valuable to manufacturers of products related to gene therapy, to sponsors of trials, or to participants in the dialogue about h o w gene therapy should be regulated. Those "Points to Consider" documents most relevant to gene therapy are listed in Table 2. This discussion will focus on the investigational n e w drug (IND) stage of regulation, since there is no experience yet with licensing of gene therapy products. C B E R encourages early interaction between sponsors and the C B E R staff, for example via meetings held before submission of a formal I N D application ("pre-IND" meetings). I N D review is intended to be a dynamic process, with exchange of ideas and information between the sponsors and the C B E R staff. The goal is for F D A to voice concerns early and to resolve potential problems. For this reason, C B E R is willing to review proposals in draft form,
especially proposals for novel therapies like gene therapy. C B E R does not require that the protocol have prior approval from other bodies such as the R A C before initiating such discussions. If sponsors begin such discussion early, problems can surface, advice can be given, and thus unanticipated delays in beginning a clinical trial can be avoided. It should be emphasized that not all the data discussed below need be available at the time the initial I N D application is submitted or even in some cases before initiation of clinical trials. C B E R bases regulatory judgments on in-house review of the laboratory and clinical data presenting results of preclinical research and of tests on the actual products and preparations to be administered to patients, to determine if they meet applicable standards. I N D applications from commercial sponsors, NIHsupported researchers, and any other sponsors are all treated in the same manner.
SOMATIC CELL THERAPY A N D G E N E THERAPY Somatic cell therapy can be defined as the administration to humans of living cells which have been manipulated ex vivo, while gene therapy is a medical intervention designed to modify the genetic material of living cells in patients or cells for administration to patients. Gene therapy can be pursued by a variety of technologies. The approach already used in a few patients is part of the broader category of somatic cell therapy. In
Table 2. Points to Consider Documents Relevant to Gene Therapy Issued by the Center for Biologics Evaluation and Research Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant D N A Technology (1985) Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1987) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1987) Points to Consider in the Collection, Processing, and Testing of Ex-Vivo-Activated Mononuclear Leukocytes for Administration to Humans (1989) To receive a copy of any of these Points to Consider documents, contact: Congressional, Consumer Affairs, and International Affairs Staff, HFB-142, Suite 109, Metro Park North III, 5600 Fishers Lane, Rockville, M D 20857; Telephone (301) 295-8228, F A X (301) 295-8266.
REGULATORY CONCERNS IN HUMAN GENE THERAPY this type of gene therapy, cells removed from a patient are manipulated by insertion of a gene. The modified autologous cells are then readministered. Somatic cell therapies differ from simple transplantation in that the cells are manipulated in some substantial way. For example, the cells m a y be grown in tissue culture in the presence of growth factors and caused to expand greatly in numbers. They m a y be induced to differentiate either by soluble factors or by contact with other cells. The cells m a y be subjected to selection, in cases in which a subpopulation of the original cells is the desired therapeutic population. Selection can involve treatments with antibody plus complement or with toxins, outgrowth in the presence of selective growth factors, and so forth. In the cases of differentiation or selection, the cell population after the manipulation steps m a y have phenotypic and functional properties different from those of the starting population. The patient m a y receive his/her o w n manipulated cells (autologous), cells from another h u m a n (allogeneic), or cells from an animal of a different species (xenogeneic). T h e precise boundary between transplantation and procedures involving sufficient manipulation in vitro to warrant designation as somatic cell therapy is not yet clear. A variety of cellular therapies are in development. A n extensive list of examples, some already in or nearly ready for human trials, can be taken from the scientific literature. This includes myoblast transfer for muscular dystrophy,(,_3) pancreatic islet cell implantation for diabetes,(4) and implantation of dopamine secreting cells for Parkinson's disease.(5,6) There are, of course, additional approaches to gene therapy that are not forms of somatic cell therapy and m a y be proposed for clinical use in the future, such as use of vectors administered directly to the recipient.(7,8) Such an approach would bypass the in vitro cellular manipulation steps, but would still raise m a n y of the same concerns that will be discussed here. The cells undergoing genetic alteration would n o w be recipient cells in vivo, which could in some cases be sampled and analyzed to address some of the same issues. Other approaches m a y emerge in the future as the technology develops. Discussion in this paper of the approach currently in clinical use is not intended to imply restriction of gene therapy proposals solely to somatic cell therapy approaches. In the area of gene therapy, proposed uses for correction of genetic defects include replacement of an enzyme,(9J0) clotting factor,'"~13) a 1-antitrypsin for emphysema/8* or the cystic fibrosis gene product.(,4) Other applications include production of a therapeutic biological such as CD4(l516) or a cytokine in situ, to permit production in the recipient of a product that is needed at high concentrations over a long period of time, and thus is meant to avoid repeated administration of an exogenous product such as a recombinant protein. In addition to gene therapies, another proposed use of this technology is gene marking: cells are genetically altered by insertion of a marker gene that is not intended to have a therapeutic effect on the patient, but that provides a w a y to identify and track the administered cells.(l7) These types of therapies and protocols m a y become c o m m o n in the near future. The question then arises as to h o w F D A will regulate the relevant products. Regulatory concerns in gene therapy fall into three categories, and each will be discussed. First, there are concerns c o m m o n to all biological products. Second, there are concerns that apply to
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somatic cell therapies in general. Finally, there are unique concerns that apply to therapy involving genetic alteration. For each of these areas, the type of questions and concerns F D A raises will be presented, along with the scientific basis for the concerns. The regulatory approach in this area is of course evolving, and input is welcome from the scientific community as approaches are formulated.
R E G U L A T O R Y CONCERNS T H A T APPLY T O ALL BIOLOGICAL PRODUCTS The area of conventional regulatory issues applying to all biological products is extremely important. Gene therapy proposals can pose risks due not only to esoteric issues, but to conventional ones. Consider, for example, a carefully conceived and ethically justified proposal that uses an elegantly transduced cell population. If that cell preparation has not been adequately tested for contamination with infectious agents, the patient could be harmed. If a gene therapy results in production of hormones, lymphokines, or toxins, then the biological properties of those molecular species themselves are very important. In thinking about more specialized concerns unique to gene therapy such as use of retroviral vectors, tissue-targeted viral vectors, or homologous recombination, the established criteria for evaluating biological products must not be neglected. This article will not go into great detail, but certain general points should be made since researchers m a y not be familiar with regulatory matters. Preclinical safety studies prior to I N D trials should include both in vitro testing and animal testing in an informative (not necessarily perfect) animal model. Safety testing should cover and exceed the dose range that is proposed for the h u m a n trials. Although proof of efficacy is obtained during clinical trials, preclinical studies should present evidence for a rationale for efficacy of the therapy. Potency testing requires some form of quantitative assay for a relevant property. Biologicals are complex materials that cannot be completely defined. History has shown that h o w a biological product is prepared is very important; in-process control is, therefore, an important aspect of defining and characterizing the product, and analyzing the end product m a y not be sufficient. For example, poor control of a production process can lead to introduction of an adventitious agent such as a virus for which no reliable assay is available, or to lability of the product, as well as to inadvertent changes in properties that escape detection. For that reason, in the case of biologies both the product and the process of its production are regulated. In an I N D , descriptions of the methods and reagents should be submitted, along with quality control test results not only on the product but on cell banks that are used in its preparation and on key intermediates in the production of the product. D u e to the importance of control of the production process, not only the biological product but also the production establishment must be licensed prior to marketing. Another aspect of process control is the testing of lot-to-lot reproducibility. Quantitative assays for appropriate properties of the material or correlates of its action are needed to confirm that at least in some ways the same material is being m a d e in each batch. Sometimes the mechanism of action of a proposed therapy is unknown. Then in vitro evidence of product potency
246 may not be available, and what to test on a lot-to-lot basis as a potency assay m a y be unclear. Still, quantitative assays of properties that m a y correlate with product action can be performed, and provide a database for eventual determination of what measurable properties of the product predict therapeutic efficacy.
R E G U L A T O R Y CONCERNS C O M M O N T O SOMATIC CELL THERAPIES There is precedent and FDA experience in dealing with therapies based on cell populations, and a Points to Consider document is available dealing with the special case of activated mononuclear leukocytes (see Table 1). Preclinical testing will be discussed first, followed by comments about lot release testing, and then concerns about cell performance in vivo. Preclinical testing includes the research phase and uses preparations analogous to the ones intended for clinical use but not necessarily the actual clinical preparations, while lot release testing is performed on all clinical batches. For any cellular therapy, the cells to be used as a starting population should be described as to autologous, allogeneic, or xenogeneic origin and tissue origin. Concerns about h u m a n or animal donor qualification, cell collection procedures, and quality control of media and components used in culturing the cells are important. Sterility is critical, both for the cell population and for intermediates in its preparation; this involves freedom from bacteria, fungi, mycoplasma, and adventitious viruses. Once a cell population has been obtained, it should be characterized at various steps of processing for a number of features, including viability and presence of relevant cell types as defined by appropriate phenotyping methods. Properties such as morphological characteristics, cell-surface markers detected by antibodies, receptors for hormones or growth factors, and biochemical features can be used as phenotypic markers. Heterogeneity of the cell population should be assessed by quantitative phenotyping. Heterogeneity of cell populations can be quite important; if each of several cultures derived from the same starting material shows activity in a qualitative assay and reaches the same total number of cells, that does not necessarily mean that the cultures are biologically equivalent. Cultures m a y have been partially overgrown with other cell types, such as subpopulations in the starting culture or contaminating populations from other cultures in the laboratory. Thus, markers of identity are needed, including both general phenotypes and in some cases patient-specific markers. In some cases, only a small percentage of cells bearing the markers m a y be necessary for acceptable activity. The relevant cell functions or products can provide markers for the potency of the cells. Products made by the cells and required for therapeutic activity should be shown to be biologically active and should be quantitated, to determine whether the level produced is adequate to produce the desired effect in vivo. All biologically active forms of the product should be considered, for example, membrane and secreted forms of the same protein. Types of cell function other than secretion of a product m a y require measurement by assays such as cellular cytotoxicity, proliferation in response to stimuli, differentiation potential, or bioassay in animals.
EPSTEIN The preclinical safety evaluation should include examination of growth control of the cells and confirmation of any expected dependence on exogenous growth factors. Animal models are often helpful for in vivo safety evaluations and should be considered. Even when there is no animal model for efficacy of the therapy, some type of preclinical safety data from in vivo animal testing should be provided, and adverse effects noted in any animal experiments should be reported. While efficacy m a y depend on only one or a few particular products of the cells, the cells m a y produce many other molecular species. Other products known to be produced by the cells should be identified when possible, and possible adverse effects of these and or unidentified species sought in animal tests. Lot release testing, that is, the testing performed on all batches of cells before use in patients, cannot use all the tests described above, particularly in the case of small batches of cells for use in single patients. S o m e types of characterization m a y use too m a n y cells or m a y be too cumbersome or time consuming for routine use. However, a number of quality control tests should be performed shortly before administration to confirm that the cell population is as expected, and should be repeated after thawing and possibly passaging of frozen cells. These should ordinarily include viability, sterility, identity, and function of some kind. Cells can be introduced into recipients in a variety of ways, including simple infusion or implantation and more complex modes such as use of solid supports coated with growth factors to favor cell growth or tissue formation/12'I8) Cells m a y not behave the same w a y in a recipient as in tissue culture. In vivo behavior of the cells can be analyzed either in animals or in clinical trials or both, depending on the availability of informative animal models. Evidence should be provided as to whether the cells survive and continue to function in vivo. If the ability of the cells to function in vitro is due to specialized conditions in culture, such as addition of an exogenous growth factor, then perhaps those conditions would have to be provided in vivo at adequate levels to maintain function. In some cases, cell survival and function might be suppressed by other influences in vivo, including rejection by the recipient's i m m u n e system. The time course of cell performance in vivo is important: H o w long the cells continue to function might determine the interval between treatments, and very transient function m a y be useful in some situations but not in others. In some cases, the cells m a y display particular trafficking patterns or homing to a particular organ due to interactions with certain other cells. Although it m a y not be technically feasible to study in all systems, cell localization in vivo should be considered, since it might have effects on safety and might influence whether the cells are able to exert their therapeutic effect. For example, if cells are intended to localize to a tumor site and kill the tumor, the fraction of the cells that reach that site should be estimated and information sought as to the fate of the remaining cells. Toxicity from cells that localize to unintended sites should be considered. In addition, systemic effects of the cells or their products could be very different from local effects. If a soluble growth factor is normally secreted at low levels and acts locally to exert an autocrine or paracrine effect, it might be harmful if it is produced at substantial levels by cells in the circulation. I m m u n e responses to the cells or their products can have important effects on results of therapy. The safety question of
REGULATORY CONCERNS IN HUMAN GENE THERAPY tumorigenicity should also be raised. These two issues apply to all somatic cell therapies, but will be discussed below in the particular context of gene therapy.
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its biological activity, and the amount produced. Expressed levels should be compared to the level required for effectiveness. Normal physiological levels m a y not be required; for example, 1 0 % of normal A D A levels can provide adequate function for i m m u n e competence.(28) If there are multiple REGULATORY CONCERNS ABOUT biological forms of the gene product being expressed, they G E N E THERAPY should all be included in the analysis and quantitated if possible. W h e n feasible, assays for expression of the inserted gene should Gene therapy will be considered here in the context of cellularbe used as lot-to-lot tests as an indication of potency. therapy reflecting the current state of the art. Not all the points Stability of gene expression should be examined. A s in the below would apply to all alternative approaches, and the appro- case of other cell functions, very transient expression would be priate testing would differ, but m a n y of these concerns are unsuitable for some therapeutic purposes. Eventually, it should broadly applicable. Note that m a n y issues related to genetic be possible to ask whether the inserted gene is expressed subject alteration of production cells and to recombinant D N A manipu- to normal regulatory controls or is expressed in an aberrant lation are addressed by the "Points to Consider' document on fashion. Improperly regulated expression of a gene product products m a d e by recombinant D N A technology (Table 2). would lead to safety concerns in some cases. Currently, retroviral vectors are being used to introduce Both the cell type chosen for gene therapy and the use of genetic material into cells in vitro.(19_21) Homologous recombi- appropriate regulatory sequences such as promoters in the nation has also been proposed and is being used in some construct m a y be employed to try to target gene expression to experimental sy stems.(22_25) This method targets gene insertion desired sites; in the future, injectable vector tropism might to the corresponding locus of the host and disables or replaces accomplish this. Genetically modified vascular grafts have been the defective gene. A s mentioned above, there is the future proposed for delivery of products that affect diseases related to possibility of direct administration of vectors to the patient(7'8) the circulatory system, or for delivery to organs just downstream which might h o m e to particular target cell types based on of the site of secretion/29' Transduction of liver cells with a receptor tropism or other factors. Use of liposomes to deliver retroviral vector containing a liver-specific promoter has been proposed(30) to achieve expression of phenylalanine hydroxygenetic material is also being considered. Vectors or other genetic constructs used in gene therapy or in lase expression in the liver. preparation of cells for gene therapy should be characterized Since there is little clinical experience, m a n y of the risks of structurally. Important information includes the sources of gene therapy are theoretical at this point; these include inservectors and genetic inserts, detailed diagrams of the steps used to tional mutagenesis and infectious spread of retroviral vectors. It arrive at the final construct, a description of virus stocks and of is hoped that data from early trials will contribute to an cell banks including the packaging cell line used to prepare accumulating data base, and that, with this information, the risks vector-containing supernatants, and information about regula- can be weighed in the context of possible benefit, alternative tory elements present in the vector. Lot release protocols for therapies, nature of the patient population, and severity of the vectors, media, and transduced cells should be included in the disease. Another important safety concern is whether the cells are IND. Quantitative assays of k n o w n sensitivity should be used to tumorigenic. Testing of cell populations for tumorigenicity is assess whether replicating virus is present or is generated, both covered in the "Points to Consider" on cell lines. If the starting in the cultures from which the supernatants are prepared and then cell population used in preparing a cell therapy product was from data on cell preparations and animal or h u m a n trials. Even derived from a tumor explant, there could be contaminating replication-competent murine retroviruses do not appear highly tumor cells in the preparation. In that case, tumor cells should be pathogenic in primates/26) Still, there is concern about introduc- quantitated and the effectiveness of removal shown before ing replication-competent retroviral material into humans, due readministration. The therapeutic cells themselves could also be to possibilities including mutation or recombination with endog- tumorigenic, due to changes in tissue culture, aberrant growth enous sequences. Safety concerns in this area have been ad- regulation in vivo, or insertional mutagenesis. Insertional mutagenesis involving insertion of a vector or a dressed by recent developments in packaging cell line technology,(27) reducing the likelihood of recombination events fragment of vector into the genome could transform a cell if the gene altered by the insertion affects growth control. Integration regenerating a proliferating virus. Genetic constructs can be integrated into cellular D N A at could also disrupt or alter some other important cell function. either random or targeted insertion sites. In the case of homolo- There are a few tests that can be performed to assess these risks. gous recombination/22,23* the gene is inserted into the genome If the cells have been transformed by the genetic manipulation at a k n o w n site, and this can be confirmed by sequencing of leading to uncontrolled growth, they should not ordinarily be regions flanking the integrated copy. In the case of retroviral used (although transformed cell lines of xenogeneic origin might vectors with random gene insertion, the number of copies of the sometimes be encapsulated and used in somatic cell therapy). If gene in a cell should be estimated. In both cases, information the cells are dependent on exogenous growth factors such as should be provided about whether the gene recombines, rear- interleukin-2 (IL-2) in culture, maintenance of this dependence ranges, or mutates rapidly; and h o w long the inserted gene should be confirmed. However, testing for effects on every remains stable and intact. The issue of insertional mutagenesis locus in the cellular genome that could be disrupted by insertional mutagenesis is impossible, and the magnitude of risk is will be discussed below. During preclinical studies, cells should be tested for expres- currently unknown. Note that other accepted treatments, such as sion of the inserted gene—for production of the correct product, some chemotherapy drugs, also carry a risk of mutagenesis.
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EPSTEIN
Immunological interactions between the cells or their prodACKNOWLEDGMENTS ucts and the recipient could alter the safety or efficacy of therapy. Preclinical studies in animal models using analogous The author thanks Drs. Janet Woodcock, Kurt Gunter, animal cells may sometimes be of value, but this question must William Egan, and Joy Cavagnaro for critical review of the also be dealt with by patient monitoring, since immunogenicity manuscript; members of the C B E R scientific research and in animal models may not reflect human responses. Immunolog- regulatory staff who reviewed drafts of the Points to Consider ical parameters to be considered include: antigenic differences document on somatic cell therapy and gene therapy; the staff of between donor and host; immune or allergic reactions to the cells the Division of Cytokine Biology and in particular Dr. Jay Siegel or their products, including the possibility of rejection of the for their work on the Points to Consider document on activated cells; and graft-versus-host reactions or autoimmune reactions mononuclear leukocytes; and Emily McFadden for excellent caused by the cells. secretarial assistance. If the infused cells are making an enzyme, receptor, or other structural protein that the host does not normally express, the host may not be tolerant to it. This is not a very well-explored REFERENCES area, but some purified or recombinant proteins administered as such have elicited immune responses/31_33) Patients deficient in 1. KUNKEL, L.M. (1989). The Wellcome lecture, 1988. Muscular a component may respond to native material, but in addition, dystrophy: a time of hope. Proc. R. Soc. Lond. [Biol] 237, 1-9. non-self epitopes due to mutations, polymorphism, glycosyla2. PARTRIDGE, T.A., M O R G A N , J.E, C O U L T O N , G.R., HOFFtion differences, aggregation, or damaged molecules m a y M A N , E.P., and K U N K E L , L.M. (1989). Conversion of mdx present immunogenic sites even in pateints who do express a myofibres from dystrophin-negative to -positive by injection of form of the same protein. If an immune response to the cells or normal myoblasts. Nature 337, 176-179. their products, either humoral or cellular, is observed, it would 3. L A W , P.K., BERTORINI, T.E., G O O D W I N , T.G., C H E N , M., FANG, Q., LI, H.-J., KIRBY, D.S., F L O R E N D O , J.A., HERbe valuable to know which antigens are recognized and whether ROD, H.G., and G O L D E N , G.S. (1990). Dystrophin production the response alters the safety or efficacy of the treatment. If the induced by myoblast transfer therapy in Duchenne muscular dysantigen is an integral part of the cell or a required product, it may trophy. Lancet 336, 114-115. not be possible to avoid the response except by immunosuppres4. SULLIVAN, S.J., MAKI, T., B O R L A N D , K.M., M A H O N E Y , sion of the recipient. However, if a component of the medium M.D., S O L O M O N , B.A., M U L L E R , T.E., M O N A C O , A.P., and or other additive is involved, it may be possible to remove CHICK, W.L. (1991). Biohybrid artificial pancreas: Long-term it. Experience with allergy to insulin preparations, either conimplantation studies in diabetic, pancreatectomized dogs. Science ventional or recombinant, provides examples of various 252,718-721. kinds/34"36* 5. WIDNER, H., BRUNDIN, P., R E H N C R O N A , S., GUSTAVII, A future approach to greater safety in gene therapy may be B., F R A C K O W I A K , R., LEENDERS, K.L., S A W L E , G., development of ways to down-regulate gene expression, or R O T H W E L L , J.C., M A R S D E N , CD., and BJORKLUND, A. inactivate or kill the cells, if adverse effects develop. Possibili(1991). Transplanted allogeneic fetal dopamine neurons survive ties include use of cells dependent on a growth factor that can be and improve motor function in idiopathic Parkinson's disease. withdrawn, sensitivity of the cells (inherently or after transducTransplant. Proc. 23, 793-795. 6. JAEGER, C.B., GREENE, L.A., TRESCO, PA., W I N N , S.R., tion with additional genes) to drugs, and possibly induction of and AEBISCHER, P. (1990). Polymer encapsulated dopaminergic transcription of an anti-sense insert. Another approach is physcell lines as "alternative neural grafts" Progr. Brain Res. 82, ical isolation of the cells by encapsulation, perhaps making them 41-46. removable. Investigation of such approaches is encouraged but 7. NABEL, E.G., PLAUTZ, G., B O Y C E , F.M., STANLEY, J.C., their use is not required. and NABEL, G.J. (1989). Recombinant gene expression in vivo A final area of concern in gene therapy protocols is patient within endothelial cells of the arterial wall. Science 244, 1342follow-up. Since this area is so new^ long-term follow-up is 1344. needed for both safety and efficacy, and the plans for patient 8. ROSENFELD, M.A., SIEGFRIED, W., Y O S H I M U R A , K , follow-up studies should be described in an IND. Evidence for Y O N E Y A M A , K , F U K A Y A M A , M., STIER, L.E., P A A K K O , viral replication should be sought. In vitro tests are often short P.K., GILARDI, P., STRATFORD-PERRICAUDET, L.D., term; patient follow-up will allow an opportunity to see whether PERRICAUDET, M., JALLAT, S., PAVIRANI, A., LECOCQ, replication-competent virus is generated during prolonged periJ.-P., and CRYSTAL, R.G. (1991). Adenovirus-mediated transfer ods during which sequences are at risk for recombination in vivo. of a recombinant alphal-antitrypsin gene to the lung epithelium in Neoplasms that develop in patients can be tested for presence of vivo. Science 252, 431-434. the inserted gene. Therapeutic activity as monitored by testing of 9. KANTOFF, P.W., GILLIO, A.P., MCLACHLIN, J.R., BORDIG N O N , C , EGLITIS, M.A., KERNAN, N.A., M O E N , R.C, some cell function or product should be followed until loss of the KOHN, D.B., YU, S.-F., KARSON, E., KARLSSON, S., ZWIEactivity is seen. Perhaps the therapy could be administered at BEL, J.A., GILBOA, E., BLAESE, R.M., NIENHUIS, A., longer intervals, or only once. O'REILLY, R.J., and ANDERSON, W.F. (1987). Expression of In conclusion, the specifics of gene therapy are novel, the human adenosine deaminase in nonhuman primates after retrovirusprocedures and products complicated, and human applications mediated gene transfer. J. Exp. Med. 166, 219-234. are of historic importance. This discussion is intended to raise 10. WILSON, J.M., DANOS, O., G R O S S M A N , M., RAULET, the issues of concern in the preclinical regulatory evaluation of D.H., and MULLIGAN, R.C. (1990). Expression of human adeproposed gene therapy protocols, and to summarize the types of nosine deaminase in mice reconstituted with retrovirus-transduced data that may address those issues. hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 87, 439443.
REGULATORY CONCERNS IN HUMAN GENE THERAPY 11. ARMENTANO, D., THOMPSON, A.R., DARLINGTON, G.,
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Amphotropic murine leukemia retrovirus is not an acute pathogen for primates. Hum. Gene Therapy 1, 15-30. and W O O , S.L.C (1990). Expression of human factor IX in rabbit hepatocytes by retrovirus-mediated gene transfer: Potential for gene 27. MILLER, A.D. (1990). Retrovirus packaging cells. Hum. Gene Therapy 1, 5-14. therapy of hemophilia B. Proc. Natl. Acad. Sci. U S A 87, 614128. JENKINS, T., RABSON, A.R., NURSE, G.T., et al. (1976). 6145. Deficiency of adenosine deaminase not associated with severe 12. ST. LOUIS, D. and V E R M A , I.M. (1988). An alternative apcombined immunodeficiency. J. Pediatr. 89, 732-736. proach to somatic cell gene therapy. Proc. Natl. Acad. Sci. USA 29. WILSON, J.M., BIRINYI, L.K., S A L O M O N , R.N., LIBBY, P., 85,3150-3154. C A L L O W , A.D., and MULLIGAN, R.C. (1989). Implantation of 13. PALMER, T.D., T H O M P S O N , A.R., and MILLER, A.D. vascular grafts lined with genetically modified endothelial cells. (1989). Production of human factor IX in animals by geneticaly Science 244, 1344-1346. modified skin fibroblasts: potential therapy for hemophilia B. 30. PENG, H., A R M E N T A N O , D., M A C K E N Z I E - G R A H A M , L., Blood 73, 438-445. SHEN, R.-F., DARLINGTON, G., LEDLEY, F.D., and W O O , 14. D R U M M , M.L., POPE, H.A., CLIFF, W.H., R O M M E N S , J.M., S.L.C. (1988). Retroviral-mediated gene transfer and expression of M A R V I N , S.A., TSUI, L.-C, COLLINS, F.S., FRIZZELL, human phenylalanine hydroxylase in primary mouse hepatocytes. R.A., and WILSON, J.M. (1990). Correction of the cystic fibrosis Proc. Natl. Acad. Sci. USA 85, 8146-8150. defect in vitro by retrovirus-mediated gene transfer. Cell 62, 31. KAPLAN, S.L., U N D E R W O O D , L.E., A U G U S T , G.P., BELL, 1227-1233. J.J., BLETHEN, S.L., BLIZZARD, R.M., B R O W N , D.R., FO15. B U O N O C O R E , L., and ROSE, J.K. (1990). Prevention of HIV-1 LEY, T.P., HINTZ, R.L., H O P W O O D , N.J., JOHANSEN, A., glycoprotein transport by soluble C D 4 retained in the endoplasmic KIRKLAND, R.T., PLOTNICK, L.P., ROSENFELD, R.G., and reticulum. Nature 345, 625-628. V A N W Y K , J.J. (1986). Clinical studies with recombinant-DNA16. M O R G A N , R.A., L O O N E Y , D.J., M U E N C H A U , D.D., derived methionyl human growth hormone in growth hormone W O N G - S T A A L , F., G A L L O , R.C, and A N D E R S O N , W.F. deficient children. Lancet i, 697-700. (1990). Retroviral vectors expressing soluble CD4: a potential gene 32. GRIBBEN, J.G., DEVEREUX, S., T H O M A S , N.S.B., KEIM, therapy for AIDS. AIDS. Res. Hum. Retroviruses 6, 183-191. M., JONES, H.M., GOLDSTEIN, A.H., and LINCH, D.C. 17. ROSENBERG, S.A., AEBERSOLD, P., CORNETTA, K , (1990). Development of antibodies to unprotected glycosylation KASID, A., M O R G A N , R.A., M O E N , R., K A R S O N , E.M., sites on recombinant human GM-CSF. Lancet 335, 434-437. LOTZE, M.T., Y A N G , J.C, TOPALIAN, S.L., MERINO, M.J., CULVER, K., MILLER, A.D., BLAESE, R.M., and ANDER- 33. McMillan, c.w., shapiro, s.s., whitehurst, d., HOYER, L.W., RAO, A.V., LAZERSON, J., and THE HEMOSON, W.F. (1990). Gene transfer into humans—Immunotherapy PHILIA STUDY GROUP. (1988). The natural history of factor of patients with advanced melanoma, using tumor-infiltrating lymVIILC inhibitors in patients with hemophilia A: A national cooperphocytes modified by retroviral gene transduction. N. Engl. J. ative study. II. Observations on the initial development of factor Med. 323, 570-578. VIILC inhibitors. Blood 71, 344-348. 18. T H O M P S O N , J.A., HAUDENSCHILD, C.C, A N D E R S O N , K.D., DIPIETRO, J. M., A N D E R S O N , W.F., and MACIAG, T. 34. GIN, H., and AUBERTIN, J. (1987). Generalized allergy due to zinc and protamine in insulin preparation treated with insulin pump. (1989). Heparin-binding growth factor 1 induces the formation of Diabetes Care 10, 789-790. organoid neovascular structures in vivo. Proc. Natl. Acad. Sci. 35. G R A M M E R , L.C, ROBERTS, M., B U C H A N A N , T.A., U S A 86, 7928-7932. FITZSIMONS, R., METZGER, B.E., and PATTERSON, R. 19. S H I M O T O H N O , K., and TEMIN, H.M. (1981). Formation of (1987). Specificity of immunoglobulin E and immunoglobulin G infectious progeny virus after insertion of herpes simplex thymidine against human (recombinant D N A ) insulin in human insulin allergy kinase gene into D N A of an avian retrovirus. Cell 26, 67-77. and resistance. J. Lab. Clin. Med. 109, 141-146. 20. MILLER, A.D., JOLLY, D.J., FRIEDMANN, T., and V E R M A , I.M. (1983). A transmissable retrovirus vector expressing human 36. GANZ, M.A., U N T E R M A N , T., ROBERTS, M., UY, R., SAHGAL, S., SAMTER, M., and G R A M M E R , L.C. (1990). ResisHPRT: gene transfer into cells obtained from humans deficient in tance and allergy to recombinant human insulin. J. Allergy Clin. HPRT. Proc. Natl. Acad. Sci. USA 80, 4709-4713. Immunol. 86, 45-51. 21. C O N E , R.D., and M U L L I G A N , R.C. (1984). High-efficiency gene transfer into mammalian cells: Generation of helper-free Address reprint requests to: Dr. Suzanne Epstein recombinant retrovirus with broad mammalian host range. Proc. Natl. Acad. Sci. U S A 81, 6349-6353. FDA, CBER, OBR, DBB 22. M A N S O U R , S.L., T H O M A S , K.R., and CAPECCHI, M.R. Building 29, Room 522, HFB-710 (1988). Disruption of the proto-oncogene int-2 in mouse embryoBethesda, M D 20892 derived stem cells: A general strategy for targeting mutations to non-selectable genes. Nature 336, 348-352. Received for publication May 8, 1991; accepted after revision 23. T H O M A S , K.R., and CAPECCHI, M.R. (1987). Site-directed July 17, 1991. mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503-512. 24. JOHNSON, R.S., SHENG, M., G R E E N B E R G , M.E., KOLOD- NOTE ADDED IN PROOF NER, R.D., P A P A I O A N N O U , V.E., and SPIEGELMAN, B.M. (1989). Targeting of nonexpressed genes in embryonic stem cells Direct all inquiries other than reprint requests to: Division of Biological Investigational N e w Drugs via homologous recombination. Science 245, 1234-1236. R o o m 1-29, HFB-230 25. ADAIR, G.M., NAIRN, R.S., WILSON, J.H., SEIDMAN, 12420 Parklawn Dr. M.M., B R O T H E R M A N , K.A., M A C K I N N O N , C , and SCHEERER, J.B. (1989). Targeted homologous recombination at Rockville, M D 20852 the endogenous adenine phosphoribosyltransferase locus in Chi(301)295-8416 nese hamster cells. Proc. Natl. Acad. Sci. U S A 86, 4574-4578. The Points to Consider in H u m a n Somatic Cell Therapy and 26. CORNETTA, K., M O E N , R.C, CULVER, K , M O R G A N , Gene Therapy (1991) is being announced in the Federal Register. R.A., McLACHLIN, J.R., STURM, S., SELEGUE, J., LOND O N , W., BLAESE, R.M., and ANDERSON, W.F. (1990).
R e g u l a t o r y C o n c e r n s in H u m a n
Gene
Therapy
SUZANNE L. EPSTEIN
ABSTRACT Gene therapy in humans is now being undertaken in an investigational setting. Such therapy involves the administration of biological products to h u m a n patients. A document entitled, "Points to Consider in H u m a n Somatic Cell Therapy and G e n e Therapy" has been prepared by the Center for Biologies Evaluation and Research ( C B E R ) of the Food and D r u g Administration ( F D A ) and is published elsewhere in this issue. This paper provides explanatory material about the C B E R regulatory process and the scientific and regulatory basis for the requests for data in that document.
handled on a case-by-case basis but follows a common set of principles. In the case of gene therapy, the field is evolving so rapidly that any set of standards might be out of date before it The two institutions that have major regulatory responsibility over human gene therapy are the N I H and the F D A . The could be announced, and so the approach must be a case-by-case N I H has previously published its "Points to Consider" approach that is responsive to the specific proposals and the changing scientific information available. This paper does not document. The F D A publishes a draft of its proposed "Points to Consider" on pages 251-256 of this issue. In this constitute a formal policy statement, but rather is a summary of issues in this area and of FDA's approach to these issues. accompanying article, Epstein provides the background
OVERVIEW S U M M A R Y
information on which the F D A "Points" are based.
R E G U L A T O R Y RESPONSIBILITIES INTRODUCTION In many cases, trials of human gene therapy are subject not to F D A oversight but also to review by institutional review Many OF THE medical interventions that can beonly characterized as gene therapy involve the administration of boards and, at least in the case of federally funded studies, the biological products to humans. This article describes in general NIH Recombinant D N A Advisory Committee (RAC) and its terms some of the regulatory and scientific considerations Human Gene Therapy Subcommittee. The focus and philosophy pertinent to Food and Drug Administration (FDA) review of of these review processes may at times be different from that of the FDA. Table 1 summarizes some differences between the applications in this area. Details can be found in the F D A documents mentioned below and in the "Points to Consider in roles and responsibilities of the F D A and the R A C . Most Human Somatic Cell Therapy and Gene Therapy" published importantly, the F D A examines product development and manelsewhere in this issue. The present discussion is limited to ufacturing, quality control issues, and safety testing involving somatic cell gene therapy; regulation of gene therapy intended to primary data about manufactured batches of material to be used modify genetic material of recipient germ cells would involve clinically. F D A regulation and review applies to both the product and the process of its production. Another distinction is additional considerations. The regulation of biological products has not historically lent that many (not all) meetings of the R A C are public, while the itself to checklists or rigid sets of rules. The products are too F D A provides confidential review since some sponsors consider diverse, and the same sets of tests are not appropriate or even the information they submit to be proprietary. It should also be mentioned that the "Points to Consider in the possible for all products. Regulation of biologies is therefore
Molecular Immunology Laboratory, Division of Biochemistry and Biophysics, FDA, CBER, Bethesda, M D 20892.
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EPSTEIN
244 Table 1. Regulation of Gene Therapy: NIH Recombinant DNA Advisory Committee and Its Human Gene Therapy Subcommittee Compared to FDA NIH-RAC
FDA
Confidential (some meetings open) Staff available for interaction and consultation on day-to-day basis Main areas of expertise, and of information scrutinized: Basic sciences Basic sciences Clinical medicine Clinical medicine Regulatory issues Law Manufacturing processes Ethics Quality control Theology Preclinical testing Preclinical testing Open, public (some meetings closed) Six meetings per year
Design and Submission of H u m a n Somatic-Cell Gene Therapy Protocols" issued by the N I H R A C is a distinct document from the F D A "Points to Consider on H u m a n Somatic Cell Therapy and Gene Therapy." The F D A has issued "Points to Consider" documents in a variety of areas, and the term was not changed due to overlapping use by the R A C . The Center for Biologies Evaluation and Research ( C B E R ) is the F D A component that has reviewed existing gene therapy protocols. C B E R has several mechanisms for facilitating the review process. A variety of documents are available, including those called "Points to Consider," which are not regulations but rather present the current thinking of the C B E R staff about important issues in a given area of product development and testing. These documents can be valuable to manufacturers of products related to gene therapy, to sponsors of trials, or to participants in the dialogue about h o w gene therapy should be regulated. Those "Points to Consider" documents most relevant to gene therapy are listed in Table 2. This discussion will focus on the investigational n e w drug (IND) stage of regulation, since there is no experience yet with licensing of gene therapy products. C B E R encourages early interaction between sponsors and the C B E R staff, for example via meetings held before submission of a formal I N D application ("pre-IND" meetings). I N D review is intended to be a dynamic process, with exchange of ideas and information between the sponsors and the C B E R staff. The goal is for F D A to voice concerns early and to resolve potential problems. For this reason, C B E R is willing to review proposals in draft form,
especially proposals for novel therapies like gene therapy. C B E R does not require that the protocol have prior approval from other bodies such as the R A C before initiating such discussions. If sponsors begin such discussion early, problems can surface, advice can be given, and thus unanticipated delays in beginning a clinical trial can be avoided. It should be emphasized that not all the data discussed below need be available at the time the initial I N D application is submitted or even in some cases before initiation of clinical trials. C B E R bases regulatory judgments on in-house review of the laboratory and clinical data presenting results of preclinical research and of tests on the actual products and preparations to be administered to patients, to determine if they meet applicable standards. I N D applications from commercial sponsors, NIHsupported researchers, and any other sponsors are all treated in the same manner.
SOMATIC CELL THERAPY A N D G E N E THERAPY Somatic cell therapy can be defined as the administration to humans of living cells which have been manipulated ex vivo, while gene therapy is a medical intervention designed to modify the genetic material of living cells in patients or cells for administration to patients. Gene therapy can be pursued by a variety of technologies. The approach already used in a few patients is part of the broader category of somatic cell therapy. In
Table 2. Points to Consider Documents Relevant to Gene Therapy Issued by the Center for Biologics Evaluation and Research Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant D N A Technology (1985) Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1987) Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1987) Points to Consider in the Collection, Processing, and Testing of Ex-Vivo-Activated Mononuclear Leukocytes for Administration to Humans (1989) To receive a copy of any of these Points to Consider documents, contact: Congressional, Consumer Affairs, and International Affairs Staff, HFB-142, Suite 109, Metro Park North III, 5600 Fishers Lane, Rockville, M D 20857; Telephone (301) 295-8228, F A X (301) 295-8266.
REGULATORY CONCERNS IN HUMAN GENE THERAPY this type of gene therapy, cells removed from a patient are manipulated by insertion of a gene. The modified autologous cells are then readministered. Somatic cell therapies differ from simple transplantation in that the cells are manipulated in some substantial way. For example, the cells m a y be grown in tissue culture in the presence of growth factors and caused to expand greatly in numbers. They m a y be induced to differentiate either by soluble factors or by contact with other cells. The cells m a y be subjected to selection, in cases in which a subpopulation of the original cells is the desired therapeutic population. Selection can involve treatments with antibody plus complement or with toxins, outgrowth in the presence of selective growth factors, and so forth. In the cases of differentiation or selection, the cell population after the manipulation steps m a y have phenotypic and functional properties different from those of the starting population. The patient m a y receive his/her o w n manipulated cells (autologous), cells from another h u m a n (allogeneic), or cells from an animal of a different species (xenogeneic). T h e precise boundary between transplantation and procedures involving sufficient manipulation in vitro to warrant designation as somatic cell therapy is not yet clear. A variety of cellular therapies are in development. A n extensive list of examples, some already in or nearly ready for human trials, can be taken from the scientific literature. This includes myoblast transfer for muscular dystrophy,(,_3) pancreatic islet cell implantation for diabetes,(4) and implantation of dopamine secreting cells for Parkinson's disease.(5,6) There are, of course, additional approaches to gene therapy that are not forms of somatic cell therapy and m a y be proposed for clinical use in the future, such as use of vectors administered directly to the recipient.(7,8) Such an approach would bypass the in vitro cellular manipulation steps, but would still raise m a n y of the same concerns that will be discussed here. The cells undergoing genetic alteration would n o w be recipient cells in vivo, which could in some cases be sampled and analyzed to address some of the same issues. Other approaches m a y emerge in the future as the technology develops. Discussion in this paper of the approach currently in clinical use is not intended to imply restriction of gene therapy proposals solely to somatic cell therapy approaches. In the area of gene therapy, proposed uses for correction of genetic defects include replacement of an enzyme,(9J0) clotting factor,'"~13) a 1-antitrypsin for emphysema/8* or the cystic fibrosis gene product.(,4) Other applications include production of a therapeutic biological such as CD4(l516) or a cytokine in situ, to permit production in the recipient of a product that is needed at high concentrations over a long period of time, and thus is meant to avoid repeated administration of an exogenous product such as a recombinant protein. In addition to gene therapies, another proposed use of this technology is gene marking: cells are genetically altered by insertion of a marker gene that is not intended to have a therapeutic effect on the patient, but that provides a w a y to identify and track the administered cells.(l7) These types of therapies and protocols m a y become c o m m o n in the near future. The question then arises as to h o w F D A will regulate the relevant products. Regulatory concerns in gene therapy fall into three categories, and each will be discussed. First, there are concerns c o m m o n to all biological products. Second, there are concerns that apply to
245
somatic cell therapies in general. Finally, there are unique concerns that apply to therapy involving genetic alteration. For each of these areas, the type of questions and concerns F D A raises will be presented, along with the scientific basis for the concerns. The regulatory approach in this area is of course evolving, and input is welcome from the scientific community as approaches are formulated.
R E G U L A T O R Y CONCERNS T H A T APPLY T O ALL BIOLOGICAL PRODUCTS The area of conventional regulatory issues applying to all biological products is extremely important. Gene therapy proposals can pose risks due not only to esoteric issues, but to conventional ones. Consider, for example, a carefully conceived and ethically justified proposal that uses an elegantly transduced cell population. If that cell preparation has not been adequately tested for contamination with infectious agents, the patient could be harmed. If a gene therapy results in production of hormones, lymphokines, or toxins, then the biological properties of those molecular species themselves are very important. In thinking about more specialized concerns unique to gene therapy such as use of retroviral vectors, tissue-targeted viral vectors, or homologous recombination, the established criteria for evaluating biological products must not be neglected. This article will not go into great detail, but certain general points should be made since researchers m a y not be familiar with regulatory matters. Preclinical safety studies prior to I N D trials should include both in vitro testing and animal testing in an informative (not necessarily perfect) animal model. Safety testing should cover and exceed the dose range that is proposed for the h u m a n trials. Although proof of efficacy is obtained during clinical trials, preclinical studies should present evidence for a rationale for efficacy of the therapy. Potency testing requires some form of quantitative assay for a relevant property. Biologicals are complex materials that cannot be completely defined. History has shown that h o w a biological product is prepared is very important; in-process control is, therefore, an important aspect of defining and characterizing the product, and analyzing the end product m a y not be sufficient. For example, poor control of a production process can lead to introduction of an adventitious agent such as a virus for which no reliable assay is available, or to lability of the product, as well as to inadvertent changes in properties that escape detection. For that reason, in the case of biologies both the product and the process of its production are regulated. In an I N D , descriptions of the methods and reagents should be submitted, along with quality control test results not only on the product but on cell banks that are used in its preparation and on key intermediates in the production of the product. D u e to the importance of control of the production process, not only the biological product but also the production establishment must be licensed prior to marketing. Another aspect of process control is the testing of lot-to-lot reproducibility. Quantitative assays for appropriate properties of the material or correlates of its action are needed to confirm that at least in some ways the same material is being m a d e in each batch. Sometimes the mechanism of action of a proposed therapy is unknown. Then in vitro evidence of product potency
246 may not be available, and what to test on a lot-to-lot basis as a potency assay m a y be unclear. Still, quantitative assays of properties that m a y correlate with product action can be performed, and provide a database for eventual determination of what measurable properties of the product predict therapeutic efficacy.
R E G U L A T O R Y CONCERNS C O M M O N T O SOMATIC CELL THERAPIES There is precedent and FDA experience in dealing with therapies based on cell populations, and a Points to Consider document is available dealing with the special case of activated mononuclear leukocytes (see Table 1). Preclinical testing will be discussed first, followed by comments about lot release testing, and then concerns about cell performance in vivo. Preclinical testing includes the research phase and uses preparations analogous to the ones intended for clinical use but not necessarily the actual clinical preparations, while lot release testing is performed on all clinical batches. For any cellular therapy, the cells to be used as a starting population should be described as to autologous, allogeneic, or xenogeneic origin and tissue origin. Concerns about h u m a n or animal donor qualification, cell collection procedures, and quality control of media and components used in culturing the cells are important. Sterility is critical, both for the cell population and for intermediates in its preparation; this involves freedom from bacteria, fungi, mycoplasma, and adventitious viruses. Once a cell population has been obtained, it should be characterized at various steps of processing for a number of features, including viability and presence of relevant cell types as defined by appropriate phenotyping methods. Properties such as morphological characteristics, cell-surface markers detected by antibodies, receptors for hormones or growth factors, and biochemical features can be used as phenotypic markers. Heterogeneity of the cell population should be assessed by quantitative phenotyping. Heterogeneity of cell populations can be quite important; if each of several cultures derived from the same starting material shows activity in a qualitative assay and reaches the same total number of cells, that does not necessarily mean that the cultures are biologically equivalent. Cultures m a y have been partially overgrown with other cell types, such as subpopulations in the starting culture or contaminating populations from other cultures in the laboratory. Thus, markers of identity are needed, including both general phenotypes and in some cases patient-specific markers. In some cases, only a small percentage of cells bearing the markers m a y be necessary for acceptable activity. The relevant cell functions or products can provide markers for the potency of the cells. Products made by the cells and required for therapeutic activity should be shown to be biologically active and should be quantitated, to determine whether the level produced is adequate to produce the desired effect in vivo. All biologically active forms of the product should be considered, for example, membrane and secreted forms of the same protein. Types of cell function other than secretion of a product m a y require measurement by assays such as cellular cytotoxicity, proliferation in response to stimuli, differentiation potential, or bioassay in animals.
EPSTEIN The preclinical safety evaluation should include examination of growth control of the cells and confirmation of any expected dependence on exogenous growth factors. Animal models are often helpful for in vivo safety evaluations and should be considered. Even when there is no animal model for efficacy of the therapy, some type of preclinical safety data from in vivo animal testing should be provided, and adverse effects noted in any animal experiments should be reported. While efficacy m a y depend on only one or a few particular products of the cells, the cells m a y produce many other molecular species. Other products known to be produced by the cells should be identified when possible, and possible adverse effects of these and or unidentified species sought in animal tests. Lot release testing, that is, the testing performed on all batches of cells before use in patients, cannot use all the tests described above, particularly in the case of small batches of cells for use in single patients. S o m e types of characterization m a y use too m a n y cells or m a y be too cumbersome or time consuming for routine use. However, a number of quality control tests should be performed shortly before administration to confirm that the cell population is as expected, and should be repeated after thawing and possibly passaging of frozen cells. These should ordinarily include viability, sterility, identity, and function of some kind. Cells can be introduced into recipients in a variety of ways, including simple infusion or implantation and more complex modes such as use of solid supports coated with growth factors to favor cell growth or tissue formation/12'I8) Cells m a y not behave the same w a y in a recipient as in tissue culture. In vivo behavior of the cells can be analyzed either in animals or in clinical trials or both, depending on the availability of informative animal models. Evidence should be provided as to whether the cells survive and continue to function in vivo. If the ability of the cells to function in vitro is due to specialized conditions in culture, such as addition of an exogenous growth factor, then perhaps those conditions would have to be provided in vivo at adequate levels to maintain function. In some cases, cell survival and function might be suppressed by other influences in vivo, including rejection by the recipient's i m m u n e system. The time course of cell performance in vivo is important: H o w long the cells continue to function might determine the interval between treatments, and very transient function m a y be useful in some situations but not in others. In some cases, the cells m a y display particular trafficking patterns or homing to a particular organ due to interactions with certain other cells. Although it m a y not be technically feasible to study in all systems, cell localization in vivo should be considered, since it might have effects on safety and might influence whether the cells are able to exert their therapeutic effect. For example, if cells are intended to localize to a tumor site and kill the tumor, the fraction of the cells that reach that site should be estimated and information sought as to the fate of the remaining cells. Toxicity from cells that localize to unintended sites should be considered. In addition, systemic effects of the cells or their products could be very different from local effects. If a soluble growth factor is normally secreted at low levels and acts locally to exert an autocrine or paracrine effect, it might be harmful if it is produced at substantial levels by cells in the circulation. I m m u n e responses to the cells or their products can have important effects on results of therapy. The safety question of
REGULATORY CONCERNS IN HUMAN GENE THERAPY tumorigenicity should also be raised. These two issues apply to all somatic cell therapies, but will be discussed below in the particular context of gene therapy.
247
its biological activity, and the amount produced. Expressed levels should be compared to the level required for effectiveness. Normal physiological levels m a y not be required; for example, 1 0 % of normal A D A levels can provide adequate function for i m m u n e competence.(28) If there are multiple REGULATORY CONCERNS ABOUT biological forms of the gene product being expressed, they G E N E THERAPY should all be included in the analysis and quantitated if possible. W h e n feasible, assays for expression of the inserted gene should Gene therapy will be considered here in the context of cellularbe used as lot-to-lot tests as an indication of potency. therapy reflecting the current state of the art. Not all the points Stability of gene expression should be examined. A s in the below would apply to all alternative approaches, and the appro- case of other cell functions, very transient expression would be priate testing would differ, but m a n y of these concerns are unsuitable for some therapeutic purposes. Eventually, it should broadly applicable. Note that m a n y issues related to genetic be possible to ask whether the inserted gene is expressed subject alteration of production cells and to recombinant D N A manipu- to normal regulatory controls or is expressed in an aberrant lation are addressed by the "Points to Consider' document on fashion. Improperly regulated expression of a gene product products m a d e by recombinant D N A technology (Table 2). would lead to safety concerns in some cases. Currently, retroviral vectors are being used to introduce Both the cell type chosen for gene therapy and the use of genetic material into cells in vitro.(19_21) Homologous recombi- appropriate regulatory sequences such as promoters in the nation has also been proposed and is being used in some construct m a y be employed to try to target gene expression to experimental sy stems.(22_25) This method targets gene insertion desired sites; in the future, injectable vector tropism might to the corresponding locus of the host and disables or replaces accomplish this. Genetically modified vascular grafts have been the defective gene. A s mentioned above, there is the future proposed for delivery of products that affect diseases related to possibility of direct administration of vectors to the patient(7'8) the circulatory system, or for delivery to organs just downstream which might h o m e to particular target cell types based on of the site of secretion/29' Transduction of liver cells with a receptor tropism or other factors. Use of liposomes to deliver retroviral vector containing a liver-specific promoter has been proposed(30) to achieve expression of phenylalanine hydroxygenetic material is also being considered. Vectors or other genetic constructs used in gene therapy or in lase expression in the liver. preparation of cells for gene therapy should be characterized Since there is little clinical experience, m a n y of the risks of structurally. Important information includes the sources of gene therapy are theoretical at this point; these include inservectors and genetic inserts, detailed diagrams of the steps used to tional mutagenesis and infectious spread of retroviral vectors. It arrive at the final construct, a description of virus stocks and of is hoped that data from early trials will contribute to an cell banks including the packaging cell line used to prepare accumulating data base, and that, with this information, the risks vector-containing supernatants, and information about regula- can be weighed in the context of possible benefit, alternative tory elements present in the vector. Lot release protocols for therapies, nature of the patient population, and severity of the vectors, media, and transduced cells should be included in the disease. Another important safety concern is whether the cells are IND. Quantitative assays of k n o w n sensitivity should be used to tumorigenic. Testing of cell populations for tumorigenicity is assess whether replicating virus is present or is generated, both covered in the "Points to Consider" on cell lines. If the starting in the cultures from which the supernatants are prepared and then cell population used in preparing a cell therapy product was from data on cell preparations and animal or h u m a n trials. Even derived from a tumor explant, there could be contaminating replication-competent murine retroviruses do not appear highly tumor cells in the preparation. In that case, tumor cells should be pathogenic in primates/26) Still, there is concern about introduc- quantitated and the effectiveness of removal shown before ing replication-competent retroviral material into humans, due readministration. The therapeutic cells themselves could also be to possibilities including mutation or recombination with endog- tumorigenic, due to changes in tissue culture, aberrant growth enous sequences. Safety concerns in this area have been ad- regulation in vivo, or insertional mutagenesis. Insertional mutagenesis involving insertion of a vector or a dressed by recent developments in packaging cell line technology,(27) reducing the likelihood of recombination events fragment of vector into the genome could transform a cell if the gene altered by the insertion affects growth control. Integration regenerating a proliferating virus. Genetic constructs can be integrated into cellular D N A at could also disrupt or alter some other important cell function. either random or targeted insertion sites. In the case of homolo- There are a few tests that can be performed to assess these risks. gous recombination/22,23* the gene is inserted into the genome If the cells have been transformed by the genetic manipulation at a k n o w n site, and this can be confirmed by sequencing of leading to uncontrolled growth, they should not ordinarily be regions flanking the integrated copy. In the case of retroviral used (although transformed cell lines of xenogeneic origin might vectors with random gene insertion, the number of copies of the sometimes be encapsulated and used in somatic cell therapy). If gene in a cell should be estimated. In both cases, information the cells are dependent on exogenous growth factors such as should be provided about whether the gene recombines, rear- interleukin-2 (IL-2) in culture, maintenance of this dependence ranges, or mutates rapidly; and h o w long the inserted gene should be confirmed. However, testing for effects on every remains stable and intact. The issue of insertional mutagenesis locus in the cellular genome that could be disrupted by insertional mutagenesis is impossible, and the magnitude of risk is will be discussed below. During preclinical studies, cells should be tested for expres- currently unknown. Note that other accepted treatments, such as sion of the inserted gene—for production of the correct product, some chemotherapy drugs, also carry a risk of mutagenesis.
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Immunological interactions between the cells or their prodACKNOWLEDGMENTS ucts and the recipient could alter the safety or efficacy of therapy. Preclinical studies in animal models using analogous The author thanks Drs. Janet Woodcock, Kurt Gunter, animal cells may sometimes be of value, but this question must William Egan, and Joy Cavagnaro for critical review of the also be dealt with by patient monitoring, since immunogenicity manuscript; members of the C B E R scientific research and in animal models may not reflect human responses. Immunolog- regulatory staff who reviewed drafts of the Points to Consider ical parameters to be considered include: antigenic differences document on somatic cell therapy and gene therapy; the staff of between donor and host; immune or allergic reactions to the cells the Division of Cytokine Biology and in particular Dr. Jay Siegel or their products, including the possibility of rejection of the for their work on the Points to Consider document on activated cells; and graft-versus-host reactions or autoimmune reactions mononuclear leukocytes; and Emily McFadden for excellent caused by the cells. secretarial assistance. If the infused cells are making an enzyme, receptor, or other structural protein that the host does not normally express, the host may not be tolerant to it. This is not a very well-explored REFERENCES area, but some purified or recombinant proteins administered as such have elicited immune responses/31_33) Patients deficient in 1. KUNKEL, L.M. (1989). 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