Cellular & Gene Therapy ProductsThe Center for Biologics Evaluation and Research (CBER) regulates human gene therapy prod-ucts - products that introduce genetic material into the body to replace faulty or missing genetic material, thus treating or curing a disease or abnormal medical condition. CBER uses both the Public Health Service Act and the Federal Food Drug and Cosmetic Act as enabling statutes for oversight.

FDA has not yet approved any human gene therapy product for sale. However, the amount of gene-related research and development occurring in the United States continues to grow at a fast rate and FDA is actively involved in overseeing this activity. FDA has received many requests from medical researchers and manufacturers to study gene therapy and to develop gene therapy products. 

Such research could lead to gene-based treatments for cancer, cystic fibrosis, heart disease, he-mophilia, wounds, infectious diseases such as AIDS, and graft-versus-host disease.

Compliance & Enforcement Letters to Industry / Healthcare Providers / Clinical Investigators Compliance Actions (Biologics)

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Gene Therapy Patient Tracking

Genetic Modification Clinical Research Information Sytem (GeMCRIS) (National Insti - tutes of Health)

Gene Therapy Patient Tracking System   (PDF - 100KB) NIH and FDA Launch New Human Gene Transfer Research Data System (National Insti -

tutes of Health) Office of Biotechnology Activities: Recombinant DNA (National Institutes of Health)

-Product Development & Approval Biologic Forms Cellular, Tissue, and Gene Therapies Advisory Committee Development & Approval Process (CBER) References for the Regulatory Process for the Office of Cellular, Tissue and Gene Thera -

pies

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Donor Eligibility Testing HCT/P Donors for Relevant Communicable Disease Agents and Diseases

Testing HCT/P Donors: Specific Requirements

-Regulatory Information Application of Current Statuatory Authorities to Human Somatic Cell Therapy Products

and Gene Therapy Products   (PDF - 489KB) Cellular & Gene Therapy Guidances References for the Regulatory Process for the Office of Cellular, Tissue and Gene Thera -

pies Proposed Approach to Regulation of Cellular and Tissue-Based Products   (PDF - 433KB) Reinventing the Regulation of Human Tissue

Safety & Availability Safety & Availability (Biologics)

Special Issues Cloning

-Publications & Presentations Workshops, Meetings Human Gene Therapies: Novel Product Development Q&A with Celia M. Witten, Ph.D,

M.D.

Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy[PDF version of this document]

Comments and suggestions regarding this document may be submitted at anytime to Dano B. Murphy, HFM-17, Center for Biologics Evaluation and Research, Food and Drug Administra-tion, 1401 Rockville Pike, Rockville, MD 20852-1448. For questions regarding this document, contact Suzanne L. Epstein, Ph.D., 301-827-0450, FAX 301-827-0449, e-mail Epsteins@CBER.FDA.GOV.

U.S. Department of Health and Human ServicesFood and Drug AdministrationCenter for Biologics Evaluation and ResearchMarch 1998

 

 

TABLE OF CONTENTS

OVERVIEW (1998)

 

I. INTRODUCTION

 

A. Definitions of Somatic Cell Therapy and Gene TherapyB. Types of TherapiesC. Regulatory ConsiderationsD. General Considerations

 

II. DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR AD - MINISTRATION

 

A. Collection of CellsB. Cell Culture ProceduresC. Cell Banking Systems Procedures: Generation and Characterization of Master

Cell Banks (MCB), Working Cell Banks (WCB), and Producer CellsD. Materials Used During Manufacturing

 

III. CHARACTERIZATION AND RELEASE TESTING OF CELLULAR GENE THER - APY PRODUCTS

 

A. Cell IdentityB. PotencyC. ViabilityD. SterilityE. PurityF. General Safety TestG. Frozen Cell Banks

 

IV. ADDITIONAL APPLICATIONS: ADDITION OF RADIOISOTOPES OR TOXINS TO CELL PREPARATIONS

 

V. PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF VECTORS FOR GENE THERAPY

 

A. Vector Construction and CharacterizationB. Vector Production SystemC. Master Viral BanksD. Lot-to-Lot Release Testing and Specifications for Vectors

 

VI. ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THER - APY

 

A. Additional Considerations for the Use of Plasmid Vector ProductsB. Additional Considerations for the Use of Retroviral Vector ProductsC. Additional Considerations for the Use of Adenoviral VectorsD. Other Gene Delivery Systems

 

VII. MODIFICATIONS IN VECTOR PREPARATIONS

 

VIII. PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES

 

A. General PrinciplesB. Animal Species Selection and Use of Alternative Animal ModelsC. Somatic Cell and Gene-Modified Cellular TherapiesD. Direct Administration of Vectors In Vivo

 

IX. CONCLUSION

 

 

Guidance for Industry1

FDA Guidance for Human Somatic Cell Therapy and Gene Therapy

 

 OVERVIEW (1998)

Since the issuance of the "Points to Consider (PTC) in Human Somatic Cell Therapy and Gene Therapy"in 1991, the range of gene therapy proposals has expanded to include additional classes of vectors and use of vectors in vivo via direct vector administration to patients. This guidance document updates and replaces the 1991 PTC with new information intended to provide manu-facturers with current information regarding regulatory concerns for production, quality control testing, and administration of recombinant vectors for gene therapy; and of preclinical testing of both cellular therapies and vectors. These guidances are not regulations, but rather represent is-sues that the Center for Biologics Evaluation and Research (CBER) staff believes should be con-sidered at this time.

Virus or DNA preparations used as preventive vaccines are not covered by this document, though there is some overlap in the issues. Separate guidance on use of plasmid products to pre-vent infectious diseases is available from the Office of Vaccines Research and Review, CBER (301) 594-2090. One pertinent document is the "Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications (December, 1996)", (61 FR 68269). Somatic cell therapies are affected by the evolving criteria for infectious disease testing. For additional guid-ance refer to the following documents:

Points to Consider in the Manufacture and Testing of Therapeutic Products for Human Use De-rived from Transgenic Animals, (8/22/95), (60 FR 44036);

PHS Guidelines on Infectious Disease Issues in Xenotransplantation, August, 1996, (61 FR 49920) and January, 1997, (62 FR 3563);

Guidance on Applications for Products Comprised of Living Autologous Cells Manipulated ex vivo and Intended for Structural Repair or Reconstruction (May, 1996), May 28, 1996, (61 FR 26523).

A Proposed Approach to the Regulation of Cellular and Tissue-based Products, February 28, 1997, (62 FR 9721).

 

 

I.  II. INTRODUCTION

 

A.B. Definitions of Somatic Cell Therapy and Gene Therapy

Recently, various innovative therapies involving the ex vivo manipulation and subsequent reintroduction of somatic cells into humans have been used or pro-posed. Somatic cell therapy is the administration to humans of autologous, allo-geneic, or xenogeneic living cells which have been manipulated or processed ex vivo. Manufacture of products for somatic cell therapy involves the ex vivo propa-gation, expansion, selection (see: "A Proposed Approach to the Regulation of Cel-lular and Tissue-based Products", Feb. 28, 1997, (62 FR 9721)), or pharmacologic treatment of cells, or other alteration of their biological characteristics. Such cel-lular products might also be used for diagnostic or preventive purposes. Manufac-turers should review policy and regulations to determine how a particular somatic cell therapy or gene therapy product is regulated.

Recently, various innovative therapies involving the introduction of somatic cells into humans have been used or proposed. For the purpose of this Guidance, the term somatic cell therapy refers to the administration to humans of autologous, al-logeneic, or xenogeneic living non-germline cells, other than transfusable blood products, for therapeutic, diagnostic, or preventive purposes.

Gene therapy is a medical intervention based on modification of the genetic mate-rial of living cells. Cells may be modified ex vivo for subsequent administration to humans, or may be altered in vivo by gene therapy given directly to the subject. When the genetic manipulation is performed ex vivo on cells which are then ad-ministered to the patient, this is also a form of somatic cell therapy. The genetic manipulation may be intended to have a therapeutic or prophylactic effect, or may provide a way of marking cells for later identification. Recombinant DNA materi-als used to transfer genetic material for such therapy are considered components of gene therapy and as such are subject to regulatory oversight.

This document does not discuss genetic manipulation aimed at the modification of germ cells.

 

C. Types of Therapies

Examples of somatic cell therapies include implantation of cells as an in vivo source of a molecular species such as an enzyme, cytokine or coagulation factor; infusion of activated lymphoid cells such as lymphokine activated killer cells and

tumor-infiltrating lymphocytes (addressed in a separate Points to Consider docu-ment: see below); and implantation of manipulated cell populations, such as hepa-tocytes, myoblasts, or pancreatic islet cells, intended to perform a complex bio-logical function.

Initial approaches to gene therapy have involved the alteration and administration of somatic cells. However, additional approaches such as the direct administration to patients of retroviral vectors or other forms of genetic material have been used. The concerns described below apply regardless of the method used, though the applicable tests may be different.

Cells for therapeutic purposes may be delivered in various ways. For example, they may be infused, injected at various sites or surgically implanted in aggre-gated form or along with solid supports or encapsulating materials. Any matrices, fibers, beads, or other materials which are used in addition to the cells may be cat-egorized as excipients, additional active components, or medical devices.

Because of the complexities of potential interactions with the cells and other con-stituents, additional components should be considered as part of the final biologi-cal product for purposes of preclinical evaluation.

 

D. Regulatory Considerations

All gene therapy products and most somatic cell therapy products are regulated by the FDA. See "A Proposed Approach to the Regulation of Cellular and Tissue-Based Products," February 28, 1997, (62 FR 9721) as well as subsequent regula-tions and policy issued in this area.

IND applications for somatic cell and gene therapies should follow the same for-mat and contain the same sections as IND's for any investigational biological product, as described in 21 CFR 312.23. Forms and guidance documents are available from CBER by phone, FAX or E-mail as listed at the end of this docu-ment. The particular information required will depend upon the experimental sys-tem and the phase of study. For those therapies for which patient entry criteria in-clude results of a genetic test, information should be submitted to the IND docu-menting and validating the test method.

Biological products are often complex mixtures that cannot be completely de-fined. Quality control of the manufacturing process as well as the final product is necessary. Poor control of production processes can lead to the introduction of ad-ventitious agents or other contaminants, or to inadvertent changes in the proper-ties or stability of the biological product that may not be detectable in final prod-uct testing. For these reasons, the methods and reagents involved in the produc-tion process should be defined. Also, cell banks and key intermediates in the pro-

duction process should be subject to quality control. Lot-to-lot reproducibility of both the final product and of critical materials such as vector-containing super-natants should be examined. Existing general regulations (21 CFR 210, 211, 312, and 600) may be relevant and should be consulted for guidance.

Exploratory phase I trials for somatic cell and gene therapy products should be based on data that assure reasonable safety and rationale. Less data may be sub-mitted to support beginning exploratory trials than may be submitted at later stages of product development, especially in the case of severe or life-threatening diseases. The review of data to support initiation of phase I trials focuses on safety, although some demonstration of rationale should also be provided.

Data from further product testing should be available at later stages of product de-velopment. A quantitative potency assay reflective of bioactivity in vivo should be developed and product stability should be studied to assure product integrity. In addition to safety, evidence of clinical efficacy is required for licensure.

If product formulation is changed during product development, a comparison of the different formulations should be made by quantitative assays of biological po-tency and, when appropriate, preclinical safety evaluation. If the product used in later phase trials differs in major ways from that used during earlier trials and if the results of the earlier trials are essential to the final product evaluation, product comparability should be demonstrated or the sponsor should assess whether ear-lier trials may need to be repeated (see: "FDA Guidance Concerning Demonstra-tion of Comparability of Human Biological Products, Including Therapeutic Biotechnology-derived Products", 4/26/96, (61 FR 10426)).

For vectors intended to be used in a number of different IND's, manufacturing in-formation can be submitted in a master file to simplify the filing process. Neither a master file nor the product it describes is "approved" or "disapproved". Rather the master file contains information and data that supplement the IND. Use of the same product for different patient populations may raise different issues or may indicate different levels of acceptable risk, but use of the master file can help identify common issues and facilitate their efficient resolution. Multiple IND sponsors can be authorized to cross-reference a master file, thus reducing redun-dant submissions as well as retaining desired confidentiality.

 

E. General Considerations

Some of the issues regarding cellular and gene therapy products overlap with those discussed in other Points to Consider documents. It is suggested that the most recent versions of Points to Consider or other Guidance documents be re-viewed. Other Guidance documents should be consulted as is appropriate, for ex-ample:

Application of Current Statutory Authorities to Human Somatic Cell Therapy Products and Gene Therapy Products, October 14, 1993, (58 FR 53248).

Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by Recombinant DNA Technology (1985), and Supplement: Nucleic Acid Characterization and Genetic Stability (1992), July 27, 1992, (57 FR 33201).

Points to Consider in the Characterization of Cell Lines Used to Produce Biologi-cals (1993), August 12, 1993, (58 FR 42974).

Points to Consider in the Manufacture and Testing of Monoclonal Antibody Prod-ucts for Human Use (1997), February 28, 1997, (62 FR 9196).

Points to Consider in the Collection, Processing, and Testing of Ex-Vivo-Acti-vated Mononuclear Leukocytes for Administration to Humans (1989), November 2, 1989, (54FR 46303).

The following sections indicate areas of concern and questions to be addressed by manufacturers of such products when filing applications. Initial clinical trials should be preceded by submission of data adequate to assure a reasonable degree of safety. A description of the methods used, actual data from appropriate tests, and evidence of assay validation should be included.

It may not be practical or possible to address all of the issues discussed below for a given system. In some instances, tests mentioned will be inapplicable or inap-propriate, or alternative procedures may be more appropriate. The methods and procedures mentioned are suggestions: sponsors may propose alternative tech-niques which will be acceptable if these issues are adequately addressed and sup-ported by data and rationale. In addition, all of the information discussed below may not be necessary before clinical trials are initiated. Sponsors are encouraged to consult with CBER staff for discussion.

The guidance suggested in this document is based in part on an assessment of the available experience with cell and gene therapy products and methods of produc-tion. Modifications of procedures will occur with time, and alternate control pro-cedures will be needed. The principles included here can serve as guidance for de-veloping these procedures.

Table of Contents

 

 

III. DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR ADMINISTRATION

 

A.B. Collection of Cells

The following information should be provided:

 

1. Cell types: The type(s) of cell to be used should be classified as autolo-gous, allogeneic, or xenogeneic in origin. The tissue source and other rele-vant identifying information should be provided.

 

2. Donor selection criteria: Any relevant characteristics of the donor(s) should be specified, including age and sex. As stated in the "Points to Consider in the Collection, Processing, and Testing of Ex-Vivo-Activated Mononuclear Leukocytes for Administration to Humans," as a minimum, allogeneic donors should meet the standards for blood donors (21 CFR 640.3), the testing and acceptance procedures should be described, and any deviations should be justified. Where applicable, additional Public Health Service recommendations regarding organ and tissue donors should be incorporated. Exclusion criteria should focus on the presence or likeli-hood of infection by HIV-1 and HIV-2, hepatitis B and C viruses, HTLV-1, and other infectious agents. Serological, diagnostic, and clinical history data to be obtained from donors should be specified. Provision for follow-up of donors will be appropriate in some cases and methods of obtaining donor data and record keeping should be thoroughly described.

If autologous cells are used, please refer to "A Proposed Approach to the Regulation of Cellular and Tissue-based Products", February 28, 1997, (62 FR 9721) for additional guidance on adventitious agent testing and label-ing. If animal species other than humans are used, a description should be provided of the origin, relevant genetic traits, husbandry, and health status of the herd or colony (see also "PHS Guidelines on Infectious Disease Is-sues in Xenotransplantation", August, 1996, 61 FR 49920 and January, 1997, (62 FR 3563).

 

3. Tissue typing: If allogeneic donors are to be used, typing for polymor-phisms such as blood type should be included when appropriate. The im-portance of matching for histocompatibility antigens (HLA class I and/or II, and perhaps minor antigens in some cases) between donor and recipient

should be addressed, and typing procedures and acceptance criteria pro-vided.

Should it be indicated or necessary to use mixtures of cells from multiple donors, special attention should be paid to possible cell interactions that could result in immune responses or other changes that might alter the per-formance of the cells. Characterization of multiple-donor cell mixtures may be problematic. Multiple-donor cell mixture products would not meet the criteria set forth in the "Proposed Approach to the Regulation of Cellu-lar and Tissue-based Products", February 28, 1997, (62 FR 9721) for regu-lation as human cellular or tissue-based products under section 361 of the Public Health Service Act (the PHS Act). Such products would be subject to regulation under the Federal Food, Drug, and Cosmetic Act and section 351 of the PHS Act.

 

4. Procedures: The procedures for the collection of cells, including the loca-tion of the facility, and any devices or materials used, should be submitted.

 

C. Cell Culture Procedures

 

1. Quality control procedures: In general, cell culture operations should be carefully managed in terms of quality of materials, manufacturing con-trols, and equipment validation and monitoring. See I, C, General Consid-erations.

 

2. Culture media: Acceptance criteria should be established for all media and components, including validation of serum additives and growth factors, as well as verification of freedom from adventitious agents. Records should be kept detailing the components used in the culture media, includ-ing their sources and lot numbers. Medium components which have the potential to cause sensitization, for example certain animal sera, selected proteins, and blood group substances, should be avoided. For growth fac-tors, measures of identity, purity, and potency should be established to as-sure the reproducibility of cell culture characteristics. More detailed dis-cussions of specifications for medium components and biologicals added to cultures are presented in the "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)" and the "Points to Con-

sider in the Collection, Processing, and Testing of Ex-Vivo-activated Mononuclear Leukocytes for Administration to Humans (1989)."

As stated in the "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals," it is recommended that penicillin and other beta-lactam antibiotics be avoided during production, due to the risk of se-rious hypersensitivity reactions in patients.

 

3. Adventitious agents in cell cultures: Documentation should be provided that cells are handled, propagated, and subjected to laboratory procedures under conditions designed to minimize contamination with adventitious agents. During long term culturing, cells should be tested periodically for contamination. Testing should ensure that cells are free of bacteria, yeast, mold, mycoplasma, and adventitious viruses. For a discussion of adventi-tious agent testing and details regarding virus testing and mycoplasma testing, the "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals" (1993), should be consulted.

 

4. Monitoring of cell identity and heterogeneity: Both manufacturing and testing procedures should be implemented which ensure the control of cell cultures with regard to identity and heterogeneity.

Cell culturing practices and facilities should be designed to avoid contami-nation of one cell culture with another.

During cell culturing, extensive drift in the properties of a cell population, or overgrowth by a different cell type originally present in low numbers, may occur. To detect such changes, cell identity should be assessed quan-titatively, for example, by monitoring cell surface antigens or biochemical markers. The method of identification chosen should also be able to detect contamination or replacement by other cells in use in the facility. Accept-able limits for culture composition should be defined. Quantitative assays of functional potency may sometimes provide a method for population phenotyping. The desired function should be monitored when the cells are subjected to manipulation, and the tests carried out periodically to assure that the desired trait is retained. Identity testing should in some cases in-clude verification of donor-recipient matching and immunological pheno-typing.

 

5. Characterization of therapeutic entity: If the intended therapeutic effect is based on a particular molecular species synthesized by the cells, enough structural and biological information should be provided to show that an appropriate and biologically active form is present.

 

6. Culture longevity: The essential characteristics of the cultured cell popula-tion (phenotypic markers such as cell surface antigens, functional proper-ties, activity in bioassays, as appropriate) should be defined, and the stabil-ity of these characteristics established with respect to time in culture. This profile should be used to define the limits of the culture period.

 

D. Cell Banking System Procedures: Generation and Characterization of Mas-ter Cell Banks (MCB), Working Cell Banks (WCB), and Producer Cells

Cell banking systems are appropriate for use with some somatic cell therapy prod-ucts that are made repeatedly from the same cell source, and with packaging or producer cells used to make gene therapy vectors, for example, bacterial cells pro-ducing a plasmid or mammalian cells producing a recombinant viral vector. These cell stocks should be handled by a formal cell banking system (often a two-tiered system). Specific guidance for the establishment of Master Cell Banks and Work-ing Cell Banks is provided in the "Points to Consider in the Characterization of Cell Lines Used to Produce Biological (1993)", 58 FR 42974. In addition, 21 CFR 610.18 may be applicable. The cell bank system used should be described as fol-lows:

 

1. Origin and history of cells: A description should be provided.

 

2. Procedures: The procedure for freezing and for recovering the cells should be described. Components used (such as DMSO or glycerol) should be specified. The number of vials preserved in a single lot and the storage conditions should be specified.

 

3. Characterization: The identity of the cells should be confirmed by appro-priate genotypic and/or phenotypic markers, and the fraction of the cell population having such identity markers measured as an indication of pu-rity. In the case of transduced or vector-producing cells, vector retention

and identity should be confirmed by restriction mapping or assay of the bioactivity of protein expressed by an inserted gene.

 

4. Testing for contaminating organisms: MCB's should be shown to be free of contaminating biological agents, including fungi, viruses other than a vector, mycoplasma, bacteria other than an intended bacterial host strain, and replication-competent viruses related to vector in certain cases (see section VI, B and C).

In the case of MCB's consisting of bacteria carrying plasmids of interest, testing for bacteriophage is not required but the possible presence of bac-teriophage should be considered, since it could adversely affect stability and yield.

 

5. Expiration dating: Product development plans should include accumula-tion of data demonstrating how long and under what conditions the cells can remain frozen and still be acceptably active when thawed.

 

6. Tests on thawed cells: Tests of viability, cell identity, and function should be repeated after thawing and/or expansion. The yield of viable cells and of quantitative functional equivalents should be compared to those values before freezing. Sterility should be confirmed using aliquots of the frozen cells.

Working Cell Banks, if used, should undergo limited testing for identity by phe-notypic or genotypic markers. Vector retention and identity should be confirmed as in MCB's by restriction mapping or assay of secreted protein activity. They should also be shown to be free of microbial and viral contamination.

For producer cells, extended culture of end-of-production cells should be per-formed on a one-time-basis to evaluate whether new contaminants are induced by growth conditions or if vector integrity is compromised (See "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use," 1997 revision, 62 FR 9196). Sponsors should propose a schedule of testing at steps which will be most informative and sensitive.

It may not be feasible to use cell banking practices with cell therapies made dif-ferently for each patient, for example autologous cells for treatment of individual patients. However, consideration should be given to testing of the final cellular product for crucial characteristics.

 

E. Materials Used During Manufacturing

Materials used during in vitro manipulation procedures, for example antibodies, cytokines, serum, protein A, toxins, antibiotics, other chemicals, or solid supports such as beads can effect the safety, purity, and potency of the final therapeutic product. These components should be clearly identified and a qualification pro-gram with set specifications should be established for each component to deter-mine its acceptability for use during the manufacturing process. When using reagent grade material, the qualification program should include testing for safety, purity, and potency of the component where appropriate. Abbreviated testing may be appropriate for use of clinical grade components. Materials of animal origin will in some cases need to be tested for adventitious agents. The country of origin should be certified when there is risk of transmissible agents causing spongiform encephalopathy.

Limits should be established for the concentrations of all production components that may persist in the final product. The methods used to remove them and the results of quantitative testing (including a description of methods and sensitivity) to show the effectiveness of their removal should be provided. Some added com-ponents, by virtue of binding or uptake, may be present in measurable amounts when the cells are administered. In such cases, consideration should be given to assessing toxicity of these components in animals or other appropriate systems.

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IV. CHARACTERIZATION AND RELEASE TESTING OF CELLULAR GENE THERAPY PRODUCTS

Applies to cellular products including ex vivo transduced cells for gene therapy.

The final biological product to be administered, as well as the production process and materials used, should be subjected to quality control testing. The specifications to be ap-plied to the final product and to other elements of the production process, along with the range of acceptable values for each, should be specified.

One lot of a biological product is considered to be a quantity of material that has been thoroughly mixed in a single vessel. This concept can be applied to somatic cell and gene therapy for purposes of planning lot testing procedures. This means that each cell popula-tion, vector preparation, or other product for such therapies prepared as a unique final mixture should be subjected to appropriate lot release testing. Preparations intended

solely for individual recipients differ from products prepared as large batches, and appro-priate lot release criteria should be chosen to fit the practical constraints of each protocol. Lot-to-lot variation provides a measure of the reproducibility of the procedures.

 

A.B. Cell Identity

Quantitative testing by phenotypic and/or biochemical assays should be used to confirm cell identity and assess heterogeneity (21 CFR 610.14).

 

C. Potency

The relevant function of the cells, if known, and/or relevant products biosynthe-sized by the cells should be defined and quantitated as a measure of potency (21.CFR 610.10).

 

D. Viability

The viability of the cells should be quantitated and a lower limit for acceptability established.

 

E. Adventitious Agent Testing

Tests should demonstrate that the cells are not contaminated with adventitious agents such as bacteria, fungi, (21 CFR 610.12), and mycoplasma, (Points to Con-sider in the Characterization of Cell Lines Used to Produce Biologicals, (1993), Attachment #2, (58 FR 42974) and viruses. The agency is considering proposed rulemaking to allow for validation of a mycoplasma free manufacturing process in cases where the final cell therapy product is too short lived to complete ade-quately sensitive testing prior to administration to patients.

 

F. Purity

Purity (21 CFR 610.13) or validation of endotoxin testing by LAL or other ac-ceptable assays should be established. The suitability and appropriateness of methods of endotoxin testing should be considered on a case-by-case basis. The

test used should be validated to show that the cell preparation does not interfere with endotoxin detection. See "Guideline on validation of the limulus amebocyte lysate test as an end-product endotoxin test for human and animal parenteral drugs, biological products, and medical devices", December, 1987.

 

G. General Safety Test

The general safety test (21 CFR 610.11) must be performed on the final product. When appropriate, modified procedures may be developed according to 21 CFR 610.9. Please note that the agency is considering proposed rulemaking to amend the GST rules and scope of applicability especially for cell therapy products.

 

H. Frozen Cell Banks

When cell populations frozen for subsequent administration are thawed, ex-panded, and then administered to patients, lot release testing on the thawed cells is needed, and can be adapted from Part II, C on cell banking practices.

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V. ADDITIONAL APPLICATIONS: ADDITION OF RADIOISOTOPES OR TOX-INS TO CELL PREPARATIONS

Therapeutic or diagnostic applications may be proposed involving cells which are modi-fied by radiolabeling or pre-loading with bioactive materials such as toxins. Thus, the cell implant may be used as a delivery system not only for its own products and functions but also for other products. Novel safety concerns may arise related to the site of cell implan-tation and localization of the radionuclide or toxin, or due to metabolic properties of the cells. These should be anticipated and addressed where possible.

Similar special issues have been raised in the past by use of radiolabeled or toxin-conju-gated antibodies, and are addressed in the "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use" (1997). Although the applica-tion to somatic cell therapies may differ, that document should be consulted.

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VI. PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF VEC-TORS FOR GENE THERAPY

The information requested below may be difficult to acquire in some systems. Sponsors may present alternative methods and data to CBER staff for review. The types of infor-mation which will assure adequate safety depend in part on the nature of the proposed clinical trial, such as: route and frequency of administration, and the intended patient population.

 

A.B. Vector Construction and Characterization

Vector source materials should be characterized and documented thoroughly. Vi-ral vectors or plasmids should be generated from cloned and characterized con-structs, and subjected to confirmatory identity tests. Information supplied should include vector derivation, including descriptions of any vectors, helper viruses, and producer cell lines used for preparation of the final construct. Known regula-tory elements such as promoters or enhancers contained within the construct should be identified.

Early in product development, vector characterization consisting of sequence data of appropriate portions of vectors and/or restriction mapping supplemented by protein characterization is acceptable. For later phases of product development and licensure, more extensive sequencing information should be provided. When sequencing of the entire vector is not feasible due to the size of the construct, it may be sufficient to sequence the genetic insert plus flanking regions and any sig-nificant modifications to the vector backbone or sites known to be vulnerable to alteration during the molecular manipulations. Vector sequences which modulate vector-host interactions should be described if known, and stability of the host cell/vector system considered.

 

C. Vector Production System

The vector production system is composed of the host cell, final gene construct or when appropriate vector intermediate, used to produce the vector (for example, retroviral producer cell). The procedure for selection of the final gene construct, method of transfer of the gene construct into the host cell and selection and char-acterization of the recombinant host cell clone including vector copy number, and the physical state of the final vector construct inside the host cell (i.e. integrated or extra chromosomal), should be described in detail. In addition, a detailed de-

scription of procedures for the propagation and expansion of the recombinant host cell clone, establishment of the seed stock and qualification of the seed stock should be provided. For information on cell banking procedures refer to Section II, C.

 

D. Master Viral Banks

When a virus, with or without a therapeutic gene, is used as a seed in the manu-facture of a therapeutic vector, it is recommended that a Master Viral Bank be created and characterized. This would include vectors derived from adenovirus, adeno-associated virus, herpes virus, poxviruses, and other lytic and non-lytic viruses. The sponsor should describe the source materials, (i.e. plasmids, vectors, oligomers, etc.) and molecular methods used to produce the source or seed vector. The genetic integrity and stability (i.e. identity) of the seed vector should be con-firmed and bioactivity of the vector seed should be demonstrated. In the absence of bioactivity data, expression of the gene should be assessed.

Master seed stocks should also be demonstrated to be free of adventitious agents, including virus, bacteria, fungi, and mycoplasma. In the case of replication-defec-tive or replication-selective vectors, Master Viral Banks should be demonstrated to be free of replication-competent viruses, which may arise as a result of contam-ination or recombination during the generation of the MVB. Testing for other in-appropriate viruses will depend upon the vector and feasibility of assays in the presence of vector virus. The "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)" (58 FR 42974), should be considered as background information.

 

E. Lot-to-Lot Release Testing and Specifications for Vectors

General testing recommendations are discussed in the sections that follow. Not all tests listed will be applicable to every vector class. Sponsors should choose appro-priate testing protocols, and consult CBER if there are questions as to the applica-bility of a specific test. Note that if drug substance (defined as bulk product not necessarily in final formulation) and drug product (defined as product in its final formulation) are the same, then only a single set of tests is necessary.

Any standard assays for the properties listed below can be used if they are quanti-tative, and are of adequate specificity and sensitivity. Assay methods should be validated by testing of known amounts of reference lots, spiked samples, or other appropriate measures, and data documenting assay performance submitted to the IND.

 

 

1. Tests of drug substance (bulk product not necessarily in final formulation):

 

a. Purity (21 CFR 610.13)

 

i. Test for total DNA or RNA content if appropriate to vector composition, e.g. A260/A280.

 

ii. Test for homogeneity of size and structure, supercoiled vs. linear, e.g. agarose gel electrophoresis.

 

iii. Test for contamination with RNA or with host DNA, e.g. gel electrophoresis, including test with bacterial host-spe-cific probe.

 

iv. Test for proteins if present as a contaminant, e.g. silver stained gel.

 

v. Test for non-infectious virus in cases in which that would be a contaminant, such as empty capsids. See: Section VI C.

 

vi. Tests for toxic materials involved in production.

 

b. Identity (21 CFR 610.14)

Test for vector identity by methods such as restriction enzyme mapping with multiple enzymes or PCR should be performed on the drug substance (see 21 CFR 610.14). In the case of a facility making multiple constructs, it should be verified that the identity testing is capable of distinguishing the constructs and detecting cross-contamination.

 

c. Adventitious agents

As methods of testing for adventitious agents become increasingly sensitive and specific over time, sponsors are encouraged to accu-mulate data validating testing methods other than those indicated, to permit future updating of this policy. In cases in which a vector product interferes with appropriate assays, for example, a lytic vi-ral vector that kills indicator cells in an assay for adventitious virus, some information may be obtained by parallel mock cultures using the same media and other reagents to allow outgrowth of a contaminant, or by assays in the presence of neutralizing antibody. The following tests should be performed:

 

 

i. Sterility test (21 CFR 610.12), for aerobic and anaerobic bacteria and fungi.

 

ii. Mycoplasma testing, as specified in the "Points to Consider in the Characterization of Cell Lines Used to Produce Bio-logicals (1993)", Attachment #2, (58 FR 42974), which specifies the procedures for detecting mycoplasma contam-ination.

 

iii. Testing for adventitious viruses, in some cases, source ma-terials or cell lines used in vector production introduce the risk of contamination with adventitious viruses. In other cases, adventitious virus can be introduced during product manufacture. Testing for an appropriate range of possible contaminating viruses is recommended, as discussed exten-sively in the "Points to Consider in the Characterization of

Cell Lines Used to Produce Biologicals (1993)" 58 FR 42974, and the ICH draft guideline Q5A, "Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin," Step 4, approved by ICH, 3/5/97. Testing for replication-competent retrovirus and adenovirus is discussed below.

 

d. Potency (21 CFR 610.10)

Potency assays should be validated during the product develop-ment process. Expression of the inserted gene can be determined by transfection of appropriate cells and demonstration of active gene product by an appropriate assay, characterized as to its sensi-tivity and specificity. Whenever possible, a potency assay should measure the biological activity of the expressed gene product, not merely its presence. For example, if enzymatic activity is the basis of the proposed therapy, an enzyme activity assay detecting con-version of substrate to product would be preferred over an im-munological assay detecting epitopes on the enzyme. If no quanti-tative potency assay is available, then a qualitative potency test should be performed.

 

2. Tests of drug product (product in its final formulation): The vector product in final container form should be tested for the properties listed below by quantitative, validated assays. Tests for endotoxin and general safety if performed on a drug product (final product) need not be performed on drug substance (bulk product).

 

a. Sterility (21 CFR 610.12), identity (21 CFR 610.14), and potency (21 CFR 610.10).

 

b. Purity (21 CFR 610.13) or validation of endotoxin testing by LAL or other acceptable assay (see "Guideline on validation of the limu-lus amebocyte lysate test as an end-product endotoxin test for hu-man and animal parenteral drugs, biological products, and medical devices, December, 1987).

 

c. General safety, as per 21 CFR 610.11. Note that this test is not needed for therapeutic DNA plasmid products because they are among the specified biotechnology products (Federal Register no-tice, Vol. 61, No. 94, May 14, 1996), even if liposomes are added.

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VII. ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THERAPY

 

A.B. Additional Considerations for the Use of Plasmid Vector Products

Many product and quality control considerations covered in the general sections above are appropriate to plasmid DNA products. The "Points to Consider on Plas-mid DNA Vaccines for Preventive Infectious Disease Indications (1996)", 61 FR 68269, may also be a useful reference. In general, complete sequencing of the plasmid should be performed. Plasmids should be characterized and specifications set with regard to the presence of RNA, protein, and bacterial host DNA contami-nants, quantities of linear and supercoiled DNA in the preparation, and presence of toxic chemicals. Toxic chemicals such as ethidium bromide should be avoided during production.

As stated in the "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals," it is recommended that penicillin and other beta-lactam an-tibiotics be avoided during production, due to the risk of serious hypersensitivity reactions in patients. If antibiotic selection is used during production, it is prefer-able not to use selection markers which confer resistance to antibiotics in signifi-cant clinical use, in order to avoid unnecessary risk of spread of antibiotic resis-tance traits to environmental microbes. Also, residual antibiotic in the final prod-uct should be quantitated when possible, and the potential for allergy considered. See 21 CFR 610.61(m) concerning labeling requirements for approved biological products if antibiotics are used during manufacture. Concerning environmental impact and the use of drug resistance traits, consult the NIH Guidelines for Re-search Involving Recombinant DNA Molecules, Section III-A-1-a (59 FR 34496, amended 61 FR 59732). Non-antibiotic selection systems can also be used.

Plasmid vectors may be administered in conjunction with lipid preparations, local anesthetics, or other chemicals intended to facilitate DNA uptake. If such a facili-tating agent is added during formulation, a specification for its amount and iden-

tity in the final product should be established. If toxic organic solvents such as chloroform are used in producing a lipid component, then processing should re-move them and lot release specifications should include testing for residual sol-vent.

 

C. Additional Considerations for the Use of Retroviral Vector Products

 

1. Testing for replication competent retrovirus: The following testing scheme for detection of replication competent retroviruses (RCR) summarizes cur-rent recommendations based on information available at this time. This scheme is currently being reevaluated, and modified guidance will be made public when available. Testing at multiple stages in production is recommended due to the limited knowledge of the risks of retrovirus ex-posure and the possibility of generation of recombinant RCR at any point in the production process. Alternative assays (e.g. marker rescue) are ac-ceptable if sensitivity is comparable to the PG4 S+L- assay. Testing should be complete prior to patient administration, particularly if cells can be cry-opreserved; otherwise testing should be performed concurrently. In order to gain biological information about events during production, molecular characterization of any RCR detected in clinical lots is also recommended.

 

a. Master Cell Bank of vector-producing cells (one time testing):

 

i. Supernatant testing, 5% of the total supernatant from cul-ture of cells for a master cell bank should be tested by am-plification on a permissive cell line (e.g., Mus dunni) in-cluding several blind passages followed by the PG4 S+L- or alternative assay.

 

ii. Producer cell testing, 1% of pooled producer cells or 108 cells, whichever is fewer, should be cocultured with a per-missive cell line (e.g., Mus dunni) including several blind passages. Supernatant from the coculture should be tested by PG4 S+L- or alternative assay.

 

b. Working cell bank (one time testing): Either supernatant testing or cocultivation of cells is recommended, using conditions described for master cell bank testing.

 

c. Lot testing of vector products:

 

i. Clinical grade supernatant, 5% of the supernatant should be tested by amplification on a permissive cell line (e.g., Mus dunni) including several blind passages, followed by the PG4 S+L- or alternative assay.

 

ii. Testing of end of production cells, 1% of total pooled end of production cells or 108 cells, whichever is fewer, should be cocultured with a permissive cell line (e.g., Mus dunni), and then amplified by several blind passages. Supernatant from the coculture should be tested by S+L- or alternative assay.

 

d. Lot testing of ex vivo transduced cells:

 

i. 1% of pooled transduced cells or 108 cells, whichever is fewer, should be cocultured with a permissive cell line (e.g., Mus dunni) including several blind passages. Super-natant from the coculture should be tested by PG4 S+L- or alternative assay.

 

ii. 5% of supernatant from the transduced cells should be tested by amplification on a permissive cell line (e.g., Mus dunni) including several blind passages. Supernatant from the coculture should be tested by PG4 S+L- assay.

 

2. Patient monitoring: Patients given retrovirus-related products should be monitored for RCR exposure. Please consult CBER for guidance.

 

D. Additional Considerations for the Use of Adenoviral Vectors.

 

1. Measurement of particles vs. infectious units: Patient doses of adenovirus-based gene therapy vectors are presently based upon some method of enu-merating viral particles, plaque forming units (PFU), or infectious units (IU) measured in cell lines complementing the replication defect (not to be confused with measurement of RCA; see Section 2 below). Given the po-tential toxicity of the adenoviral particles themselves, CBER recommends that patient dosing be based on particle number. This recommendation also reflects that particle number can be readily and reproducibly mea-sured. However, since it is quite possible that some outcomes are a func-tion of the number of infectious units administered, it is important for in-vestigators or sponsors to develop in vitro infectivity assays which are re-producible and informative, and to set appropriately tight specifications on the ratio of infectious particles to total viral particles.

Adenovirus particle measurement is commonly based on genomic DNA quantitation. Using the absorbance at 260 nm in the presence of sodium dodecyl sulfate or other virus lysing agents, the maximum number of ade-noviral particles can be calculated from OD260. The presence of non-aden-ovirus nucleic acid may yield inaccurate particle numbers and should be minimized during the manufacturing and viral purification process. Elec-tron microscope particle count has also been used for viral particle enu-meration.

Presently the titer of an adenovirus vector preparation usually refers to the infectious titer. The cell used for determining the titer is often the producer cell line. Differences in viral vectors may lead to changes in growth prop-erties and kinetics. The plaque method is efficient for adenoviral vectors with an easily complemented replication defect. Vectors with multiple replication defects may be more readily titered by alternative methods such as use of fluorescent antibodies. Both assays require optimization for time of adsorption, need for deaggregation of virus, stability, etc. Inclusion of a standard wild type virus control may facilitate the comparison of titers between different vectors and laboratories.

It is currently recommended that a ratio in the product of viral particles to biologically active virus of less than 100:1 be employed in phase I studies. The purpose of this specification is to ensure the consistent manufacture of

recombinant viruses and the highest bioactivity/particle/ patient dose and to limit possible toxicity due to viral structural proteins. As new assays are developed and validated, their comparison to old ones and their use in product characterization is encouraged.

 

2. Detection of replication-competent adenovirus: The presence of RCA in clinical lots of adenovirus vector raises a variety of safety concerns, in-cluding the possibility of adenovirus infection, unintended vector replica-tion due to the presence of wild-type helper function, and exacerbation of host inflammatory responses. The safety risks entailed by these events and other potential adverse events will differ depending on the indication and the patient population. Preclinical safety studies are inherently limited in assessment of RCA-related risks since there are no animal models that support extensive replication of human wild-type or replication competent recombinant adenovirus.

Therefore, adenovirus vectors intended to be replication-defective should be examined for the presence of replication-competent adenovirus (RCA). RCA may arise at multiple steps during the manufacturing process, through recombination with host sequences or by contamination. The amount of RCA generated during manufacture will be influenced by the overall design of the vector. Use of replication-selective adenovirus vec-tors raises additional considerations and may call for additional or differ-ent testing strategies. Such cases should be discussed with CBER.

Detection of RCA in final vector product by a cell culture/cytopathic ef-fect method is preferred at this time. Validation of assay sensitivity by spiking decreasing numbers of wild type adenovirus particles into the test inoculum is recommended. Input multiplicity of infection (MOI) should be carefully chosen because of toxicity of higher doses of virus inoculum un-related to the presence of RCA. It should also be noted that too high an in-put MOI may lead to suppression of RCA outgrowth by the vector. One or two blind passages on cells permissive for growth of the RCA, for exam-ple A549 cells, may be performed to amplify RCA before reading out on an indicator cell line. In addition, it is recommended that the assay be quantitative, that is, able to determine the number of RCA present in any patient dose.

Previous recommendations from the FDA have been that patient doses should contain no more than 1 pfu of RCA or equivalent in patients in whom adenovirus infection would be considered a potential risk. How-ever, the agency recognizes that current production techniques in combi-nation with proposed dosing schemes may make this recommendation pro-hibitively burdensome. Therefore, if sponsors wish to propose a different

specification, data should be provided demonstrating that the level of RCA present represents an acceptable risk for the intended patient population, route of administration, and dose. In order to gain biological information about events during production, molecular characterization of any RCA present in clinical lots is also recommended at this time, and should be as thorough as is practical until more is known about the types of recombina-tion that are occurring.

 

3. Adeno-associated virus: Because of the association of AAV with aden-ovirus, testing for AAV is currently recommended in the Master Cell Bank, the Master Virus Seed Stock, and the final product.

 

E. Other Gene Delivery Systems

Other gene delivery systems including additional types of viral or nucleic acid vectors are currently under development. Sponsors using new systems are encour-aged to contact CBER early in product development, to facilitate a safe and effi-cient development process.

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VIII. MODIFICATIONS IN VECTOR PREPARATIONS

In the past, CBER has considered any change in a vector to result in a new product, and has requested submission of a new IND. There is now an accumulating body of scientific evidence that will permit flexibility in this policy. CBER's goal is to facilitate progress to-wards effective therapies by abbreviating testing and reducing documentation, whenever this can be done while preserving patient safety.

Two aspects of IND submission are affected by consideration of a product as a modified vector: the decision as to whether a new IND should be submitted, and the decision as to what data should be submitted for review. Certain changes, for example minor modifica-tions in the genetic insert or changes in the antibiotic resistance gene, do not necessarily call for a new IND or for full product retesting. In all cases, derivation of a new vector should be described and the vector should meet the specifications for release testing. The other data which should be collected will vary with the degree and nature of the modifi-cations to the vector. The need for additional preclinical testing is determined by the like-lihood of altered vector biology, not just the number of nucleotide changes. In some

cases, tissue localization, germ line alteration, and animal pharmacology/ toxicology studies may be optional. Instead, the relevant safety studies could focus on specific safety concerns related to changes in the vector.

When a number of related vectors involving minor modifications are studied, they may be considered members of a panel, analogous to panels of monoclonal antibodies de-scribed in the "Points to Consider in the Manufacture and Testing of Monoclonal Anti-body Products for Human Use," 1997 revision, 62 FR 9196. As stated, such panels could be studied under a single IND and submitted for approval in a single license application. Phase 3 clinical trials should include some experience with all panel members, and effi-cacy established for the overall panel.

Vector modifications should be discussed with CBER case by case. If a sponsor wishes to abbreviate testing and IND submission for a product or product series, the sponsor should verify with CBER the adequacy of the proposed abbreviated testing scheme prior to initi-ating clinical trials. Data forthcoming from sponsors can help establish whether particular changes in vectors alter their behavior when they are compared in vivo, and therefore whether complete testing should continue to be performed.

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IX. PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES

 

A.B. General Principles

Preclinical studies are intended to define the pharmacologic and toxicologic ef-fects predictive of the human response, not only prior to initiation of clinical tri-als, but also throughout drug development. The goals of these studies include the following: to define safe starting doses and escalation schemes for clinical trials, to identify target organs for toxicity and parameters to monitor in patients receiv-ing these therapies, and to determine populations which may be at greater risk for toxicities of a given cellular or gene therapeutic.

Design of preclinical studies should take into consideration: 1) the population of cells to be administered or the class of vector used, 2) the animal species and physiologic state most relevant for the clinical indication and product class, and 3) the intended doses, route of administration, and treatment regimens. Parameters which should be studied will be discussed below.

Due to the unique and diverse nature of the products employed in cellular and gene therapies, conventional pharmacology and toxicity testing may not always be appropriate to determine the safety and biologic activity of these agents. Issues such as species specificity of the transduced gene, permissiveness for infection by viral vectors, and comparative physiology should be considered in the design of these studies. Available animal models mimicking the disease indication may be useful in obtaining both sufficient safety and efficacy data prior to entry of these agents into clinical trials.

The ICH Draft Guideline S6, "Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals," (Step 4, approved by ICH, 7/16/97) discusses in Sec-tion 3.1 the flexible application of Good Laboratory Practices in testing of biotechnology products. Although pivotal safety studies in support of marketing (e.g. carcinogenicity, reproductive toxicology) are expected to be conducted in compliance with the regulations as outlined in 21 CFR part 58, it is recognized that studies in support of entry into clinical trials may not always strictly adhere to GLP. In these cases, the principles of the regulation should be followed as closely as possible, and where deviations occur, they should be evaluated for impact on the expected clinical application, and discussed in the report submitted to the agency.

If a product is comparable to agents for which there is wide previous clinical ex-perience, or for which the insertion of a different expression cassette is not ex-pected to influence the toxicity or the dissemination of the vector, less extensive preclinical testing may suffice (see Section VII, above).

It is recommended that plans for preclinical studies be discussed with representa-tives from CBER prior to their initiation. Clinical plans requiring rapid enrollment of patients should be anticipated and preceded by adequate preclinical testing.

 

C. Animal Species Selection and Use of Alternative Animal Models

It is recognized that animal models of disease may not be available for every cel-lular or gene therapy system. Preclinical pharmacologic and safety testing of these agents should employ the most appropriate, pharmacologically relevant animal model available. A relevant animal species would be one in which the biological response to the therapy would be expected to mimic the human response. For ex-ample, a vector expressing a human cytokine would best be tested in an animal species in which that cytokine binds to the corresponding cytokine receptor with affinity comparable to that seen with human receptors, and initiates a pharmaco-logic response comparable to that expected in humans.

 

D. Somatic Cell and Gene-Modified Cellular Therapies

 

1. In vivo biological/pharmacological activity: The transduction procedure, dose of expanded or genetically modified cells, and route of administra-tion planned for the clinical trial should be evaluated preclinically. Phar-macologic studies in animals may provide useful information regarding the in vivo function, survival time and appropriate trafficking of the modi-fied cells.

 

2. Toxicologic testing: Safety testing of expanded, activated, or genetically modified somatic cells should be conducted in an appropriate animal model. Data on distribution, trafficking, and persistence of these cells in vivo mentioned above should be evaluated for safety implications as well. At a minimum, treated animals should be monitored for general health sta-tus, serum biochemistry, and hematologic profiles. Target tissues should be examined microscopically for histopathological changes.

 

E. Direct Administration of Vectors In Vivo

A number of different vectors are currently in development for direct administra-tion to human subjects. Direct administration of any of these vectors presents a number of safety concerns that will be addressed here. All toxicity and localiza-tion studies, including studies of gonadal tissue described below, should use the final formulated product, since added materials such as liposomes, or changes in pH or salt content, may alter the toxicity or distribution pattern.

Specific concerns for each vector subclass will generally be handled on a case-by-case basis, and discussion with CBER is encouraged.

 

1. Route of administration: The route of administration of vectors can influ-ence toxicity in vivo. Safety evaluation in the preclinical studies should be conducted by the identical route and method of administration as in the clinical trial whenever possible. When this is difficult to achieve in a small animal species, a method of administration similar to that planned for use in the clinic is advised. For example, intrapulmonary instillation of aden-oviral vectors by intranasal administration in cotton rats or mice is an ac-ceptable alternative to direct intrapulmonary administration through a bronchoscope.

 

2. Selection of animal species: The species of animal chosen for preclinical toxicity evaluations should be selected for its sensitivity to infection and pathologic sequelae induced by the wild-type virus related to a vector, as well as its utility as a model of biologic activity of the vector construct. Rodent models, rather than non-human primates may be useful if they are susceptible to pathology induced by the virus class. When evaluating the activity of a vector in an animal model of the clinical indication, safety data can be gathered from the same model to assess the contribution of disease-related changes in physiology or underlying pathology to the re-sponse to the vector.

 

3. Selection of dose to be employed: The doses of vectors studied preclini-cally should be selected based on preliminary activity data from studies both in vitro and in vivo. A no-effect level dose, an overtly toxic dose, and several intermediate doses should be determined, and appropriate controls, such as naive or vehicle-treated animals, should be included. For products in limited supply or with inherently low toxicity, a maximum feasible dose may be administered as the highest level tested in the preclinical studies. Preclinical safety evaluations should include at least one dose equivalent to and at least one dose escalation level exceeding those proposed for the clinical trial; the multiples of the human dose required to determine ade-quate safety margins may vary with each class of vector employed and the relevance of the animal model to humans. Scaling of doses based on either body weight or total body surface area as appropriate facilitates compar-isons across species. Information generated can be used to determine the margin of safety of the vector for use in the clinical trial, as well as to gauge an acceptable dose-escalation scheme.

 

4. Toxicologic testing: Treated animals should be monitored for general health status, serum biochemistry, and hematology, and tissues should be examined for pathological changes in histology.

 

5. Distribution of vector out of the site of administration: Localization stud-ies, designed to determine the distribution of the vector after administra-tion to its proposed site, are also recommended. Whenever possible, the intended route of administration should be employed. Additional groups of animals may be treated intravenously, as a "worst-case" scenario repre-senting the effects of widespread vector dissemination. Transfer of the

gene to normal, surrounding, and distal tissues as well as the target site should be evaluated using the most sensitive detection methods possible, and should include evaluation of gene persistence. Dose levels selected should follow those used in toxicity testing. When aberrant or unexpected localization is observed, studies should be conducted to determine whether the gene is expressed and whether its presence is associated with patho-logic effects.

 

a. Expression of gene product and induction of immune responses

Expression of the therapeutic gene product in intended or unin-tended tissues may result in unexpected toxicities, which should therefore be addressed in preclinical studies. Inflammatory, im-mune, or autoimmune responses induced by the gene product may be of concern. Animal studies should be conducted over a suffi-cient duration of time to allow development of such responses. Host immune responses against viral or transgene proteins may limit their usefulness for repeated administration in the clinic.

 

b. Vector localization to reproductive organs

With vectors for direct administration, the risk of vector transfer to germ cells should be considered. Animal testicular or ovarian sam-ples should be analyzed for vector sequences by the most sensitive method possible. If a signal is detected in the gonads, further stud-ies should be conducted to determine if the sequences are present in germ cells as opposed to stromal tissues, using techniques that may include but are not limited to cell separations or in situ PCR, or other techniques. Semen samples for analysis can be collected from mature animals including mice (G.D. Snell, et. al., Anat. Rec., 90:243-253, 1944; J.C. Kile, Jr., Anat. Rec., 109:109-117, 1951), for determination of vector incorporation into germ cells.

 

6. Host immune status and effects on gene therapy vectors: Immune status of the intended recipients of a gene therapy should be considered in the risk-benefit analysis of a product, particularly for viral vectors. If exclusion of immunocompromised patients would unduly restrict a clinical protocol, immune suppressed, genetically immunodeficient, or newborn animals may be used in preclinical studies, to evaluate any potential safety risks.

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X. CONCLUSION

As new classes of gene therapies and somatic cell therapies are developed, concerns and methods of testing will most likely change. The above guidance outlines the types of is-sues that should be examined and provides a framework for analysis of new technologies as they emerge. CBER encourages comments and suggestions from the academic and commercial communities and other interested parties during the evolution of policy in this area.

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1 This guidance document represents the Agency's current thinking on the development and regu-lation of somatic cell therapy products. It does not create or confer any rights for or on any per-son and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of applicable statutes, regulations, or both. For additional copies of this guidance, contact the Office of Communication, Training and Manufacturers As-sistance, HFM-40, Center for Biologics Evaluation and Research, Food and Drug Administra-tion, 1401 Rockville Pike, Rockville, MD 20852-1448. Send one self-addressed adhesive label to assist that office in processing your request. The document may also be obtained by mail by call-ing the CBER Voice Information System at 1-800-835-4709 or 301-827-1800, or by calling the FAX Information System at 1-888-CBER-FAX or 301-827-3844. Persons with access to the in-ternet may obtain the document at "http://www.fda.gov/cber/guidelines.htm".

Impact of Immunity Induced by Viral and DNA ProductsPrincipal Investigator: Suzanne L. Epstein, PhDOffice / Division / Lab: OCTGT / DCGT / GTIB

General Overview

Influenza causes high rates of serious illness and deaths each year in the US and around the world. Current influenza vaccines depend upon surveillance (monitoring the influenza viruses causing infections in people around the world), strain selection (choosing which influenza viruses will be represented in the upcoming year's vaccine, based on predictions of what viruses will circulate), and production of new vaccines (a time-consuming process with limited capac-ity). New strategies are needed to control seasonal influenza, unexpected influenza outbreaks, avian influenza viruses such as H5N1, and pandemics such as the one caused by H1N1. The goals of strategies would be to slow virus spread in the community and to reduce serious illness, hospitalization, and the number of deaths.

Our research program studies new approaches to influenza vaccination that help control infection regardless of which influenza virus strain emerges. The experimental 'one-size-fits-all' vaccines we study could be available off the shelf. They could be rapidly deployed to help control an emerging influenza virus for which there is no vaccine or that has genetically "drifted" (changed) so that it no longer matches available conventional vaccines. These new vaccines are designed to offer some protection by reducing the severity of disease and the spread of infection, while a spe-cific vaccine against the new or mutated influenza virus is produced.

The 'one-size-fits-all' vaccine would protect against a variety of different influenza viruses by stimulating the immune system to target those viral proteins that are similar ("conserved") among all influenza A viruses.

Vaccine candidates include plasmid DNA (small rings of DNA derived from bacteria) or aden-oviruses (a type of non-replicating virus used to express vaccine proteins in the recipient) modi-fied to express influenza proteins, as well as weakened influenza viruses similar to those used in a current vaccine. We are studying the immune responses and protection various combinations of these vaccines induce in mice. We also study vaccines in ferrets, an animal in which influenza can spread and cause disease similarly to the way it does in humans. We have found that the ex-perimental vaccines can protect against influenza infections that are lethal to unvaccinated ani-mals, and that they protect against a broad range of virus strains including highly pathogenic H5N1 (bird flu).

FDA regulates two major categories of products based on viruses and plasmid DNA: vaccines and gene therapy used to treat diseases that are currently untreatable. In the case of vaccines, im-mune responses are desirable for protection, and we need to identify the most effective responses as markers of effectiveness. In the case of gene therapy, immune responses generated by a first treatment can stimulate an immune response that can block the activity of repeat treatment. Un-derstanding the immune responses induced by vaccines and gene therapy products can help sci-entists design ways to make them safer and more effective. Such information obtained during laboratory studies of these products in animals and during clinical studies in humans will im-prove the ability of FDA regulators to make decisions about these products.

Scientific Overview

Control of influenza by vaccination is made particularly difficult by the rapid evolution of the vi-ral glycoproteins hemagglutinin and neuraminidase, which allows the virus to escape from neu-tralizing antibodies. This lab explores new approaches to influenza A vaccination that provide broad heterosubtypic protection and are not based on strain-matching. Thus, they do not depend on predicting which strain or even subtype of influenza will circulate. Heterosubtypic protection is seen in a variety of animal models (mice, ferrets, chickens, pigs, cotton rats). It often permits low-grade infection, but reduces morbidity, mortality, and virus shedding. The new experimental vaccines we study are intended for use when strain-matched vaccines are not available, when they are insufficient in supply, or when the circulating virus has drifted. This protection could be supplemented by strain-matched vaccines, once available.

We have compared a variety of vaccination strategies in order to optimize the potency of induc-tion of heterosubtypic immunity. These vaccine candidates include cold-adapted (ca) viruses and DNA prime-recombinant adenovirus (rAd) boost vaccination to the conserved antigens nucleo-protein (NP), matrix-2 (M2), or A/NP+M2. Both ca and DNA-rAd vaccinations induced anti-body and T cell responses, and protected against lethal H1N1 challenge. A/NP+M2 DNA-rAd protected against challenge with highly pathogenic A/Vietnam/1203/2004 (H5N1), but ca vac-cine did not. Thus while ca vaccines may provide some heterosubtypic immunity, in this model vaccination focusing immunity on conserved antigens was more effective for protection against a divergent, highly pathogenic virus.

Recently we have characterized immunization to nucleoprotein (NP) and matrix 2 (M2) by DNA priming followed by various types of boosting in mice and ferrets. DNA vaccination was fol-lowed by boosting with antigen-matched recombinant adenovirus (rAd) given by systemic (intra-muscular) or mucosal (intranasal) routes, or cold-adapted (ca) influenza virus given intra-nasally. All three types of prime-boost protected against mortality following virulent H1N1 and H5N1 challenges. Intranasal rAd boosting gave the best protection against morbidity. Mice vaccinated this way had minimal symptoms after challenge infections that were lethal to controls. Compared to other boosts, mucosal rAd induced stronger IgA responses, greater and more lasting virus-spe-cific activated T-cell responses in the lung, and better protection against morbidity following challenge even eight months post-boost. The immune response measures in mice that differed among vaccinations and correlated with outcome provide candidate immune correlates for fur-ther study.

We also studied prime-boost vaccination in ferrets, an animal model resembling human influenza with regard to virus spread and symptoms. In addition, these animals are outbred and thus are ge-netically diverse. Both mucosal and parenteral rAd boosting protected ferrets from lethal H5N1 challenge. The results indicate that vaccination focused on conserved antigens offers powerful protection.

This program has also included epidemiological analysis to look for evidence of human hetero-subtypic immunity. Archival records from the 1957 pandemic suggested the possibility of such protection, and we are conducting a surveillance study during the pandemic of 2009 to see if het-erosubtypic immunity can play a role in protection.

Publications

J Immunol 2011 Feb 15;186(4):2422-9TLR3-specific double-stranded RNA oligonucleotide adjuvants induce dendritic cell cross-presentation, CTL responses, and antiviral protection. Jelinek I, Leonard JN, Price GE, Brown KN, Meyer-Manlapat A, Goldsmith PK, Wang Y, Ven-zon D, Epstein SL, Segal DM

Expert Rev Vaccines 2010 Nov;9(11):1325-41Cross-protective immunity to influenza A viruses. Epstein SL, Price GE

PLoS One 2010 Oct 4;5(10):e13162Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses. Price GE, Soboleski MR, Lo CY, Misplon JA, Quirion MR, Houser KV, Pearce MB, Pappas C, Tumpey TM, Epstein SL

Vaccine 2010 Aug 16;28(36):5817-27Genetic control of immune responses to influenza A matrix 2 protein (M2). Misplon JA, Lo CY, Gabbard JD, Tompkins SM, Epstein SL

Vaccine 2009 Nov 5;27(47):6512-21Vaccination focusing immunity on conserved antigens protects mice and ferrets against vir-ulent H1N1 and H5N1 influenza A viruses. Price GE, Soboleski MR, Lo CY, Misplon JA, Pappas C, Houser KV, Tumpey TM, Epstein SL

Vaccine 2008 Apr 16;26(17):2062-72Comparison of vaccines for induction of heterosubtypic immunity to influenza A virus: Cold-adapted vaccine versus DNA prime-adenovirus boost strategies. Lo CY, Wu Z, Misplon JA, Price GE, Pappas C, Kong WP, Tumpey TM, Epstein SL

Emerg Infect Dis 2007 Mar;13(3):426-35Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1. Tompkins SM, Zhao ZS, Lo CY, Misplon JA, Liu T, Ye Z, Hogan RJ, Wu Z, Benton KA, Tumpey TM, Epstein SL

J Infect Dis 2006 Jun 1;193(11):1613-4Heterosubtypic immunity to influenza: Right hypothesis, wrong comparison - Reply to Carrat and Lavenu Epstein SL

J Infect Dis 2006 Jan 1;193(1):49-53Prior H1N1 Influenza Infection and Susceptibility of Cleveland Family Study Participants

during the H2N2 Pandemic of 1957: An Experiment of Nature. Epstein SL

Vaccine 2005 Nov 16;23(46-47):5404-10Protection against multiple influenza A subtypes by vaccination with highly conserved nu-cleoprotein. Epstein SL, Kong WP, Misplon JA, Lo CY, Tumpey TM, Xu L, Nabel GJ

Mol Ther 2005 Jul;12(1):5-8Critical Path Workshop on the Development of Cellular and Gene Therapy Products. Bloom ET, Epstein SL, Simek SL

Proc Natl Acad Sci U S A 2004 Jun 8;101(23):8682-6Protection against lethal influenza virus challenge by RNA interference in vivo. Tompkins SM, Lo CY, Tumpey TM, Epstein SL

Safety and Effectiveness of Gene TherapyPrincipal Investigator: Andrew Byrnes, PhDOffice / Division / Lab: OCTGT / DCGT / GTIB

General Overview

Gene therapy holds great promise for treating cancer, inherited disorders, and other diseases. Gene therapy, also often referred to as 'gene transfer,' uses carriers called 'vectors' to deliver genes to tissues where they are needed. The genes in gene therapy vectors code for specific pro-teins that may treat disease. Gene therapy vectors can be engineered to produce a variety of ther-apeutic proteins, and researchers are investigating the safety and effectiveness of a variety of dif-ferent types of vectors in hundreds of clinical trials in the US.

We are studying one type of commonly-used gene therapy vector that is made from a disabled cold virus -- the adenovirus vector. While adenovirus vectors are very efficient at delivering genes, adenovirus vectors can cause toxic effects that limit the amount of vector that doctors can give to patients. We are particularly interested in how to safely deliver large amounts of aden-ovirus vectors intravenously, with the goal of targeting tumors and other tissues. Another of our major goals is to develop animal models that reliably predict the safety and effectiveness of ade-novirus vectors in humans.

Our lab and others have shown that when adenovirus vectors are injected intravenously, cells in the liver called Kupffer cells rapidly ingest and dispose of them. This not only prevents the vec-tor from reaching its intended tissue but also kills the Kupffer cells themselves, damaging the

liver. We are studying how Kupffer cells recognize adenovirus and how adenovirus kills these cells. In addition, we are trying to improve adenovirus gene therapy by preventing Kupffer cells from removing vector from the circulation. Our laboratory is also studying how both non-animal and animal models can be used to predict whether particular adenoviruses can be safely used in humans. New adenovirus gene therapy vectors are tested in animals before human clinical trials begin, and it is important for both researchers and the FDA to know how well these animal stud-ies can predict safety.

Our studies will help us to understand the mechanisms for adenovirus vector removal by Kupffer cells, the resulting damage to liver, and the relevance of animal models to human outcomes. This new knowledge will enable researchers to design safer and more effective gene therapy vectors.

Scientific Overview

Adenovirus vectors have shown considerable promise in animal models and are currently being used in numerous clinical trials, especially for the therapy of cancer. We are interested in im-proving the safety and efficacy of adenovirus vectors, especially when administered through the vascular system. Certain properties of adenovirus vectors make them hazardous to administer in-travenously in large doses, and our laboratory is trying to understand and fix this problem.

One of our major areas of interest is the innate immune response to adenovirus vectors. These rapid responses can cause serious toxicity and may severely limit the doses of adenovirus vectors that are safe to use. In addition, we are also studying how cells in the liver recognize adenovirus, since this is the major site of adenovirus removal from the circulation. A better understanding of these mechanisms will help us to develop strategies to improve vector efficacy and reduce toxic-ity. We will also gain a better understanding of the advantages and disadvantages of using differ-ent species of animals to predict the behavior of adenovirus vectors in humans, which is particu-larly relevant to the regulatory work of the FDA.

Our research on vector biodistribution is focused on how Kupffer cells and other liver cells rec-ognize adenoviruses after they are injected intravenously. We are also interested in how aden-ovirus vectors interact with blood proteins, and how this in turn influences the clearance of vec-tor by the liver. We recently showed that both scavenger receptors on Kupffer cells and natural antibodies in the blood contribute to the clearance of adenovirus vectors by Kupffer cells. We are currently studying how natural antibodies interact with adenovirus.

Our research on the innate immune response to adenovirus vectors includes not just studies on cytokine and chemokine responses, but also some less well-studied (but no less important) medi-ators. We have recently characterized the roles of complement and platelet activating factor and discovered how adenovirus vectors activate these two pathways. One particularly interesting finding is that the mechanisms of complement activation in animal models are completely differ-ent from the mechanisms that occur in in vitro studies. This unexpected observation has signifi-cant implications for how we should model adenovirus interactions with the complement system. Another interesting finding is that high doses of adenovirus vectors rapidly induce a burst of

platelet activating factor in vivo that can lead to shock. As part of this work we showed that there are practical ways to block the effects of platelet activating factor and reduce toxicity due to this pathway.

Currently we are studying additional novel mediators and pathways that control innate immune responses, and how this contributes to toxicity caused by adenovirus vectors. Understanding these mediators and pathways is an essential step toward our goal of developing safer vectors and new ways to limit vector-induced toxicity.

Publications

Mol Ther 2010 Mar;18(3):609-16Induction of Shock After Intravenous Injection of Adenovirus Vectors: A Critical Role for Platelet-activating Factor. Xu Z, Smith JS, Tian J, Byrnes AP

J Virol 2009 Jun;83(11):5648-58Adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo. Tian J, Xu Z, Smith JS, Hofherr SE, Barry MA, Byrnes AP

Neuroreport 2008 Aug 6;19(12):1187-92Th1 cytokines are upregulated by adenoviral vectors in the brains of primed mice Lee MB, McMenamin MM, Byrnes AP, Charlton HM, Wood MJA

Hum Gene Ther 2008 May;19(5):547-54Interaction of Systemically Delivered Adenovirus Vectors with Kupffer Cells in Mouse Liver. Smith JS, Xu Z, Tian J, Stevenson SC, Byrnes AP

J Virol Methods 2008 Jan;147(1):54-60A quantitative assay for measuring clearance of adenovirus vectors by Kupffer cells. Smith JS, Xu Z, Byrnes AP

Genomics 2006 Apr;87(4):552-9Quality prediction of cell substrate using gene expression profiling. Han J, Farnsworth RL, Tiwari JL, Tian J, Lee H, Ikonomi P, Byrnes AP, Goodman JL, Puri RK

Virology 2006 Mar 30;347(1):183-90Heparin-binding and patterns of virulence for two recombinant strains of Sindbis virus. Bear JS, Byrnes AP, Griffin DE

IDrugs 2005 Dec;8(12):993-6Challenges and future prospects in gene therapy. Byrnes AP

Mol Ther 2006 Jan;13(1):108-17Rapid Kupffer cell death after intravenous injection of adenovirus vectors. Manickan E, Smith JS, Tian J, Eggerman TL, Lozier JN, Muller J, Byrnes AP

Mol Ther 2004 Jun;9(6):932-41Severe pulmonary pathology after intravenous administration of vectors in cirrhotic rats. Smith JS, Tian J, Lozier JN, Byrnes AP

Arch Virol 2004 2004;(18):21-33Emergence and virulence of encephalitogenic arboviruses. Griffin DE, Byrnes AP, Cook SH

Gene Ther 2004 Mar;11(5):431-8Unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver dis-ease. Smith JS, Tian J, Muller J, Byrnes AP

Cell-specific And Gene-specific Targeting of Gene Therapy VectorsPrincipal Investigator: Jakob Reiser, PhDOffice / Division / Lab: OCTGT / DCGT / GTIB

General Overview

Gene therapy holds great potential for treating a variety of serious diseases, some of which are as yet incurable. One strategy for delivering therapeutic genes is to use a virus to carry them into cells. Such gene delivery vehicles are referred to as vectors. Viruses used as vectors are specially modified so they do not reproduce inside the target cells.

Over the past decade, a variety of researchers have developed gene therapy vectors based on hu-man immunodeficiency virus (HIV). But while HIV-derived vectors are promising, they also pose a risk. These vectors might insert the therapeutic genes into chromosomes at sites that be-have like "on switches" that activate cancer-causing genes. This problem occurred previously during a study in France, triggering leukemia in children being treated with gene therapy for an inherited disease of the immune system.

Our goal is to improve the safety of HIV-based vectors so they can be used in the future to treat

patients without these risks. Therefore, we are trying to engineer such vectors so they insert genes directly into well-defined sites on human chromosomes that lack cancer-causing on-switches.

These studies will help researchers to design safer and more effective HIV-based gene therapy vectors. The knowledge gained from our research will also allow us to evaluate the risks associ-ated with the use of HIV vectors and to provide timely advice on product development for FDA sponsors.

Scientific Overview

Lentiviruses are complex retroviruses that include human immunodeficiency viruses such as HIV-1. Gene therapy vectors based on HIV-1 are being developed to deliver therapeutic genes. While these vectors are promising, they pose risks in that they may activate oncogenes during random integration into the host genome. Our goal is to develop safer HIV-1-based lentiviral (LV) vectors by limiting their integration to well-defined sites in the human genome and by nar-rowing their tissue tropism.

Toward this goal, we have designed integrase-defective LV vectors capable of integrating at spe-cific sites in the mouse genome through homologous recombination. To do this, we are pursuing a targeted trapping approach using a promoterless secretion trap vector containing genomic se-quences corresponding to genomic loci to be targeted. We rely on the endogenous promoter of the gene to be targeted to drive the expression of a selectable marker gene including a drug resis-tance gene and a cell surface marker-encoding gene. In parallel, we are investigating the capacity of phage PhiC31 integrase to mediate region-specific vector integration.

Targeted gene delivery involves broadening the tropism of vectors to transduce previously non-permissive cells or replacing the viral tropism to transduce specific target cell exclusively. These approaches offer potential advantages of enhanced therapeutic effects and reduced side effects. We are in the process of designing improved targetable LV vectors that more efficient and more flexible than the currently available ones. These vectors will either bear a cell-binding domain linked to the vector's membrane plus a separate fusion domain derived from Sindbis virus or vesicular stomatitis virus or a molecular bridge consisting of soluble avian leukosis/sarcoma virus receptors fused to cell-specific ligands. Our initial focus will be interleukin 13 (IL13)-dis-playing vectors to target IL13 receptor-over-expressing cancer cells in vitro and in vivo. If the performance of the system (specificity, vector titers) turns out to be poor, we plan to improve it using directed evolution approaches.

Our research allows us to evaluate the technologies used to construct, manufacture and test novel LV vectors (purity) and to investigate the ability of these vectors to target specific cell types in animal models (potency and safety). The information gained from these studies will enable us to identify potential risks associated with making and evaluating LV vectors for clinical use.

We use a broad range of biological approaches, including cell culture, molecular biological and

virological techniques, to construct, manufacture and test new HIV-1-based vectors. A special emphasis is on targetable vectors for cell-specific transduction and on vectors capable of site-specific integration.

Publications

Retrovirology 2010 Jan 25;7:3Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sind-bis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus. Zhang XY, Kutner RH, Bialkowska A, Marino MP, Klimstra WB, Reiser J

J Virol Methods 2009 May;157(2):113-21Simplified lentivirus vector production in protein-free media using polyethylenimine-medi-ated transfection. Kuroda H, Kutner RH, Bazan NG, Reiser J

Nat Protoc 2009;4(4):495-505Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Kutner RH, Zhang XY, Reiser J

Cloning Stem Cells 2009 Mar;11(1):167-76Generation of domestic transgenic cloned kittens using lentivirus vectors. Gómez MC, Pope CE, Kutner RH, Ricks DM, Lyons LA, Ruhe MT, Dumas C, Lyons J, Dresser BL, Reiser J

BMC Biotechnol 2009 Feb 16;9:10Simplified production and concentration of HIV-1-based lentiviral vectors using HYPER-Flask vessels and anion exchange membrane chromatography. Kutner RH, Puthli S, Marino MP, Reiser J

J Gene Med 2008 Nov;10(11):1163-75A comparative analysis of constitutive and cell-specific promoters in the adult mouse hip-pocampus using lentivirus vector-mediated gene transfer. Kuroda H, Kutner RH, Bazan NG, Reiser J

J Immunol 2008 Sep 1;181(5):3049-56TRIF and IRF-3 binding to the TNF promoter results in macrophage TNF dysregulation and steatosis induced by chronic ethanol. Zhao XJ, Dong Q, Bindas J, Piganelli JD, Magill A, Reiser J, Kolls JK

Stem Cells Dev 2008 Jun;17(3):441-50Optimized lentiviral transduction of mouse bone marrow-derived mesenchymal stem cells. Ricks DM, Kutner R, Zhang XY, Welsh DA, Reiser J

J Neurochem 2008 Feb;104(4):1055-64Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain. Zhang XY, Dinh A, Cronin J, Li SC, Reise

Viral Safety Studies in Xenotransplantation and Gene Therapy ProductsPrincipal Investigator: Carolyn A. Wilson, PhDOffice / Division / Lab: OCTGT / DCGT / GTIB

General Overview

Gammaretroviruses are simple animal viruses that are related to HIV, the cause of AIDS.

These viruses might pose risks in several regulated biologics products, both as contaminants and in their use for gene therapy, in which they act as carriers, or "vectors" to deliver genes that treat a disease by producing a therapeutic molecule. For example, a derivative of a mouse gam-maretrovirus was used for gene therapy given to children in two clinical trials in Europe for X-linked Severe Combined Immunodeficiency Disease (X-SCID, more commonly known as the "Bubble Boy Disease"). Although this therapy appeared to provide clinical benefit to most of the children, some patients developed the blood cancer leukemia. This demonstrated that these ani-mal viruses can cause disease in humans, although except for the X-SCID clinical trials, gam-maretroviruses used for gene therapy have not caused cancer.

In addition, pig gammaretroviruses are potential contaminants in some other products, such as proteins derived from pig plasma. These viruses are of particular concern in xenotransplantation products, that is, the use of living cells, tissues, or organs for treatment of human disease.

We study the gammaretrovirus porcine endogenous retrovirus (PERV), which occurs in all pigs. Using molecular biology, tissue culture, and animal models, we study the conditions that affect virus infection and transmission, especially those factors that influence the ability of PERV to in-fect human cells. The goal of this work is to develop methods for preventing transmission of PERV, as well as to generate data that guides regulatory policies for effectively screening ani-mals, xenotransplantation products obtained from those animals, and recipients of those prod-ucts,for risk of PERV transmission.

Through a collaborative program with the National Toxicology Program, our laboratory is also in the process of assessing preclinical models that may prove useful for assessing the tumorigenic potential of retroviral vectors.

In addition to our studies on gammaretroviruses, our laboratory uses retroviral vector technology

to study a CDC Category A potential agent of bioterrorism, Ebola virus (an agent of viral hemor-rhagic fever). Category A agents are high-priority agents considered to pose a major threat to na-tional security. We are trying to identify regions of the Ebola virus envelope that are highly con-served (do not readily mutate from generation to generation) and critical for infection. Based on that information we will try to develop antiviral or vaccine strategies that can protect against a broad range of varieties of the Ebola virus.

Scientific Overview

Our laboratory is primarily interested in studying the host and cellular determinants of viral entry for porcine endogenous retroviruses (PERV). PERV is a gammaretrovirus found in the genome of pigs and at least two types of PERV (PERV-A and PERV-B) can infect human cells, while PERV-C is pig-tropic. We use retroviral vectors carrying chimeric or mutated PERV envelopes to study the sequences on the PERV envelope required for viral entry. In addition, we are study-ing the PERV-A receptor on both permissive and non-permissive host cells to improve our un-derstanding of the cellular factors that influence the human tropism of PERV.

In addition, we are involved in a study sponsored by the National Toxicology Program aimed at identifying the best preclinical model to assess tumorigenic potential of retroviral vectors. The study is designed to use large group sizes in order to gain statistical confidence in the sensitivity of the model for detecting tumorigenic events. The study will be performed in several phases in order to allow for in vivo assessment of various types of retroviral vectors (gammaretrovirus, lentivirus, and spumavirus), each with an assortment of backbone configurations that will allow determination of the tumorigenic impact of various vector modifications, such as presence or ab-sence of the retroviral U3, internal enhancer elements, etc.

Finally, our laboratory has recently become interested in studying the category A agent, Ebolavirus. We are trying to identify highly conserved regions that are critical for entry in order to determine if these regions would make good antiviral or vaccine targets.

Publications

J Virol Methods 2011 Jun;174(1-2):99-109Development and characterization of rabbit and mouse antibodies against ebolavirus enve-lope glycoproteins. Ou W, Delisle J, Konduru K, Bradfute S, Radoshitzky SR, Retterer C, Kota K, Bavari S, Kuhn JH, Jahrling PB, Kaplan G, Wilson CA

Virology 2010 Jan 5;396(1):135-42Phenylalanines at positions 88 and 159 of Ebolavirus envelope glycoprotein differentially impact envelope function. Ou W, King H, Delisle J, Shi D, Wilson CA

Retrovirology 2009 Jan 14;6:3Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry. Marcucci KT, Argaw T, Wilson CA, Salomon DR

Methods Mol Biol 2009;506:477-88The US and EU regulatory perspectives on the clinical use of hematopoietic stem/progeni-tor cells genetically modified ex vivo by retroviral vectors. Wilson CA, Cichutek K

Cell Mol Life Sci 2008 Nov;65(21):3399-412Porcine endogenous retroviruses and xenotransplantation. Wilson CA

J Virol 2008 Aug;82(15):7483-91Identification of residues outside of the receptor binding domain that influence infectivity and tropism of porcine endogenous retrovirus. Argaw T, Figueroa M, Salomon DR, Wilson CA

Virology 2008 Jun 5;375(2):637-45Functional hierarchy of two L domains in porcine endogenous retrovirus (PERV) that in-fluence release and infectivity. Marcucci KT, Martina Y, Harrison F, Wilson CA, Salomon DR

Xenotransplantation 2007 Jul;14(4):309-15No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support. Levy MF, Argaw T, Wilson CA, Brooks J, Sandstrom P, Merks H, Logan J, Klintmalm G

Virus Res 2006 Nov;121(2):205-14Identification of two amino acid residues on Ebola virus glycoprotein 1 critical for cell en-try. Mpanju OM, Towner JS, Dover JE, Nichol ST, Wilson CA

J Virol 2006 Apr;80(7):3135-46Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. Martina Y, Marcucci KT, Cherqui S, Szabo A, Drysdale T, Srinivisan U, Wilson CA, Patience C, Salomon DR

Virology 2006 Mar 5;346(1):108-17The infectivity and host range of the ecotropic porcine endogenous retrovirus, PERV-C, is modulated by residues in the C-terminal region of its surface envelope protein. Gemeniano M, Mpanju O, Salomon DR, Eiden MV, Wilson CA

Am J Transplant 2005 Aug;5(8):1837-47Pseudotyping of porcine endogenous retrovirus by xenotropic murine leukemia virus in a pig islet xenotransplantation model. Martina Y, Kurian S, Cherqui S, Evanoff G, Wilson C, Salomon DR

Mol Ther 2004 Dec;10(6):976-80Workshop on long-term follow-up of participants in human gene transfer research. Nyberg K, Carter BJ, Chen T, Dunbar C, Flotte TR, Rose S, Rosenblum D, Simek SL, Wilson C

J Gen Virol 2004 Jan;85(Pt 1):15-9Limited infection without evidence of replication by porcine endogenous retrovirus in guinea pigs. Argaw T, Colon-Moran W, Wilson CA

Sites: AG Vektorologie und Experimentelle Gentherapie - Development of technologies that exploit ade-

noviruses as highly efficient vectors for gene therapy. Includes a range of publications with ab-stract links.

Alberta Gene Therapy Group - A network of researchers looking to foster collaboration in several therapeutic fields.

Amaxa Biosystems - NucleofectorTM technology allows non-viral gene transfer directly into the nucleus of primary cells and cell lines, thereby providing rapid target gene validation, acceleration of drug development and new applications for gene therapy.

American Society of Gene Therapy - Organization dedicated to promoting gene therapy research through public education, scientific meetings and scientific committees targeting specific therapeu-tic areas and also vector technology.

Amsterdam Molecular Therapeutics - Focused on developing gene therapies for lipoprotein lipase disorder, inflammatory bowel disease and neuroregeneration. Also provides contract development and manufacturing of gene and cell-based products in their GMP facility.

Ark Therapeutics - A gene medicine company developing a broad range of products based on vascular and endothelial molecular biology to treat vascular and circulatory diseases and cancer.

BioVex - Gene delivery technology allowing pharmaceutical and genomics companies to assess the therapeutic value of identified genes and also allows genes to be used as therapeutic products to treat diseases from cancer to nervous and immune system disorders

Cell Genesys - Focused on the development and commercialization of cancer vaccines and gene therapies to treat various cancers including leukemia, hemophilia A and B, restenosis after angio-plasty and Parkinson's disease.

Center for Cell and Gene Therapy - Clinical and fundamental research, job opportunities, faculties' interests and links to gene therapy sites - joint project of Baylor College of Medicine, Texas Chil-dren's Hospital, and The Methodist Hospital in Houston, TX.

Center for Gene Therapy at the University of Michigan Medical Center - The center links basic sci-ence, clinical investigation and technology transfer endeavors in the area of gene therapy, while also serving as a resource for information and education.

Centre for Gene Therapeutics - McMaster University - The mission of the Centre is to investigate, create and implement approaches utilizing the delivery of genes as therapeutic agents in the treat-ment of human and animal disease.

Ceregene, Inc. - Pioneering genetic therapies for neurologic disorders. The Children's Hospital at Westmesd - Gene Therapy Research Unit - A joint initiative of The New

Children's Hospital and the Children's Medical Research Institute is undertaking gene therapy clin-

ical trials in Australia. Research projects include cancer immunotherapy, vector development, HIV infection, targeting gene delivery to specific tissue types.

Collateral Therapeutics - A product-driven cardiovascular gene therapy company, focused on the discovery, development and commercialization of novel and innovative non-surgical gene therapy products.

Copernicus Therapeutics, Inc. - Developing human gene therapy products for cystic fibrosis and hemophilia B and DNA vaccinations. The company creates proprietary PLASmin Complexes which are efficient non-viral vectors and REPLIsome vectors which allow replication of non-viral vectors.

Crucell N.V. - Discover, develop and produce a variety of biopharmaceuticals for the treatment of human diseases. Includes technology and investors data, and careeer opportunities.

Eleanor Roosevelt Institute - Conducts basic biomedical research into therapies for Down Syn-drome, ALS, cancer, and Rec8 Syndrome. Includes map of chromosone 21, employment details and links to related resources.

enGene, Inc. - Develop technology to manufacture therapeutic proteins through genetic modifica-tion of gastrointestinal cells to find a cure for diabetes and its complications.

European Society for Gene Therapy - ESGT - Organises annual meetings in Europe to promote gene therapy research.

Finnish Gene Therapy Society - Fosters the exchange of ideas between research groups and indi-vidual scientists, organizes scientific meetings and training courses and informs general public about basic aspects and recent developments in gene therapy.

Gene Express, Inc. - Provide gene assays to assist pharmaceutical and biotech companies with new drug development and multi-gene expression analysis services to researchers. Includes tech-nology and investor data, and careers.

Gene Therapy - Human Genome Project - Definition, hurdles, ethical issues, and links to more re-sources on gene therapy provided by the Oak Ridge National Laboratory.

Gene Therapy Advisory Committee - GTAC - Advises on the ethical acceptability of proposals for gene therapy research on humans taking account of the scientific merits and the potential benefits and risks, and provides advice to UK Health Ministers on developments in gene therapy research.

Gene Therapy and Your Child - Development information for the clinical use of gene therapy for children with severe illnesses.

Gene Therapy at MD Anderson Cancer Center - The human cancer gene prevention and therapy program conducts basic research, clinical trials and vector development.

Gene Therapy Systems - Commercial firm providing reagents for gene delivery and gene therapy research, including unique pGeneGrip fluorescently labeled plasmid vectors and GenePORTER cationic lipid transfection reagent.

GeneBrowser - Commercial site focusing on the current state of retroviral vector development for gene therapy with numerous helpful links - from Nature Technology Corp.

Genteric, Inc. - A rapidly growing biopharmaceutical company that develops novel gene-based methods to treat human diseases.

GenVec - An emerging biopharmaceutical company developing novel gene therapies for diseases where local delivery of a therapeutic gene has potential benefits over currently available treat-ments. New product development initiatives have been formalized for coronary artery disease with BioBypass angiogen, restenosis prevention and cancer.

Goldyne Savad Institute of Gene Therapy - Aims to enhance the development of safe and effec-tive gene therapy by supporting research projects, from vector development and construction to production of biologicals for human use and their application in GCP clinical trials.

Harvard Gene Therapy Initiative - Provides plasmid construction and virus production services for gene therapy in both research and therapeutic applications.

Henogen - A GMP biomanufacturing company located in Belgium, able to produce, purify and aseptically fill clinical batches of therapeutic proteins, monoclonal antibodies, plasmid DNA, vacci-nal viruses or viral vectors for gene therapy.

Inherinet - Consortium of researchers from European institutions focused on developing gene therapy of hematopoietic stem cells for inherited diseases.

Introgen Therapeutics, Inc. - A leader in the development of gene therapy products to treat a vari-ety of cancers.

InvivoGen - Tools for gene therapy and cancer research including plasmid and cloning vectors, gene collections, selection antibiotics, mycoplasma curing kit, and prodrugs.

Jesse's Journey - The Foundation for Gene and Cell Therapy - A registered charitable organiza-tion raising funds for research in gene and cell based therapies focusing on Duchenne muscular dystrophy and other neuromuscular diseases.

The Journal of Gene Medicine - An information source on all aspects of human gene therapy. It offers an international comprehensive summary of both published and ongoing clinical trials. Also includes reviews on basic and therapeutic issues.

Molecular Medicine Bioservices, Inc. - A development and manufacturing company based in San Diego dedicated to the production of high-quality, clinical grade, gene and cell based therapeutics and reagents.

Molecular Virology Lab at Leiden University - The group performs basic research on viruses in or-der to facilitate the development of efficient gene-transfer vectors for use in gene therapy. Current research concerns the human adenoviruses and the systems required for producing recombinant-adenovirus vectors.

MolecularTrucks, Inc - Sells AAV purification kits of different serotypes for gene therapy research. Includes online buying facilities.

National Gene Vector Laboratories (NGVL) - An interactive group of academic production and pharmacology/toxicology laboratories whose primary goal is to provide eligible investigators with clinical grade vectors for phase I/II gene therapy clinical trials and to provide support for relevant pharmacology/toxicology studies leading up to clinical gene transfer protocols.

Office of Biotechnology Activities at the NIH - Monitors scientific progress and ethical issues in ba-sic and clinical research involving recombinant DNA and human gene transfer at NIH funded insti-tutions.

Oxagen Limited - Perform genetic analysis on collections of family samples so as to understand the mechanisms and causes of common diseases, in order to identify new drug targets, the vali-dation of targets and the development of diagnostics.

Oxford Biomedica - Focuses on developing gene transfer technologies for human gene therapy, gene-based immunotherapy, and gene-based drug discovery for cancer, AIDS, neurodegenera-tive and cardiovascular disease.

Oxford University GeneMedicine Research Group - Focused on the development gene therapies for diseases of the airway, such as asthma, lung cancer and in particular, cystic fibrosis (CF). Work includes formulation development, reagent production, pre-clinical testing, and human clini-cal trials.

Pittsburgh Human Gene Therapy Center - Fosters the growth and development of multidisci-plinary preclinical and clinical research programs aimed at the use of gene transfer technologies for the treatment of human disease.

Polyplus Transfection - Dedicated to the development, manufacturing and marketing of modular transfection reagents. Expertise includes in vitro and in vivo gene, oligonucleotide and siRNA de-livery, transient and stable cell transfection, automation and protein production in animal cells.

Puresyn, Inc. - Develops separation and purification processes for manufacturing gene delivery products such as supercoiled plasmids, synthetic oligonucleotides, recombinant adenovirus and adeno-associated virus.

Qugen Therapeutics Pte Ltd - Focuses on the development of novel gene-based technologies and products providing first proof of principle data in pre-clinical models and then moving these tech-nologies into the clinical setting.

Replicor, Inc. - Development of technologies controlling DNA replication for applications in a plat-form for gene therapy vectors and in the discovery of new therapeutic agents for cancer and tis-

sue regeneration. Retroscreen Ltd. - Virological research and clinical trials company that work on most model sys-

tems and carry out projects from laboratory based assays to full ethics committee approved clini-cal trials.

Salus Therapeutics, Inc. - Developed a series of proprietary technologies for the discovery, devel-opment and delivery of nucleic acid-based therapeutics, including antisense and gene therapy drugs.

Selective Genetics - Aims to commercialize site-specific gene therapeutics and DNA devices for tissue repair and regeneration.

Targeted Genetics Corporation - Develops gene delivery technologies and therapeutic products based on gene delivery.

Transgene S.A. - Gene therapy company based in France is developing products for cancer and cystic fibrosis. Vector platforms include adenovirus, retrovirus, vaccinia virus, cellular and syn-thetic vectors.

UCSD Program in Human Gene Therapy - Facilitates the application of basic and pre-clinical re-search to the treatment of human disease through vector development and gene discovery and characterization.

University of Minnesota Gene Therapy Center - Clinical gene therapy research programs focusing on metabolic disorders.

University of North Carolina Chapel Hill Gene Therapy - Working to translate gene therapy re-search from the laboratory bench into Phase I clinical trials for the treatment of hematologic and endocrine diseases. The center has a GMP facility for clinical grade gene vector production.

Valentis, Inc. - Developing gene-based therapeutics or "gene medicines" for the treatment of cer-tain cancers, neuromuscular, cardiovascular, rheumatological and pulmonary diseases.

Virapur - Sells virus purification kits for gene therapy and virus research which do not require ce-sium gradients.

VirRx, Inc. - A biotechnology company with a primary interest in cancer gene therapy.