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 Administration, 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 Services
Food and Drug Administration
Center for Biologics Evaluation and Research
March 1998
TABLE OF CONTENTS
OVERVIEW (1998)
- INTRODUCTION
- Definitions of Somatic Cell Therapy and Gene Therapy
- Types of Therapies
- Regulatory Considerations
- General Considerations
- DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR ADMINISTRATION
- Collection of Cells
- Cell Culture Procedures
- Cell Banking Systems Procedures: Generation and
Characterization of Master Cell Banks (MCB), Working Cell
Banks (WCB), and Producer Cells
- Materials Used During Manufacturing
- CHARACTERIZATION AND RELEASE TESTING OF CELLULAR GENE THERAPY PRODUCTS
- Cell Identity
- Potency
- Viability
- Sterility
- Purity
- General Safety Test
- Frozen Cell Banks
- ADDITIONAL APPLICATIONS: ADDITION OF RADIOISOTOPES OR TOXINS TO CELL PREPARATIONS
- PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF VECTORS FOR GENE THERAPY
- Vector Construction and Characterization
- Vector Production System
- Master Viral Banks
- Lot-to-Lot Release Testing and Specifications for
Vectors
- ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THERAPY
- Additional Considerations for the Use of Plasmid Vector
Products
- Additional Considerations for the Use of Retroviral
Vector Products
- Additional Considerations for the Use of Adenoviral Vectors
- Other Gene Delivery Systems
- MODIFICATIONS IN VECTOR PREPARATIONS
- PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES
- General Principles
- Animal Species Selection and Use of Alternative Animal
Models
- Somatic Cell and Gene-Modified Cellular Therapies
- Direct Administration of Vectors In Vivo
- 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
manufacturers 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 issues that
the Center for Biologics Evaluation and Research (CBER) staff
believes should be considered 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 prevent
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 guidance refer to the
following documents:
Points to Consider in the Manufacture and Testing of Therapeutic
Products for Human Use Derived 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).
- INTRODUCTION
- 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 proposed. Somatic cell
therapy is the administration to humans of autologous,
allogeneic, or xenogeneic living cells which have been
manipulated or processed ex vivo. Manufacture of products
for somatic cell therapy involves the ex vivo propagation,
expansion, selection (see: "A Proposed Approach to the
Regulation of Cellular and Tissue-based Products", Feb. 28,
1997, (62 FR 9721)), or pharmacologic treatment of cells, or
other alteration of their biological characteristics. Such
cellular products might also be used for diagnostic or
preventive purposes. Manufacturers 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, allogeneic, 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 material 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 administered 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 materials 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.
- 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 document: see below); and
implantation of manipulated cell populations, such as
hepatocytes, myoblasts, or pancreatic islet cells, intended
to perform a complex biological 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 aggregated 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 categorized as excipients,
additional active components, or medical devices.
Because of the complexities of potential interactions with
the cells and other constituents, additional components
should be considered as part of the final biological product
for purposes of preclinical evaluation.
- 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
regulations and policy issued in this area.
IND applications for somatic cell and gene therapies should
follow the same format 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 document. The particular information
required will depend upon the experimental system and the
phase of study. For those therapies for which patient entry
criteria include results of a genetic test, information
should be submitted to the IND documenting and validating
the test method.
Biological products are often complex mixtures that cannot
be completely defined. 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 adventitious agents or other contaminants, or to
inadvertent changes in the properties or stability of the
biological product that may not be detectable in final
product testing. For these reasons, the methods and
reagents involved in the production process should be
defined. Also, cell banks and key intermediates in the
production process should be subject to quality control.
Lot-to-lot reproducibility of both the final product and of
critical materials such as vector-containing supernatants
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 submitted 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 development. 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 potency
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 earlier trials may need to
be repeated (see: "FDA Guidance Concerning Demonstration 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 information 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 redundant submissions as well as retaining
desired confidentiality.
- 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 reviewed. Other Guidance documents should be consulted
as is appropriate, for example:
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 Biologicals (1993), August 12, 1993, (58 FR
42974).
Points to Consider in the Manufacture and Testing of
Monoclonal Antibody Products for Human Use (1997), February
28, 1997, (62 FR 9196).
Points to Consider in the Collection, Processing, and
Testing of Ex-Vivo-Activated 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
inappropriate, or alternative procedures may be more
appropriate. The methods and procedures mentioned are
suggestions: sponsors may propose alternative techniques
which will be acceptable if these issues are adequately
addressed and supported 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 production.
Modifications of procedures will occur with time, and
alternate control procedures will be needed. The principles
included here can serve as guidance for developing these
procedures.
Table of Contents
- DEVELOPMENT AND CHARACTERIZATION OF CELL POPULATIONS FOR ADMINISTRATION
- Collection of Cells
The following information should be provided:
- Cell types: The type(s) of cell to be used should
be classified as autologous, allogeneic, or xenogeneic
in origin. The tissue source and other relevant
identifying information should be provided.
- 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 likelihood 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 labeling. 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 Issues in
Xenotransplantation", August, 1996, 61 FR 49920
and January, 1997, (62 FR 3563).
- Tissue typing: If allogeneic donors are to be
used, typing for polymorphisms such as blood type
should be included when appropriate. The importance
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 provided.
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 inimmune responses or other changes that might
alter the performance 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 Cellular and Tissue-based Products",
February 28, 1997, (62 FR 9721) for regulation 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.
- Procedures: The procedures for the collection of
cells, including the location of the facility, and any
devices or materials used, should be submitted.
- Cell Culture Procedures
- Quality control procedures: In general, cell
culture operations should be carefully managed in
terms of quality of materials, manufacturing controls,
and equipment validation and monitoring. See I, C,
General Considerations.
- 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, including 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
factors, measures of identity, purity, and potency
should be established to assure the reproducibility of
cell culture characteristics. More detailed
discussions 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 Consider 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 serious
hypersensitivity reactions in patients.
- 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
adventitious 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.
- 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 contamination 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 quantitatively, 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. Acceptable 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 include verification of donor-recipient matching
and immunological phenotyping.
- 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.
- Culture longevity: The essential characteristics
of the cultured cell population (phenotypic markers
such as cell surface antigens, functional properties,
activity in bioassays, as appropriate) should be
defined, and the stability of these characteristics
established with respect to time in culture. This
profile should be used to define the limits of the
culture period.
- Cell Banking System Procedures: Generation and Characterization of Master Cell Banks (MCB), Working Cell Banks (WCB), and Producer Cells
Cell banking systems are appropriate for use with some
somatic cell therapy products 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 producing 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 Working 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 follows:
- Origin and history of cells: A description should be provided.
- 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.
- Characterization: The identity of the cells
should be confirmed by appropriate genotypic and/or
phenotypic markers, and the fraction of the cell
population having such identity markers measured as an
indication of purity. 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.
- 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 bacteriophage
should be considered, since it could adversely affect
stability and yield.
- Expiration dating: Product development plans
should include accumulation of data demonstrating how
long and under what conditions the cells can remain
frozen and still be acceptably active when thawed.
- 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 phenotypic 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 performed 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 differently 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.
- 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 program with set
specifications should be established for each component to
determine 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 components, 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.
Table of Contents
- 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 applied 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 population, 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 appropriate 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.
- Cell Identity
Quantitative testing by phenotypic and/or biochemical assays
should be used to confirm cell identity and assess
heterogeneity (21 CFR 610.14).
- Potency
The relevant function of the cells, if known, and/or
relevant products biosynthesized by the cells should be
defined and quantitated as a measure of potency (21.CFR
610.10).
- Viability
The viability of the cells should be quantitated and a lower
limit for acceptability established.
- 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 Consider 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 adequately sensitive testing prior to
administration to patients.
- Purity
Purity (21 CFR 610.13) or validation of endotoxin testing by
LAL or other acceptable 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.
- 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.
- Frozen Cell Banks
When cell populations frozen for subsequent administration
are thawed, expanded, 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.
Table of Contents
- ADDITIONAL APPLICATIONS: ADDITION OF RADIOISOTOPES OR TOXINS TO CELL PREPARATIONS
Therapeutic or diagnostic applications may be proposed involving
cells which are modified 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 implantation 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-conjugated antibodies, and are addressed in
the "Points to Consider in the Manufacture and Testing of
Monoclonal Antibody Products for Human Use" (1997). Although the
application to somatic cell therapies may differ, that document
should be consulted.
Table of Contents
- PRODUCTION, CHARACTERIZATION AND RELEASE TESTING OF VECTORS 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 information 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.
- Vector Construction and Characterization
Vector source materials should be characterized and
documented thoroughly. Viral vectors or plasmids should be
generated from cloned and characterized constructs, 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 regulatory 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 significant
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.
- 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
characterization 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 description 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.
- Master Viral Banks
When a virus, with or without a therapeutic gene, is used as
a seed in the manufacture 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 confirmed 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-defective or
replication-selective vectors, Master Viral Banks should be
demonstrated to be free of replication-competent viruses,
which may arise as a result of contamination or
recombination during the generation of the MVB. Testing for
other inappropriate 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.
- 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
appropriate testing protocols, and consult CBER if there are
questions as to the applicability 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 quantitative, 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.
- Tests of drug substance (bulk product not necessarily in final formulation):
- Purity (21 CFR 610.13)
- Test for total DNA or RNA content if
appropriate to vector composition,
e.g. A260/A280.
- Test for homogeneity of size and
structure, supercoiled vs. linear,
e.g. agarose gel electrophoresis.
- Test for contamination with RNA or
with host DNA, e.g. gel
electrophoresis, including test with
bacterial host-specific probe.
- Test for proteins if present as a
contaminant, e.g. silver stained
gel.
- Test for non-infectious virus in cases
in which that would be a contaminant,
such as empty capsids. See: Section VI
C.
- Tests for toxic materials involved
in production.
- 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.
- Adventitious agents
As methods of testing for adventitious agents
become increasingly sensitive and specific over
time, sponsors are encouraged to accumulate 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 viral 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:
- Sterility test (21 CFR 610.12), for
aerobic and anaerobic bacteria and
fungi.
- Mycoplasma testing, as specified in
the "Points to Consider in the
Characterization of Cell Lines Used
to Produce Biologicals (1993)",
Attachment #2, (58 FR 42974), which
specifies the procedures for
detecting mycoplasma contamination.
- Testing for adventitious viruses, in
some cases, source materials 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
extensively 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.
- Potency (21 CFR 610.10)
Potency assays should be validated during the
product development 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 sensitivity 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 conversion of substrate to
product would be preferred over an immunological
assay detecting epitopes on the enzyme. If no
quantitative potency assay is available, then a
qualitative potency test should be performed.
- 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).
- Sterility (21 CFR 610.12), identity (21 CFR
610.14), and potency (21 CFR 610.10).
- Purity (21 CFR 610.13) or validation of
endotoxin testing by LAL or other acceptable
assay (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).
- 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 notice, Vol. 61, No. 94, May 14, 1996),
even if liposomes are added.
Table of Contents
- ISSUES RELATED TO PARTICULAR CLASSES OF VECTORS FOR GENE THERAPY
- 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 Plasmid 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
contaminants, 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
antibiotics be avoided during production, due to the risk of
serious hypersensitivity reactions in patients. If
antibiotic selection is used during production, it is
preferable not to use selection markers which confer
resistance to antibiotics in significant clinical use, in
order to avoid unnecessary risk of spread of antibiotic
resistance traits to environmental microbes. Also, residual
antibiotic in the final product 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
Research 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 facilitating
agent is added during formulation, a specification for its
amount and identity in the final product should be
established. If toxic organic solvents such as chloroform
are used in producing a lipid component, then processing
should remove them and lot release specifications should
include testing for residual solvent.
- Additional Considerations for the Use of Retroviral Vector Products
- Testing for replication competent retrovirus: The
following testing scheme for detection of replication
competent retroviruses (RCR) summarizes current
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 exposure and the possibility of
generation of recombinant RCR at any point in the
production process. Alternative assays (e.g. marker
rescue) are acceptable if sensitivity is comparable to
the PG4 S+L- assay. Testing should be complete prior
to patient administration, particularly if cells can
be cryopreserved; 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.
- Master Cell Bank of vector-producing cells
(one time testing):
- Supernatant testing, 5% of the total
supernatant from culture of cells for a
master cell bank 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.
- Producer cell testing, 1% of pooled
producer cells or 108 cells,
whichever is fewer, should be
cocultured with a permissive cell
line (e.g., Mus dunni) including
several blind passages. Supernatant
from the coculture should be tested
by PG4 S+L- or alternative assay.
- Working cell bank (one time testing):
Either supernatant testing or cocultivation of
cells is recommended, using conditions described
for master cell bank testing.
- Lot testing of vector products:
- 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.
- 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.
- Lot testing of ex vivo transduced cells:
- 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. Supernatant from the
coculture should be tested by PG4 S+L-
or alternative assay.
- 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.
- Patient monitoring: Patients given retrovirus-related
products should be monitored for RCR exposure.
Please consult CBER for guidance.
- Additional Considerations for the Use of Adenoviral Vectors.
- Measurement of particles vs. infectious units:
Patient doses of adenovirus-based gene therapy vectors
are presently based upon some method of enumerating
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 potential 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 measured. However, since
it is quite possible that some outcomes are a function
of the number of infectious units administered, it is
important for investigators or sponsors to develop in
vitro infectivity assays which are reproducible 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 adenoviral
particles can be calculated from OD260. The presence
of non-adenovirus nucleic acid may yield inaccurate
particle numbers and should be minimized during the
manufacturing and viral purification process.
Electron microscope particle count has also been used
for viral particle enumeration.
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 properties 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.
- Detection of replication-competent adenovirus:
The presence of RCA in clinical lots of adenovirus
vector raises a variety of safety concerns, including
the possibility of adenovirus infection, unintended
vector replication 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 vectors raises additional
considerations and may call for additional or
different testing strategies. Such cases should be
discussed with CBER.
Detection of RCA in final vector product by a cell
culture/cytopathic effect 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 unrelated to the presence of RCA. It should
also be noted that too high an input 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 example 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. However, the
agency recognizes that current production techniques
in combination with proposed dosing schemes may make
this recommendation prohibitively 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 recombination that are occurring.
- Adeno-associated virus: Because of the
association of AAV with adenovirus, testing for AAV is
currently recommended in the Master Cell Bank, the
Master Virus Seed Stock, and the final product.
- 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 encouraged to
contact CBER early in product development, to facilitate a
safe and efficient development process.
Table of Contents
- 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 towards 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
modifications 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 modifications to the
vector. The need for additional preclinical testing is determined
by the likelihood 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 described in the
"Points to Consider in the Manufacture and Testing of Monoclonal
Antibody 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 efficacy 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
initiating 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.
Table of Contents
- PRECLINICAL EVALUATION OF CELLULAR AND GENE THERAPIES
- General Principles
Preclinical studies are intended to define the pharmacologic
and toxicologic effects predictive of the human response,
not only prior to initiation of clinical trials, 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
receiving 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 Section 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 experience, or for which the insertion of
a different expression cassette is not expected 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 representatives from CBER prior to their
initiation. Clinical plans requiring rapid enrollment of
patients should be anticipated and preceded by adequate
preclinical testing.
- Animal Species Selection and Use of Alternative Animal Models
It is recognized that animal models of disease may not be
available for every cellular 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 example,
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
pharmacologic response comparable to that expected in
humans.
- Somatic Cell and Gene-Modified Cellular Therapies
- In vivo biological/pharmacological activity: The
transduction procedure, dose of expanded or
genetically modified cells, and route of
administration planned for the clinical trial should
be evaluated preclinically. Pharmacologic studies in
animals may provide useful information regarding the
in vivo function, survival time and appropriate
trafficking of the modified cells.
- 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 status, serum biochemistry, and
hematologic profiles. Target tissues should be
examined microscopically for histopathological
changes.
- Direct Administration of Vectors In Vivo
A number of different vectors are currently in development
for direct administration to human subjects. Direct
administration of any of these vectors presents a number of
safety concerns that will be addressed here. All toxicity
and localization 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.
- Route of administration: The route of
administration of vectors can influence 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
adenoviral vectors by intranasal administration in
cotton rats or mice is an acceptable alternative to
direct intrapulmonary administration through a
bronchoscope.
- 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 response to
the vector.
- Selection of dose to be employed: The doses of
vectors studied preclinically 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 adequate 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 comparisons 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.
- 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.
- Distribution of vector out of the site of
administration: Localization studies, designed to
determine the distribution of the vector after
administration 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 representing 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 pathologic effects.
- Expression of gene product and induction of immune responses
Expression of the therapeutic gene product in
intended or unintended tissues may result in
unexpected toxicities, which should therefore be
addressed in preclinical studies. Inflammatory,
immune, or autoimmune responses induced by the
gene product may be of concern. Animal studies
should be conducted over a sufficient 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.
- 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
samples should be analyzed for vector sequences
by the most sensitive method possible. If a
signal is detected in the gonads, further
studies 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.
- 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.
Table of Contents
- 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 issues 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.
Table of Contents
1
This guidance document represents the Agency's current thinking on
the development and regulation of somatic cell therapy products. It
does not create or confer any rights for or on any person 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 Assistance, HFM-40, Center for Biologics Evaluation
and Research, Food and Drug Administration, 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 calling 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 internet may obtain the document at "http://www.fda.gov/cber/guidelines.htm".
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