Guidance for Industry 
 
Considerations for Plasmid DNA 
Vaccines for Infectious Disease 
Indications 
 
 
 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Training and 
Manufacturers Assistance (HFM-40), 1401 Rockville Pike, Suite 200N, Rockville, MD 20852-
1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 
http://www.fda.gov/cber/guidelines.htm. 
 
For questions on the content of this guidance, contact the Office of Vaccines Research and 
Review at (301) 827-0655. 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
November 2007
  Contains Nonbinding Recommendations 
 
 
Table of Contents 
 
I.  INTRODUCTION........................................................................................................................1 
II.  BACKGROUND..........................................................................................................................2 
III.  MANUFACTURING ISSUES....................................................................................................2 
A.  Product Manufacture......................................................................................................2 
B.  Bulk Plasmid Product Release Testing..........................................................................3 
C.  Final Product Release Testing........................................................................................4 
IV.  DNA VACCINE MODIFICATIONS.........................................................................................4 
A.  Changes to the Insert or Vector......................................................................................4 
B.  DNA Sequence Analysis..................................................................................................5 
V.  PRECLINICAL IMMUNOGENICITY AND SAFETY..........................................................5 
A.  General Considerations...................................................................................................5 
B.  Immunogenicity................................................................................................................5 
C.  Cytokines..........................................................................................................................6 
D.  Prime/Boost Strategies.....................................................................................................6 
E.  Autoimmunity..................................................................................................................6 
F.  Local Reactogenicity and Systemic Toxicity Studies....................................................7 
G.  Biodistribution, Persistence, and Integration Analysis................................................7 
VI.  REFERENCES:  REGULATIONS AND APPLICABLE GUIDANCE 
DOCUMENTS, AND RELEVANT PUBLICATIONS............................................................9 
i  Contains Nonbinding Recommendations 
 
 
Guidance for Industry 
 
Considerations for Plasmid DNA Vaccines 
for Infectious Disease Indications 
 
This guidance represents the Food and Drug Administrations (FDAs) current thinking on this 
topic.  It does not create or confer any rights for or on any person and does not operate to bind 
FDA or the public.  You can use an alternative approach if the approach satisfies the 
requirements of the applicable statutes and regulations.  If you want to discuss an alternative 
approach, contact the appropriate FDA staff.  If you cannot identify the appropriate FDA staff, 
call the appropriate number listed on the title page of this guidance. 
 
 
I.  INTRODUCTION 
 
This guidance describes our current recommendations concerning preclinical development and 
testing of DNA vaccines to prevent infectious diseases. This guidance supercedes the 1996 
Points to Consider document.  In addition, this guidance finalizes the draft guidance of the same 
title dated February 2005. 
 
For the purposes of this guidance, DNA vaccines are defined as purified plasmid preparations 
containing one or more DNA sequences capable of inducing and/or promoting an immune 
response against a pathogen.  Typically, these plasmids possess DNA sequences necessary for 
selection and replication in bacteria.  In addition, they contain eukaryotic promoters and 
enhancers as well as transcription termination/-polyadenylation sequences to promote gene 
expression in vaccine recipients, and may contain immunomodulatory elements.  DNA vaccines 
are biological products as set forth in section 351 of the Public Health Service Act (PHS ACT) 
(42 U.S.C. 262) and are regulated by FDAs Center for Biologics Evaluation and Research 
(CBER).  The principal regulations applicable to DNA vaccines are located in Title 21 Code of 
Federal Regulations (CFR) Parts 210, 211, 312, 600, 601, and 610.  Other guidance documents 
are available from CBER and may contain information that is relevant to DNA vaccines.  Some 
of these documents are listed below and additional guidance documents may be found on the 
CBER website (http://www.fda.gov/cber/guidelines.htm) or on the website of FDAs Center for 
Drugs Evaluation and Research (http://www.fda.gov/cder/guidance/index.htm). 
 
Investigational New Drug Applications (INDs) for DNA vaccines designed to prevent or treat 
infectious diseases should be submitted to CBERs Office of Vaccines Research and Review 
(OVRR) where primary review responsibility is assigned.  Plasmid DNA products intended for 
non-infectious therapeutic indications are not addressed in this guidance.  INDs for these 
products should be submitted to CBERs Office of Cellular, Tissue and Gene Therapies 
(OCTGT). 
 
1  Contains Nonbinding Recommendations 
 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities. Instead, guidances describe FDAs current thinking on a topic and should be 
viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  
The use of the word should in FDAs guidances means that something is suggested or 
recommended, but not required. 
 
 
II.  BACKGROUND 
 
In December 1996, FDA issued a guidance document, Points to Consider on Plasmid DNA 
Vaccines for Preventive Infectious Disease Indications, (1996 Points to Consider document) to 
assist the developers of DNA vaccines.  That document delineated the manufacturing, 
preclinical, and clinical issues relevant to the development of DNA vaccines, and described 
potential safety concerns that we, CBER, recommended vaccine developers address prior to the 
initiation of phase 1 clinical studies.  The recommendations involving DNA vaccine manufacture 
and testing provided in that document were based on our experiences with other types of 
vaccines and DNA-based products, including gene therapy agents. 
 
In the intervening years, we have permitted the initiation of phase 1 clinical studies of DNA 
vaccines for a number of infectious disease indications including malaria, hepatitis B, and human 
immunodeficiency virus (HIV).  The initiation of phase 1 clinical studies is predicated on the 
manufacturers and/or sponsors of vaccine clinical studies documenting the quality and 
consistency of plasmid manufacture, combined with extensive preclinical safety studies.  
Considerable preclinical and clinical experience on plasmid DNA vaccines has been 
accumulated since the issuance of the 1996 Points to Consider document.  This experience has 
been taken into consideration in revising our recommendations concerning preclinical testing of 
DNA vaccines. 
 
 
III.  MANUFACTURING ISSUES 
 
The following sections describe the manufacturing information that should be provided for new 
DNA vaccine products for clinical study under an IND (21 CFR Part 312). 
 
A.  Product Manufacture 
 
You should describe in the manufacturing summary all components used during 
manufacture as well as those present in the final product.  You should provide detailed 
descriptions of the plasmid construction, including the source and diagrams of all 
plasmids used, and all intermediate recombinant DNA cloning procedures.  You should 
provide the DNA sequence of the entire plasmid present in the Master Cell Bank (MCB) 
along with an annotated sequence identifying all open reading frames including any 
unexpected open reading frames and/or other sequence elements.  During intermediate  
2  Contains Nonbinding Recommendations 
 
 
steps in the production process, various methods can be used for identity testing, 
including restriction enzyme mapping and polymerase chain reaction (PCR).  However, 
complete sequencing of the bulk plasmid vaccine is prefered. 
 
You should describe the genotype, source of the bacterial cells, and the procedures to 
construct master and working cell banks used for production.  Specific guidance for the 
establishment of MCBs and Working Cell Banks (WCBs) is described in CBERs Points 
to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993).  
You should test both the MCBs and WCBs to ensure that they are free from 
bacteriophage and other adventitious agent contamination.  We recommend that you 
establish the genetic stability of the plasmid DNA in the WCB. 
 
The description of the manufacturing process should be sufficiently detailed to enable an 
assessment of the safety of the product.  If lots produced for preclinical safety studies are 
manufactured differently from lots proposed for clinical use, you should clearly 
document these manufacturing changes. 
 
B.  Bulk Plasmid Product Release Testing 
 
If the bulk and final product are the same (i.e., if production runs yield one lot and no 
further steps in formulation are performed), then testing as described below may be 
redundant and unnecessary.  You should test bulk plasmid products for the properties 
described below, and you should use standard assay(s) of adequate specificity and 
sensitivity.  It is understood that in the early stages of product development, these assays 
may utilize a research grade internal standard.  You should evaluate assay methods by 
testing known amounts of reference materials or spiked samples, or by other appropriate 
measures, and submit to CBER data documenting assay performance.  In addition to bulk 
and final product release testing, we recommend that you also perform in-process testing 
to ensure manufacturing consistency and product safety.  Prior to the initiation of phase 1 
clinical studies, you should initiate stability testing as early as possible to support use of 
the product for the duration of the proposed clinical investigation. 
 
Typically, the bulk release criteria will include tests for visual appearance and plasmid 
concentration. We recommend that the fraction of plasmid in supercoiled conformation 
be included in the bulk release criteria, and that you establish a minimum specification 
for supercoiled plasmid content (preferably >80%).  You may select different release 
criteria and specifications if they provide data to establish that the selected criteria are 
predictive of immunogenicity or other intended biological activities. 
 
You should evaluate bulk plasmid preparations for the presence of bacterial host cell 
macromolecules including DNA, RNA, and protein and set preliminary limits for the 
maximum level of each of these macromolecules (preferably <1%).  As product 
development progresses, the level of host cell material should be further reduced, as 
technically and logistically feasible.  You should perform a test for pyrogenic substances 
and include the test results with the final lot release documentation.  The Limulus 
3  Contains Nonbinding Recommendations 
 
 
Amebocyte Lysate (LAL) test is a sensitive indicator of the presence of bacterial 
endotoxins and endotoxin contamination.  The amount of endotoxin in plasmid DNA 
vaccine preparations should not exceed 40 EU/mg plasmid. 
 
You should include a test to establish the identity of the bulk product.  For example, 
agarose gel electrophoresis of plasmid DNA after restriction enzyme digestion is one test 
that can be used to identify and distinguish individual plasmids.  When a single 
manufacturing facility is used to manufacture more than one DNA vaccine product, the 
identity test(s) should be capable of uniquely identifying each plasmid produced in the 
facility. 
 
You should develop a potency assay.  During early clinical development, sponsors will 
have considerable flexibility in the selection of potency assays.  This flexibility could 
include in vitro measures of transfection efficiency that monitor the transcription and/or 
translation of the encoded gene(s) or in vivo assays of DNA vaccine immunogenicity.  
We recommend that quantitative potency assays that evaluate relevant biological activity 
be developed as product development proceeds.  Whenever possible, evidence that the 
selected potency assay correlates with the immunogenicity or protective activity observed 
in clinical trials should be provided.  We recommend that you maintain retention samples 
of each lot to facilitate comparisons between lots as assay development progresses.  You 
may choose to discuss the selection and implementation of a potency assay with CBER to 
ensure acceptability of the design. 
 
C.  Final Product Release Testing  
 
You should test the final DNA vaccine product for potency, general safety, sterility, 
purity, quantity, and identity.  The test methods and specifications may be the same as 
those employed for the bulk product release.  If the plasmid product is lyophilized we 
recommend that you perform a test for residual moisture.  You should perform a test for 
the presence of endotoxin on each lot of final product.  In addition to final product release 
testing, you should also perform in-process testing to ensure manufacturing consistency 
and product safety. 
 
You should establish acceptance criteria and acceptable limits and report the results for 
each lot of vaccine to be used for clinical studies. 
 
 
 IV.  DNA VACCINE MODIFICATIONS 
 
A.  Changes to the Insert or Vector 
 
If the DNA sequence of the insert gene and/or backbone vector of a DNA vaccine are 
changed, we recommend that you consult with CBER to discuss whether the nature 
and/or magnitude of the change(s) warrant the conduct of additional preclinical studies 
and/or the submission of a new IND.  You should provide to CBER a description of the 
4  Contains Nonbinding Recommendations 
 
 
changes in manufacturing process and the results from preclinical safety evaluations of 
the new (modified) DNA vaccine. 
 
B.  DNA Sequence Analysis 
 
An issue of product identity of particular relevance to DNA vaccines concerns the degree 
to which plasmids should be sequenced before the initiation of phase 1 clinical studies.  
In 1996, we recommended that manufacturers provide (at a minimum) the sequence of 
the protein-encoding gene insert.  Based on evidence that the plasmid backbone may 
influence vaccine activity, and recognizing that technological advances since 1996 have 
facilitated DNA sequencing, we recommend that manufacturers provide the complete 
sequence of the plasmid before initiating phase 1 clinical studies.  As noted in Section 
III.A. of this document, the sequence should be fully annotated, and should identify any 
unexpected open reading frames and/or other sequence elements.  Some DNA vaccines 
contain a complex mixture of plasmids, with each plasmid carrying a gene encoding a 
different antigenic protein.  You should grow each plasmid separately.  We advise that 
the identity and amount of each plasmid component in the vaccine preparation be 
determined to ensure lot to lot consistency. 
 
 
V.  PRECLINICAL IMMUNOGENICITY AND SAFETY 
 
A.  General Considerations 
 
Preclinical safety evaluation is required for all new vaccines, including DNA vaccines, 
prior to their use in clinical studies (21 CFR 312.23(a)(8)).  The Good Laboratory 
Practice (GLP) regulations (21 CFR Part 58) apply to the conduct of non-clinical 
laboratory safety studies that support or are intended to support applications such as INDs 
and biological license applications.  We recommend that you perform preclinical safety 
studies on every novel DNA vaccine or DNA vaccine/adjuvant combination.  We 
recommend that all preclinical toxicity and biodistribution/persistence studies evaluate 
the formulation and method of administration proposed for the clinical study.  Additional 
safety evaluations may be necessary when changes are made, for example, to the 
formulation or route of administration.  We recommend that you consult with CBER 
prior to submission of your IND to discuss the adequacy of your preclinical safety studies 
and prior human experience to support proceeding with the investigational vaccine 
product to Phase I clinical trials. 
 
B.  Immunogenicity 
 
We recommend that vaccine immunogenicity be assessed in a relevant animal model 
whenever possible.  This may include the evaluation of antigen-specific antibody titers, 
seroconversion rates, activation of cytokine secreting cells, and/or measures of cell-
mediated immune responses.  Unless they are also safety studies, pre-clinical 
immunogencity studies are not subject to the GLP regulations, which do not apply to 
5  Contains Nonbinding Recommendations 
 
 
basic exploratory studies carried out to determine whether a test article has potential 
utility (21 CFR 58.3(d)).  For DNA vaccines that encode multiple antigens, you should 
assess the immune response generated against a representative subset of the encoded 
antigens. 
 
C.  Cytokines 
 
For DNA vaccines that contain immunomodulatory genes (such as cytokine-encoding 
genes), we encourage preclinical studies in animal species responsive to the encoded 
human cytokine(s) or models using homologous animal gene(s).  Such studies should 
assess whether modulation of cellular or humoral components of the immune system 
might result in unintended adverse consequences, such as generalized 
immunosuppression, chronic inflammation, autoimmunity or other immunopathology.  
We recommend that you consult with CBER concerning the availability and suitability of 
animal models to conduct such testing. 
 
D.  Prime/Boost Strategies 
 
When more than one type of vaccine is used in a sequential immunization protocol, we 
recommend that you submit information supporting the safety and tolerability of the 
dose, schedule, and route of administration of each component proposed for use in the 
heterologous prime-boost regimen.  If existing data are deemed adequate to characterize 
the potential risks of the prime-boost regimen to study participants, additional toxicology 
studies may not be necessary.  We recommend that sponsors consult with CBER for 
recommendations on the need for additional toxicology information to support the 
clinical plans. 
 
E.  Autoimmunity 
 
Published preclinical studies indicate that DNA vaccination can activate autoreactive B 
cells to secrete IgG anti-DNA autoantibodies.  However, the magnitude and duration of 
this response appears to be insufficient to cause disease in normal animals or accelerate 
disease in autoimmune-prone mice.  These preclinical studies suggest that systemic 
autoimmunity is unlikely to result from DNA vaccination.  Similarly, the absence of an 
immune response against cells expressing the vaccine-encoded antigen (including muscle 
cells and dendritic cells) suggests that an autoimmune response directed against tissues in 
which such cells reside is unlikely.  Yet the possibility persists that DNA vaccines might 
idiosyncratically cause or worsen organ-specific autoimmunity by encoding antigens 
(including cryptic antigens) that cross-react with self.  Thus, we no longer recommend 
that preclinical studies be performed to specifically assess whether vaccination causes 
autoimmune disease, but recommend that the general welfare of animals in preclinical 
immunogenicity and toxicity studies continue to be carefully monitored. 
 
In cases where an immune response is induced by a transgene product encoding self-
antigen (such as a cytokine, chemokine, surface receptor/ligand, or cryptic self-antigen), 
6  Contains Nonbinding Recommendations 
 
 
we recommend that you examine potential cross-reactivity with the corresponding 
endogenous protein.  If a persistent immune response against an endogenous protein is 
detected, we recommend that you evaluate potential adverse effects by studying the 
analogous animal gene in a relevant animal model.  We further recommend that you 
monitor whether an immune response against the self-antigen is elicited during the 
clinical trial, and carefully evaluate the effect of this response on trial participants. 
 
F.  Local Reactogenicity and Systemic Toxicity Studies 
 
Studies designed to assess systemic toxicity and local site reactogenicity may be 
combined.  We recommend that these studies include at least one immunization beyond 
that planned for clinical use.  We recommend that you use the highest dose of vaccine 
planned for clinical use.  An accelerated schedule of vaccine delivery will be considered 
by FDA.  We recommend that the assessments written into the preclinical study protocols 
include toxicity to potential target organs, including the hematopoietic and immune 
systems.  We recommend that preclinical studies also include clinical pathology 
assessments (serum chemistry, hematology, and coagulation tests), and histopathology, 
encompassing both gross and microscopic assessment of tissues.  For additional 
guidance, sponsors are referred to the WHO Guidelines on Non-clinical Evaluation of 
Vaccines for recommendations concerning the choice of animal model and study design 
for evaluating local reactogenicity and systemic toxicity 
(www.who.int/entity/biologicals/publications/en/). 
 
For studies of injection site reactogenicity, you should include detailed clinical 
observations of the injection site(s) following each vaccine administration and 
histological evaluations of injection-site tissue obtained from biopsies or term necropsy 
samples.  You should evaluate both short-term and persistent toxicity, preferably by 
studying separate cohorts of animals 2 to 3 days and 2 to 3 weeks after the final 
vaccination. 
 
G.  Biodistribution, Persistence, and Integration Analysis 
 
Plasmid biodistribution, persistence and integration studies were initially recommended 
to examine whether subjects in DNA vaccine trials were at heightened risk from the long-
term expression of the encoded antigen, either at the site of injection or an ectopic site, 
and/or plasmid integration.  Theoretical concerns regarding DNA integration include the 
risk of tumorigenisis if insertion reduces the activity of a tumor suppressor or increases 
the activity of an oncogene.  In addition, DNA integration may result in chromosomal 
instability through the induction of chromosomal breaks or rearrangements. 
 
A typical biodistribution/persistence study assesses the presence of plasmid collected 
from a panel of tissues at multiple time points ranging from a few days to several months 
post administration.  The panel of tissues typically includes the blood, heart, brain, liver, 
kidney, bone marrow, ovaries/testes, lung, draining lymph nodes, spleen, muscle at the 
site of administration and subcutis at the injection site.  Plasmid levels are typically 
7  Contains Nonbinding Recommendations 
 
 
evaluated using a quantitative real time polymerase chain reaction assay (Q-PCR) 
validated for sensitivity, specificity and the absence of inhibitors.  We recommend that 
the sensitivity of this assay be sufficient to quantify <100 copies of plasmid per 
microgram of host DNA.  A claim of non-persistence requires that the amount of 
plasmid at each site falls below this limit of quantification. 
 
Studies examining plasmid biodistribution/persistence indicate that DNA vaccines 
prepared from a common plasmid vector but encoding different antigens behave 
similarly.  Conventional intramuscular, subcutaneous, intradermal, and particle-mediated 
delivery of DNA plasmids rarely results in the long-term persistence of vector DNA at 
ectopic sites.  However, tissue at or near the site of administration frequently contains 
thousands of copies of plasmid per microgram of host DNA for periods exceeding 60 
days.  Studies assessing the nature of this DNA indicates that the vast majority is not 
integrated. 
 
Based on these findings, biodistribution studies may be waived for DNA vaccines 
produced by inserting a novel gene into a plasmid vector previously documented to have 
an acceptable biodistribution/integration profile.  Biodistribution studies will still be 
necessary for DNA vaccines utilizing novel vectors, formulations, methods of delivery, 
routes of administration, or any other modifications expected to significantly impact 
cellular uptake and/or biodistribution.  Before conducting biodistribution/persistence 
studies, we recommend that you contact FDA for advice concerning the need for these 
studies. 
   
Based on published studies analyzing the frequency with which DNA plasmids persist 
and integrate, FDA believes that integration studies are warranted only when plasmid 
persists in any tissue of any animal at levels exceeding 30,000 copies per ug of host DNA 
by study termination.  If the persistence of  DNA plasmid exceeds this threshold, 
sponsors should evaluate whether the DNA has integrated into the genome of the 
vaccinated animals.  A typical integration study will assess all tissue(s) containing 
persisting DNA plasmid.  We recommend that at least four independent DNA samples be 
analyzed.  Each sample may include DNA pooled from several different donors.  Q-PCR 
is generally used to detect and quantify the amount of plasmid DNA present in each 
genomic DNA preparation.  Unintegrated plasmid DNA may be separated from high 
molecular weight genomic DNA by gel purification.  Concatamer may be eliminated by 
restriction endonuclease digestion targeting a rare motif present in the DNA plasmid.  
Specifically designed PCR primers may be used to confirm integration and identify 
genomic integration sites. 
 
 
8  Contains Nonbinding Recommendations 
 
 
VI.  REFERENCES:  REGULATIONS AND APPLICABLE GUIDANCE 
DOCUMENTS, AND RELEVANT PUBLICATIONS 
 
  U.S. CODE OF FEDERAL REGULATIONS 
 
21 CFR PART 50    Protection of Human Subjects 
21 CFR PART 56    Institutional Review Boards 
21 CFR PART 58   Good Laboratory Practice for Nonclinical Laboratory Studies 
21 CFR PART 210   Current Good Manufacturing Practice in Manufacturing, Processing, 
  Packing, or Holding of Drugs; General 
21 CFR PART 211   Current Good Manufacturing Practice for Finished Pharmaceuticals 
21 CFR PART 312   Investigational New Drug Application 
21 CFR PART 600   Biological Products: General 
21 CFR PART 601   Licensing 
21 CFR PART 610   General Biological Products Standards 
 
  POINTS TO CONSIDER DOCUMENTS   
 
Points to Consider in the Production and Testing of New Drugs and Biologicals Produced by 
Recombinant DNA Technology (4/85). 
 
Supplement to the Points to Consider in the Production and Testing of New Drugs and 
Biologicals Produced by Recombinant DNA Technology: Nucleic Acid Characterization and 
Genetic Stability (4/92). 
 
Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (7/93). 
 
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human 
Use (2/97). 
 
  INTERNATIONAL CONFERENCE ON HARMONIZATION OF 
TECHNICAL REQUIREMENTS FOR REGISTRATION OF 
PHARMACEUTICALS FOR HUMAN USE (ICH) DOCUMENTS 
 
ICH; Guideline for Industry:  Detection of Toxicity to Reproduction for Medicinal Products 
(9/94). 
 
ICH; Guideline for Industry:  Detection of Toxicity to Reproduction for Medicinal Products:  
Addendum on Toxicity to Male Fertility (4/96). 
 
ICH; Quality of Biotechnological Products:  Stability Testing of Biotechnological/Biological 
Products (2/04). 
 
9  Contains Nonbinding Recommendations 
 
 
  FDA GUIDANCE DOCUMENTS 
 
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 (12/87). 
 
Guideline for the Determination of Test Residual Moisture in Dried Biological Products (1/90). 
 
FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, 
Including Therapeutic Biotechnology-Derived Products (4/96). 
  
Guidance for Industry:  Guidance for Human Somatic Cell Therapy and Gene Therapy (3/98). 
 
Guidance for Industry:  Content and Format of Chemistry, Manufacturing and Controls 
Information and Establishment Description Information for a Vaccine or Related Product (1/99). 
 
Guidance for Industry:  Sterile Drug Products Produced by Aseptic Processing - Current Good 
Manufacturing Practice (9/04). 
 
Guidance for industry:  Considerations for Developmental Toxicity Studies for Preventive and 
Therapeutic Vaccines for Infectious Disease Indications (2/06). 
 
Draft Guidance for Industry:  INDs  Approaches to Complying with CGMP During Phase 1 
(1/06) (This draft guidance when finalized will represent FDAs current thinking on this topic). 
 
  PUBLICATIONS RELEVANT TO THE ISSUE OF PLASMID DNA 
BIODISTRIBUTION, PERSISTENCE, AND INTEGRATION ANALYSIS: 
 
Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P, Lew D. Plasmid 
DNA malaria vaccine:  the potential for genomic integration after intramuscular injection.  Hum 
Gene Ther.  1999; 10(5):759-68.
 
Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Schock 
HB, Zhang H, Faris JE, Way PA, Beare CM, Bagdon WJ, Nichols WW.  Plasmid DNA 
vaccines:  assay for integration into host genomic DNA.  Dev Biol (Basel). 2000; 104: 33-43. 
 
Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Beare 
CM, Bagdon WJ, Nichols WW.  Plasmid DNA vaccines:  investigation of integration into host 
cellular DNA following intramuscular injection in mice.  Intervirology. 2000; 43(4-6): 258-72. 
 
Parker SE, Monteith D, Horton H, Hof R, Hernandez P, Vilalta A, Hartikka J, Hobart P, Bentley 
CE, Chang A, Hedstrom R, Rogers WO, Kumar S, Hoffman SL, Norman JA.  Safety of a GM-
CSF adjuvant-plasmid DNA malaria vaccine.  Gene Ther 2001; 8:1011-1023. 
 
10  Contains Nonbinding Recommendations 
 
 
Pilling AM, Harman RM, Jones SA, McCormack NA, Lavender D, Haworth R.  The assessment 
of local tolerance, acute toxicity, and DNA biodistribution following particle-mediated delivery 
of a DNA vaccine to minipigs.  Toxicol Pathol. 2002 May-Jun; 30(3): 298-305. 
 
Kim BM, Lee DS, Choi JH, Kim CY, Son M, Suh YS, Baek KH, Park KS, Sung YC, Kim WB.  
In vivo kinetics and biodistribution of a HIV-1 DNA vaccine after administration in mice.  Arch 
Pharm Res. 2003 Jun; 26(6): 493-8. 
 
Bureau MF, Naimi S, Torero Ibad R, Seguin J, Georger C, Arnould E, Maton L, Blanche F, 
Delaere P, Scherman D.  Intramuscular plasmid DNA electrotransfer: biodistribution and 
degradation.  Biochim Biophys Acta.  2004 Jan 20; 1676(2): 138-48.  
 
Wang Z, Troilo PJ, Wang X, Griffiths TG II, Pacchione SJ, Barnum AB, Harper LB, Pauley CJ, 
Niu Z, Denisova L, Follmer TT, Rizzuto G, Ciliberto G, Fattori E, Monica NL, Manam S, 
Ledwith BJ.  Detection of integration of plasmid DNA into host genomic DNA following 
intramuscular injection and electroporation.  Gene Ther.  2004 Apr; 11(8): 711-21.  
 
Sheets RL, Stein J, Manetz TS, Duffy C, Nason M, Andrews C, Kong WP, Nabel GJ Gomez PL. 
Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory 
Syndrome, or West Nile Virus is similar, without integration, despite differing plasmid 
backbones or gene inserts.  Toxicol. Sci.  2006; 91(2):610-19. 
 
Sheets RL, Stein J., Manetz TS, Andrews C, Bailer R, Rathmann J, Gomez PL.  Toxicological 
safety evaluation of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory 
Syndrome, or West Nile Virus is similar despite differeing plasmid backbones or gene-inserts. 
Toxicol. Sci.  2006; 91(2):620-30. 
 
  PUBLICATIONS RELEVANT TO THE ISSUE OF PLASMID DNA 
MODIFICATIONS:  
 
Krieg AM, Wu T, Weeratna R, Efler SM, Love-Homan L, Yan L, Yi AK, Short D, Davis HL.  
Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs.  Proc 
Natl Acad Sci USA.  1998; 95(21): 12631-6. 
 
Krieg AM, Davis HL.  Enhancing vaccines with immune stimulatory CpG DNA.  Curr Opin Mol 
Ther.  2001; 3(1):15-24. 
 
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