Guidance for Industry 

Gene Therapy Clinical Trials – 

Observing Subjects for Delayed 

Adverse Events 

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 Cellular, Tissues, and Gene 

Therapies at 301-827-5102. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

November 2006 
Contains Nonbinding Recommendations 

Table of Contents

I. INTRODUCTION............................................................................................................. 1

II. BACKGROUND ............................................................................................................... 2

A. Potential Risks of Delayed Adverse Events Following Exposure to Gene 

Transfer Technology............................................................................................. 2

B. Previous FDA Recommendations........................................................................ 3

C. Concerns Raised by the Gene Therapy Community ......................................... 3

III. DEFINITIONS AND ABBREVIATIONS...................................................................... 4

IV. PRECLINICAL DATA USED FOR ASSESSMENT OF DELAYED RISKS IN 

GENE THERAPY CLINICAL TRIALS........................................................................ 6

A. Criteria to Assess Potential Delayed Risks of Gene Therapy ........................... 6

B. Considerations for Preclinical Study Design to Assess Vector Biodistribution 

and Persistence.................................................................................................... 10

1. Animal Study Design.................................................................................... 10 

2. Tissue Collection and Analysis..................................................................... 11 

3. Other Considerations .................................................................................... 11 

C. Vector Integration Potential and Reactivation as Risks for Delayed Adverse 

Events................................................................................................................... 11

V. RECOMMENDATIONS FOR PROTOCOLS FOR LONG-TERM FOLLOW-UP 

OBSERVATIONS: CLINICAL CONSIDERATIONS.............................................. 14

A. Decision to Conduct Long-term Follow-up Observations............................... 14

B. Suitability of Clinical Trial Populations for Long-term Follow-up 

Observations........................................................................................................ 15

C. Recommended Duration of Follow-up Observations...................................... 15

D. Elements of Follow-up Observations ................................................................ 16

E. Informed Consent in Trials Involving Long-term Follow-up Observations. 19

F. Special Considerations Regarding Integrating Vectors.................................. 19

1. Data Collection ............................................................................................. 19 

2. Data Reporting.............................................................................................. 21 

3. Informed Consent in Trials Involving Retroviral Vectors............................ 21 

VI. REFERENCES................................................................................................................ 23

i 
Contains Nonbinding Recommendations 

Guidance for Industry 

Gene Therapy Clinical Trials - Observing Subjects for Delayed 

Adverse Events 

This guidance represents the Food and Drug Administration’s (FDA’s) 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 provides to you, sponsors of gene therapy studies, recommendations regarding the 

design of studies to include the collection of data on delayed adverse events in subjects who have 

been exposed to investigational gene therapy products. We, FDA, are providing: (1) 

recommended methods to assess the risk of gene therapy-related delayed adverse events 

following exposure to investigational gene therapy products, (2) recommended methods to 

determine the likelihood that long-term follow-up observations on study subjects will provide 

scientifically meaningful information, and (3) specific advice regarding the duration and design 

of long-term follow-up observations.1 When a gene therapy clinical trial presents long-term 

risks to human subjects, a gene therapy clinical trial must provide for long-term follow-up 

observations in order to mitigate those risks. Without such long-term follow-up observations, 

 

1

 This guidance does not cover the following topics: 

• Inadvertent germline gene transfer. (The term “germline” is used to designate genetic material destined to be 

transferred to gametes). For a discussion of risks associated with inadvertent germline gene transfer for gene 

therapy products, we refer you to the following meeting transcripts: 

- December 15-16, 1997, Recombinant DNA Advisory Committee (RAC) meeting

(http://www4.od.nih.gov/oba/rac/minutes/12151697.htm), - March 11-12, 1999, RAC meeting (http://www4.od.nih.gov/oba/rac/minutes/3-99RAC.htm), and 

- November 16-17, 2000, Biological Response Modifiers Advisory Committee (BRMAC) meeting 

(http://www.fda.gov/cber/advisory/ctgt/ctgtmain.htm. November 17, 2000, 3664t2_b.pdf). 

• Vaccines used to prevent infectious diseases even if you use products analogous to those used for gene therapy 

(consult the Office of Vaccines Research and Review, Center for Biologics Evaluation and Research (CBER)). 

• Post-marketing or licensure requirements for performing long-term follow-up studies of subjects. The specific 

information needed for a licensure or post-marketing study will vary, and therefore, will be addressed with 

individual sponsors. 

• Replication-competent non-transgene-containing viruses used as agents to mediate oncolysis. Due to the 

diversity of the viral agents employed, we recommend that you discuss with the Office of Cellular, Tissues, and 

Gene Therapies (OCTGT, CBER) the potential for risks of delayed adverse events. 

• Risks due to shedding of vector to close contacts, the public, or the environment. The specifics of how and 

whether to address these risks in your clinical trial design should be discussed with OCTGT, CBER. For 

general information, see “Guidance for Industry: Environmental Assessment of Human Drug and Biologics 

Applications, Revision 1” dated July 1998 (http://www.fda.gov/cber/gdlns/environ.pdf).

1 
Contains Nonbinding Recommendations 

the study would expose the subjects to an unreasonable and significant risk of illness or injury 

(21 Code of Federal Regulations (CFR) 312.42(b)(1)(i) and (b)(2)(i)). 

Exposure to gene transfer technology means any exposure to gene therapy products or to cells or 

tissue that has been transduced with gene therapy products ex vivo by any route of 

administration. Except as noted below, this guidance applies to all subjects in clinical studies 

using gene transfer technology. The recommendations in this guidance are limited to the 

performance of long-term observations for evidence of delayed adverse events, i.e., adverse 

events that occur more than one year after exposure to the investigational gene therapy product. 

This guidance finalizes the draft guidance entitled “Guidance for Industry: Gene Therapy 

Clinical Trials – Observing Participants for Delayed Adverse Events” dated August 2005. This 

guidance also supplements the recommendations for study subject long-term follow-up in the 

“Guidance for Industry: Supplemental Guidance on Testing for Replication Competent 

Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients 

in Clinical Trials Using Retroviral Vectors” (Retroviral Vector guidance), dated November 2006 

(Ref. 1). 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s 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 FDA’s guidances means that something is suggested or 

recommended, but not required. 

II. BACKGROUND 

A. Potential Risks of Delayed Adverse Events Following Exposure to Gene 

Transfer Technology 

Study subjects exposed to gene transfer technology may be at risk of delayed adverse 

events as a consequence of persistent biological activity of the genetic material or other 

components of the products used to carry the genetic material. The persistent biological 

activity may be necessary for the product to provide a continuing clinical benefit. 

However, persistent biological activity could have adverse effects upon normal cell 

function, placing subjects at risk for development of adverse events, some of which may 

be delayed by months or years. 

Factors likely to increase the risk of delayed adverse events following exposure to gene 

transfer technology include persistence of the viral vector, integration of genetic material 

into the host genome, prolonged expression of the transgene, and altered expression of 

the host’s genes. Persistence of the viral vector, sometimes associated with latency, 

could permit continued expression of the gene or delayed effects of viral infection. 

Integration of genetic material from a viral vector into the host cell genomic DNA raises 

the risk of malignant transformation (see Section V.F for a discussion of risks of 

malignancy associated with retroviral vectors). Prolonged expression of the transgene 

2 
Contains Nonbinding Recommendations 

may also be associated with long-term risks resulting from unregulated cell growth and 

malignant transformation, autoimmune-like reaction to self antigens, and unpredictable 

adverse events. Altered expression of the host genes could also result in unpredictable 

and undesirable biologic events. 

B. Previous FDA Recommendations 

We previously issued a guidance related to retroviral vector-mediated gene therapy (Ref. 

1). We considered retroviruses to carry the highest known risk because of a reported case 

of new malignancy associated with a preclinical gene therapy study following exposure 

to cells transduced by a retroviral vector (Ref. 2), and therefore included in that guidance 

specific recommendations on performing long-term observations of subjects in trials of 

retroviral-mediated gene therapies. 

We then sought additional information regarding gene-therapy related delayed adverse 

events following exposure to other gene-therapy products. We convened three separate 

meetings of our Biological Response Modifiers Advisory Committee (BRMAC) to solicit 

advice about long-term risks to subjects in gene therapy clinical trials exposed to other 

gene therapy products. The BRMAC meetings were held on November 17, 2000; April 

6, 2001; and October 24, 2001.2 Since 2001, and after reviewing BRMAC’s 

recommendations, we have advised sponsors of studies involving gene transfer 

technology to submit to us their plans for long-term follow-up observations. We 

typically advised sponsors to observe subjects for potential gene therapy-related delayed 

adverse events for a 15 year period, and to include a minimum of five years of annual 

examinations, followed by ten years of annual queries, either in person or by 

questionnaire, of study subjects. 

C. Concerns Raised by the Gene Therapy Community 

Members of the gene therapy community asked that the issue of long-term follow-up 

following exposure to gene transfer technology be discussed in a public forum. 

Accordingly, in June 2004 a public workshop was held in association with the annual 

meeting of the American Society of Gene Therapy (ASGT). The workshop was entitled 

“Long-Term Follow-Up of Participants in Human Gene Transfer Research” and was cosponsored by the ASGT, Biotechnology Industry Organization (BIO), CBER, the NIH 

Office of Biotechnology Activities (OBA), and Pharmaceutical Research and 

Manufacturers of America (PhRMA). The workshop included a forum in which invited 

speakers discussed the challenges associated with long-term follow-up of subjects in gene 

therapy clinical studies. The workshop organizers published a summary of the discussion 

(Ref. 3). 

2

 If you desire background information regarding prior recommendations from the BRMAC about gene therapy 

trials and long-term follow-up observations, we refer you to the transcripts for the November 17, 2000; April 6, 

2001; and October 24, 2001, BRMAC meetings. The references can be located at 

http://www.fda.gov/cber/advisory/ctgt/ctgtmain.htm by searching under the year of the meeting. 

3 
Contains Nonbinding Recommendations 

Key issues identified by workshop participants include the following: 

• Not all gene therapy products present the same risks of delayed adverse events. 

Uniform recommendations for long-term follow-up for all gene therapy products did 

not take product characteristics into account. 

• Some study subjects appear unsuitable for meaningful long-term follow-up 

observations because of high short-term mortality, poor general health, or exposure to 

mutagenic agents. 

• Our recommendations regarding the duration and design of long-term follow-up have 

not been sufficiently specific. 

These issues are addressed in Sections IV and V of this guidance. 

III. DEFINITIONS AND ABBREVIATIONS 

The following definitions apply to this guidance: 

Gene therapy products: 

All products that mediate their effects by transcription and/or translation of transferred 

genetic material and/or by integrating into the host genome and that are administered as 

nucleic acids, viruses, or genetically engineered microorganisms. The products may be 

used to modify cells in vivo or transferred to cells ex vivo prior to administration to the 

recipient. 

Gene transfer: 

The transfer of genetic material into a cell. 

Gene transfer system: 

The combination of the vector, regulating elements, vector formulation, and the route and 

method of vector delivery. 

Gene transfer technology: 

The use of genetic material either alone or in a suitable transfer medium, such as lipids, 

viruses, or other microorganisms, to mediate an effect by transcription, translation, or 

integration into the host genome or any combination of these processes. Exposure to 

gene transfer technology may result from direct administration of the product to a study 

subject or through use of cells or tissues exposed to such products ex vivo prior to 

administration to a study subject. 

IND: 

Investigational New Drug Application, as described in 21 CFR Part 312. 

Integration (of DNA): 

The process whereby exogenous DNA sequences become incorporated into a genome. 

4 
Contains Nonbinding Recommendations 

Latency (of a viral infection): 

A period of time during which a virus is present in the host without producing overt 

clinical symptoms. 

Long-term follow-up observations: 

Long-term follow-up observations are extended assessments that continue some of the 

scheduled observations of a customary clinical trial. Long-term follow-up observations 

are an integral portion of the study of investigational products, such as gene therapy, that 

are considered to present a high risk of producing delayed adverse events. 

Maximum feasible dose (MFD) (in preclinical studies): 

The highest dose that can be administered to a non-human animal. Limitations may be 

due to animal size, administration site, or product characteristics. The MFD may not be 

equivalent to the clinically relevant dose. 

Persistence:

With respect to transferred genetic material, the continued presence of genetic sequences 

in the host after acute exposure to a transfecting agent, whether due to integration of the 

genetic sequence into the host genome or to latent infection with the viral vector bearing 

the genetic sequence. 

Preclinical Study: 

An investigational study performed in non-human animals or in isolated cells or tissue 

from humans or other animals. Preclinical studies may be performed prior to or during 

clinical studies. 

Reactivation (of a viral infection): 

The re-emergence of a symptomatic or asymptomatic viral infection following a period of 

latency. 

Transgene: 

An exogenous gene that is introduced into a host cell. 

Vector Sequences: 

Refers to specific sequences of nucleotides, either DNA or RNA, that have been 

introduced into a gene therapy vector. The sequence includes all components of the gene 

therapy vector, the vector backbone, transgene(s), and regulatory elements. 

Viral Vector: 

A virus that has been modified to transfer genetic material. 

5 
Contains Nonbinding Recommendations 

IV. PRECLINICAL DATA USED FOR ASSESSMENT OF DELAYED RISKS IN 

GENE THERAPY CLINICAL TRIALS 

 A. Criteria to Assess Potential Delayed Risks of Gene Therapy

We generally will not require long-term follow-up observations following exposure to 

gene transfer technology when the risk of delayed adverse events is low. To assess the 

risk related to your product, we recommend that you use available preclinical and clinical 

evidence. To assess the risks of delayed adverse events, you may use current information 

about your product and similar products based on studies that you and others have 

performed. As more data accumulates, it is important to reassess the risk to your subjects 

and, if appropriate, revise your protocol as it relates to long-term follow-up observations. 

We consider the assessment of risks to be a continuous process. New information may 

support the need for long-term follow-up observations or the revision of an existing 

study. For example, if recently reported evidence suggests a newly identified risk 

associated with your product or similar products, long-term follow-up observations may 

be necessary to mitigate long-term risks to subjects receiving these vectors. Similarly, if 

sufficient data accumulate to suggest that your product is not associated with delayed 

risks, it may be appropriate to reduce or eliminate provisions for long-term follow-up 

observations. 

Pertinent previous preclinical and clinical experience with your product or similar 

products is highly relevant in the assessment of delayed adverse events. Experience with 

products in the same vector class, administered by a similar route, and given for the same 

clinical indication may contribute helpful information. 

We recommend you refer to the series of questions in Figure 1, “Framework to Assess 

the Risk of Gene Therapy-Related Delayed Adverse Events” to help you assess the level 

of risk. When the risk of delayed adverse events is low based on your answers to these 

questions, a plan for long-term follow-up observations may not be necessary to mitigate 

risks to subjects. Evidence from preclinical studies will help you answer questions 1 – 3. 

Include all of the primary data relevant to the assessment of the risk of delayed events 

when you submit your IND to FDA (see 21 CFR 312.23(a)(8), (10)(iv), (11)). 

We suggest you use the framework in Figure 1 by answering the questions in sequence as 

follows: 

• Question 1: “Is your gene therapy product used only for ex vivo modification of 

cells?” 

If the answer is “no,” go to Question 2. If the answer is “yes,” go to Questions 3 and 

4. 

6 
Contains Nonbinding Recommendations 

• Question 2: “Do preclinical study results show persistence of vector sequences?” 

If the answer is “no,” the risk of gene therapy-related delayed adverse events is low, 

and long-term follow-up observations may not be needed. If the answer is “yes,” go 

to questions 3 and 4. 

If it is unknown whether your vector persists, for the purpose of assessing risk, we 

recommend that you either assume that it does persist, or perform a preclinical study 

to assay for vector persistence in a relevant animal species. Please refer to Section 

IV.B, “Considerations for Preclinical Study Design to Assess Vector Biodistribution 

and Persistence,” for help with preclinical trial design and details on the use and 

expected sensitivity of polymerase chain reaction (PCR) assay for biodistribution 

studies. In assays performed after the final administration of vector, persistence is 

indicated by detectable levels of vector sequences above the threshold level in the 

PCR assay and absence of an apparent downward trend over several time points. In 

contrast, persistence is unlikely if you cannot detect vector sequences with a sensitive 

PCR assay or if the assay for vector sequences demonstrates a downward trend over 

time. We encourage you to consult with OCTGT, CBER for specific advice about 

determination of persistence and biodistribution in your test system. 

• Question 3: “Are vector sequences integrated?” 

If the answer is “no,” go to question 4. If the answer is “yes,” we would require that 

clinical protocols with the product include clinical long-term follow-up observations. 

• Question 4: “Does the vector have potential for latency and reactivation?” 

If the answer is “no,” the risk is low that exposure to your gene transfer technology 

will be followed by gene therapy-related delayed adverse events. Long-term followup observations may not be needed. If the answer is “yes,” we would require that all 

your clinical protocols with the product include clinical long-term follow-up 

observations. 

 Laboratory and preclinical evidence of the low risk of delayed adverse events following 

exposure to a similar product may show that long-term follow-up observations are not 

needed. If you provide data from a similar product, we can assess the relevance to your 

product if you provide a clear explanation. 

We provide the following two examples: 

• Your product is a plasmid and the similar product is also a plasmid, but has different 

coding sequences for the proposed therapeutic gene product. The similar product has 

been used in preclinical and clinical studies, administered by an identical route and in 

an identical final formulation to that proposed in the prospective studies. Reference 

to a published study demonstrating lack of persistence of the vector for the similar 

7 
Contains Nonbinding Recommendations 

product may adequately address concerns regarding the persistence of the proposed 

vector. 

• Your proposed product and the similar product differ only with respect to route of 

administration. The similar product was administered into tumors (intratumorally). 

The proposed product is to be given intravenously. There is a published study 

demonstrating the lack of persistence of the vector when administered intratumorally. 

The data from the studies with the similar product are not sufficiently relevant, since 

there was no intended systemic exposure to the product. Thus, there is insufficient 

similarity to conclude that long-term follow-up observations are not necessary to 

mitigate long-term risks to subjects. In the absence of relevant data from a study 

involving a similar product, we recommend that you assess the risk of vector 

persistence in a preclinical study with the proposed product administered by the 

intravenous route. 

If you believe you have evidence from studies on a similar product that is adequate to 

support conclusions that the vector is unlikely to persist in human hosts and that the 

vector’s DNA does not integrate into the human genome, you may decide to submit a 

clinical protocol that does not provide for long-term follow-up observations. We will 

review such submissions and, if we disagree based upon our review of your submission 

or other additional information, we may conclude that long-term follow-up observations 

for delayed adverse events are necessary to mitigate long-term risks, and that without 

long-term follow-up observations, the study presents an unreasonable and significant risk 

to study subjects (21 CFR 312.42(b)(1)(i) and (b)(2)(i)). 

We provide the following examples of evidence that might cause us to require you to 

perform long-term follow-up observations for delayed adverse events: 

• A preclinical toxicology study indicates that expression of the transgene is associated 

with delayed toxicity. 

• The transgene provides functional replacement of a host gene; the transgene product 

is potentially immunogenic. 

• Data collected in your short-term clinical study indicate vector persistence, even 

though data from your preclinical studies suggested that the vector did not persist.

8 
Contains Nonbinding Recommendations 

Figure 1. Framework to Assess the Risk of Gene Therapy-Related Delayed Adverse Events. 

1 If you have evidence that suggests that the vector may integrate or if the vector was 

intentionally designed to facilitate integration (please refer to Table 1, Section IV.C), the answer 

is “yes.” If you have no evidence regarding integration, we recommend that you include 

preclinical study in your development plan to address this question. 

2

 If you or others identify an increased risk of delayed adverse events from persistent gene 

expression or from exposure to your product based on additional information reported after your 

protocol is accepted, you should plan to perform long-term follow-up observations even if the 

answer to these questions is “No”. See Section IV.A of the text for examples. 

3 See Section V of the text for recommendations on how to perform clinical long-term follow-up 

observations. 

9 
Contains Nonbinding Recommendations 

B. Considerations for Preclinical Study Design to Assess Vector Biodistribution 

and Persistence 

As discussed in Section IV.A, vector persistence heightens the risk of delayed adverse 

events following exposure to gene transfer technology. Indeed, the longer the vector 

persists, the greater the duration and degree of risk of delayed adverse events. We 

recommend that you perform preclinical biodistribution studies using methods that are 

shown to be sensitive and quantitative to detect vector sequences. Such studies would be 

designed to determine the distribution of your vector in nontarget tissues and the 

persistence of the vector in both nontarget and target tissues following direct in vivo 

administration of the vector product. If possible and applicable, we recommend that the 

studies employ an animal species that permits vector transduction and/or vector 

replication and that the animal species be biologically responsive to the specific transgene 

of interest (Ref. 4). The duration of the preclinical studies will vary, depending on the 

animal model employed. Projections of delayed adverse reactions in human subjects may 

be derived from assessment of data from appropriate long-term observational studies in 

animals, when possible. 

A biodistribution study in animals can be performed either as a separate study or as a 

component of a toxicology study. Consider the following points in your animal study 

design to permit evaluation of vector localization and persistence (Ref. 5). 

 1. Animal Study Design 

• Use the product in the final formulation proposed for the clinical study 

because changes in the final formulation may alter biodistribution patterns. 

• Use both genders or justify the use of a single gender. 

• Use at least 5 animals per gender per group per sacrifice time point for 

rodents, and between 3-5 animals per gender per group per sacrifice time 

point for nonrodents. 

• Consider factors in the study design that might influence or compromise the 

vector distribution and/or persistence such as the animal’s age and physiologic 

condition. 

• Use the intended clinical route of vector administration if possible. 

• Assess vector biodistribution in a vehicle control group and a group of 

animals that receives the MFD or clinically relevant dose (defined in Section 

III). Studies at additional dose levels might provide dose-dependent 

information. 

• Include appropriate safety endpoints in your biodistribution study in order to 

assess any potential correlation between vector presence/persistence and 

adverse findings if safety endpoints have not been evaluated already in a 

separate toxicity study using the same animal model. These safety endpoints 

should include clinical observations, body weights, clinical pathology, gross 

organ pathology, and histopathology. 

10 
Contains Nonbinding Recommendations 

• Include several sacrifice intervals to characterize the kinetics of vector 

distribution and persistence. We recommend sacrifice at the expected time of 

peak vector detection and at several later time points to evaluate clearance of 

vector sequences from tissues. 

2. Tissue Collection and Analysis 

• Sample and analyze the following panel of tissues, at a minimum: blood, 

injection site(s), gonads, brain, liver, kidneys, lung, heart, and spleen. 

Consider other tissues for evaluation, depending on the vector type and the 

transgene, as well as the route of administration (e.g., draining lymph nodes 

and contralateral sites for subcutaneous/intramuscular injection, bone marrow, 

eyes, etc.). 

• Choose a method for tissue collection that avoids the potential for 

contamination among different tissue samples. 

• Use a quantitative, sensitive PCR assay to analyze the samples for vector 

sequences. You should submit data to your IND to demonstrate that your 

assay methodology is capable of specifically detecting vector sequence in both 

animal and human tissues. We recognize that PCR technology is constantly 

changing, and encourage you to discuss the assay methology with us before 

initiating sample analysis. Current recommendations include the following: 

− The assay should have a demonstrated limit of quantitation of <50 copies 

of vector/1 μg genomic DNA, so that your assay can detect this limit with 

95% confidence. 

− Use a minimum of three samples per tissue. One sample of each tissue 

should include a spike of control DNA, including a known amount of the 

vector sequences, in order to assess the adequacy of the PCR assay 

reaction. The spike control will determine the specified PCR assay 

sensitivity. 

− Provide a rationale for the number of replicates for testing per tissue, 

taking into account the size of the sample relative to the tissue you are 

testing. 

3. Other Considerations 

We encourage you to discuss with FDA your study design before starting the trial 

to ensure that the trial will adequately assess both biodistribution and vector 

persistence. There are many variables that will affect the outcome and 

interpretation of the in vivo assessment of each vector type. 

C. Vector Integration Potential and Reactivation as Risks for Delayed Adverse 

Events 

Three gene therapy vectors currently under study (i.e., Gammaretrovirus, 

Lentivirus, and Herpesvirus) possess characteristics that we consider to pose high 

11 
Contains Nonbinding Recommendations 

risks of delayed adverse events. Accordingly, we believe that clinical long-term 

follow-up observational studies would be necessary to mitigate long-term risks to 

subjects receiving these vectors. Gammaretrovirus and Lentivirus have a 

documented ability to integrate and Herpesvirus has a documented potential for 

latency and reactivation. In this section, we discuss those risks and the relatively 

low risks associated with gene transfer technology with vectors that lack those 

properties. 

Most vectors used in gene therapy clinical trials can be categorized according to 

their propensity to integrate into host cell DNA. Please refer to Table 1, 

“Integration Properties of Current Commonly Used Gene Therapy Vectors in 

Clinical Trials.” As shown in Table 1 and reflected in the answer to question 3 in 

Figure 1, “Framework to Assess the Risk of Gene Therapy-Related Delayed 

Adverse Events,” vectors that have a potential to integrate present sufficient risk 

that long-term follow-up observations are necessary to mitigate long-term risks to 

subjects receiving these vectors. 

Because of its potential for latency and reactivation, a Herpes virus-based gene 

transfer vector also presents a risk of delayed adverse events related to its use as a 

vector in gene therapy products. During latency, the virus and its gene products 

remain inactive. Reactivation may be delayed for months or years following 

initial exposure. 

We are aware that the potential of vectors to integrate may be modified to 

increase their utility as gene therapy agents. For example, an adenovirus vector 

can be modified to induce integration of its DNA (Refs. 5-9). Another example 

would be changes in the methods used to introduce plasmid DNA vectors into 

cells that result in higher integration frequencies (Ref. 10). In those cases where a 

modification of the gene therapy system may have altered the persistence or 

integration properties, we recommend that you take one of the following actions: 

• Submit data to your IND from preclinical studies to assess vector persistence 

in an appropriate model. As stated in Section IV.B.3, we encourage you to 

discuss with FDA your study design before starting the trial. 

• If the vector is not persistent, the predicted risk of delayed adverse events 

would be low. Long-term follow-up observations would be at your 

discretion. 

• If the vector is persistent, we recommend that you perform preclinical 

studies to assess vector integration, as well as the potential for vector 

latency and reactivation. 

− If the studies show no evidence for persistence due to integration of 

the genetic material or development of latency, the predicted risk of 

delayed adverse events would be low. Long-term follow-up 

observations would be at your discretion. 

12 
Contains Nonbinding Recommendations 

− If the studies show no evidence for integration of the genetic material 

but studies for latency and reactivation are inconclusive, cannot be 

performed, or show evidence of latency and/or reactivation, the 

predicted risk of delayed adverse events is indeterminate. We would 

require long-term follow-up observations. 

• If preclinical studies of vector integration are not feasible, if the genetic 

material integrates, or if the vector is shown to persist in a latent state that 

may be reactivated, the risk of delayed adverse events is high or unknown, 

and long-term follow-up observations in study subjects are warranted. 

• If vector integration studies are not performed, we recommend that you 

provide other evidence to support an assessment that your vector does not 

pose high risks of delayed adverse events, including the following: 

• A discussion of why vector integration studies were not performed. 

• The evidence supporting your assessment of the risk of delayed adverse 

events posed by your product. 

Plasmids, poxvirus, adenovirus, and adeno-associated virus-based vectors (AAV) 

are vectors that do not have a propensity to integrate or reactivate following 

latency and, in the absence of evidence to the contrary, present a low risk of gene 

therapy-related delayed adverse events. However, even if your vector has a low 

propensity to integrate or reactivate, preclinical or clinical data showing 

persistence of the vector raise concerns about a risk of delayed adverse events, 

and follow-up observations would be necessary to mitigate long-term risks to 

subjects receiving these vectors. For example, if an AAV vector is shown to have 

persistent transgene expression, the risk of a delayed aberrant immune response 

should be considered because of the potential for autoimmune phenomena. 

We also note that some vectors currently considered to pose delayed risks might 

be modified in order to reduce those risks. Therefore, data supporting claims of a 

decreased risk for delayed adverse events with novel vector types could provide 

the basis for reassessing the need for performing long-term follow-up 

observations in subjects exposed to those vectors. 

13 
Contains Nonbinding Recommendations 

Table 1. Integration Properties of Current Commonly Used Gene Therapy 

Vectors in Clinical Trials. 

Vector Type Propensity to Integrate1 Long-term Follow-up 

observations2

Plasmid No No 

PoxVirus No No 

Adenovirus No No 

Adenoassociated virus3No No 

Herpesvirus No, but may undergo 

latency/reactivation 

Yes 

Gammaretrovirus Yes Yes 

Lentivirus Yes Yes 

1

Based on vector design (i.e., lack of any known mechanism to facilitate 

integration), as well as cumulative preclinical and clinical evidence suggesting 

that vector does not integrate or integrates only at very low frequencies. 

2

Specific circumstances showing persistent expression of the transgene, in the 

absence of integration, may be the basis for a conclusion that long-term follow-up 

observations are necessary to mitigate long-term risks to subjects receiving these 

vectors. This would depend on additional criteria, such as the transgene 

expressed or clinical indication, as described in the text. 

3

Rep-negative vectors only. 

V. RECOMMENDATIONS FOR PROTOCOLS FOR LONG-TERM FOLLOW-UP 

OBSERVATIONS: CLINICAL CONSIDERATIONS 

In this section, we recommend elements appropriate to the design and conduct of long-term 

follow-up observations. 

A. Decision to Conduct Long-term Follow-up Observations 

The recommendations in this section apply to protocols for which long-term follow-up 

observations appear advisable. Long-term follow-up observations may be necessary to 

mitigate long-term risks to subjects receiving these vectors if: 

• The answers to the questions posed in Section IV, Figure 1. “Framework to Assess 

the Risk of Gene Therapy-Related Delayed Adverse Events” lead you to decide that 

the risks associated with your product are high or uncertain. 

• The information about your product, taken as a whole, shows that long-term followup observations would mitigate the risks to human subjects. For examples of such 

circumstances please refer to the final paragraphs in Section IV.A. 

14 
Contains Nonbinding Recommendations 

In selected instances where we would generally require long-term follow-up 

observations, you may determine that the observations would have no scientific value 

based on the suitability of your clinical trial population. If you make that determination 

and decide not to conduct long-term follow-up, you should include in your IND the 

justification for your decision not to continue to observe your subject population. 

The sections below provide information on criteria you may choose to use to determine 

the suitability of monitoring your clinical trial population to collect scientifically 

informative data by the performance of long-term follow-up observations. We also 

discuss our recommendations for the minimum duration of follow-up observations and 

the minimum observations to be made during long-term follow-up. 

B. Suitability of Clinical Trial Populations for Long-term Follow-up 

Observations

Long-term follow-up observations may have reduced utility in assessing and mitigating 

subject risk when the population selected for the trial has characteristics, such as short 

life expectancy, multiple morbidities, and exposure to other agents, that also could cause 

delayed adverse events. Thus, for example, long-term follow-up observations might have 

little impact if the subjects have widespread disease, or extensive exposure to agents with 

potential for delayed adverse events such as radiation or chemotherapy. In contrast, longterm follow-up observations could have greater value in assessing and mitigating the 

risks to subjects who have limited disease or are disease-free, and who have few comorbidities and limited exposures to other agents with potential for delayed adverse 

events. In those cases where the gene therapy intervention alters life expectancy or comorbidities, initial assessments regarding the suitability of long-term follow-up 

observations in a particular clinical trial may need to be reconsidered. 

C. Recommended Duration of Follow-up Observations 

The duration of long-term follow-up observations should be sufficient to observe the 

subjects for risks that may be due to the characteristics of the product, the nature of the 

exposure, and the anticipated time of occurrence of delayed adverse events. The 

BRMAC on November 17, 2000, April 6, 2001, and October 24, 2001, discussed several 

different time periods for the performance of long-term follow-up observations, including 

a 15 year period (See Section II.B for reference). Based on the BRMAC advice, we also 

recommend a minimum 15 year time period for follow-up observations. However, we 

recognize that shorter periods of observation may be appropriate in individual trials based 

on supporting evidence. Elements that will influence the determination of the duration of 

long-term follow-up observations include the following: 

• The observed duration of in vivo vector persistence; 

• The observed duration of in vivo transgene expression; 

• The prior, concomitant, and post gene therapy exposures of the study population; 

• The expected survival rates in the study population; and 

15 
Contains Nonbinding Recommendations 

• Other factors that may be relevant to the feasibility and scientific value of conducting 

long-term follow-up observations. 

D. Elements of Follow-up Observations 

 Our recommendations on the nature of the follow-up observations are also based on the 

recommendations and discussions at the November 17, 2000, April 6, 2001, and October 

24, 2001, BRMAC meetings (See Section II.B for references). As more clinical data 

accumulate, our recommendations regarding the duration of long-term follow-up 

observations may change. 

It is important that the design of long-term follow-up observations be appropriate to 

detect potential gene therapy-related delayed adverse events in the study subjects enrolled 

in your clinical studies. In this document, we provide recommendations for general 

minimum elements for the long-term follow-up component of your study protocol. 

The investigator is required to prepare and maintain adequate and accurate case histories 

that record all observations and other data pertinent to the investigation on each subject 

administered the investigational drug or employed as a control in the investigation (see

21 CFR 312.62(b)). These records would include a baseline history prior to exposure to 

the product in which all diseases, conditions and physical abnormalities are recorded. 

You are encouraged to develop a template for health care providers who are not 

investigators or subinvestigators (for example, the subject's physician, physician 

assistant, or nurse practitioner) to use in recording and reporting such observations to the 

investigator. Case histories should also include information from scheduled visits by a 

health care professional and test results for persistent vector sequences. The use of 

surrogate tests may be used to indicate vector persistence if direct sequence testing would 

require an invasive procedure for the subject. 

In addition, for at least the first five years we recommend that you do the following: 

• Implement methods for detection of gene therapy-related delayed adverse events; 

• Assure that investigators maintain in the case history a detailed record of all 

exposures to mutagenic agents and other medicinal products and have ready access to 

information about their adverse event profiles; 

• Design a plan for scheduled visits with a health care provider to elicit and record new 

findings for each study subject, including history, physical examination, or laboratory 

testing at minimum intervals of one year; 

• Establish a method for investigators to record the emergence of new clinical 

conditions, including: 

− New malignancy(ies) 

− New incidence or exacerbation of a pre-existing neurologic disorder 

− New incidence or exacerbation of a prior rheumatologic or other autoimmune 

disorder 

− New incidence of a hematologic disorder; and 

16 
Contains Nonbinding Recommendations 

• Design a plan to elicit the cooperation of study subjects and their health care 

providers in reporting delayed adverse events, including unexpected illness and 

hospitalization. 

For the subsequent ten years, at a minimum, we recommend that you ensure that your 

investigators: 

• Contact subjects at a minimum of once a year. At your discretion, unless the longterm follow-up observation plan provides for additional, specific screening, you may 

arrange to contact subjects by telephone or written questionnaire rather than by office 

visits with a health care provider. 

• Continue appropriate follow-up methods as indicated by previous test results. For 

example, it would be appropriate to monitor for vector sequences in subjects who had 

previous test results demonstrating vector persistence. 

Perform all long-term follow-up observations according to FDA regulations governing 

clinical trials (See http://www.fda.gov/oc/gcp/regulations.html). We provide additional 

specific recommendations and requirements for data collection and reporting of adverse 

events for long-term follow-up clinical observations as follows: 

1. Detection of Adverse Events: To facilitate detection of delayed adverse events, 

we recommend that the protocol identify suitable health care professionals whose 

observations would be used in the assessment of the occurrence of adverse events 

in the study population. Suitable health care professionals might include 

physicians, physician’s assistants, and nurse practitioners who were not otherwise 

associated with the clinical trial. You may arrange to have such individuals 

notified to provide prompt reports of adverse events to the investigators. 

To increase subject compliance and improve the quality of data collection, we 

suggest that you encourage study subjects to monitor themselves and assist in 

reporting adverse events. Devices that study subjects could use to report events to 

the investigator include subject diaries of health-related events, informational 

brochures, and laminated, wallet-sized cards with investigator contact 

information. 

2. IND Safety Reports: You must follow applicable reporting requirements outlined 

in 21 CFR 312.32 for adverse experiences associated with the use of the product. 

As the long-term follow-up observations proceed, you must also notify each 

participating investigator of any adverse experience associated with the use of the 

gene therapy product that is both serious and unexpected (21 CFR 

312.32(c)(1)(i)(A)), as well as any new observations discovered by, or reported 

to, you (21 CFR 312.55(b)). In each IND Safety Report (required to be provided 

to investigators and FDA), you must identify all safety reports previously filed 

concerning a similar adverse experience, and analyze the significance of the 

adverse experience in light of the previous, similar reports (21 CFR 

17 
Contains Nonbinding Recommendations 

312.32(c)(1)(ii)). You must promptly investigate all safety information you 

receive (21 CFR 312.32(d)(1)). If the relationship of the adverse experience to 

the gene therapy product is uncertain, we may recommend that you perform 

additional investigations and revise your Informed Consent Document and 

Investigator Brochure to inform all study subjects of the risk of the adverse 

experiences. We may also request that investigators contact previously treated 

study subjects to inform them of the new risk.

3. Annual Reports to the IND/Summary Information: While the IND is in effect and 

until long-term follow-up observations are concluded, you must file an annual 

report. In that report, submit information obtained during the previous year's 

clinical and nonclinical investigations, including, among other things, a summary 

of all IND safety reports submitted during the past year, and a narrative or tabular 

summary showing the most frequent and most serious adverse experiences by 

body system (21 CFR 312.33(b)(1) and (2)). 

4. Amendments to Your Clinical Protocol: If clinical data suggest that your product 

is not associated with delayed risks, you may want to consider changing the 

clinical protocol regarding long-term follow-up of study subjects. However, 

before implementation of this change, you must submit to FDA a protocol 

amendment to your IND indicating the relevant changes (21 CFR 312.30(b)(1), 

(d), and (e)). 

5. Scheduled Physical Examinations: We recommend that long-term follow-up 

observations include scheduled physical examinations performed by a health care 

professional at least once a year during the first five years, unless the assessed 

risks associated with your protocol indicate that they should be done more 

frequently. For example, if a subject exposed to your product or an analogous 

product develops a rapidly progressive, potentially reversible delayed adverse 

event, and there is a reasonable possibility that the event may have been caused 

by the product, it may then become advisable to perform observations on a semiannual or quarterly basis. Such periodic evaluation should include a brief history 

and focused examination designed to determine whether there is any evidence of 

emergence of clinically important adverse events. Appropriate laboratory 

evaluations, such as a hematology profile, should be included with the periodic 

physical examination. Long-term follow-up observations are intended for study 

purposes only, not to provide evaluation and treatment of health care problems 

that are not associated with the use of the product. 

6. Vector Sequences: During long-term follow-up, we recommend that you test 

study subjects at least annually for persistent vector sequences until they become 

undetectable. The assay should be sufficiently sensitive to detect vector 

sequences. We recommend that you sample the likely population of transduced 

cells without being overly invasive (e.g., peripheral blood is a suitable sample to 

test for presence of hematopoietic stem cells, rather than bone marrow biopsy). In 

those cases where the transduced cell population may require an invasive 

18 
Contains Nonbinding Recommendations 

procedure, we recommend that you consider, instead, measuring a surrogate that 

may indicate vector persistence (e.g., the level of transgene product or some 

clinical effect). Data demonstrating the lack of detectable vector may provide a 

rationale to revise the long-term follow-up elements of your study as an 

amendment to your IND. In any such protocol amendment, include an assessment 

of risks associated with your product and an evaluation of the impact of the 

waning persistence of the vector on those risks (21 CFR 312.30(b), (d)(2)). 

E. Informed Consent in Trials Involving Long-term Follow-up Observations 

The informed consent document must describe, among other things, the purposes of the 

research, the expected duration of the subject's participation and the procedures to be 

followed (21 CFR 50.25(a)(1)). Accordingly, the informed consent document must 

explain the purpose and duration of long-term follow-up observations, the time intervals 

and the locations at which you plan to request the subjects to have scheduled study visits 

or be contacted by other means, and details as to what those contacts will involve (21 

CFR 50.25). 

We provide additional informed consent recommendations for retroviral vectors in 

Section V.F.3 below. 

F. Special Considerations Regarding Integrating Vectors 

 

The recommendations in this section apply exclusively to subjects in clinical trials who 

received integrating vectors, such as retroviral vectors or cells modified ex vivo by 

retroviral vectors. In at least two preclinical studies performed in mice, integration of 

genetic material from a retroviral vector into mouse cell DNA was reported to cause 

malignant transformation (Refs. 11 and 12). In addition, in one clinical study, three out 

of a total of 11 human subjects with X-linked Severe Combined Immunodeficiency (XSCID) have developed clonal T-cell proliferation after receiving hematopoietic cells that 

had been modified ex vivo with a retroviral vector (Refs. 13 and 14). One of the three 

subjects died (Ref. 14). These leukemias were the result of the retroviral vector-derived 

DNA integrating into the subjects’ cellular DNA. The observation that children with XSCID developed a malignancy after exposure to a retroviral vector (Ref. 13) has 

prompted us to provide additional recommendations for collection of data in studies in 

which subjects are exposed to integrating vectors, at this time best exemplified by 

retroviral vectors, including products derived from either gammaretroviruses or 

lentiviruses. 

1. Data Collection 

We recommend that you perform assays to assess the pattern of vector integration 

sites in relevant surrogate cells (e.g., determine whether cells carrying integrated 

vector sequences are polyclonal, oligoclonal, or monoclonal, with respect to 

vector integration patterns). We consider an assessment of the vector integration 

pattern to be relevant in subjects in gene therapy clinical trials involving 

19 
Contains Nonbinding Recommendations 

integrating vectors if: (1) the target cells are known to have a high replicative 

capacity and long survival, and (2) a suitable surrogate is accessible for assay. 

For example, hematopoietic stem cells have a high replicative capacity and long 

survival; peripheral blood could serve as a surrogate for testing for vector 

persistence if hematopoietic stem cells were the target of your gene therapy. In 

those cases where peripheral blood is the surrogate, analyses on purified subsets 

of hematopoietic cells (e.g., lymphocytes vs. granulocytes) may be performed, if 

deemed appropriate to the study by you or FDA. As an alternative example, if the 

integrating vector is used for in vivo transduction of liver hepatocytes, you may 

not need to perform this analysis, since terminally differentiated hepatocytes are 

non-dividing cells under normal circumstances, and there is no reasonable 

surrogate that allows for non-invasive testing of vector persistence. Please refer 

to the following recommendations for developing methods and plans for 

performing these analyses. 

(a) The choice of method to assess the pattern of vector integration sites 

should be based upon data with appropriate positive and negative controls 

(i.e., target cells with a known number and sites of vector copies 

integrated vs. target cells with no vector integrants). Studies should be 

performed to provide information about the assay sensitivity, specificity, 

and reproducibility. 

(b) We recommend that you perform an analysis to assess the pattern of 

vector integration sites if at least 1% cells in the surrogate sample are 

positive for vector sequences by PCR. As an alternative, you may base 

the decision to analyze for clonality of vector integration sites on an 

evaluation of the sensitivity of the assay system used to detect clonality. 

(c) We recommend that you test for vector sequences by PCR in subject 

surrogate samples obtained at intervals of no greater than six months for 

the first five years and then no greater than yearly for the next ten years, or 

until such time that no vector sequences are detectable in the surrogate 

sample. 

(d) We recommend that you perform an analysis to determine the site of 

vector integration if the analysis of a subject’s surrogate cells suggests a 

predominant clone (e.g., oligoclonal pattern of vector insertions) or 

monoclonality. In addition, if you detect a predominant integration site, 

test for persistence by performing another analysis for clonality no more 

than three months later. 

(e) When the nucleotide sequence adjacent to the site of the vector integration 

has been determined, we recommend that you compare the identified 

integration site sequence with known human sequences in the human 

genome database and other databases that document oncogenes to 

20 
Contains Nonbinding Recommendations 

determine whether the identified sequences are known to be associated 

with any human cancers. 

(f) While we recognize that oligoclonality or even monoclonality itself will 

not a priori result in a malignancy (Refs. 15 and 16), we also recognize 

that these changes increase the risk of a malignancy, and therefore, we 

recommend that you institute a plan to monitor the subject closely for 

signs of malignancy if any of the following conditions pertain: 

• Persistent monoclonality; 

• Clonal expansion (e.g., the per cent cells positive for a particular 

vector integration site is shown to increase over multiple timepoints); 

or 

• Evidence of vector integration near or within a locus known to have 

oncogenic activity. 

(g) To screen for specific disease entities, we recommend that you use 

established methods and/or seek advice from clinicians with expertise in 

screening for the health care risks to which, according to your evidence, 

your subjects may be exposed. 

2. Data Reporting 

If no evidence of oligo- or monoclonality is observed, we recommend that you 

report a summary of all analyses for the pattern of vector integration sites in 

narrative or tabular form in the annual report to your IND (21 CFR 312.33(b)(5)). 

However, if evidence of oligo- or monoclonality is observed, submit this essential 

information in an information amendment to the IND (21 CFR 312.31(a)). We 

recommend that you submit this amendment within 30 days. 

3. Informed Consent in Trials Involving Retroviral Vectors 

Each subject in an investigation must be provided with a description of any 

reasonably foreseeable risks from participating in the investigation (21 CFR 

50.25(a)(2)). Investigators must submit for Institutional Review Board approval 

the informed consent documents (21 CFR 56.109(b) and (c), 312.66). For all 

clinical trials in which subjects are exposed to retroviral vectors, the informed 

consent documents should include, in layman’s language, a complete and accurate 

disclosure of the development of leukemia in the children with X-SCID. We 

recommend that you include the following information, where applicable, in 

language understandable to the study subjects, in the section describing the risks 

associated with the study agent: 

• Description of study agent - The study involves giving a person some cells 

that have been changed by a retroviral vector. A retroviral vector is a virus 

that can insert genetic material into cells. 

21 
Contains Nonbinding Recommendations 

• Mechanism of action for retroviral vectors - When retroviral vectors enter a 

normal cell in the body, the deoxyribonucleic acid (DNA) of the vector inserts 

itself into the normal DNA in that cell. This process is called DNA 

integration. 

• Effect of DNA integration - Most DNA integration is expected to cause no 

harm to the cell or to the patient. However, there is a chance that DNA 

integration might result in abnormal activity of other genes. In most cases, 

this effect will have no health consequences. 

• Discussion of cancer occurring in animal studies - In some cases, abnormal 

activity of a normal gene may cause an uncontrolled growth of the cell that 

sometimes results in a cancer. This type of event has occurred in animal 

studies in which retroviral vector DNA integration appeared to cause cancers 

in mice and monkeys. 

• Discussion of delayed adverse event, leukemia-like malignancy, occurring in 

human studies - It is important that you know about some cancers that 

occurred in another gene therapy research study. The study, conducted in 

France, involved a disease called X-linked Severe Combined 

Immunodeficiency (SCID). Years after receiving cells that were modified by 

a retroviral vector, a significant number of the children in this small study 

developed a leukemia-like malignant disease (cancer). At least one child died 

from the cancer. A group of experts in this field studied the results from tests 

performed on these children’s blood cells. They concluded that the leukemialike malignancy was caused by the retroviral vector DNA. However, most of 

the children with X-linked SCID who have received experimental gene 

therapy have not been found to have a leukemia-like disease at this time. 

Although they appear healthy, we still do not know whether they, too, will 

develop a malignant growth. 

• Risk of malignancy for this study - We do not know if the retroviral vector 

used in this protocol might cause a new malignancy. However, you should be 

aware that the DNA contained in retroviral vectors will integrate into your 

DNA and that under some circumstances, this has been known to cause 

malignant (cancerous) growth months to years later.

22 
Contains Nonbinding Recommendations 

VI. REFERENCES 

1. FDA Guidance for Industry: Supplemental Guidance on Testing for Replication 

Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During 

Follow-up of Patients in Clinical Trials Using Retroviral Vectors, November 2006 

(http://www.fda.gov/cber/gdlns/retrogt1000.pdf). 

2. Donahue, R.E., et al., Helper virus induced T cell lymphoma in nonhuman primates 

after retroviral mediated gene transfer. Journal of Experimental Medicine 1992; 

176:1125-1135. 

3. Nyberg, K., et al., Workshop on long-term follow-up of participants in human gene 

transfer research. Molecular Therapy 2004; 10(6):976-980. 

4. FDA Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene 

Therapy, March 1998 (http://www.fda.gov/cber/gdlns/somgene.pdf). 

5. Bauer, S., Current FDA approach for preclinical vector biodistribution studies, March 

12, 1999: Recombinant DNA Advisory Committee Meeting 

(http://www4.od.nih.gov/oba/rac/minutes/3-99RAC.htm#IX). 

6. Shayakhmetov, D.M., et al., A high-capacity, capsid-modified hybrid 

adenovirus/adeno-associated virus vector for stable transduction of human 

hematopoietic cells. Journal of Virology 2002; 76(3):1135-1143. 

7. Goncalves, M.A., et al., Stable transduction of large DNA by high-capacity adenoassociated virus/adenovirus hybrid vectors. Virology 2004; 321(2):287-96. 

8. Picard-Maureau, M., et al., Foamy virus—adenovirus hybrid vectors. Gene Therapy 

2004; 11(8):722-28. 

9. Yant, S.R., et al., Transposition from a gutless adeno-transposon vector stabilizes 

transgene expression in vivo. Nature Biotechnology 2002; 20(10):999-1005. 

10. Wang, Z., et al., Detection of integretation of plasmid DNA into host genomic DNA 

following intramuscular injection and electroporation. Gene Therapy 2004; 

11(8):711-21. 

11. Li, Z., et al., Murine leukemia induced by retroviral gene marking. Science 2002; 

296:497. 

12. Modlich, U., et al., Leukemias following retroviral transfer of multidrug resistance 1 

(MDR1) are driven by combinatorial insertional mutagenesis. Blood 2005; 

105(11):4235. 

13. Hacein-Bey-Abina, S., et al., LMO2-associated clonal T cell proliferation in two 

patients after gene therapy for SCID-X1. Science 2003; 302(5644): 415-9. 

14. Couzin, J., Kaiser, J., As Gelsinger Case Ends, Gene Therapy Suffers Another Blow. 

Science 2005; 307:1028. 

15. Ott M.G., et al., Correction of X-linked chronic granulomatous disease by gene 

therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. 

Nature Medicine 2006; 12(4):401-9. 

16. Schmidt M., et al., Clonality analysis after retroviral-mediated gene transfer to 

CD34+ cells from the cord blood of ADA-deficient SCID neonates. Nature Medicine 

2003; 9(4):463-68. 

23