Long Term Follow-Up After 
Administration of Human Gene 
Therapy Products 
 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
Additional copies of this guidance are available from the Office of Communication, Outreach 
and Development (OCOD), 10903 New Hampshire Ave., Bldg. 71, Rm. 3128, Silver Spring, 
MD 20993-0002, or by calling 1-800-835-4709 or 240-402-8010, or email ocod@fda.hhs.gov, or 
from the Internet at https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-
regulatory-information-biologics/biologics-guidances. 
 
For questions on the content of this guidance, contact OCOD at the phone numbers or email 
address listed above. 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Biologics Evaluation and Research 
January 2020  Contains Nonbinding Recommendations 
 
Table of Contents 
 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  SCOPE ............................................................................................................................... 2 
III.  BACKGROUND ............................................................................................................... 2 
A.  Potential Risks of Delayed Adverse Events Following Exposure to Human 
Gene Therapy Products ........................................................................................ 2 
B.  History .................................................................................................................... 3 
C.  Experience Gained Through Long Term Follow-up of Subjects in Gene 
Therapy Trials ....................................................................................................... 4 
D.  Long Term Follow-up for Novel Gene Therapy Products ................................ 5 
IV.  PRECLINICAL DATA USED FOR ASSESSMENT OF DELAYED RISKS IN 
GENE THERAPY CLINICAL TRIALS ........................................................................ 5 
A.  Criteria to Assess Potential Delayed Risks of Gene Therapy Products ........... 5 
B.  Considerations for Preclinical Study Design to Assess Biodistribution and 
Persistence of Gene Therapy Product ................................................................. 9 
C.  Vector Persistence, Integration, and Reactivation and Genome Modification: 
Assessing Long Term Risks................................................................................ 11 
D.  Considerations for Preclinical Evaluation of Products that Involve Genome 
Editing .................................................................................................................. 15 
V.  RECOMMENDATIONS FOR PROTOCOLS FOR LONG TERM FOLLOW-UP 
OBSERVATIONS: CLINICAL CONSIDERATIONS ............................................... 15 
A.  Goals of the Long Term Follow-up Observations ............................................ 15 
B.  Clinical Trial Populations for Long Term Follow-up Observations .............. 16 
C.  Duration of Long Term Follow-up Observations ............................................ 16 
D.  Elements of Long Term Follow-up Observations ............................................ 17 
E.  Informed Consent in Trials Involving Long Term Follow-up Observations 21 
F.  Special Considerations Regarding Integrating Vectors .................................. 22 
G.  Special Considerations Regarding Product Involving Genome Editing ........ 26 
VI.  GENERAL CONSIDERATIONS FOR POST-MARKETING MONITORING 
PLANS FOR GENE THERAPY PRODUCTS ............................................................ 26 
VII.  LONG TERM FOLLOW-UP UNDER SPECIAL CIRCUMSTANCES .................. 27 
VIII.  DEFINITIONS ................................................................................................................ 28 
IX.  REFERENCES ................................................................................................................ 30 
APPENDICES ............................................................................................................................. 33 
 
i  Contains Nonbinding Recommendations 
 
Long Term Follow-Up After Administration of Human Gene 
Therapy Products  
 
 
Guidance for Industry 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or 
Agency) on this topic.  It does not establish any rights for any person and is not binding on FDA 
or the public.  You can use an alternative approach if it satisfies the requirements of the 
applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff 
responsible for this guidance as listed on the title page. 
 
 
I.  INTRODUCTION 
 
We, FDA, are providing you, a sponsor who is developing a human gene therapy product (GT 
Product),1 recommendations regarding the design of long term follow-up studies (LTFU 
observations) for the collection of data on delayed adverse events following administration of a 
GT product.  Often, GT products are designed to achieve therapeutic effect through permanent or 
long-acting changes in the human body.  As a result of long term exposure to an investigational 
GT product, study subjects may be at increased risk of undesirable and unpredictable outcomes 
that may present as delayed adverse event(s).  To understand and mitigate the risk of a delayed 
adverse event, subjects in gene therapy trials may be monitored for an extended period of time, 
which is commonly referred to as the long term follow-up (LTFU) period (of a clinical study).  
LTFU observations are extended assessments that continue some of the scheduled observations 
of a clinical trial past the active follow-up period, and are an integral portion of the study of 
some investigational GT products.  LTFU observations are important to monitor long term safety 
of GT products.  For GT products that present long term risks to subjects, LTFU/surveillance 
plan(s) should also be put in place post-licensure for monitoring of delayed adverse events (for 
details we refer you to section VI. of this document).  Not all GT products will require LTFU 
observations; a risk assessment should be performed by a sponsor based on several factors as 
outlined in this guidance.  
 
In this guidance, we provide a brief introduction of the product characteristics, patient-related 
factors, and the preclinical and clinical data that should be considered when assessing the need 
for LTFU observations for your GT product.  We also provide recommendations for the study 
design of LTFU observations, with specific considerations for different GT products and 
recommendations on patient monitoring for licensed GT products.  Definitions of terms used 
throughout this guidance are provided in section VIII. of this document.  
 
This guidance finalizes the draft guidance of the same title dated July 2018 and supersedes the 
document entitled Guidance for Industry:  Gene Therapy Clinical Trials  Observing Subjects 
                                                 
1 See section VIII. Definitions:  Human gene therapy product.  
1  Contains Nonbinding Recommendations 
 
for Delayed Adverse Events dated November 2006 (Ref. 1) (2006 Delayed Adverse Events).  
This guidance is also intended to supplement the guidance entitled Testing of Retroviral Vector-
Based Human Gene Therapy Products for Replication Competent Retrovirus during Product 
Manufacture and Patient Follow-up; Guidance for Industry dated January 2020.2 
 
FDAs guidance documents, including this guidance, do not establish legally enforceable 
responsibilities.  Instead, guidances describe the 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.  SCOPE 
 
This guidance applies to all gene therapy clinical studies and to licensed GT products for which 
LTFU observations are warranted based on analyses of available preclinical and clinical safety 
data for the GT product that raises concerns for delayed adverse events.  The recommendations 
in this guidance apply to human GT products that produce long lasting genetic effects and the 
performance of LTFU observations for evidence of delayed adverse events, i.e., adverse events 
that occur past the active follow-up period after exposure to the GT product, as described in the 
main study protocol.3 
 
 
III.  BACKGROUND 
 
A.  Potential Risks of Delayed Adverse Events Following Exposure to Human 
Gene Therapy Products  
 
Characteristics unique to human GT products that may be associated with delayed 
adverse events include: 
 
1.  The integration activity of the GT product:  The biological activity of 
retroviral vectors4 (e.g., vectors derived from gammaretrovirus, lentivirus, 
foamy virus etc.) and transposon elements is imparted by an integration 
event in the genome.  In general, such integration is not directed to 
specific sites in the human genome, and this raises the potential for 
disruption of critical host (human) genes at the site of integration, or 
activation of proto-oncogenes near the integration site(s) and, thereby, the 
risk for malignancies. 
  
                                                 
2 Testing of Retroviral Vector- Based Human Gene Therapy Products for Replication Competent Retrovirus during 
Product Manufacture and Patient Follow-up; Guidance for Industry is available at this website: 
https://www.fda.gov/media/113790/download.  
3 This guidance does not apply to vaccines for infectious disease indications, bacteriophage products, live 
biotherapeutic products, fecal microbiota for transplantation (FMT) products and allergenic products.    
4 See section VIII. Definitions:  Vector. 
2  Contains Nonbinding Recommendations 
 
2.  Genome editing activity:  Genome editing-based GT products impart their 
biological activity through site-specific changes in the human genome, but 
may also have off-target effects on the genome (Ref. 2).  Similar to 
integrating vectors, genome editing may produce undesirable changes in 
the genome (whether ex vivo or in vivo), with the risk of malignancies, 
impairment of gene function, etc. 
 
3.  Prolonged expression:  A GT product where the transgene (therapeutic 
gene) encodes growth factors, such as vascular endothelial growth factor 
(VEGF) or proteins associated with cell division such as p53, may raise 
the potential for unregulated cell growth and malignancies due to 
prolonged exposure to the therapeutic protein.  Similarly, transgenes 
encoding immune recognition factors may introduce the risk for 
autoimmune-like reactions (to self-antigens) upon prolonged exposure.  
For GT products that carry transcriptional regulatory elements (e.g., 
microRNA) or immune-modulatory proteins (e.g., cytokines) there is also 
the risk of unknown pleotropic effects, including altered expression of 
host (human) genes that could result in unpredictable and undesirable 
outcomes.  
 
4.  Latency:  When the GT product has the potential for latency, such as a 
herpesvirus, there is the potential for reactivation from latency and the risk 
of delayed adverse events related to a symptomatic infection. 
 
5.  Establishment of persistent infections:  GT products that are  replication 
competent viruses and bacteria, such as listeria-based bacterial vectors, 
have the potential to establish persistent infections in 
immunocompromised patients leading to the risk of developing a delayed 
but serious infection.   
 
In addition to product-related factors, the long term risk profile of a GT product should 
also take into consideration the target cell/tissues/organ, and the patient population (age, 
immune status, risk of mortality etc.), and the relevant disease characteristics.   
 
B.  History  
 
The recommendations for LTFU monitoring in the 2006 Delayed Adverse Events 
guidance (Ref. 1) were based on extensive discussions among gene therapy stakeholders, 
and cumulative preclinical and clinical experience with GT products (Refs. 3, 4, 5) as 
summarized in this section.  To discuss and solicit advice about long term risks to 
subjects exposed to such products, three separate meetings of the FDA advisory 
committee, Biological Response Modifiers Advisory Committee (BRMAC), were 
convened on November 17, 2000, April 6, 2001, and October 24, 2001 (Ref. 6).   
 
A public workshop entitled Long-term Follow-Up of Participants in Human Gene 
Transfer Research was also held in June 2001, in association with the annual meeting of 
3  Contains Nonbinding Recommendations 
 
the American Society of Gene Therapy (ASGT).  The workshop included a forum in 
which invited speakers discussed the challenges associated with LTFU of subjects in 
gene therapy clinical studies.  The workshop organizers published a summary of the 
discussion (Ref. 7).  
 
Taking these discussions into consideration, we provided detailed recommendations in 
the 2006 Delayed Adverse Events guidance document on the duration and design of 
LTFU observations (Ref. 1).  The Agency advised sponsors to observe subjects for 
delayed adverse events for as long as 15 years following exposure to the investigational 
GT product, specifying that the LTFU observation should include a minimum of five 
years of annual examinations, followed by ten years of annual queries of study subjects, 
either in person or by questionnaire.  
 
Herein, we update our recommendations in the guidance taking into account the clinical 
experience gained since 2006 in LTFU of investigational GT products (Refs. 8, 9 and as 
described in the following section), and the development of novel GT products with 
emerging technologies such as genome-editing that may be associated with an increased 
risk of delayed adverse events (as described in section III.D of this document).  
 
C.  Experience Gained Through Long Term Follow-up of Subjects in Gene 
Therapy Trials 
 
To date, leukemias have been reported in more than one trial where subjects have 
received genetically-modified cells that were manufactured using gammaretroviral 
vectors (Refs. 10-13).  Advances in analytical approaches for integration site analysis in 
patient samples collected during LTFU have provided insight into the possible 
mechanisms involved in the occurrence of such delayed adverse events (Refs. 10-16).   
 
Past clinical experience in LTFU monitoring, and significant improvements in analytical 
approaches to investigate the integration site have contributed greatly towards our 
understanding of the risks associated with integrating gene therapy vectors (Ref. 17).  
Such risks can be mitigated through improvements in vector design and the duration and 
design of LTFU observations.  Because integrating gene therapy vectors can persist in the 
body over the life-span of the patients transduced cells, vectors with an improved risk 
profile were desired, and have subsequently been developed for clinical use (Refs. 18, 
19).  These include gammaretroviral and lentiviral vectors modified:  
 
1.  To reduce the risk of activating host genes adjacent to the integration site 
(e.g., self-inactivating (SIN) vectors and vectors containing insulator 
sequences);  
 
2.  To be less genotoxic (e.g., carrying non-viral physiological promoters to 
drive the expression of the therapeutic gene); and  
 
3.  To reduce the potential for recombination, and thereby, the risk of 
generating replication competent, pathogenic variants. 
4  Contains Nonbinding Recommendations 
 
D.  Long Term Follow-up for Novel Gene Therapy Products 
 
Novel GT products developed as a result of emerging technologies, such as transposon-
based gene insertion and genome editing, also raise concerns for delayed adverse events 
due to the unique genome modifying activity of such products.  Specifically, a vector 
with a transposon element can insert transgenes into the host chromosome randomly by a 
direct cut-and-paste mechanism, mediated by the transposases (enzyme) activity in the 
product (Ref. 20).  A GT product with genome editing components can give rise to non-
specific off-target changes in the genome (Ref. 2), and may be associated with unknown 
and unpredictable risks for developing delayed adverse events in study subjects and 
patients (once approved), the extent of which will vary depending on the targeting 
mechanisms accompanying these components.  The LTFU observations for these novel 
GT products should be designed to take into account product-specific characteristics, the 
basic and translational knowledge generated in the field, and the product-specific 
preclinical data generated to enable investigational new drug application (IND) studies, 
as described in the following section.  
 
 
IV.  PRECLINICAL DATA USED FOR ASSESSMENT OF DELAYED RISKS IN 
GENE THERAPY CLINICAL TRIALS  
 
A.  Framework to Assess Potential Delayed Risks of Gene Therapy Products 
 
To assess the risk of delayed adverse events for a GT product, we recommend that you 
use available preclinical and clinical evidence, and current information about your 
product and similar products based on studies that you and others have performed.  In 
general, when the risk of delayed adverse events is low following exposure to a GT 
product, LTFU observations are not recommended.  We consider the assessment of risk 
to be a continuous process; as more data accumulates, we recommend that you reassess 
the risk to your subjects and, if appropriate, revise your existing LTFU observations 
study protocol or initiate a LTFU observations study protocol, if previously allowed to 
proceed without an LTFU observations study protocol.  
 
Preclinical and clinical experience with your product or similar products may be 
considered relevant in the assessment of the risk for delayed adverse events.  For 
example, experience with GT products in the same vector type5, administered by a 
similar route, or given for the same clinical indication may contribute helpful 
information. However, for novel products such information may not be available or 
applicable, or may be limited, in which case data from well-designed preclinical studies 
(as described in section IV.B of this document) should be used in assessing the risk of 
delayed adverse events.  Primary data and information relevant to the assessment of the 
risk of delayed events should be submitted in your IND along with other preclinical data 
(see 21 CFR 312.23(a)(8), 312.23(a)(10)(iv), and 312.23(a)(11)).  
 
                                                 
5 See Table 1 for examples of vector types. 
5  Contains Nonbinding Recommendations 
 
GT product knowledge is critical in assessing the level of risk for delayed adverse events 
and the need for LTFU observations.  To help you in this process, we refer you to section 
III.A of this document, and to the series of questions in Figure 1, Framework to Assess 
the Risk of Gene Therapy-Related Delayed Adverse Events. 
 
Figure 1.  Framework to Assess the Risk of Gene Therapy-Related Delayed Adverse Events 
 
 
1 If you have evidence that suggests that the product may integrate or if the product was intentionally 
designed to facilitate integration (please refer to Table 1, section IV.C of this document); the answer is 
yes.  
2 See section V. of the text for recommendations on how to perform clinical LTFU observations. 
 
Note, that evidence from preclinical studies will help you answer questions 3 through 5 
below and in Figure 1.  When the risk of delayed adverse events is low based on your 
answers to these questions, a plan for LTFU observations may not be necessary to 
mitigate risks to subjects.  
 
We suggest you use the framework in Figure 1 by answering the questions in sequence as 
follows:  
 
Question 1:  Does your GT product utilize genome-editing technology? 
 
If the answer is no, go to Question 2.  If the answer is yes, all your clinical 
protocols proposing administration of the GT product should include LTFU 
observations for appropriate human subject protections (see section V. for 
recommendations on how to perform clinical LTFU observations).  
 
Question 2:  Is your vector used only for ex vivo modification of cells?  
 
6  Contains Nonbinding Recommendations 
 
If the answer is no, go to Question 3.  If the answer is yes, go to Question 4.  
 
Question 3:  Do preclinical study results show persistence of the GT product? 
 
If the answer is no, the risk of product-related delayed adverse events is low, 
and LTFU observations may not be needed.  If the answer is yes, go to 
Question 4.  
 
If it is unknown whether your GT product persists, for the purpose of assessing 
the risk of delayed adverse events, we recommend that you either assume that the 
GT product does persist, or perform preclinical studies to assay for the GT 
product persistence in a relevant animal species (as described in section IV.B of 
this document and Ref. 21).  For recommendations concerning the design and 
details of such preclinical studies, please refer to section IV.B of this document. 
Specifically, we recommend the polymerase chain reaction (PCR) assay for 
determining vector persistence in biodistribution studies.  Following 
administration of the product, persistence is indicated by detectable levels of GT 
product sequences above the threshold level  (<50 copies/g genomic DNA) of 
the PCR assay, and absence of an apparent downward trend over several time 
points. In contrast, persistence is unlikely if product sequences cannot be detected 
with a sensitive assay such as PCR or if the assay for GT product sequences 
demonstrates a downward trend over time.  We encourage you to consult with the 
Office of Tissues and Advanced Therapies (OTAT) at the Center for Biologics 
Evaluation and Research (CBER) for specific advice regarding determination of 
GT product persistence and biodistribution in your test system.  
 
Question 4:  Are your vector sequences integrated or is the human genome 
otherwise genetically altered?  
 
If the answer is no, go to Question 5.  If you have evidence that suggests that 
the product may integrate or if the product was intentionally designed to facilitate 
integration (please refer to Table 1, section IV.C of this document); the answer is 
yes.  If the answer is yes, all your clinical protocols proposing administration 
of the GT product should include LTFU observations for appropriate human 
subject protections (see section V. for recommendations on how to perform 
clinical LTFU observations).  
 
Question 5:  Does the GT product have the potential for latency and 
reactivation?  
 
If the answer is no, the risk of product-related delayed adverse events is low, 
and LTFU observations may not be needed.  If the answer is yes, all your 
clinical protocols with the GT product should include LTFU observations for 
appropriate human subject protections (see section V. for recommendations on 
how to perform clinical LTFU observations).  
 
7  Contains Nonbinding Recommendations 
 
Laboratory and preclinical evidence of a low risk of delayed adverse events following 
exposure to a similar GT product may show that LTFU observations for your GT product 
are not needed.  When such data/information is made available for review, we can assess 
their relevance to your product if you provide adequate details and a clear explanation of 
similarities and differences between the two products.  For additional guidance, we 
provide the following two examples:  
 
  Your GT 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 in your program. In 
this case, reference to a published study demonstrating lack of persistence 
of the vector sequence for the similar (plasmid) product may adequately 
address concerns regarding the persistence of the proposed vector (your 
plasmid). 
 
  Your GT product and the similar product differ only with respect to route 
of administration.  The similar product was administered into tumors 
(intratumorally).  Your GT product is to be administered intravenously. 
There is a published study demonstrating the lack of persistence of the 
similar product when administered intratumorally.  In this case, the data is 
not sufficiently relevant to the GT product under study, since there was no 
intended systemic exposure to the product.  Thus, there is insufficient 
similarity to conclude that LTFU observations are not warranted in your 
proposed study to mitigate the 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 product persistence in a preclinical study with 
the proposed GT 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 either the GT product is unlikely to persist in human hosts, or 
the vector sequence does not integrate into the human genome and the GT product does 
not have the potential for latency and reactivation, you may decide to submit a clinical 
protocol that does not provide for LTFU observations.  We will review such submissions 
and, if based upon our review of your submission or other additional information, we 
conclude that LTFU observations for delayed adverse events are necessary to mitigate 
long term risks, and that without LTFU observations, the study presents an unreasonable 
and significant risk to study subjects, we may place your study on clinical hold (21 CFR 
312.42(b)(1)(i) and 312.42(b)(2)(i)).  
 
We provide the following examples of evidence obtained from investigation of a product 
that may warrant our recommendation of LTFU observations for delayed adverse events:  
 
  A preclinical toxicology study indicates that expression of the therapeutic 
gene (the transgene in your product) is associated with delayed toxicity.  
8  Contains Nonbinding Recommendations 
 
  The therapeutic gene provides functional replacement of a host gene that 
is otherwise not expressed, and the therapeutic protein is potentially 
immunogenic.  
 
  Data collected in a clinical study with your GT product indicates product 
persistence, even though data from your preclinical studies suggested that 
the product did not persist.  
 
  Data collected in a clinical study with your GT product identifies an 
increased risk of delayed adverse events. 
 
B.  Considerations for Preclinical Study Design to Assess Biodistribution and 
Persistence of Gene Therapy Product 
 
As discussed in section III.A of this document, product persistence heightens the risk of 
delayed adverse events following exposure to the GT product.  Indeed, the longer the GT 
product persists, the greater the duration and degree of risk of delayed adverse events.  
We recommend that you perform preclinical biodistribution studies using methods shown 
to be sensitive and quantitative to detect product sequences.  Such studies would be 
designed to determine the distribution of your product in non-target tissues and the 
persistence of the product in both non-target and target tissues following direct in vivo 
administration of the product.6  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 or 
to therapeutic components in the product (e.g., for products that may not contain 
transgenes and only genome editing components) (Ref. 21).  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 such observational 
studies are possible.  
 
A biodistribution study in animals can be performed either as a separate study or as a 
component of a pharmacology or toxicology study.7  We recommend that you consider 
the following points in your animal study design to permit evaluation of GT product 
localization and persistence (Ref. 22).  
 
   
                                                 
6 In contrast to biodistribution assessment, shedding describes how the vector product is excreted or released from 
the patients body (Guidance for Industry: Design and Analysis of Shedding Studies for Virus or Bacteria-Based 
Gene Therapy and Oncolytic Products; August 2015 available at https://www.fda.gov/media/89036/download ).   
7 For additional information on this topic refer to the 2018 International Pharmaceutical Regulators Programme 
(IPRP) reflection paper, titled, Expectations for Biodistribution (BD) Assessments for Gene Therapy (GT) 
Products (http://development.iprp.backend.dev6.penceo.com/sites/default/files/2018-
09/IPRP_GTWG_ReflectionPaper_BD_Final_2018_0713.pdf). 
9  Contains Nonbinding Recommendations 
 
1.  Animal Study Design  
 
a.  Use the GT product in the final formulation for the clinical study 
because changes in the final formulation may alter biodistribution 
pattern.  
b.  Use both sexes or justify the use of a single sex.  
c.  Use at least 5 animals per sex per group per sacrifice time point for 
rodents, and between 3-5 animals per sex per group per sacrifice 
time point for non-rodents.  
d.  Consider factors in the study design that might affect the GT 
product distribution and/or persistence such as the animals age 
and physiologic condition.  
e.  Use the intended clinical route of GT product administration, if 
possible.  
f.  Assess GT product biodistribution in a vehicle control group and a 
group of animals that receives the maximum proposed clinical 
dose level.  Studies at additional dose levels might provide 
information on dose-dependent effects of your product. 
g.  Inclusion of appropriate safety endpoints in your biodistribution 
study may be helpful to assess any potential correlation between 
product presence/persistence and adverse findings .  These 
endpoints can include clinical observations, body weights, clinical 
pathology, gross organ pathology, and histopathology.  
h.  Include several sacrifice intervals to characterize the kinetics of 
GT product distribution and persistence.  We recommend sacrifice 
of animals at the expected time of peak GT product detection and 
at several later time points to evaluate clearance of product 
sequences from tissues.  
 
2.  Tissue Collection and Analysis  
 
a.  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 product, vector type and tropism, and transgene(s), as well as 
the route of administration (e.g., draining lymph nodes and 
contralateral sites for subcutaneous/intramuscular injection, bone 
marrow, eyes, etc.).  
b.  Choose a method for tissue collection that avoids the potential for 
cross contamination among different tissue samples (i.e., in order 
of least to highest expected presence of vector).  
c.  Use a quantitative, sensitive assay such as quantitative PCR 
(qPCR), 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 analytical 
10  Contains Nonbinding Recommendations 
 
technologies are constantly changing, and encourage you to 
discuss the assay methodology with us before initiating sample 
analysis.  Our current qPCR recommendations include the 
following:  
 
i.  The assay should have a demonstrated limit of quantitation 
of <50 copies/g genomic DNA, so that your assay can 
detect this limit with 95% confidence. 
ii.  The DNA samples should be run in triplicate for each 
tissue. To aid the interpretation of the qPCR assay results, 
one replicate of each tissue sample should include a spike 
of control DNA, including a known amount of the vector 
sequences. The spike control will determine the specified 
qPCR assay sensitivity.  
    
iv.  In the final study report, individual animal data should be 
provided. The method for how values below the Limit of 
Quantitation of the assay are categorized and calculation of 
the median or mean value should be specified. 
 
3.  Other Considerations  
 
There are many variables that will affect the outcome and interpretation of 
the in vivo assessment of each GT product type.  Hence, we encourage 
you to discuss with OTAT the study design for your GT product in a pre-
IND meeting before initiating the preclinical biodistribution study to 
ensure that both biodistribution and persistence will be adequately 
assessed.8 
 
C.  Vector Persistence, Integration, and Reactivation and Genome Modification: 
Assessing Long Term Risks 
 
GT products may or may not use technologies that modify the host genome.  For products 
that do, such as integrating vectors (gammaretrovirus, lentivirus, foamy virus etc.), 
herpesvirus capable of latency-reactivation, and genome editing products (as described 
under sections III.A and III.D of this document, respectively), there is the risk of delayed  
adverse events.  Accordingly, as depicted in Table 1 of this document and in the answer 
to Question 4 in Figure 1, it is important to conduct LTFU observations to mitigate 
delayed risks to subjects receiving GT products with integrating activity.  
 
                                                 
8 The preclinical program for any investigational product should be individualized with respect to scope, complexity, 
and overall design, to maximize the contribution and predictive value of the resulting data for clinical safety and 
therapeutic activity.  We encourage sponsors to explore opportunities for reducing, refining, and replacing animal 
use in the preclinical program.  For example, it may be appropriate to use in vitro or in silico testing to complement 
or replace animal studies.  Sponsors are encouraged to submit proposals and justify any potential alternative 
approaches, which we will evaluate for equivalency to animal studies.  
11  Contains Nonbinding Recommendations 
 
We are aware that the potential of vectors to integrate may be modified to increase their 
utility as gene therapy agents; for example, a vector can be modified to induce integration 
of its DNA (Refs. 23-26).  Another example would be changes in the methods used to 
introduce plasmid DNA vectors into cells that result in higher integration frequencies 
(Ref. 27).  In those cases where a modification of the GT product may have altered its 
persistence or integration properties, we recommend that you submit data to your IND 
from preclinical studies to assess vector persistence in an appropriate model and take one 
of the following actions:  
 
1.  If the vector is not persistent, the predicted risk of delayed adverse events 
would appear to be low in which case LTFU observations may not be 
needed.  
 
2.  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.  
 
3.  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.  LTFU observations may not be needed. 
 
4.  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.  LTFU observations may be 
recommended for human subject protections. 
 
5.  If preclinical studies of vector integration are not feasible, if the 
therapeutic gene/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, then LTFU observations in study subjects are 
recommended for human subject protection. 
 
6.  If vector integration studies are not performed, we recommend that you 
provide other evidence to support an assessment that your product does 
not pose high risks of delayed adverse events, including the following: 
 
a.  A discussion of why vector integration studies were not performed.  
b.  The evidence supporting your assessment of the risk of delayed 
adverse events posed by your product. 
 
As stated in section IV.B.3 of this document, we encourage you to discuss with FDA 
your study design before starting the trial.  
 
GT products that are based on vectors such as plasmids, poxvirus, adenovirus, and adeno-
associated virus vectors (AAV) that do not have a propensity to integrate or reactivate 
12  Contains Nonbinding Recommendations 
 
following latency, generally present a lower risk of delayed adverse events.  Clinical data 
from LTFU observations of subjects that have received plasmids, poxvirus, adenovirus, 
and AAV in trials conducted since 2006, further supports the assessment of lower risk for 
these GT products.  However, vector or product-specific modifications may alter the risk 
profile of products that are currently considered lower risk, for example a plasmid that is 
modified to carry genome editing components.  Conversely, gene therapy vectors 
currently considered to pose delayed risks might be modified in order to reduce those 
risks.  Hence, data supporting decreased or increased risk for delayed adverse events with 
novel GT products or vector types could provide the basis for sponsors to reassess our 
recommendations for performing LTFU observations.  We encourage you to consult with 
OTAT regarding a reassessment of our recommendations for performing LTFU 
observations. 
   
13  Contains Nonbinding Recommendations 
 
Table 1.  Propensity of Commonly Used Gene Therapy Products/Vectors to Modify the 
Host Genome  
 
Product/Vector 
Propensity to Modify Genome1  Long Term Follow-up Observations2 
Type 
Plasmid  No  No 
RNA  No  No 
Poxvirus  No  No 
Adenovirus  No  No 
Adeno- 
No  Product specific 
associated virus3 
No, but may undergo 
Herpesvirus  Yes 
latency/reactivation 
Gammaretrovirus  Yes  Yes 
Lentivirus  Yes  Yes 
Transposon 
Yes  Product specific 
elements   
Microbial vectors 
No, but may persist and undergo 
for gene therapy  Product specific 
reactivation 
(MVGT)4 
Genome editing  Yes; permanent changes to the 
Yes  
products  host genome  
1 Based on product design (i.e., lack of any known mechanism to facilitate integration or genome editing), as well as 
cumulative preclinical and clinical evidence suggesting that a GT product does not integrate into or edit the genome 
or integrates in/modifies the genome at very low frequencies.  
2 Specific circumstances that indicate persistent expression of the transgene, in the absence of integration or genome 
editing, may be the basis for a conclusion that LTFU observations are recommended 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 this section.  
3 Replication-negative vectors only.  
4 For additional guidance we refer you to Recommendations for Microbial Vectors used for Gene Therapy; 
Guidance for Industry dated September 2016, 
https://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/CellularandG
eneTherapy/default.htm. 
   
14  Contains Nonbinding Recommendations 
 
D.  Considerations for Preclinical Evaluation of Products that Involve Genome 
Editing  
 
Genome editing, whether ex vivo or in vivo, introduces the risk for delayed adverse 
effects, due to 1) the permanent nature of change; 2) the potential for off-target genome 
modifications that can lead to aberrant gene expression, chromosomal translocation, 
induce malignancies, etc.; 3) the risk for insertional mutagenesis when integrating vectors 
are used to deliver the genome editing components, and the associated risk of 
tumorigenicity; and/or 4) the possibility of an immune response to the genome-editing 
components or the expressed transgene.  Preclinical safety evaluation of genome editing 
products should consider:  1) the technology used to edit the genome; 2) the target cell 
types that are modified; 3) the genomic sites that are modified, 4) the vector used to 
deliver the genome-editing components; and 5) the clinical route of administration.  
Preclinical studies evaluating these factors can inform the scope of the clinical LTFU 
observations. 
 
For guidance on the biodistribution studies when considering the vector type in the 
genome edited product, and the related long term risks with integrating vectors, we refer 
you to sections IV.B and IV.C of this document. 
 
 
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 LTFU 
observations for delayed adverse events in study subjects receiving investigational GT products. 
Typically, LTFU observations are conducted under a protocol (LTFU protocol) that is separate 
from the main study protocol or as an extension of the main protocol study, and may begin 
immediately after the first subject completes their last visit in the main study and enrolls in the 
LTFU study.   
 
A.  Goals of the Long Term Follow-up Observations 
 
The objective of LTFU observations in clinical development of a GT product is to 
identify and mitigate the long term risks to the patients receiving the GT product.  The 
LTFU protocol for gene therapy trials is primarily designed to capture delayed adverse 
events in study subjects as well as to understand the persistence of the GT product.  As a 
sponsor, you may consider designing the LTFU protocol to assess the long term clinical 
efficacy, and durability of your product.  For additional guidance on trial design for GT 
products we refer you to FDAs guidance document Considerations for the Design of 
Early-Phase Clinical Trials of Cellular and Gene Therapy Products; Guidance for 
Industry dated August 2015 (Ref. 28).   
 
   
15  Contains Nonbinding Recommendations 
 
B.  Clinical Trial Populations for Long Term Follow-up Observations 
 
When a GT product is deemed to pose a risk for delayed adverse events (based on the 
recommendations/discussions provided under sections III and IV of this document) and a 
decision to perform LTFU observations is made, all study subjects who receive the GT 
product are expected to be enrolled in the LTFU protocol (as discussed in Section V.C of 
this guidance) after signing an informed consent document.  LTFU observations may 
have reduced utility in assessing and mitigating subject risk when the population selected 
for the trial has characteristics that could confound the observation of the delayed adverse 
events, such as short life expectancy, multiple co-morbidities, and exposure to other 
agents such as radiation or chemotherapy.  In contrast, LTFU 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 co-morbidities and limited exposures to other agents 
with potential for delayed adverse events.  Hence, characteristics of the patient population 
and the disease to be treated should be considered when designing a LTFU protocol. 
 
C.  Duration of Long Term Follow-up Observations  
 
It is important that the design of LTFU observations be appropriate to detect potential 
gene therapy-related delayed adverse events in the subjects enrolled in your clinical 
studies.  The duration of LTFU should be sufficient to observe the subjects for risks that 
may be due to the characteristics of the product, the nature of the exposure (e.g. route of 
administration and the biodistribution profile), and the anticipated time of occurrence of 
delayed adverse events.  Elements that will influence the determination of the duration of 
LTFU observations include the following:  
 
  The observed duration of in vivo product persistence.  
  The observed duration of transgene expression. 
  Product characteristics in vivo. 
  Route of administration. 
  The expected survival rates and the known background rates of the events 
of interest occurring in the study population. 
  Other factors that may be relevant to the feasibility and scientific value of 
conducting LTFU observations; for example, the durability of the clinical 
effect. 
 
In general, our current recommendations for the duration of a LTFU protocol based on 
product type are as follows: 
 
  Fifteen years for integrating vectors such as gammaretroviral and lentiviral 
vectors and transposon elements. 
  Up to fifteen years for herpes virus vectors (or oncolytics) that are capable 
of establishing latency. 
  Up to fifteen years for microbial vectors that are known to establish 
persistent infection.  
   
16  Contains Nonbinding Recommendations 
 
  Up to fifteen years for genome editing products.  
  Up to five years for AAV vectors. 
 
Additionally, a risk-based approach for determining the duration of a LTFU protocol may 
be considered for vectors capable of latency (e.g., Herpesvirus), long term expression 
without integration (e.g., AAV), or vectors carrying gene editing components. 
 
Although these recommendations are broadly based on GT product type, you should also 
consider the elements listed above, in this section, as it applies to your GT product, 
disease characteristics, and the patient population, in addition to the discussions in 
sections III. and IV. of this document.  
 
To reduce the unnecessary burden to study subjects and to you as the study sponsor, it 
may be appropriate to modify the duration of the LTFU observation based on your 
ongoing assessment of product persistence, transgene expression, and clinical findings.  If 
you intend to modify the duration of the follow-up, you may submit an amendment to 
your IND justifying the change to your LTFU protocol, and communicate with FDA to 
reach a final decision (we refer you to section V. of this document for additional guidance 
regarding amendments to the clinical protocol). 
 
FDA  recommends sponsors make every effort to prevent patient loss to follow-up to the 
extent feasible for completion of LTFU observations. 
 
D.  Elements of Long Term Follow-up Observations 
 
We recommend that at least the following general elements be part of the LTFU protocol: 
 
  You should establish a dedicated clinical LTFU protocol detailing patient 
visit schedules, sampling plan (for patient test samples, such as blood), 
methods of monitoring tests, and clinical events of interest that will be 
monitored over the entire LTFU observation.  
 
  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 
investigational product in which all diseases, conditions and physical 
abnormalities are recorded.  A template for health care providers (HCPs) 
who are not investigators or sub-investigators (for example, the subjects 
physician, physician assistant, or nurse practitioner) to use in recording 
and reporting such observations to the investigator may be helpful for such 
HCPs.  Case histories should also include information from scheduled 
visits with a HCP and test results for persistent vector sequences.  The use 
of surrogate tests may be necessary to indicate vector persistence if direct 
sequence testing involves an invasive procedure for the subject.  If 
17  Contains Nonbinding Recommendations 
 
surrogate tests are considered, we recommend that you consult with FDA 
regarding the types and characteristics of the surrogate tests you intend to 
use before including them in your study.  
 
In addition, for the first five years or more (as applicable to your product), we 
recommend that you do the following:  
 
  Ensure that investigators maintain, in the case history, a detailed record of 
exposures to mutagenic agents and other medicinal products, and have 
ready access to information about their adverse event profiles. 
 
  Establish a method for investigators to record the emergence of new 
clinical conditions, such as:  
-  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. 
-  New incidence of infection (potentially product-related) 
 
  Design a plan for scheduled visits with an HCP to elicit and record new 
findings for each study subject, including history, physical examination, or 
laboratory testing, as applicable to the study population. 
-  Such a plan needs to facilitate reporting of delayed adverse events, 
including unexpected illness and hospitalization by study subjects 
and HCPs.  
 
For the subsequent period of LTFU (applicable to products for which such length LTFU 
is needed), 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 LTFU protocol provides for additional specific screening, you may 
arrange to contact subjects by telephone or written questionnaire rather 
than by office visits with an HCP.  
 
  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 LTFU observations according to FDA regulations governing clinical trials 
(Ref. 29).  
 
We provide additional specific recommendations and requirements for data collection, 
recording, and reporting of adverse events for LTFU observations as follows: 
18  Contains Nonbinding Recommendations 
 
1.  Detection of Adverse Events and Coordination of Data Collection9   
 
a.  To facilitate detection of delayed adverse events, we recommend 
that the LTFU protocol identify suitable HCPs (e.g., subjects 
primary healthcare provider) whose observations would be used in 
the assessment of the occurrence of adverse events in the study 
population.  Suitable HCPs might include physicians, physicians 
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. However, the investigators are responsible for 
reporting appropriate adverse events to the sponsor.  (21 CFR 
312.64(b)).   
 
b.  To increase subject compliance and improve the quality of data 
collection, we suggest that you encourage study subjects to be 
proactive in reporting adverse events.  Tools 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.  
 
c.  To determine the causality of potential related adverse events (such 
as tumor formation) associated with your GT product, you should 
propose a clinical program for follow-up procedures.  Such a 
program would lay out the efforts that would be needed among the 
study subjects, HCPs, investigators, and the sponsor for study 
coordination.  This includes the collection of tissue samples for 
follow-up analysis, obtaining informed consent for a biopsy or 
autopsy (see section V.E. of this document), communicating with 
the study subject, and preserving and analyzing the tissues/samples 
according to the LTFU protocol.  You may propose specific tests 
to enable causality analyses such as general blood work, 
cytogenetic and histological analysis, PCR, HLA typing, or deep 
sequencing. 
 
2.  IND Safety Reports  
 
You must follow applicable reporting requirements outlined in 21 CFR 
312.32 for adverse events associated with the use of the investigational 
product.  As the LTFU observations proceed, you must notify FDA and 
each participating investigator of any serious and unexpected suspected 
adverse reaction (21 CFR 312.32(c)(1)(i)), and findings from other studies 
(21 CFR 312.32(c)(1)(ii)).  In each IND Safety Report (required to be 
                                                 
9 To improve the detection of adverse events as well as improve the coordination of data collection for LTFU, any 
follow up procedures involving study subjects who are children and/or otherwise unable to communicate, should 
include the study subjects legally authorized representative, e.g. guardians, as defined in 21 CFR 50.3. 
19  Contains Nonbinding Recommendations 
 
provided to investigators and FDA), you must identify all safety reports 
previously filed concerning a similar adverse finding, and analyze the 
significance of the adverse finding in light of the previous, similar reports 
(21 CFR 312.32(c)(1)).  You must promptly investigate all safety 
information you receive (21 CFR 312.32(d)(1)).  If the relationship of the 
adverse event to the GT product is uncertain, additional investigations 
may be needed.  You must also revise your informed consent document 
and Investigator Brochure to include the new adverse event(s) that may be 
associated with the product or study procedures (21 CFR Part 50, 21 CFR 
312.55(b)).  You must inform all clinical investigators of the newly 
identified risk (21 CFR 312.32(c)(1)).  
 
3.  Annual Reports to the IND/Summary Information  
 
  While the IND is in effect and LTFU observations are ongoing, you must 
file an annual report (21 CFR 312.33).  It is recommended that the annual 
report contain an independent section with a subtitle for Long Term 
Follow-Up (See Appendix 1 of this document).  In that report, you should 
submit information obtained during the previous year's clinical and 
nonclinical investigations, including, 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)).  If adverse reactions are reported 
and determined to be related to your product or delivery procedure, you 
should provide causal analyses based on evidence from clinical, 
laboratory, molecular, cytogenetic, histological, or HLA analysis, or deep 
sequencing data. Please refer to Appendix 2 of this document for the 
LTFU Annual Report Template. You may reference existing IND or 
Development Safety Update Report (DSUR) subsections (e.g. preclinical 
and clinical updates) for supporting information. In lieu of annual reports, 
you may submit aDSUR.  In this case, you should provide the LTFU 
information in a subsection with a subtitle for Long-Term Follow-Up in 
your DSUR report (Ref. 30).  
 
4.  Amendments to the Clinical Protocol  
 
  If clinical data suggest that your GT product is not associated with delayed 
risks or there is no evidence of vector persistence, you may want to 
consider revising the clinical protocol regarding LTFU of study subjects. 
However, before implementation of this change, we recommend that you 
consult with FDA and provide your rationale with supporting clinical and 
laboratory data (we refer you to section V.C of this document for 
additional guidance).  You must submit to FDA a protocol amendment to 
your IND indicating the relevant changes (21 CFR 312.30(b)(1), (d), and 
(e)). 
 
20  Contains Nonbinding Recommendations 
 
 
5.  Scheduled Physical Examinations  
 
We recommend that LTFU observations include scheduled physical 
examinations performed by a HCP once a year during the first five years 
(or until the completion of LTFU if the LTFU is less than five years), 
unless the assessed risks associated with your GT product indicate that 
they should be done more frequently.  For example, if a subject exposed to 
your GT 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 semi-annual or quarterly basis, or more 
frequently as clinically indicated.  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.  LTFU observations 
are intended to collect data on delayed adverse events related to the GT 
product, and are not intended to provide evaluation or treatment data for 
the underlying disease.  
 
6.  GT Product Persistence 
 
During LTFU observations, we recommend that you test study subjects at 
least annually for persistent vector sequences until they become 
undetectable.  More frequent testing may be necessary as outlined in 
section V.G of this document.  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 
collecting the transduced cell population may involve an invasive 
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 LTFU protocol as a protocol 
amendment to your IND.  In any such protocol amendment, include an 
assessment of risks associated with your GT product and an evaluation of 
the impact of the waning persistence of the vector on those risks (21 CFR 
312.30(b) and (d)(2)).  
 
E.  Informed Consent in Trials Involving Long Term Follow-up Observations  
 
Each subject in a clinical investigation must be provided with a description of any 
reasonably foreseeable risks from participating in the investigation (21 CFR 50.25(a)(2)).  
The informed consent document must describe, among other things, the purposes of the 
21  Contains Nonbinding Recommendations 
 
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 LTFU 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).  
 
When appropriate, the informed consent document must be updated to describe any 
adverse reactions that may be associated with the product from your trial or other human 
or animal (preclinical) studies (21 CFR 50.25(b)(5)).  If the sponsor intends to store blood 
or tissue samples for future testing, the informed consent document must convey this 
information (21 CFR 50.25(a)(1)).  The informed consent  should also convey that an 
autopsy may be requested to test vector persistence, transgene expression, and related 
adverse reactions at the molecular, cellular or tissue level if there are deaths during the 
LTFU observation.  Sponsors must ensure that investigators submit the informed consent 
documents for Institutional Review Board review and approval (21 CFR 
312.53(c)(1)(vi)(d)). 
 
We provide additional informed consent recommendations for retroviral vectors in 
section V.F.3 of this document. 
 
F.  Special Considerations Regarding Integrating Vectors  
 
The recommendations in this section apply exclusively to subjects in clinical trials who 
received GT products that are integrating vectors, such as transposon elements, 
gammaretroviral, lentiviral, other retroviral vectors, or GT products that are cells modified 
ex vivo by integrating vectors or transposon-based vectors.  See section VI. for post 
licensure considerations.  Because of the risk of developing leukemias and premalignant 
conditions (clonal cell expansion) due to integration of gammaretroviral vectors and 
lentiviral vectors (as described in sections III.B and III.C of this document), we are also 
providing additional recommendations (as listed below) for collection of data in studies 
in which subjects are exposed to integrating vectors.   
 
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 the 
dominant clone(s) persist, assess what the relative contribution of the 
dominant clone(s) is, and whether the clonal outgrowth observed results in 
any (hematologic) malignancies).  We consider an assessment of the 
vector integration pattern to be relevant in subjects in gene therapy clinical 
trials involving integrating vectors when:  (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 
22  Contains Nonbinding Recommendations 
 
cells are 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.  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 subjects 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 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, 31-33), 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:  
23  Contains Nonbinding Recommendations 
 
 
i.  Persistent monoclonality;  
ii.  Clonal expansion (e.g., the percent cells positive for a 
particular vector integration site is shown to increase over 
multiple time points); or  
iii.  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.  
 
For retroviral (e.g., gammaretroviral and lentiviral) vector-based GT products, additional 
follow-up monitoring for the presence of replication competent retrovirus (RCR) may be 
necessary.  For details regarding duration of the follow-up monitoring for RCR and 
methods, please refer to the document Testing of Retroviral-Based Human Gene 
Therapy Product for Replication Competent Retrovirus During Product Manufacture and 
Patient Follow-up; Draft Guidance for Industry dated July 2018.  
 
We recommend that GT products with transposon elements be monitored in a similar 
way as gammaretroviral or lentiviral vectors.  This recommendation is based on the 
potential safety risk of insertional mutagenesis due to the random integration directed by 
the transposon, and due to the potential for remobilization of a transposon (secondary 
transposition-insertion event) as a result of the continuing presence of the transposase 
enzyme in target cells.  Yet, if your GT product contains transposon elements you may 
propose shorter LTFU observation by providing adequate supporting data/information 
related to your product. 
 
2.  Data Reporting  
 
If no evidence of oligoclonality 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 oligoclonality 
or monoclonality is observed, you must 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 of receiving 
the report of such an observation.  
 
3.  Informed Consent in Trials Involving Retroviral Vectors  
 
Please see section V.E for general consideration of LTFU observation 
informed consent.  In accordance with 21 CFR 50.25(a)(2), for all clinical 
trials in which subjects are exposed to retroviral vectors, the informed 
consent documents must include current, complete and accurate disclosure 
of the development of leukemias in the clinical trials where such adverse 
24  Contains Nonbinding Recommendations 
 
events were reported.  Further, the information that is given to the subject 
or his/her representative must be in language understandable to the subject 
or representative (21 CFR 50.20).  We provide the following list as 
information and language we recommend be included in the informed 
consent document, where applicable, in the section describing the risks 
associated with the study agent:  
 
a.  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.  
b.  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.  
c.  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.  However, in some cases, abnormal activity of a 
gene may cause unpredictable harm such as the development of 
cancer. 
d.  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. 
Clinical studies were conducted in France and United Kingdom to 
treat 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).  One child died from the cancer.  A group of 
experts in this field studied the results from tests performed on 
these childrens blood cells.  They concluded that cancer was 
caused by the retroviral vector DNA. Still, most of the children 
with X-linked SCID who have received experimental gene therapy 
have not been found to have cancer at this time.  Although they 
appear healthy, we still do not know whether they, too, will 
develop cancer.  
e.  Risk of malignancy for this study - We do not know if the 
retroviral vector used in this protocol might cause cancer.  
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 cancer months 
to years later.  
 
   
25  Contains Nonbinding Recommendations 
 
G.  Special Considerations Regarding Product Involving Genome Editing  
 
While the general principles for LTFU observations of GT products also apply to LTFU 
observations of genome editing products, we recommend that you consider the following:   
 
1.  Propose a specific plan to monitor for delayed adverse events based on the 
off-target activities noted in your preclinical studies (e.g., in vivo, in vitro 
and in silico analysis such as INDEL, (insertion and deletion of bases in a 
genome).  For example, if the off-target activity involves a tumor 
suppression gene in liver cells, you may propose a monitoring plan for 
evaluation of occurrence of liver cancer as part of the LTFU observation.  
 
2.  Propose a monitoring plan regarding the adverse events from the specific 
organ system that the genome editing targets, that may include history and 
physical examination, general and specific laboratory tests, and imaging 
studies.   
 
3.  If direct monitoring of the target tissue is not ethical or feasible, such as, 
the brain tissue, you may propose an alternative plan for monitoring of the 
products effects. 
 
4.  Quantitate the relationship between the off-target and on-target activities, 
and use the measured level of on-target activity to predict the level of off-
target activity and, if appropriate, establish a follow-up plan;  
 
5.  If the genome editing product is delivered via systemic administration, 
clinical safety monitoring may be directed not only to off-target activity of 
the target organ or tissue, but also to other off-target effects that may occur 
in other tissues and organs.  Accordingly, consider including appropriate 
monitoring tests with a rationale for the proposed monitoring in your 
LTFU protocol. 
 
 
VI.  GENERAL CONSIDERATIONS FOR POST-MARKETING MONITORING 
PLANS FOR GENE THERAPY PRODUCTS  
 
The number of subjects receiving GT products is typically limited during clinical investigations. 
In addition, the recommended LTFU (e.g., 15-year period) will often not elapse for all subjects 
who received an investigational GT product in the pre-marketing program before the product is 
licensed.  Considering that, the safety data generated during clinical trials may not capture all 
possible delayed adverse events.  Therefore, continuing LTFU observations is often essential 
even after a products licensure.  Consequently, we recommend that at the time of your BLA 
submission you submit a Pharmacovigilance Plan (PVP) as described in the FDA Guidance for 
Industry; E2E Pharmacovigilance Planning (Ref. 34).  Of note, the ongoing or the planned LTFU 
study (under an IND) could be a component of the PVP (post-licensure). The contents of PVP 
for a particular GT product will depend on its safety profile and will be based on data, which 
26  Contains Nonbinding Recommendations 
 
includes the pre-licensure clinical safety database, published literature, and known product-class 
effects, among other considerations.  
 
Routine surveillance for licensed biological products includes adverse event (AE) reporting in 
accordance with 21 CFR 600.80 (reporting of expedited and non-expedited AEs as well as 
periodic safety reports).  Submission of reports for serious, life-threatening and unexpected 
adverse events may also be required in an expedited manner beyond routine required reporting. 
 
Additional pharmacovigilance elements may be needed, such as those described in the FDA 
Good Pharmacovigilance Practices and Pharmacoepidemiologic Assessment; Guidance for 
Industry dated March 2005 (Ref. 35), for LTFU of patients treated with GT products.  For 
instance, we may recommend that you establish a registry, or use an existing patient registry, to 
systematically capture and track data from treated patients with solicited sample collection (if 
applicable), and follow-up of adverse events to resolution or stabilization to collect additional 
pertinent data.  It may be appropriate to establish a registry system to specifically capture adverse 
event data from treated patients who receive a GT product.  This registry system can be a part of 
the PVP plan and reviewed at the time of licensure.  
 
For any proposed or required post-marketing studies or clinical trials, we recommend that you 
include in your BLA submission the study protocol, statistical analysis plan, and a projected 
schedule of anticipated study milestones.  Your study protocol should include specific adverse 
events of interest that you intend to evaluate, and the duration of observation for all patients 
enrolled in your post-marketing study. LTFU data collected post-licensure should be submitted 
in the BLA annual report (See Appendix 1 of this document). 
 
During our review of your BLA, we will also assess whether a Risk Evaluation and Mitigation 
Strategy (REMS) is necessary to ensure that the benefits of your product outweigh its risks.  If 
you consider that risk mitigation measures are necessary for the safe use of your product, you 
may voluntarily submit your proposed REMS as described in Format and Content of a REMS 
Document; Draft Guidance for Industry; Drug Safety dated October 2017 (Ref. 36).  
 
If at the time of BLA approval FDA determines it is necessary to initiate LTFU observations, 
you should submit the study protocol to your existing IND, with a cross-reference letter to the 
BLA submission explaining that the protocol was submitted to the IND. 
 
 
VII.  LONG TERM FOLLOW-UP UNDER SPECIAL CIRCUMSTANCES 
 
A sponsor may cease to operate or may decide to inactivate, transfer or withdraw an IND before 
completion of LTFU observations for all subjects exposed to the GT product under its IND.  
Under such circumstances, prior to inactivating, transferring or withdrawing an IND, or ceasing 
to operate, we recommend that a sponsor consult with OTAT on the plans for completion of 
LTFU observation.  
 
 
27  Contains Nonbinding Recommendations 
 
VIII.  DEFINITIONS 
 
The following definitions apply to this guidance:  
 
Engineered site-specific endonucleases:  Enzymes that are capable of precisely cleaving 
(cutting) DNA based on specific recognition of the DNA sequence at or near the site of DNA 
cleavage.  
 
Genome editing:  A process by which DNA sequences are added, deleted, or replaced at 
specified location(s) in the genome using site-specific nuclease-dependent or nuclease-
independent technologies.  
 
Gene transfer:  The transfer of genetic material into a cell.  
 
Human gene therapy:  Human gene therapy seeks to modify or manipulate the expression of a 
gene or to alter the biological properties of living cells for therapeutic use.   
 
Human gene therapy product:  FDA generally considers human gene therapy products to 
include all products that mediate their effects by transcription or translation of transferred genetic 
material or by specifically altering host (human) genetic sequences.  Some examples of gene 
therapy products include nucleic acids (e.g., plasmids, in vitro transcribed ribonucleic acid 
(RNA)), genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-
specific nucleases used for human genome editing,10 and ex vivo genetically modified human 
cells. Gene therapy products meet the definition of biological product in section 351(i) of the 
Public Health Service (PHS) Act (42 U.S.C. 262(i)) when such products are applicable to the 
prevention, treatment, or cure of a disease or condition of human beings.11 
 
Integration (of DNA):  The process whereby exogenous DNA sequences become incorporated 
into a genome. 
 
Latency (of a viral infection):  A period of time during which a virus is present in the host 
without producing overt clinical symptoms.  
 
Persistence:  With respect to transferred or altered genetic material, the continued presence of 
transferred or modified genetic sequences in the host after acute exposure to a gene therapy 
agent, whether due to integration of the genetic sequence into the host genome, deletion, 
insertion, or otherwise modified following genome editing, or to latent infection with the viral 
vector bearing the genetic sequence.  
 
Reactivation (of a viral infection):  The re-emergence of a symptomatic or asymptomatic viral 
infection following a period of latency.  
                                                 
10 Human Genome Editing: Science, Ethics, and Governance.  The National Academies Press; 2017.  
https://www.nap.edu/read/24623/chapter/1#xvii. 
11 See Federal Register Notice:  Application of Current Statutory Authorities to Human Somatic Cell Therapy 
Products and Gene Therapy Products (58 FR 53248, October 14, 1993), 
https://www.fda.gov/media/76647/download. 
28  Contains Nonbinding Recommendations 
 
 
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 product and includes the vector backbone, transgene(s), and 
regulatory elements.  
 
Vector:  A vehicle consisting of, or derived from, biological material that is designed to deliver 
genetic material.  Examples include plasmids, viruses, and bacteria that have been modified to 
transfer genetic material. 
29  Contains Nonbinding Recommendations 
 
IX.  REFERENCES 
 
1.  Guidance for Industry:  Gene Therapy Clinical Trials  Observing Subjects for Delayed 
Adverse Events, November 2006.  https://www.fda.gov/media/72225/download  
2.  Human Genome Editing:  Science, Ethics, and Governance, National Academy Press, 
Washington D.C., 2017.  
3.  Donahue, RE, et al., Helper virus induced T cell lymphoma in nonhuman primates after 
retroviral mediated gene transfer, Journal of Experimental Medicine, 1992;176:1125-
1135. 
4.  Hacein-Bey-Abina, S, et al., Sustained correction of X-linked severe combined 
immunodeficiency by ex vivo gene therapy, N. Engl. J. Med, 2002; 346:1185-1193. 
5.  Biological Response Modifiers Advisory Committee (BRMAC), Meeting Minutes, 
Department of Health and Human Services, Food and Drug Administration, CBER, 
October 10, 2002. 
6.  Biological Response Modifiers Advisory Committee, Meeting Minutes Department of 
Health and Human Services (BRMAC), Food and Drug Administration, CBER, 
November 17, 2000; April 6, 2001; and October 24, 2001. 
7.  Nyberg, K, et al., Workshop on long-term follow-up of participants in human gene 
transfer research, Molecular Therapy, 2004; 6:976-980. 
8.  Cornetta, K, et al., Screening Clinical Cell Products for Replication Competent 
Retrovirus: The National Gene Vector Biorepository Experience, Mol. Ther. Methods 
Clin. Dev., 2008; 10:371-378. 
9.  Marcucci, KT, et al., Retrovial and Lentiviral Safety Analysis of Gene-Modified T Cell 
Products and Infused HIV and Oncology Patients, Mol. Ther., 2018; 26(1):269-279. 
10.  Hacein-Bey-Abina, S, et al., LMO2-associated clonal T cell proliferation in two patients 
after gene therapy for SCID-X1, Science, 2003; 302:415-419. 
11.  Hacein-Bey-Abina, S, et al., Insertional oncogenesis in 4 patients after retrovirus-
mediated gene therapy of SCID-X1, J. Clin Invest, 2008; 118:3132-3142. 
12.  Braun, et al., Gene Therapy for Wiskott-Aldrich SyndromeLong term Efficacy and 
Genotoxicity, Science Translational Medicine, 2014; 6:227. 
13.  Cavazzana-Calvo, et al., Gene therapy of human severe combined immunodeficiency 
(SCID)-X1 disease, Science, 2000; 288:669. 
14.  Howe, et al., Insertional mutagenesis combined with acquired somatic mutations causes 
leukemogenesis following gene therapy of SCID-X1 patients, J Clin Invest, 2008; 
118:143-150. 
15.  Cavazzana-Calvo, et al., Transfusion independence and HMGA2 activation after gene 
therapy of human -thalassaemia, Nature, 2010; 467:318-322. 
16.  Cavazzana-Calvo, et al., Haematopoietic stem cell transplantation for SCID patients: 
where do we stand?, British Journal of Haematology, 2013; 160:146-152. 
17.  Niedere, HA and CRM Bangham, Integration site and clonal expansion in human chronic 
retroviral infection and gene therapy, Viruses, 2014; 6:4140-4164. 
30  Contains Nonbinding Recommendations 
 
18.  Sakuma, T, et al., Lentiviral vectors: Basic to translational Biochem J, 2012; 443:603-
618. 
19.  Maetzig, T, et al., Gammaretroviral vectors: Biology, Technology and Application, 
Viruses, 2011; 3:677-713. 
20.  Aronovich, E, et al., The Sleeping Beauty transposon system: a non-viral vector for gene 
therapy, Hum Mol Genet, 2011; 20:R14-R20. 
21.  Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene 
Therapy Products, November 2013. https://www.fda.gov/media/87564/download  
22.  Bauer, S, Current FDA approach for preclinical vector biodistribution studies, 
Recombinant DNA Advisory Committee Meeting, March 12, 1999. 
23.  Shayakhmetov, DM, 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. 
24.  Goncalves, MA, et al., Stable transduction of large DNA by high-capacity adeno-
associated virus/adenovirus hybrid vectors, Virology, 2004; 321(2):287-296. 
25.  Picard-Maureau, M, et al., Foamy virusadenovirus hybrid vectors, Gene Therapy, 
2004; 11(8):722-728.  
26.  Yant, SR, et al., Transposition from a gutless adeno-transposon vector stabilizes 
transgene expression in vivo, Nature Biotechnology, 2002; 20(10):999-1005. 
27.  Wang, Z, et al., Detection of integration of plasmid DNA into host genomic DNA 
following intramuscular injection and electroporation, Gene Therapy, 2004; 11(8):711-
721.  
28.  Guidance for Industry: Considerations for the Design of Early-Phase Clinical Trials of 
Cellular and Gene Therapy Products, August 2015. 
https://www.fda.gov/media/106369/download  
29.  E6 (R2) Good Clinical Practice: Integrated Addendum to ICH E6 (R1): Guidance for 
Industry, March 2018. https://www.fda.gov/media/93884/download 
30.  E2F Development Safety Update Report; Guidance for Industry, August 2011.  
https://www.fda.gov/media/71255/download 
31.  Ott, MG, 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):401-409. 
32.  Fraietta, JA, et al., Disruption of TET2 promotes the therapeutic efficacy of CD19-
targeted T cells, Nature, 2018; 558(7709):307-312. 
33.  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):463-468. 
34.  E2E Pharmacovigilance Planning; Guidance for Industry, April 
2005. https://www.fda.gov/media/71238/download  
31  Contains Nonbinding Recommendations 
 
35.  Guidance for Industry: Good Pharmacovigilance Practices and Pharmacoepidemiologic 
Assessment, March 2005. https://www.fda.gov/media/71546/download  
36.  Format and Content of a REMS Document; Draft Guidance for Industry, October 2017.*  
https://www.fda.gov/media/77846/download 
 
*When finalized, this guidance will represent FDAs current thinking on this topic. 
   
   
32  Contains Nonbinding Recommendations 
 
APPENDICES 
 
APPENDIX 1:  INFORMATION FOR LONG TERM FOLLOW-UP (LTFU) 
OBSERVATION ANNUAL REPORT  
Category  Required LTFU Data  Rationale 
The placement of this title will 
Long Term Follow-Up Observation Annual 
Protocol Title  facilitate FDA to search for 
Report 
LTFU data in our database 
Total length (years)  
Starting date 
This will serve as a brief 
LTFU Protocol Status  Total number of subjects enrolled 
summary. 
Subjects that have completed LTFU observation 
Remaining subjects on LTFU observation 
This is the focus of the product 
Vector persistence 
safety assessment in the LTFU 
Clonality analyses 
protocol and provides important 
Product Information  RCR 
information for monitoring, and 
On and off-target analyses for products that involve 
for determination of the length of 
genome editing 
the LTFU observation. 
This provides data and signals to 
Preclinical  New preclinical data 
guide the direction of LTFU 
Information  Relevant findings from the literature 
observation. 
This is the focus of the product 
safety assessment in LTFU 
Any related delayed adverse event with brief 
observation, and serves as a 
narrative 
guide for the types of AE, organ 
Oncological, neurological, hematological, auto-
systems, and methodology to 
immune or other disorder 
attribute AE/Serious Adverse 
Causal analyses based on evidence from clinical, 
Event (SAE) to the GT product. 
Clinical Information  laboratory, molecular, cytogenetic, histological, 
 
HLA analysis, deep sequencing data 
The durability of clinical effect 
Serious adverse events 
also allows for an assessment of 
Evidence for persistence of the product/therapeutic 
product efficacy in the LTFU 
protein/sequences, and durability of the clinical 
observation report, but inclusion 
effects 
of such data is at the sponsors 
discretion.    
This will provide an opportunity 
Rationale for modifying LTFU observation  for revising the content and 
Revision of LTFU  FDA agreement to revised LTFU protocol: synopsis  length of the LTFU observation 
protocol  of meeting(s) discussion/email communication  based on data collected in the 
Discussion and date of discontinuation  studies or other relevant 
information. 
 
 
 
   
33  Contains Nonbinding Recommendations 
 
APPENDIX 2:  SAMPLE TEMPLATE:  LONG TERM FOLLOW-UP (LTFU) 
OBSERVATION ANNUAL REPORT  
Category  List of LTFU data  Annual reporting  
Protocol title  Long Term Follow-Up  [product name]: LTFU2017 
Observation Annual Report  annual report for protocol [#] 
LTFU protocol  Total length (years):  15 years 
status     
Starting date:  October 30, 2009 
Total number of subjects  30 
enrolled: 
Subjects that have completed  0 
LTFU observation: 
Remaining subjects on LTFU  20 (2 deaths, 5 lost to follow-up, 
observation:  3 drop outs) 
Product  Vector persistence:  PCR1 of [name] transgene 
information    positive in 17 of 20 subjects still 
  on study at 5 yrs and 3 subjects 
  at 7 yrs. 
Clonality analyses:  No clones more than 1% for 
  more than 1 testing period 
RCR  ND2, request to discontinue RCR 
  testing 
On and off-target analyses for  NA3 
products that involve genome 
editing 
Preclinical  New preclinical data  Final study report for large 
information    reproductive toxicity study in 
  normal SD rats (study report 
  [#]). Published in [journal 
  citation]. 
  No additional studies ongoing at 
  this time. 
 
Relevant findings from the  No new literature on [x] disease 
literature  at this time. 
Clinical  Any related delayed adverse  One case of rash that resolved 
information  event with brief narrative  with steroids. No other 
  symptoms. PCR of rash biopsy 
  was negative for vector. 
Oncological, neurological,  Secondary tumor on left ear, 
hematological, auto-immune or  negative for vector sequences by 
other disorder  PCR. Unrelated, melanoma.  
Causal analyses based on  NA 
evidence from clinical, 
34  Contains Nonbinding Recommendations 
 
laboratory, molecular, 
cytogenetic, histological, HLA 
analysis, deep sequencing data 
Serious adverse events  2 deaths due to sepsis, related to 
underlying disease.  
No other unexpected SAE 
reported 
Evidence for persistence of the  20 subjects are still on study with 
product/therapeutic  vector persists in BM and PBMC 
protein/sequences, and durability  samples, and clinical benefit 
of the clinical effects  observed. All twenty subjects 
have reconstituted immune 
system, with some b cell aphasia 
and low platelet counts in three 
subjects, however no 
transfusions needed to date. 
 
Revision of LTFU  Rationale for modifying LTFU  All RCR testing results negative 
Protocol  observation  (n=150 samples). Risk 
assessment determined very low 
risk of RCR developing in 
subjects at this time. 
FDA agreement to revised  LTFU  Revision to LTFU discussed 
protocol: synopsis of meeting(s)  during pre-BLA meeting [date]. 
discussion/email communication  RCR testing will no longer 
performed for LTFU protocol [#] 
  Discussion and date of  NA 
discontinuation 
1 polymerase chain reaction 
2 none detected (ND) 
3 not applicable (NA) 
35