Testing of Retroviral Vector-Based 
Human Gene Therapy Products for 
Replication Competent Retrovirus 
During Product Manufacture and 
Patient Follow-up 
 
 
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.  BACKGROUND ............................................................................................................... 2 
III.  RECOMMENDATIONS FOR PRODUCT TESTING ................................................. 3 
A.  Material for Testing .............................................................................................. 3 
1.  Vector Producer Cell Master Cell Bank ........................................................... 4 
2.  Vector Producer Cell Working Cell Bank ........................................................ 5 
3.  Retroviral Vector Supernatant Product and End of Production Cells ............... 5 
4.  Ex Vivo Transduced Cells ................................................................................ 5 
B.  Amounts for Testing ............................................................................................. 7 
1.  Supernatant Testing .......................................................................................... 7 
2.  Cell Testing ....................................................................................................... 7 
C.  Assays for Testing ................................................................................................. 7 
IV.  RECOMMENDATIONS FOR PATIENT MONITORING ......................................... 8 
A.  RCR Testing Schedule .......................................................................................... 9 
B.  Recommended Assays ........................................................................................... 9 
V.  DOCUMENTATION OF RCR TESTING RESULTS ............................................... 10 
VI.  POST-LICENSURE CONSIDERATIONS .................................................................. 10 
VII.  REFERENCES ................................................................................................................ 11 
APPENDIX .................................................................................................................................. 13 
i  Contains Nonbinding Recommendations 
 
Testing of Retroviral Vector-Based Human Gene Therapy Products 
for Replication Competent Retrovirus During Product Manufacture 
and Patient Follow-up  
 
 
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 
 
The potential pathogenicity of replication competent retrovirus (RCR) requires vigilant testing to 
exclude the presence of RCR in retroviral vector-based human gene therapy products1 (Ref. 1).  
We, FDA, are providing you, sponsors of retroviral vector-based human gene therapy products, 
recommendations regarding the testing for RCR during the manufacture of retroviral vector-
based gene therapy products, and during follow-up monitoring of patients who have received 
retroviral vector-based gene therapy products.2  Recommendations include the identification and 
amount of material to be tested as well as general testing methods.  In addition, recommendations 
are provided for monitoring patients for evidence of retroviral infection after administration of 
retroviral vector-based gene therapy products.   
 
The Retroviridae family is composed of two subfamilies:  Orthoretrovirinae, which consists of 
six genera of viruses:  Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, 
Epsilonretrovirus, and Lentivirus, and Spumaretrovirinae (foamy viruses) which has recently 
been updated to consist of five genera of viruses:  Bovispumavirus, Equispumavirus, 
Felispumavirus, Prosimiispumavirus, and Simiispumavirus (Refs. 2, 3).  RCR may potentially be 
generated during the manufacture of a retrovirus vector from any of these genera.  At this time, 
the most common retrovirus-based vectors are constructed from gammaretroviruses or 
lentiviruses, and therefore further details are provided for these genera.  Historically, lentivirus 
RCR is referred to as replication competent lentivirus (RCL).3   
 
This guidance supersedes the guidance entitled, Guidance for Industry:  Supplemental Guidance 
on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy 
                                                 
1 Retroviral vector-based human gene therapy products include retroviral vector supernatant that can be directly 
administered and ex vivo genetically modified (transduced) cells. 
2 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.    
3 RCR and RCL are synonymous for the purposes of this guidance. 
1  Contains Nonbinding Recommendations 
 
Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors dated 
November 2006 (2006 RCR Guidance) (Ref. 4).  This guidance also supplements the following 
two final guidances:  the Long Term Follow-Up After Administration of Human Gene Therapy 
Products; Guidance for Industry dated January 2020 (Long Term Follow-up Guidance) and 
Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy 
Investigational New Drug Applications (INDs); Guidance for Industry dated January 2020 
(CMC Guidance).4  This guidance finalizes the draft guidance of the same title dated July 2018.   
 
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.  BACKGROUND 
 
FDAs Center for Biologics Evaluation and Research (CBER) recommendations for RCR testing 
during retroviral vector production and patient monitoring were originally developed at a time 
when clinical experience was limited to a small number of studies using gammaretrovirus 
vectors (Ref. 5).  At that time, the overriding safety concerns associated with the use of retroviral 
vectors were exemplified by the findings of an animal study involving administration of 
gammaretroviral vector-transduced bone marrow progenitor cells that had been inadvertently 
exposed to high-titer RCR, and administered to severely immunosuppressed rhesus monkeys 
(Ref. 1).  In this setting, 3/10 animals developed lymphomas and died within 200 days.  The 
RCR was presumed to be etiologically associated with the disease by virtue of the presence of 
multiple murine RCR sequences in the lymphomas and an inverse correlation between anti-
retroviral antibodies and development of disease (Refs. 6, 7).  In contrast, another study in 
moderately-immunosuppressed cynomolgus monkeys exposed intravenously to RCR showed no 
signs of disease (Refs. 8, 9). 
 
More than two decades of experience has generated a substantial amount of data on the safety of 
retroviral vectors in clinical applications for gene therapy, including experience with different 
vector designs, vector producing cells, RCR detection assays, and lack of positive results from 
RCR testing of vector lots, ex vivo transduced cells, and patient samples collected during 
monitoring.  These data have provided the basis for public discussions, including Retroviral 
Breakout Sessions at the 1996 and 1997 FDA/National Institutes of Health (NIH) Gene Therapy 
Conferences, the 2010 Cellular, Tissue, and Gene Therapies Advisory Committee meeting (Ref. 
10), and the 2014 American Society of Gene and Cellular Therapy (ASGCT) Breakout Session 
on Replication Competent Virus (Ref. 11).  In addition, FDA scientists published an evaluation 
of RCR testing methods associated with the use of retroviral vectors (Ref. 12).  During this time, 
the gene therapy community has improved retroviral vector design to reduce the likelihood of 
                                                 
4 The Long Term Follow-up Guidance is available at this website: https://www.fda.gov/media/113768/download 
The CMC Guidance is available at this website:  https://www.fda.gov/media/113760/download 
 
 
2  Contains Nonbinding Recommendations 
 
generating RCR during the manufacturing process (Refs. 13, 14).  For instance, the likelihood 
that recombination will generate RCR is reduced by manufacturing vectors using a split plasmid 
design, where the vector genome is on a separate plasmid from the envelope gene and packaging 
functions.  RCR generation can be further reduced by using more than two plasmids for vector 
production.  Lentiviral vectors have been further modified to remove genes encoding viral 
accessory and regulatory proteins, which would cripple the functionality of an RCR in the event 
an RCR may be generated (Refs. 15, 16). 
 
Summary of Revisions from the 2006 RCR Guidance: 
 
With consideration of the accrued scientific evidence of safety associated with retroviral vector 
design and testing, we are revising our current recommendations for RCR testing during 
retroviral vector-based gene therapy product manufacture and patient monitoring.  More 
specifically, we are no longer recommending RCR testing on working cell banks for retroviral 
producer cells.  We have also revised our recommendations regarding the amount of vector that 
should be tested (section III.B and Appendix 1-1 of this document).  Briefly, we are 
recommending that you test a sufficient amount of vector to demonstrate that your vector 
contains <1 RCR per patient dose, when applicable to your clinical manufacturing practice.  
Additionally, we are recommending that all retroviral vector transduced cell products be tested 
for RCR, including those cultured for 4 days or less.  We have found no convincing evidence 
that the length of culture time influences the likelihood of RCR development in transduced cells.  
However, if you have accumulated manufacturing and clinical experience that demonstrates that 
your transduced cell product is consistently RCR-negative (section III.A.4 of this document), we 
recommend that you provide this data to support reduction or elimination of testing ex vivo 
genetically modified cells for RCR.  We have also revised our recommendations regarding 
assays for testing (section III.C of this document).  Specifically, alternative methods may be 
appropriate for lot release testing of ex vivo transduced cells in lieu of culture based methods, 
particularly when there are time constraints present.  Finally, we have revised our advice for 
active monitoring of patients following administration of retroviral vector-based products 
(section IV of this document), and added post-licensure considerations for RCR testing and risk 
assessment (section VI of this document).  Because considerations for RCR assay development 
and testing schedule during retroviral vector-based gene therapy product manufacture and patient 
monitoring are generally product-specific, FDA recommends communication with the Office of 
Tissues and Advanced Therapies (OTAT) at CBER, preferably early, i.e., during your pre-IND 
meeting, as well as throughout your product development. 
 
 
III.  RECOMMENDATIONS FOR PRODUCT TESTING 
 
A.  Material for Testing 
 
Generally, retroviral vectors are manufactured by collection of supernatant following 
transient or stable production from cultured cells.  RCR may develop at any step during 
manufacturing, from the initial transfection or transduction steps through production of 
the retroviral vector supernatant.  In addition, if the retroviral vector is used for ex vivo 
genetic modification of cells, the expansion of ex vivo transduced cells in culture 
3  Contains Nonbinding Recommendations 
 
provides the potential for amplification of an RCR contaminant that may be below the 
level of detection in the retroviral vector supernatant.  Therefore, current 
recommendations include testing of material from multiple stages of product manufacture 
(see Table of this document). 
 
When the vector is produced by transient transfection, all cell banks should be qualified 
according to section V.A.2.c.iii.a of the CMC Guidance.  Retroviral vector RCR-specific 
testing requirements are outlined below for the vector supernatant (section III.A.3 of this 
document), end of production cells (section III.A.3 of this document), and ex vivo 
transduced cells (section III.A.4 of this document), if applicable. 
 
We recommend use of a stably-transfected Vector Producer Cell (VPC) bank system, 
when possible, in order to ensure an adequate and consistent supply of retroviral vector.  
The generation of a Master Cell Bank (MCB) for the VPC allows for the collection of 
cells of uniform composition derived from a single cell clone.  The Working Cell Bank 
(WCB) is derived from the MCB, following expansion by serial subculture to a specified 
passage number (refer to Points to Consider in the Characterization of Cell Lines Used 
to Produce Biologicals dated May 1993)5.  When the vector is produced using VPC 
banks, RCR-specific testing of the VPC MCB (section III.A.1 of this document) is 
recommended in addition to vector supernatant (section III.A.3 of this document), end of 
production cells (section III.A.3 of this document), and ex vivo transduced cells (section 
III.A.4 of this document), if applicable. 
 
1.  Vector Producer Cell Master Cell Bank 
 
Both cells and supernatant from the VPC MCB should be tested for RCR using a 
cell line selected based on the tropism of the parental virus used to generate the 
vector.  For example, VPC containing envelopes such as gibbon ape leukemia 
virus (GALV) envelope or vesicular stomatitis virus glycoprotein (VSV-G) are 
typically tested on a human cell line.  Other retroviral envelopes should be tested 
on a cell line permissive for infection by the relevant RCR. 
 
If the VPC MCB was produced using a retroviral vector pseudotyped with an 
envelope distinct from the clinical vector product, for example, an ecotropic 
Murine Leukemia Virus (MLV), the potential exists for introduction of an RCR 
with that distinct envelope.  Even though an ecotropic MLV RCR may present a 
minimal direct safety risk to humans, the presence of any replication-competent 
genome in the VPC MCB is problematic because of the increased probability of 
generating an RCR with a human host range through recombination with elements 
within the VPC. 
 
Therefore, in cases where VPC are derived, at any step, by transduction with an 
ecotropic retroviral vector, testing of the MCB for the presence of ecotropic RCR 
is recommended, in addition to amphotropic RCR testing.  For example, VPC 
possibly containing ecotropic MLV envelope should be tested for RCR on an 
                                                 
5 https://www.fda.gov/media/76255/download 
4  Contains Nonbinding Recommendations 
 
appropriate cell line which is permissive to infection by ecotropic MLV-like RCR 
(Ref. 17). 
 
2.  Vector Producer Cell Working Cell Bank 
 
The risk of generating RCR during vector production with a VPC WCB that is 
derived from an appropriately qualified VPC MCB is relatively low based on our 
accumulated experience.  Therefore, we do not recommend RCR testing of the 
VPC WCB except when RCR testing of the VPC MCB is insufficient based on 
recommendations described herein. 
 
3.  Retroviral Vector Supernatant Product and End of Production Cells 
 
Both retroviral vector supernatant lots and end of production (EOP) cells should 
be tested for RCR.  EOP cells are defined as cells from which a single bulk 
harvest of retrovirus-containing supernatant is taken or cells from which the last 
of a serial set of supernatant harvests is taken.  This recommendation is based on 
data and experience reported at the 1997 FDA/NIH Gene Therapy Conference, 
where it was reported that RCR in vector production lots was not always 
consistently detected in both vector supernatant and EOP cells.  These data 
support the position that dual testing provides a complementary approach to 
assuring RCR-free retroviral vector. 
 
4.  Ex Vivo Transduced Cells 
 
If the retroviral vector is used for ex vivo genetic modification of cells, it is 
possible that RCR may be present in your vector at undetectable levels, which 
could be amplified during the manufacture of ex vivo transduced cells.  Therefore, 
we recommend that each lot of ex vivo retroviral transduced cells be tested for 
RCR.  This recommendation applies regardless of the length of time that the cells 
are cultured after transduction, because the length of culture time (e.g., greater 
than 4 days) has not been shown to strongly influence the likelihood of RCR 
development. 
 
However, experience with vectors that have been deliberately designed to 
minimize the likelihood of recombination suggests that amplification of RCR in 
transduced cells is unlikely for many vectors.  If you have accumulated 
manufacturing and clinical experience that demonstrates that your transduced cell 
product is consistently RCR-negative, we recommend that you provide this data 
to support reduction or elimination of testing ex vivo genetically modified cells 
for RCR.  We recommend you include a discussion of safety features in the vector 
design that reduces the likelihood of generating RCR, a description of vector 
testing in accordance with current guidance, and your experience manufacturing 
RCR-free cell products.  You may provide information supporting removal of 
RCR testing for lot release of ex vivo transduced cells in your IND (i.e., in the 
section titled:  Manufacturing Process Development Section 3.2.S.2.6 or 3.2.P.2.3 
5  Contains Nonbinding Recommendations 
 
of the electronic Common Technical Document (eCTD)) or discuss with the FDA 
during your pre-IND meeting. 
 
If the cumulative information sufficiently supports discontinuation of RCR testing 
as part of your ex vivo retroviral transduced cell product release testing, we 
recommend archiving a sample for at least 6 months after the product expiration 
date.  We recommend that you retain a sufficient amount (section III.B.2 and 
Appendix of this document) of the cell product to perform RCR testing in the 
future if necessary (section IV of this document).  Samples should be archived 
with appropriate safeguards to ensure long-term storage (e.g., a monitored freezer 
alarm storage system) and an efficient system for the prompt linkage and retrieval 
of the stored samples with the medical records of the patient and the production 
lot records.  We do not recommend archiving if the ex vivo transduced cells are 
tested for RCR at product release. 
 
Table.  Recommendations for Product Testing 
 
Material to be Tested  Testing for  Testing for 
Expected RCR  Ecotropic RCR   
Frequency of 
Cells and  Cells and 
Testing 
Supernatant 1  Supernatant  
MCB  One-time     
-Derived by transduction    Yes  Yes 
with ecotropic vector     
-Derived by transfection  Yes  NA2 
of retroviral vector     
plasmid   
Vector Harvest Material   Product     NA 
-EOP cells   release  Yes   
-Vector supernatant  Yes 
Ex vivo Transduced  Product    Yes, c ells  NA 
Cells  release  only   
  OR archive3 
1 RCR testing should be based on the type of vector envelopes used.  Consult text in 
section III.A.1 of this document for details. 
2 NA, not applicable. 
3 If an agreement reached with FDA to discontinue testing; consult text in section III.A.4 of this document. 
 
   
6  Contains Nonbinding Recommendations 
 
B.  Amounts for Testing 
 
1.  Supernatant Testing 
 
In all cases, we recommend testing at least 5% of the total supernatant by 
amplification on a permissive cell line.  Historically, we have recommended an 
alternative testing volume of 300 mL for vector lots over 6 liters.  This volume 
was set based on our experience at the time with gammaretrovirus vector 
production lot size, reference material, and patient dosing.  However, this 
recommendation may not be applicable to all current clinical manufacturing 
practices.  Current manufacturing experience indicates that <1 RCR/dose 
equivalent is an achievable level for retroviral vector preparations intended for 
clinical use.  Therefore, we now recommend that sufficient supernatant be tested 
to ensure a 95% probability of detection of RCR if present at a concentration of 1 
RCR/dose equivalent.  A more detailed explanation of the rationale and the 
mathematical formulas applied to achieve this level of testing confidence is found 
in the example in Appendix 1-1 of this document.  You should detail the amount 
to be tested, and provide a justification for the proposed testing volume, in the 
description of RCR testing procedures included in your IND (in the eCTD 
section:  Analytical Procedures 3.2.P.5.2). 
 
To support the underlying assumption that a single retrovirus will be detected, one 
should determine a volume in which a single RCR can be detected by an 
individual RCR assay.  Based on the determination of this volume, the total test 
volume should then be divided into replicate samples, each containing the volume 
demonstrated to detect a single RCR.  When large volumes or high titer retroviral 
vector preparations are used, interference in RCR detection may occur.  Sponsors 
are encouraged to develop more sensitive detection methods that overcome the 
interference effect of high titer retroviral vector preparations in order to use the 
alternative approach. 
 
2.  Cell Testing 
 
We recommend that you test 1% or 108 (whichever is less) pooled vector-
producing cells or ex vivo transduced cells by co-culture with a permissive cell 
line.  This recommendation is unchanged from previous recommendations and is 
consistent with public consensus expressed at the 1996 and 1997 FDA/NIH Gene 
Therapy Conferences.  
 
C.  Assays for Testing 
 
Vector supernatant assays should include culture of supernatant on a permissive cell line 
in order to amplify any potential RCR present.  Similarly, cell testing should be 
accomplished by co-culture with a permissive cell line in order to amplify any potential 
RCR present.  Typically, RCR culture assays have included a minimum of five passages.  
Assay optimization may allow for fewer passages if demonstrated to achieve similar 
7  Contains Nonbinding Recommendations 
 
assay sensitivity.  Sponsors are encouraged to develop RCR assays that support virus 
entry, amplification, and particle production specific to vector design (e.g., Mus dunni for 
ecotropic MLV (Ref. 17), C8166 cells for VSV-G pseudotyped HIV-1 (Ref. 18), or 
293F-DCSIGN cells for E1001 enveloped HIV-1 (Ref. 19), noting that certain production 
systems may require more than one amplification cell line.  The amplified material may 
then be detected in an appropriate indicator cell assay (e.g., PG-4 S+L- (Ref. 20), XC 
(Ref. 21), or by PERT (Ref. 22), or by psi-gag or VSV-G polymerase chain reaction 
(PCR) (Ref. 23), or by a commercially available p24 ELISA.  All assays should include 
relevant positive and negative controls to assess specificity, sensitivity, and 
reproducibility of the detection method employed.  Each lot of retroviral vector 
supernatant should be tested for inhibitory effects on detection of RCR by using positive 
control samples that are added to vector supernatant.  
 
Alternative methods, such as PCR, may be appropriate for lot release testing of ex vivo 
transduced cells in lieu of culture based methods; particularly, when time constraints are 
present.  
 
Any alternative methods should be developed in consultation with CBER.  Data on 
sensitivity, specificity and reproducibility should be provided to support the use of 
alternative methods. 
 
For assay development, you should develop a standard virus stock for use as a positive 
control and for method validation.  The standard virus stock can be used for 
determination of the volume in which a single RCR can be determined.  
Gammaretrovirus RCR standard virus stocks have been developed, their infectious titers 
(TCID ) have been determined, and are available through the American Type Culture 
50
Collection (ATCC) (4070A virus) or from the National Gene Vector Biorepository 
(NGVB) (GALV SEATO virus).  Refer to Appendices 1-2 and 1-3 of this document for 
detailed information about the gammaretrovirus RCR standard and how it can be used to 
determine the replicate size and number for RCR detection.  Standard virus stocks have 
not yet been developed for other retrovirus vectors.  We recommend that you develop an 
in-house standard virus stock that represents your clinical vector attributes, including, the 
genetic background, envelope protein, and deletion of accessory proteins.  The standard 
virus stock should be characterized for growth kinetics in the cells used for the RCR 
assay and tested for stability.  The standard virus stock should be used to validate the 
RCR assay prior to licensure.  For more information on assay validation, please refer to 
FDAs Analytical Procedures and Methods Validation for Drugs and Biologics; 
Guidance for Industry, dated July 2015.6  
 
 
IV.  RECOMMENDATIONS FOR PATIENT MONITORING  
 
Previous FDA guidance for active patient monitoring recommended RCR testing and/or 
archiving of patient samples at regular intervals for fifteen (15) years (Refs. 4, 24).  To date, 
                                                 
6 https://www.fda.gov/regulatory-information/search-fda-guidance-documents/analytical-procedures-and-methods-
validation-drugs-and-biologics 
8  Contains Nonbinding Recommendations 
 
RCR or delayed adverse events related to RCR have not been reported in patients who have 
received retrovirus-based gene therapies (Refs. 5, 25, 26, 27, 28).   
 
A.  RCR Testing Schedule 
 
We recommend the monitoring schedule to include analysis of patient samples at the 
following time points:  pre-treatment, followed by testing at three, six, and twelve months 
after treatment, and yearly for up to fifteen (15) years.  If any post-treatment samples are 
positive, further analysis of the RCR, and more extensive patient follow-up should be 
undertaken, in consultation with CBER.  However, if all post-treatment assays for an 
individual patient are negative during the first year, collection of the yearly follow-up 
samples may be discontinued for that individual, and yearly review of medical history 
will generally be sufficient for the patient.   
 
After you have accumulated patient monitoring data with your product, you may provide 
a rationale to discontinue all active testing of patient samples for RCR in the safety 
monitoring section of your clinical protocol.  The rationale may include a discussion of 
safety features in the vector design that reduce the likelihood of generating RCR, as well 
as results of your previous clinical testing experience.  
 
If the data sufficiently support discontinuation of active testing of patient samples, we 
recommend a yearly review of medical history be collected in lieu of patient samples, as 
a part of long-term follow-up.  This history should be targeted towards determination of 
clinical outcomes suggestive of retroviral disease, such as cancer, neurologic disorders, or 
hematologic disorders.  As part of the long-term follow-up protocol, a yearly long-term 
follow-up clinical report7 should be submitted to the IND.   
 
Relevant clinical samples should be collected and tested for RCR upon development of 
an adverse event suggestive of a retrovirus-associated disease.  If patients die of a 
suspected retrovirus-associated disease or develop neoplasms within 15 years during a 
gene therapy trial, you should assay for RCR in a biopsy sample of the neoplastic tissue 
or the pertinent autopsy tissue.  Sample collection and storage should be compatible with 
the expected testing strategy.  Additional recommendations for long-term follow-up of 
patients in clinical trials using retroviral vectors are discussed in the Long Term Follow-
up Guidance. 
 
B.  Recommended Assays 
 
We recommend two methods that are currently in use for detecting evidence of RCR 
infection in patients:  1) serologic detection of RCR-specific antibodies; and 2) analysis 
of patient peripheral blood mononuclear cells by PCR for RCR-specific DNA sequences.  
The choice of assay may depend on the vector, mode of vector administration, and the 
clinical indication.  For example, it has been shown that direct administration of VPC or 
repeat direct injection of a vector can result in vector-specific antibodies that do not 
                                                 
7 For more information, refer to section V.D.3 and Appendices of the Long Term Follow-up Guidance 
(Recommendations for Protocols for Long Term Follow-Up Observations:  Clinical Considerations). 
9  Contains Nonbinding Recommendations 
 
correlate with the presence of RCR (Refs. 29, 30).  Therefore, in cases where vector or 
VPCs are directly administered, a PCR assay may be preferable over serologic 
monitoring.  Additionally, monitoring of patient samples by PCR may be preferable over 
serologic monitoring if the patients are immunocompromised to an extent that antibody 
production may be minimal or not at all.  In either situation, all confirmed positive results 
should be pursued by direct culture assay to obtain and characterize the infectious viral 
isolate. 
 
 
V.  DOCUMENTATION OF RCR TESTING RESULTS 
 
RCR testing results from production lots and patient monitoring should be documented in 
amendments to the IND file.  Positive results from patient monitoring should be reported 
immediately as an adverse experience in the form of an IND safety report (21 CFR 312.32).  
Negative results should be reported by way of the IND annual report (21 CFR 312.33) or 
Development Safety Update Report (DSUR), if used in lieu of reporting in annual reports.  
In addition, to enhance the accumulation of data on RCR testing assays, CBER encourages 
members of the gene therapy community to publish data and/or discuss data publicly 
regarding their experience with different vector producer cell lines, patient monitoring, and 
safety. 
 
 
VI.  POST-LICENSURE CONSIDERATIONS 
 
We recommend that labeling for retroviral vector-based gene therapy products incorporate 
relevant data and information to clearly present the immediate and long-term risks associated 
with RCR.  As a critical safety test for retroviral vectors, testing for RCR during vector 
manufacture and release should continue after licensure.   
 
At the time of submission of your Biologics License Application (BLA),8 you should have 
accumulated sufficient manufacturing and clinical safety data to determine whether there is a 
significant risk of RCR developing with your product.  This risk assessment may be used to 
propose that periodic patient monitoring for RCR would not be warranted for your product post-
licensure.  However, you should include a provision in the BLA to collect relevant clinical 
samples from patients for RCR testing upon development of an adverse event suggestive of a 
retrovirus-associated disease.  In the event patients die from a disease potentially associated with 
a retrovirus or develop neoplasms within 15 years following product administration, every effort 
should be made to assay for RCR in a biopsy sample of the neoplastic tissue or the pertinent 
autopsy tissue.  
 
These post-licensure considerations should also include continued long-term patient follow-up, 
up to fifteen (15) years after dosing, in accordance with the Long Term Follow-up Guidance.  
For more information, refer to section VI of the Long Term Follow-up Guidance (General 
Considerations for Post-Marketing Monitoring Plans for Gene Therapy Products).    
                                                 
8 21 CFR 601.2. 
10  Contains Nonbinding Recommendations 
 
VII.  REFERENCES  
 
1.  Donahue, RE, et al., Helper virus induced T cell lymphoma in nonhuman primates after 
retroviral mediated gene transfer, J Exp Med, 1992; 176(4):1125-1135. 
2.  Fields Virology. In: BN Fields, DM Knipe, PM Howley, editors, 2013, Wolters Kluwer 
Health/Lippincott Williams & Wilkins: Philadelphia, 1424-1473. 
3.  Khan, et al., Spumaretroviruses: Updated taxonomy and nomenclature, Virology, 2018; 
516C:158-164. 
4.  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. 
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/supplemental-
guidance-testing-replication-competent-retrovirus-retroviral-vector-based-gene-therapy 
5.  Gunter, KC, AS Khan, and PD Noguchi, The safety of retroviral vectors, Hum Gene Ther, 
1993; 4(5):643-645. 
6.  Purcell, DF, et al., An array of murine leukemia virus-related elements is transmitted and 
expressed in a primate recipient of retroviral gene transfer, J Virol, 1996; 70(2):887-897. 
7.  Vanin, EF, et al., Characterization of replication-competent retroviruses from nonhuman 
primates with virus-induced T-cell lymphomas and observations regarding the mechanism of 
oncogenesis, J Virol, 1994; 68(7):4241-4250. 
8.  Cornetta, K, et al., No retroviremia or pathology in long-term follow-up of monkeys exposed 
to a murine amphotropic retrovirus, Hum Gene Ther, 1991; 2(3):215-219. 
9.  Kantoff, PW, et al., Correction of adenosine deaminase deficiency in cultured human T and 
B cells by retrovirus-mediated gene transfer, Proc Natl Acad Sci U.S.A., 1986; 83(17):6563-
6567. 
10. Briefing Document - Testing for Replication Competent Retrovirus (RCR)/Lentivirus (RCL) 
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11  Contains Nonbinding Recommendations 
 
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12  Contains Nonbinding Recommendations 
 
APPENDIX 
 
1-1.  Derivation of Recommendation for Test Volume for RCR Detection 
 
Assuming the RCR are present in the production lot at a concentration (c) and that an assay will 
detect a single retrovirus in the sample, the probability (p) of detecting retrovirus in a volume 
(Vt) is given by the formula:  p = 1-exp(-cVt), because the number of RCR in Vt follows a 
Poisson distribution with a parameter cVt.  Solving for Vt, one gets the following equation: 
 
Vt= - (1/c) ln (1-p) 
 
where ln denotes the natural logarithm. 
 
Value for p 
For the use of this formula, it is recommended that the value for p be set at 0.95.  With 
the recommended replicate size and number defined in Appendix 1-3 of this document, p 
becomes the probability of detecting an RCR in the production lot. 
 
Value for c 
We recommend that the value for c be set no higher than 1 RCR/dose equivalent.  If the 
concentration of RCR in the production lot is 1 RCR/dose equivalent or greater, then the 
probability of detection is at least 0.95.  If the production lot contains RCR at a 
concentration of <1 RCR/dose equivalent, the RCR may not be detected and would be 
administered to the patient.  We also recommend that a dose equivalent be defined as the 
maximum amount of vector expected to be administered at one time.  For ex vivo 
genetically modified cells, a dose equivalent is the amount of vector used to transduce the 
maximum number of target cells for each production lot. 
 
Value for Vt 
 
With the recommended value for p and c, the total volume of retroviral supernatant to be 
tested, independent of lot size, is calculated as follows: 
 
Vt = - (1 / (1 RCR/dose equivalent)) ln (1 -0.95) 
 
Direct administration example: 
If your product is administered at 1x1010 TU (transducing unit) 
Vt = - (1 / (1/1x1010 TU)) ln (1 -0.95) = 3x1010 TU 
 
Ex vivo genetic modification example: 
If you aim to transduce up to 1x108 cells at an MOI (multiplicity of infection) of 
0.5 with a titer of 1x107 TU/mL: 
Dose equivalent = (1x108 cells) (0.5 TU/cell) / (1x107 TU/mL) = 5 mL 
Vt = - (1 / (1/5 mL)) ln (1 -0.95) = 15 mL 
 
13  Contains Nonbinding Recommendations 
 
Proposals to use smaller volumes should be developed and reviewed in consultation with 
CBER. 
 
1-2.  Empirical Determination of Assay Sensitivity 
 
In collaboration with the ATCC, a standard gammaretroviral stock (ATCC # VR-1450) has been 
established for use in determination of sensitivity and validation of assays used to detect the 
presence of RCR which would be produced from VPC containing an amphotropic envelope.  
This stock can be used to determine the relative assay sensitivity for detecting RCR.  This 
information can subsequently be used to determine the size of replicates of retroviral supernatant 
to be tested that will ensure detection of a single retrovirus and thus, the number of replicates to 
ensure an adequate total volume, Vt, as specified in this guidance (Appendix 1-3 of this 
document).  The virus stock is derived from a cell line which has been transfected with a 
molecular clone encoding MoMLV with a substitution of the envelope coding region from the 
4070A strain of amphotropic MLV (Ref. 31).  Therefore, this virus stock represents a typical 
recombinant virus that could be generated in a retroviral packaging cell line containing coding 
sequences for a MLV envelope. 
 
The standard virus stock and its infectious titer can be used as a positive control to empirically 
determine the relative sensitivity of assay methods used for detection of RCR in retroviral 
vectors.  In particular, this stock will allow investigators to determine the largest test volume in 
which a single RCR can be detected.  The determination should be performed in the presence of 
a retroviral vector supernatant typical of a production lot in order to control for inhibitory effects 
of the retroviral vector particles on detection of RCR.  Availability of this standard should allow 
individual investigators to establish this methodology in their own laboratories, as well as allow 
exploration of alternative methods for detection of RCR. 
 
1-3.  Formula to Determine Replicate Size and Number 
 
Depending on the volume in which a single RCR can be detected by an individual RCR assay (as 
determined by use of the RCR standard, Appendix 1-2 of this document), it may be necessary to 
divide the total test volume into several replicate samples to ensure the detection of RCR in the 
sample.  The number of replicates (r), can be determined using the formula, 
 
r = Vt / Vs 
 
where Vs is the volume in which one RCR can be consistently detected (Appendix 1-1 of this 
document for determination of Vt). 
 
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