Guest Column | July 25, 2018

FDA's New Guidance On Replication Competent Retrovirus Testing During Gene Therapy Manufacture

By Stephen Mason, Greenleaf Health


The FDA defines human gene therapy products as 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, genetically modified microorganisms (e.g., viruses, bacteria, fungi), engineered site-specific nucleases used for human genome editing, 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.

Although the journal Science reports that the first gene therapy clinical trial was conducted in 1990 (Ref. 1),1 the FDA only approved the first gene therapy product on August 30, 2017. Two more approvals quickly followed, reflecting a suddenly rapid advancement in this field. On July 11, 2018, the FDA issued for public comment six draft guidance documents intended to serve as part of a modern, comprehensive framework for how the Center for Biological Evaluation and Research (CBER) will help advance the field of gene therapy.

This is the second article in a six-part series summarizing each of the draft guidance documents. The first article reviewed Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). This article will discuss Testing of Retroviral Vector-Based Human Gene Therapy Products for Replication Competent Retrovirus During Product Manufacture and Patient Follow-up.


The second guidance, when finalized, will replace the 2006 guidance titled 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.  Since the original guidance was issued, investigators have developed additional data on the safety of retroviral vectors in clinical applications for gene therapy, through 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.

The draft guidance document provides recommendations regarding the testing for RCR during the manufacture of retroviral vector-based gene therapy products, and for follow-up monitoring of patients who have received retroviral vector-based gene therapy products.  Specifically, the draft guidance:

  • No longer recommends RCR testing on working cell banks for retroviral producer cells.
  • Revises previous recommendations regarding the amount of vector that should be tested.  Sponsors should:
    • Test a sufficient amount of vector to demonstrate that the vector contains <1 RCR per patient dose.
    • Test all retroviral vector transduced cell products for RCR, including those cultured for four days or less.
  • Recognizes that manufacturing and clinical experience demonstrating that a transduced cell product is consistently RCR-negative may support reduction or elimination of testing ex vivo genetically modified cells for RCR.
  • Revises recommendations for active monitoring of patients following administration of retroviral vector-based products, including:
    • The patient monitoring schedule should include an analysis of patient samples at the following time points: pre-treatment, followed by testing at three, six, and 12 months after treatment, and yearly for up to 15 years.
    • If all post-treatment assays are negative during the first year, collection of the yearly follow-up samples may be discontinued.  
    • If any post-treatment samples are positive, further analysis of the RCR and more extensive patient follow-up should be undertaken in consultation with the FDA.
  • Adds post-licensure considerations for RCR testing and risk assessment, including:
    • Labeling for retroviral vector-based gene therapy products should incorporate relevant data and information to clearly present the immediate and long-term risks associated with RCR.
    • Testing for RCR during vector manufacture and release should continue after licensure, although a sponsor may propose that periodic patient monitoring for RCR would not be warranted post-licensure based on accumulated manufacturing and clinical safety data.  

The draft guidance recommends 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 polymerase chain reaction (PCR) for RCR-specific DNA sequences.  The choice of assay may depend on the vector, mode of vector administration, and the clinical indication.  All confirmed positive results should be pursued by direct culture assay to obtain and characterize the infectious viral isolate.

The guidance recommends that RCR testing results from production lots and patient monitoring should be documented in amendments to the IND file.  Positive patient monitoring results (indicating the presence of RCR) should be reported “immediately” as an adverse experience in an IND safety report. Negative results should be reported by way of the IND annual report.

The FDA will accept comments on the draft guidance through October 10, 2018. You can submit comments electronically at

The next article summarizes the draft guidance Long Term Follow-Up After Administration of Human Gene Therapy Products.


  1. Blaese et al. T Lymphocyte-Directed Gene Therapy for ADA− SCID: Initial Trial Results After 4 Years, Science 20 Oct 1995: Vol. 270, Issue 5235, pp. 475-480 DOI: 10.1126/science.270.5235.475

About The Author:

Stephen Mason is senior VP of regulatory policy in the Drug and Biological Products group at Greenleaf Health, Inc. He has experience from FDA, Capitol Hill, and within the innovator and generic pharmaceutical industries. He specializes in regulatory and legislative policy development and analysis. You can reach Mason at