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U.S. Department of Health and Human Services

Vaccines, Blood & Biologics

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Draft Guidance for Industry: Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products

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This guidance document is for comment purposes only.

Submit one set of either electronic or written comments on this draft guidance by the date provided in the Federal Register notice announcing the availability of the draft guidance.  Submit electronic comments to http://www.regulations.gov.  Submit written comments to the Division of Dockets Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852.  You should identify all comments with the docket number listed in the notice of availability that publishes in the Federal Register.

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 240-402-7800, or email ocod@fda.hhs.gov, or from the Internet at http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/default.htm.

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
July 2014

Table of Contents

I.......... Introduction
II........ Scope
III....... Background
IV....... Why Collect Shedding Data during Clinical Development?
V........ Design of Shedding Studies: Guiding Principles 
A.          Biological Characteristics
B.           Route of Administration
VI....... Collection of Shedding Data in Preclinical Studies
VII...... Collection of Shedding Data in Clinical Studies
A.          When to Collect Shedding Data in Clinical Studies?
B.           Study Design
VIII.... Analytical Assays to Measure Shedding 
IX....... Analysis of Shedding Data 
A.          The Nature of the Shed Material
B.           The Extent of Shedding
X......... What to include in a Clinical Shedding Study Report
XI....... Assessing the potential for Transmission to Untreated Individuals Due to Shedding 
A.          What Information in the Shedding Data Can be Used to Assess Potential for Transmission to Untreated Individuals?
B.           Monitoring Untreated Individuals for Transmission
XII...... References

This draft guidance, when finalized, will represent the Food and Drug Administration’s (FDA’s) current thinking on this topic.  It does not create or confer any rights for or on any person and does not operate to bind FDA or the public.  You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations.  If you want to discuss an alternative approach, contact the appropriate FDA staff.  If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.


I. Introduction

The Center for Biologics Evaluation and Research (CBER)/Office of Cellular, Tissue, and Gene Therapies (OCTGT) is issuing this guidance to provide sponsors of virus or bacteria-based gene therapy products (VBGT products)1 and oncolytic viruses or bacteria (oncolytic products)2 with recommendations on how to conduct shedding studies during preclinical and clinical development.  For purposes of this guidance, the term “shedding” means release of oncolytic or VBGT products from the patient through one or all of the following ways:  excreta (feces); secreta (urine, saliva, nasopharyngeal fluids etc.); or through the skin (pustules, sores, wounds).  Shedding is distinct from biodistribution because the latter describes how a product is spread within the patient’s body from the site of administration while the former describes how it is excreted or released from the patient’s body.  Shedding raises the possibility of transmission of product-based viruses and bacteria from treated to untreated individuals.  When finalized, this guidance will represent FDA’s current thinking on how and when shedding data should be collected for VBGT products and oncolytic products during preclinical and clinical development and how shedding data can be used to assess the potential for transmission to untreated individuals.

FDA’s guidance documents, including this guidance, do not establish legally enforceable responsibilities.  Instead, guidances describe the FDA’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited.  The use of the word should in FDA’s guidances means that something is suggested or recommended, but not required.

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II. Scope

The products covered by this guidance are oncolytic and VBGT products that OCTGT reviews.  The focus of this guidance is shedding studies, including both how and when shedding data should be collected and how shedding data can be used to assess the potential for transmission to untreated individuals.

This guidance does not cover plasmids, peptides, and genetically modified mammalian cells that OCTGT also reviews because, unlike virus or bacteria-based products, plasmids, peptides, and genetically modified mammalian cells are neither infectious nor transmissible.  This guidance also does not address collection or submission of adverse event information, including those adverse events that could be attributed to shedding.  Please see the regulations at Title 21 of the Code of Federal Regulations (CFR) Part 312, specifically 21 CFR 312.32 and 21 CFR Part 600, specifically 21 CFR 600.80, for information on the collection and submission to FDA of adverse event information. 

Finally, while assessment of shedding can be utilized to understand the potential risk to the environment, the scope of this document does not include shedding as it may relate to potential environmental concerns with respect to a specific oncolytic or VBGT product.  For more information on this topic, you may wish to consult the draft guidance entitled, “Guidance for Industry:  Determining the Need for and Content of Environmental Assessments for Gene Therapies, Vectored Vaccines and Related Recombinant Viral or Microbial Products” dated June 2014. 3   When finalized, this guidance will represent FDA’s current thinking on this topic. 

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III. Background

Oncolytic and VBGT products are derived from infectious viruses or bacteria.  In general, these product-based viruses and bacteria are not as infectious or as virulent as the parent strain of virus or bacterium because of, in part, the derivation methods and/or modifications made during product development that lead to attenuation.  Hence, it is likely that these products are shed to a lesser extent than during natural infection by the parent strain.  Nonetheless, the possibility that infectious product-based viruses and bacteria may be shed by a patient treated with an oncolytic or VBGT product raises safety concerns related to the risk of transmission to untreated individuals.  To understand this risk, shedding studies that are conducted in the target patient population(s) may be appropriate before licensure.

Typically, clinical shedding studies are not stand-alone studies but are integrated into the design of a safety or efficacy trial.  Because there are many product-specific factors and patient-specific factors, as described throughout this guidance, that can influence the design of a shedding study, sponsors should consult with OCTGT in the early stages of product development for specific recommendations as to their product.

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IV. Why collect shedding data during clinical development?

Shedding studies of oncolytic or VBGT products are conducted to provide information about the likelihood of transmission to untreated individuals and about measures to prevent such transmission.  Shedding data collected during clinical development should provide a clear and comprehensive understanding of the shedding profile of oncolytic or VBGT products in the target patient population(s).  Note that it may be appropriate to describe this data in the package insert for an approved Biologics License Application (BLA).  

Shedding of each oncolytic or VBGT product should be studied during clinical development because the shedding profile is not always predictable from historical or preclinical data.  For these products, sponsors should consider product-specific variables and patient-specific factors (as described below and throughout this guidance) that could affect shedding.  For example, the biological characteristics and route of administration (entry) of oncolytic or VBGT products can be different from that of the parent strain of viruses and bacteria.  Specifically, these products may be:

        • Derived from laboratory-adapted wild-type, attenuated or engineered strains that may not have been characterized in humans in prior studies.
        • Replication competent or incompetent viruses; viruses that can infect a host cell and amplify to produce progeny are replication competent and those that can infect a host cell but cannot amplify and produce progeny are replication incompetent.
        • Dividing and/or auxotrophic bacteria; auxotrophic bacteria are unable to synthesize an organic molecule required for their growth and division but when this molecule is available with the other nutrients they require, growth and division of the bacteria may occur.
        • Introduced into the human body through unnatural routes and hence, the infectivity, replication, persistence and shedding from the human body may be different than that of the parental strains.
        • Engineered to carry transgenes; e.g., tropism-altering genes, immune modifying gene(s) or genes that enhance oncolysis.

Also, oncolytic or VBGT products may be derived from human-specific strains and therefore, animal models may not adequately predict shedding in humans.  In addition to product-associated variables that are described above, patient-specific factors, such as differences in the immune status at the time of treatment, can contribute to the potential for product shedding (for more details refer to section VII.B.).

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V. Design of Shedding Studies: Guiding Principles 

The main considerations in the design of shedding studies are:  the choice of clinical samples that are collected from subjects in a trial (feces, urine, nasal swabs etc.); the periodicity of sample collection and duration of the monitoring period; and the assay methodology selected to test for the presence of the shed oncolytic or VBGT product in the clinical sample (Ref. 1).

To guide the design of shedding studies, the following should be considered:

A. Biological Characteristics

  • Replication competence:  The ability of the oncolytic or VBGT product to multiply and amplify in the human host greatly affects how it is disseminated in the body and can, potentially, increase the extent and duration of shedding.
  • Immunogenicity:  When the oncolytic or VBGT product is derived from viruses or bacteria that elicit a strong immune response, the product may be more rapidly cleared from circulation than a poorly immunogenic product, and may be shed for a shorter duration.  Similarly, when a product is administered multiple times, the product may be shed for a shorter duration in the later dose cycles than after the immune-priming first or early doses.
  • Persistence and latency:  The duration of a shedding study may be longer if the oncolytic or VBGT product exhibits persistence or latency-reactivation in the host, as in the case of herpes virus-based products that are capable of latency (period of time during which a virus is present in the host without producing overt clinical symptoms).  Shedding of such products may be intermittent and unpredictable.
  • Tropism:  Tropism of the product may affect what samples should be collected to assess shedding.  For example, oncolytic or VBGT products that are engineered to carry tropism modifying gene(s) or mutation(s) may exhibit an altered shedding profile than the parent virus because of retargeting of the product to different tissues or organs.

B.  Route of Administration

In addition to the tropism of the oncolytic or VBGT product, the route of product administration should be considered in the selection of sample types to collect in a shedding study.  For example, to assess shedding in patients administered an oncolytic virus by the intradermal route, we recommend the collection of skin swabs at the site of injection in addition to the other samples routinely assessed for shedding, such as urine, feces and saliva.  Similarly, we recommend the collection of nasopharyngeal washes when an oncolytic virus is administered by inhalation or via the intranasal route.

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VI. Collection of Shedding Data in Preclinical Studies

Shedding data that are collected in animals following administration of the oncolytic or VBGT product can help estimate the likelihood and potential shedding profile in humans, particularly when there is concern about transmission to untreated individuals. 4  The decision to assess shedding in preclinical studies is based on the biological characteristics, derivation, and genetic make-up of the oncolytic or VBGT product.  For example, preclinical shedding data may be requested for an oncolytic or a replication competent VBGT product, if:

  • Humans have not been previously exposed to the product, as in the case of a non-human bacterial or viral strain.
  • The product has been administered to humans, but has been modified to achieve a different in vivo tropism than the parent strain.
  • The product has been previously administered to humans; however, a change in the route of administration is proposed.

The use of the animal species/model(s) is an important factor that can affect the biological relevancy of the shedding profile generated in the animal.  Considerations include the permissiveness or susceptibility of the animal to infection from the oncolytic or VBGT product under investigation, and any preexisting immunity the animal has to the product.

Collection of shedding data is an endpoint that can be included in preclinical studies designed to collect other data, such as safety and biodistribution.  The decision to include an assessment of the shedding profile of an oncolytic or VBGT product in an animal study will depend on various product-specific factors, as described above and in sections I. and V. of this document.  We recommend that sponsors initiate communication with the Pharmacology/Toxicology staff in OCTGT early in their product development program to discuss the need for generating a shedding profile for their oncolytic or VBGT product in animals, and the planned methodology to collect this shedding data.  

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VII. Collection of Shedding Data in Clinical Studies

Shedding data collected in clinical studies provides a shedding profile of a product in the target patient population and is used to estimate the potential of transmission to untreated individuals.  Depending on the shedding profile, it may be appropriate to include the information on shedding in the Investigator Brochure and in the Informed Consent for Investigational New Drug studies.  Depending on the shedding profile, it also may be appropriate to include shedding data in the package insert for licensed products to inform patients and physicians if shedding could occur with the use of an oncolytic or VBGT product, the potential for transmission of the product to untreated individuals, and of the measures to take to prevent such transmission.

A. When to Collect Shedding Data in Clinical Studies?

  • For products classified as replication competent, we recommend that sponsors begin collecting shedding data in Phase 1 trials.  Considering that shedding of such products is associated with a higher potential for release of infectious product-based viruses or bacteria, sponsors may need to continue collecting shedding data during Phase 2 trials to better characterize shedding before pivotal studies are conducted.
  • For VBGT products that are classified either as replication incompetent or replication deficient (i.e., a virus or bacteria that can infect a host (cell) but cannot amplify and produce progeny), we recommend collecting shedding data later in product development (e.g., during Phase 2 trials), after a dose and regimen have been determined.  Compared to shedding of replication competent products, shedding of replication incompetent or replication deficient products is expected to be low, for a limited duration, and associated with a lower potential for release of infectious product-based viruses or bacteria.
  • After shedding data is collected in early phase studies, it is not uncommon that dose, route, regimen (frequency of product administration, concomitant therapy, preconditioning regimens, etc.), or indication are modified.  These changes can alter how product is shed.  In such cases, shedding data collected in early clinical trials may not be adequate or relevant to predict the shedding profile of the product in its current state, and additional data should be collected in subsequent clinical trials where the route, dose regimen, and indication are the same as in the pivotal trial.

B. Study Design

The plan to collect shedding data in clinical studies can be based on prior clinical experience with the same or similar product, but when there is no such experience, as in the case of first-in-human oncolytic or VBGT products, the shedding profile generated in animals can be informative (see section VI. of this guidance).  We recommend that sponsors prospectively design and incorporate the sampling plan in the clinical study to collect shedding data.

There are three critical choices in the design of a sampling plan:

  • Frequency of sample collection;
  • Duration of sample collection; and
  • Type(s) of samples collected.

However, there are many aspects that can influence these choices, as described earlier in section V. and further elaborated below.

  • Frequency of sample collection:  Shedding is most likely to occur in the period immediately following product administration, irrespective of replication competence of the oncolytic or VBGT product.  A second peak of shedding may be noted in the days/weeks after administration of a replication competent product as a result of its multiplication/amplification in vivo.  Accordingly, sampling should start immediately after product administration, with frequent sampling during the initial weeks following treatment to capture the shedding pattern accurately (e.g., sampling on day 1, 3, 7, 10 and then weekly).  Analysis of samples should continue until three consecutive data points at or below the limit of detection (LOD) of the shedding assay are collected.  If the level of shedding does not reach the LOD of the assay but there is a continual decreasing trend, collection should continue until the results demonstrate that a plateau has been reached in at least three consecutive data points.
  • The duration of sample collection (monitoring period for shedding):
    • In general, when the oncolytic or VBGT product is a replication competent virus or bacteria, the monitoring period for shedding is longer (for sample collection) as compared to when the product is replication incompetent or replication deficient.  This is because you will want to capture the second peak of shedding associated with multiplication or amplification in vivo.
    • The immune status of the patient population should be considered.  Patients who are immune compromised may have an extended or even different shedding profile than their immune competent counterparts.  For replication competent products, the immune competence of the patient population is a relevant factor because many of these products are used in cancer patients who have had immunosuppressive chemotherapy.  When treated with replication competent oncolytic or VBGT products, immunosuppressed patients may become persistently infected and may shed the product for extended periods of time (Ref. 2).  
    • When product is administered in multiple cycles, the duration of shedding may be shortened in later cycles because of product-specific immune responses.  Data from single dose administration of product may be used to guide the timing of sample collection following multiple administrations. 
    • If an oncolytic product is based on a herpes virus that has the potential for latency reactivation, we recommend the collection of additional samples for shedding analysis when clinical signs warrant, i.e., when patients show signs of herpes virus infection due to reactivation.

  • Type(s) of sample:  The types of clinical samples collected to assess shedding include, but are not limited to, urine, fecal swabs, and saliva.  Other clinical samples may be collected depending on the route of administration of the product or the tropism of the product-based virus or bacteria (as described in section V. of this guidance).  The natural route of transmission and shedding of the parent virus or bacterium from which the product is derived from also is considered in the choice of clinical samples.  For example, if a herpes virus-based oncolytic product is administered intradermally for treatment of skin cancers, there is the potential of transmission of the herpes virus-based product through infected scabs/skin secretions because that is the natural route of transmission for herpes viruses.  Hence, in the case of herpes virus-based oncolytic products, skin swabs or dressing from injection sites should be analyzed for shedding.  Likewise, if an adenovirus-based oncolytic product is administered intranasally, there is the possibility of transmission of the adenovirus-based product through respiratory secretions, therefore, nasopharyngeal swabs or washes should be collected in the shedding study.  For tropism-modified products, knowledge about the route of transmission and shedding of the parent virus or bacterium may not be relevant or sufficient to guide in sample collection.  For such cases, it may be appropriate to use the biodistribution or shedding data from preclinical studies of the product to estimate the likely sites of shedding and to guide the type of sample collection.
  • Blood is not typically analyzed for shedding but should be collected as part of pharmacokinetic analysis to understand the extent of product dissemination from the site of administration and the rapidity of product clearance.  This information can be particularly useful to assess the extent of product shedding when limited vascular spread is expected for products administered locally (intratumoral, intramuscular, intracranial, subretinal routes, etc.).

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VIII. Analytical Assays To Measure Shedding

An analytical assay that measures shedding is designed to detect product in the clinical sample, either by detection of nucleic acids or for the presence of infectious viral particles or dividing bacteria.  Based on the design and output (nature of the assay readout), shedding assays can vary greatly in their performance and suitability.  Hence, the choice of a shedding assay can greatly affect the quality of the data collected, and is important in the generation of meaningful shedding data, i.e., data that provides a complete shedding profile for a product and can be used to estimate the potential of transmission to untreated individuals.

We recommend that sponsors consider the following in the selection of the analytical assay to measure shedding:

    • At least one of the assays used to measure shedding should be quantitative. We recommend that sponsors report the extent of shedding of oncolytic or VBGT products in terms of the number of genome copies or infectious units to provide a quantitative assessment of shedding.  Often an assay with a quantitative readout, like quantitative polymerase chain reaction (qPCR), is used because of the ease of performing/standardizing the assay, high throughput format, rapid turnaround time, and assay sensitivity.
    • For replication competent products, detection of nucleic acids should be followed up with infectivity or growth-based assays. Replication competent products are capable of growth or multiplication in humans and if shed, can be infectious.  Since only infectious viruses or bacteria are potentially transmissible, we recommend that sponsors follow a step-wise approach for the analysis of shedding of replication competent products.  Specifically, clinical samples that are scored positive for product-specific nucleic acids should be analyzed for infectivity in cell culture (for viruses) or growth in differential media (for auxotrophic bacteria), followed by a selective method for product identification.  There are many different approaches to assess infectivity or growth with assays that have a quantitative read-out (Ref. 3).  For example:
    • For detection of infectious viruses:  Assays that measure infectivity in terms of Tissue Culture Infectious Dose 50 (TCID50), plaque-forming units (PFU), focus-forming units (FFU).
    • For detection of dividing bacteria:  Assays that measure bacterial growth in colony forming units (CFU).

Similarly, if shedding of conditionally replicating oncolytic or VBGT products is noted by qPCR assay at a level significantly above the LOD, we recommend that sponsors further characterize the shed material to confirm infectivity or growth because such products could be shed in their infectious form even if replication is confined, mostly, to tumors or to a particular tissue-type.

Sponsors may justify limiting the shedding analysis of a replication competent or conditionally replicating product to qPCR assay, if:

  • The signal noted in the qPCR assay is at or below the LOD of the infectivity or growth-based assay; or
  • The cell culture step in an infectivity assay is demonstrated to be unsuitable for the analysis of clinical samples with complex composition such as excreta due to adverse effects on cell viability.
  • Shedding analysis of replication incompetent or deficient products by qPCR may be adequate.  Most VBGT products are replication incompetent or replication deficient; for example, adeno-associated virus (AAV) vectors, E1-deleted adenovirus (Ad) vectors, and some herpes virus vectors (HSV).  Replication incompetent or replication deficient products are incapable of multiplying or dividing in humans, and therefore, are shed to a lower extent and in a form that is noninfectious.  Hence, qPCR may be adequate as the primary assay to assess shedding of replication incompetent or replication deficient products.  If shedding is noted by qPCR assay at a level significantly above the LOD, we recommend that sponsors further characterize the shed material for infectivity or growth to confirm the absence of any potential replication-competent variants of the product that may have emerged.
  • Use of a qualitative assay to assess shedding may be justified.  When a qualitative shedding assay is demonstrated to be more sensitive than a quantitative assay in the detection of a specific product, a sponsor may justify the use of the former.  However, if there is detectable shedding with a qualitative assay, a follow-up quantitative analysis should be performed to understand the extent of shedding in positively scored samples.
  • Shedding assay(s) should be demonstrated to be specific, sensitive, reproducible and accurate.  We recommend testing of clinical samples in a shedding assay in duplicates or triplicates to determine reproducibility.  The specificity of the assay should be well understood to avoid false-positive or false-negative results, particularly since retesting is not always feasible with clinical samples that are limited in quantity, like nasal or skin swabs.  The sensitivity of the assay should be determined in terms LOD and the limit of quantitation (LOQ), if using a quantitative assay.  While the Agency does not expect shedding assays to be validated, the assays should be qualified to meet minimal performance capabilities and be suitable for the intended purpose.

The effect of sample type and composition on assay performance should be well understood.  Clinical samples such as feces and urine are rich in complex organic matter that can adversely affect the performance of an assay and lead to an underestimation of shedding.  Also, samples such as feces, saliva and nasal swab are rich not only in host proteins and nucleic acids but also in the body’s natural flora and in circulating strains of viruses and bacteria from the environment.  Thus, the assay conditions should be optimized to selectively analyze for the product under investigation.  For that, the specificity of the reagents used in the assay should be assessed and the quality of the reagents should be controlled.  Certified and contaminant-free reagents should be used in the analysis of clinical samples in a shedding assay.

Interference from clinical sample matrix can lead to a false negative result or an underestimation of the amount of shedding.  For example, clinical samples like urine, saliva, and feces are rich in proteases, nucleases, ions and salts that can affect the amplification process in a polymerase chain reaction (PCR); specifically, nucleases in saliva/feces can degrade template DNA, bile salts in feces or urea in urine can affect the activity of thermostable DNA polymerases in the PCR mixture.  When PCR inhibition is suspected due to interference from components in a clinical sample, the clinical sample can be diluted to a limited extent to reduce the interfering component.  Since dilution of the clinical sample also leads to template dilution, the sensitivity of PCR assay should be assessed in each assay run.  For that, each diluted sample should be tested in parallel with one that is spiked with a reference standard or positive internal control prior to dilution.  If interference cannot be decreased by limited sample dilution, alternative or additional extraction procedures should be considered to remove the interfering component(s) in the clinical sample.  

An underestimate of the level of shedding may also result due to degradation of viral or bacterial nucleic acids in enzyme-rich clinical samples such as feces and saliva during storage, handling/shipping and nucleic acid extraction.  To account for such effects, we recommend that mock/donor sample types be spiked, soon after collection, with the reference standard or internal positive control and the percent recovery of the reference standard or internal positive control should be determined on a one-time basis.  Sample collection, storage, shipping, extraction and analysis should be performed with the same methodology as that planned for the clinical (test) samples.

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IX. Analysis of Shedding Data 

In order to assess the potential of transmission to untreated individuals due to shedding, the analysis of shedding data for oncolytic or VBGT products should address the following:

A. The Nature of the Shed Material

When clinical samples are scored positive for product in a shedding assay, the subsequent analysis of these samples should provide answers to the following questions:

  1. Do the clinical samples contain genomes indicative of the presence of product-based infectious viruses or bacteria or do the clinical samples contain mostly degraded genome fragments associated with product-based non-infectious viruses or bacteria?  
    An example of a clinical sample containing genomes indicative of the presence of infectious product-based viruses is one in which product-specific nucleic acids are amplified (by PCR) after treatment with nucleases.  Under such conditions, the only genomes that are amplifiable are the ones protected within intact nuclease-resistant viral particles that are potentially infectious.  When nuclease treatment is not feasible, amplification of a full-length viral genome by long PCR would suggest the presence ofnfectious viral particles.  Conversely, if only small genome fragments are amplifiable, then the clinical sample likely contains non-infectious product-based viruses.
  2. Can infectivity or growth attributable to the product-based viruses or bacteria be detected in the clinical sample?

Ideally, the shedding assay should be able to discern complete genomes from degraded genome fragments, or infectious from noninfectious product-based viruses, or dividing from non-dividing product-based bacteria in the samples.  We refer you to section VIII. for details on shedding assays and for our recommendations.  If qPCR is the only assay you have relied on in your shedding analysis of replication competent products, or the shedding assay screens for small genome fragments of the product, then we will assume that the shed material in the positively scored samples is infectious.

B. The Extent of Shedding

In the analysis of shedding data, the extent of shedding noted for each sample type as a factor of time, dose (amount of product administered) and regimen (number of doses) should be reported for all the patients monitored in the study.  Raw data in the shedding report should be accompanied by a corresponding analysis that is comprehensive and describes the following:

  1. The number of patients that are shedding as a percentage of the total patients in the study for each sample type, dose and regimen studied.
  2. The duration of shedding, including the first and last day of shedding, and the peak period(s) of shedding in each sample type.  The period when shedding stops in most patients in the study also should be clearly identified.
  3. The clinical sample(s) where shedding was consistently noted (type and time point) and samples that were consistently negative for all the patients in the study.
  4. The quantity of product shed in a clinical sample.  The amount shed should be reported taking into account the final volume/mass of the clinical sample, e.g., 10 PFU of virus per mL of urine, or 10 CFU of bacteria per mL of urine, or 10 genome copies per microgram of stool.  When assessing the quantity of shedding, you should factor in the stability of the product in the clinical sample, and whether there could be an underestimate of the level of shedding because of loss during sample storage, handling and shipping (for details, please refer to section VIII. of this guidance).

Note that your analysis of shedding data should be accompanied by a summary of the shedding profile of the product in patients treated for a specific indication.  While it is common practice in clinical development of oncolytic and VBGT products to study a product for different indications in multiple trials, the shedding pattern may be distinct in each study population.  We recommend against pooling shedding data from multiple trials in which the same product is studied for different indications because results from a shedding study in a given indication may not be generalizable to other indications.

Finally, we recommend that the shedding data be submitted in a format as described in the next section.

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X. What to Include in a Clinical Shedding Study Report

In order to address the potential for transmission to untreated individuals due to shedding of oncolytic or VBGT products from the patient, the following should be provided to us for review as the information is obtained during product development, and a full report should be provided in the BLA:

  1. Shedding report that provides a comprehensive shedding profile of the product in the target patient population(s) which includes the following:
      1. Background information on the product:  Derivation history; biological characteristics of parent viral or bacterial strain; route of transmission of parent strain; replication competence; attenuation; and tropism of the product.
      2. A summary of the biodistribution profile in animal models and the findings from preclinical shedding studies, if conducted.
      3. The rationale for both the clinical shedding study design (i.e., choice of clinical samples, frequency and procedures of collection and storage), and the analytical method selected to assess shedding.
      4. Your data collection/sampling plan, and your procedures for storage, shipping and handling of the product.
      5. An assay description that includes the following: 
          1. Test sample preparation or nucleic acid extraction procedures including the dilution factor and amount of nucleic acid extracted per sample.
          2. If you use qPCR assays, provide for each assay the sample volume, amount of nucleic acid per reaction, cycle numbers, primers, and size of the amplified DNA.
          3. If you use infectivity/growth-based assays, provide the permissive cell line/growth media, the conditions for adsorption and infection or growth, and the nature of the read-out (TCID50, FFU, PFU or CFU; or Cycle threshold (Ct) for assays with qPCR read-out).
          4. Assay qualification, controls and sensitivity:  Description of the qualification studies, standards, spikes, controls, number of replicates, assay variability and sensitivity (LOD and LOQ, if applicable).
      1. Analysis of shedding data: 
          1. Tabulation and/or graphical representation of the shedding data.
          2. Analysis of the data and summary of the findings from the study.
      1. Your estimate of the potential for transmission to untreated individuals of the product.

See section VIII. for a discussion of analytical assay types and conditions, section VII.B. for a discussion of clinical sample collection, section IX. for analysis of shedding data, and section XI. for details on assessing the potential for transmission to untreated individuals due to shedding.

  1. As needed, data and analysis of clinical monitoring for transmission to untreated individuals in the target patient population (refer to section XI.B. for additional information).
  2. Other relevant information on the ability of the oncolytic or VBGT product, or its parental/related strain of viruses or bacteria to potentially infect humans and cause disease.  When there is the potential to cause disease in humans, the following should be discussed:
      1. The spectrum of disease symptoms caused by the parental strain, including atypical presentation of the disease, or occurrence of asymptomatic shedding;
      2. The attenuation of the product compared to the parent strain of the virus or bacterium circulating in the community;
      3. The natural or acquired immunity of the general population that could potentially protect against infection from  the shed product;
      4. The therapeutic options to treat the infection/disease in case of transmission to untreated individuals of the shed product, and
      5. Preventive/containment measures that can limit spread of the shed product beyond the treated individual to minimize exposure of third parties, particularly, immune-compromised adults, neonates and seniors.  Note that the data you collect on onset and duration of shedding can inform appropriate preventive/containment measures.  For example, if peak shedding occurs soon after treatment when the patient is monitored in a health-care setting, then the possibility of transmission is mainly confined to health care professionals (HCP) and individuals that come into close contact with the patient.  If shedding is prolonged or if there is a second peak of shedding in the days following discharge of a patient from a health care setting, there is the possibility of transmission to contacts beyond the health care and home setting. 

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XI. Assessing the Potential for Transmission to Untreated Individuals Due to Shedding

Our current understanding is that in most cases, the potential for transmission to untreated individuals is extremely low when oncolytic or VBGT products are shed because of the derivation methods and/or modifications that are designed to attenuate the product when compared to the parent strain of virus or bacterium.  Nevertheless, you should discuss the potential for transmission based on the analysis of the shedding data collected in the clinical studies and taking into consideration the factors described below.

A. What Information in the Shedding Data Can be Used to Assess Potential for Transmission to Untreated Individuals?

  • Whether product was shed.
  • Whether the shed product was determined to be infectious.
  • Whether the amount of infectivity in the clinical samples was comparable to that needed to initiate infection in a third party.  For example, adenovirus infective dose is reportedly >150 PFU when given intra-nasally (Ref. 4) or lower when aerosolized (Ref. 5).  The minimum infectious human dose may vary when viruses or bacteria are administered or acquired through different routes, or among different strains, but for many disease-causing viruses and bacteria, the minimum infectious dose in humans may be undefined.
  • Whether the clinical sample containing the shed product represents the natural route of transmission.  For example, a respiratory virus that is shed in feces may not be as infectious and transmissible when compared to that shed in nasopharyngeal secretions.

B. Monitoring Untreated Individuals for Transmission

Because transmission to untreated individuals is an extremely low probability event, monitoring such individuals for transmission is usually not required during the clinical development of a product.  However, if we believe there is a potential for transmission, additional data will be needed to assess that possibility; in which case, we recommend that you consult with OCTGT in connection with developing a monitoring plan.

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XII. References

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1. Gene therapy products are all products that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms.  The products may be used to modify cells in vivo or transferred to cells ex vivo before administration to the recipient.  See section III of FDA’s guidance document entitled “Guidance for Industry:  Gene Therapy Clinical Trials - Observing Subjects for Delayed Adverse Events” dated November, 2006. 

2. Oncolytic products refer to replication competent viruses or dividing bacteria that are used as therapeutic agents to mediate lysis of tumor cells.  Some oncolytic products carry foreign genes (immune modifying genes, genes that enhance oncolysis etc.), and mediate part of their anti-tumor effect by transcription and/or translation of these foreign genes in the host.  Hence, oncolytic products that carry foreign genes can also be classified as gene therapy products.

3. Available at http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/

4. For general information about preclinical assessment of these products see FDA’s guidance entitled “Guidance for Industry:  Preclinical Assessment of Investigational Cellular and Gene Therapy Products” dated November 2013, at: http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/

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