Biodistribution & Shedding Assay Services
FREQUENTLY ASKED QUESTIONS
What is a biodistribution assay?
Biodistribution (BD) assays determine the amount of viral DNA/RNA in different cells and organs in an organism. Presence of vector sequences is most commonly tested by Q-PCR, although immunohistochemistry (IHC) has also been applied largely in the assessment of the BD and safety of vectors in gene therapy.
Knowledge about species infectivity, tissue tropism, and cellular infectivity of different vectors is very important for assessing the BD of a cell and gene therapy product. Two main types of BD study design are:
- Invasive: In this kind of tracking a radiolabeled isotope is usually tagged to the product whose BD is to be investigated which is injected intravenously in to the trial animal, which is sacrificed and its organs are placed in gamma radiation counters to measure the radiation.
- Non-invasive: The BD and persistence of a specific viral coat can be analyzed by engineering a fluorescent protein (e.g. GFP) or ECL tags into the CDS which can be visualized using IHC methods such as optical or fluorescence live imaging.
Why is biodistribution assessment critical for early-phase clinical trials of a biotherapeutic?
Biodistribution studies are performed to determine the in vivo distribution or localization of a drug product to support early biotherapeutic drug development. Analysis of the persistence of the vector/virus (gene therapy product) to target and nontarget tissues following direct in vivo administration in animals can be useful for the identiﬁcation of potential target organs of toxicity. This assessment is thus an important part of the overall safety profile of the gene therapy product.
AAV-derived vectors are among the most promising gene therapy tools because of their replication incompetence and ability to mediate long-term transgene expression. Various routes of delivery like intramuscular, intravenous, intra-arterial, and intracranial and high capsid diversity make biodistribution and shedding studies a major investigational ﬁeld for the safety assessment of all proposed early-phase clinical trials for a biotherapeutic.
This diagram shows where BD/shedding assays sit in the development pipeline of a typical VGT product.
What is shedding and why is shedding investigation so crucial?
Shedding is the dissemination of the Adeno-associated virus (AAV) vector through secretions or excreta from the animal model or patient. The assessment of shedding can be utilized to estimate the likelihood of transmission of vectors healthcare workers and family members and others in contact. According to recent reports AAV vector can persist for several years as intact particles in the subretinal space of dogs and nonhuman primates raises the possibility of low levels of replication, which has never been formally excluded in vivo. An alternative explanation is the ability for AAV vector particles to resist clearance when aggregated in sheltered areas after local administration at high concentration in virtual spaces for ex subretinal space. Structures of the genitourinary tract, like testis, contribute signiﬁcantly to vector shedding in the semen. All this urges the requirement for comprehensive shedding investigations.
However, the obvious concern is if the shed viral particle infectious? An in-vitro infectivity assay can be performed to test the infectivity in the biological fluids but the sensitivity is usually low for several reasons, such as the lack of a highly permissive in vivo cell line for transduction for a given AAV serotype and the interference mediated by the milieu for ex urine.
How do the biodistribution and shedding assays differ?
In terms of AAV, the basic difference between a biodistribution (BD) and shedding assay is the state of viral genome. In the BD assay, AAV genome we quantify is integrated in the host genome while in a shedding assay, a free viral particle is to be detected and if possible quantified.
Most prevalent assays for the detection of shed vector are PCR and infectivity. Quantitative polymerase chain reaction (Q-PCR) is most common although it has a limitation to distinguish between intact virion and noninfectious or degraded AAV. DNA is extracted from samples (e.g. urine, saliva, semen) and then either PCR or Q-PCR is performed.
What challenges are associated with BD/shedding assays?
A big challenge in preclinical and clinical studies is that biodistribution and shedding features of viral vectors have been evolving with the advent of newer gene therapy technologies that have resulted in vectors with higher titers, increased transduction efﬁciencies, and broader tissue/ organ tropism. These developments have adding to existing concerns regarding the risk of germ line transmission.
If the amount of AAV vector detected by Q-PCR is below the limit of detection of the infectivity assay, one might not choose to further characterize the shed AAV vector by infectivity assays due to the constraints of assay sensitivity.
What are some other considerations?
- Select primer sequences that do not amplify endogenous sequences as it could interfere with the efﬁciency and speciﬁcity of the reaction.
- To validate the speciﬁcity of the assay and exclude false positive results, analysis also must be done with ﬂuid extracted DNA alone.
- As PCR inhibition can occur in some tissue types (e.g. semen or feces) the sensitivity of the assay must be determined by analyzing serial dilutions of a surrogate DNA in isolated control sample DNA.
- Since shedding samples can have a wide breath of inhibition, like in feces samples, donor to donor variability should be analyzed.
Analysis of in vivo distribution of an AAV vector in gonads (testes or ovaries) is of specific importance because detection of the vector in gonad tissue indicates the potential for germ line integration and propagation to the descendants.
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- FDA Inspected
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What regulatory considerations are there for in vivo preclinical assessments of biologics?
FDA recommends considering following points in animal study design for evaluation of gene therapy product biodistribution and persistence.
- Gene therapy product must be used for the final formulation for the clinical study because changes in final formulation may alter biodistribution pattern.
- The intended clinical route of vector administration should be preferred.
- Appropriate safety endpoints should be included in biodistribution study to assess any potential correlation between product presence and its adverse reactions. These endpoints can include clinical observations, body weights, clinical pathology, gross organ pathology, and histopathology.
- Sacrifice of animals is recommended at the expected time of peak gene therapy product detection and at several later time points to evaluate clearance of product at the peak product detection and at several later time points to evaluate clearance of product.
FDA guidelines published/updated in year 2020 about “Long Term Follow-up After Administration of Human Gene Therapy Products” briefly discussed the requirement to develop a robust and sensitive PCR method for assessing BD in clinical samples like blood or clinical autopsy samples.
These guidelines can be extrapolated to non-clinical studies like validating the method for an ideal sensitivity of 50 copies per µg of genomic DNA. In addition, MIQE guidelines published in year 2009 can be explored for developing an ideal PCR method for BD.
“The biodistribution data, coupled with other preclinical safety endpoints such as clinical pathology and histopathology, help determine whether vector presence or gene expression correlates with any tissue-specific detrimental effects in animals.” (FDA Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products, November 2013)