ICH S12 – Biodistribution considerations for gene therapy products.


Biodistribution aims to understand the distribution of therapeutic genetic material in various target and non-target tissues. This data is crucial for correlating with mRNA expression data to determine optimal dose strategies, refine vector constructs, and interpret pharmacology and toxicology data. The objective is to ensure the vector achieves the desired expression in target tissues while minimizing the presence and expression in non-target tissues, thereby reducing the risk of off-target effects.

The nonclinical biodistribution assessment must be available when evaluating the nonclinical pharmacology and toxicology findings and should be completed before initiating the clinical trial. 

Quantitative PCR and digital PCR are considered standard assays for biodistribution studies. In 2003, the FDA and EMA, under the guidance of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), published the S12 guideline, “Nonclinical Biodistribution Considerations for Gene Therapy Products.” This guidance provides a comprehensive framework for conducting nonclinical biodistribution studies, ensuring consistency in gene therapy development.


This is a summary of the text, including our notes as a provider of biodistribution services. The original guidelines:   FDA, EMA

The scope of the S12 guidelines


Products mediating effect by expression: Gene therapy products that achieve their therapeutic effect through the expression (transcription or translation) of transferred genetic materials.


  • Purified nucleic acids such as plasmids and RNA.
  • Genetically modified microorganisms: Viruses, bacteria, and fungi modified to express transgenes, including those that edit the host genome.
  • Ex Vivo genetically modified human cells reintroduced to the patient.
  • In Vivo genome editing products with the intention to alter the host cell genome in vivo such as those delivering a nuclease and guide RNA by nonviral methods.
  • Oncolytic viruses: Although not currently always considered a gene therapy product, the principles of the guidance also apply to oncolytic viruses not genetically modified to express a transgene.


  • Prophylactic vaccines: vaccines designed to prevent diseases rather than treat or cure genetic conditions.
  • Chemically synthesized oligonucleotides: Oligonucleotides or their analogs that are chemically synthesized and not produced using biotechnology-based manufacturing processes.

The release of gene therapy products from the body, known as shedding, is discussed in other guidelines.

Definition of biodistribution

Nonclinical biodistribution is defined as the in vivo distribution, persistence, and clearance of a gene therapy product at the administration site and in both target and non-target tissues. Biodistribution involves using analytical methods to detect the gene therapy product and the transferred genetic material in collected samples. Additionally, it may include the detection of expressed messenger RNA (mRNA) from the gene therapy product.

TATAA Pro Perspectives
Our approach

At TATAA Biocenter, we bring over two decades of expertise in extraction and assay design. Sample extraction is crucial for biodistribution studies, and we test and optimize extraction efficiency for each tissue and target since target modifications, lengths, and secondary structures can significantly impact extraction efficiency.

Our qPCR and dPCR assays are designed and optimized to precisely identify the test item, whether it’s to detect a short target like siRNA or miRNA using our TATAA-invented Two-Tailed primer approach or to differentiate between transgenes and highly homologous endogenous sequences at the DNA or transcribed (mRNA) level. The assay optimization also includes tests to detect and mitigate matrix interference on the assay.

Our approach provides reliable, high-quality data that supports robust scientific conclusions.

A nonclinical biodistribution study should be conducted in a biologically relevant animal species or model, using a gene therapy product representative of the intended clinical product in terms of formulation and titer. The study should employ the same route of administration (ROA) intended for human use and dose levels sufficient to characterize the biodistribution profile comprehensively.

The highest dose level evaluated should match the expected maximum dose level in toxicology studies, usually limited by factors such as animal size, ROA/anatomic target, or gene therapy product concentration. It is crucial that the dose level for BD evaluation equates to or exceeds the anticipated maximum clinical dose level but does not surpass the highest dose level used in toxicology studies.

Biodistribution data from the same gene therapy product with a different transgene may be utilized if the tissue tropism is similar (e.g., using a fluorescent marker protein). Similarly, data from the same gene therapy product intended for a different clinical indication might be applicable. However, factors such as dose levels, dosing regimen, ROA, and changes in the promoter must be considered in this decision.

In cases where no biologically relevant animal models exist, such as when the vector targets a molecule absent in animal cells, a comprehensive discussion and justification for an alternative approach to evaluate nonclinical biodistribution is necessary.

Ensuring the quality, integrity, and reliability of the data is essential. While nonclinical biodistribution studies not conducted in compliance with Good Laboratory Practice (GLP) are generally acceptable, all in-life evaluations and sample collection procedures must adhere to GLP standards when the biodistribution evaluation is part of a GLP-compliant toxicology study.

TATAA Pro Perspectives
Validation for biodistribution

As a GLP-accredited laboratory, we conduct assays in a GLP-like manner. This means we handle samples using the same processes, control the work environment by GLP standards (including instrument service programs, temperature control, staff training, IT security, and more), and ensure that sample handling is time-stamped to maintain the integrity of the samples and the data.

Sample collection
  • Minimize the risk of contamination.
  • Ensure predefined protocols are in place before starting the study to guarantee the results’ consistency, reliability, and reproducibility.
  • Store (retain) the samples to preserve their integrity for further analysis and verification. This applies to both the vehicle control and the administered gene therapy product.
  • Maintain documentation on the order of sample collection.

Select time points to reflect the time-related changes in the gene therapy products. Additional time points can be included to capture the steady-state period, estimate persistence, and evaluate GT products after repeat administration when applicable.

For replication-competent vectors, time points should cover detecting the second peak level due to vector replication and the subsequent clearance phase.

The samples should include:

  • Injection site(s)
  • Gonads
  • Adrenal gland
  • Brain
  • Spinal cord (cervical, thoracic, and lumbar)
  • Liver
  • Kidney
  • Lung
  • Heart
  • Spleen
  • Blood

This panel can be expanded depending on vector type/tissue tropism, the expression product, ROA, disease pathophysiology, and animal sex and age. The final sample collection panel should be guided by an understanding of the gene therapy product, the target clinical population, the ROA, and existing nonclinical data.

TATAA Pro Perspectives
Sample collection for biodistribution

In discussions with the sponsor, we consider alternative sample methods. Sometimes, an RNA stabilizing reagent is preferred, while other times, snap-frozen samples are more suitable. The choice also depends on the capabilities of the sample collection site.

For validation purposes, we need blank samples in addition to surrogate matrices. Surrogate matrices resemble real animal matrices but are used to reduce the number of animals required. Blank samples are obtained from untreated animals, and even with good surrogates, they are essential for thorough validation.

Assay methodologies

Biodistribution studies necessitate quantifying the amount of genetic material (DNA/RNA) of the gene therapy product in different tissues and biofluids and, if appropriate, the expression product (mRNA, miRNA, or other). qPCR or dPCR is considered the standard. When cellular content varies significantly (e.g., in blood), DNA/RNA concentration could be stated as copy number per microliter.
To validate the experiment’s efficiency, a known quantity of nucleic acid should be spiked into the sample. Measuring the recovery of the spiked material will assess the assay’s ability to detect the target sequence accurately.

A comprehensive description of the methodology, including performance parameters such as sensitivity and reproducibility, should be provided.

TATAA Pro Perspectives
Data quality – Our spike approach

Efficiency in experiments is crucial for using data and drawing reliable conclusions. As S12 suggests, spiking should be applied to evaluate efficiency. We use spikes in the extraction, RT step, and PCR assay to ensure the test item is efficiently extracted and that the matrix does not interfere with downstream steps.

The test item itself often needs to be spiked in during the extraction validation since the extraction performance can vary significantly based on the test item’s length, modifications, and secondary structures. The test item itself is used for test items like adeno-associated virus vectors (AAV) and adenovirus vectors (AD) since a capsid surrounds the nucleic acid and extracts more like a protein than naked DNA/RNA. Another challenging test item to extract is siRNA, where sometimes the best approach is not to do an extraction but to use the homogenate as it is.

We always include our RNA spike, which is quality-controlled in-house, to ensure the RT step is efficient (for siRNA and other RNA samples and when no extraction is performed, of course). Additionally, we use our DNA spike to ensure the PCR reaction is not inhibited.

Messenger RNA

The level of expression product in vector-positive tissues/biofluids can contribute to the characterization of the safety and activity profiles. The necessity of such an assay should be decided based on a risk-based approach:

  • Depending on how much and for how long the gene therapy product is present in the tissue or biofluid.
  • Considering the patient population and their vulnerability.
  • Other safety concerns, such as whether the vector is a virus and the risks associated with the expression product, should be addressed.
Immunological considerations

Preexisting immunity in animals, especially in non-human primates (NHPs), against the gene therapy product could affect the biodistribution profile. Screening animals for preexisting immunity should be considered.
Post-administration, an immune response may result in a biodistribution profile that cannot be reliably interpreted.
Immunosuppression for the sole purpose of biodistribution is not recommended.

TATAA Pro Perspectives
Cytokine profiling

We use Olink Target 48 Cytokine panels to monitor innate immune response activation through multiplex measurements of 42 key cytokines using only microliters of plasma and blood. The panel is available for mouse and human samples and is generally compatible with non-human primates (NHPs).

Ex vivo genetically modified cells

When assessing the biodistribution of gene therapy products consisting of ex vivo genetically modified cells, key factors include the cell type, route of administration (ROA), and the impact of gene modifications on cell distribution, such as changes in cell adhesion molecule expression. Graft-versus-host disease (GVHD) in animals can complicate the biodistribution assessment of modified human T cells. Generally, biodistribution assessment is not critical for hematopoietic cells due to their expected widespread distribution after systemic administration. However, biodistribution assessment in relevant animal models should be considered if specific organ or tissue targeting is anticipated.

Gonadal tissues

It is essential to conduct a biodistribution assessment of the administered gene therapy products in the gonads for both sexes unless the target clinical population is restricted to one sex. The presence of the gene therapy products in the gonads may necessitate additional studies to determine gene therapy product levels in germ cells (e.g., oocytes, sperm) or nongermline cells in the animals. Persistent detection of the gene therapy products in nongermline cells within gonadal tissues may warrant further consideration of its potential effects on reproduction.

Triggers for additional nonclinical biodistribution studies
  • Significant changes such as ROA, increased dose levels, or another clinical indication.
  • Significant changes in the vector structure.
  • Changes in the manufacturing process that affect formulation or relative quality attributes.


Contact us for a consultation on biodistribution assay design.





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