Biodistribution in cell and gene therapies and ATMPs requires optimized extraction protocols tailored for each test item. The extraction properties of short oligos like siRNA, miRNA, and ASO, as well as modified oligos, low-abundant targets, and capsid-enclosed targets such as AAVs and ADs, need a thorough evaluation. The downstream RT-PCR and quantitative PCR must also be optimized to detect these test items within set performance criteria, meeting regulatory requirements. For comparing different constructs, these methods must perform equally well across all items.
Biodistribution studies for cell and gene therapy products play a crucial role in understanding the delivery of the drug in on- and off-target tissues and is a safety requirement by the FDA and EMA.
Bioanalysis aims to understand the distribution of therapeutic genetic material, assess the tissue specificity of the vector, and establish correlations with the levels of transgene mRNA and protein (the therapeutic product) in both target and non-target tissues.
Clinical biodistribution studies aim to determine drug exposure and persistence in target tissues and their correlation with efficacy and safety. Blood or serum samples are typically collected as surrogate measurements, as obtaining biopsies can be demanding.
In the initial preclinical stages, developers assess biodistribution to refine the vector’s tissue specificity as part of lead candidate selection. Information on expected target nucleic acid levels is valuable for subsequent assay validation. In preclinical trials, the FDA and EMA require biodistribution studies to determine dose ranges and establish the clinical dose for first-in-human trials (FIH). Initially, non-regulatory biodistribution studies are conducted to gather information about expected transgene levels in both target and non-target organs and biofluids. These insights simplify the transfer of methods to full validation approaches.
The biodistribution of in vivo expressed mRNA provides insights into the tissue specificity of regulatory elements and potential off-target expression. This correlation between the administered dose and the therapeutic product in target and non-target tissues provides data for determining a safe and effective clinical dose.
Biodistribution assessments are essential to investigational and final drug or biological approval applications submitted to regulatory agencies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA). They are critical in determining the clinical dose for initial human trials.
Biodistributon at TATAA
We optimize the extraction methods for your chosen tissues or biofluids to effectively extract
- Modified oligonucleotides
- Short targets such as siRNA and miRNA
- Low abundance targets
- GC-rich sequences
- Sequences with secondary structures
Our optimized methods apply to various sample types, including
- Biofluids
- Tissues with varying lipid content, enzyme-rich tissues, tissues with low cellular content, and tissues with high fibrous or connective tissue content
- Matrices rich in PCR inhibitory agents such as salts, mucins, urea, or fat
Assay design
Our primers and probes are designed to quantify the target without detecting endogenous genes or transcripts. Whenever possible, at least one of the primers targets non-natural sequences, such as vector backbones, non-natural exon junctions, promoter regions, endogenous genes, or codon-optimized sequences.
To maintain the highest standards of accuracy, we include RNA spikes to check for matrix interference during the RT step and DNA spikes to monitor interference in qPCR/dPCR analysis.
We optimize our assays to ensure that all constructs are quantified equally when comparing different constructs, whether they vary in modifications, lengths, or other factors.
Assay validation
Each assay is validated according to regulatory requirements, which include a comprehensive description of performance parameters such as sensitivity and reproducibility.
Validation is performed on blank samples (untreated tissues/biofluids) and, if possible, partly on surrogate matrices that resemble real matrices. Even with good surrogates, blank samples are essential for validation.
Whether GLP is required or not, 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 according to 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 both the samples and the data.
