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The Significance of Genomics Analysis in Gene Therapy Development

The Significance of Genomics Analysis in Gene Therapy Development

Gene therapies offer great hope in the treatment of rare and inherited diseases, many of which are currently incurable. In recent years, initial success and promising data have spurred a major interest in gene therapy, and development has grown exponentially. By its very nature, development of a gene therapy is a complex undertaking that requires rigorous study to ensure its safety. Among the key issues that need to be addressed are:

  • Quantifying and characterizing viral vectors
  • Targeting to the correct tissue
  • Understanding the likelihood and risk of shedding
  • In the case of gene-editing, evaluating for on- and off-target editing

Here, we will explore common and emerging applications of different genomics instruments and analyses for providing insight into these issues in both preclinical and clinical studies for gene therapy development.

Primary uses of genomics in gene therapy development

Quantifying and characterizing viral vectors

A primary challenge in gene therapy viral vector development is establishing and optimizing a process for large scale production.1 Adeno-associated virus (AAV) vectors are commonly used in gene therapy due to their, lack of known pathogenicity and capacity to achieve efficient and persistent gene transfer.1

qPCR remains the most widely used and accepted method of quantifying AAV vectors but is limited by its DNA amplification efficiency and its reliance on a standard curve.[i] Moreover, practical use of qPCR in clinical trials is restricted by limited sample availability.2

In the last few years, a powerful technique for AAV vector quantification has emerged. Research has shown that ddPCR yields accurate estimations of per-cell vector copy number (VCN) without reliance on a reference standard curve and with high sensitivity and a wide dynamic range of detection.2 In one direct comparison of qPCR and ddPCR, ddPCR was found to be up to four times more sensitive in the absolute quantification of single-stranded AAV vector genomes.3 This level of accuracy and precision is necessary in gene therapy development for assessing treatment potency and determining correct dosing.

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In another study comparing these two methods of nucleic acid quantification, ddPCR was found to be more robust and to have lower assay variance. This same study demonstrated the added value of transmission electron microscopy (TEM) analysis as a complementary method for evaluating viral structure and presence of aggregates or protein impurities, which is important for validating sample quality for clinical testing.1

Accurate quantification is essential as AAV vectors can cause toxic effects which may be dose limiting. To improve vector efficacy, optimize dosing, and reduce toxicity, it may also be useful to gain insight into the innate immune response to AAV vectors using flow cytometry and immunohistochemistry (IHC).

Targeting therapy to the correct tissue

While AAV vectors are very efficient at delivering genes, it may be difficult to target these vectors to the correct tissue. Thus, understanding and measuring biodistribution of a gene therapy is critical in both preclinical and clinical development. Given its high sensitivity, ddPCR can be used in both preclinical and clinical studies for assessing both the presence of AAV vectors and the VCN in particular tissues or biofluids.

Beyond VCN, knowledge of vector expression and potency are also essential for gene therapy development to inform candidate efficacy, safety, and pharmacodynamic profiles. Reverse-transcription ddPCR (RT-ddPCR) can be used to quantify target mRNA expression of transgenes.4

Assessing likelihood and risk of shedding

Shedding refers to the release of virus- or bacteria-based gene therapy (VBGT) products from a patient through biofluids or feces or through the skin, which raises the possibility of transmission of these products from treated to untreated individuals. Assessment of shedding can be utilized to understand the potential for transmission to third parties and the potential risk to the environment. The FDA has published a guidance document on design and analysis of preclinical and clinical shedding studies for assessing the risk of transmission to untreated individuals.5 ddPCR is useful for such studies, where high sensitivity and absolute quantification are important. Quantitative assessments of genome copy number should be accompanied by studies of growth or infectivity.

Evaluating on- and off-target gene editing

As gene editing technologies such as CRISPR/Cas9 and other sequence specific nucleases continue to advance, there is an increased need for methods to detect on- and off-target mutations. The most widely used methods for detection of on-target mutations are all PCR-based and tend to underestimate the frequency of on-target activity due to decreased sensitivity for large deletions and inefficient amplification of large insertions.6 of PCR amplicons provides direct information on the nature, diversity, and frequency of mutations, allowing for detailed characterization of on-target gene editing.

Off-target mutations are more challenging to detect because their number and position are unknown, though many CRISPR/Cas9 tools include information about potential off-target sites in the genome of interest.6 The massively parallel sequencing capability of NGS makes it an attractive option for detecting off-target mutations when there are many target sites and samples.

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Genomic analyses for adoptive cell therapies

Genomic analysis may also be useful in cell-based gene therapies such as chimeric antigen receptor (CAR) or T cell receptor (TCR)-engineered T cell products. For example, qPCR and ddPCR can be performed on peripheral blood mononuclear cells (PMBCs) to measure vector copy number VCN as an estimate of CAR vector delivery efficiency and CAR-T cell representation.7 These assessments can be performed longitudinally in both the preclinical and clinical settings. In multiple types of B cell cancers, qPCR measurements of VCN have been shown to correlate with CAR-T cell expansion kinetics, persistence, clinical response, and severity of side effects.8 However, qPCR cannot differentiate subtle copy number differences and cannot determine CAR-T cell phenotype or whether the CAR is expressed. ddPCR is more sensitive and precise and studies have shown that it can enable single-cell VCN measurements.3 If there is an antibody specific to the chimeric antigen receptor, flow cytometry analysis can also be used to quantify CAR-T cells.

Given that the CAR vector is randomly inserted into the genome during transduction, researchers may also want to assess the presence and genomic location of the integrated vector. Integration site analysis—which involves DNA fragmentation, PCR amplification, and NGS—can be used to map sites of insertional mutagenesis. It can also be used to characterize any biases in integration loci among different CAR vector delivery techniques.3

T-cell receptor (TCR) sequencing (TCRseq) is another popular assay for evaluating clonal diversity and kinetics in CAR-T cell studies. Moreover, TCR sequences can be used to track CAR-T cells after therapy and can be performed in bulk or at the single-cell level.9


Developing reproducible, scalable gene therapy products that are safe and effective for clinical use is complex. At Precision for Medicine, our specialty lab services leverage cutting-edge research, established technologies and proprietary approaches to support the entire development lifecycle. We have extensive experience in gene therapy development, helping developers advance promising candidates from preclinical through pivotal clinical studies. To learn more about how Precision for Medicine accelerates gene therapy development, view our gene therapy services.


1. Dobnik D, et al. Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol. 2019;10:1570.

2. Lin HT, et al. Application of droplet digital PCR for estimating vector copy number states in stem cell gene therapy. Hum Gene Ther Methods. 2016;27(5):197-208.

3. Lock M (2020). Viral Quantification — Adeno-Associated Virus Vector Genome Titer Assay. Available at

4. Clarner P, et al. Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors. Mol Ther Methods Clin Dev. 2021;23:68-77.

5. U.S. Food and Drug Administration. Design and Analysis of Shedding Studies for Virus or Bacteria-Based Gene Therapy and Oncolytic Products: Guidance for Industry, August 2015. Available at

6. Zischewski J, Fisher R, Bortesi L. Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases. Biotechnol Adv. 2017;35(1):95-104.

7. Lu A, et al. Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products. J Transl Med. 2020;18(1):191.

8. Hu Y, Huang J. The chimeric antigen receptor detection toolkit. Front Immunol. 2020;11:1770.

9. Pasetto A, Lu YC. Single-cell TCR and transcriptome analysis: An indispensable Tool for Studying T-Cell Biology and Cancer Immunotherapy. Front Immunol. 2021;12:689091.