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.[i] 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.[i]
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.[ii]
In the last few years, has emerged as a powerful technique for AAV vector quantification. 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.[ii] 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.[iii] This level of accuracy and precision is necessary in gene therapy development for assessing treatment potency and determining correct dosing.