Germline Integration Assessment In Preclinical Gene Therapy Studies

By Hazel Clay, Francesca Barone, Kaylyn Koenig, Rebekah McMinn, Petronella Magunda, and Norbert Makori

In recent years, gene therapy products have progressed significantly from preclinical to clinical development, increasing the likelihood of approvals for treating genetic disorders. A critical aspect of ensuring the safety of these therapies is determining that the gene product cannot be transmitted to the offspring of treated patients, which requires investigating the potential for germline integration. This process essentially aims to confirm the absence of DNA integration that could pose a risk for germline modification.

Preclinical studies in sexually mature nonhuman primates play a vital role in this evaluation. For male animals, semen samples are collected at multiple time points during the study, while for females, ovaries are harvested at necropsy for oocyte isolation. These studies typically involve 6–12 months of observation following gene product administration. In females, the ovaries are manually disrupted and mechanically denuded at necropsy to remove extraneous tissues, yielding an average of 60 follicular oocytes per ovary under microscopy. Between 50 and 100 oocytes are deemed sufficient for DNA extraction and genomic analysis. In males, semen samples are processed through various buffer wash steps, producing DNA yields ranging from 120 to 2808 ng.

These in vivo methods, coupled with robust DNA isolation techniques, provide a reliable framework for genomic analysis, enabling a thorough evaluation of the potential for germline integration in gene therapy studies.