EXPLORE OUR SCIENTIFIC CONTRIBUTIONS AT THE ASGCT MEETING 

On Wednesday, May 14, 2025, 3:45 p.m. – 4:00 p.m., Exhibit Hall Theater, Norbert Makori, PhD, Vice President of Toxicology, will present on "Nonhuman Primate Research Models in Gene and Cell Therapy: Fetal, Infant to Mature Animal Utility."
 

ABSTRACTS:

Introduction: Gene therapies targeting rare genetic conditions and disorders are on the rise. While somatic or tumor cell targets are often the aim of these therapies, risk of germline integration of the vector target sequence in gametic cells is of recent safety and regulatory awareness. With the intent to confirm zero target germline integration, in vivo sample collection must be performed with precision and optimal nucleic acid isolations are required.

Methods: Preclinical gene therapy studies are conducted on sexually mature laboratory animals, including nonhuman primates (NHPs), canines, and minipigs. A 6-12 month observation period follows gene therapy administration. Semen is collected at various study timepoints for downstream nucleic acid isolations. Two sperm cell separation techniques were compared for feasibility and preservation of nucleic acids present: (1) whole cryopreservation method, where whole semen samples were collected, flash frozen, and then thawed prior to washes and nucleic acid isolation; and the (2) swim-up method, where fresh semen was incubated with a ‘swim-up’ buffer where the sperm cells are separated from the extraneous cells contained in semen; the sperm fraction is then flash frozen and preserved for nucleic acid isolations. For females, ovaries are collected at necropsy and the tissue is manually disrupted under a microscope to isolate individual oocytes; extraneous cellular/tissue debris is removed and ‘clean’ oocytes are collected and flash frozen for downstream isolation procedures. DNA and RNA are isolated from both frozen sperm and oocytes.

Results: Semen yielded an average of 10⁸ purified sperm cells per collection, which yielded between 120 and 4404 ng of DNA. Of the two methods evaluated, the whole cryopreservation method resulted in higher sperm cell isolation and subsequent DNA yields as compared to the swim-up method. Notably fewer gametic cells were obtained from female ovaries; averaging 72 oocytes per ovary. Accordingly, lower DNA yields of 140-892 ng and RNA yields of 72-800 ng were obtained.

Conclusions: Successful and meticulous collection of gametes from in vivo test models will be applied to ongoing and future gene therapy studies to assess potential vector target germline integration. These collections and isolations procedures pave the way for and impact preclinical industry standards for sufficient assessments of gene therapies with regard to risk of potential germline integration. 

Introduction: The disease targeted for treatment by the gene or cell therapy under development generally correlates with the age of the nonhuman primate model at administration for safety and efficacy. Animal ages vary from in utero (fetuses) to sexually mature. This presentation will discuss experience working with the different age groups, including some of the challenges and opportunities, as well as potential combinations to minimize animal use.
 

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