Monitoring Fetal Somatic Cell Genome Editing In Vivo with Total-Body Positron Emission Tomography: Studies in Rhesus Macaques ( Macaca mulatta )

Abstract

Translational development of somatic cell genome editing requires monitoring the extent of editing in the body at a given time, the specificity of editing, durability, and the potential for adverse events. A noninvasive approach that can identify edited cells in vivo is beneficial for addressing these and related questions. These studies used total-body positron emission tomography (PET) to identify somatic cell gene editing in vivo in fetal rhesus macaques. Rhesus dams were screened to confirm they were seronegative for AAV serotype 9 and SaCas9 antibodies, then selected for the study. Fetuses were administered a dual imaging vector (AAV9/SaCas9 and AAV9/PCSK9 gRNA/HSV-sr39TK) in utero using an ultrasound-guided fetal intrahepatic approach in the second trimester (10¹² vector genomes/fetus). After maternal intravenous administration of 9-(4-(18)F-Fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG) (∼3 mCi/kg), PET imaging was performed in the second and third trimesters. PET imaging provided evidence of editing in the fetal liver, which was sustained. Appropriate insertion of the promoterless HSV-sr39TK reporter in frame with the PCSK9 gene was confirmed in the fetal liver near term using RNA sequencing, and correctly targeted insertions were observed. These studies have shown that total-body PET can provide insights into gene-edited somatic cells in utero and without evidence of adverse effects.