(39) Developing an Approach for In Utero Gene Editing: Lessons Learned

Cystic fibrosis is a genetic disorder resulting from mutations in the CFTR gene, and CRISPR/Cas9 genome editing offers potential treatment. Our objective was to develop CRISPR/Cas9 constructs targeting the F508 codon of the CFTR gene, deliver them to wild-type fetal sheep in utero, and assess the efficiency of CRISPR/Cas9-mediated genome editing in vivo to provide insights into its future feasibility.

Study Design:

Several CRISPR/Cas9 orthologs were designed to target the F508 codon. The gene editing efficiency was determined in fetal sheep trophoblasts utilizing T7 endonuclease I assay. The CRISPR/Cas9 ortholog with the highest nuclease activity, smallest size and cuts closest to F508 codon was packaged into a single AAV⁹. Four fetuses from two twin sheep pregnancies at day 70 of gestation were injected with 0.5 mL of 1.5 x 10¹² viral particles injected via the umbilical vein. The fetuses were harvested on day 135.

Eleven tissues of interest underwent genomic DNA extraction, PCR amplicons flanking the CRISPR/Cas9 target site were generated and amplicons were subjected to deep sequencing by Quintara Biosciences, NGS Division. NHEJ and HDR frequencies were calculated using the CRISPResso webtool.

Results: All 4 fetuses successfully received injections and survived to term.  No evidence of adverse effects were observed in treated fetuses. Amplicon NextGen sequencing revealed that CRISPR/Cas9 construct was unsuccessful at inducing indel formation in the target site. There was no significant evidence of NHEJ nor HDR.

Conclusion:

Despite promising in vitro work, in vivo delivery of CRISPR-Cas9 systems remains complex. Off-target effects may cause unsuccessful results when using the CRISPR/Cas9 system, despite its high specificity in targeting specific genes. It is also possible that the stage of fetal development at which the gene editing is performed may impact its efficacy.

Strategies such as lipid nanoparticles for packaging and delivery as well as ongoing work in organoids is expected to provide valuable insights into the safety and efficacy of CRISPR-Cas9 editing prior to further in vivo work.