O-069 Mito-TALENs reduce mitochondrial DNA mutation load following pronuclear transfer

Study question

Can mito-TALENs selectively eliminate mitochondrial DNA (mtDNA) carryover following pronuclear transfer (PNT) in a mouse model carrying a heteroplasmic mtDNA mutation?

Summary answer

Mito-TALENs effectively reduced mitochondrial DNA mutation load in embryos following pronuclear transfer, without compromising developmental outcomes.

What is known already

Maternally inherited mitochondrial diseases can arise from mutations in mtDNA. PNT can reduce the transmission risk by replacing the cytoplasm of an affected zygote with that of a healthy donor. However, a small amount of mutant mtDNA, known as carryover, remains in the reconstructed embryo, potentially leading to disease reversion if it re-expands overtime. To address this, ongoing efforts focus on eliminating residual pathogenic mtDNA. Mitochondrial-targeted TALENs (mito-TALENs) have been shown to selectively reduce mutant mtDNA in various models. Combining PNT with mito-TALENs may further reduce heteroplasmy and mitigate mtDNA genetic drift, offering a promising strategy to prevent disease recurrence.

Study design, size, duration

A murine model with a heteroplasmic mutation in the mitochondrially encoded tRNA alanine gene (mt-Ta, m.5024C>T) was used. Oocytes and zygotes from wild-type (WT, n = 143) and mutant (n = 161) female mice were retrieved, and divided into experimental groups: PNT, PNT with mRNA injection (mito-TALEN or GFP), mRNA injection alone, and unmanipulated controls. Mito-TALEN monomers were designed to target the mutant allele. GFP mRNA injection was used as control for embryo development. mRNAs were produced in vitro.

Participants/materials, setting, methods

Oocytes and zygotes were collected from superovulated WT and mutant female mice. Oocytes were fertilised using piezo-ICSI. PNT was performed between mutant and WT zygotes. mRNA was injected into WT, mutant, and PNT zygotes. Resulting embryos were cultured to the blastocyst stage in vitro. mtDNA heteroplasmy was analysed across different groups using digital PCR (dPCR). Assay validation was performed with synthetic templates of known mutation loads (0.01–50%). Results were compared with next-generation sequencing (NGS).

Main results and the role of chance

dPCR reliably detected mutation loads as low as 0.1%. Unmanipulated mutant embryos had mean heteroplasmy levels of 68.59% (SD = 7.76%; n = 31). Mito-TALEN mRNA injection significantly reduced mean mutant load to 40.23% (SD = 9.90%, n = 32, p < 0.0001), whereas GFP mRNA injection had no effect (70.09%, SD = 9.25%, n = 14) showing similar heteroplasmy levels to unmanipulated mutant embryos. Blastocyst rates were comparable across groups, including unmanipulated and mRNA-injected (mito-TALEN and GFP) WT embryos, indicating no adverse effects of mRNA injections. Colocalisation of mito-TALENs with mitochondria was confirmed using MitoTracker staining.

PNT was carried out using mutant zygotes as karyoplasts (nuclear donors) and WT zygotes as cytoplasts (mitochondrial donors). Control PNT blastocysts exhibited mtDNA carryover levels of 2.42% (SD = 0.82%, n = 25), while PNT blastocysts injected with mito-TALEN mRNA showed significantly reduced carryover levels to 0.86% (SD = 0.42%, n = 24), with some as low as 0.33% (p < 0.0001). No differences in development rates were observed between groups.

At high mutation levels (20–80%), dPCR and NGS results were strongly concordant. However, in PNT embryos, dPCR accurately quantified minor alleles below 1%, whereas NGS produced more false positives and failed to detect mutations below this threshold. In WT samples, dPCR detected minimal mutant alleles (∼0.07%), while NGS reported ∼0.6% mutation loads.

Limitations, reasons for caution

This study describes preliminary results and is limited by its small sample size. While dPCR accurately quantified minor alleles, NGS failed to detect mutation levels below 1%, highlighting the need for caution when using less sensitive methodologies for mtDNA mutation load quantification, particularly for nuclear transfer techniques.

Wider implications of the findings

Our results demonstrate that the sequential application of PNT and mito-TALENs effectively reduces mtDNA carryover, offering a promising strategy for safer application of nuclear transfer. dPCR provides precise mtDNA quantification, outperforming NGS at low mutation levels with superior accuracy and fewer false positives.

Trial registration number

No