The paper in Nature is here: http://go.nature.com/2gnnHBd
With the long-term objective of developing and establishing technologies for germline gene therapy in humans, in our recent report published in Nature we chose a heteroplasmic mutation in the MYBPC3 gene that causes hypertrophic cardiomyopathy (HCM) with significant risk of heart failure and sudden death. Inherited MYBPC3 mutations account for approximately 40% of all genetic defects leading to HCM1 and the condition affects 1 in 500 people. Because of its delayed manifestation, this mutation escapes natural selection and is often transmitted to the next generation resulting in high frequency in some patient populations. This is just one of more than 10,000 monogenic inherited disorders identified to date. Current treatment options for HCM provide mostly symptomatic relief without addressing causation. Whereas we couldn’t address the MYBPC3 mutation in somatic cells of the affected patient, we could develop the means to prevent transmission of founder mutations from parent to child using CRISPR-based gene editing approaches that have become highly efficient and effective in the last few years2-4. In particular, a target site including the mutation is subjected to CRISPR-Cas9 induced double strand breaks (DSBs) that are subsequently repaired by endogenous homology-directed (HDR) mechanisms.
Inspired by a desire to prevent transmission of disease from parents to children and rid future generations of the burdens of disease, in 2015, one male HCM patient was recruited by cardiology physician collaborators who agreed to participate. Shortly thereafter, we initiated collaborations with Dr. Jin-Soo Kim’s team in Seoul National University and Dr. Juan Carlos Izpisua Belmonte’s team at Salk Institute for CRISPR-Cas9 construct design. With their strong technical support and expertise, we first tested CRISPR-Cas9 targeting and HDR efficiency in patient induced, pluripotent stem (iPS) cells guiding the selection of a CRISPR-Cas9 construct with high efficiency for HDR-based gene correction. Attention then turned to studies involving human sperm, oocytes and embryos after three separate committees reviewed and approved progressing into basic science experiments with human gametes.
We tested efficacy of CRISPR-based MYBPC3 gene correction in human zygotes produced by fertilizing healthy donor oocytes with mutation-carrying, patient sperm. Three days-post injection, individual embryonic blastomeres were isolated and analyzed for targeting efficiency and HDR rate. Although targeting efficiency and HDR in zygote-injected embryos was much higher than observed in patient iPS, it was associated with mosaicism wherein some blastomeres carried more than one genotype (Fig. 1).
Mosaicism has been reported previously in CRISPR-Cas9 injected human embryos5-7 and is unacceptable in clinical applications. To overcome this challenge, based on the premise that mosaicism is secondary to the creation of multiple DSBs, we co-injected the CRISPR-Cas9 enzyme and the mutation-carrying sperm into unfertilized donor oocytes. Mosaicism was not observed in any cell of the multicellular embryos examined, including 72% of them which carried exclusively mutation-free DNA (Fig. 2).
A distinguishing feature of this study was the long extent of 'team work'. A total of 31 coauthors from three countries and ten institutions came together, each bringing unique insights, expertise and skills. From study design, patient recruiting, oocyte retrieving, CRISPR preparation and injecting, genome characterization, results analysis and finally manuscript writing, each coauthor made indispensable contributions.
In summary, this manuscript in Nature describes CRISPR-Cas9-based correction of a gene mutation in human preimplantation embryos with precise targeting accuracy. We hope this technique can one day be applied to all inherited genetic disorders affecting millions of people worldwide. Indeed, it might even result in elimination of some of these diseases. However, safety and efficacy must be much more clearly defined before clinical applications are initiated.
Our paper: Ma, H. et al. Correction of a pathogenic gene mutation in human embryos. Nature 548, 413–419 (2017) doi:10.1038/nature23305.
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