editing methodologies. It is possible that continuing research may yield new methodologies that rapidly supersede the safety and efficacy of current editing approaches. Non-Heritable Genome Editing: The Use of Genome Editing in Somatic Cells One potential alternative to HHGE for the treatment of genetic dis- eases is somatic genome editing. This section discusses some of the relative advantages and disadvantages of somatic editing in comparison with HHGE. The initial applications of genome editing in humans occurred in somatic cells, the cells that make up all of the cells of the body except sperm, eggs, and their precursor cells. The effects of genome editing carried 4 It has also been proposed that genome editing could be used as an alternative to MRT to prevent the transmission of mitochondrial DNA (mtDNA) disease (Reddy et al., 2015). This study used mito chondrial targeted restriction endonucleases or TALENS and showed they could be used to potentially lower mutation load. However, this procedure led to net depletion of mtDNA and thus was not suitable for oocytes with a very high level of heteroplasmic or homoplasmic mtDNA mutations. A recent paper reports the use of base editing on mtDNA (Mok et al., 2020) and may represent a new approach to addressing mitochondrial disease. A detailed analysis of mtDNA editing requires a separate study, in the context of existing treat- ments for mitochondrial diseases and options such as MRT. http://www.nap.edu/25665 Heritable Human Genome Editing Copyright National Academy of Sciences. All rights reserved. THE STATE OF THE SCIENCE 63 out in somatic cells are generally limited to the individual treated and would not be transmissible to that person’s offspring. (The special circumstance of editing somatic cells that are located in an individual’s reproductive system, such as editing in the testes to treat infertility, is discussed later in this chapter.) Despite the cost that would be associated with any clinical use of HHGE and the complex social, ethical, and scientific issues that heritable genome editing raises, the potential limitations associated with somatic edit ing, discussed below, represent one reason that HHGE has been proposed as a theoretical alternative for parents wishing to have a genetically-related child who does not have the disease-causing genotype. Somatic genome editing is an option for treating patients with monogenic disorders, but it remains in early stages of clinical use, and much more expe- rience will be needed to assess its safety and efficacy. The first clinical trial, initiated in 2009, tested the safety of using ZFNs to prevent the progression to AIDS in people infected by HIV (Tebas et al., 2014); and multiple trials using ZFNs, TALENs, and CRISPR systems are currently in progress.5 With significant funding across multiple companies, somatic genome editing is likely to lead to numerous human trials in the coming decade. The simplest targets for somatic editing are ones in which cells can be removed from a patient, treated outside the body, and returned (ex vivo genome editing) (Li et al., 2020). At present, the primary conditions that can be approached in this way are diseases resulting from mutations in HSCs. For example, promising results have been reported for patients af- fected with SCD and beta-thalassemia who were treated with CRISPR-Cas reagents to induce expression of fetal hemoglobin,6 although long-term follow-up will be needed before conclusions can be drawn regarding its successes and limitations. Trials are also under way using genome editing to enhance the activity of CAR T cells for cancer immunotherapy (Bailey and Maus, 2019; Stadtmauer et al., 2020). For many other envisioned somatic therapies, the genome editing re- agents will need to be delivered directly to a patient’s cells and tissues (in vivo genome editing). When a disease affects multiple organs, the challenge of delivery is magnified. Only in a few cases is the target tissue readily accessible. One favorable example is the eye, where direct injection of a viral vector carrying CRISPR-Cas reagents is feasible and is being applied for a rare retinal blindness condition.7 The liver is also relatively accessible, and ZFNs are being employed to enhance a gene addi tion therapy in trials targeting hemophilia and metabolic disease.8 5 See clinicaltrials.gov. 6 See, for example, clinical trial numbers NCT03745287 and NCT03655678. 7 See clinical trial NCT03872479. 8 See clinical trial numbers NCT02695160, NCT03041324, and NCT02702115. http://www.nap.edu/25665 Heritable Human Genome Editing Copyright National Academy of Sciences. All rights reserved. 64 HERITABLE HUMAN GENOME EDITING One feature of many of the above cases is that they rely on disruption of genome sequences by NHEJ. As noted above, this pathway is more active in most cells after a double-strand break is introduced than HDR. Treat- ments relying on HDR are in development, but attaining therapeutically relevant efficiencies remains challenging. For quite a number of genetic conditions, a non-disease-causing allele could be created via base editing and such approaches are being pursued actively. While somatic genome editing avoids some of the challenging issues raised by HHGE—because somatic editing involves treating existing patients who can typically consent and because the resulting genetic changes would not be passed on to subsequent generations—somatic editing has some disadvantages. First, because editing does not alter the germline, a patient receiving somatic therapy for a genetic disease could still transmit the disease-causing mutation to future children. Additionally, because only a fraction of targeted cells might be edited, eliminating cells with the disease genotype or positive selection for the edited cells might be needed to increase the fraction of stems cells that have been edited. For example, protocols for somatic editing of hemato- poietic stem cells (HSCs) commonly include cytotoxic chemotherapy to eliminate native HSCs before infusion of edited cells. These treatments confer risk of harm. Somatic genome editing therapies are also likely to be very expensive, although costs are unknown and likely to vary (Rockoff, 2019). Heritable Genome Editing: The Use of Genome Editing in Zygotes At present, the primary approach that could be used for undertaking HHGE would involve genome editing in zygotes. Because edits introduced would be present in every cell in the body, and the resulting genetic modifi- cations could be passed on to subsequent generations, it would be critically important to obtain the desired genetic change at the target site and ensure an absence of editing-induced changes elsewhere in the genome. There are unique challenges in characterizing the editing events in zygotes and early embryos, as well as important gaps in understanding how to precisely con- trol genome editing in these cells.9 9 Genome editing technologies have also been adapted to affect the epigenetic state of somatic cells by altering DNA methylation (Kang et al., 2019) and histone modifications ( Pulecio et al., 2017). Extensive epigenetic remodeling occurs during early development, and it is not clear whether epigenome editing would be heritable or how it would operate in zygotes and early embryos. Much more research on epigenome editing in embryos would need to be under taken before it could be considered as an intervention for congenital imprinting disorders (Eggermann et al., 2015). http://www.nap.edu/25665 Heritable Human Genome Editing Copyright National Academy of Sciences. All rights reserved. THE STATE OF THE SCIENCE 65 A zygote—the single, fertilized cell that results from the combination of parental gametes (the egg and sperm)—is the earliest stage of embryonic development. At first the maternal and paternal chromosomes remain in two distinct pronuclei in
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