Heritable Human Genome Editing (2020) / Chapter Skim
Currently Skimming:
2 The State of the Science
2 The State of the Science
Pages 35-94
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From page 35... ...
Part I of this chapter describes what is known about the genetics of diseases caused by mutations in a single gene -- a category known as monogenic diseases. It then discusses potential reproductive options for parents at risk of passing on a disease genotype, including the use and current limitations of in vitro fertilization (IVF)
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From page 36... ...
Part V reflects on the complexities of human genetics beyond monogenic diseases and looks ahead to other circumstances for which HHGE has been proposed. It describes what is known about the genetics of polygenic diseases, a category that includes many common diseases in which multiple genetic variants contribute to overall disease risk.
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From page 37... ...
Advances in DNA sequencing over the ensuing decade dramatically increased sequence production and reduced its cost by more than a millionfold. This advance led to brute-force methods of disease gene discovery in which sequencing of all ~20,000 protein-coding genes in the human genome in many unrelated patients with the same clinical disease could identify genes that are mutated more often than expected by chance, and also permitted routine establishment of clinical diagnosis of individuals with monogenic diseases.
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From page 38... ...
The genetic variation in the human population arises from several factors. Genetic variants originally arise as alterations to the genome sequence that arise during DNA replication or other natural processes.
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From page 39... ...
, in which the mutant protein acquires a deleterious function not found in the normal protein. Still other monogenic diseases are X-linked, due to a mutation in a gene found only on the X chromosome.
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From page 40... ...
In some cases, complexities may be layered over the descriptions above. Monogenic diseases may have incomplete penetrance: only a subset of people who inherit the same disease genotype will actually have the disease.
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From page 41... ...
According to the World Health Organization (2019a) , the global prevalence of all monogenic diseases at birth is about 1 in 100, and monogenic conditions have been reported to "collectively contribute to disease in ~0.4 percent of children and young adults" (Posey et al., 2019)
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From page 42... ...
For a dominant condition, only one such variant is enough for the individual to be affected, while recessive conditions require pathogenic variants to be present on both copies of a chromosome pair.
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From page 43... ...
FIGURE 2-2 Circumstances in which parents would not be able to produce an embryo unaffected by a genetic disease include those in which one parent is homozygous for a dominant genetic disorder or both parents are homozygous or compound heterozygous for a recessive genetic disorder.
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From page 44... ...
Preconception Genetic Testing Some prospective parents know that they are at higher risk of having a child with a serious genetic disease because one of them has a genetic disease, because they have a family history of a genetic disease, because they underwent genetic testing for a targeted set of diseases that are at higher frequency in a particular ancestry group (e.g., Finnish or Ashkenazi Jewish individuals) , or as a result of population genetic screening or testing.
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From page 45... ...
Prenatal Genetic Testing Some prospective parents have a strong desire to have a child who is genetically-related to both parents -- that is, conceived from their egg and sperm. In the early phase of genetic testing, prenatal screening became available as an option to avoid having a child with a serious monogenic disease and is the method of choice for some people.
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From page 46... ...
Our growing knowledge of the genetic basis of human disease is leading, in some cases, to therapeutics that can ameliorate or even prevent the serious effects of certain genetic diseases. Some prospective parents at risk of passing on a genetic disorder may choose to proceed to have children, depending on the effectiveness, accessibility, and affordability of the treatment options.
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From page 47... ...
IVF tends to place substantial physical, financial, and emotional burdens on the prospective parents. The cost of cycles of IVF may be covered by the health systems of some countries (as is the case in Israel, France, and the Netherlands)
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From page 48... ...
. Demand for PGT for monogenic diseases has been steadily increasing.
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From page 49... ...
It is very hard to find such data on cumulative success rates per couple. For one PGT clinic in the United Kingdom in 2016, the live birth rate for couples with single-gene disorders was 39 percent per couple starting treatment, 54 percent per couple reaching transfer, and 70 percent when the couple had two or more unaffected embryos available (Braude, 2019)
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From page 50... ...
For such couples, HHGE would represent a major new option because it could make it possible for the first time for them to have a child genetically-related to both parents but without the disease-causing genotype. Couples for Whom Unaffected Embryos Are Unlikely to Be Obtained by Cycles of In Vitro Fertilization in Conjunction with Preimplantation Genetic Testing For other couples at risk of having affected offspring, some fraction of their embryos will be genetically unaffected (e.g., an average of 50 percent in the case of one parent with an autosomal dominant disease and 75 percent when both parents are heterozygous for recessive disease mutations)
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From page 51... ...
In contrast, when couples can produce both genetically affected and unaffected embryos, subjecting all zygotes to editing would often subject unaffected zygotes to editing. Polar body genotyping could, in certain cases, provide a reliable way of distinguishing zygotes that do and do not have a disease-causing genotype (see Figure 2-4)
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From page 52... ...
In practice, determining whether PB1 carries two copies of the nondisease-causing allele is not entirely straightforward. Polymerase chain reaction (PCR)
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From page 53... ...
. The technique is also used for preimplantation diagnosis of monogenic diseases (Griesinger et al., 2009)
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From page 54... ...
to cut specific DNA sequences and can be used to readily edit the genomes of living human cells (Doudna and Charpentier, 2014; Hsu et al., 2014; Karvelis et al., 2017)
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From page 55... ...
FIGURE 2-5 The CRISPR-Cas9 genome editing system pairs a DNA-cutting enzyme, such as Cas9, with a gRNA molecule that binds to the sequence of the gene to be edited. After the Cas9 protein cuts both DNA strands, the cell detects and repairs the doublestrand break via any one of several different mechanisms.
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From page 56... ...
, simply reconnects the broken ends. This process often results in the addition or deletion of DNA sequences (indels)
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From page 57... ...
The sequence changes introduced from a template can be as subtle as changing one or a few base pairs or can involve DNA sequence insertions or deletions of hundreds or thousands of base pairs. With both NHEJ and HDR, changes are induced specifically at the site of the break made by the editing nuclease.
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From page 58... ...
In somatic genome editing, if an intervention derives a clinical benefit by introducing genetic alteration that breaks a gene or a regulatory element, this is acceptable even if the resulting DNA sequence is rarely if ever found in the human population because the change is limited to that tissue in that individual. However, this would not be acceptable for HHGE because the consequences of such genetic alteration in every tissue and at all stages of development could be expected to be deleterious in many cases.
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From page 59... ...
The tools of base editing are undergoing rapid development. Early experiments showed that sequence changes were often produced at off-target sites in DNA and even in RNA, in some cases at non-targeted sequences.
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From page 60... ...
. In addition to concerns about potential off-target events, current base editors can make only certain types of DNA sequence changes, specifically, transition mutations (changing C to T, G to A, A to G, or T to C)
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From page 61... ...
The unedited strand is cut by the modified Cas9, triggering the cell to repair the G-U mismatch to A-U, which becomes A-T upon DNA replication. Adenine base editors operate via a similar mechanism to convert targeted A-T pairs to G-C.
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From page 62... ...
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 diseases is somatic genome editing.
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From page 63... ...
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 experience will be needed to assess its safety and efficacy.
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From page 64... ...
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 hematopoietic stem cells (HSCs)
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From page 65... ...
introducing them into the pronuclei or cytoplasm of the fertilized egg. Introducing these reagents can be done by direct mechanical injection or by electroporation, both of which have been used in human embryos without significant damage (Ma et al., 2017)
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From page 66... ...
Several recent and not yet peer-reviewed preprints also report significant unintended editing near the target site in human embryos, including chromosomal modifications (Alanis-Lobato et al., 2020; Liang et al., 2020; Zuccaro et al., 2020)
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From page 67... ...
. Beyond the questions of efficiency and repair pathways at the target site, there is also a possible issue with having to target two different disease-causing variants in prospective parents in whom one parent is compound heterozygous for mutations in the same gene that each cause a dominant disease or in which different alleles are present in the same gene in cases of recessive disease.
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From page 68... ...
Before any use of genome editing in human embryos intended for establishment of a pregnancy, careful experimentation and optimization would need to be undertaken to select the reagents that would provide the greatest specificity in the genetic context of the prospective parents. Devising genome editing tools that induce only very low levels of off-target modifications in human zygotes appears feasible but would need to be validated in each specific case.
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From page 69... ...
Preventing mosaicism requires a very high efficiency of the desired ontarget modification in the one-cell zygote and restriction of editing activity beyond that stage. Research in somatic cells suggests that the Cas9-gRNA complex is rather short-lived, but the lifetime of the complex in human embryos has not been well characterized.
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From page 70... ...
Figure 2-8 70 Mosaicism inner cell mass trophectoderm inefficient editing microinjection zygote two cell four cell eight cell mosaic blastocyst FIGURE 2-8 Mosaic embryos in which cells contain different genetic material can arise, for example, when edits occur in only one cell at the two-cell stage.
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From page 71... ...
. The epigenetic remodeling that takes place in human embryos is likely to be more complex than in mice, due to the inbred genome of laboratory mice and the influence of genetic variation in humans on epigenetic variation (Delahaye et al., 2018)
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From page 72... ...
might also inform characterizations of early human development. Further fundamental research and refined assess ment methodologies will be needed to establish whether developmental milestones, including epigenetic and transcriptomic profiles, are comparable to those of unedited human embryos.
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From page 73... ...
The differences in methodologies mean that successes or failures in the use of somatic genome editing are unlikely to have direct relevance to the prospects for HHGE in humans -- the editing reagents and delivery methods used will likely differ, as will the cellular context and repair mechanisms in zygotes versus somatic cells. Nevertheless, clinical trials of somatic editing therapies represent the use of genome editing in human primary cells, rather than in cells maintained in laboratory culture.
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From page 74... ...
These include raising the efficiency of on-target editing and preventing unintended on-target events. This will likely require developing an understanding of how DNA repair processes operate in zygotes, how outcomes depend on the timing of introducing the editing reagents, and what formats of template DNA and delivery are most effective.
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From page 75... ...
Many human embryo genome editing studies have also used non-viable tripronuclear embryos (Li et al., 2017; Liang et al., 2015; Tang et al., 2018; Zhou et al., 2017) , but their abnormal chromosome content and aberrant developmental course make them unsuitable for preclinical characterization of genome editing in human zygotes as part of a potential translational pathway to HHGE.
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From page 76... ...
Implications of In Vitro Stem Cell–Mediated Gametogenesis for Heritable Genome Editing In the vast majority of cases, in vitro stem cell–mediated gametogenesis would eliminate a need for heritable genome editing as a means of preventing the transmission of monogenic diseases. For those circumstances in which a couple could produce an embryo without the disease-causing genotype, the ability to screen a large number of embryos created from male and female gametes produced from the prospective parents' somatic cells would enable suitable embryos to be identified.
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From page 77... ...
It is unclear at present which, if any, of these approaches might reach a stage of development at which clinical application could be considered. Genome Editing in Spermatogonial Stem Cells Sperm cell genomes cannot be edited directly using current technology.
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From page 78... ...
. These studies involve the completion of gametogenesis in vivo by gonadal transfer or in vitro by co-culture with neonatal testicular somatic cells but establish the principle that mouse stem cells could be converted into functional male gametes.
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From page 79... ...
(2) Alternatively, human embryonic stem cells can be derived from embryos following nuclear transfer into enucleated oocytes (ntESCs)
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From page 80... ...
Further research would be required in mammalian models, including non-human primates, to develop this as a potential method of producing human gametes. ADDITIONAL COMPONENTS OF ANY CLINICAL TRANSLATIONAL PATHWAY FOR HERITABLE HUMAN GENOME EDITING In addition to the scientific and technical considerations discussed above, any potential clinical use of HHGE would entail the incorporation of detailed plans for obtaining informed consent and for monitoring the effects of genome editing.
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From page 81... ...
Prospective parents would also need to be aware of the risk that they may give birth to a seriously ill or disabled child, and of the possibility that they may be faced with the difficult decision of whether or not to terminate a pregnancy should prenatal testing identify genetic or physical anomalies. The advantages and disadvantages of alternative routes to parenthood that would avoid the transmission of genetic disease would need to be carefully discussed as part of the consent process.
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From page 82... ...
and the report Ethical Research Involving Children (Graham et al., 2013) , assent or informed consent would need to be obtained from children born following HHGE according to the children's age and developmental level.
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From page 83... ...
The section below briefly reviews monitoring that has been done with other ARTs and then turns to the consideration for HHGE. Monitoring Children Born Through Assisted Reproductive Technologies Regarding the follow-up of children born following HHGE, the closest parallels are studies of children born through ARTs, such as IVF and ICSI, who are genetically-related to their parents; children born through ARTs using donated eggs, sperm, or embryos and who therefore lack a genetic connection to one or both parents; and children born following PGT in combination with IVF/ICSI.
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From page 84... ...
However, HHGE is distinct from PGT in that it produces an alteration to the genetic make-up of their children whereas PGT does not. Long-Term Follow-Up of Children Born Following Heritable Human Genome Editing As with informed consent, a broad approach is described for the longterm follow-up of children born following HHGE, as the most appropriate specific assessments will be dependent upon a number of factors, including
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From page 85... ...
Challenges include the small number of children who would be born following initial uses of HHGE, the need to standardize both the genetic disorders under investigation and the genome editing undertaken, and the absence of meaningful comparison groups, all of which would restrict the reliability, validity, and generalizability of the findings. In the United Kingdom, children conceived through MRT, the closest parallel to HHGE, are assessed from the prenatal period onward.
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From page 86... ...
This section discusses the potential use of HHGE in more complex circumstances: to prevent the transmission of polygenic diseases, to affect characteristics not associated with disease, and in the special circumstance of male infertility. Human genetics is complex.
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From page 87... ...
Complex, Non-Disease Traits Geneticists have also identified many genetic variants associated with personal characteristics, and genome editing has been proposed as a potential way to alter these. For example, genetic variants are associated with increased muscle strength or with an improved ability to increase strength 12 Other types of muti-genic disease inheritance are possible, including digenic, in which mutations in two different genes are required for the disease (Deltas, 2018)
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From page 88... ...
The male patient in this circumstance exists and could provide informed consent to the procedure, and the clinical intent here is to treat a condition having a negative impact on that person's life, as with somatic genome editing. However, because the cells targeted for genome editing are reproductive, the correction of an infertility-causing mutation would produce a genetic change that is heritable.
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From page 89... ...
The key messages arising from this analysis are provided below. Knowledge of Human Genetics Limits Potential Applications of Heritable Human Genome Editing For monogenic diseases, the use of HHGE to change a genetic variant that causes the disease to a non-pathogenic DNA sequence that is common in the population could prevent the disease being transmitted to offspring, if it is possible to efficiently and reliably make precise genomic changes without undesired changes affecting human embryos.
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From page 90... ...
This research would necessarily involve the use of human embryos because there are differences in the DNA repair mechanisms used by different cell types and there are important species-specific differences in early development which limits the usefulness of research in model organisms and other human cell types. Genome editing conducted in stem cells that are induced to form male, and potentially female, gametes provides a theoretical alternative approach
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From page 91... ...
Existing knowledge is not sufficient to predict the effects of making genetic changes in these circumstances. Even for monogenic diseases, sound evidence for a causative role of an identified genetic variant would be needed prior to genome editing.
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From page 92... ...
Limitations Associated with the Development of Genome Editing in Stem Cell–Derived Gametes Studies in animal models reveal that the use of genome editing in g amete precursor cells and the production of male gametes able to produce healthy embryos without a disease-causing genotype are a nearer-term prospect than the production of female gametes. The production of gametes from stem cells in vitro, if successful, would offer an alternative to HHGE in most envisaged cases.
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From page 93... ...
Recommendation 2: Extensive societal dialogue should be under taken before a country makes a decision on whether to permit clini cal use of heritable human genome editing (HHGE)
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