Mitochondrial Replacement Techniques Ethical, Social, and Policy Considerations (2016) / Chapter Skim
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2 Science and Policy Context
2 Science and Policy Context
Pages 27-78
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INTRODUCTION TO REPRODUCTIVE BIOLOGY AND MEDICINE A prefatory summary of concepts in reproductive biology and medicine is provided here to inform subsequent discussions of mitochondrial biology and genetics, mtDNA disease, and MRT in this chapter and the ethical 27
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The pronuclear genetic material first replicates before the respective nuclear membranes dissolve, followed by fusion of the male and female genetic material and equivalent division of genetic and cellular material to form the two-cell embryo. The resultant embryo will undergo rapid cell division and differentiation, acting as the fundamental precursor cells for all the cells of the human body (see Figure 2-1)
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At this stage, the early zygote contains both the male and female pronuclei and is therefore termed di-pronucleate, or 2-PN. The pronuclear genetic material first replicates before the respective nuclear membranes dissolve, followed by fusion of the male and female genetic material in the late zygote, and equivalent division of genetic and cellular material to form the two-cell embryo.
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PGD is a technique performed in the setting of IVF to test for a known inherited genetic disease and to allow selection of embryos for transfer to the uterus of the woman who will carry the pregnancy, with the goal of establishing a viable pregnancy and preventing transmission of that disease.2 Once a viable pregnancy has been achieved, additional prenatal diagnostic testing is essential to confirm the genetic information obtained by PGD, entailing chorionic villus sampling of fetal placental tissue, amniocentesis of discarded fetal cells, or cell-free DNA screening. The use of PGD for preventing transmission of mtDNA disease is discussed later in this chapter.
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The space between the inner and outer membrane is termed the intermembrane space. Oxidative phosphorylation takes place by pumping protons across the inner membrane into the intermembrane space, forming the electromotive force used to drive adenosine triphosphate (ATP)
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In mtDNA disease, these coupled reactions -- in addition to OXPHOS -- are also disrupted and can contribute to the observed disease clinical phenotypes. 6 OXPHOS is the process by which the respiratory chain generates a proton gradient across the mitochondrial inner membrane via transfer of electrons from a higher-energy donor to lower-energy cellular intermediates, terminating with formation of the terminal electron acceptor, oxygen.
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Compared with the only 2 copies of the 23 nuclear chromosomes in almost all somatic cells, mtDNA is found in high copy number,8 ranging from 2 to 10 copies per mitochondrion and 100 to 8 The copy number is the number of mtDNA molecules per cell.
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Replication of mtDNA occurs continuously throughout the cell cycle and autonomously from nDNA, which is replicated once per cell cycle; the resulting mtDNA molecules are partitioned randomly into the daughter cells during cell division.9 While mtDNA encodes for products that are essential for the production of cellular energy, it is generally agreed that nDNA plays the predominant role in determining characteristics of anatomy, physiology, personality, and the like. Mode of Inheritance As noted previously, mtDNA is solely maternally inherited in humans (see Figure 2-2)
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. The crosstalk between the nuclear and mitochondrial genomes is an important consideration in evaluations of MRT, as its disruption could have potentially deleterious effects on overall mitochondrial and cellular health (see the section on complexities related to mitochondrial genetics later in this chapter)
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From an evolutionary standpoint, the persistence of certain maternally transmitted homoplasmic mtDNA mutations has resulted in the formation of stable population subgroups, known as haplogroups, sharing the same collection of fixed mtDNA variants, or haplotypes. As the females who migrated out of Africa helped colonize the globe and novel mtDNA mutations were acquired, new haplogroups branched out from the original "macrohaplogroups" (Wallace and Chalkia, 2013)
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More than 275 disease-causing mtDNA mutations have been reported across every mtDNA gene since the first pathogenic mtDNA mutation was identified in 1988 (Saneto and Sedensky, 2013)
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In general, mtDNA diseases tend to have later onset and to be associated with relatively milder symptoms relative to nDNA-based mitochondrial diseases, whose onset is typically earlier (often in infancy or childhood) and which
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The principal effect of defective mtDNA is disruption of respiratory chain activity; consequent depletion of ATP levels and energy production; and eventual dysfunction and failure of cellular, tissue, and organ function. Age of onset, clinical presentation, natural history, and penetrance16 of mtDNA diseases are extremely variable, both within and across mtDNA mutations.
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40 MITOCHONDRIAL REPLACEMENT TECHNIQUES S t r o k e , a t a x i a , e p i l e p s y, e n c e p h a l o p a t h y, and migraines O p t i c n e u r o p a t h y, r e t i n o p a t h y, and external Deafness opthalmoplegia Cardiomyopathy and conduction defects Liver failure Diabetes Anaemia mellitus Intestinal Renal failure pseudoobstruction and diarrhea Muscle weakness, exercise intolerance, c r a m p s , a t r o p h y, and hypotonia Peripheral neuropathy FIGURE 2-3 Potential manifestations of mtDNA diseases. SOURCE: Adapted by permission from Macmillan Publishers Ltd: Nature Reviews Genetics, copyright 2005.
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(2015b) extrapolated from the point prevalence of pathogenic mtDNA mutations to estimate how many women may be at risk of transmitting mtDNA disease and thus could potentially benefit from MRT.
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Furthermore, for many women at risk of transmitting pathogenic mtDNA mutations, diagnostic techniques aimed at reliably preventing transmission of pathogenic mtDNA to future offspring (e.g., PGD or prenatal diagnosis) are not viable options, as discussed below.
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, and more recent work has shown that it can effectively reduce heteroplasmy levels and prevent transmission of pathogenic mtDNA in mouse and mammalian oocytes and one-cell embryos. As a result, heteroplasmy shift has been proposed as an alternative to MRT for preventing maternal transmission of pathogenic mtDNA mutations that would preclude the need for the contribution of a second woman's genetic material (Reddy et al., 2015)
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, which could result in higher than expected heteroplasmy levels of pathogenic mtDNA in critical tissues of offspring born following PGD. Relatedly, while PGD may reliably reduce heteroplasmy levels of pathogenic mtDNA and prevent manifestation of mtDNA disease in offspring, females born as a result of PGD may still be at risk of transmitting mtDNA disease to offspring because of higher than expected heteroplasmy levels in their oocytes.
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. 18 This report uses the term "nonpathogenic mtDNA" to describe mtDNA contributed from the female oocyte provider, with the understanding that following genetic testing of provided oocytes for known pathogenic mutations, any provided mtDNA would be presumed -- but given the rapidly expanding and shifting knowledge of mitochondrial biology and genetics, could not be assumed -- to be free of pathogenic mtDNA mutations.
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MRT replaces pathogenic mtDNA from the intended mother with nonpathogenic mtDNA from an oocyte provider. For simplicity, reproductive partners are not shown and are assumed not to carry pathogenic mtDNA mutations.
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Maternal Spindle Transfer MST would entail removal of the nDNA (specifically, the metaphase II spindle-chromosome complex,21 or MII-SCC) from the intended mother's oocyte and its subsequent fusion to an oocyte provided by another woman that contained nonpathogenic mtDNA and from which the nDNA had been removed.22 The reconstructed oocyte would then be fertilized with the intended father's, or another man's, sperm and cultured in vitro to the blastocyst stage.
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3. The reconstructed oocyte contains the intended mother's nDNA and oocyte provider's nonpathogenic mtDNA.
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. These data confirm that, while MRT would likely prevent significant mtDNA carryover and heteroplasmy in somatic tissues and organs of offspring born as a result of MRT, oocytes of females born as a result of MRT could harbor significant and clinically relevant levels of carried-over mtDNA.
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. As in MST, a small amount of cytoplasm would be transferred within the extracted karyoplast containing the pronuclei and would likely contain a variable, nonzero amount of the intended mother's pathogenic mtDNA.
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5. The reconstructed zygote contains male and female nuclear DNA from the intended mother and sperm provider and nonpathogenic mtDNA from the oocyte provider.
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(2014) compared mtDNA carryover and developmental competence in mouse oocytes and zygotes subjected to MST and PNT, respectively.
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More extensive preclinical research is needed in human oocytes and zygotes, however, to determine the feasibility, efficacy, and safety of PBT and whether these potential advantages would in fact be realized. RISKS RELATED TO MRT: SCIENTIFIC COMPLEXITIES AND TECHNICAL UNKNOWNS AND UNCERTAINTIES The clear benefit of successful implementation of MRT would be to give women who carry pathogenic mtDNA mutations the option of hav 25 One case report documents PNT attempted in human zygotes with the intent of producing viable human offspring (Zhang et al., 2003)
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Heteroplasmy: Threshold Effect and Mitotic Segregation As previously described, heteroplasmy is the state in which a cell, tissue, or individual contains more than one type of mtDNA genotype. In most cases, cells containing pathogenic mtDNA mutations manifest cellular dysfunction only when the levels of pathogenic mtDNA molecules accumulate to a certain threshold level at which clinical symptoms of mtDNA disease develop (threshold effect)
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Because of poorly understood bottleneck effects, however, some offspring may have higher-than-expected levels of pathogenic mtDNA molecules in some tissues that could exceed the threshold level required to manifest disease. This phenomenon is exemplified by cases of cytoplasm transfer,26 a procedure used for treatment of idiopathic infertility that involved injection of cytoplasm from oocytes provided by other women into the oocytes of intended mothers (Barritt et al., 2001a,b; Brenner et al., 2000, 2001, 2004; Cohen et al., 1997, 1998; Huang et al., 1999; Lanzendorf et al., 1999)
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Studies of outbred strains of model organisms, for example, have identified specific mtDNA variants that are "compatible" only with certain nuclear genome backgrounds (see Reinhardt et al.
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the uncertainty of techniques such as PGD, amniocentesis, and chorionic villus sampling (CVS) for validating efficacy of MRT -- namely for quantifying pathogenic mtDNA carryover and heteroplasmy load; and (3)
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Similar concerns arise regarding the ability of PGD, and correspondingly amniocentesis and CVS, to predict accurately the expected level of heteroplasmy in the tissues of offspring born as a result of MRT. As discussed earlier, current standards of care for the use of PGD to prevent transmission of mtDNA disease stipulate that heteroplasmy levels must be less than 5 percent to mitigate the chance of mtDNA disease in offspring.
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Federal funding for MRT research would likely be unavailable because of current legislative restrictions against funding research on human embryos. In the event that MRT were to move into clinical investigations, FDA has asserted regulatory jurisdiction, and a careful stepwise process, which would include FDA oversight and institutional review board (IRB)
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Oversight of MRT would likely involve the same statutes and regulations that apply to IVF, PGD, preimplantation genetic screening (PGS) , and cloning.
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for failure to report (Knowles and Kaebnick, 2007; SART, 2016) .27 Preimplantation Genetic Diagnosis (PGD)
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pointed to the 2001 final rule on regulation of HCT/Ps (21 CFR § 1271) , as well as a 1993 Federal Register notice that clarified FDA's authority over human somatic cell therapy and gene therapy products (58 FR § 53248)
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Twenty-nine countries prohibit germline modification; the salient laws or regulations of 10 more countries, including the United States, are either ambiguous or would restrict but not fully prohibit it. This opposition to germline modification exists even in countries that allow other types of research involving human embryos: 13 of the countries that ban germline modification permit human embryonic stem cell research, and the United Kingdom permits MRT but prohibits all other types of germline modification (Araki and Ishii, 2014)
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Act. In a 2001 letter to researchers, FDA asserted regulatory authority over "human cells used in therapy involving the transfer of genetic material by means other than the union of gamete nuclei," and noted that this genetic material includes cell nuclei, oocyte nuclei, and ooplasm containing mitochondrial genetic material.
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, and on December 18, 2015, the U.S. Congress passed an omnibus spending bill for fiscal year 201629 that would seem to forestall FDA consideration of any application to try such a technique in human clinical investigations, that is, in investigations involving transfer to a woman for gestation of the modified embryo.
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. State Laws Many states have laws regarding cloning, embryo research, stem cells, and other areas relevant to MRT.
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Under this law, it appears that some versions of MRT would be permissible (PNT would involve transfer of a nucleus into a zygote, not an oocyte) , but some would not (MST would involve transfer of nuclear genetic material into an oocyte)
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38 45 CFR Part 46. 39 In practice, some institutions use the committees established to review embryonic stem cell research to review all embryo research, but this is not required by law.
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. For example, if FDA approved MRT for the intended use of preventing the transmission of known pathogenic mtDNA mutations, a clinician could use the technique for the off-label indication of treating infertility.
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. In addition, CDC collects and publishes pregnancy success data for ART techniques, which could include MRT because it is among the "treatments or procedures which include the handling of human oocytes or embryos" (42 U.S.C.
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Each of the major medical societies has programs or documents that describe and periodically update the factors most salient to good practice in their field. At times these societies also have stepped in to help maintain high standards in fields that escape some of the formal mechanisms that exist for this purpose, such as surgery (which often innovates without the formal clinical investigations that trigger IRB review)
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2004. Role of the mitochondrial genome in assisted reproductive technologies and embryonic stem cell-based therapeutic cloning.
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2010. Pronuclear transfer in human embryos to prevent transmission of mito chondrial DNA disease.
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2015b. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.
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2014. Limitations of preimplantation genetic diagnosis for mitochondrial DNA diseases.
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2013. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants.
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2005. Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation.
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2014. Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases.
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