<\/p>\n
<\/p>\n Abstract<\/strong><\/p>\n Monogenic disorders occur at a high frequency in human populations and are commonly inherited through the germline. Unfortunately, once the mutation has been transmitted to a child, only limited treatment options are available in most cases. However, means of correcting disease-causing nuclear and mitochondrial DNA mutations in gametes or preimplantation embryos have now been developed and are commonly referred to as germline gene therapy (GGT). We will discuss these novel strategies and provide a path forward for safe, high-efficiency GGT that may provide a promising new paradigm for preventing the passage of deleterious genes from parent to child.<\/p>\n <\/p>\n<\/div>\n Main<\/strong><\/p>\n<\/div>\n<\/section>\n There are over 10,000 identified single-gene (monogenic) mutations so far that are linked to human diseases. A genetic mutation is typically presented as an alteration of the genetic code (or sequence) that ultimately results in abnormal proteins or other gene products. While some gene mutations are rare, the cumulative effect of thousands of different gene mutations on human populations is immense, affecting hundreds of millions of people worldwide. Genetic conditions are also a leading cause of early pregnancy loss, infant mortality and birth defects1<\/a><\/sup>.<\/p>\n Many gene mutations are passed down to offspring (so-called heritable, or germline, mutations) and thus persist in certain human lineages and cause familial diseases (Box\u00a01<\/a>). The high frequency of genetic disorders has spurred an interest in novel strategies for prevention of the transmission of pathogenic mutations from parent to child. While germline gene therapy (GGT) has proven to be an ethically sensitive subject, exploration of therapeutic possibilities of GGT is not premature given the dramatic evolution of the molecular diagnostics of most genetic disorders, and the ability to correct these defects in the germline2<\/a><\/sup>.<\/p>\n Experimental gene therapy approaches that involve treatment of affected somatic tissues or organs in patients are referred to as somatic gene therapy. However, most pathogenic gene mutations, once passed down to children, incite irreversible damage to tissues and organs and cause diseases such as hypertrophic cardiomyopathy, Huntington\u2019s disease or BRCA-associated cancers. In these cases, somatic gene therapy is unlikely to reverse the pathology of the disease unless therapy is attempted earlier, before onset of disease. There have been proof-of-concept studies in mouse models of genetic disease that have demonstrated that the success of fetal gene therapy for selected congenital genetic disorders, such as tyrosinemia type 1 and neuronopathic Gaucher disease, is possible3<\/a>,4<\/a><\/sup>. The efficacy of somatic gene therapy, if feasible at all, is also complicated by the necessity of targeting billions of cells in solid tissues and organs. Lastly, confining treatments to somatic cells of individual patients will not prevent transmission of defective genes from parents to children.<\/p>\n Gene therapy directed at reproductive cells (sperm and eggs) or preimplantation embryos, termed GGT, has the potential to correct disease-causing mutations to wild-type variants early in development when the mutation is present in one of few embryonic cells. In addition, GGT will not only prevent passage of genetic disease to a child, but to all future generations.<\/p>\n Gene therapy in general utilizes several alternative strategies that were initially developed for clinical applications on somatic tissues. One of the first approaches involves inserting a synthetic copy of a normal gene into a nonspecific location within the genome in addition to the mutant, endogenous copy (or copies) of the gene. The transgene is usually delivered and integrated into the target genome using viral vectors. This approach is undesirable for GGT due to safety and efficacy concerns related to using viruses and random integration of transgenes. In addition, this form of gene therapy results in genetic modifications of the human genome that are deemed ethically unacceptable for GGT.<\/p>\n Another alternative approach is based on inducing lesions in genes that are already mutated using genome editing in hopes of altering the gene to modulate disease. Genome-editing tools, including RNA-guided CRISPR\u2013Cas9, are highly effective for site-specific DNA breaks leading to targeted insertion and deletion (indel) mutations at specific loci in animals and human cells (see details below)5<\/a><\/sup>. There are a few cases in which the generation of novel mutations has been proposed for therapeutic applications in somatic cells. In particular, strategies to modify mutations causing Duchenne muscular dystrophy (DMD) were tested in mouse models. Cas9-mediated excision of exon 23 of the dystrophin (Dmd<\/i>) gene, which harbors the nonsense mutation responsible for the mouse DMD phenotype, restored expression of a truncated version of the dystrophin protein and improved skeletal muscle strength to varying degrees6<\/a><\/sup>.<\/p>\n Another example is that of the C\u2013C chemokine receptor type 5 (CCR5<\/i>) gene encoding a human cell surface protein that has been implicated in HIV-1 entry. A naturally occurring 32-base-pair (bp) deletion (\u039432) in this gene in some human populations is believed to confer resistance for homozygous carriers to some HIV-1 strains, with a few other health- or immune-system-related implications7<\/a>,8<\/a><\/sup>. Therefore, it has been proposed that inducing similar mutations in patients with HIV could be therapeutic. A clinical trial transplantation of autologous CD4+<\/sup>\u00a0T cells in which\u00a0CCR5<\/i>\u00a0was disrupted to HIV-infected individuals resulted in HIV rebound in all individuals who received the cells, likely due to low gene-editing efficiency9<\/a><\/sup>.<\/p>\n The main limitation of conventional gene editing is that there are only a handful of cases among thousands of pathogenic germline mutations for which inducing additional de novo mutations could be therapeutic. This concept is unlikely to meet the high efficacy and safety requirements for GGT because of the uncertainty associated with inducing novel gene mutations in children. Indeed, public response to a recent case of\u00a0CCR5<\/i>\u00a0disruption in human embryos that reportedly resulted in birth of twin girls in China10<\/a><\/sup>\u00a0was unanimously negative. The statement released by the organizing committee of the Second International Summit on Human Genome Editing in Hong Kong on 29 November 2018 summarized that \u201ceven if the modifications are verified, the procedure was irresponsible and failed to conform with international norms\u201d10<\/a><\/sup>.<\/p>\n We will discuss below in more detail recently developed gene therapy strategies that are based on the replacement of mutated genes with normal wild-type copies (such as for mitochondrial DNA (mtDNA) mutations) or actual repair of endogenous mutant sequences and conversion of them back to normal using endogenous DNA-repair mechanisms. We believe these technologies meet the stringent safety and efficacy standards expected for GGT and are ethically acceptable since they do not induce new genetic modifications.<\/p>\n In this regard, we would like to note that GGT may utilize various genetic manipulation methods, including gene editing and gene replacement, to achieve the therapeutic goal, which is to modify genes such that they become the wild-type variants that are common in the human population; thus, in our opinion, if GGT is carried out such that it avoids off- and on-target mutations, it should provide no long-lasting negative alteration to the germline.<\/p>\n <\/p>\n