Human germline engineering is the process by which the genome of an individual is edited in such a way that the change is heritable. This is achieved through genetic alterations within the germ cells, or the reproductive cells, such as the egg and sperm.[1] Human germline engineering should not be confused with gene therapy. Gene therapy consists of altering somatic cells, which are all cells in the body that are not involved in reproduction. While gene therapy does change the genome of the targeted cells, these cells are not within the germline, so the alterations are not heritable and cannot be passed on to the next generation.[1]

The first attempt to edit the human germline was reported in 2015, when a group of Chinese scientists used the gene editing technique CRISPR/Cas9 to edit single-celled, non-viable embryos to see the effectiveness of this technique.[2] This attempt was rather unsuccessful; only a small fraction of the embryos successfully spliced the new genetic material and many of the embryos contained a large amount of random mutations.[2][3] The non-viable embryos that were used contained an extra set of chromosomes, which may have been problematic. In 2016, another similar study was performed in China which also used non-viable embryos with extra sets of chromosomes. This study showed very similar results to the first; there were successful integrations of the desired gene, yet the majority of the attempts failed, or produced undesirable mutations.[3]

The most recent, and arguably most successful, experiment in August 2017 attempted the correction of the heterozygous MYBPC3 mutation associated with Hypertrophic Cardiomyopathy in human embryos with precise CRISPRCas9 targeting. 52% of human embryos were successfully edited to retain only the wild type normal copy of MYBPC3 gene, the rest of the embryos were mosaic, where some cells in the zygote contained the normal gene copy and some contained the mutation.[4]

In November 2018, researcher Jiankui He claimed that he had created the first human genetically edited babies, known by their pseudonyms, Lulu (Chinese: ) and Nana (Chinese: ).[5][6]{

Human genetic modification is the direct manipulation of the genome using molecular engineering. The two different types of gene modification is "somatic gene modification" and "germline genetic modification." Somatic gene modification adds, cuts, or changes the genes in cells of a living person. Germline gene modification changes the genes in sperm, eggs, and embryos. These modifications would appear in every cell of the human body.

Human germline engineering is modifying the genes in the human sex cells that can be passed on to the future generations. This process is done by a complicated but an accurate technique that contains an enzyme complex called CRISPR/Cas9 clustered regularly interspaced short palindromic repeats, this enzyme can be found in many bacteria immune system, in which they use it to fight off any harmful infections.[7]

CRISPR is a repeated, short sequence of RNA that match with the genetic sequence that the scientists are aiming to modify or engineer. CRISPR works in rhythm with Cas9, an enzyme that splices the DNA. First, the CRISPR/Cas9 complex searches through the cell's DNA until it finds and binds to a sequence that matches the CRISPR, then, the Cas9 splices the DNA. After that, the scientist inserts a piece of DNA before the cell starts repairing the spliced part, said John Reidhaar-Olson, a biochemist at Albert Einstein College of Medicine in New York[8]. The main purpose of human germline engineering is to enable the scientists to discover the unknown functions of the genes by eliminating specific DNA fragments and observing the consequences in the targeted cell. Also, scientists use CRISPR technology to fix the gene mutations and to treat or eliminate some diseases that can be passed on to the offsprings[9].

CRISPR/cas9 is a genome editing tool that allows scientists to edit the genome by adding or removing sections of DNA. It contains an enzyme and RNA, the enzyme acting like scissors to alter the DNA while the RNA acts as a guide for those enzymes. This system is currently the fastest and cheapest way to genetically engineer on the market today and its uses are endless. The RNA in the CRISPR/cas9 allows researchers to target specific sequences in the genome making it possible for them to alter one sequence and not the others surrounding them. This is a new technology for scientists in the genomic altering field.[10]

Although the CRISPR/cas9 cannot yet be used in humans[citation needed], it allows scientists to target genes more effectively in diploid cells of mammals in order to one day be used in human research. Clinical trials are being conducted on somatic cells, but CRISPR could make it possible to modify the DNA of spermatogonial stem cells. This could eliminate certain diseases in human, or at least significantly decrease a disease's frequency until it eventually disappears over generations.[11] Cancer survivors theoretically would be able to have their genes modified by the CRISPR/cas9 so that certain diseases or mutations will not be passed down to their offspring. This could possibly eliminate cancer predispositions in humans.[11] Researchers hope that they can use the system in the future to treat currently incurable diseases by altering the genome altogether.

The Berlin Patient has a genetic mutation in the CCR5 gene (which codes for a protein on the surface of white blood cells, targeted by the HIV virus) that deactivates the expression of CCR5, conferring innate resistance to HIV. HIV/AIDS carries a large disease burden and is incurable (see Epidemiology of HIV/AIDS). One proposal is to genetically modify human embryos to give the CCR5 32 allele to people.

There are many prospective uses such as curing genetic diseases and disorders. If perfected, somatic gene editing can promise helping people who are sick. In the first study published regarding human germline engineering, the researchers attempted to edit the HBB gene which codes for the human -globin protein.[2] Mutations in the HBB gene result in the disorder -thalassaemia, which can be fatal.[2] Perfect editing of the genome in patients who have these HBB mutations would result in copies of the gene which do not possess any mutations, effectively curing the disease. The importance of editing the germline would be to pass on this normal copy of the HBB genes to future generations.

Another possible use of human germline engineering would be eugenic modifications to humans which would result in what are known as "designer babies". The concept of a "designer baby" is that its entire genetic composition could be selected for.[12] In an extreme case, people would be able to effectively create the offspring that they want, with a genotype of their choosing. Not only does human germline engineering allow for the selection of specific traits, but it also allows for enhancement of these traits.[12] Using human germline editing for selection and enhancement is currently very heavily scrutinized, and the main driving force behind the movement of trying to ban human germline engineering.[13]

The ability to germline engineer human genetic codes would be the beginning of eradicating incurable diseases such as HIV/AIDS, sickle-cell anemia and multiple forms of cancer that we cannot stop nor cure today.[14] Scientists having the technology to not only eradicate those existing diseases but to prevent them altogether in fetuses would bring a whole new generation of medical technology. There are numerous disease that humans and other mammals obtain that are fatal because scientists have not found a methodized ways to treat them. With germline engineering, doctors and scientists would have the ability to prevent known and future diseases from becoming an epidemic.

The topic of human germline engineering is a widely debated topic. It is formally outlawed in more than 40 countries. Currently, 15 of 22 Western European nations have outlawed human g
ermline engineering.[15] Human germline modification has for many years has been heavily off limits. There is no current legislation in the United States that explicitly prohibits germline engineering, however, the Consolidated Appropriation Act of 2016 banned the use of U.S. Food and Drug Administration (FDA) funds to engage in research regarding human germline modifications.[16] In recent years, as new founding is known as "gene editing" or "genome editing" has promoted speculation about their use in human embryos. In 2014, it has been said about researchers in the US and China working on human embryos. In April 2015, a research team published an experiment in which they used CRISPR to edit a gene that is associated with blood disease in non-living human embryos. All these experiments were highly unsuccessful, but gene editing tools are used in labs.

Scientists using the CRISPR/cas9 system to modify genetic materials have run into issues when it comes to mammalian alterations due to the complex diploid cells. Studies have been done in microorganisms regarding loss of function genetic screening and some studies using mice as a subject. RNA processes differ between bacteria and mammalian cells and scientists have had difficulties coding for mRNA's translated data without the interference of RNA. Studies have been done using the cas9 nuclease that uses a single guide RNA to allow for larger knockout regions in mice which was successful.[17] Altering the genetic sequence of mammals has also been widely debated thus creating a difficult FDA regulation standard for these studies.

The lack of clear international regulation has lead to researchers across the globe attempting to create an international framework of ethical guidelines. Current framework lacks the requisite treaties among nations to create a mechanism for international enforcement. At the first International Summit on Human Gene Editing in December 2015 the collaboration of scientists issued the first international guidelines on genetic research.[18] These guidelines allow for the pre-clinical research into the editing of genetic sequences in human cells granted the embryos are not used to implant pregnancy. Genetic alteration of somatic cells for therapeutic proposes was also considered an ethnically acceptable field of research in part due to the lack of ability of somatic cells to transfer genetic material to subsequent generations. However citing the lack of social consensus, and the risk of inaccurate gene editing the conference called for restraint on any germline modifications on implanted embryos intended for pregnancy.

With the international outcry in response to the first recorded case of human germ line edited embryos being implanted by researcher He Jiankui, scientists have continued discussion on the best possible mechanism for enforcement of an international framework. On March 13th 2019 researchers Eric Lander, Franoise Baylis, Feng Zhang, Emmanuelle Charpentier, Paul Bergfrom along with others across the globe published a call for a framework that does not foreclose any outcome but includes a voluntary pledge by nations along with a coordinating body to monitor the application of pledged nations in a moratorium on human germline editing with an attempt to reach social consensus before moving forward into further research.[19] The World Health Organization announced on December 18th 2018 plans to convene an intentional committee on clinical germline editing.[20]

As it stands, there is much controversy surrounding human germline engineering. Editing the genes of human embryos is very different, and raises great social and ethical concerns. The scientific community, and global community, are quite divided regarding whether or not human germline engineering should be practiced or not. It is currently banned in many of the leading, developed countries, and highly regulated in the others due to ethical issues.[21] The large debate lies in the possibility of eugenics if human germline engineering were to be practiced clinically. This topic is hotly debated because the side opposing human germline modification believes that it will be used to create humans with "perfect", or "desirable" traits.[21][22][23][24][25] Those in favor of human germline modification see it as a potential medical tool, or a medical cure for certain diseases that lie within the genetic code.[22] There is a debate as to if this is morally acceptable as well. Such debate ranges from the ethical obligation to use safe and efficient technology to prevent disease to seeing actual benefit in genetic disabilities.[26] While typically there is a clash between religion and science, the topic of human germline engineering has shown some unity between the two fields. Several religious positions have been published with regards to human germline engineering. According to them, many see germline modification as being more moral than the alternative, which would be either discarding of the embryo, or birth of a diseased human.[22][24][25] The main conditions when it comes to whether or not it is morally and ethically acceptable lie within the intent of the modification, and the conditions in which the engineering is done.

The process of modifying the human genome has raised ethical questions. One of the issues is off target effects, large genomes may contain identical or homologous DNA sequences, and the enzyme complex CRISPR/Cas9 may unintentionally cleave these DNA sequences causing mutations that may lead to cell death.[27]

Another very interesting point on the debate of whether or not it is ethical and moral to engineer the human germline is a perspective of looking at past technologies and how they have evolved. Dr. Gregory Stock discusses the use of several diagnostic tests used to monitor current pregnancies and several diagnostic tests that can be done to determine the health of embryos.[23] Such tests include amniocentesis, ultrasounds, and other preimplantation genetic diagnostic tests. These tests are quite common, and reliable, as we talk about them today; however, in the past when they were first introduced, they too were scrutinized.[23]

One of the main arguments against human germline engineering lies in the ethical feeling that it will dehumanize children. At an extreme, parents may be able to completely design their own child, and there is a fear that this will transform children into objects, rather than human beings.[23][24][25] There is also a large opposition as people state that by engineering the human germline, there is an attempt at "playing God", and there is a strong opposition to this. One final, and very possible issue that causes a strong opposition of this technology is one that lies within the scientific community itself. Inevitably, this technology would be used for enhancements to the genome, which would likely cause many more to use these same enhancements. By doing this, the genetic diversity of the human race and the human gene pool as we know it would slowly and surely diminish.[23] Despite the controversy surrounding the topic of human germline engineering, it is slowly and very carefully making its way into many labs around the world. These experiments are highly regulated, and they do not include the use of viable human embryos, which allows scientists to refine the techniques, without posing a threat to any real human beings.[23]

The creation of genetically modified humans may have been performed in the mid-1990s, in which a 1997 study published in The Lancet claimed, the first case of human germ-line genetic modification resulting in normal healthy children..[28][29] In November 2018, researcher Jiankui He claimed that he had created the first human genetically edited babies, known by their pseudonyms, Lulu (Chinese: ) and Nana (Chinese: ).[5][6] Researcher Alcino J. Silva has discovered an impact the CCR5 gene has has on the memory function the brain.[30] Silva speculates the brain function of Lulu and Nana likely has been impacted but that the exact consequences of the edit are impossible to predict. Studies have sh
own mice who have had the CCR5 gene have shown a marked improvement in the function of their memory and brain recovery after stroke.

The first known publication of research into human germline editing was by a group of Chinese scientists in April 2015 in the Journal "Protein and Cell".[31] The scientists used tripronuclear (3PN) zygotes, zygotes fertilized by two sperm and therefore non-viable, to investigate CRISPR/Cas9-mediated gene editing in human cells, something that had never been attempted before. The scientists found that while CRISPR/Cas9 could effectively cleave the -globin gene (HBB), the efficiency of homologous recombination directed repair of HBB was highly inefficient and did not do so in a majority of the trials. Problems arose such as off target cleavage and the competitive recombination of the endogenous delta-globin with the HBB lead to unexpected mutation. The results of the study indicated that repair of HBB in the embryos occurred preferentially through alternative pathways. In the end only 4 of the 54 zygotes carried the intended genetic information, and even then the successfully edited embryos were mosaics containing the preferential genetic code and the mutation. The conclusion of the scientists was that further effort was needed in to improve the precision and efficiency of CRISPER/Cas9 gene editing.

In March 2017 a group of Chinese scientists claimed to have edited three normal viable human embryos out of six total in the experiment.[32] The study showed that CRISPR/Cas9 is could effectively be used as a gene-editing tool in human 2PN zygotes, which could lead potentially pregnancy viable if implanted. The scientists used injection of Cas9 protein complexed with the relevant sgRNAs and homology donors into human embryos. The scientists found homologous recombination-mediated alteration in HBB and G6PD. The scientists also noted the limitations of their study and called for further research.

In August 2017 a group of scientists from Oregon published an article in "Nature" journal detailing the successful use of CRISPR to edit out a mutation responsible for congenital heart disease.[33] The study looked at heterozygous MYBPC3 mutation in human embryos. The study claimed precise CRISPR/Cas9 and homology-directed repair response with high accuracy and percision. Double-strand breaks at the mutant paternal allele were repaired using the homologous wild-type gene. By modifying the cell cycle stage at which the DSB was induced, they were able to avoid mosaicism, which had been seen in earlier similar studies, in cleaving embryos and achieve a large percentage of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of unintended mutations. The scientists concluded that the technique may be used for the correction of mutations in human embryos. The claims of this study were however pushed back on by critics who argued the evidence was overall unpersuasive.

In June 2018 a group of scientists published and article in "Nature" journal indicating a potential link for edited cells having increased potential turn cancerous.[34] The scientists reported that genome editing by CRISPR/Cas9 induced DNA damage response and the cell cycle stopped. The study was conducted in human retinal pigment epithelial cells, and the use of CRISPR lead to a selection against cells with a functional p53 pathway. The conclusion of the study would suggest that p53 inhibition might increase efficiency of human germline editing and that p53 function would need to be watched when developing CRISPR/Cas9 based therapy.

In November 2018 a group of Chinese scientists published research in the journal "Molecular Therapy" detailing their use of CRISPR-Cas9 technology to correct a single mistaken amino acid successfully in 16 out of 18 attempts in a human embryo.[35] The unusual level of precision was achieved by the use of a base editor (BE) system which was constructed by fusing the deaminase to the dCas9 protein. The BE system efficiently edits the targeted C to T or G to A without the use of a donor and without DBS formation. The study focused on the FBN1 mutation that is causative for Marfan syndrome. The study provides proof positive for the corrective value of gene therapy for the FBN1 mutation in both somatic cells and germline cells. The study is noted for its relative precision which is a departure from past results of CRISPER-Cas9 studies.

The most controversial research to date has been the work of He Jiankui who presented his research at Second International Summit on Human Genome Editing in November 2018.[36] Jianku claimed to have implanted embryos that were successfully modified with a mutation in the CCR5 gene with the intent of preventing HIV transmission. The result of his experiment was the birth of two female children code named Lulu and Nana. The reaction against the announcement was swift and met with widespread international denunciation. Further details of Jianku's research have yet to be published aside from what was announced at the summit. Since the reveal of the research Jiankui's position at Southern University of Science and Technology has been terminated and he has been under a state of house arrest for his work and may even face the death penalty.[37]

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