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Changing the code of life

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Changing the code of life



Congratulations!
 
On 7 October,  the Royal Swedish Academy of Sciences announced that it had awarded the 2020 Nobel Prize for Chemistry to two women scientists: Emmanuelle Charpentier (L), a French microbiologist, geneticist and biochemist,  who is now the director of the Max Planck Unit for the Science of Pathogens in Berlin, Germany, and Jennifer Doudna (R), an American biochemist  who is a professor of chemistry, biochemistry and molecular biology at UC Berkeley.

The scientists developed a simple, cheap, yet powerful, and precise technique for editing DNA, which is called CRISPR-Cas9 (an acronym for Clustered Regularly Interspaced Short Palindromic Repeats) and popularly referred to as a pair of ‘genetic-scissors’. The technology endows science and scientists with extraordinary powers to manipulate genes to cure genetic diseases, improve crops to withstand drought, mould and pests, and affect climate change, and is considered to be the most important discovery in the history of biology. The Nobel citation refers to Charpentier’s and Doudna’s scientific contribution as a, “tool for rewriting the code of life”, which has “a revolutionary impact on the life sciences, by contributing to new cancer therapies and may make the dream of curing inherited diseases come true”.

For more than four years HealthPad has been following and publishing Commentaries on the scientists’ work. Our Commentaries have a large and growing global following of leading physicians, scientists, policy makers, journalists and students. The Commentaries listed below about CRISPR techniques, which we re-publish to celebrate Charpentier’s and Doudna’s Nobel Prize, have had more than 120,000 views.
 
Gene editing positioned to revolutionise medicine
1 Feb 2017
 
Gene editing battles
15 Mar 2017
 
Who should lead MedTech?
18 Jul 18
Base-editing next-generation genome editor with delivery challenges
17 oct 2018
CRISPR-Cas9 genome editing a 2-edged sword
31 Oct 2018
Will China become a world leader in health life sciences and usurp the US?
27 Feb 2019
view in full page
6 years ago

CRISPR-Cas9 genome editing a 2-edged sword

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bioethics
commentary
CRISPR
CRISPR technology
genome editing
incurable inherited diseases
  • CRISPR-Cas9 genome editing technology discovered in 2012 has revolutionized biological science and brought hope to millions of people born with incurable inherited killer diseases
  • In July 2018 the UK’s Nuffield Council on Bioethics endorsed the technology to make changes at the cell level in the human body that are heritable
  • This alarms bioethicists because there is no universally agreed regulation for CRISPR and the technology is cheap, easy-to-use and accessible and the line between “therapy” and “enhancement” is blurred
  • CRISPR was invented in the West but is rapidly being transformed into therapies in China where regulation is less than stringent
  • Will genome editing be used to enhance off-springs that satisfy parents’ preferences for children with specific characteristics?
 
 
CRISPR-Cas9 genome editing a 2-edged sword 
 

The genie is out of the bottle!
 
On the 17th July 2018 the UK’s Nuffield Council on Bioethics published a report entitled, Genome Editing and Human Reproduction: Social and Ethical Issues, which concluded that germline editing, a process by which every cell in the human body could be altered in such a way that the change is heritable, is “morally permissibly” under certain circumstances. The Council was referring to developments of an invention made in 2012 by scientists Jennifer Doudna, and Emmanuelle Charpentier. They discovered how to exploit an oddity in the immune system of bacteria to edit genes, which resulted in CRISPR-Cas9, (an acronym for Clustered Regularly Interspaced Short Palindromic Repeats), which is generally considered the most important invention in the history of biology. Since its discovery, modified versions of the technology have found a widespread use to engineer genomes and to activate or to repress the expression of genes. Clinical studies testing CRISPR-Cas9 in humans are underway.
 
In this Commentary

In this Commentary we: (i) describe CRISPR-Cas9 and indicate how it has impacted medicine, biotechnology and agriculture, but suggest that it is most famous for its potential to modify human embryos to provide therapies for inherited killer diseases for which there are no known cures, (ii) suggest that although the technology is gaining regulatory support for its use in humans, there is no universal regulatory agreement. Some countries remain opposed to using CRISPR to edit human embryos while in China regulations is less than stringent. This patchy and loose state of affairs raise concerns among bioethicists, (iii) describe a non-profit agency that has significantly increased the accessibility of the technology, which has helped to democratise CRISPR, but also makes it easier for less stringently controlled laboratories to acquire it, (iv) briefly describe the Chinese scientists first use of the technology in humans and some of the unintended consequences which resulted. We provide examples of research that followed and briefly describe the US-China race to transform CRISPR into viable therapies, and suggest that China, helped by laxed regulation, is winning the race, (v) suggest that these factors, plus the fact CRISPR blurs the distinction between ‘therapy’ and ‘enhancement’, seems to convince bioethicists that the technology at some point in the future will be used to create ‘designer babies’, (vi) conclude by noting that for millennia people have been using radical and painful methods to modify their own and their children’s bodies and this seems to suggest that in time, germline editing will be perceived as a logical extension of these customs and practices, the genie is out the bottle and customize children are likely to become the norm.
 
CRISPR-Cas9

CRISPR is a mechanism deployed by bacteria to identify the DNA of invading viruses and is used by scientists to target a specific gene. Cas-9 is an enzyme, which acts like a pair of molecular scissors to cut out a piece of DNA and, if need be, replace it with a new gene. The process is faster, cheaper and easier to use than traditional genetic modification and has been likened to editing a Word document on a computer. Thus, gene editing has been taken away from highly skilled and tightly regulated molecular biologists and made more widely available. This not only democratizes science but also heightens ethical concerns.
 
CRISPR technologies impact medicine biotechnology and agriculture
 
Since the breakthrough was made in 2012, CRISPR-Cas9 has quickly development into a powerful, cheap and accessible tool in genetics. The technology is programmable, efficient, precise and scalable and has driven significant advances across medicine, biotechnology and agriculture throughout the world. As the world’s population and average temperatures increase, the demand for larger, more nutritious harvests and climate-adaptable crops will grow. The application of CRISPR technology to agriculture allows for an efficient and accurate mode of genetic manipulation to meet these increasing needs. The technology also has been used in the fight against malaria. According to a 2018 World Health Organization report, in 2016 there were 216m cases of malaria worldwide and 445,000 deaths from the disease. Malaria is spread by the female Anopheles-gambiae mosquito, which is one of 3,500 species of mosquitoes. Scientists have used CRISPR technology to edit the genes of this specific type of mosquito to avoid the malaria causing parasite. In a study carried out at Imperial College London and published in the September 2018 edition of Nature Biotechnology researchers succeeded in destroying a population of trapped Anopheles mosquitoes by using CRISPR  technology to genetically alter cells  to spread a genetic modification that blocks female reproduction so, over time, the malaria spreading Anopheles mosquitoes die out. The research demonstrates how a specific CRISPR application can propagate a particular suite of genes throughout an entire population or species and empower scientists in the war against diseases. “It provides hope in the fight against a disease that has plagued mankind for centuries,” says Andrea Crisanti, lead author of the Imperial study.
 
But the one application, which has made CRISPR famous is the modification of the human genome, which promises to cure some of the world’s deadliest diseases for which there are no known therapies. There are some 10,000 genetic diseases of which less than 6% have approved treatments.
 
Regulatory support
 
CRISPR genome editing technologies have been gaining regulatory acceptance for their use in humans and an increasing number of scientists in the US, UK and China have reached conclusions similar to those of the Nuffield Council, and suggest that if germline editing is shown to be safe and there are no medical alternatives, it should be permitted to prevent children being born with fatal diseases. In 2017, the UK’s Human Fertilization and Embryology Authority approved an application to use genome editing, which allows scientists to change an organism’s DNA in research on human embryos. Also, in 2017 a report from the US National Academy of Sciences (NAS) stated that clinical trials for editing-out heritable diseases could be permitted in the future for serious conditions under stringent oversight. At the same time as the Nuffield Council published its findings, - July 2018 - the US Food and Drug Administration (FDA) Commissioner Scott Gottlieb announced a new regulatory framework for genome editing for rare diseases. The following month, - August 2018 - the FDA along with the US National Institutes of Health (NIH) issued joint guidelines for a new streamlined process for assessing the safety of gene-therapy human clinical studies.  And in an August 2018 New England Journal of Medicine editorial Gottlieb and NIH Director Francis Collins argue that, “there is no longer sufficient evidence to claim that the risks of gene therapy are entirely unique and unpredictable - or that the field still requires special oversight that falls outside our existing framework for ensuring safety.”
 
No international regulatory framework for CRISPR triggers concerns
 
Despite increasing support for genome editing, to-date no internationally agreed regulatory framework exists that addresses the ensuing scientific, socio-ethical and legal challenges CRISPR technologies pose for regenerative and personalised medicine. Regulation is on a country-by-country basis and most nations struggle to assess whether gene editing may or may not be different from classical genetic engineering. Several nations remain opposed to the use of the technology in humans. The most contentious issue is human germline editing.

In Canada human germline editing is a criminal offence and sanctions range from fines of US$400,000 and up to ten years imprisonment. However, there is mounting pressure from Canadian scientists to change the law. France restricts genome editing research and supports the Oviedo Convention, which is the first multilateral binding instrument entirely devoted to bio-law. It came into force in 1999, backed by the Council for Europe and aims to prohibit the misuse of innovations in biomedicine. The treaty states that, “An intervention seeking to modify the human genome may only be undertaken for preventive, diagnostic, or therapeutic purposes and only if its aim is not to introduce any modification in the genome of any descendants”. In Germany germline editing is constrained by its 1990 Embryo Protection Act, which prohibits the generation and use of embryos for basic research, and also prohibits the harvesting of embryonic cells. South Korea’s Bioethics and Biosafety Act prohibits genetic experimentation, which modifies human embryos. Western observers suggest that regulation in China is “thin” and tends to be at the provincial and hospital levels. It has been reported that Chinese hospital review boards have approved clinical studies involving gene-editing and cancer patients without fully understanding the nature and power of the technology.
The “dark-side” of CRISPR technology

Weak regulation raises concerns about the level of ethical conduct in clinical studies and the potential dangers this holds for future therapies. Cognisant of CRISPR’s powerful capabilities, its relative cheapness and accessibility, (see below) James Clapper, the former US Director of National Intelligence describes CRISPR-Cas9 gene editing in the 2016 and 2017 Agency’s Worldwide Threat Assessment reports submitted to the US Congress as, “a potential weapon for mass destruction”. Jennifer Doudna, one of the inventors of CRISPR-Cas9 says that there are things which you would not want the technology used for and, “most of the public does not appreciate what is coming”. These sentiments resonate with bioethicists concerned about the absence of stringent universal regulation and the technology getting into the “wrong hands” and resulting in “designer babies”, an escalation of societal inequalities and increased safety and biosecurity issues.
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The global competition to translate genomic data into personal medical therapies

PART 1
 

and

PART 2
 
 
Democratizing the CRISPR technology
 
Notwithstanding, many scientists view the ease of access to CRISPR technologies as a significant driver of cutting-edge research and the speed at which therapies for life-threatening diseases will enter clinics. The organization most responsible for CRISPR’s widespread accessibility is Addgene, a self-sustaining, non-profit plasmid repository, which facilitates the exchange of genetic material between laboratories throughout the world. (A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA. It can replicate independently and is used in the laboratory manipulation of genes). It is free for scientists to deposit plasmids in Addgene and a nominal fee is charged for requests. This allows for maintenance and growth of the repository without reliance on grants or external funding. Founded in 2004, Addgene has significantly reduced the frustration scientists experience sharing plasmids with one another. The organization has developed into an important one-stop-shop for depositing, storing, and distributing plasmids globally and this has significantly enabled the democratization of CRISPR technologies. More than 6,300 CRISPR-related plasmids have been developed by over 330 academic laboratories throughout the world and deposited with Addgene. Since 2013, the organization has distributed over 100,000 CRISPR plasmids to some 3,400 laboratories in more than 75 countries. 
 
Mixed results when CRISPR was first used in humans
 
CRISPR technology was first used in humans in China, when a group of scientists led by Junjiu Huang from Sun Yat-sen University in Guangzhou, attempted to modify the gene responsible for β-thalassemia, a potentially fatal blood disorder. Although the genomes of human embryos edited by the scientists could not be developed into a foetus, the researchers had difficulties publishing their findings because of ethical concerns. After being rejected by the journals Science and Nature their paper was published in 2015 in the journal Protein & Cell. The work triggered an international debate, but the research had a low success rate: only 4 of the 54 embryos that survived the technique carried the repaired genes. Huang and his colleagues identified two challenges. One was unintended genetic modifications - off target effects - when CRISPR either changes a gene scientist did not want changed or it fails to change a gene that they did. The second was that embryos, which did not get edited correctly mixed with those that did and became what is referred to as a “mosaic”.  
 
New study discovers the deletion of thousands of DNA bases
 
Initially, these anomalies were thought to be minimal and improvements to the technique were thought to be able to reduce them so that they were virtually undetectable. Indeed, since 2015 the science of human genome editing has advanced significantly and there has been an explosion of research. In 2017 alone, there were some 3,500 research papers published on CRISPR technologies but concerns about CRISPR’s accuracy remain. During the past three years of intense research CRISPR-Cas9 became popularly perceived as a technique that can edit genetic code to correct defects inside individual cells and prevent and heal many intractable illnesses. Notwithstanding, also there has been a growing concern among scientists that because Cas9 enzymes reprogram the DNA of a cell, which is the fundamental building block for the development of an organism, the technique, if inaccurate, may cause more harm than good. Recent research supports this view. A study published in the July 2018 edition of  the journal Nature Biotechnology discovered deletions of thousands of DNA bases, including at spots far from the edit. Some of the deletions can silence genes that should be active and activate genes that should be silent, including cancer-causing genes. This suggests that previous methods for detecting off-target mutations may have underestimated their true scale and therefore the potential for unintended consequences when using CRISPR technologies might be higher than originally thought. This finding poses a significant challenge for developing policy associated with CRISPR because you do not know what off-target effects will occur in humans until you use the technology.
 
Who is developing CRISPR-Cas9 therapies?
 
Notwithstanding, CRISPR–Cas9 is fast entering mainstream R&D and is perceived as a principal technology for treating diseases with a genetic basis and is increasingly playing a significant role in drug discovery. Scientists use the technology to either activate or inhibit genes and can determine the genes and proteins that cause or prevent specific diseases and thereby identify targets for potential therapies. Notwithstanding, drug development is a long and expensive process: it can take more than a decade and cost some US$2bn for researchers to move from the discovery of a target molecule to the production of a clinically approved therapy.  So, it could be some time before the first drugs using CRISPR–Cas9 gene editing make it to the clinics. Notwithstanding, a lot has been achieved in a relatively short time.
 
Research examples

UK examples of research using CRISPR technology include scientists from the Huntington’s Disease Centre at University College London’s Institute of Neurology, who in 2017 completed the first human genetic engineering study, which targeted the cause of Huntington’s disease and successfully lowered the level of the harmful huntingtin protein that irreversibly damages the brains of patients suffering from this incurable degenerative condition.  In another study using CRISPR technology and published in a 2017 edition of the New England Journal of Medicine, researchers from Barts Health NHS Trust and Queen Mary University London  made a significant step towards finding a cure for haemophilia A, a rare incurable life threatening-blood disorder, which is caused by the failure to produce certain proteins required for blood clotting. 
 
Human clinical studies
 
Although CRISPR has proved its worth as a research tool, its use as a therapeutic is still uncertain. This is partly because the technology is so new there is a dearth of data upon which to base clinical evaluations. Notwithstanding, since Chinese scientists first used CRISPR to edit a human embryo's genome, new and more accurate variants of CRISPR have been developed. At about the same time - 2015 - that Huang published his findings using CRISPR for the first time in humans, two children with Acute Lymphoblastic Leukaemia, an incurable cancer, were treated at Great Ormond Street Hospital (GOSH) in London with a version of CRISPR called CAR-T cell therapy. This entails extracting blood cells from patients, then using CRISPR technologies to edit the T cells outside the body - ex vivo gene therapy - in order to transform the cells into enhanced cancer fighters before reintroducing them back into the patient’s blood stream. The treatment proved to be such a success that in 2018 CAR-T cell therapy was made available on the NHS. A US clinical study using the same technique started in August 2018 for people with Acute Lymphoblastic Leukaemia. 
 
Over the past three years scientists in China have used newer versions of CRISPR to genetically engineer cells of at least 86 cancer and HIV patients. These cases form part of eleven human clinical studies using CRISPR-Cas9 technologies, ten of which are being undertaken in China. Another development of CRISPR is ‘base-editing’, which chemically modifies rather than cuts DNA. An August 2018 edition of the journal Molecular Therapy, describes how scientists in China used  base editing, to remodel the DNA of human embryos to treat patients with the Marfan syndrome, which is a relatively common inherited connective tissue disorder with significant morbidity and mortality. A further milestone for the technology was reported 2018 when a study, led by Zheng Hu of the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China, was the first to edit human cells while inside the body in an attempt to eliminate the human papilloma virus, which is the main cause of cervical cancer.
 
Company activity and clinical studies
 
Since the first publications in 2012 showcasing CRISPR-Cas9 as a gene editing tool, a number of companies have been set up to leverage the technology to develop innovative therapies. For example,  Editas Medicine, was founded in 2013 by Feng Zhang, Jennifer Doudna, David Liu, George Church, and J.Keith Joung. However, just a few weeks after the company’s formation, Doudna stopped all involvement with Editas after Zhang was granted a number of CRISPR patents and issues concerning intellectual property began to appear. In October 2018 Editas filed an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA) for a clinical study of a CRISPR genome editing medicine called EDIT-101 for the treatment of Leber Congenital Amaurosis type 10 (LCA10). This is a serious eye disorder that affects the retina, which is the specialized tissue at the back of the eye that detects light and colour. People with LCA10 typically have severe visual impairment from infancy.

In 2018 the European Patent Office granted Cellectis, a French biopharmaceutical company, the first patent to use CRISPR technology in human T cells.The patent will protect the application of CRISPR gene editing for T cell research until 2034, meaning every other company employing similar systems will need a license from Cellectis. Also, in 2018 CRISPR Therapeutics, co-founded by Emmanuelle Charpentier began a clinical study using CRISPR genome editing technologies and a similar ex vivo approach to target the blood disorder β-thalassemia. As yet no CRISPR therapies have reached the clinic.
 
US-China competition
 
There is intense and growing scientific competition between the US and China. Although CRISPR was invented in the West, it is more rapidly being transformed in China into therapies that can be used in clinics. An article in a January 2018 edition of the Wall Street Journal suggests that regulation governing genome editing of human embryos in China is much less stringent than in the West where researchers have to pass muster with hospital review boards, ethics committees and government agencies before receiving approval. In China it is not unusual simply for hospital committees to give such permissions. According to Carl June, director of translational research at the Abramson Cancer Center, University of Pennsylvania and well-known for his research into T-cell therapies for the treatment of cancer, “We are at a dangerous point in losing our lead in biomedicine. It is hard to know what the ideal is between moving quickly and making sure patients are safe”. Western scientists believe that the less that stringent regulation in China gives Chinese researchers a significant competitive advantage in the race to get CRISPR therapies into clinics and bioethicists believe that loose regulation will result in unintended consequences that will harm patients and lead to “designer babies”, which could set-back the field for everyone.
 
Blurred line between therapy and enhancement
 
What makes regulation challenging is that CRISPR technologies blur the distinction between “therapy” and “enhancement”. Indeed, the 2018 Nuffield Council report referred to at the beginning of this Commentary suggests that such a distinction between therapy and enhancement cannot be expected to hold. Thus, it seems reasonable to assume that sometime in the future, CRISPR technologies, which are cheap, easy to use and accessible could be used to genetically enhance off-springs. In the first instance this solely might be focused on eradicating life-threatening diseases, but in the longer term it seems probable, especially in the absence of any universally agreed and tightly administered regulations, that genome editing will be used to create off-springs, which satisfy parents’ preferences for children with specific characteristics. Further, CRISPR technology is becoming popular among DIY scientists and biohackers – people who experiment on themselves - which exacerbates the concerns of bioethicists.
 
People have been radically altering bodies for millennia
 
Another reason to believe that germline editing will be used for ‘cosmetic’ enhancements rather than medical therapies is that for millennia people have used radical techniques to modify their own and their children’s bodies for cosmetic rather than therapeutic purposes. Here we illustrate the point with a few examples.
 
From the Song dynasties, which ruled China between 960 and 1279 until the early 20th century, the Chinese practiced the custom of breaking their first daughter’s toes and tightly binding them under the soles of their feet in order to stunt growth so that when the girl grew up she would walk diffidently, which was perceived as attractive. In England during the Victorian era between the mid 19th and the beginning of the 20th century, women, to make themselves attractive to men, corseted their bodies so tightly to create twelve-inch waists that their internal organs were redistributed with potentially dangerous consequences. Girls as young as 4 from the Kayan tribe of Myanmar use heavy brass coils to elongate their necks; a painful tradition dating back to the 11th century. The brass coils, that weigh an average of 10 kilos, deform their collar bones and neck and shoulder muscles. The Mursi tribe in Africa cut the lower lips of girls and insert plates to stretch the lips up to 12 cm in diameter.
 
In the 1970s and 1980s elective cosmetic surgical procedures gained popularity among wealthy people on the East and West coasts of America in order to enhance their appearance. The trend soon became global through the explosion of mass media. According to the International Society of Aesthetic Plastic Surgery in 2017 there was a 9% overall annual increase in surgical and nonsurgical cosmetic procedures globally. The US was the leader, accounting for 17.9% of all procedures. The top five countries were the US, Brazil, Japan, Italy and Mexico, which together accounted for 41.4% of all cosmetic surgical procedures worldwide. Russia, India, Turkey, Germany and France completed the top ten countries. In 2017, 400,000 American women elected to have breasts augmentation surgery; a 41% increase since 2000. About 1m rhinoplasties are carried out each year, with high volumes in Brazil and Mexico. The International Society of Aesthetic Plastic Surgery also reported that in 2016 surgeons in South Korea carried out the most cosmetic surgical procedures per capita: 20 per 1,000 people. V-shaped chins, with minimal jaw or cheekbone, round skulls, lifted lip corners, petite lips and slight puffiness under the eyes have been popular surgeries in South Korea, but recently the demand for such procedures has decreased while simpler and less invasive surgeries have increased. The Society also reported that labiaplasty showed the biggest (45%) increase since 2015. Lower body lift procedures increased by 29%, while upper body lift, breast augmentation using fat transfer, and buttock lifts increased by some 20%.

Such examples suggest that body enhancements, using a range of techniques, have been practiced in many cultures throughout the world for millennia. Thus, it seems reasonable to assume that in the absence of stringent regulation CRISPR will be perceived by some as just another enhancement technique.
 
Takeaways

The discovery of CRISPR Cas9 has revolutionized the way we think about developing therapies for the world’s deadliest diseases. This powerful technology has significant advantages over traditional medical technologies; it is cheap, easy-to-use and accessible, and these factors have helped to drive CRISPR’s global acceptance and use as a tool for new and innovative therapies. Over the past three years CRISPR R&D and clinical studies have developed at a pace and bring huge promise and significant hope to millions of people living with conditions with high rates of morbidity and mortality. Notwithstanding, bioethicists warn that with the absence of stringent universally agreed regulation, all these advantages could easily pivot into significant disadvantages and lead to parents enhancing the genetic composition of their children to make them taller, more intelligent etc. This could be a small step away from reigniting the ‘Charles Galton movement’. Galton was an English scholar and cousin of Charles Darwin. He lived during the Victorian era and died in 1911. Among other things, Galton studied anthropology and sociology and suggested that the elevated social position and heightened intelligence of the English upper classes and the criminality and lack of intelligence of the English under classes were all inherited traits and the result of superior and inferior genetic make-up respectively. According to Galton societies could be improved by selective breeding. Bioethicists are concerned that CRISPR technologies could be used for a 21st century version of Galtonism.
 
The genie is truly out of the bottle.
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6 years ago

Base-editing next-generation genome editor with delivery challenges

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  • ‘Base editing’ is a more efficient version of CRISPR Cas-9 technology
  • CRISPR Cas-9 is a ground-breaking gene editing technology that was discovered in 2012
  • CRISPR Cas-9 operates like molecular scissors to cut and remove mutant strands of DNA and creates space for functioning genes to be inserted
  • CRISPR technologies raise hope for new therapies to replace traditional medicines and provide a one-time procedure to cure devastating inherited disorders that have no cure or few treatment options
  • Recent studies suggest that CRISPR Cas-9 is not as accurate as initially thought and can introduce thousands of unintended ‘off-target’ mutations into the genome
  • Base editing significantly reduces ‘off-target’ mutations because it does not cut the DNA but uses a chemical process to convert just one letter (base) of DNA into another
  • 66% of genetic illnesses involve mutations where there is a change in a single letter of DNA
  • A significant challenge for base editing is in the delivery of the technique
 
Base-editing next-generation genome editor with delivery challenges

Since the first human genome was sequenced in 2003 there has been a revolution in human genomics, which has transformed the way we think about diseases and their causes and has paved the way for the development of therapies that target both the illness and the patient. It has also led to the introduction of the genome-editing tool CRISPR Cas-9 in 2012. This transformed gene editing from a devilishly difficult task to an easy and inexpensive “day-to-day” laboratory technology, which allows scientists to cut-out and change sections of DNA at specific sites in an organism or cell. CRISPR technology revolutionized genetic research and raised hope that it could provide a powerful therapeutic tool for millions of people living with inherited debilitating diseases for which there are either no cures or few treatment options. Recently, next-generation gene-editing technologies have been developed, which have reignited the hope that gene therapies could eventually replace traditional medicines and be used by physicians in clinics as a one-time procedure to cure some of the most devastating inherited disorders. Notwithstanding, scientists have cautioned that the therapeutic use of CRISPR technologies have significant technical, safety, regulatory, ethical and delivery obstacles to overcome before they can be used as therapies.
 
In this Commentary
 
This Commentary describes a new and expanded gene editing technology called base editing or chemical surgery, which compliments CRISPR Cas-9, but instead of cutting strands of DNA it provides a more accurate and predictable means to rewrite single letters (bases) of DNA and RNA. This enables scientists to make more targeted and precise alterations to DNA and RNA with less unintended consequences, referred to as “off-target” effects.  Base editing has significant therapeutic potential for thousands of human disorders known to be caused by a single genetic error and range from sickle-cell anaemia to metabolic disorders to cystic fibrosis, which currently lack options. The new base editing techniques are described in three research papers, which appeared in scientific journals in late 2017. One was published in the November 2017 edition of the journal ‘Protein and Cell’, another in the October 2017 edition of the ‘Nature’ and a third in the October 2017 edition of the journal ‘Science’. Research reported in these papers represents an important advance in our ability to alter single letters (bases) in peoples’ DNA and RNA. Notwithstanding, scientists caution that before base editing techniques become standard clinical practice the technology will require more research, extensive clinical studies and significant advances in delivery methods.
 
CRISPR and intellectual property battles

Base editing is a development of CRISPR Cas-9 technology, which was developed by a group of researchers from University College Berkeley, the Max-Plank Institute, Harvard University and The Massachusetts Institute of Technology (MIT) and others. The Broad Institute, a non-profit disease research facility established jointly by Harvard University and MIT, obtained the basic US patents on CRISPR Cas-9 in February 2017 after a heated patent dispute between two of the technology’s originators. On one side Jennifer Doudna of University College Berkeley and Emmanuelle Charpentier of the Max-Planck Institute in Berlin. On the other side Feng Zhang of the Broad Institute. While the Broad Institute has been considered the winning party in the US, the European intellectual property landscape is a different story.  Due to technical errors associated with listed CRISPR inventions and claimed priority dates, the European patents filed by the Broad Institute have been revoked. 

The Broad Institute is expected to appeal the decision and the gene-editing intellectual property battles continue. Notwithstanding, this has not slowed the development and commercialization of the technology.
 
Technologies to edit the genetic code and some ethical challenges

CRISPR Cas-9, discovered in 2012, is a particularly versatile and inexpensive gene editing technology. Since its discovery it has been used extensively by scientists throughout the world in an attempt to further their understanding of the role played by genes in disease. The technology works by slicing through the two strands of bases that spiral to create DNA’s famous double-helix and is especially useful when the goal is to insert or delete DNA bases. CRISPR acts like a genetic GSP: a guide molecule made of RNA that allows a specific site of interest on the DNA double helix to be targeted. The RNA molecule is attached to a bacterial enzyme called Cas-9 that works like a pair of “molecular scissors”, which can cut out strands of DNA at an exact point. This allows scientists to target and remove faulty genetic material and create space for functioning genes to be inserted in a similar way a word processor allows you to correct and enhance documents. Although CRISPR Cas-9 is increasingly being used in studies of genetic disorders, it has been challenging for the technology to fix a point mutation, caused by a change in a single DNA letter in a given gene. Further the technology’s cutting mechanism can result in “off-target” activity, which either can make changes to a gene you do not want changed or fail to change a gene that you do. This represents a significant challenge for scientists, and a major concern for the technology’s therapeutic applications.

For example, research published in the July 2018 edition of  the journal Nature Biotechnology discovered unintended deletions of thousands of DNA bases, including at spots far from the edit. Another study reported in the May 2017 edition the journal Nature Methods found that CRISPR Cas-9 introduced hundreds of unintended mutations into the genome. And a third study published in December 2017 in the Proceedings of the US National Academy of Sciences suggested that genetic variation between patients may affect the efficacy and safety of CRISPR-based treatments enough to warrant custom treatments. In addition to these technical concerns, ethical concerns about the technique also have been raised. In the March 2015 edition of the journal Nature, Michael Werner, the executive director of the Washington DC based Alliance for Regenerative Medicine suggested that ethical and safety issues should put germline editing research (a process by which every cell in the human body could be altered in such a way that the change is heritable) off limits because, “It’s still a little premature to say that we’ve resolved all these safety issues now,” says Werner. Notwithstanding, in July 2018 the UK’s Nuffield Council on Bioethics suggested that germline editing is “morally permissibly” under certain circumstances.
 
CRISPR triggers intense commercial activity

Despite safety and ethical concerns about CRISPR, genome editing has rapidly become a large fast-growing global market. In late 2012, Charpentier  suggested to a few colleagues, including Doudna, Zhang and George Church, professor of genetics at Harvard University Medical School who is credited with developing the first direct genomic sequencing method in 1984, that they should start a company to accelerate the gene editing technology into clinics.  They did not, but later the same scientists and others started separate genome editing companies. Four have become publicly traded companies and have successfully raised billions. For example, in 2013 Charpentier founded Crispr Therapeutics. Based in Switzerland, the company has become a US$2bn Nasdaq traded company. The other three, all based in the US are: Editas Medicine, which has a market cap of US$1.3bn and was founded by Zhang, Church and David Liu, Professor of Chemistry at Harvard University, a core member of the Broad Institute, and the first to describe base editing in research published in the May 2016 edition of the journal Nature. Doudna is  a founding member of Intellia Therapeutics, which today has a market cap of US$1bn,  and Juno Therapeutics, which has a market cap of US$10bn was founded in 2013 through a collaboration of the Fred Hutchinson Cancer Research Center, Memorial Sloan-Kettering Cancer Center and the Seattle Children’s Research Institute.

Since their inceptions, big pharma companies have been competing to invest in them. In January 2018 Celgene, which already owned 9.7% of Juno agreed to acquire the rest of its stock for US$9bn in cash in order to gain access to Juno's pipeline of CAR-T cancer drugs. This technology entails extracting blood cells from patients, then using CRISPR to edit T cells (immune cells) outside the body - ex vivo gene therapy - in order to transform the cells into enhanced cancer fighters before reintroducing them back into the patient’s blood stream. Earlier Bayer, a German pharmaceutical company, acquired a US$35m equity stake in Crispr Therapeutics, which it increased in January 2018. In 2016 Bayer invested US$335m over 5-years in a joint venture with Crispr Therapeutics called Casebia, with the intention to discover, develop and commercialize new breakthrough therapeutics to cure blood disorders, blindness, and congenital heart disease. Casebia also expects to develop new delivery mechanisms for CRISPR technologies, which will be critical to future drugs meant to target cells in the human body. Crispr Therapeutics retains a 50% interest in the joint venture, and also gains access to Bayer’s state-of-the-art delivery technologies and protein engineering knowhow.
According to market analysis the global genome editing market is expected to grow at a CAGR of 14.5% to reach US$6.3bn by 2022. Market drivers include rising government funding and the growth in the number of genomic projects, high and increasing prevalence of debilitating and often fatal diseases, technological advancements, increasing production of genetically modified crops, and growing application areas of genomics.
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Tens of thousands of devastating diseases are the result of a single minute error in one letter the human genome
 
While big pharma competes to commercialize CRISPR Cas-9 technologies, scientists compete to develop ever-more versatile and efficient versions of the technology. One result of the competition among scientists is “base editing”, which is predicated upon the same basic mechanism as the standard CRISPR technology but differs because it does not require the DNA to be physically cut. Instead, base editing uses a chemical process to directly convert a single base (letter) of DNA to another without deleting and inserting random bases in the process.  Think of base editing as similar to changing one letter in a vast WORD document. The technique allows scientists to edit the body’s genes one letter at a time with exquisite precision.  Base editing rewrites single errors in the genetic code instead of cutting and replacing whole strands of DNA. The technique is not a replacement for CRISPR, but a complementary technology for altering the genome in an attempt to correct disease. Converting one letter to another may not sound significant until you consider that there are billions of letters in the human genome, and tens of thousands of diseases can be traced to a single minute error in just one letter in the human genome. Indeed, of more than 50,000 genetic changes currently known to be associated with disease in humans, 32,000 are caused by the simple substitution of one base letter for another. Base editing is significantly more efficient than standard CRISPR systems at making single base substitutions.
 
DNA molecules as a sequence of letters

Your genes are an instruction manual for your body. Hidden inside every cell in your body is a chemical called DNA. Genes are short sections of DNA, which are the biological templates your body uses to make the structural proteins and enzymes needed to build and maintain your tissues and organs. Genes influence how you look on the outside and how you function on the inside. The DNA that makes up all genomes is composed of four related chemicals called nucleic acids: (i) adenine ‘A’, (ii) guanine ’G’, (iii) cytosine ‘C’, and (iv) thymine ‘T’.  A sequence of DNA is a string of these nucleic acids (also called “bases” or “base pairs”). These bases connect in a specific way: ‘A’ always pairs with ‘T’, and ‘C’ always pairs with ‘G’. The letters represent the “alphabet” scientists use to write genetic code. The principal biological function of a base is to bond nucleic acids together. Nucleic acids are complex organic substances present in living cells, especially DNA or RNA. There are some 24,000 genes in the human genome, which are bundled into 23 pairs of chromosomes all coiled up in the nucleus of every one of your cells. There are about 37trn cells in the human body. Only about 1.5% of your genetic code, or genome, is made up of your genes. Another 10% regulates your genes to ensure that they turn on and off in the right cells at the right time.
 
The November 2017 Protein and Cell study

In the April 2015 edition of the journal Protein and Cell, scientists led by Junjiu Huang from Sun Yat-sen University in Guangzhou, China, reported research where he and his colleagues used CRISPR Cas-9 to correct abnormal β-thalassemia genes in human embryos without much success. Researchers suggested, “our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR Cas-9 platform, a prerequisite for any clinical applications of CRISPR Cas-9-mediated editing”. In the November 2017 edition of Protein and Cell Huang and colleagues demonstrated that they had enhanced the fidelity of CRISPR and used the new base editing technique for the first time in human embryos to repair a faulty gene that gives rise to β-thalassemia. They suggested that, “their study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by a base editor system”.
 
Β-thalassemia

Β-thalassemia is a serious blood disorder, common in China and southeast Asia, which can be caused by a single mutation in the DNA code. The disorder reduces the production of haemoglobin, which is an iron-containing protein in red blood cells that carries oxygen to cells throughout the body. Without treatment, patients with a severe type of β-thalassemia, usually die before age 5. Correcting this mutation in human embryos may cure people with the disorder and also prevent the disease being passed on to future generations.

Innovative approach

Humans carry two copies of every gene, and in many cases both versions have to be “healthy” to avoid disease. Because it is challenging for researchers to find a lot of embryos, which all have a rare double mutation, Huang’s team created a batch of cloned embryos, then took skin cells from patients with β-thalassemia, removed their DNA-containing nuclei, and introduced them into donor eggs that had their own nuclei removed. The eggs then developed into early stage embryos, which carried the β-thalassemia mutation. Despite the study’s success to effectively edit the embryos and repair the mutations it was only about 20% efficient. Huang noted that the base editing technique he and his colleagues used was not uniform across all cells in the embryos, and their endeavours only sometimes repaired one faulty gene instead of 2. This created what is called “mosaic embryos”, which have both normal and mutant cells and result in a patchwork of cells with different genetic make-up and is potentially dangerous. 

Huang concluded that more research is required to improve the safety of the study’s base editing approach. Notwithstanding, scientists believe Huang’s research represents a significant advance, and that base editing techniques hold out the potential to treat and prevent a number of serious and debilitating inherited human diseases, which are more common than some people realise. For example, 1 in 25 children are born with some genetic disorder, which includes β-thalassemia, cystic fibrosis, genetic blindness, sickle cell anaemia, muscular dystrophy, and Tay-Sachs disease.
 
The October 2017 Nature study

In October 2017, David Liu, and colleagues from the Broad Institute published a paper describing their latest and improved base editing research in the journal Nature. Liu's group genetically transformed base pairs at a target position in the genome of living cells with more than 50% efficiency, with virtually no detectable ‘off-target’ effects such as random insertions, deletions, translocations, or other base-to-base conversions. The work of Liu and his team is significant because it, “introduced point mutations more efficiently and cleanly, and with less off-target genome modification than a current Cas-9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells”. This clears the path for scientists to use base editing to address many more single-letter mutations than was previously possible. “What we’ve developed is a base editor, a molecular machine, that is a programmable, irreversible, efficient, and an extremely clean way to correct mutations in the genome of living cells,” says Liu.
 
Delivery is the challenge

Notwithstanding, Liu suggests that the status of base editing is like Amazon without UPS, its principal delivery agent, “Creating a machine that makes the genetic change you need to treat a disease is an important step forward, but it’s only one part of what’s needed to treat a patient. We still have to deliver that machine”, says Liu, and further, “We have to test its safety, we have to assess its beneficial effects in animals and patients and weigh them against any side-effects. We need to do many more things. But having the machine is a good start.” Liu is hopeful that base editing of DNA and RNA could be used as complementary approaches for a “broad set of potential therapeutic applications.” He and his colleagues are exploring base editing to fix blood and neurological disorders as well as hereditary deafness and blindness.
 
The October 2017 Science study and the advantages of editing RNA
 
In a paper published in the October 2017 edition of the journal Science, Feng Zhang, of the Broad Institute who is one of the original architects of CRISPR, and senior author describes a variant of base editing, which acts on RNA in human cells instead of DNA. RNA acts as a temporary genetic messenger within cells and naturally degrades in the body. This means that editing RNA instead of DNA does not result in a permanent change to a person’s genome, and therefore has significant potential as a tool for both research and disease treatment. Zhang’s base editing technique makes a temporary correction of a disease-causing mutation without permanent alteration to the genome. According to Zhang, editing DNA is, “permanent and very difficult to reverse, which poses a safety concern, while editing RNA is not.” Zhang’s approach is a potentially safer option when it comes to gene-fixing therapeutics, although any treatment using the technique would need to be administered repeatedly. But Zhang believes repetition could be an advantage because it allows for a therapy to be “upgraded” as scientific knowledge increases and provides a better understanding of specific disease states. The system can change single RNA nucleotides in mammalian cells in a programmable and precise fashion and has the ability to reverse disease-causing mutations at the RNA level, as well as other potential therapeutic and basic science applications.
 
Zhang and colleagues made one RNA-editing enzyme into a programmable gene-editing tool. “There are 12 possible base changes you can do,” says Omar Abudayyeh, a researcher at the Broad Institute and one of the paper’s authors. Having edited one, “we’re now thinking about the ways to do the other eleven.” By operating on RNA, Zhang and his colleagues avoid ‘off-target’ effects. “With RNA, you have to think about ‘off-targets’ a little differently.” says Abudayyeh. “If some of the RNA gets edited incorrectly the cell will have at least some amount of the right protein. If things go really wrong, the edit is reversible. You can always remove the system and the RNA will eventually degrade and recycle and revert back to normal,” says Abudayyeh. Liu and his team call their new creation REPAIR. They tested it on human cells growing in dishes and edited up to about 27% of the RNAs of two genes. The researchers did not find any ’off-target’ effects and suggest, “REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology.”
 
Delivery challenges

Liu and other medical researchers have stressed the significant challenges associated with delivering CRISPR technologies, which have yet to be resolved before gene editing techniques become viable therapies. The conundrum researchers face is that your body’s biological barriers, which protect you from diseases are the same barriers that create significant obstacles for the delivery of genetic editors. Let us explain. Your DNA is like Fort Knox gold in that it is extremely well protected. For a harmful agent to access your DNA it first has to get under your skin and into your bloodstream. It then has to travel through your bloodstream without being detected by your immune system, which is comprised of networks of cells, tissues, and organs that work together to protect your body. One of the important cells involved in your immune system are white blood cells, also called leukocytes, which come in two basic types that combine to seek out and destroy disease-causing agents. Assuming the harmful agent successfully gets past all these biological barriers, it then has to penetrate your cell membrane and find a way to the nucleus of the cell. These biological defences help to keep you healthy by preventing harmful agents penetrating and transforming your cells into disease-making entities. But, they are the same obstacles that prevent scientists getting gene editors to the right place at the right time in the right quantity. Although delivery technologies are improving, Crispr Therapeutics, Editas Medicine, and Intellia Therapeutics, as well as, Casebia are all investing in delivery mechanisms, which remain significant challenges to overcome before gene editing becomes a regular therapy.  This is not only a concern for private companies, but also for the public sector. In January 2018 the US National Institutes of Health announced it will be awarding US$190m in research grants over the next six years, in part to “remove barriers that slow the adoption of genome editing for treating patients”.
 
Takeaways

Researchers have made substantial scientific advances in embryo gene editing technologies, which have significant potential for next-generation therapeutics. Base editing, described in this Commentary, is one advance, which has the potential to provide effective therapies for a range of disorders known to be caused by the mutation of a single letter in a gene, which currently have either little or no means of a cure. This is important because about 66% of genetic illnesses in humans involve mutations where there is a change in a single letter (or base). Notwithstanding, before such technologies become regular therapies in clinics there are major technical challenges, which need to be overcome in the delivery mechanism for these gene editors.
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