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4 years, 12 months ago

For better or worse we all now live in CRISPR’s world

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  • Prime editing devised by researchers at the Broad Institute led by David Liu is a significant advance of the original CRISPR gene editing tool discovered in 2012
  • CRISPR can cut and edit your DNA to correct defects inside your body’s cells to prevent and heal a range of incurable diseases and has revolutionized biomedicine
  • The original CRISPR is fraught with inaccuracies referred to as off target effects
  • Prime editing substantially reduces CRISPR’s off target effects and has the potential to correct up to 89% of known disease-causing genetic variations
  • CRISPR also has the capacity to edit genes in an embryo in such a way that the change is heritable
  • In 2018 Chinese researcher He Jiankui “created” the world’s first CRISPR babies
  • This triggered international criticism from scientists and bioethicists
  • A principal concern is that CRISPR is easy-to-use, cheap, regularly used in thousands of laboratories throughout the world and there is no internationally agreed and enforceable regulatory framework for its use
 
For better or worse we all now live in CRISPR’s world
 
In 2012 the world of biomedicine changed when a revolutionary gene editing technology known as CRISPR-Cas9 (an acronym for Clustered Regularly Interspaced Short Palindromic Repeats) was discovered. The technology harnesses your body’s naturally occurring immune system that bacteria use to fight-off viruses and has the potential to forever change the fundamental nature of humanity. Since its discovery CRISPR has been developing at lightning speed primarily because it is simple and affordable and today is used in thousands of laboratories throughout the world.
 
In this Commentary
 
In this Commentary we describe prime editing, which is the latest advance of the CRISPR's tool box, devised bya team of researchers, led by Andrew Anzalone, a Jane Coffin Childs postdoctoral fellow from the Broad Institute of MIT and Harvard and published in the October 2019 edition of Nature. Prime editing is significant because it provides a means to eliminate the unintentional consequences of CRISPR and therefore bring the technique closer for use in clinics. But this is still a long way off.
 
We also review a case where an ambitious scientist “created” the first CRISPR babies. This immediately triggered international criticism and a call for tighter regulatory control of the technology. Scientists and bioethicists are concerned that CRISPR can easily be used to create heritable DNA changes, which ultimately could lead to ‘designer babies’.
 
These two accounts of CRISPR might seem “opposites” and not sit well together in a single Commentary. Notwithstanding, what prompted putting them together was John Travis, the News Managing Editor of the well-known scientific journal Science, who soon after CRISPR’s discovery in 2012  said, “For better or worse we all now live in CRISPR’s world”. 
 
CRISPR and your DNA

CRISPR is different to traditional gene therapy, which uses viruses to insert new genes into cells to try and treat diseases and has caused some safety challenges. CRISPR, which avoids the use of viruses, was conceived in 2007 when a yogurt company identified an unexpected defence mechanism that its bacteria used to fight off viruses. Subsequent research made a surprising observation that bacteria could remember viruses. CRISPR has been likened to a pair of microscopic scissors that can cut and edit your DNA to correct defects inside your body’s cells to prevent and heal a range of intractable diseases. The standard picture of DNA is a double helix, which looks similar to a ladder that has been twisted. The steps in this twisted ladder are DNA base pairs. The fundamental building blocks of DNA are the four bases adenine (A), cytosine (C), guanine (G) and thymine (T). They are commonly known by their respective letters, A, C, G and T. Three billion of these letters form the complete manual for building and maintaining  your body, but tiny errors can cause disease.  For example, a mutation that turned one specific A into a T results in the most common form of sickle cell disease.
 
The original CRISPR
 
The original CRISPR tool, which is the first and most popular gene editing system, uses a guide RNA (principally a messenger carrying instructions from your DNA for controlling the synthesis of proteins) to locate a mutated gene plus an enzyme, like Cas9, to cut the double-stranded gene helix and create space for functioning genes to be inserted. However, a concern about CRISPR is that the editing could go awry and cause unintended changes in DNA that could trigger health problems. Findings of a study published in the July 2018 edition of  the journal Nature Biotechnology found that such inaccuracies, referred to as off-target effects, were substantially higher than originally reported and some were thought to silence genes that should be active and activate genes that should be silent. These off-target effects, such as random insertions, deletions, translocations, or other base-to-base conversions, pose significant challenges for developing policy associated with the technology.

Subsequently however, the paper was retracted, and an error correction was posted on a scientific website. Contrary to their original findings, the authors of the Nature Biotechnology paper restated that the CRISPR-Cas9 gene editing approach, "can precisely edit the genome at the organismal level and may not introduce numerous, unintended, off-target mutations".
 
Base editing

Notwithstanding, researchers remained concerned about CRISPR’s off target effects and several devised a technique, referred to as base editing, to reduce these. Base editing is described in three research papers published in 2017: one in the November edition of the journal ‘Protein and Cell’, another in the October edition of ‘Science’ the and a third by researchers from the Broad Institute, in the October edition of the journal ‘Nature’. Base editing takes the original CRISPR-Cas9 and fuses it to proteins that can make four precise DNA changes: it can change the letters C-to-T, T-to-C, A-to-G and G-to-A. The technique genetically transforms base pairs at a target position in the genome of living cells with more than 50% efficiency and virtually no detectable off-target effects. Despite its success, there remained  other types of point mutations that scientists wanted to target for diseases.

 

Prime editing
 
Prime editing is different to previous gene editing systems in that it uses RNA to direct the insertion of new DNA sequences in human cells. According to David Liu,  the senior author of the 2019 Nature paper and a world renowned authority on genetics and next-generation therapeutics, “a major aspiration in the molecular life sciences is the ability to precisely make any change to the genome in any location. We think prime editing brings us closer to that goal”.  Because prime editing provides a means to be more precise and more efficient in editing human cells in a versatile way, which eliminates many of CRISPR’s unintentional errors, it significantly expands the scope of gene editing for biological and therapeutic research.
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There are around 75,000 different mutations that can cause disease in people and prime editing has the potential to correct up to 89% of known disease-causing genetic variations. According to Liu, "Prime editing is the beginning, rather than the end, of a long-standing aspiration in the molecular life-sciences to be able to make any DNA change in any position of a living cell or organism, including potentially human patients with genetic diseases". Liu’s team at the Broad Institute intends to continue optimizing prime editing. In their October 2019 Nature paper researchers reported that they can precisely correct mutant genes, which cause sickle cell anaemia and Tay Sachs disease.
 
Sickle cell anaemia and Tay Sachs
 
Sickle cell anaemia is an inherited form of anaemia. This is when there are not enough healthy red blood cells  (haemoglobin) to carry adequate oxygen throughout your body. The condition is the most common inherited blood disorder in the US, affecting 70,000 to 80,000 and further it is estimated  each year some 300,000 babies are born with the disorder worldwide. Tay-Sachs disease is a rare and fatal nerve condition often caused by the addition of four extra letters of code.  Although anyone can be a carrier of  the disease it is much more common among people of Ashkenazi (Eastern European) Jewish descent. In the Ashkenazi Jewish population, the disease incidence is about 1 in every 3,500 new-borns and the carrier frequency is 1 in every 29 individuals.
 
Some moral and ethical implications of CRISPR
 
Being able to modify your DNA with CRISPR tools has transformed scientific research and is revolutionising medicine although it will be some time before the technology is regularly used in clinics. In addition to its potential benefits there are significant moral and ethical challenges associated with the technology, especially when it is used for germline engineering, which is the process by which your genome is edited in such a way that the change is heritable. Inappropriate use of germline editing could dent the progress of the CRISPR technology.
 
The first CRISPR babies
 
One well publicized  inappropriate use of CRISPR is a team in China, led by He Jiankui of the Southern University of Science and Technology in Shenzhen, which in November 2018 “created” the first gene edited twins, known by their pseudonyms Lulu and Nana. He edited the twins’ cells to be immune to HIV infection when they were embryos, therefore ensuring that every cell in their bodies were changed, including their reproductive ones, which means their edited genomes can be passed on to their children and grandchildren, despite the fact that scientists cannot be sure what the long term effects of such lasting modifications might be. The twins are the first CRISPR babies and the first humans to have every cell in their body genetically modified using the technology.
 
In 2015 Chinese researchers were the first to edit the genes of a human embryo in a laboratory dish. Although the embryos did not go to term, the experiment triggered an international outcry from bioethicists, who argued that CRISPR should not be used to make babies. Notwithstanding, He Jiankui did just this.
 
He  employed CRISPR to alter a gene in IVF embryos to disable the production of an immune cell surface protein, CCR5, which HIV uses to establish an infection before insemination. CCR5 is a well-studied genetic mutation, and there is scientific and medical value in understanding how CRISPR can be used to disable and prevent HIV/AIDS. He believed that the use of CRISPR technology was medically appropriate and expected his experiment, “to produce an IVF baby naturally immunized against AIDS”. But more contentiously, He created twins who could pass the protective mutation to future generations. It is CRISPR’s ability to easily and cheaply edit human embryos, eggs, or sperm in order to create irrevocable changes and the potential for designer babies, which raises concerns.  
 
He defended his work at a Hong Kong genomics conference in late November 2018, but there was immediate and significant international criticism about the scientific and ethical legitimacy of his experiments, which broached China’s guidelines as well as international ethical and regulatory norms. A Chinese government investigation found He to have violated state law in pursuit of “personal fame and fortune”.  His endeavours cost him his university position and the leadership of a biotech company he founded, which had successfully raised US$43m start-up capital and was advised by Craig Mello, professor  of the University of Massachusetts Medical School and Nobel Laureate for medicine in 2006 for his genetics research.
 
Opacity and scientific competition
 
Some scientists are reluctant to be critical of He and suggest his studies, which resulted in the first CRISPR babies,  simply signal the “next chapter in the technology’s story”. He Jiankui appears to be an ambitious scientist desperate to become the first to conduct the gene editing experiment on humans, but who made some significant errors of judgement by initiating his study prematurely and by withholding information from regulatory authorities and his university. A generous interpretation might suggest that He was motivated by science and humanity. Through a Beijing-based organization, which helps Chinese people with HIV, he recruited couples for his experiment where only the fathers were living with HIV infections, which they managed by antiviral drugs. Eight couples agreed to participate, although one subsequently withdrew.
 
Since He’s statement at the Hong Kong conference he has disappeared, but the background to his studies has been well documented. In late 2017, He, who specialized in sequencing DNA, began his efforts to produce human babies from gene edited embryos and before and during his study it is reported that he sought advice from international experts in the field and communicated openly with international colleagues about his plans. Notwithstanding, it is alleged that He faked a blood test for one of the fathers in the study, aware that in China the HIV status of the father would disqualify him from participating in fertility treatments. Also, He failed to appropriately inform the hospital where the twins were edited and implanted of the status of his experiments.

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Fierce competition among scientists is not uncommon and competition fuels opacity among scientists in their battle to become the first to make a discovery. Indeed, it is not uncommon for scientists to shield their ideas and research. This does not condone He’s actions, but it might help to explain them. Generally speaking, scientific opacity is not created by ambitious scientists alone, but it is partly created by scientific funding bodies and research institutions. Such opacity is a significant obstacle to open collaboration. In addition to wanting to be the first, He’s intentions might also have been an attempt to spare children of parents with HIV/AIDS  from inheriting the disease.
CRISPR is not yet safe
 
Be that as it may, many scientists agree that CRISPR is not yet safe and precise enough to be used in human embryos. In the March 2019 edition of Nature a group of 18 prominent CRISPR scientists and bioethicists from seven countries called for a global moratorium on heritable genome editing until the establishment of an international framework that would compel countries to establish both scientific safety and broad societal agreement before allowing the technology to progress.  "We call for a global moratorium on all clinical uses of human germline editing; that is, changing heritable DNA (in sperm, eggs or embryos) to make genetically modified children" , the scientists wrote.

Opposition to germline editing is mixed
 
However, opposition to germline editing is mixed. In February 2017 the US National Academies of Sciences, Engineering, and Medicine (NASEM) published a report, which did not call for an international ban of germline editing, but instead suggested that it "might be permitted" if strict criteria were met. In July 2018, the UK’s Nuffield Council of Bioethics published a report on heritable genome editing and suggested that under certain circumstances it could be morally permissible, even in cases of human enhancement. 

Given that CRISPR is cheap, easy-to-use and already an effective tool in thousands of laboratories throughout the world, it seems reasonable to assume that standards and laws are unlikely to prevent a determined scientist and desperate patients from using the technology prematurely. Indeed, science and medicine have a history of researchers attracting public criticism for undertaking experiments prematurely only to have those experiments become common medical practices: in-vitro fertilization  (IVF) is one such example. Although IVF has a chequered history today it accounts for millions of births worldwide and  1% to 3% of all births every year in the US and Europe.
 
Germline engineering and somatic genetic modification
 
Here we describe the difference between germline and somatic adjustments. The former uses CRISPR to modify DNA in such a way that the change is heritable. The latter uses CRISPR to modify the DNA of people with incurable diseases in a way that such modifications are limited to the people treated and not passed on to future generations. Broadly speaking, your body has two kinds of cells: somatic and germ cells. The vast majority are somatic. These cells make up your body and are responsible for forming all your familiar structures: such as your skin, blood, muscles and organs etc. Your somatic cells die when you die so there is no chance of them creating a new organism. However, germ cells are different. Early in your development your germ cells  are sequestered: they divide more slowly and under restricted circumstances. Germ cells cannot become a physical feature such as an ear or a finger, but they do make the only bits of you, which can form a new person: your eggs and your sperm. Every cell in your body holds your DNA in an unbroken lineage stretching back millions of years and thousands of generations, but only the germline has a chance to go forward. Human germline modification means deliberately changing the genes passed on to children and future generations and thereby creating genetically modified people. Somatic genetic modification is different. It adds, cuts, or changes the genes in some of your cells, typically to alleviate a medical condition. The use of human genome editing to make edits in somatic cells for purposes of treating genetically inherited diseases is already in clinical studies. If perfected, somatic gene editing (gene therapy) holds promise for helping people who are sick, affecting only an individual consenting patient. With the exception of He’s studies, human clinical studies with CRISPR have been limited to somatic cells. In effect, this renders CRISPR no more consequential than any other experimental drug or treatment. Any CRISPR-made somatic cell changes are a genetic dead-end and are not heritable. However, germline cells have the possibility of immortality, with the potential to affect thousands of people over the course of several generations. Tampering with germline cells is therefore a much more serious proposition.
 
Clinical studies of gene therapies
 
Gene therapy is primarily available in a research setting. The US Food and Drug Administration (FDA) has approved only a limited number of gene therapy products for sale in the US.According to the US National Institutes of Health, which serves as a clearinghouse for biomedical research worldwide, there are over 800 clinical studies currently underway to test gene therapy as a treatment for genetic conditions. The list includes a relatively small number of CRISPR studies as a treatment for cancers of the lung, bladder, cervix and prostate, the majority of which are in China where doctors appear to be leading the race to treat cancer by editing genes. For the past two decades China has been investing heavily in biomedicine. It is one way that China is able to compete with the West and demonstrate its technological prowess in the 21st century. Also, it is important for China to keep its vast population healthy in the 21st century. Given the somewhat ambiguous state of CRISPR technology it seems reasonable to assume that the first therapeutic applications of CRISPR will be in diseases where cells can be taken out of your body, edited, checked to ensure they are safe and then reintroduced. This suggests blood disorders such as sickle cell or thalassemia.
 
Takeaways
 
Bioethicist Henry T (Hank) Greely, professor at Stanford University, California, US, compares CRISPR to the Model T Ford, which was not the first automobile, but because of its simplicity of production, dependability and affordability it transformed society. CRISPR is not the first gene editing technology, but it is cheap and easy to use and is on the cusp of transforming biomedicine. A significant challenge is getting CRISPR tools, which are capable of performing gene edits, into the right place and to ensure they are safe. Prime editing is a smart, innovative and a substantial step forward in achieving this. Indeed, David Liu and his colleagues from the Broad Institute  have expanded the gene editing toolbox to facilitate ever-more precise editing ability and efficiency. Significantly, the overwhelming majority of human genetic disorders are due to the types of mutation that prime editing is able to correct, which stands the technique in good stead to be useful in therapies for intractable diseases. Notwithstanding, it is one thing to cut out sequences of DNA that cause genetic diseases and another to make genetic changes that are passed down to all later generations. Because CRISPR is cheap, easy-to-use, in the hands of scientists throughout the world, and already has been used to create babies with heritable traits, the technology provokes deep ethical and societal debate about what is, and what is not acceptable in efforts to prevent disease. Given that CRISPR has the potential to change the nature of humanity, it is incumbent on all citizens, not just scientists, bioethicists and regulators, to call for open and inclusive processes associated with all aspects of CRISPR.
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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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