The field of genetics is a relatively young science, beginning with Gregor Mendel’s studies on the inheritance of traits in pea plants. The term “genetic engineering” was first coined by Jack Williamson in his science fiction novel “Dragon’s Island,” published in 1951. This was a year before DNA’s role in heredity was confirmed by Alfred Hershey and Martha Chase, and two years before the discovery of the double-helix structure of DNA by Franklin, Crick and Watson.
Since then, the concept of editing our genes has fascinated both scientists and popular culture – sci-fiction stories such as Aldous Huxley’s Brave New World (1932), Frank Herbert’s The White Plague (1982), Gattaca (1997), and even Marvel’s X-men comics all explore the concept of gene-editing and mutations in humans, and the ethical, philosophical and social quandaries that follow.
Gene editing is now used almost ubiquitously in scientific research. From the development of knockout mouse models by Mario R. Capecchi, Martin Evans, and Oliver Smithies in 1989, to CRISPR gene editing technology pioneered by Jennifer Doudna, Emmanuelle Charpentier and Feng Zhang in 2012. In this blog post we will be focussing on the explosion of gene editing in scientific research from the 1960s onwards – and explore how those experiments not only pushed the field forward, but how they also held us back.
The early days
By the mid-1960s, the topic of gene editing began to gain prominence. During this time, Stanfield Rogers and his team at the Oak Ridge National Laboratory in Tennessee studied the Shope papilloma virus, which causes warts in rabbits when applied topically to the skin. They found that infection with the virus led to the appearance of an enzyme called arginase in infected cells, which was not present in normal rabbit liver cells. This suggested that the virus genome contained a gene for arginase, which was being introduced into infected cells through a process called transduction.
Other researchers found that the arginase activity in papillomas from infected rabbits was very similar to the endogenous human liver enzyme. This led Rogers and his team to conclude that the virus may induce arginase activity in infected cells, and so Rogers and his team decided to try and modify the RNA genome of tobacco mosaic virus (TMV) by adding poly(A) tails and used the genetically altered TMV genome to infect tobacco plants – the first gene-editing experiment.
Unfortunately, Rogers’ early gene therapy model proved unsuccessful. But despite the failure of these early experiments, Rogers’ work using viruses as vectors for introducing new genetic information into human cells inspired the development of more successful vector systems using other classes of viruses. These vector systems have played a crucial role in the field of gene therapy.
The first recombinant DNA molecules were created in 1973 by researchers at Stanford University and the University of California, San Francisco, modifying genetic material to create desired characteristics in living organisms or their products. This process involves the insertion of DNA fragments containing the desired gene sequence into an appropriate vector which is then introduced into a host organism. This host organism is then grown to produce multiple copies of the incorporated DNA fragment, and clones containing the relevant DNA fragment are selected and harvested.
This technology was used to introduce new genes or regulatory elements into an organism’s genome or to decrease or block the expression of endogenous genes. The regulation and safe use of this technology was discussed at the Asilomar Conference in 1975. Despite initial challenges, this technology has since been used to develop a variety of products, including hormones, vaccines, therapeutic agents, and diagnostic tools, since the mid-1980s.
Insulin: The first consumer GMO drug
In the mid-late 1970s, scientists began to consider the potential of using recombinant DNA technology, to produce human insulin in bacterial cells. The process involved taking the human insulin gene, inserting it into a circle of bacterial DNA called a plasmid, and placing it into bacteria. The modified bacteria would then be grown in large vats of broth and produce pure human insulin, which could be purified and given to patients. However, at the time, researchers did not know the DNA sequence of the insulin gene or even which chromosome it was located on. They only knew the molecular structure of insulin, which had been determined by British biochemist Fred Sanger in 1955.
Herbert Boyer, a biochemist from the University of California, San Francisco, teamed up with Arthur Riggs, a geneticist from the City of Hope National Medical Center, and Keiichi Itakura, an organic chemist and expert in chemical DNA synthesis, to try to synthesize a gene for insulin using the protein’s known structure. The process of chemically synthesizing DNA was slow and involved building the sequence one nucleotide at a time.
Boyer and American venture capitalist Bob Swanson decided to start their own company, Genentech, one of the first biotechnology companies. With investment from Genentech, they pressed on with the project and were able to produce insulin in bacteria for the first time in 1979. They chemically synthesized DNA to create two separate synthetic genes, one for each of the two insulin amino acid chains. These genes were made and purified separately and then combined to produce the full insulin protein.
Unfortunately, the initial yields of insulin were small, so the team at Genentech had to find a way to increase production. They were eventually able to modify the bacteria’s control genes to encourage higher insulin production. The project was then transferred to the pharmaceutical company Eli Lilly for large-scale production. Clinical trials in 1981 proved the safety and effectiveness of this artificial insulin, which was called Humulin. In 1982, it was approved for use in people with diabetes and became the first genetically engineered drug. It virtually eliminated the problems caused by allergies and impurities in animal insulin, which had been used up until this point.
A few years later, after the human insulin gene was discovered and sequenced, production switched to using the actual human gene instead of the synthetic version. Today, human insulin produced through genetic engineering is widely used to treat diabetes and has greatly improved the lives of people with this condition.
The first genetically engineered vaccine: Hepatitis B
The Recombivax HB vaccine for hepatitis B was the first vaccine to be produced using recombinant DNA technology and was approved for human use in several countries in 1986. If left untreated, infection with hepatitis B virus (HBV) can cause severe liver damage and increased risk of developing liver cancer.
The first commercial HBV vaccine, licensed in 1981, was based on inactivated virus collected from the plasma of HBV-infected donors. However, plasma products at the time had been associated with HIV-1 and HCV transmission, and vaccine supply was limited by the availability of chronic HBV carriers. Therefore, the use of recombinant DNA technology was an attractive option for the development of a vaccine that solved both of these problems – and hepatitis B surface antigen (HBsAg) was an attractive target, given that it was encoded by a single gene and thought to be closely involved in interactions with host cells.
In 1979, William Rutter, who had been involved in research on recombinant insulin and growth hormone, Pablo Valenzuela at the University of California, San Francisco, and others, successfully cloned HBsAg into Escherichia coli expression vectors. This demonstrated the possibility of using recombinant HBsAg as an HBV vaccine. They then cloned HBsAg into yeast expression vectors a few years later, in 1982.
The Recombivax HB vaccine was developed using yeast cells that produced HBsAg particles, which were purified and used to immunize chimpanzees. The resulting immune response was similar to that seen in humans infected with HBV. Since then, the Recombivax HB vaccine has been successful in reducing the incidence of hepatitis B and has been used to immunize millions of people worldwide.
Unauthorised gene therapy: treating thalassemia
A few years later, Martin J. Cline, Chief of the Division of Hematology-Oncology at UCLA attempted unauthorized gene therapy treatment of two thalassemia patients, one in Italy and one in Israel, with genetically altered bone marrow cells. Thalassemia is a hereditary blood disorder, caused by faulty haemoglobin synthesis, and can cause anaemia and fatigue. Cline had originally submitted a treatment protocol to two committees at UCLA, one responsible for biosafety and the other, UCLA’s Institutional Review Board (IRB), responsible for overseeing the human experimentation aspects of the proposal. However, in order to bypass the requirement for review by the biosafety committee, Cline altered the experimental design so that the DNA used was in a non-recombinant form. This change was made moot when the IRB rejected the proposal.
After the rejection, Cline arranged to perform the treatments overseas. The Israeli authorities granted approval after confirming with Cline and UCLA that the revised protocol did not involve potentially hazardous recombinant DNA (rDNA). However, after gaining clearance, Cline reverted to the original protocol involving rDNA. When Cline’s deceptions were discovered, various punitive actions were taken by UCLA. Cline’s unauthorized experiments set the gene therapy movement back several years, as they raised concerns about the safety and ethics of gene therapy and prompted a need for more stringent regulations and oversight in the field.
The first success story: Ashanthi DeSilva
Ashanthi DeSilva was diagnosed with an incurable gene-based immune deficiency called adenosine deaminase (ADA) deficiency at the age of two. ADA deficiency is a rare genetic disorder that affects the immune system, making it difficult for the body to fight off infections. This condition was the inspiration for the 2001 film “Bubble Boy” – a story about a man born with “no immune system” who is forced to live his life inside a physical bubble.
To treat ADA deficiency, patients are given regular injections of PEG-ADA, an artificial form of the ADA enzyme. The injections are designed to boost T-cell numbers, which tend to decline in people with ADA deficiency. However, after two years of treatment, Ashanthi’s T-cell count was no longer responding to the injections. Without a breakthrough in the treatment of ADA deficiency, Ashanthi’s prospects were bleak.
In 1990, Ashanthi’s parents connected with William French Anderson, an American geneticist who was lobbying for permission to conduct human gene therapy trials. After a lengthy approval process, Anderson received permission to proceed with the trial in 1990, and Ashanthi’s parents agreed to have her implanted with the corrected gene via a viral vector. The trial was a success, with Ashanthi’s T-cell count going up significantly and her health improving over the following two years. She experienced no significant side effects and was able to attend school and live a relatively normal life.
This was the first gene therapy trial; it was revolutionary and opened the door to the development of other gene therapy treatments for a wide range of genetic and rare diseases. It also sparked significant interest among researchers in the biotech and speciality pharma industries, who saw the potential for gene therapy to transform the way in which incurable and difficult-to-treat conditions were addressed.
A major setback: The death of Jesse Gelsinger
Jesse Gelsinger was a patient suffering from a rare metabolic disorder called ornithine transcarbamylase deficiency syndrome (OTCD). OTCD is a condition in which ammonia builds up in the blood to lethal levels. Babies born with OTCD often fall into comas shortly after birth and suffer brain damage, with half of them dying within a month. Jesse’s milder version of the disorder was diagnosed when he was two years old, and he managed it with a low-protein diet and a regimen of nearly 50 pills a day. However, he still experienced occasional health crises, including a severe reaction to the disorder when he was 17.
In 1999, a doctor told Jesse about a clinical trial for a potential OTCD treatment being developed at the University of Pennsylvania, Jesse was eager to participate. The treatment involved injecting patients with working copies of the OTC gene, which produces an enzyme that prevents ammonia build-up, delivered by an adenovirus vector. The virus, which researchers had thought had been altered to be harmless, would infect the patients’ liver cells and integrate the added gene into their chromosomal DNA. Jesse was the 18th person to receive the modified virus, but he had a much worse reaction than previous patients, who had only experienced flu-like symptoms. Jesse became disoriented, developed jaundice, had an intense inflammatory response, and developed a dangerous blood-clotting disorder followed by kidney, liver, and lung failure. Four days after receiving the shot, Jesse was declared brain dead and taken off life support.
Jesse’s death set off a crisis in the field of gene therapy, with the term gene therapy becoming almost taboo. Biochemist Jennifer Doudna, who later discovered the CRISPR-Cas9 gene-editing mechanism said in an interview, “We were all very much aware of what happened there and what a tragedy that was. That made the whole field of gene therapy go away, mostly, for at least a decade. Even the term gene therapy became kind of a black label. You didn’t want that in your grants. You didn’t want to say, ‘I’m a gene therapist’ or ‘I’m working on gene therapy.’ It sounded terrible.”
Thanks to Jennifer Doudna, Emmanuelle Charpentier, and Feng Zhang, the gene-editing field was brought back to life with the discovery of the CRISPR-Cas9 system in 2012. Their papers published in Science and Nature Biotechnology demonstrated how this revolutionary technology could precisely edit genes, allowing researchers to make changes to an organism’s DNA with unprecedented accuracy and efficiency. CRISPR-Cas9 has since become a widely used tool in biomedical research, agriculture, and industrial biotechnology, opening up new avenues for treating genetic disorders, developing new medicines, and engineering crops with desirable traits.
A modern controversy: Treating thalassemia with gene therapy (again)
In a 2015 paper, Junjiu Huang of Sun Yat-sen University in Guangzhou and his team reported the first instance of editing the genomes of human embryos, which led to many high-profile debates within the scientific community about the ethical implications of such work.
The team used non-viable embryos, which cannot result in a live birth, obtained from local fertility clinics for the study. Non-viable embryos are created for in vitro fertilization but have an extra set of chromosomes following fertilization by two sperm, making it impossible for them to result in a live birth. However, they do undergo the first stages of development. Huang’s team attempted to modify the gene responsible for β-thalassaemia, a potentially fatal blood disorder, using the gene-editing technique known as CRISPR/Cas9.
Huang and his colleagues set out to see whether the repaired gene would be retained in the cells produced during the subsequent stages of embryo development. They injected 86 embryos and waited 48 hours for the CRISPR/Cas9 system and replacement genetic material to act and for the embryos to grow to about eight cells each. Of the 71 embryos that survived, 54 were genetically tested, revealing that just 28 were successfully spliced, and only a fraction of those contained the replacement genetic material.
The study’s results showed significant obstacles to using CRISPR/Cas9 for medical purposes, with a low success rate and a surprising number of “off-target” mutations caused by the CRISPR/Cas9 complex acting on other parts of the genome – a major safety concern, as these mutations could lead to unintended genetic modifications.
The study’s lead author, Junjiu Huang, believes that the low success rate and the off-target effects demonstrate that the technology is not yet ready for use in medical applications and should serve as a warning to any practitioners who think it is ready for testing to eradicate disease genes. George Daley, a stem-cell biologist at Harvard Medical School, also commented on the study, stating that “their study should be a stern warning to any practitioner who thinks the technology is ready for testing to eradicate disease genes.”
The paper by Huang’s team reignited a debate on human-embryo editing, with reports that other groups in China were also experimenting on human embryos. While gene editing in embryos could potentially eradicate devastating genetic diseases before a baby is born, it also raises ethical concerns as the genetic changes, which are germline modifications, are heritable and could have unpredictable effects on future generations. Additionally, there are concerns that any gene-editing research on human embryos could lead to unsafe or unethical uses of the technique.
He Jiankui: The CRISPR baby scientist
In even more recent news, you may have heard about the now infamous scientist He Jiankui, who did not heed Huang’s warnings. His controversial “CRISPR baby experiment” led to his imprisonment in China, but after he was released, he fled to Hong Kong in a desperate bid for freedom. However, just last month it was reported that Hong Kong authorities revoked his visa. This is probably the most dramatic and controversial gene editing tale of our time.
He Jiankui is a Chinese scientist who edited the genomes of human embryos in 2018, sparking a scientific and bioethical controversy. Working at the Southern University of Science and Technology (SUSTech) in Shenzhen, China, He Jiankui edited the embryos’ genomes to remove the CCR5 gene in an attempt to confer genetic resistance to HIV. He announced the birth of the genome-edited babies in a series of five YouTube videos on 25 November 2018, and the first babies, twin girls named Lulu and Nana, were born in October 2018. He was widely condemned by scientists immediately for unethical practices that involved risky procedures without fully informed consent from the families involved.
He edited the embryos’ genome, specifically targeting a gene called CCR5, using CRISPR/Cas9 technology. CCR5 is used by HIV to enter cells, and He aimed to reproduce the phenotype of a specific mutation in the CCR5 gene, known as CCR5-Δ32, which confers resistance to HIV. However, instead of introducing this known mutation, He introduced a frameshift mutation intended to make the CCR5 protein non-functional. He claims that the genetically modified embryos were tested for off-target errors and chimerism, and the resulting babies, Lulu and Nana, were born healthy.
After presenting his research at the Second International Summit on Human Genome Editing at the University of Hong Kong on 28 November 2018, Chinese authorities suspended his research activities the following day. On 30 December 2019, Chinese authorities announced that he was found guilty of forging documents and unethical conduct, and he was sentenced to three years in prison with a three-million-yuan fine (USD 430,000). He’s colleagues Zhang Renli and Qin Jinzhou received an 18-month prison sentence, a 500,000-yuan fine, and were banned from working in assisted reproductive technology for life.
Recently, He Jiankui was granted a visa under Hong Kong’s new talent scheme to attract international professionals to the city. However, the visa was revoked by Hong Kong immigration officials less than a day after it was revealed he had been granted one, due to allegations that he had lied on his application form. A criminal investigation has been launched, and future applicants under the visa scheme must now declare if they have a criminal record, a requirement that did not exist when He submitted his forms.
Since He’s release from prison in April 2022, he has set up an independent laboratory in Beijing focusing on developing affordable therapies for rare genetic diseases, such as Duchenne muscular dystrophy. He has also relaunched himself on to the international academic and media circuit, speaking at a bioethics event at the University of Kent in February 2023. However, his actions in 2018 continue to receive widespread criticism, and the impact of human gene editing on resistance to HIV infection and other body functions in experimental infants remains controversial. The World Health Organization has issued three reports on the guidelines of human genome editing since 2019, and the Chinese government has prepared regulations since May 2019.
Where do we go from here?
“With genetic technology we assume control over the hereditary blueprints of life itself. Can any reasonable person believe for a moment that such unprecedented power is without substantial risk?” – Jeremy Rifkin (prominent leader of the anti-biotech movement and author of Who should play God?)
One disease in which gene-editing has absolutely made concrete and incredible advances is sickle-cell disease. 2021 was a year full of multiple gene-therapy strategies for sickle-cell disease being developed, with several clinical trials demonstrating the therapeutic potential of gene-editing to treat sickle-cell disease. In December of 2020, researchers at Boston Children’s Hospital reported on the success of their virus gene therapy in six sickle cell disease patients treated over 6 months. In June of 2021, a team of researchers from the Broad Institute and St. Jude Children’s Research Hospital demonstrated that a base editor could efficiently correct the sickle cell disease mutation in mice. Speaking on the gene-editing technology CRISPR/Cas9, Selim Corbacioglu, a haematologist at University Hospital Regensburg in Germany said in an article for Nature, “I think that is the cure that eventually closes the book on sickle-cell disease.”
In our interview with Dominique Goodson – sickle cell warrior, advocate and president of the online platform SCDForum – we asked her what she thought about the potential of gene-editing technologies to treat sickle-cell disease. She expressed her optimism, saying “I think these therapies have great potential. It could possibly be that cure that most warriors are looking for. Especially if the research comes back and it actually works, where they are taking your own genes and not someone else’s genes, so your body is not rejecting it or you’re not doing it where you have to do a bone marrow transplant and you might run into the host versus graft disease. It may potentially be better health wise and less complications in the future from it. I think that if this works, it’s going to be amazing for the community because this is a hard condition to live with, just with all the things we have to deal with. Not just with the disease itself, but outside of the disease there is a lot to deal with. And if this is going to be the cure for it to end all the pain that comes with sickle cell and the other crap that comes with it too, it’s going to be amazing – it’s going to be something that will change the community forever.”
Clearly, gene editing has come a long way since its early beginnings in the 1960s and 1970s. From the development of recombinant DNA technology and vector systems for gene therapy to the Nobel Prize-winning discovery of CRISPR gene editing, the field has made significant progress in the understanding and manipulation of genetics. While gene editing has the potential to cure genetic diseases and improve human health, it also raises ethical concerns about its potential for unintended consequences and abuse. As the field continues to advance, it will be important to carefully consider the ethical implications of gene editing and ensure that it is used responsibly and for the benefit of society.