How Does Gene Therapy Work?
Gene replacement therapy targets inherited disease at the root cause—your DNA—by inserting healthy genes into malfunctioning body cells.
What is Gene Therapy?
Gene therapy uses viral or synthetic vectors to deliver healthy sections of DNA into cells. The new DNA integrates with the genome and instructs cells how to make specific proteins.
By fixing up genetic mutations, gene therapy has the potential to cure hundreds of serious diseases. We're talking cancer, heart disease, diabetes, cystic fibrosis, haemophilia, and AIDS.
Let's look a bit closer at the rationale. Your DNA lives inside every cell of your body, bound up into 46 chromosomes. These chromosomes contain around 20,000 genes, which each provide the instructions your cells need to make life-sustaining proteins.
In health, your cells make around 20,000 different proteins which keep you alive on a moment-to-moment basis. Proteins like:
- Insulin - helps turns glucose into energy
- Haemoglobin - transports oxygen in the blood
- Neurexin - connects neurons at the synapse
In short, your body needs to make a constant supply of proteins to stay alive. But if you inherit a faulty protein-coding gene, your cells are left stumped.
The lack of an essential protein starts a chain reaction that manifests in chronic disease.
Sometimes, a disease-mutation inherited from one parent isn't a problem. You still have a working copy of the gene from your other parent, rendering you a carrier of disease. But faulty genes coming from both parents can lead to catastrophic failure.
Take cystic fibrosis. It's caused by a single genetic mutation that affects the production of a protein called CFTR. As a consequence, chloride isn't moved around cells, water isn't attracted to the cell surface, and organ mucus becomes thick and sticky. This has disastrous effects on the lungs.
There are two main types of gene therapy:
- Somatic Gene Therapy. This is the most common definition of DNA therapy. It targets a subset of relevant body cells to treat disease in an individual patient. The majority of therapies are still in clinical development.
- Germline Gene Therapy. This targets eggs and sperms (germ cells) to prevent disease being on passed to future offspring. It's illegal in most countries because of the unknown risks it poses to future generations.
Technically, genetic vaccines are a type of gene therapy, because they introduce new genes into body cells.
However, DNA and RNA vaccines target only brief viral protein production, for a few days at most.
This contrasts with somatic gene therapy, where the new genes enter the nucleus, integrate with the host DNA, and produce human proteins for life.
The idea is elegant in theory, curing disease at the fundamental level. And yet, after 30 years of clinical trials, gene therapy is still in its infancy. Aspects of the execution have been described as "crude" and, as we'll see in a moment, vulnerable to catastrophic failure.
Even so, experts believe gene therapy will become a staple of 21st century medicine.
The First Gene Therapy Trial
In the 1980s, Ashanthi DeSilva was born without the ability to make a protein called ADA. She'd inherited faulty ADA genes from both her mother and father, leaving her with a critical shortage.
No-one knew this at the time. At birth, Ashanthi seemed like a healthy baby. But as she became exposed to common bacteria and viruses in the environment, her deficiency became manifest.
"Ashi had her first infection at just two days old. By the time she was walking, she was constantly hacking and dripping with coughs and colds." - Ricki Lewis, The Forever Fix: Gene Therapy and The Boy Who Saved It
The protein ADA plays a crucial role in our resistance to infections. Without it, Ashanthi was unable to produce healthy white blood cells, and her body quickly became overwhelmed by viruses and bacteria.
Most infants with ADA deficiency don't survive past their second birthday. Ashanthi was lucky; she was diagnosed with Severe Combined Immunodeficiency (SCID) and put on ADA protein replacement therapy, sourced from cows and infused into her bloodstream on a routine basis.
But for Ashanthi, the protein replacement therapy was only partially effective. Still, it kept her alive until she was four years old and recruited for the world's first gene therapy trial.
How Does Gene Therapy Work?
Just like the genetic vaccines against COVID, gene therapy needs a vector—a protective shell that can carry the new DNA into cells. Since viruses evolved to do this naturally, they can be modified and repurposed to deliver healthy genes right into the nucleus.
Step 1. Harvest target cells from the patient. Ashanthi's doctors drew a sample of malfunctioning white blood cells and replicated them in culture to scale the operation.
Step 2. Insert the therapeutic transgene into a viral vector. Healthy ADA-coding genes were inserted into the DNA of the viral carrier, while key viral genes were knocked out to neutralise the virus.
Step 3. Mix the modified virus with the cell culture. With its biological entry codes, the virus transfects the cells, sneaking its genetic payload into the nucleus for DNA integration.
Step 4. Return the cell culture to the patient. With the new genes on board, the cells are returned to the patient. Ashanthi received an infusion of a pint or so of murky fluid containing 10 million edited white blood cells.
The very first gene therapy trial was a success. Ashanthi's modified white blood cells began to produce ADA immediately, enabling her to make functional antibodies. The edited cells also went on to replicate with the new genes intact, providing a long-term benefit.
Although she needed 10 more infusions, Ashanthi suffered no side effects, and was cured of SCID over the next two years. Today, she's alive and well, married and with a Masters in Public Policy.
Ashanthi's treatment prompted researchers to treat newborn babies with ADA deficiency in the same way. They even took white blood cells straight from the umbilical cord, enabling the infants to produce healthy immune cells from the start of their lives.
That was more than 30 years ago. At the time, you'd be forgiven for thinking that gene therapy was on a fast-track to success, that we'd soon see it curing all kinds of gene-driven diseases. But you'd be wrong.
The Breakthrough Myth
When it comes to invention, Hollywood has taught us to imagine the lone scientist working late at night, hunched over a microscope. Suddenly, a test tube starts fizzing and he's got his winning formula.
The unsexy reality is it often takes many teams of researchers decades to develop new treatments and introduce them safely into widespread use.
When the first bone marrow transplant was performed in 1957, the survival rate was less than 1%. It was 30 years before immune suppressing drugs emerged, bringing the survival rate to 35-90% depending on the donor.
Chemotherapy was another slow burner. Chemotherapy was invented when, during post-World War I autopsies, doctors found that soldiers poisoned with mustard gas had too few white blood cells. Even though the gas was a harmful toxin, it prompted researchers to investigate if it could reduce the excess white blood cells in leukaemia patients.
The first chemotherapies using mustard gas were introduced in the 1940s. However, toxicity issues persisted, and it was several decades before mustard gas became a viable treatment for leukaemia.
Gene therapy, which must be safely customised for each new target disease, is turning out to be no different. Although some 4,000 patients received gene therapy in the decade after Ashanthi's successful treatment, the introduction of new protocols revealed the fatal risks of gene therapy.
When Gene Therapy Goes Wrong
As gene therapy trials scaled up, the use of different viral vectors dealt some massive blows. Here are some of the most famous instances of gene therapy trials going horribly wrong.
In 1999, Jesse Gelsinger was an 18-year-old with a partial OTC deficiency, meaning he couldn't digest protein in his diet. Most babies born with the full-blown deficiency suffer excessive build-up of ammonia and half of them die within a month.
Jesse's partial deficiency meant he could manage his condition with a low-protein diet and nearly 50 pills a day. He signed up to a gene therapy trial, not because he could be cured (he was told the effects would be transient) but to help babies born with the fatal form of the disease.
The trial was designed to test the adenovirus 5 (Ad5) vector for gene delivery in vivo, where cells are transfected inside the body.
The therapy was untested in humans, with the exception of the prior 18 trial participants. As the eighteenth and final patient in the trial, Jesse received the highest dose of the virus: 3.8x1013 (380,000,000,000,000) viral particles, infused directly into his liver.
Jesse had a violent immune response to the Ad5 vector. His immune system produced fever, blood clots, and widescale inflammation. Brain damage and organ failure ensued, and four days later, he died.
An FDA investigation found multiple factors in the death of Jesse Gelsinger. As a result of high ammonia levels from his OTC deficiency, Jesse had inadequate liver function, which should have excluded him from treatment. And two previous participants had suffered serious reactions to the adenovirus, which should have stopped the trial in its tracks.
Then there was the stark failure of informed consent. Jesse hadn't been told that in animal studies using a first generation vector, two lab monkeys had died from blood clots and liver inflammation. When Jesse's family later sued the trial organisers, they quickly settled out of court.
No matter how promising the technology, if trials are poorly designed and executed, the results can be devastating.
Even with a good design, clinical trials are inherently dangerous—that's why trials are necessary. Yet the warning signs in Jesse's case makes his death an even crueller tragedy.
The 1999 incident led to tighter regulations for gene therapy. Yet Jesse was not the last to suffer from this experimental treatment, and three years later, gene therapy hit the headlines again for the worst possible reasons.
The SCID Kids
"Most gene therapies are designed to achieve permanent or long-lasting effects in the human body, and this inherently increases the risk of delayed adverse events." - Gene Therapy Needs a Long Term Approach, Nature
In 2002, a gene therapy trial in France sought to treat babies suffering from X-SCID, an X-chromosome form of Ashanthi's disease. Initially, the infants responded well and the trial was deemed a success.
But in the years that followed, 4 out of 9 children developed leukaemia, a cancer leading to the excessive production of abnormal white blood cells.
It was a baffling result. The FDA temporarily halted all gene therapy trials in the US until the cause could be identified.
Research revealed the adenovirus vector had inserted the healthy genes alongside cancer-causing genes. The oncogenes were activated, causing leukaemia to develop.
It's pretty damning when your experimental therapy starts giving children cancer. The episode was widely publicised, and leukaemia claimed the life of one of the children.
Gene therapy was halted in its tracks. On examination of the cells of gene therapy recipients, several multistep mechanisms were found in which DNA insertions can activate harmful genes, or block the action of beneficial tumour-suppressing genes.
An independent investigation concluded that all integrating gene delivery models present the risk of such insertional mutagenesis.
It was a major blow to the field. The team raced to find a solution, not least because children were still dying of X-SCID, but the lead researcher himself was dying of cancer. Soon, they found success adding genetic insulators to the therapeutic DNA.
Gene therapy trials resumed with the addition of such safety features, and the evolution of gene therapy continued.
Gene therapy saw major successes in the years that followed, saving the lives of numerous children born with rare inherited disorders. Here's one such example.
When Amy and Brad Price decided to have children, they had no idea they were both carriers of a single defective gene called ARSA. This produced a 1 in 4 chance that their offspring would develop metachromatic leukodystrophy (MLD), a degenerative disease that destroys the brain's white matter, quickly leading to paralysis, dementia, and death.
The first time they heard about MLD, they already had two children. One day, their three-year-old daughter, Liviana, began showing distressing symptoms. "Mommy," she said one day, "my legs don't work."
Liviana was diagnosed with MLD, but, being symptomatic, was well past the window for treatment. Testing of her baby brother, Giovanni, showed he had also inherited the disease.
This is a common pattern in MLD. Families are only alerted to the danger when an older sibling begins to show symptoms.
Without an early diagnosis, most firstborns with MLD pass away by five years old. But it does provide a vital warning sign so that gene therapy can save their younger siblings.
This is exactly what happened to the Prices. "Without her we wouldn't have known," said their mother, Amy. "Liviana was given to us to save Giovanni."
In 2011, Italian doctors drew stem cells from Giovanni and transfected them with an HIV viral vector carrying replacement ARSA genes. Five days later, the modified cells were returned to his body, and he was able to start making his missing enzyme.
Ten years later, Giovanni has well surpassed the life expectancy for infantile MLD, and by all appearances, is cured.
Brad and Amy Price have since had six more children, and when another daughter, Cecilia, was diagnosed with MLD, she too benefited from the life-saving gene therapy.
Cases like this provide hope to families in the face of rare childhood diseases. In recent years, gene therapy trials have successfully treated SCID, haemophilia, leukaemia, and blindness caused by retinitis pigmentosa.
The Unknown Unknowns
Some gene therapy trials produce confounding results, and it can be years before the explanations become clear. As we saw with Jesse Gelsinger, promising gene therapies can become lethal when the dosage is scaled, and this continues to block the technology today.
Successful gene therapy also depends on the type of viral vector used. Today, most trials use one of four viral families.
As of 2021, a question mark hangs over the safety of widely used adeno-associated virus (AAV) vectors. Celebrated for their ability to cross the blood-brain barrier (and circumvent the need to drill into the skull to treat neurological disorders), AAVs have produced major wins—and major fails.
In 2014, one major win was a pilot study that tested AAV9 on 15 infants with spinal muscular atrophy (SMA). The virus proved highly effective at spreading through neural tissues to deliver genes to the brain and spine. The infants responded well to the treatment and the trial was deemed a success.
But by 2018, the same AAV9 vector began causing severe toxicity issues in animal studies. As doses were increased, the usually harmless virus caused severe liver and motor neuron damage, and some animals were euthanised. Researchers were unable to link the reaction to the viral itself, yet a further trial using different genes produced similar results.
"This is a big deal, potentially... [But] the community should not overreact." - Terence Flotte on Severe Toxicity in Nonhuman Primates and Piglets with Systemic High-Dose Administration of Adeno-Associated Virus Serotype 9–Like Vectors
In a technology as unpredictable as gene therapy, overreaction may well be justified. The FDA thought so, and effectively halted a human trial using high doses of AAV9 for muscular dystrophy (MD) as a precaution.
Three years later, the FDA's investigations into AAV9 toxicity are ongoing. Explanations range from DNA damage, to stress on the endoplasmic reticulum (a cell organelle used in protein synthesis), to overexpression of transgenes in liver cells.
Since then, an 18-month follow-up study has revealed AAV9 toxicity in human trials. In a study of children with Batten disease, the viral vector AAVrh10 caused acute side effects, as well as chronic lesions in the brain.
In all, 149 gene therapies using AAV vectors have seen serious adverse events occurring at a rate of 35%, with some resulting in deaths of participants.
The FDA will put its safety concerns to gene therapy developers in September, with the key question in mind: should there be a limit on AAV dose in gene therapy?
Meanwhile, 2021 saw the emergence of a new threat: for the first time in gene therapy history, a study using a lentivirus vector led to three participants developing early-stage cancers. The phase III trial targeting children with cerebral adrenoleukodystrophy (CALD) was halted by the FDA and is now under investigation.
It's possible that the lentivirus isn't to blame, but rather the unique addition of a promoter region having especially broad activity. Lentiviruses have been used to treat 300 gene therapy patients, targeting a dozen different conditions. If developers can untangle the mechanism, lentiviruses may still be in the clear.
Each new gene therapy poses complex challenges, with a unique set of protocols designed around the ideal vector, dosage, and safe DNA insertion site. Key risks include an overactive immune response, gene expression in the wrong cell types, and insertional mutagenesis.
For some, the answer to viral vectors is to use synthetic vectors. Non-viral vectors come in various forms: polymers, lipids, and ultrasound-mediated microbubbles. These technologies can be less effective at delivering genes, but they do appear safer and cheaper while presenting no DNA size limit.
In 2020, Pfizer and Moderna used lipid nanoparticles (LNPs) as the genetic vector in their mRNA vaccines against COVID. There's now a wealth of observational and clinical data available on using LNPs in humans.
But the gene delivery rate of LNPs may be insufficient for some gene therapy targets. And there's a limit on LNP dose too: in mRNA vaccines, lipid nanoparticles generate low level inflammation which kick starts the immune response. But this isn't what you want in gene therapy, especially when treating immunocompromised kids.
The US now performs two-thirds of gene therapy trials in the world, and by 2025, the FDA is expected to approve 10-20 new gene therapies per year.
The march of progress continues. Besides single-gene mutations that cause rare childhood disease, gene therapy is now being targeted at widespread ailments: cancer, cardiovascular, infectious, and inflammatory diseases.
This changes the risk-benefit ratio considerably. Children with months to live are justifiably put on last-ditch experimental treatments. But should adults with chronic disease risk everything for a cure?
The risk-benefit problem not unique to emerging gene therapies. It's what many people, including trial participants, don't fully recognise. And until gene therapies pass the test of time, they will continue to pose gambles in the steady search for cures.