How Does Gene Therapy Work?

How Does Gene Therapy Work?

What is Gene Therapy?

DNA is a complicated beast. We still don't know what most of it does. However, we do know that less than 2% of our DNA encodes the recipes for life-sustaining proteins. And when one of these recipes is faulty, we see the physical manifestation of disease.

Faulty genes are implicated in more than 6,000 disorders—including cancer, heart disease, and diabetes—with more described in the literature every year. And while some mutations are present since birth, other changes are triggered by interactions with our environment.

Gene therapy is a single or multi-dose treatment that replaces the disease-causing DNA in target cells. The new genes can integrate fully as chromosomal DNA or persist separately as episomal DNA, both of which remain in the cell nucleus.

Gene therapy re-writes tiny parts of our source code so we can produce essential proteins, preventing a cascade of malfunctions that otherwise result in serious disease.

Since 1990, gene therapy has been targeted at rare childhood diseases caused by single-gene mutations. The scope was gradually expanded over the course of more than 3,000 clinical trials. Today, this "living drug" is now used to treat certain cancers and metabolic disorders, as well as neuronal, immune, and infectious diseases.

In time, gene therapy could widely replace chemotherapy and radiotherapy—treatments which neither cure nor prevent the genetic errors that cause cancer, but often produce many side effects.

The scope for gene therapy is huge: almost every cell in the body contains protein-coding genes that are critical to their function.

But what are the risks of introducing new DNA into the body? Let's look a bit closer at the biology.

The Structure of DNA

Your DNA lives inside the nucleus of almost every cell in your body, presenting under the microscope as thin strands of chromatin. If we were to zoom in, we'd see these fine strands are actually the DNA double helix: a twisted ladder whose rungs are made of bases (A, C, G, T). Genes are made of thousands of bases in sequence.

When cells divide, DNA coils up into 46 chromosomes to prevent it from tangling or becoming damaged. This is a good opportunity to see how bases, genes, chromosomes, and DNA relate to each other.

Illustration of how genes coil into chromosomes

The DNA alphabet has only four letters—adenine (A), cytosine (C), guanine (G), and thymine (T)—which, in their thousands, produce entire genes stored in the double helix.

The Human Genome Project revealed we have ~20,000 genes which encode ~20,000 proteins. Examples of proteins include haemoglobin, antibodies, and hormones.

Types of Gene Therapy

Faulty genes can lead to minor or catastrophic failure.

Consider cystic fibrosis, which is often caused by a nonsense mutation. An early stop signal (made up of just three bases) means the genetic sequence is cut short and cells can no longer produce the protein CFTR.

Without CFTR in certain cells, chloride isn't moved around correctly, water isn't attracted to the cell surface, and organ mucus becomes thick and sticky. This causes progressive damage to the lungs and digestive system.

There are two main types of gene therapy. Like all gene therapies in the US, the treatment for cystic fibrosis uses somatic gene therapy:

  • Somatic Gene Therapy replaces DNA in target body cells to cure in an individual, but their offspring still inherit the original mutation.
  • Germline Gene Therapy is risk laden and illegal in most countries. It replaces DNA in germ cells (eggs and sperm) to prevent disease in future offspring.

In many cases, gene therapy uses modified viruses to carry replacement DNA into the cell nucleus. Although they pose complex risk factors in gene therapy, viral vectors are highly effective at entering cells and delivering a genetic payload. It's literally what they evolved to do.

Gene therapy commonly uses modified viruses as vectors to deliver the replacement DNA to malfunctioning cells

Gene therapy commonly uses modified viral vectors to deliver replacement DNA to malfunctioning cells.

Increasingly, synthetic vectors are used in gene therapy research.

Gene Therapy vs Genetic Vaccines

The genetic vaccines developed against COVID are based on the same principles and technologies as gene therapy.

However, DNA vaccines (using viral vectors) and mRNA vaccines (using lipid nanoparticle vectors) target only the short-term expression of viral genes. This contrasts with the long-term expression of human genes, often via complete DNA integration, targeted in gene therapy.


Gene Therapy Genetic Vaccines
Source Human Viral
Delivery Intravenous Intramuscular
Dosage High Low
Integration Yes No
Expression Permanent Temporary

The First Gene Therapy Trial

Ashanthi DeSilva was born without the ability to make a protein called ADA. She'd inherited faulty genes from both her mother and father, leaving her with a critical shortage. Not that anyone 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. After being diagnosed with Severe Combined Immunodeficiency (SCID), Ashanthi was put on protein replacement therapy to compensate for her missing protein. Although only partially effective, this kept her alive until she was four years old and recruited for the world's first gene therapy trial.

Each gene therapy uses a different set of protocols depending on the nature of the disorder. In Ashanthi's case, a retrovirus was used to transfect cells in vitro, and then infused into her bloodstream.

Gene therapy explained: illustration of viral vector transporting transgenic genes into patient's cells

The first gene therapy trial: (1) Malfunctioning white blood cells were harvested and replicated in culture to provide a therapeutic dose. (2) The retroviral vector was modified with the addition of ADA-coding genes, and the deletion of viral replication genes. (3) Transfection saw the virus inject its genetic payload into the nucleus. (4) The genetically modified cells were returned to the patient via intravenous infusion.

The first human gene therapy trial was a success. Millions of modified white blood cells began to produce ADA immediately, enabling Ashanthi to make functional antibodies. The edited cells also went on to replicate with the new genes intact, providing long-term benefits.

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 genetic diseases. But you'd be wrong.

The Scientific 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 scientific breakthrough myth, as illustrated by Rick and Morty

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 match).

Chemotherapy was another slow burner. The cancer treatment was invented after World War I autopsies revealed that soldiers poisoned with mustard gas had low white blood cell counts.

By World War II, researchers had transformed mustard agents into chemotherapies. They were trialled on cancer patients in the 1940s, and though some saw temporary benefits, problems with chemoresistance and chemotoxicity emerged. It was several more decades before mustard gas provided viable treatments, some of which are still used to this day.

Gene therapy, which must be tailored safely to 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 treatment, the introduction of new protocols revealed some 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 infants 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 had never been tested in humans before, and as the eighteenth and final patient in the trial, Jesse received the highest dose of the virus: 3.8x1013 (380 trillion) 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. They used a retrovirus vector to transport the DNA, which carries the equipment to integrate its genes into chromosomes.

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 retrovirus vector had inserted the healthy genes into chromosomes alongside cancer-causing genes. The oncogenes were activated, causing leukaemia to develop.

Several multistep mechanisms were identified where DNA insertions could activate harmful genes, or block the action of beneficial tumour-suppressing genes.

How gene therapy can cause insertional mutagenesis by activating dormant oncogenes in DNA

In the X-SCID trials, the viral vector genes included an enhancer region to boost transcription of the therapeutic genes. However, each virus species has preferential integration sites; in this case near specific oncogenes. The enhancer impacted native promoter regions thousands of bases downstream, thereby activating cancer-causing genes.

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.

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.

How to prevent insertional mutagenesis with genetic insulators

Genetic insulators prevent enhancer regions from triggering dormant stretches of DNA down the line.

Gene therapy trials resumed with the addition of such safety features, and the evolution of gene therapy continued.

Success Stories

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, she was already 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 a retrovirus 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. But there's a lot more work to do yet.

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; a factor that still blocks the therapy to this day.

Successful gene therapy also depends on the type of viral vector used. Most trials use just a few types of viruses:

Adenovirus Illustration AAV Illustration Retrovirus Illustration
Adenovirus AAV Retrovirus
90-100nm 25nm 80-100nm
DNA DNA RNA
In Vivo In Vivo Ex Vivo
Episomal Integration Chromosomal Integration Chromosomal Integration

In 2021, a question mark hangs over the safety of widely-used 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 had major wins—and major fails.

In 2014, a pilot study tested AAV9 on 15 infants with spinal muscular atrophy (SMA). The virus proved highly effective at spreading through neural tissues to deliver genes. 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 virus itself, yet a further trial using a different gene formula 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 AAV9–Like Vectors

Given the stakes, the FDA halted a human trial using high doses of AAV9 for muscular dystrophy (MD), and three years later, 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 AAV toxicity in human trials. In a study of children with Batten disease, an AAVrh10 vector caused acute side effects and chronic lesions in the brain.

In all, 149 gene therapies using AAV vectors have seen serious adverse events at a rate of 35%, with some resulting in death.

The FDA is now putting its safety concerns to gene therapy developers 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 (a type of retrovirus) led to three participants developing early-stage cancers. The phase III trial on children with cerebral adrenoleukodystrophy (CALD) was halted FDA and is now under investigation.

Some suggest the lentivirus isn't to blame, but rather the unique addition of a promoter region having especially broad activity. Historically, 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.

Final Thoughts

Each new gene therapy poses complex mechanics, with a unique set of protocols designed around each genetic template, vector, and dosage. Major challenges include an overactive immune response, gene expression in the wrong cell types, and insertional mutagenesis following integration.

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 are 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. There's also a question over dosage. Lipid nanoparticles generate low level inflammation which is ideal for helping vaccines kick-start the immune system. But, like viral vectors, scaling the dose of LNPs could lead to excessive inflammation and become life-threatening.

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. 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 diseases enter high risk clinical trials for a cure?

The risk-benefit equation isn't unique to gene therapy. As we've seen with public reactions to genetic vaccines, it's something many people can't appreciate—even when the answer lies in their favour. But some early gene therapies present unknown risks. Until they pass the test of time, each breakthrough trial poses a gamble in the search for a cure.

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