How Does Gene Therapy Work?

How Does Gene Therapy Work?

Gene therapy delivers new DNA to faulty body cells, repairing our biological blueprint when it becomes damaged by mutation.

The idea of treating disease at the level of DNA is eloquent, but to the uninitiated the idea begs more than a few questions. How exactly do you insert DNA into cells? How does it change your body? And what happens when gene therapy goes wrong?

Genes: The Source Code of Life

Almost every cell in your body contains your complete DNA, which is made up of more than 20,000 genes.

The relationship between DNA and chromosomes

The relationship between DNA and chromosomes. Genes are long stretches of DNA, which bundle into chromosomes when cells divide.

Genes store the recipes for making protein molecules, which in turn have many critical roles around the body. Examples of proteins include myosin, antibodies, and oxytocin. Your genes are in use all the time, storing vital instructions to keep your body ticking over.

Each cell type references different batches of genes to make proteins relevant to their role in the body

Each cell type references different batches of genes to make proteins relevant to their role in the body.

But when a gene mutates, the instructions can become corrupt and lose all meaning. Proteins are produced insufficiently or abnormally, and in the case of cancer, cell division goes into overdrive.

This is what gene therapy aims to fix. It can replace or block the action of faulty genes, or even destroy faulty cells en masse with the addition of self-destruct genes.

How Does DNA Mutate?

Let's zoom in on DNA to see how base sequences give rise to genetic information. Taking the form of a double helix, DNA is made up of four bases and a sugar-phosphate backbone.

In correct base pairing, adenine (A) connects to thymine (T), while cytosine (C) connects to guanine (G). This gives us a DNA sense strand which contains the genetic sequence, and a DNA anti-sense strand which contains its inverse counterpart.

This double-stranded storage system makes our genes more resilient and more easily repaired than a single strand.

DNA base pairs (A, C, G, T) attached to the sugar-phosphate backbone, making up the entire double helix

The DNA base pairs (A, C, G, T) are attached to a sugar-phosphate backbone, making up the entire double helix.

A mutation means one or more DNA bases have been substituted, added, or deleted within a gene. This is the trigger. The gun doesn't actually fire until the gene is expressed.

Gene expression is the conversion of genetic information into protein products. When cells need to produce proteins, they transcribe the gene into a disposable copy known as messenger RNA (mRNA). This sequence is translated into long chains of amino acids which fold up to make proteins.

Mutations take effect when DNA is expressed into proteins

Mutations take effect when DNA is expressed into proteins.

Base additions or deletions tend to have more significant effects on protein production. Since DNA bases are translated in groups of three (called codons), a missing base can create a frameshift mutation that displaces every base thereafter.

A frameshift mutation caused by a single base deletion can corrupt the entire genetic sequence that follows

A frameshift mutation caused by a single base deletion can corrupt the entire genetic sequence that follows. The new codon sequence is translated into a new chain of amino acids.

Genetic mutation isn't all bad. In fact, it's the initiating factor for evolution by natural selection. But mutation is a trial-and-error process—and where there's error, there's potential for disease.

So how do these errors creep into the code?

  • Foetal development. Tiny mistakes can occur during DNA replication every time a cell divides. They're much more prevalent during the rapid growth phase of foetal development, and are passed on to all daughter cells thereafter.
  • Environmental mutagens. Our DNA continues to mutate throughout the lifetime, exacerbated by environmental factors like UV light, cigarette smoke, and even viruses.
  • Genetic inheritance. Genes come in pairs, one from each parent, and any variation produces a different allele (version). While a single disease allele may not matter (see dominant and recessive inheritance), faulty alleles from both parents leads to full blown disease.

All in all, genetic mutations are known to cause more than 6,000 diseases and counting. They include disorders present since birth, as well as diseases that develop over the lifetime, such as diabetes, heart disease, and cancer.

What is Gene Therapy?

Gene therapy is designed to make minor but critical edits to faulty DNA and restore normal cell function. All platforms in the US fall under the domain of somatic gene therapy, which targets only diseased body cells. This is distinct from germline gene therapy which may one day edit sperm and egg cells, but is for now illegal due to the considerable risks.

The first gene therapy was approved by the FDA in 2017 to treat an eye disorder that causes progressive blindness. Since then, more than 20 other products have made it into general use.

Eventually, gene therapy will override many traditional treatments, from protein replacement (eg, insulin injections for diabetes), to drug therapy (eg, SSRIs for depression), to cancer surgery (eg, tumour removal).

Most gene therapies take one of three approaches:

  • Gene replacement adds healthy genes to treat loss-of-function diseases like cystic fibrosis.
  • Gene inhibition blocks faulty genes to treat overactive expression that causes cancer.
  • Suicide genes are destructive genes that program apoptosis (cell death) in cancerous tumours.

Notably, gene therapy only targets faulty body cells. It doesn't re-write all the DNA in all body cells. The operational scale makes it a very precise approach to tackling disease at the level of DNA, with the aim of providing a permanent cure.

One major challenge of gene therapy is to ensure the new DNA is conserved through many cycles of cell replication. It's essential that the new genes are copied in dividing cells to produce lasting therapeutic effects.

But we can't go in guns blazing. Scaling the delivery of gene therapy can induce a deadly immune response, while boosting gene expression can activate dormant cancer-causing DNA.

This is why most gene therapies are at the pre-clinical stage (animal studies) or the clinical stage (human trials), where they're examined for efficacy, side effects, adverse events, and long term outcomes.

And there are many complex variables within the platform itself. Depending on the disease, faulty genes may be swapped out entirely, repaired using selective reverse mutation, or deactivated through gene regulation. This necessitates different forms of genetic material, including DNA, mRNA, siRNA, miRNA, and others.

There's no one-size-fits-all solution to curing disease at the genetic level. This is why it will be many years before we have a full suite of gene therapy treatments.

Consider Type 1 diabetes: a lifelong disease triggered by the destruction of insulin-producing cells in the pancreas. Since insulin is a critical hormone that allows glucose to enter cells, diabetics must inject synthetic insulin at every meal to stay alive.

In a landmark 2013 study, a single injection of therapeutic DNA enabled diabetic rats to produce insulin for up to six weeks. Nearly ten years later, however, gene therapies to restore natural insulin production in humans are still in development.

The principles of gene therapy are elegant. Yet in practice, some elements of the execution have been described as crude—and as we'll see in a moment, vulnerable to catastrophic failure. Nonetheless, experts believe gene therapy will eventually become a staple of 21st century medicine.

How Does Gene Therapy Work?

While each gene therapy is highly customised to each disease, the process of gene therapy can be broken down into three broad steps.

1. Vector Creation

First we need a vector; a nanoscale vehicle that transports the genetic payload into cells. These come in two flavours: viral vectors and non-viral vectors:

Viral vectors exploit the natural drive for viruses to invade cells, deposit their genes for replication, and even integration with the host DNA.

In preparation for gene therapy, the viral replication genes are edited out. This creates space in the viral genome to add therapeutic human genes.

However, each family of viruses comes with benefits and risks. Choosing the best viral vector and dosage is a balancing act that determines the rate of DNA delivery, immune response, and risk of integration errors.

Viral vectors in gene therapy include adeno-associated viruses (AAVs), adenoviruses, and retroviruses

The most common viral vectors in gene therapy include adeno-associated viruses (AAVs), adenoviruses, and retroviruses.

Non-viral vectors include synthetic nanomolecules which can be designed from scratch to deliver the new DNA.

While non-viral vectors reduce the risk of an overactive immune response, the tend to have lower rates of gene delivery. Nonetheless, synthetic vectors are still widely researched in gene therapy, which laid the groundwork for using lipid nanoparticle (LNP) vectors in COVID-19 mRNA vaccines. More on this in a moment.

Examples of non-viral vectors in gene therapy include liposomes, dendrimers, and metal-based nanoparticles

Examples of non-viral vectors include liposomes, dendrimers, and metal-based nanoparticles.

2. Gene Delivery

Next, the vector is introduced to target cells to deliver the genetic cargo. The is called transduction when using viral vectors, or transfection when using non-viral vectors. It can occur inside or outside the body:

  • In vivo gene therapy is an infusion of vectors into the bloodstream, so they can target cells in the body.
  • In situ gene therapy is an infusion of vectors directly into a target organ like the brain or liver.
  • Ex vivo gene therapy takes place in a cell culture in the lab, before the modified cells are returned to the patient.

When a vector meets a cell, it must successfully gain entry at the cell membrane and deposit the DNA payload, all without triggering a major intracellular immune response that destroys the cell.

In gene therapy, transduction is the introduction of foreign DNA into a cell by a viral vector

In gene therapy, transduction is the introduction of foreign DNA into a cell by a viral vector. The virus binds with a cell receptor to fuse with the lipid cell membrane. It's then absorbed by a lipid bubble called an endosome which carries it to the nucleus.

3. Gene Integration

Inside the cell, vectors must escape the endosome bubble to release their genetic payload. Viruses inject genes into the nucleus via a nuclear pore, and the therapeutic genes can take up residence in one of two forms:

  • Chromosomal integration sees the new genes fully incorporated into the host cell DNA. While this carries a risk of altering existing genes, it's more reliable for creating permanent gene expression.
  • Episomes are short loops of extra-chromosomal DNA, which can attach to chromosomes during cell division to create long-lasting gene expression.
DNA integration in gene therapy

Integration sees the new DNA incorporated into chromosomes, or persist separately as episomes. The vector escapes the endosome and injects the DNA through a nuclear pore, where it takes up residence in the nucleus.

Gene Therapy vs Genetic Vaccines

Are DNA and mRNA vaccines, like the ones developed against the SARS-CoV-2 virus, considered a type of gene therapy?

While genetic vaccines are based on the same principles and technology, they don't produce permanent gene expression, which is the primary goal of gene therapy.

DNA vaccines use viral vectors to deliver select viral genes to the cell nucleus

DNA vaccines use viral vectors to target the cell nucleus.

mRNA vaccines use non-viral vectors to deliver select viral genes to the cell cytoplasm

mRNA vaccines use non-viral vectors to target the cell cytoplasm.

So the main difference between genetic vaccines and gene therapy is the persistence of foreign genes and the duration of protein production. Genetic vaccines have a short-term action by design.

The same result would be a disaster in gene therapy, which must achieve permanent gene expression to provide a cure. Indeed, this is incredibly difficult to achieve, and remains one of the biggest challenges in gene therapy.

Gene therapy vs genetic vaccines

Gene therapy vs genetic vaccines. The main difference between gene therapy and genetic vaccines is the duration of DNA expression.

The First Gene Therapy Trials

In 1986, Ashanti DeSilva was born without the ability to make a protein called ADA, which plays a crucial role in white blood cells.

At birth, Ashanti seemed like a healthy baby. But as she became exposed to bacteria and viruses in the environment, her ADA 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

Most infants with ADA deficiency don't survive past their second birthday. Ashanti was lucky. After being diagnosed with Severe Combined Immunodeficiency (SCID), she was put on protein replacement therapy using ADA sourced from cows.

Although the treatment was only partially effective, it kept Ashanti alive until she was four years old and recruited for one of the first gene therapy trials.

A sample of Ashanti's white blood cells was cultured in the lab and transfected with modified retroviruses carrying healthy ADA-coding genes. The sample was then infused into her bloodstream over the course of 20 minutes.

Gene therapy explained: illustration of viral vector transporting transgenes into cells ex vivo

In Ashanti's ex vivo gene therapy, a retrovirus vector was modified with the deletion of viral replication genes and the addition of healthy ADA genes.

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

Although she needed 10 more infusions, Ashanti suffered no side effects from the ex vivo therapy, and was cured of SCID over the next two years. Today she's alive and well, married and with a Masters in Public Policy.

Ashanti'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.

When Gene Therapy Goes Wrong

The first spectacular failure of gene therapy came nine years later, in 1999. It involved 18-year-old Jesse Gelsinger, who had a partial OTC deficiency which meant he couldn't effectively break down ammonia, a waste product of protein.

Without treatment, babies born with severe OTC deficiency die in the first few weeks of life. Jesse signed up to a gene therapy trial to help such infants, knowing he wouldn't be cured himself as the gene therapy effects would be transient.

The trial used the adenovirus 5 (Ad5) vector for delivery in situ, sending the virus directly to the liver for transfection. It had never been tested in humans before.

As the eighteenth and final patient in the trial, Jesse received the highest dose of the virus: 3.8x1013 (380 trillion) viral particles. It caused a violent immune response.

Jesse suffered fever, blood clots, and widescale inflammation. Brain damage and organ failure ensued. Four days later, he died.

Some shocking revelations followed. The FDA investigation found that two other participants, who received lower doses than Jesse, had already suffered serious reactions to the adenovirus. This should have stopped the trial in its tracks.

Prior to the human trial, animal studies using a first generation vector saw two lab monkeys die from blood clots and liver inflammation. Jesse was never told this. When the family sued the trial organisers, they quickly settled out of court.

No matter how promising the technology, if gene therapy trials are poorly designed and executed, the results can be devastating.

Even with a good design, testing new drugs is inherently dangerous—that's why trials are necessary. Informed consent and proper regulation are critical parts of the process.

Although the tragedy led to tighter clinical trial regulations, Jesse was not the last to suffer from the experimental platform. Three years later, gene therapy hit the headlines again for the worst possible reasons.

The SCID Kids

Later trials would reveal the long term effects of gene therapy gone wrong.

"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, another form of Ashanti's severe immunodeficiency disorder. They also received a retrovirus vector targeting complete chromosomal integration.

Initially, the infants responded well and the life-saving trial was deemed a success. But in the years that followed, 4 of the 9 children developed leukaemia, a cancer leading to the excessive production of abnormal white blood cells.

It was a baffling result. The FDA halted all gene therapy trials in the US until the cause could be identified.

Researchers realised the retrovirus vector had selectively inserted the new genes alongside cancer-causing genes.

Such genes start out as healthy DNA code used in cellular growth and repair. But if they mutate or express at high levels, they become cancerous oncogenes.

In the SCID trial, the therapeutic DNA included an enhancer region to boost expression. But the enhancer effect drifted thousands of bases downstream to activate dormant oncogenes as well.

How gene therapy caused insertional mutagenesis in the SCID kids

How gene therapy caused insertional mutagenesis in the SCID kids.

It's pretty damning when your experimental therapy starts giving children cancer. The episode was widely publicised in the media, and leukaemia eventually 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 trial 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.

Their answer was to add an insulator to the therapeutic DNA. Genetic insulators serve as a barrier to limit the downstream effect of enhancers.

Genetic insulators prevent enhancer regions from activating downstream DNA and causing insertional mutagenesis

Genetic insulators prevent enhancer regions from activating downstream DNA and causing insertional mutagenesis.

Clinical trials resumed with the addition of this safety feature, and the evolution of gene therapy continued.

When Gene Therapy Saves Lives

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. Their offspring would have a 1 in 4 chance of developing metachromatic leukodystrophy (MLD), a degenerative disease that destroys the brain's white matter, leading to paralysis, dementia, and death.

The first time they heard about MLD, Brad and Amy already had two children. One day, their three-year-old daughter, Liviana, complained that her legs didn't work.

Doctors soon diagnosed Liviana with MLD. But since she was already symptomatic, she was past the window for treatment. Testing of her baby brother, Giovanni, showed he had also inherited the disease.

This is common with MLD; families are only alerted to the faulty gene when an older sibling begins to show symptoms. While Liviana passed away from MLD, it provided a vital warning sign so her younger brother could be treated before the disease took hold.

In 2011, Italian doctors drew stem cells from Giovanni and transfected them ex vivo with a retrovirus vector carrying new ARSA genes. Five days later, the modified cells were returned to his body and started producing the missing enzyme.

Now a teenager, Giovanni has well surpassed the life expectancy for infantile MLD, and by all appearances is cured. Brad and Amy Price went on to have six more children, and when another daughter, Cecilia, was diagnosed with MLD, she also 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 (RP).

Is Gene Therapy Safe?

Successful gene therapy is heavily dependent on choosing the right viral vector, and delivering it at a safe but effective dose.

Take the widely-used adeno-associated virus (AAV) vectors. They were once celebrated for their ability to cross the blood-brain barrier, circumventing the need to drill into the skull to treat neurological disorders. But recent animal and human studies have thrown trusted AAV vectors into dispute.

In 2018, an AAV9 vector caused severe toxicity in primates and pigs, leading to liver and motor neuron damage.

The FDA halted human trials using high doses of AAV9 for muscular dystrophy (MD), and investigations into AAV toxicity are ongoing.

It's suspected that high-dose AAV gene therapy may cause DNA damage, stress to the endoplasmic reticulum (a cell organelle used in protein synthesis), and overexpression of transgenes in liver cells leading to liver failure.

To date, 149 gene therapies using AAV vectors have produced serious adverse events at a rate of 35%, with some resulting in death.

Another vector threat emerged in 2021. For the first time in gene therapy history, use of a lentivirus (a type of retrovirus) led to three children developing early-stage leukaemia.

Some suggest the lentivirus isn't to blame, but rather the unique addition of a promoter region triggering genes downstream. Lentiviruses have been used to treat more than 300 gene therapy patients for a dozen different conditions. If research can untangle the mechanism, lentiviruses may still be in the clear.

Discouraged by the risks of viral vectors, some researchers have turned to synthetic vectors like polymers, liposomes, and ultrasound-mediated microbubbles.

While these nanoscale carriers are currently less effective at delivering genes into cells, they are safer and cheaper, while presenting no DNA size limit.

But there's still a question of dosage. Consider the genetic mRNA vaccines that use small doses of lipid nanoparticles to deliver viral genes. This generates low level inflammation, which is ideal for kick-starting the immune system.

But gene therapy is about producing lasting genetic changes. This necessitates much higher doses of both vectors and genes. Scaling the delivery of synthetic vectors could also lead to life-threatening inflammation as seen in viruses.

The safety of gene therapy really depends on the genetic template, vector type, and dosage. Tweaking any variable in the wrong direction can lead to an excessive immune response, gene overexpression, or insertional mutagenesis.

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. Beyond rare childhood diseases, therapies are now targeted at common cancer, cardiovascular, infectious, and inflammatory diseases.

This changes the risk-benefit ratio considerably. Children with months to live may understandably be offered last-ditch experimental treatments. Yet the same may not apply to adults with chronic conditions and for which other treatments exist.

Gene therapy is a breakthrough proposition. But not all therapies pose equal risks. Until those risks are understood, each new trial poses a certain gamble in the search for a cure.

Rebecca Casale Author Bio

Rebecca Casale is a science blogger based in Auckland. If you like her content, please share it with your friends. If you don't like it, why not punish your enemies by sharing it with them?