How Does Gene Therapy Work?

How Does Gene Therapy Work?

Gene therapy works by delivering short snippets of DNA into malfunctioning body cells. It's an emerging technology designed to correct critical errors in our biological blueprint.

To the uninitiated, it begs more than a few questions. How do you get new DNA into cells? How does it change our source code? And what happens when gene therapy goes wrong?

Buckle up for the insanely bumpy ride of gene therapy development.


What are Genes?

Genes are long sequences of DNA bases (A, C, G and T) within the famous double helix. Humans have more than 20,000 genes, which are like data files stored across 46 chromosomes folders.

Genes vs DNA vs Chromosomes

Genes coil into chromosomes to prevent tangling during cell division.

Our entire genome—that is, our complete gene set—lives in all 100 trillion body cells, with the exception of red blood cells. This is why you can sequence your entire genome from almost any cell sample.

Each gene is a unique blueprint for making protein molecules. Proteins are the heavy lifters, with many structural, catalytic, and signalling roles around the body. Examples of proteins include myosin, antibodies, and oxytocin.

Cells vs Genes vs Proteins

Genes coil into chromosomes to prevent tangling during cell division.

Different cell types reference different genes relevant to their role in the body.

How Does DNA Mutate?

Mutations aren't all bad. In fact, genetic mutation is the mechanism by which all life adapts and evolves. But it's a trial-and-error process, and where there's an error, there's potential for disease.

An error means one or more DNA bases have been substituted, added, or deleted within a genetic sequence. For instance, a single base substitution in the HBB gene interferes with the production of haemoglobin which causes sickle cell anaemia.

Additions or deletions tend to be more disastrous, though. Since DNA bases are translated in groups of three, an extra base creates a frameshift that displaces every base thereafter. The entire gene sequence becomes corrupt.

The structure of DNA bases (A, C, G, T) create genes

The precise sequence of DNA bases gives us functional genes that encode proteins.

Such mutations can be inherited from our parents at the moment of conception. Inheriting a faulty gene from one parent may not matter, as long as we inherit a working copy from the other (see dominant and recessive inheritance). But faulty copies from both parents leads to full blown disease.

Mutations can also arise spontaneously in the womb. Mistakes in DNA replication occur at a much higher rate during this rapid growth phase, and can be evident in just some or all body cells.

But we're never free from the risk of genetic mutation. Our DNA can be damaged throughout the lifetime as a result of exposure to UV light, cigarette smoke, and even viruses. Damage to our DNA can cause more than 6,000 diseases, including diabetes, heart disease, and cancer.

Traditional treatments for DNA-related diseases include:

  • Replacing missing proteins, eg insulin injections
  • Introducing new stem cells, eg bone marrow transplant
  • Destroying diseased cells, eg tumour surgery
  • Preventing viral integration, eg antiretroviral drugs

What is Gene Therapy?

Gene therapy can make minor but critical edits to faulty DNA and restore normal bodily function. There are two broad types:

  • Somatic gene therapy targets diseased body cells, but the faulty genes can still be passed to future offspring.
  • Germline gene therapy targets sperm and egg cells, but is illegal due to the considerable unknown risks.

The first gene therapy was approved by the FDA in 2017. Since then, more than 20 other products have made it to market. They use at least three different approaches:

Gene therapy can introduce healthy replacement genes, inhibit over-active disease genes, or program apoptosis of runaway disease cells

Three major approaches to gene therapy.

So gene therapy only targets diseased cells, and does not re-write all the DNA in your body. In fact, it's a very precise approach to tackling disease at the fundamental level, with the aim (but not often the result) 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 into 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 (using animal studies) or the clinical stage (in human trials), where they're being examined for short and long term effects.

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?

Each gene therapy is highly tailored, with different treatment protocols required for different types of genetic errors. But we can break the process down into three broad steps.

1. Vector Loading

First we need a vector—a nanoscale vehicle that transports the genetic payload into cells.

  • Viral vectors exploit the natural drive for viruses to invade cells and deposit their genes. With genetic editing, unwanted viral genes can be snipped out and replaced with therapeutic human genes.
  • Non-viral vectors include synthetic molecules which can be designed from scratch to deliver new genes. This category includes the transfer of naked DNA using electrical pulses or high pressure gas.
Gene therapy vectors: viral vs non-viral

Like genetic vaccines, gene therapy uses both viral and non-viral vectors to deliver genes to cells.

2. Cell Transfection

Next, we introduce our vector to our target cells for transfection, which means delivering the genes into cells.

The vector must be able to gain entry at the cell membrane and drop its payload in the nucleus, all without triggering a major intracellular immune response which destroys the cell.

Gene therapy transfection

Therapeutic genes are delivered into the cell by transfection. The viral vector binds with a specific cell receptor to fuse with the cell membrane. It's then absorbed into a lipid carrier called an endosome which transports it through the cytoplasm. Endosomal escape allows the virus to reach the nucleus.

Cell transfection can occur inside or outside the body:

  • In vivo gene therapy infuses vectors into the bloodstream, so they can track down and transfect target cells in the body.
  • In situ gene therapy infuses vectors directly to a target organ like the brain or liver.
  • Ex vivo gene therapy keeps vectors out of the body entirely. Transfection occurs in a cell culture in the lab, before the edited cells are returned to the patient.

3. DNA Integration

When it arrives at the nucleus, the virus capsule breaks down and injects its genetic payload through a nuclear pore. The new genes take up residence in one of two forms:

  • Chromosomal integration stitches the new DNA into chromosomes, right alongside existing genes. This carries a risk of altering healthy genes, but is better at producing permanent gene expression.
  • Non-integrating episomes set up home as circular DNA, also known as plasmids. Episomes don't integrate with chromosomal DNA but with new techniques can still be conserved during cell replication.
Gene therapy

Depending on the type of vector, the new transgenes can (a) integrate with chromosomes or (b) persist as episomes in the nucleus. With the right techniques, both forms of DNA can be replicated and expressed long term.

What's in a Vector?

Viral vectors are highly effective at delivering genes in cells. It's literally what they evolved to do.

But each family of viruses comes with a unique set of benefits and risks. Choosing the right type and dosage is a fine balancing act that determines the rate of gene delivery, the immune response, and the risk of chromosomal integration errors.

Comparison of viral vectors used in gene therapy: adenoviruses, adeno-associated viruses, and retroviruses (including lentiviruses)

The most common viral vectors in gene therapy are adenoviruses, adeno-associated viruses, and retroviruses (including lentiviruses).

Meanwhile, synthetic non-viral vectors like liposomes are less toxic, but currently have lower rates of gene delivery. While it's an ongoing technical challenge in gene therapy, synthetic vectors as they stand today have proven valuable in vaccine development.

Genetic Vaccines vs Gene Therapy

The genetic vaccines that emerged from the COVID pandemic are based on the same technology and principles as gene therapy.

For instance, the DNA vaccines made by AstraZeneca and Johnson & Johnson contain adenovirus vectors which deliver select SARS-CoV-2 genes to cells. Our cells use the genetic information to produce viral antigens which train up our immune system.

Adenovirus (Ad) vectors in DNA vaccines

DNA vaccines use modified adenoviruses as vectors to deliver SARS-CoV-2 spike genes into cells.

Similarly, the mRNA vaccines made by Pfizer-BioNtech and Moderna contain synthetic vectors called lipid nanoparticles to transport the SARS-CoV-2 viral genes.

Lipid nanoparticle (LNP) vectors in mRNA vaccines

mRNA vaccines use lipid nanoparticles as vectors to deliver SARS-CoV-2 spike genes into cells.

The biggest difference between genetic vaccines and gene therapy is that vaccines target short-term gene expression.

Viral proteins are produced by cells only briefly, in order to trigger an adaptive immune response. Then the foreign genes and proteins are destroyed.

The same result would be a disaster in gene therapy, which is necessarily riskier than genetic vaccines because it targets long-term gene expression in many cases.

Gene therapy vs genetic vaccines

Gene therapy vs genetic vaccines.

Delivering sufficient genes, to the right cells, and achieving long-term expression are all major challenges in gene therapy.

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, an X-chromosome form of Ashanti's disease. They also received a retrovirus vector which integrated the new DNA into their 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 halted all gene therapy trials in the US until the cause could be identified.

Researchers found the retrovirus vector had inserted the healthy genes into chromosomes alongside cancer-causing genes.

We all possess such dormant genes, which are normally used in growth and repair. However, when activated by viral genes, they go haywire. In this case, oncogenes were activated by the retrovirus vector—and the children developed leukaemia.

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

The X-SCID gene therapy trials used an enhancer region to boost transcription of the therapeutic genes. But with the retrovirus having a preferential integration site near oncogenes, the enhancer also activated promoter regions thousands of bases downstream to trigger cancer.

It's pretty damning when your experimental therapy starts giving children cancer. The episode was widely publicised, 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 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 activating genes down the line.

Clinical trials resumed with the addition of such safety features, 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.

Becky Casale Author Bio

Becky 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?