Gene Therapy: Vectors, Viruses, and Why CRISPR Will Change Everything

by L. E. Carmichael

Welcome to Part 2 of my series on gene therapy! If you haven't already, I recommend that you read Part 1, Introduction to Gene Therapy: It Sounds Simple, But It's Sure Not Easy, before continuing with this post.

Ready? Here we go.

Viruses: FedEx For Genes

Gene therapy involves repairing or replacing a faulty gene that has led to disease. In order to do that, one major hurdle must be overcome: getting new DNA into the patient's cells. Cells, however, are designed to keep things out. That's why they have wrappers, called cell membranes. Scientists needed vectors: gene delivery systems capable of crossing the membrane.

In the early days of gene therapy research (by which I mean the 80s), the obvious way to cross the membrane was to use a virus. Viruses are highly efficient invaders - they have to be, because they are not capable of copying their own DNA. In other words, the only way a virus can reproduce and spread is to invade a host cell, hijack its equipment, and force the cell to package new viruses that can carry on the cycle of infection.

But what if scientists could replace some of the virus's DNA with a therapy gene? The virus would invade a patient's cells as per usual, but instead of causing infection, deliver some healthy human DNA. Sort of like molecular FedEx. Some viruses even contain DNA sequences that match sequences found in human DNA. These complementary sequences would prompt the host cell to incorporate the therapy gene into its own genome. Not only would the patient be cured, but the cure would be permanent.

What's that line about how it seemed like a good idea at the time?

The Problem With Viruses

For starters, they are viruses. The human immune system is designed to find and destroy viruses, before they invade the body's cells. This happens all the time, without our even realizing it. But sometimes, the immune system gets a little carried away.

That's exactly what happened to Jesse Gelsinger in 1999. The 18-year-old was part of a clinical trial of a gene therapy for a genetic disease known as OTC. When he received the treatment, his immune system had a massive over-reaction to the viral vector and began attacking his own cells. He died just a few days later.

The Other Problem With Viruses

Remember I said that some viruses can insert their own DNA - or therapy DNA - into the human genome, making a therapy permanent? Early gene therapy research occurred before we sequenced the entire human genome. Scientists didn't realize that complementary DNA sequences could occur in more than one location in a person's DNA... or that some of those locations were inside other genes. No good providing DNA to cure one genetic disease if your cure is going to knock out another gene. Especially if that gene helps to controls cell division... because uncontrolled cell division leads to cancer.

That's what happened to a number of young patients during an early clinical trial for the immune system deficiency SCID. And since those kids had compromised immune systems, they had no natural defences against the cancer their cure had created.

A Light at the End of the Tunnel

After these tragedies, scientists spent a lot of time searching for viral vectors that would NOT cause such horrific side effects. One type belongs to a family of viruses known as AAV. A gene therapy for Leber congenital amaurosis is built around AAV. It replaces a faulty gene in retina cells that causes children to go blind. And after decades of research, it looks as though this therapy will soon be available to the public. Developed by Spark Therapeutics, the treatment just received a unanimous endorsement from an FDA review panel, meaning approval for the therapy could be just around the corner - a literal light at the end of the tunnel for these patients.

So What is CRISPR, and Why Is Everyone So Excited About It?

A major downside of AAV viruses is that they do not incorporate their genetic package into the patient's existing genome. Which means they don't cause cancer, but also that the therapy gene can't be reliably passed on to daughter cells. That's OK for retinal cells, which don't divide. It's a lot less useful for treating diseases in other parts of the body.

CRISPR, on the other hand, works in all types of cells. It allows scientists to edit a patient's existing genes - correcting the typos that cause disease - and the changes are permanent. Here's how it works and why it's such a big deal.

Here's another really great video explaining CRISPR and its applications that unfortunately I was not able to embed.

And yes. You still have to get the components for CRISPR into the cell in the first place. One group of scientists has just found a way to do this without using viruses. They used gold particles instead.

As you can see, CRISPR could lead to incredible breakthroughs, and not just in gene therapy. But there are concerns as well. Stay tuned for Part 3, where I will explore what's perhaps the biggest one.

Introduction to Gene Therapy: It Sounds Simple, But It's Sure Not Easy

by L. E. Carmichael

Muscular dystrophy, hemophilia, Leber congenital amaurosis (LCA), cancer... they all have one thing in common. They are genetic diseases, ultimately caused by missing or malfunctioning genes in the patient's DNA. Until recently, we could treat the symptoms of many genetic diseases, but not the causes. There was no way to reach inside a person's genes and repair the faulty code that started the whole problem.

Research into gene therapy aims to change that.

What is gene therapy?

Genes are blueprints for proteins. A complete set of working proteins makes a working human. In contrast, missing or malfunctioning proteins sometimes lead to disease. Many genes and proteins have to be affected to lead to diseases like cancer. Other diseases, like LCA and Lesch-Nyhan syndrome, result from a single broken gene.

The logic behind gene therapy is very simple: if a missing or malfunctioning gene is causing disease, a working copy of the gene could cure the patient. There are two ways to provide a working copy:

1. Fix the gene that's already in the patient's DNA. This is easiest when the malfunction is caused by what's known as a "point mutation," which is basically a typo in the genetic code. Not all such typos are dangerous, but the right typo in the wrong place can knock out the entire gene.
2. Provide a new copy of the entire gene. This strategy applies for genes with larger insertions - extra code - or deletions - missing code.

Once a working gene is present in the patient's cells, his or her body can make a functional protein... and in some cases, that may be enough to cure the disease.

That Does Sound Simple... So Why Is Gene Therapy So Hard?

Lots of reasons, but we're going to focus on two:

1. Cells have lifespans. Dying cells are replaced by new cells, which are created by cell division. During this process, the cell's genome (all the DNA a person was born with) is copied so that each new daughter cell gets a complete set of instructions. There is no guarantee that a therapy gene that's simply floating around in the cell will end up in both daughters - to ensure that the shiny new gene will always be passed on, making the cure permanent, that gene has to become part of the original genome. Which means that, either
   1. the patient's original gene has to be edited in place, or
   2. the complete new gene has to embed itself into the patient's existing DNA.
2. According to one estimate, there are 37.2 TRILLION cells in the human body. Not every disease affects every cell in the body. For example, LCA is a form of blindness caused by a faulty gene in the cells of the retina. To cure LCA, only retina cells would need to be treated. Far fewer than the whole 37.2 trillion, but still enough that getting a working gene into EVERY retinal cell is still a huge challenge.

So How Do Scientists Solve These Problems?

Stay tuned! In Part 2, I will explain what CRISPR is and why scientists are so excited about it. In Part 3, we'll tackle the "every cell" problem... and some awesome/scary breakthroughs that have recently hit the news.

In the meantime, you or your teenager might want to check out my YA science book, Gene Therapy. It's a couple years old now, but is still a solid introduction to the history and science of this cutting edge medicine.