Patrick Hsu
CRISPR technology is now targeting RNA-based diseases
TEDxSanDiego 2018

Nature has a lot to teach us. Throughout the history of biology and medicine some of the most impactful and surprising therapies have come from diverse branches of the Tree of Life.

In 1928 the Scottish microbiologist Alexander Fleming returned from an occasion to find on his lab bench a Petri dish containing a fungal mold that could kill an infectious bacterial culture. He discovered penicillin, setting into motion the antibiotic revolution.

Nature has also hidden many secret tools into bacteria themselves. I’m a molecular biologist and a bioengineer, and we are developing one of these tools in my lab to study genetic disease and, hopefully, to create new kinds of medicines.

Over the last few years we’ve heard a lot about CRISPR as a technology for changing DNA directly inside of living cells through a process called genome editing. What you may not know is that CRISPR wasn’t originally developed by humans. Actually, it’s not that new. CRISPR was created by a bacteria.

Bacteria have been using various CRISPR systems for a very long time to defend themselves against foreign attackers. Such an invasion could come in the form of a bacteriophage, a bacterial form of a virus that is trying to inject its own genetic code into this much larger bacterial host.

These bacteria then will summon their CRISPR systems to recognize this foreign genetic information, to take a snapshot or a memory of it, and turn it back against the threat as a template for search and destroy.

You can think of CRISPR systems as a very elegant and effective immune defense system. Over the last decade or so, the CRISPR field has been unraveling many of the details of how these work.

This knowledge has allowed us to start to manipulate them intentionally. By taking CRISPR systems out of their native bacterial environment, we can put them into human cells and simultaneously recode this original antiviral memory with a human sequence. Now we can target nearly any sequence in our own genetic code at will.

Because genetic mutations can lead to disease, this has led to a lot of excitement that fixing mutations with CRISPR could lead to new kinds of genetically targeted medicines. CRISPR systems that target DNA with Cas9, which I helped to develop as a PhD student, has gotten almost all of the attention.

This particular flavor of CRISPR, which is being used by hundreds of labs around the world, comes originally from bacteria that can live in your skin or in your throat. There’s also a whole world of bacteria out there. Some of them are found primarily in soil, or water, or even inside of our own gut.

It turns out these bacteria have specialized due to their different environments and they’ve adapted their CRISPR systems as well. Some of these CRISPR systems don’t target DNA, but rather a different kind of molecule. These CRISPR systems target RNA.

What is RNA? RNA is the dynamic counterpart to your relatively static DNA sequence. Your DNA genome, more or less, is the same in all the cells of your body and it doesn’t change throughout your lifetime. You can think of your DNA genome like a dictionary or a phrase book that contains all of the words that can be used by your body’s cells in order to compose RNA messages so that those cells can have an actual conversation.

For example, this is how cells can respond to infections, by turning on inflammatory signals. RNA can also help carry the instructions to turn a stem cell into a specific kind of cell, like a skin cell or a brain cell. In this way, a whole organism or a person like you and me can form.

On the other hand, RNA can also cause cells to weaken or die. RNA targeting CRISPR systems are generating a lot of excitement, because there’s a whole set of diseases where it’s the RNA messages that go wrong inside of our selves. These disorders then might respond better to RNA based solutions.

This discovery and development of RNA targeting CRISPR has come about really in just the last two years. Why? Well, it turns out there’s been an explosion in genome sequence databases of a whole variety of organisms over just the last few years, largely due to the rapidly falling cost of DNA sequencing. This is helping some scientists, for example, to map out the diverse communities of bacteria that live in our gut called the microbiome, to understand how these niches are shaped by infections or diseases like Crohn’s or irritable bowel syndrome.

Now, because RNA messages can go up and down over time, and they’re highly dynamic, the idea is are there some RNA messages that can cause imbalances that can lead to disease. Being able to target these genetic defects directly is driving the clinical development of a class of drugs called gene therapies.

We thought to look in these new genome sequences potentially for new CRISPRs. We recently set out to do exactly that in my lab when we took a computational survey of these genome sequences and genome fragments and recently identified a new RNA targeting CRISPR system. It’s highly compact and it contains a single molecular machine that can be programmed by a guide RNA to find a matching target RNA.

It turns out that one variant of the system which comes from bacteria that live in the gut of New Zealand sheep turns out to work really well when transferred into human cells. We adapted this signature defense enzyme of the system into a technology we call CasRX, which of course stands for Caster Team DNLS for monococcus flavinations XVD 2002.

Using CasRX, we can now directly target specific RNA messages inside of our own cells at will, so that now we can directly target them, regulate their levels, and also to change imbalances found in disease. The hope is, can this be a new form of RNA based gene therapy? By targeting RNA, we won’t have to make permanent changes to our DNA, which could be safer.

Furthermore, RNA is dynamic, meaning it goes up and down over time, and it’s different in the different cells of our body. Eventually we might be able to control when and where a CRISPR therapeutic is acting so that it’s turned only in the cells that carry that toxic RNA.

One place in the body where RNA messages can instruct cells to weaken or die is in the brain. For example, in a kind of dementia called frontal temporal dementia, or FTD. The specific genetic defects that cause FTD vary from patient to patient, but often are thought to affect the balance of two RNA messages in that cell that encode for an important protein in the brain called tau. This imbalance in tau over the course of the patient’s lifetime progressively diminishes the health of the neurons in the brain, eventually leading to neurodegeneration and memory loss.

We thought we might be able to use CasRX to correct this imbalance and hopefully treat this disease. How would we do this? Well, once again we can reach into nature’s toolbox to find solutions to these challenges.

Viruses again are a part of our story. Most of you may think of viruses for their more negative disease-causing properties, but for gene therapies we’re instead using viruses for good. We can cut out the viral genome and actually replace it with CasRX, and then take advantage of an important property of many viruses that then infect human cells. Now we can use these engineering viruses to deliver a CRISPR therapeutic into cells of a patient.

We recently tested this in the lab, but we can’t just go to a person and ask for a sample of their brain, so instead we took advantage of a technology called induced pluripotent stem cells where you can take skin cells from a patient and reprogram it into a stem cell. Because the DNA of the cells hasn’t changed, these stem cells carry the same genetic defect that is found in these patients. Using a combination of chemical and genetic instructions we can instruct this stem cell to turn into a brain cell. In this way, we can study FTD directly in the lab.

We packaged CasRX into a therapeutic virus called AAV and delivered it to these cells and showed that we could correct this imbalanced tau so that they looked more like cells from healthy people.

What are some of the next steps for this type of therapeutic proof of concept? First, we want to move into animal models of the disease. This will allow us to look at things you can’t tell just by looking at the cells.

For example, if you take animals that have the same genetic defect found in FTD, we can dose them with the CasRX virus, and put them through a battery of memory tests to look at the progression, or maybe reversal, of pathology in the brain. This would also help us understand the amount of virus that would be safe and effective, and hopefully inform the dosage that might be used in initial human clinical trials.

CRISPR technologies are opening up a whole world of possibilities for manipulating our own genetic information and also providing new avenues for combating debilitating RNA based diseases, such as by removing toxic RNAs from our brain, or defending against RNA based viruses like influenza, zika, Ebola, and the like.

Furthermore, because changes to RNA are temporary, quite different from the permanent changes caused by targeting DNA, we’ll eventually be able to create more advanced gene therapies where we can control the timing of the drug.

There’s still so much to do. Diseases that are caused by defects with one gene will be first in the line for gene therapy, because we know what genetic defect we need to target and usually where, in which organ, needs to be targeted. Many diseases are caused by many genes. It’s a complex interaction both with each other but also with the environment.

For example, in many dementias like Alzheimer’s disease. This is something that I’m personally extremely passionate about. Two of my grandparents have Alzheimer’s. We’re working hard in my lab to try to figure out which gene combinations matter, so someday this too could be next in line for gene therapy development.

We’re just at the beginning of finding surprising new tools in unexpected places by searching nature’s already existing toolbox. Only, we want to do so now not via serendipity but in a systematic data-driven way guided by genome technologies.

My lab continues to plumb the depths of the CRISPR world and other unexplored organisms, and the weird maybe unknown enzymes that they’re using. By continuing to interpret and improve upon the complex biological language that nature uses to operate, we’ll continue to find ways to improve human health, from next generation antibiotics, drug delivery systems, to genetic engineering tools.

The next time you look in your fridge and you see some mold, or you find yourself eating some yogurt, look closer. Nature is full of many secrets.

Thank you.