Can we cure genetic diseases by rewriting DNA? | David R. Liu

The most important gift your mother and father ever gave you

was the two sets of three billion letters of DNA

that make up your genome.

But like anything with three billion components,

that gift is fragile.

Sunlight, smoking, unhealthy eating,

even spontaneous mistakes made by your cells,

all cause changes to your genome.

The most common kind of change in DNA

is the simple swap of one letter, or base, such as C,

with a different letter, such as T, G or A.

In any day, the cells in your body will collectively accumulate

billions of these single-letter swaps, which are also called "point mutations."

Now, most of these point mutations are harmless.

But every now and then,

a point mutation disrupts an important capability in a cell

or causes a cell to misbehave in harmful ways.

If that mutation were inherited from your parents

or occurred early enough in your development,

then the result would be that many or all of your cells

contain this harmful mutation.

And then you would be one of hundreds of millions of people

with a genetic disease,

such as sickle cell anemia or progeria

or muscular dystrophy or Tay-Sachs disease.

Grievous genetic diseases caused by point mutations

are especially frustrating,

because we often know the exact single-letter change

that causes the disease and, in theory, could cure the disease.

Millions suffer from sickle cell anemia

because they have a single A to T point mutations

in both copies of their hemoglobin gene.

And children with progeria are born with a T

at a single position in their genome

where you have a C,

with the devastating consequence that these wonderful, bright kids

age very rapidly and pass away by about age 14.

Throughout the history of medicine,

we have not had a way to efficiently correct point mutations

in living systems,

to change that disease-causing T back into a C.

Perhaps until now.

Because my laboratory recently succeeded in developing such a capability,

which we call "base editing."

The story of how we developed base editing

actually begins three billion years ago.

We think of bacteria as sources of infection,

but bacteria themselves are also prone to being infected,

in particular, by viruses.

So about three billion years ago,

bacteria evolved a defense mechanism to fight viral infection.

That defense mechanism is now better known as CRISPR.

And the warhead in CRISPR is this purple protein

that acts like molecular scissors to cut DNA,

breaking the double helix into two pieces.

If CRISPR couldn't distinguish between bacterial and viral DNA,

it wouldn't be a very useful defense system.

But the most amazing feature of CRISPR

is that the scissors can be programmed to search for,

bind to and cut

only a specific DNA sequence.

So when a bacterium encounters a virus for the first time,

it can store a small snippet of that virus's DNA

for use as a program to direct the CRISPR scissors

to cut that viral DNA sequence during a future infection.

Cutting a virus's DNA messes up the function of the cut viral gene,

and therefore disrupts the virus's life cycle.

Remarkable researchers including Emmanuelle Charpentier, George Church,

Jennifer Doudna and Feng Zhang

showed six years ago how CRISPR scissors could be programmed

to cut DNA sequences of our choosing,

including sequences in your genome,

instead of the viral DNA sequences chosen by bacteria.

But the outcomes are actually similar.

Cutting a DNA sequence in your genome

also disrupts the function of the cut gene, typically,

by causing the insertion and deletion of random mixtures of DNA letters

at the cut site.

Now, disrupting genes can be very useful for some applications.

But for most point mutations that cause genetic diseases,

simply cutting the already-mutated gene won't benefit patients,

because the function of the mutated gene needs to be restored,

not further disrupted.

So cutting this already-mutated hemoglobin gene

that causes sickle cell anemia

won't restore the ability of patients to make healthy red blood cells.

And while we can sometimes introduce new DNA sequences into cells

to replace the DNA sequences surrounding a cut site,

that process, unfortunately, doesn't work in most types of cells,

and the disrupted gene outcomes still predominate.

Like many scientists, I've dreamed of a future

in which we might be able to treat or maybe even cure

human genetic diseases.

But I saw the lack of a way to fix point mutations,

which cause most human genetic diseases,

as a major problem standing in the way.

Being a chemist, I began working with my students

to develop ways on performing chemistry directly on an individual DNA base,

to truly fix, rather than disrupt, the mutations that cause genetic diseases.

The results of our efforts are molecular machines

called "base editors."

Base editors use the programmable searching mechanism of CRISPR scissors,

but instead of cutting the DNA,

they directly convert one base to another base

without disrupting the rest of the gene.

So if you think of naturally occurring CRISPR proteins as molecular scissors,

you can think of base editors as pencils,

capable of directly rewriting one DNA letter into another

by actually rearranging the atoms of one DNA base

to instead become a different base.

Now, base editors don't exist in nature.

In fact, we engineered the first base editor, shown here,

from three separate proteins

that don't even come from the same organism.

We started by taking CRISPR scissors and disabling the ability to cut DNA

while retaining its ability to search for and bind a target DNA sequence

in a programmed manner.

To those disabled CRISPR scissors, shown in blue,

we attached a second protein in red,

which performs a chemical reaction on the DNA base C,

converting it into a base that behaves like T.

Third, we had to attach to the first two proteins

the protein shown in purple,

which protects the edited base from being removed by the cell.

The net result is an engineered three-part protein

that for the first time allows us to convert Cs into Ts

at specified locations in the genome.

But even at this point, our work was only half done.

Because in order to be stable in cells,

the two strands of a DNA double helix have to form base pairs.

And because C only pairs with G,

and T only pairs with A,

simply changing a C to a T on one DNA strand creates a mismatch,

a disagreement between the two DNA strands

that the cell has to resolve by deciding which strand to replace.

We realized that we could further engineer this three-part protein

to flag the nonedited strand as the one to be replaced

by nicking that strand.

This little nick tricks the cell

into replacing the nonedited G with an A

as it remakes the nicked strand,

thereby completing the conversion of what used to be a C-G base pair

into a stable T-A base pair.

After several years of hard work

led by a former post doc in the lab, Alexis Komor,

we succeeded in developing this first class of base editor,

which converts Cs into Ts and Gs into As

at targeted positions of our choosing.

Among the more than 35,000 known disease-associated point mutations,

the two kinds of mutations that this first base editor can reverse

collectively account for about 14 percent or 5,000 or so pathogenic point mutations.

But correcting the largest fraction of disease-causing point mutations

would require developing a second class of base editor,

one that could convert As into Gs or Ts into Cs.

Led by Nicole Gaudelli, a former post doc in the lab,

we set out to develop this second class of base editor,

which, in theory, could correct up to almost half of pathogenic point mutations,

including that mutation that causes the rapid-aging disease progeria.

We realized that we could borrow, once again,

the targeting mechanism of CRISPR scissors

to bring the new base editor to the right site in a genome.

But we quickly encountered an incredible problem;

namely, there is no protein

that's known to convert A into G or T into C

in DNA.

Faced with such a serious stumbling block,

most students would probably look for another project,

if not another research advisor.

(Laughter)

But Nicole agreed to proceed with a plan

that seemed wildly ambitious at the time.

Given the absence of a naturally occurring protein

that performs the necessary chemistry,

we decided we would evolve our own protein in the laboratory

to convert A into a base that behaves like G,

starting from a protein that performs related chemistry on RNA.

We set up a Darwinian survival-of-the-fittest selection system

that explored tens of millions of protein variants

and only allowed those rare variants

that could perform the necessary chemistry to survive.

We ended up with a protein shown here,

the first that can convert A in DNA

into a base that resembles G.

And when we attached that protein

to the disabled CRISPR scissors, shown in blue,

we produced the second base editor,

which converts As into Gs,

and then uses the same strand-nicking strategy

that we used in the first base editor

to trick the cell into replacing the nonedited T with a C

as it remakes that nicked strand,

thereby completing the conversion of an A-T base pair to a G-C base pair.

(Applause)

Thank you.

(Applause)

As an academic scientist in the US,

I'm not used to being interrupted by applause.

(Laughter)

We developed these first two classes of base editors

only three years ago and one and a half years ago.

But even in that short time,

base editing has become widely used by the biomedical research community.

Base editors have been sent more than 6,000 times

at the request of more than 1,000 researchers around the globe.

A hundred scientific research papers have been published already,

using base editors in organisms ranging from bacteria

to plants to mice to primates.

While base editors are too new

to have already entered human clinical trials,

scientists have succeeded in achieving a critical milestone towards that goal

by using base editors in animals

to correct point mutations that cause human genetic diseases.

For example,

a collaborative team of scientists led by Luke Koblan and Jon Levy,

two additional students in my lab,

recently used a virus to deliver that second base editor

into a mouse with progeria,

changing that disease-causing T back into a C

and reversing its consequences at the DNA, RNA and protein levels.

Base editors have also been used in animals

to reverse the consequence of tyrosinemia,

beta thalassemia, muscular dystrophy,

phenylketonuria, a congenital deafness

and a type of cardiovascular disease --

in each case, by directly correcting a point mutation

that causes or contributes to the disease.

In plants, base editors have been used

to introduce individual single DNA letter changes

that could lead to better crops.

And biologists have used base editors to probe the role of individual letters

in genes associated with diseases such as cancer.

Two companies I cofounded, Beam Therapeutics and Pairwise Plants,

are using base editing to treat human genetic diseases

and to improve agriculture.

All of these applications of base editing

have taken place in less than the past three years:

on the historical timescale of science,

the blink of an eye.

Additional work lies ahead

before base editing can realize its full potential

to improve the lives of patients with genetic diseases.

While many of these diseases are thought to be treatable

by correcting the underlying mutation

in even a modest fraction of cells in an organ,

delivering molecular machines like base editors

into cells in a human being

can be challenging.

Co-opting nature's viruses to deliver base editors

instead of the molecules that give you a cold

is one of several promising delivery strategies

that's been successfully used.

Continuing to develop new molecular machines

that can make all of the remaining ways

to convert one base pair to another base pair

and that minimize unwanted editing at off-target locations in cells

is very important.

And engaging with other scientists, doctors, ethicists and governments

to maximize the likelihood that base editing is applied thoughtfully,

safely and ethically,

remains a critical obligation.

These challenges notwithstanding,

if you had told me even just five years ago

that researchers around the globe

would be using laboratory-evolved molecular machines

to directly convert an individual base pair

to another base pair

at a specified location in the human genome

efficiently and with a minimum of other outcomes,

I would have asked you,

"What science-fiction novel are you reading?"

Thanks to a relentlessly dedicated group of students

who were creative enough to engineer what we could design ourselves

and brave enough to evolve what we couldn't,

base editing has begun to transform that science-fiction-like aspiration

into an exciting new reality,

one in which the most important gift we give our children

may not only be three billion letters of DNA,

but also the means to protect and repair them.

Thank you.

(Applause)

Thank you.