GENE EDITING
Curing Genetic Blindness With Gene Editing
From darkness to light, this is how GE revolutions are shaping the way we see.
Growing up, one of my favourite days of the year was Halloween. The sheer excitement of being able to put on my new costume, see friends, and go trick-or-treating was an experience that could never be replicated.
This was my experience:
For thousands of others, it was this:
You see, thousands of children are born with some form of genetic blindness (or severe vision loss).
For us, a day filled with excitement and happiness is, for them, another day in darkness.
Thousands are thousands too much. The struggle isn’t solely with Halloween, it’s with life. That’s what we need to change.
But, as of right now, is there hope for vision in those born with genetic blindness?
In this article, I will touch on several research papers and patents discussing potential therapeutic solutions for genetic blindness (specifically, LCA10) + applications for future trials. There’s a lot to dive into, so let’s get into it.
Overview of Gene Editing (GE)
Every living being is defined by its genes. These genes dictate our appearance, health and other traits. Sequence changes in our genes determine, for example, if we have freckles, the colour of our eyes, lactose levels, etc.
This is when gene editing comes into play. CRISPR-Cas9 is the fastest, easiest, and most cost-efficient gene-editing tool. It uses two main components:
The first component is CRISPR — a region of DNA with two distinct characteristics: nucleotide repeats and spacers.
The second is Cas9 — a protein that works alongside ribonucleic acid (RNA) strands to target + cleave specified DNA regions.
Scientists can then design a RNA that acts as a “guide” to match the gene they want to edit. After that, they attach it to Cas9 where it is taken to the host. The guide RNA (gRNA) will then be injected into the host to snip off a gene and replace it. With this technology, scientists can practically edit any gene they want.
With gene editing, we can edit our traits — literally. But it isn’t limited to that — gene editing can also play a role in disease detection and prevention.
The Problem
Take a look around you. What colours do you see?
Black? White? Green?
Whatever it may be, you “seeing” is simply your eyes interpreting lights.
That black keyboard is black because your eyes are absorbing lights off that object to express that colour (and suppress all others).
Anything you see is through your eyes absorbing lights and expressing them in a certain colour — that’s, in a nutshell, how we see.
If we look into why this is the case, we need to take a quick dive into our eyes — and specifically, the retina.
The cones and rods we see in the image above are known as photoreceptor cells. These cells play that important role of converting light into specific electric signals (or visual information). These signals are sent to your brain, which instructs your eyes to then interpret those lights in colour.
Now, what if your eyes aren’t able to perceive these lights?
If your eyes aren’t able to interpret the lights in colour, that’s where blindness (or severe vision loss) comes into play.
Leber congenital amaurosis (LCA), for example, is an inherited retinal disease present in two to three out of every 100,000 babies, which causes newborn babies to be born with severe vision loss (or in severe cases, blindness).
Essentially, their photoreceptor cells aren’t healthy or developing at a normal rate. There may be fewer, shorter, or damaged cells in these cases.
These unhealthy/dysfunctional photoreceptor cells are caused by several genomic mutations. In fact, many genetic forms of blindness are caused by these specific genetic mutation (i.e. a mutation to a certain gene in the eye). The solution to these mutations (generally) is to identify that mutation and modify that gene (by insertion, deletion, etc.). This is an extremely general process + disease, though, so we need to look a little deeper: LCA10 — the most common form of LCA.
LCA10
Mutations ~15 genes result in LCA10. The CEP290 protein is the most frequently mutated LCA gene, which represents ~15% of all cases of this disease.
The CEP290 protein plays a role in the formation and stability of cilia in photoreceptor cells.
The cilia is crucial to photoreceptor development because it acts as the gateway for proteins — helps grow + produce more photoreceptor cells.
The mutation that occurs in the CEP290 gene is a c.2991+1655A to G mutation (i.e. Adenine → Guanine mutation).
For another image (above), we can see how the photoreceptors are much thinner with the CEP290 mutation compared to normal photoreceptor cells.
In trials conducted by Editas Medicine, all subjects with LCA10 had this mutation at least once in the CEP290 gene.
The c.2991+1655A to G mutation in the CEP290 gene creates a cryptic splice site in intron 26 — a genetic mutation that inserts, deletes or changes a number of nucleotides in that specific site (in our case, insertion).
This creates an extra exon (known as exon X) and introduces a premature stop codon to the sequence. Essentially, this disrupts the function of the protein + significantly reduces levels of natural (or wild-type) CEP290 proteins. This reduction in proteins results in ceased development/production of photoreceptor cells.
This is the reason why CEP290 gene mutations are linked to retinal dystrophy — because photoreceptors cells are vulnerable to disruptions of cilia function.
Unfortunately, there are currently no approved therapeutic solutions to LCA10 — leaving many children in the dark and cold world of blindness.
However, some have shown promising results…
Potential Solution #1: EDIT-101, Editas Medicine
Resources (Research Paper, Video, and Patent):
Goal of solution: Introduce or delete bases in the genetic sequence of the CEP290 gene. Both approaches are intended to destroy the cryptic splice site resulting from the c.2991+1655A to G mutation, and in turn, increase + restore development of photoreceptor cells.
What’s interesting is that the outer segments of photoreceptor cells (which contain the CEP290 gene) are turned over every ~two weeks. Therefore, if we could potentially restore expression of the CEP290 gene, it would repair the outer segments of the photoreceptor cells.
In addition, what’s interesting with photoreceptor cells is that only 10% of the photoreceptors need to be active in order for you to see. In other words, the therapy, theoretically, only needs to be 10% effective in restoring the CEP290 expression.
I’ll get into why this is a difficult process, although it may sound easy in a future section.
Graphical Abstract:
The image above shows us the current stage of the problem (left) v. stage with the use of EDIT-101.
(Left) The point mutation in intron 26 causes an extra Exon (Exon X) to form between Exon 26 and 27 (the cryptic splice site). This results in a CEP290 protein that is truncated and non-functional.
(Right) With the use of EDIT-101 (the two gRNAs and Cas9), we are able to delete the point where that mutation occurs (a.k.a target point) and, in turn, prevent Exon X from forming in between Exon 26 and 27. This will allow us to maintain full-length and functional CEP290 expressions → create functional photoreceptor cells.
Process Overview:
The Process
CRISPR/Cas9 systems involve two things: gRNA and Cas9.
For the applications of EDIT-101, they needed to identify gRNA’s that would specifically guide the system to the ends of the point mutation.
Through studies, they narrowed their search down to seven pairs of gRNA
Out of these seven pairs, the most effective and active (for deletion rates) were gRNAs 323+64.
In order to verify the final effectiveness of this pair, they conducted another study with these conclusions:
On the right side, you can see the point mutation (in Intron 26). The grey represents wild-type (i.e. natural) CEP290 expression compared to mutated expressions.
With the point mutation (left), mutant expression > wild-type.
With the use of gRNAs 323+64 (in EDIT-101), mutant expression < wild-type.
This study was used as validation for the effectiveness of these two gRNAs.
Verification.
Now, with effective guides, they wanted to make sure that they were specific.
They conducted this process to test for guide specificity:
The most important test was Digenome-Sequencing — helps find where an enzyme (gRNA) cuts the DNA. This helps determine the chances of off-target effects (out of the possible ~150 off-target effects that they predicted).
For Digenome-Sequencing, they add the Cas9 + gRNA system to the genomic DNA where an off-target effect could potentially occur. After 16 hours, they conduct whole genome sequencing (WGS) on the DNA to see whether any off-target effects took place.
These were their findings on the test:
To the left, we have the control group, which shows that as the size of the guide increases, so do the rates of off-target effects. In the middle and right, we can see the guide 64 and 323 which has a much lower rate of off-target effects compared to the control group.
After this, they used human retina from cadaver (3–5 hours post-mortem) and created punches in the retina, which was cultured for 28 days (and transduced with clinical molecule: EDIT-101).
After 28 days, the DNA was extracted, and they verified that most of the ~150 off-target effects they previously predicted did not occur.
This is good. Not only were the guides shown to be effective, but they were also specific — limiting the chances of off-target effects.
Then, they wanted to see the relationship between the amount of dose (of EDIT-101) and editing of the CEP290 protein. They conducted this test on mice.
Through their experiment, what they realized was that, at a certain point, high doses of EDIT-101 actually reduced edits in the protein:
Shouldn’t a higher dose result in a greater number of edits?
No, the reason for why this is the case is because of protein over expression. The whole goal of this experiment is to correct the point mutation in the CEP290 gene which would, in turn, increase its production.
However, research has shown that over expression of CEP290 is associated with cellular toxicity. So, what we need to do in this case is to find the optimal point where we aren’t under expressing or over expressing this protein.
This is really one of the reasons why it’s difficult to achieve that >10% repair rate of photoreceptor cells — because there is that risk of over expression.
Examination.
IVT injection is the method that will be used to insert EDIT-101 into the retina. The challenge with this is that,
- We’re dealing with the eye — very sensitive, and
- This method doesn’t have much validation.
So, to validate whether this type of in vivo application could potentially work on humans, they tested it on non-human primates:
Their research concluded that, in all the animal subjects, tolerability of EDIT-101 was seen to be relatively high.
Their final experiment was then conducted on human patients. Because the complex was being delivered to the eye, they couldn’t remove the eye to see whether the gene was properly edited (as done with Digenome-Sequencing).
Instead, patients were tested on whether they could perceive different levels of light (which was previously impossible when the patient has LCA10).
These were their findings:
As you can see, from pretest → Day 59, the levels of light perceived in the retina were shown to be gradually increasing. In other words, the expression of the CEP290 protein was being restored and the development of photoreceptors were starting to show.
Challenges to Implementation
Delivery.
The delivery process of IVT injections is very risky because they’re delivering the CRISPR/Cas9 complex directly into the retina, which could cause adverse effects (such as mild anterior chamber inflammation — type of inflammation in the eye) which were seen in some subjects.
The typical method of delivery in GE would be through ex vivo therapies where cells are extracted, edited, and then inserted into the patient. The method for EDIT-101 is in vivo+ direct insertion into the eye, which is a novel approach.
Over-Expression.
This is extremely important for LCA10, because it has been shown that overexpression of CEP290 is associated with cellular toxicity. Hence, we need to find the optimal point where we aren’t underexpressing or overexpressing the CEP290 protein, but at the same time, allowing +10% of photreceptors to regain proper function.
Future of EDIT-101
With the trials starting to show success, it is providing validation for the potential use of EDIT-101 in LCA10 cases. However, there are still many challenges to implementation that we need to be aware of.
Currently, Editas Medicine is in early-stage clinical trials (Phase 1/2) where they are working to further evaluate the “safety, tolerability, and efficacy” of EDIT-101. To learn more about their research and pipeline, check out here.
Onto the next potential solution!
Potential Solution #2: QR-110, ProQR
Resources (Research Paper):
Goal of solution: The same as EDIT-101. Introduce or delete bases in the genetic sequence of the CEP290 gene. Both approaches are intended to destroy the cryptic splice site resulting from the c.2991+1655A to G mutation, and in turn, increase + restore development of photoreceptor cells.
Graphical Abstract:
The main goal of removing the cryptic splice cite formed between Exons 26 and 27 is very similar to EDIT-101. However, the major difference between EDIT-101 and QR-110 is how that process comes into motion.
The Process
29 oligonucleotides were designed in this experiment and screened with the goal of repairing the c.2991+1665A to G mutation and, in turn, increase production of wild-type CEP290.
An oligonucleotide’s function is to detect specific sequences of DNA and RNA (acts as a gRNA). In this experiment, it was to detect the LCA10 target point mutation (c.2991+1665A to G).
Out of the 29 oligonucleotides, they conducted another approach to identify the three best-performing oligonucleotides (leads 1–3). Specifically, they wanted to determine which lead would be more tolerable through IVT injection.
To determine this, they conducted studies on non-human primates.
They started off with a rabbit, which is known to be highly sensitive for IVT injections and often develops severe inflammation in the eyes.
Their testing concluded that Lead 2 was better tolerated than Lead 3.
Another test was conducted on another non-human primate — a monkey.
Monkeys were given an IVT injection of 60-100 μg of oligonucleotide per eye and examined for ~28 days. They concluded that the dose of Lead 2 was generally tolerated in monkeys. This provided validation for lead 2 (which is QR-110) as the best-performing oligonucleotide.
It was also shown that QR-110 (Lead 2) achieved a greater percentage of edits (on CEP290) compared to Lead 3:
For a visual representation, below, are the sequences for QR-110 (Lead 2), Lead 1, Lead 3, etc.:
They then tested QR-110 in a clinical setting and were able to demonstrate relatively high levels of CEP290 re-expression:
As seen above, QR-110 treatment resulted in a relatively high increase of CEP290 expression. With this, they knew that QR-110 was effective in targetting + destroying the cryptic splice site. However, as discussed with EDIT-101, the biggest challenge was application.
QR-110 Application Effectiveness
When discussing effectiveness, researchers wanted to ensure that QR-110 was being delivered and distributed to the proper location of the retina through IVT injections. They tested this on rabbits and mice.
After injecting QR-110, they examined the layers of retina in the subjects.
Their images used a fluorescent dye (6-Carboxyfluorescein or 6-FAM) and checked whether QR-110 diffused through the layers of the retina. Their studies indicated a rapid absoprtion of QR-110 in the retina and long retention in the retina.
Below, you can see the images take in the studies. As you’ll see, after 60 days, QR-110 fully diffuses into the retinal layers:
Results
10 μM of QR-110 treatment was shown to significantly increase the frequency (+50% increase) and average length of cilia (+27% increase). At this dose, the cilia of ~80% of photoreceptor cells had cilia length >1 μm, which is a functioning/healthy length.
As we can see in the image above, the cell % and length after the insertion of QR-110 was very similar (and in some cases, greater) than the control group (i.e. healthy group).
These studies acted as final validation for the effectiveness and safety of QR-110 applications.
The Future of GE Applications for LCA10
GE applications for LCA10 are primarily in early clinical trials. There are a lot of challenges regarding applications (as previously highlighted) — the major concern being implementaiton of these therapies and the potential dangers/risks of IVT injection.
Both EDIT-101 (below) and QR-110 are in early stage clinical trials.
The Future of GE
Vision is something we take for granted, but for those born with LCA (and other forms of genetic blindness), vision is something much greater.
GE isn’t only playing a role in restoring vision, it’s being applied for many other problems too. For example, kidney transplants.
Recently, scientists performed the first pig → human kidney transplant. They used GE technologies (specifically, CRISPR/Cas9) to turn off the genes in the pig kidney that would usually be rejected by the human body.
This allowed them to transplant that pig kidney into a human patient. The kidney was placed on the outside the body (shown below) and began functioning as normally as a human kidney.
Plus, with ~100,000 patients on the kidney transplant waitlist, this type of method could play a role in making kidney (and other organ) transplants more readily available to them.
So I say,
For the many patients in dire search of an organ transplant,
And for that kid who has had to live a life in darkness because of their vision,
There is hope for a better future.
