Using Gene Editing to Cure Hemophilia
You’re riding your bike. What if you fell. Maybe you scraped your knee. For most of us it might be a little scraped. If someone had hemophilia, instead of just bleeding externally, they would have internal bleeding, and this does not only happen after a fall. 60- 70% of hemophilia A cases have spontaneous and unprovoked, internal and external bleeding episodes that occur often. If it happens too often and bleeding is not controlled as soon as possible, it can cause permanent damage such as joint deformity and reduced mobility and can even be fatal.
What is Hemophilia?
Hemophilia is a normally inherited bleeding disorder where your blood does not clot normally because it does not have enough blood- clotting proteins.
It is caused by a deficiency in blood coagulation factors. These are basically genes that clot blood and encode key proteins in blood clotting pathways.
This disorder is an x- linked recessive disorder which means it is associated with mutations in genes in the x chromosome.
Chromosomes are inside all of us. They are what makes us, us. They are made of a protein and a single molecule of DNA. Humans have 23 pairs of chromosomes, so 46 in total.
22 of those 23 pairs look the same in both men and women. The 23rd pair is called the sex chromosome. This is what differentiates men and women. Females have two X chromosomes and males have one X and one Y chromosome.
If a man carries the mutation, they will be affected because they only have one X chromosome. Since it is recessive, if a female has one X chromosome with the mutation and one without, they will normally be unaffected.
There are two main types of hemophilia, hemophilia A and hemophilia B. Hemophilia A is caused by a deficiency of clotting factor 8 and hemophilia B is caused by a deficiency of factor 9. Both factors are made in the liver. Clotting factors are certain proteins in our body that cause blood to be thick. If there isn’t enough clotting factor, the blood is not thick enough to clot when there is any bleeding.
Current Treatments
Right now, the current treatments involve an infusion of recombinant factor 8 or factor 9. Recombinant factors are lab made products created with recombinant DNA technology which means they are not made from human blood.
This is extremely expensive, and the cost of hemophilia medication can range from 270,000 to 1 million dollars annually. In fact, children with hemophilia have been called million-dollar babies because that’s how much it can cost to help them survive. To put that in perspective, in 2020, the average household income in the US was 98 thousand. Most households can not afford the price of hemophilia.
Another problem with current treatments is that these factors have a short half-life (around 24 hours), so patients have to receive the treatment 2–3 times per week and some patients even generate a neutralizing antibody against the infused recombinant factor and develop immunity. This form of treatments is just not sustainable.
We needed to have a curative approach instead of a preventative one.
That is where gene editing comes in.
In gene editing, the mutated gene is revised, removed, or replaced. Gene editing can potentially be a life- time cure for hemophilia patients and early administration of the treatment to children could completely get rid of the need to do coagulation factor infusions.
Genome editing in general can be achieved by using technologies such as CRISPR Cas9 to create double stranded breaks at specific locations. CRISPR Cas9 specifically is comprised of a Cas9 molecule and a strand of guide RNA. If the genome is a word document on your computer, the guide RNA acts as a command find or search function and the Cas9 enzyme acts as a deleting tool or an enter/ space key as it makes a break in the DNA.
When these breaks happen, the cell tries to repair it using either non homologous end joining (NHEJ) or Homology Directed Repair (HDR).
Non homologous end joining is an error prone but efficient repair system which joins the two ends where the break occurred without using a DNA template and
Homology directed repair is a more specific system that uses a template to repair the DNA.
For a more detailed explanation of CRISPR, read this article!
These systems together literally edit genes. Just think about it. If you edit out a hereditary disease before the child is born, you can completely eradicate that disease! Even though this is definitely illegal right now… the possibility is there!
How can we use gene editing to address hemophilia?
Looking back on Hemophilia, we know that it is caused by a deficiency in clotting factors 8 and 9. We know that those clotting factors are made in the liver. So, what if we could design a gene that creates the clotting factor, and using CRISPR Cas9, insert it into the liver?
In a study done in 2019, scientists developed a genome editing therapy for Hemophilia A and did exactly that.
The B Domain Deleted Factor 8 gene is a coagulation factor 8 gene that has a deleted B domain. This basically means that this gene has been shown to create more clotting factor 8.
In order to test this, scientists created mice models. They used CRISPR Cas9 technology to delete a portion of the F8 gene in the mouse and then they had the factor 8 knockout mouse which is a model for hemophilia A.
Now, they had to get it inside of the cells and for that, they needed a vehicle or a vector. Researchers focused on using Adeno associated viruses as vectors.
Adeno viruses are a common family of viruses that are extremely similar to the flu. If you’ve had a fever, cough, or even a sore throat, it is very likely you have had it.
Adeno associated virus is that naturally occurring virus, but scientists have transformed it into a gene transfer vehicle or a vector used in gene therapy. They replace the viral DNA with new, non-viral, DNA and it is no longer considered a virus as the viral components have been replaced.
The scientists constructed 2 adeno associate virus vectors- one for the b domain deleted factor 8 gene, and one for the CRISPR cas9 guide RNA.
Then they injected both vectors through the tail of the mouse and the guide RNA located the specific spot in the liver that it was programmed to find (the albumin locus).
The Cas9 enzyme then cut at the DNA and releases the B Domain Deleted Factor gene into the liver. Then nonhomologous end joining kicked in to knock the new gene into the DNA.
The scientist tested bleeding times for each mouse and there were significantly lower blood losses in the hemophiliac mice that were administered this treatment. That meant that the bleeding disorder in the hemophilia A mouse model was cured by the treatment.
The Impact
This treatment doesn’t only change the lives of the hundreds of thousands of people who live with hemophilia, but it changes the way we approach gene editing. The technology of using adeno associated virus vectors along with CRISPR Cas9 can be used in so many ways such as curing blindness and deafness and even neurodegenerative disorders.
This treatment doesn’t only change the lives of the hundreds of thousands of people who live with hemophilia, but it improves AAV-mediated gene therapy approaches and supports the potential of a similar gene-editing approach that uses both CRISPR Cas9 and AAV vectors.
Paper Referenced
Hi! I hope you learned something and enjoyed reading this article! If you would like to connect, please reach out to me on Linkedin!
Talk to you soon :)
