Remember physical education back in middle school? The reaction depends on the person, but imagine running that mile? Whether it was dread or excitement that filled you, most of us remember running around the track four times within a certain time range to pass. After a certain point, sprinting can start to hurt your feet or lungs, and we may think that running is torture sometimes, but it’s important to be grateful for the ability to run. Unlike at least 400,000 people living worldwide with hemophilia, anyone without this disease is able to run without internal bleeding from the impact of running. While the severity varies, many people with hemophilia get swollen joints and bleeding that cannot be quickly repaired because of this genetic disorder.
Introduction to Hemophilia
Hemophilia is a genetic disease, occurring for 1 in 5,000 males around the world. Mostly males are affected because of the inheritance pattern of this disease, and women are mainly carriers, but women can be affected as well. Hemophilia is an x-linked recessive disease. It’s important to know that males have one X and one Y chromosome, while females have two X chromosomes. Because the disease is recessive, if a female only has one mutated gene for hemophilia, she will be a carrier (females need two genes to have hemophilia which is much more rare). However, if men inherit one copy of the gene, they will have hemophilia, since they only have one X chromosome and no dominant/ non-mutated gene to cancel out the mutated and recessive hemophilia gene.
There are two types of hemophilia (A and B), but I will be focusing on hemophilia A, which is more severe and accounts for about 85% of all hemophilia cases. Hemophilia A is a genetic mutation occurring at the end of the long tail of the X chromosome (Xq28 is the exact chromosomal location), where the F8 gene is located. Within this gene, there are thousands of different known mutations that can occur, but the most common is an inversion of intron 22.
An inversion is a specific type of DNA mutation where a section of DNA is flipped or rotated 180°, and when this happens, the ends of the DNA do not match up properly and the codon sequences and proteins produced can be altered. In severe hemophilia A, the section of gene F8 from exons 1 to 22 is rotated. In this article, I will be outlining the procedure and results of using gene editing technology to rotate this inversion and to revert the gene back to the normal sequence.
In hemophilia A specifically, the inversion causes coagulation factor VIII not to be produced in sufficient quantities. If someone has hemophilia, they will often have internal bleeding, many bruises, and deep cuts that won’t heal very quickly because their blood does not clot easily. Factor VIII is a main protein for the process of blood clotting. It is made by liver cells and remains inactive in the bloodstream when it is bound to factor von Willebrand, another biological molecule. When factor VIII breaks apart from this molecule, it activates, and then interacts with other proteins to form a blood clot. While the symptoms depend on the severity of hemophilia, people with severe hemophilia have a lot of pain and issues because of the lack of the protein.
However, the quality of life for patients can be greatly improved with even a small increase in the amount of protein produced. Hemophilia is also a good target for gene editing therapies since the mutation that decreases the production of factor VIII is usually a single mutation. Single gene disorders are more realistic and relatively simpler to treat than multi gene disorders, but we still have to be careful during the process, making sure to use the most effective primers, looking at off target possibilities, and more during the process.
Utilizing Gene Editing and Biotechnology to Develop Treatments
In humans, multiple different treatments for in vivo and ex vivo gene editing have been tested. I focused on using TALENs (Transcription activator-like effector nuclease) combined with induced pluripotent stem cells for ex vivo editing. The use of AAV vectors and CRISPR was also an option, but this technology is only being developed in mice right now and AAV vectors may not be able to invert such a long section of DNA while iPSCs have great potential for curing hemophilia.
Before we dive into the process, TALENs is a type of gene editing technology that uses proteins and nucleases to cut DNA at a certain location by targeting a sequence of base pairs that corresponds to the sequence of 33 amino acids that make up the protein. TALENs are much more economically viable and feasible compared to ZFNs, but not as simplistic or affordable as CRISPR.
IPSCs (induced pluripotent stem cells) are somatic cells that are reprogrammed to have the abilities of embryonic cells. For any Harry Potter fans, they are kind of like polyjuice potion for cells: when engineered properly and carefully, they can shift body cells (usually blood cells or skin cells) into embryonic cells that can be used for any purpose. Since these specialized cells can essentially become any other cell, they are extremely useful and helpful for drug development, regenerative medicine, and of course, gene therapy, and more.
For my purposes, I used Benchling software to simulate the mutation and the process of correcting it. As mentioned previously, I focused on one specific mutation (the most common inversion of intron 22), but some papers and studies have explored other mutations as well. In Benchling, I replicated the technique by first creating the inversion and then using the correct primers and enzymes to revert the inverted section. Not sponsored, but Benchling is a great free tool for digital gene editing and allowed me to mostly replicate Wu’s experiment fully online.
Based on the research done in three different papers, TALENs seems to be the best current method for treatment, but in a short while, CRISPR may prove to be better. With CRISPR, we would also be using AAV vectors most likely, which don’t have the best success rate right now because of off-target effects and the size of the mutation. The inversion 22 mutation in hemophilia a is a large mutation in the F8 gene. Out of the 26 exons that make up the gene, exon 1 to exon 22 are reversed. This changes the DNA sequence and does not allow the produced proteins to be secreted into the bloodstream to activate and then to clot blood.
Intron 22 is the largest intron, and there are other genes within this one to also know about. F8A is a small gene within the first section of intron 22 (called int22h1). F8B is also located in this intron. The interesting part about intron 22 is that there are two genes, int22h2 and int22h3, located outside of the F8 gene itself. Crossing over can occur between int22h1 and one of the other two sections, leading to the sequence being reversed and the inversion occurring. Because these important sections of the gene are out of place (and most of the gene itself is reversed), the factor VIII protein is at extremely low levels in the body (less than 1%).
In one of the studies, the iPSCs were generated from urine cells that developed into human embryonic stem cell clones about three weeks later. After analyzing the colonies of cells, specific colonies were chosen to be used for the experiment when they had the proper proteins, such as OCT4, SSEA4, and others.
Another good method of creating iPSCs is to use epithelial (skin) cells or skin fibroblasts. Blood cells are also commonly used. By introducing certain reprogramming genes, including the POU5F1 gene that encodes the previously mentioned OCT4 protein, these cells can act as any type of cell that scientists want to test on. Yamanaka’s lab showed that introducing the OCT4, MYC, SOX2, and KLF4 genes to somatic cells produced iPSCs. The proteins NANOG, LIN28, and GLIS1 are also important and can be used in place of some genes to create iPSCs. This discovery was important and advanced regenerative medicine as it made it possible for scientists to test discoveries with embryonic stem cells without using actual human embryos.
Using TALENs, specific nucleases create double strand breaks at points of the DNA. In the study that used urine cells as the base of the iPSCs, they used two TALENs — L1R1 and L2R2 — to cut the intersection of exon 22 and intron 22, where the mutation inversion begins. They used the primers F1R1, F2R2, F3, and R3 as well.
This study also used PGK-Neo cassettes inserted into linearized NheI as the donor vector type. The coding sequence of exons 23 to 26 (non-mutated section) was put into the vector to be moved into the space between exon and intron 22. The cassette, coding sequence, and primer make up the vector that can be cut out, copied, and pasted to another location in the DNA.
Since the mutation causes the large section of the gene (between exon 1 to exon 22) to be inverted, instead of moving this section that makes up 22 out of 26 exons of the total gene (over 85% of the total gene), they decided to fix the order by moving exons 23 to 26, a much easier task in terms of gene editing. Within the iPSCs, this method fixed the transcription of the mRNA of the F8 gene and allowed the factor VIII protein to be secreted!
Challenges of Using Gene Editing as a Treatment
As of now, the method of using TALENs seems to be the most effective and realistic current way of combating inv22 in severe hemophilia A, but there are some complications and promising CRISPR techniques also in development. With any gene editing, there is always the possibility of off target effects when the double strand break occurs in the wrong location. This could lead to another section of the gene being mutated — sometimes harmless, but sadly, sometimes fatal. Using iPSCs is also a challenge as they have the risk of tumors being formed when the cell undergoes mitosis rapidly. When the DNA heals itself with non homologous end joining, errors can occur and have the same consequences as off target effects where the DNA sequence could be mutated and essential protein production could be altered. Using homology directed repair is less error prone, but is also less efficient and still needs to be thoroughly tested.
We’ve all heard of CRISPR constantly in terms of gene editing and for good reasons: it’s simple, affordable, efficient, and accurate. Based on an experiment done by Dr. Chen and colleagues, it is possible to use CRISPR and AAV vectors to correct the mutation in mice and produce sufficient amounts of the factor VIII protein from the liver. The scientists cautioned against off target effects once again and some difficulties with using AAV vectors. Since this trial is only being done in mice right now, there are still many stages of trial and error that this research must get through, but with some advancements, CRISPR could be the best solution in the future.
In the end, many more treatments will likely be developed soon, but it’s important to try to come up with effective treatments fast to improve thousands of lives. This specific mutation (inversion 22) accounts for 40–50% of severe hemophilia A cases. By coming up with a cure, we are able to use our technological advances to help thousands of people suffering from this disease. Once these research proposals discussed in this article pass through clinical trials, these can be amazing approaches to diseases that have been thought to be untreatable for a long time. Knowledge really is power, and if humans can safely and ethically use genetic modification techniques to help others, why not? This is a debate for another time, but the treatments of hemophilia A being produced are hopeful, and hopefully they soon become a reality.
If you don’t have hemophilia, be grateful the next time you go out to run or walk without internal bleeding. Genetic disorders are often overlooked as they are seen as incurable, but through modern scientific discoveries, there is hope that soon all genetic disorders will be permanently curable.
Sources
Research Papers Used
- In Situ genetic correction of F8 intron 22 inversion in hemophilia A patient-specific iPSCs
- Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs
- Genome-editing technologies for gene correction of hemophilia
- Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII
- Factor VIII genetic mutations and protein alterations in hemophilia A: A review
- Hemophilia: A Practical Approach to Genetic Testing
- Gene therapy for hemophilia: what does the future hold?
Other Sources
Thank you for reading! Please reach out to me to talk or ask any questions at my email address: riadani27@gmail.com.
