Hemophilia A, also known as Classic Hemophilia, is a congenital, inherited, X-linked recessive disease caused by an F8 gene mutation located at Xq28, leading to a deficiency of clotting factor VIII (8). This type of hemophilia is five times more common than Hemophilia B, affecting approximately 1 in every 5,000 babies born [1], while Hemophilia B affects about 1 in 25,000. Hemophilia A commonly affects males more.

Hemophilia B, very similar to its counterpart Hemophilia A, is also an inherited X-linked recessive disease. It is caused by an F9 gene mutation located in Xq27, resulting in a deficiency of clotting factor IX (9). Hemophilia B is more common in males, affecting approximately 1 in 25,000-30,000 individuals [2]. An interesting fact about Hemophilia B is its association with royal families, earning it the nickname "The Royal Disease." It affected members of royal families, including Victoria of England, who passed it on to her son Leopold, leading to its presence in subsequent generations before eventually disappearing.

Differences between Hemophilia A & B. (source)

Furthermore, Hemophilia C is caused by a factor XI deficiency, specifically a lack of clotting factor XI (11) located at 4q32-35. Unlike Hemophilia A and B, it is not X-linked recessive but is autosomal recessive [3]. This means that instead of affecting a gene in the sex chromosome, it impacts chromosome 4. Hemophilia C affects both genders equally, and approximately 1 in 100,000 people in a population have Hemophilia C.

Acquired hemophilia is caused by autoantibodies. Autoantibodies are misguided antibodies that respond to your own body’s proteins instead of antigens [4] . In acquired hemophilia, the autoantibodies suddenly inhibit clotting factor VIII (8), hampering clotting function. Acquired hemophilia affects 1 in 1,000,000 of a population.

Normal Blood Coagulation pathways

The blood coagulation pathway is a complex cascade of events crucial for hemostasis. Triggered by vessel injury, it involves intrinsic and extrinsic pathways converging at Factor X activation. Factor X converts prothrombin to thrombin, which, in turn, converts fibrinogen to fibrin, forming a stable blood clot. Numerous clotting factors, including platelets, play pivotal roles. Regulatory mechanisms prevent excessive clotting, with anticoagulants like protein C and antithrombin inhibiting various factors. The balance between coagulation and anticoagulation ensures proper blood clot formation, preventing excessive bleeding or thrombosis. Dysregulation in this pathway can lead to bleeding disorders or clotting abnormalities.

Fig: Upon the lysis of blood cells, cell proteins leak out, initiating a chain reaction of aggregation. This process ultimately results in the formation of a clot containing fibrin and platelets. In Hemophilia patients, this specific blood coagulation process is absent due to various mutations, resulting in blood loss after any injury. In extreme cases, it can lead to fatality. (source)

Biology of Hemophilia

Extrinsic pathway starts when the tissue factor gets exposed by the injury of the endothelium. Tissue factor turns inactive factor 7 to activated factor 7a. (A for active) The tissue factor goes and binds with the factor 7a to form a complex that turns factor 10 to factor 10a.

The intrinsic pathway starts with platelets near the blood vessel injury activating factor 12 into factor 12a which then activates factor 11 to factor 11a and then activates factor 9 to factor 9a. Factor 9a works together with factor 8a to activate factor 10 into factor 10a.

Common Pathway: Factor 10a and factor 5a (cofactor) turns factor 2 (prothrombin) into factor 2a (thrombin). Thrombin then turns factor 1 (fibrinogen and soluble) into factor 1a (fibrin) which is insoluble and precipitates outside the blood at the injury. Thrombin also turns factor 13 into factor 13a together factor 13a and fibrin form a stable clot.

Biology of Hemophilia: Intrinsic and Extrinsic pathways of Hemophilia [Osmosis.org]

Coagulation Disorders

A common deficiency is a lack of factor 8, resulting in hemophilia A, characterized by only having von Willebrand factor. Another common deficiency is a lack of factor 9, causing Hemophilia B. Deficiency of von Willebrand factor leads to Von Willebrand disease, where factor 8 breaks down faster and can become deficient. Acquired causes of hemophilia include liver failure, as the liver synthesizes various factors. Vitamin K deficiency is another cause, as it is essential for the synthesis and release of factors 2, 7, 9, and 10. Autoimmunity against a clotting factor and disseminated intravascular coagulation, which consumes clotting factors, are additional causes of acquired hemophilia.

Etiology of Hemophilia

Males and Females

Males have sex chromosomes XY, while females have sex chromosomes XX. Children inherit one chromosome from each parent. Hemophilia A is more common in males because males only need one copy of a defective X chromosome, whereas females need two copies for symptoms. Carriers inherit only one defective X chromosome and often have minimal hemophilia symptoms. Carriers can pass the affected X chromosome with the clotting factor mutation to their children. There is a 50% chance of passing hemophilia to a baby boy and 0% chance for a girl if the father does not have hemophilia.

Fig: The possible outcomes of children with a carrier mother with a healthy father (left) and a hemophilia father with a healthy mother (right) (source)

Epidemiology of Hemophilia

Sex

More females and males in the HTC PP (Hemophilia Treatment Center Population Profile) tend to get hemophilia than in the Registry. In a recent CDC community count data, 6513 female hemophiliacs were in the Registry and 39881 in HTC PP. Also according to CDC’s data, 17626 males in the Registry and 46125 males in the HTCPP. Generally, the male population has more hemophilia than females.

Ethnicity

In terms of ethnicity, Hispanics, Latinos, and individuals of Spanish origin have a higher prevalence of hemophilia in the HTC PP compared to the Registry. According to the CDC, there are 4,162 Hispanic, Latino, and Spanish Origin individuals with hemophilia in the Registry, whereas there are 14,664 in the HTC PP. Similarly, other ethnicities also show a higher number of hemophilia cases in the HTC PP compared to the Registry, with 19,977 in the Registry and 71,342 in the HTC PP. Overall, individuals of other ethnicities have a higher incidence of hemophilia than those of Hispanic, Latino, and Spanish origin.

Race

Continuing with the discussion on race, CDC data indicates that among different racial groups, Whites have the highest number of individuals with hemophilia compared to Black or African American and other races. Specifically, Whites account for a total of 87,415 hemophiliacs, whereas Black or African American individuals have 10,478, and those from other races have 12,252. The table provides a comparison of hemophilia counts between the Registry and HTC Population Profile for Whites, Black or African American, and other races.

Patient Geographic Distribution

Geographically in the United States, states with the highest number of hemophilia patients, exceeding 1000, include California, Texas, Michigan, Illinois, Indiana, Ohio, Pennsylvania, New York, North Carolina, Florida, and Georgia. Following closely, states with 700 to 999 patients are Wisconsin and New Jersey. Subsequently, states with 500-699 hemophiliacs are Massachusetts, Minnesota, and Virginia. States with 300 to 499 hemophiliacs include Washington, Oregon, Arizona, Colorado, Missouri, Kentucky, Tennessee, and Maryland.

Genetic Engineering

Genetic engineering is a complex process involving the deliberate manipulation of an organism's genetic material. The process typically begins with the identification and isolation of a gene of interest. Using molecular tools like restriction enzymes, scientists cut the gene from its source and often modify it by adding, deleting, or altering specific segments. The modified gene is then inserted into the genome of the target organism using vectors such as plasmids or viral carriers. Once integrated, the gene becomes part of the organism's DNA and directs the production of proteins with desired functions.

This technology finds applications in various fields, from creating genetically modified crops with improved traits to developing gene therapies for treating genetic diseases. The process of genetic engineering raises ethical considerations, and ongoing research aims to refine techniques and ensure responsible applications in agriculture, medicine, and other domains.

The first approved gene therapy for Hemophilia A, Valoctocogene roxaparvovec, uses a modified adeno-associated virus to carry factor VIII to liver cells, addressing the bleeding disorder. Etranacogene dezaparvovec is the first gene therapy for Hemophilia B, utilizing a virus to deliver factor IX to liver cells. These advanced therapies come at a high cost, with Valoctocogene roxaparvovec priced between 2 to 3 million dollars.

Gene Editing Tools

There are three types of genome editing tools: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and and clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9). ZFNs are fusions of nonspecific DNA cleavage domains of the Fok I restriction endonuclease that recognize a specific DNA sequence and cut it. TALEN gene editing is a fusion of the Fok I cleavage domain and DNA binding domains made from TALE proteins.

TALE recognizes a single base pair and makes a double-strand break, similar to how ZFNs work. CRISPR/Cas9 consists of a Cas9 nuclease from bacteria and two RNAs: CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). The RNA complex allows Cas9 to cut DNA near a PAM (protospacer-adjacent motif) site. Then the body undergoes either nonhomologous end-joining or homology-directed repair (using a template).

Gene therapy Strategies

There are two types of gene therapy strategies.

For in-vivo gene transfer, genetic material like viral vectors, nanoparticles, and ribosomes is delivered directly to the patient, and genetic modification occurs in situ or in the natural or original position.

For ex-vivo gene transfer, cells are harvested from the patient and modified by gene delivery tools such as genome editing technologies, recombinant viruses, or viral vectors in vitro, which is performed elsewhere outside a living organism. The modified cells are then tested for off-target effects and cultured before being returned to the patient.

Wider application of Genetic Engineering

Genetic Engineering can be applied to various aspects, not just the treatment of diseases. It is used to modify crops, making them resistant to bacterial infections, herbicides, droughts, and diseases, thereby increasing yield per acre and helping farmers boost profits. Additionally, genetic engineering can enhance the taste, growth rate, nutritional content, and shelf life of crops.

In vivo and Ex vivo Strategy Diagram (source)

However, Genetic Engineering is banned in nearly fifty countries due to ethical and safety concerns. There is worry about non-GMO crops cross-pollinating with GMO crops, potentially disrupting the ecosystem. Concerns also exist regarding the ethical implications of genetic engineering in humans, where altering genes in sperm or egg cells may impact the treated individual and their future offspring. The concept of designer babies, enhancing "normal" people to create "perfect" humans, raises ethical questions, particularly regarding access and affordability, with only the wealthy able to afford such enhancements.

Gene delivery Agents

The most efficient method for gene delivery is transduction, where naturally occurring viruses are modified and used as vectors. Viruses, having evolved to transfer their DNA into cells, make effective carriers. The widely preferred choice for gene therapy in monogenic disorders is the Adeno-associated viral vector (AAV). Approved AAV vector treatments include Luxturna for Leber congenital amaurosis, Zolgensma for spinal muscular atrophy, and Glybera for familial lipoprotein lipase deficiency.

Parvovirus

AAV vectors are derived from parvovirus, known for being the safest virus vector. It poses no harm to humans, induces minimal immune response, and cannot replicate independently, relying on another virus for replication. The recombinant AAV vector lacks wild-type viral coding sequences, reducing the chance of triggering a cell-mediated immune response. This vector efficiently targets both dividing and postmitotic cells, and its manipulability allows for codon optimization, enhancing gene expression and translation efficiency. The primary limitation of the AAV vector is its confined space, allowing packaging of only 5.0 kb (kilobase) of DNA.

Gene Therapy in Hemophilia

In hemophilia B, AAV gene therapy is effective due to the relatively small size of the factor 9 gene and a less complex expression pathway compared to factor 8. A study at Children’s Hospital of Philadelphia with 7 participants showed initial success, but one experienced a decline in factor 9 levels, attributed to a cytotoxic T-cell response.

Diagram showing steps of how the AAV vector gene therapy works (source)

A trial sponsored by St. Jude Children’s Research Hospital and University College London achieved sustained factor 9 expression using AAV serotype 9 capsid, a noninvasive vector administration, and a mini-factor 9 expression. Over 10 years, participants experienced reduced spontaneous bleeding, improved quality of life, and no long-lasting liver damage or toxicity. However, transaminitis, elevated liver enzymes, remains a concern, especially at high vector doses. Ongoing studies demonstrate varying degrees of factor 9 activity, indicating progress in gene transfer technology for hemophilia B.

Hemophilia A: Unlike Hemophilia B, Hemophilia A faces challenges due to the limited packaging capacity of AAV vectors and poor expression profile of factor 8. To address this, the factor 8 B-domain, unnecessary for cofactor activity, is removed to reduce its size, and factor 8 expression is enhanced by reorganizing the wild-type cDNA of human factor 9. In the first clinical trial using AAV 5 pseudotyped vectors for Hemophilia A, 13 men with severe hemophilia A were tested.

Pros and Cons of Hemophilia Gene Therapy

Advantages of Gene Therapy:

Gene therapy holds immense promise in treating genetic disorders by addressing the root cause at the genetic level. It provides a targeted approach, potentially offering cures for diseases that were once considered incurable. The treatment can be personalized, tailored to an individual's genetic makeup, leading to more effective and precise interventions. Successful gene therapy may eliminate the need for ongoing treatments, providing long-term solutions.

Disadvantages of Gene Therapy:

Despite its potential, gene therapy poses challenges and concerns. Safety issues include the risk of unintended side effects, immune responses, and long-term unknown consequences. Ethical considerations arise due to the manipulation of genes, raising concerns about creating "designer babies" and altering human traits. The effectiveness of gene therapy varies, and its high cost limits accessibility. Delivery challenges, potential overstimulation of cellular processes, and regulatory hurdles add complexity to the development and adoption of gene therapies. [8]

Advantages and Limitations of Gene Therapy

Next generation Gene Therapy for Hemophilia

In the next-generation gene therapy for hemophilia, transgene engineering strategies are employed to enhance factor 8 synthesis while minimizing the risk of anti-AAV neutralizing antibodies and reducing cellular stress. A pre-clinical study using a chimeric human-porcine transgene in humanized liver mice demonstrated a ten to a hundred-fold increase in factor 8 synthesis with reduced cellular stress. This chimeric transgene achieves highly sufficient levels of factor 8, allowing for effective and long-term treatment with a lower AAV dose. The ultimate goal of gene therapy is to advance the understanding of transgene insertion and liver-targeted gene therapies, aiming to create the safest, most durable, and stable gene therapy for replacing or supplying missing or defective proteins in various medical conditions.

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Impact Statement

My name is Matthew Liang. I chose to research on Hemophilia. I went into this program interested in genetic engineering and how gene therapy can help out patients with serious genetic disorders. Hemophilia interested me because it is a type of genetic disorder that has already gotten an approved gene therapy treatment, allowing me to research and explore more about how gene therapy works and deeper understanding of what gene therapy provides. ERP strengthened my skills in how to research such as knowing what sources to look for, how to structure the research, how citing works, the overall process of research, and even how to write more sophistically. I now know more about what research is like. During my research, I learned a lot about the etiology, epidemiology, types, biology, and coagulation pathways of Hemophilia. And also learned more about genetic engineering such as its processes and benefits and how it can help treat this disease.

References

1. Haemophilia

2. Hemophilia C (Factor XI Deficiency): What It Is, Symptoms & Treatment.

3. Acquired hemophilia A: Updated review of evidence and treatment guidance

4. Acquired hemophilia

5. Women and Girls with Hemophilia Receiving Care at Specialized Hemophilia Treatment Centers in the United States - PMC

6. Bleeding Disorders Data Visualization

7. Gene therapy for hemophilia

8. Historic Overview of Genetic Engineering Technologies for Human Gene Therapy - PMC

9. Gene therapy for hemophilia

10. Hemophilia A gene therapy: current and next-generation approaches

11. What is Hemophilia | CDC

12. Hemophilia Life Expectancy and Primary Causes of Death

_By: Matthew Liang_

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of Elio Academy.