Gene Therapy for Hemophilia

Creative Commons Attribution License 4.0 Abstract Hemophilia is an X-linked recessive genetic disorder in which the body has an inability to clot, leading to an increased risk of mortality for individuals if a bleeding episode were to occur. Traditional hemophilia treatments, such as prophylactic factor replacement therapy of recombinant factor VIII and IX, have been proven to be costly and do not provide long-lasting protection during bleeding episodes. In recent years, the use of Adeno-associated viral vectors (AAV) gene therapy has been explored as a potential alternative due to efficient gene delivery and tissue tropism, overall safety and efficacy, and longer-lasting effects. However, concerns over inhibitor development persist due to the treatment complications. This review article seeks to provide an overview of the current state of AAV-based gene therapy as a treatment for hemophilia. Gene Therapy for Hemophilia


Introduction I. Background Information
Hemophilia is a bleeding disorder associated with di culties in blood clotting. These complications are caused by the low levels of factor VIII, associated with Hemophilia A, or factor IX, associated with Hemophilia B.
The amount of clotting factor de ciency determines the severity of hemophilia for each patient [1][2][3] . Hemophilia a ects over 30,000 individuals in the United States. Over half ( around 60%) of that population is diagnosed with severe hemophilia while fewer su er from moderate and mild hemophilia. Hemophilia A is four times more common than Hemophilia B and is one of the most widely distributed bleeding disorders in the population 4 . There are three classes of hemophilia: mild, moderate, and severe. The severity of hemophilia is de ned by the percentage of clotting factor de ciency. A healthy range of factor VIII and IX is between 50-150%factor activity. Individuals with mild hemophilia have 5-40% factor activity where bleeding proceeds injury, trauma, or surgery. Moderate hemophilia has 1-5% factor activity where minor injuries or spontaneous bleeding occurs. Severe hemophilia, the most common, is classi ed by <1% factor activity where frequent exterior or interior bleeds may physically disable the individual 5 . Current non-gene therapy includes methods such as direct infusion of the exogenous proteins 6 . These speci c methods that are non-gene therapeutic do not provide long-lasting e ects due to inhibitors and antibodies. Replacement therapy is e ective until an alloantibody is formed against the exogenous clotting factors 7 .

II. Emerging Technologies
When researching gene therapies for hemophilia, there were an abundant amount of other gene therapies shown to be e ective and helpful. Some studies used the method by adding more precursor proteins to make the factors FVIII and FIX, the liver will be able to produce thrombin and platelets for hemophilic patients. In other words, these studies experimented by adding to the protein pathway rather than the genes of the factors; they added factors to help build the precursors of the factors or necessary stabilizers for the factors. One of these methods is called transgene therapies.
There are many transgene therapies that evolved for various diseases as one study suggests 8 . One of the transgene therapies is the FIX-Pauda and robinhood gene therapy which in short is the transfer of FIX from a patient who has an abundance of FIX, FIX-Padua proband, to a patient with a low abundance, HB patients. This will help hemophilia patients regain their FIX levels. The limitation to this technique is the safety issue of immunogenicity and thrombogenicity as the doses were of concern.
Including other gene therapies, there are additional useful therapies not using the Another method explored was activating more of the existing Factor VIII if they are turned o in presence of Thrombosis, such as an inducible vector. Factor VIII is a cofactor in the coagulation cascade and is primarily produced by endothelial cells. A study by Alam et. al. observed that because thrombin regenerates itself with the increased amount of platelets and itself in the blood in the coagulation cascade it is important to have thrombin in the body. By endothelial injury the release of thrombin will amplify the activation of Factor VIII by re-detachment of Factor VIII from the von Willebrand factor and Factor VIII complex. Two patients who had undergone both arterial and venous thrombotic events before observing an increase in Factor VIII 9 . Von Willebrand Factor (vWF) is a major contributing factor to the availability of Factor VIII (FVIII) as vWF is a speci c carrier protein that protects FVIII of proteolytic enzymes and from degradation 10 . This insight of the activation of Factor VIII suggests that there might be an activation method to make the Factor VIII protein more readily available to the coagulation cascade and help with blood clotting.
This activating more of the existing Factor VIII if they are turned o in presence of thrombosis can prove to be a consideration in hemophiliac studies. Another study also included alternating the amino acids on Factor VIII will make it more readily activated. A study by Nogami et.al. conducted an in vivo experiment which included altering the 372 amino acid position of an Arginine protein on the factor VIII protein to a Histidine protein to make it more detachable from the vWF-FVIII complex.
Although this is a novel in vivo approach, the study saw little to no change in the cleavage rate with the amino acid change suggesting that there may be other properties associated with the thrombin and the vWF-FVIII complex cleavage mechanism 11 . This inducible mechanism can also be used in the AAV vector that is an emerging technology. These mechanisms are SIN vectors which contain an inducible package of proteins that can turn the expression of the vector on and o determined by a protein presence.
Particularly the protein would function as an antibiotic such as doxycycline or a small molecule 45 .
Furthermore, another method that was proven to be e ective is site-speci c bioconjugation which alters the activation of Factor VII which triggers the whole cascade of coagulation as suggested by Lieser et. al 12  property, it can also be used as a perioperative treatment plan in the means of managing bleeding before surgery based on the patient's needs. In terms of Hemophilia, this study and product took into consideration the intensity of bleeding of patients with hemophilia and treated people with severe hemophilia (two or more bleeds per week) two times weekly 30-40 IU/kg. Patients with a low bleeding tendency were treated every 5 days or twice weekly just as the high intensity patients as weekly treatments did not show e ciency. At the end of the study, there was a decrease of bleeds per week in patients in total 14 . In research studies, intensity or severity of hemophilia is de ned by measuring FVIII or FIX activity as suggested by a review paper by Samelson-Jones et. al.. This is again based on the potency of protein factor products and monitored post-infusion processes. Additionally, two methods to test (if this information is going to be included this information, it would be helpful to elaborate on what these tests look like) out this are run through One-stage clotting assays (OSAs) or Chromogenic substrate assays (CSAs) which can help de ne or identify the severity of hemophilia in the patient 15 .
Lastly, there are a number of non-viral ways to transduct genes. Some of those methods include electroporation, cationic proteins, cell-penetrating peptides, nanoparticles, CRISPR editing genes, and virus-like particles (VLPs) 45 .

III. AAV Gene Therapy
The most popular and most successful emerging therapy to treat hemophilia relies on the use of an Adeno-Associated Viral Vector (AAV) as a vehicle for gene delivery of factor VIII and IX. AAV-gene therapy is popular due to its e cacy, relatively low invasiveness, minor side e ects in animal models, and its ability to provide long-lasting expression of factor VIII and IX. Despite these advantages, however, the presence of neutralizing antibodies from previous exposure to AAV remains a challenge that obstructs this therapy from being widely accepted. With minor concerns over inhibitor development, AAV-gene therapy is proving to be a potential treatment for hemophilia patients.

AAV Vector Advantages and Uses
A particular gene therapy that is a gene therapy model that is being explored right now and very successful in various clinical trials. Adeno-associated virus (AAV) vector is a new technology that is emerging into the scienti c community as an e ective method to amend the existing molecular DNA sequence of cells, without incorporating the virus's DNA into the host's DNA sequence 16 . There are many advantages to AAV which are used for a plethora of diseases within the body including neurodegenerative diseases, cancer, and genetic diseases as a whole. This gene therapy model is being explored currently in various methods and research papers to observe new DNA appending techniques 17 . This gene therapy is nonpathogenic and has many uses to express genes of interest.
Additionally, there are new genetic sequencing methods that allow for the design of vectors/plasmids and target speci c parts of the body. Because of its great amendable characteristics to genetic engineering and repurposing makes it easy to design relative to previous gene therapy techniques suggested by a review by Andari et. al. 18 . This not only makes it easy to design but increases the cell speci city, cell or organ targeting, and transduction e ciency. AAV has shown to be non-pathogenic and induces a minimal in ammatory response in mouse models and early human clinical trials. Furthermore, AAV has fewer biosafety hazards, unlike other gene therapies suggested by a review by Aschauer et.al., while having a low immunogenicity and limiting the risk of insertional mutagenesis or other mutational changes when replicating 17 . Other gene therapies focused on providing the proteins necessary for transduction while we wanted to focus on the core protein that is stopping transduction and see what was lacking in the pathway. The coagulation cascade is a concept that is still being explored as many elements play a role in the triggering events. A general outlook on the cascade is started with a stimulus from the external environment such as a cut or broken tissue increasing the amount of the protein thrombin to form the extrinsic pathway. A set of triggering events will then increase the von Willebrand factor and factor VIII to interact with factor IX. Factor VIII and IX are the main factors necessary for transduction which will trigger factor 10 to convert prothrombin to thrombin. Thrombin activates factors V, VIII, IX and XI, to promote its own generation. Thrombin then will then activate soluble brinogen to convert into an insoluble version of itself called brin to make the webbing of the blood clot in the location of the cut or tissue breakage. There is a plethora of gene therapy methods explored that focus on providing the proteins necessary for transduction, and the main protein factors being factors VIII and IX. AAV gene therapy focuses on promoting factors VIII and IX which stop transduction and make up hemophilia 19, 20 . Additionally, the size of the target sequence of the factor needed to insert into the is important to consider 42  As AAV2 was kept as one type of control, changing the serotype of the vector greatly changed its tropism and e ectiveness to help the body. Vector serotypes 1, 5, and 4 were tested to be more e cient in transducing cells in the murine nervous system or within the mouse model. Because the liver is shown to have more a nity to particular capsid proteins of di erent serotypes, vectors 8 and 9 had more a nity to the liver greater than that of serotype AAV 2. AAV vector 8 was very readily transduced regardless of the method it was transferred through; it was equally e cient in both the

Limiting Factor
However, the transduction of AAV particles into the liver is limited by the ability for ssDNA to be converted into dsDNA, as the viral mechanism is dependent on host machinery 23 . To solve this problem, a study by Nathwani et. al. devised a liver-restricted mini-human factor IX (hFIX) expression cassette that allows AAV DNA to be packaged as dimers 24 . As a result, hFIX expression in mice models produced a 20-fold increase compared to similar single-stranded AAV vectors (ssAAV). Advantage of AAV technology is that di erent serotypes have the capability of targeting speci c tissues. AAV2 serotype has been shown to minimize tissue tropism as its primary transduction comes from hepatocytes. This also was bene cial in that it allowed researchers to use fewer AAV particles in hemophilia patients, thus lowering cytotoxicity.

Gene Target Sequence
After designing and analyzing which AAV vector to use for such an experiment, a good gene target sequence is necessary to insert into the AAV vector to express the needed vectors. Many pharmaceutical companies such as BioMarin Pharmaceuticals, Spark Therapeutics, P zer, and UniQure 25 .
The following companies have already designed and inserted the target FVIII or FIX gene sequence gene therapy products into the vectors and are in clinical trials evaluated in phase III studies. A particular review paper by Doshi et. al. 26 discussed clinical trials that suggested broadening the gene therapy vector applicability with patients who had pre-existing neutralizing alloantibodies to the vector or in other words the clotting factors. Here the vector genome is manipulated so that the needed gene target sequence can replace the vector sequence under a tissue-speci c promoter to make a recombinant AAV vector (rAVV). In totality, the completed modi ed AAV vector in order included the ITR, promoter, inhibitor, transgene, polyA, and ITR. The results varied based on the way the AAV vector was given via skeletal muscle, liver-directed, and intramuscular trials.

AAV Administration
Re ning administration of AAV gene therapy so as to optimize "uptake" of vectors, induce expression, provide longer-lasting e ects, and reduce liver damage/minimize immune system e ects.  22 . This method also retains the AAV gene expression for a longer period of time in the body.

Dosage
Scientists must determine the correct dose of AAV vector particles injected into the patients, as a low concentration may not produce a robust amount of expression, while a dosage that is too high may cause an immune response and liver damage. Scientists have sought to address this issue by increasing the transduction of the vector into cells such that fewer vector particles are needed and thus limiting host immunity.

Minimizing Immunogenicity
There are two ways to decrease immunogenicity; one such measure refers to a process known as Site Directed Mutagenesis. Two vector serotypes currently being used in clinical studies, rAAV-DJ and rAAV-LK03 are chimeric receptors of natural AAV serotypes. A study by Ran et. al. (2020) showed that site-directed mutagenesis (rAAV-DJ-S269T) yielded higher transduction e ciency compared to wild-type AAV vectors, likely due to evasion of the host immune response 27 . The second method of re ning the AAV vector for administration is the use of directed evolution 28 , where a wild-type AAV vector is used to generate large mutant capsid libraries and AAV2 variants with enhanced properties, such as immune evasion, non-infection of resistant cell types, and tissue transport.

Inhibitors
The immune system develops antibodies to defend against foreign agents.
Patients with hemophilia sometimes develop antibodies to FVIII in response to treatments and medication. Polyclonal high-a nity immunoglobulin G (IgG), or an inhibitor, prohibits clotting factor activity that further promotes bleeding without clot formation. Type I and Type II inhibitors vary based upon the extent of inhibition of clotting factors. The development of inhibitors is a widely understood phenomenon that continues to burden treatment e ciency. Its e ects are being studied through both genetic and environmental factors that contribute to formation. The detection and quanti cation of FVIII inhibitors include the Bethesda assay and the Nijmegen-modi ed Bethsda assay 29, 30 .
Inhibitor formation contains treatment-related risk factors that include age and intensity at rst exposure, prophylaxis, and the type of treatment (recombinant or plasma derived). The e ect of age was observed in a population of hemophilia A patients with the onset of FVIII therapy within 1 year of age. It was found that patients that started therapy earlier in life are more prone to inhibitor development. However, there is a lack of knowledge on the e ects of delayed treatment initiation due to the tendency for patients to require therapy in early age 31 . The intensity of treatment measured by scheduled gaps between series of exposures showed that shorter gaps were re ective of increased risk of inhibitor development. Prophylaxis is a treatment that exposes patients to antigens to minimize the possibility of immune response to additional treatment. It was found that prophylaxis reduces probability for the development of inhibitors 32 .
Pre-existing immunity from neutralizing antibodies or inhibitors can be overcome by creating alternate AAV serotypes 33 . The success of serotype-switching is contingent on tropism similarities of the new serotype and the lack of crossreactivity between the serotypes. The criteria has been proven di cult upon the high rate of crossreactivity in trials that prohibit successful e orts 34 .
Immune tolerance induction (ITI) is currently the only proven treatment to eliminate inhibitor development. Despite a 60-80% success rate, the high cost creates nancial di culties for patients in need of this treatment. There is potential for gene therapy to serve as an ITI-type therapy to eradicate the need for ITI due to the continual production of clotting factors 35 .

Alternatives to Gene Therapies
While AAV-gene therapy has made the most progress in the future treatment for hemophilia, new research has come out in an e ort to bypass the concerns of AAV explained previously. A recent study published by Song et. al. (2022)

Conclusion
The purpose of this review article is to provide an outlook on the current state of gene therapy as a treatment for Hemophilia. Results of recent clinical trials have proven to be e ective in restoring clotting ability in hemophilic patients. By identifying key concerns with gene therapy, scientists are taking steps to account for these problems by utilizing the following collection of sources and information to make an impact on their research The ndings that we make will help other researchers improve upon this research to have a collection and a review composed of AAV vectors to use in their clinical studies. Results in uence researchers to use the AAV gene therapy because of its e ectiveness in transducing genes into hepatocytes. Just as in hemophilia, AAV gene therapy is a useful technique applicable to many other diseases, which can lead to more breakthroughs and for other studies. This results in longer lasting and consistent treatment plans after insertion due to the limited amount of insertional mutability 17 .
Predicting or compiling e ective AAV vectors and their key qualities can advance further studies that want to use such gene manipulating technology that is not as biohazardous and feasible scheduled usage as it is e ective for months 17 . With a comprehensive collection of data from various clinical trials, researchers in the future will also have a good understanding of challenges of such treatment that requires deliberation before pursuing treatment about options with the type of AAV vectors to use and understanding the best way to infuse into the body 18 . Our research can also advise a potential gene sequence for further research to use when developing the precise gene sequence to use in future studies.