





































� 

Berkeley
Pharma Tech
Journal of Medicine

Correspondence:�
venkatarepaka@gmail.com

Keywords:

Thrombin 

Hemophilia

Factors VIII and FIX   

Hepatocytes                             

AAV vector

Inhibitors

Viral Vectors

Submitted May 3, 2022�  
Accepted�June 8, 2022      

Published July 19, 2022

Full Open Access

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
�ïw�Venkata Repakau�Daniel Lomboy, Priscilla Castro



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 patient1-3. 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 population4. 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 individual5. Current non-gene 
therapy includes methods such as direct infusion of the exogenous 
proteins6. 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 factors7.

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

Berkeley Pharma Tech Journal of Medicine | 121



study suggests8. 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 VIII9. 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
degradation10. 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 mechanism11. This inducible mechanism can also be used in the
AAV vector that is an emerging technology. These mechanisms are SIN

Berkeley Pharma Tech Journal of Medicine | 122



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 molecule45.

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. al12. Another type of
therapy that was proven successful by clinical trials of di�erent companies as
suggested by a review article by Sadiki et. al.. Site-speci�c bioconjugation is
the concept of aiding the speci�c proteins needed for delivery of drug
carriers and moieties of proteins within the body. This technology increases
protein circulation, activity, target speci�city, and protein properties as a
whole. There are many site-speci�c bioconjugations which include
unnatural amino acid incorporation, Sortase-mediated ligation, and
SpyCatcher/SpyTag bioconjugation. All of these methods make alterations
to the amino acids present in a particular drug carrier making the
permeability of that drug easier in the body. This method is favorable
because it changes the way particular factors and proteins are delivered to
the body without recombinant engineering13. Jivi is an antihemophilic
factor (recombinant) gene or a factor VIII concentrate. This study focused
on reducing the amount of times patients need to have routine prophylaxis
by making an on-demand treatment to control the bleeding. They used
recombinant DNA technology in Baby Hamster Kidney (BHK) cells to
generate the recombinant B-domain deleted human coagulation Factor VIII
(BDD-rFVIII) to obtain the activated molecule. Due to its on-demand
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 total14. In research studies, intensity or severity of hemophilia is
de�ned by measuring FVIII or FIX activity as suggested by a review paper

Berkeley Pharma Tech Journal of Medicine | 123



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 patient15.

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 sequence16. 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

Berkeley Pharma Tech Journal of Medicine | 124



DNA appending techniques17. 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 replicating17.

Figure 1: Structure of an AAV vector. GOI, the gene of interest, target sequence that
is to be inserted into the vector.

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

Berkeley Pharma Tech Journal of Medicine | 125



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 hemophilia19, 20.

Figure 2: Blood Coagulation Cascades -- the Intrinsic and Extrinsic Pathways. By Joe D - Own 

work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=1983833

Vector and Plasmid Design
From various clinical trials, we will compose a collection of e�ective AAV 
vectors that can transduce the Factors 8/9 into the hepatocytes. AAV is a 
very broad type of vector that has many versions of itself that exist in 
nature21. The key di�erences between such vectors can include the length of 
gene it can hold, various repair enzymes, and the type of capsid proteins 
present for replication. All of these elements make up the speci�c AAV 
vectors’ characteristics of replication speed and the size of the vectors. 
Additionally, the size of the target sequence of the factor needed to insert 
into the is important to consider42. There is more research done on 
Hemophilia B than hemophilia A due to the smaller size of factor 9 (c. 1.6 
kb) and factor 8 (c. 7 kb). The whole size of AAV vectors (c. 4.7 kb) limits

Berkeley Pharma Tech Journal of Medicine | 126



the packaging capacity so this is another limitation of clinical studies. A
review paper by Neinhus et. al. suggested that the recombinant AAV2
serotype was used in initial studies because it was the �rst AAV vector
serotype to be characterized. This serotype was a single-stranded vector and
was 7 kb in length21. This length included 145 base pairs of inverted
terminal repeats (ITRs) which are the regions of the vector that must be
preserved for the vector to allow packaging and further sequencing. This
virus also included adeno-viral proteins in the form of capsid proteins for
packaging and to form viral vectors. Another comprehensive study by Sands
et. al. showed the various types of AAV vectors’ serotypes tested through
liver-mediated infusion. It was known that the AAV2 serotype was very
versatile and could pack many genes with other supporting capsid proteins.
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
intraportal vein injection and by intravenous (tail vein) injection.
Additionally, when comparing the e�ciency of the rAAV-8 of FIX
administered through the portal vein injection and tail vein injection in vivo
experiments in mice, there was an equal amount of expression of the FIX
factor in the plasma throughout 20 weeks of observation time. The factor
peaked during the 4 weeks mark and gradually decreased its expression by
75% as by the end of the 20 weeks. The vectors were prepared with the help
of a native liver cell from the mouse with the double stranded vector and 10
μg of the factor plasmid. In conclusion, this study has resulted in the type of
capsid proteins for each serotype making the AAV vector serotype have a
di�erent a�nity, transduction e�ciency, and tropisms in the body22.

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 machinery23. To solve this problem, a study by Nathwani

Berkeley Pharma Tech Journal of Medicine | 127



et. al. devised a liver-restricted mini-human factor IX (hFIX) expression
cassette that allows AAV DNA to be packaged as dimers24. 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.

Figure 3: Stages in AAV vector delivery and transgene expression, listed in
combination with some resulting proteins involved in the process.

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 UniQure25.
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

Berkeley Pharma Tech Journal of Medicine | 128



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. The site of infection can greatly
increase or decrease the e�ciency of the transduction of AAV in the body.
Intraportal vein injection includes an infusion directly to the portal vein of
the liver21. According to a study by Nienhuis et. al. from the University of
London, rAAV2 was shown to have the same e�ciency as the portal vein
method and the peripheral vein infusion method. Initially, it was thought
that a portal vein injection was needed to secure the liver transduction in the
mice but this was not true. The transduction was largely a�ected by the
types of capsid proteins present in the AAV vector serotype. Based on the
study by Doshi et. al. suggesting that patients who already had existing
neutralizing antibodies (NAbs) in the liver—directed gene therapy showed
that the AAV transduction e�ciency was hindered by pre-existing NAbs.
(26) Although NAbs can be present in 30-70% of the population,
immunosuppressors were used in subsequent trials involving the liver-
directed transduction. Contrastingly, the intramuscular trial did not have
this e�ect. Another method of vector infusion is direct intraparenchymal
injection. A study by Sands et. al. suggested that a direct injection of an
AAV2 vector into the liver parenchyma which results in a relatively
widespread transduction throughout the liver22. This method also retains
the AAV gene expression for a longer period of time in the body.

Berkeley Pharma Tech Journal of Medicine | 129



Figure 4: The Three Types of AAV Administration Methods 

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 response27. The second method of re�ning the 
AAV vector for administration is the use of directed evolution28, 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

Berkeley Pharma Tech Journal of Medicine | 130



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 assay29, 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 age31. 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 inhibitors32.

Pre-existing immunity from neutralizing antibodies or inhibitors can be
overcome by creating alternate AAV serotypes33. 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�orts34.

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 factors35.

Berkeley Pharma Tech Journal of Medicine | 131



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) shows a promising alternative to AAV gene therapy 
known as Ultrasound Mediated Gene Delivery (UMGD), where the 
plasmid containing the functional Factor VIII gene is injected directly into 
Hemophilia A patients. UMGD of reporter plasmids targeting mice livers 
achieved high levels of transgene expression predominantly in hepatocytes, 
while maintaining minimal transaminase levels following injection37. 
However, these results have yet to be proven in human clinical trials and are 
still years away. Similar to AAV vectors, lentiviral vectors do not contain 
viral genes and promote long-term gene expression which make both 
vectors strong candidates for gene therapy. Despite increased stability upon 
genomic integration, lentiviral vectors activate the innate immune response 
to limit its capabilities in increasing clotting factor levels. The adaptive 
immune system, however, is unresponsive upon cell transduction38. 
Hemostatic agents have been engineered to overcome FVIII and FIX 
activity in hemophilic patients as an alternate protein therapy. The 
hyperactive FVIIa variant has undergone experimentation as a transgene for 
AAV-based liver-directed gene therapy. This resulted in the usage of a lower 
vector dose while maintaining a similar response to hemostatic conditions. 
Although clinical trials have proceeded into late stages, it has halted due to 
immunogenicity speculation. If continued in the future, the FVIIa 
transgene may be able to lower the vector dose for patients. 
Protein-engineered bypassing agents have prolonged the half-life of 
coagulation factors downstream of clotting factors that are designed to resist 
inactivation via conformational changes to protein structures. This 
treatment is undergoing early-stage clinical trials and has proven e�cacy as a 
protein therapy for acute hemorrhagic conditions39.

Berkeley Pharma Tech Journal of Medicine | 132



Table 1: Comparison  between AAV, lentivirus, and adenovirus vectors.

Inhibitors
Additionally, there are structural, functional, and expression di�erences
between the AAV vector, lentivirus, and adenoviral vectors to account for.
AAV is a smaller vector consisting of approximately 25 nm, whereas
lentivirus is 80 nm to 120 nm, and adenovector is approximately 95 nm.
AAV alongside lentiviruses carry single-stranded DNA, whereas adenoviral
vectors carry double-stranded DNA40. Double-stranded AAV vectors were
also formulated by mutation of the inverted terminal repeat (ITR). These
vectors were shown to have better transduction in many other cell lines
including in vivo experiments with hepatocytes. In comparison to the
ssAAV, the dsAAV gene helped maintain better transduction and better
DNA stability when evaluated 6 months after the initial in vivo
experiment41. AAV has a lower packaging capacity consisting of
approximately 4.7 kb while both lentiviral and adenoviral vectors have
approximately 8 kb40. This may be a limitation due to the large gene target
sequence of factors 8 and 9. Adenoviruses and AAV vectors are not
enveloped which may cause more e�cient transduction of viral DNA. The
lentiviral vector is enveloped which keeps it well protected, but concurrently
if the envelope is compromised, the virus can disintegrate. Additionally, the
lentivirus is double-stranded and embeds itself well into the host’s DNA.
The integration of the viral DNA into the host’s DNA will prelude
long-term transgene expression. The major disadvantage of this mechanism
is that it can disrupt the host’s genome by insertional mutagenesis. AAV and
adenovirus cannot integrate themselves into the host cell’s DNA. The
adenoviral vector is mostly used for short term uses as it is e�ective in
transient expression unlike the persistent gene expression of the AAV and

Berkeley Pharma Tech Journal of Medicine | 133



lentiviral vectors. Lastly, immunogenicity decreases of each vector
adenovirus, AAV, and lentivirus respectively40.

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 mutability17.
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
months17. 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 body18. 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.

Berkeley Pharma Tech Journal of Medicine | 134



References
1. Nilsson IM, Berntorp E, Lofqvist T,
Pettersson H. Twenty-�ve years' experience of
prophylactic treatment in severe hemophilia. A
and B. J Intern Med. 1992; 232: 25–32.

2. Lisowski, L., Staber, J. M., Wright, J. F., &
Valentino, L. A. (2021). The intersection of
vector biology, gene therapy, and hemophilia.
Research and practice in thrombosis and
haemostasis, 5(6), e12586.
https://doi.org/10.1002/rth2.12586

3. What is hemophilia. Centers for Disease
Control and Prevention.
https://www.cdc.gov/ncbddd/hemophilia/fact
s.html. Published July 17, 2020. Accessed
March 29, 2022.

4. Fast facts. National Hemophilia Foundation.
https://www.hemophilia.org/bleeding-disorde
rs-a-z/overview/fast-facts. Accessed March 29,
2022.

5. TBH Creative info@tbhcreative.com.
Severity of hemophilia. Indiana Hemophilia &
Thrombosis Center.
https://www.ihtc.org/severity-of-hemophilia.
Accessed March 29, 2022.

6. Peyvandi F, Garagiola I, Seregni S. Future of
coagulation factor replacement therapy. J
Thromb Haemost. 2013;11 Suppl 1:84-98.
doi:10.1111/jth.12270

7. Witmer C, Young G. Factor VIII inhibitors
in hemophilia A: rationale and latest evidence.
Ther Adv Hematol. 2013;4(1):59-72.
doi:10.1177/2040620712464509

8. Samelson-Jones BJ, Arruda VR.
Protein-Engineered Coagulation Factors for
Hemophilia Gene Therapy. Mol Ther Methods

Clin Dev. 2018;12:184-201. Published 2018
Dec 31. doi:10.1016/j.omtm.2018.12.007

9. Alam A, Doshi H, Patel DN, Patel K, James
D, Almendral J. Myocardial infarction and
factor VIII elevation in a 36-year-old man. Proc
(Bayl Univ Med Cent). 2021;35(1):93-95.
Published 2021 Sep 15.
doi:10.1080/08998280.2021.1973285

10. Castaman G. Treatment of von Willebrand
disease with FVIII/VWF concentrates. Blood
Transfus. 2011;9 Suppl 2(Suppl 2):s9-s13.
doi:10.2450/2011.003S

11. Nogami K, Zhou Q, Wakabayashi H, Fay
PJ. Thrombin-catalyzed activation of factor
VIII with His substituted for Arg372 at the P1
site. Blood. 2005;105(11):4362-4368.
doi:10.1182/blood-2004-10-3939

12. Rachel M. Lieser, Daniel Yur, Millicent O.
Sullivan, and Wilfred Chen. Bioconjugate
Chemistry. 2020 31 (10), 2272-2282. DOI:
10.1021/acs.bioconjchem.0c00456

13. Sadiki A, Vaidya SR, Abdollahi M, et al.
Site-speci�c conjugation of native antibody.
Antib Ther. 2020;3(4):271-284.
doi:10.1093/abt/tbaa027

14. Protect VIII study design. Jivi US HCP.
https://www.jivihcp.com/en/protect-viii-trial.
Accessed March 29, 2022.

15. Samelson-Jones BJ, Arruda VR.
Protein-Engineered Coagulation Factors for
Hemophilia Gene Therapy. Mol Ther Methods
Clin Dev. 2018;12:184-201. Published 2018
Dec 31. doi:10.1016/j.omtm.2018.12.007

16. Curiel DT, Fisher PB, Tew KD, Kaliberov
SA. Chapter Seven - Cancer Treatment with

Berkeley Pharma Tech Journal of Medicine | 135



Gene Therapy and Radiation Therapy. In:
Advances in Cancer Research. Amsterdam:
Elsevier Academic; 2012:221-263.

17. Aschauer DF, Kreuz S, Rumpel S (2013)
Analysis of Transduction E�ciency, Tropism
and Axonal Transport of AAV Serotypes 1, 2,
5, 6, 8 and 9 in the Mouse Brain. PLoS ONE
8(9): e76310.
https://doi.org/10.1371/journal.pone.007630

18. El Andari, J. and Grimm, D. (2021),
Production, Processing, and Characterization
of Synthetic AAV Gene Therapy Vectors.
Biotechnol. J., 16: 2000025.
https://doi.org/10.1002/biot.202000025

19. Factor VIII (FVIII). Diapharma.
https://diapharma.com/factor-viii/. Published
November 15, 2021. Accessed March 29, 2022.

20. FVIII: A crucial hemostatic factor to
control bleeding. EDUC8.
https://www.educ8-h2020.eu/esrs-blog/fviii-a-
crucial-hemostatic-factor-to-control-bleeding.
Accessed March 29, 2022.

21. Nienhuis AW, Nathwani AC, Davido�
AM. Gene Therapy for Hemophilia. In:
Molecular Therapy. Vol 25. 5th ed. CellPress;
2017:1163-1167.

22. Sands M.S. (2012) AAV-Mediated
Liver-Directed Gene Therapy. In: Snyder R.,
Moullier P. (eds) Adeno-Associated Virus.
Methods in Molecular Biology (Methods and
Protocols), vol 807. Humana Press.
https://doi.org/10.1007/978-1-61779-370-7_6

23. Fisher KJ, Gao GP, Weitzman MD,
DeMatteo R, Burda JF, Wilson JM.
Transduction with recombinant
adeno-associated virus for gene therapy is

limited by leading-strand synthesis. J Virology.
1996;70(1):520-532.
doi:10.1128/jvi.70.1.520-532.1996

24. Nathwani AC, Gray JT, Ng CY, et al.
Self-complementary adeno-associated virus
vectors containing a novel liver-speci�c human
factor IX expression cassette enable highly
e�cient transduction of murine and
nonhuman primate liver. Blood.
2006;107(7):2653-2661.
doi:10.1182/blood-2005-10-4035

25. Breakthroughs in Gene Therapy for
Hemophilia. Ashpublications.org.
https://ashpublications.org/ashclinicalnews/ne
ws/4099/Breakthroughs-in-Gene-Therapy-for-
Hemophilia. Accessed March 29, 2022.

26. Doshi BS, Arruda VR. Gene therapy for
hemophilia: what does the future hold?
Therapeutic Advances in Hematology.
September 2018:273-293.
doi:10.1177/2040620718791933

27. Ran, G., Chen, X., Xie, Y., Zheng, Q., Xie,
J., Yu, C., Pittman, N., Qi, S., Yu, F. X.,
Agbandje-McKenna, M., Srivastava, A., &
Ling, C. (2020). Site-Directed Mutagenesis
Improves the Transduction E�ciency of
Capsid Library-Derived Recombinant AAV
Vectors. Molecular therapy. Methods & clinical
development, 17, 545–555.
https://doi.org/10.1016/j.omtm.2020.03.007

28. Maheshri, N., Koerber, J., Kaspar, B. et al.
Directed evolution of adeno-associated virus
yields enhanced gene delivery vectors. Nat
Biotechnol 24, 198–204 (2006).
https://doi.org/10.1038/nbt1182

29. Gouw SC, van den Berg HM. The
multifactorial etiology of inhibitor

Berkeley Pharma Tech Journal of Medicine | 136



development in hemophilia: genetics and
environment. Semin Thromb Hemost.
2009;35(8):723-734.
doi:10.1055/s-0029-1245105

30. Witmer C, Young G. Factor VIII inhibitors
in hemophilia A: rationale and latest evidence.
Ther Adv Hematol. 2013;4(1):59-72.
doi:10.1177/2040620712464509

31. Lorenzo JI, López A, Altisent C, Aznar JA.
Incidence of factor VIII inhibitors in severe
haemophilia: The importance of patient age.
British J Haematology. 2001;113(3):600-603.
doi:10.1046/j.1365-2141.2001.02828.x

32. Gouw SC, van der Bom JG, Marijke van
den Berg H. Treatment-related risk factors of
inhibitor development in previously untreated
patients with hemophilia A: the CANAL
cohort study. Blood. 2007;109(11):4648-4654.
doi:10.1182/blood-2006-11-056291

33. Louis Jeune V, Joergensen JA, Hajjar RJ,
Weber T. Pre-existing anti-adeno-associated
virus antibodies as a challenge in AAV gene
therapy. Hum Gene Ther Methods.
2013;24(2):59-67. doi:10.1089/hgtb.2012.243

34. Boutin S, Monteilhet V, Veron P, et al.
Prevalence of serum IgG and neutralizing
factors against adeno-associated virus (AAV)
types 1, 2, 5, 6, 8, and 9 in the healthy
population: implications for gene therapy
using AAV vectors. Hum Gene Ther.
2010;21(6):704-712.
doi:10.1089/hum.2009.182

35. Patel SR, Lundgren TS, Spencer HT,
Doering CB. The immune response to the
FVIII gene therapy in preclinical models. Front
Immun. 2020;11.
doi:10.3389/�mmu.2020.00494

36. Song S, Lyle MJ, Noble-Vranish ML, et al.
Ultrasound-mediated gene delivery of factor
VIII plasmids for hemophilia A gene therapy in
mice. Mol Ther Nucleic Acids.
2022;27:916-926. Published 2022 Jan 10.
doi:10.1016/j.omtn.2022.01.006.

37. Ultrasound-mediated gene delivery of
factor VIII plasmids for hemophilia A gene
therapy in mice, Mol Ther Nucleic Acids,
Volume 27, 2022, Pages 916-926, ISSN
2162-2531,
https://doi.org/10.1016/j.omtn.2022.01.006.

38. Vandendruessche T, Thorrez L,
Acosta-Sanchez A, et al. E�cacy and safety of
adeno-associated viral vectors based on
serotype 8 and 9 vs. lentiviral vectors for
hemophilia B gene therapy. J Thromb Haemost.
2006;5(1):16-24.
doi:10.1111/j.1538-7836.2006.02220.x

39. Samelson-Jones BJ, Arruda VR.
Protein-Engineered Coagulation Factors for
Hemophilia Gene Therapy. Mol Ther Methods
Clin Dev. 2018;12:184-201. Published 2018
Dec 31. doi:10.1016/j.omtm.2018.12.007

40. Tosolini AP and Sleigh JN (2020)
Intramuscular Delivery of Gene Therapy for
Targeting the Nervous System. Front. Mol.
Neurosci. 13:129. doi:
10.3389/fnmol.2020.00129

41. Wang, Z., Ma, HI., Li, J. et al. Rapid and
highly e�cient transduction by
double-stranded adeno-associated virus vectors
in vitro and in vivo. Gene Ther 10, 2105–2111
(2003). https://doi.org/10.1038/sj.gt.3302133

42. Paul Batty, David Lillicrap, Advances and
challenges for hemophilia gene therapy, Hum

Berkeley Pharma Tech Journal of Medicine | 137



Mol Gen, Volume 28, Issue R1, October 2019,
Pages R95–R101,
https://doi.org/10.1093/hmg/ddz157

43. Batty, Paul; Lillicrap, David Hemophilia
Gene Therapy: Approaching the First Licensed
Product, HemaSphere: March 2021 - Volume 5
- Issue 3 - p e540
doi: 10.1097/HS9.0000000000000540

44.  Ohmori, T. Advances in gene therapy for 
hemophilia: basis, current status, and future 
perspectives. Int J Hematol 111, 31–41 (2020). 
https://doi.org/10.1007/s12185-018-2513-4

45.  James W. Larrick, M.D. Ph.D., interview, 
April 27, 2022.

Berkeley Pharma Tech Journal of Medicine | 138


	Front_Page_Repaka
	body



