Download - ALS 320 Christmas Disease Report Final Draft
A survey of recent discoveries: Identification of an innovative
diagnostic tool for Hemophilia BALS 320: Medical Diagnostics, Fall 2015- Section 1, Team 2
Jesse Forchap, Brandy Fugate, Benjamin Blackwell,
Anthony Park, Caitie Staat, and Khaled Hamad
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Abstract
Hemophilia B is a rare blood clotting disorder that affects an estimated 1 in every 34,000
males in the United States. Hemophilia B is caused by a mutation in the Factor IX gene, which
interferes with the coagulation cascade. The molecular basis of Hemophilia B is complex,
revolving around proper folding of Factor IX such that its activated form can properly participate
in the coagulation cascade. As a result, there are a variety of diagnostic tools used to measure
Factor IX in the blood, but all of these devices are presently used exclusively in professional
point-of-care settings. There has been little effort put forth in the search for an at-home device
that patients can use to monitor and control their coagulation factor levels in the blood. By
presenting a summary of the currently understood pathways, preventions, treatments, and
diagnostic tools involved in Hemophilia B management, we hope to encourage further
investigation into a more convenient diagnostic device and potential new treatment options for
sufferers of Hemophilia B.
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An Introduction to Hemophilia B
Hemophilia B, also known as Christmas disease, is a rare blood clotting disorder caused
by deficiency in the Factor IX blood-clotting factor. The term “Christmas disease” is derived
from the first patient, Stephen Christmas, who was diagnosed with the disease in the early
1950s.1 Factor IX (FIX) is a blood-clotting factor produced in the liver, and is a critical
component of the blood clotting cascade.2 The FIX gene is located on the X chromosome, and
mutations in this gene are the primary cause of Hemophilia B. In some rare cases, the disease
may be acquired during the latter part of a patient’s life as an auto immune disorder. Christmas
disease is the second most common type of Hemophilia; Hemophilia A is most common and is
caused by a deficiency in Factor VIII production.3
Patients with Hemophilia B suffer from joint damage, general organ deterioration, and
abnormal bleeding after minor operations, such as tooth extraction.1 However, the degree of
bleeding varies depending on the individual and the severity of the disease. According to the
National Hemophilia Foundation, there are three main classes of Hemophilia B determined by
FIX plasma levels.4 Severe cases are defined by FIX levels less than 1% of the normal range, in
which patients experience spontaneous bleeding in their joints and muscles. This class of severity
is easily detected through apparent physiological symptoms. FIX levels between 1% - 5% of
normal define moderate cases, and patients in this class generally have bleeding episodes after
injury. Detection of moderate cases is less apparent than severe cases, in that bleeding events are
less apparent. In mild cases, FIX levels are above 5 % but less than 40 % of normal. Mild cases
of Hemophilia B can go undetected for years due to the absence of typical symptoms, as only
severe injury or trauma results in excessive bleeding.
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The FIX gene is located on the long arm of the X chromosome. Females with an FIX
variant on only one X chromosome are considered carriers, but do not express symptoms.2
Female carriers may rarely experience abnormal bleeding episodes after childbirth and require
FIX replacement therapy. 2 Hemophilia B is more prevalent in males because of its X-linked
nature. In the U.S., Hemophilia B occurs in approximately 1 of 34,000 males, and the number of
patients in the U.S. as of 2010 was approximately 4,000. Annual Hemophilia B expenditure in
the U.S. is approximately $58,000 per patient which results in a total of $246.2 million per year
if all patients were to be treated. This cost could increase over the coming years as our
population continues to rise, bringing attention to the importance of Hemophilia B within the
American health care system.5
Current diagnostic methods are primarily quantitative. A variety of these methods will be
discussed in depth. Monitoring of FIX levels comprises the basis of Hemophilia B prognosis.
Presently, FIX levels can only be measured in a professional point-of-care setting, which is
inconvenient to patients. Additionally, treatments for modulation of FIX activity rely on an
approximation of FIX levels in the blood. We hope to encourage additional research by
examining current treatment methods for Hemophilia B and proposing novel diagnostics that
may assist in simplifying treatment and improving quality of life. First, we will delve into current
preventative measures and treatment strategies used to mitigate the symptoms and complications
associated with Hemophilia B.
Preventative Measures and Current Treatment Strategies
While Hemophilia B is rare, it is important that potential parents affected by this disease
examine preventative measures when considering pregnancy. Preimplantation Genetic Diagnosis
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(PGD) is an innovative technique that assists in informing couples prior to conception of the
chances that their child will develop Hemophilia B.6 PGD consists of two main steps: in-vitro
fertilization (IVF) is first utilized, and a biopsy of the embryo is subsequently genetically
analyzed. IVF is accomplished through hyperstimulation of the ovaries followed by retrieval of
the egg, fertilization of the egg, and development of the embryo. Once developed, a small sample
cell is taken from the embryo. Fluorescent In Situ Hybridization (FISH) and Polymerase Chain
Reaction (PCR) are used for genetic evaluation of this sample.6 Embryos that are discovered to
be unaffected by Hemophilia B are then implanted into the mother’s uterus.6 Parents may opt to
conceive naturally and either accept the potential challenges and risks of giving birth to a baby
who has the genetic disorder, or proceed with prenatal diagnosis, then terminate the pregnancy if
Hemophilia B is confirmed.7 However, termination of a viable pregnancy based on a Hemophilia
diagnosis raises strong ethical and cultural concerns.
Hemophilia B is principally treated by managing clotting FIX. Replacement FIX therapy
is the preferred method of treatment for Hemophilia B. Concentrated FIX is regularly injected
into the vein multiple times per week, temporarily replacing the patient’s clotting factor.8
Clotting factor concentrates were previously made via reconstitution of donated human blood.
Donated blood was screened to prevent disease transmission, though risk of contracting an
infectious disease still existed. Clotting factors are now being procured from genetically
modified cells introduced to a hamster cell line. These are known as recombinant clotting
factors.9 The FIX gene is incorporated into the hamster cell genome, and the protein is produced
in large amounts using the cell line. This is less risky because it utilizes animal cells that are free
of human diseases.9 Clotting factors produced through cell lines can be convenient as they have a
long shelf life and can thus be made more readily available to patients.10
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Depending on the severity and pattern of bleeding, there are two classifications of
therapies used. Replacement therapy is used to prevent regular bleeding, and is therefore
prophylactic. Children with severe Hemophilia B use prophylactic therapy on a regular basis.11
There are two sub-types of prophylactic therapy: primary, which young children start using to
lessen or prevent diseases in the joints and will be continued for life, and secondary, which is
used frequently for a limited period of time when bleeding begins.8 The benefits of using
prophylactic therapy include a lower risk of spontaneous bleeding, the ability to participate in
sports, and lower risk of damage to the joints.8,11 The disadvantages of using prophylaxis include
constant injections and inevitably higher expenses.8,11 However, on-demand therapy can be used
in place of prophylaxis for the purpose of occasional or sporadic bleeding, as needed. On-
demand therapy is less rigorous and less expensive than prophylactic therapy. 8
One of the most severe complications that can develop through treatment of Hemophilia
B is antibody resistance to clotting factor.12 FIX can be destroyed by these antibodies, which then
prevents replacement therapy from working properly. These antibodies are known as inhibitors,
and they develop in 1.5-3% of Hemophilia B patients.8,12 Figure 1 depicts the Bethesda assay
used to detect the inhibitor concentration (or amount of antibodies) in the blood.8
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Figure 1: Bethesda units (BUs) and factor present in blood post-injection. A higher BU value indicates a
higher concentration of inhibitors present in the blood, which results in less active FIX post-injection.
When the inhibitor concentration increases, additional FIX must be injected into the patient to maintain
healthy FIX levels. Adapted from www.sevensecure.com/inhibitor/inhibitor-education.aspx
Stemming from our conversation on current treatments, we will now discuss the basis of
Hemophilia B on a molecular level, and how the various blood-clotting factors play their roles in
the clotting cascade.
The Molecular Pathway of Hemophilia B
The pathway of Hemophilia B was first elucidated in 1986 by Diuguid, et al. at the New
England Medical Center and Tufts University School of Medicine in Boston.2 The FIX protein is
synthesized exclusively in hepatocytes and is produced as a zymogen, or an inactive precursor.2,13
In the unadulterated pathway, FIX is first translated by ribosomes in hepatocytes, and is
structured as a single chain plasma glycoprotein peptide sequence.14 The FIX molecule is
comprised of three main units: the signal peptide, which facilitates the transport of the protein
across the plasma membrane, the propeptide sequence, and the final protein product.2 Before any
posttranslational modifications are made, the signal peptide is first cleaved from the FIX
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molecule once it has exited the nucleus. Next, a series of modifications occur including
glycosylation, cleavage of the propeptide, beta-hydroxylation, and vitamin K-dependent
carboxylation.2,14 Once these posttranslational modification reactions are completed, the FIX
zymogen is released from the hepatocyte.
It is important to note that this pathway occurs on the platelet surface at the site of
damage along a blood vessel.15 Effective completion of the blood coagulation cascade can only
occur on cell surface membranes.16 In order for FIX to be activated, it must be converted into a
serine protease by Factor XIa in the presence of Ca 2+ .14 When FIX experiences these conditions,
the hydrolysis of two peptide bonds buried inside the Factor IX molecule occurs, which allows
for the release of the activation peptide.14 This results in procurement of Factor IXaβ, which is the
active and final form of FIX. It is comprised of a single heavy chain and a single light chain,
with the heavy chain containing the active site.14 Factor IXaβ activates Factor X along with
assistance from Factor VIIIa.16 This occurs though complexing of Factor IXaβ and Factor VIIIa on
the membrane surface, whose new product is able to generate Factor Xa.16 Factor Xa then
complexes with Factor Va, which promotes activation of prothrombin. Finishing out the pathway,
the now active thrombin cleaves fibrinogen present near the platelet surface, which then
polymerizes to form a fibrin clot.16 Figure 2 illustrates the previously described pathway.
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Figure 2: A display of the entire coagulation cascade from initial injury to final clot formation. This figure
shows all key players involved in the formation of a clot. Adapted from the Hemophilia Report 2014.6
There are two types of defects that have been characterized in Hemophilia B:
independent deletion mutations and point mutations.16 This will be discussed in depth in the next
section. For the sake of simplicity we will discuss only one of the many possible mutations that
leads to Hemophilia B that disrupts γ-carboxylation. However, this discussion is linked to a
broader context in that many mutations that cause Hemophilia B interfere with γ-carboxylation.16
FIX is vitamin K-dependent, meaning that it requires a carboxylase to bind to it in the presence
of vitamin K in order to become secreted. In the point mutation being discussed, an arginine
becomes a serine at the -1 residue, as discussed in the article by Diuguid, et al. This leads to
interference of γ-carboxylation, and ultimately causes failure of the cleavage of FIX’s
propeptide.2 FIX subsequently misfolds and cannot activate Factor X because it is incapable of
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complexing with Factor XIa on the cell membrane. From this point, Factor X cannot go on to
promote activation of prothrombin. Thus, the clot cascade fails and excessive bleeding occurs at
the site of injury.
This mutation example can be extended to some other deletion mutations that cause a
similar non-recognition of the FIX molecule by the Vitamin K-dependent kinase within
hepatocytes. From the elucidated pathway of Factor IX, and subsequently Hemophilia B, we can
derive some key biomarkers that can be utilized in the diagnosis and monitoring of this disease.
Genetics Research and Biomarkers of Hemophilia B
Hemophilia B is caused by mutations in the Factor IX gene, and there are more than
1,100 such mutations that have been identified.17 The most common FIX mutation that causes
Hemophilia B is substitution of a single DNA base pair, although other mutations may still
occur.18 These mutations are linked to a decreased quantity of active FIX and lead to defective
blood clotting and excessive bleeding. Therefore, severity of Hemophilia B chiefly depends on
the severity of the mutation.
Identification of FIX mutations within their locus on the X chromosome is essential
because identified regions can be used as genetic markers to treat Hemophilia B. Two common
types of genetic screens performed to identify heterogeneous mutations in FIX are Restriction
Fragment Length Polymorphism (RFLP) analysis and haplotyping. RFLP analysis is a technique
that exploits variations in homologous DNA sequences. A haplotype is a group of genes within
an organism that were inherited together from a single parent. Both of these analyses can offer
prevention through screening by detecting X-linked gene carriers.
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Figure 3 Informativeness of the RFLP markers in different population groups of India.19 Panels A, B and
C respectively represent the Eastern, Southern and Western regions. RFLP markers analyzed in each bar
diagram are denoted by DdeI (D), HhaI (Hh), Hpy188I (Hp), MnlI (M), TaqI (T), and XmnI (X).19 Panel
D shows informativeness for North India obtained from a separate study (Chowdhury et al. 2001).19
Informativeness for each marker was calculated as the percentage of females heterozygous in each
cluster.19 Adapted from Mukherjee, S. et al., 2006.19
For RFLP analysis, Mukherjee, et al. collected data from eight different populations that
were composed of 107 normal females from different geographical areas with clear family
histories of Hemophilia B, and 13 carriers that were unrelated to each other.19 Then, DdeI, XmnI,
MnlI, TaqI, and HhaI restriction sites were used to identify the genetic markers (Figure 3).19 In
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addition to RFLP analysis, researchers also used Single Nucleotide Polymorphisms (SNPs) to
screen regions of the FIX gene.19 The results from this study on various populations in India
showed that only two SNPs were found as possible candidates for differentiating normal groups
from carrier groups, whereas RFLP markers on specific groups were effective in differentiating
between carrier groups and normal groups.19 The study also mentioned that additional genetic
markers were necessary for more efficient analysis because the variability of heterozygosity was
quite high.19
Another study done in Sweden focused on discovering the origin of high occurrences of
certain FIX mutations in specific subgroups of the population.17 Their objective was to analyze
and classify FIX mutations within Swedish families into two different types of mutations. These
were classified as independent recurrent mutations (RMs) or common mutation events, also
known as identical by descent (IBD).17 By resequencing and performing haplotype analyses on
86 Swedish families with 74 genetic markers, researchers found that both RMs and IBDs were
present at the same proportion -slightly over 50%- in patients with the mild severity phenotype
of hemophilia B.17
Genetic marker analyses of the FIX gene on different population groups from different
geographical regions showed a wide range of variability and specificity based on types of
analysis performed. In juxtaposition to the RFLP markers used in the study performed by
Mukherjee, et al. (Markers 20-30), the study performed in Sweden used the M1 marker.20 Since
genetic markers vary greatly from population to population, discovery of new genetic markers by
performing different experimental methods such as Random Amplified Polymorphic DNA
(RAPD), Amplified Fragment Length Polymorphism (AFLP) and Quantitative Trait Locus
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(QTL) analyses could potentially lead to more efficacious and specified genetic markers of
Hemophilia B.21
Coagulation FIX, translated from the FIX gene, is the most well-researched biomarker
used for detecting Hemophilia B.18 FIX is associated with an activated partial thromboplastin
time (aPTT) of the intrinsic pathway.22 As was stated before, in the normal coagulation cascade,
FIX is activated by Factor XIa and activates Factor X with assistance from Factor VIIIa and other
molecules.23 Although levels of Factor XI, Factor X and Factor VIII could be possible candidates
for alternative biomarkers of Hemophilia B, FIX is still strongly recommended since other
factors are placed either upstream or downstream of the FIX activation within coagulation
pathway. Therefore, FIX is the most logical biomarker to utilize for Hemophilia B at the present
time. We will now move into the diagnostic methods that use FIX to identify Hemophilia B.
Diagnostics for Hemophilia B
Various types of tests have been implemented for screening and diagnosis of Hemophilia
B, though quantitative tests are predominantly employed in regular practice. The Hemochron®
Signature Elite, a handheld point-of-care testing device, can perform various coagulation tests
and is commonly used in clinical settings.24 For initial screening, activated partial thromboplastin
time (aPTT) is most often utilized, and is a one-stage clotting assay.25 Both aPTT and an
alternative screening coagulation test, prothrombin time (PT), can be determined by the
Hemochron® system.24 Both tests are performed using similar mechanisms, but each requires
different sample types and reagents.
Sample separation is not required in the Hemochron® system, as this device is a whole-
blood system. Venous or finger-stick samples are analyzed.24 Testing occurs within disposable
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cuvettes preloaded with reagents; these reagents increase the sensitivity of each assay.26,27 PT
sample tubes are preloaded with dried thromboplastin,26 whereas sample tubes for aPTT testing
are preloaded with platelet factor substitutes and kaolin reagents that standardize the activation
of clotting factors.27 Respective stabilizers and buffers are also included in both types of sample
tubes.
When a sample is introduced to the system, the device dispenses 15 microliters of that
sample into a cuvette. The device automatically performs reagent mixing. An incubation period
is not required for aPTT testing.27 As the cuvette is moved rapidly back and forth within the
instrument, motion of the sample is monitored and measured by LED optical detectors in line
with the sample. As the sample clots, the overall motion of the sample is reduced. When the
movement has decreased below a certain threshold, the device identifies that an endpoint has
been reached. The time required to clot is reported respectively for each test. Results of both tests
can be obtained in approximately two minutes.26,27 PT results are represented in terms of the
International Normalized Ratio (INR), a ratio of the patient’s resulting PT compared to a normal
range that is designated internationally.26 Activated PTT results are expressed in terms of their
plasma-equivalent values, which are automatically calculated by the system.27
The primary test currently administered for final diagnosis of Hemophilia B is a FIX
assay. One type of analyzer developed for coagulation automation utilizes electromagnetic
mechanical detection methods to detect plasma levels of activated FIX.28 Siemens has developed
one such analyzer called the Sysmex® CS-5100 System. Although this device is not currently
available in the U.S., other devices that employ this type of technology are commonly
utilized.28,29 The Sysmex® System, similar to the Hemochron® device, measures motion within
the sample provided.29 FIX deficient plasma is the primary reagent added to the sample plasma in
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these assays.30 Factors within specimens given by healthy patients are able to correct for deficient
factors within the reagent, and clotting will occur. However, in Hemophilia B patients, clotting
will not occur as both the reagent and sample plasma have factor deficiencies.31 Fibrin formation
is measured as it surrounds an iron ball that is also added to the plasma.32 Electromagnetic action
moves the iron ball within the plasma. Mobility of the ball is reduced as a clot accumulates
around it. The device detects the consequent lack of movement and provides an output value.32
Final diagnosis of Hemophilia B can be assigned a level of severity: mild, moderate, or severe.4
Disease severity along with associated signs and symptoms is outlined in Table 1.
Table 1: Clinically assigned severity of Hemophilia B.
Severity Factor IX levels Relative signs of deficiency
Mild >5% FIXa activity in plasma Prolonged bleeding post-surgery
Moderate 1% – 5% FIX activity in plasma Excessive bleeding after injury; occasionally spontaneous bleeding
Severe <1% FIX activity in plasma Frequent spontaneous bleeding episodes
Clinically assigned severity of Hemophilia B is listed according to incremental levels of Factor IX
activitya.8 Various physiological signs may also correspond with the severity of the disease.
FIX is a reliable biomarker that is specific to Hemophilia B. However, diagnostics are
presently only available in professional settings, which may be due to rarity of the disease and
the resultantly small market size. Development of a portable device designed to measure
activated FIX levels would benefit Hemophilia B patients, as the availability of a point-of-care
diagnostic tool is a critically unmet need. Many patients with mild or moderate disease diagnoses
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may not be aware of less obvious internal bleeding, placing them at risk for development of
comorbidities such as hemarthroses (bleeding in joints), renal disease, and cardiovascular
complications.8 Routine, prophylactic treatment for management of bleeding reduces the
likelihood of developing these conditions.8 Rapid testing methods could therefore assist in
reducing the occurrence of associated diseases.
Further research should be done to determine whether or not an at-home diagnostic
device could be applied to routine patient care, thereby reducing the long-term side effects of
bleeding. Traditional assay techniques may be utilized to quickly measure levels of FIX in the
blood. This would assist a patient in determining when treatment is necessary. Some patients
naturally develop inhibitors against FIX added to the bloodstream,12 indicating that development
of an immunoassay might be an effective way to construct a new diagnostic tool. Potential
integration of a color change mechanism might be utilized to determine how much of the analyte
has bound to a test line. This could provide a partially quantitative result, allowing patients to
identify when immediate injections of FIX replacement are required.
Looking Ahead: Future Treatments of Hemophilia B
Patients suffering from Hemophilia B are currently unable to measure the
concentration of FIX at home. Current available options include waiting for physiological
symptoms to arise, which can increase the chances of unnecessary bodily harm, or to inject FIX
multiple times a week and assume that levels of FIX are stable. A new possible delivery method
that can solve these issues is an implantable closed loop device, which has proven successful in
diabetic patients.
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Figure 4 Implantable Glucose Device33 retrieved from Implantable closed-loop glucose-sensing and
insulin delivery. Adapted from Renard E., 2002. 33
The glucose device shown in Figure 4 stabilizes patient’s blood glucose levels over
longer periods of time compared to regular insulin injections.33 This device contains an
intravenous glucose sensor, compartment to store insulin, and a catheter. The glucose sensor uses
an enzymatic reaction involving glucose-oxidase that is monitored by an oxygen sensor; the
sensor measures changes in oxygen levels, and sends a signal through a subcutaneous connector
that relays signals from the sensor to the compartment filled with insulin.33 The device then uses
a mathematical algorithm to release a specific concentration of insulin through the catheter33.
Implementing a similar device into a Hemophilia B patient’s treatment regimen would reduce
their risk of joint degradation or other associated diseases by maintaining FIX levels in the blood.
However, there is currently no sensor on the market that can measure FIX in solution.
One way to improve quality of life of Hemophilia B patients is by reducing the number of
injections needed per week. It has been proven that PEGylation of certain proteins can increase
the half-life of a protein in solution. PEGylation is performed by covalently binding a
polyethylene glycol molecule to the desired protein.34 The addition of polyethylene glycol
External Programmer using telemetry
Central port refill
Intraperitoneal catheter
Abdominal connecting lead (subcutaneous)
Subcutaneous connector
Intravenous enzymatic glucose sensor
Slideport
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decreases the chances of protein metabolism, propensity for immunogenic response, and
clearance.34 By PEGylating FIX concentrates, we can expect to see a decrease in the number of
injections per week, and a reduction in the immunogenic response against FIX. However,
PEGylation has some potential side effects. PEGylation has increased coagulation of proteins in
some cases, which increases the chances of clot formation in blood vessels, causing thrombosis.34
Further studies should be conducted to see the effects PEGylation may have on FIX coagulation
in particular.
An alternative treatment to injecting FIX concentrates is gene therapy. Gene therapy
provides a way of introducing a wildtype FIX gene into a cell to be expressed if the host gene is
mutated or defective. The gene can either be integrated directly into a cell’s chromosome, having
a long-lasting effect, or can be delivered to the nucleus, which has a short-lasting effect because
the gene is attached to a free-floating plasmid.35 The type of delivery method will therefore
predict whether the gene therapy will result in a treatment or a cure. There are three possible
methods that could be used to deliver the FIX gene to its target: adeno-associated vectors, naked
plasmids, and lentiviral vectors. By using adeno-associated virus vectors (AAVs), the FIX gene
can be inserted inside a virus that delivers the FIX gene to the nucleus where the gene is
expressed by the host’s cellular machinery (Figure 5).13 Unfortunately, there have been instances
of immunogenicity occurring using this method, as the virus is identified as non-self by the host
immune system.35
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Figure 5 Gene therapy using Adeno-associated virus vector. Adapted from Carr, et al. 2015. 13
Utilization of naked plasmids is an alternative approach to avoiding immunogenic
response since they are a non-viral vector, although this method has a less permanent effect than
AAV. Naked plasmids cannot insert the gene into the host cell’s target chromosome, so the
expression of Factor IX will fade with time. It is also difficult to target specific cells using this
method.1
Lentiviral vectors can incorporate FIX gene into the host cell, which means FIX
expression would not fade over time.35 Lentiviral vectors have been used in both in-vivo and ex-
vivo studies. Utilization of a lentiviral vector produced an immunogenic response similar to the
AAV vector in-vivo.35 However, when the lentivirus was transduced via ex-vivo techniques it did
not initiate an immunogenic response.35 Thus, ex-vivo transduction of FIX gene may be a
potential treatment for Hemophilia B in the future.
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Conclusion
While the cause of Hemophilia B has been known since the mid-1950s and treatments
have subsequently been produced, there has yet to be any significant development of an at-home
diagnostic tool to improve patient quality of life. The multitude of diagnostic tools that have thus
far been produced are exclusively purposed for professional point of care or lab-based settings.
We hope that from the information and suggestions put forth in this paper, further research may
be performed to develop an at-home diagnostic tool to assist patients in maintaining persistent,
healthy FIX blood levels.
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