1. Researchers have identified a potential new diagnostic tool for Hemophilia B that could allow patients to monitor their coagulation factor levels at home. 2. Hemophilia B is caused by a deficiency in Factor IX and affects approximately 1 in 34,000 males in the US. Current diagnostic methods can only be done in a clinical setting. 3. The document discusses Hemophilia B treatment options, the molecular pathway of blood clotting, genetic research into Hemophilia B biomarkers, and encourages further research into more convenient diagnostic tools.

1. A survey of recent discoveries:1
Identification of an innovative2
diagnostic tool for Hemophilia B3
ALS 320: Medical Diagnostics, Fall 2015- Section 1, Team 24
Jesse Forchap, Brandy Fugate, Benjamin Blackwell,5
Anthony Park, Caitie Staat, and Khaled Hamad6
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WORD COUNT: 4,0318
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2. Abstract17
Hemophilia B is a rare blood clotting disorder that affects an estimated 1 in every 34,00018
males in the United States. Hemophilia B is caused by a mutation in the Factor IX gene, which19
interferes with the coagulation cascade. The molecular basis of Hemophilia B is complex,20
revolving around proper folding of Factor IX such that its activated form can properly participate21
in the coagulation cascade. As a result, there are a variety of diagnostic tools used to measure22
Factor IX in the blood, but all of these devices are presently used exclusively in professional23
point-of-care settings. There has been little effort put forth in the search for an at-home device24
that patients can use to monitor and control their coagulation factor levels in the blood. By25
presenting a summary of the currently understood pathways, preventions, treatments, and26
diagnostic tools involved in Hemophilia B management, we hope to encourage further27
investigation into a more convenient diagnostic device and potential new treatment options for28
sufferers of Hemophilia B.29
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31
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33
34
35
36

3. An Introduction to Hemophilia B37
Hemophilia B, also known as Christmas disease, is a rare blood clotting disorder caused38
by deficiency in the Factor IX blood-clotting factor. The term “Christmas disease” is derived39
from the first patient, Stephen Christmas, who was diagnosed with the disease in the early40
1950s.1 Factor IX (FIX) is a blood-clotting factor produced in the liver, and is a critical41
component of the blood clotting cascade.2 The FIX gene is located on the X chromosome, and42
mutations in this gene are the primary cause of Hemophilia B. In some rare cases, the disease43
may be acquired during the latter part of a patient’s life as an auto immune disorder. Christmas44
disease is the second most common type of Hemophilia; Hemophilia A is most common and is45
caused by a deficiency in Factor VIII production.346
Patients with Hemophilia B suffer from joint damage, general organ deterioration, and47
abnormal bleeding after minor operations, such as tooth extraction.1 However, the degree of48
bleeding varies depending on the individual and the severity of the disease. According to the49
National Hemophilia Foundation, there are three main classes of Hemophilia B determined by50
FIX plasma levels.4 Severe cases are defined by FIX levels less than 1% of the normal range, in51
which patients experience spontaneous bleeding in their joints and muscles. This class of severity52
is easily detected through apparent physiological symptoms. FIX levels between 1% - 5% of53
normal define moderate cases, and patients in this class generally have bleeding episodes after54
injury. Detection of moderate cases is less apparent than severe cases, in that bleeding events are55
less apparent. In mild cases, FIX levels are above 5 % but less than 40 % of normal. Mild cases56
of Hemophilia B can go undetected for years due to the absence of typical symptoms, as only57
severe injury or trauma results in excessive bleeding.58

4. The FIX gene is located on the long arm of the X chromosome. Females with an FIX59
variant on only one X chromosome are considered carriers, but do not express symptoms.260
Female carriers may rarely experience abnormal bleeding episodes after childbirth and require61
FIX replacement therapy. 2 Hemophilia B is more prevalent in males because of its X-linked62
nature. In the U.S., Hemophilia B occurs in approximately 1 of 34,000 males, and the number of63
patients in the U.S. as of 2010 was approximately 4,000. Annual Hemophilia B expenditure in64
the U.S. is approximately $58,000 per patient which results in a total of $246.2 million per year65
if all patients were to be treated. This cost could increase over the coming years as our66
population continues to rise, bringing attention to the importance of Hemophilia B within the67
American health care system.568
Current diagnostic methods are primarily quantitative. A variety of these methods will be69
discussed in depth. Monitoring of FIX levels comprises the basis of Hemophilia B prognosis.70
Presently, FIX levels can only be measured in a professional point-of-care setting, which is71
inconvenient to patients. Additionally, treatments for modulation of FIX activity rely on an72
approximation of FIX levels in the blood. We hope to encourage additional research by73
examining current treatment methods for Hemophilia B and proposing novel diagnostics that74
may assist in simplifying treatment and improving quality of life. First, we will delve into current75
preventative measures and treatment strategies used to mitigate the symptoms and complications76
associated with Hemophilia B.77
Preventative Measures and Current Treatment Strategies78
While Hemophilia B is rare, it is important that potential parents affected by this disease79
examine preventative measures when considering pregnancy. Preimplantation Genetic Diagnosis80

5. (PGD) is an innovative technique that assists in informing couples prior to conception of the81
chances that their child will develop Hemophilia B.6 PGD consists of two main steps: in-vitro82
fertilization (IVF) is first utilized, and a biopsy of the embryo is subsequently genetically83
analyzed. IVF is accomplished through hyperstimulation of the ovaries followed by retrieval of84
the egg, fertilization of the egg, and development of the embryo. Once developed, a small sample85
cell is taken from the embryo. Fluorescent In Situ Hybridization (FISH) and Polymerase Chain86
Reaction (PCR) are used for genetic evaluation of this sample.6 Embryos that are discovered to87
be unaffected by Hemophilia B are then implanted into the mother’s uterus.6 Parents may opt to88
conceive naturally and either accept the potential challenges and risks of giving birth to a baby89
who has the genetic disorder, or proceed with prenatal diagnosis, then terminate the pregnancy if90
Hemophilia B is confirmed.7 However, termination of a viable pregnancy based on a Hemophilia91
diagnosis raises strong ethical and cultural concerns.92
Hemophilia B is principally treated by managing clotting FIX. Replacement FIX therapy93
is the preferred method of treatment for Hemophilia B. Concentrated FIX is regularly injected94
into the vein multiple times per week, temporarily replacing the patient’s clotting factor.895
Clotting factor concentrates were previously made via reconstitution of donated human blood.96
Donated blood was screened to prevent disease transmission, though risk of contracting an97
infectious disease still existed. Clotting factors are now being procured from genetically98
modified cells introduced to a hamster cell line. These are known as recombinant clotting99
factors.9 The FIX gene is incorporated into the hamster cell genome, and the protein is produced100
in large amounts using the cell line. This is less risky because it utilizes animal cells that are free101
of human diseases.9 Clotting factors produced through cell lines can be convenient as they have a102
long shelf life and can thus be made more readily available to patients.10103

6. Depending on the severity and pattern of bleeding, there are two classifications of104
therapies used. Replacement therapy is used to prevent regular bleeding, and is therefore105
prophylactic. Children with severe Hemophilia B use prophylactic therapy on a regular basis.11106
There are two sub-types of prophylactic therapy: primary, which young children start using to107
lessen or prevent diseases in the joints and will be continued for life, and secondary, which is108
used frequently for a limited period of time when bleeding begins.8 The benefits of using109
prophylactic therapy include a lower risk of spontaneous bleeding, the ability to participate in110
sports, and lower risk of damage to the joints.8,11 The disadvantages of using prophylaxis include111
constant injections and inevitably higher expenses.8,11 However, on-demand therapy can be used112
in place of prophylaxis for the purpose of occasional or sporadic bleeding, as needed. On-113
demand therapy is less rigorous and less expensive than prophylactic therapy. 8114
One of the most severe complications that can develop through treatment of Hemophilia115
B is antibody resistance to clotting factor.12 FIX can be destroyed by these antibodies, which116
then prevents replacement therapy from working properly. These antibodies are known as117
inhibitors, and they develop in 1.5-3% of Hemophilia B patients.8,12 Figure 1 depicts the118
Bethesda assay used to detect the inhibitor concentration (or amount of antibodies) in the blood.8119

7. 120
Figure 1: Bethesda units (BUs) and factor present in blood post-injection. A higher BU value indicates a121
higher concentration of inhibitors present in the blood, which results in less active FIX post-injection.122
When the inhibitor concentration increases, additional FIX must be injected into the patient to maintain123
healthy FIX levels. Adapted from www.sevensecure.com/inhibitor/inhibitor-education.aspx124
Stemming from our conversation on current treatments, we will now discuss the basis of125
Hemophilia B on a molecular level, and how the various blood-clotting factors play their roles in126
the clotting cascade.127
The Molecular Pathway of Hemophilia B128
The pathway of Hemophilia B was first elucidated in 1986 by Diuguid, et al. at the New129
England Medical Center and Tufts University School of Medicine in Boston.2 The FIX protein is130
synthesized exclusively in hepatocytes and is produced as a zymogen, or an inactive131
precursor.2,13 In the unadulterated pathway, FIX is first translated by ribosomes in hepatocytes,132
and is structured as a single chain plasma glycoprotein peptide sequence.14 The FIX molecule is133
comprised of three main units: the signal peptide, which facilitates the transport of the protein134
across the plasma membrane, the propeptide sequence, and the final protein product.2 Before any135
posttranslational modifications are made, the signal peptide is first cleaved from the FIX136

8. molecule once it has exited the nucleus. Next, a series of modifications occur including137
glycosylation, cleavage of the propeptide, beta-hydroxylation, and vitamin K-dependent138
carboxylation.2,14 Once these posttranslational modification reactions are completed, the FIX139
zymogen is released from the hepatocyte.140
It is important to note that this pathway occurs on the platelet surface at the site of141
damage along a blood vessel.15 Effective completion of the blood coagulation cascade can only142
occur on cell surface membranes.16 In order for FIX to be activated, it must be converted into a143
serine protease by Factor XIa in the presence of Ca 2+ .14 When FIX experiences these conditions,144
the hydrolysis of two peptide bonds buried inside the Factor IX molecule occurs, which allows145
for the release of the activation peptide.14 This results in procurement of Factor IXaβ, which is the146
active and final form of FIX. It is comprised of a single heavy chain and a single light chain,147
with the heavy chain containing the active site.14 Factor IXaβ activates Factor X along with148
assistance from Factor VIIIa.16 This occurs though complexing of Factor IXaβ and Factor VIIIa on149
the membrane surface, whose new product is able to generate Factor Xa.
16 Factor Xa then150
complexes with Factor Va, which promotes activation of prothrombin. Finishing out the pathway,151
the now active thrombin cleaves fibrinogen present near the platelet surface, which then152
polymerizes to form a fibrin clot.16 Figure 2 illustrates the previously described pathway.153

9. 154
Figure 2: A display of the entire coagulation cascade from initial injury to final clot formation. This figure155
shows all key players involved in the formation of a clot. Adapted from the Hemophilia Report 2014.6
156
There are two types of defects that have been characterized in Hemophilia B:157
independent deletion mutations and point mutations.16 This will be discussed in depth in the next158
section. For the sake of simplicity we will discuss only one of the many possible mutations that159
leads to Hemophilia B that disrupts γ-carboxylation. However, this discussion is linked to a160
broader context in that many mutations that cause Hemophilia B interfere with γ-carboxylation.16161
FIX is vitamin K-dependent, meaning that it requires a carboxylase to bind to it in the presence162
of vitamin K in order to become secreted. In the point mutation being discussed, an arginine163
becomes a serine at the -1 residue, as discussed in the article by Diuguid, et al. This leads to164
interference of γ-carboxylation, and ultimately causes failure of the cleavage of FIX’s165
propeptide.2 FIX subsequently misfolds and cannot activate Factor X because it is incapable of166

10. complexing with Factor XIa on the cell membrane. From this point, Factor X cannot go on to167
promote activation of prothrombin. Thus, the clot cascade fails and excessive bleeding occurs at168
the site of injury.169
This mutation example can be extended to some other deletion mutations that cause a170
similar non-recognition of the FIX molecule by the Vitamin K-dependent kinase within171
hepatocytes. From the elucidated pathway of Factor IX, and subsequently Hemophilia B, we can172
derive some key biomarkers that can be utilized in the diagnosis and monitoring of this disease.173
Genetics Researchand Biomarkers of Hemophilia B174
Hemophilia B is caused by mutations in the Factor IX gene, and there are more than175
1,100 such mutations that have been identified.17 The most common FIX mutation that causes176
Hemophilia B is substitution of a single DNA base pair, although other mutations may still177
occur.18 These mutations are linked to a decreased quantity of active FIX and lead to defective178
blood clotting and excessive bleeding. Therefore, severity of Hemophilia B chiefly depends on179
the severity of the mutation.180
Identification of FIX mutations within their locus on the X chromosome is essential181
because identified regions can be used as genetic markers to treat Hemophilia B. Two common182
types of genetic screens performed to identify heterogeneous mutations in FIX are Restriction183
Fragment Length Polymorphism (RFLP) analysis and haplotyping. RFLP analysis is a technique184
that exploits variations in homologous DNA sequences. A haplotype is a group of genes within185
an organism that were inherited together from a single parent. Both of these analyses can offer186
prevention through screening by detecting X-linked gene carriers.187

11. 188
Figure 3 Informativeness of the RFLP markers in different population groups of India.19
Panels A, B and189
C respectively represent the Eastern,Southern and Western regions. RFLP markers analyzed in each bar190
diagram are denoted by DdeI (D),HhaI (Hh), Hpy188I (Hp), MnlI (M), TaqI (T), and XmnI (X).19
Panel191
D shows informativeness for North India obtained from a separate study (Chowdhury et al. 2001).19
192
Informativeness for each marker was calculated as the percentage of females heterozygous in each193
cluster.19
Adapted from Mukherjee, S. et al., 2006.19
194
195
For RFLP analysis, Mukherjee, et al. collected data from eight different populations that196
were composed of 107 normal females from different geographical areas with clear family197
histories of Hemophilia B, and 13 carriers that were unrelated to each other.19 Then, DdeI, XmnI,198
MnlI, TaqI, and HhaI restriction sites were used to identify the genetic markers (Figure 3).19 In199

12. addition to RFLP analysis, researchers also used Single Nucleotide Polymorphisms (SNPs) to200
screen regions of the FIX gene.19 The results from this study on various populations in India201
showed that only two SNPs were found as possible candidates for differentiating normal groups202
from carrier groups, whereas RFLP markers on specific groups were effective in differentiating203
between carrier groups and normal groups.19 The study also mentioned that additional genetic204
markers were necessary for more efficient analysis because the variability of heterozygosity was205
quite high.19206
Another study done in Sweden focused on discovering the origin of high occurrences of207
certain FIX mutations in specific subgroups of the population.17 Their objective was to analyze208
and classify FIX mutations within Swedish families into two different types of mutations. These209
were classified as independent recurrent mutations (RMs) or common mutation events, also210
known as identical by descent (IBD).17 By resequencing and performing haplotype analyses on211
86 Swedish families with 74 genetic markers, researchers found that both RMs and IBDs were212
present at the same proportion -slightly over 50%- in patients with the mild severity phenotype213
of hemophilia B.17214
Genetic marker analyses of the FIX gene on different population groups from different215
geographical regions showed a wide range of variability and specificity based on types of216
analysis performed. In juxtaposition to the RFLP markers used in the study performed by217
Mukherjee, et al. (Markers 20-30), the study performed in Sweden used the M1 marker.20 Since218
genetic markers vary greatly from population to population, discovery of new genetic markers by219
performing different experimental methods such as Random Amplified Polymorphic DNA220
(RAPD), Amplified Fragment Length Polymorphism (AFLP) and Quantitative Trait Locus221

13. (QTL) analyses could potentially lead to more efficacious and specified genetic markers of222
Hemophilia B.21223
Coagulation FIX, translated from the FIX gene, is the most well-researched biomarker224
used for detecting Hemophilia B.18 FIX is associated with an activated partial thromboplastin225
time (aPTT) of the intrinsic pathway.22 As was stated before, in the normal coagulation cascade,226
FIX is activated by Factor XIa and activates Factor X with assistance from Factor VIIIa and other227
molecules.23 Although levels of Factor XI, Factor X and Factor VIII could be possible candidates228
for alternative biomarkers of Hemophilia B, FIX is still strongly recommended since other229
factors are placed either upstream or downstream of the FIX activation within coagulation230
pathway. Therefore, FIX is the most logical biomarker to utilize for Hemophilia B at the present231
time. We will now move into the diagnostic methods that use FIX to identify Hemophilia B.232
Diagnostics for Hemophilia B233
Various types of tests have been implemented for screening and diagnosis of Hemophilia234
B, though quantitative tests are predominantly employed in regular practice. The Hemochron®235
Signature Elite, a handheld point-of-care testing device, can perform various coagulation tests236
and is commonly used in clinical settings.24 For initial screening, activated partial thromboplastin237
time (aPTT) is most often utilized, and is a one-stage clotting assay.25 Both aPTT and an238
alternative screening coagulation test, prothrombin time (PT), can be determined by the239
Hemochron® system.24 Both tests are performed using similar mechanisms, but each requires240
different sample types and reagents.241
Sample separation is not required in the Hemochron® system, as this device is a whole-242
blood system. Venous or finger-stick samples are analyzed.24 Testing occurs within disposable243

14. cuvettes preloaded with reagents; these reagents increase the sensitivity of each assay.26,27 PT244
sample tubes are preloaded with dried thromboplastin,26 whereas sample tubes for aPTT testing245
are preloaded with platelet factor substitutes and kaolin reagents that standardize the activation246
of clotting factors.27 Respective stabilizers and buffers are also included in both types of sample247
tubes.248
When a sample is introduced to the system, the device dispenses 15 microliters of that249
sample into a cuvette. The device automatically performs reagent mixing. An incubation period250
is not required for aPTT testing.27 As the cuvette is moved rapidly back and forth within the251
instrument, motion of the sample is monitored and measured by LED optical detectors in line252
with the sample. As the sample clots, the overall motion of the sample is reduced. When the253
movement has decreased below a certain threshold, the device identifies that an endpoint has254
been reached. The time required to clot is reported respectively for each test. Results of both tests255
can be obtained in approximately two minutes.26,27 PT results are represented in terms of the256
International Normalized Ratio (INR), a ratio of the patient’s resulting PT compared to a normal257
range that is designated internationally.26 Activated PTT results are expressed in terms of their258
plasma-equivalent values, which are automatically calculated by the system.27259
The primary test currently administered for final diagnosis of Hemophilia B is a FIX260
assay. One type of analyzer developed for coagulation automation utilizes electromagnetic261
mechanical detection methods to detect plasma levels of activated FIX.28 Siemens has developed262
one such analyzer called the Sysmex® CS-5100 System. Although this device is not currently263
available in the U.S., other devices that employ this type of technology are commonly264
utilized.28,29 The Sysmex® System, similar to the Hemochron® device, measures motion within265
the sample provided.29 FIX deficient plasma is the primary reagent added to the sample plasma266

15. in these assays.30 Factors within specimens given by healthy patients are able to correct for267
deficient factors within the reagent, and clotting will occur. However, in Hemophilia B patients,268
clotting will not occur as both the reagent and sample plasma have factor deficiencies.31 Fibrin269
formation is measured as it surrounds an iron ball that is also added to the plasma.32270
Electromagnetic action moves the iron ball within the plasma. Mobility of the ball is reduced as a271
clot accumulates around it. The device detects the consequent lack of movement and provides an272
output value.32 Final diagnosis of Hemophilia B can be assigned a level of severity: mild,273
moderate, or severe.4 Disease severity along with associated signs and symptoms is outlined in274
Table 1.275
276
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 IX277
activitya
.8
Various physiological signs may also correspond with the severity of the disease.278
279
FIX is a reliable biomarker that is specific to Hemophilia B. However, diagnostics are280
presently only available in professional settings, which may be due to rarity of the disease and281
the resultantly small market size. Development of a portable device designed to measure282
activated FIX levels would benefit Hemophilia B patients, as the availability of a point-of-care283

16. diagnostic tool is a critically unmet need. Many patients with mild or moderate disease diagnoses284
may not be aware of less obvious internal bleeding, placing them at risk for development of285
comorbidities such as hemarthroses (bleeding in joints), renal disease, and cardiovascular286
complications.8 Routine, prophylactic treatment for management of bleeding reduces the287
likelihood of developing these conditions.8 Rapid testing methods could therefore assist in288
reducing the occurrence of associated diseases.289
Further research should be done to determine whether or not an at-home diagnostic290
device could be applied to routine patient care, thereby reducing the long-term side effects of291
bleeding. Traditional assay techniques may be utilized to quickly measure levels of FIX in the292
blood. This would assist a patient in determining when treatment is necessary. Some patients293
naturally develop inhibitors against FIX added to the bloodstream,12 indicating that development294
of an immunoassay might be an effective way to construct a new diagnostic tool. Potential295
integration of a color change mechanism might be utilized to determine how much of the analyte296
has bound to a test line. This could provide a partially quantitative result, allowing patients to297
identify when immediate injections of FIX replacement are required.298
Looking Ahead: Future Treatments of Hemophilia B299
Patients suffering from Hemophilia B are currently unable to measure the300
concentration of FIX at home. Current available options include waiting for physiological301
symptoms to arise, which can increase the chances of unnecessary bodily harm, or to inject FIX302
multiple times a week and assume that levels of FIX are stable. A new possible delivery method303
that can solve these issues is an implantable closed loop device, which has proven successful in304
diabetic patients.305

17. 306
Figure 4 Implantable Glucose Device33
retrieved from Implantable closed-loop glucose-sensing and307
insulin delivery. Adapted from Renard E., 2002. 33
308
The glucose device shown in Figure 4 stabilizes patient’s blood glucose levels over309
longer periods of time compared to regular insulin injections.33 This device contains an310
intravenous glucose sensor, compartment to store insulin, and a catheter. The glucose sensor uses311
an enzymatic reaction involving glucose-oxidase that is monitored by an oxygen sensor; the312
sensor measures changes in oxygen levels, and sends a signal through a subcutaneous connector313
that relays signals from the sensor to the compartment filled with insulin.33 The device then uses314
a mathematical algorithm to release a specific concentration of insulin through the catheter33.315
Implementing a similar device into a Hemophilia B patient’s treatment regimen would reduce316
their risk of joint degradation or other associated diseases by maintaining FIX levels in the blood.317
However, there is currently no sensor on the market that can measure FIX in solution.318
One way to improve quality of life of Hemophilia B patients is by reducing the number of319
injections needed per week. It has been proven that PEGylation of certain proteins can increase320
the half-life of a protein in solution. PEGylation is performed by covalently binding a321
polyethylene glycol molecule to the desired protein.34 The addition of polyethylene glycol322
External Programmer
using telemetry
Central
port
refill
Intraperitoneal
catheter
Abdominal connecting
lead (subcutaneous)
Subcutaneous
connector
Intravenous
enzymatic
glucose sensor
Slideport

18. decreases the chances of protein metabolism, propensity for immunogenic response, and323
clearance.34 By PEGylating FIX concentrates, we can expect to see a decrease in the number of324
injections per week, and a reduction in the immunogenic response against FIX. However,325
PEGylation has some potential side effects. PEGylation has increased coagulation of proteins in326
some cases, which increases the chances of clot formation in blood vessels, causing327
thrombosis.34 Further studies should be conducted to see the effects PEGylation may have on328
FIX coagulation in particular.329
An alternative treatment to injecting FIX concentrates is gene therapy. Gene therapy330
provides a way of introducing a wildtype FIX gene into a cell to be expressed if the host gene is331
mutated or defective. The gene can either be integrated directly into a cell’s chromosome, having332
a long-lasting effect, or can be delivered to the nucleus, which has a short-lasting effect because333
the gene is attached to a free-floating plasmid.35 The type of delivery method will therefore334
predict whether the gene therapy will result in a treatment or a cure. There are three possible335
methods that could be used to deliver the FIX gene to its target: adeno-associated vectors, naked336
plasmids, and lentiviral vectors. By using adeno-associated virus vectors (AAVs), the FIX gene337
can be inserted inside a virus that delivers the FIX gene to the nucleus where the gene is338
expressed by the host’s cellular machinery (Figure 5).13 Unfortunately, there have been instances339
of immunogenicity occurring using this method, as the virus is identified as non-self by the host340
immune system.35341

19. 342
Figure 5 Gene therapy using Adeno-associated virus vector. Adapted from Carr, et al. 2015. 13
343
344
Utilization of naked plasmids is an alternative approach to avoiding immunogenic345
response since they are a non-viral vector, although this method has a less permanent effect than346
AAV. Naked plasmids cannot insert the gene into the host cell’s target chromosome, so the347
expression of Factor IX will fade with time. It is also difficult to target specific cells using this348
method.1349
Lentiviral vectors can incorporate FIX gene into the host cell, which means FIX350
expression would not fade over time.35 Lentiviral vectors have been used in both in-vivo and ex-351
vivo studies. Utilization of a lentiviral vector produced an immunogenic response similar to the352
AAV vector in-vivo.35 However, when the lentivirus was transduced via ex-vivo techniques it353
did not initiate an immunogenic response.35 Thus, ex-vivo transduction of FIX gene may be a354
potential treatment for Hemophilia B in the future.355
356

20. Conclusion357
While the cause of Hemophilia B has been known since the mid-1950s and treatments358
have subsequently been produced, there has yet to be any significant development of an at-home359
diagnostic tool to improve patient quality of life. The multitude of diagnostic tools that have thus360
far been produced are exclusively purposed for professional point of care or lab-based settings.361
We hope that from the information and suggestions put forth in this paper, further research may362
be performed to develop an at-home diagnostic tool to assist patients in maintaining persistent,363
healthy FIX blood levels.364
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