This case study describes a 23-year-old male with deep vein thrombosis found to have elevated factor IX levels. Testing found a point mutation causing increased factor IX activity. Animal studies using a modified factor IX gene showed promise for gene therapy at lower doses to treat hemophilia B while avoiding immune responses. Further research aims to develop this technique to safely and effectively deliver clotting factors.

1. Sadia Islam

2. Thrombophilia
Venous thromboembolism <45 years of
age usually associated with family hx
Recurrent episodes
Commonly associated with a gain-of-
function mutation in the factor V gene
(factor V Leiden) or in the prothrombin
gene variant 20210A.

3. Elevated IX levels
Elevated level of IX independent risk
factor for venous thrombosis.
The prevalence of elevated factor
IX is 20% among patients with venous
thrombosis and 5% in the general
population.

5. Case Study
23 year old Caucasian male
Family origins – northeast region of Italy
Femoral – popliteal – deep – vein thrombosis
in the right leg
Confirmed on compression ultrasonography
Occurred a few days after mild muscular
stretching
LMWH at a dose of 100U/kg of body weight
twice daily and warfarin adjusted to achieve
INR of 2.0-3.0

6. Coagulation and
thrombophilia screening
Normal levels of protein C, protein S, and
antithrombin.
Factor V Leiden and Prothrombin variant
G20210A not detected.
Lupus anticoagulant, anticardiolipin,
hyperhomocysteinemia were all negative.
FVIII and FXI had normal levels
Whole family tested

7. Factor IX results
Factor IX activity level – 776% of normal
activity
Factor IX antigen – 92%
Ratio of antigen to activity was 8.4

8. Family results
Mother – normal antigen levels, 337% activity
level.
Brother – normal activity level, 551% activity level.
All other family members had normal results.

9. DNA analysis
Point mutation in the factor IX gene (G31134T
transversion) that caused a substitution of
leucine for arginine at position 338.
A wide range of patients from the same
geographical area with thrombophilia tested,
none were identified with this mutation

12. Current treatment
The disease is currently treated with
protein replacement therapy -
intravenous infusion of clotting factor
concentrates.
 (>US$200,000 per year for an adult with severe
disease treated prophylactically),
About 20% of the world’s haemophilia
population can afford these regimens.
For the poorest, the disease remains lethal
in childhood or adolescence.

13. Indications for Gene terapy
 The therapeutic window is wide-
increasing factor IX levels to >2% of
normal levels will improve phenotype
 Levels up to 150% are still within the
normal range
 End points are easy to measure.

14. Gene therapy
Patients are injected with vectors – (non
disease carrying viruses) - to carry DNA
sequences that hold a genetic code that
enables clotting factor production.
Trials of AAV (Adeno-associated viral -8
vector ) administration to the liver have
been somewhat successful for severe
haemophilia B.

15. Current issues
Immune response against the vectors used to
carry the DNA
Some patients can develop antibodies that
attack and destroy the clotting factors
before they have a chance to work.
Severity of response has so far defeated its
benefits
Patients with more severe haemophilia may
need frequent sessions, which is expensive
and time consuming

16. Vectors appear to trigger a strong dose
dependant immune response, the higher the
vector dose, the stronger the immune
response is.
 Severe Haemophilia High dose  severe response
Typically, for each specific combination of
vector, transgene and target tissue, one or
two of the following problems predominate
as the major clinical obstacle.

17. Gene silencing
Gene silencing can result, for example, from
promoter methylation leading to loss of
therapeutic transgene expression.
First documented clinically in a study of
retrovirus-mediated gene transfer for X linked‑
chronic granulomatous disease
Subject initially benefited from therapeutic
intervention but later died from complications
associated with the underlying disease itself.
It was shown that 60% of granulocytes
harboured the therapeutic gene, but less
than 10% expressed it, at the time of death.

18. Insertional mutagenesis
Results from random integration of the
therapeutic vector genome within host DNA.
First reported in a clinical study for retrovirus-
mediated gene transfer for X linked severe‑
combined immunodeficiency, in which
vector integration led to malignancy.
No vector-induced malignancy has been
reported in clinical gene transfer studies in
which adeno-associated virus (AAV), were
used although they also show evidence of
integration.

19. Immunotoxicity & Phenotoxicity
Refers to harmful immune responses to either
the vector or the transgene product.
Activation of T cells directed against AAV
capsid antigen has been documented in
several clinical studies
Associated with loss of therapeutic efficacy.
Transgene-driven immune responses in
several animal models of gene transfer with
AAV.
Can result from the overexpression or ectopic
expression of the donated gene.

20. Adeno-associated viral -8 –
vector or AAV-8 vector

21. Horizontal transmission of donated
DNA
The risk of vector shedding after in vivo
gene transfer is proportional to time since
administration and to the vector dose
administered.
Human studies indicate that vector can
be found in body fluids (such as serum
and urine) for several weeks after systemic
vector administration.

23. Next step
Researchers have now set out to find a
technique that delivers lower vector doses to
reduce immune response, while effectively
producing clotting factors.

24. Factor IX Padua Studies
The team set out to see whether the
increased blood clotting ability of FIX-
Padua would allow lower vector doses to
be delivered, achieving an effective
treatment without triggering a strong
immune response.
FIX-Padua was administered via injection
to three dogs who had severe forms of
haemophilia B similar to those found in
humans.

26. Results
One dog already possessed inhibitory antibodies
prior to treatment having been exposed to
clotting factors before, while two of the dogs had
never been exposed to clotting factors and had
no inhibitory antibodies.
The two dogs that had no prior inhibitory
antibodies showed significant improvement in
their haemophilia within 1 week, easing from
severe to mild.
In addition, the dogs had no bleeding episodes
for up to 2 years.
They had no immune response to FIX-Padua and
there was also no sign of thrombosis.

28. Results
The third dog that had pre-existing inhibitory
antibodies also showed significant improvement
following treatment with FIX-Padua.
The animal's haemophilia also eased from severe
to mild, and this persisted for up to 3 years.
The inhibitory antibodies that the dog already
possessed were eradicated by the injection -
something the researchers say has never been
seen in an animal model before.
The team also tested FIX-Padua in mouse models
of haemophilia and found it produced similar
results.

30. Isolation of FIX
FIX was isolated by means of affinity
chromatography and ion exchange
chromatography
Genomic DNA obtained and sequenced.
Direct detection of R338L mutation was
performed with the use of PCR
Human embryonic kidney 293 cells transduced
by lentivirus vectors that expressed wild type FIX
or FIX with R338L mutation with a use of a
mutagenesis kit.