4. BLEEDING PHENOTYPE
• Do not bleed from superficial cuts or small vessels (e.g. nosebleeds)
• Delayed bleeding
• Deep muscle and joint bleeding
5. CURRENT TREATMENTS FOR HAEMOPHILIA
Factor replacement therapy:
1. Treat bleeds as they occur- “on demand” therapy
2. Treat bleeds before they occur- “prophylactic” therapy
6. DISADVANTAGES OF PROPHYLACTIC THERAPY
• Regular injections: Factor VII three times per week, Factor IX two times per week
• Need to start at early age
• Difficult in young children
• May need portacath
• Expensive (~£180,000 per year)
7. WHY GENE THERAPY?
• Continuous, 24/7, endogenous expression of protein at therapeutic levels
following a single gene transfer manoeuvre
• Single gene defect (deficiency of Factor VIII or IX gene)
• Therapeutic goal is modest- an increase in plasma Factor VIII or IX levels above
1% would be sufficient ameliorate the bleeding phenotype
8. GENE THERAPY FOR HAEMOPHILIA A
• Less success compared to haemophilia B due to the large size (7kb) of
Factor VIII
• Current approaches include:
The co-administration of two AAV vectors separately encoding the FVIII
heavy- and light chains
Use of AAV vectors encoding the B domain deleted Factor VIII (BDD-FVIII)
variant that is around 4.4 kb in size.
Use of lentiviral vectors to target haematopoietic stem cells.
11. STUDY DESIGN
• 10 men with severe haemophilia B each received a single dose of vector,
administered through a peripheral vein.
• 6 patients enrolled in an initial phase 1 dose-escalation trial, with 2 patients
each receiving a low, intermediate, or high dose.
• 4 additional patients later enrolled who received the high dose.
12. RESULTS
• Persistent stable FIX expression in all 10
subjects (1 to 6%) up to 4 years later.
• Of 7 patients receiving prophylactic treatment
before gene therapy, 4 were able to stop and
most others were able to increase the interval
between prophylactic infusions.
• Significant reduction in amount of factor IX
concentrate administered over the duration of
the study resulting in a financial savings of
more than £1.75 million based on 2014 prices.
13. CONCLUSION
• Gene therapy for the treatment of haemophilia patients may soon reach the
clinic.
• Gene therapy can be less invasive compared to protein replacement therapy
that requires multiple weekly injections.
• Offers the potential of a cure for haemophilia rather than just disease
management.
• More economical because the number of treatment events should be limited,
potentially to a single treatment.
14. REFERENCES
• Long-Term Safety and Efficacy of Factor IX Gene Therapy in Hemophilia B, N
Engl J Med 2014; 371:1994-2004
• Current status of haemophilia gene therapy
High, K H; Nathwani, A; Spencer, T; Lillicrap, D (2014)
Haemophilia : the official journal of the World Federation of Hemophilia vol.
20 Suppl 4 p. 43-9
• Gene therapy for haemophilia
Murphy, Samuel L; High, Katherine A (2008)
British journal of haematology vol. 140 (5) p. 479-87
• Gene therapy returns to centre stage
Naldini, Luigi (2015)
Nature Publishing Group,vol. 526 (7573) p. 351-360
Editor's Notes
Haem A: 1 in 10000
Haem B: 1 in 30000
Clinically indistinguishable, pattern of bleeding the same
Mild, moderate and severe forms of the disease.
Mild (6-40% factor levels)- Excessive bleeding only after major trauma or surgery
Moderate (2-5% factor levels)- Prolonged or excessive bleeding after minor trauma, rarely bleeds into joints
Severe (<1% factor levels)- spontaneous’ bleeds, joints and muscle, easy bruising, bleeding often delayed but prolonged, bleeding time normal
Clinically indistinguishable, pattern of bleeding the same
Extrinsic pathway: TF complexes with factor 7 converting it to 7a. TF and 7a convert F9 to 9a which converts F10 to 10a involving calcium and phospholipids. F10a converts F2 to thrombin which converts fibrinogen to fibrin. Once Thrombin is activated there is a feedback loop from thrombin that converts F5 to 5a and 8 into 8a. So the loss of factor 8 results in the loss of an important feedback loop.
Intrinisc: Xa interacts with tissue factor pathway inhibitor (TFPI) and turns off the extrinsic arm. However, by this point there is enough thrombin around to kick off coagulation along the intrinsic arm (with XI, IX and VIII) – and from there on out, the intrinsic arm supplies the rest of the fibrin.
Primary haemostasis is intact and sufficient for small vessels
Primary haemostasis works initially, Inadequate thrombin generation leads to Poor fibrin mesh, Increased susceptibility to fibrinolysis.
Tissues with low TF expression, More dependent on intrinsic pathway feedback
Haemophilia A
Less success due to large size of factor 8 which far exceeds the normal packaging capacity of AAV vectors.
Two AAV vectors
The co-administration of two AAV vectors separately encoding the FVIII heavy- and light chains separately encoding the FVIII heavy- and light chains whose intracellular association in vivo leads to the formation of a functional molecule. The alternative two AAV vector approach exploits the tendency of these vectors to form head to tail concatamers. Therefore, by splitting the expression cassette such that one AAV vector contains a promoter and part of the coding sequence, as well as a splice donor site, whereas the other AAV vector contains the splice acceptor site and the remaining coding sequence. Following in vivo head to tail concatemerization a functional transcript is created that is capable of expressing full-length FVIII protein
B domain deleted
Use of AAV vectors encoding the B domain deleted Factor XIII (BDD-FXIII) variant that is around 4.4 kb in size. This was made after it was found that the B-domain was found to be redundant for maintaining haemostasis.
Use of lentiviral vectors
They have a larger capacity so can carry the factor 8 gene. Although FVIII expression is generally considered to be liver specific, many studies have shown that different cell types are capable of synthesizing functional FVIII protein. Virtually any cell type with access to the bloodstream can be targeted for gene transfer. With respect to retroviral gene transfer, the haematopoietic stem cell (HSC) is efficiently modified and transplanted, and has, therefore, been a reasonable target for haemophilia A gene therapy.
Limitations of previous studies
Two early trials of hemophilia B gene therapy that used either intramuscular or liver-targeted delivery of AAV factor IX vectors based on AAV serotype 2, did not achieve stable expression of factor IX in the plasma of patients with severe hemophilia B. Many studies have shown that the intramuscular route is not an effective route of administartion
Liver toxicity was observed in patients in the liver-targeted study, an adverse event that may have been due to the activation of capsid specific T cells after the infusion of the high vector dose.
Efficient transduction with AAV is, however, limited by the need to convert its single-stranded (ss) genome into transcriptionally active double-stranded (ds) forms in target cells because of its dependence on host-cell–mediated DNA synthesis of the leading strand or annealing of complementary genomes derived from separate virions.
This vector has several features to combat these issues, including a liver-specific promoter, designed self-complementary in a tail-to-tail dimer to increase transcriptional activation and transduction efficiency, and codon optimization for sequences frequently found among highly expressed eukaryotic genes. Additionally, the AAV2 vector was pseudotyped with an AAV8-capsid to blunt immune responses, since AAV8 has lower sero prevalence in humans than AAV2 so there’ll be reduced production of neutralizing antibodies. The transgene also contains the liver specific promoter LP1, which limits the FIX gene expression to only hepatocytes.
Toxicity in study
The major vector-related adverse event was a dose-dependent, asymptomatic increase in the serum Alanine aminotransferase levels associated with a decline in factor IX levels, suggesting a loss of transduced hepatocytes. Expansion of the high dose cohort showed that this adverse event was common, occurring in four of the six patients. The increase in serum ALT levels occurred consistently at 7 to 10 weeks after gene transfer, thus defining the critical period of monitoring and pharmacologic intervention. The precise pathophysiological basis for the hepatocellular toxicity, which has also been observed with other serotypes and genomic configurations, remains unclear. Apart from the dose of vector, there were no identifiable clinical differences between the patients who had an increase in the ALT level and those who did not have an increase. In any case, prednisolone was effective in limiting the hepatocellular toxicity as well as preserving the expression of transgenic factor IX, especially when such treatment was initiated early.
As expected, long-lasting AAV8 capsid-specific humoral immunity developed in all the patients who underwent gene transfer. Although the increase in anti-AAV8 IgG levels did not have direct clinical consequences as far as we can tell, its persistence at high titers would preclude subsequent gene transfer with vector of the same serotype if transgene expression fell below therapeutic levels. However, preclinical studies suggest that it may be possible to achieve successful transduction in patients with preexisting antiAAV8 antibodies after the infusion of AAV vectors pseudotyped with alternative serotypes, such as AAV5, that have low cross-reactivity with antiAAV8 antibodies.
