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The Science Behind Sanofi's Hemophilia Pipeline

Sanofi scientists and bioengineers are exploring several different ways to help make life more predictable and manageable for people living with hemophilia. They are researching three investigational approaches to hemophilia treatment: a new class of extended half-life factor replacement therapy, a potentially transformative RNA Interference (RNAi) therapy, and a potentially curative gene therapy. Let's take a look at the science behind the research.

Hemophilia is a genetic condition

Hemophilia is a rare, genetic condition in which a person has insufficient levels of a specific blood-clotting factor. People with missing or defective factor VIII have hemophilia A. People with missing or defective factor IX have hemophilia B. Without enough clotting factor, a person can experience spontaneous or prolonged bleeding, often into the joints and soft tissues.

Many people with hemophilia are treated with replacement blood-clotting factors, given by intravenous (IV) infusion. The frequency of IV infusions depends on the type and severity of hemophilia a person has, and their treatment regimen.

Hemophilia results from an imbalance of pro- and anti-clotting proteins

To stop bleeding from a damaged vessel, the body relies on a balance between pro-coagulant and anti-coagulant proteins that regulate clotting.1,2  In people with hemophilia there is an imbalance: a lack of pro-coagulant proteins leads to insufficient production of thrombin.3,4

  • Thrombin initiates clot formation. It is responsible for many clotting events, including the formation of the fibrin mesh that forms the beginning of a seal.5
  • Antithrombin limits thrombin generation and coagulation, which is important for keeping blood flowing. 
Injured blood vessel illustration1
Illustration of thrombin circulating2
Thrombin activates platelets3


(1) In red: blood vessel and erythrocytes (red blood cells). (2 and 3) In response to an injury in a blood vessel, platelets (gray) are activated by thrombin (blue) to produce clotting factors

1. How factor replacement therapies work

Clotting factors are cleared from the blood

All replacement factors circulate in the blood until: 

  • they are activated to help stop bleeding, or
  • they are broken down and cleared as part of a natural process. 

This clearance happens so quickly that for some people with hemophilia A, standard, prophylactic half-life factor replacement has to be given every second day, and sometimes more often, so that clotting-factor levels do not fall too low.

Extended half-life factor replacement therapies keep factors circulating longer

Hemophilia researchers figured out how replacement factors can stay in the bloodstream longer and remain available for clotting. The idea of "extended half-life" factor replacement therapies is to help factor VIII or IX “hitch a ride” on proteins that use a slower recycling pathway.

Factor VIII has a chaperone, called von Willebrand Factor

But there is a built-in limit on how long inactive (stand-by) clotting factors can circulate. Factor VIII usually travels with a companion called von Willebrand factor (vWF), which protects it from being broken down.6,7

Every 15–19 hours, half of the vWF gets cleared from the blood, taking factor VIII (yellow) along with it. That is why making factor VIII replacement therapies that are effective for longer than 19 hours is a big hurdle for researchers.

Illustration of factor VIII protected by von Willebrand Factor

Von Willebrand factor (light blue) protects factor VIII (yellow) from being broken down by certain proteins (green and dark blue) 

Scientists have discovered a potential alternative to von Willebrand Factor

Scientists at Sanofi have been developing a replacement factor VIII that enjoys the kind of protection vWF gives, without getting caught up in the vWF recycling pathway.8

Illustration of VWF unable to bind to replacement factor VIII1


When replacement factor VIII (yellow) is bound to a vWF domain (teal), the vWF circulating in the blood (teal and light blue) cannot bind to it

In this approach, inactive factor VIII is equipped with: 

  • A vWF domain – a small part of the protein that occupies vWF’s full spot on replacement factor VIII. This acts as a decoy, so the body's own vWF does not bind to the factor.
  • XTEN, a bioengineered molecule that fends off proteins that try to break down and clear factor VIII.
  • A linker molecule that releases the decoy when thrombin activates the replacement factor for clotting.
  • An Fc domain, or protein fragment, that allows the factor to bind to the right recycling pathway so it can stay in circulation longer.

The ambition of this novel investigational design is to make it possible to sustain high levels of replacement factor VIII in the blood over several days.

2. How RNAi therapies are designed to work

Scientists observed that patients who have lower levels of anti-thrombin in their blood produce more thrombin than others. They found that having lower levels of antithrombin-3 can restore thrombin production and make clotting more effective.9,10,11

A new approach in hemophilia research harnesses a natural biological process: RNA interference, or RNAi.12,13,14 Sanofi scientists are investigating ways to use this process to slow down the body's manufacture of antithrombin-3 proteins.

In the nucleus, sequences of DNA (green) act as the blueprint. From this, a template called messenger RNA (mRNA, purple) is produced. The mRNA template travels to a protein factory, where it is used to synthesize a protein (blue)

  • Sequences of DNA (genes) act as a blueprint for making antithrombin-3.
  • The instructions in this gene are converted into a protein by way of RNA. This happens when the DNA is copied, producing a template: a single-stranded messenger RNA (mRNA) molecule.15
  • The mRNA template is used by the body to synthesize antithrombin-3 proteins.
  • Without mRNA templates, the proteins cannot be made.

A single strand of small, interfering RNA (siRNA, green) in a complex of enzymes and other proteins (teal)

Building on this knowledge, scientists engineered a molecule that intercepts antithrombin-3 mRNA templates and destroys them. The molecule is made of small, "interfering" RNA (siRNA), which can partner up with mRNA-eating enzymes inside the cell.16,17

How this experimental approach works18,19

  • The siRNA lures an mRNA molecule on its way to deliver antithrombin-3 templates to a protein-building factory.
  • The mRNA binds to the siRNA.
  • Enzymes break down the mRNA and clear it, leaving the siRNA strand free to attract another mRNA.
  • Antithrombin-3 templates are destroyed before they make it to protein factories, slowing down protein production.
  • Antithrombin 3 levels remain low, so more thrombin is available to initiate blood clotting. 


(1) Small, interfering RNA (siRNA, green) lures an mRNA molecule (purple) on its way to deliver antithrombin-3 templates to a protein-building factory. (2) The mRNA binds to the siRNA (white), then (3) enzymes break down the mRNA and clear it. (4) The siRNA strand is free to attract another mRNA. Antithrombin-3 templates are destroyed before they make it to protein factories, slowing down protein production

Using this approach, Sanofi researchers have developed a potential treatment that is under investigation for prophylactic use by people with hemophilia A or B, with or without inhibitors (antibodies that attack replacement factors).20

3. Gene therapies aim to cure disease with a one-time treatment

In hemophilia research, the goal of gene therapy is to cure the disease by correcting the single DNA mutation that prevents patients from manufacturing enough factor VIII or IX. This research is ongoing.21

Gene therapies exploit the ability of retroviruses to insert DNA into their host’s genome. Instead of the pathological DNA, scientists program a retrovirus to insert replacement genes, like a patch on the genome.22 In hemophilia, these genes would be delivered to the liver, where all blood-clotting factors are made.23

Gene therapy research in hemophilia

A type of retrovirus called a lentivirus are being investigated as a DNA delivery service. Sanofi scientists are pursuing lentiviruses as delivery vehicles because24:

  • They are large and can carry a lot of information: They are big enough to hold both the corrected DNA sequence of factor VIII, biochemical switches to turn them on or off, and biological information that would prevent white blood cells from attacking the retrovirus.
  • They can integrate into the genome in the liver cells, so that when the cells divide the corrected DNA is reproduced consistently. 

This is one of many experimental approaches to gene therapy at Sanofi.

References

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  2. Saulius Butenas, Cornelis van’t Veer, Kenneth G. Mann; “Normal” Thrombin Generation. Blood 1999; 94 (7): 2169–2178
  3. Siegemund T, Petros S, Siegemund A, Scholz U, Engelmann L. Thrombin generation in severe haemophilia A and B: the endogenous thrombin potential in platelet-rich plasma. Thromb Haemost. 2003;90(5):781-786. doi:10.1160/TH03-01-0027
  4. Brummel-Ziedins KE, Whelihan MF, Gissel M, Mann KG, Rivard GE. Thrombin generation and bleeding in haemophilia A. Haemophilia. 2009;15(5):1118-1125. doi:10.1111/j.1365-2516.2009.01994.
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  11. Asakura H, Jokaji H, Saito M, et al. Study of the balance between coagulation and fibrinolysis in disseminated intravascular coagulation using molecular markers. Blood Coagul Fibrinolysis. 1994;5(5):829-832. doi:10.1097/00001721-199410000-00022
  12. Bartz S, Jackson AL. How will RNAi facilitate drug development?. Sci STKE. 2005;2005(295):pe39. Published 2005 Aug 2. doi:10.1126/stke.2952005pe39
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  15. YourGenome: What is gene expression? (2016) Creative Commons 4.0 CC-BY license [online] Available at: https://www.yourgenome.org/facts/what-is-gene-expression [Accessed 23 July 2020].
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Karin Knobe
Inside Hemophilia Research at Sanofi
Committed to Hemophilia Research

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