On Wednesday we were saddened learned that double Nobel laureate Fred Sanger had died, so it was fitting that yesterday also saw the announcement of an important scientific advance that owes everything to the molecular biology revolution he helped to launch – one that may improve the lives of many thousands of people with Hemophilia A.

Hemophilia A is caused by a deficiency in the production of coagulation factor VIII, which leads to an increased risk of bleeding, and is due to defects in the gene located on the X-chromosome that lead to either insufficient production of factor VIII or production of defective factor VIII. Patients with severe Hemophilia A require frequent intravenous injections of recombinant factor VIII to prevent serious bleeding. The BBC reported on Wednesday that a team of scientists based in the US and France have developed a gene therapy that successfully treated hemophilia in 2 dogs, and continued to prevent serious bleeds more than 2 years following treatment. Their therapeutic strategy involved isolating bone marrow hematopoietic stem cells (stem cells that give rise to all types of blood cell – including red blood cells, white blood cells and the platelets that are crucial to clotting) and transforming them with a lentiviral vector containing a gene encoding factor VIII under the control of a promoter that had previously been shown to drive expression of the drives the expression the target gene in the platelets of mice and dogs. The transformed hematopoietic stem cells were then infused back into the same dog from which they had been isolated. Writing in the Nature Communications paper (1) reporting this study lead author Dr David Wilcox of the Medical College of Wisconsin and his colleagues discuss why dogs were the ideal subjects for preclinical evaluation of this therapy (for a great article on the crucial role of dogs in haemophilia research see this article from the magazine HEMAWARE).

A canine model for haemophilia A exists, which results from a genetic mutation causing a large inversion of the FVIII gene (that resembles a molecular genetic defect found in about 40% of humans with the severe haemophilia A)4. Likewise, canine haemophilia A is essentially identical to the human disease in its clinical presentation characterized by severe-intermittent episodes of joint bleeding and haemorrhage. Protein replacement therapy is the most common treatment of severe bleeding episodes for haemophilia A but it has been confounded by the formation of inhibitory antibodies to transfused human FVIII in 30% of patients5,6. Similarly, 100% of dogs utilized from the Chapel Hill colony for this study develop inhibitory antibodies after being transfused with human FVIII (ref. 7), albeit severe bleeding is successfully treated with canine FVIII supplements. Thus, canine haemophilia A appears to be an ideal system to determine whether platelets can be used successfully to deliver human FVIII to the site of a vascular injury as a feasible approach to improve haemostasis within a ‘large-animal’ model of haemophilia A with the ability to form inhibitory antibodies to human FVIII.”

The above quotation also refers to an advantage of the technique they used over previous gene therapy methods developed to treat hemophilia A, one that the BBC article surprisingly didn’t pick up on. The BBC article mentions that an advantage that targeting expression of factor VIII to the platelets over previous studies where factor VIII was expressed in the liver of dogs with hemophilia A is that it would be suitable for patients who have damaged livers, but this is not the main advantage. The production of inhibitory antibodies against recombinant factor VIII by the patient is a problem that reduces the effectiveness of current therapies in about a third of people with hemophilia A, and this was also a problem in gene therapy techniques previously tested in clinical trials where factor VIII is secreted into the bloodstream from tissues such as the liver. Dr Wilcox and colleagues had the idea that by targeting expressing factor VIII specifically to platelets it would not be exposed to and blocked by inhibitory antibodies.

The test this theory they turned to a genetically modified mouse model of hemophilia A, which had already proven very useful in earlier stages of the development of gene therapy to treating hemophilia A. In a study undertaken in GM mice (2) which had been immunized so that they produced inhibitory antibodies against human factor VIII, Dr Wilcox and colleagues at the Medical college of Wisconsin demonstrated that the their lentiviral vector that directed factor VIII expression specifically to the platelets resulted in the expression of therapeutic levels of factor VIII associated with platelets in the blood, even 6 months after treatment.

What’s more, they showed that this was possible using a nonmyeloablative conditioning regime before infusing the transformed hematopoietic stem cells. Conditioning regimes reduce the immune response to a transplant (and in cancers such as leukemia also eradicate the cancerous cells, using drugs such as the nitrogen mustards that we discussed earlier this week) but the myeloablative conditioning regimes that are very effective in treating leukemia carry significant risks, for example from infections following the procedure. Nonmyeloablative conditioning that does not completely destroy the patient’s reduces the risk of infection and transplant related death, and is thus more appropriate for conditions that are not immediately life threatening. This study paved the way for the evaluation of platelet specific factor VIII therapy in dogs that was reported on Wednesday. It is noteworthy that in the dogs, which were also treated using a nonmyeloablative pre-transplant conditioning regimen, no inhibitory antibodies were detected against human factor VIII (unlike with previous gene therapy techniques), indicating that when associated with platelets it is sequestered from the immune system.

Almost 2 years ago we reported on the success of a small clinical trial of gene therapy in the treatment of hemophilia B following studies in mice and monkeys. We hope that with the development of a gene therapy technique that requires a milder conditioning regime and can avoid inhibitory antibodies this success will soon be repeated in hemophilia A.

Paul Browne

1) Du LM, Nurden P, Nurden AT, Nichols TC, Bellinger DA, Jensen ES, Haberichter SL, Merricks E, Raymer RA, Fang J, Koukouritaki SB, Jacobi PM, Hawkins TB, Cornetta K, Shi Q, Wilcox DA. “Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.” Nat Commun. 2013 Nov 19;4:2773. doi: 10.1038/ncomms3773. Pubmed 24253479

2) Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, Wilcox DA, Shi Q. “Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.” J Thromb Haemost. 2012 Aug;10(8):1570-80. doi: 10.1111/j.1538-7836.2012.04791.x.PubMed 22632092. PMC3419807