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At sites of vascular injury, the hemostasis mechanism operates to form a hemostatic plug and minimize the blood loss. Platelets (a blood cell type) and coagulation factors (circulating proteins) take major parts in the hemostasis mechanism. Hemophilia is a genetic disorder in which blood clotting is impaired. A deficiency of clotting factor VIII is known as hemophilia A, while a deficiency of factor IX is known as hemophilia B. About 5,000 hemophilia patients are currently registered in Japan. The common treatment for hemophilia is replacement of the blood-clotting factors. Although administration of blood clotting factors once came with a risk of infection by a plasma-derived disease such as HCV and HIV, today the risk of infection is much reduced, thanks to the development of recombinant (genetically modified) clotting factors. Clotting factor concentrates have been improved, but even now the half-life of clotting factor concentrates remain as short as a few hours. Consequently, patients who desire to use clotting factor prophylactically must use it within a short time window, which diminishes their quality of life.
Our research center is developing a gene therapy for hemophilia. Hemophilia is considered to be a good target for gene therapy for two reasons: 1) hemophilia results from an abnormality in a single gene; and 2) a small amount of clotting factor can improve a patient’s condition. Because it is difficult to correct genetic abnormalities at the cellular level, we are using a method in which additional normal coagulation factors are expressed in the patient’s body. To deliver coagulation factor genes to a patient’s body, we use two methods. One is direct vector infusion, which entails infusing a vector(gene carrier)into patients. The infused vector carries the clotting factor gene to the patient’s cells, where it is directly expressed. The other method is cellular therapy, which entails transplantation of cells that can express normal clotting factor to the patient’s body. The clotting factor expressed by the transplanted cells then circulates in the patient’s bloodstream with endogenously expressed clotting factors.
To date, adeno-associated virus (AAV) vectors have been used in clinical trials worldwide. Various serotypes of AAV are known. Using AAV8, we are able to efficiently introduce genes into the liver, which then physiologically expresses the encoded coagulation factor. Importantly, the expression of the coagulation factor persists for long periods. We have already succeeded in using this gene therapy to treat hemophilia in a monkey model. For a clinical trial in a few years, we are now trying to generate a vector having sufficient purity and safety.
In the United Kingdom, six hemophilia B patients received gene therapy through direct infusion of a vector, and most of the patients showed good clinical outcomes. Unfortunately, however, patients that produced a neutralizing antibody against AAV did not show improvement of their condition. We determined that about 30-40% of hemophilia patients have this antibody. However, we have since developed a new vector infusion method, which is the first in the world that can avoid the antibody reaction. With this method, the vector is administered directly into the portal vein using a catheter.
For cellular therapy, we use a third-generation simian immunodeficiency virus (SIV) vector that has no pathogenicity or self-propagation ability.
We previously confirmed the long-term expression of inserted genes using this vector.
In addition to their ability to attach to sites of vascular injury, platelets are able to release coagulation factors, and we developed a new cellular therapy based on that ability.
But because platelets are an anuclear cell with a short lifetime, it is difficult to generate gene-modified platelets.
We therefore transduced SIV harboring the gene encoding the targeted protein with a platelet-specific promoter into hematopoietic stem cells to produce gene-modified platelets. These platelets expressed the encoded coagulation factor and released it at sites of vascular injury, reducing the bleeding tendency in a mouse model of hemophilia A. We have also applied the same method to suppress platelet proteins using RNA interference.
One of the most important factors worsening hemophilia patients’quality of life is hemophilia arthropathy, which is associated with repeated joint bleeding.
We have established a new method for preventing and treating hemophilia arthropathy using mesenchymal stem cells (MSCs) transduced with SIV encoding coagulation factor.
MSCs can be obtained from bone marrow cells or adipose tissue. Intra-articular injection of the transduced MSCs significantly ameliorated the hemarthrosis and hemophilic arthropathy induced by knee joint needle puncture in hemophiliac mice. Because cell administration can be achieved using minimally invasive intra-articular puncture, we believe this cellular therapy has the potential for clinical application.
Our research has been performing this research with the assistance of the Ministry of Health, Labor and Welfare. In an effort to provide gene therapy to hemophilia patients as soon as possible, we will push forward with this research.