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Stem cells showing promising results in developing a treatment for Haemophilia

Stem cells showing promising results in developing a treatment for Haemophilia

April 28, 2023
Dr. Lana du Plessis
April 28, 2023
Dr. Lana du Plessis

History of Haemophilia

The timeline history of Haemophilia follows an intricate and interesting path through history.

The first case of Haemophilia dates back to ancient Egypt, although some authors claim the first reference came from Hebrew texts in the second century A.D. Haemophilia only became well known when Queen Victoria from England transmitted her Haemophilia A gene to several royal houses in Europe, including her latest son Leopold who died at 30 of a bleeding episode following a mild knee injury.

In haemophilia, one inherits an abnormal gene from a parent so you don’t make sufficient quantities of one of the clotting factors. There are 3 types of Haemophilia, A, B and C, depending on which clotting factor is decreased. Haemophilia A is the most common type of Haemophilia and you do not have sufficient clotting factor 8 (Factor VIII). Haemophilia B is when you don’t have enough clotting factor 9 (Factor IX). Haemophilia C is also known as factor 11 (Factor XI) deficiency or Rosenthal syndrome.

Haemophilia A, the most common affects 1 in 10,000 male babies. Clinically, patients with mild deficiency (5–40 % activity of Factor VIII), usually tend to bleed only after major surgical procedures. Patients with moderate deficiency (1–5 % activity of Factor VIII) and severe (<1 % activity) usually become symptomatic after minor surgical procedures or spontaneously bleed. Approximately 70–80 % of bleeding episodes result in severe bleeding in the joints. Progressive Haemophilia joint arthritis is the most important long-time complication.

The first successful blood transfusion was performed post-World War II on an 11-year-old boy and thereafter increased access of Haemophiliac patients to blood or plasma transfusions improved their life- expectancy to 30 years of age.

The life expectancy of Haemophiliacs improved further when Judith G. Pool in 1964 discovered a frozen fraction of plasma contained proportionally greater quantities of Factor VIII. In the 1970s. Nilsson and Ahlberg in Sweden pioneered the regular administration of Factor VIII in a prophylactic study, and life expectancy was increased to 68 years of age. Infusion and transfusion of plasma derivatives were not safe, and complications started to appear soon thereafter as they were obtained by donations from poor people, paid for donating, and these people had a higher frequency of infectious agents in their blood.

The Human Immunodeficiency Virus (HIV) appeared in the 1980’s, and the first haemophilic HIV patient in the US was reported in 1982. In the US the HIV incidence increased to 60 cases per million in 1990.  HIV accounted for 25% of deaths in Haemophiliacs in the 1990s in Netherlands. Two years later it was discovered that 60 % of the US haemophilic patients, and 80 % of all patients ever treated with clotting factor concentrates were already infected with hepatitis C.

The genetic sequence of Factor VIII gene was elucidated in mid-1980 and this led to the production of recombinant Factor VIII (rFactor VIII). The new rFactor VIII was expressed in culture and the first patient treated was in 1987. Thereafter, no donations were required as the use of the recombinant rFactor VIII became the treatment of choice. Since 1985 there were no further reports of viral transmission linked to the use of rFactor VIII in the developed world. These recombinant clotting factors have increased the life expectancy of all Haemophiliacs worldwide. The life expectancy for a mild to moderate Haemophiliac is 70 years of age, whereas for severe cases of Haemophilia, it is estimated to be at least 15 years less.

Since the 1990s risk of blood-borne infections has been controlled with the use of recombinant replacement therapy, as well as with the introduction of more sensitive immunoassays for the serological markers associated with transfusion-transmitted viruses (TTVs). A new complication has emerged in Haemophilia, that of the development of inhibitors (a neutralising immunoglobulin directly acting against Factor VIII) which is now the most frequent and serious complication. Academics are now focused on comparing different treatment options and their association with the emergence of these antibodies. Since 2000, researchers have focused their efforts on the costs and cost-effectiveness of long-term treatment.

There is currently no cure for Haemophilia.

Currently Haemophilia treatment for severe cases is regular injection of clotting factor. The cost of treatment is extremely expensive, approximately $400,000 per patient (R7,2 million per patient) per year and the majority of patients in the world with the disorder don’t have access to this treatment. In addition, about a third of patients develop antibodies to the treatment.

Haemophilia is a monogenic disorder, has a broad therapeutic window, has very good animal models, and is therefore ideal for gene and/or cell therapy. Gene therapy using adeno-associated virus (AAV) vector is an option and has shown promise for long-term therapy, but AAV-vector based approaches carry with them problems, including possible tissue damage and immunogenicity. The clotting factors are mainly produced in the liver, so liver transplantation is an alternative long-term treatment option. However, there is a scarcity of donor organs and the need for constant immunosuppression represents a major drawback. A number of these gene therapy candidates for Haemophilia A, such as SB-525 by Sangamo Therapeutics and PfizerAMT-180 by uniQure; and BioMarin’s valoctocogene roxaparvovec, or BMN 270, are under development and clinical in testing. All of them are based on an adeno-associated virus (AAV) vector, to deliver a functional version of the F8 gene to the patient’s cells. The risks associated with AAV-vector-based therapy are far from elucidated and carry many risks.

Therefore, being able to cure haemophilia with stem cell transplants would be a significant breakthrough.

The first research strategy would be to engineer mesenchymal stem cells (MSCs), a type of adult stem cell so that they produce high levels of factor VIII. The cells, acting as a carrier for the gene, would then be transplanted into the patient. Even though the approach might not be successful at curing the disease, it would at least solve the antibody problem so that the current treatment would be effective.

In some instances, haematopoietic stem cell transplant (HSCT) has become an emerging therapy approach for Haemophilia patients.

In the first case where a boy underwent allogeneic (from a donor) HSCT at age 4, the boy presented with increasing bleeding symptoms and prolonged PTT. He was eventually diagnosed with mild Haemophilia A with a residual FVIII-activity of 18 percent in the absence of FVIII-inhibitors. Additional examinations revealed MDS-RAEB (myelodysplastic syndrome-refractory anaemia with excess blasts) and this leads to low levels of any type of blood cell. After HSCT, the FVIII activity spontaneously increased to a maximal level of 45 percent, remained elevated for four weeks, but ultimately declined to pre-HSCT levels two months later. The child’s recovery post-procedure went well, with no associated toxicity or immune system rejection. At the last follow-up of the boy’s condition, his factor VIII activity was at 19.6% and he did not have any bleeding symptoms. He was not cured but remained in a stable condition.

In the first testing of this treatment in animals, the researchers used stem cells from the father’s bone marrow, which were engineered to produce high levels of factor VIII. MSCs were selected because they are home to sites of injury or inflammation. In two of the treated animals, the cells homed to the sites of injury and stopped the bleeding and in addition, all spontaneous bleeding events ceased. The existing joint damage was completely reversed, thus improving the normal posture and gait of the crippled animals, and they resumed normal activity. Currently, scientists are working to find better ways of administering cells and to understand a paradox of the treatment—while the stem cells were able to stop the bleeding, the treatment induced an immune response in the animals.

Because the gene for Factor VIII is enormous, 26 exons, it cannot readily be delivered by a virus for genetic engineering purposes, some scientists have tried truncating the gene. Stem cells can overcome this bottleneck. Sinusoidal endothelial cells in the liver are the main producers of Factor VIII. Thus, using stem cells that can differentiate into endothelial cells can be a great solution for Haemophilia overall. Bone marrow has a similar capacity to transdifferentiate into sinusoidal endothelial cells, which can compensate for the deficiency of factor VIII. They even have the potential to treat chronic liver failure and liver injury.

Thus, researchers at the Boston Children’s Hospital’s Department of Cardiac Surgery laboratory came up with a strategy to test this use of stem cells in Haemophilia. They reprogrammed the epithelial cells obtained from urine samples of Haemophiliacs into induced pluripotent stem cells. They used gene-editing on the iPS cells, inserting multiple copies of the normal gene for factor VIII using a so-called piggyBac transposon system. Finally, they used the edited iPS cells to create large quantities of endothelial cells, which line blood vessels and naturally secrete factor VIII protein.

A bioengineered blood vessel secreting factor VIII (shown in green), seven days after implantation of treated human cells in mice with Haemophilia.

Courtesy of Melero-Martin Lab/Boston Children’s Hospital.

A Factor VIII for Haemophilia A, named ET-3 has been purified and demonstrated to be indistinguishable and superior to human Factor VIII with respect to intracellular processing, activation and stability, and in vivo efficacy. ET-3 specific activity is approximately 50% greater for the engineered protein, its half-life following activation is approximately 3-fold longer, and it engages the unfolded protein response to a lesser extent than human Factor VIII. Transplant of ET-3 lentiviral-modified CD34+ cells into mice resulted in ET-3 expression in all mice comparable with normal levels. The goal of this project remains to obtain clinical approval for a haematopoietic stem cell transplant gene therapy clinical trial for haemophilia A.

Another group used a combination of ex vivo-engineered stem cells for their approach. First, they collected placenta-derived mesenchymal stromal cells (PMSCs) and endothelial colony-forming cells (ECFCs) from discarded human term placenta and ECFCs from umbilical cord blood, respectively. These cells were then genetically engineered with a version of FVIII to express a functional Factor VIII, using lentiviral-based gene therapy. The genetically modified cells were transplanted alone or in combination into immune-deficient mice to check which one of the regimens allowed cells to last longer inside the body. The results showed that the co-transplant of PMSCs and ECFCs resulted in the best and longer transplants, stably producing a functional Factor VIII inside the mice’s bodies over at least six months. In addition, the transplant was more successful if performed a few days after birth, compared with transplants in adults.

Haemophilia B on the other hand can be used in viral genetic delivery because it is a smaller gene, it has 8 exons. Salk Institute researchers have combined CRISPR-Cas9 gene editing with stem cell technology to make an autologous cell therapy for the genetic blood clotting disorder Haemophilia B. In mice, in vivo tests showed that gene-edited, stem cell–derived liver cells were viable and functional in Haemophiliac mice for nearly a year, after a single injection.

Other approaches to stem cell therapy have been tested at the Salk Institute, using cells from donor livers or derived from autologous stem cells. Three major sources of hepatocytes exist, heterologous cadaveric hepatocytes, pluripotent stem cell-derived hepatic-like cells (HLCs), derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), and induced HLCs (Heps) derived by direct reprogramming of fibroblasts into HLCs. These sources have their own respective advantages and disadvantages.

In order to test two different approaches to long-term cell therapy, the Salk Institute team first developed a new, quadruple knockout mouse model of Haemophilia B that was able to accommodate the engraftment and expansion of human hepatocytes (hHeps).

They first transplanted cadaveric, cryopreserved induced pluripotent stem cell-derived hepatic-like cells, directly into the spleens of the Haemophiliac animals. These transplanted cells readily engrafted and remained healthy, functional, and non-tumorigenic. They have tested hepatocytes from multiple donors and sources and have not seen any adverse reactions in the more than 40 animals tested.

As an alternative to using heterologous donor hepatocytes, the Salk Institute team developed an approach based on the use of patients’ own, gene-corrected, and in vitro–differentiated cells. The aim was to generate hepatocyte-like cells (HLCs) from Factor IX gene-corrected iPSCs derived from peripheral blood-derived mononuclear cells (PBMCs) from Haemophiliac patients. The outcomes were as good as with the donor HLCs. The only drawback however was engraftment of these cells was not as good as in the cadaveric study. Encouragingly, data from prior studies of severe Haemophilia patients have suggested that low levels of 15% to 20% of Factor IX levels are sufficient to stop joint bleeding and might be therapeutically relevant.

The researchers concluded that the major benefits of the autologous cell therapy approach include the ability of IPSCs to support homology-directed repair recombination and gene editing. Additionally, because the cell therapy is derived from the patient’s own cells, there should be no risk of an immune reaction or the need for long-term immunosuppressive drugs.

In light of the recent findings of these different techniques of stem cell transplantation in Haemophilia patients, these promising results show that genetically engineered stem cells can potentially achieve stable, long-term engraftment in Haemophilia patients.


  • Sokal EM, Lombard C, Mazza G. Mesenchymal stem cell treatment for hemophilia: a review of current knowledge. J Thromb Haemost. 2015 Jun;13 Suppl 1: S161-6. doi: 10.1111/jth.12933. PMID: 26149017.

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