Fetal Treatment of Genetic Disorders

Rationale, Experimental Work, and Barriers to Success

Conceptualization of hematopoietic stem cell transplantation dates back to 1945 when Owen found that dizygotic cattle twins shared placental circulation. Later, Billingham et al. demonstrated that early fetal exposure to antigens allowed for tolerance to the exposed antigen, meaning that postnatal treatment was better tolerated. These investigators isolated cells from various organs in one strain of mice, and these were then injected into fetuses of a different strain. After birth, these mice were able to tolerate skin grafts from mice of the transplanted strain. This early study set the groundwork for future research exploring the fetal environment as an avenue for tolerance to foreign antigens.

IUHCT was later performed in anemic mice with a mutation in the c-kit gene, which is responsible for the differentiation, proliferation, and survival of hematopoietic stem cells. Using a transplacental injection of hematopoietic stem cells, it was shown that stem cells from either fetal livers or adult bone marrow were able to correct the anemia. Mice that were more anemic supported the engraftment of stem cells, whereas mice that were not anemic did not engraft. Further studies demonstrated that engraftment of stem cells after IUHCT could be achieved in mice that had no stem cells or mice with severe combined immunodeficiency (SCID), in which a genetic defect prevents the proper maturation of T and B cells. Mice that were deficient in stem cells demonstrated donor-derived multilineage engraftment, whereas SCID-transplanted mice were only able to produce donor-derived lymphocytes. These were the first studies to show that IUHCT could lead to successful engraftment if the host was deficient in that particular lineage of cell, whereas host cell competition limited the effectiveness of IUHCT and explained why IUHCT in immunocompetent hosts had not led to successful engraftment.

Studies of stem cell engraftment after IUHCT in animals without inherent defects in their hematopoiesis showed engraftment below clinically relevant levels including mice, dogs, goats, and primates. Further research has improved techniques for delivery of transplanted cells. Intrahepatic and intravascular cell delivery allows greater numbers of hematopoietic stem cells to be transplanted and results in increases in overall chimerism. Reaching levels of 1%–2% engraftment result in donor-specific tolerance of donor cells. Mice that reach these levels of chimerism have been found to be able to accept donor skin grafts and do not show immunoreactivity to donor antigens.

Even in the fetal environment, an immune response can still limit donor cell engraftment. In fetal mice, although the fetal immune system matures later than in humans, the main culprit for rejection of transplanted cells has been shown to be the maternal immune system. A barrier to engraftment are maternal T cells that cross the placenta. In human fetuses, in which there is earlier maturation of the immune system, it has been demonstrated that the trafficking of maternal antigens to the fetus makes fetal T cells less reactive to the mother. Thus, transplantation of maternal stem cells into fetuses should overcome the maternal immune response to the transplantation, particularly because the human fetus should have T cells specific for these maternal antigens.

Studies of In Utero Stem Cell Transplantation in Large Animal Models

One of the major early successes with IUHCT was in the sheep model. Using an intraperitoneal injection of fetal stem cells, allogeneic engraftment could be shown in 75% of recipients with engraftment as high as 30%. The sheep continued to have chimerism 9 months after transplantation and never developed evidence of graft-versus-host disease. This early success leads to a great deal of enthusiasm; however, initial clinical application of IUHCT failed for numerous diseases.

Studies in the pig model were more encouraging, and it was demonstrated that IUHCT recipients were able to develop donor-specific tolerance and to tolerate donor-matched kidney transplants without immunosuppression. In studies of a canine model of leukocyte adhesion deficiency (CLAD), dogs underwent IUHCT via an intraperitoneal injection, which resulted in low levels of engraftment in two dogs and improvement in the disease phenotype. The dogs were then able to undergo a booster transplant to improve their chimerism to clinically relevant levels. Around this time, discoveries made in the murine model of IUHCT were used to further improve donor cell engraftment, specifically the use of maternal cells. Dogs were then treated with maternally derived donor stem cells via intracardiac injection. Chimerism was significantly improved with the new approach, and 21 of 24 dogs had engraftment greater than 1% with an average of 11%. This verified that the barriers found in the mouse model were limiting in the large animal models as well.

Early Clinical Experience in Humans

There has been little clinical success in IUHCT in humans due in large part to the unforeseen barriers to engraftment that were only identified after the early trials. At this time, 26 cases of IUHCT have been attempted in human fetuses for a variety of diseases with only bare lymphocyte syndrome and X-linked SCID, demonstrating clinically relevant engraftment and disease amelioration. It appears that in all successful cases, engraftment occurred due to the lack of host immune cell competition. Despite limited success, numerous lessons were learned from these early experiences and multiple improvements have been made based in part on the identified barriers found in animal studies. Fetal access has improved significantly. Ultrasound guidance allows for safe access to the umbilical vein and allows for a more efficient intravascular delivery of hematopoietic stem cells. In addition, it is now recognized that a protocol based on transplantation of a high dose of maternal-derived stem cells, injected intravascularly, has the best chance of allowing engraftment. Even if clinically significant levels are not achieved, a low level of chimerism could allow a postnatal “booster” transplantation with minimal conditioning. This approach has been used in mouse models of thalassemia and sickle cell disease.

Hemoglobinopathies

Hemoglobinopathies are a group of clinical disorders caused by genetic defects that cause either an abnormal structure of hemoglobin or insufficient production. IUHCT can potentially correct or lessen the disease burden of any disease that results from defective hematopoiesis, including hemoglobinopathies. Hemoglobinopathies are ideal targets for IUHCT by virtue of their overall prevalence in the general population and their deficiency arising from within the hematopoietic stem cell population. Postnatal allogeneic stem cell transplantation for sickle cell disease has been demonstrated as curative for patients with symptomatic sickle cell disease and thalassemia. The most prevalent clinically severe hemoglobinopathy is sickle cell anemia. Its clinical manifestations and disease burden vary greatly from individual to individual. The disease is characterized by vasoocclusive crises that require prolonged hospitalization. The overall cost of sickle cell disease in the United States was estimated at $460,151 per person and this disorder afflicts approximately 100,000 Americans. There have been several advances that have improved and prolonged the lives of patients with sickle cell disease ; however, hematopoietic stem cell transplantation remains the only curative treatment. Unfortunately, postnatal stem cell transplantation carries a lifetime risk of graft-versus-host disease and requires myeloablative preconditioning. Although clinical experience with high chimerism levels is sparse, in the preclinical and few clinical cases, graft-versus-host disease is rarely observed when the amount of mature T cells is controlled for.

Thalassemias are also common; gene frequencies are estimated to range from 2.5% to 15% in the tropics and subtropics. α-Thalassemia major manifests with severe anemia in utero, including hydrops fetalis and fetal demise. If left untreated, α-thalassemia major (ATM) is lethal, but fetal therapy with in utero transfusions can be lifesaving for these fetuses, and fetuses who are treated with in utero transfusions can survive with reasonable neurologic outcomes. This disease represents an ideal target for in utero therapy because it is fatal in utero without treatment. Because the fetus already needs to receive an invasive procedure in utero to survive, the HSC transplantation can be performed at the same time as the transfusions.

Lysosomal Storage Disorders

Lysosomal storage disorders are a group of diseases that result from a genetic deficiency in an enzyme required for the normal metabolic functions of the lysosome. Lysosomes are therefore unable to break down their complex substrates, which then accumulate within the cells leading to cellular dysfunction. Consequences of the disease include intellectual disability, skeletal dysplasias, pulmonary insufficiency, and, in severe cases, hydrops fetalis and in utero fetal demise. Patients with certain lysosomal storage disorders can be treated after birth with enzyme replacement therapy (ERT). The deficient enzyme is transfused and taken up by various cells, decreasing the extent of cellular damage. This approach is limited, however, and ERT does not appear to cross the blood-brain barrier and does not significantly improve the central nervous system deterioration. Additionally, the deficient enzyme is seen as foreign by the patient’s immune system. Postnatal therapy is limited by the development of antibodies against the exogenous enzyme, which leads to decreased effectiveness of the enzyme and allergic responses. As a result, immunosuppression is required for continued enzyme therapy.

To improve outcomes in patients with lysosomal storage disorders, stem cell transplantation has been performed in multiple lysosomal storage disorders including Hurler disease, Batten disease, metachromatic leukodystrophy, Krabbe disease, and I-cell disease. Clinical results have varied based on the disease, which stem cells were transplanted, age at transplantation, and chimerism levels, but overall clinical results have been promising. In Hurler disease specifically, improvements were noted in survival, cognitive development, preservation of hearing, corneal clouding, and respiratory support requirements. Earlier age at transplantation and posttransplant enzyme levels were predictive of outcome improvement.

In utero hematopoietic stem cell transplantation coupled with in utero enzyme replacement therapy could greatly improve outcomes for this group of patients for multiple reasons. First, many affected fetuses die before birth or before enzyme replacement therapy can be initiated. Second, fetal accumulation of toxic metabolic by-products leads to numerous developmental insults before the possible initiation of enzyme replacement. Neurologic outcomes may be improved with earlier therapy, particularly during fetal development, either by providing the enzyme before formation of the blood brain barrier or by early receptor-mediated transport. Third, fetal exposure to the exogenous enzyme may induce tolerance, which would make postnatal therapy more efficacious.