Trial Information

AMD3100 in Combination With G-CSF to Mobilize Peripheral Blood Stem Cells in Patients With Fanconi Anemia(FA): A Phase I/II Study

Fanconi anemia is a rare autosomal recessive syndrome comprised of progressive bone marrow
failure, congenital anomalies and a predisposition to malignancy. The heterozygote rate in
the United States may be as high as 1 in 300. The mean age for the onset of aplastic anemia
is approximately eight years. Although improved supportive care has prolonged the survival
of these patients from only a few years from the diagnosis of bone marrow failure, the mean
age of death is still approximately 24 years of age. Most patients die from complications of
bone marrow failure including bleeding, or infection, or from malignancy or complications of
stem cell transplantation. In a recent 20 year review of patients in the Fanconi anemia
registry, the actual risk of developing leukemia or other cancers was approximately 30%.

The diagnosis of Fanconi anemia initially rested upon finding the combination of bone marrow
failure with congenital anomalies. These anomalies include cafe au lait spots and/or hypo
pigmentation of the skin, short stature, upper limb malformations (often involving the thumb
or radius), renal and gastrointestinal abnormalities, microcephaly, and characteristic
facies with a broad nasal base, epicanthal folds, narrow set and small eyes and
micrognathia. The bone marrow failure is characterized by slow progression to severe bone
marrow aplasia and pancytopenia, stress erythropoiesis with fetal features including
macrocytosis, elevated hemoglobin F, and i antigen expression. Attempts to culture bone
marrow progenitors in vitro from patients with Fanconi anemia demonstrates decreased numbers
of myeloid and erythroid colonies (CFU) consistent with clinical bone marrow failure.

Fanconi anemia cells appear to have a defect in DNA repair that leads to increased
spontaneous chromosomal breakage. This feature increases the susceptibility of Fanconi
anemia cells to DNA bifunctional cross-linking agents such as mitomycin C and diepoxybutane
(DEB). The diagnosis of Fanconi anemia now relies upon detecting increased chromosomal
breakage after in vitro treatment with DEB. 11 Similarly, cells cultured from patients with
Fanconi anemia display increased susceptibility to the cytotoxicity of mitomycin C. More
recently, cells from patients with Fanconi anemia have been demonstrated to display G2 phase
prolongation/arrest, increased sensitivity to toxicity by oxygen, defective p53 induction
and increased apoptosis.

Fanconi anemia can be classified into at least thirteen complementation groups by somatic
cell hybrids. The complementation is based upon correction of the chromosomal sensitivity to
cross-linking agents in hybrid cells. Twelve independent genes have been cloned and
characterized within these 13 complementation groups. A loss of function in any of these
genes including FANC A, B, C, D2, E, F, G, J, L, M, N, and FANC D1 (which is BRCA2) can
cause Fanconi anemia. However, complementation groups A, C, and G account for approximately
80-85% of patients with Fanconi anemia in the United States. Discrete mutations in these
genes have been identified in families with the disorder. Expression of the complementary
cDNA gene in the respective Fanconi anemia cells in vitro corrects the increased chromosomal
breakage from DEB and the increased sensitivity to mitomycin C. Expression of gene products
in bone marrow progenitors from patients with Fanconi anemia increases survival in in vitro
assays.

The current treatment for Fanconi anemia relies upon hematological support in the form of
red blood cell and platelet transfusions. Aplastic anemia will transiently respond to
androgen therapy in 50% of children. G-CSF has also been utilized in published studies from
our own group to improve the number of myeloid cells in the peripheral circulation. Bone
marrow transplantation has cured some patients of their bone marrow failure; however, there
appears to be more toxicity to the conditioning regimens and there may be increased numbers
of solid tumors post transplant compared to patients without the disorder. Survival five
years after a matched sibling transplant now exceeds 65% and after an unrelated donor
transplant 30%. More recent studies in unrelated donor transplant for Fanconi anemia at
Cincinnati Children's Hospital and the University of Minnesota have reported survival rates
approaching those observed in matched sibling donor transplants. However, graft failure
resulting in death remains a major obstacle. The availability of sufficient numbers of
(previously purified and cryopreserved) autologous HSC for re-infusion after graft failure
may prevent this complication.

Finally, gene therapy approaches are being pursued, but to date, there is no evidence for
cure with this approach in humans, although correction has been reported in murine models.
These studies are hampered by the fact that mouse knockouts of FA genes do not develop
spontaneous aplastic anemia and thus are not phenocopies of the human disease. Thus in
previously reported mouse-studies, gene therapy approaches required ablative total body
irradiation of the recipient mice to ensure engraftment of the gene corrected stem cells.

An obvious limitation of Fanconi anemia hematopoietic stem cell gene transfer is that the
necessary target for genetic manipulation, the hematopoietic stem cell (or its surrogate,
CD34+ cell) is progressively lost during the development of aplastic anemia. Thus at the
time of severe aplasia and the greatest need for treatment, target stem cells for genetic
modification are deficient. Collection of a meaningful number of HSC prior to the onset of
aplastic anemia for eventual use in a therapeutic gene therapy trial will be explored in the
study outlined here. Key questions remaining are whether corrected HSC from Fanconi anemia
patients will engraft after autologous re-infusion without any cyto-reductive treatment of
the recipient and, if engrafted, whether the corrected cells will demonstrate a
proliferative advantage over uncorrected stem cells.