Reduced incidence of thrombosis in mice with hereditary spherocytosis following neonatal treatment with normal hematopoietic cells.

Thrombosis is a life-threatening complication of hemolytic anemia in humans. Cardiac thrombi are present in all adult alpha-spectrin-deficient (sph/sph) mice with severe hereditary spherocytosis, providing a model for events preceding thrombosis. The current study evaluated (1) the timing of thrombosis initiation and (2) the effect of postnatal transplantation of normal cells on life span and thrombotic incidence in adult mice. Thrombi are detected histologically following necropsy in untreated sph/sph mice of various ages and are not observed until 6 weeks of age. Thrombotic incidence increases from 50% at 6 to 7 weeks of age to 100% at 9 weeks of age. As a potential therapy, nonablated sph/sph neonates were transfused with either genetically marked normal peripheral blood (PB), bone marrow (BM), or both and assessed for donor cells and thrombosis. A single transfusion of PB, with or without BM, significantly increases the percentage of sph/sph mice that survive to weaning (4 weeks of age). Replacement in all sph/sph recipients is limited to red blood cells (RBCs). RBCs derived from donor PB are lost within 5 weeks. PB plus BM prolongs high-level donor PB cell production better than BM alone. Thrombotic incidence is significantly reduced in all sph/sph mice treated with PB, BM, or both. Hence, the presence of normal blood cells in the peripheral circulation of neonatal and adult sph/sph mice rescues the former and abrogates the development of thrombosis in the latter. (Blood. 2001;97:3972-3975)

In utero fetal liver cell transplantation without toxic irradiation alleviates lysosomal storage in mice with mucopolysaccharidosis type VII.

Lysosomal storage diseases, such as Mucopolysaccharidosis type VII (MPS VII), cause progressive loss of mobility and intellect and result in early death. Treatment of progressive diseases must occur before the blood-brain barrier closes. In MPS VII mice, normal donor hematopoietic cells secrete the missing enzyme beta-glucuronidase (GUSB) that reverses disease manifestations. Correction of lysosomal storage is limited to the visceral organs unless transplantation is preceded by high-dose irradiation. We hypothesize that irradiation opens the blood-brain barrier allowing passage of corrective cells. Here we transplanted genetically myeloablated MPS VII fetuses to determine whether earlier treatment without toxic irradiation is systemically corrective. Cells with a selective advantage in utero were identified. Donor fetal liver cells (FLC), a substitute for difficult to obtain murine cord blood cells, were increased 10-fold in the host peripheral blood over equivalent numbers of adult marrow cells injected simultaneously and were stable long term in both primary and secondary hosts. GUSB- MPS VII fetuses injected with GUSB+ FLC were assessed longitudinally after birth. Donor FLC replaced host stem cell descendants, prolonged life dramatically, and reduced bone dysplasia and lysosomal storage in all tissues long term. GUSB, donor leptomeningeal cells, and microglia were present in the brain at 11 months postinjection. Lysosomal storage in cortical neurons and glia, although not completely corrected, was reduced. We conclude that in utero intervention without toxic pretreatment in this model reduces the storage disease long term and improves the length and quality of life despite exerting only minor effects on the brain.

Amelioration of severe hereditary spherocytosis in nonablated adult mice by marrow transplantation.

Human severe hereditary spherocytosis (sHS) is life threatening and transfusion dependent. sHS is lethal within 6 days of birth for 99% of jaundiced (ja/ja) mice, making these mice excellent models for early therapeutic interventions. Nonablated ja/ja neonates simultaneously transfused and given intravenous injections of normal marrow become chimeric for donor cells. Significant improvement of red blood cell parameters occurs but is temporary because the donor marrow-derived cells gradually disappear from the circulation. The average lifespan, however, is increased to 8.7 months. We postulate that donor cells are diluted by rapidly proliferating host cells during postnatal growth. Here, we test this hypothesis by determining whether treatment of adults improves long-term therapy. Nonablated ja/ja adults rescued by a single neonatal transfusion were injected intravenously with 1 x 10(10) normal, genetically marked donor marrow cells/kg body weight. Donor cell implantation and blood parameters were monitored periodically and tissue histopathology was determined at necropsy.sHS recipients with 100% donor erythroid cells have significantly improved red blood cell counts throughout life when compared with ja/ja controls transfused once at birth. Total serum iron and bilirubin levels are corrected in ja/ja marrow recipients. Donor-implanted HS mice necropsied at 16 to 21 months of age have normal mean cell hemoglobin concentration and dramatically decreased tissue iron deposits. Reticulocyte counts but not red cell counts normalize, suggesting the HS mice reset their response to hypoxia. Nonablative transplantation performed after cessation of host postnatal red blood cell amplification can be therapeutic long term for transfusion-dependent hemolytic anemias.

Multiple high cell dose injections of normal marrow into newborn jaundiced mice dramatically prolong life despite transient repopulation.

Jaundiced (ja/ja) mice have a severe hemolytic anemia caused by deficiency of the erythroid cytoskeletal protein beta-spectrin. Unless they are transfused, 99% of the mutant mice die after birth. Here, we test a new therapy involving multiple, high cell dose marrow injections into newborn non-ablated recipients. The ja/ja and normal newborn mice were injected intravenously with a total of 8.7 x 10(6) genetically marked +/+ marrow cells/g body weight. Donor and host red blood cells were quantified and the status of the recipients monitored. The jaundiced but not the normal recipients had up to 57% replacement with donor red cells by 9 weeks. The treatment significantly increased red cell counts and extended the average lifespan to 5 months beyond that previously reported for ja/ja mice transfused at birth. Replacement was limited to red cells. The donor cells disappeared in three of five mutant mice alive beyond 27 weeks. Marrow from a 48-month-old ja/ja recipient no longer positive for donor cells was injected into a secondary host. The recipient acquired the blood phenotype of the primary ja/ja host. The possibility that the marker was not well tolerated following multiple cell injections was investigated in normal adult mice injected with a total of 5.3 x 10(6) marrow cells/g body weight. Recipients became chimeric (>38% donor red and white cells) long-term (>12 months). The results indicate donor stem cells (a) prolong life in the jaundiced mice, but (b) do not survive long-term when injected into newborn mice. We conclude that destructive mechanisms may not be limited to ja/ja red cells.

An alternative first exon in the distal end of the erythroid ankyrin gene leads to production of a small isoform containing an NH2-terminal membrane anchor.

Mouse erythroid ankyrin is encoded by the Ank1 gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3'-untranslated regions. They are encoded by a new first exon located in intron 39 of the Ank1 gene and three previously described Ank1 exons (40, 41, and 42). The 5'-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

High-frequency genetic reversion mediated by a DNA duplication: the mouse pink-eyed unstable mutation.

The mouse pink-eyed unstable (p(un)) mutation, affecting coat color, exhibits one of the highest reported reversion frequencies of any mammalian mutation and is associated with a duplication of genomic DNA at the p locus. In this study, genomic clones containing the boundaries of the p(un) duplication were isolated and characterized. The structure of these sequences and their wild-type and revertant counterparts were analyzed by restriction mapping, PCR product analysis, DNA sequence analysis, and pulsed-field gel electrophoresis. DNA from p(un) was distinguished from wild-type and revertant DNA by a head-to-tail tandem duplication of approximately 70 kilobases. No differences were detected between revertant and wild-type DNAs. Thus, the reversion in phenotype of p(un) mice is coupled with the loss of one copy of an approximately 70-kilobase duplicated segment. Testable models are presented to account for p(un) reversion.

Bone marrow replacement in the treatment of hemolytic disease in mice.

Bone marrow replacement therapy following whole-body x- or gamma-irradiation has until now proven to be of limited value in the treatment of individuals with hemolytic disease. The large doses of radiation required for destruction of defective erythropoietic tissues coupled with their resultant high mortality appears to limit its usefulness. Techniques have been developed by the authors to limit the extent of exposure and to improve survival following irradiation. These techniques include shielding of all parts of the body except the hind limbs, prophylactic use of antibiotics, and preparatory blood transfusion to suppress the development of indigenous defective erythrocytes. Using these combined techniques we were able to establish high rates of survival, successful engraftment, and long-term clinical improvement in mice with several hemolytic disorders emanating from hereditary defects in spectrin production and incorporation. Evidence is presented indicating that complete bone marrow replacement occurs even in nonirradiated portions of the erythron and that only donor type red blood cells appear in the circulation.