Mary E. Fabry

Permanent and panerythroid correction of murine β thalassemia by multiple lentiviral integration in hematopoietic stem cells

Achieving long-term pancellular expression of a transferred gene at therapeutic level in a given hematopoietic lineage remains an important goal of gene therapy. Advances have recently been made in the genetic correction of the hemoglobinopathies by means of lentiviral vectors and large locus control region (LCR) derivatives. However, panerythroid β globin gene expression has not yet been achieved in β thalassemic mice because of incomplete transduction of the hematopoietic stem cell compartment and position effect variegation of proviruses integrated at a single copy per genome. Here, we report the permanent, panerythroid correction of severe β thalassemia in mice, resulting from a homozygous deletion of the β major globin gene, by transplantation of syngeneic bone marrow transduced with an HIV-1-derived [β globin gene/LCR] lentiviral vector also containing the Rev responsive element and the central polypurine tract/DNA flap. The viral titers produced were high enough to achieve transduction of virtually all of the hematopoietic stem cells in the graft with an average of three integrated proviral copies per genome in all transplanted mice; the transduction was sustained for >7 months in both primary and secondary transplants, at which time ≈95% of the red blood cells in all mice contained human β globin contributing to 32 ± 4% of all β-like globin chains. Hematological parameters approached complete phenotypic correction, as assessed by hemoglobin levels and reticulocyte and red blood cell counts. All circulating red blood cells became and remained normocytic and normochromic, and their density was normalized. Free α globin chains were completely cleared from red blood cell membranes, splenomegaly abated, and iron deposit was almost eliminated in liver sections. These findings indicate that virtually complete transduction of the hematopoietic stem cell compartment can be achieved by high-titer lentiviral vectors and that position effect variegation can be mitigated by multiple events of proviral integration to yield balanced, panerythroid expression. These results provide a solid foundation for the initiation of human clinical trials in β thalassemia patients.

Transferrin therapy ameliorates disease in [beta]-thalassemic mice

Individuals with β-thalassemia develop progressive systemic iron overload, resulting in high morbidity and mortality. These complications are caused by labile plasma iron, which is taken up by parenchymal cells in a dysregulated manner; in contrast, erythropoiesis depends on transferrin-bound iron uptake via the transferrin receptor. We hypothesized that the ineffective erythropoiesis and anemia observed in β-thalassemia might be ameliorated by increasing the amount of circulating transferrin. We tested the ability of transferrin injections to modulate iron metabolism and erythropoiesis in Hbbth1/th1 mice, an experimental model of β-thalassemia. Injected transferrin reversed or markedly improved the thalassemia phenotype in these mice. Specifically, transferrin injections normalized labile plasma iron concentrations, increased hepcidin expression, normalized red blood cell survival and increased hemoglobin production; this treatment concomitantly decreased reticulocytosis, erythropoietin abundance and splenomegaly. These results indicate that transferrin is a limiting factor contributing to anemia in these mice and suggest that transferrin therapy might be beneficial in human β-thalassemia.

Mechanisms for sickle red blood cell retention in choroid.

Purpose. Although sickle (SS) red cell-mediated vaso-occlusion in retina and resultant retinopathy is well documented, the effects of SS red cells on choroidal vasculature are poorly understood. The intent of this study was to determine, using a rat model, the conditions under which retention of sickle erythrocytes in choroid occur and if that retention can be inhibited. Methods. Sickle red cells were density separated into high density (SS4) or normal density, reticulocyte-enriched fractions (SS2). Red cells were labeled with FITC and administered IV to anesthetized Sprague Dawley rats. Rats were made either hypoxic or were given TNF-a intraperitoneally 5 hours before intravenous administration of red cells. Five minutes after administration of red cells, rats were exsanguinated, the retinas removed, and choroids prepared as flat-mounts. The number of red cells retained in five high power fields of choroid was then determined. In other experiments, SS red cells were preincubated with the cyclic peptide TBC772 [inhibits binding of a4ß1 (VLA-4) and a4ß7 to their ligands], a control peptide TBC1194, or a VLA-4 neutralizing antibody before administration to the rat or antibodies against VLA-4 ligands were delivered IV before administration of SS red cells. Results. Hypoxic conditions before administration of SS red cells significantly stimulated retention of SS4 cells (P = 0.0003), but did not significantly increase retention of SS2 cells. Administration of TNF-a significantly increased retention of all types of SS red cells (P < 0.001). Preincubation of cells with anti-VLA-4 or TBC 772 inhibited retention of SS red cells in choriocapillaris of TNF-a-treated rats (P < 0.0001). Complete inhibition of cytokine-stimulated retention was also accomplished by IV administration of mono-clonal antibodies against fibronectin or its CS-1 domain, a ligand for VLA-4. Conclusions. The mechanisms for retention of SS red cells in retina and choroid appear identical: hypoxia-mediated retention of dense red cells and adherence of red cells in reticulocyte-rich fractions after cytokine stimulation. TNF-a-stimulated retention of SS red cells in choroid appears to be mediated by VLA-4, presumably on the surface of some reticulocytes. This increased retention was inhibited by a VLA-4 antagonist (TBC772), a VLA-4 neutralizing antibody or by blocking one of VLA-4s ligands, the CS-1 portion of fibronectin.

Nonperfusion of retina and choroid in transgenic mouse models of sickle cell disease

PURPOSEnTo determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease.nnnMETHODSnMice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections.nnnRESULTSnHRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction.nnnCONCLUSIONSnNonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.