beta-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human beta-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the beta-globin expression cassette. Thus far, mouse studies have shown correction of the beta-thalassemia intermedia phenotype and a partial, variable correction of beta-thalassemia major phenotype. We tested a lentiviral vector carrying the human beta-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human beta-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.
Based on a prospective cohort study of 1056 patients with sickle cell anemia (Hb SS) initiated in 1959, we investigated the influence of calendar era, age, sex, and prior medical conditions on the subsequent development of irreversible organ damage and survival using the Cox regression model with time-dependent covariates adjusting for all prior occurrences. We studied 30 acute clinical events, and focused on 8 prototypic forms of irreversible organ damage. Childhood survival to age 20 years has improved from 79% for those born before 1975 to 89% for children born in or after 1975. Bone infarction was a significant risk factor for avascular necrosis (p = 0.01), and infantile dactylitis was a significant risk factor for stroke (p = 0.01). Prior hospitalized vaso-occlusive sickle crisis in adults was significantly associated with the increased rate of avascular necrosis (p < 0.001), leg ulcers (p < 0.001), sickle chronic lung disease (p < 0.001), renal failure (p < 0.005), and early death (p < 0.001). The diagnosis of clearly evident clinical conditions such as leg ulcer, osteonecrosis, and retinopathy predicted an increased likelihood of developing a more lethal form of organ damage and earlier death: 77% of patients with chronic lung disease, 75% of those with renal insufficiency, and 51% of those with stroke had a prior chronic condition. Of the 232 patients who died, 73% had 1 or more clinically recognized forms of irreversible organ damage. By the fifth decade, nearly one-half of the surviving patients (48%) had documented irreversible organ damage. End-stage renal disease (glomerulosclerosis), chronic pulmonary disease with pulmonary hypertension, retinopathy, and cerebral microinfarctions are manifestations of arterial and capillary microcirculation obstructive vasculopathy. The current study underscores the need for preventive therapy to ameliorate the progression of the sickle vasculopathy.
The 3'-and 5'-terminal nucleotide sequences of the defective interfering (DI) RNAs present in a preparation of DI influenza virus were determined. It was found that all DI RNAs possessed identical terminal sequences for at least the first 13 nucleotides at the 5' end and at least the last 12 nucleotides at the 3' end. The sequence of the DI RNAs is (5')A-GU-A-G-A-A-AC-A-A-G-G-... -C-C-U-G-C-U-U-U-C-G-C-U-OH(3'). In addition, the same sequences were present at the 3' and 5' termini of the viral polymerase genes (P1, P2, and P3) from which these DI RNAs originate. These results indicate that DI RNAs of influenza virus are formed by an internal deletion of the genomic RNA.Defective interfering (DI) animal viruses are noninfectious viruses that interfere with the replication of standard viruses (1, 2). DI influenza virus is produced after repeated undiluted passage of the virus (3), and over 99% of the infectious virus population is replaced by DI virus (4). The DI influenza virus has small virus-specific RNA molecules not present in the standard virus (4-7). Recently it has been directly demonstrated that the ribonucleoprotein complexes of these new deleted forms of the viral RNA specifically cause interference (8).In a previous report (9) the sequence relationships among these DI-RNAs and their relationship to the standard eight viral genes were studied by oligonucleotide mapping. Because each clone of influenza virus produces a unique set of DI RNAs when passaged (4), several DI RNAs are present in each DI virus preparation. All the DI RNAs studied thus far (9) have been found to be forms of one of the viral polymerase genes (P1, P2, or P3) from which sequences have been deleted. Comparison of the oligonucleotide maps of DI RNAs and P genes revealed that the DI RNAs originating from the same polymerase gene were related in one of two ways. Either they contained completely overlapping regions or each contained both overlapping and nonoverlapping regions (9). This latter group of DI RNAs could not be formed from the progenitor RNA by a mechanism of common initiation and simple deletion from one end as reported for vesicular stomatis virus (10) and Sendai virus (11). We therefore proposed (9) internal deletion of the progenitor virion RNA as one possible model for the formation of influenza DI RNAs.We have now determined the sequences of the 3' and 5' ends of three DI RNAs present in a DI influenza virus preparation as well as the sequences of the 3' and 5' ends of the polymerase genes. The sequence analysis of the DI RNAs and their progenitor genes indicates a model for the formation of influenza DI RNAs quite different from that proposed for other negative-stranded viruses (1, 11). (Mr 1 X 106). The third, Li, is also a deleted form of the P3 gene (9). MATERIALS AND METHODSPreparation of 3'-and 5'-Labeled RNAs. Viral RNA (vRNA) was extracted from a suspension of purified virus (13). The RNA was labeled at its 3' end as described (14) in a reaction containing 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesu...
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