To study telomere length dynamics in hematopoietic cells with age, we analyzed the average length of telomere repeat sequences in diverse populations of nucleated blood cells. More than 500 individuals ranging in age from 0 to 90 yr, including 36 pairs of monozygous and dizygotic twins, were analyzed using quantitative fluorescence in situ hybridization and flow cytometry. Granulocytes and naive T cells showed a parallel biphasic decline in telomere length with age that most likely reflected accumulated cell divisions in the common precursors of both cell types: hematopoietic stem cells. Telomere loss was very rapid in the first year, and continued for more than eight decades at a 30-fold lower rate. Memory T cells also showed an initial rapid decline in telomere length with age. However, in contrast to naive T cells, this decline continued for several years, and in older individuals lymphocytes typically had shorter telomeres than did granulocytes. Our findings point to a dramatic decline in stem cell turnover in early childhood and support the notion that cell divisions in hematopoietic stem cells and T cells result in loss of telomeric DNA.

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 Ϸ95% of the red blood cells in all mice contained human ␤ globin contributing to 32 ؎ 4% of all ␤-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 ␣ 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 ␤ thalassemia patients.

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IntroductionFanconi anemia (FA) is an autosomal recessive disease of childhood characterized by progressive pancytopenia, various developmental abnormalities, and a predisposition to acute myeloid leukemia. 1 Most individuals with FA, however, succumb to the complications of aplastic anemia. 2 FA cells demonstrate increased sensitivity to DNA cross-linking agents such as mitomycin C (MMC), diepoxybutane, and cisplatin, 2,3 a feature that serves as the basis for an important diagnostic test. FA cells treated with these cross-linking agents show a striking increase in double-strand DNA breaks and inhibited growth with cell cycle arrest in G 2 . 2 To date at least 7 potential FA genes have been indicated by complementation studies, and most of these genes, FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG, whose mutations account for 6 of the complementation groups, have now been characterized. [4][5][6][7][8][9][10] Despite the variety of genes involved in this disorder, mutations in FANCA and FANCC account for approximately 80% of all patients with FA. 11 Murine Fancc, being highly similar to the human ortholog, is able to complement human cells deficient in FANCC, restoring MMC resistance. 12 Fancc-deficient mouse strains were generated through gene targeting. Both had similar phenotypes, 13,14 demonstrating compromised gametogenesis, and an increase in the number of chromosomal aberrations, both spontaneously and after exposure to MMC. However, the targeted lines recapitulated neither the developmental nor the hematologic defects typical of human FA. 13,14 The reason for this interspecies discordance is unknown, but it has limited the utility of the mutant mice as potential models of FA.A number of hypotheses regarding the nature of the primary defect in FA have been suggested, including the proposal that FA proteins constitute a DNA damage recognition and signaling pathway, whose impairment is manifested by chromosomal instability and increased sensitivity to interstrand DNA cross-linking agents. 15 Although a reduced ability to process DNA cross-links is clearly evident, it has also been proposed that an abnormal reduction of MMC in FA cells leads to the production of reactive species that in turn generate cross-links and other types of oxidative lesions. 16 Thus, FA might also result, at least in part, from an abnormal regulation of cell redox state or of the cellular response to oxidative stress or both. In support of this notion, addition of Cu/Zn superoxide dimutase (SOD) to the culture medium of FA cells was reported to attenuate chromosomal breakage as well as MMC cytotoxicity, 16 an effect also observed in FA cells overexpressing thioredoxin. 17 In keeping with an inability to regulate either production, or the consequences of reactive oxygen species (ROS), some FA cells were shown to be hypersensitive to oxygen. 16,18 Thus, cells grew slowly at elevated oxygen levels (eg, 35%) and For personal use only. on May 9, 2018. by guest www.bloodjournal.org From tended to arrest at G 2 , whereas at low oxygen conc...