Arthur Bank

Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia

The β-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of β-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound βE/β0-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The βE-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated βE-globin with partial instability. When this is compounded with a non-functional β0 allele, a profound decrease in β-globin synthesis results, and approximately half of βE/β0-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral β-globin gene transfer, an adult patient with severe βE/β0-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl−1, of which one-third contains vector-encoded β-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells

Pre-clinical studies indicate that efficient retrovirus-mediated gene transfer into hematopoietic stem cells and progenitor cells can be achieved by co-localizing retroviral particles and target cells on specific adhesion domains of fibronectin. In this pilot study, we used this technique to transfer the human multidrug resistance 1 gene into stem and progenitor cells of patients with germ cell tumors undergoing autologous transplantation. There was efficient gene transfer into stem and progenitor cells in the presence of recombinant fibronectin fragment CH-296. The infusion of these cells was associated with no harmful effects and led to prompt hematopoietic recovery. There was in vivo vector expression, but it may have been limited by the high rate of aberrant splicing of the multidrug resistance 1 gene in the vector. Gene marking has persisted more than a year at levels higher than previously reported in humans.

Amputation and adriamycin in primary osteosarcoma.

Abstract Adriamycin has been found effective in metastatic osteogenic sarcoma. To determine its efficacy in osteosarcoma without detectable metastases, 21 patients were given adjuvant adriamycin th...

An Ikaros-Containing Chromatin-Remodeling Complex in Adult-Type Erythroid Cells

ABSTRACT We have previously described a SWI/SNF-related protein complex (PYR complex) that is restricted to definitive (adult-type) hematopoietic cells and that specifically binds DNA sequences containing long stretches of pyrimidines. Deletion of an intergenic DNA-binding site for this complex from a human β-globin locus construct results in delayed human γ- to β-globin switching in transgenic mice, suggesting that the PYR complex acts to facilitate the switch. We now show that PYR complex DNA-binding activity also copurifies with subunits of a second type of chromatin-remodeling complex, nucleosome-remodeling deacetylase (NuRD), that has been shown to have both nucleosome-remodeling and histone deacetylase activities. Gel supershift assays using antibodies to the ATPase-helicase subunit of the NuRD complex, Mi-2 (CHD4), confirm that Mi-2 is a component of the PYR complex. In addition, we show that the hematopoietic cell-restricted zinc finger protein Ikaros copurifies with PYR complex DNA-binding activity and that antibodies to Ikaros also supershift the complex. We also show that NuRD and SWI/SNF components coimmunopurify with each other as well as with Ikaros. Competition gel shift experiments using partially purified PYR complex and recombinant Ikaros protein indicate that Ikaros functions as a DNA-binding subunit of the PYR complex. Our results suggest that Ikaros targets two types of chromatin-remodeling factors—activators (SWI/SNF) and repressors (NuRD)—in a single complex (PYR complex) to the β-globin locus in adult erythroid cells. At the time of the switch from fetal to adult globin production, the PYR complex is assembled and may function to repress γ-globin gene expression and facilitate γ- to β-globin switching.

A Phase I/II Clinical Trial of β‐Globin Gene Therapy for β‐Thalassemia

Abstract: Recent success in the long‐term correction of mouse models of human β‐thalassemia and sickle cell anemia by lentiviral vectors and evidence of high gene transfer and expression in transduced human hematopoietic cells have led to a first clinical trial of gene therapy for the disease. A LentiGlobin vector containing a β‐globin gene (βA‐T87Q) that produces a hemoglobin (HbβA‐T87Q) that can be distinguished from normal hemoglobin will be used. The LentiGlobin vector is self‐inactivating and contains large elements of the β‐globin locus control region as well as chromatin insulators and other features that should prevent untoward events. The study will be done in Paris with Eliane Gluckman as the principal investigator and Philippe Leboulch as scientific director.

Globin composition and synthesis of hemoglobins in developing fetal mice erythroid cells.

Fetal mouse erythropoiesis proceeds initially in yolk-sac blood islands (8 to 12 days) and, subsequently, in liver (12 to at least 16 days). Yolksac cells synthesize three hemoglobins, Hb EI, Hb EII and Hb EIII. Hb EI has x- and y-globin chains; Hb EII has α and y; HB EIII, α and z. No detectable β-globin is formed in these cells. Liver erythroid cells form only adult hemoglobin, composed of α- and β-chains.

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

Abstract We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32 P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.

Multiple hematopoietic defects and delayed globin switching in Ikaros null mice

We have previously reported the structure of a chromatin remodeling complex (PYR complex) with Ikaros as its DNA binding subunit that is specifically present in adult murine and human hematopoietic cells. We now show that homozygous Ikaros “knockout” (null) mice lack the PYR complex, demonstrating the requirement for Ikaros in the formation of the complex on DNA. Heterozygous Ikaros null mice have about half as much PYR complex, indicating a dosage effect for both Ikaros and PYR complex. We also show that Ikaros null mice have multiple hematopoietic cell defects including anemia and megakaryocytic abnormalities, in addition to previously reported lymphoid and stem cell defects. The null mice also have a delay in murine embryonic to adult β-globin switching and a delay in human γ to β switching, consistent with a previously suggested role for PYR complex in this process. Lastly, cDNA array analyses indicate that several hematopoietic cell-specific genes in all blood lineages are either up- or down-regulated in 14-day embryos from Ikaros null as compared with wild-type mice. These results indicate that Ikaros and PYR complex function together in vivo at many adult hematopoietic cell-specific genes and at intergenic sites, affecting their expression and leading to pleiotropic hematopoietic defects.

Polyribosomes and control of protein synthesis: Effects of sodium fluoride and temperature in reticulocytes

Reticulocytes from phenylhydrazine-treated rabbits were pre-incubated with 0·01 M -sodium fluoride to inhibit protein synthesis and dissociate polyribosomes to 80 s ribosomes. Upon incubation of these cells without fluoride, protein synthesis is restored and reaches rates comparable to control cells (not pre-incubated with fluoride) within three minutes at 37°C when only a fraction of the polyribosomes have re-formed. In these cells, polyribosome content and polyribosome size may continue to increase for 60 to 120 minutes at 37°C without a change in rate of protein synthesis. As polyribosome content increases, the specific activity of the polyribosomes remains constant. These data indicate that in these cells polyribosome content is not the factor which limits the rate of protein synthesis. An estimate of the time for globin chain synthesis indicates that it increases progressively as polyribosome content increases. The protein synthesized during polyribosome formation in fluoride pre-incubated cells is predominantly globin and, by sedimentation and chromatographic characteristics, indistinguishable from the protein synthesized in control cells. The effect of the temperature of incubation on protein synthesis by reticulocytes was examined. In contrast to the alterations in polyribosomes with a constant rate of protein synthesis during recovery from the “fluoride effect”, increasing the temperature of incubation of reticulocytes between 17 and 37°C is associated with no change in polyribosome size or content, but an increase in polyribosome specific activity and in rate of protein synthesis. The data are interpreted in terms of mechanisms which might alter polyribosome size, content and/or function in reticulocytes which synthesize no RNA.

Accumulation of α- and β-globin messenger RNAs in mouse erythroleukemia cells

Abstract The accumulation of α- and β-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific α- and β-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me 2 SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of α-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in β-globin mRNA sequences is not detected until 20–24 hr after culture. In cells exposed to hemin, both α- and β-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of αβ-mRNA is maintained during induction. In maximally induced cells, the αβ-globin mRNA ratios are approximately 1 in cells induced by Me 2 SO and HMBA, and 0.66 and 0.3–0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of α- and β-like genes. In addition, the relative accumulation of α- and β-globin mRNAs in induced cells differs with various types of inducers.