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Tamon Nishino

Publications and source records attributed to Tamon Nishino.

6 recordsLinked to original sources
PubMed2006

Partial correction of murine beta-thalassemia with a gammaretrovirus vector for human gamma-globin.

Several studies have demonstrated that recombinant lentivirus vectors containing extended globin gene expression cassettes and regulatory elements can ameliorate the pathogenic sequela in murine models of beta-thalassemia and sickle cell disease. Similarly promising results have not yet been obtained with recombinant gammaretrovirus vectors. Of these two vector classes, only gammaretroviruses have been tested extensively in clinical trials, with a proven ability to transduce long-term reconstituting hematopoietic stem cells with an exceedingly low incidence of serious side effects. Toward the continuing goal of developing retrovirus vectors for the treatment of the beta-chain hemoglobinopathies, we report here the assessment of a recombinant gammaretrovirus vector for human gamma-globin in murine models of beta-thalassemia. In the beta-thalassemia intermedia Hbbth-3/+ model, we observed a dose-dependent but transient increase in total hemoglobin and red blood cells, with a 2.5 +/- 0.2 g/dL increase in hemoglobin for transduction rates > or = 33%. In the severe beta-thalassemia major Hbbth-3/Hbbth-3 model, we observed a modest but statistically significant increase in survival, from a median of 15 days to 30 days (P = 0.001). These studies provide the first evidence that globin gene transfer vectors based on recombinant gammaretroviruses may provide a viable option for the treatment of the beta-chain hemoglobinopathies.

Animals↗
PubMed2006

Effects of human gamma-globin in murine beta-thalassaemia.

Murine models of beta-thalassaemia have been used to test therapeutic globin gene vectors. However, the level of gamma-globin expression necessary to achieve full phenotypic correction in these models is unclear. In order to address this issue, we carried out breeding and transplantation studies in murine models of beta-thalassaemia intermedia (Hbb(th-3)/+) and severe beta-thalassaemia major (Hbb(th-3)/Hbb(th-3)) using transgenic lines expressing various levels of human gamma-globin. Expression of gamma-globin RNA at a modest 7-14% of total alpha-globin RNA resulted in the selective survival of HbF(+) erythrocytes, a fivefold increase in total HbF, and a phenotypic improvement in the beta-thalassaemia intermedia model. Full normalisation of erythrocyte indices in this model required gamma-globin RNA expression at 27% of alpha-globin, resulting in an average 40% (6.8 g/dl) HbF. Studies using the homozygous Hbb(th-3) model of lethal beta-thalassaemia major demonstrated that even this high level of gamma-globin expression, for reasons related to the function of the hybrid globin tetramers, could only prolong, but not fully support, survival. Taken together, these results indicate that only the heterozygous Hbb(th-3) model of beta-thalassaemia intermedia can be reliably used for the pre-clinical assessment of gamma-globin gene therapy vectors, as well as other means of gamma-globin gene induction.

Animals↗
PubMed2002

Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo.

We have previously described the development of oncoretrovirus vectors for human gamma-globin using a truncated beta-globin promoter, modified gamma-globin cassette, and alpha-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken beta-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of gamma-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the gamma-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the gamma-globin cassette from the insulated vector was expressed in 49% +/- 20% of RBCs long term. RNase protection analysis indicated that the insulated gamma-globin cassette was expressed at 23% +/- 16% per copy of mouse alpha-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for gamma-globin expression approaching the therapeutic range for sickle cell anemia and beta thalassemia.

3T3 Cells↗
PubMed2002

Hematopoietic stem cell gene therapy.

Gene therapy applications that target hematopoietic stem cells (HSCs) offer great potential for the treatment of hematologic disease. Despite this promise, clinical success has been limited by poor rates of gene transfer, poor engraftment of modified cells, and poor levels of gene expression. We describe here the basic approach used for HSC gene therapy, briefly review some of the seminal clinical trials in the field, and describe several recent advances directed toward overcoming these limitations.

Animals↗
PubMedDate not supplied

Selection with a regulated cell growth switch increases the likelihood of expression for a linked gamma-globin gene.

Several lines of evidence indicate that in vivo drug selection can be used to overcome the low rates of gene transfer and engraftment encountered in many hematopoietic stem cell gene therapy settings. However, whether selection imposed on one transcription cassette effects the likelihood of expression from a second, independent transcription cassette within the same vector has been less well studied. In order to address this issue, we engineered an oncoretrovirus vector to express two separate transcription units: (i) a bicistronic cassette encoding both GFP and a pharmacologically regulated cell growth switch based on the thrombopoietin receptor Mpl; and (ii) a highly position-dependent second cassette encoding human gamma-globin. Studies in cell cultures and in mice transplanted with transduced marrow indicated that selective expansion increased by more than 9-fold the fraction of erythroid cells expressing the linked but separate expression cassette for gamma-globin. This increase was far greater then that observed for the bicistronic GFP gene, and cannot be explained by a simple increase in the fraction of cells containing provirus. These results suggest that selective expansion favors erythroid stem/progenitor cells with provirus integrated at chromosomal sites which are relatively resistant to silencing position effects.

Animals↗