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Biomedical subjects

David W Emery

Publications and source records attributed to David W Emery.

12 recordsLinked to original sources
PubMed2007

The transcription factor KLF11 can induce gamma-globin gene expression in the setting of in vivo adult erythropoiesis.

Previous studies in a fetal erythroid cell line demonstrated that the transcription factor, Krüppel-like factor 11 (KLF11), could specifically induce transcription from a gamma-globin gene promoter, and that this induction was mediated through a specific canonical CACCC cis-DNA binding motif. We report here that ectopic expression of KLF11 can also induce fetal gamma-globin gene expression in the setting of adult erythropoiesis both in vitro and in vivo. Studies in an adult-stage murine erythroleukemia (MEL) cell line demonstrated that retrovirus vector-mediated transduction of KLF11 could increase both the amount of expression from a basally active, but not from a overtly silenced, recombinant gamma-globin transgene, as well as the frequency of cells expressing this transgene. A similar pattern of gamma-globin gene induction was also observed both in vitro and in vivo following KLF11 transduction of bone marrow from mice containing a basally active gamma-globin transgene. These studies provide the first evidence that ectopic expression of a transcription factor can induce gamma-globin gene expression in vivo during adult erythropoiesis.

Animals↗
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

Integration bias of gammaretrovirus vectors following transduction and growth of primary mouse hematopoietic progenitor cells with and without selection.

The recent recognition that recombinant retrovirus vectors can induce oncogenic transformation has stimulated much interest in the pattern of vector integration sites. We report here on the integration pattern of a gammaretrovirus reporter vector following transduction and ex vivo culture of primary mouse bone marrow progenitor cells in the absence and presence of drug selection. Using a novel method of cloning junction fragments, we observed no bias for integrations within genes, but did observe a bias for integrations within gene-dense regions and especially near transcriptional start sites of highly active genes, similar to previous reports in other cell types. We also document a novel bias for integrations within or near a class of genes that encode nuclear-localized proteins. We found that drug selection resulted in an increase in the frequency of recovered integration events that were located within the beginning of genes, integration events that were located in less gene-dense regions, and integration events that were oriented in an antisense direction relative to flanking gene transcription. Taken together, these studies provide new insights into the nature of retrovirus vector integration patterns in primary cells and demonstrate that selection based on vector expression can bias the integration site repertoire.

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↗
PubMed2005

Simultaneous sequence transfer into two independent locations of a reporter vector using MultiSite Gateway technology.

The bacteriophage lambda recombination system is increasingly used for recombinant DNA applications that involve the frequent transfer of sequences into and between shuttle and reporter vectors. This approach bypasses the need for restriction endonucleases or ligases and, as such, is easily scalable and automated. However this system has not yet been tested for the ability to support the simultaneous introduction of donor fragments into two separate target sites of a single reporter plasmid. This attribute would greatly facilitate studies of cis-regulatory elements that only function in specific combinations, such as a class of regulatory elements known as chromatin insulators. With the goal of facilitating a screen for chromatin insulators, we sought to determine whether the commercially available MultiSite Gateway Technology recombination system could be used to simultaneously insert candidate insulator elements into two separate locations of a functional reporter plasmid. We show that this application is both highly efficient and specific, generating the desired recombination products nearly three quarters of the time without disrupting the specificity of the reporter system. As such, these studies establish a novel application of the MultiSite Gateway Technology for the generation of recombinant reporter plasmids where the constituent elements function in a combinatorial fashion.

Bacteriophage lambda↗
PubMed2004

Differences of globin transgene expression in stably transfected cell lines and transgenic mice.

Previous studies demonstrated that DNase I hypersensitive site -40 (HS-40) of the alpha-globin locus is capable of greatly enhancing expression of a hybrid beta/gamma-globin transcriptional unit in plasmid-transfected murine erythroleukemia (MEL) cells. However, as reported here, this same gamma-globin gene expression cassette was only transcribed at trace amounts in erythroid cells of transgenic mice. This lack of expression was not directly attributable to the beta/gamma-globin transcriptional unit, since this same unit linked to a composite beta-globin locus control region was expressed at high levels in transgenic mice. This lack of expression was also not directly attributable to chromosomal position effects, since addition of chromatin insulators failed to increase the frequency of expression. DNase I hypersensitivity and chromatin immunoprecipitation assays demonstrated that the lack of expression was correlated with a closed chromatin structure. We hypothesize that transgenes undergo dynamic changes in chromatin conformation following chromosomal integration and that the discrepant results reported here can be attributed to the relatively high level of chromatin remodeling that occurs in the transgenic mouse model, coupled with the relative inability of the HS-40 element to maintain an open chromatin state under such conditions.

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

Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors.

The expression of integrated oncoretrovirus vectors is subject to the inhibitory effects of surrounding chromatin. A previous report from our laboratory indicated that such position effects can be overcome by flanking a reporter vector with the cHS4 chromatin insulator. To characterize this activity more thoroughly, we switched the promoter-gene combinations in the reporter vector and analyzed expression of these vectors flanked with the cHS4 fragment in both orientations following bone marrow transduction and transplantation in mice. The results indicate that the cHS4 fragment can function in both orientations and can insulate both the virus long-terminal-repeat (LTR) promoter and an internal phosphoglycerate kinase (Pgk) promoter. However, insulation of the LTR promoter diminished when the orientation of the cHS4 fragment placed the CTCF-binding core element immediately proximal to the U3 region, suggesting a minimal distance requirement. Moreover, placement of the cHS4 fragment in the U3 region of the 3' LTR dramatically decreased the level of expression from an internal Pgk promoter, presumably by blocking interaction with the 3' LTR enhancer. Finally, sorting studies suggest that the severity of position effects or autonomous promoter silencing increases as transduced progenitors differentiate into mature progeny. These findings have direct implications for the use of chromatin insulators such as cHS4 in oncoretrovirus vectors.

3' Untranslated Regions↗
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↗
PubMed2002

Use of CD9 expression to enrich for porcine hematopoietic progenitors.

OBJECTIVE: The aim of this study was to develop novel markers for enrichment of hematopoietic progenitors from bone marrow of swine. MATERIALS AND METHODS: We previously showed that pig bone marrow contains a "side population" (SP) of Hoechst dye-effluxing cells that resembles the hematopoietic stem cell (HSC)-containing murine SP and therefore represents a putative pig stem cell population. We screened a panel of monoclonal antibodies for those that allowed positive or negative enrichment of porcine SP cells and tested one of these for enrichment of hematopoietic progenitors in short-term and long-term in vitro assays. We then screened an expression library to clone the gene whose product is recognized by this antibody. RESULTS: Among a panel of 35 monoclonal lines screened, we found three that were useful for positive enrichment of SP cells and seven for negative enrichment. The 4-6 monoclonal line, allowing around 10-fold negative enrichment of SP cells, recognized the product of the porcine CD9 gene. Hematopoietic progenitors measured by short-term colony-forming unit and long-term cobblestone area-forming cell assays were around 10-fold enriched in the CD9(negative/low) fraction and were significantly depleted in the CD9(high) fraction. CONCLUSIONS: The antibody against the porcine CD9 gene product may be of use for enrichment of porcine hematopoietic stem cells. This approach to identify novel markers for enrichment of hematopoietic progenitors may be applicable to other mammalian species.

Amino Acid Sequence↗
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↗