Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects.

The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer. Similar studies showed that four copies of a 90-bp fragment containing the cHS4 enhancer-blocking activity actually repressed vector green fluorescent protein (GFP) expression. In contrast, a 400-bp fragment containing the 250-bp core plus 3' flanking sequences protected vector expression to the same degree as the full-length 1.2-kb fragment. The 400-bp fragment activity was confirmed in a lentiviral vector expressing human beta-globin in murine erythroid leukemia (MEL) cells. Taken together, these studies indicate that the insulating activity of the 250-bp cHS4 core can be influenced by distance, and identify an extended core element that confers full barrier activity in the setting of two different classes of retroviral vectors.

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.

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.

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.

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.

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.

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.