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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.

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 ratesor = 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.

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.

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed in vitro and in vivo . To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken β-globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.

Hemophilia B, or factor IX deficiency, is an X-linked recessive disorder occurring in about 1 in 25,000 males. Affected individuals are at risk for spontaneous bleeding into many organs; treatment mainly consists of the transfusion of clotting factor concentrates prepared from human blood or recombinant sources after bleeding has started. Small- and large-animal models have been developed and/or characterized that closely mimic the human disease state. As a preclinical model for gene therapy, recombinant adeno-associated viral vectors containing the human or canine factor IX cDNAs were infused into the livers of murine and canine models of hemophilia B, respectively. There was no associated toxicity with infusion in either animal model. Constitutive expression of factor IX was observed, which resulted in the correction of the bleeding disorder over a period of over 17 months in mice. Mice with a steady-state concentration of 25% of the normal human level of factor IX had normal coagulation. In hemophilic dogs, a dose of rAAV that was approximately 1/10 per body weight that given to mice resulted in 1% of normal canine factor IX levels, the absence of inhibitors, and a sustained partial correction of the coagulation defect for at least 8 months.

An E1a-deleted adenovirus vector constitutively express- adenovirus) and Ad/RSV-hAAT, the level and duration of ing native adenovirus E3-gp19K (Ad.RSV-gp19K) was con- serum hAAT protein were unrelated to gp19K protein structed in order to determine whether or not E3-gp19K expression. Evaluation of MHC I abundance on hepatomediated interference with antigen presentation would cytes following in vivo transduction demonstrated that result in prolonged transgene expression in vivo. Cultured recombinant adenovirus rapidly increased the abundance fibroblasts infected with Ad.RSV-gp19K produced a native of surface MHC I molecules on hepatocytes, and surface size gp19K protein and had decreased cell surface levels MHC I molecules were reduced earlier and to a greater of MHC I as shown by immunoprecipitation and flow cyto- extent following wild-type adenovirus infection compared metry. The congenic mouse strains Balb/b (H-2 b MHC I with hepatocytes transduced with control or Ad.RSVwith high gp19K affinity), Balb/k (H-2 k MHC I with no gp19K gp19K recombinant adenovirus. This difference in surface affinity), and Balb/c (H-2 d MHC I with moderate gp19K MHC I down-regulation may be related to the different proaffinity) were chosen for in vivo experiments because of moters (RSV-LTR versus the native E3 promoter), and will their range of gp19K affinities. Following transduction of be an important consideration in the development of newer mice from each strain with Ad.RSV-gp19K and Ad/RSV- generation adenovirus vectors designed to evade host hAAT (a reporter adenovirus), or Ad/RSV-cFIX (control immune responses.

The development of oncoretrovirus vectors for human gamma-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of gamma-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the beta-globin locus control region to enhance the expression of an Agamma-globin gene with a truncated -382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for Agamma-globin linked to various combinations of the HPFH2 enhancer, the alpha-globin HS40 enhancer, and several versions of the promoter from Agamma-globin or beta-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoter-autonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a -201 bp Agamma-globin gene promoter with the Greek HPFH -117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248+/-99% per copy of mouse alpha-globin (62% of total alpha-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for Agamma-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the Agamma-globin promoter in the context of a gene transfer vector.

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 γ-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 γ-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.

We have previously described the development of oncoretrovirus vectors for human γ-globin using a truncated β-globin promoter, modified γ-globin cassette, and α-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 β-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 γ-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the γ-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 γ-globin cassette from the insulated vector was expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated γ-globin cassette was expressed at 23% ± 16% per copy of mouse α-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 γ-globin expression approaching the therapeutic range for sickle cell anemia and β thalassemia.

Although gene transfer vectors based on recombinant retroviruses have been known to have the potential to lead to insertional mutagenesis, that integrations can lead to oncogenic transformation of transduced target cells in a gene therapy setting has led to intensive research on integration site choice. Several groups have reported that oncoretroviral vectors have a slight preference for landing within genes, a strong preference for integration within the region immediately around transcriptional start sites, and a preference for actively transcribed genes. Using primary mouse bone marrow progenitors as target cells for an oncoretroviral vector, we addressed three questions: 1) Are genes preferentially the targets; 2) Are regions surrounding transcriptional starts favored; and 3) Does selection for the vector bias the repertoire of integration sites? We obtained bone marrow from 5-FU treated mice, prestimulated the cells for two days, transduced by co-cultivation on irradiated ecotropic producer lines at a low multiplicity of infection. The retrovirus has a bacterial drug resistance cassette for Zeocin in the long terminal repeat, an MSCV LTR transcribing a GFP reporter, and a separate internal Pgk-Neo cassette (used to select for expression). Progenitor cell colonies were recovered in methylcellulose +/|[minus]| G418 seven days after transduction. Integration junctions were cloned directly from genomic DNA with the Zeocin cassette present in the LTRs, rather than by a PCR based strategy, enabling us to obtain larger genomic segments. Junction sequences were mapped by BLAST to build 33 of the mouse genome in the ENSEMBL database. We identified 130 integration sites from unselected pools and 130 integration sites from selected pools of progenitor cells. We also mapped an equivalent number of simulated integrations; this dataset was treated as the experimental dataset was, with unmappable sites (such as those that landed entirely in gaps or repetitive elements) removed. We have found that MSCV integrations do not occur more frequently into genes (defined as within known transcripts introns and exons, 42%) than random (41%), differing from what was previously reported for MLV integration into HeLa cells. Comparing the integrations that are within 5 kb upstream of a transcription start site, there is a highly significant difference between our experimental dataset (18%) and the simulated dataset (4%), P = < .0001. With the progenitor colonies having undergone selection, the frequency of landing within introns has increased from 35% to 43%. When the overall distribution of integrations is compared to gene density mapped onto the mouse chromosomes, it appears that oncoretroviruses favor gene-rich regions of the mouse genome. Though MSCV retroviruses do not have a preference for integrating within genes, they favor the 5 kb region upstream of transcriptional starts, 18% in this setting, versus 8.5% observed previously in HeLa cells. These results suggest that there may be less of a bias for oncoretrovrus integration within genes in mouse bone marrow progenitor cells compared to other cells types tested to date, and that the use of selection can bias the repertoire of integration sites that are observed following oncoretrovirus transduction.

Recombinant retroviruses are being developed as gene transfer vectors for a wide range of research and therapeutic applications. However, vector expression is subject to the inhibitory effects of chromatin surrounding the sites of integration, and vector sequences can activate oncogenes surrounding the site of integration. In order to address these limitations, we have been investigating a class of DNA elements called chromatin insulators. We have previously demonstrated that a prototypic insulator cHS4 can increase the frequency of expression for reporter genes and human γ-globin in mice transplanted with transduced marrow, and that this activity may be influenced by distance. In order to test the role of distance directly, the frequency of GFP expression from a vector flanked with various forms of the cHS4 element was assessed in myeloid progenitor colonies. Linking the cHS4 core to a 950bp native sequence or 720bp spacer increased the frequency of expression 2.1±0.3 fold (p 590bp from the target promoter in order to provide insulating activity. In order to further test the efficacy of the cHS4 element, we analyzed an insulated vector for human γ-globin in mouse models of β-thalassemia. In the heterozygous Hbbth-3/+ model there was a modest improvement in RBC counts (3.8±0.1×106/ul vs 2.6±1.1 for mock) and total hemoglobin (6.7±0.2 g/dl vs. 4.7±1.9 for mock) for mice with ≥25% g(+) RBC, although this effect was only transient. In the homozygous Hbbth-3/Hbbth-3 model there was increased survival to 34±20 days vs 16±7 days for mock (p