High-Efficiency Gene Transfer into Normal and Adenosine Deaminase-Deficient T Lymphocytes Is Mediated by Transduction on Recombinant Fibronectin Fragments

Primary human T lymphocytes are powerful targets for genetic modification, although the use of these targets in human gene therapy protocols has been hampered by low levels of transduction. We have shown previously that significant increases in the transduction of hematopoietic stem and progenitor cells with retroviral vectors can be obtained by the colocalization of the retrovirus and target cells on specific fibronectin (FN) adhesion domains (H. Hanenberg, X. L. Xiao, D. Dilloo, K. Hashino, I. Kato, and D. A. Williams, Nat. Med. 2:876–882, 1996). We studied the transfer of genes into primary T lymphocytes by using FN-assisted retroviral gene transfer. Activated T lymphocytes were infected for three consecutive days on the recombinant FN fragment CH-296 with a retroviral vector encoding the murine B7-1 protein. Transduced lymphocytes were analyzed for murine B7-1 expression, and it was found that under optimal conditions, 80 to 89% of the CD3⁺ lymphocytes were transduced. Gene transfer was predominantly augmented by the interaction between VLA-4 on the T lymphocytes and the FN adhesion site CS-1. Adenosine deaminase (ADA)-deficient primary T lymphocytes transduced on CH-296 with a retrovirus encoding murine ADA (mADA) exhibited levels of mADA activity severalfold higher than the levels of the endogenous human ADA protein observed in normal human T lymphocytes. Strikingly, the long-term expression of the transgene was dependent on the activation status of the lymphocytes. This approach will have important applications in human gene therapy protocols targeting primary T lymphocytes.     

Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells.

The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Generation of murine stromal cell lines supporting hematopoietic stem cell proliferation by use of recombinant retrovirus vectors encoding simian virus 40 large T antigen.

The bone marrow is a complex microenvironment made up of multiple cell types which appears to play an important role in the maintenance of hematopoietic stem cell self-renewal and proliferation. We used murine long-term marrow cultures and a defective recombinant retrovirus vector containing the simian virus 40 large T antigen to immortalize marrow stromal cells which can support hematopoiesis in vitro for up to 5 weeks. Such cloned cell lines differentially supported stem cells which, when transplanted, allowed survival of lethally irradiated mice, formed hematopoietic spleen colonies in vivo, and stimulated lymphocyte proliferation in vitro. Molecular and functional analyses of these cell lines did not demonstrate the production of any growth factors known to support the proliferation of primitive hematopoietic stem cells. All cell lines examined produced macrophage colony-stimulating factor. The use of immortalizing retrovirus vectors may allow determination of unique cellular proteins important in hematopoietic stem cell proliferation by the systematic comparison of stromal cells derived from a variety of murine tissues.     

A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in high-level gene expression in human T lymphocytes

We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) immediate-early promoter in a self-inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRalphaLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters-murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241-4250, 1994). We found that the combination of Rh-MLV LTR and a partial gag sequence of MoMLV (Deltagag(871-1612)) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Deltagag(871-1612) were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells.     

Evaluation of Tat-encoding bicistronic human immunodeficiency virus type 1 gene transfer vectors in primary canine bone marrow mononuclear cells

Tat-encoding human immunodeficiency virus type 1 (HIV-1) gene transfer vectors were evaluated in primary canine bone marrow mononuclear cells. Tat vectors provided higher levels of gene expression than vectors with internal promoters. The HIV-1 vector was also more efficient than Moloney murine leukemia virus (MoMLV) vectors for transduction of canine bone marrow mononuclear cells in vitro. Transplantation experiments in dogs with transduced autologous marrow cells confirmed the superiority of HIV-1 vectors over MoMLV vectors for gene transfer into canine bone marrow cells. Tat vectors may be useful not only for providing high levels of therapeutic gene expression in hematopoietic cells but also for study of the biological effects of Tat in those tissues in the canine model.     

The Moloney murine leukemia virus repressor binding site represses expression in murine and human hematopoietic stem cells

The Moloney murine leukemia virus (MLV) repressor binding site (RBS) is a major determinant of restricted expression of MLV in undifferentiated mouse embryonic stem (ES) cells and mouse embryonal carcinoma (EC) lines. We show here that the RBS repressed expression when placed outside of its normal MLV genome context in a self-inactivating (SIN) lentiviral vector. In the lentiviral vector genome context, the RBS repressed expression of a modified MLV long terminal repeat (MNDU3) promoter, a simian virus 40 promoter, and three cellular promoters: ubiquitin C, mPGK, and hEF-1a. In addition to repressing expression in undifferentiated ES and EC cell lines, we show that the RBS substantially repressed expression in primary mouse embryonic fibroblasts, primary mouse bone marrow stromal cells, whole mouse bone marrow and its differentiated progeny after bone marrow transplant, and several mouse hematopoietic cell lines. Using an electrophoretic mobility shift assay, we show that binding factor A, the trans-acting factor proposed to convey repression by its interaction with the RBS, is present in the nuclear extracts of all mouse cells we analyzed where expression was repressed by the RBS. In addition, we show that the RBS partially repressed expression in the human hematopoietic cell line DU.528 and primary human CD34(+) CD38(-) hematopoietic cells isolated from umbilical cord blood. These findings suggest that retroviral vectors carrying the RBS are subjected to high rates of repression in murine and human cells and that MLV vectors with primer binding site substitutions that remove the RBS may yield more-effective gene expression.     

Comparison of promoter suppression in avian and murine retrovirus vectors.

Previously, we described "promoter suppression" in infectious retrovirus vectors with two genes and an internal promoter. Here, we examined several parameters of promoter suppression and found that the amount of suppression in an integrated retrovirus vector was dependent both on whether the vector was derived from spleen necrosis virus or murine leukemia virus and on which internal promoter was present in the vector. Murine leukemia virus vectors showed less suppression than analogous spleen necrosis virus vectors. Furthermore, the amount of suppression was not dependent on either the relative strengths of the promoters nor the distance between the promoters. Moreover, we found that in vectors in which one promoter was suppressed, there was an inverse correlation between the DNaseI sensitivity of the chromatin surrounding a promoter and the suppression of its expression.     

Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.     

应用绿色荧光蛋白研究外源基因在造血细胞的表达调控

以增强型绿色荧光蛋白为报道分子,研究双基因逆转录病毒载体介导小鼠骨髓细胞的基因转移中,SV(simianvirus)40启动子和内部核糖体进入位点(internalribosomeentrysite,IRES)对双基因共表达的影响。构建双基因中间序列分别为SV40启动子和IRES的载体pLESN和pLEIN。经包装获得较高滴度的病毒上清,以共培养的方式感染5-氟尿嘧啶预刺激的小鼠骨髓细胞。流式细胞仪检测表明转染效率约25%,PCR证明EGFP基因整合至骨髓细胞基因组。半固体培养转基因细胞7d,LEIN组获得具有G418抗性的集落中98%表达绿色荧光,而LESN组54%。结果表明:在双基因逆转录病毒载体介导的小鼠骨髓细胞的基因转导中,IRES与内部SV40启动子相比,更能保证双基因的共同表达。     

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.     

A mouse c-myc retrovirus transforms established fibroblast lines in vitro and induces monocyte-macrophage tumors in vivo.

Activation of the c-myc proto-oncogene, in the form of DNA rearrangements that lead to constitutive expression, has been implicated in the genesis of a wide range of tumors. Therefore, it is of great interest to determine the influence of c-myc oncogene activation on cellular growth control, especially in primary cells. To facilitate the efficient transfer of an activated c-myc oncogene, we developed a mouse retrovirus that contains the c-myc protein-coding sequences and which can be transmitted in the presence of a Moloney murine leukemia virus helper or established as a helper-free stock with a retrovirus-packaging cell line. The virus can transform established lines of mouse fibroblasts to anchorage-independent growth; the transformed cells are tumorigenic in nude mice. However, the virus was not capable of inducing foci of transformed cells on confluent monolayers. In addition to studies on established cell lines, the effect of the c-myc retrovirus on primary cells was examined. Infection of bone marrow cells gave rise to partially transformed mononuclear phagocytes which were entirely dependent upon an exogenous supply of the monocyte-specific colony-stimulating factor CSF-1 for proliferation. Infection in vivo induced monocyte-macrophage tumors with a latency period of 8 to 10 weeks.     

Expression vectors for human adenosine deaminase gene therapy

Somatic gene transfer offers a possible new approach for treatment of human genetic disease. Defects affecting blood-forming tissues are candidates for therapies involving transfer of genetic information into hematopoietic stem cells. Adenosine deaminase (ADA) deficiency is being used as a model disease for which gene transfer techniques can be developed and evaluated. We describe here the construction and testing of 20 retroviral vectors for their ability to transfer and express human ADA in vitro and in vivo via a mouse bone marrow transplantation model. After infection of primary bone marrow with one fo these vectors (p delta NN2ADA), human ADA was detected in 60-85% of spleen colonies at day 14 and maintained long term in the blood of fully reconstituted mice. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.