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PubMed2000

Elevated tumor necrosis factor-alpha activation of human immunodeficiency virus type 1 subtype C in Southern Africa is associated with an NF-kappaB enhancer gain-of-function.

The human immunodeficiency virus type 1 (HIV-1) epidemic within southern Africa is predominantly associated with the HIV-1C subtype. Functional analysis of the enhancer region within the long terminal repeat (LTR) indicates that HIV-1C isolates have >/=3 NF-kappaB binding sites, unlike other subtypes, which have only 1 or 2 sites. A correlation was shown between NF-kappaB enhancer configuration and responsiveness to the proinflammatory cytokine tumor necrosis factor (TNF)-alpha within the context of naturally occurring subtype LTRs, subtype-specific NF-kappaB enhancer regions cloned upstream of an isogenic HXB2 core promoter or a heterologous SV40 minimal promoter, and full-genome subtype clones. In all cases, TNF-alpha activation was correlated with the subtype configuration of the NF-kappaB enhancer. Whether the naturally occurring gain-of-function in the NF-kappaB enhancer of HIV-1C observed in this study can provide a selective advantage for the virus in vivo remains to be determined and warrants further study.

Binding Sites↗
PubMed2006

Mechanism for the establishment of transcriptional HIV latency in the brain in a simian immunodeficiency virus-macaque model.

BACKGROUND: The brain is considered to be a reservoir of latent human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). We examined the mechanism by which innate immune responses contribute to the establishment of this reservoir. METHODS: Gene-specific RNA and DNA were quantitated using real-time reverse-transcription polymerase chain reaction (RT-PCR). Protein expression was examined using Western blot analysis. Binding to and regulation of the SIV long terminal repeat (LTR) was examined using electrophoretic mobility shift assay, luciferase reporter constructs, and chromatin immunoprecipitation assay. RESULTS: Interferon-beta (IFN-beta) and myxovirus A (MxA) mRNA are produced in the brain during acute SIV infection. IFN-beta both suppresses SIV LTR activity and induces expression of the dominant-negative isoform of CCAAT/enhancer-binding protein-beta (C/EBP-beta). C/EBP-beta and its dominant-negative isoform respectively enhance and suppress histone acetylation at the SIV LTR and are present at the SIV LTR in vivo. SIV DNA persists when viral RNA is undetectable in the brain, and activation of the LTR is suppressed at the level of histone acetylation. CONCLUSION: Innate immune responses to virus infection that suppress acute virus replication in the brain also facilitate transcriptional latency of SIV. These data provide the first mechanistic model of HIV latency in the brain.

Animals↗
PubMed2000

In vitro cellular tropism of human T cell leukemia virus type 2.

Human T cell leukemia virus type 1 (HTLV-1) and 2 (HTLV-2) are distinct oncogenic retroviruses that infect several cell types, but display their biologic/pathogenic activity only in T lymphocytes. HTLV-1 is associated with adult T cell leukemia, a malignancy of mature CD4(+) T cells, and a chronic neurological disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 is less pathogenic and has been associated with a few cases of a variant of hairy cell leukemia and neurological disease. Previous studies have indicated that in vivo HTLV-1 has a preferential tropism for CD4(+) T cells, whereas HTLV-2 in vivo tropism is less clear, but appears to favor CD8(+) T cells. The molecular mechanism that determines the cellular tropism of HTLV-1 and HTLV-2 has not been precisely determined. However, one study by our group has provided evidence that HTLV-1-enhanced viral transcription in CD4(+) T cells may be responsible for its tropism. In an effort to understand HTLV-2 tropism we tested the ability of HTLV-2 to infect, replicate in, and transform purified CD4(+) or CD8(+) T cells in cell culture. After cocultures of purified primary human CD4(+) and CD8(+) T cells with an HTLV-2-producer cell line we measured viral transcription by reverse transcription PCR analysis, virus production by p19(gag) ELISA, proviral integration by DNA slot-blot analysis, surface phenotype by FACS analysis, and cellular transformation. We also measured HTLV-2 long terminal repeat-directed transcription in the presence and absence of Tax in purified CD4(+) and CD8(+) T cells, using transient transfection assays. Our data indicate that CD4(+) and CD8(+) T cells are equally susceptible to HTLV-2 infection. We observed no significant difference in viral transcription based on mRNA and virus production in CD4(+) and CD8(+) T cell cocultures. Although LTR transcription was enhanced 12- to 16-fold in the presence of Tax, there was no significant difference in CD4(+) or CD8(+) T cells. Interestingly, we show that HTLV-2 preferentially transforms CD8(+) T cells in culture. Together, our data indicate that, unlike HTLV-1, HTLV-2 cell tropism is not due to inhibition of viral infection and inefficient gene expression in CD4(+) versus CD8(+) T cells, and likely involves unique interactions with viral and CD8(+) T cell-specific proteins.

CD4-Positive T-Lymphocytes↗
PubMed1999

Extracellular HIV type 1 Tat protein induces CD69 expression through NF-kappaB activation: possible correlation with cell surface Tat-binding proteins.

The HIV-1 Tat protein, essential for HIV-1 gene expression and viral replication, is known to be secreted by infected cells and has pleiotropic effects on various cell functions. It seems that extracellular Tat may exert its functions on cellular targets by at least two different mechanisms, namely, by adsorptive endocytosis, and by a possible interaction with cell surface receptor(s). Here we report that extracellular Tat activates AIM/CD69 gene transcription through an NF-kappaB-dependent pathway in the erythroleukemia cell line K562. Tat induces NF-kappaB binding to DNA as a result of IkappaBalpha phosphorylation and degradation, which depend on the intracellular redox state. We found that the second Tat-coding exon is required for CD69 gene trans-activation, but not for HIV LTR gene transcription. Fluorescein-labeled Tat proteins were used to study cell surface binding sites and cellular uptake of the proteins. Full-length Tat protein has specific binding sites on the surface of K562 cells, whereas truncated Tat1-48, which is efficiently internalized by the cells, does not bind to the cell surface. Our results suggest that extracellular Tat may activate a cell surface-mediated pathway that induces intracellular signal transduction in K562 cells, leading to the activation of NF-kappaB and the transcription of NF-kappaB-dependent genes, such as CD69.

Antigens, CD↗
PubMed2000

An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method.

The two-plasmid rescue method of constructing Ad vectors, which relies on either homologous or Cre-mediated recombination between two plasmids cotransfected into 293 or 293Cre4 cells, respectively, offers advantages over other approaches because of its simplicity. We have improved the efficiency of vector construction by both homologous and Cre-mediated recombination by replacing the single ITR in the shuttle plasmid with a head-to-head ITR junction. We have also expanded the versatility of this method by incorporating a Cre expression cassette into the plasmids to permit high-efficiency Cre-mediated vector rescue using 293 cells, abrogating the need for Cre-expressing cell lines. This new system retains the simplicity of the original but results in an approximately 100-fold increase in the number of recombinant viruses produced, all of which contain the foreign DNA insert, and allows high-efficiency Cre-mediated vector isolation using any E1-complementing cell line.

Adenoviridae↗
PubMed2000

Lentiviral and murine retroviral transduction of T cells for expression of human CD40 ligand.

Efficient transduction of primary human T cells is an important goal toward treating a number of genetic defects. Patient T cells could be harvested by leukapheresis, transduced, and returned to the donor. A wide range of secreted or cell surface therapeutic proteins may be delivered in this way. The ability to produce antibodies is the consequence of interactions between T cells and B cells and lack of expression of CD40 ligand (CD40L) on T cells causes X-linked hyper-IgM syndrome (XHIM). We are investigating delivery of a normal CD40 ligand to treat this disorder. We tested promoters driving the expression of either reporter genes such as enhanced green fluorescent protein (eGFP) or human CDC40L. Using murine retroviruses, the best able to drive gene expression in T cells was the cytomegalovirus (CMV) promoter enhancer element; however, transduction efficiency was low. To achieve efficient, high-level gene expression we tested lentiviral gene delivery vectors. At a low multiplicity of infection (MOI) (0.5-2) a large fraction of target cells was transduced by lentiviral vectors (40-93%), and the strength of gene expression was high, as determined by flow cytometric analysis. We monitored the expression of eGFP or human CD40L on T cell lines and untransformed primary human T cells from normal and CD40L-deficient patients. We achieved efficient gene expression without an extended exposure to virus, and without the need for selection. These results are encouraging for efficient lentivirus-mediated transduction of refractory human cells to achieve therapeutic gene delivery.

Animals↗
PubMed2001

Simplified retroviral vector gcsap with murine stem cell virus long terminal repeat allows high and continued expression of enhanced green fluorescent protein by human hematopoietic progenitors engrafted in nonobese diabetic/severe combined immunodeficient mice.

Despite efforts toward improvements in retrovirus-mediated gene transfer, stable high-level expression of a therapeutic gene in human hematopoietic stem cells remains a great challenge. We have evaluated the efficiency of different viral long terminal repeats (LTRs) in long-term expression of a transgene in vivo, using severe combined immunodeficiency (SCID)-repopulating cell assays. Vectors used were variants of the simplified retroviral vector GCsap with the different LTRs of Moloney murine leukemia virus (MLV), myeloproliferative sarcoma virus (MPSV), and murine stem cell virus (MSCV). The enhanced green fluorescent protein (EGFP) gene was used as a marker to assess levels of transduction efficiency. CD34+ cells isolated from human cord blood were transduced by exposure to virus-containing supernatants on fibronectin fragments and in the presence of stem cell factor, interleukin 6, Flt-3 ligand, and thrombopoietin, and then transplanted into nonobese diabetic/SCID mice. Engraftment of human cells highly expressing EGFP, with differentiation along multiple cell lineages, was demonstrated for up to 18 weeks posttransplant, although the three different vectors showed different transduction frequencies (MLV, <0.1-33.2%; MPSV, <0.1-22.8%; MSCV, 0.3-51.7%). Of importance is that high-level transduction frequencies in human progenitor cells were also confirmed by colony-forming cell assays using bone marrow from transplanted mice, in which EGFP-expressing, highly proliferative potential colonies were observed by fluorescence microscopy. In these mice the vector carrying the MSCV LTR generated more EGFP-expressing human cells than did either of the other two constructs, indicating that GCsap carrying the MSCV LTR may be an efficient tool for stem cell gene therapy.

Animals↗
PubMed2003

Pathways of removal of free DNA vector ends in normal and DNA-PKcs-deficient SCID mouse hepatocytes transduced with rAAV vectors.

Elucidation of the mechanisms of transformation of single-stranded (ss) recombinant adeno-associated virus (rAAV) vector genomes into a variety of stable double-stranded (ds) forms is key to a complete understanding of rAAV vector transduction in vivo. Ds monomer genome formation and cellular ds DNA break (DSB) repair pathways that remove free vector ends toxic to cells, presumably play a central role in this process. By delivering rAAV and naked ds linear DNA vectors into livers of DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-deficient severe combined immunodeficiency (SCID) and wild-type mice, we demonstrate the presence of three major pathways for free ds vector end removal: (1) DNA-PKcs-dependent self-circularization, (2) DNA-PKcs-independent self-circularization, and (3) DNA-PKcs-independent concatemerization. By using the DNA-PKcs-independent pathways, mouse hepatocytes efficiently removed free ds rAAV vector ends even in the absence of DNA-PKcs. Our studies suggest a hierarchical organization of these processes; self-circularization is the preferred pathway over concatemerization, although the former has a limited capacity to remove free vector ends. These studies shed new light on the molecular mechanisms of rAAV vector transduction in vivo.

3' Untranslated Regions↗
PubMed2004

Enhanced transgene expression in quiescent and activated human CD8+ T cells.

The level of expression of retroviral vector-encoded proteins in T cells, decreasing during periods of quiescence, could be an obstacle to their clinical utility. To identify promoter systems that could increase the strength and persistence of transgene expression in primary human CD8(+) T cells, we designed a panel of Moloney retroviral vectors to express a destabilized enhanced green fluorescent protein (d4EGFP) reporter protein (t(1/2) = 4 hr). We found that the promoters phosphoglycerate kinase (Pgk), beta-actin, and long terminal repeat (LTR) produced the highest levels of d4EGFP expression in proliferating T cells, but that expression dramatically declined in quiescent cells with all promoters. To improve gene expression, we examined the effect of the beta-interferon (IFN) scaffold attachment region (SAR). This SAR augmented expression from mammalian promoters in cycling T cells, but had a small effect on maintenance of expression in resting T cells. However, when the SAR was combined with the LTR promoter, it significantly enhanced expression in resting and cycling cells. These data support use of the IFN-beta SAR with the LTR in Moloney retroviral vectors to help sustain gene expression in resting primary human CD8(+) T cells and to enhance gene expression in activated T cells.

CD8-Positive T-Lymphocytes↗
PubMed2004

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Cells, Cultured↗
PubMed1999

DNA from both high-capacity and first-generation adenoviral vectors remains intact in skeletal muscle.

Previous studies of the use of adenoviral vectors in animal models of gene therapy have focused on the immune response against transduced cells as the major limiting factor to long-term transgene expression. In this study we eliminated the variable of immunity induced by expression of the transgene in order to investigate vector DNA stability of both first-generation and high-capacity adenoviral vectors after gene transfer to skeletal muscle. Transgene expression from a high-capacity adenoviral vector remained at a high level for at least 20 weeks and was accompanied by persistence of intact vector genomes. In contrast, transgene expression from a first-generation adenoviral vector markedly diminished by 6 weeks after gene transfer and was accompanied by mild and variable inflammatory cell infiltrates. Surprisingly, despite this loss of transgene expression, the first-generation adenoviral vector genomes persisted like the high-capacity adenoviral vector genomes. Therefore, in the absence of immunity to transgene proteins, loss of expression from the first-generation vector was due to inhibition of transgene expression rather than to the elimination of vector-containing cells. DNA stability and persistent expression of the high-capacity adenoviral vector supports the potential of this vector for clinical applications of muscle gene transfer.

Adenoviridae↗
PubMed1999

A method for efficient extraction of bovine papilloma virus-based minichromosomes that preserves native chromatin structure.

Aiming to create an adequate model for investigation of the molecular mechanisms involved in transcriptional regulation by steroid hormones, a number of cell lines carrying bovine papilloma virus (BPV) based constructs containing the mouse mammary tumor virus long terminal repeat (LTR) were established (Ostrowski et al., Mol. Cell. Biol. 3, 2945-2957, 1983). However, all our attempts to extract from the cells such minichromosomes as nucleoprotein complexes using a method previously described (Ostrowski, Nucleic Acids Res. 15, 6957-6971, 1987) failed. Here, we show that this failure was attributable to DNA rearrangements in most of the cell lines, resulting in the integration of the BPV-based constructs into the host cell genome. We have identified two cell lines where the constructs are episomal. Micrococcal nuclease digestion of the nuclei demonstrated the presence of nucleosomes positioned over the episomal MMTV LTR. We managed to optimize conditions for preparation of nuclei and minichromosomes, which allowed extraction of approximately 40% of the minichromosomes, most of them being in circular superhelical form. Our data show clearly that the main factor preventing the release of minichromosomes from the nuclei is the presence of polyamines in the cell lysis buffer. The organization of MMTV promoter chromatin was unaffected by the extraction procedure, suggesting that these minichromosomes could be valuable templates for in vitro transcription studies and to identify proteins involved in chromatin remodeling during transcription.

Animals↗
PubMed1998

Immunization with both T cell-dependent and T cell-independent vaccines augments HIV viral load secondarily to stimulation of tumor necrosis factor alpha.

Vaccination of HIV-infected individuals increases HIV viral load, reduces CD4 cell counts, and might influence disease progression. Because these deleterious effects are postulated to be secondary to a direct activation of T lymphocytes induced by the immunogen, we compared immunologic and virologic effects of a T cell-dependent and a T cell-independent vaccine. Seventeen HIV-infected children were immunized with influenza (FLU) (T cell-dependent) or pneumococcal (PNEUMO) (T cell-independent) vaccines. HIV viral load and type 1 (IL-2 and IFN-gamma) and type 2 (IL-4 and IL-10) cytokine production were evaluated before and 7, 14, and 28 days after vaccination. Slopes of CD4 cell counts analyzed 6 months before and 6 months after vaccination were not significantly different. HIV viral load increased in both groups of children despite the fact that type 1 cytokine production and the type 1-to-type 2 ratio increased in FLU-vaccinated but not in PNEUMO-vaccinated patients. Thus, an increase in HIV viral load in the absence of T cell activation (as measured by cytokine production) was observed in PNEUMO-vaccinated children. Because polysaccharides of the bacterial cell wall stimulate TNF-alpha production by monocyte-macrophages and TNF-alpha was shown to stimulate HIV replication directly on activation of NF-kappa b after binding the long terminal repeat (LTR) sequences of HIV, we measured TNF-alpha production and observed a significant increase in both groups of vaccines. These data suggest that an increase in HIV viral load can be observed in vaccinated HIV-infected children even independent of direct antigen-induced activation of T lymphocytes, and that augmented production of TNF-alpha might play a role in this phenomenon.

Bacterial Vaccines↗
PubMed2006

Estradiol negatively regulates HIV-LTR promoter activity in glial cells.

HIV-associated dementia results from neuronal loss and an alteration of neuronal function due to a loss of synapses. While HIV infection in astrocytes is limited, astrocytes exhibit a chronic nonproductive infection that can lead to the release of neurotoxic proteins. Additionally, infection can disrupt the normal neurotrophic role of astrocytes that results in neuronal death. Gonadal steroid hormones are known to act as trophic and protective factors in the brain under a variety of normal and pathological conditions. In the present study, to determine if estrogen plays a role in the ability of Tat to function as a transcriptional activator within astrocytes, we examined the effect of estrogen on regulation of viral transcription. We utilized an immortalized human astrocyte cell line (SVGA) stably transfected with a reporter plasmid containing the HIV-1IIIB LTR driving the chloramphenicol acetyltransferase (CAT) gene. The amount of transcriptional activity was measured by quantifying the amount of CAT produced. We determined that 17beta-estradiol treatment (1 nM) had no effect on basal LTR activity. Following transfection with a Tat-expressing plasmid, there was a 100-fold increase in CAT production. This induction was reduced by 40% in cells pretreated with 17beta-estradiol. 17beta- Estradiol only suppressed transcription stimulated by Tat. Furthermore, we determined that this effect was specific to 17beta-estradiol and estrogen receptor agonists. This activity was limited to astrocytes as no effect was observed in a monocytic cell line. Finally, the mechanism of action did not involve an alteration in levels of Cdk9 or Cyclin T1 proteins necessary for Tat activation of the HIV-1 LTR. This study demonstrates a novel activity of 17beta-estradiol in glial cells that could play a role in the maintenance of neuronal health during HIV infection of the central nervous system.

Analysis of Variance↗
PubMed1996

A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography.

The extensive testing of adeno-associated virus (AAV) as a vector for human gene therapy has been hampered by low efficiency of the current packaging system, which is based on transient transfection with plasmid DNAs and infection with adenovirus in permissive cells. In an effort to resolve this problem, HeLa cell-based packaging cell lines were established. These packaging cells carry multiple copies of the AAV genome lacking the inverted terminal repeat (ITR) sequences. The AAV genes were silent in these cells but inducibly expressed by adenovirus infection. When the AAV vector plasmid containing the neoR gene flanked by the ITRs was also integrated into these cells, efficient production of the recombinant AAV particles occurred after adenovirus infection. AAV vector particles in cell lysates could be concentrated by sulfonated cellulose column chromatography. Using the packaging cells and the column chromatography technique, it is possible to prepare AAV vectors with the titer of higher than 10(8) cfu/ml or 5 x 10(10) particles/ml. This new strategy should be useful for testing AAV vectors in vivo.

Cellulose↗
PubMed1997

HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells.

Novel hybrid vectors, which incorporate critical elements of both herpes simplex virus type 1 (HSV-1) amplicon vectors and adeno-associated virus (AAV) vectors, are able to sustain transgene expression in dividing glioma cells for over 2 weeks. These vectors combine the high infectibility and large transgene capacity of HSV-1 vectors with the potential for episomal amplification and chromosomal integration of AAV vectors. The hybrid vectors contain the HSV-1 origin of DNA replication, oriS, and the DNA cleavage/packaging signal, pac, which allow amplicon replication and packaging in HSV-1 virions. The lacZ reporter gene under control of the CMV IE1 promoter is flanked by AAV inverted terminal repeat (ITR) sequences, which facilitate replication and genomic integration of this cassette in the host cell nucleus. Constructs were generated with or without the AAV rep gene (rep+ and rep-) to assess its importance in extending transgene expression. Expression of Rep proteins was confirmed by Western blot analysis. An HSV-1 amplicon construct containing the reporter gene, but no AAV sequences, was used as a control. Constructs were packaged into HSV-1 virions with or without helper virus and these vector stocks were used to infect human U87 glioma cells in culture. The hybrid vectors supported transgene retention and expression for over 2 weeks, whereas the control amplicon vector lost the transgene after 10 days. Expression was somewhat longer for the rep+ as compared to the rep- hybrid vectors. Toxicity due to the HSV-1 helper virus was eliminated using helper virus-free amplicon vector stocks. Transgene constructs could also be packaged in AAV virions, using AAV and adenovirus or HSV-1 helper functions. These HSV/AAV hybrid vectors should allow long-term, nontoxic gene delivery of DNA constructs to both dividing and nondividing cells.

Animals↗
PubMed1998

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

Adenoviridae↗
PubMed1998

A retroviral vector that directs simultaneous expression of alpha and beta T cell receptor genes.

The transfer of alpha/beta T cell receptor (TCR) genes into T lymphocytes or their precursors could provide a means to increase frequency of tumor- or pathogen-specific cytotoxic T lymphocytes. To begin to address this possibility, we have used class I MHC-restricted alpha/beta TCR cDNAs to develop a retroviral TCR expression vector. Alpha- and beta-chain cDNAs were inserted into a derivative of the LN series of retroviral vectors, with the retroviral LTR directing expression of TCR-beta and an internal cytomegalovirus promoter/enhancer driving TCR-alpha expression. The variable region fragments can be replaced using unique restriction sites that have been introduced into the proximal constant regions. We have used this vector system to transfer two different pairs of alpha/beta TCR genes into an alpha- and beta-chain-deficient T cell hybridoma. TCR- hybridoma cells were transduced by coculture with pools of virus-producing cells, and fluorescence-activated cell sorting was used to enrich for cells expressing the transduced TCR. Transduction with either alpha/beta TCR restores stable, long-lived expression of the alpha/beta TCR complex. TCR-mediated signal transduction is also reconstituted, as demonstrated by the ability of transduced cells to secrete IL-2 following stimulation with Vbeta-specific antibodies. Our results suggest that alpha/beta T cell receptor gene transfer could provide a basis for new approaches to immunotherapy, and that further studies examining the in vivo fate of transduced TCR are possible.

Cytomegalovirus↗