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Results for “transducer selectivity”

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At least 91 records · Page 5Linked to original sources
PubMed1991

Ultrasonography of the pancreas.

Disorders of the pancreas occur frequently, but they are difficult to diagnose because of anatomic inaccessibility of the pancreas, vague clinical signs and examination findings, and inconsistent laboratory results. Ultrasonography is the imaging modality of choice to evaluate the pancreas. Ultrasonographic examination of the pancreas is difficult due to ultrasound wave propagation interference by adjacent bowel gas, abdominal pain accompanying pancreatitis, and inability to image the normal pancreas consistently. These three problems can be overcome by adhering to a systematic scanning method, proper transducer selection, and proper patient preparation. Scanning techniques used to image the pancreas, and ultrasonographic findings of canine and feline pancreatitis and canine pancreatic neoplasia, are explained.

Acute Disease↗
PubMed2002

Ex vivo selection for oncoretrovirally transduced green fluorescent protein-expressing CD34-enriched cells increases short-term engraftment of transduced cells in baboons.

In an effort to improve hematopoietic stem cell gene transfer rates using gibbon ape leukemia virus (GALV)-pseudotype retroviral vectors in baboons, we have studied preselection of transduced green fluorescent protein (GFP)-expressing CD34-enriched marrow cells. Three animals were transplanted with GFP-selected (GS) CD34-enriched marrow. To ensure engraftment, preselected GFP-positive cells were infused together with unselected neo-transduced cells. After transduction on fibronectin, cells were cultured for an additional 2 days to allow for expression of GFP. GFP-expressing cells were enriched by fluorescence-activated cell sorting and infused together with cells from the unselected fractions after myeloablative irradiation of the recipient. Three other animals were transplanted with GFP-transduced CD34-enriched cells without prior GFP selection (GU). At 4 weeks after transplant, the percentage of GFP-expressing white blood cells was significantly higher in the GS group (6.6%) than in the GU group (1.3%) (p < 0.002). The higher gene transfer levels in the animals transplanted with GS cells gradually declined, and by day 100 after transplant, gene transfer levels were similar in both groups. PCR analysis performed on genomic DNA isolated from peripheral blood cells demonstrated that the decline in GFP-positive cells was due to the loss of gene-marked cells and not due to loss of expression. These results show that transplantation of CD34-positive marrow cells selected for GFP-positive cells after transduction provides high levels of transduced granulocytes in the short term. However, using this experimental design with concomitant infusion of unselected cells and the use of oncoretroviral vectors, preenrichment of vector-expressing, transduced CD34-enriched cells does not improve long-term persistence and expression.

Animals↗
PubMed2002

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

For skin gene therapy, achieving prolonged high-level gene expression in a significant percentage of keratinocytes (KC) is difficult because we cannot selectively target KC stem cells. We now demonstrate that topical colchicine treatment can be used to select, in vivo, KC progenitor cells transduced with the multidrug resistance gene (MDR1). When human skin equivalents containing MDR1-transduced KC were grafted onto immunocompromised mice, topical colchicine treatments significantly increased (7-fold) the percentage of KC expressing MDR1, compared to vehicle-treated controls, for up to 24 wk. Topical colchicine treatment also significantly enhanced the amount of MDR1 protein expressed in individual KC. Furthermore, quantitative real-time PCR analysis of MDR1 transgene copy number demonstrates that topical colchicine treatment selects and enriches for KC progenitor cells in the skin that contain and express MDR1. For clinical skin gene therapy applications, this in vivo selection approach promises to enhance both the duration and expression level of a desired therapeutic gene in KC, by linking its expression to the MDR1 selectable marker gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Limiting numbers of G156A O(6)-methylguanine-DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection.

The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (triangle upMGMT) transduced LTRC present in 5 x 10(4) to 100 x 10(4) marrow cells infused into nonmyeloablated mice by the administration of O(6)-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, triangle upMGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, triangle upMGMT(+) CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% triangle upMGMT(+) CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows triangle upMGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that triangle upMGMT may be a potent, dominant, selectable marker for use in dual gene therapy.

Animals↗
PubMed1994

Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer.

We have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent-activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony-forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. Our results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and should provide a unique tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.

Animals↗
PubMed1998

Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein.

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

Cells, Cultured↗
PubMed1999

Selection of keratinocytes transduced with the multidrug resistance gene in an in vitro skin model presents a strategy for enhancing gene expression in vivo.

In gene therapy studies, achieving prolonged, high-level gene expression in a significant percentage of cells has been difficult. One solution to enhance expression would be to select for cells expressing both the desired gene and a linked selectable marker gene in a bicistronic vector. As a potential target tissue, the skin is easily accessible for safe topical application of a selecting agent that could lead to significant gene expression in a high percentage of keratinocytes. To test the feasibility of such an approach, a skin raft culture model was developed. Human keratinocytes were transduced with the multidrug resistance (MDR) gene, which confers resistance to a variety of cytostatic and antimitotic compounds, such as colchicine. While growing on acellular dermis, transduced keratinocytes were treated with various doses of colchicine (10-50 ng/ml). Colchicine treatment increased the percentage of keratinocytes expressing MDR to almost 100% in raft cultures, Significantly, keratinocytes in colchicine-treated, MDR-transduced raft cultures were able to proliferate normally and form a stratified, differentiated epidermis. This model suggests that topical selection for MDR-expressing keratinocytes in vivo should be feasible without hampering the biologic integrity of skin. Thus, topical selection leading to enhanced expression of a desired gene, linked to a resistance gene, holds future promise for skin gene therapy.

Animals↗
PubMed2004

Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo.

In gene therapy, a clinically relevant therapeutic effect requires long-term expression of the desired gene at a level sufficient to correct or at least alleviate the underlying gene defect. One approach to achieve persistent as well as high-level transgene expression in a significant percentage of target cells would be to select cells expressing both the desired transgene and a linked selectable gene--such as the human multi-drug resistance (MDR1) gene--in a bicistronic vector. Because of its accessibility, the skin is a very attractive target tissue to select genetically modified cells, allowing topical application of a selecting agent, thus minimizing potential toxic side effects. Among the potential selecting drugs, agents that block cell division, such as colchicine, are of particular interest because the use of anti-mitotic drugs takes advantage of the rapid keratinocyte (KC) turnover in the epidermis and the need for continued proliferation to substitute the KC lost due to selection. Before assessing the therapeutic benefit of such an approach, several key questions need to be answered in preclinical models: (1) Does topical colchicine application achieve the desired in vivo effect by blocking KC mitosis without eliciting unwanted toxic side effects? (2) Are MDR-transduced (MDR+) human KC still able to proliferate and differentiate when treated with colchicine? (3) Can MDR+ KC be enriched by topical selection? (4) Does topical selection result in persistent transgene expression by selecting KC stem cells expressing MDR? To answer these questions and to test the feasibility of such an approach both an in vitro skin equivalent and an in vivo human skin graft model were developed in which MDR+ KC were treated with different dosages of colchicine. Quantitative and qualitative analyses of MDR expression in human KC showed that topical colchicine treatment selects high-level transgene expression in a high percentage of KC. Moreover, determination of transgene copy numbers demonstrated that MDR+ KC progenitor cells were enriched by topical selection resulting in long-term expression of the transgene in the skin. Thus, in summary, these models demonstrate that topical selection of MDR+ KC is a safe approach to efficiently enhance long-term gene expression in the skin and holds future promise for clinical gene therapy applications.

Administration, Topical↗
PubMed1997

Selection of retrovirally transduced cells to enhance the efficiency of gene therapy.

Retrovirally mediated gene transfer is the approach of choice for many laboratories interested in gene therapy. One problem, however, is that the efficiency of therapeutic gene transfer to some important target cells is low. A strategy employed by a number of investigators to overcome this shortcoming is to construct recombinant viruses that employ selection modalities. These strategies can be classified broadly into either metabolic or cell-surface selection strategies. This article examines each selection approach and notes their important strengths and weaknesses. Further, the various gene products used in each approach to effect sorting of transduced cells is detailed.

Animals↗
PubMed1997

Murine hematopoietic reconstitution after tagging and selection of retrovirally transduced bone marrow cells.

A major problem facing the effective treatment of patients with cancer is how to get the specific antitumor agent into every tumor cell. In this report we describe the use of a strategy that, by using retroviral vectors encoding a truncated human CD5 cDNA, allows the selection of only the infected cells, and we show the ability to obtain, before bone marrow transplantation, a population of 5-fluouracil-treated murine bone marrow cells that are 100% marked. This marked population of bone marrow cells is able to reconstitute the hematopoietic system in lethally irradiated mice, indicating that the surface marker lacks deleterious effects on the functionality of bone marrow cells. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD5-expressing cells. Nevertheless, a significant proportion of the hematopoietic cells no longer expresses the surface marker CD5 in the 9-month-old recipient mice. This transcriptional inactivity of the proviral long terminal repeat (LTR) was accompanied by de novo methylation of the proviral sequences. Our results show that the use of the CD5 as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, which can entirely reconstitute the hematopoietic system in mice. These results should now greatly enhance the power of studies aimed at addressing questions such as generation of cancer-negative hematopoiesis.

Animals↗
PubMed2002

Selective expansion of transduced cells for hematopoietic stem cell gene therapy.

Although gene transfer into hematopoietic stem cells holds a considerable therapeutic potential, clinical trials targeting this cell compartment have achieved limited success. Poor transduction efficiency with gene transfer vectors used in human studies has hindered delivering therapeutic genes to clinically relevant numbers of target cells. One way to overcome the low-efficiency problem is by selecting or expanding the number of genetically modified cells to a suprathreshold level to achieve clinical efficacy. This approach may be further classified into 2 categories: one is to transfer a drug resistance gene and eliminate unmodified cells with cytotoxic drugs, and the other is to confer a direct growth advantage on target cells. This review aims at an overview of recent advances involving these strategies, with some details of "selective amplifier genes," a novel system that we have developed for specific expansion of genetically modified hematopoietic cells.

Animals↗
PubMed1995

Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity.

Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon gamma (IFN-gamma) activation sites, which were first identified in the promoters of IFN-gamma-inducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-gamma, interleukin (IL) 6, granulocyte-macrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT--AA motif with a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.

Base Sequence↗
PubMed2003

Discovery of JSI-124 (cucurbitacin I), a selective Janus kinase/signal transducer and activator of transcription 3 signaling pathway inhibitor with potent antitumor activity against human and murine cancer cells in mice.

Constitutively activated, tyrosine-phosphorylated signal transducer and activator of transcription (STAT) 3 plays a pivotal role in human tumor malignancy. To discover disrupters of aberrant STAT3 signaling pathways as novel anticancer drugs, we developed a phosphotyrosine STAT3 cytoblot. Using this high throughput 96-well plate assay, we identified JSI-124 (cucurbitacin I) from the National Cancer Institute Diversity Set. JSI-124 suppressed the levels of phosphotyrosine STAT3 in v-Src-transformed NIH 3T3 cells and human cancer cells potently (IC(50) value of 500 nM in the human lung adenocarcinoma A549) and rapidly (complete inhibition within 1-2 h). The suppression of phosphotyrosine STAT3 levels resulted in the inhibition of STAT3 DNA binding and STAT3-mediated but not serum response element-mediated gene transcription. JSI-124 also decreased the levels of tyrosine-phosphorylated Janus kinase (JAK) but not those of Src. JSI-124 was highly selective for JAK/STAT3 and did not inhibit other oncogenic and tumor survival pathways such as those mediated by Akt, extracellular signal-regulated kinase 1/2, or c-Jun NH(2)-terminal kinase. Finally, JSI-124 (1 mg/kg/day) potently inhibited the growth in nude mice of A549 tumors, v-Src-transformed NIH 3T3 tumors, and the human breast carcinoma MDA-MB-468, all of which express high levels of constitutively activated STAT3, but it did not affect the growth of oncogenic Ras-transformed NIH 3T3 tumors that are STAT3 independent or of the human lung adenocarcinoma Calu-1, which has barely detectable levels of phosphotyrosine STAT3. JSI-124 also inhibited tumor growth and significantly increased survival of immunologically competent mice bearing murine melanoma with constitutively activated STAT3. These results give strong support for pharmacologically targeting the JAK/STAT3 signaling pathway for anticancer drug discovery.

3T3 Cells↗
PubMed2000

In vivo selection of hepatocytes transduced with adeno-associated viral vectors.

A murine model for hereditary tyrosinemia Type I (HTI) was evaluated for in vivo gene therapy with adeno-associated viral (AAV) vectors expressing the enzyme fumarylacetoacetate hydrolase. Transduction of a limited number of hepatocytes was accomplished following infusion of vector into the portal circulation. Corrected hepatocytes were expanded in vivo by withdrawing a drug which prevents the accumulation of toxic metabolites. The liver was eventually repopulated with hepatocytes harboring a functional and apparently integrated AAV provirus. Recipient animals regained normal liver function and architecture and the underlying metabolic derangements were normalized. After 9 months, vector-treated animals showed benign hepatomas, whereas in untreated animals areas of marked dysplasia were present within hepatomas.

Animals↗
PubMed2005

An inducible caspase 9 safety switch for T-cell therapy.

The efficacy of adoptive T-cell therapy as treatment for malignancies may be enhanced by genetic modification of infused cells. However, oncogenic events due to vector/transgene integration, and toxicities due to the infused cells themselves, have tempered enthusiasm. A safe and efficient means of removing aberrant cells in vivo would ameliorate these concerns. We describe a "safety switch" that can be stably and efficiently expressed in human T cells without impairing phenotype, function, or antigen specificity. This reagent is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow conditional dimerization using a small molecule pharmaceutical. A single 10-nM dose of synthetic dimerizer drug induces apoptosis in 99% of transduced cells selected for high transgene expression in vitro and in vivo. This system has several advantages over currently available suicide genes. First, it consists of human gene products with low potential immunogenicity. Second, administration of dimerizer drug has no effects other than the selective elimination of transduced T cells. Third, inducible caspase 9 maintains function in T cells overexpressing antiapoptotic molecules. These characteristics favor incorporation of inducible caspase 9 as a safety feature in human T-cell therapies.

Animals↗