The development over the past decade of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. However, despite substantial progress, a number of key technical issues need to be resolved before gene therapy can be safely and effectively applied in the clinic. Future technological developments, particularly in the areas of gene delivery and cell transplantation, will be critical for the successful practice of gene therapy.

https://science.sciencemag.org/content/sci/260/5110/926.full.pdf

The Basic Science of Gene Therapy

Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.

Biochemical, cellular, and pharmacological aspects of the multidrug transporter

The analysis of how human cancers evade chemotherapy has revealed a rich variety of cell-based genetic changes resulting in drug resistance. One of the best studied of these genetic alterations is increased expression of an ATP-dependent plasma membrane transport system, known as P-glycoprotein, or the multidrug transporter. This transporter actively effluxes a large number of natural product, hydrophobic, cytotoxic drugs, including many important anticancer agents. This review focuses on the genetic and molecular genetic analysis of the human multidrug transporter, including structure-function analysis, pre- and posttranslational regulation of expression, the role of gene amplification in increased expression, and the properties of transgenic and "knock-out" mice. One important feature of the MDR gene is its potential for the development of new selectable vectors for human gene therapy.

/pdf/genetic-analysis-of-the-multidrug-transporter-49u8fa4pxx.pdf

Genetic Analysis of the Multidrug Transporter

We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells. Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner. Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells. In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells. In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus. On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus. When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells.

Direct contact between hematopoietic cells and viral packaging cell lines or other sources of stroma has been shown to increase the efficiency of retroviral-mediated gene transfer into these target cells compared with infection with viral supernatant. We have investigated the role of defined bone marrow extracellular matrix molecules (ECM) in this phenomenon. Here we report that infection of cells adhering to the carboxy-terminal 30/35-kD fragment of the fibronectin molecule (30/35 FN), which contains the alternatively spliced CS-1 cell adhesion domain, significantly increases gene transfer into hematopoietic cells. Two retroviral vectors differing in recombinant viral titer were used. Gene transfer into committed progenitor cells and long-term culture-initiating cells, an in vitro assay for human stem cells, was significantly increased when the cells were infected while adherent to 30/35 FN-coated plates compared with cells infected on BSA-coated control plates or plates coated with other bone marrow ECM molecules. Although gene transfer into committed progenitor cells and to a lesser degree into long-term culture-initiating cells was increased on intact fibronectin as well, increased gene transfer efficiency into hematopoietic cells on 30/35 FN was dependent on CS-1 sequence since infection on a similar FN fragment lacking CS-1 (42 FN) was suboptimal. 30/35 FN has previously been shown by our laboratory and other investigators to mediate adhesion of primitive murine and human hematopoietic stem cells to the hematopoietic microenvironment. Additional studies showed that neither soluble 30/35 FN nor nonspecific binding of hematopoietic cells to poly-L-lysine-coated plates had any appreciable effect on the infection efficiency of these cells. Our findings indicate that hematopoietic stem cell adhesion to specific ECM molecules alters retroviral infection efficiency. These findings should aid in the design of gene transfer protocols using hematopoietic progenitor and stem cells for somatic gene therapy.

/pdf/bone-marrow-extracellular-matrix-molecules-improve-gene-2hss1ek87a.pdf

Bone marrow extracellular matrix molecules improve gene transfer into human hematopoietic cells via retroviral vectors.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

https://www.pnas.org/content/pnas/89/20/9676.full.pdf

Transfer and expression of the human multiple drug resistance gene into live mice.

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells

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Transfer and expression of the human multiple drug resistance gene into live mice.

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells