Poor expression of MDR1 transgene in HeLa cells by bicistronic Moloney murine leukemia virus-based vector.

Abstract:

:Cotransfer of a therapeutic gene together with the human MDR1 gene provides an opportunity to increase the number of transduced marrow cells, expressing the therapeutic gene, by in vivo selection for MDR1. We have used an Lg-MDR1-IRES-neo (LgMIN) retroviral vector, containing MDR1 and neo genes, separated by the EMCV IRES. Human HeLa or canine CTAC cells, transduced with GALV env pseudotyped LgMIN at an MOI of less than 0.01 to ensure 1 proviral copy/genome, were selected with either G418 for neo expression or colchicine for MDR1 expression. The titer determined on HeLa cells with G418 selection was eight-fold higher than that with colchicine selection. In contrast, the same viral supernatant exhibited only a 1.4-fold difference between neo- and MDR1-based viral titer values for CTAC cells. The transduced HeLa cells, with one intact proviral copy per genome, exhibited a 55-fold higher resistance to G418 but only a 4-fold higher resistance to colchicine and a 2-fold higher resistance to Taxol compared with nontransduced cells. About 23% of the transduced cell population did not express vector-derived P-glycoprotein (P-gp) as detected by anti-human P-gp MAb MRK-16. This could explain the difference in viral titers obtained on CTAC cells but not that obtained on HeLa cells. The vector-mediated increase in expression of P-gp was about 20-fold higher in CTAC cells as compared with HeLa cells. These results indicated suppression of expression of vector-derived MDR1 in HeLa cells, in contrast with CTAC cells. To investigate further the possible reasons for this difference, genomic DNA was isolated from the G418-resistant individual colonies of infected cells and analyzed by PCR for full-length proviral MDR1. For transduced CTAC and HeLa cells, selected at a G418 concentration of 1 mg/ml, PCR detected aberrant forms of MDR1 in 17 to 25% of colonies tested. The aberrant forms consisted of MDR1 genes with 2- and 0.7-kb deletions. DNA sequencing across the 2-kb and the 0.7-kb deletion junction suggests cryptic splicing in the producer cell line as the origin of these deletions. The 2-kb deletion corresponds to MDR1 mRNA cryptic splicing via donor (codon 113) and acceptor (codon 773). The 0.7-kb deletion corresponds to splicing via the same donor and a different acceptor (codon 344). When transduced HeLa cells were selected at a higher concentration of G418 (3 mg/ml), the aberrant forms were detected at an increased frequency of about 50% of colonies tested. These results indicate that vector-derived MDR1 is a poor selective marker in HeLa cells but not in CTAC cells and that deletions, which inactivated the MDR1 gene in a bicistronic Mo-MuLV vector, may provide an advantage for expression of the second transgene in HeLa cells.

journal_name

Hum Gene Ther

journal_title

Human gene therapy

authors

Zaboikin MM,Schuening FG

doi

10.1089/hum.1998.9.15-2263

subject

Has Abstract

pub_date

1998-10-10 00:00:00

pages

2263-75

issue

15

eissn

1043-0342

issn

1557-7422

journal_volume

9

pub_type

杂志文章
  • Antitumor therapy based on cellular competition.

    abstract::A major obstacle for the efficacy of cancer gene therapy is the need to transduce a high proportion of tumor cells with genes that directly or indirectly cause their death. During the formation of certain organs, cells compete among themselves to colonize the whole tissue. We reasoned that cell competition could be us...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2008.144

    authors: Martinez-Quintanilla J,Cascallo M,Fillat C,Alemany R

    更新日期:2009-07-01 00:00:00

  • Quantification and characterization of autotransduction in retroviral vector producer cells.

    abstract::Gene therapy has evolved into a tempting strategy for the management of cancer and other life-threatening diseases. Various approaches employ retroviral vectors to deliver the therapeutic gene. The profound knowledge about retrovirus biology allows the generation of increasingly advanced vector systems as well as an a...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2007.071

    authors: Brandtner EM,Kodajova P,Knapp E,Ertl R,Tabotta W,Salmons B,Günzburg WH,Hohenadl C

    更新日期:2008-01-01 00:00:00

  • Plasmid DNA encoding targeted naturally processed peptides generates protective cytotoxic T lymphocyte responses in immunized animals.

    abstract::Genetic immunization has been widely applied in efforts to find novel and efficient mechanisms of stimulating the immune response. An effective attack against viral pathogens or tumors often requires activation of T cell-mediated immunity and the generation of cytotoxic T cells. Intramuscular immunization with plasmid...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1998.9.3-325

    authors: Hedley ML,Strominger JL,Urban RG

    更新日期:1998-02-10 00:00:00

  • Antigen-specific tolerance of human alpha1-antitrypsin induced by helper-dependent adenovirus.

    abstract::As efficient and less toxic virus-derived gene therapy vectors are developed, a pressing problem is to avoid immune response to the therapeutic gene product. Secreted therapeutic proteins potentially represent a special problem, as they are readily available to professional antigen-presenting cells throughout the body...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2006.036

    authors: Cerullo V,McCormack W,Seiler M,Mane V,Cela R,Clarke C,Rodgers JR,Lee B

    更新日期:2007-12-01 00:00:00

  • A mutant Tat protein provides strong protection from HIV-1 infection in human CD4+ T cells.

    abstract::Here we show potent inhibition of HIV-1 replication in a human T cell line and primary human CD4(+) cells by expressing a single antiviral protein. Nullbasic is a mutant form of the HIV-1 Tat protein that was previously shown to strongly inhibit HIV-1 replication in nonhematopoietic cell lines by targeting three steps...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2012.176

    authors: Apolloni A,Lin MH,Sivakumaran H,Li D,Kershaw MH,Harrich D

    更新日期:2013-03-01 00:00:00

  • Use of nonintegrating lentiviral vectors for gene therapy.

    abstract::Vectors based on lentiviruses have become potent tools for efficient gene transfer to multiple cell types both in vitro and in vivo. In part this is attributable to the stability of transduction afforded by integration into the target cell genome. However, evidence indicates that episomal forms of the vector can also ...

    journal_title:Human gene therapy

    pub_type: 杂志文章,评审

    doi:10.1089/hum.2007.013

    authors: Philpott NJ,Thrasher AJ

    更新日期:2007-06-01 00:00:00

  • Inhibition of atrogin-1/MAFbx expression by adenovirus-delivered small hairpin RNAs attenuates muscle atrophy in fasting mice.

    abstract::Atrogin-1 or muscle atrophy F-box (MAFbx) is a major atrophy-related E3 ubiquitin ligase highly expressed in skeletal muscle during muscle atrophy and other disease states such as sepsis, cancer cachexia, and fasting. In this paper, we report experiments inhibiting MAFbx activity in fasting mice and in the skeletal my...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2010.057

    authors: Cong H,Sun L,Liu C,Tien P

    更新日期:2011-03-01 00:00:00

  • Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice.

    abstract::At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, trea...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2006.137

    authors: Baba Y,Yazawa T,Kanegae Y,Sakamoto S,Saito I,Morimura N,Goto T,Yamada Y,Kurahashi K

    更新日期:2007-02-01 00:00:00

  • Long-term efficacy of adeno-associated virus serotypes 8 and 9 in hemophilia a dogs and mice.

    abstract::We reported total correction of blood coagulation plasma factor VIII (FVIII) activity, using adeno-associated virus serotype 8 (AAV8) vectors for liver-specific gene transfer in hemophilia A mice. We now show, irrespective of immunosuppression or route of administration, total long-term correction of hemophilia A mice...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2006.17.427

    authors: Sarkar R,Mucci M,Addya S,Tetreault R,Bellinger DA,Nichols TC,Kazazian HH Jr

    更新日期:2006-04-01 00:00:00

  • A method of limited replication for the efficient in vivo delivery of adenovirus to cancer cells.

    abstract::Replication-deficient viral vectors are currently being used in gene transfer strategies to treat cancer cells. Unfortunately, viruses are limited in their ability to diffuse through tissue. This makes it virtually impossible to infect the majority of tumor cells in vivo and results in inadequate gene transfer. This p...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1998.9.8-1209

    authors: Han JS,Qian D,Wicha MS,Clarke MF

    更新日期:1998-05-20 00:00:00

  • Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

    abstract::Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initiall...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1998.9.6-771

    authors: Glimm H,Flügge K,Möbest D,Hofmann VM,Postmus J,Henschler R,Lange W,Finke J,Kiem HP,Schulz G,Rosenthal F,Mertelsmann R,von Kalle C

    更新日期:1998-04-10 00:00:00

  • Generation of CD34+ cells from CCR5-disrupted human embryonic and induced pluripotent stem cells.

    abstract::C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndr...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2011.126

    authors: Yao Y,Nashun B,Zhou T,Qin L,Qin L,Zhao S,Xu J,Esteban MA,Chen X

    更新日期:2012-02-01 00:00:00

  • A "distant" bystander effect of suicide gene therapy: regression of nontransduced tumors together with a distant transduced tumor.

    abstract::Antitumor gene therapy using herpes simplex type 1 thymidine kinase (TKh) and ganciclovir (GCV) treatment has revealed an important intratumoral bystander effect. A whole tumor can be eliminated when only a fraction of its tumor cells express TKh. We now report that the bystander effect not only acts within a tumor, b...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1997.8.15-1807

    authors: Kianmanesh AR,Perrin H,Panis Y,Fabre M,Nagy HJ,Houssin D,Klatzmann D

    更新日期:1997-10-10 00:00:00

  • Efficiencies of transgene expression in nociceptive neurons through different routes of delivery of adeno-associated viral vectors.

    abstract::Transferring therapeutic genes into the nociceptive system, including dorsal root ganglia (DRGs) and the spinal cord, is potentially a powerful approach for the treatment of chronic pain in humans. Adeno-associated viral vectors (AAVs) are particularly useful in delivering foreign genes to targeted tissues because the...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/104303403765701187

    authors: Xu Y,Gu Y,Wu P,Li GW,Huang LY

    更新日期:2003-06-10 00:00:00

  • Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1.

    abstract::Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and la...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2017.252

    authors: VanLith C,Guthman R,Nicolas CT,Allen K,Du Z,Joo DJ,Nyberg SL,Lillegard JB,Hickey RD

    更新日期:2018-11-01 00:00:00

  • Direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12.

    abstract::Mice bearing breast tumors were treated with a single dose of an adenovirus expressing interleukin-12 (AdmIL-12.1) injected intratumorally, which produced regressions in greater than 75% of the treated tumors; approximately one-third of the animals remained tumor free. Complete regression was associated with immunity ...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1996.7.16-1995

    authors: Bramson JL,Hitt M,Addison CL,Muller WJ,Gauldie J,Graham FL

    更新日期:1996-10-20 00:00:00

  • Hearing on the possible uses and misuses of genetic information.

    abstract::In summary, I will reiterate the five points I would like to leave with you today: First, the biological revolution has extraordinary power to do good. As long as the use of our new genetic knowledge is guided by the traditional ideals of the healing professions--to help improve the human condition without doing harm-...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1992.3.1-51

    authors: Healy B

    更新日期:1992-02-01 00:00:00

  • Adenovirus-mediated hepatic gene transfer in mice: comparison of intravascular and biliary administration.

    abstract::Recombinant adenoviruses have received much attention as a potential vector for gene therapy because of their ability to transduce many cell types with high efficiencies in vivo. After intravenous infusion, the majority of the vector is found in hepatocytes, but vector DNA is found to varying degrees in other tissues....

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1996.7.14-1693

    authors: Peeters MJ,Patijn GA,Lieber A,Meuse L,Kay MA

    更新日期:1996-09-10 00:00:00

  • Nuclease-deficient CRISPR-based approaches for in vitro and in vivo gene activation.

    abstract::CRISPR-based technology has been adapted to achieve a wide range of genome modifications including transcription regulation. The focus of this review is on the application of CRISPR-based platforms such as nuclease-deficient Cas9 and Cas12a, to achieve targeted gene activation. We review studies to date that have empl...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2020.241

    authors: Lek A,Ma K,Woodman K,Lek M

    更新日期:2021-01-15 00:00:00

  • DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

    abstract::La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an ag...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/10430349950017653

    authors: Schuh T,Schultz J,Moelling K,Pavlovic J

    更新日期:1999-07-01 00:00:00

  • Helper-dependent adenoviral vectors containing modified fiber for improved transduction of developing and mature muscle cells.

    abstract::Adenoviruses (Ads) have shown great utility as vectors for the delivery of genes to mammalian cells, partly because of their ability to infect a wide range of different cell types independent of the replicative state of the cell. However, Ads do not transduce mature muscle efficiently because of low levels of the natu...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/104303404772679986

    authors: Bramson JL,Grinshtein N,Meulenbroek RA,Lunde J,Kottachchi D,Lorimer IA,Jasmin BJ,Parks RJ

    更新日期:2004-02-01 00:00:00

  • The effects of human serum and cerebrospinal fluid on retroviral vectors and packaging cell lines.

    abstract::Human serum is known to inactivate many retroviruses, including murine leukemia viruses (MLV). Exposure of vectors based on MLV to human serum components would presumably decrease the efficiency of gene transfer in vivo. Human serum also lyses xenogeneic cells, which would affect the survival of retroviral vector pack...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1995.6.5-635

    authors: Russell DW,Berger MS,Miller AD

    更新日期:1995-05-01 00:00:00

  • Enhanced Transduction of Macaca fascicularis Hematopoietic Cells with Chimeric Lentiviral Vectors.

    abstract::Recent marketing approval for genetically engineered hematopoietic stem and T cells bears witness to the substantial improvements in lentiviral vectors over the last two decades, but evaluations of the long-term efficacy and toxicity of gene and cell therapy products will, nevertheless, require further studies in nonh...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2018.179

    authors: Sii-Felice K,Castillo Padilla J,Relouzat F,Cheuzeville J,Tantawet S,Maouche L,Le Grand R,Leboulch P,Payen E

    更新日期:2019-10-01 00:00:00

  • Regulatory and ethical issues for phase I in utero gene transfer studies.

    abstract::Clinical gene transfer research has involved adult and child subjects, and it is expected that gene transfer in fetal subjects will occur in the future. Some genetic diseases have serious adverse effects on the fetus before birth, and there is hope that prenatal gene therapy could prevent such disease progression. Res...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2011.062

    authors: Strong C

    更新日期:2011-11-01 00:00:00

  • Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated virus partially restores cystic fibrosis transmembrane conductance regulator function to polarized human cystic fibrosis airway epithelial cells.

    abstract::We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.2005.16.1116

    authors: Liu X,Luo M,Zhang LN,Yan Z,Zak R,Ding W,Mansfield SG,Mitchell LG,Engelhardt JF

    更新日期:2005-09-01 00:00:00

  • Herpesvirus vector-mediated gene delivery to human monocytes.

    abstract::In vitro delivery of interferon-alpha (IFN-alpha) to cultured human monocytes by means of a replication-incompetent herpesvirus vector inhibits human immunodeficiency virus (HIV) replication. To explore the possibility of IFN-alpha gene delivery by vector-infected human monocytes, monocytes were isolated and the cultu...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1996.7.11-1331

    authors: Weir JP,Dacquel EJ,Aronovitz J

    更新日期:1996-07-10 00:00:00

  • Activation of a diphtheria toxin A gene by expression of human immunodeficiency virus-1 Tat and Rev proteins in transfected cells.

    abstract::Expression of a gene encoding the diphtheria toxin A (DT-A) fragment, controlled by tissue specific regulatory elements, has previously been used to kill selected cell populations. Here, we have examined the feasibility of controlling DT-A expression using regulatory systems from the human immunodeficiency virus (HIV-...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1991.2.1-53

    authors: Harrison GS,Maxwell F,Long CJ,Rosen CA,Glode LM,Maxwell IH

    更新日期:1991-04-01 00:00:00

  • Progress in adeno-associated virus type 2 vector production: promises and prospects for clinical use.

    abstract::Vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) are among the most promising gene delivery vehicles currently being developed. These vectors are not only capable of transducing a large variety of human cell types in vitro and in vivo, but in immunocompetent animal models can establish l...

    journal_title:Human gene therapy

    pub_type: 杂志文章,评审

    doi:10.1089/10430349950016799

    authors: Grimm D,Kleinschmidt JA

    更新日期:1999-10-10 00:00:00

  • Synthesis and processing of genetically modified human proinsulin by rat myoblast primary cultures.

    abstract::Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/hum.1996.7.1-71

    authors: Simonson GD,Groskreutz DJ,Gorman CM,MacDonald MJ

    更新日期:1996-01-01 00:00:00

  • Transduction of long-term and mobilized peripheral blood-derived NOD/SCID repopulating cells by foamy virus vectors.

    abstract::Foamy virus (FV) vectors are a promising gene delivery system for use in hematopoietic stem cell gene therapy. Previous FV vector marking studies in the NOD/SCID xenotransplantation model used umbilical cord blood (UCB)-derived SCID repopulating cells (SRCs) that were assayed 5-10 weeks posttransplantation. We now rep...

    journal_title:Human gene therapy

    pub_type: 杂志文章

    doi:10.1089/10430340460732481

    authors: Josephson NC,Trobridge G,Russell DW

    更新日期:2004-01-01 00:00:00