Intrabody-mediated knockout of the high-affinity IL-2 receptor in primary human T cells using a bicistronic lentivirus vector

Richardson, JH; Hofmann, Wolfgang; Sodroski, J; Marasco, W.A.

doi:10.1038/sj.gt.3300644

Cited by 28 publications

(16 citation statements)

References 35 publications

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“…Because of this, we evaluated levels of WT and class I mutant gene expression across numerous cell lines using a single infection strategy. For this, single-round HIV-1 carrying the bacterial CAT gene in the viral nef position and either WT or class I IN mutant D116A (2,7,14) was pseudotyped with the HIV-1 envelope as previously described (20,42). Four different CD4-positive Tcell lines, C8166 (45), , Jurkat (59), and CEM-12D7 (43), each of which was previously used to study HIV-1 IN mutant replication (7,15,16,28,50,53), were infected with equal RT-cpm of virus.…”

Section: Resultsmentioning

confidence: 99%

“…Single-round HIV-1 carrying the pac gene in place of nef and either WT or D116A IN were pseudotyped with either the HIV-1 or VSV-G glycoprotein essentially as previously described (42). Cells infected with equal RT-cpm of envelope-matched viruses were extensively washed, incubated with predetermined levels of puromycin for 3 days, and then serially diluted into 96-well plates in the presence of drug.…”

Section: Resultsmentioning

confidence: 99%

“…Whereas CAT viruses were prepared as previously described using two plasmids (20), pac viruses required three or four different plasmids depending on the identity of the viral envelope (22,42). Transfected cell supernatants were tested for Mg 2ϩ -dependent 32 P-RT activity as previously described (15) and for p24 levels by enzyme-linked immunosorbent assay as recommended by the manufacturer (NEN Life Science Products, Boston, Mass.).…”

mentioning

confidence: 99%

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Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

Nakajima

Engelman

2001

J Virol

103

154

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Retroviruses carry two enzymes, reverse transcriptase (RT) and integrase (IN), which function early in the viral life cycle. Soon after infection, RT converts genomic RNA into linear double-stranded cDNA. This DNA, which contains a copy of the viral long terminal repeat (LTR) at each end, is the substrate for IN-mediated DNA recombination. IN initially processes the 3Ј ends of the cDNA adjacent to phylogenetically conserved CA dinucleotides and then inserts these cleaved ends into a target DNA site in a cell chromosome. The cisacting end regions important for integration define the viral attachment (att) sites, which are comprised of U3 and U5 sequences in the upstream and downstream LTRs, respectively. (For a recent review of retroviral integration, see reference 6.)In addition to the linear DNA product of reverse transcription, various types of circular DNA form in retroviral-infected cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

Nakajima

Engelman

2001

J Virol

103

154

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“…Strategies for achieving retrovirus-mediated, multi-gene transduction include use of internal ribosomal entry sequences (IREs). [1][2][3][4] The IRES vectors allow for the transduction of up to two genes simultaneously, in cis, on a single vector. Other retroviral vectors for transducing two genes into individual cells have contained internal promoters 1,5,6 to drive gene expression.…”

Section: Introductionmentioning

confidence: 99%

Cotransduction of nondividing cells using lentiviral vectors

Frimpong

Spector

2000

Gene Ther

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“…Successful transduction has been demonstrated with, for example, retrovirus and lentivirus vectors (BouHamdan et al, 1999;Chen et al, 1994b;Marasco et al, 1999;Richardson et al, 1998). To date the first clinical studies involving patients with ovarian cancer are ongoing using adenovirus vectors to express anti-erbB-2 intrabodies (Alvarez et al, 2000;Deshane et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

Artsaenko

Tessmann

Sack³

et al. 2003

Journal of General Virology

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The hepatitis C virus (HCV) NS3 protein possesses both protease and helicase activities and is essential for virus replication and maturation. Specific inhibition of NS3 enzymatic activity can be achieved by antibody binding. Transduction of hepatocytes with encoding cDNA leading to intracellular expression of antibody fragments is expected to terminate HCV replication in infected cells. The objective of the present study was the generation of human antibody fragments that neutralize the viral NS3 helicase activity for gene therapeutic applications and drug design. A human immunoglobulin phage-display library cloned from bone marrow aspirate of patients infected with HCV was used for affinity selection against HCV NS3 helicase. Antibody fragments with high affinity to HCV helicase were isolated. To evaluate the inhibitory potential of isolated single-chain antibody fragments, a helicase-mediated, DNA-unwinding enzymatic assay was developed in ELISA format. Recombinant protein comprising the full-length HCV NS3 helicase domain was expressed in the baculovirus expression system. Recombinant antibodies that inhibit the HCV helicase at nanomolar concentrations, with efficacies ranging from 20 % to complete abrogation of enzymatic unwinding activity, were identified. These antibody fragments may be useful for novel gene therapeutic strategies that employ intracellular immunization and may provide new insights into the design of small molecule inhibitors of essential HCV proteins.

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Intrabody-mediated knockout of the high-affinity IL-2 receptor in primary human T cells using a bicistronic lentivirus vector

Cited by 28 publications

References 35 publications

Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

Human Immunodeficiency Virus Type 1 Replication in the Absence of Integrase-Mediated DNA Recombination: Definition of Permissive and Nonpermissive T-Cell Lines

Cotransduction of nondividing cells using lentiviral vectors

Abrogation of hepatitis C virus NS3 helicase enzymatic activity by recombinant human antibodies

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