T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

Swan, Christina H.; Bühler, Bernd; Tschan, Mario P.; Barbas, Carlos F.; Torbett, Bruce E.

doi:10.1038/sj.gt.3302801

Cited by 72 publications

(81 citation statements)

References 79 publications

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“…To inhibit intracellular TLRs, which cannot be targeted by classical mAbs, antimalarial drugs and short DNA sequences are used, but they often lack sufficient specificity [12,16,17]. By contrast, ER-retained intrabodies do act specifically [18] and are capable of persistently blocking receptor functions if the intrabody gene is stably expressed upon retroviral or lentiviral virus infection [29,30]. Here, we developed an ER intrabody (αT9ib) for the specific knockdown of the intracellularly localized TLR9.…”

Section: Discussionmentioning

confidence: 99%

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Reimer

Somplatzki

Zegenhagen

et al. 2013

Cellular and Molecular Biology Letters

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Toll-like receptor 9 (TLR9) is a component of the innate immune system, which recognizes the DNA of both pathogens and hosts. Thus, it can drive autoimmune diseases. Intracellular antibodies expressed inside the ER block transitory protein functions by inhibiting the translocation of the protein from the ER to its subcellular destination. Here, we describe the construction and characterization of an anti-TLR9 ER intrabody (αT9ib). The respective single-chain Fv comprises the variable domains of the heavy and light chain of a monoclonal antibody (mAb; 5G5) towards human and murine TLR9. Co-expression of αT9ib and mouse TLR9 in HEK293 cells resulted in co-localization of both molecules with the ER marker calnexin. Co-immunoprecipitation of mouse TLR9 with αT9ib indicated that αT9ib interacts with its cognate antigen. The expression of αT9ib inhibited NF-κB-driven reporter gene activation upon CpG DNA challenge but not the activation of TLR3 or TLR4. Consequently, TLR9-driven TNFα production was inhibited in RAW264.7 macrophages upon transfection with the αT9ib expression plasmid. The αT9ib-encoding open reading frame was integrated into an adenoviral cosmid vector to produce the recombinant adenovirus (AdV)-αT9ib. Transduction with AdVαT9ib specifically inhibited TLR9-driven cellular TNFα release. These data strongly indicate that αT9ib is a very promising experimental tool to block TLR9 signaling.

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Section: Discussionmentioning

confidence: 99%

Molecular cloning and characterization of a novel anti-TLR9 intrabody

Reimer

Somplatzki

Zegenhagen

et al. 2013

Cellular and Molecular Biology Letters

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“…Virus particles were generated as described elsewhere (Swan et al, 2006). SAEC and NCI-H661 cells were incubated with viral supernatants for 3 days in the presence of polybrene (4 μg/ml; Sigma).…”

Section: Lentivirus Production and Cell Transductionmentioning

confidence: 99%

Heterodimerization controls localization of Duox-DuoxA NADPH oxidases in airway cells

Luxen

Noack

Frausto

et al. 2009

Journal of Cell Science

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Duox NADPH oxidases generate hydrogen peroxide at the air-liquid interface of the respiratory tract and at apical membranes of thyroid follicular cells. Inactivating mutations of Duox2 have been linked to congenital hypothyroidism, and epigenetic silencing of Duox is frequently observed in lung cancer. To study Duox regulation by maturation factors in detail, its association with these factors, differential use of subunits and localization was analyzed in a lung cancer cell line and undifferentiated or polarized lung epithelial cells. We show here that Duox proteins form functional heterodimers with their respective DuoxA subunits, in close analogy to the phagocyte NADPH oxidase. Characterization of novel DuoxA1 isoforms and mispaired Duox-DuoxA complexes revealed that heterodimerization is a prerequisite for reactive oxygen species production. Functional Duox1 and Duox2 localize to the leading edge of migrating cells, augmenting motility and wound healing. DuoxA subunits are responsible for targeting functional oxidases to distinct cellular compartments in lung epithelial cells, including Duox2 expression in ciliated cells in an ex vivo differentiated lung epithelium. As these locations probably define signaling specificity of Duox1 versus Duox2, these findings will facilitate monitoring Duox isoform expression in lung disease, a first step for early screening procedures and rational drug development.

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“…Various intrabodies against HIV-1 proteins including Tat, Vif, Reverse Transcriptase and Integrase have been shown to inhibit virus replication in gene-modified cells in vitro (Goncalves et al, 2002;Kitamura et al, 1999;Levy-Mintz et al, 1996;Mhashilkar et al, 1995;Shaheen et al, 1996). Moreover, intrabodies against the viral co-receptors CXCR4 and CCR5 have been designed that retain these proteins in the ER (BouHamdan et al, 2001;Cordelier et al, 2004;Swan et al, 2006). Although these approaches efficiently inhibited HIV-1 replication in cell culture systems, intrabody techniques have not been further evaluated in vivo.…”

Section: Antiviral Proteinsmentioning

confidence: 99%