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Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

ABSTRACT: In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proximal part) and two variants (in forward and reverse orientation) of a previously characterized synthetic promoter derived from elements of transcription factor genes. We transduced a number of cell lines and primary cells in vitro. In addition, hematopoietic stem cells were transduced with MSPs and transferred into lethally irradiated recipient mice. Fluorescence activated cell sorting analysis was performed to determine the GFP expression in different cell populations both in vitro and in vivo. We showed that MSPs can efficiently be used for lentiviral gene delivery and that the 150-bp proximal part of the CD68 promoter provides primarily macrophage-specific expression of GFP. We propose that this is the best currently available MSP to use for directing transgene expression to macrophage populations in vivo using lentiviral vectors.

SUBMITTER: Levin MC

PROVIDER: S-EPMC3697098 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

Levin M C MC Lidberg U U Jirholt P P Adiels M M Wramstedt A A Gustafsson K K Greaves D R DR Li S S Fazio S S Linton M F MF Olofsson S-O SO Borén J J Gjertsson I I

Gene therapy 20111201 11

In gene therapy, tissue-specific promoters are useful tools to direct transgene expression and improve efficiency and safety. Macrophage-specific promoters (MSPs) have previously been published using different delivery systems. In this study, we evaluated five different MSPs fused with green fluorescent protein (GFP) to delineate the one with highest specificity using lentiviral delivery. We compared three variants of the CD68 promoter (full length, the 343-bp proximal part and the 150-bp proxim ...[more]

PMID: 22130447

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Project description:Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2C226,228S that induces TM dysfunction and outflow resistance.

Dataset Information

Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

Publications

Evaluation of macrophage-specific promoters using lentiviral delivery in mice.

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