Differences in transductional tropism of adenoviral and lentiviral vectors in the rat brainstem

Abstract

Adenoviral vectors (AVVs) and lentiviral vectors (LVVs) are highly useful research tools which can be used to investigate the function of specific cell phenotypes in the brain. The transductional tropism of viral vectors has a critical impact upon the transgene expression in different brain areas. This largely depends on the properties of the viral particles, which for AVVs are most commonly analogous to the serotype 5 adenovirus and, in the case of LVVs, are determined by the envelope used for pseudotyping, for example the vesicular stomatitis virus coat (VSVG). We have created a matching set of shuttle plasmids that allow a one-step transfer of an entire expression cassette between the backbones of AVVs and LVVs. This has permitted a fair assessment of the impact of the vector type on tropism for both AVVs and LVVs. Thus, the aims of this study were twofold: (i) to develop and demonstrate the validity of a transgene 'swap' system between AVVs and LVVs; and (ii) using this system, to assess the tropism of AVVs and LVVs for neuronal versus glial cell types. We have constructed AVVs and VSVG-coated LVVs to express monomeric red fluorescent protein (mRFP) driven by the human cytomegalovirus promoter (hCMV). Transgene expression in neurones and glia in the hypoglossal and dorsal vagal motor nuclei of the rat brainstem was compared by determining the colocalization with immunostaining for the neuronal marker NeuN (neuronal nuclear antigen) and the glial marker GFAP (glial fibrillatory acidic protein). We found that 55% of mRFP-expressing cells transduced with AVVs were immunopositive for GFAP, while only 38% were NeuN-immunoreactive. In contrast, when the same expression cassette was delivered by VSVG-coated LVVs, the neurone/glia ratio of mRFP expression was reversed with 56% of mRFP-positive cells identified as neurones and 26% as glia. Thus, the present study provides compelling evidence that VSVG-coated LVVs significantly shift transgene expression towards neurones while transduction with AVVs favours glia.

Calcineurin Triggers Reactive/Inflammatory Processes in Astrocytes and Is Upregulated in Aging and Alzheimer's Models

“…Interestingly, neurons in the Ad-LacZ and AdaCaN treatment conditions showed similar intensities of calcineurin labeling (Fig. 2, C vs F ), suggesting that the Ad-aCaN construct was not taken up by neurons as readily as by astrocytes, an observation consistent with the preferential uptake of adenovirus by glial cells (Duale et al, 2005). Together, the results indicate that the effects of Ad-aCaN on astrocytes were mediated primarily by uptake and overexpression of calcineurin in astrocytes and by direct activation of intracellular astrocytic pathways regulating the activation response.…”

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“…However, the speed and quality of the method depend on the effectiveness of the viral penetration into the target cell, as well as on the activity of the promoter used for transgenic expression (transcriptional tropism). Some types of viral vectors, including alphaviruses, adeno-associated viruses (AAV), AVV, and LVV (of which AAV, LVV, and AAB are used most widely at the present time), [17][18][19] were tested as a means of delivering genes into brain cells.…”

Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy

“…These lead promoter sequences were inserted into pTYF-Sw-linker, a lentiviral shuttle plasmid, upstream of EGFP. 30 Conventional (Figure 2a), bi-directional (Figure 2b) and IRES-based designs (Figure 2c) for TSTA were constructed for 2TPH and 3.6TPH promoters within LVV shuttle plasmids.…”

“…To construct pTYF-G4BS-2TPH-EGFP-IRES-GAL4|p65, initially, IRES2 was PCR amplified from pIRES2-EGFP (Clontech Laboratories Inc., Mountain View, CA, USA) and inserted into pTYF-Swlinker at XmaI (SmaI) and SacII sites 30 to obtain pTYF-Sw-IRES. An NheI/ GAL4|p65-SV40pA/NheI fragment was excised from pTYF-mCMV/SYN-EGFP and inserted into pTYF-Sw-IRES at the SpeI site downstream of IRES.…”