Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

Xu, Rui‐hua; Janson, Christopher G.; Mastakov, Mihail Y.; Lawlor, P.; Young, Deborah; Mouravlev, Alexandre; Fitzsimons, Helen L.; Choi, Kai Luk; Ma, Hsu; Dragunow, Mike; Leone, Paola; Chen, Q; Dicker, Bridget; During, Matthew J.

doi:10.1038/sj.gt.3301529

Cited by 167 publications

(131 citation statements)

References 56 publications

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“…Klein, Meyer and colleagues have demonstrated transduction of cultures of astrocytes, microglia and cortical neurons using AAV2 or AAV8 with the CBA promoter [12,17,18]. In a quantitative comparison of 10 promoters in AAV2 vectors, in cultures of rat hippocampal, cortical, striatal and nigral cells, the 1.8kb NSE promoter and the 2.5kb EF1α promoter mediated the strongest expression; unfortunately, these were two of the largest promoters tested [35]. Even so, the NSE promoter has been used in AAV vectors for efficient and long-term expression of GFP in neurons in vivo [19].…”

Section: Discussionmentioning

confidence: 99%

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

Royo¹,

Vandenberghe²,

Ma³

et al. 2008

Brain Research

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Most current methods of gene delivery for primary cultured hippocampal neurons are limited by toxicity, transient expression, the use of immature neurons, and/or low efficiency. We performed a direct comparison of seven serotypes of adeno-associated virus (AAV) vectors for genetic manipulation of primary cultured neurons in vitro. Serotypes 1, 2, 7, 8 and 9 mediated highly efficient, nontoxic, stable long-term gene expression in cultured cortical and hippocampal neurons aged 0-4 weeks in vitro; serotypes 5 and 6 were associated with toxicity at high doses. AAV1 transduced over 90% of all cells with approximately 80% of the transduced cells being neurons. The method was readily adapted to a high-throughput format to demonstrate neurotrophin-mediated neuroprotection from glutamate toxicity in cultured neurons at 2 weeks in vitro. These vectors should prove highly useful for efficient overexpression or downregulation of genes in primary neuronal cultures at any developmental stage.

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Section: Discussionmentioning

confidence: 99%

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

Royo¹,

Vandenberghe²,

Ma³

et al. 2008

Brain Research

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“…[11][12][13][14][15][16] Another strategy is to combine both components into one self-regulating virus so that target cells only need to be infected by one virus to allow regulatable expression. 7,[17][18][19][20][21][22][23][24][25][26] Recombinant adeno-associated viral (rAAV) vectors have several properties that make them one of the most promising vehicles for gene delivery to the CNS. 27 These vectors have been reported to infect and transduce both dividing and nondividing cells, including neurons, with minimal cellular toxicity or host immune response.…”

Section: Introductionmentioning

confidence: 99%

“…27 In the CNS, significant long-term transduction of neurons by rAAV has been observed for up to 1 year. 25,28 Until recently, however, the use of rAAV was hampered by the lack of methods for producing high titer vectors that were not contaminated with adenovirus or herpes simplex virus. With the development of helper virus-free packaging systems 29,30 and a new purification protocol, 31,32 the production of large-scale high titer rAAV free of contaminating helper virus has become routine.…”

Section: Introductionmentioning

confidence: 99%

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

et al. 2004

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Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

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“…29,32 However, when using AAV carrying the GFAP promoter, most transduced brain cells appeared neuronal. 33,34 As the AAV ITR sequences could function directly as a promoter for expression of a reporter gene, 35 overriding the GFAP promoter by AAV ITRs could possibly lead to neuronal expression. 36 This notion is not explicitly supported by the observation from the current study using an expression cassette in which the GFAP promoter has been used together with the AAV ITRs.…”

Section: Astrocytic Specificitymentioning

confidence: 99%

Astrocytic expression of transgene in the rat brain mediated by baculovirus vectors containing an astrocyte-specific promoter

Wang

2006

Gene Ther

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Therapeutic gene expression in glial cells has been tested for the treatment of neurological diseases in animal models. Many of such studies used the promoter of the glial fibrillary acidic protein (GFAP) to restrict gene expression to astrocytes. We have investigated in the current study whether it is possible to improve the transcriptional activity of the cellular promoter, while maintaining its cell-type specificity. We constructed an expression cassette containing a hybrid cytomegalovirus (CMV) enhancer/GFAP promoter and placed it into baculovirus vectors, a type of viral vectors capable of transducing astrocytes. In another vector design, we used inverted terminal repeats (ITRs) from adeno-associated virus (AAV) to flank the expression cassette. The recombinant baculoviruses with the hybrid promoter improved gene expression levels over two orders of magnitude in glial cell lines and by 10-fold in the rat brain when compared to the baculoviruses with the GFAP promoter alone. The expression was further improved by ITR flanking, reaching levels higher than that mediated by the baculovirus vectors with the CMV immediate-early enhancer/ promoter (CMV promoter). Using these recombinant baculoviruses, we observed extended in vivo transgene expression in the rat brain at 90 days postinjection, by which time the gene expression from baculovirus vectors with the GFAP or CMV promoter had already become undetectable. The astrocyte specificity of the GFAP promoter was preserved in the engineered expression cassette with the CMV enhancer and the AAV ITRs, as demonstrated by immunohistological analysis of brain samples and an axonal retrograde transport assay. Taken together, our findings suggest that these baculovirus vectors may serve as useful tools for astrocyte-specific gene expression in the brain.

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Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

Cited by 167 publications

References 56 publications

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

Specific AAV serotypes stably transduce primary hippocampal and cortical cultures with high efficiency and low toxicity

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

Astrocytic expression of transgene in the rat brain mediated by baculovirus vectors containing an astrocyte-specific promoter

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