Engineering adeno-associated viruses for clinical gene therapy

Kotterman, Melissa A.; Schaffer, David V.

doi:10.1038/nrg3742

Cited by 687 publications

(568 citation statements)

References 75 publications

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“…Gene therapy often aims at reintroducing the wild‐type sequence of a deficient gene in the affected organs, tissues or cell populations. Therefore, the target sequence needs to be driven by effective tissue‐specific promoters, and packed into appropriate vectors such as viruses for their efficient delivery to and into the target cells (Kotterman and Schaffer, 2014). …”

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confidence: 99%

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Rudeck

Etard

Khan

et al. 2016

Genesis

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Summary: Gene therapeutic approaches to cure genetic diseases require tools to express the rescuing gene exclusively within the affected tissues. Viruses are often chosen as gene transfer vehicles but they have limited capacity for genetic information to be carried and transduced. In addition, to avoid off‐target effects the therapeutic gene should be driven by a tissue‐specific promoter in order to ensure expression in the target organs, tissues, or cell populations. The larger the promoter, the less space will be left for the respective gene. Thus, there is a need for small but tissue‐specific promoters. Here, we describe a compact unc45b promoter fragment of 195 bp that retains the ability to drive gene expression exclusively in skeletal and cardiac muscle in zebrafish and mouse. Remarkably, the described unc45b promoter fragment not only drives muscle‐specific expression but presents heat‐shock inducibility, allowing a temporal and spatial quantity control of (trans)gene expression. Here, we demonstrate that the transgenic expression of the smyd1b gene driven by the unc45b promoter fragment is able to rescue the embryonically lethal heart and skeletal muscle defects in smyd1b‐deficient flatline mutant zebrafish. Our findings demonstrate that the described muscle‐specific unc45b promoter fragment might be a valuable tool for the development of genetic therapies in patients suffering from myopathies. genesis 54:431–438, 2016. © 2016 The Authors. Genesis Published by Wiley Periodicals, Inc.

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confidence: 99%

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Rudeck

Etard

Khan

et al. 2016

Genesis

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“…Attempts to tackle this challenge either focused on restricting transgene expression through tissue-specific promoters and/or post-translational targeting strategies 3 leaving the problem of off-target delivery untouched or on altering AAVs' cell entry features by modifying the viral capsid. The latter approaches, referred here as cell entry targeting, either use adaptors possibly shielding the viral capsid from natural receptor binding and bridging between viral capsid and the chosen target receptor (non-genetic approaches) or permanently change the capsid by genetic engineering 4,5 . As of yet, genetic engineering employing DNA shuffling of capsid-encoding genes from different serotypes and directed evolution as means to select mosaic AAV capsids with novel features has demonstrated its promise as powerful strategy to improve, but not to restrict, gene delivery to a tissue of choice [5][6][7] .…”

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confidence: 99%

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

et al. 2015

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We describe receptor-targeted adeno-associated viral (AAV) vectors that allow genetic modification of rare cell types ex vivo and in vivo while showing no detectable off-targeting. Displaying designed ankyrin repeat proteins (DARPins) on the viral capsid and carefully depleting DARPin-deficient particles, AAV vectors were made specific for Her2/neu, EpCAM or CD4. A single intravenous administration of vector targeted to the tumour antigen Her2/neu was sufficient to track 75% of all tumour sites and to extend survival longer than the cytostatic antibody Herceptin. CD4-targeted AAVs hit human CD4-positive cells present in spleen of a humanized mouse model, while CD8-positive cells as well as liver or other off-target organs remained unmodified. Mimicking conditions of circulating tumour cells, EpCAM-AAV detected single tumour cells in human blood opening the avenue for tumour stem cell tracking. Thus, the approach developed here delivers genes to target cell types of choice with antibody-like specificity.

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“…Therefore, in order to elucidate the function of tumor cell derived-VEGF in breast cancer biology, VEGF165 was selected as the target gene in the present study, and the VEGF165-EGFP fusion gene expression vector was constructed. As a newly developed technology, recombinant expression through viral vectors have been used in numerous laboratory experiments and clinical trials (27)(28)(29). There are mainly five types of viral vectors that are commonly used for recombinant expression, namely adenovirus, adeno-associated virus, herpes simplex virus, retrovirus and lentivirus (14).…”

Section: Discussionmentioning

confidence: 99%

Construction and expression of a lentivirus expression vector carrying the VEGF165-EGFP fusion gene in breast cancer MCF-7 cells

Luo¹,

Huang²,

Hou³

et al. 2017

Oncology Letters

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Abstract. Vascular endothelial growth factor (VEGF)165 is one of the most abundant and potent angiogenic factors in both physiological and pathological conditions. However, the function and mechanism of VEGF165 in tumors and their environment remain to be elucidated. In the present study, a lentivirus vector (LV) that contained the VEGF165-enhanced green fluorescent protein (EGFP) fusion gene was constructed and transfected into the human breast cancer cell line MCF-7. Following transfection, the expression of VEGF165 in MCF-7 cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Further cellular localization of VEGF165 was observed through fluorescence microscopy. The titer of the recombinant lentivirus was 5.44x10 7 TU/ml in the LV-VEGF165-EGFP group and 5.00x10 8 TU/ml in the LV-EGFP negative control group. RT-qPCR and western blotting demonstrated that the expression of VEGF165 was significantly increased in the LV-VEGF165-EGFP group compared with the control group. The present study lays the foundation for in vitro and in vivo studies on tumor cell derived-VEGF165. Furthermore, the present fusion gene expression vector may provide a potential approach for gene therapy treatment of cancer and other diseases that require regulation of angiogenesis.

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Engineering adeno-associated viruses for clinical gene therapy

Cited by 687 publications

References 75 publications

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

A compact unc45b‐promoter drives muscle‐specific expression in zebrafish and mouse

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

Construction and expression of a lentivirus expression vector carrying the VEGF165-EGFP fusion gene in breast cancer MCF-7 cells

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