Limits...
Development of dual-activity vectors by co-envelopment of adenovirus and SiRNA in artificial lipid bilayers.

Yilmazer A, Tian B, Kostarelos K - PLoS ONE (2014)

Bottom Line: We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid.Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency.Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine Laboratory, Faculty of Life Sciences, University College London, London, United Kingdom.

ABSTRACT
Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.

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The biological activity of co-enveloped Ad and siRNA in DC-Chol:DOPE.The naked Ad, DC-Chol:DOPE enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. (a) Luciferase assay was performed in order to measure the percentage of luciferase expression. (b) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus Ad.
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pone-0114985-g004: The biological activity of co-enveloped Ad and siRNA in DC-Chol:DOPE.The naked Ad, DC-Chol:DOPE enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. (a) Luciferase assay was performed in order to measure the percentage of luciferase expression. (b) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus Ad.

Mentions: A luciferase expressing human lung carcinoma cell line (A549-luc-C8) was used to study the dual-activity vectors in vitro. This cell line is stably expressing luciferase gene, therefore we chose siLuc to silence luciferase and Ad-β-Gal to deliver β-Gal gene. Cells were incubated with the vectors for 3 h and 24 h after gene silencing was analyzed by luciferase assay. As seen in Fig. 4, percentage of relative luciferase activity was around 40–50% when cells were transfected by DC-Chol:DOPE-Ad+siRNA or DC-Chol:DOPE-siRNA. When the same cell lysates were analyzed for β-Gal activity, DC-Chol:DOPE envelopment showed a lower gene expression profile compared to naked Ad treated cells. Inclusion of siRNA in the dual-activity vector system did not alter the biological activity of enveloped Ad vectors, similar levels of β-Gal expression and luciferase silencing were obtained (Fig. 4). Negative control experiments were performed with a siNeg sequence, and the results indicated that the presence of siRNA in dual-activity vectors did not have any effect on the gene delivery capacity of these vectors (S2 Figure).


Development of dual-activity vectors by co-envelopment of adenovirus and SiRNA in artificial lipid bilayers.

Yilmazer A, Tian B, Kostarelos K - PLoS ONE (2014)

The biological activity of co-enveloped Ad and siRNA in DC-Chol:DOPE.The naked Ad, DC-Chol:DOPE enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. (a) Luciferase assay was performed in order to measure the percentage of luciferase expression. (b) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus Ad.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264847&req=5

pone-0114985-g004: The biological activity of co-enveloped Ad and siRNA in DC-Chol:DOPE.The naked Ad, DC-Chol:DOPE enveloped Ad, co-enveloped Ad-siRNA and enveloped siRNA are the vectors examined in this study. A549-luc-A9 cells were transfected with these vectors and after 24 h, cells were lysed. (a) Luciferase assay was performed in order to measure the percentage of luciferase expression. (b) β-Gal expression was assessed by β-Gal assay. * p<0.05 versus Ad.
Mentions: A luciferase expressing human lung carcinoma cell line (A549-luc-C8) was used to study the dual-activity vectors in vitro. This cell line is stably expressing luciferase gene, therefore we chose siLuc to silence luciferase and Ad-β-Gal to deliver β-Gal gene. Cells were incubated with the vectors for 3 h and 24 h after gene silencing was analyzed by luciferase assay. As seen in Fig. 4, percentage of relative luciferase activity was around 40–50% when cells were transfected by DC-Chol:DOPE-Ad+siRNA or DC-Chol:DOPE-siRNA. When the same cell lysates were analyzed for β-Gal activity, DC-Chol:DOPE envelopment showed a lower gene expression profile compared to naked Ad treated cells. Inclusion of siRNA in the dual-activity vector system did not alter the biological activity of enveloped Ad vectors, similar levels of β-Gal expression and luciferase silencing were obtained (Fig. 4). Negative control experiments were performed with a siNeg sequence, and the results indicated that the presence of siRNA in dual-activity vectors did not have any effect on the gene delivery capacity of these vectors (S2 Figure).

Bottom Line: We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid.Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency.Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.

View Article: PubMed Central - PubMed

Affiliation: Nanomedicine Laboratory, Faculty of Life Sciences, University College London, London, United Kingdom.

ABSTRACT
Gene therapy with human adenovirus type 5 (Ad5) has been extensively explored for the treatment of diseases resistant to traditional therapies. Intravenous administration leads to rapid clearance from blood circulation and high liver accumulation, which restrict the use of Ad-based vectors in clinical gene therapy protocols that involve systemic administration. We have previously proposed that such limitations can be improved by engineering artificial lipid envelopes around Ad and designed a variety of artificial lipid bilayer envelopes around the viral capsid. In this study, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within the same lipid bilayer. Such a dual-activity vector can offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, Ribo green and dot blot assays showed that siRNA and Ad virions can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors.

Show MeSH
Related in: MedlinePlus