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Engineering fusogenic molecules to achieve targeted transduction of enveloped lentiviral vectors.

Lei Y, Joo KI, Wang P - J Biol Eng (2009)

Bottom Line: Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM.Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency.Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089, USA.

ABSTRACT

Background: Lentiviral vectors with broad tropism are one of the most promising gene delivery systems capable of efficiently delivering genes of interest into both dividing and non-dividing cells while maintaining long-term transgene expression. However, there are needs for developing lentiviral vectors with the capability to deliver genes to specific cell types, thus reducing the "off-target" effect of gene therapy. In the present study, we investigated the possibility of engineering the fusion-active domain of a fusogenic molecule (FM) with the aim to improve targeted transduction of lentiviral vectors co-displaying an anti-CD20 antibody (alphaCD20) and a FM.

Results: Specific mutations were introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to generate several mutant FMs. Lentiviral vectors incorporated with alphaCD20 and one of the engineered FMs were successfully produced and demonstrated to be able to preferentially deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM. Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency.

Conclusion: The fusion domain of Sindbis virus glycoprotein is amenable for engineering and the engineered proteins provide elevated capacity to mediate lentiviral vectors for targeted transduction. Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

No MeSH data available.


Related in: MedlinePlus

Co-expression of αCD20 and a FM on the viral vector surface. (a, left) Schematic diagrams to illustrate the interaction between αCD20 on the vector surface and CD20 on the cell surface, and the immuno-fluorescent staining scheme. (a, right) Acquired confocal images of labeled viral vector binding to cells. (b) FACS analysis of 293T or 293T/CD20 cells incubated with FUGW/αCD20+FM or FUGW/Ab+FM. The binding of virus to 293T/CD20 cells was detected by FACS staining with antibody against the FM. Solid line, analysis on cells incubated with indicated viral vectors; shaded area (control), analysis on cells without incubation with vectors.
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Figure 3: Co-expression of αCD20 and a FM on the viral vector surface. (a, left) Schematic diagrams to illustrate the interaction between αCD20 on the vector surface and CD20 on the cell surface, and the immuno-fluorescent staining scheme. (a, right) Acquired confocal images of labeled viral vector binding to cells. (b) FACS analysis of 293T or 293T/CD20 cells incubated with FUGW/αCD20+FM or FUGW/Ab+FM. The binding of virus to 293T/CD20 cells was detected by FACS staining with antibody against the FM. Solid line, analysis on cells incubated with indicated viral vectors; shaded area (control), analysis on cells without incubation with vectors.

Mentions: In order for our targeting system to work, lentiviral vectors must be enveloped with both αCD20 for binding, and a FM for fusion. We employed a confocal imaging method to analyze the co-incorporation of the αCD20 and the FM. The target cells were incubated with viral vectors at 4°C for 1 hour, followed by sequential staining of the FM (blue color) and the αCD20 (red color). To label the core of the vectors, we adapted a previously reported method to synthesize viral vectors encapsulated with a protein (GFP-Vpr) consisting of GFP fused with viral protein R (Vpr) [32]. It has been shown that the provision of GFP-Vpr in trans during transfection can allow the fluorescent protein to be incorporated into the core of HIV-based lentiviral vectors through the interaction between Vpr and the P6 region of the HIV gag protein [32]. We harvested GFP-Vpr-tagged lentiviral vectors bearing αCD20 and SINmu and incubated them with 293T/CD20 cells at 4°C for 1 hour. After extensive washing, the treated cells were subjected to immuno-fluorescence staining and imaging. GFP-labeled signals were detected on the surface of 293T/CD20 cells and their signals were co-localized with signals for both αCD20 and SINmu (Fig. 3a, top), while no fluorescence signals were obtained for 293T cells lacking the expression of CD20 (Fig. 3a, bottom). The co-localization of GFP, αCD20 and SINmu suggested that the cells can produce lentiviral vectors displaying both αCD20 and a FM in a single virion. Similar results were also observed for vectors bearing the other type of FMs (SGN, SGM, or AGM) (data not shown).


Engineering fusogenic molecules to achieve targeted transduction of enveloped lentiviral vectors.

Lei Y, Joo KI, Wang P - J Biol Eng (2009)

Co-expression of αCD20 and a FM on the viral vector surface. (a, left) Schematic diagrams to illustrate the interaction between αCD20 on the vector surface and CD20 on the cell surface, and the immuno-fluorescent staining scheme. (a, right) Acquired confocal images of labeled viral vector binding to cells. (b) FACS analysis of 293T or 293T/CD20 cells incubated with FUGW/αCD20+FM or FUGW/Ab+FM. The binding of virus to 293T/CD20 cells was detected by FACS staining with antibody against the FM. Solid line, analysis on cells incubated with indicated viral vectors; shaded area (control), analysis on cells without incubation with vectors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2698826&req=5

Figure 3: Co-expression of αCD20 and a FM on the viral vector surface. (a, left) Schematic diagrams to illustrate the interaction between αCD20 on the vector surface and CD20 on the cell surface, and the immuno-fluorescent staining scheme. (a, right) Acquired confocal images of labeled viral vector binding to cells. (b) FACS analysis of 293T or 293T/CD20 cells incubated with FUGW/αCD20+FM or FUGW/Ab+FM. The binding of virus to 293T/CD20 cells was detected by FACS staining with antibody against the FM. Solid line, analysis on cells incubated with indicated viral vectors; shaded area (control), analysis on cells without incubation with vectors.
Mentions: In order for our targeting system to work, lentiviral vectors must be enveloped with both αCD20 for binding, and a FM for fusion. We employed a confocal imaging method to analyze the co-incorporation of the αCD20 and the FM. The target cells were incubated with viral vectors at 4°C for 1 hour, followed by sequential staining of the FM (blue color) and the αCD20 (red color). To label the core of the vectors, we adapted a previously reported method to synthesize viral vectors encapsulated with a protein (GFP-Vpr) consisting of GFP fused with viral protein R (Vpr) [32]. It has been shown that the provision of GFP-Vpr in trans during transfection can allow the fluorescent protein to be incorporated into the core of HIV-based lentiviral vectors through the interaction between Vpr and the P6 region of the HIV gag protein [32]. We harvested GFP-Vpr-tagged lentiviral vectors bearing αCD20 and SINmu and incubated them with 293T/CD20 cells at 4°C for 1 hour. After extensive washing, the treated cells were subjected to immuno-fluorescence staining and imaging. GFP-labeled signals were detected on the surface of 293T/CD20 cells and their signals were co-localized with signals for both αCD20 and SINmu (Fig. 3a, top), while no fluorescence signals were obtained for 293T cells lacking the expression of CD20 (Fig. 3a, bottom). The co-localization of GFP, αCD20 and SINmu suggested that the cells can produce lentiviral vectors displaying both αCD20 and a FM in a single virion. Similar results were also observed for vectors bearing the other type of FMs (SGN, SGM, or AGM) (data not shown).

Bottom Line: Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM.Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency.Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089, USA.

ABSTRACT

Background: Lentiviral vectors with broad tropism are one of the most promising gene delivery systems capable of efficiently delivering genes of interest into both dividing and non-dividing cells while maintaining long-term transgene expression. However, there are needs for developing lentiviral vectors with the capability to deliver genes to specific cell types, thus reducing the "off-target" effect of gene therapy. In the present study, we investigated the possibility of engineering the fusion-active domain of a fusogenic molecule (FM) with the aim to improve targeted transduction of lentiviral vectors co-displaying an anti-CD20 antibody (alphaCD20) and a FM.

Results: Specific mutations were introduced into the fusion domain of a binding-deficient Sindbis virus glycoprotein to generate several mutant FMs. Lentiviral vectors incorporated with alphaCD20 and one of the engineered FMs were successfully produced and demonstrated to be able to preferentially deliver genes to CD-20-expressing cells. Lentiviral vectors bearing engineered FMs exhibited 8 to 17-fold enhanced transduction towards target cells as compared to the parental FM. Different levels of enhancement were observed for the different engineered FMs. A pH-dependent study of vector transduction showed that the broader pH range of the engineered FM is a possible mechanism for the resulted increase in transduction efficiency.

Conclusion: The fusion domain of Sindbis virus glycoprotein is amenable for engineering and the engineered proteins provide elevated capacity to mediate lentiviral vectors for targeted transduction. Our data suggests that application of such an engineering strategy can optimize the two-molecular targeting method of lentiviral vectors for gene delivery to predetermined cells.

No MeSH data available.


Related in: MedlinePlus