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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

Transduction of engineered lentiviral vectors bearing both an antibody and FM to cell lines. 293T and 293T/CD20 cells (2 × 105) were transduced with 2 ml of fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). Transduction of 293T cells was included as control. (a) iMFI on 293T and 293T/CD20 cells transduced by the fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). (b) Titers of fresh viral vectors (FUGW/αCD20+FM) on 293T and 293T/CD20 cells. (c) p24 amount of fresh viral vectors (FUGW/αCD20+FM, FUGW/Ab+FM, and FUGW/VSVG)
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Figure 4: Transduction of engineered lentiviral vectors bearing both an antibody and FM to cell lines. 293T and 293T/CD20 cells (2 × 105) were transduced with 2 ml of fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). Transduction of 293T cells was included as control. (a) iMFI on 293T and 293T/CD20 cells transduced by the fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). (b) Titers of fresh viral vectors (FUGW/αCD20+FM) on 293T and 293T/CD20 cells. (c) p24 amount of fresh viral vectors (FUGW/αCD20+FM, FUGW/Ab+FM, and FUGW/VSVG)

Mentions: We then investigated how lentiviral vectors bearing different FMs would transduce cells by using 293T/CD20 as the target cell line, and the 293T parental cell line as the negative control. Six days post-transduction, cells were analyzed by flow cytometry. To quantify the difference of our targeted transduction system, we utilized a metric that incorporates both the efficiency and magnitude of the GFP signals detected from the transduced cells [33]. The transduction magnitude was obtained by the mean fluorescence intensity (MFI) of the transduced cells. By multiplying the MFI by transduction efficiency, we derived a metric termed integrated MFI (iMFI) that reflects the total intensity of the GFP signals from the virus transduced cells. As indicated in Fig. 4(a), FUGW/αCD20+AGM displayed the highest iMFI in the target cells (293T/CD20), followed by FUGW/αCD20+SGM, FUGW/αCD20+SGN, and FUGW/αCD20+SINmu. When the same set of viral vectors were used to transduce the non-target cells (293T), much lower iMFI signals were detected. The specific transduction titers of these viral vectors against 293T and 293T/CD20 cells were measured in Fig. 4(b); 8-17-fold increase of preferential transduction of CD20-expressing cells was achieved, depending on which FM was used (Fig. 4b). To confirm that the specific transduction was mediated by the incorporated antibody on the vector surface, we made control vectors bearing a FM and Ab. Spin-transduction of these vectors to 293T/CD20 and 293T cells showed low iMFI signals (Fig. 4a). In addition, to eliminate the possibility that the difference was due to a variance in viral production from the producing cells, we performed an enzyme linked immunosorbent assay (ELISA) to detect the p24 levels in the viral supernatants. As indicated in Fig. 4(c), the p24 levels between the different lentiviral vectors were in the similar range. When comparing the ability of various FMs to mediate transduction, the FM with higher efficiency for targeted transduction would always result in higher background transduction.


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

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

Transduction of engineered lentiviral vectors bearing both an antibody and FM to cell lines. 293T and 293T/CD20 cells (2 × 105) were transduced with 2 ml of fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). Transduction of 293T cells was included as control. (a) iMFI on 293T and 293T/CD20 cells transduced by the fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). (b) Titers of fresh viral vectors (FUGW/αCD20+FM) on 293T and 293T/CD20 cells. (c) p24 amount of fresh viral vectors (FUGW/αCD20+FM, FUGW/Ab+FM, and FUGW/VSVG)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Transduction of engineered lentiviral vectors bearing both an antibody and FM to cell lines. 293T and 293T/CD20 cells (2 × 105) were transduced with 2 ml of fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). Transduction of 293T cells was included as control. (a) iMFI on 293T and 293T/CD20 cells transduced by the fresh viral vectors (FUGW/αCD20+FM, or FUGW/Ab+FM). (b) Titers of fresh viral vectors (FUGW/αCD20+FM) on 293T and 293T/CD20 cells. (c) p24 amount of fresh viral vectors (FUGW/αCD20+FM, FUGW/Ab+FM, and FUGW/VSVG)
Mentions: We then investigated how lentiviral vectors bearing different FMs would transduce cells by using 293T/CD20 as the target cell line, and the 293T parental cell line as the negative control. Six days post-transduction, cells were analyzed by flow cytometry. To quantify the difference of our targeted transduction system, we utilized a metric that incorporates both the efficiency and magnitude of the GFP signals detected from the transduced cells [33]. The transduction magnitude was obtained by the mean fluorescence intensity (MFI) of the transduced cells. By multiplying the MFI by transduction efficiency, we derived a metric termed integrated MFI (iMFI) that reflects the total intensity of the GFP signals from the virus transduced cells. As indicated in Fig. 4(a), FUGW/αCD20+AGM displayed the highest iMFI in the target cells (293T/CD20), followed by FUGW/αCD20+SGM, FUGW/αCD20+SGN, and FUGW/αCD20+SINmu. When the same set of viral vectors were used to transduce the non-target cells (293T), much lower iMFI signals were detected. The specific transduction titers of these viral vectors against 293T and 293T/CD20 cells were measured in Fig. 4(b); 8-17-fold increase of preferential transduction of CD20-expressing cells was achieved, depending on which FM was used (Fig. 4b). To confirm that the specific transduction was mediated by the incorporated antibody on the vector surface, we made control vectors bearing a FM and Ab. Spin-transduction of these vectors to 293T/CD20 and 293T cells showed low iMFI signals (Fig. 4a). In addition, to eliminate the possibility that the difference was due to a variance in viral production from the producing cells, we performed an enzyme linked immunosorbent assay (ELISA) to detect the p24 levels in the viral supernatants. As indicated in Fig. 4(c), the p24 levels between the different lentiviral vectors were in the similar range. When comparing the ability of various FMs to mediate transduction, the FM with higher efficiency for targeted transduction would always result in higher background transduction.

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