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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

Examination of addition of NH4Cl or solubleαCD20 on the targeted transduction results. (a) NH4Cl was added into indicated viral supernatants during transduction for 8 hours, after which, the supernatants were replaced with fresh media. GFP expression was analyzed 3 days post-transduction. (b) Various amount of soluble αCD20 or isotype control were added into indicated viral supernatants during transduction for 8 hours, after which, the supernants were replaced with fresh media.
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Figure 5: Examination of addition of NH4Cl or solubleαCD20 on the targeted transduction results. (a) NH4Cl was added into indicated viral supernatants during transduction for 8 hours, after which, the supernatants were replaced with fresh media. GFP expression was analyzed 3 days post-transduction. (b) Various amount of soluble αCD20 or isotype control were added into indicated viral supernatants during transduction for 8 hours, after which, the supernants were replaced with fresh media.

Mentions: Our transduction experiment clearly showed the important role of FMs in determining the overall vector infectivity and that the newly engineered FMs exhibited improved ability to induce targeted transduction as compared to original FM (SINmu) (Fig. 4). We designed an experiment to investigate the possible underlying mechanism responsible for their differences. Lentiviral vectors, FUGW/αCD20+SINmu, FUGW/αCD20+SGN, FUGW/αCD20+SGM, and FUGW/αCD20+AGM, were incubated with 293T/CD20 cells in the absence or presence of a graded concentration of ammonium chloride (NH4Cl); NH4Cl is known to be able to neutralize the acidic endosomal environment [34]. The changes in transduction were measured by flow cytometry. As shown in Fig. 5(a), the four vectors displaying various FMs behaved differently in response to different concentration of NH4Cl. FUGW/αCD20+SINmu was the most sensitive to the neutralization treatment and transduction efficiency dropped the fastest as a result of the changes in pH environment, followed by FUGW/αCD20+SGN, FUGW/αCD20+SGM, and FUGW/αCD20+AGM. It appeared that the engineered vector that was more resistant to the NH4Cl treatment could transduce target cells with a higher efficiency.


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

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

Examination of addition of NH4Cl or solubleαCD20 on the targeted transduction results. (a) NH4Cl was added into indicated viral supernatants during transduction for 8 hours, after which, the supernatants were replaced with fresh media. GFP expression was analyzed 3 days post-transduction. (b) Various amount of soluble αCD20 or isotype control were added into indicated viral supernatants during transduction for 8 hours, after which, the supernants were replaced with fresh media.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Examination of addition of NH4Cl or solubleαCD20 on the targeted transduction results. (a) NH4Cl was added into indicated viral supernatants during transduction for 8 hours, after which, the supernatants were replaced with fresh media. GFP expression was analyzed 3 days post-transduction. (b) Various amount of soluble αCD20 or isotype control were added into indicated viral supernatants during transduction for 8 hours, after which, the supernants were replaced with fresh media.
Mentions: Our transduction experiment clearly showed the important role of FMs in determining the overall vector infectivity and that the newly engineered FMs exhibited improved ability to induce targeted transduction as compared to original FM (SINmu) (Fig. 4). We designed an experiment to investigate the possible underlying mechanism responsible for their differences. Lentiviral vectors, FUGW/αCD20+SINmu, FUGW/αCD20+SGN, FUGW/αCD20+SGM, and FUGW/αCD20+AGM, were incubated with 293T/CD20 cells in the absence or presence of a graded concentration of ammonium chloride (NH4Cl); NH4Cl is known to be able to neutralize the acidic endosomal environment [34]. The changes in transduction were measured by flow cytometry. As shown in Fig. 5(a), the four vectors displaying various FMs behaved differently in response to different concentration of NH4Cl. FUGW/αCD20+SINmu was the most sensitive to the neutralization treatment and transduction efficiency dropped the fastest as a result of the changes in pH environment, followed by FUGW/αCD20+SGN, FUGW/αCD20+SGM, and FUGW/αCD20+AGM. It appeared that the engineered vector that was more resistant to the NH4Cl treatment could transduce target cells with a higher efficiency.

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