Limits...
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

Targeted transduction of CD20-positive human primary B cells. (a) Fresh unfractionated human PBMCs (1 × 106) were spin-transduced twice with indicated vectors (FUGW/αCD20+FM, 2.5 × 106 TU, or FUGW/VSVG, 25 × 106 TU). 48 hours later, cells were collected and analyzed by FACS (b) iMFI on fresh unfractionated human PBMCs transduced by the indicated viral vectors (FUGW/αCD20+FM, and FUGW/VSVG).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2698826&req=5

Figure 6: Targeted transduction of CD20-positive human primary B cells. (a) Fresh unfractionated human PBMCs (1 × 106) were spin-transduced twice with indicated vectors (FUGW/αCD20+FM, 2.5 × 106 TU, or FUGW/VSVG, 25 × 106 TU). 48 hours later, cells were collected and analyzed by FACS (b) iMFI on fresh unfractionated human PBMCs transduced by the indicated viral vectors (FUGW/αCD20+FM, and FUGW/VSVG).

Mentions: One of the advantages of using targeted vectors is their potential ability to transduce specific cell types in a mixed population without the need to isolate the target cells. We tested whether our targeted vectors with engineered FMs can specifically transduce primary B cells in an unfractionated primary cell population. One million of fresh, unfractionated human peripheral blood mononuclear cells (PBMC) were transduced twice with concentrated FUGW/αCD20+FM (2.5 × 106 TU), or FUGW/VSVG (25 × 106 TU). The cells were analyzed by flow cytometry 2 days post-transduction. As shown in Fig. 6(a), transduction of bulk PBMC populations with targeted vectors resulted in specific modification of CD20-positive PBMC, whereas no GFP signals were detected in CD20-negative cells. In the control experiment where FUGW/VSVG was used for transducing bulk PBMC populations, GFP signals were detected in both CD20-positive and CD20-negative cells and a higher iMFI signal was detected in the CD20-negative PBMC as compared to CD20-positive PBMC. Quantification of total expression intensity indicated that vector FUGW/αCD20+AGM attributed the highest iMFI in the CD20-positive PBMC, followed by FUGW/αCD20+SGM, FUGW/αCD20+SIN, and FUGW/αCD20+SGN (Fig. 6b). This was in good agreement with the specific transduction against 293T/CD20 cells (Fig. 4b). The stable integration of the GFP gene was confirmed by the genomic PCR analysis (data not shown).


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

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

Targeted transduction of CD20-positive human primary B cells. (a) Fresh unfractionated human PBMCs (1 × 106) were spin-transduced twice with indicated vectors (FUGW/αCD20+FM, 2.5 × 106 TU, or FUGW/VSVG, 25 × 106 TU). 48 hours later, cells were collected and analyzed by FACS (b) iMFI on fresh unfractionated human PBMCs transduced by the indicated viral vectors (FUGW/αCD20+FM, and FUGW/VSVG).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Targeted transduction of CD20-positive human primary B cells. (a) Fresh unfractionated human PBMCs (1 × 106) were spin-transduced twice with indicated vectors (FUGW/αCD20+FM, 2.5 × 106 TU, or FUGW/VSVG, 25 × 106 TU). 48 hours later, cells were collected and analyzed by FACS (b) iMFI on fresh unfractionated human PBMCs transduced by the indicated viral vectors (FUGW/αCD20+FM, and FUGW/VSVG).
Mentions: One of the advantages of using targeted vectors is their potential ability to transduce specific cell types in a mixed population without the need to isolate the target cells. We tested whether our targeted vectors with engineered FMs can specifically transduce primary B cells in an unfractionated primary cell population. One million of fresh, unfractionated human peripheral blood mononuclear cells (PBMC) were transduced twice with concentrated FUGW/αCD20+FM (2.5 × 106 TU), or FUGW/VSVG (25 × 106 TU). The cells were analyzed by flow cytometry 2 days post-transduction. As shown in Fig. 6(a), transduction of bulk PBMC populations with targeted vectors resulted in specific modification of CD20-positive PBMC, whereas no GFP signals were detected in CD20-negative cells. In the control experiment where FUGW/VSVG was used for transducing bulk PBMC populations, GFP signals were detected in both CD20-positive and CD20-negative cells and a higher iMFI signal was detected in the CD20-negative PBMC as compared to CD20-positive PBMC. Quantification of total expression intensity indicated that vector FUGW/αCD20+AGM attributed the highest iMFI in the CD20-positive PBMC, followed by FUGW/αCD20+SGM, FUGW/αCD20+SIN, and FUGW/αCD20+SGN (Fig. 6b). This was in good agreement with the specific transduction against 293T/CD20 cells (Fig. 4b). The stable integration of the GFP gene was confirmed by the genomic PCR analysis (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