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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

Schematic representation of key constructs in this study encoding the FM derived from the Sindbis virus glycoprotein, lentiviral transfer vector FUGW, membrane-bound human/mouse chimeric antibody against CD20 (αCD20), and accessory proteins for surface expression of antibody (Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader peptide of Sindbis virus glycoprotein; E1: E1 protein of the Sindbis virus glycoprotein for mediating fusion; E2: E2 protein of the Sindbis virus glycoprotein for binding to viral receptor; HA tag: 10-amino acid epitope hemagglutinin sequences (MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green fluorescent protein; WPRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3 region with deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α: human elongation factor 1α promoter; αCD20κ and αCD20λ: light chain and heavy chain of human/mouse chimeric antibody against CD20; TM: human antibody transmembrane domain; Igα and Igβ: human antibody accessory proteins Igα and Igβ. For the FM constructs (SINmu, SGN, SGM and AGM), the amino acid sequences at E1 226 region are shown. The sequence starts at amino acid 225 and ends with amino acid 234 of the wild-type E1 protein. Specific amino acids involved in generating new FMs are shown underlined in bold.
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Figure 1: Schematic representation of key constructs in this study encoding the FM derived from the Sindbis virus glycoprotein, lentiviral transfer vector FUGW, membrane-bound human/mouse chimeric antibody against CD20 (αCD20), and accessory proteins for surface expression of antibody (Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader peptide of Sindbis virus glycoprotein; E1: E1 protein of the Sindbis virus glycoprotein for mediating fusion; E2: E2 protein of the Sindbis virus glycoprotein for binding to viral receptor; HA tag: 10-amino acid epitope hemagglutinin sequences (MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green fluorescent protein; WPRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3 region with deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α: human elongation factor 1α promoter; αCD20κ and αCD20λ: light chain and heavy chain of human/mouse chimeric antibody against CD20; TM: human antibody transmembrane domain; Igα and Igβ: human antibody accessory proteins Igα and Igβ. For the FM constructs (SINmu, SGN, SGM and AGM), the amino acid sequences at E1 226 region are shown. The sequence starts at amino acid 225 and ends with amino acid 234 of the wild-type E1 protein. Specific amino acids involved in generating new FMs are shown underlined in bold.

Mentions: We previously demonstrated that cell-specific targeted transduction can be achieved by lentiviral vectors enveloped with αCD20 and a FM [21]. The FM used in that study was a mutant viral glycoprotein derived from the Sindbis virus. The Sindbis envelope glycoprotein consists of two domains; E1 is responsible for mediating the fusion between the virus and target cell and E2 is responsible for directing the binding of the virus to the cellular antigen on the target cell surface [28]. Chen and co-workers reported a fusion-competent, but binding-deficient form of the Sindbis envelope glycoprotein which was generated by inserting a ZZ binding domain into the E2 region of the envelope protein [13,14]. We further modified this binding-deficient envelope protein by replacing the ZZ binding domain with a HA tag; the resulting protein was designated SINmu [21]. In addition, Kielian and co-workers have previously shown that a mutation on E1, at region 266, of the Sindbis virus envelope protein results in viruses that were less dependent on cholesterol for transduction [23]. Their work identified three distinct mutants with enhanced efficiency to transduce cholesterol-depleted cells [23]. We reasoned that incorporation of these mutations into SINmu might endow new FMs with enhanced efficiency to induce fusion of antibody-displaying lentiviral vectors to accomplish targeted transduction. To test this hypothesis, we generated three FMs designated as SGN, SGM and AGM (Fig. 1). These FMs differed from the parental FM (SINmu) by three amino acids at region 226 of the E1 protein.


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

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

Schematic representation of key constructs in this study encoding the FM derived from the Sindbis virus glycoprotein, lentiviral transfer vector FUGW, membrane-bound human/mouse chimeric antibody against CD20 (αCD20), and accessory proteins for surface expression of antibody (Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader peptide of Sindbis virus glycoprotein; E1: E1 protein of the Sindbis virus glycoprotein for mediating fusion; E2: E2 protein of the Sindbis virus glycoprotein for binding to viral receptor; HA tag: 10-amino acid epitope hemagglutinin sequences (MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green fluorescent protein; WPRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3 region with deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α: human elongation factor 1α promoter; αCD20κ and αCD20λ: light chain and heavy chain of human/mouse chimeric antibody against CD20; TM: human antibody transmembrane domain; Igα and Igβ: human antibody accessory proteins Igα and Igβ. For the FM constructs (SINmu, SGN, SGM and AGM), the amino acid sequences at E1 226 region are shown. The sequence starts at amino acid 225 and ends with amino acid 234 of the wild-type E1 protein. Specific amino acids involved in generating new FMs are shown underlined in bold.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Schematic representation of key constructs in this study encoding the FM derived from the Sindbis virus glycoprotein, lentiviral transfer vector FUGW, membrane-bound human/mouse chimeric antibody against CD20 (αCD20), and accessory proteins for surface expression of antibody (Igαβ). CMV: human cytomegalovirus immediate-early gene promoter; E3: leader peptide of Sindbis virus glycoprotein; E1: E1 protein of the Sindbis virus glycoprotein for mediating fusion; E2: E2 protein of the Sindbis virus glycoprotein for binding to viral receptor; HA tag: 10-amino acid epitope hemagglutinin sequences (MYPYDVPDYA); Ubi: human ubiquitin-C promoter; GFP: green fluorescent protein; WPRE: woodchuck regulatory element; LTR: long-terminal repeat; ΔU3: U3 region with deletion to disable the transcriptional activity of integrated viral LTR promoter; EF1α: human elongation factor 1α promoter; αCD20κ and αCD20λ: light chain and heavy chain of human/mouse chimeric antibody against CD20; TM: human antibody transmembrane domain; Igα and Igβ: human antibody accessory proteins Igα and Igβ. For the FM constructs (SINmu, SGN, SGM and AGM), the amino acid sequences at E1 226 region are shown. The sequence starts at amino acid 225 and ends with amino acid 234 of the wild-type E1 protein. Specific amino acids involved in generating new FMs are shown underlined in bold.
Mentions: We previously demonstrated that cell-specific targeted transduction can be achieved by lentiviral vectors enveloped with αCD20 and a FM [21]. The FM used in that study was a mutant viral glycoprotein derived from the Sindbis virus. The Sindbis envelope glycoprotein consists of two domains; E1 is responsible for mediating the fusion between the virus and target cell and E2 is responsible for directing the binding of the virus to the cellular antigen on the target cell surface [28]. Chen and co-workers reported a fusion-competent, but binding-deficient form of the Sindbis envelope glycoprotein which was generated by inserting a ZZ binding domain into the E2 region of the envelope protein [13,14]. We further modified this binding-deficient envelope protein by replacing the ZZ binding domain with a HA tag; the resulting protein was designated SINmu [21]. In addition, Kielian and co-workers have previously shown that a mutation on E1, at region 266, of the Sindbis virus envelope protein results in viruses that were less dependent on cholesterol for transduction [23]. Their work identified three distinct mutants with enhanced efficiency to transduce cholesterol-depleted cells [23]. We reasoned that incorporation of these mutations into SINmu might endow new FMs with enhanced efficiency to induce fusion of antibody-displaying lentiviral vectors to accomplish targeted transduction. To test this hypothesis, we generated three FMs designated as SGN, SGM and AGM (Fig. 1). These FMs differed from the parental FM (SINmu) by three amino acids at region 226 of the E1 protein.

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