Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes

doi: 10.1016/j.omtm.2019.08.001

Figure Lengend Snippet: Schematic Representation of Wild-Type and Engineered Viral Envelope Glycoproteins (A) Amino-acid sequence alignment of glycoprotein cytoplasmic tail: murine leukemia virus (4070A), endogenous feline retrovirus (RD114), and gibbon ape leukemia virus SEATO strain (GaLV). (B) Amino-acid sequence alignment of the cytoplasmic tail region of the glycoproteins used in LV production and schematic representation of the envelope expression cassette. The black arrows indicate the protease cleavage site. E, ectodomain; M, transmembrane domain; T, cytoplasmic tail after R-peptide cleavage; R, R-peptide; CMV, cytomegalovirus promoter; INT, intron; Sp, spacer; pAn, polyadenylation site.

Article Snippet: Plasmids coding for envelope glycoproteins are as follows: pMD2.G (Addgene #12259, kindly provided by Dr. Didier Trono) codes for VSV-G; pCMV-GaLV-TR codes for a modified glycoprotein of the GaLV SEATO strain and results from the removal of 19 nt prior to the start codon of the GaLV glycoprotein from phGaLV10A1 by inverse PCR; phGaLV10A1 was kindly provided by Dr. Otto Merten (Généthon, Évry, France); pCMV-RD114-TR codes for a modified RD114 glycoprotein, amplified from the pLTR-RD114A plasmid (Addgene #17576, kindly provided by Dr. Jakob Reiser) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-RDpro codes for RDpro glycoprotein, which was chemically synthesized (GeneScript, Piscataway, NJ, USA) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-4070A codes for the amphotropic MLV glycoprotein amplified from pMonoZeo-4070A and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-GaLV-TR pro , pCMV-RD114-TR pro , and pCMV-4070A pro code for the respective modified viral glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by that of the HIV-1 Gag matrix-capsid, SQNYPIVQ, by inverse PCR from the parental plasmids; pCMV-GaLV-TR synt , pCMV-RD114-TR synt , and pCMV-4070A synt code for the respective viral modified glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by synthetic peptide GSGIFLETSL by performing two inverse PCRs from the parental plasmids; pCMV-GaLV-TR ΔR , pCMV-RD114-TR ΔR , and pCMV-4070A ΔR code for the respective truncated glycoproteins in which the R-peptide was deleted from the cytoplasmic tail of the glycoproteins and replaced by a STOP codon by inverse PCR.

Techniques: Sequencing, Virus, Expressing

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes

doi: 10.1016/j.omtm.2019.08.001

Figure Lengend Snippet: Transient LV Production Titers with the Engineered Envelope Glycoproteins (A and B) Transient production titers of LVs pseudotyped with 4070A, 4070A-derived, RD114-TR, RD114-TR-derived, RDpro, GaLV-TR, GaLV-TR-derived, or VSV-G glycoproteins using the (A) HIV-1 wild-type protease or (B) HIV-1 T26S protease. The bars represent the transducing units (TUs), and the circles represent the physical particles (PPs). The titer values presented are the means ± SD of 3 independent experiments (n = 3). Statistical analysis for the comparison of LV titers was performed by using an unpaired Student t test (two-tailed). *p < 0.05; **p < 0.01; ***p < 0.001. The ratios of TUs to PPs are indicated above each set of LV titers.

Article Snippet: Plasmids coding for envelope glycoproteins are as follows: pMD2.G (Addgene #12259, kindly provided by Dr. Didier Trono) codes for VSV-G; pCMV-GaLV-TR codes for a modified glycoprotein of the GaLV SEATO strain and results from the removal of 19 nt prior to the start codon of the GaLV glycoprotein from phGaLV10A1 by inverse PCR; phGaLV10A1 was kindly provided by Dr. Otto Merten (Généthon, Évry, France); pCMV-RD114-TR codes for a modified RD114 glycoprotein, amplified from the pLTR-RD114A plasmid (Addgene #17576, kindly provided by Dr. Jakob Reiser) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-RDpro codes for RDpro glycoprotein, which was chemically synthesized (GeneScript, Piscataway, NJ, USA) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-4070A codes for the amphotropic MLV glycoprotein amplified from pMonoZeo-4070A and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-GaLV-TR pro , pCMV-RD114-TR pro , and pCMV-4070A pro code for the respective modified viral glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by that of the HIV-1 Gag matrix-capsid, SQNYPIVQ, by inverse PCR from the parental plasmids; pCMV-GaLV-TR synt , pCMV-RD114-TR synt , and pCMV-4070A synt code for the respective viral modified glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by synthetic peptide GSGIFLETSL by performing two inverse PCRs from the parental plasmids; pCMV-GaLV-TR ΔR , pCMV-RD114-TR ΔR , and pCMV-4070A ΔR code for the respective truncated glycoproteins in which the R-peptide was deleted from the cytoplasmic tail of the glycoproteins and replaced by a STOP codon by inverse PCR.

Techniques: Derivative Assay, Comparison, Two Tailed Test

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes

doi: 10.1016/j.omtm.2019.08.001

Figure Lengend Snippet: Representative Pictures of HEK293T Cells Transiently Expressing Envelope Glycoproteins 80× bright-field microscopy. Scale bars, 45 μm.

Article Snippet: Plasmids coding for envelope glycoproteins are as follows: pMD2.G (Addgene #12259, kindly provided by Dr. Didier Trono) codes for VSV-G; pCMV-GaLV-TR codes for a modified glycoprotein of the GaLV SEATO strain and results from the removal of 19 nt prior to the start codon of the GaLV glycoprotein from phGaLV10A1 by inverse PCR; phGaLV10A1 was kindly provided by Dr. Otto Merten (Généthon, Évry, France); pCMV-RD114-TR codes for a modified RD114 glycoprotein, amplified from the pLTR-RD114A plasmid (Addgene #17576, kindly provided by Dr. Jakob Reiser) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-RDpro codes for RDpro glycoprotein, which was chemically synthesized (GeneScript, Piscataway, NJ, USA) and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-4070A codes for the amphotropic MLV glycoprotein amplified from pMonoZeo-4070A and cloned into the vector resultant from phGaLV10A1 restriction with EcoRI and KasI enzymes; pCMV-GaLV-TR pro , pCMV-RD114-TR pro , and pCMV-4070A pro code for the respective modified viral glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by that of the HIV-1 Gag matrix-capsid, SQNYPIVQ, by inverse PCR from the parental plasmids; pCMV-GaLV-TR synt , pCMV-RD114-TR synt , and pCMV-4070A synt code for the respective viral modified glycoproteins in which the viral protease cleavage sequence VQALVLTQ of the 4070A glycoprotein cytoplasmic tail was replaced by synthetic peptide GSGIFLETSL by performing two inverse PCRs from the parental plasmids; pCMV-GaLV-TR ΔR , pCMV-RD114-TR ΔR , and pCMV-4070A ΔR code for the respective truncated glycoproteins in which the R-peptide was deleted from the cytoplasmic tail of the glycoproteins and replaced by a STOP codon by inverse PCR.

Techniques: Expressing, Microscopy