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

Abstract

Lentiviral vectors (LVs) are excellent tools for gene transfer into mammalian cells. It is noteworthy that the first gene therapy treatment using LVs was approved for commercialization in 2017. The G glycoprotein from rhabdovirus vesicular stomatitis virus (VSV-G) is the glycoprotein most used to pseudotype LVs, due to its high efficiency in transducing several cell types and its resistance to viral vector purification and storage conditions. However, VSV-G expression induces cytotoxicity, which limits LV production to short periods. As alternative to VSV-G, γ-retrovirus glycoproteins (4070A derived, GaLV derived, and RD114 derived) have been used to pseudotype both γ-retroviral vectors (RVs) and LVs. These glycoproteins do not induce cytotoxicity, allowing the development of stable LV producer cells. Additionally, these LV pseudotypes present higher transduction efficiencies of hematopoietic stem cells when compared to VSV-G. Here, new 4070A-, RD114-TR-, and GaLV-TR-derived glycoproteins were developed with the aim of improving its cytoplasmic tail R-peptide cleavage and thus increase LV infectious titers. The new glycoproteins were tested in transient LV production using the wild-type or the less active T26S HIV-1 protease. The GaLV-TR-derived glycoproteins were able to overcome titer differences observed between LV production using wild-type and T26S protease. Additionally, these glycoproteins were even able to increase LV titers, evidencing its potential as an alternative glycoprotein to pseudotype LVs.

Semi-Replication-Competent Retroviral Vectors Expressing Gibbon Ape Leukemia Virus Fusogenic Membrane Glycoprotein (GALV FMG) Gene for Cancer Gene Therapy

“…These titers were a little higher than those obtained with MoMLV-gag-pol-IRES-EGFP and pCLXSN-GALV-IRES-EGFP (2×10 6 TU/ml). This result is consistent with previous reports that truncation of the GALV R peptide increases the incorporation of envelope protein into infectious viral particles (27,28). s-RCR from MoMLV-gag-pol-IRES-EGFP and pCLXSN-GALV-IRES-EGFP showed reduced viral spread upon passage of the transfected cells (Fig.…”

CAR-NK cells: A promising cellular immunotherapy for cancer

“…Similarly, lentivirus pseudotyped with envelop protein of Gibbon ape leukaemia virus also efficiently transduce primary NK cells. [64] …”

“…Similarly, lentivirus pseudotyped with envelop protein of Gibbon ape leukaemia virus also efficiently transduce primary NK cells. [64] Given the challenges with genetic transduction in primary NK cells, transfection methods such as electroporation and lipofection have also been used to deliver exogenous genes into NK cells. Compared to viral transduction, transfection of NK cells is associated with more rapid expression of the transgene with lower level of apoptosis, less variability among individuals, and higher gene transfer efficiency.…”

The stability of envelope-pseudotyped lentiviral vectors

“…A range of different envelope proteins have been employed for production of envelopepseudotyped lentiviral vectors. These include the envelope glycoprotein of vesicular stomatitis virus (the VSV-G protein), the envelope protein of nonhuman retroviruses (e.g., the ecotropic retrovirus murine leukemia virus (MULV), the gibbon ape leukemia virus (GALV), the feline endogenous RD114 retrovirus, Moloney MULV 4070A, Moloney MULV strain 10A1, as well as the rabies virus glycoprotein, and the measles virus hemagglutinin and fusion glycoproteins [6][7][8][9][10][11][12][13][14]. All but one of these viruses is capable of binding receptors on human cells: solely the ecotropic MULV recognizes a receptor on murine cells only.…”