Designing Lentiviral Gene Vectors

Gene therapy relies on the delivery of therapeutic genes into patients’ cells. The microdevices used to reach the cells and to transfer the gene payload are called gene vectors. Viral packaging machinery is often utilized to generate the particles transporting the cargo genes. Lentiviruses, a subgroup of retroviruses, are highly suitable for remodeling into gene transfer vectors because they offer the stability of transgene expression, the ability to reach the nuclei of the therapeutically important non-dividing cells and are known to have a low immunogenic profile. Well studied members of the lentiviruses include human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2), feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV). It is important not to confuse “gene delivery vectors” and “gene cloning vectors”. While the former are microparticles delivering genes, the latter are replicating vehicles for the amplification of nucleic acid sequences. “Gene delivery vectors” and “gene cloning vectors” coincide when the naked DNA of replicating bacterial plasmids or replication competent viruses is used for gene delivery into cells. Viral gene delivery vectors are normally nonreplicating and should correctly be referred to as “viral vectors”, not “viruses”. Particles of viral vectors can be referred to as “virions” or “transducing particles”, because viral gene transfer is traditionally described as “transduction”. Replication deficient viral gene vector particles are similar to “defective interfering particles”, that is, faulty non-self-viable virions arising during natural viral infections and competing with non-defective virions, which were described in virology literature many years ago. Native lentiviral envelope proteins, which determine the cell range of viral infectivity (tropism) and mediate the fusion of viral and cellular membranes, are always composed from two non-covalently attached subunits, one of which (e.g. gp41 glycoprotein in HIV-1) is membrane-embedded and the other is an external subunit (e.g. gp120 glycoprotein in HIV-1). This arrangement makes lentiviruses notoriously unstable because of their tendency to shed the external subunit of the envelope protein. As the virion’s stability is a pre-requisite for the effective purification and concentration of viral vector preparations, in