HB9-mCherry Lentivirus

The most commonly used lentiviral vectors are those based on the human immunodeficiency virus (HIV). Of course, concerns about the production of replication-competent viral particles have forced the development of complex lentiviral vectors with multiple safeguards. Similar to γ-retroviral vectors, split-component systems that separate essential viral genes from cis-regulatory sequences reduce the chances of viral recombination and subsequent reactivation. Nevertheless, the U.S. Food and Drug Administration (FDA) requires that therapeutic products generated by lentiviral transduction be thoroughly tested for the presence of replication-competent virus. Recently, hybrid lentiviral vectors derived from non-human lentiviruses (simian, equine, feline, caprine, and bovine) have been proposed as potentially safer alternatives, as their parental viruses are pathogenic in humans but can efficiently transduce human cells when optimized. The construction of CAR-T cells by lentiviral vectors is similar in some respects to γ-retroviral vectors. Packaging of the recombinant viral genome into viral particles requires the concomitant expression of the viral genes gag, pol, and env, which encode the viral capsid protein, replicase, and envelope glycoprotein, respectively, in trans from a heterologous subgenomic helper plasmid that does not contain any packaging signals. In addition to gag, pol, and env, lentiviral vectors require the transactivator rev protein, which binds to the rev response element (RRE) and enhances the nuclear export and expression of gag-pol transcripts. The packaging construct expressing gag-pol is transfected with a vector carrying the CAR gene and the env and rev packaging constructs. In fact, activated T cells are more susceptible to lentiviral infection than resting T cells, and anti-CD3 and anti-CD28 antibodies can activate T cells out of the G0 phase and make them more sensitive to lentiviral transduction. Productive transduction and genomic integration allow stable expression of the CAR gene in T cells, and lentivirally generated CAR-T cells targeting the CD19 antigen have shown significant antitumor efficacy in clinical trials.