Overview | Duke Viral Vector Core

Lentiviral Vectors

Lentiviral vectors (LVs) are HIV-1-based gene delivery platforms that can stably deliver gene-of-interest into cells in-vitro and in-vivo in highly efficient way. LV genome encoded by approximately nine kb positive ssRNA molecule, two of which homodimerizing and package in protein-enveloped viral particles. Following attachment and entry into host cells, viral reverse transcription step occurs in the host’s cytoplasm. During this process the viral dsDNA synthesized and the DNA transported into the nucleus. In the nucleus DNA undergo integration into the host chromosome. Gene delivery is stable because the recipient DNA stably integrates in the chromosome and is copied along with the DNA of the cell every time the cell divides. One of the discriminating features of LVs is their ability to integrate into non-dividing cells, in contrast to other vectors that either don’t integrate efficiently into chromosomal DNA (e.g. Adenoviral and Adenoviral-Associated vectors), or can only integrate upon cell division (e.g. simple Retroviral vectors).

To generate lentiviral particles calcium-phosphate or polyethylenimine (PEI) transient transfection protocols are employed. Human embryonic kidney (HEK) 293T cells, expressing a polyomavirus-derived Large-T antigen used for transfection of three cassettes: packaging, envelope and expression cassettes. The Envelope cassette delivers envelope protein (often the vectors pseudotyped with Vesicular Stomatitis Virus G protein (VSV-G) in trans. Packaging cassette, which is devoid of all of the accessory genes of the HIV-1 excluding the Tat and the Rev, is termed second-generation packaging cassette to distinguish it from the initial packaging construct, containing all of the HIV-1 accessory genes. Rev protein is crucial for exporting the fully length, or partially spliced RNAs from the nucleus into the cytoplasm, while Tat protein requires if the mRNA expressed from the UTR-3’ promoter located in the 5’LTR. If synthetic promoters, such as CMV or RSV employed, Tat expression is not required. Gag-Pol genes supply all necessary structural and enzymatic proteins requiring for the viral life cycle. Third cassette that requires for generating lentiviral vector is an expression cassette that is flanked by LTRs and the Psi-sequence of HIV. The LTRs are necessary to integrate the therapeutic gene into the genome of the target cell, while the Psi-sequence acts as a signal sequence and is necessary for packaging RNA with the reporter or therapeutic gene into viral particles. PBS and PPT cis-elements plays crucial role in the RT-process requiring for synthesis of the dsDNA of the vector. Since deletion in the 3’-LTR will translocate into the 5’LTR upon the RT-reaction, the virus particles that produced are replication deficient or in other words self-inactivated (SIN), so are designed to be unable to continue to infect their host after they deliver their therapeutic cargo.