Synapsin-Null Lentivirus

Lentiviruses (LVs) are produced as replication-defective viral particles whose RNA genome contains a so-called self-inactivating (SIN) 3′ long terminal repeat (LTR) that, after reverse transcription into a linear double-stranded DNA genome, produces a U3-deleted, enhancer- and promoter-less 5′ long terminal repeat (LTR). The ΔU3 long terminal repeat retains the minimum 18 bp long HIV-1 sequence required for integration, as well as the R region and the U5 region containing the polyadenylation sequence of the vector. Thus, the SIN lentiviral provirus is transcriptionally inactive, allowing the vector to carry a completely independent transgene expression cassette. In addition, the lentiviral genome retains the cis-acting viral sequences required for viral encapsidation, reverse transcription, and integration in the host cell genome, and lacks all other viral regulatory elements and genes, which are replaced by the transgene vector. These essential sequences include the packaging signal (Ψ), the primer binding site (PBS) and polypurine sequence (PPT) required for reverse transcription, the major HIV-1 intron with donor and acceptor splice sites, and the Rev response element (RRE) required for Rev-mediated nuclear export of the unspliced ​​intact genomic transcript. During the packaging process, two copies of the genomic transcript are selected from the cellular RNA pool through the interaction of the Ψ sequence located in the 5′ untranslated region with the Gag nucleocapsid domain. The lentiviral Ψ is a complex sequence that includes part of the transcriptional activation response element (TAR) of the U5 region, the PBS, and part of the gag gene, all of which are required for efficient packaging. The binding of Rev to the RRE enhances encapsidation by 70-fold, so its incorporation into the lentivirus (LV) is also required for packaging. Overall, the size of the complete LV Ψ is approximately 1.7 kb; attempts to remove or shorten its components result in reduced viral titers.