Synapsin-mCherry Lentivirus

For use in gene therapy, the lentiviral genome has been modified in several ways to maximize safety. These modifications have been divided into three iterations. In the current third-generation system, the components of the viral genome are divided into packaging plasmids, transfer plasmids, and envelope plasmids, which are primarily transfected into HEK 293T cells for virion production. The packaging plasmid contains all the elements required for viral production, including genes for the structural and enzymatic proteins gag and pol, as well as the viral regulatory gene rev. Alternatively, rev can also be expressed from a second packaging plasmid, resulting in a four-plasmid system. The transfer plasmid contains the transgene of interest as well as the necessary cis-acting elements, such as the psi(y) RNA packaging signal and the 3′ and modified 5′ LTR elements. This modified 5′ LTR replaces the endogenous HIV promoter with a constitutively active tat-independent promoter, allowing the tat gene to be deleted from the packaging plasmid. The env-encoded protein is expressed on a separate plasmid sequence and can be expressed by altering the tropism of the virus (a process called pseudotyping). This multi-plasmid system ensures that only the gene of interest and the cis-acting sequences are present in subsequent transductions. Nonessential viral proteins, such as accessory proteins, are completely deleted, which helps reduce immunogenicity, and the deletion of these coding sequences further reduces the likelihood of recombination, resulting in replication-competent lentivirus. Further safety improvements have been made in second- and third-generation systems, including the development of a self-inactivating transfer vector. This is achieved by deleting the U3 region of the 3′ LTR of the transfer vector, which is transferred to the 5′ LTR after reverse transcription and integration, effectively reducing the activity of the LTR promoter. This deletion does not significantly affect viral titers, and in addition, it reduces promoter interference, thereby avoiding unnecessary transgene silencing. The packaging of viral genes into separate plasmids and the use of self-inactivating transfer vectors further reduce the likelihood of generating replication-competent lentivirus.