EF1α-SpCas9 Lentivirus

Lentiviral vectors (LV) have been chosen as gene delivery vehicles for their ability to transduce all types of non-latent or slowly proliferating cells, making them extremely attractive for clinical applications. LVs belong to the same family of retroviridae as gamma-retroviruses. They contain an RNA genome that is reverse transcribed into DNA in transduced cells. The first generation of retroviral vectors laid the foundation for important principles that ensure their safe use. First, there is the possibility of recombination events during vector manufacturing, which can render the virus replication-competent. To avoid this, the viral genome needs to be split into different expression constructs. Second, enhancer and promoter sequences in the long terminal repeats (LTRs) are deleted to generate so-called self-inactivating (SIN) vectors; this is a safety measure to avoid activation of surrounding (oncogenic) genes, as reported in some gamma-retroviral vector clinical trials. Third, the integration of heterologous envelope glycoproteins onto the vector surface can expand or restrict the host range of the vector, a process called pseudotyping. LVs underwent “generations” of modifications. They are classified according to the packaging plasmid used for production. The first generation of lentiviruses contained HIV gag, pol, regulatory genes, and accessory genes. Second-generation lentiviruses enable the removal of helper genes without any negative impact on their infectivity or vector yield. In addition, the safety profile is improved because the resulting replication-competent lentiviruses are free of virulence factors. The safety profile of third-generation lentiviruses is further improved because the promoter in the LTR can now be activated independently of the HIV protein TAT. An additional safety measure is the inactivation of integrase without affecting reverse transcription and trafficking to the nucleus. These non-integrating LVs have been effectively used in post-mitotic tissues, especially in combination with gene editing tools (zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)).