Life cycle of retrovirus. Infection of the host cell by the retrovirus begins when the viral envelope proteins bind to their receptors expressed on the cell membrane of the host cell (1). The viral and cell membranes are fused and the capsid including the viral RNA is released into the cytoplasm (2). Viral RNA is reverse transcribed to DNA and is transported to the nucleus upon mitosis (3). Viral cDNA is integrated to the host cell DNA, thus forming the provirus (4). Provirus is transcribed to mRNA by the host cell’s transcription machinery (5). The mRNA is translated, leading to the production of viral proteins (6). The viral proteins are further processed by viral protease and subsequently are encapsulated together with new viral RNA in order to form new viral particles. The newly formed viral particles exit the host cell through budding (8) and they undergo maturation in order to initiate the infection of a host cell (9) (Figure created with Xxxxxxxxx.xxx). Retroviruses are often used in CAR T-cell therapy to achieve stable expression of the CAR receptor. However, the ability of the retrovirus to replicate is undesirable and potentially dangerous. For this reason, replication defective retroviral vectors are used in which the gag, pol, pro and env genes are replaced by the DNA sequence of interest. Since these proteins are necessary for viral infection, dedicated packaging cells that provide the missing proteins in trans are used to produce the retroviral vector for delivery and integration into T-cells. The retroviral vector contains a cassette for transgene expression typically driven by the 5’ LTR promoter. Further optimisation was achieved via the development of U3-deleted self-inactivating (SIN) vectors, the use of cellular promoters rather than viral promoter elements and deletion of coding regions of the viral genome. This was an important improvement due to reports of severe adverse events related to oncogenesis in stem cells after retroviral insertion93,94. Various classes of virus vectors have been used to stably integrate CAR encoding DNA into the T-cell genome such as retrovirus, lentivirus and adeno-associated virus (AAV). Lentivirus and retrovirus are the most commonly used vectors in the generation of CAR T-cells. A key advantage for lentiviral vectors is the fact that T-cell activation or proliferation is not essential for genomic integration however, lentiviral transduction of quiescent T-cells is relatively inefficient. In addition, lentiviral vectors produce a...
