Over the past few decades, retroviral vectors have been used in gene therapy clinical trials for the treatment of a number of inherited diseases and cancers.
The earliest retrovirus vectors were based on simple oncogenic gammaretroviruses such as Moloney murine leukemia virus MMLV which, when pseudotyped with envelope proteins from other viruses such as the gibbon ape leukemia virus envelope protein GALV or vesicular stomatitis virus G protein VSV-G , can efficiently introduce genes to a wide range of host cells. However, gammaretroviral vectors have the disadvantage that they are unable to efficiently transduce nondividing or slowly dividing cells.
As a result, specific protocols have been developed to activate cells through the use of growth factors and cytokines. The three major structural genes present in the genome are the gag group-specif antigen , pol polymerase , and env envelope.
The other regulatory genes are tat HIV transactivator and rev regulator of expression of virion protein. In addition, four types of accessory genes are also present in the HIV genome: vif viral infectivity , vpr virus protein R , vpu virus protein U , and nef negative factor.
Replication, integration, and packaging of lentiviruses are mediated by cis-acting elements of the lentiviral genome. Retroviruses are characterized by their ability to reverse transcribe their RNA genome into a cDNA copy that can be integrated into the host genome. They consist of two copies of positive sense ssRNA molecules in the genome.
Retroviruses can be classified into three subfamilies based on the different types of accessory and regulatory genes present in the genome. They are oncoviruses, lentiviruses, and spumaviruses. Oncoviruses are subclassified into three categories based on the morphology; B-type, C-type, and D-type. The different subfamilies of retroviruses with examples are described in table 1. Retroviruses consist of reverse transcriptase enzyme for the reverse transcription of the RNA genome.
The inner core of the retrovirus is surrounded by an outer protein layer, consisting of the matrix MA protein. The envelope of the retrovirus is derived from the host cell membrane. Retroviruses infect dividing cell lines and primary cells, stem cells, and hematopoietic cells. Lentivirus: Lentivirus is a type of retrovirus, consisting of long incubation periods and causing chronic, progressive, and typically fatal diseases in animals.
Retroviral Replication. Infection begins when the viral Env protein interacts with the cellular receptor and enters the cell. After the accumulation of newly synthesized viral proteins and viral genomic RNA, the components are packaged and bud from the cell, acquiring a cellularly-derived membrane. The particle matures when the Gag and Gag-Pol polyproteins are cleaved by the viral protease. Figures adapted from [ 1 , 2 , 4 , 5 ].
Figure 4. Making Lentiviral and Retroviral Vectors. The packaging cells produce infectious particles, whose genome only encodes sequences from the transfer plasmid, which can be used to transduce the target cells. Retroviral vector production right occurs in a similar manner to lentivirus production, with the main difference being that the initial step is transfection with a single plasmid.
The transfer plasmid alone is transfected into a packaging cell line Phoenix that already contains the helper constructs. Figure 5. Lentiviral Plasmids. Most lentiviral systems use a transfer plasmid and two helper plasmids. Their features are described in detail in the text. Newer systems may employ three helper plasmids with the rev gene being encoded on a separate plasmid.
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