Biomedical Engineering Reference
In-Depth Information
vector can hold and deliver a large transgene or gene regulatory cassette; (iii) whether the
vector is stable or transient; (iv) whether it is safe and free from host immune reaction;
(v) whether the transfection is very efficient; and (vi) whether high titers canbe produced.
12.1.3.1 Retroviruses
The original retrovirus used as a vector was the Moloney murine leukemia virus (MMLV).
Retroviruses are RNA viruses that work in dividing cells but not in non-dividing cells. The
carrying capacity of retroviruses is about 10 kb when fully deleted. Retroviruses are easily
produced in large amounts by being secreted from packaging cells and can be concentrated.
The retrovirus integrates into a host chromosome and can produce very long-term stable
transgene expression. Over time, however, gene silencing does occur. One of the biggest
drawbacks is random insertion, which could lead to mutagenesis. For experimental use
retroviruses have been widely employed, but for human use they are risky.
12.1.3.2 Lentivirus
Lentiviruses are retroviruses derived from human immundodeficiency virus (HIV) or feline
immundodeficiency virus (FIV), simian immundodeficiency virus (SIV) or equine infectious
anemia virus (EIAV). Like other retroviruses, they integrate into the host chromosome and
are actively taken up by dividing cells and provide long-lasting transgenic expression. They
have a large carrying capacity of about 10 kb, and can also enter non-dividing cells. The
transduction efficiency is 20-50%. Therefore, lentivirus vectors are suitable for gene deliv-
ery in cancer cells and cancer stem cells where the goal is to stop cell division. They have
advantages for delivering transgenes that can induce differentiation or apotosis. For produc-
ing lentivirus, three components are used: (i) the expressing plasmid, which contains the
transgene or shRNA; (ii) a packaging plasmid; and (iii) an envelope plasmid. 293T/293FT
cells are normally used for packaging lentivirus via transient transfection. In the develop-
ment of lentivirus vectors, the first-generation lentiviral vector had three plasmids: (i) the
expressing plasmid with
cis
-acting elements of HIV and the gene of interest; (ii) packaging
plasmid with all HIV viral genes, except the envelope gene; and (iii) envelope plasmid
vesicular stomatitis virus (VSV G). Although the envelope gene is separated from helper
plasmid, first-generation lentivirus contains HIV accessory genes in its packaging plasmid
and has serious safety concerns. The second-generation lentiviral vector is improved by
removing accessory genes from the packaging plasmid, which increases the safety. The
common packaging plasmid contains both psPAX2 and pCMV-dR8.2 dvpr with compo-
nents of gag, rev, cPPT and RRE. The third-generation lentiviral vector is self-inactivating
(SIN) vector, which is removed from the enhancer region of 3' U3 of long terminal repeat
(LTR), leading to a transcriptionally inactive vector, but offering maximal biosafety. To
decrease the chance of retroviral recombination, the packaging plasmid is split into two
separate plasmids, one containing rev and the other containing gag-pol. Since it needs the
transfection of four different plasmids in 293T/293FT cells, the packaging efficiency is
lower than the first- and second-generation systems.
12.1.3.3 Herpes simplex virus (HSV)
HSV is a dsDNA virus with a very large capacity (20-30 kb). Since HSV normally infects
neurons its potential use has been mostly studied in neuronal cells. It infects non-dividing
