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. 2014 Feb;25(1):14-32.
doi: 10.1089/hgtb.2013.016. Epub 2013 Nov 1.

Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

Free PMC article

Lentiviral Vector-Mediated RNA Silencing in the Central Nervous System

Thomas H Hutson et al. Hum Gene Ther Methods. .
Free PMC article


RNA silencing is an established method for investigating gene function and has attracted particular interest because of the potential for generating RNA-based therapeutics. Using lentiviral vectors as an efficient delivery system that offers stable, long-term expression in postmitotic cells further enhances the applicability of an RNA-based gene therapy for the CNS. In this review we provide an overview of both lentiviral vectors and RNA silencing along with design considerations for generating lentiviral vectors capable of RNA silencing. We go on to describe the current preclinical data regarding lentiviral vector-mediated RNA silencing for CNS disorders and discuss the concerns of side effects associated with lentiviral vectors and small interfering RNAs and how these might be mitigated.


<b>FIG. 1.</b>
FIG. 1.
Basic structure of the wild-type HIV-1 virus. The diagram illustrates the key structural features of the wild-type HIV-1 virus. The surface glycoprotein (SU, gp120) and transmembrane glycoprotein (TM, gp41) are responsible for binding to the target cell and initiating cell entry. The matrix proteins (MA, p17) separate the outer lipid envelope from the inner capsid core (CA, p24). The capsid forms a cone-shaped inner core containing the condensed ribonucleoprotein complex, which is composed of two copies of the single-stranded viral RNA genome and nucleocapsid protein (NC, p7) that facilitate reverse transcription. The inner core also contains several viral enzymes including reverse transcriptase (RT, p66/p51), integrase (IN, p31), and protease (PR, p10) that are essential for viral replication. Color images available online at
<b>FIG. 2.</b>
FIG. 2.
Structure of the HIV-1 proviral genome and the third-generation lentiviral vector system. (A) The HIV-1 proviral genome contains nine genes and several cis-acting sequences. (B) To reduce the risk of generating replication-competent viruses, essential viral genes are provided in trans and separated onto three helper plasmids, which prevents them being incorporated into the vector genome. Third-generation lentiviral vectors are generated with four plasmids: the packaging plasmid, envelope plasmid, Rev plasmid, and transfer plasmid. (C) The transfer plasmid, which carries the viral vector genome, contains only essential cis-acting viral sequences. In the example shown the vector would encode a CMV-driven transgene and an H1-driven shRNA cassette. In this plasmid the 5′ LTR U3 region is replaced by a heterologous promoter such as the RSV promoter, whereas on the 3′ LTR the U3 region bears a deletion that removes transcriptional activity; after reverse transcription in the transduced cell the LTRs on the double-stranded DNA viral genome have the structure of the plasmid 3′ LTR. CMV, cytomegalovirus promoter; cPPT, central polypurine tract; cTS, central termination sequence; dLTR, long terminal repeat from which 400 bp in the U3 region has been deleted; GLS, gag leader sequence; ψ, encapsidation sequence; LTR, long terminal repeat; Poly-A, polyadenylylation signal; RRE, Rev response element; RSV, Rous sarcoma virus promoter; shRNA, short hairpin RNA; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element. Color images available online at
<b>FIG. 3.</b>
FIG. 3.
Fate of the lentiviral genome after transduction by integration-proficient and deficient lentiviral vectors. (A) Integration-proficient vectors pseudotyped with VSV-G are thought to enter host cells via the endosomal pathway. Once inside the cytosol the RNA genome is reverse transcribed in a multiprotein complex known as the preintegration complex (PIC). The PIC is then thought to translocate to the nucleus via the nuclear pore complex whereupon the viral vector DNA is integrated into the host cell DNA. The transcriptional activity of the viral vector LTR is minimal because of extensive deletions in the U3 region. Instead, shRNA expression proceeds from the internal RNA polymerase III (Pol III) promoter. The shRNA is then exported to the cytosol, where the loop is removed by Dicer and the antisense strand is loaded into the RNA-induced silencing complex (RISC) complex. (B) Integration-deficient vectors gain access to the cell the same way, whereupon reverse transcription and nuclear translocation proceed normally. However, the PIC contains a mutant integrase, thereby inhibiting integration. Instead, the viral vector DNA forms double-stranded circles after self-ligation or recombination of the LTRs (partial deletion of the U3 region in self-inactivating vectors, denoted as dLTR). This is then followed by shRNA expression and gene silencing as normal. Color images available online at
<b>FIG. 4.</b>
FIG. 4.
The mammalian RNA silencing pathway. The diagram summarises the major features of the RNA silencing pathway in mammalian cells. The pink boxes on the right represent the endogenous miRNA pathway, while the blue boxes on the left indicate the points at which the endogenous silencing pathway can be subverted to produce knockdown of specified target mRNAs. Artificial miRNA-like constructs, known as shRNA-miRs, are transcribed from RNA Pol II or III promoters and enter the miRNA pathway at the start. Artificial shRNAs are generally expressed from RNA Pol III promoters and enter the miRNA pathway at the Dicer stage. Lastly, conventional ∼21–23 nucleotide siRNAs only need to be unwound in the cytoplasm before they enter the RISC to mediate gene silencing. Color images available online at

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