Plasmidderived Short Hairpin RNAs

In 2002, several groups simultaneously introduced the concept of plasmid-induced gene silencing by RNAi (summarized by Shi8). The most widely used approach converts the siRNA sequence into a DNA sequence encoding the sense strand of the siRNA, a loop and the antisense strand of the siRNA. Transcription from this DNA template results in a self-complementary RNA molecule referred to as short hairpin RNA (shRNA). These shRNA molecules are recognized as substrates by Dicer and are then processed to give siRNA-type molecules (Figure 12.1).

Usually, shRNA molecules are transcribed intracellularly under the control of RNA polymerase III promoters. These promoters are evolutionary optimized to generate high levels of short, precisely defined RNA molecules. Unlike RNA polymerase II transcripts, the resulting transcripts are neither capped nor polyadenylated. Most commonly used are the promoter of the U6 component of the splicosome9 11 and the H1 promoter of the RNA component of RNaseP.12 The U6 or the H1 promoter preferentially initiate transcription at guanine or adenine, respectively, and recognize a series of five to six thymines as a termination signal. It is therefore essential not to use sequences that include longer stretches of consecutive uridines in either of the siRNA strands.

Figure 12.1

Vector expression of short hairpin RNAs (shRNAs). The self-complementary RNA molecules can be expressed under control of polymerase III promoters, like the U6 or H1 promoter. The shRNAs are then processed by the RNase Dicer to yield the siRNA. (Figure adapted with kind permission of the publisher from Jens Kurreck, Oligonucleotide als zellbiologische Werkzeuge, in ed. F. Lottspeich and J. Engels, Bioanalytik, Spektrum Verlag, Germany, 2006.)

Figure 12.1

Vector expression of short hairpin RNAs (shRNAs). The self-complementary RNA molecules can be expressed under control of polymerase III promoters, like the U6 or H1 promoter. The shRNAs are then processed by the RNase Dicer to yield the siRNA. (Figure adapted with kind permission of the publisher from Jens Kurreck, Oligonucleotide als zellbiologische Werkzeuge, in ed. F. Lottspeich and J. Engels, Bioanalytik, Spektrum Verlag, Germany, 2006.)

The exact design of the shRNA determines its efficiency. First, the size and the nucleotide sequence of the loop were shown to play an important role.12 More recently, Siolas et al.13 demonstrated that the length of the duplex greatly influences the potency of the shRNA as well. Chemically synthesized shRNAs 29 nucleotides in length were found to be much more potent inducers of RNAi than were siRNAs or shRNAs with the standard 19-mer helix. A possible mechanistic explanation for this higher efficiency is that these longer shRNAs are initially processed by Dicer to give 21-mers. According to the current model, Dicer is involved in the loading process of siRNAs into RISC,14 thus explaining the higher potency of Dicer substrates compared to traditional 21-mer siRNAs.

Transcription with RNA polymerase II yields RNAs that contain a cap at the 5'-end and a poly-A tail at the 3'-end. Only a few reports of work employing RNA polymerase II promoters to generate shRNAs have therefore been published to date. Since the cap and poly-A tail are not compatible with the RNAi machinery, this approach requires a setup with an optimized version of the cytomegalovirus promoter, as well as a minimized poly-A cassette to generate functional shRNA molecules.15

Transient transfection of cells with shRNA-expressing plasmids enables the induction of RNAi over an extended period of time compared to the application of chemically synthesized siRNAs. The transfection efficiency of plasmids, however, is usually significantly lower than that of oligonucleotides, resulting in insufficient knockdown. A simple calculation demonstrates this problem: if a plasmid encoding an shRNA that mediates an 80% knockdown of the target gene is transfected into 60% of the cells, the observed decrease in the protein level will be 48%. For most applications, this extent of silencing will be insufficient to investigate gene functions.

To overcome this problem, viral vectors can be used that transfer the shRNA expression cassettes into almost 100% of the targeted cells (vide infra) or stable cell lines can be selected, which integrate the shRNA-encoding sequence and its promoter into their genomes. For this purpose, the plasmid usually contains an antibiotic resistance cassette. In one of the first examples, stable cell lines were selected in which the expression of the tumour-suppressor gene p53 was found to be almost completely inhibited, even after two months.12 It has been observed that the amount of shRNA in stably transfected cells is dramatically reduced compared to the amount of shRNA transiently expressed.16 The high efficiency, however, means that even low levels of active shRNAs will be sufficient for good knockdown of the target gene.

The plasmid-based RNAi approach also allows the combination of two or more siRNAs. This strategy is desirable for various applications. First, it allows the simultaneous knockdown of two target genes. Second, combination of two or more potent drugs is a common strategy in conventional antiviral therapy to prevent the emergence of escape mutants. It will therefore also be advantageous to combine multiple shRNAs for RNAi-based treatments of viral infections.

Several reports in the literature have shown that extended inhibition of viruses by means of single-gene RNAi will result in the emergence of escape mutants that are no longer susceptible to the used siRNA. This problem is particularly relevant for RNA viruses with an error-prone replication machinery. A single point mutation in the target site was found to be sufficient to render the respective siRNA inactive.17 19 Interestingly, human immunodeficiency virus 1 (HIV-1) can escape RNAi silencing not only through mutations in the siRNA target site, but also through base substitutions that alter the local RNA structure, rendering the target site inaccessible for the siRNA.20 In one case, an extended deletion in the HIV-nef gene was observed.19 This gene is obviously dispensable for virus replication under certain conditions. It is thus advisable to target regions of the viral genome that are essential for the viral life cycle.

As a strategy to prevent viral escape, we developed an siRNA double expression vector (SiDEx), which generates two shRNAs simultaneously.21 This vector contains two expression cassettes, each consisting of a U6 promoter and the shRNA-encoding complementary DNA (cDNA). At first, we demonstrated that the two U6 promoters do not interfere with one another and both shRNAs inhibit the expression of their target. We then artificially introduced a point mutation into the viral target gene fused to green fluorescent protein (GFP) as a reporter so that the respective mono-expression vector lost its silencing capacity. The second shRNA of the double-expression vector, however, compensated this loss of activity and maintained high inhibitory activity. In subsequent experiments we demonstrated the high efficiency of the double expression cassette in inhibiting replication of infectious coxsackievirus B3.22

Berkhout et al. used lentiviral vectors to inhibit HIV-1 replication and found a double shRNA-expression vector to delay virus escape.23 Combination of shRNAs was furthermore found to have an additive inhibitory effect on HIV-1, so that a lentiviral vector was developed that expresses three different shRNAs targeting gag and two different sites of pol from separate H1 promoters. In an alternative approach, three RNA-based silencing technologies were combined: A triple combination of an shRNA targeting HIV-1 rev and tat mRNAs, a decoy binding to trans-activation responsive element (TAR) and a ribozyme cleaving the mRNA of the CC chemokine receptor 5 (CCR5) receptor was found to mediate long-term inhibition of HIV-1 in haematopoietic progenitor cells.24

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