Small Silencing RNAs and Gene Therapy


Within one decade, ribonucleic acid interference (RNAi), that is, the sequence‐specific knockdown of gene expression triggered by small silencing RNAs, has rapidly matured from a biological curiosity into our single most promising biotherapeutic for a wide array of human diseases. Its exciting looming clinical translation is particularly accelerated by the increasingly pursued juxtaposition of RNAi technologies with established gene therapy methodologies. Fostering this mutual attraction of two potent clinical modalities and paving the way to fully harness their therapeutic power are the abilities of viral gene transfer vectors to mediate stable, efficient and tailored transduction of RNAi into recipient cells. Finally, moreover adding to the enormous promise of combining small silencing RNAs and gene therapy are latest findings on the role of endogenous microRNAs for various human diseases, further enlarging our already fertile chest of tools and targets for intervention and fortifying the optimism that RNAi gene therapies will soon become a clinical reality.

Key Concepts:

  • Expression of RNAi triggers from viral gene transfer vehicles is a potent strategy to optimise the issues of in vivo delivery and specificity.

  • Vice versa, the development of expressible, vector‐compatible RNAi triggers adds a crucial therapeutically relevant template to our arsenal of gene therapy vectors.

  • The combination of RNAi and gene therapy thus synergistically enhances the power and promise of each individual approach.

  • Beyond exploiting RNAi to directly target and destroy disease‐associated genes, cellular miRNAs can also be used to segregate vectors or viruses.

  • This allows to either direct transgene expression to a certain subset of cells in the body, or to increase the specificity and safety of oncolytic viruses.

  • Further improvements of RNAi gene therapy vectors concern the promoter, which can be constitutive or inducible, weak or strong, or specific or ubiquitous.

  • Concurrent advances in viral vector genome designs and structures help to maintain a threshold of RNAi expression required for potent gene silencing.

  • Latest improvements in the field of RNAi gene therapy vectors comprise combinatorial approaches either aiming at enhancing custom vector properties or juxtaposing various RNAi triggers and other gene silencers, or co‐targeting different viral or cellular genes, to avert target viral escape by mutation.

Keywords: RNA interference; RNAi; small silencing RNAs; short hairpin RNA; microRNA; gene therapy; AAV vectors; posttranscriptional regulation; molecular evolution; combinatorial RNAi

Figure 1.

The mutual attraction between RNAi and gene therapy. As discussed in the text, both technologies synergistically benefit from each other, in that small silencing RNAs add another utmost potent class of transgenes to conventional gene therapy vectors. Vice versa, the latter provide a unique and extremely powerful and versatile means of delivering and expressing small silencing RNAs to or in cells, respectively.

Figure 2.

Viral vector options to express small silencing RNAs in cells. (a) Owing to the availability of a large battery of different viruses as templates, RNAi vectors can be designed to mediate short‐ (top, AAV as example) or long‐term (bottom, lentiviral vectors) RNAi expression in target cells. Note that the depicted scenario applies to rapidly cycling cells (such as embryonic stem cells), in which nonintegrating vectors such as AAV are lost during cell division. In contrast, AAV/RNAi vectors can also yield very long‐term (at least one year) RNAi expression in quiescent cells, such as hepatocytes in an intact liver, by assuming a stable episomal vector form. (b) One of the most important and unique benefits from the combination of RNAi with gene therapy is the versatility of the viral vectors and their genomes. As indicated in the figure and as discussed in the text, by switching either the promoter or the capsid, or both together, one can easily retarget a given small RNA expression cassette to a different cell type, or modulate the intracellular expression levels at will.

Figure 3.

Examples of the most prominent current RNAi vector applications. Shown from top to bottom are depictions of the use of viral vectors to express (a) shRNAs (perfectly matched dsRNAs, two red bars) or miRNA scaffolds (imperfect duplexes, red and blue bars), resulting in reduction of expression of a target protein (symbolised by red ball); (b) miRNA sponges/decoys, resulting in an increase of protein expression (or a reduction of pathogenic viral replication, see text); (c) miRNA‐tagged therapeutic transgenes, resulting in segregated gene expression, determined by the presence or absence of the miRNA in target cells or (d) miRNA‐tagged oncolytic viral genomes, resulting in selective killing of target (tumour) cells (see text for further details).

Figure 4.

Improvements of RNAi vector genomes. (a) Schematic depiction of the various promoter systems currently in use for vector‐mediated small silencing RNA expression: (1) RNA polymerase III promoters (e.g. U6, H1 or 7SK) or RNA polymerase II promoters (e.g. CMV) for (2) shRNA or (3) miRNA expression (sometimes in combination with reporter or further therapeutic genes, shown in (4)). (b) Various modifications of basic promoters (see (a)) to achieve inducibility. Examples (from top to bottom) are the placement of elements from the tetracycline system (tet operator sequence) upstream, downstream or both ((1)–(3)) of an RNA polymerase III or II (4) promoter. Another variation is the use of the reverse tetracycline activator‐based system within the RNA polymerase III or II promoters ((5)–(6)), or of a KRAB‐based system (see text and references therein for details) (7). (c) Latest generation of double‐stranded AAV vectors for RNAi expression. Shown on top is the wild‐type AAV genome with its two genes flanked by packaging signals (orange boxes) and a size of approximately 4.5 kb. Shown below is a conventional RNAi vector in which for example an shRNA expression cassette is co‐packaged together with a stuffer sequence (in order to maintain a proper total genome size and effective packaging). Depicted underneath is the latest creation of dsAAV vectors in which a single RNAi sequence is cloned between two modified packaging signals, resulting in eventual encapsidation of a self‐complementary genome containing two inverted copies of the RNAi cassette and mediating highly rapid and potent RNAi expression.

Figure 5.

Combinatorial approaches to further enhance RNAi specificity and safety. (a) Shown are the three major strategies for molecular evolution of viral vector capsids towards custom properties: (A) DNA family shuffling, that is the in vitro recombination of various related parental viral capsid sequences based on partial homologies; (B) peptide display, that is the presentation of randomised short peptides on the capsid surface, with the hope that some of them will mediate more efficient binding to a given target cell and (C) randomised PCR, where entire capsids are randomly mutagenised and then screened for improved properties. As also indicated, it is moreover possible to combine the various schemes, either pair‐wise ((D)–(F)) or even all three (G), resulting in even higher diversities and thus increased chances of selecting the desired novel particles. (b) Similarly, different approaches can be juxtaposed on the RNAi vector genome level, permitting combinatorial gene silencing which is especially critical for viral targets. For instance, multiple shRNAs/miRNAs can be expressed against the same target sequence (to increase potency), from either different vectors (1) or from a single genome (2). Alternatively, the shRNAs/miRNAs can be directed against a combination of viral (red) and cellular (orange/black) targets, again from multiple (3) or one (4) genome(s), to concurrently increase specificity and efficacy, by preventing viral escape by target site mutation.



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Further Reading

Grimm D (2009) Small silencing RNAs: state‐of‐the‐art. Advanced Drug Delivery Reviews 61: 672–703.

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Grimm, Dirk(Jun 2010) Small Silencing RNAs and Gene Therapy. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0022396]