Therapeutic application of RNAi: is mRNA targeting finally ready for prime time?

Abstract

With unprecedented speed, RNA interference (RNAi) has advanced from its basic discovery in lower organisms to becoming a powerful genetic tool and perhaps our single most promising biotherapeutic for a wide array of diseases. Numerous studies document RNAi efficacy in laboratory animals, and the first clinical trials are underway and thus far suggest that RNAi is safe to use in humans. Yet substantial hurdles have also surfaced and must be surmounted before therapeutic RNAi applications can become a standard therapy. Here we review the most critical roadblocks and concerns for clinical RNAi transition, delivery, and safety. We highlight emerging solutions and concurrently discuss novel therapeutic RNAi-based concepts. The current rapid advances create realistic optimism that the establishment of RNAi as a new and potent clinical modality in humans is near.

Figure 1. Mechanism of RNAi.

dsRNA is cleaved at specific sites by Dicer to form siRNA. siRNA can also be produced either in vitro, after which it can be conjugated to other molecules for efficient delivery into the target cells, or within cells, via DNA-based vectors encoding shRNA. siRNA binds to RISC, the action of which exposes the antisense strand of siRNA and allows it to recognize mRNA with a complementary sequence. Upon mRNA binding to RISC, the mRNA is cleaved and degraded, resulting in the posttranslational silencing of gene expression. Figure modified from ref. .

Figure 2. Classes of AAV vectors for RNAi expression.

(i) Wild-type AAV genome. The 2 AAV genes (rep and cap, encoding replication and capsid proteins) are flanked by inverted terminal repeats (ITRs) that serve as replication and packaging signals. (ii) In a conventional single-stranded AAV vector genome, rep and cap are replaced by an shRNA expression cassette. A stuffer sequence is needed to increase the genome size to the packaging optimum. (iii) In an advanced double-stranded AAV vector, the total size of shRNA and stuffer is reduced to less than half the size of wild-type AAV, and 1 ITR is partially deleted (indicated by asterisk). As a result, the vector undergoes a single replication cycle in cells during virus production, leading to duplication of the shRNA/stuffer cassette. In infected cells, the 2 shRNA copies rapidly anneal with each other, which results in instant and robust RNAi expression. (iv) Alternatively, the stuffer DNA can be replaced with further copies of the same (or other) shRNA cassettes to potentiate the RNAi effect or to create a coRNAi vector directed against multiple targets.

Figure 3. Levels of control over RNAi expression with viral vectors.

Through binding to cellular receptors, the viral capsid (a) will determine the tropism of the RNAi vector, i.e., the tissue and cell type that will be infected. This occurs regardless of the vector insert. In a conventional shRNA expression cassette (i), the promoter (b) can further contribute to specificity by being active only in desired tissue or cell types. Alternatively, promoters can be made regulatable via exogenous triggers. Ideally, both properties are combined to permit spatiotemporal control over shRNA expression. Moreover, the shRNA itself (c) is a major determinant of specificity and control and should be designed to selectively bind to the target mRNA. (ii) Theoretically, it should be possible to create hybrid vector genomes in which an shRNA cassette is fused with a binding site for a particular miRNA (d; black box). This would allow the restriction of shRNA expression only to cells in which this miRNA is not expressed, thus helping to minimize off-target effects. (iii) Alternatively, the hybrid genome (or a vector expressing a cDNA; green box) could be fused with multiple tandem sites for miRNA binding and then be used to sequester, and thus inactivate, this miRNA from the cellular pool. This strategy is useful to block miRNAs that are involved in pathogenic processes such as tumorigenesis.