Abstract
Introduction
An important observation first discovered in Drosophila was that long double-stranded RNAs were processed into short 19- to 23-base duplexes with 2 nucleotide 3′ single-stranded overhangs by the RNAse III enzyme called Dicer. These smaller RNAs are termed small interfering RNAs or siRNAs.
In 2001 Tuschl and colleagues, as well as Morgan and collaborators, provided the first study illustrating that siRNAs could be artificially introduced into mammalian cells to trigger post-transcriptional gene silencing (Caplen et al., 2001; Elbashir et al., 2001). The demonstrations that RNAi could be used to selectively silence gene expression in human cells led to the rapid formation of several biotech companies devoted to developing siRNAs as therapeutics for a variety of diseases.
Large pharmaceutical companies were slow to get involved in siRNA drug development, perhaps owing to the slow progression of the DNA antisense field and concerns about how such relatively high-molecular-weight, nucleic-acid-based drugs could be delivered in vivo.
First Move
The first major entry into the field by big pharma was Merck, which purchased the siRNA biotech company SIRNA for a fee of slightly over $1 billion. This acquisition led to several other major pharmaceutical companies, including Roche, Pfizer, and Novartis, jumping into the game, although this past year Roche and Pfizer divested their RNAi investments, and Novartis and Merck downsized their investments.
The reason is not lack of success in the development of siRNA-based drugs because since the discovery of RNAi only 13 years ago, there have been more than 30 clinical trials involving 21 different siRNA biological drugs developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders.
Advancements in bioengineering and nanotechnology have led to improved control of delivery and release of some siRNA therapeutics. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules.
A great feature about siRNAs as drugs is their 2-dimensional design. Once a target mRNA is identified, a series of siRNAs complementary to various regions of the target can be rapidly synthesized and tested in cell culture. Lead candidates most often have low nanomolar and often picomolar IC50 values.
Variations on the 19+2 siRNA structure have also been successfully developed. We developed what we call Dicer substrates, which are asymmetric 25/27 base duplexes processed in cells by the enzyme Dicer (Amarzguioui et al., 2006). The dicing step mimics the natural process that microRNAs (miRNAs) undergo, providing a tight coupling with the effector proteins of RNAi.
In addition, dicing allows molecules useful for targeted delivery such as aptamers to be attached to one of the strands of the duplex wherein the active siRNA is processed from the complex and enters the RNAi pathway (Neff et al., 2011). Backbone modifications have been introduced into siRNAs to enhance stability and to suppress toll-like receptor activation (TLR). These primarily include 2′ OMe and 2′ Fluoro modifications to the ribose moiety as well as phorphorothioate inter-nucleotide linkages.
Unfortunately, the field of siRNA therapeutics suffered a setback following a demonstration that siRNAs designed to knockdown the VEGFA or VEGF receptor for treatment of adult macular degeneration were doing so via activation of toll-like receptor 3, and not by a sequence-specific degradation mechanism (Kleinman et al., 2008). These siRNAs were the first to hit clinical trials, but now are out of the picture. These findings should serve as a good lesson to the field of RNAi therapeutics that the use of naked, unmodified siRNAs should be avoided for most clinical applications, unless triggering of TLRs is part of the therapeutic strategy.
Nanoparticle Delivery
The first demonstration that an siRNA delivered to human subjects was directing target-specific cleavage of the mRNA came from a clinical trial using a liganded nanoparticle delivery vehicle for an siRNA targeting ribonucleotide reductase in treatment of solid cancers (Davis et al., 2010). Fortunately, the majority of the major players in RNAi therapeutics have a delivery vehicle program as part of their ongoing siRNA drug development.
While siRNAs are delivered to cells in double-stranded RNA form, short hairpin RNAs (shRNAs) are transcribed within the cellular nucleus, transported into the cytoplasm, and processed by cellular machinery into an siRNA-like form.
The first issued U.S. patent in the RNAi field covered both the research and therapeutic uses of shRNAs. ShRNAs are usually transcribed from a Pol III promoter but can also be transcribed with Pol II promoters. As was the case with siRNAs described above, a major setback for the use of shRNAs was reported when it was revealed that high level expression of most shRNAs in mouse liver resulted in acute liver toxicity and often death of the treated animals (Grimm et al., 2006).
Again, this was a valuable lesson for those of us using shRNAs therapeutically in that too much is not a good thing, and that controlled or moderated levels of shRNA expression are necessary to avoid such toxicities. Fortunately, our first therapeutic application of a Pol III-expressed shRNA did not prove to be toxic owing to its relatively low level of expression.
Early on we adopted an shRNA strategy for inhibiting human immunodeficiency virus-1 (HIV-1) by incorporating an shRNA targeting the HIV-1 tat/rev transcripts in the backbone of a lentiviral vector. This construct has been used to transduce hematopoietic stem cells that have been infused into acquired immune deficiency syndrome/lymphoma patients. We have observed long-term expression of the shRNA in primary blood mononuclear cells (PBMCs) from the treated patients, indicating no toxicity associated with this constitutive expression of an shRNA in this first human clinical trial (DiGiusto et al., 2010).
Any treatment of RNAi-based therapeutics would be remiss were it not to mention the importance of the discovery of miRNAs (Lee et al., 1993). It took almost a decade since the first description of miRNAs in Caenorhabditis elegans to realize their significance in human disease, particularly in cancers (Calin et al., 2002).
Rational Approaches
Our current knowledge of the RNAi pathway in humans comes primarily from studies of miRNA genesis. Since miRNAs are often dys-regulated in cancers and other diseases, restoring or inhibiting miRNA function are now considered to be rational therapeutic approaches. As with the siRNA therapeutic field, miRNA therapeutics are becoming the foundation for new biotech development.
MiRNAs can be delivered using the same strategies that have been developed for siRNAs, and they can also be expressed using vector-delivered gene constructs. Unlike siRNAs that are designed to fully base pair with their targets and direct site-specific cleavage of the mRNA, miRNAs function by binding to target transcripts (primarily in 3′ untranslated regions) via a short complementary sequence of 6–8 nucleotides wherein they direct the RNA-induced silencing complex to inhibit translation of the targeted message.
A single miRNA can affect the expression of well over 100 different transcripts, making them powerful modulators of cellular physiology. This promiscuous function of miRNAs also means that therapeutic applications of miRNAs have to be dealt with very cautiously since delivery to nondiseased cells or tissue could result in inhibition of vital protein expression. The same can also be said for siRNAs since they can also function as promiscuous miRNAs if not properly designed and backbone modified.
In closing it is safe to say that the world of small RNAs has opened up a whole universe of therapeutic opportunities. We are probably at the very earliest stages in our understanding of all the roles that these small regulators of gene expression can play.
The disappointing pullout of the large pharmaceutical companies from small RNA-based therapeutics should not be viewed as a lack of faith in their potential as novel drugs, but most likely reflects the fact that we still are on the early phase of the learning curve with respect to their delivery and global effects on cellular physiology.
It is important for basic scientists and biotech to continue to pursue investigations into RNAi mechanisms and delivery of small RNAs. Certainly, there will come a time when a major form of treatment for diseases of aberrant gene expression will be with small RNAs.