RNA Interference in Mammalian Cells

The formal description of RNA interference (RNAi) as a biological response to double-stranded RNA resulted from a desire to understand a number of intriguing observations arising from the use of antisense RNAs in Caenorhabditis elegans (1). This led ultimately to the discovery that worms could be programmed to silence genes by exposing animals to homologous double-stranded RNAs (dsRNAs; termed triggers) (1). It is now clear that an RNAi pathway is present in many if not most eukaryotes (2). A biochemical understanding of the RNAi pathway was crucial to realizing that dsRNAs shorter than 30 bp could be used to specifically trigger an RNAi response in mammals. Tuschl and colleagues demonstrated that transfection of mammalian cells with small interfering RNAs (siRNAs) could specifically induce RNAi and thus cracked the barrier to the use of RNAi as a genetic tool in mammals (3). It took a remarkably short period of time for siRNAs to be adopted as a standard component of the molecular biology toolkit.

The introduction of siRNAs into mammalian cells can be achieved through standard transfection. The strength and duration of the silencing response is determined by several factors. On a population basis, the overall efficiency of transfection is a major determinant, which must be addressed by optimizing conditions. In each individual cell, silencing depends upon a combination of the amount of siRNA that is delivered and upon the potential of that individual siRNA to suppress its target (the potency). Even a relatively poor siRNA can silence its target provided that sufficient quantities are delivered. However, essentially “forcing” the system with such reagents is likely to lead to numerous undesired effects (4).

The discovery of the endogenous triggers of the RNAi pathway in the form of microRNAs (miRNAs) that are encoded in the genome suggested that RNAi might be triggered in mammalian cells by providing synthetic genes that express mimics of such endogenous triggers. A number of laboratories simultaneously took related approaches to this goal by expressing mimics of miRNAs in the form of short hairpin RNAs (shRNAs) from RNA polymerase III or RNA polymerase II promoters (5,6,7,8) or expressed separate sense and antisense transcripts from Pol III promoters (9,10). The shRNAs themselves varied in size and design, with stems ranging from 19–29 nucleo-tides in length and with various degrees of structural similarity to natural miRNAs or siRNAs. All of these approaches were effective to varying degrees, and indeed, at present, no real consensus has developed on the most effective way to present expressed siRNAs into the RNAi pathway, although most investigators utilize short hairpins of 19–25 bp in length transcribed by Pol III promoters.

Since these triggers are encoded by DNA vectors, they can be delivered to cells in any of the innumerable ways that have been devised for delivery of DNA constructs that enable ectopic messenger RNA (mRNA) expression. These include standard transient trans-fection, stable transfection, and delivery with viral vectors ranging from retrovi-ruses to adenoviruses. Expression can also be driven by either constitutive or inducible promoter systems (11,12,13).

RNAi and Human Immunodeficiency Virus Therapeutics

Human immunodeficiency virus (HIV) was the first infectious agent targeted by RNAi, perhaps because the life cycle and pattern of gene expression of HIV is well understood. Synthetic siRNAs and expressed shRNAs have been used to target virtually all of the HIV-encoded RNAs in cell lines, including tat, rev, gag, pol, nef, vif, env, vpr, and the long terminal repeat (LTR) (10)(14,15,16,17). Subsequent work showed a host of other viruses, including hepatitis B virus (HBV), hepatitis C virus (HCV), poliovirus, respiratory syncytial virus (RSV), and others, were targetable by RNAi (recently reviewed in Reference (18).Despite the early successes of RNAi-mediated inhibition of HIV-encoded RNAs in cell lines, targeting the virus directly represents a substantial challenge for clinical applications, because the high viral mutation rate will lead to mutants that can escape being targeted (19,20,21,22), although a clever recent strategy takes

advantage of escape mutants in critical genes by targeting the mutants directly (23). The problem of viral resistance mutants to RNAi is not limited to HIV, as other RNA viruses with RNA-dependent RNA polymerases or reverse transcriptases also share this propensity to produce populations of mutants during replicative cycles (21)(24,25,26). An alternative approach to avoid this problem is to target cellular transcripts that encode functions required for HIV-1 entry and replication. To this end, cellular cofactors such as NFκB, the HIV receptor CD4, and the co-receptors CCR5 and CXCR4 have all been down-regulated with the result of blocking viral replication or entry (15,16)(27,28,29). The macrophage-tropic CCR5 co-receptor holds particular promise as a target. This receptor is not essential for normal immune function, and individuals homozygous for a 32-bp deletion in this gene are resistant to HIV infection, whereas individuals who are heterozygous for this deletion have delayed progression to autoimmune deficiency syndrome (AIDS) (30,31,32). Andersen and Akkina (27) used a lentiviral vector to transduce a combination of anti-CCR5 and CXCR4 shRNAs in human lymphocytes. Down-regulation of these receptors resulted in virtually complete inhibition of viral infectivity relative to controls. However, since CXCR4 is essential for hematopoietic stem cell homing to marrow and subsequent T cell differentiation (33,34,35), targeting this receptor is not a good choice for an anti-HIV therapy nor is targeting the essential CD4 receptor, with the exception of dendritic cells where the DC-SIGN receptor can be targeted by siRNAs to prevent infection (36). Targeting only the CCR5 co-receptor may also present problems, since HIV-1 switches to CXCR4 tropism during the course of AIDS, creating a more virulent infection (37). Thus, there are drawbacks in targeting cellular HIV cofactors, and viral targets will need to be included in any successful strategy using RNAi. A possible solution to using shRNAs against essential cellular targets is to incorporate them into a Tat inducible promoter system (38). This strategy is yet to be applied to cellular targets essential for HIV replication, but should be used in the future. Finally, it may be possible to use RNAi to prevent viral transmission by using siRNAs as a microbicide (39).