Lisa Scherer

Approaches for the sequence-specific knockdown of mRNA

Over the past 25 years there have been thousands of published reports describing applications of antisense nucleic acid derivatives for targeted inhibition of gene function. The major classes of antisense agents currently used by investigators for sequence-specific mRNA knockdowns are antisense oligonucleotides (ODNs), ribozymes, DNAzymes and RNA interference (RNAi). Whatever the method, the problems for effective application are remarkably similar: efficient delivery, enhanced stability, minimization of off-target effects and identification of sensitive sites in the target RNAs. These challenges have been in existence from the first attempts to use antisense research tools, and need to be met before any antisense molecule can become widely accepted as a therapeutic agent.

Interplay between HIV-1 infection and host microRNAs

Using microRNA array analyses of in vitro HIV-1-infected CD4+ cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4+CD8− PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3′-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3′-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

Optimization and characterization of tRNA-shRNA expression constructs

Expression of short hairpin RNAs via the use of PolIII-based transcription systems has proven to be an effective mechanism for triggering RNAi in mammalian cells. The most popular promoters for this purpose are the U6 and H1 promoters since they are easily manipulated for expression of shRNAs with defined start and stop signals. Multiplexing (the use of siRNAs against multiple targets) is one strategy that is being developed by a number of laboratories for the treatment of HIV infection since it increases the likelihood of suppressing the emergence of resistant virus in applications. In this context, the development of alternative small PolIII promoters other than U6 and H1 would be useful. We describe tRNALys3-shRNA chimeric expression cassettes which produce siRNAs with comparable efficacy and strand selectivity to U6-expressed shRNAs, and show that their activity is consistent with processing by endogenous 3′ tRNAse. In addition, our observations suggest general guidelines for expressing effective tRNA-shRNAs with the potential for graded response, to minimize toxicities associated with competition for components of the endogenous RNAi pathway in cells.

Recent applications of RNAi in mammalian systems.

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incorporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new therapeutic modality.

Therapeutic applications of RNA interference: recent advances in siRNA design.

Publisher Summary This chapter focuses on providing information for the design of constructs that mediate effective silencing. The chapter emphasizes recent research in mammalian systems to provide information most easily translated to the therapeutic setting. The basic siRNA expression cassette is described and initial studies on the effects of chemical backbone modifications that are being tested for applications involving exogenous delivery are reviewed. Synthetic siRNAs are the method of choice for exogenous, short-term applications. Currently, unmodified siRNAs mediate RNAi effects that typically peak at 2–3 days post-transfection. The most common design for synthetic siRNAs mimics the endogenous siRNAs produced by Dicer cleavage of trigger dsRNA, where the sense and antisense strands are 21–23 nucleotides long. The chapter also discusses the important features of shRNAs and the use of a variety of promoters, including those that express shRNAs as part of larger transcripts, which is referred to as hybrid RNAi molecules. The effects of RNAi/target mismatches that have implications for specificity and escape of rapidly evolving targets are considered. Understanding the basis for processing efficiency could be very important in a therapeutic setting where stable expression levels are typically much lower than in the transient transfections used for screening.

Optimized Lentiviral Vectors for HIV Gene Therapy: Multiplexed Expression of Small RNAs and Inclusion of MGMTP140K Drug Resistance Gene

Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMTP140K marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.Gene therapy with hematopoietic stem and progenitor cells is a promising approach to engineering immunity to human immunodeficiency virus (HIV) that may lead to a functional cure for acquired immunodeficiency syndrome (AIDS). In support of this approach, we created lentiviral vectors with an engineered polycistronic platform derived from the endogenous MCM7 gene to express a diverse set of small antiviral RNAs and a drug resistance MGMTP140K marker. Multiple strategies for simultaneous expression of up to five RNA transgenes were tested. The placement and orientation of each transgene and its promoter were important determinants for optimal gene expression. Antiviral RNA expression from the MCM7 platform with a U1 promoter was sufficient to provide protection from R5-tropic HIV in macrophages and resulted in reduced hematopoietic toxicity compared with constructs expressing RNA from independent RNA polymerase III promoters. The addition of an HIV entry inhibitor and nucleolar TAR RNA decoy did not enhance antiviral potency over constructs that targeted only viral RNA transcripts. We also demonstrated selective enrichment of gene-modified cells in vivo using a humanized mouse model. The use of these less toxic, potent anti-HIV vectors expressing a drug selection marker is likely to enhance the in vivo efficacy of our stem cell gene therapy approach in treating HIV/AIDS.

Chapter 14:RNA Based Therapies for Treatment of HIV Infection

Controlling human immunodeficiency virus (HIV) infection continues to be a major challenge, both in underdeveloped and developed nations, despite the fact that the currently employed drug cocktails have markedly changed the profile of progression to acquired immune deficiency syndrome (AIDS) in HIV-...

Cancer Therapeutic Applications of Ribozymes and RNAi

Ribozymes are ribonucleic acid (RNA) molecules capable of acting as enzymes even in the complete absence of proteins. They have the catalytic activity of breaking and/or forming covalent bonds with extraordinary specificity, accelerating the rate of these reactions. The ability of RNA to serve as a catalyst was first shown for the self-splicing group I intron of Tetrahymena and the RNA moiety of RNAse P (1–3). Subsequent to the discovery of these two RNA enzymes, RNA-mediated catalysis has been associated with the self-splicing group II introns of yeast, fungal, and plant mitochondria (as well as chloroplasts) (4); single-stranded plant viroid and virusoid RNAs (5–7); hepatitis delta virus (8); and a satellite RNA from Neurospora mitochondria (9). It is rather clear that the RNA component of the larger ribosomal subunit is functioning as a peptidyltransferase as well (10–13). The potential functioning of spliceosomal smaller nuclear (sn)RNAs as a ribozyme in complex with the premessenger RNA (pre-mRNA) to catalyze pre-mRNA splicing has also been proposed (14). It is highly likely that additional RNA catalytic motifs and new roles for RNA-mediated catalysis will also be found as more is learned about the genomes of a variety of organisms.