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Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1β), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

The present global health emergency involving the emergence and rapid spread of a novel coronavirus has prompted the world scientific community to consider how it can help to fight this growing viral pandemic. With few safe and effective drugs available to combat this threat to humanity and the normal functioning of our society, the oligonucleotide research community is uniquely positioned to apply its technology and expertise to help alleviate the crisis, thanks to its capacity for rational drug design, swift development cycles, and pursuing targets undruggable by conventional treatment strategies.

The manifestation of RNA interference (RNAi)-based therapeutics lies in safe and successful delivery of small interfering RNAs (siRNAs), the molecular entity that triggers and guides sequence-specific degradation of target mRNAs. Optimizing the chemistry and structure of siRNAs to achieve maximum efficacy is an important parameter in the development of siRNA therapeutics. The RNAi protein machinery can tolerate a variety of non-canonical modifications made to siRNAs, each of which imparts advantageous properties. Here, we review these modifications to siRNAs in pre-clinical and clinical studies.

Nucleic acid aptamers are in vitro-selected small, single-stranded DNA or RNA oligonucleotides that can specifically recognize their target on the basis of their unique 3-dimensional structures. Recent advances in the development of escort aptamers to deliver and enhance the efficacy of other therapeutic agents have drawn enthusiasm in exploiting cell-type-specific aptamers as drug delivery vehicles. This review mainly focuses on the recent developments of aptamer-mediated targeted delivery systems. We also place particular emphasis on aptamers evolved against cell membrane receptors and possibilities for translation to clinical applications.

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

During the life cycles of most retroviruses and lentiviruses, dimerization and packaging of two copies of viral genomic RNA is required for the subsequent conversion of RNA into double stranded DNA by reverse transcriptase. For human immunodeficiency virus type 1 (HIV-1), dimerization is mediated by interactions of the stem-loop structures in the dimerization-packaging, or psi (Psi) domain. We have tethered anti-HIV gag ribozymes and small antisense RNAs to the HIV Psi domain in an HIV-1 lentiviral vector to facilitate copackaging of these replication inhibitors with HIV genomic RNAs during HIV infectious challenge. In order to maximize the base pairing of the ribozymes or antisense segments to the HIV-1 genomic target, sequences in HIV-1 were identified that are highly accessible to antisense pairing. Ribozymes or antisense RNAs designed to target these sequences were inserted in the lentiviral vector at the same relative distance to the Psi element as the HIV-1 target sites. Packaged vectors were transduced into CEM cells followed by challenges with HIV-1. Only the constructs that harbored short antisense segments complementary to HIV-1 gag produced replication incompetent HIV-1. These results demonstrate that a short stretch of antisense pairing downstream of the dimerization domain in an HIV-based vector can drive dimerization and provide a powerful approach for inhibition of HIV-1.

Short interfering RNA (siRNA)-mediated knockdown of deleterious endogenous transcripts has potential applications for the treatment of hereditary diseases. In situations where the mutant and wildtype transcripts cannot be discriminated from one another by siRNAs, it may be necessary to simultaneously carry out gene replacement with a modified form of the target RNA that is resistant to siRNA activity. To test this possibility, we have taken advantage of a potent siRNA that knocks down EGFP mRNA. In this system, wild-type EGFP expression is suppressed by the siRNA, whereas an EGFP construct with codon modifications in the target region that is otherwise fully functional is not downregulated. When expression of the wild-type message is inhibited, EGFP expression can be simultaneously restored by transfecting these cells with the codon-modified version of EGFP. These studies provide a detailed methodology and system for testing this strategy with RNA interference (RNAi).

Chronic myeloid leukemia (CML) is characterized by a reciprocal chromosomal translocation between chromosomes 9 and 22 t(9;22)(q34;q11) that causes fusion of the bcr and abl genes. Transcription and splicing of the fusion gene generate two major splice variants of the bcr/abl transcript that encode an oncoprotein with tyrosine kinase activity. We have taken advantage of lentiviral vectormediated delivery of anti-bcr/abl short hairpin RNAs (shRNA) to downregulate the bcr/abl transcript in Philadelphia chromosome-positive (Ph+) K562 leukemia cells. This downregulation caused complete inhibition of proliferation of these cells and was accompanied by >90% inhibition of the bcr/abl transcript and p210 protein. These results demonstrate the feasibility of using a lentiviral vector to stably transduce therapeutic shRNAs into leukemia cells for the potential ex vivo purging of Ph+ cells in an autologous hematopoietic cell transplant setting. Furthermore, the robust expression of the shRNAs from our lentiviral vector suggests that this system could be generally useful for the expression of other shRNAs.

The CCR5 beta-chemokine receptor is the coreceptor for macrophage-tropic (M-tropic) strains of HIV-1 and appears to be the principal coreceptor during early stages of human immunodeficiency virus-1 (HIV-1) infection. Approximately 1%-2% of the Western European Caucasian population is homozygous for a 32-bp deletion in the coding region of the CCR5 gene, rendering them less susceptible to HIV infection. These individuals still harbor a normal immune response, thereby making CCR5 an attractive cellular target for anti-HIV therapies. Based on the natural population studies, reduction in CCR5 expression should not affect the physiologic function of the modified cells but should interfere with their susceptibility to HIV-1 infection. To downregulate this receptor, we have designed a hammerhead ribozyme (RZ) that specifically targets the CCR5 mRNA and lacks complementarity to other members of the chemokine receptor gene family. For expression of this highly specific ribozyme, we have taken advantage of the stable transcripts afforded by transcription from the RNA polymerase III (pol III)-based adenoviral VA1 gene. Importantly, the VA1-chimeric ribozyme is stably expressed with a half-life of almost 6 hours. Using this expression system, we show up to 70% downregulation of the elevated levels of CCR5 receptor in the HOS-CD4.CCR5 cell line. The monocytic cell line PM1 was stably transduced with the chimeric VA1 ribozyme constructs. In these cells, substantial resistance to challenge with an M-tropic but not a T-tropic HIV viral strain was observed, demonstrating specificity in downregulating the CCR5 coreceptor. The VA1-CCR5 ribozyme chimeras described in this study should prove useful in both studies of CCR5 receptor function and therapeutic intervention of monocytotropic HIV-1 infection. The VA1 vector described in this study is well suited for the stable cytoplasmic expression of other ribozyme constructs as well.

We have synthesized and tested a cis-cleaving ribozyme designed to have thermodynamically stable stem-loop structures. This cis-ribozyme cleaves very efficiently in vitro, with a cleavage rate of about 0.5/min. Surprisingly, during the course of in vitro transcription and cleavage of our ribozyme, a product of unusual mobility accumulates and coincides with a sharp decline in the rate of formation of cleavage products. Analyses of this electrophoretic variant demonstrated that it is formed by interactions of the cleavage products. Despite the fact that the products and ribozyme transcript are of identical sequence, the cleavage products interact only with one another and not with the uncleaved precursor. This suggests a significant structural difference between the cleaved and uncleaved ribozyme transcripts. Testing of this cis-ribozyme in both yeast and mammalian cells shows no significant cleavage activity in vivo. We conclude that the structure of the ribozyme flanking sequences is important for optimizing the rate of ribozyme cleavage, but this enhanced rate does not necessarily correlate with enhanced in vivo function.

Retroviruses require a specific host cellular tRNA primer for initiation of first-strand DNA synthesis. This primer is bound by viral proteins and copackaged into virions. We have exploited this property in the design and testing of an antiviral ribozyme fused to tRNA(3Lys), the primer used for lentiviral replication, including human immunodeficiency virus (HIV-1 and HIV-2). The chimera consists of tRNA(3Lys) covalently attached to a hammerhead ribozyme, which is targeted to the region immediately upstream of the primer binding site of the HIV-1 genome. The tRNA-ribozyme chimeric transcript is catalytically active in vitro and is efficiently bound by HIV reverse transcriptase with an affinity similar to that of tRNA(3Lys). We have expressed the chimeric RNAs from either the tRNA(3Lys) intragenic RNA polymerase III promoter or from a human U6 snRNA promoter. The U6 promoter results in up to 10-fold enhanced expression of the tRNA-ribozyme. Most importantly, the tRNA(3Lys)-ribozymes are encapsidated in HIV-1 virions such that they are effective in substantially reducing the level of infectious virus produced from cells cotransfected with HIV-1 proviral DNA. These results demonstrate the feasibility of using this novel strategy to reduce HIV infectivity and more generally indicate the potential power of using the retroviral primer tRNAs as tools for expressing and delivering ribozymes and other antiretroviral RNAs to the virion capsid.

Hammerhead ribozymes have been proposed as potential therapeutic agents for the treatment of viral and other diseases. However, a clear understanding of the cleavage reaction in vivo is not available at present. In these studies, we chose the yeast Saccharomyces cerevisiae as a model system to study the effects of hammerhead cleavage on gene expression in vivo. Several reporter genes were employed to monitor the self-cleaving characteristics of three different ribozymes. We show that these ribozymes decrease expression of some reporter genes by interfering with splice site selection or translation initiation and not by in vivo cleavage of the RNA transcripts. In fact, it appears that although these ribozymes can efficiently self-cleave the RNA in vitro, they are not able to function in vivo. We have identified a yeast splicing protein that interacts in vivo with our cis-ribozyme by specifically recognizing the ribozyme structure (Lin and Rossi, 1996). This interaction does not occur if different secondary structures are used in place of the ribozyme. The binding of this protein to the ribozyme can account for the inability of ribozymes to efficiently cleave in yeast. Remarkably, when yeast extracts are added to in vitro trans-cleavage reactions, the cleavage ability of the ribozyme is hampered, whereas the addition of mammalian extracts yields an enhancement of the reactions. These results confirm the presence of factor(s) that can block ribozyme function in the yeast intracellular environment.

To examine hammerhead ribozyme activity in the nuclear environment, we have used nuclei isolated from HTLV-I tax transformed fibroblasts to evaluate ribozymes targeted against HTLV-I tax RNA. The ribozyme activity in nuclei suspension was strongly dependent on the resistance of the particular ribozyme to endogenous nucleases. A ribozyme containing exclusively 2'-deoxynucleotides in its stems cleaved target RNA by its catalytic activity in the absence of proteins and caused degradation in their presence by induction of nuclear RNase H activity. A ribozyme containing 2'-amino- and 2'-fluoropyrimidine nucleosides in combination with terminal phosphorothioate linkages was significantly more stable in nuclei suspension and also exhibited a more than threefold higher cleavage efficacy than its unmodified counterpart. The increased resistance against nuclease degradation is mainly due to terminal phosphorothioate linkages, suggesting that both 5' and 3'-exonucleases are primarily responsible for the nuclear degradation of oligonucleotides.

We have analyzed expression of anti-HIV-1 hammerhead ribozymes in the context of retroviral vectors. To determine optimal vector designs for ribozyme expression, we compared three vectors, each of which contained the same pair of anti-HIV-1 hammerhead ribozymes in tandem. Despite the presence of vastly different amounts of vector-derived flanking sequences, the ribozymes produced by each vector had similar cleavage activity when assayed in vitro. The ribozyme vectors were packaged into amphotropic virion and used to transduce human CEM T lymphocytes. Analysis by Northern blot and RNAse protection assays demonstrated that the highest steady-state levels of ribozyme-containing transcripts were produced by a vector in which the ribozymes were expressed under transcriptional control of the vector MoMuLV LTR. Despite these differences in the levels of ribozyme transcripts achieved by the vectors, their ability to confer resistance to HIV-1 replication was similar. Therefore, other factors than the absolute levels of ribozymes play a role in determining the effectiveness of ribozyme vectors to inhibit HIV-1. These may include structural features of the transcripts that affect the antisense effects of the ribozyme constructs, the actual catalytic activity of the ribozymes, their RNA folding, the binding of proteins, and the intracellular localization. Greater understanding of these factors may permit more effective application of ribozymes to inhibit gene expression.