November 15, 2008 (Vol. 28, No. 20)

Lloyd Dunlap

Gene-Function Analysis Use on Rise as a Result of Increased Knowledge

There are now more than 500 known human miRNAs, with ongoing discovery directed toward an estimated 10,000. More than 50% of human miRNA genes are located in cancer-associated genomic regions. miRNA expression patterns are associated with a number of tumor types, as well as regulating processes such as cellular development, metabolism, and viral infection.

The SMI Group Conference entitled “RNAi, siRNA, and miRNA”, held in London, addressed developments in understanding the biology of RNAi and its potential applications in gene-function analysis and therapy, as well as siRNA and new developments in the world of miRNAs.

Representing Beckman Research Institute’s City of Hope, John Rossi, Ph.D., dean of the graduate school of biological sciences, spoke on “Small RNA-Mediated Transcriptional Gene Silencing and Transcriptional Activation.” The City of Hope—one of five institutes endowed when the Beckman family sold their interest to SmithKline—focuses on research for catastrophic diseases.

Dr. Rossi answered the question about whether RNAi participates in mammalian transcriptional gene silencing by citing the work of Kuwabara and others that a small modulatory dsRNA specifies the fate of adult neural stem cells. The experimental rationale employed in Dr. Rossi’s work involved using the RASSF1A gene—which encodes a tumor suppressor heavily methylated and epigenetically silenced in many primary tumors—as a model system.

Further, HeLa cells provided a model system for testing the hypothesis of siRNA-directed methylation of human DNA, since this gene is fully unmethylated and transcriptionally active in these cells. Finally, Dr. Rossi’s work expressed short hairpin RNA that can be processed into siRNAs targeting methylation-sensitive CpG islands in the RASSF1A CpG and assayed for introduction of methylation.

Dr. Rossi determined that siRNA-guided methylation reduced expression in RASSF1A by 60–70%. In other areas such as the pcDNA vector, mutant siRNA, and HeLa cells where no methylation occurred, showed no reduction in transcription.

Although fewer than 1-in-50 clones showed DNA methylation, the population of cells showed 60–70% inhibition of gene expression, indicating that silencing may be associated with histone methylation as in fission yeast.

The City of Hope team then created a CCR5 promoter-EGFP fusion cell line and transfected siRNAs targeting the promoter region. They followed up with silencing analyses and ChIP analyses of CCR5 and RASSF1A-silenced promoters and identified Argonaute 1 and H3K27 trimethyl histone marks associated with silencing.

Argonaute 1 was heavily enriched at the targeted loci—by as much as 185-fold initially, before leveling off to about 20-fold. “Without Argonaute 1 you can’t silence CCR5 promoters,” explained Dr. Rossi.

CCR5, a chemokine receptor, is absent in many Scandinavians who are highly resistant to the HIV virus. The resistant population has been found to be homozygous for deletion of CCR5 receptors. “We are trying to understand the mechanism for permanently shutting down CCR5 to prevent many viral infections,” he added.

Understanding the functional requirements for TGS is critical to using this approach in a clinical setting. Most recently, the Rossi lab demonstrated an endogenous miRNA shown to regulate expression of an RNA Polymerase III subunit, providing the first demonstration of a miRNA silencing a promoter in mammalian cells.

Knock-down and viability vs. siRNA concentration, and plate-to-plate variability in knock-down at 500 nM siRNA concentration

Genome-Wide RNAi Screening

Johan Pihl, product manager for Cellaxess®HT at Cellectricon (Sweden), spoke on “An Automated High Throughput System for Genome-Wide RNAi Screening.” Cellectricon launched its first product in 2003 and has a subsidiary in Gaithersburg, MD. “We identify bottlenecks in drug discovery and apply our unique screening systems to solve problems,” said Pihl.

The company’s two product platforms are Dynaflow® microfluidic screening platform for ion channel research and the Cellaxess® capillary electrode electroporation platform for “efficient transfection of biologically relevant cell lines and primary cell types with high viability.” Pihl characterizes recently introduced Cellaxess HT as “the world’s first high-throughput electroporation system for genome-wide RNAi & cDNA screening.”

The Cellaxess transfection process enables electroporation directly in microtiter plates and culture dishes. The process handles even hard-to-transfect cell lines and primary cells and can be integrated with standard liquid handling processes, facilitating electroporation of cells in HT formats.

“Cellaxess HT transfects a very wide range of cell types and lines previously not possible to screen on,” Pihl observes. A key characteristic of the process is its low toxicity and resulting high cell viability due to less cell processing and low current, compared to conventional electroporation.

Developed in collaboration with leading pharma and research groups for RNAi and cDNA screening, Cellaxess is a focused system for reagent-free delivery of genetic material to primary cells and cell lines with “true high-throughput capabilities” via a fully automated workstation for transfection in the 384-well format.

The process can be integrated with LAS systems from all major providers, and features throughput of up to 50,000 wells per day, depending on configuration. The system is plug-and-play for automated RNAi delivery, with no optimization of liquid handling or transfection parameters required. Transfection kits, good for 115,000 wells, include an electroporation module, 384-well plates, and HTS-compatible buffers.

Pihl provided the conference with four examples of results achievable with the Cellaxess process. The first, a demonstration of gene silencing in HeLa-S3 cells by knockdown of PLK-1, assessed viability 72 hours post-transfection using nuclear staining and automated microscopy, and found 95% viability and 88% knockdown. PLK-1 is a cell-cycle associated gene, upregulated and essential for a variety of cancer cell lines.

Similar results were achieved in the other three cases—gene silencing in differentiated 3T3-L1 adipocytes where the lamin A/C (1) gene was chosen for evaluation of knockdown; human primary vein endothelial cells where knockdown was evaluated by RT-PCR; and neuro-2a cells. In all cases viability was 90% or better and knockdown 83%, except in the case of the lamin A/C (1) gene where knockdown was 75%.

SNALP-Formulated siRNAs

Current research under way by Ines Royaux, Ph.D., senior scientist, Johnson & Johnson Pharmaceutical Research Division, focuses on liver-specific knockdown using SNALP-formulated siRNAs to study pathways in lipoprotein secretion and steatosis. “As the application of RNA interference in vivo further develops, we are pursuing several promising technologies for systemic delivery,” she noted.

The goal of the current study was to evaluate the stable nucleic acid particles (SNALP) systemic RNAi delivery platform to knockdown genes encoding for key enzymes in triglyceride synthesis. SNALP-formulated siRNAs were found to effectively knockdown mRNA levels by more than 90% in liver compared to a SNALP-delivered scrambled siRNA control.

The knockdown in liver was further investigated following a three-week high-fat feeding challenge. The results demonstrated the efficient in vivo SNALP-mediated delivery of siRNA by systemic route and the utility of aiming at new targets to reduce fat storage in liver and improve hepatic steatosis.

Dr. Royaux has also been investigating peptide-mediated delivery of siRNA across the blood-brain barrier to the brain, local delivery of RNAi using viral vectors such as lentiviral and HSV-1-based vectors, and liposome formulations for system delivery of siRNA. The goal is to develop a general system by using a targeting ligand.

Human LNA Test

Reporting on advances in locked nucleic acid (LNA) development, Troels Koch, Ph.D., vp, research at Santaris Pharma, spoke at the conference regarding “Potent Single-Stranded Inhibition of Coding and Non-Coding RNA.”

Dr. Koch’s participation at the meeting came less than two weeks after his company announced that its LNA drug, SPC3649 (LNA-antimiR™-122) had become the first miRNA medicine to be tested in humans. The first clinical trial will involve 48 male volunteers.

“LNA’s high binding strength to its complementary target is an important component to the high potency, which in turn, allows significant reductions in dosage concentrations combined with good specificity,” Dr. Koch explained. Bio-stability is also much improved compared to benchmark phosphorothioate chemistry. LNA oligonucleotides can be designed as both gap-mers for targeting mRNA and mix-mers for targeting miRNA.

The former segments of LNA nucleotides are posted at either end of the DNA/PS chain, while a mix-mer—as the name implies—has LNA nucleotides interspersed in several locations. Biodistribution of a 16-mer gap-mer in mice 24 hours after a single intravenous dose was broad and included bone marrow (5%), kidney (21%), liver (11%), lymph node (14%), and spleen (4%).

Dr. Koch described a study in mice with a 16-mer LNA against ApoB-100 mRNA, the major apolipoprotein of low-density lipoproteins, which is responsible for carrying cholesterol to tissues. Mice dosed three consecutive days at 25 mg/kg showed an 80% reduction in ApoB mRNA and a 70% reduction in total plasma cholesterol.

The effect was of long duration, moderating to about 50% after approximately three weeks. Dr. Koch also reported a significant potency increase of ApoB mRNA down regulation in mouse liver going from the 16-mer to a 12-mer. The 12-mer showed significantly higher potency for ApoB mRNA down-regulation and the corresponding reductions in plasma cholesterol was also more efficacious. In fact, a total dose of 3 mg/kg was enough to reduce total plasma cholesterol by 50%.

Dr. Koch explained that the potency induction by LNA observed for mRNA down-regulations was also an important property for potent miRNA inhibition. The potency for miRNA inhibition of LNA mix-mers closely followed the affinity of the mix-mers for the mature miRNA. The most potent 15-mer, SPC3649, was 10–20 times more potent for miR122 inhibition compared to a 16-mer with 10 degrees lower Tm.

Dr. Koch showed that, following the inhibition of miR122 expression, total plasma cholesterol was reduced in mice and in nonhuman primates. In the monkey study, four groups of five African green monkeys were dosed at 1, 3, and 10 mg/kg levels on days 1, 3, and 5, while the fourth group acted as the control.

Relative plasma cholesterol reduction was dose-related generally with the lowest level reached in all three groups between day 20 and day 40. Liver biopsies at day 6 demonstrated localization of the LNA-antimiR in hepatocytes, and complete duplex formation between SPC3649 and miR122.

It was also shown that inhibition of miR122 by SPC3649 led to a potent inhibition of HCV replication in HUH7 hepatocytes. The latter has interest for HCV treatments since miR122 is a host factor and thus represents a potential new approach for treating HCV patients.

Finally, LNAs are generally well tolerated, with low acute toxicity compared to PS oligonucleotides, and with no clinically meaningful biochemical, hematologic, or histopathologic toxic effects in rodents or monkeys at clinically relevant doses.

As a result of the improved affinity, pharmacokinetics, and toxicity profiles, LNAs possesses attractive drug-like properties and a useful therapeutic index while providing siRNA-like potencies without the complications of complex delivery systems. The conclusion in the presentation was that LNA serves as a common platform for potent inhibition of both mRNA and microRNA.

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