September 15, 2008 (Vol. 28, No. 16)
Players Are Buoyed by a Fast-Growing Field with a Tremendous Amount of Potential
One of the most dynamic areas of research is the study of RNA interference (RNAi). This is a huge field when you consider all the different aspects such as gene silencing and miRNA research. “It’s very complicated, but there is a lot of potential in it,” explains Martin Kreutz, Ph.D., R&D scientist, miRNA research at Qiagen.
“Qiagen has developed a robust and accurate method for transcriptome-wide miRNA quantification using SYBR Green detection-based, real-time PCR,” reports Dr. Kreutz, who will be speaking at Select Biosciences “RNAi Europe” to be held in Stockholm later this month. “The miScript System is highly specific, sensitive, and requires very small amounts of input RNA,” he adds.
The miScript System is a three-component system that comprises the miScript Reverse Transcription Kit, miScript SYBR Green PCR Kit, and miScript Primer Assay. The miScript Reverse Transcription Kit enables reverse transcription of miRNAs, mRNAs, and other noncoding RNAs into cDNA in a single step. The miScript Primer Assay used in combination with the miScript SYBR Green PCR Kit enables real-time PCR of miRNA using SYBR Green detection.
“In contrast to other systems, the cDNA generated using the miScript Reverse Transcription Kit can be used for detection of multiple miRNAs,” says Dr. Kreutz. “A single cDNA preparation can be used with different miRNA primer assays for detection of hundreds of miRNAs, allowing comprehensive expression profiling of known miRNAs. Our system is also well suited to accommodate new developments like 3´ heterogeneity.”
The miScript Reverse Transcription Kit also converts other noncoding RNAs and cellular mRNA into cDNA. This allows the detection of noncoding RNAs and mRNAs by real-time PCR using the appropriate primers (e.g., QuantiTect Primer Assays for mRNA detection).
Dr. Kreutz notes that one of the most interesting aspects of miScript is that it is not only useful for miRNAs but also other noncoding RNAs. “You are not restricted to working with miRNAs—you can also use these samples for multiple targets,” Dr. Kreutz adds. “You can look at a miRNA and its potential target mRNA in one sample. If you store these samples at -20ºC, you can even come back and review the cDNA for a miRNA that was not in your focus of interest when creating that cDNA, or a miRNA that was not even described then.”
Targeted Genome Editing
Rational genome engineering in mammalian cells has great potential across multiple research avenues. To capitalize on these opportunities, Sangamo Biosciences and Sigma-Aldrich recently partnered to commercialize the CompoZr™ technology, which enables high-frequency genome editing via the application of designed zinc finger nucleases (ZFNs).
“Drawing from our work with transformed cell lines, primary human cells, and multipotent stem cells, we will present several examples of single, double, and triple gene knockout, as well as targeted gene insertion into native chromosomal loci,” says Trevor Collingwood, Ph.D., manager of strategic development at Sigma-Aldrich.
Within these ZFNs, the DNA-binding specificity of the zinc finger protein determines the site of nuclease action. Such engineered ZFNs are able to recognize and bind to a specified locus and evoke a double-strand break in the targeted DNA with high efficiency and base-pair precision.
“The cell then employs the natural DNA repair processes of either homology-directed repair or nonhomologous end joining to heal the targeted break,” adds Dr. Collingwood. “These two pathways provide the investigator with the ability to provoke three unique outcomes in genome editing—gene correction, gene deletion, and targeted gene addition.”
The ability to design ZFNs to target specific endogenous loci with high precision enables the researcher to insert transgenes at defined locations, or even to tag endogenous genes with fluorescent markers to follow their expression. The speed and efficiency of this process enables multiple genomic changes in the same cell.
“For example,” Dr. Collingwood explains, “a triple gene knockout in CHO cells is a particularly exciting demonstration of the potential for serious custom engineering of cell lines using CompoZr.”
Dr. Collingwood explains that commercial applications of the ZFN technology initially focused on the optimization of cell lines such as CHO cells for enhanced bioproduction of recombinant therapeutics. Activity is now increasing in the areas of functional genomics and cell-based screening.
An important new application of CompoZr is in the field of animal models such as the rat, where existing methods are lacking. But the technology is not limited to mammalian species; recent publications have demonstrated the use of ZFNs for targeted gene knockout in zebrafish. Together, these applications offer new and enabling approaches to animal and cell-based systems for drug screening and toxicology, as well as recreating models of human disease, Dr. Collingwood says.
“The technology is still in its early days, but this is only the beginning,” notes Dr. Collingwood. “Our goal is to increase ZFN design capacity and throughput to the point where CompoZr technology can be applied by the researcher on a genome-wide scale.”
Extended Gene Silencing
Bio-Rad has collaborated with Integrated DNA Technologies to develop RNAi tool sets. The results of this two-year collaboration, which pairs IDT’s siRNA design and synthesis proficiency with Bio-Rad’s transfection and amplification expertise, delivers validated Dicer-Substrate 27-mer small interfering RNA (siRNA) duplexes for RNAi applications. “We’ll be presenting silencing longevity and potency data for Dicer-Substrate 27-mer siRNAs and matched 21-mer siRNAs,” reports Eli Hefner, Ph.D., senior scientist at Bio-Rad. “We will also be discussing our Dicer-Substrate 27-mer (siLentMer) product validation procedure,”
Dr. Hefner points out that not everyone has heard of synthetic Dicer-Substrates. Dicer-Substrate siRNAs (DsiRNAs), which have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration, had their beginnings with John Rossi at City of Hope. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RNA-induced silencing complex and serve as a sequence-specific guide to target degradation of complementary mRNA species.
“Despite the continuous evolution of sequence selection algorithms, many synthetic siRNAs still fail to achieve the desired level of silencing,” adds Dr. Hefner. “The only way to truly know that your siRNA will work is to buy one that was pretested. Even when an siRNA is capable of reducing the target gene mRNA to low levels, the corresponding protein concentration might not be affected. This can occur when the window of effective siRNA operation is shorter than the protein half life, hence the desire to identify RNAi-inducing molecules such as the siLentMer Dicer-Substrates that have a longer window of operation.”
Bio-Rad’s siLentMer-validated siRNAs help solve this dilemma by delivering effective gene silencing for an extended period of time using low siRNA concentrations, the company reports. According to Dr. Hefner and senior product manager Christina Whitman, they are functionally tested via RT-qPCR for 85% mRNA knockdown, and many will demonstrate sustained silencing for up to 6–9 days.
“We validate by testing in-house, and we are stringent with our passing criteria,” says Whitman. “For some experiments, synthetic siRNAs are able to achieve 70 percent silencing, which might be okay depending on your protein stability. With the validated siLentMer Dicer-substrates you are guaranteed the highest possible silencing of 85 percent or higher.”
siRNA Specificity Enhancement
In the last two years, Applied Biosystems has focused on improving siRNA by improving specificity, says Susan Magdaleno, Ph.D., senior manager, scientist at RNAi technologies. “Over the course of that time, we’ve found better predictive algorithms for siRNA as well as a novel arrangement of chemical modifications in the siRNA duplex that significantly reduce off-target effects as measured by microarray and cell-based assays. The result are siRNAs called Silencer® Select siRNAs, which are chemically modified siRNAs that are up to 100-fold more potent than first- or second-generation designs. You get higher specificity siRNA performance but not at the expense of siRNA potency.”
Dr. Magdaleno notes that these improvements to the siRNA duplex have a number of benefits. “One of the most important aspects is the chemical modification that enhances the specificity of the siRNA without compromising on the potency of the siRNA,” she adds. “The combination of the chemical modification with higher potency siRNA together results in siRNAs that demonstrate coherence of cellular phenotypes between different siRNAs sequences targeting the same mRNA that hasn’t been achievable until now. The end effect will be greater confidence in RNAi experimental results.”
Dr. Magdaleno’s presentation will describe the development of the chemically modified siRNA that resulted in Silencer Select siRNA. “The methodology we used during the development is important, because it was a great learning experience. We were constantly asking questions—how do you define an off-target event? How do you reduce it? How do we know we reduced it? We have identified a combination of bases that when modified, enhances specificity, but at the moment we don’t yet understand how the modification improves specificity. We know that the reagents researchers use are critically important in their experiments. They must have confidence in the technology they are using.”
“The availability of cellular models with silenced genes offers the possibility of hands-on studies based on RNAi. Access to such cell lines helps the researcher focus on experimental questions without the need to first construct a cellular model,” says Jean-François Têtu, Ph.D., product specialist at tebu-bio. “Tebu-bio offers SilenciX human adherent cells that have been modified by nonviral and safe transfection for long-term silencing of genes by RNAi.”
According to Dr. Têtu, SilenciX was developed in collaboration with Denis Biard from Commissariat à l’Energie Atomique. “SilenciX is the first ready-to-use cellular model that is stable and has guaranteed knock down at more that 70 percent extinction at the RNA level.”
This third-generation cell line is a robust cell model they produced with a new algorithm called designer of small interfering RNA that Ecole de Mines de Paris developed. This algorithm for predicting siRNA efficiency is based on a simple linear model.
As an RNA duplex progresses from discovery phase to the clinic several hundred grams of the duplex, manufactured to GMP standards, need to be sourced over the lifetime of the trials. “Since 1999, Avecia Biotechnology has been a supplier of GMP oligonucleotides,” reports Kevin Fettes, process development group leader.
“Recently, as the therapeutic potential of RNAi has become established, the demand for GMP materials to be used in clinical trials has increased,” Fettes says. “My presentation will discuss the technology used to manufacture RNA duplexes and considerations that need to be addressed as demand for the duplex increases throughout clinical trials and as process validation and commercial launch approach,” he says.
Avecia recently installed new large-scale high-pressure purification equipment for RNA/aptamers and the capacity to manufacture hundreds of kilograms of commercial product per year, according to Fettes. “The objective of our process-development group at Avecia is to provide efficient, robust, and scalable processes that can be used for clinical manufacture, process validation, and beyond,” he adds.