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Sunday, May 17, 2009

Improved personalized cancer treatment with new RNAi delivery

In technology that promises to one day allow drug delivery to be tailored to an individual patient and a particular cancer tumor, researchers at the University of California, San Diego School of Medicine, have developed an efficient system for delivering siRNA into primary cells. The work will be published in the May 17 in the advance on-line edition of Nature Biotechnology.

The team solved the problem of delivery of siRNAs into cells by making a PTD fusion protein with a double-stranded RNA-binding domain, termed PTD-DRBD, which masks the siRNA's negative charge. This allows the resultant fusion protein to enter the cell and deliver the siRNA into the cytoplasm where it specifically targets mRNAs from cancer-promoting genes and silences them.

The researchers have a startup, Traversa Therapeutics, which is commercializing this work.

Traversa's siRNA delivery technology is specifically designed to avoid the physical size and bioavailability problems inherent in the Liposome/cationic-lipid approach. The technology is non-cytotoxic, delivers to the entire cell population and all cell-types tested, and is dramatically smaller than a liposome. The Company expects the technology to provide improved pharmacokinetics, distribution and bioavailability over other methods. The technology supports delivery to primary and tumor cells, T cells, B cells, Macrophage, neuronal cells and human stem cells, where other approaches have failed. This ability to induce RNA interference in entire cell populations and all cell types in a non-cytotoxic fashion is unique to Traversa's technology and provides the Company's competitive advantage.

Traversa's siRNA delivery technology (PTD-DRBD) is a protein comprised of multiple Peptide Transduction Domains (PTD) linked to a Double-stranded RNA Binding Domain (DRBD). The PTD portion of the protein induces delivery into the cell through a fluid-uptake mechanism that all cells perform, called macropinocytosis. The DRBD portion of the protein initially binds to the siRNA, and later releases the siRNA once inside the cell.

RNA Interference (RNAi) is a recently discovered natural biological process. The Central Dogma of biology is that DNA makes RNA, and RNA subsequently makes protein. Because undesired proteins are the cause of most human disease, pharmaceutical drugs typically target select proteins and block their function. RNAi works upstream from the manufacture of protein in cells, silencing genes and thereby blocking the creation of these disease-causing proteins before they are made.

This breakthrough discovery is being harnessed by RNAi researchers to develop an entirely new class of human therapeutic that could potentially treat sixty percent of all human disease – the Interfering RNA. This new class of drugs brings with it enormous potential:

- Significantly improved specificity of target molecules
- Greater efficacy with fewer side effects
- New drugs for rare or difficult to treat diseases
- Reduced drug discovery timelines
- Faster response to pandemic infection

Interfering RNAs have tremendous selectivity, degrade only target RNAs, and yield specific gene silencing. However, due to their relatively large size (~14,000-18,000 Daltons), they require an additional delivery technology in order to enter cells and produce their intended effect.

"RNAi has an unbelievable potential to manage cancer and treat it," said Steven Dowdy, PhD, Howard Hughes Medical Institute Investigator and professor of cellular and molecular medicine at UC San Diego School of Medicine. "While there's still a long way to go, we have successfully developed a technology that allows for siRNA drug delivery into the entire population of cells, both primary and tumor-causing, without being toxic to the cells."

For many years, Dowdy has studied the cancer therapy potential of RNA inhibition which can be used to silence genes through short interfering, double-stranded RNA fragments called siRNAs. But delivery of siRNAs has proven difficult due to their size and negative electrical charge – which prohibits them from readily entering cells.

A small section of protein called a peptide transduction domain (PTD) has the ability to permeate cell membranes. Dowdy and colleagues saw the potential for PTDs as a delivery mechanism for getting siRNAs into cancer cells. He and his team had previously generated more than 50 "fusion proteins" using PTDs linked to tumor-suppressor proteins.

"Simply adding the siRNAs to a PTD didn't work, because siRNAs are highly negatively charged, while PTDs are positively charged, which results in aggregation with no cellular delivery," Dowdy explained. The team solved the problem by making a PTD fusion protein with a double-stranded RNA-binding domain, termed PTD-DRBD, which masks the siRNA's negative charge. This allows the resultant fusion protein to enter the cell and deliver the siRNA into the cytoplasm where it specifically targets mRNAs from cancer-promoting genes and silences them.

To determine the ability of this PTD-DRBD fusion protein to deliver siRNA, the researchers generated a human lung cancer reporter cell line. Using green and fluorescent protein and analyzing the cells using flow cytometry analysis, they were able to determine the magnitude of RNA inhibitory response and the percentage of cells undergoing this response. They found that the entire cellular population underwent a maximum RNAi response. Similar results were obtained in primary cells and cancer cell lines.

"We were subsequently able to introduce gene silencing proteins into a large percentage of various cell types, including T cells, endothelial cells and human embryonic stem cells," said Dowdy. "Importantly, we observed no toxicity to the cells or innate immune responses, and a minimal number of transcriptional off-target changes."

These RNAi methods can be continually tweaked to combat new mutations – a way to overcome a major problem associated with current cancer therapies. "Such therapies can't be used a second time if a cancer tumor returns, because the tumor has mutated the target gene to avoid the drug binding," said Dowdy. "But since the synthetic siRNA is designed to bind to a single mutation and only that mutation on the genome, it can be easily and rapidly changed while maintaining the delivery system – the PTD-DRBD fusion protein."

"Cancer is a complex, genetic disease that is different in every patient," Dowdy added. "This is still in early stages, but I believe the siRNA-induced RNAi approach to personalized cancer treatment is the only thing on the table."

Wednesday, May 6, 2009

DNA Boxes Could Deliver Drugs

Chemistry World is reporting that Danish researchers have made a nano-sized box out of DNA that can be locked or opened in response to 'keys' made from short strands of DNA. By changing the nature or number of these keys, it should be possible to use the boxes as sensors, drug delivery systems or even molecular computers.

To make the box shape, the team took a long, circular single strand of DNA from a virus that infects bacteria called bacteriophage M13. This M13 sequence is a cheap source of single-stranded DNA and is convenient size for building with. To turn this ring of DNA into a box, the team used a computer to work out exactly the right combination of short strands of complementary DNA which could 'staple' the appropriate areas of the ring together to get the desired box shape. When they mixed the M13 strand with the 220 short 'staple strands' and heated them up for an hour, the boxes neatly self-assembled.

Kjems reveals that the group have already had some success with putting cargo inside the boxes, including enzymes and quantum dots. 'It's quite big (about 30nm) inside - it could fit virus particles or quite big enzymes and other macromolecules.' In terms of applications, Kjems can foresee three main purposes for the box: 'One is as a calculator or logic gate; the second is for controlled release, for example of drugs, in response to external stimuli; and the last is as a sensor - where the thing you are sensing causes the box to open or close and give a readout.'

The DNA origami technique is quite straightforward, Mao comments, so could be applied to all sorts of similar structures. The fact that the box can be easily opened and closed also makes it ideal for moving guest molecules around. 'I'm really looking forward to seeing what the group do next,' he adds.

MIT Technology Review also has coverage.

Deoxyribose sugar cubes: Because complementary regions of DNA like to pair up, researchers were able to design a long strand of DNA that, combined with many tiny DNA staples, would automatically assemble itself into a nano-sized box. This technique is known as DNA origami. Here, the boxes were imaged using cryo-electron tomography to confirm their cubelike structures and hollow interior.
Credit: : Ebbe S. Andersen, Aarhus University

21 pages of supplemental information from the Journal Nature article.

The abstract in the journal Nature. [Nature 459, 73-76 (7 May 2009) | doi:10.1038/nature07971; Received 9 November 2008; Accepted 6 March 2009]
Self-assembly of a nanoscale DNA box with a controllable lid

The unique structural motifs and self-recognition properties of DNA can be exploited to generate self-assembling DNA nanostructures of specific shapes using a 'bottom-up' approach1. Several assembly strategies have been developed for building complex three-dimensional (3D) DNA nanostructures. Recently, the DNA 'origami' method was used to build two-dimensional addressable DNA structures of arbitrary shape that can be used as platforms to arrange nanomaterials with high precision and specificity. A long-term goal of this field has been to construct fully addressable 3D DNA nanostructures. Here we extend the DNA origami method into three dimensions by creating an addressable DNA box 42 36 36 nm3 in size that can be opened in the presence of externally supplied DNA 'keys'. We thoroughly characterize the structure of this DNA box using cryogenic transmission electron microscopy, small-angle X-ray scattering and atomic force microscopy, and use fluorescence resonance energy transfer to optically monitor the opening of the lid. Controlled access to the interior compartment of this DNA nanocontainer could yield several interesting applications, for example as a logic sensor for multiple-sequence signals or for the controlled release of nanocargos.


The DNA origami design software program with documentation and tutorials is
available here: