Enhancing RNA Interference

Enhancing RNA Interference

Tuesday, 25 June 2013

Nanoparticles that deliver short strands of RNA offer a way to treat cancer and other diseases by shutting off malfunctioning genes. Although this approach has shown some promise, scientists are still not sure exactly what happens to the nanoparticles once they get inside their target cells.

A new study from MIT sheds light on the nanoparticles' fate and suggests new ways to maximize delivery of the RNA strands they are carrying, known as short interfering RNA (siRNA).

Lipid nanoparticles (carrying siRNA) are 

shown as they are transported inside cells 

using endocytic vesicles. Credit: Daria 

Alakhova and Gaurav Sahay. 

"We've been able to develop nanoparticles that can deliver payloads into cells, but we didn't really understand how they do it," says Daniel Anderson, the Samuel Goldblith Associate Professor of Chemical Engineering at MIT.

"Once you know how it works, there's potential that you can tinker with the system and make it work better."

Anderson, a member of MIT's Koch Institute for Integrative Cancer Research and MIT's Institute for Medical Engineering and Science, is the leader of a research team that set out to examine how the nanoparticles and their drug payloads are processed at a cellular and subcellular level. Their findings appear in the June 23 issue of Nature Biotechnology. Robert Langer, the David H. Koch Institute Professor at MIT, is also an author of the paper.

One RNA-delivery approach that has shown particular promise is packaging the strands with a lipid like material; similar particles are now in clinical development for liver cancer and other diseases.

Through a process called RNA interference, siRNA targets messenger RNA (mRNA), which carries genetic instructions from a cell's DNA to the rest of the cell. When siRNA binds to mRNA, the message carried by that mRNA is destroyed. Exploiting that process could allow scientists to turn off genes that allow cancer cells to grow unchecked.

Scientists already knew that siRNA-carrying nanoparticles enter cells through a process, called endocytosis, by which cells engulf large molecules. The MIT team found that once the nanoparticles enter cells they become trapped in bubbles known as endocytic vesicles. This prevents most of the siRNA from reaching its target mRNA, which is located in the cell's cytosol (the main body of the cell).

This happens even with the most effective siRNA delivery materials, suggesting that there is a lot of room to improve the delivery rate, Anderson says.

"We believe that these particles can be made more efficient. They're already very efficient, to the point where micrograms of drug per kilogram of animal can work, but these types of studies give us clues as to how to improve performance," Anderson says.

Molecular traffic jam

The researchers found that once cells absorb the lipid-RNA nanoparticles, they are broken down within about an hour and excreted from the cells.

They also identified a protein called Niemann Pick type C1 (NPC1) as one of the major factors in the nanoparticle-recycling process. Without this protein, the particles could not be excreted from the cells, giving the siRNA more time to reach its targets.

"In the absence of the NPC1, there's a traffic jam, and siRNA gets more time to escape from that traffic jam because there is a backlog," says Gaurav Sahay, an MIT postdoc and lead author of the Nature Biotechnology paper.

In studies of cells grown in the lab without NPC1, the researchers found that the level of gene silencing achieved with RNA interference was 10 to 15 times greater than that in normal cells.

Lack of NPC1 also causes a rare lysosomal storage disorder that is usually fatal in childhood. The findings suggest that patients with this disorder might benefit greatly from potential RNA interference therapy delivered by this type of nanoparticle, the researchers say. They are now planning to study the effects of knocking out the NPC1 gene on siRNA delivery in animals, with an eye toward testing possible siRNA treatments for the disorder.

The researchers are also looking for other factors involved in nanoparticle recycling that could make good targets for possibly slowing down or blocking the recycling process, which they believe could help make RNA interference drugs much more potent. Possible ways to do that could include giving a drug that interferes with nanoparticle recycling, or creating nanoparticle materials that can more effectively evade the recycling process.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Gaurav Sahay, William Querbes, Christopher Alabi, Ahmed Eltoukhy, Sovan Sarkar, Christopher Zurenko, Emmanouil Karagiannis, Kevin Love, Delai Chen, Roberto Zoncu, Yosef Buganim, Avi Schroeder, Robert Langer & Daniel G Anderson

Nature Biotechnology (2013), doi:10.1038/nbt.2614

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mRNA Offers A Safer Way to Reprogram Cells

New technique could revert cells to immature state that can develop into any cell type
Saturday, 24 July 2010

In recent years, scientists have shown that they can reprogram human skin cells to an immature state that allows the cells to become any type of cell. This ability, known as pluripotency, holds the promise of treating diseases such as diabetes and Parkinson's disease by transforming the patients' own cells into replacements for the non-functioning tissue.

However, the techniques now used to transform cells pose some serious safety hazards. To deliver the genes necessary to reprogram cells to a pluripotent state, scientists use viruses carrying DNA, which then becomes integrated into the cell's own DNA. However, this so-called DNA-based reprogramming carries the risk of disrupting the cell's genome and leading it to become cancerous.

Now, for the first time, MIT researchers have shown that they can deliver those same reprogramming genes using RNA, the genetic material that normally ferries instructions from DNA to the cell's protein-making machinery. This method could prove much safer than DNA-based reprogramming, say the researchers, Associate Professor of Electrical and Biological Engineering Mehmet Fatih Yanik and electrical engineering graduate student Matthew Angel.

Yanik and Angel describe the method, also the subject of Angel's master's thesis, in the July 23 issue of the journal PLoS ONE.

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MIT researchers used RNA to induce these fibroblast cells to express four genes necessary to reprogram cells to an immature state. Credit: Yanik Laboratory, MIT.

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However, the researchers say they cannot yet claim to have reprogrammed the cells into a pluripotent state. To prove that, they would need to grow the cells in the lab for a longer period of time and study their ability to develop into other cell types — a process now underway in their lab. Their key achievement is demonstrating that the genes necessary for reprogramming can be delivered with RNA.

"Before this, nobody had a way to transfect cells multiple times with protein-encoding RNA," says Yanik. (Transfection is the process of introducing DNA or RNA into a cell without using viruses to deliver them.)

In 2006, researchers at Kyoto University showed they could reprogram mouse skin cells into a pluripotent, embryonic-like state with just four genes. More recently, other scientists have achieved the same result in human cells by delivering the proteins encoded by those genes directly into mature cells, but that process is more expensive, inefficient and time-consuming than reprogramming with DNA.

Yanik and Angel decided to pursue a new alternative by transfecting cells with messenger RNA (mRNA), a short-lived molecule that carries genetic instructions copied from DNA.

However, they found that RNA transfection poses a significant challenge: When added to mature human skin cells, mRNA provokes an immune response meant to defend against viruses made of RNA. Repeated exposure to long strands of RNA leads cells to undergo cell suicide, sacrificing themselves to help prevent the rest of the body from being infected.

Yanik and Angel knew that some RNA viruses, including hepatitis C, can successfully suppress that defensive response. After reviewing studies of hepatitis C's evasive mechanisms, they did experiments showing they could shut off the response by delivering short interfering RNA (siRNA) that blocks production of several proteins key to the response.

Once the defence mechanism is shut off, mRNA carrying the genes for cell reprogramming can be safely delivered. The researchers showed that they could induce cells to produce the reprogramming proteins for more than a week, by delivering siRNA and mRNA every other day.

Source: Massachusetts Institute of Technology
Contact: Jennifer Hirsch

Reference:
Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins
Matthew Angel and Mehmet Fatih Yanik.
PLoS ONE 23 July, 2010, 5(7): e11756. doi:10.1371/journal.pone.0011756
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siRNA Nanoparticles Can Silence Cancer Genes in Humans

Caltech-led researchers unveil scientific results from siRNA Phase I clinical trial in cancer patients
Sunday, 21 March 2010

A California Institute of Technology-led team of researchers and clinicians has published the first proof that a targeted nanoparticles — used as an experimental therapeutic and injected directly into a patient's bloodstream—can traffic into tumours, deliver double-stranded small interfering RNAs (siRNAs), and turn off an important cancer gene using a mechanism known as RNA interference (RNAi). Moreover, the team provided the first demonstration that this new type of therapy, infused into the bloodstream, can make its way to human tumours in a dose-dependent fashion — i.e., a higher number of nanoparticles sent into the body leads to a higher number of nanoparticles in the tumour cells.

These results, published in the March 21 advance online edition of the journal Nature, demonstrate the feasibility of using both nanoparticles and RNAi-based therapeutics in patients, and open the door for future "game-changing" therapeutics that attack cancer and other diseases at the genetic level, says Mark Davis, the Warren and Katharine Schlinger Professor of Chemical Engineering at Caltech, and the research team's leader.

The discovery of RNA interference, the mechanism by which double strands of RNA silence genes, won researchers Andrew Fire and Craig Mello the 2006 Nobel Prize in Physiology or Medicine. The scientists first reported finding this novel mechanism in worms in a 1998 Nature paper. Since then, the potential for this type of gene inhibition to lead to new therapies for diseases like cancer has been highly touted.

"RNAi is a new way to stop the production of proteins," says Davis. What makes it such a potentially powerful tool, he adds, is the fact that its target is not a protein. The vulnerable areas of a protein may be hidden within its three-dimensional folds, making it difficult for many therapeutics to reach them. In contrast, RNA interference targets the messenger RNA (mRNA) that encodes the information needed to make a protein in the first place.

"In principle, that means every protein now is druggable because its inhibition is accomplished by destroying the mRNA. And we can go after mRNAs in a very designed way given all the genomic data that are and will become available," says Davis.

Still, there have been numerous potential roadblocks to the application of RNAi technology as therapy in humans. One of the most problematic has been finding a way to ferry the therapeutics, which are made up of fragile siRNAs, into tumour cells after direct injection into the bloodstream. Davis, however, had a solution. Even before the discovery of RNAi, he and his team had begun working on ways to deliver nucleic acids into cells via systemic administration. They eventually created a four-component system — featuring a unique polymer — that can self-assemble into a targeted, siRNA-containing nanoparticle. The siRNA delivery system is under clinical development by Calando Pharmaceuticals, Inc., a Pasadena-based nanobiotech company.

"These nanoparticles are able to take the siRNAs to the targeted site within the body," says Davis. Once they reach their target — in this case, the cancer cells within tumours — the nanoparticles enter the cells and release the siRNAs.

The scientific results described in the Nature paper are from a Phase I clinical trial of these nanoparticles that began treating patients in May 2008. Phase I trials are, by definition, safety trials; the idea is to see if and at what level the drug or other therapy turns harmful or toxic. These trials can also provide an in-human scientific proof of concept — which is exactly what is being reported in the Nature paper.

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This electron micrograph shows the presence of numerous siRNA-containing targeted nanoparticles both entering and within a tumour cell. Credit: Caltech/Swaroop Mishra.

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Using a new technique developed at Caltech, the team was able to detect and image nanoparticles inside cells biopsied from the tumours of several of the trial's participants. In addition, Davis and his colleagues were able to show that the higher the nanoparticle dose administered to the patient, the higher the number of particles found inside the tumour cells — the first example of this kind of dose-dependent response using targeted nanoparticles.

Even better, Davis says, the evidence showed the siRNAs had done their job. In the tumour cells analyzed by the researchers, the mRNA encoding the cell-growth protein ribonucleotide reductase had been degraded. This degradation, in turn, led to a loss of the protein.

More to the point, the mRNA fragments found were exactly the length and sequence they should be if they had been cleaved in the spot targeted by the siRNA, notes Davis.

"It's the first time anyone has found an RNA fragment from a patient's cells showing the mRNA was cut at exactly the right base via the RNAi mechanism," he says.

"It proves that the RNAi mechanism can happen using siRNA in a human."

"There are many cancer targets that can be efficiently blocked in the laboratory using siRNA, but blocking them in the clinic have been elusive," says Antoni Ribas, associate professor of medicine and surgery at UCLA's Jonsson Comprehensive Cancer Center.

"This is because many of these targets are not amenable to be blocked by traditionally designed anti-cancer drugs. This research provides the first evidence that what works in the lab could help patients in the future by the specific delivery of siRNA using targeted nanoparticles. We can start thinking about targeting the un-targetable."

"Although these data are very early and more research is needed, this is a promising study of a novel cancer agent, and we are proud of our contribution to the initial clinical development of siRNA for the treatment of cancer," says Anthony Tolcher, director of clinical research at South Texas Accelerated Research Therapeutics (START).

"Promising data from the clinical trials validates our years of research at City of Hope into ribonucleotide reductase as a target for novel gene-based therapies for cancer," adds co-author Yun Yen, associate director for translational research at City of Hope.

"We are seeing for the first time the utility of siRNA as a cancer therapy and how nanotechnology can target cancer cells specifically."

The Phase I trial — sponsored by Calando Pharmaceuticals — is proceeding at START and UCLA's Jonsson Comprehensive Cancer Center, and the clinical results of the trial will be presented at a later time.

"At the very least, we've proven that the RNAi mechanism can be used in humans for therapy and that the targeted delivery of siRNA allows for systemic administration," Davis says.

"It is a very exciting time."

Reference:
Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticlesMark E. Davis, Jonathan E. Zuckerman, Chung Hang J. Choi, David Seligson, Anthony Tolcher, Christopher A. Alabi, Yun Yen, Jeremy D. Heidel & Antoni Ribas
Nature advance online publication 21 March 2010, doi:10.1038/nature08956
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