This is a PTD-DRBD fusion protein. Credit: Dowdy Lab/UC San Diego 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. "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."
Monday, May 25, 2009
The future of personalized cancer treatment: An entirely new direction for RNAi delivery
The illustration shows a cross-section of part of the cochlea, the fluid-filled part of the inner ear that converts vibrations from incoming sounds into nerve signals that travel to the brain via the auditory nerve. University of Utah and Baylor College of Medicine researchers found evidence that stereocilia -- bundles of tiny hair-like tubes atop "hair cells" in the cochlea -- dance back and forth to mechanically amplify incoming sounds via what is known as the "flexoelectric effect." Credit: William Brownell, Baylor College of Medicine. Utah and Texas researchers have learned how quiet sounds are magnified by bundles of tiny, hair-like tubes atop "hair cells" in the ear: when the tubes dance back and forth, they act as "flexoelectric motors" that amplify sound mechanically. "We are reporting discovery of a new nanoscale motor in the ear," says Richard Rabbitt, the study's principal author and a professor and chair of bioengineering at the University of Utah College of Engineering. "The ear has a mechanical amplifier in it that uses electrical power to do mechanical amplification." "It's like a car's power steering system," he adds. "You turn the wheel and mechanical power is added. Here, the incoming sound is like your hand turning the wheel, but to drive, you need to add power to it. These hair bundles add power to the sound. If you did not have this mechanism, you would need a powerful hearing aid." The new study is scheduled for publication Wednesday, April 22 in PLoS ONE, a journal published by the Public Library of Science. The first author is Katie Breneman, a bioengineering doctoral student at the University of Utah. The study was coauthored by William Brownell, a professor of otolaryngology (ear, nose and throat medicine) at Baylor College of Medicine in Houston. The researchers speculate flexoelectrical conversion of electricity into mechanical work also might be involved in processes such as memory formation and food digestion. Dancing Cells and Hair-like Tubes in Your Ears Previous research elsewhere indicated that hair cells within the cochlea of the inner ear can "dance" - elongate and contract - to help amplify sounds. The new study shows sounds also may be amplified by the back-and-forth flexing or "dancing" of "stereocilia," which are the 50 to 300 hair-like nanotubes projecting from the top of each hair cell. Such flexing converts an electric signal generated by incoming sound into mechanical work - namely, more flexing of the stereocilia - thereby amplifying the sound by what is known as a flexoelectric effect. "Dancing hairs help you hear," says Breneman. The study "suggests sensory cells in the ear are compelled to move when they hear sounds, just like a music aficionado might dance at a concert. In this case, however, they'll dance in response to sounds as miniscule as the sound of your own blood flow pulsating in your ear." In a yet-unpublished upcoming study, Rabbitt, Breneman and Brownell find evidence the hair cells themselves - like the stereocilia bundles atop those cells - also amplify sound by getting longer and shorter due to flexoelectricity. Rabbitt and Brownell estimate the combined flexoelectric amplification - by both hair cells and the hair-like stereocilia atop hair cells - makes it possible for humans to hear the quietest 35 to 40 decibels of their range of hearing. Rabbitt says the flexoelectric amplifiers are needed to hear sounds quieter than the level of comfortable conversation. "The beauty of the amplifier is that it allows you to hear very quiet sounds," Brownell says. Rabbit says that because hair cells die as people age, older people often "need a hearing aid because amplification by the hair cells is not working." Because hair-like stereocilia also are involved in our sense of balance, the flexing of stereocilia not only contributes to hearing, but "also likely is involved in our sense of gravity, motion and orientation - all the things needed to have balance," Rabbitt says. The new study is part of an effort by researchers to understand the amazing sensitivity of human hearing. Rabbitt says the hair cells are so sensitive they can detect sounds almost as small as those caused by Brownian motion, which is the irregular movement of particles suspended in gas or liquid and bombarded by molecules or atoms. Richard Rabbitt, professor and chair of bioengineering at the University of Utah, led a study indicating that a mechanism known as "flexoelectricity" works within the cochlea of the ear to amplify quiet sounds. Bundles of tiny hair-like tubes called stereocilia dance back and forth atop "hair cells" in the cochlea, serving as "flexoelectric motors" to amplify sound mechanically. Rabbitt says it is like power steering for your hearing. Credit: University of Utah College of Engineering. Hair cells are inside the inner ears of many animals. They are within the ear's cochlea, which is the spiral, snail-shell-shaped cavity where incoming sound vibrations are converted into nerve impulses and sent to the brain. Incoming sounds must be amplified because incoming sound waves are "damped" by fluid that fills the inner ear. Hair cells are about 10 microns wide, and 30 to 100 microns long. By comparison, a human hair is roughly 100 microns wide. A micron is one-millionth of a meter. The hair-like stereocilia tubes poking out the top of a hair cell are each a mere 1 to 10 microns long and about 200 nanometers wide, or 200 billionths of a meter wide. Brownell says the new study shows how the flexoelectric effect "can account for the amplification of sound in the cochlea." Stereocilia essentially are membranes that have been rolled into tiny tubes, so "the fact that a membrane can generate acoustic [mechanical] energy is novel," says Brownell. "Imagine hearing a soap bubble talk." Flexoelectricity in a membrane was noted a few decades ago when a researcher in Europe showed that flexing or bending a simple membrane in a laboratory generated an electrical field. Then, in 1983, Brownell showed that a hair cell from a guinea pig's ear changed in length when an electric field was applied to it in a lab dish. The length of stereocilia changes along the coiled length of the cochlea. Different lengths are sensitive to different frequencies of sound. And different animals have different ranges of stereocilia lengths. Breneman and colleagues devised math formulas and used computer simulations to arrive at the new study's key finding: The flexoelectric amplifier can explain why varying lengths of stereocilia predict which sound frequencies are heard most easily by a variety of animals, from humans to bats, mice, turtles, chickens and lizards. "They found that a longer stereocilium was more efficient if it was receiving low-frequency sounds," while shorter stereocilia most efficiently amplified high-frequency sound, Brownell says. Breneman says scientists now know of five ways the ears amplify sound, and "what makes this one unique is that it would be present in the stereocilia bundles of all hair cells, not only outer hair cells." The cochleae of humans and other mammals have "inner hair cells" that sense sound passively and active "outer hair cells" that amplify sounds. Other higher animals have hair cells, without a distinction between inner and outer. Because the new study shows the dancing hair-like stereocilia act like an amplifier on any hair cell, "it explains how this amplifier may work in all higher animals like birds and reptiles, not just humans," Rabbitt says. How the Amplifier Works in the Inner Ear - and Perhaps Elsewhere When sound enters the cochlea and reaches the hair cells, sound pressure makes the hair-like stereocilia tubes "pivot left or right similar to the way a signpost bends in heavy wind," Breneman says. The tops of the tubes are connected to each other by protein filaments. Where each filament comes in contact with the top end of a stereocilium tube, there is an "ion channel" that opens and closes as the bundle of stereocilia sway back and forth. When the channel opens, electrically charged calcium and potassium ions flow into the tubes. That changes the electric voltage across the membrane encasing each stereocilium, making the tubes flex and dance even more. Such flexoelectricity amplifies the sound and ultimately releases neurotransmitter chemicals from the bottom of the hair cells, sending the sound's nerve signal to the brain, Breneman says. "We've got these nanotubes - stereocilia - moving left and right and converting electrical power [from ions] into mechanical amplification of sound-induced vibrations in the ear," Rabbitt says. He says the "flexoelectric motor" is the collective movement of the stereocilia in response to sound. Brownell says the new study - showing that sound is amplified by "dancing" membrane tubes atop hair cells - adds to growing evidence that membranes do not "just sit there," but instead are "dynamic structures capable of doing work using a mechanism called flexoelectricity." Brownell and Rabbitt note that stereocilia involved in amplifying hearing have similarities with other tube-like structures in the human body, such as villi in the gut, dendritic spines on the signal-receiving ends of nerve cells and growth cones on the signal-transmitting axon ends of growing nerve cells. So they speculate flexoelectricity may play a role in how villi in the intestines help absorb food and how nerves grow and repair themselves. "There is some evidence that dendrites and axons change their diameter during intracellular voltage changes, and that could well have flexoelectric origins," says Rabbitt. "Any time you have a membrane with small diameter - like in axons, dendrites and synaptic vesicles [located between nerve cells], there will be large flexoelectric forces and effects. Therefore, the flexoelectric effect may be at work in things like learning and memory. But that's pretty speculative."Power steering for your hearing: Ears have tiny 'flexoelectric' motors to amplify sound
Small molecules might block mutant protein production in Huntington's disease
Dr. David Corey, professor of pharmacology and biochemistry (center) and from left, Dr. Masayuki Matsui, postdoctoral researcher, and Dr. Jiaxin Hu, assistant instructor in pharmacology Molecules that selectively interfere with protein production can stop human cells from making the abnormal molecules that cause Huntington's disease, researchers at UT Southwestern Medical Center have found. These man-made molecules also were effective against the abnormal protein that causes Machado-Joseph disease, a neurological condition similar to Huntington's. The work has been done only in cultured cells, and it will take years before the effectiveness of this process can be tested in patients, the researchers cautioned. "I wouldn't want to give Huntington's patients or gene carriers any false hope, but I am excited about where this work might go in the future," said Dr. David Corey, professor of pharmacology and biochemistry at UT Southwestern and senior author of the study, which appears online May 3 in Nature Biotechnology. The researchers' approach relies on interfering with the steps by which genetic information in cells is "translated" from DNA to make proteins, which carry out vital biological functions. Huntington's and Machado-Joseph are fatal inherited diseases caused by abnormal repeats of a small segment in a person's DNA, or genetic code, represented by the letters CAG. These mutations result in the body producing malfunctioning proteins that cause the diseases. The more repeats, the worse the disease, and the earlier in life it appears. A person with the disease carries one normal copy of the gene and one mutated copy in his or her cells. In Huntington's, this CAG repeat occurs in a gene called huntingtin, and in Machado-Joseph, it occurs in a gene called ataxin-3. A person with Huntington's can have up to 100 CAG repeats. CAG repeats are involved in several other neurodegenerative diseases, including Fragile X syndrome, the most common form of mental retardation, and myotonic dystrophy. While these genes are best known for the devastating effects of their mutated forms, their normal forms are essential for embryonic development, nerve function and other bodily processes. Any treatment that interferes with the mutant forms must leave the normal forms as unaffected as possible, Dr. Corey said. "Attempting to intervene is very risky, but because the problem is important, it's worth doing," he said. In the current study, the researchers created short lengths of molecules that resemble ribonucleic acid (RNA), the chemical cousin of DNA. These mimics, called PNAs and LNAs, were specifically designed to bind to CAG repeats, preventing cells from creating the abnormal proteins. The researchers also designed short lengths of RNA called small interfering RNA, or siRNA, to interfere with CAG repeats. In cells from Huntington's patients, the PNAs, LNAs and siRNAs decreased the amount of mutant protein produced, in some cases up to 100 percent. The effect was greatest when the compounds interfered with long lengths of CAG repeats; the effectiveness varied, however, among cells taken from different patients. Some forms of these compounds left the normal forms of huntingtin and ataxin-3 proteins undisturbed, but other compounds partly or completely blocked their formation. In some cells, some of the RNA mimics drastically cut the production of both mutant and normal proteins - an undesirable effect, Dr. Corey said. These findings indicate that further tweaking of the molecular structures of the RNA mimics will be needed to minimize the effects on normal proteins. "It is encouraging that small chemical changes could substantially enhance selectivity," Dr. Corey said. "If we can test a handful of compounds and identify better ones, we have reason to believe that more testing will continue to produce significant improvement." Because this study was done in cultured cells, and not in whole animals or humans, it does not indicate how much of the abnormal proteins must be blocked to treat the disease effectively, he said. "Fifty percent inhibition might be enough, but that remains to be determined," Dr. Corey said. In future studies, the researchers plan to try these RNA mimics in whole animals, using several different mutations of the genes. Laurie Tompkins, who oversees neurogenetics grants at the National Institutes of Health's National Institute of General Medical Sciences, said the ability to control individual genes makes this work stand out. "By exploiting processes that occur in normal cells, Dr. Corey has come up with a clever way to do this that may well lead to new ways to combat Huntington's and other related diseases," she said.
The image shows a hot spring in Yellowstone National Park, Montana, a site where bacteria containing chlorosomes can be found in the brightly colored mats. At the upper left is a thin-section electron micrograph of the green sulfur bacterium Chlorobaculum tepidum, showing chlorosomes along the periphery of the cells as light-colored ovals. The next image is an electron micrograph of an isolated chlorosome from the bchQRU mutant, and the next image is a cryo-electron micrograph of the same. Finally, the last panel at the right shows a molecular model of the chlorophylls in the chlorosome. Individual chlorophyll molecules are illustrated in green and show their hydrophobic tails pointing outward. Credit: Image by Donald Bryant, Penn State University, courtesy of Proceedings of the National Academy of Sciences (PhysOrg.com) -- An international team of scientists has determined the structure of the chlorophyll molecules in green bacteria that are responsible for harvesting light energy. The team's results one day could be used to build artificial photosynthetic systems, such as those that convert solar energy to electrical energy. A research paper about the discovery will be published on 4 May 2009 in the Proceedings of the National Academy of Sciences. The scientists found that the chlorophylls are highly efficient at harvesting light energy. "We found that the orientation of the chlorophyll molecules make green bacteria extremely efficient at harvesting light," said Donald Bryant, Ernest C. Pollard Professor of Biotechnology at Penn State and one of the team's leaders. According to Bryant, green bacteria are a group of organisms that generally live in extremely low-light environments, such as in light-deprived regions of hot springs and at depths of 100 meters in the Black Sea. The bacteria contain structures called chlorosomes, which contain up to 250,000 chlorophylls. "The ability to capture light energy and rapidly deliver it to where it needs to go is essential to these bacteria, some of which see only a few photons of light per chlorophyll per day." The Nuclear Magnetic Resonance (NMR) results enabled the scientists to determine that the chlorophyll molecules (shown in green and orange) in green bacteria are arranged in helical spirals, and are positioned at an angle to the long axis of the nanotubes. Credit: Image by Donald Bryant, Penn State University, courtesy of Proceedings of the National Academy of Sciences "Each chlorosome in a green bacterium has a unique organization," said Bryant. "They are like little andouille sausages. When you take cross-sections of andouille sausages, you see different patterns of meat and fat; no two sausages are alike in size or content, although there is some structure inside, nevertheless. Chlorosomes in green bacteria are like andouille sausages, and the variability in their compositions had prevented scientists from using X-ray crystallography to characterize the internal structure." To get around this problem, the team used a combination of techniques to study the chlorosome. They used genetic techniques to create a mutant bacterium with a more regular internal structure, cryo-electron microscopy to identify the larger distance constraints for the chlorosome, solid-state nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the chlorosome's component chlorophyll molecules, and modeling to bring together all of the pieces and create a final picture of the chlorosome. First, the team created a mutant bacterium in order to determine why the chlorophyll molecules in green bacteria became increasingly complex over evolutionary time. To create the mutant, they inactivated three genes that green bacteria acquired late in their evolution. The team suspected that the genes were responsible for improving the bacteria's light-harvesting capabilities. "Essentially, we went backward in evolutionary time to an intermediate state in order to understand, in part, why green bacteria acquired these genes," Bryant said. The team found that the more evolved, wild-type bacteria grow faster at all light intensities than the mutant form. "Indeed, the reason that chlorophylls became more complex was to increase light-harvesting efficiency," said Bryant. Next, the team isolated chlorosomes from the mutant and the wild-type forms of the bacteria and used cryo-electron microscopy -- a type of electron microscopy that is performed at super-cold cryogenic temperatures -- to take pictures of the chlorosomes. The pictures revealed that chlorophyll molecules inside chlorosomes have a nanotube shape. "They are like Russian dolls, with one concentric tube fitting inside the next," said Bryant. "The mutant bacterium's chlorosomes contain only one set of tubes, whereas the wild-type chlorosomes contain many tubes, each arranged in a unique pattern, like those andouille sausages." The team then went a step further and used solid-state NMR spectroscopy -- a technique in which samples are spun very rapidly and exposed to a magnetic field -- to look deep inside the chlorosome. This technique enables researchers to understand the relationships between atomic nuclei in a sample and, ultimately, to acquire structural information about the molecules of interest. "The NMR data revealed to us that the individual chlorophyll molecules in green bacteria are arranged in dimers -- molecules consisting of two identical simpler molecules -- with their long hydrophobic, or water-repellent, tails sticking out of either side," said Bryant. "We also learned precisely how the chlorophyll molecules attach to one another, and we were able to measure the distance between chlorophyll molecules. The cryo-electron microscopy pictures showed gross structural details and distances, and the NMR results allowed us to quantify these distances as well, and confirmed to us that what were were seeing was, in fact, stacks of the chlorophyll molecules all lined up," he said. The NMR results also enabled the scientists to determine that the chlorophyll molecules in green bacteria are arranged in helical spirals. In the mutant bacteria, the chlorophyll molecules are positioned at a nearly 90-degree angle in relation to the long axis of the nanotubes, whereas the angle is less steep in the wild-type organism. "It's the orientation of the chlorophyll molecules that is the most important thing here," said Bryant. The last steps for the team were to pull together all of their data and to create a detailed computer model of the structure. "At first it seems counterintuitive that green bacteria have managed to evolve a better light-harvesting system by increasing disorder in the chlorosome structure," said Bryant. "Most people would think that if you make something that is more highly ordered, you'll end up with something that works better. But this is clearly a case where that isn't true. If all of the chlorophylls are identically arranged in a chlorosome, then the energy from the photon, once it is absorbed, is going to wander around over all of those chlorophylls, which could take a long time. In the wild-type form, you have these different domains where chlorophyll molecules are located and, therefore, the ability of photon energy to migrate becomes restricted. In other words, the energy in an individual photon visits a smaller number of chlorophylls, and that's an advantage to the organism because the energy can get to where it needs to go faster. Speed is the name of the game that green bacteria play with light. The organisms have only a couple of nanoseconds for the energy to get someplace useful or else the energy is going to be lost. The speed required can be a problem for bacteria that receive only a few photons of light per chlorophyll per day." Bryant said that the team's results may one day be used to build artificial photosynthetic systems that convert solar energy to electricity. "The interactions that lead to the assembly of the chlorophylls in chlorosomes are rather simple, so they are good models for artificial systems," he said. "You can make structures out of these chlorophylls in solution just by having the right solution conditions. In fact, people have done this for many years; however, they haven't really understood the biological rules for building larger structures. I won't say that we completely understand the rules yet, but at least we know what two of the structures are now and how they relate to the biological system as a whole, which is a huge advance."Scientists determine the structure of highly efficient light-harvesting molecules in green bacteria
Research team finds important role for junk DNA
Princeton scientists are probing the genetics of the pond organism Oxytricha, shown here in the process of reproducing. (Photo: Robert Hammersmith) (PhysOrg.com) -- Scientists have called it "junk DNA." They have long been perplexed by these extensive strands of genetic material that dominate the genome but seem to lack specific functions. Why would nature force the genome to carry so much excess baggage? Now researchers from Princeton University and Indiana University who have been studying the genome of a pond organism have found that junk DNA may not be so junky after all. They have discovered that DNA sequences from regions of what had been viewed as the "dispensable genome" are actually performing functions that are central for the organism. They have concluded that the genes spur an almost acrobatic rearrangement of the entire genome that is necessary for the organism to grow. It all happens very quickly. Genes called transposons in the single-celled pond-dwelling organism Oxytricha produce cell proteins known as transposases. During development, the transposons appear to first influence hundreds of thousands of DNA pieces to regroup. Then, when no longer needed, the organism cleverly erases the transposases from its genetic material, paring its genome to a slim 5 percent of its original load. "The transposons actually perform a central role for the cell," said Laura Landweber, a professor of ecology and evolutionary biology at Princeton and an author of the study. "They stitch together the genes in working form." The work appeared in the May 15 edition of Science. In order to prove that the transposons have this reassembly function, the scientists disabled several thousand of these genes in some Oxytricha. The organisms with the altered DNA, they found, failed to develop properly. Other authors from Princeton's Department of Ecology and Evolutionary Biology include: postdoctoral fellows Mariusz Nowacki and Brian Higgins; 2006 alumna Genevieve Maquilan; and graduate student Estienne Swart. Former Princeton postdoctoral fellow Thomas Doak, now of Indiana University, also contributed to the study.Landweber and other members of her team are researching the origin and evolution of genes and genome rearrangement, with particular focus on Oxytricha because it undergoes massive genome reorganization during development. In her lab, Landweber studies the evolutionary origin of novel genetic systems such as Oxytricha's. By combining molecular, evolutionary, theoretical and synthetic biology, Landweber and colleagues last year discovered an RNA (ribonucleic acid)-guided mechanism underlying its complex genome rearrangements. "Last year, we found the instruction book for how to put this genome back together again -- the instruction set comes in the form of RNA that is passed briefly from parent to offspring and these maternal RNAs provide templates for the rearrangement process," Landweber said. "Now we've been studying the actual machinery involved in the process of cutting and splicing tremendous amounts of DNA. Transposons are very good at that." The term "junk DNA" was originally coined to refer to a region of DNA that contained no genetic information. Scientists are beginning to find, however, that much of this so-called junk plays important roles in the regulation of gene activity. No one yet knows how extensive that role may be. Instead, scientists sometimes refer to these regions as "selfish DNA" if they make no specific contribution to the reproductive success of the host organism. Like a computer virus that copies itself ad nauseum, selfish DNA replicates and passes from parent to offspring for the sole benefit of the DNA itself. The present study suggests that some selfish DNA transposons can instead confer an important role to their hosts, thereby establishing themselves as long-term residents of the genome.
