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FOXO1: Single gene switch to convert human gastrointestinal cells to insulin-producing cells

FOXO1: Single gene switch to convert human gastrointestinal cells to insulin-producing cells | Amazing Science | Scoop.it

By switching off a single gene, scientists have converted human gastrointestinal cells into insulin-producing cells, demonstrating in principle that a drug could retrain cells inside a person’s GI tract to produce insulin. The finding raises the possibility that cells lost in type 1 diabetes may be more easily replaced through the reeducation of existing cells than through the transplantation of new cells created from embryonic or adult stem cells. The new research was reported in the online issue of the journal Nature Communications.


"People have been talking about turning one cell into another for a long time, but until now we hadn't gotten to the point of creating a fully functional insulin-producing cell by the manipulation of a single target," said the study's senior author, Domenico Accili, MD, the Russell Berrie Foundation Professor of Diabetes (in Medicine) at Columbia University Medical Center (CUMC).


The finding raises the possibility that cells lost in type 1 diabetes may be more easily replaced through the reeducation of existing cells than through the transplantation of new cells created from embryonic or adult stem cells.


For nearly two decades, researchers have been trying to make surrogate insulin-producing cells for type 1 diabetes patients. In type 1 diabetes, the body's natural insulin-producing cells are destroyed by the immune system.


Although insulin-producing cells can now be made in the lab from stem cells, these cells do not yet have all the functions of naturally occurring pancreatic beta cells.


This has led some researchers to try instead to transform existing cells in a patient into insulin-producers. Previous work by Dr. Accili's lab had shown that mouse intestinal cells can be transformed into insulin-producing cells; the current Columbia study shows that this technique also works in human cells.


The Columbia researchers were able to teach human gut cells to make insulin in response to physiological circumstances by deactivating the cells' FOXO1 gene. Accili and postdoctoral fellow Ryotaro Bouchi first created a tissue model of the human intestine with human pluripotent stem cells. Through genetic engineering, they then deactivated any functioning FOXO1 inside the intestinal cells. After seven days, some of the cells started releasing insulin and, equally important, only in response to glucose.


The team had used a comparable approach in its earlier, mouse study. In the mice, insulin made by gut cells was released into the bloodstream, worked like normal insulin, and was able to nearly normalize blood glucose levels in otherwise diabetic mice: New Approach to Treating Type I Diabetes? Columbia Scientists Transform Gut Cells into Insulin Factories. That work, which was reported in 2012 in the journal Nature Genetics, has since received independent confirmation from another group.

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Peter Phillips's curator insight, July 2, 2014 6:43 PM

New hope for diabetics - without a transplant.

Eric Chan Wei Chiang's curator insight, July 13, 2014 10:08 AM

These findings indicate that gastrointestinal cells and insulin producing β cells in the pancreas probably differentiated from the same line of cells during development. Insulin production in gastrointestinal cells is probably deactivated by the FOXO1 gene.

 

This opens up new possibilities as there is already a proof of concept for treating HIV with induced pluripotent stem cells. http://sco.lt/7yg3g9

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Silkworms and robot work together to weave Silk Pavilion

Silkworms and robot work together to weave Silk Pavilion | Amazing Science | Scoop.it

Researchers at MIT Media Lab's Mediated Matter group have created a dome from silk fibers woven by a robotic arm, which was then finished by live silkworms. 


The project is intended to explore how digital and biological fabrication techniques can be combined to produce architectural structures. The team programmed the robotic arm to imitate the way a silkworm deposits silk to build its cocoon. The arm then deposited a kilometer-long silk fiber across flat polygonal metal frames to create 26 panels. These panels were arranged to form a dome, which was suspended from the ceiling.


6500 live silkworms were then placed on the structure. As the caterpillars crawled over the dome, they deposited silk fibres and completed the structure.


The Silk Pavilion was designed and constructed at the MIT Media Lab as part of a research project to explore ways of overcoming the existing limitations of additive manufacturing at architectural scales.


Mediated Matter group director Neri Oxman believes that by studying natural processes such as the way silkworms build their cocoons, scientists can develop ways of "printing" architectural structures more efficiently than can be achieved by current 3D printing technologies.


“In traditional 3D printing the gantry-size poses an obvious limitation; it is defined by three axes and typically requires the use of support material, both of which are limiting for the designer who wishes to print in larger scales and achieve structural and material complexity,” Oxman said earlier this year.


“Once we place a 3D printing head on a robotic arm, we free up these limitations almost instantly." Their research also showed that the worms were attracted to darker areas, so fibers were laid more sparsely on the sunnier south and east elevations of the dome.


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Synthetic biology: How best to build a living cell

Synthetic biology: How best to build a living cell | Amazing Science | Scoop.it
Experts weigh in on the biggest obstacles in synthetic biology — from names to knowledge gaps — and what it will take to overcome them.


The engineering slant of synthetic biology has brought impressive accomplishments. These include whole-cell biosensors; cells that synthesize antimalaria drugs; and bacterial viruses designed to disperse dangerous, tenacious biofilms.


To design these, engineers are trained to model systems as black boxes, abstractly linking inputs and outputs. They can often control a system with only a limited understanding of it. But synthetic-biology projects are frequently thwarted when engineering runs up against the complexity of biology.


Synthetic biology would benefit greatly from deeper insights into the mechanisms of biological systems. Such approaches have already yielded insights into how organized processes in cells work because of, rather than in spite of, noisy gene expression. Synthetic biology is also informing biology, helping to reveal how a gene product can amplify or inhibit its own expression and so allow cells to flip between stable states. Much more remains to be explored and discovered.


The biggest challenge for synthetic biology is how to extend beyond projects that focus on single products, organisms and processes. Right now, most applications engineer bacteria that start a synthesis with glucose and turn out biofuels or fine chemicals, such as vanillin or artemesinin. A broader scope could help to build a 'greener' economy, in which more organisms make a greater range of chemicals.


The chemical industry is a marvel of efficiency, taking raw materials such as oil and converting them into a wide range of products, including plastics and pharmaceuticals. This is possible in part because feedstocks can be interconverted through various large-scale reactions for which catalysts and processes have been optimized over several decades.


Synthetic biology could unlock the large-scale use of carbon sources from lignocellulose to coal. Synthetic 'bioalchemy' would reformat the basic elements of life to take advantage of abundant supplies of formerly rare intermediates such as the nylon precursor adipate, which is used to synthesize antibiotics. Metabolic engineering is already capable of syntheses that use glucose or other standard carbon sources as precursors, but the co-culture of synthetically modified organisms would make these processes more efficient. The ability to engineer photosynthetic organisms might even allow light to be used as the ultimate energy source and carbon dioxide as the ultimate carbon source.

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Therapeutic siRNA Interventions: What we have learned

Therapeutic siRNA Interventions: What we have learned | Amazing Science | Scoop.it

Treatments based on RNA interference are improving now that technologies are delivering longer-lasting gene silencing.


The 2006 Nobel Prize in Physiology or Medicine was awarded jointly to Andrew Z. Fire and Craig C. Mello for their 1998 discovery of RNA interference (RNAi), gene silencing by double-stranded RNA.

Today, RNAi-based therapeutics are in Phase II and Phase III clinical trials. The rapid development of this technology demonstrates its enormous potential for treatment of a range of diseases.

A major hurdle for clinical applications is the safe and effective delivery of small interfering RNA (siRNA). Unlike biologics that target membrane proteins, siRNA molecules need to enter the cytosol of diseased cells to work. In addition, unlike small molecules that diffuse freely across the cell membrane, siRNA molecules are large and negatively charged. They cannot easily and independently cross the cell membrane.

Current siRNA nanoparticle delivery platforms in clinical trials, such as cationic lipoplexes and polyplexes, induce transient gene silencing; they lack a sustained siRNA release property. In vitro studies have indicated that efficacy, in general, lasts less than two weeks at the cellular level.

A new lipid-polymer hybrid nanoparticle combines a cationic liposome system with a controlled-release polymer technology, allowing siRNA encapsulation along with sustained release. Encapsulation of the siRNA would be very low if it depended solely on the noncharged, controlled-release polymer technology. Sustained delivery allows for longer activity, and, potentially, subsequent lower dosage and injection frequencies.

An in vitro proof-of-concept study showed that the lipid-polymer hybrid nanoparticle slowly releases the siRNA over the course of a month, allowing sustained knockdown of PHB1, a protein involved in cell proliferation, apoptosis, chemoresistance, and other biological processes in lung carcinoma cells.

“It takes a long time to discover a drug or small molecule to target a protein of interest, plus there are many undruggable proteins. The beautiful thing about RNAi technology is you can target any protein you want by silencing the gene,” explains Jinjun Shi, Ph.D., assistant professor, Laboratory for Nanoengineering and Drug Delivery, Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School.

The new lipid-polymer hybrid nanoparticle technology is initially intended for use in fundamental research and target validation. The goal is to eventually extend its application to the clinic as a vehicle for delivering therapeutic siRNAs and, perhaps, for co-delivering chemotherapeutics and siRNAs for synergistic cancer treatment.


Via Integrated DNA Technologies
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p110δ inhibitors found to stimulate immunity against many cancer types

p110δ inhibitors found to stimulate immunity against many cancer types | Amazing Science | Scoop.it

new study, published in Nature, provides the first evidence that such drugs can significantly restrict tumor growth and spread and reduce the chances of relapse for a broad range of cancers. The researchers at UCL, the Babraham Institute and Queen Mary University of London, together with scientists from Genentech, South San Francisco, showed that inhibition of the p110δ enzyme helps to boost the body's immune system to kill tumor cells. The research was funded by Cancer Research UK, the Biotechnology and Biological Sciences Research Council and the Wellcome Trust.


"Our study shows that p110δ inhibitors have the potential to offer effective immunity to many types of cancer by unleashing the body's own immune response," says study co-leader Professor Bart Vanhaesebroeck of the UCL Cancer Institute, who first discovered the p110δ enzyme in 1997. "p110δ is highly expressed and important in white blood cells, called 'leukocytes'. Given that leukemias are the result of leukocytes becoming cancerous, they are a natural target for p110δ inhibitors. Now, we have shown that blocking p110δ also has the remarkable effect of boosting the body's immune response against leukemias as well as other cancers."


The team showed that inhibiting p110δ in mice significantly increased cancer survival rates across a broad range of tumor types, both solid and haematological cancers. For example, mice in which p110δ was blocked survived breast cancer for almost twice as long as mice with active p110δ. Their cancers also spread significantly less, with far fewer and smaller tumors developing. Survival after surgical removal of primary breast cancer tumors was also vastly improved, which has important clinical implications for stopping breast cancer from returning following surgery. The team's data further show that following p110δ inhibition, the immune system could develop an effective memory response to completely fight off the cancer.


Lead author Dr Khaled Ali, who is now based at Amgen, San Francisco, says: "When we first introduced tumors in p110δ-deficient mice, we expected them to grow faster because p110δ is important for the immune system. Instead, some tumors started shrinking. When we investigated this unexpected effect, we found that p110δ is especially important in so-called regulatory T cells which are suppressive immune cells that the tumors engage to protect themselves against immune attack."


The p110δ enzyme is a member of the PI3-kinase family, and is sometimes called PI3Kδ. p110δ and the other PI3Ks are hot drug targets for the pharmaceutical industry as they are implicated in many cancers and are readily druggable.

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Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing

Actionable Diagnosis of Neuroleptospirosis by Next-Generation Sequencing | Amazing Science | Scoop.it

Because more than 100 different infectious agents can cause encephalitis, establishing a diagnosis with the use of cultures, serologic tests, and pathogen-specific PCR assays can be difficult. Unbiased next-generation sequencing has the potential to revolutionize our ability to discover emerging pathogens, especially newly identified viruses [5-8]. However, the usefulness of next-generation sequencing for the diagnosis of infectious diseases in a clinically relevant timeframe is largely unexplored. [9] A group of scientists and medical doctors now used unbiased next-generation sequencing to identify a treatable, albeit rare, bacterial cause of meningoencephalitis. In the case they showed, the results of next-generation sequencing contributed directly to a dramatic effect on the patient's care, resulting ultimately in a favorable outcome. Thus, unbiased next-generation sequencing coupled with a rapid bioinformatics pipeline provided a clinically actionable diagnosis of a specific infectious disease from an uncommon pathogen that eluded conventional testing for months after the initial presentation. This approach facilitated the use of targeted and efficacious antimicrobial therapy.

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Scientists Successfully Transplant and Grow Stem Cells in Pigs

Scientists Successfully Transplant and Grow Stem Cells in Pigs | Amazing Science | Scoop.it
One of the biggest challenges for medical researchers studying the effectiveness of stem cell therapies is that transplants or grafts of cells are often rejected by the hosts. This rejection can render experiments useless, making research into potentially life-saving treatments a long and difficult process. Now, researchers at the University of Missouri have shown that a new line of genetically modified pigs will host transplanted cells without the risk of rejection.


"The rejection of transplants and grafts by host bodies is a huge hurdle for medical researchers," said R. Michael Roberts, Curators Professor of Animal Science and Biochemistry and a researcher in the Bond Life Sciences Center. "By establishing that these pigs will support transplants without the fear of rejection, we can  move stem cell therapy research forward at a quicker pace."


In a published study, the team of researchers implanted human pluripotent stem cells in a special line of pigs developed by Randall Prather, an MU Curators Professor of reproductive physiology. Prather specifically created the pigs with immune systems that allow the pigs to accept all transplants or grafts without rejection. Once the scientists implanted the cells, the pigs did not reject the stem cells and the cells thrived. Prather says achieving this success with pigs is notable because pigs are much closer to humans than many other test animals.


"Many medical researchers prefer conducting studies with pigs because they are more anatomically similar to humans than other animals, such as mice and rats," Prather said. "Physically, pigs are much closer to the size and scale of humans than other animals, and they respond to health threats similarly. This means that research in pigs is more likely to have results similar to those in humans for many different tests and treatments."


"Now that we know that human stem cells can thrive in these pigs, a door has been opened for new and exciting research by scientists around the world," Roberts said. "Hopefully this means that we are one step closer to therapies and treatments for a number of debilitating human diseases."


Roberts and Prather published their study, "Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency" in the Proceedings of the National Academy of Sciences (PNAS).

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CRTC2: Scientists Unravel the Molecular Secret of Short, Intense Workouts

CRTC2: Scientists Unravel the Molecular Secret of Short, Intense Workouts | Amazing Science | Scoop.it

In the last few years, the benefits of short, intense workouts have been extolled by both researchers and exercise fans as something of a metabolic panacea capable of providing greater overall fitness, better blood sugar control and weight reduction—all of it in periods as short as seven minutes a few times a week.


Now, in a new study, scientists from the Florida campus of The Scripps Research Institute (TSRI) confirm that there is something molecularly unique about intense exercise: the activation of a single protein.


The study, published recently by The EMBO Journal, revealed the effects of a protein known as CRTC2.


The scientists were able to show that following high-intensity exercise, which enlists the sympathetic nervous system’s “fight or flight” response, CRTC2 integrates signals from two different pathways—the adrenaline pathway and the calcium pathway, to direct muscle adaptation and growth only in the contracting muscle.


Using mice genetically modified to conditionally express CRTC2, the scientists showed that molecular changes occurred that emulated exercised muscles in the absence of exercise.


In the genetically altered animal models, this resulted in a muscle size increase of approximately 15 percent. Metabolic parameters, indicating the amount of fuel available to the muscles, also increased substantially—triglycerides went up 48 percent, while glycogen supplies rose by a startling 121 percent.

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Cellular traffic control system mapped for the first time

Cellular traffic control system mapped for the first time | Amazing Science | Scoop.it

Cells regulate the uptake of nutrients and messenger cargos and their transport within the cell. This process is known as endocytosis and membrane traffic. Different cargos dock onto substrate specific receptors on the cell membrane. Special proteins such as kinases, GTPases and coats, activate specific entry routes and trigger the uptake of the receptors into the cell. For their uptake, the receptors and docked cargos become enclosed by the cell membrane. In the next steps, the membrane invaginates and becomes constricted. The resulting vesicle is guided via several distinct stations, cellular organelles, to its final destination in the cell.


For her study, Dr. Prisca Liberali, senior scientist in the team of Professor Lucas Pelkmans, sequentially switched off 1200 human genes. Using automated high-throughput light microscopy and computer vision, she could monitor and compare 13 distinct transport paths involving distinct receptors and cellular organelles. Precise quantifications of thousands of single cells identified the genes required for the different transport routes. Surprisingly, sets of transport routes are co-regulated and coordinated in specific ways by different programs of regulatory control.


Subsequently, Dr. Liberali calculated the hierarchical order within the genetic network and thereby identified the regulatory topology of cellular transport. "The transport into the cell and within the cells proceeds analogously to the cargo transport within a city" describes the scientist. "Like in a city, the traffic on the routes within a cell and their intersections is tightly regulated by traffic lights and signs to guide the cargo flow."


Thanks to this unique quantitative map, the fine regulatory details of transport paths and processes within a cells could be mapped for the first time. Particularly the genes that encode for these traffic lights and switches are often de-regulated in disease. With this map, it is now possible to predict how this leads to traffic jams in the cells, causing the disease phenotype. Alternatively, since many drugs have been developed to target these traffic lights and switches, the map can be used to come up with possible drug combinations to target unwanted traffic, such as viruses, to the waste disposal system of the cell.

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ComplexInsight's curator insight, June 10, 2014 2:44 AM

Mapping the fine regulatory details of transport paths and processes within cells is key to understanding gene and protein functions, cancer, viral interactions and potential treatments.  Interesting read.

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Flow-through peptide synthesis for cell-based assays on Teflon-coated paper

Flow-through peptide synthesis for cell-based assays on Teflon-coated paper | Amazing Science | Scoop.it

In classical solid-phase synthesis, the growing peptide chain is fixed to a solid support—usually a polymer bead—so that the reagents can be rapidly and easily washed away after each step. Parallel solid-phase synthesis, known as SPOT synthesis, was developed as an alternative in the 1990s. This allows a large number of peptides to be obtained on a planar support with a small surface area. SPOT synthesis has since been adapted for other applications, such as cell-based screening.


The problem is that existing SPOT systems are not well-suited for chemical reactions. When individual drops of reagent are added by pipette, they wet small areas of the membrane—the SPOTs. The circular spot of solvent absorbed by the membrane determines the size of the "reaction vessel". Unlike in classical solid-phase synthesis, this limits the amounts of reagent, and flow-through conditions are not possible. This significantly limits the possible yields of the reactions.


A team headed by Frédérique Deiss and Ratmir Derda at the University of Alberta (Canada) has now found an elegant solution to this problem. The researchers used a Teflon coating to form a pattern of solvent-repellent barriers on a paper support. The pattern restricts the liquids to specific Teflon-free zones on the paper, forming small "reaction vessels" that can hold a larger volume than the usual SPOTs. This not only allows for the use of excess volumes of reagents, but also allows for a flow-through reaction because the larger volume ensures for gravity-driven flow of the reagent solution through the paper. The flow rate can be varied by using paper of different porosity. This significantly improves yields.


There is an additional advantage to this method: the paper can be stacked or folded into thicker three-dimensional structures. The researchers were able to identify various peptides among those immobilized on the surface that support cell adhesion, growth, or differentiation in a three-dimensional environment.


More information: Deiss, F., Matochko, W. L., Govindasamy, N., Lin, E. Y. and Derda, R. (2014), "Flow-Through Synthesis on Teflon-Patterned Paper To Produce Peptide Arrays for Cell-Based Assays." Angew. Chem. Int. Ed.. doi: 10.1002/anie.201402037

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Strange Brews: The Genes for Craft Beer Elucidated by Deep Sequencing of Yeast

Strange Brews: The Genes for Craft Beer Elucidated by Deep Sequencing of Yeast | Amazing Science | Scoop.it

After thousands of years of unwitting domestication, brewing yeasts — the microorganisms that ferment a brewer’s tepid slop of grain, water and hops into beer — are as diverse as the beer they make. And now two research teams, from White Labs and a Belgian genetics laboratory, are mapping outtheir sprawling genealogy, creating the first genetic family tree for brewing yeasts and the beers they make.


The laboratories have sequenced the DNA of more than 240 strains of brewing yeasts from around the world. Alongside samples from breweries like Sierra Nevada, Duvel Moortgat and Stone, “we’ve thrown in a few wine, bakers, bio-ethanol and sake yeasts to compare,” saidKevin Verstrepen, director of the lab in Belgium.

By getting a line-by-line reading of the 12 million molecules that make up the DNA of each yeast, Dr. Verstrepen said, the researchers will be able not only to tell how closely related two yeasts are (is Sam Adams’s closer to Stone’s, or Sierra Nevada’s?) but to answer other important questions: which breweries started with the same strains of yeasts, how these organisms evolved over time and, of course, how all of it translates to taste.

“Yeasts can make over 500 flavor and aroma compounds,” said Chris White, the founder of White Labs, affecting a beer’s alcohol level, clarity and texture. But while brewing yeast is one of the best-studied organisms in molecular and cell biology, exactly how its genes translate to brewing properties is still poorly understood.

By comparing the DNA of hundreds of yeasts, along with information on how they act and brew differently, “we’ll have a unique window into the genetic code,” said Mr. Prahl, who is leading the experiment at White Labs. He is comparing each yeast’s sequencing information with brewing data on more than 2,000 batches of beer — including the four he was tasting.

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Molecular crowding: Simple nucleic acid and protein folding may be sped up by 400,000 percent inside cells

Molecular crowding: Simple nucleic acid and protein folding may be sped up by 400,000 percent inside cells | Amazing Science | Scoop.it

Crowding has notoriously negative effects at large size scales, blamed for everything from human disease and depression to community resource shortages. But relatively little is known about the influence of crowding at the cellular level. A new JILA study shows that a crowded environment has dramatic effects on individual biomolecules.


In the first data on the underlying dynamics (or kinetics)of crowded single biomolecules , reported in Proceedings of the National Academy of Sciences,* JILA researchers found that crowding leads to a 35-fold increase in the folding rate of RNA (ribonucleic acid), while the unfolding rate remains relatively stable.


RNA is a long chain-like molecule that contains genetic information, makes proteins and catalyzes biological reactions. It must fold into the correct 3D shape to function properly. The new results show that while RNA usually spends most of its time unfolded, in a crowded situation it folds much more often, although it remains folded for the usual period of time during each round.


"Cells are 25 to 35 percent filled with 'stuff'—proteins, nucleic acids, lipids, etc.—and the effect of crowding on simple reactions like folding of nucleic acids and proteins is not well understood," JILA/NIST Fellow David Nesbitt says. "Almost all detailed kinetic data comes from in vitro studies, that is, not in a living cell.


"But our work at the single-molecule level suggests that the rates and equilibrium constants (where folding and unfolding rates are equal) for simple nucleic acid folding processes may be shifted by up to 400,000 percent or more from what one might expect from such uncrowded solution studies."


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Genome scientist Craig Venter in deal to make humanized pig organs

Genome scientist Craig Venter in deal to make humanized pig organs | Amazing Science | Scoop.it

Genome pioneer J. Craig Venter is teaming up with a unit of United Therapeutics Corp to develop pig lungs that have been genetically altered to be compatible with humans, a feat that, if successful, could address the urgent need for transplant organs for people with end-stage lung disease.


Venter's privately held company Synthetic Genomics Inc on Tuesday said it has entered a multiyear deal with United Therapeutics' Lung Biotechnology Inc to develop the so-called humanized pig organs.


Humans, pigs and most other mammals share about 90 percent of the same genes. What Venter's team will do is to determine which aspects of the pig genome need to be altered to make porcine lungs compatible with humans, avoiding the rejection response that occurs even in human-to-human transplants.


"We're going to start with generating a brand new super-accurate sequence of the pig genome, and then go through in detail and compare it to the human genome," Venter, the founder and chief executive of Synthetic Genomics Inc, stated recently.


"The goal is to go in and edit, and where necessary, rewrite using our synthetic genomic tools, the pig genes that seem to be associated with immune responses," said Venter, who is best known for his role in mapping the human genome over a decade ago and who created synthetic life in 2010.


"We want to get it so there is no acute or chronic rejection," he said.

Venter's team is tasked with editing and rewriting the pig genome and providing the United Therapeutics group with a series of altered cells. United Therapeutics will take those cells and transplant them into pig eggs, generating embryos that develop and are born with humanized lungs.


If all goes well, Venter thinks his team will be able to deliver the cells in a few years. Testing the humanized organs in clinical trials to ensure they are safe in people will take many more years.


Via Ray and Terry's
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Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips

Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips | Amazing Science | Scoop.it
A diagnostic tool that's about the size of a credit card has identified a highly prized gut microbe.


Isolating microbes carrying genes of interest from environmental samples is important for applications in biology and medicine. However, this involves the use of genetic assays that often require lysis of microbial cells, which is not compatible with the goal of obtaining live cells for isolation and culture.


A recent development by Caltech describes the design, fabrication, biological validation, and underlying physics of a microfluidic SlipChip device that addresses this challenge. The device is composed of two conjoined plates containing 1000 microcompartments, each comprising two juxtaposed wells, one on each opposing plate. Single microbial cells are stochastically confined and subsequently cultured within the microcompartments. Then, each microcompartment is split into two replica droplets, both containing microbial culture, and then controllably separate the two plates while retaining each droplet within each well. The inventors experimentally describe the droplet retention as a function of capillary pressure, viscous pressure, and viscosity of the aqueous phase. Within each pair of replicas, one can be used for genetic analysis, and the other preserves live cells for growth. This microfluidic approach provides a facile way to cultivate anaerobes from complex communities. The researchers validate this method by targeting, isolating, and culturing Bacteroides vulgatus, a core gut anaerobe, from a clinical sample. To date, this methodology has enabled isolation of a novel microbial taxon, representing a new genus.


This approach could also be extended to the study of other microorganisms and even mammalian cells or tissue samples, and may enable targeted retrieval of solutions in applications including digital PCR, sequencing, single cell analysis, and protein crystallization.

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Melanie Patterson's curator insight, July 2, 2014 5:57 PM

This is clever.  Rustem Ismagilov's group (formerly from the UofC) is using their slip chip invention to solve problems - I never expected him to start growing gut microbes.  Great story. 

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Observing kinase activity in live single cells

Observing kinase activity in live single cells | Amazing Science | Scoop.it

Ongoing efforts have shown that multicellular systems are best understood as a combination of heterogeneous single cell behaviors. Intrinsic noise generates cell-to-cell variation that can be critical for cellular survival, development and differentiation. In response to changing environments, cells also generate complex signaling dynamics that encode relevant information for gene expression, proliferation or stress responses. Indeed, bulk population dynamics are often qualitatively different from single cell behaviors. As a result, live-cell microscopy has acquired a central role to study single cell biology. Dynamic single cell reporters are essential for live-cell microscopy.


However, the number and type of molecular events that can be dynamically monitored in an individual cell is small. Such reporters have led to the successful measurement of metabolic state, transcription factor localization and even protein activities in live single cells. In the latter category, kinase activities are of particular interest. Kinases are known to regulate multiple and diverse biological functions, including the cell cycle, the innate immune response, development and cell differentiation.


Recently, researchers have developed a novel technology to generate single cell reporters for kinase activity. Their approach is based on the concept of converting phosphorylation into a nucleocytoplasmic shuttling event. In fact, there are numerous examples of phosphorylation-regulated nucleo-cytoplasmic translocation in naturally occurring proteins. The scientists hypothesized that by understanding this phenomena they could synthetically engineer single color kinase reporters for single cells. Therefore, after exploring the sequence space using the JNK (c-Jun N terminal Kinase) substrate c-Jun, they defined a set of rules that they can use to engineer single cell kinase activity reporters. They named these reporters Kinase Translocation Reporters (KTR). In addition, they showed that KTR technology is generalizable by implementing KTR sensors for JNK, p38, ERK and PKA, thus covering different types of kinases. The researchers also used this technology to show that multiplexing capabilities go beyond any current method. In particular, they measured JNK, p38 and ERK activities simultaneously in live single cells.


This technology opens the possibility of analyzing multiple signaling networks, cell cycle and a broad range of kinase-mediated processes simultaneously in live single cells.

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Three-dimensional light-sensitive mini retina grown from human iPS cells in the lab

Three-dimensional light-sensitive mini retina grown from human iPS cells in the lab | Amazing Science | Scoop.it
Researchers at Johns Hopkins have constructed a functioning segment of a human retina out of stem cells that is able to respond to light.


The eye is often compared to a camera, but although its basic design is as simple as an old-fashioned box Brownie, its detailed structure is more complex than the most advanced electronics. This means that, unlike simpler organs, studies of retinal disease rely heavily on animal studies, and treating such illnesses is extremely difficult. One ray of hope in the field comes from researchers at Johns Hopkins, who have constructed a functioning segment of a human retina out of stem cells that is able to respond to light.


The retina is the complex lining of the human eye that acts like the the film (or the imaging sensor, for the younger crowd) in a camera. It’s made of some 10 layers of tissue, including structural membranes, nerve ganglia, and photoreceptor cells; the rods that detect black and white images and work best in low light, and the cones, which detect color. If scientists could recreate this structure in the laboratory, it would be a major breakthrough in treating eye diseases.


The Johns Hopkins researchers’ approach was to use human-induced pluripotent stem cells (iPS). In other words, adult cells were induced to revert to stem cells, from which any of the 200 specialized cells in the human body can be derived. The Johns Hopkins team programmed the stem cells to grow into retinal progenitor cells in a culture dish.


These cells developed into retina cells, much in the same way and at the same rate as in a human embryo. As they did so, the cells differentiated into the some of the seven different kinds of cells that make up the retina and organized themselves into the three-dimensional outer segment structures necessary for the photoreceptors to work.


"We knew that a 3D cellular structure was necessary if we wanted to reproduce functional characteristics of the retina," says M Valeria Canto-Soler, an assistant professor of ophthalmology at the Johns Hopkins University School of Medicine, "but when we began this work, we didn't think stem cells would be able to build up a retina almost on their own. In our system, somehow the cells knew what to do."


Growing retina segments has been achieved before, but where Johns Hopkins’ work stands out is that these mini-retinas actually function. When the mini-retinas reached the equivalent of 28-weeks of development, the researchers hooked the photoreceptor cells up to electrodes and flashed pulses of light at them. According to the scientists, the cells displayed the same photochemical reactions as in a normal retina – especially in regard to the rods that make up the majority of the photoreceptors.

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Food for Africa: Genetically-engineered carotene-enriched 'super' banana to face first human trial

Food for Africa: Genetically-engineered carotene-enriched 'super' banana to face first human trial | Amazing Science | Scoop.it

A super-enriched banana genetically engineered to improve the lives of millions of people in Africa will soon have its first human trial, which will test its effect on vitamin A levels, Australian researchers said Monday.

The project plans to have the special banana varieties -- enriched with alpha and beta carotene which the body converts to vitamin A -- growing in Uganda by 2020.


The bananas are now being sent to the United States, and it is expected that the six-week trial measuring how well they lift vitamin A levels in humans will begin soon.


"Good science can make a massive difference here by enriching staple crops such as Ugandan bananas with pro-vitamin A and providing poor and subsistence-farming populations with nutritionally rewarding food," said project leader Professor James Dale.


The Queensland University of Technology (QUT) project, backed by the Bill and Melinda Gates Foundation, hopes to see conclusive results by year end.


"We know our science will work," Professor Dale said.

"We made all the constructs, the genes that went into bananas, and put them into bananas here at QUT."


Dale said the Highland or East African cooking banana was a staple food in East Africa, but had low levels of micro-nutrients, particularly pro-vitamin A and iron.


"The consequences of vitamin A deficiency are dire with 650,000-700,000 children world-wide dying ... each year and at least another 300,000 going blind," he said.


Researchers decided that enriching the staple food was the best way to help ease the problem.


While the modified banana looks the same on the outside, inside the flesh is more orange than a cream color, but Dale said he did not expect this to be a problem.


He said once the genetically modified bananas were approved for commercial cultivation in Uganda, the same technology could potentially be expanded to crops in other countries -- including Rwanda, parts of the Democratic Republic of Congo, Kenya and Tanzania.

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Design of self-assembling protein nanomachines: A nanocage builds itself from engineered components

Design of self-assembling protein nanomachines: A nanocage builds itself from engineered components | Amazing Science | Scoop.it
Biological systems produce an incredible array of self-assembling protein tools on a nanoscale, such as molecular motors, delivery capsules and injection devices. Inspired by sophisticated molecular machines naturally found in living things, scientists want to build their own with forms and functions customized to tackle modern day challenges. A new computational method, proven to accurately design protein nanomaterials that arrange themselves into a symmetrical, cage-like structure, may be an important step toward that goal.
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Gene editing tool CRISPR-CAS can write HIV out of the picture

Gene editing tool CRISPR-CAS can write HIV out of the picture | Amazing Science | Scoop.it

The latest form of genetic engineering can give human cells a rare mutation that keeps them HIV-free.


Take a hot new method that's opened up a new era of genetic engineering, apply it to the wonder stem cells that in 2012 won their discoverer a Nobel prize, and you might just have a tool to cure HIV infection. That's the hope of researchers led by Yuet Kan of the University of California, San Francisco – and they have proved the basic principle, altering the genome of induced pluripotent stem cells (iPSCs) to give them a rare natural mutation that allows some people to resist HIV.


Kan's work relies on "genome editing" – snipping out a particular DNA sequence and replacing it with another. It's much more precise than traditional forms of genetic engineering, in which sequences are added to the genome at random locations.


To alter the stem cells, Kan's team turned to the CRISPR-Cas9 system, asuper-efficient method of genome editing based on an ancient bacterial "immune system". In bacteria, the system takes fragments of DNA from invading viruses and splices them into the cell's own DNA, where they act like "wanted" posters, allowing the viruses to be recognised and attacked in future.


About 1 per cent of people of European descent are resistant to HIV, because they carry two copies of a mutation in the gene for a protein called CCR5. The virus must lock onto this protein before it can invade white blood cells, and the mutations prevent it from doing so.


Using a bone marrow transplant from a naturally HIV-resistant person,Timothy Ray Brown was famously "cured" of HIV infection. Kan's goal is to achieve the same result without the need to find compatible HIV-resistant bone marrow donors – who are in vanishingly short supply.


It's fairly easy to make iPSCs from a person's cells, which then have the potential to grow into any type of cell in the body. So if iPSCs could be given two copies of the protective mutation, it should be possible to make personalised versions of the therapy that cleared HIV from Brown's body. Kan's team has now shown that CRISPR-Cas9 can efficiently make the necessary genome edit. As expected, white blood cells grown from these altered stem cells were resistant to HIV upon testing.


"It's a really fantastic application of the tool," says Philip Gregory, chief scientific officer with Sangamo BioSciences of Richmond, California. However, he warns that there is a long way to go before it can be turned into a practical therapy.

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Cancer and AIDS are widely thought to be the bane of mankind’s existence. Any therapy which could restrict either of these diseases is significant! Read about more novel therapies here:

http://www.scoop.it/t/biotech-and-beyond/?tag=Novel+Therapies

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Synthetic Biology Students Build the First Eukaryotic Chromosome from Scratch

Synthetic Biology Students Build the First Eukaryotic Chromosome from Scratch | Amazing Science | Scoop.it

In March undergraduate students in Johns Hopkins University's Build a Genome course announced they had made a yeast chromosome from scratch—and history, too. It is the first time anyone has synthesized the chromosome of a complex organism, a landmark achievement in the field of synthetic biology. It is also a triumph for the movement known as DIY biology.


The target was chromosome 3, which controls the yeast's sexual reproduction and has 316,617 base pairs of the DNA alphabet—A for adenine, G for guanine, C for cytosine and T for thymine. To synthesize it, the students took a shortcut: they built only the sections considered essential or nonrepetitive. The resulting chromosome had a more manageable 272,871 base pairs. And as reported inScience, the yeast with the new genes thrived just as well as regular yeast did in terms of size and growth.


“They are going strong,” says biologist Jef Boeke of New York University, who helped lead the research as part of the Synthetic Yeast 2.0 project—an effort to build a synthetic genome for yeast that would give scientists nearly complete control of it. Boeke and others plan to grow this batch for thousands of generations over the next several years to see how they evolve over time, which will give scientists a better understanding of fundamental biology, from the role of “junk DNA” to the absolute minimum of genetic code necessary for survival. “The questions are endless,” Boeke says.


The current work is just 3 percent of the way toward creating an entirely synthetic yeast genome (there are 16 chromosomes in total) and will take many more years to finish. If finished, synthetic yeast could be second on the list of organisms with genomes built from scratch—the J. Craig Venter Institute built a bacterium's genome in 2010.


It could also be a breakthrough in humanity's millennia-long cohabitation withSaccharomyces cerevisiae, which is responsible for bread and wine. Yeasts today churn out human proteins for medicines, biofuels and other specialty products. Being able to fine-tune the microscopic fungus's genetics could lead to better beer or sustainable chemicals, according to Boeke. And after yeast? “The fruit fly? The worm? We're not sure what is next.”

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FOXM1 and CENPF: Synergy between Two Genes Drives Aggressive Prostate Cancer

FOXM1 and CENPF: Synergy between Two Genes Drives Aggressive Prostate Cancer | Amazing Science | Scoop.it

Two genes work together to drive the most lethal forms of prostate cancer, according to new research by investigators in the Columbia University Department of Systems Biology.  These findings could lead to a diagnostic test for identifying those tumors likely to become aggressive and to the development of novel combination therapy for the disease.


The two genes—FOXM1 and CENPF—had been previously implicated in cancer, but none of the prior studies suggested that they might work synergistically to cause the most aggressive form of prostate cancer. The study was published today in the online issue ofCancer Cell.


“Individually, neither gene is significant in terms of its contribution to prostate cancer,” said co-senior author Andrea Califano, the Clyde and Helen Wu Professor of Chemical Biology in Biomedical Informatics and Chair of the Department of Systems Biology. “But when both genes are turned on, they work together synergistically to activate pathways associated with the most aggressive form of the disease.”


“Ultimately, we expect this finding to allow doctors to identify patients with the most aggressive prostate cancer so that they can get the most effective treatments,” said co-senior author Cory Abate-Shen, the Michael and Stella Chernow Professor of Urologic Sciences and also a member of the Department of Systems Biology. “Having biomarkers that predict which patients will respond to specific drugs will hopefully provide a more personalized way to treat cancer.”


Scientists widely recognize that cancer is characterized by multiple genetic changes. “However, distinguishing the handful of genes that are driving the cancer from the many genes whose altered expression does not contribute directly to the cancer has proven to be a daunting task,” said Dr. Califano. “It becomes even more difficult when genes work together synergistically, because they must be analyzed in pairs rather than one by one. For instance, the approximately 1,000 genes that have been linked to cancer can be combined into about 500,000 gene pairs, each of which may represent a synergistic tumor driver. This is an enormous number that defies our best statistical tools and requires sophisticated systems biology approaches.”


Using the high-performance computing cluster housed in the Department of Systems Biology, the analysis identified FOXM1 and CENPF as a synergistic driver pair in aggressive prostate cancer in both mice and humans, as these regulators jointly control genetic programs associated with the most prominent tumor hallmarks in both species. Individually, the aberrant expression of these genes does not activate these programs. When acting together, however, the two genes can wreak havoc in the cancer cell and turn it into a very aggressive tumor.


To validate the roles of FOXM1 and CENPF, the researchers silenced the expression of the genes in four human prostate cancer cell lines, first individually and then together. Silencing either gene alone had only a modest effect on the ability of the cells to form tumors. However, co-silencing both genes at once completely stopped the growth of tumors in a mouse. This observation is consistent with a synergistic interaction, where the joint effect of both genes is much greater than the sum of their individual effects. 


The researchers then analyzed prostate cancers from a group of more than 900 patients who had undergone prostate removal surgery. This analysis showed a striking correlation between the co-expression of FOXM1 and CENPF and the poorest disease outcome. In sharp contrast, expression of either gene alone did not correlate with aggressive disease. In addition, tumors in which neither gene was aberrantly expressed had the best prognosis.

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New method reveals that single protein interaction is key to embryonic stem cell differentiation

New method reveals that single protein interaction is key to embryonic stem cell differentiation | Amazing Science | Scoop.it
Proteins are responsible for the vast majority of the cellular functions that shape life, but like guests at a crowded dinner party, they interact transiently and in complex networks, making it difficult to determine which specific interactions are most important.


Researchers from the University of Chicago have pioneered a new technique to simplify the study of protein networks and identify the importance of individual protein interactions. By designing synthetic proteins that can only interact with a pre-determined partner, and introducing them into cells, the team revealed a key interaction that regulates the ability of embryonic stem cells to change into other cell types. They describe their findings June 5 in Molecular Cell.


"Our work suggests that the apparent complexity of protein networks is deceiving, and that a circuit involving a small number of proteins might control each cellular function," said senior author Shohei Koide, PhD, professor of biochemistry & molecular biophysics at the University of Chicago.


For a cell to perform biological functions and respond to the environment, proteins must interact with one another in immensely complex networks, which when diagrammed can resemble a subway map out of a nightmare. These networks have traditionally been studied by removing a protein of interest through genetic engineering and observing whether the removal destroys the function of interest or not. However, this does not provide information on the importance of specific protein-to-protein interactions.


To approach this challenge, Koide and his team pioneered a new technique that they dub "directed network wiring." Studying mouse embryonic stem cells, they removed Grb2, a protein essential to the ability of the stem cell to transform into other cell types, from the cells. The researchers then designed synthetic versions of Grb2 that could only interact with one protein from a pool of dozens that normal Grb2 is known to network with. The team then introduced these synthetic proteins back into the cell to see which specific interactions would restore the stem cell's transformative abilities.


"The name, 'directed network wiring,' comes from the fact that we create minimalist networks," Koide said. "We first remove all communication lines associated with a protein of interest and add back a single line. It is analysis by addition."


Despite the complexity of the protein network associated with stem cell development, the team discovered that restoring only one interaction—between Grb2 and a protein known as Ptpn11/Shp2 phosphatase—was enough to allow stem cells to again change into other cell types.


"We were really surprised to find that consolidating many interactions down to a single particular connection for the protein was sufficient to support development of the cells to the next stage, which involves many complicated processes," Koide said. "Our results show that signals travel discrete and simple routes in the cell."


Koide and his team are now working on streamlining directed network wiring and applying it to other areas of study such as cancer. With the ability to dramatically simplify how scientists study protein interaction networks, they hope to open the door to new research areas and therapeutic approaches.


"We can now design synthetic proteins that are far more sophisticated than natural ones, and use such super-performance proteins toward advancing science and medicine," he said.

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Shutting down the cloaking system of SARS could pave the way to new vaccines for SARS and MERS

Shutting down the cloaking system of SARS could pave the way to new vaccines for SARS and MERS | Amazing Science | Scoop.it

A Purdue University-led research team has figured out how to disable a part of the SARS virus responsible for hiding it from the immune system; a critical step in developing a vaccine against the deadly disease.

"This is a first step toward creating a weakened and safe virus for use in an attenuated live vaccine," said Mesecar, Purdue's Walther Professor of Cancer Structural Biology and professor of biological sciences and chemistry. "This also could serve as a molecular roadmap for performing similar studies on other coronaviruses, like MERS, because this enzyme appears to be common to all viruses within this family."


Mesecar and his team captured the molecular structure of a key SARS enzyme, papain-like protease, and revealed how it strips a host cell of the proteins ubiquitin and ISG15, which are involved in triggering an immune response.


"With most viruses, when a cell is infected it sends out an alarm triggering an immune response that fights the infection, but successful viruses are able to trick the immune system," Mesecar said. "By clipping off these two proteins, SARS short circuits the host cell's signaling pathways and prevents it from alerting the immune system to its presence. By removing these proteins, the enzyme serves as a biological cloaking system for the SARS virus that allows it to live and replicate undetected."

The disruption in its natural signaling pathways also causes an infected cell to miscommunicate with the cells around it, which leads to a response that eventually kills those cells, he said.


"Some treatments prevent a virus from replicating and stop further infection, but that doesn't necessarily prevent a harmful reaction to the virus," Mesecar said. "Sometimes it is the confusion in cellular communication that makes a virus lethal."


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Researchers use light to coax stem cells to repair teeth

Researchers use light to coax stem cells to repair teeth | Amazing Science | Scoop.it

A Harvard-led team is the first to demonstrate the ability to use low-power light to trigger stem cells inside the body to regenerate tissue, an advance they reported in Science Translational Medicine.  The research, led by David J. Mooney, Robert P. Pinkas Family Professor of Bioengineering at the Harvard School of Engineering and Applied Sciences (SEAS), lays the foundation for a host of clinical applications in restorative dentistry and regenerative medicine more broadly, such as wound healing, bone regeneration, and more.


The team used a low-power laser to trigger human dental stem cells to form dentin, the hard tissue that is similar to bone and makes up the bulk of teeth. What’s more, they outlined the precise molecular mechanism involved, and demonstrated its prowess using multiple laboratory and animal models. 


A number of biologically active molecules, such as regulatory proteins called growth factors, can trigger stem cells to differentiate into different cell types. Current regeneration efforts require scientists to isolate stem cells from the body, manipulate them in a laboratory, and return them to the body—efforts that face a host of regulatory and technical hurdles to their clinical translation. But Mooney’s approach is different and, he hopes, easier to get into the hands of practicing clinicians.


“Our treatment modality does not introduce anything new to the body, and lasers are routinely used in medicine and dentistry, so the barriers to clinical translation are low,” said Mooney, who is also a Core Faculty Member at the Wyss Institute for Biologically Inspired Engineering at Harvard. “It would be a substantial advance in the field if we can regenerate teeth rather than replace them.” 

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Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases

Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases | Amazing Science | Scoop.it

The capacity of designed nucleases, like ZFNs and TALENs, to generate DNA double-stranded breaks (DSBs) at desired positions in the genome has created optimism for therapeutic translation of locus-directed genome engineering. ZFNs and TALENs are chimeric nucleases composed of a custom-designed DNA binding domain fused to the DNA-cleavage domain from the FokI endonuclease that upon dimer formation cleaves the DNA. ZFN- and TALEN-induced DSBs trigger genome editing through cellular repair mechanisms involving either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR) with an available DNA donor template. Designer nucleases have broad applications in biological experimentation (Urnov et al., 2010;Bogdanove and Voytas, 2011) and have been successfully utilized for the production of gene knockout model animals (Doyon et al., 2008Geurts et al., 2009Tesson et al., 2011) and in emerging gene therapies (Perez et al., 2008Li et al., 20112013Sun et al., 2012).


The safety of designer nucleases is of major concern in relation to their use in treatment of human diseases. Thus far, ZFNs and TALENs have been administered to cells by transfection or electroporation of nucleic acids, DNA or RNA, encoding a pair of nuclease proteins (Urnov et al., 2005Miller et al., 2011Carlson et al., 2012) or by exploiting viral gene vehicles such as integrase-deficient lentiviral vectors (IDLVs) (Lombardo et al., 2007), adeno-associated virus-derived vectors (AAV vectors) (Ellis et al., 2013), or adenoviral vectors (Holkers et al., 2013). Successful administration of ZFN- or TALEN-encoding genes leads to high intracellular levels of nucleases and furthermore imposes a risk of random insertion in the genome, resulting potentially in prolonged nuclease expression and accumulating events of off-target cleavage. Ideally, ZFNs and TALENs are provided in a ‘hit-and-run’ fashion allowing short-term and dose-controllable nuclease activity without losing the effectiveness of creating locus-directed DSBs. Towards this goal, ZFNs have been fused to destabilizing domains regulated by small molecules to attenuate ZFN toxicity (Pruett-Miller et al., 2009).


Moreover, by exploiting the cell-penetrating capability of ZFNs, targeted gene disruption has recently been achieved by direct cellular delivery of purified ZFN proteins (Gaj et al., 2012). Although such approach may require multiple treatments due to the reduced cellular uptake of proteins (Mellert et al., 2012), recent findings suggest that ZFN uptake may be further improved by ligand-mediated endocytosis (Chen et al., 2013). However, for gene correction by homology-directed repair such strategies would need to be combined with other means of delivering the donor template.


It has been known for decades that retroviruses can tolerate the incorporation of heterologous proteins (Jones et al., 1990Weldon et al., 1990). Lentiviral particles (LPs) have been engineered to carry foreign proteins for the purpose of visualizing the intracellular behavior of the virus during infection (McDonald et al., 2002Jouvenet et al., 2008) and altering the viral integration profile (Bushman, 1994Goulaouic and Chow, 1996Bushman and Miller, 1997), as well as for ferrying antiviral (Okui et al., 2000Ao et al., 2008) and antitumor (Link et al., 2006Miyauchi et al., 2012) protein therapeutics. As the delicate structural composition of HIV-1-derived lentiviral particles is easily disturbed by an inappropriate load of nonviral proteins, leading to suboptimal vector yields and/or reduced transduction capability, various strategies for transducing heterologous protein cargo have been scrutinized. In early strategies, the accessory HIV-1 protein Vpr was adapted as a carrier of fused proteins (Wu et al., 1995). Recently, Vpr fusions have been shown also to ferry Cre recombinase (Michel et al., 2010) and I-SceI meganuclease (Izmiryan et al., 2011) into transduced cells. However, HIV-1 virions incorporate relatively few copies of Vpr (estimated 700 copies Vpr per virion [Swanson and Malim, 2008]), and the therapeutic potential of such approach may be hampered further by the known toxicity of the Vpr protein (Tachiwana et al., 2006).


Alternatively, nonviral proteins may be packaged in LPs as part of the Gag polypeptide, as was previously shown for reporter proteins like GFP (Aoki et al., 2011) and the apoptosis-inducing caspase 3 protein (Miyauchi et al., 2012). During virion maturation, Gag is processed by the viral proteins into shorter proteins constituting the structural—and most abundant—proteins of the virus particle. It is estimated that each virion contains 5000 copies of Gag and 250 copies of GagPol (Swanson and Malim, 2008). A research team recently adapted LPs for the delivery of the piggybac DNA transposase (Cai et al., 2014). The transposase was released from Gag in the virus particles in a protease-dependent manner and found to be able to facilitate efficient DNA transposition in transduced cells. In yet another strategy, heterologous proteins fused to the integrase in the Pol region of the GagPol polypeptide were successfully delivered by protein transduction (Schenkwein et al., 2010).


This present study describes the use of lentivirus-derived particles as carriers of designer nucleases for safe administration of ZFN and TALEN proteins fused to lentiviral Gag precursors. The researchers produce ZFN-loaded lentiviral particles that induce high-efficiency gene disruption with a favorable on-target/off-target ratio in safe genomic harbors like the CCR5 locus. Also, gene disruption and repair is evident in cells treated with particles carrying TALEN proteins. Successful incorporation of nuclease proteins within lentiviral particles allows co-delivery of nucleases and the donor template for homology-directed repair. The obtained findings demonstrate targeted and programmable gene repair in the human genome by delivery of both ‘scissors’ and ‘patch’ in a single combined protein and gene vehicle.

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