Salk Scientists Discover a Key to Mending Broken Hearts

Researchers regenerate and heal mouse hearts by using the molecular machinery the animals had all along

Friday, 07 November 2014

Researchers at the Salk Institute have healed injured hearts of living mice by reactivating long dormant molecular machinery found in the animals’ cells, a finding that could help pave the way to new therapies for heart disorders in humans.

An injured zebrafish heart showing proliferating

cells in the wounded area of the heart (red) and

cardiac muscle cells (green). Credit: Courtesy of

the Salk Institute for Biological Studies. 

The new results, published November 6, 2014 in the journal Cell Stem Cell, suggest that although adult mammals don’t normally regenerate damaged tissue, they may retain a latent ability as a holdover, like their distant ancestors on the evolutionary tree. When the Salk researchers blocked four molecules thought to suppress these programs for regenerating organs, they saw a drastic improvement in heart regeneration and healing in the mice.

The findings provide proof-of-concept for a new type of clinical treatment in the fight against heart disease, which kills about 600,000 people each year in the United States – more than AIDS and all cancer types combined, according to the U.S. Centers for Disease Control and Prevention.

“Organ regeneration is a fascinating phenomenon that seemingly recapitulates the processes observed during development. However, despite our current understanding of how embryogenesis and development proceeds, the mechanisms preventing regeneration in adult mammals have remained elusive,” says the study’s senior author Juan Carlos Izpisua Belmonte, a professor in the Gene Expression Laboratory at Salk and holder of the Roger Guillemin Chair.

Within the genomes of every cell in our bodies, we have what information we need to generate an organ. Izpisua Belmonte’s group has for many years focused on elucidating the key molecules involved in embryonic development as well as those potentially underlying healing responses in regenerative organisms such as the zebrafish.

From left: Alejandro Ocampo, Concepcion

Rodriguez Esteban, Juan Carlos Izpisua

Belmonte, Ignacio Sancho-Martinez, Tomoaki

Hishida and Eric Vazquez. Credit: Courtesy of

the Salk Institute for Biological Studies. 

Indeed, back in 2003, Izpisua Belmonte’s laboratory first identified the signals preceding zebrafish heart regeneration. And in a 2010 Nature paper, the researchers described how regeneration occurred in the zebrafish. Rather than stem cells invading injured heart tissue, the cardiac cells themselves were reverting to a precursor-like state (a process called ‘dedifferentiation’), which, in turn, allowed them to proliferate in tissue.

Although in theory it might have seemed like the next logical step to ask whether mammals had evolutionarily conserved any of the right molecular players for this kind of regenerative reprogramming, in practice it was a scientific risk, recalls Ignacio Sancho-Martinez, a postdoctoral researcher in Izpisua Belmonte’s lab.

“When you speak about these things, the first thing that comes to peoples’ minds is that you’re crazy,” he says.

“It’s a strange sounding idea, since we associate regeneration with salamanders and fish, but not mammals.”

Most other studies have looked to the hearts of neonatal mammals for molecular clues about proliferation, to no avail.

“Instead, we thought, ‘If fish know how to do it, there must be something they can teach us about it,’” says the study’s first author Aitor Aguirre, a postdoctoral researcher in Izpisua Belmonte’s group.

In a dish, heart muscle cells return to a precursor-

like state after pro-regenerative treatment with

microRNA inhibitors. Green shows a

disorganized cardiomyocyte cytoskeleton

indicative of cell dedifferentiation; red shows

mitochondrial organization. Credit: Courtesy of

the Salk Institute for Biological Studies. 

The team decided to focus on microRNAs, in part because these short strings of RNA control the expression of many genes. They performed a comprehensive screen for microRNAs that were changing in their expression levels during the healing of the zebrafish heart and that were also conserved in the mammalian genome.

Their studies uncovered four molecules in particular – MiR-99, MiR-100, Let-7a and Let-7c – that fit their criteria. All were heavily repressed during heart injury in zebrafish and they were also present in rats, mice and humans.

However, in studies of mammalian cells in a culture dish and studies of living mice with heart damage, the group saw that the levels of these molecules were high in adults and did not decline with injury. So the team used adeno-associated viruses specific for the heart to target each of those four microRNAs, suppressing their levels experimentally.

Injecting the inhibitors into the hearts of mice that had suffered a heart attack triggered the regeneration of cardiac cells, improving numerous physical and functional aspects of the heart, such as the thickness of its walls and its ability to pump blood. The scarring caused by the heart attack was much reduced with treatment compared to controls, the researchers found.

The improvements were still obvious three and six months after treatment – a long time in a mouse’s life.

“The good thing is that the success was not limited to the short term, which is quite common in cardiac regenerative biology,” Sancho-Martinez says.

The new study focused only on a handful of 70 some microRNA candidates that turned up in the group’s initial screen. These other molecules will likely also play a part in heart cell proliferation, healing scars and promoting the formation of new blood vessels – all processes critical for heart repair, Sancho-Martinez says. The data are available so that other research groups can focus on molecules that interest them.

The next step for Izpisua Belmonte’s team is to move into larger animals and see whether “regenerative reprogramming” can work in larger hearts, and for extended periods after treatment, says Sancho-Martinez. And, although the virus packaging disappeared from the animals’ bodies by 2 weeks after treatment, the scientists are working on a new way to deliver the inhibitors to avoid the need for viruses altogether.

Source: Salk Institute

Reference:

In vivo reprogramming with conserved regenerative effectors promotes robust mammalian heart regeneration

Authors: Aitor Aguirre, Nuria Montserrat, Serena Zachiggna, Emmanuel Nivet, Tomoaki Hishida, Marie Nicole Krause, Leo Kurian, Alejandro Ocampo, Eric Vazquez-Ferrer, Concepcion Rodriguez-Esteban, Sachin Kumar, James J. Moresco, John R. Yates III, Josep Maria Campistol, Ignacio Sancho-Martinez, Mauro Giacca and Juan Carlos Izpisua Belmonte

Cell Stem Cell, Volume 15, Issue 5, p589–604, 6 November 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.10.003

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Human Skin Cells Reprogrammed Directly into Brain Cells

Human Skin Cells Reprogrammed Directly into Brain Cells

Wednesday, 22 October 2014

Scientists have described a way to convert human skin cells directly into a specific type of brain cell affected by Huntington's disease, an ultimately fatal neurodegenerative disorder. Unlike other techniques that turn one cell type into another, this new process does not pass through a stem cell phase, avoiding the production of multiple cell types, the study's authors report.

Scientists have described a way to convert

human skin cells directly into a specific type of

brain cell affected by Huntington's disease, an

ultimately fatal neurodegenerative disorder.

Unlike other techniques that turn one cell type

into another, this new process does not pass

through a stem cell phase, avoiding the

production of multiple cell types, the study's

authors report. The researchers, at Washington

University School of Medicine in St. Louis,

demonstrated that these converted cells survived

at least six months after injection into the brains

of mice and behaved similarly to native cells in

the brain. Human skin cells (top) can be

converted into medium spiny neurons (bottom)

with exposure to the right combination of

microRNAs and transcription factors, according

to work by Andrew Yoo and his research team. 

Credit: Yoo lab.

The researchers, at Washington University School of Medicine in St. Louis, demonstrated that these converted cells survived at least six months after injection into the brains of mice and behaved similarly to native cells in the brain.

"Not only did these transplanted cells survive in the mouse brain, they showed functional properties similar to those of native cells," said senior author Andrew S. Yoo, PhD, assistant professor of developmental biology.

"These cells are known to extend projections into certain brain regions. And we found the human transplanted cells also connected to these distant targets in the mouse brain. That's a landmark point about this paper."

The work appears Oct. 22 in the journal Neuron.

The investigators produced a specific type of brain cell called medium spiny neurons, which are important for controlling movement. They are the primary cells affected in Huntington's disease, an inherited genetic disorder that causes involuntary muscle movements and cognitive decline usually beginning in middle-adulthood. Patients with the condition live about 20 years following the onset of symptoms, which steadily worsen over time.

The research involved adult human skin cells, rather than more commonly studied mouse cells or even human cells at an earlier stage of development. In regard to potential future therapies, the ability to convert adult human cells presents the possibility of using a patient's own skin cells, which are easily accessible and won't be rejected by the immune system.

Scientists have described a way to convert

human skin cells directly into a specific type of

brain cell affected by Huntington's disease, an

ultimately fatal neurodegenerative disorder.

Unlike other techniques that turn one cell type

into another, this new process does not pass

through a stem cell phase, avoiding the

production of multiple cell types, report

researchers at Washington University School of

Medicine in St. Louis. The investigators,

including Andrew Yoo, PhD, (from left) Michelle

Richner and Matheus Victor, demonstrated that

these converted cells survived at least six months

after injection into the brains of mice and

behaved similarly to native cells in the brain. 

Credit: Daniel Abernathy.

To reprogram these cells, Yoo and his colleagues put the skin cells in an environment that closely mimics the environment of brain cells. They knew from past work that exposure to two small molecules of RNA, a close chemical cousin of DNA, could turn skin cells into a mix of different types of neurons.

In a skin cell, the DNA instructions for how to be a brain cell, or any other type of cell, are neatly packed away, unused. In past research published in Nature, Yoo and his colleagues showed that exposure to two microRNAs called miR-9 and miR-124 altered the machinery that governs packaging of DNA. Though the investigators still are unravelling the details of this complex process, these microRNAs appear to be opening up the tightly packaged sections of DNA important for brain cells, allowing expression of genes governing development and function of neurons.

Knowing exposure to these microRNAs alone could change skin cells into a mix of neurons, the researchers then started to fine tune the chemical signals, exposing the cells to additional molecules called transcription factors that they knew were present in the part of the brain where medium spiny neurons are common.

"We think that the microRNAs are really doing the heavy lifting," said co-first author Matheus B. Victor, a graduate student in neuroscience.

"They are priming the skin cells to become neurons. The transcription factors we add then guide the skin cells to become a specific subtype, in this case medium spiny neurons. We think we could produce different types of neurons by switching out different transcription factors."

Yoo also explained that the microRNAs, but not the transcription factors, are important components for the general reprogramming of human skin cells directly to neurons. His team, including co-first author Michelle C. Richner, senior research technician, showed that when the skin cells were exposed to the transcription factors alone, without the microRNAs, the conversion into neurons wasn't successful.

The researchers performed extensive tests to demonstrate that these newly converted brain cells did indeed look and behave like native medium spiny neurons. The converted cells expressed genes specific to native human medium spiny neurons and did not express genes for other types of neurons. When transplanted into the mouse brain, the converted cells showed morphological and functional properties similar to native neurons.

To study the cellular properties associated with the disease, the investigators now are taking skin cells from patients with Huntington's disease and reprogramming them into medium spiny neurons using the approach described in the new paper. They also plan to inject healthy reprogrammed human cells into mice with a model of Huntington's disease to see if this has any effect on the symptoms.

Source: Washington University School of Medicine

Contact: Julia Evangelou Strait

Reference:

Generation of human striatal neurons by microRNA-dependent direct conversion of fibroblasts

Victor MB, Richner M, Hermanstyne TO, Ransdell JL, Sobieski C, Deng PY, Klyachko VA, Nerbonne JM, Yoo AS

Neuron. Oct. 22, 2014, Volume 84, Issue 2, p311–323

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Researchers Identify How Heart Stem Cells Orchestrate Regeneration

Exosomes, tiny 'bubbles' secreted by specialized cardiac stem cells, appear to carry 'instructions' that spur heart cells to regenerate following heart attack

Wednesday, 07 May 2014

Investigators at the Cedars-Sinai Heart Institute – whose previous research showed that cardiac stem cell therapy reduces scarring and regenerates healthy tissue after a heart attack in humans – have identified components of those stem cells responsible for the beneficial effects.

In a series of laboratory and lab animal studies, Heart Institute researchers found that exosomes, tiny membrane-enclosed "bubbles" involved in cell-to-cell communication, convey messages that reduce cell death, promote growth of new heart muscle cells and encourage the development of healthy blood vessels.

"Exosomes were first described in the mid-1980s, but we only now are beginning to appreciate their potential as therapeutic agents. We have found that exosomes and the cargo they contain are crucial mediators of stem cell-based heart regeneration, and we believe this might lead to an even more refined therapy using the 'active ingredient' instead of the entire stem cell," said Eduardo Marbán, MD, PhD, director of the Cedars-Sinai Heart Institute and a pioneer in developing investigational cardiac stem cell treatments.

"The concept of exosome therapy is interesting because it could potentially shift our strategy from living-cell transplantation to the use of a non-living agent," he added.

"Stem cells must be carefully preserved to keep them alive and functioning until the time of transplant, and there are some risks involved in cell transplantation. In contrast, exosome therapy may be safer and simpler and based on a product with a longer shelf life."

In lab experiments, the researchers isolated exosomes from specialized human cardiac stem cells and found that exosomes alone had the same beneficial effects as stem cells. Exosomes also produced the same post-heart attack benefits in mice, decreasing scar size, increasing healthy heart tissue and reducing levels of chemicals that lead to inflammation. Even when exosomes were injected in mice after heart attack scars were well-established, and traditionally viewed as "irreversible," they brought about multiple structural and functional benefits.

Exosomes transport small pieces of genetic material, called microRNAs, which enable cells to communicate with neighbouring cells to change their behaviour. The researchers pinpointed one such microRNA – one that is especially plentiful in cardiac stem cell exosomes – as responsible for some of the benefits. It is likely, they believe, that this and other microRNAs in the exosomes work together to produce the regenerative effects.

"The exosomes appear to contain the signalling information needed to regenerate healthy heart tissue, they are naturally able to permeate cells, and they have a coating that protects their payloads from degradation as they shuttle from cell to cell," said Marbán, senior author of an article in the May 6, 2014 Stem Cell Reports.

"Injecting exosomes derived from specialized cardiac stem cells may be an attractive alternative to the transplantation of living cells."

Marbán and his clinical and research teams in 2009 performed the first procedure in which a heart attack patient's heart tissue was used to grow specialized stem cells that were injected back into the heart. In 2012, they reported results of a clinical trial that found significant reduction in the size of heart attack-caused scars in patients who underwent the experimental stem cell procedure, compared to others who did not.

They also published findings from an animal study showing that the effect of stem cell therapy following heart attack is indirect – the stem cells themselves do not survive long after being placed in the heart, but they cause enduring effects by stimulating the rapid growth of surviving heart tissue and attracting stem cells already in the heart, which mature into functional heart cells.

The new study sheds light on the underlying mechanisms, crediting stem cell exosomes and the communications cargo they carry for orchestrating regeneration to repair heart attack damage.

The process to grow cardiac-derived stem cells was developed earlier by Marbán when he was on the faculty of Johns Hopkins University. The university has filed for a patent on that intellectual property and has licensed it to Capricor Inc., a biotechnology company in which Marbán is a founder and equity holder. Cedars-Sinai has filed for a patent for the exosome discovery and has licensed it to Capricor. The company provided no funding for this study.

Source: Cedars-Sinai Medical Center 

Contact: Sally Stewart

Reference:

Exosomes as Critical Agents of Cardiac Regeneration Triggered by Cell Therapy

Ahmed Gamal-Eldin Ibrahim, Ke Cheng, Eduardo Marbán

Stem Cell Reports, 6 May 2014, Volume 2, Issue 5, p606–619

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Protein Switch Dictates Cellular Fate

Stem cell or neuron

Thursday, 13 February 2014

Human neural progenitor cells isolated under

selective culture conditions from the developing

human brain and directed through lineage

differentiation. Neural progenitor cells are

stained green; differentiated astrocytes are

orange. Nuclei are stained blue. Credit: Image

courtesy of the National Institute of Neurological

Disorders and Stroke. 

Researchers at the University of California, San Diego School of Medicine have discovered that a well-known protein has a new function: It acts in a biological circuit to determine whether an immature neural cell remains in a stem-like state or proceeds to become a functional neuron.

The findings, published in the February 13 online issue of Cell Reports, more fully illuminate a fundamental but still poorly understood cellular act – and may have significant implications for future development of new therapies for specific neurological disorders, including autism and schizophrenia.

Postdoctoral fellow Chih-Hong Lou, working with principal investigator Miles F. Wilkinson, PhD, professor in the Department of Reproductive Medicine and a member of the UC San Diego Institute for Genomic Medicine, and other colleagues, discovered that this critical biological decision is controlled by UPF1, a protein essential for the nonsense-mediated RNA decay (NMD) pathway.

NMD was previously established to have two broad roles. First, it is a quality control mechanism used by cells to eliminate faulty messenger RNA (mRNA) – molecules that help transcribe genetic information into the construction of proteins essential to life. Second, it degrades a specific group of normal mRNAs. The latter function of NMD has been hypothesized to be physiologically important, but until now it had not been clear whether this is the case.

Wilkinson and colleagues discovered that in concert with a special class of RNAs called microRNA, UPF1 acts as a molecular switch to determine when immature (non-functional) neural cells differentiate into non-dividing (functional) neurons. Specifically, UPF1 triggers the decay of a particular mRNA that encodes for a protein in the TGF-b signalling pathway that promotes neural differentiation. By degrading that mRNA, the encoded protein fails to be produced and neural differentiation is prevented. Thus, Lou and colleagues identified for the first time a molecular circuit in which NMD acts to drive a normal biological response.

NMD also promotes the decay of mRNAs encoding proliferation inhibitors, which Wilkinson said may explain why NMD stimulates the proliferative state characteristic of stem cells.

"There are many potential clinical ramifications for these findings," Wilkinson said.

"One is that by promoting the stem-like state, NMD may be useful for reprogramming differentiated cells into stem cells more efficiently.”

"Another implication follows from the finding that NMD is vital to the normal development of the brain in diverse species, including humans. Humans with deficiencies in NMD have intellectual disability and often also have schizophrenia and autism. Therapies to enhance NMD in affected individuals could be useful in restoring the correct balance of stem cells and differentiated neurons and thereby help restore normal brain function."

Source: University of California - San Diego 

Contact: Scott LaFee

Reference:

Posttranscriptional Control of the Stem Cell and Neurogenic Programs by the Nonsense-Mediated RNA Decay Pathway

Chih H. Lou, Ada Shao, Eleen Y. Shum, Josh L. Espinoza, Lulu Huang, Rachid Karam, and Miles F. Wilkinson 

Cell Reports, 13 February 2014, 10.1016/j.celrep.2014.01.028

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Insulin-producing Beta Cells from Stem Cells

Scientists decipher early molecular mechanisms of differentiation

Thursday, 23 January 2014

Endodermal cells, they form organs such as lung,

liver and pancreas. Credit: IDR, Helmholtz

Zentrum München.

The Wnt/β-catenin signalling pathway and microRNA 335 are instrumental in helping form differentiated progenitor cells from stem cells. These are organized in germ layers and are thus the origin of different tissue types, including the pancreas and its insulin-producing beta cells. With these findings, Helmholtz Zentrum München scientists have discovered key molecular functions of stem cell differentiation which could be used for beta cell replacement therapy in diabetes. The results of the two studies were published in the renowned journal Development.

The findings of the scientists of the Institute of Diabetes and Regeneration Research (IDR) at Helmholtz Zentrum München (HMGU) provide new insights into the molecular regulation of stem cell differentiation. These results reveal important target structures for regenerative therapy approaches to chronic diseases such as diabetes.

During embryonic development, organ-specific cell types are formed from pluripotent stem cells, which can differentiate into all cell types of the human body. The pluripotent cells of the embryo organize themselves at an early stage in germ layers: the endoderm, mesoderm and ectoderm. From these three cell populations different functional tissue cells arise, such as skin cells, muscle cells, and specific organ cells.

Various signalling pathways are important for this germ layer organization, including the Wnt/β-catenin signalling pathway. The cells of the pancreas, such as the beta cells, originate from the endoderm, the germ layer from which the gastrointestinal tract, the liver and the lungs also arise. Professor Heiko Lickert, director of the IDR, in collaboration with Professor Gunnar Schotta of LMU München, showed that the Wnt/β-catenin signalling pathway regulates Sox17, which in turn regulates molecular programs that assign pluripotent cells to the endoderm, thus inducing an initial differentiation of the stem cells.

In another project Professor Lickert and his colleague Professor Fabian Theis, director of the Institute of Computational Biology (ICB) at Helmholtz Zentrum München, discovered an additional mechanism that influences the progenitor cells. miRNA-335, a messenger nucleic acid, regulates the endodermal transcription factors Sox17 and Foxa2 and is essential for the differentiation of cells within this germ layer and their demarcation from the adjacent mesoderm. The concentrations of the transcription factors determine here whether these cells develop into lung, liver or pancreas cells. To achieve these results, the scientists combined their expertise in experimental research with mathematical modelling.

"Our findings represent two key processes of stem cell differentiation," said Lickert.

"With an improved understanding of cell formation we can succeed in generating functional specialized cells from stem cells. These could be used for a variety of therapeutic approaches. In diabetes, we may be able to replace the defective beta cells, but regenerative medicine also offers new therapeutic options for other organ defects and diseases."

Diabetes is characterized by a dysfunction of the insulin-producing beta cells of the pancreas. Regenerative treatment approaches aim to renew or replace these cells. An EU-funded research project ('HumEn'), in which Lickert and his team are participating, shall provide further insights in the field of beta-cell replacement therapy.

The aim of research at Helmholtz Zentrum München, a partner in the German Center for Diabetes Research (DZD), is to develop new approaches for the diagnosis, treatment and prevention of major common diseases such as diabetes mellitus.

Source: Helmholtz Zentrum München

Contact: Heiko Lickert

References:

Wnt/β-catenin signalling regulates Sox17 expression and is essential for organizer and endoderm formation in the mouse 

Silvia Engert, Ingo Burtscher, W. Perry Liao, Stanimir Dulev, Gunnar Schotta and Heiko Lickert

Development, 2013, 140:3128-3138, doi:10.1242/dev.088765

miR-335 promotes mesendodermal lineage segregation and shapes a transcription factor gradient in the endoderm

Dapeng Yang, Dominik Lutter, Ingo Burtscher, Lena Uetzmann, Fabian J. Theis, and Heiko Lickert

Development, 2014, 141, 514-525, doi:10.1242/dev.104232

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Reading DNA, Backward and Forward

Reading DNA, Backward and Forward

Tuesday, 25 June 2013

MIT biologists have discovered a mechanism that allows cells to read their own DNA in the correct direction and prevents them from copying most of the so-called "junk DNA" that makes up long stretches of our genome.

Only about 15 percent of the human genome consists of protein-coding genes, but in recent years scientists have found that a surprising amount of the junk, or intergenic DNA, does get copied into RNA — the molecule that carries DNA's messages to the rest of the cell.

Scientists have been trying to figure out just what this RNA might be doing, if anything. In 2008, MIT researchers led by Institute Professor Phillip Sharp discovered that much of this RNA is generated through a process called divergent expression, through which cells read their DNA in both directions moving away from a given starting point.

In a new paper appearing in Nature on June 23, Sharp and colleagues describe how cells initiate but then halt the copying of RNA in the upstream, or non-protein-coding direction, while allowing it to continue in the direction in which genes are correctly read. The finding helps to explain the existence of many recently discovered types of short strands of RNA whose function is unknown.

"This is part of an RNA revolution where we're seeing different RNAs and new RNAs that we hadn't suspected were present in cells, and trying to understand what role they have in the health of the cell or the viability of the cell," says Sharp, who is a member of MIT's Koch Institute for Integrative Cancer Research.

"It gives us a whole new appreciation of the balance of the fundamental processes that allow cells to function."

Graduate students Albert Almada and Xuebing Wu are the lead authors of the paper. Christopher Burge, a professor of biology and biological engineering, and undergraduate Andrea Kriz are also authors.

Choosing direction

DNA, which is housed within the nucleus of cells, controls cellular activity by coding for the production of RNAs and proteins. To exert this control, the genetic information encoded by DNA must first be copied, or transcribed, into messenger RNA (mRNA).

When the DNA double helix unwinds to reveal its genetic messages, RNA transcription can proceed in either direction. To initiate this copying, an enzyme called RNA polymerase latches on to the DNA at a spot known as the promoter. The RNA polymerase then moves along the strand, building the mRNA chain as it goes.

When the RNA polymerase reaches a stop signal at the end of a gene, it halts transcription and adds to the mRNA a sequence of bases known as a poly-A tail, which consists of a long string of the genetic base adenine. This process, known as polyadenylation, helps to prepare the mRNA molecule to be exported from the cell's nucleus.

By sequencing the mRNA transcripts of mouse embryonic stem cells, the researchers discovered that polyadenylation also plays a major role in halting the transcription of upstream, noncoding DNA sequences. They found that these regions have a high density of signal sequences for polyadenylation, which prompts enzymes to chop up the RNA before it gets very long. Stretches of DNA that code for genes have a low density of these signal sequences.

The researchers also found another factor that influences whether transcription is allowed to continue. It has been recently shown that when a cellular factor known as U1 snRNP binds to RNA, polyadenylation is suppressed. The new MIT study found that genes have a higher concentration of binding sites for U1 snRNP than noncoding sequences, allowing gene transcription to continue uninterrupted.

A widespread phenomenon

The function of all of this upstream noncoding RNA is still a subject of much investigation.

"That transcriptional process could produce an RNA that has some function, or it could be a product of the nature of the biochemical reaction. This will be debated for a long time," Sharp says.

His lab is now exploring the relationship between this transcription process and the observation of large numbers of so-called long noncoding RNAs (lncRNAs). He plans to investigate the mechanisms that control the synthesis of such RNAs and try to determine their functions.

"Once you see some data like this, it raises many more questions to be investigated, which I'm hoping will lead us to deeper insights into how our cells carry out their normal functions and how they change in malignancy," Sharp says.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Albert E. Almada, Xuebing Wu, Andrea J. Kriz, Christopher B. Burge & Phillip A. Sharp

Nature (2013) doi:10.1038/nature12349

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Enhancing RNA Interference

Enhancing RNA Interference

Tuesday, 25 June 2013

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

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

Lipid nanoparticles (carrying siRNA) are 

shown as they are transported inside cells 

using endocytic vesicles. Credit: Daria 

Alakhova and Gaurav Sahay. 

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

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

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

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

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

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

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

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

Molecular traffic jam

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

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

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

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

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

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

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

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

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

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

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How Many Genes in the Human Genome? Human Genome Far More Active than Thought

GENCODE Consortium discovers far more genes than previously thought

Friday, 07 September 2012

The GENCODE Consortium expects the human genome has twice as many genes than previously thought, many of which might have a role in cellular control and could be important in human disease. This remarkable discovery comes from the GENCODE Consortium, which has done a painstaking and skilled review of available data on gene activity.

Among their discoveries, the team describes more than 10,000 novel genes; identify genes that have 'died' and others that are being resurrected. The GENCODE Consortium reference gene catalogue has been one of the underpinnings of the larger ENCODE Project and will be essential for the full understanding of the role of our genes in disease.

The GENCODE Consortium is part of the ENCODE Project that, today, publishes 30 research papers describing findings from their nearly decade-long effort to describe comprehensively all the active regions of our human genome. ENCODE was launched in 2003 after the completion of the Human Genome Project, and brought together an international group of scientists tasked with identifying and describing all functional regions of the human genome sequence.

"We have uncovered a staggering array of genes in our genome, simply because we can examine many genomes in a detail that was not possible a decade ago," says Dr. Jennifer Harrow, GENCODE principle investigator from the Wellcome Sanger Institute. 

"As sequencing technology improves, so we have much more data to explore.

"But our work remains a skilled effort to annotate correctly our human genome - or, more precisely, our human genomes, for each of us differ. These vast texts of genetic information will not give up their secrets easily. GENCODE has made amazing strides to enable immediate access of its reference gene set by other researchers."

The team more accurately described the genes that contain the genetic code to make proteins: they found 20,687 such protein-coding genes, a value that has not changed greatly from previous work. The new set captures far more of the alternative forms of these genes found in different cell types.

More significant are their findings on genes that do not contain genetic code to make proteins - non-coding genes - and the graveyard of supposedly 'dead' genes from which some are emerging, resurrected from the catalogue of pseudogenes.

They mapped and described 9,277 long non-coding genes, a relatively new type that acts, not through producing a protein, but directly through its RNA messenger. Long non-coding RNAs derived from these genes can play a significant part in human biology and disease, but they remain only poorly understood.

"If the Human Genome Project was the baseline for genetics, ENCODE is the baseline for biology, and GENCODE are the parts that make the human biological machine work. Our list is essential to all those who would fix the human machine," said Dr. Tim Hubbard.

The new map of such genetic components gives researchers more avenues to explore in their quest to understand human biology and human disease. Remarkably, the team thinks their job is not complete and believes that there may be another 10,000 of these genes yet to be uncovered.

"Our initial work from the Human Genome Project suggested there were around 20,000 protein-coding genes and that value has not changed greatly," says Professor Roderic Guigo, GENCODE principle investigator from Centre for Genomic Regulation, Barcelona.

"However GENCODE has shown that long non-coding RNAs are far more numerous and important than previously thought"

"The limited knowledge we have of the class of long non-coding RNAs suggests they might play a major role in regulating the activity of other genes. If this is generally true of this group, we have much more to explore than we imagined."

As dramatic, GENCODE has catalogued for the first time a set of more than 11,000 pseudogenes by examining the entire human genome. There is some emerging evidence that many of these genes, too, might have some biological activity.

The GENCODE team predicts that at least 9% of pseudogenes may be active with some controlling the activity of other genes. Pseudogenes have been implicated in many biological activities, such as the prevention of certain elements known to be involved in the development of cancer.

"At the announcement of the Human Genome Project draft sequence, we emphasized this was the end of the beginning, that 'at present most genes - probably tens of thousands - remain a mystery'", says Dr. Tim Hubbard, lead principle investigator of GENCODE from the Wellcome Trust Sanger Institute. 

"Today, we describe many thousands of genes for the first time."

"If the Human Genome Project was the baseline for genetics, ENCODE is the baseline for biology, and GENCODE are the parts that make the human biological machine work. Our list is essential to all those who would fix the human machine."

The GENCODE human reference set will be updated every three months to ensure that models are continually refined and assessed based on new experimental data deposited in the public databases.

Source: Wellcome Trust Sanger Institute 

Contact: Don Powell

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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at

http://cellnews-blog.blogspot.com/

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