Researchers at the University of Delaware have discovered a novel technique–that acts like a “spell-checker” for correcting a misspelling in the DNA code–to repair the defective gene that causes spinal muscular atrophy (SMA). This hereditary neuromuscular disease is the number-one genetic killer of children under two years old.

Babies born with Type 1 SMA, the most severe form of the disease, can’t walk, crawl, sit unsupported, lift their heads, or breathe normally. Fifty percent die before their second birthday.

“Think of it like a spell-check program–we’re erasing the wrong letter in the DNA code and putting the right one in,” said Eric Kmiec, professor of biological sciences at UD.

Kmiec, who holds 14 patents for gene-editing technologies at the University, collaborated with research scientist Darlise DiMatteo and undergraduate Stephanie Callahan on the discovery in his laboratory at the Delaware Biotechnology Institute.

The technique has shown promising results in tests in mice and is now poised for development by OrphageniX Inc., based in Wilmington, Del. The start-up company was incorporated in 2005 to commercialize UD-patented technologies for repairing genes that cause rare, hereditary, “orphan” diseases, so named because they have not been “adopted” by the pharmaceutical industry for the development of treatments.

According to the Families of Spinal Muscular Atrophy, an international, nonprofit organization, the disease affects one in 6,000 babies born, and one in 40 people is a genetic carrier.

A genetic ‘bandage’

Spinal muscular atrophy is caused by a mutation in the SMN1 gene, which affects the motor neurons, the nerve cells in the spinal cord that control the muscles of the rib cage and limbs, which are essential for breathing, swallowing, sitting and walking.

Each gene is made up of a length of DNA, a code composed of the four chemical units that make up the genetic alphabet: A for adenine, G for guanine, C for cytosine and T for thymine.

In spinal muscular atrophy, a defect occurs in the SMN1 gene. There’s a letter out of place–a T (thymine) occurs where there should be a C (cytosine). As a result, the gene doesn’t make a protein that the motor nerves in the spinal cord need to survive, which leads to the gradual atrophy, or wasting, of the muscles.

To replace the function of the defective SMN1 gene, the UD research team used a gene in the human body that is nearly an exact copy (SMN2). Then they introduced a small fragment of this healthy gene’s DNA–a genetic “bandage” referred to as an oligonucleotide–into a diseased cell, triggering the cell to heal itself.

Tests of the technique in mice with spinal muscular atrophy, conducted by Jackson Laboratory in Bar Harbor, Maine, showed “very promising results” with the development of healthy muscle in the animals, Kmiec said.

“Babies with SMA die early in life,” Kmiec noted. “But if we can deliver the healing agent to the appropriate cell, we can help address this horrible disease. We’re not looking at a cure, but we hope this technique could lead to a series of treatments that could alleviate the symptoms and improve the quality of life of patients,” Kmiec said.

The technique, known as targeted gene alteration (TGA), is among a group of UD-patented technologies under development by OrphageniX, a pre-clinical development stage biotechnology company that has moved quickly out of the starting gate since its launch in February 2007.

“OrphageniX plans to develop a treatment for spinal muscular atrophy with help from expert consultants in the field,” Michael Herr, chief executive officer, said.

The development of a treatment for SMA would advance to clinical testing within a year from funding by either investors or commercial collaborators, Herr noted.

Patients with the less severe, Type III form of spinal muscular atrophy would be targeted for initial human trials. Although individuals with Type III SMA suffer from a range of muscle weakness and fatigue quickly, the disease generally is not life-threatening at this stage.

Herr said that OrphageniX is committed to helping people by commercializing scientific breakthroughs, but he noted that, “we must also provide an adequate return to investors for OrphageniX to succeed.”

Truly translational research

For his latest research to be truly “translational,” extending from the lab bench to the bedside, Kmiec said it has been critical to involve people like Darlise DiMatteo, who have a keen understanding of spinal muscular atrophy.

DiMatteo, who joined Kmiec’s research team a year ago, formerly worked at Nemours Alfred I. duPont Hospital for Children, where she conducted research studies of muscular dystrophy and SMA for more than a decade. The world-renowned children’s hospital continues to be an important partner on the project, Kmiec said.

“We’ve received significant assistance from Drs. Vicky Funanage and Wenlan Wang at A. I. duPont Hospital,” Kmiec noted. “They would be a natural choice for clinical trials in SMA.”

“I love coming to work knowing that this research could make a difference for families affected by this disease,” DiMatteo said. “It’s intriguing–why does a deficit in this particular protein cause this disease? And why do humans have an SMN2 gene that’s almost identical to SMN1 when animals don’t have that kind of backup? The effort will have been worth it if we can help find the answers.”

The research also has had a profound effect on Stephanie Callahan, an undergraduate student at UD who helped carry out the laboratory experiments, working under DiMatteo’s guidance.

Callahan had the opportunity to participate in the project through a summer internship in the IDeA Network of Biomedical Research Excellence (INBRE) program offered by the Delaware Biotechnology Institute when she was a student at Delaware Technical and Community College. Now she’s finishing up her degree in biological sciences with a concentration in biotechnology and wants to pursue her master’s degree at UD. After completing her education, she hopes to get a job doing research in industry, perhaps at a pharmaceutical company.

“It really opened my eyes to the possibilities and the potential applications of what you can do in the lab,” Callahan said. “It’s been a great experience for me.”

Kmiec said the research so far has all the elements of a “real Delaware story”–connecting UD, A. I. duPont Hospital for Children, tobacco settlement funding awarded by the state, and a start-up company fueled by Delaware investors–and he’s excited about the future.

“Publishing an article in a research journal is not the accomplishment–that is what some of us are paid to do, and my colleagues do this as well as I,” Kmiec said. “But the fact that the research program is translational and is working in that direction with outside validation and support is the real news. I hope our experience will help UD and other researchers like us realize their technology possibilities,” he added.

“What we’ve discovered–this gene spell-check–sounds very simple, where you erase one letter and put the right one in,” Kmiec noted, “but finding the pathway has taken a long time, since 1994. Now, with this latest development, we’ve taken a laser shot out of the primordial soup. It’s a chance finally to make a difference for families with this disease.”

The research is published in the Jan. 14 online edition of Experimental Cell Research. The study was supported by $477,500 in National Tobacco Settlement funds to the state of Delaware. The research grant was awarded through the Delaware Health Fund.

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Deposits of nearly pure silica discovered by the Mars Exploration Rover Spirit in Gusev Crater formed when volcanic steam or hot water (or maybe both) percolated through the ground. Such deposits are found around hydrothermal vents like those in Yellowstone National Park. That’s the conclusion of planetary scientists working with data collected by the rover’s Miniature Thermal Emission Spectrometer (Mini-TES), developed at Arizona State University.

The silica discovery, announced briefly by NASA in 2007, is fully described in a multi-author paper that appears in the May 23, 2008 issue of the scientific journal Science. The lead author is Steven Squyres of Cornell University, principal investigator for the rover science payload.

The silica finding turns a spotlight on an important site that may contain preserved traces of ancient Martian life.

“On Earth, hydrothermal deposits teem with life and the associated silica deposits typically contain fossil remains of microbes,” says Jack Farmer, professor of astrobiology in ASU’s School of Earth and Space Exploration, part of the College of Liberal Arts and Sciences. Farmer is one of the paper’s co-authors.

“But we don’t know if that’s the case here,” Farmer notes, “because the rovers don’t carry instruments that can detect microscopic life.” He adds, “What we can say is that this was once a habitable environment where liquid water and the energy needed for life were present.”

NASA landed the two Mars rovers, Spirit and Opportunity, on opposite sides of the planet in January 2004 to look for rocks showing the presence of water. As of now, the rovers are more than four Earth years into a mission designed to last just three months. Despite dust collecting on their solar panels and mechanical wear-and-tear, both are continuing to explore.

Dawning realization

The silica discovery unfolded in slow motion as Spirit emerged from hibernation after its second Martian winter. The rover spent those months on the edge of a football-field-size feature dubbed Home Plate.

Home Plate lies in the Columbia Hills, a range of low hills in the middle of Gusev Crater, which spans 100 miles (170 kilometers) wide. The Hills rise about 300 feet (100 meters) above the flat lava plain that fills Gusev, but their structure and origin remain unclear to scientists.

“We were going back to an area of exposed soil called the Tyrone site, which we didn’t have time to investigate before winter began,” notes Steven Ruff, a faculty research associate at ASU’s Mars Space Flight Facility. Ruff is another of the paper’s co-authors.

The Tyrone soil proved rich in sulfate minerals, a phenomenon seen by Spirit at other locations in the Columbia Hills, where Spirit has been exploring since late 2004. While sulfates can form in several ways, water is involved in most.

“While parked next to Tyrone, we used the Mini-TES to look at some nearby light-toned and knobby outcrops,” says Ruff.

Ruff is the geologist in charge of day-to-day operations for Mini-TES, which was designed by ASU’s Philip Christensen, a Regents’ Professor of Geological Sciences and director of the Mars Space Flight Facility. Each rover is outfitted with a Mini-TES.

Silica surprise

Ruff continues, “It wasn’t clear what we were seeing in the knobby outcrops because they were contaminated with dust and wind-blown soil. But I thought they might be silica-rich.” Additional surveys with Mini-TES identified other outcrops, similarly contaminated but likewise hinting at silica.

As it happened, the rover’s jammed right front wheel inadvertently produced the “Aha!” moment. Ruff and others on the science team noticed that the stuck wheel had gouged a trench a few inches deep through the soil as the rover drove ahead in reverse, dragging the crippled wheel behind.

“The trench looked bright white,” Ruff recalls, “but we thought initially it was just more sulfate minerals.”

While parked nearby, however, Ruff got curious. “We aimed Mini-TES at the trench and it showed a clear silica spectrum. This prompted us to drive back to it, where the rover’s Alpha Particle X-Ray Spectrometer told us the white soil was more than 90 percent silica. That’s a record high for silica on Mars.”

Fumaroles and hot springs

Making such pure silica requires a lot of water, says Ruff. “On Earth, the only way to have this kind of silica enrichment is by hot water reacting with rocks.”

This, Ruff says, links the silica with Home Plate, which the rover team already knew was a volcanic feature. “Home Plate came from an explosive volcanic event with water or ice being involved,” he explains. “We saw where rocks were thrown into the air and landed to make small indentations in the soft, wet ash sediment around the vent.”

Once alerted what to look for, the scientists found more silica in many places nearby.

As Ruff explains, “It’s not just the soil in a trench in one place. It’s a broader story of outcrops that extend 50 meters [about 150 feet] away from Home Plate. It’s not a small scale, modest phenomenon.”

The combination of geothermal heat and water produces a hydrothermal system like that which powers the hot springs, geysers, mudpots, and fumaroles (steam vents) of Yellowstone National Park.

Capturing evidence

Astrobiologist Farmer helped with the mineral identification by supplying a variety of high silica rock samples from his laboratory collection. They included rocks from hot spring and fumarole deposits in Yellowstone and New Zealand. These rocks provided reference spectra for Mini-TES. “The best fit we got was with siliceous sinter,” he says, referring to deposits of “opal,” a type of amorphous silica laid down by hot springs.

Farmer explains that hydrothermal systems generally precipitate silica and other minerals as heated groundwater rises, cools, and gives off dissolved gases. “If there were organisms living there,” he says, “our terrestrial experience shows that microbes can easily be entrapped and preserved in the deposits.” Silica, he notes, is an excellent medium for capturing and preserving traces of microbial life.

Whether Mars ever had life is unknown. But if there was once a Martian biosphere, both Ruff and Farmer say the deposits around fumaroles and hot springs are ideal places to start hunting for it.

Although the microscopic imagers on the current rovers cannot resolve the microbial remains seen in terrestrial hot spring deposits, Farmer notes that the new microscopic imagers now in development for future rovers should let scientists detect such features in situ.

Says Farmer, “We just need to deliver such instruments to the right place. The discoveries at Home Plate have helped us know where to go next.”

Ruff adds, “This discovery has us really excited. This site is clearly the best example of a habitable environment that we’ve found in Gusev.”

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Researchers from the University of Melbourne, Australia, and the University of Texas, USA, have extracted genes from the extinct Tasmanian tiger (thylacine), inserted it into a mouse and observed a biological function — this is a world first for the use of the DNA of an extinct species to induce a functional response in another living organism.

The results showed that the thylacine Col2a1 gene has a similar function in developing cartilage and bone development as the Col2a1 gene does in the mouse.

“This is the first time that DNA from an extinct species has been used to induce a functional response in another living organism,” said Dr Andrew Pask, RD Wright Fellow at the University of Melbourne’s Department of Zoology who led the research.

“As more and more species of animals become extinct, we are continuing to lose critical knowledge of gene function and their potential.”

“Up until now we have only been able to examine gene sequences from extinct animals. This research was developed to go one step further to examine extinct gene function in a whole organism,” he said.

“This research has enormous potential for many applications including the development of new biomedicines and gaining a better understanding of the biology of extinct animals,” said Professor Richard Behringer, Deputy Head of the Department of Molecular Genetics, M.D. Anderson Cancer Center, at the University of Texas, who is the corresponding author on the paper.

The last known Tasmanian tiger died in captivity in the Hobart Zoo in 1936. This enigmatic marsupial carnivore was hunted to extinction in the wild in the early 1900s.

Researchers say fortunately some thylacine pouch young and adult tissues were preserved in alcohol in several museum collections around the world.

The research team used thylacine specimens from Museum Victoria in Melbourne Australia to examine how the thylacine genome functioned.

The research team isolated DNA from 100 year old ethanol fixed specimens. After authenticating this DNA as truly thylacine, it was inserted into mouse embryos and its function examined.

The thylacine DNA was resurrected, showing a function in the developing mouse cartilage, which will later form the bone.

“At a time when extinction rates are increasing at an alarming rate, especially of mammals, this research discovery is critical,” says Professor Marilyn Renfree, Federation Fellow and Laureate Professor in the University of Melbourne’s Department of Zoology, the senior author on the paper.

“For those species that have already become extinct, our method shows that access to their genetic biodiversity may not be completely lost.”

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