USF Health preclinical study tests several formulations of chitosan-enriched siRNA nanoparticles intended to improve gene therapy targeting neurodegenerative diseases

Neurologist Juan Sanchez-Ramos, MD, PhD, director of USF Health’s HDSA Huntington’s Disease Center of Excellence, examines patient and clinical trial participant Brittany Bosson.

Huntington’s disease (HD) is a hereditary brain disease that typically strikes adults in the prime of life – leading to progressive deterioration of movement, mood and thinking. While some drugs temporarily alleviate symptoms, currently no therapies prevent, slow or stop the course of HD.

Juan Sanchez-Ramos, MD, PhD, the Helen Ellis Professor of Neurology and director of  the HDSA Huntington’s Disease Center of Excellence, University of South Florida Health (USF Health), sees firsthand how this devastating illness – sometimes described as a mix of Parkinson’s disease, ALS and Alzheimer’s disease — affects patients and their families.  For the last several years, even as he leads clinical trials evaluating potential new drugs, the physician-scientist has worked with a mouse model of HD to develop and test a nanoparticle system that can precisely deliver gene therapy from the nose to areas of the brain most affected by HD.

He is closer than ever before to a viable noninvasive treatment – one that could be administered by nasal spray or drops, rather than spinal puncture or direct injection into the brain.

In a preclinical study published Oct. 27 in Nanomedicine: Nanotechnology, Biology and Medicine, senior author Dr. Sanchez-Ramos and colleagues build on their earlier findings demonstrating that chitosan-enriched, manganese-coated nanoparticles loaded with small interfering RNA (siRNA) could be successfully delivered by nose drops to targeted parts of the brain affected by HD.  In a Huntington’s disease mouse model the nanoparticles reduced expression of the mutated HTT gene that causes HD by at least 50% in four regions: the olfactory bulb, striatum, hippocampus and cortex. The defective HTT gene leads to production of a toxic form of protein, known as the huntingtin protein. In essence, this new treatment silences the genetic message “telling” a cell to generate more huntingtin proteins. To ultimately benefit patients, the abnormal protein production must be reduced enough to block or slow the dysfunction and eventual loss of nerve cells accounting for clinical symptoms.

“Our nose-to-brain approach for delivery of gene therapies is non-invasive, safe and effective,” said Dr. Sanchez-Ramos, a co-inventor of the novel anti-HTT siRNA nanoparticle delivery system patented by USF.

Searching for ways to optimize HD gene silencing

For the latest study, reported in Nanomedicine, Dr. Sanchez-Ramos collaborated with researchers from the USF Health Department of Neurology and the University of Massachusetts Medical School’s RNA Therapeutics Institute.  Seeking to optimize HD gene silencing when the siRNA is delivered by a nasal route, the team tested different formulations and sizes of the nanoparticles in a mouse model expressing the human HD gene. Among their findings:

— Four different versions of the nanoparticles tested lowered HD gene expression in the brain by 50%. However, lowering levels of the toxic huntingtin protein in brain tissue took longer, with the highest reduction of the protein (53%) seen in the olfactory bulb at the base of the brain and the lowest (38%) in the cerebral cortex, the brain’s outer layer. Also, simply administering “naked” siRNA through the nose (without the protective chitosan encasement) did little to reduce HD gene expression even though previous research has shown similar naked siRNA injected directly into the brain was highly effective.

— Enclosing the siRNA in chitosan protected the silencing RNA from being prematurely degraded “en route” to its HD brain targets. The compound chitosan is derived from the hard outer skeleton of shellfish or the external skeleton of insects. Encapsulating siRNA into a chitosan nanoparticle allowed the silencing RNA to be enriched to higher doses without damaging the molecule, resulting in significant reduction in HD gene expression, the researchers report.

— Increasing the number siRNA nanoparticles within a defined dose of nose drops is a key to improving therapeutic potential. “The ability to fabricate concentrated NP (nanoparticle) preparations without damaging siRNA content is a critical factor for successful intranasal delivery of gene silencing agents,” the researchers concluded.

A major challenge of gene therapy for HD and other neurodegenerative diseases has been getting the molecules intended to replace a missing gene or suppress an overactive gene past the blood-brain barrier, a kind of defensive wall that selectively filters which molecules can enter the brain from circulating blood.

But over the last several years, research progressed in overcoming this barrier and promising laboratory findings set the stage for clinical trials in patients with HD.

For example, led by Dr. Sanchez-Ramos, USF Health is the only Florida site participating in the Roche-sponsored GENERATION HD1 Study. This pivotal phase 3 international clinical trial is testing whether a huntingtin-lowering, antisense oligonucleotide drug can halt underlying pathology of the disease enough to improve symptoms in adult patients. The injectable drug, administered directly into the cerebral-spinal fluid, successfully bypasses the blood-brain barrier and stopped disease progression in laboratory models. However, the investigational drug must be administered every two months by lumbar puncture at the clinic.

The normal huntingtin gene contains a DNA alphabet that repeats the letters C-A-G as many as 26 times, but people who develop Huntington’s disease have an excessive number of these consecutive C-A-G triplet repeats — greater than 39.| Graphic by Sandra C. Roa

Working toward a simpler, noninvasive treatment

With a chronic illness that gradually encompasses the entire central nervous system, like HD, even minimally-invasive injections with fine needles or infusions may pose risks of infection or other complications associated with neurosurgical procedures, Dr. Sanchez-Ramos said. So, he continues to work toward a noninvasive nose-to-brain treatment that would be simpler to repeat and well-tolerated by patients over their lifetime.

Dr. Sanchez-Ramos says the idea for incorporating nontoxic amounts of manganese chelate into the chitosan-based nanoparticles to help gene therapy delivery was sparked by early studies investigating how welders exposed to high levels of neurotoxic manganese oxide from welding fumes developed Parkinson’s disease symptoms.  It turns out that the olfactory nerve has an affinity for the chemical manganese.

“Manganese is good at guiding our nanoparticles from the nasal passages to the olfactory nerves and transporting the particles directly to structures deep in the brain… Realizing that was one of our biggest breakthroughs,” he said.  Manganese also permits the nanoparticles to be visualized by MRI imaging, so that their distribution and accumulation in different regions of brain can be tracked.

The nose-to-brain method of delivering the manganese-containing siRNA nanoparticles needs to be tested in a larger-brain animal model before moving to human trials.

The USF Health study was supported by a grant from the National Institute of Health’s National Institute of Neurological Disorders and Stroke.

As his preclinical research on nose-to-brain delivery of gene therapy for Huntington’s disease progresses, Dr. Sanchez-Ramos serves as Florida principal investigator for a worldwide clinical trial testing an injectable drug designed to slow the progression of Huntington’s disease.

-Photos by Allison Long, USF Health Communications and Marketing

The founding director of the USF Health Heart Institute has a passion for innovation, translational medicine and entrepreneurship.

Samuel A. Wickline, MD, has parlayed his expertise in harnessing nanotechnology for molecular imaging and targeted treatments into an impressive $1-million portfolio of National Institutes of Health awards, multiple patents and four start-up biotechnology companies.

“We’ve developed nanostructures that can carry drugs or exist as therapeutic agents themselves against various types of inflammatory diseases, including, cancer, cardiovascular disease, arthritis and even infectious diseases like HIV,” said Dr. Wickline, who arrived at USF Health last month from the Washington University School of Medicine in St. Louis.

Dr. Wickline: “Innovation is not just about having a new idea, it’s about having a useful idea.”

 Dr. Wickline comments on how being a physician adds perspective to the science he conducts.

At Washington University, Dr. Wickline, a cardiologist, most recently was J. Russell Hornsby Professor in Biomedical Sciences and a professor of medicine with additional appointments in biomedical engineering, physics, and cell biology and physiology.

“I like the challenge of building things,” he said.

In St. Louis, he built a 29-year career as an accomplished physician-scientist keenly interested in translating basic science discoveries into practical applications to benefit patients. He served as chief of cardiology at Jewish Hospital, developed one of the first cardiac MRI training and research programs in the country, helped establish Washington University’s first graduate program in biomedical engineering, and led a university consortium that works with academic and industry partners to develop medical applications for nanotechnology.

At USF, there will be no shortage of challenging opportunities to build.

Building the USF Health Heart Institute

A major part of Dr. Wickline’s new job is helping to design, build and equip the Heart Institute. Most importantly, he will staff the state-of-the-art facility with a critical interdisciplinary mix of top biomedical scientists (including immunologists, molecular biologists, cell physiologists and genomics experts), who investigate the root causes of heart and vascular disease with the aim of finding new ways to detect, treat and prevent them. The Heart Institute will be co-located with new Morsani College of Medicine in downtown Tampa; construction on the combined facility is expected to begin later this year.

“I have been impressed by the energy and commitment here at the University of South Florida to invest substantial resources in a heart institute,” Dr. Wickline said. “I believe we have a lot to offer in terms of bench-to-bedside research that could solve some of the major cardiovascular problems” like atherosclerosis or heart failure.

“We want to put together a program that supplies the appropriate core facilities to attract the best and brightest researchers to this cardiovascular institute.”

Cardiovascular disease is the leading cause of death in the United States and worldwide, so exploring potential new treatment options is critical. One of the Heart Institute’s driving themes will be advancing concepts and findings that prove promising in the laboratory into projects commercialized for clinical use, Dr. Wickline said.

“Our goal is to make a difference in the lives of patients,” he said. “Innovation is not just about having a new idea, it’s about having a useful idea.”

Dr. Wickline also serves as associate dean for cardiovascular research and a professor of cardiovascular sciences at the Morsani College of Medicine. He holds the Tampa General Endowed Chair for Cardiovascular Research created last year with a gift from USF’s primary teaching hospital.

With Washington University colleague Hua Pan, PhD, a biomedical engineer and expert in molecular biology, Dr. Wickline is re-building his group at USF. Dr. Pan was recently recruited to USF as an assistant professor of medicine to continue her collaborations with Dr. Wickline.

 An example of Dr. Wickline’s group using nanotechnology to help combat atherosclerosis.

Dr. Wickline’s lab focuses on building nanoparticles to deliver drugs or other therapeutic agents to specific cell types, or targets.

Designing nanoparticles to “kill the messenger”

Dr. Wickline’s lab focuses on building nanoparticles – shaped like spheres or plates, but 10 to 50 times smaller than a red blood cell – to deliver drugs or other therapeutic agents through the bloodstream to specific cell types, or targets. These tiny carrier systems can effectively deliver a sizeable dosage directly to a targeted tissue, yet only require small amounts of the treatment in the circulation to reduce the risk of harmful side effects.

Some types of nanoparticles can carry image-enhancing agents that allow researchers to quantify where the illuminated particles travel, serving as beacons to specific molecules of interest, and enabling one to determine whether a therapeutic agent has penetrated its targeted site, Dr. Wickline said.

Dr. Wickline also is known for designing nanoparticles derived from a component of bee venom called melittin. While bee venom itself is toxic, Dr. Wickline’s laboratory has detoxified the molecule and modified its structure to produce a formula that allows the nanoparticles to carry small interfering (siRNA), also known as “silencing RNA,” or other types of synthetic DNA or RNA strand.

Among other functions, siRNA can be used to inhibit the genes that lead to the production of toxic proteins. Many in the nanotechnology research and development community are working to make siRNA treatment feasible as what Dr. Wickline calls “a message killer,” but the challenges have been daunting.

“The big challenge in the field of siRNA, and many companies have failed at this, is how to get the nanostructure to the cells so that the siRNA can do what it’s supposed – hit its target and kill the messenger — without being destroyed along the way, or having harmful side effects,” Dr. Wickline said. “We figured out how to engineer into a simple peptide all of the complex functionality that allows that to happen.”

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 Dr. Wickline comments on the underlying similarities between cardiovascular disease and cancer.

Different targets, same delivery vehicle

In a recent series of experiments in mice, Dr. Wickline and colleagues have shown that silencing RNA messages delivered by nanoparticle to a specific type of immune cell known as a macrophage – a “big eater” of fat – actually shrinks plaques that accumulate inside the walls of the arteries during atherosclerosis, one of the main causes of cardiovascular disease. The build-up of atherosclerotic plaques with fat-laden macrophages narrows, weakens and hardens arteries, eventually reducing the amount of oxygen-rich blood delivered to vital organs.

This type of plaque-inhibiting nanotherapy could be useful in aggressive forms of atherosclerosis where patients have intractable chest pain or after an acute heart attack or stroke to prevent a secondary cardiac event, Dr. Wickline said.

In another study, Washington University School of Medicine researchers investigated the potential of the siRNA nanoparticle designed by co-investigators Dr. Pan and Dr. Wickline in treating the inflammation that may lead to osteoarthritis, a degenerative joint disease that is a major cause of disability in the aging population. The nanoparticles — injected directly into injured joints in mice to suppress the activity of the molecule NF-κB — reduced local inflammation immediately following injury and reduced the destruction of cartilage. The findings were reported September 2016 in the Proceedings of the National Academy of Sciences.

Previously, Dr. Wickline said, the Washington University group had shown that nanoparticles delivered through the bloodstream inhibited inflammation in a mouse model of rheumatoid arthritis. And, another laboratory at the University of Kentucky is studying whether locally injected siRNA nanoparticles can quell the bacterial inflammation that can lead to a serious gum disease known as periodontitis. Other collaborating labs are using these nanoparticles in pancreatic, colon, and ovarian cancers with good effects.

“The specific targets in these cases may be different, but the nice thing about this kind of delivery system for RNA interference is that the delivery agent itself, the nanostructures, are the same,” Dr. Wickline said. “All we have to do is change out a little bit of the genetic material that targets the messages and we’re set up to go after another disease. So it’s completely modular and nontoxic.”

The St. Louis-based biotechnology company Trasir Therapeutics is developing these peptide-based nanocarriers for silencing RNA to treat diseases with multiple mechanisms of inflammation. Dr. Wickline co-founded the company in 2014 and continues to serve as its chief scientific officer.

Dr. Wickline with colleague Hua Pan, PhD, a biomedical engineer with expertise in molecular biology.

 Inhibiting chronic inflammation without getting rid of beneficial immune responses.

Calming the destructive cycle of inflammation

Dr. Wickline’s work is supported by several NIH RO1 grants, including one from the National Heart, Lung and Blood Institute to develop and test nanotherapies seeking to interrupt inflammatory signaling molecules and reduce the likelihood of thrombosis in acute cardiovascular syndromes.

In essence, Dr. Wickline said, he is interested in suppressing chronic inflammation, without disrupting the beneficial functions of surveillance by which the immune system recognizes and destroys invading pathogens or potential cancer cells.

“If you can inhibit the ongoing inflammation associated with (inappropriate) immune system response, you inhibit the positive feedback cycle of more inflammation, more plaques, more damage and more danger,” he said. “If you can cool off inflammation by using a message killer that says (to macrophages) ‘don’t come here, don’t eat fat, don’t make a blood clot’ – that’s what we think could be a game changer.”

Another NIH grant has funded collaborative work to develop an image-based nanoparticle that detects where in a compromised blood vessel too much blood clotting (hypercoagulation) occurs, and delivers potent anti-clotting agent only to that site. Formation of abnormal blood clots can trigger a heart attack when a clot blocks an artery that leads to heart muscle, or a stroke when a clot obstructs an artery supplying blood to the brain.

Because this site-specific nanotherapy targets only areas of active clotting, it may provide a safer, more effective approach against cardiac conditions like atrial fibrillation and acute heart attack than existing anticoagulant drugs such as warfarin and newer blood thinners like Xarelto® (rivaroxoban) or Eliquis® (apixiban), all which work systemically and come with raised risk for serious bleeding, Dr. Wickline said.

In a study published last year in the journal Arteriosclerosis, Thrombosis, and Vascular Biology, Dr. Wickline and colleagues found that nanoparticles delivering a potent inhibitor of thrombin, a coagulant protein in blood that plays a role in inflammation, not only reduced clotting risk but also rapidly healed blood vessel endothelial barriers damaged during plaque growth.

The preclinical work showed the experimental treatment “is actually an anti-atherosclerotic drug as well as an anti-clotting drug, so there are many potential applications,” Dr. Wickline said.

Dr. Wickline received his MD degree from the University of Hawaii School of Medicine. He completed a residency in internal medicine, followed by clinical and research fellowships in cardiology at Barnes Hospital and Washington University, where he joined the medical school faculty in 1987.

He has authored more than 300 peer-reviewed papers and holds numerous U.S. patents. Dr. Wickline is a fellow of the American College of Cardiology and the American Heart Association, and a 2014 recipient of the Washington University Chancellor’s Award for Innovation and Entrepreneurship.

– Photos by Sandra C. Roa and Eric Younghans

Led by neurologist Dr. Juan-Sanchez-Ramos, the mouse-model study will refine a noninvasive nose-to-brain delivery system using manganese nanoparticles

Huntington’s disease (HD) is an incurable, hereditary brain disorder that typically strikes adults in the prime of their lives – gradually affecting movement, mood and mental activity. Involuntary “dance-like” movements, known as chorea, are the most common motor symptoms.  Patients also commonly develop depression and suicidal thoughts, and increasing difficulty with cognitive function makes it difficult to hold a job.

The one drug currently approved by the Food and Drug Administration to alleviate chorea does not change the course of HD.

Dr. Juan Sanchez-Ramos, professor of neurology at the USF Health Morsani College of Medicine, is the lead investigator for a new $2.3-million NIH grant studying a non-invasive drug delivery system designed to safely and effectively transport large therapeutic molecules (nucleic acids) from nose to brain.

 Listen to Dr. Sanchez-Ramos talk about a major obstacle to gene therapy.

Where’s the cure?

When the single lethal gene for HD was discovered in 1993, USF Health neurologist Juan-Sanchez, MD, PhD, promised some patients he would help find a cure or effective treatment for the rare, but ravaging, disease that runs in families.   At the time, he was a clinical team member of the U.S.-Venezuela Collaborative Research Project, a landmark study that identified and documented cases of HD and the disease’s progression in a unique community of families in Lake Maracaibo, Venezuela.

While celebrating the gene’s discovery with other clinicians in a village, he asked some HD patients gathered why they were not applauding the breakthrough. They answered with a typical Venezuelan gesture, “¿Y la cura?’” Dr. Sanchez-Ramos said. Translation: “So, where’s the cure?”

The pledge he made early in his career got a major boost last month when USF Health was awarded a new five-year, $2.3 million grant from the National Institutes of Health’s National Institute of Neurological Disorders and Stroke. Principal investigator Dr. Sanchez-Ramos and his team — using a mouse model for Huntington’s disease — will assess and refine a new nanoparticle carrier system they’ve designed to transport therapeutic gene-silencing molecules from the nasal passages to the brain.  The interdisciplinary team includes researchers from the USF Department of Neurology, USF Nanomedicine Research Center, Moffitt Cancer Center and the University of Massachusetts Medical School’s RNA Therapeutics Institute.

From left, the USF team of investigators includes Gary Martinez, PhD (Moffitt Cancer Center); Dr. Sanchez-Ramos; Vasyl Sava, PhD; Xiaoyuan Kong; Subhra Mohapatra, PhD; Shijiie Song, MD; and Shyam Mohapatra, PhD. Not pictured are Neil Aronin, MD, and Anastasia Khvorova, PhD, both of the University of Massachusetts RNA Therapeutics Institute.

 Dr. Sanchez-Ramos comments on the nose-to-brain nanocarrier delivery system his team will be studying and refining.

Delivering therapeutic molecules for a global brain disease

“This NIH study will allow us to test exactly how the nanoparticles get from the nose to the brain, how they are disseminated from the olfactory bulb to other parts of the brain, and how long they stay before dissipating,” said Dr. Sanchez-Ramos, professor of neurology and director of the Huntington’s Disease Center of Excellence at the USF Health Morsani College of Medicine.

“We want all parts of the brain to be exposed to these gene silencing molecules, because Huntington’s is a global brain disease; as the disease advances, no part of the brain is spared”.

There is still much work to be done but, if proven successful, the nose-to-brain approach could be used to non-invasively (via nasal spray or drops) deliver all kinds of drugs, including DNA therapy and nerve growth factors, which would otherwise be blocked from entering the brain by the blood-brain barrier.

“It could have applications for modifying a wide range of brain disorders,” Dr. Sanchez-Ramos said.

Gene-silencing technology without neurosurgery

The normal huntingtin gene contains a DNA alphabet that repeats the letters C-A-G as many as 26 times, but people who develop HD have an excessive number of these consecutive C-A-G triplet repeats — greater than 39. The defective gene leads to a toxic huntingtin protein, which appears to play a critical role in nerve cell function.  HD is autosomal dominant, meaning if one parent has a copy of the faulty gene each child’s chance of inheriting the disease is 50 percent. The disease emerges slowly, usually between ages 30 and 50 (average age of diagnosis in the United States is 38), but onset can be earlier or later.  Research suggests that the greater the number of C-A-G repeats the earlier symptoms tend to appear and the faster they progress.

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Gene therapy is not new to HD or other neurodegenerative diseases. In the past, Dr. Sanchez-Ramos said, it primarily involved replacing a missing gene or delivering therapeutic molecules to help enhance cell survival.  More recently, research applications using small interfering RNA, or siRNA, continue to advance gene therapy’s potential use to modulate the expression of genes, including silencing or suppressing overactive genes.

“Researchers have already found that you can silence the Huntington’s disease gene in animal models,” Dr. Sanchez-Ramos said, “but no one has yet delivered these gene-silencing molecules other than surgically — either by stereotaxic injection of viral vectors, or by direct infusion into the brain or cerebrospinal fluid.

“The neurosurgical approach is just not feasible for patients with a chronic illness that gradually encompasses the entire central nervous system.”

Overcoming a major obstacle: the blood-brain barrier 

Preliminary mouse model experiments indicate the unique nanocarrier system designed by the USF researchers will overcome the major obstacle of invasive delivery as well as bypass the blood-brain barrier, a gatekeeper between the blood and brain tissue that selectively filters which molecules can enter the brain.

USF has patent pending for the system, which incorporates manganese-containing nanoparticles that rapidly target brain tissue after simple nasal administration.  The biodegradable nanoparticles encapsulate gene-silencing molecules made to inhibit the activity of the HD gene.

“The system transports the nanoparticles from nose to brain where siRNA (the gene-silencing molecule) is released and triggers the dissolving of messenger RNA so that it cannot go on to produce the abnormal protein that causes Huntington’s disease,” Dr. Sanchez-Ramos said.  “Our approach is promising, reasonable and safe.”

Dr. Sanchez-Ramos directs the Huntington’s Disease Society of America Center of Excellence at USF, where he cares for patients, many of whom are enrolled in clinical trials offered through the center. Kristy Yehle, right, participates in Enroll-HD, an international observational study for Huntington’s disease families.

In their series of NIH-supported studies, the USF researchers will visualize and track nose-to-brain transport of the manganese-containing nanoparticles in the mice using magnetic resonance imaging. (The contrast agent safely injected into patients undergoing some MRI tests contains manganese.)

Dr. Sanchez suspects that the nanoparticles may access the deeper regions of the brain through spaces surrounding the brain’s neurons and blood vessels rather than by the olfactory nerves alone, but the experiments will help quantify how the nanocarrier system works.  The study will also evaluate the effectiveness of the gene-silencing molecules in reducing or preventing motor and behavioral symptoms in the HD mice and look for ways to optimize the distribution and dosing.

On the threshold of a cure

The Huntington’s Disease Society of America (HDSA) Center of Excellence at USF, one of the largest regional referral centers in the Southeast, has treated more than 600 patients and their families since earning the HDSA designation more than 10 years ago. Many patients enroll in clinical studies testing investigational drugs and tracking the natural history of the disease in search of biomarkers.

Early in his career, while working as part of an international research team in Venezuela, Dr. Sanchez-Ramos promised some patients he would help find a cure or effective treatment for Hurtington’s disease.

At USF’s center, Dr. Sanchez-Ramos listens to their stories about struggling with and overcoming the challenges of living with HD and their determination to live each day to the fullest. The clinician-scientist remembers the promise he made in Venezuela.  He remains optimistic that research by USF and others combining nanomedicine and gene-silencing technology will lead to human trials, and ultimately, effective therapies to prevent HD or delay its progression.

“We’ve found a way to hit this single-gene disease with global symptoms at its source – by knocking out the abnormal gene expression,” Dr. Sanchez-Ramos said.

“I’m more hopeful than ever that we’re on the threshold of a cure for Huntington’s disease.”

Photos by Eric Younghans and animated graphic by Sandra Roa, USF Health Communications and Marketing

A Dana-Farber/Boston Children’s and VA Hospital/University of South Florida team created the antiviral therapy, which harnesses the power of stapled peptide and nanoparticle technologies to thwart the respiratory virus

Boston, MA, and Tampa, FL (April, 17, 2014) — New therapies are needed to prevent and treat respiratory syncytial virus (RSV) – a potentially lethal respiratory infection that can severely affect infants, young children and the elderly.

Despite a wide range of anti-RSV efforts, there are no vaccines or drugs on the market to effectively prevent or treat the infection.

Now researchers at the Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and Harvard Medical School in Boston, MA, and the James A. Haley VA Hospital and the University of South Florida (USF) in Tampa, FL, have developed novel double-stapled peptides that inhibit RSV in cells and in mice. The team also showed that this peptide’s capacity to block infection was significantly boosted when delivered to the lungs by miniscule, biodegradable particles known as nanoparticles.

The team’s findings are reported online today in The Journal of Clinical Investigation. 

Shyam Mohapatra, PhD, leads the VA/University of South Florida research team with expertise in nanoparticle technology. He holds a test tube of nanoparticle solution.

RSV employs a fusion protein with a helical structure to enable the virus to bind to and penetrate epithelial cells lining the nose and lungs.

The Dana-Farber/Boston Children’s/Harvard laboratory led by co-senior author Loren Walensky, MD, PhD, used their chemical strategy known as hydrocarbon stapling to make “double-stapled” RSV peptides. Stapling helps the peptides retain their natural helical shape and resist degradation by the body’s enzymes while disrupting the fusion process needed for RSV to infect host cells.

The VA/USF group led by co-senior author Shyam Mohapatra, PhD, tested these double-stapled peptides, alone and in combination with propriety nanoparticles, in mice to demonstrate significant inhibition of RSV infection.

“This is an exciting advance in the fight against respiratory syncytial virus infection,” said Dr. Mohapatra, director of the USF Nanomedicine Research Center and the USF Health Morsani College of Medicine’s Division of Translational Medicine, and a research career scientist at James A. Haley VA Hospital.

“We found that double-stapled peptide interference targeting the virus fusion protein can be administered in the form of a nasal drop or spray.  The treatment suppressed viral entry and reproduction, including spread from nose to lungs, providing substantial protection from infection when administered several days before viral exposure.”

“Designing therapeutic peptides based on a virus’ very own fusion apparatus was previously exploited to block HIV-1 infection, but this class of drugs was severely limited by the pharmacologic liabilities of peptides in general, including loss of bioactive structure and rapid digestion in the body,” said Dr. Walensky, associate professor of pediatrics at Harvard Medical School, pediatric hematologist/oncologist at Dana-Farber/Boston Children’s and principal investigator in Dana-Farber’s Linde Program in Cancer Chemical Biology.

“Peptide stapling restores the natural helical shape, which also inhibits proteolysis, providing a new opportunity to take advantage of a well-validated mechanism of action to thwart viruses like RSV that otherwise lack drugs for preventing or treating infection.”

Loren Walensky, MD, PhD, leads the Dana-Farber/Boston Children’s/Harvard laboratory with expertise in stapled peptide technology. The screen image shows the chemical structure of a stapled peptide, with the arrow pointing to the hydrocarbon staple (in yellow).

Dr. Mohapatra and his team developed nose drops containing the Walensky laboratory’s double-stapled peptides after combining them with TransGenex’s chitosan nanoparticles that stick to mucous-producing cells lining the lungs.

First, the researchers treated mice intranasally with stapled peptide nose drops, both before and during infection with RSV.  The treated mice showed significantly lower levels of virus in the nose and lungs, and less airway inflammation, compared to untreated mice.

Then, double-stapled peptides encapsulated in nanoparticles were delivered to the lungs via the trachea to test whether the combination could further increase the effectiveness of this experimental therapy.  The nanoparticle preparation markedly improved delivery of the peptides to the lungs, and the combination worked better and longer in preventing RSV pneumonia than the double-stapled peptide alone.

The researchers say to the best of their knowledge this preclinical study is the first to combine peptide stapling and nanoparticle technologies to maximize the delivery, persistence, and effectiveness of an antiviral therapy.

RSV is the most common virus causing lung and airway infections in infants and young children. Most have had this infection by age 2, and it can be especially serious, even deadly, in high-risk groups, such as babies born prematurely and those whose immune systems do not work well. The virus hospitalizes thousands of infants each year for pneumonia or brochiolitis and has been associated with a significantly greater risk of developing asthma later in life.  The elderly are also at high risk of complications from RSV infection.

“This is a new way forward in the development of strategies to prevent RSV infection,” said Terrence Dermody, MD, the Dorothy Overall Wells professor of pediatrics and director of the Division of Pediatric Infectious Diseases at Vanderbilt University School of Medicine, who was not involved with the research. “The authors are to be complimented on the clever design, interdisciplinary approach and extension from cell-culture experiments to animal studies. I am particularly excited about the possible application of this technology to other viruses.”

The study was supported in part by grants from the National Institutes of Health, Research Career Scientist and VA Merit Review Awards from the U.S. Department of Veterans Affairs, and a Burroughs Wellcome Fund Career Award.

Article citation:
Gregory H. Bird, Sandhya Boyapalle,Terianne Wong, Kwadwo Opoku-Nsiah, Raminder Bedi, W. Christian Crannell, Alisa F. Perry, Huy Nguyen, Vivianna Sampayo,  Ankita Devareddy, Subhra Mohapatra,  Shyam S. Mohapatra and Loren D. Walensky,  “Mucousal delivery of a double-stapled RSV peptide prevents nasopulmonary infection,”   Journal of Clinical Investigation, 2014;124(5): doi:10.1172/JCI71856.

About Dana-Farber/Boston Children’s Cancer and Blood Disorders Center

Dana-Farber/Boston Children’s Cancer and Blood Disorders Center brings together two internationally known research and teaching institutions that have provided comprehensive care for pediatric oncology and hematology patients since 1947.  The Harvard Medical School affiliates share a clinical staff that delivers inpatient care at Boston Children’s Hospital and outpatient care at the Dana-Farber Cancer Institute’s Jimmy Fund Clinic. Dana-Farber/Boston Children’s brings the results of its pioneering research and clinical trials to patients’ bedsides through five  clinical centers: the Blood Disorders Center, the Brain Tumor Center, the Hematologic Malignancies Center, the Solid Tumors Center, and the Stem Cell Transplant Center.

About USF Health

USF Health’s mission is to envision and implement the future of health. It is the partnership of the USF Health Morsani College of Medicine, the College of Nursing, the College of Public Health, the College of Pharmacy, the School of Biomedical Sciences and the School of Physical Therapy and Rehabilitation Sciences; and the USF Physician’s Group. The University of South Florida is a Top 50 research university in total research expenditures among both public and private institutions nationwide, according to the National Science Foundation. For more information, visit www.health.usf.edu

Media contacts:
Anne DeLotto Baier, USF Health Communications, University of South Florida
(813) 974-3303 or abaier@health.usf.edu

Irene Sege, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center
617-919-7379 or irene.sege@childrens.harvard.edu