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