- Use of the delivery system in mouse models results in unprecedented siRNA penetration across the intact blood brain barrier.
- Technology could offer potential for a variety of human neurological disorders
In the past few decades, researchers have identified biological pathways leading to neurodegenerative diseases and developed promising molecular agents to target them. However, the translation of these findings into clinically approved treatments has progressed at a much slower rate, in part because of the challenges scientists face in delivering therapeutics across the blood-brain barrier (BBB) and into the brain. To facilitate successful delivery of therapeutic agents to the brain, a team of bioengineers, physicians, and collaborators at Brigham and Women’s Hospital and Boston Children’s Hospital created a nanoparticle platform, which can facilitate therapeutically effective delivery of encapsulated agents in mice with a physically breached or intact BBB. In a mouse model of traumatic brain injury (TBI), they observed that the delivery system showed three times more accumulation in brain than conventional methods of delivery and was therapeutically effective as well, which could open possibilities for the treatment of numerous neurological disorders. Findings were published in Science Advances.
Previously developed approaches for delivering therapeutics into the brain after TBI rely on the short window of time after a physical injury to the head, when the BBB is temporarily breached. However, after the BBB is repaired within a few weeks, physicians lack tools for effective drug delivery.
“It’s very difficult to get both small and large molecule therapeutic agents delivered across the BBB,” said corresponding author Nitin Joshi, PhD, an associate bioengineer at the Center for Nanomedicine in the Brigham’s Department of Anesthesiology, Perioperative and Pain Medicine. “Our solution was to encapsulate therapeutic agents into biocompatible nanoparticles with precisely engineered surface properties that would enable their therapeutically effective transport into the brain, independent of the state of the BBB.”
The technology could enable physicians to treat secondary injuries associated with TBI that can lead to Alzheimer’s, Parkinson’s, and other neurodegenerative diseases, which can develop during ensuing months and years once the BBB has healed.
“To be able to deliver agents across the BBB in the absence of inflammation has been somewhat of a holy grail in the field,” said co-senior author Jeff Karp, PhD, of the Brigham’s Department of Anesthesiology, Perioperative and Pain Medicine. “Our radically simple approach is applicable to many neurological disorders where delivery of therapeutic agents to the brain is desired.”
Rebekah Mannix, MD, MPH, of the Division of Emergency Medicine at Boston Children’s Hospital and a co-senior author on the study, further emphasized that the BBB inhibits delivery of therapeutic agents to the central nervous system (CNS) for a wide range of acute and chronic diseases. “The technology developed for this publication could allow for the delivery of large number of diverse drugs, including antibiotics, antineoplastic agents, and neuropeptides,” she said. “This could be a game changer for many diseases that manifest in the CNS.”
The therapeutic used in this study was a small interfering RNA (siRNA) molecule designed to inhibit the expression of the tau protein, which is believed to play a key role in neurodegeneration. Poly(lactic-co-glycolic acid), or PLGA, a biodegradable and biocompatible polymer used in several existing products approved by the U.S. Food and Drug Administration, was used as the base material for nanoparticles. The researchers systematically engineered and studied the surface properties of the nanoparticles to maximize their penetration across the intact, undamaged BBB in healthy mice. This led to the identification of a unique nanoparticle design that maximized the transport of the encapsulated siRNA across the intact BBB and significantly improved the uptake by brain cells.
A 50 percent reduction in the expression of tau was observed in TBI mice who received anti-tau siRNA through the novel delivery system, irrespective of the formulation being infused within or outside the temporary window of breached BBB. In contrast, tau was not affected in mice that received the siRNA through a conventional delivery system.
“In addition to demonstrating the utility of this novel platform for drug delivery into the brain, this report establishes for the first time that systematic modulation of surface chemistry and coating density can be leveraged to tune the penetration of nanoparticles across biological barriers with tight junction,” said first author Wen Li, PhD, of the Brigham’s Department of Anesthesiology, Perioperative and Pain Medicine.
In addition to targeting tau, the researchers have studies underway to attack alternative targets using the novel delivery platform.
“For clinical translation, we want to look beyond tau to validate that our system is amenable to other targets,” Karp said. “We used the TBI model to explore and develop this technology, but essentially anyone studying a neurological disorder might find this work of benefit. We certainly have our work cut out, but I think this provides significant momentum for us to advance toward multiple therapeutic targets and be in the position to move ahead to human testing.”
This work was supported by the National Institutes of Health (HL095722), Fundac¸a~o para a Cie^ncia e a Tecnologia through MIT-Portugal (TB/ECE/0013/2013), and the Football Players Health Study at Harvard, funded by a grant from the National Football League Players Association.
Karp has been a paid consultant and or equity holder for multiple biotechnology companies (listed here). Joshi, Karp, Mannix, Li, Qiu and Langer have one unpublished patent based on the nanoparticle work presented in this manuscript.
Paper cited: Li, W et al. “BBB pathophysiology independent delivery of siRNA in traumatic brain injury” Science Advances DOI: TB/ECE/0013/2013
Mission to MAARS: Long Non-Coding RNA May Play a Key Role in Cardiovascular Disease
Atherosclerosis is marked by the buildup of inflammatory cells which narrow arteries to the point of chest pain and muscle weakness. Severe cases result in lesions and internal ruptures of arteries or even thrombosis in coronary arteries. One way that investigators are working to understand how atherosclerosis occurs and progresses is by looking at long non-coding RNAs (lncRNAs), strands of RNA that are not translated into proteins and which may play integral but understudied roles in cell regulation and disease progression. Through utilization of genetically modified high-risk atherosclerotic mice, a research team from Brigham and Women’s Hospital identified and characterized Macrophage-Associated Atherosclerosis lncRNA Sequence (MAARS), which is expressed specifically in macrophages in atherosclerotic plaques and contributes to the progression of the disease. Results are published in Nature Communications.
“We hypothesized, given the unknown role of lncRNAs, that some may be highly expressed in the blood vessel wall during the process of atherosclerosis,” said Mark Feinberg, MD, senior author and member of the Brigham’s Division of Cardiovascular Medicine. “We want to identify who these actors are, what they are doing, and how we can understand their function in a way that provides a foundation for future therapeutic opportunities.”
Feinberg and colleagues used genetically modified mice prone to atherosclerosis and placed them on a high cholesterol diet, which boosted their cholesterol to 500-1,000 units, up from a normal level of around 200 units. The mice were observed on their high cholesterol diet for 12 weeks, then placed on a normal diet and observed while their cholesterol levels returned to normal. Researchers isolated the innermost lining of the blood vessel walls of these mice and sent the samples for RNA sequencing to identify the presence of lncRNAs.
Among the list of present lncRNAs was MAARS, which piqued the interest of researchers with its specificity to macrophages and expression pattern. As atherosclerosis developed in the mice over the initial 12 weeks, the presence of MAARS increased 270-fold; once fed normal diets, the presence of MAARS decreased from its heightened expression by 60 percent. Targeted interruption of MAARS’s function reduced atherosclerotic lesion formation by 52 percent by decreasing macrophage cell death and increasing efferocytosis — the clearance of dead cellular debris — from these lesions. These effects were largely independent of effects on circulating cholesterol.
Researchers found an important relationship between MAARS and an RNA-binding protein known as HuR. In the vessel wall, MAARS interacts with HuR, which plays a critical role in cell death. If MAARS is deliberately inhibited, HuR is released from the nucleus into the cytoplasm and the macrophages continue their cleaning. This chain of events leads to more active macrophages able to clean up more plaque and debris produced by atherosclerosis.
“lncRNAs play a really important role in cardiovascular disease,” said Feinberg. “We had no idea what we were going to find, and we ended up identifying a lncRNA that has a crucial role in macrophages and pathways that could have therapeutic potential. We are shedding light on new players in old signaling pathways. It is so exciting to add more nuance to this area of research, since that means future studies will have that much more to work with.”
This work was supported by the National Institutes of Health (HL115141, HL134849, HL148207, HL148355, HL153356), the American Heart Association (18SFRN33900144 and 20SFRN35200163, 18POST34030395), the Arthur K. Watson Charitable Trust, the Dr. Ralph and Marian Falk Medical Research Trust, a Sarnoff Cardiovascular Research Foundation Fellowship, and the Swiss National Science Foundation (P2BEP3_162063).
Paper Cited: Feinberg, Mark et al. “A macrophage-specific lncRNA regulates apoptosis and atherosclerosis by tethering HuR in the nucleus” Nature Communications DOI: 10.1038/s41467-020-19664-2
Breast Cancer Study Uncovers How Macrophages May Contribute to a Therapeutic Weak Spot
Breast cancer, the second most common cancer in the United States, can result from a number of cellular misregulations, such as deficiencies in the DNA-repairing breast cancer gene, BRCA. Typically, BRCA-associated breast cancer is treated with poly ADP ribose polymerase (PARP) inhibitors and, recently, clinical trials have investigated pairing PARP inhibitor therapy with immunotherapy. Based on preclinical data, it is expected that the combination will recruit and activate T cells — immune cells that can kill tumor cells. Despite interest in this combination, researchers are already looking ahead for ways to get even more benefit from PARP inhibitors plus checkpoint inhibitors in breast cancer patients. That is precisely what a team of Dana-Farber/Brigham and Women’s Cancer Center researchers have devoted their time to: identifying ways to boost the response to PARP inhibitors. The team found macrophage-mediated immune suppression to be the weak spot of PARP inhibition treatment. Findings are published in Nature Cancer.
“The question that drove our research was: How can we overcome PARP inhibitor resistance to turn this treatment into a homerun?” said Jennifer Guerriero, PhD, senior author and member of the Brigham’s Division of Breast Surgery and director of the Dana-Farber Breast Tumor Immunology Laboratory. “Our findings suggest that there’s something in the tumor microenvironment limiting the ability for T cells to be activated, and that something else is likely macrophages, which we found become highly suppressive after PARP inhibitor therapy.”
Initial results of the combination of a PARP inhibitor and a checkpoint inhibitor in small numbers of patients with metastatic breast cancer have shown this combination to be active. A national pre-surgical trial led by DFCI investigators has recently opened and will examine this targeted combination in patients with genetic mutations sensitive to PARP inhibitors.
Similar to BRCA proteins, PARP proteins act to repair damaged DNA; in tumors, inhibiting DNA repairs means cancer cell death, so the combined elimination of BRCA and PARP repair mechanisms induce cancer cell death. PARP inhibitors recruit T cells, which are required for the body to recognize the presence of cancerous cells.
Like T cells, macrophages are another type of immune cell, which is recruited to wounds to patch them up. With cancer, macrophages are recruited to tumor sites, which are viewed as wounds to macrophages, and repair, strengthen, and, consequentially, exacerbate the tumor state. The team found an abundance of macrophages expressing a receptor necessary for their survival, CSF-1R, to be present in cancerous tissue after PARP inhibition treatment. Therefore, they hypothesized that targeting CSF-1R-positive macrophages (a particularly suppressive macrophage type) in combination with PARP inhibition would lead to an enhanced anti-tumor response.
Since CSF-1R-positive macrophages exacerbate the tumor state, disabling these macrophages seemed an important therapeutic target for investigators. Using a triple-negative breast cancer BRCA-deficient mouse model, the team characterized these suppressive macrophages by assessing T cell and macrophage responses to different therapies and combinations of therapies.
When PARP and CSF-1R inhibition therapies were combined, there were dramatic anti-tumor responses seen with significant increase in overall survival. Furthermore, the triple combination of PARP inhibitor, CSF-1R inhibitor, and SREBP1 (a key regulator of lipid metabolism) inhibition was able to completely eliminate tumors in the aggressive triple-negative breast cancer mouse model. Researchers inferred from this therapeutic success that the PARP inhibitor directly activates macrophages to be suppressive in the tumor microenvironment.
While breast cancer tissue is often characterized before treatment, biopsies of tissue after treatment begins could provide more nuance to the characterization of these actors. Importantly, elucidating the mechanisms for PARP and macrophages will be critical in developing effective therapies and moving forward with clinical translation.
“At Dana-Farber/Brigham and Women’s Cancer Center we have the opportunity to work closely with our clinical colleagues and ask these really important questions that will be critical to identify better biomarkers, so we can identify which patients will respond to which therapies,” said Guerriero. “I am very optimistic about the use of PARP inhibitors — they are a game changer for patients with BRCA-deficient cancers, and their application is not just limited to breast cancers.”
Funding for this research was provided by the Dana-Farber Cancer Institute/Eli Lilly & Co. Research Collaboration, the Dana-Farber/Harvard Cancer Center (DF/HCC) Specialized Program of Research Excellence (SPORE) in Breast Cancer P50 CA1685404 Career Enhancement Award, the Susan G. Komen Foundation Career Catalyst Award CCR18547597, the Terri Brodeur Breast Cancer Foundation, the Breast Cancer Research Foundation, the Ludwig Center at Harvard, the Center for Cancer Systems Pharmacology NCI U54-CA225088, Eli Lilly, NIH/NHLBI (K08 HL128802 R01 CA090687 and P50 CA1685404), the Rob and Karen Hale Distinguished Chair in Surgical Oncology, the Spanish Ministerio de Economia, Industria y Competitividad (grant SAF2017-83565-R) and the Fundación Cientifica de la Asociacion Española Contra el Cancer (AECC) (grant PROYEI6018YELA).
Paper Cited: Mehta, A, et al. “Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer” Nature Cancer DOI: 10.1038/s43018-020-00148-7
MGB Study Finds Majority of COVID-19 Patients Died in Hospital
During the spring, less than 5 percent of Mass General Brigham patients diagnosed with COVID-19 died at home, highlighting the importance of planning ahead for end-of-life care
There have been over 280,000 deaths in the United States due to COVID-19, with the infectious nature of the disease preventing many patients from receiving end-of-life care at home. Researchers from Brigham and Women’s Hospital and collaborators found that 95.5 percent of individuals who died with a diagnosis of COVID-19 in the Mass General Brigham health system between February 18 and May 18, 2020 did so in the hospital. To better characterize the intensity of end-of-life care and promote discussions about at-home care, the researchers analyzed specific death settings, determining that roughly 40 percent of hospital deaths occurred in the intensive care unit. Findings were published as a letter to the editor in the Journal of Palliative Medicine.
“Surveys have shown that most patients prefer to die at home, but there is also an ongoing debate about whether that is the best option for patients when resources for at-home care are limited,” said corresponding author Isaac Chua, MD, MPH, of the Division of General Internal Medicine and Primary Care at the Brigham. “The purpose of this study was to open up a larger conversation about whether we need to provide more advanced care at home, so that if patients want to die at home, they would have that option.”
The researchers state that the large percentage of patients that died in the intensive care unit (ICU) suggests that end-of-life care was intense for the plurality of those who died in the hospital. Roughly a third of the other deaths occurred in the general ward, and less than one fifth (17.8 percent) occurred in an inpatient palliative care unit. Based on the overwhelming number of patients who died in the hospital and ICU, it is likely that most patients who expressed a desire to die at home were unable to do so.
While the study population only included 16 patients who died outside of the hospital, making observations about this group hard to generalize, two patterns emerged. First, more than 93 percent of the patients who died outside of the hospital were white, whereas white individuals accounted for only about 61 percent of those who died in the hospital. Secondly, the population was on average older, with a median age of 91.2, compared to 77.8 among those who died in hospital.
Chua hypothesizes that the higher median age among those who died outside the hospital possibly reflects that older adults may have been more prepared to make end-of-life decisions, facilitating earlier plans for a safe discharge to the home while minimizing risk of infecting other household members. In contrast, a younger patient may have been more likely to continue to pursue hospital-based care.
“Most COVID-19 patients likely have not been thinking about their own mortality, and so they may be undecided about what they want,” Chua said. “There’s a lot of uncertainty and a lot of stress.”
Clinicians face uncertainty as well. “Especially at the beginning of the pandemic, the prognostic factors for good and bad outcomes were unknown,” Chua said. “It’s hard to create a pathway for sound end-of-life care because of how novel this virus is, how little we know about it, and how hard it is to plan ahead.”
Notably, 61 percent of the COVID-19 patients received a form of sub-specialty palliative care, which Chua says is a positive finding reflective of MGB institutions’ commitment to implement palliative care services amidst the COVID-19 crisis. Still, patient preferences surrounding end-of-life care for COVID-19 remain understudied, and hospital systems are still exploring ways to provide patient-centered care at home for acute and subacute illnesses.
“Our health care system needs to be thoughtful about different patient trajectories, and patients should really think through what they would want, too,” Chua said. “Even if one isn’t anticipating a poor outcome, being able to have these difficult conversations early on in a hospitalization enables the hospital team to figure out what it can do to plan ahead and be as patient-centered as possible, coordinating resources to align care with patient priorities.”
There was no dedicated funding organization for this study.
Paper cited: Chua, IS et al. “Place of Death and End-of-Life Care Utilization among COVID-19 Decedents in a Massachusetts Health Care System” Journal of Palliative Medicine DOI: 10.1089/jpm.2020.0674
Blood Test for Alzheimer’s Disease Predicts Future Cognitive Decline in Healthy People
Higher baseline levels of an N-terminal fragment of tau (NT1) in blood samples from participants in the Harvard Aging Brain Study were associated with increased risk of progression to cognitive decline and Alzheimer’s disease
Today, a clinician can order a blood test to check a patient’s cholesterol or hemoglobin A1c levels — biomarkers that help predict an individual’s risk of cardiovascular disease or diabetes, respectively. But despite decades of advances in the understanding of Alzheimer’s disease (AD), a blood test for predicting its risk remains elusive. Imaging scans of the brain and lumbar punctures that collect cerebrospinal fluid can offer diagnoses, but such tests are expensive and cumbersome for patients. Two years ago, investigators at Brigham and Women’s Hospital reported the development of a blood test for a fragment of the protein tau, a hallmark of AD. Now, that test for levels of N-terminal fragment of tau (NT1) has been evaluated in participants in the Harvard Aging Brain Study (HABS), a cohort of cognitively normal, older adults who are followed closely over time. In Nature Communications, the authors report that baseline NT1 levels in the blood were highly predictive of the risk of cognitive decline and AD dementia.
“Our findings indicate that measuring a tau fragment in plasma can help predict which elderly people are likely to decline and how quickly they are likely to decline,” said corresponding author Dennis Selkoe, MD, co-director of the Ann Romney Center for Neurologic Diseases. “We’re excited because there are currently no commercially available blood tests to predict risk of AD in still-healthy individuals. Having such a blood test allows us to better screen people for enrollment in AD prevention trials and represents progress toward diagnostic tests for AD in medical care.”
Selkoe cautions that a commercial test for routine clinical care likely remains several years away. But for clinical trials that seek to evaluate preventive treatments for AD, such as the large-scale clinical trials led by co-author Reisa Sperling, MD, MMSc, director of the Center for Alzheimer Research and Treatment at the Brigham NT1 levels could be measured before a participant enrolls in a the trial, and potentially also as a longitudinal measure to assess treatment response. The test ultimately represents a far less costly and less invasive alternative to imaging and lumbar punctures.
The current study, led by first author Jasmeer Chhatwal, MD, PhD, now an attending physician and scientist in the Massachusetts General Hospital Department of Neurology, evaluated the predictive value of NT1 among 236 cognitively normal participants in HABS. Participants were on average 74 years old when they entered HABS and were followed for an average of five years. Blood samples were collected in the first year.
The research team found that higher levels of NT1 in blood samples taken at the beginning of the trial were strongly associated with future clinical progression. The team divided participants into those with high, medium and low NT1 levels, finding that for the group with the highest levels, the risk of advancing to mild cognitive impairment (MC I) or AD dementia was 2.4-fold. NT1 levels predicted decline across multiple areas of memory, including episodic memory — remembering specific events or experiences such as a person’s birthday or a family visit — and also predicted how fast the participant’s cognition would decline. Imaging data showed that higher baseline NT1 blood levels were associated with elevated brain levels of β-amyloid plaques and the accumulation of tangles of tau — both classical signs of AD.
The authors note that relatively few participants in HABS progressed to AD, an important limitation of this cohort. They found that another brain protein — known as NfL — which has been studied by other groups, may also be associated with cognitive decline, especially among people who already show signs of cognitive deficits. NfL was a less strong predictor than NT1 in the study.
“The NT1 tau fragment may be a reflection of damage to neurons and synapses, allowing us to use blood samples to detect what is happening in a patient’s brain years before they begin experiencing symptoms,” said Selkoe. “This could give us an invaluable window of time in which to evaluate interventions for preventing cognitive decline and AD dementia.”
The Harvard Aging Brain Study is funded by the National Institute on Aging (P01AG036694) with additional support from several philanthropic organizations. Additional funding for this work was provided by the National Institutes of Health (R01 AG 006173; K23 AG049087; R01 AG062667) and the Doris Duke Charitable Foundation.
Paper cited: Chhatwal, Jasmeer P et al. “Plasma N-terminal tau fragment levels predict future cognitive decline and neurodegeneration in healthy elderly individuals” Nature Communications DOI: 10.1038/s41467-020-19543-w
New Treatment in Development for Irritable Bowel Syndrome with Constipation
- Preclinical studies find that an innovative bilayered capsule can overcome current challenges for delivering drugs to the colon
- Findings suggest new solution for relieving abdominal pain associated with IBS-C
- Clinical trials planned to begin within next 18 months
Patients suffering from irritable bowel syndrome with constipation (IBS-C) have long needed an upgrade in treatment. Rapid-release, cramp-inducing doses of chenodeoxycholic acid (CDC) have previously shown promise in treating constipation, but further development has been hampered by the abdominal pain associated with the sudden release of CDC. Researchers at Brigham and Women’s Hospital and the Massachusetts Institute of Technology (MIT) devised a plan to deliver CDC in a bilayered capsule, finding that this mode of delivery could decrease colon cramping and thus produce a better patient experience. In preclinical studies, the team found evidence that this bilayered delivery system has the potential to reduce cramping and provide constipation relief. Findings are published in Clinical and Translational Gastroenterology.
“We know bile acids are capable of helping with motility, but what has been attempted in the past is giving a bolus — a boatload of bile acid all at once. This manifests in increased bowel movements, but also pain,” said Giovanni Traverso, MD, PhD, of the Brigham’s Division of Gastroenterology, Hepatology and Endoscopy and the Department of Mechanical Engineering at MIT. “Could we take this endogenous, natural product and deliver it in a way that overcomes this risk of contractions?”
The liver produces bile acids to aid in the digestive process, regulating intestinal motility, fluid homeostasis, and humoral activity. Bile acids, such as pro-motility CDC, have been previously studied in patients for their pro-motility effects and recognized to enhance water ingression and bowel motility. The challenge has been how to administer these in ways that minimize potential side effects; to accomplish this, researchers developed a bilayered delivery system.
The bilayered delivery mechanism was tested in swine models for half-life, colon cramping and whether it caused a similar dosage spike to single-layered delivery. Immediate release of the pill’s surface layer established a healthy local concentration of CDC, which in turn offered enough colonic fluid to initiate the pill’s second, slower-release layer of CDC. This biphasic release of CDC established a low-dose, long-lasting presence of bile acid over time, avoiding the dosage spike and decreasing cramping.
Several important limitations exist for this study, including lack of similar studies for comparison and shortcomings of the swine model; the team was not able to measure levels of abdominal pain in the swine model, and only rectal contractions were measured, as opposed to a full colonic evaluation.
Within the next 18 months, clinical trials will begin for the bilayered delivery of CDC to IBS-C patients, with pill production regulated by the team’s newly founded Bilayer Therapeutics.
Traverso envisions applications for this therapeutic far beyond IBS-C. Bile acids are involved in metabolic diseases, including diabetes and liver cytopathy.
“While further studies are necessary to demonstrate the therapeutic potential of these systems in humans, our findings suggest that controlled delivery of bile acids to the colon may represent a novel approach to treating gastrointestinal diseases such as constipation,” said co-author Joshua Korzenik, MD, of the Brigham’s Gastroenterology and Hepatology Division.
Funding for this work was provided by the Alexander von Humboldt Foundation (Feodor Lynen Fellowship) and the Department of Mechanical Engineering at Massachusetts Institute of Technology. Traverso, Korzenik and co-authors are co-inventors on a patent application describing the bile acid delivery systems described here within. Traverso, Korzenik and co-authors have a financial interest in Bilayer Therapeutics, Inc. This company is developing oral bile acid delivery systems.
Paper Cited: Steiger C et al. “Controlled Delivery of Bile Acids to the Colon” Clinical and Translational Gastroenterology DOI: 10.14309/ctg.0000000000000229