Kenneth Adler

Idiopathic pulmonary fibrosis (IPF) is an ultimately fatal disease whose only curative treatment is lung transplant. IPF is characterized by formation of fibrotic lesions in the lung, eventually resulting in scarring and progressive loss of lung function. Despite some newer treatments, IPF patients still have a median survival rate of only 3-5 years once diagnosed. Clearly, new therapeutic approaches are needed to treat this devastating disease. A promising avenue of approach is stem cell therapy. In the past 8 years, our lab has been developing lung spheroid cells (LSCs) as a novel source of therapeutic lung cells, and FDA approval of clinical trials with LSC treatment of patients with IPF are being pursued. Nonetheless, stem cell-based therapy faces several important limitations. Live cells need to be carefully preserved and processed before usage, and cell transplantation carries certain immunogenicity and tumorigenicity risks. Importantly, live stem cells cannot be delivered to the lung via inhalation, which is the most convenient and effective route to deliver therapeutics to the lung. Recently, we and others have made the novel and exciting observation that many adult stem cells exert their beneficial effects mainly through secretion of regenerative factors that go on to promote endogenous repair. In the preliminary studies that form the basis for this application, we have discovered that secretions from cultured LSCs are just as, if not more, effective than the LSCs themselves in attenuating and resolving IPF in rodent models of the disease. In the quest for active components in the LSC secretions, we found that LSCs secrete large numbers of exosomes (30-150 nm vesicles secreted by numerous cell types). We have shown that exosomes derived from LSCs (LSC-Exo) are therapeutic and regenerative to the injured lung, suggesting these nanostructures are largely responsible for the reparative response to LSC secretions in rodent models of IPF. It is known that exosomes carry microRNAs (miRs) cargoes that could play important roles in cell-cell communication and tissue repair, and indeed we found that LSC-Exo are highly enriched with miR-30a and Let-7. In this proposed study, we plan to determine safety and efficacy as well as medium effective dose of LSC-Exo required for lung repair in rodent models of IPF, to determine the major recipient cells of LSC-Exo in the lung, and determine that the relevant molecular target(s) of exosomal mediated repair and recovery. We hypothesize that key miRs withing LSC-Exo such as miR-30a and Let-7 are mediators of the TGF-beta signaling pathway, using the data produced by scRNA-Seq we will finally determine whether further miR enrichment in these exosomes achieves optimal lung repair. The development of cell-free or non-living therapeutics derived from stem cells has the potential to revolutionize current regenerative medicine practice.

The high probability of breakdown in the functioning of the central nervous system (CNS) during late stages of aging, as in Alzheimer??????????????????s disease and various dementias is a major concern for the elderly. Triggers that initiate age-associated diseases and neurological conditions are for the most part unknown. A key to these associations could be the population of ependymal cells in the brain. Ependymal cells form a monolayer that functions as a barrier between the cerebrospinal fluid (CSF) and the overlying cellular compartments of the brain. As such, they regulate CSF production, circulation, and filtering, and thus ependymal cells are a key component of the newly described ???????????????glymphtic-lymphatic?????????????????? system. This system is purported to control CSF-vascular interactions in the brain parenchyma and thus contribute to the overall clearance of the brain of toxicants and metabolic byproducts and allow entry of immune cells into the brain. The ependymal layer appears damaged in the aged brain, yet whether the damage is caused by malfunctioning signals in the overlying CNS tissue, or if ependymal damage causes defects in neurons and glia in the CNS remain unknown. We have developed several genetic mouse models which suggest the ependymal layer may be the root of many problems in the brain interstitium related to various neurodegenerative diseases and during normal aging in the CNS. We will use these models to study this novel concept. Studies in our mouse models have revealed a previously unknown expression and clearance of mucins by ependymal cells in the CNS. Since mucins function to protect against inflammation and infectious diseases in other tissues, our results have led to the central hypothesis that mucin secretion by ependymal cells is required for maintenance and functional integrity of homeostasis in the forebrain during aging, and that disruption of mucin secretion can lead to aberrant function and disease in the CNS. Our project uses a variety of genetic mice, together with cellular, molecular, and biochemical approaches to test our hypothesis. Potential for Broader Impact: Our approaches to understand how aging affects the brain through its monolayer of ependymal cells have wide implications. Disruption of filtration and protective functions of ependymal cells may be the root of a range of pathological conditions that emerge during late stages of aging. Therefore, undertaking the basic cellular mechanisms that control aging of the brain is critical to understanding not only how healthy aging may be controlled by ependymal cells, but also how abnormalities in ependymal aging may lead to devastating diseases such as Alzheimer??????????????????s. Moreover, the mechanisms we study can be harnessed to develop novel aging therapeutics by targeting ependymal functions selectively.

Uveitis, the most common form of intraocular inflammation, accounts for 10-15% of preventable blindness in the US. Treatment options are limited to cortiscosteroids and/or immunosuppressants, but both of these therapies show limited efficacy and are associated with numerous adverse and potentially serious side effects. New therapeutic targets leading to effective and safe treatments are urgently required. A novel potential therapeutic target to treat uveitis is Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS). Uveitis, both acute and chronic, is characterized by an inflammatory response involving an influx of leukocytes, production of proinflammatory cytokines, and activation of NF-????????B. Interestingly, these same responses in another organ, the lung, characterize pulmonary inflammation and development of acute respiratory distress syndrome (ARDS). We have found that inhibiting MARCKS in the lung via administration of a MARCKS-inhibitory peptide, named BIO-11006, ameliorates inflammation and reverses development of ARDS in mouse models of the disease, and does so by inhibiting NF-????????B activation, leukocyte influx, and pro-inflammatory cytokine production. Given similarities in the inflammatory processes in the eye in uveitis and the lung in ARDS, we looked preliminarily at the effects of treating uveitis in the lipopolysaccharide (LPS) rabbit model of the disease with intravitreally-injected BIO-11006. This treatment dramatically blocked leukocyte influx and attenuated inflammation in the LPS rabbit model. In this application, we wish to expand on these preliminary findings and provide proof-of-concept that inhibition of MARCKS function by intravitreally ?????????????????? injected BIO-11006 can ameliorate inflammation in the eye. The hypothesis is that treatment of either LPS-inflamed rabbit eyes (a model of acute uveitis) or experimental autoimmune uveitis (EAU) rat eyes (a model of chronic uveitis) with BIO-11006 will inhibit or reverse, in a concentration- and time-dependent manner, the inflammatory response. The aims are: 1) To determine whether treatment of rabbits with intravitreal injection of BIO-11006 at selected time points after development of acute uveitis via LPS administration will attenuate, in a time- and concentration-dependent manner, the acute inflammatory response; and, 2) To determine whether treatment of rats with intravitreal injection of BIO-11006 at selected time points after development of chronic uveitis via sensitization with Interphotoreceptor Retinoid Binding Protein (IRBP) will attenuate, in a time- and concentration-dependent manner, the chronic inflammatory response. Successful proof-of-concept in these studies can set the stage for further development of a new treatment for uveitis in either injectable or topical application (e.g. eye drop) form.

New therapies directed at NSCLC metastasis are clearly needed. An emerging therapeutic target in NSCLC is myristoylated alanine-rich C-kinase substrate (MARCKS). Levels of phosphorylated MARCKS (p-MARCKS) correlate with NSCLC aggressiveness, metastatic behavior and stage.1-3 We demonstrated, using inhibitory peptides, that blocking MARCKS phosphorylation and function dramatically reduces metastasis and tumor growth in mouse NSCLC models.3,4 An inhaled MARCKS inhibitory peptide, BIO-11006, has shown significant efficacy in a phase 2a clinical trial in advanced NSCLC (https://clinicaltrials.gov/ct2/show/NCT03472053?term=biomarck&rank=3). In this trial, patients were treated with either standard of care alone (SOC: pemetrexed/carboplatin) or SOC + inhaled BIO-11006 administered BID. With all 60 patients enrolled, results after 3 months showed statistically significant (p=0.02) improvement of Overall Response Rate (ORR) in the BIO-11006/SOC group compared to SOC alone. BIO-11006/SOC also showed less Disease Progression (DP) than SOC alone (7% to 17% respectively) and more Partial Response (PR) than SOC (40% to 30%). Thus, MARCKS appears to be a novel, effective therapeutic target in this disease; however, exact mechanism(s) of action of BIO-11006 and MARCKS inhibition have not been elucidated. Dissection of these molecules and pathways can reveal novel therapeutic targets.

More than 30 million people in the US suffer from asthma and incidence is increasing. Despite increasing prevalence, current treatments for asthma are inadequate and can exhibit deleterious off-target effects. Treatment strategies generally rely upon combinations of palliative administration of ????????-agonists to relax constricted airway smooth muscle, neutralizing individual inflammatory mediators and/or general dampening of the immune response with glucocorticosteroids. However, targeting single mediators in an inflammatory response only removes a fraction of the inflammatory ????????????????pool??????????????? of mediators. Therefore, a more desirable approach is to specifically target the inflammatory cells that are the source of the mediators, rather than the mediators those cells produce. Mast cells are key effector cells in allergic asthma where they infiltrate the airway smooth muscle bundles of asthmatic patients directly contributing to airway hyperresponsiveness and inflammation. We have identified a method to specifically target mast cells and basophils, which are the principle inflammatory cells that trigger an immediate allergic response. Our mast cell-targeting oligonucleotide eliminates the expression of the receptor for IgE that recognize allergens and activate mast cells. Thus, by eliminating the expression of this receptor on the surface of mast cells, we have identified the first drug that can specifically target these cell types and reduce their responsiveness to allergens. We now plan to test the efficacy of this innovative drug with delivery to the lungs in a mouse model of asthma to assess allergen-induced inflammation. This study will provide critical pilot data for drug development and commercialization of this novel therapeutic strategy.

The adult central nervous system (CNS) lacks endogenous mechanisms for replacing its damaged or diseased tissue, such as the neural circuits susceptible to epilepsy. Use of embryonic neural stem cells for replacement of CNS tissue poses a number of ethical and methodological limitations. Alternatively, the discovery of adult neural stem cells has raised hope for utilization of endogenous mechanisms for cell replacement in the CNS. This endeavor requires a comprehensive understanding of molecular mechanisms that distinguish adult stem cells from their embryonic counterparts. We have identified a Fork head transcription factor, FoxJ1, that is specifically expressed by cells forming the adult stem cell niche. Moreover, we recently discovered that FoxJ1 expression is ?turned on? when embryonic neural stem cells initiate their transformation into their adult form. This proposal is designed to test our hypothesis that FoxJ1 expression and activity is essential for the transformation of embryonic neural stem cells into their adult form in the CNS.

The objective of the proposed project is to advance the development of novel peptides targeting MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein as platform drug molecules for the treatment of Acute Lung Injury (ALI) and its more severe form, Acute Respiratory Distress Syndrome (ARDS), thereby providing the first pharmacologic therapeutic for this condition. ALI/ARDS is a major cause of respiratory failure; worldwide, over 1 million cases occur annually, with ~ 200,000 adult and 15,000 pediatric cases per year in the U.S. This disorder is characterized by a large influx of neutrophils to the lung as part of an acute inflammatory response, injury to epithelial and endothelial cell barriers, and pulmonary edema,all of which lead to respiratory failure. Treatment options are presently limited to mechanical ventilation, a supportive therapy which still results in an in-hospital mortality rate of ~40% in the U.S., with few survivors discharged to home due to residual pulmonary and brain damage incurred during the acute illness. There currently are no drugs approved for treatment of patients with ALI/ARDS. The annual cost to the U.S. healthcare system due to prolonged hospitalization and intensive medical intervention is over $5 billion dollars. Thus, the potential impact of developing a new drug that could reverse ALI/ARDS would be immense. BioMarck Inc has generated preliminary data in mice with ALI induced by IT instillation of LPS showing that inhalation of aerosolized N-terminal peptide inhibitors of MARCKS not only completely prevents ALI/ARDS when administered prior to the insult, but also reverses the inflammation and injury up to 36 hrs AFTER LPS instillation. Additional preliminary data show that the inhaled peptide, specifically BIO-11006 (an investigational drug with an active IND under which the FDA allows clinical trials in COPD patients) is rapidly metabolized after inhalation to a shorter, longer-lived peptide metabolite. We have synthesized this metabolite and labeled it BIO-10901. In ALI/ARDS, where the lung is severely inflamed and injured, metabolism of BIO-11006 could be severely compromised; thus, we believe that direct inhalation of the metabolite, BIO-10901, will provide better efficacy than BIO-11006. We propose here to determine if BIO-10901, shows more efficacy than the parent BIO-11006 in reversing the progression of ALI/ARDS in the mouse LPS model (Aim #1A) and in a more patho-physiologically-relevant model, bacterial pneumonia, which replicates the most common cause of ALI/ARDS in human patients (Aim #1B). We hypothesize that BIO-10901 will be clearly superior to BIO-11006 in these experiments, and will exhibit a consistent pattern of 20% or greater reduction in inflammatory measures and disease progression in comparison to the reductions provided by BIO-11006.

Subject: An important key to neutrophil adhesion, migration, and effector function activation is the point of convergence of pro-inflammatory receptor signaling and cytoskeletal organization. In this application, we present the concept that a key molecule is the Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) protein. Since MARCKS associates with cell membranes and the actin cytoskeleton in a manner regulated by PKC phosphorylation and Ca+2/calmodulin binding, MARCKS represents a potential point of convergence of signaling and cytoskeletal organization in the mechanism activating many neutrophil functions. In studies that form the basis for this application, a synthetic cell permeant peptide corresponding to the aminoterminal 24 aa of MARCKS that inhibits MARCKS binding to membranes [named the MANS (myristoylated N-terminal Sequence) peptide (1-3)], significantly inhibits human neutrophil migration and adhesion in response to fMLF, IL-8, or LTB4 (4), equine neutrophil migration in response to LTB4 (see preliminary data), and also significantly inhibits human neutrophil degranulation in response to phorbol ester (PMA) stimulation (3). This same peptide attenuates migration of neutrophils into the lung in vivo in 2 separate models of pulmonary inflammation in mice. An inhibitor of MARCKS function derived from the MANS peptide is currently in Phase II clinical trials in people for treatment of COPD. Significance: Neutrophil recruitment into inflamed tissue and activation are key events in inflammation. However, massive migration of neutrophils into tissues and subsequent wholesale activation by pro-inflammatory mediators is quite damaging to tissues. Neutrophils account for much of the damage during inflammation in many important diseases of horses and other species. Our laboratory focuses on identifying key molecules that regulate neutrophil function to identify new targets for anti-inflammatory therapy that may have clinical applications to treat serious equine diseases such as sepsis/endotoxemia, laminitis, pneumonia, heaves, colitis, ischemic colic, and arthritis. Hypothesis and Objectives; The specific hypothesis to be tested is: MARCKS protein is essential for equine neutrophil adhesion, migration, and effector function activation. We will test this hypothesis with the following four Objectives: 1) Determine whether MARCKS protein regulates equine neutrophil adhesion 2) Determine whether MARCKS protein regulates equine neutrophil migration 3) Determine whether MARCKS protein regulates production of reactive oxygen intermediates by equine neutrophils 4) Determine whether MARCKS protein regulates equine neutrophil degranulation. Study Design: Standard assays established in our laboratory will be used to determine the effects of MARCKS protein inhibition by treatment with the MANS peptide on freshly isolated equine neutrophil migration, adhesion, respiratory burst activity, and degranulation stimulated by key neutrophil activators. In all cases, vehicle and a randomly scrambled peptide called RNS will serve as control. Budget and Timeline: $45,475 direct costs ($49,113 total costs) per year for 1 year. Expected Results: We expect that MARCKS protein will be essential for one or more of the neutrophil functions investigated in this proposal and that the MANS peptide will inhibit these functions. Anticipated Outcomes: If our hypothesis is correct, we will determine whether the MARCKS protein is a key element in the mechanism regulating equine neutrophil function and establish whether the MANS peptide effectively inhibits equine neutrophil functions. Potential Impact on Animal Health: We expect to establish proof of principle that inhibition of the MARCKS by the cell permeant MANS peptide or its derivatives is an exciting new strategy for treating inflammation in horses.

Supplement for Pre-doctoral student Teresa Green.

Neutrophil recruitment into inflamed tissue is a key event in inflammation. However, massive migration of neutrophils into tissues and subsequent wholesale activation is quite damaging to tissues. Indeed, neutrophils account for much of the damage during inflammatory diseases. Our laboratory focuses on studying molecules that regulate neutrophil function to identify new targets to treat canine inflammatory diseases such as pneumonia, chronic bronchitis, and chronic rhinitis. We propose that a key molecule in the mechanism of neutrophil activation is the Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) protein. In studies that form the basis for this application, a synthetic cell permeant peptide corresponding to the aminoterminal 24 aa of MARCKS that inhibits MARCKS binding to membranes (named the MANS [myristoylated N-terminal Sequence] peptide), significantly inhibits human neutrophil migration and adhesion in response to fMLF, IL-8, or LTB4, canine neutrophil migration in response to LTB4, and human neutrophil degranulation in response to phorbol ester (PMA) stimulation. This same peptide attenuates migration of neutrophils into the lung in vivo in 2 models of pulmonary inflammation in mice. The specific hypothesis to be tested is: MARCKS protein is essential for canine neutrophil adhesion, migration, and effector function activation. If our hypothesis is correct, we will have discovered a novel function for MARCKS protein in the mechanism regulating canine neutrophil functions and establish proof of principle that inhibition of the MARCKS by the cell permeant MANS peptide or its derivatives is an exciting new strategy for treating inflammation in dogs.