Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion.

RNA-binding proteins (RBPs) modulate alternative splicing outcomes to determine isoform expression and cellular survival. To identify RBPs that directly drive alternative exon inclusion, we developed tethered function luciferase-based splicing reporters that provide rapid, scalable and robust readouts of exon inclusion changes and used these to evaluate 718 human RBPs. We performed enhanced cross-linking immunoprecipitation, RNA sequencing and affinity purification-mass spectrometry to investigate a subset of candidates with no prior association with splicing. Integrative analysis of these assays indicates surprising roles for TRNAU1AP, SCAF8 and RTCA in the modulation of hundreds of endogenous splicing events. We also leveraged our tethering assays and top candidates to identify potent and compact exon inclusion activation domains for splicing modulation applications. Using these identified domains, we engineered programmable fusion proteins that outperform current artificial splicing factors at manipulating inclusion of reporter and endogenous exons. This tethering approach characterizes the ability of RBPs to induce exon inclusion and yields new molecular parts for programmable splicing control.

An Activity-Based Nanosensor for Minimally-Invasive Measurement of Protease Activity in Traumatic Brain Injury.

Current screening and diagnostic tools for traumatic brain injury (TBI) have limitations in sensitivity and prognostication. Aberrant protease activity is a central process that drives disease progression in TBI and is associated with worsened prognosis; thus direct measurements of protease activity could provide more diagnostic information. In this study, a nanosensor is engineered to release a measurable signal into the blood and urine in response to activity from the TBI-associated protease calpain. Readouts from the nanosensor were designed to be compatible with ELISA and lateral flow assays, clinically-relevant assay modalities. In a mouse model of TBI, the nanosensor sensitivity is enhanced when ligands that target hyaluronic acid are added. In evaluation of mice with mild or severe injuries, the nanosensor identifies mild TBI with a higher sensitivity than the biomarker GFAP. This nanosensor technology allows for measurement of TBI-associated proteases without the need to directly access brain tissue, and has the potential to complement existing TBI diagnostic tools.

Infusible Extracellular Matrix Biomaterial Promotes Vascular Integrity and Modulates the Inflammatory Response in Acute Traumatic Brain Injury.

Traumatic brain injury (TBI) affects millions of people each year and, in many cases, results in long-term disabilities. Once a TBI has occurred, there is a significant breakdown of the blood-brain barrier resulting in increased vascular permeability and progression of the injury. In this study, the use of an infusible extracellular matrix-derived biomaterial (iECM) for its ability to reduce vascular permeability and modulate gene expression in the injured brain is investigated. First, the pharmacokinetics of iECM administration in a mouse model of TBI is characterized, and the robust accumulation of iECM at the site of injury is demonstrated. Next, it is shown that iECM administration after injury can reduce the extravasation of molecules into the brain, and in vitro, iECM increases trans-endothelial electrical resistance across a monolayer of TNFα-stimulated endothelial cells. In gene expression analysis of brain tissue, iECM induces changes that are indicative of downregulation of the proinflammatory response 1-day post-injury/treatment and neuroprotection at 5 days post-injury/treatment. Therefore, iECM shows potential as a treatment for TBI.

Engineered nanomaterials that exploit blood-brain barrier dysfunction for delivery to the brain.

The blood-brain barrier (BBB) is a highly regulated physical and functional boundarythat tightly controls the transport of materials between the blood and the brain. There is an increasing recognition that the BBB is dysfunctional in a wide range of neurological disorders; this dysfunction can be symptomatic of the disease but can also play a role in disease etiology. BBB dysfunction can be exploited for the delivery of therapeutic nanomaterials. Forexample, there can be a transient, physical disruption of the BBB in diseases such as brain injury and stroke, which allows temporary access of nanomaterials into the brain. Physicaldisruption of the BBB through external energy sources is now being clinically pursued toincrease therapeutic delivery into the brain. In other diseases, the BBB takes on new properties that can beleveraged by delivery carriers. For instance, neuroinflammation induces the expression ofreceptors on the BBB that can be targeted by ligand-modified nanomaterials, and theendogenous homing of immune cells into the diseased brain can be hijacked for the delivery ofnanomaterials. Lastly, BBB transport pathways can be altered to increase nanomaterial transport. In this review, we will describe changes that can occur in the BBB in disease, and how these changes have been exploited by engineered nanomaterials forincreased transport into the brain.

Analysis of PEG-lipid anchor length on lipid nanoparticle pharmacokinetics and activity in a mouse model of traumatic brain injury.

Traumatic brain injury (TBI) affects millions of people worldwide, yet there are currently no therapeutics that address the long-term impairments that develop in a large portion of survivors. Lipid nanoparticles (LNPs) are a promising therapeutic strategy that may address the molecular basis of TBI pathophysiology. LNPs are the only non-viral gene delivery platform to achieve clinical success, but systemically administered formulations have only been established for targets in the liver. In this work, we evaluated the pharmacokinetics and activity of LNPs formulated with polyethylene glycol (PEG)-lipids of different anchor lengths when systemically administered to a mouse model of TBI. We observed an increase in LNP accumulation and activity in the injured brain hemisphere compared to the uninjured contralateral brain hemisphere. Interestingly, transgene expression mediated by LNPs was more durable in injured brain tissue compared to off-target organs when compared between 4 and 24 hours. The PEG-lipid is an important component of LNP formulation necessary for the stable formation and storage of LNPs, but the PEG-lipid structure and content also has an impact on LNP function. LNP formulations containing various ratios of PEG-lipid with C18 (DSPE-PEG) and C14 (DMG-PEG) anchors displayed similar physicochemical properties, independent of the PEG-lipid compositions. As the proportion of DSPE-PEG was increased in formulations, blood circulation times of LNPs increased and the duration of expression increased. We also evaluated diffusion of LNPs after convection enhanced delivery (CED) in healthy brains and found LNPs distributed >1 mm away from the injection site. Understanding LNP pharmacokinetics and activity in TBI models and the impact of PEG-lipid anchor length informs the design of LNP-based therapies for TBI after systemic administration.

Intravascularly infused extracellular matrix as a biomaterial for targeting and treating inflamed tissues.

Decellularized extracellular matrix in the form of patches and locally injected hydrogels has long been used as therapies in animal models of disease. Here we report the safety and feasibility of an intravascularly infused extracellular matrix as a biomaterial for the repair of tissue in animal models of acute myocardial infarction, traumatic brain injury and pulmonary arterial hypertension. The biomaterial consists of decellularized, enzymatically digested and fractionated ventricular myocardium, localizes to injured tissues by binding to leaky microvasculature, and is largely degraded in about 3 d. In rats and pigs with induced acute myocardial infarction followed by intracoronary infusion of the biomaterial, we observed substantially reduced left ventricular volumes and improved wall-motion scores, as well as differential expression of genes associated with tissue repair and inflammation. Delivering pro-healing extracellular matrix by intravascular infusion post injury may provide translational advantages for the healing of inflamed tissues 'from the inside out'.

Heads Up! Interlinked Amyloidogenic and Axonal Transport Pathways in Concussion-Induced Neurodegeneration.

Mild traumatic brain injury (mTBI), a condition in which brain function is transiently disrupted by a mechanical force, is a major risk factor for developing Alzheimer's disease (AD) and other neurodegenerative conditions. In this commentary, we summarize recent findings in human neurons derived from induced pluripotent stem cells, detailing early neuronal events following mild injury that may seed future neurodegeneration. In particular, we discuss interlinked relationships between mTBI and several biological pathways hypothesized to underlie AD progression, including amyloidogenic cleavage of amyloid precursor protein (APP), impairment of axonal transport, and the development of APP-associated axonal swellings. We also describe the implications of these findings for future mechanistic and translational studies.

Porous Silicon Nanoparticles Targeted to the Extracellular Matrix for Therapeutic Protein Delivery in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major cause of disability and death among children and young adults in the United States, yet there are currently no treatments that improve the long-term brain health of patients. One promising therapeutic for TBI is brain-derived neurotrophic factor (BDNF), a protein that promotes neurogenesis and neuron survival. However, outstanding challenges to the systemic delivery of BDNF are its instability in blood, poor transport into the brain, and short half-life in circulation and brain tissue. Here, BDNF is encapsulated into an engineered, biodegradable porous silicon nanoparticle (pSiNP) in order to deliver bioactive BDNF to injured brain tissue after TBI. The pSiNP carrier is modified with the targeting ligand CAQK, a peptide that binds to extracellular matrix components upregulated after TBI. The protein cargo retains bioactivity after release from the pSiNP carrier, and systemic administration of the CAQK-modified pSiNPs results in effective delivery of the protein cargo to injured brain regions in a mouse model of TBI. When administered after injury, the CAQK-targeted pSiNP delivery system for BDNF reduces lesion volumes compared to free BDNF, supporting the hypothesis that pSiNPs mediate therapeutic protein delivery after systemic administration to improve outcomes in TBI.

Targeting the Extracellular Matrix in Traumatic Brain Injury Increases Signal Generation from an Activity-Based Nanosensor.

Traumatic brain injury (TBI) is a critical public health concern and major contributor to death and long-term disability. After the initial trauma, a sustained secondary injury involving a complex continuum of pathophysiology unfolds, ultimately leading to the destruction of nervous tissue. One disease hallmark of TBI is ectopic protease activity, which can mediate cell death, extracellular matrix breakdown, and inflammation. We previously engineered a fluorogenic activity-based nanosensor for TBI (TBI-ABN) that passively accumulates in the injured brain across the disrupted vasculature and generates fluorescent signal in response to calpain-1 cleavage, thus enabling in situ visualization of TBI-associated calpain-1 protease activity. In this work, we hypothesized that actively targeting the extracellular matrix (ECM) of the injured brain would improve nanosensor accumulation in the injured brain beyond passive delivery alone and lead to increased nanosensor activation. We evaluated several peptides that bind exposed/enriched ECM constituents in the brain and discovered that nanomaterials modified with peptides that target hyaluronic acid (HA) displayed widespread distribution across the injury lesion, in particular colocalizing with perilesional and hippocampal neurons. Modifying TBI-ABN with HA-targeting peptide led to increases in activation in a ligand-valency-dependent manner, up to 6.6-fold in the injured cortex compared to a nontargeted nanosensor. This robust nanosensor activation enabled 3D visualization of injury-specific protease activity in a cleared and intact brain. In our work, we establish that targeting brain ECM with peptide ligands can be leveraged to improve the distribution and function of a bioresponsive imaging nanomaterial.

Pharmacokinetic Analysis of Peptide-Modified Nanoparticles with Engineered Physicochemical Properties in a Mouse Model of Traumatic Brain Injury.

Peptides are used to control the pharmacokinetic profiles of nanoparticles due to their ability to influence tissue accumulation and cellular interactions. However, beyond the study of specific peptides, there is a lack of understanding of how peptide physicochemical properties affect nanoparticle pharmacokinetics, particularly in the context of traumatic brain injury (TBI). We engineered nanoparticle surfaces with peptides that possess a range of physicochemical properties and evaluated their distribution after two routes of administration: direct injection into a healthy mouse brain and systemic delivery in a mouse model of TBI. In both administration routes, we found that peptide-modified nanoparticle pharmacokinetics were influenced by the charge characteristics of the peptide. When peptide-modified nanoparticles are delivered directly into the brain, nanoparticles modified with positively charged peptides displayed restricted distribution from the injection site compared to nanoparticles modified with neutral, zwitterionic, or negatively charged peptides. After intravenous administration in a TBI mouse model, positively charged peptide-modified nanoparticles accumulated more in off-target organs, including the heart, lung, and kidneys, than zwitterionic, neutral, or negatively charged peptide-modified nanoparticles. The increase in off-target organ accumulation of positively charged peptide-modified nanoparticles was concomitant with a relative decrease in accumulation in the injured brain compared to zwitterionic, neutral, or negatively charged peptide-modified nanoparticles. Understanding how nanoparticle pharmacokinetics are influenced by the physicochemical properties of peptides presented on the nanoparticle surface is relevant to the development of nanoparticle-based TBI therapeutics and broadly applicable to nanotherapeutic design, including synthetic nanoparticles and viruses.

Selective Permeabilization of Gram-Negative Bacterial Membranes Using Multivalent Peptide Constructs for Antibiotic Sensitization.

The drug-impermeable bacterial membrane in Gram-negative pathogens limits antibiotic access to intracellular drug targets. To expand our rapidly waning antibiotic arsenal, one approach is to improve the intracellular delivery of drugs with historically poor accumulation in Gram-negative bacteria. To do so, we engineered macromolecular potentiators to permeabilize the Gram-negative membrane to facilitate drug influx. Potentiators, known as WD40, were synthesized by grafting multiple copies of a cationic α-helical antimicrobial peptide, WLBU2, onto a dextran polymer scaffold. WD40 enabled drug uptake in the model pathogen P. aeruginosa, a capability that was not observed with unmodified WLBU2 peptide. WD40 was able to reduce minimum inhibitory concentrations of a drug panel by up to 3 orders of magnitude. Hydrophobic and highly three-dimensional antibiotics exhibited the greatest potentiation. Antibiotic activity was potentiated in several clinical strains and resulted in sensitization of drug-resistant strains to rifampin, a drug not previously used for Gram-negative infections.

Nanomedicine for Acute Brain Injuries: Insight from Decades of Cancer Nanomedicine.

Acute brain injuries such as traumatic brain injury and stroke affect 85 million people a year worldwide, and many survivors suffer from long-term physical, cognitive, or psychosocial impairments. There are few FDA-approved therapies that are effective at preventing, halting, or ameliorating the state of disease in the brain after acute brain injury. To address this unmet need, one potential strategy is to leverage the unique physical and biological properties of nanomaterials. Decades of cancer nanomedicine research can serve as a blueprint for innovation in brain injury nanomedicines, both to emulate the successes and also to avoid potential pitfalls. In this review, we discuss how shared disease physiology between cancer and acute brain injuries can inform the design of novel nanomedicines for acute brain injuries. These disease hallmarks include dysregulated vasculature, an altered microenvironment, and changes in the immune system. We discuss several nanomaterial strategies that can be engineered to exploit these disease hallmarks, for example, passive accumulation, active targeting of disease-associated signals, bioresponsive designs that are "smart", and immune interactions.

Peptide Spiders: Peptide-Polymer Conjugates to Traffic Nucleic Acids.

Therapeutic nucleic acids hold great promise for the treatment of genetic diseases, yet the delivery of this highly charged macromolecular drug remains a challenge in the field. Peptides are promising agents to mediate nucleic acid delivery because they can encode a biological function to overcome the trafficking barriers. Electrostatic nanocomplexes of nucleic acid and peptides can achieve effective delivery, but the balance between their stability and biological function must be finely tuned. In this work, we explore two peptide building blocks that have been studied in the literature: targeting ligands and intracellular trafficking peptides. We grafted these peptides on a polyethylene glycol (PEG) backbone with eight sites for substitution to create so-called "peptide spiders". These conjugates achieve stability via the well-known hydrophilic shielding effect of PEG. In addition, the coordination of peptide building blocks into multimers may create new biological properties, such as the well-known phenomena of increased binding avidity with multivalent ligands. In this work, we linked two trafficking peptides to the PEG backbone using either nonreducible or reducible chemistries and investigated the ability of these materials to carry silencing RNAs into mammalian cells. We then investigated these nanomaterials for their pharmacokinetic properties and silencing of undruggable targets in a mouse model of cancer. While reducible linkages were more potent at silencing in vitro, this effect was reversed when applied in the context of living animals. This work offers an insight into peptide-based delivery materials and investigates peptide-polymer linkages.

An Activity-Based Nanosensor for Traumatic Brain Injury.

Currently, traumatic brain injury (TBI) is detected by medical imaging; however, medical imaging requires expensive capital equipment, is time- and resource-intensive, and is poor at predicting patient prognosis. To date, direct measurement of elevated protease activity has yet to be utilized to detect TBI. In this work, we engineered an activity-based nanosensor for TBI (TBI-ABN) that responds to increased protease activity initiated after brain injury. We establish that a calcium-sensitive protease, calpain-1, is active in the injured brain hours within injury. We then optimize the molecular weight of a nanoscale polymeric carrier to infiltrate into the injured brain tissue with minimal renal filtration. A calpain-1 substrate that generates a fluorescent signal upon cleavage was attached to this nanoscale polymeric carrier to generate an engineered TBI-ABN. When applied intravenously to a mouse model of TBI, our engineered sensor is observed to locally activate in the injured brain tissue. This TBI-ABN is the first demonstration of a sensor that responds to protease activity to detect TBI.

Tumor-Targeting, MicroRNA-Silencing Porous Silicon Nanoparticles for Ovarian Cancer Therapy.

Silencing of aberrantly expressed microRNAs (miRNAs or miRs) has emerged as one of the strategies for molecular targeted cancer therapeutics. In particular, miR-21 is an oncogenic miRNA overexpressed in many tumors, including ovarian cancer. To achieve efficient administration of anti-miR therapeutics, delivery systems are needed that can ensure local accumulation in the tumor environment, low systemic toxicity, and reduced adverse side effects. In order to develop an improved anti-miR therapeutic agent for the treatment of ovarian cancer, a nanoformulation is engineered that leverages biodegradable porous silicon nanoparticles (pSiNPs) encapsulating an anti-miR-21 locked nucleic acid payload and displaying a tumor-homing peptide for targeted distribution. Targeting efficacy, miR-21 silencing, and anticancer activity are optimized in vitro on a panel of ovarian cancer cell lines, and a formulation of anti-miR-21 in a pSiNP displaying the targeting peptide CGKRK is identified for in vivo evaluation. When this nanoparticulate agent is delivered to mice bearing tumor xenografts, a substantial inhibition of tumor growth is achieved through silencing of miR-21. This study presents the first successful application of tumor-targeted anti-miR porous silicon nanoparticles for the treatment of ovarian cancer in a mouse xenograft model.

iRGD-guided Tumor-penetrating Nanocomplexes for Therapeutic siRNA Delivery to Pancreatic Cancer.

Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with αvß3/5 integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti-Kras siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer. Mol Cancer Ther; 17(11); 2377-88. ©2018 AACR.

Ultrasensitive tumour-penetrating nanosensors of protease activity.

The ability to identify cancer lesions with endogenous biomarkers is currently limited to tumours ~1 cm in diameter. We recently reported an exogenously administered tumour-penetrating nanosensor that sheds, in response to tumour-specific proteases, peptide fragments that can then be detected in the urine. Here, we report the optimization, informed by a pharmacokinetic mathematical model, of the surface presentation of the peptide substrates to both enhance on-target protease cleavage and minimize off-target cleavage, and of the functionalization of the nanosensors with tumour-penetrating ligands that engage active trafficking pathways to increase activation in the tumour microenvironment. The resulting nanosensor discriminated sub-5 mm lesions in human epithelial tumours and detected nodules with median diameters smaller than 2 mm in an orthotopic model of ovarian cancer. We also demonstrate enhanced receptor-dependent specificity of signal generation in the urine in an immunocompetent model of colorectal liver metastases, and in situ activation of the nanosensors in human tumour microarrays when re-engineered as fluorogenic zymography probes.

Porous Silicon Nanoparticle Delivery of Tandem Peptide Anti-Infectives for the Treatment of Pseudomonas aeruginosa Lung Infections.

There is an urgent need for new materials to treat bacterial infections. In order to improve antibacterial delivery, an anti-infective nanomaterial is developed that utilizes two strategies for localization: i) a biodegradable nanoparticle carrier to localize therapeutics within the tissue, and ii) a novel tandem peptide cargo to localize payload to bacterial membranes. First, a library of antibacterial peptides is screened that combines a membrane-localizing peptide with a toxic peptide cargo and discovers a tandem peptide that displays synergy between the two domains and is able to kill Pseudomonas aeruginosa at sub-micromolar concentrations. To apply this material to the lung, the tandem peptide is loaded into porous silicon nanoparticles (pSiNPs). Charged peptide payloads are loaded into the pores of the pSiNP at ≈30% mass loading and ≈90% loading efficiency using phosphonate surface chemistry. When delivered to the lungs of mice, this anti-infective nanomaterial exhibits improved safety profiles over free peptides. Moreover, treatment of a lung infection of P. aeruginosa results in a large reduction in bacterial numbers and markedly improves survival compared to untreated mice. Collectively, this study presents the selection of a bifunctional peptide-based anti-infective agent and its delivery via biodegradable nanoparticles for application to an animal model of lung infection.

Comparison of Modular PEG Incorporation Strategies for Stabilization of Peptide-siRNA Nanocomplexes.

Nanoparticulate systems have shown great promise in overcoming the considerable trafficking barriers associated with systemic nucleic acid delivery, which must be addressed to unlock the full potential of technologies such as RNAi and gene editing in vivo. In addition to mediating the cytoplasmic delivery of nucleic cargo and shielding it from nuclease degradation and immunostimulation, nucleic-acid-containing nanomaterials delivered intravenously must also be stable in the bloodstream after administration to avoid toxicity and off-target delivery. To this end, the hydrophilic molecule polyethylene glycol (PEG) has been deployed in many different nanoparticle systems to prevent aggregation and recognition by the reticuloendothelial system. However, the optimal strategy for incorporating PEG into self-assembled nucleic acid delivery systems to obtain nanoparticle stability while retaining important functions such as receptor targeting and cargo activity remains unclear. In this work, we develop substantially improved formulations of tumor-penetrating nanocomplexes (TPNs), targeted self-assembled nanoparticles formulated with peptide carriers and siRNA that have been shown to mitigate tumor burden in an orthotopic model of ovarian cancer. We specifically sought to tailor TPNs for intravenous delivery by systematically comparing formulations with three different classes of modular PEG incorporation (namely PEG graft polymers, PEG lipids, and PEGylated peptide), each synthesized using straightforward bioconjugation techniques. We found that the addition of PEG lipids or PEGylated peptide carriers led to the formation of small and stable nanoparticles, but only nanoparticles formulated with PEGylated peptide carriers retained substantial activity in a gene silencing assay. In vivo, this formulation significantly decreased accumulation in off-target organs and improved initial availability in circulation compared to results from the original non-PEGylated particles. Thus, from among a set of candidate strategies, we identified TPNs with admixed PEGylated peptide carriers as the optimal formulation for systemic administration of siRNA on the basis of their performance in a battery of physicochemical and biological assays. Moreover, this optimized formulation confers pharmacologic advantages that may enable further translational development of tumor-penetrating nanocomplexes, highlighting the preclinical value of comparing formulation strategies and the relevance of this systematic approach for the development of other self-assembled nanomaterials.