Systemic in utero gene editing as a treatment for cystic fibrosis.

In utero gene editing has the potential to modify disease causing genes in multiple developing tissues before birth, possibly allowing for normal organ development, disease improvement, and conceivably, cure. In cystic fibrosis (CF), a disease that arises from mutations in the cystic fibrosis transmembrane conductance regulator ( ) gene, there are signs of multiorgan disease affecting the function of the respiratory, gastrointestinal, and reproductive systems already present at birth. Thus, treating CF patients early is crucial for preventing or delaying irreversible organ damage.

Next generation triplex-forming PNAs for site-specific genome editing of the F508del CFTR mutation.

Background: Cystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF.

Enhancing in vivo cell and tissue targeting by modulation of polymer nanoparticles and macrophage decoys.

The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters.

Polymer nanoparticles deliver mRNA to the lung for mucosal vaccination.

An inhalable platform for messenger RNA (mRNA) therapeutics would enable minimally invasive and lung-targeted delivery for a host of pulmonary diseases. Development of lung-targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here, we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung.

Rational nanoparticle design: Optimization using insights from experiments and mathematical models.

Polymeric nanoparticles are highly tunable drug delivery systems that show promise in targeting therapeutics to specific sites within the body. Rational nanoparticle design can make use of mathematical models to organize and extend experimental data, allowing for optimization of nanoparticles for particular drug delivery applications. While rational nanoparticle design is attractive from the standpoint of improving therapy and reducing unnecessary experiments, it has yet to be fully realized.

delivery of miRNA induces epigenetic alterations and corrects pulmonary pathology in congenital diaphragmatic hernia.

Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy as gas exchange is managed by the placenta, but impaired lung function results in critical illness from the time a baby takes its first breath. MicroRNA (miR) 200b and its downstream targets in the TGF-β pathway are critically involved in lung branching morphogenesis.

Nucleic Acid Delivery to the Vascular Endothelium.

This Review examines the state-of-the-art in the delivery of nucleic acid therapies that are directed to the vascular endothelium. First, we review the most important homeostatic functions and properties of the vascular endothelium and summarize the nucleic acid tools that are currently available for gene therapy and nucleic acid delivery. Second, we consider the opportunities available with the endothelium as a therapeutic target and the experimental models that exist to evaluate the potential of those opportunities.

In vivo correction of cystic fibrosis mediated by PNA nanoparticles.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We sought to correct the multiple organ dysfunction of the F508del CF-causing mutation using systemic delivery of peptide nucleic acid gene editing technology mediated by biocompatible polymeric nanoparticles. We confirmed phenotypic and genotypic modification in vitro in primary nasal epithelial cells from F508del mice grown at air-liquid interface and in vivo in F508del mice following intravenous delivery.

Inhalable polymer nanoparticles for versatile mRNA delivery and mucosal vaccination.

Unlabelled: An inhalable platform for mRNA therapeutics would enable minimally invasive and lung targeted delivery for a host of pulmonary diseases. Development of lung targeted mRNA therapeutics has been limited by poor transfection efficiency and risk of vehicle-induced pathology. Here we report an inhalable polymer-based vehicle for delivery of therapeutic mRNAs to the lung.

Escaping the endosome: assessing cellular trafficking mechanisms of non-viral vehicles.

Non-viral vehicles hold therapeutic promise in advancing the delivery of a variety of cargos in vitro and in vivo, including small molecule drugs, biologics, and especially nucleic acids. However, their efficacy at the cellular level is limited by several delivery barriers, with endolysosomal degradation being most significant. The entrapment of vehicles and their cargo in the acidified endosome prevents access to the cytosol, nucleus, and other subcellular compartments.

Nanoparticle-mediated convection-enhanced delivery of a DNA intercalator to gliomas circumvents temozolomide resistance.

In patients with glioblastoma, resistance to the chemotherapeutic temozolomide (TMZ) limits any survival benefits conferred by the drug. Here we show that the convection-enhanced delivery of nanoparticles containing disulfide bonds (which are cleaved in the reductive environment of the tumour) and encapsulating an oxaliplatin prodrug and a cationic DNA intercalator inhibit the growth of TMZ-resistant cells from patient-derived xenografts, and hinder the progression of TMZ-resistant human glioblastoma tumours in mice without causing any detectable toxicity. Genome-wide RNA profiling and metabolomic analyses of a glioma cell line treated with the cationic intercalator or with TMZ showed substantial differences in the signalling and metabolic pathways altered by each drug.

PEGylation of poly(amine-co-ester) polyplexes for tunable gene delivery.

There is growing interest in PEGylation of cationic polymeric vehicles for gene delivery in order to improve vehicle stability and reduce toxicity, but little is known about the effects of PEG coatings on transfection. We used a polymer from the poly(amine-co-ester) (PACE) family blended with PEG-conjugated PACE at different ratios in order to explore the effects of polyplex PEGylation on the transfection efficiency of plasmid DNA, mRNA, and siRNA in vitro and mRNA in vivo. We discovered that concentrations of PACE-PEG as low as 0.

Nanoparticles for delivery of agents to fetal lungs.

Fetal treatment of congenital lung disease, such as cystic fibrosis, surfactant protein syndromes, and congenital diaphragmatic hernia, has been made possible by improvements in prenatal diagnostic and interventional technology. Delivery of therapeutic agents to fetal lungs in nanoparticles improves cellular uptake. The efficacy and safety of nanoparticle-based fetal lung therapy depends on targeting of necessary cell populations.

Polymeric vehicles for nucleic acid delivery.

Polymeric vehicles are versatile tools for therapeutic gene delivery. Many polymers-when assembled with nucleic acids into vehicles-can protect the cargo from degradation and clearance in vivo, and facilitate its transport into intracellular compartments. Design options in polymer synthesis yield a comprehensive range of molecules and resulting vehicle formulations.

Poly(Lactic-co-Glycolic Acid) Nanoparticle Delivery of Peptide Nucleic Acids In Vivo.

Many important biological applications of peptide nucleic acids (PNAs) target nucleic acid binding in eukaryotic cells, which requires PNA translocation across at least one membrane barrier. The delivery challenge is further exacerbated for applications in whole organisms, where clearance mechanisms rapidly deplete and/or deactivate exogenous agents. We have demonstrated that nanoparticles (NPs) composed of biodegradable polymers can encapsulate and release PNAs (alone or with co-reagents) in amounts sufficient to mediate desired effects in vitro and in vivo without deleterious reactions in the recipient cell or organism.

High-throughput quantitative microscopy-based half-life measurements of intravenously injected agents.

Accurate analysis of blood concentration and circulation half-life is an important consideration for any intravenously administered agent in preclinical development or for therapeutic application. However, the currently available tools to measure these parameters are laborious, expensive, and inefficient for handling multiple samples from complex multivariable experiments. Here we describe a robust high-throughput quantitative microscopy-based method to measure the blood concentration and circulation half-life of any fluorescently labeled agent using only a small (2 µL) amount of blood volume, enabling additional end-point measurements to be assessed in the same subject.

Poly(amine-co-ester) nanoparticles for effective Nogo-B knockdown in the liver.

Degradable poly(amine-co-ester) (PACE) terpolymers hold tremendous promise for siRNA delivery because these materials can be formulated into delivery vehicles with highly efficient siRNA encapsulation, providing effective knockdown with low toxicity. Here, we demonstrate that PACE nanoparticles (NPs) provide substantial protein knockdown in human embryonic kidney cells (HEK293) and hard-to-transfect primary human umbilical vein endothelial cells (HUVECs). After intravenous administration, NPs of solid PACE (sPACE)-synthesized with high monomer content of a hydrophobic lactone-accumulated in the liver and, to a lesser extent, in other tissues.

Debugging the genetic code: non-viral delivery of therapeutic genome editing technologies.

Efforts to precisely correct genomic mutations that underlie hereditary diseases for therapeutic benefit have advanced alongside the emergence and improvement of genome engineering technologies. These methods can be divided into two main classes: active nucleasebased platforms including the popular CRISPR/Cas9 system and oligo/polynucleotide strategies including triplex-forming oligonucleotides (TFOs), such as peptide nucleic acids (PNAs). These technologies have been successful in cell culture and in animals, but important challenges remain before these tools can be translated into the clinic; they must be effectively delivered to and taken up by specific cell types of interest, achieve correction levels in target cells that significantly ameliorate the disease phenotype, and demonstrate minimal off-target and toxicity effects.

Tunability of Biodegradable Poly(amine- co-ester) Polymers for Customized Nucleic Acid Delivery and Other Biomedical Applications.

Gene therapy promises to treat diseases that arise from genetic abnormalities by correcting the underlying cause of the disease rather than treating the associated symptoms. Successful transfer of nucleic acids into cells requires efficient delivery vehicles that protect the cargo and can penetrate the appropriate cellular barriers before releasing their contents. Many viral vectors and synthetic polycationic vectors for nucleic acid delivery do not translate well from in vitro to in vivo applications due to their instability and toxicity.

Nanoparticle targeting to the endothelium during normothermic machine perfusion of human kidneys.

Ex vivo normothermic machine perfusion (NMP) is a new clinical strategy to assess and resuscitate organs likely to be declined for transplantation, thereby increasing the number of viable organs available. Short periods of NMP provide a window of opportunity to deliver therapeutics directly to the organ and, in particular, to the vascular endothelial cells (ECs) that constitute the first point of contact with the recipient's immune system. ECs are the primary targets of both ischemia-reperfusion injury and damage from preformed antidonor antibodies, and reduction of perioperative EC injury could have long-term benefits by reducing the intensity of the host's alloimmune response.

Dynamics of Tissue-Induced Alignment of Fibrous Extracellular Matrix.

Aligned fibers of extracellular matrix (ECM) affect the direction, efficiency, and persistence of migrating cells. To uncover the mechanisms by which multicellular tissues align their surrounding ECM before migration, we used an engineered three-dimensional culture model to investigate the dynamics of ECM alignment around tissues of defined geometry. Analysis of ECM alignment over time revealed that tissues rapidly reorganize their surrounding matrix, with a characteristic time that depends on the type of cell and the initial tissue geometry.

A 3D Culture Model to Study How Fluid Pressure and Flow Affect the Behavior of Aggregates of Epithelial Cells.

Cells are surrounded by mechanical stimuli in their microenvironment. It is important to determine how cells respond to the mechanical information that surrounds them in order to understand both development and disease progression, as well as to be able to predict cell behavior in response to physical stimuli. Here we describe a protocol to determine the effects of interstitial fluid flow on the migratory behavior of an aggregate of epithelial cells in a three-dimensional (3D) culture model.

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix.

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition.

Interstitial fluid pressure regulates collective invasion in engineered human breast tumors via Snail, vimentin, and E-cadherin.

Many solid tumors exhibit elevated interstitial fluid pressure (IFP). This elevated pressure within the core of the tumor results in outward flow of interstitial fluid to the tumor periphery. We previously found that the directionality of IFP gradients modulates collective invasion from the surface of patterned three-dimensional (3D) aggregates of MDA-MB-231 human breast cancer cells.