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. In vivo treatment resulted in a partial gain of CFTR function in epithelia as measured by in situ potential differences and Ussing chamber assays and correction of CFTR in both airway and GI tissues with no off-target effects above background. Our studies demonstrate that systemic gene editing is possible, and more specifically that intravenous delivery of PNA NPs designed to correct CF-causing mutations is a viable option to ameliorate CF in multiple affected organs.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

BACKGROUND: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of ß-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. METHODS AND FINDINGS: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. CONCLUSIONS: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

A narrative review of in utero gene therapy: advances, challenges, and future considerations.

The field of in utero gene therapy (IUGT) represents a crossroad of technologic advancements and medical ethical boundaries. Several strategies have been developed for IUGT focusing on either modifying endogenous genes, replacing missing genes, or modifying gene transcription products. The list of candidate diseases such as hemoglobinopathies, cystic fibrosis, lysosomal storage disorders continues to grow with new strategies being developed as our understanding of their respective underlying molecular pathogenesis increases. Treatment in utero has several distinct advantages to postnatal treatment. Biologic and physiologic phenomena enable the delivery of a higher effective dose, generation of immune tolerance, and the prevention of phenotypic onset for genetic diseases. Therapeutic technology for IUGT including CRISPR-Cas9 systems, zinc finger nucleases (ZFN), and peptide nucleic acids (PNAs) has already shown promise in animal models and early postnatal clinical trials. While the ability to detect fetal diagnoses has dramatically improved with developments in ultrasound and next-generation sequencing, treatment options remain experimental, with several translational gaps remaining prior to implementation in the clinical realm. Complicating this issue, the potential diseases targeted by this approach are often debilitating and would otherwise prove fatal if not treated in some manner. The leap from small animals to large animals, and subsequently, to humans will require further vigorous testing of safety and efficacy.

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. This study aimed to determine the relative distribution of nanoparticles of a variety of compositions and sizes in the lungs of fetal mice delivered through intravenous and intra-amniotic routes. Intravenous delivery of particles was more effective than intra-amniotic delivery for epithelial, endothelial and hematopoietic cells in the fetal lung. The most effective targeting of lung tissue was with 250nm Poly-Amine-co-Ester (PACE) particles accumulating in 50% and 44% of epithelial and endothelial cells. This study demonstrated that route of delivery and particle composition impacts relative cellular uptake in fetal lung, which will inform future studies in particle-based fetal therapy.

Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair.

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of ß-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

Alginate microparticles loaded with basic fibroblast growth factor induce tissue coverage in a rat model of myelomeningocele.

BACKGROUND/PURPOSE: We sought to develop a minimally invasive intra-amniotic therapy for prenatal treatment of myelomeningocele (MMC) in an established rat model. METHODS: Time-dated pregnant rats were gavage-fed retinoic acid to induce MMC. Groups received intraamniotic injections at E17.5 with alginate particles loaded with fluorescent dye, basic fibroblast growth factor (Alg-HSA-bFGF), fluorescently tagged albumin (Alginate-BSA-TR), free bFGF, blank alginate particles (Alg-Blank), or PBS. Groups were analyzed at 3 h for specific particle binding or at term (E21) to determine MMC coverage. RESULTS: Alginate microparticles demonstrated robust binding to the MMC defect 3 h after injection. Of those specimens analyzed at E21, 150 of 239 fetuses (62.8%) were viable. Moreover, 18 of 61 (30%) treated with Alg-HSA-bFGF showed evidence of soft tissue coverage compared to 0 of 24 noninjected (P = 0.0021), 0 of 13 PBS (P = 0.0297), and 0 of 42 free bFGF (P = P < 0.0001). Scaffolds of aggregated particles associated with disordered keratinized tissue were observed covering the defect in 2 of 18 (11%) Alg-BSA-TR and 3 of 19 (16%) Alg-Blank specimens. CONCLUSIONS: Injection of microparticles loaded with bFGF resulted in significant soft tissue coverage of the MMC defect compared to controls. Alginate microparticles without growth factors might result in scaffold development over the fetal MMC. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A.

Peptide Nucleic Acids as a Tool for Site-Specific Gene Editing.

Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated gene editing in cell free extracts, PNAs have been used to successfully correct human disease-causing mutations in cell culture and in vivo in preclinical mouse models. Gene correction via PNAs has resulted in clinically-relevant functional protein restoration and disease improvement, with low off-target genome effects, indicating a strong therapeutic potential for PNAs in the treatment or cure of genetic disorders. This review discusses the progress that has been made in developing PNAs as an effective, targeted agent for gene editing, with an emphasis on recent in vivo, nanoparticle-based strategies.

Adjuvant hypofractionated partial-brain radiation therapy for pediatric Ewing sarcoma brain metastases: case report.

This case report demonstrates that hypofractionated partial-brain radiation therapy with limited margins is a reasonable approach following gross tumor resection of Ewing sarcoma metastases to the brain. The patient presented with 2 intracranial metastases treated with gross-total resection followed by radiation therapy to 30 Gy in 5 fractions. The patient experienced symptomatic treatment-related inflammatory changes with resolution after receiving dexamethasone. He remains alive at 21 months of follow-up with no evidence of disease.