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1.
EMBO Rep ; 22(12): e53471, 2021 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-34698433

RESUMEN

Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell-cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood-brain barrier integrity four days after a controlled cortical impact injury.


Asunto(s)
Angiopoyetinas , Células Endoteliales , Células Endoteliales/metabolismo , Neovascularización Fisiológica , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transducción de Señal
2.
Nature ; 552(7685): 415-420, 2017 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-29236688

RESUMEN

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.


Asunto(s)
Bioingeniería , Evolución Molecular Dirigida , Genoma Viral , Nucleocápside/genética , Nucleocápside/metabolismo , ARN Viral/metabolismo , Ensamble de Virus , Animales , Sistemas de Liberación de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , Aptitud Genética , Terapia Genética , Virus de la Inmunodeficiencia Bovina/química , Virus de la Inmunodeficiencia Bovina/genética , Ratones , Modelos Moleculares , Nucleocápside/química , ARN Mensajero/metabolismo , Selección Genética
3.
J Biol Chem ; 296: 100657, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33857478

RESUMEN

The integrin αvß6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvß6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvß6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvß6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvß6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvß6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvß6 in vivo, expanding opportunities for cancer targeting and therapy.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Neoplasias/metabolismo , Fragmentos de Péptidos/metabolismo , Radiofármacos/metabolismo , Suero/química , Proteínas del Envoltorio Viral/metabolismo , Ciclización , Virus de la Fiebre Aftosa/metabolismo , Humanos , Células K562 , Neoplasias/diagnóstico por imagen , Neoplasias/patología
4.
Angew Chem Int Ed Engl ; 60(39): 21211-21215, 2021 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-34328683

RESUMEN

The coronavirus disease 2019 (COVID-19) pandemic has devastated families and disrupted healthcare, economies and societies across the globe. Molecular recognition agents that are specific for distinct viral proteins are critical components for rapid diagnostics and targeted therapeutics. In this work, we demonstrate the selection of novel DNA aptamers that bind to the SARS-CoV-2 spike glycoprotein with high specificity and affinity (<80 nM). Through binding assays and high resolution cryo-EM, we demonstrate that SNAP1 (SARS-CoV-2 spike protein N-terminal domain-binding aptamer 1) binds to the S N-terminal domain. We applied SNAP1 in lateral flow assays (LFAs) and ELISAs to detect UV-inactivated SARS-CoV-2 at concentrations as low as 5×105  copies mL-1 . SNAP1 is therefore a promising molecular tool for SARS-CoV-2 diagnostics.


Asunto(s)
Aptámeros de Nucleótidos/química , COVID-19/diagnóstico , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/análisis , COVID-19/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Modelos Moleculares , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología
5.
Soft Matter ; 16(15): 3762-3768, 2020 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-32239011

RESUMEN

Cell therapy for spinal cord injuries offers the possibility of replacing lost cells after trauma to the central nervous system (CNS). In preclinical studies, synthetic hydrogels are often co-delivered to the injury site to support survival and integration of the transplanted cells. These hydrogels ideally mimic the mechanical and biochemical features of a healthy CNS extracellular matrix while also providing the possibility of localized drug delivery to promote healing. In this work, we synthesize peptide-functionalized polymers that contain both a peptide sequence for incorporation into self-assembled peptide hydrogels along with bioactive peptides that inhibit scar formation. We demonstrate that peptide hydrogels formulated with the peptide-functionalized polymers possess similar mechanical properties (soft and shear-thinning) as peptide-only hydrogels. Small angle neutron scattering analysis reveals that polymer-containing hydrogels possess larger inhomogeneous domains but small-scale features such as mesh size remain the same as peptide-only hydrogels. We further confirm that the integrated hydrogels containing bioactive peptides exhibit thrombin inhibition activity, which has previously shown to reduce scar formation in vivo. Finally, while the survival of encapsulated cells was poor, cells cultured on the hydrogels exhibited good viability. Overall, the described composite hydrogels formed from self-assembling peptides and peptide-modified polymers are promising, user-friendly materials for CNS applications in regeneration.


Asunto(s)
Células Inmovilizadas/metabolismo , Hidrogeles/química , Péptidos/química , Células Madre/metabolismo , Trombina/química , Animales , Células Inmovilizadas/citología , Humanos , Ratones , Células Madre/citología
6.
Angew Chem Int Ed Engl ; 59(32): 13430-13436, 2020 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-32378290

RESUMEN

The use of π-conjugated polymers (CPs) in conductive hydrogels remains challenging due to the water-insoluble nature of most CPs. Conjugated polyelectrolytes (CPEs) are promising alternatives because they have tunable electronic properties and high water-solubility, but they are often difficult to synthesize and thus have not been widely adopted. Herein, we report the synthesis of an anionic poly(cyclopentadienylene vinylene) (aPCPV) from an insulating precursor under mild conditions and in high yield. Functionalized aPCPV is a highly water-soluble CPE that exhibits low cytotoxicity, and we found that doping hydrogels with aPCPV imparts conductivity. We also anticipate that this synthetic strategy, due to its ease and high efficiency, will be widely used to create families of not-yet-explored π-conjugated vinylene polymers.


Asunto(s)
Ciclopentanos/química , Hidrogeles/química , Polímeros/química , Animales , Ciclopentanos/síntesis química , Ciclopentanos/toxicidad , Conductividad Eléctrica , Hidrogeles/síntesis química , Ratones , Células 3T3 NIH , Oxidación-Reducción , Polímeros/síntesis química , Polímeros/toxicidad
7.
Bioconjug Chem ; 30(2): 350-365, 2019 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-30398844

RESUMEN

The nonviral delivery of exogenous nucleic acids (NA) into cells for therapeutic purposes has rapidly matured into tangible clinical impact. Synthetic polymers are particularly attractive vectors for NA delivery due to their relatively inexpensive production compared to viral alternatives and their highly tailorable chemical properties; indeed, many preclinical investigations have revealed the primary biological barriers to nonviral NA delivery by systematically varying polymeric material properties. This review focuses on applications of pH-sensitive chemistries that enable polymeric vectors to serially address multiple biological barriers to NA delivery. In particular, we focus on recent innovations with in vivo evaluation that dynamically enable colloidal stability, cellular uptake, endosomal escape, and nucleic acid release. We conclude with a summary of successes to date and projected areas for impactful future research.


Asunto(s)
Preparaciones de Acción Retardada/química , Técnicas de Transferencia de Gen , Ácidos Nucleicos/administración & dosificación , Polímeros/química , Animales , Preparaciones de Acción Retardada/metabolismo , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ácidos Nucleicos/genética , Polímeros/metabolismo
8.
Proc Natl Acad Sci U S A ; 113(9): 2514-9, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26888285

RESUMEN

A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.


Asunto(s)
Axones , Neuronas Motoras/metabolismo , Péptidos/metabolismo , Médula Espinal/metabolismo , Humanos , Integrasas/metabolismo , Neuronas Motoras/citología , Transporte de Proteínas , Médula Espinal/citología
9.
Biomacromolecules ; 18(9): 2723-2731, 2017 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-28813139

RESUMEN

A PEG-based cyclized vinyl polymer was synthesized via one-step RAFT polymerization and used as a precursor of injectable hydrogels. Dithiol linkers including laminin-derived peptides containing IKVAV and YIGSR sequences and DTT were used for gelation. Fast and adjustable gelation rate was achieved through nucleophile-initiated thiol-Michael reaction under physiological conditions. Low swelling ratio and moderate degradation rate of the formed hydrogels were observed. 3D encapsulation of neural progenitor cells in the synthetic hydrogel showed good cell viability over 8 days. The long-term cell survival and proliferation were promoted by the introduction of laminin-derived peptides. This hydrogel platform based on peptide-cross-linked, cyclized vinyl polymers can be used as a universal hydrogel template for 3D cell encapsulation.


Asunto(s)
Hidrogeles/química , Laminina/química , Nanopartículas/química , Oligopéptidos/química , Fragmentos de Péptidos/química , Compuestos de Vinilo/química , Proliferación Celular , Supervivencia Celular , Células HeLa , Humanos , Hidrogeles/efectos adversos , Hidrogeles/síntesis química , Laminina/administración & dosificación , Nanopartículas/efectos adversos , Células-Madre Neurales/efectos de los fármacos , Oligopéptidos/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Compuestos de Vinilo/efectos adversos , Compuestos de Vinilo/síntesis química
10.
Small ; 12(20): 2750-8, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-27061622

RESUMEN

The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery.


Asunto(s)
Técnicas de Transferencia de Gen , Poliaminas/química , Espectroscopía de Resonancia Magnética , Metacrilatos/química , Estructura Molecular , Ácidos Nucleicos/química , Nylons/química , Polielectrolitos , Espectroscopía Infrarroja por Transformada de Fourier
11.
Proc Natl Acad Sci U S A ; 110(10): 4075-80, 2013 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-23431182

RESUMEN

Neurological diseases and trauma often cause demyelination, resulting in the disruption of axonal function and integrity. Endogenous remyelination promotes recovery, but the process is not well understood because no method exists to definitively distinguish regenerated from preexisting myelin. To date, remyelinated segments have been defined as anything abnormally short and thin, without empirical data to corroborate these morphological assumptions. To definitively identify regenerated myelin, we used a transgenic mouse with an inducible membrane-bound reporter and targeted Cre recombinase expression to a subset of glial progenitor cells after spinal cord injury, yielding remarkably clear visualization of spontaneously regenerated myelin in vivo. Early after injury, the mean length of sheaths regenerated by Schwann cells and oligodendrocytes (OLs) was significantly shorter than control, uninjured myelin, confirming past assumptions. However, OL-regenerated sheaths elongated progressively over 6 mo to approach control values. Moreover, OL-regenerated myelin thickness was not significantly different from control myelin at most time points after injury. Thus, many newly formed OL sheaths were neither thinner nor shorter than control myelin, vitiating accepted dogmas of what constitutes regenerated myelin. We conclude that remyelination, once thought to be static, is dynamic and elongates independently of axonal growth, in contrast to stretch-based mechanisms proposed in development. Further, without clear identification, past assessments have underestimated the extent and quality of regenerated myelin.


Asunto(s)
Vaina de Mielina/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Axones/patología , Axones/fisiología , Enfermedades Desmielinizantes/patología , Enfermedades Desmielinizantes/fisiopatología , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Ratones Transgénicos , Modelos Neurológicos , Vaina de Mielina/patología , Plasticidad Neuronal/fisiología , Oligodendroglía/patología , Oligodendroglía/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células de Schwann/patología , Células de Schwann/fisiología , Traumatismos de la Médula Espinal/patología
12.
13.
ACS Cent Sci ; 10(3): 666-675, 2024 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-38559305

RESUMEN

The Stimulator of Interferon Genes (STING) pathway is a promising target for cancer immunotherapy. Despite recent advances, therapies targeting the STING pathway are often limited by routes of administration, suboptimal STING activation, or off-target toxicity. Here, we report a dendritic cell (DC)-targeted polymeric prodrug platform (polySTING) that is designed to optimize intracellular delivery of a diamidobenzimidazole (diABZI) small-molecule STING agonist while minimizing off-target toxicity after parenteral administration. PolySTING incorporates mannose targeting ligands as a comonomer, which facilitates its uptake in CD206+/mannose receptor+ professional antigen-presenting cells (APCs) in the tumor microenvironment (TME). The STING agonist is conjugated through a cathepsin B-cleavable valine-alanine (VA) linker for selective intracellular drug release after receptor-mediated endocytosis. When administered intravenously in tumor-bearing mice, polySTING selectively targeted CD206+/mannose receptor+ APCs in the TME, resulting in increased cross-presenting CD8+ DCs, infiltrating CD8+ T cells in the TME as well as maturation across multiple DC subtypes in the tumor-draining lymph node (TDLN). Systemic administration of polySTING slowed tumor growth in a B16-F10 murine melanoma model as well as a 4T1 murine breast cancer model with an acceptable safety profile. Thus, we demonstrate that polySTING delivers STING agonists to professional APCs after systemic administration, generating efficacious DC-driven antitumor immunity with minimal side effects. This new polymeric prodrug platform may offer new opportunities for combining efficient targeted STING agonist delivery with other selective tumor therapeutic strategies.

14.
Cancer Res ; 84(7): 1149-1164, 2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38270917

RESUMEN

Adult diffuse gliomas commonly recur regardless of therapy. As recurrence typically arises from the peritumoral edema adjacent to the resected bulk tumor, the profiling of somatic mutations from infiltrative malignant cells within this critical, unresected region could provide important insights into residual disease. A key obstacle has been the inability to distinguish between next-generation sequencing (NGS) noise and the true but weak signal from tumor cells hidden among the noncancerous brain tissue of the peritumoral edema. Here, we developed and validated True2 sequencing to reduce NGS-associated errors to <1 false positive/100 kb panel positions while detecting 97.6% of somatic mutations with an allele frequency ≥0.1%. True2 was then used to study the tumor and peritumoral edema of 22 adult diffuse gliomas including glioblastoma, astrocytoma, oligodendroglioma, and NF1-related low-grade neuroglioma. The tumor and peritumoral edema displayed a similar mutation burden, indicating that surgery debulks these cancers physically but not molecularly. Moreover, variants in the peritumoral edema included unique cancer driver mutations absent in the bulk tumor. Finally, analysis of multiple samples from each patient revealed multiple subclones with unique mutations in the same gene in 17 of 22 patients, supporting the occurrence of convergent evolution in response to patient-specific selective pressures in the tumor microenvironment that may form the molecular foundation of recurrent disease. Collectively, True2 enables the detection of ultralow frequency mutations during molecular analyses of adult diffuse gliomas, which is necessary to understand cancer evolution, recurrence, and individual response to therapy. SIGNIFICANCE: True2 is a next-generation sequencing workflow that facilitates unbiased discovery of somatic mutations across the full range of variant allele frequencies, which could help identify residual disease vulnerabilities for targeted adjuvant therapies.


Asunto(s)
Edema Encefálico , Neoplasias Encefálicas , Glioma , Adulto , Humanos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Edema Encefálico/genética , Edema Encefálico/diagnóstico , Edema Encefálico/patología , Glioma/patología , Edema , Mutación , Microambiente Tumoral
15.
J Control Release ; 354: 188-195, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36596342

RESUMEN

Gene therapy approaches that utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) ribonucleases have tremendous potential to treat human disease. However, CRISPR therapies delivered by integrating viral vectors are limited by potential off-target genome editing caused by constitutive activation of ribonuclease functions. Thus, biomaterial formulations are being used for the delivery of purified CRISPR components to increase the efficiency and safety of genome editing approaches. We previously demonstrated that a novel peptide identified by phage display, TAxI-peptide, mediates delivery of recombinant proteins into neurons. In this report we utilized NeutrAvidin protein to formulate neuron-targeted genome-editing nanoparticles. Cas12a ribonucleases was loaded with biotinylated guide RNA and biotinylated TAxI-peptide onto NeutrAvidin protein to coordinate the formation a targeted ribonuclease protein (RNP) complex. TAxI-RNP complexes are polydisperse with a 14.3 nm radius. The nanoparticles are stable after formulation and show good stability in the presence of normal mouse serum. TAxI-RNP nanoparticles increased neuronal delivery of Cas12a in reporter mice, resulting in induced tdTomato expression after direct injection into the dentate gyrus of the hippocampus. TAxI-RNP nanoparticles also increased genome editing efficacy in hippocampal neurons versus glia. These studies demonstrate the ability to assemble RNP nanoformulations with NeutrAvidin by binding biotinylated peptides and gRNA-loaded Cas12a ribonucleases into protein nanoparticles that target CRISPR delivery to specific cell-types in vivo. The potential to deliver CRISPR nanoparticles to specific cell-types and control off-target delivery to further reduce deleterious genome editing is essential for the creation of viable therapies to treat nervous system disease.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Ratones , Animales , Humanos , Edición Génica/métodos , Ribonucleasas , Péptidos , Neuronas
16.
Nat Rev Mater ; 7(4): 314-331, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38464996

RESUMEN

Despite the rising global incidence of central nervous system (CNS) disorders, CNS drug development remains challenging, with high costs, long pathways to clinical use and high failure rates. The CNS is highly protected by physiological barriers, in particular, the blood-brain barrier and the blood-cerebrospinal fluid barrier, which limit access of most drugs. Biomaterials can be designed to bypass or traverse these barriers, enabling the controlled delivery of drugs into the CNS. In this Review, we first examine the effects of normal and diseased CNS physiology on drug delivery to the brain and spinal cord. We then discuss CNS drug delivery designs and materials that are administered systemically, directly to the CNS, intranasally or peripherally through intramuscular injections. Finally, we highlight important challenges and opportunities for materials design for drug delivery to the CNS and the anticipated clinical impact of CNS drug delivery.

17.
J Neurosci ; 29(20): 6722-33, 2009 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-19458241

RESUMEN

Progenitors that express NG2-proteoglycan are the predominant self-renewing cells within the CNS. NG2 progenitors replenish oligodendrocyte populations within the intact stem cell niche, and cycling NG2 cells are among the first cells to react to CNS insults. We investigated the role of NG2 progenitors after spinal cord injury and how bone morphogen protein signals remodel the progressive postinjury (PI) niche. Progeny labeled by an NG2-specific reporter virus undergo a coordinated shift in differentiation profile. NG2 progeny born 24 h PI produce scar-forming astrocytes and transient populations of novel phagocytic astrocytes shown to contain denatured myelin within cathepsin-D-labeled endosomes, but NG2 progenitors born 7 d PI differentiate into oligodendrocytes and express myelin on processes that wrap axons. Analysis of spinal cord mRNA shows a temporal shift in the niche transcriptome of ligands that affect PI remodeling and direct progenitor differentiation. We conclude that NG2 progeny are diverse lineages that obey progressive cues after trauma to replenish the injured niche.


Asunto(s)
Antígenos/metabolismo , Proteoglicanos/metabolismo , Traumatismos de la Médula Espinal/cirugía , Nicho de Células Madre/patología , Trasplante de Células Madre/métodos , Células Madre/fisiología , Análisis de Varianza , Animales , Animales Recién Nacidos , Antígenos/genética , Apoptosis/genética , Apoptosis/fisiología , Caspasa 3/metabolismo , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Fluorescentes Verdes/genética , Ratones , Proteína Básica de Mielina/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Proteoglicanos/genética , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/metabolismo , Transducción de Señal , Traumatismos de la Médula Espinal/fisiopatología , Nicho de Células Madre/fisiopatología , Células Madre/metabolismo , Factores de Tiempo , Transfección
18.
J Control Release ; 322: 149-156, 2020 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-32198024

RESUMEN

Traumatic brain injury (TBI) is largely non-preventable and often kills or permanently disables its victims. Because current treatments for TBI merely ameliorate secondary effects of the initial injury like swelling and hemorrhaging, strategies for the induction of neuronal regeneration are desperately needed. Recent discoveries regarding the TBI-responsive migratory behavior and differentiation potential of neural progenitor cells (NPCs) found in the subventricular zone (SVZ) have prompted strategies targeting gene therapies to these cells to enhance neurogenesis after TBI. We have previously shown that plasmid polyplexes can non-virally transfect SVZ NPCs when directly injected in the lateral ventricles of uninjured mice. We describe the first reported intracerebroventricular transfections mediated by polymeric gene carriers in a murine TBI model and investigate the anatomical parameters that dictate transfection through this route of administration. Using both luciferase and GFP plasmid transfections, we show that the time delay between injury and polyplex injection directly impacts the magnitude of transfection efficiency, but that overall trends in the location of transfection are not affected by injury. Confocal microscopy of quantum dot-labeled plasmid uptake in vivo reveals association between our polymers and negatively charged NG2 chondroitin sulfate proteoglycans of the SVZ extracellular matrix. We further validate that glycosaminoglycans but not sulfate groups are required for polyplex uptake and transfection in vitro. These studies demonstrate that non-viral gene delivery is impacted by proteoglycan interactions and suggest the need for improved polyplex targeting materials that penetrate brain extracellular matrix to increase transfection efficiency in vivo.


Asunto(s)
Lesiones Traumáticas del Encéfalo , Células-Madre Neurales , Animales , Lesiones Traumáticas del Encéfalo/terapia , Ventrículos Laterales , Ratones , Neurogénesis , Transfección
19.
bioRxiv ; 2020 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-33501432

RESUMEN

Angiopoietin 1 and 2 (Ang1 and Ang2) modulate angiogenesis and vascular homeostasis through engagement of their very similar F-domain modules with the Tie2 receptor tyrosine kinase on endothelial cells. Despite this similarity in the underlying receptor binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of protein kinase B (AKT), strengthens cell-cell junctions and enhances endothelial cell survival while Ang2 antagonizes these effects 1-4 . To investigate the molecular basis for the opposing effects, we examined the protein kinase activation and morphological phenotypes produced by a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: scaffolds presenting 4 F-domains have Ang2 like activity, upregulating pFAK and pERK but not pAKT, and failing to induce cell migration and tube formation, while scaffolds presenting 6 or more F-domains have Ang1 like activity, upregulating pAKT and inducing migration and tube formation. The scaffolds with 8 or more F-domains display superagonist activity, producing stronger phenotypes at lower concentrations than Ang1. When examined in vivo , superagonist icosahedral self-assembling nanoparticles caused significant revascularization in hemorrhagic brains after a controlled cortical impact injury.

20.
Biomaterials ; 192: 235-244, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30458359

RESUMEN

Endocytosed biomacromolecule delivery systems must escape the endosomal trafficking pathway in order for their cargo to exert effects in other cellular compartments. Although endosomal release is well-recognized as one of the greatest barriers to efficacy of biologic drugs with intracellular targets, most drug carriers have relied on cationic materials that passively induce endosomal swelling and membrane rupture with low efficiency. To address the endosome release challenge, our lab has developed a diblock copolymer system for nucleic acid delivery that selectively displays a potent membrane-lytic peptide (melittin) in response to the pH drop during the endosomal maturation. To further optimize this system, we evaluated a panel of peptides with reported lytic activity in comparison to melittin. Nineteen different lytic peptides were synthesized and their membrane-lytic properties at both neutral and acidic pH characterized using a red blood cell hemolysis assay. The top five performing peptides were then conjugated to our pH-sensitive diblock copolymer via disulfide linkers and used to deliver a variety of nucleic acids to cultured mammalian cells as well as in vivo to the mouse brain. We demonstrate that the sharp pH-transition of VIPER compensates for potential advantages from pH-sensitive peptides, such that polymer-peptide conjugates with poorly selective but highly lytic peptides achieve safe and effective transfection both in vitro and in vivo. In addition, peptides that require release from polymer backbones for lysis were less effective in the VIPER system, likely due to limited endosomal reducing power of target cells. Finally, we show that certain peptides are potentiated in lytic ability by polymer conjugation and that these peptide-polymer constructs are most effective in vivo.


Asunto(s)
Preparaciones de Acción Retardada/química , Meliteno/química , Ácidos Nucleicos/administración & dosificación , Polímeros/química , Animales , Femenino , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Micelas , Transfección
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