RESUMEN
Despite the rapid and broad implementation of CRISPR-Cas9-based technologies, convenient tools to modulate dose, timing, and precision remain limited. Building on methods using synthetic peptide nucleic acids (PNAs) to bind RNA with unusually high affinity, we describe guide RNA (gRNA) spacer-targeted, or 'antispacer', PNAs as a tool to modulate Cas9 binding and activity in cells in a sequence-specific manner. We demonstrate that PNAs rapidly and efficiently target complexed gRNA spacer sequences at low doses and without design restriction for sequence-selective Cas9 inhibition. We further show that short PAM-proximal antispacer PNAs achieve potent cleavage inhibition (over 2000-fold reduction) and that PAM-distal PNAs modify gRNA affinity to promote on-target specificity. Finally, we apply antispacer PNAs for temporal regulation of two dCas9-fusion systems. These results present a novel rational approach to nucleoprotein engineering and describe a rapidly implementable antisense platform for CRISPR-Cas9 modulation to improve spatiotemporal versatility and safety across applications.
Asunto(s)
Ácidos Nucleicos de Péptidos , ARN Guía de Kinetoplastida , Sistemas CRISPR-Cas , Edición Génica/métodos , Ácidos Nucleicos de Péptidos/farmacología , ARN Guía de Kinetoplastida/genéticaRESUMEN
The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.
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Fármacos Anti-VIH/administración & dosificación , Sistemas de Liberación de Medicamentos , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Animales , Fármacos Anti-VIH/farmacocinética , Simulación por Computador , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Ratones , Ratones Endogámicos BALB C , NanopartículasRESUMEN
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.
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Fibrosis Quística/terapia , ADN/genética , Edición Génica/métodos , Terapia Genética/métodos , Ácidos Nucleicos de Péptidos/genética , Talasemia beta/terapia , Animales , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , ADN/metabolismo , Modelos Animales de Enfermedad , Marcación de Gen/métodos , Técnicas de Transferencia de Gen , Humanos , Ratones , Nanopartículas/química , Nanopartículas/metabolismo , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/metabolismo , Reparación del ADN por Recombinación , Talasemia beta/genética , Talasemia beta/metabolismo , Talasemia beta/patologíaRESUMEN
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.
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ADN/metabolismo , Edición Génica/métodos , Ácidos Nucleicos de Péptidos/farmacología , Animales , ADN/química , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/uso terapéuticoRESUMEN
Since their invention in 1991, peptide nucleic acids (PNAs) have been used in a myriad of chemical and biological assays. More recently, peptide nucleic acids have also been demonstrated to hold great potential as therapeutic agents because of their physiological stability, affinity for target nucleic acids, and versatility. While recent modifications in their design have further improved their potency, their preclinical development has reached new heights due to their combination with recent advancements in drug delivery. This review focuses on recent advances in PNA therapeutic applications, in which chemical modifications are made to improve PNA function and nanoparticles are used to enhance PNA delivery.
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Sistemas de Liberación de Medicamentos/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/uso terapéutico , Elementos sin Sentido (Genética) , Estabilidad de Medicamentos , Edición Génica , Humanos , MicroARNs , Estructura Molecular , Nanopartículas/administración & dosificación , Nanopartículas/química , Ácidos Nucleicos de Péptidos/química , Ácidos Nucleicos de Péptidos/farmacocinética , SolubilidadAsunto(s)
Infecciones por VIH , VIH-1 , Animales , Fármacos Anti-VIH , Modelos Animales de Enfermedad , RatonesRESUMEN
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. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. METHODS: Here, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). RESULTS: Treatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. CONCLUSIONS: These findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.
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Through preimplantation genetic diagnosis, genetic diseases can be detected during the early stages of embryogenesis, but effective treatments for many of these disorders are lacking. Gene editing could allow for correction of the underlying mutation during embryogenesis to prevent disease pathogenesis or even provide a cure. Here, we demonstrate that administration of peptide nucleic acids and single-stranded donor DNA oligonucleotides encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles to single-cell embryos allows for editing of an eGFP-beta globin fusion transgene. Blastocysts from treated embryos exhibit high levels of editing (~94%), normal physiological development, normal morphology, and no detected off-target genomic effects. Treated embryos reimplanted to surrogate moms show normal growth without gross developmental abnormalities and with no identified off-target effects. Mice from reimplanted embryos consistently show editing, characterized by mosaicism across multiple organs with some organ biopsies showing up to 100% editing. This proof-of-concept work demonstrates for the first time the use of peptide nucleic acid (PNA)/DNA nanoparticles as a means to achieve embryonic gene editing.
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Peptide nucleic acids (PNAs) can target and stimulate recombination reactions in genomic DNA. We have reported that γPNA oligomers possessing the diethylene glycol γ-substituent show improved efficacy over unmodified PNAs in stimulating recombination-induced gene modification. However, this structural modification poses a challenge because of the inherent racemization risk in O-alkylation of the precursory serine side chain. To circumvent this risk and improve γPNA accessibility, we explore the utility of γPNA oligomers possessing the hydroxymethyl-γ moiety for gene-editing applications. We demonstrate that a γPNA oligomer possessing the hydroxymethyl modification, despite weaker preorganization, retains the ability to form a hybrid with the double-stranded DNA target of comparable stability and with higher affinity than that of the diethylene glycol-γPNA. When formulated into poly(lactic-co-glycolic acid) nanoparticles, the hydroxymethyl-γPNA stimulates higher frequencies (≥ 1.5-fold) of gene modification than the diethylene glycol γPNA in mouse bone marrow cells.
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Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.
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Neoplasias de la Mama , Amplificación de Genes , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Daño del ADN , Femenino , Genómica , Humanos , OligonucleótidosRESUMEN
Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.
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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. For example, poly(lactic-co-glycolic acid) (PLGA) NPs can encapsulate and deliver PNAs and accompanying reagents to mediate gene editing outcomes in cells and animals, or PNAs alone to target oncogenic drivers in cells and correct cancer phenotypes in animal models. In this chapter, we provide a primer on PNA-induced gene editing and microRNA targeting-the two PNA-based biotechnological applications where NPs have enhanced and/or enabled in vivo demonstrations-as well as an introduction to the PLGA material and detailed protocols for formulation and robust characterization of PNA/DNA-laden PLGA NPs.
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Nanopartículas/química , Ácidos Nucleicos de Péptidos , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , ADN/genética , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Edición Génica , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Interferencia de ARNRESUMEN
Nanoparticles (NPs) are promising vehicles for drug delivery because of their potential to target specific tissues [1]. Although it is known that NP size plays a critical role in determining their biological activity, there are few quantitative studies of the role of NP size in determining biodistribution after systemic administration. Here, we engineered fluorescent, biodegradable poly(lactic-co-glycolic acid) (PLGA) NPs in a range of sizes (120-440nm) utilizing a microfluidic platform and used these NPs to determine the effect of diameter on bulk tissue and cellular distribution after systemic administration. We demonstrate that small NPs (â¼120nm) exhibit enhanced uptake in bulk lung and bone marrow, while larger NPs are sequestered in the liver and spleen. We also show that small NPs (â¼120nm) access specific alveolar cell populations and hematopoietic stem and progenitor cells more readily than larger NPs. Our results suggest that size of PLGA NPs can be used to tune delivery to certain tissues and cell populations in vivo.
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Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Nanopartículas , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Microfluídica , Tamaño de la Partícula , Distribución TisularRESUMEN
Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48â¯h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.
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Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacocinética , Fármacos Anti-VIH/farmacología , Cristalografía por Rayos X , Sistemas de Liberación de Medicamentos/métodos , Diseño de Fármacos , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/química , Humanos , Ratones , Ratones Endogámicos BALB C , Nanopartículas/uso terapéutico , Nanopartículas/virología , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/farmacocinética , Inhibidores de la Transcriptasa Inversa/farmacologíaRESUMEN
Metallic stents cause vascular wall damage with subsequent smooth muscle cell (SMC) proliferation, neointimal hyperplasia, and treatment failure. To combat in-stent restenosis, drug-eluting stents (DES) delivering mTOR inhibitors such as sirolimus or everolimus have become standard for coronary stenting. However, the relatively non-specific action of mTOR inhibitors prevents efficient endothelium recovery and mandates dual antiplatelet therapy to prevent thrombosis. Unfortunately, long-term dual antiplatelet therapy leads to increased risk of bleeding/stroke and, paradoxically, myocardial infarction. Here, we took advantage of the fact that nitric oxide (NO) increases Fas receptors on the SMC surface. Fas forms a death-inducing complex upon binding to Fas ligand (FasL), while endothelial cells (ECs) are relatively resistant to this pathway. Selected doses of FasL and NO donor synergistically increased SMC apoptosis and inhibited SMC growth more potently than did everolimus or sirolimus, while having no significant effect on EC viability and proliferation. This differential effect was corroborated in ex vivo pig coronaries, where the neointimal formation was inhibited by the drug combination, but endothelial viability was retained. We also deployed FasL-NO donor-releasing ethylene-vinyl acetate copolymer (EVAc)-coated stents into pig coronary arteries, and cultured them in perfusion bioreactors for one week. FasL and NO donor, released from the stent coating, killed SMCs close to the stent struts, even in the presence of flow rates mimicking those of native arteries. Thus, the FasL-NO donor-combination has a potential to prevent intimal hyperplasia and in-stent restenosis, without harming endothelial restoration, and hence may be a superior drug delivery strategy for DES.
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Células Endoteliales/citología , Proteína Ligando Fas/farmacología , Miocitos del Músculo Liso/citología , Óxido Nítrico/farmacología , Sirolimus/farmacología , Animales , Reactores Biológicos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Vasos Coronarios/citología , Células Endoteliales/efectos de los fármacos , Everolimus/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Miocitos del Músculo Liso/efectos de los fármacos , Compuestos Nitrosos/farmacología , Polímeros/química , PorcinosRESUMEN
Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.
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Terapias Fetales/métodos , Edición Génica/métodos , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/terapia , Nanopartículas/administración & dosificación , Reparación del Gen Blanco/métodos , Animales , ADN de Cadena Simple/administración & dosificación , ADN de Cadena Simple/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ácidos Nucleicos de Péptidos/administración & dosificación , Ácidos Nucleicos de Péptidos/genética , Embarazo , Seguridad , Útero , Globinas beta/genética , Talasemia beta/sangre , Talasemia beta/genética , Talasemia beta/terapiaRESUMEN
New methods for long-lasting protection against sexually transmitted disease, such as the human immunodeficiency virus (HIV), are needed to help reduce the severity of STD epidemics, especially in developing countries. Intravaginal delivery of therapeutics has emerged as a promising strategy to provide women with local protection, but residence times of such agents are greatly reduced by the protective mucus layer, fluctuating hormone cycle, and complex anatomical structure of the reproductive tract. Polymeric nanoparticles (NPs) capable of encapsulating the desired cargo, penetrating through the mucosal surfaces, and delivering agents to the site of action have been explored. However, prolonged retention of polymer carriers and their enclosed materials may also be needed to ease adherence and confer longer-lasting protection against STDs. Here, we examined the fate of two poly (lactic acid)-hyperbranched polyglycerols (PLA-HPG) NP formulations - 1) nonadhesive PLA-HPG NPs (NNPs) and 2) surface-modified bioadhesive NPs (BNPs) - loaded with the antiretroviral elvitegravir (EVG) after intravaginal administration. BNP distribution was widespread throughout the reproductive tract, and retention was nearly 5 times higher than NNPs after 24 h. Moreover, BNPs were found to be highly associated with submucosal leukocytes and epithelial cell populations for up to 48 h after topical application, and EVG was retained significantly better in the vaginal lumen when delivered with BNPs as opposed to NNPs over a 24 h period. Our results suggest that bioadhesive PLA-HPG NPs can greatly improve and prolong intravaginal delivery of agents, which may hold potential in providing sustained protection over longer durations.
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Fármacos Anti-VIH/administración & dosificación , Preparaciones de Acción Retardada/química , Nanopartículas/química , Quinolonas/administración & dosificación , Administración Intravaginal , Animales , Fármacos Anti-VIH/farmacocinética , Femenino , VIH/efectos de los fármacos , Infecciones por VIH/prevención & control , Humanos , Ratones Endogámicos C57BL , Quinolonas/farmacocinética , Vagina/metabolismo , Vagina/virologíaRESUMEN
MicroRNAs (miRs) are frequently overexpressed in human cancers. In particular, miR-210 is induced in hypoxic cells and acts to orchestrate the adaptation of tumor cells to hypoxia. Silencing oncogenic miRs such as miR-210 may therefore offer a promising approach to anticancer therapy. We have developed a miR-210 inhibition strategy based on a new class of conformationally preorganized antisense γ peptide nucleic acids (γPNAs) that possess vastly superior RNA-binding affinity, improved solubility, and favorable biocompatibility. For cellular delivery, we encapsulated the γPNAs in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Our results show that γPNAs targeting miR-210 cause significant delay in growth of a human tumor xenograft in mice compared to conventional PNAs. Further, histopathological analyses show considerable necrosis, fibrosis, and reduced cell proliferation in γPNA-treated tumors compared to controls. Overall, our work provides a chemical framework for a novel anti-miR therapeutic approach using γPNAs that should facilitate rational design of agents to potently inhibit oncogenic microRNAs.
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Glioblastoma multiforme (GBM) is a fatal brain tumor characterized by infiltration beyond the margins of the main tumor mass and local recurrence after surgery. The blood-brain barrier (BBB) poses the most significant hurdle to brain tumor treatment. Convection-enhanced delivery (CED) allows for local administration of agents, overcoming the restrictions of the BBB. Recently, polymer nanoparticles have been demonstrated to penetrate readily through the healthy brain when delivered by CED, and size has been shown to be a critical factor for nanoparticle penetration. Because these brain-penetrating nanoparticles (BPNPs) have high potential for treatment of intracranial tumors since they offer the potential for cell targeting and controlled drug release after administration, here we investigated the intratumoral CED infusions of PLGA BPNPs in animals bearing either U87 or RG2 intracranial tumors. We demonstrate that the overall volume of distribution of these BPNPs was similar to that observed in healthy brains; however, the presence of tumors resulted in asymmetric and heterogeneous distribution patterns, with substantial leakage into the peritumoral tissue. Together, our results suggest that CED of BPNPs should be optimized by accounting for tumor geometry, in terms of location, size and presence of necrotic regions, to determine the ideal infusion site and parameters for individual tumors.
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Neoplasias Encefálicas/metabolismo , Convección , Sistemas de Liberación de Medicamentos , Ácido Láctico/administración & dosificación , Nanopartículas/administración & dosificación , Ácido Poliglicólico/administración & dosificación , Animales , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/farmacocinética , Humanos , Masculino , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas Endogámicas F344 , Ratas Desnudas , Ratas Sprague-Dawley , Carga TumoralRESUMEN
The blood disorder, ß-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the γ position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human ß-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing γPNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% ß-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation γPNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.