RESUMEN
Monogenic blood diseases are among the most common genetic disorders worldwide. These diseases result in significant pediatric and adult morbidity, and some can result in death prior to birth. Novel ex vivo hematopoietic stem cell (HSC) gene editing therapies hold tremendous promise to alter the therapeutic landscape but are not without potential limitations. In vivo gene editing therapies offer a potentially safer and more accessible treatment for these diseases but are hindered by a lack of delivery vectors targeting HSCs, which reside in the difficult-to-access bone marrow niche. Here, we propose that this biological barrier can be overcome by taking advantage of HSC residence in the easily accessible liver during fetal development. To facilitate the delivery of gene editing cargo to fetal HSCs, we developed an ionizable lipid nanoparticle (LNP) platform targeting the CD45 receptor on the surface of HSCs. After validating that targeted LNPs improved messenger ribonucleic acid (mRNA) delivery to hematopoietic lineage cells via a CD45-specific mechanism in vitro, we demonstrated that this platform mediated safe, potent, and long-term gene modulation of HSCs in vivo in multiple mouse models. We further optimized this LNP platform in vitro to encapsulate and deliver CRISPR-based nucleic acid cargos. Finally, we showed that optimized and targeted LNPs enhanced gene editing at a proof-of-concept locus in fetal HSCs after a single in utero intravenous injection. By targeting HSCs in vivo during fetal development, our Systematically optimized Targeted Editing Machinery (STEM) LNPs may provide a translatable strategy to treat monogenic blood diseases before birth.
Asunto(s)
Edición Génica , Células Madre Hematopoyéticas , Nanopartículas , Animales , Células Madre Hematopoyéticas/metabolismo , Edición Génica/métodos , Nanopartículas/química , Ratones , Femenino , Embarazo , Lípidos/química , Antígenos Comunes de Leucocito/metabolismo , Antígenos Comunes de Leucocito/genética , Humanos , Terapia Genética/métodos , Sistemas CRISPR-Cas , LiposomasRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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.
Asunto(s)
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 , SolubilidadRESUMEN
Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem-loop sequence, which is bound to the stem-loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3' end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3'hExo, forms a ternary complex with SLBP and the stem-loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3'hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3'hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.
Asunto(s)
Región de Flanqueo 3' , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Estabilidad del ARN , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Células HeLa , Histonas/genética , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3' end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.
Asunto(s)
ARN Helicasas DEAD-box/química , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Unión al ARN/química , Proteínas de Pez Cebra/química , Secuencia de Aminoácidos , Animales , ARN Helicasas DEAD-box/metabolismo , Factor 3 de Iniciación Eucariótica/química , Factor 3 de Iniciación Eucariótica/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
RESUMEN
Structural fetal diseases, such as congenital diaphragmatic hernia (CDH) can be diagnosed prenatally. Neonates with CDH are healthy in utero 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-ß pathway are critically involved in lung branching morphogenesis. Here, we characterize the expression of miR200b and the TGF-ß pathway at different gestational times using a rat model of CDH. Fetal rats with CDH are deficient in miR200b at gestational day 18. We demonstrate that novel polymeric nanoparticles loaded with miR200b, delivered in utero via vitelline vein injection to fetal rats with CDH results in changes in the TGF-ß pathway as measured by qRT-PCR; these epigenetic changes improve lung size and lung morphology, and lead to favorable pulmonary vascular remodeling on histology. This is the first demonstration of in utero epigenetic therapy to improve lung growth and development in a pre-clinical model. With refinement, this technique could be applied to fetal cases of CDH or other forms of impaired lung development in a minimally invasive fashion.
RESUMEN
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.
Asunto(s)
Fibrosis Quística , Nanopartículas , Animales , Anticuerpos , Fenómenos Químicos , Células Epiteliales , Nanopartículas/químicaRESUMEN
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.
RESUMEN
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.
RESUMEN
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.
Asunto(s)
Hernias Diafragmáticas Congénitas , Nanopartículas , Surfactantes Pulmonares , Animales , Células Endoteliales , Femenino , Pulmón , Ratones , EmbarazoRESUMEN
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 3â¯h for specific particle binding or at term (E21) to determine MMC coverage. RESULTS: Alginate microparticles demonstrated robust binding to the MMC defect 3â¯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 (Pâ¯=â¯0.0021), 0 of 13 PBS (Pâ¯=â¯0.0297), and 0 of 42 free bFGF (Pâ¯=â¯Pâ¯<â¯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.
Asunto(s)
Alginatos/farmacología , Terapias Fetales/métodos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Meningomielocele/terapia , Líquido Amniótico , Animales , Materiales Biocompatibles/farmacología , Femenino , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Embarazo , RatasRESUMEN
The untranslated regions (UTRs) of many mRNAs contain sequence and structural motifs that are used to regulate the stability, localization, and translatability of the mRNA. It should be possible to discover previously unidentified RNA regulatory motifs by examining many related nucleotide sequences, which are assumed to contain a common motif. This is a general practice for discovery of DNA-based sequence-based patterns, in which alignment tools are heavily exploited. However, because of the complexity of sequential and structural components of RNA-based motifs, simple-alignment tools are frequently inadequate. The consensus sequences they find frequently have the potential for significant variability at any given position and are only loosely characterized. The development of RNA-motif discovery tools that infer and integrate structural information into motif discovery is both necessary and expedient. Here, we provide a selected list of existing web-accessible algorithms for the discovery of RNA motifs, which, although not exhaustive, represents the current state of the art. To facilitate the development, evaluation, and training of new software programs that identify RNA motifs, we created the UAlbany training UTR (TUTR) database, which is a collection of validated sets of sequences containing experimentally defined regulatory motifs. Presently, eleven training sets have been generated with associated indexes and "answer sets" provided that identify where the previously characterized RNA motif [the iron responsive element (IRE), AU-rich class-2 element (ARE), selenocysteine insertion sequence (SECIS), etc.] resides in each sequence. The UAlbany TUTR collection is a shared resource that is available to researchers for software development and as a research aid.
Asunto(s)
Biología Computacional , Bases de Datos de Ácidos Nucleicos , Procesamiento Postranscripcional del ARN , ARN no Traducido/genética , Algoritmos , Animales , Inteligencia Artificial , Humanos , Internet , New York , Programas InformáticosRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
Neoplasias Encefálicas , Hipofraccionamiento de la Dosis de Radiación , Radioterapia Adyuvante/métodos , Sarcoma de Ewing/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/cirugía , Niño , Humanos , MasculinoRESUMEN
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.
Asunto(s)
Edición Génica/métodos , Terapia Genética/métodos , Células Madre Hematopoyéticas/metabolismo , Ácidos Nucleicos de Péptidos/genética , Talasemia beta/terapia , Animales , Línea Celular , ADN/administración & dosificación , ADN/genética , Modelos Animales de Enfermedad , Hemoglobinas/análisis , Humanos , Inyecciones Intravenosas , Ratones , Ratones Transgénicos , Nanopartículas/administración & dosificación , Ácidos Nucleicos de Péptidos/administración & dosificación , Proteínas Proto-Oncogénicas c-kit/metabolismo , Factor de Células Madre/administración & dosificación , Factor de Células Madre/metabolismo , Globinas beta/genética , Talasemia beta/sangre , Talasemia beta/genéticaRESUMEN
Triplex-forming oligonucleotides (TFOs) are capable of coordinating genome modification in a targeted, site-specific manner, causing mutagenesis or even coordinating homologous recombination events. Here, we describe the use of TFOs such as peptide nucleic acids for targeted genome modification. We discuss this method and its applications and describe protocols for TFO design, delivery, and evaluation of activity in vitro and in vivo.