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1.
J Proteome Res ; 23(6): 2067-2077, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38776430

RESUMO

Engineered macromolecules offer compelling means for the therapy of conventionally undruggable interactions in human disease. However, their efficacy is limited by barriers to tissue and intracellular delivery. Inspired by recent advances in molecular barcoding and evolution, we developed BarcodeBabel, a generalized method for the design of libraries of peptide barcodes suitable for high-throughput mass spectrometry proteomics. Combined with PeptideBabel, a Monte Carlo sampling algorithm for the design of peptides with evolvable physicochemical properties and sequence complexity, we developed a barcoded library of cell penetrating peptides (CPPs) with distinct physicochemical features. Using quantitative targeted mass spectrometry, we identified CPPS with improved nuclear and cytoplasmic delivery exceeding hundreds of millions of molecules per human cell while maintaining minimal membrane disruption and negligible toxicity in vitro. These studies provide a proof of concept for peptide barcoding as a homogeneous high-throughput method for macromolecular screening and delivery. BarcodeBabel and PeptideBabel are available open-source from https://github.com/kentsisresearchgroup/.


Assuntos
Peptídeos Penetradores de Células , Proteômica , Humanos , Proteômica/métodos , Peptídeos Penetradores de Células/química , Algoritmos , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Ensaios de Triagem em Larga Escala/métodos , Substâncias Macromoleculares/química , Substâncias Macromoleculares/análise
2.
Cancer ; 126(13): 3114-3121, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32320050

RESUMO

BACKGROUND: Constitutional or somatic mosaic epimutations are increasingly recognized as a mechanism of gene dysregulation resulting in cancer susceptibility. Beckwith-Wiedemann syndrome is the cancer predisposition syndrome most commonly associated with epimutation and is extremely variable in its phenotypic presentation, which can include isolated tumors. Because to the authors' knowledge large-scale germline DNA sequencing studies have not included methylation analysis, the percentage of pediatric cancer predisposition that is due to epimutations is unknown. METHODS: Germline methylation testing at the 11p15.5 locus was performed in blood for 24 consecutive patients presenting with hepatoblastoma (3 patients) or Wilms tumor (21 patients). RESULTS: Six individuals with Wilms tumor and 1 patient with hepatoblastoma were found to have low-level gain of methylation at imprinting control 1, and a child with hepatoblastoma was found to have loss of methylation at imprinting control 2. The loss of methylation at imprinting control 2 was found to be maternally inherited, despite not being associated with any detectable genomic alteration. CONCLUSIONS: Overall, 33% of patients (8 of 24 patients) with Wilms tumor or hepatoblastoma were found to have an epigenetic susceptibility that was detectable in the blood. It is interesting to note that low-level gain of methylation at imprinting control 1 predominantly was detected in females with bilateral Wilms tumors. Further studies in larger cohorts are needed to determine the efficacy of testing all patients with Wilms tumor or hepatoblastoma for 11p15.5 epimutations in the blood as part of DNA analysis because this hallmark of predisposition will not be detected by sequencing-based approaches and detecting a cancer predisposition may modify treatment.


Assuntos
Síndrome de Beckwith-Wiedemann/sangue , Metilação de DNA/genética , Impressão Genômica/genética , Hepatoblastoma/sangue , Tumor de Wilms/sangue , Adolescente , Adulto , Síndrome de Beckwith-Wiedemann/genética , Síndrome de Beckwith-Wiedemann/patologia , Criança , Pré-Escolar , Cromossomos Humanos Par 11/genética , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa/genética , Hepatoblastoma/genética , Hepatoblastoma/patologia , Humanos , Lactente , Masculino , Proteínas de Neoplasias/genética , Tumor de Wilms/genética , Tumor de Wilms/patologia , Adulto Jovem
3.
J Clin Oncol ; : JCO2400826, 2024 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-39121437

RESUMO

PURPOSE: Revumenib, an oral, small molecule inhibitor of the menin-lysine methyltransferase 2A (KMT2A) interaction, showed promising efficacy and safety in a phase I study of heavily pretreated patients with KMT2A-rearranged (KMT2Ar) acute leukemia. Here, we evaluated the activity of revumenib in individuals with relapsed/refractory (R/R) KMT2Ar acute leukemia. METHODS: AUGMENT-101 is a phase I/II, open-label, dose-escalation and expansion study of revumenib conducted across 22 clinical sites in five countries (ClinicalTrials.gov identifier: NCT04065399). We report results from the phase II, registration-enabling portion. Individuals age ≥30 days with R/R KMT2Ar acute leukemia or with AML and nucleophosmin 1 (NPM1) mutation were enrolled. Revumenib was administered once every 12 hours, at 163 mg (95 mg/m2 if weight <40 kg) with a strong cytochrome P450 inhibitor, in 28-day cycles. The primary end points were the rate of complete remission (CR) or CR with partial hematologic recovery (CR + CRh) and safety. At a prespecified interim analysis, safety was assessed in all KMT2Ar treated patients; efficacy was assessed in those with centrally confirmed KMT2Ar. The separate NPM1 cohort of the trial is ongoing. RESULTS: From October 1, 2021, to July 24, 2023, N = 94 patients (median [range] age, 37 [1.3-75] years) were treated. Grade ≥3 adverse events included febrile neutropenia (37.2%), differentiation syndrome (16.0%), and QTc prolongation (13.8%). In the efficacy-evaluable patients (n = 57), the CR + CRh rate was 22.8% (95% CI, 12.7 to 35.8), exceeding the null hypothesis of 10% (P = .0036). Overall response rate was 63.2% (95% CI, 49.3 to 75.6), with 15 of 22 patients (68.2%) having no detectable residual disease. CONCLUSION: Revumenib led to high remission rates with a predictable safety profile in R/R KMT2Ar acute leukemia. To our knowledge, this trial represents the largest evaluation of a targeted therapy for these patients.

4.
Mol Ther ; 19(1): 172-80, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20859257

RESUMO

Triplex-forming peptide nucleic acids (PNAs) are powerful gene therapy agents that can enhance recombination of short donor DNAs with genomic DNA, leading to targeted and specific correction of disease-causing genetic mutations. Therapeutic use of PNAs is severely limited, however, by challenges in intracellular delivery, particularly in clinically relevant targets such as hematopoietic stem and progenitor cells. Here, we demonstrate efficient and nontoxic PNA-mediated recombination in human CD34(+) cells using poly(lactic-co-glycolic acid) (PLGA) nanoparticles for intracellular oligonucleotide delivery. Treatment of progenitor cells with nanoparticles loaded with PNAs and DNAs targeting the ß-globin locus led to levels of site-specific modification in the range of 0.5-1% in a single treatment, without detectable loss in cell viability, resulting in a 60-fold increase in modified and viable cells as compared to nucleofection. As well, the differentiation capacity of the progenitor cells treated with nanoparticles did not change relative to untreated progenitor cells, indicating that nanoparticles are safe and minimally disruptive delivery vectors for PNAs and DNAs to mediate gene modification in human primary cells. This is the first demonstration of the use of biodegradable nanoparticles to deliver genome-editing agents to human primary cells, and provides a strong rationale for systemic delivery of complex nucleic acid mixtures designed for gene correction.


Assuntos
Antígenos CD34/biossíntese , Células-Tronco Hematopoéticas/fisiologia , Nanopartículas/administração & dosagem , Ácidos Nucleicos Peptídicos/administração & dosagem , Recombinação Genética , Reparo Gênico Alvo-Dirigido , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA/genética , Marcação de Genes/métodos , Técnicas de Transferência de Genes , Genoma , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ácido Láctico/farmacologia , Nanopartículas/química , Oligonucleotídeos/farmacologia , Tamanho da Partícula , Ácidos Nucleicos Peptídicos/genética , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Receptores CCR5/genética , Globinas beta/genética
5.
Leukemia ; 33(8): 1934-1943, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30760869

RESUMO

Acute myeloid leukemias (AML) are characterized by mutations of tumor suppressor and oncogenes, involving distinct genes in adults and children. While certain mutations have been associated with the increased risk of AML relapse, the genomic landscape of primary chemotherapy-resistant AML is not well defined. As part of the TARGET initiative, we performed whole-genome DNA and transcriptome RNA and miRNA sequencing analysis of pediatric AML with failure of induction chemotherapy. We identified at least three genetic groups of patients with induction failure, including those with NUP98 rearrangements, somatic mutations of WT1 in the absence of apparent NUP98 mutations, and additional recurrent variants including those in KMT2C and MLLT10. Comparison of specimens before and after chemotherapy revealed distinct and invariant gene expression programs. While exhibiting overt therapy resistance, these leukemias nonetheless showed diverse forms of clonal evolution upon chemotherapy exposure. This included selection for mutant alleles of FRMD8, DHX32, PIK3R1, SHANK3, MKLN1, as well as persistence of WT1 and TP53 mutant clones, and elimination of FLT3, PTPN11, and NRAS mutant clones. These findings delineate genetic mechanisms of primary chemotherapy resistance in pediatric AML, which should inform improved approaches for its diagnosis and therapy.


Assuntos
Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Criança , Resistencia a Medicamentos Antineoplásicos , Genes do Tumor de Wilms , Genes p53 , Humanos , Leucemia Mieloide Aguda/genética
6.
Nat Commun ; 7: 13304, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27782131

RESUMO

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.


Assuntos
Edição de Genes/métodos , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Ácidos Nucleicos Peptídicos/genética , Talassemia beta/terapia , Animais , Linhagem Celular , DNA/administração & dosagem , DNA/genética , Modelos Animais de Doenças , Hemoglobinas/análise , Humanos , Injeções Intravenosas , Camundongos , Camundongos Transgênicos , Nanopartículas/administração & dosagem , Ácidos Nucleicos Peptídicos/administração & dosagem , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/administração & dosagem , Fator de Células-Tronco/metabolismo , Globinas beta/genética , Talassemia beta/sangue , Talassemia beta/genética
7.
Adv Healthc Mater ; 4(3): 361-6, 2015 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-25156908
8.
Nat Commun ; 6: 6952, 2015 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-25914116

RESUMO

Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Terapia Genética/métodos , Ácidos Nucleicos Peptídicos/uso terapêutico , Animais , Linhagem Celular , Cloretos/metabolismo , Proteínas de Ligação a DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ácido Láctico , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Mucosa Respiratória/metabolismo
9.
Methods Mol Biol ; 1176: 89-106, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25030921

RESUMO

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.


Assuntos
Marcação de Genes/métodos , Genoma/genética , Conformação de Ácido Nucleico , Animais , Expressão Gênica , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mutagênese , Oligonucleotídeos/química , Oligonucleotídeos/genética , Ácidos Nucleicos Peptídicos , Recombinação Genética
10.
Curr Gene Ther ; 14(5): 331-42, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25174576

RESUMO

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.


Assuntos
DNA/genética , Marcação de Genes , Proteínas de Fluorescência Verde/genética , Nanopartículas/química , Ácidos Nucleicos Peptídicos/genética , Globinas beta/genética , Animais , Medula Óssea/metabolismo , DNA/administração & dosagem , DNA/química , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ácido Láctico , Camundongos , Camundongos Transgênicos , Nanopartículas/administração & dosagem , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/química , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Edição de RNA , Doadores de Tecidos
11.
Artif DNA PNA XNA ; 4(2): 49-57, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23954968

RESUMO

The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.


Assuntos
Nanocápsulas/química , Ácidos Nucleicos Peptídicos/genética , Sequência de Bases , Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Células HeLa , Humanos , Cinética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Tamanho da Partícula , Ácidos Nucleicos Peptídicos/química , Receptores CCR5/genética , Receptores CCR5/metabolismo , Transfecção/métodos
12.
Mol Ther Nucleic Acids ; 2: e135, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24253260

RESUMO

Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ(-/-) mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP-treated PBMCs displayed significantly higher levels of CD4(+) T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP-encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance.Molecular Therapy-Nucleic Acids (2013) 2, e135; doi:10.1038/mtna.2013.59; published online 19 November 2013.

13.
J Control Release ; 155(2): 312-6, 2011 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-21620910

RESUMO

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60bp "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in the treatment or cure of inherited disorders of the blood such as ß-thalassemia or sickle cell anemia. Gene editing in HSPCs and differentiated T cells could also help combat HIV infection by modifying the HIV co-receptor CCR5, which is necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. Here, we review the use of engineered biodegradable polymer nanoparticles for site-specific genome editing in human hematopoietic cells, which represent a promising approach for ex vivo and in vivo gene therapy.


Assuntos
Portadores de Fármacos/química , Ácidos Nucleicos Peptídicos/administração & dosagem , Polímeros/química , Reparo Gênico Alvo-Dirigido , Animais , Materiais Biocompatíveis/química , DNA/administração & dosagem , DNA/química , DNA/efeitos dos fármacos , Técnicas de Transferência de Genes , Genoma , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Nanopartículas/química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética
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