RESUMO
A novel method for the parallel synthesis of peptide-biocargo conjugates was developed that utilizes affinity purification for fast isolation of the conjugates in order to avoid time consuming HPLC purification. The methodology was applied to create two libraries of cell-penetrating peptide (CPP)-PNA705 conjugates from parallel-synthesized peptide libraries. The conjugates were tested for their ability to induce splicing redirection in HeLa pLuc705 cells. The results demonstrate how the novel methodology can be applied for screening purposes in order to find suitable CPP-biocargo combinations and further optimization of CPPs.
Assuntos
Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/metabolismo , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismo , Splicing de RNA , RNA Mensageiro/metabolismo , Peptídeos Penetradores de Células/química , Cromatografia Líquida de Alta Pressão , Células HeLa , Humanos , Conformação Molecular , Biblioteca de Peptídeos , Splicing de RNA/genética , RNA Mensageiro/genéticaRESUMO
Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.
Assuntos
Peptídeos Penetradores de Células/química , Lipopeptídeos/química , Oligonucleotídeos Antissenso/administração & dosagem , Processamento Alternativo , Animais , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/toxicidade , Células Cultivadas , Meios de Cultura , Meios de Cultura Livres de Soro , Endocitose , Células HeLa , Humanos , Cinética , Luz , Lipopeptídeos/metabolismo , Lipopeptídeos/toxicidade , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Nanoestruturas/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/metabolismo , Espalhamento de Radiação , Soluções , TemperaturaRESUMO
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease caused by frameshift or nonsense mutations in the DMD gene, resulting in the loss of dystrophin from muscle membranes. Exon skipping using splice-switching oligonucleotides (SSOs) restores the reading frame of DMD pre-mRNA by generating internally truncated but functional dystrophin protein. To potentiate effective tissue-specific targeting by functional SSOs, it is essential to perform accelerated and reliable in vitro screening-based assessment of novel oligonucleotides and drug delivery technologies, such as cell-penetrating peptides, before their in vivo pharmacokinetic and toxicity evaluation. We have established novel canine immortalized myoblast lines by transducing murine cyclin-dependent kinase-4 and human telomerase reverse transcriptase genes into myoblasts isolated from beagle-based wild-type or canine X-linked muscular dystrophy in Japan (CXMDJ) dogs. These myoblast lines exhibited improved myogenic differentiation and increased proliferation rates compared with passage-15 primary parental myoblasts, and their potential to differentiate into myotubes was maintained in later passages. Using these dystrophin-deficient immortalized myoblast lines, we demonstrate that a novel cell-penetrating peptide (Pip8b2)-conjugated SSO markedly improved multiexon skipping activity compared with the respective naked phosphorodiamidate morpholino oligomers. In vitro screening using immortalized canine cell lines will provide a basis for further pharmacological studies on drug delivery tools.
Assuntos
Quinase 4 Dependente de Ciclina/genética , Distrofina/genética , Morfolinos/farmacologia , Distrofia Muscular de Duchenne/terapia , Telomerase/genética , Animais , Linhagem Celular , Cães , Éxons/genética , Terapia Genética , Humanos , Camundongos , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mioblastos/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Peptídeos/genética , Peptídeos/farmacologia , Sítios de Splice de RNA/genéticaRESUMO
The full therapeutic potential of oligonucleotide (ON)-based agents has been hampered by cellular delivery challenges. Cell-penetrating peptides (CPP) represent promising delivery vectors for nucleic acids, and their potential has recently been evaluated using a functional splicing redirection assay, which capitalizes on the nuclear delivery of splice-correcting steric-block ON analogues such as peptide nucleic acids (PNA). Despite encouraging in vitro and in vivo data with arginine-rich CPP-steric block conjugates, mechanistic studies have shown that entrapment within the endosome/lysosome compartment after endocytosis remains a limiting factor. Previous work from our group has shown that CPP oligomerization greatly improves cellular delivery and increases transfection of plasmid DNA. We now report the chemical synthesis and the evaluation of multivalent CPP-PNA constructs incorporating monomeric (p53(mono)) and dendrimer-like tetrameric (p53(tet)) forms of the p53 tetramerization domain containing peptide, a 10 arginine CPP domain (R10), and a splice redirecting PNA (PNA705). These CPP-PNA conjugates were termed R10p53(tet)-PNA705 and R10p53(mono)-PNA705, referring to their oligomerization state. The present study demonstrates that the splicing redirection efficiency of PNA705 is much greater in the context of the tetrameric R10p53(tet)-PNA705 construct than for the monomeric and occurs at nanomolar concentrations, demonstrating that multivalency is an important factor in delivering PNA into cells.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Dendrímeros/química , Dendrímeros/farmacologia , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Peptídeos Penetradores de Células/síntese química , Dendrímeros/síntese química , Técnicas de Transferência de Genes , Vetores Genéticos , Células HeLa , Humanos , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/genéticaRESUMO
Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO) to cell penetrating peptides (CPP) has recently been proposed as a new delivery strategy. It is particularly suitable to interfere sequence-specifically with pre-mRNA splicing thus offering various applications in fundamental research and in therapeutics. The chemical synthesis of these CPP conjugates as well as methodologies to monitor their cellular uptake and their efficiency in a reliable and easy to implement assay of splicing correction will be described.
Assuntos
Oligonucleotídeos/administração & dosagem , Ácidos Nucleicos Peptídicos/administração & dosagem , Peptídeos/química , Separação Celular , Citometria de Fluxo , Células HeLa , Humanos , Microscopia de Fluorescência , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the award of the 2017 Oligonucleotide Therapeutic Society Lifetime Achievement Award. This acts as a prelude to the rewarding collaborative studies in the Gait and Wood research groups aimed toward the enhanced delivery of charge neutral ON drugs and the development of a series of Arg-rich cell-penetrating peptides called Pip (peptide nucleic acid/phosphorodiamidate morpholino oligonucleotide [PNA/PMO] internalization peptides) as conjugates of such ONs. In this review we concentrate on these developments toward the treatment of the neuromuscular diseases Duchenne muscular dystrophy and spinal muscular atrophy toward a platform technology for the enhancement of cellular and in vivo delivery suitable for widespread use as neuromuscular and neurodegenerative ON drugs.
Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Doenças Neuromusculares/tratamento farmacológico , Peptídeos Penetradores de Células/genética , Humanos , Morfolinos/genética , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/uso terapêuticoRESUMO
Charge neutral steric block oligonucleotide analogues, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO), have promising biological and pharmacological properties for antisense applications, such as for example in mRNA splicing redirection. However, cellular uptake of free oligomers is poor and the utility of conjugates of PNA or PMO to cell penetrating peptides (CPP), such as Tat or Penetratin, is limited by endosomal sequestration. Two new families of arginine-rich CPPs named (R-Ahx-R)(4) AhxB and R(6)Pen allow efficient nuclear delivery of splice correcting PNA and PMO at micromolar concentrations in the absence of endosomolytic agents. The in vivo efficacy of (R-Ahx-R)(4) AhxB PMO conjugates has been demonstrated in mouse models of Duchenne muscular dystrophy and in various viral infections.
Assuntos
Oligonucleotídeos/química , Peptídeos/química , Animais , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos/métodos , Humanos , Estrutura Molecular , Ácidos Nucleicos Peptídicos/administração & dosagem , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacocinética , Peptídeos/administração & dosagem , Peptídeos/farmacocinéticaRESUMO
New candidates for development as potential drugs or virucides against HIV-1 infection and AIDS continue to be needed. The HIV-1 RNA leader sequence has many essential functional sites for virus replication and regulation that includes several highly conserved sequences. The review describes the historical context of targeting the HIV-1 RNA leader sequence with antisense phosphorothioate oligonucleotides, such as GEM 91, and goes on to describe modern approaches to targeting this region with steric blocking oligonucleotide analogues having newer and more advantageous chemistries, as well as recent studies on siRNA, towards the attainment of antiviral activity. Recent attempts to obtain improved cell delivery are highlighted, including exciting new developments in the use of peptide conjugates of peptide nucleic acid (PNA) as potential virucides.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Oligodesoxirribonucleotídeos Antissenso/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Regiões 5' não Traduzidas/efeitos dos fármacos , Regiões 5' não Traduzidas/genética , Fármacos Anti-HIV/química , HIV-1/genética , Humanos , Oligodesoxirribonucleotídeos Antissenso/química , RNA Interferente Pequeno/química , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Tionucleotídeos/administração & dosagem , Tionucleotídeos/químicaRESUMO
We synthesized and evaluated by surface plasmon resonance 64 LNA/2'-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2'-O-methyl nucleoside analogues where one or two LNA units are located on the 3' side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay.
Assuntos
Aptâmeros de Nucleotídeos/química , Repetição Terminal Longa de HIV , Oligonucleotídeos Antissenso/análise , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Células HeLa , Humanos , Cinética , Luciferases/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos , Oligonucleotídeos Antissenso/químicaRESUMO
Oligonucleotides composed of 2'-O-methyl and locked nucleic acid residues complementary to HIV-1 trans-activation responsive element TAR block Tat-dependent trans-activation in a HeLa cell assay when delivered by cationic lipids. We describe an improved procedure for synthesis and purification under highly denaturing conditions of 5'-disulphide-linked conjugates of 3'-fluorescein labelled oligonucleotides with a range of cell-penetrating peptides and investigate their abilities to enter HeLa cells and block trans-activation. Free uptake of 12mer OMe/LNA oligonucleotide conjugates to Tat (48-58), Penetratin and R9F2 was observed in cytosolic compartments of HeLa cells. Uptake of the Tat conjugate was enhanced by N-terminal addition of four Lys or Arg residues or a second Tat peptide. None of the conjugates entered the nucleus or inhibited trans-activation when freely delivered, but inhibition was obtained in the presence of cationic lipids. Nuclear exclusion was seen for free delivery of Tat (48-58), Penetratin and R9 conjugates of 16mer phosphorothioate OMe oligonucleotide. Uptake into human fibroblast cytosolic compartments was seen for Tat, Penetratin, R9F2 and Transportan conjugates. Large enhancements of HeLa cell uptake into cytosolic compartments were seen when free Tat peptide was added to Tat conjugate of 12mer OMe/LNA oligonucleotide or Penetratin peptide to Penetratin conjugate of the same oligonucleotide.
Assuntos
Produtos do Gene tat/antagonistas & inibidores , Repetição Terminal Longa de HIV/efeitos dos fármacos , HIV-1/genética , Oligonucleotídeos/metabolismo , Oligonucleotídeos/farmacologia , Ácidos Nucleicos Peptídicos/metabolismo , Ácidos Nucleicos Peptídicos/farmacologia , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Dissulfetos/química , Fibroblastos/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Oligonucleotídeos/síntese química , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Ácidos Nucleicos Peptídicos/síntese química , Tionucleotídeos/síntese química , Tionucleotídeos/química , Tionucleotídeos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3' end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed 'crook' siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics.
Assuntos
Reação em Cadeia da Polimerase/métodos , Interferência de RNA , RNA Interferente Pequeno/análise , RNA Interferente Pequeno/química , Linhagem Celular , Primers do DNA , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus , Fenótipo , RNA Mensageiro/metabolismo , Moldes Genéticos , TransfecçãoRESUMO
The trans-activation response (TAR) RNA stem-loop that occurs at the 5' end of HIV RNA transcripts is an important antiviral target and is the site of interaction of the HIV-1 Tat protein together with host cellular factors. Oligonucleotides and their analogues targeted to TAR are potential antiviral candidates. We have investigated a range of cell penetrating peptide (CPP) conjugates of a 16mer peptide nucleic acid (PNA) analogue targeted to the apical stem-loop of TAR and show that disulfide-linked PNA conjugates of two types of CPP (Transportan or a novel chimeric peptide R6-Penetratin) exhibit dose-dependent inhibition of Tat-dependent trans-activation in a HeLa cell assay when incubated for 24 h. Activity is reached within 6 h if the lysosomotropic reagent chloroquine is co-administered. Fluorescein-labelled stably-linked conjugates of Tat, Transportan or Transportan TP10 with PNA were inactive when delivered alone, but attained trans-activation inhibition in the presence of chloroquine. Confocal microscopy showed that such fluorescently labelled CPP-PNA conjugates were sequestered in endosomal or membrane-bound compartments of HeLa cells, which varied in appearance depending on the CPP type. Co-administration of chloroquine was seen in some cases to release fluorescence from such compartments into the nucleus, but with different patterns depending on the CPP. The results show that CPP-PNA conjugates of different types can inhibit Tat-dependent trans-activation in HeLa cells and have potential for development as antiviral agents. Endosomal or membrane release is a major factor limiting nuclear delivery and trans-activation inhibition.
Assuntos
Antivirais/química , Antivirais/farmacologia , Produtos do Gene tat/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Antivirais/análise , Permeabilidade da Membrana Celular , Cloroquina/farmacologia , Fluoresceínas/química , Corantes Fluorescentes/química , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Membranas Intracelulares/química , Microscopia Confocal , Ácidos Nucleicos Peptídicos/análise , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The unabated increase in spread of HIV infection worldwide has redoubled efforts to discover novel antiviral and virucidal agents that might be starting points for clinical development. Oligonucleotides and their analogs targeted to form complementary duplexes with highly conserved regions of the HIV RNA have shown significant antiviral activity, but to date clinical studies have been dominated by RNase H-inducing oligonucleotide analog phosphorothioates (GEM 91 and 92) that have specificity and efficacy limitations. However, they have proven the principle that oligonucleotides can be safe anti-HIV drugs. Newer oligonucleotide analogs are now available, which act as strong steric block agents of HIV RNA function. We describe our ongoing studies targeting the HIV-1 trans-activation responsive region (TAR) and the viral packaging signal (psi) with steric block oligonucleotides of varying chemistry and demonstrate their great potential for steric blocking of viral protein interactions in vitro and in cells and describe the first antiviral studies. Peptide nucleic acids (PNA) disulfide linked to cell-penetrating peptides (CPP) have been found to have particular promise for the lipid-free direct delivery into cultured cells and are excellent candidates for their development as antiviral and virucidal agents.
Assuntos
Fármacos Anti-HIV , Sistemas de Liberação de Medicamentos/métodos , Oligonucleotídeos/uso terapêutico , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacologia , Células Cultivadas , HIV/genética , Repetição Terminal Longa de HIV/efeitos dos fármacos , Humanos , Oligonucleotídeos/farmacologia , Ácidos Nucleicos Peptídicos/uso terapêutico , RNA Viral/efeitos dos fármacosRESUMO
We report the synthesis of a novel 2'-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and incorporation into a series of small steric blocking OMe oligonucleotides targeting the apical stem-loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. Binding to TAR RNA is substantially enhanced for certain single site substitutions in the centre of the oligonucleotide, and doubly substituted anti-TAR OMe 9mers or 12mers exhibit remarkably low binding constants of <0.1 nM. G-clamp-containing oligomers achieved 50% inhibition of Tat-dependent in vitro transcription at approximately 25 nM, 4-fold lower than for a TAR 12mer OMe oligonucleotide and better than found for any other oligonucleotide tested to date. Addition of one or two OMe G-clamps did not impart cellular trans-activation inhibition activity to cellularly inactive OMe oligonucleotides. Addition of an OMe G-clamp to a 12mer OMe-locked nucleic acid chimera maintained, but did not enhance, inhibition of Tat-dependent in vitro transcription and cellular trans-activation in HeLa cells. The results demonstrate clearly that an OMe G-clamp has remarkable RNA-binding enhancement ability, but that oligonucleotide effectiveness in steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone.
Assuntos
Fármacos Anti-HIV/farmacologia , Produtos do Gene tat/antagonistas & inibidores , HIV-1 , Oligorribonucleotídeos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Produtos do Gene tat/genética , HIV-1/genética , Células HeLa , Humanos , Metilação , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Compostos Organofosforados/química , RNA Viral/química , RNA Viral/metabolismo , Ribonucleosídeos/síntese química , Ribonucleosídeos/química , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Mixmer oligonucleotides consisting of residues of both 2'-O-methylnucleosides (OMe) and locked nucleic acids (LNA) were designed targeting two stem-loops in the 5'-UTR of HIV-1 RNA, the transactivation response region (TAR), which is the site of binding of the Tat protein, and the SL3 loop, which is the primary packaging element that binds the Gag polyprotein. These oligonucleotides were found to inhibit syncitia formation dose- and sequence-dependently when delivered to HeLa T4 LTR beta-Gal cells and subsequently infected with HIV-1.
Assuntos
Fármacos Anti-HIV/farmacologia , Antivirais/farmacologia , Oligonucleotídeos/química , Oligonucleotídeos/farmacologia , RNA/química , Ativação Transcricional , Regiões 5' não Traduzidas , Sequência de Bases , Relação Dose-Resposta a Droga , Produtos do Gene gag/genética , Infecções por HIV/tratamento farmacológico , Repetição Terminal Longa de HIV , HIV-1/genética , HIV-1/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/síntese química , Elementos de Resposta , beta-Galactosidase/metabolismoRESUMO
We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO as cargo with both C- and N-termini unfunctionalized. The synthetic methods involve conjugation in solution phase, followed by rapid purification via biotin-streptavidin immobilization and subsequent reductive release into solution, avoiding the need for painstaking high-performance liquid chromatography purifications. The synthesis methods were applied for screening of PMO conjugates of a 16-member library of variants of a 10-residue ApoE peptide, which was suggested for blood-brain barrier crossing. In this work the conjugate library was tested in an exon skipping assay using skeletal mouse mdx cells, a model of Duchene's muscular dystrophy where higher activity peptide-PMO conjugates were identified compared with the starting peptide-PMO. The results demonstrate the power of the parallel synthesis methods for increasing the speed of optimization of peptide sequences in conjugates of PMO for therapeutic screening.
Assuntos
Peptídeos Penetradores de Células/síntese química , Morfolinos/síntese química , Distrofia Muscular de Duchenne/terapia , Sequência de Aminoácidos , Animais , Apolipoproteínas E/química , Linhagem Celular , Peptídeos Penetradores de Células/metabolismo , Química Click , Éxons , Terapia Genética , Humanos , Camundongos , Morfolinos/metabolismo , TransfecçãoRESUMO
Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.
Assuntos
Agamaglobulinemia/tratamento farmacológico , Doenças Genéticas Ligadas ao Cromossomo X/tratamento farmacológico , Morfolinos/administração & dosagem , Atrofia Muscular Espinal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Nanopartículas , Peptídeos/administração & dosagem , Sequência de Aminoácidos , Animais , Células Cultivadas , Modelos Animais de Doenças , Camundongos , Dados de Sequência Molecular , Morfolinos/uso terapêutico , Peptídeos/químicaRESUMO
Development of artificial nucleic acids for therapeutic applications warrants that the oligomers be endowed with high specificity, enzymatic stability and with no/reduced off-target effects. The balance between strength of the duplex with target RNA and enzyme stability is therefore the key factor for the designed modification. The chiral serinol derivative combines the attributes of amino- and methoxy- substitution when at 2'- position and at 3'- and 5'- ends, effectively balancing the duplex stability and resistance to hydrolytic enzymes. The biological effect seen is the remarkable improvement in splice correction by the steric blocking antisense oligonucleotide with just 4 modified units, i.e ~20% substitution with R-aminomethoxypropyloxy (R-AMP)-thymidine within the 2'-OMe 18mer sequence.
Assuntos
Materiais Biomiméticos/química , DNA/química , Propanolaminas/química , Propilenoglicóis/química , Splicing de RNA , RNA/química , RNA/genética , Sequência de Bases , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , DNA/genética , Células HeLa , Humanos , Serina/química , Estereoisomerismo , Uridina/análogos & derivados , Uridina/químicaRESUMO
The chemistry of the oligonucleotide backbone is crucial to obtaining high activity in vivo in exon skipping applications. Apart from the ability to bind strongly and sequence-specifically to pre-mRNA targets, the type of backbone also influences cell delivery, in vivo pharmacology, bio-distribution, toxicology, and ultimately the therapeutic use in humans. Reviewed here are classes of oligonucleotide commonly used for exon skipping applications, namely negatively charged backbones typified by RNA analogues having 2'-O-substitution and a phosphorothioate linkage and charge-neutral backbones such as PNA and PMO. Also discussed are peptide conjugates of PNA and PMO that enhance cellular and in vivo delivery and their potential for drug development. Finally, the prospects for development of other analogue types in exon skipping applications are outlined.
Assuntos
Éxons , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/genética , Processamento Alternativo , Animais , Humanos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , Peptídeos/química , Peptídeos/genética , Precursores de RNA/genéticaRESUMO
Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic AO delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart. We have previously reported impressive heart activity including high-splicing efficiency and dystrophin restoration following a single administration of an arginine-rich cell-penetrating peptide (CPPs) conjugated to a phosphorodiamidate morpholino oligonucleotide (PMO): Pip5e-PMO. However, the mechanisms underlying this activity are poorly understood. Here, we report studies involving single dose administration (12.5 mg/kg) of derivatives of Pip5e-PMO, consecutively assigned as Pip6-PMOs. These peptide-PMOs comprise alterations to the central hydrophobic core of the Pip5e peptide and illustrate that certain changes to the peptide sequence improves its activity; however, partial deletions within the hydrophobic core abolish its efficiency. Our data indicate that the hydrophobic core of the Pip sequences is critical for PMO delivery to the heart and that specific modifications to this region can enhance activity further. The results have implications for therapeutic PMO development for DMD.