Delivery of nucleic acids by cell-penetrating peptides: application in modulation of gene expression
Date
2011-05-18
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Abstract
Viimaste aastakümnete teaduslike saavutuste abil on loodud suur hulk erinevaid meetodeid, mis võimaldavad reguleerida geeniekspressiooni. Enamik geeniekspressiooni mõjutamiseks kasutatavaid molekule põhinevad nukleiinhapetel (NH-del) ja nende analoogidel, varieerudes lühikestest oligonukleotiididest (ON) kuni suurte plasmiidse DNA (pDNA) molekulideni. Oma füsikokeemilistest omadustest tingituna ei ole need molekulid ise võimelised bioloogilisi membraane läbima ning oma toimekohtadeni jõudmiseks vajavad nad transportvektorite abi.
Rakku sisenevad peptiidid (RSP-d) on üks grupp mitteviiruslikke transportvektoreid, mis on leidnud laialdast kasutust alates nende avastamisest 1994. a. RSP-d on katioonsed ja/või amfipaatsed peptiidid, mis koosnevad 5–40 aminohappest ning on võimelised rakkudesse transportima erinevaid bioloogiliselt aktiivseid kargomolekule, nende hulgas pDNA-d, splaissingut korrigeerivaid ON-e (SKO) ja väikeseid interfereeruvaid RNA-sid (siRNA), seda nii in vitro kui in vivo tingimustes.
Käesoleva töö eesmärk oli disainida ja iseloomustada uusi RSP-del põhinevaid vektoreid, mis moodustaksid mitte-kovalentselt nanopartikleid erinevate NH-del põhinevate molekulidega, seal hulgas pDNA-d geenide transportimiseks, SKO-sid splaissingu moduleerimiseks ning siRNA-sid geeniekpressiooni vaigistamiseks. Modifikatsioonidena lisasime RSP-dele stearhappe ja/või uudse endosomotroopse modifikatsiooni või muutsime RSP aminohappelist järjestust. Kokkuvõttes moodustavad kirjeldatud peptiidid mitteviiruslike transportvektorite platvormi erinevate NH-del ja nende analoogidel põhinevate molekulide transpordiks, mida saab kasutada geeniteraapia eesmärkidel ning geeniekpressiooni moduleerimisel, nii in vitro kui in vivo tingimustes.
Scientific advances over the last couple of decades have witnessed the emergence of a wide variety of methods to manipulate gene expression. Nucleic acids and their analogues, ranging from shorter oligonucleotides to full-size plasmids, form the battery of molecules that can be utilized for gene expression modulation. Unfortunately, the physicochemical properties of these molecules impede their translocation across biological membranes, thus, in order to reach their active sites within cells, they require assistance in their intracellular transport. Cell-penetrating peptides (CPPs) are one class of non-viral delivery vectors, which have been shown to facilitate the delivery of various types of bioactive cargos, including plasmid DNA (pDNA), splice-correcting oligonucleotides (SCOs) and small interfering RNAs (siRNAs), both in vitro and in vivo. This thesis aims for the development and characterization of novel CPP-based vectors with improved delivery properties for nucleic acid-based molecules. That includes the delivery of pDNA for gene transfer, SCOs for splicing correction and siRNAs for gene silencing, by non-covalent nanoparticle formation approach, both in vitro and in vivo. We modify our CPPs by adding stearic acid and/or a novel endosomotropic modification or by making amino acid substitutions in the peptide backbone. Conclusively, these novel vectors show remarkable delivery potential for the delivery of gene modulating compounds both in vitro and in vivo.
Scientific advances over the last couple of decades have witnessed the emergence of a wide variety of methods to manipulate gene expression. Nucleic acids and their analogues, ranging from shorter oligonucleotides to full-size plasmids, form the battery of molecules that can be utilized for gene expression modulation. Unfortunately, the physicochemical properties of these molecules impede their translocation across biological membranes, thus, in order to reach their active sites within cells, they require assistance in their intracellular transport. Cell-penetrating peptides (CPPs) are one class of non-viral delivery vectors, which have been shown to facilitate the delivery of various types of bioactive cargos, including plasmid DNA (pDNA), splice-correcting oligonucleotides (SCOs) and small interfering RNAs (siRNAs), both in vitro and in vivo. This thesis aims for the development and characterization of novel CPP-based vectors with improved delivery properties for nucleic acid-based molecules. That includes the delivery of pDNA for gene transfer, SCOs for splicing correction and siRNAs for gene silencing, by non-covalent nanoparticle formation approach, both in vitro and in vivo. We modify our CPPs by adding stearic acid and/or a novel endosomotropic modification or by making amino acid substitutions in the peptide backbone. Conclusively, these novel vectors show remarkable delivery potential for the delivery of gene modulating compounds both in vitro and in vivo.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
dissertatsioonid, geeniteraapia, rakumembraan, bioloogiline transport, penetratsioon, peptiidid, nukleiinhapped, gene therapy, cell membrane, membrane transport, penetration, peptides, nucleic acids