A novel strategy for peptide-mediated cellular delivery and induction of endosomal escape
Date
2011-07-21
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Oligonukleotiididel põhinev geeni regulatsioon omab suurt potentsiaali geeni teraapias. Kuid kuna suured ja hüdrofiilsed nukleiinhapete polümeerid ei suuda läbida plasmamembraani, on vaja efektiivset ja mitte toksilist transportvektorit, et soodustada nende transporti rakku. Rakku sisenevad peptiidid (RSP-d) on grupp transportvektoreid, mida on laialdaselt kasutatud nukleiinhapete rakku viimiseks, k.a. plasmiidne DNA (pDNA) ja splaissingut korrigeerivad oligonukleotiidid (SKO), nii in vitro kui in vivo tingimustes kasutades kovalentset või mitte-kovalentset peptiid:oligonukleotiid kompleksi moodustamise strateegiat.
Käesoleva töö peamiseks eesmärgiks oli disainida ja sünteesida uued ja efektiivsed peptiidsed vektorid nukleiinhapete transpordiks rakku ning hinnata nende rakendusvõimalusi in vitro kui ka in vivo tingimustes, kasutades mitte kova¬lentset koinkubatsiooni strateegiat. Me kasutasime kahte metoodikat: geeni transfektsiooni plasmiidse DNAga ja splaissingu korrigeerimist oligonukleotiididega. Antud töös me esitleme mitmeid uusi ja võrreldes algse TP10ga täiustatud transpordi võimega RSPsid, mis saadi stearüülhappe (stearüleerimine) ja/või fosforüülrühma (fosforüleerimine) lisamisel või peptiidi peaahelas aminohappe asenduse tulemusena. Kõik nimetatud modifikatsioonid tehti ees¬märgiga, et parandada transporditavate molekulide rakku sisenemist ja et soodustada nende vabanemist rakusisestest endotsütootilistest vesiikulitest.
Esimeses ja teises töös me näitasime, et kimäärse ja amfipaatse TP10 ning mitteamfipaatse ja sünteetilise (RXR)4 peptiidi stearüülhappega N-terminaalse modifitseerimise tulemuseks on peptiid, mis on võimeline efektiivselt ja mitte toksiliselt nukleiinhappeid sihtkohta transportima. Stearüleeritud TP10 säilitas oma efektiivsuse isegi seerumi juuresolekul.
Kolmandas töös me esitleme keemiliselt modifitseeritud stearüül-TP10-l põhinevaid transpordivektoreid efektiivseks SKO rakku viimiseks, mis saadi asendades peptiidi peaahelas 8 positsioonil olev isoleutsiin treoniiniga ja modifitseerides vastavalt türosiini ja /või treoniini nende fosforüleeritud monomeeridega.
Kokkuvõttes, väike modifikatsioon peptiidi peaahelas võib oluliselt parandada RSP, kui transpotvektori omadusi ja soodustada ravimite efektiivset rakku viimist. Peptiid:oligonukleotiid komplekside vabanemine endosoomidest on siiani olnud peamine takistus molekulide sihtmärgini jõudmisel. Käeosolevas töös disainitud ja modifitseeritud RSP-d transportisid oligonukleotiide efektiiv¬selt rakku nii in vitro kui ka in vivo tingimustes soodustades transporditavate molekulide vabanemist rakusisestest endotsütootilistest vesiikulitest. Käesoleva töö tulemusena saadi mitmed perspektiivikad RSP-d, mida tulevikus on võimalik kasutada geeni teraapias.
Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for these large-size and hydrophilic nucleic acid polymers, consequently an efficient and non-toxic delivery vector is needed to promote their deliver into the cell. Cell-penetrating peptides are a group of delivery vectors that have been extensively exploited for intracellular delivery of nucleic acids, including plasmid DNA (pDNA) and splice-correction oligonucleotides in vitro and in vivo using covalent and non-covalent strategies for attaching the cargo. The main aims of this thesis were to design and to synthesize novel and effi¬cient peptide vectors for nucleic acids delivery and evaluate them for in vitro applications using non-covalent co-incubation strategy. We used two metho¬dologies: gene transfection with plasmid DNA and splice-correction assay with oligonucleotides. In this thesis we present several novel CPPs with improved delivery properties, compared with parent peptides, TP10 and (RXR)4, which were acquired by adding stearic acid (stearylation) and/or phosphoryl group (phosphorylation) modifications or by performing amino acid substitution in backbone of the peptide. All these modifications were done in order to facilitate better uptake of cargo into the cell and provide more efficient endosomal escape from intracellular compartments. In papers I–II, we showed that N-terminal modification with stearic acid of chimeric and amphipatic TP10 or non-amphipatic and synthetic (RXR)4 peptides resulted in efficient peptide vector for nucleic acids delivery in non-toxic fashion and in case of stearylated TP10 was preserved efficacy even in the presence of serum. In paper III, we presented chemically modified stearyl-TP10 based delivery vectors for efficient SCO delivery, which were obtained by replacement of Ile8 by threonine in the backbone of the peptide and by modifying of tyrosine and/or threonine by their phosphorylated monomers, respectively. In conclusion, minor modifications in peptide backbone can improve the delivery properties of CPPs in order to promote effective intracellular delivery of therapeutic molecule and we believe that our studies help to overcome the difficulty of endosomal escape providing highly potential and efficient CPPs for oligonucleotides delivery both in vitro and in vivo.
Oligonucleotide-based gene regulation has a high potential in gene therapy, but the plasma membrane is impermeable for these large-size and hydrophilic nucleic acid polymers, consequently an efficient and non-toxic delivery vector is needed to promote their deliver into the cell. Cell-penetrating peptides are a group of delivery vectors that have been extensively exploited for intracellular delivery of nucleic acids, including plasmid DNA (pDNA) and splice-correction oligonucleotides in vitro and in vivo using covalent and non-covalent strategies for attaching the cargo. The main aims of this thesis were to design and to synthesize novel and effi¬cient peptide vectors for nucleic acids delivery and evaluate them for in vitro applications using non-covalent co-incubation strategy. We used two metho¬dologies: gene transfection with plasmid DNA and splice-correction assay with oligonucleotides. In this thesis we present several novel CPPs with improved delivery properties, compared with parent peptides, TP10 and (RXR)4, which were acquired by adding stearic acid (stearylation) and/or phosphoryl group (phosphorylation) modifications or by performing amino acid substitution in backbone of the peptide. All these modifications were done in order to facilitate better uptake of cargo into the cell and provide more efficient endosomal escape from intracellular compartments. In papers I–II, we showed that N-terminal modification with stearic acid of chimeric and amphipatic TP10 or non-amphipatic and synthetic (RXR)4 peptides resulted in efficient peptide vector for nucleic acids delivery in non-toxic fashion and in case of stearylated TP10 was preserved efficacy even in the presence of serum. In paper III, we presented chemically modified stearyl-TP10 based delivery vectors for efficient SCO delivery, which were obtained by replacement of Ile8 by threonine in the backbone of the peptide and by modifying of tyrosine and/or threonine by their phosphorylated monomers, respectively. In conclusion, minor modifications in peptide backbone can improve the delivery properties of CPPs in order to promote effective intracellular delivery of therapeutic molecule and we believe that our studies help to overcome the difficulty of endosomal escape providing highly potential and efficient CPPs for oligonucleotides delivery both in vitro and in vivo.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Keywords
peptiidid, bioloogiline transport, oligonukleotiidid, peptides, biological transport, oligonucleotides