Development of cell-penetrating peptides for gene delivery: from transfection in cell cultures to induction of gene expression in vivo
Kuupäev
2016-05-13
Autorid
Ajakirja pealkiri
Ajakirja ISSN
Köite pealkiri
Kirjastaja
Abstrakt
Geeniteraapia on nukleiinhapetel põhinev ravistrateegia haiguste raviks, mis ilmnevad vigase geeniekspressiooni tõttu. Terapeutiline geen sisestatakse plasmiidsesse ekspressiooni vektorisse (pDNA), et saavutada rakku sisenema ning seetõttu on edukaks geeniteraapia rakendamiseks vajalik tema transport rakkudesse. Üheks selliseks transportvektorite klassiks on rakku sisenevad peptiidid (RSPd), mis on kuni 30 aminohapet pikad katioonsed ja/või amfipaatsed peptiidsed ühendid ning nad on võimelised rakkudesse viima väga erinevaid makromolekule, seal hulgas nukleiinhappeid.
Selle väitekirja eesmärk on rakukultuuris efektiivse RSP arendamine nukleiinhappe transportimiseks loomsesse haigusmudelisse. Selleks on esmalt iseloomustatud RSP vahendatud geenitransporti rakukultuuris, näiteks, geeni transpordi efektiivsus, ohutus ning rakkudesse sisenemismehhanism. Seejärel me hindasime RSP potentsiaali transportida geeni erinevatesse kudedesse pärast süsteemset manustamist. Kuigi saavutasime kõrged geeniekspressiooni tasemed erinevates kudedes, ilmnes vajadus täienduste järgi, seal hulgas spetsiifiline transport kasvaja koesse. Selle jaoks kasutasime erinevaid strateegiaid, seal hulgas polüetüleenglükooli (PEG) molekuli või spetsiifiliste peptiidjärjestuste konjugeerimine RSPle. PEG molekuli lisamine võimaldas suurendada RSP/pDNA komplekside biosobivust in vivo ning maskeerida geeni transporti erinevatesse kudedesse ja rakkudesse. Selleks, et aktiveerida geenitransport ainult kasvaja koes, konjugeeriti PEG molekul peptiidile läbi spetsiifilise järjestuse, mida on võimelised lõikama kasvajas üle-ekspresseeritud ensüümid ning mille tulemusena PEG eemaldatakse ning RSP viib pDNA kasvajakoes olevatesse rakkudesse, kus see ekspresseeritakse. Lisaks optimiseerisime ka RSP/pDNA komplekside formulatsiooni, et parandada geeni transporti ilma PEG molekuli lisamiseta. Me iseloomustasime peptiidi katioonse laengu ning rasvhappejäägi pikkuse olulisust RSP-vahendatud geenitranspordil in vivo tingimustes. Kokkuvõtteks, välja töötatud RSPdel põhinev geeni transportsüsteemi omab potentsiaali geeniteraapia läbiviimiseks olulistes haigusmudelites.
Gene therapy is considered to have great therapeutic potential for a wide variety of diseases that occur due to malfunctioning genes. To achieve therapeutic effects, genetic material needs to reach target cells, and thus must overcome complex intra and extracellular barriers. Because the properties of nucleic acids, such as their high molecular weight and negative net charge, prohibit translocation over cell membranes, the successful application of gene therapy relies on the development of gene delivery vehicles. Cell-penetrating peptides (CPPs) are one class of non-viral transport vectors that have been exploited to deliver nucleic acids into cells. CPPs can be up to 30 amino acids long, typically cationic and/or amphipathic, and can facilitate the delivery of large nucleic acid molecules such as plasmid DNA (pDNA) into cells. The main purpose of the research presented in this dissertation was to develop an efficient CPP in cell culture that is applicable for systemic gene delivery in vivo, and has potential to treat diseases caused by aberrant gene expression, such as cancer. First, we characterized various aspects of peptide based gene delivery, such as potential gene induction efficacy and the uptake mechanisms in cell culture. Next we evaluated the potential for CPP-mediated pDNA delivery after systemic administration in mice and found that improvements were required, including the need to achieve tumor specific gene delivery. For that, we evaluated various strategies such as the conjugation of either targeting peptides or polyethyleneglycol (PEG) molecules to the CPPs. The latter strategy improved the biocompatibility of CPP/pDNA complexes and permitted us to shield the universal transfection property of CPPs, which could be further activated specifically in tumor tissues and induce gene expression. We also optimized the complex formulation to improve their gene delivery properties without PEGylation and characterized other CPP properties such as cationic charge density and fatty acid modification. We found both to be important aspects that govern CPP-mediated gene delivery not only in cell culture, but also in vivo. In conclusion, the potential of CPP-based gene delivery system could be further extended for gene therapy applications in relevant disease models.
Gene therapy is considered to have great therapeutic potential for a wide variety of diseases that occur due to malfunctioning genes. To achieve therapeutic effects, genetic material needs to reach target cells, and thus must overcome complex intra and extracellular barriers. Because the properties of nucleic acids, such as their high molecular weight and negative net charge, prohibit translocation over cell membranes, the successful application of gene therapy relies on the development of gene delivery vehicles. Cell-penetrating peptides (CPPs) are one class of non-viral transport vectors that have been exploited to deliver nucleic acids into cells. CPPs can be up to 30 amino acids long, typically cationic and/or amphipathic, and can facilitate the delivery of large nucleic acid molecules such as plasmid DNA (pDNA) into cells. The main purpose of the research presented in this dissertation was to develop an efficient CPP in cell culture that is applicable for systemic gene delivery in vivo, and has potential to treat diseases caused by aberrant gene expression, such as cancer. First, we characterized various aspects of peptide based gene delivery, such as potential gene induction efficacy and the uptake mechanisms in cell culture. Next we evaluated the potential for CPP-mediated pDNA delivery after systemic administration in mice and found that improvements were required, including the need to achieve tumor specific gene delivery. For that, we evaluated various strategies such as the conjugation of either targeting peptides or polyethyleneglycol (PEG) molecules to the CPPs. The latter strategy improved the biocompatibility of CPP/pDNA complexes and permitted us to shield the universal transfection property of CPPs, which could be further activated specifically in tumor tissues and induce gene expression. We also optimized the complex formulation to improve their gene delivery properties without PEGylation and characterized other CPP properties such as cationic charge density and fatty acid modification. We found both to be important aspects that govern CPP-mediated gene delivery not only in cell culture, but also in vivo. In conclusion, the potential of CPP-based gene delivery system could be further extended for gene therapy applications in relevant disease models.
Kirjeldus
Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Märksõnad
rakku sisenevad peptiidid, nukleiinhapped, geenid, plasmiidivektorid, bioloogiline transport, transfektsioon, rakukultuurid, in vivo meetod, geeniteraapia, cell-penetrating peptides, nucleic acids, genes, plasmid vectors, biological transport, transfection, cell cultures, in vivo, gene therapy