How, why, what and where: Mechanisms behind CPP/cargo nanocomplexes
Date
2016-05-17
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Abstract
Mitmed haigused on põhjustatud vigadest geenides või geeni produkti töötlemisel rakus. Haigusseisundite leevendamiseks või haiguse raviks oleks võimalik rakendada geeniteraapiat. Geeniteraapia on nukleiinhappemolekulide (NH-de) rakendamine eesmärgiga vigast või puuduvat geeni asendada, vaigistada või korrigeerida geeni produkti töötlust rakus. NH-de ehk DNA ja RNA molekulide ja nende teisendite kasutamist piiravad NH-de omadused nagu suur negatiivsete laengute tihedus, suurus ja kättesaadavus ensüümidele, mille tõttu nad tavaliselt rakkudes ja kudedes kiirelt lagundatakse ning rakumembraane iseseisvalt ei läbi. Selleks, et NH-d oleks võimalik laialdasemalt rakendada, on vajalik turvalise ja efektiivse meetodi olemasolu, mis võimaldaks neid õigetesse kudedesse, rakkudesse ja rakusisestesse organellidesse transportida.
Lisaks viirustel põhinevatele meetoditele ja füüsikalistele meetoditele on üheks võimalikuks transpordiviisiks ka keemiliste meetodite kasutamine. Keemilised meetodid hõlmavad enda all nii rakkude keemilist mõjutamist, mis soodustaks NH-de rakku sisenemist kui ka keemiliste transportvektorite, mis koos NH-ga rakumembraane läbivad, kasutamist. Üheks suure potentsiaaliga keemiliseks vektoriks on rakku sisenevad peptiidid (RSP-d, cell-penetrating peptide, CPP – inglise k.). RSP-d on aminohapetest koosnevad, valkudest lühemad järjestused. Nende eriliseks omaduseks on see, et nad suudavad läbida rakumembraane ja läbi membraanide transportida ka molekule, mis on nendest kordades suuremad, kaasa arvatud NH-sid. RSP-d võivad ka ise avaldada bioloogilist efekti, aga RSP-dega on erinevatesse rakkudesse ja loom-mudelitesse viidud nii suuri DNA molekule, väikseid geenivaigistamist vahendavaid RNA molekule kui ka valke.
RSP-des olevate aminohapete positiivse laenguga kõrvalahelate olemasolu võimaldab NH-ga kokku segades moodustada nanopartikleid. Nanopartiklid on väiksed RSP-dest ja NH-test koosnevad osakesed. Doktoritöö eesmärgiks on selgitada neid füüsikalisi ja keemilisi parameetreid ja rakus toimuvaid protsesse, mis on seotud nanopartiklite transpordiga ning kohaletoimetatud NH-de bioloogilise aktiivsusega.
Many diseases are caused by defects in genetic information of a living organism. One approach to alleviate the disease state or cure it permanently is the use of gene therapy. Gene therapy is the harnessing of nucleic acid (NA) molecules to alter gene expression or express a gene lacking in the cell. In gene therapy, the limited delivery of therapeutic NAs is the major obstacle curbing their use in medicine. The clinical use of NAs is restricted due to their low uptake into cells, because of their high molecular weight, negative charge, hydrophilic nature and lack of efficient uptake pathway for NAs. In addition to these, NAs are prone to enzymatic degradation by enzymes in the serum, extracellular matrix and in the cells. In order to increase the NAs use for therapeutic and biotechnological approaches, several methods have been developed. Viral vectors are considered the most efficient type of delivery method, but there are several concerns connected to innate properties of viruses, that have led to the development of physical and chemical approaches for delivery. One of highly potential approaches is the use of cell-penetrating peptides (CPPs) - a class of chemical vectors, that are able to cross cell membranes and carry into the cell different types of cargoes, including NAs. CPPs are short amino acid sequences that usually contain positively charged amino acids in their sequence. When CPPs are mixed with NAs, they are able to form non-covalently associated nanoparticles. The physico-chemical properties of CPP/NA nanoparticles and how these are linked to interactions with cells, intracellular trafficking and final biological effect gained from delivered cargo is an important field of study that could give us the basis for further development of chemical delivery vectors. These vectors could be used for biotechnological approaches, such as protein production in cultured cells, and gene therapy approaches.
Many diseases are caused by defects in genetic information of a living organism. One approach to alleviate the disease state or cure it permanently is the use of gene therapy. Gene therapy is the harnessing of nucleic acid (NA) molecules to alter gene expression or express a gene lacking in the cell. In gene therapy, the limited delivery of therapeutic NAs is the major obstacle curbing their use in medicine. The clinical use of NAs is restricted due to their low uptake into cells, because of their high molecular weight, negative charge, hydrophilic nature and lack of efficient uptake pathway for NAs. In addition to these, NAs are prone to enzymatic degradation by enzymes in the serum, extracellular matrix and in the cells. In order to increase the NAs use for therapeutic and biotechnological approaches, several methods have been developed. Viral vectors are considered the most efficient type of delivery method, but there are several concerns connected to innate properties of viruses, that have led to the development of physical and chemical approaches for delivery. One of highly potential approaches is the use of cell-penetrating peptides (CPPs) - a class of chemical vectors, that are able to cross cell membranes and carry into the cell different types of cargoes, including NAs. CPPs are short amino acid sequences that usually contain positively charged amino acids in their sequence. When CPPs are mixed with NAs, they are able to form non-covalently associated nanoparticles. The physico-chemical properties of CPP/NA nanoparticles and how these are linked to interactions with cells, intracellular trafficking and final biological effect gained from delivered cargo is an important field of study that could give us the basis for further development of chemical delivery vectors. These vectors could be used for biotechnological approaches, such as protein production in cultured cells, and gene therapy approaches.
Description
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Keywords
rakku sisenevad peptiidid, nukleiinhapped, nanokompleksid, bioloogiline transport, plasmiidivektorid, geenivaigistus, cell-penetrating peptide, nucleic acids, nanocomplexes, biological transport, plasmid vectors, gene silencing