Condensation of DNA for Gene Delivery: Studies on Vector-Nucleic Acid Interactions and Transfection Efficiency
Doktorsavhandling, 2010

This thesis concerns the delivery of nucleic acids into cells and tissues with the aim to change or correct gene expression. The field of gene delivery has the ultimate goal of producing genetic drugs for clinical applications, and the activity in this area is intense although so far the use is limited to research purposes. The main bottleneck is the lack of suitable delivery vectors. Viral vectors have undisputed efficiency but have shown to impose large safety risks, why non-viral methods are being increasingly explored. In this work, two classes of non-viral vectors, dendrimers and cell-penetrating peptides (CPPs), have been investigated for their interactions with DNA and their ability to mediate gene delivery into cultured cells with the aim of deducing relations between vector structure and biological activity. In addition, methods allowing biophysical characterization of vector-DNA complexes with focus on their dynamic nature have been developed. For dendrimeric vectors, the effect of surface-modifications aiming to increase their biocompatibility was studied. The results showed that although these modifications render dendrimers completely non-toxic, they also abolish the ability to mediate transfection. By biophysical studies, this could be related to a decreased DNA binding capacity of the modified dendrimers, which highlights a previously largely neclected issue in the dendrimer field. CPPs are promising vectors given their inherent ability to stimulate uptake into mammalian cells and deliver macromolecular cargo. Here, peptides with varying content of arginines and lysines were examined and it was found that arginine residues enhance the uptake efficiency compared to lysines. Arginines also showed to promote more efficient condensation of DNA, and the arginine-enriched variant of the peptide was the only one that displayed successful gene delivery. However, arginines alone were not sufficient since other CPPs with equal numbers of arginines were non-active in this respect. Hydrophobic residues in the peptide were found to be equally important, with one possible mechanism being to provide stability upon interaction with negatively charged cell surface residues. In conclusion, the thesis contributes with biophysical insights into the complicated process of gene delivery and gives clues for future development of new and more efficient vectors.

dendrimer

endocytosis

uptake

gene delivery

optical spectroscopy

structure-activity relationship

DNA

cell-penetrating peptide

KC
Opponent: Professor Astrid Gräslund, Stockholms universitet

Författare

Kristina Fant

Chalmers, Kemi- och bioteknik, Fysikalisk kemi

Stimulated endocytosis in penetratin uptake: Effect of arginine and lysine

Biochemical and Biophysical Research Communications,; Vol. 371(2008)p. 621-625

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DNA condensation by PAMAM dendrimers: Self-assembly characteristics and effect on transcription

Biochemistry,; Vol. 47(2008)p. 1732-1740

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Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency

Molecular Pharmaceutics,; Vol. 7(2010)p. 1734-1746

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GENETISKA SJUKDOMAR ORSAKAS av fel i vår arvsmassa. Genom att föra in korrekta gener i sjuka celler kan dessa bli botade. Denna process kallas för genöverföring eller transfektion, och är den här avhandlingens fokus. Ännu finns ingen säker och effektiv metod för att utföra behandlingen på patienter, men tekniken är redan ett viktigt forskningsverktyg som har bidragit enormt till vår förståelse av olika geners funktion och reglering. På molekylär nivå består det vi kallar för gener av bitar av DNA. Eftersom dessa är stora och negativt laddade stängs de effektivt ute från celler. Därför behövs en vektor, en bärarmolekyl, som kan packa ihop DNA-molekylerna så att de kan ta sig in. Genom flera decennier av forskning vet man idag ganska mycket om vilka hinder som behöver övervinnas på vägen. Däremot är det fortfarande oklart hur vektorer ska designas för att klara av detta. I den här avhandlingen undersöks just hur vektorers kemiska struktur påverkar deras förmåga att hjälpa DNA in i celler. Dessutom har olika metoder utvecklats för att studera hur vektor/DNA-komplex förändras med tiden, till exempel då de kommer i närheten av en cells yta. Detta är viktigt för en detaljerad förståelse av hur genöverföring går till, vilket kan ge ledtrådar till var i processen dagens vektorer misslyckas och därmed hur de kan förbättras. Den forskning som presenteras här har bidragit med biofysikaliska insikter i processen, och är ett steg på vägen mot klinisk användning av genetiska läkemedel.

Genetic diseases are caused by errors in our gene pool. By transferring correct genes into diseased cells these can be cured. This process is called gene delivery or transfection, and is the topic of this thesis. So far, there is no safe, efficient method to perform the treatment in patients. However the technique is already an important research tool that has contributed greatly to our understanding of the function and regulation of various genes. At a molecular level, what we call "genes" consist of pieces of DNA. These are large and negatively charged so they are efficiently excluded from cells. Therefore, a vector (a carrier molecule) is needed that can compact the DNA molecules so that they can get inside. Through decades of research efforts we know today a great deal about the barriers that need to be overcome. However, it is still unclear how a vector should be designed to achieve this. This thesis studies how the chemical structure of vectors affects their ability to help DNA enter cells. In addition, methods have been developed to study how vector/DNA complexes change over time, for example as they get in vicinity of a cell surface. This is important for a detailed understanding of the gene delivery process, which in turn can give clues to at what step today's vectors fail and consequently how they can be improved. The research that is presented here contributes with biophysical insights in this process, and is one step on the road to clinical use of genetic drugs.

Ämneskategorier

Fysikalisk kemi

Läkemedelskemi

Kemi

ISBN

978-91-7385-435-1

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 3116

KC

Opponent: Professor Astrid Gräslund, Stockholms universitet