Thesis (doctoral)--Eberhard-Karls-Universität zu Tübingen, 2002.

Atypical gene expression has a major influence on the disease profile of several severe human disorders. Oligonucleotide (ON) based therapeutics has opened an avenue for compensating deviant protein expression by acting on biologically important nucleic acids, mainly RNAs. Antisense ONs (asONs) can be designed to target complementary specific RNA sequences and thereby to influence the corresponding protein synthesis. However, cellular uptake of ONs is poor and is, together with the target specificity of the asONs, the major limiting factor for the development of ON based therapeutics. In this thesis, the mechanisms of well-characterized cell-penetrating peptides (CPPs) are evaluated and CPPs are adapted for cellular ON-delivery. The functionality of ON derivatives in cells is investigated and by optimization of asONs, targeting pre-messenger RNA, high efficiency and specificity is achieved. The optimization of the asONs is based on sequence design and through the choice of nucleic acid analogue composition. It is concluded that asONs, partly composed of locked nucleic acids are attractive for splice-switching applications but these mixmers must be designed with limited number of locked nucleic acid monomers to avoid risk for off-target activity. A protocol allowing for convenient characterization of internalization routes for CPPs is established and utilized. A mechanistic study on cellular CPP uptake and translocation of associated ON cargo reveals the importance of the optimal combination of for example charge and hydrophobicity of CPPs for efficient cellular uptake. Formation of non-covalent CPP:ON complexes and successful cellular delivery is achieved with a stearylated version of the well-recognized CPP, transportan 10. The results illustrate that CPPs and ON derivatives have the potential to become winning allies in the competition to develop therapeutics regulating specific protein expression patterns involved in the disease profile of severe human disorders. / At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted.Peper 5: In press. / VINNOVA-SAMBIO Multidisciplinary BIO

Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.

No description available.

Zugl.: Frankfurt (Main), Universiẗat, Diss., 2008.

Univ., Diss., 2009--Marburg.

No description available.

No description available.

Two microarray designs were developed and produced to screen for multiple bovine pathogens commonly found in the cattle industry today. The first microarray was designed, built, and processed in-house using conventional material and equipment and targeted Pasteurella multocida, Manheimia haemolytica, Histophilus somni, and Arcanobacterium pyogenes. For each pathogen, 12 perfect-match oligonucleotide probes, which were also designed in-house, targeted different sections of the respective 16S ribosomal genes, and were coupled with 12 corresponding mismatched probes for background. These arrays were able to produce distinct hybridization patterns for each pathogen that were easily visible without the need for computer analysis. However, the need for PCR amplification of the 16S gene prior to hybridization motivated us to explore more efficient array options. The second designed microarray, a custom Affymetrix GeneChip, targeted Escherichia coli, Salmonella typhimurium, and Salmonella dublin in addition to the previously mentioned pathogens and was more successful in overall performance than the "in-house" arrays. In addition to the 16S gene, oligonucleotide probes targeted other genes (from 2 to >4500, depending on whether the genome was sequenced) that were unique to each pathogen. This array also differed from the "in-house" arrays in that mismatched probes were not designed. The different probe sets performed at different detection limits as P. multocida, A. pyogenes, S. typhimurium, and S. dublin were detected with as little as 250ng of hybridized genomic DNA (gDNA), while M. haemolytica, H. somni, and E. coli required as much as 1μg gDNA. These pathogens were also spiked into bovine tissue to simulate multiorgan infections in which they were individually detected with the microarray design.

Cell-penetrating peptides are a class of peptides which have achieved a lot of recognition due to their vector abilities. Since their discovery over a decade ago, there has been an uncertainty concerning the mechanism by which they are internalized into the cells. Early studies claimed the uptake to be receptor- and energy independent, whereas more recent studies have shifted the general view to a more endocytotic belief, without prior binding to a receptor. As an increasing amount of reports emerges claiming the uptake to be endocytic, there is still a discrepancy concerning which endocytic mechanism that is responsible for the internalization and how to exploit the endocytic machinery for improved delivery. The main aim of this thesis was to elucidate the internalization mechanism for a series of cell-penetrating peptides derived from naturally occurring proteins, such as the prion protein which is thought to be the infectious particle in prion disorders. Furthermore, applications in gene regulation and improvement of delivery efficacy by induction of endosomolysis were examined. The results obtained confirm the uptake of cell-penetrating peptides to be endocytic; however the internalization mechanism appears to be peptide dependent where macropinocytosis is the most widespread endocytic component responsible for the internalization. The results further demonstrate that the biological response can be increased manifold by the induction of endosomolysis, either by using lysosomotropic agents or peptides able to alter their secondary structure upon protonation with concomitant endosomolysis. Altogether the results prove that enhanced delivery using cell-penetrating peptides can be achieved by exploiting the intrinsic endocytic mechanisms involved in the translocation process.