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Gene therapy tools: oligonucleotides and peptides
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)ORCID iD: 0000-0001-8813-1096
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases.

The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2016. , 66 p.
Keyword [en]
Gene therapy, oligonucleotide, peptide, RNA-cleaving deoxyribozyme, deoxyribozyme, DNAzyme, cell-penetrating peptide, CPP, enzyme, enzyme kinetics, kinetic assay, assay, telomerase, telomerase inhibitor, imetelstat, GRN163L
National Category
Biochemistry and Molecular Biology Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-132271ISBN: 978-91-7649-460-8OAI: oai:DiVA.org:su-132271DiVA: diva2:950969
Public defence
2016-09-30, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2016-09-07 Created: 2016-08-04 Last updated: 2016-08-24Bibliographically approved
List of papers
1. A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes
Open this publication in new window or tab >>A High-Throughput Kinetic Assay for RNA-Cleaving Deoxyribozymes
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 8, e0135984Article in journal (Refereed) Published
Abstract [en]

Determining kinetic constants is important in the field of RNA-cleaving deoxyribozymes (DNAzymes). Using todays conventional gel assays for DNAzyme assays is time-consuming and laborious. There have been previous attempts at producing new and improved assays; however these have drawbacks such as incompatibility with structured DNAzymes, enzyme or substrate modifications and increased cost. Here we present a new method for determining single-turnover kinetics of RNA-cleaving DNAzymes in real-time and in a high-throughput fashion. The assay is based on an intercalating fluorescent dye, PicoGreen, with high specificity for double-stranded DNA and heteroduplex DNA-RNA in this case formed between the DNAzyme and the target RNA. The fluorescence decreases as substrate is converted to product and is released from the enzyme. Using a Flexstation II multi-mode plate reader with built in liquid handling we could automate parts of the assay. This assay gives the possibility to determine single-turnover kinetics for up to 48 DNAzymes simultaneously. As the fluorescent probe is extrinsic there is no need for enzyme or substrate modifications, making this method less costly compared to other methods. The main novelty of this assay is the possibility of using full-length mRNA as the DNAzyme target.

National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-120911 (URN)10.1371/journal.pone.0135984 (DOI)000360069400083 ()
Available from: 2015-09-24 Created: 2015-09-18 Last updated: 2016-08-11Bibliographically approved
2. Quantitative Microplate Assay for Real-Time Nuclease Kinetics
Open this publication in new window or tab >>Quantitative Microplate Assay for Real-Time Nuclease Kinetics
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, e0154099Article in journal (Refereed) Published
Abstract [en]

Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies.

National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-130869 (URN)10.1371/journal.pone.0154099 (DOI)000374898500143 ()27101307 (PubMedID)
Available from: 2016-06-07 Created: 2016-06-07 Last updated: 2016-08-11Bibliographically approved
3. Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides
Open this publication in new window or tab >>Optimized luciferase assay for cell-penetrating peptide-mediated delivery of short oligonucleotides
2015 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 484, 136-142 p.Article in journal (Refereed) Published
Abstract [en]

An improved assay for screening for the intracellular delivery efficacy of short oligonucleotides using cell-penetrating peptides is suggested. This assay is an improvement over previous assays that use luciferase reporters for cell-penetrating peptides because it has been scaled up from a 24-well format to a 96-well format and no longer relies on a luciferin reagent that has been commercially sourced. In addition, the homemade luciferin reagent is useful in multiple cell lines and in different assays that rely on altering the expression of luciferase. To establish a new protocol, the composition of the luciferin reagent was optimized for both signal strength and longevity by multiple two-factorial experiments varying the concentrations of adenosine triphosphate, luciferin, coenzyme A, and dithiothreitol. In addition, the optimal conditions with respect to cell number and time of transfection for both short interfering RNA (siRNA) and splice-correcting oligonucleotides (SCOs) are established. Optimal transfection of siRNA and SCOs was achieved using the reverse transfection method where the oligonucleotide complexes are already present in the wells before the cells are plated. Z' scores were 0.73 for the siRNA assay and 0.71 for the SCO assay, indicating that both assays are suitable for high-throughput screening.

Keyword
Cell-penetrating peptide (CPP), Luciferase, Short interfering RNA (siRNA), Splice-correcting oligonucleotide (SCO), High-throughput screening (HTS)
National Category
Chemical Sciences Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-119526 (URN)10.1016/j.ab.2015.05.023 (DOI)000357967500023 ()
Available from: 2015-08-21 Created: 2015-08-17 Last updated: 2016-08-04Bibliographically approved
4. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides
Open this publication in new window or tab >>Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides
2015 (English)In: Nucleic Acid Therapeutics, ISSN 2159-3337, E-ISSN 2159-3345, Vol. 25, no 6, 306-310 p.Article in journal (Refereed) Published
Abstract [en]

At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

National Category
Biological Sciences Cell and Molecular Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-124718 (URN)10.1089/nat.2015.0558 (DOI)000365529900003 ()26479411 (PubMedID)
Available from: 2016-01-18 Created: 2016-01-04 Last updated: 2016-08-11Bibliographically approved

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