Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Splice-switching efficiency and specificity for oligonucleotides with locked nucleic acid monomers
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Show others and affiliations
2008 (English)In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 412, 307-313 p.Article in journal (Refereed) Published
Abstract [en]

The use of antisense oligonucleotides to modulate splicing patterns has gained increasing attention as a therapeutic platform and, hence, the mechanisms of splice-switching oligonucleotides are of interest. Cells expressing luciferase pre-mRNA interrupted by an aberrantly spliced beta-globin intron, HeLa pLuc705, were used to monitor the splice-switching activity of modified oligonucleotides by detection of the expression of functional luciferase. It was observed that phosphorothioate 2'-O-methyl RNA oligonucleotides containing locked nucleic acid monomers provide outstanding splice-switching activity. However, similar oligonucleotides with several mismatches do not impede splice-switching activity which indicates a risk for off-target effects. The splice-switching activity is abolished when mismatches are introduced at several positions with locked nucleic acid monomers suggesting that it is the locked nucleic acid monomers that give rise to low mismatch discrimination to target pre-mRNA. The results highlight the importance of rational sequence design to allow for high efficiency with simultaneous high mismatch discrimination for splice-switching oligonucleotides and suggest that splice-switching activity is tunable by utilizing locked nucleic acid monomers.

Place, publisher, year, edition, pages
2008. Vol. 412, 307-313 p.
Keyword [en]
locked nucleic acid (LNA), mismatch, RNA, splice-switching activity, splice-switching oligonucleotide
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:su:diva-13896DOI: 10.1042/BJ20080013ISI: 000256491900012OAI: oai:DiVA.org:su-13896DiVA: diva2:180416
Available from: 2008-05-16 Created: 2008-05-16 Last updated: 2015-04-21Bibliographically approved
In thesis
1. Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
Open this publication in new window or tab >>Specificity of antisense oligonucleotide derivatives and cellular delivery by cell-penetrating peptides
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2009. 64 p.
Keyword
cell-penetrating peptide, splice-switching oligonucleotide, oligonucleotide derivative
National Category
Neurosciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-31226 (URN)978-91-7155-950-0 (ISBN)
Public defence
2009-12-22, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Projects
VINNOVA-SAMBIO Multidisciplinary BIO
Note
At the time of doctoral defense, the following papers were unpublished and had s status as follows: Paper 4: Accepted. Peper 5: In press.Available from: 2009-11-30 Created: 2009-11-08 Last updated: 2015-04-21Bibliographically approved
2. Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates
Open this publication in new window or tab >>Cell-penetrating peptides, novel synthetic nucleic acids, and regulation of gene function: Reconnaissance for designing functional conjugates
2008 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Our genome operates by sending instructions, conveyed by mRNA, for the manufacture of proteins from chromosomal DNA in the nucleus of the cell to the protein synthesizing machinery in the cytoplasm. Alternative splicing is a natural process in which a single gene can encode multiple related proteins. During RNA splicing, introns are selectively removed resulting in alternatively spliced gene products. Alternatively spliced protein products can have very different biological effects, such that one protein isoform is disease-related while another isoform is desirable. Splice switching opens the door to new drug targets, and antisense oligonucleotides (asONs), designed to switch splicing, are effective drug candidates. Cellular uptake of oligonucleotides(ONs) is poor, therefore utilization of cell-penetrating peptides (CPPs), well recognized for intracellular cargo delivery, is a promising approach to overcome this essential issue. Most CPPs are internalized by endocytosis, although the mechanisms involved remain controversial.

Here, evaluation of CPP-mediated ON delivery over cellular membranes has been performed. A protocol that allows for convenient assessment of CPP-mediated cellular uptake and characterization of corresponding internalization routes is established. The protocol is based on both fluorometric uptake measurements and a functional splice-switching assay, which in itself is based on biological activity of conveyed ONs. Additionally, splice switching ONs (SSOs) have been optimized for high efficiency and specificity. Data suggest that SSO activity is improved for chimeric phosphorothioate SSOs containing locked nucleic acid (LNA) monomers. It is striking that the LNA monomers in such chimeric constructs give rise to low mismatch discrimination of target pre-mRNA, which highlight the necessity to optimize sequences to minimize risk for off-target effects.

The results are important for up-coming work aimed at developing compounds consisting of peptides and novel synthetic nucleic acids, making these entities winning allies in the competition to develop therapeutics regulating protein expression patterns.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 45 p.
Keyword
Cell-penetrating peptide, nucleic acids, alternative splicing, mismatch discrimination
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-7491 (URN)978-91-7155-580-9 (ISBN)
Presentation
(English)
Supervisors
Available from: 2008-04-14 Created: 2008-04-14 Last updated: 2015-04-21Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Guterstam, PeterLindgren, MariaJohansson, HenrikLangel, Ülo
By organisation
Department of Neurochemistry
In the same journal
Biochemical Journal
Neurosciences

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 92 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf