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A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
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2011 (English)In: Molecular Therapy, ISSN 1525-0016, E-ISSN 1525-0024, Vol. 19, no 8, 1457-1467 p.Article in journal (Refereed) Published
Abstract [en]

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.

Place, publisher, year, edition, pages
2011. Vol. 19, no 8, 1457-1467 p.
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:su:diva-56148DOI: 10.1038/mt.2011.10ISI: 000293378500010PubMedID: 21343913OAI: oai:DiVA.org:su-56148DiVA: diva2:409783
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2017-12-11Bibliographically approved
In thesis
1. CELL PENETRATING PEPTIDES: CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT
Open this publication in new window or tab >>CELL PENETRATING PEPTIDES: CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT
2011 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Cell penetrating peptides (CPPs) have been extensively studied and exploited as drug delivery vectors for a wide variety of therapeu-tic cargos. However, several issues remain to be addressed regarding the enhancement of their efficiency and stability. In addition, to be available for patients, CPP-based therapeutics have to be formulated into suitable pharmaceutical forms that can be readily manufactured, transported, stored and conveniently used.In this thesis, three chemically modified CPPs are developed having superior delivery properties for several nucleic acid-based the-rapeutic cargoes including: plasmids, small interfering RNA (siRNA) and splice switching oligonucleutides (SSOs), in different in-vitro and in-vivo models. In Paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (TP10) can form stable nanopar-ticles with plasmids that efficiently transfect different cell types and can mediate efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, stearyl-TP10 is further modified with pH titratable trifluoromethylquinoline moieties to facilitate endosomal release. The new peptide, denoted PepFect 6 (PF6), elicited robust RNAi responses when complexed with siRNA in several cell models and promoted strong RNAi responses in differ-ent organs following systemic delivery in mice without any associated toxicity. In paper III , a new peptide with ornithine modification, PF14, is shown to efficiently deliver SSOs in different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne‟s muscular dystrophy (DMD). Additionally, we have developed a method for incorporating this delivery system into solid formulation that could be suitable for several therapeutic appli-cations. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocparticles in solution even when stored at elevated temperatures for several weeks.Taken together, these results demonstrate that certain chemical modifications could drastically enhance the activity and stability of CPPs in-vitro and in-vivo. Moreover, we show that CPP-based thera-peutics could be formulated into convenient and manufacturable do-sage forms.

Place, publisher, year, edition, pages
Stockholm: Universitetetsservice US-AB, 2011. 48 p.
National Category
Natural Sciences
Research subject
Neurochemistry and Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56364 (URN)978-91-7447-216-5 (ISBN)
Presentation
2011-03-21, Heilbronnsalen, Svante Arrhenius väg 21A, Stockholm, 12:15 (English)
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Supervisors
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2015-04-21Bibliographically approved
2. Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
Open this publication in new window or tab >>Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted.

Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University Stockholm University, 2012. 86 p.
Keyword
cell penetrating peptides, gene therapy, scavenger receptors, pharmaceutical formulation, solid dispersion
National Category
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-75537 (URN)978-91-7447-464-0 (ISBN)
Public defence
2012-06-11, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
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Supervisors
Note
At the time of doctoral defence the following paper was unpublished and had a status as follows: Paper nr 4: SubmittedAvailable from: 2012-05-10 Created: 2012-04-20 Last updated: 2012-04-24Bibliographically approved

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