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CELL PENETRATING PEPTIDES: CHEMICAL MODIFICATION AND FORMULATION DEVELOPMENT
Stockholm University, Faculty of Science, Department of Neurochemistry. (Ülo Langel)
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: urn:nbn:se:su:diva-56364ISBN: 978-91-7447-216-5OAI: oai:DiVA.org:su-56364DiVA: diva2:410663
Presentation
2011-03-21, Heilbronnsalen, Svante Arrhenius väg 21A, Stockholm, 12:15 (English)
Opponent
Supervisors
Available from: 2011-04-14 Created: 2011-04-14 Last updated: 2015-04-21Bibliographically approved
List of papers
1. Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
Open this publication in new window or tab >>Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 9, 3972-3987 p.Article in journal (Refereed) Published
Abstract [en]

While small interfering RNAs (siRNAs) have been rapidly appreciated to silence genes, efficient and non-toxic vectors for primary cells and for systemic in vivo delivery are lacking. Several siRNA-delivery vehicles, including cell-penetrating peptides (CPPs), have been developed but their utility is often restricted by entrapment following endocytosis. Hence, developing CPPs that promote endosomal escape is a prerequisite for successful siRNA implementation. We here present a novel CPP, PepFect 6 (PF6), comprising the previously reported stearyl-TP10 peptide, having pH titratable trifluoromethylquinoline moieties covalently incorporated to facilitate endosomal release. Stable PF6/siRNA nanoparticles enter entire cell populations and rapidly promote endosomal escape, resulting in robust RNAi responses in various cell types (including primary cells), with minimal associated transcriptomic or proteomic changes. Furthermore, PF6-mediated delivery is independent of cell confluence and, in most cases, not significantly hampered by serum proteins. Finally, these nanoparticles promote strong RNAi responses in different organs following systemic delivery in mice without any associated toxicity. Strikingly, similar knockdown in liver is achieved by PF6/siRNA nanoparticles and siRNA injected by hydrodynamic infusion, a golden standard technique for liver transfection. These results imply that the peptide, in addition to having utility for RNAi screens in vitro, displays therapeutic potential.

National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56149 (URN)10.1093/nar/gkq1299 (DOI)000290589500046 ()21245043 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2015-04-21Bibliographically approved
2. PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
Open this publication in new window or tab >>PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 12, 5284-5298 p.Article in journal (Refereed) Published
Abstract [en]

Numerous human genetic diseases are caused by mutations that give rise to aberrant alternative splicing. Recently, several of these debilitating disorders have been shown to be amenable for splice-correcting oligonucleotides (SCOs) that modify splicing patterns and restore the phenotype in experimental models. However, translational approaches are required to transform SCOs into usable drug products. In this study, we present a new cell-penetrating peptide, PepFect14 (PF14), which efficiently delivers SCOs to different cell models including HeLa pLuc705 and mdx mouse myotubes; a cell culture model of Duchenne's muscular dystrophy (DMD). Non-covalent PF14-SCO nanocomplexes induce splice-correction at rates higher than the commercially available lipid-based vector Lipofectamine™ 2000 (LF2000) and remain active in the presence of serum. Furthermore, we demonstrate the feasibility of incorporating this delivery system into solid formulations that could be suitable for several therapeutic applications. Solid dispersion technique is utilized and the formed solid formulations are as active as the freshly prepared nanocomplexes in solution even when stored at an elevated temperatures for several weeks. In contrast, LF2000 drastically loses activity after being subjected to same procedure. This shows that using PF14 is a very promising translational approach for the delivery of SCOs in different pharmaceutical forms.

National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-56147 (URN)10.1093/nar/gkr072 (DOI)000292564900040 ()21345932 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2015-09-04Bibliographically approved
3. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
Open this publication in new window or tab >>A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo
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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.

National Category
Chemical Sciences
Identifiers
urn:nbn:se:su:diva-56148 (URN)10.1038/mt.2011.10 (DOI)000293378500010 ()21343913 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2015-03-27Bibliographically approved

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