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PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation
Stockholm University, Faculty of Science, Department of Neurochemistry.
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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.

Place, publisher, year, edition, pages
2011. Vol. 39, no 12, 5284-5298 p.
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-56147DOI: 10.1093/nar/gkr072ISI: 000292564900040PubMedID: 21345932OAI: oai:DiVA.org:su-56147DiVA: diva2:409782
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)
Opponent
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)
Opponent
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
3. Rational design of novel cell-penetrating peptides
Open this publication in new window or tab >>Rational design of novel cell-penetrating peptides
2012 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Although cell-penetrating peptides (CPPs) have been an active field of research for over 20 years there are still properties such as efficiency and stability that must be improved for CPPs to reach their full potential as delivery vectors.

In this thesis two peptides were designed for non-covalent oligonucleotide delivery, in an attempt to overcome two major issues hampering wider use of CPPs, namely low stability and endosomal entrapment.

In Paper I we designed a new peptide named PepFect14, which has unnatural amino-acid ornithine as the source of positive charge, to address the stability issue. PepFect14 was shown to deliver SCOs in a highly efficient manner into different cell-lines. Furthermore, formulation of non-covalent PepFect14:SCO complexes into solid form, suitable for storage and transportation, was developed. The formulated complexes were stable for weeks and retained high activity.

In Paper II we modified PepFect14 with endosomolytic groups to facilitate endosomal escape, the resulting peptide was named PepFect15. Delivery of SCO and miRNA was efficiently mediated by PepFect15 into HeLa cells and endosomolytic property was validated. Additionally, uptake of Pepfect15:SCO complexes was shown to be mediated by scavenger receptor class A.

Place, publisher, year, edition, pages
Stockholm: Stockholm University, 2012
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-80474 (URN)978-91-7447-475-6 (ISBN)
Presentation
2012-03-30, Heilbronnsalen, Svante Arrhenius väg 21A, Stockholm, 13:30 (English)
Opponent
Supervisors
Available from: 2012-10-30 Created: 2012-09-20 Last updated: 2017-11-28Bibliographically approved
4. Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
Open this publication in new window or tab >>Cell-penetrating peptides for oligonucleotide delivery: Design and uptake mechanisms
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The use of oligonucleotides for gene therapy has the potential to efficiently treat a plethora of diseases with minimal side effects. However, the use of oligonucleotides is hampered by the properties of these molecules, which make it essentially impossible for them to permeate the cellular membrane. Therefore, a great deal of research has been focused on developing delivery vectors, which can efficiently and safely deliver oligonucleotides into cells. Cell-penetrating peptides (CPPs) constitute a class of delivery vectors that have received much attention since they were discovered over 20 years ago. CPPs can deliver a wide variety of cargos into cells, such as small molecules, proteins, oligonucleotides and particles, in an efficacious and non-toxic manner.

In this thesis two new CPPs for oligonucleotide delivery were designed. The purpose of the design was to create CPPs, which form stable complexes with oligonucleotides and have endosomolytic properties. The new peptides showed superior potency in intracellular oligonucleotide delivery compared to previously reported CPPs. These results demonstrate that it is possible to drastically improve the efficiency of existing CPPs with relatively simple modifications.

It is well known that CPPs use endocytosis to gain entry into cells, however, why cells endocytose CPPs has never been clearly established. In this thesis we determine that several CPP:oligonucleotide complexes interact with scavenger receptors, and that this interaction leads to endocytosis. The results presented in this thesis provides a deeper understanding of how CPPs function and thereby insights how to improve CPP design.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2014. 62 p.
Keyword
Cell-penetrating peptides, gene therapy, scavenger receptors
National Category
Chemical Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-108344 (URN)978-91-7649-023-5 (ISBN)
Public defence
2014-11-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-11-06 Created: 2014-10-21 Last updated: 2015-04-21Bibliographically approved

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