Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity

Saher, Osama; Rocha, Cristina S. J.; Zaghloul, Eman M.; Wiklander, Oscar P. B.; Zamolo, Susanna; Heitz, Marc; Ezzat, Kariem; Gupta, Dhanu; Reymond, Jean‐Louis; Zain, Rula; Hollfelder, Florian; Darbre, Tamis; Lundin, Karin E.; Andaloussi, Samir El; Cm, Smith

doi:10.1016/j.ejpb.2018.09.002

Cited by 20 publications

(34 citation statements)

References 50 publications

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“…187.15-5LNA (5 -aGacTuaCcaCuuCc) was used for the BTK gene cell models [21]. The G2-RR peptide dendrimer ( Figure 1) used in the study was synthesized using solid phase synthesis while applying Fmoc/Boc protection strategy, as reported previously [17]. Lipofectin (a liposomal formulation of a 1:1 (w/w) ratio of DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and DOPE (dioleoyl phosphotidylethanolamine)), and Lipofectamine 2000 (L2000) were both purchased from Invitrogen (Stockholm, Sweden).…”

Section: Methodsmentioning

confidence: 99%

“…The G2-RR PDLO-complex formulation procedures, N/P ratio, and Lipofectin amounts were the same as described previously [17]. All formulations were prepared in HEPES buffered glucose (HEPES buffered glucose (HBG) buffer/20 mM HEPES, 5% glucose, pH = 7.4).…”

Section: On Transfection Of Reporter Cell Lines Under Serum Conditionsmentioning

confidence: 99%

“…Although transfection under serum conditions is closer to the in vivo situation, serum has been often reported to negatively affect the transfection efficacy by various mechanisms [18]. Interestingly, the transfection efficiency of the G2-RR PDLO-complex under serum conditions was enhanced upon the addition of sucrose to the formulations [17]. protocol.…”

Section: Introductionmentioning

confidence: 99%

“…We previously described the synthesis and testing of 20 different peptide dendrimer structures for their ability to deliver splice-switching ONs. The ternary complexes of the Peptide Dendrimer/Lipid/ON were named after the initial of each component i.e., Peptide dendrimer/lipid/ON (PDLO)-complexes [17].…”

Section: Introductionmentioning

confidence: 99%

“…In this report, we investigated the serum associated transfection efficacy of PDLO-complexes consisting of the reported peptide dendrimer (G2-RR) with Lipofectin (as a lipid component) and the splice-switching ONs to be delivered [17]. To enhance the transfection efficacy under these conditions, we focused on screening 18 different excipients, consisting of sugars and polymers.…”

Section: Introductionmentioning

confidence: 99%

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Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations

et al. 2019

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Non-viral transfection vectors are commonly used for oligonucleotide (ON) delivery but face many challenges before reaching the desired compartments inside cells. With the support of additional compounds, it might be more feasible for a vector to endure the barriers and achieve efficient delivery. In this report, we screened 18 different excipients and evaluated their effect on the performance of peptide dendrimer/lipid vector to deliver single-stranded, splice-switching ONs under serum conditions. Transfection efficiency was monitored in four different reporter cell lines by measuring splice-switching activity on RNA and protein levels. All reporter cell lines used had a mutated human β-globin intron 2 sequence interrupting the luciferase gene, which led to an aberrant splicing of luciferase pre-mRNA and subsidence of luciferase protein translation. In the HeLa Luc/705 reporter cell line (a cervical cancer cell line), the lead excipients (Polyvinyl derivatives) potentiated the splice-switching activity up to 95-fold, compared to untreated cells with no detected cytotoxicity. Physical characterization revealed that lead excipients decreased the particle size and the zeta potential of the formulations. In vivo biodistribution studies emphasized the influence of formulations as well as the type of excipients on biodistribution profiles of the ON. Subsequently, we suggest that the highlighted impact of tested excipients would potentially assist in formulation development to deliver ON therapeutics in pre-clinical and clinical settings.

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Section: Methodsmentioning

confidence: 99%

Section: On Transfection Of Reporter Cell Lines Under Serum Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations

et al. 2019

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Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Kuhn

Klein

Danaf

et al. 2019

Adv Funct Materials

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Phosphorodiamidate morpholino oligomers (PMOs) are oligonucleotide analogs that can be used for therapeutic modulation of pre-mRNA splicing. Similar to other classes of nucleic acid-based therapeutics, PMOs require delivery systems for efficient transport to the intracellular target sites. Here, artificial peptides based on the oligo(ethylenamino) acid succinyltetraethylenpentamine (Stp), hydrophobic modifications, and an azide group are presented, which are used for strain-promoted azide-alkyne cycloaddition conjugation with splice-switching PMOs. By systematically varying the lead structure and formulation, it is determined that the type of contained fatty acid and supramolecular assembly have a critical impact on the delivery efficacy. A compound containing linolenic acid with three cis double bonds exhibits the highest splice-switching activity and significantly increases functional protein expression in pLuc/705 reporter cells in vitro and after local administration in vivo. Structural and mechanistic studies reveal that the lipopeptide PMO conjugates form nanoparticles, which accelerate cellular uptake and that the content of unsaturated fatty acids enhances endosomal escape. In an in vitro Duchenne muscular dystrophy exon skipping model using H2K-mdx52 dystrophic skeletal myotubes, the highly potent PMO conjugates mediate significant splice-switching at very low nanomolar concentrations. The presented aminoethylene-lipopeptides are thus a promising platform for the generation of PMO-therapeutics with a favorable activity/toxicity profile.

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Peptides as a material platform for gene delivery: Emerging concepts and converging technologies

2020

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Novel peptide-dendrimer/lipid/oligonucleotide ternary complexes for efficient cellular uptake and improved splice-switching activity

Cited by 20 publications

References 50 publications

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations

Sugar and Polymer Excipients Enhance Uptake and Splice-Switching Activity of Peptide-Dendrimer/Lipid/Oligonucleotide Formulations

Supramolecular Assembly of Aminoethylene‐Lipopeptide PMO Conjugates into RNA Splice‐Switching Nanomicelles

Peptides as a material platform for gene delivery: Emerging concepts and converging technologies

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