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Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

Järver P, Zaghloul EM, Arzumanov AA, Saleh AF, McClorey G, Hammond SM, Hällbrink M, Langel Ü, Smith CI, Wood MJ, Gait MJ, El Andaloussi S - Nucleic Acid Ther (2015)

Bottom Line: Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems.However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs.To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

View Article: PubMed Central - PubMed

Affiliation: 1 Medical Research Council , Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom .

ABSTRACT
Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

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Nanoparticle tracking analysis (NTA) and splice-correction efficiency of phosphorodiamidate morpholinos (PMOs) formulated by PepFect 6 (PF6) and PF14 shown in U2OS cells stably transfected with a luciferase reporter cloned into a mutated Bruton's tyrosine kinase (BTK) intron as an X-linked agammaglobulinemia (XLA) cell model. (A) NTA of PMO formulated by PF6 and PF14 at two different doses; low dose is the dose used in the cell transfections experiments, while high dose is 10 times more than the low one (1 and 10 μM respectively). (B) Splice-correction efficiency by PMOs formulated with PF6 at molar ratio (MR) 5 and PF14 at MR 2 and 3. The upper band represents the aberrantly spliced luciferase (luc) RNA, which includes a pseudoexon sequence, while the lower band is for the corrected form.
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f1: Nanoparticle tracking analysis (NTA) and splice-correction efficiency of phosphorodiamidate morpholinos (PMOs) formulated by PepFect 6 (PF6) and PF14 shown in U2OS cells stably transfected with a luciferase reporter cloned into a mutated Bruton's tyrosine kinase (BTK) intron as an X-linked agammaglobulinemia (XLA) cell model. (A) NTA of PMO formulated by PF6 and PF14 at two different doses; low dose is the dose used in the cell transfections experiments, while high dose is 10 times more than the low one (1 and 10 μM respectively). (B) Splice-correction efficiency by PMOs formulated with PF6 at molar ratio (MR) 5 and PF14 at MR 2 and 3. The upper band represents the aberrantly spliced luciferase (luc) RNA, which includes a pseudoexon sequence, while the lower band is for the corrected form.

Mentions: We have previously demonstrated that in addition to electrostatic interactions, the overall hydrophobicity of CPPs plays also an important role for complex formation [33,38]. Given the previous success of the TP10 derived peptides PF6 and PF14 to efficiently convey small interfering RNAs and SSOs into various refractory cells in vitro, we here wanted to evaluate whether such peptides could be exploited also for noncovalent delivery of PMOs. This would open a new avenue for PMO sequence screenings in vitro, since there is a current lack of efficacious systems for PMO delivery. As seen in Figure 1A, both peptides formed a homogenous population of particles with a mean size of around 100 nm. Interestingly, and in contrast to their behavior when used as delivery vehicles for anionic ONs, where particles start aggregating at higher concentrations, the size distribution of complexes does not significantly change by increasing the dose of the formulated ON (Fig. 1A).


Peptide nanoparticle delivery of charge-neutral splice-switching morpholino oligonucleotides.

Järver P, Zaghloul EM, Arzumanov AA, Saleh AF, McClorey G, Hammond SM, Hällbrink M, Langel Ü, Smith CI, Wood MJ, Gait MJ, El Andaloussi S - Nucleic Acid Ther (2015)

Nanoparticle tracking analysis (NTA) and splice-correction efficiency of phosphorodiamidate morpholinos (PMOs) formulated by PepFect 6 (PF6) and PF14 shown in U2OS cells stably transfected with a luciferase reporter cloned into a mutated Bruton's tyrosine kinase (BTK) intron as an X-linked agammaglobulinemia (XLA) cell model. (A) NTA of PMO formulated by PF6 and PF14 at two different doses; low dose is the dose used in the cell transfections experiments, while high dose is 10 times more than the low one (1 and 10 μM respectively). (B) Splice-correction efficiency by PMOs formulated with PF6 at molar ratio (MR) 5 and PF14 at MR 2 and 3. The upper band represents the aberrantly spliced luciferase (luc) RNA, which includes a pseudoexon sequence, while the lower band is for the corrected form.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4376484&req=5

f1: Nanoparticle tracking analysis (NTA) and splice-correction efficiency of phosphorodiamidate morpholinos (PMOs) formulated by PepFect 6 (PF6) and PF14 shown in U2OS cells stably transfected with a luciferase reporter cloned into a mutated Bruton's tyrosine kinase (BTK) intron as an X-linked agammaglobulinemia (XLA) cell model. (A) NTA of PMO formulated by PF6 and PF14 at two different doses; low dose is the dose used in the cell transfections experiments, while high dose is 10 times more than the low one (1 and 10 μM respectively). (B) Splice-correction efficiency by PMOs formulated with PF6 at molar ratio (MR) 5 and PF14 at MR 2 and 3. The upper band represents the aberrantly spliced luciferase (luc) RNA, which includes a pseudoexon sequence, while the lower band is for the corrected form.
Mentions: We have previously demonstrated that in addition to electrostatic interactions, the overall hydrophobicity of CPPs plays also an important role for complex formation [33,38]. Given the previous success of the TP10 derived peptides PF6 and PF14 to efficiently convey small interfering RNAs and SSOs into various refractory cells in vitro, we here wanted to evaluate whether such peptides could be exploited also for noncovalent delivery of PMOs. This would open a new avenue for PMO sequence screenings in vitro, since there is a current lack of efficacious systems for PMO delivery. As seen in Figure 1A, both peptides formed a homogenous population of particles with a mean size of around 100 nm. Interestingly, and in contrast to their behavior when used as delivery vehicles for anionic ONs, where particles start aggregating at higher concentrations, the size distribution of complexes does not significantly change by increasing the dose of the formulated ON (Fig. 1A).

Bottom Line: Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems.However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs.To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

View Article: PubMed Central - PubMed

Affiliation: 1 Medical Research Council , Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom .

ABSTRACT
Oligonucleotide analogs have provided novel therapeutics targeting various disorders. However, their poor cellular uptake remains a major obstacle for their clinical development. Negatively charged oligonucleotides, such as 2'-O-Methyl RNA and locked nucleic acids have in recent years been delivered successfully into cells through complex formation with cationic polymers, peptides, liposomes, or similar nanoparticle delivery systems. However, due to the lack of electrostatic interactions, this promising delivery method has been unsuccessful to date using charge-neutral oligonucleotide analogs. We show here that lipid-functionalized cell-penetrating peptides can be efficiently exploited for cellular transfection of the charge-neutral oligonucleotide analog phosphorodiamidate morpholino. The lipopeptides form complexes with splice-switching phosphorodiamidate morpholino oligonucleotide and can be delivered into clinically relevant cell lines that are otherwise difficult to transfect while retaining biological activity. To our knowledge, this is the first study to show delivery through complex formation of biologically active charge-neutral oligonucleotides by cationic peptides.

Show MeSH
Related in: MedlinePlus