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Despite the advances in gene therapy and in oligonucleotide (ON) chemistry, efficient cellular delivery remains an obstacle. Most current transfection reagents suffer from low efficacy or high cytotoxicity. In this report, we describe the synergism between lipid and dendrimer delivery vectors to enhance the transfection efficiency, while avoiding high toxicity. We screened a library of 20 peptide dendrimers representing three different generations and evaluated their capability to deliver a single-stranded splice-switching ON after formulating with lipids (DOTMA/DOPE). The transfection efficiency was analyzed in 5 reporter cell lines, in serum-free and serum conditions, and with 5 different formulation protocols. All formulations displayed low cytotoxicity to the majority of the tested cell lines. The complex sizes were < 200 nm; particle size distributions of effective mixtures were < 80 nm; and, the zeta potential was dependent on the formulation buffer used. The best dendrimer enhanced transfection in a HeLa reporter cell line by 30-fold compared to untreated cells under serum-free conditions. Interestingly, addition of sucrose to the formulation enabled - for the first time - peptide dendrimers/lipid complexes to efficiently deliver splice-switching ON in the presence of serum, reaching 40-fold increase in splice switching. Finally, in vivo studies highlighted the potential of these formulae to change the biodistribution pattern to be more towards the liver (90% of injected dose) compared to the kidneys (5% of injected dose) or to unformulated ON. This success encourages further development of peptide dendrimer complexes active in serum and future investigation of mechanisms behind the influence of additives on transfection efficacy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)(4) peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)(4) peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine 2000. We show that stearyl-(RxR)(4) mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)(4) or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine 2000, we show that stearyl-(RxR)(4) is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)(4) complexed with 2'-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)(4) promotes dose-dependent splice correction in parity with (RxR)(4)-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)(4) an intriguing vector for future in vivo experiments.

Peptides, polypeptides and proteins have been extensively studied for their various structural and functional roles in living organisms. However, breakthrough discoveries in the last decades identified some peptide-based matrices that posses the ability to traverse biological membranes, and many peptides, polypeptides and even complete proteins have been shown to have such properties. Hence, these matrices have been successfully used for the intracellular delivery of many therapeutic cargos including small molecules, proteins, peptides, oligonucleutides, plasmids and nanoparticles both in vitro and in vivo. Being neither toxic nor carcinogenic and meanwhile efficient in delivery, they are recognized as very promising vectors to overcome the shortcomings of the available technologies. The characteristics of these peptide-based matrices and their applications in drug delivery are here briefly illustrated together with current challenges and future prospects.

Background: In amyloid-positive individuals at risk for Alzheimer's disease (AD), high soluble 42-amino acid amyloid-β (Aβ42) levels are associated with normal cognition. It is unknown if this relationship applies longitudinally in a genetic cohort.Objective: To test the hypothesis that high Aβ42 preserves normal cognition in amyloid-positive individuals with Alzheimer's disease (AD)-causing mutations (APP, PSEN1, or PSEN2) to a greater extent than lower levels of brain amyloid, cerebrospinal fluid (CSF) phosphorylated tau (p-tau), or total tau (t-tau).Methods: Cognitive progression was defined as any increase in Clinical Dementia Rating (CDR = 0, normal cognition; 0.5, very mild dementia; 1, mild dementia) over 3 years. Amyloid-positivity was defined as a standard uptake value ratio (SUVR) ≥1.42 by Pittsburgh compound-B positron emission tomography (PiB-PET). We used modified Poisson regression models to estimate relative risk (RR), adjusted for age at onset, sex, education, APOE4 status, and duration of follow-up. The results were confirmed with multiple sensitivity analyses, including Cox regression.Results: Of 232 mutation carriers, 108 were PiB-PET-positive at baseline, with 43 (39.8%) meeting criteria for progression after 3.3±2.0 years. Soluble Aβ42 levels were higher among CDR non-progressors than CDR progressors. Higher Aβ42 predicted a lower risk of progression (adjusted RR, 0.36; 95% confidence interval [CI], 0.19-0.67; p = 0.002) better than lower SUVR (RR, 0.81; 95% CI, 0.68-0.96; p = 0.018). CSF Aβ42 levels predicting lower risk of progression increased with higher SUVR levels.Conclusion: High CSF Aβ42 levels predict normal cognition in amyloid-positive individuals with AD-causing genetic mutations. [ABSTRACT FROM AUTHOR]

Nucleic acids and their synthetic oligonucleotide (ON) analogs are a group of gene therapeutic compounds which hold enormous clinical potential. Despite their undoubted potential, clinical translation of these molecules, however, has been largely held back by their limited bioavailability in the target tissues/cells. To overcome this, many different drug delivery systems have been devised. Among others, short delivery peptides, called cell-penetrating peptides (CPPs), have been demonstrated to allow for efficient delivery of nucleic acids and their ON analogs, in both cell culture and animal models. In this review, we provide brief overview of the latest advances in nucleic acid delivery with CPPs, covering the two main vectorization strategies, covalent conjugation and nanoparticle formation-based approach. In conclusion, CPP-based drug delivery systems have the capacity to overcome the hurdle of delivery and thus have the potential to facilitate the clinical translation of nucleic acid-based therapeutics. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]