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

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 succinyl‐tetraethylenpentamine (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

Fine tuning of phosphorothioate inclusion in 2'-O-methyl oligonucleotides contributes to specific cell targeting for splice-switching modulation

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery

Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides

The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs

Delivery of oligonucleotide-based therapeutics : challenges and opportunities

Funding Information: This work was supported by funding from Cooperation of Science and Technology (COST) Action CA17103 (networking grant to V.A-G). V.A-G holds a Miguel Servet Fellowship from the ISCIII [grant reference CPII17/00004] that is part-funded by the European Regional Development Fund (ERDF/FEDER) and also acknowledges funding from Ikerbasque (Basque Foundation for Science). S.M.H is funded by the Medical Research Council and Muscular Dystrophy UK. A.A-R receives funding from amongst others the Duchenne Parent Project, Spieren voor Spieren, the Prinses Beatrix Spierfonds, Duchenne UK and through Horizon2020 project BIND. A.G and R.W.J.C are supported by several foundations including the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid, Stichting Blinden-Penning, Landelijke Stichting voor Blinden en Slechtzienden, Stichting Oogfonds Nederland, Stichting Macula Degeneratie Fonds, and Stichting Retina Nederland Fonds (who contributed through UitZicht 2015-31 and 2018-21), together with the Rotterdamse Stichting Blindenbelangen, Stichting Blindenhulp, Stichting tot Verbetering van het Lot der Blinden, Stichting voor Ooglijders, and Stichting Dowilvo; as well as the Foundation Fighting Blindness USA, grant no. PPA-0517-0717-RAD. R.A.M.B is supported by Hersenstichting Nederland Grant DR-2018-00253. G.G. is supported by Ministry of Research and Innovation in Romania/National Program 31N/2016/PN 16.22.02.05. S.A is supported by Project PTDC/BBB-BMD/6301/2014 (Funda??o para a Ci?ncia e a Tecnologia?MCTES, Portugal). L.R.D. is supported by Fundaci?n Ram?n Areces Grant XVII CN and Spanish Ministry of Science and Innovation (MICINN, grant PID2019-105344RB-I00). T.L is supported by Estonian Research Council grant PSG226. S.K is supported by the Friedrich-Baur-Stiftung. C.F is funded by The Danish Council for Independent Research, Technology and Production Sciences (grant number DFF-4184-00422). W.vRM is supported by ZonMw Programme Translational Research 2 [Project number 446002002], Campaign Team Huntington and AFM Telethon [Project number 20577]. S.E.B is supported by the H2020 projects B-SMART, Grant number 721058, and REFINE, Grant number 761104. A.T.G is supported by the Institut National de la sant? et la recherche m?dicale (INSERM) and the Association Monegasque contre les myopathies (AMM). L.E. is founded by the Association Monegasque contre les myopathies (AMM). Publisher Copyright: © 2021 The Authors. Published under the terms of the CC BY 4.0 licenseNucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.publishersversionPeer reviewe

PepFect 14, a novel cell-penetrating peptide for oligonucleotide delivery in solution and as solid formulation

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

Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo

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

mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells

mRNA vaccines have recently proved to be highly effective against SARS-CoV-2. Key to their success is the lipid-based nanoparticle (LNP), which enables efficient mRNA expression and endows the vaccine with adjuvant properties that drive potent antibody responses. Effective cancer vaccines require long-lived, qualitative CD8 T cell responses instead of antibody responses. Systemic vaccination appears to be the most effective route, but necessitates adaptation of LNP composition to deliver mRNA to antigen-presenting cells. Using a design-of-experiments methodology, we tailored mRNA-LNP compositions to achieve high-magnitude tumor-specific CD8 T cell responses within a single round of optimization. Optimized LNP compositions resulted in enhanced mRNA uptake by multiple splenic immune cell populations. Type I interferon and phagocytes were found to be essential for the T cell response. Surprisingly, we also discovered a yet unidentified role of B cells in stimulating the vaccine-elicited CD8 T cell response. Optimized LNPs displayed a similar, spleen-centered biodistribution profile in non-human primates and did not trigger histopathological changes in liver and spleen, warranting their further assessment in clinical studies. Taken together, our study clarifies the relationship between nanoparticle composition and their T cell stimulatory capacity and provides novel insights into the underlying mechanisms of effective mRNA-LNP-based antitumor immunotherapy

Nukleiinhapete transport rakku sisenevate peptiididega geeniekspressiooni moduleerimise eesmärgil

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Viimaste aastakümnete teaduslike saavutuste abil on loodud suur hulk erinevaid meetodeid, mis võimaldavad reguleerida geeniekspressiooni. Enamik geeniekspressiooni mõjutamiseks kasutatavaid molekule põhinevad nukleiinhapetel (NH-del) ja nende analoogidel, varieerudes lühikestest oligonukleotiididest (ON) kuni suurte plasmiidse DNA (pDNA) molekulideni. Oma füsikokeemilistest omadustest tingituna ei ole need molekulid ise võimelised bioloogilisi membraane läbima ning oma toimekohtadeni jõudmiseks vajavad nad transportvektorite abi. Rakku sisenevad peptiidid (RSP-d) on üks grupp mitteviiruslikke transportvektoreid, mis on leidnud laialdast kasutust alates nende avastamisest 1994. a. RSP-d on katioonsed ja/või amfipaatsed peptiidid, mis koosnevad 5–40 aminohappest ning on võimelised rakkudesse transportima erinevaid bioloogiliselt aktiivseid kargomolekule, nende hulgas pDNA-d, splaissingut korrigeerivaid ON-e (SKO) ja väikeseid interfereeruvaid RNA-sid (siRNA), seda nii in vitro kui in vivo tingimustes. Käesoleva töö eesmärk oli disainida ja iseloomustada uusi RSP-del põhinevaid vektoreid, mis moodustaksid mitte-kovalentselt nanopartikleid erinevate NH-del põhinevate molekulidega, seal hulgas pDNA-d geenide transportimiseks, SKO-sid splaissingu moduleerimiseks ning siRNA-sid geeniekpressiooni vaigistamiseks. Modifikatsioonidena lisasime RSP-dele stearhappe ja/või uudse endosomotroopse modifikatsiooni või muutsime RSP aminohappelist järjestust. Kokkuvõttes moodustavad kirjeldatud peptiidid mitteviiruslike transportvektorite platvormi erinevate NH-del ja nende analoogidel põhinevate molekulide transpordiks, mida saab kasutada geeniteraapia eesmärkidel ning geeniekpressiooni moduleerimisel, nii in vitro kui in vivo tingimustes.Scientific advances over the last couple of decades have witnessed the emergence of a wide variety of methods to manipulate gene expression. Nucleic acids and their analogues, ranging from shorter oligonucleotides to full-size plasmids, form the battery of molecules that can be utilized for gene expression modulation. Unfortunately, the physicochemical properties of these molecules impede their translocation across biological membranes, thus, in order to reach their active sites within cells, they require assistance in their intracellular transport. Cell-penetrating peptides (CPPs) are one class of non-viral delivery vectors, which have been shown to facilitate the delivery of various types of bioactive cargos, including plasmid DNA (pDNA), splice-correcting oligonucleotides (SCOs) and small interfering RNAs (siRNAs), both in vitro and in vivo. This thesis aims for the development and characterization of novel CPP-based vectors with improved delivery properties for nucleic acid-based molecules. That includes the delivery of pDNA for gene transfer, SCOs for splicing correction and siRNAs for gene silencing, by non-covalent nanoparticle formation approach, both in vitro and in vivo. We modify our CPPs by adding stearic acid and/or a novel endosomotropic modification or by making amino acid substitutions in the peptide backbone. Conclusively, these novel vectors show remarkable delivery potential for the delivery of gene modulating compounds both in vitro and in vivo

Transfection of infectious RNA and DNA/RNA layered vectors of semliki forest virus by the cell-penetrating peptide based reagent PepFect6.

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells