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Currently, messenger RNA (mRNA)-based lipid nanoparticle formulations revolutionize the clinical field. Cationic polymer-based complexes (polyplexes) represent an alternative compound class for mRNA delivery. After establishing branched polyethylenimine with a succinylation degree of 10% (succPEI) as highly effective positive mRNA transfection standard, a diverse library of PEI-like peptides termed sequence-defined oligoaminoamides (OAAs) was screened for mRNA delivery. Notably, sequences, which had previously been identified as potent plasmid DNA (pDNA) or small-interfering RNA (siRNA) carriers, displayed only moderate mRNA transfection activity. A second round of screening combined the cationizable building block succinoyl tetraethylene pentamine and histidines for endosomal buffering, tyrosine tripeptides and various fatty acids for mRNA polyplex stabilization, as well as redox-sensitive units for programmed intracellular release. For the tested OAA carriers, balancing of extracellular stability, endosomal lytic activity, and intracellular release capability was found to be of utmost importance for optimum mRNA transfection efficiency. OAAs with T-shape topology containing two oleic acids as well-stabilizing fatty acids, attached via a dynamic bioreducible building block, displayed superior activity with up to 1000-fold increased transfection efficiency compared to their non-reducible analogs. In the absence of the dynamic linkage, incorporation of shorter less stabilizing fatty acids could only partly compensate for mRNA delivery. Highest GFP expression and the largest fraction of transfected cells (96%) could be detected for the bioreducible OAA with incorporated histidines and a dioleoyl motif, outperforming all other tested carriers as well as the positive control succPEI. The good in vitro performance of the dynamic lead structure was verified in vivo upon intratracheal administration of mRNA complexes in mice.

When optimizing nanocarriers, structural motifs that are beneficial for the respective type of cargo need to be identified. Here, succinoyl tetraethylene pentamine (Stp)-based lipo-oligoaminoamides (OAAs) were optimized for the delivery of plasmid DNA (pDNA). Structural variations comprised saturated fatty acids with chain lengths between C2 and C18 and terminal cysteines as units promoting nanoparticle stabilization, histidines for endosomal buffering, and disulfide building blocks for redox-sensitive release. Biophysical and tumor cell culture screening established clear-cut relationships between lipo-OAAs and characteristics of the formed pDNA complexes. Based on the optimized alternating Stp-histidine backbones, lipo-OAAs containing fatty acids with chain lengths around C6 to C10 displayed maximum gene transfer with around 500-fold higher gene expression than that of C18 lipo-OAA analogues. Promising lipo-OAAs, however, showed only moderate in vivo efficiency. In vitro testing in 90% full serum, revealing considerable inhibition of lytic and gene-transfer activity, was found as a new screening model predictive for intravenous applications in vivo.

The programmable endonuclease activity and simple usage of CRISPR/Cas9 have revolutionized the field of genome editing. The binding of single guide RNA (sgRNA) by the Cas9 protein results in the formation of negatively charged ribonucleoprotein (RNP) complexes. The presence of this functional complex inside cells is imperative for the intended specific genome modifications. The direct intracellular delivery of Cas9/sgRNA RNP complexes is of great advantage. In this work, a compound library of sequence-defined oligo(ethylenamino) amides containing structural motifs for stable nanoparticle formation, cellular uptake, and endosomal release was used for the screening and development of suitable Cas9 RNP delivery vehicles. Lipid-containing oligoaminoamides (lipo-OAAs) were identified as the most efficient carriers for intracellular Cas9/sgRNA delivery and gene disruption. Fluorescence correlation spectroscopy measurements indicated that the lipo-OAAs only interact with sgRNA-loaded Cas9 protein, which suggests exclusive ionic interaction with the negatively charged RNPs. The type of contained fatty acid turned out to have a critical impact on the knock out efficiency: the presence of one hydroxy group in the fatty acid dramatically changes the properties and performance of the resulting Cas9/sgRNA lipo-OAA complexes. The lipo-OAA-containing hydroxy-stearic acid (OHSteA) was superior to the analogues with saturated or unsaturated fatty acids without hydroxylation; it formed smaller and more defined nanoparticles with Cas9/sgRNA and improved the cellular uptake and endosomal release, which altogether resulted in an increased nuclear association and the highest gene knock out levels. The efficient and adaptable delivery platform has high potential for the future development of therapeutics based on precise genome modifications.

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g 123 I, biological half-life 3 hours) by 123 I-scintigraphy. Therapy studies with three cycles of polyplexes and 131 I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

We report novel pH-reversibly surface-shielded polyplexes with enhanced gene transfer activity upon systemic administration. A four-arm-structured sequence-defined cationic oligomer KK[HK[(H-Sph-K)3HC]2]2 was designed and synthesized on solid-phase, containing additional lysine residues not only for improved pDNA polyplex stability, but also providing attachment points for subsequent polyplex functionalization with amine-reactive shielding polymers. Herein, the surface of polyplexes was shielded with hydrophilic polymers, monovalent PEG or monovalent and multivalent pHPMA, optionally attached to the polyplex via the acid-labile linker AzMMMan. Overall, surface modification with PEG or pHPMA resulted in a decrease in the zeta potential of polyplexes, consistent with the degree of surface shielding. At pH 6.0, only polyplexes modified via the acid-labile linkage showed an increase in zeta potential, consistent with a "deshielding" in acidic environment, expected as beneficial for endosomal escape. Shielding was more efficient for multivalent pHPMA (20kDa, 30kDa) as compared to monovalent pHPMA (10kDa, 20kDa, 30kDa) or PEG (5kDa). In vitro transfection studies revealed higher gene expression by the polyplexes with the acid-labile shield as compared to their irreversibly shielded counterparts. Intravenous administration of AzMMMan-pHPMA modified polyplexes in an in vivo tumor mouse model mediated enhanced gene expression in the subcutaneous tumor and reduced undesirable expression in the liver.

Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.

Due to its minimal size and lack of bacterial backbone sequences, minicircle (MC) DNA presents a promising alternative to plasmid DNA (pDNA) for non-viral gene delivery in terms of biosafety and improved gene transfer. Here, luciferase pDNA (pCMV-luc) and analogous MC DNA (MC07.CMV-luc) were formulated into polyplexes with c-Met targeted, PEG-shielded sequence-defined oligoaminoamides, or linear PEI (linPEI) as standard transfection agent. Distinct physicochemical and biological characteristics were observed for polyplexes formed with either pDNA or MC DNA as vectors. The carriers were found to dominate the shape of polyplexes, whereas the DNA type was decisive for the nanoparticle size. c-Met-targeted, tyrosine trimer-containing polyplexes were optimized into compacted rod structures with a size of 65-100 nm for pDNA and 35-40 nm for MC. Notably, these MC polyplexes display a lack of cell cycle dependence of transfection and a ∼200-fold enhanced gene transfer efficiency in c-Met-positive DU145 prostate carcinoma cultures over their tyrosine-free pDNA analogues.

Nucleic acid molecules are important therapeutic agents in the field of antisense oligonucleotide, RNA interference, and gene therapies. Since nucleic acids are not able to cross cell membranes and enter efficiently into cells on their own, the development of efficient, safe, and precise delivery systems is the crucial challenge for development of nucleic acid therapeutics. For the delivery of nucleic acids to their intracellular site of action, either the cytosol or the nucleus, several extracellular and intracellular barriers have to be overcome. Multifunctional carriers may handle the different special requirements of each barrier. The complexity of such macromolecules however poses a new hurdle in medical translation, which is the chemical production in reproducible and well-defined form. Solid-phase assisted synthesis (SPS) presents a solution for this challenge. The current review provides an overview on the design and SPS of precise sequence-defined synthetic carriers for nucleic acid cargos.

The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g 123 I, biological half-life 3 hours) by 123 I-scintigraphy. Therapy studies with three cycles of polyplexes and 131 I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

Cationic polymers present a versatile platform for the nonviral delivery of therapeutic nucleic acids. In order to achieve effective nucleic acid transfer, polymeric carriers ought to comprise multiple functionalities. Precise chemistries for site-specific placements of the different delivery modules within the carriers present the basis for uncovering structure-activity relationships required for further optimization. Here we present the design and systematic evaluation of a library of 42 sequence-defined oligo(ethanamino)amides generated by solid-phase assisted syntheses. The carriers contained two- or four-arm topologies of different artificial oligoamino acid domains for nucleic acid complexation, terminated by cysteines for disulfide-triggered polyplex stabilization, linked with monodisperse polyethylene glycol (PEG) for surface shielding and terminal folic acid for receptor specific cellular uptake. Additional functional elements included histidines for endosomal escape and/or tyrosine trimers for enhanced hydrophobic polyplex stabilization. In vitro screening of the oligomer library identified a folate-PEG-linked two-arm oligocation structure comprising histidines and tyrosine trimers as the most effective class of carriers for the delivery of pDNA and siRNA.

The synthesis of precise gene delivery vehicles by solid-supported chemistry is an effective way to establish structure–activity relationships and optimize existing transfection carriers. Sequence-defined cationic oligomers with different topologies were modified with twin disulfide-forming cysteine–arginine–cysteine (CRC) motifs. The influence of this motif versus single disulfide on the biophysical properties and biological performance of polyplexes was investigated, with pDNA and siRNA as nucleic acid cargoes. Clear differences between structures with isolated cysteines and CRC motifs were observed with respect to properties like nucleic acid binding, serum stability, response to reducing agents, and gene transfer/silencing. The main observed effect of the CRC motif was to increase polyplex stability. The consequences for nucleic acid delivery were less predictable and depended on oligomer topology. For some oligomers intrinsically forming stable polyplexes (i.e., already in the absence of CRC motif), this further stabilization resulted in a reduction or even loss in transfection efficiency. For PEGylated and targeted oligomers with intrinsically less stable polyplex structures, this modification led to a significant enhancement in transfection efficiency.

The delivery of nucleic acids represents a therapeutic approach for the precise adjustment of cellular dysregulations on the genetic level. However, safe and efficient usage of therapeutic nucleic acids remains a challenging task since several barriers have to be overcome within the delivery pathway. Despite a lower efficiency compared to viral vectors, nonviral transfer systems also offer several advantages such as high control over structural design and properties, flexibility regarding cargo type and capacity and scalable manufacturing technologies. Polymers with protonatable amines are widely used as synthetic delivery systems since these compounds can fulfill several prerequisites such as nucleic acid binding, cellular uptake and endosomal release, as a result of their basic characteristics. To facilitate the individual delivery steps efficiently, sequential protonation in different pH ranges has to be provided by the transfecting agents. Herein solid-phase supported synthesis was used to set up a library of structurally related oligomers containing different oligoamino acid building blocks1xSolid-phase synthesis of sequence-defined T-, i-, and U-shape polymers for pDNA and siRNA delivery. Schaffert, D., Troiber, C., Salcher, E.E., Frohlich, T., Martin, I., Badgujar, N., Dohmen, C., Edinger, D., Klager, R., Maiwald, G., Farkasova, K., Seeber, S., Jahn-Hofmann, K., Hadwiger, P., and Wagner, E. Angew. Chem., Int. Ed. Engl. 2011; 50: 8986–8989Crossref | PubMed | Scopus (95)See all References and basic α-amino acids such as histidine and pyridylalanine. The impact of individual protonation characteristics on different stages of gene transfer, such as pDNA binding potency, endosomal buffering and mediation of transgene expression, was evaluated to identify favourable pKa ranges and an optimal balance between high basicity for stable nucleic acid binding and residual buffer capacity for endosomal escape. Consistent with previous observations2xFine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes. Lachelt, U., Kos, P., Mickler, F.M., Herrmann, A., Salcher, E.E., Rodl, W., Badgujar, N., Brauchle, C., and Wagner, E. Nanomedicine (N. Y., NY, U. S.). 2014; 10: 35–44Abstract | Full Text | Full Text PDF | PubMed | Scopus (63)See all References the length of continuous diaminoethane motifs in oligoamino acid building blocks vastly determined the protonation characteristics having impact on the separate gene delivery steps. Oligoamine segments with only two protonatable amines exhibited low pDNA complexation capability but high endosomal buffering, whereas segments with three secondary amines provided good complexation but low buffer capacity. Since both parameters are important requirements, oligomers exclusively containing these building blocks as protonatable elements did not induce high transgene expression. Different basicities have to be combined and balanced in order to address all distinct barriers within the delivery pathway individually. Consequently, the combination of a potent pDNA binder with additional elements for endosomal buffering greatly enhanced the final transgene expression. Surprisingly, the exact location of maximal buffer capacity within the endosomal pH range seemed to be critical for successful gene transfer. We suggest that endosomal escape of pDNA polyplexes via the hypothesized proton-sponge effect is favoured only in a narrow window of the endosomal pH range.References

Electrochemotherapy is now well established ablative local tumor treatment for veterinary as well as human cancer. Electrochemotherapy combines the use of permeabilizing electric pulses applied directly at the tumor site with the injection of chemotherapeutic drugs, cisplatin or bleomycin. Increased membrane permeability enables enhanced entry of chemotherapeutic drug into the cells and resulted in higher tumor cell kill. Up to 80% complete responses are obtained after single electrochemotherapy treatment of cutaneous tumors of different histology (Yarmush et al. Annu Rev Biomed Eng. 2014; 16:295-320). To add a systemic component to this very effective local treatment, in preclinical studies it was combined with several different immune therapies, including gene therapy with plasmid DNA encoding interleukin-12 (IL-12) (Radiol Oncol. 2012;46:302-11). In our on-going clinical research, the effect of IL-12 gene therapy combined with electrochemotherapy was evaluated in 18 client- owned dogs with mastocytomas. Written consent for participation was obtained from the owners and the study was approved by the competent authorities (Veterinary administration and Administration for biotechnology of the Ministry of agriculture and environment; Republic Ethical Committee for the experiments involving animals). Electrochemotherapy using intratumoral injection of bleomycin and application of electric pulses was preformed prior to gene therapy. Plasmid encoding IL-12 was injected peritumorally into the skin and immediately thereafter electric pulses were applied. Local response of tumors was evaluated by measurements of tumors’ size. Systemic response was assessed by determination of IL-12 and IFN-γ in patients’ sera. Safety of gene therapy was evaluated by qRT-PCR measurement for the presence of plasmid DNA at the site of application and possible horizontal gene transfer by in vitro transformation of plasmid DNA encoding IL-12 into the bacteria isolated from the dog's (patient’s) skin. One month after the therapy, 11 dogs (61%) had a complete response, 4 (22%) partial response, and 3 (16%) stable disease. At the end of observation period (median 25 months) 16 dogs (89%) had complete response and 2 had to be euthanized due to the progression of disease. In all dogs, except two, serum IFN-γ or IL-12 levels were detected, without systemic toxicity. At the site of DNA plasmid application, except for one patient, the plasmid was not detected at 1 week post-treatment. No horizontal gene transfer was detected; plasmid was not detected in any of the residential bacteria and in vitro transformation of plasmid into isolated strains was negative. Collectively, the results of our on-going clinical trial demonstrate that IL-12 gene therapy combined with electrochemotherapy is safe, feasible and effective treatment for canine mastocytoma. Citation Format: Maja Cemazar, Jerneja Ambrozic Avgustin, Gregor Sersa, Darja Pavlin, Ana Krhac Levacic, Natasa Tesic, Mitja Rak, Ursa Lampreht, Natasa Tozon. Interleukin-12 gene therapy combined with local ablative technique electrochemotherapy for treatment of canine mastocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 292. doi:10.1158/1538-7445.AM2015-292