Native chemical ligation for conversion of sequence-defined oligomers into targeted pDNA and siRNA carriers.

Native chemical ligation (NCL) was established for the conversion of sequence-defined oligomers of different topologies into targeted and PEG shielded pDNA and siRNA carriers. From an existing library of non-targeted oligoethanamino amides, six oligomers containing N-terminal cysteines were selected as cationic cores, to which monodisperse polyethylene glycol (PEG) containing terminal folic acid as targeting ligand (or terminal alanine as targeting negative control ligand) were attached by NCL. Ligated conjugates plus controls (in sum 18 oligomers) were evaluated for pDNA or siRNA gene delivery. Biophysical characteristics including nucleic acid binding in the absence or presence of serum, as well as biological activities in cellular uptake and gene transfer (or gene silencing, respectively) were determined. In most cases, the folic acid-PEG-ligated oligomers displayed a strongly improved cellular binding, uptake and gene transfer into receptor-positive KB cells as compared to the alanine-PEG controls. Changing the topological structures by increasing the number of cationic arms, adding tyrosine trimers as polyplex stabilizing domains, or histidines facilitating endosomal escape resulted in beneficial gene transfer characteristics. The screen revealed different requirements for pDNA and siRNA delivery. A folate-PEG ligated histidinylated four-arm oligomer was most effective for pDNA delivery but inactive for siRNA, whereas a folate-PEG-ligated three-arm oligomer with tyrosine trimer modifications was most effective in siRNA mediated gene silencing. The results demonstrate the site-selective NCL reaction as powerful method to modify existing oligomers. Thus multifunctional targeted carriers can be obtained with ease and used to identify lead structures for subsequent in vivo delivery.

Gene silencing and antitumoral effects of Eg5 or Ran siRNA oligoaminoamide polyplexes.

Two antitumoral siRNAs (directed against target genes Eg5 and Ran) complexed with one of three sequence-defined cationic oligomers were compared in gene silencing in vitro and antitumoral in vivo efficacy upon intratumoral injection. Two lipo-oligomers (T-shape 49, i-shape 229) and the three-arm oligomer 386 were chosen because of their high efficiency in previous marker gene silencing screens. The oligomers showed very similar target-specific gene knockdown in murine neuroblastoma cells. Silencing persisted only for a short period (maximum on day 1 at mRNA and day 2 at protein level) triggering siRNA specific in vitro tumor cell killing. The fastest onset of protein knockdown and strongest antitumoral effect was mediated by oligomer 386. Tumor growth reduction in vivo was evaluated in the subcutaneous Neuro2A mouse model. Intratumoral injections of either Eg5 or Ran siRNA/oligomer 49 polyplexes led to reduced tumor growth and prolonged survival of mice compared to control siRNA and buffer treatment. Target knockdown was evidenced in tumors by mitotic Aster formation for Eg5 knockdown and apoptotic TUNEL stain for Ran knockdown. Ran siRNA displayed better antitumoral efficacy and was chosen for in vivo comparison of the oligomers. A very clear order of antitumoral activity (oligomer 386 > 49 > 229) was observed. In summary, the similar in vitro gene silencing efficiencies on mRNA level by the tested oligomers did not correlate with the observed therapeutic effects in vivo. Oligomer 386 with the fastest onset of protein knockdown and best in vitro cell killing mediated the best in vivo antitumoral efficacy.

Formulation development of lyophilized, long-term stable siRNA/oligoaminoamide polyplexes.

Polyplexes based on precise oligoaminoamides exhibited promising results in non-viral siRNA delivery. However, one serious limitation is insufficient stability of polyplexes in liquid, which raises the demand for lyophilized, long-term stable formulations. Two different siRNA/oligoaminoamide polyplexes were prepared. Freeze-thaw experiments were performed, in order to test various formulations containing sucrose, trehalose, lactosucrose, and hydroxypropyl-ß-cyclodextrin for their cryoprotective potential and to investigate the influence of the oligoaminoamide structure on particle stability. Selected formulations were lyophilized and tested for storage stability up to 6 months. Moreover, reconstitution of the lyophilisates in reduced volume as a technique to prepare higher concentration formulations was studied. Samples were analyzed for particle size, gene silencing, cytotoxicity, turbidity, subvisible particles, osmolarity, residual moisture content, glass transition temperature, and morphology. Depending on the oligoaminoamide, siRNA polyplexes maintained particle size and gene silencing efficiency in the absence or presence of low amounts (7%) of stabilizers after freeze-thawing, lyophilization, and reconstitution. Particle stability was highly dependent on the oligoaminoamide used, but independent of the presence of cysteines that form intra-particular disulfide bridges. In contrast to all other excipients, hydroxypropyl-ß-cyclodextrin did not provide sufficient stability. For lyophilized 5%/10% sucrose and 7% lactosucrose formulations, long-term stability was demonstrated at 40 °C with retained particle size, retained gene silencing activity, unchanged turbidity values, low numbers of subvisible particles, low residual moisture level, and sufficiently high glass transition temperature. Hence, this work is a promising approach in order to provide long-term stable siRNA polyplex formulations that are ready to use after a simple reconstitution step.

Comparison of four different particle sizing methods for siRNA polyplex characterization.

The ability to reliably determine the size of siRNA polyplexes is the key for the rational design of particles and their formulation, as well as, their safe application in vivo. At the moment, no standard technique for size measurements is available. Each method has different underlying principles and hence may give different results. Here, four different analytical methods were evaluated for their suitability to analyze the characteristics of homogeneous and heterogeneous siRNA polyplexes: dynamic light scattering (DLS), atomic force microscopy (AFM), nanoparticle trafficking analysis (NTA), and fluorescence correlation spectroscopy (FCS). Three different siRNA polyplex compositions generated with different, precise, and hydrophobically modified oligoaminoamides were used in this study. All of the evaluated methods were suitable for analysis of medium sized, homogeneous siRNA polyplexes (~120 nm). Small particles (<40 nm) could not be tracked with NTA, but with the other three methods. Heterogeneous polyplexes were generally difficult to analyze. Only by visualization with AFM, the heterogeneity of those polyplexes was observable. FCS was the only method suitable for measuring polyplex stability in 90% fetal bovine serum. Physico-chemical characteristics of polyplexes are important quality criterions for successful in vivo application and future formulation development. Therefore, a comprehensive analysis by more than one method is of particular importance.

Stabilizing effect of tyrosine trimers on pDNA and siRNA polyplexes.

Nine sequence-defined, polycationic oligomers were synthesized containing motifs of three consecutive tyrosines (Y3) as stabilizing components for pDNA and siRNA polyplex assembly. For pDNA, a combination of terminal oligotyrosines and cysteines was necessary and sufficient for stable polyplex formation. Stable siRNA binding required a combination of terminal cysteines and oligotyrosines, as well as a central hydrophobic modification (oligotyrosines or fatty acids). The phenolic group within the aromatic amino acids of Y3 containing oligomers further increased the endosomal buffer capacity. As a result, the new class of oligotyrosine containing oligomers was efficient in pDNA and siRNA transfection, in most cases superior to a previously established cysteine-containing, dioleic acid modified oligomer without the Y3 motif. Additionally, increased serum stability of the new oligomers with terminal Y3 motifs was demonstrated by gel shift and fluorescence correlations spectroscopy (FCS). In vivo stability and biodistribution was monitored by intravenous administration of chemically stabilized Cy7 siRNA either as free form, or complexed with the nine Y3 containing oligomers or control oligomers. Oligomer 332, with the overall most beneficial in vitro and in vivo characteristics, was applied in RAN siRNA polyplexes for intratumoral treatment of neuroblastoma-bearing mice. This resulted in significantly reduced tumor growth compared to animal treated with control siRNA polyplexes.

Nanosized multifunctional polyplexes for receptor-mediated siRNA delivery.

Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery in vitro and in vivo. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptide-siRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability in vivo, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability in vivo and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney.

New sequence-defined polyaminoamides with tailored endosomolytic properties for plasmid DNA delivery.

Heterogeneity of polymeric carriers is one of the most elusive obstacles in the development of nonviral gene delivery systems, concealing interaction mechanisms and limiting the use of structure-activity relationship studies. In this report, novel sequence-defined polyaminoamides, prepared by solid-phase assisted synthesis, were used to establish first structure-activity relationships for polymer-based plasmid DNA delivery. By combining a cationic building block with hydrophobic modifications and bioreversible disulfide cross-linking sites, transfection polymers with tailored lytic and DNA binding properties were designed. These polymers demonstrated clear correlation between structure and performance in lysis and DNA binding assays. In vitro studies showed negligible toxicity and highly efficient gene transfer, demonstrating the potential of this platform in the fast, combinatorial development of new transfection polymers.

Structure-activity relationships of siRNA carriers based on sequence-defined oligo (ethane amino) amides.

Sequence defined oligo (ethane amino) amides produced by solid-phase supported synthesis using different building blocks and molecular shapes were tested for structure-activity relationships in siRNA delivery. Efficient reporter gene knockdown was obtained in a variety of cell lines using either branched three-armed structures, or lipid-modified structures with i-shape, T-shape, U-shape configuration. For the majority of structures (apart from U-shapes), the presence of 2 or 3 cysteines was strictly required for polyplex stabilization and silencing activity. Although all four building blocks contain the ethylenediamine proton sponge motif, only oligomers assembled with the tetraethylenepentamine based amino acids (Stp, Gtp, Ptp) but not with the triethylenetetramine based amino acid (Gtt) were able to mediate efficient gene silencing. For the lipopolymeric structures, out of the tested saturated (from C4 to C18) and unsaturated (C18) fatty acid moieties, two proximate oleic acids or linolic acids provided the oligomers with the best gene silencing activity and also pH specific lytic activity at pH 5.5, presumably facilitating endosomal escape of the polyplexes. Evaluation of oligomer chain length revealed a minimal number of at least two oligo (ethane amino) building blocks per oligomer arm as necessary for the vast majority of structures, but only marginal changes were found with higher numbers (structures with up to 60 ethane amino nitrogens were evaluated). Two promising carriers (T-shape 49, i-shape 229) were also evaluated for EG5 siRNA delivery. This resulted in tumor cell cycle arrest, and appearance of mitotic monoastral spindles both in vitro and in vivo upon systemic delivery. Repeated intratumoral treatment with EG5 siRNA polyplexes significantly reduced Neuro2A-eGFPLuc tumor growth in a siRNA-specific manner.

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)ß(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.

Nucleic acid carriers based on precise polymer conjugates.

Polymer polydispersity, random conjugation of functional groups, and poorly understood structure-activity relationships have constantly hampered progress in the development of nucleic acid carriers. This review focuses on the synthetic concepts for the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport. Dendrimers, defined peptide carriers, sequence-defined polyamidoamines assembled by solid-phase supported synthesis, and precise lipopeptides or lipopolymers have been characterized for pDNA and siRNA delivery. Conjugation techniques such as click chemistries and peptide ligation are available for conjugating polymers with functional transport elements such as targeting or shielding domains and for direct covalent modification of therapeutic nucleic acids in a site-specific mode.