493. Nonviral Gene Transfer by Sequence-Defined Proton-Sponges with Combined Nucleic Acid Binding and Endosomal Buffering: Balancing Basicities
Authors: Ulrich Lächelt, Ana Krhac Levacic, Dominik Wendel, Ernst Wagner
Journal: Molecular Therapy (2016)

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 blocks1Schaffert D. Troiber C. Salcher E.E. Fröhlich T. Martin I. Badgujar N. Dohmen C. Edinger D. Kläger R. Maiwald G. Farkasova K. Seeber S. Jahn-Hofmann K. Hadwiger P. Wagner E. Solid-phase synthesis of sequence-defined T-, i-, and U-shape polymers for pDNA and siRNA delivery.Angew. Chem., Int. Ed. Engl. 2011; 50: 8986-8989Crossref PubMed Scopus (141) Google Scholar 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 observations2Lächelt U. Kos P. Mickler F.M. Herrmann A. Salcher E.E. Rödl W. Badgujar N. Bräuchle C. Wagner E. Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes.Nanomedicine (N. Y., NY, U. S.). 2014; 10: 35-44Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar 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