Multifunctional protein nanocarriers for targeted nuclear gene delivery in nondividing cells

Abstract

Compartmentalization within eukaryotic cells hinders the efficient delivery of therapeutic agents to the cell nucleus. Here we describe novel multifunctional DNA carriers (MDCs) that self-assemble with DNA to form structured nanoparticles that possess virus-like functions for cellular trafficking. MDCs contain, in fusion, the DNA-compacting sperm chromatin component protamine, alpha-melanocyte-stimulating hormone for cell-targeted internalization, the endosome-translocation domain of diphtheria toxin, and an optimized nuclear localization sequence to confer recognition by the nuclear import machinery. The structure of the MDC-DNA particles was examined using atomic force microscopy, and the functionality of each domain assessed using in vitro techniques, including a reconstituted nuclear transport assay in semi-intact cells relying on the use of quantitative confocal laser scanning microscopy. The nanoparticles were internalized in cell-specific fashion and subsequently exited the endosome into the cytoplasm. Notably, the nanoparticles interact with cellular nuclear transport proteins as shown in direct binding assays and are actively trafficked into the cell nucleus of nondividing cells, resulting in 3- to 4-fold higher reporter gene expression in growth-arrested human embryonic kidney cells, as well as lower cytotoxicity, than lipid and polyethyleneimine vectors. The importance of each functional domain was examined by comparing MDCs with different domain compositions as controls, as well as using antibodies to block particular pathways. MDCs that utilize cellular signaling pathways have enormous potential to safely and efficiently deliver therapeutic transgenes into the nucleus of nondividing cells.

Chronic Kidney Diseases and Nanoparticle Therapeutics

“…Multifunctional DNA carriers (MDCs) which self-assemble with DNA to form structured nanoparticles that possess virus-like functions for cellular trafficking [60]. MDCs interact with cellular nuclear transport proteins gene expression in growth-arrested human embryonic kidney cells.…”

Artificial Viruses: Exploiting Viral Trafficking for Therapeutics

“…(2) to promote robust cell-specific attachment and internalization. An extensive range of targeting ligands have been used to effect cell-specific drug and DNA uptake, including peptides and full-length proteins [23][24][25], vitamins [26,27], carbohydrates [28,29], and hormones [30][31][32]. Antibodies have also been used to attain cellspecific drug and DNA uptake, by attaching to the vector's surface either a monoclonal antibody [33][34][35] or a recombinant single chain variable fragment (scFv) protein that comprises the variable regions of the heavy and light chains of an immunogloblin [36][37][38].…”

“…A number of other virus-derived sequences have been exploited to enhance endosome escape of drugs and DNA, including the influenza fusogenic peptide INF-7 [55], the penton base protein of adenovirus [56], and the gp41 transmembrane protein from the human immunodeficiency virus (HIV) [57]. Endosome disruption moieties are not exclusive to viruses, with poreforming and fusogenic proteins identified in bacteria [58], plants [59], and bee venom [60], some of which have been used to facilitate the endosome escape of non-viral vectors [32,58]. Furthermore, it is possible to mimic the fusogenic properties of HA-2 to create engineered amphipathic peptide sequences that elicit endosome escape of non-viral vectors [61,62], with the added advantage of these small peptides being potentially less immunogenic than full protein domains of viral or bacterial origin [63].…”

“…The building of an artificial virus requires that all the components mentioned above be assembled together with the drug/DNA cargo into a single nano-sized particle whilst maintaining the functionalities of the individual components. A number of different approaches have been undertaken to combine functional components into a single virus-like vector, including the use of multifunctional fusion proteins [32], liposomes with embedded functional protein domains [165,166], and assemblies of synthetic polymers and peptides [167]. As highlighted in this review, an abundance of peptide-or protein-based transport domains from a diverse range of organisms have been characterized and may be assembled together into single multifunctional fusion proteins.…”

“…A recently described multidomain fusion protein, termed a "multifunctional DNA carrier" (MDC), was assembled from the sperm chromatin component protamine for DNA compaction, the -melanocyte-stimulating hormone for celltargeted internalization, the endosome fusogenic domain of diphtheria toxin, and the optimized T-ag NLS to confer recognition by the nuclear import machinery [32]. The MDC proteins were shown to spontaneously assemble with plasmid DNA to produce nano-sized complexes in which all domains were fully functional.…”