Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Identification of a subunit of a novel Kleisin-beta/SMC complex as a potential substrate of protein phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates. PP2A is required for chromosome segregation, but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-beta, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.     

Methylation of the protein phosphatase 2A catalytic subunit is essential for association of Balpha regulatory subunit but not SG2NA, striatin, or polyomavirus middle tumor antigen

Binding of different regulatory subunits and methylation of the catalytic (C) subunit carboxy-terminal leucine 309 are two important mechanisms by which protein phosphatase 2A (PP2A) can be regulated. In this study, both genetic and biochemical approaches were used to investigate regulation of regulatory subunit binding by C subunit methylation. Monoclonal antibodies selectively recognizing unmethylated C subunit were used to quantitate the methylation status of wild-type and mutant C subunits. Analysis of 13 C subunit mutants showed that both carboxy-terminal and active site residues are important for maintaining methylation in vivo. Severe impairment of methylation invariably led to a dramatic decrease in Balpha subunit binding but not of striatin, SG2NA, or polyomavirus middle tumor antigen (MT) binding. In fact, most unmethylated C subunit mutants showed enhanced binding to striatin and SG2NA. Certain carboxy-terminal mutations decreased Balpha subunit binding without greatly affecting methylation, indicating that Balpha subunit binding is not required for a high steady-state level of C subunit methylation. Demethylation of PP2A in cell lysates with recombinant PP2A methylesterase greatly decreased the amount of C subunit that could be coimmunoprecipitated via the Balpha subunit but not the amount that could be coimmunoprecipitated with Aalpha subunit or MT. When C subunit methylation levels were greatly reduced in vivo, Balpha subunits were found complexed exclusively to methylated C subunits, whereas striatin and SG2NA in the same cells bound both methylated and unmethylated C subunits. Thus, C subunit methylation is critical for assembly of PP2A heterotrimers containing Balpha subunit but not for formation of heterotrimers containing MT, striatin, or SG2NA. These findings suggest that methylation may be able to selectively regulate the association of certain regulatory subunits with the A/C heterodimer.     

Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells

Synthetic vectors were evaluated for their ability to mediate efficient mRNA transfection. Initial results indicated that lipoplexes, but not polyplexes based on polyethylenimine (PEI, 25 and 22 kDa), poly(L-lysine) (PLL, 54 kDa) or dendrimers, mediated efficient translation of mRNA in B16-F10 cells. Significant mRNA transfection was achieved by lipoplex delivery in quiescent (passage 0) human umbilical vein endothelial cells (HUVEC), and by passage 4, 10.7% of HUVEC were transfected compared to 0.84% with DNA. Lack of expression with PEI 25 kDa/mRNA or PLL 54 kDa/mRNA in a cell-free translation assay and following cytoplasmic injection into Rat1 cells indicated that these polyplexes were too stable to release mRNA. In contrast, polyplexes formed using smaller PEI 2 kDa and PLL 3.4 kDa gave 5-fold greater expression in B16-F10 cells compared to DOTAP, but were dependent on chloroquine for transfection activity. Endosomolytic activity was incorporated by conjugating PEI 2 kDa to melittin and resulting PEI 2 kDa-melittin/mRNA polyplexes mediated high transfection levels in HeLa cells (31.1 +/- 4.1%) and HUVEC (58.5 +/- 2.9%) in the absence of chloroquine, that was potentiated to 52.2 +/- 2.7 and 71.6 +/- 1.7%, respectively, in the presence of chloroquine. These results demonstrate that mRNA polyplexes based on peptide-modified low molecular weight polycations can possess versatile properties including endosomolysis that should enable efficient non-viral mRNA transfection of quiescent and post-mitotic cells.     

A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry

In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i. e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Expression of protein phosphatase 2A mutants and silencing of the regulatory B alpha subunit induce a selective loss of acetylated and detyrosinated microtubules

Carboxymethylation and phosphorylation of protein phosphatase 2A (PP2A) catalytic C subunit are evolutionary conserved mechanisms that critically control PP2A holoenzyme assembly and substrate specificity. Down-regulation of PP2A methylation and PP2A enzymes containing the B alpha regulatory subunit occur in Alzheimer's disease. In this study, we show that expressed wild-type and methylation- (L309 Delta) and phosphorylation- (T304D, T304A, Y307F, and Y307E) site mutants of PP2A C subunit differentially bind to B, B', and B'-type regulatory subunits in NIH 3T3 fibroblasts and neuro-2a (N2a) neuroblastoma cells. They also display distinct binding affinity for microtubules (MTs). Relative to controls, expression of the wild-type, T304A and Y307F C subunits in N2a cells promotes the accumulation of acetylated and detyrosinated MTs. However, expression of the Y307E, L309 Delta, and T304D mutants, which are impaired in their ability to associate with the B alpha subunit, induces their loss. Silencing of B alpha subunit in N2a and NIH 3T3 cells is sufficient to induce a similar breakdown of acetylated and detyrosinated MTs. It also confers increased sensitivity to nocodazole-induced MT depolymerization. Our findings suggest that changes in intracellular PP2A subunit composition can modulate MT dynamics. They support the hypothesis that reduced amounts of neuronal B alpha-containing PP2A heterotrimers contribute to MT destabilization in Alzheimer's disease.     

A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential

BACKGROUND: Poor endosomal release is one major barrier of gene delivery. Endosomolytic polyethylenimine-melittin conjugates have shown to enhance gene transfer efficiency; however, cytotoxicity due to their general membrane-destabilizing properties limits their application. To overcome this drawback we grafted a polycation with a masked pH-responsive melittin derivate and investigated lytic activity, gene transfer efficiency and cytotoxicity of the resulting conjugate. METHODS: Melittin (Mel) was modified with dimethylmaleic anhydride (DMMAn) and covalently coupled to poly-L-lysine (PLL). The membrane lytic activity was analyzed after incubation at neutral or endosomal pH. PLL-DMMAn-Mel polyplexes were generated in HEPES-buffered glucose and tested in transfection experiments using luciferase as reporter gene. Cellular cytotoxicity was analyzed by measurement of membrane integrity and metabolic activity. RESULTS: Covalent attachment of DMMAn-modified melittin to PLL resulted in a pH-responsive conjugate. No lytic activity was observed at neutral pH; after acidic cleavage of the protecting groups at pH 5 lytic activity was regained. Acute toxicity was greatly reduced (as compared to PLL-Mel or even unmodified PLL) and high gene expression levels (up to 1800-fold higher than unmodified PLL) were obtained. CONCLUSIONS: Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also strongly reduces the acute toxicity of melittin and PLL. Hence this modification might be useful for optimizing polycationic gene carriers.