Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells in vitro and in vivo

The potential of two small poly-L-lysines ( sPLLs), low molecular weight sPLL ( LMW-L) containing 7 - 30 lysine residues and L18 with 18 lysine repeats, to enhance the efficiency of liposome-mediated gene transfer ( GT) with cationic lipid DOCSPER {[}1,3- dioleoyloxy- 2-( N-5-carbamoyl-spermine)-propane] in vascular smooth muscle cells ( SMCs) was investigated. Dynamic light scattering was used for determination of particle size. Confocal microscopy was applied for colocalization studies of sPLLs and plasmid DNA inside cells. GT was performed in proliferating and quiescent primary porcine SMCs in vitro and in vivo in porcine femoral arteries. At low ionic strength, sPLLs formed small complexes with DNA ( 50 100 nm). At high ionic strength, large complexes ( 11 mu m) were observed without any significant differences in particle size between lipoplexes ( DOCSPER/ DNA) and lipopolyplexes ( DOCSPER/ sPLL/ DNA). Both sPLLs were colocalized with DNA inside cells 24 h after transfection, protecting DNA against degradation. DOCSPER/ sPLL/ DNA formulations enhanced GT in vitro up to 5- fold, in a porcine model using local periadventitial application up to 1.5- fold. Both sPLLs significantly increased liposome- mediated GT. Poly-L-lysine L18 was superior to LMW-L since it enabled maximal GT at a 10-fold lower concentration. Thus, sPLLs may serve as enhancers for GT applications in SMCs in vitro and in vivo using local delivery. Copyright (c) 2007 S. Karger AG, Basel

Enhanced Gene Delivery Mediated by Low Molecular Weight Chitosan/DNA Complexes: Effect of pH and Serum

This study was designed to systematically evaluate the influence of pH and serum on the transfection process of chitosan-DNA complexes, with the objective of maximizing their efficiency. The hydrodynamic diameter of the complexes, measured by dynamic light scattering (DLS), was found to increase with salt and pH from 243 nm in water to 1244 nm in PBS at pH 7.4 and aggregation in presence of 10% serum. The cellular uptake of complexes into HEK 293 cells assessed by flow cytometry and confocal fluorescent imaging was found to increase at lower pH and serum. Based on these data, new methodology were tested and high levels of transfection (>40%) were achieved when transfection was initiated at pH 6.5 with 10% serum for 8-24 h to maximize uptake and then the media was changed to pH 7.4 with 10% serum for an additional 24-40 h period. Cytotoxicity of chitosan/DNA complexes was also considerably lower than Lipofectamine. Our study demonstrates that the evaluation of the influence of important parameters in the methodology of transfection enables the understanding of crucial physicochemical and biological mechanisms which allows for the design of methodologies maximising transgene expression

Evidence for Impaired CARD15 Signalling in Crohn's Disease without Disease Linked Variants

BACKGROUND:Sensing of muramyl dipeptide (MDP) is impaired in Crohn's disease (CD) patients with disease-linked variants of the CARD15 (caspase activation and recruitment domain 15) gene. Animal studies suggest that normal CARD15 signalling prevents inflammatory bowel disease, and may be important for disease development in CD. However, only a small fraction of CD patients carry the disease linked CARD15 variants. The aim of this study was thus to investigate if changes could be found in CARD15 signalling in patients without disease associated CARD15 variants. METHODOLOGY/PRINCIPAL FINDINGS:By mapping the response to MDP in peripheral monocytes obtained from CD patients in remission not receiving immunosuppresives, an impaired response to MDP was found in patients without disease linked CARD15 variants compared to control monocytes. This impairment was accompanied by a decreased activation of IkappaB kinase alpha/beta (IKKalpha/beta), the initial step in the nuclear factor kappaB (NFkappaB) pathway, whereas activation of mitogen-activated protein (MAP)-kinases was unaffected. MDP additionally stimulates the inflammasome which is of importance for processing of cytokines. The inflammasome was constitutively activated in CD, but unresponsive to MDP both in CD and control monocytes. CONCLUSIONS/SIGNIFICANCE:These results suggest that inhibited MDP-dependent pathways in CD patients not carrying the disease-associated CARD15 variants might be of importance for the pathogenesis of CD. The results reveal a dysfunctional immune response in CD patients, not able to sense relevant stimuli on the one hand, and on the other hand possessing constitutively active cytokine processing

Prostate Cancer-Specific and Potent Antitumor Effect of a DD3-Controlled Oncolytic Virus Harboring the PTEN Gene

Prostate cancer is a major health problem for men in Western societies. Here we report a Prostate Cancer-Specific Targeting Gene-Viro-Therapy (CTGVT-PCa), in which PTEN was inserted into a DD3-controlled oncolytic viral vector (OV) to form Ad.DD3.E1A.E1B(Δ55)-(PTEN) or, briefly, Ad.DD3.D55-PTEN. The woodchuck post-transcriptional element (WPRE) was also introduced at the downstream of the E1A coding sequence, resulting in much higher expression of the E1A gene. DD3 is one of the most prostate cancer-specific genes and has been used as a clinical bio-diagnostic marker. PTEN is frequently inactivated in primary prostate cancers, which is crucial for prostate cancer progression. Therefore, the Ad.DD3.D55-PTEN has prostate cancer specific and potent antitumor effect. The tumor growth rate was almost completely inhibited with the final tumor volume after Ad.DD3.D55-PTEN treatment less than the initial volume at the beginning of Ad.DD3.D55-PTEN treatment, which shows the powerful antitumor effect of Ad.DD3.D55-PTEN on prostate cancer tumor growth. The CTGVT-PCa construct reported here killed all of the prostate cancer cell lines tested, such as DU145, 22RV1 and CL1, but had a reduced or no killing effect on all the non-prostate cancer cell lines tested. The mechanism of action of Ad.DD3.D55-PTEN was due to the induction of apoptosis, as detected by TUNEL assays and flow cytometry. The apoptosis was mediated by mitochondria-dependent and -independent pathways, as determined by caspase assays and mitochondrial membrane potential

Disease-specific oligodendrocyte lineage cells arise in multiple sclerosis

Multiple sclerosis (MS) is characterized by an immune system attack targeting myelin, which is produced by oligodendrocytes (OLs). We performed single-cell transcriptomic analysis of OL lineage cells from the spinal cord of mice induced with experimental autoimmune encephalomyelitis (EAE), which mimics several aspects of MS. We found unique OLs and OL precursor cells (OPCs) in EAE and uncovered several genes specifically alternatively spliced in these cells. Surprisingly, EAE-specific OL lineage populations expressed genes involved in antigen processing and presentation via major histocompatibility complex class I and II (MHC-I and -II), and in immunoprotection, suggesting alternative functions of these cells in a disease context. Importantly, we found that disease-specific oligodendroglia are also present in human MS brains and that a substantial number of genes known to be susceptibility genes for MS, so far mainly associated with immune cells, are expressed in the OL lineage cells. Finally, we demonstrate that OPCs can phagocytose and that MHC-II-expressing OPCs can activate memory and effector CD4-positive T cells. Our results suggest that OLs and OPCs are not passive targets but instead active immunomodulators in MS. The disease-specific OL lineage cells, for which we identify several biomarkers, may represent novel direct targets for immunomodulatory therapeutic approaches in MS

Self-assembling poly(l-lysine)/DNA complexes capable of integrin-mediated cellular uptake and gene expression

Integrin-mediated delivery of genes is evaluated using a synthetic vector formed by self-assembly of DNA with an oligolysine- peptide sequence containing RGD (referred to as K16-RGD). The RGD peptide binds plasmid DNA effectively and inhibits ethidium bromide/DNA fluorescence at N-to-P ratios of less than 1.0. At N:P ratio 1.0, peptide/DNA complexes formed show a mixture of normal DNA migration and retention at the origin when analysed by agarose electrophoresis. At N:P ratio of 1.2, the complexes have a slight positive surface charge (5 mV) and in the absence of serum they show 10-fold increase uptake into 293 cells, compared with control poly(L-lysine)/DNA vectors, together with a 100-fold increase in transfection. In the presence of serum, RGD-mediated uptake is decreased about 3-fold, but the targeted vectors achieve over 150 times greater transfection than poly(L-lysine)/DNA controls. Transfection could be inhibited by addition of competing RGD, and to a lesser extent RGE, peptides. The targeted vector is believed to achieve cell uptake and transfection by binding av integrins in the cell surface, and the approach could be employed to promote internalisation of vectors following their binding to other, high affinity, receptors, in a system analogous to adenovirus entry

DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4·10−5 g/ml and (3) both components should be mixed together in deionised water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the ζ-potential which finally resulted in formation of a “protein layer” and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the transfection efficiency of DNA complexes with copolymers was always higher than that of the cationic homopolymer slightly increasing with increasing content of HPMA in the copolymers but being about 10–100-times lower than the complexes DNA–poly(l-lysine. In the cytoplasmic injections, it was observed that DNA complexes produced greater gene expression than a direct microinjection of free DNA. The block copolymer complexes were also found to be more efficient than the corresponding simple polycation complexes. In the nuclear microinjection, precisely the opposite behaviour was observed. </p

DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4·10−5 g/ml and (3) both components should be mixed together in deionised water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the ζ-potential which finally resulted in formation of a “protein layer” and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the transfection efficiency of DNA complexes with copolymers was always higher than that of the cationic homopolymer slightly increasing with increasing content of HPMA in the copolymers but being about 10–100-times lower than the complexes DNA–poly(l-lysine. In the cytoplasmic injections, it was observed that DNA complexes produced greater gene expression than a direct microinjection of free DNA. The block copolymer complexes were also found to be more efficient than the corresponding simple polycation complexes. In the nuclear microinjection, precisely the opposite behaviour was observed. </p