Quorum-sensing-based toolbox for regulatable transgene and siRNA expression in mammalian cells.

Technologies for regulated expression of multiple transgenes in mammalian cells have gathered momentum for bioengineering, gene therapy, drug discovery, and gene-function analyses. Capitalizing on recently developed mammalian transgene modalities (QuoRex) derived from Streptomyces coelicolor, we have designed a flexible and highly compatible expression vector set that enables desired transgene/siRNA control in response to the nontoxic butyrolactone SCB1. The construction-kit-like expression portfolio includes (i) multicistronic (pTRIDENT), (ii) autoregulated, (iii) bidirectional (pBiRex), (iv) oncoretro- and lentiviral transduction, and (v) RNA polymerase II-based siRNA transcription-fine-tuning vectors for straightforward implementation of QuoRex-controlled (trans)gene modulation in mammalian cells.

Poly(ethylene glycol) hydrogels formed by conjugate addition with controllable swelling, degradation, and release of pharmaceutically active proteins.

Hydrogels were formed by conjugate addition of polyethylene glycol (PEG) multiacrylates and dithiothreitol (DTT) for encapsulation and sustained release of protein drugs; human growth hormone (hGH) was considered as an example. Prior to encapsulation, the hGH was precipitated either by Zn2+ ions or by linear PEG, to protect the hGH from reaction with the gel precursors during gelation. Precipitation by Zn2+ ions yielded precipitates that dissolved slowly and delayed release from even highly permeable gels, whereas linear PEG yielded rapidly dissolving precipitates.

Engineered Streptomyces quorum-sensing components enable inducible siRNA-mediated translation control in mammalian cells and adjustable transcription control in mice.

Background: Recent advances in functional genomics, gene therapy, tissue engineering, drug discovery and biopharmaceuticals production have been fostered by precise small-molecule-mediated fine-tuning of desired transgenes.

Methods: Capitalizing on well-evolved quorum-sensing regulatory networks in Streptomyces coelicolor we have designed a mammalian regulation system inducible by the non-toxic butyrolactone SCB1. Fusion of the S.

The effect of the linker on the hydrolysis rate of drug-linked ester bonds.

Tailoring the length of a sulfide containing linker adjusts the hydrolysis of a drug-linked ester bond to values appropriate for once-a-week administrations. A model drug of paclitaxel was coupled using a hydrolyzable linker to a poly(ethylene glycol) macromonomer, via a conjugate addition reaction between a thiol and an acrylamide. The macromonomers were synthesized in three steps with an average overall yield of 70%.

Evaluation of pH-dependent membrane-disruptive properties of poly(acrylic acid) derived polymers.

Anionic pH-sensitive membrane-disruptive polymers have evolved as a new class of bioactive excipients for the cytosolic delivery of therapeutic macromolecules. A large variety of anionic copolymers and analogues of poly(acrylic acid) (PA) was investigated and compared to a cationic PA copolymer. The pH-responsive membrane-disruptive properties were characterized by employing three in vitro models, such as pH dependent shift of pyrene fluorescence, liposome leakage and lysis of red blood cells.

Streptomyces-derived quorum-sensing systems engineered for adjustable transgene expression in mammalian cells and mice.

Prokaryotic transcriptional regulatory elements have been adopted for controlled expression of cloned genes in mammalian cells and animals, the cornerstone for gene-function correlations, drug discovery, biopharmaceutical manufacturing as well as advanced gene therapy and tissue engineering. Many prokaryotes have evolved specific molecular communication systems known as quorum-sensing to coordinate population-wide responses to physiological and/or physicochemical signals. A generic bacterial quorum-sensing system is based on a diffusible signal molecule that prevents binding of a repressor to corresponding operator sites thus resulting in derepression of a target regulon.

Comparative transfection studies of human ovarian carcinoma cells in vitro, ex vivo and in vivo with poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes.

Background: Poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) can be used successfully for in vitro transfection of different cell lines, including the OVCAR-3 human ovarian carcinoma cell line. The aim of this study was to investigate whether it is possible to transfect OVCAR-3 cells in vivo with polyplexes containing p(DMAEMA).

Methods: In order to understand the generally observed gap between in vitro and in vivo transfection, we gradually went from in vitro to in vivo transfection of OVCAR-3 cells, while keeping the exposure conditions the same, as far as possible.

Copolymers of 2-(dimethylamino)ethyl methacrylate with ethoxytriethylene glycol methacrylate or N-vinyl-pyrrolidone as gene transfer agents.

Random copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate (triEGMA) or N-vinylpyrrolidone (NVP) of different molecular weights and compositions were synthesized, characterized and evaluated as polymeric transfectants in vitro. All synthesized copolymers (comonomer fraction up to 66 mol%) were able to bind to DNA, yielding polymer-plasmid complexes (polyplexes). However, the polymer-plasmid ratio at which small complexes (size 0.

Thermosensitive polymers as carriers for DNA delivery.

Copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) and N-isopropylacryl amide (NIPAAm) of various monomer ratios and molecular weights were evaluated as carrier systems for DNA delivery. All copolymers, even with a low DMAEMA content of 15 mol%, were able to bind to DNA at 25 degrees C. Light-scattering measurements indicate that complexation is accompanied by precipitation of the (co)polymer in the complex caused by a drop of the lower critical solution temperature of the (co)polymer.

A versatile method for the conjugation of proteins and peptides to poly[2-(dimethylamino)ethyl methacrylate].

Random copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with aminoethyl methacrylate (AEMA) were synthesized by radical polymerization. The amount of incorporated primary amino groups could be controlled by the feed ratio of AEMA to DMAEMA, and was varied from 2 to 6 mol %. Subsequently, protected thiol groups were introduced in a derivatization step with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and subsequent treatment with dithiothreitol (DTT).

Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery.

A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.

2-(Dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents.

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is able to bind to DNA by electrostatic interactions. At a polymer/plasmid ratio above 2 (w/w) positively charged complexes were formed with a size around 0.2 microm.

Effect of size and serum proteins on transfection efficiency of poly ((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles.

Purpose: The aim of this study was to gain insight into the relation between the physical characteristics of particles formed by a plasmid and a synthetic cationic polymer (poly(2-dimethylamino)ethyl methacrylate, PDMAEMA) and their transfection efficiency.

Methods: The PDMAEMA-plasmid particles were characterized by dynamic light scattering (size) and electrophoretic mobility measurements (charge). The transfection efficiency was evaluated in cell culture (COS-7 cells) using a pCMV-lacZ plasmid coding for beta-galactosidase as a reporter gene.