Hanno V. J. Kolbe

Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology

Non-enzymatic deamidation of asparagine and glutamine residues in proteins and peptides are reviewed by first outlining the well-described reaction mechanism involving cyclic imide intermediates, followed by a discussion of structural features which influence the reaction rate. The second and major part describes analytical techniques that allow studying deamidation in proteins using recombinant human growth hormone and recombinant hirudin as examples. Finally, the significance of non-enzymatic deamidation with respect to the production of pharmaceutical proteins is discussed.

Enhanced in vitro and in vivo cationic lipid-mediated gene delivery with a fluorinated glycerophosphoethanolamine helper lipid.

One of the main drawbacks of synthetic, non‐viral gene vectors is their relatively low in vivo efficiency when compared with viral vectors. The present paper describes the use of a partially fluorinated glycerophosphoethanolamine (F‐PE), a close analog of DOPE, which, as a helper lipid with the cationic lipopolyamine pcTG90, increases its in vitro and in vivo gene transfer capability to a larger extent than DOPE.

Multiple systemic expression of human interferon-β in mice can be achieved upon repeated administration of optimized pcTG90-lipoplex

The possibility of achieving multiple systemic expression of human interferon-β in mice upon repeated intravenous administration of cationic liposome–DNA complex (lipoplex) was investigated. Lipoplexes containing the pentammonio lipid pcTG90 were first optimized by selecting the most efficient ratio of pcTG90 to phosphatidylethanolamine (DOPE) and the N/P ratio of cationic lipid nitrogen to DNA phosphate. Highest levels and reproducibility of gene expression were obtained using pcTG90/DOPE (1:2) liposomes complexed with the IFNB1 gene containing plasmid pTG14169 at a N/P ratio of 10. Following lipoplex administration, an early but transient human interferon-β expression in serum was observed. Importantly, repeated systemic gene expression could be achieved upon re-administration with a minimal time interval of 14 days between two injections. For an interval period of 6 days, subsequent gene expression was inhibited by a first administration of lipoplexes containing either a luciferase reporter gene plasmid or an empty plasmid, but was not inhibited when free (non-complexed) plasmid pTG14169 was first injected. Multiple injections of pcTG90-lipoplex performed once every other month resulted in three subsequent peaks of systemic IFNB1 gene expression in mice. In conclusion, our study demonstrates the feasibility of expanding the therapeutic window of a cytokine using repetitive intravenous administration of lipoplex.

Effect of liposome-encapsulated clodronate pretreatment on synthetic vector-mediated gene expression in mice

One of the main limitations for the use of synthetic vectors in gene therapy is their relatively low in vivo efficiency when compared with viral vectors. Here, we describe a pretreatment protocol with liposome-encapsulated clodronate in mice by which gene expression levels of a luciferase reporter gene could be increased up to nine-fold in the lung, after intravenous (i.v.) injection of glycerolipoplexes. Optimal results were obtained if mice were pretreated with liposome-encapsulated clodronate 1 day before injection of lipoplexes. The enhancement effect could be observed for lipoplexes prepared with different multivalent cationic glycerolipids. Most remarkably, polyplexes behaved in the opposite way. Liposome-encapsulated clodronate pretreatment strongly reduced reporter gene expression after i.v. injection of polyethylenimine-polyplexes (ExGen500).

Direct lipid quantitation of cationic liposomes by reversed-phase HPLC in lipoplex preparation process.

The proposed reversed-phase high-performance liquid chromatography method with ultraviolet detection provides a simple and rapid procedure to separate and quantitate lipids from cationic liposomes used in gene transfer. We describe experimental conditions which do not require lipid extraction from liposomes prior to sample analysis. Evaluation of the method reported here showed suitable lipid separation capacity and quantitation accuracy from cationic liposomes composed of either the pentammonio lipid pcTG90 and dioleoyl phosphatidylethanolamine, or 1, 2-dioleoyl-3-trimethylamonium propane and cholesterol. Detection limits were in the range of 0.5-1 microg depending on the lipid. This quantitative method has proven useful in lipoplex formulation processing development and its application may be extended to a wide range of lipid-based gene and drug delivery systems.

Solvoplex: A New Type of Synthetic Vector for Intrapulmonary Gene Delivery

A novel type of synthetic vector, termed solvoplex, is described that can greatly enhance gene expression in lung after intrapulmonary delivery. Solvoplexes consist of plasmid DNA and organic solvents. Several organic solvents were analyzed, and luciferase reporter gene expression was observed after intrapulmonary delivery of solvoplexes containing DPSO (di-n-propylsulfoxide), TMU (tetramethylurea), or BMSO (butylmethylsulfoxide). Expression levels correlated with the amount of solvent used at constant DNA amounts. Highest expression was obtained in the lung after intratracheal injection with 15% DPSO resulting in an increase up to 440-fold compared with DNA alone. DPSO-solvoplexes (15%) gave higher reporter gene expression than polyplexes (ExGen 500) or lipoplexes (DOTAP-cholesterol or DOTAP-DOPE). Solvoplex-mediated gene expression did not depend on the delivery mode, and was observed in both mice and rats. Readministration of DPSO-solvoplexes was possible. A second injection after 4 weeks resulted in expression levels similar to the first administration. Histological analyses using lacZ and GFP reporter genes demonstrated gene expression in the lung airway epithelium after intratracheal and microspray delivery. When luciferase expression levels in lung homogenates were compared with adenovirus vectors, DPSO-solvoplexes were 4- or 100-fold less efficient, depending on the promoter used in the viral vector. A quantitative histological comparison between solvoplexes and adenovirus vectors in the best expressing regions revealed that solvoplexes yielded about 2% LacZ-positive cells in the lung airway epithelium, and adenovirus vectors about 20%. Using the microsprayer system, we demonstrated that DNA remained intact in solvoplexes on spraying and that reporter gene expression was observed in mice after intrapulmonary delivery of a solvoplex spray. DNA in DPSO-solvoplexes remained stable and functional after prolonged storage at room temperature.

Synthesis and stability study of the new pentammonio lipid pcTG90, a gene transfer agent

Abstract The cationic lipid pcTG90 has been prepared from ( S )-1-aminopropane-2,3-diol by N -acylation with N -protected 18-amino-3,7,11,15-tetraazaoctadecanoic acid and O -acylation with oleic acid. The former acid could be obtained from 1,3-propanediamine via tetraprotected caldopentamine. The stability of the cationic lipid in HEPES buffer has been studied.

Isolation of recombinant partial gag gene product p18 (HIV-1Bru) from Escherichia coli

The membrane-associated structural protein, p18, of the human immunodeficiency virus (HIV-1), has been expressed in Escherichia coli. The recombinant protein was purified by cation-exchange chromatography on S Sepharose followed by cation-exchange high-performance liquid chromatography (HPLC) on Sulfoethyl Aspartamide. The isolation of 28.7 mg of recombinant p18 from 16.71 of cell culture represents an overall yield of ca. 20%. Recombinant p18 was characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, reversed-phase HPLC, amino acid composition and amino acid sequence analysis of the N-terminus. Edman degradation of peptides generated by trypsin or Staphylococcus aureus V8 proteolytic digestion, including the C-terminus, confirmed the amino acid sequence to be that predicted from the cDNA. A C-terminally cleaved form of recombinant p18, p18LM, was separated in the cation-exchange HPLC step and was partially characterized in parallel with the intact molecule. By Western blotting it was shown that recombinant p18 in addition to the cleaved form p18LM is recognized by a monoclonal antibody which was generated against the natural protein from HIV-1.