The immunogenicity of antibody aggregates in a novel transgenic mouse model.

Purpose: Protein aggregates have been discussed as a potential risk factor related to immunogenicity. Here we developed a novel human IgG transgenic (tg) mouse system expressing a mini-repertoire of human IgG1 antibodies (Abs) for the assessment of immunogenic properties of human mAb preparations.

Methods: Transgenic mice were generated using germline versions of the human Ig heavy chain γ1 (IgH-γ1), and the human Ig light chain (IgL) κ and λ genes.

Structure and function of purified monoclonal antibody dimers induced by different stress conditions.

Purpose: To investigate structure and function of different monoclonal antibody (MAb) dimers.

Methods: MAb dimers were induced by process-related, low pH and UV light stress. Dimers were isolated and purified by chromatography and extensively characterized by biochemical, structural and functional methods.

Optimizing targeted gene delivery: chemical modification of viral vectors and synthesis of artificial virus vector systems.

In comparison to classical medicines, gene therapy has the potential to mediate the highest possible level of therapeutic specificity. Every normal or diseased cell can switch on or off a gene expression cassette in a tissue-, disease-, and time-dependent fashion, by use of specific transcription factors that are active only in a given unique situation. In practice, we face the problem in realizing the concept: the delivery of nucleic acids into target cells is very ineffective and presents a formidable challenge.

DNA polyplexes based on degradable oligoethylenimine-derivatives: combination with EGF receptor targeting and endosomal release functions.

Combination of the degradable polymeric gene carriers OEI-HD-1 and LT- OEI-HD-1 with an EGF targeting conjugate resulted in strongly (up to 900-fold) enhanced polyplex activity in EGF-receptor rich HUH7 hepatocellular carcinoma cells. The targeting ligand effect was DNA dose dependent, could be blocked by competitive receptor binding with unbound EGF ligand, and was not observed in receptor-negative control cells. Measures which enhance intracellular endosomal escape, either photochemically enhanced intracellular release (PCI) or the incorporation of a novel membrane-active melittin analog NMA-3, further enhanced gene transfer activity of EGF/OEI-HD-1 polyplexes.

Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes.

Melittin-polyethylenimine (PEI) conjugates have been shown to enhance gene transfer efficiency of polyplexes due to their membrane-destabilizing properties. Inherent lytic activity at neutral pH however also provokes high cytotoxicity due to plasma membrane damage. In order to shift the lytic activity towards the endosomal membrane, several melittin analogs were designed.

Targeting of Polyplexes: Toward Synthetic Virus Vector Systems.

Dominating issues in gene vector optimization are specific in recognizing the target cells and exploiting the proper intracellular trafficking routes. Any progress in this area will result in improved specific gene transfer, reduce the required therapeutic vector doses and, in consequence, lower the overall toxicity to the host. To provide polyplexes with the ability to distinguish between non-target and target cells, cell-binding ligands have been incorporated which recognize target-specific cellular receptors.

Targeting of polyplexes: toward synthetic virus vector systems.

Dominating issues in gene vector optimization are specific in recognizing the target cells and exploiting the proper intracellular trafficking routes. Any progress in this area will result in improved specific gene transfer, reduce the required therapeutic vector doses and, in consequence, lower the overall toxicity to the host. To provide polyplexes with the ability to distinguish between non-target and target cells, cell-binding ligands have been incorporated which recognize target-specific cellular receptors.

C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes.

Background: One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency.

Methods: The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI.

Toward synthetic viruses: endosomal pH-triggered deshielding of targeted polyplexes greatly enhances gene transfer in vitro and in vivo.

Nonviral vectors should undergo "virus-like" changes compatible with the steps of gene delivery. Poly(ethylene) glycol (PEG) shielding of DNA/polycation polyplexes protects from nonspecific interactions with the extracellular environment. pH-triggered removal of the shield within the endosome may be advantageous.

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.