No abstract

For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.

Background Efficient and target‐specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non‐target cell types. Methods Polycation‐based vectors, including adenovirus‐enhanced transferrinfection (AVET) and transferrin‐polyethylenimine (Tf‐PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. Results Gene transfer after intratumoral application was 10–100 fold more efficient with Tf‐PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf‐PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. Conclusion For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs. Copyright © 1999 John Wiley & Sons, Ltd.

Based on observations that DBA/2 mice develop a highly specific response towards an HLA-Cw3-derived epitope, consisting entirely of CD8+CD62L-Vb10+ cells, we have established an in vivo mouse model for screening a variety of immunization approaches. Responder cells were readily detectable in small samples of the peripheral blood using three-color FACS analysis. This permitted multiple, sequential determination of CD8+ T-cell responses in living animals at a very high degree of precision. In vivo electroporation delivery of expression construct plasmids, outclassed the other approaches tested. Dominant, specific responses were induced already upon a single administration. Both the peak and the longevity of the response resembled those that are generated by the most active viral infections. The induced CTLs rejected epitope-bearing tumor cells in vivo and released interferon-g upon stimulation with the correct MHC::peptide combination in vitro. The potent in vivo response was not influenced by known modulators of the innate immune system, such as CpG DNA and LPS content. In vivo electroporation thus deserves consideration in the future in antitumor and antiviral immunization approaches, where CD8+ T cells play a predominant role.

The ovine mammary gland turned out to be a very well suited as a model system for evaluation and optimization of various gene transfer protocols.