Spezifische DNA-Antikörper Konjugate zur selektiven Adressierung von humanen Zellen

Targeted delivery of genes into specific cells is still a challenging task for gene therapeutic approaches. One is based upon the fact, that cancer cells and healthy cells show different receptor expression patterns. The difference in surface receptor expression can be used to selectively address cells. In this thesis DNA, encoding for a gene, was introduced into the nucleus of human cancer cells. The DNA was conjugated with an antibody fragment that is recognized and bound by receptors on the cell surface. It is important to modify the DNA sequence-specifically in a region that is not encoding for the gene, to avoid complications during transcription of the gene. This was achieved by combination of the SNAP-tag technology with sequence-specific modification of DNA with a DNA-methyltransferase (MTase). The SNAP-tag is a mutant of the human O6-alkylguanine-DNA alkyltransferase, which can be genetically fused to the desired antibody fragment and reacts specifically with para-substituted benzylguanine derivatives (BG). DNA-MTases catalyze the sequence-specific transfer of a methyl group from the natural cofactor S-adenosyl-L-methionine (AdoMet) to DNA, within their recognition sequence. However DNA-MTases can be used as tools for specific labeling of DNA with synthetic cofactors. Here two types of cofactor analogues, N-adenosylaziridine cofactors and double-activated cofactors, which carry a BG, have been developed. They were used to couple BG sequence-specifically to DNA, which is then reacted with a SNAP-tag fusion protein to yield the desired DNA-protein conjugate.The method was developed with the model compounds pBR322 and HisSNAP-YFP. For the sequence-specific modification of DNA four cofactor analogues with different linker lengths were developed: 6BGAz, 6BG8Az, AdoYnBG3 and AdoYnBG35. Experiments with AdoYnBG35 (longest linker) showed the best coupling efficiency with DNA-MTase M.BseCI and the SNAP-tag proteins. For experiments with human cancer cells plasmid DNA with the gene for green fluorescent protein (GFP) and one (pGFP, 1 x) or ten recognition sites (pGFP+I, 10 x) for M.BseCI were used. Both plasmids, pGFP (1 x) and pGFP+I (10 x), were modified sequence-specifically with M.BseCI and AdoYnBG35. The antibody-SNAP protein scFv-425-SNAP was used for labeling of the BG-modified DNA. It is recognized specifically by cells which overexpress the epidermal growth factor receptor (EGFR). Cellular experiments were therefore carried out with EGFR+ cell lines A431 (epidermal cancer) and MDA-MB-468 (breast cancer). For cellular binding experiments the DNA was additionally modified with the fluorophore 5(6)-Carboxytetramethylrhodamine (TAMRA) sequence-specifically, to visualize binding of the conjugate to the cell surface. The fluorescent conjugates scFv-425-pGFP-TAMRA (1 x) and scFv-425-pGFP+I-TAMRA (10 x) were incubated with the cells and were observed through FACS and confocal microscopy. Binding as well as internalization of the conjugates were noticed with EGFR+ cells. Subsequent transfection experiments with the conjugates scFv-425-pGFP (1 x) and scFv-425-pGFP+I (10 x), without fluorophore, did not show GFP expression initially. However, the application of selection pressure through G814 antibiotic yielded GFP expression.This method for the specific conjugation of DNA with proteins is an effective and easy way to perform sequence-specific modification of (long) DNA with a defined stoichiometry. In addition labeling of DNA within an essential DNA sequence can be avoided. Endogenous substances (DNA and protein) assure sufficient biocompatibility of the conjugates, while targeted addressing of cells suppresses unspecific reactions