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@PHDTHESIS{Hanz:672823,
author = {Hanz, Gisela Maria},
othercontributors = {Weinhold, Elmar and Albrecht, Markus},
title = {{S}pezifische {DNA}-{A}ntikörper {K}onjugate zur
selektiven {A}dressierung von humanen {Z}ellen},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2016-08324},
pages = {1 Online-Ressource (IX, 184 Seiten) : Illustrationen,
Diagramme},
year = {2016},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2016},
abstract = {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.},
cin = {152620 / 150000},
ddc = {540},
cid = {$I:(DE-82)152620_20140620$ / $I:(DE-82)150000_20140620$},
pnm = {OPPa117b - Targeted delivery of innovative chimeric
antibody/nucleic acid therapeutics to cancer cells (ZUK2-SF)
/ ERS Seed Fund (ZUK2) - Exploratory Research Space: Seed
Fund (2) als Anschubfinanzierung zur Erforschung neuer
interdisziplinärer Ideen (ZUK2-SF) / Zukunftskonzept II -
Zukunftskonzept der RWTH Aachen University im Rahmen der
zweiten Exzellenzinitiative (ZUK2)},
pid = {G:(DE-82)ZUK2-SF-OPPa117b / G:(DE-82)ZUK2-SF /
G:(DE-82)ZUK2},
typ = {PUB:(DE-HGF)11},
urn = {urn:nbn:de:hbz:82-rwth-2016-083245},
url = {https://publications.rwth-aachen.de/record/672823},
}
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