Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.

Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials.

A nuclear localization signal can enhance both the nuclear transport and expression of 1 kb DNA.

Although the entry of DNA into the nucleus is a crucial step of non-viral gene delivery, fundamental features of this transport process have remained unexplored. This study analyzed the effect of linear double stranded DNA size on its passive diffusion, its active transport and its NLS-assisted transport. The size limit for passive diffusion was found to be between 200 and 310 bp. DNA of 310-1500 bp entered the nuclei of digitonin treated cells in the absence of cytosolic extract by an active transport process. Both the size limit and the intensity of DNA nuclear transport could be increased by the attachment of strong nuclear localization signals. Conjugation of a 900 bp expression cassette to nuclear localization signals increased both its nuclear entry and expression in microinjected, living cells.

DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA.

The nuclear entry of exogenous DNA in mammalian cells is critical for efficient gene transfer. A novel technique was developed for the covalent attachment of cationic peptides to double-stranded DNA using a cyclo-propapyrroloindole cross-linker. The attachment of the SV40 large T antigen nuclear localization signal peptide induced the nuclear accumulation of the conjugated DNA in digitonin-permeabilized cells via the classical pathway for the nuclear transport of karyophilic proteins. Increased nuclear uptake of the modified DNA, however, did not occur after it was microinjected into the cytoplasm of cultured cells. This demonstration that the covalent modification of DNA with a signal peptide alters its behavior and interaction with other cellular factors portends the potential of DNA vector chemistry to enhance the efficiency of cellular gene transfer.

Nuclear import of DNA in digitonin-permeabilized cells.

DNA can enter intact mammalian nuclei with varying degrees of efficiency in both transfected and microinjected cells, yet very little is known about the mechanism by which it crosses the nuclear membrane. Nucleocytoplasmic transport of fluorescently labeled DNA was studied using a digitonin-permeabilized cell system. DNA accumulated in the nucleus with a punctate staining pattern in over 80% of the permeabilized HeLa cells. Nuclear localization of the labeled DNA was energy dependent and occurred through the nuclear pore, but did not require the addition of soluble cytoplasmic protein factors necessary for protein import.

Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation.

Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.

Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle.

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.

Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle.

Plasmid pRSVL persisted and expressed luciferase for at least 19 months in mouse skeletal muscle after intramuscular injection. Other injected plasmids also stably expressed long-term suggesting that any plasmid DNA could stably persist and express in muscle. Plasmid DNA was demonstrated by quantitative PCR in some of the muscle DNA samples for at least 19 months after injection. The methylation pattern of the plasmid DNA remained in its bacterial form indicating that the foreign DNA did not replicate in the muscle cells. The electroporation of total cellular DNA from injected muscles into bacteria indicated that the plasmid DNA was extrachromosomal. Chromosomal integration of plasmid DNA was searched for by electroporating the injected muscle DNA into bacteria after restriction enzyme digestion and ligation. No plasmids containing plasmid/chromosome junctions were observed in over 1800 colonies examined. Lack of integration increases the theoretical safety of this gene transfer technique. Long-term stability of plasmid DNA in muscle indicates that muscle is an attractive target tissue for the introduction of extrachromosomal plasmid or viral DNA for the purpose of gene therapy.