Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP).

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

The acquisition of narrow binding specificity by polyspecific natural IgM antibodies in a semi-physiological environment.

Natural IgM antibodies (Abs) play an important role in clearing pathogens, enhancing immune responses, and preventing autoimmunity. However, the molecular mechanisms that mediate the functions of natural IgM Abs are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs are commonly polyspecific and recognize a variety of antigens (Ags) with no apparent structural homology. It is generally believed that polyspecificity is an inherent property of natural Abs. However, there is increasing evidence that polyspecificity may be induced by mild denaturing conditions. In this study, we compared the specificity of three polyspecific IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All three Abs lost their polyspecificity in serum. They no longer reacted with conventional screening Ags, including hapten-BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with cognate peptide Ags selected from a T7 phage library. The acquisition of narrow specificity by polyspecific IgM in serum was also observed with muscle tissue sections used as a source of endogenous Ags. The loss of polyspecificity by different Abs was apparently dependent on the presence of different serum constituents. The results of this study suggest that the seemingly inherent polyspecificity of many natural IgM Abs may be largely an in vitro phenomenon related to the lack of normal serum components in the medium. Potential mechanisms underlying the loss of polyreactivity are discussed.

In vivo selection and validation of liver-specific ligands using a new T7 phage peptide display system.

In vivo phage display is a powerful source of new peptide ligands for specific organ targeting by drugs and gene therapy vectors. Since the introduction of this methodology a decade ago, a number of peptides that preferentially react with organ-specific endothelium and parenchymal markers have been selected. One organ that has been conspicuously missing from these selection studies is the liver, which possesses a multitude of acquired and hereditary disorders and represents a highly important therapeutic target. Herein, we set out to fill this gap by introducing a novel peptide display system containing cloned sequences in the tail fiber protein (p17) of phage T7. The p17 display effectively avoids the innate immune system and is well suited both for selection of new liver-specific ligands and for validation of protein sequences that have been implicated in liver targeting by the use of conventional biochemical methods.

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy.

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.

Sequence-specific binding of normal serum immunoglobulin M to exposed protein C-termini.

Both the timely clearance of degraded endogenous structures and the presence of secreted natural immunoglobulin M (IgM) are needed to avoid autoimmunity. These requirements may be causally related provided that natural IgM preferentially reacts with degraded antigens and, by activating complement, mediates their non-inflammatory clearance through complement receptors. We have previously shown that normal serum IgM reacts in vivo and in vitro with virtually all randomly generated C-terminal peptides displayed on T7 phage. The resultant multivalent IgM-peptide complexes activate complement and are detected by a loss of phage infectivity. A striking feature of these reactions is that different C-terminal peptides ( approximately 3-4 amino acids) specifically react with different 'C-terminal' IgM (C-IgM) antibodies. This suggests that degraded supramolecular structures, expressing elevated levels of identical C-termini as a result of proteolysis, denaturation and abnormal exposure of repetitive protein constituents, may be preferential targets of C-IgM-mediated complement activation in the physiological environment. The specificity of C-IgM-peptide reactions is much higher than one would expect, assuming that normal serum IgM mostly comprises polyspecific natural antibodies. However, it is possible that polyspecific IgM is not adequately registered by our 'functional' phage-inactivation assays. In this study, we resolve the issue of C-IgM specificity by directly characterizing the binding reactivity of normal serum IgM with phage-displayed C-terminal peptides.

The effect of cell division on the cellular dynamics of microinjected DNA and dextran.

Gene delivery is a multistep process that is being studied to increase its efficiency, a major hurdle for effective gene therapy. Our study focused on the nuclear entry step by microinjecting a mixture of fluorescent dextran and the pEYFP-Nuc plasmid (encoding a nuclear-targeted, enhanced GFP) into the cytoplasm of nondividing and dividing cells that were selected using non-chemical means. After 10 and 1000 ng/microl of plasmid DNA (pDNA) were cytoplasmically injected, 28% and 50% of the cells that had not divided expressed GFP, respectively, compared with 50% and 90% for the cells that had divided. This result suggested that pDNA can enter the nonmitotic nuclei of mononucleated cells, albeit at a lower efficiency than mitotic nuclei. The ability of pDNA to enter the intact nuclei of nondividing cells is consistent with our previous experience using multinucleated myotubes and digitonin-permeabilized cells in culture and using intravascular naked pDNA delivery in vivo. An explanation for the small effect of cell division was provided by studies using fluorescently labeled molecules and confocal fluorescent microscopy. They showed that the bulk of large dextran, and similarly pDNA, was excluded from re-formed nuclei after mitosis, thereby limiting the effect of cell division on the nuclear entry of pDNA.