Hapten-Binding Bispecific Antibodies for the Targeted Delivery of SiRNA and SiRNA-Containing Nanoparticles.

Hapten-binding bispecific antibodies (bsAbs) are effective and versatile tools for targeting diverse payloads, including siRNAs, to specific cells and tissues. In this chapter, we provide examples for successful SiRNA delivery using this powerful targeting platform. We further provide protocols for designing and producing bsAbs, for combining bsAbs with SiRNA into functional complexes, and achieving specific mRNA knockdown in cells by using these functional complexes.

Comparison of the lateral tail vein and the retro-orbital venous sinus routes of antibody administration in pharmacokinetic studies.

In pharmacokinetic studies, intravenous (i.v.) administration of antibodies to mice is usually done via the lateral tail vein. This approach can cause stress to the mice and has a high rate of failure because it is challenging to perform correctly. Administration via the retro-orbital venous sinus has been suggested as a good alternative to tail vein i.v. administration of antibodies. Evidence is still needed, however, to determine whether the route of administration has an effect on the absorption or the pharmacokinetic activity of the injected antibody. The authors compared serum concentrations and pharmacokinetic parameters of a therapeutic antibody administered via tail vein injection or via retro-orbital injection. The findings suggest that there is no difference in the absorption or pharmacokinetic activity of therapeutic antibodies when administered via the lateral tail vein versus the retro-orbital venous sinus.

Efficient immunoglobulin gene disruption and targeted replacement in rabbit using zinc finger nucleases.

Rabbits are widely used in biomedical research, yet techniques for their precise genetic modification are lacking. We demonstrate that zinc finger nucleases (ZFNs) introduced into fertilized oocytes can inactivate a chosen gene by mutagenesis and also mediate precise homologous recombination with a DNA gene-targeting vector to achieve the first gene knockout and targeted sequence replacement in rabbits. Two ZFN pairs were designed that target the rabbit immunoglobulin M (IgM) locus within exons 1 and 2. ZFN mRNAs were microinjected into pronuclear stage fertilized oocytes. Founder animals carrying distinct mutated IgM alleles were identified and bred to produce offspring. Functional knockout of the immunoglobulin heavy chain locus was confirmed by serum IgM and IgG deficiency and lack of IgM(+) and IgG(+) B lymphocytes. We then tested whether ZFN expression would enable efficient targeted sequence replacement in rabbit oocytes. ZFN mRNA was co-injected with a linear DNA vector designed to replace exon 1 of the IgM locus with ∼1.9 kb of novel sequence. Double strand break induced targeted replacement occurred in up to 17% of embryos and in 18% of fetuses analyzed. Two major goals have been achieved. First, inactivation of the endogenous IgM locus, which is an essential step for the production of therapeutic human polyclonal antibodies in the rabbit. Second, establishing efficient targeted gene manipulation and homologous recombination in a refractory animal species. ZFN mediated genetic engineering in the rabbit and other mammals opens new avenues of experimentation in immunology and many other research fields.

A novel regulator of telomerase. S100A8 mediates differentiation-dependent and calcium-induced inhibition of telomerase activity in the human epidermal keratinocyte line HaCaT.

Recently we reported a differentiation-dependent inhibition of telomerase activity in human epidermis. Consistent with this observation we found that in keratinocyte cultures calcium-induced differentiation correlates with a decline in telomerase activity. To get further support for a role of calcium in the regulation of telomerase and to elucidate the underlying molecular mechanisms we investigated the effect of calcium on telomerase in the human epidermal keratinocyte line HaCaT. Treatment with thapsigargin, which increases intracellular calcium concentrations, inhibited telomerase activity without down-regulating the expression of hTERT (human telomerase reverse transcriptase). This observation together with the fact that increasing calcium reduced telomerase activity in cell-free extracts suggests that calcium directly interacts with the telomerase complex. This interaction could be mediated by the calcium-binding protein S100A8 as indicated by its ability to mimic the inhibitory effect of calcium. S100A8-induced reduction in telomerase activity was abrogated by S100A9. The ratio of both proteins remained constant in cells treated with thapsigargin, but their interactions were altered similarly in intact cells after thapsigargin treatment and in cell-free extracts in response to calcium. We hypothesize that calcium binds to S100A8/S100A9 complexes and alters their composition, thus enabling S100A8 to interact with the telomerase complex and inhibit its activity.

MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes.

MRP14 (S100A9) is the major calcium-binding protein of neutrophils and monocytes. Targeted gene disruption reveals an essential role of this S100 protein for transendothelial migration of phagocytes. The underlying molecular mechanism comprises major alterations of cytoskeletal metabolism. MRP14, in complex with its binding partner MRP8 (S100A8), promotes polymerization of microtubules. MRP14 is specifically phosphorylated by p38 mitogen-activated protein kinase (MAPK). This phosphorylation inhibits MRP8/MRP14-induced tubulin polymerization. Phosphorylation of MRP14 is antagonistically regulated by binding of MRP8 and calcium. The biologic relevance of these findings is confirmed by the fact that MAPK p38 fails to stimulate migration of MRP14(-/-) granulocytes in vitro and MRP14(-/-) mice show a diminished recruitment of granulocytes into the granulation tissue during wound healing in vivo. MRP14(-/-) granulocytes contain significantly less polymerized tubulin, which subsequently results in minor activation of Rac1 and Cdc42 after stimulation of p38 MAPK. Thus, the complex of MRP8/MRP14 is the first characterized molecular target integrating MAPK- and calcium-dependent signals during migration of phagocytes.

Transgenic mice reveal novel activities of growth hormone in wound repair, angiogenesis, and myofibroblast differentiation.

An increasing number of patients are being treated with growth hormone (GH) for the enhancement of body growth but also as an anti-aging strategy. However, the side effects of GH have been poorly defined. In this study we determined the effect of GH on wound repair and its mechanisms of action at the wound site. For this purpose, we performed wound healing studies in transgenic mice overexpressing GH. Full thickness incisional and excisional wounds of transgenic animals developed extensive, highly vascularized granulation tissue. However, wound bursting strength was not increased. Wound closure was strongly delayed as a result of enhanced granulation tissue formation and impaired wound contraction. The latter effect is most likely due to a significantly reduced number of myofibroblasts at the wound site. By using in vitro studies with stressed collagen lattices, we identified GH as an inhibitor of transforming growth factor beta-induced myofibroblast differentiation, resulting in a reduction in fibroblast contractile activity. These results revealed novel roles of GH in angiogenesis and myofibroblast differentiation, which are most likely not mediated via insulin-like growth factors at the wound site. Furthermore, our data suggested that systemic GH treatment is detrimental for wound healing in healthy individuals.

Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.