A novel ß-glucan-oligonucleotide complex selectively delivers siRNA to APCs via Dectin-1.

Delivering therapeutic nucleic acids to targeted cells and organs has been a challenge for decades. A novel technology to deliver oligonucleotide therapeutics to immune cells is here described. In this approach, a macromolecular complex of oligonucleotides and the ß-1,3-glucan schizophyllan (SPG) is selectively delivered to cells expressing a lectin receptor, Dectin-1, via SPG-Dectin-1 interaction. Detailed investigation of Dectin-1-expressing cells revealed that Dectin-1 is expressed in all subsets of monocytes as well as dendritic cell (DC) populations, including conventional DCs (cDCs) and plasmacytoid DCs (pDCs), in humans. The expression patterns in mice and humans are comparable, except for the expression in pDCs. The results indicate that Dectin-1 is expressed on cells capable of professional antigen presentation, except for B cells. We chose CD40 as a target gene for small interfering RNA (siRNA) as CD40 expression in antigen-presenting cells (APCs), particularly in DCs, plays critical roles in regulating immune responses. Dose-dependent cellular uptake of siCD40-SPG complexes was confirmed in cells expressing Dectin-1. Gene silencing activity was confirmed in vitro by the reduction of CD40 mRNA and by the site-specific cleavage of CD40 mRNA as determined by the 5' RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) technique. In vivo activity of siCD40-SPG complexes was demonstrated as the reduced CD40 protein expression in monocytes and DCs in mice. Furthermore, the in vivo activity of siCD40-SPG targeting human CD40 was confirmed in cynomolgus monkeys by the 5'RLM-RACE technique. In conclusion, we have demonstrated the receptor-ligand binding-mediated delivery of siRNA targeting immune-regulating monocytes and DCs via the interaction of SPG and its receptor, Dectin-1. As monocytes and DCs play central roles in inducing and controlling immune responses, Dectin-1-targeted delivery of nucleic acids should provide a useful tool for developing drugs to treat a wide range of diseases, including autoimmune diseases, allergy, and cancer, as well as transplantation.

Pharmacologic expansion of donor-derived, naturally occurring CD4(+)Foxp3(+) regulatory T cells reduces acute graft-versus-host disease lethality without abrogating the graft-versus-leukemia effect in murine models.

Adoptive transfer of regulatory T cells (Tregs) prevents graft-versus-host disease (GVHD) in mouse models, indicating a pivotal role for Tregs in controlling GVHD. The present study demonstrates the efficacy of Tregs pharmacologically induced in vivo in GVHD prevention. A single i.v. administration of a liposomal formulation of α-galactosylceramide (RGI-2001) at the time of allogeneic bone marrow transplantation with spleen cells significantly prolonged the survival of mice experiencing lethal acute GVHD. RGI-2001 expanded donor-derived CD4(+)Foxp3(+) Tregs in the spleen, lymph nodes, and bone marrow in a dose-dependent manner. On day 15 posttransplantation, the spleens of mice treated with RGI-2001 (1 µg/kg) contained 5-fold higher percentages or 10-fold higher numbers of CD4(+)Foxp3(+) Tregs compared with the spleens of untreated mice. Host-specific immunosuppression was introduced in treated mice, whereas the responsiveness to third-party alloantigens and leukemia cells was maintained. Using Foxp3:GFP reporter mice as donors, it was clearly shown that RGI-2001 expanded the pre-existing naturally occurring Tregs (nTregs) in donor spleen cells. Finally, RGI-2001 synergized with a subtherapeutic dose of rapamycin in nTreg expansion and further prolonged survival. Our results provide the first demonstration of the efficacy of nTregs pharmacologically expanded in vivo in preventing acute GVHD without abrogation of the beneficial graft-versus-leukemia effect.

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas: (monoclonal antibodylmouse embryos/teratocarcinoma/leukemic cell/cell surface marker).

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.