Microsphere-based delivery and ex vivo manipulation of dendritic cells for autoimmune therapies

Abstract

The present invention relates to tolerogenic mammalian dendritic cells (iDCs) and methods for the production of tolerogenic DCs. In addition, the present invention provides methods for administration of tolerogenic dendritic cells as well as particles containing oligonucleotides to mammalian subjects. Enhanced tolerogenicity in a host can be useful for treating inflammatory and autoimmune related diseases, such as type 1 diabetes.

1 - 20 . (canceled) 
     
     
         21 . A pharmaceutical composition suitable for injection comprising: a therapeutically effective amount of a population of manipulated tolerogenic dendritic cells comprising oligonucleotides comprising a nucleic acid sequence set forth as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, or combinations thereof in a fluidic phase. 
     
     
         22 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells are isolated from a donor. 
     
     
         23 . The pharmaceutical composition of  claim 22 , wherein the donor is a human, a non-human primate, or a mouse. 
     
     
         24 . The pharmaceutical composition of  claim 22 , wherein the population of tolerogenic dendritic cells are isolated from bone marrow, thymus, lymph node, skin, pancreas, peripheral blood or any combination thereof. 
     
     
         25 . The pharmaceutical composition of  claim 22 , wherein the tolerogenic dendritic cells are for autologous use, for allogeneic use, for xenogeneic use or any combination thereof after isolation from the donor. 
     
     
         26 . The pharmaceutical composition of  claim 22 , wherein the tolerogenic dendritic cells have been expanded to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 passage number before delivery to the subject. 
     
     
         27 . The pharmaceutical composition of  claim 22 , wherein the tolerogenic dendritic cells have been frozen and thawed after isolation from a donor. 
     
     
         28 . The pharmaceutical composition of  claim 27 , wherein the tolerogenic dendritic cells have been frozen for about 1 day, 1 week, 1 month, 1 year, 2 years, or 3 years after isolation from the donor. 
     
     
         29 . The pharmaceutical composition of  claim 22 , wherein the tolerogenic dendritic cells have been treated with oligonucleotides, growth factors, serum, cytokines, or any combination thereof after isolation from the donor. 
     
     
         30 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express one or more marker selected from the group consisting of CD19+, CD27+, CD38+, CD24+ and any combination thereof. 
     
     
         31 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express one or more marker selected from the group consisting of CD1B+, CD5+, CD19+, IL10+ and any combination thereof. 
     
     
         32 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express one or more marker selected from the group consisting of MHCII+, CD11c+, CD80+, CD40+, CD86+, and any combination thereof. 
     
     
         33 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells have a purity level of at least 70% as measured by expression of one or more marker selected from the group consisting of CD19+, CD27+, CD38+, CD24+, CD1B+, CD5+, CD19+, IL10+, MHCII+, CD11c+, CD80+, CD40+, CD86+, and any combination thereof. 
     
     
         34 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express one or more marker selected from the group consisting of CD11+, CD45+; CD83+HLA−DR+CD11c+; MHCII+, CD11c+, CD80+, CD40+, CD86+; CD1B+, CD5+, CD19+, IL10+; CD19+, CD27+, CD38, CD24+, CD27+, and any combination thereof. 
     
     
         35 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express high levels of one or more of CD1d, CD24, CD38, IgD, CD40, or any combination thereof. 
     
     
         36 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express medium levels of one or more of CD1d, CD24, CD38, IgD, or CD40, or any combination thereof. 
     
     
         37 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells express low levels of one or more of CD10, CD1d, CD24, CD38, IgD, or CD40, or any combination thereof. 
     
     
         38 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells are capable of inducing expression of FoxP3 in the naive T-cells, blocking the conversion of naive T-cells to TH17 T-cells, capable of deleting effector T-cells, retain their tolerogenic phenotype upon stimulation with at least one TLR agonist, increase expression of costimulatory molecules, do not transiently increase their oxygen consumption rate upon stimulation with at least one TLR agonist, capable of converting B-cells to regulatory B-cells, or any combination thereof. 
     
     
         39 . The pharmaceutical composition of  claim 21 , further comprising small molecules, hormones, lipids, proteins such as growth factors, cytokines, chemokines or combinations thereof. 
     
     
         40 . The pharmaceutical composition of  claim 21 , further comprising an additional cell population known to promote ex-vivo survival of the population of tolerogenic dendritic cells. 
     
     
         41 . The pharmaceutical composition of  claim 40 , wherein the additional cell population is from a feeder layer. 
     
     
         42 . The pharmaceutical composition of  claim 40 , wherein the additional cell population is a population of stromal cells, non-tolerogenic dendritic cells, cells isolated from spleen, cells isolated from bone marrow. 
     
     
         43 . The pharmaceutical composition of  claim 40 , wherein the additional cell population is a population of cells containing a functional moiety, wherein the function moiety is an antigen that can be recognized, a fluorescent protein, a fluorescent quantum dot, a radio-active isotope or a combination thereof. 
     
     
         44 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells are at least 50% viable. 
     
     
         45 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells are at least 70% viable. 
     
     
         46 . The pharmaceutical composition of  claim 21 , comprising a level of endotoxin that is less than about 5 EU/kg body weight. 
     
     
         47 . The pharmaceutical composition of  claim 21 , wherein the pharmaceutical composition is configured for intravenous, intramuscular, subcutaneous, intraperitoneal, intrathecal, epidural, intra-arterial, intra-articular, intranodal injection. 
     
     
         48 . The pharmaceutical composition of  claim 21 , comprising a semi solid suspension in the fluidic phase, a gel suspension in the fluidic phase, or any combination thereof. 
     
     
         49 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells comprises about 0.05×10 6 , 0.1×10 6 , 0.15×10 6 , 0.2×10 6 , 0.25×10 6 , 0.3×10 6 , 0.4×10 6 , 0.5×10 6 , 0.6×10 6 , 0.7×10 6 , 0.8×10 6 , 0.9×10 6 , 0.05×10 7 , 0.1×10 7 , 0.15×10 7 , 0.2×10 7 , 0.25×10 7 , 0.3×10 7 , 0.4×10 7 , 0.5×10 7 , 0.6×10 7 , 0.7×10 7 , 0.8×10, 0.9×10 7 , 0.05×10 8 , 0.1×10 8 , 0.15×10 8 , 0.2×10 8 , 0.25×10 8 , 0.3×10 8 , 0.4×10 8 , 0.5×10 8 , 0.6×10 8 , 0.7×10 8 , 0.8×10 8 , or 0.9×10 8  cells. 
     
     
         50 . The pharmaceutical composition of  claim 21 , wherein the population of tolerogenic dendritic cells comprises between about 1×10 5  and about 6.4×10 7  cells.