CA2449186A1 - Generation of therapeutic regulatory dc by multiple gene silencing through induction of rna interference - Google Patents

Abstract

Methods of generating regulatory dendritic cells (DC) capable of stimulating and suppressing immune responses are disclosed. Compositions for inducing differentiation, transformation, and growth of said regulatory DC are thought. Delivery of nucleic acids capable of modifying dendritic cell function are further disclosed.

Description

DESCRIPTION
Field of the invention The present invention pertains to the field of immunology, more particularly to the field of generating immune regulatory DC. Said DC are a useful treatment for treatment of a variety of disorders associated by over-activation or pathological inhibition of immune responses.
Background It is known in the art that immune therapy offers exciting prospects for patients with cancer, autoimmunity and transplanted organs. Recently many researches have investigated the practical utility of dendritic cells (DC) as tools for immune modulation.
The ability of DC to act both as augmenters and inhibitors of immune response has prompted investigation into their therapeutic use in experimental models and clinically.
A shortcoming of DC-therapeutics is the present inability to gene-specifically modify the DC in an effective manner. It is the object of the present invention to disclose novel methods of manipulating DC, either through endowing stimulatory or inhibitory capacities. This is accomplished through silencing of immune regulatory genes, co-transfection with immune stimulatory genes, multiple targeting of synergistic targets, and cell-specific transfection.
Control of Immunity by DC
Stimulation and control of T cell [1], B cell [2], NK cell [3, 4] arid NKT
cell [5] function is co-ordinated directly and indirectly by the dendritic cell (DC). Acting as the most potent of all antigen presenting cells (APC), the DC is uniquely able to influence the immune response through possessing 3 broadly defined molecular signals: 1) Direct molecules for stimulation of the T cell receptor TCR. These include MHC I, MHC
II, and CD 1 d for stimulation of the conserved NKT cell TCR [6]. 2) Membrane-bound costimulatory signals (ie CD40, CD80/86 and OX-40L) [7]. 3) Soluble stimulatory molecules (ie IL-12, LIGHT) [8]. Additionally, the biology of the DC is uniquely formed for its ability to activate T cell responses. Generally immature DC are found in the periphery, constantly patrolling for foreign antigens. Immature DC are highly phagocytic, but possess low T cell activatory activity. Upon recognition of various foreign entities, DC mature, upregulate expression of lymph node homing receptors, and migrate into T cell-rich areas for stimulation of immunity [9]. Phagocytosed material is stored inside DC endosomes and upon activation, the pre-formed endosomes are rapidly exported to the cell surface where the MHC II-Ag complexes activate T cells [9]. On the other hand, the ability of the immature DC to constantly phagocytose self antigens, leads to its ability to generated tolerance to "self' and thus prevent autoimmunity [10].
The ability of DC to control whether a stimulatory or inhibitory response will follow antigenic immunization depends of which of the 3 signals described above are present, and in what concentration they are present. For example, we have previously generated tolerogenic DC (Tol-DC) through the inhibition of the IKK-(3 pathway using LF-15015, an analogue of the immune-suppressive drug deoxyspergualin [11].
These Tol-DC are inhibitors of T cell activation, inhibitors of costimulated T
cells, and induce production of T regulatory cells. Interestingly, the expression of MHC II, CD40, CD$6 and IL-12 was suppressed. Supporting the idea that Tol-DC possess less of the Signals 1,2 or 3 comes from experiments with KLH-pulsed DC from CD40-knockout which induced antigen-specific T regulatory (Treg) cells in vivo [12]. On the other hand, DC
transfected to expression high levels of one or more of the 3-Signals can be used for stimulating potent immune responses against viruses, bacteria, or cancer-antigens [13].
Such stimulatory DC are particularly beneficial in clinical circumstances where a predisposition exists for weakened immune response such as cancer, in which DC
vaccination upregulates beneficial Thl immunity [14].
Generation of Stimulatory DC Through Various Manipulations The fact that DC are potent stimulators of immunity has prompted their use for treatment of conditions that require activation of T cells such as cancer. The inherent immunogenicity of DC can be upregulated by stimulation of these cells through various receptors, such as the Toll-like receptor (TLR) family. Ligands of these receptors such as long double-stranded RNA (TLR-3) [15], unmethylated cpg motifs (TLR-9) [16], and imiquimod (TLR-?) [17] are successful for stimulation. Host factors, however, play certain roles that can inhibit ability of DC to stimulate immune response. For example, melanoma is known to secrete high amounts of vascular endothelial growth factor (VEGF) that inhibits DC maturation through blocking NF-kB activation [18].
Alternatively, prostate cancer secretes a soluble DC-apoptosis inducing factor [19]. In order to generate more potent DC, investigators have transfected DC with antigenic mRNA [20], IL-12 gene [21], flt-3L gene [22], yr GM-CSF plasmid [23]. These have produced increased immune stimulatory potency of the DC, however further work needs to be performed for optimization. Consequently, a more detailed understanding of the receptor-ligand interactions associated with inhibition of DC will assist in developing modified DC that are successful for usage in these conditions. A potential modification of the DC for use in cancer would be blocking the receptors associated with immune suppressive signals, such as the IL-10 [24] or VEGF receptors [25].
Generation of Stimulatory DC Through Various Manipulations An immature DC is classified phenotyically as having low expression of Signal 1,2, and 3, and functionally as a poor stimulatory of mixed lymphocyte reaction (MLR) [26].
Typically, immature DC are known to be tolerogenic, inhibiting immune response through providing weak Signal 1,2 and/or 3 [27]. While immature DC are highly phagocytic and can be pulsed with specific antigens for a "tolerogenic vaccine", a concern is that these DC will mature upon in vivo administration. Generation of maturation-resistant DC was reported by Lutz et al through culture in low dose GM-CSF
in absence of IL-4 [28]. Further studies have generated immature DC through culture with inhibitory cytokines such as IL-10 [29] and TGF-(3 [30], although maturation-resistance was not evaluated. Recent studies have noted that Tol-DC and immature DC
may not be exactly the same. For example Sato et al demonstrated that for maximum tolerogenicity, DC must be raised under conditions that inhibit Signal 2 and 3, while stimulation with LPS/TNF-alpha is needed for induction of high level of Signal 1 [31 ].

In light of this, it will be important to specifically be able to silence certain immune stimulatory genes, while at the same time not alter the basal immune inhibitory genes found in the DC.
DC can either be transfected with certain molecules, or endogenous signals from the DC
can be inhibited by either pharmaceutical, genetic, or cell-culture techniques. There are a multitude of reports describing gene-transfection of DC. We ourselves have used FasL-transfected DC for the induction of donor-reactive apoptosis [32]. Others have reported that augmenting the levels of immune inhibitory cytokines through transfection with IL-4 [33], IL-10 [34] or TGF-(3 [35], allows the DC to inhibit graft rejection, or to protect from autoimmune diseases.
Gene Manipulation of DC
Antisense oligonucleotides (AO) are sequences of DNA designed to block target genes by annealing with target mRNA, forming a RNA-DNA duplex, which is recognized and cleaved by the enzyme RNase H [36]. AO was the first therapeutic modality to offer the hope of gene-specific suppression. Disadvantages of the original AO technology included susceptibility of the nucleotides to intracellular, and in vivo degradation.
Overcoming this problem through the use of morpholino and phosphothiorate backbones has led to widespread interested in AO therapies [37, 38]. Unfortunately, the problems of non-specific suppression, and longevity of suppressing effect remain significant drawbacks that impede the wide-spread clinical use of AO as drugs. In fact, a recent Phase III trial of AO for colitis has demonstrated no significant benefit [39].
Despite these drawbacks, the ability of AO to specifically inhibit genes of interest has stimulated the interest of immunologists. Due to the importance of cytokines in controlling immune functions, immunomodulation using AO to cytokines has been proposed [40]. An interesting early experiment targeted the T cell stimulatory cytokine IL-2 in the context of allograft rejection. Using osmotic pumps to deliver AO
to IL-2, Qu et al have increased allograft survival by blocking IL-2 production in murine cardiac S
allograft recipients [41]. Targeting other genes important for immune function has also been performed. Blocking expression of the LPS co-receptor CD14 using AO, increased survival in murine models of scepticemia [42). By targeting expression of intercellular adhesion molecule-1 with AO, Toda et al reduced leukocyte-induced damage to ischemic lungs [43].
As DC are the most potent immune regulatory cell, application of AO to this cell is of particular interest. DC transfection with AO has been reported using either electroporation or liposomal methods. Interestingly, one of the first gene targets using AO on DC was the peptide transporter, Tr~anspo~t Associated Frotein (TAP).
Inhibition of this protein on DC was able to endow the cells with more potent antigen presenting function [44]. Following this finding, targeting of the MHC invariant chain with AO was also performed. Through facilitating less competition for the MHC binding groove, DC
with suppressed invariant chain where able to prime immune responses better than control DC [45]. Another AO approach to generating DC with heightened immune stimulating capability was performed by suppressing the inhibitory cytokine IL-10. It is known that during DC differentiation, IL-10 acts in a negative autocrine manner to regulate the maturation, and T cell-stimulating capacity of DC. Taking this into account, Igietseme et al compared the T cell-activating function of wild-type DC, DC
from IL-10 knockout mice, and DC treated with AO to IL-10. The knockout and the AO-suppressed IL-10 DC possessed greater T cell stimulating function, and also invoked the generation of a Thl phenotype (ie high IFN-y, low IL-4) [46].
Inhibitory DC would have practical applications for treatment of transplant rejection and autoimrnunity. To this end, inhibition of the T cell costimulatory molecules CD80 and CD86 was reported by AO. DC in which either CD80 or CD86 was suppressed possessed inhibited allostimulatoxy activity, induced high IL-4, low IFN-g production from T cells, increased the percentage of apoptotic T cells in culture, and inhibited the generation of CD8 CTL in vivo [47). Importantly, the administration of AO
manipulated DC to cardiac recipients was able to prolong allograft survival, although only modestly [47]. Targeting of other DC-bound immune stimulatory molecules was also performed.

Gorczynski et al demonstrated that suppressing expression of MD-1 on DC
resulted in inhibition of allostimulatory activity, Thl>Th2 cytokine switch and prolongation of allograft survival [48]. These effects were dependent on ability of suppressed MD-1 to increase expression of the DC inhibitory molecules OX-2. In addition to this, AO
inhibition of the novel costimulatory molecule B7H3 on DC has resulted in DC
with similar inhibitory functions as described above [49J. The ability to induce immune modulation through suppressing DC genes suggests a novel and practical method of altering immune function. The recent observation that administration of manipulated DC
can not only inhibit the generation of immune response, but can also inhibit a T cell response after initiation, suggests the practicality of DC immunotherapy [12].
However, the fact that AO possess temporally limited effects provides the concern that DC may start to re-express the immune stimulatory genes after being placed in vivo.
In such a situation, the administered DC may actually serve the counter-purpose of being immune stimulatory. Although AO are theoretically promising, clinical applications have not been beneficial. Additionally, several problems are intrinsic to AO
therapeutics: 1. Large quantities are needed for effects; 2. Lack of specificity in some cases [S0, 51] and; 3.
Poor transfection into target cells [S0, 51]. For example, in the study cited above using IL-2 specific AO to block graft rejection, a very high dose of AO was needed to be administered using continuous intravenous osmotic pump in order to achieve a modest graft survival benefit over untreated controls [41]. Similarly, although AO
have entered Phase III clinical trials, there was no significant difference over placebo [52]. For these reasons, novel methods therapeutically applicable gene-specific silencing are desired.
RNA interference (RNAi) RNAi is a process by which a double-stranded RNA (dsRNA) selectively inactivates homologous mRNA transcripts. The initial suggestion that dsRNA may possess such a gene silencing effect came from work in Petunias in which overexpression of the gene responsible for purple pigmentation actually caused the flower to lose their endogenous color [53]. This phenomenon was termed co-suppression since both the inserted gene transcript and the endogenous transcript Were suppressed. In 1998, Fire et~ al injected C.

elegans with RNA in sense, antisense and the combination of both in order to suppress expression of several functional genes. Surprisingly, injection of the combined sense and antisense RNA led to more potent suppression of gene expression than sense or antisense used individually. Inhibition of gene expression was so potent that approximately 1-3 molecules of duplexed RNA per cell were effective at knocking down gene expression.
Interestingly, suppression of gene expression would migrate from cell to cell and would even be passed from one generation of cells to another. This seminal paper was the first to describe RNAi [54]. One problem present at the initial description of RNAi, and subsequent papers following, was that in order to induce RNAi, long pieces 200-800 base pairs, of dsRNA had to be used. This is impractical for therapeutic uses due to the sensitivity of long RNA to cleavage by RNAses found in the plasma and intracellularly.
In addition, long pieces of dsRNA induce a panic response in eukaryotic cells, part of which includes nonspecific inhibition of gene transcription but production of interferon-a [55]. In 2001, it was demonstrated that subsequent to entry of long dsRNA
duplex into the cytoplasm, a ribonuclease III type enzymatic activity cleaves the duplex into smaller, 21-23 base-pairs which are active in blocking endogenous gene expression.
These small pieces of RNA, termed small interfering RNA (siRNA) are capable of blocking gene expression in mammalian cells without triggering the nonspecific panic response [56].
Therefore, there are 2 methods of inducing RNAi, the naturally occurring method that takes place when viral or long double-stranded RNA enters the cell. Upon crossing the membrane, the dsRNA is recognized by: 1) 2'S' OS, an enzyme that turns on an enzymatic cascade leading to inhibition of protein synthesis, 2) Activation of the protein kinase R (PKR) which also results in non-specific shut-down of cellular activity, and 3) DICER, a nuclease cuts the dsRNA into 21-23 base-pairs that are active in blocking endogenous gene expression (Figure lA). This method of gene-silencing is not advantageous for research or experimental purposes due to the non-specificity of effects.
However, theoretically it is conceivable that administration of long dsRNA
targeting cancer immune suppressive genes would have the two-fold effect of non-specifically blocking tumor proliferation, as well as silencing the immune suppressive genes. The other method of inducing RNAi is through administration of pre-formed, synthetic siRNA of 21-23 nucleotide base-pairs. This approach only targets the endogenous RNA
transcript and does not possess indiscriminate inhibitory effects (Figure 1B).
Several recent studies have demonstrated the utility and practicality of siRNA
mediated gene silencing for blocking expression of disease-associated genes in vitro.
Novina et al demonstrated inhibition of HIV entry and replication using siRNA specific for CD4 and gag, respectively [57]. Suppression of human papilloma virus gene expression in tissue biopsies from women with cervical carcinoma was reported using siRNA specific fox the E6 and E7 genes [58). Furthermore, induction of leukemic cell line apoptosis and complete inhibition of bcr-abl expression was achieved using siRNA [59]. The first report of siRNA used in animal models is from McCaffrey et al who suppressed expression of luciferase in mice by administration of siRNA using a hydrodynamic transfection method [60]. A subsequent study using HeLa cells xenografted on nude mice compared efficacy of gene suppression between AO and siRNA. Consistent with in vitro suggestions, in vivo siRNA administration resulted in a more potent and longer lasting suppression of gene expression than obtained with AO [61].
Silencing gene expression through siRNA is superior to conventional gene or antibody blocking approaches due to the following: 1) Blocking efficacy is more potent [61]; 2) Targeting gene expression is more specific [62]; 3) Inhibitory effects can be pass for multiple generations [63]; 4) In vitYO transfection efficacy is higher and can be expressed in a stable manner [64]; 5) In vivo use is more practical and safer due to lower concentration needed and no neutralizing antibody; 6) Tissue or cell specific gene targeting is possible using specific promoter vector [65, 66] or specific antibody conjugated liposome; 7) Simultaneously targeting multiple genes or multiple exons silencing is possible for increasing efficacy [67].
DESCRIPTION OF THE INVENTION
The disclosed invention teaches methodologies for manipulation of DC in order to stimulate or inhibit immune responses. One embodiment of the present invention is generation of an inhibitory regulatory DC (iREG-DC) population that is suppressive to T
cell responses. More specifically, the invention teaches that iREG-DC can be produced through selective silencing of a single or multiple immune stimulatory genes using the technique of RNAi. Additionally, the inhibitory capacity of iREG-DC can be further augmented through transfectian with genes known to inhibit immune responses.
Genes encoding molecules that both stimulate and inhibit ability of the DC to activate T cells are well known and described in the art. The advantage of generating iREG-DC
resides in their ability to act as an antigen-specific activator of T cells when the iREG-DC is pulsed with antigen. Delivery of antigens into iREG-DC can be performed before or subsequent to gene silencing and/or transfection. Methods of introducing antigens to DC
include but are not limited to co-culture with antigenic proteins, peptides, or mRNA
encoding the antigen of interest. An alternative approach for delivery of antigen includes transfection of iREG-DC with a plasmid encoding the antigenic protein or derivatives thereof. Ability of iREG-DC to inhibit immune responses can be assessed both in vitro and based on in vivo efficacy. During production of iREG-DC parameters such as ability to induce Treg formation, ability to inhibit MLR, and capacity for inducing a Thl>Th2 shift in T cell cytokine profile can be used to determine the regulatory abilities of iREG-DC.
Generation of iREG-DC can be initiated with a bone marrow culture for production of bone marrow-derived DC. Said cultures have been extensively described in the art. One example of generating BM-DC involves: l) Extraction of bone marrow from femurs and tibia of given mice followed by purification of mononuclear cells through either density gradient methods such as Ficoll, or lysis of erthrocytes using a hypotonic lysis buffer solution; 2) Said mononuclear cells are then cultured in a media suitable for sustaining cellular viability. Suitable culture media include RPMI, DMEM, Opti-MEM
supplemented with fetal calf serum or the AIM-V which does not require serum supplementation. In order to induce proliferation of the monocytic progenitor cells granulocyte-monocyte colony stimulating factor is added to the culture media at a concentration between 5 ng/ml to 100 ng/ml, but preferably, 10 ng/ml.
Interleukin-4 is simultaneously added to the culture in order to suppress macrophage overgrowth. After 5-12 days, of culture, but preferable 7-days, a population of cells arises that possesses a high concentration of DC as witnessed by CD1 lc expression; 3) The generated DC
population is then transfected with siRNA in order to silence immune stimulatory genes.
The siRNA may be delivered in the form of free oligonucleotides, siRNA-expression cassettes, siRNA-expression plasmids, or siRNA-carrying viral vectors.
Suitable targets for silencing include gene encoding adhesion molecules, membrane-bound immune stimulatory molecules, cytokines, and immune-stimulatory transcription factors such as NF-kB, STAT4, T-bet, and GATA-3; 4) In order to increase the efficacy of suppression by iREG-DC simultaneous cotransfection, or transfection subsequent to gene silencing may be performed with genes encoding immune suppressive molecules, said molecule may include TGF-b, IL-10, thrombospondin, indolamine-dioxygenase, or galectin-3; 5) Antigen-specificity of iREG-DC can be endowed through pulsing said cells with antigens of interest. The pulsing procedure could comprise of addition of exogenous antigens in the form of proteins, peptides, or aggregated peptides.
Alternatively, genetic pulsing of iREG-DC can be performed through transfecting said cells using mRNA
encoding the antigen of interest, or a plasmid that once internalized will transcribe the given antigen. Advantages of genetic pulsing include the preferential localization of the antigens in the "intracellular" pathway of antigen presentation. Antigens entered through this route preferentially become expressed on MHC I and target CD8+ T cells.
The pulsing procedure can be performed both before and/or after the silencing/transfection step of gene manipulation described. In some circumstances it may be advantageous to add antigens to the DC preparation early in the culture time since immature DC
possess a higher endocytic rate compared to mature ones. Once iREG-DC are generated, they can be used to treat a variety of diseases associated with immune hyperactivation.
It will be obvious to one skilled in the art that many variations of the above iREG-DC
generating approach can be performed without departing from the scope, or spirit of the invention disclosed. For example, DC progenitors can be purified from a variety of sources besides the bone marrow. For clinical use, DC are generally obtained from monocytic cultures whose starting source is monocytes obtained from peripheral blood mononuclear cells that are subsequently cultured in IL-4 and GM-CSF. Other methods of generating DC include addition of calcium ionophore or the peptide EPI.b recently.
Using the example of multiple sclerosis (MS) as a prototypic autoimmune disease, iR.EG-DC can be used as a cellular therapy by pulsing said cells with antigenic targets important in MS. It is known both in clinical MS, and in animal models that myelin basic protein (MBP) acts as an autoantigen. Clinical trials attempting to tolerize recipients to MBP
have shown some success in ameliorating disease progression, however effects are transient and inconsistent. Administration to the patient a composition of MBP-pulsed iREG-DC can be a useful method of specifically inactivating pathogenic T
cells, while at the same time inducing production of T-regulatory cells that would prevent reoccurrence of autoimmune attack. Such an approach to treatment of autoimmunity is more beneficial than commonly used approaches such as immune suppressants that non-specifically inhibit all T cell activity, leading to increased susceptibility to pathogens and neoplasms.
Another embodiment of the invention involves generation of iREG-DC in vivo. It is known that a variety of adjuvants such as Complete Freund's Adjuvant (CFA) possess the ability to initiated macrophage and dendritic cell homing and uptake of exogenous entities. Based on our findings that siRNA can be uptaken by DC in absence of specialized transfection reagents, we have admixed CFA with siRNA for immune stimulatory gene with the antigen we sought to modify. The mixture of CFA, siRNA, and antigen subsequently initiate an immune regulatory process that results in the generation of T regulatory cells and inhibition of antigen-specific response.
Such a tolerance-inducing vaccine or "ToleroVax" can be used for inhibiting immune responses to a variety of autoimmune disease including but not limited to: rheumatoid arthritis, Stevens-Johnson syndrome, juvenile rheumatoid arthritis, psoriatic arthritis, allergies, psoriasis, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, multiple sclerosis, allergic encephalomyelitis, systemic lupus erythematosus, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, scleroderma, Wegener's granulomatosis, chronic active hepatitis, myasthenia gravis, idiopathic spree, lichen planes, Crohn's disease, Graves ophthalmopathy, sarcoidosis, contact dermatitis primary biliary cirrhosis, primary juvenile diabetes, dry eye associated with Sjogren's syndrome, uveitis posterior, and interstitial lung fibrosis.
Another embodiment of the invention is preparation of immune stimulatory DC
termed sREG-DC. Using the culture procedures described above for generation of iREG-DC, sREG-DC are produced by silencing immune suppressive genes and/or transfection with immune stimulatory genes. Similar to the preparation of i~EG-DC, sIREG-DC can be pulsed with a variety of antigens in order to increase the magnitude and specificity of immune responses. sREG-DC are of great benefit for viral infections such as HIV where it is preferential to induce responses in a polyvalent fashion due to the great variability of antigens on the viral surface. Immunological depression in neoplasia results in an inability to mount successful responses. In addition, the ability of cancers to suppress the antigen-presenting compartment of the immune response often makes conventional vaccination strategies unsuccessful.
Examples 1. Incorporation of siIZNA into DC by endocytosis DC were cultured from bone marrow progenitors in the presence of GM-CSF and IL-4. 1 x 106 day-7 cultured DC were liposomely transfected with unlabeled (control siRlVA), or fluorescein labeled (Fl-siRNA) siRNA specific for luciferase sequence at indicated concentration (upper panel). Alternately, these siRNAs were added to DC
culture without transfection reagent at day 5. DC were activated with LPSlTNFa on day 8 and the transfection efficacy assessed by flow cytametry on day 9. The data on Figure 1.
indicates that DC can be efficiently (80-90%) transfected by phagocytosis.
2. Silencing of DC by siRNA-containing organ storage solution DC were cultured for 4 days in 6-well plate. SEC-GAPDH was added in the culture without transfection reagents. DC were collected at indicated times and stained with anti-GAPDH mAb. The expression of GAPDH was analyzed using flow cytometry and compared with control DC (bolded lines) and silenced DC (fine lines) (Figure 2).
Incorporation of fluorescent (Fl) siRNA into perfused kidney. Fl-siRNA
targeting luciferase was admixed with Ringer's lactated solution at a concentration of 100 nMol.
A total of 4 ml of the said admixture was flushed through a kidney from an anaesthetized BALB/c mouse. Subsequently the kidney was clamped for half an hour.
Fluorescence was detected by confocal microscopy. A significant increase in green staining was observed in the perfused kidney (siPerfuseTM Treated) compared to controls (Figure 3).
Silencing of GAPDH in a perfused kidney model. siRNA targeting GAPDH was designed and was admixed with Ringer's lactated solution at a concentration of nMol. A total of 4 ml of the said admixture was flushed through a kidney from an anaesthetized BALB/c mouse. Subsequently the kidney was clamped for half an hour.
GAPDH expression was detected by immunohistochemistry. A significant decrease in GAPDH staining was observed in the perfused kidney compared to control (Figure 4).
3. Mufti-gene silencing in DC
pSilencer-IL12 and pSilencer-GAPDH were co-transfected to 7-day cultured DC by GenePorter. 48 hours after gene silencing, the control siRN.A transfected DC
(control-DC, upper panel) and siRNA transfected DC (lower panel) were staining with anti-mouse IL-12 and GAPDH (bolded lines) respectively. The isotype controls are shown as broken lines (Figure 5).
4. Gene-silencing of MHC II
A siRNA construct derived from SEQ 1D NO 2, specifically targeting MHC II
alpha chain was inserted into the commercially available pSilencer and added to marine BM-DC culture at the concentration of lug/ml at day 4. On Day 7 cells were activated with LPS and TNF at a concentration of 10 ng/ml. MHC II expression was detected by flow cytometry on 24-48 hours later. A profound inhibition of MHC II expression was observed in the siRNA-pSilencer treated groups (Figure 6).

5. Gene-silencing of IL-12 p35 A siRNA construct derived from SEQ ID NO 1, specifically targeting IL-12p35 was inserted into the commercially available pSilencer and added to murine BM-DC
culture at the concentration of lug/ml at day 4. On Day 7 cells were activated with LPS and TNF at a concentration of 10 nglml. IL-12 expression was detected by flow cytometry on 24-48 hours later using intracellular staining. A profound inhibition of IL-12 expression was observed in the siRNA-pSilencer treated groups (Figure 7).

6. Gene-silencing of CD40 using SEC
SEC were generated as PCR products consisting of a hairpin siRNA template flanked by promoter and terminator sequences. Once the SEC is transfected into cells, the hairpin siRNA is expressed from the PCR product and leads to gene silencing. siRNA
expression cassette kit (Ambion Inc, Texas USA) was used to generate SEC
targeting CD40. Briefly, 4 targets of 21 nucleotide long were selected from cDNA
(GeneBank).
The active sequences are designated SEQ ID NOS 3-6. 3 PCR reactions were performed to generate SEC. The first reaction formed half of the hairpin siRNA (sense), the second reaction made the precursor SEC (second half of the siRNA hairpin), the last reaction added the terminator and restriction sites for cloning to the SEC. CD40-SEC(four different target sites: SEQ 1D NOS 3-6) were added into DC culture at 150ng/ml at day6 followed by transfection of 300ng SEC at day 7. DC were activated with LPS
with a period of 20 hours. FAGS was performed at day 8. Varying degrees of CD40 inhibition were seen by flow cytometry (Figure 8).

7. Gene-silencing of IL-12 using SEC
SEC were generated as PCR products consisting of a hairpin siRNA template flanked by promoter and terminator sequences. Once the SEC is transfected into cells, the hairpin siRNA is expressed from the PCR product and leads to gene silencing. siRNA
expression cassette kit (Ambion Inc, Texas USA) was used to generate SEC
targeting IL-12. Briefly, 4 targets of 21 nucleotide long were selected from cDNA
(GeneBank). The active sequences are designated SEQ 1D NOS 15-18. 3 PCR reactions were performed to generate SEC. The first reaction formed half of the hairpin siRNA (sense), the second reaction made the precursor SEC (second half of the siRNA hairpin), the last reaction added the terminator and restriction sites for cloning to the SEC. IL-12 SEC(four different target sites: SEQ ID NOS 15-18) were added into DC culture at 150ng/ml at day 6 followed by transfection of 304ng SEC at day 7. DC were activated with LPS
with a period of 20 hours. FACS was performed at day 8. Varying degrees of IL-12 inhibition were seen by flow cytometry (Figure 9).

8. pInterference Plasmid siRNA sequence of a variety of specificities can be inserted in our plnterference plasmid (Figure 10) for expansion and utilization in mammalian cells. Mun I and Hindi III
restriction sites serve as the area for integrating SEC DNA that was described in SEQ ID
NOS 3-18. The pInterference plasmid was used to deliver SEC DNA targeting SEQ
ID
4, which specifically inhibits expression of CD40. Treatment with plnterference containing mismatched DNA did not modify expression of CD40 (Figure 1 la). In contrast plnterference containing the CD40-specific SEQ ID 4 was able to markedly reduce CD40 expression (Figure l lb).

9. Inhibition of ReIB expression siRNA targeting ReIB specific SEC ID NOS 19-22 was administered to DC using GeneSilence reagent. Assessment of RelB expression was performed using RT-PCR.
A
23%, 69%, 95%, and 15% reduction in ReIB mRNA was observed compared to control cells using SEC ID 19, 20, 21, and 22, respectively Sequence Listings Sequence ID: 1 SEQUENCE CHARACTERISTICS: IL-12 sense construct LENGTH: 65 TYPE: DNA
ORGANISM: Synthetic gatcccgCCTGCTGAAGACCACAGATttcaagagaATCTGTGGTCTTCAGCAGGttttttgga as Sequence m: 2 SEQUENCE CHARACTERISTICS: MHC II alpha Sense construct LENGTH: 64 TYPE: DNA
ORGANISM: Synthetic gatcccGACGACATTGAGGCCGACCttcaagagaGGTCGGCCTCAATGTCGTCttttttggaa a Sequence ID: 3 SEQUENCE CHARACTERISTICS: CD40(1), Sense SEM Template LENGTH: 55 TYPE. DNA
ORGANISM: Synthetic ACACTACACAAATGTTCCACTGGGCTGAGAACCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 4 SEQUENCE CHARACTERISTICS: CD40(2), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic CCTCTACACAAAAGGTACAGACAGTGTCTGACCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 5 SEQUENCE CHARACTERISTICS: CD40(3), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic AAACTACACAAATTTCTGTAGGACCTCCAAGCCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 6 SEQUENCE CHARACTERISTICS: CD40(4), Sease SEA Template LENGTH: 54 TYPE: DNA
ORGANISM: Synthetic GTGCTACACAAACACTGAGATGCGACTCTCTCGGTGTTTCGTCCTTTCCACAA
G
Sequence ID: 7 SEQUENCE CHARACTERISTICS: CD80(1), Sense SEM Template LENGTH: 54 TYPE: DNA
ORGANISM: Synthetic CTCCTACACAAAGAGCCTTGGACATGGAAACCGGTGTTTCGTCCTTTCCACAA
G
Sequence II?: 8 SEQUENCE CHARACTERISTICS: CD80(2), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic GAGCTACACAAACTCATCTTCATGAGGAGAGCCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 9 SEQUENCE CHARACTERISTICS: CD80(3), Sense SEM Template LENGTH: 54 TYPE: DNA

ORGANISM: Synthetic AGACTACACAAATCTTATACTCGGGCCACACCGGTGTTTCGTCCTTTCCACAA
G
Sequence m: 10 SEQUENCE CHARACTERISTICS: CD80(4), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic GTTCTACACAAAAACCAAGAGAAGCGAGGCTCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 11 SEQUENCE CHARACTERISTICS: CD86(1), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic CTGCTACACAAACAGCTCACTCAGGCTTATGCCGGTGTTTCGTCCTTTCCACA
AG
Sequence lD: 12 SEQUENCE CHARACTERISTICS: CD86(2). Sense SEM Template LENGTH: 54 TYPE: DNA
ORGANISM: Synthetic AATCTACACAAAATTGATCCTGTGGGTGGCTCGGTGTTTCGTCCTTTCCACAA
G
Sequence ID: 13 SEQUENCE CHARACTERISTICS: CD86(3), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic TTCCTACACAAAGAATGAAAGAGAGAGGCTGCCGGTGTTTCGTCCTTTCCAC
AAG
Sequence 117: 14 SEQUENCE CHARACTERISTICS: CD86(4), Sense SEM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic ATCCTACACAAAGATAGTCTCTCTGTCAGCGCCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 15 SEQUENCE CHARACTERISTICS: II.-12(1) Sense, S8M Template LENGTH: 54 TYPE: DNA
ORGANISM: Synthetic AGACTACACAAATCTGTGGTCTTCAGCAGGTCGGTGTTTCGTCCTTTCCACAA
G
Sequence ID: 16 SEQUENCE CHARACTERISTICS: IL-12(2) Sense, SFM Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic ACCCTACACAAAGGTCATCATCAAAGACGTCCGGTGGTTCGTCCTTTCCACA
AG
Sequence 1D: 1?
SEQUENCE CHARACTERISTICS: IL-12(3) Sense, SFM Template LENGTH: 55 TYPE: DNA

ORGANISM: Synthetic ATCCTACACAAAGATCTGCTGATGGTTGTGACCGGTGTTTCGTCCTTTCCACA
AG
Sequence 117: 18 SEQUENCE CHARACTERISTICS: IL-12(4) Sens a SEbt Template LENGTH: 55 TYPE: DNA
ORGANISM: Synthetic GCTCTACACAA.AAGCAGGATGCAGAGCTTCACCGGTGTTTCGTCCTTTCCACA
AG
Sequence ID: 19 SEQUENCE CHARACTERISTICS: RelB Targetl, sense LENGTH: 19 TYPE: DNA
ORGANISM: Mouse GACCATAGATGAATTGGAA
Sequence ID: 20 SEQUENCE CHARACTERISTICS: RelB Target2, sense LENGTH: 19 TYPE: DNA
ORGANISM: Mouse GGACATATCCGTGGTGTTC
Sequence 117: 21 SEQUENCE CHARACTERISTICS: RelB Target3, sense LENGTH: 19 TYPE: DNA

ORGANISM: Mouse CATCGGAGCTGCGGATTTG
Sequence ID: 22 SEQUENCE CHARACTERISTICS: ReIB Target4, sense LENGTH: 19 TYPE: DNA
ORGANISM: Synthetic GCAGATCGCCATTGTGTTC
Sequence 117: 23 SEQUENCE CHARACTERISTICS: CD40 (target a) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AATTCTCAGC:CCAG'I'GGAAC~A.
Sequence ID: 24 SEQUENCE CHARACTERISTICS: CD40 (target b) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AATCAGACACTGTCTGTACCT
Sequence ID: 25 SEQUENCE CHARACTERISTICS: CD40 (target c) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AACTTGC'rAGGTCCTACACiAAA
Sequence ID: 26 SEQUENCE CHARACTERISTICS: CD40 (target d) LENGTH: 21 TYPE: DNA

ORGANISM: Mouse AAAGAGACrTCGCA'I'C'CCACi I'G
Sequence ID: 27 SEQUENCE CHARACTERISTICS: CD80 (target a) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAGTTTCCATGTCCAAGGt~'TC.
Sequence ID: 28 SEQUENCE CHARACTERISTICS: CD80 (target b) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAC:TCTCC~TCATGAAGATGAG
Sequence ID: 29 SEQUENCE CHARACTERISTICS: CD80 {target c) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAG'I'G'I"GGCC:C CiAG' I:'A'a'A A GA
Sequence ff~: 30 SEQUENCE CHARACTERISTICS: CD80 (target d) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAAGCCTC:GC:TTCTCTTGGTTA
Sequence ID: 31 SEQUENCE CHARACTERISTICS: CD86 (target a) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AACATAAGCCTGAGTGAGCTG
Sequence ID: 32 SEQUENCE CHARACTERISTICS: CD86 (target b) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAACiCt:AC.'CC;AC'AGGA'l:'CAA'1:' Sequence lD: 33 SEQUENCE CHARACTERTSTICS: CD86 (target c) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AACAGCCTC1~C'1'C'~1"I"fCA'1'~l'C
Sequence ID: 34 SEQUENCE CHARACTERISTICS: CD86 (target d) LENGTH: 21 TYPE: DNA
ORGANISM: Mouse AAt;GCTGACAGAGAGACTATC
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Claims (57)

1. A dendritic cell (DC) modified genetically through administration of a construct, said construct selected from a group comprising of: double stranded RNA (dsRNA), dsRNA
of less than 26 base pairs, and a vector that induces expression of dsRNA.

2. The DC of claim 1 wherein said vector is selected from a group comprising of: a plasmid, a short interfering RNA expression cassette, or a viral vector.

3. The vector of claim 2 wherein said vector is pInterference.

4. The DC of claim 1 wherein gene expression is modified through simultaneously inhibiting a plurality of 2 or more genes.

5. The DC of claim 1 wherein gene expression is modified through sequentially inhibiting a plurality of 2 or more genes.

6. A stimulating regulatory dendritic cell (sREG-DC) generated by silencing of immune inhibitory genes through methods that induce the process of RNA interference.

7. The sREG-DC of claim 6 wherein silencing is induced through administration of an RNA-duplex with homology to the immune inhibitory gene.

8. The RNA-duplex of claim 7 wherein the size of said duplex is between 19-27 base pairs.

9. The RNA-duplex of claim 7 wherein the size of said duplex is between 19-22 base pairs.

10. The sREG-DC of claim 6 wherein said immune inhibitory genes are selected from a group consisting of indoleamine 2,3-dioxygenase, interleukin 10, TGF-.beta.3, STAT-6, and GATA-3.

11. An inhibiting regulatory dendritic cell (iREG-DC) generated by silencing of immune stimulatory genes through methods that induce the process of RNA interference.

12. The iREG-DC of claim 11 wherein silencing is induced through administration of an RNA-duplex with homology to the immune inhibitory gene.

13. The RNA-duplex of claim 12 wherein the size of said duplex is between 19-27 base pairs.

14. The RNA-duplex of claim 12 wherein the size of said duplex is between 19-22 base pains.

15. The sREG-DC of claim 11 wherein said immune stimulatory genes are selected from a group consisting of IL-l,IL-2, IL,-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-19, IL-21, TNF-.alpha., IFN-.gamma., T-bet, Rel-b, Rel-c, NF-.kappa.B p50, NF-.kappa.B p65, STAT4, and T-bet.

16. The sREG-DC of claim 1 wherein expression of an immune stimulatory gene is induced for amplification of immune stimulatory activity.

17. The sREG-DC of claim 16 wherein said immune stimulatory gene is selected from a group comprising: IL-1, IL-2, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15, IL-17, IL-19, IL-21, TNF-.alpha., IFN-g, T-bet, Rel-b, Rel-c, NF-kB p50, NF-kB p65, STAT4, and T-bet.

18. The sREG-DC of claim 16 wherein immune stimulatory gene expression is accomplished through transfection of nucleic acids encoding said genes.

19. The sREG-DC of claim 16 wherein expression of said genes is induced through manipulation of culture conditions.

20. The sREG-DC of claim 19 wherein manipulation of culture conditions is accomplished through addition of agonists of toll-like receptors.

21. The iREG-DC of claim 11 wherein expression of an immune suppressive gene is induced for promotion of immune inhibitory activity.

22. The iREG-DC of claim 21 wherein said immune suppressive gene is selected from a group comprising: indoleamine 2,3-dioxygenase, interleukin 10, TGF-.beta., STAT-6, and GATA-3.

23. The iREG-DC of claim 21 wherein immune suppressive gene expression is accomplished through transfection of nucleic acids encoding said genes.

24. The iREG-DC of claim 21 wherein expression of said genes is induced through manipulation of culture conditions.

25. The iREG-DC of claim 24 wherein DC are cultured in media containing low concentrations of GM-CSF.

26. The iREG-DC of claim 24 wherein DC are cultured in media containing immune suppressive cytokines or hormones.

27. The iREG-DC of claim 26 wherein said immune suppressive cytokines include IL-4, IL-10, IL-13, IL-20, TGF-b, VEGF, and glucocorticoids.

28. A method of treating a patient suffering from cancer comprising stimulation of said patient's immune response through administration of sREG-DC.

29. The method of claim 28 wherein sREG-DC are co-administered with a tumor antigen.

30. The method of claim 28 wherein sREG-DC are administered intratumorally.

31. The method of claim 28 wherein sREG-DC are pulsed with a tumor antigen.

32. The method of claim 28 wherein said cancer is selected from a group comprising:
cancer of the cancers of the lung, breast, ovary, cervix, colon, head and neck, pancreas, prostate, stomach, bladder, kidney, bone liver, esophagus, brain, testicle, uterus.

33. A method of treating a patient suffering from an autoimmune disease comprising administration of said patient with iREG-DC in a concentration sufficient to induce alleviation of autoimmunity.

34. The method of claim 33 wherein iREG-DC are pulsed with an antigen known to be an inciting factor in the autoimmune process.

35. The method of claim 33 wherein said autoimmune disease is selected from a group comprising of rheumatoid arthritis, Stevens-Johnson syndrome, juvenile rheumatoid arthritis, psoriatic arthritis, allergies, psoriasis, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, multiple sclerosis, allergic encephalomyelitis, systemic lupus erythematosus, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, scleroderma, Wegener's granulomatosis, chronic active hepatitis, myasthenia gravis, idiopathic sprue, lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis, contact dermatitis primary biliary cirrhosis, primary juvenile diabetes, dry eye associated with Sjogren's syndrome, uveitis posterior, and interstitial lung fibrosis.

36. A vector for inducing expression of immune modulatory genes in dendritic cells.

37. A method of delivering nucleic acids capable of inducing RNA interference by addition of said nucleic acids to an organ storage solution.

38. The use of the organ storage solution from claim 37 for preservation of skin, heart, pancreas, lung, liver, kidney, cellular, and neuronal grafts.

39. A method of delivering nucleic acids capable of inducing RNA interference by incorporation of said nucleic acids into a carrier that can specifically target various cell types.

40. The method of claim 39 wherein said carrier is a liposome.

41. The method of claim 39 wherein said carrier is an immunoliposome.

42. A method of modifying dendritic cells by inducing RNA interference to a plurality of 2 or more immune associated genes.

43. A construct for inducing RNA interference in DC while simultaneously expressing an antigen to which immune modulation is desired.

44. A tolerogenic vaccine comprised of iREG-DC pulsed with an antigen to whom tolerance is desired.

45. A method of generating sREG-DC and iREG-DC in vivo through administration of an siRNA construct specific for immune modulatory genes from claims 10 and 15.

46. An immunoliposome loaded with a plurality of 2 or more siRNA sequences specific to immunomodulatory genes from claims 10 and 15.

47. An siRNA expression vector that upon transfection induces expression of siRNA
targeting the immunomodulatory genes from claims 10 and 15.

48. A composition for inhibiting expression of the interleukin 12 p35 gene comprised of nucleic acid with the sequence of SEQ ID NOS 1.

49. A composition for inhibiting expression of the MHC II gene comprised of nucleic acid with the sequence of SEQ ID NOS 2.

50. A composition for inhibiting expression of the CD40 gene comprised of nucleic acid with the sequence of SEQ ID NOS 3-6.

51. A composition for inhibiting expression of the CD80 gene comprised of nucleic acid with the sequence of SEQ ID NOS 7-10.

52. A composition for inhibiting expression of the CD86 gene comprised of nucleic acid with the sequence of SEQ ID NOS 11-14.

53. A composition for inhibiting expression of the IL-12 gene comprised of nucleic acid with the sequence of SEQ ID NOS 15-18.

54. An siRNA construct for inhibition of ReIB in which one chain of the double-stranded duplex possesses homology to the target nucleic acids described as SEQ ID NOS
19-22.

55. A composition useful for the inhibition of CD40 expression comprising a double stranded RNA duplex possessing Watson-Crick base pairing homology with a DNA
target selected from the group of SEQ ID NOS 23-26.

56. A composition useful for the inhibition of CD80 expression comprising a double stranded RNA duplex possessing Watson-Crick base pairing homology with a DNA
target selected from the group of SEQ ID NOS 27-30.

57. A composition useful for the inhibition of CD86 expression comprising a double stranded RNA duplex possessing Watson-Crick base pairing homology with a DNA
target selected from the group of SEQ ID NOS 31-34.