# Patent

## Title
Lentiviral transduced dendritic cells for RNAi

## Abstract

The invention provides methods to prepare and use lentivirus transduced mammalian dendritic cells with altered levels of T cell co-stimulatory molecules.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of the filing date of U.S. application Ser. No. 61/312,457, filed on Mar. 10, 2010, the disclosure of which is incorporated by reference herein in its entirety.


BACKGROUND
Dendritic cells (DC) are professional antigen-presenting cells of the immune system. They have the ability to take up, process and present antigens (Ag) to resting lymphocytes in draining lymph nodes. The importance of DC in initiation of T cell immunity and tolerance is firmly established (Steinman et al., 2003). Functionally mature DC are highly efficient at priming adaptive immune responses against viruses, pathogens and endogenous tumors (Banchereau and Steinman, 1998); whereas steady state “immature” DC are capable of uptaking and presenting self-Ag to tolerize autoreactive T cells (Wilson et al., 2003; Bonifaz et al., 2002; Hawiger et al., 2001; Scheinecker et al., 2002; Steinman et al., 2000). Phenotypic analysis of surface expression of co-stimulatory molecules has been routinely used in defining mature and “immature” DC. The ability to use DC in the induction of antigen (Ag)-specific tolerance, Ag-specific immunity or specific differentiation of T helper subsets holds great promises in DC-based immunotherapy of various diseases such as cancer, viral infections, allergy, as well as autoimmunity. The increasing number of co-stimulatory molecules identified to date, however, highlights the complex regulation of co-stimulatory signals.
RNA interference (RNAi) is an innate cellular process that involves multiple RNA-protein interactions (Fire, 1999; Hannon, 2002). Its gene silencing activity is activated when a double-stranded RNA (dsRNA) molecule of greater than 19 duplex nucleotides enters the cells, causing degradation of both the dsRNA and single stranded RNA (endogenous mRNA) of identical sequences (Fire, 1999; Hannon, 2002). Transient transfection of short synthetic dsRNA in DC demonstrated the feasibility of applying this technique in studying DC functions (Li et al., 2004; Gu et al., 2006; Hill et al., 2003; Laderach et al., 2003; Liu et al., 2004; Orabona et al., 2005; Li et al., 2007). A single lentiviral vector system has been established to stably express short hairpin RNA (shRNA) to induce RNA interference (RNAi) in cell lines and primary T cells (An et al., 2003; Qin et al., 2003).
SUMMARY OF THE INVENTION
The increasing number of co-stimulatory molecules identified on dendritic cells (DC) to date highlights the complex regulation of co-stimulatory signals in T cell activation. As described herein, a single lentiviral vector system to stably express short hairpin RNA (shRNA) to induce RNA interference (RNAi) was employed to regulate different levels of surface expression of a co-stimulatory molecule stably and reproducibly in dendritic cells. First, as described herein, it was demonstrated that lentiviral vectors induced RNA interference in DC without functional impairments. Then CD40 was employed as a target co-stimulatory molecule to demonstrate the feasibility of using lentiviral vectors in delivering different shRNA target sequences to genetically modify DC so as to express different levels of CD40. Functional data demonstrated that quantitative expression of CD40 on LPS-stimulated DC had different functional outcomes on Ag-specific T cell responses in vitro and in vivo. Collectively, this system allows the examination of functional significance(s) of the quantitative and/or qualitative expression of a single or multiple co-stimulatory molecule(s) on DC, provides a platform to genetically modify primary DC to express defined, quantitative expression levels of a protein of interest, and allows the genetic engineering of DC to define their functional properties (such as tolerance, Th1 inducing or Th2 inducing) in DC-based therapies.
The invention provides methods of making lentivirus transduced DC, e.g., primary DC, mature DC or immature DC, useful in the immunotherapy of a large number of immune and inflammatory disorders (e.g., AIDS, cancers, allergy, autoimmune diseases, and graft rejections in transplantation), such in suppression of immune responses in autoimmune disease, allergy or transplantation, as well as in anti-cancer and anti-viral immunotherapies.
In one embodiment, the invention provides a recombinant lentivirus having a genome with sequences, e.g., sequences useful to generate short hairpin RNA (shRNA), selected to alter the expression of a co-stimulatory molecule on DC. Co-stimulatory molecules for DC include but are not limited to ICAM-1, ICOS-L, B7.1 (CD80), B7.2 (CD86), B7RP1, 4-1BB-L, PD-L, OX40L or CD40, or other molecules that bind to molecules on T cells, molecules such as LFA-1, CD28, ICOS, CTLA-4, PD-1, 4-1BB (CD137), OX40 (CD134), or CD40L. In one embodiment, the lentivirus vector comprises a plurality of different sequences for one co-stimulatory molecule, e.g., each sequence with sequence similarity to different regions of the gene of the co-stimulatory molecule. In one embodiment, the lentivirus vector comprises a plurality of different sequences for different co-stimulatory molecules, e.g., each sequence has sequence similarity to a different co-stimulatory molecule gene. In one embodiment, the stable dendritic cells, when presenting antigen, alter the induction of Th1 cells. In one embodiment, the stable dendritic cells, when presenting antigen, alter the induction of Th2 cells.
In one embodiment, a recombinant, e.g. helper-free, lentivirus expresses sequences in DC that yield shRNA sequences specific for CD40 nucleic acid sequences, e.g., human CD40 sequences, which is turn induce RNAi in those cells, thereby altering the expression of CD40. In one embodiment, the shRNA sequences correspond to sequences in the coding region of the co-stimulatory molecule gene, e.g., CD40 gene. In another embodiment, the shRNA sequences correspond to sequences in the 3′ non-coding region of the co-stimulatory molecule gene, e.g., CD40 gene.
In one embodiment, the expression of the shRNA sequences decreases surface expression of the co-stimulatory molecule. In one embodiment, the decreased expression of the co-stimulatory molecule results in DC that, when exposed to an antigen and antigen-specific T cells, suppresses the proliferation of those T cells. In one embodiment, the antigen is an auto-antigen. In one embodiment, the antigen is an allo-antigen. In one embodiment, the antigen is an allo-antigen specific for a tissue or organ to be transplanted. In one embodiment, the antigen is an allergy specific antigen. In one embodiment, the antigen is an auto-antigen associated with an autoimmune disease. In one embodiment, the antigen is an exogenous antigen. As used herein, an “exogenous” antigen is one not normally encoded by the genome of and expressed in a mammal.
In one embodiment, the decreased expression of the co-stimulatory molecule results in DC that, when exposed to an antigen and antigen-specific T cells, tolerizes those T cells. In one embodiment, the decreased expression of the co-stimulatory molecule results in DC that, when exposed to an antigen and antigen-specific T cells, results in apoptosis of those T cells. In one embodiment, the administration of the dendritic cells of the invention to a mammal results in T cell tolerization, the down regulation of priming or activity of T cells, the inhibition of at least one symptom of an indication or disease, or any combination thereof.
In one embodiment, the expression of the sequences in the lentivirus vector in DC increases surface expression of a co-stimulatory molecule. In one embodiment, the increased expression of the co-stimulatory molecule results in DC that, when exposed to an antigen and antigen-specific T cells, enhances the proliferation of those T cells. In one embodiment, the antigen is a tumor specific antigen. In one embodiment, the antigen is a pathogen specific antigen, e.g., a viral, bacterium or fungal specific antigen
The invention provides a method of making stably transduced mammalian dendritic cells. The method includes providing a population of mammalian dendritic cells and stably transducing the cells with a recombinant helper-free lentivirus comprising a genome having a transcription cassette comprising nucleic acid sequences for a T cell co-stimulatory molecule, the expression of which is capable of inducing RNAi in the dendritic cells and altering the immune properties of the dendritic cells. In one embodiment, the co-stimulatory molecule comprises CD40, ICOS-L, ICAM-1, B7.1 (CD80), B7.2 (CD86), B7RP1, 4-1BB-L, PD-L, or OX40L. In one embodiment, the cells are human cells. In one embodiment, the nucleic acid sequences encode short hairpin RNA, e.g., of lengths of about 15 to about 1000 nucleotides in length, or any integer between 15 and 1000, e.g., 20, 30, 40, 50, 60, 70 or 100 nucleotides. In one embodiment, the stably transduced dendritic cells have decreased levels of one or more T cell co-stimulatory molecules relative to corresponding cells that are not transduced by the recombinant helper-free lentivirus, e.g., when cells are contacted with an agent that stimulates expression of the co-stimulatory molecules.
Further provided is a population of isolated stably lentivirus transduced mammalian dendritic cells comprising a recombinant lentivirus comprising a transcription cassette comprising nucleic acid sequences for a T cell co-stimulatory molecule, the expression of which is capable of inducing RNAi in the dendritic cells and altering the immune properties of the dendritic cells. In one embodiment, the co-stimulatory molecule comprises CD40, ICOS-L, ICAM-1, B7.1 (CD80), B7.2 (CD86), B7RP1, 4-1BB-L, PD-L, or OX40L. In one embodiment, the cells are human cells. In one embodiment, the nucleic acid sequences encode short hairpin RNA, e.g., of lengths of about 15 to about 1000 nucleotides in length, or any integer between 15 and 1000, e.g., 20, 30, 40, 50, 60, 70 or 100 nucleotides. In one embodiment, the stably transduced dendritic cells have decreased levels of the T cell co-stimulatory molecule relative to corresponding cells that are not transduced by the recombinant helper-free lentivirus.



BRIEF DESCRIPTION OF FIGURES
FIG. 1. RNAi sequences against mouse CD40 gene (NM—011611) (SEQ ID NO:11). pLKOCD40 clones (66243, nucleotide 2136-2156, GCAGGGTACTGGCTAAATAAA; SEQ ID NO:1), (66244, nucleotide 1606-1626, CCAAAGGATAATGAGATGTTA; SEQ ID NO:2) were obtained from Open Biosystems (Huntsville, Ala.); FG12-siCD40#1, nucleotide 2502-2522, TACCATTTAGTTCTTGAATAA; SEQ ID NO:3; FG12-siCD40#2, nucleotide 1590-1610, AAAGGTGGTCAAGAAACCAAA; SEQ ID NO:4; FG12-siCD40#3, nucleotide 2449-2469 GTGGTGTATTGTAGAAATTAT; SEQ ID NO:5; FG12-siCD40#4, nucleotide 1431-1451, CTGGACAAGCTGTGAGGATAA; SEQ ID NO:6.
FIG. 2. Efficient lentiviral vectors transduction in bone marrow-derived dendritic cell. A) Phenotype of bone marrow-derived dendritic cells before and after LPS stimulation. Mean Fluorescence Intensity (MFI) of CD40 was indicated. B) Lentiviral vectors were highly efficient in transducing DC. Over 85% of CD11c+ was transduced and expressed EGFP reporter gene. Percentages of EGFP-expressing cells were maintained upon LPS stimulation. C) The shRNA-expressing lentiviral vectors that down-regulate CD40 expression at different levels specifically. The CD40-silencing constructs, FG12H1Si#4 and 66243, were capable of mediating 2 to 4-fold down-regulation of the Mean Fluorescence Intensity of surface CD40 expression respectively when compared to the mock and irrelevant FG12H1SiLuc control. The MFI of CD40 was indicated for each DC group. The gene silencing of CD40 was specific because surface expression of C086 was not affected in these transduced DC.
FIG. 3. Lentiviral vectors deliver short interfering RNA into bone marrow derived dendritic cells without functional impairments. A) Lentiviral vectors were highly efficient in transducing DC. Over 85% of CD11c+ was transduced. B) Bone marrow derived DC were immature in phenotype (no/low expression of CD40, CD86 co-stimulatory molecules). Upon LPS stimulation, both mock and the transduced DC upregulated surface expression of CD40 and CD86. The shRNA-expressing lentiviral vectors that down-regulate CD40 expression at different levels specifically. The CD40-silencing constructs, FG12H1Si#4, 66243, were capable of mediating 3-fold and 10-fold down-regulation of the mean florescence intensity of surface CD40 expression respectively when compared to the mock and irrelevant FG12H1SiLuc control. The gene silencing of CD40 was specific because surface expression of CD86 was not affected in these transduced DC. C) Mock and shRNA-transduced DC produced comparable level of IL-1β upon activation. No spontaneous DC activation (IL-1β production) was observed (data not shown) in the supernatant of a panel of lentiviral vectors target different molecules was examined. In the presence of LPS stimulation, Mock and shRNA-transduced DC produced comparable level of IL-1β as detected by ELISA. “Other siRNA sequences” included the CD80, CD86 shRNA sequences that did not mediate down-regulation of CD80, CD86. ShRNA-transduced DC were not impaired in LPS-induced IL-12 production. Mock and shRNA-transduced DC were analyzed for IL-12 production using intracellular cytokine staining, and analyzed in flow cytometry.
FIG. 4. Lentiviral vectors deliver short interfering RNA into bone marrow derived dendritic cells without impairing antigen presenting ability of these DC. A) Mock-transduced and shRNA-transduced C57/BL6 DC induced similar allogeneic proliferative T cell response in vitro. B6 GMDC was transduced with either 66243 alone or a combination of 66243 and FG12SiCD40. After puromycin selection, these cells were then treated with Mitomycin C and co-cultured with allogeneic Balb/c T cells for three days. The proliferation of T cells was measured by CFSE dilution. T cells cultured with medium or mock-transduced DC were used as control. C57/BL6 T cell cultured with C57/BL6 DC as syngeneic control. DC of CD40−/− immunodeficient was used as a control in this assay to demonstrate allogeneic T cell proliferative response in vitro is independent of CD40. B) ShRNA-transduced DC were capable of priming Ag-specific T cell responses in vitro. DO11.10 T cells were purified by positive selection of CD90.1 via AutoMacs. Purified T cells were labeled with CFSE and co-cultured with primary DC at a ratio of 1:10 with OVA peptide SIINFEKL (SEQ ID NO:9) or ISQAVHAAHAEINEAGR (SEQ ID NO:10). OVA-specific T cell proliferation was measured by CFSE dilution after 3 days of culture. C) Genetic modified DC did not support T cell priming in vivo. DC were transduced with FG12H1SiLuc and 66243, respectively on D2 culture. Transduced cells were selected by puromycin on D7, and matured by 1 μg/mL LPS 18 hours before harvest. Harvested DC were phenotyped, and were used to immunize mice (i.v. 1×106 per mouse). 5 days after immunization, lymph node cells were obtained from immunized mice, and were labelled with CFSE. They were co-cultured with primary DC at a ratio of 10:1 with or with the presence of OVA protein. The proliferation of OVA-specific T cell was measured by CFSE dilution after 4 days of co-culture.
FIG. 5. Different levels of CD40 surface expression on the LPS-matured DC will have different impacts on naïve T cell activation in vitro. A) Down-regulation of CD40 suppressed OVA-specific T cell proliferation in vitro. DO11.10 T cells were purified and labeled with CFSE. They were co-cultured with primary DC (mock or transduced) at a ratio of 1:10 with OVA peptide SIIFEKL (SEQ ID NO:17; PI, irrelevant peptide control) or ISQAVHAAHAEINEAGR (SEQ ID NO:10; PII, Antigenic peptide for DO11.10 T cells). OVA-specific T cell proliferation was measured by CFSE dilution after 3 days of culture. Empty LKO and LKOsiEGFP (that has a shRNA sequence against irrelevant EGFP) vectors were used as specificity control for 66243 and 66244 constructs. Transduced DC (CD40med, CD40low) were selected by puromycin before co-culture. B) CD40low but not CD40med DC induced T cell apoptosis in vitro. Naïve OVA-specific DO11.10 T cells were purified and co-cultured with Ag-pulsed primary DC (mock or transduced) as described in A. Apoptotic cells were identified by Annexin V staining in FACS on Day 3.
FIG. 6. Schematic of CD40 switch in DC.



DETAILED DESCRIPTION OF THE INVENTION
In contrast to transient transfection studies, the lentiviral vector system supports stable and long-term expression of shRNA in the modified cells that might prove useful in development of stable, genetically defined DC to study the role of co-stimulatory molecules in functional definition of DC in vivo. As the sequence encoded in the dsRNA contributes to the potency of the dsRNA in degradation of target mRNA (Reynolds et al., 2004), the choice of shRNA target sequences in the lentiviral vector system may allow regulation of different levels of surface expression of a co-stimulatory molecule stably and reproducibly. For instance, inhibiting expression of certain co-stimulatory molecules in dendritic cells when those cells are presenting antigen to T cells, may suppress an immune response and/or tolerize those T cells, which may be useful in diseases such as autoimmune diseases or aberrant immune responses to exogenous antigens, e.g., those causing allergic disorders.
The invention thus provides a population of isolated stably lentivirus transduced mammalian dendritic cells comprising a recombinant lentivirus comprising a transcription cassette comprising nucleic acid sequences for a T cell co-stimulatory molecule, the expression of which is capable of inducing RNAi in the dendritic cells and altering the immune properties of the dendritic cells. In one embodiment, the co-stimulatory molecule comprises CD40, ICOS-L, ICAM-1, B7.1 (CD80), B7.2 (CD86), B7RP1, 4-1BB-L, PD-L, or OX40L. In one embodiment, the cells are human cells. In one embodiment, the nucleic acid sequences encode short hairpin RNA, e.g., of lengths of about 15 to about 1000 nucleotides in length, or any integer between 15 and 1000, e.g., 20, 30, 40, 50, 60, 70 or 100 nucleotides. In one embodiment, the stably transduced dendritic cells have decreased levels of the T cell co-stimulatory molecule relative to corresponding cells that are not transduced by the recombinant helper-free lentivirus.
The invention also provides a method to induce suppression of antigen specific T cells in a mammal. The method includes administering to a mammal having or suspected of having antigen specific T cells, an effective amount of a population of isolated stably lentivirus transduced mammalian dendritic cells comprising a recombinant lentivirus comprising a transcription cassette comprising nucleic acid sequences for a T cell co-stimulatory molecule, the expression of which is capable of inducing RNAi in the dendritic cells, which population is exposed to the antigen. In one embodiment, the antigen is an auto-antigen. In one embodiment, the antigen is an exogenous antigen.
The invention provides a method to induce allo-specific T cell proliferation or suppression. The method includes contacting dendritic cells with an effective amount of a recombinant DNA having a nucleic acid sequence for a co-stimulatory molecule. In one embodiment, the expression of the recombinant DNA in dendritic cells induces RNA interference. In one embodiment, the expression of the recombinant DNA in dendritic cells increases expression or activity of the co-stimulatory molecule.
Also provided is a method to prime or inhibit a specific T cell response, comprising contacting dendritic cells with an effective amount of a recombinant DNA having a nucleic acid sequence for a co-stimulatory molecule. In one embodiment, the expression of the recombinant DNA in dendritic cells induces RNA interference. In one embodiment, the expression of the recombinant DNA in dendritic cells increases expression or activity of the co-stimulatory molecule.
Further provided is a method to increase or decrease CD40 expression in dendritic cells, comprising contacting dendritic cells with an effective amount of a recombinant DNA having a nucleic acid sequence for CD40. In one embodiment, the expression of the recombinant DNA in dendritic cells induces RNA interference. In one embodiment, the expression of the recombinant DNA in dendritic cells increases expression or activity of CD40. In one embodiment, the expression of the recombinant DNA in dendritic cells decreases expression or activity of CD40.
A. Exemplary Autoimmune Diseases
Autoimmune diseases are characterized by an abnormal immune response involving either cells or antibodies, that are in either case directed against normal autologous tissues. Autoimmune diseases in mammals can generally be classified in one of two different categories: cell-mediated disease (i.e., T-cell) or antibody-mediated disorders. Non-limiting examples of cell-mediated autoimmune diseases include multiple sclerosis, rheumatoid arthritis, Hashimoto thyroiditis, type I diabetes mellitus (Juvenile onset diabetes) and autoimmune uveoretinitis (see Table 1). Antibody-mediated autoimmune disorders include, but are not limited to, myasthenia gravis, systemic lupus erythematosus (or SLE), Graves' disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia, autoimmune asthma, cryoglobulinemia, thrombic thrombocytopenic purpura, primary biliary sclerosis and pernicious anemia (see Table 1). The antigen(s) associated with systemic lupus erythematosus is small nuclear ribonucleic acid proteins (Snurps), Graves' disease is the thyrotropin receptor, thyroglobulin and other components of thyroid epithelial cells (Akamizu et al., 1996; Kellerman et al., 1995; Raju et al., 1997; and Texier et al., 1992), pemphigus is cadherin-like pemphigus antigens such as desmoglein 3 and other adhesion molecules (Memar et al., 1996: Stanley, 1995; Plott et al., 1994; and Hashimoto, 1993), and thrombic thrombocytopenic purpura is antigens of platelets.
Other autoimmune diseases and their specific autoantigens and/or target tissues are disclosed in Schwartz, R. S. et al. in Fundamental Immunology, Third Edition, Paul, W. E., Ed., Raven










TABLE 1







Disease Model
Specific Autoantigen









Multiple Sclerosis
MBP



Rheumatoid Arthritis
Collagen



Autoimmune Thyroiditis
Thyroglobulin



Myasthenia Gravis
Acetylcholine receptor



Autoimmune uvoretinitis
S-antigen



Systemic Lupus Erythematosus
DNA



Diabetes
islet cell extract



Chronic Active Hepatitis
Liver extract



Adrenalitis
Adrenal gland extract



Polymyositis
Muscle extract



Autoimmune hemolytic anemia
Hematopoietic cells



Rheumatic carditis
Heart extract



Scleroderma
Skin cell extract










An autoimmune disease is a malfunction of the immune system of mammals, including humans. In a mammal afflicted with such a disease, the immune system treats autologous tissues (self or endogenous antigens) and substances as if they were foreign and dangerous, and evokes the immune defense that is usually reserved for use against exogenous and dangerous substances (e.g., foreign tissues or invading organisms), including sensitization of T cells and synthesis of high affinity antibodies. Table 2 provides exemplary recombinant or purified autoantigens recognized by autoantibodies associated with human autoimmune disorders.








TABLE 2





Autoantigen
Autoimmune disease















A. Cell or organ-specific autoimmunity








Acetylcholine receptor
Myasthenia gravis


Actin
Chronic active hepatitis, primary



biliary cirrhosis


Adenine nucleotide translocator
Dilated cardiomyopathy, 


(ANT)
myocarditis


β-Adrenoreceptor
Dilated cardiomyopathy


Aromatic L-amino acid decarboxylase
Autoimmune polyendocrine



syndrome type 1 (APS-1)


Asialoglycoprotein receptor
Autoimmune hepatitis


Bactericidal/permeability-increasing
Cystic fibrosis vasculitides


protein (Bpi)



Calcium-sensing receptor
Acquired hypoparathyroidism


Cholesterol side-chain cleavage
APS-1


enzyme (CYPlla)



Collagen type IV α3-chain
Goodpasture syndrome


Cytochrome P450 2D6 (CYP2D6)
Autoimmune hepatitis


Desmin
Crohn disease, coronary artery



disease


Desmoglein 1
Pemphigus foliaceus


Desmoglein 3
Pemphigus vulgaris


F-actin
Autoimmune hepatitis


GM gangliosides
Guillain-Barre syndrome


Glutamate decarboxylase (GAD65)
Type 1 diabetes, stiff man syndrome


Glutamate receptor (GLUR)
Rasmussen encephalitis


H/K ATPase
Autoimmune gastritis


17-a-Hydroxylase (CYP17)
APS-1


21-Hydroxylase (CYP21)
Addison Disease


IA-2 (ICA512)
Type 1 diabetes


Insulin
Type 1 diabetes, insulin hypo-



glycemic syndrome (Hirata disease)


Insulin receptor
Type B insulin resistance, acanthosis,



systemic lupus erythematosus (SLE)


Intrinsic factor type 1
Pernicious anemia


Leukocyte function-associated antigen
Treatment-resistant Lyme arthritis


(LFA-1)



Myelin-associated glycoprotein
Polyneuropathy


(MAG)



Myelin basic protein
Multiple sclerosis, demyelinating



diseases


Myelin oligodendrocyte glycoprotein
Multiple sclerosis


(MOG)



Myosin
Rheumatic fever


p-80-Coilin
Atopic dermatitis


Pyruvate dehydrogenase complex-E2
Primary biliary cirrhosis


(PDC-E2)



Sodium iodide symporter (NIS)
Graves disease, autoimmune



hypothyroidism


SOX-10
Vitiligo


Thyroid and eye muscle shared protein
Thyroid associated ophthalmopathy


Thyroglobulin
Autoimmune thyroiditis


Thyroid peroxidases
Autoimmune Hashimoto thyroiditis


Thyrotropin receptor
Graves disease


Tissue transglutaminase
Coeliac disease


Transcription coactivator p75
Atopic dermatitis


Tryptophan hydroxylase
APS-1


Tyrosinase
Vitiligo, metastatic melanoma


Tyrosine hydroxylase
APS-1







B. Systemic autoimmunity








ACTH
ACTH deficiency


Aminoacyl-tRNA histidyl synthetase
Myositis, dermatomyositis


Aminoacyl-tRNA synthetase (several)
Polymyositis, dermatomyositis


Cardiolipin
SLE


Carbonic anhydrase II
SLE, Sjogren syndrome,



systemic sclerosis


Autoantigen
Autoimmune disease


Collagen (multiple types)
Rheumatoid arthritis (RA), SLE,



progressive systemic sclerosis


Centromere-associated proteins
Systemic sclerosis


DNA-dependent nucleosome-
Dermatomyositis


stimulated ATPase



Fibrillarin
Scleroderma


Fibronectin
SLE, RA, morphea


Glucose-6-phosphate isomerase
RA


β2-Glycoprotein 1 (β2-GPI)
Primary antiphospholipid syndrome


Golgin (95, 97, 160, 180)
Sjogren syndrome, SLE, RA


Heat shock protein
Various immune-related disorders


Hemidesmosomal protein 180
Bullous pemphigoid, herpes



gestationis, cicatricial pemphigoid


Histone H2A-H2B-DNA
SLE


IgE receptor
Chronic idiopathic urticaria


Keratin
RA


Ku-DNA-protein kinase
SLE


Ku-nucleoprotein
Connective tissue syndrome


La phosphoprotein (La 55-B)
Sjogren syndrome


Myeloperoxidase
Necrotizing and crescentic



glomerulonephritis (NCGN),



systemic vasculitis


Proteinase 3 (PR3)
Wegener granulomatosis,



Churg-Strauss syndrome


RNA polymerase I-III (RNP)
Systemic sclerosis, SLE


Signal recognition protein (SRP54)
Polymyositis


Topoisomerase-I (Scl-70)
Scleroderma, Raynaud syndrome


Tubulin
Chronic liver disease, visceral



leishmaniasis


Autoantigen
Autoimmune disease


Vimentin
Systemic autoimmune disease







C. Plasma protein and cytokine autoimmunity








C1 inhibitor
Autoimmune C1 deficiency


C1q
SLE, membrane proliferative


Cytokines (IL-1α, IL-β, IL-6,
RA, Systemic sclerosis, normal


IL-10, LIF)
subjects


Factor II, factor V, factor VII,
Prolonged coagulation time


factor VIII, factor IX, factor X,



factor XI, factor XII, thrombin, vWF



Glycoprotein IIb/IIIg and Ib/IX
Autoimmune thrombocytopenia



purpura


IgA
Immunodeficiency


Oxidized LDL (OxLDL)
Atherosclerosis







D. Cancer and paraneoplastic autoimmunity








Amphiphysin
Neuronopathy, small lung cell



cancer


Cyclin B1
Hepatocellular carcinoma


DNA topoisomerase II
Liver cancer


Desmoplakin
Paraneoplastic pemphigus


Gephyrin
Paraneoplastic stiff man syndrome


Hu proteins
Paraneoplastic encephalomyelitis


Neuronal nicotinic acetylcholine
Subacute autonomic neuropathy,


receptor
cancer


p53
Cancer, SLE


p62 (IGF-II mRNA-binding protein)
Hepatocellular carcinoma (China)


Recoverin
Cancer-Associated retinopathy


Ri protein
Paraneoplastic opsoclonus



myoclonus ataxia


β IV spectrin
Lower motor neuron syndrome


Autoantigen
Autoimmune disease


Synaptotagmin
Lambert-Eaton myasthenic



syndrome


Voltage-gated calcium channels
Lambert-Eaton myasthenic



syndrome


Yo protein
Paraneoplastic cerebellar



degeneration









B. Exemplary Exogenous Antigens

Allergic diseases within the scope of the invention include allergic rhinitis, allergic asthma, atopic dermatitis, allergic gastroentheropathy, anaphylaxis, urticaria and angioedema. Allergens within the scope of the invention include, but are not limited to, protein antigens of Alternaria altemata (Alt a I), Artemisia vulgaris (Art v II), Aspergillus fumigatus (Asp f II), Dermatophagoides far. (Der f I), Dermatophagoides pteron. (Der p I, Der p III, Der p IV, Der p VI and Der p VIII), and domestic animals such as Felis domesticus (Fel d I), cows, pigs, poultry, mice, hamsters, rabbits, rats, guinea pigs, dogs and horses. Common fungal antigens include those of Basidiomycetes such as Ustilago, Ganoderma, Alternaria, Cladosporium, Aspergillus, Sporobolomyces, Penicillium, Epicoccum, Fusarium, Phoma, Borrytis, Helminthosporium, Stemphylium and Cephalosporium; Phycomycetes such as Mucor and Rhizopus; and Ascomycetes such as Eurotium and Chaetomium. 
Pollinating plants which may have protein antigens associated with allergies include club mosses, ferns, conifers, flowering plants, grasses, sedges, palms, cattails, nettles, beeches, chenopods, sorrels, willows, poplars, maples, ashes, ragweeds (antigen E, antigen K and Ra3) and sages, or proteinaceous plant products such as those found in latex products.
Hymenoptera insects that may have protein antigens associated with allergies include the honeybee, yellow jacket, hornet, wasp and fire ant, although protein antigens of other insects are also within the scope of the invention.
Allergies associated with foods may be the result of protein antigens in crustaceans (e.g., shrimp, lobster and crab), mollusks (e.g., clams), fish, legumes (e.g., peanut, pea, beans, and licorice), seeds (e.g., sesame, cottonseed, caraway, mustard, flaxseed, and sunflower), nuts, berries, egg white, buckwheat and milk.
C. Pharmaceutical Compositions and Routes of Administration
The dendritic cells of the invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration including parenteral, e.g., intravenous or intra-arterial, intradermal, subcutaneous, oral or nasal (e.g., inhalation), transdermal (topical), transmucosal, nasal, pulmonary, ocular, gastrointestinal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Alternate routes of administration include intraperitoneal, intra-articular, intracardiac, intracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, intraventricular, and the like.
The present dendritic cells may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 90% of the weight of a given unit dosage form. The amount of cells in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The cells may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the particle or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms of the cells of the invention may be provided in an instant release, controlled release, sustained release, or target particle-delivery system. Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, suppositories, ovules, implants, amorphous or crystalline powders, aerosols, and lyophilized formulations. Depending on the route of administration used, special devices may be required for application or administration of the particle, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks, or presented in the form of implants and pumps requiring incision. Pharmaceutical dosage forms are often composed of the cells, an excipient(s), and a container/closure system. One or multiple excipients, also referred to as inactive ingredients, can be added to the dendritic cells of the invention to improve or facilitate manufacturing, stability, administration, and safety of the particle, and can provide a means to achieve a desired release profile. Therefore, the type of excipient(s) to be added to the cells can depend on various factors, such as, for example, the physical and chemical properties of the particle, the route of administration, and the manufacturing procedure. Pharmaceutically acceptable excipients are available in the art, and include those listed in various pharmacopoeias. (See, e.g., USP, JP, EP, and BP, FDA web page (www.fda.gov), Inactive Ingredient Guide 1996, and Handbook of Pharmaceutical Additives, ed. Ash; Synapse Information Resources, Inc. 2002.)
Pharmaceutical dosage forms of cells of the present invention may be manufactured by any of the methods well-known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-making, tabletting, suspending, extruding, spray-drying, levigating, emulsifying, (nano/micro-) encapsulating, entrapping, or lyophilization processes. As noted above, the compositions of the present invention can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.
Proper formulation is dependent upon the desired route of administration. For intravenous injection, for example, the composition may be formulated in aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidine, or citrate for adjustment of the formulation pH, and a tonicity agent, such as, for example, sodium chloride or dextrose. For transmucosal or nasal administration, semisolid, liquid formulations, or patches may be used, possibly containing penetration enhancers. Such penetrants are generally known in the art. For oral administration, the dendritic cells can be formulated in liquid or semi-solid dosage forms and as instant or controlled/sustained release formulations. Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, emulsions and the like. The dendritic cells may also be formulated in rectal compositions, such as suppositories or retention enemas.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Depending on the injection site, the vehicle may contain water, synthetic or vegetable oil, and/or organic co-solvents. In certain instances, such as with lyophilized product or a concentrate, the parenteral formulation would be reconstituted or diluted prior to administration. Depot formulations, providing controlled or sustained release of an invention dendritic cell, may include injectable suspensions of nano/micron or nano/micro or non-micronized crystals. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, poly(ol) (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the particle into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the particle can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the particle in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; dissolution retardant; anti-adherants; cationic exchange resin; wetting agents; antioxidants; preservatives; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a preservative; a colorant; a sweetening agent such as sugars such as dextrose, sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring, each of these being synthetic and/or natural. Oral compositions can also include a self-assembling molecule expressed in a food plant that is then fed to the intended recipient.
For administration by inhalation, the dendritic cells are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Systemic administration can also be by transmucosal means. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. The active agents can also be prepared in the form of suppositories or retention enemas for rectal delivery.
In one embodiment, the compositions are prepared with carriers that protect rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
When a composition of the present invention is used for administration to an individual animal, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. The composition may also contain variable but small quantities of endotoxin-free formaldehyde, and preservatives, which have been found safe and not contributing to undesirable effects in the organism to which the composition is administered.
Supplementary compounds can also be incorporated into the compositions. Carrier molecules may be genes, polypeptides, antibodies, liposomes or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Further examples of known carriers include polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles. Carriers may also include pharmaceutically acceptable salts such as mineral acid salts (for example, hydrochlorides, hydrobromides, phosphates, sulphates) or the salts of organic acids (for example, acetates, propionates, malonates, benzoates). Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. Various formulations and particle delivery systems are available in the art, and a thorough discussion of pharmaceutically acceptable carriers are available in the art (see, e.g., USIP. Remington; The Science and Practice of Pharmacology (Lippincott Williams & Wilkins, 21st ed. 2005); and Ansel & Stoklosa, Pharmaceutical Calculations (Lippincott Williams & Wilkins, 11th ed., 2001).
Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present dendritic cells can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as antimicrobial agents can be added to optimize the properties for a given use.
Useful dosages of the dendritic cells can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The concentration of the cells in a liquid composition may be from about 0.01-95 wt-% or more, e.g., from about 0.1-80 wt-%.
The amount of cells required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
In general, however, a suitable dose may be in the range of from about 102 to about 1010 cells, e.g., from about 104 to about 108 cells. The cells are conveniently administered in unit dosage form; for example, containing 102 to 108 cells, or conveniently 104 to 106 cells, or conveniently 106 to 107 cells per unit dosage form.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
The invention will be described by the following non-limiting examples.
Example I
The feasibility of using lentiviral vectors was established in delivering stable shRNA in the manipulation of DC functions in vitro and in vivo. A number of shRNA target sequences was used to demonstrate that lentiviral vectors delivered RNA interference in DC without functional impairments. For example, CD40 was used as a target co-stimulatory molecule to demonstrate the feasibility of generating DC that expresses different levels of CD40. Functional data further demonstrate that quantitative expression of CD40 on LPS-stimulated DC have different functional outcome on Ag-specific T cell responses in vitro.
Materials and Methods
Mice. C57BL/6 and BALB/c mice were purchased from Animal Care Services (The University of Manitoba). CD40−/− gene-targeted mice were purchased from Jackson Laboratory (Bar Harbor, Me., USA). The University of Manitoba's Review Board has approved all animal studies. DO11.10 TCR transgenic mice for I-Ad/OVA323-339 (Robertson et al., 2000) were kindly provided by Dr. Xi Yang (University of Manitoba, Department of Medical Microbiology). The University of Manitoba's Review Board approved all animal studies.
Plasmids, lentiviral vectors production. 293T cells used for virus production and titration were cultured in IMDM (HyClone, Logan, Utah) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, Utah), and 1% penicillin/streptomycin/glutamate (PSG) (Gibco, Grand Island, N.Y.). VSV-G pseudotyped lentiviral vectors (SIN18-cppt-RhMLV-E, Cppt-2E in short, FUGW, TRIP-GFP, PGK-C) were prepared in 293T cells, concentrated, and titered as described previously. FG12h1siluc and Cloning of the shRNA sequences into lentiviral vectors was described previously (Qin et al., 2003; Stewart et al., 2003). pLKOCD40 clones (66243, GCAGGGTACTGGCTAAATAAA (SEQ ID NO:1)), (66244, CCAAAGGATAATGAGATGTTA (SEQ ID NO:2)) were obtained from Open Biosystems (Huntsville, Ala.); FG12-siCD40#1, TACCATTTAGTTCTTGAATAA (SEQ ID NO:3); FG12-siCD40#2, AAAGGTGGTCAAGAAACCAAA (SEQ ID NO:4); FG12-siCD40#3, GTGGTGTATTGTAGAAATTAT (SEQ ID NO:5); FG12-siCD40#4, CTGGACAAGCTGTGAGGATAA (SEQ ID NO:6); FG12-siCD80, TTACAACTCTCCTCATGAA (SEQ ID NO:7); and FG12-siCD86, TCTACGACTTCACAATGTT (SEQ ID NO:8). The locations of the CD40 siRNA sequences on CD40 mRNA are shown in FIG. 1.
DC preparation, transduction and immunizations. The murine BMDC were generated from bone marrow precursors as previously described (Inaba et al., 1992). Briefly, mouse bone marrow cells were extracted from the femura and tibiae of C57/B6 mice, and were plated at 1×106 cells/mL in RPMI 1640 (HyClone) medium supplemented with PSG (Invitrogen), 2-ME, 10% FBS (HyClone), and 20 ng/mL GM-CSF (PetroTech). Culture medium was changed every 2 days. If required, LPS (Sigma) was added to the culture at 10 μg/mL 18 hours before cells were harvested. Transduction of DC were carried out by adding of viral supernatant (at various MOIs, 0.28 mL per well) in the presence of 8 μg/mL of Polybrene (Sigma, St. Louis, Mo.) to the adherent cells on D2 BM culture. Cells were incubated for 3 hours at 37° C. and 5% CO2. Virus-containing supernatant was removed at the end of transduction. Transduced cells were cultured in 0.5 mL GMCSF-conditioned medium as described above. Mock DC or transduced DC selected by puromycin were pulsed with OVA protein (Sigma) overnight before LPS stimulation. They were harvested on D9, and were washed at least three times using PBS. 1×106 DC were injected i.v. Immunized mice were sacrificed 5 days after immunization, and T cells were sorted to detect OVA-specific immune response.
Flow cytometry. Fluorochrome-conjugated antibodies (CD11c, CD40, CD86, CD80, IL-12) were all purchased from BioLegend (San Diego, Calif.). The fluorescence profile was generated on a BD FACSCalibur Flow Cytometer. 10000 events of gated population was collected and analyzed. Histogram and density plots were produced by the FCS Express version 2.0 (De Novo Software, ON). In intracellular staining of IL-12, DCs were treated with BFA (10 μg/mL) (Sigma) 6 hours before harvest to block the secretion of IL-12. DCs were then collected and stained with Phycoerythrin conjugated (PE) anti-mouse IL-12 as indicated.
Lymphocyte proliferation assay. BALB/c splenic and lymph node T cells were sorted using CD90 (Thy1.2) MicroBeads (Miltenyi Biotec) The T cells of allogeneic animals were resuspended in PBS at 2×107/ml, and were then mixed with equal volume of 3 μM CFSE. The reaction was quenched using 100% FBS. These labeled cells were then washed carefully before use. Irradiated DC and CFSE labeled T cells were cocultured on a round bottom 96-well plate (Corning) at different ratios for 3 days. The supernatant of culture was collected for cytokine detection using ELISA, the proliferation of T cells was measured by CFSE dilution. In the in vitro assay of Ag-specific T cell priming, 1×106 lentiviral vector transduced DC were used to immunize mice via i.v. after they were pulsed with OVA protein (Sigma, St. Louis, Mo.), and stimulated by LPS. Immunized mice were sacrificed 5 days after immunization, and T cells were sorted using CD90 (Thy1.2) MicroBeads (Miltenyi Biotec, Auburn, Calif.). Purified T cells were labeled with CFSE, and were co-cultured with OVA pulsed DC for 96 hours. T cell proliferation was evaluated by the dilution of CFSE dye in FACS.
Cytokine production detected by ELISA. The IL-113 production in the supernatant of DC culture was quantified as described previously (Hoeck and Woisetschlager, 2001). Monoclonal anti-mouse IL-1β (Biolegend, San Diego Calif.) was used as capture antibody and biotinylated anti-mouse IL-1β was used as detecting antibody. Binding was detected by streptavidin-alkaline phosphatase and PNPP substrate (Sigma, St. Louis, Mo., USA). The sensitivity limit of ELISA was 0.016 ng/mL. Each data point represents reading from a minimum of three independent assays performed in triplicate.
Results and Discussion
Efficient transduction of DC using lentiviral vectors was established. A lentiviral vector was employed that expressed an EGFP transgene (Cppt2E) (Tran and King, 2007), and a shRNA-expressing lentiviral vector that concomitantly expressed the EGFP reporter gene (FG12H1Siluc, FG12SiCD40#4) to optimize transduction efficiency of DC in the transduction protocol. DC were derived from bone marrow cultured in GMCSF-conditioned medium in vitro. These CD11c+ DC expressed no/low surface expression of co-stimulatory molecules (CD86, CD40) (FIG. 2A mock; CD80, data not shown), thus resembling an “immature” DC phenotype. Upon LPS stimulation, they undergo a maturation process that up-regulated surface expression of these co-stimulatory molecules (FIG. 2A mock). Transduction efficiency was evaluated by the percentage of EGFP+CD11c+ cells by flow cytometry. Efficient gene transfer and transgene expression of EGFP were established in the transduced DC. Greater than 80% CD11c+ DC transduced in the transduction protocol (FIG. 2B).
Although lentiviral transduced DC that were genetically modified to over-express specific Ag has been well documented in its ability to prime Ag-specific immune responses in vitro and (He et al., 2005; Kung et al., 2005), introduction of shRNA into DC, however, raises concern as to whether the presence of “foreign” RNA may trigger pathogen pattern-recognition receptors that will alter normal DC properties. A current model suggests that TLR-induced immunostimulation by RNA is determined by the length, sequence, form of RNA delivered, and the cell type studied (Robbins and Rossi, 2005). It is therefore important to examine functional characteristics of gene silencing and the potential immunostimulation in a system-specific manner. A number of lentiviral vectors were constructed and identified that expressed shRNA sequences against different target sequences/genes, such as the irrelevant luciferase gene (FG12H1SiLuc, irrelevant target specificity control), and DC co-stimulatory molecules (CD40, CD86 and CD80). The CD40-silencing constructs, FG12H1Si#4, 66243, were capable of mediating 3-fold and 10-fold down-regulation of the mean florescence intensity of surface CD40 expression respectively when compared to the mock and irrelevant FG12H1SiLuc control (FIG. 2C). The gene silencing of CD40 was specific because surface expression of CD86 was not affected in these transduced DC (FIG. 2C). The lentiviral vectors that expressed CD86 and CD80 shRNA target sequences did not induce any CD86 and CD80 gene silencing respectively in the transduced DC (data not shown), suggesting that the predicted sequences were ineffective in gene silencing of these two genes.
This panel of lentiviral vectors that had different gene silencing capability was used to examine whether lentiviral vector-mediated delivery of the shRNA would induce unwanted, non-specific DC stimulations or functional impairments in the DC maturation process. In exposure to external stimulations by toll-like receptors, the immature DC undergoes a maturation program that up-regulates surface expression of their co-stimulatory molecules, and production of pro-inflammatory cytokines (such as IL-1beta and IL-12). These mature DC are functionally active in presenting Ag and priming Ag-specific T cells in vitro and in vivo. Bone marrow derived DC were transduced with these lentiviral vectors, and examined for any changes in their phenotype, cytokine production and their ability to induce Ag-specific T cell proliferation in vitro and in vivo. Mock-transduced DC (not transduced by any lentiviral vectors) was used as a negative control. No spontaneous maturation or activation was detected in all of the transduced DC when compared to the mock-transduced DC control. The transduced DC expressed no/low surface expression of co-stimulatory molecules (CD86, CD80 and CD40) (FIG. 2A, top panel). They did not produce any detectable IL-1β or IL-12 (data not shown).
Next, LPS was added to these DC cultures to test whether the shRNA-transduced DC were impaired in DC maturation. No impairment of LPS-induced maturation was detected in these transduced cells when compared to the mock control. LPS stimulated both mock and transduced DC to up-regulate co-stimulatory molecules (such as CD40 and CD86) on the surface (FIG. 2A), and to produce IL-1β and IL-12. The FG12SiCD40#4 transduced DC (that expressed an effective RNAi sequence to down-regulate CD40 expression) produced similar responses in IL-1β, IL-12 and CD86 upregulation upon LPS stimulation when compared to the mock, FG12H1SiLuc controls and other shRNA lentiviral vectors that were not effective in silencing CD80, CD86 or CD40. Collectively, the data established that lentiviral vector delivery (and de novo expression) of these shRNA sequences in the transduced DC did not induce or impair DC activation/maturation, and was independent of the gene silencing activities.
FG12siLuc and 66243 vectors were used to further examine the functional ability of these transduced DC to induce allo-specific T cell proliferation in vitro, and to prime an OVA-specific T cell response in vivo. The CD40 co-stimulatory molecule has previously been shown to be essential in the activation of naïve allogeneic T cells in vivo but not in vitro (Haase et al., 2004). This was confirmed using CD40−/− DC in an allogeneic MLR in vitro (FIG. 3A), and therefore used this system to examine any functional impairment of the transduced DC unrelated to the down-regulation of CD40 expression we observed in the 66243-transduced DC. The mock and the transduced DC showed no difference in the allogeneic T cell proliferative responses, as determined by CFSE dilution (FIG. 3A) and 3H-thymine proliferation assays (data not shown). In addition, further enhancement of CD40 down-regulation by co-transduction of two CD40-silencing shRNA vectors (66243+FG12siCD40) did not alter the activation of allogeneic T cells in vitro (FIG. 3A).
FG12siLuc-transduced DC were used to test the ability of the shRNA-transduced DC to prime an Ag-specific T cell response in vitro. Purified T cells of the OVA-DO.11 TCR transgenic animals were used as responder T cells in a co-culture assay to test whether shRNA-transduced DC were impaired in their ability to prime naive Ag-specific T cells in vitro. Both the mock and the FG12H1SiLuc transduced DC induced OVA-specific T cell proliferation in vitro (FIG. 3B). To further test their ability to prime OVA-specific T cells in vivo, mock transduced DC and FG12siLuc-transduced DC were pulsed with ovalbumin (OVA) protein Ag, and adoptively transferred into syngeneic naïve recipient C57BL/6 mice. The immunized animals were sacrificed and analyzed for OVA-specific T cells in a proliferation assay. As expected, no OVA-specific T cell proliferation was observed in the naïve animals, and in the immunized animals when the OVA Ag was absent in the assay. Induction of OVA-specific T cells was observed in both groups of the mice that were immunized with the Mock-transduced DC and the FG12siLuc-transduced DC (FIG. 3C), demonstrating that delivery and expression of a shRNA sequence by lentiviral vectors did not induce any apparent changes in Ag-priming ability of DC non-specifically in vivo.
If the choice of the sequence encoded in the dsRNA contributes to the potency of the dsRNA in degradation of target mRNA (Reynolds et al., 2004), the use of these sequences in the lentiviral vector system would allow us to regulate different levels of surface expression of a co-stimulatory molecule reproducibly and stably. The CD40 co-stimulatory molecule was used to test whether quantitative expression of this molecule will affect DC function in vitro. CD40 shRNA-containing lentiviral vectors (66243, 66244) was used in the established transduction protocol to generate DC that expressed medium (CD40med) and low levels of CD40 (CD40low), respectively, on the cell surface (FIG. 4). They were pulsed with OVA peptide I (irrelevant control), or OVA peptide II (OVA antigenic peptide specific to DO.11 TCR). Purified T cells of the OVA-TCR transgenic animals were used as responder T cells in a co-culture assay to test whether shRNA-transduced DC were impaired in their ability to induce Ag-specific T cell proliferation in vitro. Levels of CD40low down-regulation were found correlated to the suppression of naive OVA-specific T cell proliferation in the OVA transgenic T cells in vitro (FIG. 4A). Interestingly, only CD40low DC induce significant apoptosis in vitro (FIG. 4B). The data demonstrated that lentiviral vector-mediated RNA interference supports quantitative analysis of a co-stimulatory molecule in DC function. Different levels of CD40 surface expression on the LPS-matured DC will have different impacts on naïve T cell activation in vitro.
Conclusions
Dendritic cells (DC) are specialized regulator of innate and adaptive immunity. Advances in using lentiviral vectors to deliver short hairpin RNA (shRNA) in specific gene silencing provide a unique tool to generate stable genetically modified DC to direct different cellular responses (such as Th differentiations or immunological tolerance) in vivo. This approach allows the examination of the importance of quantitative expression of co-stimulatory molecule(s), such as CD40, in determining different functional properties of DC to tolerize, activate or polarize antigen-specific Th1/Th2 immune responses in vivo. The ability to regulate types of T cell responses against specific antigens is useful in the development of vaccines and therapeutic approaches to infectious diseases.
Example II
Replication-incompetent HIV-1 based lentiviral vector is now emerging as a promising delivery system to genetically modify DC through antigen recognition, co-stimulatory molecules and/or polarization signals for the manipulations of Ag-specific immunity in vivo. The following will discuss some of the recent advances in the uses of lentiviral vectors in DC-based immunotherapy.
Manipulation of Immune Responses by Genetically Engineered Dendritic Cells
Dendritic cells (DC) are professional antigen presenting cells (APC) that initiate and regulate immune responses. They present antigenic peptides in the context of major histocompatibility complex (MHC)/Peptide complexes (Signal 1) to engage cognate T cell receptors of T cells to define antigen (Ag) specificity. They possess a number of co-stimulatory molecules (Signal 2) to regulate optimal T cell activation. Binding of B7-1 and B7-2, for examples, to CD28 or CTLA-4 respectively on T cells will provide the co-stimulatory activity for naïve T cell activation or inhibitory activity of T cell activation. A third signal (the polarization signal), which comes from cytokines or chemokines released by the APC, will regulate differentiation of naïve T cells into distinct types of T helper cells (O'Sullivan et al., 2003; Sharpe et al., 2002; Kalinksi et al., 2005; and Reis et al., 2006). Tight regulation of these signals upon DC/T cell interaction will ultimately determine the functional ability of DC to induce Ag-specific tolerance, Ag-specific immunity or specific differentiation of T helper subsets.
The ability to generate DC of defined therapeutic property therefore holds great promises in transplantation, as well as immunotherapy of cancer, infectious diseases, allergy and autoimmunity. Genetic manipulation of DC is an attractive approach because molecules such as protein antigens (Ag), co-stimulatory molecules or cytokines can be expressed and manipulated (in the forms of DNA, RNA, short interference RNA or short hairpin RNA) directly in the DC by viral and non-viral gene delivery systems. Indeed, different strategies have been developed to modify DC ex vivo for manipulations of Ag-specific immunity in vivo (Tables 3 and 4).







TABLE 3







Induction of anti-tumor and anti-viral responses using


genetically modified DC











Di-
Targeting
Targeting
Delivery 



seases
strategy
molecules
system
Ref





Tumor
Signal 1
TAA encoding
liposome
Pecher et al.,




cDNA

2002




TAA encoding
Liposome/
Pecher et al.,




cDNA
Adenovirus
2001




TAA encoding
Electro-
Kyte et al.,




mRNA
poration
2007




TAA encoding
Adenovirus
Tsao et al.,




cDNA

2007




TAA encoding
lentivirus
Mossoba et al.,




cDNA

2008




Mutated mRNA
Electro-
Abdel-Wahab




of TAA
poration
et al., 2003



Signal 2
TAA + CD40L
Adenovirus
Zhang et al.,






2003




OX40L
Retrovirus
Yanagita et al.,






2004




CD40L/RANKL/
Adenovirus
Yurkovetsky




4-1BBL

et al., 2006




CD80
Avipox vector
Tsang et al., 






2001




CD70
Electro-
Keller et al.,





poration
2009




CD54 (ICAM-1)
Avipox vector
Yang et al.,






2005




CD58 (LFA-3)
Avipox vector
Yang et al., 






2005


Virus
Signal 1
Influenza antigen




infection

M1 mRNA/
Electro-
Strobel et al.,




cDNA
poration
2000



Signal 1
CMV antigen IE1
rAAV
Yu et al., 2008



Signal 1
HIV-1
Sendai virus/
Hosoya et al.,





adenovirus
2008



Signal 1
HCV-S/HCV-NS
Lentivirus
Jirmo et al.,






2010



Signal 1
HCV NS cDNA/ 
Electro-
Yu et al., 2008




mRNA
poration




Signal 1
HCV NS
Adenovirus
Zabaleta et al.,






2008



Signal 1
HIV-1 Gag and
Electro-
Van Gulck




Env proteins.
poration
et al., 2006



Signal 1
HBsAg
Adenovirus
Huang et al.,






2006





Abbreviations: Tumor Associated Antigen (TAA); Interferon-gamma-Inducible Protein-10 (IP-10); secondary lymphoid tissue chemokine (SLC); monokine induced by IFN-gamma (Mig); recombinant adeno-associated virus (rAAV); HBV surface surface antigen (HbsAg); HCV structural antigen (HCV-S) and non-structural antigen (HCV-NS)













TABLE 4







Induction of immunological tolerance in transplantation or autoimmune diseases












Diseases
DC source
Delivery system
Targeting molecules
Therapeutic effect
Ref





Transplantation
Donor/in
adenovirus
sTNFRI
Prolonged cardiac allograft
Wang et al., 2006



vitro


survival




Donor/in
adenovirus
IL-10
Prolonged cardiac allograft
Zhang et al., 2004



vitro


survival




Donor/in
Lipofectamine
Fast
Prolonged cardiac allograft
Min et al., 2000



vitro


survival




Donor/in
Electroporation
CTLA-4
Prolonged islet transplant
O'Rourke et al., 2000



vitro


survival




Donor/in
Retrovirus
TGF-β
Prolonged heart transplant
Takayama et al., 2002



vitro


survival




Recipient/in
Passive pulsing
NF-κB ODN
Prolonged cardiac allograft
Tiao et al., 2004



vitro


survival




Recipient/in
adenovirus
MHC-I
Prolonged cardiac allograft
Fry et al., 2002



vitro


survival



Autoimmune
In vitro
Lentivirus
RelB-specific shRNA
Prevent development of
Zhang et al., 2009


disease



EAMG




In vitro
Lentivirus
IL-4
Prevent NOD
Creusot et al., 2008



In vitro
Adenovirus
Galectin-1
delayed onset of NOD
Perone et al., 2006



In vivo
Lentivirus
BAFF
Ameliorate RA
Lai et al., 2008



In vitro
Electroporation
MOG + TRAIL
Prevent EAE
Hirata et al., 2005



In vitro
Adenovirus
Suppressive TCR
Abrogated EAE
Mahnke et al., 2003





mimic peptide





In vitro
Electroporation
c-MIR (MHC-II
resistance to EAE
Ohmura-Hoshino et al., 2003





inhibitor)




Allergic disease
In vitro
Lentivirus
IL-10
Reversed experimental asthma
Henry et al., 2008


Inflammation
In vitro
Adenovirus
IL-4
Inhibited inflammation DTH
Kim et al., 2007





Abbreviations: soluble TNF-α receptor type I (sTNFRI); Fas ligand (FasL); oligodeoxyribonucleotides (ODN); B cell-activating factor (BAFF); myelin oligodendrocyte glycoprotein (MOG); TNF-Related Apoptosis Inducing Ligand (TRAIL); experimental autoimmune myasthenia gravis (EAMG); non-obese diabetic (NOD); autoimmune arthritis (RA); Prevent experimental autoimmune encephalomyelitis (EAE); inflammation of delayed-type hypersensitivity (DTH)






Lentiviral Vectors and Dendritic Cell Transductions

Replication-incompetent HIV-1 based lentiviral vectors are capable of stable transduction of both dividing and relatively quiescent cells. This feature is advantageous over retroviral vectors derived from oncoretroviruses such as Moloney murine leukemia virus (MoMLV) because these retroviral vectors require proliferation of the target cells for integration (Case et al., 1999; Roe et al., 1993). Safety features that safeguard the use of lentiviral vectors in gene therapy in vivo are built into the vector production system. Firstly, accessory genes (vif, vpr, vpu and nef) that are not essential for transduction were eliminated from a packaging construct (Kafri et al., 1997; Kim et al., 1998; Zuffrex et al., 1967). Secondly, the uses of three-plasmid (or four-plasmid) expression system that consists of packaging, envelope and vector constructs have minimized the possibility for generating replication-competent virus through recombination (Mochizuki et al., 1998; Naldini et al., 1996; Naldini et al., 1996). The latter possibility is further minimized by the deletions of the TATA box and binding sites for transcription factors Sp1 and NF-kB, in the U3 region of the 3′ long terminal repeat (LTR) of the HIV-1 vectors to inactivate the LTR promoter activities (Mixoshi et al., 1998; Zuffrex et al., 1995). We prepared replication-incompetent, vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors by 3-plasmids transfection of highly susceptible 293T cells to obtain high virus titers (typically 108 IU/mL) for primary cell transductions. In the arguably closest model system for human hematopoietic stem cell gene therapy, we demonstrated transduction of autologous CD34+ stem cells and long-term expression of a foreign green florescent protein (GFP) gene in all hematopoietic cell lineages in vivo without the induction of anti-GFP immune responses in the myeloablated rhesus macaques (Kung et al., 2003). The animals remained healthy with no evidence of hematopoietic abnormalities or malignancies in a 5-year long-term study (Kung et al., 1993).
Lentiviral Vectors in Genetic Modifications of DC Ex Vivo
Ex vivo tissue culture conditions for DC and the preparation of high-titer lentiviral vector have been well documented and optimized in recent years (Tuyaerts et al., 2008; Kutner et al., 2009; Ansorge et al., 2009). Lentiviral vector is now emerging as a promising gene delivery vector system for DC because of the relatively high efficiency in the transduction of immature and differentiating DC (Zhang et al., 2009; Veron et al., 2001). Long-term, stable transductions of human monocyte-derived DC, human CD34+-derived DC, and mouse bone marrow derived DC (BMDC) have been reported (Breckpot et al., 2005). In the development of anti-cancer immunotherapy, DCs transduced with a lentiviral vector that expressed a surrogate tumor associated antigen (TAA) ovalbumin (OVA) have been shown to effectively process and present the OVA antigens, and induced OVA-specific T cell responses. A single immunization of the lentiviral vector-transduced DC in a mouse melanoma model induced protective immunity against a lethal dose of the OVA-expressing B16 melanoma cells challenge, as well as therapeutic anti-tumor immunity against the established B16 tumors (He et al., 2005). Other clinically relevant TAAs, such as TRP-2, MAGE-3, Melan/MART-1, and tyrosinase, have been inserted into lentiviral vectors. DCs transduced with these lentiviral vectors effectively presented the TAA to activate established T cell lines or clones specific for the epitopes derived from these TAA (Metharom et al., 2005; Breckpot et al., 2004; Lopes et al., 2006; Metharom et al., 2001; Pullaers et al., 2000). As lentiviral vector-transduced DC induces stronger anti-tumor immunity when compared to peptide/protein-pulsed DC (He et al., 2005), strategies that aim at further stimulating innate immunity to produce inflammatory cytokines for T cell activations may augment the therapeutic efficacy of the TAA-transduced DC in the treatment of cancer. Lentiviral vector transduced DC that over-expressed Toll-like receptor (TLR) or TLR adaptor proteins (e.g., MyD88 and TRIF/TICAM-1) were able to enhance tumor-specific lysis and delay the growth of pre-existing tumor (Akazawa et al., 2007). Gene silencing of A20, a ubiquitin-modifying enzyme that negatively regulated receptor-interacting protein and TNFR-associated factors 2 and 6, in DC resulted in spontaneous production of proinflammatory cytokines such as IL-6, TNF and IL-12p40 (Song et al., 2005). It is also possible to enhance antigen presentation ability of DC by prolonging their life span via gene silencing of the mitogen-inducible nuclear orphan receptor (MINOR) whose expression leads to apoptosis (Wang et al., 2009), or by augmenting co-stimulatory signals on DC using lentiviral vectors to over-express co-stimulatory molecules such as CD40L (Koya et al., 2003) or gp34/OX40L (Kobayashi et al., 2004). Successful cancer vaccine strategy may require not only the induction of potent tumor antigen-specific immune responses, but also a mechanism to overcome tolerance/suppressive mechanisms. Efforts have been made to overcome such tolerance by enhancing Ag presentation abilities of the transduced DC. In a mouse model of aggressive renal cell carcinoma pulmonary metastases, massive metastases resulted in tumor-induced immune tolerance and tumor-related death (Zhang et al., 2005). Zhang et al., transduced hematopoietic stem cells (HSC) to express a surrogate TAA Ag (influenza hemagglutinin, HA), and transplanted these lentiviral transduced HSC in the animals with established pulmonary metastases to provide a constant pool of HA-expressing DC (and progenitors). Upon in vivo GM-CSF-induced DC expansion and adoptive transfer of HA-specific T cells, in vivo derived HA-expressing DC overcame tumor-induced immune suppression, and promoted HA-specific T cell activation that translated into improved survival of the tumor-bearing animals (Zhang et al., 2005). Multiple myeloma (MM) cell lysate-pulsed DC of the MM patients induced higher frequency of CD4+CD25highFoxP3high Treg cells, and suppressed activation of T cells (Wang et al., 2009). Han et al. reported that lentiviral transduction of DC from the MM patients to over-express calnexin (CNX), a chaperone protein involved in the proper folding and maturation of glycoproteins, was able to overcome such suppression. As over-expression of CNX in DC upregulated class I MHC and exhibited increased functional avidity (Wang et al., 2009), the ability to activate T cells at a lower threshold may be sufficient to induce the expansion of high-avidity effector T cells in the immunosuppressive tumor microenvironment (Han et al., 2008; Wang et al., 2009).
Lentiviral vectors have also been developed for the immunotherapy of viral infections. Immunizing mice with the lentiviral vector-transduced DC that expressed the immunodominant epitopes of lymphocytic choriomeningitis virus (LCMV) glycoprotein gp33-41 protected the immunized mice from a lethal dose of LCMV challenge (Zarei et al., 2004). Expression of multiple hepatitis C virus non-structural proteins (NS2, NS3, NS4a, b and NS5a, b) in monocyte-derived DC stimulated Ag-specific CD8+ T cell responses against NS3, NS4a and NS5b proteins, suggesting a potential use of lentiviral vectors in anti-HCV vaccine development (Jirmo et al., 2010).
The plasticity of DC functions supports the development of DC-based immunotherapy that suppresses Ag-specific immune responses in autoimmune diseases, allergy or transplantation tolerance. BMDC that were genetically engineered to secrete neuropeptide vasoactive intestinal peptides (VIP) expressed lower levels of CD40 and CD86, produced little IL-12p70 but higher level of IL-10 upon LPS stimulation (Tuscano et al., 2010). A single administration of the VIP-expressing DC decreased clinical symptoms in the experimental autoimmune encephalomyelitis and increased survival rate in the mouse model of peritoneal sepsis (Tuscano et al., 2010). In an experimental autoimmune myasthenia gravis (EAMG) model, administration of lentiviral-transduced DC that encoded short hairpin RNA (shRNA) against RelB gene suppressed the pre-existing immune responses, were able to induce IL-10 and IL-4 production in vitro and in vivo, and decreased anti-AChR IgG, IgG1, IgG2b Ab levels in serum (Zhang et al., 2009). The transduction of DC with lentiviral vectors that expressed IL-10 rendered them tolerogenic in a therapeutic mouse model of asthma (Henry et al., 2008), allergic contact dermatitis (Iglesias et al., 2006).
Direct Injections of Lentiviral Vectors to Genetically Modify DC In Vivo
VSV-G has been widely used in psuedotyping lentiviral vector particles because of the stability, the high titers and the effective transduction of cells of broad tropism. The VSV-G pseudotyped lentiviral vectors are efficient in the transduction of ex vivo manipulated DC for adoptive transfer in vivo, as well as inducing strong Ag-specific cellular and humoral responses in their direct injections in experimental animals in vivo (Dullaers et al., 2006; Breckpot et al., 2007; Esslinger et al., 2003; Palmowski et al., 2004; Rowe et al., 2006; Iglesias et al., 2006; Kim et al., 2005; He et al., 2006a; Chapatte et al., 2006). Intravenous injection of lentiviral vectors transduced cells in liver and MHC II-positive APC in spleen (VandenDriessche et al., 2002), and supported long-term gene expression in APC in spleen. Using a GFP-expressing lentiviral vector in the examination of the long-term transgene expression in DC subsets in spleen, Arce et al. demonstrated that intravenous injection of lentiviral vectors transduced proliferating DC precursors that maintained in spleen for at least 2 months. The persistence of the transduced DC precursors led to prolonged Ag presentation and effective T-cell memory (Arce et al., 2009). Esslinger et al. reported that resting DC at the site of subcutaneous injection was selectively transduced. The modified DC homed to draining lymph nodes and spleen where they primed antigen-specific T cells (Breckpot et al., 2007). Skin-derived DC (CD11c+B220−CD8−CD11b+DEC205+) subset, but not langerhans cells or langerin-expressing dermal DC, is the major APC that induces optimal CD8+ T cell responses after intradermal lentiviral-mediated immunization (He et al., 2006a; He et al., 2006b; Furmanov et al., 2010).
Direct administration of lentiviral vectors encoding TAA genes induced more potent TAA-specific immune responses, when compared to the DC that were transduced with lentiviral vectors ex vivo. There were higher number of IFN-γ producing cytotoxic T lymphocyte (CTL), stronger cytolytic activity, that in turn correlated well with a better protective immunity and prolonged survival time (Breckpot et al., 2007). Direct injection of HIV-1 polyepitopes-encoding lentiviral vectors into HLA-A2 or HLA-B7 transgenic mice induced broad CTL response against all 13 epitopes in the HLA-A2 transgenic and 8 out of 12 epitopes in the HLA-B7 transgenic mice along with augmented number of IFN-γ producing T cells (Iglesias et al., 2007). In a pilot study that utilized lentiviral vectors pseudotyped with two VSV-G of non-cross-reactive serotypes in a prime/boost vaccination strategy, Beignon et al. demonstrated that direct injections of the simian immunodeficiency virus (SIV) GAG-encoding lentiviral vectors in cynomolgus macaques elicited robust humoral and cellular immune responses against SIV GAG, and showed protection against SIVmac251 challenge (Beignan et al., 2009).
Use of Genetically Modified DC in Expansion of Specific T Cell Subsets In Vitro
The ability to expand a specific T cell subset with defined functional property in vitro will facilitate greatly the uses of these T cells in clinical applications in vivo (Kalinksi et al., 2010). For example, the αβTCR+CD4−CD8− double-negative (DN) T regulatory cells are a small population of T cells (of low 1-5% frequency) that are capable of suppressing Ag-specific T cells in inducing immunological tolerance in allograft transplantation (Zhang et al., 2000; Thomson et al., 2007). In a 2C-TCR transgenic mouse model, Thomson et al., transduced recipient-derived mature DC with a lentiviral vector that expressed the allogeneic major histocompatibility complex class I Ld antigen (LV-Ld). These modified DC were able to expand effectively the DN Treg cells, to maintain their ability to suppress CD8+ T cells in vitro and to prolong the survival of the Ld+ skin grafts in vivo (Thomson et al., 2007).
Use of shRNA of Different Gene Silencing Potency to Define DC Functions In Vivo
The increasing number of co-stimulatory molecules identified to date highlights the importance and complexity of co-stimulatory signals in defining DC functions. CD40 is upregulated in the mature immunogenic DC to induce optimal T cell activation. We established a lentiviral system to genetically modulate surface expression levels of CD40 on primary DC (Zhang et al., 2009). Quantitative surface expressions of CD40 were genetically defined by the use of shRNA sequences that target different regions of CD40 mRNA and of different gene silencing potencies. Using the transduced LPS-stimulated DC that expressed different level of CD40 on the cell surface, we demonstrated that the expression levels of CD40 on DC were pivotal in determining the different functional properties of DC to tolerize, activate or polarize antigen-specific Th1/Th2 immune responses in vivo (Zhang et al, unpublished). It thus presented a “switch” that may prove useful in delivering a specific function/therapeutic property of the genetically modified DC in the treatment of immune disorders, such as Type I diabetes.
Use of Non-Integrating Lentiviral Vectors in Transient Genetic Modifications
An important feature of lentiviral vectors is the ability of the viral integrase protein to integrate the gene(s) of interest in the host genome, thus supporting the efficient and long-term gene expression of the transgene in the transduced cells. Although the preferred sites of integrations seem to differ between the lentiviral and retroviral vectors (Felice et al., 2009), there will always be a concern that the vector integrations may pose a risk of the insertional mutagenesis. In applications that do not require a long-lived stable gene expression in dividing cells, non-integrated lentiviral vectors may provide a safer alternative to the current lentiviral-based therapies in driving the expression of the therapeutic gene. Towards this end, integration-deficient lentiviral vectors (that harbored class I mutation of the HIV-1 integrase) were developed for vaccination, and gene therapy in muscle cells and central nervous system (Apolonia 2007; Negri et al., 2007; Rahim et al., 2006). Integrase defective lentiviral vectors were effective in the transduction of human monocyte-derived DC (Negri et al., 2010) and mouse BMDC (Coutant et al., 2008). In a mouse model of West Nile Virus (WNV) infection, a single administration of nonintegrative lentiviral vectors that expressed a secreted form of the envelope of a WNV strain induced robust B cell response, and protected the immunized animals from a lethal WNV challenge (Coutant et al., 2008). Human monocyte-derived DC transduced by the influenza matrix M1 protein-encoding integration defective lentiviral vectors ex vivo were able to induce expansion of M1 primed CD8+ T cells in vitro (Negri et al., 2010).
Development of Novel Lentiviral Vectors in DC-Specific Transductions In Vivo
The successful demonstration of direct administration of the VSV-G pseudotyped lentiviral vectors in the genetic modification of DC in vivo raises the exciting potential of direct administration of the lentiviral vectors in the clinical DC-based applications. However, given the broad cell tropisms of the VSV-G envelope, novel strategies that target genetic modifications or transgene expression to DC (or DC subset) specifically may be highly desirable. It may avoid unwanted “off-target” cell activation, reduce the doses of the vector administration needed in the therapy and also allow specific manipulation of a therapeutic DC function. Two major strategies have been developed to target specific genetic modification of DC. First, transcriptional targeting of DC can be achieved by the incorporation of a DC-specific promoter in the lentiviral vectors. Cui et al. (2002) used the human HLA-DRα promoter in the transduction of human pluripotent CD34+ cells to regulate transgene expression in the HLA-DR+ cells that included differentiated DC (Cui et al., 2002). A DC-STAMP promoter was used to express high level of Ag to eliminate the reactive CD4 and CD8 T cells in peripheral organs and thymus, which led to induction of T cell tolerance in a mouse model of autoimmune diabetes (Dresch et al., 2008). Recently, Zhang et al. cloned a 1.2 kb CD11c promoter into a lentiviral vector to drive transgene expression in DC cells in vitro and in the generation of DC-specific transgenic mice (Zhang et al., 2009). Second, novel designs of DC-specific viral envelope proteins used in the pseudotyping of lentiviral vectors. Towards this end, Morizono et al. modified the sindbis virus envelope protein to express either a Fc-binding protein A domain, a biotin-adaptor peptide or arginine-glycine-aspartic acid RGD peptide. Such modifications supported conjugations of the pseudotyped lentiviral vectors with antibodies of defined Ag specificities, avidin-conjugated specific antibodies or integrin-expressing cells, respectively, for targeted cell type-specific transduction (Morizono et al., 2009; Morizono et al., 2009; Morizono et al., 2005). Mutant sindbis virus envelope glycoproteins were also engineered to bind to the DC-SIGN protein on DC (Dai et al., 2009; Yang et al., 2008; Morizono et al., 2011). A single immunization of these pseudotyped vectors was capable in eliciting Ag-specific immune responses in vivo (Dai et al., 2009; Yang et al., 2008).
Conclusions and Future Perspectives
Deregulation of the host immune system accounts for a large number of immune and inflammatory disorders (e.g., AIDS, cancers, allergy, autoimmune diseases, and graft rejections in transplantation). At present, non-Ag specific approaches with broad immunomodulatory effects (e.g., corticosteroids as anti-inflammatories, cyclosporine to inhibit all T cell activity) are widely used to modify human immune responses. A better understanding of the basic mechanisms underlying DC functional abilities to regulate initiation and activation of the immune system will allow development of selective therapeutic approaches that turn on or off specific and appropriate immune responses without interfering with the remaining human immune capacity. Replication-incompetent HIV-1 based lentiviral vectors are relatively simple to prepare in a standard laboratory (Kung et al., 2009). They present an exciting candidate gene therapy vector for hematopoietic progenitor cells, DC and other primary cell types (Kung et al., 2003; Kung et al., 2000; Tran et al., 2007).
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by targeting mRNAs for translational repression or degradation. Studies of miRNA identified selective expression of specific miRNA in specific cell types (Carissimi et al., 2009; O'Connell et al., 2010). Lentiviral vectors that expressed specific miRNA target sequence were capable of inducing either stable knockdown of the miRNA of interest or “de-targeting” transgene expression in specific cell type, depending on the expression levels of the mRNA target sequences in the transduced cells (Genter et al., 2009; Brown et al., 2007; Brown et al., 2007). Direct injection of the miR-142-regulated lentiviral vector that expressed a model GFP Ag in non-APC induced GFP-specific regulatory T cells, and promoted immunologic tolerance in mice (Annoni et al., 2009). Future identifications of miRNAs that are either absent in DC or play a regulatory role in defining specific DC function allows development of novel lentiviral vectors that target these miRNA in DC-based immunotherapy.
Recent studies revealed that crosstalk between DC and non-T lymphocytes (such as NK and NKT cells) played an important role in the regulation and induction of optimal immune response. Vaccination with mature DC transfected with mRNA encoding for TAA and the pro-inflammatory cytokines interleukin (IL)-12 and IL-18 induced optimal anti-tumor immune responses mediated by both increased NK and TAA-specific cytotoxic T cell activities (Bontkes et al., 2005). Similarly, loading TAA genetic modified DC with invariant natural killer T (NKT) cell activating factor, alpha-galactosylceramide, displayed synergic effects of TAA-specific cytotoxic T lymphocytes and NKT cells against metastatic melanoma (Fukushima et al., 2009). Genetic modifications of DC that enhance interaction/activation of these non-T lymphocytes may prove useful in further improving the efficacy of such DC-based immunotherapy.
In vivo administration of VSV-G pseudotyped lentiviral vectors activated splenic plasmacytoid DC to produce a robust IFNαβ response (Brown et al., 2007), suggesting lentiviral vector preparations can be immunogenic (Pichlmair et al., 2007). In ex vivo DC transductions, however, there are contrasting reports on the effects of lentiviral vectors on DC activation. Chen et al. (2004) reported lentiviral vector transduced human monocyte-derived DC displayed lower level of CD80, CD86, ICAM-1 and DC-SIGN, and a diminished capacity to induce Th1 differentiation in vitro. Gruber et al. (2000) reported that transduction of human monocyte-derived DC did not alter their viability, phenotype, maturation and capacity to stimulate T cell proliferation (Gruber et al., 2000). Veron et al demonstrated that lentiviral vector transduction of CD34+ progenitor-derived pDC did not induce IFNα production when compared to the non-transduced pDC cells (Veron et al., 2009). Breckpot et al. (2010) demonstrated that activation of mouse DC by lentiviral vectors was dependent on cell entry and reverse transcription. Moreover, such activation is dependent on TLR3 and TLR7-mediated TLR signaling (Breckpot et al., 2010). It is possible that differences in the lentiviral vector preparations, doses of the vector used in DC transduction, DC subsets, as well as the differentiation or maturation status of the DC used in the transduction will determine the extent of lentiviral vector-induced activation in the transduced DC. A good understanding of the magnitude of such lentiviral vector-induced DC activation in a specific treatment module is important in the optimal uses of these lentiviral vector-transduced DC in immunotherapy.
Advances in the novel design of the lentiviral vectors, envelope proteins for pseudotyping, and in vivo administrations of these vectors will continue to open up new opportunities and applications of the genetic engineering of DC in immunotherapy.
In summary, DC are professional antigen presenting cells (APC) that initiate and regulate immune responses. Molecules such as Ag, costimulatory molecules or cytokines can be expressed and manipulated (in the forms of DNA, RNA, siRNA or shRNA) directly in the DC to mediate specific modifications. Different strategies have been developed to modify DC ex vivo for manipulations of Ag-specific immunity in vivo. Replication-incompetent HIV-1 based lentiviral vectors are capable of stable transduction of both dividing and relatively quiescent cells. Safety features that safeguard the use of lentiviral vectors in gene therapy in vivo are built into the vector production system. Long-term, stable transduction of human monocyte-derived DC, human CD34+-derived DC, and mouse bone marrow derived DC have been reported.
DCs transduced with the lentiviral vector that expressed tumor associated antigen (TAA) or viral Ag have been shown to effectively process and present these Ag, and induced Ag-specific T cell responses. Strategies that aim at further stimulating innate immunity to produce inflammatory cytokines for T cell activations may augment the therapeutic efficacy of the TAA-transduced DC in the treatment of cancer. It is also possible to enhance antigen presentation ability of DC by prolonging their life span via gene silencing of the mitogen-inducible nuclear orphan receptor (MINOR) whose expression leads to apoptosis, or augmenting co-stimulatory signals on DC using lentiviral vectors to over-express costimulatory molecules such as CD40L or gp34/OX40L. The plasticity of DC functions supports the development of DC-based immunotherapy that suppresses Ag-specific immune responses in autoimmune diseases, allergy or transplantation tolerance. Moreover, the ability to expand a specific T cell subset (such as Treg, Th1, Th2 or Th17) with defined functional property in vitro will facilitate greatly the uses of these cells in clinical applications in vivo.
Quantitative surface expression of key molecule (such as CD40) can be genetically defined by the use of shRNA sequences that target different regions of CD40 mRNA and of different gene silencing potency. Expression levels of CD40 on DC are pivotal in determining the different functional properties of DC to tolerize, activate or polarize antigen-specific Th1/Th2 immune responses in vivo.
In applications that do not require a long-lived stable gene expression in dividing cells, non-integrated lentiviral vectors may provide a safer alternative to the current lentiviral-based therapies in driving the expression of the therapeutic gene. Integrase defective lentiviral vectors were effective in the transduction of human monocyte-derived DC and mouse BMDC, and the induction of anti-viral immunity.
Two major strategies have been developed to target specific genetic modification of DC. Transcriptional targeting of DC can be achieved by the incorporation of a DC-specific promoter (e.g., HLA-DRalpha, CD11c, DC-STAMP promoters) in the lentiviral vectors. Novel designs of DC-specific viral envelope proteins used in the pseudotyping of lentiviral vectors.
Replication-incompetent HIV-1 based lentiviral vectors emerge as an exciting candidate gene therapy vector for hematopoietic progenitor cells and DC. A better understanding of the basic mechanisms underlying DC functional abilities to regulate initiation and activation of the immune system allows development of selective therapeutic approaches that turn on or off specific and appropriate immune responses without interfering with the remaining human immune capacity. Identification of miRNAs that are either absent in DC or play a regulatory role in defining specific DC function allows development of novel lentiviral vectors that target these miRNA in DC-based immunotherapy. Genetic modifications of DC that enhance interaction/activation of these non-T lymphocytes may prove useful in further improving the efficacy of such DC-based immunotherapy. Advances in the novel design of the lentiviral vectors, envelope proteins for pseudotyping, and in vivo administrations of these vectors will continue to open up new opportunities and applications of the genetic engineering of DC in immunotherapy of a large number of immune and inflammatory disorders (e.g., AIDS, cancers, allergy, autoimmune diseases, and graft rejections in transplantation)
Example III
Exemplary sequences useful for the co-stimulatory sequences in lentiviruses of the invention are provided below.
NM—000074
Homo sapiens CD40 ligand (CD40LG), mRNA.


    
    
        1 actttgacag tcttctcatg ctgcctctgc caccttctct gccagaagat accatttcaa
        61 ctttaacaca gcatgatcga aacatacaac caaacttctc cccgatctgc ggccactgga
        121 ctgcccatca gcatgaaaat ttttatgtat ttacttactg tttttcttat cacccagatg
        181 attgggtcag cactttttgc tgtgtatctt catagaaggt tggacaagat agaagatgaa
        241 aggaatcttc atgaagattt tgtattcatg aaaacgatac agagatgcaa cacaggagaa
        301 agatccttat ccttactgaa ctgtgaggag attaaaagcc agtttgaagg ctttgtgaag
        361 gatataatgt taaacaaaga ggagacgaag aaagaaaaca gctttgaaat gcaaaaaggt
        421 gatcagaatc ctcaaattgc ggcacatgtc ataagtgagg ccagcagtaa aacaacatct
        481 gtgttacagt gggctgaaaa aggatactac accatgagca acaacttggt aaccctggaa
        541 aatgggaaac agctgaccgt taaaagacaa ggactctatt atatctatgc ccaagtcacc
        601 ttctgttcca atcgggaagc ttcgagtcaa gctccattta tagccagcct ctgcctaaag
        661 tcccccggta gattcgagag aatcttactc agagctgcaa atacccacag ttccgccaaa
        721 ccttgcgggc aacaatccat tcacttggga ggagtatttg aattgcaacc aggtgcttcg
        781 gtgtttgtca atgtgactga tccaagccaa gtgagccatg gcactggctt cacgtccttt
        841 ggcttactca aactctgaac agtgtcacct tgcaggctgt ggtggagctg acgctgggag
        901 tcttcataat acagcacagc ggttaagccc accccctgtt aactgcctat ttataaccct
        961 aggatcctcc ttatggagaa ctatttatta tacactccaa ggcatgtaga actgtaataa
        1021 gtgaattaca ggtcacatga aaccaaaacg ggccctgctc cataagagct tatatatctg
        1081 aagcagcaac cccactgatg cagacatcca gagagtccta tgaaaagaca aggccattat
        1141 gcacaggttg aattctgagt aaacagcaga taacttgcca agttcagttt tgtttctttg
        1201 cgtgcagtgt ctttccatgg ataatgcatt tgatttatca gtgaagatgc agaagggaaa
        1261 tggggagcct cagctcacat tcagttatgg ttgactctgg gttcctatgg ccttgttgga
        1321 gggggccagg ctctagaacg tctaacacag tggagaaccg aaaccccccc cccccccccg
        1381 ccaccctctc ggacagttat tcattctctt tcaatctctc tctctccatc tctctctttc
        1441 agtctctctc tctcaacctc tttcttccaa tctctctttc tcaatctctc tgtttccctt
        1501 tgtcagtctc ttccctcccc cagtctctct tctcaatccc cctttctaac acacacacac
        1561 acacacacac acacacacac acacacacac acacacacac agagtcaggc cgttgctagt
        1621 cagttctctt ctttccaccc tgtccctatc tctaccacta tagatgaggg tgaggagtag
        1681 ggagtgcagc cctgagcctg cccactcctc attacgaaat gactgtattt aaaggaaatc
        1741 tattgtatct acctgcagtc tccattgttt ccagagtgaa cttgtaatta tcttgttatt
        1801 tattttttga ataataaaga cctcttaaca ttaa (SEQ ID NO:12)

NM—005191

Homo sapiens CD80 molecule (CD80), mRNA.

        1 gacaagtact gagtgaactc aaaccctctg taaagtaaca gaagttagaa ggggaaatgt
        61 cgcctctctg aagattaccc aaagaaaaag tgatttgtca ttgctttata gactgtaaga
        121 agagaacatc tcagaagtgg agtcttaccc tgaaatcaaa ggatttaaag aaaaagtgga
        181 atttttcttc agcaagctgt gaaactaaat ccacaacctt tggagaccca ggaacaccct
        241 ccaatctctg tgtgttttgt aaacatcact ggagggtctt ctacgtgagc aattggattg
        301 tcatcagccc tgcctgtttt gcacctggga agtgccctgg tcttacttgg gtccaaattg
        361 ttggctttca cttttgaccc taagcatctg aagccatggg ccacacacgg aggcagggaa
        421 catcaccatc caagtgtcca tacctcaatt tctttcagct cttggtgctg gctggtcttt
        481 ctcacttctg ttcaggtgtt atccacgtga ccaaggaagt gaaagaagtg gcaacgctgt
        541 cctgtggtca caatgtttct gttgaagagc tggcacaaac tcgcatctac tggcaaaagg
        601 agaagaaaat ggtgctgact atgatgtctg gggacatgaa tatatggccc gagtacaaga
        661 accggaccat ctttgatatc actaataacc tctccattgt gatcctggct ctgcgcccat
        721 ctgacgaggg cacatacgag tgtgttgttc tgaagtatga aaaagacgct ttcaagcggg
        781 aacacctggc tgaagtgacg ttatcagtca aagctgactt ccctacacct agtatatctg
        841 actttgaaat tccaacttct aatattagaa ggataatttg ctcaacctct ggaggttttc
        901 cagagcctca cctctcctgg ttggaaaatg gagaagaatt aaatgccatc aacacaacag
        961 tttcccaaga tcctgaaact gagctctatg ctgttagcag caaactggat ttcaatatga
        1021 caaccaacca cagcttcatg tgtctcatca agtatggaca tttaagagtg aatcagacct
        1081 tcaactggaa tacaaccaag caagagcatt ttcctgataa cctgctccca tcctgggcca
        1141 ttaccttaat ctcagtaaat ggaatttttg tgatatgctg cctgacctac tgctttgccc
        1201 caagatgcag agagagaagg aggaatgaga gattgagaag ggaaagtgta cgccctgtat
        1261 aacagtgtcc gcagaagcaa ggggctgaaa agatctgaag gtcccacctc catttgcaat
        1321 tgacctcttc tgggaacttc ctcagatgga caagattacc ccaccttgcc ctttacgtat
        1381 ctgctcttag gtgcttcttc acttcagttg ctttgcagga agtgtctaga ggaatatggt
        1441 gggcacagaa gtagctctgg tgaccttgat caaggtgttt tgaaatgcag aattcttgag
        1501 ttctggaagg gactttagag aataccagtg ttattaatga caaaggcact gaggcccagg
        1561 gaggtgaccc gaattataaa ggccagcgcc agaacccaga tttcctaact ctggtgctct
        1621 ttccctttat cagtttgact gtggcctgtt aactggtata tacatatata tgtcaggcaa
        1681 agtgctgctg gaagtagaat ttgtccaata acaggtcaac ttcagagact atctgatttc
        1741 ctaatgtcag agtagaagat tttatgctgc tgtttacaaa agcccaatgt aatgcatagg
        1801 aagtatggca tgaacatctt taggagacta atggaaatat tattggtgtt tacccagtat
        1861 tccatttttt tcattgtgtt ctctattgct gctctctcac tcccccatga ggtacagcag
        1921 aaaggagaac tatccaaaac taatttcctc tgacatgtaa gacgaatgat ttaggtacgt
        1981 caaagcagta gtcaaggagg aaagggatag tccaaagact taactggttc atattggact
        2041 gataatctct ttaaatggct ttatgctagt ttgacctcat ttgtaaaata tttatgagaa
        2101 agttctcatt taaaatgaga tcgttgttta cagtgtatgt actaagcagt aagctatctt
        2161 caaatgtcta aggtagtaac tttccatagg gcctccttag atccctaaga tggctttttc
        2221 tccttggtat ttctgggtct ttctgacatc agcagagaac tggaaagaca tagccaactg
        2281 ctgttcatgt tactcatgac tcctttctct aaaactgcct tccacaattc actagaccag
        2341 aagtggacgc aacttaagct gggataatca cattatcatc tgaaaatctg gagttgaaca
        2401 gcaaaagaag acaacatttc tcaaatgcac atctcatggc agctaagcca catggctggg
        2461 atttaaagcc tttagagcca gcccatggct ttagctacct cactatgctg cttcacaaac
        2521 cttgctcctg tgtaaaacta tattctcagt gtagggcaga gaggtctaac accaacataa
        2581 ggtactagca gtgtttcccg tattgacagg aatacttaac tcaataattc ttttcttttc
        2641 catttagtaa cagttgtgat gactatgttt ctattctaag taattcctgt attctacagc
        2701 agatactttg tcagcaatac taagggaaga aacaaagttg aaccgtttct ttaataa (SEQ ID NO:13)

NM—001252

Homo sapiens CD70 molecule (CD70), mRNA

        1 ccagagaggg gcaggctggt cccctgacag gttgaagcaa gtagacgccc aggagccccg
        61 ggagggggct gcagtttcct tccttccttc tcggcagcgc tccgcgcccc catcgcccct
        121 cctgcgctag cggaggtgat cgccgcggcg atgccggagg agggttcggg ctgctcggtg
        181 cggcgcaggc cctatgggtg cgtcctgcgg gctgctttgg tcccattggt cgcgggcttg
        241 gtgatctgcc tcgtggtgtg catccagcgc ttcgcacagg ctcagcagca gctgccgctc
        301 gagtcacttg ggtgggacgt agctgagctg cagctgaatc acacaggacc tcagcaggac
        361 cccaggctat actggcaggg gggcccagca ctgggccgct ccttcctgca tggaccagag
        421 ctggacaagg ggcagctacg tatccatcgt gatggcatct acatggtaca catccaggtg
        481 acgctggcca tctgctcctc cacgacggcc tccaggcacc accccaccac cctggccgtg
        541 ggaatctgct ctcccgcctc ccgtagcatc agcctgctgc gtctcagctt ccaccaaggt
        601 tgtaccattg cctcccagcg cctgacgccc ctggcccgag gggacacact ctgcaccaac
        661 ctcactggga cacttttgcc ttcccgaaac actgatgaga ccttctttgg agtgcagtgg
        721 gtgcgcccct gaccactgct gctgattagg gttttttaaa ttttatttta ttttatttaa
        781 gttcaagaga aaaagtgtac acacaggggc cacccggggt tggggtggga gtgtggtggg
        841 gcgtagtggt ggcaggacaa gagaaggcat tgagcttttt ctttcatttt cctattaaaa
        901 aatacaaaaa tca (SEQ ID NO:14)

NG—012083

Homo sapiens intercellular adhesion molecule 1 (ICAM1),

    
    

















gene
5001 . . . 20775




/gene = “ICAM1”




/gene_synonym = “BB2; CD54; P3.58”




/note = “intercellular adhesion molecule 1”




/



mRNA
join (5001 . . . 5386, 8925 . . . 9188, 17641 . . . 17946,




18193 . . . 18480, 18563 . . . 18817, 18943 . . . 19188,




19275 . . . 20775)



exon
5001 . . . 5386



CDS
join (5320 . . . 5386, 8925 . . . 9188, 17641 . . . 17946,




18193 . . . 18480, 18563 . . . 18817, 18943 . . . 19188,




19275 . . . 19447)



exon
8925 . . . 9188



exon
17641 . . . 17946



STS
17695 . . . 18437


“





exon
18193 . . . 18480



STS
18348 . . . 18667



exon
18563 . . . 18817



exon
18943 . . . 19188



STS
19058 . . . 19738



exon
19275 . . . 20775



STS
19458 . . . 19682



STS
19672 . . . 20386



STS
19727 . . . 19866



STS
20472 . . . 20606


”





gene
21134 . . . 22682




gene = “ICAM4”



mRNA
join (21134 . . . 21566, 21696 . . . 22682)




gene = “ICAM4”



mRNA
join (21134 . . . 21566, 21696 . . . 21998, 22146 . . . 22682)




gene = “ICAM4”



mRNA
join (21134 . . . 21566, 21773 . . . 21998, 22146 . . . 22682)




gene = “ICAM4”



CDS
join (21173 . . . 21566, 21773 . . . 21998, 22146 . . . 22344)




gene = “ICAM4”



CDS
join (21173 . . . 21566, 21696 . . . 21998, 22146 . . . 22264)




gene = “ICAM4”



CDS
join (21173 . . . 21566, 21696 . . . 22015)




gene = “ICAM4”



STS
21874 . . . 22676



STS
22013 . . . 22262



STS
22047 . . . 22240



STS
22073 . . . 22282









Origin

    
    
        1 attttatctg ctcgtccagc atggtcagcc ctccactttt taaattttat tttatttatt
        61 tttttgagac agagtctcac tctgttgtcc aggttggagt ccagtggcgt gatttcggct
        121 cactgcaacc tctacttccc aggttcgagc aattctcctg cctcagcttc ccgagtagct
        181 gggattacag gcccgcgtcc ccacacctag ctaatttttg tatttttagt agagacaggg
        241 tttcaccatg ttggccaggc tggtctcgaa ctcctgacct caggtgattc tcctgccttg
        301 gcctcccaaa gtgctgagat tacaggtgtg agccactgca cacggcctta aattttattt
        361 attatttatt tatttattta tttagagact tagtctcact ctgttgccca ggctggagtg
        421 cagtggcatg gtctcggctc actgcactcc acctcctggg ttcacgccat tctcctgcct
        481 cagcctcccg agtagctggg actacaggcg cccaccacca ctcccggcta atttttgtat
        541 ttttagtaga gatggggttt cactgtgtta gccaggatag tctcgatctc ctgacctcgt
        601 gatccgcctg cctcggcctc ccaaagtgct gggattactt attttgtttt ttgtagagac
        661 aggttctcac tgtgttgccc aggctggtct tgaactcctg atctcaagtg atcttcccac
        721 ctcagtctct caaagggctg ggattacagg ggtgagccac tgcaccccac cttccctcta
        781 ctttttgacg gtttccttct gctatgaatg tgcatgtcca gttgtctgct tcttagaact
        841 gatatttacc ttcctcatcc atcagccatt ggaggaggac tgggaccgct cagattattg
        901 atctgaccca ttctttcggc agggtttcct ggtggctgtc ttccatcacc aaaactggaa
        961 tcagaagagt ttccatagcc cttttttttt ccccacatct ttgctgaagc agagttttga
        1021 aaaacaaaac cacaaactaa gctattcccc agaagaaatc tgtaatcaaa gataagctct
        1081 gccgggcaca gtggctcacg ccttttggag gccaaggcgg gcggatcacc tgaggtcagg
        1141 agttctagac ctgccaggcc aacatggtaa aacctcatct ctactaaaaa tacaaaaatt
        1201 agctagatgt ggtggtgggt acctgtagtc tcagctacct gggaggctga ggcaagagaa
        1261 tcgcttgaac ctgggaagta gaggttgcag tgagccgaga ttgcaccact gcactccagc
        1321 ctgggcgacg gagtgagacg acctcacaaa aatttacata aataaaatga aaagtaaaat
        1381 aaaaatacaa aagttggccg ggtgcgtttg ctcacgcctg taatcccagc actttgggag
        1441 ggtgaggcag gcagataatg aggtaagaag atcgagacca tcctggctaa cacggtgaaa
        1501 ccctgtctct actaaaaata caaaaaatta gctgtgcgtg gtgacacgca cctgtagtcc
        1561 cagctatttg ggaggctgag gcaggagaat cacttgaacc tgggaggtgg aggttgcagt
        1621 gagccgagat cgcaccactg cactccagcc tgggccacag agtgagactc catcttgaaa
        1681 aaaaaaaaaa atacaaaagt tagccagggg tgttggtggg tgcctgtaat cccagctatt
        1741 tgggaggcta aggcagaaga atttcttgaa cctaggaaac ggaggttgca gtgagccgag
        1801 atcacacctc tgtactccag cctggacaac agagcgagac tttgtctcaa aaaaaaaaaa
        1861 aaaaaaaaaa ctaaataggc cgggagcagt ggctcatgcc tataatccca gccctttggg
        1921 aggccaaggc aggtggatca cttgaggtca ggagtttgag accaggctgg ccaacatggt
        1981 gtaaccccgt ctctactaaa aacacaaaaa ttagccgggt ctggtggcgt atgtctgtaa
        2041 tcccagctac tcgggaggct gaggcaggag aatcacttaa acctgggagg caggggttgc
        2101 agtgagctga gatcgtgcca ctgcactcta gccagggtga cagagtgaaa ctctgtctca
        2161 aaaaattaaa aaagaaattc agcaagtaat gagttaagga attcgaatat taaggcgagt
        2221 gacaaggaac gcccaggatg tggcccagga tggagtaggg gggacactca tttaggagaa
        2281 agctcaggcc acaagacagg aggagccagc cttgttgggg ttgaagggaa gagcattcca
        2341 ggctgaggga actgcaaggc gtttgcatgg gacactatgg gatggcttct gcccttggtg
        2401 ggcagcctct ggtctgaggc cattctttgg cctgcctgac tgtctggcaa ccgggaggaa
        2461 gccctgccct tcctggagac agaaacaaag gtctaggaaa tatctgcttc ccttttcctt
        2521 gaaaaacgct taagggaacg gaggactggg aggtgccgtc tctctctgcc agcctgcccc
        2581 ctaccatagc catcccactc ccatctcaga aagtgacccg ccatcctcca aaaggctcgg
        2641 accctgatca aggagtcatc ccccttgtcc cagcacctcc agttggccca gcctccaaaa
        2701 cggatgtcaa attcagccct ttctccaagg acactgccca gtccaggccc cactatcatt
        2761 catctggact agaacagtca cctcctctcc catctcctgg ctgcagctct tgaagcctca
        2821 actgggcccc tgtgaacact tgagttaggg caaggtcctt cctctgctca gaaccctcta
        2881 tacctcccac ctcgctgggc ataaaagcca aagtcctggc caggcacggt ggctcacatc
        2941 tgttatccca gcactttggg aggccaaggg gggcggatca ctagaggtca ggagttagag
        3001 accaacatgg tgaaacccca tctctactaa aaatacaaaa attagctagg cgtggtgacg
        3061 cacccctgta gtaccagcta ctcggtaggc tgaggtggga gaatcgcttg aacctgggag
        3121 gcagagtttg cagtgagccg agatcacacc actgtgctcc agcctgggtg acagaacgag
        3181 actggggttc agaaacaaac aaacaaaaca acaaagtcct cctcaggtga caggaacttg
        3241 cacctatctg ccctgtcatc tccctgcccg ctcctctcct cgaatctctc ctttgctaag
        3301 cctgctccag ccacactgtt ctcctggctg ttcctttttt tttttttttt tttttttttt
        3361 tgagtctcac tctcacccag gctggagtgc agtgcctcta tcttggctca ctgcaacctc
        3421 cgcctgccgg gttcaagaga ttctcctgca tcagcctccc aagtaggtgg aattacaggt
        3481 gtgcaccacc acacccggct aatttttgta ttttgcatag agatgggggt ctccctatgt
        3541 tgcccaggct ggtcttgaac tcctgggctc aagtgatcct cccatctcgg cctcccaaaa
        3601 tgctgggatt acaggtggga gccgcgccca ggtggatttt tgtctgactc tgttcattcc
        3661 tgtgtcccca gtacctggaa ggacgccaag cacacagtag gcgcttaaaa aacattgagc
        3721 cacatgttga gaaaagaacg gcaccattgt ggctgcaagt gggacttggg ccgcgcgggg
        3781 gagctcgcgc acctcgggcc ggggcaagag ctcagtggaa cccgcccgag gaagaacccg
        3841 tggcgcagga ttttcccagg ccttctgagg accaggggcg tcccccgtcc caccctgtga
        3901 ctttgctcag gccgttccgg ggcgggaatt cagaactcct cagcccccca agaaaaaaat
        3961 atccccgtgg aaattccttg ggaatgaccg aggcggggga aatatgcgtc tctggatggc
        4021 cagtgactcg cagccccctt ccccgatagg aagggcctgc gcgtccgggg acccttcgct
        4081 tccccttctg ctgcgcgacc tccctggccc ctcggagatc tccatggcga cgccgcgcgc
        4141 gccccacaac aggaaagcct taggcggcgc ggcttggtgc tcggagactt aagagtaccc
        4201 agcctcgacg tggtggatgt cgagtcttgg ggtcacacgc acaggcggtg gccaagcaaa
        4261 cacccgctca tatttagtgc atgagcctgg gttcgagttg ccggagcctc gcgcgtaggg
        4321 caggggttcg agcgcccctt ctccctgcct cgcctctgcg cctgggggct gctgcctcag
        4381 tttcccagcg acaggcaggg atttcgagcg tccccctccc ctccctcgtc aagatccaag
        4441 ctagctgcct cagtttcccc gcggagcctg ggacgccagc ggaggggctc ggcgcgtagg
        4501 gatcacgcag cttccttcct ttttctggga gctgtaaaga cgcctccgcg gccaaggccg
        4561 aaaggggaag cgaggaggcc gccggggtga gtgccctcgg gtgtagagag aggacgccga
        4621 tttccccgga cgtggtgaga ccgcgcttcg tcactcccac ggttagcggt cgccgggagg
        4681 tgcctggctc tgctctggcc gcttctcgag aaatgcccgt gtcagctagg tgtggacgtg
        4741 acctaggggg aggggcatcc ctcagtggag ggagcccggg gaggattcct gggcccccac
        4801 ccaggcaggg ggctcatcca ctcgattaaa gaggcctgcg taagctggag agggaggact
        4861 tgagttcgga ccccctcgca gcctggagtc tcagtttacc gctttgtgaa atggacacaa
        4921 taacagtctc cactctccgg ggaagttggc agtatttaaa agtacttaat aaaccgctta
        4981 gcgcggtgta gaccgtgatt caagcttagc ctggccggga aacgggaggc gtggaggccg
        5041 ggagcagccc ccggggtcat cgccctgcca ccgccgcccg attgctttag cttggaaatt
        5101 ccggagctga agcggccagc gagggaggat gaccctctcg gcccgggcac cctgtcagtc
        5161 cggaaataac tgcagcattt gttccggagg ggaaggcgcg aggtttccgg gaaagcagca
        5221 ccgccccttg gcccccaggt ggctagcgct ataaaggatc acgcgcccca gtcgacgctg
        5281 agctcctctg ctactcagag ttgcaacctc agcctcgcta tggctcccag cagcccccgg
        5341 cccgcgctgc ccgcactcct ggtcctgctc ggggctctgt tcccaggtga gtcggggtgg
        5401 ggattgccgt cgggccagtt ctccgaagcc ccgggaggac cggctcccgg gtcaggtcat
        5461 gcatgcttag gtagctgttt atgggaagga ggggctagag acagcgattg aaaggcaaca
        5521 gccagtaggt tcgaatccag accctgcata cctccacgtg tggccttggg ctatagattg
        5581 cagctttaaa aaagggtagg gggttggaga tggaggggag gggcgggcct cgttttgttg
        5641 cccaggccgg tcttgaactc cgggggtcta gccttacctc ctgcctcagc ctcccgagta
        5701 gctgggatga gaggtgtgaa ccaccgcctt gcttggctag attgcgtctc ttacagtttc
        5761 tcagctgtaa aacgggaaac gttatagcgg ccacctggca gggtatcttg gcccagcgca
        5821 gcacctggcc ccaggactcg atcatgatgg tttgggaact tggctctgtg ccaacccaac
        5881 aaggcttaag ggacccccac ccccctcaag atgtatattc tgttcctcat cctctctgcc
        5941 cctggggaag tccagggctg cttctacttg ggggaattcc agagctgact tatccgtggc
        6001 ccaaagctga gaagtgggac gccccagcac accctccccc agctccagcc cagctaggga
        6061 agagggaagg ggtcagaggg tctttcatgg tggtgtaagt ttggggaacc aggagggtgg
        6121 gagattgaca gcttggttaa cagctcaaca aagcctgaga tccaggccag cacggtagtt
        6181 catgccagta atcccaacac tttaggagcc ccaggcgggc gaatcactta aggtcaggag
        6241 tttgagacca gcctggccaa catggcaaca tcccgtctct actaaaaata caaaaattag
        6301 ctggcatggt ggtgggcgcc tgtgatccca gctgctcggg aggctgaggg aggaaaatcc
        6361 cttaagccca cgaggctgag gttgcagtga accaagattg tgccactgca ctccagcctg
        6421 ggagacatag cgagattctg tctcaaaaaa caaagcgttc tgatccggac tcagacccag
        6481 atcgcactgc tttctagctg agtaaccatt tctctctatg aaatgggaat ggtcccagaa
        6541 tctcccttgg agaatgtatg gagccagtgt cctcacaccc ccatccaaga tagaacaaat
        6601 ctgagacagg aatctttgag tgaggcagtg ctgggctcag acattttttc ccaccttcgg
        6661 aggcagcaga atctgaggga cctgatccaa ataagcccct tctttctttc ttttcttttc
        6721 tttttttttt tttttttttt ttgagacgga gtctcactct gtcgcccagg ctggagtgca
        6781 gtggcgtgat ctcggctcac tgcaacctct gcctcccagg ttcaaacgat tctcctgcct
        6841 cagcctccct gagtagctgg gactacaggc atgtgccatc acacccggct aatcactgtg
        6901 ttagccagga tggtctcgat ttcctgacct catgatctgc ccaccctgcc tcccaaagtg
        6961 ctgggattac atgcgtgagc cacagtgccc accccgtaag ccccttcttt cttacctgca
        7021 aggtagccag ttgctaccca tcctgtgctg agttacttgt attagcaagg gatggggtgg
        7081 ctatactcac ccaccttaca gatggggaaa ttgaggccca aagaggggga aactacgtgt
        7141 ctcagggagt gaggagccag tctgattcct ggagggctga ctgtctccac ctgacttctt
        7201 aggagggagg agggcaccaa cttcacatta aaatctggtt ggacacagtg gctcacacct
        7261 gtaatcctgg cattttggga ggcttaggcg ggaggatcac ttgaggccag gagtttgaga
        7321 ccagccttag caacatagtg agaccccatc tctacaaaaa tgtttttcag ggccaggcgc
        7381 ggtggctcac acctataatc ccagcacttt gggaggctga ggcgggcgga ttacctgagg
        7441 tcaggagttt gagaccagcc tgaccaacat ggagaaaccc cgtctctact aaaagtacaa
        7501 aattacccgg gcgtggtggc gcatgcctgt aatcccagct actcgggagg ctgaggcagg
        7561 agaatcgctt gaacctggga ggcggaggtt gcggtgaact gagatcgtgc cattgcactc
        7621 cagcctgggc aacaagagct aaactccgtc tcaaaaaaaa aaaaatgttt ttcaaatatt
        7681 agccgggtat ggtggtgtcc tgtagtccca gctacttggg aggctgagat gggaggatca
        7741 cttgagccca ggagttcaag gttacagtga gctatgattg tgccactgta ttccagcctg
        7801 ggtaacagag ggagacccgt ttaaaaaaaa aaaagtgatg gctaaagtcc ttccatggct
        7861 ccctattgcc ctcagtataa agaacacatg tggctgggcg tggtggttca cgcctgtaat
        7921 cccagcactt tgggaggctg aggcgggcgg atcacttgag gccaggagtt tgagagcagg
        7981 ctggccgacg tggcgaaacc ccgtctctat taaaaataca aaaattagct gggcgtggtg
        8041 gtgcttgcct gtaatcccag atactctgga ggctgaggca ggagaatcac ttgaacccgg
        8101 gaggcaaagg ttgcagtgag ctgagattgc gccactgcac tccagtctga gtgacaaagc
        8161 gagactccat ctcaaaaaaa aaaaaaataa aagaacacat ctttagcatg gccttcagtg
        8221 ctcacgggat cttcctgaat taatctcccc ctcttcatcc ttgctcactc agctccagcc
        8281 accctgcccc gggacatctg tacttgcctg gaacttattt cccttttctc cggacagcca
        8341 gccctttctc gtcatttaga tctctgctga aacattaccc tgtcaccaaa gcactgtcta
        8401 ttctatcacc ctgttttgtt tttgtcaaag ctcatattaa catcagttat taattatctt
        8461 gtttgctcat aatttttttt tttttttttt tggagacaga gtctcgttct gttgctcagg
        8521 ctggagtgca gtggcacaat cttggctcac tgtaacctcc acctcccagg ttcaagtgat
        8581 tcttgtgcct cagcctccca aatagctagg actacaggca cgtcccacca tgcccagcta
        8641 atttttgtat ttttagtgga gacggggctt tgtcatgttg gccaggctga tctcaaattc
        8701 ctgacctcaa gtgatctgcc cgccttggcc tcccaaagtg ctgggattac aggcgtgagc
        8761 caccacaccc ggcctgctca tgaattttct ctttaacttc cacatcgaag gcaaagtatt
        8821 gtcttgttaa ggctgtgcct ccagcaccca gcacaggctg ggcgcacatt cccttgatga
        8881 acctgatttg taatgcctgt cgcctcttcc ctcgtttctt ctaggacctg gcaatgccca
        8941 gacatctgtg tccccctcaa aagtcatcct gccccgggga ggctccgtgc tggtgacatg
        9001 cagcacctcc tgtgaccagc ccaagttgtt gggcatagag accccgttgc ctaaaaagga
        9061 gttgctcctg cctgggaaca accggaaggt gtatgaactg agcaatgtgc aagaagatag
        9121 ccaaccaatg tgctattcaa actgccctga tgggcagtca acagctaaaa ccttcctcac
        9181 cgtgtactgt gagtaactga gcccggaggg ctggactagg cagacccggt gggagagacg
        9241 tgcaggggca cctgcagagg cctgggggaa tctttgccac ttgctcgtag ggtcaaggag
        9301 gggctccttg cagggcaggt ggggacatcc ttggaaagtc cctttgtgaa tttctttggg
        9361 tacaattaaa gtatttacag gctgggtgcg gtggctcatg cctgtaatcc cagcactttg
        9421 agaggctgag gctggcggat cacctgagat caggagttta agtttcgcca acatggcgaa
        9481 accctgtctc tgctgaaaat acaaaaatca gccgggcatg gtgtcaagcg cctgtaatcc
        9541 cagctacttg gaaggctgag gcaggagaac gcttgaacct gagaggcaga gattgcagtg
        9601 agccgcgatc gtgccagtgc actccagtct ggataacaga gcaagattcc atctcaagaa
        9661 aaaaaaaatg ccatctctct atgcctcact ctttgaacat atgacacggt cctgcttcag
        9721 acactttaat aaaagatgca aattaagcca agtgtggtgg cttgtaccta taatcccaac
        9781 tactccagag gctgaggcag aaggatggtt tgagcccagg agtttgagac cagcctgggc
        9841 aacagagtga gaccctgttt ctttcttttt tttttttttt tttttgagac ggagtctcac
        9901 tctgtcgccc aggctggagt gcagtggtgt gatctcggct cactgcaagc tccgcctccc
        9961 gggttcacgc cattctcctg cctcagcctc ccgagtagct gggactacag gcgcctgcca
        10021 ccatgcccgg ctcatttttt tgtattttta gtagagacgg ggtttcactg tgttagccag
        10081 gatggtctca atctcctgac ctagtggtcc gcccgcctcg gcctcccaaa gtgctgggat
        10141 tacaggtgta agccactgtg cccatccaag accctgtttc taccggaaaa aaaaagtaaa
        10201 taatttagct gggcatcgtg gtgtgcacct gtaatcccag ctgctcctga ggctgtgatg
        10261 ggaggattgc tttaacccag gggttcgaat cctaggagtt cgaatccatc ctaggcaaca
        10321 tagcaaaacc ccatttttat ttaaaaaaaa aaaaaaagat atgagttaaa actagccctg
        10381 ggatggcatt tttcacatat tggtaacaaa caaaagaatt gatggccggg cgcagtggct
        10441 cacgcctgta atcccagcac tttgggaggc cgaggcgggc ggatcacaag gtcaggagat
        10501 cgagaccatc ctggctaaca tggtgaaacc ccgtctctac taaaactaca aaaaattagc
        10561 cgggcatggt ggcaggcgcc tgtggtccca gctactcagg aggctgaggc aggagaatgg
        10621 catgaacccg ggaggcagag cttgcagtga gccaagatcg tgccactgca ctccagcctg
        10681 ggcgacagag caagactcca tctcaaaaaa aaaaaaaaaa aaaagaattg ataacagctg
        10741 tgctgccaag gctattggaa cgtaggaggt cctaggacag tgctgttggg agcataaata
        10801 agcccaaccc tgtggcggga aattgggcat cagttctcaa aatgtcatgg gctgggcacg
        10861 gtggctcacg cctgtaatcc cagcactttg ggaggctgag ggaggcggat cacttgaggt
        10921 caggagttcg agaccagcct gaccaccatg gagaaacccc gtctctatta aaaatacaaa
        10981 aaaaactagc caggcatagt ggcacatacc tgtaatccca gctactcggg aggctgaggc
        11041 aggagaatct cttgaaactg ggaggcagag gttgcggtga gctgagattg cgccactgca
        11101 ctccagcttg ggcaacaaga gcaaaactcc atctcaaaaa aaaaaaaaaa agaaataaaa
        11161 gaaggtatgt tgaatatgag tggtatgcca ccctcacatt agggaagggc agtttcgggg
        11221 aggctgtatt tatgtataaa atagccctaa aaggaagtgg gagaaatgac aatattagct
        11281 ggctatgaga agagaggctg ggaggctgtg ggagagggct tgggtgtgga gaattctttt
        11341 tgttttttcc tttttttgag acagagtttc actcttgttg cccaggctgg agtgcaatgg
        11401 cacgatctca gctcaccgca accttcacct cctgagttta agtgattctc cggcctcagc
        11461 ctcccgacta gctgggatta caggcatggg ccactacgcc tggcaaattt tgtattttta
        11521 gtagagacag ggtttctcca tgttggtcag gctggtcttg aactctgacc tcaggtgatc
        11581 cgcccgcctc ggcctcccaa agtgctggga ttacaacgtg agccactttg cctggctgag
        11641 aattcttttt ttgttgttgt ctttttgaga tggagttttg ctgtgtcccc agcctggagt
        11701 gcaatggtgt aatctcagct cactgcaacc tctccctccc gggttcaagc aattctcctg
        11761 cctcagcctc ccaagtagct ggaattacag gcgcccagca ccacgcccgg ctaatttttg
        11821 tatttttagt agagacggga tttcatcatg ttggccaggc tggtcttgat ctcctgacct
        11881 tgtgatctgc ccgcctcggc ctcccaaagt gctgggatta caggcgtgag ccactgcccc
        11941 cagccgagaa tttctctttg cgtccttcct actttgggga cttcgaatgg tgggaaagag
        12001 ttatcaaggc caaaataagg aattcaaatg aaaacaaaac aaaatcaaag aagaaaaaac
        12061 agaagagcac tgggcaggct aggcacgtgg ctcatgccta taatcccagt gatttagaag
        12121 gccgaagtag aaggatcgct tggaggccag gagttggaca ccagcctggg caacatagca
        12181 agaccccata tctacaaaaa ataaaaaacc taaccaagcg tgctggcata ctagtagtcc
        12241 cagctactca ggaggctgag gtgggaggat cacctgagcc tgggaggtcc aggctgtagt
        12301 gagccgtgat gacaccactg cactccagcc tgggtgacag agaaagaccc tgtctctaaa
        12361 aaataaaaac tggccaagta gctttgggat tagccttggg ttccagtccc agcaaggcct
        12421 ttaatagctt gggacatgac ttctgcattt actttgcaat caggtgagac ctcctctgat
        12481 ggggaaaatg acacggtgag tgacaaagga tgttctccta tcattgtgtc agggcaagga
        12541 agcctctggg taaatgatca aatgatcagc tttgcttctg atttggaggg tgggtgagca
        12601 gatgctgacc ttcccaggtg agggaagtcc ccgaacattc ccagcagctt ctggaaaccc
        12661 cagggaaacc tctttgaagg tcttttctgc atctctgcct gataggtctt tttttttttt
        12721 tttttttttt tctttttttt gacacacagt cttgctctgt cgcccaggct ggatcactgc
        12781 aacctccatc tcccgggttc aagcagttct cctgcctcat cctctccagt agctgggatt
        12841 acaggcacct ggcaccacgc ctggctaatt tttgtatttt tagtagagac agggtttcgc
        12901 caagttggcc aggctggtct cgaactctag acctctggtg atccacccgc ctcagcctcc
        12961 cgaagtgctg ggattacagg cttgagccac cacgcgcggc tctgcctgat agctgagagc
        13021 atagaactcc aggtttgaga cctggctctg ccacatttct ccctctatga ctgtgggtgc
        13081 cccactttgc cttagttttt acctctgtga aatggagcag atggctggca caggtagcaa
        13141 aggagtaaaa gttatgtggg agggtggtac ctgagagaga ctctagcttg gtcttgcccc
        13201 acccctggtg taaacataaa gaagcctccc tggatggctc aatcttctcc aaaaaggtta
        13261 gaggtgtaat tcctagagga ggcgaccact agctgggctt tgaaggatgt gtaggagttc
        13321 ataaggacag gcattctggg caggaggaac agcctgggca aaagttggga gcagggagaa
        13381 atcttgatgg aggcaggagg aggaggaggt aggttggtgt aggccaggtg cagtggctca
        13441 cacctgtaat cccagtgctt tgggaggcca aagcaagagg attgtttgag cccaggagtt
        13501 cgagaccagc ctgggcaaca tagcgagacc ctgtctctaa gaaaaaataa aaaaattagg
        13561 gtacagtggc atatgcttgt attctcaact actctggagg ctcaagtggg aggatcactt
        13621 gaacccagga atttttttgt ttttgttttt gtttttttga gatggagtct cactctgtgg
        13681 cccaggctgg agtgcagtgg tgccatcttg gctcactgca gcctcctcca cctcctgggt
        13741 tcaaccgatt ctgctgcctg agcctcccga gttgctggga ttaaggtgcc caccatcatg
        13801 cccagctaat ttttttgtat ttttagtaga gatagggttt accatgttag ccaggctggt
        13861 cttgaacgcc tgaccgcaag agatcctcct gcctcagttt cccaaagtgc tgggattata
        13921 ggtgtgagcc actgagcctg gtcaagccca ggaatttgag gttacagtca gctatgattg
        13981 caccactgca ttccagccca ggtgacagag agagacactg cccctaaaaa aaaaaaaaaa
        14041 attgattgat gggaggaagg gtgaggttgg cagagccttg aatgccaggt ggaggagctg
        14101 ggactttcct tcttggggtg atagggagtc atggagggtt tctgagcagg ccagggatta
        14161 gatagctgaa ggctggattt actggaagcc aatgagcagt tggctatggt ccttgtccac
        14221 gcggcccatg ttgtgggcag tgaccgtatt caagaaggga aggacagaca agtatttgaa
        14281 tacttcagtg accaggattt ggtaaaggac tgcaggtcag ggtcaagaag aggtgagagc
        14341 aggacagact tcctccccgc tgcaccaggc agctgagctg ggtttcctct aggggctgag
        14401 gtttgagggt acctcaagtt ctgcaagagt ctataggagg tggtaagaga gaagagctgg
        14461 aggtcagagt tttcttgact atatatatat atattttttt gtttttgttt ttaacagctt
        14521 aacagctttc tgttttattt ttagagacag ggtctcaggg tctcactttg tcacccaggc
        14581 tggagtgcag tggtacaatc gtagctgact gcagcctcaa actcccaggc tcaagaaatc
        14641 ctcctcccac ccttagcctc ctgagtagca gggactacag gtgtgagcca gcaggaagcc
        14701 cagctggttt tttttttttc tttggtgttt tttgtttgtt tgtttgagac cggagtttcg
        14761 ctcttattgc ccaggctgaa gtgcaatggc aggatcttag ctcaccacaa cctccgaccc
        14821 ccaggatcaa gctattctct tgcctcagcc acctgagtag ctgggattac aggcatgcga
        14881 caccacacaa ggctaatttt gtatttttag taaagacagg gtttctccat gttggtcagg
        14941 ctggtctcga actcccaacc tcagatgatc cacctgcctc ggcctcccaa agtgctgaga
        15001 ttacaggcat gagccaccgt gcccggcctt tttttttttt tttttttttt tgagacagag
        15061 tctcactctg tcgcccaggc aggagtgcag tggttcgatc tgggctcact gcaagctccg
        15121 cctcccgggt tcactccatc ctcctgcctt agcctcctga gtagctggga ctacaggcgc
        15181 ccaccaccac gcctggctaa ttttttgtat ttttagtaga gacggggttt caccgtgtta
        15241 gccaggatgg tcttgatctc ctgacctcgt gatccgcccg ccttggcctc ccaaagtgct
        15301 gggattacag gcgtgagcca ccatgtctgg cctggccagg ctggtcttga actcctgact
        15361 tccggtgatc catctgttct ggcctcccaa agtgctggga ttacaggcat aagccaccac
        15421 gccatgccga agcccagctt gtttttaatt tttttttttt tttttggaga aatgaggtct
        15481 tgcaatgttg cccaagctag ccttgaactc ctggcctcaa atgaccccgc cttggcatcc
        15541 caaagtactg ggattacaga tgtgagccac catgccccag ccttgctttc ttgagatacg
        15601 atttagaata ccataagatt catccctttt aagcacataa ttcaatgact tctgtacaaa
        15661 caaccatgac tacaatctaa ttttaaaata tttcaatcac tctaaaaaag aaacctcctg
        15721 cttatgtaca gcgactctgt ctacctctta agtgaattct cctaccttta atagccctat
        15781 tttacagttc aggaaactga ggttcagaga gacaaagtca cttacccaca gcaaagaagc
        15841 aaggctgggt atcaaatgca ggaccccccc ggtcctgatg cttttttttt tttttttttt
        15901 ttcctctgag agagactctc actctgtcac tcagtctaga gtacagtggc gcgatctcag
        15961 ttcactgcaa tctctgcctc ctgggctgaa gtaatccttt cctcacaagt aaacctcagc
        16021 ctctcaagta gctgggacta caggcacaca acaccacgcc tggctaattt ttgtattttt
        16081 aggtagagac ggggtttcac tatattggcc aggctggtct tgaactcctg acctcaggtg
        16141 atccgcctgc ctcggccccc caaagtgttg ggattacagg cgtgagccac cacacgcagc
        16201 ctttttgtta ttagactctg tcattactga cttttttttt tttttaatag aaacagggtc
        16261 tttctttccc aggctaaagt acagtggcat gatcacagtt cactatagcc ttaaactcct
        16321 gggctcaagt gatcctcctg cctcagcctc ccaagtagca gggactacag gtgtgcacca
        16381 ccacacccag ttaaccattc attcattcat tcattcattt attttgagat ggagtctcgc
        16441 tctgtcacct aggctggagt gcagtggcac gatctcagct cactgcaacc tccacctccc
        16501 aggttcaaga gattctcctg cctcagcctc ccgagtagct gagactacag gcgtgcacca
        16561 ccatgccaga ctaatttttg tatttttaat agagacgggg tttcactctg ttggtcaggc
        16621 ttatctcgaa ctcctgactt cgtggatcca ccctccttgg cctcccaaag tgctgggatt
        16681 aaaggcgtga gccaccgcgc cctgccaacc tttttttaat ttttcttaga gatgggggtc
        16741 tccctatgtt gcccaggctt gtcttgaact cctggcctca agtgaccctc ttgccttggc
        16801 ctcacaaagt gctaggatta cagcctgagc catcacacct ggccaacagg tttttttttt
        16861 tgttttgttt tgttttttaa agaatgtcta ggccaggctc atttactttc acctgtaatc
        16921 ccagcacttt gggaggccgg ggtgggcaga tcacttgagg tcaggaattc gagaccagcc
        16981 tgggcaacat gctgaaaccc cgtttctact aaaaatacaa aaattagctg ggtgtggtga
        17041 cacgtgcctg taatcccagc tactcaggag gttgaggcag gagaattgct tgaacccagg
        17101 aggcagaggt tgcagtgagc caagatcatg ccatcaccct ccagcctggg cgacagaagg
        17161 agactcagtc taaaaactta attaattaat taattaaaaa taaaaataca aaaattaacc
        17221 tggtgtggtg gtgtgtgcct gtaatctaag ctactcagga ggctgaggca ggagaatccc
        17281 ctgaatccca gaggcagagg ttgcagtgag ccaagatcga gccactgttt gcccagtcta
        17341 gtgcactggg ctgctgaatt tatttgacca gacacctagc aatagacttt gaagttcttt
        17401 tccacttttc actctaagat gctgctgtca tgaataagga atattttgat ccccttcaca
        17461 aacactcggg gccctcttac cagttttcac tgaagatctt gacattccta tctgcttagg
        17521 tgtctgggcg tgtttggggg agatactgaa gaggtagggc tcccaggcag gtgcagttcg
        17581 tctgttaggc aggcagcaag gtccacttca ccagacaccc ccacctctgt tttcctgcag
        17641 ggactccaga acgggtggaa ctggcacccc tcccctcttg gcagccagtg ggcaagaacc
        17701 ttaccctacg ctgccaggtg gagggtgggg caccccgggc caacctcacc gtggtgctgc
        17761 tccgtgggga gaaggagctg aaacgggagc cagctgtggg ggagcccgct gaggtcacga
        17821 ccacggtgct ggtgaggaga gatcaccatg gagccaattt ctcgtgccgc actgaactgg
        17881 acctgcggcc ccaagggctg gagctgtttg agaacacctc ggccccctac cagctccaga
        17941 cctttggtga ggattgaaga agccagcagg gagaaggtgg gggtggggta tcctgcaatg
        18001 cggtgcctgt ggccacagga tcttttgaga tgggtgtggc cccggctaag gggtgcatgt
        18061 gttctaggcg tatgtgacct aggctgctga gtggccctgg aagaggatct cgcaggaggg
        18121 ggaatgaaat gccccagaga agggcttcgg gacgtccatc cctgtctgct cacacctttc
        18181 ttctctccct agtcctgcca gcgactcccc cacaacttgt cagcccccgg gtcctagagg
        18241 tggacacgca ggggaccgtg gtctgttccc tggacgggct gttcccagtc tcggaggccc
        18301 aggtccacct ggcactgggg gaccagaggt tgaaccccac agtcacctat ggcaacgact
        18361 ccttctcggc caaggcctca gtcagtgtga ccgcagagga cgagggcacc cagcggctga
        18421 cgtgtgcagt aatactgggg aaccagagcc aggagacact gcagacagtg accatctaca
        18481 gtaagaaggg gcaggggcgg agtggggctt cttgggggtg tgacctgaac ccggggcggg
        18541 gctcactgtg tgcctattcc aggctttccg gcgcccaacg tgattctgac gaagccagag
        18601 gtctcagaag ggaccgaggt gacagtgaag tgtgaggccc accctagagc caaggtgacg
        18661 ctgaatgggg ttccagccca gccactgggc ccgagggccc agctcctgct gaaggccacc
        18721 ccagaggaca acgggcgcag cttctcctgc tctgcaaccc tggaggtggc cggccagctt
        18781 atacacaaga accagacccg ggagcttcgt gtcctgtgtg agtggggctg ctggtcaatg
        18841 gcccctatcc cccaaggccc aatctccctg aaggtcccat aaggtcttgc ctccaagtcc
        18901 tgcccccacc cacctccatg tcatctcatc gtgtttttcc agatggcccc cgactggacg
        18961 agagggattg tccgggaaac tggacgtggc cagaaaattc ccagcagact ccaatgtgcc
        19021 aggcttgggg gaacccattg cccgagctca agtgtctaaa ggatggcact ttcccactgc
        19081 ccatcgggga atcagtgact gtcactcgag atcttgaggg cacctacctc tgtcgggcca
        19141 ggagcactca aggggaggtc acccgcaagg tgaccgtgaa tgtgctctgt gagtgagccg
        19201 gcgggcagag ctgggtgggg gcaggggcca tggacctaat gcaatcctca ccgcctgttg
        19261 tatcctcccc acagcccccc ggtatgagat tgtcatcatc actgtggtag cagccgcagt
        19321 cataatgggc actgcaggcc tcagcacgta cctctataac cgccagcgga agatcaagaa
        19381 atacagacta caacaggccc aaaaagggac ccccatgaaa ccgaacacac aagccacgcc
        19441 tccctgaacc tatcccggga cagggcctct tcctcggcct tcccatattg gtggcagtgg
        19501 tgccacactg aacagagtgg aagacatatg ccatgcagct acacctaccg gccctgggac
        19561 gccggaggac agggcattgt cctcagtcag atacaacagc atttggggcc atggtacctg
        19621 cacacctaaa acactaggcc acgcatctga tctgtagtca catgactaag ccaagaggaa
        19681 ggagcaagac tcaagacatg attgatggat gttaaagtct agcctgatga gaggggaagt
        19741 ggtgggggag acatagcccc accatgagga catacaactg ggaaatactg aaacttgctg
        19801 cctattgggt atgctgaggc cccacagact tacagaagaa gtggccctcc atagacatgt
        19861 gtagcatcaa aacacaaagg cccacacttc ctgacggatg ccagcttggg cactgctgtc
        19921 tactgacccc aacccttgat gatatgtatt tattcatttg ttattttacc agctatttat
        19981 tgagtgtctt ttatgtaggc taaatgaaca taggtctctg gcctcacgga gctcccagtc
        20041 ctaatcacat tcaaggtcac caggtacagt tgtacaggtt gtacactgca ggagagtgcc
        20101 tggcaaaaag atcaaatggg gctgggactt ctcattggcc aacctgcctt tccccagaag
        20161 gagtgatttt tctatcggca caaaagcact atatggactg gtaatggtta caggttcaga
        20221 gattacccag tgaggcctta ttcctccctt ccccccaaaa ctgacacctt tgttagccac
        20281 ctccccaccc acatacattt ctgccagtgt tcacaatgac actcagcggt catgtctgga
        20341 catgagtgcc cagggaatat gcccaagcta tgccttgtcc tcttgtcctg tttgcatttc
        20401 actgggagct tgcactatgc agctccagtt tcctgcagtg atcagggtcc tgcaagcagt
        20461 ggggaagggg gccaaggtat tggaggactc cctcccagct ttggaagcct catccgcgtg
        20521 tgtgtgtgtg tgtatgtgta gacaagctct cgctctgtca cccaggctgg agtgcagtgg
        20581 tgcaatcatg gttcactgca gtcttgacct tttgggctca agtgatcctc ccacctcagc
        20641 ctcctgagta gctgggacca taggctcaca acaccacacc tggcaaattt gatttttttt
        20701 ttttttccag agacggggtc tcgcaacatt gcccagactt cctttgtgtt agttaataaa
        20761 gctttctcaa ctgcctcagc cttgtgtgag ttgaggggag gtgtcacatc cagctggagt
        20821 cctttctaag cagccacagc ctgatcctcc cacttcctcc cccaagaaaa cattgtgggt
        20881 tgatggccat accctgaggt tctggtccaa atcggacttt ctatgacctt ctgggtctct
        20941 agtgaaaact aaagactcct ctccagaaaa aaacatttgg tttctaatga ggcctggaat
        21001 cttattcttg acctggggag cggaatccct ttttgcagta ctcccgggcc ctctgttggg
        21061 gcctcccctt cctctccagg gtggagtcga ggaggcgggg ctgcgggcct ccttatctct
        21121 agagccggcc ctggctctct ggcgcggggc cccttagtcc gggctttttg ccatggggtc
        21181 tctgttccct ctgtcgctgc tgtttttttt ggcggccgcc tacccgggag ttgggagcgc
        21241 gctgggacgc cggactaagc gggcgcaaag ccccaagggt agccctctcg cgccctccgg
        21301 gacctcagtg cccttctggg tgcgcatgag cccggagttc gtggctgtgc agccggggaa
        21361 gtcagtgcag ctcaattgca gcaacagctg tccccagccg cagaattcca gcctccgcac
        21421 cccgctgcgg caaggcaaga cgctcagagg gccgggttgg gtgtcttacc agctgctcga
        21481 cgtgagggcc tggagctccc tcgcgcactg cctcgtgacc tgcgcaggaa aaacacgctg
        21541 ggccacctcc aggatcaccg cctacagtga gggacagggg ctcggtcccg gctggggtga
        21601 ggggaggggg ctggaagagg tgggggaagg gtagttgaca gtcgctctat agggagcgcc
        21661 cgcggacctc actcagaggc tcccccttgc cttagaaccg ccccacagcg tgattttgga
        21721 gcctccggtc ttaaagggca ggaaatacac tttgcgctgc cacgtgacgc aggtgttccc
        21781 ggtgggctac ttggtggtga ccctgaggca tggaagccgg gtcatctatt ccgaaagcct
        21841 ggagcgcttc accggcctgg atctggccaa cgtgaccttg acctacgagt ttgctgctgg
        21901 accccgcgac ttctggcagc ccgtgatctg ccacgcgcgc ctcaatctcg acggcctggt
        21961 ggtccgcaac agctcggcac ccattacact gatgctcggt gaggcacccc tgtaaccctg
        22021 gggactagga ggaagggggc agagagagtt atgaccccga gagggcgcac agaccaagcg
        22081 tgagctccac gcgggtcgac agacctccct gtgttccgtt cctaattctc gccttctgct
        22141 cccagcttgg agccccgcgc ccacagcttt ggcctccggt tccatcgctg cccttgtagg
        22201 gatcctcctc actgtgggcg ctgcgtacct atgcaagtgc ctagctatga agtcccaggc
        22261 gtaaaggggg atgttctatg ccggctgagc gagaaaaaga ggaatatgaa acaatctggg
        22321 gaaatggcca tacatggtgg ctgacgcctg taatcccagc actttgggag gccgaggcag
        22381 gagaatcgct tgagcccagg agttcgagac cagcctggac aacatagtga gaccccgtct
        22441 atgcaaaaaa tacacaaatt agcctggtgt ggtggcccgc acctgtggtc ccagctaccc
        22501 gggaggctga gttgggagga tcctttgagc cctgaaagtc gaggttgcag tgagccttga
        22561 tcgtgccact gcactccagc ctgggggaca gagcacgacc ctgtctccaa aaataaaata
        22621 aaaataaaaa taaatattgg cgggggaacc ctctggaatc aataaaggct tccttaacca
        22681 gcctctgtcc tgtgacctaa gggtccgcat tactgccctt cttcggagga actggtttgt
        22741 ttttgttgtt gttgttgttt ttgcgatcac tttct (SEQ ID NO:15)

BC005930

Homo sapiens CD58 molecule, mRNA

        1 gtaggcggtg cttgaactta gggctgcttg tggctgggca ctcgcgcaga ggccggcccg
        61 acgagccatg gttgctggga gcgacgcggg gcgggccctg ggggtcctca gcgtggtctg
        121 cctgctgcac tgctttggtt tcatcagctg tttttcccaa caaatatatg gtgttgtgta
        181 tgggaatgta actttccatg taccaagcaa tgtgccttta aaagaggtcc tatggaaaaa
        241 acaaaaggat aaagttgcag aactggaaaa ttctgaattc agagctttct catcttttaa
        301 aaatagggtt tatttagaca ctgtgtcagg tagcctcact atctacaact taacatcatc
        361 agatgaagat gagtatgaaa tggaatcgcc aaatattact gataccatga agttctttct
        421 ttatgtgctt gagtctcttc catctcccac actaacttgt gcattgacta atggaagcat
        481 tgaagtccaa tgcatgatac cagagtatta caacagccat cgaggactta taatgtactc
        541 atgggattgt cctatggagc aatgtaaacg taactcaacc agtatatatt ttaagatgga
        601 aaatgatctt ccacaaaaaa tacagtgtac tcttagcaat ccattattta atacaacatc
        661 atcaatcatt ttgacaacct gtatcccaag cagcggtcat tcaagacaca gatatgcact
        721 tatacccata ccattagcag taattacaac atgtattgtg ctgtatatga atggtatgta
        781 tgctttttaa aacaaaatag tttgaaaact tgcattgttt tccaaaggtc agaaaatagt
        841 ttaaggatga aaataaagtt tgaaatttta gacattcgaa aaaaaaaaaa aaaaagaaaa
        901 aaaaaaa (SEQ ID NO:16)
    
    


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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

## Claims

1. A method of making stably transduced mammalian dendritic cells with altered immune properties, comprising providing a population of mammalian dendritic cells; stably transducing the cells with a recombinant helper-free lentivirus comprising a genome having a transcription cassette comprising nucleic acid sequences for CD4, the expression of which sequences in the dendritic cells is capable of inducing RNAi, thereby decreasing cell surface expression of CD40; and selecting transduced dendritic cells with decreased levels of CD40 expression that result in antigen-specific T cell tolerization or Th2 polarization in vivo relative to corresponding dendritic cells that are not transduced with the recombinant helper-free lentivirus, wherein the nucleic acid sequences for CD40 comprise CTGGACAAGCTGTGAGGATAA (SEQ ID NO:6), GCAGGGTACTGGCTAAATAAA (SEQ ID NO:1) or CCAAAGGATAATGAGATGTTA (SEQ ID NO:2).

2. The method of claim 1 wherein the cells are human cells.

3. The method of claim 1 wherein the cells are immature primary cells.

4. The method of claim 1 further comprising contacting the transduced cells with an agent that stimulates expression of one or more co-stimulatory molecules.

5. The method of claim 4 further comprising contacting the transduced cells with an antigen.

6. A stably transduced mammalian dendritic cell prepared by the method of claim 1.

7. A population of isolated stably lentivirus transduced mammalian dendritic cells comprising a recombinant lentivirus comprising a transcription cassette comprising nucleic acid sequences for CD40, the expression of which sequences in the dendritic cells is capable of inducing RNAi, thereby decreasing cell surface expression of CD40, which population is selected to have decreased levels of CD40 expression that result in antigen-specific T cell tolerization or Th2 polarization in vivo relative to corresponding dendritic cells that are not transduced with the recombinant helper-free lentivirus, wherein the nucleic acid sequences for CD40 comprise CTGGACAAGCTGTGAGGATAA (SEQ ID NO:6), GCAGGGTACTGGCTAAATAAA (SEQ ID NO:1) or CCAAAGGATAATGAGATGTTA (SEQ ID NO:2).

8. The dendritic cells of claim 7 wherein the cells are human cells.

9. The dendritic cells of claim 7 wherein the stably transduced dendritic cells have decreased levels of CD40 relative to corresponding cells that are not transduced by the recombinant helper-free lentivirus.

10. The method of claim 1 wherein the nucleic acid sequences for CD40 comprise CTGGACAAGCTGTGAGGATAA (SEQ ID NO:6), GCAGGGTACTGGCTAAATAAA (SEQ ID NO:1) or CCAAAGGATAATGAGATGTTA (SEQ ID NO:2).

11. The dendritic cells of claim 7 wherein the nucleic acid sequences for CD40 comprise GCAGGGTACTGGCTAAATAAA (SEQ ID NO:1) or CCAAAGGATAATGAGATGTTA (SEQ ID NO:2).