Generation and use of engineered cells

Abstract

The present disclosure relates to genetically modified dendritic cells or their precursors expressing at least one antigen-derived peptide and at least one immunomodulatory molecule, their medical uses and methods of preparation. The present invention also relates to the production of CD49b producing IL-10 + LAG‑3+Tr1 cells or antigen-specific FOXP3 + In vitro methods for T cells, and related medical uses and pharmaceutical compositions.

Description

Technical Field

[0001] The present disclosure relates to genetically modified dendritic cells or their precursors that express at least one antigen-derived peptide and at least one immunomodulatory molecule, their medical uses and methods of preparation. The present invention also relates to CD49b that produces IL-10. + LAG-3+Tr1 cells or antigen-specific FOXP3 + In vitro methods for T cells, and related medical uses and pharmaceutical compositions. Background Art

[0002] Identification of novel approaches aimed at selectively controlling antigen (Ag)-specific pathogenic T cell responses and promoting / restoring tolerance in T-cell-mediated diseases represents one of the grand goals of managing autoimmune diseases and organ transplantation in humans. In line with this, a new type of vaccination (also known as “inverse vaccination” or “tolerogenic vaccination”) aims to induce or restore an immune state in an anergic setting, which may be directed against foreign Ag (i.e., protein therapeutics, allergens, or transgenics) or against autologous Ag (1). The overall goal of tolerogenic strategies is to suppress adverse reactions by deleting / suppressing / deviancing Ag-specific Teff cells and to support Ag-specific T regulatory cells (Tregs)—forkhead box P3 (FOXP3)-expressing Tregs (FOXP3 + Induction and / or expansion of Treg)(2) or IL-10 producing T regulatory type 1 (Tr1) cells(3). Many different reverse vaccination approaches have been proposed:

[0003] i) Non-Ag-specific immunotherapy using monoclonal antibodies targeting different cell populations (i.e., anti-CD3, anti-CD20, anti-CD52, CTLA-4Ig) or pro-inflammatory cytokines (i.e., anti-TNFα, anti-IL-1β) or immunomodulatory compounds (i.e., rapamycin, mycophenolate mofetil),

[0004] ii). Ag-specific immunotherapy using autologous Ag or allergens.

[0005] As agents of tolerogenic strategies, regulatory cells have been proposed as tools for cell therapy. There is growing evidence that different subsets of dendritic cells (DCs), whether naturally occurring or experimentally induced, play a key role in maintaining tissue homeostasis and promoting tolerance (see (4-7)), thereby acting as regulatory cells. The regulatory capacity of DCs depends on their immature state and they can be induced by immune mediators, genetic manipulations, or signals from other immune cells. Tolerogenic DCs (tolDCs) present Ag and prime Ag-specific T cells and can also induce Ag-specific Tregs (8). A better understanding of the biology of tolDCs and the mechanisms that regulate their induction, activity, and plasticity, as well as the development of protocols suitable for the in vitro generation of tolDCs, offers the potential to translate their use into immunotherapies for immune-mediated diseases (8-12). DCs represent selected tolerogenic cells that meet the goal of promoting / restoring Ag-specific tolerance because they i) promote Ag-specific Tregs; ii) regulate Ag-specific pathogenic T cells; iii) generate a tolerogenic microenvironment rich in anti-inflammatory mediators that supports the maintenance of long-term Ag-specific anergy. Proof-of-principle clinical trials completed so far have demonstrated the safety and feasibility of tolDC-based immunotherapy in autoimmune diseases and in the prevention of graft rejection after organ transplantation (10, 11, 13-15). However, the stability of infused DCs and their maintenance of tolerance remain issues to be addressed for improving the safety and efficacy of successful DC-based cell therapies.

[0006] Optimal tolDC should present Ag in an unactivated state or in a microenvironment rich in anti-inflammatory cytokines or inhibitory molecules. In order to stabilize these conditions, the inventors proposed a novel strategy based on state-of-the-art lentiviral vector (LV) technology that would ensure the generation of stable and effective tolerogenic DCs. Lentiviral vectors (LV) will transduce human DC precursors (16) and induce strong and lasting anti-tumor T cell responses (17). In addition, LV-mediated DC transduction does not lead to significant changes in DC activation (18), which supports the possibility of LV-mediated stable and effective Ag presentation to generate immunogenic or tolerogenic DCs. Therefore, to date, LV has been used to genetically modify DCs for DC immunogenicity-based therapies. DCs transduced with LV encoding tumor-associated Ags generate tumor-specific CD8 + T cells (17). DCs transduced by LVs are able to express CD4 T cells only when LV-mediated Ag has reached the class II MHC presentation pathway. +LV encoding the invariant chain (Ii) fused to ovalbumin (OVA) (LV.IiOVA) was injected into transduced DCs in vivo, which acquired the ability to present the encoded OVA in competition with class II MHC and promoted the proliferation of OVA-specific CD4 + The ability of LV to transduce T cells (19). Direct in vivo administration of LV to transduced DCs offers some advantages: it does not require cell manipulation, and the vector itself triggers acute inflammation, which provides an adjuvant effect; however, it does not provide highly specific cell targeting. In contrast, in vitro LV-mediated DC transduction can significantly improve safety by reducing off-target transduction and by the limited lifespan of the transferred cells. In addition, in vitro administration of generated LV-transduced DCs allows repeated cell administration.

[0007] A variety of reagents have been used to differentiate human tolDC in vitro (20). To define the optimal tolerogenic DC for use in vivo, a comparative analysis of different subsets of tolDC differentiated in vitro was recently reported, which examined their stability, cytokine production profiles, and suppressive activity (20, 21). The results indicate that IL-10-regulated mature DC are the most suitable cells for tolerogenic DC-based therapies. The inventors' team contributed to the identification of IL-10 as a key factor promoting effective tolerogenic DC differentiation and described a cell subset named DC-10 that can be induced by peripheral blood mononuclear cells in the presence of IL-10 and is characterized by the ability to secrete large amounts of IL-10. DC-10 are mature myeloid cells that express immunoregulatory molecules, including HLA-G, ILT3, and ILT4, which are potent inducers of Ag-specific Tr1 cells in vitro (22, 23). DC-10 are stable cells because they retain their tolerogenic activity even when activated (24). Interestingly, stimulation of allergen-specific T cells with autologous DC-10 promoted their conversion to IL-10-producing suppressor T cells (25), suggesting that DC-10 is a good candidate for converting effector T cells into Tregs. Overexpression of IL-10-converted murine bone marrow-derived DC in tolDC prevented allergic contact dermatitis after in vivo transfer (26). Other candidates for conferring tolerogenicity to DCs are induction of indoleamine 2,3-dioxygenase (IDO1), a tryptophan degrading enzyme that is an immunomodulator in several pathological conditions. IDO expression has been promoted by several means in antigen-presenting cells, including fibrinogen and myeloid DCs ((27), WO2013 / 040552, WO2018037108, and WO2017192786). Overall, these approaches failed to promote stable and sustained overexpression of IDO in treated cells.

[0008] One of the main goals of clinicians is to find alternative treatments, to prevent graft rejection after organ transplantation. Improvements in immunosuppressive therapy for preventing rejection after allogeneic organ transplantation have shown the benefit of limiting acute rejection, but the side effects associated with long-term immunosuppressive regimens (see the drug table 1 of following approvals) represent one of the main causes of chronic transplant failure. Standard immunosuppressive regimens are effective. However, they require long-term treatment, and these long-term treatments are associated with many side effects, and compared to general populations, the life expectancy of transplant patients at present, including the life expectancy of renal transplant patients, is still significantly shorter (van Sandwijk MS etc., Neth J Med.2013). Immunosuppressive therapy is given every day, and annual costs of $14,000 are caused.

[0009] Table 1: Approved drugs

[0010]

[0011] Alternative therapies based on regulatory cell immunotherapy have entered the clinical arena in the last decade with the goal of reducing immunosuppression (28). Among them are therapies based on T regulatory cells (Tregs). To date, up to 30 different clinical trials have been completed or are being conducted using polyclonal freshly isolated or in vitro expanded Tregs to prevent transplant rejection (29). Ongoing clinical trials using Treg-based therapies have demonstrated the safety and some clinical benefits of this approach. However, several unresolved issues remain to be addressed:

[0012] - the potential for polyclonal ex vivo expanded Tregs to mediate pan immunosuppression in vivo (30); for this reason, preclinical studies are ongoing to generate antigen-specific Tregs to limit this side effect;

[0013] -The possibility that infused Tregs are unstable under strong inflammatory conditions in vivo and adopt pathogenic effector T phenotype and function, potentially mediating graft rejection;

[0014] Long-lasting Tregs have an overall impact on impeding immunity against infections and malignancies (29).

[0015] An interesting alternative and complementary approach to Treg-based therapies is the myeloid regulatory cell (MRC)-based therapy. Compared to Treg, MRC (i.e., Mreg and TolDC) exert immunomodulatory effects through different mechanisms, including depletion of Ag-specific effector T cells and promotion of tissue repair and regeneration processes. In addition, MRC induce Ag-specific Treg in vivo in a physiological manner. There are almost no patients who have been treated with MRC (i.e., Mreg or TolDC). Therefore, to date, the published data on a small number of transplanted patients have demonstrated the safety of this approach and show that infusion of donor-derived Mreg in patients with renal transplantation can reduce the immunosuppressive regimen and induce Treg in vivo (31).

[0016] Therefore, there remains a need for cell therapies for the treatment of autoimmune diseases, inflammatory diseases, and graft-versus-host disease. SUMMARY OF THE INVENTION

[0018] Ag presentation by immature DCs is a well-known natural mechanism for inducing peripheral immune tolerance (32), and the inventors proposed to utilize immunomodulatory manipulation to ensure Ag presentation by immature genetically modified DCs.

[0019] In the present invention, it has surprisingly been found that genetically modified dendritic cells or their precursors, which have been modified with a nucleic acid comprising a combination of: i) a sequence encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or protein or antigenic fragment thereof; and ii) a sequence encoding at least one immunomodulatory protein, are particularly advantageous for therapeutic applications.

[0020] The nucleic acid may further comprise at least one miRNA target sequence.

[0021] MiRNAs are small noncoding RNAs that negatively regulate the expression of specific target genes at the post-transcriptional level (33). When miRNAs are partially complementary to target messenger RNA (mRNA) sequences in the 3' untranslated region (3'UTR), they reduce target mRNA stability and inhibit translation. Alternatively, when miRNAs are almost perfectly complementary to their mRNA targets, they cleave the mRNA, triggering its large-scale destruction, resulting in loss of protein expression. MiRNAs have different expression profiles in different tissues and cell types, differentially regulating the transcriptional profiles and cellular functions of genes, thereby providing cell-specific and developmental stage-specific regulation of gene expression (34). miRNAs play an important role in controlling many processes within the immune system, including cell differentiation and homeostasis, cytokine responses, interactions with pathogens, and tolerance induction. DC development, differentiation, and function are regulated by specific expression profiles of miRNAs. Specifically, miR-155 and miR-146a expression are associated with DC maturation in humans and mice (35-38). Thus, by inserting 2x miR-155 and 2x miR146a target sequences (miR155T.mir146aT) into the 3'UTR region of an LV cassette (LV.IiAg), encoding the invariant chain (Ii) fused to a selected portion of the desired Ag, the inventors achieved suppression of transgene expression and, therefore, Ag presentation in LV-DCs that enter the activation program.

[0022] The methods provided herein are designed to induce a tolerogenic response to LV-encoded Ag. The efficacy of LV-mediated gene transfer into DCs and their precursors provides several clinically applicable opportunities to exploit the functional plasticity of DCs to design specific immunotherapies for tolerance induction in autoimmunity and transplantation.

[0023] According to an embodiment of the present invention, LV-IL-10 engineered DC (DC IL-10 ) can be used to prevent graft rejection after organ transplantation.

[0024] LV-mediated IL-10 gene transfer in DCs (DCs IL-10 ) has the potential to overcome the major limitations of Treg-based therapies and be more effective than other MRCS because it would result in a drug product that:

[0025] - Induction of allospecific immune anergy in effector T cells;

[0026] - Promotes self-reinforcing peripheral regulation and physiologically induces allospecific Tregs in vivo;

[0027] - have a limited lifespan in the body (up to 14 days), limiting the overall long-term impact on immunity against infections and malignancies;

[0028] - Promotes stable overexpression of IL-10, ensuring a specific IL-10-rich microenvironment that regulates T cells, myeloid cells, and innate cells, maintaining long-term tolerance.

[0029] The present invention provides methods for inducing tolerance to antigens or suppressing immune responses to antigens by regulatory immune cells, wherein the immune cells are genetically modified by newly developed tolerogenic vectors, preferably LVs encoding Ag-derived peptides or antigenic peptides (e.g., epitopes), which allow expression of Ag-derived peptides or antigenic peptides and pro-tolerogenic molecules. In some embodiments, the tolerogenic cells are delivered to an individual, and the presentation of Ag induces tolerance to Ag and / or suppresses immune responses to Ag. In some embodiments, the tolerogenic cells are used to promote in vitro Ag-specific Tregs, which are suitable for cell-based methods.

[0030] The present invention provides a method for inducing tolerance to Ag in an individual, the method comprising generating engineered immune cells with a vector, preferably a lentiviral vector (LV), to provide expression of Ag-derived peptides (epitopes) and tolerogenic molecules. The inventors have developed several LV-based gene transfer tools that allow for the coordinated expression of two transgenes (bidirectional (bd) LV (39, 40), WO2004094642 incorporated by reference) and / or targeted transgene expression to specific cell subpopulations by exploiting endogenous miRNA-mediated post-transcriptional regulation (miRNA-regulated LV (41, 42), WO2010125471 incorporated by reference). Furthermore, the inventors generated LVs encoding the invariant chain (Ii) fused to Ag and under the control of the phosphoglycerate kinase (PGK) ubiquitous promoter (PGK.Ii-Ag) (43), which ensured stable presentation of the encoded Ag in the presence of MHC class I as endogenous Ag, while also allowing Ag processing and presentation in the presence of MHC class II as exogenous Ag, resulting in CD4 + and CD8 + T cell stimulation.

[0031] The advantages of the present invention are:

[0032] - Vector-mediated transduction of DC precursors or DCs allows stable expression of the encoded peptide, which renders the resulting DCs more efficient in presenting Ag to T cells;

[0033] - Inclusion of miRNA target sequences allows post-transcriptional negative regulation of the encoded Ag, which can limit Ag presentation by DC-Ag.miRNAs at immature stages and prevent Ag presentation in the inflammatory microenvironment;

[0034] - Vector-mediated stable overexpression of IDO or IL-10 ensures Ag presentation in an IDO- or IL-10-enriched microenvironment;

[0035] - Human DC precursors can be stably transduced with vectors, particularly LVs;

[0036] - Populations of engineered DCs with diverse specificities can be used;

[0037] - Different engineered DCs can be combined to maximize tolerogenic activity;

[0038] - DC-IL-10 / Ag and DC-Ag.miRNA-T (or DC-Ag.miRNA, i.e., containing miRNA target sequences) promote the differentiation of Ag-specific Tr1 cells in vivo;

[0039] - Engineered DCs are short-lived cells, allowing for multiple DC injections;

[0040] -Highly versatile generation of Ii-Ag constructs driving specific Ag expression.

[0041] According to the present invention, strong suppression of T effector cells and / or strong activation of T regulatory cells is provided, as exemplified by three different methods.

[0042] According to the preferred embodiment of LV described herein:

[0043] - The promoter can be ubiquitous (e.g. PGK)

[0044] - When the method is DC-IL-10 / Ag or DC-IDO / Ag, the vector can be bidirectional.

[0045] According to a preferred embodiment, a clinical protocol based on the use of the tolerogenic DCs of the present invention will provide:

[0046] - Modified autologous / allogeneic DCs will be administered to patients via one or more infusions to re-establish / induce stable tolerance to a specific antigen;

[0047] - Modification of autologous / allogeneic DCs by transduction with single LVs or mixtures of LVs encoding different fragments of antigens (from known antigen libraries) and / or different tolerogenic molecules in order to reconstitute / induce tolerogenic responses covering a variety of specificities.

[0048] According to another preferred embodiment, DC-IL-10 / Ag can be used simultaneously in vitro to generate T regulatory type 1 cells (Tr1) that are specific for the antigen according to the protocol described in WO2007131575 (incorporated by reference). Such antigen-specific Tr1 cells can be purified in vitro according to the protocol described in WO2013192215 (incorporated by reference) and then infused into the patient in combination with an infusion of the modified tolerogenic DCs of the present invention, thereby maximizing the tolerogenic response to the antigen.

[0049] Then, the present invention provides a genetically modified dendritic cell or a precursor cell thereof modified with a nucleic acid construct, wherein the nucleic acid construct comprises:

[0050] a nucleic acid sequence a) encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or protein or an antigenic fragment thereof, said sequence a) being operably linked to a first promoter and optionally to a first transcriptional regulatory sequence, and

[0051] - a nucleic acid sequence b) encoding at least one immunomodulatory protein, said sequence b) optionally being operably linked to a second promoter and optionally to a second transcriptional regulatory sequence.

[0052] The precursor cells are precursor cells of dendritic cells and are also genetically modified.

[0053] Then, the genetically modified dendritic cells or their precursors constitutively express at least one antigen-derived peptide (or antigenic peptide or protein or antigenic fragment thereof) and at least one immunomodulatory molecule. Such modified cells present at least one molecule on the cell surface or intracellularly or produce and / or secrete at least one molecule.

[0054] Modifications can be introduced by transduction, transformation, or electroporation.

[0055] The first promoter and the second promoter may be the same or different.

[0056] Promoters include promoters from the following families: phosphoglycerate kinase 1 (PGK), cytomegalovirus (CMV), spleen focus forming virus (SSPV), human elongation factor 1 alpha (EF1α), myeloid-related protein 8 (MRP8), myeloid-specific promoter (MSP), CAG promoter consisting of the CMV immediate early enhancer linked to the chicken β-actin promoter, synthetic myeloid-specific promoter (146gp61), mouse mammary tumor virus (MMTV), CD11b, protein-tyrosine kinase (c-Fes), cytochrome B-245 β chain (CYBB), and receptor tyrosine kinase (TEK).

[0057] The first transcription regulatory sequence and the second transcription regulatory sequence can be the same or different.

[0058] Antigenic peptides or proteins or antigenic fragments thereof are also referred to as antigenic peptides or antigenic protein variants.

[0059] The nucleic acid may also comprise a sequence encoding an immunodominant peptide and its variable flanking regions, each consisting of 5-10 amino acids.

[0060] Preferably, the sequence a) further comprises at least one miRNA target sequence at its 3' end.

[0061] Preferably, the nucleic acid construct further comprises a sequence encoding Vpx.

[0062] Preferably, the nucleic acid construct further comprises a sequence encoding a marker, preferably a selectable marker, preferably, the marker is GFP, ΔNGFR, or ΔCD19.

[0063] Preferably, the human invariant chain is lip33, lip41, lip35 or lip43.

[0064] Preferably, the antigenic peptide or protein or antigenic fragment thereof is derived from an autologous antigen and / or a harmless antigen and / or an allergen.

[0065] Preferably, the antigenic peptide or protein or antigenic fragment thereof is selected from the group of immunodominant peptides or variants thereof as described in Table 2. The variant is an antigenic variant.

[0066] In a preferred embodiment, the immunomodulatory protein is selected from the group consisting of IL-10, indoleamine 2,3-dioxygenase (IDO), PDL-1, PDL-2, ILT-3, ILT-4, HO-1, ICOS-L Gal9, HVME, HLA-G, HLA-E, IL-35, TGF-b, CTLA-4Ig, PGE2, TNFRs, Arg1, preferably IL-10, indoleamine 2,3-dioxygenase (IDO) or a mixture thereof.

[0067] In a preferred embodiment, at least one miRNA target sequence is selected from the group targeting the following targets: miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-21, miR-29a, miR-29b, miR-29c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a, miR-125b, miR-126, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424, preferably miR155, miR146a or a mixture thereof, and preferably the miRNA target sequence is repeated. Preferably, the miR155 target sequence is repeated twice, and the miR146a target sequence is repeated twice.

[0068] Preferably, the genetically modified dendritic cell or its precursor is a cell that exhibits at least one of the following properties: modulation of CD4 + and CD8 + T cell responses; modulation of antigen-specific CD4 T cells in vitro and / or in vivo + and CD8 + T cell proliferation; conducive to the generation of regulatory DCs; conducive to the expansion of antigen-specific Tr1 and / or FOXP3 + Treg cells, which are tolerogenic, present antigens in the context of class I and class II MHC.

[0069] Preferably, the nucleic acid construct is inserted into a vector, preferably a lentiviral vector, more preferably a unidirectional or bidirectional vector.

[0070] In a preferred embodiment, the genetically modified dendritic cells or their precursors according to the present invention are used for medical purposes, preferably for preventing and / or treating a disorder selected from the group consisting of graft-versus-host disease, organ rejection, autoimmune diseases, allergic diseases, inflammatory or autoinflammatory diseases, and immune responses induced by gene therapy.

[0071] Preferably, the autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune bowel disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune myositis, psoriasis, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, autoimmune hepatitis, alopecia areata, pemphigus vulgaris, vitiligo, aplastic anemia, autoimmune uveitis, alopecia areata, amyotrophic lateral sclerosis (Lou Gehrig's disease), ankylosing spondylitis, anti-GBM nephritis, antiphospholipid syndrome, osteoarthritis, autoimmune active chronic hepatitis, autoimmune inner ear disease (AIED), Balo disease, Behcet's disease, Berger's disease, Disease, bullous pemphigoid, cardiomyopathy, chronic fatigue immune dysfunction syndrome, Churg-Strauss Syndrome, cicatricial pemphigoid, cold agglutinin disease, cranial arteritis colitis, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis and JDM, Devic disease, eczema, essential mixed cryoglobulinemia, eosinophilic fasciitis, fibromyalgia-fibromyositis, fibrosing alveolitis, giant cell arteritis, glomerulonephritis, Goodpasture's disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hepatitis, Hughes syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, irritable bowel syndrome, Kawasaki disease, lichen planus, lupus hepatitis, lupus / SLE, Lyme disease, Meniere's disease Disease), Mixed Connective Tissue Disease, Myositis: Juvenile Myositis (JM), Juvenile Dermatomyositis (JDM), and Juvenile Polymyositis (JPM), Osteoporosis, Pars Planitis, Pemphigus Vulgaris, Polyglandular Autoimmune Syndrome, Polymyalgia Rheumatica, Polymyositis, Primary Biliary Cirrhosis, Primary Sclerosing Cholangitis, Psoriasis, Raynaud's Syndrome, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Scleritis, Scleroderma, Viscosity Syndrome, Still's Disease, Stiff Man Syndrome, Sydenham's Chorea, Takayasu Arteritis, Temporal Arteritis,Ulcerative colitis, uveitis, vasculitis, Wegener's Granulomatosis and Wilson's syndrome, preferably, the autoimmune disease is a vasculitis, such as catastrophic anti-phospholipid syndrome (also known as Asherson's syndrome), giant cell arteritis and anti-ANCA vasculitis or myasthenia gravis, refractory celiac disease, autoimmune uveitis, such as Behcet's disease, pemphigus vulgaris, giant cell myocarditis, Graves' disease, Addison's disease, Granulomatosis with polyangiitis.

[0072] Preferably, the allergic disease is asthma, atopic allergy or atopic dermatitis.

[0073] Preferably, the inflammatory or autoinflammatory disease is a chronic inflammatory disease, preferably the chronic inflammatory disease is selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, celiac disease.

[0074] In a preferred embodiment, the genetically modified dendritic cells of the invention or their precursors are used to prevent an immune response to protein replacement therapy, preferably for the treatment of lysosomal storage disorders or hemophilia.

[0075] The present invention also provides a nucleic acid construct comprising:

[0076] a nucleic acid sequence a) encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or protein or an antigenic fragment thereof, said sequence a) being operably linked to a first promoter and optionally to a first transcriptional regulatory sequence, and

[0077] - a nucleic acid sequence b) encoding at least one immunomodulatory protein, said sequence b) optionally being operably linked to a second promoter and optionally to a second transcriptional regulatory sequence.

[0078] As indicated above, the first promoter and the second promoter may be the same or different.

[0079] As indicated above, the first transcriptional regulatory sequence and the second transcriptional regulatory sequence can be the same or different.

[0080] Preferably, the human invariant chain is lip33, lip41, lip35 or lip43.

[0081] The present invention also provides a vector comprising the nucleic acid construct as defined above, preferably the vector is a lentiviral vector, preferably the vector is a unidirectional or bidirectional vector, preferably the vector is produced using enveloped viral particles expressing Vpx, and / or the vector is produced using packaging cells, wherein the packaging cells are genetically engineered to reduce the expression of CD47.

[0082] Preferably, the vector is an expression vector.

[0083] The present invention also provides an in vitro method for producing a genetically modified dendritic cell or a vector thereof as defined above, comprising the steps of:

[0084] a. Isolation of PBMCs from subjects;

[0085] b. Isolation of CD14 from the isolated PBMC + cell;

[0086] c. Incubate the isolated CD14 with an effective amount of Vpx + cell;

[0087] d. Transduce the isolated CD14 with the vector of the present invention + cell.

[0088] Preferably, step d is carried out in the presence of an effective amount of at least one agent, preferably the agent is IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-10, preferably the amount of IL-4, the amount of GM-CSF or the amount of IL-10 is 1-1000 ng.

[0089] Preferably, PBMCs are isolated from peripheral blood or leukapheresis.

[0090] Also preferably, the vector is a lentiviral vector, and preferably the amount of the lentiviral vector is 1-100 MOI.

[0091] Preferably, an effective amount of Vpx is added on day 0 of the culture and continued for about 1 to 8 hours, preferably about 6 to 8 hours.

[0092] The present invention also provides genetically modified dendritic cells or precursor cells thereof obtainable by the above method.

[0093] The present invention also provides a method for producing CD49b that produces IL-10. + LAG-3 + An in vitro method for Tr1 cells comprising the steps of:

[0094] a) isolating PBMCs from a blood sample of a subject;

[0095] b) exposing the isolated PBMCs to an effective amount of the genetically modified dendritic cells or precursor cells thereof as defined above under appropriate culture conditions.

[0096] Preferably, the ratio of PBMC:genetically modified dendritic cells or their precursors is about 5:1-10:1.

[0097] The present invention also provides IL-10 producing CD49b obtainable by the method as defined above. + LAG-3 + Tr1 cells are preferably used for medical purposes.

[0098] Preferably, the IL-10-producing CD49b + LAG-3 + Tr1 cells will be expressed at 1x10 4 Up to 20x10 7 Different concentration ranges are infused, preferably 3x10 5 Up to 20x10 6 cells.

[0099] The present invention also provides a method for producing antigen-specific FOXP3 + An in vitro method for T cells comprising the steps of:

[0100] a) isolating PBMCs from a blood sample of a subject;

[0101] b) exposing the isolated PBMCs to an effective amount of the genetically modified dendritic cells or precursor cells thereof as defined above under appropriate culture conditions.

[0102] Preferably, the genetically modified dendritic cells or precursors thereof express at least indoleamine 2,3-dioxygenase (IDO).

[0103] The present invention also provides antigen-specific FOXP3 obtainable according to the method as defined above. + T cells, preferably for medical use.

[0104] Preferably, the pro-specific FOXP3 + T cells were expressed at 1x10 4 Up to 20x10 7 Different concentration ranges are infused, preferably 3x10 5 Up to 20x10 6 cells.

[0105] The present invention also provides a pharmaceutical composition comprising the genetically modified cells of the present invention or the IL-10-producing CD49b as defined above. + LAG-3 +Tr1 cells or antigen-specific FOXP3 as defined above + T cells or any combination thereof and a pharmaceutically acceptable carrier.

[0106] Preferably, the composition further comprises a therapeutic agent.

[0107] The therapeutic agent can be any agent known to those skilled in the art to treat at least one of the conditions of the present invention, such as, but not limited to, immunosuppressants, steroids, rapamycin, mycophenolate mofetil, rituximab, methotrexate, fludarabine, anti-inflammatory agents, anti-allergic agents.

[0108] Other therapeutic agents include, but are not limited to, immunosuppressants (e.g., antibodies against other lymphocyte surface markers (e.g., CD40, α-4 integrin) or antibodies against cytokines), other fusion proteins (e.g., CTLA-4-Ig TNFR-Ig ), TNF-a blockers such as Enbrel, Remicade, Cimzia, and Humira, cyclophosphamide (CTX) (i.e. Revimmune TM ), methotrexate (MTX) (ie ), belimumab (ie ), or other immunosuppressive drugs (e.g., cyclosporin A, FK506-like compounds, rapamycin compounds or steroids), antiproliferative agents, cytotoxic agents or other compounds that may contribute to immunosuppression.

[0109] In some embodiments, the other therapeutic agent inhibits or reduces T cell activation and cytokine production through different pathways. In one such embodiment, the other therapeutic agent is a CTLA-4 fusion protein, such as CTLA-4Ig (abatacept). CTLA-4Ig competes with the co-stimulatory receptor CD28 on T cells for binding to CD80 / CD86 (B7-1 / B7-2) on antigen presenting cells, thereby inhibiting T cell activity. In some embodiments, the other therapeutic agent is a CTLA-4Ig fusion protein, known as belatacept.

[0110] Belatacept contains two amino acid substitutions (L104E and A29Y) that significantly increase its affinity for CD86 in vivo. In another embodiment, the additional therapeutic agent is Maxy-4.

[0111] In another embodiment, the second therapy is a second agent that induces IDO expression. Second therapies that induce IDO expression are described in Johnson, et al., Immunotherapy, 1(4):645-661 (2009) and U.S. Patent Nos. 6,395,876 and 6,451,840. In one embodiment, the second therapy that induces IDO expression is a nanoparticle loaded with an expression vector encoding an ID01 or ID02 polypeptide.

[0112] In another embodiment, the second therapeutic agent preferably treats chronic metastatic rejection or GvHD, thereby effectively targeting the treatment regimen to acute or chronic suppression of rejection or GvHD. In another embodiment, the second therapeutic agent is a TNF-α blocker.

[0113] In another embodiment, the second therapeutic agent increases the amount of adenosine in serum, see, e.g., WO 08 / 147482. In some embodiments, the second therapeutic agent is CD73-Ig, recombinant CD73, or another agent that increases CD73 expression (e.g., a cytokine or monoclonal antibody or small molecule), see, e.g., WO 04 / 084933. In another embodiment, the second therapeutic agent is interferon beta.

[0114] In some embodiments, the composition is administered in conjunction with or following a compound that increases Treg activity or production.

[0115] Exemplary Treg enhancing agents include, but are not limited to, glucocorticoids fluticasone, salmeterol, antibodies to IL-12, ΓENγ, and IL-4; vitamin D3, and dexamethasone, and combinations thereof. Antibodies or their pro-inflammatory molecules can be combined or used interchangeably with the disclosed compositions. For example, the antibody can bind to IL-6, IL-23, IL-22, or IL-21.

[0116] As used herein, the term "rapamycin compound" includes the neutral tricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs, and other macrolide compounds, which are believed to have the same mechanism of action as rapamycin (e.g., inhibiting cytokine function). The language "rapamycin compound" includes compounds that have structural similarity to rapamycin, for example, compounds with a similar macrocyclic structure that have been modified to improve their therapeutic efficacy. Exemplary rapamycin compounds are known in the art.

[0117] The expression "FK506-like compound" includes FK506, and FK506 derivatives and analogs, e.g., compounds having structural similarity to FK506, e.g., compounds having a similar macrocyclic structure, which have been modified to enhance their therapeutic efficacy. Examples of FK506-like compounds are known in the art. Preferably, the expression "rapamycin compound" as used herein does not include FK506-like compounds.

[0118] Other suitable therapeutic agents include, but are not limited to, anti-inflammatory agents. The anti-inflammatory agent can be non-steroidal, steroidal, or a combination thereof. One embodiment provides an oral composition comprising about 1% (w / w) to about 5% (w / w), particularly about 2.5% (w / w) or an anti-inflammatory agent. Representative examples of non-steroidal anti-inflammatory agents include, but are not limited to, oxicams such as piroxicam, isoxicam, tenoxicam, sudoxicam; salicylates such as aspirin, disalcid, benoylate, trilisate, safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac ), furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, such as mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acid; acid); propionic acid derivatives such as ibuprofen, naproxen, benoxaprofen, flurbiprofen, ketoprofen, fenoprofen, fenbufen, indoprofen, pirprofen, carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, aminoprofen, and tiaprofenic; pyrazoles such as phenylbutazone, oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures of these nonsteroidal anti-inflammatory agents may also be used.

[0119] Representative examples of steroidal anti-inflammatory agents include, but are not limited to, corticosteroids such as hydrocortisone, hydroxytriamcinolone, alpha-methyldexamethasone, dexamethasone phosphate, beclomethasone dipropionate, clobetasol valerate, desonide, desoxymethasone, deoxycorticosterone acetate, dexamethasone, diclosone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine, fluphenaz ... butylesters), fluocortolone, fluprednisone acetate (flumethiazine), flurandrenolide, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, fluocinolone, fludrocortisone, difluorosone diacetate, fluradrenolone, fludrocortisone, diflurosone diacetate, fluradrenolone acetonide, medroxyprogesterone, amcinafel, amcinafel, betamethasone and its other esters, chlorprednisone, chlorprednisone acetate acetate), clocortelone, clescinolone, diclosporin, diflurprednate, fluclofenac, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone cyclopentylpropionate, hydrocortisone carbamate, methylprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate, triamcinolone.

[0120] The present invention also provides genetically modified dendritic cells or precursors thereof modified with a nucleic acid construct comprising a nucleic acid sequence encoding IL-10, said sequence being operably linked to a promoter and optionally operably linked to a transcriptional regulatory sequence and / or optionally operably linked to a marker, preferably a selectable marker.

[0121] The present invention also provides a genetically modified dendritic cell or a precursor cell thereof modified by a nucleic acid construct, wherein the nucleic acid construct comprises:

[0122] a nucleic acid sequence a) encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or protein or an antigenic fragment thereof, said sequence a) being operably linked to a first promoter and optionally to a first transcriptional regulatory sequence, and

[0123] - a nucleic acid sequence encoding at least one miRNA target sequence, optionally operably linked to a second promoter and optionally linked to a second transcriptional regulatory sequence.

[0124] As indicated above, the first promoter and the second promoter may be the same or different.

[0125] As indicated above, the first transcriptional regulatory sequence and the second transcriptional regulatory sequence can be the same or different.

[0126] Preferably, the human invariant chain is lip33, lip41, lip35 or lip43.

[0127] Preferably, the genetically modified dendritic cells or their precursors as defined above are used for organ and / or bone marrow transplantation and / or for preventing and / or treating graft-versus-host disease, or for preventing and / or treating a disease selected from the group consisting of autoimmune diseases, allergic diseases, inflammatory diseases, and immune responses induced by gene therapy.

[0128] Still preferably, the genetically modified cells are obtained by transduction with a single vector or a mixture of vectors (eg, lentiviral vectors) encoding different fragments of the antigen (according to a library of known antigens).

[0129] In a preferred embodiment, the genetically modified dendritic cells or their precursors are used to prevent immune responses against self-antigens, preferably for the treatment of autoimmune and autoinflammatory diseases.

[0130] In a preferred embodiment, genetically modified dendritic cells are used to prevent immune responses against allogeneic suppression, preferably for the treatment of organ transplantation.

[0131] It will be appreciated by those skilled in the art that, for nucleic acids encoding proteins or polypeptides, mutations leading to conservative amino acid substitutions can be made in the nucleic acids to provide functionally equivalent variants, or homologues of proteins or peptides. In some aspects, sequence changes leading to conservative amino acid substitutions of the nucleic acids are disclosed.

[0132] The invention is illustrated by way of non-limiting examples with reference to the following figures.

[0133] Figure 1Lentiviral vector design. Bidirectional LV construct designed for transduction of DC precursors. CLIP = class II-related invariant chain peptide; li = invariant chain; PGK = phosphoglycerate kinase.

[0134] Figure 2 LV.DCs were generated by LV.II OVA-mediated gene transfer into bone marrow-derived DCs. Bone marrow (BM) cells were differentiated into DCs in the presence of GM-CSF and transduced with the indicated LVs on day 2. Untransduced DCs (UNT) served as controls. On day 8 of differentiation, expression of CD11c, CD80, and CD86 was analyzed by FACS. The percentage of positive cells is indicated.

[0135] Figure 3 DC-IL-10 / OVA exhibited low stimulatory activity. Bone marrow (BM) cells were differentiated into DCs using GM-CSF and transduced with LV-IiOVA, LV-IL-10 / OVA, and LV-IDO / OVA on day 2. As a control, untransduced DCs (UNT) were generated. eFluor-labeled OTII CD4 + T cells, and proliferation was measured by dye dilution 3 days later.

[0136] Figure 4 DC-IL-10 / OVA promotes antigen-specific hyporesponsiveness. Bone marrow (BM) cells were differentiated into DCs using GM-CSF and transduced with LV-IL-10 / OVA on day 2. As a control, DC-OVA was generated. OTII CD4 + T cells were cultured for 7 days. After culturing, cells generated with DC-OVA [T(DC-OVA] and cells generated with DC-IL-10 / OVA [T(DC-IL-10 / OVA] were labeled with eFluor and stimulated with DC-OVA, and proliferation was measured by dye dilution after 4 days.

[0137] Figure 5 Activation-dependent upregulation of miR155 and miR146a restricts OVA expression and presentation by DC-OVA miRNA. Bone marrow (BM) cells were differentiated into DCs with GM-CSF and transduced with LV.OVA.miRNA or LV-IiOVA on day 2. DCs were kept unactivated or activated with LPS for 24 hours. As controls, DCs pulsed with OVA peptide and untransduced DCs (DC UNT) were used. eFluor-labeled OTII CD4 + T cells. After 3 days, proliferation was measured by dye dilution.

[0138] Figure 6Administration of LV-DCs promotes the expansion of OVA-specific T cells. Chimeric mice were generated by injecting bone marrow cells derived from OT-II / Fir-Tiger (5%) and CD45.1 (95%) and received four DC injections (DC-IL-10 / OVA, DC-IDO / OVA, DC-OVA.miRNA) and DC-OVA or DC-GFP as controls (A). Five weeks after the last DC injection, mice were sacrificed and the percentage of CD45.2 OTII firTiger CD4 T cells in the spleen was determined by FACS (B). *p<0.05, Mann-Whitney U test.

[0139] Figure 7 IL-10-producing Tr1 cells were induced by DC-IL-10 / OVA and DC-OVA miRNA. Chimeric mice were obtained by injecting bone marrow cells derived from OT-II / Fir-Tiger (5%) and CD45.1 (95%), which received 4 injections of the indicated LV-DCs. Five weeks after the last DC injection, mice were sacrificed and CD49b expression in the spleen was determined by FACS. + LAG-3 + Tr1 (A), IL10-producing Tr1 cells (GFP + ) (B) Percentage. *<0.05, Mann-Whitney U test.

[0140] Figure 8 Low proliferative responsiveness of T cells activated in vivo by LV-DC. Chimeric mice were generated by injecting bone marrow cells derived from OT-II / Fir-Tiger (5%) and CD45.1 (95%), which received four LV-DC injections. Five weeks after the last LV-DC injection, mice were sacrificed and CD4 purified from spleen was detected by efluor670. + T cells were stained and restimulated with DC-OVA (T:DC ratio of 10:1). After 4 days, proliferation was measured by dye dilution. Data are reported as stimulation index [(% of T cells that split DC-OVA) / (% of T cells that split untreated DC)]. **<0.005, *<0.05, Mann-Whitney U test.

[0141] Figure 9 .Generate OVA-specific CD4 + and CD8 + LV constructs for T cell proliferation. Bone marrow (BM) cells were differentiated into DCs with GM-CSF and on day 2 were treated with LV constructs encoding IiOVA. 315-363 and IiOVA 242-363 LV transduction, the IiOVA 315-363Contains OTII CD4 + T cell recognition epitope, and IiOVA 242-363 Contains OTII CD4 + and OTI CD8 + Epitopes recognized by T cells (A). DC GFP and non-transduced DCs (DCs UT ) was used as control. The eFluor-labeled OTII CD4 + or OTICD8 + T cells. After 3 days, proliferation was measured by dye dilution (B).

[0142] Figure 10 .DC-IL-10 / InsB promotes CD4 + Bone marrow (BM) isolated from NOD mice was differentiated into DCs in the presence of GM-CSF and treated with LV-IiInsB on day 2. 4-29 、LV-IiInsB 4-29 -miRNA, LV-IL-10 / InsB 4-29 and LV-IDO / InsB 4-29 Transduction. As a control, DC-OVA was generated. eFour-labeled splenic CD4 isolated from diabetic NOD mice were stimulated with the indicated LV-DCs. + T cells. After 3 days of co-culture, proliferation was measured by dye dilution. The percentage of expanded cells is indicated.

[0143] Figure 11 In vivo localization and life cycle of LV-DCs. Bone marrow (BM) isolated from Balb / c mice was differentiated into DCs in the presence of GM-CSF and transduced with LVs encoding luciferase on day 2. LV-DCs (5x10 6 ) were injected intravenously and intraperitoneally into Balb / c recipient mice. Biodistribution and LV-DC survival were monitored by bioluminescence imaging (BLI) at the indicated time points.

[0144] Figure 12 Autologous LV-DC cell therapy protects NOD mice from T1D onset. Bone marrow (BM) isolated from NOD mice was differentiated into DCs in the presence of GM-CSF and treated with LV-IiOVA, LV-IiInsB on day 2. 4-29 、LV-IiInsB 4-29 -miRNA, LV-IL-10 / InsB 4-29 and LV-IDO / InsB 4-29Transduction to produce DC-OVA, DC-InsB, DC-InsB.miRNA, DC-IL-10 / InsB, and DC-IDO / InsB. 10-week-old NOD female mice received intravenous injections of DC-OVA (n=3), DC-InsB (n=10), DC-InsB.miRNA (n=6), DC-IL-10 / InsB (n=9), and DC-IDO / InsB (n=11) three times weekly, and blood glucose levels were monitored three times weekly to assess T1D development. **p<0.005, log-rank (Mantel-Cox) test.

[0145] Figure 13 Develop a protocol for efficient transduction of human DCs using bidirectional LVs. CD14 cells isolated from peripheral blood of healthy subjects (n=8) were pretreated with Vpx-VLPs. + Cells were transduced with LV-ΔNGFR / GFP (LV-DC vpx) for 6-8 hours and then transduced with LV-ΔNGFR / GFP on days 0, 2, and 5 during DC differentiation. As a control, DCs transduced with LV-ΔNFGR / GFP (LV-DC) were differentiated from the same donor. A. Protocol for LV-mediated transduction of monocyte-derived DCs. B. Quantification of transduction efficiency based on ΔNGFR expression on differentiated DCs.

[0146] Figure 14 Pretreatment with Vpx and LV-mediated transduction did not activate human LV-DCs. CD14 isolated from peripheral blood of healthy subjects (n=6) were pretreated with Vpx-VLPs. + Cells were transduced for 6-8 hours and then transduced with LV-ΔNGFR / GFP (LV-DC vpx) on days 0, 2, and 5 during DC differentiation. As a control, DCs transduced with LV-ΔNFGR / GFP (LV-DC) were differentiated from the same donor. Activation of LV-DC was monitored by expression of CD86. LV-DC were transduced in the absence of VPX (white markers) or in the presence of VPX (black markers).

[0147] Figure 15 .DC IL-10 CD14 isolated from the peripheral blood of healthy subjects (n=11) was pretreated with Vpx-VLP. + Cells were transfected for 6-8 hours and then treated with LV-ΔNGFR / GFP (DC) on day 0 during DC differentiation. GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 As controls, DC-10 cells differentiated from the same donor in the presence of GM-SCF / IL-4 and IL-10 and non-transduced DCs (DC UTA. Quantification of transduction efficiency based on ΔNGFR expression on differentiated DCs. B. Expression of the indicated surface markers was assessed by FACS. *P < 0.05, ** < 0.01, Wilcoxon signed-rank test.

[0148] Figure 16 In the absence of IL-12, DC IL-10 CD14 isolated from the peripheral blood of healthy subjects (n=5) were pretreated with Vpx-VLP to secrete high levels of IL-10. + Cells were transfected for 6-8 hours and then treated with LV-ΔNGFR / GFP (DC) on day 0 during DC differentiation. GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 As controls, DC-10 cells differentiated from the same donor in the presence of GM-SCF / IL-4 and IL-10 and non-transduced DCs (DC UT The resulting cells were either left unactivated or activated with LPS / IFNg for 48 hours. IL-10 (A) and IL-12 (B) levels in the culture supernatants were measured by ELISA (n=5). *P<0.05, **<0.01, Wilcoxon signed-rank test.

[0149] Figure 17 .DC IL-10 Induction of allogeneic CD3 + CD14 T cells isolated from peripheral blood of healthy subjects (n=11) were pretreated with Vpx-VLP. + Cells were transfected for 6-8 hours and then treated with LV-ΔNGFR / GFP (DC) on day 0 during DC differentiation. GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 As controls, DC-10 cells differentiated from the same donor in the presence of GM-SCF / IL-4 and IL-10 and non-transduced DCs (DC UT ). Allogeneic CD3 + T cells were labeled with eFlour and stimulated with the indicated DCs for 5 days. The percentage of proliferating cells was calculated based on the dilution of the proliferation dye. Total CD3 + (A) CD3 + CD4 + (B) and CD3 + CD8 + Proliferation of T (C) cells. *P < 0.05, **P < 0.01, Wilcoxon signed-rank test.

[0150] Figure 18 .DCIL-10 Promotes allospecific anergy to CD4 + CD14 T cells isolated from peripheral blood of healthy subjects (n=7) were pretreated with Vpx-VLP. + Cells were transfected for 6-8 hours and then treated with LV-ΔNGFR / GFP (DC) on day 0 during DC differentiation. GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 As a control, DC-10 and non-transduced DC (DC) from the same donor were differentiated in the presence of GM-SCF / IL-4 and IL-10. UT ) was used as control. Allogeneic CD4 + T cells were cultured for 10 days. After 10 days, T cells were labeled with eFlour and restimulated with mature DCs (mDCs) that were homologous to the DCs used for the initial immunization. The percentage of proliferated cells was calculated based on the dilution of the proliferation dye (n=7). **<0.005, *<0.05, Mann-Whitney test.

[0151] Figure 19 .DC IL-10 CD14 T cells isolated from the peripheral blood of healthy subjects (n=7) were pretreated with Vpx-VLPs to promote allogeneic specific IL-10 production. + Cells were transfected for 6-8 hours and then treated with LV-ΔNGFR / GFP (DC) on day 0 during DC differentiation. GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 As controls, DC-10 cells differentiated from the same donor in the presence of GM-CSF / IL-4 and IL-10 and non-transduced DCs (DC UT ). Allogeneic CD3 + T cells for 10 days. A. After 10 days, Tr1 cells (CD49b + LAG-3 + ) percentage (n=7). B. After 10 days, cells were restimulated with mature DC (mDC) that were homologous to the DC used for priming, and IL-10 levels were assessed by ELISA 48 hours later (n=7). **<0.005, *<0.05, Mann-Whitney test.

[0152] Figure 20 Adoptive transfer of DCs IL-10 Delaying graft-versus-host disease. Balb / c bone marrow (BM) cells were differentiated into DCs with GM-CSF and treated with LV-ΔNGFR / GFP (DCs) on day 2. GFP ) or LV-ΔNGFR / IL-10 (DCIL-10 Balb / c mice were lethally irradiated and intravenously injected with C57Bl / 6BM cells (10 7 ) and spleen cells (5x10 6 On day 2, DCs were adoptively transferred into mice. GFP or DC IL-10 (2x10 6 ), and the mice were monitored for weight loss (A) and survival (B).

[0153] Figure 21 A protocol for efficient transduction of human DCs with bidirectional LVs encoding a given antigen. CD14 isolated from peripheral blood of healthy subjects was cultured in serum-free medium. + The cells were pretreated with Vpx-VLP (2 μl / well) for 6-8 hours and then transduced with LV on day 0 during human DC differentiation to obtain human (h)LV-DC. Half of the medium was replaced (LV dilution) on day 1. DCs were differentiated in the presence of IL-4 (100 ng / ml) and GM-CSF (100 ng / ml).

[0154] Figure 22 DC-SIGN expression can be used to monitor LV-DC differentiation in vitro. CD14 isolated from the peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 was treated with Vpx-VLP. + Cells were cultured for 6-8 hours and then transduced with LV-ΔNGFR / Ag (DC-Ag), LV-IL-10 / Ag (DC-IL-10 / Ag), or LV-IDO / Ag (DC-IDO / Ag). As a control, DCs transduced with LV encoding human CLIP (DC-CLIP) were differentiated in parallel. DC differentiation was monitored by expression of DC-SIGN and CD14.

[0155] Figure 23 .DC-IL-10 / Ag transduction efficiency. CD14 isolated from peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + The cells were incubated for 6-8 hours and then transduced with LV-ΔNGFR / Ag (DC-Ag) and LV-IL-10 / Ag (DC-IL-10 / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIPA. Quantification of DC-Ag transduction efficiency based on ΔNGFR expression. B. To monitor DC-IL-10 / Ag transduction efficiency, DCs were left unstimulated or stimulated with LPS (200 ng / ml) and IFN-γ (50 ng / ml) for 24 hours. At 6 hours, brefeldin was added to the cells, and IL-10 expression was quantified by intracytoplasmic staining. The percentage of positive cells is indicated.

[0156] Figure 24 .DC-IDO / Ag transduction efficiency. CD14 isolated from peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + Cells were transduced with LV-DNFGR / Ag, LV-IDO / Ag (DC-IDO / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP A. Quantification of DC-Ag transduction efficiency based on ΔNGFR expression. B. Quantification of DC-IDO / Ag transduction efficiency based on IDO expression in the prepared samples. The percentage of positive cells is indicated.

[0157] Figure 25 DC-IL-10 / Ag expression of DC-10 related markers. CD14 isolated from the peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + The cells were incubated for 6-8 hours and then transduced with LV-ΔNGFR / Ag (DC-Ag) and LV-IL-10 / Ag (DC-IL-10 / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP ) of non-transduced DCs (DC UT The expression of the indicated surface markers CD14, CD163+CD141+, ILT4, and HLA-G were assessed by FACS.

[0158] Figure 26 DC-IL-10 / Ag secretes high levels of IL-10 during or after activation. CD14 isolated from the peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + The cells were incubated for 6-8 hours and then transduced with LV-ΔNGFR / Ag (DC-Ag) and LV-IL-10 / Ag (DC-IL-10 / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP ) and non-transduced DCs (DCUT The resulting cells were either left unactivated or activated with LPS / IFNγ (200 ng / ml LPS and 50 ng / ml IFN-γ) for 48 hours. IL-10 levels in the culture supernatants were measured by ELISA. ***P < 0.0001, ****< 0.0001, Mann-Whitney test.

[0159] Figure 27 DC-IL-10 / Ag secreted low levels of IL-12 after activation. CD14 isolated from the peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + The cells were incubated for 6-8 hours and then transduced with LV-ΔNFGR / Ag (DC-Ag) and LV-IL-10 / Ag (DC-IL-10 / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP ) and non-transduced DCs (DC UT The resulting cells were activated with LPS / IFNγ (200 ng / ml LPS and 50 ng / ml IFN-γ) for 48 hours. IL-12 levels in the culture supernatants were measured by ELISA. *P < 0.05, Mann-Whitney test.

[0160] Figure 28 .DC-IL-10 / Ag induces autologous CD3 + CD14 T cells isolated from peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + Cells were transduced with LV-ΔNFGR / AgNFGR / Ag (DC-Ag) and LV-IL-10 / Ag (DC-IL-10 / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP ). HLA-DQ8 donors were stimulated with LV-DCs encoding insulin B peptide (InsB), and specifically, cells transduced with LV-ΔNFGR / InsB (DC-InsB) or cells transduced with LV-IL-10 / InsB (DC-IL-10 / InsB) were generated, and HLA-DQ2.5 donors were transduced with LVs encoding glia peptide (Glia), and specifically, cells transduced with LV-ΔNFGR / Glia (DC-Glia) or cells transduced with LV-IL-10 / Glia (DC-IL-10 / Glia) were generated. Autologous CD3 +T cells were labeled with eFlour and stimulated with the indicated DCs at a 10:1 ratio for 6 days. The percentage of proliferating cells was calculated based on the dilution of the proliferation dye. *P < 0.05, Wilcoxon signed-rank test.

[0161] Figure 29 DC-IL-10 / Ag promotes Ag-specific Tr1 cells. CD14 isolated from the peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + Cells were transduced for 6-8 hours and then transduced with LV-ΔNFGR / Ag NFGR / Ag (DC-Ag) (A), LV-IL-10 / Ag (DC-IL-10 / Ag) (B) on day 0 during DC differentiation. HLA-DQ8 donors were stimulated with LV-DCs encoding insulin B peptide (InsB) and, in particular, cells transduced with LV-ΔNFGR / InsB (DC-InsB) or cells transduced with LV-IL-10 / InsB (DC-IL-10 / InsB) were generated. HLA-DQ2.5 donors were transduced with LVs encoding glia peptide (Glia) and, in particular, cells transduced with LV-ΔNFGR / Glia (DC-Glia) or cells transduced with LV-IL-10 / Glia (DC-IL-10 / Glia) were generated. Autologous CD3 + T cells were labeled with eFlour and stimulated with the indicated DCs. After 10 days, the proliferation of Tr1 cells (CD49b + LAG-3 + ). The % positive cells are expressed as one of the four tested donors.

[0162] Figure 30 .DC-IDO / Ag induces autologous CD3 + Ag-specific proliferation of T cells. CD14 T cells isolated from peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + Cells were cultured for 6-8 hours and then transduced with LV-ΔNFGR / Ag (DC-Ag) or LV-IDO / Ag (DC-IDO / Ag) on ​​day 0 during DC differentiation. As a control, LV-CLIP-transduced DCs (DCs) differentiated from the same donor were used. CLIP). HLA-DQ8 donors were stimulated with LV-DCs encoding insulin B peptide (InsB), and specifically, cells transduced with LV-ΔNFGR / InsB (DC-InsB) or cells transduced with LV-IL-10 / InsB (DC-IL-10 / InsB) were generated, and HLA-DQ2.5 donors were transduced with LVs encoding glia peptide (Glia), and specifically, cells transduced with LV-ΔNFGR / Glia (DC-Glia) or cells transduced with LV-IL-10 / Glia (DC-IL-10 / Glia) were generated. Autologous CD3 + T cells were labeled with eFlour and stimulated for 6 days with the indicated DCs at a ratio of 10:1. The percentage of proliferating cells based on the dilution of the proliferation dye is shown.

[0163] Figure 31 .DC-IDO / Ag promotes FOXP3 + CD14 T cells isolated from peripheral blood of healthy subjects with HLA-DQ2.5 or HLA-DQ8 were treated with Vpx-VLP. + Cells were transduced for 6-8 hours and then transduced with LV-ΔNFGR / Ag (DC-Ag) and LV-IDO / Ag (DC-IDO / Ag) on ​​day 0 during DC differentiation. HLA-DQ8 donors were stimulated with LV-DCs encoding insulin B peptide (InsB) and, in particular, cells transduced with LV-IDO / InsB (DC-IDO / InsB) were generated. HLA-DQ2.5 donors were transduced with LVs encoding glia peptide (Glia) and, in particular, cells transduced with LV-IDO / Glia (DC-IDO / Glia). Autologous CD3 + T cells were labeled with eFlour and stimulated with the indicated DCs. Ten days later, Treg cells (FOXP3+CTLA-4+) were assessed by FACS staining.

[0164] Figure 32 .DC IL-10 In vitro promotion of allogeneic specific Tr1 cells. CD14 isolated from peripheral blood of healthy subjects were pretreated with Vpx-VLP on day 0. + cells, and during DC differentiation, LV-ΔNGFR / GFP (DC GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 ) transduction. In parallel, non-transduced DCs (DC UT On day 7, DCs were used to stimulate allogeneic CD4 +T cells were isolated from peripheral blood and cultured at a ratio of 10:1. After 10 days, DCs were purified using CD4 Miltenyi microbeads. UT [T(DC UT )]、DC GFP [T(DC GFP )] or DC IL-10 [T(DC IL-10 T cells were cultured and stained with a proliferation dye, then restimulated with LPS-matured CD4 T cells (differentiated from the same donor as the DC used for primary stimulation). After 3 days, proliferation was assessed by flow cytometry. The percentage of proliferating cells at the culture endpoint was calculated by total proliferation dye dilution. Each point represents a single donor, and data are presented as mean ± STD. *P ≤ 0.05 (Wilcoxon matched pairs test, two-tailed).

[0165] Figure 33 .DC IL-10 In vitro promotion of allogeneic specific Tr1 cells. CD14 isolated from peripheral blood of healthy subjects were pretreated with Vpx-VLP on day 0. + cells, and during DC differentiation, LV-ΔNGFR / GFP (DC GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 ) transduction. In parallel, non-transduced DCs (DC UT On day 7, DCs were used to stimulate allogeneic CD4 + T cells were isolated from peripheral blood and cultured at a ratio of 10:1. After 10 days, DCs were purified using CD4 Miltenyi microbeads. UT [T(DC UT )]、DC GFP [T(DC GFP )] or DC IL-10 [T(DC IL-10 )] T cells cultured and their suppressive activity was assessed. + Autologous CD4 + T cells were stained with proliferation dye and then stimulated with mDC (differentiated from the same donor used in the primary stimulation) at a 1:1 ratio of T (DC IL-10 ) cells. The percentage of proliferating cells at the culture endpoint (left) was calculated using the total proliferation dye dilution. Each point represents a single donor, and data are shown as mean ± standard deviation (A). One representative donor is shown (B).

[0166] Figure 34 .DC IL-10Prevents allogeneic specific T cell reactivation in humanized mice (huMice). NSG mice are transplanted with 2-4x10 5 CD34 + Reconstructed humanized mice were immunized with irradiated allogeneic APCs by intravenous injection. On day 7, only autologous non-transduced DCs (DCs UT ) or use DC IL-10 (DC UT +DC IL10 ) or DC GFP (DC UT +DC GFP ) enhanced immune humanized mice; showed PB CD4 + Kinetics of cell proliferation.

[0167] Figure 35 .DC IL-10 CD14 isolated from the peripheral blood of healthy subjects was pretreated with Vpx-VLP on day 0. + cells and treated with LV-ΔNGFR / IL-10 (DC IL-10 On day 7, DCs were activated with LPS, heat-killed Listeria monocytogenes, flagellin-containing S. typhimurium, PoliI:C, ODN2006 (CpG), or a cocktail of cytokines (IL-1b, TNF-a, and IL-6). IL-10 After 24 hours, expression of the indicated surface markers, CD1a (A), CD141 (B), and CD83 (C), was assessed by FACS. Each point represents a single donor, and data are presented as mean ± STD. *P ≤ 0.05 (Wilcoxon matched-pairs test, two-tailed).

[0168] Figure 36 .DC IL-10 CD14 isolated from the peripheral blood of healthy subjects was pretreated with Vpx-VLP on day 0. + cells and treated with LV-ΔNGFR / IL-10 (DC IL-10 On day 7, DCs were activated with LPS, heat-killed Listeria monocytogenes, flagellin-containing S. typhimurium, Poli I:C, ODN2006 (CpG), or a cocktail of cytokines (IL-1b, TNF-a, and IL-6). IL-10After 24 hours, expression of the indicated surface markers, HLA-G (A) and ILT4 (B), was assessed by FACS. Each point represents a single donor, and data are presented as mean ± STD. *P ≤ 0.05 (Wilcoxon matched-pairs test, two-tailed).

[0169] Figure 37 .DC IL-10 CD14 isolated from the peripheral blood of healthy subjects was pretreated with Vpx-VLP on day 0. + cells, and during DC differentiation, LV-ΔNGFR / GFP (DC GFP ) or LV-ΔNGFR / IL-10 (DC IL-10 On day 5, DCs were stimulated with LPS and LPS or Poli I:C. IL-10 and DC GFP On day 7, DCs were used to stimulate allogeneic CD4 + T cells were isolated from peripheral blood and cultured at a ratio of 10:1. After 10 days, mDCs were purified using CD4 Miltenyi microbeads. GFP [T(mDC GFP )]、DC Il-10 [T(DC Il-10 )] or stimulated DC IL-10 [T(stimDC IL-10 )] T cells cultured. A. Frequency of Tr1 cells. Primed T cells were stained with CD3, CD4, CD45RA, CD49b and LAG-3 to assess the percentage of Tr1 cells by flow cytometry. BT cell proliferation. Primed T cells were stained with proliferation dyes and then restimulated with LPS-matured DCs (differentiated from the same donor as the DCs used for primary stimulation). After 3 days, proliferation was assessed by flow cytometry. The percentage of proliferating cells in the precursor population was calculated by peak analysis (right), while the percentage of proliferating cells at the end of culture (left) was calculated by overall proliferation dye dilution. C. Cytokine production profile. IL-10 production in cell culture supernatants was assessed by ELISA. D. Inhibitory activity. CD4 T cells used in primary stimulation were stimulated with LPS-matured DCs (differentiated from the same donor as the DCs used for primary stimulation). + Autologous CD4 + T cells, in a 1:1 ratio of T (DC IL-10 ) or T(stimDC IL-10) cells. The percentage of proliferating cells at the end of culture (left) was calculated by total proliferation dye dilution. Each point represents a single donor, and data are shown as mean ± STD. White circular symbols represent DCs activated with LPS. IL-10 The red circular symbols represent DCs treated with Poli I:C. IL-10 Stimulated cells.

[0170] Figure 38 . Time course analysis of Vpx-transduced human DCs using bidirectional LV. CD14 cells isolated from peripheral blood of healthy subjects were pretreated with 5 μl Vpx-VLP. + Cells were transduced for 1-6 hours and then with LV-ΔNGFR / GFP during DC differentiation on day 0. Transduction efficiency was quantified based on ΔNGFR expression on differentiated DCs.

[0171] Figure 39 Increased transgene expression by LV particles without human CD47. On day 0 during DC differentiation, CD14 cells isolated from peripheral blood of healthy subjects were transduced with LV.PGK.GFP. + LV.PGK.GFP was generated using packaging cell lines overexpressing human CD47 (high CD47 LV) or knocking out CD47 (no CD47 LV). Classical LV was used as a control. Compared to high huCD47 (huCD47-high) LV particles or wt LV particles carrying normal huCD47 levels, LV particles without human CD47 increased transduction efficiency, expressed as % GFP+ cells, normalized by % GFP 293T.

[0172] DETAILED DESCRIPTION OF THE INVENTION

[0173] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, molecular biology, histology, immunology, and oncology that are within the capabilities of various skilled artisans in the art and which are described in the literature.

[0174] Immunomodulatory molecules

[0175] Immunomodulatory molecules are substances (proteins or small molecules) that regulate immune responses.

[0176] An immune response is a process mediated by cells of the immune system that react to antigens. Immune responses can include immunity to pathogenic microorganisms and their products, or autoimmunity to self-antigens, allergies to antigens that cause allergies, and transplant rejection to alloantigens. In this process, the main cells involved are T cells and B cells, as well as antigen-presenting cells, including macrophages and dendritic cells. The immune response can be measured by the proliferation of T cells and the secretion of cytokines such as IL-2, IL-4, IL-10, and IFNg.

[0177] Immunomodulatory molecules include receptors such as PDL-1, PDL-2, ILT-3, ILT-4, HO-1, ICOS-L Gal9, HVME, HLA-G, HLA-E; soluble mediators such as IL-10, IL-35, TGF-α, CTLA-4Ig, PGE2, TNFRs; enzymes such as IDO, Arg1; drugs such as rapamycin, dexamethasone, vitamin D3, corticosteroids. Preferred immunomodulatory molecules are IL-10 and / or IDO.

[0178] As used herein, the term "enhance" may refer to an action that improves, boosts, increases, or otherwise increases the presence or activity of a specific target. For example, enhancing an immune response may refer to any action that will result in an improvement, boost, increase, or otherwise increase an immune response. In other examples, enhancing the expression of a nucleic acid may include, but is not limited to, increasing the transcription of the nucleic acid, increasing mRNA abundance (e.g., increasing mRNA transcription), reducing mRNA degradation, increasing mRNA translation, etc. In other examples, enhancing the expression of a protein may include, but is not limited to, increasing the transcription of a nucleic acid encoding the protein, increasing the stability of the mRNA encoding the protein, increasing the translation of the protein, increasing the stability of the protein, etc.

[0179] MicroRNA (miRNA) is a small non-coding RNA that regulates cellular gene expression by post-transcriptional silencing. When miRNA is partially complementary to the target mRNA sequence, they generally reduce target mRNA stability and inhibit translation. On the contrary, when miRNA is almost completely complementary to its mRNA target, they cut mRNA, triggering its large-scale destruction. MiRNA can achieve tissue-specific regulation of the transgenic of systemic delivery and ubiquitin expression at the post-transcriptional level. MiRNA has different expression profiles in different tissues and cell types, and it differentially regulates the transcriptional profile and cellular function of cellular genes, including APC and immune activation. Therefore, the method provided herein adopts immune-related miRNA (for example, APC-specific miRNA) to silence the transgenic expression in immune cells (for example, APC).

[0180] The miR or miRNA target sequence or "seed sequence" is essential for the binding of the miRNA to the mRNA. The target sequence or seed sequence is a conserved heptametrical sequence that is primarily located at positions 2-7 of the 5' end of the miRNA. Even if the base pairing between the miRNA and its target mRNA is not a perfect match, the "seed sequence" must be completely complementary.

[0181] miRNA target sequences are sequences that regulate mRNA expression and subsequently protein expression.

[0182] miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-21, miR-29a, miR-29b, miR-29c, miR-30b, miR-31, miR-34a, miR-92a-1, miR-106a, miR-125a, miR-125b, miR-126, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424. More preferably, miR155 and miR146a are each repeated twice.

[0183] "Receptor antigen" refers to an antigen expressed by a recipient. As used herein, "effector cell" refers to a cell that mediates an immune response to an antigen. Examples of effector cells include, but are not limited to, T cells and B cells.

[0184] As used herein, the term "immune response" includes T cell-mediated and / or B cell-mediated immune responses. Exemplary immune responses include T cell responses, for example, cytokine production and cytotoxicity, and B cell responses, for example, antibody production. In addition, the term immune response includes an immune response indirectly affected by T cell activation (for example, antibody production (humoral response)) and activated cytokine response cells (for example, macrophages). The immune cells involved in the immune response include: lymphocytes, such as B cells and T cells (CD4+, CD8+, Th1 and Th2 cells): antigen presenting cells (for example, professional antigen presenting cells, such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells, such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; bone marrow cells, such as macrophages, eosinophils, mast cells, basophils and granulocytes.

[0185] An antigen is a substance that causes the immune system to react, for example, by producing T cells that recognize peptides derived from a protein substance, and B cells that produce antibodies against the substance. An antigen can have one or more epitopes.

[0186] Antigen-derived peptides or antigenic peptides or proteins are peptides or proteins derived from antigens that are processed and presented in competition with class I or class II MHC molecules and T cells. They are typically composed of 9-12 amino acids. They contain at least one immunodominant peptide or epitope. Antigen-derived peptide fragments or antigenic peptide or antigenic protein fragments are fragments that are shorter than the antigenic peptide or protein and have the antigenicity of the peptide or protein.

[0187] As used herein, an immunodominant peptide (or "epitope") is a portion of an antigen that can elicit an immune response, including a B and / or T cell response. An antigen may have one or more immunodominant peptides. Most antigens have many epitopes, i.e., they are multivalent. In some embodiments, the size of an epitope is about 10 amino acids. Preferably, the immunodominant peptide or epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably 6-14 amino acids, more preferably about 7-12 amino acids, and most preferably about 8-10 amino acids. It is understood by those skilled in the art that in some cases, the three-dimensional structure of the molecule, rather than a specific linear sequence, is the primary criterion for antigenic specificity and, therefore, distinguishes one immunodominant peptide or epitope from another.

[0188] In the present invention, in order to correctly process and present the immunodominant peptide, the construct will contain a nucleotide sequence encoding the immunodominant peptide and variable flanking regions, each flanking region consisting of 5-10 amino acids. For example, for diabetes, the immunodominant peptide is insulin B 9-23 , while the construct includes the gene encoding insulin B 4-29 nucleotide sequence.

[0189] In the present invention, the antigenic peptide or antigenic fragment thereof is derived from a polypeptide associated with an abnormal physiological response, including but not limited to autoimmune diseases, allergic reactions, and other diseases of the present invention.

[0190] Modified antigen-derived peptides or antigenic peptides

[0191] In the present invention, the antigen-derived peptide (or antigenic peptide) or immunodominant peptide or epitope can be modified, for example, to enhance T cell recognition. Such modifications include, but are not limited to, citrullination, deamidation, methylation, carbamylation, glycosylation, acylation, acetylation, formylation, amidation, and hydroxylation.

[0192] For example, an antigen-derived peptide or immunodominant peptide or epitope for rheumatoid arthritis is advantageously modified to be a citrullinated peptide or glycosylated.An antigen-derived peptide or immunodominant peptide or epitope for celiac disease (gliadin) is advantageously modified to be a deamidated peptide.

[0193] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. An immune response can involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that any macromolecule, including nearly any protein or peptide, can be an antigen. Furthermore, an antigen can be derived from recombinant or genomic DNA. Those skilled in the art will appreciate that any DNA comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response is used herein and therefore encodes the term "antigen" as used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be apparent that antigens can be synthesized or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids. "Antigen presenting cells" (APCs) are cells capable of activating T cells and include, but are not limited to, monocytes / macrophages, B cells, and dendritic cells (DCs).

[0194] invariant chain

[0195] The invariant chain (li; CD74) has multiple functions but is best characterized as a major class II MHC (MHCII) chaperone. It is a type II protein consisting of a short cytoplasmic tail, a transmembrane region, and a luminal domain that can be further divided into a membrane-proximal disordered region, a major MHCII interacting sequence (CLIP), and a C-terminal trimerization domain (44, 45). Mice express two li isoforms, p31 and p41, the latter resulting from alternative splicing (46). In humans, the corresponding isoforms are called p33 and p41. In addition, approximately 20% of li mRNA is translated from upstream start codons that generate p35 and p43 isoforms. They have a 16-amino acid cytoplasmic stretch that includes a strong di-arginine (RxR) ER retention motif (47-49).

[0196] In conjunction with MHC II, synthetic Ii can be viewed as: (i) a guard that controls access to the MHC II groove; (ii) a scaffold that assists in the folding and pairing of α and β MHC II chains; and (iii) a leader that directs MHC II to the endosomal pathway. It is well established that these Ii functions depend primarily on the ability of its CLIP domain to occupy the MHC II peptide groove. Several reports have shown that Ii proteolysis in endosomes allows HLA-DM to disengage from the CLIP groove and catalyze the binding of nominally antigenic peptides (reviewed in (50)).

[0197] The invariant chain of the MHC II molecule (Ii, invariant chain, MHC II gamma chain) is the most commonly described sequence in the literature that can mediate targeting. Various variants of the invariant chain in humans are described, and are also known as IiP33, IiP41, IiP35 and IiP43 (51), which are suitable as targeting modules. Another sequence suitable as a targeting module for the purposes of the present invention is the beta chain of the MHC II molecule (52). Fragments of these sequences are also suitable as targeting modules.

[0198] The invariant chain is a protein encoded by the CD74 gene in humans. It is a polypeptide involved in the formation and trafficking of class II MHC proteins. Nascent class II MHC proteins in the ER bind to fragments of the invariant chain (CLIP), thereby shaping the peptide binding groove and preventing the formation of a closed conformation. The invariant chain promotes the release of class II MHC from the endosomes of the ER, which contain endocytosed antigenic proteins (from the exogenous pathway).

[0199] Here, the term invariant chain includes all naturally occurring or artificially produced full-length or fragmented homologous genes and proteins that have a certain similarity to the human invariant chain.

[0200] Vpx

[0201] Myeloid cells, such as dendritic cells and macrophages, are poorly responsive to vector transduction, particularly lentiviral vector transduction, due to the myeloid-specific restriction factor SAMHD1. SIVmac / HIV-2 and related viruses alleviate SAMHD1-mediated restriction by encoding Vpx, a virion packaging accessory protein that induces SAMHD1 degradation after infection. HIV-1 does not encode Vpx and is therefore unable to package proteins.

[0202] Appropriately, the Vpx packaging motif can be packaged in a lentiviral vector virion, for example, it can be placed in the p6 region of a Gag / Pol expression vector, which is used to generate a lentiviral vector virion that in turn packages a high copy number of Vpx. Alternatively, Vpx can be provided to DCs or their precursor cells by pre-processing virus-like particles (VLPs) containing Vpx.

[0203] markers

[0204] In the present invention, the marker is preferably a selective marker, such as ΔNGFR as described herein, the coding sequence of which is included in the nucleic acid construct to enable selection of transduced cells. An alternative could be a truncated form of CD19, in which deletion of the CD19 cytoplasmic domain eliminates the signal transduction pathway

[93] .

[0205] Bicistronic constructs

[0206] Bicistronic vectors or constructs are constructs in which two factors are expressed using multiple promoters or by including an internal ribosome entry site (IRES) element. An IRES element is a nucleotide sequence that allows translation to begin in the middle of a messenger RNA (mRNA) sequence.

[0207] carrier

[0208] In addition to the major elements described above for the vector, the vector also includes conventional control elements that are operatively linked to the nucleic acid sequence in a manner that allows transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with a virus produced by the present disclosure. As used herein, "operatively linked" sequences include expression control sequences that are contiguous with the gene of interest, as well as expression control sequences that act in trans or are located at a distance from the gene of interest.

[0209] Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, when desired, sequences that enhance secretion of the encoded product. Many expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be used. As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be "operably" linked when they are covalently linked in a manner that places the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence. If translation of the nucleic acid sequence into a functional protein is desired, two DNA sequences are said to be operably linked when: induction of the promoter in the 5' regulatory sequence results in transcription of the coding sequence, and the nature of the linkage between the two DNA sequences does not (1) result in the introduction of frameshift mutations, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, if the promoter region is capable of effecting transcription of the DNA sequence, the promoter region will be operatively connected to the nucleic acid sequence so that the resulting transcript can be translated into the desired protein or polypeptide. Similarly, when two or more coding regions are connected in such a way that their transcription from a common promoter results in the expression of two or more proteins that have been translated in frame. In some embodiments, operatively connected coding sequences produce fusion proteins. In some embodiments, operatively connected coding sequences produce functional RNA (e.g., shRNA, miRNA, miRNA inhibitors).

[0210] For nucleic acids encoding proteins, a polyadenylation sequence is typically inserted after the nucleic acid sequence.

[0211] Another vector element that can be used is the internal ribosome entry site (IRES). The IRES sequence is used to produce more than one polypeptide from a single gene transcript. The IRES sequence will be used to produce proteins that contain more than one polypeptide chain. The selection of these and other common vector elements is conventional, and a variety of such sequences exist.

[0212] The precise nature of the regulatory sequences required for gene expression in a host cell may vary between species, tissues, or cell types, but generally should include 5' non-transcribed and 5' non-translated sequences (where necessary) involved in the initiation of transcription and translation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, an enhancer element, and the like. In particular, such 5' non-transcribed regulatory sequences will include a promoter region comprising a promoter sequence for transcriptional control of an operably linked gene. As needed, the regulatory sequences may also include an enhancing sequence or an upstream activator sequence. Optionally, the vectors disclosed herein may include a 5' leader or signal sequence. The selection and design of appropriate vectors are within the ability and discretion of those of ordinary skill in the art.

[0213] Examples of constitutive promoters include, but are not limited to, the retroviral sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al., Cell, 41:521-530 (1985)], the SV 40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter [Invitrogen].

[0214] Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or a particular physiological state (e.g., the acute phase, a particular differentiation state of the cell, or only replicating cells). Inducible promoters and inducible systems are available from a variety of commercial sources, including but not limited to: Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by those skilled in the art. Examples of inducible promoters regulated by exogenously supplied promoters include: the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), tetracycline repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), tetracycline inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), RU486 inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Other types of inducible promoters that can be used in this context are those that are regulated by specific physiological states (e.g., temperature, acute phase, a specific differentiation state of the cell, or only replicating cells).

[0215] In another embodiment, the natural promoter of the nucleotide sequence will be used. When it is desired that the expression of the nucleic acid should mimic natural expression, a natural promoter can be used. When the expression of the nucleic acid must be regulated temporally or developmentally, or regulated in a tissue-specific manner, or when regulating in response to a specific transcriptional stimulus, a natural promoter can be used. In another embodiment, other natural expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences also can be used to simulate natural expression.

[0216] In some embodiments, regulatory sequences confer tissue-specific gene expression capability. In some cases, tissue-specific regulatory sequences are combined with tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: liver-specific thyroxine-binding globulin (TBG) promoter, insulin promoter, glucagon promoter, somatostatin promoter, pancreatic polypeptide (PPY) promoter, synaptophysin-1 (Syn) promoter, creatine kinase (MCK) promoter, mammalian desmin (DES) promoter, α-myosin heavy chain (α-MHC) promoter, or cardiac troponin T (cTnT) promoter. Other exemplary promoters include the β-actin promoter, the hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); the α-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), the osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); the bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11: 654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161: 1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal, such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13: 503-15 (1993)), neurofilament light chain gene promoter In some embodiments, the promoter is the muscle-specific promoter Desmin460 or the truncated muscle creatine kinase (tMCK) promoter.

[0217] It will be appreciated by those skilled in the art that, for nucleic acids encoding proteins or polypeptides, mutations leading to conservative amino acid substitutions can be carried out in the nucleic acids to provide functionally equivalent protein or peptide variant homologues. In some aspects, sequence changes including those leading to conservative amino acid substitutions of the nucleic acid sequence are disclosed.

[0218] dendritic cells

[0219] Dendritic cells are professional antigen-presenting cells of the immune system that have the ability to process and present antigens to T cells.

[0220] The term "dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cells found in lymphoid or non-lymphoid tissues. These cells are characterized by their unique morphology and high levels of surface MHC class II expression. DC can be isolated from a variety of tissue sources. DC are highly capable of sensitizing MHC-restricted T cells and are very efficient in presenting antigens to T cells in situ.

[0221] Antigens can be self-antigens expressed during T cell development and tolerance, as well as foreign antigens present during normal immune processes.

[0222] Dendritic cell precursors

[0223] Dendritic cell precursors are cells that express CD14 (CD14 + ).

[0224] Autoantigens

[0225] Self-antigens (self-Ags), also known as immunodominant peptides, are typically normal proteins or protein complexes that are recognized by the immune system of patients with autoimmune diseases. Under normal conditions, these antigens do not promote an immune response, but in autoimmune diseases, they promote T cell responses that lead to tissue damage. Table 2 provides a list of known immunodominant peptides.

[0226] Self-Ag includes self-Ag in T1D, including nonspecific islet cell Ag (ICA), insulin, glutamic acid decarboxylase 65 (GAD 65), insulinoma antigen-2 (IA-2), heat shock protein (HSP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), imogen-38, and β-cell-specific self-Ags, such as zinc transporter-8 (ZnT8), pancreatic duodenal homeobox factor 1 (FDX1), chromogranin A (CHGA), and islet amyloid polypeptide (IAPP); self-Ags in MS, including myelin basic protein (MBP); proteolipid protein (PLP); myelin oligodendrocyte glycoprotein (MOG); myelin-associated antigen (MAG), myelin-associated oligodendrocyte basic protein (MOBP) and 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase); S100β protein, and transaldolase H; self-Ags in RA, including the Fc-portion of immunoglobulins; citrullinated antigens, carbamylated antigens, collagen, 65-kDa thermophilic antigen Shock proteins, cartilage glycoprotein-aggregan 1, aggrecan core protein precursor (ACAN), α-fibrinogen (FGA), vimentin (VIM); self-Ag in IBD, including zymogen granule membrane glycoprotein 2 (GP2); tropomyosin (TM), carcinoembryonic antigen (CEA); self-Ag in vasculitis, including beta-2-glycoprotein 1 (b2GPI), myeloperoxidase (MPO); proteinase 3 / myeloblastin (PR3); self-Ag in myasthenia gravis, including nicotinic acetylcholine receptor (nAChR, muscle-specific kinase (MuSK); self-Ag in autoimmune uveitis retinal S-antigen (PDSAg), heterogeneous nuclear ribonucleoprotein (Hnrph3), inner photoreceptor retinoid binding protein (IRBP), cellular retinal aldehyde-binding protein (cRALBP); self-Ag in pemphigus vulgaris, including desmoglein-31 (Dsg 1), Desmoglein-3 (Dsg 3), Pemphaxin (PX).

[0227] Table 2: Known immunodominant peptides

[0228]

[0229]

[0230]

[0231]

[0232] Variants of such known immunodominant peptides are also encompassed by the present invention, wherein the variants retain the antigenicity of the immunodominant peptide.

[0233] harmless antigens

[0234] Innocent antigens are substances that are present in the body and do not usually promote an active immune response (food antigens, including prolamins, ovalbumin, peanut-derived proteins, milk-derived proteins, wheal-derived proteins, etc.).

[0235] Allergens

[0236] Allergens are usually harmless substances that can trigger an immune response and cause an allergic reaction. Allergens include gluten from cereals, peanut-derived proteins, timothy grass allergens (Phl p 1, 2, 5a, 5b, 6), venom-derived proteins, Bet v 1 from birch pollen (Betula verrucosa), Der p 1 and Der p 2 from house dust mites (Dermatophagoides pteronyssinus), Pyr c 5 from pears (Pyrus communis), and Cor a 1 from hazelnuts (Corylus avellana).

[0237] Regulation of CD4+ and CD8+ T cell responses

[0238] Regulation of CD4+ and CD8+ T cell responses refers to the effects on T cells to produce varying levels of pro-inflammatory (i.e., IFN-g, IL-2, GM-CSF) or anti-inflammatory (i.e., IL-10, TGF-β) cytokines, granzymes, and expressed receptors (i.e., CD69, CD25, CTLA-4).

[0239] The levels of pro-inflammatory and anti-inflammatory cytokines can be measured by any method known in the art.

[0240] Antigen-specific CD4 + and CD8 + Regulation of T cell proliferation in vitro and / or in vivo

[0241] Antigen-specific CD4 + and CD8 + Modulation of T cell proliferation in vitro and / or in vivo refers to the property of a cell to inhibit the activation and proliferation of T cells.

[0242] Generate regulatory DCs

[0243] Generating regulatory DCs refers to methods that modulate DCs to regulate them so that they secrete high levels of anti-inflammatory cytokines (i.e., IL-10) and low levels of pro-inflammatory cytokines (i.e., IL-12, TNF-α, etc.) and express tolerance molecules (i.e., HLA-G, IL 4, IDO).

[0244] Favors antigen-specific Tr1 and / or FOXP3+ Expansion of Treg cells

[0245] Favors antigen-specific Tr1 and / or FOXP3 + Expansion of Treg cells refers to the property of cells that induce / convert CD4 T cells with pathogenic activity into regulatory cells capable of suppressing T cell responses in vitro and / or in vivo.

[0246] Tolerogenic cells

[0247] Tolerogenic cells are cells that promote the generation of regulatory cells in vitro and / or in vivo.

[0248] Antigen presentation in the context of class I and class II MHC

[0249] Antigen presentation in the context of class I and class II MHC activates CD4 through its TCR in an antigen-specific manner + and CD8 + The properties of T cells.

[0250] Immunotherapeutic agents

[0251] They are a class of molecules that can induce, enhance or suppress immune responses, including rapamycin, dexamethasone, vitamin D3, etc.

[0252] Cell nomenclature

[0253] LV-DCs are dendritic cells that have been transduced with lentiviral vectors (LV).

[0254] tolDCs are dendritic cells with tolerogenic activity.

[0255] LV.IiOVA is a LV encoding the invariant chain fused to the OVA peptide.

[0256] LV.OVA.miRNA is a unidirectional LV encoding the invariant chain fused to the OVA peptide and the target sequences of miRNA155 and miRNA146a.

[0257] LV-IL-10 / OVA is a bidirectional LV that co-encodes the invariant chain and IL-10 fused to the OVA peptide.

[0258] LV-IDO / OVA is a bidirectional LV that co-encodes the invariant chain and IDO fused to the OVA peptide.

[0259] DC-OVA are dendritic cells that have been transduced with LV encoding the invariant chain fused to the OVA peptide (LV-liOVA).

[0260] DC-OVAmiRNA are dendritic cells that have been transduced with LV encoding the invariant chain fused to the OVA peptide and the target sequences for miRNA155 and miRNA146a.

[0261] DC-IL-10 / OVA are dendritic cells that have been transduced with LV co-encoding the invariant chain and IL-10 fused to the OVA peptide (LV-IL-10 / OVA).

[0262] DC-IDO / OVA are dendritic cells that have been transduced with LV co-encoding the invariant chain and IDO fused to the OVA peptide (LV-IDO / OVA).

[0263] OTII CD4 + T cells are CD4+ T cells isolated from TCR transgenic mice that recognize OVA 323-339 Peptide.

[0264] OTI CD8 + T cells are CD8+ T cells isolated from TCR transgenic mice that recognize OVA 242-353 Peptide.

[0265] OVA peptide-pulsed DCs are dendritic cells that have been pulsed with OVA peptide.

[0266] DC-UnT are untransduced cells.

[0267] DC-GFP or DC GFP are dendritic cells that have been transduced with LV encoding GFP (LV.GFP).

[0268] DC-InsB are dendritic cells that have been transduced with LV encoding the invariant chain fused to InsB (LV.InsB).

[0269] DC-InsB.miRNA are dendritic cells that have been transduced with LV encoding the invariant chain fused to InsB and the target sequences for miRNA155 and miRNA146a (LV.InsB.miRNA).

[0270] DC-IL-10 / InsB are dendritic cells that have been transduced with LV encoding the invariant chain and IL-10 fused to the InsB peptide (LV.IL-10 / InsB).

[0271] DC-IDO / InsB are dendritic cells that have been transduced with LV encoding the invariant chain and IDO fused to InsB (LV.IDO / InsB).

[0272] LV-ΔNGFR / GFP is a bidirectional LV that co-encodes ΔNGFR and GFP.

[0273] LV-GFP is a unidirectional LV that encodes GFP.

[0274] LV-IL-10 is a bidirectional LV that co-encodes ΔNGFR and IL-10.

[0275] DC IL-10 are dendritic cells that have been transduced with LV encoding ΔNGFR and IL-10.

[0276] DC-10 differentiates from CD14 in the presence of IL-10, IL-4, and GM-CSF. + cells of dendritic cells.

[0277] Allogeneic CD3 + T cells are T cells that are specific for self-Ag.

[0278] Allospecific non-reactive CD4 + CD4 T cells are specific for self-Ag + T cells, which do not proliferate.

[0279] Mature DCs (mDCs) are differentiated from CD14 in the presence of IL-4 and GM-CSF. + cells and dendritic cells activated with LPS.

[0280] Allogeneic (allo-) mature DCs are differentiated from allogeneic CD14 in the presence of IL-4 and GM-CSF. + cells and dendritic cells activated with LPS.

[0281] Allospecific IL-10-producing TrI cells are T cells specific for self-Ag that produce IL-10 and express CD49b and LAG-3, are anergic and suppress T cell responses.

[0282] LV-ΔNGFR / Ag is a bidirectional LV that co-encodes the invariant chain fused to an antigen-derived peptide and ΔNGFR.

[0283] LV-IL-10 / Ag is a bidirectional LV that co-encodes the invariant chain and IL-10 fused to an antigen-derived peptide.

[0284] LV-CLIP is a bidirectional LV that co-encodes the invariant chain CLIP peptide and ΔNGFR.

[0285] DC-IDO / Ag are dendritic cells that have been transduced with LV encoding the invariant chain and IDO fused to an antigen-derived peptide.

[0286] hLV-DCs are differentiated from human CD14 + cells and dendritic cells transduced with LV.

[0287] DC UT Differentiated from allogeneic CD14 in the presence of IL-4 and GM-CSF + cells of dendritic cells.

[0288] T(DC UT ) cells, by using allogeneic DC UT Culture CD4 + T cells were generated on day 10 of T cell culture.

[0289] T(DC GFP ), by using allogeneic DC GFP Culture CD4 + T cells were generated on day 10 of T cell culture.

[0290] T(DC IL-10 ), by using allogeneic DC IL-10 Culture CD4 + T cells were generated on day 10 of T cell culture.

[0291] T(stimDC IL-10 ), by using allogeneic DC IL-10 Culture CD4 + T cells were generated on day 10, and the DC IL -10 Stimulated with LPS or Poli I:Cc.

[0292] In some aspects, the present disclosure provides transfected or transduced host cells. The term "transfection" or "transduction" is used to refer to the uptake of foreign DNA by cells, and when foreign DNA is introduced into the cell membrane, the cell is "transfected" or "transduced". Many transfection / transduction techniques are well known in the art. See, for example, Graham et al. (1973) "Virology" (Virology), 52:456, Sambrook et al. (1989) "Molecular Cloning, a laboratory manual", Cold Spring Harbor Laboratory Press, New York, Davis et al. (1986) "Basic Methods in Molecular Biology" (Basic Methods in Molecular Biology), Elsevier Press (Elsevier), and Chu et al. (1981) Gene 13:197. This type of technology can be used to introduce one or more exogenous nucleic acids (such as nucleotide integration vectors and other nucleic acid molecules) into suitable host cells.

[0293] "Host cell" refers to any cell that has or is capable of having a substance of interest. Typically, the host cell is a mammalian cell. The host cell can serve as a recipient of DNA constructs, plasmids, accessory function vectors, or other transfer DNA associated with lentiviral vector production. The term includes the descendants of the original cell that has been transfected / transduced. Therefore, as used herein, "host cell" can refer to a cell that has been transfected / transduced with an exogenous DNA sequence. It should be understood that due to natural, accidental, or deliberate mutations, the descendants of a single parent cell may not necessarily be completely identical to the original parent in morphology, genome, or total DNA complement.

[0294] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that during storage or transfer of such clonal populations, spontaneous or induced changes may occur in the karyotype. Thus, cells derived from a reference to a cell line may not be exactly identical to the progenitor cell or culture, and reference to a cell line includes such variants.

[0295] As used herein, the term "recombinant cell" or "genetically modified cell" refers to a cell into which an exogenous DNA segment has been introduced, such as a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid (eg, RNA).

[0296] As used herein, the term "vector" refers to any genetic element capable of replicating and transferring gene sequences between cells when associated with appropriate control elements, such as plasmids, phages, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, and the like. Thus, the term encompasses cloning and expression vectors, as well as viral vectors, preferably lentiviral vectors. In some embodiments, useful vectors are contemplated to be vectors in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by an introduced synthetic structure or a synthetic structure of the cell required to initiate specific transcription of a gene. The phrases "operably located at," "under the control of," or "under transcriptional control" refer to a promoter that is in the correct position and orientation relative to a nucleic acid to control RNA polymerase initiation and gene expression. The term "expression vector or construct" refers to any type of genetic construct comprising a nucleic acid in which part or all of a nucleic acid coding sequence is capable of being transcribed. In some embodiments, expression includes transcription of a nucleic acid, for example, the production of a biologically active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA, miRNA inhibitor) by a transcribed gene.

[0297] In the present invention, the term "indoleamine dioxygenase" or "IDO" refers to IDO1 (indoleamine 2,3-dioxygenase, EC 1.13.1 1.52) or IDO2 (indoleamine-pyrrole 2,3 dioxygenase-like 1, EC 1.13.11.-), which are two different proteins that can catabolize tryptophan and can be expressed by APC.

[0298] "Immune tolerance" means the lack of response to an antigen (self or foreign) and includes natural tolerance or induced tolerance (ie, deliberate manipulation of the immune system).

[0299] "Self-antigen" means any molecule or chemical group of an organism that acts as an antigen in inducing a T effector cell response or antibody formation in another organism, but to which the healthy immune system of the parent organism is tolerant. In certain circumstances, for example, when a subject has an autoimmune disease or is susceptible to an autoimmune disease, the parent organism is intolerant to the self-antigen and has a specific adaptive immune response against the self-antigen.

[0300] "Exogenous therapeutic agent" means any therapeutic agent that originates from outside the subject and is used to treat the subject.

[0301] The term "co-culture" means culturing two (or more) cell types in the presence of each other.

[0302] It will be understood by those skilled in the art that the compositions and methods described herein can be used in conjunction with current therapeutic methods for treating diseases and conditions described elsewhere herein. As a non-limiting example, the cells of the present invention can be used in conjunction with immunosuppressive drug therapy. The advantage of using cells in conjunction with immunosuppressive drugs is that by using the methods of the present invention to improve the severity of the immune response in the subject, the amount of immunosuppressive drug therapy used and / or the frequency of administration of immunosuppressive drug therapy can be reduced for such transplant recipients. The benefit of reducing the use of immunosuppressive drug therapy is to alleviate the general immunosuppression and undesirable side effects associated with immunosuppressive drug therapy. It is also expected that the cells of the present invention can be given to the recipient repeatedly or as a "one-time" therapy to prevent or treat diseases or conditions, such as autoimmune diseases or conditions, inflammatory diseases or conditions, or diseases or conditions associated with transplants, such as host rejection of donor tissue or graft-versus-host disease. Giving cells to the recipient of a transplant once eliminates the need for chronic immunosuppressive drug therapy. However, if necessary, cells can be given multiple times.

[0303] Based on the disclosure provided herein, dendritic cells or their precursors can be obtained from any source, for example, from a tissue donor, a transplant recipient, or other unrelated sources (completely different individuals or species). The cell can be autologous relative to T cells (obtained from the same host), or it can be allogeneic relative to T cells. In the case where dendritic cells or their precursors are allogeneic, the cell can be autologous relative to the transplant to which the T cells respond, or the cell can be obtained from a mammal that is allogeneic relative to the source of the T cells or the source of the transplant to which the T cells respond. In addition, T cells can be xenogeneic relative to T cells (obtained from animals of different species), for example, mouse cells can be used to inhibit the activation and proliferation of human T cells.

[0304] Another aspect of the present invention includes a route for administering cells to a subject. Cells can be administered by a route that is suitable for the environment. Cells can be administered systemically, i.e., parenterally, by intravenous or intraperitoneal injection, or can be targeted to specific tissues or organs, such as bone marrow, by subcutaneous implantation of cells or by injection of cells into connective tissue (e.g., muscle).

[0305] Cells can be suspended in an appropriate diluent at a concentration of approximately 1x10 4 to about 20x10 7 cells / ml, preferably about 5x10 6 Cells / ml. Suitable excipients for injectable solutions are excipients that are biologically and physiologically compatible with the cells and receptors, such as buffered saline solutions or other suitable excipients. Compositions for administration can be formulated, manufactured, and stored according to standard methods consistent with appropriate sterility and stability.

[0306] The dosage of cells varies within a wide range and can be adjusted to the requirements of the subject in each particular case. The number of cells used depends on the weight and condition of the recipient, the number and / or frequency of administration, and other variables known to those skilled in the art.

[0307] Autoimmune diseases

[0308] Autoimmune diseases are conditions caused by an abnormal immune response to self-antigens and include: type 1 diabetes, autoimmune bowel disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune myositis, psoriasis, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, autoimmune hepatitis, alopecia areata, pemphigus vulgaris, vitiligo, aplastic anemia, and autoimmune uveitis.

[0309] Autoimmune diseases also include: alopecia areata, amyotrophic lateral sclerosis (Lou Gehrig's disease), ankylosing spondylitis, anti-GBM nephritis, antiphospholipid syndrome, osteoarthritis, asthma, atopic allergy, atopic dermatitis, autoimmune active chronic hepatitis, autoimmune inner ear disease (AIED), Barlow disease, Behçet's disease, Berger's disease, bullous pemphigoid, cardiomyopathy, chronic fatigue immune dysfunction syndrome, Tschag-Strauss syndrome, cicatricial pemphigoid, cold agglutinin disease, cranial arteritis colitis, CREST syndrome, Crohn's disease, Dagdalar disease, dermatomyositis and JDM, Devic's disease, eczema, essential mixed cryoglobulinemia, eosinophilic fasciitis, fibromyalgia–fibromyositis, fibrosing alveolitis, giant cell arteritis, glomerulonephritis, Goodpasture's disease, and Guillain-Barré syndrome. symptoms, Hashimoto's thyroiditis, hepatitis, Hughes syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, irritable bowel syndrome, Kawasaki disease, lichen planus, lupus hepatitis, lupus / SLE, Lyme disease, Meniere's disease, mixed connective tissue disease, myositis / JM, JDM, and JPM, osteoporosis, pars planitis, pemphigus vulgaris, polyglandular autoimmune syndromes, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, Raynaud's syndrome, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, scleritis, scleroderma, sticky blood syndrome, Still's disease, stiff-person syndrome, Sydenham's chorea, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, Wegener's granulomatosis, and Wilson's syndrome.

[0310] Preferred autoimmune diseases include: vasculitis, such as refractory antiphospholipid antibody syndrome (also known as Asherson's syndrome), giant cell arteritis and anti-ANCA vasculitis, myasthenia gravis, refractory celiac disease, autoimmune uveitis, such as Behçet's disease, pemphigus vulgaris, giant cell myocarditis, Graves' disease, Addison's disease, and granulomatosis with polyangiitis.

[0311] Materials and Methods

[0312] All protocols were approved by the Institutional Review Board, and samples were collected with written informed consent in accordance with the Declaration of Helsinki.

[0313] Cell preparation and cell lines. Bone marrow cells isolated from Balb / c, C57Bl / 6, or NOD mice were maintained in culture in the presence of rmGM-CSF (25 ng / mL; R&D Systems) for 8 days to differentiate into DCs.

[0314] Peripheral blood mononuclear cells (PBMCs) were prepared by centrifugation on a gradient. CD4 T cell isolation kit (Miltenyi Biotec) was used to purify CD4 T cells. + T cells were isolated to obtain a purity of >95%. Then, CD4 + CD45RO on T cells + Cell depletion. CD4 + CD45RA + The ratio is always greater than 90%. + and CD3 + CD14 was purified by positive selection using microbeads (Miltenyi Biotec) + and CD3 + T cells were obtained with a purity of >95%.

[0315] CD14 cells were isolated from PBMCs by positive selection using CD14 microbeads (Miltenyi Biotec) according to the manufacturer's instructions. +Monocytes. In the presence of 10 ng / ml rhIL-4 (R&D Systems) and 100 ng / ml rhGM-CSF (Genzyme) and 10 ng / ml rhIL-10 (BD Bioscience), cells were cultured in RPMI 1640 (Lonza) (DC medium) for 7 days to differentiate DC-10. The RPMI 1640 was supplemented with 10% fetal bovine serum (FBS) (Lonza) or 5% human serum (HS) (EuroClone), 100 U / ml penicillin / streptomycin (Lonza, Italy), and 2 mM L-glutamine (Lonza, Italy). On day 5, cells cultured with rhIL-4 and rhGM-CSF were matured for an additional 2 days with 1 μg / ml LPS (Sigma) to generate mDCs. On day 7, DCs were collected, phenotyped, and used to stimulate T cells.

[0316] In some experiments, HLA-DQ8 was expressed in the presence of 10 ng / ml rhIL-4 and 100 ng / ml rhGM-CSF (Miltenyi Biotec) with or without 10 ng / ml rhIL-10 (CellGenix). + or HLA-DQ2.5 + CD14 + Cells were cultured at a density of 10^6 cells / ml in serum-free DC medium (CellGenix) supplemented with penicillin / streptomycin (Euroclone). On day 3, cells were supplemented with 1 ml of serum-free medium plus 20 ng / ml rhIL-4 and 200 ng / ml rhGM-CSF (Miltenyi Biotec). Immature DCs were harvested on day 7 for subsequent phenotypic and functional analysis.

[0317] DNA extraction and HLA-DQ screening. To select HLA-DQ8 + and / or HLA-DQ2.5 + Genomic DNA was extracted from 200 μl of whole blood from healthy donors using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer's instructions. The presence of the HLA-DQ8 or -DQ2.5 allele was determined by PCR using the Eu-GEN kit (Eurospital) according to the manufacturer's instructions.

[0318] Plasmid construction. Synthetic and encoding InsB 4-29 or OVA 315-353The coding sequence of the mouse invariant chain (CD74) was fused to the sequence (GeneArt) and cloned into multiple LV backbones: hPGK.XXX.WPRE (84) to obtain LV-IiOVA and LV-IiInsB; hPGK.XXX.WPRE miR155T.mir146aT to obtain LV-IiOVA miRNA and LV-IiInsB miRNA, and cloned into bidirectional backbones: hPGK.XXX.WPRE.mCMVIL10.SV40PA (85) and hPGK.XXX.WPRE.mCMVIDO.SV40PA to obtain LV-IL-10 / OVA and LV-IL-10 / InsB and LV-IDO / OVA and LV-IDO / InsB, respectively.

[0319] The coding sequence of human IL-10 was extracted from pH15C (ATCC no. 68192), and a 549 bp fragment was cloned into the multiple cloning site of pBluKSM (Invitrogen) to obtain pBluKSM-hIL-10. A 555 bp fragment was obtained by excising hIL-10 from pBluKSM-hIL-10 and ligated with 1074.1071.hPGK.GFP.WPRE.mhCMV.dNGFR.SV40PA (85) (herein named LV-ΔNGFR) to obtain LV-IL-10 / ΔNGFR. A bidirectional promoter (human PGK promoter + minimal core elements of the CMV promoter in reverse) allowed co-expression of both transgenes. The LV-IL-10 / ΔNGFR sequence was verified by pyrosequencing (Primm).

[0320] Synthesis and encoding of insulin B peptide 4-29 (InsB 4-29 ) or α2-gliadin 51-80 sequences were fused to the coding sequence of the human invariant chain p33 isoform (GeneArt) and cloned into the following bidirectional backbone: hPGK.XXX.WPRE.mCMV.YYYY.SV40PA to obtain LV-lip33Ag / ΔNGFR, LVlip33Ag / IL-10, or LVlip33Ag / IDO. As a control, the sequence encoding the antigen was replaced with the class II related invariant chain peptide (CLIP). The sequence of the resulting plasmid was verified by pyrosequencing (GATC).

[0321] Vector production and titration. VSV-G pseudotyped third generation bdLV was produced by transient four-plasmid co-transfection into 293T cells with Ca3PO4 and concentrated by ultracentrifugation (40). Titers were estimated by limiting dilution, vector particles were measured by immunocapture of HIV-1 Gag p24 antigen (NEN Life Science), and vector infectivity was calculated as the ratio of titer to particles. Titers ranged from 5x10 8 Up to 6x10 9 Transducing units / ml, infectivity is 5x10 4 to 10 5 Transducing units / ng of p24.

[0322] Transduction of Dendritic Cells. Bone marrow cells isolated from Balb / c, C57Bl / 6, or NOD mice were differentiated into DCs in the presence of GM-CSF (25 ng / mL; R&D Systems) and transduced with LV at a multiplicity of infection (MOI) of 3 on day 2.

[0323] CD14 cells were inoculated as described above in the presence of 1-5 μl of virus-like particles (VIP) containing viral protein X (VPX). + Monocytes were transduced 6 hours later with LV at a multiplicity of infection (MOI) of 5. After 14-18 hours, half the medium was replenished. The efficiency of transduction of cells was assessed by flow cytometry on transduced controls based on cell surface expression of ΔNGFR.

[0324] Cytokine determination. Monocyte-derived DCs were harvested on day 7, washed with PBS, and replated at 500,000 cells / ml in fresh medium alone or supplemented with 200 ng / ml LPS and 50 ng / ml IFNγ. After 48 hours, supernatants were collected and cytokine concentrations were determined by ELISA.

[0325] Proliferation and inhibition assays. To assess Ag-specific proliferation of CD4+ and CD8+ T cells, OTII and OTI cells were labeled with eFluor-670 proliferation dye (Invitrogen) according to the manufacturer's instructions. eFluor-labeled T cells were seeded alone in a U-bottom 96-well plate in a final volume of 200 μl or in the presence of LV-DC (T:DC ratio of 10:1). After 5 days, T cell proliferation was assessed by flow cytometry. Cells were acquired using a BD FACSCanto II analyzer and analyzed using Flow Jo software.

[0326] To assess Ag-specific proliferation, CD4+ T cells (autologous to monocyte-derived DCs) were thawed, incubated at 37°C for 1-2 hours, and labeled with efluor-450 proliferation dye (Invitrogen) according to the manufacturer's instructions. 150,000 eFluor-labeled CD4+ T cells were seeded into 96-well round-bottom plates alone in a final volume of 200 μl, or in the presence of DCs transduced with LV-lip33Ag / ΔNGFR or LVlip33Ag / IL-10 or control LV-lip33-CLIP (T:DC ratio of 10:1). After 6 days, the proliferation of CD4+ T cells was assessed by flow cytometry. Cells were acquired using a BD FACSCanto II analyzer and analyzed using Flow Jo software.

[0327] Flow cytometry analysis. The phenotype of mouse BM-LVDC was determined by flow cytometry on day 8 after differentiation. To detect cell surface antigens, the following monoclonal antibodies (mAbs) were used: anti-CD11c-V450 (e-bioscience), anti-CD86-Pe-Cy7 (BD Biosciences), anti-CD80-PerCPCy5.5 (BD Biosciences), and anti-IAb-Pe (BD Biosciences). OTII cells were identified using anti-CD4-Pe-Cy7 (BD Biosciences) and anti-CD45.2-Pacific Blue (BD Biosciences).

[0328] The phenotype of monocyte-derived human LV-DCs was determined by flow cytometry on day 7. To detect cell surface antigens, the following monoclonal antibodies (mAbs) were used: anti-DC-SIGN-Pe (BD Biosciences), anti-CD14-FITC (BD Biosciences), anti-HLADR-APC-Cy7 (BD Biosciences), anti-CD86-PercP-Cy5.5 (BD Biosciences), anti-CD83-BV421 (BD Biosciences), anti-DNGF-APC (Miltenyi Biotech), and anti-CD11c-Pe-Cy7 (BD Biosciences). Cell surface expression of tolerogenic molecules was also determined: anti-HLAG-PE (ExBio), anti-ILT4-APC (R&D Systems), anti-CD163-PCy5.5 (BD Biosciences), and anti-CD141-BV421 (BD Biosciences). Cell viability was assessed using a live / dead cell viability assay (Thermo Fisher) according to the manufacturer's instructions. To assess the frequency of IL-10-producing DCs, LV-DCs were stimulated for 14-16 hours with LPS 200 ng / ml and IFNg 50 ng / ml + brefeldin A (10 μg / ml). As previously described (Levings JI 2001), intracellular expression of IL-10 was determined using anti-IL-10-Pe (BD Pharmingen). To assess the frequency of IDO-expressing DCs, cells were fixed with 2% formaldehyde solution (Thermo Fisher) for 20 minutes and permeabilized with PBS 2% FBS PBS containing 0.5% saponin (Sigma) for 10 minutes, followed by intracellular staining with anti-human IDO Pe (e Biosciences).

[0329] To detect FOXP3 after surface staining (clone 259D, Biogen, USA), cells were fixed, permeabilized, and stained with a FOXP3 staining buffer combination according to the manufacturer's instructions (e Biosciences, USA). To express granzyme B after surface staining (clone MHGB04, Invitrogen, USA), cells were fixed, permeabilized, and stained with BD Cytofix / Cytoperm TM The kit was used for staining according to the manufacturer's instructions (catalog number 554714, Biogen, USA). Samples were collected using a BD FACS Canto II or BD-LSR Fortessa analyzer and analyzed using Flow Jo software.

[0330] Mice. C57Bl / 6, female NOD (NOD / LtJ) and Balb / c mice (Charles River Laboratories) were purchased and housed under specific pathogen-free conditions. The inventors crossed and generated Foxp3 reporter mice in the inventors' laboratory. The inventors used age- and sex-matched littermates between 8 and 12 weeks of age. Chimeric mice were generated by transplanting CD45.1 (95%) and CD45.2 OTII / FirTiger (5%) BM cells into lethally irradiated CD45.1 mice. OTII / FirTiger CD4+ T cells are the ones that recognize OVA, respectively. 323-339 and TCR transgenic cells expressing RFP and GFP as reporter genes for foxp3 and il10.

[0331] NOD mice were considered diabetic when blood glucose measurements were ≥250 mg / dl on two consecutive days as determined by the Bayer BREEZE blood glucose monitoring system (Bayer). All procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the San Raffaele Institute in Milan (IUCAC 416 and 604).

[0332] GvHD model: Balb / c mice were lethally irradiated and intravenously injected with C57Bl / 6BM cells (10 7 ) and spleen cells (5x10 6 On day 2, DCs were adoptively transferred into mice. GFP or DC IL-10 (2x10 6 ), and the mice were monitored for weight loss and survival.

[0333] Method for generating human Treg cells in vitro. To induce Ag-specific CD4+ Treg cells, T cells autologous to monocyte-derived DCs were thawed, incubated at 37°C for 1-2 hours, and labeled with efluor-450 proliferation dye (Invitrogen) according to the manufacturer's instructions. 6eFluor-labeled CD4+ T cells were seeded into 24-well round-bottom plates in a final volume of 2 ml in the presence of DCs transduced with LV-lip33Ag / DNGFR, LVlip33Ag / IL-10, LVlip33Ag / IDO, or control LV-lip33-CLIP (T:DC ratio of 10:1). After 10 days, the proliferation of CD4+ T cells was assessed by flow cytometry, and in the case of LVlip33Ag / IL-10, the presence of TR1 cells was assessed by the co-expression of CD49b and LAG-3 on proliferating cells, and in the case of LVlip33Ag / IDO, the presence of FOXP3+ Tregs was assessed by the co-expression of FOXP3 and CTLA-4 on proliferating cells. Cells were acquired using a BD FACSCanto II analyzer and analyzed using Flow Jo software.

[0334] Vpx-VLP production. Concentrated Vpx-incorporated virus-like particles (VLPs) were generated by transient Ca3PO4 transfection of two plasmids (a plasmid expressing VSV-G and a simian immunodeficiency virus-derived packaging plasmid, SIV3+) into 293T cells and concentrated by ultracentrifugation as described (86). Titers were estimated by limiting dilution. Titers ranged from 5 × 10 8 Up to 6x10 9 Transduction units / ml.

[0335] T cell differentiation and suppression assay. 10 6 CD4 + T cells and 10 5 Allogeneic DCs were cultured in X-VIVO 15 medium (Lonsard, Switzerland) (10:1, T:DC), supplemented with 5% human serum (Sigma Aldrich, California, USA) and 100 U / ml penicillin / streptomycin (Lonsard, Switzerland). After 10 days, the primary T cells were collected and purified using CD4 microbeads (Miltenyi Biotec, Germany). UT Stimulated T cells are called T (DC UT ) cells, and DC GFP Stimulate those T cells called T (DC GFP ) cells. IL-10 Cultured T cells are called T (DC IL-10 ) cells, while DCs stimulated with LPS- or Poli I:C IL-10 Those T cells cultured are called T (stimDC IL-10 )cell.

[0336] Cell proliferation dye The primed T cells were stained with 670 (e Biosciences, CA, USA) and then compared with DCs from the same donor used for priming. UT Co-plating (10:1, T:DC).After 3 days of stimulation, T cells were collected, washed, and proliferation was assessed by flow cytometry.

[0337] In order to evaluate T(DC IL-10 ) and T(stimDC IL-10 ) cells, we used cell proliferation dyes 450 (e Biosciences, California, USA) against all CD4 T cells autologous to the T cells used in priming. + T cells (responder cells) were stained and stained with mature DCs from the same donor used for priming UT Cell proliferation dye T (DC) stained with 670 (e Biosciences, California, USA) IL-10 ) or T(DC IL -10 *) Cells were added at a 1:1 ratio with responder cells (total T:DC ratio was 10:1). After 4 days, the percentage of dividing responder T cells was calculated by dilution of proliferation dye using flow cytometry.

[0338] DC stimulation. In some experiments, DCs were collected on day 7 and replated alone or in the presence of the following stimuli: 1 μg / ml LPS (Sigma-Aldrich, CA, USA), 10 8 Cells were cultured with 10 μg / ml of heat-killed Listeria monocytogenes (code tlrl-hklm, InvivoGen, CA, USA), 1 μg / ml of flagellin S. typhimurium (code tlrl-stfla, InvivoGen, CA, USA), 10 μg / ml of Poli (I:C) (code tlrl-pic, InvivoGen, CA, USA), 5 μM of ODN2006 (CpG) (code tlrl-2006, InvivoGen, CA, USA), or 10 ng / ml of a mixture of IL-1b, TNF-a, and IL-6 cytokines (R&D Systems, MN, USA). After 24 hours, supernatants were collected to assess cytokine secretion profiles by ELISA, and cells were analyzed by flow cytometry.

[0339] Modulation of immune responses in humanized mice. 2-5 day old NSG (NOD.Cg-Prkdc scid Il2rg tm1WjI / SzJ, JAX mouse strain) mice were sublethally irradiated (1.5 cGy) and injected intrahepatically with 10 5 CD34+ cells (purity ≥95%, Lonsard) were obtained as previously described (Santoni de Sio et al. JACI 2018).

[0340] Starting from 8 weeks after transplantation, the percentage of total human cells and T cells in peripheral blood was monitored by flow cytometry. Once the human graft was stable and T cell repopulation could be clearly detected (usually around 11-13 weeks after transplantation), 5x10 6 Humanized mice (huMice) were immunized with allogeneic CD3- T cells magnetically separated from human peripheral blood (DynabeadsCD3 - Thermo Fisher Scientific). One week later, the percentage of human T cells was assessed by flow cytometry, and the humanized mice were randomly assigned to experimental groups and injected with 3x10 5 Untransduced dendritic cells (DCs) UT ), or 3x10 5 Untransduced dendritic cells plus 3x10 5 GFP or IL-10 transduced dendritic cells (DCs GFP and DC IL-10 ), which were differentiated from CD14+ monocytes isolated from the same donor used for CD3-purification. T cell proliferation in peripheral blood was assessed by Ki67 staining.

[0341] Packaging cell lines CD47 high (CD47 hi) and CD47 free (CD47 free) were generated. Plasmids expressing Cas9 and sgRNA as previously described (87) were used to disrupt CD47 expression in 293T cells. The CRISPR sequences used to generate sgRNA were: CD47A (CTACTGAAGTATACGTAAAGTGG) (SEQ ID NO: 115), B (CTTGTTTAGAGCTCCATCAAAGG) (SEQ ID NO: 116), C (ATCGAGCTAAAATATCGTGTTGG) (SEQ ID NO: 117).

[0342] Gene disruption and mismatch-selective endonuclease assays were performed by calcium phosphate-mediated transient transfection of the indicated amounts of the desired sgRNA-expressing plasmid and the Cas9-expressing plasmid. The mismatch-selective endonuclease assay was used to measure the extent of mutations caused by nonhomologous end joining (NHEJ) at the Cas9 target site as described (88). PCR was performed using primers flanking the sgRNA binding site in the CD47 gene (forward: 5'-TTCCTTTCCAGGATCAGCTCAGC-3' (SEQ ID NO: 118); reverse: 5'-TTGATTCAAAGGAGTACCTATCCC-3' (SEQ ID NO: 119)).

[0343] For SIN-RV genome transfer, PGK.CD47 (Genewiz), encoding a human codon-optimized version of the CD47 cDNA synthesized from a gene, was exchanged with GFP to become pRT43.3.PGK.GFP (BamHI-NotI) (89) and used to generate 293T CD47 high cells. 293T cells were transfected with pRT43.3.PGK.CD47, the packaging plasmid pCMV-Gag / Pol (Moroni leukemia virus), and pMD2.G as described (89). 293T cells expressing CD47 high and CD47 null were used to generate LVs as described above.

[0344] Statistical Analysis. Mean values ​​are reported as mean ± SEM. The inventors used the Mann-Whitney test and ANOVA test to determine the statistical significance of the data. The inventors defined significance as *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, and ***P < 0.0001. The inventors used Prism program 5.0 (GraphPad Software, Inc.) for statistical calculations.

[0345] sequence

[0346] In the proteins described below, immunodominant peptides or epitopes are highlighted in bold, while deamidated residues are highlighted in grey.

[0347] LV constructs of murine DCs

[0348] Invariant chain (m-Ii, CD74) (DNA) (SEQ ID NO: 120)

[0349] atggatgaccaacgcgacctcatctctaaccatgaacagttgcccatactgggcaaccgccctagagagccagaaaggtgcagccgtggagctctgtacaccggtgtctctgtcctggtggctctgctcttggctgggcaggccaccactgcttacttcctgtaccagcaacagggccgcctagacaagctgaccatcacctcccagaacctgcaactggagagccttcgcatgaagcttccgaaatctgccaaacctgtgagccagatgcggatggctactcccttgctgatgcgtccaatgtccatggataacatgctccttgggcctgtgaagaacgttaccaagtacggcaacatgacccaggaccatgtgatgcatctgctcacgaggtctggacccctggagtacccgcagctgaaggggaccttcccagagaatctgaagcatcttaagaactccatggatggcgtgaactggaagatcttcgagagctggatgaagcagtggctcttgtttgagatgagcaagaactccctggaggagaagaagcccaccgaggctccacctaaagagccactggacatggaagacctatcttctggcctgggagtgaccaggcaggaactgggtcaagtcaccctg

[0350] Invariant chain (m-Ii, CD74) (protein) (SEQ ID NO:121)

[0351] MDDQRDLISNHEQLPILGNRPREPERCSRGALYTGVSVLVALLLAGQATTAYFLYQQQGRLDKLTITSQNLQLESLRMKLPKSAKPVSQMRMATPLLMRPMSMDNMLLGPVKNVTKYGNMTQDHVMHLLTRSGPLEYPQLKGTFPENLKHLKNSMDGVNWKIFESWMKQWLLFEMSKNSLEEKKPTEAPPKEPLDMEDLSSGLGVTRQELGQVTL

[0352] Invariant chain fused in-frame to:

[0353] >OVA 315 - 353, STOP(DNA)(SEQ ID NO:122)

[0354] tgtggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagcacatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgcaagctga

[0355] OVA 315 - 353, STOP(Protein)(Epitope OVA 323-339 )(SEQ ID NO:123)

[0356]

[0357] >OVA 242 - 353.STOP(DNA)(SEQ ID NO:124)

[0358] tgcatgttggtgctgttgcctgatgaagtctcaggccttgagcagcttgagagtataatcaactttgaaaaactgactgaatggaccagttctaacgttatggaagagaggaagatcaaagtgtacttacctcgcatgaagatggaggaaaaatacaacctcacatctgtcttaatggctatgggcattactgacgtgtttagctcttcagccaatctgtctggcatctcctcagcagagagcctgaagatatctcaagctgtccatgcagcacatgcagaaatcaatgaagcaggcagagaggtggtagggtcagcagaggctggagtggatgctgccagctga

[0359] OVA 242 - 353.STOP(Protein)(Epitope: OVA 257-264 ; OVA 323-339 )(SEQ ID NO:125)

[0360]

[0361] >InsB 4 - 29.STOP(SEQ ID NO:126)

[0362] cagcacctttgtggttcccacctggtggaggctctctacctggtgtgtggggagcgtggcttcttctacacacccatgtaa

[0363] InsB 4-29.STOP(Protein)(Epitope InsB 9-23 )(SEQ ID NO:127)

[0364]

[0365] >InsB 4-29R22E.STOP(DNA)(SEQ ID NO:128)

[0366] cagcacctttgtggttcccacctggtggaggctctctacctggtgtgtggggagcgtggcttcttctacacacccatgtaa

[0367] InsB 4-29R22E.STOP(Protein)(Epitope InsB 9-23R22E )(SEQ ID NO:129)

[0368]

[0369] >GAD65 500-585(DNA)(SEQ ID NO:130)

[0370] Cacacaaatgtctgcttctggtttgtacctcctagtttgcgcactctggaagacaatgaagagagaatgagccgcctctcaaaggtggcgccagtgattaaagccagaatgatggagtatgggaccacaatggtcagctaccaacccttaggggacaaggtcaacttcttccgcatggtcatctcaaaccctgcagcaactcaccaagacattgacttccttattgaagaaatcgaacgcctcggacaagatttgt

[0371] GAD65 500-585(Protein)(Epitope: GAD 509-528 ; GAD 524-543 ,GAD 561-575 )(SEQ ID NO:131)

[0372]

[0373] >GAD65 202-225(DNA)(SEQ ID NO:132)

[0374] actaacatgttcacctatgagatcgcccctgtatttgtgctgctagaatatgttacactaaagaaaatgagataa

[0375] GAD65 202-225 (protein) (epitope: GAD 206-220 ) (SEQ ID NO: 133)

[0376]

[0377] >IGRP191-218(DNA)(SEQ ID NO:134)

[0378] gaggcctttgaacacactccaggagtccacatggccagcttgagtgtgtacctgaagaccaacgtcttcctcttcctgtttgcctaa

[0379] IGRP195-214 (protein) (epitope: IGRP 195-214 ) (SEQ ID NO: 135)

[0380]

[0381] W1E14 (DNA) (SEQ ID NO: 136)

[0382] gaggtggaggaccctcaggtggcccagctggagctgggcggcggccctggcgccggcgacctgcagaccctggccctgtggagcagaatggaccagctggccaaggagctgaccgccgagtga

[0383] WE14 (fusion protein) (combination N-ter C pep–ChrA) (SEQ ID NO: 137)

[0384]

[0385] LV constructs of human DCs

[0386] Human invariant chain (hu-Ii, p33, clip )(DNA)(SEQ ID NO:138)

[0387] atggatgaccagcgcgaccttatctccaacaatgagcaactgcccatgctgggccggcgccctggggccccggagagcaagtgcagccgcggagccctgtacacaggcttttccatcctggtgactctgctcc tcgctggccaggccaccaccgcctacttcctgtaccagcagcagggccggctggacaaactgacagtcacctcccagaacctgcagctggagaacctgcgcatgaagcttcccaagcctcccaagcctgtgagc aagatgcgcatggccaccccgctgctgat gcaggcg ctgcccatgggagccctgccccaggggcccatgcagaatgccaccaagtatggcaacatgacagaggaccatgtgatgcacctgctccagagtcactggaactggaggacccgtcttctgggctgggtgtga

[0388] Human invariant chain (hu-Ii, p33, clip )(protein)(SEQ ID NO:139)

[0389]

[0390] Replace the Clip sequence with a sequence encoding the Ag of interest

[0391] hu.InsB 4-29(DNA)(SEQ ID NO:140)

[0392] caacacctgtgcggctcacacctggtggaagctctctacctagtgtgcggggaacgaggcttcttctacacacccaag

[0393] hu.InsB 4-29 (protein) (epitope InsB 9-23 ) (SEQ ID NO: 141)

[0394]

[0395] hu.InsB 4-29(-14E-21E-22E)(DNA)(SEQ ID NO:142)

[0396] caacacctgtgcggctcacacctggtggaagaactctacctagtgtgcggggaagaaggcttcttctacacacccaag

[0397] hu.InsB 4-29(-14E-21E-22E)(Protein)(Epitope InsB 9-2314E-21E-22E ) (SEQ ID NO: 143)

[0398]

[0399] hu.PPI proinsulin / proinsulin 71-96 (DNA) (epitope PPI 71-96 ) (SEQ ID NO: 144)

[0400] ggccctggtgcaggcagcctgcagcccttggccctggaggggtccctgcagaagcgtggcattgtggaacaatgctgt

[0401] hu.PPI (protein) (epitope C19A3) (SEQ ID NO: 145)

[0402]

[0403] hu.PPI proinsulin / proinsulin 13-28 (DNA) (PPI 13-28 ) (SEQ ID NO: 146)

[0404] ctgctggccctctggggacctgacccagccgcagcctttgtgaaccaa

[0405] hu.PPI(protein)(epitope PPI 17-24 ) (SEQ ID NO: 147)

[0406]

[0407] I-A2 801-821(DNA)(SEQ ID NO:148)

[0408] Gagagcggctgcaccgtcatcgtcatgctgaccccgctggtggaggatggtgtcaagcagtgt

[0409] I-A2 (protein) (epitope: I-A2 805-820 ; I-A2 806-814 ) (SEQ ID NO: 149)

[0410]

[0411] a2-gliadin 51-80> (SEQ ID NO: 150)

[0412] tctcagcagccctacctgcaactgcagccctttccacagcctgagctgccctatcctcagcctcagcctagctttccacctcagcag

[0413] a2-gliadin 51-80>(protein)(epitope a2-gli 55-76 ) (SEQ ID NO: 151)

[0414]

[0415] T regulatory cell epitope (Tregitope) 289 (DNA) (SEQ ID NO: 152)

[0416] Gaggagcagtacaacagcacctacagagtggtgagcgtgctgaccgtgctgcaccaggactgg

[0417] T regulatory cell epitope 289 (protein) (SEQ ID NO: 153)

[0418] EEQYNSTYRVVSVLTVLHQDW

[0419] Clone into:

[0420] -Unidirectional LV backbone (SEQ ID NO: 154)

[0421] LV.PGK. Ii

[0422] ccacggg gttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaa cgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctaccc ttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggac gtgacaaacggaagccgcac

[0423] PGK. IiAg.miR 155T. miR146aT(SEQ ID NO:155)

[0424]

[0425]

[0426]

[0427] LV fragment (SEQ ID NO:156)

[0428] bd.ΔNGFR.PGK.GFP

[0429]

[0430]

[0431] bd.IL10.PGK.GFP(SEQ ID NO:157)

[0432]

[0433]

[0434] bd.huIDO.PGK.IiAg(SEQ ID NO:158)

[0435]

[0436]

[0437]

[0438] huIL-10(DNA)(SEQ ID NO:159)

[0439] atgcacagctcagcactgctctgttgcctggtcctcctgactggggtgagggccagcccaggccagggcacccagtctgagaacagctgcacccacttcccaggcaacctgcctaacatgcttcgagatctccgagatgccttcagcagagtgaagactttctttcaaatgaaggatcagctggacaacttgttgttaaaggagtccttgctggaggactttaagggttacctgggttgccaagccttgtctgagatgatccagttttacctggaggaggtgatgccccaagctgagaaccaagacccagacatcaaggcgcatgtgaactccctgggggagaacctgaagaccctcaggctgaggctacggcgctgtcatcgatttcttccctgtgaaaacaagagcaaggccgtggagcaggtgaagaatgcctttaataagctccaagagaaaggcatctacaaagccatgagtgagtttgacatcttcatcaactacatagaagcctacatgacaatgaagatacgaaactga [[ID=​​​​​​​​​

[0444] huIDO (protein) (SEQ ID NO:162)

[0445] MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPASQQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG*

[0446] gagpol polyprotein simian immunodeficiency virus (Vpx) (DNA) (SEQ ID NO:163)

[0447]

[0448] gagpol polyprotein Simian immunodeficiency virus (Vpx) (protein) (SEQ ID NO: 164)

[0449] MGARNSVLSGKKADELEKIRLRPGGKKKYMLKHVVWAANELDRFGLAESLLENKEGCQKILSVLAPLVPTGSENLKSLYNTVCVIWCIHAEEKVKHTEEAKQIVQRHLVVETGTAETMPKTSRPTA PSSGRGGNYPVQQIGGNYVHLPLSPRTLNAWVKLIEEKKFGAEVVPGFQALSEGCTPYDINQMLNCVGDHQAAMQIIRDIINEEAADWDLQHPQPAPQQGQLREPSGSDIAGTTSSVDEQIQWMYRQ QNPIPVGNIYRRWIQLGLQKCVRMYNPTNILDVKQGPKEPFQSYVDRFYKSLRAEQTDAAVKNWMTQTLLIQNANPDCKLVLKGLGVNPTLEEMLTACQGVGGPGQKARLMAEALKEALAPVPIPF AAAQKRGPRKPIKCWNCGKEGHSARQCRAPRRQGCWKCGKMDHVMAKCPDRQAGFLGLGPWGKKPRNFPMAQVHQGLTPTAPPEDPAVDLLKNYMQLGKQQRESREKPYKEVTEDLLHLNSLFGGDQ Example

[0450] Alternative strategies for generating tolerogenic DC (tolLV DC) have been developed based on LV-mediated gene transfer of specific Ag-derived peptides plus target sequences of miR155 and miR146a, known regulators of DC maturation (DC-Ag.miRNA), accompanied by IL-10 (DC-IL-10 / Ag) or IDO (DC-IDO / Ag). Figure 1 To determine the mode of action of tolLV-DC, the inventors used ovalbumin (OVA) as a model Ag. 315-353 Fusion of Ii LV containing TCR transgenic OTII CD4 + T cell recognition of OVA 323-339 LV was used to transduce bone marrow (BM) cells during DC differentiation. 315-353 .miR155T.miR146aT、LV.IL-10.IiOVA315-353 、LV.IDO.IiOVA 315-353 , and control LV.IiOVA 315-353 Used to obtain the following: DC-OVA, DC-OVA.miRNA, DC-IL-10 / OVA, DC-IDO / OVA. LV-DC is CD11c + and expressed CD80, CD86, and class II MHC at the same levels as untransduced DCs. Figure 2 DC-OVA promotes OTIICD4 + T cell proliferation, while DC-IL-10 / OVA induced low OTII CD4 + T cell proliferation response. In contrast, DC-IDO / OVA-induced proliferation was comparable to that induced by DC-OVA ( Figure 3 ). It is noteworthy that T cells generated with DC-IL-10 / OVA were not responsive to secondary OVA stimulation ( Figure 4 ), indicating that DC transduction with LV.IL-10.IiOVA promotes a DC population that is functionally super-imposable to DC-10, a cell population generated in vitro in the presence of IL-10 that effectively promotes unstressed Ag-specific T cells ((22); WO2007 / 131575; US2016 / 0046910A1). DC-OVA.miRNA promotes OTII CD4 + T cells proliferate, but upon LPS activation, miR155- and miR146a-mediated posttranscriptional regulation abrogates their ability to promote OTII CD4 + T cell proliferation capacity ( Figure 5 ), which indicates that DC-OVA miRNA is only expressed in the immature-like CD4 + T presents OVA.

[0451] To investigate the mechanism of action of LV-DCs, we generated chimeric mice by transplanting CD45.1 (95%) and CD45.2 OTII / FirTiger (5%) bone marrow (BM) cells into lethally irradiated CD45.1 mice. 323-339 and TCR transgenic cells expressing RFP and GFP as reporter genes for foxp3 and il10. At full reconstitution, repeated injections of different subsets of LV-DE cells with approximately 5% circulating OTII / FirTiger CD4 + T cell chimeric mice ( Figure 6, left panel). Five weeks after the last DC injection, OVA-specific CD45.2 OTII CD4 + The frequency of T cells in the spleens of mice treated with DC-OVA was significantly higher than that of mice injected with DC-GFP ( Figure 6 , right panel). In addition, OVA-specific CD4 + The expansion of T cells was evident in mice that received different tolerogenic LV-DC encoding OVA, but not GFP. In addition, we observed an increase in CD4 T cells in mice treated with DC-OVA or tolerogenic LV-DC expressing OVA and tolerogenic molecules. + The expansion of memory T cells (not shown) indicates that in vivo priming of OVA-specific T cells occurs. The inventors then investigated the induction of OVA-specific Tregs in treated chimeric mice using reporter genes and the expression of Tr1-specific markers CD49b and LAG-3 (90) as well as the expression of CD25 of Foxp3 Tregs. The results showed that injection of DC-OVA.miRNA or DC-IL-10 / OVA promoted significantly higher IL-10-producing CD49b compared to that observed in mice treated with DC-GFP or DC-OVA. + LAG-3 + Expansion of Tr1 cells ( Figure 7 In contrast, LV-DC treatment did not significantly induce FOXP3 + Expansion of Tregs (not shown). After in vitro restimulation with DC-OVA, T cells isolated from mice treated with tolerogenic LV-DCs were hypo-responsive, compared to the results observed with T cells isolated from mice injected with DC-OVA, which were expressed via OTII CD4 + The low proliferation capacity of T cells was demonstrated ( Figure 8 ).

[0452] With the goal of modulating CD4+ and CD8+ T cell responses, the inventors generated OVA encoding 242-353 LVs containing the TCR transgenic OTII CD4 + T cell recognition of OVA 323-339 and TCR transgenic OTI CD8 + OVA of T cells 257-264 (SIINFEKL). BM cells were transfected with LV.IiOVA 315-353 or LV.IiOVA 242-353 Transduction and transfer of engineered DC-OVA 315-353 and DC-OVA 242-353Used to stimulate OTII and OTI cells. DC-OVA promoted the proliferation of OTI CD4+ T cells, while DC-OVA242-353 (but not DC-OVA315-353) promoted the proliferation of OTI cells ( Figure 9 These data suggest that LV-DCs can be engineered to modulate CD4 + and CD8+ T cell responses.

[0453] These results indicate that LV-mediated gene transfer of Ag fused to the invariant chain allows DCs to present and promote Ag-specific CD4 + and CD8 + Furthermore, the addition of tolerogenic elements (miRNA target sequences, IL-10, or IDO) to the LV backbone ensures the presentation of encoded Ag by DC or immature-like DC in the presence of high levels of IL-10 and IDO, favoring the generation of regulatory DC that promote Ag-specific T cell hyporesponsiveness and, in the case of DC-OVA.miRNA or DC-IL-10 / OVA, the expansion of Ag-specific Tr1 cells.

[0454] To investigate the efficacy of LV-DCs in regulating diabetic T cell responses in vitro and in vivo, LVs encoding the cytokine InsB were generated. 4-29 Fusion Li, which contains only the diabetic peptide InsB 9-23 Alternatively, the LVs were combined with miRNA155 and 146a target sequences, IL-10 or IDO, and used to transduce BM cells isolated from NOD mice (during DC differentiation). 4- 29 .miR155T.miR146aT、LV.IL-10.IiInsB 4-29 、LV.IDO.IiInsB 4-29 and LV.liInsB as a control 9-23 and LV.IiOVA 315-35 Used to obtain DC-InsB.miRNA, DC-IL-10 / InsB, DC-IDO / InsB, DC-InsB and DC-OVA. LV-DC is CD11c + and expressed MHC class II IA at levels similar to those of untransduced DCs. g7 and CD86 (not shown). When stimulated with DC-InsB, CD4 isolated from diabetic NOD mice + T cells proliferated but not when stimulated with DC-OVA ( Figure 10Similar to the results obtained with OVA, DC-IL-10 / InsB promoted the proliferation of low CD4 + In contrast, T cells stimulated with DC-InsB miRNA and DC-IDO / InsB proliferated as much as cells stimulated with DC-InsB ( Figure 10 ).

[0455] The inventors next investigated the biodistribution and survival of LV-DCs in vivo. Therefore, on day 2 of DC differentiation, BM cells isolated from Balb / c mice were transduced with LVs encoding luciferase. luc The drug was injected intravenously (iv) or intraperitoneally (ip) into Balb / c recipient mice, and the biodistribution and LV-DCs were monitored by bioluminescence imaging (BLI). luc Survival, as expected, of LV-DCs after intravenous or intraperitoneal injection luc From day 6, LV-DCs were injected intravenously. luc localized in the spleen, and from day 2, LV-DCs injected intraperitoneally luc Localized in the spleen. The injected cells gradually disappeared on the 8th to 10th day ( Figure 11 ). The efficacy of LV-DC in preventing the development of T1D was investigated by intraperitoneal injection of cells. To this end, 10-week-old NOD female mice were repeatedly injected with DC-InsB, DC-InsB miRNA, DC-IL-10 / InsB, DC-IDO / InsB, and DC-OVA. The results showed that constitutive expression of IDO by DC-IDO / InsB significantly reduced the development of T1D in NOD mice compared to control mice treated with DC-OVA (p=0.0028) or DC-InsB (p=0.0407) ( Figure 11 Administration of DC-IL-10 / InsB resulted in milder but nonsignificant disease control, whereas DC-InsB miRNA-treated NOD mice exhibited delayed T1D onset compared to DC-OVA-treated controls.

[0456] Treated mice were sacrificed 15 weeks after the last DC injection, and the frequency of Tregs in spleen and pancreatic lymph nodes was analyzed. + LAG-3 + Tr1 cells or CD25 + Foxp3 + Specific induction of Tregs ( Figure 12 ), and CD4 isolated from LV-DC treated mice + T cells against InsB 9-23The high variability of the proliferative response of LV-DCs was not related to the injected LV-DC subtype (not shown).

[0457] In summary, the inventors developed an efficient and powerful method to generate stable and potent tolerogenic DCs by cutting-edge technology based on LVs encoding specific self-Ags and tolerogenic molecules.

[0458] To translate the LV-based approach to human cells, the inventors first developed an efficient protocol to facilitate bdLV-mediated transduction of human DCs. To this end, CD14 + Cells were pretreated with or without virus-like particles containing the simian immunodeficiency virus (SIV)-derived accessory protein Vpx VPL, which counteracted SAMHD1-mediated restriction. Figure 13 Pretreatment with Vpx-VPL improved transduction efficiency at all time points analyzed, with higher efficiencies achieved when cells were pretreated with Vpx-VPL on day 0 ( Figure 13 Importantly, Vpx-VPL pretreatment at day 0 did not affect the activation of cells generated by the end of culture ( Figure 14 Time course analysis showed that one hour of exposure to Vpx-VPL was sufficient to achieve 95% transduction efficiency ( Figure 38 ).

[0459] Utilizing established protocols to generate engineered human LV-DCs, the inventors first investigated the use of LV co-encoding IL-10 and ΔNGFR to generate DCs. IL-10 The ability of the ΔNGFR was previously used to generate Tr1-like (CD4 IL-10 ) cells ((40, 85) WO2016146542). CD14 + Cells were treated with Vpx-VPL for 6-8 hours and then with LV-IL-10 / ΔNGFR (DC) on day 0 of DC differentiation. IL-10 ) or LV-GFP / ΔNGFR(DC GFP As a control, the inventors used GM-CSF and IL-4 or GM-CSF, IL-4 and IL-10 to culture CD14 + The cells were derived from DC-10 cells differentiated from the same donor and non-transduced DC (DC UT Both vectors efficiently transduced human DCs, achieving up to 98% transduction, as shown by ΔNGFR expression ( Figure 15 Left panel). Analysis of the expression of DC-10 related markers showed that DC IL-10 express CD14, CD16, CD141, CD163, ILT4, and HLA-G, while DC GFPControl DC UT Not expressed ( Figure 15 Compared to DC-10, DC IL-10 Secrete significantly higher levels of IL-10 at steady state and upon activation. Importantly, similar to DC-10, DC IL-10 After activation, IL-12 is not produced ( Figure 16 ). Similar to DC-10, DC IL-10 In allogeneic T cells (CD4 + and CD8 + T cells) promotes hyporesponsiveness ( Figure 17 ). The inventors then compared DC IL-10 Compared to the ability of DC-10 to promote the non-reactive allogeneic specific Tr1 cells. IL-10 Stimulate allogeneic CD4 + T cells for 10 days, or DC GFP 、DC UT and DC-10 as controls. UT Primed T cells (T-DCs UT ) or use DC GFP Primed T cells (T-DCs GFP ), use DC IL-10 CD4 for primary immunization + T cells (T-DCs IL-10 ), similar to cells activated with DC-10 (T-DC-10), restimulated with the same self-Ag used for their priming, were anergic ( Figure 18 ). In addition, compared to T-DC UT and T-DC GFP cells, T-DC IL-10 and T-DC-10 cells contained a significantly higher proportion of Tr1 cells ( Figure 19 , left). Compared with T-DC-10, T-DC UT and T-DC GFP T-DCs, when restimulated with the same self-Ag used for their priming IL-10 cells secreted significantly higher levels of IL-10 ( Figure 19 , right).

[0460] Together, these findings indicate that LV-mediated IL-10 gene transfer transforms human DCs into DC-10-like cells, conferring the ability to regulate allogeneic T cells and promote the differentiation of anergic allospecific Tr1 cells.

[0461] To study DC IL-10To investigate the ability of murine DCs to prevent graft-versus-host disease (GvHD), we generated murine DCs by transducing BM cells isolated from Balb / c mice with LV-IL-10 / ΔNGFR during DC differentiation. IL-10 As a control, BM cells (DCs) transduced with LV-GFP / ΔNGFR were generated. GFP ). Then, mouse DC IL-10 and DC GFP Adoptive transfer into Balb / c mice that were lethally irradiated and injected with allogeneic (C57Bl / 6) BM cells and splenocytes. GFP Treated mice developed lethal GvHD, whereas a single injection of DC IL-10 Significantly delayed GvHD ( Figure 20 ).

[0462] To generate Ag-specific human LV-DCs, the inventors designed LV constructs encoding the human CLIP sequence (invariant chain p33 binding domain of class II MHC molecules) fused to a self-Ag-derived peptide. 4-29 Sequence fusion Iip33 (LV.InsB 4-29 ) or fused with α2-gliadin 51-80 lip33 (LV.Glia 51-80 ). Monocytes were transduced into DCs using the optimized protocol with LV, which predicted that CD14 cells were pretreated with Vpx-VPL in serum-free medium. + Precursor ( Figure 21 After differentiation, DC differentiation was monitored by the expression of DC-SIGN. Figure 22 As shown, human LV-transduced cells are DC-SIGN + , and in the case of cells transduced with LV-IL-10 / Ag (DC-IL-10 / Ag), the resulting DCs also expressed CD14. In the case of control LV-ΔNGFR / Ag transduced cells, the transduction efficiency was assessed by ΔNGFR expression. In the case of cells transduced with LV-IL-10 / Ag, the transduction efficiency was monitored by intracytoplasmic staining for IL-10. Specifically, DC-IL-10 / Ag and cells transduced with LV-CLIP (DC-IL-10 / Ag) showed a significant difference in transduction efficiency. CLIP DCs transduced with LV-ΔNGFR / Ag (DC-Ag) or LV-ΔNGFR / Ag (DC-Ag) (as a control) were left unstimulated or activated with LPS / IFN-g for 24 hours and stained for IL-10. The results showed that DC-IL-10 / Ag expressed IL-10 at steady state and after activation, but only 4-10% of DCs expressed IL-10 after stimulation. CLIP and DC-Ag expressing IL-10 ( Figure 23In the case of LV-IDO / Ag transduced cells, transduction efficiency was monitored by IDO expression. Figure 24 As shown, DC-IDO / Ag expressed IDO, while DC CLIP and DC-Ag barely expressed IDO.

[0463] Similar to DC IL-10 , DC-IL-10 / Ag expressed high levels of DC-10-related markers, including CD14, CD141, CD163, and ILT4, but did not express HLA-G ( Figure 25 ). In contrast, DC-IDO / Ag and DC UT 、DC CLIP The cytokine production profile of DC-IL-10 / Ag showed that it was similar to DC IL-10 These cells secrete significantly higher levels of IL-10 and lower levels of IL-12 at steady state or after LPS / IFN-g stimulation. Figure 26 and Figure 27 ). DC-IDO / Ag showed the same UT 、DC CLIP The cytokine profile was super-imposable to that of DC-Ag (not shown).

[0464] Functional characterization of DC-IL-10 / Ag revealed that concomitant overexpression of IL-10 downregulated the proliferation of Ag-specific T cells in association with DC-Ag, which continuously induced Ag-specific proliferative responses in HLA-restricted T cells. Figure 28 Importantly, stimulation of autologous T cells with DC-IL-10 / Ag for 10 days promoted the induction of Ag-specific anergic T cells containing a high frequency of CD49b+LAG-3+Tr1 cells ( Figure 29 DC-IDO / Ag promoted the proliferation of autologous T cells, similar to the proliferation induced by control DC-Ag ( Figure 30 ), and stimulation of autologous T cells with DC-IDO / Ag for 10 days promoted the induction of a cell population containing a high proportion of FOXP3+CTLA4+ cells ( Figure 31 ).

[0465] Together, these data demonstrate that DCs engineered with LVs encoding the invariant chain (Ii) fused to specific Ags coupled to multiple target sequences of miR155 and miR146a, known regulators of DC maturation (DC-Ag.miRNA), or to IL-10 (DC-IL-10 / Ag) or IDO (DC-IDO / Ag) generate tolerogenic DC populations capable of modulating Ag-specific T cell responses and promoting the cellular differentiation of Ag-specific Tr1 cells or FOXP3+ T cells in vitro and in vivo.

[0466] Based on the above evidence, exemplary methods can be used to obtain strong suppression of T effector cells and / or strong activation of T regulatory cells.

[0467] As a DC similar to the DC-10 IL-10 We stimulated allogeneic CD4 + T cells for 10 days to study their ability to promote allogeneic specific Tr1 cells in vitro. In all tested donors, compared with DC UT and DC GFP Primary T cells (respectively [T(DC UT ) and T(DC GFP ) cells]), using DC IL-10 Primary immunization CD4 + T cells ([T(DC IL-10 ) cells]) contain a higher proportion of Tr1 cells ( Figure 19 T cells (DCs) were restimulated with mature DCs (mDCs) that were autologous to the DCs used for priming. IL-10 ) cells, compared to (T-DC UT ) and (T-DC GFP ) cells proliferate at a lower level ( Figure 32 ), compared with (T-DC UT ) and (T-DC GFP ) cells produced significantly higher levels of IL-10, but similar levels of IFN-γ. Finally, using mDCs from the same donor as the DCs used for priming, T(DCs IL-10 ) cells suppress autologous CD4 + T cell proliferation was inhibited by an average of 67% ( Figure 33 Taken together, these findings suggest that LV-mediated IL-10 gene transfer in human DCs promotes IL-10 The production of TNF-α induced by TNF-α induced the formation of TNF-α, which confers the ability to regulate allogeneic T cell responses and promote the differentiation of allogeneic specific Tr1 cells in vitro.

[0468] To evaluate DC IL-10In vivo regulatory activity, the inventors utilized the recently developed human umbilical cord blood CD34 + Intrahepatic injection of human CD34 cells into sublethally irradiated neonatal NSG mice + Cell-permissive human CD45 + T cells efficiently engraft into the bone marrow (BM) and differentiate into lymphoid (B, T effector, and T regulatory) and myeloid mature cells in the periphery (91). Mice reconstituted with huNSC were immunized by intravenous injection of irradiated allogeneic human APCs and then immunized with autologous DCs only. UT Or use DC IL-10 (DC UT +DC IL10 ) or DC GFP (DC UT +DC GFP ) Enhance immunity for 7 days ( Figure 34 ). Use DC IL-10 Allogeneic DC UT Induced CD4 + In vivo proliferation of T cells was assessed by Ki67 staining ( Figure 34 These data indicate that human DC IL-1 Modulates allogeneic T cell responses in vivo.

[0469] One of the key aspects of DC-based cell products is their stability (i.e., maintenance of Tr1 cell induction and stimulatory activity, expression of specific markers, and secretion of cytokines after activation). Therefore, the inventors evaluated the stability of DCs after in vitro stimulation with different TLR agonists (i.e., LPS, Listeria, flagellin, Poli I:C, and CpG) or a mixture of proinflammatory cytokines (IL-1b, TNF-a, and IL-6). IL-10 Similar to the data obtained previously in DC-10 (92), after activation, DC IL-10 Stably express CD163 and CD141 ( Figure 35 In contrast, CD16 expression was affected by activation with LPS or Listeria ( Figure 35 ). Compared with unstimulated DC IL-10 Activated DC IL-10 No significant changes in CD14 and CD1a expression were observed ( Figure 35 ). DCs stimulated with LPS, Listeria, and CpG IL-10 After 4 h, the expression of CD86 was significantly upregulated, while no effect was observed on the expression of CD83 and HLA-DR ( Figure 35 ). Although DC IL-10HLA-G expression in WT cells remained stable after activation (not shown), but ILT4 expression was significantly increased or decreased after stimulation with LPS and Listeria or Poli I:C and CpG, respectively ( Figure 36 Since ILT4 plays an important role in DC-10-mediated Tr1 cell induction (22), the inventors selected LPS and Poli I:C to stimulate DC IL-10 The tolerogenic activity of activated DCs was studied in vitro. IL-10 In steady state and after LPS / IFNg stimulation, DCs secrete a large amount of IL-10, but no IL-12. IL-10 The percentage of induced Tr1 cells in culture was lower, but still higher than that of DCs. GFP Culture ( Figure 36 When restimulated with mDCs autologous to the DCs used for priming, T(stim-DC IL-10 ) were nonallergic, even compared to T(unstim-DC IL-10 ), its anergy was not obvious. Similarly, compared with T(unstim-DC IL-10 ), T(stim-DC IL-10 ) had a lower level of IL-10 production, but compared with T(DC GFP ), whose level is higher ( Figure 37 Ignoring the observed differences in Tr1 marker expression, anergy, and cytokine production, T(stim-DC IL-10 ) and T(unstim-DC IL-10 )quite( Figure 37 In conclusion, we conclude that by activating DC IL-10 induced Tr1 cells and unstimulated DCs IL-10 TLR stimulation does not alter DC IL-10 tolerance capacity.

[0470] The interaction of CD47 on LV particles with its ligand Sirp-α on target cells impairs transduction efficiency by reducing LV particle uptake via phagocytosis. Therefore, the inventors tested whether the expression of CD47 on LV particles alters the transduction efficiency of human DCs. To this end, they performed transduction of DC precursors using LV particles with different levels of human CD47 (huCD47) on their surface (high huCD47 LV > LV > no HuCD47 LV). Interestingly, LV-mediated transduction of DC precursors expressing Sirp-α was significantly increased when using LV particles without huCD47 ( Figure 39 ).

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[0564] 93.Sato SJ Immunol.1997;159:3278-3287 Sequence Listing <110> Ospedale San Raffaele SRL Telethon Foundation San Raffaele Foundation Center <120> Generation and use of engineered cells <130> PCT 140607 <150> EP18178595 <151> 2018-06-19 <160> 164 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 1 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 2 Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Glu Gly 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 3 Ser His Leu Val Glu Glu Leu Tyr Val Leu Val Cys Gly Glu Glu Gly 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 4 Gly Gln Val Glu Leu Gly Gly Gly Asn Ala Val Glu Val Leu Lys 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 5 Leu Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln 1 5 10 15 <210> 6 <211> 18 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 6 Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly 1 5 10 15 Ile Val <210> 7 <211> 8 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 7 Trp Gly Pro Asp Pro Ala Ala Ala 1 5 <210> 8 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 8 Ser His Leu Val Glu Ala Leu Val Leu Val Cys Gly Glu Arg Gly 1 5 10 15 <210> 9 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 9 Val Met Asn Ile Leu Leu Gln Tyr Val Val 1 5 10 <210> 10 <211> 18 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 10 Thr Ala Gly Thr Thr Val Tyr Gly Ala Phe Asp Pro Leu Leu Ala Val 1 5 10 15 Ala Asp <210> 11 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 11 Val Asn Phe Phe Arg Met Val Ile Ser Asn Pro Ala Ala Thr His Gln 1 5 10 15 Asp Ile Asp Phe Leu Ile 20 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 12 Val Ile Val Met Leu Thr Pro Leu Val 1 5 <210> 13 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 13 Ser Phe Tyr Leu Lys Asn Val Gln Thr Gln Glu Thr Arg Thr Leu Thr 1 5 10 15 Gln Phe His Phe 20 <210> 14 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 14 Gln His Leu Gln Lys Asp Tyr Arg Ala Tyr Tyr Thr Phe 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 15 Tyr Thr Phe Leu Asn Phe Met Ser Asn Val Gly Asp Pro 1 5 10 <210> 16 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 16 Arg Val Leu Asn Ile Asp Leu Leu Trp Ser Val Pro Ile 1 5 10 <210> 17 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 17 Asp Trp Ile His Ile Asp Thr Thr Pro Phe Ala Gly Leu 1 5 10 <210> 18 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 18 Trp Ser Lys Met Asp Gln Leu Ala Lys Glu Leu Thr Ala Glu 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 19 Val Ala Ala Asn Ile Val Leu Thr Val 1 5 <210> 20 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 20 Met Glu Phe Leu Glu Arg Thr Tyr Leu Val Asn Asp Lys Ala Ala Lys 1 5 10 15 Met His Ala Phe 20 <210> twenty one <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty one Tyr Leu Val Asn Asp Lys Ala Ala Lys Met His Ala Phe Thr Leu Glu 1 5 10 15 Ser Val Glu Leu 20 <210> twenty two <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty two Ser Leu Trp Leu Ser Ser Lys Pro Pro Ser Lys Arg Leu Thr Phe Gly 1 5 10 15 Trp His Arg Ala 20 <210> twenty three <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty three Pro Pro Ser Lys Arg Leu Thr Phe Gly Trp His Arg Ala Glu Ile Leu 1 5 10 15 Gly Ala Leu Leu 20 <210> twenty four <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> twenty four Phe Ile Phe Ser Ile Leu Val Leu Ala Ser Thr Ile Thr Ile Leu Lys 1 5 10 15 Asp Phe Ser Ile 20 <210> 25 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 25 Leu Lys Asp Phe Ser Ile Leu Leu Met Glu Gly Val Pro Lys Ser Leu 1 5 10 15 Asn Tyr Ser Gly 20 <210> 26 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 26 Ser Leu Asn Tyr Ser Gly Val Lys Glu Leu Ile Leu Ala Val Asp Gly 1 5 10 15 Val Leu Ser Val 20 <210> 27 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 27 Phe Gly Trp His Arg Ala Glu Ile Leu Gly Ala Leu Leu Ser Ile Leu 1 5 10 15 Cys Ile Trp Val 20 <210> 28 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 28 Thr Met Asn Gln Val Ile Leu Ser Ala His Val Ala Thr Ala Ala Ser 1 5 10 15 Arg Asp Ser Gln 20 <210> 29 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 29 Lys Phe Gly Ala Asp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu 1 5 10 15 Leu Ala Asp Ala 20 <210> 30 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 30 Asn Pro Val Glu Ile Arg Arg Gly Val Met Leu Ala Val Asp Ala Val 1 5 10 15 Ile Ala Glu Leu 20 <210> 31 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 31 Gln Ser Ile Val Pro Ala Leu Glu Ile Ala Asn Ala His Arg Lys Pro 1 5 10 15 Leu Val Ile Ile Ala 20 <210> 32 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 32 Leu Val Leu Asn Arg Leu Lys Val Gly Leu Gln Val Val Ala Val Lys 1 5 10 15 Ala Pro Gly Phe 20 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 33 Ile Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys Ile Pro Ala Leu 1 5 10 15 Asp Ser Leu Thr 20 <210> 34 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 34 Val Asn Met Val Glu Lys Gly Ile Ile Asp Pro Thr Lys Val Val Arg 1 5 10 15 Thr Ala Leu Leu 20 <210> 35 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 35 Ser Pro Ser Leu Trp Glu Ile Glu Phe Ala Lys Gln Leu Ala Ser Val 1 5 10 15 <210> 36 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 36 Gln Asp Phe Thr Asn Arg Ile Asn Lys Leu Lys Asn Ser 1 5 10 <210> 37 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> MOD_RES <222> (6) <223> x is citrulline <400> 37 Gln Asp Phe Thr Asn Xaa Ile Asn Lys Leu Lys Asn Ser 1 5 10 <210> 38 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 38 Val Val Leu Leu Val Ala Thr Glu Gly Arg Val Arg Val Asn Ser Ala 1 5 10 15 Tyr Gln Asp Lys 20 <210> 39 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <220> <221> MOD_RES <222> (10)..(10) <223> x is citrulline <400> 39 Val Val Leu Leu Val Ala Thr Glu Gly Xaa Val Arg Val Asn Ser Ala 1 5 10 15 Tyr Gln Asp Lys 20 <210> 40 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 40 Ser Ala Val Arg Ala Arg Ser Ser Val Pro Gly Val Arg 1 5 10 <210> 41 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 41 Ser Ala Val Arg Ala Cys Ile Thr Ser Ser Val Pro Gly Val Arg 1 5 10 15 <210> 42 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 42 Gln Tyr Met Arg Ala Asp Gln Ala Ala Gly Gly Leu Arg 1 5 10 <210> 43 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 43 Gln Tyr Met Cys Ile Thr Ala Asp Gln Ala Ala Gly Gly Leu Arg 1 5 10 15 <210> 44 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 44 Ala Gly Phe Lys Gly Glu Gln Gly Pro Lys Gly Glu Pro 1 5 10 <210> 45 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 45 Ala Gly Phe Lys Gly Gly Gly Glu Gln Gly Pro Lys Gly Glu Pro 1 5 10 15 <210> 46 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 46 Met Glu Val Gly Trp Tyr Arg Pro Pro Phe Ser Arg Val Val His Leu 1 5 10 15 Tyr Arg Asn Gly Lys 20 <210> 47 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 47 His Cys Leu Gly Lys Trp Leu Gly His Pro Asp Lys Phe 1 5 10 <210> 48 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 48 Glu Asn Pro Val Val His Phe Phe Lys Asn Ile Val Thr Pro Arg 1 5 10 15 <210> 49 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 49 Lys Tyr Leu Ala Thr Ala Ser Thr Met Asp His Ala Arg His Gly Phe 1 5 10 15 Leu Pro Arg His 20 <210> 50 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 50 Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe Gly 1 5 10 15 Tyr Gly Gly <210> 51 <211> 25 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 51 Ala Gln Gly Thr Leu Ser Lys Ile Phe Lys Leu Gly Gly Arg Asp Ser 1 5 10 15 Arg Ser Gly Ser Pro Met Ala Arg Arg 20 25 <210> 52 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 52 Tyr Leu Ser Gly Ala Asp Leu Asn Leu 1 5 <210> 53 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 53 Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro 1 5 10 <210> 54 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 54 Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Gln 1 5 10 15 Pro <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 55 Gln Leu Ile Pro Cys Met Asp Val Val Leu 1 5 10 <210> 56 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 56 Tyr Leu Gln Leu Gln Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro 1 5 10 15 <210> 57 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 57 Pro Phe Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro 1 5 10 15 <210> 58 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 58 Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln 1 5 10 15 <210> 59 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 59 Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln 1 5 10 15 <210> 60 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 60 Tyr Pro Gln Pro Gln Leu Pro Tyr Pro Gln Pro Gln Pro Phe Arg 1 5 10 15 <210> 61 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 61 Tyr Pro Ser Gly Gln Gly Ser Phe Gln Pro Ser Gln Gln Asn Pro 1 5 10 15 <210> 62 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 62 Gly Ser Phe Gln Pro Ser Gln Gln Asn Pro Gln Ala Gln Gly Ser 1 5 10 15 <210> 63 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 63 Gln Ala Gln Gly Ser Val Gln Pro Gln Gln Leu Pro Gln Phe Glu 1 5 10 15 <210> 64 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 64 Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu Pro Tyr 1 5 10 <210> 65 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 65 Pro Gln Pro Glu Leu Pro Tyr Pro Gln Pro Glu 1 5 10 <210> 66 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 66 Gln Gln Pro Phe Pro Gln Pro Glu Gln Pro Phe Pro 1 5 10 <210> 67 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 67 Phe Pro Gln Pro Glu Gln Pro Phe Pro Trp Gln Pro 1 5 10 <210> 68 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 68 Gln Gly Ile Ile Gln Pro Glu Gln Pro Ala Gln Leu 1 5 10 <210> 69 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 69 Ser Gly Glu Gly Ser Phe Gln Pro Ser Gln Glu Asn Pro Gln 1 5 10 <210> 70 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 70 Phe Pro Glu Gln Pro Glu Gln Pro Tyr Pro Glu Gln 1 5 10 <210> 71 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 71 Lys Val Ser Phe Phe Cys Lys Asn Lys Glu Lys Lys Cys Ser Tyr 1 5 10 15 <210> 72 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 72 Val Pro Val Lys Lys Ala Thr Val Val Tyr Gln Gly Glu Arg Val 1 5 10 15 <210> 73 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 73 Ser Cys Lys Leu Val Pro Val Lys Lys Ala Thr Val Val Tyr Gln Gly 1 5 10 15 Glu Arg Val Lys Ile Gln 20 <210> 74 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 74 Met Ile Ser Pro Val Leu Ile Leu Phe Ser Ser Phe Leu Cys His Val 1 5 10 15 Ile Ala Gly <210> 75 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 75 Gln Ala Thr Gln Lys Ile Thr Tyr Arg Ile Ser Gly Val Gly Ile Asp 1 5 10 15 Gln <210> 76 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 76 Pro Phe Gly Ile Phe Val Val Asp Lys Asn Thr Gly Asp Ile Asn Ile 1 5 10 15 Thr <210> 77 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 77 His Leu Asn Ser Lys Ile Ala Phe Lys Ile Val Ser Gln Glu Pro Ala 1 5 10 15 Gly <210> 78 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 78 Gly Thr Pro Met Phe Leu Leu Ser Arg Asn Thr Gly Glu Val Arg Thr 1 5 10 15 Leu <210> 79 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 79 Gln Cys Glu Cys Asn Ile Lys Val Lys Asp Val Asn Asp Asn Phe Pro 1 5 10 15 Met <210> 80 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 80 Ser Val Lys Leu Ser Ile Ala Val Lys Asn Lys Ala Glu Phe His Gln 1 5 10 15 Ser <210> 81 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 81 Asn Val Arg Glu Gly Ile Ala Phe Arg Pro Ala Ser Lys Thr Phe Thr 1 5 10 15 Val <210> 82 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 82 Ile Tyr Val Asn Val Glu Pro Thr Phe Gln Arg Thr Leu His Lys Thr 1 5 10 15 Lys <210> 83 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 83 Gly Glu Ile Arg Thr Met Asn Asn Phe Leu Asp Arg Glu Ile Tyr Val 1 5 10 15 Asn Val Glu Pro 20 <210> 84 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 84 Met Asn Asn Phe Leu Asp Arg Glu Ile Tyr Asn Val Glu Pro Thr Phe 1 5 10 15 Gln Arg Thr <210> 85 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 85 Asp Arg Glu Ile Tyr Val Asn Val Glu Pro Thr Phe Gln Arg Thr Leu 1 5 10 15 His Lys Thr Lys 20 <210> 86 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 86 Thr Leu Thr Leu Leu Pro Leu Leu Ala Asn Asn Arg Glu Arg Arg Gly 1 5 10 15 Ile Ala Leu Asp 20 <210> 87 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 87 Asn Arg Glu Arg Arg Gly Ile Ala Leu Asp Gly Lys Ile Lys His Glu 1 5 10 15 Asp Thr Leu <210> 88 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 88 Leu Leu Ala Asn Asn Arg Glu Arg Arg Gly Ile Ala Leu Asp Gly Lys 1 5 10 15 Ile Lys His Glu 20 <210> 89 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 89 Ala Ser Ser Thr Ile Ile Lys Glu Gly Ile Asp Arg Thr Val Leu Gly 1 5 10 15 Ile Leu Val Ser 20 <210> 90 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 90 Tyr Gln Ile Lys Val Lys Leu Thr Val Ser Gly Phe Gly Glu Leu Thr 1 5 10 15 Ser Ser Glu <210> 91 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 91 Met Ala Ala Ser Gly Lys Thr Ser Lys Ser Glu Pro Asn His Val Ile 1 5 10 15 Phe Lys Lys <210> 92 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 92 Gln Val Gln Pro Val Asp Gly Val Val Leu Val Asp Pro Asp Leu Val 1 5 10 15 Lys Gly Lys Lys 20 <210> 93 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 93 Val Tyr Val Thr Leu Thr Cys Ala Phe Arg Tyr Gly Gln Glu Asp Val 1 5 10 15 Asp Val Ile Gly 20 <210> 94 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 94 Leu Thr Phe Arg Arg Asp Leu Tyr Phe Ser Arg Val Gln Val Tyr Pro 1 5 10 15 Pro Val Gly Ala 20 <210> 95 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 95 Pro Phe Leu Leu Thr Phe Pro Asp Tyr Leu Pro Cys Ser Val Met Leu 1 5 10 15 Gln Pro Ala Pro 20 <210> 96 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 96 Gln Asp Ser Gly Lys Ser Cys Gly Val Asp Phe Glu Val Lys Ala Phe 1 5 10 15 Ala Thr Asp Ser 20 <210> 97 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 97 Thr Asp Ala Glu Glu Asp Lys Ile Pro Lys Lys Ser Ser Val Arg Tyr 1 5 10 15 Leu Ile Arg Ser 20 <210> 98 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 98 Phe Met Ser Asp Lys Pro Leu His Leu Ala Val Ser Leu Asn Arg Glu 1 5 10 15 Ile Tyr Phe His 20 <210> 99 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 99 Gly Glu Pro Ile Pro Val Thr Val Thr Val Thr Asn Asn Thr Glu Lys 1 5 10 15 Thr Val Lys Lys 20 <210> 100 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 100 Ile Lys Ala Cys Val Glu Gln Val Ala Asn Val Val Leu Tyr Ser Ser 1 5 10 15 Asp Tyr Tyr Val 20 <210> 101 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 101 Gly Lys Ile Lys His Glu Asp Thr Asn Leu Ala Ser Ser Thr Ile Ile 1 5 10 15 Lys Glu Gly Ile 20 <210> 102 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 102 Gly Glu Leu Thr Ser Ser Glu Val Ala Thr Glu Val Pro 1 5 10 <210> 103 <211> 11 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 103 Leu Thr Ser Ser Glu Val Ala Thr Glu Val Pro 1 5 10 <210> 104 <211> 38 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 104 Phe Lys Asp Tyr Ser Ser Val Val Arg Pro Val Glu Asp His Arg Gln 1 5 10 15 Val Val Glu Val Thr Val Gly Leu Gln Leu Ile Gln Leu Ile Asn Val 20 25 30 Asp Glu Val Asn Gln Ile 35 <210> 105 <211> 17 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 105 Leu Gly Thr Trp Thr Tyr Asp Gly Ser Val Val Ala Ile Asn Pro Glu 1 5 10 15 Ser <210> 106 <211> 41 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 106 Val Lys Lys Ile His Ile Pro Ser Glu Lys Ile Trp Arg Pro Asp Leu 1 5 10 15 Val Leu Tyr Asn Asn Ala Asp Gly Asp Phe Ala Ile Val Lys Phe Thr 20 25 30 Lys Val Leu Leu Gln Tyr Thr Gly His 35 40 <210> 107 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 107 Ile His Ile Pro Ser Glu Lys Ile Trp Arg Pro Asp Leu Val Leu Tyr 1 5 10 15 <210> 108 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 108 Leu Gly Thr Trp Thr Tyr Tyr Asp Gly Val Val Ala Ile Asn Pro Glu Ser 1 5 10 15 <210> 109 <211> 18 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 109 Asp Pro Thr Tyr Leu Asp Ile Thr Tyr His Phe Val Met Gln Arg Leu 1 5 10 15 Pro Leu <210> 110 <211> 18 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 110 Asp Thr Pro Tyr Leu Asp Ile Thr Tyr His Phe Val Met Gln Arg Leu 1 5 10 15 Pro Leu <210> 111 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 111 Val Ile Val Glu Leu Ile Pro Ser Thr Ser Ser Ala Val 1 5 10 <210> 112 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 112 Lys Ser Tyr Cys Glu Ile Ile Val Thr His Phe Pro Phe Asp Glu Gln 1 5 10 15 Asn Cys Ser Met Lys Leu Gly 20 <210> 113 <211> 20 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 113 Pro Arg Trp Asn Gly Glu Lys Leu Tyr Gln Glu Ala Arg Lys Ile Val 1 5 10 15 Gly Ala Met Val 20 <210> 114 <211> 32 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 114 Asp Leu Gly Trp Gly Val Val Gly Thr His Ala Ala Pro Ala His Gly 1 5 10 15 Gln Ala Leu Gly Ala Val Gly His Trp Leu Val Leu Leu Trp Gln Leu 20 25 30 <210> 115 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 115 ctactgaagt atacgtaaag tgg 23 <210> 116 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 116 cttgtttaga gctccatcaa agg 23 <210> 117 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 117 atcgagctaa aatatcgtgt tgg 23 <210> 118 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 118 ttcctttcca ggatcagctc agc 23 <210> 119 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 119 ttgattcaaa ggagtaccta tccc 24 <210> 120 <211> 645 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 120 atggatgacc aacgcgacct catctctaac catgaacagt tgcccatact gggcaaccgc 60 cctagagagc cagaaaggtg cagccgtgga gctctgtaca ccggtgtctc tgtcctggtg 120 gctctgctct tggctgggca ggccaccact gcttacttcc tgtaccagca acagggccgc 180 ctagacaagc tgaccatcac ctcccagaac ctgcaactgg agagccttcg catgaagctt 240 ccgaaatctg ccaaacctgt gagccagatg cggatggcta ctcccttgct gatgcgtcca 300 atgtccatgg ataacatgct ccttgggcct gtgaagaacg ttaccaagta cggcaacatg 360 acccaggacc atgtgatgca tctgctcacg aggtctggac ccctggagta cccgcagctg 420 aaggggacct tcccagagaa tctgaagcat cttaagaact ccatggatgg cgtgaactgg 480 aagatcttcg agagctggat gaagcagtgg ctcttgtttg agatgagcaa gaactccctg 540 gaggagaaga agcccaccga ggctccacct aaagagccac tggacatgga agacctatct 600 tctggcctgg gagtgaccag gcaggaactg ggtcaagtca ccctg 645 <210> 121 <211> 215 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 121 Met Asp Asp Gln Arg Asp Leu Ile Ser Asn His Glu Gln Leu Pro Ile 1 5 10 15 Leu Gly Asn Arg Pro Arg Glu Pro Glu Arg Cys Ser Arg Gly Ala Leu 20 25 30 Tyr Thr Gly Val Ser Val Leu Val Ala Leu Leu Leu Ala Gly Gln Ala 35 40 45 Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys Leu 50 55 60 Thr Ile Thr Ser Gln Asn Leu Gln Leu Glu Ser Leu Arg Met Lys Leu 65 70 75 80 Pro Lys Ser Ala Lys Pro Val Ser Gln Met Arg Met Ala Thr Pro Leu 85 90 95 Leu Met Arg Pro Met Ser Met Asp Asn Met Leu Leu Gly Pro Val Lys 100 105 110 Asn Val Thr Lys Tyr Gly Asn Met Thr Gln Asp His Val Met His Leu 115 120 125 Leu Thr Arg Ser Gly Pro Leu Glu Tyr Pro Gln Leu Lys Gly Thr Phe 130 135 140 Pro Glu Asn Leu Lys His Leu Lys Asn Ser Met Asp Gly Val Asn Trp 145 150 155 160 Lys Ile Phe Glu Ser Trp Met Lys Gln Trp Leu Leu Phe Glu Met Ser 165 170 175 Lys Asn Ser Leu Glu Glu Lys Lys Pro Thr Glu Ala Pro Pro Lys Glu 180 185 190 Pro Leu Asp Met Glu Asp Leu Ser Ser Gly Leu Gly Val Thr Arg Gln 195 200 205 Glu Leu Gly Gln Val Thr Leu 210 215 <210> 122 <211> 126 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 122 tgtggcatct cctcagcaga gagcctgaag atatctcaag ctgtccatgc agcacatgca 60 gaaatcaatg aagcaggcag agaggtggta gggtcagcag aggctggagt ggatgctgca 120 agctga 126 <210> 123 <211> 41 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 123 Cys Gly Ile Ser Ser Ala Glu Ser Leu Lys Ile Ser Gln Ala Val His 1 5 10 15 Ala Ala His Ala Glu Ile Asn Glu Ala Gly Arg Glu Val Val Gly Ser 20 25 30 Ala Glu Ala Gly Val Asp Ala Ala Ser 35 40 <210> 124 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 124 tgcatgttgg tgctgttgcc tgatgaagtc tcaggccttg agcagcttga gagtataatc 60 aactttgaaa aactgactga atggaccagt tctaacgtta tggaagagag gaagatcaaa 120 gtgtacttac ctcgcatgaa gatggaggaa aaatacaacc tcacatctgt cttaatggct 180 atgggcatta ctgacgtgtt tagctcttca gccaatctgt ctggcatctc ctcagcagag 240 agcctgaaga tatctcaagc tgtccatgca gcacatgcag aaatcaatga agcaggcaga 300 gaggtggtag ggtcagcaga ggctggagtg gatgctgcca gctga 345 <210> 125 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 125 Cys Met Leu Val Leu Leu Pro Asp Glu Val Ser Gly Leu Glu Gln Leu 1 5 10 15 Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp Thr Ser Ser Asn 20 25 30 Val Met Glu Glu Arg Lys Ile Lys Val Tyr Leu Pro Arg Met Lys Met 35 40 45 Glu Glu Lys Tyr Asn Leu Thr Ser Val Leu Met Ala Met Gly Ile Thr 50 55 60 Asp Val Phe Ser Ser Ser Ala Asn Leu Ser Gly Ile Ser Ser Ala Glu 65 70 75 80 Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 85 90 95 Glu Ala Gly Arg Glu Val Val Gly Ser Ala Glu Ala Gly Val Asp Ala 100 105 110 Ala Ser <210> 126 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 126 cagcaccttt gtggttccca cctggtggag gctctctacc tggtgtgtgg ggagcgtggc 60 ttcttctaca cacccatgta a 81 <210> 127 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 127 Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys 1 5 10 15 Gly Glu Arg Gly Phe Phe Tyr Thr Pro Met 20 25 <210> 128 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 128 cagcaccttt gtggttccca cctggtggag gctctctacc tggtgtgtgg ggagcgtggc 60 ttcttctaca cacccatgta a 81 <210> 129 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic<00|03116><400> 129 Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys 1 5 10 15 Gly Glu Glu Gly Phe Phe Tyr Thr Pro Met<00|03120>20 25 <210> 130 <211> 256<00031|23><212> DNA <213> Artificial sequence <220> <2|23> Synthetic <400> 130 cacacaaatg tctgcttctg gtttgtacct cctagtttgc gcactctgga agacaatgaa 60 gagagaatga gccgcctctc aaaggtggcg ccagtgatta aagccagaat gatggagtat 120 gggaccacaa tggtcagcta ccaaccctta ggggacaagg tcaacttctt ccgcatggtc |80 Note: There seems to be some incorrect formatting in the original text like "00|03116", "00|03120", "00031|23", "2|23" which are left as-is in the translation as per the instruction to preserve all 7 - digit tags exactly. It's possible there are errors in the original text formatting.atctcaaacc ctgcagcaac tcaccaagac attgacttcc ttattgaaga aatcgaacgc 240 ctcggacaag atttgt 256 <210> 131 <211> 85 <212> PRT <213> Synthetic sequence <220> <223> Synthetic <400> 131 His Thr Asn Val Cys Phe Trp Phe Val Pro Pro Ser Leu Arg Thr Leu 1 5 10 15 Glu Asp Asn Glu Glu Arg Met Ser Arg Leu Ser Lys Val Ala Pro Val 20 25 30 Ile Lys Ala Arg Met Met Glu Tyr Gly Thr Thr Met Val Ser Tyr Gln 35 40 45 Pro Leu Gly Asp Lys Val Asn Phe Phe Arg Met Val Ile Ser Asn Pro 50 55 60 Ala Ala Thr His Gln Asp Ile Asp Phe Leu Ile Glu Glu Ile Glu Arg 65 70 75 80 Leu Gly Gln Asp Leu 85 <210> 132 <211> 75 <212> DNA <213> Synthetic sequence <220> <223> Synthetic <400> 132 actaacatgt tcacctatga gatcgcccct gtatttgtgc tgctagaata tgttacacta 60 aagaaaatga gataa 75 <210> 133 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 133 Thr Asn Met Phe Thr Tyr Glu Ile Ala Pro Val Phe Val Leu Leu Glu 1 5 10 15 Tyr Val Thr Leu Lys Lys Met Arg 20 <210> 134 <211> 87 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 134 gaggcctttg aacacactcc aggagtccac atggccagct tgagtgtgta cctgaagacc 60 aacgtcttcc tcttcctgtt tgcctaa 87 <210> 135 <211> 28 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 135 Glu Ala Phe Glu His Thr Pro Gly Val His Met Ala Ser Leu Ser Val 1 5 10 15 Tyr Leu Lys Thr Asn Val Phe Leu Phe Leu Phe Ala 20 25 <210> 136 <211> 123 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 136 gaggtggagg accctcaggt ggcccagctg gagctgggcg gcggccctgg cgccggcgac 60 ctgcagaccc tggccctgtg gagcagaatg gaccagctgg ccaaggagct gaccgccgag 120 tga 123 <210> 137 <211> 40 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 137 Glu Val Glu Asp Pro Gln Val Ala Gln Leu Glu Leu Gly Gly Gly Pro 1 5 10 15 Gly Ala Gly Asp Leu Gln Thr Leu Ala Leu Trp Ser Arg Met Asp Gln 20 25 30 Leu Ala Lys Glu Leu Thr Ala Glu 35 40 <210> 138 <211> 435 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 138 atggatgacc agcgcgacct tatctccaac aatgagcaac tgcccatgct gggccggcgc 60 cctggggccc cggagagcaa gtgcagccgc ggagccctgt acacaggctt ttccatcctg 120 gtgactctgc tcctcgctgg ccaggccacc accgcctact tcctgtacca gcagcagggc 180 cggctggaca aactgacagt cacctcccag aacctgcagc tggagaacct gcgcatgaag 240 cttcccaagc ctcccaagcc tgtgagcaag atgcgcatgg ccaccccgct gctgatgcag 300 gcgctgccca tgggagccct gccccagggg cccatgcaga atgccaccaa gtatggcaac 360 atgacagagg accatgtgat gcacctgctc cagagtcact ggaactggag gacccgtctt 420​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Ala Thr Thr Ala Tyr Phe Leu Tyr Gln Gln Gln Gly Arg Leu Asp Lys 50 55 60 Leu Thr Val Thr Ser Gln Asn Leu Gln Leu Glu Asn Leu Arg Met Lys 65 70 75 80 Leu Pro Lys Pro Pro Lys Pro Val Ser Lys Met Arg Met Ala Thr Pro 85 90 95 Leu Leu Met Gln Ala Leu Pro Met Gly Ala Leu Pro Gln Gly Pro Met 100 105 110 Gln Asn Ala Thr Lys Tyr Gly Asn Met Thr Glu Asp His Val Met His 115 120 125 Leu Leu Gln Ser His Trp Asn Trp Arg Thr Arg Leu Leu Gly Trp Val 130 135 140 <210> 140 <211> 78 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 140 caacacctgt gcggctcaca cctggtggaa gctctctacc tagtgtgcgg ggaacgaggc 60 ttcttctaca cacccaag 78 <210> 141 <211> 26 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 141 Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys 1 5 10 15 Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys 20 25 <210> 142 <211> 78 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 142 caacacctgt gcggctcaca cctggtggaa gaactctacc tagtgtgcgg ggaagaaggc 60 ttcttctaca cacccaag 78 <210> 143 <211> 26 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 143 Gln His Leu Cys Gly Ser His Leu Val Glu Glu Leu Tyr Leu Val Cys 1 5 10 15 Gly Glu Glu Gly Phe Phe Tyr Thr Pro Lys 20 25 <210> 144 <211> 78 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 144 ggccctggtg caggcagcct gcagcccttg gccctggagg ggtccctgca gaagcgtggc 60 attgtggaac aatgctgt 78 <210> 145 <211> 26 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 145 Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu 1 5 10 15 Gln Lys Arg Gly Ile Val Glu Gln Cys Cys 20 25 <210> 146 <211> 48 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 146 ctgctggccc tctggggacc tgacccagcc gcagcctttg tgaaccaa 48 <210> 147 <211> 16 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 147 Leu Leu Ala Leu Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln 1 5 10 15 <210> 148 <211> 63 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 148 gagagcggct gcaccgtcat cgtcatgctg accccgctgg tggaggatgg tgtcaagcag 60 tgt 63 <210> 149 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 149 Glu Ser Gly Cys Thr Val Ile Val Met Leu Thr Pro Leu Val Glu Asp 1 5 10 15 Gly Val Lys Gln Cys 20 <210> 150 <211> 87 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 150 tctcagcagc cctacctgca actgcagccc tttccacagc ctgagctgcc ctatcctcag 60 cctcagccta gctttccacc tcagcag 87 <210> 151 <211> 29 <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 151 Ser Gln Gln Pro Tyr Leu Gln Leu Gln Pro Phe Pro Gln Pro Glu Leu 1 5 10 15 Pro Tyr Pro Gln Pro Gln Pro Ser Phe Pro Pro Gln Gln 20 25 <210> 152 <211> 63 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 152 gaggagcagt acaacagcac ctacagagtg gtgagcgtgc tgaccgtgct gcaccaggac 60 tgg 63 <210> 153 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> synthetic <400> 153 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 1 5 10 15 Leu His Gln Asp Trp 20 <210> 154 <211> 7862 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 154 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180 tttgccttcc tgtttttgct cacccagaaa cgctgggtgaa agtaaagat gctgaagaatc 240 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300 gttttcgcc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 360 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 420 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 480 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 540 tgacaacgat cggaggaccg aaggagctaa ccgcttttt gcacaacatg ggggatcatg 600 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 660 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 720 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 780 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 840 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 900 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 960 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1020 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1140 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1200 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1260 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1320 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1440 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1500 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1560 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1620 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1800 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1860 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1920 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2040 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2100 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2160 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttggc 2220 cattgcatac gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 2280 taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 2340 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 2400 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2460 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 2520 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2580 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2640 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 2700 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 2760 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgcccc 2820 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 2880 tagtgaaccg gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact 2940 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc 3000 ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa 3060 aatctctagc agtggcgccc gaacagggac ctgaagcga aaggaacc agagctctct 3120 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagg gcgaggggcg gcgactggtg 3180 agtacgccaa aaattttgac tagcggaggc taggaggaga gagatggggtg cgagagcgtc 3240 agtattaagc gggggagaat tegtcggcga tgggaaaaa ttcggttaag gccaggggga 3300 aagaaaaaat aataatttaa acatagta tgggcagca gggagctga acgattcgca 3360 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa 3420 ccatccctc agacaggagatc agagaactt agatcattat atatacagt agcaccctc 3480 tattgtgtgc atcaaggat agagaataaa gatcaagg aagctttaga agatagag 3540 gagagcaa aaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg 3600 yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah tgaaccatta ggagtagcac ccaccaggc aaagagaga gtggtgcaga gagaaaaaag 3720 agcagtggga ataggactt tgttccttgg gttcttggga gcagcaggaa gcactatggg 3780 cgcagcctca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca 3840 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg 3900 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca 3960 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa 4020 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg 4080 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa 4140 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa 4200 ttggtttaac ataacaaatt ggctgtggta tataaatta ttcataatga tagtaggagg 4260 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg 4320 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga 4380 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg 4440 atctcgacgg tatcggttaa cttttaaaag aaaagggggg attggggggt acagtgcagg 4500 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 4560 tacaaaaatt caaaatttta tcgatcacga gactagcctc gagaagcttg atatcgaatt 4620 cccacggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc 4680 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc 4740 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg 4800 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga 4860 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg 4920 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcggg gcgcgccgag 4980 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg 5040 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc 5100 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccatg gatgaccaac 5160 gcgacctcat ctctaaccat gaacagttgc ccatactggg caaccgccct agagagccag 5220 aaaggtgcag ccgtggagct ctgtacaccg gtgtctctgt cctggtggct ctgctcttgg 5280 ctgggcaggc caccactgct tacttcctgt accagcaaca gggccgccta gacaagctga 5340 ccatcacctc ccagaacctg caactggaga gccttcgcat gaagcttccg aaatctgcca 5400 aacctgtgag ccagatgcgg atggctactc ccttgctgat gcgtccaatg tccatggata 5460 acatgctcct tgggcctgtg aagaacgtta ccaagtacgg caacatgacc caggaccatg 5520 tgatgcatct gctcacgagg tctggacccc tggagtaccc gcagctgaag gggaccttcc 5580 cagagaatct gaagcatctt aagaactcca tggatggcgt gaactggaag atcttcgaga 5640 gctggatgaa gcagtggctc ttgtttgaga tgagcaagaa ctccctggag gagaagaagc 5700 ccaccgaggc tccacctaaa gagccactgg acatggaaga cctatcttct ggcctgggag 5760 tgaccaggca ggaactgggt caagtcaccc tgtgtggcat ctcctcagca gagagcctga 5820 agatatctca agctgtccat gcagcacatg cagaaatcaa tgaagcaggc agagaggtgg 5880 tagggtcagc agaggctgga gtggatgctg caagctgata agtcgacaat caacctctgg 5940 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 6000 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 6060 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 6120 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 6180 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 6240 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 6300 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 6360 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 6420 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 6480 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattcgagct cggtaccttt 6540 aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 6600 actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc 6660 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 6720 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 6780 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag 6840 tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 6900 tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 6960 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 7020 tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccag ttccgcccat 7080 tctccgcccc atggctgact aattttttt atttatgcag aggccgaggc cgcctcggcc 7140 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcgtcgag 7200 acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac 7260 aacgtcgtga ctgggaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 7320 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 7380 gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg 7440 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 7500 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 7560 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 7620 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 7680 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 7740 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 7800 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 7860 cc 7862 <210> 155 <211> 8085<s <212> DNA <213> Artificial sequence[[ID=r <220><0003s36><223> Synthetic <400> 155 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120 It should be noted that there may be some inaccuracies in the tags in the original text. For example, and seem to have some incorrect capitalization. It is recommended to double-check the original text for accuracy.aaaggaag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180 tttgccttcc tgtttttgct cacccagaaa cgctgggtgaa agtaaagat gctgaagaatc 240 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300 gttttcgcc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 360 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 420 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 480 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 540 tgacaacgat cggaggaccg aaggagctaa ccgcttttt gcacaacatg ggggatcatg 600 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 660 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 720 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 780 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 840 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 900 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 960 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1020 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1140 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1200 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1260 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1320 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1440 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1500 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1560 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1620 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1800 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1860 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1920 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2040 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2100 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2160 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttggc 2220 cattgcatac gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 2280 taccgccatg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 2340 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 2400 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 2460 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 2520 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 2580 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 2640 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 2700 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 2760 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgcccc 2820 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 2880 tagtgaaccg gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact 2940 agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc 3000 ccgtctgttg tgtgactctg gtactagag atccctcaga cccttttagt cagtgtggaa 3060 aatctctagc agtggcgccc gaacagggac ctgaagcga aaggaacc agagctctct 3120 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagg gcgaggggcg gcgactggtg 3180 agtacgccaa aaattttgac tagcggaggc taggaggaga gagatggggtg cgagagcgtc 3240 agtattaagc gggggagaat tegtcggcga tgggaaaaa ttcggttaag gccaggggga 3300 aagaaaaaat aataatttaa acatagta tgggcagca gggagctga acgattcgca 3360 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa 3420 ccatccctc agacaggagatc agagaactt agatcattat atatacagt agcaccctc 3480 tattgtgtgc atcaaggat agagaataaa gatcaagg aagctttaga agatagag 3540 gagagcaa aaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg 3600 yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah yeah tgaaccatta ggagtagcac ccaccaggc aaagagaga gtggtgcaga gagaaaaaag 3720 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg 3780 cgcagcctca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca 3840 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg 3900 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca 3960 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa 4020 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg 4080 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa 4140 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa 4200 ttggtttaac ataacaaatt ggctgtggta tataaatta ttcataatga tagtaggagg 4260 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg 4320 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacccg acaggcccga 4380 aggaatagaa gaagaaggtg gagagagaga cagagacaga tccattcgat tagtgaacgg 4440 atctcgacgg tatcggttaa cttttaaaag aaaagggggg attggggggt acagtgcagg 4500 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 4560 tacaaaaatt caaaatttta tcgatcacga gactagcctc gagaagcttg atatcgaatt 4620 cccacggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc 4680 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc 4740 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg 4800 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga 4860 caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg 4920 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcggg gcgcgccgag 4980 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg 5040 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc 5100 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccatg gatgaccaac 5160 gcgacctcat ctctaaccat gaacagttgc ccatactggg caaccgccct agagagccag 5220 aaaggtgcag ccgtggagct ctgtacaccg gtgtctctgt cctggtggct ctgctcttgg 5280 ctgggcaggc caccactgct tacttcctgt accagcaaca gggccgccta gacaagctga 5340 ccatcacctc ccagaacctg caactggaga gccttcgcat gaagcttccg aaatctgcca 5400 aacctgtgag ccagatgcgg atggctactc ccttgctgat gcgtccaatg tccatggata 5460 acatgctcct tgggcctgtg aagaacgtta ccaagtacgg caacatgacc caggaccatg 5520 tgatgcatct gctcacgagg tctggacccc tggagtaccc gcagctgaag gggaccttcc 5580 cagagaatct gaagcatctt aagaactcca tggatggcgt gaactggaag atcttcgaga 5640 gctggatgaa gcagtggctc ttgtttgaga tgagcaagaa ctccctggag gagaagaagc 5700 ccaccgaggc tccacctaaa gagccactgg acatggaaga cctatcttct ggcctgggag 5760 tgaccaggca ggaactgggt caagtcaccc tgtgtggcat ctcctcagca gagagcctga 5820 agatatctca agctgtccat gcagcacatg cagaaatcaa tgaagcaggc agagaggtgg 5880 tagggtcagc agaggctgga gtggatgctg caagctgata agtcgacaat caacctctgg 5940 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 6000 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 6060 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 6120 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 6180 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 6240 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 6300 attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 6360 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 6420 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 6480 agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattcgagct cgctagctaa 6540 cccctatcac aattagcatt aacgatcccc tatcacaatt agcattaaac cggtccccta 6600 tcacaattag cattaatcac cccctatcac aattagcatt aacccggggt aaaacccatg gaattcagtt ctcacgataa cccatggaat tcagttctca acgcgtaacc catggaattc agttctcatc acaacccatg gaattcagtt ctcaggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactgga gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggacccact gcttaagcct caataagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat 7020 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat gaatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aataagca atagcatcac aaatttcaca aataagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg 7320 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa 7380 gtagtgagga ggcttttttg gaggcctagg cttttgcgtc gagacgtacc caattcgccc 7440 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa 7500 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 7560 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 7620 tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc 7680 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt 7740 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc 7800 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt 7860 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt 7920 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt 7980 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa 8040 aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcc 8085 <210> 156 <211> 9213 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 156 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 60 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 120 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 180 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc 240 gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 300 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 360 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 420 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg 480 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 540 It should be noted that there may be some inaccuracies in the translation of the specific sequence content, but it is translated according to the requirements. And the "0" in "gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc 240" in the original text seems to be a typo, here it is translated as "240" as it is, but it may need to be further confirmed in the actual context.tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 600 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 660 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 720 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 780 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 840 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 900 tttcccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 960 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1020 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1080 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1140 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1200 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1260 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1320 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1380 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1440 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1500 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1560 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1620 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1680 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1740 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1800 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 1860 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1920 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1980 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2040 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2100 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2160 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2220 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2280 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2340 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2400 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2460 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2520 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2580 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2640 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2700 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 2760 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 2820 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 2880 tcattaatgc agctggcacg acaggtttc cgactggaaa gcgggcagtg agcgcaacgc 2940 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3000 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3060 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 3120 ttggccattg catacgttgt atccatatca tatatgtac atttatattg gctcatgtcc 3180 aacattaccg ccatgttgac attgattatt gactagttat tatagtaat caattacggg 3240 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 3300 gcctggctga ccgcccaacg acccccgcc attgacgtca ataatgacgt atgttcccat 3360 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 3420 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 3480 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 3540 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 3600 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 3660 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 3720 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 3780 tcgtttagtg aaccggggtc tctctggtta gaccagatct gagcctggga gctctctggc 3840 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 3900 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 3960 tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc 4020 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 4080 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 4140 gcgtcagtat taagcgggggg agaattagat cgcgatggga aaaattcgg ttaggccag 4200 ggggaaaaaaaataaaaaacata tagtagggc aagcagggag ctagaacgat 4260 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaata ctgggacagc 4320 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatatat acagtagca 4380 ccccttattg tgtgcatca aggatagaga taaagacac caggaagct tagacaga 4440 taggaggaga gcaaaaaaa agtaagacca ccgcacagca agcggccgct gatcttcaga 4500 cctggaggag gagatatgag ggacaatttgg agagtgaat taddaata taagtagta 4560 aaaattgaac cattaggagt agcacccacc aaggxaaga gagagtggt gcagagagaa 4620 aaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact 4680 atgggcgcag cctcaatgac gctgacggta caggccagac aattattgtc tgtatagtg 4740 cagcagcaga acaatttgct gaggggtatt gaggcgcac agcatctgtt gcaactcaca 4800 gtctggggca tcaagcagct ccaggcaag atcctggctg tggaagata cctaaaggat 4860 caacagctcc tgggattg gggttgctt ggaaaactca ttgcaccac tgctgtgcct 4920 tggaatgcta gttggagtaa taatctctg gaacagattg gatcacacg acctggatgg 4980 agtgggacag agaattac aattacaca gcttaataca ctcttaatt gagaatcgc 5040 aaaaccagca agaaagaat gaacagaat tattggatt agaatgg gcaagttttgt 5100 ggaattggtt taacaataca aattggctgt ggtatataa attattcata atgatagtag 5160 gaggcttggt aggtttaaga atagttttg ctgtactttc tatagtgaat agagttaggc 5220 agggatattc accattatcg ttcagacccc accccaccac cccgaggga cccgacaggc 5280 ccgaaggaat agagagaa ggtgagaga gagacagaga cagatccatt cgattagtga 5340 acggatctcg acggtatcgg ttaactttta aaagaaaagg ggggattggg gggtacagtg 5400 cagggaaag atagtagac atataagca caggacataca aactaaaaaaaaac 5460 aaattacaa aattcaaat tttcgatc acgagactag cctcgagaga tctgatcata 5520 atcagccata ccacattgt agaggtttta cttgctttaa aaaacctccc acacctccc 5580 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 5640 aatggttaca aataaggcaa tagcatcaca aatttcacaa ataaggcatt tttttcactg 5700 cattctagtt ttggtttgtc caaactcatc aatgtatctt atcatgtctg gatctcaaat 5760 ccctcggaag ctgcgcctgt catcgaattc ctgcagcccg gtgcatgact aagctagctc 5820 agttagcctc ccccatctcc cctagaggat ccccctgttc cacctcttga aggctatgta 5880 ggccacaagg cccacaacca cagcagccag gatggagcaa tagacaggga tgaggttgtc 5940 ggtggtgcct cgggtcacca cgggctggga gctgcccatc actgtggtca ccacacctgc 6000 caccgtgctg gctatgaggt cttgttctgg aggtgcctca ggctcctggg tgctgggggc 6060 tgtgctgtcc gagccctctg ggggtgtgga ccgtgtaatc caacggccag ggatctcctc 6120 gcactcggcg tcggcccagc gtgtgcactc gcggagctgg cgctcggtgt cctcgcacac 6180 ggtgcagggc aggcacgggt ccacgtggtt ggcctcgtcg gaatacgtgc cgtcggggca 6240 ctcctcgcac acggtgttct gcttgtcctg gcaggagaac acgaggcccg agcccgcctc 6300 gcacacgcgg cacgcctcgc agcgcccagt cgtctcatcc tggtagtagc cgtaggcgca 6360 gcggcacacg gcgtcgtcgg cctccacgca cggcgccgac atgctctgga gccccacgca 6420 ctcggtgcac ggcttgcacg gctcggtcgc gctcaccacg tcggagaacg tcacgctgtc 6480 caggcagggc tcacacacgg tctggttggc tccacaaggc tgggccacac cctcgcccag 6540 gttgcaggct ttgcagcact caccgctgtg tgtgtacagg cctgtggggc atgcctcctt 6600 ggcacctcca agggacaccc ccagaagcag caacagcagc aggcgcggcc cgtccatggc 6660 gcggccggtg gcacctgccc ccatcgcccg cctcccgcgg cagcgctcga cttccagctc 6720 ggtccgcttt gcggactgat ggggctgcgc tgcgctgcgc tccagcgccc cccctgcccg 6780 ccggagctgg ccgcggcccg aattccgcgg aggctggatc ggtcccggtg tcttctatgg 6840 aggtcaaaac agcgtggatg gcgtctccag gcgatctgac ggttcactaa acgagctctg 6900 cttatatagg cctcccaccg tacacgccta ccctcgagaa gcttgatatc gaattcccac 6960 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 7020 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 7080 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 7140 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 7200 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 7260 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcggggcgcg ccgagagcag 7320 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 7380 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 7440 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggtcgcca ccatggtgag 7500 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt 7560 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct 7620 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac 7680 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga 7740 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga 7800 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg 7860 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga 7920 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa 7980 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta 8040 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag 8100 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga 8160 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaagcggccg 8220 cgtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta 8280 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc 8340 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga 8400 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac 8460 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc 8520 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc 8580 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg 8640 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc 8700 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctag agcctcttcc 8760 gcgtcttcgc cttcccgggt cgagctcggt acctttaaga ccaatgactt acaaggcagc 8820 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca 8880 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc 8940 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg 9000 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag 9060 acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc ttattattca 9120 gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt ttattgcagc 9180 ttataatggt tacaaataaa gcaatagcat cac 9213 <210> 157 <211> 9109 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 157 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 60 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 120 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 180 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc 240 gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 300 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 360 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 420 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg 480 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 540 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 600 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 660 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 720 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 780 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 840 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 900 tttcccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 960 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1020 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1080 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1140 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1200 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1260 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1320 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1380 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1440 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1500 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1560 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1620 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1680 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1740 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1800 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 1860 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1920 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1980 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2040 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2100 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2160 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2220 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2280 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2340 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2400 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2460 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2520 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2580 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2640 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2700 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 2760 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 2820 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 2880 tcattaatgc agctggcacg acaggtttc cgactggaaa gcgggcagtg agcgcaacgc 2940 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3000 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3060 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 3120 ttggccattg catacgttgt atccatatca tatatgtac atttatattg gctcatgtcc 3180 aacattaccg ccatgttgac attgattatt gactagttat tatagtaat caattacggg 3240 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 3300 gcctggctga ccgcccaacg acccccgcc attgacgtca ataatgacgt atgttcccat 3360 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 3420 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 3480 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg 3540 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 3600 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 3660 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 3720 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 3780 tcgtttagtg aaccggggtc tctctggtta gaccagatct gagcctggga gctctctggc 3840 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 3900 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 3960 tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc 4020 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 4080 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 4140 gcgtcagtat taagcgggggg agaattagat cgcgatggga aaaattcgg ttaggccag 4200 ggggaaaaaaaataaaaaacata tagtagggc aagcagggag ctagaacgat 4260 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaata ctgggacagc 4320 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatatat acagtagca 4380 ccccttattg tgtgcatca aggatagaga taaagacac caggaagct tagacaga 4440 taggaggaga gcaaaaaaa agtaagacca ccgcacagca agcggccgct gatcttcaga 4500 cctggaggag gagatatgag ggacaatttgg agagtgaat taddaata taagtagta 4560 aaaattgaac cattaggagt agcacccacc aaggxaaga gagagtggt gcagagagaa 4620 aaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact 4680 atgggcgcag cctcaatgac gctgacggta caggccagac aattattgtc tgtatagtg 4740 cagcagcaga acaatttgct gaggggtatt gaggcgcac agcatctgtt gcaactcaca 4800 gtctggggca tcaagcagct ccaggcaag atcctggctg tggaagata cctaaaggat 4860 caacagctcc tgggattg gggttgctt ggaaaactca ttgcaccac tgctgtgcct 4920 tggaatgcta gttggagtaa taatctctg gaacagattg gatcacacg acctggatgg 4980 agtgggacag agaattac aattacaca gcttaataca ctcttaatt gagaatcgc 5040 aaaaccagca agaaagaat gaacagaat tattggatt agaatgg gcaagttttgt 5100 ggaattggtt taacaataca aattggctgt ggtatataa attattcata atgatagtag 5160 gaggcttggt aggtttaaga atagttttg ctgtactttc tatagtgaat agagttaggc 5220 agggatattc accattatcg ttcagacccc accccaccac cccgaggga cccgacaggc 5280 ccgaaggaat agagagaa ggtgagaga gagacagaga cagatccatt cgattagtga 5340 acggatctcg acggtatcgg ttaactttta aaagaaaagg ggggattggg gggtacagtg 5400 cagggaaag atagtagac atataagca caggacataca aactaaaaaaaaac 5460 aaattacaa aattcaaat tttcgatc acgagactag cctcgagaga tctgatcata 5520 atcagccata ccacattgt agaggtttta cttgctttaa aaaacctccc acacctccc 5580 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 5640 aatggttaca aataaggcaa tagcatcaca aatttcacaa ataaggcatt tttttcactg 5700 cattctagtt ttggtttgtc caaactcatc aatgtatctt atcatgtctg gatctcaaat 5760 ccctcggaag ctgcgcctgt catcgaattc ctgcagcccg gtgcatgact aagctagcag 5820 ttcagttccg gatcttcatg gtcatgtagg cctcgatgta gttgatgaag atgtcgaact 5880 cgctcatggc cttgtagatg cccttttcct gcagcttgtt gaaggcgttt ttgacctgtt 5940 ccacggcctt gctcttgttc tcgcagggca gaaatctgtg gcaccgcctc agccgcagcc 6000 gcagggtttt caggttctcg cccaggctgt tcacgtgggc cttgatgtcg gggtcctggt 6060 tctcggcctg gggcatcact tcttccaggt agaactggat catctcgctc agggcctggc 6120 agcccaggta gcccttgaaa tcttccagca ggctctcttt cagcagcagg ttgtccagct 6180 ggtccttcat ctggaagaat gttttcactc tgctgaaggc gtccctcagg tcccgcagca 6240 tgttgggcag gttgccgggg aagtgggtgc agctgttctc gctctgggtg ccctggccag 6300 gagaggctct gacgccggtc agcagcacca ggcagcacag cagggcggag ctgtgcatag 6360 tcggtccgct ttgcggactg atggggctgc gctgcgctgc gctccagcgc cccccctgcc 6420 cgccggagct ggccgcggcc cgaattccgc ggaggctgga tcggtcccgg tgtcttctat 6480 ggaggtcaaa acagcgtgga tggcgtctcc aggcgatctg acggttcact aaacgagctc 6540 tgcttatata ggcctcccac cgtacacgcc taccctcgag aagcttgata tcgaattccc 6600 acggggttgg ggttgcgcct tttccaaggc agccctgggt ttgcgcaggg acgcggctgc 6660 tctgggcgtg gttccgggaa acgcagcggc gccgaccctg ggtctcgcac attcttcacg 6720 tccgttcgca gcgtcacccg gatcttcgcc gctacccttg tgggcccccc ggcgacgctt 6780 cctgctccgc ccctaagtcg ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa 6840 acggaagccg cacgtctcac tagtaccctc gcagacggac agcgccaggg agcaatggca 6900 gcgcgccgac cgcgatgggc tgtggccaat agcggctgct cagcggggcg cgccgagagc 6960 agcggccggg aaggggcggt gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc 7020 ctgcccgcgc ggtgttccgc attctgcaag cctccggagc gcacgtcggc agtcggctcc 7080 ctcgttgacc gaatcaccga cctctccc cagggggatc caccatggat gaccaacgcg 7140 acctcatctc taaccatgaa cagttgccca tactgggcaa ccgccctaga gagccagaaa 7200 ggtgcagccg tggagctctg tacaccggtg tctctgtcct ggtggctctg ctcttggctg 7260 ggcaggccac cactgcttac ttcctgtacc agcaacaggg ccgcctagac aagctgacca 7320 tcacctccca gaacctgcaa ctggagagcc ttcgcatgaa gcttccgaaa tctgccaaac 7380 ctgtgagcca gatgcggatg gctactccct tgctgatgcg tccaatgtcc atggataaca 7440 tgctccttgg gcctgtgaag aacgttacca agtacggcaa catgacccag gaccatgtga 7500 tgcatctgct cacgaggtct ggacccctgg agtacccgca gctgaagggg accttcccag 7560 agaatctgaa gcatcttaag aactccatgg atggcgtgaa ctggaagatc ttcgagagct 7620 ggatgaagca gtggctcttg tttgagatga gcaagaactc cctggaggag aagaagccca 7680 ccgaggctcc acctaaagag ccactggaca tggaagacct atcttctggc ctgggagtga 7740 ccaggcagga actgggtcaa gtcaccctgt gcatgttggt gctgttgcct gatgaagtct 7800. caggccttga gcagcttgag father actttgaaaa actgactgaa tggaccagtt ctaacgttat ggaagagagg aagatcaaag tgtacttacc tcgcatgaag atggaggaaa aatacaacct cacatctgtc ttaatggcta tgggcattac tgacgtgttt agctcttcag 8040. ccaatctgtc tggcatctcc tcagcagaga gcctgaagat atctcaagct gtccatgcag cacatgcaga aatcaatgaa gcaggcagag aggtggtagg gtcagcagag gctggagtgg atgctgccac ctgataagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 8280 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 8340 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 8400. ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 8460 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc 8520 tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 8580 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 8640 ctctagagcc tcttccgcgt cttcgccttc ccgggtcgag ctcggtacct ttaagaccaa 8700 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag 8760 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt 8820 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc 8880 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta 8940 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat 9000 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga 9060 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcac 9109 <210> 158 <211> 9577 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 158 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat 60 caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc 120 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg 180 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc 240 gagacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt 300 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 360 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 420 tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg 480 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 540 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 600 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 660 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 720 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 780 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 840 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 900 tttcccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 960 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1020 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1080 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1140 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1200 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1260 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1320 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1380 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1440 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1500 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1560 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1620 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1680 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1740 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1800 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 1860 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 1920 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 1980 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2040 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2100 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2160 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2220 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2280 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2340 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2400 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2460 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2520 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2580 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2640 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2700 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 2760 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 2820 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 2880 tcattaatgc agctggcacg acaggtttc cgactggaaa gcgggcagtg agcgcaacgc 2940 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3000 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3060 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc 3120 ttggccattg catacgttgt atccatatca tatatgtac atttatattg gctcatgtcc 3180 aacattaccg ccatgttgac attgattatt gactagttat tatagtaat caattacggg 3240 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 3300 gcctggctga ccgcccaacg acccccgcc attgacgtca ataatgacgt atgttcccat 3360 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc 3420 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga 3480 cggtaaatgg cccgcctggc attatgccca gtacatgacc tttgggact ttcctacttg 3540 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat 3600 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 3660 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc 3720 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc 3780 tcgtttagtg aaccggggtc tctctggtta gaccagatct gagcctggga gctctctggc 3840 3900 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 3960 tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc 4020 tctctcgacg caggactcgg cttgctgaag cgcgcacgg aagaggcgag gggcggcgac 4080 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 4140 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag 4200 ggggaaagaa aaaatataaa taaaacata tagtatggc aagcagggag ctagaacgat 4260 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaata ctgggacagc 4320 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatatat acagtagca 4380 ccccttattg tgtgcatca aggatagaga taaagacac caggaagct tagacaga 4440 taggaggaga gcaaaaaaa agtaagacca ccgcacagca agcggccgct gatcttcaga 4500 cctggaggag gagatatgag ggacaatttgg agagtgaat taddaata taagtagta 4560 aaaattgaac cattaggagt agcacccacc aaggxaaga gagagtggt gcagagagaa 4620 aaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact 4680 atgggcgcag cctcaatgac gctgacggta caggccagac aattattgtc tgtatagtg 4740 cagcagcaga acaatttgct gaggggtatt gaggcgcac agcatctgtt gcaactcaca 4800 gtctggggca tcaagcagct ccaggcaag atcctggctg tggaagata cctaaaggat 4860 caacagctcc tgggattg gggttgctt ggaaaactca ttgcaccac tgctgtgcct 4920 tggaatgcta gttggagtaa taatctctg gaacagattg gatcacacg acctggatgg 4980 agtgggacag agaattac aattacaca gcttaataca ctcttaatt gagaatcgc 5040 aaaaccagca agaaagaat gaacagaat tattggatt agaatgg gcaagttttgt 5100 ggaattggtt taacaataca aattggctgt ggtatataa attattcata atgatagtag 5160 gaggcttggt aggtttaaga atagttttg ctgtactttc tatagtgaat agagttaggc 5220 agggatattc accattatcg ttcagacccc accccaccac cccgaggga cccgacaggc 5280 ccgaaggaat agagagaa ggtgagaga gagacagaga cagatccatt cgattagtga 5340 acggatctcg acggtatcgg ttaactttta aaagaaaagg ggggattggg gggtacagtg 5400 cagggaaag atagtagac atataagca caggacataca aactaaaaaaaaac 5460 aaattacaa aattcaaat tttcgatc acgagactag cctcgagaga tctgatcata 5520 atcagccata ccacattgt agaggtttta cttgctttaa aaaacctccc acacctccc 5580 ctgaacctga aacataaaat gatgcaatt gttgttgtta acttgtttat tgcagcttat 5640 aatggttaca aaggcaa tagcatcaca aatttcacaa ataggcatt ttttcactg 5700 cattctagtt ttggtttgtc caaactcatc aatgtatctt atcatgtctg gatctcaaat 5760 ccctcggaag ctgcgcctgt catcgaattc ctgcagcccg gtgcatgact aagctagcag 5820 tctaaggcca actcagaaga gctttctcgg ttgtatcttt cacactcctt aggaaagtca 5880 tgggattcgt acccccagtc cctctgcttt ccacatttga gggctcttcc gacttgtcgc 5940 catcagtggg cttcttcttc gaaggtttca taatgtaagt atctactatt gcgaggtgga 6000 actttctcac agagaccaga ccattcacac actcgttata agctttcgtc aagtcttcat 6060 tgtgtcttga aatgacaaac tcacggactg ggggagctga ctctaagaag aaaaggaagt 6120 tccggtgggc tggaggcatg tactctctca tttcctggag gaattctgca ggagattctt 6180 tgccagcctc gtgttttat cccagaagga catcaagact ctggaagatg ctgctctggc 6240 ctgcactgcc ccctgaaac atttttgggg tgtcccagac cccctcatac agcagacctt 6300 ctggcagctt ggagctgcat ttccagccag acagatatat gcggagaacg tggaaaaacg 6360 tgtctgggtc cacaaagtca cgcatcctct taaaaatttc cttggctttc tccagactgg 6420 tagctatgtc gtgcagtgcc ttttccaatg ctttcaggtc ttgacgctct actgcactgg 6480 atacagtggg gattgctttg attgcaggag aagctgcgat ttccaccaat agagagacga 6540 ggaagaagcc cttgtcgcag tccccaccag gaaatgagaa cagaatgtcc atgttctcgt 6600 atgtcatggg cccattgggg tcctttttct tccagtttgc caggacacag tctgcataag 6660 acagaatagg aggcaggccc aacttctctg agagctcgca gtagggaaca gcaatattgc 6720 ggggcagcac ctttcgaaca tcgtcatccc ctcggttcca cacatacgcc atggtgatgt 6780 accccagggc caggtgtgcc aggcgctgta acctgtgtcc tctcagtccg tccgtgctca 6840 gtgtgggcag cttttcaact tcttctcgaa gctgcccgtt ctcaatcagc acaggcagat 6900 ttctagccac aaggacccag gggctgtatg cgtcgggcag ctccaccagt ggatgtggta 6960 gagcaaagcc cacatcttca tctatgtggt ggtcttcaag gattcttcta gaaccttctg 7020 taggagatat tttactgagt gccatagtcg gtccgctttg cggactgatg gggctgcgct 7080 gcgctgcgct ccagcgcccc ccctgcccgc cggagctggc cgcggcccga attccgcgga 7140 ggctggatcg gtcccggtgt cttctatgga ggtcaaaaca gcgtggatgg cgtctccagg 7200 cgatctgacg gttcactaaa cgagctctgc ttatataggc ctcccaccgt acacgcctac 7260 cctcgagaag cttgatatcg aattcccacg gggttggggt tgcgcctttt ccaaggcagc 7320 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc 7380 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct 7440 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg 7500 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac gtctcactag taccctcgca 7560 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc 7620 ggctgctcag cggggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg 7680 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct 7740 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag 7800 ggggatccac catggatgac caacgcgacc tcatctctaa ccatgaacag ttgcccatac 7860 tgggcaaccg ccctagagag ccagaaaggt gcagccgtgg agctctgtac accggtgtct 7920 ctgtcctggt ggctctgctc ttggctgggc aggccaccac tgcttacttc ctgtaccagc 7980 aacagggccg cctagacaag ctgaccatca cctcccagaa cctgcaactg gagagccttc 8040 gcatgaagct tccgaaatct gccaaacctg tgagccagat gcggatggct actcccttgc 8100 tgatgcgtcc aatgtccatg gataacatgc tccttgggcc tgtgaagaac gttaccaagt 8160 acggcaacat gacccaggac catgtgatgc atctgctcac gaggtctgga cccctggagt 8220 acccgcagct gaaggggacc ttcccagaga atctgaagca tcttaagaac tccatggatg 8280 gcgtgaactg gaagatcttc gagagctgga tgaagcagtg gctcttgttt gagatgagca 8340 agaactccct ggaggagaag aagcccaccg aggctccacc taaagagcca ctggacatgg 8400 aagacctatc ttctggcctg ggagtgacca ggcaggaact gggtcaagtc accctgtgtg 8460 gcatctcctc agcagagagc ctgaagatat ctcaagctgt ccatgcagca catgcagaaa 8520 tcaatgaagc aggcagagag gtggtagggt cagcagaggc tggagtggat gctgcaagct 8580 gataagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta 8640 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta 8700 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt 8760 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg 8820 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt 8880 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag 8940 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg acgtcctttc 9000 catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc 9060 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctagagcctc 9120 ttccgcgtct tcgccttccc gggtcgagct cggtaccttt aagaccaatg acttacaagg 9180 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact 9240 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct 9300 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc 9360 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc 9420 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta 9480 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg 9540 cagcttataa tggttacaaa taaagcaata gcatcac 9577 <210> 159 <211> 537 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 159 atgcacagct cagcactgct ctgttgcctg gtcctcctga ctggggtgag ggccagccca 60 ggccagggca cccagtctga gaacagctgc acccacttcc caggcaacct gcctaacatg 120 cttcgagatc tccgagatgc cttcagcaga gtgaagactt tctttcaaat gaaggatcag 180 ctggacaact tgttgttaaa ggagtccttg ctggaggact ttaagggtta cctgggttgc 240 caagccttgt ctgagatgat ccagttttac ctggaggagg tgatgcccca agctgagaac 300 caagacccag acatcaaggc gcatgtgaac tccctggggg agaacctgaa gaccctcagg 360 ctgaggctac ggcgctgtca tcgatttctt ccctgtgaaa acaagagcaa ggccgtggag 420 caggtgaaga atgcctttaa taagctccaa gagaaaggca tctacaaagc catgagtgag 480 tttgacatct tcatcaacta catagaagcc tacatgacaa tgaagatacg aaactga 537 <210> 160 <211> 178 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 160 Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val 1 5 10 15 Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His 20 25 30 Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe 35 40 45 Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu 50 55 60 Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys 65 70 75 80 Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro 85 90 95 Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu 100 105 110 Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg 115 120 125 Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn 130 135 140 [[ID=·16]]Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu 145 150 155 160 Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile 165 170 175 Arg Asn <210> 161 <211> 1212 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 161 atggcccatg ccatggaaaa cagctggacc atcagcaaag agtaccacat cgacgaggaa 60 gtgggcttcg ccctgcctaa tcctcaagag aacctgcctg acttctacaa cgactggatg 120 tttatcgcca aacatctgcc cgacctgatc gagagcggcc agctggagaga aagagtggaa 180 aagctgaaca tgctgagcat cgaccacctg accgaccaca agtctcagag actggccaga 240 ctggtgctgg gctgtatcac catggcctac gtgtgggggaa aaggccatgg cgacgtgcgg 300 aaagtgctgc ccagaaatat cgccgtgcct tactgccagc tgtccaagaa gctggaactg 360 cctcctatcc tggtgtacgc cgattgcgtg ctggccaact ggaagaa ggaccccaac 420 aagcccctga cctacgagaa catggacgtg ctgtttagct tccgcgacgg cgattgcagc 480 aagggattct tcctggtgtc cctgctggtg gaaatcgccg ctgcctctgc catcaaagtg 540 atccccaccg tgttcaaggc catgcagatg caagagcggg acaccctgct gaaggccctg 600 ctggaaattg cctcctgcct ggaaaaagcc ctccaggtgt tccaccagat ccacgaccac 660 gtgaacccca aggccttctt cagcgtgctg cggatctatc tgtctggctg gaagggcaat 720 ccccagctgt ctgacggcct ggtgtatgaa ggcttctggg aagatcccaa agagttcgct 780 ggcggctctg ccggacagtc tagtgtgttc cagtgcttcg atgtgctgct gggcatccag 840 caaacagccg gcggaggaca tgctgctcag tttctgcaag acatgcggcg gtacatgcct 900 ccagctcacc ggaactttct gtgcagcctg gaaagcaacc ccagcgtgcg ggaattcgtg 960 ctgtctaaag gcgacgccgg actgagagaa gcctacgatg cctgtgtgaa ggctctggtg 1020 tctctgcgga gctaccacct ccagatcgtg accaagtaca ttctgatccc cgccagccag 1080 cagcctaaag agaacaagac cagcgaggac ccctccaagc tggaagcaaa aggcacaggc 1140 ggaaccgatc tgatgaactt cctgaaaacc gtgcggtcca ccaccgagaa gtctctgctg 1200 aaagagggct ga 1212 <210> 162 <211> 403 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 162 Met Ala His Ala Met Glu Asn Ser Trp Thr Ile Ser Lys Glu Tyr His 1 5 10 15 Ile Asp Glu Glu Val Gly Phe Ala Leu Pro Asn Pro Gln Glu Asn Leu 20 25 30 Pro Asp Phe Tyr Asn Asp Trp Met Phe Ile Ala Lys His Leu Pro Asp 35 40 45 Leu Ile Glu Ser Gly Gln Leu Arg Glu Arg Val Glu Lys Leu Asn Met 50 55 60 Leu Ser Ile Asp His Leu Thr Asp His Lys Ser Gln Arg Leu Ala Arg 65 70 75 80 Leu Val Leu Gly Cys Ile Thr Met Ala Tyr Val Trp Gly Lys Gly His 85 90 95 Gly Asp Val Arg Lys Val Leu Pro Arg Asn Ile Ala Val Pro Tyr Cys 100 105 110 Gln Leu Ser Lys Lys Leu Glu Leu Pro Pro Ile Leu Val Tyr Ala Asp 115 120 125 Cys Val Leu Ala Asn Trp Lys Lys Lys Asp Pro Asn Lys Pro Leu Thr 130 135 140 Tyr Glu Asn Met Asp Val Leu Phe Ser Phe Arg Asp Gly Asp Cys Ser 145 150 155 160 Lys Gly Phe Phe Leu Val Ser Leu Leu Val Glu Ile Ala Ala Ala Ser 165 170 175 Ala Ile Lys Val Ile Pro Thr Val Phe Lys Ala Met Gln Met Gln Glu 180 185 190 Arg Asp Thr Leu Leu Lys Ala Leu Leu Glu Ile Ala Ser Cys Leu Glu 195 200 205 Lys Ala Leu Gln Val Phe His Gln Ile His Asp His Val Asn Pro Lys 210 215 220 Ala Phe Phe Ser Val Leu Arg Ile Tyr Leu Ser Gly Trp Lys Gly Asn 225 230 235 240 Pro Gln Leu Ser Asp Gly Leu Val Tyr Glu Gly Phe Trp Glu Asp Pro 245 250 255 Lys Glu Phe Ala Gly Gly Ser Ala Gly Gln Ser Ser Val Phe Gln Cys 260 265 270 Phe Asp Val Leu Leu Gly Ile Gln Gln Thr Ala Gly Gly Gly His Ala 275 280 285 Ala Gln Phe Leu Gln Asp Met Arg Arg Tyr Met Pro Pro Ala His Arg 290 295 300 Asn Phe Leu Cys Ser Leu Glu Ser Asn Pro Ser Val Arg Glu Phe Val 305 310 315 320 Leu Ser Lys Gly Asp Ala Gly Leu Arg Glu Ala Tyr Asp Ala Cys Val 325 330 335 Lys Ala Leu Val Ser Leu Arg Ser Tyr His Leu Gln Ile Val Thr Lys 340 345 350 Tyr Ile Leu Ile Pro Ala Ser Gln Gln Pro Lys Glu Asn Lys Thr Ser 355 360 365 Glu Asp Pro Ser Lys Leu Glu Ala Lys Gly Thr Gly Gly Thr Asp Leu 370 375 380 Met Asn Phe Leu Lys Thr Val Arg Ser Thr Thr Glu Lys Ser Leu Leu 385 390 395 400 Lys Glu Gly <210> 163 <211> 4346 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 163 atgggcgcga gaaactccgt cttgtcaggg aagaaagcag atgaattaga aaaaattagg 60 ctacgacccg gcggaaagaa aaagtacatg ttgaagcatg tagtatgggc agcaaatgaa 120 ttagatagat ttggattagc agaaagcctg ttggagaaca aagaaggatg tcaaaaaata 180 ctttcggtct tagctccatt agtgccaaca ggctcagaaa atttaaaaag cctttataat 240 actgtctgcg tcatctggtg cattcacgca gaagagaaag tgaaacacac tgaggaagca 300 aaacagatag tgcagagaca cctagtggtg gaaacaggaa cagcagaaac tatgccaaaa 360 acaagtagac caacagcacc atctagcggc agaggaggaa attacccagt acaacaaata 420 ggtggtaact atgtccacct gccattaagc ccgagaacat taatgcctg ggtaaaattg 480 atagaggaaa agaatttgg agcagaagta gtgccaggat ttcaggcact gtcagaagggc 540 tgcaccccct atgacattaa tcagatgtta aattgtgtgg gagaccatca agcggctatg 600 steps gagattatta aatgaggag gctgcagatt gggacttgca gcaccacaa 660 ccagctccac aaaaggaca gcttagggag ccgtcaggat cagatattgc aggaacaact 720 agttcagtag atgacaat ccagtggatg tacagacac agaccccat accagtaggc 780 aacatttaca ggagatggat ccaactgggg ttgcaaaat gtgtcagaat gtataaccca 840 aaaacattc tagagtaaaaaagggcca aaagagccat ttcagagcta tgtagacagg 900 ttctacaaaa gcttaagagc agaaaaca gatgcagcag windowgaattg gatgactca 960 acacctgctga ttcaaatgc taacccagat tgcaagctag tgctgaaggg gctgggtgtg 1020 aatcccaccc taggagaat gctgacggct tgtcaggag tgggggacc aggagagg 1080 gctagattaa tggcagaagc cctgaaagag gccctcgcac cagtgccaat cccttttgca 1140 gcagcccaga agaggggacc aagaaagcca attaagtgtt ggaattgtgg gaaggaggga 1200 cactctgcaa ggcaatgcag agccccaga agacagggat gctggaatg tggaaaatg 1260 gaccatgtta tggccaatg cccagacaga caggcggtt ttttaggct tggtccatgg 1320 ggaaagaagc cccgcaattt cccatggct caagtgcatc aggggctgac gccaactgct 1380 cccccagagg acccagctgt ggatctgcta agaactaca tgcagttggg caagcagcag 1440 aggaaagca gagaagcc ttacaggag gtgacagagg atttgctgca cctcaatct 1500 ctctttggag gagaccagta gtcactgctc atattgaagg acagcctgta gaagtattat 1560 tggatacagg ggctgatgat tctattgtaa caggaataga gttaggtcca cattataccc 1620 aaaatagt aggaata ggaggtttta ttatacta agaataaaaaaaaaa 1680 taagtttt agggcaaagg atttaaggga caatcatgac agggacact ccgattaca 1740 ttttggtag gatttgcta acagctctgg ggatgtctct aaatcttccc atagctagg 1800 tagagctgt aaagtcacc ttaaagccag gaaggttgg accaaattg aagcagtggc 1860 cattatcaa agaaagata gttgcattaa gagaaatctg tgaaagatg gaaaaagatg 1920 gtcagttgga ggaagctccc ccgaccaatc catacacac cccacattt gccataaga 1980 aaaaagaata gacaatgg agaatgctga tagattttag gaacataat agggtcactc 2040 aggactttac agaagtccaa ttaggatac cacaccctgc aggacttac aaaaggaaaa 2100 ggattacagt actgatata ggtgatgcat atttctccat acctctagat gagaattta 2160 ggcagtacac tgcctttact ttaccacg taaataatgc agagccagga aacgataca 2220 tttataaggt tctgcctcag ggatggaagg gtcaccagc catcttccaa tacactatga 2280 gatagtgct agaacccttc aggaaggcaa atccagatgt gaccttagctc cagtatatgg 2340 atgacatctt atagctagt gaggacag acctggaca tgacaggta gttttacagc 2400 taaaggaact cttaaatagc atagggttct ctaccccaga agagaatttc caaaagatc 2460 ccccattca atggatgggg tacgaattgt ggccgacaaa atggagttg siaagatag 2520 agttgccaca agagagacc tggacagtga atgatataca gaagttagta ggagtattaa 2580 attgggcagc tcaatttat ccaggtataa aaaccaaca tctctgtagg tttattagag 2640 gaaaaatgac tctaacagag gaagttcagt ggactgagat ggcagaagca gaatgagg 2700 aaaatagat attctcagt caggacaag aaggatgtta tchaccaga ggcaagccat 2760 tagagccac ggtaataaag agtcaggaca atcagtggtc ttaayaattt caccaagaag 2820 aaaatact gaaagtagga aaatttgcaa agatagaa tacacatacc aatggagtta 2880 gactattagc acatgtaata cagaaaatag gaaggaagc atagtgatc tggggacagg 2940 tcccaaaatt ccacttacca gttgagagggg atgtaggg acagtggtgg acagactatt 3000 ggcaggtaac ctggataccg gagtgggat ttatctcaac gccaccacta gtaagattag 3060 tcttcaatct agtgaggac cctatagagg gagagaaac cttattaca gatgatcat 3120 gtataaaca gtcaaagaa gggaaagcag gatatacac agataggggc aagacaaag 3180 taaaagtgtt agaacagact actaatcaac aagcagaatt agaagcatttt ctcatggcat 3240 tgacagactc agggccaag acaatatta tagtagattc acatagtt atgggaataa 3300 taacaggatg ccctacagaa tcagagagca ggctagttaa ccaaata gagaaatga 3360 ttaaaagtc agaatttat gtagcatggg taccacca caaggtata ggaggaaacc 3420 aagaaataga ccacctagtt agtcagggga ttagacaagt tctctcttg gaaaagatag 3480 agccagcaca agagaacat gataaatacc atagtaatgt aaagaattg gtattcaaat 3540 ttggattacc cagaatagtg gccagacaga tagtagacac ctgtgataaa tgtcatcaga 3600 aaggagaagc tatacatggg caggtaatt cagatctagg gacttggcaa atggactgta 3660 cccatctaga aggaaaaata gtcatagttg cagtacatgt agctagtgga ttcatagag 3720 cagaagtaat tccacagag acaggagac agacagcact atttctgtta aaattggcag 3780 gcagatggcc tattacacat ctacacacag ataatggtgc taactttgcc tcgcaagaag 3840 taaagatggt tgcatggtgg gcagggatag agcacacctt tggggtacca tacaatccac 3900 agagtcagggg agtagtggaa gcaatgaatc accacctgaa aaatcaata gatagaatca 3960 gggaacaagc aaattcagta gaaccatag tattaatggc agttcattgc atgaatttta 4020 aaagaagggg aggaataggg gatatgactc cagcagaaag attaattaac atgatcacta 4080 cagaacaaga aatacaattt caacaatcaa aaaactcaaa atttaaaaat tttcgggtct 4140 attacagaga aggcagagat caactgtgga agggacccgg tgagctattg tggaaagggg 4200 aaggagcagt catcttaaag gtagggacag acattaaggt agtacccaga agaaaggcta 4260 aaattatcaa agattatgga ggaggaaaag aggtggatag cagttcccac atggaggata 4320 ccggagaggc tagagaggtg gcatag 4346 <210> 164 <211> 506 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 164 Met Gly Ala Arg Asn Ser Val Leu Ser Gly Lys Lys Ala Asp Glu Leu 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr Met Leu Lys 20 25 30 His Val Val Trp Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu Ala Glu He Val Val Trp Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu Ala Glu Ser Leu Leu Glu Asn Lys Glu Gly Cys Gln Lys Ile Leu Ser Val Leu 50 55 60 Ala Pro Leu Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Tyr Asn 65 70 75 80 Thr Val Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys His 85 90 95 Thr Glu Glu Ala Lys Gln Ile Val Gln Arg His Leu Val Val Glu Thr 100 105 110 Gly Thr Ala Glu Thr Met Pro Lys Thr Ser Arg Pro Thr Ala Pro Ser 115 120 125 Ser Gly Arg Gly Gly Asn Tyr Pro Val Gln Gln Ile Gly Gly Asn Tyr 130 135 140 Val His Leu Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu 145 150 155 160 Ile Glu Glu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala 165 170 175 Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys 180 185 190 Val Gly Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile Ile Asn 195 200 205 Glu Glu Ala Ala Asp Trp Asp Leu Gln His Pro Gln Pro Ala Pro Gln 210 215 220 Gln Gly Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala Gly Thr Thr 225 230 235 240 Ser Ser Val Asp Glu Gln Ile Gln Trp Met Tyr Arg Gln Gln Asn Pro 245 250 255 Ile Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln Leu Gly Leu Gln 260 265 270 Lys Cys Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Val Lys Gln 275 280 285 Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser 290 295 300 Leu Arg Ala Glu Gln Thr Asp Ala Ala Val Lys Asn Trp Met Thr Gln 305 310 315 320 Thr Leu Leu Ile Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys 325 330 335 Gly Leu Gly Val Asn Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln 340 345 350 Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala Leu 355 360 365 Lys Glu Ala Leu Ala Pro Val Pro Ile Pro Phe Ala Ala Ala Gln Lys 370 375 380 Arg Gly Pro Arg Lys Pro Ile Lys Cys Trp Asn Cys Gly Lys Glu Gly 385 390 395 400 His Ser Ala Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys 405 410 415 Cys Gly Lys Met Asp His Val Met Ala Lys Cys Pro Asp Arg Gln Ala 420 425 430 Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro 435 440 445 Met Ala Gln Val His Gln Gly Leu Thr Pro Thr Ala Pro Pro Glu Asp 450 455 460 Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly Lys Gln Gln 465 470 475 480 Arg Glu Ser Arg Glu Lys Pro Tyr Lys Glu Val Thr Glu Asp Leu Leu 485 490 495 His Leu Asn Ser Leu Phe Gly Gly Asp Gln 500 505

Claims

1. A genetically modified dendritic cell or a precursor cell thereof modified by a nucleic acid construct, wherein the construct comprises: - a nucleic acid sequence a) encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or antigenic protein or an antigenic fragment thereof, wherein the human invariant chain is lip33, lip41, lip35 or lip43, said sequence a) being operably linked to a first promoter and optionally to a first transcriptional regulatory sequence, and - a nucleic acid sequence b) encoding at least one immunomodulatory protein, said sequence b) optionally being operably linked to a second promoter and optionally being operably linked to a second transcriptional regulatory sequence, wherein said immunomodulatory protein is IL-10.

2. The genetically modified dendritic cell or its precursor cell according to claim 1, wherein: The sequence a) further comprises at least one miRNA target sequence at its 3' end.

3. The genetically modified dendritic cell or its precursor cell according to claim 1 or 2, wherein: The first promoter and the second promoter are the same or different.

4. A genetically modified dendritic cell or a precursor cell thereof as claimed in any one of the preceding claims, wherein: The nucleic acid construct further comprises a sequence encoding a marker.

5. The genetically modified dendritic cell or its precursor cell according to claim 4, wherein: The marker is a selective marker.

6. A genetically modified dendritic cell or a precursor cell thereof as claimed in any one of the preceding claims, wherein: The antigenic peptide or antigenic protein or antigenic fragment thereof is derived from an autologous homologous antigen and / or a harmless antigen and / or an allergen.

7. A dendritic cell or a precursor cell thereof genetically modified as claimed in any one of the preceding claims, wherein: The antigenic peptide or antigenic protein or antigenic fragment thereof is selected from the group of immunodominant peptides described in any one of SEQ ID NOs: 1-114.

8. The genetically modified dendritic cell or its precursor cell according to any one of claims 2 to 7, wherein: The at least one miRNA target sequence is selected from the group consisting of: miR-15a, miR-16-1, miR-17, miR-18a, miR-19a, miR-20a, miR-19b-l, miR-21, miR-29a, miR-29b, miR-29c, miR-30b, miR-31, miR-34a, miR-92a-l, miR-106a, miR-125a, miR-125b, miR-126, miR-142-3p, miR-146a, miR-150, miR-155, miR-181a, miR-223 and miR-424.

9. The genetically modified dendritic cell or precursor cell thereof according to any one of claims 2 to 8, wherein at least one miRNA target sequence is selected from miR155, miR146a or a mixture thereof.

10. The genetically modified dendritic cell or precursor cell thereof according to claim 8 or 9, wherein the miRNA target sequence is repeated.

11. A genetically modified dendritic cell or a precursor cell thereof as claimed in any one of the preceding claims, wherein: The nucleic acid construct is inserted into a vector.

12. The genetically modified dendritic cell or precursor cell thereof according to claim 11, wherein the vector is a lentiviral vector.

13. The genetically modified dendritic cell or precursor cell thereof according to claim 11 or 12, wherein the vector is a unidirectional or bidirectional vector.

14. Use of the genetically modified dendritic cell or its precursor cell according to any one of claims 1 to 13 for preparing a medicament for preventing and / or treating organ rejection.

15. Use of the genetically modified dendritic cell or its precursor cell according to any one of claims 1 to 13 for preparing a medicament for preventing and / or treating a condition selected from the group consisting of graft-versus-host disease, autoimmune diseases, allergic diseases, inflammatory diseases, and immune responses induced by gene therapy.

16. The use according to claim 15, wherein The autoimmune disease is selected from the group consisting of type 1 diabetes, autoimmune bowel disease, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, autoimmune myositis, psoriasis, Addison's disease, Graves' disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, celiac disease, autoimmune hepatitis, alopecia areata, pemphigus vulgaris, vitiligo, aplastic anemia, autoimmune uveitis, alopecia areata, amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease), ankylosing spondylitis, anti-GBM nephritis, antiphospholipid syndrome, osteoarthritis, autoimmune active chronic hepatitis, autoimmune inner ear disease (AIED), Barlow's disease, Behcet's disease, Berger's disease, bullous pemphigoid, cardiomyopathy, chronic fatigue immune dysfunction syndrome, Tschag-Strauss syndrome, cicatricial pemphigoid, cold agglutinin disease, cranial arteritis colitis, CREST syndrome, Crohn's disease, Daigol's disease, dermatomyositis, and JDM, Devic disease, eczema, basic mixed cryoglobulinemia, eosinophilic fasciitis, fibromyalgia–fibromyositis, fibrosing alveolitis, giant cell arteritis, glomerulonephritis, Goodpass disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hepatitis, Hughes syndrome, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, irritable bowel syndrome, Kawasaki disease, lichen planus, lupus hepatitis, Lyme disease, Meniere's disease, mixed connective tissue disease, myositis, osteoporosis, Pars planitis, pemphigus vulgaris, polyglandular autoimmune syndrome, polymyalgia rheumatica, polymyositis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, Raynaud's syndrome, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, scleritis, scleroderma, sticky blood syndrome, Still's disease, stiff-man syndrome, Sydenham's chorea, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, Wegener's granulomatosis, and Wilson's syndrome.

17. The use according to claim 15, wherein the autoimmune disease is giant cell arteritis, anti-ANCA vasculitis, myasthenia gravis, refractory celiac disease, autoimmune uveitis, pemphigus vulgaris, giant cell myocarditis, Graves' disease or Addison's disease.

18. The use according to claim 15, wherein the autoimmune disease is granulomatosis with polyangiitis.

19. The use according to claim 15, wherein Allergic diseases are asthma, atopic allergy or atopic dermatitis.

20. The use according to claim 15, wherein The inflammatory disease is a chronic inflammatory disease.

21. The use according to claim 20, wherein the chronic inflammatory disease is inflammatory bowel disease or celiac disease.

22. The use according to claim 20, wherein the chronic inflammatory disease is Crohn's disease or ulcerative colitis.

23. The use according to claim 15, for preventing an immune response to protein replacement therapy.

24. The use according to claim 23, wherein the protein replacement therapy is used to treat a lysosomal storage disorder or hemophilia.

25. A nucleic acid construct comprising: - a nucleic acid sequence a) encoding a chimeric protein consisting of a human invariant chain fused to at least one antigenic peptide or antigenic protein or an antigenic fragment thereof, wherein the human invariant chain is lip33, lip41, lip35 or lip43, said sequence a) being operably linked to a first promoter and optionally to a first transcriptional regulatory sequence, and - a nucleic acid sequence b) encoding at least one immunomodulatory protein, said sequence b) optionally being operably linked to a second promoter and optionally being operably linked to a second transcriptional regulatory sequence, wherein said immunomodulatory protein is IL-10.

26. A vector comprising the nucleic acid construct as defined in claim 25.

27. The vector of claim 26, wherein the vector is a lentiviral vector.

28. The vector according to claim 26 or 27, wherein the vector is a unidirectional or bidirectional vector.

29. The vector of claim 26, wherein the vector is produced using enveloped viral particles expressing Vpx, and / or the vector is produced using packaging cells, wherein the packaging cells are genetically engineered to reduce expression of CD47.

30. An in vitro method for producing a genetically modified dendritic cell or a precursor cell thereof as defined in any one of claims 1 to 13, comprising the steps of: a. Isolation of PBMCs from subjects; b. Isolation of CD14 from the isolated PBMC + cell; c. Incubate the isolated CD14 with an effective amount of Vpx + cell; d. transducing the isolated CD14 with a vector as described in any one of claims 26-29 + cell.

31. The in vitro method of claim 30, wherein: Step d is performed in the presence of an effective amount of at least one agent.

32. The in vitro method of claim 31, wherein the agent is IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF) or IL-10.

33. The in vitro method of claim 32, wherein the amount of IL-4, the amount of GM-CSF or the amount of IL-10 is 1-1000 ng.

34. The in vitro method according to any one of claims 30 to 33, wherein: The PBMCs are isolated from peripheral blood or by leukapheresis.

35. The in vitro method according to any one of claims 30 to 33, wherein The vector is a lentiviral vector.

36. The in vitro method of claim 35, wherein the amount of the lentiviral vector is 1-100 MOI.

37. A genetically modified dendritic cell or a precursor cell thereof obtained by the method of any one of claims 30 to 33.

38. A CD49b that produces IL-10 + LAG-3 + An in vitro method for Tr1 cells, comprising the steps of: a) isolating PBMCs from a blood sample of a subject; b) exposing the isolated PBMC to an effective amount of the genetically modified dendritic cells or precursor cells thereof as defined in any one of claims 1 to 13 or 37 under suitable culture conditions.

39. The in vitro method of claim 38, wherein: The ratio of PBMC: genetically modified dendritic cells or their precursors is about 5:1 - 10:

1.

40. A CD49b that produces IL-10 obtainable by the method of claim 38 or 39 + LAG-3 + Tr1 cells.

41. A CD49b producing IL-10 obtainable by the method of claim 38 or 39 + LAG-3 + Use of Tr1 cells for preparing a drug for preventing and / or treating a disease selected from the group consisting of graft-versus-host disease, autoimmune diseases, allergic diseases, inflammatory diseases, and immune responses induced by gene therapy.

42. A CD49b that produces IL-10 obtainable by the method of claim 38 or 39 + LAG-3 + Use of Tr1 cells for preparing a medicament for preventing and / or treating organ rejection.

43. A method for producing antigen-specific FOXP3 + An in vitro method for T cells comprising the steps of: a) isolating PBMCs from a blood sample of a subject; b) exposing the isolated PBMC to an effective amount of the genetically modified dendritic cells or precursor cells thereof as defined in any one of claims 1 to 13 or 37 under suitable culture conditions.

44. The in vitro method of claim 43, wherein The genetically modified cells express at least indoleamine 2,3-dioxygenase (IDO).

45. Antigen-specific FOXP3 obtainable by the method of claim 43 or 44. + T cells.

46. ​​Use of IL-10-producing FOXP3+ T cells obtainable by the method of claim 43 or 44 for preparing a medicament for preventing and / or treating a condition selected from the group consisting of graft-versus-host disease, autoimmune diseases, allergic diseases, inflammatory diseases, and immune responses induced by gene therapy.

47. Use of IL-10-producing FOXP3+ T cells obtainable by the method of claim 43 or 44 for preparing a medicament for preventing and / or treating organ rejection.

48. A pharmaceutical composition comprising a genetically modified cell as defined in any one of claims 1 to 13 or 37 or a CD49b producing IL-10 as defined in claim 40 + LAG-3 + Tr1 cells or antigen-specific FOXP3 as defined in claim 45 + T cells or any combination thereof and a pharmaceutically acceptable carrier.

49. The pharmaceutical composition of claim 48, further comprising a therapeutic agent.

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