DESCRIPTION

TREATMENTS FOR SYSTEMIC LUPUS ERYTHEMATOSUS

This application claims benefit of priority to U.S. Provisional Application Serial No. 61/885,859, filed October 2, 2013, the entire contents of which are hereby incorporated by reference.

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under grant no. GM 103456 awarded by the National Institutes Health. The government has certain rights in the invention BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of medicine, autoimmune disease and molecular biology. More particularly, it concerns use of pharmaceutical agents to treat systemic lupus erythematosus.

2. Description of Related Art

Systemic lupus erythematosus (SLE) is an autoimmune disease that wreaks havoc through the body's own immune system. The disease is estimated to affect nearly 1 million Americans, primarily women between the ages of 20-40. SLE has an incidence of about 1- in-700 women between the ages of 20 and 60. SLE can affect any organ system and can cause severe tissue damage.

SLE is associated with the production of antinuclear antibodies, circulating immune complexes, and activation of the complement system. Numerous autoantibodies of differing specificity are present in SLE. SLE patients often produce autoantibodies having anti-DNA, anti-Ro, and anti-platelet specificity and that are capable of initiating clinical features of the disease, such as glomerulonephritis, arthritis, serositis, complete heart block in newborns, and hematologic abnormalities. These autoantibodies are also possibly related to central nervous system disturbances.

Untreated lupus can be fatal as it progresses from attack of skin and joints to internal organs, including lung, heart, and kidneys (with renal disease being the primary concern). Lupus mainly appears as a series of flare-ups, with intervening periods of little or no disease manifestation. Kidney damage, measured by the amount of proteinuria in the urine, is one of the most acute areas of damage associated with pathogenicity in SLE, and accounts for at least 50% of the mortality and morbidity of the disease.

Currently, there are no really curative treatments for patients who have been diagnosed with SLE. From a practical standpoint, physicians generally employ a number of powerful immunosuppressive drugs such as high-dose corticosteroids, e.g., prednisone, or azathioprine or cyclophosphamide, which are given during periods of flare-ups, but may also be given persistently for those who have experienced frequent flare-ups. Even with effective treatment, which reduces symptoms and prolongs life, many of these drugs have potentially harmful side effects to the patients being treated. In addition, these immunosuppressive drugs interfere with the person's ability to produce all antibodies, not just the self-reactive anti- DNA antibodies Immunosuppressants also weaken the body's defense against other potential pathogens, thereby making the patient extremely susceptible to infection and other potentially fatal diseases, such as cancer. In some of these instances, the side effects of current treatment modalities, combined with continued low-level manifestation of the disease, can cause serious impairment and premature death.

Methods for treatment of SLE involving antibodies are also described. For example, monoclonal antibodies against anti-DNA antibodies (the monoclonal antibodies being referred to therein as anti-idiotypic antibodies) are used to remove the pathogenic anti-DNA antibodies from the patient's system. However, the removal of large quantities of blood for treatment can be a dangerous, complicated process. High-dose intravenous immune globulin (IVIG) infusions have also been used in treating certain autoimmune diseases. Up until the present time, treatment of SLE with IVIG has provided mixed results, including both resolution of lupus nephritis (Akashi et al, 1990), and in a few instances, exacerbation of proteinuria and kidney damage (Jordan et al, 1989). In short, improved methods of treating SLE are desperately needed.

SUMMARY OF THE INVENTION

Thus, in accordance with the present disclosure, there is provided a method of treating a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function. Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration. Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus. The method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab). The one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor. The inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E. The inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor. The inhibitor may be given more than once, including chronic administration.

Also provided is a method of reducing flare duration or severity in a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function. Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration. Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus. The method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab). The one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor. The inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E. The inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor. The inhibitor may be given more than once, including chronic administration.

Other embodiments of the disclosure are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Examples section are understood to be embodiments of the disclosure that are applicable to all aspects of the disclosure.

The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions and kits of the disclosure can be used to achieve methods of the disclosure.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." It is also contemplated that anything listed using the term "or" may also be specifically excluded.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any foam of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-E. CpG-induced p38 MAPK activation is down-regulated in Bankl ' ^' B cells. (FIG. 1A) Proliferation assay of CFSE-labeled splenic B cells from Bankl ^'1' mice and its littermate control ( ^+/+ ) mice following 2 μΜ CpG or 20 μg/ml LPS stimulation for 48, 72 and 96h. Propidium iodide (Pl)-negative viable cells were gated and analyzed by flow cytometry. Data shown is representative of 3 independent experiments. (FIGS. 1B-C) Cell extracts from WT and Bankl KO splenic B cells stimulated with 10 μg/mL anti-mouse IgM F(ab') ₂ (anti-IgM) (FIG. IB) or 2 μΜ CpG (FIG. 1C) and analyzed by Western blot with specific antibodies for ERK, ΓΝΚ, p38 and Ι Β and the corresponding phospho-specific antibodies. (FIG. ID) Quantification of densitometry analysis of phospho-p38 Western blot data is shown as mean ± SEM. (FIG. IE) Cell extracts from splenic B cells stimulated with 20 μg/mL LPS and 1 μg/mL of the TLR7 agonist (R848) were analyzed by Western blot with specific antibodies to p38. WB data shown is representative of 2 independent experiments.

FIGS. 2A-C. CpG-induced IL-6 production is downregulated in Bankl ' ^' B cells, but Tlr9 gene expression is not affected. (FIG. 2A) IL-6 and IL-10 were measured by ELISA of WT ( ^+/+ ) and Bankl KO ( ^_/~ ) B-cell supernatants collected at the indicated times following CpG, anti-IgM+ CpG or CpG+ anti-mouse CD40 (10 μg/ml). Data shown are mean ± SD of 3 replicates, and is representative of 4 independent experiments. (FIG. 2B) Splenic B cells from Bankl ^+I+ and Bankl ^'1' mice were stimulated with 2 μΜ CpG for the indicated times. Relative expression of the TLR9 gene was analyzed by Taqman real-time PCR. Data shown is the mean ± SEM of 3 replicates from a representative experiment out of 3 performed. (FIG. 2C) Bankl ^+/+ and Bankl ^' ' ^' splenic B cells were stimulated with LPS (20 μg/ml) or R848 (1 μg/mL), or left unstimulated for the times shown. Data are the representative of two independent experiments with 3 replicates each. Bars represent mean ± SD.

FIGS. 3A-D. MK2 and MNK1/2 inhibitors suppress IL-6 cytokine secretion in mouse splenic B cells. Mouse C57BL/6J (WT) splenic B cells (1 * 10 ⁶ cells/ml) were seeded in triplicate in RPMI 1640 complete medium in a 48-well tissue culture plate. The cells were pretreated with and without (FIG. 3 A) MK2 specific inhibitor, PF3644022 (1 and 10 μΜ) for an hour, (FIG. 3B) MNK1 inhibitor, CGP57380 (10 and 20 μΜ) for 30 min, and (FIG. 2C) MNKs inhibitor cercosporamide (20 and 50 μΜ) for an hour before stimulation of CpG (2 μΜ) for 24h. The control well contains CpG and cell culture grade DMSO (<0.2% v/v) only. The culture supernatants were collected after 24h of stimulation and the capture ELISA for IL-6 was performed. The culture supernatants were placed in triplicate in a 96-well ELISA plate and IL-6 ELISA was performed. Bars represent mean ± SD from two independent experiments. Unpaired t-test was performed and all the treated groups were compared with control, **p<0.001 and ***p<0.0001. (FIG. 3D) Viability of cells after above treatment was tested by propidium iodide (PI) labeling, the whole cells were gated and analyzed by flow cytometry, the figure shows (Pl)-negative viable cells.

FIGS. 4A-D. Reduced IL-6 secretion can be attributed to downregulation of the phosphorylation of the p38-MNKl/2-eIF4E cascade in Bankl ^' ' ^' B cells following CpG stimulation and not to IL-6 gene transcription and IL-6 mRNA stability. (FIG. 4A) Splenic B cells from Bankl ^+/+ and Bankl ^'1' mice were stimulated with 2 μΜ CpG for indicated time shown on the plot. Relative expression of the IL-6 gene was determined using Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from 1 out of 3 independent experiments. (FIG. 4B) Splenic B cells from Bankl ^+/+ and Bankl ^'1' littermate mice were stimulated with 2 μΜ CpG for 20h followed by the addition of 1 μg/mL of actinomycin D (Act D). Relative expression of the IL-6 gene was analyzed with RT-PCR, and % of IL-6 mRNA was determined. Data shown is the mean of 3 replicates from one representative experiment out of three performed. The thin dotted line indicates 50% degradation of IL-6 mRNA. (FIG. 4C) Bank ^+/+ or ^_/~ splenic B cells were stimulated with 2 μΜ CpG for the indicated times and tested by Western blot using p-MNKl/2, MNK1/2, p-eIF4E, and eIF4E specific antibodies. The data are representative of three independent experiments. (FIG. 4D) Quantification analysis of phospho-MNKl/2 and phospho-eIF4E Western blot data by densitometry. The relative intensities of phospho-MNKl/2 and phospho-eIF4E were quantified using ImageJ software (freely available through the National Institutes of Health). The plots are from 3 independent experiments and data are expressed as mean ± SD.

FIGS. 5A-C. Bankl deficiency does not affect 4E-BP1 phosphorylation controlled through the AKT-mTORCl signaling cascade. Splenic B cells from Bankl+/+ and Bankl-/- littermate mice were stimulated with (FIG. 5A) CpG (2 μΜ), (FIG. 5B) anti- mouse CD40 (10 μg/ml), and (FIG. 5C) CpG (2 μΜ) in combination with anti-mouse CD40 (10 μg/ml) at 37 °C in a water bath for the indicated time points. Cytoplasmic cell extracts were prepared by using B cell lysis buffer containing appropriate amount of protease inhibitor and sodium vanadate. Equal amount of cell lysate protein (approx. 2x l0 ⁶ cell) was loaded in each lane and western blot was performed as written in the material and methods. The data are representative of two independent experiments. The relative intensities of the phospho-bands were quantified as described in FIGS. 4A-D.

FIGS. 6A-C. Cell viability and CFSE-labeled cell proliferation between Bankl ^+/+ and Bankl ^'A splenic B cells. CFSE-labeled splenic B cells were treated with the indicated stimuli for the indicated time, harvested and analyzed by flow cytometry. Debris and doublet cells were discriminated and viable cells (propidium iodide-negative) were gated. % of viability was determined. Histograms represent overlaid data from un-stimulated CFSE- labeled B cells, stimulated Bankl ^+/+ (solid line) and Bankf B cells (dash line).

FIGS. 7A-B. Immunoglobulin isotype switch or expression of BLIMP1 (Prdml) is not significantly affected by BANK1 deficiency. (FIG. 7A) Splenic B cells (l x lO ⁶ cells/ml) from Bankl ^+/+ and BankP ^A littermate mice were seeded in RPMI 1640 complete medium in a 48 well tissue culture plate. The cells were either stimulated with CpG (2 μΜ) or left unstimulated for 6 days. The supernatants were analyzed for immunoglobulin isotype antibodies. The data are presented as O.D. except for IgG2a/2c where data are represented as ng/ml. Data are from 4 independent experiments. Bars represent mean ± SD. Significance of IgG2a/2c between Bankl ^+/+ and BankT ^1' was tested using an unpaired t-test. p<0.05% was considered significant. (FIG. 7B) The relative expression of Prdml was determined with Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from one representative out of three independent experiments.

FIGS. 8A-B. Increased production of IL-6 by splenic purified B cells from BANKIFL-Tg with CpG or anti-CD40 + CpG stimulation. The cytokine IL-6 was measured with ELISA in supernatants of BANKIFL-Tg mice and compared to WT controls from purified B-cells collected at indicated times after CpG stimulation (FIG. 8A) or CpG and anti-CD40 stimulation (FIG. 8B). Data shown are mean ± SD of 3 replicates, and representative of 3 independent experiments.

FIGS. 9A-C. Deficiency of BANK1 modifies several phenotypes present in

B6.Slel ^{z z} .Yaa lupus prone mouse. (FIG. 9A) Serum from 25 week old Be.Slel^Bankl ' ^' .yaa mice and genotype littermates heterozygous and sufficient for Bankl was measured for presence of IgG anti-dsDNA antibodies using ELISA in serial dilutions. Black circles show positive control sera. (FIG. 9B) A pair of littermates of B6.Slel ^{z z} Bank ^~ .yaa and B6.Slel ^z/z Bankl ^+M .yaa mice of 12 weeks of age was used in FIGS. 9B-C. Stark differences in populations of B and T cells in the spleen and lymph nodes between B6.Slel.yaa Bankl- sufficient and 5aw 7-deficient mice were observed. (FIG. 9C) Picture of the inguinal lymph nodes of the same mice as in FIG. 9B.

FIG. 10. Deficiency of Bankl restores IL-6 levels in serum of lupus-prone mice to normal. Serum from 25 week-old B6.Slel ^{z/ '} Bankl ^{'1' '} .yaa mice and genotype littermates sufficient for Bankl were measured for presence of IL-6 using ELISA (*p<0.05).

FIG. 11. Inhibitors of the translation initiation pathway are capable of inhibiting IL-6 production induced with CpG by BANK1 human transgenic B cells. Left panel - Twelve weeks old hBanklTg/wt.mBankl-/- female mice were used for total splenocyte isolation. 0.5 x 10 ⁶ Spleen cells/500 μΐ CRPMI pre-treated with and without the MNK1 inhibitor CGP57380 at 5, 10 and 20 μΜ; and PatA 0.1 to 0.005 μΜ concentrations for 30 min. Then these cells were stimulated with and without CpG and in combination with anti-CD40 ligand for 24h. After stimulation, supernatants were checked for inhibition of IL-6 using ELISA, and cells were processed for staining and cell viability (right panel) checked with FACSCalibur. P values: ** p <0.01 ; **** p <0.0001.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

As discussed above, there remains a critical need for improved therapies for SLE. Because of the role of CpG-induced signaling in autoimmunity, and the putative role of BANK1 as a TIR-containing adaptor, the inventors hypothesized that BANK1 may participate in CpG-induced signaling. Their results established a function for BANK1 in CpG-induced responses that have important implications for the role of BANK1 in infections and autoimmunity, where BANK1 has been established as a susceptibility gene (Kozyrev et ah, 2008). In particular, the inventors have connected BANK1 function with IL-6 translation through the eIF4E pathway, and propose eIF4E as a target for treating SLE. These and other aspects of the disclosure are described in greater detail below.

I. Systemic Lupus Erythematosus

A. Disease Manifestations

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease (or autoimmune connective tissue disease) that can affect any part of the body. The disease occurs nine times more often in women than in men, especially in women in child-bearing years ages 15 to 35, and is also more common in those of non-European descent.

As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage. SLE can induce abnormalities in the adaptive and innate immune system, as well as mount Type III hypersensitivity reactions in which antibody-immune complexes precipitate and cause a further immune response. SLE most often damages the joints, skin, lungs, heart, blood components, blood vessels, kidneys, liver and nervous system. The course of the disease is unpredictable, often with periods of increased disease activity (called "flares") alternating with suppressed or decreased disease activity. A flare has been defined as a measurable increase in disease activity in one or more organ systems involving new or worse clinical signs and symptoms and/or laboratory measurements. It must be considered clinically significant by the assessor and usually there would be at least consideration of a change or an increase in treatment (Ruperto et ah, 2010).

SLE has no cure, and leads to increased morbidity and early mortality in many patients. The most common causes of death in lupus patients include accelerated cardiovascular disease (likely associated with increased inflammation and perhaps additionally increased by select lupus therapies), complications from renal involvement and infections. Survival for people with SLE in the United States, Canada, and Europe has risen to approximately 95% at five years, 90% at 10 years, and 78% at 20 years in patients of European descent; however, similar improvements in mortality rates in non-Caucasian patients are not as evident. Childhood systemic lupus erythematosus generally presents between the ages of 3 and 15, with girls outnumbering boys 4: 1, and typical skin manifestations being butterfly eruption on the face and photosensitivity.

SLE is one of several diseases known as "the great imitators" because it often mimics or is mistaken for other illnesses. SLE is a classical item in differential diagnosis, because SLE symptoms vary widely and come and go unpredictably. Diagnosis can thus be elusive, with some people suffering unexplained symptoms of untreated SLE for years. Common initial and chronic complaints include fever, malaise, joint pains, myalgias, fatigue, and temporary loss of cognitive abilities. Because they are so often seen with other diseases, these signs and symptoms are not part of the American College of Rheumatology SLE classification criteria. When occurring in conjunction with other signs and symptoms, however, they are suggestive.

The most common clinical symptom which brings a patient for medical attention is joint pain, with the small joints of the hand and wrist usually affected, although nearly all joints are at risk. Between 80 and 90% of those affected will experience joint and/or muscle pain at some time during the course of their illness. Unlike rheumatoid arthritis, many lupus arthritis paitents will have joint swelling and pain, but no X-ray changes and minimal loss of function. Fewer than 10% of people with lupus arthritis will develop deformities of the hands and feet. SLE patients are at particular risk of developing articular tuberculosis. An association between osteoporosis and SLE has been found, and SLE may be associated with an increased risk of bone fractures in relatively young women.

Over half (65%) of SLE sufferers have some dermatological manifestations at some point in their disease, with approximately 30% to 50% suffering from the classic malar rash (or butterfly rash) associated with the name of the disorder. Some may exhibit chronic thick, annual scaly patches on the skin (referred to as discoid lupus). Alopecia, mouth ulcers, nasal ulcers, and photosensitive lesions on the skin are also possible manifestations. Anemia may develop in up to 50% of lupus cases. Low platelet and white blood cell counts may be due to the disease or as a side effect of pharmacological treatment. People with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum. Abnormalities associated with antiphospholipid antibody syndrome include a paradoxical prolonged partial thromboplastin time (which usually occurs in hemorrhagic disorders) and a positive test for antiphospholipid antibodies; the combination of such findings has earned the term "lupus anticoagulant- positive." SLE patients with anti-phospholipid autoantibodies have more ACR classification criteria of the disease and may suffer from a more severe lupus phenotype.

A person with SLE may have inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis. The endocarditis of SLE is characteristically noninfective (Libman-Sacks endocarditis), and involves either the mitral valve or the tricuspid valve. Atherosclerosis also tends to occur more often and advances more rapidly than in the general population. Lung and pleura inflammation can cause pleuritis, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome.

Painless hematuria or proteinuria may often be the only presenting renal symptom. Acute or chronic renal impairment may develop with lupus nephritis, leading to acute or end- stage renal failure. Because of early recognition and management of SLE, end-stage renal failure occurs in less than 5% of cases. A histological hallmark of SLE is membranous glomerulonephritis with "wire loop" abnormalities. This finding is due to immune complex deposition along the glomerular basement membrane, leading to a typical granular appearance in immunofluorescence testing.

Neuropsychiatric syndromes can result when SLE affects the central or peripheral nervous systems. The American College of Rheumatology defines 19 neuropsychiatric syndromes in systemic lupus erythematosus. The diagnosis of neuropsychiatric syndromes concurrent with SLE is one of the most difficult challenges in medicine, because it can involve so many different patterns of symptoms, some of which may be mistaken for signs of infectious disease or stroke. The most common neuropsychiatric disorder people with SLE have is headache, although the existence of a specific lupus headache and the optimal approach to headache in SLE cases remains controversial. Other common neuropsychiatric manifestations of SLE include cognitive dysfunction, mood disorder (including depression), cerebrovascular disease, seizures, polyneuropathy, anxiety disorder, cerebritis, and psychosis. CNS lupus can rarely present with intracranial hypertension syndrome, characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space-occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents. More rare manifestations are acute confusional state, Guillain-Barre syndrome, aseptic meningitis, autonomic disorder, demyelinating syndrome, mononeuropathy (which might manifest as mononeuritis multiplex), movement disorder (more specifically, chorea), myasthenia gravis, myelopathy, cranial neuropathy and plexopathy. Neural symptoms contribute to a significant percentage of morbidity and mortality in patients with lupus. As a result, the neural side of lupus is being studied in hopes of reducing morbidity and mortality rates. The neural manifestation of lupus is known as neuropsychiatric systemic lupus erythematosus (NPSLE). One aspect of this disease is severe damage to the epithelial cells of the blood-brain barrier.

SLE causes an increased rate of fetal death in utero and spontaneous abortion (miscarriage). The overall live-birth rate in SLE patients has been estimated to be 72%. Pregnancy outcome appears to be worse in SLE patients whose disease flares up during pregnancy. Neonatal lupus is the occurrence of SLE symptoms in an infant born from a mother with SLE, most commonly presenting with a rash resembling discoid lupus erythematosus, and sometimes with systemic abnormalities such as heart block or hepatosplenomegaly. Neonatal lupus is usually benign and self-limited.

Fatigue in SLE is probably multifactorial and has been related to not only disease activity or complications such as anemia or hypothyroidism, but also to pain, depression, poor sleep quality, poor physical fitness and lack of social support.

Different clinical measurements have been used to determine whether a SLE patients is having a clinic flare. One of the most common measurements is the Systemic Lupus Erythematosus Disease Activity Index SELENA Modification (world-wide-web at rheumatology.org/Practice/Clinical/Indexes/Systemic_Lupus_Er ythematosus_Disease_Activi ty_Index_SELENA_Modification/). This scale uses a point system to calculate when the accumulated significance of recent changes in various indicators translates into a mild/moderate (SLEDA Index of 3-1 1 point change) or a severe (12 of more point change) flare. Although helpful in defining clinical flares in therapeutic and observational SLE clinical trials, this information only defines a flare state and does not help predict or identify patients who likely have an impending flare (an important clinical problem). In addition, no consensus, objective molecular test or tests are consistently associated individually with increased disease activity, nor with imminent SLE disease flare. Having such a molecular test would be greatly beneficial to SLE clinical care to help guide therapy, prevent damage, and minimize therapeutic toxicity.

B. Diagnosis

Antinuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti-ENA) responses form the mainstay of SLE serologic testing. Several techniques are used to detect ANAs. Clinically the most widely used method is indirect immunofluorescence. The pattern of fluorescence suggests the type of antibody present in the patient's serum. Direct immunofluorescence can detect deposits of immunoglobulins and complement proteins in the patient's skin. When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus.

ANA screening yields positive results in many connective tissue disorders and other autoimmune diseases, and may occur in healthy individuals. Subtypes of antinuclear antibodies include anti-Smith and anti-double-stranded DNA (dsDNA) antibodies (which are linked to SLE) and anti-histone antibodies (which are linked to drug-induced lupus). Anti- dsDNA antibodies are relatively specific for SLE; they are present in up to 50% of cases depending on ethnicity, whereas they appear in less than 2% of people without SLE. The anti-dsDNA antibody titers also tend to reflect disease activity, although not in all cases. Other ANA that may occur in SLE sufferers are anti-Ul RNP (which also appears in systemic sclerosis), anti-Ro (or anti-SSA) and anti-La (or anti-SSB; both of which are more common in Sj5gren's syndrome). anti-Ro and anti-La, when present in the maternal circulation, confer an increased risk for heart conduction block in neonatal lupus. Other tests routinely performed in suspected SLE are complement system levels (low levels suggest consumption by the immune system), electrolytes and renal function (disturbed if the kidneys are involved), liver enzymes, and complete blood count. II. IL-6, BANK1 and eIF4

A. IL-6

Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory and an antiinflammatory cytokine. In humans, it is encoded by the IL-6 gene. IL-6 is secreted by T cells and macrophages to stimulate immune response, e.g., during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium Streptococcus pneumoniae.

IL-6 is also considered a myokine, a cytokine produced from muscle, and is elevated in response to muscle contraction. It is significantly elevated with exercise, and precedes the appearance of other cytokines in the circulation. During exercise, it is thought to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery. In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine. IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on

TNF-alpha and IL-1, and activation of IL-lra and IL-10.

IL-6 is one of the most important mediators of fever and of the acute phase response.

It is capable of crossing the blood-brain barrier and initiating synthesis of PGE ₂ in the hypothalamus, thereby changing the body's temperature setpoint. In muscle and fatty tissue,

IL-6 stimulates energy mobilization that leads to increased body temperature. IL-6 can be secreted by macrophages in response to specific microbial molecules, referred to as pathogen-associated molecular patterns (PAMPs). These PAMPs bind to a highly important group of detection molecules of the innate immune system, called pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and intracellular compartments and induce intracellular signaling cascades that give rise to inflammatory cytokine production.

IL-6 is also essential for hybridoma growth and is found in many supplemental cloning media such as briclone. Inhibitors of IL-6 (including estrogen) are used to treat postmenopausal osteoporosis. IL-6 is also produced by adipocytes and is thought to be a reason why obese individuals have higher endogeneous levels of CRP. Intranasally administered IL-6 has been shown to improve sleep-associated consolidation of emotional memories.

IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells.

IL-6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL-6Ra chain (CD 126), and the signal-transducing component gpl30 (also called CD130). CD130 is the common signal transducer for several cytokines including leukemia inhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. In contrast, the expression of CD 126 is restricted to certain tissues. As IL-6 interacts with its receptor, it triggers the gpl30 and IL-6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular regions of gpl30 to initiate a signal transduction cascade through certain transcription factors, Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs).

IL-6 is probably the best-studied of the cytokines that use gpl30, also known as IL-6 signal transducer (IL6ST), in their signalling complexes. Other cytokines that signal through receptors containing gpl30 are Interleukin 1 1 (IL-1 1), Interleukin 27 (IL-27), ciliary neurotrophic factor (CNTF), cardiotrophin- 1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), oncostatin M (OSM), Kaposi's sarcoma-associated herpesvirus interleukin 6-like protein (KSHV-IL6). These cytokines are commonly referred to as the IL-6 like or gpl30 utiliing cytokines.

In addition to the membrane-bound receptor, a soluble form of IL-6R (sIL-6R) has been purified from human serum and urine. Many neuronal cells are unresponsive to stimulation by IL-6 alone, but differentiation and survival of neuronal cells can be mediated through the action of sIL-6R. The sIL-6R/IL-6 complex can stimulate neurites outgrowth and promote survival of neurons and, hence, may be important in nerve regeneration through remyelination.

IL-6 is relevant to many diseases such as diabetes, atherosclerosis, depression, Alzheimer's Disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, Behcet's disease, and rheumatoid arthritis. Advanced/metastatic cancer patients have higher levels of IL-6 in their blood. Hence there is an interest in developing anti-IL-6 agents as therapy against many of these diseases. The first such is tocilizumab, which has been approved for rheumatoid arthritis. Another, ALD518, is in clinical trials.

B. BANK1

Human and mouse Bankl encode the B cell scaffold protein with ankyrin repeats ! (BANK1). BANK1 is a tyrosine kinase substrate that becomes extensively tyrosine phosphorylated and is capable of binding the Src family kinases Lyn and Blk (Castillejo- Lopez et ah, 2012; Yokoyama et ah, 2002) and promotes tyrosine phosphorylation of inositol 1,4,5-trisphosphate receptors. While apparently involved in BCR signaling, the function of BANK1 during signaling induced by CpG, an agonist of the major toll-like receptor, TLR9 expressed in B cells, is not known. It also is involved in B-cell receptor induced Ca ²⁺ mobilization from intracellular stores and promotes Lyn-mediated phosphorylation of IP3 receptors 1 and 2.

The gene is located on the long arm of chromosome 4 (4q24) on the Watson (plus) strand and is 284,206 bases in length. The gene encodes a protein of 785 amino acids (molecular weight 89.335 kDa) and four isoforms are known. The gene is expressed in B-cell but not T-cell or myeloid cell lines. The greatest expression is in CD19 ⁺ B-cells with very low expression in other cell populations.

It has been proposed that BANK1 acts as an adaptor or scaffold protein in the same family as the B cell adapter for PI3K (BCAP) and the Drosophila homologue Dof (Yokoyama et al, 2002). Consistent with this hypothesis, the inventors' recent studies have shown that exon 2 of human BANK1 encodes a highly hydrophobic domain, which renders the protein susceptible to aggregation (Kozyrev et al, 2012); scaffold and adaptor proteins are known to form complex structures to facilitate intracellular signaling at the proper time and differentiation stage. Furthermore, exon 2 also encodes a predicted N-terminal toll/IL-1 receptor (TIR) domain that is shared by BCAP (Troutman et al, 2012) and used in the interaction of BCAP with the adaptors MyD88 and TIRAP.

TLR9 is the major endosomal TLR in B cells that recognizes viral nucleic acids, and TLR9 signaling is believed to have an important role in autoimmunity (Christensen et al, 2005). TLR9 signaling is stimulated by hypomethylated DNA oligonucleotides or CpG (Bernasconi et al, 2003), leading to a pro-inflammatory response (Sun et al, 2007). CpG- induced signaling activates mitogen activated protein kinase (MAPK) pathways, including p38, JNK and ERK. Stimulation of p38 and ERK signaling by growth factors, stress or viral infections can induce transcriptional activation, but can also induce two pathways of post- transcriptional regulation of protein synthesis: (a) control of mRNA stabilization (Neininger et al, 2002 and Bollig et al, 2003) by the mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase) MK2, and (b) the transient formation of the heterotrimeric eIF4E/eIF4F/eIF4G translation initiation complex through phosphorylation of eIF4E (Banerjee et al, 2002; Morley, 1997).

In mice, the only kinases known to phosphorylate eIF4E are MNK1 and MNK2.

MNK2 is constitutively active, while MNK1 is regulated by the MAP kinases (Sonenberg, 2008). A second axis of control of eIF4E activation is through the AKT/mTORCl pathway. This pathway regulates the phosphorylation of 4E-BP1, the eIF4E binding protein. Under non-phosphorylated conditions, 4E-BP 1 retains eIF4E (Gingras et al, 1999 and Richter and Sonenberg, 2005). Once 4E-BP1 becomes phosphorylated by mTORCl, it releases eIF4E, which is in turn phosphorylated by MNK1/2 (Livingstone et al, 2009).

C. eIF4

Eukaryotic initiation factor (elF) complex 2 forms a ternary complex with GTP and the initiator Met-tRNA - this process is regulated by guanine nucleotide exchange and phosphorylation and serves as the main regulatory element of the bottleneck of protein expression. Before translation can progress to the elongation stage, a number of initiation factors must facilitate the synergy of the ribosome and the mRNA and ensure that the 5' UTR of the mRNA is sufficiently devoid of secondary structure. Binding in this way is facilitated by group 4 eukaryotic initiation factors; eIF4 has implications in the normal regulation of translation as well as the transformation and progression of cancerous cells; as such, it represents an interesting field of research.

Eukaryotic translation initiation factor 4E, also known as eIF4E, is a protein that in humans is encoded by the EIF4E gene. eIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of a multiprotein complex termed eIF4F. The eIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. The other subunits of eIF4F are (a) a 50-kD polypeptide, termed eIF4A, that possesses ATPase and RNA helicase activities, and (b) a 220-kD polypeptide, eIF4G. eIF4E has been shown to interact with eIF4Al, eIF4EBP3, eIF4EBP l, eIF4EBP2, eukaryotic translation initiation factor 4γ, eIF4G2 and eIF4ENIF l.

eIF4E's function is to bind an mRNA cap and ultimately bring it to the ribosome. eIF4E is part of the eIF4F pre-initiation complex, which is made up of eIF4E, and eIF4G (eIF4F is sometimes considered to have additional protein components). Almost all cellular proteins require eIF4E in order to be translated into protein. eIF4E binds the first nucleotide on the 5' end of an mRNA molecule (known as the cap): a 7 methyl guanosine (m7G). It sandwiches m7G between 2 tryptophan residues, and other amino acids are involved in the binding.

Some viruses cut eIF4G in such a way that the eIF4E binding site is removed and the virus is able to translate its proteins without eIF4E. Also, some cellular proteins (the most notable being heat shock proteins) do not require eIF4E in order to be translated. Both viruses and cellular proteins achieve this through an IRES structure in the RNA.

Fragile X mental retardation protein (FMR1) acts to regulate translation of specific mRNAs through its binding of eIF4E. FMRP acts by binding CYFIP 1 , which directly binds eIF4e at a domain that is structurally similar to those found in 4E-BPs including EIF4EBP3, EIF4EBP 1, and EIF4EBP2. The FMRP/CYFIP 1 complex binds in such a way as to prevent the eIF4E-eIF4G interaction, which is necessary for translation (biology) to occur. The FMRP/CYFIP l/eIF4E interaction is strengthened by the presence of mRNA(s). In particular, BC1 RNA allows for an optimal interaction between FMRP and CYFIP 1. RNA-BC1 is a non-translatable, dendritic mRNA, which binds FMRP to allow for its association with a specific target mRNA. BC1 may function to regulate FMRP and mRNA interactions at synapse(s) through its recruitment of FMRP to the appropriate mRNA. In addition, FMRP may recruit CYFIP 1 to specific mRNAs in order to repress translation. The FMRP-CYFIP 1 translational inhibitor is regulated by stimulation of neuron(s). Increased synaptic stimulation resulted in the dissociation of eIF4E and CYFIP 1, allowing for the initiation of translation. III. Treating SLE

A. Treatments Targeting eIF4E

The present disclosure contemplates the use of inhibitors of eIF4 to treat SLE. In general, there are two categories of inhihitors - those that inhibit eIF4A/E/G expression, and those that do not inhibit expression but instead inhibit the activity of eIF4A/E/G. As discussed below, inhibitors may be biological - nucleic acid or peptides - or pharmaceutical (i.e., small molecules).

1. Biological Inhibitors

Antisense Constructs. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

Ribozymes. Another general class of inhibitors is ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et ah, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et ah, 1981 ; Michel and Westhof, 1990; Reinhold- Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et ah, 1981). For example, U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990). It has also been shown that ribozymes can elicit genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that was cleaved by a specific ribozyme.

RNAi. RNA interference (also referred to as "RNA-mediated interference" or RNAi) is another mechanism by which protein expression can be reduced or eliminated. Double- stranded RNA (dsRNA) has been observed to mediate the reduction, which is a multi-step process. dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp et al, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction. RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp, 1999; Sharp et al, 2000; Elbashir et al, 2001). It is generally accepted that RNAi acts post-transcriptionally, targeting RNA transcripts for degradation, and possibly by inhibiting translation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher et al, 2000).

siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et al, 1998). Of particular interest are those siRNAs that span an exon-intron junction. The making of siRNAs has been mainly through direct chemical synthesis; through processing of longer, double-stranded R As through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive. Chemical synthesis proceeds by making two single-stranded RNA-oligomers followed by the annealing of the two single-stranded oligomers into a double-stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889, 136, 4,415,732, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).

Several further modifications to siRNA sequences have been suggested in order to alter their stability or improve their effectiveness. It is suggested that synthetic complementary 21-mer RNAs having di-nucleotide overhangs (i.e., 19 complementary nucleotides + 3' non-complementary dimers) may provide the greatest level of suppression. These protocols primarily use a sequence of two (2'-deoxy)thymidine nucleotides as the di- nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA. The literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight (<20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.

Chemically synthesized siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et al. (2001) wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen et al, 2000; Elbashir et al, 2001).

WO 99/32619 and WO 01/68836 suggest that RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference. The enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. See U.S. Patent 5,795,715. The contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene. The length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length. An important aspect of this reference is that the authors contemplate digesting longer dsRNAs to 21-25 mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo. They do not describe or present data for synthesizing and using in vitro transcribed 21-25 mer dsRNAs. No distinction is made between the expected properties of chemical or enzymatically synthesized dsRNA in its use in RNA interference.

Similarly, WO 00/44914, incorporated herein by reference, suggests that single strands of RNA can be produced enzymatically or by partial/total organic synthesis. Preferably, single-stranded RNA is enzymatically synthesized from the PCR® products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides. WO 01/36646, incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures. This reference also provides that in vitro synthesis may be chemical or enzymatic, for example using cloned RNA polymerase (e.g., T3, T7, SP6) for transcription of the endogenous DNA (or cDNA) template, or a mixture of both. Again, no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.

U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized. The templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence. The templates can be attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.

In a specific embodiment, the inventors propose to inhibit eIF4A/E/G expression in adult tissues in vitro using siRNA or shRNA in an adenoviral vector. A GFP marker can be utilized to determine cells that take up the vector, and thus permit checking for appropriate inhibition of eIF4A/E/G production. The use of an inducible promoter allows for induction of the siRNA or shRNA only under specific growth conditions, thereby permitting reversible inhibition of eIF4A/E/G.

Peptide Fragments. The present invention contemplates the design, production and use of various eIF4A/E/G peptides. In general, the peptides will be 50 residues or less, again, comprising no more than 20 consecutive residues of eIF4A/E/G. The overall length may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. Ranges of peptide length of 4-50 residues, 5-50 residues, 6-50 residues, 7-50 residues, 7-25, residues, 4- 20 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 residues are contemplated. The number of consecutive eIF4A/E/G residues may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Ranges of consecutive residues of 4-20 residues, 5-20 residues, 6-20 residues, 7-20 residues and 4-15 residues, 5-15, residues, 6-15 residues or 7-15 residues are contemplated.

The present invention may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof. While L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in some proteins produced by exotic sea- dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain. The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L- glyceraldehyde is levorotary).

One form of an "all-D" peptide is a retro-inverso peptide. Retro-inverso modification of naturally-occurring polypeptides involves the synthetic assemblage of amino acids with a- carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence. A retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds ( H-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.

As mentioned above, the present invention contemplates fusing or conjugating a cell delivery domain (also called a cell delivery vector, or cell transduction domain). Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007). Of particular interest are poly-D-Arg and poly-D-Lys sequences (e.g., dextrorotary residues, eight residues in length), while others are shown in Table 1, below. TABLE 1

CDD/CTD PEPTIDES SEQ ID NO

QAATATRG SAASRPTERPRAPARSASRPRRPVE 5

RQIKI WF QNRRMKWKK 6

RRMKWKK 7

RRWRRWWRRWWRRWRR 8

RGGRLSYSRRRFSTSTGR 9

YGRK RRQRRR 10

RKKRRQRRR 11

YARAAARQARA 12

RRRRRRRR 13

KKKKKKKK 14

GWTLNSAGYLLGKINLKALAALAKXIL 15

LLILLRRRIR Q ANAH SK 16

SRRHHCRSKAKRSRHH 17

NRARRNRRRVR 18

RQLRIAGRRLRGRSR 19

KLIKGRTPIKFGK 20

RRIPNRRPRR 21

KLALKLALKALKAALKLA 22

KLAKLAKKLAKLAK 23

GALFLGFLGAAGSTNGAWSQPKKKRKV 24

KETWWETWWTEWSQPKKKRKV 25

GALFLGWLGAAGSTMGAKKKRKV 26

MGLGLHLLVLAAALQGAKSKRKV 27

AAVALLPAVLLALLAPAAANYKKPKL 28

MANLGYWLLALFVTMWTDVGLCKKRPKP 29

LGTYTQDFNKFHTFPQTAIGVGAP 30

DP GDP GVTVTVTVTVTG GDPXPD 31

PPPPPPPPPPPPPP 32

VRLPPPVRLPPPVRLPPP 33

PRPLPPPRPG 34

SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 35

TRSSRAGLQFPVGRVHRLLR 36

GIGKFLHSAKKFGKAFVGEIMNS 37

KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 38

ALWMTLLKKVLKAAAKAALNAVLVGANA 39

GIGAVL VLTTGLPALISWI RKRQQ 40

INLKALAALAKKIL 41

GFFALIP IISSPLP TLLSAVGSALGGSGGQE 42

LAKWALKQGFAKLKS 43

SMAQDIISTIGDLVKWIIQTVNXFTKK 44

LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPRTES 45

LKKLLKKLLKKLLKKLLKKL 46

KLKLKLKLKLKLKLKLKL 47

PAWRKAFRWAWRMLKKAA 48

Also as mentioned above, peptides modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino- and/or carboxyl-terminal residues.

It will be advantageous to produce peptides using the solid-phase synthetic techniques

(Merrifield, 1963). Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973. These synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide. The peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.

Aside from the 20 standard amino acids can can be used, there are a vast number of "non-standard" amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid. Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism. Nonstandard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.

Linkers or cross-linking agents may be used to fuse peptides to other proteinaceous sequences. Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety. The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols. The cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking polypeptides. In instances where a particular peptide does not contain a residue amenable for a given cross-linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.

Dominant negative mutants. Dominant negative proteins are mutant defective proteins with can negate the effects of normal, functional proteins when both are present in the same environment. In many cases, dominant negative proteins homo-multimerize and are thus able to "poison" a complext that contains one or more functional proteins. In designing dominant negative molecules, one may design molecules that interact with one binding partner but fail to interact with another, such as due to a conformational change or single site mutation or trunctation. Another option is to design a molecule that binds normally, but lacks a subsequent function such as an enzymatic activity (phosphorylation, cleavage, etc.). Yet another option is to remove a sequence from the molecule that would permit its translocation to another portion of the cell. Other possibilities also exist. 2. Pharmaceutical Inhibitors

An inhibitor of eIF4A function is hippuristinol, a small molecule found in the coral Ms hippuris. Its structure is shown below:

Another inhibitor of eIF4A is pateamine A (PatA), a natural product first isolated from marine sponges. This compound has attracted considerable attention as a potential anticancer agent. Recently, researchers have shown that PatA inhibits cap-dependent eukaryotic translation initiation. While PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. Its structure is shown below:

Another eIF4A inhibitor is 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2).

An inhibitor of eIF4E function is ribavirin. Ribavirin is a guanosine (ribonucleic) analog used to stop viral RNA synthesis and viral mRNA capping; simply put, it is a nucleoside inhibitor. Its brand names include Copegus™, Rebetol™, Ribasphere™, Vilona™, and Virazole™, and it is an anti-viral drug indicated for severe RSV infection (individually), (notably for persistent) hepatitis C infection (can be used in conjunction with peg-interferon a2b or peg-interferon a2a), and some other viral infections. Ribavirin is a prodrug, which when metabolized resembles purine RNA nucleotides. In this form it interferes with RNA metabolism required for viral replication.

Ribavirin is active against a number of DNA and RNA viruses. It is a member of the nucleoside antimetabolite drugs that interfere with duplication of viral genetic material. Ribavirin is active against influenzas, flaviviruses, and agents of many viral hemorrhagic fevers. In Europe and the U.S. the oral (capsule or tablet) form of ribavirin is used in the treatment of hepatitis C, in combination with pegylated interferon drugs. Ribavirin is the only known treatment for a variety of viral hemorrhagic fevers, including Lassa fever, Crimean-Congo hemorrhagic fever, Venezuelan hemorrhagic fever, and Hantavirus infection, although data regarding these infections are scarce and the drug might be effective only in early stages.

The primary observed serious adverse side effect of ribavirin is hemolytic anemia, which may worsen preexisting cardiac disease. The mechanism for this effect is due to ribavirin's buildup inside erythrocytes. Oxidative damage to erythrocyte cell membrane is usually inhibited by glutathione; however, with reduced ATP levels caused by ribavirin, glutathione levels are impaired, permitting oxidative erythrocyte cell lysis. The gradual loss of erythrocytes leads to anemia. The anemia is dose-dependent and may sometimes be compensated by decreasing dose. Ribavirin is also a teratogen in some animal species and thus poses a theoretical reproductive risk in humans, remaining a hazard as long as the drug is present, which can be as long as 6 months after a course of the drug has ended.

The aerosol form has been used in the past to treat respiratory syncytial virus-related diseases in children. However, its efficacy has been called into question by multiple studies, and most institutions no longer use it. It is still used in some cases. In Mexico, ribavirin ("ribavirina") has been sold for use against influenza. Studies have been mixed, but the derivative viramidine may have more promise. It has been used (in combination with ketamine, midazolam, and amantadine) in treatment of rabies. This drug is also used to control the life span of enterovirus 71 which causes hand, foot, and mouth disease. Notably, for severe RSV in children, the drug is delivered as aerosol particles for 12-18 hours daily. The method is very expensive, inconvenient and used rarely.

Physically ribavirin is similar to the sugar D-ribose from which it is derived. It is freely soluble in water, and is re-crystallized as fine silvery needles from boiling methanol. The three free sugar hydroxyls make the pure drug hydrophilic enough that it is only sparingly soluble in anhydrous ethanol. Classically, ribavirin is prepared from natural D- ribose by blocking the 2', 3' and 5' OH groups with benzyl groups, then derivatizing the OH with an acetyl group which acts as a suitable leaving group upon nucleophilic attack. The ribose Γ carbon attack is accomplished with a 1,2,4 triazole-3 -carboxymethyl ester, which directly attaches the nitrogen of the triazole to the carbon of the ribose, in the proper 1-β- D isomeric position. The bulky benzyl groups hinder attack at the other sugar carbons. Following purification of this intermediate, treatment with ammonia in methanolic conditions then simultaneously deblocks the ribose hydroxyls, and converts the triazole carboxymethyl ester to the carboxamide. Following this step, ribavirin may be recovered in good quantity by cooling and crystallization.

Ribavirin is possibly best viewed as a ribosyl purine analogue with an incomplete purine 6-membered ring. This structural resemblance historically prompted replacement of the 2' nitrogen of the triazole with a carbon (which becomes the 5' carbon in an imidazole), in an attempt to partly "fill out" the second ring, but to no great effect. Such 5' imidazole riboside derivatives show antiviral activity with 5' hydrogen or halide, but the larger the substituent, the smaller the activity, and all proved less active than ribavirin. Note that two natural products were already known with this imidazole riboside structure: substitution at the 5' carbon with OH results in pyrazomycin/pyrazofurin, an antibiotic with antiviral properties but unacceptable toxicity, and replacement with an amino group results in the natural purine synthetic precursor 5-aminoimidazole-4-carboxamide-l- -D-ribofuranoside (AICAR), which has only modest antiviral properties.

Derivatization of the triazole 5' carbon, or replacement with a nitrogen (i.e., the 1,2,4,5 tetrazole 3-carboxamide), also results in substantial loss of activity, as does alkyl derivatization of the 3' carboxamide nitrogen. The 2' deoxyribose version of ribavirin (the DNA nucleoside analogue) is not active as an antiviral, suggesting strongly that ribavirin requires RNA-dependent enzymes for its antiviral activity. Antiviral activity is retained for acetate and phosphate derivation of the ribose hydroxyls, including the triphosphate and 3',5' cyclic phosphates, but these compounds are no more active than the parent molecule, reflecting the high efficiency of esterase and kinase activity in the body.

The most successful ribavirin derivative to date is the 3-carboxamidine derivative of the parent 3-carboxamide, first reported in 1973 and now called taribavirin (former names viramidine and ribamidine). This drug shows a similar spectrum of antiviral activity to ribavirin, which is not surprising as it is now known to be a pro-drug for ribavirin. Viramidine, however, has useful properties of less erythrocyte-trapping and better liver- targeting than ribavirin. The first property is due to viramidine's basic amidine group which inhibits drug entry into RBCs, and the second property is probably due to increased concentration of the enzymes which convert amidine to amide, in liver tissue. Viramidine is in phase III human trials and may one day be used in place of ribavirin, at least against certain kinds of viral hepatitis. Viramidine's slightly superior toxicological properties may eventually cause it to replace ribavirin in all uses of ribavirin.

Ribavirin is absorbed from the GI tract probably by nucleoside transporters. Absorption is about 45%, and this is modestly increased (to about 75%) by a fatty meal. Once in the plasma, ribavirin is transported through the cell membrane also by nucleoside transporters. Ribavirin is widely distributed in all tissues, including the CSF and brain. The pharmacokinetics of ribavirin is dominated by trapping of the phosphate form inside cells, particularly red blood cells (RBCs) which lack the enzyme to remove the phosphate once it has been added by kinases, and therefore attain high concentrations of the drug. Most of the kinase activity which converts the drug to active nucleotide form, is provided by adenine kinase. This enzyme is more active in virally infected cells.

The volume of distribution of ribavirin is large (2000 L/kg) and the length of time the drug is trapped varies greatly from tissue to tissue. The mean half-life for multiple doses in the body is about 12 days, but very long-term kinetics are dominated by the kinetics of RBCs (half-life 40 days). RBCs store ribavirin for the lifetime of the cells, releasing it into the body's systems when old cells are degraded in the spleen. About a third of absorbed ribavirin is excreted into the urine unchanged, and the rest is excreted into urine as the de-ribosylated base 1,2,4-triazole 3-carboxamide, and the hydrolysis product of this, 1,2,4-triazole 3- carboxylic acid.

U.S. Patent Publication 20100144805 describes pharmaceutical small-molecule inhibitors of eIF4E that block the

wherein Ri is a hydrazone thiazole moiety of the structure:

or a barbituric acid moiety (or derivative thereof) of the structure:

R2 is hydrogen, hydroxyl or a nitro group present in one, two or three locations on the ring to which it is attached; R ₃ is a group individually present in one, two or three locations on the ring, wherein the group may be halo, hydrogen, conjugated or unconjugated aryl or heteroaryl, a alicyclic or polycyclic group, or R ₃ , taken with the ring to which it is attached, forms a conjugated ring structure, e.g., a naphthalene ring; R4 is hydrogen, carboxyl, a lower alkyl ester, e.g. :

< ) < _\ or carbonyl (in which case the dotted bond is present); R ₅ is N (in which case the dotted bond is present), NH, or carbonyl; and R-6 is NH or carbonyl.

Other possible inhibitors include MNK1/2 inhibitors (cercosporamide, S209, CGP57380, PF3644022) and mTOR ATP-active site inhibitors (rapamycin, RAD001, CCI- 779, PP242, Torinl, WYE-132, Ku-0063794, Palomid 529, and AZD8055, ΓΝΚ128, AZD2014 Palomid 529 BEZ235, PF-04691502, XL765).

B. Combination Therapies

The present invention also contemplates the treatment of SLE using standard therapeutic approaches where indicated in combination with the eIF4E therapies of the present disclosure. In general, the treatment of SLE involves treating elevated disease activity and trying to minimize the organ damage that can be associated with this increased inflammation and increased immune complex formation/deposition/complement activation. The compositions would be provided in a combined amount effective to exert a combined effect on the damaged tissue. This process may involve contacting the cells with the eIF4E inhibitor, in combination with a second therapeutic agent or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the eIF4E inhibitor and the other includes the second agent.

Alternatively, treatment with the eIF4E inhibitor may precede or follow the additional agent treatment by intervals ranging from minutes to weeks. In embodiments where the second agent is applied separately to the target, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent would still be able to exert an advantageously combined effect on the target. In such instances, it is contemplated that one would contact the target with both modalities within about 12-24 hr of each other and, more preferably, within about 6-12 hr of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (Yokoyama et al, 2002, Kozyrev et al, 2012, Troutman et al, 2012, Christensen et al, 2005, Bernasconi et al, 2003 and Sun et al, 2007) to several weeks (Yokoyama et al, 2002, Kozyrev et al, 2012, Troutman et al, 2012, Christensen et al, 2005, Bernasconi et al, 2003, Sun et al, 2007 and Neininger et al, 2002) lapse between the respective administrations. It also is conceivable that more than one administration of either the eIF4E inhibitor in combination with a second therapeutic agent will be desired. Various combinations may be employed, where the eIF4E inhibitor is "A" and the second therapeutic agent is "B", as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B Other combinations are contemplated.

Foundational treatment for combination therapies as described above can include corticosteroids and anti-malarial drugs. Certain types of lupus nephritis such as diffuse proliferative glomerulonephritis require round of cytotoxic drugs. These drugs include, most commonly, cyclophosphamide and mycophenolate. Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used for SLE 'off-label' In November 2010, an FDA advisory panel recommended approving belimumab (Benlysta) as a treatment for elevated disease activity seen in autoantibody- positive lupus patients. The drug was approved by the FDA in March 201 1.

Due to the variety of symptoms and organ system involvement with SLE, its severity in an individual must be assessed in order to successfully treat SLE. Mild or remittent disease may, sometimes, be safely left minimally treated with hydroxychloroquine alone. If required, nonsteroidal anti-inflammatory drugs and low dose steroids may also be used. Hydroxychloroquine (HCQ) is an FDA-approved antimalarial used for constitutional, cutaneous, and articular manifestations. Hydroxychloroquine has relatively few side effects, and there is evidence that it improves survival among people who have SLE and stopping HCQ in stable SLE patients led to increased disease flares in Canadian lupus patients. Disease-modifying antirheumatic drugs (DMARDs) are oftentimes used off-label in SLE to decrease disease activity and lower the need for steroid use. DMARDs commonly in use are methotrexate and azathioprine. In more severe cases, medications that aggressively suppress the immune system (primarily high-dose corticosteroids and major immunosuppressants) are used to control the disease and prevent damage. Cyclophosphamide is used for severe glomerulonephritis, as well as other life-threatening or organ-damaging complications, such as vasculitis and lupus cerebritis. Mycophenolic acid is also used for treatment of lupus nephritis, but it is not FDA-approved for this indication. Depending on the dosage, people who require steroids may develop Cushing's symptoms of truncal obesity, purple striae, buffalo hump and other associated symptoms. These may subside if and when the large initial dosage is reduced, but long-term use of even low doses can cause elevated blood pressure, glucose intolerance (including metabolic syndrome and/or diabetes), osteoporosis, insomnia, avascular necrosis and cataracts.

Numerous new immunosuppressive drugs are being actively tested for SLE. Rather than suppressing the immune system nonspecifically, as corticosteroids do, they target the responses of individual types of immune cells. Belimumab, or a humanized monoclonal antibody against B-lymphocyte stimulating factor (BlyS or BAFF), is FDA approved for lupus treatment and decreased SLE disease activity, especially in patients with baseline elevated disease activity and the presence of autoantibodies. Addition drugs, such as abatacept, epratuzimab, etanercept and others, are actively being studied in SLE patients and some of these drugs are already FDA-approved for treatment of rheumatoid arthritis or other disorders. Since a large percentage of people with SLE suffer from varying amounts of chronic pain, stronger prescription analgesics (pain killers) may be used if over-the-counter drugs (mainly nonsteroidal anti-inflammatory drugs) do not provide effective relief. Potent NSAIDs such as indomethacin and diclofenac are relatively contraindicated for patients with SLE because they increase the risk of kidney failure and heart failure.

Moderate pain is typically treated with mild prescription opiates such as dextropropoxyphene and co-codamol. Moderate to severe chronic pain is treated with stronger opioids, such as hydrocodone or longer-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone. The fentanyl duragesic transdermal patch is also a widely used treatment option for the chronic pain caused by complications because of its long-acting timed release and ease of use. When opioids are used for prolonged periods, drug tolerance, chemical dependency, and addiction may occur. Opiate addiction is not typically a concern, since the condition is not likely to ever completely disappear. Thus, lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.

Intravenous immunoglobulins may be used to control SLE with organ involvement, or vasculitis. It is believed that they reduce antibody production or promote the clearance of immune complexes from the body, even though their mechanism of action is not well- understood. Unlike immunosuppressives and corticosteroids, IVIGs do not suppress the immune system, so there is less risk of serious infections with these drugs. Avoiding sunlight is the primary change to the lifestyle of SLE sufferers, as sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. These two problems can lead to patients becoming housebound for long periods of time. Drugs unrelated to SLE should be prescribed only when known not to exacerbate the disease. Occupational exposure to silica, pesticides and mercury can also make the disease worsen.

Renal transplants are the treatment of choice for end-stage renal disease, which is one of the complications of lupus nephritis, but the recurrence of the full disease in the transplanted kidney is common in up to 30% of patients.

Antiphospholipid syndrome is also related to the onset of neural lupus symptoms in the brain. In this form of the disease the cause is very different from lupus: thromboses (blood clots or "sticky blood") form in blood vessels, which prove to be fatal if they move within the blood stream. If the thromboses migrate to the brain, they can potentially cause a stroke by blocking the blood supply to the brain. If this disorder is suspected in patients, brain scans are usually required for early detection. These scans can show localized areas of the brain where blood supply has not been adequate. The treatment plan for these patients requires anticoagulation. Often, low-dose aspirin is prescribed for this purpose, although for cases involving thrombosis anticoagulants such as warfarin are used.

C. Pharmaceutical Formulations and Delivery

Where therapeutic applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.

The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

For oral administration the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences," 15 ^th Ed., 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

In a particular embodiment, microbes can be engineered to deliver drugs according to the present disclosure. Lactobacillus strains have been used for the purpose (drug delivery of paclitaxel). L. acidophilus and L. rhamnosus can form minicells in modified MRS broth. Nanoparticle size of obtained minicells is ~400 nm in diameter. These minicells have been packaged paclitaxel (10 μg/ml) and cephalosporin (10 μg/ml). Alternatively, for protein drugs and certain small molecules, synthetic machinery can be embedded in microorganisms for the production of drugs. Delivery of the microorganism therefore constitutes administration of the drug itself.

IV. Examples

The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 - MATERIALS AND METHODS

Mice. Bankl ' mice were kindly provided by Dr T. Kurosaki (Riken Research Centre for Allergy and Immunology, Kyoto, Japan) and were backcrossed nine generations onto the C57BL/6J background. C57BL/6J mice were purchased from Jackson Laboratory, Bar Harbor, Maine, USA. Mice were maintained under specific pathogen free (SPF) barrier conditions. This study was approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.

Antibodies and reagents. Phospho-specific antibodies to p38 (Thrl 80/Tyrl82, clonel2F8 #4631), JNK (Thrl83/Tyrl85, clone 81E11, #4668), ERK (Thr202/Tyr204, clone D13.14.4E, #4370), I /c B a (Ser32, clone 14D4, #2859), eIF4E (Ser209, #9741), M K1/2 (Thrl97/202, #211 1), 4E-BP1 (Thr37/46, clone 236B4, #2855), mTOR (Ser2448, clone D9C2, #5536), and AKT (Ser473, clone 193H12, #4058) and phosphorylation-state- independent antibodies to p38 (#9212), JNK, ERK, I /c B a , eIF4E (clone C46H6, #2067), MNK1 (clone C4C1, #2195), 4E-BP1 (clone 53H1 1, #9644), mTOR (clone 7C10, #2983), and AKT (#9272), were purchased from Cell Signaling Technology (Danvers, MA). Phosphorylation-state-independent antibody for MNK2 (S-20) was purchased from Sigma- Aldrich (St. Louis, MI).

Purification of splenic B cells. Splenic B cells were purified by negative selection using magnetic bead separation. Briefly, spleen cells from Bankl ^+/+ and Bankl ^"7" littermates were labeled with a cocktail of biotin-conjugated antibodies for 15 minutes. Cells were incubated an additional 15 minutes with anti-biotin micro beads (B-Cell Isolation Kit, mouse; Miltenyi Biotech, Auburn, CA) at 4° C. The labeled non-B-cells were depleted by magnetic retention in a MACS column while unlabeled B cells were recovered. The purity of the resulting cell population was typically more than 95% B220 ⁺ CD3 ^" as assessed by flow cytometry analysis.

Cell Culture. Cell culture was performed with RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), 2-ME (50 μΜ) and antibiotics for all experiments, except for p38 analyses and the Akt axis where cells were serum-starved two hours prior to stimulation. Purified splenic B cells (10 ⁶ cells/mL) were treated with 10 μg/ml F(ab')2 fragment of anti-mouse IgM (Jackson Immunoresearch, West Grove, PA), 2 μΜ CpG (ODN Type B; 1668, Invivogen, San Diego, CA) or combination of anti-mouse IgM and CpG, 20 μg/mL TLR4 agonist LPS or 1 μg/mL of the TLR7 agonist R848 (Invivogen, San Diego, CA), or were left unstimulated. Supernatants were collected at 0, 12, 24 and 48 hr, and 72 and 96 hours for anti-CD40 stimulation (low-endotoxin, azide-free anti-mouse CD40, Biolegend, San Diego, CA) was included (10 μg/ml final concentration), with or without CpG. Western Blot. Stimulated splenic B cells were lysed in buffer containing 1% TritonX-100, 50 mM Tris pH 7.4, 50 mM NaCl, 1 mM EDTA, 2 mM Na ₃ V0 ₄ , and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, ΓΝ). LDS sample buffer and reducing agent (Life Technologies, Carlsbad, CA) were added. Samples were then boiled, and protein was separated with NuPAGE 4-12% Bis-Tris Gels (Life Technologies, Carlsbad, CA). Proteins were blotted onto polyvinylidene difluoride membranes, blocked with 5% skimmed milk and the immunoblots were processed with specific antibodies diluted with 5% BSA or 5% skimmed milk solution, and detected with Novex ECL substrate (Invitrogen, Carlsbad CA).

ELISA Assays. Cytokine secretion by in vitro stimulated B cells was measured using the ELISA kit for IL-6 and IL-10 purchased from BD Bioscience (San Jose, CA). For this, 96-well plates were coated with antibody against mouse IL-6 or mouse IL-10 overnight at 4°C. Plates were then blocked for 1 h at room temperature with 10% FBS in PBS. Supernatants were then added to the plate and left for 2 h at room temperature. Following washing, biotinylated anti-IL-6 or anti-IL-10 was added and plates incubated for further an hour at room temperature. Streptavidine alkaline phosphatase was then added following incubation at room temperature and washing with tetramethylbenzidine liquid substrate. Absorbance was read at 450 nm, from which absorbance was subtracted at 570 nm within 30 min of stopping reaction. Cytokine concentration was determined by extrapolation from the standard curve. Standard curve was generated using recombinant mouse IL-6 or recombinant mouse IL-10 (BD Bioscience, San Jose, CA).

Splenic B cells (0.5 l0 ⁶ cells/well) were stimulated for 6 days and supernatants were analyzed for immunoglobulin isotype antibodies as per the instructions from BD Pharmingen™ mouse immunoglobulin isotype ELISA kit (BD Biosciences, San Diego, CA, US). The inventors quantified IgG2a and IgG2c isotype antibodies using mouse IgG2a Ready-Set-Go ELISA kit (eBioscience Inc. San Diego CA, US) and mouse IgG2c ELISA Quantification Set (Bethyl Laboratories, Inc. TX, US) respectively and data are presented as ng/ml. For other antibodies, only OD's are shown.

Determination of Cell Viability and Proliferation Assay. Purified splenic B cells were resuspended in pre-warmed PBS/ 0.1% BSA and labeled with 2 μΜ CFSE (Life Technologies, Carlsbad, CA), and incubated at 37 °C in a water bath for 10 min. Five volumes of culture medium were added for quenching. After incubating 5 min on ice, the cell pellet was collected by centrifugation, washed 3 times with fresh complete RPMI medium and resuspended to the appropriate density. Cells were then stimulated with 10 μg/ml F(ab')2 fragment of anti-IgM, 2 μΜ CpG, 10 μg/ml anti-CD40, 20 μg/ml LPS, and 1 μg/ml R848, respectively, or the shown combinations and cultured for 48, 72 and 96 hr. After harvest, cells were stained with propidium iodide (PI, final concentration of 1 μg/ml; eBioscience Inc. San Diego CA, US) and acquired by LSR II or FACScalibur (BD bioscience, San Jose, CA) 36,000 events in total. For flow cytometry, debris and doublet cells were discriminated and viable cells (PI- negative) were gated and % of cell viability was determined. The histograms shown with FL1 channel represent overlaid data from un-stimulated CFSE-labeled B cells, stimulated Bankl ^+/+ and Bankl ^7" B cells, and the y-axis is presented as % viability of maximum. The analysis was conducted using FlowJo software (Tree Star Inc., Ashland, OR).

Inhibition of IL-6 production from splenic B cells. The MNK1 specific inhibitor,

CGP57380 [4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine] was obtained from Calbiochem®-Millipore (Billerica, MA), and MNKs inhibitor cercosporamide and MK2 inhibitor, PF-3644022 [(10R)-9,10,1 l,12-tetrahydro-10-methyl-3-(6-methyl-3-pyridinyl)-8H- [l,4]diazepino[5',6':4,5]thieno[3,2-f ]quinolin-8-one hydrate] were purchased from Sigma- Aldrich (St. Louis, MI). The purified splenic B cells (5 x 10 ⁵ cells/ well) were seeded in complete RPMI1640 medium in a 48 well tissue culture plate. Then the cells were pretreated with or without inhibitor for 30 min or lh, respectively, before CpG stimulation for 24 hr time at which cells were harvested and subjected to determine % of viability by using propidium iodide staining (final concentration 1 μg/ml) and flow cytometry analysis. Supernatants were collected and stored at -80 °C until use and IL-6 was measured using a capture ELISA. The control well contained CpG and cell culture grade DMSO (Pierce, Rockford, IL) only (17).

Taqman RT-PCR and IL-6 mRNA Stability Assay. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) from triplicate cultures of 5 x 10 ⁵ splenic B cells in the presence or absence of CpG treatment for the indicated times. After quantification and quality control of the RNA with a Nanodrop spectrophotometer (Thermo Scientific, West Palm Beach, FL), 400 ng RNA was subjected to first-strand cDNA synthesis for qRT-PCR (Origene, Rockvile MD). 5 ng total cDNA/RNA was used per reaction in TaqMan Fast Universal PCR Master Mix (Life Technologies, Carlsbad, CA). The qRT-PCR reactions were performed on a 7900 HT Fast Real-Time PCR system. The primers and probes for mouse TLR9 (Assay ID: Mm00446193_ml), Prdml (Mm00476128_ml) and IL-6 (Assay ID: Mm00446190_ml) and internal control gene 18S rRNA (4319413E) were purchased from Taqman Gene expression Assays (Life Technologies, Carlsbad, CA). For the IL-6 mRNA stability assay, 5 x 10 ⁵ splenic B cells were cultured in triplicate and treated with 2 μΜ CpG for 20h followed by addition of 1 μ^ηιΐ actinomycin D (Sigma- Aldrich, St. Louis, MI) for 0, 50, 100, 200 min. RNA was isolated and IL-6 transcription was determined with Taqman qRT-PCR using the primers as described above.

EXAMPLE 2 - RESULTS

BANK1 Deficiency Reduces CpG-Induced p38 Activation in Splenic B Cells. To discern the signaling cascades affected by BANK1, the inventors tested if BANK1 deficiency altered B cell proliferation after stimulation with CpG. Using purified splenic B-cells from Bankr ^7" and littermate control ( ^+/+ ) mice, they did not observe any differences at any of the time points tested in proliferation with CpG or LPS, a TLR4 agonist (FIG. 1A). The inventors also did not observe differences in cell proliferation when using anti-IgM, anti-IgM+CpG, anti-CD40, CpG + anti-CD40, R848, R848+anti-CD40, LPS, LPS + anti-CD40, or anti- IgM+LPS (FIGS. 6A-C). They then tested the effects of BANK 1 deficiency on IgM-induced activation of NF κ B by analyzing phosphorylation and degradation of I κ B a , and activation of the MAPK pathways by studying phosphorylation of ERK and p38. BANK1 deficiency did not affect phosphorylation of MAPKs, or I κ B a following stimulation with anti-IgM alone (FIG. IB).

After CpG stimulation, p38 phosphorylation was importantly reduced (FIGS. 1C and ID), but ERK, ΓΝΚ and I /c B a phosphorylation were unchanged, in Bankl ^7" cells compared to cells from WT mice (FIG. 1C) or from Bankl ^+/+ littermates (data not shown). For all pathways, stimulation with anti-IgM+CpG (data not shown) yielded results identical to CpG alone (FIG. 1C). BANK1 deficiency had no effect on p38 signaling induced by LPS or R848 (FIG. IE). Therefore, BANK1 specifically modulates CpG-induced p38 signaling in vitro.

BANK1 Deficiency Reduces CpG-Stimulated IL-6 Secretion. It is known that the

MAPK p38-signaling pathway regulates the production of several cytokines including IL-6, TNFa, IFNy, and IL-10 (Enslen et al, 1998, Nagaleekar et al, 201 1 and Foey et al, 1998). Having shown that BANK1 deficiency reduced p38 phosphorylation after CpG stimulation, the inventors asked whether reduction in p38 phosphorylation leads to modulation of IL-6 or IL-10 secretion by Bankl ^7" B cells. As expected (Vanden and Bishop, 2008, Poudrier et al, 1998 and Barr et al, 2007), anti-IgM alone did not induce detectable IL-6 or IL-10 in splenic B-cells from WT, Bankl ^+/+ or Bankl ^7" littermates (data not shown), while CpG induced detectable levels of IL-10 and IL-6 production. Bankl ^7" B-cells showed consistently reduced IL-6 but normal IL-10 secretion in response to CpG alone or the combination of CpG and BCR ligation (FIG. 2A).

In general, it has been observed that the combination of anti-CD40 and CpG leads to the most optimal secretion of IL-6 by B cells (Saito et al , 2002 and Haxhinasto and Bishop, 2004), and BANKl deficiency has been shown to lead to activation of Akt following stimulation through CD40. When the inventors investigated production of IL-6 by combining anti-CD40 and CpG, they clearly observed that the amount of IL-6 secreted was importantly increased when adding anti-CD40 to CpG as compared to CpG alone in Bankl ^+/+ B cells. Deficiency of BANKl reduced the levels of secreted IL-6 (FIG. 2A). The inventors tested if stimulation with CpG would lead to a reduction in cell viability by BANKl deficient cells and hence to reduced IL-6 secretion. This was not the case (FIG. 6). The inventors did observe slightly fluctuated cell viability when combining CpG with anti-CD40 in BANKl deficient cells, but proliferation was normal. Hence, the inventors conclude that reduced secretion of IL-6 is not due to changes in cell viability in BANKl deficient B cells.

CpG is a known agonist of Tlr9. To test if the reduced expression of IL-6 and reduced phosphorylation of p38 could be due to reduced expression of Tlr9, the inventors tested mRNA expression. Tlr9 gene expression was no different between Bankl ^+/+ and Bankl ^"7" mice (FIG. 2B). Next, the inventors examined whether BANKl deficiency had an effect on the production of IL-6 induced by TLR7 and TLR4. However, LPS and the TLR7 agonist R848 induced subtle amounts of IL-6 (Vanden and Bishop, 2008 and Poudrier et al, 1998) as compared to CpG stimulation and BANKl deficiency had no effect (FIG. 2C). Taken together, these results show that BANKl deficiency reduces secretion of IL-6 in response to CpG stimulation, overcomes the effect of anti-CD40 and is not due to changes in cell viability or to reduced Tlr9 expression.

Bankl affects translation initiation via the MNK1/2 and eIF4E pathway but not mRNA stability, which is controlled by MK2. The inventors suspected that BANKl deficiency could decrease IL-6 production by reducing IL-6 translation initiation and/or mRNA stability, since p38 directly controls these via the kinases MNK1/2 and MK2, respectively (Raught and Gingras, 1999; Ronkina et al, 2007). MK2 inhibitors are known to reduce IL-6 secretion by human peripheral blood mononuclear cells (Mourey et al, 2010). Consistent with this, the inventors observed reduced IL-6 production in CpG-stimulated WT mouse B cells treated with the MK2 inhibitor PF3644022 (FIG. 3 A). In addition, there was a significant and reproducible, though less marked reduction of IL-6 secretion after treatment with the MNKl/2/eIF4E inhibitors CGP57380 and cercosporamide using doses that minimally affect cell viability (FIGS. 3B-D). These results show that MNK1/2 and MK2 inhibitors can reduce IL-6 secretion in mouse B cells and confirm that both MNK1/2 and MK2 are important for CpG-stimulated IL-6 secretion from mouse splenic B cells.

Since CpG-induced IL-6 production was reduced in Bankl ^7" cells, the inventors tested if IL-6 gene expression was altered by the deficiency of BANK1. As shown in FIG. 4A, IL-6 gene expression showed no differences between BANK 1 -sufficient and BANK 1 -deficient B cells following CpG stimulation. Since the p38-MK2 signal pathway is responsible in maintaining mRNA stability, the inventors tested the effect of BANK1 deficiency on MK2- mediated IL-6 secretion by testing the stability oiIL-6 mRNA. CpG-stimulated Bankl ^+/+ and Bankr ^7" splenic B-cells showed comparable IL-6 mRNA stability (FIG. 4B). Next, the inventors tested the effects of BANK1 on the activation of the p38-MNKl/2-mediated signaling pathway. MNK1/2 regulates the translation initiation factor eIF4E through phosphorylation (Raught and Gingras, 1999, Andersson and Sundler, 2006 and Shveygert et al, 2010). The inventors therefore analyzed phosphorylation of MNK1/2 and eIF4E. CpG- induced MNK1/2 and eIF4E phosphorylation were consistently reduced in Bankl ^7" cells (FIGS. 4C-D). These results show that BANK1 influences IL-6 secretion by its effects on the p38-regulated MNKl/2/eIF4E/eIF4G pathway of translation initiation while BANK1 does not affect IL-6 secretion via the MK2 pathway.

BANK1 has no effect on the AKT-mTORCl-4E-BPl Signaling Cascade. Activation of eIF4E is also controlled via the AKT-mTORCl-4E-BPl signaling cascade. Activation of this pathway leads to phosphorylation of 4E-BP1, which in turn releases eIF4E for it to be phosphorylated by MNK1/2 and initiate translation. In addition, stimulation through CD40 has been reported to lead to increased AKT phosphorylation in Bankl ^"7" purified B cells (Aiba et al, 2006). The cascade was induced with CpG alone (FIG. 5A), and the phosphorylation of AKT, mTOR and 4E-BP1 was strongly induced when anti-CD40 was included alone or in combination with CpG (FIGS. 5B and 5C). The inventors did observe an increase, albeit weak, in phospho-AKT following anti-CD40 and anti-CD40+CpG treatment on Bankl ^7" B cells (FIGS. 5B and 5C) (31). However, the inventors did not observe any differences in the downstream mTORCl to 4E-BP1, when the inventors used anti-CD40 alone or combined with CpG between Bankl ^7" and Bankl ^+/+ B cells or on phospho-AKT when using CpG alone (FIGS. 5A-C). Overall, these results show that BANK1 acts only in the MNK1/2 and eIF4E arm of p38 signaling to induce IL-6 secretion following CpG stimulation, and that absence of BANK1 does not affect activation of AKT, mTORCl or 4E- BP1 of induction of translation initiation. BANKl deficiency leads to a tendency towards increased production of IgG2a/c subclass antibodies but does not affect expression of the Blimpl gene (Prdml). Finally, the inventors tested if BANKl had an effect on the in vitro secretion of antibodies following CpG stimulation. These results show a non-significant tendency towards increased, rather than decreased secretion of IgG2a and IgG2c antibodies by Bankl ^"7" B cells. Further, the inventors did not observe any difference in the expression of the Prdml gene that codifies for the transcription factor Blimpl (FIGS. 7A-B). These results suggest that secretion of IgG2a/2c antibodies is not significantly affected by reduced secretion of IL-6 by Bankl ^7" B cells.

Production of IL-6 is increased following CpG stimulation in a transgenic for the human BANKl gene. The inventors have shown that deficiency of BANKl specifically affects the phosphorylation of p38 and the downstream signaling of the translation initiation complex molecule eIF4E leading to decreased production of IL-6 protein (Wu et al, 2013). To show that the opposite occurs when one has over-expression of BANKl, the inventors produced a mouse transgenic for the human full-length isoform of BANKl . BANKl is expressed only in B cells thanks to the CD 19 promoter included in the construct. They observed increased levels of IL6 following in vitro stimulation with either CpG or CD40+CpG stimulation splenic B cells of BANKIFL-Tg mice as compared to normal C57B1/6 mice (WT) (FIGS. 8A-B).

Deficiency of Bankl suppresses the development of lupus-like disease. In order to analyze whether deficiency of BANKl can influence autoimmunity, and abrogate or delay the development of lupus, and to show whether BANKl could have a role in TLR7 signaling, the inventors crossed the BANKl -deficient animals with B6.Slel ^z/z .yaa, a congenic mouse containing the NZW locus Slel that leads to several lupus-related B cell phenotypes (Morel et al, 2001; Shi et al, 2007; Sobel et al, 2002; Vuyyuru et al, 2009). The yaa region in male animals amplifies the phenotypic effect, leading to the development of kidney disease in male mice (Hwang et al, 2012; Subramanian et al, 2006).

Recently, it has been shown that the yaa "mutation" is the result of a duplication of the genomic region in the X chromosome containing the TLR7 and TLR8 genes, which has naturally translocated onto the Y chromosome of the BXSB mouse (Subramanian et al, 2006), previously known as the BXSB.yaa strain Izui et al, 2000). The inventors performed experiments on the strain Be.Slel^Bankl ' .yaa and used as control the B6.Slelz/zBankl ^+/+ .yaa littermate. The results show that the deficiency of BANKl does suppress the development of lupus phenotypes (FIGS. 9A-C). Serum 11-6 is reduced in lupus prone mice deficient for Bankl. Among the lupus phenotypes is the increase in serum IL-6, which promotes IgG antibody production and inflammation. Recently, Darise it has been shown that the KO of IL-6 normalizes all the lupus phenotypes of the B6.Slel.yaa mouse. The inventors now show that lack of Bankl in this mouse led to a reduction of the in vivo serum levels of IL-6 excessively produced due to the presence of the yaa genomic segment, as detected in serum of the B6.Slel ^z/z Bankl ^"A .yaa mice (FIG. 10). In conclusion, deficiency of Bankl reduces the effects induced by the presence of the yaa translocation (or TLR7 duplication) supporting that Bankl has an effect on TLR7 signaling.

Translation inhibitors can reduce the production of IL-6 induced by CpG in B cells from the BANKl Tg mouse. There are several inhibitors of translation initiation, such as Pateamine A (PatA), a small molecule isolated from the marine sponge Mycale s., and with immunosuppressive properties (Low et al, 2005; Di Marco et al, 2012). PatA alters the activity of the mRNA helicase EIF4A, needed for the assembly of the 40s ribosome subunit (Cencic et al, 2012). Other inhibitors, such as Cercosporamide or CGP57380, inhibit MNK1/2 (Altman et al, 2013), the kinase that following p38 activation, phosphorylates eIF4E. Inhibition of assembly of the complex by PatA at low doses has been shown to inhibit the production of pro-inflammatory cytokines (Di Marco et al, 2012; Gingras et al, 1999), but the effects of M K1 inhibition on IL-6 production, have never been shown in mice.

The inventors therefore tested the effect of Pateamine and the MNK1 inhibitor

CGP57380 in vitro, purifying splenic B cells from the transgenic mice following stimulation with CpG to detect IL-6 production and avoiding levels of the drugs that may be toxic to the cells (FIG. 1 1). FIG. 11 shows how 0.01 μΜ Pateamine is non-toxic (right panel) and is capable of inhibiting IL-6 secretion following CpG stimulation in purified B cells from the transgenic BANKIFL-Tg mice. In these experiments, the transgenic animals were crossed onto the Bankl-/- mice in order to have the human trans gene without the expression of the endogenous murine Bankl .

EXAMPLE 3 - DISCUSSION The inventors show here for the first time that the B cell adaptor with ankyrin repeats

BANKl influences signaling leading to the formation of the translation initiation eIF4E complex following stimulation with CpG, and that BANKl deficiency results in reduction of the translation of the proinflammatory cytokine IL-6. They have also shown that mRNA stability of IL-6 is not affected, nor the second axis of regulation of translation initiation via AKT, which is also induced by CpG and strongly amplified with anti-CD40 treatment. Clearly, the combination of CpG and anti-CD40 treatment leads to stronger signaling of the AKT axis followed by strong activation of mTOR and 4E-BP1. However, absence of BANKl has no effect on the signaling pathway through this axis, except weak activation of AKT. While the secretion of IL-6 is optimal (Poudrier et al, 1998, Barr et al, 2007 and Barr et al, 2010) with the combination of CpG and anti-CD40, BANKl deficiency leads to its reduction, and anti-CD40 cannot overcome this effect. These results clearly show that overall BANKl controls the production of IL-6 via the p38-MNKl/2-eIF4E pathway. At the same time, activation of the CD40-induced pathway leads to strong phosphorylation of the AKT axis, which would eventually promote translation initiation of IL-6 through phosphorylation of 4E-BP 1 and the release of eIF4E for it to become phosphorylated by MNK1/2. However, the inventors clearly observe that this is not the case.

The involvement of BANKl in modulating the IL-6 response after CpG stimulation has important implications in the pathogenesis of autoimmunity and viral infection. Sera from autoimmunity -prone animals and cerebrospinal fluid from SLE patients have elevated levels of IL-6 (Alcocer-Varela et al, 1992 and Stuart et al, 1995) and peripheral blood cells from SLE patients spontaneously secrete increased levels of IL-6 (Linker-Israeli et al, 1991). Autoimmunity is ameliorated by IL-6 ablation (Barr et al, 2012). It is also important to note that IL-6, coordinated with IL-21, has been reported to control the differentiation towards plasma cells (Eto et al, 2011). More recently, B cell derived IL-6 was shown to function in an autocrine manner and trigger receptor revision through re-expression of RAG in the post germinal center response (Yan et al, 2012).

IL-6 has also important roles in infectious diseases. It has been reported that B cells release IL-6 to promote a follicular helper T cell (T _FH ) response to viral infection. B cell- derived IL-6 was necessary and sufficient to induce IL-21 from CD4+ T cells in vitro and to support TFH cell development in vivo upon acute influenza virus infection (Karnowski et al, 2012). IL-6 is also involved in the induction but not maintenance of plasma cells (Cassese et al, 2003). BANKl deficient mice have normal T cell-dependent humoral responses following immunization with NP-CGG (Aiba et al, 2006). One explanation for the unchanged primary humoral response reported by Aiba et al in Bankl-/- mice is likely to be the lack of TLR agonist challenge and IL-6 production by B cells in their in vivo system. Therefore, it will be worth to address if infection with DNA- or RNA-viruses, instead of NP- CGG immunization can initiate TLR9 or TLR7 activation that would probably result in reduced IL-6 production by Bankl-/- B cells, and suppressed germinal center responses. Regarding humoral responses, at this point the inventors are unable to explain the tendency towards increased IgG2a/2c production induced by CpG and they do not know if this bears any relationship with the increased serum IgG2a observed by Aiba et al (Aiba et al, 2006).

BANKl has been genetically associated with SLE and other autoimmune diseases, and here the inventors observe that BANKl deficiency decreased IL-6 secretion by altering the translation initiation pathway upon CpG stimulation. These results therefore suggest that BANKl could be involved in controlling disease development through the control of IL-6 secretion. While there are several mechanisms through which IL-6 production is regulated, it is clear that the production of IL-6 is controlled through a multitude of pathways and through different genes.

BANKl was found to contain a conformational modular TIR domain at the N- terminus, similar to its relative, the molecule BCAP. BCAP is required for TLR-mediated activation of PI3K and AKT in macrophages (Troutman et al, 2012). In contrast, CpG- induced AKT activation is normal in Bankl-/- B cells (FIG. 5A). The data suggest that BCAP, rather than BANKl, may play a critical role in TLR-mediated activation of AKT, and further, BCAP and CD19 have complementary roles in BCR-mediated-PI3K activation (Aiba et al, 2008). While BANKl appears to mediate AKT activation upon CD40 ligation in B cells (Aiba et al, 2006; FIG. 5B), these results support a specific role for BANKl in transducing CpG-induced signals via p38-MNKl/2 and the translation initiation factor eIF4E in B cells. Thus, BANKl and BCAP rather than playing redundant roles, appear to have very different ones.

BANKl, with a putative TIR domain, would be prone to bind molecules containing TIR domains, and explain the very specific role of BANKl in p38 signaling and translation initiation. BANKl is an adaptor molecule with a modular structure. It has up to 23 tyrosines susceptible of phosphorylation, and their substrate could alternate among a variety of molecules forming specific complexes during CpG-induced activation, different from those occurring following BCR-induced ligation, for instance. One of those complexes induced by CpG could include p38. How BANKl controls the p38-MNKl/2 pathway downstream of TLR9 is at present not known, but is a subject of study in the inventors' laboratory.

The inventors have described that BANKl shows an interaction with the Src tyrosine kinase BLK (1), and that BLK serves to promote the interaction between BANKl and phospholipase C y2, a key molecule in signal transduction during BCR ligation (Bernal- Quiros et al, 2013). This phenomenon is apparently not linked to CpG-induced signaling, as the inventors do not observe phosphorylation of BANKl or changes in binding of BANKl to BLK following CpG stimulation (data not shown). Nevertheless, the inventors provide here a role for BANK1 in CpG-induced signaling in the production of IL-6, which could be highly relevant in the study of autoimmunity and inflammation.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

V. References

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Aiba et al, Blood 11 1 : 1497-503, 2008.

Aiba et al, Immunity 24: 259-68, 2006.

Akashi et al, J. Rheumatol 17:375-379, 1990.

Alcocer-Varela <?i iz/., Lupus 1 : 1 11-7, 1992.

Altman et al, Blood 121 , 3675-81, 2013.

Andersson and Sundler, Cytokine 33: 52-7, 2006.

Banerjee et al, J. Virol 76: 5937-48, 2002.

Barr et al, Euro. J. Immunol 37: 3040-53, 2007.

Barr et al, J. Immunol. 185: 2783-9, 2010.

Barr el/., J. Exp. Med. 209: 1001-10, 2012.

Bernal-Quiros et al, PLoS ONE 8: e59842, 2013.

Bernasconi et al, Blood 101 : 4500-4, 2003.

Bodanszky et al, J. Antibiot., 29(5):549-53, 1976.

Bollig et al, Biochem. Biophys. Res. Comm. 301 : 665-70, 2003.

Bosher and Labouesse, Nat. Cell. Biol, 2:E31-E36, 2000.

Caplan et al . Gene, 252(l-2):95-l 05, 2000.

Cassese et al, J. Immunol. 171 : 1684-90, 2003.

Castillejo-Lopez et al, Annals Rheumatic Dis. 71 : 136-42, 2012.

Cencic et al, Meth. Enzymol. 51 1, 437-61, 2012.

Christensen et al, J. Exp. Med. 202: 321 -31, 2005.

Cook e? al, Cell, 27:487-496, 1981.

Di Marco et al, Nature Commun. 3, 896, 2012.

Elbashir et al, Nature, 411(6836):494-498, 2001.

Emlen et al, J. Biol. Chem. 273 : 1741-8, 1998.

Eto et al., PLoS ONE 6: el 7739, 2011.

Fire et al, Nature, 391 :806-81 1 , 1998.

Fischer, Med. Res. Rev., 27(6):755-796, 2007.

Foey et al, J. Immunol. 160: 920-8, 1998. Forster and Symons, Cell, 49:211-220, 1987.

Gerlach et al, Nature (London), 328:802-805, 1987.

Gingras et al, Genes & Devel. 13 : 1422-37, 1999.

Gingras et al, Ann. Rev.Bbiochem. 68, 913-63, 1999.

Grishok et al, Science, 287:2494-2497, 2000.

Haxhinasto and Bishop, J. Biol. Chem. 279: 2575-82, 2004.

Hwang et al, J. Immunol 189, 5786-96, 2012.

Izui et al , Int. Rev. Immunol. 19, 447-72, 2000.

Jordan et al, Clin. Immunol. Immunopathol. 53 : S164-169, 1989.

Joyce, Nature, 338:217-244, 1989.

Karnowski et al, J. Exp. Med. 209: 2049-64, 2012.

Ketting e/ a/., Cell, 99: 133-141, 1999.

Kim and Cook, Proc. Natl Acad. Sci. USA, 84:8788-8792, 1987.

Kozyrev <¾ «/., Genes Immun. 13: 129-38, 2012.

Kozyrev et al, Nat. Genet. 40: 211-6, 2008.

Lin and Avery, Nature, 402: 128-129, 1999.

Linker- Israeli et al, J. Immunol. 147: 1 17-23, 1991.

Livingstone et al, Chemistry & Biology 16: 1240-9, 2009.

Low et al, Molecular Cell 20, 709-22, 2005.

Merrifield, J. Am. Chem. Soc, 85:2149-2154, 1963.

Michel and Westhof, J. Mol. Biol , 216:585-610, 1990.

Montgomery et al, Proc. Natl. Acad. Sci. USA, 95: 155-2-15507, 1998.

Morel et al, Proc Natl Acad Sci USA 98, 1787-92, 2001.

Morley, Biochem. Soc. Trans. 25: 503-9, 1997.

Mourey et al, J. Pharmacol. Exp. Ther. 333 : 797-807, 2010.

Nagaleekar et al, J. Immunol. 186: 4140-6, 201 1.

Neininger et al, J. Biol Chem. 277: 3065-8, 2002.

Peptide Synthesis, 1985

Poudrier et al, Euro. J. Immunol. 28: 3371-83, 1998.

Protective Groups in Organic Chemistry, 1973

Raught and Gingras, Int 'lJ. Biochem. & Cell Biol. 3 1 : 43-57, 1999.

Reinhold-Hurek and Shub, Nature, 357: 173-176, 1992.

Richter and Sonenberg, Nature 433 : 477-80, 2005. Ronkina et al, i 27: 170-81, 2007.

Rowlett et al, American J. Physiology 294: G452-9, 2008.

Ruperto et al, Lupus 0: 1-10, 2010.

Saito h a/., J. Clinical Invest. 109: 1453-62, 2002.

Sarver et al, Science, 247: 1222-1225, 1990.

Scanlon et al, Proc. Natl. Acad. Sci. USA, 88: 10591-10595, 1991.

Sharp and Zamore, Science 287:2431-2433, 2000.

Sharp, Genes Dev., 13 : 139-141, 1999.

Shi et al. Arthritis Rheum 56, 3057-69, 2007.

Shveygert ei a/., Mol. Cell. Biol. 30: 5160-7, 2010.

Sobel et al, J. Immunol. 169, 2694-700, 2002.

Solid Phase Peptide Synthelia, 1984

Sonenberg, Biochem. Cell Biol. 86: 178-83, 2008.

Stuart et al, Clin. Exp. Rheumatol. 13 : 17-22, 1995.

Subramanian et al, Proc Natl Acad Sci USA 103, 9970-5, 2006.

Sun et al, lnflamm. Allergy Drug Targets 6: 223-35, 2007.

Tabara ei a/., Cell, 99: 123-132, 1999.

Troutman et al , Proc. Natl. Acad. Sci. USA, 109: 273-8, 2012.

U.S. Patent 4,415,732

U.S. Patent 4,458,066

U.S. Patent 5,354,855

U.S. Patent 5,795,715

U.S. Patent 5,889, 136

U.S. Patent 5,889, 155

U.S. Patent 6,261,569

U.S. Patent Publication 20100144805

Vanden Bush et al, Euro. J. Immunol. 38: 400-9, 2008.

Vuyyuru et al. , J. Immunol. 183, 5716-27, 2009.

Wincott et al, Nucleic Acids Res., 23(14):2677-2684, 1995.

WO 00/44914

WO 01/36646

WO 01/68836

WO 99/32619 Wu etal, J. Immunol 191, 6110-6, 2013. Yan etal, J. Autoimmun.38: 1-9, 2012. Yokoyama et al , EMBO J.21: 83-92, 2002.