WO2010151827A1 - X-box binding proteins (xbp-1) variants and methods of using the same - Google Patents

Abstract

Provided is a variant of X-box binding protein-1 (Xbp1) that cannot be spliced in response to activation of the UPR. This variant contains a silent mutation which removes the IRE1 recognition site. Also provided is the use of this variant in the treatment of UPR-activating diseases.

Description

X-BOX BINDING PROTEIN 1 (XBP1) VARIANTS AND METHODS OF USING THE SAME

Inventors: David Kiewlich

[001] This application claims benefit of priority to U.S. Provisional Serial No. 61/220,486, filed June 25, 2009, the entire contents of which are hereby incorporated by reference.

FIELD

[002] Provided herein are variants of X-box Binding Protein 1 , Xbp1 , and methods of using the same. More particularly, provided is an Xbp1 variant comprising silent mutations removing the IRE1 recognition site thereby preventing Xbp1 mRNA splicing.

REFERENCE TO SEQUENCE LISTING

[003] This application incorporates by reference the attached sequence listing in both paper and electronic copy in .txt format. Applicant further certifies that the content contained in the paper and electronic copies are identical.

BACKGROUND

[004] X-Box Binding protein 1 (Xbp1 ) is a basic leucine zipper-containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by misfolded proteins in the endoplasmic reticulum. The UPR is a survival response that is activated when there is an overload of unfolded or misfolded proteins in the endoplasmic reticulum (EnR). Although the detection machinery is not fully understood, the UPR can be activated by hypoxia or nutrient starvation, common conditions in solid tumors, or through the action of drugs, such as tunicamycin (Tm). Tm blocks the glycosylation of proteins, which, in turn, interferes with protein folding. Once the UPR is initiated, a 26 bp fragment from the Xbp1 mRNA is removed, generating a translationally frameshifted protein designated Xbp1 S. Involved in the splicing of the Xbp1 mRNA is Inositol Requiring Enzyme 1 (IRE1 ), a transmembrane RNase which recognizes a specific site on the Xbp1 mRNA sequence. The protein encoded by the spliced Xbp1 mRNA, Xbp1 S, is larger and thought to be more transcriptionally active than the protein produced by the unspliced Xbp1 mRNA, Xbp1 U.

DESCRIPTION OF THE DRAWINGS

[005] Figure 1 depicts an exemplary mutation of the IRE1 recognition sequence to generate non-spliceable Xbp1 mRNA. The mRNA sequence around the splicing site for Xbp1 with the translation for Xbp1 U is shown. The bases involved in the IRE1 recognition site are bolded and underlined. The 26 base pair "intron" that is spliced out is boxed. The bases changed to make the Xbp1 ns gene are shown under the sequence. Note that the base changes are silent mutations in Xbp1 U.

[006] Figure 2 shows protein expression of Xbpi s, Xbpiwt and Xbp1 ns MCF7 pools of stable cells. MCF7 cell pools, transduced with Tet-Advantage only (TetON), or, Tet-Advantage along with Xbpi ns, Xbpiwt or Xbpi s lentivirus were treated with 1 μg/ml of DOX for 3 days. Cells were then incubated with Tm for 4 hours to induce splicing of Xbp1 mRNA. Protein expression was determined by western analysis with Anti-FLAG antibody, and equal loading was determined by anti-actin antibodies.

DETAILED DESCRIPTION

[007] It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[008] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleotide" is a reference to one or more nucleotide and includes equivalents thereof known to those skilled in the art, and so forth.

[009] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

[010] All publications and patents mentioned herein are hereby incorporated herein by reference for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[011] As used herein, various terms are defined below.

[012] A "nucleic acid" denotes deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues. The term nucleic acid, depending on context, is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[013] "Nucleic acid derived from a gene" denotes a nucleic acid for whose synthesis the gene, or a subsequence thereof, has ultimately served as a template. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA, and the like, are derived from the gene and detection of such derived products is indicative of the presence and/or abundance of the original gene and/or gene transcript in a sample.

[014] A polypeptide, nucleic acid, or other component is "isolated" when it is partially or completely separated from components with which it is normally associated (other peptides, polypeptides, proteins (including complexes, for example, polymerases and ribosomes which may accompany a native sequence), nucleic acids, cells, synthetic reagents, cellular contaminants, cellular components, etc.), for example, such as from other components with which it is normally associated in the cell from which it was originally derived. A polypeptide, nucleic acid, or other component is isolated when it is partially or completely recovered or separated from other components of its natural environment such that it is the predominant species present in a composition, mixture, or collection of components (i.e., on a molar basis it is more abundant than any other individual species in the composition). In some instances, the preparation consists of more than about 60%, 70% or 75%, typically more than about 80%, or more than about 90% of the isolated species.

[015] A "substantially pure" nucleic acid (e.g., RNA or DNA), polypeptide, protein, or composition also means where the object species (e.g., nucleic acid or polypeptide) comprises at least about 50, 60, 70, 80, 90, or 95 percent by weight of all the macromolecular species present in the composition. An object species can also be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of derivatives of a single macromolecular species.

[016] The term "purified" generally means that the nucleic acid, polypeptide, or protein is at least about 50% pure, about 60% pure, about 70% pure, about 75% pure, about 85% pure, about 95% pure, and about 99% pure.

[017] The term "silent mutation" when used with reference, for example, to an mRNA sequence, denotes that the nucleic acid sequence has been modified but does not alter the amino acid sequence upon translation. [018] A "variant" of a specified nucleic acid comprises an nucleic acid sequence which differs from that of the specified nucleotide sequence by virtue of at least one "nucleic acid modification" as herein defined. Such a modification can include a deletion, an insertion or a replacement of one nucleic acid for another nucleic acid.

[019] The term "vector" may refer to, depending on context, cloning vectors, expression vectors, or both. The term vector and the term "plasmid" are used interchangeably.

[020] The term "expression vector" or "expression plasmid" denotes the vehicle by which an expression cassette can be introduced into a host cell, so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence.

[021] The terms "express" and "expression" mean allowing or causing the information in a gene or DNA sequence to become manifest, for example, producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product" such as a protein. The expression product itself, for example, the resulting protein, may also be said to be "expressed." An expression product can be characterized as intracellular, extracellular, or secreted.

[022] The term "host cell" means any suitable cell line capable of expressing a protein of interest, for example Xbp1 .

[023] X-Box Binding protein 1 (Xbp1 , cDNA sequence provided in SEQ ID NO:1 ) is a basic leucine zipper-containing transcription factor that plays a key role in the unfolded protein response (UPR), a cell program that is activated by unfolded or misfolded proteins in the endoplasmic reticulum.

[024] Disclosed herein is an isolated Xbp1 variant comprising a mutation in the IRE1 recognition site which thereby prevents mRNA splicing. In some embodiments, the mutation is a silent mutation which allows the variant to behave similarly to Xbp1 U. These silent mutations will produce Xbp1 U protein, but render the mRNA resistant to being spliced preventing expression of Xbp1 S protein from the recombinant mRNA. This would allow the recombinant Xbp1 U to act as a dominant negative, suppressing the transcriptional activities of Xbp1 S protein.

[025] In some embodiments, the mutation is depicted in Figure 1 and is shown in SEQ ID NO:2. Other embodiments are shown in SEQ ID NOS: 3-10, which comprise single codon mutations that interfere with Xbp1 mRNA splicing. In other embodiments, a combination of mutations may be used to prevent Xbp1 mRNA splicing. Thus, in some embodiments, one or more of the mutations may be used in combination further interfering with IRE1 recognition and therefore Xbp1 mRNA splicing.

[026] The Xbp1 variant may be generated through any suitable means capable of creating a mutation in a DNA sequence, for example by oligonucleotide synthesis, site-directed mutagenesis, DNA shuffling, and PCR site-directed mutagenesis. Methods of Use

[027] As used herein, various terms are defined below.

[028] The term "treating" or "treatment" includes any process, action, application, therapy, or the like, wherein a subject (or patient), including a human being, is provided medical aid with the object of improving the subject's condition, directly or indirectly, or slowing the progression of a condition or disorder in the subject.

[029] The phrase "therapeutically effective" means the amount of agent administered that will achieve the goal of improvement in a disease, condition, and/or disorder severity, while avoiding or minimizing adverse side effects associated with the given therapeutic treatment.

[030] The term "pharmaceutically acceptable" means that the subject item is appropriate for use in a pharmaceutical product.

[031] Over-expression of the unspliced form of Xbp1 protein acts as a dominant negative on the activity of Xbp1 S protein, likely through competition for binding sites in the genome. Accordingly, one embodiment provides for a method of treating UPR-activating diseases comprising administering a therapeutically effective amount of human X-box binding protein-1 (Xbp1 ) variant, wherein the variant prevents Xbp1 splicing by blocking the IRE1 recognition site. Thus, in some embodiments, the Xbp1 variant can be provided as a dominant negative therapeutic.

[032] Xbp1 mRNA is spliced in response to cytotoxic stresses such as hypoxia or nutrient deprivation, through a cell program called the unfolded protein response (UPR). If splicing is prevented, the cells are unable to respond to the stress and will enter apoptosis. [033] UPR-activating diseases include malignancies, including but not limited to acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, cancer of the bile duct, bladder cancer, bone cancer, osteosarcomal malignant fibrous histiocytomal brain stem gliomal brain tumor, breast cancer, bronchial adenomas, carcinoid tumors, adrenocotrical carcinoma, central nervous system lymphoma, cancer of the sinuse, cancer of the gall bladder, gastric cancer, cancer of the salivary glands, cancer of the esophagus, neural cell cancer, intestinal cancer, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, B-cell lymphoma, T-cell lymphoma, endometrial cancer, epithelial cancer, endometrial cancer, intraocular melanoma, retinoblastoma, hairy cell leukemia, liver cancer, Hodgkin's disease, Kaposi's sarcoma, lung cancer, non-Hodgkin's lymphoma, melanoma, multiple myeloma, neuroblastoma, prostate cancer, retinoblastoma, Ewing's sarcoma, vaginal cancer, Waldenstrom's macroblubulinemia, adenocarcinomas, ovarian cancer, chronic lymphocytic leukemia, pancreatic cancer, and Wilm's tumor.. Specifically, B-cell lymphoma, which is principally dependent on the transcriptional activity of Xbp1 S protein, would be a particularly attractive target for a dominant negative Xbp1 U therapeutic.

[034] The term "combination therapy" or "co-therapy" means the administration of two or more therapeutic agents to treat a disease, condition, and/or disorder. Such administration encompasses co-administration of two or more therapeutic agents in a substantially simultaneous manner or administration of each type of therapeutic agent in a sequential manner.

[035] Combination therapy includes administration of a single pharmaceutical dosage formulation which contains the Xbp1 variant and one or more additional therapeutic agents, as well as administration of Xbp1 variant and each additional therapeutic agents in its own separate pharmaceutical dosage formulation. For example, Xbp1 variant and a therapeutic agent may be administered to the patient together in a single dosage composition or each agent may be administered in separate dosage formulations.

[036] Where separate dosage formulations are used, the Xbp1 variant and one or more additional therapeutic agents may be administered at essentially the same time (e.g., concurrently) or at separately staggered times (e.g., sequentially).

Pharmaceutical compositions

[037] The Xbp1 non-spliceable variant as described herein may be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be non-pyrogenic. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers may be employed including, but not limited to saline, glycine, or the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, and the like. The concentration of Xbp1 variant in such pharmaceutical formulation may vary widely, and may be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration.

[038] The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which may be used pharmaceutically. Pharmaceutical compositions of the invention may be administered by subcutaneous means.

[039] Formulations suitable for subcutaneous, intravenous, intramuscular, and the like deliveries; suitable pharmaceutical carriers; and techniques for formulation and administration may be prepared by any of the methods well known in the art (see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 20^th edition, 2000). Determination of a Therapeutically Effective Dose

[040] The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to the amount of an agent that may be used to effectively treat a disease (e.g., breast cancer) compared with the efficacy that is evident in the absence of the therapeutically effective dose.

[041] The therapeutically effective dose may be estimated initially in animal models (e.g., rats, mice, rabbits, dogs, or pigs). The animal model may also be used to determine the appropriate concentration range and route of administration. Such information may then be used to determine useful doses and routes for administration in humans.

[042] The exact dosage may be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration may be adjusted to provide sufficient levels of the agent or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.

EXAMPLES

[043] It will be apparent to those skilled in the art that the examples and embodiments described herein are by way of illustration and not of limitation, and that other examples may be used without departing from the spirit and scope of the present invention, as set forth in the claims.

[044] Using the Tet-Advantage system of ClonTech, we created lentiviruses to enable creation of cell lines with DOX-regulated expression of Xbp1 proteins. The endogenous gene for Xbp1 , called Xbpi wt, (which expresses both Xbp1 U and Xbp1 S protein), was cloned from MCF7 total RNA. The spliced Xbp1 gene sequence, called Xbp1 s, (which expresses only the spliced Xbp1 S protein) was cloned from Tunicamycin (Tm)-treated MCF7 total RNA. [045] To create the non-spliceable gene sequence (called Xbp1 ns) that is only capable of expressing the smaller Xbp1 U protein, we analyzed the recognition site for IRE1 as described in Yoshida, et.al., 2001 , and predicted that three silent mutations would disrupt the IRE1 recognition site and enable expression of only unspliceable Xbp1 mRNA (See Figure 1 ). This change was made to the Xbp1 wt sequence through site-directed mutagenesis and the resulting Xbp1 gene was designated Xbp1 ns (Not Splicable). A FLAG tag was added to the N-terminus of all genes for ease of detection of the various Xbp1 protein forms.

[046] Shown in Figure 2 are lysates of transduced pools of cells analyzed by western blots for induction and expression of the Xbp1 transgenes.

[047] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

ClaimsWhat is claimed is:

1 . An isolated nucleic acid molecule encoding a modified X-box binding protein 1 (Xbp1 ) comprising a mutation removing the IRE1 recognition site.

2. The isolated nucleic acid molecule of claim 1 , wherein the mutation is a silent mutation.

3. The isolated nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of those shown in SEQ ID NOS:2-10.

4. A recombinant DNA characterized in that the DNA molecule according to claim 1 is inserted into a vector DNA.

5. An isolated host cell comprising the recombinant DNA according to claim 4.

6. A method of treating U PR-activating diseases comprising administering the isolated nucleic acid molecule of claim 1 .

7. The method of claim 6, wherein the U PR-activating disease is a malignancy.