Ubiquitinating gabpa or gabpb prevents activation of the mutant TERT promoter

Abstract

Methods and compositions for preventing activation of mutant TERT promoters are described, including for inhibiting cancer cells. In some cases, a GABPA-interacting domain or GABPB-interacting domain is linked to a ubiquitin ligase to prevent activation of mutant TERT promoters by a GABP complex.

Description

UBIQUITINATING GABPA OR GABPB PREVENTS ACTIVATION OF THE MUTANT TERT PROMOTERCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 547,934, filed November 9, 2023 and U.S. Provisional Patent Application No. 63 / 547,979, filed November 9, 2023, which is incorporated by reference for all purposes.REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0002] The Sequence Listing written in file 081906-1471360-UCSF254210PC.xml, created on November 7, 2024, 35,000 bytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference in its entirety for all purposes.BACKGROUND

[0003] A fundamental characteristic of most cells is their limited lifespan and replicative capacity, in part because of telomeric shortening with each cell division. Telomeric erosion occurs due to incomplete DNA replication by DNA polymerase. Below a certain threshold of telomere length, telomeres become dysfunctional, and cells are triggered to enter replicative senescence. Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, the enzyme responsible for telomere maintenance. TERT is generally not expressed in normal somatic cells, functioning to prevent uncontrolled replication. Cancer cells, however, can achieve replicative immortality through reactivation of TERT expression.

[0004] TERT reactivation in cancer cells promotes cellular immortalization and the resultant malignant phenotype by stabilizing telomere length and buffering entry into replicative senescence. Many cancer types acquire a point mutation in the TERT promoter which allows for TERT reactivation. Consequently, TERT expression is detectable in up to 90% of human primary cancers.

[0005] The point mutation in the TERT promoter of cancer cells creates a de novo binding motif for E26 transformation-specific transcription factors, such as GA binding protein (GABP). GABP is a multimeric protein complex composed of the DNA-binding GABP subunit A (GABP A) and a transactivating subunit chosen from either GABP subunit Bl (GABPB1) or GABP subunit B2 (GABPB2). The GABP complex has been identified as activating mutant TERT promoters in cancer. However, no known classes of drugs or biologies exist that can target the GABP complex to prevent TERT reactivation in cancer.BRIEF SUMMARY

[0006] Provided herein is a polypeptide comprising a GABPA-interacting domain optionally linked to a ubiquitin ligase. Also provided herein is a polypeptide comprising a GABPB- interacting domain optionally linked to a ubiquitin ligase.

[0007] In some embodiments, the GABPA-interacting domain comprises an Ankyrin repeat sequence from GABPB1L or GABPB2. In some embodiments, the GABPA-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NO: 1 or SEQ ID NO: 2, or a GABPA-binding fragment thereof. In some embodiments, the polypeptide comprises no more than 170, 180, 190, or 200 contiguous amino acids of GABPB1L.

[0008] In some embodiments, the GABPB-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NO: 19, or a GABPA-binding fragment thereof. In some embodiments, the GABPB-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23. In some embodiments, the polypeptide comprises no more than 120, 130, 140 or 150 contiguous amino acids of human GABPA.

[0009] In some embodiments, the GABPA-interacting domain or GABPB-interacting domain is fused to a heterologous amino acid sequence. In some embodiments, the GABPA-binding domain or GABPB-interacting domain is fused to a ubiquitin ligase. In some embodiments, the ubiquitin ligase comprises an E3 ligase.

[0010] In some embodiments, the polypeptide further comprises a nuclear localization sequence.

[0011] Also provided herein is a nucleic acid comprising a polynucleotide encoding the polypeptide as described above or elsewhere herein. In some embodiments, a promoter is operably linked to the polynucleotide.

[0012] Also provided herein is a vector comprising the nucleic acid as described above or elsewhere herein. In some embodiments, the vector is a viral vector.

[0013] Also provided herein is a cell comprising the nucleic acid as described above or elsewhere herein or the vector as described above or elsewhere herein.

[0014] Also provided herein is a method of inhibiting expression of TERT in a cell having a TERT promoter mutation, the method comprising, introducing the polypeptide as described above or elsewhere herein in the cell, or introducing the nucleic acid or vector as described above or elsewhere herein into the cell such that the polypeptide is expressed and binds a GAB PA or GABP protein in the cell. In some embodiments, the cell is a human cell and the introducing comprises administering the polypeptide, nucleic acid or vector to the human such that the polypeptide, nucleic acid or vector is delivered to the cell. In some embodiments the cell is a cancer cell. In some embodiments the cancer cell is a glioblastoma or a glioma.

[0015] Also provided herein is a pharmaceutical composition comprising a pharmaceutically- acceptable excipient and the polypeptide, nucleic acid, of vector as described above or elsewhere herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description(s) of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0017] FIG. 1 shows the effect of two constructs on TERT and GABPB 1 expression in wildtype and mutated TERT promoter cell lines.

[0018] FIG. 2 depicts various constructs of the full length GABPB1L, the minimal domain of GABPB 1 found to interact with GABPA, and two embodiments of the minimal domain fused to a ubiquitin ligase and optionally a nuclear localization sequence.

[0019] FIG. 3 shows the effect of various constructs on TERT expression in wildtype and mutated TERT promoter cell lines.

[0020] FIG. 4 shows the effect of various constructs on GABPA protein levels in mutated TERT promoter cell lines.

[0021] FIG. 5 depicts retroviral vectors for the delivery of a control protein or a GABPA- interacting domain as described herein.

[0022] FIG. 6 depicts retroviral vectors for the delivery of a GABPA-interacting domain optionally fused to a ubiquitin ligase as described herein.

[0023] FIG. 7 shows the effect on TERT and GABPB 1 expression in mutated TERT promoter glioblastoma cells transduced with replicated retroviruses containing various constructs.

[0024] FIG. 8 shows the effect of three constructs on TERT expression in mutated TERT promoter cell lines.

[0025] FIG. 9 depicts protein interaction between GABPB-interacting domains and GABPB 1 or GABPB2.

[0026] FIG. 10 shows the effect of GABPB-interacting domain fragments on GABPB 1 expression in mutated TERT promoter cell lines.

[0027] FIG. 11 shows the effect of various constructs on GABPB protein levels in mutated TERT promoter cell lines.

[0028] FIG. 12 shows the effect of various constructs on TERT expression in mutated TERT promoter cell lines.DEFINITIONS

[0029] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.

[0030] The use of any and all examples or exemplary language (e.g., “for example” or “such as”) provided herein, is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0031] The terms “may,” “may be,” “can,” and “can be,” and related terms are intended to convey that the subject matter involved is optional (that is, the subject matter is present in some examples and is not present in other examples), not a reference to a capability of the subject matter or to a probability, unless the context clearly indicates otherwise.

[0032] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as "including," "comprising,” or "having" certain elements are also contemplated as "consisting essentially of and "consisting of’ those certain elements. As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0033] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0034] The term “GABPA-interacting domain” refers to an amino acid portion of a protein capable of binding with GA binding protein subunit A (GABPA). Interaction of a polypeptide with GABPA can be measured by, for example, immunoprecipitation.

[0035] “Polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0036] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine, pyrroline-carboxy-lysine, and selenocysteine.

[0037] The term “conservative amino acid substitutions” refers to the substitution (conceptually or otherwise) of an amino acid from one such group with a different amino acid from the same group. One example of substitutions is based on analyzing the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (see, e.g., Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other and, therefore, resemble each other most in their impact on the overall protein structure (see, e.g., Schulz, G. E. and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag). One example of a set of amino acid groups defined in this manner include: (i) a charged group, consisting of Glu and Asp, Lys, Arg and His; (ii) a positively-charged group, consisting of Lys, Arg and His; (iii) a negatively-charged group, consisting of Glu and Asp; (iv) an aromatic group, consisting of Phe, Tyr and Trp; (v) a nitrogen ring group, consisting of His and Trp; (vi) a large aliphatic nonpolar group, consisting of Vai, Leu and He; (vii) a slightly-polar group, consisting of Met and Cys; (viii) a small-residue group, consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and Pro; (ix) an aliphatic group consisting of Vai, Leu, He, Met and Cys; and (x) a small hydroxyl group consisting of Ser and Thr. Other examples of conservative substitutions based on shared physical properties are the substitutionswithin the following groups : 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0038] As used herein the phrase “heterologous” refers to what is not found in nature. The term "heterologous sequence" refers to a sequence not normally found in a given cell in nature. As such, a heterologous nucleotide or protein sequence may be: (a) foreign to its host cell (i.e., is exogenous to the cell); or (b) be naturally found in the host cell but positioned outside of its natural locus.

[0039] As used herein, the phrase “introduced” or “introducing” in the context of introducing a polypeptide, nucleic acid, or a vector refers to the translocation of the polypeptide, nucleic acid sequence, or vector from outside a cell to inside the cell. In some cases, introducing refers to transducing or infecting a cell or a population of cells with a viral vector or viral particle carrying one or more non-viral nucleic acids. In some cases, introducing refers to the expression of a protein encoded by a nucleic acid translocated into the cell. In some cases, translocation of the nucleic acid from outside the cell to inside the nucleus of the cell occurs. Various methods of such translocation are contemplated, including, but not limited to, viral infection, electroporation, transfection, transduction, contact with nanowires or nanotubes, receptor mediated internalization, translocation via cell penetrating peptides, liposome mediated translocation, translocation via lipid nanoparticles, and the like.

[0040] As used herein, a “cell” can be in vivo, ex vivo or in vitro, and includes any cell that may have a TERT promoter mutation, for example, a cancer cell. As used herein the term “cancer cell” refers to cells that exhibit markedly increased proliferation as compared to non- cancerous somatic cells. In some embodiments, the cells are human cancer cells.

[0041] The term “nucleic acid” or “nucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof, for example, “polynucleotides,” in either single- or double-stranded form. The nucleic acid molecule may be derived from a variety of sources, including DNA, cDNA, synthetic DNA, RNA, or combinations thereof. Such nucleic acid sequences may comprise genomic DNA which may or may not include naturally occurringintrons. Moreover, such genomic DNA may be obtained in association with promoter regions, introns, or poly A sequences.

[0042] “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., SEQ ID NO: 1 or 2), which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0043] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 95% identity, optionally 96%, 97%, 98%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. For an amino acid sequence, optionally, identity exists over a region that is at least about 50 amino acids in length, or more preferably over a region that is 100 to 150 or 200 or more amino acids in length, or where not indicated over the entire length of the reference sequence.

[0044] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0045] A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 50 to 600, usually about 75 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art.

[0046] An algorithm for determining percent sequence identity and sequence similarity is the BLAST 2.0 algorithms, e.g., as described in, and Altschul et al. (1990) J. Mol. Biol. 215:403-410 (see also Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402) . Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, and N=-4.

[0047] A “promoter” is defined as one or more a nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

[0048] A promoter is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence, for example, a polynucleotide. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0049] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. A “vector” as used here refers to a recombinant construct in which a nucleic acid sequence of interest is inserted into the vector. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".DETAILED DESCRIPTION OF THE INVENTION

[0050] The inventors have discovered that administration of a GA binding protein subunit A (GABPA)-interacting domain or a GA binding protein subunit B (GABPB)-interacting domain can prevent mutant telomerase reverse transcriptase (TERT) promoter activation. TERT expression is used to maintain telomere length and enable neoplastic cell division, for example in cancer cells. Moreover, polypeptide constructs have been developed from portions of GABPA- interacting domains and GAB PB -interacting domains that can be fused to a ubiquitin ligase to tag GABPA or GABPB for degradation by the ubiquitin proteasome system.I. PolypeptidesA. GABPA-interacting Domains

[0051] This disclosure provides for polypeptides comprising a GABPA-interacting domain. GABPA is the DNA-binding subunit of the GA binding protein (GABP) complex. In addition to the GABPA subunit, the GABP complex has a transactivating subunit comprised of GA binding protein subunit B (GABPB), either isoform GA binding protein subunit Bl (GABPB1) or isoform GA binding protein subunit B2 (GABPB2). GABPB 1 has two isoforms, one of which is the tetramer-forming GABPB IL. Portions of GABPB IL or GABPB2 are effective in interacting with GABPA and thus can be used alone or optionally fused, for example to a ubiquitin ligase to inhibit GABPA activation of mutant TERT promoters. For example, GABPA-interacting domains include but are not limited to (a) SEQ ID NO: 1, representing the sequence of human GABPB IL (SEQ ID NO: 3) from amino acids 5-166 (where numbering starts at the first amino acid of GABPB IL) and (b) SEQ ID NO: 2, representing amino acids 5-166 of human GABPB2 (SEQ ID NO: 4).

[0052] Further, as shown herein, smaller portions of GABPB IL (SEQ ID NO: 3) or GABPB2 (SEQ ID NO: 4) may be used. In some embodiments, the GABPA-interacting domain is at least 80, 85, 90, 95%, 98%, 99% or 100% identical with SEQ ID NO: 1 (i.e., amino acids 5-166 of SEQ ID NO: 3) or SEQ ID NO: 2, or a GABPA-binding fragment thereof. In some embodiments, the GABPA-interacting domain comprises the Ankyrin repeat sequence in GABPB IL (SEQ ID NO: 3) or GABPB2 (SEQ ID NO: 4) or a GABPA-binding fragment thereof. In some embodiments, the GABPA-binding fragment comprises amino acids 5-159, 5-160, 5-161, 5-162, 5-163, 5-164 or 5-165 of SEQ ID NO: 3 or 4. In some embodiments, the GABPA-interacting domain comprises the Ankyrin repeat sequence in GABPB1L (SEQ ID NO: 1) or GABPB2 (SEQ ID NO: 2) with deletions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 3 or SEQ ID NO: 4, optionally also having a deletion of 1, 2, 3, 4, 5, 6, or 7 amino acids from the C-terminus of SEQ ID NO: 3 or SEQ ID NO: 4.

[0053] In addition, larger portions of GABPB1L (SEQ ID NO: 3) or GABPB2 (SEQ ID NO: 4) may be used. In some embodiments, the GABPA-interacting domain comprises the Ankyrin repeat sequence in GABPB1L (SEQ ID NO: 1) or GABPB2 (SEQ ID NO: 2) and has the addition of 1, 2, 3, or 4 amino acids that correspond to the amino acids on the N-terminus of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the GABPA-interacting domain comprises no more than 170, 180, 190, or 200 contiguous amino acids of GABPB1L (SEQ ID NO: 3) or GABPB2 (SEQ ID NO: 4).

[0054] In addition to GABPB IL and GABPB2, GABPA-interacting domains, or fragments thereof, from the proteins in Table 1 can also be used as described herein as a GABPA- interacting domain (e.g., fused to a ubiquitin ligase). Such proteins include, but are not limited to, those provided in Table 1.Table 1 . Table of proteins having GABPA-interacting domainsabove, is provided as part of a fusion protein with one or more heterologous amino acid sequence. The heterologous amino acid sequence can comprise a separate functional domain, including but not limited to: an enzyme or other catalytically active protein or peptide, including but not limited to a ligase, protease, or nuclease (for example a Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN), Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) / Cas-type nuclease, or other nuclease with gene-editing functionality), or an antibody or a binding-fragment thereof, transcriptional activator or repressor, or other binding protein that can alter DNA transcription, replication, repair, recombination processes, or a combination thereof (e.g., a CRISPRi construct). In some embodiments, the translational fusion protein comprises the GABPA-interacting domain and a ubiquitin ligase. In other embodiments, the GABPA-interaction domain is covalently or non- covalently linked to a heterologous polypeptide, for example a ubiquitin ligase. Ubiquitin ligases are enzymes that induce ubiquitination and subsequent degradation of proteins by the ubiquitin proteasome system. For example, the ubiquitin ligase disclosed herein may be an El, E2, or E3 ubiquitin ligase. In some embodiments, the ubiquitin ligase is a human ubiquitin ligase.

[0056] In some embodiments, the ubiquitin ligase is an E3 ubiquitin ligase. In some embodiments, the E3 ligase is a cullin ring ubiquitin ligase. E3 ubiquitin ligases can include, butare not limited to: beta-Transducin repeat containing protein (BTRCP), F-Box and WD repeat domain-containing protein-7 (FBW7), S phase kinase associated protein-2 (SKP2), von Hippel Lindau protein (VHL), speckle-type POX protein (SPOP), suppressor of cytokine signaling-2 protein (SOCS2), ankyrin repeat and suppressor of cytokine signaling box-containing-1 protein (ASB1), and C-terminus of Hsc70-interacting protein (CHIP). An exemplary ubiquitin ligase can include, for example, SPOP (SEQ ID NO: 9). Other exemplary ubiquitin ligases are described in, for example, Bekes, et al., Nature Reviews Drug Discovery volume 21, pages 181-200 (2022). In some embodiments, the ubiquitin ligase sequence further comprises a flexible polypeptide linker sequence which may be codon optimized, as described herein, to remove a splice site present in the linker sequence.

[0057] In some embodiments, the GABPA-interacting domain is covalently or non-covalently linked to a heterologous polypeptide, for example a ubiquitin ligase interacting protein. Ubiquitin ligase interacting are proteins or portions of a protein capable of binding a ubiquitin ligase as described above or elsewhere herein. Exemplary ubiquitin ligase interacting proteins, include, but are not limited to those provided in Table 2.Such proteins include, but are not limited to, those provided in Table 2.Table 2. Table of ubiquitin ligase interacting proteins

[0058] In some embodiments, polypeptides disclosed herein further comprise a nuclear localization sequence (NLS). ANLS is an amino acid sequence that acts as a signal to mediate the transport of the polypeptide described herein from the cytoplasm into the nucleus of a cell, for example, a cell having a TERT promoter mutation. In some embodiments, a NLS is obtained from a ubiquitin ligase. An exemplary nuclear localization sequence can include, for example SEQ ID NO: 11. Other exemplary nuclear localization sequences are described in, for example, Lu, etal., Cell Communication and Signaling volume 19, article number: 60 (2021).

[0059] In some embodiments, polypeptides disclosed herein further comprise one or more amino acid substitutions to improve GABPA-binding. Such substitutions may be identified by one skilled in the art using programs such as AlphaFold® and Struct-Evo®. As used herein, substitution refers to removing at least one amino acid residue in a protein wildtype or starting sequence and inserting a different amino acid in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. As used herein, an improvement in binding (e.g., binding between a GABPA-interacting domain and GABPA) is not meant to denote a particular value, for example, a substitution may result in a 1% improvement in binding or a 10% improvement in binding.B. GABPB-interacting Domains

[0060] Also provided herein are polypeptides comprising a GABPB-interacting domain. As described above, GABPA and GABPB (either the GABPB1 or GABPB2 isoform) interact to form a functional complex which activates mutant TERT promoters. Portions of GABPA are effective in interacting with GABPB and can be used alone, or optionally fused to another polypepetide, for example a ubiquitin ligase, to inhibit GABPB activation of mutant TERT promoters. For example, GABPB-interacting domains include, but are not limited to, SEQ ID NO: SEQ ID NO: 19, representing the sequence of human GABPA.

[0061] As used herein, GABPB refers to both the GABPB 1 or GABPB2 isoform. Thus, for example, a GABPB-interacting domain may interact with either GABPB 1 or GABPB2. In someembodiments, the GABPB-interacting domain described herein interacts with GABPB1 (SEQ ID NO: 1) at amino acids 1-166, 5-166, 1-157, or 5-157. In some embodiments, the GABPB- interacting domain described herein interacts with GABPB2 (SEQ ID NO: 2) at amino acids 1- 166, 5-166, 1-157, or 5-157.

[0062] Further, as shown herein, smaller portions of GABPA (SEQ ID NO: 19) may be used as a GABPB-interacting domain. In some embodiments, the GABPB-interacting domain is at least 80, 85, 90, 95%, 98%, 99% or 100% identical with SEQ ID NO: 21 (i.e., amino acids 316- 454 of SEQ ID NO: 19) or SEQ ID NO: 22 (i.e., amino acids 316-429 of SEQ ID NO: 19), or a GABPB-binding fragment thereof. In some embodiments, the GABPB-binding fragment comprises amino acids 316-454 or 316-429 of SEQ ID NO: 19. In some embodiments, the GABPB-interacting domain comprises no more than 120, 130, 140 or 150 contiguous amino acids of human GABPA (SEQ ID NO: 19).

[0063] In addition, larger portions of GABPA (SEQ ID NO: 19) may be used. In some embodiments, the GABPB-interacting domain comprises the full length GABPA (SEQ ID NO: 19). In some embodiments, the GABPB-interacting domain comprises the full length GABPA and has the addition of 1, 2, 3, or 4 amino acids that correspond to the amino acids on the N- terminus of SEQ ID NO: 19.

[0064] In some embodiments, the GABPB-interacting domain comprises one or more substitutions to improve GABPB binding. In some embodiments, the GABPB-interacting domain comprises GABPA (SEQ ID NO: 19) and further comprises one or more substitutions at amino acid residues K364, R376, R379, Y380, Y381, Y382, or K389. In some embodiments, the GABPB-interacting domain comprises GABPA (SEQ ID NO: 19) and further comprises one or more substitutions selected from K364A, R376A, R379A, Y380A, Y381A, Y382A, or K389A. In some embodiments, the GABPB-interacting domain comprises GABPA (SEQ ID NO: 19) and further comprises R376A and R379A substitutions. In some embodiments, the GABPB- interacting domain is at least 80, 85, 90, 95%, 98%, 99% or 100% identical with SEQ ID NO: 23.

[0065] In some embodiments, the GABPB-interacting domain, for example as described above, is provided as part of a fusion protein with one or more heterologous amino acid sequence. In some embodiments, the translational fusion protein comprises the GABPB-interacting domain and a ubiquitin ligase. In other embodiments, the GABPB-interaction domain is covalently or non-covalently linked to a heterologous polypeptide, for example a ubiquitin ligase. Discussion relating to ubiquitin ligases above and elsewhere herein apply to the present polypeptide comprising a GABPB-interacting domain and a ubiquitin ligase.

[0066] In some embodiments, polypeptides comprising a GABPB-interacting domain and a ubiquitin ligase disclosed herein further comprise a nuclear localization sequence (NLS). In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 11. Discussion relating to NLS above and elsewhere herein apply to the present polypeptide comprising a GABPB-interacting domain and a ubiquitin ligase.II. Nucleic Acids, Vectors, and Cells

[0067] Nucleic acids encoding the GABPA-interacting domain or fusion proteins comprising the GABPA-interacting domain and optionally a ubiquitin ligase are also provided. In certain embodiments, a nucleic acid comprising a polynucleotide encoding the polypeptide as described herein is used for expression of the polypeptide in vitro (e g., for production of the polypeptide), ex vivo, or in vivo. Such nucleic acids may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or analogs thereof. In some embodiments, nucleic acids comprise a promoter operably linked to the polynucleotide. Exemplary nucleic acids encoding a GABPA-interacting domain are provided in SEQ ID NOS: 5, 6, 7 or 8.

[0068] Also provided are nucleic acids encoding the GABPB-interacting domain or fusion proteins comprising the GABPB-interacting domain and optionally a ubiquitin ligase. In certain embodiments, a nucleic acid comprising a polynucleotide encoding the polypeptide as described herein is used for expression of the polypeptide in vitro (e.g., for production of the polypeptide), ex vivo, or in vivo. Such nucleic acids may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or analogs thereof. In some embodiments, nucleic acids comprise a promoter operably linked to the polynucleotide. An exemplary nucleic acid encoding a GABPB-interacting domain are provided in SEQ ID NOs: 20, 24, or 25.

[0069] The nucleic acids may be introduced into cells ex vivo or in vivo, or administered to a subject in vivo. For production of the polypeptide in vitro, one can use prokaryotic or eukaryotic cells for polypeptide production.

[0070] Nucleic acids for administration to a subject can be formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier can vary depending on the mode of administration, which can be as described herein for administration of the polypeptide.

[0071] DNA or RNA compositions can be administered once, or multiple times as needed to induce the desired response. Multiple administrations can be administered, for example, biweekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.

[0072] Nucleic acids can be administered in solution (e.g., a phosphate-buffered saline solution) by injection, usually by an intravenous, subcutaneous, or intramuscular route. Dosages depend on the route of administration and can be readily determined by one of skill in the art.

[0073] In some embodiments, the nucleic acid comprising a polynucleotide encoding the polypeptide comprising a GABPA-interacting domain or GABPB -interacting domain optionally fused to a ubiquitin ligase is provided, or administered, as a vector. In some embodiments, the vector is a viral vector, wherein the nucleic acid comprising a polynucleotide encoding the polypeptide comprising a GABPA-interacting domain or GABPB -interacting domain optionally fused to a ubiquitin ligase can be contained within a viral vector and administered as a viral particle. Exemplary viral vectors include but are not limited to adenovirus vectors (e.g., Ad2, Ad5, Ad7), adeno-associated viral vectors, herpes simplex viral vectors, retroviral vectors, pox viral vectors (such as vaccinia and avian poxvirus vectors, such as the fowlpox and canarypox vectors), lentiviral vectors, alphavirus vectors, poliovirus vectors, measles vectors, and other positive and negative stranded RNA viruses, viroids, and virusoids, or portions thereof. In some embodiments, the nucleic acid may be codon optimized for delivery as a viral vector.

[0074] In some embodiments, the retroviral vector is a replicating retroviral vector. Replicating retroviral vectors have viral genes necessary for replication. Thus, replicating retroviral vectors can replicate and spread in dividing host cells, leading to persistent expression of genetic information contained in the replicating retroviral vector.

[0075] In other embodiments, the nucleic acid may be administered as a non-viral vector, including, but not limited to, as a plasmid, in a nanoparticle, (e.g., a lipid nanoparticle), or in a liposome.

[0076] Provided herein are cells comprising the nucleic acid as described herein, or the vector as described herein. In some embodiments, the cell is a human cell, optionally comprising a TERT promoter mutation. In some embodiments, the cell is a cancer cell. The nucleic acid or vector may be introduced to the cell using one of the methods as described herein.III. Methods and Pharmaceutical Compositions

[0077] Also provided herein are methods of inhibiting expression of TERT in a cell having a TERT promoter mutation comprising introducing a polypeptide, nucleic acid, or vector as described herein into the cell. The polypeptides or polynucleotides encoding the polypeptides containing a GABPA-interacting domain or GABPB-interacting domain optionally fused to a ubiquitin ligase can be used to inhibit TERT expression in a cell having a TERT promoter mutation. Polynucleotides may be introduced to the cell as a nucleic acid or in a vector containing the nucleic acid. The polypeptide or polynucleotide encoding the polypeptide is introduced into the cell such that the polypeptide is expressed by the cell and binds a GABPA or GABPB protein therein. In some embodiments, the cell is in a human. Accordingly, the polypeptides or polynucleotides described herein can be administered to a human having or suspected of having a TERT promoter mutation in an appropriate dosage to ameliorate or treat at least one symptom thereof. Thus, also provided herein are methods of inhibiting expression of TERT in a cell having a TERT promoter mutation comprising introducing a polypeptide or polynucleotide as described herein into the cell.

[0078] The TERT promoter mutation may be any alteration that results in the addition of a second binding motif for E26 transformation-specific transcription factors within the TERT promoter, including, but not limited to, point mutations, translocations, duplications, or deletions. TERT promoter mutations are discussed, for example, in Bell, et al., Mol. Cancer. Res., volume 14, pages 315-323 and Barger et al., Nature Comms., 13: 5430 (2022). Exemplary TERT promoter mutations include, but are not limited to, those listed in Table 3.Table 3. Table of TERT promoter mutations

[0079] As described herein, the polypeptide or polynucleotide encoding a polypeptide may be introduced to a cell such that the polypeptide is expressed and binds a GABPA or GABPB protein therein. In some embodiments, the cell having a TERT promoter mutation is a cancer cell. In some embodiments, the cancer type includes, but is not limited to bladder cancer, connective tissue cancer, skin cancer, liver cancer, thyroid cancer, ovarian cancer, cervical cancer, adrenal cancer, gastric cancer, head and neck cancer, bone cancer, ocular cancer, esophageal cancer, lung cancer, mesothelioma, neuroblastoma, and central nervous system cancer including glioblastoma, meningioma, and medulloblastoma.

[0080] Also provided are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and the polypeptide as described herein comprising at least a GABPA- interacting domain or GABPB -interacting domain and optionally fused to a ubiquitin ligase. In some embodiments, the pharmaceutical composition comprises a nucleic acid or vector encoding the polypeptide as described herein. The pharmaceutical composition can additionally contain other therapeutic agents that are suitable for treating or preventing a given disorder.Pharmaceutically carriers can enhance or stabilize the composition, or to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0081] A pharmaceutical composition as described herein can be administered by a variety of methods known in the art. The route and / or mode of administration vary depending upon the desired results. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. The pharmaceutically acceptable carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., polypeptide described herein, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0082] Typically, a therapeutically effective dose or efficacious dose of the polypeptides, nucleic acids, or vectors described herein is employed in the pharmaceutical compositions. The polypeptides, nucleic acids, or vectors can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the desired response (e.g., a therapeutic response). In determining a therapeutically or prophylactically effective dose, a low dose can be administered and then incrementally increased until a desired response is achieved with minimal or no undesired side effects. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0083] Actual dosage levels of the active ingredients in the pharmaceutical compositions can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration,without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.EXAMPLES

[0084] Reactivation of telomerase reverse transcriptase (TERT) is necessary to overcome replicative senescence and prevent cell apoptosis. TERT mutations are the most common noncoding mutation in cancer. These mutations reactivate telomerase, allowing for indefinite maintenance of telomere length and enabling cellular immortalization. See, for example, Kim, et al., Science volume 266, pages 2011-2015 (1994). The transcription factor binding site created by the promoter mutations specifically recruit the E26 transformation-specific transcription factor GA-binding protein (GABP), a multimeric transcription factor composed of the GABPA and GABPB subunits. GABPB is encoded by either GABPB1, expressed as both a dimerforming GABPB1S and tetramer-forming GABPB IL, or its paralogue GABPB2. See, for example, Bell et al., Science volume 348, pages 1036-1039 (2015).

[0085] Recent developments in the design of proteolysis-targeting chimera protein degraders have allowed specific targeting of transcription factors for degradation, for example, by fusion of a ligand for a protein of interest with a ubiquitin ligase. See, for example, Bekes, et al., Nature Reviews Drug Discovery volume 21, pages 181-200 (2022). Three different GABP complexes, the GABPB 1 S dimer, the GABPB IL heterotetramer, and the GABPB2 heterotetramer, can maintain activation of the mutant TERT promoter. However, in each, GABPA binding to the promoter is necessary. Thus, determining a minimal domain which could inhibit GABPA binding to the promoter and optionally be fused to a ubiquitin ligase was critical to evaluating GABPA as a therapeutic target.Methods

[0086] Cell Culture - LN-18, U-251, LN-229, UM-UC-3, and SF7996 cells were cultured in DMEM and Ham’s F12 50 / 50 mixed media (Corning, Cat # MT10090CV) with 10% fetal bovine serum (Gibco, Cat # 10082-147) and lx penicillin / streptomycin (Gibco, Cat # 15-140- 122). Human embryonic kidney 293T cells (ATCC), were cultured in Dulbecco's modified Eagle's medium-nutrient mixture supplemented with 10 % fetal bovine serum (Seradigm, VWR) and 1 % Penicillin / streptomycin (Corning, Inc.). Cell cultures were maintained at 37° Celsius in a humidified incubator with 5% CO2. Medium was changed every 3-5 days depending on cell density. Cells were split at a ratio of 1 :3-l : 10 when they reached 80% to 90% confluence. Cell lines were authenticated by short tandem repeat analysis and confirmed to be Mycoplasma freeby PCR using the method previously described in Uphoff, et al, Cancer Cell Culture volume 731, pages 93-103 (2011).

[0087] RT-qPCR - Total RNA was purified with Quick-RNA Microprep (Zymo Research, #R1051) or Miniprep (Zymo Research, #R1058) kits. cDNA was generated by converting 1000 ng of Dnase-treated RNA to cDNA using the iScript cDNA Synthesis Kit (BioRad, #1708891). This cDNA was then diluted 1 :5 using nuclease free water, and 2 pl was used as the template material for qPCR reactions alongside standard curves for each target gene. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed using the POWER SYBR Green Master Mix (Applied Biosystems, #4367659). Dissociation curves were performed to confirm PCR specificity. Relative expression levels were calculated using QuantStudio 5 (Thermo Scientific) measured against GUSB.

[0088] Production of Lentiviral Particles and in vitro Lentiviral Transduction -Replication-deficient lentivirus was produced by transient transfection of psPAX2 (Addgene plasmid ID 12260), pDM2G (Addgene plasmid ID 12259) and the transfer plasmid containing the GABPA-interacting domain (N174-MCS-IRES-PURO, Addgene plasmid ID 81068) into HEK293T cells. Viral supernatant was collected at 72-hours post transfection and filtered through a 0.22-micron PES membrane filter. Cell lines were transduced with serially diluted viral supernatant containing polybrene (EMD Millipore, #TR-1003-G) at a final concentration of 4 pg / mL. Media was replaced with complete media containing 1.0 pg / mL of puromycin and selection was conducted for 72 hours. The cells transduced with the titration that resulted in approximately 30% survival following antibiotic selection were expanded and used for downstream experiments.

[0089] Western Blot - Whole cell protein extracts were prepared using Mammalian Protein Extraction Reagent (Thermo Scientific, #78501) with IX Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, #78440) and 0.2 units / mL final of Turbo Nuclease (Sigma, #T4330-50KU). Extracts were incubated on ice for 10 minutes, centrifuged at 4° Celsius for 10 minutes at 16,000 g, and the supernatant was isolated. Protein concentration was determined by bicinchoninic acid protein assay (Pierce, #23225). Equal quantities of protein (20- 50 pg) were fractionated on 10% Bis-Tris SDS-polyacrylamide gel electrophoresis gels (Invitrogen, #NPO315BOX) and transferred to PVDF membranes (Bio-Rad, #1620177). Alpha-actinin served as the loading control for each experiment. Efficient transfer and equal loading were confirmed with Ponceau S staining of the membrane. Membranes were blocked with 5% nonfat dry milk (Bio-Rad, #1706404) in lx Tris-buffered saline Tween-20 (TBST) (Pierce, #28360) for 1 hour at room temperature while rocking. The membranes were incubated with primary antibodies at 4° Celsius overnight: anti-GABPA (Santa Cruz, sc-22810), at a 1 :500 dilution in 5% nonfat dry milk in lx TBST; anti-alpha-actinin (Cell Signaling, #6487), at a 1 :2000 dilution in 5% BSA in lx TBST. The next day, membranes underwent three cycles of washing with lx TBST for 5 minutes while rocking at room temperature. The membranes were the incubated for 1 hour at room temperature with a horseradish peroxidase-linked anti-rabbit immunoglobulin G secondary antibody (Cell Signaling, #7074), at a 1:5000 dilution in 5% nonfat dry milk in lx TBST. Enhanced chemiluminescence (Thermo Scientific, #32109) was used for protein detection.

[0090] Plasmid Construction - The B1DN10 SPOP fused via a linker sequence was optimized for mammalian gene expression and virus stability by mammalian codon optimization and removal of cryptic splice sites and repeats. This resulted in the generation of two vectors that differ by one amino acid in the linker sequence. The single amino acid substitution or mutation removes a high scoring cryptic splice site that could potentially destabilize the vector. The P2A B1DN10 SPOP sequences were synthesized by TWIST Biosciences and Gibson assembly was utilized to place these sequences into the retroviral replicating vector plasmid pAC3_P2A, creating pAC3_P2A_BlDN10_SPOP(wildtype linker) and pAC3_P2A_BlDN10_SPOP(mutant linker).

[0091] Vector Production - Retroviral replicating vectors encoding the B IDNIO spop genes were produced via transient calcium phosphate co-transfection of 293T cells with viral plasmid DNA. Virus containing supernatant was collected at approximately 36-48 hours after initial transfection, clarified by centrifugation and filtered through a 0.45 pm filter. The resulting supernatants were stored in aliquots at -80° Celsius prior to determining vector titers.

[0092] Functional Vector Titer Determination - Functional viral titers in transducing units per mL (TU / mL) were determined via flow-cytometry following transduction of U251 cells with various volumes (0.002-0.1 mL) of vector. 100,000 U251 cells were seeded into each well of a 6 well TC plate. 24 hours later, representative wells were counted to determine the number of cellson the day of transduction. The medium in remaining wells was replaced with 1 mL of medium containing 4 mg / mL of polybrene. Different volumes of vector supernatant (0.002, 0.005, 0.01, 0.025, 0.05, or 0.1 mL) were added to individual wells and the cells were incubated for 2 hours before adding an additional 1 mL of complete medium. The cells were incubated for 24 hours at 37° Celsius. After 24 hours, the medium was replaced with Azidothymidine (AZT, Sigma Aldrich, A2169) containing medium (50 pM) to prevent additional vector replication and spread, and cells were incubated for another 24 hours. Cells were harvested and processed for titer determination via intracellular detection of the viral gag protein. Cells were fixed with 4% paraformaldehyde for 20 min, prior to permeabilization with 0.1% TritonX in PBS for 30 minutes. Cells were washed thoroughly with staining buffer (0.2% BSA in PBS) and a 1 / 8000 dilution of a rabbit anti GAG antibody (Abeam, ab 100970) was added to le6cells in a final volume of 0.1 mL and the cells were incubated at room temperature with shaking for 30 minutes.

[0093] Cells were washed thoroughly with staining buffer and a 1 / 2000 dilution of secondary antibody, Goat anti rabbit IgG conjugated to APC (Invitrogen, A10931) was added to the cells in a final volume of 0.1 mL and the cells were incubated at room temperature with shaking for 30 min in the dark. Cells were washed thoroughly and the percent of GAG positive cells was determined by flow cytometry to detect the APC / GAG positive cells at each vector supernatant volume. Titer was determined using the following calculation:(Number of cells on day of transduction / 100) X (% of positive cells) Amount applied to the well (mL)

[0094] Assessing Vector Replication and Kinetics - Vector replication and kinetics were determined following transduction of U251 cells at a multiplicity of infection (MOI, number of vector particles per cell) of 0.01. U251 cells were seeded at 100,000 cells per well of a 6 well plate and incubated for 24 hours at 37° Celsius in an humidified incubator. Representative wells were counted to determine the cell number per well and this number was used to determine the volume of vector needed to achieve an MOI of 0.01. The cells were incubated for a further 24 - 48 hours, cells were split at a 1 : 10 ratio on reaching confluence and at regular intervals after with the remaining 90% of cells processed as previously described for the detection of intracellular viral GAG protein. The increase in percent GAG positive cells over time was graphed.

[0062] Assessing Vector Stability - Vector replication and kinetics were determined following transduction of U251 cells at an MOI of 0.1. U251 cells were seeded at 100,000 cells per well of a 6 well plate and incubated for 24 hours at 37° Celsius in a humidified incubator. Representative wells were counted to determine the cell number per well and this number was used to determine the volume of vector needed to achieve an MOI of 0.1. Cells were incubated for a total of 7 days following transduction with splits at 1 : 10 ratio at regular intervals when confluency was reached. Each 7-day interval was considered a single virus passage, and on day 6 of each passage, naive U251 cells were seeded at 100,000 cells per well of a 6 well plate. The vector supernatant from the transduced cells was used to transduce the naive cells on day 7 at a 1 : 10 dilution. The day 7 transduced cells were harvested, and genomic DNA (gDNA) was isolated using the Qiagen DNeasy Blood and Tissue Kit. For vector stability this process is repeated for up to 10 passages.

[0095] The gDNA is analyzed for vector stability, e.g., detection of transgene dropouts by PCR amplification of the vector DNA, specifically the region spanning the transgene insertion site using the following primers:Forward (3’end of env); 5’-CTGATCTTACTCTTTGGACCTTG-3’(SEQ ID NO: 26)Reverse (5’UTR); 5’-CCCCTTTTTCTGGAGACTAAATAA-3’ (SEQ ID NO: 27)

[0096] The resulting PCR products were run on a 1% agarose gel containing ethidium bromide and the banding pattern was analyzed following exposure of the gel to UV light. Vector instability or transgene dropout is identified by band sizes lower than the expected size seen in the positive plasmid DNA controls.ResultsGABPA-Interacting Domains

[0097] To determine the minimal domain of GABPB1 that could interact with GABPA, two polypeptides spanning the Ankyrin repeat domain of GABPB 1 were designed. As shown in FIG. 1, GABPB1 amino acids 5-166 (SEQ ID NO: 1) and amino acids 5-157 transgenes were generated and lentivirally transduced into mutated TERT promoter and wildtype TERT promoter cell lines. The construct containing GABPB 1 amino acids 5-166 (SEQ ID NO: 1) significantly reduced TERT expression in the mutant TERT promoter cells, indicating that the construct couldfunction as a dominant negative, while the amino acids 5-157 construct did not significantly reduce TERT expression (FIG. 1). TERT expression was confirmed by GABPB1 expression, which is inversely correlated with the GABP complex function. The construct containing GABPB1 amino acids 5-166 (SEQ ID NO: 1) inhibited GABP function in both mutated TERT promoter and wildtype TERT promoter cell lines, while the amino acid 5-157 construct did not (FIG. 1).

[0098] A fusion protein was designed using the GABPB1 minimal domain construct (SEQ ID NO: 1), a flexible polypeptide linker, and an ubiquitin ligase binding domain, specifically the cullin 3-binding BTB domain of speckle-type POX protein (SPOP) (SEQ ID NO: 9) (FIG. 2). As shown in FIG. 2, the fusion protein optionally contained a nuclear localization sequence (SEQ ID NO: 11). To test the degrader function of the fusion protein, three mutated TERT promoter glioblastoma cell lines (U-251, LN-229, and SF7996) and one TERT promoter wildtype cell line (LN- 18) were lentivirally transduced with an empty vector, the minimal GABPB1 domain (SEQ ID NO: 1), or the fusion protein as described above, with or without the nuclear localization sequence (FIG. 3). Each of the minimal GABPB1 domain, the fusion protein with the nuclear localization sequence, and the fusion protein without the nuclear localization sequence significantly reduced TERT expression levels in the mutated TERT promoter glioblastoma cell lines (FIG. 3).

[0099] Subsequent western blot analysis of GABPA protein levels showed that the fusion protein with the nuclear localization sequence resulted in the greatest level of GABPA protein reduction, presumably because it can interact with and tag for ubiquitination GABPA subunits within both the cytoplasm and nucleus (FIG. 4). To evaluate the fusion protein function using a clinically relevant delivery method, mutated TERT promoter glioblastoma cells were transduced with replicating retroviral vectors containing the minimal GABPB1 domain (SEQ ID NO: 1), or the fusion protein with the nuclear localization sequence, with or without a mutation in the linker region to eliminate a high scoring splice site (VI 791) (FIG. 5-6). Retroviral vector replication and spread in U251 cells was not adversely affected by insertion of the B1DN10 or the B1DN10_SPOP sequences, with greater than 80 % of cells positive for retroviral GAG 15 days post transduction at an MOI of 0.01. The stability of each vector was confirmed by serial passage of the vector on naive U251 cells. No evidence of transgene drop-out was observed for theB 1 DN 10 or B 1 DN 1 O SPOP encoding vectors up to passage 5 and assessment will be continued to passage 10. Furthermore, when delivered by replicating retrovirus, the fusion protein with the nuclear localization sequence, with or without the linker mutation, decreased TERT expression significantly compared to a control and the GABPB1 minimal domain alone (FIG. 7). Likewise, GABPB1 expression was increased 2.7-fold in the cells transduced with the fusion protein compared to the control or GABPB1 minimal domain alone (FIG. 7).GABPB-Interacting Domains

[0100] To generate a GABPB-interacting domain that acts as a dominant negative, the DNA binding abilities of GABPA were ablated. GABPA residues critical for DNA-binding were identified using mutagenesis and two mutant GABPA proteins were generated from the full length GABPA (SEQ ID NO: 19). The mutant GABPA proteins were as follows:Mutant 1 : R376A, R379AMutant 2: R376A, R379A, Y380F, Y381F, Y382F, K364A, K389A

[0101] TERTp mutant human GBM cell lines were stably infected to measure the effect of the above mutants acting as GABPA dominant negatives on TERT expression. The R376A and R379A mutations (Mutant 1 above; SEQ ID NO: 23) were sufficient and more effective at creating a GABPA dominant negative (FIG. 8).

[0102] Next, to identify the minimal portion of GABPA that could still act as a dominant negative, GBM cells were infected with an empty vector, a wildtype GABPA, the Mutant 1 variant, or the Mutant 2 variant in the following sequence length variants: full length GABPA (SEQ ID NO: 19), residues 316-454 (SEQ ID NO: 21), and residues 316-429 (SEQ ID NO: 22).

[0103] Both the 316-454 and 316-429 GABPA fragments were able to act as dominant negatives (FIGs. 9-10). Function was determined by conducting qPCR for GABPB1 expression, which is inversely related to GABP function (i.e., if GABP is inhibited, then GABPB1 expression will increase). This effect is because GABP binds to and represses the GABPB1 promoter in a negative feedback loop.

[0104] To improve the effect of this dominant negative, the same sequence of SPOP used in with the GABPA-interacting domain above was fused to the GABPA fragment of residues 316-454 in the Mutant 1 variant. The function of this degrader was assessed by lentivirally transducing GBM cell lines and conducting a western blot for GABPB 1 and GABPB2 as well as conducting qPCR to assess TERT expression. As shown in Figures 11-12, this degrader is able to significantly reduce the level of GABPB 1 protein expression and this leads to a significant reduction in TERT expression.

[0105] Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only and are not meant to be limiting in any way.

[0106] All documents (for example, patents, patent applications, books, journal articles, or other publications) cited herein are incorporated by reference in their entirety and for all purposes, to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent such documents incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any contradictory material.INFORMAL SEQUENCE LISTINGSEQ ID NO: 1 human GABPB1 (Asp5-Thrl66):DLGKKLLEAARAGQDDEVRILMANGAPFTTDWLGTSPLHLAAQYGHYSTTEVLLRAGV SRDARTKVDRTPLHMAASEGHASIVEVLLKHGADVNAKDMLKMTALHWATEHNHQEVVELLIKYGADVHTQSKFCKTAFDISIDNGNEDLAEILQIAMQNQINTSEQ ID NO: 2 human GABPB2 (Asp5-Vall66):DLGKRLLEAARKGQDDEVRTLMANGAPFTTDWLGTSPLHLAAQYGHYSTAEVLLRAG VSRDARTKVDRTPLHMAAADGHAHIVELLVRNGADVNAKDMLKMTALHWATERHHRDVVELLIKYGADVHAFSKFDKSAFDIALEKNNAEILVILQEAMQNQVNVSEQ ID NO: 3 human GABPB1L (Metl-Val396):MSLVDLGKKLLEAARAGQDDEVRILMANGAPFTTDWLGTSPLHLAAQYGHYSTTEVLLRAGVSRDARTKVDRTPLHMAASEGHASIVEVLLKHGADVNAKDMLKMTALHWATEHNHQEVVELLIKYGADVHTQSKFCKTAFDISIDNGNEDLAEILQIAMQNQINTNPESPDTVTIHAATPQFIIGPGGVVNLTGLVSSENSSKATDETGVSAVQFGNSSTSVLATLAALAEASAPLSNSSETPVVATEEVVTAESVDGAIQQVVSSGGQQVITIVTDGIQLGNLHSIPTSGIGQPIIVTMPDGQQVLTVPATDIAEETVISEEPPAKRQCIEIIENRVESAEIEEREALQKQLDEANREAQKYRQQLLKKEQEAEAYRQKLEAMTRLQTNKEAVSEQ ID NO: 4 human GABPB2 (Metl-Ser449):MSLVDLGKRLLEAARKGQDDEVRTLMANGAPFTTDWLGTSPLHLAAQYGHYSTAEVLLRAGVSRDARTKVDRTPLHMAAADGHAHIVELLVRNGADVNAKDMLKMTALHWATERHHRDVVELLIKYGADVHAFSKFDKSAFDIALEKNNAEILVILQEAMQNQVNVNPERANPVTDPVSMAAPFIFTSGEVVNLASLISSTNTKTTSGDPHASTVQFSNSTTSVLATLAALAEASVPLSNSHRATANTEEIIEGNSVDSSIQQVMGSGGQRVITIVTDGVPLGNIQTSIPTGGIGQPFIVTVQDGQQVLTVPAGKVAEETVIKEEEEEKLPLTKKPRIGEKTNSVEESKEGNERELLQQQLQEANRRAQEYRHQLLKKEQEAEQYRLKLEAIARQQPNGVDFTMVEEVAEVDAVVVTEGELEERETKVTGSAGTTEPHTRVSMATVSSSEQ ID NO: 5 Nucleotides encoding SEQ ID NO: 1:GATCTCGGAAAAAAGCTCCTGGAGGCCGCACGGGCCGGCCAGGACGACGAAGTGCGGATCCTGATGGCAAACGGGGCTCCATTCACCACCGACTGGCTGGGGACATCTCCCCTCCATCTCGCTGCTCAATATGGGCATTATTCCACCACCGAAGTGCTGCTCCGCGCCGGGGTCTCTCGGGACGCACGCACAAAAGTGGACCGGACCCCCCTCCACATGGCCGCCTCTGAGGGCCACGCCTCCATCGTCGAAGTGCTGCTGAAACACGGTGCCGACGTGAACGCTAAAGACATGCTGAAGATGACAGCCCTCCACTGGGCCACAGAGCATAACCATCAAGAGGTGGTCGAGCTGCTGATTAAGTACGGCGCCGACGTCCACACACAGAGCAAGTTCTGTAAAACAGCTTTTGACATCAGTATCGACAATGGCAACGAAGATCTGGCCGAGATTCTGCAAATCGCCATGCAGAATCAGATCAATACCSEQ ID NO: 6 Nucleotides encoding SEQ ID NO: 2:GACTTGGGAAAGAGGTTGCTAGAAGCAGCAAGAAAAGGCCAAGATGATGAAGTGA GAACGTTGATGGCAAATGGCGCCCCATTCACCACAGACTGGCTTGGAACATCACCCCTCCACCTTGCAGCTCAATATGGTCATTATTCCACAGCAGAAGTACTCCTTCGAGCA GGTGTTAGCAGGGATGCCCGGACTAAAGTAGACAGGACCCCCTTGCACATGGCTGC AGCCGATGGACATGCGCACATCGTGGAACTGCTTGTTCGGAATGGTGCAGATGTGA ATGCCAAGGACATGCTGAAGATGACAGCTTTGCATTGGGCCACAGAGCGCCACCAT CGAGATGTCGTAGAGTTACTTATCAAATATGGAGCTGATGTCCATGCTTTCAGCAAATTTGATAAATCAGCCTTTGACATAGCTCTGGAGAAAAACAATGCTGAGATTTTGGTC ATCCTCCAGGAAGCAATGCAGAATCAGGTGAATGTTSEQ ID NO: 7 Nucleotides encoding SEQ ID NO: 3:ATGTCCCTGGTAGATTTGGGAAAGAAGCTTTTAGAAGCGGCACGAGCAGGTCAAGA TGATGAAGTTCGTATTTTGATGGCAAATGGAGCTCCCTTTACTACAGACTGGCTGGGAACTTCTCCACTTCATCTAGCAGCACAGTATGGTCATTATTCCACCACAGAGGTACT GCTGCGAGCTGGTGTGAGCAGAGATGCCAGAACCAAAGTGGACCGAACACCATTACATATGGCAGCTTCTGAGGGCCATGCCAGCATAGTAGAGGTTTTACTTAAGCATGGTG CTGATGTCAATGCAAAGGACATGTTAAAGATGACAGCTCTCCATTGGGCCACAGAACACAATCATCAAGAGGTGGTGGAACTTTTAATCAAATATGGTGCTGATGTACACACG CAAAGTAAATTTTGTAAAACTGCATTTGATATTTCAATAGACAATGGAAATGAAGAT TTAGCAGAGATATTACAGATTGCTATGCAGAACCAAATCAACACAAACCCAGAGAG TCCTGACACTGTGACAATACATGCTGCAACACCACAGTTTATCATTGGACCTGGAGG GGTGGTGAACCTAACAGGTCTGGTATCTTCAGAAAATTCATCCAAGGCAACAGATGAAACGGGTGTATCTGCTGTTCAGTTTGGAAACTCTTCTACATCAGTATTAGCTACATT AGCTGCCTTAGCTGAAGCATCTGCTCCATTGTCCAATTCTTCAGAAACTCCAGTAGT GGCCACAGAAGAAGTAGTTACTGCAGAATCTGTGGATGGTGCCATTCAGCAAGTAG TTAGTTCAGGGGGTCAGCAAGTCATCACAATAGTTACAGATGGAATTCAGCTTGGAA ATTTGCACTCTATTCCAACCAGTGGAATTGGTCAGCCCATCATTGTGACCATGCCAGATGGACAACAAGTATTAACAGTACCAGCAACAGACATTGCTGAAGAAACTGTTATA AGTGAAGAACCACCAGCTAAGAGACAATGTATCGAAATAATTGAAAACCGGGTGGA ATCTGCAGAAATAGAAGAGAGAGAAGCTCTTCAGAAACAGCTGGATGAAGCAAATC GAGAAGCACAAAAATATCGACAGCAGCTCCTAAAGAAAGAACAGGAAGCAGAGGCCTACAGACAGAAGTTGGAAGCTATGACTCGTCTTCAGACTAATAAAGAAGCTGTTTA ASEQ ID NO: 8 Nucleotides encoding SEQ ID NO: 4:ATGTCTTTGGTGGACTTGGGAAAGAGGTTGCTAGAAGCAGCAAGAAAAGGCCAAGA TGATGAAGTGAGAACGTTGATGGCAAATGGCGCCCCATTCACCACAGACTGGCTTG GAACATCACCCCTCCACCTTGCAGCTCAATATGGTCATTATTCCACAGCAGAAGTACTCCTTCGAGCAGGTGTTAGCAGGGATGCCCGGACTAAAGTAGACAGGACCCCCTTG CACATGGCTGCAGCCGATGGACATGCGCACATCGTGGAACTGCTTGTTCGGAATGGTGCAGATGTGAATGCCAAGGACATGCTGAAGATGACAGCTTTGCATTGGGCCACAGA GCGCCACCATCGAGATGTCGTAGAGTTACTTATCAAATATGGAGCTGATGTCCATGCTTTCAGCAAATTTGATAAATCAGCCTTTGACATAGCTCTGGAGAAAAACAATGCTGA GATTTTGGTCATCCTCCAGGAAGCAATGCAGAATCAGGTGAATGTTAATCCAGAGAGAGCCAACCCTGTGACTGACCCTGTGAGTATGGCTGCTCCATTCATCTTCACGTCGGGTGAGGTTGTTAACCTCGCAAGCCTTATTTCTTCAACCAACACCAAAACAACCTCAGGTGACCCCCATGCCTCAACAGTACAGTTTTCAAATTCTACCACCTCAGTGCTGGCTACCCTTGCAGCTCTTGCTGAGGCATCAGTCCCCCTCTCCAACTCACACAGAGCCACAGCCAATACAGAGGAAATTATAGAAGGAAATTCCGTTGACTCATCAATCCAGCAAGTAATGGGGAGTGGAGGCCAGAGGGTCATCACCATAGTGACTGATGGAGTCCCTCTGGGTAATATCCAAACTTCAATCCCTACTGGAGGCATTGGCCAGCCATTTATTGTAACTGTGCAAGATGGACAGCAAGTTCTAACTGTACCTGCTGGTAAGGTTGCAGAGGAGACTGTAATTAAAGAGGAAGAAGAAGAGAAGTTGCCACTAACAAAGAAACCAAGGATAGGAGAGAAGACAAACAGTGTGGAGGAAAGCAAGGAAGGCAATGAAAGAGAGCTACTACAGCAACAACTCCAGGAGGCCAATCGAAGAGCCCAGGAATACCGACACCAGCTCCTAAAGAAAGAGCAGGAAGCAGAACAGTACCGTCTTAAGCTGGAGGCCATAGCCCGACAGCAGCCCAATGGAGTTGATTTCACCATGGTTGAAGAGGTGGCTGAGGTAGATGCTGTAGTAGTCACAGAGGGGGAGTTGGAAGAGAGAGAGACAAAAGTGACTGGGTCA GCAGGGACCACAGAGCCTCACACTAGAGTTTCCATGGCAACTGTTTCATCTTAASEQ ID NO: 9 SPOP (Serl67-Arg375):SVNISGQNTMNMVKVPECRLADELGGLWENSRFTDCCLCVAGQEFQAHKAILAARSPVFSAMFEHEMEESKKNRVEINDVEPEVFKEMMCFIYTGKAPNLDKMADDLLAAADKYALERLKVMCEDALCSNLSVENAAEILILADLHSADQLKTQAVDFINYHASDVLETSGWKSMVVSHPHLVAEAYRSLASAQCPFLGPPRKRLKQSSEQ ID NO: 10 Nucleotides encoding SEQ ID NO: 9:AGTGTCAACATTTCTGGCCAGAATACCATGAACATGGTAAAGGTTCCTGAGTGCCGGCTGGCAGATGAGTTAGGAGGACTGTGGGAGAATTCCCGGTTCACAGACTGCTGCTTGTGTGTTGCCGGCCAGGAATTCCAGGCTCACAAGGCTATCTTAGCAGCTCGTTCTCCGGTTTTTAGTGCCATGTTTGAACATGAAATGGAGGAGAGCAAAAAGAATCGAGTTGAAATCAATGATGTGGAGCCTGAAGTTTTTAAGGAAATGATGTGCTTCATTTACACGGGGAAGGCTCCAAACCTCGACAAAATGGCTGATGATTTGCTGGCAGCTGCTGACAAGTATGCCCTGGAGCGCTTAAAGGTCATGTGTGAGGATGCCCTCTGCAGTAACCTGTCCGTGGAGAACGCTGCAGAAATTCTCATCCTGGCCGACCTCCACAGTGCAGATCAGTTGAAAACTCAGGCAGTGGATTTCATCAACTATCATGCTTCGGATGTCTTGGAGACCTCTGGGTGGAAGTCAATGGTGGTGTCACATCCCCACTTGGTGGCTGAGGCATACCGCTCTCTGGCTTCAGCACAGTGCCCTTTTCTGGGACCCCCACGCAAACGCCTGAAGCAATCCSEQ ID NO: 11 NLS of SPOP (Lys366-Ser371):KRLKQSSEQ ID NO: 12 Nucleotides encoding SEQ ID NO: 11:AAACGCCTGAAGCAATCCSEQ ID NO: 13 Linker present in SEQ ID NO: 9:S VNI SGQNTMNMVK VPECRL ADELGGSEQ ID NO: 14 Nucleotides encoding SEQ ID NO: 13:AGCGTGAATATCAGCGGCCAGAATACCATGAATATGGTGAAGGTCCCTGAGTGCAG ACTGGCCGACGAGTTGGGCGGASEQ ID NO: 15 Mutated linker:SVNISGQNTMNMIKVPECRLADELGGSEQ ID NO: 16 Nucleotides encoding SEQ ID NO: 15:AGCGTGAATATCAGCGGCCAGAATACCATGAATATGATCAAGGTCCCTGAGTGCAG ACTGGCCGACGAGTTGGGCGGASEQ ID NO: 17 human GABPB1S (Metl-Lys360):MSLVDLGKKLLEAARAGQDDEVRILMANGAPFTTDWLGTSPLHLAAQYGHYSTTEVLL RAGVSRDARTKVDRTPLHMAASEGHASIVEVLLKHGADVNAKDMLKMTALHWATEH NHQEVVELLIKYGADVHTQSKFCKTAFDISIDNGNEDLAEILQIAMQNQINTNPESPDTV TTHAATPQFnGPGGVVNLTGLVSSENSSKATDETGVSAVQFGNSSTSVLATLAALAEAS APLSNSSETPVVATEEVVTAESVDGAIQQVVSSGGQQVITIVTDGIQLGNLHSIPTSGIGQP IIVTMPDGQQVLTVPATDIAEETVISEEPPAKRQCIEIIENRVESAEIEVRSLLPGVLCRSHP KSEQ ID NO: 18 Nucleotides encoding SEQ ID NO: 17:ATGTCCCTGGTAGATTTGGGAAAGAAGCTTTTAGAAGCGGCACGAGCAGGTCAAGA TGATGAAGTTCGTATTTTGATGGCAAATGGAGCTCCCTTTACTACAGACTGGCTGGG AACTTCTCCACTTCATCTAGCAGCACAGTATGGTCATTATTCCACCACAGAGGTACT GCTGCGAGCTGGTGTGAGCAGAGATGCCAGAACCAAAGTGGACCGAACACCATTAC ATATGGCAGCTTCTGAGGGCCATGCCAGCATAGTAGAGGTTTTACTTAAGCATGGTG CTGATGTCAATGCAAAGGACATGTTAAAGATGACAGCTCTCCATTGGGCCACAGAA CACAATCATCAAGAGGTGGTGGAACTTTTAATCAAATATGGTGCTGATGTACACACG CAAAGTAAATTTTGTAAAACTGCATTTGATATTTCAATAGACAATGGAAATGAAGAT TTAGCAGAGATATTACAGATTGCTATGCAGAACCAAATCAACACAAACCCAGAGAG TCCTGACACTGTGACAATACATGCTGCAACACCACAGTTTATCATTGGACCTGGAGG GGTGGTGAACCTAACAGGTCTGGTATCTTCAGAAAATTCATCCAAGGCAACAGATG AAACGGGTGTATCTGCTGTTCAGTTTGGAAACTCTTCTACATCAGTATTAGCTACATT AGCTGCCTTAGCTGAAGCATCTGCTCCATTGTCCAATTCTTCAGAAACTCCAGTAGT GGCCACAGAAGAAGTAGTTACTGCAGAATCTGTGGATGGTGCCATTCAGCAAGTAGTTAGTTCAGGGGGTCAGCAAGTCATCACAATAGTTACAGATGGAATTCAGCTTGGAA ATTTGCACTCTATTCCAACCAGTGGAATTGGTCAGCCCATCATTGTGACCATGCCAG ATGGACAACAAGTATTAACAGTACCAGCAACAGACATTGCTGAAGAAACTGTTATA AGTGAAGAACCACCAGCTAAGAGACAATGTATCGAAATAATTGAAAACCGGGTGGA ATCTGCAGAAATAGAAGTAAGGAGTCTTTTACCCGGTGTGCTTTGCCGCAGTCATCC AAAATAASEQ ID NO: 19 human GABPA full length:MTKREAEELIEIEIDGTEKAECTEESIVEQTYAPAECVSQAIDINEPIGNLKKLLEPRLQCS LDAHEICLQDIQLDPERSLFDQGVKTDGTVQLSVQVISYQGIEPKLNILEIVKPADTVEVV IDPDAHHAESEAHLVEEAQVITLDGTKHITTISDETSEQVTRWAAALEGYRKEQERLGIPYDPIQWSTDQVLHWVVWVMKEF SMTDIDLTTLNISGRELC SLNQEDFFQRVPRGEILWSHLELLRKYVLASQEQQMNEIVTIDQPVQIIPASVQSATPTTIKVINSSAKAAKVQRAPRISGEDRSSPGNRTGNNGQ1QLWQFLLELLTDKDARDCISWVGDEGEFKLNQPELVAQKWGQRKNKPTMNYEKLSRALRYYYDGDMICKVQGKRFVYKFVCDLKTLIGYSAAELNRLV TECEQKKLAKMQLHGIAQPVTAVALATASLQTEKDNSEQ ID NO: 20 Nucleotides encoding SEQ ID NO: 19:ATGACTAAAAGAGAAGCAGAGGAGCTGATAGAAATTGAGATTGATGGAACAGAGAAAGCAGAGTGCACAGAAGAAAGCATTGTAGAACAAACCTACGCGCCAGCTGAATGTGTAAGCCAGGCCATAGACATCAATGAACCAATAGGCAATTTAAAGAAACTGCTAGAACCAAGACTACAGTGTTCTTTGGATGCTCATGAAATTTGTCTGCAAGATATCCAGCTGGATCCAGAACGAAGTTTATTTGACCAAGGAGTAAAAACAGATGGAACTGTACAGCTTAGTGTACAGGTAATTTCTTACCAAGGAATTGAACCAAAGTTAAACATCCTTGAAATTGTTAAACCTGCGGACACTGTTGAGGTTGTTATTGATCCAGATGCCCACCATGCTGAATCAGAAGCACATCTTGTTGAAGAAGCTCAAGTGATAACTCTTGATGGCACAAAACACATCACAACCATTTCAGATGAAACTTCAGAACAAGTGACAAGATGGGCTGCTGCACTGGAAGGCTATAGGAAAGAACAAGAACGCCTTGGGATACCCTATGATCCCATACAGTGGTCCACAGACCAAGTCCTGCATTGGGTGGTTTGGGTAATGAAGGAATTCAGCATGACCGATATAGACCTCACCACACTCAACATTTCGGGGAGAGAATTATGTAGTCTCAACCAAGAAGATTTTTTTCAGCGGGTTCCTCGGGGAGAAATTCTCTGGAGTCATCTGGAACTTCTCCGAAAATATGTATTGGCAAGTCAAGAACAACAGATGAATGAAATAGTTACAATTGATCAACCTGTGCAAATTATTCCAGCATCAGTGCAATCTGCTACACCTACTACCATTAAAGTTATAAATAGTAGTGCGAAAGCAGCCAAAGTACAAAGAGCGCCGAGGATTTCAGGAGAAGATAGAAGCTCACCTGGGAACAGAACAGGAAACAATGGCCAAATCCAACTATGGCAGTTTTTGCTAGAACTTCTTACTGATAAGGACGCTCGAGACTGCATTTCTTGGGTTGGTGATGAAGGTGAATTTAAGCTAAATCAGCCTGAACTGGTTGC ACAGAAATGGGGACAGCGTAAAAATAAGCCTACGATGAACTATGAGAAACTCAGTCGTGCATTAAGATATTATTACGATGGGGACATGATTTGTAAAGTTCAAGGCAAGAGATTTGTGTACAAGTTTGTCTGTGACTTGAAGACTCTTATTGGATACAGTGCAGCGGAGTTGAACCGTTTGGTCACAGAATGTGAACAGAAGAAACTTGCAAAGATGCAGCTCCAT GGAATTGCCCAGCCAGTCACAGCAGTAGCTCTGGCTACTGCTTCTCTGCAAACGGAA AAGGATAATTGASEQ ID NO: 21 human GABPA (Met316-Asn454):MNNGQIQLWQFLLELLTDKDARDCISWVGDEGEFKLNQPELVAQKWGQRKNKPTMNYEKLSRALRYYYDGDMICKVQGKRFVYKFVCDLKTLIGYSAAELNRLVTECEQKKLAKMQLHGIAQPVTAVALATASLQTEKDNSEQ ID NO: 22 human GABPA (Met316-Met429):MNNGQIQLWQFLLELLTDKDARDCISWVGDEGEFKLNQPELVAQKWGQRKNKPTMNYEKLSRALRYYYDGDMICKVQGKRFVYKFVCDLKTLIGYSAAELNRLVTECEQKKLAKMSEQ ID NO: 23 human GABPA full length (R376A and R379A)MTKREAEELIEIEIDGTEKAECTEESIVEQTYAPAECVSQAIDINEPIGNLKKLLEPRLQCSLDAHEICLQDIQLDPERSLFDQGVKTDGTVQLSVQVISYQGIEPKLNILEIVKPADTVEVVIDPDAHHAESEAHLVEEAQVITLDGTKHITTISDETSEQVTRWAAALEGYRKEQERLGIPYDPIQWSTDQVLHWVVWVMKEF SMTDIDLTTLNISGRELC SLNQEDFFQRVPRGEILWSHLELLRKYVLASQEQQMNEIVTIDQPVQIIPASVQSATPTTIKVINSSAKAAKVQRAPRISGEDRSSPGNRTGNNGQIQLWQFLLELLTDKDARDCISWVGDEGEFKLNQPELVAQKWGQRKNKPTMNYEKLSAALAYYYDGDMICKVQGKRFVYKFVCDLKTLIGYSAAELNRLVTECEQKKLAKMQLHGIAQPVTAVALATASLQTEKDNSEQ ID NO: 24 nucleotides encoding SEQ ID NO: 23:ATGACTAAAAGAGAAGCAGAGGAGCTGATAGAAATTGAGATTGATGGAACAGAGAAAGCAGAGTGCACAGAAGAAAGCATTGTAGAACAAACCTACGCGCCAGCTGAATGTGTAAGCCAGGCCATAGACATCAATGAACCAATAGGCAATTTAAAGAAACTGCTAGAACCAAGACTACAGTGTTCTTTGGATGCTCATGAAATTTGTCTGCAAGATATCCAGCTGGATCCAGAACGAAGTTTATTTGACCAAGGAGTAAAAACAGATGGAACTGTACAGCTTAGTGTACAGGTAATTTCTTACCAAGGAATTGAACCAAAGTTAAACATCCTTGAAATTGTTAAACCTGCGGACACTGTTGAGGTTGTTATTGATCCAGATGCCCACCATGCTGAATCAGAAGCACATCTTGTTGAAGAAGCTCAAGTGATAACTCTTGATGGCACAAAACACATCACAACCATTTCAGATGAAACTTCAGAACAAGTGACAAGATGGGCTGCTGCACTGGAAGGCTATAGGAAAGAACAAGAACGCCTTGGGATACCCTATGATCCCATACAGTGGTCCACAGACCAAGTCCTGCATTGGGTGGTTTGGGTAATGAAGGAATTCAGCATGACCGATATAGACCTCACCACACTCAACATTTCGGGGAGAGAATTATGTAGTCTCAACCAAGAAGATTTTTTTCAGCGGGTTCCTCGGGGAGAAATTCTCTGGAGTCATCTGGAACTTCTCCGAAAATATGTATTGGCAAGTCAAGAACAACAGATGAATGAAATAGTTACCATTGATCAACCTGTGCAAATTATTCCAGCATCAGTGCAATCTGCTACACCTACTACCATTAAAGTTATAAATAGTAGTGCGAAAGCAGCCAAAGTACAAAGAGCGCCGAGGATTTCAGGAGAAGATAGAAGCTCACCTGGGAACAGAACAGGAAACAATGGCCAAATCCAACTATGGCAGTTTTTGCTAGAACTTCTTACTGATAAGGACGCTCGAGACTGCATTTCTTGGGTTGGTGATGAAGGTGAATTTAAGCTAAATCAGCCTGAACTGGTTGCACAGAAATGGGGACAGCGTAAAAATAAGCCTACGATGAACTATGAGAAACTCAGTGCCGCATTAGCCTATTATTACGATGGGGACATGATTTGTAAAGTTCAAGGCAAGAGATTTGTGTACAAGTTTGTCTGTGACTTGAAGACTCTTATTGGATACAGTGCAGCGGAGTTGAACCGTTTGGTCACAGAATGTGAACAGAAGAAACTTGCAAAGATGCAGCTCCATGGAATTGCCCAGCCAGTCACAGCAGTAGCTCTGGCTACTGCTTCTCTGCAAACGGAAAAGGATAATTGASEQ ID NO: 25 nucleotides encoding SEQ ID NO: 21:ATGAACAATGGCCAAATCCAACTATGGCAGTTTTTGCTAGAACTTCTTACTGATAAGGACGCTCGAGACTGCATTTCTTGGGTTGGTGATGAAGGTGAATTTAAGCTAAATCAGCCTGAACTGGTTGCACAGAAATGGGGACAGCGTAAAAATAAGCCTACGATGAACTATGAGAAACTCAGTCGTGCATTAAGATATTATTACGATGGGGACATGATTTGTAAAGTTCAAGGCAAGAGATTTGTGTACAAGTTTGTCTGTGACTTGAAGACTCTTATTGGATACAGTGCAGCGGAGTTGAACCGTTTGGTCACAGAATGTGAACAGAAGAAACTTGCAAAGATGCAGCTCCATGGAATTGCCCAGCCAGTCACAGCAGTAGCTCTGGCTACTGCTTCTCTGCAAACGGAAAAGGATAATTGA

Claims

WHAT IS CLAIMED IS:

1. A polypeptide comprising a GABPA-interacting domain and a ubiquitin ligase.

2. The polypeptide of claim 1, wherein the GABPA-interacting domain comprises an Ankyrin repeat sequence from GABPB1 or GABPB2.

3. The polypeptide of claim 1, wherein the GABPA-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NO: 1 or SEQ ID NO:2, or a GABPA-binding fragment thereof.

4. The polypeptide of any one of claims 1-3, wherein the ubiquitin ligase comprises an E3 ligase.

5. The polypeptide of any one of claims 1-4, wherein the polypeptide further comprises a nuclear localization sequence.

6. A polypeptide comprising a GABPA-interacting domain at least 80, 85, 90, or 95% identical to SEQ ID NO: 1 or SEQ ID NO:2, or a GABPA-binding fragment thereof, wherein the polypeptide comprises no more than 170, 180, 190 or 200 contiguous amino acids of human GABPB1L.

7. The polypeptide of claim 6, wherein GABPA-interacting domain is fused to a heterologous amino acid sequence.

8. A nucleic acid comprising a polynucleotide encoding the polypeptide of any one of claims 1-7.

9. The nucleic acid of claim 8, comprising a promoter operably linked to the polynucleotide.

10. A vector comprising the nucleic acid of any one of claims 8 or 9.

11. The vector of claim 10, wherein the vector is a viral vector.

12. A cell comprising the nucleic acid of any one of claims 8 or 9 or the vector of any one of claims 10 or 11.

13. A method of inhibiting expression of TERT in a cell having a TERT promoter mutation, the method comprising, introducing the polypeptide of any one of claims 1-7 in the cell or introducing the nucleic acid of any one of claims 8 or 9 or the vector of any one of claims 10 or 11 into the cell such that the polypeptide is expressed and binds a GABPA protein in the cell.

14. The method of claim 13, wherein the cell is in a human and the introducing comprises administering the polypeptide, nucleic acid or vector to the human such that the polypeptide, nucleic acid or vector is delivered to the cell.

15. The method of claim 13 or 14, wherein the cell is a cancer cell.

16. The method of claim 15, wherein the cancer cell is a glioblastoma or a glioma.

17. A pharmaceutical composition comprising a pharmaceutically-acceptable excipient and the polypeptide of any one of claims 1-7, the nucleic acid of any one of claims 8 or 9 or the vector of any one of claims 10 or 1118. A polypeptide comprising a GABPB-interacting domain and a ubiquitin ligase.

19. The polypeptide of claim 18, wherein the GABPB-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NO: 19 or a GABPB-binding fragment thereof.

20. The polypeptide of claim 18, wherein the GABPB-interacting domain is at least 80, 85, 90, or 95% identical to SEQ ID NOs: 21, 22, or 23.

21. The polypeptide of any one of claims 18-20, wherein the ubiquitin ligase comprises an E3 ligase.

22. The polypeptide of any one of claims 18-21, wherein the polypeptide further comprises a nuclear localization sequence.

23. A polypeptide comprising a GABPB-interacting domain at least 80, 85, 90, or 95% identical to SEQ ID NO: 1, or a GABPB-binding fragment thereof, wherein the polypeptide comprises no more than 120, 130, 140 or 150 contiguous amino acids of human GABPA.

24. The polypeptide of claim 23, wherein GABPB-interacting domain is fused to a heterologous amino acid sequence.

25. A nucleic acid comprising a polynucleotide encoding the polypeptide of any one of claims 18-24.

26. The nucleic acid of claim 25, comprising a promoter operably linked to the polynucleotide.

27. A vector comprising the nucleic acid of any one of claims 25 or 26.

28. The vector of claim 27, wherein the vector is a viral vector.

29. A cell comprising the nucleic acid of any one of claims 25 or 26 or the vector of any one of claims 27 or 28.

30. A method of inhibiting expression of TERT in a cell having a TERT promoter mutation, the method comprising, introducing the polypeptide of any one of claims 18- 24 in the cell or introducing the nucleic acid of any one of claims 25 or 26 or the vector of any one of claims 27 or 28 into the cell such that the polypeptide is expressed and binds a GABPB protein in the cell.

31. The method of claim 30, wherein the cell is in a human and the introducing comprises administering the polypeptide, nucleic acid or vector to the human such that the polypeptide, nucleic acid or vector is delivered to the cell.

32. The method of claim 30 or 31, wherein the cell is a cancer cell.

33. The method of claim 32, wherein the cancer cell is a glioblastoma or a glioma.

34. A pharmaceutical composition comprising a pharmaceutically-acceptable excipient and the polypeptide of any one of claims 18-24, the nucleic acid of any one of claims 25 or 26 or the vector of any one of claims 27 or 28.

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