Compositions and methods for treating p53-mediated cancers - Patents.com

Abstract

A novel class of fusion proteins is disclosed that recruit the cell's innate chaperone machinery, specifically the Hsp70-mediated system, to specifically restore p53 function.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 253,759, filed October 8, 2021, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a sequence listing, which has been submitted herewith in electronically readable XML format via EFS-Web and is incorporated herein by reference in its entirety. The electronic sequence listing file, filed electronically on October 7, 2022 and named "269548-517365_Sequence-listing.XML", has a file size of 142,953 bytes.

[0003] The present disclosure relates to the use of chaperone fusion proteins for the treatment of p53-mediated cancers. [Background technology]

[0004] Cancer is one of the leading causes of death worldwide. Approximately 38.4% of men and women will be diagnosed with cancer at some point in their lives (based on 2013-2015 data: NIH National Cancer Research). Reducing the burden of cancer will have a profound impact on society as a whole in improving the overall quality of life.

[0005] Traditional cancer treatments include surgery, radiation, drug therapy, and other therapies. These approaches may cure, shrink, or stop the progression of cancer. However, once cancer invasion or metastasis is observed, systemic treatment with drug therapy would be the first line of defense to ameliorate symptoms and improve quality of life. Many drugs have been developed in the past 20-30 years to treat cancer, but most of them have not been satisfactory. Recent developments using immunotherapy are very exciting, but most research is yet to be completed. The main reason for the difficulties associated with cancer treatment is that cancer (tumor) cells are derived from normal cells and they share many characteristics, which makes it difficult for drugs to specifically recognize only tumor cells. Nevertheless, decades of efforts by countless scientists have identified many common and unique features in cancer cells. For example, more than 50% of human cancers are known to have mutations in the p53 gene. p53 exerts its major function as a transcription factor inducing cell cycle arrest, senescence, and apoptosis in response to genotoxic stress signals (Vogelstein, B. et al., (2000) Nature 408, 307-310; Petitjean, A. et al., (2007) Oncogene 26, 2157-2165). Given the high frequency of p53 mutations in cancer, it is natural to expect that intervention against p53 mutants could develop a promising strategy for cancer treatment. However, this hope has not yet been realized.

[0006] All proteins expressed in cells need to be correctly folded into their intended structure to function properly. An increasing number of diseases and disorders have been shown to be associated with improper folding of proteins and / or improper deposition and aggregation of proteins and lipoproteins, as well as infectious proteinaceous substances. Examples of diseases caused by misfolding, also known as conformational diseases or proteopathies, include Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTLD). Mutant proteins aggregate in cells, resulting in typical cytotoxic cytoplasmic inclusions.

[0007] The wild-type p53 protein contains 393 amino acids and is composed of several structural and functional domains. The N-terminal region consists of a transactivation domain and a proline-rich region. The tetramerization domain and the basic domain are located at the C-terminus (Romer, L. et al., (2006) Angew Chem Int Ed Engl 45, 6440-6460; Joerger, AC and Fersht, AR (2008) Annu Rev Biochem 77, 557-582; Joerger, AC and Fersht, AR (2010) Cold Spring Harb Perspect Biol 2, a000919). The majority of p53 somatic mutations in cancer cells (about 74%) are missense mutations in the DNA-binding domain, called "hot spots," that result in single amino acid substitutions in the p53 protein, such as R175, G245, R248, R249, R273, and R282. These mutations can be divided into two classes: 1) DNA contact mutations (R248, R273, and R282) that result in loss of DNA binding residues, and 2) conformational mutations (R175, R245, and R249) that cause structural changes in p53, resulting in misfolding of the p53 protein (Strano, S. et al. (2007) Oncogene 26, 2212-2219; Freed-Pastor, WA and Prives, C. (2012) Genes Dev 26, 1268-1286). Since p53 normally functions as a tetramer, mutant p53 can suppress the tumor suppressor function of the remaining wild-type p53 counterpart or other p53 family members, especially p63 and p73, through a "dominant-negative" mechanism (Brosh, R. and Rotter, V. (2009) Nat Rev Cancer 9, 701-713; Muller, PA and Vousden, KH (2014) Cancer Cell 25, 304-317). Therefore, restoring p53 function or suppressing mutant p53 activity is considered to have great potential as a novel and effective strategy.Only recently have compounds emerged that target mutant oncogenic p53 proteins (Yu, X., Narayanan, S. et al. (2014) Apoptosis 19, 1055-1068). Aiming to screen for compounds that specifically target mutant p53 for the growth inhibition caused by the R273H mutation, Bykov et al. found that the compound 2,2-bis(hydroxymethyl)-1-azabicyclo[2,2,2]octan-3-one, named PRIMA-1 (p53-reactivation and induction of massive apoptosis-1, APR-017), promoted p53 refolding by modifying thiol groups in mutant p53 and induced several p53 target genes (Bykov, VJ et al., (2002) Nat Med 8, 282-288; Bykov, VJ et al., (2002) Carcinogenesis 23, 2011-2018). PRIMA-1 promotes apoptosis in human cancer cells with mutated p53 (Bykov, VJ et al., (2005) Oncogene 24, 3484-3491; Lambert, JM et al., (2009) Cancer Cell 15, 376-388), and has become one of the first compounds to advance into phase I and II clinical trials for hematological malignancies and prostate cancer.

[0008] Cancer-associated missense mutations in the p53 gene often result in conformational changes that increase the free energy of the protein and separate mutant and wild-type p53. Suppression of such mutant p53 is one of the promising strategies to restore p53 function. In line with this strategy, it has been shown that lowering the temperature or refolding mutant p53 with chemical chaperones such as glycerol restores normal p53 function (Brown, CR et al., (1997) J Clin Invest 99, 1432-1444; Ohnishi, T. et al., (1999) Int J Radiat Biol 75, 1095-1098; Ohnishi, T., Ohnishi, K. et al., (1999) Radiat Res 151, 498-500).

[0009] Heat shock 70 kDa proteins (herein referred to as "Hsp70s") constitute a ubiquitous class of chaperone proteins in cells of various species (Tavaria et al., (1996) Cell Stress Chaperones 1, 23-28). Hsp70 requires assistant proteins called co-chaperone proteins, such as J-domain proteins and nucleotide exchange factors (NEFs), to function (Hartl et al., (2009) Nat Struct Mol Biol 16, 574-581). In the current model of the Hsp70 chaperone mechanism for folding proteins, Hsp70 cycles between ATP- and ADP-bound states, and the J-domain protein binds another protein (called a "client protein") that requires folding or refolding and interacts with the ATP-bound form of Hsp70 (Hsp70-ATP) (Young (2010) Biochem Cell Biol 88, 291-300; Mayer, (2010) Mol Cell 39, 321-331). Binding of the J-domain protein-client protein complex with Hsp70-ATP stimulates ATP hydrolysis, which causes a conformational change in the Hsp70 protein, closing the helix lid, thereby stabilizing the interaction between the client protein and Hsp70-ADP, as well as triggering the release of the J-domain protein, which is then free to bind another client protein.

[0010] Thus, according to this model, J domain proteins play an important part within the Hsp70 machinery by acting as bridges and facilitating the capture of various client proteins and their submission to the Hsp70 machinery for folding or refolding into the appropriate conformation (Kampinga & Craig (2010) Nat Rev Mol Cell Biol 11, 579-592). The J domain family is widely conserved in species ranging from prokaryotes (DnaJ proteins) to eukaryotes (Hsp40 protein family). The J domain (approximately 60-80 aa) is composed of four helices: I, II, III, and IV. Helices II and III are connected via a mobile loop that contains an "HPD motif," which is highly conserved across J domains and is thought to be important for activity (Tsai & Douglas, (1996) J Biol Chem 271, 9347-9354). Mutations within the HPD sequence have been found to abolish J domain function.

[0011] Given the background provided above on proteopathies, it seems clear that reducing the levels of misfolded proteins could serve as a means to treat, prevent, or otherwise ameliorate the symptoms of these devastating disorders, and that mobilizing the cells' innate ability to repair protein misfolding would be a logical choice to pursue. Summary of the Invention

[0012] The present inventors have developed a novel class of fusion proteins that recruit the cells' innate chaperone machinery, specifically the Hsp70-mediated system, to specifically reduce the level or restore the function of mutant p53. Unlike in the previous work by the present inventors, which used fusion proteins containing fragments of Hsp40 protein (also called J protein), a co-chaperone that interacts with Hsp70, to enhance protein secretion and expression, the present work uses J domain-containing fusion proteins to reduce the level or restore the function of mutant p53 protein. In this regard, the present inventors have made the surprising discovery that the elements of the J domain required for function are quite different from the use of the J domain in enhancing protein expression and secretion, demonstrating a separate mechanism for the mode of action of the present fusion proteins. The fusion proteins described herein contain a J domain and a domain with affinity for p53. The presence of the p53-binding domain in the fusion protein results in the specific restoration of mutant p53 protein.

[0013] E1. Thus, in a first aspect, disclosed herein is an isolated fusion protein comprising a J domain of a J protein and a p53-binding domain. E2. The fusion protein of E1, wherein the J domain of the J protein is of eukaryotic origin. E3. The fusion protein according to any one of E1 to E2, wherein the J domain of the J protein is of human origin. E4. The fusion protein according to any one of E1 to E3, wherein the J domain of the J protein is localized in the cytoplasm. E5. The fusion protein according to any one of E1 to E4, wherein the J domain of the J protein is selected from the group consisting of SEQ ID NOs: 1 to 50. E6. The fusion protein of any one of E1 to E5, wherein the J domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49. E7. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID NO:5. E8. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID NO: 10. E9. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID NO: 16. E10. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID NO: 25. E11. The fusion protein of any one of E1 to E6, wherein the J domain comprises the sequence of SEQ ID NO: 31. E12. The p53-binding domain has a K for p53 of 1 μM or less, e.g., 300 nM or less, 100 nM or less, 30 nM or less, 10 nM or less, as measured, for example, using an ELISA assay. D The fusion protein according to any one of E1 to E11, comprising: E13. The fusion protein according to any one of E1 to E12, wherein the p53-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 51 to 56. E14. The fusion protein according to any one of E1 to E13, wherein the p53-binding domain comprises a sequence of SEQ ID NO: 51 to 53. E15. The fusion protein according to any one of E1 to E13, wherein the p53-binding domain comprises the sequence of SEQ ID NO:51. E16. The fusion protein of any one of E1 to E13, wherein the p53-binding domain comprises the sequence of SEQ ID NO:52. E17. The fusion protein according to any one of E1 to E16, comprising multiple p53-binding domains. E18. The fusion protein according to any one of E1 to E17, which consists of two p53-binding domains. E19. The fusion protein according to any one of E1 to E18, which consists of three p53-binding domains. E20. The following constructs: a. DNAJ-XT, b. DNAJ-XTXT, c. DNAJ-XTXTXT, d. TX-DNAJ, e. TXTX-DNAJ, f. TXTXTX-DNAJ, g. TX-DNAJ-XT, h. TX-DNAJ-XTXT, i. TDNAJ-X-TTTTTDNAJ-XT, j. TXTX-DNAJ-X-TT, k. TTDNAJ-XTX-TTTTTDNAJ-XT, l. TXTX-DNAJ-XTXTXT, m. TXTXTX-DNAJ-XT, n. TXTXTX-DNAJ-XTXT, o. TXTXTX-DNAJ-XTXTXT, p.DnaJ-X-DnaJ-XTXT, q. TX-DnaJ-X-DnaJ, r. TXTX-DnaJ-X-DnaJ, and s. TX-TDnaJ-X-TDnaJ-X-TTTT one of the following: Where: T is the p53 binding domain, DNAJ is the J domain of the J protein, The fusion protein of any one of E1 to E19, wherein X is an optional linker. E21. The fusion protein according to any one of E1 to E20, comprising a J domain sequence of SEQ ID NO: 5 and a p53-binding domain sequence of SEQ ID NO: 52. E22. The fusion protein of any one of E1 to E21, comprising a J domain sequence of SEQ ID NO:5 and two copies of the p53-binding domain sequence of SEQ ID NO:52. E23. The fusion protein according to any one of E1 to E22, comprising a sequence selected from the group consisting of SEQ ID NOs: 80 to 91, and 100 to 107. E24. The fusion protein according to any one of E1 to E23, comprising a sequence selected from the group consisting of SEQ ID NO:80. E25. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:81. E26. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:82. E27. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 83. E28. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 84. E29. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:85. E30. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:86. E31. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:87. E32. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:88. E33. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:89. E34. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 90. E35. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO:91. E36. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 100. E37. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 101. E38. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 102. E39. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 103. E40. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 104. E41. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 105. E42. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 106. E43. The fusion protein according to any one of E1 to E23, comprising the sequence of SEQ ID NO: 107. E44. The fusion protein of any one of E1-E43, further comprising a targeting reagent. E45. The fusion protein of any one of E1 to E44, further comprising an epitope. E46. The fusion protein of E45, wherein the epitope is a polypeptide selected from the group consisting of SEQ ID NOs: 68 to 74. E47. The fusion protein of any one of E1-E46, further comprising a cell permeability agent. E48. The fusion protein of E47, wherein the cell permeation agent is selected from the group consisting of SEQ ID NOs: 75-78. E49. The fusion protein of any one of E1 to E48, further comprising a signal sequence. E50. The fusion protein of E49, wherein the signal sequence comprises a peptide sequence selected from the group consisting of SEQ ID NOs: 94 to 96. E51. The fusion protein according to any one of E1 to E50, which is capable of restoring p53 function in a cell. E52. The fusion protein of any one of E1-E51, which is capable of reducing a p53-mediated neoplasia. E53. A nucleic acid sequence encoding the fusion protein according to any one of E1 to E52. E54. The nucleic acid sequence of E53, wherein the nucleic acid is DNA. E55. The nucleic acid sequence of E54, wherein the nucleic acid is RNA. E56. The nucleic acid sequence of any one of E53 to E55, wherein the nucleic acid comprises at least one modified nucleic acid. E57. The nucleic acid sequence of any one of E53 to E56, further comprising a promoter region, a 5'UTR, a 3'UTR, such as a poly(A) signal. E58. The nucleic acid sequence of E57, wherein the promoter region comprises a sequence selected from the group consisting of a CMV enhancer sequence, a CMV promoter, a CBA promoter, a UBC promoter, a GUSB promoter, an NSE promoter, a synapsin promoter, a MeCP2 promoter, and a GFAP promoter. E59. A vector comprising the nucleic acid sequence according to any one of E53 to E58. E60. The vector of E59, selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus (vaccinia or myxoma), paramyxovirus (measles, RSV, or Newcastle disease virus), baculovirus, reovirus, alphavirus, and flavivirus. E61. The vector of E59 or E60, which is an AAV. E62. A viral particle comprising a capsid and a vector according to any one of E59 to E61. E63. The viral particle of E62, wherein the capsid is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotyped AAV, rhesus-derived AAV, AAVrh8, AAVrh10, and AAV-DJan AAV capsid mutants, AAV hybrid serotypes, organotropic AAV, cardiotropic AAV, and cardiotropic AAVM41 mutants. E64. The viral particle of E62 or E63, wherein the capsid is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAVrhlO. E65. The viral particle according to any one of E62 to E64, wherein the capsid is AAV2. E66. The viral particle according to any one of E62 to E64, wherein the capsid is AAV5. E67. The viral particle according to any one of E62 to E64, wherein the capsid is AAV8. E68. The viral particle according to any one of E62 to E64, wherein the capsid is AAV9. E69. The viral particle according to any one of E62 to E64, wherein the capsid is AAVrh10. E70. A pharmaceutical composition comprising an agent selected from the group consisting of a fusion protein according to any one of E1 to E52, a cell expressing the fusion protein according to E1 to E52, a nucleic acid according to any one of E53 to E58, a vector according to any one of E59 to E61, and a viral particle according to any one of E62 to E69, and a pharma- ceutically acceptable carrier or excipient. E71. A method for reducing toxicity of p53 protein in a cell, comprising contacting the cell with an effective amount of one or more agents selected from the group consisting of a fusion protein described in any one of E1-E52, a cell expressing a fusion protein of E1-E52, a nucleic acid described in any one of E53-E58, a vector described in any one of E59-E61, a viral particle described in any one of E62-E69, and a pharmaceutical composition described in E70. E72. The method of E71, wherein the cell is in a subject. E73. The method of E72, wherein the subject is a human. E74. The method of any one of E71-E73, wherein the subject is identified as having cancer. E75. Malignant neoplasms of cancer declared or presumed to be primary at the following sites: malignant neoplasms of the lips, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of the mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female genital tract, including ovarian cancer; malignant neoplasms of the male genital tract, including prostate cancer The method of E74, wherein the tumor is selected from the group consisting of malignant neoplasms; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; malignant neoplasms of poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; and neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes. E76. The method of any one of E71-E75, wherein there is a decrease in the amount of abnormal p53 protein in the cell when compared to a control cell. E77. A method of treating, preventing, or delaying the progression of cancer in a subject in need thereof, comprising administering an effective amount of one or more agents selected from the group consisting of a fusion protein described in any one of E1-E52, a cell expressing a fusion protein of E1-E52, a nucleic acid described in any one of E53-E58, a vector described in any one of E59-E61, a viral particle described in any one of E62-E69, and a pharmaceutical composition described in E70. E78. Malignant neoplasms in which p53 disease is stated or presumed to be primary at the following sites: malignant neoplasms of the lips, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of the mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female genital tract, including ovarian cancer; malignant neoplasms of the male genital tract, including prostate cancer. The method of E77, wherein the tumor is selected from the group consisting of: malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; malignant neoplasms of poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; and neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes. E79. Use of one or more of a fusion protein described in any one of E1-E52, a cell expressing a fusion protein described in E1-E52, a nucleic acid described in any one of E53-E58, a vector described in any one of E59-E61, a viral particle described in any one of E62-E69, and a pharmaceutical composition described in E70 in the preparation of a medicament useful for preventing or delaying the progression of a p53 disease in a subject. E80. The use according to E79, wherein the p53 disease is cancer. E81. Malignant neoplasms of cancer declared or presumed to be primary at the following sites: malignant neoplasms of the lips, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of the mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female genital tract, including ovarian cancer; malignant neoplasms of the male genital tract, including prostate cancer The use described in E80, selected from the group consisting of malignant neoplasms; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; malignant neoplasms of poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; and neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes. [Brief description of the drawings]

[0014] [Figure 1A] Figure 1 shows a Clustal Omega sequence alignment of representative human J domain sequences, with the highly conserved HPD domain shown in the highlighted box. [Figure 1B] FIG. 1 shows a Clustal Omega sequence alignment of representative human J domain sequences. [Diagram 2] FIG. 1 shows several illustrative fusion protein constructs containing a J domain and a p53-binding domain. [Diagram 3] FIG. 1 shows the effect of expressing wild-type or the R175 or R249S mutants of p53, either alone or co-expressing Construct 2, in either the absence or presence of 100 nM doxorubicin, on immunodetectable levels of p53 or p21. [Figure 4]FIG. 1 shows ELISA-based quantification of p21 in HCT-116 cells stably transfected with the R249S mutant of p53, either alone or in the presence of Construct 2, treated with or without 1 μM doxorubicin. [Diagram 5] FIG. 1 shows the effect on cytotoxicity of expressing Construct 2 in HCT-116 cells stably transfected with constructs expressing either the R175H or R249S mutants of p53, using LDH activity as a surrogate for cytotoxicity. [Figure 6] FIG. 1 shows the effect on cytotoxicity of expressing Construct 3 in cells transfected with constructs expressing either wild-type, R175H mutant, or R249 mutant of p53, using LDH activity as an indicator of cytotoxicity. [Figure 7] FIG. 1 shows the effect on cytotoxicity of expressing construct 16 or construct 17 in cells transfected with constructs expressing either the wild type, the R175H mutant, or the R249 mutant of p53, using LDH activity as an indicator of cytotoxicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0016] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0017] As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including, but not limited to, both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics. Standard one-letter or three-letter codes are used to name amino acids.

[0018] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for a subject vector. A host cell includes the progeny of a single host cell. The progeny are not necessarily completely identical (in morphology or in genome of total DNA complement) to the original parent cell, due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a vector of this invention.

[0019] "Isolated" when used to describe various polypeptides disclosed herein means a polypeptide that has been identified and separated and / or recovered from components of its natural environment. The contaminant components of its natural environment are typically materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. As will be apparent to those skilled in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart. In addition, a "concentrated," "separated," or "diluted" polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is generally greater than that of its naturally occurring counterpart. Generally, a polypeptide produced by recombinant means and expressed in a host cell is considered to be "isolated."

[0020] An "isolated" polynucleotide, or a nucleic acid encoding a polypeptide, or a nucleic acid encoding another polypeptide, is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule that is normally associated with the natural source of the nucleic acid encoding the polypeptide. An isolated nucleic acid molecule encoding a polypeptide is in a form or configuration other than that in which it is found in nature. Thus, an isolated nucleic acid molecule encoding a polypeptide is distinguished from the nucleic acid molecule encoding that particular polypeptide when it is present in a natural cell. However, an isolated nucleic acid molecule encoding a polypeptide includes a nucleic acid molecule encoding a polypeptide that is contained in a cell that normally expresses the polypeptide, where, for example, the nucleic acid molecule is in a chromosomal or extrachromosomal location that is different from the location in the natural cell.

[0021] The terms "polynucleotide", "nucleic acid", "nucleotide", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a label component.

[0022] As defined herein, the term "p53 disorder" or "p53-mediated disease" refers to disorders associated with p53-mediated neoplasia, particularly caused by mutant p53 proteins. Examples of p53 disorders, preferably with respect to cancer and other neoplasms, include, but are not limited to, malignant neoplasms declared or presumed to be primary at the following sites: malignant neoplasms of the lips, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of mesothelium and soft tissue, including sarcoma; breast cancer; malignant neoplasms of the genital tract; malignant neoplasms of the female genital organs, including ovarian cancer; malignant neoplasms of the male genital organs, including prostate cancer; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; malignant neoplasms of poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes.

[0023] A "vector" is a nucleic acid molecule, preferably self-replicating in a suitable host, that transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for the insertion of DNA or RNA into a cell, replicating vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" usually implies a suitable host cell configured with an expression vector that can function to produce a desired expression product.

[0024] The term "operably linked" refers to the juxtaposition of the described components, in which they are in a relationship that allows them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences. An "operably linked" sequence can include both expression control sequences that are in close proximity to a gene of interest, and expression control sequences that act in trans or at a distance to control the gene of interest. The term "expression control sequence" refers to polynucleotide sequences that are necessary to affect the expression and processing of the coding sequence to which they are ligated. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and, if necessary, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosomal binding sites, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequence" is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. Unless otherwise stated, a description or reference herein to inserting a nucleic acid molecule encoding a fusion protein of the present invention into an expression vector means that the inserted nucleic acid is also operably linked within the vector with a functional promoter and other transcriptional and translational control elements required for expression of the encoded fusion protein when the expression vector containing the inserted nucleic acid molecule is introduced into a compatible host cell or compatible cell of an organism.

[0025] "Recombinant" when applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and / or ligation steps, and other procedures, resulting in a construct that is potentially expressible in a host cell.

[0026] The terms "gene" and "gene fragment" are used interchangeably herein. They refer to a polynucleotide that contains at least one open reading frame that is capable of encoding a specific protein after being transcribed and translated. A gene or gene fragment can be genomic DNA or cDNA, and it can cover the entire coding region or a segment thereof, so long as the polynucleotide contains at least one open reading frame. A "fusion gene" is a gene that is composed of at least two heterologous polynucleotides that are linked together.

[0027] The terms "disease" and "disorder" are used interchangeably to refer to pathological conditions identified by accepted standards of medical care and practice in the art.

[0028] As used herein, the term "effective amount" refers to an amount of a treatment that is sufficient to reduce or ameliorate the severity and / or duration of a disease or one or more symptoms thereof; prevent the development of a harmful or pathological condition; cause regression of a pathological condition; prevent the recurrence, development, occurrence, or progression of one or more symptoms associated with a pathological condition; detect a disorder; or enhance or improve the prophylactic or therapeutic effects of a treatment (e.g., the administration of another prophylactic or therapeutic agent).

[0029] As used herein, the term "J domain" refers to fragments that retain the ability to accelerate the catalytic activity of Hsp70 and its cognate intrinsic ATPases. The J domains of various J proteins have been determined (see, e.g., Kampinga et al. (2010) Nat. Rev., 11:579-592; Hennessy et al. (2005) Protein Science, 14:1697-1709, each of which is incorporated by reference in its entirety) and are characterized by several hallmarks: they contain four alpha helices (I, II, III, IV) and usually have a highly conserved tripeptide sequence motif of histidine, proline, and aspartic acid (referred to as the "HPD motif") between helices II and III. Typically, the J domain of a J protein is between 50 and 70 amino acids in length, and the site of interaction (binding) of the J domain with the Hsp70-ATP chaperone protein is believed to be a region extending from within helix II, and the HPD motif is required for stimulation of Hsp70 ATPase activity. As used herein, the term "J domain" is intended to include naturally occurring J domain sequences and functional variants thereof that retain the ability to accelerate the intrinsic ATPase activity of Hsp70, which can be measured using methods well known in the art (see, e.g., Horne et al. (2010) J. Biol. Chem., 285, 21679-21688, which is incorporated herein by reference in its entirety). A non-limiting list of human J domains is provided in Table 1.

[0030] Detailed Description The present inventors have found that the fusion protein construct comprising the J domain of J protein and p53 binding domain can be contacted with cells to some extent, resulting in the unexpected effect of restoring mutant p53 function.Mutant p53 is believed to cause some destructive neoplasia.Therefore, the present invention provides useful compositions and methods for treating p53 disorder, for example, in subjects who need it.

[0031] To overcome the problems associated with chaperone-based therapy, we investigated whether it would be possible to design artificial chaperone proteins with high specificity. We designed a series of fusion protein constructs containing an effector domain (J domain sequence) for Hsp70 binding / activation, and a domain that confers specificity for the p53 protein. The resulting fusion proteins act to accelerate the catalytic activity of Hsp70 and its cognate intrinsic ATPase, resulting in increased protein folding, restored function, and / or accelerated clearance.

[0032] I. Fusion Protein Constructs a. J domains useful in the present invention The J domains of various J proteins have been determined. See, for example, Kampinga et al., Nat. Rev., 11:579-592 (2010); Hennessy et al., Protein Science, 14:1697-1709 (2005). The J domains useful in preparing the fusion proteins of the present invention are key to defining the features of the J domain that primarily accelerate Hsp70 ATPase activity. Thus, the isolated J domains useful in the present invention include a polypeptide domain characterized by having four alpha helices (I, II, III, IV) and a highly conserved tripeptide sequence of histidine, proline, and aspartic acid (referred to as the "HPD motif") between helices II and III. Typically, the J domain of a J protein is between 50 and 70 amino acids in length, the site of interaction (binding) of the J domain with the Hsp70-ATP chaperone protein is believed to be a region extending from within helix II, and the HPD motif underlies the prototypical activity. Exemplary J domains include, but are not limited to, the J domains of DnaJB1, DnaJB2, DnaJB6, DnaJC6, the J domain of the SV40 large T antigen, and the J domain of the mammalian cysteine ​​string protein (CSP-α). Amino acid sequences for these and other J domains that may be used in the fusion proteins of the invention are provided in Table 1. The conserved HPD motif is highlighted in bold. In one embodiment, the fusion proteins disclosed herein comprise a J domain selected from the group consisting of SEQ ID NOs: 1-50. In another embodiment, the fusion proteins disclosed herein comprise a J domain that comprises a consensus HPD motif. In one particular embodiment, the J domain is selected from the group consisting of SEQ ID NOs: 1-15, 17-50. In certain embodiments, the fusion protein comprises a J domain selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 16, 24, 25, 31, and 49.

[0033] [Table 1-1]

[0034] [Table 1-2]

[0035] [Table 1-3]

[0036] b. p53 binding domain The fusion protein also contains at least one p53-binding domain, which may be a single or multimeric polypeptide linked to a J domain to form the fusion protein.

[0037] Ideally, the p53 binding domain will have sufficient affinity to be able to bind p53 protein when present in a cell at pathological levels. Thus, in one embodiment, the fusion protein has a K for a p53 reporter construct, e.g., 2 μM or less, 1 μM or less, 500 nM or less, 300 nM or less, 100 nM or less, 30 nM or less, when tested by ELISA on a 96-well microtiter plate. D The p53 binding domain has the following structure:

[0038] It is noted that the Hsp70 machinery is believed to engage only misfolded proteins. However, in some cases, it may be preferable for the fusion protein to engage only misfolded forms of p53. Thus, in some embodiments, the p53 binding domain preferentially binds misfolded forms of p53. Although not necessarily, the ability of the p53 binding domain to have a higher affinity for misfolded forms (e.g., R175, R245, and R249 mutant forms of p53) would allow the fusion protein to more selectively engage only pathogenic forms to the Hsp70 machinery.

[0039] In another embodiment, the fusion protein also contemplates the use of a p53 binding domain that is chemically conjugated with a J domain. The p53 binding domain can be directly conjugated with a J domain. Alternatively, it can be conjugated with a J domain by a linker. For example, there are many chemical crosslinkers known to those skilled in the art that are useful for crosslinking a p53 binding domain with a J domain, or a targeting domain with a fusion protein that includes a p53 binding domain and a J domain. For example, the crosslinker is a heterobifunctional crosslinker that can be used to link molecules in a stepwise manner. Heterobifunctional crosslinkers provide the ability to design more specific coupling methods for protein conjugation, thereby reducing the occurrence of undesirable side reactions such as homoprotein polymers. A variety of heterobifunctional crosslinkers are known in the art, including succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC); 4-succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)-toluene (SMPT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-[3-(2-pyridyldithio)propionate]hexanoate (LC-SPDP). Crosslinkers with N-hydroxysuccinimide moieties can be obtained as N-hydroxysulfosuccinimide analogs, which generally have higher water solubility. In addition, crosslinkers with disulfide bridges in the linking chain can alternatively be synthesized as alkyl derivatives to reduce the amount of linker cleavage in vivo. In addition to heterobifunctional crosslinkers, several other crosslinkers exist, including homobifunctional crosslinkers and photoreactive crosslinkers.Disuccinimidyl suberate (DSS), bismaleimidohexane (BMH), and dimethylpimelimidate·2HCl are examples of useful homobifunctional crosslinkers for use in this disclosure, while bis-[B-(4-azidosalicylamido)ethyl]disulfide (BASED) and N-succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) are examples of useful photoreactive crosslinkers. For a recent review of protein coupling techniques, see Means et al. (1990) Bioconj. Chem. 1:2-12, incorporated herein by reference.

[0040] [Table 2]

[0041] c. Optional Linker The fusion proteins described herein may optionally contain one or more linkers. The linker may be peptidic or non-peptidic. The purpose of the linker is, inter alia, to provide an appropriate distance between functional domains in the protein (e.g., between the J domain and the p53 binding domain, between the tandem arrangement of the p53 binding domain, between the J domain and the p53 binding domain and either the optional targeting reagent, or between the J domain and the p53 binding domain and either the optional detection domain or epitope) for optimal function of each of the domains. Obviously, the linker preferably does not interfere with the function of each of the J domains, the target protein binding domains of the fusion proteins of the present invention. The linker, when present in the fusion proteins of the present invention, may be selected to attenuate the cytotoxicity caused by the target protein (p53 protein), and may be omitted if direct attachment achieves the desired effect. The linker present in the fusion protein of the present invention may comprise one or more amino acids encoded by a nucleotide sequence present on a segment of nucleic acid per cloning site of an expression vector into which a nucleic acid segment encoding a protein domain or the entire fusion protein as described herein is inserted in frame. In one embodiment, the peptide linker is between 1 and 20 amino acids in length. In another embodiment, the peptide linker is between 2 and 15 amino acids in length. In yet another embodiment, the peptide linker is between 2 and 10 amino acids in length. It is within the knowledge and skill of those skilled in the art to select one or more polypeptide linkers to generate a fusion protein according to the present invention. See, for example, Arai et al., Protein Eng., 14(8):529-532 (2001); Crasto et al., Protein Eng., 13(5):309-314 (2000); George et al., Protein Eng., 15(11):871-879 (2003); Robinson et al., Proc. Natl. Acad. Sci. USA, 95:5929-5934 (1998) (each of which is incorporated herein by reference in its entirety). Examples of linkers of two or more amino acids that can be used in preparing a fusion protein according to the present invention include, but are not limited to, those provided in Table 3 below.

[0042] [Table 3]

[0043] d. Targeting Reagents The fusion protein disclosed herein may further comprise a targeting moiety. As used herein, the terms "targeting moiety" and "targeting reagent" are used interchangeably and refer to a substance associated with a fusion protein that enhances the binding, transport, accumulation, residence time, bioavailability, or modifies the biological activity or therapeutic effect of the fusion protein in a cell or in the subject's body. The targeting moiety may have functionality at the tissue, cell, and / or subcellular level. The targeting moiety can direct the localization or intracellular distribution of the fusion protein to a specific cell, tissue, or organ, for example, by administering the fusion protein to a subject. In one embodiment, the targeting moiety is located at the N-terminus of the fusion protein. In another embodiment, the targeting moiety is located at the C-terminus of the fusion protein. In yet another embodiment, the targeting moiety is located internally. In another embodiment, the targeting moiety is attached to the fusion protein via chemical conjugation.

[0044] Targeting moieties may include, but are not limited to, organic or inorganic molecules, peptides, peptidomimetics, proteins, antibodies or fragments thereof, growth factors, enzymes, lectins, antigens or immunogens, viruses or components thereof, viral vectors, receptors, receptor ligands, toxins, polynucleotides, oligonucleotides or aptamers, nucleotides, carbohydrates, sugars, lipids, glycolipids, nucleoproteins, glycoproteins, lipoproteins, steroids, hormones, growth factors, chemoattractants, cytokines, chemokines, drugs, or small molecules, among others.

[0045] In exemplary embodiments of the present invention, the targeting moiety enhances the binding, transport, accumulation, residence time, bioavailability, or modifies the biological activity or therapeutic effect of the platform, or its associated ligand and / or active substance, in target cells or tissues, such as nerve cells, the central nervous system, and / or the peripheral nervous system.Thus, the targeting moiety may have specificity for cell receptors associated with the central nervous system, or otherwise be associated with enhancing delivery to the CNS through the blood-brain barrier (BBB).Consequently, such a ligand may be both a ligand and a targeting moiety.

[0046] In some embodiments, the targeting moiety can be a cell penetrating peptide, for example, as described in U.S. Patent No. 10,111,965, which is incorporated herein by reference in its entirety. In another embodiment, the targeting moiety can be an antibody or an antigen-binding fragment or single-chain derivative thereof, for example, as described in U.S. Patent Application No. 16 / 131,591, which is incorporated herein by reference in its entirety. In a further embodiment, the targeting moiety can be an amino acid sequence for a nuclear localization signal or nuclear export signal.

[0047] The targeting moiety can be linked to the platform for targeted cell delivery by directly or indirectly binding to its core. For example, in embodiments in which the core comprises a nanoparticle, the conjugation of the targeting moiety to the nanoparticle can utilize a similar functional group used to tether PEG to the nanoparticle. Thus, the targeting moiety can be directly bound to the nanoparticle through the functionalization of the targeting moiety. Alternatively, the targeting moiety can be indirectly bound to the nanoparticle through the conjugation of the targeting moiety to a functionalized PEG, as discussed above. The targeting moiety can be attached to the core by covalent, non-covalent, or electrostatic interactions. In one embodiment, the targeting moiety is a peptide. In certain embodiments, the targeting moiety is a peptide that is covalently attached to the N-terminus of the fusion protein.

[0048] e. Epitope In certain embodiments, the fusion proteins of the present invention contain an optional epitope or tag that can confer additional properties to the fusion protein. As used herein, the terms "epitope" and "tag" are used interchangeably to refer to an amino acid sequence, typically 300 amino acids or less in length, that is typically attached to the N-terminus or C-terminus of the fusion protein. In one embodiment, the fusion proteins of the present invention further comprise an epitope that is used to facilitate purification. Examples of such epitopes useful for purification, provided in Table 4 below, include human IgG1 Fc sequence (SEQ ID NO: 68), FLAG epitope (DYKDDDDK, SEQ ID NO: 69), His6 epitope (SEQ ID NO: 70), c-myc (SEQ ID NO: 71), HA (SEQ ID NO: 72), V5 epitope (SEQ ID NO: 73), or glutathione-s-transferase (SEQ ID NO: 74). In another embodiment, the fusion proteins of the present invention further comprise an epitope that is used to increase the half-life of the fusion protein when administered to a subject, e.g., a human. An example of such an epitope useful for increasing half-life includes a human Fc sequence. Thus, in one particular embodiment, the fusion protein comprises a human Fc epitope in addition to the J domain and the p53 binding domain. The epitope is located at the C-terminus of the fusion protein.

[0049] [Table 4]

[0050] f. Cell-penetrating peptides In still other embodiments, the fusion proteins described herein may further comprise a cell-penetrating peptide. Cell-penetrating peptides are known to carry conjugated cargo, whether it is a small molecule, peptide, protein, or nucleic acid, into cells. Non-limiting examples of cell-penetrating peptides in the fusion proteins of the present invention include polycationic peptides, such as HIV TAT peptide 49-57, polyarginine, and penetratin pAntan (43-58), amphipathic peptides, such as pep-1, hydrophobic peptides, such as C405Y, and the like. See Table 5 below.

[0051] [Table 5]

[0052] Thus, in one embodiment, the fusion protein comprises a cell-penetrating peptide and a fusion protein, the cell-penetrating peptide being selected from the group consisting of SEQ ID NOs: 75-78, and the fusion protein comprising a J domain and a p53-binding domain. In one embodiment, the fusion protein is selected from the group consisting of SEQ ID NOs: 80-91 and 100-107. In another embodiment, the fusion protein comprises a signal sequence of SEQ ID NO: 75, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In another embodiment, the fusion protein comprises a cell-penetrating peptide of SEQ ID NO: 76, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In yet another embodiment, the fusion protein comprises a cell-penetrating peptide of SEQ ID NO: 77, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In yet another embodiment, the fusion protein comprises a cell-penetrating peptide of SEQ ID NO: 78, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. Cells expressing the present fusion protein constructs containing a cell-permeable peptide can be administered to a subject, e.g., a human subject (e.g., a patient having or at risk of suffering from a p53 disorder). The fusion protein is secreted from the cell and helps restore p53 function and / or reduces p53 aggregation.

[0053] g. Arrangement of J domain and p53-binding domain The fusion proteins described herein can be configured in a number of ways. In one embodiment, the p53 binding domain is attached C-terminal to the J domain. In another embodiment, the p53 binding domain is attached N-terminal to the J domain. The p53 binding domain and the J domain, in either configuration, can be optionally separated via a linker as described above.

[0054] In some embodiments, a J domain may be attached to multiple p53 binding domains, for example, two p53 binding domains, three p53 binding domains, four p53 binding domains, or more. A p53 binding domain may be attached to the N-terminal side of the J domain. Alternatively, a p53 binding domain may be attached to the C-terminal side of the J domain. In yet another embodiment, a p53 binding domain may be attached to the N-terminal and C-terminal sides of the J domain. Each of the multiple p53 binding domains may be the same p53 binding domain. In another embodiment, each of the multiple p53 binding domains in the fusion protein may be a different p53 binding domain (i.e., different sequence).

[0055] In some embodiments, the fusion protein is selected from the following groups: a. DNAJ-XT, b. DNAJ-XTXT, c. DNAJ-XTXTXT, d. TX-DNAJ, e. TXTX-DNAJ, f. TXTXTX-DNAJ, g. TX-DNAJ-XT, h. TX-DNAJ-XTXT, i. TXTX-DNAJ-X-TT, j. TTDNAJ-XTX-TTTTTDNAJ-XT, k. TXTX-DNAJ-XTXTXT, l. TXTXTX-DNAJ-XT, m. TXTXTX-DNAJ-XTXT, n. TXTXTX-DNAJ-XTXTXT, o. DnaJ-X-DnaJ-XTXT, p.TX-DnaJ-X-DnaJ, q. TXTX-DnaJ-X-DnaJ, and r. TX-TDnaJ-X-TDnaJ-X-TTTT may include a structure selected from Where: T is the p53 binding domain, DNAJ is the J domain of the J protein, X is an optional linker.

[0056] In one embodiment, the fusion protein comprises a J domain selected from the group consisting of SEQ ID NO:5, 6, 10, 24, and 31. In one particular embodiment, the fusion protein comprises a J domain of SEQ ID NO:5.

[0057] In another embodiment, the p53-binding domain is selected from the group consisting of SEQ ID NOs: 51 to 56. In one particular embodiment, the p53-binding domain is selected from the group consisting of SEQ ID NOs: 51 to 52.

[0058] In yet another embodiment, the fusion protein comprises a J domain of SEQ ID NO:5 and a p53 binding domain of SEQ ID NO:51. In another embodiment, the fusion protein comprises a J domain of SEQ ID NO:5 and at least two copies of a p53 binding domain of SEQ ID NO:52.

[0059] Non-limiting examples of fusion protein constructs comprising a J domain and a p53 binding domain are depicted diagrammatically in Figure 2 and also shown below in Table 6. In another embodiment, the particular fusion protein construct is selected from the group consisting of SEQ ID NOs: 80-91.

[0060] [Table 6-1]

[0061] [Table 6-2]

[0062] [Table 6-3]

[0063] II. Nucleic Acids Encoding Fusion Protein Constructs According to another aspect of the present invention, an isolated nucleic acid is provided, comprising a polynucleotide selected from (a) a polynucleotide encoding a fusion protein according to any of the preceding embodiments, or (b) a complement of the polynucleotide of (a). The present invention provides isolated nucleic acids encoding fusion proteins comprising a J domain and a p53 binding domain, as well as sequences complementary to such nucleic acid molecules encoding fusion proteins, as well as homologous variants thereof. In another aspect, the present invention encompasses methods for making nucleic acids encoding fusion proteins disclosed herein, sequences complementary to nucleic acids encoding fusion proteins, as well as homologous variants thereof. The nucleic acid according to this aspect of the present invention can be pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0064] In yet another aspect, a method of producing a fusion protein is disclosed, comprising the steps of (a) synthesizing and / or assembling nucleotides encoding the fusion protein, (b) incorporating the encoding gene into an expression vector suitable for a host cell, (c) transforming a suitable host cell with the expression vector, and (d) culturing the host cell under conditions that cause or allow the fusion protein to be expressed in the transformed host cell, thereby producing a biologically active fusion protein, which is recovered as an isolated fusion protein by standard protein purification methods known in the art.Standard recombinant techniques in molecular biology are used to generate the polynucleotides and expression vectors of the invention.

[0065] According to the present invention, the nucleic acid sequence encoding the fusion protein disclosed herein (or its complement) is used to generate a recombinant DNA molecule that directs the expression of the fusion protein in a suitable host cell. Several cloning strategies are suitable for carrying out the present invention, many of which are used to generate constructs that include genes encoding the fusion protein of the present invention or its complement. In some embodiments, a cloning strategy is used to produce a gene encoding the fusion protein of the present invention or its complement.

[0066] In certain embodiments, the nucleic acid encoding the one or more fusion proteins is an RNA molecule, which can be pre-messenger RNA (pre-mRNA), messenger RNA (mRNA), RNA, genomic DNA (gDNA), PCR amplified DNA, complementary DNA (cDNA), synthetic DNA, or recombinant DNA.

[0067] In various embodiments, the nucleic acid is an mRNA that is introduced into a cell to transiently express a desired polypeptide. As used herein, "transient" refers to the expression of an unintegrated transgene for a period of hours, days, or weeks, the period of expression being shorter than the period for the expression of a polynucleotide when integrated into a genome or contained within a stable plasmid replicon in a cell.

[0068] In certain embodiments, the mRNA encoding the polypeptide is an in vitro transcribed mRNA.As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that is synthesized in vitro.Generally, the in vitro transcribed RNA is produced from an in vitro transcription vector.The in vitro transcription vector comprises a template that is used to produce the in vitro transcribed RNA.

[0069] In certain embodiments, the mRNA may further comprise a 5' cap or modified 5' cap and / or poly(A) sequence. As used herein, a 5' cap (also called an RNA cap, an RNA 7-methylguanosine cap, or an RNA m7G cap) is a modified guanine nucleotide that is added to the "front" or 5' end of a eukaryotic messenger RNA immediately after the start of transcription. The 5' cap is linked to the first transcribed nucleotide and comprises a terminal group that is recognized by the ribosome and protected from Rnase. The capping moiety may be modified to modulate the functionality of the mRNA, for example, its stability or efficiency of translation. In certain embodiments, the mRNA comprises a poly(A) sequence of between about 50 and about 5000 adenines. In one embodiment, the mRNA comprises a poly(A) sequence of between about 100 and about 1000 bases, between about 200 and about 500 bases, or between about 300 and about 400 bases. In one embodiment, the mRNA comprises a poly(A) sequence of about 65, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 bases or more. The poly(A) sequence can be chemically or enzymatically modified to modulate the functionality of the mRNA, e.g., localization, stability, or efficiency of translation.

[0070] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined below.These terms include polynucleotides in which one or more nucleotides are added, deleted, or replaced with different nucleotides compared to the reference polynucleotide.In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to the reference polynucleotide, so that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0071] In certain embodiments, the nucleic acid sequence comprises a nucleotide sequence encoding a gene of interest (e.g., a fusion protein comprising a J domain and a p53 binding domain) within a nucleic acid cassette. As used herein, the term "nucleic acid cassette" or "expression cassette" refers to a genetic sequence within a vector that can express an RNA and then a polypeptide. In one embodiment, the nucleic acid cassette contains a gene of interest, e.g., a polynucleotide of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, e.g., a promoter, an enhancer, a poly(A) sequence, and a gene of interest, e.g., a polynucleotide of interest. A vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector so that the nucleic acid within the cassette can be transcribed into RNA, translated into a protein or polypeptide as necessary, undergo appropriate post-translational modifications required for activity in transformed cells, and translocated to an appropriate compartment for biological activity by targeting to an appropriate intracellular compartment or by secretion to an extracellular compartment.Preferably, the cassette has 3' and 5' ends adapted for immediate insertion into a vector, e.g., it has a restriction endonuclease site at each end.The cassette can be removed and inserted into a plasmid or viral vector as a single unit.

[0072] Illustrative ubiquitous expression control sequences suitable for use in certain embodiments include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and P1 from vaccinia virus. l promoter, elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (b-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter (Okabe et al. (1997) FEBS Lett. 407:313-9), the b-actin promoter and myeloproliferative sarcoma virus enhancer, the (MND) U3 promoter with the negative control region deleted and replaced with the dl587rev primer binding site (Haas et al., Journal of Virology. 2003;77(l7):9439-9450).

[0073] In one embodiment, at least one element for enhancing transgene target specificity and expression, such as a promoter (see, e.g., Powell et al. (2015) Discovery Medicine 19(102):49-57, the contents of which are incorporated herein by reference in their entirety), may be used with the polynucleotides described herein. Promoters that drive expression in most tissues include, but are not limited to, human elongation factor la-subunit (EFla), immediate early cytomegalovirus (CMV), chicken β-actin (CBA) and its derivatives CAG, β-glucuronidase (GUSB), or ubiquitin C (UBC). Tissue-specific expression elements are used to restrict expression to certain cell types, including, but not limited to, neural promoters that may be used to restrict expression to, for example, neurons, astrocytes, or oligodendrocytes. Non-limiting examples of tissue-specific expression elements for neurons include, but are not limited to, the promoters of neuron-specific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), synapsin (Syn), methyl-CpG binding protein 2 (MeCP2), CaMKII, mGluR2, NFL, NFH, ηβ2, PPE, Enk, and EAAT2. Non-limiting examples of tissue-specific expression elements for astrocytes include the promoters of glial fibrillary acidic protein (GFAP) and EAAT2. Non-limiting examples of tissue-specific expression elements for oligodendrocytes include the promoter of myelin basic protein (MBP). Yu et al. ((2011) Molecular Pain, 7:63, incorporated by reference in its entirety) evaluated the expression of eGFP under the CAG, EFIa, PGK, and UBC promoters in rat DRG cells and primary DRG cells using lentiviral vectors and found that UBC showed weaker expression than the other three promoters, with only 10-12% glial expression for all promoters.Soderblom et al. (E.Neuro 2015, incorporated by reference in its entirety) evaluated eGFP expression in AAV8 with the CMV and UBC promoters and AAV2 with the CMV promoter after injection into the motor cortex. Intranasal administration of plasmids containing the UBC or EFIa promoters showed higher sustained airway expression than expression with the CMV promoter (see, e.g., Gill et al. (2001) Gene Therapy, vol. 8, pp. 1539-1546, incorporated by reference in its entirety). Husain et al. ((2009) Gene Therapy, incorporated by reference in its entirety) evaluated ΗβΗ constructs with the hGUSB promoter, HSV-1 LAT promoter, and NSE promoter and found that the ΗβΗ constructs showed weaker expression than NSE in mouse brain. Passini and Wolfe (J. Virol. 2001, 12382-12392, incorporated by reference in its entirety) evaluated the long-term effects of HβH vectors after intraventricular injection in neonatal mice and found persistent expression for at least one year. Xu et al. ((2001) Gene Therapy, 8, 1323-1332, incorporated by reference in its entirety) found low expression in all brain regions when the NF-L and NF-H promoters were used compared to CMV-lacZ, CMV-luc, EF, GFAP, hENK, nAChR, PPE, PPE+wpre, NSE(0.3kb), NSE(1.8kb), and NSE(1.8kb+wpre). Xu et al. found that the promoter activity, in descending order, was NSE (1.8 kb), EF, NSE (0.3 kb), GFAP, CMV, hENK, PPE, NFL, and NFH. NFL is a 650 nucleotide promoter, and NFH is a 920 nucleotide promoter. Both are absent from the liver, but NFH is abundant in proprioceptive sensory neurons, brain, and spinal cord, and NFH is present in the heart. Scn8a is a 470 nucleotide promoter that is expressed in DRG, spinal cord, and brain, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells, cortex, thalamus, and hypothalamus.(See, e.g., Drews et al. 2007 and Raymond et al. 2004, incorporated by reference in their entireties).

[0074] III. Vectors Containing Nucleic Acids Encoding Fusion Proteins Vectors comprising the nucleic acid according to the present invention are also provided. Such vectors preferably contain additional nucleic acid sequences, such as elements (e.g., promoter and / or terminator sequences) necessary for transcription / translation of the nucleic acid sequence encoding the phosphatase. The vector may also contain a nucleic acid sequence encoding a selection marker (e.g., an antibiotic) to select or maintain a host cell transformed with the vector. The term "vector" refers to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. The vector may contain sequences directing autonomous replication in the cell or may contain sequences sufficient to allow integration into the host cell DNA. In certain embodiments, non-viral vectors are used to deliver one or more polynucleotides contemplated herein to diseased cells (e.g., neuronal cells). In one embodiment, the vector is an in vitro synthesized or synthetically prepared mRNA encoding a fusion protein comprising a J domain and a p53 binding domain. Examples of non-viral vectors include, but are not limited to, mRNA, plasmids (eg, DNA or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

[0075] Examples of vectors include, but are not limited to, plasmids, autonomously replicating sequences, and transposable elements, such as piggyBac, Sleeping Beauty, Mosl, Tcl / mariner, Tol2, mini-Tol2, Tc3, MuA, Himar I, Frog Prince, and their derivatives. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, such as lambda phage or M13 phage, and animal viruses. Examples of viruses useful as vectors include, but are not limited to, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses (vims)), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors include, but are not limited to, pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V 5-DEST™, pLenti6 / V 5-DEST™, and pLenti6.2 / V 5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequence of the polypeptide disclosed herein can be ligated into such an expression vector for expression of the polypeptide in mammalian cells.

[0076] In certain embodiments, the vector is an episomal vector, or a vector that is maintained extrachromosomally. As used herein, the term "episomal" refers to a vector that can replicate without integration into the host chromosomal DNA and without being gradually lost from dividing host cells, i.e., the vector replicates extrachromosomally or episomally.

[0077] Vectors can contain one or more recombination sites for any of a variety of site-specific recombinases.It should be understood that the target site for site-specific recombinase is in addition to any site required for integration of vector, for example, retroviral or lentiviral vector.As used herein, the term "recombination sequence", "recombination site" or "site-specific recombination site" refers to the specific nucleic acid sequence that recombinase recognizes and binds to.

[0078] For example, one recombination site for Cre recombinase is loxP, a 34 base pair sequence containing two 13 base pair inverted repeats (which serve as recombinase binding sites) flanking an 8 base pair core sequence (see Figure 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Suitable recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod et al., 1996), FI, F2, F3 (Schlake and Bode, 1994), FyFs (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

[0079] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme integrase, e.g., phi-c3l. (The pC3l SSR mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). Both attB and attP, named for the attachment sites for phage integrase in the bacterial and phage genomes, respectively, contain imperfect inverted repeats that are presumably bound by the φ031 homodimer (Groth et al., 2000). The product sites, attL and attR, are effectively additional tpQA 1-mediated recombination (Belteki et al., 2003), making the reaction irreversible. In catalyzing insertion, attB-bearing DNA has been found to insert into genomic attP sites more easily than attP sites do (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, a typical strategy is to position an attP-bearing "docking site" to a defined locus by homologous recombination, which is then partnered with an attB-bearing incoming sequence as an insertion.

[0080] As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a start codon, such as ATG, of a cistron (protein coding region), thereby resulting in cap-independent translation of a gene. See, for example, Jackson et al., 1990 Trends Biochem Sci 15(12):477-83, and Jackson and Kaminski 1995 RNA 1(10):985-1000. In certain embodiments, a vector comprises one or more polynucleotides of interest that encode one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences can be separated by one or more IRES sequences, or polynucleotide sequences that encode self-cleaving polypeptides. In one embodiment, the IRES used in the polynucleotides contemplated herein is the EMCV IRES.

[0081] As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of mRNA with the small subunit of the ribosome, increasing translation (Kozak, 1986, Cell 44(2):283-92, and Kozak, 1987, Nucleic Acids Res.15(20):8125-48). In certain embodiments, the vector comprises a polynucleotide having a consensus Kozak sequence and a polynucleotide encoding a fusion protein comprising a J domain and a p53 binding domain. Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of the polyadenylation signal. In certain embodiments, the vector comprises a polyadenylation sequence 3' to the polynucleotide encoding the polypeptide to be expressed.

[0082] Examples of viral vector systems suitable for use in certain embodiments contemplated herein include, but are not limited to, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia virus vectors.

[0083] In various embodiments, one or more polynucleotides encoding a fusion protein comprising a J domain and a p53 binding domain are introduced into a cell, for example, a neuronal cell, by transducing the cell with a recombinant adeno-associated virus (rAAV) comprising one or more polynucleotides. AAV is a small (about 26 nm), replication-defective, mainly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells, and can integrate its genome into the genome of a host cell. Recombinant AAV (rAAV) is typically composed, at a minimum, of a transgene and its control sequence, and 5' and 3' AAV inverted terminal repeat (ITR). The ITR sequence is about 145 bp long. In certain embodiments, the rAAV comprises ITR and capsid sequences isolated from AAV1, AAV2 (e.g., as described in US6962815B2, which is incorporated herein by reference in its entirety), AAV3, AAV4, AAV5 (e.g., as described in US7479554B2, which is incorporated herein by reference in its entirety), AAV6, AAV7, AAV8 (e.g., as described in US7282199B2, which is incorporated herein by reference in its entirety), AAV9 (e.g., as described in US9737618B2, which is incorporated herein by reference in its entirety), AAV rh10 (e.g., as described in US9790472B2, which is incorporated herein by reference in its entirety), or AAV10. In one embodiment, the vector of the invention is encapsulated into a capsid selected from the group consisting of AAV2, AAV5, AAV8, AAV9, and AAV rh10. In one embodiment, the vector is encapsulated in AAV2. In one embodiment, the vector is encapsulated in AAV5. In one embodiment, the vector is encapsulated in AAV8. In one embodiment, the vector is encapsulated in AAV9. Yet, in one embodiment, the vector is encapsulated in AAV rh10.

[0084] In some embodiments, a chimeric rAAV is used, in which the ITR sequence is isolated from one AAV serotype and the capsid sequence is isolated from a different AAV serotype. For example, a rAAV with an ITR sequence from AAV2 and a capsid sequence from AAV6 is called AAV2 / AAV6. In certain embodiments, a rAAV vector can comprise an ITR from AAV2 and a capsid protein from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV comprises an ITR sequence from AAV2 and a capsid sequence from AAV6. In a preferred embodiment, the rAAV comprises an ITR sequence from AAV2 and a capsid sequence from AAV2.

[0085] In some embodiments, engineering and selection methods can be applied to the AAV capsid to make it more likely to transduce a cell of interest.

[0086] The construction, production and purification of rAAV vectors are disclosed, for example, in U.S. Pat. Nos. 9,169,494; 9,169,492; 9,012,224; 8,889,641; 8,809,058; and 8,784,799, each of which is incorporated by reference in its entirety.

[0087] IV. Delivery In certain embodiments, one or more polynucleotides encoding fusion proteins comprising J domain and p53 binding domain are introduced into cells by non-viral or viral vectors. Illustrative methods of non-viral delivery of polynucleotides contemplated in certain embodiments include, but are not limited to, electroporation, sonoporation, lipofection, microinjection, particle gun, virosome, liposome, immunoliposome, nanoparticle, polycation or lipid nucleic acid conjugate, naked DNA, artificial virion, DEAE-dextran mediated transfer, gene gun, and heat shock.

[0088] Examples of polynucleotide delivery systems suitable for use in certain contemplated embodiments include, but are not limited to, delivery systems provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, and Copernicus Therapeutics Inc. Lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides have been described in the literature. See, for example, Liu et al., (2003) Gene Therapy 10:180-187; and Balazs et al., (20W) Journal of Drug Delivery 2011:1-12. Antibody-targeted, bacterial-derived, non-viable nanocell-based delivery is also contemplated in certain embodiments.

[0089] In certain embodiments, viral vectors containing contemplated polynucleotides may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection), intrathecal injection, intraventricular injection, or local application, as described below. Alternatively, vectors may be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirate, tissue biopsy, etc.) or hematopoietic stem cells of a universal donor, and then the cells are re-implanted into the patient.

[0090] In one embodiment, a viral vector containing a polynucleotide encoding a fusion protein disclosed herein is directly administered to an organism for transduction of cells in vivo.

[0091] Alternatively, naked DNA can be administered. Administration can be by any of the routes that are normally used to introduce molecules into ultimate contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation.Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and although more than one route can be used to administer a particular composition, a particular route can provide a more immediate and effective response than another route.

[0092] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into cells, such as neural cells or neural stem cells, by transducing the cells with a retrovirus, such as a lentivirus, that contains one or more polynucleotides. As used herein, the term "retrovirus" refers to an RNA virus that transcribes its genomic RNA into a linear double-stranded DNA copy, and then covalently integrates the genomic DNA into the host genome. Illustrative retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentivirus. As used herein, the term "lentivirus" refers to a group (or genus) of complex lentiviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred.

[0093] Lentiviral vectors preferably contain some safety enhancements as a result of modifying the LTR. "Self-inactivating" (SIN) vectors refer to replication-defective vectors, such as those in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter that drives the transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, promoters of the viruses Simian Virus 40 (SV40) (e.g., early or late), Cytomegalovirus (CMV) (e.g., immediate early), Moloney Murine Leukemia Virus (MoMLV), Rous Sarcoma Virus (RSV), and Herpes Simplex Virus (vims) (HSV) (thymidine kinase). In certain embodiments, lentiviral vectors are produced according to known methods. See, e.g., Kutner et al., BMC Biotechnol. 2009;9:10 Doi:10.1186 / 1472-6750-9-10; Kutner et al., Nat. Protoc. 2009;4(4):495-505 Doi:l0.l038 / nprot.2009.22.

[0094] According to certain embodiments contemplated herein, most or all of the viral vector backbone sequence is derived from lentivirus, such as HIV-1.However, it should be understood that many different sources of retrovirus and / or lentivirus sequence can be used, or a large number of combined substitutions and changes in a portion of lentivirus sequence can be accommodated without impairing the ability of transfer vector to perform the functions described herein.In addition, various lentivirus vectors are known in the art, see Naldini et al. (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136 (many of which can be adapted to make the viral vector or transfer plasmid contemplated herein).

[0095] In various embodiments, one or more polynucleotides encoding the fusion proteins disclosed herein are introduced into target cells by transducing the cells with an adenovirus containing one or more polynucleotides. Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. High titers and high levels of expression have been obtained using such vectors. This vector can be produced in large quantities in a relatively simple system. Most adenovirus vectors are engineered so that the transgene replaces the Ad Ela, Elb, and / or E3 genes, and then the replication-defective vector is propagated in human 293 cells that supply the missing gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing differentiated cells such as those found in liver, kidney, and muscle. Conventional Ad vectors have a large carrying capacity.

[0096] The generation and propagation of current adenoviral vectors, which are replication-deficient, can utilize a unique helper cell line, named 293, which is transformed with Ad5 DNA fragments from human embryonic kidney cells and constitutively expresses the El protein (Graham et al., 1977). Because the E3 region is dispensed with from the adenoviral genome (Jones & Shenk, 1978), current adenoviral vectors with the help of 293 cells carry foreign DNA in either the El, E3, or both regions (Graham & Prevec, 1991). Adenoviral vectors have been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies in administering recombinant adenoviruses to different tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), intramuscular injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993), and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993). An example of the use of Ad vectors in clinical trials included polynucleotide therapy for antitumor immunization using intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

[0097] In various embodiments, one or more polynucleotides encoding the fusion proteins of the invention are introduced into a target cell of a subject by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, containing the one or more polynucleotides.

[0098] Mature HSV virions are composed of an enveloped icosahedral capsid, which contains a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. In one embodiment, the HSV-based viral vector is defective in one or more essential or non-essential HSV genes. In one embodiment, the HSV-based viral vector is replication-defective. Most replication-defective HSV vectors contain deletions that remove one or more immediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in immediate-early genes selected from the group consisting of ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantage of HSV vectors is their ability to enter latency, which can result in long-term DNA expression, and their large viral DNA genome, which can accommodate exogenous DNA inserts up to 25 kb. HSV-based vectors are described, for example, in U.S. Pat. Nos. 5,837,532, 5,846,782, and 5,804,413, and International Patent Applications WO91 / 02788, WO96 / 04394, WO98 / 15637, and WO99 / 06583, each of which is incorporated by reference in its entirety.

[0099] V. Cells expressing the fusion protein In yet another aspect, the invention provides a cell expressing the fusion protein described herein. The cell may be transfected with a vector encoding the fusion protein as described above. In one embodiment, the cell is a prokaryotic cell. In another embodiment, the cell is a eukaryotic cell. In yet another embodiment, the cell is a mammalian cell. In a particular embodiment, the cell is a human cell. In another embodiment, the cell is a human cell derived from a patient suffering from or at risk of suffering from p53-mediated neoplasia, including, but not limited to, cancer and other neoplasms, including, but not limited to, malignant neoplasms declared or presumed to be primary at the following sites: malignant neoplasms of the lip, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and and other malignant neoplasms of the skin; malignant neoplasms of mesothelium and soft tissue, including sarcomas; malignant neoplasms of the breast; malignant neoplasms of the female genital organs, including ovarian cancer; malignant neoplasms of the male genital organs, including prostate cancer; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; malignant neoplasms of poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes. The cells may be liver parenchymal or muscle cells.

[0100] The cells expressing the fusion protein can be useful in producing the fusion protein. In this embodiment, the cells are transfected with a vector that overexpresses the fusion protein. The fusion protein can optionally contain an epitope, for example, a human Fc domain or a FLAG epitope as described herein, which facilitates purification (using a protein A column or an anti-FLAG antibody column, respectively). The epitope can be connected to the rest of the fusion protein via a linker or a protease substrate sequence so that the epitope can be removed from the fusion protein during or after purification.

[0101] In an alternative embodiment, cells expressing secreted forms of the fusion protein can be used. For example, the fusion protein construct can be designed to have a signal sequence at the N-terminus. Representative signal sequences are shown in Table 7 below.

[0102] [Table 7]

[0103] Thus, in one embodiment, the fusion protein comprises a signal sequence and a fusion protein, the signal sequence being selected from the group consisting of SEQ ID NOs: 94-96, and the fusion protein comprising a J domain and a p53 binding domain. In one embodiment, the signal sequence is selected from the group consisting of SEQ ID NOs: 94-96, and the fusion protein is selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In another embodiment, the fusion protein comprises a signal sequence of SEQ ID NO: 94, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In another embodiment, the fusion protein comprises a signal sequence of SEQ ID NO: 95, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. In another embodiment, the fusion protein comprises a signal sequence of SEQ ID NO: 96, and a fusion protein selected from the group consisting of SEQ ID NOs: 80-91, 100-107. Cells expressing a fusion protein construct comprising a signal sequence can be administered to a subject, e.g., a human subject (e.g., a patient having or at risk of suffering from a p53 disorder). The fusion protein is secreted from cells and helps restore p53 function and / or reduces p53 aggregation.

[0104] As described above, in certain embodiments, the fusion protein can further comprise a cell-penetrating peptide.The cell expressing the fusion protein comprising signal sequence and cell-penetrating peptide can secrete the fusion protein lacking signal sequence.The secreted fusion protein also comprising cell-penetrating peptide can then enter nearby cells, and has the potential to restore p53 function and / or reduce p53 aggregation in those cells.

[0105] VI. How to use In another embodiment, the present invention provides a method for achieving beneficial effects in disorders and / or in p53 disorders, which are disorders or conditions mediated by mutant p53. The p53 disorder is cancer. Cancer can include any of the following: malignant neoplasms declared or presumed to be primary at the following sites: malignant neoplasms of the lips, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female reproductive tract, including ovarian cancer. Malignant neoplasms; malignant neoplasms of the male reproductive organs, including prostate cancer; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; poorly defined, secondary, and unspecified sites; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes.

[0106] In some embodiments, the invention provides a method for treating a subject, such as a human, having a p53 disease, disorder, or condition, comprising administering to the subject a therapeutically or prophylactically effective amount of a fusion protein, a nucleic acid encoding such a fusion protein, or a viral vector encoding such a fusion protein described herein, wherein said administration results in an improvement in one or more biochemical or physiological parameters or clinical endpoints associated with the p53 disease, disorder, or condition.

[0107] In another embodiment, the present invention provides a method for restoring p53 function and / or reducing p53 aggregation in cells.The cells can be cultured cells or isolated cells.The cells can also be derived from a subject, for example, a human subject.In one embodiment, the human subject suffers from or is at risk of suffering from a p53 disorder disease, including one or more types of cancer.

[0108] The activity of p53 protein can be detected in several ways. In one example, the transcriptional activity of p53 can be monitored by downstream genes such as p21. In another embodiment, p53 function is monitored by a reporter gene assay that includes an artificial reporter construct.

[0109] Thus, in one embodiment, the method comprises contacting a cell with an amount of a fusion protein, or a nucleic acid, vector, or viral particle encoding said fusion protein, effective to restore p53 activity by at least 10%, e.g., at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, as compared to untreated or control cells.

[0110] As shown in Example 1 below, expression of a fusion protein comprising a J domain and a p53 binding domain has been found to restore levels of p21 expression. As such, in another embodiment, the method includes contacting a cell with an amount of the fusion protein, a cell expressing the fusion protein, or a nucleic acid, vector, or viral particle encoding the fusion protein effective to restore p53 protein function by at least 10%, e.g., at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, as compared to untreated or control cells.

[0111] VII. Pharmaceutical Compositions The compositions contemplated herein may include one or more fusion proteins comprising a J domain and a p53 binding domain, a polynucleotide encoding such a fusion protein, a vector comprising the same, a genetically modified cell, etc., as contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated as a pharma- ceutical or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if necessary, the composition may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active substances. There is virtually no limit to other components that may also be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended treatment.

[0112] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0113] As used herein, a "pharmaceutically acceptable carrier," "diluent," or "excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary medicine. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn (com) starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn (com) oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

[0114] VIII. Dosage The dosage of the compositions described herein (e.g., compositions comprising fusion protein constructs, nucleic acids, or gene therapy viral particles) can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of symptoms; the frequency of treatment, and the type of co-treatment, if any; and the clearance rate of the compound in the animal to be treated. The compositions described herein can be initially administered at an appropriate dosage, which can be adjusted as necessary depending on clinical response. In some embodiments, the dosage of the composition is a prophylactically or therapeutically effective amount.

[0115] IX. Kits Kits are contemplated that include (a) a pharmaceutical composition comprising a fusion protein construct described herein that restores p53 function in a cell or subject, a nucleic acid encoding such a fusion protein, or a viral particle that includes such a nucleic acid, and (b) a package insert that includes instructions for carrying out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising a composition described herein that restores p53 function in a cell or subject described herein, (b) an additional therapeutic agent, and (c) a package insert that includes instructions for carrying out any of the methods described herein. EXAMPLES

[0116] To test whether the J domain could be specifically engineered to promote proper folding of mutant proteins, we designed and tested several fusion protein constructs designed to target the p53 protein.

[0117] Example 1: Fusion Protein Design A. Method General Techniques and Materials The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of one of ordinary skill in the art. Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, 2001; Current protocols in molecular biology, FM Ausubel et al., eds., 1987; Methods in Enzymology series, Academic Press, San Diego, Calif.; PCR 2: a practical approach, MJ MacPherson, BD Hames, and GR Taylor, eds., Oxford University Press, 1995; Antibodies, a laboratory manual, Harlow, E. and Lane, D., eds., Cold Spring Harbor Laboratory, 1988; Goodman & Gilman's The Pharmacological Basis of Therapeutics, 11th ed., McGraw-Hill, 2005; and Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 4th ed., John Wiley & Sons, 2006. Sons, Somerset, NJ, 2000, the contents of which are incorporated herein by reference in their entirety. HEK-293 cells (human embryonic kidney cells) were purchased from American Type Culture Collection (Manassas, VA). Anti-FLAG antibody was purchased from Thermo Fisher Scientific. Rabbit anti-GFP antibody was purchased from GenScripts (Piscataway, NJ).To facilitate purification and characterization, some of the fusion protein constructs used in this Example 1 contain, in addition to the sequences provided in SEQ ID NOs:80-91, the FLAG epitope of SEQ ID NO:69, either at the C- or N-terminus of the protein, plus a short linker sequence.

[0118] Protein expression and detection in HCT-116 cells Expression vector plasmids encoding various protein constructs were transfected into HCT-116 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Cell lysates were analyzed for expressed proteins using immunoblot assays. Media samples were centrifuged to remove debris before analysis. Cells were lysed in lysis buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM EDTA, 2% SDS) containing 2 mM PMSF and protease cocktail (Complete Protease Inhibitor Cocktail; Sigma). After brief sonication, samples were analyzed for expressed proteins using immunoblot assays. For immunoblot analysis, samples were boiled in SDS sample buffer and run on polyacrylamide electrophoresis. Separated protein bands were then transferred to PVDF membranes.

[0119] The expressed proteins were detected using chemiluminescent signals. Briefly, the blots were reacted with a primary antibody capable of binding a specific protein (e.g., p21). After washing away the unreacted primary antibody, an enzyme-linked secondary antibody (e.g., HRP-linked anti-IgG antibody) was reacted with the primary antibody molecules bound to the blot. After rinsing, a chemiluminescent reagent was added, and the resulting chemiluminescent signal in the blot was captured on an X-ray film.

[0120] ELISA HCT-116 cells were homogenized in lysis buffer (50 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitor cocktail, 2 mM PMSF, 10 mM NaF, and 2 mM Na3VO4. After brief sonication, debris was removed by centrifugation. Protein concentration was measured by BCA assay kit (Pierce). Supernatants were subjected to ELISA (Human Total p21 / CIP1 / CDKN1A DuoSet IC ELISA: R&D Systems) using the manufacturer's standard protocol.

[0121] Cytotoxicity Assay (LDH Assay) HCT-116 cells or HCT-116 cells stably expressing p53R249S were seeded onto 48-well culture plates. On day 1, the medium was replaced with fresh medium containing 8ug / ml polybrene and lentiviral particles expressing construct 2 or a control lentivirus encoding no substantial genes. After 72 hours, the medium was replaced with fresh medium containing 1uM doxorubicin and the cells were cultured for an additional 12 hours. For some studies, construct 2 was expressed by lipofection. 24 hours after transfection, the medium was replaced with fresh medium and the cells were cultured for an additional 24-48 hours. The medium was then collected and subjected to LDH assay (LDH-Cytox™ Assay Kit: BioLegend: 426401) using the manufacturer's protocol.

[0122] B. Reporter Constructs We first investigated whether the fusion molecules of the present invention targeting p53 could restore its function in cultured cells. To this end, we created p53 constructs containing wild-type p53, as well as p53 containing R175H and R249S mutations, known as "conformational mutations" (see Table 8 below). HCT-116 cells were cultured and transfected with plasmids encoding wild-type p53 or p53 mutants containing R175H (SEQ ID NO: 98) or R249S (SEQ ID NO: 99). As shown in Figure 3, we found that when mutant p53 containing R175H (left) or R249S (right) was expressed instead of wild-type p53, downstream induction of p21 by p53 was abolished (e.g., compare lane 2 vs. 3).

[0123] [Table 8]

[0124] C. Fusion Protein Constructs To determine whether the fusion proteins of the present invention can be used to restore p53 function, initial experiments were first performed by co-expression of fusion proteins containing a J domain sequence derived from human Hsp40 J domain protein conjugated with a peptide sequence recognizing mutant p53 (data not shown). A plasmid encoding construct 2 (JB1-CDB3) was transfected into HCT-116 cells together with a reporter construct (p53R175H or p53R249S). After expression, cells were lysed in lysis buffer and subsequently subjected to immunoblotting assays using anti-p21 or anti-p53 antibodies. Expression of wild-type p53 resulted in induction of p21, whereas mutants (R175H or R249S) failed to do so. Surprisingly, cells expressing construct 2 showed substantial restoration of p21 expression, whereas p53 protein levels themselves were not significantly altered (Figure 3).

[0125] To further validate the results, stable cells expressing mutant p53(R249S) were established by standard methods. Initial HCT-116 cells or HCT-116 cells stably expressing p53R249S were cultured and incubated with lentivirus expressing construct 2. After incubation, cells were stimulated with 1 uM doxorubicin for 12 hours. Cells were lysed and protein concentration was measured by BCA assay. Equal amounts of cell lysates were subjected to ELISA to quantify p21 expression. As shown in Figure 4, cells stably expressing mutant p53 retained lower p21 expression levels (1st bar vs. 2nd bar). Incubation with doxorubicin strongly induced p21 expression (3rd bar), but more weakly in cells expressing mutant p53 (4th bar). Surprisingly, construct 2 completely restored p21 induction (5th bar).

[0126] Finally, the cytotoxic activity of p53 was evaluated. HCT-116 cells stably expressing either the R175H or R249S p53 mutants were cultured, and construct 2 was expressed using lentivirus as described above. After expression, the cells were cultured for an additional 12 hours in the presence of 1 μM doxorubicin. The medium was collected, and an LDH cytotoxicity assay was performed. As shown in Figure 5, cells expressing construct 2 were more sensitive to the cytotoxic effect of doxorubicin. These data suggested that the p53 conformational problem could be resolved using an Hsp70-mediated pathway.

[0127] Similarly, additional constructs were made and tested: cells expressing either the R175H or R249S mutant forms of p53 exhibited a lower cytotoxic effect compared to wild-type p53, while construct 3 (Figure 6), construct 16, or construct 17 (Figure 7) either partially restored the cytotoxic effect.

[0128] We then designed a series of fusion protein constructs, as presented in Table 9.

[0129] [Table 9-1]

[0130] [Table 9-2]

[0131] Example 2: AAV vectors encoding fusion protein constructs An exemplary gene therapy vector is constructed with an AAV9 vector carrying a codon-optimized cDNA encoding the fusion protein construct of Table 9. The cDNA encoding the construct is located downstream of a Kozak sequence and is polyadenylated by the bovine growth hormone polyadenylation (BGHpA) signal. The entire cassette is flanked by two non-coding terminal inverted sequences of AAV-2.

[0132] Recombinant AAV vectors are prepared using a baculovirus expression system similar to that described above (Urabe et al., 2002; Unzu et al., 2011 (reviewed in Kotin, 2011)). Briefly, three recombinant baculoviruses are used to infect SF9 insect cells, one encoding REP for replication and packaging, one encoding CAP-5 for the capsid of AAV9, and one carrying an expression cassette. Purification is performed using AVB Sepharose high-speed affinity media (GE Healthcare Life Sciences, Piscataway, NJ). Vectors are titrated using QPCR with primer-probe combinations for the transgene, and titers are expressed as genome copies per ml (GC / ml). Vector titers are approximately 8×10 13 ~2×10 14 Between GC / ml.

[0133] Other Aspects All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. If a term in this application is found to be defined differently than in a document incorporated herein by reference, the definition provided herein shall serve as the definition for that term.

[0134] While the invention has been described with respect to particular embodiments thereof, it will be understood that the invention can be further modified and that this application is intended and claimed to cover within its scope any variations, uses, or adaptations of the invention which generally follow the principles of the invention and which come within known or customary practice within the art and which may be applicable to the essential features set forth above, including departures from the present disclosure.

Claims

1. An isolated fusion protein comprising a J domain of a J protein and a p53-binding domain.

2. 2. The fusion protein of claim 1, wherein the J domain of the J protein is selected from the group consisting of SEQ ID NOs: 1-15 and 17-50.

3. 3. The fusion protein of claim 2, wherein the J domain comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 5, 6, 10, 24, 25, 31, and 49.

4. The fusion protein of any one of claims 1 to 3, wherein the p53-binding domain comprises a sequence selected from the group consisting of SEQ ID NOs: 51 to 56.

5. The fusion protein of any one of claims 1 to 3, comprising multiple p53-binding domains.

6. A fusion protein according to any one of claims 1 to 3, comprising a J domain sequence of SEQ ID NO: 5 and a p53 binding domain of SEQ ID NO:

52.

7. 2. The fusion protein of claim 1, comprising a sequence selected from the group consisting of SEQ ID NOs: 80-91 and 100-107.

8. The fusion protein of any one of claims 1 to 3 and 7, further comprising a targeting reagent, an epitope, a cell penetration agent or a signal sequence.

9. The fusion protein of any one of claims 1 to 3 and 7, which is capable of restoring p53 function in a cell.

10. A nucleic acid sequence encoding the fusion protein of any one of claims 7.

11. The nucleic acid sequence of claim 10, further comprising a promoter region, a 5'UTR and a 3'UTR.

12. A vector comprising the nucleic acid sequence of claim 10.

13. A viral particle comprising a capsid and the vector of claim 12.

14. A pharmaceutical composition for use in the prevention or treatment of a p53-mediated disease, comprising an agent selected from the group consisting of a fusion protein according to any one of claims 1 to 3 and 7, a cell expressing the fusion protein according to any one of claims 1 to 3 and 7, a nucleic acid according to claim 10, a vector according to claim 12, a viral particle according to claim 13, and a pharmaceutically acceptable carrier or excipient.

15. The pharmaceutical composition of claim 14, wherein the p53-mediated disease is cancer or neoplasm.

16. The cancer or neoplasm is a malignant neoplasm declared or presumed to be primary in the following locations: malignant neoplasms of the lip, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of the mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female genital organs, including ovarian cancer; malignant neoplasms of the male genital organs, including prostate cancer.

16. The pharmaceutical composition of claim 15, wherein the cancer is selected from the group consisting of malignant neoplasms; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; ill-defined, secondary, and unspecified site malignant neoplasms; malignant neoplasms of lymphatic, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; and neoplasms of uncertain or unknown behavior, including myelodysplastic syndromes.

17. 19. Use of one or more of the fusion protein of any one of claims 1 to 3 and 7, a cell expressing the fusion protein of any one of claims 1 to 3 and 7, a nucleic acid of claim 10, a vector of claim 12, a viral particle of claim 13, and a pharmaceutical composition of claim 15 in the manufacture of a medicament useful for preventing or slowing the progression of a p53-mediated disease in a subject.

18. The use of claim 17, wherein the p53-mediated disease is cancer or other neoplasm.

19. The cancer or neoplasm is a malignant neoplasm declared or presumed to be primary in the following locations: malignant neoplasms of the lip, oral cavity, and pharynx, including head and neck cancer; malignant neoplasms of the digestive tract, including esophageal, colon, liver, or pancreatic cancer; malignant neoplasms of the respiratory and intrathoracic organs, including lung cancer; malignant neoplasms of bone and articular cartilage, including osteosarcoma; melanoma and other malignant neoplasms of the skin; malignant neoplasms of the mesothelium and soft tissue, including sarcoma; malignant neoplasms of the breast; malignant neoplasms of the female reproductive organs, including ovarian cancer; malignant neoplasms of the male reproductive organs, including prostate cancer.

19. The use of claim 18, wherein the cancer is selected from the group consisting of malignant neoplasms of the organs; malignant neoplasms of the urinary tract, including bladder cancer; malignant neoplasms of the eye, brain, and other parts of the central nervous system, including glioblastoma; malignant neoplasms of the thyroid and other endocrine glands, including thyroid cancer; ill-defined, secondary, and unspecified site malignant neoplasms; malignant neoplasms of lymphoid, hematopoietic, and related tissues, including multiple myeloma, lymphocytic leukemia, or myeloid leukemia; and neoplasms of indeterminate or unknown behavior, including myelodysplastic syndromes.

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