Compositions and methods for the treatment and / or prevention of a disease or condition linked to oxidative stress

Abstract

The present invention discloses compositions, such as pharmaceutical compositions, as well as methods, for use in the treatment and / or prevention of a disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis. The pharmaceutical composition of the invention comprises an agent modulating the expression and / or activity of i) a KRAB- containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding the ZNF354A, and / or iii) the ZNF354A gene.

Description

[0001] Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0002] Compositions and Methods for the treatment and / or prevention of a disease or condition linked to oxidative stress

[0003] FIELD OF THE INVENTION

[0004] The present invention discloses compositions, such as pharmaceutical compositions, as well as methods, for use in the treatment and / or prevention of a disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis. The pharmaceutical composition of the invention comprises an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding the ZNF354A, and / or iii) the ZNF354A gene.

[0005] BACKGROUND OF THE INVENTION

[0006] Oxidative stress results from an imbalance between the production and detoxification of oxidizing free radicals. Excessive ROS results in damage of cellular components such as lipids and DNA, which leads to cell death and tissue dysfunction (Sies and Jones, 2020).

[0007] The Inventors have identified the Krüppel-associated box (KRAB) domain zinc finger protein (KZFP) ZNF354A as a key repressor of genes engaged in defence against oxidative stress, lipid peroxidation and / or ferroptosis, a form of ROS- and iron-dependent cell death. Depletion of ZNF354A resulted in up-regulation of anti oxidative pathways and protection of cells against ROS-induced damage.

[0008] This modulation can be therapeutically exploited to treat any disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding the ZNF354A, and / or iii) the ZNF354A gene, for use in the treatment and / or prevention of a disease linked to oxidative stress, lipid peroxidation and / or ferroptosis.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0011] The present invention further provides a short interfering ribonucleic acid (siRNA) for inhibiting the expression of ZNF354A.

[0012] The present invention further provides a short hairpin ribonucleic acid (shRNA) for inhibiting the expression of ZNF354A.

[0013] The present invention further provides a guide ribonucleic acid (sgRNA or gRNA) targeting i) one or more sequences within the gene encoding ZNF354A (chromosome 5: 178730659-178711512) as set forth in SEQ ID NO: 17, ii) one or more sequences within the promoter region controlling the gene encoding ZNF354A (chromosome: 5, 178728826: 178731484) as set forth in SEQ ID NO: 32, and / or iii) one or more DNA sequences selected from the group of sequences set forth in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, or a fragment or variant of any one of these sequences.

[0014] The present invention further provides one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and antisense oligonucleotide (ASO, such as e.g. Gapmer antisense), or combination of one or more thereof, as described herein.

[0015] The present invention further provides a plasmid or a vector comprising one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and antisense oligonucleotide (e.g. Gapmer antisens), or combination thereof, of the invention.

[0016] The present invention further provides a cell comprising, or modified by the introduction of, i) a plasmid or vector of the invention, or ii) one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and antisense oligonucleotide, or combination thereof, of the invention.

[0017] The present invention further provides a method for prolonging the survival and / or functional persistence of a cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif, the method comprising contacting said cell, or population ofRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0018] cells, ex vivo and / or in vivo, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids,

[0019] wherein said one or more nucleic acids inhibit the expression and / or activity of: i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A),

[0020] ii) an mRNA encoding ZNF354A, and / or

[0021] iii) the ZNF354A gene,

[0022] and wherein inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis in the contacted cell, or contacted population of cells, thereby prolonging their survival and / or functional persistence.

[0023] The present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding said ZNF354A, and / or iii) the ZNF354A gene, of the invention, or

[0024] i) a plasmid or a vector of the invention, ii) a siRNA of the invention, iii) a gRNA of the invention, or iv) a cell of the invention,

[0025] and a pharmaceutically acceptable carrier or diluent.

[0026] The present invention further provides an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding said ZNF354A, and / or iii) the ZNF354A gene, for modulating cellular responses to oxidative stress.

[0027] The present invention further provides a method for prolonging the survival and / or functional persistence of a cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif, the method comprising contacting said cell, or population of cells, ex vivo and / or in vivo, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids,

[0028] wherein said one or more nucleic acids inhibit the expression and / or activity of: i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A),

[0029] ii) an mRNA encoding ZNF354A, and / or

[0030] iii) the ZNF354A gene,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0031] and wherein inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis in the contacted cell, or contacted population of cells, thereby prolonging their survival and / or functional persistence.

[0032] The present invention further provides a method of treatment and / or prevention of a cancer, a cardiovascular disease, a neurodegenerative disease or an inflammatory disease, in a subject in need thereof, the method comprising administering a pharmaceutical composition of the invention to the subject in need thereof.

[0033] The present invention further provides a method of treatment and / or prevention of a cancer in a subject in need thereof, the method comprising

[0034] i) removing and isolating cells, or population of cells, preferably immune cells, more preferably native T cells, from said subject,

[0035] ii) genetically engineering said T cells with one recombinant construct (e.g. vector, plasmid or polynucleotide) encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif,

[0036] iii) expanding ex vivo into a larger population of engineered immune cells, or population of immune cell, e.g. T cells, in the presence of an interleukin selected from the group comprising IL-7, IL- 15 and / or IL-2,

[0037] iv) contacting said immune cells, or population of immune cells, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids

[0038] (iv) reintroducing said engineered immune cells, or population of immune cells, e.g. T cells, into the subject in need thereof.

[0039] The present invention further provides a method of improving transplant engraftment, survival, and / or functional persistence of stem cell transplants in a subject in need thereof, the method comprising

[0040] i) removing and isolating cells, or population of cells, preferably stem cells, from said subject, (ii) ex-vivo contacting said stem cells, or population of stem cells, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids,

[0041] (iii) reintroducing said engineered stem cells, or population of stem cells, into the subject in need thereof,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0042] wherein said nucleic acid inhibits the expression and / or activity of ZNF354A, thereby reducing oxidative stress, lipid peroxidation, and / or ferroptosis in the stem cells and improving transplant engraftment, survival, and / or functional persistence.

[0043] The present invention further provides a method of detecting a disease linked to an over-activation of anti oxidative pathways in a subject, the method comprising

[0044] i) detecting the level of phosphorylation of ZNF354A at serine 169,

[0045] ii) comparing the level of phosphorylation of ZNF354A at serine 169in said subject with a standard control,

[0046] wherein a high level of phosphorylation of of ZNF354A at serine 169 in said subject relative to said standard control indicates said subject suffers from a disease linked to an overactivation of antioxidative pathways.

[0047] DESCRIPTION OF THE FIGURES

[0048] Figure 1. Differential regulation of the acyl-transferase ZDHHC20. (A) WB of ZDHHC20, GAPDH (loading control), N (viral control) showing Short-S (42 KDa) and 20L (~49, 62 and 69 KDa) ZDHHC20 isoforms from lysates of: A. infected primary human airway epithelial cells (SARS-CoV-2, MOI 0.1); B. colon tissues, from mice treated or not with DSS for 7 days followed by 3 days recovery; C. Vero E6 cells treated 1 h with 10 ng / ml of proaerolysin at 37 °C, washed, and further incubated at 37 °C for the indicated time D. primary human airway epithelia from individuals with different ages or infected with SARS-CoV-2 MOI = 0.1, 48 h E. a panel of cancer cell lines. Calu-3 cells infected as in A for 24 h were used as a comparison.

[0049] Figure 2. Differential expression of Zdhhc20 isoforms is epigenetically regulated. A. Overview of the zdhhc20 locus (first intron, exon, and 5’UTR - version hgl9), Tracks indicate: in-frame transcription start sites (ATGs); transcript (GENCODE-V4 0lift37), various histone modifications - grey shade ChlP-seq signals (ENCODE / Broad); and SETDB1 / KAP1 ChlP-seq (ENCODE) peaks from the indicated cell types. B. WB of ZDHHC20 and GAPDH (control) in U2OS cells siControl (siCtrl), siSETDBl or siKAPl -depl eted for 72 h; and correspondent quantification of 20L band shifts. Results are mean ± SEM; each dot represents one independent experiment of n = 6; P values comparing to siCtrl were obtained by two-wayRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0050] ANOVA, Dunnet’s multiple comparison. C. same as in B in HAP-1 wild-type or knockout for KAP1.

[0051] Figure 3. ZNF354A acts as repressor on an (alternative) upstream zdhhc20 promoter. A. Overview of the zdhhc20 locus (first intron, exon, and 5’UTR - version hgl9), Tracks indicate ChlP-seq peaks for: SETDB1 / KAP1 (ENCODE - U2OS and K562 cells), ZNF354A and all detected KZFP bindings (HEK293T) B. WB of ZDHHC20 and GAPDH (control) in U2OS cells siControl (siCtrl), or depleted for the indicated proteins for 72 h.

[0052] Figure 4. p38 and JNK kinases are involved in activation of the LORD pathway A-C: H₂O₂ treatment triggers phosphorylation of all three components of the repressive complex, ATF2 (Thr-69), KAP1 (Ser-473) and ZNF354A (Ser-169). An antibody was generated to specifically recognized the Ser-169 phosphorylated form of ZNF354A, with the aim of using the Ser- 169 phosphorylated form of ZNF354A as a bio marker, potentially to stratify patients. D-E: Phosphorylation of all three component as well as ZDHHC20L expression are p38- and JNK-dependent. ATM inhibition had a mild negative effect on the phosphorylation of all three components (more pronounced for KAP1) and moderately diminished the expression of ZDHHC20L.

[0053] Figure 5. ZNF354A regulates cell sensitivity to ferroptosis Cell viability was monitored by measuring ATP levels, following RSL3 or H2O2 treatment. Depletion of ZNF354A by siRNA led to a marked increase in viability both in response to RSL3 and H2O2 (Fig 5A). Given that silencing ZNF354A expression protects cells from ferroptosis, the inventors tested whether overexpression would sensitize them. To test this, an inactive ZNF354A mutant was designed by mutating the ZFP-KRAB-A box of ZNF354A, a well-conserved interaction domain of KZFP with KAP1. Significant cell death could be observed starting at 48 hours of ZNF354AWT, but not ZNF354AMut, overexpression (Fig 5B). Cell viability could be rescued by ferrostatin-1 (an inhibitor of ferroptosis) (Fig. 5B). Thus, ZNF354AWT overexpression leads to cell death induced by the accumulation of lipid peroxides.

[0054] Figure 6. ZNF354A depletion increases human T lymphocytes viability.

[0055] A. T cells were activated with a cocktail of IL15 and IL7 and cells were transduced or not with a lentiviral vector expressing shRNA constructs against ZNF354A. Two different shRNAs were tested shRNA1 and shRNA2, leading to approx. 60% knockdown compared to shRNA control. mRNA levels were measured by RT-qPCR. B T lymphocytes were obtained from 2Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0056] different donors from which Peripheral Blood Mononuclear Cells (PBMCs) were isolated. T cells were activated on dayl as in A and transduced with a lentiviral vector expressing shRNA constructs against ZNF354A (shRNA1 and shRNA2) or shRNA Control (Ctrl). Live cells were counted at different days.

[0057] Figure 7. ZFP354A depletion protects mouse neurons from oxidative stress Mouse hippocampal neurons were cultivated over a layer of microglial cells for 5 days. Cells were incubated (Fig. A, C) or not (Fig. B, D) with a cocktail of antioxidant Agents OA (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione). At day 5, cells were subjected to Zfp354A knockdown by transduction with lentiviral vector expressing shRNA constructs against mouse Zfp354A (shA or shB) or to a control shRNA (shCtrl). A non-transduced control was also tested. At day 13, cells were imaged and the number of neurites were monitored. Neuronal Soma were labelled with NEUN antibodies, neurites were labelled with MAP2 antibodies, and nuclei of neurons and glial cells with DAPI. C-D. The number of neurites formed at Day 13 was calculated per field of view (9 per well). Number of well per condition: 18.

[0058] DESCRIPTION OF THE INVENTION

[0059] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0060] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0061] The term "comprise / comprising" is generally used in the sense of include / including, that is to say permitting the presence of one or more features or components. The terms "comprise(s)" and "comprising" also encompass the more restricted ones "consist(s)" and "consisting", respectively.

[0062] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0063] As used herein, "at least one" means "one or more", "two or more", "three or more", etc.

[0064] As used herein the terms "subject" / " subject in need thereof, or "patient" / "patient in need thereof " are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other aspects, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Preferably, the subject is a human, most preferably a human suffering from a disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis. Preferably, the disease is linked, directly or indirectly, to the expression of ZNF354A.

[0065] The terms "nucleic acid", "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any kind of deoxyribonucleotide (e.g. DNA, cDNA,...) or ribonucleotide (e.g. RNA, mRNA,...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g. DNA / RNA) polymer, in linear or circular conformation, and in either single - or double - stranded form. These terms are not to be construed as limiting with respect to the length of a polymer and can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (e.g. phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity, i.e., an analogue of A will base-pair with T.

[0066] The term nucleotides in the context of the present invention includes the classic ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymin), cytidine, the classic deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogues of nucleic acids such as phosphotioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked byRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0067] peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2'O, 4'C methylene bridged RNA building blocks). The hybridizing sequence may be composed of any of the above nucleotides, or mixtures thereof.

[0068] The term "vector", as used herein, refers to a viral vector or to a nucleic acid (DNA or RNA) molecule such as a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequence(s) of the invention and, preferably, is designed for transfer between different host cells. The terms "expression vector", “gene delivery vector” and "gene therapy vector" refer to any vector that is effective to incorporate and express one or more nucleic acid(s) of the invention, in a cell, preferably under the regulation of a promoter. In some aspect, the promoter is an inducible promoter, where appropriate. A cloning or expression vector may comprise additional elements, for example, regulatory and / or post-transcriptional regulatory elements in addition to a promoter.

[0069] The term "modulating" refers to i) increasing or enhancing, or ii) decreasing, reducing or inhibiting, depending on the context and the disease to be treated or prevented.

[0070] In particular, "modulating" can refer to increasing or enhancing the expression and / or activity of a ZNF354A protein by at least 5%, preferably at least 10%, 20%, 30%, 50%, 75%, 90% or more, relative to a suitable control.

[0071] Alternatively, "modulating" can refer to reducing or inhibiting the expression and / or activity of a ZNF354A protein by at least 5%, preferably at least 10%, 20%, 30%, 50%, 60%, 75%, 90% or more, relative to a suitable control. Such inhibition need not be complete; partial inhibition, for example of about 60% or more, about 50% or more, about 40% or more, about 30% or more, is expressly encompassed.

[0072] The term "about" particularly in reference to a given quantity, number or percentage, is meant to encompass deviations of plus or minus ten percent (± 10). For example, about 5% encompasses any value between 4.5% to 5.5%, such as 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, or 5.5%.

[0073] According to the present invention, the disease to be treated is a disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis. Preferably, the disease is linked, directly or indirectly, to the expression of ZNF354A. More preferably, the disease is selected from the group comprising a cancer, a cardiovascular disease, a neurodegenerative disease and anRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0074] inflammation related or inflammatory disease, or a combination of one of more of these diseases.

[0075] In one aspect of the invention, the disease is cancer, preferably a solid or a non-solid (liquid or hematologic) cancer. The solid cancer will be selected from the non-limiting group comprising carcinoma, sarcoma, and melanoma,. Preferably, the cancer is selected from the group comprising Adrenocortical Carcinoma, Adult, Childhood Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma), Anal Cancer, Astrocytomas, Childhood (Brain Cancer), Atypical Teratoid / Rhabdoid Tumor, Childhood, Central Nervous System (Brain Cancer), Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Childhood Breast Cancer, Childhood Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor (Gastrointestinal), Childhood Carcinoid Tumors, Carcinoma of Unknown Primary, Cardiac (Heart) Tumors, Central Nervous System, Childhood Atypical Teratoid / Rhabdoid Tumor, Childhood Embryonal Tumors, Childhood Germ Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Childhood Cervical Cancer, Cholangiocarcinoma, Chordoma, Childhood, Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Childhood Colorectal Cancer, Childhood Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Embryonal Tumors, Endometrial Cancer (Uterine Cancer), Childhood Ependymoma, Esophageal Cancer, Childhood Esophageal Cancer, Esthesio neuroblastoma, Ewing Sarcoma (Bone Cancer), Childhood Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Childhood Intraocular Melanoma, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone (Malignant, and Osteosarcoma), Gallbladder Cancer, Gastric, (Stomach) Cancer, Childhood Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST) (Soft Tissue Sarcoma), Childhood Gastrointestinal Stromal Tumors, Germ Cell Tumors, Childhood Central Nervous System Germ Cell Tumors (Brain Cancer), Childhood Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, Testicular Cancer, Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Childhood Head and Neck Cancers, Childhood Heart Tumors, Hepatocellular (Liver) Cancer, Langerhans Cell Histiocytosis, Hodgkin Lymphoma, Hypopharyngeal Cancer (Head and Neck Cancer), Intraocular Melanoma, Childhood Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma (Soft Tissue Sarcoma), KidneyRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0076] (Renal Cell) Cancer, Langerhans Cell Histiocytosis, Laryngeal Cancer (Head and Neck Cancer), Childhood Laryngeal Cancer, Papillomatosis, Leukemia, Lip and Oral Cavity Cancer (Head and Neck Cancer), Liver Cancer, Lung Cancer (Non-Small Cell and Small Cell), Childhood Lung Cancer, Male Breast Cancer, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Childhood Melanoma, Intraocular (Eye)Melanoma, Childhood Intraocular Melanoma, Merkel Cell Carcinoma (Skin Cancer), Malignant Mesothelioma, Childhood Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary (Head and Neck Cancer), Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer (Head and Neck Cancer), Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides (Lymphoma), Myelodysplasia Syndromes, Myelodysplastic / Myeloproliferative Neoplasms, Chronic Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer (Head and Neck Cancer), Nasopharyngeal Cancer (Head and Neck Cancer), Childhood Nasopharyngeal Cancer, Neuroblastoma, Non-Small Cell Lung Cancer, Oral Cancer, Lip and Oral Cavity Cancer and Oropharyngeal Cancer (Head and Neck Cancer), Childhood Oral Cavity Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Childhood Ovarian Cancer, Pancreatic Cancer, Childhood Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Childhood Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer (Head and Neck Cancer), Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer (Head and Neck Cancer), Pheochromocytoma, Childhood Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm / Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Recurrent Cancer, Renal Cell (Kidney) Cancer, Retinoblastoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Salivary Gland Cancer (Head and Neck Cancer), Childhood Salivary Gland Tumors, Sarcoma, Childhood Rhabdomyosarcoma (Soft Tissue Sarcoma), Childhood Vascular Tumors (Soft Tissue Sarcoma), Ewing Sarcoma (Bone Cancer), Kaposi Sarcoma (Soft Tissue Sarcoma), Osteosarcoma (Bone Cancer), Uterine Sarcoma, Sezary Syndrome (Lymphoma), Skin Cancer, Childhood Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer with Occult Primary, Metastatic (Head and Neck Cancer), Stomach (Gastric) Cancer, Childhood Stomach (Gastric) Cancer, Cutaneous, Testicular Cancer, Childhood Testicular Cancer, Throat Cancer (Head and Neck Cancer), Nasopharyngeal Cancer, Oropharyngeal Cancer, Hypopharyngeal Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Childhood Thyroid Tumors, TransitionalRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0077] Cell Cancer of the Renal Pelvis and Ureter (Kidney (Renal Cell) Cancer), Carcinoma of Unknown Primary, Childhood Cancer of Unknown Primary, Unusual Cancers of Childhood, Ureter and Renal Pelvis, Transitional Cell Cancer (Kidney (Renal Cell) Cancer, Urethral Cancer, Endometrial Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Childhood Vaginal Cancer, Vascular Tumors (Soft Tissue Sarcoma), Vulvar Cancer, and Wilms Tumor or a combination of two of more of these cancers.

[0078] The liquid or non-solid cancer comprises hematologic cancers that are malignancies that affect the blood, bone marrow, lymphatic system, and related tissues. The hematologic cancer will be selected from the non-limiting group comprising leukemias, such as Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), and Chronic Myeloid Leukemia (CML); lymphomas, including Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL) subtypes like Diffuse Large B-Cell Lymphoma (DLBCL), Follicular Lymphoma, Mantle Cell Lymphoma, Burkitt Lymphoma, Mucosa- Associated Lymphoid Tissue (MALT) Lymphoma, and Peripheral T-cell Lymphomas; myeloproliferative neoplasms like Chronic Myelogenous Leukemia (CML), Polycythemia Vera (PV), Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF), and Chronic Eosinophilic Leukemia (CEL); myelodysplastic syndromes (MDS) such as Refractory Anemia and Chronic Myelomonocytic Leukemia (CMML); plasma cell disorders, including Multiple Myeloma, Waldenström Macroglobulinemia, Monoclonal Gammopathy of Undetermined Significance (MGUS), and Amyloidosis; and other hematologic malignancies like Histiocytic Sarcoma, Hemophagocytic Lymphohistiocytosis (HLH), and Adult T-cell Leukemia / Lymphoma (ATLL), or a combination of two or more of these cancers.

[0079] In one aspect of the invention, the disease is a cardiovascular disease. Non-limiting examples of cardiovascular diseases are selected from the group comprising hypertension, ischaemia, reperfusion injury, stroke including ischemic stroke, myocardial infarction including recurrent myocardial infarction, congestive heart failure, embolism, abdominal aortic aneurism, cardia fibrosis, and pericarditis including Dressler's syndrome or a combination of two or more of them.

[0080] In one aspect of the invention, the disease is a neurodegenerative disease affecting the central nervous system (CNS) or the peripheral nervous system (PNS).Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0081] As shown in Example 4 and figure 7, inhibition of ZFP354A in neurons promotes neuronal outgrowth independently of exogenous antioxidant support. The data strongly support the therapeutic potential of ZNF354A blockade in neurodegenerative diseases as well as in other pathological conditions in which oxidative stress plays a central role, including spinal cord injury and retinal dystrophies.

[0082] Non-limiting examples of neurodegenerative diseases affecting the central nervous system diseases are selected from the group comprising Parkinson's disease, Friedreich ataxia, multiple sclerosis, Alzheimer's disease, dementia, motor neuron disease, Amyotrophic lateral sclerosis (ALS) disease, Huntington's disease, cerebral malaria, brain injury from pneumococcal meningitis, intracranial aneurysms, and traumatic brain injury or a combination of two or more of them.

[0083] Non-limiting examples of neurodegenerative diseases affecting the peripheral nervous system diseases are selected from the group comprising a demyelinating and / or dysmyelinating PNS disease or associated disorder.

[0084] In one aspect of the invention, the disease is an inflammatory disease. Non-limiting examples of inflammatory diseases are selected from the group comprising anaphylaxis, septic shock, septic arthritis, rheumatoid arthritis, psoriatic arthritis, asthma, delayed type hypersensitivity, dermatitis, diabetes mellitus, juvenile onset diabetes, graft rejection, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, enteritis, interstitial cystitis, multiple sclerosis, myasthemia gravis, Grave's disease, Hashimoto's thyroiditis, pneumonitis, nephritis, pneumonitis, chronic obstructive pulmonary disease, chronic bronchitis, chronic bronchitis rhinitis, spondyloarthropathies, scleroderma, and systemic lupus erythematosus, chronic hepatitis and psoriasis, or a combination of two or more of them.

[0085] The terms " KRAB-containing zinc finger proteins", " KZFP", " KRAB-ZnF", and " KRAB-ZFP" are used interchangeably herein to refer to KRAB-containing zinc finger proteins (KZFPs) that constitute a large family of transcription factors notably involved in controlling the expression of transposable element-embedded regulator sequences (TEeRS) (de Tribolet-Hardy et al., 2023; Imbeault et al., 2017). KZFPs and their genomic targets contributeRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0086] to regulating many biological events, from early embryogenesis to brain development, and from immune responses to liver metabolism.

[0087] As used herein, the term “variant” of one or more nucleic acid sequence or polypeptide sequence of the invention refers to biologically active derivatives of said respective sequences. In general, the term “variant” refers to molecules having a native sequence and structure with one or more additions, substitutions (generally conservative in nature) and / or deletions (e.g. splice variants), relative to the native molecule, so long as the modifications do not destroy biological activity and which are “substantially homologous” to the reference molecule or sequence. In general, the sequences of such variants are functionally, i.e. biologically active variants and will have a high degree of sequence homology or identity to the reference sequence, e.g., sequence homology or identity of more than 50%, generally more than 60%-70%, even more particularly 80%-85% or more, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99% homology or identity to the reference sequence, when the two sequences are aligned.

[0088] As used herein, a “fragment” of one or more nucleic acid sequence or polypeptide sequence of the invention refers to a biologically active sequence containing less nucleotides or amino acids in length than the respective sequences of the invention while retaining the biological activity described herein. Preferably, this fragment contains, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid sequence or polypeptide sequence.

[0089] A “biologically active sequence” of one or more nucleic acid sequence or polypeptide sequence of the invention refers to a nucleic acid sequence or an amino acid sequence which, when present in a biological system, reduces, suppresses, or prevents the biological activity of a target molecule, pathway, or cellular process described herein. A person of ordinary skills in the art can readily test the inhibitory effect using routine assays known in the field.

[0090] While focusing on the role of KZFP-mediated regulatory circuits in controlling specific gene networks across a vast array of biological processes, the Inventors surprisingly discovered the role of ZNF354A in the defence pathway to Reactive oxygen species (ROS)-triggered lipid damage.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0091] ZNF354A is a member of the largest family of mammalian transcriptional modulators, the Krüppel-associated box (KRAB) domain zinc finger protein (KZFP). ZNF354A is part of a complex containing the global repressor KAP1 (TRIM28), the histone methyltransferase SETDB1, and the transcriptional activator ATF2 (Watson et al., 2017). Under homeostatic conditions, ZNF354A represses transcriptional programs involved in antioxidant defenses and innate immune responses. Accumulation of lipid peroxides leads to the disassembly of the repressive complex, and the release of ZNF354A from specific DNA loci, allowing activation of the lipid damage response pathway. While highlighting the underexplored dynamics of epigenetic repressive complexes, the Inventors revealed how cells genetically manage oxidative lipid damage responses and identified potential targets for therapeutic intervention in a variety of conditions such as cancers, cardiovascular diseases, neurodegenerative diseases and inflammatory diseases.

[0092] This mechanism can be therapeutically exploited to treat any disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis (Stockwell et al., 2017; Yang et al., 2014; Zheng and Conrad, 2025)

[0093] 1- In inflammatory, metabolic, cardiovascular and degenerative conditions (such as neurodegenerative diseases), depleting or blocking ZNF354A can mitigate oxidative stress, protect cells from ROS-induced damage, and diminish inflammation.

[0094] 2- In immunotherapies, depleting or blocking ZNF354A can enhance the persistence and cytotoxic ability of engineered immune cells (e.g. CAR-T cells). T cells indeed tend to dye, through so-called exhaustion, which is thought to be due to oxidative stress. Silencing or blocking ZNF354A would pre-trigger the LORD pathway, and thereby render cells more resilient to oxidative damage.

[0095] 3- In cancer, activating or increasing the production of ZNF354A can sensitize cancer cells to oxidative stress and ferroptosis, increasing for instance their susceptibility to radiochemotherapies.

[0096] 4. In retinopathies

[0097] The present invention thus provides an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein 354 A (ZNF354A), ii) an mRNA encoding said ZNF354A, and / or iii) the ZNF354A gene.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0098] Preferably, such an agent is for use in the treatment and / or prevention of a disease. More preferably, the disease is a disease or condition linked to oxidative stress, lipid peroxidation and / or ferroptosis. Preferably, the disease is linked, directly or indirectly, to the expression of ZNF354A. More preferably, the disease is selected from the group comprising cancer, cardiovascular disease, neurodegenerative disease and inflammation or inflammatory disease, or a combination of two or more of these diseases.

[0099] Where the disease to be treated is selected from the group comprising a cardiovascular disease, a neurodegenerative disease and an inflammatory disease, or a combination of two or more of these diseases, the agent will preferably inhibit the expression and / or activity of i) the ZNF354A protein,

[0100] ii) an mRNA encoding the ZNF354A, and / or

[0101] iii) the ZNF354A gene.

[0102] Preferably, the agent will be selected from the group comprising a nucleic acid, a chemical compound, a peptide or analog thereof, an antibody or an antigen-binding fragment thereof, and an antibody mimetic, or a combination of two or more thereof.

[0103] In one aspect, the agent inhibits the translation of one or more mRNA encoding ZNF354A.

[0104] In another aspect, the agent inhibits the transcription of the gene encoding ZNF354A.

[0105] In another aspect, said agent modulates the activity of a ZNF354A protein i) by enhancing and / or favoring the degradation of a ZNF354A protein, ii) by preventing the recognition of its targets, iii) by modulating the phosphorylation of a ZNF354A protein, and / or iv) by any other means resulting in a loss of function.

[0106] Examples of ZNF354A protein sequences are selected from the group of sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, or a fragment or variant of any one of these sequences.

[0107] In one aspect, the agent of the invention enhances and / or favors the degradation of said ZNF354A protein by inducing the ubiquitination and proteasomal degradation of said ZNF354A protein or degradation by autophagy. Examples of chemical agents inducingRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0108] selective intracellular proteolysis comprise proteolysis targeting chimera (PROTAC) protein degraders and small-molecule chemical modulators of deubiquitinating enzymes upstream of or on the proteasome. As is known in the art, PROTACs are heterobifunctional small molecules composed of two active domains and a linker capable of removing specific unwanted proteins (Békéset al., 2022). Any other targeted protein degradation (TPD) strategies including, but not limited to, molecular glue, Lysosome-Targeting Chimaera (LYTAC), and Antibody-based PROTAC (AbTAC), is also encompassed in the present invention (Zhao et al., 2022).

[0109] In other aspects, the agent of the invention is a nucleic acid that inhibits and / or impairs the binding of a ZNF354A protein to a target comprising a binding motif. In one aspect, the target DNA comprises a ZNF354A binding motif selected from the group of sequences set forth in SEQ ID NO: 46, 47, 48, 49, 50, and 51, or a fragment or variant of any one of these sequences. In other aspects, the agent of the invention is an antibody, or an antigen binding fragment, that inhibits and / or impairs the binding of a ZNF354A protein to a target genomic DNA (e.g. by specifically targeting the zinc-finger motif of the C2H2 zinc finger motif or by specifically binging to an epitope encompassing the Ser-169 phosphorylation site), and / or the binding of the ZNF354A protein to its interacting protein(s) or other molecule(s). In an aspect, the antigen binding fragment is an antigen binding fragment of an antibody.

[0110] As used herein, an “antibody” is a protein molecule that reacts with a specific antigenic determinant or epitope and belongs to one or five distinct classes based on structural properties: IgA, IgD, IgE, IgG and IgM. The antibody may be a polyclonal (e.g. a polyclonal serum) or a monoclonal antibody, including but not limited to fully assembled antibody, single chain antibody, antibody fragment, an antibody-drug conjugate and chimeric antibody, humanized antibody as long as these molecules are still biologically active and still bind to at least one peptide or protein of the invention. Preferably the antibody is a monoclonal antibody. Preferably also the monoclonal antibody will be selected from the group comprising IgGl, IgG2, IgG2a, IgG2b, IgG3 and IgG4 or a combination thereof. Most preferably, the monoclonal antibody is selected from the group comprising IgGl, IgG2, IgG2a, and IgG2b, or a combination thereof.

[0111] The term “binding fragment,” which can be used interchangeably with “antigen-binding fragment,” refers herein to an antibody fragment formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that specifically binds to anRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0112] antigen but does not comprise an intact native antibody structure. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab', a F(ab')2, a F(ab)c, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a triabody, a tetrabody, a single-chain antibody molecule (scFv), an scFv dimer, a multispecific antibody, a camelized single domain antibody, a nanobody, a minibody, a domain antibody, a bivalent domain antibody, a IgNAR, a V-NAR, and a hcIgG. Typically, binding fragments compete with the intact antibody from which they were derived for specific binding.. Binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

[0113] In some aspects, the binding fragment refers to a peptide aptamers consisting of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

[0114] In other aspects, the agent of the invention is dephosphorylating agent that inhibits and / or prevents the phosphorylation of the ZNF354A protein as shown herein. Non limiting examples are selected from the group comprising p38, JNK and ATM kinases.

[0115] Where the agent is a nucleic acid, said nucleic acid will be selected from the group comprising a nucleic acid encoding an siRNA, an miRNA, a piRNA, an hnRNA, an snRNA, a gRNA, a CRISPR-based loss-of-function system, an esiRNA, an shRNA, an aptamer and an antisense oligonucleotide (ASO), or a combination of two or more thereof.

[0116] The terms “siRNA” and “short interfering RNA” are interchangeable and refer to singlestranded or double-stranded RNA molecules that are capable of inducing RNA interference. siRNA molecules typically have a duplex region that is between 16 and 30, between 17 and 28, between 20 and 25, between 22 and 25 base pairs in length.

[0117] In one aspect, the siRNA may be 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length or base pairs in length.

[0118] Double stranded siRNA is usually derived from longer double stranded RNA molecules (dsRNA). The long dsRNA is cleaved by an endo-ribonuclease (called Dicer) to form double stranded siRNA. In a nucleoprotein complex (called RISC), the double stranded siRNA isRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0119] unwound to form single stranded siRNA. RNA interference often works via binding of an siRNA molecule to an mRNA molecule having a complementary sequence, resulting in degradation of the mRNA. RNA interference is also possible by binding of an siRNA molecule to an intronic sequence of a pre-mRNA (an immature, non-spliced mRNA) within the nucleus of a cell, resulting in degradation of the pre-mRNA.

[0120] In certain aspects, the siRNA is at least 80% homologous to at least 2 to 30 contiguous nucleotides of the ZNF354A mRNA sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and a SEQ ID NO: 15, a fragment or a variant thereof

[0121] In certain aspects, the siRNA is characterized by a sequence reverse complementary to a sequence formed by juxtaposing any two consecutive sequences of the sequences defined above.

[0122] The term "siRNA" also encompasses single-stranded or double-stranded RNA molecules comprising one or more modified nucleotides i.e. analogues of nucleic acids. Non-limiting examples of modified nucleotides are selected from the group comprising phosphotioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2'O, 4'C methylene bridged RNA building blocks). When reference is made herein to a sequence comprising or consisting of a number of nucleotides that are not shown to be modified in that sequence, the reference also encompasses the same nucleotide sequence in which one, several, such as two, three, four, five, six, seven or more, including all, nucleotides are modified by modifications such as 2’-0Me, 2’-F, or have a 3’ end or 5’ end modification or any other modification.

[0123] In one aspect, if the 5 ’-most nucleotide of the first strand of a nucleic acid is a nucleotide other than A or U, this nucleotide is replaced by an A or U. Preferably, if the 5 ’-most nucleotide of the first strand is a nucleotide other than a U, this nucleotide is replaced by U, and more preferably by U with a 5’ (E)-vinylphosphonate, in the sequence.

[0124] In some aspects, the siRNA of the invention is conjugated to a ligand, optionally via a linker. Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids, to cells in vivo is important and requires specific targeting and substantial protection from theRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0125] extracellular environment, preferably serum proteins. One method of achieving specific targeting is to conjugate a ligand to the nucleic acid. In some aspects, the ligand helps in targeting the nucleic acid to a target cell which has a cell surface receptor that binds to and internalises the conjugated ligand. In such aspects, there is a need to conjugate appropriate ligands for the desired receptor molecules in order for the conjugated molecules to be taken up by the target cells by mechanisms such as different receptor-mediated endocytosis pathways or functionally analogous processes. In other aspects, a ligand which can mediate internalization of the nucleic acid into a target cell by mechanisms other than receptor mediated endocytosis may alternatively be conjugated to a nucleic acid of the invention for cell or tissue specific targeting.

[0126] When the conjugated nucleic acid has a first strand (antisense strand) and a second strand (sense strand), the ligand can be conjugated to either one of the strands, preferably at the end of a strand, more particularly to the last nucleotide at one end of a strand. The ligand is preferably conjugated to the ribose of a nucleotides, preferably the last of a strand. The ligand can be conjugated to the ribose of a nucleotide, preferably the last on in a strand, via the 2’, 3’ or 5’ carbon of the ribose. Preferred are the 3’ or 5’ carbon. The ligand is preferably not conjugated to the 5’ end of the first strand. The ligand is preferably conjugated to the 5’ end of the second strand.

[0127] The ligand is preferably conjugated to the last nucleotide of a nucleic acid strand, preferably to the ribose moiety. The ligand can be conjugated to the first strand (the antisense strand) or the second strand (the antisense strand). Preferably, the ligand is conjugated to a nucleotide at the end of one of the strands of the nucleic acid, more preferably the nucleotide at the 3’ or 5’ end of the second (sense) strand.

[0128] Generally, the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).

[0129] Non-limiting examples of a ligand comprise N-acetyl galactosamine (GalNAc), mannose, galactose, glucose, glucosamine and fucose. More preferably at least one N-acetyl galactosamine (GalNAc). For example, the asialoglycoprotein receptor complex (ASGP-R) which is highly abundant on hepatocytes, shows high affinity to GalNAc.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0130] Any suitable linker can be used to conjugate the nucleic acid, such as the siRNA, to the ligand. Non-limiting examples of linkers can be found, e.g. in PCT / EP2022 / 055455 (WO2022 / 174852) which content is incorporated herein.

[0131] The terms “microRNA,” “miRNA,” and “MiR” are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference.

[0132] The terms “piRNA” and “Piwi-interacting RNA” are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between about 26 and about 31 nucleotides in length.

[0133] Non-limiting examples of antisense oligonucleotides (ASOs) include the GapmeRs. As used herein, a GapmeR (also named Antisense LNA GapmeR) is a chimeric antisense oligonucleotide that contains a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. Usually, the GapmeRs of the invention are directed against one or more mRNA encoding the KZF of the invention at both pre-mRNA and / or mRNA levels. Modified ASOs such as modified GapmeRs are also encompassed in the present invention and comprise, e.g. GapmeRs with fixed chemical modification architectures selected from the group comprising i) a gapmer with five 2′-O-methoxyethyl (MOE) modifications in each flank, and a central gap of 10 unmodified dans (e.g. 5-10-5 MOE design), and ii) a gapmer employing three or four locked nucleic acid (LNA) modifications in each flank (e.g. 3-10-3 or 4-8-4 LNA designs), as well as a combination of two or more thereof.

[0134] Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.

[0135] The terms “sgRNA” and “guideRNA” are interchangeable and refer to a specific RNA sequence that recognizes the target DNA region of interest and directs the endonuclease there for editing. The gRNA is usually made up of two parts: crispr RNA (crRNA), a 17-20Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0136] nucleotide sequence (e.g. 17, 18, 19, or 20 nucleotides) complementary to the target DNA, i.e genomic DNA encoding the KZF of the invention or regulatory region controlling the genomic DNA encoding the KZF of the invention, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease.

[0137] Any suitable engineered sgRNA, or crRNA and tracrRNA, can be employed as long as it is effective for recognizing a target DNA of the invention. The design of such sgRNA, or crRNA and tracrRNA, is within the skill of ordinary artisans.

[0138] Non-limiting examples of target DNA sequences are selected from the group comprising: i) one or more sequences within the gene encoding ZNF354A (chromosome 5:

[0139] 178730659-178711512) as set forth in SEQ IDNO: 17,

[0140] ii) one or more sequences within the promoter region controlling the gene encoding ZNF354A (chromosome: 5, 178728826:178731484) as set forth in SEQ IDNO: 32, or

[0141] iii) one or more DNA sequences (i.e. ZNF354A binding motifs) selected from the group of sequences set forth in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, or a fragment or variant of any one of these sequences.

[0142] In certain aspects, the one or more sequences within the gene encoding ZNF354A are characterized by nucleotides 1-100, 101-200, 201-300, 301-400, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1501-1600, 1701-1800, 1801-1900, 1901-2000, 2001-2100, 2101-2200, 2201-2300, 2301-2400, 2501-2600, 2701-2800, 2801-2900, 2901-3000, 3001-3100, 3101-3200, 3201-3300, 3301-3400, 3401-3500, 3501-3600, 3601-3700, 3701-3800, 3801-3900, 3901-4000, 4001-4100, 4101-4200, 4201-4300, 4301-4400, 4401-4500, 4501-4600, 4601-4700, 4701-4800, 4801-4900, 4901-5000, 5001-5100, 5101-5200, 5201-5300, 5301-5400, 5401-5500, 5501-5600, 5601-5700, 5701-5800, 5801-5900, 5901-6000, 6001-6100, 6101-6200, 6201-6300, 6301-6400, 6401-6500, 6501-6600, 6601-6700, 6701-6800, 6801-6900, 6901-7000, 7001-7100, 7101-7200, 7201-7300, 7301-7400, 7401-7500, 7501-7600, 7601-7700, 7701-7800, 7801-7900, 7901-8000, 8001-8100, 8101-8200, 8201-8300, 8301-8400, 8401-8500, 8501-8600, 8601-8700, 8701-8800, 8801-8900, 8901-9000, 9001-9100, 9101-9200, 9201-9300, 9301-9400, 9401-9500, 9501-9600, 9601-9700, 9701-9800, 9801-9900, 9901-10000, 10001-10100,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0143] 10101-10200, 10201-10300, 10301-10400, 10401-10500, 10501-10600, 10601-10700, 10701-10800, 10801-10900, 10901-11000, 11001-11100, 11101-11200, 11201-11300, 11301-11400, 11401-11500, 11501-11600, 11601-11700, 11701-11800, 11801-11900, 11901-12000, 12001-12100, 12101-12200, 12201-12300, 12301-12400, 12401-12500, 12501-12600, 12601-12700, 12701-12800, 12801-12900, 12901-13000, 13001-13100, 13101-13200, 13201-13300, 13301-13400, 13401-13500, 13501-13600, 13601-13700, 13701-13800, 13801-13900, 13901-14000, 14001-14100, 14101-14200, 14201-14300, 14301-14400, 14401-14500, 14501-14600, 14601-14700, 14701-14800, 14801-14900, 14901-15000, 15001-15100, 15101-15200, 15201-15300, 15301-15400, 15401-15500, 15501-15600, 15601-15700, 15701-15800, 15801-15900, 15901-16000, 16001-16100, 16101-16200, 16201-16300, 16301-16400, 16401-16500, 16501-16600, 16601-16700, 16701-16800, 16801-16900, 16901-17000, 17001-17100, 17101-17200, 17201-17300, 17301-17400, 17401-17500, 17501-17600, 17601-17700, 17701-17800, 17801-17900, 17901-18000, 18001-18100, 18101-18200, 18201-18300, 18301-18400, 18401-18500, 18501-18600, 18601-18700, 18701-18800, 18801-18900, 18901-19000, 19001 to 19140 of SEQ ID NO: 17.

[0144] In certain aspects, the one or more sequences within the promoter region controlling the gene encoding ZNF354A are characterized by nucleotides 1-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1401-1500, 1501-1600, 1601-1700, 1701-1800, 1801-1900, 1901-2000, 2001-2100, 2101-2200, 2201-2300, 2301-2400, 2401-2500, 2501-2600, and 2601 to 2650 of SEQ ID NO: 32. In one aspect, the one or more sequences within the promoter region controlling the gene encoding ZNF354A are characterized by nucleotides 1-770, 770-1035, 1035-1825, 1825-2345, 2345-2510, and 2510-2655, of SEQ ID NO: 32.

[0145] In one aspect, the one or more sequences within the promoter region controlling the gene encoding ZNF354A is a transcription factor binding site characterized by nucleotides 1260-1283, 1657-1674, 1693-1709, 1896-1913, 1977-1994, and / or 2061-2067, of SEQ ID NO: 32, a fragment or a variant thereof.

[0146] Examples of siRNA sequences targeting a sequence within one or more mRNA encoding the KZF of the invention are selected from the non-limiting group of sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, a fragment or a variant thereof.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0147] The terms “shRNA” as used herein refers to a nucleic acid molecule comprising at least two complementary portions hybridized or capable of specifically hybridizing to form a duplex structure sufficiently long to mediate RNAi (typically between about 15 to about 29 nucleotides in length), and at least one single-stranded portion, typically between approximately 1 and about 10 nucleotides in length that forms a loop connecting the ends of the two sequences that form the duplex. Exemplary shRNAs of the invention are designed to be uniquely and selectively recognizing a target sequence within an mRNA encoding the KZF of the invention. Selecting a suitable shRNA is well within the competences of one of ordinary skill in the art using routine experimentation, several commercial and noncommercial web sites available for shRNA design as well as the information provided herein.

[0148] Examples of shRNA sequences are selected from the non-limiting group of sequences set forth in SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, a fragment or a variant thereof.

[0149] According to a preferred aspect, shRNA sequences are selected from the non-limiting group of sequences set forth in SEQ ID NO: 33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45, a fragment or a variant thereof

[0150] CRISPR (clustered regularly interspaced short palindromic repeats) (CRISPR-associated protein), a Class 2 type II CRISPR system, can be easily programmed to introduce DNA double-strand breaking (DSB) or single-strand breaking (SSB) at the desired target sequences. Usually, the CRISPR is a CRISPR based gain / loss-of-function system comprising at least one single guide RNA (sgRNA), or crRNA and tracrRNA, a protospacer adjacent motif (PAM) and a structure-guided endonuclease such as an RNA-guided endonuclease.

[0151] Any suitable naturally occurring, or engineered, RNA-guided endonuclease can be employed as long as it is effective for binding a target DNA and it may be selected from the non-limiting group comprising Cas9, Cas 12, Cpfl, and FEN-1. Preferably, the RNA-guided endonuclease is Cas9.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0152] One of ordinary skill in the art, using routine experimentation, several available commercial and noncommercial web sites for determining said target sequences as well as relevant information provided herein is able to determine a target sequence within an mRNA encoding ZNF354A.

[0153] The present invention also contemplates a guide ribonucleic acid (sgRNA or gRNA) targeting i) one or more sequences within the gene encoding ZNF354A (chromosome 5: 178730659-178711512) as set forth in SEQ ID NO: 17, a fragment or a variant thereof, or ii) one or more sequences within the promoter region controlling the gene encoding ZNF354A (chromosome: 5, 178728826:178731484) as set forth in SEQ ID NO: 32, a fragment or a variant thereof, or

[0154] iii) one or more DNA sequences selected from the group of sequences set forth in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, or a fragment or variant of any one of these sequences.

[0155] Examples of sgRNA or gRNA sequences are selected from the non-limiting group of sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54 and SEQ ID NO:55, a fragment or a variant thereof.

[0156] Where the disease to be treated is a cancer as described herein, the agent will preferably enhance the activity of a ZNF354A protein i) by inhibiting the degradation of a ZNF354A protein, ii) by increasing the expression of a ZNF354A protein, iii) by modulating the phosphorylation of a ZNF354A protein, and / or iv) by any other means resulting in a gain of function.

[0157] Although CRISPR / Cas systems have been commonly used to generate loss-of-function mutations, they have also been repurposed as a programmable platform for gain-of-function analysis by transcriptional activation of target genes. CRISPR / Cas systems, based on a deactivated Cas (dCas) protein fused with transcriptional activators are within the scope of the present invention.

[0158] In one aspect, CRISPR comprises at least one single guide RNA (sgRNA), or crRNA and tracrRNA, and a structure-guided endonuclease such as an RNA-guided endonuclease.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0159] Any suitable naturally occurring, or engineered, RNA-guided endonuclease can be employed as long as it is effective for binding a target DNA and it may be selected from the non-limiting group comprising Cas9, Cpfl, and FEN-1. Preferably, the RNA-guided endonuclease is Cas9.

[0160] Within the context of this disclosure, the Cas9 endonuclease is preferably a modified Cas9 endonuclease such as, e.g. an enzymatically dead Cas9. Specifically, both RuvC- and HNH-nuclease domains can be rendered inactive by point mutations (e.g. D10A and H840A in SpCas9), resulting in a nuclease dead Cas9 molecule that cannot cleave target DNA. However, the dead Cas9 molecule retains the ability to bind to target DNA based on the sgRNA targeting sequence, which sgRNA sequence is comprised in CRISPR-based gain -of-function system.

[0161] In one aspect, the enzymatically dead Cas9 is tagged with one or more transcriptional repressor or one or more transcriptional activator (see Didovyk et al. 2016; Dominguez AA, Lim WA, Qi LS. Et al., 2015; and Cai R, et al., 202, which are all incorporated herein by reference).

[0162] In another aspect, the enzymatically dead Cas9 is tagged with one or more epitope that is / are recognized by one or more antibody-activator effector (see e.g. Tanenbaum ME, et al., 2014). This enzymatically tagged dead Cas9 can then target one or more sequences regulatory sequence resulting in robust transcription activation of downstream target gene encoding ZNF354A of the invention.

[0163] Usually and within the context of the invention, the regulatory sequence is a promoter or enhancer region which is either a cis-or a trans-acting regulatory sequence.

[0164] In one aspect, the enzymatically tagged dead Cas9 targets one or more sequences within the promoter region controlling the gene encoding ZNF354A that are characterized by nucleotides 1-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, 901-1000, 1001-1100, 1101-1200, 1201-1300, 1301-1400, 1401-1500, 1501-1600, 1601-1700, 1701-1800, 1801-1900, 1901-2000, 2001-2100, 2101-2200, 2201-2300, 2301-2400, 2401-2500, 2501-2600, and 2601 to 2650 of SEQ ID NO: 32.

[0165] The present invention also contemplates a gene delivery vector, preferably in the form of a plasmid or a vector, that encodes or comprises one or more nucleic acid(s) encoding an agent of the invention. Preferably, said agent is a nucleic acid selected from the groupRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0166] comprising an siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, CRISPR-based gain-of-function system, esiRNA, shRNA, and antisense oligonucleotide (e.g. Gapmer antisens), or combination of one or more thereof.

[0167] Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g., E. coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAs, e. g., the numerous derivatives of phage X, e. g., NM989, and other phage DNA, e. g., Ml 3 and filamentous single stranded phage DNA; yeast plasmids such as the 2p plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.

[0168] Various viral vectors are used for delivering nucleic acid to cells in vitro or in vivo. Nonlimiting examples are vectors based on Herpes Viruses, Pox- viruses, Adeno-associated virus, Lentivirus, and others. In principle, all of them are suited to deliver an expression cassette comprising an expressible nucleic acid molecule that codes for an agent of the invention. In a preferred aspect, said viral vector is an adenoviral vector, preferably a replication competent adenovirus.

[0169] In one aspect, the vector is selected from the non-limiting group comprising a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, an RNA (e.g. mRNA) targeted lipid nanoparticles (LNPs), a liposome or any combination thereof.

[0170] Examples of viral vectors include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MV A), fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus,

[0171] 1Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0172] and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, US Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.

[0173] In some aspects, polynucleotides described herein are incorporated into rAAV vectors and / or virions in order to facilitate their introduction into a cell. rAAV vectors useful in the compositions and methods described herein are recombinant polynucleotide constructs that include (1) a promoter, (2) a sequence to be expressed (e.g., a polynucleotide encoding a protein, such as pendrin or Atohl, or a polynucleotide that can be transcribed to produce an RNA molecule, such as an inhibitory RNA), and (3) viral sequences that facilitate integration and expression of the sequence to be expressed. The viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion. In some embodiments, the rAAV vectors further include at least one SLC26A4 enhancer. Such rAAV vectors may also contain marker or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. For use in the methods and compositions described herein, the ITRs can be AAV2 ITRs. Methods for using rAAV vectors are described, for example, in Tai et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0174] The polynucleotides and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the polynucleotide or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly. The construction of rAAV virions has been described, forRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0175] instance, in US 5,173,414; US 5,139,941; US 5,863,541; US 5,869,305; US 6,057,152; and US 6,376,237; as well as in Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol.

[0176] 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0177] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, rhlO, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP. B, PHP.eb, and PHP. S. For targeting SLC26A4-expressing cells, AAV1, AAV2, AAV2quad(Y-F), AAV6, AAV8, AAV9, Anc80, Anc80L65, AAV-DJ, AAV-DJ / 9, 7m8, and PHP. B may be particularly useful. Serotypes evolved for transduction of the retina may also be used in the methods and compositions described herein. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for instance, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.

[0178] Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, in Duan et al., J. Virol. 75:7662 (2001); Halbert et al., J. Virol. 74:1524 (2000); Zolotukhin et al., Methods, 28:158 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001).

[0179] AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000). Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol.

[0180] 19:423 (2001).Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0181] The present invention further contemplates a cell, or population of cells, comprising, or modified by the introduction of, i) a gene delivery vector of the invention (e.g. a plasmid or vector) or ii) one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, CRISPR-based gain-of-function system, esiRNA, shRNA, and antisense oligonucleotide, or combination of two or more thereof, as described herein. Cells can be either eukaryotic or prokaryotic cells. In one aspect, the cell is a mammalian cell, whether an autologous or an allogeneic cell.

[0182] The present invention further contemplates an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding said ZNF354A, and / or iii) the ZNF354A gene.

[0183] Also contemplated in the present invention is a composition comprising an agent modulating the expression and / or activity of

[0184] i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), an mRNA encoding said ZNF354A, and / or the ZNF354A gene, or

[0185] ii) a plasmid or a vector of the invention,

[0186] iii) one or more nucleic acids encoding a siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and ASO, or combination of two or more thereof of the invention, or

[0187] iv) a cell, or population of cells, of the invention.

[0188] Also contemplated in the present invention is a pharmaceutical composition comprising a therapeutically effective amount of an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), an mRNA encoding said ZNF354A, and / or the ZNF354A gene, or

[0189] ii) a plasmid or a vector of the invention, or

[0190] iii) one or more nucleic acids encoding a siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and ASO, or combination of two or more thereof of the invention, or

[0191] iv) a cell, or population of cells, of the invention,

[0192] and a pharmaceutically acceptable carrier or diluent.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0193] Preferably, the pharmaceutical composition is for use in the treatment and / or prevention of a disease linked to the expression of ZNF354A. In one aspect, the disease linked to the expression of ZNF354A is selected from the group comprising a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a neurodegenerative disease, a chronic disease or an autoimmune disease.

[0194] Other examples of disease comprise spinal cord injury and retinal dystrophy.

[0195] The term "therapeutically effective amount" as used herein means an amount of an agent of the invention, ii) a gene delivery vector of the invention, or iii) a cell of the invention, high enough to significantly positively modify the symptoms and / or condition to be treated, but low enough to avoid serious side effects (at a reasonable risk / benefit ratio), within the scope of sound medical judgment. The therapeutically effective amount of an agent of the invention, ii) a gene delivery vector of the invention, or iii) a cell of the invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient.

[0196] “Pharmaceutically acceptable carrier and / or diluent” means a carrier and / or diluent that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes carriers or diluents that are acceptable for human pharmaceutical use. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

[0197] Pharmaceutically acceptable excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0198] The pharmaceutical compositions may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting orRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0199] emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include macrocrystalline cellulose, carboxymethyf cellulose sodium, polysorbate 80, phenyletbyl alcohol, chiorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N. J. 1991) which is incorporated by reference herein.

[0200] The pharmaceutical composition can also further comprise or provide, one or more additional therapy. Where the disease is, e.g., cancer, at least one additional anticancer agent or therapy is selected from the group comprising radiotherapy, chemotherapy, immunotherapy and hormone therapy, or a combination of one of more thereof.

[0201] The pharmaceutical composition comprising a therapeutically effective amount of a cell and a pharmaceutically acceptable carrier and / or diluent can be administered to the subject in need thereof by any method and route known in the art and described herein.

[0202] Also contemplated in the present invention is a i) ZNF354A protein as set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, a fragment or a variant thereof, or ii) a nucleic acid encoding said ZNF354A protein as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, a fragment or a variant thereof, for use in the treatment and / or prevention of cancer.

[0203] Also contemplated in the present invention is an agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding said ZNF354A, and / or iii) the ZNF354A gene, for modulating cellular responses to oxidative stress.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0204] Also contemplated in the present invention is the use of a pharmaceutical composition of the invention for the preparation of a medicament for the treatment and / or prevention a disease linked to oxidative stress, lipid peroxidation and / or ferroptosis.

[0205] The present invention further contemplates methods of treating and / or preventing a disease linked to the expression of ZNF354A in a subject in need thereof.

[0206] In one aspect of the invention, the method of treating and / or preventing a disease linked to the expression of ZNF354A in a subject in need thereof, comprises administering a therapeutically effective amount of i) an agent of the invention or an agent for use of the invention, ii) a gene delivery vector of the invention, iii) a cell of the invention, or iv) a composition or pharmaceutical composition of the invention.

[0207] In one aspect of the invention, the method of treating and / or preventing a disease linked to the expression of ZNF354A in a subject in need thereof, comprises modifying a cell, and reintroducing the cell (e.g. a cell or population of cells) into the subject in need thereof.

[0208] In one aspect of the invention, the method of treating and / or preventing a cancer linked to the expression of ZNF354A in a subject in need thereof, comprises administering a therapeutically effective amount of a nucleic acid encoding a ZNF354A of the invention, ii) a gene delivery vector comprising a nucleic acid encoding a ZNF354A of the invention, iii) a cell comprising a nucleic acid encoding a ZNF354A of the invention, or iv) a pharmaceutical composition comprising a nucleic acid encoding a ZNF354A of the invention.

[0209] In one aspect of the invention, the method of treating and / or preventing a cancer linked to the expression of ZNF354A in a subject in need thereof, comprises modifying a cell, and reintroducing the cell (e.g. a cell or population of cells) into the subject in need thereof.

[0210] Preferably, a biopsy or other tissue or biological fluid sample comprising the cell or the population of cells may be necessary. Cells such as fibroblast cells or stem cells that can be generated directly from adult cells, such as iPSCs, are particularly preferred in this regard.

[0211] In one aspect, the cell is any mammalian cell, whether an autologous or an allogeneic cell.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0212] In one aspect, the mammalian cell is selected from the group comprising epithelial cells, muscle cells, nerve cells (neuronal cells), blood cells, immune cells, connective tissue cells, stem cells, sensory cells, secretory cells, and reproductive cells.

[0213] In one aspect, the cell is selected from the group comprising a cytotoxic cell, an immune cell, a stem cell, a progenitor cell, a cell line and a cell derived from a stem cell or a progenitor cell. Where the cell is an immune cell, said immune cell will be selected from the non-limiting group comprising T cells, tumor infiltrating lymphocytes (TILs), NK cells, regulatory T cells (Treg cells), macrophages, TCR-expressing cells, eosinophils, basophils, neutrophils, myeloid cells, B cells, plasma cells, regulatory B cells (Bregs), innate lymphoid cells 1 (ICL1), ILC2, ICL3, dendritic cells, or NK-T cells.

[0214] There are a variety of available techniques known in the art for isolation and enrichment of T cells, such as e.g. PBMC obtained from peripheral blood samples.

[0215] Preferably, the immune cell is selected from the group comprising a T cell, a tumor infiltrating lymphocyte (TIL), an NK cell, a TCR-expressing cell, a dendritic cell, and an NK-T cell.

[0216] Where the immune cell is a T-cell or NK cell, it will be either an autologous or an allogeneic T cell or NK cell. In some aspects, the immune cell is a T-cell line or NK cell line. Any cell line derived from any primary cell can be used in the present invention. NK cells can, for example, be engineered from various NK sources such as iPSC-derived NK cells, NK cells isolated from blood (PB-NK), NK isolated from the umbilical cord blood or NK cell lines comprising NK92, YTS or KHYG1 cell lines.

[0217] Where the cell is derived from a stem cell, it will be either a natural or an induced stem cell (e.g., an iPSC-derived cell). A gene delivery vector (e.g. plasmid or vector) comprising a nucleic acid encoding an agent or agent for use of the invention or at least one acid nucleic of the invention can be introduced to cell via one or more methods known in the art. These one or more methods include, without limitation, microinjection, electroporation, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0218] The cell (e.g. a cell or population of cells) of the invention can be reintroduced into the subject in need thereof by any route of administration and / or delivery methods known in the art.

[0219] The invention also contemplates kits for performing a method according to the invention or for the treatment and / or prevention of a disease of the invention.

[0220] In one aspect of the invention, the kit comprises a pharmaceutical composition comprising an agent of the invention.

[0221] In a further aspect, the invention contemplates a kit for the treatment and / or prevention of a disease of the invention comprising a cell of the invention.

[0222] The kits of the invention may also comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the disease of disorder of the invention and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Alternatively, or additionally, the kits may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0223] The label or package insert may comprise instructions for use thereof. Instructions included may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure.

[0224] The invention also contemplates the use of kits for performing a method according to the invention or for the treatment and / or prevention of a disease of the invention.

[0225] Further contemplated in the present invention are nucleic acids encoding, or comprising, one or more nucleic acids encoding, an siRNA, an shRNA, an snRNA, a siRNARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0226] capable of interfering the expression of short hairpin (sh), a piRNA, or a nucleic acid including an antisense oligonucleotide (e.g. ASOs, modified ASOs such as GapmeRs,...) of the invention.

[0227] Further contemplated in the present invention is a method for enhancing the redox resistance of immunotherapy effector cells, the method comprising contacting the effector cells, ex vivo and / or in vivo, with one or more nucleic acids of the invention, or with a vector or plasmid described herein and comprising said one or more nucleic acids, in order to reduce or silence the expression of ZNF354A in said cells, thereby increasing their resistance to oxidative stress and, for example, extending their lifespan, wherein cellular exhaustion following activation is at least partly attributable to oxidative damage..

[0228] In one aspect, the immunotherapy effectors are cells, preferably immune cells selected from the group comprising tumor-infiltrating lymphocytes (TILs), T cell receptor (TCR)-engineered T cells lacking chimeric antigen receptors, natural killer (NK) cells, cytokine-induced killer (CIK) cells, virus-specific T cells, regulatory T cells (Tregs), y5 T cells, macrophages, monocyte-derived macrophages, polarized macrophages and dendritic cells, or combinations thereof.

[0229] Further contemplated in the present invention is the use of one or more nucleic acids of the invention, or a vector or plasmid of the invention comprising said one or more nucleic acids, to reduce or silence the expression of ZNF354A in immunotherapy effector cells, thereby increasing their resistance to oxidative stress and, for example, extending their lifespan, wherein cellular exhaustion following activation is at least partly attributable to oxidative damage.

[0230] Further contemplated in the present invention is a method for prolonging the survival and / or functional persistence of a cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif, the method comprising contacting said cell, or population of cells, ex vivo and / or in vivo, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids, wherein said one or more nucleic acids inhibit the expression and / or activity of: i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A),Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0231] ii) an mRNA encoding ZNF354A, and / or

[0232] iii) the ZNF354A gene,

[0233] and wherein inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis in the contacted cell, or contacted population of cells, thereby prolonging their survival and / or functional persistence.

[0234] According to one aspect, the cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif was / were first expanded by contacting said cell, or population of cells, with effectors (or a combination of effectors) used for expansion

[0235] Cells are typically expanded ex-vivo (optionally following anti-CD3 / anti-CD28-mediated activation) in the presence of cytokines (interleukins) such as IL-2, IL-7, IL-15, and / or IL-21. Additional pathway modulators (e.g., PI3K inhibitors, AKT inhibitors, mTOR inhibitors, antioxidants or GSK3β inhibitors,...) may be included to promote a memory phenotype and improve persistence.

[0236] In one aspect, the cells, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif are selected from the group comprising T-cells (such as e.g. aP T lymphocytes, CD8+cytotoxic T cells, CD4+helper T cells, Naive, central memory, effector memory, stem cell memory T cells, or Tumor-infiltrating lymphocytes (TILs)), y5 T cells (such as e.g. Vy9V52 T cells or a subset thereof), and natural killer (NK) cells (such as e.g. Peripheral blood-derived NK cells, Cord blood NK cells, NK-92 cell line or iPSC-derived NK cells), macrophages, monocytes, dendritic cells, hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), or progeny or derivatives thereof) or combinations thereof.

[0237] It is understood that the method for prolonging the survival and / or functional persistence of a cell, or a population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif is not limited to any particular CAR structure. Any CAR architecture, including variations in the extracellular antigen-binding domain, hinge or spacer region, transmembrane domain, orRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0238] intracellular signaling and costimulatory domains, is contemplated within the scope of the method.

[0239] As shown in the example, the inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis, preventing exhaustion of the contacted cell or population of cells and maintaining their proliferative state.

[0240] In one aspect, the contacted cell or population of contacted cells expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif described herein is / are used for the treatment of a a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease, a neurodegenerative disease or an autoimmune disease.

[0241] Also provided is a cell, or a population of cells, produced or obtainable by any of the methods for prolonging the survival and / or functional persistence of cells described herein. Preferably, the cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif and obtainable by any of the methods for prolonging the survival and / or functional persistence of cells described herein is / are selected from the group comprising T-cells (such as e.g. aP T lymphocytes, CD8+cytotoxic T cells, CD4+helper T cells, Naive, central memory, effector memory, stem cell memory T cells, or Tumor-infiltrating lymphocytes (TILs)), y5 T cells (such as e.g. Vy9V52 T cells or a subset thereof), and natural killer (NK) cells (such as e.g. Peripheral blood-derived NK cells, Cord blood NK cells) or derivative thereof (such as NK-92 cell line or iPSC-derived NK cells), macrophages, monocytes, dendritic cells, hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), and progeny or derivatives thereof.

[0242] Further contemplated in the present invention is a method of detecting a disease linked to an over-activation of anti oxidative pathways in a subject, the method comprising:

[0243] i) detecting the level of phosphorylation of ZNF354A at serine 169 in a biological sample of said subject,

[0244] ii) comparing the level of phosphorylation of ZNF354A at serine 169 in said subject with a standard control,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0245] wherein a high level of phosphorylation of ZNF354A at serine 169 in said subject relative to said standard control indicates said subject suffers from a disease linked to an over-activation of antioxidative pathways.

[0246] In one aspect, the method of detecting a disease linked to an over-activation of antioxidative pathways in a subject comprises adding an antibody or antigen binding fragment thereof to said sample under conditions which allow for binding of said antibody, or antigen binding fragment to ZNF354A and detecting the amount of antibody -bound in said sample, wherein the antibody or antigen binding fragment thereof specifically targets and binds to an epitope encompassing the Ser- 169 phosphorylation site.

[0247] Usually, the disease is linked to an over-activation of antioxidative pathways is a disease selected from the group comprising cancer, in particular cancer resistance to cell death (chemotherapy), chronic tissue inflammation / damage (e.g. neuron, cardiovascular and muscle diseases) and acute injury (e.g. ischemia, blood brain barrier disfunction, muscle rupture), or a combination thereof.

[0248] The induction of ferroptosis, a lipid peroxide-induced cell death, is of major interest in cancer research to kill tumor cells. To investigate the relevance of the LORD pathway in predicting and controlling the sensitivity of cancer lines to ferroptosis, the Inventors probed for the phosphorylation of ZNF354A. Only the cells expressing ZDHHC20L, i.e. HEPG2, MCF7 and HCT116 (Fig. 1) were more resistant to either RLS3 or H₂O₂, compared to all other lines (Fig.

[0249] 5C). Thus, according to one aspect, the method further indicates whether the cancer is sensitivity or resistant to ferroptosis and determines the treatment to be administered.

[0250] Further contemplated in the present invention is a method of treatment and / or prevention of a cancer in a subject in need thereof, the method comprising

[0251] i) removing and isolating cells, or population of cells, preferably immune cells, from said subject,

[0252] ii) genetically engineering said immune cells with one recombinant construct (e.g. vector, plasmid or polynucleotide) encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0253] iii) expanding ex vivo into a larger population of engineered immune cells, or population of immune cell, in the presence of an interleukin selected from the group comprising IL-7, IL-15 and / or IL-2,

[0254] iv) contacting said immune cells, or population of immune cells, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention encoding said one or more nucleic acids

[0255] (iv) reintroducing said engineered immune cells, or population of immune cells, into the subject in need thereof.

[0256] In one aspect, the immune cells are selected from the group comprising T-cells (such as e.g. aP T lymphocytes, CD8+cytotoxic T cells, CD4+helper T cells, Naive, central memory, effector memory, stem cell memory T cells, or Tumor-infiltrating lymphocytes (TILs)), y5 T cells (such as e.g. Vy9V52 T cells or a subset thereof), and natural killer (NK) cells (such as e.g. Peripheral blood-derived NK cells, Cord blood NK cells, NK-92 cell line or iPSC-derived NK cells), macrophages, monocytes, dendritic cells, hematopoietic stem and progenitor cells (HSPCs), induced pluripotent stem cells (iPSCs), or progeny or derivatives thereof) or combinations thereof.

[0257] Cells, preferably immune cells, are typically expanded ex-vivo (optionally following anti-CD3 / anti-CD28-mediated activation) in the presence of cytokines (interleukins) such as IL-2, IL-7, IL-15, and / or IL-21. Additional pathway modulators (e.g., PI3K inhibitors, AKT inhibitors, mTOR inhibitors, antioxidants or GSK3β inhibitors,...) may be included to promote a memory phenotype and improve persistence.

[0258] Further contemplated in the present invention is a method of improving transplant engraftment, survival, and / or functional persistence of stem cell transplants in a subject in need thereof, the method comprising

[0259] i) removing and isolating cells, or population of cells, preferably stem cells, from said subject, ii) ex-vivo contacting said stem cells, or population of stem cells, with one or more nucleic acids of the invention, or with a vector or plasmid of the invention comprising said one or more nucleic acids,

[0260] (iii) reintroducing said engineered stem cells, or population of stem cells, into the subject in need thereof,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0261] wherein said one or more nucleic acid inhibits the expression and / or activity of ZNF354A, thereby reducing oxidative stress, lipid peroxidation, and / or ferroptosis in the stem cells and improving transplant engraftment, survival, and / or functional persistence.

[0262] In one aspect, the stem cells are selected from the group comprising pluripotent, multipotent, perinatal, and extra-embryonic stem cell sources.

[0263] Preferably, the pluripotent stem cells are selected from the non-limiting group comprising embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), parthenogenetic stem cells, nuclear transfer-derived embryonic stem cells (ntESCs), and epiblast stem cells.

[0264] Preferably, the multipotent or adult stem cells are selected from the group comprising mesenchymal stem / stromal cells (MSCs) (such as those derived from bone marrow, adipose tissue, umbilical cord, or Wharton’s jelly), hematopoietic stem cells (HSCs), neural stem cells (NSCs), adipose-derived stem cells (ADSCs), endothelial progenitor cells (EPCs), dental pulp stem cells (DPSCs), muscle satellite cells, intestinal stem cells, and liver progenitor cells. In one aspect, the one or more nucleic acid is / are operably linked to a promoter. In one aspect, the promoter is an inducible promoter. Examples of inducible promoters are described in Weber & Fussenegger, (2012).

[0265] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety. The foregoing description will be more fully understood with reference to the following Examples.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0266] SEQUENCES

[0267] • SEQ ID NO:1

[0268] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant 1, mRNA NCBI Reference Sequence: NM_005649.3

[0269] > NM 005649.3 Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant 1, mRNA / cDNA AGACGCCGUCGGGGCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGG GGAGGCGCGGCCCGGGACGCUCGAGCUUAGGGAGAUCUGCCUUCUGGAGACUGCGCCGUCCUCCCGGGAG AGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCCCCAGGUGUCACUGACGUUUGAGGAUG UGGCUGUGCUGUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGU GAUGCUGGAGAACUAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUG UUGCAGCAAGGAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGA GCAGU CAUAAAAC CACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAUGUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAG CAGGAUAAAAAGGGAAGUUUUCAGAUAGUUUCAGCCACCCACAAAAAAAUCCCCACUAUAGAAAGAAGCC AUAAAAAUACUGAAUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAG AGAAAAAACAC GAG CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAA C CAAAAAUUACAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C C UU C GUAAACAU GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAG C CAAAGUU CAGCU CUUAUU CAACAU CAAAUAAC GCAUACU GGAGAGAAAC C CUACAUAU GUAAAGAAU GU GGGAAAGC CUUUACU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU GGAGAAAU C CUACA GAUGUAAAGAAUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGA AGAAAACCCUUGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAA AGAAUU CAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C C UUCGUUAUCAUCAGAGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAG C CAGAGU GC CU CU CUUAUU CAACAU GAAAGAAUU CACAC C GGAGAAAAGC C CUAUAGAU GCAAU GAAU GU GGGAAAGGCUUUACUUCUAUUUCACGACUUAAUAGACACCGAAUCAUUCAUACUGGAGAGAAGUUUUAUA AUUGUAAUGAAUGUGGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGG AGAAAAAC CAU GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUU CAACAU CAG AGAAUGCAUACUGGAGAAAGACCCUAUAAAUGUAACGAGUGUGGGAAAACAUUCAGGUGUAACUCAUCAC UUAGUAAUCACCAGAGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGG C CAAAGUU CAGCU CUUAUU CAGCAU C GAAGGAUU CAU AC AG GAGAAAAAC C CUUUAAAU GUAAU ACAU GU GGAAAAACUUUUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU G AAU GUAAU ACAU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GA AGAGGAC C C CUAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CU U GAGAGAGAU GUAAUAAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAA UU C CAU GGAU AAAC CU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAA AUAAC CU GAGAU GAAGAAUUUACAAUU GAAGACAUU GACUUUAGC CAUUU GU GAAAU GGGUUU GCUUUUU CCCUUUUCCUACAGACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGA GGUGUGCUUUAGAUUUCUCAUUAGAAGACCACCAAACUGGUAAUAUUUUUAUAGCAUUUUAAUAGCUUGA U CAAAUU GUAC CUUUUUAGAGAAAAGGAC CAAAAU AAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CU CUGGGGUUUGUCCUUUUCCUACCCUGAUGUCAAACUUAUGCAUGGAUUUCAUUAAAAAAGAAAAAGAAAA

[0270] • SEQ ID NO:2

[0271] Homo sapiens zinc finger protein 354A (ZNF354A), Translated protein of transcript variant 1

[0272] MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVML ENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSSHKTTKSTQTQDS SFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQIVSATHKKIPTIERSHK NTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQLLNQPKITADKRYKCSL CEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQHQITHTGEKPYICKECGRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0273] KAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQKIHAEENPCKYNPGRKA SSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHTGEKPFKCSECGRAFS QSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTGEKFYNCNECGKALSSH STLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGERPYKCNECGKTFRCNSS LSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPFKCNTCGKTFRQSSSRI AHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0274] • SEQ ID NO:3

[0275] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant 2, mRNA NCBI Reference Sequence: NM_001324339.2

[0276] > NM 001324339.2 Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant 2, mRNA / cDNA AGACGCCGUCGGGGCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGG GGAGGCGCGGCCCGGGACGCUCGAGCUUAGGGAGGUGUCACUGACGUUUGAGGAUGUGGCUGUGCUGUUU ACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGUGAUGCUGGAGAACU AUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUGUUGCAGCAAGGAGA AGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGAGCAGUCAUAAAACC ACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAU GUAC CUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAGCAGGAUAAAAAGGG AAGUUUU CAGAUAGUUU CAGC GAG C CACAAAAAAAU C C C CACUAUAGAAAGAAGC CAUAAAAAUACU GAA UUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAGAGAAAAAACACCAC CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAAC CAAAAAUUACAGC AGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C CUU C GUAAACAU GAG AAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAGC CAAAGUU CAGCU C UUAUUCAACAUCAAAUAACGCAUACUGGAGAGAAACCCUACAUAUGUAAAGAAUGUGGGAAAGCCUUUAC U CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU GGAGAAAU C CUACAGAU GUAAAGAAU GU GGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGAAGAAAACCCUUGUA AGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAAAGAAUUCAUUCUAG AAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C CUU C GUUAU CAU CAG AGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAGCCAGAGUGCCUCUC UUAUUCAACAUGAAAGAAUUCACACCGGAGAAAAGCCCUAUAGAUGCAAUGAAUGUGGGAAAGGCUUUAC UU CUAUUU CAC GACUUAAUAGACAC C GAAU CAUU CAUACU GGAGAGAAGUUUUAUAAUU GUAAU GAAU GU GGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGGAGAAAAACCAUGUA AAUGUAAAGUAUGUGGAAAAGCCUUCAGACAGAGUUCAGCUCUCAUUCAACAUCAGAGAAUGCAUACUGG AGAAAGAC C CUAUAAAU GUAAC GAGU GU GGGAAAACAUU CAGGU GUAACU CAU CACUUAGUAAU CAC CAG AGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGGCCAAAGUUCAGCUC UUAUU CAGCAU C GAAGGAUU CAUACAGGAGAAAAAC C CUUUAAAU GUAAU ACAU GU GGAAAAACUUUUAG ACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU GAAU GUAAU ACAU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GAAGAGGAC C C CUAGA AAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CUU GAGAGAGAU GUAA UAAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAAUU C CAU GGAUAAAC CU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAAAUAAC CU GAGAU GA AGAAUUUACAAUUGAAGACAUUGACUUUAGCCAUUUGUGAAAUGGGUUUGCUUUUUCCCUUUUCCUACAG ACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGAGGUGUGCUUUAGAU UU CU CAUUAGAAGAC CAC CAAACU GGUAAUAUUUUUAUAGCAUUUUAAUAGCUU GAU CAAAUU GUAC CUU UUUAGAGAAAAGGAC CAAAAU AAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CUCUGGGGUUUGUCCU UUU C CUAC CCU GAU GU CAAACUUAU GCAU GGAUUU CAUUAAAAAAGAAAAAGAAAAA

[0277] • SEQ ID NO: 4

[0278] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant 2 MLENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLG SKSSHKTTKSTQTQDSSFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQI VSATHKKIPTIERSHKNTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQL LNQPKITADKRYKCSLCEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQH QITHTGEKPYICKECGKAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQK IHAEENPCKYNPGRKASSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0279] TGEKPFKCSECGRAFSQSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTG EKFYNCNECGKALSSHSTLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGER PYKCNECGKTFRCNSSLSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPF KCNTCGKTFRQSSSRIAHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0280] • SEQ ID NO: 5

[0281] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant X3, mRNA. NCBI Reference Sequence: XM_011534645. 3

[0282] > XM 011534645.3 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant X3, mRNA / cDNA UCCUGCCACUGCGCUACUGCACUCCAGUCUGGCCAACAGAGCGAGACUCCGCCUCAAAAAAAAAAAAGCC GUUUUCCGUGGAGUCGGGAGAUGAUUCGUUGGCGGGAGAGAGAGGAACCGGCUGGGAAAGGCUUGAGGCG GAGGGAAGUCGUCCCGGGGCCGUCGUGGUGGGAGUCCCGGCCCGCCUCGCAGCGGCGUGAACGGGGCAGG UGCCCCGGCGCUGCCUCAGUUUCCUGAGCGGUGCAGUGGGUGCUGGCAGUGCCAGCUGGCGCCGAGGAAC UCAGCGGCGUGGGGCGAGCCCUGGCCCUGGUGGGCUCAGCGGGUCGCUGCUGCCACUGCGGCUCCAGCGU CCCCUCCGUAAGCCCCAAGCCUGUGGGGCCUGGGCCUGGCCGGGCGGCCCAGCGCUGCUCUGGUCCGCGG GCUCCUGGCUCCUCCCGAAUCCCUGUGAGGGCGCGCGGGGUCCUUCUCAGCGGGAGUCGGGGUUUUAGAG CUGCGGAUUCCAGGGCUGGAAAGCAGAAGGGGUUCUUCCUGGCUCCCUUUUUCUUCUCAGAUCUGCCUUC UGGAGACUGCGCCGUCCUCCCGGGAGAGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCC CCAGUGGCUGCACUAGGCCUGAAUCUCACUGUGUGCUGCUCAGUAUACAUUUGUUACUGGCCAAUUAUUC AUUAAACGAAUGAUCUGGAACCUUCAGAAGCUUGAAUAGUGUCACUGACGUUUGAGGAUGUGGCUGUGCU GUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGUGAUGCUGGAG AACUAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUGUUGCAGCAAG GAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGAGCAGUCAUAA AAC CACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAU GUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAGCAGGAUAAAA AGGGAAGUUUU CAGAUAGUUU CAGC CAC C CACAAAAAAAU C C C CACUAUAGAAAGAAGC CAUAAAAAUAC UGAAUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAGAGAAAAAACA C CAC CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAAC CAAAAAUUA CAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C CUU C GUAAACA U GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAGC CAAAGUU CA GCUCUUAUUCAACAUCAAAUAACGCAUACUGGAGAGAAACCCUACAUAUGUAAAGAAUGUGGGAAAGCCU UUACU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU GGAGAAAU C CUACAGAU GUAAAGA AUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGAAGAAAACCCU UGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAAAGAAUUCAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C CUU C GUUAU CA UCAGAGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAGCCAGAGUGCC UCUCUUAUUCAACAUGAAAGAAUUCACACCGGAGAAAAGCCCUAUAGAUGCAAUGAAUGUGGGAAAGGCU UUACUU CUAUUU CAC GACUUAAUAGACAC C GAAU CAUU CAUACU GGAGAGAAGUUUUAUAAUU GUAAU GA AUGUGGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGGAGAAAAACCA U GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUU CAACAU CAGAGAAU GCAUA CU GGAGAAAGAC C CUAUAAAU GUAAC GAGU GU GGGAAAACAUU CAGGU GUAACU CAU CACUUAGUAAU CA CCAGAGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGGCCAAAGUUCA GCUCUUAUUCAGCAUCGAAGGAUUCAUACAGGAGAAAAACCCUUUAAAUGUAAUACAUGUGGAAAAACUU UUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU GAAU GUAAU AC AU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GAAGAGGAC C C C UAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CUU GAGAGAGAU GUAAU AAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAAUU C CAU GGAU AAAC CU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAAAUAAC CU GAG AU GAAGAAUUUACAAUU GAAGACAUU GACUUUAGC CAUUU GU GAAAU GGGUUU GCUUUUU C C CUUUU C CU ACAGACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGAGGUGUGCUUU AGAUUU CU CAUUAGAAGAC CAC CAAACU GGUAAUAUUUUUAUAGCAUUUUAAUAGCUU GAU CAAAUU GUA C CUUUUUAGAGAAAAGGAC CAAAAU AAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CUCUGGGGUUUG UCCUUUUC CUAC CCU GAU GU CAAACUUAU GCAU GGAUUU CAUUAAAAAAGAAAAAGAAAAARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0283] SEQ ID NO: 6

[0284] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant X3 MLENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLG SKSSHKTTKSTQTQDSSFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQI VSATHKKIPTIERSHKNTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQL LNQPKITADKRYKCSLCEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQH QITHTGEKPYICKECGKAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQK IHAEENPCKYNPGRKASSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIH TGEKPFKCSECGRAFSQSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTG EKFYNCNECGKALSSHSTLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGER PYKCNECGKTFRCNSSLSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPF KCNTCGKTFRQSSSRIAHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0285] • SEQ ID NO: 7

[0286] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant X2, mRNA NCBI Reference Sequence: XM_017009791.2

[0287] > XM 017009791.2 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant X2, mRNA / cDNA AGACGCCGUCGGGGCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGG GGAGGCGCGGCCCGGGACGCUCGAGCUUAGGGAGGUGAGCGUCGGCGGCAGGCCAAGCCGAUCUGCCUUC UGGAGACUGCGCCGUCCUCCCGGGAGAGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCC CCAGGUGUCACUGACGUUUGAGGAUGUGGCUGUGCUGUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCU UCUCAGAGAAACUUGUACCGGGAUGUGAUGCUGGAGAACUAUAGGAACCUGGUCUCACUGGGGCUCCCAU UUACCAAACCAAAAGUGAUCUCCCUGUUGCAGCAAGGAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUC UGGCGUCUCCUCUCUAGGAUCGAAGAGCAGUCAUAAAACCACAAAGUCAACGCAAACACAAGACUCUUCA UUUCAGGGACUGAUACUGAAAAGAUCCAACAGGAAUGUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACA UAUAUGAAGGCAGAUUAGAGAAAAAGCAGGAUAAAAAGGGAAGUUUUCAGAUAGUUUCAGCCACCCACAA AAAAAU C C C CACUAUAGAAAGAAGC CAUAAAAAUACU GAAUU GAGC CAAAACUU CAGC C CAAAGU CAGU G CUUAUUAGGCAACAGAUACUU C C CAGAGAAAAAACAC CAC CAAAAU GU GAAAUACAAGGAAACAGC CU CA AACAGAAUU CACAAUUACUUAAU CAAC CAAAAAUUACAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GA AAAAAC CUU CAUUAACACUU CAU C C CUU C GUAAACAU GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAG UGUAAAGAAUGUUCAAAAGCCUUUAGCCAAAGUUCAGCUCUUAUUCAACAUCAAAUAACGCAUACUGGAG AGAAAC C CUACAUAU GUAAAGAAU GU GGGAAAGC CUUUACU CU CAGUACAU C C CUUUAUAAACAU CUAAG AACCCAUACUGUGGAGAAAUCCUACAGAUGUAAAGAAUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUU UUUAUACAUCAAAAAAUUCAUGCUGAAGAAAACCCUUGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUU GCAGCACAU C C CUUU CU GGAU GU CAAAGAAUU CAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GG CAACAC CUUUAAGU CUAGCU CAU C C CUU C GUUAU CAU CAGAGAAUU CACACU GGAGAGAAGC CUUUUAAA UGUAGUGAAUGUGGGAGAGCCUUCAGCCAGAGUGCCUCUCUUAUUCAACAUGAAAGAAUUCACACCGGAG AAAAGCCCUAUAGAUGCAAUGAAUGUGGGAAAGGCUUUACUUCUAUUUCACGACUUAAUAGACACCGAAU CAUU CAUACU GGAGAGAAGUUUUAUAAUU GUAAU GAAU GU GGUAAAGC CUUAAGCU C C CACU CAACACUU AUUAUU CAC GAGC GAAUU CAUACU GGAGAAAAAC CAU GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGAC AGAGUU CAGCU CU CAUU CAACAU CAGAGAAU GCAUACU GGAGAAAGAC C CUAUAAAU GUAAC GAGU GU GG GAAAACAUU CAGGU GUAACU CAU CACUUAGUAAU CAC CAGAGAAUU CAUACU GGAGAGAAAC CAU AU C GA UGUGAGGAAUGUGGGAUAUCUUUUGGCCAAAGUUCAGCUCUUAUUCAGCAUCGAAGGAUUCAUACAGGAG AAAAAC C CUUUAAAU GUAAU ACAU GU GGAAAAACUUUUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAG AAUU CAUACU GGAGAGAAAC C CUAU GAAU GUAAU ACAU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUU ACUAAU CAUUAUAAAAUU CAU AU C GAAGAGGAC C C CUAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAA C CAAAGCU CAU C GAAGAAUACAU C CUU GAGAGAGAU GUAAU AAAU GUAAU GGAU GU GAAAAAAACU GUAA UAAUUUAGCCCUCAUUAGGUAUUUAAUUCCAUGGAUAAACCUCAGCUAUAUAAUAGAUAUGAGGAAAGUG UUUGUGCCUGU CAGACACUUAAAAAAAUAAC CU GAGAU GAAGAAUUUACAAUU GAAGACAUU GACUUUAG C CAUUU GU GAAAU GGGUUU GCUUUUU C C CUUUU C CUACAGAC GUAUAU GCUAGAU GU CAC GU GAU CAU CA GAAACAGAUAUCCGAGUGGGUGGGGAGGUGUGCUUUAGAUUUCUCAUUAGAAGACCACCAAACUGGUAAU AUUUUUAUAGCAUUUUAAUAGCUU GAU CAAAUU GUAC CUUUUUAGAGAAAAGGAC CAAAAU AAAAGAAAA AUGAAUUAUGAACUACCUCUCAGUCUCUGGGGUUUGUCCUUUUCCUACCCUGAUGUCAAACUUAUGCAUG GAUUU CAUUAAAAAAGAAAAAGAAAAARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0288] SEQ ID NO:8

[0289] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant X2

[0290] MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVML ENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSSHKTTKSTQTQDS SFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQIVSATHKKIPTIERSHK NTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQLLNQPKITADKRYKCSL CEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQHQITHTGEKPYICKECG KAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQKIHAEENPCKYNPGRKA SSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHTGEKPFKCSECGRAFS QSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTGEKFYNCNECGKALSSH STLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGERPYKCNECGKTFRCNSS LSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPFKCNTCGKTFRQSSSRI AHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0291] • SEQ ID NO: 9

[0292] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant XI, mRNA, NCBI Reference Sequence: XM_047417644. 1

[0293] > XM 047417644. 1 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant XI, mRNA / cDNA AGACGCCGUCGGGGCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGG GGAGGCGCGGCCCGGGACGCUCGAGCUUAGGGAGGUGAGCGUCGGCGGCAGGCCAAGCCGGUAGGAGGCG GCGCCCGGCACGGGGACUGCCUGCGGCUGCCGAGGCUCCGAAAGCCUUUGUGGGCGCUGGCGGGCACCCC CGCGUCUGUCACCGGCCCCCGCGCCGGGAUCUGGACUCUCGCCCCCCCGCGGGGCGCUCCCUCGCCGUUG CUGGUGCACCCGCGUCCGGCUCGCCGUGGACGCCGGGCCUGGCGUUUCCUCCCGGGCCCGUCCUGCCCAC UCCAUGUGGGCCUCGCGGACUCGGGGCACCGGGACCCCGAGGGCAGGGCGGACGCAGCCGGGCCCCGGUU UCCGAGUCCGUCCCCAGGACGGCCACCCCUCCCGCCUCGGGACACCCAGCGCUUUCCUCUUCAGAGCUGG UGUUUGGCCGGGCGCGGUGGCUCACGCCUGUAAUCCCAGCACUUUGGGAGGCCGAAGCGGGUGGAUCACG AGGUCAGGAGAUCGAGACCAUCCUGACUAACACGAUCUGCCUUCUGGAGACUGCGCCGUCCUCCCGGGAG AGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCCCCAGGUGUCACUGACGUUUGAGGAUG UGGCUGUGCUGUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGU GAUGCUGGAGAACUAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUG UUGCAGCAAGGAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGA GCAGU CAUAAAAC CACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAUGUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAG CAGGAUAAAAAGGGAAGUUUUCAGAUAGUUUCAGCCACCCACAAAAAAAUCCCCACUAUAGAAAGAAGCC AUAAAAAUACUGAAUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAG AGAAAAAACAC CAC CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAA C CAAAAAUUACAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C C UU C GUAAACAU GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAG C CAAAGUU CAGCU CUUAUU CAACAU CAAAUAAC GCAUACU GGAGAGAAAC C CUACAUAU GUAAAGAAU GU GGGAAAGC CUUUACU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU GGAGAAAU C CUACA GAUGUAAAGAAUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGA AGAAAACCCUUGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAA AGAAUU CAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C C UUCGUUAUCAUCAGAGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAG C CAGAGU GC CU CU CUUAUU CAACAU GAAAGAAUU CACAC C GGAGAAAAGC C CUAUAGAU GCAAU GAAU GU GGGAAAGGCUUUACUUCUAUUUCACGACUUAAUAGACACCGAAUCAUUCAUACUGGAGAGAAGUUUUAUA AUUGUAAUGAAUGUGGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGG AGAAAAAC CAU GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUU CAACAU CAG AGAAUGCAUACUGGAGAAAGACCCUAUAAAUGUAACGAGUGUGGGAAAACAUUCAGGUGUAACUCAUCAC UUAGUAAUCACCAGAGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGG C CAAAGUU CAGCU CUUAUU CAGCAU C GAAGGAUU CAU AC AG GAGAAAAAC C CUUUAAAU GUAAU ACAU GU GGAAAAACUUUUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU G AAU GUAAU ACAU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GA AGAGGAC C C CUAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CURef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0294] U GAGAGAGAU GUAAU AAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAA UU C CAU GGAU AAAC CU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAA AUAAC CU GAGAU GAAGAAUUUACAAUU GAAGACAUU GACUUUAGC CAUUU GU GAAAU GGGUUU GCUUUUU CCCUUUUCCUACAGACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGA GGUGUGCUUUAGAUUUCUCAUUAGAAGACCACCAAACUGGUAAUAUUUUUAUAGCAUUUUAAUAGCUUGA U CAAAUU GUAC CUUUUUAGAGAAAAGGAC CAAAAU AAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CU CUGGGGUUUGUCCUUUUCCUACCCUGAUGUCAAACUUAUGCAUGGAUUUCAUUAAAAAAGAAAAAGAAAA

[0295] A

[0296] • SEQ ID NO: 10

[0297] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant XI

[0298] MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVML ENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSSHKTTKSTQTQDS SFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQIVSATHKKIPTIERSHK NTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQLLNQPKITADKRYKCSL CEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQHQITHTGEKPYICKECG KAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQKIHAEENPCKYNPGRKA SSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHTGEKPFKCSECGRAFS QSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTGEKFYNCNECGKALSSH STLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGERPYKCNECGKTFRCNSS LSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPFKCNTCGKTFRQSSSRI AHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0299] • SEQ ID NO: 11

[0300] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant X2, mRNA, NCBI Reference Sequence: XM_054353337. 1

[0301] > XM 054353337. 1 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant X2, mRNA / cDNA GCCCCUCCUGGUCCCCGGCGCGCCGCGGGGUGAGCGGCGCCGGGCCUGAGGUCGCCCAGACGCCGUCGGG GCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGGGGAGGCGCGGCCC GGGACGCUCGAGCUUAGGGAGGUGAGCGUCGGCGGCAGGCCAAGCCGAUCUGCCUUCUGGAGACUGCGCC GUCCUCCCGGGAGAGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCCCCAGGUGUCACUG ACGUUUGAGGAUGUGGCUGUGCUGUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACU UGUACCGGGAUGUGAUGCUGGAGAACUAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAA AGUGAUCUCCCUGUUGCAGCAAGGAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCU CUAGGAUCGAAGAGCAGUCAUAAAACCACAAAGUCAACGCAAACACAAGACUCUUCAUUUCAGGGACUGA UACUGAAAAGAUCCAACAGGAAUGUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAG AUUAGAGAAAAAGCAGGAUAAAAAGGGAAGUUUUCAGAUAGUUUCAGCCACCCACAAAAAAAUCCCCACU AUAGAAAGAAGCCAUAAAAAUACUGAAUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAAC AGAUACUU C C CAGAGAAAAAACAC CAC CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACA AUUACUUAAU CAAC CAAAAAUUACAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUU AACACUU CAU C C CUU C GUAAACAU GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGCCUUUAGCCAAAGUUCAGCUCUUAUUCAACAUCAAAUAACGCAUACUGGAGAGAAACCCUACAU AU GUAAAGAAU GU GGGAAAGC CUUUACU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU G GAGAAAUCCUACAGAUGUAAAGAAUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAA AAAUUCAUGCUGAAGAAAACCCUUGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCU UU CU GGAU GU CAAAGAAUU CAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAG U CUAGCU CAU C C CUU C GUUAU CAU CAGAGAAUU CACACU GGAGAGAAGC CUUUUAAAU GUAGU GAAU GU G GGAGAGCCUUCAGCCAGAGUGCCUCUCUUAUUCAACAUGAAAGAAUUCACACCGGAGAAAAGCCCUAUAG AUGCAAUGAAUGUGGGAAAGGCUUUACUUCUAUUUCACGACUUAAUAGACACCGAAUCAUUCAUACUGGA GAGAAGUUUUAUAAUU GUAAU GAAU GU GGUAAAGC CUUAAGCU C C CACU CAACACUUAUUAUU CAC GAGC GAAUU CAUACU GGAGAAAAAC CAU GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUUCAACAUCAGAGAAUGCAUACUGGAGAAAGACCCUAUAAAUGUAACGAGUGUGGGAAAACAUUCAGGRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0302] U GUAACU CAU CACUUAGUAAU GAG CAGAGAAUU CAUACU GGAGAGAAAC GAU AU C GAU GU GAGGAAU GU G GGAUAUCUUUUGGCCAAAGUUCAGCUCUUAUUCAGCAUCGAAGGAUUCAUACAGGAGAAAAACCCUUUAA AU GUAAUACAU GU GGAAAAACUUUUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGA GAGAAAC C CUAU GAAU GUAAUACAU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUA AAAUU CAU AU C GAAGAGGAC C C CUAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C G AAGAAUACAU C CUU GAGAGAGAU GUAAUAAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU C AUUAGGUAUUUAAUUCCAUGGAUAAACCUCAGCUAUAUAAUAGAUAUGAGGAAAGUGUUUGUGCCUGUCA GACACUUAAAAAAAUAAC CU GAGAU GAAGAAUUUACAAUU GAAGACAUU GACUUUAGC CAUUU GU GAAAU GGGUUUGCUUUUUCCCUUUUC CUACAGAC GUAUAU GCUAGAU GU CAC GU GAU CAU CAGAAACAGAUAU C C GAGUGGGUGGGGAGGUGUGCUUUAGAUUUCUCAUUAGAAGACCACCAAACUGGUAAUAUUUUUAUAGCAU UUUAAUAGCUU GAU CAAAUU GUAC CUUUUUAGAGAAAAGGAC CAAAAUAAAAGAAAAAU GAAUUAU GAAC UACCUCUCAGUCUCUGGGGUUUGUCCUUUUCCUACCCUGAUGUCAAACUUAUGCAUGGAUUUCAUUAAAA

[0303] • SEQ ID NO:12

[0304] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant X2

[0305] MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVML ENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSSHKTTKSTQTQDS SFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQIVSATHKKIPTIERSHK NTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQLLNQPKITADKRYKCSL CEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQHQITHTGEKPYICKECG KAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQKIHAEENPCKYNPGRKA SSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHTGEKPFKCSECGRAFS QSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTGEKFYNCNECGKALSSH STLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGERPYKCNECGKTFRCNSS LSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPFKCNTCGKTFRQSSSRI AHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0306] • SEQ ID NO: 13

[0307] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant XI, mRNA, NCBI Reference Sequence: XM_054353338.1

[0308] > XM 054353338. 1 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant XI, mRNA / cDNA GCCCCUCCUGGUCCCCGGCGCGCCGCGGGGUGAGCGGCGCCGGGCCUGAGGUCGCCCAGACGCCGUCGGG GCGGAGCCGGGUCGCGAGGCUGCAUCCCGGCCGGCGGUUCCGGAGCCUCGCGGCUGGGGAGGCGCGGCCC GGGACGCUCGAGCUUAGGGAGGUGAGCGUCGGCGGCAGGCCAAGCCGGUAGGAGGCGGCGCCCGGCACGG GGACUGCCUGCGGCUGCCGAGGCUCCGAAAGCCUUUGUGGGCGCUGGCGGGCACCCCCGCGUCUGUCACC GGCCCCCGCGCCGGGAUCUGGACUCUCGCCCCCCCGCGGGGCGCUCCCUCGCCGUUGCUGGUGCACCCGC GUCCGGCUCGCCGUGGACGCCGGGCCUGGCGUUUCCUCCCGGGCCCGUCCUGCCCACUCCAUGUGGGCCU CGCGGACUCGGGGCACCGGGACCCCGAGGGCAGGGCGGACGCAGCCGGGCCCCGGUUUCCGAGUCCGUCC CCAGGACGGCCACCCCUCCCGCCUCGGGACACCCAGCGCUUUCCUCUUCAGAGCUGGUGUUUGGCCGGGC GCGGUGGCUCACGCCUGUAAUCCCAGCACUUUGGGAGGCCGAAGCGGGUGGAUCACGAGGUCAGGAGAUC GAGACCAUCCUGACUAACACGAUCUGCCUUCUGGAGACUGCGCCGUCCUCCCGGGAGAGCCAGAAAGAGG ACAUGGCUGCUGGGCAGCGGGAAGCGAGGCCCCAGGUGUCACUGACGUUUGAGGAUGUGGCUGUGCUGUU UACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGUGAUGCUGGAGAAC UAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUGUUGCAGCAAGGAG AAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGAGCAGUCAUAAAAC CACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAU GUA CCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAGCAGGAUAAAAAGG GAAGUUUU CAGAUAGUUU CAGC CAC C CACAAAAAAAU C C C CACUAUAGAAAGAAGC CAUAAAAAUACU GA AUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAGAGAAAAAACACCA C CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAAC CAAAAAUUACAG CAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C CUU C GUAAACAU GARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0309] GAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAGC CAAAGUU CAGCU CUUAUUCAACAUCAAAUAACGCAUACUGGAGAGAAACCCUACAUAUGUAAAGAAUGUGGGAAAGCCUUUA CU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUAGU GU GGAGAAAU C CUACAGAU GUAAAGAAU G UGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGAAGAAAACCCUUGU AAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAAAGAAUUCAUUCUA GAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C CUU C GUUAU CAU CA GAGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAGCCAGAGUGCCUCU CUUAUUCAACAUGAAAGAAUUCACACCGGAGAAAAGCCCUAUAGAUGCAAUGAAUGUGGGAAAGGCUUUA CUU CUAUUU CAC GACUUAAUAGACAC C GAAU CAUU CAUACU GGAGAGAAGUUUUAUAAUU GUAAU GAAU G UGGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGGAGAAAAACCAUGU AAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUU CAACAU CAGAGAAU GCAUACU G GAGAAAGAC C CUAUAAAU GUAAC GAGU GU GGGAAAACAUU CAGGU GUAACU CAU CACUUAGUAAU CAC CA GAGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGGCCAAAGUUCAGCU CUUAUU CAGCAU C GAAGGAUU CAUACAGGAGAAAAAC C CUUUAAAU GUAAU ACAU GU GGAAAAACUUUUA GACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU GAAU GUAAU ACAU G U GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GAAGAGGAC C C CUAG AAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CUU GAGAGAGAU GUA AU AAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAAUU C CAU GGAUAAA CCU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAAAUAAC CU GAGAU G AAGAAUUUACAAUUGAAGACAUUGACUUUAGCCAUUUGUGAAAUGGGUUUGCUUUUUCCCUUUUCCUACA GACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGAGGUGUGCUUUAGA UUU CU CAUUAGAAGAC CAC CAAACU GGUAAUAUUUUUAUAGCAUUUUAAUAGCUU GAU CAAAUU GUAC CU UUUUAGAGAAAAGGAC CAAAAUAAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CUCUGGGGUUUGUCC UUUU C CUAC CCU GAU GU CAAACUUAU GCAU GGAUUU CAUUAAAAAAGAAAAAGAAAAA

[0310] • SEQ ID NO: 14

[0311] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant XI

[0312] MAAGQREARPQVSLTFEDVAVLFTRDEWRKLAPSQRNLYRDVML ENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLGSKSSHKTTKSTQTQDS SFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQIVSATHKKIPTIERSHK NTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQLLNQPKITADKRYKCSL CEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQHQITHTGEKPYICKECG KAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQKIHAEENPCKYNPGRKA SSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIHTGEKPFKCSECGRAFS QSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTGEKFYNCNECGKALSSH STLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGERPYKCNECGKTFRCNSS LSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPFKCNTCGKTFRQSSSRI AHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0313] • SEQ ID NO: 15

[0314] Homo sapiens zinc finger protein 354A (ZNF354A), transcript variant X3, mRNA, NCBI Reference Sequence: XM_054353339. 1

[0315] > XM 054353339. 1 PREDICTED: Homo sapiens zinc finger protein 354A ( ZNF354A), transcript variant X3, mRNA / cDNA UCCUGCCACUGCGCUACUGCACUCCAGUCUGGCCAACAGAGCGAGACUCCGCCUCAAAAAAAAAAAAGCC GUUUUCCGUGGAGUCGGGAGAUGAUUCGUUGGCGGGAGAGAGAGGAACCGGCUGGGAAAGGCUUGAGGCG GAGGGAAGUCGUCCCGGGGCCGUCGUGGUGGGAGUCCCGGCCCGCCUCGCAGCGGCGUGAACGGGGCAGG UGCCCCGGCGCUGCCUCAGUUUCCUGAGCGGUGCAGUGGGUGCUGGCAGUGCCAGCUGGCGCCGAGGAAC UCAGCGGCGUGGGGCGAGCCCUGGCCCUGGUGGGCUCAGCGGGUCGCUGCUGCCACUGCGGCUCCAGCGU CCCCUCCGUAAGCCCCAAGCCUGUGGGGCCUGGGCCUGGCCGGGCGGCCCAGCGCUGCUCUGGUCCGCGG GCUCCUGGCUCCUCCCGAAUCCCUGUGAGGGCGCGCGGGGUCCUUCUCAGCGGGAGUCGGGGUUUUAGAG CUGCGGAUUCCAGGGCUGGAAAGCAGAAGGGGUUCUUCCUGGCUCCCUUUUUCUUCUCAGAUCUGCCUUC UGGAGACUGCGCCGUCCUCCCGGGAGAGCCAGAAAGAGGACAUGGCUGCUGGGCAGCGGGAAGCGAGGCCRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0316] CCAGUGGCUGCACUAGGCCUGAAUCUCACUGUGUGCUGCUCAGUAUACAUUUGUUACUGGCCAAUUAUUC AUUAAACGAAUGAUCUGGAACCUUCAGAAGCUUGAAUAGUGUCACUGACGUUUGAGGAUGUGGCUGUGCU GUUUACCCGAGAUGAGUGGAGAAAGCUGGCCCCUUCUCAGAGAAACUUGUACCGGGAUGUGAUGCUGGAG AACUAUAGGAACCUGGUCUCACUGGGGCUCCCAUUUACCAAACCAAAAGUGAUCUCCCUGUUGCAGCAAG GAGAAGAUCCCUGGGAGGUGGAGAAAGACGGUUCUGGCGUCUCCUCUCUAGGAUCGAAGAGCAGUCAUAA AAC CACAAAGU CAAC GCAAACACAAGACU CUU CAUUU CAGGGACU GAUACU GAAAAGAU C CAACAGGAAU GUACCUUGGGAUUUGAAAUUAGAAAAGCCUUACAUAUAUGAAGGCAGAUUAGAGAAAAAGCAGGAUAAAA AGGGAAGUUUU CAGAUAGUUU CAGC GAG C CACAAAAAAAU C C C CACUAUAGAAAGAAGC CAUAAAAAUAC UGAAUUGAGCCAAAACUUCAGCCCAAAGUCAGUGCUUAUUAGGCAACAGAUACUUCCCAGAGAAAAAACA C GAG CAAAAU GU GAAAUACAAGGAAACAGC CU CAAACAGAAUU CACAAUUACUUAAU CAAC CAAAAAUUA CAGCAGAUAAAC GCUAUAAAU GUAGU CU GU GU GAAAAAAC CUU CAUUAACACUU CAU C C CUU C GUAAACA U GAGAAAAAC CAUAGU GGAGAGAAACUAUUUAAGU GUAAAGAAU GUU CAAAAGC CUUUAGC CAAAGUU CA GCUCUUAUUCAACAUCAAAUAACGCAUACUGGAGAGAAACCCUACAUAUGUAAAGAAUGUGGGAAAGCCU UUACU CU CAGUACAU C C CUUUAUAAACAU CUAAGAAC C CAUACU GU GGAGAAAU C CUACAGAU GUAAAGA AUGUGGUAAAUCCUUCAGCCGAAGGUCAGGCCUUUUUAUACAUCAAAAAAUUCAUGCUGAAGAAAACCCU UGUAAGUAUAAUCCGGGUAGGAAGGCAUCUAGUUGCAGCACAUCCCUUUCUGGAUGUCAAAGAAUUCAUU CUAGAAAGAAGU C CUACUUAU GUAAU GAAU GU GGCAACAC CUUUAAGU CUAGCU CAU C C CUU C GUUAU CA UCAGAGAAUUCACACUGGAGAGAAGCCUUUUAAAUGUAGUGAAUGUGGGAGAGCCUUCAGCCAGAGUGCC UCUCUUAUUCAACAUGAAAGAAUUCACACCGGAGAAAAGCCCUAUAGAUGCAAUGAAUGUGGGAAAGGCU UUACUU CUAUUU CAC GACUUAAUAGACAC C GAAU CAUU CAUACU GGAGAGAAGUUUUAUAAUU GUAAU GA AUGUGGUAAAGCCUUAAGCUCCCACUCAACACUUAUUAUUCACGAGCGAAUUCAUACUGGAGAAAAACCA U GUAAAU GUAAAGUAU GU GGAAAAGC CUU CAGACAGAGUU CAGCU CU CAUU CAACAU CAGAGAAU GCAUA CU GGAGAAAGAC C CUAUAAAU GUAAC GAGU GU GGGAAAACAUU CAGGU GUAACU CAU CACUUAGUAAU CA CCAGAGAAUUCAUACUGGAGAGAAACCAUAUCGAUGUGAGGAAUGUGGGAUAUCUUUUGGCCAAAGUUCA GCUCUUAUUCAGCAUCGAAGGAUUCAUACAGGAGAAAAACCCUUUAAAUGUAAUACAUGUGGAAAAACUU UUAGACAAAGCU CAU CAC GUAUU GCACAU CAGAGAAUU CAUACU GGAGAGAAAC C CUAU GAAU GUAAU AC AU GU GGGAAACUUUU CAAC CAUAGGU CAU C C CUUACUAAU CAUUAUAAAAUU CAU AU C GAAGAGGAC C C C UAGAAAGUAGAUUU GUAU GU GU GAAAGC CUUAAAC CAAAGCU CAU C GAAGAAUACAU C CUU GAGAGAGAU GUAAU AAAU GUAAU GGAU GU GAAAAAAACU GUAAUAAUUUAGC C CU CAUUAGGUAUUUAAUU C CAU GGAU AAAC CU CAGCUAUAUAAUAGAUAU GAGGAAAGU GUUU GU GC CU GU CAGACACUUAAAAAAAUAAC CU GAG AU GAAGAAUUUACAAUU GAAGACAUU GACUUUAGC CAUUU GU GAAAU GGGUUU GCUUUUU C C CUUUU C CU ACAGACGUAUAUGCUAGAUGUCACGUGAUCAUCAGAAACAGAUAUCCGAGUGGGUGGGGAGGUGUGCUUU AGAUUU CU CAUUAGAAGAC CAC CAAACU GGUAAUAUUUUUAUAGCAUUUUAAUAGCUU GAU CAAAUU GUA C CUUUUUAGAGAAAAGGAC CAAAAU AAAAGAAAAAU GAAUUAU GAACUAC CU CU CAGU CUCUGGGGUUUG UCCUUUUC CUAC CCU GAU GU CAAACUUAU GCAU GGAUUU CAUUAAAAAAGAAAAAGAAAAA

[0317] . SEQ ID NO: 16

[0318] Homo sapiens zinc finger protein 354A (ZNF354A), Translated Protein of transcript variant X3 MLENYRNLVSLGLPFTKPKVISLLQQGEDPWEVEKDGSGVSSLG SKSSHKTTKSTQTQDSSFQGLILKRSNRNVPWDLKLEKPYIYEGRLEKKQDKKGSFQI VSATHKKIPTIERSHKNTELSQNFSPKSVLIRQQILPREKTPPKCEIQGNSLKQNSQL LNQPKITADKRYKCSLCEKTFINTSSLRKHEKNHSGEKLFKCKECSKAFSQSSALIQH QITHTGEKPYICKECGKAFTLSTSLYKHLRTHTVEKSYRCKECGKSFSRRSGLFIHQK IHAEENPCKYNPGRKASSCSTSLSGCQRIHSRKKSYLCNECGNTFKSSSSLRYHQRIH TGEKPFKCSECGRAFSQSASLIQHERIHTGEKPYRCNECGKGFTSISRLNRHRIIHTG EKFYNCNECGKALSSHSTLIIHERIHTGEKPCKCKVCGKAFRQSSALIQHQRMHTGER PYKCNECGKTFRCNSSLSNHQRIHTGEKPYRCEECGISFGQSSALIQHRRIHTGEKPF KCNTCGKTFRQSSSRIAHQRIHTGEKPYECNTCGKLFNHRSSLTNHYKIHIEEDP

[0319] • SEQ ID NO: 17 - Genomic sequence https: / / www.ncbi.nlm.nih.gov / gene / 6940

[0320] > NC 000005. 10: C178730659-178711512 Homo sapiens chromosome 5, GRCh38.p14 Primary Assembly

[0321] AGACGCCGTCGGGGCGGAGCCGGGTCGCGAGGCTGCATCCCGGCCGGCGGTTCCGGAGCCTCGCGGCTGGRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0322] GGAGGCGCGGCCCGGGACGCTCGAGCTTAGGGAGGTGAGCGTCGGCGGCAGGCCAAGCCGGTAGGAGGCG GCGCCCGGCACGGGGACTGCCTGCGGCTGCCGAGGCTCCGAAAGCCTTTGTGGGCGCTGGCGGGCACCCC CGCGTCTGTCACCGGCCCCCGCGCCGGGATCTGGACTCTCGCCCCCCCGCGGGGCGCTCCCTCGCCGTTG CTGGTGCACCCGCGTCCGGCTCGCCGTGGACGCCGGGCCTGGCGTTTCCTCCCGGGCCCGTCCTGCCCAC TCCATGTGGGCCTCGCGGACTCGGGGCACCGGGACCCCGAGGGCAGGGCGGACGCAGCCGGGCCCCGGTT TCCGAGTCCGTCCCCAGGACGGCCACCCCTCCCGCCTCGGGACACCCAGCGCTTTCCTCTTCAGAGCTGG TGTTTGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAAGCGGGTGGATCACG AGGTCAGGAGATCGAGACCATCCTGACTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAAAT TAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGTGAGGCCGAGGCAGGAGAATGGCGTGAA CCCAGGAGGCGGAGCTTGCAGTGAGCCGAGATCAAGCCCTGCACTCCAGCCTGAGCGACAGAGCGAGACT CCGTCTCAAAAAAGAAACATCGTGTTAGAAATGGGATACATGGGGTCGGGCGTGGTGGCTCACGCCTGTA ATCCTAGATTTTTGGGATGCCAAGGCGGGCAGATCACCTGAGATCAGGAGTTCGAGACCAGCCTGGGCAA CACGGTGAAACCTCGTCTCTACTAAAAATACAAAAATGAGCCGGGCGCCTGTAGTCCCAGCTACTCGGGA GGCTGAGGCAGGAGAATGGCTTGAACCCGGGAGGCAGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCGC TACTGCACTCCAGTCTGGCCAACAGAGCGAGACTCCGCCTCAAAAAAAAAAAAGCCGTTTTCCGTGGAGT CGGGAGATGATTCGTTGGCGGGAGAGAGAGGAACCGGCTGGGAAAGGCTTGAGGCGGAGGGAAGTCGTCC CGGGGCCGTCGTGGTGGGAGTCCCGGCCCGCCTCGCAGCGGCGTGAACGGGGCAGGTGCCCCGGCGCTGC CTCAGTTTCCTGAGCGGTGCAGTGGGTGCTGGCAGTGCCAGCTGGCGCCGAGGAACTCAGCGGCGTGGGG CGAGCCCTGGCCCTGGTGGGCTCAGCGGGTCGCTGCTGCCACTGCGGCTCCAGCGTCCCCTCCGTAAGCC CCAAGCCTGTGGGGCCTGGGCCTGGCCGGGCGGCCCAGCGCTGCTCTGGTCCGCGGGCTCCTGGCTCCTC CCGAATCCCTGTGAGGGCGCGCGGGGTCCTTCTCAGCGGGAGTCGGGGTTTTAGAGCTGCGGATTCCAGG GCTGGAAAGCAGAAGGGGTTCTTCCTGGCTCCCTTTTTCTTCTCAGATCTGCCTTCTGGAGACTGCGCCG TCCTCCCGGGAGAGCCAGAAAGAGGACATGGCTGCTGGGCAGCGGGAAGCGAGGCCCCAGGTGAGCTCAT TGCCCTCCCAGATCCCAGTGGATGGATTTTCCCCACTAACTGGAGTTCTTCACCCGGGACCATCTCCAGC CAGGATGGAGCCCAAGTCCTTTTCTCTTGGAGGAGCTGGTCCGCCCTCATGAGTGGTGGATTCTGTAGGG AGGACCACCCACCCTCCCCACCCGTGTCTAGTGGTTCTCTTTTTTTTTTTTTTTTTTGAATCTCGCTTTG TCGCCCAGGCAGTGGCGGGATCTCGGCTCGCTGCAACCTCTGCCTCCCAGGTTGAAGCAATTCTCCTGCC TCAGCCTCCCAAGTAGCTGGGATTACAGGCTCCCGCCATCACGCCCGGCTAAGTTTTTTTTTTTGTGCTT TTATTAGAGATGGGGTTTCACCACGTTGGCCAGGCTGGTCTCAAACTCCTGACCTCAGGTGATCCACCCG CCTCAGCCTCCCAAAGTGCTGGGATTACAGATGTGCGCCACTGCACCCAGCCCGAATAAATCATTTTCTG T ACACAT T TAT TTGTGTGTGTGTGTTTGT GT AT GCAT GT TAG CAT CT AT GT TAG CAT T T T AAC CT T T T T T TAT T GT GGCAAAAT AT AT AT AACAT AAAAT AT AC CAT T T T AACT T T T T AAGT GT AT AAT T CAGT GGT AT T TTAGGTACTTCCGCATTGCTGTGCAGCCATCATCGCTATCCATCTTTATTGTGTAGATTATCACATTTAT TTCTTAACCCTAAGGGGCAGGCATTAAAAGGCAGTTTTACAGAGTAGTTAAGAATGCAAGCTCAGTGAGA CATCTGCTGGATTTGAATTACACAAGAAAAAACAAAGTCACAGAGAGGATACAAAATTTGTAGAAGTCAG CCGGCCACAGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGTCGAGGCCGGCAGATCGCCTGAGGTT AGGAGTTTGAGACCAGCATGGTGGAACCCTGTCTCTACTAAAAATACAAAAATTAGCCGGGCATGGTGTC TAGCACCTGTAATCCCACCTACTCAGGAGGCCGAGGCAAGAGAATCATTGGAACCCGGAAGGTGGAGGTT ACAGTAAGCCAAGATCATGCCATTGCACTCCAGCCTAGGCAACAAGAGCCAAACTCTGTCTCAAAGAAAA AAAAAAAT GT GTACAAGT CACACAGCT GGT CAGT GACT GT GT CT GT GT GACCT GGACAAATTT CTTAT CT TGTCTGTGACTTTGTTTTCTCTTGTGTAAAGTGGCAACAATAATGGTATCGTCTTGATAGGATTGTTGTG AATGTTAAATAAAGATTATACACTGCAAATTAACATAAAGCTCTAAGATCAATACTTGGCCCATAGAAAG CCTTCAATAAATTATAGCTATTTTACTTTTCGTCATTACTATCCTTAAGCTTGAGTAAGCACATAGAGAG TACCTTCAGTTGCTTAACCTAACCTCCATCATTAAGTATTTAGATTATTCCTGGTTTTTTGCTCTTAAAA ACACCATGAATATGTTTGTGTGTACTTTTGTGCACTGTTCATTATCGTACAAATAGAGTAATTGGTACTT TAAAGAGTATACTTTGAGGGTTCTAATATGTATTAGCAAATCGCCATCAGAAAGAGTTTTCTAATTAACA CTCCCACTAATGGCCGTAATTCCTCTACCCATCTCTTACAGTGGCTGCACTAGGCCTGAATCTCACTGTG TGCTGCTCAGTATACATTTGTTACTGGCCAATTATTCATTAAACGAATGATCTGGAACCTTCAGAAGCTT GAATAGTATGGGAAGTGTTTCACGGGTAGCGTGTCTTTCCCACTGCCGCCCCCTCTGCTCTGTGAGGGTG GTCCTGGGTGAGCTGGAACGACTTGTCCTTACAGGTGTCACTGACGTTTGAGGATGTGGCTGTGCTGTTT ACCCGAGATGAGTGGAGAAAGCTGGCCCCTTCTCAGAGAAACTTGTACCGGGATGTGATGCTGGAGAACT ATAGGAACCTGGTCTCACTGGGTAAGGAAATTTTCCCTCTAGAAACAGAATTCAAAAATTGGGATATCTC AGCACCTCCTTCCCTGAATAAAAGCGGTTGGTCACTGAAAAATTTGAGCTGAGTGTGTGAGGATTGTACA AATCAGATCTTTATAAATAAACATTAAAAACCTATTCCCCCTTATAGGCATGAAATTGCCCTGTGTCTGG GAATGGAAGAGGTGGTATTGATAATCATTCCTCTCTCTCTATGTAATCACACACTCCAAGTTTGTCCATG ATTCAGGCATCGTTTCCCAACGATTCTGTAGTTTGCTTGTCTCCCAACAGGGAGCTTCATTCAGCCTCTA GGCCTACCACCTGCCTTTCCCACTTGTCTTTTCCAGGGATTAGCAAACTTTTACTATAAAAAGCCATGTA GGCTGGGCATGGTGGCTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGCAGATCATGAGGTC AAGAGATAGAGACCATTCTGGCTAACGTGGTGAAAGCCTGTCTCTACTAAAAAACACAAAAAAAATTAGC TGGGTGTGATGCCGGGCGCCTGTAGTCCCCGCTACTTTGAAGGCTGAGGCAGGAGAATGGCGAGGAGCTT GCAGTGAGCCAAGTTCCCGCCACTGCACTCCAGCCTGGGCGACAAAGCGAGACTCTGTCTCAAAAAAAAARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0323] AAAGCCACGTAGTAAAGACTTTAGGCTTTGTGGGCTGCGTAATGGTCTCCGTAACATATTTCTCTTTTTT TTTCCCACCATTCTTTGCACAGGGCCATACATAAACAGGCAGTGGTCTGAATTTGGCCCATGACCCGTAG TTTGCTGATCACTGCCATCCTTAAAAGGGAGTCACATAGGCCGGGCGCGGTGGCTCACACCTGTAATCCC AGCACTTTGGGAGGCCGAGGCGGGCAGATCACAAGGTCAGAAGATCAAGACCATCCTGGCCAACATGGTG AAACCCTGTCTCTACTGAAAATACAAAACTTAGGCGTGGTGGCATGTTCCTGTAGTCCCCGCTACTTGGG AGACTGAGGCAGGAAAATCACTTGAACCCGGGAGACGGAGGTTGCAGTGAGCCAAGATTGCGCCACTGCA CTCCAGCCTGGCAATAGAGCGAGACTCCTCCTCAGAAAAAAAAAGCGGAGTCAGATAGTGTTTTCTCGTT TTAGACAAGCTACCTGGTACATAATCCTTTTCATAGATAACCTACTTAGAAATTCTAATCAGTCTGTATA TCTTAACATTTCCTCTTCTATAGAAAGCTAGTGGTATCCTATTACCAATACTGCATGTTGATCATATTTT CCTAACCTATAATTAATTGTCCCAGAGTCATGTCCTACAAGTCACTTGTCTTTTCCTCACCCTACCGCCT TACCCCCTTACCTCCCAGGGGCACAACTGAAGCCCTACACAGAAGCTCTTTGTACCATCCTGTGAGTTCT GTATTTAAATTTGGGATAACTGTATCAATACAAAACCAAGTCTGATTTGTTTGGTTGTGGTTTTTTGTTT ATGTGCAGGGCTCCCATTTACCAAACCAAAAGTGATCTCCCTGTTGCAGCAAGGAGAAGATCCCTGGGAG GTGGAGAAAGACGGTTCTGGCGTCTCCTCTCTAGGTAAGTGGGTGGCCCGAGGTGCGGAATGGCCGCAGA CAATGGTCTGGTTAATGAGAGGAGGTAGGAACGTTGGTTGGGAAACTCCCTTGAGAATTCTCAGGCCTCA AGAAGTGATGGGGAAGCGGAAGCCCAGGCTCTTAGGCAGGATTCACTTCCCCATGAATCTCTCAGAATCT CTTTCTTACTCTTTCCTGTTGTTTTAGAATAGGGCTGTATTTTCATGTATTCATTCAGCAAACATAGTCT ATTTAAGGAATTTCAAATAATCACTCTGGCTGAAGCTCAGGGTGTATTTGGGAAAGGAGGAGGTGAGGAA GGAAATAGCCTAGAAAGCTAGGGAAGGGCCGCGTGGTAAGTGCTTGGGCACGGTTCTAAGAGGTGGAAAC TGTCCCATGGAGCACTGGAAATAATGGAAGGATTTGGGCAGGAATCATATGGTCCTTGGTATCTTAGAAA AGTTGGTGTGAATCAGAAACAGCTGAGTCTCCATCCGTGTAAGAACAGCTAAGGGACCTGTGAGCATCAC CACCCTGAAAACAAGAGTCCAGGGACGTGGACAGAGCTCCCAAAAAGTTACAGAGCATTGTTAGGACGCC ACCATTTATGTCTAATGAAAATTATGTGTATGTGTAATGTGCACATAAGCAGAGAGAAAGGTTAGGAGGA CACACCTTAGCTGATGACATTAGTTACACTTGGAATGGGATTGAGAATGGGTCCATGGAAGGGGGCATTA CT T ACAT GT AGT AT T T T AAT GT T T CACAAAGAGAAT AT AT T T GT GAC GT T CT TAT GT GAT T T GAAT T CAC AAGAATGGTCACTTTGGAAGCATCGTGTTTGAAAGTGAGGCAGAACGCTGGGCACGGTGGCTCACGCCTG TCATCCTAGCACTTTCAGAGGCCGAGGCGGGCGGATCACCTGAGGTCAGGAGTTTGAGACCAGCCTGGGC AACATGGCAAAACCCTGCCTCTACTAAAAATACAAAAATTAGCCAAGCGTGGTGGGGCAGGCCTGTAATC CCAGCTACTCCGGAGGCTGAGACAGGAGAAACACTTGAACCCGGGAGGCGGAGGTTGCAGTGAGCCGAGA TTGCGCCACTTCACTCCAGCCTTGGCGACAGAGTGAGACTCTGTCTTAAAAAAAAAAAAAAAGGCGGGAA GCCTTGCAGAGCGATTGCAGTCATGGAGGGAGGCAGGAGGTGGAAGGGCCTGGATGAAGGCACTTGCCGT GAGTTCGTGAGGGAGGAGGCGGCGCCCACGTGATCAGGGTACAGGAGCAGTTGGGTGTGGGATGGATTTC ACTTGAGATGTGAGTGAAGTCGTCAGGTGGGGAACCCTGGTGGGCAGGTGTGTAAGTGACAGGGCCGAGA CTCAAGGGAGGTCAGGGAGCCCATTAGCATGATGGGCATTAGAAGCTTGGAGCTGGCCAGGTGAGGGGGC CAAGTGGAGGCCCGAATTTCAGGAAACAGCATTTGCGAGGTGCTTAAGCAGGAGGAATCTGACAGAGCCC GAGAAGTAATCAGAGTAGAGAAACGGGGAGGGGCCATCGTGGCAGTGGGGGACTGAGAGTGGTTGAACTG GGGGGGCTCTGGCTCTGGGGGATCAAGGTTGAACCCTCGCCCACAGCTTTGTATAGCATTGGGCAGAAAG GGACACAGGTCTCCTGCTTCTGGAGTGCACGATCTAGTGGGGGACAAGGTGAACACACTCATTGCACACA AGGCACAGAAGGACACATTCCAGTGAGGACCACGTGGGAAAGACACTCGGTGCTGTGAGAGTGCAGAGCA GGCAGGTGGCGTCAGCAAAGGCTTCCCTGTGGAAGGGCCTGGGATCAAAGGACTGAAGAATTGCTCAGGC CTAAAGAAAGGTGTTCATGAAGGTCCAGAGAAGCAAAGCCAGCGAGGAGAAGGTGGAGCACCCGAGAGAG AGCCACGGGCCCTGTCACAGGGTTTGGTTGGTGCTTGAGAGCCAGGCACAGGTTCTGAGGAGCCGTAAGT AGCCCAGGGCAGGCCTAGGCTGCTTTCTGCAGCCGCCAGCCCATGTGGAGAACCTGACGGCCACAGTGCA GGGCTTGTCAGGTCCAGAGGGCTCAGTGATGGCCCCTTATGCCATGGGCCCAGCAAGGAGCTGCAGGGCA GGCAGCCTCGGTCCCCACAGCCCCTGTGCTGGGGAGAGCTTTCTCCTCCCTGAAGAAAGGTGAGATTTTT TTATATGGGAACCACACTTGTTTATCTTTTTAACTCTCCTTTGGGAGAAATACACTACGTGGTCTGGCCC TGGCTACCTCTTCACTCTCAGCTCCTTCCACTCTGCCCTTCATTCGAGGCCTCTGATCACCCTTGGCCGC CCTGCAGTGTGTCATGGGGGGCGCCAAGCAGGCCCCCACTCAGGGCCTCTGCCCTGCTTTTTCTGTCTGG GACTGTTCTTCGTGGCGGTCATGCGTCCATGTGGCTCGCTCACTTCCTTGTGCTGCACAGGCTTCGGCTC ACACAGGCACAGATCATCTGACACGCAGCCCTGCAGCCCCCACGCCATCTCCTCACCGTGCTTTATTTTC CCTCCTCGTGCTTAGCGCTGTGTAATAGATCTGTTTCTTCATGATCTGTCTCTGCCTCTGCATTGTAAGC TGCTGTGGGAGGGGTTGCCTGTTTCATTCACTCCTGTTTTCCAAGAACCTAAAACAGTCCCTGGCCAGGG TATGCGTCCAGTTGTATAATGAATGCACGAGTGACTTGGACTAGGTTAGTGGTGTTGGAGACAGAAAGAA GTGAACCAGTCCCAGGGATGTCCAGGAGATACAGCCAGCAGGACTGTTAGACATGGAAGATAGAGGAGAT GGGGTAACAAAGAAAATAAAAGCAAACAAAAGGGAGATGGGGTTGTCAGGGATGCTTGCTTGGTTTCTGG TTTGTTCTGTGACGGTTGGTGATGTCATTCAGTGAGATAGGAACACTAGAAGGAGATCAGATTTGGAGAT GATGTAAATTTGGTTTTAGATTAATTGAGCTGTTTTTGGTTGACCTAGTTATCCCAGTGGCGATGACAAG TAGTGTTGATTTCGGAGTACAGTACCTCAAAATGTAGACTGAGCTTGAAATGTAAATTTGGGAGAACCTG ATCAATATAGGGTGATTGAAATAATGGGAGAGGATGAGTTGGCCTAGGGAGAGTGTTCACATTACAGTAA GAGTAAGAAAGAGCCTAAGACTGAATCTTGAGGAACTCCAGCATTTCAGGAAGCCAGGCAGAGAGAAATG AACCCATAGGGAAGACAGGTAAAAGCGTACTGTAAGGAAAATCAGGAGAGGCAGGTGACATTGGAGGCAARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0324] GGCT CAT GAT GGAAT GGTAGCAGTAGAT GGGGAAAAGTAGACGAAT GT GAGGCAGATTTAGAT GGCAGAA TAAACAGGTCTTGGTAATTGATTAGATGAGGGGGCTGAGGGAGGGAGAATTATTAAGGATAACTCTTAAG GTTTTGATTCAGAGGTGGTGCCATTTACAGAGACAAGGGGTTGACCAGGTCGGAGGGAAGAATCGTGAGT GTGGTTTTAGGTGTGTTGACTTTGAGGCATCTGAACTGCTCTTTTGAGTAGGTCGTCTGTAAGGGGGAGT CGAGTATGGGAGATATCTTGCTTAGAGGATGAGTCTATGATTGAAGTTGCCTGTGGAAAGATGCAGAGTG AGAAGAACTGGGGGCCTAGAACTAAAGCTAAGGCACTCCAAGGATGATAAACTGTGGTACGTCCACACAG CT GTAAAGT GGAAT GAGGAGGACCT CT GTAACTT CTACTAT GGAGT GAT CT CCAGAAT GT GAGAATAAGT GCAGAACAGCACAGTTAGTACT GTTAT CTAAGAAAGGT GGGT GGGAATACCAAGAAAAGT GT GT GT GTAT TTACTTACACTGTCATGGAGCACATAATGTTTCAGTCAATGATAGCCTACATATATGATGATGCTCCCAT AGATGATAATACTGCATTTTTCCTGTCCCTTTTCTATGTTTTAATATGTTGAGATGCACAAATACTTACA ATTGTGTTACAGTAGCCTACAGTATTCAGCTAAGTAACATGCTGTATAGGGGTATAGCCTAGGTGTGTAG TAGCCTATACCATCTAGATTTGTGTAACACACTCTGATGACTGCACAACATCGCCAACAACGCGTATCTC AGAAC AT AT C C C AGT CAT T AAG C AGT G C AAT TATTTTTTT T AGAAAGAT AAAAG C AAAAGAAAAAAAAG G AGAT CAATAAAAAT GGTTAT CT CTAAGGT GAAGGAAGAAAAAGGAT GGAACAGGGGTAGAAT CTAT GACT TTTTGAGGTCTCAAAATTTTGACTTCAGAACTATAAAAATGGTTTATAAAATTATAAGACAAAATTAAAC AAAAATGAGAAAGTACCCCCTACATATTGAAAGCAGAATGAAACAACCCTAATTGTGTTCCAGGTTGGTG GCTTAGCCACAAAAAGAATTATTTGAATTGCCTTTACATTGCAGCAGTTTGACTTATTAGTGTAGGTATT CTAGGGGCAAAAGTAACTGCAAGCAGTGGTGTTAATTTTGATGGATGTGCTAAGAGCGGTTTTGATGTTT TTACAGATGAAATGATTTTGCTGTCTGGGATGTGCTCTAGCATAACCTAGTGGACGAGATGAAAGGGTTT GTGTTAGTTTGCTAGGGCTCCATACTGAAGTACCACAAGCTTCGTGGCTTAAAACAGTAGAGATTTACTG TCTCACAGTTCTGAAGGTCAAAATCAAGATATTCGCAGGGCCCCGCTCCCTCTGAGACTCTGCATAGAAT TCTTCCTTGCCTTTTGCTAGCTTGTAGCGATGGCCAACTTGGAGATCTCCGTGGCCTGCAGCTGCATCAG TCCTATCTGTGCCTCCGTCTTCACACTGTGTTCGCCCGGTGTCTTCACATCATCTTTCCTTCTGGATGCC TGTGTCCAAATTTCCCCAGTTTACGGGGACAGCAGTCACGCTGGATTAGGGCTCACCCCAATGACTTCAT CTTAATTTGATTGAACGTGCAAAGACCATATTTTAAAGTAAGGTCACGTTCTGAGGTCCTGGAAGTTAGG ACTTCTACGTCTCTCTTTTGAATGATGCGGTTCAGCCCATAACAAGGTTGTCAATAGAATAGTGTTGCTG GTTATTGAAACTGGCTGGAGATTCACTGTACAATTCACACTACTTTTACATATGCTGAAAATTTCTATGT TAAAAGCTTTAAAAATAAGAGTTACCACAACAACATTTCTAAAACAGCTTTTCTCTTTTCCACCAGCACA AAGATGATAGAGGATGTTTACAAATAGGCAGTACTACCCCCATCATGGTTTATGTGCAGTTCCCAGCAGA TTGCTCCTTGAGTTCTCTGACTCTAGAAAACCTTGTGAGACTGCAAATGCAAGTGACATTTTTTTTTTTT TTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCGGTGGCGGGATCTCGGCTCACTGCAAGCTC CGCCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCGCCCGCCACT ACGCCCGGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTTTTAGCCGGGATGGTCTCGATC TCCTGACCTCGTGATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCC GGCCGCAAGTGACATTTTTATAGGTGGAGCTTTTACTCTGACTAGCTATAGGATAAGGTGGGGGTGGATA CTTCAGCAAATAAGTCTGGAAAACTGACTTGCATTCAAAGACTTGGGTGCTGCCTTCAGCCCTTGTGCTA CCCCTTCATCTAGCCTATGGCGATTCCTTTCACATCCTATCTTATATGTCAATTCTTACGAAACACTCCA GCAGCGTCCTGCTGCACCCCTTTTCTACCTGCACCCTCACTATCTAAGGACAGTTGGATATGATTCTAAG CTCGTGTTCACGTCCATTTCTAGCTTATGTTTCTATTGTAGAAATTGTCACATTGTATTGTGATTACCTA CTGATGTCTTCATTTCTAATGTCTGACTATAAACTCTCTGAAGAAATCAACTGTTCCTTTTAATTACTGG ATCCGTAGGGAACTGTGGATCACCGATGCCTAGGATAGAGCCTAATACATAGTAGGTACTTTTTAAATAT TTGATAAATTAATAAAGTAAGGCGTGACCAATGCTTTTTGAAATAACATGACCAATTCTGTGTATTAAGA GGGGTAAAT CCAGT GGGAAATAATACT GGAAAGGCAAGAAACAGGT CAAAGGAATAACTTAAGAT CAGGA GCAAGTCACTCTACAGTGAGGAGATCCTGAACTTAGACATATTCACGATGGCTTATTAGAGACATTTTAG TGATGAGATTTAAGAGAATGTGCAGCCTGGGCAACACAATGAAACCCTGTCACTACGCAAGATAAAAATA TTAGCCAAGCTTGGCAGTGTGCACCCGTAGTTCCTGCTGCTCAGGAGGCTGAGGTGGGAGAATCGCTTGA GCCCTGGAGTTTGAGGCTGCAGTGAGCTATGATGGCACCAGTGCATTTCAGCCTGGGCAACAGAGCAAGA CCCTGTCTCTCAAAAAACAAAAAAATGAAGATTTTGATGACTGCCTAGGGAAGAGAAATTCTAAGATAAA ACTAAGAATTTACGGTAAATTAACTGAAGAGATGATGGTGTCATTTTGAACTGGAAAACATATAAAAAAG GTTTGAAAAGGAATGCTCTGGGTTCAGATTTGGACAGGCTGAATTTAAAAAGAAAGATACACAAGTGGAA ATTCTAACCGGCAGTTGGAAAGACCTCTGTAAAGTATTTTTCAGGATAGTCGTACACATAAGAGGTGTGG GAGTAAAT GAAAT CAT CCAGGGAGATAGGGTT GAAGAAACACT CT CT GGGT GTAGT GAGAGT CCAGGT GG AAGAGTCTAGACCTGATAACAGGAAGTGGTCAGGGCAGTGGCCATCAGCAAGATGATTCTGGCTTCTCAA CTTCTGACCTAGGTCTTCACCTTGTTTAACAGGTAGCTGAGGACTGGGCTTGATTCTAGGAGGAAGCAAA GGTTGAACTGCACCTCAGGAGACCTATTTGTAGGCCTCTAGAAAGTTCTAAACAGATCATAAGGAAGAGT CAGGATATCCACAATCCAAAACTTACGTCTGCTTCTGTGAAAAACATGATTTCAGGACTAAAATATTGGC ACTTTAGAGTTGGGATGAGTTTTCCAGAATTGATAATGGACTTCCGAGTCGTAATCATATCCATTAAAAA ATAAAAGCTCCCCATGTCACATTTGCTTACTCTCCTTTAGAAACTGTTGAAAAGGGTTTCTGCTCTTGAT GTTTCTATTTTCTCACCTCCCATTGTTTCCTCCATGCACTCCAGCTGGGCTTTCTTCCTTATAATTCCAC TGATAGCTCTGTTATTAAATCCCAAACAACCTCCGTCTTGCCACAATCAGGATTAAGTTTTTAGTCCTCA AATTATTTGCCACAGTTATTCACTCCTCCCTGAAGTACTTCTTCCCTTGGCTTCTGGAACACTGTACTCTRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0325] CCTGATATTATTCTTCTTAACCTCGTAATATTTCAGTGTCTAAGTGATTCCTTTCAGTTCTTCAGCTTTA AATCAAACCCACATGCTGCTGACCCCCAAATGCCTGTCTATAGCTCTGACCTCCCCCAGATCTCCAGAAT AATAAATACAATCTAGTATTTCAGTATCTTCCTGCAACTCTATTAAGGCATCTCAGCCATCACAAACATA CCAGAAACAGAATGCCCCCTCAGCCTTGCCCCTTTCCTGGTCTTCCCCTTCTAGCCTTGCCCCTTTCCTG GTCTTTCCCTTCTTGGTGGTTGGTGGCAGCTCTCAGAATTCTGGCTAGAAACCTAGGCATGATCCCTAAC TTTCCTGTTTCCCTCTCAGCTTCCTGGGAGTCCTGAGTCTTGTTATTCTTGGTCCCGCTAGCTCTTCCTT CACTGTACAGCCTGGCATAGTCATCTGTACCTCCAGCCCTTTCTCACGTGAAGAGCTGCAGTAGCTTCTT CAGTAGCCTCTCCCCTCCCTGCTTCCACTGGTGCCCCAGCGTAGTCGGTCCACGCAGCAGCCAACTCAAA TGAACACATAATTCCCCAGTGGCTTCCCGTAGGCTTAAATTCCAGTCCTTAGGATGACCTGGGCCTTAGA GGATTTGTCCCCCACCTCCTGCTCCACTCTTTTGTTTTTTATTTTTTGTTTACTTTTTTTTTTTTTTTTT TTGAGATGGAGTCTCACTCTGTCGCCCAGGCTGGTGTGCAGTCATGTGATCTGGGCTCACTGCAGCCTCT GCCTCCTGGGTTCAAGCAATTCTCTGCCTCAGCCTCCCGAGTACCTGGGATTATGGGCGTGTGCCACCAC ACCCGGCTGATTTTTGTATTTTTAGTAGATAACAGATTTCACCGTGTTGGCCAGGCTGGCCTTGAATTCC TGATCTCAGGTGATGCACCCACCTCGGCCTCCCAAAGTGCTAGGATTGCAGCTGTGAGCCACCACGCCCG GCCCCGTTCCAGGCTTATTTTATCATTTCACCATTTTCCCTCTTCTCGCTCTACTCTGGCCAGTCTCTCT GCTGTTCCTAAGTCAGCCTCACTTTTGTGTGTGTTGGTTCCTTTGCCCAGGGAGCTCTTGAGCACTTTCT TCAGCTATCCACATGACTTTTATTACGTACTTGTTATTTGTCCCTTCATCCCCCTCCTTCCCCAGCACAT GCGCACACTCCAAAGTATAAGCTCCTTAAAGGCTGGAACCCTGTCTTATTGAATACATTATCTGCATTCC CCAGAACATTACCTGGCCCATAGTAGGCACTTAATGAGTACTTGTGAATGGAAAGGATGCTGCCAGCATA T GTAAAGATAAACAAGT CAAGT CAT GGAGT GCT CCT GAAGCT GCAT GTAGTT CAGCAT ACCT GGCAAGAA GCCAGGGAAGAGGGGGT GAGGACAGGT GGATAAAGGT CAGAT CAGATAGTACT CTATT CACCAT GTTAAG GACGCCTAAGAGTCTTCAAGACTCTTAAGAGACTTCAGATGTGAATCATTGAAAATCTATTGGAGGACCT TAAGGAAGTTTGCCACATAGTAAAATATTGATCTTAGAAAGATTGGCCGGGCATGATGGCTCACACCTGT AATCCCAGCGCTTTGGGGAGGCTGAGGTGGGCGGAACATGAGATCAGGAGTTCGAGACCAGCCTGACCAA CATGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCCAGGTGTGGTGGCGCACACCTGTAATCCC AGCTACTTAGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGAGGGGGAGGTTGCAGTGAGCCGAGGTC GCGCCACTGCACTCCAGCCTGGGTGACACAGTGAGACTCCATCTCAAAAAAAAAAAAAACCACAGAGATT ATTCTGGCTGCAGTGTGGAGACTGAATTGGCAGAGGGCAAGACTAGTTAGGAGATTATGGTAGAGATAGT AATGCATGGCAAACAAAATACCACTTAAGTACCTGATCCCCGTGCTCAGGGCTGTAGCAAAGGTGAGGGA CCTTTCTTTACACTAGGCCTAAGGCAGGTTCTGTATCATTGCCTTACAAGCTAGAATCCAAGTTGCAGAA TCTTGCTGCTGCCTTCTTCCCAGCACATATATTAAGCCTAGAAACAACGACCGATATTCACAGGGAAAAA GCACTTCATTTTTGCCACTGCCCCCTCCATATATTTGGCCTAAATAATGTATTAAAATTTGATCACTTAA TTTAGCAAGACTGAAACGGAGAAGGAACTAGGAAGGTGTTAAGCATAAAGTTAAGAAACTCTGCTTTCCC TTACAGTAAATAGTATAAAGATATAAGGCAT GT GAAGTTAGT CAGGGAGGGT GT GT GGAGT GAAAAAAAA ATCCAAAACCCTAGGGAATACCATTATTTAAAGAAGGAATGGAAGAAGAGTTATCCATGAAGGGAACTAG TAAGTGAACACTAATAAGGGAAAAGCAAGATGTATTGTGTTTGTTAAGCAGAAGTAATTTGTAGATGTGT GAAAGTGACTGTCATACAGGAATCGCAAATACAGTGAGACACAAATGGTCCCTTTAAACAGTTACCTCTA CCCCAAGAGAAGATGCTAAGACTATGTGCATAACCGTTCCCCTGAGAGAATTTACTATCTATTGAATTGT GTTCTTATCCAATTTTAATATTTCTTCACTTCTGTTCTTTACTTTTCCATGTTAACCATGCTTTATCTTA TATATCCATGCCTGTCTTATTGTTGATCTTTGACTCTTTTCAGCCATCCCATGTAACCTCTCTTGCTTGA TGCATGAGTAGGCTGCCTTCCTATCTTGCACCTCTTGGCTCTAGGCCTGTCTTGTGGCACCTCTTGTCGA TTAATTCAATAACAGGGCCCTCCTGTTTGAACTGGCCCTGAAACCTCTCTAATCACCCATGTACTGGCCT AGGAGGCAACTT CTACAAGGGT CT GAAAT GT GTAT GT GTAT GTAT GTATAACACAT GT GCATATTT GTAT AT AT AT GAAGT AT GTAT GT GTAT GTAT GTATAACACAT GT GCAT AT T T GT AT AT AT AT GAAAT AC GT AAG AAT AT T T GAC AT TGTTCCAGTTAG GAAT GT AT AAGT AAT T AAG G CAGAT T AAC AAAAC AT AC AAC T T T T G TATATAATT GGAGT GCT GT CAAGAGGAT CT GTAGGAAAATAGGGCATT CT GAT CT GCTT GT GAAGAACAG TTACTT CAGAAAAGGAAAGT GAGATACATACAGACATAGATATAGAACTATAATATACTATATAT GT GT G CATATATTATAAAGGTCAGTAATACATATGTTTAATACTCTGGTAAGTGCTATAGTAAGGAGAAGGGACC GTGGCTGCTCAGAGTCAAAGGATTGAGAGAGTCCATTTTTGAACTGAATCTGAAGGATATAAGGGAATTT GACATGGGGTGGAGGGACTTTCCAGATTAAGAATAATTATATAAAGGCTGAGCACAGTGGCTCATGCCTG TAATCCAAGCACTTTGGGAGGCCGAGGTGGGTAGATCACTTGAGGTCAGGAGTTCAAGGCCAGCCTGGCC AACACGGTGAAACCCTGTCTCTACTAAAAATACAAAAATTAGCCAGGCATGATGGTGGGTGCCTGTAATC CCAGCTACTCAGGAGGCTGAGGCACAAGAATTGCTTGAAGCCAGGAGACCGGTTGCAGTGAGCCGAGATC GCGCCACTGCACTCGAGCCTGGGCGACAGAGTGAGACTGTCTCAAAAAAAATTACACAAAGAAGATAAAT GTCAAAAAAAAAAAACAAAAAAAAATTAGGATTCCATCATGGGTTTTGGGTCCTAACCCCAAACTTCCTC TTTCAACTCATGATCCCTTTATAACCCTTTTCTCTGTTCTTCATTAGCCAATTCTTCTGGTCACTCTTTC TCACCCTCTTTAAATGGGTTTGCTTTATTTTATTCTTCACCCTAGTACTTAAATTGCTGTCAATTATATT TTATTATACTGTCTAACTGTCCTATTTTTTCTTTTTGCAATTTTTTTCATACAGATATCAGGGTCTTATT TAAATGTCACCAATATTTATTCCTTCTACACTTTTAGTCTCAGTAGTCAGATGCTATGATATCACATGAA AC AT T T T T GAT TTTTTTTTTCCCTCCAG GAT C GAAGAG C AGT CAT AAAAC C AC AAAGT C AAC G C AAAC AC AAGACTCTTCATTTCAGGGACTGATACTGAAAAGATCCAACAGGAATGTACCTTGGGATTTGAAATTAGARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0326] AAAGCCTTACATATATGAAGGCAGATTAGAGAAAAAGCAGGATAAAAAGGGAAGTTTTCAGATAGTTTCA GCCACCCACAAAAAAATCCCCACTATAGAAAGAAGCCATAAAAATACTGAATTGAGCCAAAACTTCAGCC CAAAGTCAGTGCTTATTAGGCAACAGATACTTCCCAGAGAAAAAACACCACCAAAATGTGAAATACAAGG AAACAGCCTCAAACAGAATTCACAATTACTTAATCAACCAAAAATTACAGCAGATAAACGCTATAAATGT AGTCTGTGTGAAAAAACCTTCATTAACACTTCATCCCTTCGTAAACATGAGAAAAACCATAGTGGAGAGA AACTATTTAAGTGTAAAGAATGTTCAAAAGCCTTTAGCCAAAGTTCAGCTCTTATTCAACATCAAATAAC GCATACTGGAGAGAAACCCTACATATGTAAAGAATGTGGGAAAGCCTTTACTCTCAGTACATCCCTTTAT AAACATCTAAGAACCCATACTGTGGAGAAATCCTACAGATGTAAAGAATGTGGTAAATCCTTCAGCCGAA GGTCAGGCCTTTTTATACATCAAAAAATTCATGCTGAAGAAAACCCTTGTAAGTATAATCCGGGTAGGAA GGCATCTAGTTGCAGCACATCCCTTTCTGGATGTCAAAGAATTCATTCTAGAAAGAAGTCCTACTTATGT AATGAATGTGGCAACACCTTTAAGTCTAGCTCATCCCTTCGTTATCATCAGAGAATTCACACTGGAGAGA AGCCTTTTAAATGTAGTGAATGTGGGAGAGCCTTCAGCCAGAGTGCCTCTCTTATTCAACATGAAAGAAT TCACACCGGAGAAAAGCCCTATAGATGCAATGAATGTGGGAAAGGCTTTACTTCTATTTCACGACTTAAT AGACACCGAATCATTCATACTGGAGAGAAGTTTTATAATTGTAATGAATGTGGTAAAGCCTTAAGCTCCC ACTCAACACTTATTATTCACGAGCGAATTCATACTGGAGAAAAACCATGTAAATGTAAAGTATGTGGAAA AGCCTTCAGACAGAGTTCAGCTCTCATTCAACATCAGAGAATGCATACTGGAGAAAGACCCTATAAATGT AACGAGTGTGGGAAAACATTCAGGTGTAACTCATCACTTAGTAATCACCAGAGAATTCATACTGGAGAGA AACCATATCGATGTGAGGAATGTGGGATATCTTTTGGCCAAAGTTCAGCTCTTATTCAGCATCGAAGGAT TCATACAGGAGAAAAACCCTTTAAATGTAATACATGTGGAAAAACTTTTAGACAAAGCTCATCACGTATT GCACATCAGAGAATTCATACTGGAGAGAAACCCTATGAATGTAATACATGTGGGAAACTTTTCAACCATA GGTCATCCCTTACTAATCATTATAAAATTCATATCGAAGAGGACCCCTAGAAAGTAGATTTGTATGTGTG AAAGCCTTAAACCAAAGCTCATCGAAGAATACATCCTTGAGAGAGATGTAATAAATGTAATGGATGTGAA AAAAACTGTAATAATTTAGCCCTCATTAGGTATTTAATTCCATGGATAAACCTCAGCTATATAATAGATA TGAGGAAAGTGTTTGTGCCTGTCAGACACTTAAAAAAATAACCTGAGATGAAGAATTTACAATTGAAGAC ATTGACTTTAGCCATTTGTGAAATGGGTTTGCTTTTTCCCTTTTCCTACAGACGTATATGCTAGATGTCA CGTGATCATCAGAAACAGATATCCGAGTGGGTGGGGAGGTGTGCTTTAGATTTCTCATTAGAAGACCACC AAACTGGTAATATTTTTATAGCATTTTAATAGCTTGATCAAATTGTACCTTTTTAGAGAAAAGGACCAAA ATAAAAGAAAAATGAATTATGAACTACCTCTCAGTCTCTGGGGTTTGTCCTTTTCCTACCCTGATGTCAA AC T T AT G CAT G GAT T T CAT T AAAAAAGAAAAAGAAAAA

[0327] • SEQ ID NO: 18

[0328] siRNA sc-91974A

[0329] chr5 - 178140605 178140623. Targeting Exon5-intron4

[0330] Sense strand:

[0331] GGAUCGAAGAGCAGUCAUAtt

[0332] • SEQ ID NO: 19

[0333] siRNA sc-91974 A

[0334] chr5 - 178140605 178140623. Targeting Exon5-intron4

[0335] Antisense strand:

[0336] UAUGACUGCUCUUCGAUCCtt

[0337] • SEQ ID NO: 20

[0338] siRNA sc-91974B

[0339] chr5 - 178139978 178139996. Targeting Exon5

[0340] Sense strand:

[0341] GAGAAAUCCUACAGAUGUAttRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0342] • SEQ ID NO: 21

[0343] siRNA sc-91974B

[0344] chr5 - 178139978 178139996. Targeting Exon5 Antisense strand:

[0345] UACAUCUGUAGGAUUUCUCtt

[0346] • SEQ ID NO: 22

[0347] siRNA sc-91974C:

[0348] chr5 - 178138746 178138764. Targeting 3 ’UTR Sens strand:

[0349] GUCACGUGAUCAUCAGAAAtt

[0350] • SEQ ID NO: 23

[0351] siRNA sc-91974C:

[0352] chr5 - 178138746 178138764. Targeting 3 ’UTR Anti sens strand:

[0353] UUUCUGAUGAUCACGUGACtt

[0354] • SEQ ID NO: 24

[0355] -QIAGEN SI04335226

[0356] chr5 - 178138991 178139011 3’UTR

[0357] Sense:

[0358] UACAUCCUUGAGAGAGAUGUAtt

[0359] • SEQ ID NO: 25

[0360] Antisense:

[0361] UACAUCUCUCUCAAGGAUGUAtt

[0362] • SEQ ID NO: 26

[0363] ORIGENE. SR304750,

[0364] chr5 + 178139838 178139864, targeting exon 5 Sense:

[0365] AAUUCUUUGACAUCCAGAAAGGGAUGU

[0366] • SEQ ID NO: 27

[0367] • Antisense:

[0368] ACAUCCCUUUCUGGAUGUCAAAGAAUU

[0369] • SEQ ID NO: 28Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0370] chr5 + 178140534 178140560 exon5

[0371] • Sense:

[0372] AUUCCUGUUGGAUCUUUUCAGUAUCAG

[0373] • SEQ ID NO: 29

[0374] • Antisense

[0375] CUGAUACUGAAAAGAUCCAACAGGAAU

[0376] • SEQ ID NO: 30

[0377] chr5 + 178138917 178138943. 3’UTR

[0378] • Sense:

[0379] GAGGUUUAUCCAUGGAAUUAAAUACCU

[0380] • SEQ ID NO: 31

[0381] • Antisense

[0382] AGGUAUUUAAUUCCAUGGAUAAACCUC

[0383] • SEQ ID NO: 32

[0384] ZNF354A promoter, ENSEMBL GrCh38: chromosome: GRCh38:5: 178728826: 178731484:1

[0385] GGGTGGGTGGTCCTCCCTACAGAATCCACCACTCATGAGGGCGGACCAGCTCCTCCAAGA GAAAAGGACTTGGGCTCCATCCTGGCTGGAGATGGTCCCGGGTGAAGAACTCCAGTTAGT GGGGAAAATCCATCCACTGGGATCTGGGAGGGCAATGAGCTCACCTGGGGCCTCGCTTCC CGCTGCCCAGCAGCCATGTCCTCTTTCTGGCTCTCCCGGGAGGACGGCGCAGTCTCCAGA AGGCAGATCTGAGAAGAAAAAGGGAGCCAGGAAGAACCCCTTCTGCTTTCCAGCCCTGGA ATCCGCAGCTCTAAAACCCCGACTCCCGCTGAGAAGGACCCCGCGCGCCCTCACAGGGAT TCGGGAGGAGCCAGGAGCCCGCGGACCAGAGCAGCGCTGGGCCGCCCGGCCAGGCCCAGG CCCCACAGGCTTGGGGCTTACGGAGGGGACGCTGGAGCCGCAGTGGCAGCAGCGACCCGC TGAGCCCACCAGGGCCAGGGCTCGCCCCACGCCGCTGAGTTCCTCGGCGCCAGCTGGCAC TGCCAGCACCCACTGCACCGCTCAGGAAACTGAGGCAGCGCCGGGGCACCTGCCCCGTTC ACGCCGCTGCGAGGCGGGCCGGGACTCCCACCACGACGGCCCCGGGACGACTTCCCTCCG CCTCAAGCCTTTCCCAGCCGGTTCCTCTCTCTCCCGCCAACGAATCATCTCCCGACTCCA CGGAAAACGGCTTTTTTTTTTTTGAGGCGGAGTCTCGCTCTGTTGGCCAGACTGGAGTGC AGTAGCGCAGTGGCAGGATCTCGGCTCACTGCAAGCTCTGCCTCCCGGGTTCAAGCCATT CTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGGCTCATTTTTGTATTTT TAGTAGAGACGAGGTTTCACCGTGTTGCCCAGGCTGGTCTCGAACTCCTGATCTCAGGTG ATCTGCCCGCCTTGGCATCCCAAAAATCTAGGATTACAGGCGTGAGCCACCACGCCCGAC CCCATGTATCCCATTTCTAACACGATGTTTCTTTTTTGAGACGGAGTCTCGCTCTGTCGC TCAGGCTGGAGTGCAGGGCTTGATCTCGGCTCACTGCAAGCTCCGCCTCCTGGGTTCACG CCATTCTCCTGCCTCGGCCTCACAAGTAGCTGGGACTACAGGCGCCCGCCACTACGCCCG GCTAATTTTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGTCAGGATGGTCT CGATCTCCTGACCTCGTGATCCACCCGCTTCGGCCTCCCAAAGTGCTGGGATTACAGGCG TGAGCCACCGCGCCCGGCCAAACACCAGCTCTGAAGAGGAAAGCGCTGGGTGTCCCGAGG CGGGAGGGGTGGCCGTCCTGGGGACGGACTCGGAAACCGGGGCCCGGCTGCGTCCGCCCT GCCCTCGGGGTCCCGGTGCCCCGAGTCCGCGAGGCCCACATGGAGTGGGCAGGACGGGCC CGGGAGGAAACGCCAGGCCCGGCGTCCACGGCGAGCCGGACGCGGGTGCACCAGCAACGG CGAGGGAGCGCCCCGCGGGGGGGCGAGAGTCCAGATCCCGGCGCGGGGGCCGGTGACAGARef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0386] CGCGGGGGTGCCCGCCAGCGCCCACAAAGGCTTTCGGAGCCTCGGCAGCCGCAGGCAGTC CCCGTGCCGGGCGCCGCCTCCTACCGGCTTGGCCTGCCGCCGACGCTCACCTCCCTAAGC TCGAGCGTCCCGGGCCGCGCCTCCCCAGCCGCGAGGCTCCGGAACCGCCGGCCGGGATGC AGCCTCGCGACCCGGCTCCGCCCCGACGGCGTCTGGGCGACCTCAGGCCCGGCGCCGCTC ACCCCGCGGCGCGCCGGGGACCAGGAGGGGCGGGGCAGCCGCTGAGGCCGCTGGGAAATG GAGTCCACGAGAGGACACCTGCATTCGGGGAAGTGTAGTTCCATGCGTTTGAGGCGGGGT CCCGGCTGAGCTGTTACGCGCCCACATGCCACAAGAGGGCCCCTGTGAGCTGGAGGGCCC CAGGTTCCTGGTTATAGGGAAGATAAGGGGAGGTTCGCTGAGGGCAGTAACTTGCAACTC TGTGTCTGTGGGAATTTCGGGAGGGAGGGCGTGGGCGGCAGGAAAACGGCCCAGAACAGA CCTAGTGCCCCTCAGTTATTCACCAGAGGGGCACTGAGCACCTGCTGTGTGCCAAGCACG GTTGCAAGTGGTTTCACATGTATCAATCAATGAAAGAAACGTCACTGCCCTGGAGAACAT TTTTTTGGGGGGTAGGGAGCAGACAATAAGCCATACACATAATTATATAACATTTTCGGG GATAAATGTGAGGAGAAACAGGGTATGTGGAGGGGTTTCGTCGCTGAGGTGAGAATGGAC GAGGACTCCCAGGAAGTGCCTGAAGCAAAGGGGAGGGAGGTGCCAGCTACTGTGAAGCCA ACGGATGTATGTATGTATATGTGTGTGTGTGTGTGTGTATATATATATATAT TTTTTTTA AACTAAATCTTCTCAGTTACAAAAAGAACACCGGCTCCATTTAGAAAGTTTGGCGCTTCC CTACTTAAGAATATGCCTTGAACATTTGAATTTGTCAATAAATATTCTCCCTCAGTGGCA TTTTTGATAGCTTCATTGC ZNF354A - entire promoter region - chr5: 178,728,826-178,731,484 Underline - Enhancer 1 - chr5: 178,728,992-178,729,587

[0387] Italic - Enhancer 2 - chr5: 178,729,863-178,730,649

[0388] Underline / italic - Promoter - chr5: 178,730,650-178,731,225

[0389] Underline - Enhancer 3 - chr5: 178,731,168-178,731,345

[0390] Experimentally confirmed TF binding motifs

[0391] chr5:178, 730, 085-178, 730, 108

[0392] chr5: 178,730,482-178,730,499

[0393] chr5:178, 730, 518-178, 730, 534

[0394] chr5: 178,730,721-178,730,738

[0395] chr5:178, 730, 803-178, 730, 819

[0396] chr5: 178,730,886-178,730,892

[0397] The TSS is underlined and the transcript is encoded on the reverse strand.

[0398] Experimentally confirmed TF binding motifs are highlighted in black.

[0399] Source: GRCh38.pl4, Ensembl release 113

[0400] Human sh RNA

[0401] • SEQ ID NO: 33

[0402] GGAUCGAAGAGCAGUCAUA

[0403] • SEQ ID NO: 34

[0404] GAGAAAUCCUACAGAUGUA

[0405] • SEQ ID NO: 35

[0406] GUCACGUGAUCAUCAGAAA SEQ ID NO: 36Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0407] GCCCTCATTAGGTATTTAATT

[0408] • SEQ ID NO: 37 GCCCAAAGTCAGTGCTTATTA

[0409] • SEQ ID NO: 38 CAGCTCTTATTCAGCATCGAA

[0410] • SEQ ID NO: 39 CAACGCAAACACAAGACTCTT

[0411] • SEQ ID NO: 40 CTCTCAGTACATCCCTTTATA

[0412] • SEQ ID NO: 41 ATCCCTTTCTGGATGTCAAAG

[0413] • SEQ ID NO: 42 AGCCCAAAGTCAGTGCTTATT

[0414] • SEQ ID NO: 43 CAAACACAAGACTCTTCATTT

[0415] • SEQ ID NO: 44 TCTCAGTACATCCCTTTATAA

[0416] • SEQ ID NO: 45 TTAGCCCTCATTAGGTATTTA

[0417] ZNF354A binding motifs M07622_2.00

[0418] • SEQ ID NO: 46

[0419] Fw.

[0420] RTTTARHCCATTTAC

[0421] Rev.

[0422] • SEQ ID NO: 47 GTAAATGGDYTAAAY

[0423] M08331_2.00

[0424] Fw.

[0425] • SEQ ID NO: 48

[0426] RTTTARDCCATTTAYRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0427] Rev.

[0428] • SEQ ID NO: 49

[0429] RTAAATGGHYTAAAY

[0430] M08914_2.00

[0431] Fw.

[0432] • SEQ ID NO: 50

[0433] DTTYHDNYCHTTTMYWTTTADTGW

[0434] Rev.

[0435] • SEQ ID NO: 51 WCAHTAAAWRKAAADGRNHDRAAH

[0436] Table 1

[0437] Code for Nucleotide representation

[0438] Letter Nucleotides it represents A Adenine (A)

[0439] C Cytosine (C)

[0440] G Guanine (G)

[0441] T Thymine (T)

[0442] W Weak (A or T)

[0443] S Strong (C or G)

[0444] M Amino (AorC)

[0445] K Keto (G or T)

[0446] R Purine (A or G)

[0447] Y Pyrimidine (CorT)

[0448] B NotA(C, G, orT)

[0449] D Not e (A, G, orT)

[0450] H NotG(A, C, orT)

[0451] V NotT(A, C, orG)

[0452] N Any nucleotide (A, C, G, or T)

[0453]

[0454] sgRNA to crispr ZNF354A human

[0455] • SEQ ID NO: 52Ref. 6.2620 PCT PCT Appn Final PAT8531PC00 AGCCCAAAGTCAGTGCTTATT

[0456] • SEQ ID NO: 53

[0457] CAAACACAAGACTCTTCATTT

[0458] • SEQ ID NO: 54

[0459] TCTCAGTACATCCCTTTATAA

[0460] • SEQ ID NO: 55

[0461] TTAGCCCTCATTAGGTATTTA

[0462] Mouse sh RNA

[0463] • SEQ ID NO: 67 (shA)

[0464] AGACCTTGACTTCAGGTATTT

[0465] • SEQ ID NO: 68 (shB)

[0466] GATGTGAATGCCTCGACTATA

[0467] • SEQ ID NO: 69 (shRNA1)

[0468] CTCTCAGTACATCCCTTTATA

[0469] • SEQ ID NO: 70 (shRNA2)

[0470] CAAACACAAGACTCTTCATTT

[0471] • SEQ ID NO: 71 (shCtrtl)

[0472] ATTCAGCCCATATCGTTTCACTCGAGTGAAACGATATGGGCTGAATATTTTRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0473] EXAMPLES

[0474] EXAMPLE 1

[0475] MATERIALS AND METHODS

[0476] Cell lines

[0477] U2OS (ATCC: HTB 96), MDA-MB-231 (ATCC: HTB 26), K-562 (ATCC: CCL 243), SUDHL4 (ATCC: CRL 2957), SUDHL5 (ATCC: CRL 2958), SUDHL6 (ATCC: CRL 2959), MCF7 (ATCC: HTB 22), SW480 (ATCC: CCL 228), LS1034 (ATCC: CRL 2158), HCT116 (ATCC: CRL 3502), HAP-1 (Horizon Discovery: HZGGHC9001), HAP-1 KO KAP1 (Horizon Discovery: HZGHC000293c003), HEPG2 (ATCC: HB 8065), Calu-3 (ATCC: HTB 55), 3T3 (ATCC: CRL 1658), VERO-E6 (ATCC: CRL 1586) cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, penicillin, and streptomycin. HELA (ATCC: CCL 2) cells were grown in MEM Media supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 1% NEAA and 1% glutamine (all media from ThermoFisher Scientific).

[0478] Antibodies and Reagents

[0479] The antibodies and reagents were acquired from: ATF2 (Abeam: ab_131484; RRID: AB_11156678, rabbit: l:2000x), phosphor-ATF2 (T69) (Abeam: 131106; RRID: AB_11157608;rabbit: ElOOOx), GAPDH (Thermofisher: 398600; RRID: AB 2533438; mouse: l:4000x), ZDHHC20 (Sigma: SAB4501054; RRID: AB_10744838; rabbit: l:2000x), HA-HRP (Roche: 112013819001; RRID: AB 390917; rat: l:5000x), KAP1 (Abeam: ab_109289; RRID: AB 10863057, rabbit: ElOOOx), phosphor-KAPl S473 was generated in D. TRONO laboratory using the following peptide krsrSgegevsgl (rabbit: l:500x), Nucleocapside N SARS-CoV-2 (Genetex: GTX135357; RRID: AB_2868464; rabbit l:4000x), FLAG (Sigma: F3165; RRID: AB 259529; mouse: l:2000x), phosphor-Threonine-Serine (BD: 612548; RRID: AB 399843; rabbit: ElOOOx), GPX4 (Cell Signalling: 52455; RRID: AB_2924984, rabbit: ElOOOx), V5-HRP (Sigma: V2260; RRID: AB_261857, mouse: l:2000x), Actin (Millipore: MAB1501; RRID: AB_2223041, mouse: l:4000x), MAT1A (Abeam: 129176; RRID: AB_11145300, rabit: l:2000x), ZNF354A (Santa Cruz: 81140; RRID: AB 1131557, mouse: ElOOOx), 4G10 phospho-Tyrosine (Millipore: 05-321; RRID: AB 309678, mouse: ElOOOx) and HRP-conjugated secondary antibodies (GE-Healthcare).Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0480] RSL-3 (Sigma: S8155), Hydrogen Peroxide 30% (Fisher: H / 1750 / 15), Rotenone (Cayman Chemical: CAY-13995), Ferrostatin-1 (Sigma: SML0583), Bodipy 581 / 591 Cll (Sigma: D3861), Liperfluo (Fisher: L248), Propidium Iodide (Thermofisher: P3566), Cell Viability assay (Promega: G9711), JQ1 (Sigma: SML-1524), and Mithramycin (LKT: M3476).

[0481] siRNA

[0482] The following human siRNA (5' >3') with the corresponding target sequences were purchased from Qiagen: ZNF354A (TACATCCTTGAGAGAGATGTA, SEQ ID NO: 56),

[0483] ZNF 317 (CATGACGGAAATCACACTAAA, SEQ ID NO: 57),

[0484] ZNF 308 (CAGTTCATTGGCAACCTCATA, SEQ ID NO: 58),

[0485] ZNF 677 (GAGATTGGTTACAGTACAAGA, SEQ ID NO: 59),

[0486] ZNF793 (ACCCTGGCATGTAAACGTTTA, SEQ ID NO: 60),

[0487] SETDB1 (TCGGGTGGTCGCCAAATACAA, SEQ ID NO: 61),

[0488] KAP1 (AGCGTCCTGGCACTAACTCAA, SEQ ID NO: 62),

[0489] ATF2 (CCGAGGTAGTCCACATACAGAA, SEQ ID NO: 63),

[0490] GPX4 (CGGCTGCGTGGTGAAGCGCTA, SEQ ID NO: 64).

[0491] The following human siRNA (5' >3') were purchased from Santa Cruz: ZNF354A (sc-91974), SP1 (sc-44221), FOXA1 (sc-37930). Mouse siRNA were purchased from Qiagen for mouse ZFP354A (TACCACTTTAATAACATTCAA, SEQ ID NO: 65).

[0492] As a control siRNA, a sequence targeting the viral glycoprotein VSV-G (5’-ATTGAACAAACGAAACAAGGA-3’, SEQ ID NO: 66) was used. Transfections of 50 nM of siRNA were carried out using TransIT-X2 (MIRUS), and the cells were analyzed at least 72 h after transfection.

[0493] Western Blotting

[0494] Cells were washed three times in lx PBS at 4°C and lysed in Buffer (lx PBS, 0.5% NP40 and protease inhibitor cocktail; Roche) for 30 min on ice. Lysates were then spin down at 10 000 g, and the protein content of supernatant were determined, the samples were boiled in Laemmli buffer for 5 min before separation via SDS-PAGE and western blotting against the different anti-bodies used in this study. Western blots were developed using the ECL protocol and imaged on a Fusion Solo from Vilber Lourmat. Densitometric analysis was performed using the software Bio-ID from the manufacturer.

[0495] PlasmidsRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0496] Plasmids expressing HA-tagged ZNF354A-HA, ZNF317-HA, ZNF677-HA, ZNF793-HA were obtained from the D. Trono lab previous generated by (Imbeault et al., 2017). Plasmids expressing FLAG-ATF2 were from Origene (RC2118983), and plasmids expressing SP1-FLAG (25543) and FOXA1-V5 (153109) from Addgene. Mutations to change ZNF354A-HA to ZNF354A-FLAG, FLAG-ATF2 to HA-ATF2, HA-ATF2 6A (T52A-T69A-T71A-T73A-S490A-S498A), HA-ATF26D (T52D-T69D-T71D-T73D-S490D-S498D), ZNF354A-HARR (D18R-E27R), ZNF354A RA (E45R-N46A), ZNF354A RR-RA (D18R-E27R- E45R-N46A) were done following Quickchange mutagenesis kit (Agilent) instructions.

[0497] Aerolysin purification and cell intoxication

[0498] Proaerolysin toxin was produced and purified by our laboratory in Aeromonas salmonicida as previously described (Howard, S. P. & Buckley, J. T. Activation of the hole-forming toxin aerolysin by extracellular processing. 1985, J Bacteriol 163, 336-340). Cells were treated one hour in complete medium with 10 ng / ml of proaerolysin at 37 °C. Cells were washed twice in complete medium and further incubated at 37 °C for indicated times.

[0499] GREAT - analysis

[0500] ChIP-seq data for ZNF354A-HA binding sites were obtained from KRABopedia (de Tribolet-Hardy et al., 2023) and submitted to the Genomic Regions Enrichment of Annotations Tool (GREAT - http: / / great.stanford.edu / public / html / ) (McLean et al., 2010). The parameters for gene regulatory domains for each gene were defined as domain that extends in both directions of the TSS until the nearest TSS up to a maximum extension of 300 kb.

[0501] Human MucilAir assays

[0502] MucilAir-human upper respiratory tissues (Epithelix, Geneve, Switzerland) were maintained according to the manufacturer’s protocol. For infection, tissues were washed apically with 200 pl of DPBS, calcium, magnesium (#14040091 — Gibco) for 20 min at 37 °C and the basal medium replaced with fresh mucilair medium. Tissues were infected at the MOI of 0.1 PFU (assuming the manufacturer’s estimations of 500000 cells per tissue). Tissues were inoculated with 200 pl of SARS-CoV-2 (passage 3) for 3 h (apically) at 33 °C. The apical inoculum was removed and infected tissues were maintained for until harvest. For harvest tissues were lysed using 300 pl lysis buffer for 30 min and processed for western blot.

[0503] Viral Stock production and titration with plaque-based assaysRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0504] All viral stocks were produced and isolated from supernatants of Vero E6 cells, cultured in T75 culture flasks to a confluency of 80-90%, and infected with an original passage 2 (P2) SARS-CoV-2 virus, for 48 or 72 h, at MOI-0.05, in 10 ml DMEM supplemented with 2.5% FCS. Original stocks were obtained from the following strain: hCoV-19 / Switzerland / GE9586 / 2020|EPI_ISL_414022|2020-02-27. Passage 3 supernatants were harvested, clear of cell debris by centrifugation (500 g 10 min) and filtration (0.45 pm), aliquoted and stored at -80 C. Viral titers were quantified by determining the number of individual plaque forming units after 48 h of infection in confluent Vero E6 cells as described in S. Mesquita et al., 2023).

[0505] SARS CoV-2 infections

[0506] All infections for experimental analysis were done using passage 3 SARS-CoV-2 stocks. Vero E6 cells or CALU-3 cells seeded to a confluency of 90 to 100%, were, washed twice in warm serum-free medium and inoculated with the indicated MOI of SARS-CoV-2, diluted in serum free medium. 1 hour after inoculation cells were washed with complete medium and infection allowed to proceed for the indicated time points in DMEM supplemented with 2.5% FCS, penicillin and streptomycin.

[0507] Drug and toxin treatments

[0508] All drug treatments were done for the indicated times in complete culture and / or infection medium. Equivalent volume of Dimethyl sulfoxide (DMSO), used as a solvent, was added for control samples. Specifically, culture media was replaced, cells were washed once in warm culture media and drugs resuspended as indicated were used as: RSL3 (in DMSO - 3 pM, increasing concentrations 0.6 to 10 pM), Ferrostatin-1 (DMSO - 1 - 15 pM), H2O2 (0.5 - 100 pM), Rotenone (1 - 10 pM).

[0509] UCSC genome browser analysis

[0510] UCSC genome browser at ZDHHC20 locus was obtained from (Human version hgl9). Specific UCSC genome browser tracks were selected for display in Fig. 1 and 2. Transcript annotation corresponds to GENCODE Genes track (version V40lift37); Histone Modifications by ChlP-seq from ENCODE / Stanford / Yale / USC / Harvard display ChIP-seq signals for H3K4me1, H3K4me3, H3K27Ac marks for HepG2 and H3K9me3 for U2OS. Transcription Factor ChIP-seq Peaks track shows transcription factor SETDB1, and KAP1 binding sites for U2OS and K562 cells based on ChIP-seq experiments from ENCODE. KZFP ChIP-seq data showRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0511] binding sites for different KZFP proteins obtained from KRABopedia (https: / / tronoapps.epfl.ch / web / krabopedia / ) (https: / / doi.org / 10.1101 / 2023.02.27.530095)

[0512] Q-RT-PCR

[0513] For the real-time PCR, RNA was extracted from a six-well dish using the RNeasy kit (Qiagen). In all, 0.5-1 mg of the total RNA extracted was used for the reverse transcription using random hexamers and superscript II (Invitrogen). A 1:40 dilution of the cDNA was used to perform the real-time PCR using using Applied Biosystems SYBR Green Master Mix on 7900 HT Fast QPCR System (Applied Biosystems) with SDS 2.4 Software. All data (always in triplicate) were normalized to Ct values from three housekeeping (HK) genes ALAS-1, Guss and TBP. Results were expressed as 2^(-ΔΔCt)*100%.

[0514] RNAseq

[0515] RNA quality was controlled on the TapeStation 4200 (Agilent), which confirmed their integrity. Libraries for mRNA-seq were prepared with the Stranded mRNA Ligation method (Illumina), according to manufacturer’s instructions, starting from 800ng RNA. Libraries, all bearing unique dual indexes, were subsequently loaded at 100 pM on a Novaseq 6000 flow cell (Illumina) and sequenced according to manufacturer instructions, yielding pairs of 60 nucleotides reads at a depth of about 70 mio reads pairs per sample. Reads were trimmed of their adapters with bclconvert v00.000.000.3.9.3 (Illumina) and quality-controlled with fastQC v0.11.9. FastQ Screen v0.14.0 tool was used for screening FASTQ files reads against multiple reference genomes.

[0516] RNAseq processing and analysis

[0517] Sequencing reads were mapped to the human hgl9 genome using hisat2 [D. Kim, B. Langmead, and S. L. Salzberg, “HISAT: a fast spliced aligner with low memory requirements,” Nature Methods, vol. 12, no. 4, pp. 357-360, Mar. 2015.] with parameters: hisat2 -k 5 —seed 42 — rna-strandness RF.

[0518] Counts on genes were generated using featureCounts [Y. Liao, G. K. Smyth, and W. Shi, “featureCounts: an efficient general purpose program for assigning sequence reads to genomic features,” Bioinformatics, vol. 30, no. 7, pp. 923-930, Apr. 2014.]. Only uniquely mapped reads were used for counting on genes.

[0519] Normalization for sequencing depth was done using the TMM method as implemented in the limma package of Bioconductor [Gentleman et al., 2004],Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0520] Differential gene expression analysis was performed using voom (Law et al., 2014) as it has been implemented in the limma package of Bioconductor. A gene was considered to be differentially expressed when the fold change between groups was bigger than 2 and the p-value was smaller than 0.05. A moderated t-test (as implemented in the limma package of R) was used to test significance. P-values were corrected for multiple testing using the Benjamini-Hochberg’s method (Benjamini and Hochberg, 1995).

[0521] Gene set enrichment analysis (GSEA) was performed using the ClusterProfiler package in R. The analysis utilized a ranked list of genes and gene set collections downloaded from MsigDB. The GSEA was conducted with the following parameters: the number of permutations was set to 10,000, minimum gene set size was 10, maximum gene set size was 800.

[0522] Flow cytometry analysis of membrane permeability and lipid peroxidation

[0523] U2OS cells (6 well plate at approximate 1 x 10^6 cells), siRNA depleted as indicated were treated with 5 pM RSL-3 for 24 h. Cells were washed twice in EBSS (10 pM Hepes) and Lipid peroxides stained by Liperfluo (Dojindo) and C11-BODIPY581 / 591 (Invitrogen). Stock solutions of Liperfluo or BODIPY, prepared in DMSO, according to manufacturer’s instructions were resuspended in EBSS-Hepes to 5 pM (Liperfluo) and 10 pM for Bodipy and used for cell incubation 1 ml per well for 30 min at 37°C. After treatment cells were washed, trypsinized and resuspended in 500 ul EBSS-Hepes. For propidium iodide analysis Liperfluor labelled cells were stained with propidium iodide prior analysis at final concentration 1 pg / ml. Analytical flow cytometry was performed using an LSRII or LSR Fortessa (BD; Becton Dickinson) instrument. For Liperfluor, and BODIPY-oxidated cells were analysed at Excitation: 488 nm, Filter: 530 / 30; For PI and BODIPY-non-oxidated Excitation: 561 nm Filter 610 / 20. 10,000 events were acquired per each replicate per sample. Results were analyzed using Flow Jo.

[0524] DSS-induced mice colitis

[0525] Six wild-type C57BL / 6j mice (8-week-old males) were used. Three (n = 3) mice were used as controls and three mice were given 3% dextran sulfate sodium in the drinking water for 7 days, then switched to regular drinking water for 3 days. Three Control 8-week male were given drinking water that did not contain DSS. During the 10-day experiment, mice were weighted and disease activity index (DAI) was performed daily based on stool consistency, presence of occult blood and body weight loss. On day 10, mice were euthanized with injection of pentobarbital and bled via cardiac puncture, and colon and intestines were collected. For animalRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0526] experimentation, all procedures were performed according to protocols approved by the Veterinary Authorities of the Canton Vaud and according to the Swiss Law (license VD 3497, EPFL).

[0527] Results

[0528] Differential regulation of the acyl-transferase ZDHCC20.

[0529] The Inventors found that the acyl-transferase ZDHHC20, which mediates post-translational lipid modification of proteins (S-acylation), is expressed as several isoforms (S. Mesquita et al, 2023). A “short” ~35 kDa isoform is produced under physiological homeostatic conditions, but following aggressions such SARS-CoV-2 infection (Fig. 1 A) or exposure to pore-forming bacterial toxins (Fig. 1C), as well as in a dextran sodium sulfate-DSS-induced mouse model of ulcerative colitis (Fig. IB), N-terminally extended Long-ZDHHC20 isoforms (20L, between 40 and 60 kD in apparent molecular weight) are produced by activation of one or several upstream transcription start sites (Fig 2A) (S. Mesquita et al, 2023). More recently we also observed that expression of 20L isoforms increases with age, as observed in primary epithelial lung tissues (Fig ID) and is detected in some cancer cell lines, including HCT116 human colorectal carcinoma, the MCF7 breast cancer (Fig 2E) and HepG2 hepatocellular carcinoma (S. Mesquita et al, 2023) cells.

[0530] Differential expression of Zdhhc20 isoforms is epigenetically regulated.

[0531] Chromatin analyses of HEPG2 cells revealed the presence of the H3Ac27 and H3K4m3 activation histone marks at the canonical zdhhc20 promoter (Fig 2A), as well as H4K4mel over an upstream region coinciding with binding sites for the FOXA1 and SP1 transcriptional activator, consistent with the presence of an enhancer (Fig 2A). In U2OS cells, we also detected the repressive mark H3K9me3 further upstream, indicative of heterochromatin (Fig 2A).

[0532] Within this region, ENCODE ChIP-seq peak data indicates that this locus is bound by the SET Domain Bifurcated Histone Lysine Methyltransferase 1 (SETDB1), which mediates trimethylation of histone 3 on lysine 9, and by KAP1 (KRAB -associated protein 1), which recruits SETDB1 to KZFPs, Krüppel-associated box (KRAB) domain-containing zinc-finger proteins (Fig 2A). Accordingly, we could demonstrate that silencing of SETDB1 or KAP1 by RNA interference induced the expression of 20L isoforms (Fig 2B), a result confirmed in a KAP1 KO HAP cells (Fig 2C). It can be concluded that the expression of 20L ZDHHC20 isoforms is repressed by the heterochromatin-inducing KAP1 / SETDB1 complex.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0533] ZNF354A acts as repressor on an upstream zdhhc20 promoter

[0534] To identify the KZFP(s) responsible for recruiting the KAP1 / SETDB 1 complex to the zdhhc20 locus, the Inventors analyzed ChIP-seq datasets generated in 293T cells overexpressing HA-tagged forms of these proteins (KRABopedia). Several KZFPs (ZNF317, 793 and 677) were found to bind at the zdhhc20 promoter, but ZNF354A recruitment coincided with two previously identified SETDB1 and H3K9me3 peaks upstream and downstream the TSS (Fig 3 AB) The Inventors tested the impact of downregulating these KZFPs by RNA interference in U2OS cells, which confirmed that specifically ZNF354A is responsible for repressing the 20L ZDHHC20 isoforms (Fig. 3B).

[0535] P38 and JNK-dependent phosphorylation controls activation of the LORD pathway The Inventors have shown that different stimuli will lead to the accumulation of lipid peroxides, which subsequently trigger the phosphorylation of the three repressive complex components ATF2-KAP1 and ZNF354A, as shown for cells treated with H2O2 (Fig. 4A). Using protein specific antibodies against a given phosphorylated residue, we found that ATF2 undergoes phosphorylation of Thr-69, KAP1 on Ser-463, andZNF354A on Ser-169 (Fig. 4A-C). The later antibody was generated by the Inventors and is not commercially available (Fig. 4C). These phosphorylation events dependent on the activity of three kinases, p38, JNK and ATM (Fig.

[0536] 4DE). Inhibitors of p38 or JNK completely abrogated the phosphorylation of ATF2, KAP1 and ZNF354A, and prevented the expression of ZDHHC20L (Fig. 4DE). Inhibiting ATM had a mild effect on these four events (Fig. 4DE). And inhibition of ERK only affected ATF2 phosphorylation (Fig. 4D).

[0537] EXAMPLE 2 - Silencing of ZNF354A increases the life span of activated T lymphocytes

[0538] MATERIALS AND METHODS BUFFY coat was obtained from 2 different donors from which Peripheral Blood Mononuclear Cells (PBMCs) and subsequently CD8 T lymphocytes were isolated.

[0539] On day 1, T cells were activated using a cytokine cocktail comprising IL-15 (1 U / ml) and IL-7 (0.155 U / ml), and subsequently transduced with a lentiviral vector encoding shRNA constructs directed against ZNF354A (shRNA1 or shRNA 2), or transduced with a control shRNA (SEQ ID NO: 71 (shCtrtl)).Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0540] Results

[0541] Two different shRNAs were tested (SEQ ID NO: 69 (shRNA1) and SEQ ID NO: 70 (shRNA2)), leading to approximately 60% knockdown as shown in Fig. 6A.

[0542] Cells were subsequently counted at different days following activation. A striking difference between control T cells, and ZNF354A silenced cells became available at day 11: the number of control cells dropped by 45 to 60%, depending on the donor, while the number of ZNF354A-silenced T cells increased by 50 to 130% depending on the donor and shRNA construct (Fig.

[0543] 6B).

[0544] Thus, silencing of ZFN354A prevents the exhaustion of activated T cells, maintaining them in a proliferative state.

[0545] EXAMPLE 3 - ZNF354A-depleted CAR-T cells demonstrate enhanced expansion kinetics and sustained cytotoxic activity

[0546] The above-described studies were extended to CAR-T cells as effector substrates using a combination of in vitro and in vivo functional assays. Unmodified CAR-T cells and ZNF354A-depleted CAR-T cells were directly compared for their proliferative capacity, serial tumor-killing ability, differentiation status, exhaustion phenotype, transcriptional profile, and metabolic state following repeated tumor rechallenge.

[0547] In vitro, ZNF354A-depleted CAR-T cells demonstrated enhanced expansion kinetics and sustained cytotoxic activity across multiple rounds of tumor rechallenge compared to unmodified CAR-T cells. Serial killing assays revealed improved persistence of effector function and delayed onset of functional exhaustion. Phenotypic analysis showed an increased proportion of memory-like T cells and reduced expression of exhaustion markers. Transcriptional profiling confirmed enrichment of gene signatures associated with sternness, persistence, and metabolic fitness. Metabolic analyses further demonstrated improved mitochondrial function and enhanced oxidative capacity consistent with superior long-term functionality.

[0548] In vivo efficacy was evaluated using human tumor xenograft models in immunodeficient mice. Human tumor cells were implanted subcutaneously, followed by adoptive transfer of CAR-T cells. Dose-titration studies first established a limiting dose threshold at which anti-tumor activity of unmodified CAR-T cells became suboptimal or undetectable. At these limitingRef. 6.2620 PCT PCT Appn Final PAT8531PC00

[0549] doses, ZNF354A-depleted CAR-T cells retained robust anti-tumor activity, resulting in significantly improved tumor control and prolonged survival relative to mice treated with unmodified CAR-T cells. Tumor regression was more durable, and relapse rates were reduced in animals receiving ZNF354A-depleted CAR-T cells.

[0550] These findings demonstrate that depletion of ZNF354A enhances CAR-T cell potency, persistence, and anti-tumor efficacy in both in vitro and in vivo settings. The same experimental framework was successfully applied to CAR-NK cells and tumor-infiltrating lymphocytes (TILs), where analogous improvements in expansion, persistence, and tumor-control capacity were observed following ZNF354A depletion.

[0551] EXAMPLE 4 - ZNF354A depletion protects neurons from oxidative stress

[0552] In this experiment, primary mouse hippocampal neurons were cultured for 13 days on a glial cell layer, in the presence or absence of an antioxidant cocktail (vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione). On day 5, cells were transduced either with shRNA targeting ZFP354A (two independent shRNAs, namely SEQ ID NO: 67 (shA) and SEQ ID NO: 68 (shB)) or with a control shRNA (sequence).

[0553] At day 13, neuronal viability and morphology were assessed. Neuronal soma were labelled using NEUN antibodies, neurites were stained with MAP2 antibodies, and nuclei were visualized with DAPI. The number of neurites per field of view (9 fields per well; 18 wells per condition) was quantified.

[0554] Results

[0555] The total number of neuronal cells remained unchanged across conditions. While a modest increase in viability was observed (data not shown), ZFP354A knockdown resulted in a marked and significant increase in neuronal sprouting, as evidenced by a substantially greater abundance of neurites compared to control cells. Importantly, this effect was observed both in the presence and absence of antioxidant treatment.

[0556] These results demonstrate that inhibition of ZFP354A promotes neuronal outgrowth independently of exogenous antioxidant support. The data strongly support the therapeutic potential of ZNF354A blockade in neurodegenerative disorders such as Parkinson’s disease,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0557] Alzheimer’s disease, and ALS, as well as in other pathological conditions in which oxidative stress plays a central role, including spinal cord injury and retinal dystrophies.

[0558] REFERENCE LIST

[0559] Bekes, M., Langley, D. R. & Crews, C. M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181-200 (2022)

[0560] Benjamini, Y., Hochberg, Y., 1995. Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple Testing. Journal of the Royal Statistical Society. Series B (Methodological) 57, 289–300.

[0561] Cai R, Lv R, Shi X, Yang G, Jin J. CRISPR / dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation. Int J Mol Sci. 2023 Oct 3;24(19): 14865.

[0562] de Tribolet-Hardy, J., Thorball, C. W., Forey, R., Planet, E., Due, J., Coudray, A., Khubieh, B., Offner, S., Pulver, C., Fellay, J., Imbeault, M., Turelli, P., Trono, D., 2023.

[0563] Genetic features and genomic targets of human KRAB-zinc finger proteins. Genome Res 33, 1409–1423. https: / / doi.org / 10.1101 / gr.277722.123

[0564] Didovyk A, Borek B, Tsimring L, Hasty J. Transcriptional regulation with CRISPR-Cas9: principles, advances, and applications. Curr Opin Biotechnol. 2016 Aug;40: 177-184. Dominguez AA, Lim WA, Qi LS. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat Rev Mol Cell Biol. 2016 Jan;17(1):5-15. doi: 10.1038 / nrm.2015.2. Epub 2015 Dec 16.

[0565] Imbeault, M., Helleboid, P.-Y., Trono, D., 2017. KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks. Nature 543, 550–554.

[0566] https: / / doi. org / 10.1038 / nature21683

[0567] McLean, C. Y., Bristor, D., Hiller, M., Clarke, S. L., Schaar, B. T., Lowe, C. B., Wenger, A. M., Bejerano, G., 2010. GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol 28, 495–501. https: / / doi.org / 10.1038 / nbt.1630 REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N. J. 1991)

[0568] S. Mesquita, F., Abrami, L., Bracq, L., Panyain, N., Mercier, V., Kunz, B., Chuat, A., Carlevaro-Fita, J., Trono, D., van der Goot, F. G., 2023. SARS-CoV-2 hijacks a cell damage response, which induces transcription of a more efficient Spike S- acyltransferase. Nat Commun 14, 7302. https: / / doi.org / 10.1038 / s41467-023-43027-2 Sies, H., Jones, D. P., 2020. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21, 363–383. https: / / doi.org / 10.1038 / s41580-020-0230-3

[0569] Stockwell, B. R., Friedmann Angeli, J. P., Bayir, H., Bush, A. I., Conrad, M., Dixon, S. J., Fulda, S., Gascon, S., Hatzios, S. K., Kagan, V. E., Noel, K., Jiang, X., Linkermann, A., Murphy, M. E., Overholtzer, M., Oyagi, A., Pagnussat, G. C., Park, J., Ran, Q., Rosenfeld, C. S., Salnikow, K., Tang, D., Torti, F. M., Torti, S. V., Toyokuni, S., Woerpel, K. A., Zhang, D. D., 2017. Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell 171, 273–285. https: / / doi. Org / 10.1016 / j.cell.2017.09.021

[0570] Tanenbaum ME, Gilbert LA, Qi LS, Weissman JS, Vale RD. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014 Oct 23; 159(3):635-46.Ref. 6.2620 PCT PCT Appn Final PAT8531PC00

[0571] Watson, G., Ronai, Z., Lau, E., 2017. ATF2, a paradigm of the multifaceted regulation of transcription factors in biology and disease. Pharmacol Res 119, 347–357. https: / / doi. Org / 10.1016 / j.phrs.2017.02.004

[0572] Weber, W.; Fussenegger, M. Emerging biomedical applications of synthetic biology.

[0573] Nat Rev Genet. 13:21-35 (2012).

[0574] Yang, W. S., SriRamaratnam, R., Welsch, M. E., Shimada, K., Skouta, R., Viswanathan, V. S., Cheah, J. H., Clemons, P. A., Shamji, A. F., Clish, C. B., Brown, L. M., Girotti, A. W., Cornish, V. W., Schreiber, S. L., Stockwell, B. R., 2014. Regulation of ferroptotic cancer cell death by GPX4. Cell 156, 317–331. https: / / doi. Org / 10.1016 / j.cell.2013.12.010

[0575] Zhao, L., Zhao, J., Zhong, K. et al. Targeted protein degradation: mechanisms, strategies and application. Sig Transduct Target Ther 7, 113 (2022)

[0576] Zheng, J., Conrad, M., 2025. Ferroptosis: when metabolism meets cell death. Physiol Rev 105, 651–706. https: / / doi.org / 10.1152 / physrev.00031.2024

Claims

Ref. 6.2620 PCT PCT Appn Final PAT8531PC00CLAIMS1. An agent modulating the expression and / or activity of i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A), ii) an mRNA encoding the ZNF354A, and / or iii) the ZNF354A gene, for use in the treatment and / or prevention of a disease linked to oxidative stress, lipid peroxidation and / or ferroptosis.

2. The agent for use of claim 1, wherein the disease linked to oxidative stress, lipid peroxidation and / or ferroptosis is selected from the group comprising a cancer, a cardiovascular disease, and a neurodegenerative disease.

3. The agent for use according to claim 2, wherein the disease linked to oxidative stress, lipid peroxidation, and / or ferroptosis is a cancer characterized by elevated acyl-transferase ZDHHC20 expression, and wherein the agent enhances the activity of a ZNF354A protein by one or more of: i) inhibiting degradation of the ZNF354A protein, ii) increasing expression of the ZNF354A protein, iii) modulating phosphorylation of the ZNF354A protein, and / or iv) any other mechanism resulting in a gain of function.

4. The agent for use according to claim 3, wherein the treatment with the agent reduces the expression or activity of N-terminally extended Long-ZDHHC20 isoforms.

5. The agent for use according to claim 2, wherein the disease linked to oxidative stress, lipid peroxidation, and / or ferroptosis is selected from a cardiovascular disease and a neurodegenerative disease, and wherein the agent inhibits the activity of a ZNF354A protein by one or more of: i) inhibiting the translation of an mRNA encoding ZNF354A, ii) inhibiting the transcription of the gene encoding ZNF354A, and / or iii) inhibiting the activity of a ZNF354A protein.

6. The agent for use of claim 5, wherein the agent inhibits the activity of a ZNF354A protein by one or more of: i) enhancing and / or favoring the degradation of aZNF354A protein, ii) preventing the recognition of its targets, iii) modulating the phosphorylation of a ZNF354A protein, and / or iv) any other means resulting in a loss of function.Ref. 6.2620 PCT PCT Appn Final PAT8531PC007. The agent for use of any one of the preceding claims, wherein the agent is selected from the group comprising a nucleic acid, a chemical compound, a peptide or analog thereof, an antibody or an antigen-binding fragment thereof, and an antibody mimetic, or a combination of two or more thereof.

8. The agent for use of claim 7, wherein the nucleic acid is selected from the group comprising a nucleic acid encoding an siRNA, an miRNA, a piRNA, an hnRNA, an snRNA, a gRNA, a CRISPR-based loss-of-function system, an esiRNA, an shRNA, and an antisense oligonucleotide (ASO), or a combination of two or more thereof.

9. The agent for use of any one of the preceding claims, wherein- the mRNA encoding ZNF354A is selected from the group comprising a sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15, a fragment or a variant thereof, or a combination of two or more thereof, and / or- the gene encoding ZNF354A is selected from the group comprising a sequence set forth in SEQ ID NO: 17, a fragment or a variant thereof, and / or- the ZNF354A protein is selected from the group comprising a sequence as set forth in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 16, a fragment or a variant thereof, or a combination of two or more thereof.

10. A short interfering ribonucleic acid (siRNA) for inhibiting the expression of ZNF354A.

11. The siRNA of claim 10, wherein the sense strand of the siRNA targets the ZNF354A mRNA sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and a SEQ ID NO: 15, a fragment or a variant thereof or a combination of two or more thereof, and wherein the sense strand of the siRNA is at least 80% homologous to at least 2 to 30 contiguous nucleotides of the ZNF354A mRNA sequence as set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and a SEQ ID NO: 15, a fragment or a variant thereof.Ref. 6.2620 PCT PCT Appn Final PAT8531PC0012. The siRNA of claim 10 or 11, wherein the siRNA is selected from the group of sequences set forth in SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, a fragment or a variant thereof.

13. The siRNA of any one of claims 10 to 12, wherein the siRNA comprises one or more modified nucleotides selected from the group comprising phosphotioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide linkage, with the nucleobase attached to the alpha-carbon of the glycine) or locked nucleic acids (LNA; 2'O, 4'C methylene bridged RNA building blocks).

14. A short hairpin ribonucleic acid (shRNA) for inhibiting the expression of ZNF354A.

15. The shRNA sequences of claim 14, which is selected from the group of sequences set forth in any one of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, a fragment or a variant thereof.

16. A guide ribonucleic acid (sgRNA or gRNA) targetingi) one or more sequences within the gene encoding ZNF354A (chromosome 5: 178730659-178711512) as set forth in SEQ ID NO: 17,ii) one or more sequences within the promoter region controlling the gene encoding ZNF354A (chromosome: 5, 178728826:178731484) as set forth in SEQ ID NO: 32, and / oriii) one or more DNA sequences selected from the group of sequences set forth in SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, and SEQ ID NO: 51, or a fragment or variant of any one of these sequences.

17. The sgRNA or gRNA of claim 16, which is selected from the group of sequences set forth in SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and SEQ ID NO: 55, a fragment or a variant thereof.Ref. 6.2620 PCT PCT Appn Final PAT8531PC0018. One or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and antisense oligonucleotide (ASO, such as e.g. Gapmer antisense), or combination of one or more thereof, as described in any one of claims 8 to 17.

19. A plasmid or a vector comprising one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and antisense oligonucleotide (ASO, such as e.g. Gapmer antisense), or combination of one or more thereof, as described in claim 18.

20. The vector of claim 19, which is selected from the group comprising a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, an RNA (e.g. mRNA) targeted lipid nanoparticles (LNPs), a liposome or any combination thereof.

21. The vector of claim 20, which is a lentiviral vector.

22. A cell comprising, or modified by the introduction of,i) a plasmid or vector of any one of claims 19 to 21, orii) one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and ASO, or combination of two or more thereof, of claim 17.

23. The cell of claim 22, wherein the cell is a eukaryotic or a prokaryotic cell.

24. The cell of claim 23, wherein the cell is a mammalian cell.

25. The cell of any one of claims 23 to 24, wherein the cell is selected from the non-limiting group comprising T cells, tumor infiltrating lymphocytes (TILs), NK cells, regulatory T cells (Treg cells), macrophages, TCR-expressing cells, eosinophils, basophils, neutrophils, myeloid cells, B cells, plasma cells, regulatory B cells (Bregs), innate lymphoid cells 1 (ICL1), ILC2, ICL3, dendritic cells, stem cells or cells derived therefrom, and NK-T cells.Ref. 6.2620 PCT PCT Appn Final PAT8531PC0026. The cell of claims 25, wherein the cell expresses at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR) or any other synthetic tumor targeting motif.

27. A method for prolonging the survival and / or functional persistence of a cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif, the method comprising contacting said cell, or population of cells, ex vivo and / or in vivo, with one or more nucleic acids of claim 17, or with a vector or plasmid of any one of claims 19 to 21 encoding said one or more nucleic acids,wherein said one or more nucleic acids inhibit the expression and / or activity of: i) a KRAB-containing zinc finger protein ZNF354A (ZNF354A),ii) an mRNA encoding ZNF354A, and / oriii) the ZNF354A gene,and wherein inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis in the contacted cell, or contacted population of cells, thereby prolonging their survival and / or functional persistence.

28. The method of claim 27, wherein the a cell, or population of cells, expressing at least one recombinant construct encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif was / were first expanded by contacting said cell, or population of cells, with an interleukin selected from the group comprising IL-7, IL-15 and / or IL-2.

29. The method according to any one of claims 27-28, wherein inhibition of ZNF354A expression and / or activity reduces oxidative stress, lipid peroxidation, and / or ferroptosis, thereby preventing exhaustion of the contacted cell or population of cells and maintaining their proliferative state.

30. The method according to any one of claims 27-28, wherein the contacted cell or population of contacted cells is / are used for the treatment of a cancer, a cardiovascular disease, or a neurodegenerative disease.Ref. 6.2620 PCT PCT Appn Final PAT8531PC0031. A cell, or population of cells, obtainable by a method according to any one of claims 27 to 30.

32. A pharmaceutical composition comprising a therapeutically effective amount of i) a plasmid or vector of any one of claims 19 to 21, orii) one or more nucleic acids encoding the siRNA, miRNA, piRNA, hnRNA, snRNA, gRNA, CRISPR-based loss-of-function system, esiRNA, shRNA, and ASO, or combination of two or more thereof, of claim 17, oriii) a cell of any of claims 22 to 26, oriv) a cell, or population of cells, obtainable by a method according to any one of claims 27 to 30,and optionally a pharmaceutically acceptable carrier, diluent and / or excipient.

33. The pharmaceutical composition of claim 32 for use in the treatment and / or prevention of a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

34. A method of treatment and / or prevention of a cancer, a cardiovascular disease, a neurodegenerative disease or an inflammatory disease, in a subject in need thereof, the method comprising administering a pharmaceutical composition according to claim 32 or 33 to the subject in need thereof.

35. A method of treatment and / or prevention of a cancer in a subject in need thereof, the method comprisingi) removing and isolating cells, or population of cells, preferably immune cells, more preferably native T cells, from said subject,ii) genetically engineering said T cells with one recombinant construct (e.g. vector, plasmid or polynucleotide) encoding a chimeric antigen receptor (CAR), a T cell receptor (TCR), or any other synthetic tumor-targeting motif,iii) expanding ex vivo into a larger population of engineered immune cells, or population of immune cell, e.g. T cells, in the presence of an interleukin selected from the group comprising IL-7, IL- 15 and / or IL-2,Ref. 6.2620 PCT PCT Appn Final PAT8531PC00iv) contacting said immune cells, or population of immune cells, with one or more nucleic acids of claim 17, or with a vector or plasmid of any one of claims 19 to 21 encoding said one or more nucleic acids,(iv) reintroducing said engineered immune cells, or population of engineered immune cells, e.g. T cells, into the subject in need thereof.

36. The agent for use of claim 2 or the method of claim 30 or the pharmaceutical composition of claim 33 or the method of claim 35, wherein the cancer is a solid cancer or a liquid (e.g. hematologic) cancer.

37. A method of improving transplant engraftment, survival, and / or functional persistence of stem cell transplants in a subject in need thereof, the method comprisingi) removing and isolating cells, or population of cells, preferably stem cells, from said subject, ii) ex-vivo contacting said stem cells, or population of stem cells, with one or more nucleic acids of claim 17, or with a vector or plasmid of any one of claims 19 to 21 encoding said one or more nucleic acids,iii) reintroducing said engineered stem cells, or population of stem cells, into the subject in need thereof,wherein said nucleic acid inhibits the expression and / or activity of ZNF354A, thereby reducing oxidative stress, lipid peroxidation, and / or ferroptosis in the stem cells and improving transplant engraftment, survival, and / or functional persistence.

38. The method of claim 37, wherein the stem cells are selected from the group comprising pluripotent, multipotent, perinatal, and extra-embryonic stem cell sources.

39. The method of claim 38, wherein the pluripotent stem cells are selected from the group comprising embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), parthenogenetic stem cells, nuclear transfer-derived embryonic stem cells (ntESCs), and epiblast stem cells.

40. The method of claim 38, wherein the multipotent or adult stem cells are selected from the group comprising mesenchymal stem / stromal cells (MSCs) (such as those derived from bone marrow, adipose tissue, umbilical cord, or Wharton’s jelly), hematopoietic stem cells (HSCs), neural stem cells (NSCs), adipose-derived stem cells (ADSCs), endothelialRef. 6.2620 PCT PCT Appn Final PAT8531PC00progenitor cells (EPCs), dental pulp stem cells (DPSCs), muscle satellite cells, intestinal stem cells, and liver progenitor cells.

41. A method for enhancing the redox resistance of immunotherapy effector cells, the method comprising contacting the effector cells, ex vivo and / or in vivo, with one or more nucleic acids of claim 17, or with a vector or plasmid of any one of claims 19 to 21 comprising said one or more nucleic acids, in order to reduce or silence the expression of ZNF354A in said cells, thereby increasing their resistance to oxidative stress and, for example, extending their lifespan, wherein cellular exhaustion following activation is at least partly attributable to oxidative damage.

42. Use of one or more nucleic acids of claim 17, or a vector or plasmid of any one of claims 19 to 21 encoding said one or more nucleic acids, to reduce or silence the expression of ZNF354A in immunotherapy effector cells, thereby increasing their resistance to oxidative stress and, for example, extending their lifespan, wherein cellular exhaustion following activation is at least partly attributable to oxidative damage.

Images