WIP1 inhibitor for the treatment of glomerular disease

Abstract

Collapsing focal segmental glomerulosclerosis (collapsing FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by rapid progression to end-stage kidney disease (ESKD). The inventors used the telomerase-induced mouse model of CG (i-TERTci mice) to identify mechanisms to inhibit CG pathogenesis. They found that inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocks disease initiation in i-TERTci mice. They found that pharmacologic inhibition of WIP1 enzymatic activity in either telomerase mice with CG or in Tg26 mice promotes partial remission of proteinuria and ameliorates renal histopathologic features. Their findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG. Thus, the present invention relates to a method of preventing or treating glomerular disease (e.g. focal segmental glomerulosclerosis, in particular collapsing glomerulopathy) in a subject in need thereof, comprising the step of administering to said subject a Wild-type p53-induced phosphatase 1 (WIP1) inhibitor.

Description

[0001] WIP1 INHIBITOR FOR THE TREATMENT OF GLOMERULAR DISEASE

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular nephrology. The present invention relates to the prevention and the treatment of glomerular disease.

[0004] BACKGROUND OF THE INVENTION:

[0005] Like the other variants of focal segmental glomerulosclerosis (FSGS),1collapsing glomerulopathy (CG) is characterized by injury to podocytes, the highly specialized epithelial cells that critically support the glomerular filtration barrier. Nonetheless, in contrast to the other variants of FSGS, CG has the peculiarity of displaying exuberant glomerular epithelial cell hyperplasia forming pseudo-crescents in association with tuft collapse.2,3These distinctive features may explain why patients with CG exhibit poor responsiveness to conventional treatments for FSGS4,5and underscore the need for innovative and specific therapies.

[0006] Understanding the pathogenetic mechanisms of CG would provide critical insights for treatment development. Advances in the field benefited from the development of several animal models of CG. The double transgenic mouse model called “i-TERT” for inducible-TERT mice, enabled conditional overexpression of the telomerase protein component TERT6. In this model, TERT overexpression induces massive proteinuria and histological features reminiscent of CG including glomerular epithelial cell hyperplasia.6Further analysis of human kidney biopsies from individuals with CG, including HIV-associated nephropathy (HIV AN),7-9showed specific upregulation of TERT that is not observed in patients with other variants of FSGS.6TERT effects on glomerular cells are independent from its role in telomere synthesis since induction of TERTC1overexpression, a catalytically inactive form of TERT that is unable to elongate the telomere sequences,10similarly leads to CG in i-TERTclmice.6Thus, TERT upregulation is sufficient to trigger CG, and the i-TERTclmouse model represents a valuable tool to test potential therapeutic approaches.

[0007] Previous studies highlighted connections between TERT non-canonical functions and Wnt signaling pathway. TERT directly modulates Wnt signaling pathway by serving as a cofactor in a P-catenin transcriptional complex.11In addition, telomerase-induced CG in i-TERTclmice induces upregulation of Wnt signaling, and inhibiting Wnt signaling by means of systemic expression of Wnt inhibitor Dkkl results in marked podocyte normalization and improved filtration barrier function in both i-TERTclmice and a mouse model of HIV AN.6This suggests that modulating Wnt signaling could be a promising strategy to improve CG outcome. A signaling pathway that efficiently modulates Wnt in adult mice involves WIP1, a protein phosphatase encoded by the Ppmld gene broadly expressed in adult mice.12WIP1 is historically described as a p53 target engaged in a negative feedback loop that represses the DNA damage response (DDR).13'17Previous studies showed that WIP1 inactivation inhibits Wnt signaling in the mouse brain, thereby altering neurogenesis.18,19

[0008] The involvement of WIP1 in renal physiopathology remained unexplored and the article from Duret et al64represents the first report highlighting the relevance of inhibiting WIP1 to improve glomerulopathies. Indeed, while a previous study showed that WIP1 overexpression modulated polarization of macrophages and subsequently promoted the expression of 2 mesenchymal markers in a co-cultured kidney tubular cell line in vitro63, this study failed to show any impact of WIP1 down-regulation on expression of these markers in their in vitro system. Beside, the results presented by Jia et al. do not include the cell types that are relevant in the context of glomerulopathies in vivo, i.e. glomerular cells. Finally, while the authors suggest in this study that TGF-0 repression resulting from WIP1 inhibition could attenuate renal pathology, the molecular data obtained in the in vivo context in the article from Duret et al64demonstrate on the contrary that WIP1 inhibition in glomerular diseases in vivo is associated with TGF- 0 activation and that such activation is necessary to ameliorate disease in vivo. Thus, Jia et al. overstate the few data they obtained in vitro and these data do not suggest that WIP1 inhibition could have a beneficial effect in glomerular diseases in vivo.

[0009] To determine whether WIP1 depletion could interfere with TERT function on kidney epithelium, the inventors generated i-TERTcl;WIPl ' ' compound mice. They demonstrated that WIP1 inactivation restrains the ability of TERTC1to induce CG. Surprisingly, repression of TERTC1function upon WIP1 depletion does not involve Wnt signaling inhibition, but instead relies on promotion of a senescence-like phenotype. They demonstrate that treatment of i- TERTC1mice and of Tg26 mice -a mouse model of HIV AN- with a WIP1 inhibitor promotes partial remission of proteinuria and improves histopathological features of CG. Histological examination as well as bulk RNA sequencing profiling further reveal that WIP1 inhibition does not promote kidney fibrosis or inflammation. Finally, they document the requirement of cytostatic functions of TGF-0 signaling for CG repression observed in the context of WIP1 depletion in i-TERTclmice. Altogether, their findings suggest that pharmacologic targeting of WIP1 may be a promising therapeutic approach for patients with CG. SUMMARY OF THE INVENTION:

[0010] The present invention is defined by the claims. In particular, the present invention relates to the prevention and the treatment of glomerular disease.

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] Collapsing focal segmental glomerulosclerosis (collapsing FSGS), also known as collapsing glomerulopathy (CG), is the most aggressive variant of FSGS and is characterized by rapid progression to end-stage kidney disease (ESKD). Understanding CG pathogenesis represents a key step for the development of targeted therapies. Previous work implicated the telomerase protein component TERT in CG pathogenesis, as transgenic TERT expression in adult mice resulted in a CG resembling that seen in human primary CG and HIV-associated nephropathy (HIV AN). The inventors used the telomerase-induced mouse model of CG (i-TERTclmice) to identify mechanisms to inhibit CG pathogenesis. They found that inactivation of WIP1 phosphatase, a p53 target acting in a negative feedback loop, blocks disease initiation in i- TERTC1mice. Repression of disease initiation upon WIP1 deficiency is associated with senescence enhancement and requires TGF-P functions. They further assessed efficacy of a pharmacologic treatment to reduce disease severity in both i-TERTclmice and in a mouse model of HIV AN (Tg26 mice). They found that pharmacologic inhibition of WIP1 enzymatic activity in either telomerase mice with CG or in Tg26 mice promotes partial remission of proteinuria and ameliorates renal histopathologic features. Histological as well as high throughput sequencing methods further showed that selective inhibition of WIP1 does not promote kidney fibrosis or inflammation. Their findings suggest that targeting WIP1 may be an effective therapeutic strategy for patients with CG.

[0013] Main definitions:

[0014] As used herein, the term “Wild type p53-induced phosphatase 1” (WIP1), also known as PP2C 5 or PPM ID, refers to a serine / threonine protein phosphatase, belonging to the 2C 5 type protein phosphatases. WIP1 is an established oncogene due to its dephosphorylation of several tumor suppressors and negative control of the DNA damage response system. It has been reported to dephosphorylate p53, ataxia telangiectasia mutated, checkpoint kinase 1 and p38 mitogen activated protein kinases, forming negative feedback loops to inhibit apoptosis and cell cycle arrest. WIP1 serves a major role in tumorigenesis, progression, invasion, distant metastasis and apoptosis in various types of human cancer. Human WIP1 gene accession number is NG 023265.1. As used herein, the term “telomerase reverse transcriptase” (TERT) is the catalytic subunit of the enzyme telomerase, which, together with the telomerase RNA component (TERC), comprises the most important unit of the telomerase complex.

[0015] As used herein, the term “senescence” refers to the molecular program implemented during biological aging leading to cell cycle arrest and gradual deterioration of organ function.

[0016] As used herein, the term “podocyte” refers to specialized visceral epithelial cells anchored to the glomerular basement membrane (GBM) that make up one of the three layers constituting the glomerular filtration barrier. This barrier retains large molecules such as proteins in the bloodstream while smaller molecules such as water, salts, and sugars are filtered to generate the urine primitive. Podocytes present cytoplasmic extensions referred as foot processes or pedicels that interdigitate with those of adjacent podocytes through specialized tight junctions named slit diaphragms (SD). The complex cytoarchitecture of podocytes increase the surface area of the cells enabling efficient ultrafiltration. Podocytes secrete and maintain the GBM. There are numerous coated vesicles and coated pits along the basolateral domain of the podocytes which indicate a high rate of vesicular traffic. Podocytes possess a well-developed endoplasmic reticulum and a large Golgi apparatus, indicative of a high capacity for protein synthesis and post-translational modifications. There is also growing evidence of a large number of multivesicular bodies and other lysosomal components seen in these cells, indicating a high endocytic activity. The podocyte slit diaphragms are composed of a number of cellsurface proteins including nephrin, podocalyxin, and P-cadherin, and prevent the passage of large macromolecules such as serum albumin and gamma globulin. Proteins that are required for the correct function of the slit diaphragm include nephrin, NEPHI, NEPH2, podocin, CD2AP and FATE The glomerular ultrafiltrate is further processed by the tubular segments of the nephron to produce urine. Podocytes are also involved in regulation of glomerular filtration rate (GFR). When podocytes contract, they cause closure of filtration slits. This decreases the GFR by reducing the surface area available for filtration.

[0017] As used herein, the term “glomerulus” is a network of small blood vessels (capillaries) known as a tuft, located at the beginning of a nephron in the kidney. Each of the two kidneys contains about one million nephrons. The tuft of capillaries is enclosed by a cup-like structure named Bowman’s capsule, and in the central region of the tuft, mesangial cells provide structural support to the glomerulus. Blood filtration occurs through the glomerular filtration barrier that consist of three layers: the fenestrated endothelial cells constituting the capillary wall, the glomerular basement membrane made of a complex mesh of extracellular matrix proteins, and the podocyte. The ultrafiltrate produced by the glomerulus is poured in the Bowman's capsule, then enters the renal tubule of the nephron. The glomerulus receives its blood supply from an afferent arteriole of the renal arterial circulation. Unlike most capillary beds, the glomerular capillaries exit into efferent arterioles rather than venules. The resistance of the efferent arterioles causes sufficient hydrostatic pressure within the glomerulus to provide the force for ultrafiltration. The glomerulus and its surrounding Bowman's capsule constitute the renal corpuscle, the basic filtration unit of the kidney. The rate at which blood is filtered through all glomeruli, and thus the measure of the overall kidney function, is called glomerular filtration rate.

[0018] As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. In some embodiments, the patient is an adult. In some embodiments, the subject is more than 15 years old. In some embodiments, the subject is more than 20 years old. In some embodiments, the subject is more than 25 years old. In some embodiments, the subject is more than 30 years old. In some embodiments, the subject is more than 35 years old. In some embodiments, the patient is an elderly. In a preferred embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with focal segmental glomerulosclerosis (FSGS). In some embodiments, the patient is an elderly. In a preferred embodiment of the invention, a subject according to the invention refers to any subject (preferably human) afflicted with or susceptible to be afflicted with collapsing glomerulopathy (CG).

[0019] As used herein, the term “proteinuria” is the presence of excess proteins in the urine. In healthy persons, urine contains very little protein, less than 150 mg / day; an excess is suggestive of illness. Excess protein in the urine often causes the urine to become foamy (although this symptom may also be caused by other conditions). Severe proteinuria can cause nephrotic syndrome in which there is worsening swelling of the body.

[0020] As used herein, the terms “glomerular diseases” or “glomerulopathy” refer to a set of diseases affecting the glomeruli of the nephron. Such diseases can include processes that are inflammatory or noninflammatory. Many diseases and conditions can damage the glomeruli. Two broad terms used to describe many forms of damage to the glomeruli are: glomerulonephritis which corresponds to inflammation (swelling) of the glomeruli and glomerulosclerosis which corresponds to scarring / hardening of the glomeruli. Glomerular diseases are classified as follows :

[0021] Primary: a kidney disease specifically affecting the glomeruli (e.g., minimal change gl omerul onephriti s)

[0022] Secondary: a disease affecting the glomeruli in the context of a systemic disease (e.g., lupus nephritis in Systemic lupus erythematosus) or a disease affecting another organ (e.g., diabetic nephropathy)

[0023] Diffuse: when > 50% of glomeruli is affected (e.g., diffuse proliferative gl omerul onephriti s)

[0024] Focal: when < 50% of glomeruli is affected (e.g., focal segmental glomerulosclerosis) Global: when the entire glomerulus is affected

[0025] Segmental: only part of the glomerulus is affected

[0026] Proliferative: when there are an increased number of cells in the glomerulus

[0027] Membranous: when there is a thickening of the glomerular basement membrane (e.g., membranous nephropathy)

[0028] Sclerosing: when there is a scarring of the glomerulus

[0029] Necrotizing: when there is a cell death within the glomerulus

[0030] Crescentic: when there is an accumulation of cells such as macrophages, fibroblasts, and epithelial cells in Bowman space (e.g. crescentic glomerulonephritis).

[0031] In a particular embodiment, the glomerular disease of the present invention is chosen among: crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy or focal segmental glomerulosclerosis.

[0032] As used herein, the term “crescentic glomerulonephritis” also known as rapidly progressive glomerulonephritis is characterized by the presence of extensive glomerular crescents (usually greater than 50%) as the principal histologic finding. Because it often clinically presents with a rapid decline in kidney function, it is also known as rapidly progressive glomerulonephritis (RPGN). As used herein, the term “lupus nephritis” is an inflammation of the kidneys caused by systemic lupus erythematosus (SLE), an autoimmune disease. It is a type of glomerulonephritis in which the glomeruli become inflamed. Since it is a result of SLE, this type of glomerulonephritis is said to be secondary, and has a different pattern and outcome from conditions with a primary cause originating in the kidney.

[0033] As used herein, the term “diabetic nephropathy” also known as diabetic kidney disease, is the chronic loss of kidney function occurring in those with diabetes mellitus. Diabetic nephropathy is the leading causes of chronic kidney disease (CKD) and end-stage renal disease (ESRD) globally. The triad of protein leaking into the urine (proteinuria or albuminuria), rising blood pressure with hypertension and then falling renal function is common to many forms of CKD. Protein loss in the urine due to damage of the glomeruli may become massive, and cause a low serum albumin with resulting generalized body swelling (edema) so called nephrotic syndrome.

[0034] As used herein, the term “focal segmental glomerulosclerosis” (FSGS) is a histopathologic finding of scarring (sclerosis) of glomeruli and damage to renal podocytes. This process damages the filtration function of the kidney, resulting in protein presence in the urine due to protein loss. FSGS is a leading cause of excess protein loss — nephrotic syndrome — in children and adults. Signs and symptoms include proteinuria and edema. Kidney failure is a common long-term complication of the disease. FSGS can be classified as primary, secondary, or genetic, depending on whether a particular toxic or pathologic stressor or genetic predisposition can be identified as the cause. Five mutually exclusive variants of focal segmental glomerulosclerosis may be distinguished by the pathologic findings seen on renal biopsy: Collapsing variant, Glomerular tip lesion variant, Cellular variant, Perihilar variant or Not otherwise specified (NOS) variant.

[0035] In a particular embodiment, the focal segmental glomerulosclerosis is collapsing glomerulopathy (CG), a collapsing variant.

[0036] As used herein, the term “collapsing glomerulopathy” (CG) has its general meaning in the art and refers to a distinct entity, so called collapsing focal segmental glomerulosclerosis (collapsing FSGS), involving extracapillary proliferation and marked dysregulation of the quiescent podocyte phenotype (Bariety J, Nochy D, Mandet C, Jacquot C, Glotz D, Meyrier A: Podocytes undergo phenotypic changes and express macrophagic-associated markers in idiopathic collapsing glomerulopathy. Kidney Int 53 : 918 -925, 1998. Srivastava T, Garola RE, Singh HK: Cell-cycle regulatory proteins in the podocyte in collapsing glomerulopathy in children. Kidney Int 70 : 529 -535, 2006). Diseased podocytes exhibit a loss and gain of markers of differentiation and proliferation, respectively. Recent studies indicate that parietal epithelial cells also may be recruited into the viscerally located proliferative lesion (Dijkman HB, Weening JJ, Smeets B, Verrijp KC, van Kuppevelt TH, Assmann KK, Steenbergen EJ, Wetzels J: Proliferating cells in HIV and pamidronate-associated collapsing focal segmental glomerulosclerosis are parietal epithelial cells. Kidney Int 70 : 338-344, 2006). Collapsing glomerulopathy (CG) can be primary or secondary (Albaqumi M, Barisoni L. Current views on collapsing glomerulopathy. J Am Soc Nephrol. 2008 Jul;19(7): 1276-81. PMID: 18287560). Secondary forms can be associated to HIV infection or other viral infections (such as hepatitis C virus infection, and parvovirus), drug addiction, pamidronate, systemic lupus erythematosus- like disorder and multiple myeloma or can be favored by genetic background.

[0037] Method of treatment of the present invention:

[0038] A method of preventing or treating glomerular disease in a subject in need thereof, comprising the step of administering to said subject a Wild-type p53-induced phosphatase 1 (WIP1) inhibitor.

[0039] In a particular embodiment, the present invention relates to method of preventing or treating focal segmental glomerulosclerosis in a subject in need thereof, comprising the step of administering to said subject a Wild-type p53-induced phosphatase 1 (WIP1) inhibitor.

[0040] In a particular embodiment, the present invention relates to method of preventing or treating collapsing glomerulopathy (CG) in a subject in need thereof, comprising the step of administering to said subject a Wild-type p53-induced phosphatase 1 (WIP1) inhibitor.

[0041] As used herein, the term “inhibitor of WIP1” refers to a natural or synthetic compound that has a biological effect to inhibit the expression and / or the activity of WIPE

[0042] In a particular embodiment, the WIP1 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. As used herein, the term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0043] In a particular embodiment, the WIP1 inhibitor is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

[0044] In a particular embodiment, the WIP1 inhibitor is a small organic molecule.

[0045] As used herein, the term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0046] In a particular embodiment, the WIP1 inhibitor is GSK2830371.

[0047] As used herein, the term “GSK2830371” refers to a highly selective WIP1 phosphatase inhibitor having the following CAS No. 1404456-53-6, the following chemical formule: C23H29CIN4O2S and the following chemical structure :

[0048] In some embodiments, the WIP1 inhibitor is an antibody.

[0049] As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93 / 1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06 / 030220 and WO 06 / 003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

[0050] In a particular, the WIP1 inhibitor is an intrabody having specificity for WIP1. As used herein, the term "intrabody" generally refers to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking / localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

[0051] In some embodiments, the WIP1 inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of WIP1.

[0052] In a particular embodiment, the inhibitor of JMY expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long doublestranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukaemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0053] In some embodiments, the WIP1 inhibitor is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0054] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0055] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534 / genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0056] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0057] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0058] The present invention also relates to a combined preparation comprising the WIP1 inhibitor for use according to the invention and a classical treatment. More particularly, the invention relates to a i) WIP1 inhibitor and a ii) classical treatment for simultaneous, separate or sequential use in the treatment of glomerular disease, as a combined preparation.

[0059] In a particular embodiment, the invention relates to a i) WIP1 inhibitor and a ii) classical treatment for simultaneous, separate or sequential use in the treatment of glomerular disease (e.g. focal segmental glomerulosclerosis (FSGS), in particular collapsing glomerulopathy), as a combined preparation.

[0060] In a particular embodiment, the invention relates to a i) WIP1 inhibitor and a ii) classical treatment for simultaneous, separate or sequential use in the treatment of collapsing glomerulopathy (CG), as a combined preparation. As used herein, the term “classical treatment” refers to any compound, natural or synthetic, immunotherapy and chemotherapy used for the treatment of a glomerular disease (e.g. focal segmental glomerulosclerosis, in particular collapsing glomerulopathy).

[0061] In some embodiment, the classical treatment refers to a treatment with a immunosuppressive agent.

[0062] Typically, the invention relates to a i) WIP1 inhibitor and a ii) immunosuppressive agent for simultaneous, separate, or sequential use in the treatment of glomerular disease. In particular , the invention relates to a i) WIP1 inhibitor and a ii) immunosuppressive agent for simultaneous, separate, or sequential use in the treatment of focal segmental glomerulosclerosis (FSGS). More particularly, a i) WIP1 inhibitor and a ii) immunosuppressive agent for simultaneous, separate, or sequential use in the treatment of collapsing glomerulopathy (CG).

[0063] As used herein the terms “immunosuppressive therapy” or “immunosuppressant” or “immunosuppressive agent” refer to drugs that inhibit or prevent the activity of the immune system.

[0064] Examples of immunosuppressive agent include but are not limited to steroid, glucocorticoids (such as prednisone, dexamethasone, and hydrocortisone), cytostatics such as alkylating agents (i.e cyclophosphamide), antimetabolites, azathioprine and mercaptopurine or cytotoxic antibiotics), antibodies, drugs acting on immunophilins (such as ciclosporin, tacrolimus, sirolimus, everolimus, zotarolimus), Interferons, Opioids, TNF binding proteins, Mycophenolate or Small biological agents (such as fmgolimod, muriocin).

[0065] In some embodiment, the classical treatment refers to a treatment with an angiotensin- converting-enzyme (ACE) inhibitors.

[0066] Typically, the invention relates to a i) WIP1 inhibitor and an ii) ACE inhibitors for simultaneous, separate, or sequential use in the treatment of glomerular disease. In particular, the invention relates to a i) WIP1 inhibitor and an ii) ACE inhibitors for simultaneous, separate, or sequential use in the treatment of focal segmental glomerulosclerosis (FSGS). More particularly, a i) WIP1 inhibitor and a ii) ACE inhibitors for simultaneous, separate, or sequential use in the treatment of collapsing glomerulopathy (CG). As used herein the term “Angiotensin-converting-enzyme inhibitors” (ACE inhibitors) refers to a class of medication used primarily for the treatment of high blood pressure and heart failure. ACE inhibitors inhibit the activity of angiotensin-converting enzyme, an important component of the renin-angiotensin system which converts angiotensin I to angiotensin II, and hydrolyses bradykinin. Therefore, ACE inhibitors decrease the formation of angiotensin II, a vasoconstrictor, and increase the level of bradykinin, a peptide vasodilator. This combination is synergistic in lowering blood pressure.

[0067] Exemple of inhibitors include but are not limited to benazepril, zofenopril, perindopril, trandolapril, captopril, fosinopril, enalapril, quinapril, moexipril, lisinopril, and ramipril.

[0068] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., an inhibitor of WIP1 alone or in a combination with a classical treatment) into the subject, such as by, oral, intravenous, intramuscular, enteral, subcutaneous, parenteral, systemic, local, spinal, nasal, topical or epidermal administration (e.g., by injection or infusion). When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0069] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0070] Pharmaceutical composition of the present invention

[0071] In a third aspect, the invention relates to a pharmaceutical composition for use in the treatment of glomerular disease. In particular, the invention relates to a pharmaceutical composition for use in the treatment of focal segmental glomerulosclerosis (FSGS). In particular, the invention relates to a pharmaceutical composition for use in the treatment of collapsing glomerulopathy (CG).

[0072] In a particular embodiment, the pharmaceutical composition according to the invention comprises a WIP1 inhibitor.

[0073] In a particular embodiment, the invention relates to a pharmaceutical composition comprising a WIP1 inhibitor and a classical treatment as described above.

[0074] In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and a classical treatment, as combined preparation for use simultaneously, separately or sequentially in the treatment of glomerular disease.

[0075] In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and a classical treatment, as combined preparation for use simultaneously, separately or sequentially in the treatment of focal segmental glomerulosclerosis. In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and a classical treatment, as combined preparation for use simultaneously, separately or sequentially in the treatment of collapsing glomerulopathy.

[0076] In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and an immunosuppressive agent, as combined preparation for use simultaneously, separately or sequentially in the treatment of glomerular disease.

[0077] In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and an immunosuppressive agent, as combined preparation for use simultaneously, separately or sequentially in the treatment of focal segmental glomerulosclerosis.

[0078] In a particular embodiment, the pharmaceutical composition according to the invention wherein the WIP1 inhibitor and an immunosuppressive agent, as combined preparation for use simultaneously, separately or sequentially in the treatment of collapsing glomerulopathy.

[0079] In another embodiment, the pharmaceutical composition according to the invention, wherein the WIP1 inhibitor is GSK2830371.

[0080] The WIP1 inhibitor as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. “Pharmaceutically” or “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0081] In certain embodiments, the pharmaceutical formulation can be suitable for parenteral administration. The terms “parenteral administration” and “administered parenterally,” as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In certain embodiments, the present invention provides a parenteral formulation comprising a WIP1 inhibitor and a classical as a combined preparation.

[0082] In certain embodiments, the present invention provides a parenteral formulation comprising a WIP1 inhibitor and a classical treatment as a combined preparation. For example, and not by way of limitation, the present invention provides a parenteral formulation comprising GSK2830371 and a classical treatment as a combined preparation. In a particular embodiment, when the WIP1 inhibitor is combined with a classical treatment, the combination is formulated for oral, cutaneous or topical use.

[0083] Method of screening of a WIP1 inhibitor

[0084] A further object of the present invention relates to a method of screening a drug suitable for the treatment of glomerular disease comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and / or expression of WIP1.

[0085] In a particular embodiment, the present invention relates to a method of screening a drug suitable for the treatment of focal segmental glomerulosclerosis comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and / or expression of WIP1.

[0086] In a particular embodiment, the present invention relates to a method of screening a drug suitable for the treatment of collapsing glomerulopathy comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and / or expression of WIP1.

[0087] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of WIP1. In some embodiments, the assay first comprises determining the ability of the test compound to bind to WIP1. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of WIP1. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of WIP1, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

[0088] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0089] FIGURES:

[0090] Figure 1: Inhibition of WIP1 phosphatase activity ameliorates disease severity in the Tg26 mouse model of HIV AN. (A) Mean number of PCNA-positive cells per glomerular cross section in kidneys from Tg26 mice treated with either DMSO or GSK2830371. (B) (n=12 for each group, half females and half males). Percentage of collapsed glomeruli in Tg26 mice injected with either DMSO or GSK2830371. Data are shown for each animal and mean value for each group is shown as a green line. ***P = 0.0002 by t-test. For each animal, all glomeruli (about 150) on the whole kidney section were analyzed. (C) Percentage of parenchyma with tubular atrophy and interstitial fibrosis (IFTA) in Tg26 mice injected with either DMSO or GSK2830371. **P = 0.0088 by Mann-Whitney test for Tg26 (+GSK2830371) versus Tg26 (+DMSO) mice. (D) Percentage of parenchyma with interstitial inflammation in Tg26 mice injected with either DMSO or GSK2830371. **P = 0.0072 by t-test for Tg26 (+GSK2830371) versus Tg26 (+DMSO) mice. (E) Analysis of urinary microalbuminuria to creatinine ratio in Tg26 mice (+DMSO) and Tg26 mice (+GSK2830371) before the start of IP treatment (dO) and at the time of kidney collection 7 days after the start of IP injections (d7) (n=8 for each group). ***P < 0.0001 by t-test for Tg26 (+GSK2830371) at d7 versus Tg26 (+GSK2830371) at dO. (F) Individual variation of urinary microalbuminuria to creatinine ratio of Tg26 mice injected with either DMSO or GSK2830371 in the time course of the experiment.

[0091] Figure 2: Treatment with selective WIP1 inhibitor does not promote kidney fibrosis nor inflammation. (A) Percentage of collapsed glomeruli in the kidneys of i-TERTci mice treated with either DMSO or GSK2830371 for 21 days. Data are shown for each animal, and mean value for each group is shown as a green line. For each animal, all glomeruli (approximately 150) present on the entire kidney section were analyzed. (B) Percentage of fibrosis area in the kidneys of i-TERTci mice treated with either DMSO or GSK2830371 for 21 days. Data are shown for each animal, and mean value for each group is shown as a green line.

[0092] EXAMPLE:

[0093] Material & Methods

[0094] Mice.

[0095] Tetracycline-regulated i-TERTcltransgenic mice, and WIPE7' mice were previously described.10,12’20Oligonucleotide pairs used to genotype these mice are provided in Supplementary Table SI. Tg26 transgenic mice contain a proviral human immunodeficiency virus (HIV) DNA construct carrying a deletion of the gag and pol genes. On the FVBN / J genetic background, heterozygous transgenic mice exhibit proteinuria and collapsing glomerulopathy (CG) starting at about 3 weeks of age.21,22

[0096] TERTci overexpression in i-TERTci and i-TERTci;WIPl- / ~ mice.

[0097] Double transgenic i-TERTclmice encompass both actin-rtTA+and tetop-TERTcl+ transgenes, allowing doxycycline-regulated and ubiquitous expression of TERTC1in these mice. Mice are maintained in a heterozygous status for each transgene. To induce TERTC1overexpression, doxycycline (2 mg / ml in 5% sucrose) (Doxycycline Hy elate, Sigma, Ref# D9891) was administered in drinking water in light-protected bottles and changed biweekly. The control mice followed the same treatment.

[0098] Treatment of i-TERTcland Tg26 mice with GSK2830371.

[0099] Two to four months old i-TERTclmice (half females, half males) were treated with doxycycline to induce TERTC1overexpression. High proteinuria levels (above 10 mg / mL) were observed at about 12 days of treatment. At this point, daily intraperitoneal (IP) injection of GSK2830371(Sigma, Ref# SML1048) (25 mg / kg body weight in 5% DMSO / 95% corn oil) was administered until the end of the experiment. Control i-TERTclmice were treated similarly with daily IP injection of DMSO (5% DMSO / 95% com oil). Animals were randomly assigned to treatment and vehicle groups. The same treatments were administered to Tg26 mice. Four- week-old heterozygous Tg26 mice (half females, half males) with proteinuria scored as 3+ (above 5 mg / mL), were randomized to receive daily IP injection of GSK2830371 or DMSO for 7 days. Kidneys collected from i-TERTcland Tg26 mice at the end of the procedures were evaluated for histopathologic injury based on a semi quantitative scale by a renal pathologist (V.D.D.), who was blinded to the treatment arms22or used for RNA-seq. Treatment of mice with Galunisertib.

[0100] Two to four months old mice were treated with doxycycline in drinking water for 18 days. Galunisertib (Sigma, Ref# SML2851) was administered by daily intraperitoneal (IP) injection (10 mg / kg body weight in 5% DMSO / 95% com oil) from the start of doxycycline treatment (day 0) until the end of the experiment (day 18). Daily IP injection of DMSO (5% DMSO / 95% corn oil) was similarly administered to control groups.

[0101] RNA-seq.

[0102] Total RNA was extracted using the RNeasy Micro Kit with DNase treatment (Qiagen, Ref# 74004). RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Only samples with RNA Integrity Number (RIN) >7 were further submitted to high throughput sequencing. Samples were subjected to high-output (2x100 bp) paired-end sequencing by BGI Global services (China). Quality of raw reads was assessed with FastQC. Clipping adaptor sequences was carried out using Trimmomatic and the trimmed reads were aligned to the mouse genome reference (mm 10).

[0103] Proteinuria analysis.

[0104] Urine was collected from each mouse before starting the experimental procedures, and in the time course of the experiments. Proteinuria levels were assessed using Bradford protein assay (BioRad, Ref# 5000002) and the Chemstrip assay (Chemstrip 10 UA, Roche).

[0105] Urinary albumin to creatinine ratio (ACR) analysis.

[0106] Urine was collected from each mouse before starting the experimental procedures, and in the time course of the experiments. Urine samples were assessed for protein content by the Albumin Creatinine Ratio Assay kit (Abeam, Ref# ab241018), or the Albuwell M (Mouse Albumin ELISA) kit (Ethos Biosciences, Ref# 1011) with Stanbio Direct Creatinine LiquiColor (Stanbio Laboratory, Procedure No 0430).

[0107] Histology.

[0108] Kidneys were fixed overnight in 10% buffered formalin and embedded in paraffin. Four pm tissue sections were stained with hematoxylin (H&E) (Microm microtech, Ref# U / C0303) and eosin (Microm microtech, Ref# U / C0363), Sirius Red (Abeam, Ref# ab 150681) or Periodic Acid-Schiff (PAS) (Sigma, Ref# 395B-1KT) for microscopic analysis. Immunohistochemistry.

[0109] Antigen retrieval was performed on 4 pm paraffin sections using Vector unmasking reagent (Vector Laboratories, Ref# H3300). Mouse monoclonal primary antibodies were detected using a biotinylated anti-mouse IgG (MOM kit, Vector Laboratories, Ref# BMK-2202) followed by streptavin-AlexaFluor647 (Jackson ImmunoResearch, Ref# 016-600-084). For immunostaining using rabbit and rat primary antibodies, kidney sections were blocked (PBS; 10 mg / ml BSA; 5% NGS; 0.01% Triton), then incubated with the primary antibody diluted in the blocking solution for overnight at 4°C. Detection was performed with a AlexaFluor594- conjugated goat anti-rabbit or AlexaFluor488-conjugated goat anti-rat secondary antibodies (1 :600) (Jackson Immunoresearch, Ref# 111-585-144 and Ref# 112-545-167). The primary antibodies used are mentioned in Supplementary Table S2. For immunohistochemistry on intestine, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes at room temperature. The chromogenic detection was achieved using DAB staining (Vector, Ref# SK4100) at room temperature. The reaction was subsequently halted by rinsing with tap water, followed by counterstaining with hematoxylin.

[0110] TUNEL assay.

[0111] TUNEL analysis was performed on 4pm paraffin sections using TUNEL apoptosis detection kit (Millipore, Ref# S7100). Slides were deparaffinized and rehydrated, treated with proteinase K, quenched in 3% hydrogen peroxide and incubated with terminal deoxynucleotidyl transferase to label fragmented DNA with UTP-digoxigenin. Finally, sections were incubated with HRP-conjugated digoxigenin-specific antibody and visualized by chromogenic detection and hematoxylin counterstain.

[0112] Senescence-associated p-galactosidase staining.

[0113] Kidneys were frozen sectioned and fixed with fixative solution from the Senescence- Associated P-Galactosidase (SA-P-Gal) Staining Kit (Cell Signaling, Ref #9860). Tissue sections were then stained at pH 6.0 for 12h at 37°C following the manufacturer's instructions, and counterstained with nuclear fast red (Sigma, Ref# N3020).

[0114] Image analysis using ImageJ software.

[0115] Stained kidney sections were sequentially scanned to acquire imaging of the entire kidney section. For glomerular signal analysis, glomeruli areas were manually demarcated on entire kidney sections, and demarcated glomeruli were defined as areas to be analyzed using ImageJ software. Signal intensity or aera covered by signal were then determined by ImageJ.

[0116] Preparation of glomerular and tubular extracts.

[0117] Kidneys were harvested and renal pelvis and inner medulla were removed using fine surgical forceps and scissors. Kidneys were then minced and incubated with digestion buffer solution I (20 ug / ml DNAse I, 1 mg / ml pronase, 100 ug / ml collagenase IV in HBSS) for 30 min at 37°C under gentle rotation. Samples were then passed through 100 Dm cell strainers and the filtrates were pressed through 40 m cell strainers. The retained glomeruli on the top of the 40 m strainers were rinsed into a clean tube and the filtrates containing the tubular fragments were collected.

[0118] Quantitative RT-PCR.

[0119] For whole kidney analysis, snap-frozen kidneys were ground in liquid nitrogen. RNA was isolated from organs or glomerular and tubular extracts by homogenization in Trizol (Thermo Fischer, Ref# 15596018), and Ipg of total RNA (whole kidney) was reverse-transcribed (QuantiTect Reverse Transcription kit, Qiagen, Ref# 205311), then subjected to qPCR (FastStart Universal SYBR Green Master (Rox), (Roche, Ref# 4913850001) using primer pairs mentioned in Supplementary Table S3. SYBR-green analysis was performed using the 7900HT Fast Real-Time PCR System machine (AB I). The expression level of each gene analyzed was normalized to the corresponding HPRT level.

[0120] Western-blot.

[0121] Samples were lysed in RIPA buffer containing protease inhibitors (BLAP) and phosphatase inhibitors (Roche, Ref# 04906837001). Total cell lysates were separated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with primary antibodies. The signals were detected with chemiluminescence reagents (Thermo Fisher Scientific, Ref# 34095). The antibodies used are mentioned in Supplementary Table S4.

[0122] Analysis of RNA-seq data.

[0123] Differentially expressed genes (DEGs) were identified using the Bioconductor package DESeq2. DEGs were considered statistically significant at an adjusted p-value (Padj) < 0.05. Gene set enrichment analysis (GSEA) was performed using the hallmark C7 collections on GSEA software (version 3.0) and MsigDB database and C2 (curated gene sets) database. The reported GSEA output was selected based on a false discovery rate (FDR) q-value < 0.25. The g:Profiler tool set was used to identify the enriched pathways using the Reactome database.

[0124] Analysis of public snRNA-seq data.

[0125] Published snRNA-seq data for normal human adult kidneys available in GEO under accession number (GSE151302) and for a COVID-19 patient autopsy kidney tissue under accession number (GSE167747) were used to analyze the expression of PPM1D gene (WIP1).

[0126] Bioinformatics Analysis of snRNA-seq data.

[0127] R package, Seurat (version 4.1.0) was used for quality control, dimensionality reduction and the clustering of the single nucleus RNA-seq datasets and cell type annotation of four merged count matrix data sets from healthy kidney (GSM4572192, GSM4572193, GSM4572194, GSM4572195 ) and GSM5557003 form COVID-19 patient (GSM5557003 ).Poor quality nuclei were excluded, such as nuclei with <500 or >5,000 unique genes expressed genes (as they are putative duplets). Nuclei were also discarded if their mitochondrial gene percentages were over 15%. Data were normalized and scaled. Dimensional reduction was performed with UMAP. Clustering was performed using the function FindCluster in Seurat to identify cell clusters for both studies. To identify the marker genes, differential expression analysis was performed by the function Find AllMarkers. Assigning cell type identity to clusters was performed based on the top differential genes from each cluster and clusters were labeled with cell type names based on the expression of putative cell type markers reported in previous study (Muto et al., 2021) (e.g. Nphs2 for podocyte, Emcn for endothelial cells, Slc34al for proximal tubular cells, etc). Specific expression of genes of interest in various formats such as Heatmap, violin plot and dot plot using ggplot2 R package. Dot plots were used to show the average expression level of the genes of interest in each cluster or cell type population.

[0128] Study approval.

[0129] All mice were treated in accordance with the Institutional Animal Care and Use Committee approved guidelines at the Universite Cote d’Azur (UCA, Nice, France) (CIEPAL-AZUR Agreements APAFIS#15232-2018051116515863v3, APAFIS#28252-2020111812195508v2, and APAFIS#33000-2021091515568721 v2). Statistics.

[0130] Data were analyzed using PRISM software. Data were first submitted to the outliers test (ROUT Method with Q=l%). Identified outliers were removed for subsequent analysis. Normal distribution of the data was then assessed using an Anderson-Darling test. When the data passed normality test, parametric tests were performed: ANOVA or two-tailed unpaired t-test. When the data did not pass normality test, non-parametric tests were performed: Kruskal-Wallis test, two-tailed Mann-Whitney test or Wilcoxon matched-pairs signed rank test. A P < 0.05 was considered statistically significant and is presented as follow: * for / J< 0.05, ** for / J< 0.01, and *** for < 0.001.

[0131] Results

[0132] WIP1 deficiency restrains the ability of TERT to induce a collapsing glomerulopathy

[0133] To determine the impact of WIP1 deficiency on TERT -induced CG, we crossed i-TERTclmice with Ppmld constitutive knockout mice (WIPE") (Data not shown) and we treated i- TERTC1;WIPE ‘ compound mice with doxycycline to induce TERTC1overexpression (Data not shown . Urinary albumin to creatinine ratio (ACR) monitoring of these mice revealed that while i-TERTclmice display massive proteinuria from day 14 of doxycycline treatment,6,23TERTC1overexpression in the setting of WIP1 deficiency in i-TERTcl;WTPE ' mice did not induce significant proteinuria (Data not shown). Kidney histology examination further showed that i- TERTC1;WIPE ‘ mice did not display the histopathological features of CG observed in i-TERTclmice (Data not shown)6

[0134] Podocytes are quiescent within the adult kidney, a pre-requisite for maintaining their specialized functions, and they express the cyclin dependent kinase (CDK)-inhibitors p27 and p57.24,25Podocyte alterations induced by TERTC1overexpression include acute cell cycle entry, which compromises their ability to support filtration barrier function.6Induction of podocyte proliferation in i-TERTclmice is accompanied with downregulation of both p27 and p57 (Data not shown). Nonetheless, such ability of TERTC1to repress p27 and p57 expression was blunted by WIP1 inactivation in i-TERTcl;WIPE ‘ (Data not shown). We next carried out quantification of global cell proliferation within glomeruli and assessed the proportion of proliferating cells that stain positive for podocyte-specific marker WT1, for activated parietal epithelial cell (PEC) marker CD44, or which are located in the PEC layer (Data not shown). This quantification revealed that PECs and podocytes contributed equally to the epithelial hyperplasia observed in glomeruli of i-TERTclmice (Data not shown). In addition, such analysis showed that WIP1 deficiency prevented the significant podocyte loss observed in i-TERTclmice (Data not shown . Finally, glomerular proliferation analysis showed that the appearance of PCNA+ cells in i-TERTclmice was blocked by WIP1 deficiency in i-TERTcl;WIPlAmice (Data not shown). Collectively, these results indicate that WIP1 inactivation interferes with the ability of TERTC1to induce CG in adult mice.

[0135] WIP1 deficiency does not impede Wnt pathway induction but promotes activation of the DNA damage response in kidneys of i-TERT01mice

[0136] On the basis of previous studies linking both telomerase and WIP1 to the Wnt program,6,10’11’18’19’23we hypothesized that the inability of TERTC1to induce CG upon WIP1 deficiency may be attributable to a defect in Wnt signaling activation. We therefore queried activation status of the Wnt pathway in kidneys of i-TERTcl;WIPlAmice. The antagonists Dickkopf proteins (Dkks) and Wnt ligands are secreted proteins that can act in a paracrine manner. Hence, to better understand the involvement of nephron compartments in repressing CG progression in i-TERTcl;WIPlAmice we prepared extracts enriched in intact glomeruli or tubules using size- selective sieving performed on fresh kidneys (Data not shown). Quantitative RT-PCR (qRT-PCR) analysis of these extracts showed no significant change in the expression levels of the Dkks tested in i-TERTcl;WIPlAcompared to i-TERTclmice (Data not shown). In addition, among Wnt ligands tested, only Wnt4 showed significant upregulation in glomerular extracts from i-TERTclmice that was not altered in i-TERTcl;WIPlAmice (Data not shown). Further western blot analysis of the level of the activated, unphosphorylated form of P-catenin (ABC) revealed its marked increase in both i-TERTcland i-TERTcl;WIPlAmice (Data not shown). Those results suggest that repression of TERTC1functions observed in a WIP1 null background cannot be attributable to a defect in Wnt signaling pathway activation.

[0137] WIP1 is a serine / threonine phosphatase described as a direct target of p5312that serves as a negative feedback regulator of the DDR by directly dephosphorylating several key components of the ataxia-telangiectasia mutated kinase (ATM)-dependent signaling network, including the sensor kinase ATM,15and its target histone H2AX (Data not shown11Hence, we sought to determine whether WIP1 depletion led to DDR promotion in i-TERTcl;WIPlAmice. Immunofluorescence analysis revealed a significant and similar increase of phosphorylated H2AX (y-H2AX) foci in podocytes of both i-TERTcland i-TERTcl;WIPlAmice (Data not shown). Assessment of phospho-ATM levels by immunofluorescence showed a concomitant significant increase of phospho-ATM foci in podocytes of i-TERTclmice, with an upward trend in i-TERTcl;WIPlAmice (Data not shown). Interestingly, WIP1 deletion on its own also promoted phospho-ATM induction in podocytes of WIPE" mice (Data not shown). This result suggests that combined WIP1 deficiency and TERTC1overexpression amplifies DDR signaling activation.

[0138] WIP1 deficiency upon TERT01overexpression does not induce apoptosis but promotes the implementation of a senescence-like program

[0139] We next aimed to determine if activation of DDR signaling led to induction of apoptosis in the kidneys of i-TERTcl;WIPlAmice. Assessment of expression level of the proapoptotic p53 target genes Bax, Bbc3 (encoding PUMA) and Noxa26'28showed no induction of their transcription in i-TERTcl;WIPlAmice (Data not shown). Further examination of the apoptotic effector Caspase-3 by western blot showed no increase of its activated cleaved form in kidneys of i-TERTcl;WIPlAmice (Data not shown). Finally, examination of DNA fragmentation by the mean of TUNEL assay showed no activation of the apoptotic process in kidneys of i- TERTC1;WIP1Amice (Data not shown). Together, those results show that activation of DDR signaling observed in the kidneys of i-TERTcl;WfPl' ' mice does not lead to apoptosis induction.

[0140] Because persistent DDR activation can lead to cellular senescence,29we next aimed to determine the status of the senescence process in the kidneys of i-TERTcl;WIPlAmice. The cyclin-dependent kinase inhibitors p21 and pl6 both represent key components of the senescence process,30,31but display differential roles in implementation of the senescence program, with p21 being described as important for the onset of senescence whereas pl6 upregulation represents a key event in maintaining deep senescence observed during aging.29Hence, we sought to determine whether p21 and pl6 were upregulated in i-TERTcl;WIPlAmice. While pl6 was not expressed in any of the mouse groups, p21 was significantly upregulated in the glomerular extracts from i-TERTcl;WIPlAmice (Data not shown). We next assessed senescence-associated P-galactosidase (SA-P-gal) activity in kidney sections from i- TERTC1;WIP1Amice. This analysis revealed significant induction of SA-P-gal activity in kidneys of i-TERTcl;WfPF ' mice when compared to the other groups (Data not shown). Such induction of SA-P-gal activity appeared to occur primarily in the tubular compartment of the nephron, with the glomeruli showing weaker induction (Data not shown). This result, together with our assessment of p21 expression level, suggests a complex interplay between the tubular and glomerular compartments upon induction of senescence markers in i-TERTcl;WIPlAmice. Nonetheless, broad induction of SA-P-gal activity in the nephron compartments was not observed in the setting of sole WIP1 deficiency in WIPE ' mice (Data not shown) suggesting that promotion of a senescence-like program requires simultaneous modulation of TERTC1and WIPl. These results indicate that the inability of TERTC1to exert its functions on kidney epithelium when WIPl is deleted is associated with the deployment of cytostatic functions of the senescence process.

[0141] WIPl deficiency upon TERTci overexpression leads to induction of senescence

[0142] Because persistent DDR activation can lead to cellular senescence (26), we next aimed to determine the status of the senescence process in the kidneys of i-TERTci;WIPl- / - mice. Expression level analysis of the cyclin-dependent kinase inhibitors p21 and pl 6, which represent key components of the senescence process (27, 28), revealed their significant upregulation in kidneys of i-TERTci;WIPl- / - mice (Data not shown). Interestingly, upregulation of the DDR senescence effector p21 was massive in kidneys of i-TERTci;WIPl- / - mice with an expression level that exceed that observed in 2 years old mice (Data not shown). Further examination of p21 by immunohistochemistry showed its marked upregulation in kidney glomeruli of i-TERTci;WIPl- / - mice (Data not shown). We next assessed senescence- associated P-galactosidase (SA-P-gal) activity in kidney sections from i-TERTci;WIPl- / - mice. This analysis revealed significant induction of SA-P-gal activity in kidneys of i-TERTci;WIPl- / - mice when compared to the other groups (Data not shown). Intriguingly, such induction of SA-P-gal activity appeared to occur primarily in the tubular compartment of the nephron rather than in glomeruli (Data not shown), suggesting that induction of senescence in i- TERTci;WIPl- / - mice spreads through all the nephron compartments. Together, these results indicate that the inability of TERTci to exert its functions on kidney epithelium when WIPl is deleted is associated to promotion of the senescence process.

[0143] Beside the DDR, WIPl was recently found to inhibit TGF-P signaling (29), a potent growth inhibition factor that exerts its cytostatic effects by upregulating cyclin-dependent kinase inhibitors including p21 (30). To assess the requirement for TGF-P activation in the impairment of TERTci functions in i-TERTci;WIPl- / - mice, we targeted TGF-P signaling pathway by intraperitoneally injecting Galunisertib, a selective TGF-P receptor type I kinase inhibitor (31). Either Galunisertib or the vehicle DMSO was injected in a daily manner into i-TERTci;WIPl- / - mice treated with doxycycline (Data not shown). Galunisertib treatment did not induce adverse effects on kidney filtration function in control actin-rtTA+ mice (Data not shown). In addition, Galunisertib treatment effectively prevented p21 upregulation and senescence induction in the kidneys of i-TERTci;WIPl- / - mice (Data not shown). Whereas proteinuria remained low in DMSO-injected i-TERTci;WIPl- / - mice, proteinuria worsened during the time course of the experiment in i-TERTci;WIPl- / - mice treated with Galunisertib (Fig. 4G). Perturbed filtration barrier function in Galunisertib-injected i-TERTci;WIPl- / - mice was associated with the onset of CG histopathological features in the kidneys of these mice (Data not shown . These results indicate that TGF-P signaling appends to DDR activation upon WIP1 deficiency to promote senescence induction, thereby restraining the ability of TERTci to induce CG in adult mice.

[0144] Pharmaceutical inhibition of WIP1 ameliorates CG progression

[0145] In addition to CG remission observed in condition of WIP1 full knockout, we found that partial deletion of WIP1 in i-TERTcl;WIPl+ / ' mice inhibited induction of massive proteinuria (Data not shown . Hence, we sought to assess the therapeutic potential of partial repression of WIP1 activity on CG progression upon its pharmacologic inhibition. Forward that goal, we targeted WIP1 phosphatase activity in the i-TERTclmodel by daily intraperitoneal (IP) injection of GSK2830371, a selective inhibitor that binds to a specific and unique flap subdomain located near the catalytic site of WIPE32Injection of mice with GSK2830371, or with the vehicle DMSO, was initiated after 12 days of doxycycline treatment (Data not shown}. GSK2830371 treatment effectively inhibited WIP1 phosphatase activity, leading to increased level of the phosphorylated form of Chk2 in kidneys of i-TERTclmice injected with GSK2830371 when compared to DMSO-injected i-TERTclmice (Data not shown}. Nonetheless, such inhibition did not impact filtration barrier function in control actin-rtTA+ mice (Data not shown}. Proteinuria continually worsened during the time course of the experiment in DMSO-injected i-TERTclmice (Data not shown}. In contrast, GSK2830371 treatment caused partial remission of proteinuria in i-TERTclmice (Data not shown}. The considerable improvement in filtration barrier function observed in GSK2830371 -treated i-TERTclmice was associated with a lower burden of glomeruli that display the characteristic abnormalities of CG defined as segmental or global collapse of the capillary walls with hypertrophy and hyperplasia of the overlying glomerular epithelial cells (Data not shown}. Interestingly, the percentage of parenchyma with tubular atrophy and interstitial fibrosis (IFTA) and of parenchyma with interstitial inflammation showed a downward trend in GSK2830371 -treated i-TERTclmice compared to DMSO-treated controls (Data not shown}. Improvement of histological features observed following WIP1 inhibition was accompanied by a significant reduction in the prevalence of proliferating cells within glomeruli of GSK2830371 -treated i-TERTclmice compared to DMSO-treated controls (Data not shown}. We next aimed to evaluate the therapeutic potential of inhibiting WIP1 in an alternative mouse model of CG. Toward that goal we used Tg26 mice that express a transgene encoding a replication-deficient version of HIV and that mirror HIV AN.21Similarly to the i-TERTclmodel, CG progression in Tg26 mice is characterized by abnormal emergence of proliferating cells within glomeruli (Data not shown). While glomerular cell proliferation and podocyte dedifferentiation was more pronounced in the Tg26 model compared to the i-TERTclmodel, the proportion of proliferating cells derived from podocytes or PECs was similar in both models (Data not shown). Daily IP injection of GSK2830371 for 7 days was performed on 4 week-old Tg26 mice with high urinary ACR. This treatment significantly restrained glomerular proliferation in Tg26 mice (Figure 1A). In addition, global glomerular histology was improved in Tg26 mice injected with GSK2830371 compared with DMSO-treated control Tg26 mice (Data not shown), and the percentage of collapsed glomeruli was significantly lower in the GSK2830371 -treated Tg26 mice compared to DMSO-treated controls (Figure IB). This was accompanied by significant decrease of the percentage of parenchyma with tubular atrophy and interstitial fibrosis (IFTA) (Figure 1C) and of parenchyma with interstitial inflammation (Figure ID). In addition, treatment of Tg26 mice with GSK2830371 significantly reduced albuminuria (Figures IE and IF). Such improvement in kidney filtration function was accompanied by the induction of lipofuscin as well as the decrease of nuclear Lamin Bl, which are hallmarks of senescence33,34, in the tubules of GSK2830371 -treated Tg26 mice (Data not shown). Taken together, these results show a striking improvement of CG histopathologic features accompanied by partial proteinuria remission upon inhibition of WIP1 phosphatase activity in both i-TERTcland Tg26 mice. Increased levels of TERT have previously been described in the kidneys of both human and mouse HIV AN.6Further examination of public data35'38revealed that TERT upregulation is a general feature of CG in humans, including that seen in COVID-19-associated nephropathy (COVAN) (Data not shown). While expression levels of WIP1 appeared unchanged in whole kidney biopsy specimens from individuals with CG, single-nucleus RNA sequencing (snRNA-seq) performed on an individual with COVAN indicated enrichment of WIP1 in specific kidney compartments (Data not shown). Hence, promoting cell-cycle arrest by means of WIP1 inhibition appears to be a relevant approach to treat patients with CG. Treatment with selective WIP1 inhibitor does not promote kidney fibrosis or inflammation

[0146] Next, we sought to determine whether a longer inhibition of WIP1 caused adverse effects on kidney histology and function. Toward that goal, we performed daily IP injections of GSK2830371 on i-TERTclmice with high levels of proteinuria (15-20 days of doxycycline treatment) and we maintained this treatment for 21 days (Data not shown). The i-TERTclmice injected with DMSO showed constant progression of their urinary ACR and 4 out of the 5 mice were euthanized before injection 14 (i 14, i.e. 29-34 days of doxycycline treatment) due to rapid deterioration of their general condition (Data not shown). In contrast, 7 of 8 i-TERTclmice injected with GSK2830371 survived beyond i 14 and showed either improvement or limited progression of their urinary ACR over the course of the experiment (Data not shown). Such improvement of filtration barrier function in GSK2830371 -treated i-TERTclmice was associated with a lower load of collapsed glomeruli (Figure 2A). Deterioration of the GSK2830371 -treated animals during the last days of the experiment leading to their euthanasia (Data not shown) was probably due to the unmitigated effects of TERTC1overexpression on the gastrointestinal tract (Data not shown)11. Besides, while p21 remained upregulated upon treatment of i-TERTclmice with GSK2830371 (Data not shown), kidneys of these mice did not show adverse increase in fibrosis (Figure 2B).

[0147] To gain insight into the molecular factors and pathways modulated in GSK2830371 -treated i- TERTC1mice we performed high-throughput sequencing (bulk RNA-seq) analysis of total kidneys from DMSO- and GSK2830371 -treated i-TERTclmice, and we compared transcriptomes of these mice to those of control actin-rtTA+ mice. Assessment of Gene Ontology term (GO term) using g:profiler highlighted enhanced extracellular matrix (ECM) remodeling in the context of GSK2830371 treatment compared to DMSO-injected i-TERTclmice (Data not shown). Gene set enrichment analysis (GSEA) further revealed 17 gene signatures present in both conditions and highlighted 3 gene signatures enriched in i-TERTclmice injected with DMSO and not found in i-TERTclmice treated with GSK2830371 (Data not shown). The gene signature related to the p53 pathway appeared to be promoted in mice treated with GSK2830371 compared to those injected with DMSO, suggesting that GSK treatment effectively inhibited the ability of WIP1 to repress p53 functions (Data not shown). Interestingly, inflammation-related gene signatures were globally repressed in i-TERTclmice treated with GSK2830371 compared to DMSO, and cytokines and chemokines appeared to be predominantly downregulated upon treatment with GSK2830371 (Data not shown). Hence, longer-term treatment with GSK2830371 does not appear to promote inflammation in the kidney of i-TERTclmice.

[0148] Cytostatic functions of TGF-P signaling are required to repress CG progression upon WIP1 depletion

[0149] Beside the DDR, WIP1 was recently found to inhibit TGF-P signaling.39The TGF-b superfamily ligands are recognized as major components of the secreted senescence-associated secretory phenotype (SASP) that act in a paracrine manner. In this context, TGF-b signaling exerts its cytostatic effects by upregulating cyclin-dependent kinase inhibitors pl 5, p21, p57 and p27.40,41Our results show that p21, p27 and p57 are upregulated in i-TERTcl;WIPlAmice (Data not shown . In addition, our bulk RNA-seq analysis confirmed that pl6 is not expressed in the kidney of i-TERTclmice in the context of WIP1 inhibition and further showed that pl 5, p21 and p27 are upregulated in this context (Data not shown . Such promotion of cytostatic components of TGF-P signaling might be related to stabilization of Smad4, the core mediator of TGF-P signaling described as a target of WIP1 (Data not shown}.39Hence, we sought to assess the requirement for TGF-P activation in the impairment of TERTC1functions in i- TERTC1;WIP1Amice. Toward that goal, we targeted TGF-P signaling pathway by intraperitoneally injecting Galunisertib, a selective TGF-P receptor type I kinase inhibitor42Either Galunisertib or the vehicle DMSO was injected in a daily manner into i-TERTcl;WIPlAmice treated with doxycycline (Data not shown}. Galunisertib treatment did not induce adverse effects on kidney filtration function in control actin-rtTA+ mice (Data not shown}. Whereas urinary ACR remained low in DMSO-injected i-TERTcl;WIPlAmice, it worsened during the time course of the experiment in i-TERTcl;WIPlAmice treated with Galunisertib (Data not shown}. Perturbed filtration barrier function in Galunisertib-injected mice was associated with the onset of CG histopathological features (Data not shown}. This occurrence of CG in i- TERTC1;WIP1Amice injected with Galunisertib was associated with prevention of SA-P-gal induction (Data not shown}. These results indicate that TGF-P signaling appends to DDR activation upon WIP1 deficiency to promote the induction of senescence-associated cytostatic factors, thereby restraining the ability of TERTC1to induce CG in adult mice.

[0150] Discussion

[0151] First characterized in the setting of HIV infection (HIV-associated nephropathy, HIV AN),7-9collapsing glomerulopathy (CG), the most aggressive variant of FSGS associated with the poorest prognosis,2has recently been described as a manifestation of COVID-19 infection (COVID-19-associated nephropathy, COVAN)43’44No specific therapy exists for CG, and patients with the primary disease are typically treated with similar immunosuppressive regimens as for other primary FSGS variants. Nonetheless, patients with CG exhibit poor responsiveness to steroid therapy and are less likely to attain durable remissions.4,5

[0152] We report here that both inactivation and inhibition of WIP1 phosphatase restrain CG initiation and progression respectively in the i-TERTclmouse model of CG as well as in the HIV AN mouse model. We found that this blockage observed in a WIP1 deficient context does not rely on Wnt / a-catenin signaling repression or on apoptosis induction; instead, it is associated with the promotion of senescence-associated cytostatic factors (Data not shown . Our results indicate that WIP1 -mediated repression of this senescence-like program in the setting of CG involves its ability to modulate both DDR and TGF-P signaling pathways (Data not shown . TGF-P is considered a potent profibrotic mediator in the context of chronic kidney disease (CKD). However, activation of TGF-P signaling does not appear to be detrimental in all kidney disease conditions.45Thus, enhanced TGF-P signaling has a protective role in some pathological settings such as crescentic glomerulonephritis.46Here, we found that the cyclin- dependent kinase inhibitors targeted by TGF-P signaling are upregulated upon WIP1 depletion in i-TERTclmice, and we showed that TGF-P signaling is required to restrain CG initiation in i-TERTcl;WIPlAmice. Thus, activation of cytostatic functions of TGF-P signaling proves to be beneficial in proliferative glomerular diseases, indicating that activation of TGF-P results in fundamental differences in disease progression depending on the pathogenic context. Interestingly, we found that pharmacologic inhibition of WIP1 did not promote interstitial fibrosis or inflammation, but instead led to their reduction. Thus, modulation of TGF-P following the inactivation of WIP1 functions does not appear to promote fibrosis or inflammation in the context of CG. Besides, several studies reported roles for WIP1 in modulating the immune system, including in the regulation of myeloid and lymphocyte differentiation, neutrophil infiltration and in the formation of neutrophil extracellular traps (NETs)47'50. Thus, the ability of WIP1 to inflect the immune system may be involved in the disease remission observed in our experimental models of CG upon WIP1 depletion. Further studies will be needed to understand how modulation of the immune response contributes to disease remission upon WIP1 inhibition in CG.

[0153] A previous study showed that the use of a cyclin-dependent kinase (CDK) inhibitor in the Tg26 mouse model of HIV AN reverses and attenuates disease progression without repressing HIV- 1 transgene expression.51The present study shows that induction of a senescence-like program in the setting of WIP1 depletion or inhibition promotes CG remission in both i-TERTcland Tg26 mice. Together, those results suggest that glomerular cell proliferation is a driving force for disease initiation and progression in CG. In addition, our data indicate that the deployment of the senescence program in the context of WIP1 depletion in CG remains partial. Indeed, most of the cytokines and chemokines found downregulated upon WIP1 inhibition in i-TERTclmice are recognized components of the secreted senescence-associated secretory phenotype (SASP).52Hence, implementation of a senescence-like phenotype that retains only the cytostatic functions of the senescence program appears to be beneficial for CG progression without promoting fibrosis or inflammation over the long term.

[0154] Maladaptive cell proliferation is observed in diverse kidney diseases53such as immune mediated mesangial cell proliferation of IgA nephropathy54and lupus nephritis.55An initial study that used CDK inhibitor showed promising results with renal functional improvement in a rat model of mesangial proliferative glomerulonephritis (GN).56Future studies should explore the potential role of WIP1 inhibitor therapy to transiently promote cytostatic functions of the senescence program in GN. Conversely, targeting senescent cells in disease contexts where cell proliferation is required to restore kidney function might be beneficial, as recently shown following acute kidney injury.57Transient treatment might be beneficial in specific subsets of CG, such as in the patient with COVID-19 and APOL1 high-risk genotype, which is a major genetic risk factor for CG58,59and CO VAN.60Examination of public data revealed that CO VAN is associated with upregulation of TERT and enrichment of WIP1 in specific nephron compartments. Interestingly, review of public databases revealed that common variants in the PPM1D locus associate with serum levels of BUN and Cystatin C in humans.61,62While studies are needed to decipher the molecular mechanisms underlying this association, these observations further implicate a role for WIP1 in kidney dysfunction.

[0155] In summary, the present study demonstrates that treatment using WIP1 inhibitor considerably ameliorates disease progression in two independent experimental models of CG. Our finding that p21 is upregulated while pl6 is not expressed suggests that the senescence-like program deployed in the context of WIP1 deficiency in CG is distinct from the one deployed in the context of kidney aging. In addition, our results suggest that targeting WIP1 in the context of CG does not promote SASP induction. Future studies are warranted to explore the potential therapeutic benefit of WIP1 inhibition in patients with CG. REFERENCES:

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Claims

CLAIMS:

1. A method of preventing or treating glomerular disease in a subject in need thereof, comprising the step of administering to said subject a Wild-type p53-induced phosphatase 1 (WIP1) inhibitor.

2. The method of preventing or treating glomerular disease according to claim 1 wherein the glomerular disease is chosen among crescentic glomerulonephritis, lupus nephritis, diabetic nephropathy or focal segmental glomerulosclerosis (FSGS).

3. The method of preventing or treating glomerular disease according to claim 2 wherein the FSGS is collapsing glomerulopathy (CG).

4. The method of preventing or treating glomerular disease according to claims 1 to 3 wherein the WIP1 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

5. The method of preventing or treating glomerular disease according to claims 1 to 3 wherein the WIP1 inhibitor is GSK2830371.

6. i) WIP1 inhibitor and a ii) a classical treatment as a combined preparation for use in the treatment of glomerular disease.

7. The combined preparation according to claim 6 wherein glomerular disease is focal segmental glomerulosclerosis, in particular collapsing glomerulopathy.

8. The combined preparation according to claims 6 to 7 wherein the WIP1 inhibitor is GSK2830371.

9. A pharmaceutical composition comprising a WIP1 inhibitor for use in the treatment of glomerular disease.

10. The pharmaceutical composition according to claim 9 comprising i) WIP1 inhibitor, ii) a classical treatment, as a combined preparation for use in the treatment of glomerular disease.

11. The pharmaceutical composition according to claims 9 to 10 wherein the WIP1 inhibitor is GSK2830371.

12. The pharmaceutical composition according to claims 9 to 10 wherein the glomerular disease is focal segmental glomerulosclerosis, in particular collapsing glomerulopathy.

13. The pharmaceutical composition according to claim 10 wherein the classical treatment is an immunosuppressive agent or an angiotensin-converting-enzyme (ACE) inhibitor.

14. The pharmaceutical composition according to claim 13 wherein the immunosuppressive agent is chosen among steroid, glucocorticoids (such as prednisone, dexamethasone, and hydrocortisone), cytostatics such as alkylating agents (i.e cyclophosphamide), antimetabolites, azathioprine and mercaptopurine or cytotoxic antibiotics), antibodies, drugs acting on immunophilins (such as ciclosporin, tacrolimus, sirolimus, everolimus, zotarolimus), Interferons, Opioids, TNF binding proteins, My cophenolate or Small biological agents (such as fmgolimod, muriocin).

15. A method of screening a drug suitable for the treatment of glomerular disease comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and / or the activity of WIPE 16. The method of screening of claim 15 wherein the glomerular disease is focal segmental glomerulosclerosis, in particular collapsing glomerulopathy.

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