Methods for treating proliferative glomerulonephritis

Abstract

The inventors explored the relevance of alpelisib in the MRL / MpJ-Faslpr / J mouse (referred to herein as MRL-lpr), a mouse model of lupus nephritis. MRL-lpr mice treated with alpelisib showed less proteinuria compared to vehicle. Even more strikingly, a reverse trajectory was shown by comparison of albuminuria before and after treatment initiation. Indeed, MRL-lpr mice treated with alpelisib showed improvement in proteinuria arguing for reversibility of the disease. At the time of euthanasia, MRL-lpr mice treated with alpelisib tended to have a lower kidney-to-body weight ratio compared to vehicle-treated animals. Kidney examination showed that alpelisib was associated with the absence of glomerular lesions and improved kidney function compared to vehicle-treated mice. The inventors concluded that alpelisib and more generally PIK3CA inhibition represent a promising drug for patients with proliferative glomerulonephritis. The present invention relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a PIK3CA inhibitor.

Description

Technical Field

[0001] Field of the Invention: The present invention relates to methods and compositions for the treatment of proliferative glomerulonephritis, such as lupus nephritis or focal segmental glomerulosclerosis.

Background Art

[0002] Background of the Invention: PIK3CA is a ubiquitously expressed lipid kinase that controls signaling pathways involved in cell proliferation, motility, survival, and metabolism 1 PIK3CA is mainly recruited through tyrosine kinase receptors. PIK3CA encodes the 110 kDa catalytic alpha subunit (p110α) of PI3K, which converts phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3; or PIP3) at the cell membrane, followed by the recruitment of PDK1, which then phosphorylates AKT at the Thr308 residue to initiate downstream cellular effects. PIK3CA also regulates many other pathways, including the Rho / Rac1 signaling cascade 2

[0003] Some subjects have somatic gain-of-function mutations in the PIK3CA gene, which leads to asymmetric overgrowth called PIK3CA-related overgrowth syndrome (PROS). PROS is a rare genetic disorder defined by tissue hypertrophy that can be local or systemic. The mutations are not inherited but occur during embryonic development, causing somatic mosaicism 3 The clinical symptoms of patients with PIK3CA gain-of-function mutations are mosaic, but also extremely extensive due to the tissues involved 4 Patients typically have complex tissue malformations including abnormal blood vessels, disorganized adipose tissue, muscle hypertrophy, and / or bone deformities 5-9 ​Due to the wide variability in clinical symptoms and the difficulty in identifying genes that often require biopsy of the affected area, the exact prevalence of PIK3CA gain-of-function mutations remains unknown. The inventors have recently created a mouse model that recapitulates the phenotype of PROS patients. The inventors have identified alpelisib, a PIK3CA inhibitor under development in oncology, as a promising therapeutic agent in the mouse model and have been permitted to use this drug to treat PROS patients in a compassionate setting. Patients treated with alpelisib showed clinical, biological, and radiological improvements.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The inventors previously reported that the first PROS patient treated with alpelisib had kidney dysfunction with nephrotic-range proteinuria. This chronic kidney disease was associated with severe vascular malformations (venous and lymphatic abnormalities, but also mixed arteriovenous shunts), lower limb paralysis with vesicoureteral reflux, severe congestive heart failure, and a complex medical situation involving the use of rapamycin (an mTOR inhibitor). This drug is well known to induce proteinuria in patients with chronic kidney disease (CKD). 10 Rapamycin was discontinued without clinical or biological changes.

[0005] Thus, there is a need to find a new treatment strategy for treating proliferative glomerulonephritis.

Means for Solving the Problems

[0006] Summary of the Invention: The present invention relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a PI3K inhibitor, particularly a PIK3CA inhibitor. In particular, the present invention is defined by the claims.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0008] Detailed Description of the Invention: The present inventors explored the relevance of alpelisib in MRL / MpJ-Faslpr / J mice (referred to herein as MRL-lpr), a mouse model of lupus nephritis. MRL-lpr mice treated with alpelisib showed less proteinuria compared to the vehicle. Even more severely, a reverse trajectory was shown by comparing albuminuria before and after treatment introduction. Indeed, MRL-lpr mice treated with alpelisib showed improvement in proteinuria, demonstrating the reversibility of the disease. At the time of sacrifice, MRL-lpr mice treated with alpelisib tended to have a lower kidney-to-body weight ratio compared to animals treated with the vehicle. Kidney examination showed that alpelisib was associated with the absence of glomerular lesions and improved kidney function compared to mice treated with the vehicle.

[0009] The present inventors concluded that alpelisib, more generally PIK3CA inhibition, represents a promising drug for patients with proliferative glomerulonephritis.

[0010] Accordingly, the present invention relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, comprising the step of administering to the subject a therapeutically effective amount of a PI3K inhibitor, particularly a PIK3CA inhibitor.

[0011] In some embodiments, the invention also relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, the method consisting essentially of administering to the subject a therapeutically effective amount of a PIK3CA inhibitor.

[0012] In some embodiments, the invention also relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, the method consisting of administering to the subject a therapeutically effective amount of a PIK3CA inhibitor.

[0013] As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, and include the treatment of a subject at risk of developing a disease or suspected of having a disease, as well as a subject diagnosed as being ill or suffering from a disease or medical condition, including suppression of clinical recurrence. Treatment can be administered to a subject having a medical disorder or who may ultimately acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or palliate one or more symptoms of the disorder or recurrent disorder, or to extend the survival of the subject beyond that expected in the absence of such treatment. A "treatment regimen" means a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. A treatment regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a treatment regimen (or a portion of a treatment regimen) used for the initial treatment of a disease. A common goal of an induction regimen is to provide a high level of drug to the subject during the initial period of the treatment regimen. An induction regimen can (in whole or in part) utilize a "loading regimen," which involves administering a larger dose of drug than the physician may use during the maintenance regimen, administering the drug more frequently than the physician may administer the drug during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used for the maintenance of a subject during the treatment of a disease, e.g., to keep the subject in remission over a long period (months or years). Maintenance therapy can use continuous treatment (e.g., administering the drug at regular intervals such as weekly, monthly, annually, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment at recurrence, or treatment upon achievement of certain predetermined criteria [e.g., pain, disease symptoms, etc.]).

[0014] As used herein, the term "Proliferative Glomerulonephritis" (PGN) refers to an increase in the cellularity of the glomeruli, which is caused by proliferation of endogenous glomerular cells, infiltration of leukocytes, or both. This occurs primarily in the context of glomerular deposition of immunoglobulins, immune complexes, or complement components. Different subtypes have been described based on histological features: mesangial cell proliferation, endocapillary proliferation, diffuse proliferation, or extracapillary proliferation (also called crescentic glomerulonephritis). In some embodiments, the proliferative glomerulonephritis is extracapillary proliferative glomerulonephritis.

[0015] In certain embodiments, the proliferative glomerulonephritis is caused by the following diseases selected from the group consisting of, but not limited to: infectious diseases (post-streptococcal glomerulonephritis, infective endocarditis, occult visceral sepsis, hepatitis B infection - with vasculitis and / or cryoglobulinemia, HIV infection, hepatitis C - with cryoglobulinemia, with membranoproliferative glomerulonephritis), multi-organ diseases (systemic lupus erythematosus, IgA nephropathy, Henoch-Schönlein purpura, systemic necrotizing vasculitis - including granulomatosis with polyangiitis Wegener type, Goodpasture syndrome, essential mixed cryoglobulinemia, malignant tumors, relapsing polychondritis, rheumatoid arthritis - with vasculitis). In some embodiments, the vasculitis is antineutrophil cytoplasmic autoantibody (ANCA) vasculitis.

[0016] In certain embodiments, the proliferative glomerulonephritis is caused by systemic lupus erythematosus.

[0017] As used herein, the term "Systemic Lupus Erythematosus" (SLE) refers to a systemic autoimmune disease that is thought to be manifested by a wide range of abnormalities in immune regulation. It is the most common type of lupus.

[0018] In certain embodiments, the proliferative glomerulonephritis is lupus nephritis.

[0019] As used herein, the term "lupus nephritis" (LN) refers to inflammation of the kidneys caused by systemic lupus erythematosus (SLE). Up to 60% of lupus patients develop LN. When the kidneys become inflamed, they no longer function properly to filter toxins, by-products, excess salts, excess body fluids, and other impurities from the blood. If left uncontrolled, LN can lead to kidney failure. Even with treatment, loss of kidney function sometimes progresses. If both kidneys fail, a subject with LN may require dialysis. Ultimately, a subject with LN may need to receive a kidney transplant. Symptoms of loss of kidney function or abnormalities include increased amounts of protein in the urine (proteinuria), foaming in the subject's urine, and / or higher levels of blood urea nitrogen (BUN). In another specific embodiment, the proliferative glomerulonephritis is focal segmental glomerulosclerosis (FSGS).

[0020] As used herein, the term "subject" refers to any mammal, such as rodents, felines, canines, and primates. In particular, in the present invention, the subject is a human who has or is susceptible to at least one of the disabling proliferative glomerulonephritides as described above.

[0021] In certain embodiments, the subject is a human who has or is susceptible to an infectious disease (post-streptococcal glomerulonephritis, infective endocarditis, occult visceral sepsis, hepatitis B infection - with vasculitis and / or cryoglobulinemia, HIV infection, hepatitis C - with cryoglobulinemia, with membranoproliferative glomerulonephritis) or a multi-organ disease (systemic lupus erythematosus, IgA nephropathy, Henoch-Schönlein purpura, systemic necrotizing vasculitis - including granulomatosis with polyangiitis Wegener type, Goodpasture syndrome, essential mixed cryoglobulinemia, malignancy, relapsing polychondritis, rheumatoid arthritis - with vasculitis). In some embodiments, the vasculitis is antineutrophil cytoplasmic autoantibody (ANCA) vasculitis.

[0022] In another embodiment, the subject is a human who has or is susceptible to proliferative glomerulonephritis.

[0023] In another embodiment, the subject is a human who has or is susceptible to membranous and extracapillary proliferative glomerulonephritis.

[0024] In another embodiment, the subject is a human who has or is susceptible to mesangial proliferative glomerulonephritis.

[0025] In another embodiment, the subject is a human who has or is susceptible to diffuse capillary proliferative glomerulonephritis.

[0026] In another embodiment, the subject is a human who has or is susceptible to systemic lupus erythematosus (SLE).

[0027] In another embodiment, the subject is a human who has or is susceptible to lupus nephritis (LN).

[0028] In some embodiments, the subject is a human who has or is susceptible to focal segmental glomerulosclerosis (FSGS).

[0029] As used herein, the term "PI3K" also refers to phosphoinositide 3-kinase, also called phosphatidylinositol 3-kinase. PI3K belongs to a family of enzymes that phosphorylate the 3'-hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns). Activation of the PI3K signaling pathway can result in the synthesis of PIP3 from PIP2. PIK3CA is mainly recruited through tyrosine kinase receptors. PIK3CA encodes the 110 kDa catalytic alpha subunit (p110α) of PI3K, which at the cell membrane converts phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3; or PIP3), followed by the recruitment of PDK1, which then phosphorylates AKT at the Thr308 residue to initiate downstream cellular effects. PIK3CA also regulates many other pathways, including the Rho / Rac1 signaling cascade.

[0030] As used herein, the term "PI3K inhibitor" refers to a natural or synthetic compound having a biological effect of inhibiting the activity or expression of PI3K. More specifically, such a compound is capable of inhibiting the kinase activity of at least one member of the PI3K family, for example, at least one member of class I PI3K. In certain embodiments, the PI3K inhibitor may be a pan-inhibitor of class I PI3K (known as p110), or an isoform specific for a class I PI3K isoform (among the four types of isoforms, p110α, p110β, p110γ, or p110δ).

[0031] In certain embodiments, the PI3K inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide. The term "peptidomimetic" refers to a small protein-like chain designed to mimic a peptide. In certain embodiments, the inhibitor of PI3K is an aptamer. An aptamer is a class of molecules that represents an alternative to antibodies in terms of molecular recognition. An aptamer is an oligonucleotide or oligopeptide sequence with the ability to recognize substantially any class of target molecules with high affinity and specificity.

[0032] In certain embodiments, the PI3K inhibitor is a small organic molecule. The term "small organic molecule" refers to a molecule of a size comparable to that of organic molecules commonly used in pharmaceuticals. This 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.

[0033] In certain embodiments, the PI3K inhibitor is a small molecule that is an isoform-selective inhibitor of PI3K selected from among the following compounds: BYL719 (alpelisib, Novartis), GDC-0032 (taselisib, Genentech / Roche), BKM120 (buparlisib), A66 (University of Auckland), GDC0941 (pictilisib, Genentech), PX-866 (Oncothyreon), dactolisib, bosteralisib (SAR245409, XL765), pilaralisib, GDC-0077 (inasidenib, Genentech / Roche), CYH33 (lisobalisib), TAK-117 / MLN1117 / INK1117 (serabelisib), BAY80-6946 (copanlisib, Bayer Healthcare), or a pharmaceutically acceptable salt thereof. In more specific embodiments, the isoform-selective inhibitor of PI3K is selected from among the following compounds: BYL719 (alpelisib, Novartis), A66 (University of Auckland), GDC-0077 (inasidenib, Genentech / Roche), CYH33 (lisobalisib), TAK-117 / MLN1117 / INK1117 (serabelisib), or a pharmaceutically acceptable salt thereof. In even more specific embodiments, the isoform-selective inhibitor of PI3K is selected from among the following compounds: BYL719 (alpelisib, Novartis), GDC-0077 (inasidenib, Genentech / Roche), TAK-117 / MLN1117 / INK1117 (serabelisib), or a pharmaceutically acceptable salt thereof. Such PI3K inhibitors are well known in the art and are described, for example, in Wang et al Acta Pharmacological Sinica (2015) 36: 1170-1176.

[0034] In certain embodiments, the PI3K inhibitor is BYL719 and its derivatives. As used herein, the term "BYL719" also referred to as alpelisib, is an ATP-competitive oral PI3K inhibitor selective for the p110α isoform activated by the mutant PIK3CA gene (Furet P., et al. 2013; Fritsch C., et al 2014). This molecule is also referred to as alpelisib and has the following formula and structure in the art C 19 H 22 F3N5O2S:

Chemical Structure

[0035] In certain embodiments, the PI3K inhibitor is GDC-0032 and its derivatives, developed by Roche. This molecule, also called taselisib, has the following formula and structure in the art C 24 H 28 N8O2:

Chemical Structure

[0036] In some embodiments, the PI3K inhibitor is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments as long as they exhibit the desired biological activity. This term includes antibody fragments containing antigen-binding domains, such as Fab’, Fab, F(ab’)2, single-domain antibodies (DAB), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, biobodies, tribodies (each an scFv-Fab fusion, bispecific or trispecific); sc-diabodies, kappa (lambda) bodies (scFv-CL fusions); BiTE (bispecific T cell engager, an scFv-scFv tandem for attracting T cells); DVD-Ig (dual variable domain antibody, a bispecific format); SIP (small immunoprotein, a type of minibody); SMIP (“small modular immunopharmaceutical” scFv-Fc dimer; DART (ds-stabilized diabody “dual affinity retargeting”); small antibody mimics containing one or more CDRs and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, which is specifically incorporated herein by reference). Diabodies are further described, inter alia, in European Patent Application Publication No. 404,097 and International Publication No. 93 / 11161; while 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 fragments can be treated to reduce disulfide bridges to produce Fab’ fragments. Papain digestion can lead to the formation of Fab fragments.Fabs, Fab’, and F(ab’)2, scFvs, Fvs, dsFvs, Fds, dAbs, TandAbs, ds-scFvs, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized recombinantly or 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 describes and enables the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Patent No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as those described in U.S. Patent Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. “Human antibodies,” such as those described in U.S. Patent Nos. 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody as described in European Patent Application Publication No. 0368684, International Publication No. 06 / 030220, and International Publication No. 06 / 003388. In certain embodiments, 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 a continuous cell line in culture. Techniques for production and isolation include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and EBV hybridoma technology.

[0037] In particular, the PI3K inhibitor is an intrabody that is specific for PI3K. As used herein, the term "intrabody" generally refers to an intracellular antibody or antibody fragment. Antibodies, particularly single-chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modifications can involve, for example, fusion to a stable intracellular protein, such as, for example, maltose binding protein, or the addition of an intracellular transport / localization peptide sequence, such as, for example, endoplasmic reticulum retention. In some embodiments, the intrabody is a single-domain antibody. In some embodiments, the antibodies according to the invention are single-domain antibodies. The term "single-domain antibody" (sdAb) or "VHH" refers to the single-chain variable domain of a type of antibody found in camelid mammals, which is naturally lacking a light chain. Such VHHs are also referred to as "nanobodies®". According to the invention, the sdAb can in particular be a llama sdAb.

[0038] In some embodiments, the PI3K inhibitor is a short hairpin RNA (shRNA), small interfering RNA (siRNA), or antisense oligonucleotide that inhibits the expression of USP14. In certain embodiments, the inhibitor of USP14 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. shRNAs are generally expressed using vectors introduced into cells, and the vectors utilize the U6 promoter to ensure that the shRNA is constantly expressed. This vector is usually passed on to daughter cells, allowing gene silencing to be inherited. The shRNA hairpin structure is cleaved by cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNA that matches the siRNA to which it is bound. Small interfering RNA (siRNA) is sometimes known as short interfering RNA or silencing RNA and is a class of double-stranded RNA molecules about 20-25 nucleotides in length that play various roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, whereby siRNA interferes with the expression of a specific gene. Antisense oligonucleotides include antisense RNA molecules and antisense DNA molecules and act to directly block the translation of target mRNA by binding to the target mRNA, thus preventing protein translation, or increasing mRNA degradation, and thus reducing the level of the target protein in the cell and, thus, its activity. For example, antisense oligonucleotides that are at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically inhibit the gene expression of genes with known sequences are well known in the art (see, e.g., U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).The antisense oligonucleotides, siRNAs, and shRNAs of the present invention can be delivered in vivo, either alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into a cell, typically a mast cell. Typically, a vector transports a nucleic acid into a cell with reduced degradation compared to the degree of degradation that would occur in the absence of the vector. Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, and other vehicles derived from viral or bacterial sources engineered by the insertion or incorporation of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid sequence. Viral vectors are a preferred type of vector and include nucleic acid sequences from, but are not limited to, the following viruses: retroviruses such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40-type viruses; polyomaviruses; Epstein-Barr virus; papillomaviruses; herpesviruses; vaccinia virus; poliovirus; and RNA viruses such as retroviruses. Although not named, other vectors known in the art can be readily used.

[0039] In some embodiments, the inhibitor of PI3K expression is an endonuclease. Over the past few years, remarkable progress in sequencing technologies has provided an unprecedented detailed overview of multiple genetic abnormalities in cancer. By greatly expanding the list of new potential cancer genes and tumor suppressor genes, these new data have characterized the normal and pathological functions of these genes and, in particular, strongly emphasized the need for a rapid and reliable strategy to evaluate their role as drivers during carcinogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies offer means to recapitulate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered endonuclease enzymes have received considerable attention in recent years. The mechanism underlying endonuclease-based genome inactivation generally requires an initial step of single- or double-strand DNA cleavage, which in turn induces two different cellular mechanisms for DNA repair, which can be utilized for DNA inactivation: error-prone non-homologous end joining (NHEJ) and high-fidelity homology-directed repair (HDR).

[0040] In certain embodiments, 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, segments of prokaryotic DNA that contain short repeats of nucleotide sequences.

[0041] In some embodiments, the endonuclease is CRISPR-cas9 from Streptococcus pyogenes. The CRISPR / Cas9 system is described in U.S. Patent No. 8,697,359 and U.S. Patent Application Publication No. 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has recently been engineered as a new and powerful tool for genome editing.It has already been successfully used to target important genes in many cell lines and organisms, including humans (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.113.160713), 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.). Some groups are currently using this method to introduce a single point mutation (deletion or insertion) in a specific target gene via a single gRNA.Instead, a pair of gRNA-guided Cas9 nucleases can be used, and 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 genomic loci.

[0042] In some embodiments, the endonuclease is CRISPR-Cpf1, a recently characterized CRISPR (Cpf1) from Provotella and Francisella 1 in Zetsche et al. ("Cpf1 is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System(2015); Cell; 163, 1-13).

[0043] In another embodiment, the present invention relates to a PI3K inhibitor for use according to the present invention, and a classical treatment as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0044] As used herein, the terms "combination treatment", "combination therapy" or "therapy combination" refer to treatment using more than one drug therapy. Combination therapy may be dual therapy or two-drug therapy.

[0045] As used herein, the term "classical treatment" is well known in the art and refers to treatments used to treat proliferative glomerulonephritis (Hahn et al 2013, Arthritis Care Res(Hoboken). 2012 Jun; 64(6): 797-808; doi: 10.1002 / acr.21664).

[0046] In the context of the present invention, classical treatments are selected from the group consisting of, but not limited to, immunosuppressants, glucocorticoids, inhibitors of MAPK, PAK, mTOR, TKI, PARP, and / or EGFR. When several inhibitors are used, a mixture of inhibitors is obtained. In the case of multi-drug therapy (e.g., dual, triple, or quadruple therapy), at least one other inhibitor can be accompanied by a PI3K inhibitor.

[0047] In certain embodiments, the PI3K inhibitor described above is combined with immunosuppressive therapy.

[0048] As used herein, the term "immunosuppressive therapy" refers to immunosuppressive treatment, which means that the subject is administered one or more immunosuppressive drugs. Immunosuppressive drugs that can be used in transplantation procedures include azathioprine (AZA), methotrexate, cyclophosphamide (CYC), FK-506 (tacrolimus), rapamycin, corticosteroids, and cyclosporine. These drugs can be used in monotherapy or in combination therapy.

[0049] In certain embodiments, the immunosuppressive treatment is carried out using azathioprine.

[0050] In certain embodiments, the immunosuppressive treatment is carried out using cyclophosphamide.

[0051] In another embodiment, the PI3K inhibitor described above is combined with glucocorticoid therapy.

[0052] As used herein, the term "glucocorticoid therapy" refers to the class of corticosteroids, which is the class of steroid hormones. Glucocorticoids are corticosteroids that bind to glucocorticoid receptors.

[0053] In certain embodiments, the glucocorticoid therapy is carried out using prednisone.

[0054] In another embodiment, the classical treatment is mycophenolate mofetil (MMF, CELLCEPT).

[0055] In certain embodiments, a PI3K inhibitor, an immunosuppressant, and a glucocorticoid can be combined as a three-drug treatment for use in the treatment of proliferative glomerulonephritis.

[0056] In certain embodiments, a PI3K inhibitor, an immunosuppressant, and a glucocorticoid can be combined as a three-drug treatment, and the PI3K inhibitor, the immunosuppressant, and the glucocorticoid are BYL719, azathioprine or cyclophosphamide, and prednisone, respectively.

[0057] In certain embodiments, a PI3K inhibitor according to the present invention, and an immunosuppressant, a glucocorticoid, an inhibitor of MAPK, PAK, mTOR, TK, PARP or EGFR, as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0058] In certain embodiments, a PI3K inhibitor according to the present invention, and an immunosuppressant, a glucocorticoid, an inhibitor of MAPK, PAK, mTOR, TK, PARP or EGFR, as a combined formulation for simultaneous, separate, or sequential use in the treatment of lupus nephritis in a subject in need thereof.

[0059] In certain embodiments, a PI3K inhibitor according to the present invention, and an immunosuppressant, a glucocorticoid, an inhibitor of MAPK, PAK, mTOR, TK, PARP or EGFR, as a combined formulation for simultaneous, separate, or sequential use in the treatment of focal segmental glomerulosclerosis (FSGS) in a subject in need thereof.

[0060] In another embodiment, the present invention relates to a combination comprising a PI3K inhibitor and at least one classical treatment selected from the group consisting of immunosuppressive agents, glucocorticoids, MAPK, PAK, mTOR, TK, PARP or EGFR inhibitors for use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0061] In another embodiment, inhibitors of PI3K, MAPK and PAK can be combined as a three-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K, MAPK, and PAK can be combined as a three-drug treatment, and the PI3K, MAPK, and inhibitors are BYL719, selumetinib, and IPA-3, respectively.

[0062] The present invention also relates to a method for treating proliferative glomerulonephritis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a PI3K inhibitor. In certain embodiments, the method according to the present invention, a combination formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis, a PI3K inhibitor and a MAPK inhibitor, a PAK inhibitor, an mTOR inhibitor, a TKI, a PARP inhibitor or an EGFR inhibitor.

[0063] As used herein, the term "MAPK" refers to mitogen-activated protein kinase, a type of protein kinase that is specific for the amino acids serine and threonine. MAPK is involved in cellular responses to diverse stimuli such as mitogens, osmotic stress, heat shock, inflammatory cytokines, etc. Six groups of MAPK have been identified to date: extracellular signal-regulated kinases (ERK1, ERK2), c-Jun N-terminal kinases (JNK), p38 isoforms (MAPK11, MAPK12, MAPK13, MAPK14), ERK5 (MAPK7), ERK3 (MAPK6) and ERK4 (MAPK4), ERK7 / 8 (MAPK15). In certain embodiments, the MAPK inhibitor is an inhibitor of ERK1 / ERK2. The inhibitors of ERK1 / ERK2 are selected from the group consisting of, but not limited to, VTX-11e, SCH772984.

[0064] In certain embodiments, the MAPK inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide. In certain embodiments, the MAPK inhibitor is a p38-MAPK inhibitor. Typically, the inhibitors of p38-MAPK are selected from the group consisting of SB 203580, SB 203580 hydrochloride, SB681323 (zilmapimod), LY2228820 dimesylate, BIRB 796 (doramapimod), BMS-582949, pamapimod, GW856553, ARRY-797AL 8697, AMG 548, CMPD-1, EO 1428, JX 401, RWJ 67657, TA 01, TA 02, VX 745, DBM 1285 dihydrochloride, ML 3403, SB 202190, SB 239063, SB 706504, SCIO 469 hydrochloride, SKF 86002 dihydrochloride, SX 011, TAK 715, VX 702, or PH-797804.

[0065] In certain embodiments, the inhibitor of MAPK is an inhibitor of MEK. MEK1 and MEK2 are members of a larger family of dual-specificity kinases (MEK1-7) that phosphorylate threonine and tyrosine residues of various MAP kinases. In certain embodiments, the inhibitor of MAPK is selected from the group consisting of trametinib (GSK1120212); selumetinib (AZD6244).

[0066] In certain embodiments, the PI3K inhibitor and the MAPK inhibitor for use according to the invention are in a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof, the PI3K inhibitor is BYL719 and the MAPK inhibitor is selumetinib.

[0067] In another embodiment, a PI3K inhibitor and a PAK inhibitor for use according to the invention as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0068] As used herein, the term "PAK" refers to p21-activated kinases that regulate cytoskeletal remodeling, phenotypic signaling, and gene expression and affect a wide variety of cellular processes such as directed motility, invasion, metastasis, growth, cell cycle progression, angiogenesis, and the like. In certain embodiments, the PAK inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide.

[0069] In certain embodiments, the inhibitor of PAK is selected from the group consisting of PP1, hPIP1, NESH, Merlin, CRIPak, LKB1, mesalamine, glaucarubinone, myricetin, β-elemene, miR-7, miR-let-7, miR-145, FRAX1036, OSU-03012, and IPA-3.

[0070] In certain embodiments, the PAK inhibitor is used with thalidomide, lenalidomide, or pomalidomide as a combined formulation for use in the treatment of proliferative glomerulonephritis.

[0071] In certain embodiments, the PI3K inhibitor and the PAK inhibitor according to the invention are a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof, the PI3K inhibitor is BYL719, and the PAK inhibitor is IPA-3.

[0072] In another embodiment, a PI3K inhibitor and an mTOR inhibitor according to the invention as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0073] As used herein, the term "mTOR" also refers to the mammalian target of rapamycin, which is known as the mechanistic target of rapamycin and FK506-binding protein 12-rapamycin-associated protein 1 (FRAP1). mTOR functions as a serine / threonine protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription. mTOR has two structurally distinct complexes: mTORC1 and mTORC2. In certain embodiments, the mTOR inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide.

[0074] In certain embodiments, the inhibitor of mTOR is selected from the group consisting of rapamycin (also known as sirolimus and described in U.S. Patent No. 3,929,992), temsirolimus, deforolimus, everolimus, tacrolimus, and rapamycin analogs or derivatives thereof, AMG954, AZD8055, AZD2014, BEZ235, BGT226, CC-115, CC-223, LY3023414, P7170, DS-7423, OSI-027, GSK2126458, PF-04691502, PF-05212384, INK128, MLN0128, MLN1117, ridafolimus, metformin, XL765, SAR245409, SF1126, VS5584, GDC0980, and GSK2126458.

[0075] As used herein, the term "rapamycin analog or derivative thereof" includes compounds having a rapamycin core structure as defined in U.S. Patent Application Publication No. 2003 / 0008923, which is incorporated herein by reference, and which may be chemically or biologically modified while still retaining mTOR inhibitory properties. Such derivatives include esters, ethers, oximes, hydrazones, and hydroxylamines of rapamycin, as well as compounds in which the functional groups on the rapamycin core structure are modified, for example, by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered to be rapamycin derivatives. Specific examples of esters and ethers of rapamycin are esters and ethers of the hydroxyl groups at positions 42 and / or 31 of the rapamycin nucleus, and esters and ethers of the hydroxyl group at position 27 (after chemical reduction of the 27-ketone). Specific examples of oximes, hydrazones, and hydroxylamines are the ketones at position 42 of the rapamycin nucleus (after oxidation of the 42-hydroxyl group) and the 27-ketone.

[0076] Examples of 42- and / or 31-esters and ethers of rapamycin are disclosed in the following patents, which are hereby incorporated by reference in their entirety: alkyl esters (U.S. Patent No. 4,316,885); aminoalkyl esters (U.S. Patent No. 4,650,803); fluorinated esters (U.S. Patent No. 5,100,883); amide esters (U.S. Patent No. 5,118,677); carbamate esters (U.S. Patent No. 5,118,678); silyl ethers (U.S. Patent No. 5,120,842); amino esters (U.S. Patent No. 5,130,307); acetals (U.S. Patent No. 551,413); aminodiesters (U.S. Patent No. 5,162,333); sulfonic acid esters and sulfuric acid esters (U.S. Patent No. 5,177,203); esters (U.S. Patent No. 5,221,670); alkoxy esters (U.S. Patent No. 5,233,036); O-aryl, -alkyl, -alkenyl and alkynyl ethers (U.S. Patent No. 5,258,389); carbonate esters (U.S. Patent No. 5,260,300); arylcarbonyl and alkoxycarbonyl carbamates (U.S. Patent No. 5,262,423); carbamates (U.S. Patent No. 5,302,584); hydroxy esters (U.S. Patent No. 5,362,718); hindered esters (U.S. Patent No. 5,385,908); heterocyclic esters (U.S. Patent No. 5,385,909); gem-disubstituted esters (U.S. Patent No. 5,385,910); aminoalkanoic acid esters (U.S. Patent No. 5,389,639); phosphorylcarbamate esters (U.S. Patent No. 5,391,730); carbamate esters (U.S. Patent No. 5,411,967); carbamate esters (U.S. Patent No. 5,434,260); amidinocarbamate esters (U.S. Patent No. 5,463,048); carbamate esters (U.S. Patent No. 5,480,988); carbamate esters (U.S. Patent No. 5,480,989); carbamate esters (U.S. Patent No. 5,489,680); hindered N-oxide esters (U.S. Patent No. 5,491,231); biotin esters (U.S. Patent No. 5,504,091); O-alkyl ethers (U.S. Patent No. 5,665,772); and PEG esters of rapamycin (U.S. Patent No. 5,780,462).

[0077] Examples of 27-esters and ethers of rapamycin are disclosed in U.S. Patent No. 5,256,790, which is hereby incorporated by reference in its entirety.

[0078] Examples of oximes, hydrazones, and hydroxylamines of rapamycin are disclosed in U.S. Patent Nos. 5,373,014, 5,378,836, 5,023,264, and 5,563,145, which are hereby incorporated by reference in their entirety. The preparation of these oximes, hydrazones, and hydroxylamines is disclosed in the patents listed above. The preparation of 42-oxorapamycin is disclosed in U.S. Patent No. 5,023,263, which is hereby incorporated by reference in its entirety.

[0079] Other compounds within the scope of "rapamycin analogs or derivatives thereof" include, for example, the "rapalogs" in International Publication No. 98 / 02441 and the references cited therein, and the compounds and classes of compounds referred to as "epirapalogs" in, for example, International Publication No. 01 / 14387 and the references cited therein.

[0080] Another compound within the scope of the "rapamycin derivative" is everolimus, a 4-O-(2-hydroxyethyl) rapamycin derived from a macrolide antibiotic produced by Streptomyces hygroscopicus (Novartis). Everolimus is also known as Certican, RAD-001, and SDZ-RAD. Another preferred mTOR inhibitor is zotarolimus (Abbott Laboratories), an antiproliferative agent. Zotarolimus is thought to inhibit smooth muscle cell proliferation with the cell growth inhibitory effect resulting from the inhibition of mTOR. Another preferred mTOR inhibitor is tacrolimus, a macrolide lactone immunosuppressant isolated from the soil fungus Streptomyces tsukubaensis. Tacrolimus is also known as FK 506, FR 900506, Fujimycin, L 679934, Tsukubanolide, Protopic, and Prograf. Other preferred mTOR inhibitors include AP-23675, AP-23573, and AP-23841 (Ariad Pharmaceuticals).

[0081] Preferred rapamycin derivatives include everolimus, CCI-779 (rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid; U.S. Patent No. 5,362,718); 7-epi-rapamycin; 7-thiomethyl-rapamycin; 7-epi-trimethoxyphenyl-rapamycin; 7-epi-thiomethyl-rapamycin; 7-demethoxy-rapamycin; 32-demethoxy-rapamycin; 2-desmethyl-rapamycin; and 42-O-(2-hydroxy)ethylrapamycin (U.S. Patent No. 5,665,772).

[0082] An additional mTORC2 inhibitor can be OSI-027 (OSI Pharmaceuticals), a small molecule mTORC2 inhibitor. OSI-027 inhibits the mTORC2 signaling complex and enables the potential for complete truncation of abnormal cell signaling through this pathway.

[0083] Also, Torquinib, an ATP-competitive mTOR kinase domain inhibitor, and an inhibitor of mTORC2 can also be used in accordance with the present invention. Exemplary Torquinibs include PP242 and PP30 (see Feldman et al. (2009) PLoS Biology 7:371) and Torin1 (see Thoreen et al. (2009) J Biol Chem 284:8023).

[0084] In certain embodiments, a PI3K inhibitor and an mTOR inhibitor for use in accordance with the present invention, as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof, wherein the PI3K inhibitor is BYL719 and the mTOR inhibitor is everolimus.

[0085] In another embodiment, a PI3K inhibitor and a tyrosine kinase inhibitor (TKI) for use in accordance with the present invention, as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0086] As used herein, the term "TKI" refers to a tyrosine kinase inhibitor. Tyrosine kinases are involved in the phosphorylation of many proteins. Examples of tyrosine kinase proteins: AATK; ABL; ABL2; ALK; AXL; BLK; BMX; BTK; CSF1R; CSK; DDR1; DDR2; EGFR; EPHA1; EPHA2; EPHA3; EPHA4; EPHA5; EPHA6; EPHA7; EPHA8; EPHA10; EPHB1; EPHB2; EPHB3; EPHB4; EPHB6; ERBB2; ERBB3; ERBB4; FER; FES; FGFR1; FGFR2; FGFR3; FGFR4; FGR; FLT1; FLT3; FLT4; FRK; FYN; GSG2; HCK; IGF1R; ILK; INSR; INSRR; IRAK4; ITK; JAK1; JAK2; JAK3; KDR; KIT; KSR1; LCK; LMTK2; LMTK3; LTK; LYN; MATK; MERTK; MET; MLTK; MST1R; MUSK; NPR1; NTRK1; NTRK2; NTRK3; PDGFRA; PDGFRB; PLK4; PTK2; PTK2B; PTK6; PTK7; RET; ROR1; ROR2; ROS1; RYK; SGK493; SRC; SRMS; STYK1; SYK; TEC; TEK; TEX14; TIE1; TNK1; TNK2; TNNI3K; TXK; TYK2; TYRO3; YES1; ZAP70. In certain embodiments, the TKI is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA or antisense oligonucleotide.

[0087] In certain embodiments, the tyrosine kinase is EGFR. As used herein, the term "EGFR" refers to the epidermal growth factor receptor, which is a member of the ErbB family of receptors, and is a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). EGFR is involved in differentiation and cell growth. An inhibitor of EGFR refers to a compound that inhibits cell growth. In certain embodiments, the inhibitor of EGFR is selected from the group consisting of gefitinib, erlotinib, afatinib, brigatinib, lapatinib, icotinib, cetuximab, osimertinib, zalutumumab, nimotuzumab, and matuzumab.

[0088] In certain embodiments, the inhibitor of EGFR is an irreversible mutant-selective EGFR inhibitor that specifically targets EGFR activating mutations that occur newly and upon acquisition of resistance. Typically, such inhibitors inhibit the most common EGFR mutations L858R, Ex19del, and T790M. Thus, in certain embodiments, the inhibitor of EGFR is EGF816, also known as nazartinib, developed by Novartis.

[0089] In certain embodiments, the tyrosine kinase is VEGF. As used herein, the term "VEGF" refers to vascular endothelial growth factor. VEGF stimulates a cellular response by binding to tyrosine kinase receptors (VEGFRs) on the cell surface, and notably, stimulates the formation of blood vessels (angiogenesis). The VEGF family includes five members in mammals: VEGF-A, placental growth factor (PGF), VEGF-B, VEGF-C, and VEGF-D. In certain embodiments, an inhibitor of VEGF refers to inhibiting the stimulation of growth cells and the formation of blood vessels. In certain embodiments, an inhibitor of VEGF is selected from the group consisting of ranibizumab (Lucentis®), aflibercept (Eylea®), and bevacizumab (Avastin®), tivozanib, lenvatinib, axitinib, imatinib, or brolucizumab (RTH258).

[0090] In another embodiment, the inhibitor is a VEGFR inhibitor. As used herein, the term "VEGFR" refers to the receptor for vascular endothelial growth factor (VEGF). There are three main subtypes of VEGFR: VEGFR1, VEGFR2, and VEGFR3. The VEGFR inhibitor is selected from the group consisting of pegaptanib, lenvatinib, motesanib, pazopanib, cabozantinib (Cabometyx®).

[0091] In some embodiments, the TKI is selected from the group consisting of gefitinib, erlotinib, dasatinib, nilotinib, bosutinib, ponatinib, luxolitinib, xalkori, cabozantinib, and sunitinib. In certain embodiments, the TKI is imatinib.

[0092] In another embodiment, a PI3K inhibitor and a PARP inhibitor for use according to the invention as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0093] As used herein, the term "PARP" refers to poly(ADP-ribose) polymerase, an enzyme involved in cellular processes such as DNA repair, genomic stability, and programmed cell death. In certain embodiments, a PARP inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamer, siRNA, or antisense oligonucleotide.

[0094] The PARP inhibitor is selected from the group consisting of iniparib (BSI 201), talazoparib (also known as BMN-673), veliparib (ABT-888), olaparib (also known as AZD-2281 and commercialized as Lynparza (registered trademark)), rucaparib (also known as Rubraca (registered trademark)), or niraparib (also known as Zejula (registered trademark)).

[0095] The inhibitors of PI3K, MAPK, PAK, mTOR, TKI, PARP, and / or EGFR described above can be used as part of a multi-drug therapy for the treatment of proliferative glomerulonephritis in a subject in need thereof.

[0096] The PI3K inhibitor can be used as a single inhibitor or in combination with other inhibitors such as inhibitors of MAPK, PAK, mTOR, TKI, PARP, and / or EGFR. When several inhibitors are used, a mixture of inhibitors is obtained. In the case of multi-drug therapy (e.g., two-drug, three-drug, or four-drug therapy), at least one other inhibitor can be accompanied by a PI3K inhibitor.

[0097] In certain embodiments, inhibitors of PI3K and MAPK can be combined as a two-drug therapy for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K and MAPK can be combined for use as a two-drug therapy, and the inhibitors of PI3K and MAPK are BYL719 and selumetinib, respectively.

[0098] In another embodiment, inhibitors of PI3K and ERK can be combined as a two-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K and ERK can be combined for use as a dual therapy, and the inhibitors of PI3K and ERK are BYL719 and VTX-11e, respectively.

[0099] In another embodiment, inhibitors of PI3K and mTOR can be combined as a two-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K and mTOR can be combined for use as a two-drug treatment, and the inhibitors of PI3K and mTOR are BYL719 and everolimus, respectively.

[0100] In another embodiment, inhibitors of PI3K and TK can be combined as a two-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K and TK can be combined for use as a two-drug treatment, and the inhibitors of PI3K and TK are BYL719 and sunitinib, respectively.

[0101] In another embodiment, inhibitors of PI3K and VEGF can be combined as a two-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K and TK can be combined for use as a dual therapy, and the inhibitors of PI3K and VEGF are BYL719 and brolucizumab (RTH258), respectively.

[0102] In another embodiment, inhibitors of PI3K, MAPK, and PAK can be combined as a three-drug treatment for use in the treatment of proliferative glomerulonephritis. In certain embodiments, inhibitors of PI3K, MAPK, and PAK can be combined as a three-drug treatment, and the inhibitors of PI3K, MAPK, and are BYL719, selumetinib, and IPA-3, respectively.

[0103] As used herein, the term "administering" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., an inhibitor of PI3K) that exists outside the body, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, the administration of the substance typically occurs after the onset of the disease or the symptom. When a disease or a symptom thereof is being prevented, the administration of the substance typically occurs before the onset of the disease or the symptom.

[0104] As used herein, the term "co-administering" refers to the administration of at least two or three active ingredients by the same route and simultaneously or substantially simultaneously. The term "separately administering" refers to the administration of at least two or three active ingredients by different routes and simultaneously or substantially simultaneously. The term "sequentially administering" refers to the administration of at least two or three active ingredients at different times, and the administration routes may be the same or different.

[0105] "Therapeutically effective amount" is intended to refer to the minimum amount of an active agent necessary to confer a therapeutic benefit on a subject. For example, a "therapeutically effective amount" for a subject is an amount that induces remission, alleviates, or causes improvement in a pathological symptom, disease progression, or physiological state associated with or subject to a disorder. It will be understood that the total daily usage of the compounds of the present invention can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound being used; the specific composition being used, the age, body weight, general health, sex, and diet of the subject; the time of administration, the route of administration, and the rate of excretion of the specific compound being used; the duration of the treatment; drugs used in combination with or concurrently with the specific compound being used; and like factors well known in the medical arts. For example, it is within the skill of those in the art to start the dosage of the compound at a level lower than that 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 product can vary widely from 0.01 to 1,000 mg per adult per day. Typically, the composition contains 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 symptomatic adjustment of the dosage to the subject being treated. Pharmaceutical products typically contain 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. The effective amount of the drug is usually supplied at dosage levels of from 0.0002 mg / kg to about 20 mg / kg body weight per day, particularly from about 0.001 mg / kg to about 7 mg / kg body weight per day.

[0106] As described above, the PIK3CA inhibitor may form a pharmaceutical composition alone or in combination with a pharmaceutically acceptable excipient and optionally a sustained release matrix, such as a biodegradable polymer, etc., in combination with classical treatments.

[0107] Accordingly, the present invention relates to a pharmaceutical composition comprising a PIK3CA inhibitor for use in the treatment of proliferative glomerulonephritis as described above.

[0108] In certain embodiments, the present invention relates to a pharmaceutical composition comprising a PIK3CA inhibitor for use in the treatment of lupus nephritis (LN).

[0109] In certain embodiments, the present invention relates to a pharmaceutical composition comprising a PIK3CA inhibitor for use in the treatment of focal segmental glomerulosclerosis (FSGS).

[0110] In a further embodiment, the present invention relates to a pharmaceutical composition comprising, as a combined preparation for treating proliferative glomerulonephritis, i) a PIK3CA inhibitor and ii) a classical treatment as described above.

[0111] "Pharmaceutically" or "pharmaceutically acceptable" refers, where appropriate, to molecular entities and compositions that do not produce adverse reactions, allergic reactions, or other undesirable reactions when administered to mammals, particularly humans. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. The pharmaceutical compositions of the invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient, alone or in combination with another active ingredient, in unit dosage form, as a mixture with conventional pharmaceutical supports, can be administered to animals and humans. Suitable unit dosage forms include 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, subcutaneous, transdermal, intrathecal and intranasal administration forms, as well as rectal administration forms. Typically, the pharmaceutical composition includes a pharmaceutically acceptable vehicle for a formulation that can be injected. These are, in particular, isotonic, sterile, physiological saline (sodium monophosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride and the like, or mixtures of such salts), or dry, in particular lyophilized compositions, which, depending on the case, enable the constitution of an injection solution upon addition of sterile water or physiological saline. Suitable pharmaceutical forms for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy injectability 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 containing the compounds of the invention as free bases or pharmaceutically acceptable salts can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose and the like. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations include preservatives to prevent the growth of microorganisms.The polypeptide (or the nucleic acid encoding it) can be formulated in a composition in its neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or iron(III) hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride and the like. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents that delay absorption such as aluminum monostearate and gelatin and the like. Sterile injectable solutions are prepared, if required, by incorporating the required amount of the active polypeptide with some of the other ingredients enumerated above in a suitable solvent and then followed by filter sterilization. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a 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 method of preparation is by vacuum drying and lyophilization techniques that yield a powder of the active ingredient plus any additional ingredients from a previously sterile filtered solution. Upon formulation, the solution is administered in a therapeutically effective amount in a manner compatible with the dosage formulation.The formulations are readily administered in a variety of dosage forms, such as the injectable solution type described above, but drug release capsules and the like can also be used. For example, for parenteral administration in an aqueous solution, the solution is, if necessary, appropriately buffered and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this context, the sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of a subcutaneous injection solution or injected at the proposed site of injection. 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 dosage appropriate for an individual subject.

[0112] A further object of the present invention relates to a method of screening for a drug suitable for the treatment of proliferative glomerulonephritis, comprising i) providing a test compound and ii) determining the ability of the test compound to inhibit the activity of PI3K.

[0113] Any biological assay well known in the art may be suitable for determining the ability of a test compound to inhibit the activity of PI3K. In some embodiments, the assay involves first determining the ability of a test compound to bind to PI3K. In some embodiments, a population of cells is then contacted and activated to determine the ability of a test compound to inhibit the activity of PI3K. In particular, the effect induced by the test compound is determined by comparison to the effect 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 similar to negative control conditions. As used herein, the terms "control substance," "control agent," or "control compound" refer to molecules that are inert or have no activity associated with the ability to modulate biological activity or expression. It should be understood that test compounds capable of inhibiting the activity of PI3K, as determined using the in vitro methods described herein, are likely to exhibit similar regulatory capabilities in in vivo applications. Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers, or nucleic acids. For example, a test compound according to the invention may be selected from a library of previously synthesized compounds, or a library of compounds whose structure has been determined in a database, or a library of newly synthesized compounds. In some embodiments, the test compound may be selected from small organic molecules.

[0114] The present invention is further illustrated by the following figures and examples. However, these examples and figures should not be construed in any way as limiting the scope of the invention.

Examples

[0115] Example 1: Materials and Methods Animal Tests R26StopFLP110 on C57BL / 6 background *(Stock #012343), R26StopCAG-EGFP (Stock #006071), and Podocin-Cre mice (Stock #008523), Tg26 / HIV mice on FVB background (Stock #022354), Pik3Calox / lox mice (Stock #017704), MRL / MpJ-Faslpr / J (Stock #000485) were obtained from the Jackson Library. NZBWF1 / OlaHsd mice were obtained from Envigo. Whenever required, at least 10 backcrosses were performed before using the mice for experiments. The mice were randomly assigned to each group in a manner where male and female, age in months, and body weight were matched, unless otherwise indicated. All animal procedures were approved by the Ministry of Higher Education, Research and Innovation (APAFIS#30133-2020111914293579 v8) and were carried out in accordance with the guidelines of the University of Paris Descartes to ensure animal welfare.

[0116] For the unilateral nephrectomy experiment, the right kidney was removed under anesthesia. For the medical treatment of the mice, 50 mg kg-1 alpelisib (MedChem Tronica) in 1% carboxymethylcellulose (Sigma Aldrich) + 0.5% Tween (Sigma Aldrich) or vehicle (1% carboxymethylcellulose + 0.5% Tween) was administered once daily by oral gavage for the indicated period. Blood and urine were obtained at the indicated times. At euthanasia, blood, urine, and kidneys were collected. In some experiments, spleen, heart, and bone marrow (BM) in the femur and tibia were also collected. Tissues were fixed in 4% paraformaldehyde, paraffin-embedded for immunohistochemical analysis, snap-frozen in optimal cutting temperature (OCT), or stored at -80 °C for mRNA or protein analysis.

[0117] Blood and urine measurements Mouse blood cell counts were analyzed using a hematology analyzer (ProCyte Dx, IDEXX Laboratories). Mouse serum creatinine, blood urea nitrogen, urinary albumin, and urinary creatinine were evaluated using an AU5800 (Beckman Coulter) automated analyzer. Serum anti-dsDNA measurements were performed using a mouse anti-dsDNA ELISA kit (LBIS) according to the manufacturer's instructions. Absorbance was measured using an Infinite M Nano (TECAN).

[0118] Histopathological and immunohistochemical analysis 4-μm kidney sections were stained with periodic acid Schiff staining (PAS), Masson trichrome staining (MT), periodic acid methenamine silver staining (PAM), or hematoxylin and eosin staining (HE) for histological analysis. The degree of glomerular lesions was evaluated using the following scoring system: 0 = no lesions, 1 = up to 25% of glomeruli affected, 2 = 25 - 50% of glomeruli affected, 3 = 50 - 75% of glomeruli affected, 4 = 75 - 100% affected. At least 50 glomeruli per sample were measured for each analysis. The indirect immunoperoxidase method was used for immunohistochemistry. Briefly, after deparaffinization, antigen retrieval was performed using citrate buffer (pH 6) or Tris-EDTA buffer (pH 9 or pH 6) with microwave or high temperature (95°C). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide, non-specific protein binding was blocked with 2.5% normal horse serum (Vector Laboratories), and endogenous biotin activity was quenched with an avidin / biotin blocking kit (Vector Laboratories). When using mouse primary antibodies in mouse tissues, endogenous mouse immunoglobulins were blocked with Klear mouse blocking reagent (Diagomics). After blocking, tissue sections were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: rabbit anti-Ki67 antibody (SP6; Thermo Fisher Scientific), rabbit anti-P-S6RP antibody (D68F8; Cell Signaling Technology), mouse anti-P-AKT (Ser473) antibody (587F11; Cell Signaling Technology), rabbit anti-P-AKT (Thr308) antibody (C31E5E; Cell Signaling Technology), mouse anti-S6RP antibody (54E2; Cell Signaling Technololgy), chicken anti-GFP antibody (Abcam), guinea pig anti-nephrin antibody (GP-N2; Progen), rabbit anti-podocin antibody (P0372; Sigma Aldrich), mouse anti-WT1 antibody (6F-H2; DAKO).

[0119] The corresponding secondary antibodies, including anti-mouse IgG, anti-rabbit IgG, anti-guinea pig IgG, biotinylated anti-rabbit IgG (Vector Laboratories), and biotinylated anti-mouse IgG (Vector Laboratories), were applied. For avidin / biotin detection, an R.T.U. Vectastain Kit (Vector Laboratories) or streptavidin (Thermo Fisher Scientific) was used. Immunoreactive antigen sites were detected using hydrogen peroxide and diaminobenzidine. For frozen immunofluorescence staining, frozen tissue sections (4 μm) were air-dried briefly and fixed in 50% methanol / 50% acetone. The sections were then blocked with 2.5% normal horse serum and incubated overnight at 4°C with the primary antibody: guinea pig anti-nephrin antibody (GP-N2; Progen). The sections were then incubated with the secondary antibodies: fluorescein isothiocyanate conjugate (FITC) anti-mouse IgG (Sigma Aldrich), FITC anti-mouse IgM (BD Biosciences), or FITC anti-mouse C3 antibody (abcam) for 40 minutes at room temperature. Images were captured with a Zeiss LSM700 confocal microscope. Immunohistochemical color development was performed using a suitable horseradish peroxidase (HRP) antibody, and images were captured with a Nikon Eclipse E800 microscope. Image J software was used for image analysis. Ilastik (Interactive Machine Learning for (Bio)Image Analysis, version 1.33post3) was used to select the positive signal regions in the glomeruli.

[0120] cDNA Synthesis and Quantitative RT-PCR Analysis Total RNA in the renal cortex was extracted using NucleoSpin RNA (Macherey Nagel). Complementary DNA was reverse transcribed using the TaqMan High Capacity cDNA RT Kit (Thermofisher). qPCR was performed using the CFX Connect Real-Time System (Bio-Rad Laboratories) with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). Expression levels were analyzed by the delta-delta Ct method. Hypoxanthine phosphoribosyltransferase (Hprt) was used as a normalization control.

[0121] Western blotting Protein extracts from renal cortex in RIPA buffer were separated by SDS-PAGE, transferred onto membranes, incubated with antibodies, and subsequent incubation with a suitable peroxidase-conjugated secondary antibody. The following primary antibodies were used: rabbit anti-P-S6RP antibody (D68F8; Cell Signaling Technology), rabbit anti-P-AKT (Ser473) antibody (D9E; Cell Signaling Technology), rabbit anti-P-AKT (Thr308) antibody (C31E5E; Cell Signaling Technology), mouse anti-S6RP antibody (54E2; Cell Signaling Technololgy), mouse anti-Akt (pan) antibody (40D4, Cell Signaling Technology), chicken anti-GFP antibody (ab13970; Abcam), mouse anti-alpha tubulin antibody (B-5-1-2; Sigma Aldrich), rabbit anti-nephrin antibody (29070; IBL), rabbit anti-podocin antibody (P0372; Sigma Aldrich). Chemiluminescence was acquired using the ChemiDoc MP (Bio-Rad Laboratories), and densitometry was performed using the Image Lab software (Bio-Rad Laboratories, version 6.0.1).

[0122] Mouse cell preparation and flow cytometry Mononuclear cells (MNCs) from the peripheral blood, spleen, BM, and lymph nodes of MRL / Lpr mice treated with either medium or apelacib were prepared essentially as previously described 12. For lymphocyte and granulocyte / macrophage lineage analysis, anti-CD3a antibody (500A2; Becton Dickinson), anti-CD4 antibody (L3T4; Becton Dickinson), anti-CD8a antibody (63-6.72; Becton Dickinson), anti-B220 antibody (RA3-6B2; Becton Dickinson), anti-CD11b antibody (M1 / 70; Becton Dickinson), anti-Ly6G antibody (RB6-8C5; Becton Dickinson), and CD16 / CD32 (2.4G2; Becton Dickinson). Cells were resuspended in 0.2 - 1 mL of PBS + 2% FCS + 2 μL of 7-aminoactinomycin D (7-AAD, Thermo Fischer Scientific). Flow cytometry was performed using an SP6800 spectral analyzer (SONY). Data were analyzed using FlowJo software (TreeStar).

[0123] Preparation of single cell suspension For imaging flow cytometry (Amnis ImageStream) or single-cell RNAseq analysis, single-cell suspensions were prepared as follows. PIK3CAWT mice and PIK3CA Podo-HT mice were intravenously injected with M-450 Dynabeads (Thermofischer, ref# 14013) and saline. Kidneys were rinsed in phosphate-buffered saline and cut into small pieces on ice in RPMI 1640 medium. The Multi Tissue Dissociation Kit 1 (Miltenyi Biotec) was used to digest the kidneys. First, to enrich glomeruli, kidney pieces were stirred in digestion buffer at 37°C for 20 minutes. Cells were then filtered (100 μm, Miltenyi Biotec) and centrifuged at 4°C at 300 g for 5 minutes. The cell pellet was resuspended in RPMI1640 medium and rinsed. The cell pellet was resuspended again in digestion buffer and digested using the Half Protocol 37C_Multi_B to create a single-cell suspension. The kidney cell suspension was filtered (70 μm, Miltenyi Biotec), centrifuged at 4°C at 300 g for 5 minutes, and resuspended in RPMI 1640 medium. The cell pellet was incubated with RBC lysis buffer (Miltenyi Biotec) on ice for 3 minutes, centrifuged at 4°C at 300 g for 5 minutes, and resuspended in 1 ml PBS + 0.04% BSA. Cell number and viability were analyzed microscopically using a 0.4% trypan blue solution. The protocol was designed to achieve a single-cell suspension with minimal experimental time and maximum possible cell viability.

[0124] Imaging flow cytometry (Amnis ImageStream) According to the single-cell suspension procedure, flow cytometry cell preparation was performed essentially according to the flow cytometry protocol by Cell Signaling Technology. Briefly, cell samples were fixed with 4% PFA for 15 minutes, permeabilized with methanol on ice, washed several times, and then resuspended in PBS. Samples were labeled with rabbit anti-P-S6RP-AF647 antibody (D68F8; Cell Signaling Technology) and / or rabbit anti-P-AktThr308-PE antibody (D25E6; Cell Signaling Technology). Rabbit IgG isotype controls (AF647 and PE; Cell Signaling Technology) were used. The resuspended cells were run on an ImageStream ISX mkII (Amnis) that combined flow cytometry and detailed cell imaging. A magnification of 40× was used for all acquisitions. Data were acquired using INSPIRE software (Amnis) and analyzed using IDEAS software (v.6.2, Amnis).

[0125] Single-cell RNA-seq barcoding and cDNA synthesis Single-cell suspensions were used as input with the 10x Chromium Controller with approximately 20,000 cells, targeting to obtain 10,000 cells per library (10x Genomics). Barcoding and cDNA synthesis were performed according to the manufacturer's instructions (10x Genomics). An equimolar pool of four individual 10X Genomics single-cell expression 3’ V3 libraries was prepared by the Imagine Genomic Core Facility and sequenced on a NovaSeq6000, Illumina using an S2 FlowCell (sequencing mode Paired-End 100+100 bases+index). A total of 2 billion reads were targeted for this pool of four libraries (500 million reads per library).

[0126] Single-cell RNA-seq pre-processing Sequencing data quality analysis was performed using FastQC v0.11.2 on four mouse samples. For read alignment and unique molecular identifier (UMI) quantification, CellRanger software v3.0.2 was used with the Mus musculus genome mm10, with default parameters and gene annotation from Ensembl 100 (+eGFP and tdTomato). Four expression matrices containing UMI counts were integrated, retaining only genes with UMI≥1 in at least one cell. The following filters were applied to generate the global matrix used in further analysis: cells with UMI≥2000, number of detected genes≥600, and cells with UMI≤60% in mitochondrial genes. For UMI normalization, Seurat 3.1.1 (Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transitomic data across different Conditions, technologies, and species. Nature biotechnology 36, 411-420(2018)) was used to apply a global scaling normalization method with a scale factor of 10,000 and log transformation of the data. This was followed by a scaling linear transformation step to avoid highly expressed genes with higher weights in downstream analysis.

[0127] Clustering and marker genes PCA was performed on the scaled data, with a Jackstraw plot to select the number of PCs to retain as input for the Seurat clustering process. The clustering process was performed using default parameters, the Louvain algorithm as the clustering method, 15 PCs, and a resolution parameter defining a cluster granularity set to 0.5. Marker genes defining each cluster were found via differential expression testing using the Wilcoxon rank sum test and a log fold change threshold of 1.

[0128] Trajectory analysis Monocle single-cell trajectories were constructed using M3Drop (Andrews, T. S. & Hemberg, M. M3Drop: dropout-based feature selection for scRNASeq. Bioinformatics 35, 2865-2867(2019)) and Monocle 2.10.1 (Qiu, X. et al. Reversed graph embeddedding resolutions complex single-cell trajectories. Nature methods 14, 979-982(2017)). Input genes for Monocle trajectory construction were selected using an unsupervised approach via M3Drop results, whereby differentially expressed genes were identified based on the Michaelis-Menten function for the relationship between mean expression and dropout rate, and the relevant genes are those that shift above the fitted curve. The default Monocle workflow was then implemented to generate trajectories.

[0129] Enrichment analysis Analysis of enriched KEGG pathways was performed in a database from Mus musculus organisms using the WebGestaltR package (Qiu, X. et al. Reversed graph embeddedding resolves complex single-cell trajectories. Nature methods 14, 979-982(2017)). GO terms and pathways were considered enriched if fold enrichment ≥ 2.0, uncorrected p-value ≤ 0.05, and minimum number of regulated genes in pathway / term ≥ 2.0.

[0130] LC-MS analysis of alpelisib Each tissue type was homogenized in 100% cold methanol with a tissue-to-vehicle ratio of 1 mg of tissue to 5 μl of methanol. After sonication for 20 seconds, the tissue extract was centrifuged at 13,000 g for 30 minutes and then injected onto a Phenomenex Kinetex XB-C18 HPLC column (100 mm x 2.1 mm) at 45 °C. Alprazolam was analyzed by reverse-phase HPLC (Shimadzu LCMS system 8040 interfaced with LabSolutions software). 20 micrograms of the tissue extract was injected onto the column, and the mobile phase used for separation consisted of two eluents: Solvent A was 0.1% formic acid in ddH2O, and Solvent B was acetonitrile with 0.1% formic acid. Compounds were separated at a flow rate of 0.6 ml / min by the following discontinuous gradient: The initial concentration of 20% in Solvent B was increased to 70% over 6 minutes, which was followed by a decrease to 20% over the next 1 minute, and the initial conditions were then maintained for 14 minutes. Alprazolam was monitored spectrophotometrically by absorbance at 205 - 600 nm (photodiode detector) and by tandem mass detection. Mass measurements were performed in positive ion mode using multiple reaction monitoring (MRM) with an electrospray ionization source. Three MRM transitions for alprazolam were used as follows: 442.1>328.0, 442.1>288.0, and 442.1>115.1. Quantification was performed by integration of the peak absorbance area using a calibration curve established with various known concentrations of alprazolam.

[0131] Human samples Human kidney samples were obtained from patients being followed at the Necker Children's Hospital. Clinical and biological data available at the time of kidney biopsy or diagnosis of lupus nephritis relapse were collected. Classical histological analysis and immunofluorescence analysis were performed using kidney biopsies fixed with formalin, alcohol, and acetic acid (AFA), which were then paraffin-embedded. A portion of the immunofluorescence test and droplet digital PCR (ddPCR) were performed using OCT frozen sections. Written informed consent was obtained from each patient.

[0132] STAR-FISH and Droplet Digital PCR STAR-FISH (Specific-To-Allele PCR - FISH) was performed as previously described13. For droplet digital PCR (ddPCR), 20-μm frozen kidney sections were placed on PEN membrane slides, rapidly stained with hematoxylin to recognize kidney structures. Laser capture microdissection (LCM) was performed using a Leica LMD7000 system. Glomeruli or tubules were collected for each patient. ddPCR (QX200 system, Bio-Rad Laboratories) was performed to specifically detect variants in pools of different cells. ddPCR Supermix (without dUTP) for probes was used according to the manufacturer's protocol. Data were analyzed using a QX200 droplet reader and Quantasoft Analysis Pro software (BioRad Laboratories; version 1.0.596).

[0133] Spatial Transcriptomics The GeoMx (trademark) digital spatial profiling experiment was performed according to the Nanostring GeoMx-NGS DSP instrument manual and as previously reported (Merritt, C. R. et al. Multiplex digital spatial profiling of protein and RNA in fixed tissue. Nature biotechnology 38, 586-599 (2020)). Briefly, 4um AFA-fixed paraffin-embedded human samples were baked at 37°C overnight and at 65°C for 1 hour, and then they were processed using a protocol on a Leica automated platform that included three main steps: 1) slide baking, 2) antigen retrieval at 100°C for 20 minutes, and 3) proteinase K treatment at 1.0 ug / ml for 15 minutes. After removing the slides from the Leica, the slides were incubated overnight with the GeoMx WTA assay probe cocktail. The next day, the slides were washed and morphological marker incubations were applied prior to loading on the GeoMx machine. Anti-pan-cytokeratin (clone AE1 / AE3, Novus Biologicals), anti-CD10 (clone EPR22867-118, Abcam), anti-CD31 (clone JC / 70A, abcam) were used as morphological markers. On the GeoMx machine, the slides were fluorescently scanned and ROI selection was completed. Barcodes were collected and subsequent barcode reading was performed on an Illumina NGS platform. Individual counts were normalized to the 75th percentile of the signal from their respective individual ROIs (Q3 normalization). Data analysis was performed with GeoMx DSP software v2.5.1.145. The Reactome database v78 was used for pathway analysis.

[0134] Data analysis and statistics Data were expressed as mean ± SEM or SD. Survival curves were analyzed using the log-rank (Mantel-Cox) test. Two-way ANOVA with Bonferroni's post hoc test was used to determine statistical significance between experimental groups. Two-group experiments were compared using two-sided Student's t-test. Statistical analysis was performed using GraphPad Prism software (version 7.0a).

[0135] Results Patients with gain-of-function mutations in PIK3CA in podocytes Renal biopsy of Patient 1 revealed the presence of complex glomerulonephritis characterized by focal segmental glomerulosclerosis (FSGS), a mixture of crescentic lesions without immune deposits, extensive fibrosis with tubular dilation (>80% of the parenchyma), cylinders, and inflammatory infiltrating cells (data not shown). Immunofluorescence testing showed the proliferation and activation of the AKT / mTOR pathway in glomerular epithelial cells (data not shown). The inventors initially thought that these lesions were caused by a complex medical situation combining vesicoureteral reflux, congestive heart failure, and rapamycin. The inventors discontinued rapamycin, but did not observe any improvement in proteinuria or renal function (data not shown). As previously reported, due to the severity of the CLOVES / PROS condition, the inventors were permitted to treat this patient with alpelisib, an approved specific PIK3CA inhibitor. This drug led to the reduction of different malformations and the complete recovery of congestive heart failure. Interestingly, the inventors observed improvement in a large amount of proteinuria and stabilization of renal function. The inventors hypothesized that this patient might carry a PIK3CA mutation in renal epithelial cells. To explore this hypothesis, the inventors first initiated by performing in situ PCR using fluorescence-labeled mismatch primers designed to specifically amplify mutant (H1047R) and wild-type PIK3CA alleles on paraffin-embedded renal biopsy sections. T-47D cells were used as a positive control (data not shown). The inventors observed the presence of PIK3CA mutant alleles in glomerular epithelial cells (data not shown). Several types of glomerular cells, including podocytes, appeared to carry the mutation. However, the inventors were unable to perform any co-immunostaining to highlight the cell types affected. To confirm the presence of the mutation, the inventors performed droplet PCR of glomeruli isolated from the renal biopsy of Patient 1 using laser capture microdissection (data not shown).No changes in any of the PIK3CA alleles were revealed by control biopsy, whereas the presence of the PIK3CA H1047R mutation was demonstrated by droplet PCR of glomeruli from Patient 1 (data not shown). The inventors concluded that Patient 1 harbored a PIK3CA gain-of-function mutation in glomerular epithelial cells.

[0136] Mouse model of PIK3CA gain-of-function mutation in podocytes Based on the glomerular profile of kidney dysfunction in Patient 1, the presence of PIK3CA variants in glomerular cells, and the dramatic reduction in proteinuria after introduction of alpelisib, the inventors decided to generate a mouse model of PIK3CA gain-of-function mutation specifically in podocytes. The inventors utilized the transgenic mouse strain R26StopFLP110 that expresses a dominant-active PIK3CA transgene after breeding with Cre recombinase mice. R26StopFLP110 * mice were mated with Podocin Cre mice to generate PIK3CA Pod animals that express the hyperactivated form of PIK3CA. To track Cre recombination, PIK3CA Pod-HET mice were then mated with Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J mice 11. These mice express a cell membrane-localized tdTomato fluorescent protein in all tissues, which is replaced by GFP after Cre recombination. At birth, PIK3CA Pod-HET mice were indistinguishable from WT control littermates (PIK3CA WT ). However, PIK3CA Pod-HET mice developed progressive albuminuria from 3 months of age, with slowly deteriorating kidney function (data not shown) and reduced survival rate (data not shown). Compared to controls, PIK3CA Pod-HETThe mice showed glomerular pathology with a mixture of crescentic and collapsing glomerulopathy as observed in patient 1 (data not shown). Light microscopy revealed hypertrophied multinucleated podocytes with intracytoplasmic vacuoles, but also protein casts and inflammatory cell infiltration (data not shown). Using immunofluorescence experiments, we demonstrated that the AKT / mTOR pathway mediates the PIK3CA Pod-HET We confirmed that PIK3CA is activated in mouse podocytes (data not shown). One of the main characteristics of podocyte injury is the loss of differentiation markers. We decided to test cell differentiation by analyzing the expression of WT1, a transcription factor that marks mature podocytes, and nephrin, podocin, and nestin, proteins that contribute to foot process formation in differentiated podocytes. Staining experiments revealed that PIK3CA Pod-HET Mice showed a marked loss of podocyte differentiation markers compared to controls (data not shown). Mechanistically, PIK3CA is involved in cell growth and proliferation. We first investigated the proliferation rate in glomeruli of different mouse models using the proliferation marker Ki-67. The number of Ki-67+ cells was low in control mice, whereas the number of Ki-67+ cells was significantly higher in PIK3CA+ mice. Pod-HET The PIK3CA1 expression level was significantly increased in the glomeruli of mice (data not shown). Next, using the Amnis ImageStream® system, the present inventors Pod-HET We confirmed that podocytes isolated from mice were hypertrophic compared to controls (Figure 1M). Flow cytometry experiments showed a correlation between podocyte size and the degree of activation of the AKT / mTOR pathway, notably the mTORC1 pathway (data not shown). These data are consistent with the fact that cells that enter the cell cycle lose their differentiation markers. Indeed, we conclude that overactivation of PIK3CA in podocytes leads to both proliferation and dedifferentiation, thereby resulting in FSGS with progressive proteinuria and renal dysfunction. This mouse model recapitulates the renal phenotype observed in patient 1.

[0137] To explore the allelic dosage effect of PIK3CA pathway hyperactivation, the inventors generated homozygous mutants for PIK3CA in podocytes (hereinafter referred to as mice in this specification). At birth, the PIK3CA PIK3CAPod-HO mice were indistinguishable from controls. However, these mice rapidly developed heavy albuminuria (data not shown) and had a decreased survival rate (data not shown). Histological examination at 12 weeks of age revealed dedifferentiation of podocytes (data not shown) along with severe proliferative glomerulonephritis (data not shown). Consistently, dedifferentiation, AKT / mTOR pathway recruitment, and proliferation were observed in podocytes (data not shown). These findings demonstrate that the cumulative activation of the PIK3CA pathway is associated with a more severe disease phenotype. Pod-HO Hyperactivation of the PIK3CA pathway in podocytes is associated with changes in cell fate determination

[0138] Podocytes are post-mitotic cells with limited potential for proliferation and regeneration. However, under certain pathological conditions, podocytes can re-enter the cell cycle and dedifferentiate (Griffin, S. V., Petermann, A. T., Durvasula, R. V. & Shankland, S. J. Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle coordination protein. Nephrol Dial Transplant 18 Suppl 6, vi8-13(2003)). To identify the early transcriptional changes in podocytes associated with PIK3CA gain-of-function mutations, the inventors performed single-cell RNA sequencing (scRNA-seq) on PIK3CA mice and PIK3CA WT mice and PIK3CA Pod-HETKidney cells from mice were mapped (data not shown). To increase the podocyte population, which accounts for only 0.18% of normal mouse kidneys (Park, J. et al. Single-cell transitomics of the mouse kidney reveals potential cellular targets of kidney disease. Science (New York, N.Y.) 360, 758-763 (2018)), the inventors concentrated glomeruli using magnetic bead injection. Two mice for each condition were pooled to cover phenotypic heterogeneity. A total of 49,600 cells were isolated and sequenced. After data normalization and filtering (data not shown), 26,508 cells were further analyzed. Unsupervised clustering of the pooled full dataset identified 20 clusters (data not shown). Using established cell type markers from the literature, the inventors annotated broad cluster classes (data not shown). The inventors annotated six podocyte clusters based on the expression of Nphs2, Synpo, Podxl, Pdpn, and EGFP (data not shown). The inventors decided to include a part of the clusters with weaker expression of podocyte markers (podocytes 4, -5, and -6), with the aim of understanding different cellular states of podocytes. Monocle single-cell trajectories from the six podocyte clusters resulted in five distinct clusters (data not shown). Podocyte 2, 4, and 5 clusters showed similar pseudo-time series distributions, while podocyte 1 and 3 clusters were different (data not shown). The podocyte 1 cluster was activated for the PI3K-AKT pathway and signaling pathways regulating pluripotency; the podocyte 3 cluster was enriched for inflammatory pathways (data not shown). PIK3CA WT In mice, the podocyte clusters accounted for approximately 10% of the total cells analyzed, while PIK3CA Pod-HET In mice, it increased to 23.7%, demonstrating podocyte proliferation (data not shown). The inventors finally, PIK3CA Pod-HETIt was observed that podocytes in mice showed increased expression of genes known to be upregulated in FSGS, such as metallothionein Mt1 and Mt2, EGF, wnt4, Col1a2, Col4a3, Col4a4, or Il18, etc., which are related to inhibition of apoptosis, dedifferentiation, and proliferation (data not shown). These results indicate that PI3K-AKT pathway activation in podocytes drives the fate conversion of podocytes.

[0139] Alpelisib is PIK3CA Pod Improve the mouse model Patient 1 showed improvement in albuminuria after the introduction of alpelisib. However, during that time, alpelisib was associated with the reduction of vascular malformations and the correction of severe congestive heart failure, adding potential confounding factors. The inventors questioned whether alpelisib could be a treatment option. The inventors first explored whether this drug could diffuse into the kidney. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed that alpelisib was detectable 2 hours after enteral tube feeding (data not shown). The inventors then orally administered alpelisib to 3-month-old PIK3CA Pod-HET mice for 2-3 weeks and observed a gradual decrease in albuminuria (data not shown) accompanied by glomerular inhibition of the AKT / mTOR pathway (data not shown) and a decrease in podocyte size (data not shown).

[0140] Alpelisib-treated PIK3CA Pod-HET Single-cell RNA sequencing (scRNA-seq) analysis of kidney cells from alpelisib-treated PIK3CA Pod-HET mice revealed that the podocyte population recovered up to 11.7% compared to vehicle-treated PIK3CA

[0141] Next, the inventors performed a pilot study by administering either vehicle or alpelisib to 3-month-old PIK3CA Pod-HO mice in which the mice had already established proteinuria. The inventors decided to use PIK3CA Pod-HO mice. This is because this disease is more severe than the heterozygous model. 3-month-old PIK3CA Pod-HO mice were treated for two consecutive weeks (data not shown). During the treatment period, the inventors observed a progressive decrease in albuminuria in the alpelisib-treated mice. The mice were then sacrificed, and histological analysis of the kidneys showed that PIK3CA Pod-HO mice treated with alpelisib had preserved glomeruli compared to PIK3CA Pod-HO mice treated with vehicle (data not shown). The podocyte differentiation marker was still expressed at the protein level in the alpelisib-treated group (data not shown). The AKT / mTOR pathway was blunted in the podocytes of PIK3CA Pod-HO mice (data not shown). Mechanistically, alpelisib treatment was associated with a decrease in glomerular proliferation (Figure 1, and data not shown).

[0142] To better compare the improvement and potential reversibility of kidney lesions, the inventors performed unilateral nephrectomy on 8-week-old PIK3CA Pod-HO mice and controls, treated them with either vehicle or alpelisib, and decided to pair-compare the kidneys at the time of sacrifice (data not shown). After nephrectomy or sham surgery, the mice were randomly treated with either vehicle or alpelisib for two weeks (data not shown). The inventors first observed that uninephrectomized PIK3CA WT mice treated with either vehicle or alpelisib had no special phenotype without proteinuria or kidney dysfunction (Figures 2A, 2B, 2C). However, after surgery, PIK3CA Pod-HOMice rapidly developed a large amount of proteinuria with severe glomerular lesions (Figures 2A, 2B, 2C, 2D). However, the amount of proteinuria was significantly reduced in the nephrectomized PIK3CA Pod-HO mice treated with alpelisib (Figures 2A and 2E). Consistently, compared to vehicle-treated mice, PIK3CA Pod-HO mice that received alpelisib had better kidney function (Figures 2B and 2C), preserved glomeruli (Figure 2D), less tubular dilation (data not shown), higher expression of podocyte markers (data not shown), and reduced podocyte AKT / mTOR pathway activation (data not shown). The inventors concluded that alpelisib can prevent and reverse the disease in a mouse model with a PIK3CA gain-of-function mutation in podocytes.

[0143] Alpelisib improves different mouse models of proliferative glomerulonephritis PIK3CA Pod The mouse model developed features of collapsing glomerulopathy and extracapillary lesions. In humans, collapsing glomerulopathy can be either primary or secondary to other diseases such as HIV or toxic exposure. Extracapillary disease is characteristic of severe autoimmune diseases that affect the kidney, such as lupus nephritis. Both types of glomerular lesions are associated with severe kidney injury and poor kidney survival.

[0144] Crescentic glomerulonephritis is characteristic of severe autoimmune diseases that affect the kidney, such as lupus nephritis. Both types of glomerular lesions are associated with significant kidney damage and poor kidney survival. To further investigate whether the AKT / mTOR pathway is activated in the glomerular lesions of patients with these disorders, the inventors performed immunofluorescence experiments. These experiments confirmed the activation of the AKT / mTOR pathway specifically in glomerular epithelial cells (data not shown), compared to kidney biopsies from individuals with other diseases. PIK3CA Pod-HETo compare the results obtained from single-cell RNA sequencing in a mouse model, the inventors further examined the spatial transcriptome changes in glomeruli in patients with extracapillary lesions using GeoMx™ Digital Spatial Profiler technology. Kidney biopsies from four patients with severe active lupus nephritis (LN) and four patients with minimal change disease (MCD) were selected for analysis (data not shown). A total of 49 glomeruli were selected as the regions of interest (ROIs) (33 ROIs for LN and 16 ROIs for MCD). More than 18,000 genes were analyzed in these ROIs (data not shown). The glomerular transcriptome of LN samples showed distinct, broader clustering patterns compared to those of MCD samples (data not shown). PIK3CA Pod-HET Consistent with single-cell RNA sequencing analysis in mice, the gene expression profiles in LN glomeruli showed a dedifferentiated state and elevated levels of inflammation-related transcripts (data not shown). The inventors stained for pAKT Ser473 separately in LN samples, classified each ROI based on the level of pAKT activity, classifying 20 ROIs as highly active and 13 ROIs as lowly active (data not shown). Higher pAKT activity correlated with gene set enrichment of the WNT pathway and significant upregulation of WNT4 (data not shown). Indeed, PIK3CA Po-HET mutant mice showed transcriptome changes closely resembling the pattern observed in LN patients.

[0145] The inventors wondered whether alpelisib could be a target in such glomerulonephritis where limited therapeutic agents are available. The inventors initiated by exploring the degree of AKT / mTOR pathway activation in Tg26 HE mice, a model of collapsing glomerulopathy. This transgenic mouse model typically develops proteinuria with severe kidney lesions by about 24 days of age and dies between 2 and 9 months. Four-week-old Tg26 mice (n = 45) were treated with vehicle (Tg26 WTn = 9, Tg26 HE n = 14) or alpelisib (Tg26 WT n = 8, Tg26 HE n = 14) were randomly assigned (data not shown). Tg26 mice treated with alpelisib HE showed lower albuminuria (data not shown), a decrease in kidney volume (data not shown), less severe glomerular lesions (data not shown), higher expression of podocyte differentiation markers (data not shown), decreased expression of fibrosis markers Col1a and Col3a (data not shown), as well as urinary tubular injury markers Lcn2 and KIM1 (data not shown), and lower expression of the inflammatory marker Tnfa (data not shown) compared to vehicle-treated mice. Mechanistically, alpelisib was associated with significant AKT / mTOR pathway inhibition (data not shown). However, kidney function did not improve significantly (data not shown). Although the results were actually promising, in order to overcome the variability of the model, the inventors decided to repeat the experiment and perform unilateral nephrectomy before treatment. This could accelerate kidney lesions and enable comparison of the contralateral kidneys. Four-week-old Tg26 HE mice and Tg26 WT mice underwent unilateral nephrectomy and then, for four weeks, were randomly assigned to vehicle (Tg26 WT n = 8, Tg26 HE n = 8) or alpelisib (Tg26 WT n = 8, Tg26 HE n = 9) treatment (data not shown). At sacrifice, the inventors observed that the surface of the kidneys of unilateral nephrectomy Tg26 WT or Tg26 treated with alpelisib HE was regular and smooth, while the surface of the kidneys of Tg26 mice treated with vehicle HE was irregular (data not shown). Albuminuria improved significantly in Tg26 mice treated with alpelisib compared to vehicle treatment (data not shown). The kidney / body weight ratio was lower in uninephrectomized Tg26 treated with alpelisib HE ​HE Improved in mice (data not shown). By kidney histology, in the uninephrectomized Tg26 mice treated with apelacib HE showed less glomerular lesions (data not shown), maintained the expression of podocyte markers (data not shown), and had reduced expression in Col1a, Col3a, and Lcn2 (data not shown). The AKT / mTOR pathway in glomeruli was significantly blunted in mice treated with apelacib (data not shown). Finally, kidney function was significantly improved in Tg26 mice treated with apelacib compared to vehicle treatment (Figure 3). HE mice (Figure 3).

[0146] To better understand the role of PIK3CA in podocytes during collapsing glomerulopathy, the inventors decided to genetically remove PIK3CA specifically in these cells. To achieve this, the inventors first mated Podocin Cre mice with PIK3CAlox / lox to generate Podo-KO these mice. These mice were then backcrossed with the FVB / N strain for 10 generations and mated with Tg26 mice to obtain PIK3CA-KO these mice. These mice were viable and had no special phenotype at birth. With aging, Tg26 PIK3CA-KO developed less albuminuria, glomerular lesions, renal fibrosis, and improved kidney function compared to Tg26 HE (data not shown). Indeed, deletion of PIK3CA in podocytes is sufficient to reduce the severity of collapsing glomerular lesions in the Tg26 HE mouse model. Next, the inventors attempted to determine whether removal of PIK3CA in podocytes in the later stages of life, when glomerular lesions are already established, could lead to improvement in kidney lesions. For this purpose, the inventors generated Tg26 HE PIK3CA iPodo-KO mice by mating tamoxifen-inducible Podocin Cre mice with PIK3CA in the FVB / N strain lox / loxBackcrossed the mice (10th generation) and mated them with Tg26 HE mice. Induced Cre recombination in Tg26 HE PIK3CA iPodo-KO mice at 3 weeks of age and sacrificed the mice at 12 weeks of age. The inventors observed that these mice showed improved glomerular lesion scores and reduced albuminuria compared to the control group (data not shown). Similarly, when Cre recombination was induced at 6 weeks of age, the kidney lesions reversed by 12 weeks of age (data not shown). However, when Cre recombination was induced at a later time point, specifically at 8 weeks of age, the disease progression was no longer affected, indicating that once the kidney lesions become too severe, it becomes difficult to reverse the phenotype (data not shown). In this model of collapsing glomerulopathy, the inventors determined that activation of PIK3CA in podocytes is significantly important and that inhibiting it holds great promise as a potential therapeutic approach.

[0147] The inventors then decided to examine the effect of PIK3CA inhibition in extracapillary glomerulonephritis, particularly in the lupus mouse model. The inventors first started with NZBWF1 / J mice, an established model of lupus-like nephritis. NZBWF1 / J mice are autoimmune and develop progressive and complex immune glomerulonephritis characterized by proteinuria and kidney dysfunction that starts around 25 weeks of age. However, this model is characterized by its variability. To accelerate and homogenize the lesions, the inventors performed unilateral nephrectomy in 30 female mice at 24 weeks of age (data not shown). Notably, the incidence and severity of symptoms were more prominent in females, and the inventors used only females for this study. The mice were then randomly assigned to either vehicle (n = 15) or alpelisib (n = 15) for 4 weeks. At the end of the treatment period, the mice were sacrificed and their kidney histology was compared to that of one removed during unilateral nephrectomy. At the time of unilateral nephrectomy, there were no phenotypic differences including proteinuria and kidney histology between the two groups (data not shown). However, at the time of sacrifice, alpelisib-treated mice showed significantly less albuminuria (Figure 4A) and lower blood urea nitrogen levels (Figure 4B). The kidney-to-body weight ratio was significantly decreased in uninephrectomized NZBWF1 / J mice treated with alpelisib (Figure 4C). At the histological level, alpelisib-treated mice had preserved glomeruli compared to vehicle (data not shown). Glomerular lesions progressed severely in vehicle-treated uninephrectomized NZBWF1 / mice, but they remained stable in the alpelisib group (Figure 4D). Glomerular AKT / mTOR pathway activation was blunted with alpelisib (Figures 4D and 4E). Col1a and Col3a mRNA expression was decreased in mice treated with alpelisib (Figures 4F and 4G) as well as TNFa (Figure 4H). Podocyte differentiation markers were significantly improved in the group of uninephrectomized NZBWF1 / J mice treated with alpelisib (data not shown). In NZBWF1 / J mice treated with alpelisib, the inventors observed partial correction of anemia and a significant increase in platelet count.The deposition of IgG, IgM, and C3 in glomeruli significantly decreased accordingly in mice receiving alpelisib (data not shown). The inventors next checked the level of dsDNA, an activity marker of SLE, and observed a tendency of decrease in NZBWF1 / J mice treated with alpelisib (data not shown). Notably, the spleen size significantly decreased in both sham-operated mice and uninephrectomized mice treated with alpelisib (data not shown).

[0148] The inventors next decided to explore the relevance of alpelisib in MRL / MpJ-Faslpr / J mice (referred to herein as MRL-lpr), another mouse model of lupus nephritis. These mice with homozygous Fas mutations usually develop an autoimmune disease similar to systemic lupus erythematosus with lymph node hyperplasia, progressive renal insufficiency, and skin lesions. Female MRL-Ipr mice die at an average age of 18 - 20 weeks. The inventors first randomly assigned MRL-lpr female mice at 8 weeks of age, prior to the full development of the disease, to either vehicle or alpelisib. The inventors confirmed that the mice already had albuminuria prior to drug administration. Alpelisib was associated with a significant extension of the mean lifespan (Figure 4I). MRL-lpr mice treated with alpelisib showed less proteinuria compared to the vehicle (Figure 4J). Even more strikingly, a reverse trajectory was shown by comparing albuminuria before and after the introduction of treatment. Indeed, MRL-lpr mice treated with alpelisib showed an improvement in proteinuria, which is relevant to the reversibility of the disease (Figures 4K and 4L). At the time of sacrifice, MRL-lpr mice treated with alpelisib tended to have a lower kidney-to-body weight ratio compared to vehicle-treated animals (data not shown). Kidney examination showed that alpelisib was associated with the absence of glomerular lesions (data not shown) and improved kidney function (data not shown) compared to vehicle-treated mice.

[0149] dsDNA in MRL-lpr mice increases over time (data not shown). To further demonstrate the potential reversibility of glomerular lesions with alpelisib, the inventors decided to randomly assign 12-week-old MRL-lpr female mice to either vehicle or alpelisib. At this age, kidney lesions are already present. The mice were then treated for 4 consecutive weeks (data not shown). Alpelisib was again associated with a decrease in proteinuria (data not shown). At the histological level, the inventors found that the glomerular lesion score improved in the alpelisib group, as well as Cola1 and Cola3 mRNA expression (data not shown). Consistently, the glomerular AKT / mTOR pathway was inhibited in the group of mice treated with alpelisib (data not shown). The expression of podocyte differentiation markers increased in alpelisib mice compared to vehicle (data not shown). Also, dsDNA antibodies were not different between the two groups at the start of treatment but decreased significantly with alpelisib treatment (data not shown).

[0150] The inventors concluded that alpelisib, more generally PIK3CA inhibition, represents a promising drug for patients with FSGS, lupus nephritis, and more generally proliferative glomerulonephritis.

[0151] Example 2: Materials and Methods To accelerate and exacerbate kidney lesions, unilateral nephrectomy was performed in NZB / NZBW F1 mice presenting with lupus with kidney injury (methodology described above). These mice were treated with different compounds: BYL719 (alpelisib, Novartis), GDC-0077 (inavolisib, Genentech / Roche), TAK-117 / MLN1117 / INK1117 (serabelisib), GDC-0032 (taselisib, Genentech / Roche), or rapamycin.

[0152] Results The results are depicted in FIGS. 5A, 5B, and 5C, demonstrating that PIK3CA inhibitors represent a promising drug for patients with proliferative glomerulonephritis.

[0153] References: Throughout this application, various references are cited that describe the state of the art to which the present invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [Table 1]

Claims

1. A pharmaceutical composition comprising a PIK3CA inhibitor for use in a method for treating proliferative glomerulonephritis in a subject requiring such treatment, comprising the step of administering a therapeutically effective amount of the PIK3CA inhibitor to the subject.

2. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the method of claim 1, which is essentially present in the step of administering a therapeutically effective amount of the PIK3CA inhibitor to a target.

3. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the method of claim 1 or 2, wherein the proliferative glomerulonephritis is caused by a disease selected from the group consisting of, but not limited to, infectious diseases (post-streptococcal glomerulonephritis, infectious endocarditis, latent visceral sepsis, hepatitis B infection with vasculitis and / or cryoglobulinemia, HIV infection, hepatitis C with cryoglobulinemia, membranoproliferative glomerulonephritis), and multi-organ diseases (including systemic lupus erythematosus, IgA nephropathy, Henoch-Schönlein purpura, systemic necrotizing vasculitis - including granulomatosis with Wegener's polyangiitis, Goodpasture syndrome, essential mixed cryoglobulinemia, malignant tumors, relapsing polychondritis, and rheumatoid arthritis with vasculitis).

4. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the method according to claim 1 or 2, wherein the proliferative glomerulonephritis is lupus nephritis.

5. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the method according to claim 1 or 2, wherein the proliferative glomerulonephritis is focal segmental glomerulosclerosis.

6. A pharmaceutical composition for use in the method of claim 1 or 2, comprising a PIK3CA inhibitor selected from the group consisting of, but not limited to, BYL719 (alpelisib, Novartis), A66 (University of Auckland), GDC-0077 (inavolisib, Genentech / Roche), CYH33 (lysovalisib), TAK-117 / MLN1117 / INK1117 (cerabelisib) or pharmaceutically acceptable salts thereof.

7. A pharmaceutical composition for use in the method according to claim 1 or 2, wherein the PIK3CA inhibitor is selected from the group consisting of BYL719 (alpelisib, Novartis), GDC-0077 (inavolisib, Genentech / Roche), TAK-117 / MLN1117 / INK1117 (cerabelisib) or pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the method according to claim 1 or 2, wherein the PIK3CA inhibitor is BYL719 and its derivatives.

9. PIK3CA inhibitors and ii) classical treatments as combined formulations for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis.

10. The combination formulation according to claim 9, wherein the aforementioned classical treatment is selected from the group consisting of, but not limited to, immunosuppressants, glucocorticoids, MAPK, PAK, mTOR, TKI, PARP and / or EGFR inhibitors.

11. A pharmaceutical composition comprising a PIK3CA inhibitor for use in the treatment of proliferative glomerulonephritis.

12. The pharmaceutical composition according to claim 11, wherein the proliferative glomerulonephritis is lupus nephritis.

13. A pharmaceutical composition comprising a PIK3CA inhibitor and a classic treatment as a combined formulation for simultaneous, separate, or sequential use in the treatment of proliferative glomerulonephritis.

14. A method for screening PIK3CA inhibitors, comprising i) providing a test compound, and ii) determining the ability of the test compound to inhibit and / or reduce the activity and / or expression of PIK3CA.

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