Minimal nephrin promoter

Abstract

(i) A promoter comprising a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17, the promoter having a length of about 1.1 kb or less.

Description

[Technical field]

[0001] The present invention relates to promoters capable of driving the expression of transgenes in podocytes. The present invention also relates to polynucleotides and vectors comprising said promoters. [Background technology]

[0002] There are many diseases that affect kidney function by attacking the glomerulus. The glomerulus filters approximately 180 liters of plasma every day, and the healthy glomerular filtration barrier has the remarkable ability to retain approximately 99.9% of large proteins, including albumin, without blockage throughout our lifetime. The glomerular filtration barrier (GFB) includes three main layers: glomerular endothelial cells, glomerular basement membrane (GBM), and podocytes.

[0003] GBM is made up of a highly cross-linked macromolecular meshwork of type IV collagen, proteoglycans and laminin. Hereditary forms of glomerular disease can be caused by genetic defects in these molecular structures. For example, Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, resulting in abnormalities in the collagen IV α345 network of the basement membrane. Alport syndrome affects approximately 1 in every 5,000 to 10,000 individuals in continental Europe and the United States. The condition usually develops during childhood and is associated with a variety of phenotypes, including progressive loss of kidney function, and may also include hearing loss and eye abnormalities. Other GBM-related disorders include Pearson syndrome and Nail-Patella syndrome (Chiang, CK and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0004] Podocytes have also been implicated as key cells in the progression of glomerular diseases. Podocytes are highly differentiated mesoderm-derived cells found only in renal glomeruli. They exhibit unique features that are important for glomerular filtration, such as foot processes and slit septa. Inherited glomerular diseases associated with podocytes include nephrotic syndrome, Fraser and Dennis-Drash syndromes, Schimke immune osteogenesis imperfecta, and Epstein and Fechtner syndromes (Chiang, CK and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0005] Therefore, glomerular cells, such as podocytes, are potential targets for gene therapy approaches.

[0006] To maximize the potential of gene therapy, a promoter sequence is required that can drive the expression of transgenes in glomerular cells, such as podocytes. Wong et al. (American Journal of Physiology Renal Physiology; 2000; 279(6); F1027-32) described a 1.25kb DNA fragment from the human nephrin promoter and 5' flanking region that can induce podocyte-specific expression. This promoter has been used to achieve kidney-specific expression of GFP using AAV9 vectors (Picconi et al.; 2014; Molecular Therapy - Methods & Clinical Development; 1, 14014). Summary of the Invention [Problem to be solved by the invention]

[0007] Currently, a significant amount of vector cargo capacity (e.g., AAV cargo capacity) is accounted for by non-coding elements, such as nephrin or podocin promoter, WPRE element and polyadenylation sequence.Therefore, there is a need in the art for a minimal promoter that can drive transgene expression in glomerular cells, such as podocytes, to provide maximum flexibility for gene therapy approaches to glomerular diseases. [Means for solving the problem]

[0008] The present invention is based on the inventors' surprising provision of a minimal promoter capable of driving the expression of a transgene in kidney cells, in particular glomerular cells, such as podocytes.

[0009] In one aspect, the invention provides (i) a promoter comprising a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4 or 17, the promoter having a length of about 1.1 kb or less.

[0010] In some embodiments, the present invention relates to a nucleic acid sequence as set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, where n1 is an integer from 1 to 430, and / or (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3≧n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061; nucleotide sequence, or a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to said nucleotide sequence. The present invention provides a promoter comprising or consisting of a nucleotide sequence thereof.

[0011] In some embodiments, (iii) the position corresponding to position 493 of SEQ ID NO:1 is G, the position corresponding to position 1080 of SEQ ID NO:1 is T, the position corresponding to position 1169 of SEQ ID NO:1 is C, and the position corresponding to position 1249 of SEQ ID NO:1 is G.

[0012] In one aspect, the present invention relates to a method for the preparation of a nucleic acid sequence as set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, where n1 is an integer between 100 and 430, and / or (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3≧n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061; Promoters are provided that comprise or consist of nucleotide sequences having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to the nucleotide sequence.

[0013] In some embodiments, (iii) position 493 of SEQ ID NO:1 is substituted with G, position 1080 of SEQ ID NO:1 is substituted with T, position 1169 of SEQ ID NO:1 is substituted with C, and position 1249 of SEQ ID NO:1 is substituted with G.

[0014] The promoter may (i) comprise a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4 or 17, and / or the promoter may have a length of about 1.1 kb or less.

[0015] The promoter may function in kidney cells. The promoter may be capable of driving expression of a transgene in the kidney. The promoter may be a kidney-specific promoter. The promoter may function in podocytes. The promoter may be capable of driving expression of a transgene in podocytes. The promoter may be a podocyte-specific promoter.

[0016] Suitably, the promoter further comprises (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 5 or 18, a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 6 or 19, and / or a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 7 or 20. In some embodiments, the promoter further comprises (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 6 or 19.

[0017] Suitably, the promoter further comprises (iii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:8 or one or more fragments thereof.

[0018] Suitably, the promoter according to any preceding claim comprises, from 5' to 3': (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4 or 17; (iii) optionally a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 8 or one or more fragments thereof; and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 5 or 18; a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 6 or 19; and / or a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 7 or 20.

[0019] In some embodiments, the promoter comprises, from 5' to 3', (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:4, and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:7. In some embodiments, the promoter is composed of, from 5' to 3', (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:4, and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:7, and the promoter has a length of about 1.1 kb or less.

[0020] In some embodiments, the promoter is comprised, from 5' to 3', of (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 17, and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20. In some embodiments, the promoter is comprised, from 5' to 3', of (i) the nucleotide sequence of SEQ ID NO: 17, and (ii) the nucleotide sequence of SEQ ID NO: 20.

[0021] In some embodiments, the promoter comprises, from 5' to 3', (i) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 17, and (ii) a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20, and the promoter has a length of about 1.1 kb or less. In some embodiments, the promoter comprises, from 5' to 3', (i) the nucleotide sequence of SEQ ID NO: 17, and (ii) the nucleotide sequence of SEQ ID NO: 20, and the promoter has a length of about 1.1 kb or less.

[0022] The promoter may have a length of about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. The promoter may have a length of 0.265 kb to 1.0 kb, 0.265 kb to 0.9 kb, 0.265 kb to 0.8 kb, 0.265 kb to 0.7 kb, 0.265 kb to 0.6 kb, 0.265 kb to 0.5 kb, 0.265 kb to 0.4 kb, or 0.265 kb to 0.3 kb.

[0023] The promoter may comprise (a) a retinoic acid receptor binding site, (b) a WT1 binding site, (c) an enhancer box, (d) a transcription factor binding region, and / or (e) a transcription initiation site.Preferably, the retinoic acid receptor binding site comprises or consists of the nucleotide sequence shown as SEQ ID NO: 10 or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO: 10.Preferably, the WT1 binding site comprises or consists of the nucleotide sequence shown as SEQ ID NO: 11 or a nucleotide sequence having one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 11.Preferably, the enhancer box comprises or consists of the nucleotide sequence shown as SEQ ID NO: 12 or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO: 12. Preferably, the transcription factor binding region comprises or consists of the nucleotide sequence shown as SEQ ID NO: 13 or 43 or a nucleotide sequence having one, two, three, four or five substitutions, deletions or insertions compared to SEQ ID NO: 13 or 43. Preferably, the transcription start site comprises or consists of an "AG" dinucleotide. Preferably, the transcription factor binding site is operably linked to the transcription start site, and optionally the transcription factor binding site is immediately (directly) upstream of the transcription start site.

[0024] The nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4 or 17 may contain one or more of: (a) a retinoic acid receptor binding site, (b) a WT1 binding site, and (c) an enhancer box. One or more of the following nucleotide sequences: (a) GGGGTCA at positions corresponding to positions 7 to 13 of SEQ ID NO: 4 or 17, (b) CGGAGGCTGGGGAGGCA at positions corresponding to positions 14 to 30 of SEQ ID NO: 4 or 17, and (c) ATGTG at positions corresponding to positions 49 to 53 of SEQ ID NO: 4 or 17 may be present in the nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO: 4 or 17.

[0025] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0026] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:15, SEQ ID NO:59 or SEQ ID NO:47.

[0027] In a preferred embodiment, the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 47. In a more preferred embodiment, the promoter comprises or consists of the nucleotide sequence of SEQ ID NO: 47.

[0028] In one aspect, the invention provides a promoter comprised of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:2.

[0029] In one aspect, the invention provides a promoter comprised of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:3.

[0030] In one aspect, the invention provides a promoter comprised of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:15.

[0031] In one aspect, the invention provides a promoter comprised of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity to SEQ ID NO:59.

[0032] In one aspect, the invention provides a promoter comprised of a nucleotide sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:47.

[0033] In one aspect, the invention provides a promoter consisting of the nucleotide sequence of SEQ ID NO:47.

[0034] In one aspect, the invention provides a polynucleotide comprising a promoter according to the invention.

[0035] The promoter may be operably linked to a protein coding sequence, which in some embodiments encodes NPHS2 or a fragment and / or variant thereof, a COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof, or CFI, CFH or FHL-1 or a fragment and / or variant thereof.

[0036] In some embodiments, the protein coding sequence encodes NPHS2 or a fragment and / or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:55 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:56 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:57 or a fragment thereof.

[0037] In some embodiments, the protein coding sequence encodes a COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs:21-23 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to one of SEQ ID NOs:24-26 or a fragment thereof.

[0038] In some embodiments, the protein coding sequence encodes CFI or a fragment and / or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:49 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:50 or a fragment thereof.

[0039] In some embodiments, the protein coding sequence encodes CFH or a fragment and / or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:51 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:52 or a fragment thereof.

[0040] In some embodiments, the protein coding sequence encodes FHL-1 or a fragment and / or variant thereof. In some embodiments, the protein coding sequence encodes an amino acid sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:53 or a fragment thereof. In some embodiments, the protein coding sequence comprises or consists of a nucleotide sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO:54 or a fragment thereof.

[0041] In some embodiments, the protein coding sequence is operably linked to one or more additional regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences.

[0042] In one aspect, the present invention provides a vector comprising a polynucleotide according to the present invention. Suitably, the vector is capable of transducing podocytes, optionally the vector is capable of specifically transducing podocytes.

[0043] Advantageously, vector is virus vector, for example, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, alphavirus vector, flavivirus vector, rhabdovirus vector, measles virus vector, Newcastle disease virus vector, poxvirus vector and picornavirus vector, preferably, vector is AAV vector.Advantageously, said virus vector is in the form of virus vector particle, preferably, said virus vector is in the form of AAV vector particle.

[0044] In some embodiments, the vector is in the form of an AAV vector particle encapsidated by AAV3B, LK03, or AAV9 capsid proteins.

[0045] In one aspect, the present invention provides a cell comprising a polynucleotide according to the invention or a vector according to the invention.

[0046] In one aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide according to the invention, a vector according to the invention or a cell according to the invention.

[0047] In one aspect, the present invention provides a polynucleotide according to the invention, a vector according to the invention or a cell according to the invention for use in medicine.

[0048] In one aspect, the invention provides the use of a promoter according to the invention to drive expression of a coding sequence. The expression may be kidney-specific. Suitably, the expression is podocyte-specific. [Brief description of the drawings]

[0049] [Figure 1]Schematic diagram of a minimal nephrin promoter. (A) An exemplary full-length nephrin promoter may be 1249 bp long (excluding the start codon) and is hereinafter referred to as the "FL" nephrin promoter. (B) An exemplary minimal nephrin promoter with a deleted 5' region may be 819 bp long (excluding the start codon) and is hereinafter referred to as the "midi" nephrin promoter. (C) An exemplary minimal nephrin promoter with a deleted 5' region and a deleted mid-region may be 265 bp long (excluding the start codon) and is hereinafter referred to as the "mini" nephrin promoter. (D) The following regions of the nephrin promoter are shown: (i) human mouse homology region, (ii) retinoic acid receptor (RAR) binding site, (iii) WT1 binding site, (iv) enhancer box, (v) transcription factor binding region, and (vi) transcription start site. [Figure 2A] Schematic diagram of lentiviral vector containing GFP operably linked to the midi nephrin promoter. (A) The pACE_hNPHS1 promoter was used as a template to introduce BamH1 and Cla1 restriction sites. (B) The final constructed vector containing GFP operably linked to the midi nephrin promoter. [Figure 2B] This is a continuation of Figure 2A. [Figure 3A] Schematic diagram of lentiviral vector containing GFP operably linked to mini-nephrin promoter. (A) pACE_hNPHS1 promoter was used as template for PCR and two sections of the promoter were gel extracted. (B) Final construction vector containing GFP operably linked to mini-nephrin promoter. [Figure 3B] This is a continuation of Figure 3A. [Figure 4]Figure 1 shows expression of GFP in CiPodocytes after transduction with lentiviral vector. Human CiPodocytes stably expressing GFP-tagged nephrin promoter were generated using a lentiviral approach. GFP expression was observed by fluorescence microscopy. (A) shows non-transduced CiPodocytes. (B) shows CiPodocytes stably expressing GFP-tagged mini-nephrin promoter. (C) shows CiPodocytes stably expressing GFP-tagged FL-nephrin promoter. [Diagram 5] Figure 1 shows expression of GFP in differentiated ciPodocytes after transduction with lentiviral vector. Differentiated conditionally immortalized podocytes (ciPodocytes) were transduced with a lentiviral vector containing GFP operably linked to the nephrin promoter. GFP expression was detected using immunoprecipitation (IP). [Figure 6] Human glomerular cells transduced with lentivirus-GFP.Nephrin promoter (FL or 265). FACS analysis showing median GFP fluorescence (AFU) of all surviving singlets of conditionally immortalized human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte analyzer is shown. Non-transduced cells (cell control) were compared to cells transduced with lentiviral constructs carrying a GFP expression cassette controlled by the full-length human nephrin promoter (hNPHS1.GFP) or the mini human nephrin promoter (265.GFP). All cells were differentiated for 10 days, trypsinized (100uL) and diluted 1:1000 in PBS, 2% FBS, DRAQ7 (150uL). Data and error bars represent 3 technical replicates (100uL, >2500 cells) ± SEM. [Figure 7-1]1 shows AAV transfer plasmids containing COL4A3, COL4A4 and COL4A5 linked to the mini-nephrin promoter. (A) is a schematic diagram of pAAV.265.Col4a3.3flag.sv40, an AAV plasmid containing COL4A3 linked to the mini-nephrin promoter. The SmaI site is indicated, and the following fragments are expected after restriction with SmaI: 1.6238bp, 2.2753bp, 3.56bp, 4.11bp, 5.11bp. (B) is a schematic diagram of pAAV.265.Col4a4.3flag.sv40, an AAV plasmid containing COL4A4 linked to the mini-nephrin promoter. The SmaI site is indicated, and the following fragments are expected after restriction with SmaI: 1.4052bp, 2.2753bp, 3.2224bp, 4.56bp, 5.11bp, 6.11bp. (C) is a schematic diagram of pAAV.265.Col4a5.3flag.sv40, an AAV plasmid containing COL4A5 linked to the mini-nephrin promoter. The SmaI site is indicated, and the following fragments are expected after restriction with SmaI: 1.4272 bp, 2.2753 bp, 3.2032 bp, 4.56 bp, 5.11 bp, 6.11 bp. (D) is a diagram showing restriction digestion with SmaI. The MW=1 Kb DNA ladder, lanes 1, 2 and 3, correspond to digestion of the plasmids shown in (A), (B) and (C), respectively. (E) is a schematic diagram showing the restriction digestion. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8-1]Podocytes transduced with AAV.COL4.Nephrin265.Sv40 virus. (A) Immunoprecipitation experiments of full-length FLAG-tagged Col4a3 (LK03) or Col4a5 (LK03) in human differentiated CiPodocytes (conditionally immortalized) pulled down with anti-FLAG antibody. Anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human FLAG IgG was used as a control. (B) Western blot of protein lysates showing expression levels of Col4a3 (LK03 capsid serotype), Col4a5 (LK03) and Col4a5 (2 / 9 capsid serotype) in human or mouse differentiated CiPodocytes. Uninfected human and mouse CiPodocytes were used as controls. (C) Confocal images showing immunofluorescence staining of transduced Col4a5 in human wild-type CiPodocytes / Col4a5 3xFlag AAV CiPodocytes with F-actin. Col4a5 is present at the cytosolic level in human differentiated podocytes infected with Col4a5 3xFlag AAV virus compared to their wild-type counterparts. [Figure 8-2] This is a continuation of Figure 8-1. [Figure 9] Figure 9 shows the expression of CFH, CFI and CFHL1 in HEK cells. Protein expression analysis by Western blot in 293T human embryonic kidney cells is shown. Cells were transfected with pAAV-265-CFH, pAAV-FL-CFI or pAAV-FL-CFHL1. NT (non-transfected 293T HEK cells). Expression of each of the transgenes is demonstrated in cell lysates and / or medium using protein-specific antibodies or anti-FLAG tag or anti-MYC tag antibodies. HEK293T cells transfected with a plasmid expressing the CFH transgene under the control of the 265 bp minimal nephrin promoter expressed and secreted human factor H. [Figure 10-1]Transduction of factor H mutant podocytes ("human early disease (ED) podocytes") with AAV2 / 9 265-CFH or transfection with a plasmid encoding CFH. (A) Analysis of human factor H concentrations using an ELISA assay. Podocytes transduced with AAV2 / 9 virus containing the CFH transgene exhibited higher concentrations of human factor H in the culture medium than non-transduced controls. (B) Average human factor H concentrations from (A). (C) Analysis of human factor H concentrations using an ELISA assay. Podocytes transfected with a plasmid expressing the CFH transgene under the control of the 265 bp minimal nephrin promoter exhibited higher concentrations of human factor H than non-transfected controls. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 11-1] FIG. 11 shows expression of CFH in the kidneys of WT mice. Gene expression profiles showing increased (A) viral ITRs, (B) human CFH cDNA, and (C) viral particles in wild-type mice injected with pAAV.NPHS1(265).hCFH.WPRE.bGH compared to mice injected with saline control. (D) Immunofluorescence imaging of kidney sections from wild-type C57BL6 mice injected with saline control (m1) or AAV gene therapy product (pAAV.NPHS1(265).hCFH.WPRE.bGH, n=3) (m2 / m3 / m4). Arrows indicate colocalization of nephrin and CFH to mouse glomeruli. Zoom=20x. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 12]Figure 1 shows expression of GFP using 265bp v 818bp v FL minimal nephrin promoter (plasmid transfection) in kidney cells. Shown is expression of eGFP in cells transfected with plasmids containing full length (PS0281), 818bp (PS0301) and 265bp (PS0282) minimal nephrin promoter driving eGFP. (A) shows transfection results in HEK293T cells. (B) shows transfection results in proximal duct epithelial cells (PTEC). (C) shows transfection results in human podocytes. (D) shows transfection results in glomerular endothelial cells (GENC). [Figure 13] Figure 1 shows expression of GFP using 265bp v FL minimal promoter (AAV transduction) in kidney cells. Shown is expression of eGFP in cells transduced with AAV particles incorporating transgene cassettes containing full length (PS0281) and 265bp (PS0282) minimal nephrin promoter driving eGFP. (A) shows transduction results in human podocytes. (B) shows transduction results in proximal duct epithelial cells (PTEC). (C) shows transduction results in glomerular endothelial cells (GENC). (D) shows transduction results in mesangial cells. [Figure 14]Expression of human podocin using the 265bp v FL minimal nephrin promoter. (A) Protein expression analysis by FAC in Lenti-X293T human embryonic kidney cells. Cells were transfected with 3ug of plasmid with PEI at a 1:3 ratio. Each plasmid expresses human podocin-HA driven by either the full-length (FL.NPSH1-podocin-HA) or 265bp (265.hNPSH1-podocin-HA) minimal nephrin promoter. Expression of human podocin is demonstrated by anti-HA antibody 2 days after transfection. Data from N=3 are presented as mean ± SD. (B) Protein expression analysis by FAC in human podocytes. Cells were transduced with AAV particles incorporating a human podocin-HA transgene cassette driven by either the full-length (FL.NPSH1-podocin-HA) or 265 bp (265.hNPSH1-podocin-HA) minimal nephrin promoter. Expression of human podocin is demonstrated by anti-HA antibody 9 days after transduction and differentiation. [Figure 15] Figure 1. In vivo expression of GFP using the 265bp v FL minimal nephrin promoter by AAV transduction. Evaluation of the 265- and FL-nephrin promoter in vivo in WT mice (C57 / Bl6) is shown. AAV2 / 9 particles expressing GFP under the control of either the full-length (FL-GFP) or the 265bp (265-GFP) minimal nephrin promoter were generated. Tail vein injection of approximately 1x10e13 AAV2 / 9 per mouse (2x mice and 1x untreated mouse per treatment group). Kidney tissue harvest was performed 4 weeks after injection. qPCR analysis was performed and GFP copy numbers were normalized to 18S (housekeeping gene). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] Detailed Description Various preferred features and embodiments of the invention are described below by way of non-limiting examples.

[0051] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0053] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0054] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, all nucleic acid sequences are written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino-terminus to carboxy-terminus orientation, respectively.

[0055] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology which are within the capabilities of those skilled in the art and such techniques are explained in the literature. See, e.g., Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13, and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM, and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM, and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis. of DNA, Academic Press. Each of these general documents is incorporated herein by reference.

[0056] promoter The present invention provides a promoter for glomerular gene therapy.

[0057] "Promoter" is used herein according to its typical meaning to refer to a sequence of DNA that controls and / or initiates transcription of RNA from a DNA sequence.

[0058] Suitably, the promoter may function in a mammalian cell, such as a human cell. The promoter may be capable of driving expression of a transgene in a mammalian cell, such as a human cell. The promoter may be a mammalian promoter, such as a human promoter.

[0059] The promoter may function in kidney cells. The promoter of the present invention may be capable of driving expression of a transgene in the kidney. Examples of kidney cells in which the promoter may function include, but are not limited to, glomerular cells.

[0060] The promoter can function in glomerular cells. The promoter of the present invention can be capable of driving the expression of transgenes in glomeruli. Mature glomeruli contain four cell types: parietal epithelial cells that form Bowman's capsule, podocytes that cover the outermost layer of the glomerular filtration barrier, fenestrated endothelial cells that are covered with glycocalyx and in direct contact with blood, and mesangial cells that are located between capillary loops (Vaughan, MR and Quaggin, SE, 2008. Journal of the American Society of Nephrology, 19(1), pp.24-33).

[0061] The promoter may function in podocytes. The promoter of the present invention may be capable of driving expression of a transgene in podocytes.

[0062] The promoter of the present invention may be a tissue-specific promoter. As used herein, a "tissue-specific promoter" is a promoter that preferentially promotes the expression of a transgene in a particular type of cell or tissue. Advantageously, a tissue-specific promoter can promote higher expression of a transgene in a cell type compared to other cell types. For example, a tissue-specific promoter may be a promoter that promotes an expression level of a transgene that is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in a cell type compared to the expression level in other cell types.

[0063] Preferably, the promoter is a kidney-specific promoter. In some embodiments, the promoter is a glomerulus-specific promoter. In some embodiments, the promoter is a podocyte-specific promoter.

[0064] The expression of the transgene can be measured by any suitable method known in the art. For example, by measuring the expression of a reporter transgene, e.g., green fluorescent protein (GFP), operably linked to a promoter, the expression of the reporter transgene correlates with the ability of the promoter to promote expression of a gene. The expression of the reporter transgene, e.g., GFP, can be determined by any suitable method, e.g., FACS. For example, a kidney-specific promoter can promote higher expression of a reporter transgene in kidney cells compared to other cell types (e.g., CNS, retina, lung, pancreas, heart or muscle cells). For example, a podocyte-specific promoter can promote higher expression of a reporter transgene in conditionally immortalized podocytes compared to other cell types. Suitable podocyte cell lines, e.g., CIHP-1, are well known to those skilled in the art. Methods for generating immortalized podocytes are well known to those skilled in the art. Suitable methods are described in Ni, L. et al., 2012. Nephrology, 17(6), pp.525-531.

[0065] Preferably, the promoter is a minimal kidney-specific promoter. In some embodiments, the promoter is a minimal glomerulus-specific promoter. In some embodiments, the promoter is a minimal podocyte-specific promoter.

[0066] The promoter may have a length of about 1.2 kb or less. Preferably, the promoter has a length of about 1.18 kb or less, about 1.17 kb or less, about 1.16 kb or less, about 1.15 kb or less, about 1.14 kb or less, about 1.13 kb or less, about 1.12 kb or less, about 1.11 kb or less, or about 1.10 kb or less. Preferably, the promoter has a length of about 1.15 kb or less.

[0067] The promoter may have a length of about 1.1 kb or less, hi some embodiments, the promoter has a length of about 1.1 kb or less, 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less.

[0068] In some embodiments, the promoter has a length of about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. In some embodiments, the promoter has a length of 818 bp or less. In some embodiments, the promoter has a length of 800 bp or less.

[0069] In some embodiments, the promoter has a length of about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less, hi some embodiments, the promoter has a length of about 0.3 kb or less.

[0070] The promoter may have a length of about 250 bp or more. In some embodiments, the promoter has a length of about 250 to 1100 bp, 250 to 1000 bp, 250 to 900 bp, 250 to 800 bp, 250 to 700 bp, 250 to 600 bp, 250 to 500 bp, 250 to 400 bp, or 250 to 300 bp.

[0071] The promoter may have a length of about 265 bp or more. In some embodiments, the promoter has a length of about 265-1100 bp, 265-1000 bp, 265-900 bp, 265-800 bp, 265-700 bp, 265-600 bp, 265-500 bp, 265-400 bp, or 265-300 bp.

[0072] In one embodiment, the promoter has a length of 250-300 bp, 250-280 bp, 255-275 bp, 260-270 bp, or about 265 bp. In one embodiment, the promoter has a length of 800-850 bp, 800-840 bp, 810-830 bp, 815-825 bp, about 819 bp, or about 818 bp.

[0073] Nephrin promoter The promoter may be a synthetic promoter. As used herein, a "synthetic promoter" is a promoter that does not occur in nature. Typically, a synthetic promoter is generated by genetic modification of an existing promoter. Thus, a synthetic promoter may be derived from an existing promoter, i.e., it may be identical to an existing promoter, except that it contains one or more genetic modifications (e.g., substitution, deletion or insertion).

[0074] Preferably, the promoter is derived from the nephrin (NPHS1) promoter. Preferably, the promoter is a minimal nephrin promoter. The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes.

[0075] The human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6):1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6):F1027-32. The NPHS1 promoter is a 1.2 kb fragment that appears to be podocyte-specific. The 1.2 kb promoter region lacks a TATA box but has recognition motifs for other transcription factors, such as PAX-2 binding elements, E-boxes and GATA consensus sequences.

[0076] Exemplary nephrin promoters are set forth as SEQ ID NO:1, SEQ ID NO:14, SEQ ID NO:44 and SEQ ID NO:45.

[0077] Exemplary Nephrin Promoter - 1,249 bp (SEQ ID NO:1) [ka]

[0078] Exemplary variant nephrin promoter - 1,192 bp (SEQ ID NO: 14) [ka] [ka]

[0079] Exemplary variant nephrin promoter - 1,192 bp (SEQ ID NO:44) [ka]

[0080] Exemplary variant nephrin promoter - 1,192 bp (SEQ ID NO:45) [ka] [ka]

[0081] Suitably, the promoter of the present invention is derived from SEQ ID NO: 1 or a variant having at least 70% identity to SEQ ID NO: 1. Suitably, the promoter of the present invention has one or more deletions compared to SEQ ID NO: 1 or a variant having at least 70% identity to SEQ ID NO: 1. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 1. Exemplary variants are SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45.

[0082] Suitably, the variant of SEQ ID NO: 1 comprises one or more of the following substitutions: A493G, C1080T, G1169C and C1249G. Suitably, the variant of SEQ ID NO: 1 comprises each of the substitutions A493G, C1080T, G1169C and C1249G (see SEQ ID NO: 14).

[0083] Suitably, the promoter of the present invention is derived from SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45, or a variant having at least 70% identity to SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45. Suitably, the promoter of the present invention has one or more deletions compared to SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45, or a variant having at least 70% identity to SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 14, SEQ ID NO: 44 or SEQ ID NO: 45.

[0084] Suitably, the promoter of the present invention is derived from SEQ ID NO: 14 or a variant having at least 70% identity to SEQ ID NO: 14. Suitably, the promoter of the present invention has one or more deletions compared to SEQ ID NO: 14 or a variant having at least 70% identity to SEQ ID NO: 14. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 14.

[0085] Advantageously, the promoter of the present invention comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1 and having one or more deletions, for example one or two deletions, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to said nucleotide sequence. Advantageously, the promoter of the present invention comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 1 and having two or more deletions, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to said nucleotide sequence. The deletions may be of any size. Preferably, the deletions are at least 50 bp, at least 100 bp, at least 150 bp, at least 200 bp, at least 250 bp, at least 300 bp, at least 350 bp or at least 400 bp in size, respectively. Preferably, the deletions are 50-500 bp, 100-500 bp, 150-500 bp, 200-500 bp, 250-500 bp, 300-500 bp, 350-500 bp or 400-500 bp in size, respectively.

[0086] In some embodiments, the promoter of the present invention is set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, where n1 is an integer from 1 to 430, and / or (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3≧n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061; nucleotide sequence, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to said nucleotide sequence. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0087] In some embodiments, (iii) the position corresponding to position 493 of SEQ ID NO:1 is G, the position corresponding to position 1080 of SEQ ID NO:1 is T, the position corresponding to position 1169 of SEQ ID NO:1 is C, and / or the position corresponding to position 1249 of SEQ ID NO:1 is G.

[0088] In a preferred embodiment, (iii) the position corresponding to position 493 of SEQ ID NO:1 is G, the position corresponding to position 1080 of SEQ ID NO:1 is T, the position corresponding to position 1169 of SEQ ID NO:1 is C, and the position corresponding to position 1249 of SEQ ID NO:1 is G.

[0089] The above nucleotide positions are referred to by reference to the positions in SEQ ID NO:1, but one of skill in the art would be able to readily identify the corresponding nucleotide positions in the variant, for example by aligning SEQ ID NO:1 and the variant sequence. For example, position 493 of SEQ ID NO:1 corresponds to position 436 of SEQ ID NO:14, position 1080 of SEQ ID NO:1 corresponds to position 1023 of SEQ ID NO:14, position 1169 of SEQ ID NO:1 corresponds to position 1112 of SEQ ID NO:14, and position 1249 of SEQ ID NO:1 corresponds to position 1192 of SEQ ID NO:14.

[0090] For example, the promoter of the present invention is set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, where n1 is an integer from 1 to 430; and / or (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3≧n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061; It may comprise or consist of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence.

[0091] In some embodiments, (iii) position 493 of SEQ ID NO:1 is substituted with G, position 1080 of SEQ ID NO:1 is substituted with T, position 1169 of SEQ ID NO:1 is substituted with C, and / or position 1249 of SEQ ID NO:1 is substituted with G.

[0092] In a preferred embodiment, (iii) position 493 of SEQ ID NO:1 is substituted with G, position 1080 of SEQ ID NO:1 is substituted with T, position 1169 of SEQ ID NO:1 is substituted with C, and position 1249 of SEQ ID NO:1 is substituted with G.

[0093] Preferably, n1 is an integer of 50 to 430, 100 to 430, 150 to 430, 200 to 430, 250 to 430, 300 to 430, 350 to 430, or 400 to 430. In some embodiments, n1 is an integer of 100 to 430. In some embodiments, n1=430, that is, positions 1 to 430 of SEQ ID NO: 1 are deleted in the promoter of the present invention.

[0094] The difference between n3 and n2 defines the size of the deletion. Preferably, n3≧n2+49, n3≧n2+99, n3≧n2+149, n3≧n2+199, n3≧n2+249, n3≧n2+299, n3≧n2+349, n3≧n2+399, n3≧n2+449, n3≧n2+499, or n3≧n2+549. In some embodiments, n3≧n2+49.

[0095] The location of the deletion is determined by the values ​​of n2 and n3. Preferably, n2 and n3 are each an integer of 550 to 1050, n2 and n3 are each an integer of 600 to 1000, n2 and n3 are each an integer of 650 to 950, n2 and n3 are each an integer of 700 to 900, and n2 and n3 are each an integer of 750 to 850.

[0096] In some embodiments, n2=508 and n3=1061, ie, positions 508 to 1061 of SEQ ID NO: 1 are deleted in the promoter of the present invention.

[0097] Promoter region The present inventors determined the region of the nephrin promoter that drives expression of the transgene.

[0098] A promoter typically comprises a "core" region and a "proximal" region. The "core promoter region" may comprise a transcription initiation site, an RNA polymerase binding site, and a general transcription factor binding site. The "proximal promoter region" may comprise, for example, the primary regulatory elements and specific transcription factor binding sites required to promote efficient and controllable transcription. The size and composition of both the core promoter region and the proximal promoter region typically vary gene-specifically. A promoter may also comprise a 5' untranslated region (5'UTR) (also known as a leader sequence) downstream of the core promoter region and upstream from the start codon. (See, for example, FIG. 1).

[0099] The promoter may be a hybrid promoter. As used herein, "hybrid promoter" includes a combination of elements from different promoters. For example, the hybrid promoter may include a proximal promoter region from one existing promoter and a core promoter region from another existing promoter to achieve desired transgene expression. Muscle hybrid promoters are described in Piekarowicz, K. et al. (2019). Methods & clinical development, 15, 157-169.

[0100] In some embodiments, a promoter of the invention comprises (i) a nucleotide sequence set forth as SEQ ID NO: 4, or a variant that is at least 70% identical to SEQ ID NO: 4. Without wishing to be bound by theory, it is believed that a nucleotide sequence having at least about 70% identity to SEQ ID NO: 4 can provide the proximal promoter region.

[0101] Exemplary Proximal Promoter Region (SEQ ID NO:4) [ka]

[0102] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 4. The promoter may comprise a variant of SEQ ID NO: 4 shown as SEQ ID NO: 17. Suitably, the variant of SEQ ID NO: 4 comprises the substitution A63G (see SEQ ID NO: 17).

[0103] In some embodiments, the promoter of the invention comprises (i) a nucleotide sequence set forth as SEQ ID NO: 17, or a variant that is at least 70% identical to SEQ ID NO: 17. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17.

[0104] Exemplary variant proximal promoter region (SEQ ID NO: 17) [ka]

[0105] In some embodiments, a promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO:5 or 18, or a variant that is at least 70% identical to SEQ ID NO:5 or 18; a nucleotide sequence set forth as SEQ ID NO:6 or 19, or a variant that is at least 70% identical to SEQ ID NO:6 or 19; and / or a nucleotide sequence set forth as SEQ ID NO:7 or 20, or a variant that is at least 70% identical to SEQ ID NO:7 or 20.

[0106] In some embodiments, a promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO:5, or a variant that is at least 70% identical to SEQ ID NO:5. Without wishing to be bound by theory, it is believed that a nucleotide sequence having at least about 70% identity to SEQ ID NO:5 can provide a core promoter region.

[0107] Exemplary core promoter region (SEQ ID NO:5) [ka]

[0108] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 5. The promoter may comprise a variant of SEQ ID NO: 5, shown as SEQ ID NO: 18. Suitably, the variant of SEQ ID NO: 5 comprises the substitution C19T (see SEQ ID NO: 18).

[0109] In some embodiments, the promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO: 18, or a variant that is at least 70% identical to SEQ ID NO: 18. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 18.

[0110] Exemplary variant core promoter region (SEQ ID NO: 18) [ka]

[0111] In some embodiments, the promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO: 6, or a variant that is at least 70% identical to SEQ ID NO: 6. Without wishing to be bound by theory, it is believed that a nucleotide sequence having at least about 70% identity to SEQ ID NO: 6 can provide the 5'UTR.

[0112] Exemplary 5'UTR (SEQ ID NO:6) [ka]

[0113] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 6. The promoter may comprise a variant of SEQ ID NO: 6, shown as SEQ ID NO: 19. Suitably, the variant of SEQ ID NO: 6 comprises the substitutions G76C and / or C156G (see SEQ ID NO: 19).

[0114] In some embodiments, the promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO: 19, or a variant that is at least 70% identical to SEQ ID NO: 19. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 19.

[0115] Exemplary variant 5'UTR (SEQ ID NO: 19) [ka]

[0116] In some embodiments, the promoter of the present invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO: 7, or a variant that is at least 70% identical to SEQ ID NO: 7. Without wishing to be bound by theory, it is believed that a nucleotide sequence having at least about 70% identity to SEQ ID NO: 7 can provide the core promoter region and 5'UTR.

[0117] Exemplary core promoter region and 5'UTR (SEQ ID NO:7) [ka]

[0118] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 7. The promoter may comprise a variant of SEQ ID NO: 7, shown as SEQ ID NO: 20. Suitably, the variant of SEQ ID NO: 7 comprises the substitutions C19T, G108C and / or C188G (see SEQ ID NO: 20). Suitably, the variant of SEQ ID NO: 7 comprises the substitutions C19T, G108C and C188G (see SEQ ID NO: 20).

[0119] In some embodiments, the promoter of the invention comprises (ii) a nucleotide sequence set forth as SEQ ID NO:20, or a variant that is at least 70% identical to SEQ ID NO:20. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:20.

[0120] Exemplary variant core promoter region and 5'UTR (SEQ ID NO:20) [ka]

[0121] In some embodiments, a promoter of the invention comprises (iii) a nucleotide sequence having at least 70% identity to SEQ ID NO:8, or one or more fragments thereof. Suitably, the promoter comprises a nucleotide sequence having at least about 70% identity to SEQ ID NO:8, or one or more fragments thereof, immediately downstream of the proximal promoter region and / or immediately upstream of the core promoter region.

[0122] The promoter may comprise a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 8 or one or more fragments thereof. The promoter may comprise the nucleotide sequence of SEQ ID NO: 8 or one or more fragments thereof.

[0123] Exemplary Optional Promoter Region (SEQ ID NO:8) [ka]

[0124] Preferably, the one or more fragments are (a) a 5'-end fragment and / or (b) a 3'-end fragment. Preferably, the 5'-end fragment may be immediately downstream of the proximal promoter region. Preferably, the 3'-end fragment may be immediately upstream of the core promoter region. For example, a promoter of the present invention may be (a) a nucleotide sequence having at least 70% of positions 1 to x of SEQ ID NO:8, and / or (b) a nucleotide sequence having at least 70% identity to position y to 554 of SEQ ID NO:8. where x and y are integers and y>x.

[0125] Fragments of SEQ ID NO:8 may be of any length. Suitably, the fragments may have a length of about 500 bp or less, 450 bp or less, 400 bp or less, 350 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.

[0126] In some embodiments, the promoter does not include SEQ ID NO:8.

[0127] In some embodiments, a promoter of the invention comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 9, or a fragment thereof. Suitably, the promoter comprises a nucleotide sequence having at least about 70% identity to SEQ ID NO: 9, or a fragment thereof, immediately upstream of the proximal promoter region.

[0128] The promoter may comprise a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 9 or a fragment thereof. The promoter may comprise the nucleotide sequence of SEQ ID NO: 9 or one or more fragments thereof.

[0129] Exemplary Optional Upstream Promoter Region (SEQ ID NO:9) [ka]

[0130] Preferably, the fragment is a 3'-end fragment. For example, the promoter of the present invention may comprise a nucleotide sequence having at least 70% identity to position z to 430 of SEQ ID NO:9, where z is an integer.

[0131] Fragments of SEQ ID NO:9 may be of any length. Suitably, the fragments may have a length of about 400 bp or less, 350 bp or less, 300 bp or less, 250 bp or less, 200 bp or less, 150 bp or less, 100 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, or 10 bp or less.

[0132] In some embodiments, the promoter does not include SEQ ID NO:9.

[0133] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 4 or 17; (iii) optionally a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:8 or one or more fragments thereof, and (ii) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:5 or 18, a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:6 or 19, and / or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0134] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 90% identity to SEQ ID NO: 4 or 17; (iii) optionally, a nucleotide sequence having at least 90% identity to SEQ ID NO:8 or one or more fragments thereof; and (ii) a nucleotide sequence having at least 90% identity to SEQ ID NO: 5 or 18, a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 19, and / or a nucleotide sequence having at least 90% identity to SEQ ID NO: 7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0135] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 4 or 17; (iii) optionally, (a) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a 5'-terminal fragment of SEQ ID NO:8, and / or (b) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a 3'-terminal fragment of SEQ ID NO:8, and (ii) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:5 or 18, a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:6 or 19, and / or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0136] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 90% identity to SEQ ID NO: 4 or 17; (iii) optionally, (a) a nucleotide sequence having at least 90% identity to a 5'-terminal fragment of SEQ ID NO:8 and / or (b) a nucleotide sequence having at least 90% identity to a 3'-terminal fragment of SEQ ID NO:8, and (ii) a nucleotide sequence having at least 90% identity to SEQ ID NO: 5 or 18, a nucleotide sequence having at least 90% identity to SEQ ID NO: 6 or 19, and / or a nucleotide sequence having at least 90% identity to SEQ ID NO: 7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0137] Promoter elements The present inventors determined the functional elements of the nephrin promoter that drive transgene expression.

[0138] The promoter of the present invention may contain one or more of the following elements: (a) a retinoic acid receptor binding site, (b) a WT1 binding site, (c) an enhancer box, (d) a transcription factor binding region, and (e) a transcription start site.

[0139] Preferably, the promoter of the present invention contains all of the following elements: (a) a retinoic acid receptor binding site, (b) a WT1 binding site, (c) an enhancer box, (d) a transcription factor binding region, and (e) a transcription start site.

[0140] Retinoic acid receptor (RAR) binding site refers to a polynucleotide sequence capable of binding to RAR alpha, RAR beta and / or RAR gamma. The RAR binding site may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 10 or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO: 10. The substitutions, deletions or insertions may be any substitutions, deletions or insertions of a single nucleotide such that the RAR binding site retains at least one of its endogenous functions.

[0141] Exemplary RAR Binding Site (SEQ ID NO:10) [ka]

[0142] WT1-binding site refers to a polynucleotide sequence that can bind to the zinc finger polypeptide encoded by WT1, the Wilms tumor suppressor gene. The WT1-binding site may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 11 or the nucleotide sequence having one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 11. The substitutions, deletions or insertions may be any substitutions, deletions or insertions of a single nucleotide such that the WT1-binding region retains at least one of its endogenous functions.

[0143] Exemplary WT1 binding site (SEQ ID NO:11) [ka]

[0144] Enhancer box refers to the DNA response element found in some eukaryotes that functions as a protein binding site.The enhancer box may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 12 or the nucleotide sequence with one or two substitutions, deletions or insertions compared to SEQ ID NO: 12.The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the enhancer box retains at least one of its intrinsic functions.

[0145] Exemplary enhancer box (SEQ ID NO: 12) [ka]

[0146] One or more of (a) a retinoic acid receptor binding site, (b) a WT1 binding site, and (c) an enhancer box may be present in the proximal promoter region. Preferably, each of (a) a retinoic acid receptor binding site, (b) a WT1 binding site, and (c) an enhancer box is present in the proximal promoter region.

[0147] In some embodiments, one or more of the following elements are present in (i) a nucleotide sequence having at least 70% identity to SEQ ID NO:4 or 17: (a) a RAR binding site at a position corresponding approximately to positions 7-13 of SEQ ID NO:4 or 17, (b) a WT1 binding site at a position corresponding approximately to positions 14-30 of SEQ ID NO:4 or 17, and (c) an enhancer box at a position corresponding approximately to positions 49-53 of SEQ ID NO:4 or 17. In some embodiments, each of the elements is present in (i) a nucleotide sequence having at least 70% identity to SEQ ID NO:4 or 17.

[0148] In some embodiments, one or more of the following nucleotide sequences are present in (i) a nucleotide sequence having at least 70% identity to SEQ ID NO:4 or 17: (a) GGGGTCA at positions corresponding to positions 7-13 of SEQ ID NO:4 or 17, (b) CGGAGCTGGGGAGGCA at positions corresponding to positions 14-30 of SEQ ID NO:4 or 17, and (c) ATGTG at positions corresponding to positions 49-53 of SEQ ID NO:4 or 17. In some embodiments, each of the nucleotide sequences is present in (i) a nucleotide sequence having at least 70% identity to SEQ ID NO:4 or 17.

[0149] Suitably, the promoter may comprise a transcription factor binding region comprising or consisting of the nucleotide sequence shown as SEQ ID NO: 13 or a nucleotide sequence having one, two, three, four or five substitutions, deletions or insertions compared to SEQ ID NO: 13. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the transcription factor binding region retains at least one of its endogenous functions. An exemplary variant is shown in SEQ ID NO: 43. Suitably, the variant of SEQ ID NO: 13 comprises the substitution C19T (see SEQ ID NO: 43).

[0150] Exemplary transcription factor binding regions (SEQ ID NO: 13) [ka]

[0151] Suitably, the promoter may comprise a transcription factor binding region comprising or consisting of the nucleotide sequence shown as SEQ ID NO: 43 or a nucleotide sequence having one, two, three, four or five substitutions, deletions or insertions compared to SEQ ID NO: 43. The substitution, deletion or insertion may be any substitution, deletion or insertion of a single nucleotide such that the transcription factor binding region retains at least one of its endogenous functions.

[0152] Exemplary transcription factor binding regions (SEQ ID NO:43) [ka]

[0153] Other suitable transcription factor binding regions will be known to those of skill in the art. For example, other suitable transcription factor binding regions include TACGAT (SEQ ID NO: 37), TATAAT (SEQ ID NO: 38), GATACT (SEQ ID NO: 39), TATGAT (SEQ ID NO: 40) and TATGTT (SEQ ID NO: 41).

[0154] Suitably, the promoter may include a transcription initiation site which comprises, or is composed of, an "AG" dinucleotide.

[0155] Preferably, the transcription factor binding site is operably linked to a transcription start site. Preferably, the transcription factor binding site may be immediately upstream of the transcription start site. Without wishing to be bound by theory, it is believed that the transcription factor binding site and the transcription start site may provide a core promoter region.

[0156] Suitably, the promoter may comprise a 5' untranslated region, which may comprise or consist of a nucleotide sequence having at least about 70%, 80%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6 or 19. The 5' untranslated region may comprise or consist of SEQ ID NO: 6 or 19.

[0157] Preferably, the 5' untranslated region is operably linked to the transcription initiation site. Preferably, the 5' untranslated region is directly downstream of the transcription initiation site.

[0158] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 4 or 17, wherein each of the following elements is present: (a) an RAR binding site at a position approximately corresponding to positions 7 to 13 of SEQ ID NO: 4 or 17, (b) a WT1 binding site at a position approximately corresponding to positions 14 to 30 of SEQ ID NO: 4 or 17, and (c) an enhancer box at a position approximately corresponding to positions 49 to 53 of SEQ ID NO: 4 or 17; (iii) optionally a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:8 or one or more fragments thereof, and (ii) a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:5 or 18, a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:6 or 19, or a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0159] In some embodiments, the promoters of the present invention are (i) a nucleotide sequence having at least 90% identity to SEQ ID NO: 4 or 17, wherein each of the following elements is present: (a) an RAR binding site at a position corresponding approximately to positions 7 to 13 of SEQ ID NO: 4 or 17, (b) a WT1 binding site at a position corresponding approximately to positions 14 to 30 of SEQ ID NO: 4 or 17, and (c) an enhancer box at a position corresponding approximately to positions 49 to 53 of SEQ ID NO: 4 or 17; (iii) optionally, a nucleotide sequence having at least 90% identity to SEQ ID NO:8 or one or more fragments thereof; and (ii) a nucleotide sequence having at least 90% identity to SEQ ID NO:5 or 18, a nucleotide sequence having at least 90% identity to SEQ ID NO:6 or 19, or a nucleotide sequence having at least 90% identity to SEQ ID NO:7 or 20. The nucleotide sequence of the present invention may comprise or consist of the nucleotide sequence of

[0160] Exemplary Promoters In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:2, or a variant that is at least 70% identical to SEQ ID NO:2.

[0161] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO:2, or a variant that is at least 70% identical to SEQ ID NO:2, and the promoter has a length of about 1.1 kb or less.

[0162] In some embodiments, the promoter of the present invention is comprised of the nucleotide sequence shown as SEQ ID NO:2, or a variant that is at least 70% identical to SEQ ID NO:2.

[0163] Exemplary minimal nephrin promoter - 819bp (SEQ ID NO:2) [ka] [ka]

[0164] Suitably the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 2. The promoter may comprise or consist of a variant of SEQ ID NO: 2 shown as SEQ ID NO: 15 or SEQ ID NO: 46.

[0165] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:15, or a variant that is at least 70% identical to SEQ ID NO:15.

[0166] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO: 15, or a variant that is at least 70% identical to SEQ ID NO: 15, and the promoter has a length of about 1.1 kb or less.

[0167] In some embodiments, the promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:15, or a variant that is at least 70% identical to SEQ ID NO:15.

[0168] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:15.

[0169] Exemplary minimal nephrin promoter variant - 819bp (SEQ ID NO:15) [ka]

[0170] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:46, or a variant that is at least 70% identical to SEQ ID NO:46.

[0171] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO: 46, or a variant that is at least 70% identical to SEQ ID NO: 46, and the promoter has a length of about 1.1 kb or less.

[0172] In some embodiments, a promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:46, or a variant that is at least 70% identical to SEQ ID NO:46.

[0173] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:46.

[0174] Exemplary minimal nephrin promoter variant - 819bp (SEQ ID NO:46) [ka]

[0175] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:58, or a variant that is at least 70% identical to SEQ ID NO:58.

[0176] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO:58, or a variant that is at least 70% identical to SEQ ID NO:58, and the promoter has a length of about 1.1 kb or less.

[0177] In some embodiments, a promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:58, or a variant that is at least 70% identical to SEQ ID NO:58.

[0178] Exemplary minimal nephrin promoter - 818bp (SEQ ID NO:58) [ka] [ka]

[0179] Suitably the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 58. The promoter may comprise or consist of a variant of SEQ ID NO: 58 shown as SEQ ID NO: 59 or SEQ ID NO: 60.

[0180] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:59, or a variant that is at least 70% identical to SEQ ID NO:59.

[0181] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO:59, or a variant that is at least 70% identical to SEQ ID NO:59, and the promoter has a length of about 1.1 kb or less.

[0182] In some embodiments, a promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:59, or a variant that is at least 70% identical to SEQ ID NO:59.

[0183] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:59.

[0184] Exemplary minimal nephrin promoter variant - 818bp (SEQ ID NO:59) [ka]

[0185] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:60, or a variant that is at least 70% identical to SEQ ID NO:60.

[0186] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO:60, or a variant that is at least 70% identical to SEQ ID NO:60, and the promoter has a length of about 1.1 kb or less.

[0187] In some embodiments, a promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:60, or a variant that is at least 70% identical to SEQ ID NO:60.

[0188] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:60.

[0189] Exemplary minimal nephrin promoter variant - 818bp (SEQ ID NO:60) [ka]

[0190] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:3, or a variant that is at least 70% identical to SEQ ID NO:3.

[0191] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO:3, or a variant that is at least 70% identical to SEQ ID NO:3, and the promoter has a length of about 1.1 kb or less.

[0192] In some embodiments, the promoter of the present invention is comprised of the nucleotide sequence shown as SEQ ID NO:3, or a variant that is at least 70% identical to SEQ ID NO:3.

[0193] Exemplary minimal nephrin promoter - 265bp (SEQ ID NO:3) [ka]

[0194] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 3. Suitably, the variant of SEQ ID NO: 3 comprises one or more of the substitutions A63G, C96T, G185C and C265G. Suitably, the variant of SEQ ID NO: 3 comprises the substitutions A63G, C96T, G185C and C265G (see SEQ ID NOs: 17-20). The promoter may comprise or consist of a variant of SEQ ID NO: 3 shown as SEQ ID NO: 16 or SEQ ID NO: 47.

[0195] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:16, or a variant that is at least 70% identical to SEQ ID NO:16.

[0196] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO: 16, or a variant that is at least 70% identical to SEQ ID NO: 16, and the promoter has a length of about 1.1 kb or less.

[0197] In some embodiments, the promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:16, or a variant that is at least 70% identical to SEQ ID NO:16.

[0198] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:16.

[0199] Exemplary minimal nephrin promoter variant - 265bp (SEQ ID NO: 16) [ka]

[0200] In some embodiments, a promoter of the present invention comprises or consists of a nucleotide sequence set forth as SEQ ID NO:47, or a variant that is at least 70% identical to SEQ ID NO:47.

[0201] In some embodiments, a promoter of the invention comprises a nucleotide sequence set forth as SEQ ID NO: 47, or a variant that is at least 70% identical to SEQ ID NO: 47, and the promoter has a length of about 1.1 kb or less.

[0202] In some embodiments, a promoter of the present invention is comprised of the nucleotide sequence set forth as SEQ ID NO:47, or a variant that is at least 70% identical to SEQ ID NO:47.

[0203] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:47.

[0204] Exemplary minimal nephrin promoter variant - 265bp (SEQ ID NO:47) [ka]

[0205] Polynucleotides The present invention provides a polynucleotide comprising a promoter of the present invention.

[0206] The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those skilled in the art. They may also be cloned by standard techniques. The polynucleotides may be isolated polynucleotides.

[0207] Longer polynucleotides are generally produced using recombinant means, for example, using polymerase chain reaction (PCR) cloning techniques. This involves creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence desired to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, performing the polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture through an agarose gel), and recovering the amplified DNA. The primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into an appropriate vector.

[0208] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention.

[0209] Protein coding sequence The promoter may be operably linked to one or more protein coding sequences.

[0210] The protein coding sequence may code for any polypeptide of interest.For example, the protein coding sequence may code for any polypeptide related to glomerular disease.The protein coding sequence may code for a polypeptide involved in GBM-related hereditary glomerular disease, such as Alport syndrome.The protein coding sequence may code for a polypeptide involved in podocyte-related hereditary glomerular disease.

[0211] Suitably, the protein coding sequence is selected from the group consisting of COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, CFHL1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3 or VEGFC polypeptide.

[0212] Suitably, the protein coding sequence encodes a polypeptide having a length of 1450 or more amino acids, 1500 or more amino acids, 1550 or more amino acids, 1600 or more amino acids or 1650 or more amino acids.

[0213] For protein-encoding polynucleotides, it will be understood by those skilled in the art that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. In addition, those skilled in the art will understand that, using routine techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the invention can be made to reflect the codon usage of any particular host organism in which the polypeptide of the invention is to be expressed.

[0214] Protein coding sequences may be codon-optimized. Different cells differ in their frequency of use of certain codons. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. Codons in a sequence can be altered to adjust them to match the relative abundance of the corresponding tRNAs, thereby increasing expression. For the same reason, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. Thus, additional translational control becomes available. Codon usage tables are known in the art for mammalian cells (e.g., humans), as well as for a variety of other organisms.

[0215] The protein-encoding nucleotide sequences disclosed herein may contain or lack a stop codon at their 3' end. Thus, the disclosure encompasses the SEQ ID NOs disclosed herein with or without the presence of a stop codon.

[0216] COL4A3, COL4A4 and COL4A5 Polypeptides The protein coding sequence may encode a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.

[0217] The COL4A3, COL4A4 and COL4A5 proteins are homologous polypeptides of approximately 170-185 kDa that contain collagenous Gly-XY repeats frequently interrupted by non-collagenous sequences forming triple helical repeats. Each polypeptide also contains a large globular non-collagenous domain at the carboxyl terminus.

[0218] Alport syndrome (AS) is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, resulting in abnormalities in the collagen IV α345 network of the basement membrane.

[0219] The COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof may be capable of forming a collagen IV α345 network.

[0220] Approximately 200-300 amino acids may be removed from each of the COL4A3, COL4A4 and COL4A5 polypeptides to generate truncated transgenes suitable for a minigene approach. The amino acids may be removed from the triple helix repeats. Preferably, no amino acids are removed from the non-collagenous regions.

[0221] In some embodiments, the COL4A3, COL4A4 and COL4A5 polypeptides are full-length polypeptides.

[0222] Preferably, the COL4A3, COL4A4 or COL4A5 polypeptide is human. An example of human COL4A3 is COL4A3 with UniProtKB accession number Q01955. An example of human COL4A4 is COL4A3 with UniProtKB accession number P53420. An example of human COL4A5 is COL4A5 with UniProtKB accession number P29400.

[0223] Suitably, the COL4A3 peptide may comprise or consist of a polypeptide sequence as set forth in SEQ ID NO: 21, or a variant that is at least 70% identical to SEQ ID NO: 21. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 21.

[0224] Suitably, the COL4A4 peptide may comprise or consist of a polypeptide sequence as set forth in SEQ ID NO: 22, or a variant which is at least 70% identical to SEQ ID NO: 22. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 22.

[0225] Suitably, the COL4A5 peptide may comprise or consist of a polypeptide sequence as set forth in SEQ ID NO: 23, or a variant that is at least 70% identical to SEQ ID NO: 23. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 23.

[0226] Exemplary COL4A3 Amino Acid Sequence - Uniprot Reference Q01955 (SEQ ID NO:21) [ka] [ka]

[0227] Exemplary COL4A4 Amino Acid Sequence - Uniprot Reference P53420 (SEQ ID NO:22) [ka]

[0228] Exemplary COL4A5 Amino Acid Sequence - Uniprot Reference P29400 (SEQ ID NO:23) [ka] [ka]

[0229] The protein coding sequence may comprise or consist of a COL4A3, COL4A4 or COL4A5 transgene.

[0230] An example of a transgene encoding COL4A3 is provided in NM_000091.5. An example of a transgene encoding COL4A4 is provided in NM_000092.5. An example of a transgene encoding COL4A5 is provided in NM_000495.5.

[0231] Suitably, the COL4A3 transgene may comprise or consist of a polynucleotide sequence as set forth in SEQ ID NO: 24, or a variant which is at least 70% identical to SEQ ID NO: 24. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 24.

[0232] Suitably, the COL4A4 transgene may comprise or consist of a polynucleotide sequence as set forth in SEQ ID NO: 25, or a variant which is at least 70% identical to SEQ ID NO: 25. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 25.

[0233] Suitably, the COL4A5 transgene may comprise or consist of a polynucleotide sequence as set forth in SEQ ID NO: 26, or a variant which is at least 70% identical to SEQ ID NO: 26. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 26.

[0234] Exemplary COL4A3 transgene sequence (SEQ ID NO:24) [ka] [ka] [ka]

[0235] Exemplary COL4A4 transgene sequence (SEQ ID NO:25) [ka] [ka]

[0236] Exemplary COL4A5 transgene sequence (SEQ ID NO:26) [ka] [ka] [ka]

[0237] COL4A3, COL4A4 or COL4A5 transgene may contain intron or intron sequence that can be used to improve gene expression. The COL4A3, COL4A4 or COL4A5 transgene may contain protein tag, such as hemagglutinin (HA) tag. HA can be used as epitope tag, and it has been shown that it does not interfere with the biological activity or biodistribution of the protein to which it is attached. The protein tag can facilitate detection, isolation and purification of the transgene. Other suitable protein tags can include Myc tag, polyhistidine tag and flag tag.

[0238] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:47 is operably linked to a protein coding sequence encoding a COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof.

[0239] In some embodiments, a promoter consisting of the nucleotide sequence of SEQ ID NO:47 is operably linked to a protein coding sequence encoding a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.

[0240] Nephrotic syndrome (NS)-related transgene The protein coding sequence may comprise or consist of the NS-associated transgene.

[0241] Nephrotic syndrome (NS) is a chronic kidney disease characterized by severe proteinuria, hypoalbuminemia, edema and hyperlipidemia. The NS-associated transgene can be a gene that is associated with a monogenic form of NS and is expressed in podocytes.

[0242] Suitable NS-related transgenes include NPHS2, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAFB, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, and TP53R. These include K, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2 and NLRP3.

[0243] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:47 is operably linked to the NS-associated transgene.

[0244] In some embodiments, a promoter comprised of the nucleotide sequence of SEQ ID NO:47 is operably linked to the NS-associated transgene.

[0245] NPHS2 The protein coding sequence may encode NPHS2 or a fragment and / or variant thereof.

[0246] "NPHS2" is the abbreviation for the polypeptide encoded by the NPHS2 gene, also known as podocin. NPHS2 is a 42 kDa hairpin-shaped membrane-bound podocyte-specific protein that is a key component of the slit diaphragm; a protein complex at the intercellular junction between the foot processes of adjacent podocytes. It localizes to lipid rafts and interacts with other important slit diaphragm proteins such as nephrin, CD2AP and TRPC6. It is essential for the maintenance of the slit diaphragm and thus the integrity of the glomerular filtration barrier.

[0247] The fragment and / or variant of NPHS2 can retain NPHS2 activity. For example, the fragment and / or variant of podocin can regulate glomerular permeability. Suitably, the fragment and / or variant of NPHS2 can have the same or similar activity as NPHS2, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% of the activity of NPHS2.

[0248] Those skilled in the art can use conservative substitutions to generate fragments and / or variants based on the known structural and functional characteristics of NPHS2 (see, for example, Tabassum, A., et al., 2014. Interdisciplinary Sciences: Computational Life Sciences, 6(1), pp.32-39) and / or based on known variants (see, for example, NCBI Gene ID: 7827 and NCBI HomoloGene: 22826). Suitably, the fragments and / or variants of NPHS2 include a transmembrane domain with two cytoplasmic domains at the N-terminus and C-terminus.

[0249] NPHS2 gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice and rats.The NPHS2 can be human NPHS2.Preferably, the NPHS2 can comprise or consist of the polypeptide sequence of UniProtKB access Q9NP85 or its fragment and / or variant.

[0250] In some embodiments, NPHS2 comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 55 or a fragment thereof. Suitably, the NPHS2 comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 55 or a fragment thereof.

[0251] In some embodiments, NPHS2 comprises or consists of SEQ ID NO:55, or a fragment thereof. [ka] Exemplary NPHS2 Amino Acid Sequence (SEQ ID NO:55)

[0252] In some embodiments, the NPHS2 transgene comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 56 or a fragment thereof. Suitably, the NPHS2 transgene comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 56 or a fragment thereof.

[0253] In some embodiments, the NPHS2 transgene comprises or consists of the nucleotide sequence of SEQ ID NO:56, or a fragment thereof. [ka] Exemplary NPHS2 transgene sequence (SEQ ID NO:56)

[0254] In some embodiments, the NPHS2 transgene comprises or consists of a nucleotide sequence that is at least 70% identical to SEQ ID NO: 57 or a fragment thereof. Suitably, the NPHS2 transgene comprises or consists of a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 57 or a fragment thereof.

[0255] In some embodiments, the NPHS2 transgene comprises or consists of the nucleotide sequence of SEQ ID NO:57, or a fragment thereof. [ka] Exemplary NPHS2 transgene sequence (SEQ ID NO:57)

[0256] In some embodiments, the promoter comprises or consists of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:47 and is operably linked to a protein coding sequence encoding NPHS2 or a fragment and / or variant thereof.

[0257] In some embodiments, a promoter consisting of the nucleotide sequence of SEQ ID NO:47 is operably linked to a protein coding sequence encoding NPHS2 or a fragment and / or variant thereof.

[0258] Vascular endothelial growth factor (VEGF) C transgene The protein coding sequence may comprise or consist of a vascular endothelial growth factor (VEGF) C transgene.

[0259] VEGFC is a lymphangiogenic growth factor that is known to signal through two receptors, VEGFR-3 (Flt4) and VEGFR-2 (Flk4). VEGFC is produced by cells in a prepropeptide form and dimerizes before being cleaved into tetramers.

[0260] The VEGFC transgene may comprise a polynucleotide encoding any form of VEGFC, for example, a prepropeptide form, a tetrameric form, an intermediate form or the fully processed mature VEGFC.

[0261] If necessary, the polynucleotides encoding different forms of VEGFC polypeptide can be used in any combination.Preferably, the VEGFC transgene comprises a polynucleotide encoding one or more polypeptides having VEGFC biological activity, i.e., peptides that can bind and activate VEGFR-2 and / or VEGRF-3.More preferably, the VEGFC transgene comprises a polynucleotide encoding a polypeptide that comprises VEGFC homology domain and has VEGFC biological activity, i.e., polypeptides that can bind and activate VEGFR-2 and / or VEGRF-3.Further details of suitable VEGFC polynucleotides and polypeptides include those described in WO2015 / 022447 and US2014 / 0087002.

[0262] The VEGFC polynucleotide may comprise the VEGFC open reading frame (ORF) sequence of SEQ ID NO: 36. The VEGFC polynucleotide may comprise a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the VEGFC ORF sequence of SEQ ID NO: 36. The variant sequence may encode a VEGFC polypeptide that retains the ability to bind and activate VEGFR-2 and VEGFR-3.

[0263] Exemplary VEGFC Polynucleotides (SEQ ID NO:36) [ka]

[0264] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or 100% identity to SEQ ID NO:47 is operably linked to the VEGF(C) transgene.

[0265] In some embodiments, a promoter consisting of the nucleotide sequence of SEQ ID NO:47 is operably linked to the VEGF(C) transgene.

[0266] Complement Proteins and Complement Inhibitors The protein coding sequence may encode a complement protein or a fragment and / or variant thereof.

[0267] As used herein, a "complement protein" is a protein that is part of the complement system. The complement system, also known as the complement cascade, is a central part of innate immunity that serves as the first line of defense against foreign and modified host cells. The complement system consists of plasma proteins that are primarily produced by the liver or membrane proteins that are expressed on the cell surface. Complement functions in plasma, tissues, or intracellularly. Complement proteins work together as a cascade to opsonize pathogens and trigger a series of inflammatory responses that help immune cells fight infection and maintain homeostasis (Merle, NS, et al., 2015. Frontiers in immunology, 6, 262).

[0268] There are three pathways of complement activation: classical, alternative, and lectin. Although the three complement pathways differ in their mechanisms of target recognition, they converge on the activation of the central component C3. This activation is followed by cleavage of C5, initiating the assembly of the membrane attack complex (MAC). Enzymatic cleavage of C3 and C5 results in the production and release of the anaphylatoxins C3a and C5a.

[0269] Suitably, the complement protein is selected from the list consisting of CFI, CFH, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin and clusterin or fragments and / or variants thereof.

[0270] The protein coding sequence may encode an inhibitor of the complement system or a fragment and / or variant thereof.

[0271] As used herein, "complement system inhibitors" or "complement inhibitors" are proteins that block the activation of the complement system. Complement is tightly controlled by these inhibitors, which essentially protect self-cells and tissues from unwanted complement activation. Complement inhibitors can regulate complement activation at various stages of the classical, lectin, and alternative pathways. Suitably, the complement inhibitor is a naturally occurring complement inhibitor or a fragment and / or variant thereof. Preferably, the complement system inhibitor is an inhibitor of the complement system in humans.

[0272] Complement inhibitors are divided into two categories: soluble inhibitors and membrane-bound inhibitors.Preferably, the inhibitors of complement system are soluble complement inhibitors.Soluble complement inhibitors include C1 inhibitor (C1INH), complement factor I (CFI), complement factor H (CFH), complement factor H-like protein 1 (FHL-1), C4 binding protein (C4BP), clusterin and vitronectin.Membrane-bound regulators include CD46, CD55, CD59, CD35 and CUB and Sushi multiple domain 1 (CSMD1).

[0273] Inhibitors of the complement system may be selected from CFI, CFH, FHL-1, C1INH, C4BP, CD46, CD55, CD59, CD35, vitronectin, clusterin and CSMD1 or fragments and / or variants thereof.

[0274] Preferably, the inhibitor of the complement system is selected from CFI, CFH and FHL-1 or fragments and / or variants thereof.

[0275] CFI The protein coding sequence can encode CFI or a fragment and / or variant thereof.

[0276] Complement factor I (CFI) is a trypsin-like serine protease that inhibits the complement system by cleaving three peptide bonds in the alpha chain of C3b and two bonds in the alpha chain of C4b, thereby inactivating these proteins.

[0277] CFI is a glycoprotein heterodimer consisting of disulfide-linked heavy and light chains. The heavy chain has four domains, namely, the FI membrane attack complex (FIMAC) domain, the CD5 domain, and the low density lipoprotein receptor 1 and 2 (LDLr1 and LDLr2) domain. The heavy chain plays an inhibitory role in keeping the enzyme inactive until it encounters a complex formed by the substrate (C3b or C4b) and cofactor proteins (Factor H, C4b binding protein, complement receptor 1, and membrane cofactor protein). When the enzyme binds to the substrate:cofactor complex, the heavy:light chain interface is disrupted and the enzyme is activated by an allosteric effect. The light chain contains only a serine protease domain. This domain contains the catalytic triad His-362, Asp-411, and Ser-507, and is responsible for the specific cleavage of C3b and C4b.

[0278] CFI or a fragment and / or variant thereof may be capable of cleaving C3b into iC3b and / or may be capable of cleaving iC3b into C3d,g.

[0279] Fragments and / or variants of CFI may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivating activity and iC3b degrading activity of native CFI. The C3b inactivating activity and iC3b degrading activity of the fragments and / or variants of CFI and native CFI may be measured using any suitable method known to one of skill in the art, for example, using a proteolytic assay.

[0280] Preferably, the CFI is a human CFI. An example of a human CFI is the CFI having UniProtKB accession number P05156.

[0281] Suitably, the CFI may comprise or consist of the polynucleotide sequence set forth as SEQ ID NO:49, or a variant that is at least 70% identical to SEQ ID NO:49.

[0282] An exemplary CFI polypeptide sequence (SEQ ID NO:49): [ka]

[0283] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:49.

[0284] An example of a nucleotide sequence encoding CFI is NM_000204.5. Suitably, the protein coding sequence encoding CFI may comprise or consist of the polynucleotide sequence set forth as SEQ ID NO:50, or a variant that is at least 70% identical to SEQ ID NO:50.

[0285] An exemplary CFI polynucleotide sequence (SEQ ID NO:50): [ka]

[0286] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:50.

[0287] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:47 is operably linked to a protein coding sequence encoding CFI or a fragment and / or variant thereof.

[0288] In some embodiments, a promoter consisting of the nucleotide sequence of SEQ ID NO:47 is operably linked to a protein coding sequence encoding CFI or a fragment and / or variant thereof.

[0289] CFH The protein coding sequence may encode CFH or a fragment and / or variant thereof.

[0290] Complement factor H (CFH) regulates complement activation on self cells and surfaces. CFH competes with complement factor B (CFB) binding to C3b, acts as a cofactor for the CFI-catalyzed proteolytic cleavage of C3b, and promotes the irreversible dissociation of C3bBb and C3b2Bb into their separate components. Thus, CFH not only inhibits the formation of convertases, but also shortens the lifetime of any convertase complexes that are formed.

[0291] CFH is a large (155 kDa) soluble glycoprotein. CFH is composed of a total of 20 domains, each of which contains approximately 60 amino acid residues, called complement control protein modules (CCPs) or short consensus repeats, linked by short linkers of 3-8 residues. The CCP modules are numbered 1-20 (from the N-terminus of the protein), with CCPs 1-4 and 19-20 associating with C3b, while CCPs 7 and 19-20 bind GAG and sialic acid.

[0292] CFH or its fragments and / or variants may have the ability to bind C3b and / or C3d, and / or act as a cofactor for the CFI-catalyzed proteolytic cleavage of C3b, and / or increase the irreversible dissociation of C3bBb and C3b2Bb into their separate components. CFH fragments and / or variants may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native CFH. The activity of CFH fragments and / or variants and native CFH may be measured using any suitable method known to those skilled in the art.

[0293] Preferably, the CFH is human CFH. An example of a human CFH is the CFH having UniProtKB accession number P08603.

[0294] Suitably, CFH may comprise or consist of the polynucleotide sequence shown in SEQ ID NO:51, or a variant which is at least 70% identical to SEQ ID NO:51. An exemplary CFH polypeptide sequence (SEQ ID NO:51): [ka]

[0295] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:51.

[0296] An example of a nucleotide sequence encoding CFH is NM_000186.4. Suitably, the protein coding sequence encoding CFH may comprise or consist of the polynucleotide sequence shown as SEQ ID NO:52, or a variant that is at least 70% identical to SEQ ID NO:52.

[0297] An exemplary CFH polynucleotide sequence (SEQ ID NO:52): [ka] [ka]

[0298] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:52.

[0299] A CFH fragment may be a splice variant, for example, complement factor H-like protein 1 (FHL-1) is a CFH gene splice variant that is nearly identical to the N-terminal 7 domains (CCP 1-7) of CFH.

[0300] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:47 is operably linked to a protein coding sequence encoding CFH or a fragment and / or variant thereof.

[0301] In some embodiments, a promoter comprised of the nucleotide sequence of SEQ ID NO:47 is operably linked to a protein coding sequence encoding CFH or a fragment and / or variant thereof.

[0302] FHL-1 The protein coding sequence may encode FHL-1 or a fragment and / or variant thereof.

[0303] FHL-1 or a fragment and / or variant thereof may have the ability to bind C3b and / or C3d. The fragment and / or variant of FHL-1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native FHL-1. The activity of the fragment and / or variant of FHL-1 and native FHL-1 may be measured using any suitable method known to the skilled artisan.

[0304] Preferably, the FHL-1 is human FHL-1. An example of human FHL-1 is FHL-1 having NCBI Reference Sequence: NP_001014975.1.

[0305] Suitably, FHL-1 may comprise or consist of a polypeptide sequence as shown in SEQ ID NO:53, or a variant which is at least 70% identical to SEQ ID NO:53.

[0306] An exemplary FHL-1 polypeptide sequence (SEQ ID NO:53): [ka]

[0307] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:53.

[0308] An example of a nucleotide sequence encoding FHL-1 is NM_001014975.2. Suitably, the protein coding sequence encoding FHL-1 may comprise or consist of the polynucleotide sequence set forth as SEQ ID NO:54, or a variant that is at least 70% identical to SEQ ID NO:54.

[0309] An exemplary FHL-1 polynucleotide sequence (SEQ ID NO:54): [ka] [ka]

[0310] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:54.

[0311] In some embodiments, a promoter comprising or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:47 is operably linked to a protein coding sequence encoding FHL-1 or a fragment and / or variant thereof.

[0312] In some embodiments, a promoter consisting of the nucleotide sequence of SEQ ID NO:47 is operably linked to a protein coding sequence encoding FHL-1 or a fragment and / or variant thereof.

[0313] Other Regulatory Elements In addition to the promoter of the present invention, the polynucleotide may contain one or more additional regulatory sequences that can act before or after transcription.Preferably, the protein coding sequence may be operably linked to one or more additional regulatory sequences.The one or more additional regulatory sequences can promote the expression of protein in glomerular cells (e.g., podocytes).

[0314] A "regulatory sequence" is any sequence that promotes the expression of a polypeptide, for example, acts to increase the expression of a transcript or to improve the stability of an mRNA. Suitable additional regulatory sequences include, for example, enhancer elements, posttranscriptional regulatory elements, and polyadenylation sites.

[0315] Suitably, the polynucleotide of the present invention does not comprise any further regulatory sequences (apart from the promoter of the present invention).

[0316] Enhancer The polynucleotide of the present invention may include an enhancer. Preferably, the enhancer is operably linked to the protein coding sequence. The enhancer can promote the expression of the protein in glomerular cells (e.g., podocytes). Preferably, the enhancer is a mammalian enhancer, e.g., a human enhancer.

[0317] An "enhancer" is a region of DNA that can increase the likelihood that transcription of a particular gene will occur upon binding of a protein (activator). Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream of its start site. Any suitable enhancer may be used, and its selection can be readily made by one of ordinary skill in the art.

[0318] The polynucleotide of the present invention may comprise a tissue-specific enhancer. As used herein, a "tissue-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in a particular cell or tissue. Advantageously, a tissue-specific enhancer can promote higher expression of a gene in a particular cell type compared to other cell types. Higher expression may be measured, for example, by measuring the expression of a transgene, such as GFP, operably linked to the enhancer, and the expression of the transgene correlates with the ability of the enhancer to promote the expression of a gene. For example, a tissue-specific enhancer may be an enhancer that promotes at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher gene expression levels in a particular cell type compared to expression levels in other cell types.

[0319] Suitable tissue-specific enhancers will be known to those skilled in the art. The enhancer may be a kidney-specific enhancer, preferably a glomerulus-specific enhancer, more preferably a podocyte-specific enhancer. Suitably, the enhancer may be operably linked to the protein coding sequence.

[0320] Suitably, the enhancer may be or may be derived from an enhancer associated with a gene selectively expressed in human kidney, glomerular cells and / or podocytes. Methods for identifying enhancer regions associated with a gene will be well known to those skilled in the art.

[0321] Preferably, the podocyte-specific enhancer is the NPHS1 or NPHS2 enhancer, or a fragment or derivative thereof. Preferably, the podocyte-specific enhancer is the NPHS1 enhancer, or a fragment or derivative thereof.

[0322] The NPHS1 enhancer has been described in Guo, G., et al., 2004. Journal of the American Society of Nephrology, 15(11), pp.2851-2856. A 186 bp fragment from the human NPHS1 promoter was capable of inducing podocyte-specific expression of a β-galactosidase transgene when placed in front of a heterologous minimal promoter in transgenic mice.

[0323] Suitably, the NPHS1 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO:27, or a variant which is at least 70% identical to SEQ ID NO:27.

[0324] An exemplary NPHS1 enhancer (SEQ ID NO:27): [ka]

[0325] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:27.

[0326] Preferably, the polynucleotide of the present invention does not contain an enhancer.

[0327] Kozak sequence The polynucleotide of the present invention may comprise a Kozak sequence. Preferably, the Kozak sequence is operably linked to a protein coding sequence. The Kozak sequence may be inserted before the start codon of a protein to improve translation initiation.

[0328] Suitable Kozak sequences will be known to those skilled in the art.

[0329] Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO:28, or a variant which is at least 65% identical to SEQ ID NO:28.

[0330] An exemplary Kozak sequence (SEQ ID NO:28): [ka]

[0331] Suitably, the variant may be at least 75%, at least 85% or at least 90% identical to SEQ ID NO:28.

[0332] Preferably, the polynucleotides of the present invention do not contain a Kozak sequence.

[0333] Post-transcriptional regulatory elements The polynucleotide of the present invention may comprise a post-transcriptional regulatory element. Preferably, the post-transcriptional regulatory element is operably linked to a protein coding sequence. The post-transcriptional regulatory element may improve gene expression.

[0334] The polynucleotide may include a Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE), suitably operably linked to a protein coding sequence.

[0335] The WPRE sequence may have substitutions, deletions or insertions within the X antigen promoter and / or the start codon of the X antigen, which may prevent the production of a functional X antigen.

[0336] Suitably, the WPRE may comprise or consist of the nucleotide sequence set forth as SEQ ID NO:29, or a variant which is at least 70% identical to SEQ ID NO:29.

[0337] An exemplary WPRE (SEQ ID NO:29): [ka]

[0338] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO:29.

[0339] Preferably, the polynucleotide of the present invention does not contain post-transcriptional regulatory elements.

[0340] Polyadenylation Signal The polynucleotide of the present invention may comprise a polyadenylation signal. Advantageously, the polyadenylation signal may be operably linked to the protein coding sequence. The polyadenylation signal may improve gene expression.

[0341] Suitable polyadenylation signals include early SV40 polyadenylation signal (SV40pA), chicken beta-globin polyadenylation signal, bovine growth hormone polyadenylation signal (bGH) or soluble neuropilin-1 polyadenylation signal.In some embodiments, the polyadenylation signal is early SV40 polyadenylation signal (SV40pA) or chicken beta-globin polyadenylation signal.Preferably, the polyadenylation signal is early SV40 polyadenylation signal (SV40pA).

[0342] Suitably, the polyadenylation signal may comprise or consist of a nucleotide sequence as set forth in SEQ ID NO: 30, or a variant which is at least 70% identical to SEQ ID NO: 30. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 30.

[0343] An exemplary SV40pA signal sequence (SEQ ID NO:30): [ka]

[0344] Suitably, the polyadenylation signal may comprise or consist of a nucleotide sequence as set forth in SEQ ID NO: 31, or a variant which is at least 70% identical to SEQ ID NO: 31. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 31.

[0345] An exemplary bGH poly(A) signal sequence (SEQ ID NO:31): [ka]

[0346] Suitably, the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 32, or a variant which is at least 70% identical to SEQ ID NO: 32. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 33.

[0347] An exemplary soluble neuropilin-1 polyadenylation signal (SEQ ID NO:32): [ka]

[0348] Suitably, the polyadenylation signal may comprise or consist of the nucleotide sequence set forth as SEQ ID NO: 42, or a variant which is at least 70% identical to SEQ ID NO: 42. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 42.

[0349] Exemplary Chicken Beta-Globin Polyadenylation Signal (SEQ ID NO:42) [ka]

[0350] vector The present invention provides a vector comprising a polynucleotide of the present invention.

[0351] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. The four main types of vectors are plasmids, viral vectors, cosmids and artificial chromosomes.

[0352] Preferably, the vector of the present invention is a viral vector. The vector of the present invention is preferably an adeno-associated viral (AAV) vector, although it is contemplated that other viral vectors may be used.

[0353] The vector of the present invention may be in the form of a viral vector particle. Preferably, the viral vector of the present invention is in the form of an AAV vector particle.

[0354] The method of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, is well known in the art.Suitable methods are described in Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436, Merten, OW, et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017, and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489.

[0355] The vectors of the invention may be capable of transducing kidney cells, hi some embodiments, the vectors of the invention are capable of specifically transducing kidney cells.

[0356] The vectors of the present invention are preferably capable of transducing glomerular cells (e.g., podocytes). In some embodiments, the vectors of the present invention are capable of specifically transducing glomerular cells (e.g., podocytes). The vectors of the present invention are preferably capable of transducing podocytes. In some embodiments, the vectors of the present invention are capable of specifically transducing podocytes.

[0357] Adeno-associated virus (AAV) vectors The vector of the invention may be an adeno-associated virus (AAV) vector. The vector of the invention may be in the form of an AAV vector particle.

[0358] AAV genome An AAV vector or vector particle may comprise the AAV genome, or a fragment or derivative thereof.

[0359] AAV genome is a polynucleotide sequence, and the sequence may code for the functions required for the production of AAV particles.These functions include those that operate in the replication and packaging cycle of AAV in host cells, including the encapsidation of the AAV genome into AAV particles.Naturally occurring AAV is replication-deficient and depends on the provision of helper functions in trans to complete the replication and packaging cycle.Therefore, the AAV genome of the AAV vector of the present invention is typically replication-deficient.

[0360] The AAV genome may be in single-stranded form (ssAAV) of either positive or negative strand, or alternatively in double-stranded form (dsAAV). The use of double-stranded form allows the avoidance of the DNA replication step in the target cell, and therefore accelerates the expression of the transgene. The maximum packaging capacity of the single-stranded form is greater than that of the double-stranded form. Preferably, the AAV genome is in single-stranded form.

[0361] Naturally occurring AAVs can be classified according to various biological systems. The AAV genome can be from any naturally occurring serotype, isolate or clade of AAV.

[0362] AAV may be referred to in terms of its serotype. Serotypes represent variant subspecies of AAV, which have characteristic reactivity that can be used to distinguish them from other variant subspecies due to their capsid surface antigen expression profile. Typically, AAV vector particles with a particular AAV serotype do not cross-react efficiently with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. In some embodiments, the AAV vector of the present invention may be AAV3B, LK03, AAV9 or AAV8 serotype. In some embodiments, the AAV vector of the present invention may be AAV3B, LK03 or AAV9 serotype.

[0363] AAV can also be referred to in terms of clades or clones. This refers to the phylogenetic relationship of AAVs from nature, and typically refers to the lineage of AAVs that can be traced back to a common ancestor and includes all its descendants. In addition, AAV can also be referred to in terms of specific isolates, i.e., genetic isolates of specific AAVs found in nature. The term genetic isolates refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, and thus defines a population that is recognizably different at the genetic level.

[0364] Typically, the AAV genome of a naturally occurring serotype, isolate or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional origin of replication and allows the integration and excision of the vector from the genome of the cell. The ITR may be the only sequence required in cis next to the therapeutic gene.

[0365] The AAV genome may also include packaging genes, such as the rep and / or cap genes, which code for packaging functions for AAV particles. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include p5, p19 and p40 promoters. For example, the p5 and p19 promoters are generally used to express the rep gene, and the p40 promoter is generally used to express the cap gene. The rep gene codes for one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene codes for one or more capsid proteins, such as VP1, VP2 and VP3 or variants thereof. These proteins constitute the capsid of the AAV particle, which determines the AAV serotype. VP1, VP2, and VP3 can be produced by alternative mRNA splicing (Trempe, JP and Carter, BJ, 1988. Journal of virology, 62(9), pp.3356-3363). Thus, VP1, VP2, and VP3 may have the same sequence, but VP2 is truncated at the N-terminus compared to VP1, and VP3 is truncated at the N-terminus compared to VP2.

[0366] The AAV genome may be the entire genome of a naturally occurring AAV. For example, an AAV vector or vector particle may be prepared by using a vector that contains the entire AAV genome.

[0367] Preferably, the AAV genome is derivatized for administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome, and derivatives that can be made by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Preferably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Preferably, the AAV genome is a derivative of AAV2.

[0368] The derivative of AAV genome includes any truncated or modified AAV genome that allows the expression of transgene from the AAV vector of the present invention in vivo.Typically, the AAV genome can be significantly truncated to contain the minimum viral sequence but retain the above functions.This is preferred for safety reasons, since it reduces the risk of the vector recombining with wild-type virus and also avoids the induction of cellular immune response due to the presence of viral gene protein in target cells.

[0369] Typically, the derivative will contain at least one inverted terminal repeat (ITR), preferably two or more ITRs, for example two ITRs or more. One or more of the ITRs may be derived from AAV genomes with different serotypes, or may be chimeric or mutated ITRs. A preferred mutated ITR is one that has a trs (terminal resolution site) deletion. This deletion allows the genome to replicate continuously, creating a single-stranded genome that contains both coding and complementary sequences, i.e., a self-complementary AAV (scAAV) genome. This allows the avoidance of DNA replication in target cells, and therefore accelerates the expression of the transgene. However, the maximum packaging capacity of scAAV is reduced. Preferably, the AAV genome is not a scAAV genome.

[0370] The AAV genome may comprise one or more ITR sequences or variants thereof from any naturally occurring serotype, isolate or clade of AAV.The AAV genome may comprise at least one, for example two, ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 or variants thereof.Preferably, the AAV genome may comprise at least one, for example two, AAV2 ITRs.

[0371] The inclusion of one or more ITRs is preferred to aid in the formation of AAV vector concatemers in the host cell nucleus, for example after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. The formation of such episomal concatemers protects the AAV vector during the life of the host cell, thereby allowing long-term expression of the transgene in vivo.

[0372] Advantageously, the ITR elements will be the only sequences that are retained from the native AAV genome in the derivative. The derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons mentioned above and also to reduce the possibility of the vector integrating into the host cell genome. Furthermore, the reduced size of the AAV genome allows for greater flexibility in incorporating other sequence elements (e.g., regulatory elements) as well as transgenes into the vector.

[0373] Thus, the derivatives of the invention may have the following portions removed: one inverted terminal repeat (ITR) sequence, the replication (rep) and the capsid (cap) genes. However, the derivatives may further comprise one or more of the rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency into a specific site on human chromosome 19 and shows a negligible frequency of random integration, therefore retention of integration capacity in AAV vectors may be acceptable in a therapeutic setting.

[0374] The present invention further encompasses the provision of sequences of the AAV genome in an order and arrangement that differs from that of the native AAV genome. The present invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from two or more viruses. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

[0375] AAV serotypes and capsid proteins The AAV vector particle may be encapsidated by a capsid protein. The serotype can facilitate transduction of glomerular cells (e.g., podocytes), e.g., specific transduction of glomerular cells (e.g., podocytes).

[0376] The AAV vector particle may be a kidney-specific vector particle. Preferably, the AAV vector particle is a glomerulus-specific (e.g., podocyte-specific) vector particle. The AAV vector particle may be encapsidated by a glomerulus-specific (e.g., podocyte-specific) capsid. The AAV vector particle may comprise a glomerulus-specific (e.g., podocyte-specific) capsid protein.

[0377] Advantageously, AAV vector particles may be in a transcapsidated form, in which the AAV genome or derivative with ITR of one serotype is packaged in the capsid of a different serotype.The AAV vector particles also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid.The AAV vector particles also include chemically modified forms with ligands adsorbed on their capsid surface.For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0378] Where the derivative comprises capsid proteins, i.e., VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAV. In particular, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones or isolates of AAV within the same vector (i.e., pseudotype vectors). The AAV vector may be in the form of a pseudotyped AAV vector particle.

[0379] Chimeric, shuffled or capsid modified derivatives will typically be selected to provide one or more desired functionalities to the AAV vector. Thus, these derivatives may exhibit increased efficiency of gene delivery, reduced immunogenicity (humoral or cellular), altered tropism range and / or improved targeting of podocytes compared to AAV vectors containing naturally occurring AAV genomes. Increased efficiency of gene delivery may be achieved by improved receptor or co-receptor binding at the cell surface, improved internalization, improved transport into cells and nuclei, improved uncoating of viral particles and improved conversion of single-stranded genome to double-stranded form. Increased efficiency may also be related to altered tropism range or targeting of podocytes, such that the vector dose is not diluted by administration to tissues that do not require it.

[0380] Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes.This may be achieved, for example, by marker rescue methods, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select for capsid sequences with desired properties.The capsid sequence of the different serotype can be altered in cells by homologous recombination to produce novel chimeric capsid proteins.

[0381] Chimeric capsid proteins also include those created by manipulation of capsid protein sequences to transfer particular capsid protein domains, surface loops or particular amino acid residues between two or more capsid proteins, for example between two or more capsid proteins of different serotypes.

[0382] Shuffled or chimeric capsid proteins may also be produced by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be produced by randomly fragmenting the sequences of related AAV genes, such as AAV genes encoding capsid proteins of several different serotypes, and then reassembling the fragments in a self-priming polymerase reaction that may also cause crossover between regions of sequence homology. A library of hybrid AAV genes produced by shuffling the capsid genes of several serotypes in this way can be screened to identify viral clones with desired functionality. Similarly, AAV capsid genes may be randomly mutated using error-prone PCR to produce a diverse library of variants that can then be selected for desired properties.

[0383] The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions or insertions to the native wild-type sequence. In particular, the capsid gene may be modified by the insertion of a sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide may advantageously function as a ligand for a particular cell type, thereby providing improved binding to the target cell or improving the specificity of the targeting of the vector to a particular cell population. The unrelated protein may also be an epitope or affinity tag that aids in the purification of the viral particle as part of the production process. The site of insertion will typically be selected so as not to interfere with other functions of the viral particle, such as the internalization, transport of the viral particle.

[0384] The capsid protein may be an artificial or mutated capsid protein. As used herein, the term "artificial capsid" means that the capsid particle comprises an amino acid sequence that does not occur in nature or that is engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein comprises a mutation or alteration in the amino acid sequence compared to the sequence of the parent capsid from which the artificial capsid amino acid sequence is derived, when the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned.

[0385] The capsid protein may contain a mutation or modification compared to a wild-type capsid protein that improves the ability to transduce podocytes compared to unmodified or wild-type viral particles. The improved ability to transduce podocytes may be measured, for example, by measuring the expression of a transgene, such as GFP, carried by the AAV vector particle, and expression of the transgene in podocytes correlates with the ability of the AAV vector particle to transduce podocytes.

[0386] The AAV vector particle may be AAV3B, LK03, AAV9, or AAV8 vector particle.The present inventors have shown that AAV vector particles with AAV3B, LK03, AAV9 and AAV8 serotypes can transduce podocytes.Preferably, the AAV vector particle is AAV3B vector particle or LK03 vector particle.More preferably, the AAV vector particle is AAV3B vector particle.

[0387] The AAV vector particle may comprise AAV3B, LK03, AAV9, or AAV8 capsid protein.Preferably, the AAV vector particle comprises AAV3B capsid protein or LK03 capsid protein.More preferably, the AAV vector particle comprises AAV3B capsid protein.

[0388] The AAV vector particle may comprise AAV3B, LK03, AAV9, or AAV8 capsid proteins VP1, VP2, and VP3.Preferably, the AAV vector particle comprises AAV3B or LK03 capsid proteins VP1, VP2, and VP3.More preferably, the AAV vector particle comprises AAV3B capsid proteins VP1, VP2, and VP3.

[0389] AAV vector particles may comprise one or more AAV2 ITR sequences and AAV3B capsid protein, LK03 capsid protein, AAV9 capsid protein, or AAV8 capsid protein.Preferably, the AAV vector particles comprise one or more AAV2 ITR sequences and AAV3B or LK03 capsid protein.More preferably, the AAV vector particles comprise one or more AAV2 ITR sequences and AAV3B capsid protein.

[0390] The AAV vector particle may have an AAV2 genome and an AAV3B capsid protein (AAV2 / 3B), an AAV2 genome and an LK03 capsid protein, an AAV2 genome and an AAV9 capsid protein (AAV2 / 9), or an AAV2 genome and an AAV8 capsid protein (AAV2 / 8).Preferably, the AAV vector particle comprises an AAV2 genome and an AAV3B or LK03 capsid protein.More preferably, the AAV vector particle comprises an AAV2 genome and an AAV3B capsid protein.

[0391] The AAVX / Y nomenclature may indicate a pseudotyped AAV, for example, when the ITR sequences are derived from AAVX and are adjacent to a cassette carrying a payload encapsidated in serotype AAVY (i.e., having AAVY capsid proteins).

[0392] AAV3B serotype The AAV vector particle may comprise AAV3B capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV3B capsid protein.

[0393] Two distinct AAV3 isolates (AAV3A and AAV3B) have been cloned. Compared to vectors based on other AAV serotypes, AAV3 vectors are thought to transduce most cell types inefficiently. However, AAV3B can efficiently transduce podocytes. AAV3B is described in Rutledge, EA, et al., 1998. Journal of virology, 72(1), pp.309-319.

[0394] The AAV vector particle may comprise AAV3B VP1 capsid protein, AAV3B VP2 capsid protein, and / or AAV3B VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV3B VP1 capsid protein, AAV3B VP2 capsid protein, and / or AAV3B VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV3B VP1, VP2, and VP3 capsid proteins.

[0395] Suitably, the AAV3B VP1 capsid protein may comprise or consist of the amino acid sequence set forth as SEQ ID NO:33, or a variant that is at least 90% identical to SEQ ID NO:33.

[0396] Exemplary AAV3B VP1 capsid protein (SEQ ID NO:33): [ka]

[0397] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:33.

[0398] Suitably, the AAV3B VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO:33, or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:33.

[0399] LK03 serotype The AAV vector particle may comprise the LK03 capsid protein. Suitably, the AAV vector particle may be encapsidated by the LK03 capsid protein.

[0400] The AAV-LK03cap sequence is composed of fragments from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, 9) and is described in Lisowski, L., et al., 2014. Nature, 506(7488), pp.382-386. The inventors have shown that the AAV-LK03 vector can achieve high transduction rate of nearly 100% in human podocytes in vitro.

[0401] The AAV vector particle may comprise LK03 VP1 capsid protein, LK03 VP2 capsid protein, and / or LK03 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by LK03 VP1 capsid protein, LK03 VP2 capsid protein, and / or LK03 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by LK03 VP1, VP2, and VP3 capsid proteins.

[0402] Suitably, the LK03 VP1 capsid protein may comprise or consist of the amino acid sequence shown as SEQ ID NO:34, or a variant which is at least 90% identical to SEQ ID NO:34.

[0403] Exemplary LK03 VP1 capsid protein (SEQ ID NO:34): [ka]

[0404] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:34.

[0405] Suitably, the LK03 VP2 and VP3 capsid proteins may be N-terminally truncated versions of SEQ ID NO:34, or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:34.

[0406] AAV9 serotype The AAV vector particle may comprise AAV9 capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV9 capsid protein.

[0407] The present inventors have shown that AAV9 vectors can achieve high transduction in human podocytes in vitro.

[0408] The AAV vector particle may comprise AAV9 VP1 capsid protein, AAV9 VP2 capsid protein, and / or AAV9 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV9 VP1 capsid protein, AAV9 VP2 capsid protein, and / or AAV9 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by AAV9 VP1, VP2, and VP3 capsid proteins.

[0409] Suitably, the AAV9 VP1 capsid protein may comprise or consist of the amino acid sequence set forth as SEQ ID NO:35, or a variant that is at least 90% identical to SEQ ID NO:35.

[0410] Exemplary AAV9 VP1 capsid protein (SEQ ID NO:35): [ka] [ka]

[0411] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:35.

[0412] Suitably, the AAV9 VP2 and VP3 capsid proteins may be N-terminally truncated versions of SEQ ID NO:35, or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:35.

[0413] Other viral vectors Retroviral and lentiviral vectors The vector of the present invention may be a retroviral or lentiviral vector.The vector of the present invention may be a retroviral or lentiviral vector particle.

[0414] Retroviral vectors may be derived or inducible from any suitable retrovirus. Many different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).

[0415] Retroviruses can be broadly classified into two categories: "simple" and "complex". Retroviruses can be further classified into seven groups. Five of these groups are oncogenic retroviruses. The remaining two groups are lentiviruses and spumaviruses.

[0416] The basic structure of retrovirus and lentivirus genomes share many common features, such as 5'LTR and 3'LTR.Between or within these are located packaging signals to allow the genome to be packaged, primer binding sites, integration sites to allow integration into the host cell genome, and gag, pol and env genes that code packaging components (these are polypeptides required for the construction of viral particles).Lentiviruses have additional features, such as the rev gene and RRE sequence in HIV, which allow the efficient transport of the RNA transcripts of integrated provirus from the nucleus to the cytoplasm of infected target cells.

[0417] In the provirus, these genes are flanked on both sides by regions called long terminal repeats (LTRs), which are involved in the integration and transcription of the provirus and can also act as enhancer-promoter sequences to control the expression of viral genes.

[0418] The LTRs are themselves identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary considerably among different retroviruses.

[0419] In a defective retroviral vector genome, gag, pol and env may be absent or not functional.

[0420] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication may be removed from the virus, rendering the viral vector replication-deficient, and a portion of the viral genome may be replaced with a library encoding candidate modulating moieties operably linked to regulatory control regions and reporter moieties in the vector genome to generate vectors containing candidate modulating moieties capable of transducing target host cells and / or integrating their genome into the host genome.

[0421] Lentiviral vectors are part of a larger group of retroviral vectors. Briefly, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immune deficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototypic "slow virus" Visna-Maedi virus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the more recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV).

[0422] The lentivirus family is different from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells.In contrast, other retroviruses, such as MLV, cannot infect non-dividing or slow-dividing cells, such as the cells that make up muscle, brain, lung and liver tissue.

[0423] A lentiviral vector, as used herein, is a vector that contains at least one component part derivable from a lentivirus, preferably the component part involved in the biological mechanism by which the vector infects cells and expresses genes or replicates.

[0424] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not naturally infect primates, especially humans). An example of a non-primate lentivirus may be any member of the Lentiviridae family that does not naturally infect primates.

[0425] As examples of lentivirus-based vectors, HIV-1 and HIV-2 based vectors are described below.

[0426] HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. Sequences spanning the gag open reading frame have been shown to be important for HIV-1 packaging. Thus, HIV-1 vectors often contain the relevant portion of gag in which the translation initiation codon is mutated. In addition, most HIV-1 vectors also contain a portion of the env gene, including the RRE. Rev binds to the RRE, allowing transport of full-length or singly spliced ​​mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, the requirement for Rev and RRE can be alleviated by using constitutive transport elements from certain simple retroviruses, e.g., Mason-Pfizer monkey virus. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0427] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient transport of full-length or singly spliced ​​viral RNA.

[0428] Preferably, the viral vectors used in the present invention have a minimal viral genome.

[0429] By "minimal viral genome" it is to be understood that the viral vector has been engineered to remove non-essential elements and retain essential elements to provide the necessary functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this methodology can be found in WO 1998 / 017815.

[0430] Preferably, the plasmid vector used to produce the viral genome in the host / packaging cell will have sufficient lentiviral genetic information to allow packaging of the RNA genome in the presence of packaging components into viral particles capable of infecting target cells but not capable of independent replication to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0431] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell will also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of said genome in the host cell / packaging cell. These regulatory sequences may be the native sequences associated with the viral sequence to be transcribed (i.e., the 5'U3 region) or they may be a heterologous promoter, such as another viral promoter (e.g., the CMV promoter).

[0432] The vector may be a self-inactivating (SIN) vector, in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated to transduce non-dividing cells in vivo with an efficiency similar to that of wild-type vectors. Transcriptional inactivation of the long terminal repeats (LTRs) in the SIN provirus should prevent recruitment by replication-competent virus. This should in turn allow regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTRs.

[0433] The vector may be integration-defective. Integration-defective lentiviral vectors (IDLV) can be generated, for example, by packaging the vector with a catalytically inactive integrase (e.g., HIV integrase with a D64V mutation at the catalytic site), or by modifying or deleting essential att sequences from the LTR of the vector, or a combination of the above.

[0434] Adenovirus Vectors The vector of the present invention may be an adenoviral vector.The vector of the present invention may be an adenoviral vector particle.

[0435] Adenoviruses are double-stranded, linear DNA viruses that do not undergo an RNA intermediate. There are more than 50 different human adenovirus serotypes, classified into six subgroups based on genetic sequence homology. The natural target of adenoviruses is the respiratory and digestive epithelia, and they generally cause only mild symptoms. Serotypes 2 and 5 (which share 95% sequence homology) are the most commonly used in adenovirus vector systems and are usually associated with upper respiratory tract infections in young people.

[0436] Adenoviruses are used as vectors for gene therapy and expression of foreign genes. Their large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and can replicate efficiently in complementation cell lines, resulting in up to 10 12 Adenoviruses can produce extremely high titers of up to 1000 ng / ml, thus making them one of the best systems to study gene expression in primary non-replicating cells.

[0437] Expression of viral or foreign genes from the adenoviral genome does not require a replicating cell. Adenoviral vectors enter cells by receptor-mediated endocytosis. Once inside the cell, adenoviral vectors rarely integrate into host chromosomes. Instead, they function episomally (independently of the host genome) as linear genomes in the host nucleus. Thus, the use of recombinant adenoviruses alleviates the problems associated with random integration into the host genome.

[0438] Herpes simplex virus vector The vector of the present invention may be a herpes simplex virus vector.The vector of the present invention may be a herpes simplex virus vector particle.

[0439] Herpes simplex virus (HSV) is a neurotropic DNA virus with advantageous properties as a gene delivery vector. HSV is highly infectious, making HSV vectors an efficient vehicle for the delivery of exogenous genetic material into cells. Viral replication is easily blocked by null mutations in immediate early genes that can be complemented in trans in vitro, allowing for the facile production of high titer, pure preparations of non-pathogenic vector. Its genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuron nuclei for the duration of the host's lifespan. The vector is non-pathogenic and cannot reactivate for long-term persistence. By utilizing latency active promoter complexes in the vector design, long-term, stable transgene expression in the nervous system can be obtained. Due to the broad expression pattern of the cellular receptors recognized by the virus, HSV vectors transduce a wide variety of tissues. An improved understanding of the processes involved in cell entry has enabled targeting of the tropism of HSV vectors.

[0440] Other viral vectors Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42.

[0441] The vector of the invention may be an alphavirus vector. The vector of the invention may be an alphavirus vector particle. The vector of the invention may be a flavivirus vector. The vector of the invention may be a flavivirus vector particle.

[0442] Self-amplifying ssRNA viruses consist of alphaviruses (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (e.g., Kunjin virus, West Nile virus, and Dengue virus) with genomes of positive polarity. Alphaviruses have been applied primarily in preclinical gene therapy research for cancer treatment. Alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors, and recombinant replication-deficient or replication-competent particles.

[0443] The vector of the present invention can be a rhabdovirus vector.The vector of the present invention can be a rhabdovirus vector particle.The vector of the present invention can be a measles virus vector.The vector of the present invention can be a measles virus vector particle.

[0444] Rhabdoviruses (e.g., rabies and vesicular stomatitis viruses) and measles virus have negative-strand genomes. Among the rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied in preclinical gene therapy studies. Measles virus (e.g., MV-Edm) has found many gene therapy applications.

[0445] The vector of the present invention may be a Newcastle Disease Virus vector.The vector of the present invention may be a Newcastle Disease Virus vector particle.

[0446] The ssRNA paramyxovirus Newcastle disease virus (NDV) has been frequently applied in cancer gene therapy because it replicates specifically in tumor cells.

[0447] The vector of the present invention may be a poxvirus vector.The vector of the present invention may be a poxvirus vector particle.

[0448] The characteristic feature of poxviruses is their dsDNA genome, which can fully accommodate more than 30 kb of foreign DNA. Poxviruses have found some applications as gene therapy vectors. For example, vaccinia virus vectors have demonstrated the potential for cancer treatment. Vaccinia virus is a large enveloped virus with a linear double-stranded DNA genome of approximately 190 kb. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which makes it useful for the delivery of large genes. A number of attenuated vaccinia virus strains suitable for gene therapy applications are known in the art, such as MVA and NYVAC strains.

[0449] The vector of the present invention may be a picornavirus vector.The vector of the present invention may be a picornavirus vector particle.

[0450] Picornoviruses are non-enveloped ssRNA viruses. Coxsackieviruses, which belong to the Picornaviridae family, have been used as oncolytic vectors.

[0451] Variants, derivatives, analogues, homologues and fragments In addition to the specific proteins and nucleotides described herein, the present invention also encompasses variants, derivatives, homologues and fragments thereof.

[0452] In the context of the present invention, a "variant" of any given sequence is a sequence in which the residues (whether amino acid residues or nucleic acid residues) of the particular sequence are modified in such a manner that the polypeptide or polynucleotide retains at least one of its intrinsic functions. A variant sequence can be obtained by adding, deleting, substituting, modifying, replacing and / or mutating at least one residue present in a naturally occurring polypeptide or polynucleotide. For example, a variant promoter sequence retains at least some level of the activity and specificity of the promoter sequence from which it is derived.

[0453] The term "derivative" as used herein in reference to a protein or polypeptide of the invention includes any substitution, mutation, modification, exchange, deletion and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0454] Typically, amino acid substitutions may be made, for example from 1, 2 or 3 to 10 or 20 substitutions, provided that the altered sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0455] Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, so long as the intrinsic function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine and tyrosine.

[0456] Conservative substitutions may be made, for example, according to the following table. Amino acids listed in the same column in column 2 and preferably in the same line in column 3 may be substituted for each other:

[0457] [Table 1]

[0458] As used herein, the term "homolog" refers to a variant having a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" can be equated with "identity."

[0459] In this context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Typically, a homologue will contain the same active site etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), it is preferred in the context of the present invention to express homology in terms of sequence identity.

[0460] In this context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, it is preferred in the context of the present invention to express homology in terms of sequence identity.

[0461] Preferably, reference to a sequence showing a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence showing that stated percent identity over the full length of the referenced SEQ ID NO.

[0462] Homology comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0463] Percent homology may be calculated over a continuous sequence, i.e., one sequence is aligned with the other sequence, and then each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called "ungapped" alignment. Typically, such ungapped alignment is performed only over a relatively small number of residues.

[0464] Although this is a very simple and consistent method, it does not take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion in an amino acid or nucleotide sequence may exclude subsequent residues or codons from alignment, thus resulting in a significant decrease in the percentage of homology when performing a global alignment. As a result, most sequence comparison methods are designed to generate optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in order to try to maximize local homology.

[0465] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, so that a sequence alignment that contains as few gaps as possible, reflecting a higher relatedness between the two compared sequences for the same number of identical amino acids or nucleotides, scores higher than one that contains many gaps. Typically, an "affine gap cost" is used, which sets a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties naturally result in optimized alignments that contain fewer gaps. Most alignment programs allow the gap penalty to be modified. However, when using such software for sequence comparison, it is preferred to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0466] Calculation of maximum homology percentage therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can carry out sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the comparison tool of the GENEWORKS suite. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0467] Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​usually used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs usually use either the public default values ​​or a custom symbol comparison table if provided (see user manual for further details). For some applications, it is preferred to use the public default values ​​for the GCG package, or in the case of other software, the default matrix, e.g., BLOSUM62.

[0468] Once the software has produced an optimal alignment, it is possible to calculate the percentage of homology, preferably the percentage of sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percentage of sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the referenced SEQ ID NO.

[0469] A "fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is of functional or, for example, interest in an assay. A "fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0470] Such variants, derivatives, homologues and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When making an insert, a synthetic DNA may be made that encodes the insert, along with 5' and 3' flanking regions that correspond to the naturally occurring sequence on either side of the insertion site. The flanking regions will contain convenient restriction sites that correspond to sites in the naturally occurring sequence, so that the naturally occurring sequence can be cleaved with the appropriate enzyme(s) and the synthetic DNA can be ligated to the cleavage. The DNA is then expressed according to the present invention to make the encoded protein. These methods are merely illustrative of the numerous standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0471] cell In one aspect, the invention provides a cell comprising a polynucleotide or vector of the invention. The cell may be an isolated cell. The cell may be a human cell, preferably an isolated human cell.

[0472] A vector containing a polynucleotide for use in the present invention may be introduced into a cell using various techniques known in the art, such as transfection, transduction and transformation. Preferably, the vector of the present invention is introduced into a cell by transfection or transduction.

[0473] The cells can be of any cell type known in the prior art.

[0474] Suitably, the cell may be a producer cell. The term "producer cell" includes cells that produce viral particles after transient transfection, stable transfection or vector transduction of all elements required to produce said viral particles, or any cell engineered to stably contain the elements required to produce viral particles. Suitable producer cells will be well known to those skilled in the art. Suitable producer cell lines include HEK293 (e.g. HEK293T), HeLa, and A549 cell lines.

[0475] Suitably, the cell may be a packaging cell. The term "packaging cell" includes cells that contain some or all of the elements required to package an infectious recombinant virus. The packaging cell may be deficient in recombinant viral vector genome. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins. Cells that contain only some of the elements required for the production of enveloped viral particles are useful as intermediate reactants in the creation of viral particle producer cell lines through subsequent steps of transient transfection, transduction or stable integration of each of the further required elements. These intermediate reactants are encompassed by the term "packaging cell". Suitable packaging cells will be well known to those skilled in the art.

[0476] Suitably, the cell may be a kidney cell or a glomerular cell, such as a podocyte. Suitably, the cell may be an immortalized kidney cell or a glomerular cell, such as an immortalized podocyte. Suitable podocyte cell lines, such as CIHP-1, are well known to those skilled in the art. Methods for generating immortalized podocytes are well known to those skilled in the art. A suitable method is described in Ni, L., et al., 2012. Nephrology, 17(6), pp.525-531.

[0477] Pharmaceutical Compositions In one aspect, the invention provides a pharmaceutical composition comprising a polynucleotide or vector of the invention or a cell of the invention.

[0478] A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharma- ceutically active substance, i.e., a vector. A pharmaceutical composition preferably comprises a pharma- ceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0479] "Pharmaceutically acceptable" includes that the formulation is sterile and pyrogen-free. The carriers, diluents, and / or excipients must be "acceptable" in the sense of being compatible with the vector and not harmful to the recipient thereof. Typically, the carriers, diluents, and excipients will be saline or injection media, which will be sterile and pyrogen-free, although other acceptable carriers, diluents, and excipients may be used.

[0480] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical arts. The choice of pharmaceutical carrier, excipient or diluent can be selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may contain any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s) as, or in addition to, the carrier, excipient or diluent.

[0481] Examples of pharma- ceutically acceptable carriers include, for example, water, saline, alcohol, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, flavor oils, fatty acid mono- and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0482] The vector, cell or pharmaceutical composition according to the present invention may be administered in a manner suitable for treating and / or preventing the diseases described herein. The appropriate dosage may be determined by clinical trials, but the amount and frequency of administration will be determined by factors such as the subject's health status, and the type and severity of the subject's disease. The pharmaceutical composition may be appropriately formulated.

[0483] The vector, cell or pharmaceutical composition according to the invention may be administered parenterally, e.g., intravenously or by infusion techniques. The vector, cell or pharmaceutical composition may be administered in the form of a sterile aqueous solution which may contain other substances, e.g., sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of 3 to 9). The pharmaceutical composition may be suitably formulated. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0484] The vectors, cells or pharmaceutical compositions according to the invention may be administered systemically, for example by intravenous injection.

[0485] The vectors, cells or pharmaceutical compositions according to the invention may be administered locally, for example by targeting administration to the kidney. Suitably, the vectors, cells or pharmaceutical compositions may be administered by injection into the renal artery, or by ureteral or subthecal injection.

[0486] The pharmaceutical composition may comprise the vector or cells of the invention in an injection medium, such as a sterile isotonic solution, which may be enclosed in an ampoule, disposable syringe or multiple dose vial made of glass or plastic.

[0487] The vector, cell or pharmaceutical composition may be administered in a single or multiple doses. In particular, the vector, cell or pharmaceutical composition may be administered in a single, one-time dose. The pharmaceutical composition may be appropriately formulated.

[0488] The vectors, cells or pharmaceutical compositions may be administered in a variety of doses (e.g., measured in vector genomes (vg) / kg). In any event, a physician will determine the actual dosage that will be most appropriate for any individual subject, which will vary with the age, weight, and response of that particular subject. Typically, however, in the case of the AAV vectors of the present invention, a dose of 10 10 ~10 14vg / kg, or 10 11 ~10 13 Doses of vg / kg may be administered.

[0489] The pharmaceutical composition may further comprise one or more other therapeutic agents.

[0490] The invention further includes the use of kits comprising the vectors, cells and / or pharmaceutical compositions of the invention. Preferably, the kits are for use in a method and are used as described herein, e.g., in the therapeutic methods described herein. Preferably, the kits include instructions for use of the components of the kit.

[0491] Methods for Treating and / or Preventing Disease In one aspect, the invention provides a vector, a cell or a pharmaceutical composition according to the invention for use as a medicament.

[0492] In one aspect, the invention provides the use of a vector, a cell or a pharmaceutical composition according to the invention in the manufacture of a medicament.

[0493] In one aspect, the present invention provides a method of administering a vector, a cell or a pharmaceutical composition according to the present invention to a subject in need thereof.

[0494] Glomerular disorders The vector, cell or pharmaceutical composition according to the invention can be used to treat a glomerular disease in a subject. Preferably, the subject is a human subject.

[0495] In one aspect, the invention provides a vector, a cell or a pharmaceutical composition according to the invention for use in the prevention or treatment of a glomerular disease.

[0496] In a related aspect, the invention provides the use of a vector, a cell or a pharmaceutical composition according to the invention for the manufacture of a medicament for preventing or treating a glomerular disease.

[0497] In a related aspect, the present invention provides a method for preventing or treating a glomerular disease comprising administering to a subject in need thereof a vector, a cell or a pharmaceutical composition according to the present invention.

[0498] Glomerular diseases can be classified as either nephrotic or nephritic. Nephrotic syndromes are often associated with factors that affect the integrity of podocyte-podocyte or podocyte-GBM interactions. In contrast, factors involved in the pathogenesis of nephritic syndromes can vary but can include circulating platelets and leukocytes, GBM, resident glomerular endothelial cells and mesangial cells. (Chiang, CK and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0499] Preferably, the glomerular disease is a hereditary glomerular disease, i.e., a genetic glomerular disease, including podocyte-associated hereditary glomerular diseases, such as nephrotic syndrome, and GBM-associated glomerular diseases, such as Alport syndrome.

[0500] Preferably, the glomerular disease is a podocyte-associated hereditary glomerular disease. Examples of podocyte-associated hereditary glomerular diseases include Finnish-type congenital nephrotic syndrome, congenital nephrotic syndrome type 2, familial nephrotic syndrome type 3, Fraser syndrome and Dennis-Drash syndrome, Schimke immune osteogenesis imperfecta, nephrotic syndrome caused by a mutation in CD2AP, nephrotic syndrome caused by a mutation in actinin-4, nephrotic syndrome caused by a mutation in TRPC6, and Epstein and Fechtner syndrome. Preferably, the glomerular disease is nephrotic syndrome.

[0501] Preferably, the glomerular disease is GBM-related hereditary glomerular disease.Podocyte-related hereditary glomerular disease includes X-linked Alport syndrome, autosomal recessive Alport syndrome, autosomal dominant Alport syndrome, thin basement membrane disease, Pearson syndrome and nail-patella syndrome.Preferably, the glomerular disease is Alport syndrome (AS).AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease and thin basement membrane nephropathy.

[0502] [Example] The present invention will now be further described by means of examples, which are meant to serve to aid those skilled in the art in practicing the present invention and are not intended to limit the scope of the invention in any way.

[0503] [Example 1] Design, construction and testing of a minimal nephrin promoter Design of a minimal nephrin promoter The human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6):1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6):F1027-32. The NPHS1 promoter is a 1.2 kb fragment that appears to be podocyte specific. It is hereinafter referred to as the "FL" nephrin promoter and is shown in FIG. 1A.

[0504] The FL nephrin promoter was first truncated to 822 bp (819 bp excluding the initiation codon) by deleting the N-terminal sequence, hereafter referred to as the "midi" nephrin promoter and shown in FIG. 1B.

[0505] The midi nephrin promoter was further truncated to 268 bp (265 bp excluding the initiation codon) by removing the putative general transcription domain from the central region, hereafter referred to as the "mini" nephrin promoter and shown in Figure 1C.

[0506] Construction of vector constructs Midi Nephrin Promoter As shown in Figure 2A, the pACE_hNPHS1 promoter was used as a template to introduce BamHI and ClaI restriction sites. The fragment was then gel extracted and digested with ClaI and BamHI at 37°C for 1 hour before being ligated into the pLenti GFP Blast vector. The ligation was further transformed into stable competent E. coli cells and the DNA was extracted and sequenced (Midi promoter). The final lentiviral vector is shown in Figure 2B.

[0507] Mini Nephrin Promoter The overhang (OH) was PCRed using the pACE_hNPHS1 promoter as shown in Figure 3A. The two pieces of the promoter containing the OH were gel extracted for NEBuilder HiFi assembly reaction into the pLenti GFP Blast vector. The ligation reaction was then cleaned using a DNA cleanup kit before being transformed into stable competent E. coli cells. DNA was extracted and sequenced. The final lentiviral vector is shown in Figure 3B.

[0508] Testing of vector constructs A minimal nephrin promoter was used to express GFP in an in vitro cell model to confirm efficacy and podocyte specificity.

[0509] pLenti GFP Blast nephrin promoter constructs (full length, Midi and Mini) were used to transfect HEK293T cells for 48 h to generate viruses, which were further used to generate human conditionally immortalized podocytes stably expressing either GFP-tagged FL NPHS1, midi or mini promoter.

[0510] Conditionally immortalized human podocytes (ciPodocytes) were transfected with lentiviral vectors to determine whether the minimal promoter could drive GFP expression. Both the midi and mini-nephrin promoters were shown to drive GFP expression. Figure 4 shows a representative fluorescent microscopy image showing GFP expression from the mini-nephrin promoter. Figure 5 shows a representative Western blot showing GFP expression from the mini-nephrin promoter. These results indicate that the mini-nephrin promoter can drive the expression of a transgene in podocytes.

[0511] Lentiviral vectors were also used to transduce human glomerular cells. ciPodocytes and glomerular endothelial cells were transduced with lentivirus containing GFP linked to a mini-nephrin reporter. Figure 6A-C shows FACS analysis showing the median GFP fluorescence (AFU) of all surviving singlets of conditionally immortalized human podocytes (LY) and glomerular endothelial cells (GEnC) using a Novocyte analyzer. Non-transduced cells (cell control) were compared to cells transduced with a lentiviral construct carrying a GFP expression cassette controlled by the full-length human nephrin promoter (hNPHS1.GFP) or the mini-human nephrin promoter (265.GFP). All cells were differentiated for 10 days, trypsinized (100uL) and diluted 1:1000 in PBS, 2% FBS, DRAQ7 (150uL). Data and error bars represent 3 technical replicates (100uL, >2500 cells) ± SEM. These results indicate the specificity of podocytes for the minimal nephrin promoter compared with glomerular endothelial cells.

[0512] [Example 2] Design, construction and testing of a minimal nephrin promoter bound to COL4A3, COL4A4 and COL4A5 Design and construction of AAV constructs The following AAV transfer plasmids containing COL4A3, COL4A4 and COL4A5 linked to the mini nephrin promoter ("265") were designed and constructed. · pAAV.265.Col4a3.3flag.sv40, an AAV plasmid containing COL4A3 linked to the mini-nephrin promoter (see Figure 7A). pAAV.265.Col4a4.3flag.sv40, an AAV plasmid containing COL4A4 linked to the mini-nephrin promoter (see Figure 7B) pAAV.265.Col4a5.3flag.sv40, an AAV plasmid containing COL4A5 linked to the mini-nephrin promoter (see Figure 7C)

[0513] SmaI digestion was performed to confirm the identity of the plasmid (see Figure 7D-E), indicating that COL4 a3, a4 and a5 with the 265 bp mini nephrin promoter and SV40 polyA tail were successfully cloned into AAV.

[0514] Testing of AAV constructs The following AAV viral vectors were prepared using standard methods: AAV.COL4A3.Nephrin265.Sv40 with the LK03 serotype AAV.COL4A5.Nephrin265.Sv40 with the LK03 serotype AAV.COL4A5.Nephrin265.Sv40 with serotype 2 / 9

[0515] Figure 8A shows immunoprecipitation experiments of full-length FLAG-tagged Col4a3 (LK03) or Col4a5 (LK03) in human differentiated ciPodocytes pulled down with anti-FLAG antibody. Anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human FLAG IgG was used as a control.

[0516] Figure 8B shows Western blots of protein lysates showing expression levels of Col4a3 (LK03 capsid serotype), Col4a5 (LK03) and Col4a5 (2 / 9 capsid serotype) in differentiated human or mouse ciPodocytes. Uninfected human and mouse cipodocytes were used as controls.

[0517] Figure 8C shows confocal images showing immunofluorescence staining of transduced Col4a5 in human wild-type CiPodocytes / Col4a5 3xFlag AAV CiPodocytes with F-actin. Col4a5 is present at the cytosolic level in human differentiated podocytes infected with Col4a5 3xFlag AAV virus compared to wild-type counterparts.

[0518] These results indicate that we successfully transduced full-length COL4a3 and COL4a5 linked to the mini-nephrin promoter into human podocytes, and that the mini-nephrin promoter drives the expression of full-length COL4a3 or COL4a5 in human podocytes.

[0519] [Example 3] Expression of CFH, CFI and CFHL1 in HEK cells material and method 60-80% confluent 293T human embryonic kidney cells grown in DMEM supplemented with 10% FBS were triple transfected with pHelper (HGTI1), one of two pAAV Rep-Cap (LK03 or AAV9) and one of three ITR expression plasmids containing 1) CFH under a 265 bp minimal nephrin promoter of SEQ ID NO: 47 (pAAV-265-CFH), 2) CFI under a full-length minimal nephrin promoter of SEQ ID NO: 14 (pAAV-FL-CFI) or 3) CFHL1 under a full-length minimal nephrin promoter of SEQ ID NO: 14 (pAAV-FL-CFHL1). All constructs were tagged with MYC and FLAG.

[0520] Transfections were performed in 150 mm culture dishes in serum-free medium in the presence of polyethyleneimine (PEI). The medium was changed to DMEM containing FBS the day after transfection. Four days after transfection, the medium and cells were harvested and processed separately. The medium was frozen and stored at -80°C. The cells were lysed using RIPA buffer supplemented with proteinase inhibitors and stored at -80°C.

[0521] Protein concentration in cell lysates was measured using the Pierce BCA Protein Assay and 10ug of total protein from each sample was loaded onto a 4-15% polyacrylamide Tris-glycine gel. A total of 2.6uL of media from each sample was loaded onto the gel. Proteins were transferred to nitrocellulose membranes using the iBlot2 dry blotting system. The following primary antibodies were used for protein detection: anti-factor H (Abcam, cat. ab124769), anti-factor I (Abcam, ab278524), anti-MYC tag (CST, cat.2276S), anti-FLAG tag (CST, cat. 14793S) and anti-GAPDH (Millipore, cat. MAB374).

[0522] result Factor H was expressed in both cell lysates and medium from 293T HEK cells transfected with the CFH expression plasmid (Figure 9, lanes 2, 3, 8, 9). Expression was detected using a factor H-specific antibody. Factor H was not detected in cell lysates or medium from untransfected cells or cells transfected with the CFI or CFHL1 expression plasmids (Figure 9, lanes 1, 4-7, 10-13). These results demonstrate that the 265 bp minimal nephrin promoter allows expression of transgenes in target cells.

[0523] [Example 4] Transduction of factor H mutant podocytes with AAV2 / 9 265-CFH or transfection with a plasmid encoding CFH Transduction of factor H mutant podocytes with AAV2 / 9 265-CFH Conditionally immortalized human podocytes carrying a mutation in endogenous factor H (Muehlig et al., 2020), grown in RPMI supplemented with 10% FBS and 1% ITS, were seeded in 6-well culture dishes and then grown at 33 °C to 70-80% confluency. Cells were incubated with AAV2 / 9 containing the CFH transgene under the control of the 265 bp minimal nephrin promoter (SEQ ID NO: 47). Cells incubated without this virus were used as non-transduced (NT) controls. On the same day after viral transduction, cells were shifted to the non-permissive temperature of 37 °C to allow cell differentiation and transgene expression. The medium was changed twice the day after transduction. On day 10 after transduction, cell medium was collected and the concentration of human factor H was measured by ELISA using an anti-factor H antibody (Abcam, cat. ab252359).

[0524] When human podocytes with a mutation in factor H were transduced with AAV2 / 9 virus containing a CFH transgene under the control of a 265 bp minimal nephrin promoter, higher concentrations of human factor H were demonstrated in the culture medium than in non-transduced cells (FIGS. 10A and 10B), demonstrating that a factor H transgene delivered by AAV and under the control of a 265 bp minimal nephrin promoter can be expressed in human podocytes.

[0525] Transfection of factor H mutant podocytes with a plasmid encoding CFH Conditionally immortalized human podocytes harboring a mutation in endogenous factor H (Muehlig et al., 2020), grown in RPMI supplemented with 10% FBS and 1% ITS, were seeded in 6-well culture dishes and then grown to 70-80% confluency at 33 °C. For plasmid transfection, cells were incubated with 1.5 ug of expression plasmid in serum-free medium in the presence of polyethylenimine. Cells without added plasmid were used as non-transfected (NT) controls. The medium was replaced with medium containing FBS the next day. Three days after transfection, the medium was collected and analyzed by ELISA (Abcam, cat. ab252359).

[0526] Podocytes transfected with a plasmid expressing the CFH transgene under the control of the 265 bp minimal nephrin promoter exhibited higher concentrations of human factor H than non-transfected controls (FIG. 10C).

[0527] [Example 5] Expression of CFH in the kidneys of WT mice material and method 100 μL of AAV2 / 9 gene therapy product (pAAV.NPHS1(265).hCFH.WPRE.bGH) or saline was administered by IV tail vein injection to wild type C57BL6 mice. The tagged wild type human CFH transgene expressed in AAV was under the control of a 265 bp minimal nephrin promoter (SEQ ID NO: 47). AAV was harvested 3 days later, purified by ultracentrifugation, and titered in PBS (approximately 1.5x10e13 / ml). All animals completed the study on day 21 and were sacrificed. Kidneys were snap frozen in liquid nitrogen and then used for RNA extraction with RNeasy Micro Kit (Qiagen Catalog No. / ID: 74004) as per manufacturer's protocol. RNA was then converted to cDNA using High-Capacity RNA-to-cDNA™ Kit (4387406) prior to qPCR analysis.

[0528] Quantitative qPCR was performed on DNA samples obtained from the kidneys of pAAV_CFH-injected mice.Standard curve qPCR using SYBR green reagent and passive ROX method was used to detect the presence of ITR in the kidney DNA samples.The viral genome per pg of DNA was calculated using a standard curve of known amounts of ITR amplicons.Finally, viral genome / cell was calculated based on the assumption that diploid mouse cells have 6 pg of DNA.

[0529] Immunofluorescence staining was performed on 5-micron-thick frozen kidney sections using anti-nephrin (PROGEN) and anti-CFH (ab124767) antibodies.

[0530] Tissues were embedded in OCT compound (VWR, Cat. No. 361603E) and flash frozen in liquid nitrogen. 10uM sections were cut using a cryostat (Thermo, Cryostar NX270). Tissue sections were fixed with 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.3% Triton-X for 15 minutes at room temperature, and then blocked with 5% BSA for 30 minutes at room temperature.

[0531] Primary antibodies (rabbit monoclonal to factor H [EPR6226], Abcam catalog number ab124769 and nephrin (NPHS1) (1243-1256) guinea pig polyclonal antibody, Origene catalog number BP5030) were diluted in 5% BSA (1:100 for anti-factor H and 1:300 for anti-nephrin). Incubation was for 1 hour at room temperature.

[0532] Secondary antibodies (goat anti-rabbit IgG (H+L) secondary antibody, Alexa Fluor Plus 488, Invitrogen, Cat. No. A32731 and goat anti-guinea pig IgG (H+L) Alexa Fluor 568, Invitrogen, Cat. No. A-11075) were diluted 1:500 in PBS and incubation was performed at room temperature for 30 min.

[0533] Slides were mounted with slide mounting medium Fluoromount-G (Southern Biotech, Cat. No. 0100-01) and imaged with a Leica DM750 fluorescent microscope at 20X magnification.

[0534] result Injection of wild-type mice with AAV containing human CFH under the 265 bp minimal nephrin promoter results in infection of the kidney with AAV and increased expression of virus and human CFH in the kidney compared to wild-type mice injected with saline controls (Figure 11A-C). Immunofluorescence staining demonstrates colocalization of nephrin and CFH in mouse glomeruli (Figure 11D).

[0535] [Example 7] Expression of GFP using the 265bp v 818bp v FL minimal nephrin promoter in kidney cells (plasmid transfection) Plasmids were constructed containing eGFP under the control of (i) the full length (FL) minimal nephrin promoter of SEQ ID NO: 14 (PS0281), (ii) the 818 bp minimal nephrin promoter of SEQ ID NO: 59 (PS0301) or (iii) the 265 bp minimal nephrin promoter of SEQ ID NO: 47 (P20282).

[0536] Podocytes, glomerular endothelial cells (GENCs) and proximal duct epithelial cells (PTECs) were seeded at a density of 1.5e5 cells per well in 6-well plates and allowed to adhere overnight. For HEK293T cells, 5e5 cells per well were used. The next day, 3ug of plasmid DNA in 150ul of DMEM was mixed with 9ul of PEI (also in 150ul of DMEM), incubated for 15 minutes at room temperature and analyzed for eGFP expression by flow cytometry after 48 hours. Untransfected cells were used as a negative control for gating the negative and positive populations.

[0537] The results in Figure 12 show that all three promoters were able to drive eGFP expression in all cell types tested. These results demonstrate that despite the removal of large regions of the nephrin promoter sequence, the full-length nephrin promoter can still be minimized to 818 bp or 265 bp to express a transgene in target cells.

[0538] [Example 8] Expression of GFP using the 265bp vFL minimal nephrin promoter in kidney cells (AAV transduction) material and method AAV production: 60–80% confluent 293T human embryonic kidney cells grown in DMEM supplemented with 10% FBS were triple transfected with pHelper (HGTI1, PS0150), Rep-Cap plasmid (LK03, PS0240) and one of two ITR expression plasmids (PS0281 and PS0282, respectively) containing either the full-length (FL) minimal nephrin promoter driving eGFP expression or the 265 bp minimal nephrin promoter. Transfections were performed on 150 mm culture dishes in the presence of polyethylenimine (PEI). The day after transfection, the medium was replaced with DMEM containing FBS. Four days after transfection, the medium and cells were collected and processed separately. PEG was added to the filtered supernatant to precipitate the viral particles, which were subsequently centrifuged at 1500 g for 20 min. The cell pellet was resuspended in TD buffer and frozen at -80°C, followed by thawing at 37°C and vortexing. The freeze-thaw cycle was repeated five times. The pellet from the PEG-treated supernatant was then added to the lysed cells, and benzonase and sodium deoxycholate were added and incubated at 37°C for 30 minutes. The sample was then centrifuged and the supernatant filtered, followed by purification by iodixanol gradient ultracentrifugation.

[0539] AAV QC: AAV samples were titrated by qPCR to determine the number of viral genomes per ml of viral supernatant. Primers designed to target the eGFP sequence were used. SDS-PAGE analysis was performed on samples to determine purity. Alkaline gel electrophoresis was used to demonstrate integration of the intact transgene cassette into the viral particles.

[0540] AAV transduction: Cells were seeded into wells of 6-well plates at a density of 1.5e5 cells / well and allowed to adhere overnight. The next day, viral particles were added at a multiplicity of infection (MOI) of 5e5 particles per cell. Cells were placed at 37 degrees to initiate differentiation and cultured for 10 days. After 10 days, cells were analyzed for eGFP expression by flow cytometry.

[0541] result The results in Figure 13 show that both promoters were able to drive eGFP expression in all cell types tested. The 265 bp minimal nephrin promoter showed higher eGFP expression levels in each of the cells tested.

[0542] These data again demonstrate that the 265 bp minimal nephrin promoter is capable of driving eGFP expression in target cells. Furthermore, this promoter may be incorporated into AAV vectors for use in gene therapy. Using the 265 bp minimal nephrin promoter instead of the full-length minimal nephrin promoter may allow more genetic material to be incorporated into the transgene cassette for AAV purposes.

[0543] [Example 9] Expression of human podocin using the 265bp vFL minimal nephrin promoter in HEK (plasmid transfection) Plasmids were constructed containing HA-tagged podocin under the control of (i) the full-length (FL) minimal nephrin promoter of SEQ ID NO: 14 (FL.NPSH1-podocin-HA) or (ii) the 265 bp minimal nephrin promoter of SEQ ID NO: 47 (265.hNPSH1-podocin-HA).

[0544] 293T human embryonic kidney cells grown in DMEM supplemented with 10% FBS, 1% penicillin / streptomycin and 1% sodium pyruvate were seeded at a density of 5e5 in 6-well culture plates. Transfection was performed the next day when the cells reached 70-80% confluency. The transfection reagent mixture was prepared by complexing 3ug of expression plasmid with polyethylenimine (PEI) in a 1:3 ratio in serum-free medium. Non-transfected controls received transfection mixtures containing PEI at the same ratio without the expression plasmid. Cells were incubated at 37°C and 5% CO2 with the transfection mixture until the next day when the medium was replaced with complete medium. Cells were harvested 2 days after transfection and analyzed by FAC for the expression of podocin by staining the HA tag with an anti-HA antibody.

[0545] The results in Figure 14A show that both promoters were able to drive human podocin-HA expression in HEK cells, demonstrating that minimizing the full-length minimal nephrin promoter to 265 bp still allows transgene expression in target cells despite removing most of the promoter sequence.

[0546] [Example 10] Expression of human podocin using the 265bp vFL minimal nephrin promoter in human podocytes (AAV transduction) material and method AAV production: 60–80% confluent 293T human embryonic kidney cells grown in DMEM supplemented with 10% FBS, 1% penicillin / streptomycin and 1% sodium pyruvate were triple transfected with pHelper (HGTI1, PS0150), pAAV Rep-Cap (LK03, PS0240) and one of two ITR expression plasmids containing either the full-length (FL) minimal nephrin promoter driving podocin-HA expression or the 265 bp minimal nephrin promoter (FL.NPSH1-podocin-HA and 265.hNPSH1-podocin-HA, respectively). Transfections were performed on 150 mm culture dishes in the presence of polyethyleneimine (PEI) at a 1:2 ratio. The day after transfection, the medium was replaced with DMEM containing FBS. Four days after transfection, the medium and cells were collected and processed separately. PEG was added to the filtered supernatant to precipitate virus particles, which were then centrifuged at 1500g for 20 min. The cell pellet was resuspended in TD buffer and frozen at -80°C, then thawed at 37°C and vortexed. Five freeze-thaw cycles were repeated. The pellet from the PEG-treated supernatant was then added to the lysed cells. Further benzonase and sodium deoxycholate were added and incubated at 37°C for 30 min. The sample was then centrifuged and the supernatant was filtered, followed by purification by iodixanol gradient ultracentrifugation.

[0547] AAV QC: AAV samples were titrated by qPCR to determine the number of viral genomes per ml of viral supernatant using primers designed to target the podocin sequence.

[0548] AAV transduction: Cells were seeded into wells of 12-well plates at a density of 0.5e5 cells / well and allowed to adhere overnight. The following day, viral particles were added at a multiplicity of infection (MOI) of 5e5 particles per cell. Cells were placed at 37 degrees to initiate differentiation and cultured for 9 days. After 9 days, cells were harvested and stained with anti-HA antibody for HA expression by flow cytometry as a measure of podocin expression.

[0549] result As demonstrated by the plasmid transfection data in HEK cells (FIG. 11A), the results shown in FIG. 14B further confirm that both promoters are capable of driving transgene expression in human podocytes. The 265 bp minimal nephrin promoter showed higher podocin expression levels in human podocytes.

[0550] [Example 11] In vivo expression of GFP using the 265bp vFL promoter by AAV transduction The 265-nephrin promoter and the FL-nephrin promoter were evaluated in vivo in WT mice (C57 / Bl6). AAV particles as described in Example 8 were administered by tail vein injection at approximately 1×10e13 AAV2 / 9 per mouse (2× mice and 1× untreated mouse per treatment group), except that AAV9 (PS0241) was used instead of LK03. Kidney tissue harvesting was performed 4 weeks after injection. qPCR analysis was performed on the kidney, and GFP copy numbers were normalized to 18S (housekeeping gene). 18S rRNA is a reliable normalization gene for real-time PCR.

[0551] The results in Figure 15 show that there was detectable GFP expression in the kidney using both the 265 bp and full-length minimal nephrin promoters. Expression in the kidney was higher using the 265 bp minimal nephrin promoter.

[0552] All publications mentioned in the above specification are incorporated herein by reference.Various modifications and alterations of the disclosed method, cell, composition and use of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.Although the present invention has been disclosed in connection with specific and preferred embodiments, it is understood that the invention described in the claims should not be unduly limited to such specific embodiments.Indeed, various modifications of the disclosed modes for carrying out the present invention will be apparent to those skilled in the art and are considered to be within the scope of the appended claims.

[0553] Embodiment Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs. 1. (i) A promoter comprising a nucleotide sequence having at least 70% identity to SEQ ID NO:4, the promoter having a length of about 1.1 kb or less. 2. As set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, where n1 is an integer between 100 and 430, and / or (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3≧n2, n2 is an integer from 508 to 1061, and n3 is an integer from 508 to 1061; A promoter comprising or consisting of a nucleotide sequence having at least 70% identity to a nucleotide sequence. 3. The promoter according to item 2, wherein (i) the promoter comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 and has a length of about 1.1 kb or less. 4. The promoter of any of the preceding items, which is a podocyte-specific promoter. 5. (ii) The promoter of any of the preceding items, further comprising a nucleotide sequence having at least 70% identity to SEQ ID NO:5, a nucleotide sequence having at least 70% identity to SEQ ID NO:6 and / or a nucleotide sequence having at least 70% identity to SEQ ID NO:7. 6. (iii) A promoter according to any of the preceding items, further comprising a nucleotide sequence having at least 70% identity to SEQ ID NO:8, or one or more fragments thereof. 7. The promoter according to any of the preceding items, comprising, from 5' to 3', (i) a nucleotide sequence having at least 70% identity to SEQ ID NO:4, (iii) optionally a nucleotide sequence having at least 70% identity to SEQ ID NO:8 or one or more fragments thereof, and (ii) a nucleotide sequence having at least 70% identity to SEQ ID NO:5, a nucleotide sequence having at least 70% identity to SEQ ID NO:6 and / or a nucleotide sequence having at least 70% identity to SEQ ID NO:7. 8. The promoter of any of the preceding items, having a length of about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. 9. A promoter according to any of the preceding items, having a length of 0.265 kb to 1.0 kb, 0.265 kb to 0.9 kb, 0.265 kb to 0.8 kb, 0.265 kb to 0.7 kb, 0.265 kb to 0.6 kb, 0.265 kb to 0.5 kb, 0.265 kb to 0.4 kb, or 0.265 kb to 0.3 kb. 10. (a) Retinoic acid receptor binding site, (b) WT1 binding site, (c) enhancer box, (d) a transcription factor binding region, and / or (e) Transcription start site 4. The promoter of any of the preceding items, comprising: 11. The promoter according to item 10, wherein the retinoic acid receptor binding site comprises or consists of the nucleotide sequence shown as SEQ ID NO:10 or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO:10. 12. The promoter according to item 10 or 11, wherein the WT1 binding site comprises or consists of the nucleotide sequence shown as SEQ ID NO: 11 or a nucleotide sequence having one, two or three substitutions, deletions or insertions compared to SEQ ID NO: 11. 13. The promoter according to any one of items 10 to 12, wherein the enhancer box comprises or consists of the nucleotide sequence shown as SEQ ID NO: 12 or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO: 12. 14. The promoter according to any one of items 1 or 3 to 13, wherein (i) a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 comprises one or more of: (a) a retinoic acid receptor binding site; (b) a WT1 binding site; and (c) an enhancer box. 15. The nucleotide sequence: (a) GGGGTCA at positions corresponding to positions 7 to 13 of SEQ ID NO:4; (b) CGGAGGCTGGGGAGGCA at positions corresponding to positions 14 to 30 of SEQ ID NO:4; (c) ATGTG at positions corresponding to positions 49 to 53 of SEQ ID NO: 4 one or more of: (i) is present in a nucleotide sequence having at least 70% identity to SEQ ID NO:4; 15. The promoter according to any one of items 1 or 3 to 14. 16. The promoter according to any one of items 10 to 15, wherein the transcription factor binding region comprises or consists of the nucleotide sequence shown as SEQ ID NO: 13 or a nucleotide sequence having one, two, three, four or five substitutions, deletions or insertions compared to SEQ ID NO: 13. 17. The promoter according to any one of items 10 to 16, wherein the transcription initiation site comprises or consists of an "AG" dinucleotide. 18. The promoter according to any one of items 10 to 17, wherein the transcription factor binding site is operably linked to the transcription start site, and optionally the transcription factor binding site is directly upstream of the transcription start site. 19. A promoter according to any of the preceding items, comprising or consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:2 or SEQ ID NO:3. 20. A promoter consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:2. 21. A promoter consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:3. 22. A polynucleotide comprising a promoter according to any one of items 1 to 21. 23. The polynucleotide according to item 22, wherein the promoter is operably linked to the protein coding sequence. 24. The polynucleotide according to item 23, wherein the protein coding sequence is operably linked to one or more further regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences. 25. A vector comprising the polynucleotide according to any one of items 22 to 24. 26. The vector according to item 25, which is capable of transducing podocytes, and optionally capable of specifically transducing podocytes. 27. The vector according to item 25 or 26, which is a viral vector, such as an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector and a picornavirus vector, preferably an AAV vector. 28. The vector according to item 27, wherein the viral vector is in the form of a viral vector particle, preferably the viral vector is in the form of an AAV vector particle. 29. The vector of any one of items 25 to 28, in the form of an AAV vector particle encapsidated by AAV3B, LK03 or AAV9 capsid proteins. 30. A cell comprising a polynucleotide according to any one of items 22 to 24 or a vector according to any one of items 25 to 29. 31. A pharmaceutical composition comprising the polynucleotide according to any one of items 22 to 24, the vector according to any one of items 25 to 29, or the cell according to item 30. 32. A polynucleotide according to any one of items 22 to 24, a vector according to any one of items 25 to 29 or a cell according to item 30 for use in medicine. 33. Use of a promoter according to any one of items 1 to 21 to drive expression of a coding sequence. 34. The use according to item 32, wherein expression is podocyte-specific.

Claims

1. (i) A promoter comprising a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17, said promoter having a length of about 1.1 kb or less.

2. As set forth in SEQ ID NO:1, (i) positions 1 to n1 of SEQ ID NO: 1 are deleted, and n1 is an integer between 100 and 430; (ii) positions n2 to n3 of SEQ ID NO: 1 are deleted, n3 is ≧n2, n2 is an integer between 508 and 1061, and n3 is an integer between 508 and 1061; and / or (iii) the position corresponding to position 493 of SEQ ID NO:1 is G, the position corresponding to position 1080 of SEQ ID NO:1 is T, the position corresponding to position 1169 of SEQ ID NO:1 is C, and the position corresponding to position 1249 of SEQ ID NO:1 is G; a nucleotide sequence, or A nucleotide sequence having at least 70% identity to the nucleotide sequence A promoter comprising or consisting of:

3. 3. The promoter of claim 2, wherein (i) the promoter comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17 and / or has a length of about 1.1 kb or less.

4. The promoter according to any one of claims 1 to 3, which is a kidney-specific promoter.

5. 5. The promoter of claim 1, further comprising: (ii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 5 or 18; a nucleotide sequence having at least 70% identity to SEQ ID NO: 6 or 19; and / or a nucleotide sequence having at least 70% identity to SEQ ID NO: 7 or 20, and optionally further comprising (iii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 8, or one or more fragments thereof.

6. 6. The promoter of claim 1, comprising from 5' to 3': (i) a nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17; (iii) optionally a nucleotide sequence having at least 70% identity to SEQ ID NO: 8 or one or more fragments thereof; and (ii) a nucleotide sequence having at least 70% identity to SEQ ID NO: 5 or 18, a nucleotide sequence having at least 70% identity to SEQ ID NO: 6 or 19, and / or a nucleotide sequence having at least 70% identity to SEQ ID NO: 7 or 20.

7. 7. The promoter of any one of claims 1 to 6, having a length of about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less, and / or having a length of 0.265 kb to 1.0 kb, 0.265 kb to 0.9 kb, 0.265 kb to 0.8 kb, 0.265 kb to 0.7 kb, 0.265 kb to 0.6 kb, 0.265 kb to 0.5 kb, 0.265 kb to 0.4 kb, or 0.265 kb to 0.3 kb.

8. (a) a retinoic acid receptor binding site, optionally comprising or consisting of a nucleotide sequence as set forth as SEQ ID NO:10, or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO:10; (b) a WT1-binding site, optionally comprising or consisting of a nucleotide sequence as set forth as SEQ ID NO:11, or a nucleotide sequence having one, two or three substitutions, deletions or insertions compared to SEQ ID NO:11; (c) an enhancer box, optionally comprising or consisting of a nucleotide sequence as set forth in SEQ ID NO: 12, or a nucleotide sequence having one or two substitutions, deletions or insertions compared to SEQ ID NO: 12; (d) a transcription factor binding region, and / or (e) Transcription start site The promoter according to any one of claims 1 to 7, comprising:

9. A nucleotide sequence having at least 70% identity to SEQ ID NO: 4 or 17, (a) a retinoic acid receptor binding site, optionally with GGGGTCA at a position corresponding to positions 7 to 13 of SEQ ID NO: 4 or 17; (b) a WT1-binding site, optionally comprising CGGAGGCTGGGGAGGCA at a position corresponding to positions 14 to 30 of SEQ ID NO: 4 or 17; and (c) an enhancer box, optionally with ATGTG at a position corresponding to positions 49 to 53 of SEQ ID NO: 4 or 17; 9. The promoter of claim 1, comprising one or more of the following:

10. wherein the transcription factor binding region comprises or consists of a nucleotide sequence as set forth as SEQ ID NO: 13 or 43 or a nucleotide sequence having one, two, three, four or five substitutions, deletions or insertions compared to SEQ ID NO: 13 or 43, and optionally (i) the transcription initiation site comprises or consists of an "AG" dinucleotide; (ii) a transcription factor binding site is operably linked to the transcription initiation site; and / or (iii) The promoter of claim 8 or 9, wherein the transcription factor binding site is directly upstream of the transcription start site.

11. A promoter according to any one of claims 1 to 10, comprising or consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:2 or SEQ ID NO:

3.

12. 12. The promoter according to any one of claims 1 to 11, comprising or consisting of a nucleotide sequence having at least 70%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO:

47.

13. A promoter consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:15, or SEQ ID NO:

59.

14. A promoter consisting of a nucleotide sequence having at least 70% identity to SEQ ID NO:

47.

15. 15. The promoter of claim 14, comprising a nucleotide sequence having at least 90%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 47, or comprising the nucleotide sequence of SEQ ID NO:

47.

16. A polynucleotide comprising a promoter according to any one of claims 1 to 15.

17. 17. The polynucleotide of claim 16, wherein the promoter is operably linked to the protein coding sequence.

18. 18. The polynucleotide of claim 16 or 17, wherein the promoter is operably linked to a protein coding sequence encoding NPHS2 or a fragment and / or variant thereof; a COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof; or CFI, CFH or FHL-1 or a fragment and / or variant thereof, preferably wherein the protein coding sequence encodes an amino acid sequence or fragment thereof that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 55, 21 to 23, 49, 51 or 53.

19. 19. The polynucleotide of any one of claims 16 to 18, wherein the protein coding sequence is operably linked to one or more further regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences.

20. 20. A vector comprising the polynucleotide of any one of claims 16 to 19.

21. 21. The vector of claim 20, capable of transducing podocytes, optionally specifically transducing podocytes.

22. 22. The vector according to claim 20 or 21, which is a viral vector, such as an adeno-associated viral (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles viral vector, a Newcastle disease viral vector, a poxvirus vector and a picornavirus vector, preferably an AAV vector, optionally wherein the viral vector is in the form of a viral vector particle, preferably wherein the viral vector is in the form of an AAV vector particle.

23. 23. The vector of any one of claims 20 to 22, in the form of an AAV vector particle encapsidated by AAV3B, LK03 or AAV9 capsid protein.

24. 24. A cell comprising a polynucleotide according to any one of claims 16 to 19 or a vector according to any one of claims 20 to 23.

25. 25. A pharmaceutical composition comprising a polynucleotide according to any one of claims 16 to 19, a vector according to any one of claims 20 to 23, or a cell according to claim 24.

26. 27. A polynucleotide according to any one of claims 16 to 19, a vector according to any one of claims 20 to 23 or a cell according to claim 24 for use in medicine.

27. Use of a promoter according to any one of claims 1 to 15, a polynucleotide according to any one of claims 16 to 19, a vector according to any one of claims 20 to 23, or a cell according to claim 24, or a composition according to claim 25, for the manufacture of a medicament for the treatment of a renal disease.

28. 28. The use of claim 27, wherein the renal disease is a glomerular basement membrane associated disease, optionally wherein the glomerular basement membrane associated disease is selected from the group consisting of Alport syndrome, Pearson syndrome, and Nail-Patella syndrome.

29. 28. The use of claim 27, wherein the renal disease is a podocyte-associated hereditary glomerular disease, optionally wherein the podocyte-associated hereditary glomerular disease is selected from the group consisting of nephrotic syndrome, Fraser syndrome, Dennis-Drash syndrome, Schimke immune dysplasia, and Epstein and Fechtner syndrome.

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