Gene therapy

Abstract

A viral vector, wherein the viral vector comprises a COL4A3, COL4A4 or COL4A5 transgene.

Description

Technical Field

[0001] The present invention relates to a viral vector containing a COL4A3, COL4A4 or COL4A5 transgene and a kidney-specific promoter, and to the use of such viral vector in the treatment of Alport syndrome.

Background Art

[0002] Alport syndrome (AS) is a hereditary disease that affects approximately 1 in 5,000 - 10,000 individuals in the general population in Europe and the United States. AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease, and thin basement membrane nephropathy. The disease typically presents in childhood and is associated with a progressive loss of kidney function, as well as a range of phenotypes that may include hearing loss and ocular abnormalities.

[0003] AS is caused by pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes, which result in abnormal collagen IVα345 networks in the basement membrane. This disease can be inherited in X-linked, autosomal dominant, or autosomal recessive forms, with the X-linked form being the most common, accounting for approximately 15% and 20% of cases for autosomal recessive and autosomal dominant, respectively.

[0004] Without treatment, kidney disease progresses from microscopic hematuria to proteinuria, progressive renal insufficiency, and end-stage renal disease in all males with X-linked AS, and in all males and females with autosomal recessive AS.

[0005] AS can be diagnosed by genetic testing, and current treatments include angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs) to delay the onset of end-stage renal disease. However, at present, there is no way to prevent end-stage renal failure, and kidney transplantation is the only option.

[0006] There are several important challenges to overcome in developing effective gene therapies for AS. First, the COL4A5, COL4A3, and COL4A4 proteins each have 1685, 1670, and 1690 amino acids, respectively, and the limited carrying capacity of adeno-associated virus (AAV) makes their transport by AAV vectors difficult. Second, a key challenge is successfully administering gene therapy to podocytes in the glomeruli of the kidney that produce collagen IV in the glomerular basement membrane.

[0007] The present invention aims to provide a novel gene therapy vector capable of efficiently delivering COL4A3, COL4A4, or COL4A5 transgenes to podocytes, thereby providing a treatment for Alport syndrome. [Overview of the Initiative]

[0008] This invention provides a viral vector comprising the COL4A3, COL4A4, or COL4A5 transgene. By using this viral vector, podocytes in the glomeruli of the kidney can be targeted to treat Alport syndrome.

[0009] While we do not wish to be constrained by theory, we believe that podocytes offer a very manageable target for gene therapy approaches to kidney disease, and that targeting podocytes with COL4A3, COL4A4, or COL4A5 can alter, or at least partially normalize, the collagen IVα345 network in the glomerular basement membrane.

[0010] In one embodiment, the present invention provides a viral vector comprising a COL4A3, COL4A4, or COL4A5 transgene.

[0011] The COL4A3 transgene may encode a polypeptide sequence having at least 70% identity with SEQ ID NO: 1, or a COL4A3 polypeptide composed of said polypeptide sequence, or a fragment thereof; the COL4A4 transgene may encode a polypeptide sequence having at least 70% identity with SEQ ID NO: 2, or a COL4A4 polypeptide composed of said polypeptide sequence, or a fragment thereof; and / or the COL4A5 transgene may encode a polypeptide sequence having at least 70% identity with SEQ ID NO: 3, or a COL4A5 polypeptide composed of said polypeptide sequence, or a fragment thereof. In some embodiments, the COL4A3 transgene encodes a full-length COL4A3 polypeptide, the COL4A4 transgene encodes a full-length COL4A4 polypeptide, and / or the COL4A5 transgene encodes a full-length COL4A5 polypeptide. Appropriately, the COL4A3, COL4A4, or COL4A5 transgene is human (it is a human COL4A3, COL4A4, or COL4A5 transgene) and / or contains a hemagglutinin (HA) tag.

[0012] Preferably, the viral vector includes a podocyte-specific promoter. Suitablely, the podocyte-specific promoter is the minimal nephrin promoter NPHS1 or the podosin promoter NPHS2. In some embodiments, the podocyte-specific promoter is the minimal nephrin promoter NPHS1.

[0013] The inventors have developed a minimal nephrine promoter that is shorter than known minimal nephrine promoters and, surprisingly, has the ability to drive the expression of transgenes in podocytes. Surprisingly, this promoter also retains podocyte specificity. By using such a minimal nephrine promoter, it is possible to minimize the size of the package and assist in the packaging of full-length COL4A3, COL4A4, or COL4A5. Accordingly, this minimal nephrine promoter NPHS1 may contain or consist of a nucleotide sequence shown as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10.

[0014] Preferably, the viral vector is an adeno-associated virus (AAV). Preferably, the AAV vector is in the form of an AAV vector particle. In some embodiments, the AAV vector particle is a podocyte-specific AAV vector. In some embodiments, the AAV vector is AAV serotype 2 / 9, LK03, or 3B.

[0015] In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene is a minigene.

[0016] In some embodiments, the viral vector further includes a Woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the viral vector does not include a Woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0017] In some embodiments, the viral vector further includes a Kozak sequence between the promoter and the COL4A3, COL4A4, or COL4A5 transgene.

[0018] Appropriately, the viral vector further includes a polyadenylation signal, such as a bovine growth hormone (bGH) polyadenylation signal or an early SV40 polyadenylation signal. In some embodiments, the polyadenylation signal is an early SV40 polyadenylation signal.

[0019] In one embodiment, the present invention provides a viral vector gene therapy in which the viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

[0020] In a preferred embodiment, the viral vector is the viral vector according to the present invention.

[0021] In one embodiment, the present invention provides a viral vector gene therapy, wherein the gene therapy is as follows: A first viral vector containing at least a portion of the COL4A3, COL4A4, or COL4A5 transgene, and A second viral vector containing at least a portion of the corresponding COL4A3, COL4A4, or COL4A5 transgene. Includes.

[0022] In a preferred embodiment, the first viral vector is a viral vector according to the present invention, and / or the second viral vector is a viral vector according to the present invention.

[0023] In one embodiment, the present invention provides a viral vector or viral vector gene therapy according to the present invention for use in the treatment or prevention of Alport syndrome.

[0024] Suitably, the viral vector or viral vector gene therapy is administered to a human patient. In some embodiments, the viral vector or viral vector gene therapy is administered systemically. In some embodiments, the viral vector or viral vector gene therapy is administered by intravenous injection. In some embodiments, the viral vector or viral vector gene therapy is administered by injection into the renal artery.

Brief Description of Drawings

[0025] [Figure 1] FIG. 1 shows an example of the DNA sequence of the minimal human nephrin promoter (NPHS1). [Figure 2] FIG. 2 shows an example of the DNA sequence of the WPRE sequence. [Figure 3] FIG. 3 shows an example of the DNA sequence of the bGH poly(A) signal sequence. [Figure 4A] FIG. 4 shows an exemplary AAV transfer plasmid 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 shown, and the following fragments, namely 1. 6238 bp, 2. 2753 bp, 3. 56 bp, 4. 11 bp, 5. 11 bp are expected after restriction with SmaI. [Figure 4B] FIG. 4 shows an exemplary AAV transfer plasmid containing COL4A3, COL4A4, and COL4A5 linked to the mini-nephrin promoter. (B) is a schematic diagram of pAAV.265.Colia4.3flag.sv40, an AAV plasmid containing COL4A4 linked to the mini-nephrin promoter. The SmaI site is shown, and the following fragments, namely 1. 4052 bp, 2. 2753 bp, 3. _2224 bp, 4. 56 bp, 5. 11 bp, 6. 11 bp are expected after restriction with SmaI. [Figure 4C] Figure 4 shows an exemplary AAV transfer plasmid containing COL4A3, COL4A4, and COL4A5 conjugated to a mininephrine promoter. (C) is a schematic diagram of pAAV.265.Col4a5.3fl-ag.sv40, an AAV plasmid containing COL4A5 conjugated to a mininephrine promoter. The SmaI site is shown, and the following fragments, namely 1.4272 bp, 2.2753 bp, 3.2032 bp, 4.56 bp, 5.11 bp, and 6.11 bp, are expected after SmaI restriction. [Figure 4D] Figure 4 shows exemplary AAV transfer plasmids containing COL4A3, COL4A4, and COL4A5 conjugated to a mininephrine promoter. (D) shows restriction digestion by SmaI. MW = 1 Kb DNA ladder, with lanes 1, 2, and 3 corresponding to the digests for the plasmids shown in (A), (B), and (C), respectively. [Figure 4E] (E) is a schematic diagram showing restricted digestion. [Figure 5A] Figure 5 shows podocytes transduced with AAV.COL4.Nephrin265.Sv40 virus. (A) shows immunoprecipitation experiments of full-length FLAG-tagged Col4a3 (LK03) or Col4a5 (LK03) in human differentiated Ci (conditionally immortalized) podocytes pulled down with anti-FLAG antibody. Anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human-FLAG IgG was used as a control. [Figure 5B] (B) is a figure showing Western blots of protein lysates in human or mouse differentiated Ci podocytes, illustrating the expression levels of Col4a3 (LK03 capsid serotype), Col4a5 (LK03), and Col4a5 (2 / 9 capsid serotype). Uninfected human and mouse Ci podocytes were used as controls. [Figure 5C]Figure 5 shows podocytes transduced using the AAV.COL4.Nephrin265.Sv40 virus. (C) shows a confocal image of immunofluorescence staining of transduced Col4a5 in human wild-type Ci podocytes / Col4a5 3xFlag AAV Ci podocytes, along with F-actin. Col4a5 is present at the cytosolic level in human differentiated podocytes infected with the Col4a5 3xFlag AAV virus compared to its wild-type counterpart. [Figure 6] Figure 6 shows schematic diagrams of minimal nephrin promoters. (A) The full-length nephrin promoter is 1249 bp long (excluding the start codon) and will hereafter be referred to as the "FL" nephrin promoter. (B) An exemplary minimal nephrin promoter with a deleted 5' region is 819 bp long (excluding the start codon) and will hereafter be referred to as the "midi" nephrin promoter. (C) An exemplary minimal nephrin promoter with a deleted 5' region and a deleted central region is 265 bp long (excluding the start codon) and will hereafter be referred to as the "mini" nephrin promoter. (D) The following regions of the nephrin promoter are shown: (i) the human-mouse homology region, (ii) the retinoic acid receptor (RAR) binding site, (iii) the WT1 binding site, (iv) the transcription factor binding region, and (iv) the transcription initiation site. [Figure 7A] Figure 7 shows a schematic diagram of a lentiviral vector containing GFP, which has been conjugated to the midinephrin promoter in a manipulable manner. (A) The BamHI and ClaI restriction sites were introduced by using the pACE_hNPHS1 promoter as a template. [Figure 7B] Figure 7 shows a schematic diagram of a lentiviral vector containing GFP conjugated to the midinephrin promoter in a manipulable manner. (B) Figure shows the final construct vector containing GFP conjugated to the midinephrin promoter in a manipulable manner. [Figure 8A]Figure 8 shows a schematic diagram of a lentiviral vector containing GFP conjugated in a manipulable manner to a mininephrine promoter. (A) Two sections of the pACE_hNPHS1 promoter were subjected to PCR and gel extraction using the pACE_hNPHS1 promoter as a template. [Figure 8B] Figure 8 is a schematic diagram of a lentiviral vector containing GFP conjugated to a mininephrine promoter in a manipulable manner. (B) This figure shows the final construct vector containing GFP conjugated to a mininephrine promoter in a manipulable manner. [Figure 9] Figure 9 shows the expression of GFP in Ci podocytes after transduction with a lentiviral vector. Human Ci podocytes that stably express a GFP-tagged nephrin promoter were generated using a lentiviral approach. GFP expression was observed by fluorescence microscopy. (A) shows untransduced Ci podocytes. (B) shows Ci podocytes that stably express a GFP-tagged mininephrin promoter. (C) shows Ci podocytes that stably express a GFP-tagged FL nephrin promoter. [Figure 10] Figure 10 shows the expression of GFP in differentiated ci podocytes after transduction with a lentiviral vector. Differentiated facultatively immortalized podocytes (ci podocytes) were transduced using a lentiviral vector containing GFP ligated to the nephrin promoter in a manipulable manner. GFP expression was detected using immunoprecipitation (IP). [Figure 11]Figure 11 shows human glomerular cells transduced using lentivirus-GFP.nephrine promoter (minimum or 265). FACS analysis was performed using Novocyte Analyser to display the median GFP fluorescence (AFU) of all live singlets of conditionally immortalized human podocytes (LYs) and glomerular endothelial cells (GEnCs). Untransduced cells (cell control) were compared to those transduced with lentivirus constructs containing GFP expression cassettes controlled by full-length human nephrine promoter (hNPHS1.GFP) or microhuman nephrine promoter (265.GFP). All cells were differentiated for 10 days, triedpsinized (100 μL), and then diluted in PBS, 2% FBS, 1:1000 DRAQ7 (150 μL). Data and error bars represent three technical replicates (100 μL, >2500 cells) ± SEM. [Modes for carrying out the invention]

[0026] Viral vector Adeno-associated virus (AAV) vector The aforementioned viral vector may be an adeno-associated virus (AAV), and suitable AAV vector serotypes include 2 / 9, LK03, and 3B.

[0027] The aforementioned viral vector may be in the form of AAV vector particles.

[0028] The AAV vector particles may have a capsid formed by a capsid protein. The serotype can promote podocyte transduction, for example, podocyte-specific transduction. Preferably, the AAV vector particles are podocyte-specific vector particles. The AAV vector particles may have a capsid formed by a podocyte-specific capsid. The AAV vector particles may contain a podocyte-specific capsid protein. Targeted transduction to the podocytes will eliminate the effect of hepatotropy after systemic application.

[0029] Suitablely, the AAV vector particle may be in a transcapsidated form, in which an AAV genome or derivative having an ITR of a certain serotype is packaged on a capsid of a different serotype. The AAV vector particle also includes a mosaic form in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV vector particle also includes a chemically modified form having ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting specific cell surface receptors.

[0030] If 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 AAVs. In particular, the present invention encompasses providing capsid protein sequences (i.e., pseudotype vectors) obtained from different serotypes, clades, clones, or isolates of AAV within the same vector. The AAV vector may be in the form of pseudotype AAV vector particles.

[0031] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide the AAV vector with one or more desired functionalities. Thus, compared to AAV vectors containing naturally occurring AAV genomes, these derivatives may exhibit increased gene delivery efficiency, decreased (humoral or cellular) immunogenicity, altered tropism range, and / or improved podocyte targeting. Increased gene delivery efficiency may be achieved through improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Increased efficiency may also be associated with altered tropism range or podocyte targeting, such that the vector dose is not diluted by administration to tissues that do not require it.

[0032] Chimeric capsid proteins include those created by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This may be carried out, for example, by a marker rescue method, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and then directed selection is used to select a capsid sequence with the desired properties. These capsid sequences of different serotypes can be modified intracellularly by homologous recombination to produce novel chimeric capsid proteins.

[0033] Chimeric capsid proteins also include those created by manipulating the capsid protein sequence to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes.

[0034] Shuffled or chimeric capsid proteins may also be constructed by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be constructed by randomly fragmenting the sequences of relevant AAV genes, such as AAV genes encoding capsid proteins of multiple different serotypes, and then subsequently reconstructing the fragments in a self-priming polymerase reaction that can cause cross-reaction between regions with sequence homology. By screening a library of hybrid AAV genes constructed in this way by shuffling several serotype capsid genes, viral clones with desired functionality can be identified. Similarly, by randomly mutating AAV capsid genes using error-prone PCR, a diverse library of variants can be constructed that can later be selected for desired properties.

[0035] The sequence of the capsid gene may also be genetically modified to introduce specific deletions, substitutions, or insertions from the natural wild-type sequence. In particular, the capsid gene may be modified by inserting a sequence of an unrelated protein or peptide within the open reading frame of the capsid code sequence or at the N- and / or C-terminus of the capsid code sequence. The unrelated protein or peptide may advantageously function as a ligand for a particular cell type, thereby providing improved binding to target cells or improving the specificity of the vector's targeting to a particular cell population. The unrelated protein may also assist in the purification of the viral particle as part of the production process, i.e., it may be an epitope or affinity tag. The insertion site is typically selected so as not to interfere with other functions of the viral particle, such as its internalization or transport.

[0036] The capsid protein may be an artificial or mutant capsid protein. As used herein, the term "artificial capsid" means that the capsid particle contains an amino acid sequence that does not exist in nature, or an amino acid sequence that has been manipulated (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein contains mutations or mutations in its amino acid sequence when the artificial capsid amino acid sequence is aligned with the parent capsid amino acid sequence, compared to the parent capsid sequence from which the artificial capsid amino acid sequence is derived.

[0037] The capsid protein may contain mutations or modifications that improve its ability to transduce podocytes compared to the wild-type capsid protein or to unmodified or wild-type virus particles. The improvement in transduction ability to podocytes may be measured, for example, by measuring the expression of a transgene delivered by the AAV vector particle, such as GFP, and the expression of said transgene in podocytes correlates with the ability of the AAV vector particle to transduce podocytes.

[0038] AAV9 serotype AAV 2 / 9 serotypes have shown significant tropism to the kidneys of neonatal and adult mice, localizing to the glomeruli and tubules (Luo et al., 2011; Picconi et al., 2014; Schievenbusch et al., 2010), and AAV2 / 9 vectors combined with renal intravenous injection have been shown to be suitable for kidney-targeted gene delivery (Rocca et al., 2014). AAV 2 / 9 is therefore a suitable vector for use in the viral vector of the present invention.

[0039] The AAV vector particles may contain the AAV9 capsid protein. Preferably, the AAV vector particles may have a capsid formed by the AAV9 capsid protein.

[0040] The AAV vector particles may contain AAV9 VP1 capsid protein, AAV9 VP2 capsid protein, and / or AAV9 VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by AAV9 VP1 capsid protein, AAV9 VP2 capsid protein, and / or AAV9 VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by AAV9 VP1, VP2, and VP3 capsid proteins.

[0041] Suitablely, the AAV9 VP1 capsid protein may include or consist of an amino acid sequence represented by SEQ ID NO: 31, or a variant that is at least 90% identical to SEQ ID NO: 31.

[0042] Exemplary AAV9 VP1 capsid protein (SEQ ID NO: 31): TIFF0007884456000001.tif66151

[0043] Appropriately, 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: 31.

[0044] Appropriately, the AAV9 VP2 and VP3 capsid proteins may be N-terminus truncations of SEQ ID NO: 31, or N-terminus truncations of 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: 31.

[0045] AAV LK03 serotype Synthetic AAV capsids such as LK03 can also be suitable vectors for use in the viral vector of the present invention. This vector has been shown to transduce human primary hepatocytes with high efficiency in vitro and in vivo. However, this vector has not been used for kidney-targeted gene delivery until now. Surprisingly, the AAV-LK03 vector can achieve a high transduction rate of nearly 100% in human podocytes in vitro and can be used for specific transduction into podocytes in vitro (see PCT / GB2020 / 050097).

[0046] The aforementioned AAV-LK03 capsid (cap) sequence consists of fragments from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, 9), and as described by Lisowski, L., et al., 2014. Nature, 506(7488), pp.382-386, 97.7% of the capsid (cap) gene sequence and 98.9% of its amino acid sequence are from AAV-3B.

[0047] The AAV vector particles may contain the LK03 capsid protein. Preferably, the AAV vector particles may have a capsid formed by the LK03 capsid protein.

[0048] The AAV vector particles may contain LK03 VP1 capsid protein, LK03 VP2 capsid protein, and / or LK03 VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by LK03 VP1 capsid protein, LK03 VP2 capsid protein, and / or LK03 VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by LK03 VP1, VP2, and VP3 capsid proteins.

[0049] Suitablely, the LK03 VP1 capsid protein may include or consist of an amino acid sequence represented by SEQ ID NO: 32, or a variant that is at least 90% identical to SEQ ID NO: 32.

[0050] Exemplary LK03 VP1 capsid protein (SEQ ID NO: 32): TIFF0007884456000002.tif66151

[0051] Appropriately, the variant may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequence number 32.

[0052] Appropriately, the LK03 VP2 and VP3 capsid proteins may be N-terminally deficient variants of SEQ ID NO: 32, or N-terminally deficient variants of 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: 32.

[0053] AAV3B serotype AAV-3B is also known for its human hepatocyte tropism and is another suitable vector for use in the viral vector of the present invention. This vector has not been used to date for kidney-targeted gene delivery.

[0054] The AAV vector particles may contain the AAV3B capsid protein. Preferably, the AAV vector particles may have a capsid formed by the AAV3B capsid protein.

[0055] Two distinct AAV3 isolates (AAV3A and AAV3B) have been cloned. Compared to vectors based on other AAV serotypes, AAV3 vectors are generally 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.

[0056] The AAV vector particles may contain AAV3B VP1 capsid protein, AAV3B VP2 capsid protein, and / or AAV3B VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by the AAV3B VP1 capsid protein, AAV3B VP2 capsid protein, and / or AAV3B VP3 capsid protein. Preferably, the AAV vector particles may have a capsid formed by the AAV3B VP1, VP2, and VP3 capsid proteins.

[0057] Appropriately, the AAV3B VP1 capsid protein may include or consist of an amino acid sequence represented by SEQ ID NO: 33, or a variant that is at least 90% identical to SEQ ID NO: 33.

[0058] Exemplary AAV3B VP1 capsid protein (SEQ ID NO: 33): TIFF0007884456000003.tif43151TIFF0007884456000004.tif25150

[0059] Appropriately, the variant may be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to sequence number 33.

[0060] Appropriately, the AAV3B VP2 and VP3 capsid proteins may be N-terminally deficient variants of SEQ ID NO: 33, or N-terminally deficient variants of 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.

[0061] AAV genome The aforementioned AAV vector or AAV vector particle may contain an AAV genome or a fragment or derivative thereof.

[0062] The AAV genome is a polynucleotide sequence that may encode functions required for the production of AAV particles. These functions include those that work in the replication and packaging cycle of AAV in host cells, including the capsid formation of the AAV genome onto the AAV particle. Naturally occurring AAVs are replication-deficient and depend on the supply of helper functions in trans for the completion of the replication and packaging cycle. Therefore, the AAV genome used in this invention is typically replication-deficient.

[0063] The AAV genome may be in a single-stranded form with either a positive or negative strand, or it may be in a double-stranded form instead. The use of a double-stranded form allows for the avoidance of the DNA replication step in target cells, thereby accelerating 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 a single-stranded form.

[0064] Naturally occurring AAVs can be classified according to various biological systems. The AAV genome may be from any naturally occurring serotype, isolate, or clade of AAV.

[0065] AAV can be referred to in terms of its serotype. A serotype corresponds to a variant subspecies of AAV, which has characteristic reactivity that can be used to distinguish it from other variant subspecies due to the expression profile of its capsid surface antigen. Typically, AAV vector particles having a particular AAV serotype do not efficiently cross-react with neutralizing antibodies specific to 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 of serotype AAV3B, LK03, AAV9, or AAV8.

[0066] AAVs can also be referred to in terms of clades or clones. This refers to the phylogenetic relationships of naturally occurring AAVs, and typically, groups of AAVs that can be traced back to a common ancestor and include all of its descendants. Furthermore, AAVs can also be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, and therefore defines a population that is distinct enough to be recognized at the genetic level.

[0067] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one reverse-terminal repeat (ITR). ITR sequences provide a functional origin for replication by acting cis-wise and enable vector integration and excision from the cell's genome. ITRs may be the only sequences required cis-wise next to therapeutic genes.

[0068] The AAV genome may also include packaging genes, such as rep and / or cap genes, which encode packaging functions for AAV particles. Promoters may be manipulably ligated to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters. For example, the p5 and p19 promoters are commonly used to express the rep gene, and the p40 promoter is commonly used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or their variants. The cap gene encodes one or more capsid proteins, such as VP1, VP2, and VP3 or their variants. 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). Therefore, VP1, VP2, and VP3 may have the same sequence, but in this case, VP2 is truncated at the N-terminus compared to VP1, and VP3 is truncated at the N-terminus compared to VP2.

[0069] 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 containing the entire AAV genome.

[0070] Preferably, the AAV genome is derivatized for administration to a patient. Such derivatization is standard practice in the art, and the present invention encompasses the use of any known derivative of the AAV genome, and derivatives that can be produced 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.

[0071] Derivatives of the AAV genome include any truncated or modified AAV genome that enables the expression of the transgene from the AAV vector of the present invention in vivo. Typically, the AAV genome can be significantly truncated to contain minimal viral sequences while retaining the above-mentioned functions. This is preferred for safety reasons, as it reduces the risk of recombination of the vector with wild-type virus and further avoids the induction of a cellular immune response due to the presence of viral gene proteins in target cells.

[0072] Accordingly, derivatives of the present invention may have the following portions removed: one reverse-terminal repeat (ITR) sequence, a replication (rep) gene, and a capsid (cap) gene. However, derivatives may further contain one or more rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV is frequently integrated into specific sites on human chromosome 19 and exhibits random integration at negligible frequencies; therefore, retention of integration ability in AAV vectors may be acceptable in a therapeutic setting.

[0073] The present invention further includes providing AAV genome sequences with different order and arrangement from those of the natural AAV genome. The present invention also includes the substitution of one or more AAV sequences or genes with a chimeric gene composed of sequences from another virus or sequences from two or more viruses. Such a chimeric gene may be composed of sequences from two or more related viral proteins of different viral species.

[0074] Minigenetic Approach COL4A3, COL4A4, and COL4A5, each consisting of approximately 1700 amino acids, are difficult to package in their full-length form into AAV vectors due to AAV packaging limitations. However, we have developed a minimal nephrine promoter that is shorter than any known minimal nephrine promoter and, surprisingly, possesses the ability to drive transgene expression in podocytes. Using such a minimal nephrine promoter, it is possible to minimize the size of the cargo and assist in the packaging of full-length COL4A3, COL4A4, or COL4A5.

[0075] One alternative option for packaging full-length COL4A3, COL4A4, or COL4A5 is to provide the COL4A3, COL4A4, or COL4A5 transgene as a minigene. This minigene approach has been successfully used in the development of gene therapies for treating Duchenne muscular dystrophy (Kodippili et al. 2018). In this approach, the transgene is truncated to fit into a vector without losing the activity of the protein encoded by the transgene.

[0076] The COL4A3, COL4A4, and COL4A5 proteins are homologous polypeptides of approximately 170–185 kDa that contain collagen Gly-XY repeat sequences, often separated by non-collagenary sequences, and form a triple helix repeat. Each polypeptide also contains a large globular non-collagenary domain at its carboxyl terminus. To create truncated transgenes suitable for the minigene approach, approximately 200–300 amino acids should be removed from each of the COL4A3, COL4A4, and COL4A5 polypeptides. These amino acids may be removed from the triple helix repeat. Preferably, these amino acids are not removed from the non-collagenary regions.

[0077] The COL4A5 minigene, having an N-terminal HA tag or an N-terminal MyC tag, may be ligated to AAV2 / 9, AAVLK03, and AAVL3 vectors containing the human minimal nephrin promoter (NPHS2).

[0078] Viral vector gene therapy The present invention provides viral vector gene therapy, wherein the viral vector comprises a COL4A3, COL4A4, or COL4A5 transgene.

[0079] The viral vector used in the aforementioned viral vector gene therapy may be any viral vector of the present invention as described herein. Therefore, when a viral vector is referred to herein, it should be understood that this may also refer to viral vector gene therapy unless otherwise indicated in the context.

[0080] Dual vector string An alternative option to the aforementioned viral vector gene therapy may be to utilize a dual-vector approach. In this approach, the viral vector gene therapy comprises a first viral vector containing at least a portion of the COL4A3, COL4A4, or COL4A5 transgene, and an optional podocyte-specific promoter, and a second viral vector containing at least a portion of the corresponding COL4A3, COL4A4, or COL4A5 transgene, and an optional podocyte-specific promoter. In other words, the transgene can be split into two separate sequences, each of which can be incorporated into the viral vector gene therapy described herein. The AAV dual-vector approach is described, for example, in McClements and MacLaren 2017 (which is incorporated herein by reference). The transgene sequences used in the dual-vector approach may have overlapping exon or intron sequences that, upon transduction, combine, for example, through homologous recombination to reform a single transgene sequence. Alternatively, these two sequences may not overlap and instead combine, for example, by an intein protein trans-splicing approach. By incorporating a splice-donating signal into one of two vectors and a splice-receiving signal into the second vector, it is possible to enable trans-splicing that produces mature mRNA after head-to-tail concatemer formation mediated by ITRs. Furthermore, these approaches can be integrated into various hybrid approaches, such as combining recombination with trans-splicing.

[0081] The first viral vector may be a viral vector according to the present invention as described herein, and / or the second viral vector may be a viral vector according to the present invention as described herein. In a preferred embodiment, both the first viral vector and the second viral vector are viral vectors according to the present invention as described herein.

[0082] COL4A3, COL4A4, and COL4A5 transgenes The COL4A3, COL4A4, or COL4A5 transgenes may contain introns or intron sequences, and may have the ability to improve gene expression using such sequences. The introns or intron sequences may be used in either the minigene or dual-vector approach. The dual-vector approach allows for recombination of the first or second portion of the transgene via homologous sequences of the introns. This is particularly useful when combining the dual-vector approach with splice donor and acceptor methods, as using exon sequences would result in the excision of a portion of the protein (which is usually undesirable).

[0083] The COL4A3, COL4A4, or COL4A5 transgene may encode a COL4A3, COL4A4, or COL4A5 polypeptide, or a fragment or derivative thereof.

[0084] The COL4A3, COL4A4, or COL4A5 polypeptides or their fragments or derivatives may be capable of forming a collagen IVα345 network. Preferably, the fragments have approximately 200-300 amino acids removed.

[0085] In some embodiments, the COL4A3, COL4A4, or COL4A5 polypeptide is a full-length polypeptide.

[0086] Preferably, the COL4A3, COL4A4, or COL4A5 polypeptide is human. An example of human COL4A3 is COL4A3 with UniProtKB access number Q01955. An example of human COL4A4 is COL4A3 with UniProtKB access number P53420. An example of human COL4A5 is COL4A5 with UniProtKB access number P29400.

[0087] Preferably, the COL4A3 peptide may include or consist of a polypeptide sequence represented as SEQ ID NO: 1, or a variant that is at least 70% identical to SEQ ID NO: 1. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0088] Preferably, the COL4A4 peptide may include or consist of a polypeptide sequence represented as SEQ ID NO: 2, or a variant that is at least 70% identical to SEQ ID NO: 2. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 2.

[0089] Preferably, the COL4A5 peptide may include or consist of a polypeptide sequence represented as SEQ ID NO: 3, or a variant that is at least 70% identical to SEQ ID NO: 3. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 3.

[0090] Exemplary COL4A3 amino acid sequence (SEQ ID NO: 1) TIFF0007884456000005.tif149151

[0091] Exemplary COL4A4 amino acid sequence (SEQ ID NO: 2) TIFF0007884456000006.tif150151

[0092] Exemplary COL4A5 amino acid sequence (SEQ ID NO: 3) TIFF0007884456000007.tif59150TIFF0007884456000008.tif90150

[0093] One example of a nucleotide sequence encoding COL4A3 is NM_000091.5. One example of a nucleotide sequence encoding COL4A4 is NM_000092.5. One example of a nucleotide sequence encoding COL4A5 is NM_000495.5.

[0094] Preferably, the COL4A3 transgene may include or consist of a polynucleotide sequence represented as SEQ ID NO: 4, or a variant that is at least 70% identical to SEQ ID NO: 4. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

[0095] Preferably, the COL4A4 transgene may include or consist of a polynucleotide sequence represented as SEQ ID NO: 5, or a variant that is at least 70% identical to SEQ ID NO: 5. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

[0096] Preferably, the COL4A5 transgene may include or consist of a polynucleotide sequence represented as SEQ ID NO: 6, or a variant that is at least 70% identical to SEQ ID NO: 6. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 6.

[0097] Exemplary COL4A3 transgene sequence (SEQ ID NO: 4) TIFF0007884456000009.tif221150TIFF0007884456000010.tif216150

[0098] Exemplary COL4A4 transgene sequence (SEQ ID NO: 5) TIFF0007884456000011.tif234150TIFF0007884456000012.tif211151

[0099] Exemplary COL4A5 transgene sequence (SEQ ID NO: 6) TIFF0007884456000013.tif235151TIFF0007884456000014.tif210151

[0100] The COL4A3, COL4A4, or COL4A5 transgenes may have optimized codons. Different cells use different codons. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a given cell type. It is possible to increase expression by modifying the codons in the sequence to match the relative abundance of the corresponding tRNA. For the same reason, it is possible to decrease expression by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, additional levels of translational regulation are possible. Codon frequency tables are publicly known in the art for mammalian cells (e.g., humans) and for various other organisms.

[0101] Regulatory array promoter The viral vector of the present invention may include a promoter for promoting the expression of the COL4A3, COL4A4, or COL4A5 polypeptide. Preferably, the promoter may be ligated to the COL4A3, COL4A4, or COL4A5 transgene in a manipulable manner.

[0102] Preferably, the promoter is operable in podocytes. Preferably, the promoter has the ability to drive the expression of the transgene in podocytes. Preferably, the viral vector of the present invention includes a podocyte-specific promoter. Appropriately, the COL4A3, COL4A4, or COL4A5 transgene is ligated to the podocyte-specific promoter in an operable manner.

[0103] As described above, the inventors have developed a minimal nephrine promoter that is shorter than known minimal nephrine promoters and, surprisingly, has the ability to drive transgene expression in podocytes. Surprisingly, this promoter also retains podocyte specificity. By using such a minimal nephrine promoter, it is possible to minimize the size of the package and assist in the packaging of full-length COL4A3, COL4A4, or COL4A5.

[0104] The use of podocyte-specific promoters, such as minimal nephrin promoters, allows for the specific targeting of the viral vector to podocytes (Moeller et al., 2002; Picconi et al., 2014). Suitable minimal nephrin promoters include NPHS1 and podosin promoter NPHS2. This enables the specific targeting of transgene expression to podocytes in the glomerular basement membrane of the kidney and minimizes off-target expression. Since podocytes are terminally differentiated, non-dividing cells, they can be targeted for stable transgene expression, and all risks of vector dilution can be reduced or avoided. In a preferred embodiment of the present invention, the promoter is NPHS1. An example of a suitable DNA sequence for the NPHS1 promoter is shown in Figure 1. As with the transgene, the promoter species is preferably matched to the patient species. For example, when treating human patients, human NHPS1 or human NPHS2 is typically used.

[0105] As used herein, “podocyte-specific promoter” may be a promoter that preferentially promotes the expression of a transgene in podocytes. Appropriately, a podocyte-specific promoter may promote higher expression of a transgene in podocytes compared to other cell types. For example, a podocyte-specific promoter may promote the expression level of a transgene at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 300%, 400%, 500%, or 1000% higher in podocytes compared to the expression level in other cell types.

[0106] The expression of the transgene may be measured by any suitable method known in the art. For example, by measuring the expression of a reporter transgene, such as GFP, which is manipulably linked to the promoter, where the expression of the reporter transgene correlates with the promoter's ability to promote gene expression. The expression of the reporter transgene, such as GFP, may be determined by any suitable method, such as FACS. For example, a podocyte-specific promoter may promote higher expression of the reporter transgene in conditionally immortalized podocytes compared to other cell types, such as glomerular endothelial cells. Suitable podocyte systems, such as CIHP-1, are well known to those skilled in the art. Methods for producing 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.

[0107] Appropriately, the promoter may be a minimal podocyte-specific promoter. The promoter may have a length of about 1.2 kb or less. Appropriately, 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. Appropriately, the promoter has a length of about 1.15 kb or less. The promoter may have a length of about 1.1 kb or less. In 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.

[0108] 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. 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. In some embodiments, the promoter has a length of about 0.3 kb or less.

[0109] The promoter may have a length of about 250 bp or more. In some embodiments, the promoter has a length of about 250-1100 bp, 250-1000 bp, 250-900 bp, 250-800 bp, 250-700 bp, 250-600 bp, 250-500 bp, 250-400 bp, or 250-300 bp. 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. 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 another embodiment, the promoter has a length of 800–850 bp, 800–840 bp, 810–830 bp, 815–825 bp, or about 819 bp.

[0110] Minimal nephrin promoter The viral vector of the present invention may contain a minimal nephrine promoter. Preferably, the minimal nephrine promoter may be ligated to the COL4A3, COL4A4, or COL4A5 transgene in a manipulable manner.

[0111] The minimal nephrin promoter may also be a minimal NPHS1 promoter. For example, the NPHS1 promoter may have a length of 1.2 kb or less. The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes.

[0112] The minimal human NPHS1 promoter is described by 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. This minimal NPHS1 is a 1.2 kb fragment and appears to be podocyte-specific. This 1.2 kb promoter region lacks the TATA box but has recognition motifs for other transcription factors, such as the PAX-2 binding element, E box, and GATA consensus sequence.

[0113] Appropriately, the minimal nephrine promoter may include or consist of a nucleotide sequence represented as SEQ ID NO: 7, or a variant that is at least 70% identical to SEQ ID NO: 7 (as also shown in Figure 1).

[0114] Exemplary minimal NPHS1 promoter (SEQ ID NO: 7): TIFF0007884456000015.tif108150

[0115] Appropriately, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to sequence number 7.

[0116] Suitablely, the minimal nephrine promoter may include or consist of a nucleotide sequence represented as SEQ ID NO: 8, or a variant that is at least 70% identical to SEQ ID NO: 8.

[0117] Exemplary minimal NPHS1 promoter (SEQ ID NO: 8): TIFF0007884456000016.tif18149TIFF0007884456000017.tif95149

[0118] Appropriately, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to sequence number 8.

[0119] In some embodiments, the minimal nephrine promoter comprises or consists of a nucleotide sequence represented as SEQ ID NO: 9, or a variant that is at least 70% identical to SEQ ID NO: 9.

[0120] Exemplary minimal nephrine promoter - 819 bp (SEQ ID NO: 9) TIFF0007884456000018.tif73149

[0121] Appropriately, 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%, and at least 99% identical to sequence number 9.

[0122] In a preferred embodiment, the minimal nephrine promoter comprises or consists of a nucleotide sequence represented as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10.

[0123] Exemplary minimal nephrine promoter - 265 bp (SEQ ID NO: 10) TIFF0007884456000019.tif25150

[0124] Appropriately, 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%, and at least 99% identical to sequence number 10.

[0125] Preferably, the minimal nephrine promoter is derived from SEQ ID NO: 8 or a variant having at least 70% identity with SEQ ID NO: 8. Preferably, the minimal nephrine promoter has one or more deletions compared to SEQ ID NO: 8 or a variant having at least 70% identity with SEQ ID NO: 8. Preferably, 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: 8. An exemplary variant is SEQ ID NO: 7.

[0126] Preferably, the minimal nephrine promoter includes or consists of a nucleotide sequence having one or more deletions, for example, one or two deletions, or a nucleotide sequence exhibiting 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. Preferably, the minimal nephrine promoter includes or consists of a nucleotide sequence having two or more deletions, or a nucleotide sequence exhibiting 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. The deletions may be of any size. Appropriately, the deletions are each 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. Appropriately, the deletions are each at least 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.

[0127] In some embodiments, the minimal nephrine promoter is the nucleotide sequence according to SEQ ID NO: 8, however here (i) The 1st to n1st positions of sequence number 8 are missing, where n1 is an integer from 1 to 430, and / or (ii) The n2-n3 position of sequence number 8 is missing, where n3 ≥ n2, n2 is an integer between 508 and 1061, and n3 is an integer between 508 and 1061. or a nucleotide sequence exhibiting 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 with the nucleotide sequence, It contains or consists of such nucleotide sequence.

[0128] For example, the minimal nephrine promoter is the nucleotide sequence according to SEQ ID NO: 8, however here (i) The 1st to n1th position of sequence number 8 is missing, where n1 is an integer from 1 to 430, and / or (ii) The n2-n3 position of sequence number 8 is missing, where n3 ≥ n2, n2 is an integer between 508 and 1061, and n3 is an integer between 508 and 1061. It may contain 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 with respect to.

[0129] Appropriately, n1 is an integer between 50 and 430, 100 and 430, 150 and 430, 200 and 430, 250 and 430, 300 and 430, 350 and 430, or 400 and 430. In some embodiments, n1 is an integer between 100 and 430. In some embodiments, n1 = 430, i.e., digits 1 through 430 of sequence number 8 are missing.

[0130] The size of the deletion is specified by the difference between n3 and n2. Suitablely, 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.

[0131] The values ​​that n2 and n3 take determine the location of the deletion. Suitablely, n2 and n3 are integers from 550 to 1050, n2 and n3 are integers from 600 to 1000, n2 and n3 are integers from 650 to 950, n2 and n3 are integers from 700 to 900, and n2 and n3 are integers from 750 to 850. In some embodiments, n2=508 and n3=1061, i.e., positions 508 to 1061 of sequence number 8 are deleted.

[0132] Minimal nephrin promoter region The inventors have determined the region of the nephrin promoter that drives the expression of the transgene.

[0133] A promoter typically includes a “core” and a “proximal” region. The “core promoter region” may include a transcription start site, an RNA polymerase binding site, and a basic transcription factor binding site. The “proximal promoter region” may include, for example, key regulatory elements and specific transcription factor binding sites required to promote effective and controllable transcription. The size and components of both the core and proximal promoter regions typically vary in a gene-specific manner. The promoter may also include a 5' untranslated region (5'UTR) (also known as a leader sequence) downstream of the core promoter region and upstream of the start codon.

[0134] The minimal nephrin promoter may be a hybrid promoter. As used herein, "hybrid promoter" includes a combination of elements derived from different promoters. For example, a hybrid promoter may include a proximal promoter region derived from one existing promoter and a core promoter from another existing promoter in order to achieve the expression of a desired transgene. Muscle hybrid promoters are described in Piekarowicz, K., et al. (2019). Methods & clinical development, 15, 157-169.

[0135] In some embodiments, the minimal nephrine promoter includes (i) a nucleotide sequence represented as SEQ ID NO: 12, or a variant that is at least 70% identical to SEQ ID NO: 12. While we do not wish to be bound by theory, it is thought that a nucleotide sequence having at least about 70% identity with SEQ ID NO: 12 could provide a proximal promoter region.

[0136] Exemplary proximal promoter region (SEQ ID NO: 12) TIFF0007884456000020.tif11150

[0137] Appropriately, 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: 12. The minimum nephrine promoter may include a variant of SEQ ID NO: 12, shown as SEQ ID NO: 13.

[0138] Exemplary variant proximal promoter region (SEQ ID NO: 13) TIFF0007884456000021.tif11150

[0139] In some embodiments, the minimal nephrine promoter includes (ii) a nucleotide sequence represented as SEQ ID NO: 14, or a variant that is at least 70% identical to SEQ ID NO: 14, a nucleotide sequence represented as SEQ ID NO: 15, or a variant that is at least 70% identical to SEQ ID NO: 15, and / or a nucleotide sequence represented as SEQ ID NO: 16, or a variant that is at least 70% identical to SEQ ID NO: 16.

[0140] In some embodiments, the minimal nephrine promoter includes (ii) a nucleotide sequence represented as SEQ ID NO: 14, or a variant that is at least 70% identical to SEQ ID NO: 14. While we do not wish to be bound by theory, it is thought that a nucleotide sequence having at least about 70% identity with SEQ ID NO: 14 could provide a core promoter region.

[0141] Exemplary core promoter region (SEQ ID NO: 14) TIFF0007884456000022.tif6150

[0142] Appropriately, 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: 14. The minimum nephrine promoter may include a variant of SEQ ID NO: 14, shown as SEQ ID NO: 17.

[0143] Exemplary variant core promoter region (SEQ ID NO: 17) TIFF0007884456000023.tif6141

[0144] In some embodiments, the minimal nephrine promoter includes (ii) a nucleotide sequence represented as SEQ ID NO: 15, or a variant that is at least 70% identical to SEQ ID NO: 15. While we do not wish to be bound by theory, it is thought that a nucleotide sequence having at least about 70% identity to SEQ ID NO: 15 could provide a 5'UTR.

[0145] Exemplary 5'UTR (SEQ ID NO: 15) TIFF0007884456000024.tif18149

[0146] Appropriately, 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: 15. The minimum nephrine promoter may include a variant of SEQ ID NO: 15 shown as SEQ ID NO: 18.

[0147] Exemplary variant 5'UTR (sequence number 18) TIFF0007884456000025.tif18154

[0148] In some embodiments, the minimal nephrine promoter includes (ii) a nucleotide sequence represented as SEQ ID NO: 16, or a variant that is at least 70% identical to SEQ ID NO: 16. While we do not wish to be bound by theory, it is thought that a nucleotide sequence having at least about 70% identity to SEQ ID NO: 16 could provide the core promoter region and the 5'UTR.

[0149] Exemplary core promoter region and 5'UTR (SEQ ID NO: 16) TIFF0007884456000026.tif18150

[0150] Appropriately, 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: 16. The minimum nephrine promoter may include a variant of SEQ ID NO: 16, shown as SEQ ID NO: 19.

[0151] Exemplary variant core promoter region and 5'UTR (SEQ ID NO: 19) TIFF0007884456000027.tif18150

[0152] In some embodiments, the minimal nephrine promoter includes (iii) a nucleotide sequence having at least 70% identity with SEQ ID NO: 20 or one or more fragments thereof. Preferably, the minimal nephrine promoter includes a nucleotide sequence having at least about 70% identity with SEQ ID NO: 20 or one or more fragments thereof immediately downstream of the proximal promoter region and / or immediately upstream of the core promoter region.

[0153] The minimal nephrine promoter may have a nucleotide sequence that is 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% identical to SEQ ID NO: 20 or one or more fragments thereof. The minimal nephrine promoter may contain the nucleotide sequence of SEQ ID NO: 20 or one or more fragments thereof.

[0154] An example of an arbitrary promoter region (SEQ ID NO: 20) TIFF0007884456000028.tif53150

[0155] Preferably, one or more of the aforementioned fragments are (a) a 5'-terminal fragment and / or (b) a 3'-terminal fragment. Preferably, the 5'-terminal fragment may be located immediately downstream of the proximal promoter region. Preferably, the 3'-terminal fragment may be located immediately upstream of the core promoter region. For example, the minimal nephrine promoter is as follows: (a) A nucleotide sequence having at least 70% of the 1-x positions of SEQ ID NO: 20, and / or (b) A nucleotide sequence having at least 70% identity with position y~554 of sequence number 20, where x and y are integers and y > x. It may include.

[0156] The fragment(s) of sequence number 20 can be of any length. Appropriately, the fragment(s) may have lengths of approximately 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.

[0157] In some embodiments, the minimum nephrine promoter does not include SEQ ID NO: 20.

[0158] In some embodiments, the minimal nephrine promoter includes a nucleotide sequence having at least 70% identity with (iv) SEQ ID NO: 21 or a fragment thereof. Preferably, the minimal nephrine promoter includes a nucleotide sequence having at least about 70% identity with SEQ ID NO: 21 or a fragment thereof immediately upstream of the proximal promoter region.

[0159] The minimal nephrine promoter may contain 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 with SEQ ID NO: 21 or a fragment thereof. The minimal nephrine promoter may contain the nucleotide sequence of SEQ ID NO: 21 or one or more fragments thereof.

[0160] Example of an arbitrary upstream promoter region (SEQ ID NO: 21) TIFF0007884456000029.tif29150TIFF0007884456000030.tif12150

[0161] Suitablely, the fragment is a 3' terminal fragment. For example, the minimal nephrine promoter may contain a nucleotide sequence having at least 70% identity with position z~430 (where z is an integer) of SEQ ID NO: 21.

[0162] The fragment of sequence number 21 can be of any length. Appropriately, the fragment may have a length of approximately 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.

[0163] In some embodiments, the minimum nephrine promoter does not include SEQ ID NO: 21.

[0164] In some embodiments, the minimum nephrin promoter is located from 5' to 3', i.e. (i) A nucleotide sequence having at least 70% identity with SEQ ID NO: 12, (iii) Optionally, a nucleotide sequence having at least 70% identity with SEQ ID NO: 20 or one or more fragments thereof, and (ii) A nucleotide sequence having at least 70% identity with SEQ ID NO: 14, a nucleotide sequence having at least 70% identity with SEQ ID NO: 15, and / or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 It contains or consists of such nucleotide sequence.

[0165] In some embodiments, the minimum nephrin promoter is located from 5' to 3', i.e. (i) A nucleotide sequence having at least 70% identity with SEQ ID NO: 12, (iii) optionally, (a) a nucleotide sequence having at least 70% identity to the 5' terminal fragment of SEQ ID NO: 20, and / or (b) a nucleotide sequence having at least 70% identity to the 3' terminal fragment of SEQ ID NO: 20, (ii) A nucleotide sequence having at least 70% identity with SEQ ID NO: 14, a nucleotide sequence having at least 70% identity with SEQ ID NO: 15, and / or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 It contains or consists of such nucleotide sequence.

[0166] Minimal nephrine promoter element The inventors have determined the functional elements of the nephrin promoter that drive the expression of the transgene.

[0167] The minimal nephrine promoter may include 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 initiation site.

[0168] Appropriately, the minimal nephrine promoter includes 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 initiation site.

[0169] A retinoic acid receptor (RAR) binding site refers to a polynucleotide sequence capable of binding RARα, RARβ, and / or RARγ. The RAR binding site may include or consist of the nucleotide sequence shown as SEQ ID NO: 22, or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 22. The substitutions, deletions, or insertions may be any single nucleotide substitutions, deletions, or insertions such that the RAR binding site retains at least one of its endogenous functions.

[0170] Exemplary RAR binding site (SEQ ID NO: 22) GGGGTCA

[0171] A WT1 binding site refers to a polynucleotide sequence capable of binding to the zinc finger polypeptide encoded by the Wilms tumor suppressor gene WT1. The WT1 binding site may include, or consist of, the nucleotide sequence shown as SEQ ID NO: 23, or a nucleotide sequence having one, two, or three substitutions, deletions, or insertions compared to SEQ ID NO: 23. The substitutions, deletions, or insertions may be any single nucleotide substitutions, deletions, or insertions such that the WT1 binding region retains at least one of its endogenous functions.

[0172] Exemplary WT1 binding site (SEQ ID NO: 23) TIFF0007884456000031.tif6141

[0173] An enhancer box refers to a DNA response element found in some eukaryotes that functions as a protein binding site. The enhancer box may include or consist of a nucleotide sequence shown as SEQ ID NO: 24, or a nucleotide sequence having one or two substitutions, deletions, or insertions compared to SEQ ID NO: 24. The substitutions, deletions, or insertions may be any single nucleotide substitutions, deletions, or insertions such that the enhancer box retains at least one of its endogenous functions.

[0174] Exemplary enhancer box (SEQ ID NO: 24) ATGTG

[0175] (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.

[0176] In some embodiments, one or more of the following elements are present in (i) a nucleotide sequence having at least 70% identity with SEQ ID NO: 12, namely (a) a RAR binding site at a position approximately corresponding to positions 7-13 of SEQ ID NO: 12, (b) a WT1 binding site at a position approximately corresponding to positions 14-30 of SEQ ID NO: 12, and (c) an enhancer box at a position approximately corresponding to positions 49-53 of SEQ ID NO: 12. In some embodiments, each of these elements is present in (i) a nucleotide sequence having at least 70% identity with SEQ ID NO: 12.

[0177] In some embodiments, one or more of the following nucleotide sequences are present in (i) a nucleotide sequence having at least 70% identity with SEQ ID NO: 12, namely (a) GGGGTCA at positions 7-13 of SEQ ID NO: 12, (b) CGGAGGCTGGGGAGGCA at positions 14-30 of SEQ ID NO: 12, and (c) ATGTG at positions 49-53 of SEQ ID NO: 12. In some embodiments, each of these nucleotide sequences is present in (i) a nucleotide sequence having at least 70% identity with SEQ ID NO: 12.

[0178] Suitablely, the minimal nephrin promoter may include a nucleotide sequence shown as SEQ ID NO: 25, or a nucleotide sequence having one, two, three, four, or five substitutions, deletions, or insertions compared to SEQ ID NO: 25, or a transcription factor-binding region composed of such nucleotide sequences. The substitutions, deletions, or insertions may be any single nucleotide substitutions, deletions, or insertions such that the transcription factor-binding region retains at least one of its endogenous functions.

[0179] Exemplary transcription factor binding region (SEQ ID NO: 25) TIFF0007884456000032.tif6141

[0180] Other suitable transcription factor binding regions are well known to those skilled in the art. For example, other suitable transcription factor binding regions include TACGAT (SEQ ID NO: 36), TATAAT (SEQ ID NO: 37), GATACT (SEQ ID NO: 38), TATGAT (SEQ ID NO: 39), and TATGTT (SEQ ID NO: 40).

[0181] Appropriately, the minimal nephrine promoter may include an "AG" dinucleotide or a transcription initiation site composed of the "AG" dinucleotide.

[0182] Preferably, the transcription factor binding site is manipulably linked to the transcription initiation site. Preferably, the transcription factor binding site may be located immediately upstream of the transcription initiation site. While we do not wish to be bound by theory, it is conceivable that the transcription factor binding site and the transcription initiation site may provide a core promoter region.

[0183] Appropriately, the minimum nephrine promoter may include a 5' untranslated region. The 5' untranslated region may contain or consist of a nucleotide sequence having at least about 70%, 80%, 90%, 95%, or 99% sequence identity with SEQ ID NO: 15. The 5' untranslated region may contain or consist of SEQ ID NO: 15.

[0184] Preferably, the 5' untranslated region is manipulably connected to the transcription initiation site. Preferably, the 5' untranslated region may be located immediately downstream of the transcription initiation site.

[0185] In some embodiments, the minimum nephrin promoter is defined as follows, from 5' to 3': (i) A nucleotide sequence having at least 70% identity with SEQ ID NO: 12, wherein each of the following elements is present: (a) a RAR binding site at a position approximately corresponding to positions 7-13 of SEQ ID NO: 12, (b) a WT1 binding site at a position approximately corresponding to positions 14-30 of SEQ ID NO: 12, and (c) an enhancer box at a position approximately corresponding to positions 49-53 of SEQ ID NO: 12. (iii) Optionally, a nucleotide sequence having at least 70% identity with SEQ ID NO: 20 or one or more fragments thereof, and (ii) A nucleotide sequence having at least 70% identity with SEQ ID NO: 14, a nucleotide sequence having at least 70% identity with SEQ ID NO: 15, or a nucleotide sequence having at least 70% identity with SEQ ID NO: 16 It contains or consists of such nucleotide sequence.

[0186] Exemplary minimal nephrin promoter In some embodiments, the minimal nephrine promoter comprises or consists of a nucleotide sequence represented as SEQ ID NO: 9, or a variant that is at least 70% identical to SEQ ID NO: 9.

[0187] In some embodiments, the minimal nephrine promoter comprises a nucleotide sequence shown as SEQ ID NO: 9, or a variant that is at least 70% identical to SEQ ID NO: 9, wherein the promoter has a length of approximately 1.1 kb or less.

[0188] In some embodiments, the minimal nephrine promoter consists of a nucleotide sequence represented as SEQ ID NO: 9, or a variant that is at least 70% identical to SEQ ID NO: 9.

[0189] Appropriately, 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: 9. The minimum nephrine promoter may include or consist of a variant of SEQ ID NO: 9, shown as SEQ ID NO: 34.

[0190] Exemplary minimal nephrine promoter variant - 819 bp (SEQ ID NO: 34) TIFF0007884456000033.tif60152TIFF0007884456000034.tif11151

[0191] In some embodiments, the minimal nephrine promoter includes or consists of a nucleotide sequence represented as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10.

[0192] In some embodiments, the minimal nephrine promoter comprises a nucleotide sequence shown as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10, wherein the promoter has a length of approximately 1.1 kb or less.

[0193] In some embodiments, the minimal nephrine promoter consists of a nucleotide sequence represented as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10.

[0194] Appropriately, 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: 10. The minimum nephrine promoter may include or consist of a variant of SEQ ID NO: 10, shown as SEQ ID NO: 35.

[0195] Exemplary minimal nephrine promoter variant - 265 bp (SEQ ID NO: 35) TIFF0007884456000035.tif23150

[0196] Minimal Podosin Promoter The viral vector of the present invention may include a minimal podosin promoter. Preferably, the minimal podosin promoter may be ligated to the COL4A3, COL4A4, or COL4A5 transgene in a manipulable manner.

[0197] The minimal podosin promoter may also be a minimal NPHS2 promoter. For example, the NPHS2 promoter may have a length of 0.6 kb or less. The NPHS2 gene encodes podosin, which is selectively expressed in podocytes.

[0198] The minimal human NPHS2 promoter is described by Oleggini R, et al., 2006. Gene Expr. 13(1):59-66. This minimal NPHS2 is a 630 bp fragment that showed expression in podocytes in vitro.

[0199] Suitablely, the minimal podosin promoter may include or consist of a nucleotide sequence represented as SEQ ID NO: 11, or a variant that is at least 70% identical to SEQ ID NO: 11.

[0200] Exemplary minimal NPHS2 promoter (SEQ ID NO: 11): TIFF0007884456000036.tif60149

[0201] Appropriately, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 11.

[0202] Other promoters Other non-podocyte-specific promoters for use in the present invention are well known to those skilled in the art. In some embodiments, the promoter may have a length of about 300 bp or less. In some embodiments, the promoter has a length of about 290 bp or less, 280 bp or less, 270 bp or less, 260 bp or less, 250 bp or less, 240 bp or less, 230 bp or less, 220 bp or less, 210 bp or less, or 200 bp or less. The use of a promoter with a length of about 300 bp or less may assist in packaging the COL4A3, COL4A4, and COL4A5 transgenes into AAV vectors in their full-length form.

[0203] Exemplary promoters with a length of approximately 300 bp or less are described by Wang, D., et al., 1999. Gene therapy, 6(4), pp.667-675. Wang et al. describe four short promoters with significantly higher activity than AAV ITR alone and with a size of 102 bp to 200 bp. These promoters are AAV-P5 (150 bp), SV40e (200 bp), TK1 (110 bp), and the second TK promoter (TK2) (102 bp), which has a further 10 bp deletion between its distal and proximal elements.

[0204] Woodchuck hepatitis virus post-transcriptional regulatory elements The viral vector may further contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Preferably, the WPRE may be ligated to the COL4A3, COL4A4, or COL4A5 transgene in a manipulable manner. The WPRE is a DNA sequence that, upon transcription, constructs a tertiary structure that enhances expression. The inclusion of the WPRE may increase the expression of the transgene delivered by the vector. The WPRE sequence may be mutated to reduce oncogenicity without significant loss of RNA-enhancing activity (as incorporated herein by reference to Schambach et al., 2005). An example of a suitable WPRE sequence is shown in Figure 2.

[0205] Appropriately, the WPRE may include or consist of a nucleotide sequence represented as SEQ ID NO: 26, or a variant that is at least 70% identical to SEQ ID NO: 26 (as also shown in Figure 2).

[0206] Exemplary WPRE (Sequence ID 26) TIFF0007884456000037.tif54151

[0207] Appropriately, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to sequence number 26.

[0208] In some embodiments, the viral vector of the present invention does not contain a WPRE sequence.

[0209] Protein tags The COL4A3, COL4A4, or COL4A5 transgenes may include a protein tag, such as a hemagglutinin (HA) tag. HA can be used as an epitope tag and has been shown not to interfere with the bioactivity or in vivo distribution of the tagged protein. The protein tag can facilitate the detection, isolation, and purification of the transgene. Other suitable protein tags include MyC tags, polyhistidine tags, and flag tags.

[0210] In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene includes one or more flag tags. In some embodiments, the COL4A3, COL4A4, or COL4A5 transgene includes three flag tags.

[0211] Kozak Array The viral vector may further include a Kozak sequence between the promoter and the COL4A3, COL4A4, or COL4A5 transgene. The Kozak sequence is known to play a major role in the initiation of the translation process and, therefore, can enhance the expression of the COL4A3, COL4A4, or COL4A5 transgene. Suitable Kozak sequences are well known to those skilled in the art.

[0212] Suitablely, the Kozak sequence may include or consist of a nucleotide sequence represented as SEQ ID NO: 27, or a variant that is at least 65% identical to SEQ ID NO: 27.

[0213] Exemplary Kossack sequence (sequence number 27) GCCGCCACCAUGG

[0214] Appropriately, the variant may be at least 75%, at least 85%, or at least 90% identical to SEQ ID NO: 27.

[0215] In some embodiments, the viral vector of the present invention does not contain a Kozak sequence.

[0216] Polyadenylation signal The viral vector may further include a polyadenylation signal, such as a bovine growth hormone (bGH) polyadenylation signal, as shown in Figure 3, for example. Preferably, the polyadenylation signal may be ligated to the COL4A3, COL4A4, or COL4A5 transgene in a manipulable manner. Polyadenylation is the addition of a poly(A) tail to messenger RNA. The poly(A) tail consists of multiple adenosine monophosphates; in other words, the poly(A) tail is a continuous RNA molecule containing only adenine bases. The poly(A) tail is important for mRNA nuclear export, translation, and stability. Inclusion of a polyadenylation signal can therefore enhance the expression of the COL4A3, COL4A4, or COL4A5 transgene.

[0217] Suitable polyadenylation signals include the initial SV40 polyadenylation signal (SV40pA), the chicken β-globin polyadenylation signal, the bovine growth hormone polyadenylation signal (bGH), or the soluble neuropilin-1 polyadenylation signal. In some embodiments, the polyadenylation signal is the initial SV40 polyadenylation signal (SV40pA) or the chicken β-globin polyadenylation signal. Preferably, the polyadenylation signal is the initial SV40 polyadenylation signal (SV40pA).

[0218] Preferably, the polyadenylation signal may include or consist of a nucleotide sequence represented as SEQ ID NO: 28, or a variant that is at least 70% identical to SEQ ID NO: 28 (as also shown in Figure 3). Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 28.

[0219] Exemplary bGH poly(A) signal sequence (SEQ ID NO: 28): TIFF0007884456000038.tif23150

[0220] Preferably, the polyadenylation signal may include or consist of a nucleotide sequence represented as SEQ ID NO: 29, or a variant that is at least 70% identical to SEQ ID NO: 29. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 29.

[0221] Exemplary soluble neuropilin-1 polyadenylation signal (SEQ ID NO: 29XX): aaataaaatacgaaatg

[0222] Preferably, the polyadenylation signal may include or consist of a nucleotide sequence represented as SEQ ID NO: 30, or a variant that is at least 70% identical to SEQ ID NO: 30. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 30.

[0223] Exemplary SV40pA signal sequence (SEQ ID NO: 30): TIFF0007884456000039.tif11149

[0224] Preferably, the polyadenylation signal may include or consist of a nucleotide sequence represented as SEQ ID NO: 41, or a variant that is at least 70% identical to SEQ ID NO: 41. Preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 41.

[0225] Exemplary chicken β-globin polyadenylation signal (SEQ ID NO: 41) TIFF0007884456000040.tif6149

[0226] Reverse terminal repeat sequence The viral vector may further include reverse terminal repeat (ITR) sequences at both ends of the vector. For example, the structure of the vector may be, in order, ITR-promoter-transgene (optionally having a protein tag)-optional WRPE-polyadenylation signal-ITR.

[0227] The aforementioned ITR may function as a promoter (Flotte, TR, et al. 1993. Journal of Biological Chemistry, 268(5), pp.3781-3790).

[0228] Typically, an AAV genome contains at least one reverse terminal repeat (ITR), preferably two or more ITRs, for example, two or more ITRs. One or more of these ITRs may originate from AAV genomes with different serotypes, or they may be chimeric or mutant ITRs. A preferred mutant ITR is one having a deletion of a terminal resolution site (trs). This deletion allows for continuous replication of the genome, creating a single-stranded genome containing both the coding and complementary sequences, i.e., a self-complementary AAV genome. This allows for avoidance of DNA replication in target cells, thereby accelerating the expression of the transgene. However, the maximum packaging capacity of scAAV is reduced. Appropriately, the AAV genome is not a scAAV genome.

[0229] The AAV genome may contain one or more ITR sequences or variants thereof from any naturally occurring serotype, isolate, or clade of AAV. The AAV genome may contain at least one, for example, two, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. Preferably, the AAV genome may contain at least one, for example, two, AAV2 ITRs.

[0230] Including one or more ITRs is preferable, for example, to assist concatemer formation of the AAV vector in the nucleus of the host cell after the conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. Such episomal concatemer formation protects the AAV vector for the lifetime of the host cell, thereby enabling long-term expression of the transgene in vivo.

[0231] Ideally, the ITR element is the only sequence retained from the natural AAV genome in the derivative. The derivative preferably does not contain the rep and / or cap genes of the natural genome, nor any other sequences of the natural genome. This is preferable for the reasons mentioned above, and also to reduce the likelihood of the vector being incorporated into the host cell genome. Furthermore, reducing the size of the AAV genome increases flexibility in incorporating not only the transgene but also other sequence elements (e.g., regulatory elements) into the vector.

[0232] Variants, derivatives, analogues, homologs, and fragments In addition to the specific proteins and nucleotides described herein, the present invention also encompasses their variants, derivatives, homologs, and fragments.

[0233] In relation to the present invention, a “variant” of any given sequence is a sequence in which a residue (whether an amino acid residue or a nucleic acid residue) of a particular sequence is modified in such a way that the polypeptide or polynucleotide retains at least one of its endogenous functions. Variant sequences can be obtained by adding, deleting, substituting, modifying, exchanging, and / or muting at least one residue present in a naturally occurring polypeptide or polynucleotide. For example, a variant promoter sequence retains at least some degree of the activity and specificity of the original promoter sequence from which the sequence was obtained.

[0234] As used herein with respect to the protein or polypeptide of the present invention, the term “derivative” includes any substitution, mutation, modification, exchange, deletion and / or addition of one (or more) amino acid residues to or from its sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0235] Typically, amino acid substitutions may range from, for example, one, two, or three substitutions to ten or twenty substitutions, provided that the modified sequence retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not exist in nature.

[0236] The proteins used in the present invention may also have deletions, insertions, or substitutions of amino acid residues that result in silent changes and functionally equivalent proteins. Planned amino acid substitutions may be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as the endogenous function is preserved. For example, aspartic acid and glutamic acid are listed as charged amino acids, lysine and arginine are listed as positively charged amino acids, and asparagine, glutamine, serine, threonine, and tyrosine are listed as amino acids with uncharged heads that have similar hydrophilic values.

[0237] Conservative substitutions may be carried out, for example, according to the table below. The amino acids listed in the same column in the second column, and preferably in the same row in the third column, can be substituted for each other:

[0238] [Table 1]

[0239] As used herein, the term "homolog" refers to a variant that has a certain degree of homology to a wild-type amino acid sequence or wild-type nucleotide sequence. The term "homology" can be considered identical to "identity."

[0240] In this context, a homologous sequence is considered to include an amino acid sequence that is at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, preferably at least 95%, 96%, 97%, 98%, or 99% identical. Typically, a homolog contains the same active site as the target amino acid sequence. While homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in relation to the present invention, homology is preferably expressed in terms of sequence identity.

[0241] In this context, homologous sequences are considered to include nucleotide sequences that are at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the target sequence, preferably at least 95%, 96%, 97%, 98%, or 99% identical. While homology can also be considered in terms of similarity, in relation to the present invention, homology is preferably expressed in terms of sequence identity.

[0242] Preferably, when referring to an sequence that indicates an identity percentage for any one of the sequence numbers detailed herein, it refers to an sequence that indicates the stated identity percentage over the full length of the sequence number referred to.

[0243] Homology comparisons can be performed by visual estimation or, more commonly, by employing readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0244] Homology percentages may be calculated over consecutive sequences; that is, one sequence is aligned with the other, and then each amino acid or nucleotide in one sequence is directly compared to its corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignment is performed only on a relatively small number of residues.

[0245] While this method is very simple and consistent, it does not take into account that, for example, in otherwise identical sequence pairs, a single insertion or deletion in an amino acid or nucleotide sequence can exclude subsequent residues or codons from the alignment, potentially leading to a significant decrease in the homology percentage when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting "gaps" during sequence alignment with the aim of maximizing local homology.

[0246] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting a higher relevance between the two compared sequences, scores higher than one with many gaps. Typically, an "affine gap cost" is used, setting a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue within that gap. This is the most commonly used gap scoring system. A high gap penalty naturally results in an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable 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 gaps and -4 for each extension.

[0247] Calculating the maximum homology percentage therefore requires first creating an optimal alignment that takes gap penalties into account. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Develeux et al. (1984) Nucleic Acids Research 12: 387). Other software that can perform sequence comparisons 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 tools in the GENEWORKS suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable 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).

[0248] While the final homology percentage can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which 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 programs in the BLAST suite). GCG Wisconsin programs typically use either public default values ​​or custom symbol comparison tables if provided (see the user manual for further details). For some applications, it is preferable to use the public default values ​​for the GCG package, or for other software, to use a default matrix such as BLOSUM62.

[0249] Once the software generates an optimal alignment, the homology percentage, preferably the sequence identity percentage, can be calculated. The software typically performs this as part of a sequence comparison and produces a numerical result. The sequence identity percentage may be calculated as the number of identical residues as a percentage of the total residues in the sequence number mentioned.

[0250] A "fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide of interest functionally or, for example, in an assay. A "fragment" therefore refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0251] Such variants, derivatives, homologs and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When making an insert, synthetic DNA encoding the insert may be made along with 5' and 3' flanking regions corresponding to the naturally occurring sequences on both sides of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the sequences such that the naturally occurring sequences are cut by a suitable enzyme(s) and the synthetic DNA is ligated to the cut sites. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely illustrative of numerous standard techniques known in the art for the manipulation of DNA sequences, and other known techniques may also be used.

[0252] cell In one aspect, the present invention provides a cell comprising the viral vector of the present invention. The cell may be an isolated cell. The cell may be a human cell, suitably an isolated human cell.

[0253] The viral vector may be introduced into the cell using various techniques known in the art, such as transfection, transduction and transformation. Suitably, the vector of the present invention is introduced into the cell by transfection or transduction.

[0254] The cell can be any cell type known in the prior art.

[0255] Suitably, the cell may be a producer cell. The term "producer cell" includes a cell that produces viral particles after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the viral particles, or any cell that has been engineered to stably contain the elements necessary to produce the viral particles. Suitable producer cells will be well known to those skilled in the art. Suitable producer cell lines include the HEK293 (e.g., HEK293T), HeLa, and A549 cell lines.

[0256] Appropriately, the cells may be packaging cells. The term “packaging cells” includes cells that contain some or all of the elements necessary for packaging an infectious recombinant virus. The packaging cells may be deficient in the recombinant viral vector genome. Typically, such packaging cells contain one or more vectors capable of expressing viral structural proteins. Cells containing only some of the elements necessary for the production of enveloped viral particles are useful as intermediate reactants in the preparation of viral particle producer cell systems through subsequent transient transfection, transduction, or stable incorporation steps of each of the further required elements. These intermediate reactants are encompassed within the term “packaging cells.” Appropriate packaging cells will be well known to those skilled in the art.

[0257] Appropriately, the cells may be kidney cells or glomerular cells, such as podocytes. Appropriately, the cells may be immortalized kidney cells or glomerular cells, such as immortalized podocytes. Suitable podocyte lines, such as CIHP-1, will be well known to those skilled in the art. Methods for producing immortalized podocytes will be 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.

[0258] As described above, the aforementioned cells have been described by referring to viral vectors, but it should be understood that viral vector gene therapy can be used as an alternative.

[0259] Methods for treating or preventing Alport syndrome In one embodiment, the present invention provides a viral vector, cells, or pharmaceutical composition according to the present invention for use as a pharmaceutical.

[0260] In one embodiment, the present invention provides the use of a viral vector, cells, or pharmaceutical composition according to the present invention in the manufacture of a pharmaceutical product.

[0261] In one embodiment, the present invention provides a method for administering a viral vector, cells, or pharmaceutical composition according to the present invention to a target that needs it.

[0262] In one embodiment, the present invention provides a viral vector, cells, or pharmaceutical composition according to the present invention for use in the prevention or treatment of Alport syndrome.

[0263] In one embodiment, the present invention provides the use of a viral vector, cell, or pharmaceutical composition according to the present invention for the manufacture of a pharmacopoeia for the prevention or treatment of Alport syndrome.

[0264] In one embodiment, the present invention provides a method for preventing or treating Alport syndrome, comprising administering a viral vector, cells, or pharmaceutical composition according to the present invention to a subject in need.

[0265] Genetic therapy of target podocytes with COL4A3, COL4A4, or COL4A5 viruses may alter, at least partially, the glomerular basement membrane of AS patients. The structural effects of such constructs on the glomerular basement membrane may be tested in vitro using human spheroid models of wild-type and Alport syndrome podocytes. These spheroid models may be examined for changes in glomerular basement membrane composition. Functional testing may also be performed using nephrons in a chip model containing glomerular endothelial cells and podocytes co-cultured on one side of a channel to produce a mature glomerular basement membrane, which can be used to measure protein permeability through the channel. Constructs may be tested in mouse α3 or α5 KO mice, or α4 spontaneous mouse mutants. Constructs may be administered by tail vein injection, and efficacy will be measured by proteinuria levels and survival.

[0266] Alport syndrome (AS) can be treated or prevented using the viral vector gene therapy of the present invention. AS patients typically present with hematuria, which may progress to proteinuria. Hematuria can be determined by the presence of red blood cells in the urine upon microscopic examination. Basal microalbuminuria levels of less than 30 mg / day are usually considered non-pathological. Levels of approximately 30 mg / day to approximately 300 mg / day are called microalbuminuria and are considered pathological. Albumin levels greater than 300 mg / day are called overt albuminuria, and proteinuria levels greater than 3.5 g / day are considered nephrotic proteinuria. Patients treated with the viral vector of the present invention may exhibit hematuria, microalbuminuria, overt albuminuria, or nephrotic proteinuria.

[0267] Treatment of patients before the onset of proteinuria may slow or prevent the progression of proteinuria, thereby delaying or preventing end-stage renal failure. Patients presenting with nephrotic proteinuria may be treated similarly. Since the collagen IVα345 network in the glomerular basement membrane is modified, normalized, or repaired by the transgene, proteinuria levels should gradually decrease after gene therapy treatment.

[0268] The patient may also, or instead, test positive for pathogenic variants of COL4A3, COL4A4, or COL4A5. COL4A3 and COL4A4 pathogenic variants may be heterozygous (autosomal dominant) or biallelelic (autosomal recessive). COL4A5 pathogenic variants may be hemizygous or heterozygous (X-linked). The patient is preferably treated with one or more viral vectors containing a transgene corresponding to the gene(s) on which the patient has the pathogenic variant. For example, a patient with a pathogenic COL4A5 variant may be treated with a viral vector containing the COL4A5 transgene. The patient may test positive for two or more pathogenic variants of COL4A3, COL4A4, or COL4A5. Such a patient may be treated with two or more viral vectors containing different transgenes, i.e., each viral vector containing a transgene corresponding to the gene on which the patient has the pathogenic variant.

[0269] In particular, the patient may have X-linked AS, which is typically associated with a pathogenic variant of COL4A5.

[0270] As used herein, the term “patient” may include any mammal, including humans. The patient may be an adult or a child, for example, a newborn or an infant. The patient may be male or female. The patient may be a male patient with X-linked AS, particularly a male patient in adolescence.

[0271] The viral vectors, cells, or pharmaceutical compositions according to the present invention may be administered parenterally, for example, intravenously or by infusion techniques. The vectors, cells, or pharmaceutical compositions may also be administered in the form of a sterile aqueous solution that may contain other substances, such as salts or glucose sufficient to make an isotonic solution with blood. The aqueous solution may be appropriately buffered (preferably to pH 3-9). The pharmaceutical compositions may be formulated as appropriate. The preparation of suitable parenteral formulations under sterile conditions is readily achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0272] The virus vector, cell or medicine may be administered systemically, such as by intravenous injection.

[0273] The virus vector, cell or pharmaceutical composition according to the present invention may be administered locally, for example by targeting the administration to the kidney. Appropriately, the virus vector, cell or pharmaceutical composition may be administered by injection into the renal artery, or by ureteral or subcapsular injection. In an embodiment of the present invention, the virus vector may be administered by injection into the renal artery. In an alternative embodiment of the present invention, the virus vector may be administered by retrograde administration, for example via the ureter using a urinary catheter.

[0274] The virus vector, cell or pharmaceutical composition may be administered as a single dose, that is to say, subsequent administration of the vector may not be required. When repeated administration is required, different viral serotypes can be used for the vector. For example, the vector used for the first administration may contain AAV-LK03 or AAV-3B, while the vector used for subsequent administrations may contain AAV 2 / 9.

[0275] The virus vector, cell or pharmaceutical composition may be administered at various doses (for example, measured in vector genomes (vg) / kg). In any case, a physician will determine the actual dosage that will be most appropriate for any individual subject, and that dosage will vary depending on the age, weight, and response of that particular subject. However, typically, in the case of the AAV vectors of the present invention, a dose of 10 10 ~10 14 vg / kg, or 10 11 ~10 13 vg / kg may be administered.

[0276] In some cases, the viral vector, cells, or pharmaceutical composition may be administered in combination with the patient's transient immunosuppression, for example, by administering the viral vector simultaneously with or after treatment with oral steroids. Immunosuppression may be desirable before and / or during gene therapy treatment to suppress the patient's immune response to the vector. However, since the AAV capsid is not encoded by the vector, it is only transiently present in the transduced cells. Therefore, the capsid is gradually degraded and removed, which means that long-term expression of the transgene may be possible with a short-term immunomodulatory regimen that blocks the immune response to the capsid until the capsid sequence is removed from the transduced cells. Immunosuppression may therefore be desirable about six weeks after the administration of the gene therapy.

[0277] The viral vector, cells, or pharmaceutical composition may be administered in combination with, or instead of, a renin-angiotensin therapeutic strategy, such as an angiotensin-converting enzyme (ACE) inhibitor, an aldosterone antagonist (e.g., spironolactone), or an angiotensin receptor blocker (ARB).

[0278] Pharmaceutical composition The viral vector may be administered in the form of a pharmaceutical composition. In other words, the viral vector may be combined with one or more pharmaceutically acceptable carriers, diluents, and / or excipients. A suitable pharmaceutical composition is preferably sterile.

[0279] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. Pharmaceutical choices of carriers, excipients, or diluents can be selected in relation to the intended route of administration and standard pharmaceutical practices. The pharmaceutical composition may contain, or in addition to, any suitable binder(s), lubricants(s), suspending agents(s), coating agents(s), or solubilizers(s).

[0280] Examples of pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0281] The pharmaceutical composition may further contain one or more other therapeutic agents.

[0282] The present invention further includes the use of a kit comprising the viral vector, cells, and / or pharmaceutical composition of the present invention. Preferably, the kit is for use in the method described above and is used as described herein, for example, in the therapeutic method described herein. Preferably, the kit includes instructions for use of the components of the kit.

[0283] As described above, methods for treating or preventing Alport syndrome have been described by referring to viral vectors, but it is understood that viral vector gene therapy can be used as an alternative.

[0284] (Examples) Alport syndrome, a disorder affecting the collagen α3α4α5(IV) network in the glomerular basement membrane, lacks a glomerular-specific therapeutic strategy. Currently, treatment focuses on targeting hypertension. Early indicators of Alport syndrome, such as increased glomerular filtration rate and microalbuminuria, are both associated with changes in the glomerular basement membrane. Collagen α3α4α5(IV) is produced by podocytes, not endothelial cells.

[0285] The objective of this study is to combine a successful strategy for treating Alport syndrome with a safe and effective gene delivery approach that can induce COL4A3, COL4A4, or COL4A5 gene expression in podocytes, preferably early in the disease and before the onset of proteinuria. [Examples]

[0286] Example 1 - Design, construction, and testing of minimal nephrine promoters conjugated to COL4A3, COL4A4, and COL4A5. Design and construction of AAV structures The following AAV transfer plasmids containing COL4A3, COL4A4, and COL4A5 conjugated to a mininephrine promoter ("265"; see Example 2 for details on the design, construction, and testing of the mininephrine promoter), namely, • This is an AAV plasmid containing COL4A3 conjugated to a mininephrine promoter, pAAV.265.Col4a3.3flag.sv40 (see Figure 4A). • This is an AAV plasmid containing COL4A4 conjugated to a mininephrine promoter, pAAV.265.Col4a4.3flag.sv40 (see Figure 4B). • This is an AAV plasmid containing COL4A5 conjugated to a mininephrine promoter, pAAV.265.Col4a5.3flag.sv40 (see Figure 4C). I designed and built it.

[0287] SmaI digestion was performed to confirm the identity of the plasmids (see Figures 4D-E). This demonstrated the successful cloning of COL4 a3, a4, and a5 into AAV, including the 265bp mininephrine promoter and SV40 polyA tail.

[0288] Testing of AAV structures The following AAV virus vectors, namely, LK03 serotype AAV.COL4A3.Nephrine 265.Sv40 LK03 serotype AAV.COL4A5.Nephrine 265.Sv40 • 2 / 9 serotype AAV.COL4A5.Nephrine 265.Sv40 It was prepared using the standard method.

[0289] Figure 5A shows immunoprecipitation experiments of full-length FLAG-tagged Col4a3 (LK03) or Col4a5 (LK03) in differentiated human ci podocytes pulled down with anti-FLAG antibody. Anti-FLAG antibody precipitated both Col4a3 and Col4a5. Human-FLAG IgG was used as a control.

[0290] Figure 5B shows Western blots of protein lysates indicating the expression levels of Col4a3 (LK03 capsid serotype), Col4a5 (LK03), and Col4a5 (2 / 9 capsid serotype) in human or mouse differentiated ci podocytes. Uninfected human and mouse ci podocytes were used as controls.

[0291] Figure 5C shows a confocal image of immunofluorescence staining of transduced Col4a5 in human wild-type Ci podocytes / Col4a5 3xFlag AAV Ci podocytes, along with F-actin. Col4a5 is present at the cytosolic level in human differentiated podocytes infected with Col4a5 3xFlag AAV virus, compared to its wild-type counterpart.

[0292] These results demonstrate that the inventors unexpectedly succeeded in transducing full-length COL4a3 and COL4a5 conjugated to the mininephrine promoter into human podocytes, and that the mininephrine promoter unexpectedly drives the expression of full-length COL4a3 or COL4a5 in the human podocytes. [Examples]

[0293] Example 2 - Design, construction, and testing of a minimal nephrine promoter Design of the Minimal Nephrine Promoter The human NPHS1 promoter is described by 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. This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific. Hereafter, it will be referred to as the "FL" nephrin promoter and is shown in Figure 6A.

[0294] First, the aforementioned FL nephrin promoter was cleaved and the N-terminal sequence was deleted, resulting in a length of 822 bp (819 bp excluding the start codon). This will hereafter be referred to as the "midi" nephrin promoter and is shown in Figure 6B.

[0295] The aforementioned midinephrin promoter was further cleaved, and the putative general transcription domain was removed from the central region, resulting in a length of 268 bp (265 bp excluding the start codon). This will hereafter be referred to as the "mini" nephrin promoter and is shown in Figure 6C.

[0296] Construction of vector structures Midinephrine promoter The BamHI and ClaI restriction sites were introduced using the pACE_hNPHS1 promoter as a template, as shown in Figure 7A. The fragments were then gel-extracted before ligation to the pLenti GFP Blast vector and further digested with ClaI and BamHI at 37°C for 1 hour. The ligated reaction product was then used to transform stable, competent Escherichia coli (E. coli) cells, and the DNA was extracted and sequenced (midi-promoter). The final lentiviral vector is shown in Figure 7B.

[0297] Mininephrine promoter Using the pACE_hNPHS1 promoter, the overhang (OH) shown in Figure 8A was subjected to PCR. Two sections of the promoter containing the OH were gel-extracted for the NEBuilder HiFi Assembly reaction to the pLenti GFP Blast vector. This ligation product was then purified using a DNA cleanup kit prior to its transformation into stable, competent Escherichia coli (E. coli) cells. The DNA was extracted and then sequenced. The final lentiviral vector is shown in Figure 8B.

[0298] Testing of vector constructs The efficacy and podocyte specificity were checked by expressing GFP in an in vitro cell model using the aforementioned minimal nephrin promoter.

[0299] We generated viruses by transfecting HEK293T cells for 48 hours using pLenti GFP Blast nephrin promoter constructs (full-length, midi, and mini), and further used these to create human conditionally immortalized podocytes that stably express either the GFP-tagged FL NPHS1, midi, or mini promoter.

[0300] The ability of the minimal promoter to drive GFP expression was determined by transfecting conditionally immortalized human podocytes (CI podocytes) with a lentiviral vector. Both the midi and mininephrine promoters were shown to drive GFP expression. Figure 9 shows a representative fluorescence microscopy image showing GFP expression from the mininephrine promoter. Figure 10 shows a representative Western blot showing GFP expression from the mininephrine promoter. These results indicate that the minimal nephrine promoter can drive the expression of the transgene in podocytes.

[0301] Human glomerular cells were transduced using the same lentiviral vector. Podocytes and glomerular endothelial cells were transduced using a lentivirus containing GFP conjugated to the mini-nephrine reporter. Figures 11A-C show FACS analysis using Novocyte Analyser to display the median GFP fluorescence (AFU) of all live singlets of conditionally immortalized human podocytes (LYs) and glomerular endothelial cells (GEnCs). Untransduced cells (cell control) were compared to cells transduced with a lentiviral construct containing a GFP expression cassette controlled by the full-length human nephrine promoter (hNPHS1.GFP) or the mini-human nephrine promoter (265.GFP). All cells were differentiated for 10 days, triedpsinized (100 μL), and then diluted in PBS, 2% FBS, 1:1000 DRAQ7 (150 μL). The data and error bars represent three technical replicates (100 μL, >2500 cells) ± SEM. These results demonstrate podocyte specificity for the minimal nephrin promoter compared to glomerular endothelial cells. [Examples]

[0302] Example 3 - Podocyte-Targeted Gene Therapy The inventors have developed a targeted gene delivery system in human and mouse podocytes using adeno-associated virus (AAV) (see PCT / GB2020 / 050097). Using a podocyte-specific promoter (nephrin), AAV serotypes 2 / 9 successfully infected podocytes in vivo and induced podosin expression. In animals where podosin was knocked down using the Cre-Loxp system (NPHS2fl / fl), proteinuria occurred, and AAV treatment successfully restored podosin expression and improved proteinuria. Furthermore, the inventors demonstrated efficient (superior efficiency to AAV2 / 9) and specific transduction of GFP into human podocytes by AAV LK03 using the same promoter.

[0303] References KODIPPILI K, HAKIM CH, PAN X, YANG HT, YUE Y, ZHANG Y, SHIN JH, YANG NN, DUAN D. Dual AAV Gene Therapy for Duchenne Muscular Dystrophy with a 7-kb Mini-Dystrophin Gene in the Canine Model. Hum Gene Ther. 2018 Mar;29(3):299-311. LUO, X., HALL, G., LI, S., BIRD, A., LAVIN, P. J., WINN, M. P., KEMPER, A. R., BROWN, T. T. & KOEBERL, D. D. 2011. Hepatorenal correction in murine glycogen storage disease type I with a double-stranded adeno-associated virus vector. Mol Ther, 19, 1961-70. MCCLEMENTS ME, MACLAREN RE. Adeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large Transgenes. Yale J Biol Med. 2017 Dec 19;90(4):611-623 MOELLER, M. J., SANDEN, S. K., SOOFI, A., WIGGINS, R. C. & HOLZMAN, L. B. 2002. Two gene fragments that direct podocyte-specific expression in transgenic mice. J Am Soc Nephrol, 13, 1561-7. PICCONI, J. L., MUFF-LUETT, M. A., WU, D., BUNCHMAN, E., SCHAEFER, F. & BROPHY, P. D. 2014. Kidney-specific expression of GFP by in-utero delivery of pseudotyped adeno-associated virus 9. Molecular Therapy. Methods & Clinical Development, 1, 14014. ROCCA, C. J., UR, S. N., HARRISON, F. & CHERQUI, S. 2014. rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study. Gene therapy, 21, 618-628. SCHAMBACH, A., BOHNE, J., BAUM, C., HERMANN, F. G., EGERER, L., VON LAER, D. & GIROGLOU, T. 2005. Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene Therapy, 13, 641. SCHIEVENBUSCH, S., STRACK, I., SCHEFFLER, M., NISCHT, R., COUTELLE, O., HOESEL, M., HALLEK, M., FRIES, JWU, DIENES, H.-P., ODENTHAL, M. & BUENING, H. 2010. Combined Paracrine and Endocrine AAV9 mediated Expression of Hepatocyte Growth Factor for the Treatment of Renal Fibrosis. Molecular Therapy, 18, 1302-1309.

[0304] Embodiment Various features and embodiments of the present invention are described herein with reference to the following numbered sections.

[0305] 1. Viral vector gene therapy, wherein the viral vector is as follows: COL4A3, COL4A4, or COL4A5 transgenes, and Any podocyte-specific promoter The viral vector gene therapy, including the said.

[0306] 2. The viral vector gene therapy according to item 1, wherein the podocyte-specific promoter is the minimal nephrin promoter NPHS1 or the podosin promoter NPHS2.

[0307] 3. The viral vector gene therapy according to item 1 or 2, wherein the viral vector is adeno-associated virus (AAV).

[0308] 4. The viral vector gene therapy according to item 3, wherein the AAV vector is AAV serotype 2 / 9, LK03, or 3B.

[0309] 5. A viral vector gene therapy according to any one of items 1 to 4, wherein the COL4A3, COL4A4, or COL4A5 transgene is a minigene.

[0310] 6. The gene therapy described above is as follows: A first viral vector containing at least a portion of the COL4A3, COL4A4, or COL4A5 transgene, and Any podocyte-specific promoter, and A second viral vector containing at least a portion of the corresponding COL4A3, COL4A4, or COL4A5 transgene, and Any podocyte-specific promoter A viral vector gene therapy as described in any one of items 1 to 4, including the one described in item 1 to 4.

[0311] 7. The viral vector gene therapy according to any one of items 1 to 6, wherein the viral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0312] 8. A viral vector gene therapy according to any one of items 1 to 7, wherein the COL4A3, COL4A4, or COL4A5 transgene is human and / or includes a hemagglutinin (HA) tag.

[0313] 9. The viral vector gene therapy according to any one of items 1 to 8, wherein the viral vector further comprises a Kozak sequence between the promoter and the COL4A3, COL4A4, or COL4A5 transgene.

[0314] 10. The viral vector gene therapy according to any one of items 1 to 9, wherein the viral vector further comprises a polyadenylation signal such as a bovine growth hormone (bGH) polyadenylation signal.

[0315] 11. Viral vector gene therapy as described in any one of items 1 to 10, for use in the treatment or prevention of Alport syndrome.

[0316] 12. The viral vector gene therapy for use according to item 11, wherein the viral vector gene therapy is administered to a human patient.

[0317] 13. The viral vector gene therapy for use according to item 11 or 12, wherein the viral vector gene therapy is administered systemically.

[0318] 14. The viral vector gene therapy for use according to any one of items 11 to 13, wherein the viral vector gene therapy is administered by intravenous injection.

[0319] 15. The viral vector gene therapy for use according to any one of items 11 to 14, wherein the viral vector gene therapy is administered by injection into the renal artery. This disclosure includes the following embodiments. [1] A viral vector comprising a COL4A3, COL4A4, or COL4A5 transgene. [2] The following (i) to (iii) are true, that is (i) The COL4A3 transgene comprises a polypeptide sequence having at least 70% identity with SEQ ID NO: 1, or encodes a COL4A3 polypeptide composed of such polypeptide sequence, or a fragment thereof. (ii) The COL4A4 transgene encodes a COL4A4 polypeptide or a fragment thereof that contains or is composed of a polypeptide sequence having at least 70% identity with SEQ ID NO: 2, and / or (iii) The COL4A5 transgene comprises a polypeptide sequence having at least 70% identity with SEQ ID NO: 3, or encodes a COL4A5 polypeptide composed of such polypeptide sequence, or a fragment thereof. The viral vector according to Embodiment 1. [3] The viral vector according to Embodiment 1 or 2, wherein (i) to (iii) below, that is, (i) the COL4A3 transgene encodes a full-length COL4A3 polypeptide, (ii) the COL4A4 transgene encodes a full-length COL4A4 polypeptide, and / or (iii) the COL4A5 transgene encodes a full-length COL4A5 polypeptide. [4] The viral vector according to any one of embodiments 1 to 3, wherein the viral vector comprises a podocyte-specific promoter. [5] The viral vector according to any one of embodiments 1 to 4, wherein the podocyte-specific promoter is minimal nephrin promoter NPHS1 or podosin promoter NPHS2, preferably minimal nephrin promoter NPHS1. [6] The viral vector according to Embodiment 5, wherein the minimal nephrine promoter NPHS1 comprises or consists of a nucleotide sequence shown as SEQ ID NO: 10, or a variant that is at least 70% identical to SEQ ID NO: 10. [7] The viral vector according to any one of embodiments 1 to 6, wherein the viral vector is adeno-associated virus (AAV). [8] The viral vector according to Embodiment 7, wherein the AAV vector is in the form of an AAV vector particle. [9] The viral vector according to Embodiment 7 or 8, wherein the AAV vector particles are podocyte-specific AAV vectors.

[10] The viral vector according to any one of embodiments 7 to 9, wherein the AAV vector is AAV serotype 2 / 9, LK03, or 3B.

[11] The viral vector according to any one of embodiments 1 to 10, wherein the COL4A3, COL4A4, or COL4A5 transgene is a minigene.

[12] The viral vector according to any one of Embodiments 1 to 11, wherein the viral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[13] The viral vector according to any one of Embodiments 1 to 11, wherein the viral vector does not contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[14] The viral vector according to any one of Embodiments 1 to 13, wherein the COL4A3, COL4A4, or COL4A5 transgene is a human COL4A3, COL4A4, or COL4A5 transgene and / or includes a hemagglutinin (HA) tag.

[15] The viral vector according to any one of Embodiments 1 to 14, wherein the viral vector further comprises a Kozak sequence between the promoter and the COL4A3, COL4A4, or COL4A5 transgene.

[16] The viral vector according to any one of embodiments 1 to 15, wherein the viral vector further comprises a polyadenylation signal such as a bovine growth hormone (bGH) polyadenylation signal or an early SV40 polyadenylation signal.

[17] The viral vector according to embodiment 16, wherein the polyadenylation signal is an initial SV40 polyadenylation signal.

[18] Viral vector gene therapy, wherein the gene therapy is as follows: A first viral vector containing at least a portion of the COL4A3, COL4A4, or COL4A5 transgene, and A second viral vector containing at least a portion of the corresponding COL4A3, COL4A4, or COL4A5 transgene. The viral vector gene therapy, including the said.

[19] The viral vector gene therapy according to Embodiment 18, wherein the first viral vector is the viral vector described in any of Embodiments 1 to 17 and / or the second viral vector is the viral vector described in any of Embodiments 1 to 17.

[20] A viral vector or viral vector gene therapy according to any one of embodiments 1 to 19 for use in the treatment or prevention of Alport syndrome.

[21] The viral vector or viral vector gene therapy for use according to Embodiment 20, wherein the viral vector or viral vector gene therapy is administered to a human patient.

[22] The viral vector or viral vector gene therapy for use according to Embodiment 20 or 21, wherein the viral vector or viral vector gene therapy is administered systemically.

[23] The viral vector or viral vector gene therapy for use according to any one of embodiments 20 to 22, wherein the viral vector or viral vector gene therapy is administered by intravenous injection.

[24] The viral vector or viral vector gene therapy for use according to any one of embodiments 20 to 23, wherein the viral vector or viral vector gene therapy is administered by injection into the renal artery.

Claims

1. A viral vector comprising a COL4A3, COL4A4, or COL4A5 transgene and a podocyte-specific promoter.

2. (i) The COL4A3 transgene encodes a COL4A3 polypeptide comprising the polypeptide sequence described in SEQ ID NO: 1, or a functional variant thereof having at least 90% identity with SEQ ID NO: 1, (ii) The COL4A4 transgene encodes a COL4A4 polypeptide comprising the polypeptide sequence described in SEQ ID NO: 2, or a functional variant thereof having at least 90% identity with SEQ ID NO: 2, or (iii) The COL4A5 transgene encodes a COL4A5 polypeptide comprising or composed of the polypeptide sequence described in SEQ ID NO: 3, or a functional variant thereof having at least 90% identity with SEQ ID NO:

3. The viral vector according to claim 1.

3. The viral vector according to claim 1 or 2, wherein (i) the COL4A3 transgene encodes a full-length COL4A3 polypeptide, (ii) the COL4A4 transgene encodes a full-length COL4A4 polypeptide, and / or (iii) the COL4A5 transgene encodes a full-length COL4A5 polypeptide.

4. The viral vector according to any one of claims 1 to 3, wherein the podocyte-specific promoter is the minimal nephrin promoter NPHS1 or the podosin promoter NPHS2.

5. The viral vector according to claim 4, wherein the podocyte-specific promoter is the minimal nephrin promoter NPHS1.

6. The viral vector according to claim 4 or 5, wherein the minimal nephrine promoter NPHS1 includes or is composed of a nucleotide sequence shown as SEQ ID NO: 10, or a functional variant that is at least 90% identical to SEQ ID NO: 10, or the minimal nephrine promoter NPHS1 includes or is composed of a nucleotide sequence shown as SEQ ID NO:

35.

7. The viral vector according to any one of claims 1 to 6, wherein the viral vector is an adeno-associated virus (AAV) vector.

8. The viral vector according to claim 7, wherein the AAV vector is in the form of an AAV vector particle.

9. The viral vector according to claim 7 or 8, wherein the AAV vector is AAV serotype 2 / 9, LK03, or 3B.

10. The viral vector according to any one of claims 1 to 9, wherein the COL4A3, COL4A4, or COL4A5 transgene is a minigene.

11. The viral vector according to any one of claims 1 to 10, wherein the viral vector further comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

12. The viral vector according to any one of claims 1 to 10, wherein the viral vector does not contain a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

13. The viral vector according to any one of claims 1 to 12, wherein the COL4A3, COL4A4, or COL4A5 transgene is a human COL4A3, COL4A4, or COL4A5 transgene, and / or comprises a hemagglutinin (HA) tag.

14. The viral vector according to any one of claims 1 to 13, wherein the viral vector further comprises a Kozak sequence between the podocyte-specific promoter and the COL4A3, COL4A4, or COL4A5 transgene.

15. The viral vector according to any one of claims 1 to 14, wherein the viral vector further comprises a polyadenylation signal.

16. The viral vector according to claim 15, wherein the polyadenylation signal is a bovine growth hormone (bGH) polyadenylation signal or an SV40 early polyadenylation signal.

17. A pharmaceutical composition comprising a viral vector according to any one of claims 1 to 16, and one or more pharmaceutically acceptable carriers, diluents and / or excipients.

18. Use of a viral vector according to any one of claims 1 to 16 in the manufacture of a medicament for the treatment or prevention of Alport syndrome.

19. The use according to claim 18, wherein the pharmaceutical is for administering the viral vector to a human patient.

20. The use according to claim 18 or 19, wherein the pharmaceutical is for systemic administration of the viral vector.

21. The use according to any one of claims 18 to 20, wherein the pharmaceutical product is for systemic administration of the viral vector by intravenous injection.

22. The use according to claim 18 or 19, wherein the pharmaceutical product is for systemic administration of the viral vector by injection into the renal artery.

23. A pharmaceutical composition for the treatment or prevention of Alport syndrome, comprising a viral vector according to any one of claims 1 to 16.

24. The pharmaceutical composition according to claim 23, wherein the viral vector is administered to a human patient.

25. The pharmaceutical composition according to claim 23 or 24, wherein the viral vector is administered systemically.

26. The pharmaceutical composition according to any one of claims 23 to 25, wherein the viral vector is administered by intravenous injection.

27. The pharmaceutical composition according to claim 23 or 24, wherein the viral vector is administered by injection into the renal artery.

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