Methods of transducing podocytes

Abstract

The present invention provides methods for transduction of human podocytes, the methods comprising contacting the podocytes with an AAV-ShH10 vector particle. The present invention also provides methods for treating and / or preventing a kidney disease, the methods comprising administering an AAV-ShH10 vector particle to a human subject in need thereof, wherein the AAV-ShH10 vector particle transduces podocytes of the subject.

Description

[0001] METHODS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for transducing human podocytes. The present invention also relates to methods for treating and / or preventing a kidney disease in a human subject by transducing podocytes of said subject.

[0004] BACKGROUND TO THE INVENTION

[0005] There are many diseases that affect kidney function by attacking the glomerulus. The glomerulus filters approximately 180 litres of plasma each day, and the healthy glomerular filtration barrier has an astonishing ability to retain about 99.9% of large proteins including albumin over our lifetimes without clogging. The glomerular filtration barrier (GFB) comprises 3 main layers: the glomerular endothelial cell, the glomerular basement membrane (GBM) and the podocyte.

[0006] The GBM is made of a highly cross-linked macromolecular meshwork of type IV collagen, proteoglycans, and laminin. Genetic forms of glomerular disease can be caused by genetic defects in these molecular structures. For example, Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes. Other GBM-associated diseases include Pierson syndrome and Nail-patella syndrome (see Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0007] The podocyte has also been implicated as a key cell in the progression of glomerular disease. Podocytes are mesodermally derived cells that are highly specialized and found only in the renal glomerulus. They exhibit unique characteristics such as foot processes and slit diaphragms, which are critical for glomerular filtration. Podocyte-associated genetic glomerular diseases include Nephrotic Syndrome, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, and Epstein and Fechtner syndrome (see Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0008] In order to maximise gene therapy potential, vector particles which can transduce human podocytes with high efficiency are required. The AAV-LK03 serotype has shown to be a highly efficient transducer of human podocytes (see Ding, W.Y., et al., 2023. Science Translational Medicine, 15(708), p.eabc8226). However, there is a need for further AAV serotypes which can transduce human podocytes with high efficiency. SUMMARY OF THE INVENTION

[0009] The present inventors surprisingly found that AAV-ShH10 vector particles are capable of transducing human podocytes with high efficiency. AAV-ShH10 vector particles were surprisingly shown to transduce human podocytes and glomerular spheroids in vitro, as demonstrated by high expression levels of a GFP reporter gene.

[0010] Whilst AAV-ShH10 vector particles have previously been shown to exhibit high rates of renal gene transfer in mice (see Furusho, T., et al., 2023. bioRxiv, 2023.07.28.548760), it has not previously been shown that AAV-ShH10 vector particles are capable of transducing podocytes. In particular, it has not previously been shown that AAV-ShH10 vector particles are capable of transducing human podocytes. For example, whilst it is known that AAV9 vector particles can transduce mouse podocytes, AAV9 vector particles only transduce human podocytes with very low efficiency.

[0011] In one aspect, the present invention provides a method for transduction of human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle.

[0012] In another aspect, the present invention provides a method for delivering a coding sequence to podocytes of a human subject, the method comprising administering an AAV-ShH 10 vector particle comprising the coding sequence to said subject, thereby transducing and expressing the coding sequence in the podocytes of said subject.

[0013] In another aspect, the present invention provides a method for treating and / or preventing a kidney disease, the method comprising administering an AAV-ShH 10 vector particle to a human subject in need thereof, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0014] In another aspect, the present invention provides an AAV-ShH 10 vector particle for use in gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0015] In another aspect, the present invention provides an AAV-ShH 10 vector particle for use in treating and / or preventing a kidney disease in a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0016] In another aspect, the present invention provides use of an AAV-ShH 10 vector particle for the manufacture of a medicament for gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject. In another aspect, the present invention provides use of an AAV-ShH10 vector particle for the manufacture of a medicament for treating and / or preventing a kidney disease in a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0017] In another aspect, the present invention provides use of an AAV-ShH10 vector particle for transducing human podocytes in vitro or ex vivo.

[0018] The AAV-ShH 10 vector particle may be encapsidated by ShH10 VP1 , VP2, and VP3 capsid proteins. In some embodiments, the ShH10 VP1 capsid protein comprises or consists of an amino acid sequence having at least 99% identity to SEQ ID NO: 1. In some embodiments, the ShH10 VP2 capsid protein comprises or consists of an amino acid sequence having at least 99% identity to SEQ ID NO: 2. In some embodiments, the ShH10 VP3 capsid protein comprises or consists of an amino acid sequence having at least 99% identity to SEQ ID NO: 3. The AAV-ShH 10 vector particle may have a total number of 60 VP1 , VP2, and VP3 subunits per capsid and comprises ShH10 VP1 , VP2, and VP3 capsid proteins in a ratio of 1 :1 :10. The AAV-ShH 10 vector particle may comprise an AAV genome that is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

[0019] In some embodiments, the AAV-ShH10 vector particle encodes a therapeutic gene product. In some embodiments, the AAV-ShH 10 vector particle encodes a polypeptide associated with a kidney disease, or an N-terminal part or C-terminal part thereof. In some embodiments, the AAV-ShH 10 vector particle comprises a coding sequence encoding a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, CFHL1 , C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MYO1 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide, or an N-terminal part or C-terminal part thereof. In some embodiments, the AAV- ShH 10 vector particle comprises a podocyte-specific promoter. In some embodiments, the AAV-ShH10 vector particle comprises a NPHS1 promoter or a NPHS2 promoter. In some embodiments, the AAV-ShH 10 vector particle comprises one or more further regulatory sequences.

[0020] The AAV-ShH 10 vector particle may be administered to the human subject in the form of a pharmaceutical composition comprising a therapeutically effective amount of the AAV-ShH10 vector particle and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition has a total volume of from about 0.1 mL to about 100 mL. In some embodiments, the AAV-ShH10 vector particle is administered to the human subject by injection into the renal artery, by intraparenchymal injection, by transparenchymal injection, by renal vein injection, or by ureteral or subcapsular injection. In some embodiments, the AAV-ShH10 vector particle is administered to the human subject by intravenous injection. In some embodiments, the human subject has or is at risk of a genetic kidney disease or a complement-mediated kidney disease.

[0021] DESCRIPTION OF DRAWINGS

[0022] Figure 1 - Transduction of human podocytes with AAV vector particles determined by fluorescence microscopy

[0023] (A-E) Images of eGFP transduced human podocytes using (A) AAV-LK03, (B) AAV9, (C) AAV-3B, (D) AAV-ShH10 and (E) AAV4, pseudotyped AAV particles (MOI=5E+5 vg per cell). AAV9 demonstrates very low transduction, with AAV4 demonstrating the next lowest level of transduction. AAV-LK03, AAV-3B and AAVShHIO demonstrate similar levels of transduction.

[0024] Figure 2 - Transduction of human and mouse podocytes with AAV vector particles determined by FACS analysis

[0025] (A) FACS analysis of human podocytes transduced with AAV particles (MOI=1 E+2, 1 E+3 and 1 E+4 vg per cell). (B) FACS analysis of mouse podocytes transduced with AAV particles (MOI=5E+4, 1 E+5 and 5E+5 vg per cell). Higher MOIs were used for mouse podocytes relative to human podocytes as they demonstrated lower transduction for most serotypes tested.

[0026] Figure 3 - Transduction of human glomerular spheroids with AAV vector particles determined by fluorescence microscopy

[0027] Glomerular spheroids were generated by co-culture of human glomerular endothelial cells with human podocytes. Transduction of spheroids was performed with AAVs pseudotyped with different capsids and spheroids were imaged after 72 hours.

[0028] Figure 4 - Transduction of mouse glomerular spheroids with AAV vector particles determined by fluorescence microscopy

[0029] Glomerular spheroids were generated by co-culture of mouse glomerular endothelial cells with mouse podocytes. Transduction of spheroids was performed with AAVs pseudotyped with different capsids and imaged after 72 hours. Only AAV-ShH10 pseudotyped AAVs were capable of high transduction in mouse glomerular spheroids. Low levels of transduction were visible with AAV-LK03, AAV9 and AAV-3B.

[0030] DETAILED DESCRIPTION

[0031] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0032] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0033] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes", "containing", or "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or steps. The terms "comprising", "comprises" and "comprised of" also include the term "consisting of".

[0034] Numeric ranges are inclusive of the numbers defining the range. As used herein the term “about” means approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical value or range, it modifies that value or range by extending the boundaries above and below the numerical value(s) set forth. In general, the terms “about” and “approximately” may be used herein to modify a numerical value(s) above and below the stated value(s) by 10%.

[0035] Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0036] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0037] All publications mentioned in the specification are herein incorporated by reference.

[0038] AAV-ShH 10 vector particles

[0039] The present invention provides a method for transduction of human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle. An AAV vector particle is encapsidated by capsid proteins and may be referred to in terms of its serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11 , and derivatives thereof.

[0040] The serotype may facilitate the transduction of specific cell types. AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV vector particles may be in the form of a pseudotyped AAV vector particle. AAV vector particles may also be chemically modified, bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor.

[0041] The VP1 , VP2 and VP3 proteins make up the capsid of an AAV particle, which determines the AAV serotype. VP1 , VP2, and VP3 may be produced by alternate mRNA splicing (see e.g. Trempe, J.P. and Carter, B.J., 1988. Journal of virology, 62(9), pp.3356-3363). Thus, VP1 , VP2 and VP3 may have identical sequences, except that VP2 is truncated at the N-terminus relative to VP1 , and VP3 is truncated at the N-terminus relative to VP2. An AAV vector particle typically comprises a total number of 60 VP1 , VP2, and VP3 subunits per capsid, wherein the VP1 , VP2, and VP3 capsid proteins are in a ratio of 1 :1 :10

[0042] Methods to generate AAV capsid variants are well known in the art (see e.g. Lee, E.J., et al., 2018. Current opinion in biomedical engineering, 7, pp.58-63). For example, the efficiency and specificity of AAV gene delivery can be improved using point mutations on the viral capsid. A second rational design approach is to introduce functional domains non-viral in nature into the AAV capsid to elicit desired functions. A third rational design approach involves using chemical biology strategies to make more precise modifications to the capsid.

[0043] The sequences of AAV capsid genes may be genetically modified to introduce specific deletions, substitutions or insertions with respect to a native AAV wild-type sequence. In particular, AAV capsid genes may be modified by the insertion of a sequence of an unrelated protein or peptide within an open reading frame of the AAV capsid coding sequence, or at the N- and / or C-terminus of the AAV capsid coding sequence. The unrelated protein or peptide may advantageously be one which acts as a ligand for a particular cell type, thereby conferring improved binding to a target cell or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein may also be one which assists purification of the viral particle as part of the production process, e.g. an epitope or affinity tag. The site of insertion will typically be selected so as not to interfere with other functions of the AAV viral particle e.g. internalisation, trafficking of the AAV viral particle, etc.

[0044] In some embodiments, a capsid protein variant has at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the reference capsid protein.

[0045] In some embodiments, a capsid protein variant has at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identity to the reference capsid protein.

[0046] In some embodiments, a capsid protein variant has 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to the reference capsid protein.

[0047] In some embodiments, a capsid protein variant has 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, 10 substitutions, 11 substitutions, 12 substitutions, 13 substitutions, 14 substitutions, 15 substitutions, 16 substitutions, 17 substitutions, 18 substitutions, 19 substitutions, 20 substitutions, 21 substitution, 22 substitutions, 23 substitutions, 24 substitutions, 25 substitutions, 26 substitutions, 27 substitutions, 28 substitutions, 29 substitutions, or 30 substitutions compared to the reference capsid protein.

[0048] In some embodiments, a capsid protein variant has an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to the reference capsid protein.

[0049] In some embodiments, a capsid protein variant has an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids compared to the reference capsid protein. In some embodiments, a capsid protein variant has a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to the reference capsid protein.

[0050] In some embodiments, a capsid protein variant has a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids compared to the reference capsid protein.

[0051] In some embodiments, compared to the reference capsid protein, a capsid protein variant has:

[0052] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0053] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0054] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0055] In some embodiments, compared to the reference capsid protein, a capsid protein variant has:

[0056] (a) 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, 10 substitutions, 11 substitutions, 12 substitutions, 13 substitutions, 14 substitutions, 15 substitutions, 16 substitutions, 17 substitutions, 18 substitutions, 19 substitutions, 20 substitutions, 21 substitution, 22 substitutions, 23 substitutions, 24 substitutions, 25 substitutions, 26 substitutions, 27 substitutions, 28 substitutions, 29 substitutions, or 30 substitutions;

[0057] (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and / or

[0058] (c) a deletion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids.

[0059] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0060] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0061] In some embodiments, compared to the reference capsid protein, a capsid protein variant has: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0062] AAV-ShH10 serotype and capsid proteins

[0063] As used herein, the term “AAV-ShH10 vector particles” may refer to AAV vector particles which have serologic cross-reactivity with the AAV6 serotype and which comprise the N451 D mutation and / or D532N mutation.

[0064] AAV-ShH10 is an AAV variant which is closely related to AAV serotype 6 (AAV6) and which is capable of efficient, selective Muller cell infection through intravitreal injection. AAV-ShH10 was generated from a shuffled library of cap genes of the naturally occurring AAV6 parent serotype. ShH10 differs from AAV6 at only four residues: 1319V, N451 D, D532N, and H642N. The N451 D mutation was sufficient to confer the intravitreal Muller tropism. The D532N mutation, located near the HSPG binding domain of the AAV6 capsid, may enable the virus to utilize a transduction pathway distinct from AAV6 (see Klimczak, R.R., et al., 2009. PloS one, 4(10), p.e7467; and US 2012 / 0164106 A1).

[0065] Serologic cross- reactivity may be determined by any method known in the art (see e.g. Gao, G., et al., 2004. Journal of virology, 78(12), pp.6381-6388; and Gao, G.P., et al., 2002. PNAS, 99(18), pp.11854-11859). For example, antisera to the AAV-ShH10 serotype may be generated by injection of AAV-ShH10 vector particles. The AAV vector particles to be tested may be pre-incubated with the AAV-ShH10 antiserum, and then incubated with indicator 84- 31 cells for 48 to 72 h. Transduction may be assessed under a UV microscope. Neutralizing antibody titers may be reported as the highest serum dilution that inhibited transduction by 50% of that seen with serum from a naive animal.

[0066] In some embodiments, AAV-ShH10 vector particles have a neutralization titer of 1 / 80 or less, 1 / 160 or less, 1 / 320 or less, 1 / 640 or less, 1 / 1280 or less, 1 / 2560 or less, 1 / 5120 or less, 1 / 10240 or less, 1 / 20480 or less, or 1 / 40960 or less when incubated with AAV-ShH10 antiserum. In some embodiments, AAV-ShH10 vector particles have a neutralization titer of 1 / 5120 or less, 1 / 10240 or less, 1 / 20480 or less, or 1 / 40960 or less when incubated with AAV- ShH10 antiserum. In some embodiments, AAV-ShH10 vector particles have a neutralization titer of about 1 / 5120, about 1 / 10240, about 1 / 20480, or about 1 / 40960 when incubated with AAV-ShH10 antiserum.

[0067] In some embodiments, AAV-ShH10 vector particles comprise the N451 D mutation. In some embodiments, AAV-ShH10 vector particles comprise the D532N mutation. In some embodiments, AAV-ShH10 vector particles comprise the N451 D mutation and the D532N mutation.

[0068] In some embodiments, AAV-ShH10 vector particles further comprise the 1319V mutation. In some embodiments, AAV-ShH10 vector particles comprise the 1319V mutation, the N451 D mutation, and the D532N mutation.

[0069] In some embodiments, AAV-ShH10 vector particles further comprise the H642N mutation. In some embodiments, AAV-ShH10 vector particles comprise the 1319V mutation, the N451 D mutation, the D532N mutation, and the H642N mutation.

[0070] AAV-ShH10 vector particles may bind Heparan sulfate proteoglycan (HSPG). Binding affinity to the receptors may be determined by any method known in the art. For example, binding affinity to HSPG may be determined by using heparin affinity chromatography (see e.g. Klimczak, R.R., et al., 2009. PloS one, 4(10), p.e7467).

[0071] AAV-ShH10 vector particles may be capable of transducing human podocytes. Transduction efficiency of human podocytes may be determined by any method known in the art (see e.g. Ding, W.Y., 2023. Science Translational Medicine, 15(708), p.eabc8226). For example, by measuring the expression of a reporter transgene, e.g. GFP, carried by the AAV-ShH 10 vector particle, wherein expression of the transgene in human podocytes correlates with the ability of the AAV vector particle to transduce human podocytes.

[0072] AAV-ShH 10 vector particles may comprise an AAV-ShH 10 capsid protein or a variant thereof. For example, AAV-ShH10 vector particles may comprise AAV-ShH10 VP1 proteins, AAV- ShH 10 VP2 proteins, and AAV-ShH 10 VP3 proteins, or variants thereof. AAV-ShH 10 vector particles may comprise a total number of 60 VP1 , VP2, and VP3 subunits per capsid. AAV- ShH10 vector particles may comprise ShH10 VP1 , VP2 and VP3 capsid proteins, or variants thereof, in a ratio of 1 :1 :10 (VP1 :VP2:VP3).

[0073] AAV-ShH10 variant capsids have been described in the art (see e.g. Pellissier, L.P., et al., 2014. Molecular Therapy-Methods & Clinical Development, 1 , p.14009; Keravala, A., et al., 2017. Investigative Ophthalmology & Visual Science, 58(8), pp.4097-4097; and WO2018 / 160686A1). Further AAV-ShH10 variant capsids may be generated by amino acid substitutions, deletions, or insertions. AAV-ShH 10 vector particles which are known in the art include AAV-ShH 10Y vector particles and AAV-ShH 10 / 7m8 vector particles.

[0074] I n some embodiments, the AAV-ShH 10 vector particle is a parental AAV-ShH 10 vector particle or a variant thereof, an AAV-ShH 10Y vector particle or a variant thereof, or an AAV- ShH 10 / 7m8 vector particle or a variant thereof. In some embodiments, the AAV-ShH 10 vector particle is a parental AAV-ShH 10 vector particle, an AAV-ShH 10Y vector particle, or an AAV- ShH 10 / 7m8 vector particle.

[0075] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of SEQ ID NOs: 1 or 5- 10. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 .

[0076] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least

[0077] 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least

[0078] 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least

[0079] 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to any of SEQ ID NOs: 1 or

[0080] 5-10. In some embodiments, the AAV-ShH 10 vector particle comprises an AAV capsid protein having at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least

[0081] 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least

[0082] 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least

[0083] 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to SEQ ID NO: 1.

[0084] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to any of SEQ ID NOs: 1 or 5-10. In some embodiments, the AAV- ShH10 vector particle comprises an AAV capsid protein having 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: 1. In some embodiments, the amino acid substitution(s) are selected from Y252F, Y273F, Y445F, Y701 F, Y705F, and Y731 F (see e.g. Song, L., et al., 2013. Cytotherapy, 15(8), pp.986- 998).

[0085] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to any of SEQ ID NOs: 1 or 5-10. In some embodiments, the AAV- ShH10 vector particle comprises an AAV capsid protein having an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 1. In some embodiments, the amino acid sequence inserted is selected from LGETTRP (SEQ ID NO: 11), NETITRP (SEQ ID NO: 12), KAGQANN (SEQ ID NO: 13), KDPKTTN (SEQ ID NO: 14), KDTDTTR (SEQ ID NO: 15), RAGGSVG (SEQ ID NO: 16), AVDTTKF (SEQ ID NO: 17), and STGKVPN (SEQ ID NO: 18), LALGETTRPA (SEQ ID NO: 19); LANETITRPA (SEQ ID NO: 20), LAKAGQANNA (SEQ ID NO: 21), LAKDPKTTNA (SEQ ID NO: 22), LAKDTDTTRA (SEQ ID NO: 23), LARAGGSVGA (SEQ ID NO: 24), LAAVDTTKFA (SEQ ID NO: 25), LASTGKVPNA (SEQ ID NO: 26), AALGETTRPA (SEQ ID NO: 27); AANETITRPA (SEQ ID NO: 28), AAKAGQANNA (SEQ ID NO: 29), AAKDPKTTNA (SEQ ID NO: 30), GLGETTRPA (SEQ ID NO: 31); GNETITRPA (SEQ ID NO: 32), GKAGQANNA (SEQ ID NO: 33), and GKDPKTTNA (SEQ ID NO: 34). In some embodiments, the amino acid sequence inserted is LGETTRP or LALGETTRPA. In some embodiments, the amino acid sequence is inserted between two adjacent amino acids selected from 450 and 451 , 451 and 452, 452 and 453, 453 and 454, 454 and 455, 455 and 456, 456 and 457, 457 and 458, 458 and 459, 459 and 460, 460 and 461 , 461 and 462, 462 and 463, or 463 and 464. In some embodiments, the amino acid sequence is inserted between amino acid residues 457 and 458, amino acid residues 458 and 459, or amino acid residues 459 and 460. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to any of SEQ ID NOs: 1 or 5-10. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 1.

[0086] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to any of SEQ ID NOs: 1 or 5-10: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to SEQ ID NO: 1 : (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0087] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to any of SEQ ID NOs: 1 or 5-10: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to SEQ ID NO: 1 : (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0088] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to any of SEQ ID NOs: 1 or 5-10: (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to SEQ ID NO: 1 : (a) no substitutions; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0089] In some embodiments, the AAV-ShH10 vector particle comprises an AAV capsid protein having, compared to any of SEQ ID NOs: 1 or 5-10: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions. In some embodiments, the AAV-ShH 10 vector particle comprises an AAV capsid protein having, compared to SEQ ID NO: 1 : (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0090] Parental ShH10 capsid proteins

[0091] I n some embodiments, the AAV-ShH 10 vector particle is a parental AAV-ShH 10 vector particle or a variant thereof.

[0092] In some embodiments, the AAV-ShH10 vector particle comprises a parental AAV-ShH10 VP1 capsid protein, or variant thereof. In some embodiments, the AAV-ShH 10 vector particle comprises a parental AAV-ShH 10 VP1 capsid protein, a parental AAV-ShH 10 VP2 capsid protein, and / or a parental AAV-ShH10 VP3 capsid protein, or variants thereof. In some embodiments, the AAV-ShH10 vector particle is encapsidated by parental AAV-ShH10 VP1 , VP2, and VP3 capsid proteins, or variants thereof. In some embodiments, the AAV-ShH10 vector particle is encapsidated by a total number of 60 VP1 , VP2, and VP3 subunits and the parental AAV-ShH10 VP1 , VP2, and VP3 capsid proteins, or variants thereof, are in a ratio of 1 : 1 :10.

[0093] A parental AAV-ShH10 is described in Klimczak, R.R., et al., 2009. PloS one, 4(10), p.e7467. An example parental AAV-ShH10 VP1 capsid sequence is provided below in SEQ ID NO: 1. Parental AAV-ShH10 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 1. An example parental AAV-ShH10 VP2 capsid sequence is provided in SEQ ID NO: 2. An example parental AAV-ShH10 VP3 capsid sequence is provided in SEQ ID NO: 3.

[0094] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP1 capsid protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.

[0095] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP1 capsid protein having at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least

[0096] 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least

[0097] 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least

[0098] 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: 1.

[0099] In some embodiments, the AAV VP1 capsid protein has 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: 1.

[0100] In some embodiments, the AAV VP1 capsid protein has an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 1.

[0101] In some embodiments, the AAV VP1 capsid protein has a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 1. In some embodiments, the AAV VP1 capsid protein has, compared to SEQ ID NO: 1 :

[0102] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0103] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0104] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0105] In some embodiments, the AAV VP1 has, compared to SEQ ID NO: 1 : (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid,

[0106] 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0107] In some embodiments, the AAV VP1 capsid protein has, compared to SEQ ID NO: 1 : (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions,

[0108] 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0109] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP1 capsid protein having the amino acid sequence of SEQ ID NO: 1.

[0110] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP2 capsid protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP2 capsid protein having at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: 2.

[0111] In some embodiments, the AAV VP2 capsid protein has 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: 2.

[0112] In some embodiments, the AAV VP2 capsid protein has an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 2.

[0113] In some embodiments, the AAV VP2 capsid protein has a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 2.

[0114] In some embodiments, the AAV VP2 capsid protein has, compared to SEQ ID NO: 2:

[0115] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0116] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0117] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0118] In some embodiments, the AAV VP2 capsid protein has, compared to SEQ ID NO: 2: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0119] In some embodiments, the AAV VP2 capsid protein has, compared to SEQ ID NO: 2: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0120] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP2 capsid protein having the amino acid sequence of SEQ ID NO: 2.

[0121] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP3 capsid protein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0122] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP3 capsid protein having at least 98.0%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least

[0123] 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99.0%, at least

[0124] 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least

[0125] 99.7%, at least 99.8%, at least 99.9% identity, or 100.0% identity to the amino acid sequence of SEQ ID NO: 3.

[0126] In some embodiments, the AAV VP3 capsid protein has 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 15 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution compared to SEQ ID NO: 3.

[0127] In some embodiments, the AAV VP3 capsid protein has an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 3. In some embodiments, the AAV VP3 capsid protein has a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid compared to SEQ ID NO: 3.

[0128] In some embodiments, the AAV VP3 capsid protein has, compared to SEQ ID NO: 3:

[0129] (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution;

[0130] (b) an insertion of 20 amino acids or less, 15 amino acids or less, 10 amino acids or less, 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or les, or 1 amino acid; and / or

[0131] (c) a deletion of 10 amino acids or less, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid.

[0132] In some embodiments, the AAV VP3 capsid protein has, compared to SEQ ID NO: 3: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) an insertion of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids; and (c) no deletions.

[0133] In some embodiments, the AAV VP3 capsid protein has, compared to SEQ ID NO: 3: (a) 50 or fewer substitutions, 40 or fewer substitutions, 30 or fewer substitutions, 20 or fewer substitutions, 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 substitution; (b) no insertions; and (c) no deletions.

[0134] In some embodiments, the AAV-ShH10 vector particle comprises an AAV VP3 capsid protein having the amino acid sequence of SEQ ID NO: 3.

[0135] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 3.

[0136] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 91% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 91 % identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 91 % identity to the amino acid sequence of SEQ ID NO: 3.

[0137] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 92% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 92% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 92% identity to the amino acid sequence of SEQ ID NO: 3.

[0138] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 93% identity to the amino acid sequence of SEQ ID NO: 3.

[0139] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 94% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 94% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 94% identity to the amino acid sequence of SEQ ID NO: 3.

[0140] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 95% identity to the amino acid sequence of SEQ ID NO: 3.

[0141] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 96% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 96% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 96% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 97% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 97% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 97% identity to the amino acid sequence of SEQ ID NO: 3.

[0142] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 98% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 98% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 98% identity to the amino acid sequence of SEQ ID NO: 3.

[0143] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99% identity to the amino acid sequence of SEQ ID NO: 3.

[0144] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.0% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.0% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.0% identity to the amino acid sequence of SEQ ID NO: 3.

[0145] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.1 % identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.1% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.1% identity to the amino acid sequence of SEQ ID NO: 3.

[0146] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.2% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.2% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.2% identity to the amino acid sequence of SEQ ID NO: 3.

[0147] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.3% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.3% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.3% identity to the amino acid sequence of SEQ ID NO: 3.

[0148] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.4% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.4% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.4% identity to the amino acid sequence of SEQ ID NO: 3.

[0149] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.5% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.5% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.5% identity to the amino acid sequence of SEQ ID NO: 3.

[0150] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.6% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.6% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.6% identity to the amino acid sequence of SEQ ID NO: 3.

[0151] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.7% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.7% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.7% identity to the amino acid sequence of SEQ ID NO: 3.

[0152] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.8% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.8% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.8% identity to the amino acid sequence of SEQ ID NO: 3.

[0153] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein having at least 99.9% identity to the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein having at least 99.9% identity to the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein having at least 99.9% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1 ; (b) an AAV VP2 capsid protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3.

[0154] In some embodiments, the AAV-ShH10 vector particle comprises: (a) an AAV VP1 capsid protein consisting of the amino acid sequence of SEQ I D NO: 1 ; (b) an AAV VP2 capsid protein consisting of the amino acid sequence of SEQ ID NO: 2; and (c) an AAV VP3 capsid protein consisting of the amino acid sequence of SEQ ID NO: 3.

[0155] ShHIOY capsid proteins

[0156] In some embodiments, the AAV-ShH10 vector particle is a AAV-ShH10Y vector particle or a variant thereof.

[0157] In some embodiments, the AAV-ShH10 vector particle comprises an AAV-ShH10Y VP1 capsid protein, or variant thereof. In some embodiments, the AAV-ShH10 vector particle comprises an AAV-ShH10Y VP1 capsid protein, an AAV-ShH10Y VP2 capsid protein, and / or an AAV-ShH10Y VP3 capsid protein, or variants thereof. In some embodiments, the AAV- ShH10 vector particle is encapsidated by AAV-ShH10Y VP1 , VP2, and VP3 capsid proteins, or variants thereof. In some embodiments, the AAV-ShH10 vector particle is encapsidated by a total number of 60 VP1 , VP2, and VP3 subunits and the AAV-ShH10Y VP1 , VP2, and VP3 capsid proteins, or variants thereof, are in a ratio of 1 :1 :10.

[0158] AAV-ShH10Y is described in Pellissier, L.P., et al., 2014. Molecular Therapy-Methods & Clinical Development, 1 , p.14009 and carries an additional tyrosine to phenylalanine mutation (Y445F) to enhance its transduction efficiency. An example AAV-ShH10Y VP1 capsid sequence is provided below in SEQ ID NO: 4, in which Y445F is underlined. AAV-ShH10Y VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 4.

[0159] ShH10 / 7m8 capsid proteins

[0160] In some embodiments, the AAV-ShH10 vector particle is a AAV-ShH10 / 7m8 vector particle or a variant thereof.

[0161] In some embodiments, the AAV-ShH10 vector particle comprises an AAV-ShH10 / 7m8 VP1 capsid protein, or variant thereof. In some embodiments, the AAV-ShH10 vector particle comprises an AAV-ShH10 / 7m8 VP1 capsid protein, an AAV-ShH10 / 7m8 VP2 capsid protein, and / or an AAV-ShH10 / 7m8 VP3 capsid protein, or variants thereof. In some embodiments, the AAV-ShH10 vector particle is encapsidated by AAV-ShH10 / 7m8 VP1 , VP2, and VP3 capsid proteins, or variants thereof. In some embodiments, the AAV-ShH10 vector particle is encapsidated by a total number of 60 VP1 , VP2, and VP3 subunits and the AAV-ShH10 / 7m8 VP1 , VP2, and VP3 capsid proteins, or variants thereof, are in a ratio of 1 :1 :10.

[0162] ShH10 / 7m8 is described in Keravala, A., et al., 2017. Investigative Ophthalmology & Visual Science, 58(8), pp.4097-4097 and WO2018 / 160686A1 and was generated by inserting an 7m8-loop in the surface exposed receptor-binding region of ShH10.

[0163] In some embodiments, an AAV-ShH10 / 7m8 VP1 capsid protein comprises a peptide insertion relative to a parental AAV-ShH10 VP1 capsid protein (e.g. SEQ ID NO: 1), wherein the peptide insertion is an 7m8 insertion peptide, and the insertion site is located between two adjacent amino acids selected from 450 and 451 , 451 and 452, 452 and 453, 453 and 454, 454 and 455, 455 and 456, 456 and 457, 457 and 458, 458 and 459, 459 and 460, 460 and 461 , 461 and 462, 462 and 463, or 463 and 464. In some embodiments, the amino acid sequence is inserted between amino acid residues 457 and 458, amino acid residues 458 and 459, or amino acid residues 459 and 460 of the parental AAV-ShH10 VP1 capsid protein (e.g. SEQ ID NO: 1). AAV-ShH10 VP2 and VP3 capsid proteins may be N-terminal truncations thereof.

[0164] The 7m8 insertion peptide may comprise or consist of an amino acid sequence selected from: LGETTRP (SEQ ID NO: 11), NETITRP (SEQ ID NO: 12), KAGQANN (SEQ ID NO: 13), KDPKTTN (SEQ ID NO: 14), KDTDTTR (SEQ ID NO: 15), RAGGSVG (SEQ ID NO: 16), AVDTTKF (SEQ ID NO: 17), and STGKVPN (SEQ ID NO: 18), LALGETTRPA (SEQ ID NO: 19); LANETITRPA (SEQ ID NO: 20), LAKAGQANNA (SEQ ID NO: 21), LAKDPKTTNA (SEQ ID NO: 22), LAKDTDTTRA (SEQ ID NO: 23), LARAGGSVGA (SEQ ID NO: 24), LAAVDTTKFA (SEQ ID NO: 25), LASTGKVPNA (SEQ ID NO: 26), AALGETTRPA (SEQ ID NO: 27); AANETITRPA (SEQ ID NO: 28), AAKAGQANNA (SEQ ID NO: 29), AAKDPKTTNA (SEQ ID NO: 30), GLGETTRPA (SEQ ID NO: 31); GNETITRPA (SEQ ID NO: 32), GKAGQANNA (SEQ ID NO: 33), and GKDPKTTNA (SEQ ID NO: 34).

[0165] In some embodiments, an AAV-ShH10 / 7m8 VP1 capsid protein comprises a peptide insertion relative to a parental AAV-ShH10 VP1 capsid protein (e.g. SEQ ID NO: 1), wherein the peptide insertion comprises or consists of the amino acid sequence LGETTRP or the amino acid sequence LALGETTRPA, and the insertion site is located between amino acid residues 456 and 457, amino acid residues 457 and 458, or amino acid residues 458 and 459 of the parental AAV-ShH10 VP1 capsid protein (e.g. SEQ ID NO: 1). AAV-ShH10 VP2 and VP3 capsid proteins may be N-terminal truncations thereof. Example AAV-ShH10 / 7m8 VP1 capsid sequences are provided below in SEQ ID NOs: 5-10, in which the 7m8 insertion peptide is underlined. AAV-ShH10 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NOs: 5-10.

[0166] AAV vector genomes

[0167] AAV-ShH10 vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of any serotype is packaged in an AAV-ShH10 capsid or variant thereof.

[0168] An AAV genome may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, an AAV genome is typically replicationdeficient.

[0169] An AAV genome may be in single-stranded form (ssAAV), either positive or negative-sense, or alternatively in double-stranded form (dsAAV). The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression. The maximum packaging capacity of the single-stranded form is larger than the double-stranded form. Suitably, an AAV genome is in single-stranded form.

[0170] Typically, an AAV genome comprises at least one inverted terminal repeat sequence (ITR). An ITR sequence acts in cis to provide a functional origin of replication and allows for integration and excision of the vector from the genome of a cell. ITRs may be the only sequences required in cis next to the therapeutic gene.

[0171] An AAV genome may also comprise packaging genes, such as rep and / or cap genes which encode packaging functions for an AAV particle. A promoter may be operably linked 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 generally used to express the rep gene, while the p40 promoter is generally used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1 , VP2 and VP3 or variants thereof.

[0172] An AAV genome may be the full genome of a naturally occurring AAV. Preferably, an AAV genome is derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art.

[0173] An AAV genome may be a derivative of any naturally occurring AAV. Suitably, an AAV genome is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Suitably, an AAV genome is a derivative of AAV2.

[0174] Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate an AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

[0175] Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV (scAAV) genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression. However, the maximum packaging capacity of a scAAV is reduced.

[0176] An AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof. An AAV genome may comprise at least one, such as two, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof. Suitably, an AAV genome may comprise at least one, such as two, AAV2 ITRs. Suitably, an AAV genome may comprise two AAV2 ITRs.

[0177] The inclusion of one or more ITRs is preferred to aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0178] Suitably, ITR elements will be the only sequences retained from the native AAV genome in a derivative. A derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the one or more coding sequences.

[0179] The following portions could therefore be removed in a derivative: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, derivatives may additionally include one or more rep and / or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting.

[0180] Coding sequences

[0181] The AAV-ShH 10 vector particle may comprise one or more coding sequences (CDS). The one or more coding sequences may encode any polypeptide or polynucleotide or interest, such as a therapeutic gene product.

[0182] The one or more coding sequences may encode a full-length polypeptide, or an N-terminal part or C-terminal part thereof. To overcome packaging capacity limitation, a full-length CDS may split into two parts and packaged into two separate AAV vectors, typically referred to as dual AAVs (see e.g. McClements, M.E. and MacLaren, R.E., 2017. The Yale journal of biology and medicine, 90(4), p.611 ; and Riedmayr, L.M., et al., 2023. Nature Communications, 14(1), p.6578). The polypeptide may be any of interest, for example a therapeutic protein.

[0183] In some embodiments, the AAV-ShH 10 vector particle encodes a polypeptide associated with a kidney disease, or an N-terminal part or C-terminal part thereof. In some embodiments, the AAV-ShH 10 vector particle encodes a polypeptide associated with a glomerular disease, or an N-terminal part or C-terminal part thereof. In some embodiments, the AAV-ShH 10 vector particle encodes a polypeptide associated with a GBM-associated genetic glomerular disease, such as Alport Syndrome, or an N-terminal part or C-terminal part thereof. In some embodiments, the AAV-ShH 10 vector particle encodes a polypeptide associated with podocyte-associated genetic glomerular disease, or an N-terminal part or C-terminal part thereof.

[0184] In some embodiments, the one or more coding sequences encode a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL-1 , C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MYO1 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide, or an N-terminal part or C-terminal part thereof.

[0185] In some embodiments, the one or more coding sequences encode a NPHS2, COL4A3, COL4A4 or COL4A5, CFI, CFH, FHL-1 , and / or NPHS1 polypeptide, or an N-terminal part or C-terminal part thereof. In some embodiments, the one or more coding sequences encode a NPHS2 polypeptide. In some embodiments, the one or more coding sequences encode a COL4A3, COL4A4 or COL4A5 polypeptide, or an N-terminal part or C-terminal part thereof. In some embodiments, the one or more coding sequences encode a CFI, CFH, and / or FHL-1 polypeptide. In some embodiments, the one or more coding sequences encode a NPHS1 polypeptide.

[0186] In other embodiments, the one or more coding sequences do not encode a NPHS2 polypeptide. In other embodiments, the one or more coding sequences do not encode a COL4A3, COL4A4 or COL4A5 polypeptide, or an N-terminal part or C-terminal part thereof. In other embodiments, the one or more coding sequences do not encode a CFI, CFH, or FHL- 1 polypeptide. In other embodiments, the one or more coding sequences do not encode a VEGFC polypeptide. In some embodiments, the one or more coding sequences do not encode a NPHS2, COL4A3, COL4A4, COL4A5, CFI, CFH, or FHL-1 polypeptide. In some embodiments, the one or more coding sequences do not encode NPHS2, CFI, CFH, or FHL- 1 , and the one or more coding sequences do not encode an N-terminal part of an COL4A3, COL4A4 or COL4A5 polypeptide, and the one or more coding sequences do not encode a C- terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide.

[0187] It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0188] The coding sequence may be codon-optimised. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells (e.g. humans), as well as for a variety of other organisms.

[0189] Nephrotic syndrome (NS)-associated transgene

[0190] The AAV-ShH 10 vector particle may comprise a coding sequence a NS-associated transgene.

[0191] Nephrotic syndrome (NS) is a chronic kidney disease characterized by significant proteinuria, hypoalbuminemia, oedema and hyperlipidemia. The NS-associated transgene may be a gene associated with a monogenic form of NS and expressed in podocytes.

[0192] Suitable NS-associated transgenes include NPHS2, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAFB, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PM M2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, AP0L1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAG 12, MY01E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XP05, TNS2 and NLRP3.

[0193] In some embodiments, the AAV-ShH10 vector particle does not comprise a NS-associated transgene.

[0194] NPHS2

[0195] The AAV-ShH10 vector particle may comprise a coding sequence encoding NPHS2, or a fragment and / or variant thereof.

[0196] “NPHS2” is the abbreviated name of the polypeptide encoded by the NPHS2 gene and is also known as podocin. NPHS2 is a 42kDa hairpin like membrane-associated podocyte-specific protein that is a key component of the protein complex at the slit diaphragm; the cell-cell junction between adjacent podocyte foot processes. It localises to lipid rafts and interacts with other important slit diaphragm proteins like nephrin, CD2AP and TRPC6. It is essential in the maintenance of the slit diaphragm, and consequently the integrity of the glomerular filtration barrier.

[0197] A fragment and / or variant of NPHS2 may retain NPHS2 activity and / or function. For example, a fragment and / or variant of podocin may regulate glomerular permeability. Suitably, a fragment and / or variant of NPHS2 may have the same or similar activity and / or function to NPHS2, e.g. may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity and / or function of NPHS2.

[0198] A person skilled in the art would be able to generate fragments and / or variants using conservative substitutions, based on the known structural and functional features of NPHS2 (see e.g. Tabassum, A., et al., 2014. Interdisciplinary Sciences: Computational Life Sciences, 6(1), pp.32-39), and / or based on known variants (see e.g. NCBI Gene ID: 7827 and NCBI HomoloGene: 22826). Suitably, a fragment and / or variant of NPHS2 comprises a transmembrane domain, with two cytoplasmic domains at the N- and C-terminus.

[0199] The NPHS2 gene is conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, and rat. The NPHS2 may be a human NPHS2. Suitably, the NPHS2 may comprise or consist of a polypeptide sequence of UniProtKB accession Q9NP85, or a fragment and / or variant thereof.

[0200] In some embodiments, the NPHS2 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 35 or a fragment thereof. Suitably, the NPHS2 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 35 or a fragment thereof.

[0201] In some embodiments, the NPHS2 comprises or consists of the amino acid sequence of SEQ ID NO: 35 or a fragment thereof.

[0202] In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding NPHS2, or a fragment and / or variant thereof. In some embodiments, the AAV- ShH10 vector particle does not comprise a coding sequence encoding NPHS2.

[0203] NPHS1

[0204] The AAV-ShH10 vector particle may comprise a coding sequence encoding NPHS1 , or a fragment and / or variant thereof.

[0205] “NPHS1” is the abbreviated name of the polypeptide encoded by the NPHS1 gene and is also known as nephrin. NPHS1 is a type-1 transmembrane protein found at the slit diaphragm of glomerular podocytes (see e.g. Li, X., et al., 2015. Journal of the American Society of Nephrology, 26(10), pp.2361 -2377). The slit diaphragm functions as an ultrafilter to exclude albumin and other plasma macromolecules in the formation of urine. Mutations in NPHS1 gene can result in nephrotic syndrome, with the most common mutations being associated with Finnish-type congenital nephrosis, which is characterized by severe proteinuria and loss of the slit diaphragm and foot processes. A fragment and / or variant of NPHS1 may retain NPHS1 activity and / or function. For example, a fragment and / or variant of NPHS1 may maintain slit diaphragm integrity and slit diaphragm- mediated signaling. Suitably, a fragment and / or variant of NPHS1 may have the same or similar activity and / or function to NPHS1 , e.g. may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity and / or function of NPHS1 .

[0206] A person skilled in the art would be able to generate fragments and / or variants using conservative substitutions, based on the known structural and functional features of NPHS1 (see e.g. Weng, Z., et al., 2018. Journal of the American Society of Nephrology, 29(9), pp.2362-2371), and / or based on known variants (see e.g. NCBI Gene ID: 4868 and NCBI HomoloGene: 20974). Suitably, a fragment and / or variant of NPHS1 comprises eight Ig-like domains, a fibronectin type Ill-like domain, and an unstructured intracellular domain (e.g. comprising a PDZ domain-binding motif).

[0207] The NPHS1 gene may be conserved in chimpanzee, Rhesus monkey, dog, cow, mouse, and rat. The NPHS1 may be a human NPHS1. Suitably, the NPHS1 may comprise or consist of a polypeptide sequence of UniProtKB accession 060500, or a fragment and / or variant thereof.

[0208] In some embodiments, the NPHS1 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 63 or a fragment thereof. Suitably, the NPHS1 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 63 or a fragment thereof. In some embodiments, the NPHS1 comprises or consists of the amino acid sequence of SEQ ID NO: 63 or a fragment thereof.

[0209] COL4A3, COL4A4 and COL.4A5 polypeptides

[0210] The AAV-ShH10 vector particle may comprise a coding sequence encoding a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment and / or variant thereof.

[0211] COL4A3, COL4A4 and COL4A5 proteins are approximately 170-185 kDa homologous polypeptides containing collagenous Gly-X-Y repeat sequences frequently interrupted by non- collagenous sequences and forming a triple helix repeat. Each polypeptide also contains a large globular non-collagenous domain at the carboxyl-terminal end.

[0212] Alport syndrome (AS) is caused by pathogenic variants in the COL4A3, COL4A4 and COL4A5 genes, which result in abnormalities of the collagen IV a345 network of basement membranes. The COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof may be capable of forming a collagen IV a345 network. Approximately 200-300 amino acids may be removed from each of the COL4A3, COL4A4 and COL4A5 polypeptides to produce a truncated coding sequence suitable for a mini-gene approach. The amino acids may be removed from the triple helix repeat. Preferably the amino acids are not removed from the non-collagenous region.

[0213] Preferably, the COL4A3, COL4A4 or COL4A5 polypeptide is human. An example human COL4A3 is the COL4A3 having the UniProtKB accession number Q01955. An example human COL4A4 is the COL4A3 having the UniProtKB accession number P53420. An example human COL4A5 is the COL4A5 having the UniProtKB accession number P29400.

[0214] Suitably, the COL4A3 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 36, or a variant which is at least 70% identical to SEQ ID NO: 36. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 36.

[0215] Suitably, the COL4A4 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 37, or a variant which is at least 70% identical to SEQ ID NO: 37. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 37.

[0216] Suitably, the COL4A5 peptide may comprise or consist of the polypeptide sequence shown as SEQ ID NO: 38, or a variant which is at least 70% identical to SEQ ID NO: 38. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 38.

[0217] In some embodiments, the AAV-ShH10 vector particle comprises a coding sequence encoding a full-length COL4A3, COL4A4 or COL4A5 polypeptide.

[0218] In other embodiments, the AAV-ShH 10 vector particle comprises a coding sequence encoding an N-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide. The N-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide may have a length of at least 250 amino acids.

[0219] In other embodiments, the AAV-ShH 10 vector particle comprises a coding sequence encoding a C-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide. The C-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide may have a length of at least 250 amino acids.

[0220] In other embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding an N-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide or a coding sequence encoding a C-terminal part of a COL4A3, COL4A4 or COL4A5 polypeptide. The N- terminal part and the C-terminal part may each have a length of at least 250 amino acids. Complement Factor I (CFI)

[0221] The AAV-ShH10 vector particle may comprise a coding sequence encoding CFI, or a fragment and / or variant thereof.

[0222] Complement factor I (CFI) is a trypsin-like serine protease that inhibits the complement system by cleaving three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b thereby inactivating these proteins.

[0223] CFI is a glycoprotein heterodimer consisting of a disulfide linked heavy chain and light chain. The heavy chain has four domains: an Fl membrane attack complex (FIMAC) domain, CD5 domain, and low density lipoprotein receptor 1 and 2 (LDLrl and LDLr2) domains. The heavy chain plays an inhibitory role in maintaining the enzyme inactive until it meets the complex formed by the substrate (either C3b or C4b) and a cofactor protein (Factor H, C4b-binding protein, complement receptor 1 , and membrane cofactor protein). Upon binding of the enzyme to the substrate:cofactor complex, the heavy:light chain interface is disrupted, and the enzyme activated by allostery. The light chain contains only the serine protease domain. This domain contains the catalytic triad His-362, Asp-411 , and Ser-507, which is responsible for specific cleavage of C3b and C4b.

[0224] The CFI or a fragment and / or variant thereof may be capable of cleaving C3b into iC3b and / or may be capable of cleaving iC3b into C3d,g. The fragment and / or variant of CFI may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b-inactivating and iC3b-degradation activity of native CFI. The C3b-inactivating and iC3b-degradation activity of the fragment and / or variant of CFI and native CFI, may be determined using any suitable method known to those of skill in the art. For example, using a proteolytic assay.

[0225] Preferably, the CFI is a human CFI. An example human CFI is the CFI having the UniProtKB accession number P05156.

[0226] In some embodiments, the CFI comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 39 or a fragment thereof. Suitably, the CFI comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 39 or a fragment thereof.

[0227] In some embodiments, the CFI comprises or consists of the amino acid sequence of SEQ ID NO: 39 or a fragment thereof. In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding CFI, or a fragment and / or variant thereof. In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding CFI.

[0228] Complement Factor H (CFH)

[0229] The AAV-ShH10 vector particle may comprise a coding sequence encoding CFH, or a fragment and / or variant thereof.

[0230] Complement factor H (CFH) regulates complement activation on cells and surfaces. CFH competes for binding of complement factor B (CFB) to C3b, acts as a cofactor for CFI- catalysed proteolytic cleavage of C3b, and accelerates the irreversible dissociation of C3bBb and C3b2Bb into their separate components. Thus, CFH not only inhibits formation of the convertases but it also shortens the lifespan of any convertase complex that forms.

[0231] CFH is a large (155 kDa) soluble glycoprotein. CFH is composed from a total of 20 domains, each containing approximately 60 amino acid residues and termed complement control protein modules (CCPs) or short consensus repeats that are joined by short linkers consisting of 3-8 residues. The CCP modules are numbered from 1-20 (from the N-terminus of the protein): CCPs 1-4 and CCPs 19-20 engage with C3b while CCPs 7 and CCPs 19-20 bind to GAGs and sialic acid.

[0232] The CFH or a fragment and / or variant thereof may be capable of binding C3b and / or C3d; and / or acting as a cofactor for the CFI-catalysed proteolytic cleavage of C3b; and / or increasing the irreversible dissociation of C3bBb and C3b2Bb into their separate components. The fragment and / or variant of CFH may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native CFH. The activity of the fragment and / or variant of CFH and native CFH may be determined using any suitable method known to those of skill in the art.

[0233] Preferably, the CFH is a human CFH. An example human CFH is the CFH having the UniProtKB accession number P08603.

[0234] In some embodiments, the CFH comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 40 or a fragment thereof. Suitably, the CFH comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 40 or a fragment thereof.

[0235] In some embodiments, the CFH comprises or consists of the amino acid sequence of SEQ ID NO: 40 or a fragment thereof. In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding CFH, or a fragment and / or variant thereof. In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding CFH.

[0236] Factor H-like protein 1 (FHL-1)

[0237] The AAV-ShH10 vector particle may comprise a coding sequence encoding FHL-1 , or a fragment and / or variant thereof.

[0238] FHL-1 or a fragment and / or variant thereof may be capable of binding C3b and / or C3d. The fragment and / or variant of FHL-1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native FHL-1 . The activity of the fragment and / or variant of FHL-1 and native FHL-1 may be determined using any suitable method known to those of skill in the art.

[0239] Preferably, the FHL-1 is a human FHL-1. An example human FHL-1 is the FHL-1 having the NCBI Reference Sequence: NP_001014975.1.

[0240] In some embodiments, the FHL-1 comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 41 or a fragment thereof. Suitably, the FHL-1 comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 41 or a fragment thereof.

[0241] In some embodiments, the FHL-1 comprises or consists of the amino acid sequence of SEQ ID NO: 41 or a fragment thereof.

[0242] In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding FHL-1 , or a fragment and / or variant thereof. In some embodiments, the AAV-ShH10 vector particle does not comprise a coding sequence encoding FHL-1.

[0243] Vascular endothelial growth factor (VEGF)C polypeptides

[0244] The AAV-ShH10 vector particle may comprise a coding sequence encoding a vascular endothelial growth factor (VEGF)C polypeptide, or a fragment and / or variant thereof.

[0245] VEGFC is a lymphangiogenic growth factor, which is known to signal via two receptors, VEGFR-3 (Flt4) and VEGFR-2 (Flk4). VEGFC is produced by cells in a prepropeptide form, which dimerises before being cleaved into a tetramer. The AAV-ShH10 vector particle may comprise a coding sequence encoding any form of VEGFC, such as the prepropeptide form, the tetramer form, the intermediate form, or fully processed mature VEGFC.

[0246] If desired, coding sequences encoding different forms of VEGFC polypeptides may be used in any combination. Preferably, the coding sequences comprise a polynucleotide encoding one or more polypeptides having VEGFC biological activity, i.e. , peptides that can bind to and activate VEGFR-2 and / or VEGRF-3. More preferably, the coding sequences comprise comprise a polynucleotide encoding a polypeptide comprising the VEGFC homology domain and having VEGFC biological activity, i.e., a polypeptide that can bind to and activate VEGFR- 2 and / or VEGRF-3. Further details of suitable VEGFC polynucleotides and polypeptides include those described in WO 2015 / 022447 and US 2014 / 0087002.

[0247] The variant sequence may encode a VEGFC polypeptide that has retained the capability to bind and activate VEGFR-2 and VEGFR-3.

[0248] In some embodiments, the VEGFC polypeptide comprises or consists of an amino acid sequence which is at least 70% identical to SEQ ID NO: 42 or a fragment thereof. Suitably, the VEGFC polypeptide comprises or consists of an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 42 or a fragment thereof.

[0249] In some embodiments, the VEGFC polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 42 or a fragment thereof.

[0250] Regulatory elements

[0251] The AAV-ShH10 vector particle may comprise one or more regulatory sequences which may act pre- or post-transcriptionally. Suitably, one or more coding sequences may be operably linked to the one or more regulatory sequences. The one or more regulatory sequences may facilitate expression of the one or more coding sequences.

[0252] “Regulatory sequences” are any sequences which facilitate expression of a polypeptide or polynucleotide encoded by a coding sequence, e.g. act to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory sequences include for example promoters, enhancer elements, post-transcriptional regulatory elements and polyadenylation sites.

[0253] In some embodiments, the AAV-ShH10 vector particle does not comprise a 5’-truncated NPHS2 promoter, a super core promoter, or a spliceosomal intron or a fragment thereof comprising a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site. Promoters

[0254] The AAV-ShH10 vector particle may comprise a promoter. Suitably, the promoter may be operably linked to one or more coding sequences. The promoter may facilitate expression of the one or more coding sequences.

[0255] A “promoter” is a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person. The promoter may be a human promoter. Suitably, the promoter is operable in human podocytes. The promoter may be capable of driving expression of a coding sequence in human podocytes. The promoter may be a minimal promoter. As used, herein, a “minimal promoter” means the minimal sequence that can act as a promoter.

[0256] The promoter may be a constitutive promoter, an inducible promoter, or a repressible promoter. The promoter may be a ubiquitous promoter or a tissue-specific promoter.

[0257] In some embodiments, the promoter is a constitutive promoter. As used herein, a “constitutive promoter” is a promoter which is always active. Suitable constitutive promoters will be known to the skilled person. Example constitutive promoters include the CMV promoter, the EF1a promoter, the CAG promoter, the PGK promoter, the U6 promoter, the T7 promoter, the SV40 promoter, and the Sp6 promoter.

[0258] In some embodiments, the promoter is an inducible promoter. An “inducible promoter” may refer to a promoter which is activated in response to specific stimuli (e.g. in response to chemicals, temperature, or light). Suitable inducible promoters will be known to the skilled person. In some embodiments, the promoter is a repressible promoter. A “repressible promoter” may refer to a promoter which is de-activated in response to specific stimuli. Suitable repressible promoters will be known to the skilled person.

[0259] In some embodiments, the promoter is a ubiquitous promoter. As used herein, a “ubiquitous promoter” is a promoter which is active in a wide range of cells and tissues.

[0260] In other embodiments, the promoter is a cell-specific or tissue-specific promoter. As used herein, a “cell-specific promoter” or “tissue-specific promoter” is a promoter which preferentially facilitates expression of a coding sequence in a specific type of cells or tissue (see e.g. Zheng, C. and Baum, B.J., 2008. Gene Therapy Protocols: Design and Characterization of Gene Transfer Vectors, pp.205-219). Suitably, a cell-specific or tissuespecific promoter may facilitate higher expression of a coding sequence in one cell-type or tissue as compared to other cell-types or tissues. For example, a cell-specific or tissue-specific promoter may be a promoter which facilitates expression of a coding sequence at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in one cell-type or tissue as compared to expression in other cell-types or tissues. Suitable tissue-specific promoters will be known to the skilled person (see e.g. Toscano, M.G., et al., 2011. Gene therapy, 18(2), pp.117-127; and Powell, S.K., et al., 2015. Discovery medicine, 19(102), p.49) and can be generated using methods known in the art (see and Shen, S.Q., et al., 2016. Genome research, 26(2), pp.238-255).

[0261] In some embodiments, the promoter is a kidney-specific promoter. Suitable kidney-specific promoters will be known to the skilled person.

[0262] In some embodiments, the promoter is a glomerular-specific promoter. Suitable glomerular- specific promoters will be known to the skilled person.

[0263] In some embodiments, the promoter is a podocyte-specific promoter. Suitable podocytespecific promoters will be known to the skilled person.

[0264] In some embodiments, the promoter is a NPHS1 or a NPHS2 promoter, or a variant thereof.

[0265] In some embodiments, the promoter is a minimal kidney-specific promoter. As used, herein, a “minimal kidney-specific promoter” means the minimal sequence that can act as a kidneyspecific promoter. In some embodiments, the promoter is a minimal glomerular-specific promoter. In some embodiments, the promoter is a minimal podocyte-specific promoter. In some embodiments, the promoter is a minimal NPHS1 or a minimal NPHS2 promoter, or a variant thereof.

[0266] In some embodiments, the promoter is a human promoter, e.g. a minimal human NPHS1 promoter or a minimal human NPHS2 promoter.

[0267] NPHS1 promoter

[0268] In some embodiments, the promoter is a NPHS1 promoter, or a variant thereof.

[0269] The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes. A human NPHS1 promoter has been described in Moeller et al. 2002 J Am Soc Nephrol, 13(6):1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6): F1027-32. This NPHS1 promoter is a 1.2kb fragment and appears to be podocyte-specific. The 1.2kb promoter region lacks a TATA box, but has recognition motifs for other transcription factors e.g. PAX-2 binding element, E-box and GATA consensus sequences. Suitably, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 43, or a variant which is at least 70% identical to SEQ ID NO: 43. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 43.

[0270] Suitably, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 64, or a variant which is at least 70% identical to SEQ ID NO: 64. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 64.

[0271] The NPHS1 promoter may be a minimal NPHS1 promoter. Suitably, the NPHS1 promoter has a length of about 1.1 kb or less, about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, about 0.7 kb or less, about 0.6 kb or less, about 0.5 kb or less, about 0.4 kb or less, or about 0.3 kb or less. Suitably, the NPHS1 promoter has a length of about 265 bp or more. In some embodiments, the NPHS1 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.

[0272] In some embodiments, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 44, or a variant which is at least 70% identical to SEQ ID NO: 44. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 44.

[0273] In some embodiments, the AAV-ShH10 vector particle does not comprise a NPHS1 promoter, or a variant thereof.

[0274] NPHS2 promoter

[0275] In some embodiments, the promoter is a NPHS2 promoter, or a variant thereof.

[0276] The NPHS2 gene encodes podocin, which is selectively expressed in podocytes. A human NPHS2 promoter has been described in Oleggini R, et al., 2006. Gene Expr. 13(1):59-66.

[0277] Suitably, the NPHS2 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 45, or a variant which is at least 70% identical to SEQ ID NO: 45. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 45.

[0278] The NPHS2 promoter may be a minimal NPHS2 promoter. Suitably, the NPHS2 promoter has a length of about 2.0 kb or less, about 1 .8 kb or less, about 1 .6 kb or less, about 1.4 kb or less, about 1.2 kb or less, about 1.0 kb or less, about 0.9 kb or less, about 0.8 kb or less, or about 0.7 kb or less. Suitably, the NPHS2 promoter has a length of about 628 bp or more. In some embodiments, the NPHS2 promoter has a length of about 628-2000 bp, 628-1800 bp, 628- 1600 bp, 628-1400 bp, 628-1200 bp, 628-1000 bp, 628-900 bp, 628-800 bp, or 628-700 bp.

[0279] In some embodiments, the NPHS2 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 46, or a variant which is at least 70% identical to SEQ ID NO: 46. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 46.

[0280] The NPHS2 promoter may be a 5’-truncated NPHS2 promoter, i.e. a truncate of a full-length NPHS2 promoter. In some embodiments, the 5’-truncated NPHS2 promoter has a length of 628 base pairs (bp) or less, 600 bp or less, 550 bp or less, 500 bp or less, 450 bp or less, 400 bp or less, 350 bp or less, 300 bp or less, 250 bp or less, or 200 bp or less. In some embodiments, the 5’-truncated podocin promoter has a length of about 100 bp or more, 150 bp or more, 200 bp or more, 250 bp or more, 300 bp or more, 350 bp or more, 400 bp or more, 450 bp or more, 500 bp or more, 550 bp or more, or 600 bp or more. In some embodiment, the 5’-truncated podocin promoter has a length of from about 150 bp to about 600 bp.

[0281] In some embodiments, the AAV-ShH10 vector particle does not comprise a 5’-truncated NPHS2 promoter.

[0282] In some embodiments, the AAV-ShH10 vector particle does not comprise a NPHS2 promoter, or a variant thereof.

[0283] Hybrid promoters

[0284] In some embodiments, the promoter is a hybrid enhancer-promoter.

[0285] In some embodiments, the hybrid enhancer-promoter is a hybrid kidney-specific promoter comprising a kidney-specific enhancer and a ubiquitous or core promoter. In some embodiments, the hybrid enhancer-promoter is a hybrid glomerular-specific promoter comprises a glomerular-specific enhancer and a ubiquitous or core promoter. In some embodiments, the hybrid enhancer-promoter is a hybrid podocyte-specific promoter comprises a podocyte-specific enhancer and a ubiquitous or core promoter.

[0286] Suitably, the enhancer may be or may be derived from an enhancer associated with a gene with selective expression in human podocytes. Methods to identify the enhancer regions associated with genes will be well known to those of skill in the art. Preferably, the enhancer is a NPHS1 or a NPHS2 enhancer, or a fragment and / or variant thereof. Preferably, the enhancer is a human enhancer, e.g. a human NPHS1 enhancer or human NPHS2 enhancer.

[0287] A NPHS1 enhancer has been described in Guo, G., et al., 2004. Journal of the American Society of Nephrology, 15(11), pp.2851-2856. A 186-bp fragment from the human NPHS1 promoter was capable of directing podocyte-specific expression of a p-galactosidase coding sequence when placed in front of a heterologous minimal promoter in transgenic mice.

[0288] Suitably, a NPHS1 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 47, or a variant which is at least 70% identical to SEQ ID NO: 47. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 47.

[0289] A NPHS2 enhancer has been described in WO 2023 / 213738. The NPHS2 enhancer comprises NPHS2 motif Lmx1b-FoxC2 and allowed the selective expression of genes of interest in podocytes and other kidney cell lines.

[0290] Suitably, a NPHS2 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 48, or a variant which is at least 70% identical to SEQ ID NO: 48. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 48.

[0291] The enhancer may be upstream of a ubiquitous promoter or core promoter. The enhancer and the ubiquitous promoter or core promoter may be operably linked. In some embodiments, the enhancer is upstream of a core promoter. In some embodiments, the enhancer is upstream of a super core promoter.

[0292] The “core promoter” is typically 80 nucleotides long, encompassing from -40 to +40 relative to the transcription start site and consists of several functional sub-regions, termed core elements or motifs. A “super core promoter” may refer to a synthetic core promoter which contains a combination of core promoter elements that drive high levels of transcription. For example, a super core promoter may contain a TATA box, an initiator motif (Inr), a motif ten element (MTE) and a downstream promoter element (DPE). Example super core promoters have been described in Juven-Gershon, T., et al., 2006. Nature methods, 3(11), pp.917-922 and Even, D.Y., et al., 2016. PloS one, 11(2), p.e0148918.

[0293] A super core promoter may comprise from 5’ to 3’: a TATA box, an initiator motif (Inr), a motif ten element (MTE), and a downstream promoter element (DPE). The super core promoter may comprise one or more further core promoter elements, such as transcription binding sites (e.g. TFIIB recognition element).

[0294] Suitably, the super core promoter is selected from super core promoter 1 (SCP1), super core promoter 2 (SCP2), or super core promoter 3 (SCP3). In some embodiments, the super core promoter is SCP1.

[0295] In some embodiments, the super core promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 49. Suitably, the super core promoter comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 49. In some embodiments, the super core promoter comprises or consists of the nucleotide sequence SEQ ID NO: 49.

[0296] In some embodiments, the hybrid enhancer-promoter comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 50. Suitably, the hybrid enhancerpromoter comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 50. In some embodiments, the hybrid enhancer-promoter comprises or consists of the nucleotide sequence SEQ ID NO: 50.

[0297] In some embodiments, the AAV-ShH10 vector particle does not comprise a super core promoter. In some embodiments, the AAV-ShH10 vector particle does not comprise a hybrid promoter. In some embodiments, the promoter is not a hybrid enhancer-promoter.

[0298] Spliceosomal introns The AAV-ShH10 vector particle may comprise a spliceosomal intron or fragment thereof. The spliceosomal intron or fragment thereof may be downstream of a promoter and upstream of a coding sequence.

[0299] A “spliceosomal intron” may refer to a non-coding sequence excised from pre-m RNAs by the spliceosome during mRNA splicing. Spliceosomal introns have been found in most eukaryotic genes and their lengths vary between species, from just tens of bases in some protists to hundreds of kilobases in some mammals (see e.g. Roy, S. W. and Gilbert, W., 2006. Nature Reviews Genetics, 7(3), pp.211-221).

[0300] The spliceosomal intron may be a naturally-occurring spliceosomal intron, a chimeric spliceosomal intron (e.g. with elements derived from two or more naturally-occurring spliceosomal introns), or a variant spliceosomal intron. Examples of suitable naturally- occurring spliceosomal introns include the rabbit beta globin intron I, the rabbit beta globin intron II, the minute virus of mice (MVM) intron, and the F.IX truncated intron 1. Examples of suitable chimeric spliceosomal introns include p-globin SD I immunoglobin heavy chain SA, Adenovirus SD I immunoglobulin SA, SV40 late SD I SA (19S / 16S), and Hybrid adenovirus SD I IgG SA (see e.g. Powell, S.K., et al., 2015. Discovery medicine, 19(102), pp.49-57). Examples of suitable variant spliceosomal introns are described below.

[0301] A spliceosomal intron typically contains the following conserved elements from 5’ to 3’: a 5’ splice donor site, also known as a 5’ splice site; a branch point sequence, comprising the branch point; a polypyrimidine tract; and a 3’ splice acceptor site, also known as a 3’ splice site (see e.g. Padgett, R.A., et al., 1986. Annual review of biochemistry, 55(1), pp.1119-1150). Suitably, the spliceosomal intron may comprise or consist of the nucleotide sequence GTRAGT(N)xYTNAY(N)ni(Y)n2(N)n3YAGG (SEQ ID NO: 65), wherein x is from about 10 to about 1000 (e.g. from about 10 to about 100), n1 is from about 2 to about 22, n2 is from about 10 to about 20, and n1 + n2 + n3 is from about 15 to about 40, or a nucleotide sequence having five or fewer, four or fewer, three or fewer, two or fewer, or one nucleotide substitution.

[0302] The spliceosomal intron may be a rabbit beta globin intron or a variant thereof. The rabbit beta globin (RBG) gene may have GenBank accession number V00882. The RBG intron I may comprise or consist of the nucleotide sequence of SEQ ID NO: 51. The RBG intron II may comprise or consist of the nucleotide sequence of SEQ ID NO: 52.

[0303] Suitably, the spliceosomal intron may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 51 or 52, or a variant which is at least 70% identical to SEQ ID NO: 51 or 52. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 51 or 52.

[0304] An example RBG intron I variant is provided below in SEQ ID NO: 53. Compared to the RBG intron I, the polypyrimidine tract sequence has been altered by substituting the native G and A residues in this region with T to increase the polypyrimidine tract consensus.

[0305] Suitably, the spliceosomal intron may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 53, or a variant which is at least 70% identical to SEQ ID NO: 53. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 53.

[0306] In some embodiments, the AAV-ShH10 vector particle comprises a spliceosomal intron fragment. The spliceosomal intron fragment may be a fragment of any naturally-occurring, chimeric, or variant spliceosomal intron, comprising or consisting of the following conserved elements from 5’ to 3’: a branch point sequence; a polypyrimidine tract; and a 3’ splice acceptor site. The spliceosomal intron fragment preferably does not comprise a 5’ splice donor site.

[0307] Suitably, the spliceosomal intron fragment may comprise or consist of the nucleotide sequence (N)xYTNAY(N)ni(Y)n2(N)n3YAGG, wherein x is from about 10 to about 1000 (e.g. from about 10 to about 100), n1 is from about 2 to about 22, n2 is from about 10 to about 20, and n1 + n2 + n3 is from about 15 to about 40, or a nucleotide sequence having five or fewer, four or fewer, three or fewer, two or fewer, or one nucleotide substitution.

[0308] An example spliceosomal intron fragment derived from the RBG intron I is provided below in SEQ ID NO: 54. Compared to the RBG intron I, the polypyrimidine tract sequence has been altered by substituting the native G and A residues in this region with T to increase the polypyrimidine tract consensus; and 22 nucleotides at the 5’ end have been deleted, including the 5’ splice donor site.

[0309] Suitably, the spliceosomal intron fragment may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 54, or a variant which is at least 70% identical to SEQ ID NO: 54. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 54.

[0310] Another example spliceosomal intron fragment derived from the RBG intron I is provided below in SEQ ID NO: 55. Compared to SEQ ID NO: 54, 12 nucleotides have been inserted at the 5’ end (including a restriction site but not including a 5’ splice donor site). Suitably, the spliceosomal intron fragment may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 55, or a variant which is at least 70% identical to SEQ ID NO: 55. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 55.

[0311] In some embodiments, the AAV-ShH 10 vector particle does not comprise a regulatory element comprising a fragment of a spliceosomal intron, wherein the regulatory element comprises a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site, and wherein the regulatory element does not comprise a 5’ splice donor site

[0312] In some embodiments, the AAV-ShH10 vector particle does not comprise a spliceosomal intron or a fragment thereof comprising a branch point sequence, a polypyrimidine tract, and a 3’ splice acceptor site.

[0313] Enhancers

[0314] The AAV-ShH 10 vector particle may comprise an enhancer. Suitably, the enhancer may be operably linked to a coding sequence. The enhancer may facilitate expression of the protein.

[0315] An “enhancer” is a region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They can be located up to 1 Mbp (1 ,000,000 bp) away from the gene, upstream or downstream from the start site. Any suitable enhancer may be used, the selection of which may be readily made by the skilled person.

[0316] In some embodiments, the enhancer is a NPHS1 or a NPHS2 enhancer, or a fragment or derivative thereof. In some embodiments, the enhancer is a human enhancer, e.g. a human NPHS1 enhancer or human NPHS2 enhancer.

[0317] The enhancer may be used with the corresponding promoter, for example the NPHS1 enhancer may be used with the NPHS1 promoter. Alternatively, the enhancer may be used with a different promoter.

[0318] Kozak sequence

[0319] The AAV-ShH 10 vector particle may comprise a Kozak sequence. Suitably, the Kozak sequence may be operably linked to a coding sequence. A Kozak sequence may be inserted before the start codon of the protein to improve the initiation of translation. Suitable Kozak sequences will be well known to those of skill in the art (see e.g. Kozak, M., 2002. Gene, 299(1-2), pp.1-34). A consensus Kozak sequence in vertebrates may have the sequence of SEQ ID NO: 56 or SEQ ID NO: 57.

[0320] Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 56 or 57, or variants thereof which have five or fewer deletions, substitutions or insertions. Suitably, the variants may have four or fewer, three or fewer, two or fewer, or one deletion(s), substitution(s) or insertion(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have one deletion and / or one substitution. Suitably, the variants may have one deletion and one substitution.

[0321] Post-transcriptional regulatory elements

[0322] The AAV-ShH10 vector particle may comprise a post-transcriptional regulatory element. Suitably, the post-transcriptional regulatory element may be operably linked to a coding sequence. Suitably, the transcriptional regulatory element may be downstream of a coding sequence.

[0323] The AAV-ShH10 vector particle may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE). Suitably, the WPRE may be operably linked to the coding sequence. Suitably, the transcriptional regulatory element may be downstream of the coding sequence.

[0324] The WPRE sequence may have mutations within the X-antigen promoter and / or the initiation codon of the X-antigen. This may prevent the production of a functional X-antigen. Suitably, the WPRE may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 58, or a variant which is at least 70% identical to SEQ ID NO: 58. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 58.

[0325] Polyadenylation sequence

[0326] The AAV-ShH10 vector particle may comprise a polyadenylation sequence. Suitably, the polyadenylation sequence may be operably linked to a coding sequence. Suitably, the polyadenylation sequence may be downstream of a coding sequence. A “polyadenylation sequence” is a region of DNA or mRNA that comprises the elements required for polyadenylation of the mRNA. The sequence elements for polyadenylation typically include a polyadenylation signal (with consensus sequence AATAAA) and a polyadenylation site (consensus sequence CA) and may also include a GT-rich downstream element.

[0327] Suitable polyadenylation sequences are well-known in the art and may include a bovine growth hormone polyadenylation sequence (bGH), a soluble neuropilin-1 polyadenylation sequence, an early SV40 polyadenylation sequence (SV40pA), and a chicken beta-globin polyadenylation sequence.

[0328] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 59, or a variant which is at least 70% identical to SEQ ID NO: 59. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 59.

[0329] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 60, or a variant which is at least 70% identical to SEQ ID NO: 60. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 60.

[0330] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 61, or a variant which is at least 70% identical to SEQ ID NO: 61. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 61.

[0331] Suitably, the polyadenylation sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO: 62, or a variant which is at least 70% identical to SEQ ID NO: 62. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 62.

[0332] Spacer sequences

[0333] The AAV-ShH10 vector particle may further comprise one or more further nucleotide sequences, for example one or more spacer sequence. As used herein, a “spacer sequence” may refer to any nucleotide sequence which is inserted between two elements, such that the elements are in a relationship permitting them to function in their intended manner. Suitably, spacer sequences may be included to provide a polynucleotide of a desired length. For example, for efficient encapsidation of AAV vectors, spacer sequences may be included if the expression cassette is less than 3.0 kb in length. Suitably, a spacer sequence has no function or activity.

[0334] The length and sequence of a spacer sequence is not particularly limited. Suitably, a spacer sequence has a length of about 1000 bp or less, about 900 bp or less, about 800 bp or less, about 700 bp or less, about 600 bp or less, about 500 bp or less, about 400 bp or less, about 300 bp or less, about 200 bp or less, about 180 bp or less, about 160 bp or less, about 140 bp or less, about 120 bp or less, or about 100 bp or less. Suitably, a spacer sequence has a length of about 1 bp or more, about 2 bp or more, about 3 bp or more, about 4 bp or more, about 5 bp or more, or about 10 bp or more. Suitably, a spacer sequence has a length of about 1 bp to about 1000 bp, about 1 bp to about 500 bp, about 1 bp to about 200 bp, or about 10 bp to about 100 bp. Suitably, a spacer sequence has an arbitrary sequence that lacks function or activity (e.g. lacks binding sites) (see e.g. Estrada, J., et al., 2016. PloS one, 11(3), p.e0151740).

[0335] Variants, derivatives, homologues and fragments

[0336] In addition to the specific polypeptides and polynucleotides mentioned herein, the invention also encompasses variants, derivatives, homologues and fragments thereof.

[0337] In the context of the invention, a “variant” of any given sequence may refer a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the given sequence.

[0338] The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one or all of its endogenous functions.

[0339] Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues. Polypeptides used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0340] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

[0341] The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool e.g. SIFT (Vaser, R., et al., 2016. Nature protocols, 11(1), pp.1-9), PolyPhen-2 (Adzhubei, I., et al., 2013. Current protocols in human genetics, 76(1), pp.7-20), CADD (Rentzsch, P., et al., 2021. Genome medicine, 13(1), pp.1- 12), REVEL (loannidis, N.M., et al., 2016. The American Journal of Human Genetics, 99(4), pp.877-885), MetaLR (Dong, C., et al., 2015. Human molecular genetics, 24(8), pp.2125- 2137), and / or MutationAssessor (Reva, B., et al., 2011. Nucleic acids research, 39(17), pp.e118-e118) or based on clinical data e.g. ClinVar (Landrum, M.J., et al., 2016. Nucleic acids research, 44(D1), pp.D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations may be considered tolerated, benign, and / or likely benign.

[0342] In the present context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express it in terms of sequence identity. In the present context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.

[0343] The term “homologue” as used herein means a variant having a certain similarity with the wild type amino acid sequence or the wild type nucleotide sequence, e.g. having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% similarity, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similarity to the subject sequence.

[0344] Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0345] Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0346] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0347] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0348] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0349] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (see e.g. Devereux, J., et al., 1984. Nucleic acids research, 12(1), pp.387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see e.g. Altschul, S.F., et al., 1990. Journal of molecular biology, 215(3), pp.403-410), BLAST 2 (see e.g. Tatusova, T.A. and Madden, T.L., 1999. FEMS microbiology letters, 174(2), pp.247-250), FASTA (see e.g. Pearson, W.R. and Lipman, D.J., 1988. PNAS, 85(8), pp.2444-2448.), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle.

[0350] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix.

[0351] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0352] The term “fragment” as used herein refers to a variant sequence that is a portion of a full- length polypeptide or polynucleotide. Fragments are typically selected regions of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay.

[0353] Such variants, derivatives, homologues and fragments may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0354] Methods of treatment

[0355] The present invention provides a method for delivering one or more coding sequences to podocytes of a human subject, the method comprising administering an AAV-ShH10 vector particle comprising the one or more coding sequences to said subject.

[0356] Following administration, the AAV-ShH 10 vector particle may transduce the podocytes of said subject and express the one or more coding sequences in the podocytes. Expression of the one or more coding sequences in the podocytes may be determined by any suitable method, for example by an enzyme-linked immunosorbent assay (ELISA) or by immunoblotting.

[0357] The methods of the invention may be used to prevent and / or treat a disease. For example, the methods of the invention may be used for gene therapy. Gene therapy may refer to methods which aim to produce a therapeutic effect through the manipulation of gene expression or through altering the biological properties of living cells.

[0358] In one aspect, the present invention provides use of an AAV-ShH 10 vector particle for transducing human podocytes in vivo.

[0359] In another aspect, the present invention provides an in vivo method for delivering one or more coding sequences to human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle comprising one or more coding sequences.

[0360] In another aspect, the present invention provides an AAV-ShH 10 vector particle for use in gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0361] In another aspect, the present invention provides use of an AAV-ShH 10 vector particle for the manufacture of a medicament for gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject. In another aspect, the present invention provides a method for gene therapy of a human subject, the method comprising administering an AAV-ShH10 vector particle to the subject, wherein the AAV-ShH10 vector particle transduces podocytes of the subject.

[0362] Human subjects

[0363] The subject may be an adult, an adolescent, or a child. In some embodiments, the subject is an adult. In other embodiments, the subject is a paediatric patient. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child.

[0364] The subject may have or may be at risk of a kidney disease. For example, the subject may have or may be at risk of a glomerular disease. For example, the subject may have or may be at risk of a podocyte-associated glomerular disease. For example, the subject may have or may be at risk of a GBM-associated glomerular disease.

[0365] In some embodiments, the subject has or is at risk of a genetic glomerular disease, i.e. a glomerular disease which is inherited. Genetic glomerular diseases include podocyte-associated genetic glomerular diseases, such as nephrotic syndrome, and GBM-associated glomerular diseases, such as Alport Syndrome.

[0366] In some embodiments, the subject has or is at risk of a podocyte-associated genetic glomerular disease. Podocyte-associated genetic glomerular diseases include Congenital nephrotic syndrome of the Finnish type, Congenital nephrotic syndrome type 2, Familial nephrotic syndrome type 3, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, Nephrotic syndrome caused by mutations in CD2AP, Nephrotic syndrome caused by mutations in actinin-4, Nephrotic syndrome caused by mutations in TRPC6, and Epstein and Fechtner syndrome. Suitably, the glomerular disease is nephrotic syndrome.

[0367] In some embodiments, the subject has or is at risk of a GBM-associated genetic glomerular disease. GBM-associated genetic glomerular diseases include X-linked Alport syndrome, Autosomal recessive Alport syndrome, Autosomal dominant Alport syndrome, Thin basement membrane diseases, Pierson syndrome, and Nail-patella syndrome. Suitably, the glomerular disease is Alport syndrome (AS). AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease and thin basement membrane nephropathy.

[0368] In some embodiments, the subject has or is at risk of a complement-mediated kidney disease. As used herein a “complement-mediated kidney disease” is a disease of the kidney which is caused by dysregulation of the complement system. The complement system can cause kidney injury in a variety of different diseases. Suitably, the complement-mediated kidney disease is caused by excessive activation of the complement system. Exemplary complement- mediated kidney diseases include IgA nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome (aHUS), shiga toxin-associated HUS, lupus nephritis, cryoglobulinemia, anti-GBM disease, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated rejection of renal transplant, membranous nephropathy, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

[0369] In some embodiments, the subject has or is at risk of diabetic nephropathy. Diabetic nephropathy, also known as diabetic kidney disease, is the chronic loss of kidney function occurring in those with diabetes mellitus.

[0370] In some embodiments, the subject has or is at risk of Fabry disease. Fabry disease is an inherited disorder that results from the accumulation of globotriaosylceramide within lysosomes. Kidney complications are common and serious effects of the disease.

[0371] Kidney diseases

[0372] The methods of the invention may be used to prevent and / or treat a kidney disease. For example, the methods of the invention may be used for kidney gene therapy.

[0373] In one aspect, the present invention provides an AAV-ShH10 vector particle for use in preventing and / or treating a kidney disease in a human subject, wherein the AAV-ShH10 vector particle transduces podocytes of the subject.

[0374] In another aspect, the present invention provides use of an AAV-ShH10 vector particle for the manufacture of a medicament for preventing and / or treating a kidney disease in a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0375] In another aspect, the present invention provides a method for preventing and / or treating a kidney disease in a human subject, the method comprising administering an AAV-ShH 10 vector particle to the subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

[0376] The kidney disease may be a glomerular disease. For example, the kidney disease may be a podocyte-associated glomerular disease. For example, the kidney disease may be a GBM- associated glomerular disease. The kidney disease may be a genetic kidney disease (see e.g. Hildebrandt, F., 2010. The Lancet, 375(9722), pp.1287-1295). The polynucleotide may correspond to the kidney disease which is intended to be treated and / or prevented. For example, when the kidney disease is Alport Syndrome, the polynucleotide may encode COL4A3, COL4A4 or COL4A5. For example, when the kidney disease is nephrotic syndrome, the polynucleotide may encode a NS-associated transgene. For example, when the kidney disease is complement-associated, the polynucleotide may encode a complement protein.

[0377] In some embodiments, the kidney disease is a genetic glomerular disease. Genetic glomerular diseases include podocyte-associated genetic glomerular diseases, such as nephrotic syndrome, and GBM -associated glomerular diseases, such as Alport Syndrome.

[0378] In some embodiments, the kidney disease is a podocyte-associated genetic glomerular disease. Podocyte-associated genetic glomerular diseases include Congenital nephrotic syndrome of the Finnish type, Congenital nephrotic syndrome type 2, Familial nephrotic syndrome type 3, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, Nephrotic syndrome caused by mutations in CD2AP, Nephrotic syndrome caused by mutations in actinin-4, Nephrotic syndrome caused by mutations in TRPC6, and Epstein and Fechtner syndrome. Suitably, the glomerular disease is nephrotic syndrome.

[0379] In some embodiments, the kidney disease is a GBM-associated genetic glomerular disease. GBM-associated genetic glomerular diseases include X-linked Alport syndrome, Autosomal recessive Alport syndrome, Autosomal dominant Alport syndrome, Thin basement membrane diseases, Pierson syndrome, and Nail-patella syndrome. Suitably, the glomerular disease is Alport syndrome (AS). AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease and thin basement membrane nephropathy.

[0380] In some embodiments, the kidney disease is a complement-mediated kidney disease. Exemplary complement-mediated kidney diseases include IgA nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome (aHUS), shiga toxin-associated HUS, lupus nephritis, cryoglobulinemia, anti-GBM disease, Anti neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated rejection of renal transplant, membranous nephropathy, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

[0381] In some embodiments, the kidney disease is diabetic nephropathy.

[0382] In some embodiments, the kidney disease is Fabry disease. Pharmaceutical compositions

[0383] The AAV-ShH10 vector particle may be administered in the form of a pharmaceutical composition. A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent i.e. the AAV-ShH10 vector particle. It preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0384] By “pharmaceutically acceptable” is included that the formulation is sterile and pyrogen free. The carrier, diluent, and / or excipient must be “acceptable” in the sense of being compatible with the vector and not deleterious to the recipients thereof. Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free; however, other acceptable carriers, diluents, and excipients may be used.

[0385] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

[0386] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0387] The pharmaceutical composition may comprise the AAV-ShH10 vector particle in infusion media, for example a sterile isotonic solution. Suitably, the pharmaceutical composition comprises an isotonic buffer (e.g. at about pH 7.4). In some embodiments, the pharmaceutical composition comprises phosphate buffered saline (PBS) buffer (e.g. pH 7.4). Optionally, the PBS is supplemented with about 200 mM NaCI. In some embodiments, the pharmaceutical composition comprises plasmalyte. In some embodiments, the pharmaceutical composition comprises about 0.001 % poloxamer 188.

[0388] The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. The pharmaceutical composition may have a total volume of from about 0.1 mL to about 100 mL. For example, the pharmaceutical composition may have a volume of from about 1 ml to about 100 ml, from about 5 ml to about 50 ml, from about 5 ml to about 25 ml, or from about 10 ml to about 25 ml. In some embodiments, the pharmaceutical composition has a volume of about 1 ml, about 2 ml, about 3 ml, about 4 ml, about 5 ml, about 6 ml, about 7 ml, about 8 ml, about 9 ml, about 10 ml, about 11 ml, about

[0389] 12 ml, about 13 ml, about 14 ml, about 15 ml, about 16 ml, about 17 ml, about 18 ml, about

[0390] 19 ml, about 20 ml, about 21 ml, about 22 ml, about 23 ml, about 24 ml, about 25 ml, about

[0391] 26 ml, about 27 ml, about 28 ml, about 29 ml, or about 30 ml. The pharmaceutical composition may comprise the AAV-ShH10 vector particle in an amount of from about 1x107vg / ml to about 1x1014vg / ml, from about 1x107vg / ml to about 5x1013vg / ml, from about 1x1010vg / ml to about 1x1013vg / ml, or from about 1x101° vg / ml to about 1x1012vg / ml.

[0392] The pharmaceutical composition may further comprise one or more other therapeutic agents.

[0393] The AAV-ShH10 vector particle or pharmaceutical composition may be provided in the form of a kit. Said kit may be used in the methods described herein, e.g., in the therapeutic methods as described herein. Preferably, said kits comprise instructions for use of the kit components.

[0394] Administration

[0395] The AAV-ShH10 vector particle may be administered in a manner appropriate for treating and / or preventing the diseases described herein. The quantity and frequency of administration will be determined by such factors as the condition of the subject, and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials. The pharmaceutical composition may be formulated accordingly.

[0396] The AAV-ShH10 vector particle may be administered parenterally, for example, intravenously, or by infusion techniques. The AAV-ShH10 vector particle may be administered in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0397] The AAV-ShH 10 vector particle may be administered systemically, for example by intravenous injection. In some embodiments, the AAV-ShH10 vector particle is administered by intravenous injection.

[0398] The AAV-ShH 10 vector particle may be administered locally, for example by targeting administration to the kidney. Suitably, the AAV-ShH 10 vector particle may be administered by injection into the renal artery, by intraparenchymal injection, by transparenchymal injection, by renal vein injection, or by ureteral or subcapsular injection. In some embodiments, the AAV- ShH10 vector particle is administered by injection into the renal artery.

[0399] The AAV-ShH10 vector particle may be administered in a single or in multiple doses. Particularly, the AAV-ShH10 vector particle may be administered in a single, one-off dose. The pharmaceutical composition may be formulated accordingly.

[0400] The AAV-ShH10 vector particle may be administered at varying doses (e.g. measured in vector genomes (vg) per kg). The physician in any event will determine the actual dosage which will be most suitable for any individual subject and it will vary with the age, weight and response of the particular subject. Typically, however, for AAV-ShH 10 vector particles, doses of 1010to 1014vg / kg, or 1011to 1013vg / kg may be administered.

[0401] In vitro and ex vivo methods

[0402] In one aspect, the present invention provides use of an AAV-ShH 10 vector particle for transducing human podocytes in vitro.

[0403] In another aspect, the present invention provides use of an AAV-ShH 10 vector particle for transducing human podocytes ex vivo.

[0404] In another aspect, the present invention provides an in vitro method for delivering one or more coding sequences to human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle comprising one or more coding sequences.

[0405] In another aspect, the present invention provides an ex vivo method for delivering one or more coding sequences to human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle comprising one or more coding sequences.

[0406] In another aspect, the present invention provides an ex vivo method for delivering one or more coding sequences to a kidney, the method comprising contacting the kidney with an AAV- ShH 10 vector particle comprising one or more coding sequences, wherein the AAV-ShH 10 vector particle transduces podocytes of the kidney.

[0407] Following delivery to the human podocytes, the AAV-ShH 10 vector particle may transduce the podocytes and express the one or more coding sequences in the podocytes. Expression of the one or more coding sequences may be determined by any suitable method, for example by enzyme-linked immunosorbent assay (ELISA) or by immunoblotting.

[0408] The in vitro methods of the invention may be used in in vitro assays. For example, the in vitro methods of the invention may be used to study expression of the one or more coding sequences in human podocytes and / or to study rescue of human podocytes, for example lacking a functional gene encoded by the AAV-ShH10 vector particle. In some embodiments, the human podocytes are conditionally immortalised human podocytes. In some embodiments, the human podocytes are mutant human podocytes (e.g. comprising a gene knockout).

[0409] The ex vivo methods of the invention may be used during ex vivo organ perfusion (see e.g. Yuzefovych, Y., et al., 2020. Frontiers in Immunology, 11 , p.265). For example, the ex vivo methods of the invention may be used to genetically engineer a human kidney prior to transplantation. The ex vivo methods of the invention may also be used in ex vivo assays. For example, to study kidney development (see e.g. Chen, T.L., et al., 2014. The Scientific World Journal, 2014(1), p.682189). In some embodiments, the human podocytes are in an ex vivo cultured intact kidney. In some embodiments, the kidney is an ex vivo cultured intact embryonic kidney.

[0410] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example: Skoog, D.A., et al. (2013) Fundamentals of Analytical Chemistry, 9th edition, Cengage learning; Walker J. M. (2009) The Protein Protocols Handbook, 3rd edition, Springer Nature; Green, M.R. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M., et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, A. J. (2013) DNA Sequencing Protocols, Humana Press; Nielsen, B.S. and Jones, J. (2021) In Situ Hybridization Protocols, Springer US; Herdewijn, P. (2010) Oligonucleotide Synthesis: Methods and Applications, Humana Press; and Luo, Y. (2019) CRISPR Gene Editing: Methods and Protocols, Springer New York. Each of these general texts is herein incorporated by reference.

[0411] EXAMPLES

[0412] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0413] Example 1 - Transduction of human / mouse podocytes and glomerular spheroids with AAV-ShH10 vector particles The objective of this study was to evaluate AAV serotypes that would provide the greatest transduction in the targeted cell / tissue (i.e. human podocytes). To assess transduction efficiency, in vitro studies were performed using human and mouse cells.

[0414] Results and discussion

[0415] Conditionally immortalised human and mouse podocytes were transduced with a range of AAV vectors encoding an eGFP transgene, each pseudotyped with a different capsid protein selected from AAV-LK03, AAV9, AAV-3B, AAV-ShH10 and AAV4. Various multiplicities of infection (MOIs) were tested, and cells were analysed 72 hours later by Fluorescent Activated Cell Sorting (FACS) and microscopy for eGFP expression. 72 hours was deemed a sufficient duration of time to allow cells to be successfully transduced and express appropriate levels of eGFP for detection. The results are shown in Figures 1 and 2.

[0416] Cells transduced at an MOI of 5E+5 viral particles per cell were used for microscopy assessment and demonstrated high transduction with AAV-LK03, AAV-3B and AAV-ShH10 pseudotyped particles (Figure 1A, C, D). Conversely, AAV4 pseudotyped particles showed very low transduction of cells relative to AAV-LK03, AAV-3B and AAV-ShH10 (Figure 1E) and AAV9 pseudotyped particles demonstrated no visible expression at all (Figure 1 B).

[0417] FACS analysis was performed on human podocytes transduced with MOIs of 1 E+2, 1 E+3 and 1 E+4 viral particles per cell. As shown in Figure 2A, AAV-LK03, AAV-3B and AAV-ShH10 pseudotyped particles demonstrated similar transduction levels with MOIs of 1 E+2, 1 E+3 and 1 E+4 particles per cell, while AAV9 pseudotyped particles demonstrated minimal levels of transduction. It was concluded from these data that AAV-LK03, AAV-3B and AAV-ShH10 pseudotyped particles were the optimal transducers of human podocytes in vitro compared to the other capsids.

[0418] FACS analysis was performed on mouse podocytes transduced with MOIs of 5E+4, 1 E+5 and 5E+5 viral particles per cell. As shown in Figure 2B, AAV-ShH10 pseudotyped particles can transduce mouse podocytes efficiently with nearly 100% transduction observed. Conversely, all other capsids demonstrated relatively low eGFP expression.

[0419] To further complement the initial experiments, 3D spheroids comprising of podocytes and glomerular endothelial cells (GENCs) were generated to mimic the glomerular structure more accurately. These were transduced with AAV vectors and analysed by microscopy after 72 hours for eGFP expression. The results are shown in Figures 3 and 4.

[0420] The glomerular spheroids were transduced with 1 E+5 viral particles per cell. The cell number was estimated to be approximately 20,000 cells per spheroid. Transductions of human spheroids with AAV-LK03, AAV-3B and AAV-ShH10 pseudotyped particles demonstrated high levels of transduction as determined by microscopy (Figure 3). AAV9 pseudotyped particles also demonstrated some transduction of spheroids, although expression is significantly lower compared to other capsids.

[0421] Transductions of mouse glomerular spheroids with AAV-ShH10 pseudotyped particles demonstrated very high transduction (Figure 4). AAV-LK03, AAV-3B and AAV9 pseudotyped particles demonstrated very low transduction of mouse spheroids. These results correlate with the experiments on cells in 2D culture.

[0422] Together, these in vitro analyses demonstrate that AAV-LK03, AAV-3B and AAV-ShH10 pseudotyped particles all transduce human podocytes equally well. AAV-ShH10 was additionally permissive in mouse podocytes in both 2D and 3D cultures. In contrast, AAV9 pseudotyped particles were poor transducers of human podocytes.

[0423] Materials and methods

[0424] AAV particles were obtained from an accredited AAV vector manufacturer (VectorBuilder GmbH, Neu-lsenburg, Germany). All studies were performed using AAV vectors containing the same expression cassette, comprising of a GFP transgene driven by a CMV promoter, followed by a WPRE sequence to increase transgene expression and a bGH PolyA tail to promote transcript stability.

[0425] AAV particles were produced via triple transfection of HEK293T cells with (i) plasmids encoding the AAV2 Rep genes with varying capsids, (ii) the ITR-containing transgene plasmid and (iii) a helper plasmid encoding adenoviral helper proteins. Capsids used in this study were AAV-LK03, AAV9, AAV-3B, AAV-ShH10 and AAV4. All capsid plasmids contained the same backbone sequence, only differing in their Cap sequence.

[0426] Podocytes were maintained in RPMI (with Glutamine) (Sigma Aldrich) supplemented with 10% FBS (Sigma Aldrich) and 5% ITS (Insulin, Transferrin, Selenium) (ThermoFisher) solution. Cells were cultured at 33°C and grown until -90% confluency, at which point they were passaged into new flasks at a split ratio of 1 :5. GENCs were maintained in EBM-2 basal medium supplemented with the EBM-2 Bulletkit (Lonza). Cells were cultured at 33°C and grown until -80-90% confluency, at which point they were passaged into new flasks at a split ratio of 1 :4. For transduction, cells were seeded into 12-well plates and left to adhere overnight. AAV particles were added the following day using a range of MOIs. Cell culture media was changed the day after transduction and then every 2-3 days until analysis. Cells were imaged on a Leica DMC4500 microscope. For each cell type exposure was maintained at the same level across all wells transduced with various AAV particles. Exposure levels were set using non transduced controls to remove background fluorescence.

[0427] For FACS analysis, cells were harvested from plates using trypsin. The trypsin was neutralised using media containing 10% serum. Cells were then centrifuged at 1 ,000g for 2 minutes and re-suspended in PBS. Centrifugation was repeated to wash away residual media and the cell pellets were re-suspended in Cells Staining Buffer to be run on the flow cytometer. The FACS voltages were set using non transduced samples to set forward and side scatter and identification of the cell population. Forward Scatter (Height) was plotted against Forward Scatter (Area) to allow exclusion of doublets. eGFP positive cells were gated on the FITC channel (B530 / 30), with the non-transduced control allowing no greater than 0.5% positive cells as a background fluorescence.

Claims

CLAIMS1. A method for transduction of human podocytes, the method comprising contacting the podocytes with an AAV-ShH 10 vector particle.

2. A method for delivering a coding sequence to podocytes of a human subject, the method comprising administering an AAV-ShH 10 vector particle comprising the coding sequence to said subject, thereby transducing and expressing the coding sequence in the podocytes of said subject.

3. A method for treating and / or preventing a kidney disease, the method comprising administering an AAV-ShH 10 vector particle to a human subject in need thereof, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

4. An AAV-ShH 10 vector particle for use in gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

5. An AAV-ShH 10 vector particle for use in treating and / or preventing a kidney disease in a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

6. Use of an AAV-ShH 10 vector particle for the manufacture of a medicament for gene therapy of a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

7. Use of an AAV-ShH 10 vector particle for the manufacture of a medicament for treating and / or preventing a kidney disease in a human subject, wherein the AAV-ShH 10 vector particle transduces podocytes of the subject.

8. Use of an AAV-ShH 10 vector particle for transducing human podocytes in vitro or ex vivo.

9. The method according to any of claims 1-3, the AAV-ShH 10 vector particle for use according to claim 4 or 5, or the use according to any of claims 6-8, wherein the AAV-ShH 10 vector particle is encapsidated by ShH10 VP1 , VP2, and VP3 capsid proteins.

10. The method according to claim 9, the AAV-ShH 10 vector particle for use according to claim 9, or the use according to claim 9, wherein the ShH10 VP1 capsid protein comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

11. The method according to claim 9 or 10, the AAV-ShH 10 vector particle for use according to claim 9 or 10, or the use according to claim 9 or 10, wherein the ShH10 VP2 capsid proteincomprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 2.

12. The method according to any of claims 9-11 , the AAV-ShH10 vector particle for use according to any of claims 9-11 , or the use according to any of claims 9-11 , wherein the ShH 10 VP3 capsid protein comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 3.

13. The method according to any of claims 9-12, the AAV-ShH10 vector particle for use according to any of claims 9-12, or the use according to any of claims 9-12, wherein the AAV- ShH10 vector particle has a total number of 60 VP1 , VP2, and VP3 subunits per capsid and comprises ShH 10 VP1 , VP2, and VP3 capsid proteins in a ratio of 1 : 1:10.

14. The method according to any of claims 1-3 or 9-13, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-13, or the use according to any of claims 6-8 or 9-13, wherein the AAV-ShH10 vector particle comprises an AAV genome that is a derivative of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

15. The method according to any of claims 1-3 or 9-14, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-14, or the use according to any of claims 6-8 or 9-14, wherein the AAV-ShH10 vector particle encodes a therapeutic gene product.

16. The method according to any of claims 1-3 or 9-15, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-15, or the use according to any of claims 6-8 or 9-15, wherein the AAV-ShH10 vector particle encodes a polypeptide associated with a kidney disease, or an N-terminal part or C-terminal part thereof.

17. The method according to any of claims 1-3 or 9-16, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-16, or the use according to any of claims 6-8 or 9-16, wherein the AAV-ShH10 vector particle comprises a coding sequence encoding a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, CFHL1 , C1 INH, C4BP, MASP2, C3, C5aR1 , C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1 , ARHGAP24, ARGHDIA, CD151 , CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1 B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1 , Smad7, TP53RK, TPRKB, VDR, WDR73, WT1 , ZMPSTE24, APOL1 , NPHS1 , TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1 , ANLN, CRB2, ITGA3, KANK1 , KANK4, MAGI2, MYO1 E, OCRL, PTPRO, SMARCAL1 , SYNPO, TBC1 D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide, or an N-terminal part or C-terminal part thereof.

18. The method according to any of claims 1-3 or 9-17, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-17, or the use according to any of claims 6-8 or 9-17, wherein the AAV-ShH10 vector particle comprises a podocyte-specific promoter.

19. The method according to any of claims 1-3 or 9-18, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-18, or the use according to any of claims 6-8 or 9-18, wherein the AAV-ShH10 vector particle comprises a NPHS1 promoter or a NPHS2 promoter.

20. The method according to claim 18 or 19, the AAV-ShH10 vector particle for use according to claim 18 or 19, or the use according to claim 18 or 19, wherein the AAV-ShH10 vector particle comprises one or more further regulatory sequences.21 . The method according to any of claims 2-3 or 9-20, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-20, or the use according to any of claims 6-7 or 9-20, wherein the AAV-ShH10 vector particle is administered to the human subject in the form of a pharmaceutical composition comprising a therapeutically effective amount of the AAV-ShH10 vector particle and a pharmaceutically acceptable carrier, diluent or excipient.

22. The method according to claim 21 , the AAV-ShH10 vector particle for use according to claim 21 , or the use according to claim 21 , wherein the pharmaceutical composition has a total volume of from about 0.1 mL to about 100 mL.

23. The method according to any of claims 2-3 or 9-22, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-22, or the use according to any of claims 6-7 or 9-22, wherein the AAV-ShH10 vector particle is administered to the human subject by injection into the renal artery, by intraparenchymal injection, by transparenchymal injection, by renal vein injection, or by ureteral or subcapsular injection.

24. The method according to any of claims 2-3 or 9-23, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-23, or the use according to any of claims 6-7 or 9-23, wherein the AAV-ShH10 vector particle is administered to the human subject by intravenous injection.

25. The method according to any of claims 2-3 or 9-24, the AAV-ShH10 vector particle for use according to any of claims 4-5 or 9-24, or the use according to any of claims 6-7 or 9-24, wherein the human subject has or is at risk of a genetic kidney disease or a complement- mediated kidney disease.

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