Methods of delivering viral vectors to kidneys

Abstract

The present invention relates to a method of delivering a viral vector to a kidney of a subject, the method comprising: (a) inserting a catheter into a renal artery; (b) optionally inflating the balloon to occlude the renal artery; and (c) injecting or infusing the viral vector into the renal artery via the catheter. The invention also relates to a viral vector for use in therapy, wherein the viral vector is delivered by said method.

Description

Field of the Invention

[0001] The present invention relates to methods for delivering viral vectors to the kidney. The present invention also relates to viral vectors for use in therapy, wherein the viral vector is delivered by the method. Background of the Invention

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

[0003] The GBM is composed of a highly cross-linked macromolecular network of type IV collagen, proteoglycans, and laminin. Genetic forms of glomerular diseases 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 in the collagen IV α345 network of the basement membrane. In all individuals in continental Europe and the United States, approximately 1 in 5,000 - 10,000 people are affected by Alport syndrome. The condition typically presents in childhood and is associated with a range of phenotypes, which include progressive loss of kidney function and may also include hearing loss and eye abnormalities. Other GBM-related diseases include Pierson syndrome and nail-patella syndrome (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

[0004] Podocytes are also considered key cells in the progression of glomerular diseases. Podocytes are highly specialized mesoderm-derived cells and are found only in the glomeruli. They exhibit unique features such as foot processes and slit diaphragms, which are crucial for glomerular filtration. Podocyte-related genetic glomerular diseases include nephrotic syndrome, Frasier syndrome, and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, and Epstein and Fechtner syndromes. (Chiang, C.K. and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539).

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

[0006] To maximize the potential of gene therapy, an optimal method for delivering viral vectors to glomerular cells (such as podocytes) is needed. However, the kidney is a difficult target for conventional intravenous delivery because the filtration selectivity of the glomerulus will prevent most molecular therapies from entering the kidney from the blood. In addition, although multiple direct kidney injection methods have been tested with viral vectors (such as adeno-associated virus (AAV) vectors), the efficiency of gene delivery has been inconsistent and is generally still too small to effectively treat diseases (see, for example, Rubin, J.D. and Barry, M.A., 2020. Molecular diagnosis & therapy, 24(4), pp.375-396).

[0007] Accordingly, there is a need for an improved method for delivering viral vectors to glomerular cells (such as podocytes). Summary of the Invention

[0009] The present inventors have developed an improved method for delivering viral vectors to the kidney (specifically glomerular cells, such as podocytes) via direct renal artery injection. This method can be performed in a minimally invasive manner and has improved safety compared to prior art direct kidney injection methods.

[0010] In addition, the present inventors have unexpectedly shown that direct renal artery injection of viral vectors can result in a significant increase in transgene expression and viral copy number in the kidney (specifically glomeruli and podocytes) when compared to intravenous injection. In addition, direct renal artery injection of viral vectors can result in transgene expression localized to the kidney with minimal expression in other tissues (such as the liver).

[0011] In one aspect, the present invention provides a method for delivering a viral vector to the kidney, the method comprising inserting a catheter into a renal artery and delivering the viral vector into the renal artery via the catheter.

[0012] The method can be a minimally invasive procedure. Suitably, the method does not include the step of occluding the renal vein. Suitably, the method does not include the step of inserting a catheter into the renal vein and / or does not include the step of directly inserting a catheter into the aorta. Suitably, the method does not include forming a closed circuit through the kidney. Suitably, the method does not include the step of clamping the renal artery, renal vein or aorta. Suitably, the total renal ischemia time is about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 10 minutes to about 30 minutes, about 15 to about 25 minutes or about 5 minutes.

[0013] The catheter can be inserted into the renal artery by any suitable method. In a preferred embodiment, the catheter is inserted into the renal artery via a percutaneous approach. Suitably, the percutaneous approach is via the carotid artery or via the femoral artery. Suitably, the insertion of the catheter via the percutaneous route is facilitated by a sheath. Suitably, the catheter is inserted into the renal artery via a guide wire.

[0014] The viral vector can be delivered to the renal artery by any suitable method. Suitably, the viral vector is injected or infused into the renal artery. In some embodiments, the viral vector is infused into the renal artery using an infusion pump under non-flow conditions. Suitably, the viral vector is delivered to the renal artery within about 1 minute to about 30 minutes. In some embodiments, the viral vector is delivered to the renal artery within about 1 minute to about 5 minutes, optionally about 2 minutes or about 4 minutes. In some embodiments, the viral vector is delivered to the renal artery within about 15 minutes to about 30 minutes or about 15 minutes to about 20 minutes, optionally about 17 minutes.

[0015] In some embodiments, the catheter is an occlusion balloon catheter. In some embodiments, the method includes the step of inflating the balloon to occlude the renal artery. Suitably, the renal artery is occluded for about 1 minute to about 25 minutes. In some embodiments, the renal artery is occluded for about 2 minutes to about 10 minutes, optionally about 5 minutes. In some embodiments, the renal artery is occluded for about 15 minutes to about 25 minutes or about 15 minutes to about 20 minutes, optionally about 20 minutes.

[0016] In one embodiment, the present invention provides a method for delivering a viral vector to a subject's kidney, the method comprising:

[0017] (a) inserting a catheter into the renal artery of the kidney;

[0018] (b) optionally inflating a balloon to occlude the renal artery; and

[0019] (c) injecting or infusing the viral vector into the renal artery via the catheter,

[0020] wherein the method is a minimally invasive procedure and wherein the method does not include the step of inserting the catheter into the renal vein of the kidney.

[0021] In one embodiment, the present invention provides a method for delivering a viral vector to a subject's kidney, the method comprising: inserting a catheter into the renal artery of the kidney and injecting or infusing the viral vector into the renal artery via the catheter, wherein the method does not include the step of occluding the renal artery or the renal vein of the kidney and wherein the method does not include the step of clamping the aorta.

[0022] The method of the present invention can result in the delivery of a viral vector to the kidney. For example, the method can result in the delivery of a viral vector to the renal cortex and / or renal medulla, particularly the renal cortex. The method can result in the delivery of a viral vector to the glomeruli. The method can result in the delivery of a viral vector to the renal podocytes. The method can result in the kidney-specific delivery of a viral vector.

[0023] The subject can be any suitable subject. The subject can be a human subject. The human subject can be an adult, adolescent, or child. The subject can have a kidney disease or can be at risk of a kidney disease. The subject can have a glomerular disease or can be at risk of a glomerular disease. The subject can have a hereditary glomerular disease or can be at risk of a hereditary glomerular disease, optionally wherein the subject has a podocyte-related hereditary glomerular disease or is at risk of a podocyte-related hereditary glomerular disease.

[0024] The viral vector can be delivered at any suitable dose. Suitably, the viral vector is delivered at a dose of from about 1x10 6 vg / kg to about 1x10 14 vg / kg or from about 1x10 6 vg / kg to about 1x10 13 vg / kg. In some embodiments, the viral vector is delivered at a dose of from about 1x10 9 vg / kg to about 1x10 12 vg / kg. In some embodiments, the viral vector is delivered at a dose of from about 3x10 9 vg / kg to about 3x10 11 vg / kg. Suitably, the viral vector is delivered at a dose of from about 1x10 8 vg to about 1x10 15 vg or from about 1x10 8 vg to about 5x10 14 vg. In some embodiments, the viral vector is delivered at a dose of from about 1x10 11 vg to about 1x10 14 vg. In some embodiments, the viral vector is delivered at a dose of from about 2x10 11 vg to about 2x10 13 vg. In some embodiments, the viral vector is delivered at a dose of from about 5x10 11 vg to about 2x10 13 vg. In some embodiments, the viral vector is delivered at a dose of from about 1x10 12 vg to about 2x10 13 vg. In some embodiments, the viral vector is delivered at a dose of from about 1x10 13 vg.

[0025] The viral vector can be any suitable viral vector. Suitably, the viral vector is capable of transducing renal cells, optionally wherein the vector is capable of specifically transducing renal cells. Suitably, the viral vector is capable of transducing glomerular cells, optionally wherein the vector is capable of specifically transducing glomerular cells. Suitably, the viral vector is capable of transducing podocytes, optionally wherein the vector is capable of specifically transducing glomerular podocytes. Suitably, the viral vector is selected from adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

[0026] In a preferred embodiment, the viral vector is an adeno-associated virus (AAV) vector particle. In some embodiments, the viral vector is in the form of an AAV vector particle capsidated with the LK03, AAV3B, or AAV9 capsid protein. In some embodiments, the viral vector is in the form of an AAV vector particle capsidated with the LK03 capsid protein.

[0027] The viral vector may comprise any suitable protein - coding sequence. Suitably, the protein - coding sequence encodes a therapeutic protein, preferably wherein the protein - coding sequence encodes a polypeptide associated with a hereditary glomerular disease, optionally a polypeptide associated with a podocyte - related hereditary glomerular disease. In some embodiments, the protein - coding sequence encodes COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL - 1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3 or VEGFC polypeptide. In some embodiments, the protein - coding sequence encodes NPHS2 or a fragment and / or variant thereof; a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof; or CFI, CFH or FHL - 1, or a fragment and / or variant thereof. In some embodiments, the protein - coding sequence does not encode a gene - editing agent. In some embodiments, the protein - coding sequence does not encode a nuclease. In some embodiments, the protein - coding sequence does not encode Cas9.

[0028] The protein - coding sequence may be operably linked to any promoter. In some embodiments, the protein - coding sequence is operably linked to a kidney - specific promoter. In some embodiments, the protein - coding sequence is operably linked to a podocyte - specific promoter. In some embodiments, the protein - coding sequence is operably linked to an NPHS1 promoter or an NPHS2 promoter. In some embodiments, the protein - coding sequence is operably linked to a minimal NPHS1 promoter. In some embodiments, the protein - coding sequence is operably linked to a constitutive promoter. In some embodiments, the protein - coding sequence is operably linked to a CMV promoter.

[0029] The protein-coding sequence can be operably linked to one or more additional regulatory elements. Suitably, the protein-coding sequence is operably linked to a post-transcriptional regulatory element and / or a polyadenylation sequence. In some embodiments, the protein-coding sequence is operably linked to the woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the protein-coding sequence is operably linked to a polyadenylation signal, such as the bovine growth hormone polyadenylation signal.

[0030] The viral vector can be in the form of a viral vector preparation. The viral vector preparation can contain any suitable amount of the viral vector and can be formulated in any suitable manner. Suitably, the viral vector preparation contains from about 1x10 7 vg / ml to about 1x10 14 vg / ml or from about 1x10 7 vg / ml to about 5x10 13 vg / ml of the viral vector. In some embodiments, the viral vector preparation contains from about 1x10 10 vg / ml to about 1x10 13 vg / ml of the viral vector. In some embodiments, the viral vector preparation contains from about 1x10 10 vg / ml to about 1x10 12 vg / ml of the viral vector. Suitably, the viral vector preparation contains an isotonic buffer, such as phosphate-buffered saline (PBS) buffer or plasmalyte. In some embodiments, the viral vector preparation contains about 0.001% poloxamer 188. Suitably, the viral vector preparation has a volume of 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.

[0031] The viral vector can be delivered to a single kidney or both kidneys of a subject. In some embodiments, the method of the invention is performed once to deliver the viral vector to a single kidney of a subject. In some embodiments, the method of the invention is performed twice to deliver the viral vector to both kidneys of a subject.

[0032] In one aspect, the invention provides a viral vector for therapy, wherein the viral vector is delivered by the method according to the invention.

[0033] In one aspect, the invention provides a viral vector for treating or preventing kidney diseases, wherein the viral vector is delivered by the method according to the invention.

[0034] In one aspect, the invention provides the use of a viral vector for the preparation of a medicament, wherein the medicament is delivered by the method according to the invention.

[0035] In one aspect, the present invention provides the use of a viral vector for the preparation of a medicament for the treatment or prevention of kidney diseases, wherein the medicament is delivered by the method according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 - Schematic diagram of an AAV vector encoding podocin under the control of the hNPHS1 promoter

[0037] ITR: Inverted terminal repeat; hNPHS1 promoter: human full-length nephrin promoter; HA: Hemagglutinin; WPRE: Woodchuck post-transcriptional regulatory element; bGH: Bovine growth hormone polyA signal.

[0038] Figure 2 - Transcriptomic analysis for assessing transduction efficiency

[0039] Pigs 3 - 6 are pigs treated with dRAi. Pigs 1 and 2 are controls. AAV: Adeno-associated virus; bGH: Bovine growth hormone; LKC: Left kidney cortex; LKM: Left kidney medulla; RKC: Right kidney cortex; RKM: Right kidney medulla.

[0040] Figure 3 - Co-localization study of nephrin and HA-tagged podocin (LKC and RKC)

[0041] (A) shows single-channel images of nephrin (left panel) and HA-tagged podocin (middle panel), and the corresponding composite image showing co-localization of nephrin and HA-tagged podocin (right panel). (B) Enlarged views of the designated areas demonstrate the expression of HA-podocin in podocytes, as shown by co-localization with the podocyte marker nephrin. HA: Hemagglutinin; LKC: Left kidney cortex; RKC: Right kidney cortex.

[0042] Figure 4 - Schematic diagram of an AAV vector encoding eGFP under the control of the CMV promoter

[0043] CMV: Cytomegalovirus; bGH: Bovine growth hormone; eGFP: Enhanced green fluorescent protein; ITR: Inverted terminal repeat; WPRE: Woodchuck post-transcriptional regulatory element.

[0044] Figure 5 - Transcriptomic analysis of mRNA GFP expression (kidneys injected with rAAV vs. non-injected kidneys) and biodistribution (colon, liver, pancreas)

[0045] GFP: Green fluorescent protein; IV: Intravenous; r.a.: Direct renal artery injection.

[0046] Figure 6-Transcriptomic analysis of the expression and biodistribution of -mRNA GFP in porcine kidneys (colon, liver, pancreas)

[0047] GFP: Green Fluorescent Protein; IV: Intravenous; LKC: Left Kidney Cortex; LKM: Left Kidney Medulla; r.a.: Direct Renal Artery Injection; RKC: Right Kidney Cortex; RKM: Right Kidney Medulla.

[0048] Figure 7 -Double immunofluorescence (anti-GFP) of porcine left kidney cortex

[0049] Composite images showing the co-localization of GFP (Green Fluorescent Protein) and nephrin in the renal cortex (63x magnification). dRAi: Direct Renal Artery Injection; IV: Intravenous.

[0050] Figure 8 -Double immunofluorescence (anti-GFP and anti-nephrin) of porcine right kidney cortex

[0051] Composite images showing the co-localization of GFP (Green Fluorescent Protein) and nephrin in the renal cortex (63x magnification). dRAi: Direct Renal Artery Injection; IV: Intravenous.

[0052] Figure 9 -Highly magnified expression within glomerular ultrastructure

[0053] Composite images showing the co-localization of GFP (Green Fluorescent Protein), nephrin, and DAPI (4’,6-diamidino-2-phenylindole) within glomerular ultrastructure.

[0054] Figure 10 -Double immunofluorescence (anti-GFP) of porcine liver

[0055] Composite images showing the co-localization of GFP (Green Fluorescent Protein) and DAPI (4’,6-diamidino-2-phenylindole) in liver tissue. Representative samples from each animal were randomly selected since all images in the high-dose and low-dose dRAi treatment groups were GFP-negative. dRAi: Direct Renal Artery Injection; IV: Intravenous.

[0056] Figure 11 -Schematic diagram of AAV vector and study design

[0057] (A) Schematic diagram of the AAV vector encoding GFP under the control of the hNPHS1 (full-length) promoter. ITR: Inverted Terminal Repeat; hNPHS1(FL) promoter: Human full-length nephrin promoter; WPRE: Woodchuck Post-Transcriptional Regulatory Element; bGH: Bovine Growth Hormone Poly A signal. (B) Schematic diagram of the study design.

[0058] Figure 12- Biodistribution of tissue samples by qPCR

[0059] AAV genomes detected per milligram (mg) of tissue after direct renal artery injection or IV injection with (dRAi+O) or without (dRAi) renal artery occlusion. (A) Treated kidney; and (B) Untreated kidney.

[0060] Figure 13 - RNAscope in situ hybridization (dRAi) vs. IV

[0061] Paraffin-embedded formalin-fixed tissues were evaluated by RNAscope in situ hybridization for the local presence of AAV mRNA in renal cortex samples after direct renal artery injection (dRAi) or IV injection without renal artery occlusion. Pigs received (A) dRAi with 1x10 12 vg; (B) dRAi with 5x10 12 vg; (C) dRAi with 1x10 13 vg; or (D) IV with 1x10 13 vg.

[0062] Figure 14 - RNAscope in situ hybridization (dRAi+O) vs. IV

[0063] Paraffin-embedded formalin-fixed tissues were evaluated by RNAscope in situ hybridization for the local presence of AAV mRNA in renal cortex samples after direct renal artery injection (dRAi+O) or IV injection with renal artery occlusion. Pigs received (A) dRAi+O with 1x10 13 vg; (B) dRAi+O with 2x10 13 vg; or (C) IV with 1x10 13 vg.

[0064] Figure 15 - Schematic of AAV vectors and study design

[0065] (A) Schematic of an AAV vector encoding podocin under the control of the hNPHS1 promoter. ITR: Inverted terminal repeat; hNPHS1: Human full-length nephrin promoter; hPodocin(WT)-HA: Wild-type human podocin with a hemagglutinin tag; WPRE: Woodchuck post-transcriptional regulatory element; bGH: Bovine growth hormone polyA signal. (B) Schematic of the study design.

[0066] Figure 16 - Biodistribution of tissue samples by qPCR

[0067] AAV genomes detected per milligram (mg) of tissue for untreated control pigs (UTC) and pigs treated with PS0438. (A) Treated kidney; (B) Untreated kidney; (C) Liver; and (D) Spleen.

[0068] Figure 17 -RNAscope in situ hybridization (dRAi+O)

[0069] Paraffin-embedded formalin-fixed tissues were evaluated by RNAscope in situ hybridization for the local presence of AAV mRNA in renal cortex samples after direct renal artery injection (dRAi+O) with renal artery occlusion. (A) Treated kidney and (B) Untreated kidney.

[0070] Figure 18 -Immunofluorescence of HA-tagged podocin and nephrin

[0071] Immunofluorescence (IF) analysis was performed on sections obtained from OCT blocks of renal cortex. Sections were stained with DAPI and antibodies targeting WT-1, HA, and nephrin. (A) Composite image of two glomeruli and corresponding single-channel images showing (B) HA-tagged podocin (green) and (C) nephrin. (D) Composite image of one glomerulus and corresponding single-channel images showing (E) HA-tagged podocin (green) and (F) nephrin.

[0072] Figure 19 -Podocin ELISA of renal cortex

[0073] Podocin was detected in renal cortex samples by ELISA for untreated control pigs (UTC) and pigs treated with PS0438, in nanograms per milligram of total protein.

[0074] Figure 20 -Double immunofluorescence of glomeruli

[0075] (A) shows a composite image of glomeruli stained with DAPI and an antibody targeting nephrin. GFP expression is absent in the negative control (PBS, left panel) and is higher after direct renal artery injection (dRAi, right panel) compared to intravenous injection (iv, middle panel). (B) shows a composite image of glomeruli stained with DAPI and an antibody targeting nephrin (left panel) or PDGFb (right panel). GFP expression co-localizes with nephrin (NPHS1) and PDGFb, confirming its localization to podocytes and mesangial cells, respectively.

[0076] Figure 21 -Western blot analysis

[0077] (A) Western blot analysis and (B) densitometry of mouse liver samples showed higher GFP expression with AAV vector administration by IV as compared to administration of AAV vector by direct renal artery injection (dRAi). DETAILED DESCRIPTION OF THE INVENTION

[0079] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used to practice or test the embodiments of the present disclosure. Those skilled in the art will understand that they can combine all features of the present invention disclosed herein without departing from the scope of the disclosed invention.

[0080] 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.

[0081] As used herein, the term "comprising" (comprising, comprises, comprised of) is synonymous with "including" (including, includes) or "containing" (containing, contains), and is inclusive or open-ended and does not exclude additional, unrecited members, elements or steps. The term "comprising" (comprising, comprises, comprised of) also includes the term "consisting of".

[0082] Numeric ranges include the numbers defining the range. As used herein, the term "about" means approximately, roughly, generally 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 stated numerical value. Generally, the terms "about" and "approximately" can be used herein to modify numerical values that are 10% above and below the stated value.

[0083] Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

[0084] The publications discussed herein are provided only 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.

[0085] All publications mentioned in the specification are incorporated herein by reference.

[0086] Glomerular and Podocyte Gene Therapy

[0087] The glomerulus is the filtering unit of the kidney. It filters approximately 180 liters of plasma per day, and the healthy glomerular filtration barrier has the remarkable ability to retain approximately 99.9% of large proteins (including albumin) throughout our lifetime without clogging. The afferent arteriole enters the glomerular capillary network (where filtration occurs), and blood leaves the glomerulus via the efferent arteriole. The glomerular filtration barrier (GFB) consists of 3 main layers: glomerular endothelial cells, the glomerular basement membrane (GBM), and podocytes.

[0088] Podocytes, the third layer of the GFB, play a key role in the maintenance of the GFB. Podocytes are highly specialized cells, composed of a cell body, primary processes, secondary processes, and foot processes that interdigitate with foot processes of adjacent podocytes to form slit diaphragms. Podocytes form an effective and dynamic sieve, which is mainly attributed to the integrity of the slit diaphragm.

[0089] Gene therapy targeting the glomerulus is challenging. For example, although lentivirus may be useful in transducing tubules, it has not shown any in vivo transduction of the glomerulus to date. Additionally, initial attempts to deliver adenovirus via the renal artery or retrogradely via the ureter seem to mainly result in tubule or interstitial transduction. Initial studies of the rodent kidney using AAV2 have shown mainly tubule transduction, with no expression in the glomerulus.

[0090] Delivery methods

[0091] In one aspect, the present invention provides a method for delivering a viral vector to the kidney via direct renal artery injection. The method may include the steps of inserting a catheter into the renal artery and injecting or infusing the viral vector into the renal artery via the catheter. In the context of the present invention, the term "delivery" may be used interchangeably with the term "administration".

[0092] The method is preferably an in vivo method (i.e., not an ex vivo method). The method can be a minimally invasive procedure, i.e., the method can be a minimally invasive approach. As used herein, "minimally invasive procedure, minimally invasive surgeries" may refer to surgical techniques that limit the size of the incision required, thereby reducing wound healing time, associated pain, and the risk of infection (see, for example, Jaffray, B., 2005. Archives of disease in childhood, 90(5), pp.537-542). In contrast, the incisions made during "open surgery" sometimes leave large wounds that can be painful and take a long time to heal. Many medical procedures are referred to as minimally invasive, including percutaneous procedures. In a preferred embodiment, the method does not include open surgery.

[0093] In some embodiments, the method does not include inserting more than one catheter. In some embodiments, the method does not include the step of inserting a catheter into the renal vein. In some embodiments, the method does not include the step of inserting a catheter directly into the aorta (e.g., into the infrarenal aorta).

[0094] In preferred embodiments, the method does not include any clamping steps (e.g., clamping the renal artery, renal vein, or aorta). In some embodiments, the method does not include the step of clamping the renal artery. In some embodiments, the method does not include the step of clamping the renal vein. In some embodiments, the method does not include the step of clamping the aorta (e.g., the infrarenal aorta).

[0095] In some embodiments, the method has a total renal ischemia time of about 1 hour or less. As used herein, "renal ischemia time" can refer to the time from severing or reducing the blood supply to the kidney to restoring the normal blood supply to the kidney. Reduced renal ischemia can result in reduced renal injury or a reduced risk of renal injury.

[0096] In some embodiments, the total renal ischemia time is about 60 minutes or less, about 50 minutes or less, about 40 minutes or less, about 30 minutes or less, about 25 minutes or less, about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less. In some embodiments, the total renal ischemia time is about 0 minutes.

[0097] In some embodiments, the total renal ischemia time is about 10 minutes to about 30 minutes, about 15 minutes to about 25 minutes, or about 20 minutes.

[0098] In some embodiments, the total renal ischemia time is about 5 minutes.

[0099] Step (a): Insertion of the catheter

[0100] In the method of the present invention, a catheter is inserted into the renal artery.

[0101] As used herein, a "catheter" can refer to a thin tube made of a medical-grade material that can be inserted into the body for a surgical procedure. Although available catheters have different levels of stiffness depending on the application, in most uses, the catheter is a thin flexible tube (soft catheter). Suitably, the catheter is a percutaneous catheter. In some embodiments, the catheter is an occlusion balloon catheter.

[0102] As used herein, "inserted into the renal artery" means that after the catheter is inserted, the distal end of the catheter is located within the renal artery. This method is different from the method in which the catheter is located below the renal artery. The renal artery typically arises from the left medial side of the abdominal aorta at a 90° angle, immediately below the superior mesenteric artery, and has a radius of approximately 0.25 cm. Before reaching the renal hilum, each renal artery divides into four or five branches. The term "renal artery" can refer to any of these branches.

[0103] The catheter can be inserted into the renal artery through any suitable route. In a preferred embodiment, the catheter is inserted into the renal artery via a percutaneous route. This route is compatible with minimally invasive surgery. The percutaneous route can be any suitable route, such as via the carotid artery or via the femoral artery. In some embodiments, the catheter is inserted into the renal artery via the carotid artery. In some embodiments, the catheter is inserted into the renal artery via the (common) femoral artery. The standard procedures for inserting a catheter via a percutaneous route are known to those skilled in the art. For example, catheterization of the carotid artery or (common) femoral artery can be performed, a sheath can be introduced, and a guide wire can be introduced to facilitate the introduction of the catheter into the renal artery.

[0104] In some embodiments, the method includes the step of inserting a catheter into the carotid artery or (common) femoral artery. In some embodiments, the insertion of the catheter via a percutaneous path is facilitated by a sheath. In some embodiments, the method includes the step of introducing a sheath into the carotid artery or (common) femoral artery. As used herein, a "sheath" can refer to a short hollow tube that can be introduced into a blood vessel to facilitate catheter insertion. The sheath can be kept in place until the surgery is completed and the catheter is removed. In some embodiments, the catheter is inserted into the renal artery via a guide wire. In some embodiments, the method includes the step of introducing a guide wire into the renal artery. As used herein, a "guide wire" can refer to a thin filament used to guide the placement of a catheter within a blood vessel.

[0105] In some embodiments, the step of inserting the catheter into the renal artery (a) includes:

[0106] (a1) catheterizing the carotid artery or (common) femoral artery;

[0107] (a2) inserting a sheath into the carotid artery or (common) femoral artery;

[0108] (a3) inserting a guide wire into the renal artery; and

[0109] (a4) inserting the catheter into the renal artery.

[0110] Step (b): Occlusion of the blood vessel

[0111] The method can optionally include the step of occluding the renal artery. This can increase the residence time of the viral vector in the kidney, thereby increasing viral transduction.

[0112] As used herein, "occlusion" of a blood vessel can refer to completely blocking blood flow through the blood vessel. The occlusion can be examined by any suitable method. For example, by injecting or infusing a contrast agent and imaging the blood flow via angiography. The occlusion can be performed by any suitable method, preferably by inflating a balloon. This method is compatible with minimally invasive surgery.

[0113] In some embodiments, the method comprises the steps of:

[0114] (a) Inserting a balloon catheter into the renal artery;

[0115] (b) Inflating the balloon to occlude the renal artery; and

[0116] (c) Injecting or infusing a viral vector into the renal artery via the catheter.

[0117] In some embodiments, the renal artery is occluded for about 1 minute or longer, about 2 minutes or longer, about 3 minutes or longer, about 4 minutes or longer, about 5 minutes or longer, about 6 minutes or longer, about 7 minutes or longer, about 8 minutes or longer, about 9 minutes or longer, about 10 minutes or longer, about 11 minutes or longer, about 12 minutes or longer, about 13 minutes or longer, about 14 minutes or longer, about 15 minutes or longer, about 16 minutes or longer, about 17 minutes or longer, about 18 minutes or longer, about 19 minutes or longer, or about 20 minutes or longer.

[0118] In some embodiments, the renal artery is occluded for about 30 minutes or shorter, about 25 minutes or shorter, about 20 minutes or shorter, about 19 minutes or shorter, about 18 minutes or shorter, about 17 minutes or shorter, about 16 minutes or shorter, about 15 minutes or shorter, about 14 minutes or shorter, about 13 minutes or shorter, about 12 minutes or shorter, about 11 minutes or shorter, about 10 minutes or shorter, about 9 minutes or shorter, about 8 minutes or shorter, about 7 minutes or shorter, about 6 minutes or shorter, about 5 minutes or shorter, about 4 minutes or shorter, about 3 minutes or shorter, about 2 minutes or shorter, or about 1 minute or shorter.

[0119] In some embodiments, the renal artery is occluded for about 1 minute to about 30 minutes or about 1 minute to about 25 minutes. In some embodiments, the renal artery is occluded for about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes.

[0120] In some embodiments, the renal artery is occluded for about 2 minutes to about 10 minutes, about 3 minutes to about 8 minutes, about 4 minutes to about 6 minutes, or about 5 minutes.

[0121] In some embodiments, the renal artery is occluded for about 10 minutes to about 30 minutes, about 15 minutes to about 25 minutes, about 15 minutes to about 20 minutes, or about 20 minutes.

[0122] In other embodiments, the method does not include the step of occluding the renal artery.

[0123] In a preferred embodiment, the method does not include the step of occluding the renal vein. Occluding the renal vein may increase the risk of renal vein thrombosis. In subjects with kidney disease, the kidneys may be at an increased risk of thrombosis.

[0124] In a preferred embodiment, the method does not include the step of occluding the aorta.

[0125] In some embodiments, the method does not include the steps of occluding the renal vein and the aorta.

[0126] In some embodiments, the method does not include the steps of occluding the renal artery, the renal vein, or the aorta.

[0127] In some embodiments, the method does not include occluding any of the blood vessels of the subject.

[0128] Step (c): Injection or infusion of the viral vector

[0129] In the method of the present invention, the viral vector is delivered into the renal artery via a catheter. The viral vector can be delivered by any suitable method, such as by injection or infusion.

[0130] Suitably, the viral vector can be delivered into the renal artery within about 1 minute to about 30 minutes. In some embodiments, the viral vector is delivered into the renal artery within about 1 minute to about 25 minutes. In some embodiments, the viral vector is delivered into the renal artery within about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, or about 30 minutes. Suitably, the viral vector is delivered at a substantially constant rate.

[0131] In some embodiments, a viral vector is injected into the renal artery via a catheter. As used herein, "injection" can refer to delivery under pressure or flow (e.g., pressing a liquid into the body through a syringe). In some embodiments, the viral vector is injected into the renal artery without using an infusion pump. In some embodiments, the viral vector is injected into the renal artery within about 5 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 15 minutes to about 20 minutes, about 16 minutes to about 18 minutes, or about 17 minutes.

[0132] In some embodiments, a viral vector is infused into the renal artery via a catheter. As used herein, "infusion" can refer to delivery at atmospheric pressure or without flow (e.g., by using an infusion pump). In some embodiments, the viral vector is infused into the renal artery under no-flow conditions using an infusion pump. In some embodiments, the viral vector is infused into the renal artery within about 5 minutes to about 30 minutes, about 10 minutes to about 25 minutes, about 15 minutes to about 20 minutes, or about 17 minutes. In some embodiments, the viral vector is infused into the renal artery within about 1 minute to about 5 minutes, about 1 minute to about 4 minutes, about 1 minute to about 3 minutes, or about 2 minutes. In some embodiments, the viral vector is infused into the renal artery within about 1 minute to about 10 minutes, about 2 minutes to about 8 minutes, about 3 minutes to about 5 minutes, or about 4 minutes.

[0133] Other method steps

[0134] The methods of the invention can include any other suitable steps, such as flushing the catheter, withdrawing the catheter, withdrawing the sheath, and / or closing any openings.

[0135] In some embodiments, the method further includes the step of flushing the catheter. Such a step can ensure that the viral vector remains within the catheter. The catheter can be flushed with any suitable solution (such as an isotonic solution). In some embodiments, the catheter is flushed with saline (e.g., heparinized saline) or phosphate-buffered saline (PBS). In some embodiments, the catheter is flushed with heparinized saline.

[0136] In some embodiments, the method includes the step of flushing the catheter before injecting or infusing the viral vector. In some embodiments, the method includes the step of flushing the catheter after injecting or infusing the viral vector. In some embodiments, the method includes the step of flushing the catheter before and after injecting or infusing the viral vector.

[0137] In some embodiments, the method further comprises the step of deflating the balloon. In some embodiments, the method further comprises the step of withdrawing the catheter. In some embodiments, the method further comprises the step of withdrawing the sheath. Appropriately, the catheter and sheath are removed slowly to avoid damaging the blood vessel. In some embodiments, the method further comprises closing the opening.

[0138] In some embodiments, the method comprises the following steps:

[0139] (a) Inserting a balloon catheter into the renal artery;

[0140] (b) Optionally inflating the balloon to occlude the renal artery;

[0141] (c) Injecting or infusing a viral vector into the renal artery via the catheter; and

[0142] (d) Flushing the catheter, optionally deflating the balloon, and withdrawing the catheter.

[0143] In some embodiments, the method does not include forming a closed circuit through the kidney. As used herein, a "closed circuit" through the kidney can isolate the viral vector preparation from the subject's systemic circulation. In some embodiments, less than about 20% v / v of the viral vector preparation circulating through the closed circuit leaks outside the closed circuit. For example, a closed circuit through the kidney can include a catheter inserted into the renal artery and a catheter inserted into the renal vein. A pump can drive fluid into the kidney via the renal artery catheter and out of the kidney via the renal vein catheter.

[0144] In some embodiments, the method does not include using a membrane oxygenation device. In some embodiments, the viral vector preparation does not pass through a membrane oxygenation device. In some embodiments, the closed circuit further includes a membrane oxygenation device.

[0145] In some embodiments, the method does not include the step of perfusing the kidney with saline before injecting or infusing the viral vector into the renal artery.

[0146] Kidney-specific delivery

[0147] The method of the present invention results in the delivery of the viral vector to the kidney.

[0148] As used herein, "delivery" of a viral vector can refer to transduction with the viral vector, which can subsequently result in the expression of RNA and / or protein encoded by the viral vector. Delivery of a viral vector to a particular organ, tissue, or cell type can be determined by any suitable method, including transcriptomic analysis and / or immunofluorescence.

[0149] The method of the present invention results in the transduction of the kidney with a viral vector, and subsequent expression of the RNA encoded by the viral vector in the kidney and / or expression of the protein encoded by the viral vector in the kidney. Transduction of a particular organ, tissue, or cell type can be determined by viral load or viral copy number, which can be determined by any suitable method (see, e.g., Dobnik, D., et al., 2019. Frontiers in microbiology, 10, p.1570). Expression of RNA in a particular organ, tissue, or cell type can be determined by transcriptomics. For example, RNA can be extracted from tissue, converted to cDNA, and quantified by qPCR. Expression of protein in a particular organ, tissue, or cell type can be determined by immunofluorescence. For example, by labeling the protein with a fluorescent marker and quantifying by fluorescence microscopy.

[0150] The method can result in the delivery of the viral vector to the renal cortex and / or renal medulla. In a preferred embodiment, the method results in the delivery of the viral vector to the renal cortex. In a more preferred embodiment, the method results in the delivery of the viral vector to the glomerulus. In an even more preferred embodiment, the method results in the delivery of the viral vector to renal podocytes.

[0151] The method of the present invention can result in kidney-specific delivery of the viral vector. As used herein, "kidney-specific delivery" can mean that the viral vector predominantly transduces the kidney, which can subsequently result in the predominant expression of the RNA and / or protein encoded by the viral vector in the kidney.

[0152] Suitably, the method results in the delivery of the viral vector to the kidney, but does not result in significant delivery of the viral vector to other tissues. In some embodiments, the other tissues are selected from one or more of the liver, colon, and pancreas. In some embodiments, the method results in the delivery of the viral vector to the kidney, but does not result in significant delivery of the viral vector to the liver.

[0153] The viral vector can be delivered to a single kidney or both kidneys of a subject. In some embodiments, the method delivers the viral vector to a single kidney of the subject. In some embodiments, the method is repeated to deliver the viral vector to both kidneys of the subject.

[0154] In one embodiment, the present invention provides a method of delivering a viral vector to a subject, the method comprising: (1) delivering the viral vector to the right kidney of the subject by the method according to the present invention; and / or (2) delivering the viral vector to the left kidney of the subject by the method according to the present invention.

[0155] In one embodiment, the present invention provides a method for delivering a viral vector to a subject, the method comprising: (1) delivering the viral vector to the right kidney of the subject by the method according to the present invention; and (2) delivering the viral vector to the left kidney of the subject by the method according to the present invention.

[0156] Steps (1) and (2) can be in any order and can be carried out over any duration. In some embodiments, steps (1) and (2) are carried out within a day. In some embodiments, steps (1) and (2) are carried out as part of the same procedure. In some embodiments, step (2) directly follows step (1) or step (1) directly follows step (2).

[0157] The method of the present invention can be carried out on the subject one or more times. The method can be repeated over any suitable time period. Suitably, the method of the present invention is carried out once on the kidney (i.e., the viral vector is administered as a single dose). In some embodiments, the method of the present invention is carried out once to deliver the viral vector to a single kidney of the subject. In some embodiments, the method of the present invention is carried out twice to deliver the viral vector to both kidneys of the subject.

[0158] Subject

[0159] The subject can be any suitable subject in need thereof. The subject can be a mammal. In a preferred embodiment, the subject is a human. The subject can be an adult, adolescent or child.

[0160] The subject can have a kidney disease or can be at risk of a kidney disease. For example, the subject can have a glomerular disease or can be at risk of a glomerular disease. For example, the subject can have a podocyte-related glomerular disease or can be at risk of a podocyte-related glomerular disease. For example, the subject can have a GBM-related glomerular disease or can be at risk of a GBM-related glomerular disease.

[0161] In some embodiments, the subject has a hereditary glomerular disease (i.e., a glomerular disease that is genetic) or is at risk of a hereditary glomerular disease. Hereditary glomerular diseases include podocyte-related hereditary glomerular diseases such as nephrotic syndrome and GBM-related glomerular diseases such as Alport syndrome.

[0162] In some embodiments, the subject has a podocyte-related hereditary glomerular disease or is at risk of a podocyte-related hereditary glomerular disease. Podocyte-related hereditary glomerular diseases include Finnish congenital nephrotic syndrome, congenital nephrotic syndrome type 2, familial nephrotic syndrome type 3, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, nephrotic syndrome caused by CD2AP mutations, nephrotic syndrome caused by actin 4 mutations, nephrotic syndrome caused by TRPC6 mutations, and Epstein and Fechtner syndromes. Suitably, the glomerular disease is nephrotic syndrome.

[0163] In some embodiments, the subject has a GBM-related hereditary glomerular disease or is at risk of a GBM-related hereditary glomerular disease. GBM-related hereditary glomerular diseases include X-linked Alport syndrome, autosomal recessive Alport syndrome, autosomal dominant Alport syndrome, thin basement membrane disease, 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.

[0164] In some embodiments, the subject has a complement-mediated kidney disease or is at risk of a complement-mediated kidney disease. As used herein, "complement-mediated kidney disease" is a kidney disease caused by a 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 an overactivation of the complement system. Exemplary complement-mediated kidney diseases include IgA nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome (aHUS), stx-associated HUS, lupus nephritis, cryoglobulinemia, anti-GBM disease, ANCA-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated renal transplant rejection, membranous nephropathy, membranoproliferative glomerulonephritis I or membranoproliferative glomerulonephritis III.

[0165] In some embodiments, the subject has diabetic nephropathy or is at risk of diabetic nephropathy. Diabetic nephropathy, also known as diabetic kidney disease, is the chronic loss of kidney function that occurs in patients with diabetes.

[0166] In some embodiments, the subject has Fabry disease or is at risk of Fabry disease. Fabry disease is a hereditary disorder caused by the accumulation of globotriaosylceramide within lysosomes. Renal complications are a common and severe consequence of the disease.

[0167] Viral vector dose

[0168] Viral vectors can be delivered at any suitable dose (e.g., measured as vector genomes (vg) or vg / kg). The dose can be determined by factors such as the condition of the subject, the age of the subject, the weight of the subject, and the type and severity of the subject's disease, and the appropriate dose can be determined by a physician. The viral vector can be formulated accordingly.

[0169] Compared to other delivery methods (e.g., systemic administration such as intravenous administration), the method of the present invention can allow for the use of lower doses of viral vectors. Thus, compared to systemic administration such as intravenous administration, the method of the present invention is associated with a lower risk of immune response and fewer off-target effects, such as lower or no liver expression of the protein encoded by the viral vector.

[0170] Suitably, the viral vector is delivered at a dose of about 1x10 6 vg / kg or higher, about 1x10 7 vg / kg or higher, about 1x10 8 vg / kg or higher, about 1x10 9 vg / kg or higher, about 1x10 10 vg / kg or higher, about 1x10 11 vg / kg or higher or about 1x10 12 vg / kg or higher. Suitably, the viral vector is delivered at a dose of about 1x10 14 vg / kg or lower or about 1x10 13 vg / kg or lower. Suitably, the viral vector is delivered at a dose of about 1x10 6 vg / kg to about 1x10 14 vg / kg. Suitably, the viral vector is delivered at a dose of about 1x10 6 vg / kg to about 1x10 13 vg / kg. Suitably, the viral vector is delivered at a dose of about 1x10 6 vg / kg to about 10x10 6 vg / kg, about 1x10 7 vg / kg to about 10x10 7 vg / kg, about 1x10 8 vg / kg to about 10x10 8 vg / kg, about 1x10 9 vg / kg to about 10x10 9 vg / kg, about 1x10 10 vg / kg to about 10x10 10vg / kg, about 1x10 11 vg / kg to about 10x10 11 vg / kg or about 1x10 12 vg / kg to about 10x10 12 vg / kg of the dose is delivered.

[0171] In some embodiments, the viral vector is at about 1x10 9 vg / kg to about 1x10 12 vg / kg of the dose is delivered. In some embodiments, the viral vector is at about 3x10 9 vg / kg to about 3x10 11 vg / kg of the dose is delivered.

[0172] Suitably, the viral vector is at about 1x10 8 vg or higher, about 1x10 9 vg or higher, about 1x10 10 vg or higher, about 1x10 11 vg or higher, about 1x10 12 vg or higher, about 1x10 13 vg or higher or about 1x10 14 vg or higher of the dose is delivered. Suitably, the viral vector is at about 1x10 15 vg or lower or about 1x10 14 vg or lower of the dose is delivered. Suitably, the viral vector is at about 1x10 8 vg to about 1x10 15 vg of the dose is delivered. Suitably, the viral vector is at about 1x10 8 vg to about 5x10 14 vg of the dose is delivered. Suitably, the viral vector is at about 1x10 8 vg to about 1x10 14 vg, about 1x10 9 vg to about 1x10 14 vg, about 1x10 10 vg to about 1x10 14 vg, about 1x10 11 vg to about 1x10 14 vg or about 1x10 12 vg to about 1x10 14 vg of the dose is delivered. Suitably, the viral vector is at about 1x10 8 vg to about 10x10 8 vg, about 1x10 9 vg to about 10x10 9vg, about 1x10 10 vg to about 10x10 10 vg, about 1x10 11 vg to about 10x10 11 vg, about 1x10 12 vg to about 10x10 12 vg, about 1x10 13 vg to about 10x10 13 vg or about 1x10 14 vg to about 10x10 14 Dose delivery of vg.

[0173] In some embodiments, the viral vector is at about 1x10 11 vg to about 1x10 14 Dose delivery of vg. In some embodiments, the viral vector is at about 1x10 11 vg, about 2x10 11 vg, about 3x10 11 vg, about 4x10 11 vg, about 5x10 11 vg, about 6x10 11 vg, about 7x10 11 vg, about 8x10 11 vg, about 9x10 11 vg, about 1x10 12 vg, about 2x10 12 vg, about 3x10 12 vg, about 4x10 12 vg, about 5x10 12 vg, about 6x10 12 vg, about 7x10 12 vg, about 8x10 12 vg, about 9x10 12 vg, about 1x10 13 vg, about 2x10 13 vg, about 3x10 13 vg, about 4x10 13 vg, about 5x10 13 vg, about 6x10 13 vg, about 7x10 13 vg, about 8x10 13 vg, about 9x10 13 vg or about 1x10 14 Dose delivery of vg.

[0174] In some embodiments, the viral vector is at about 2x1011 vg to about 2x10 13 dose delivery of vg, e.g., based on a 70 kg subject.

[0175] In some embodiments, the viral vector is at about 5x10 11 vg to about 5x10 13 vg dose delivery. In some embodiments, the viral vector is at about 1x10 12 vg to about 5x10 13 vg dose delivery. In some embodiments, the viral vector is at about 5x10 12 vg to about 5x10 13 vg dose delivery.

[0176] In some embodiments, the viral vector is at about 5x10 11 vg to about 2x10 13 vg dose delivery. In some embodiments, the viral vector is at about 1x10 12 vg to about 2x10 13 vg dose delivery. In some embodiments, the viral vector is at about 5x10 12 vg to about 2x10 13 vg dose delivery.

[0177] In some embodiments, the viral vector is at about 5x10 11 vg to about 1x10 13 vg dose delivery. In some embodiments, the viral vector is at about 1x10 12 vg to about 1x10 13 vg dose delivery. In some embodiments, the viral vector is at about 5x10 12 vg to about 1x10 13 vg dose delivery.

[0178] In some embodiments, the viral vector is at about 1x10 13 vg dose delivery.

[0179] Viral vector formulation

[0180] The viral vector can be delivered into the renal artery in the form of a viral vector preparation.

[0181] As used herein, "viral vector preparation" can refer to a composition comprising or consisting of a therapeutically effective amount of a viral vector. It preferably includes a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). "Pharmaceutically acceptable" includes that the preparation is sterile and pyrogen-free. The carrier, diluent, and / or excipient must be "acceptable" in the sense of being compatible with the viral vector and harmless to its recipient. Typically, the carrier, diluent, and excipient will be sterile and pyrogen-free normal saline or infusion medium, however, other acceptable carriers, diluents, and excipients may be used.

[0182] The viral vector can be delivered into the renal artery in the form of a sterile aqueous solution, which can contain other substances, e.g., sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution can be appropriately buffered (preferably to a pH of 3 to 9). Preparation of a suitable parenteral preparation under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0183] Suitably, the viral vector preparation contains an isotonic buffer (e.g., at about pH 7.4). In some embodiments, the viral vector preparation contains a phosphate buffered saline (PBS) buffer (e.g., pH 7.4). Optionally, the PBS is supplemented with about 200 mM NaCl. In some embodiments, the viral vector preparation contains plasmalyte. In some embodiments, the viral vector preparation contains about 0.001% poloxamer 188 (also known as Pluronic F-68).

[0184] The viral vector preparation can contain any suitable amount of the viral vector. Suitably, the viral vector preparation contains about 1x10 7 vg / ml or higher, about 1x10 8 vg / ml or higher, about 1x10 9 vg / ml or higher, about 1x10 10 vg / ml or higher, about 1x10 11 vg / ml or higher, about 1x10 12 vg / ml or higher or about 1x10 13 vg / ml or higher of the viral vector. Suitably, the viral vector preparation contains 1x10 14 vg / ml or lower or about 1x10 13 vg / ml or lower of the viral vector. Suitably, the viral vector preparation contains about 1x10 7 vg / ml to about 1x10 14 vg / ml of the viral vector. Suitably, the viral vector preparation contains about 1x10 7 vg / ml to about 5x10 13A viral vector in an amount of vg / ml. Suitably, the viral vector preparation comprises from about 1x10 7 vg / ml to about 10x10 7 vg / ml, from about 1x10 8 vg / ml to about 10x10 8 vg / ml, from about 1x10 9 vg / ml to about 10x10 9 vg / ml, from about 1x10 10 vg / ml to about 10x10 10 vg / ml, from about 1x10 11 vg / ml to about 10x10 11 vg / ml, from about 1x10 12 vg / ml to about 10x10 12 vg / ml or from about 1x10 13 vg / ml to about 10x10 13 vg / ml of the viral vector.

[0185] In some embodiments, the viral vector preparation comprises from about 1x10 10 vg / ml to about 1x10 13 vg / ml of the viral vector. In some embodiments, the viral vector preparation comprises from about 1x10 10 vg / ml to about 1x10 12 vg / ml of the viral vector.

[0186] The viral vector preparation can be of any suitable volume. Suitably, the viral vector preparation has a volume of 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 viral vector preparation 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 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL or about 30 mL.

[0187] The viral vector preparation can further comprise one or more other therapeutic agents.

[0188] The viral vector

[0189] The viral vector of the present invention is preferably an adeno-associated virus (AAV) vector, although other viral vectors are expected to be usable. Other suitable viral vectors may include lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

[0190] The viral vector of the present invention can be in the form of viral vector particles. Methods for preparing and modifying viral vectors and viral vector particles (such as those derived from AAV) are well known in the art. Suitable methods are described in Ayuso, E., etal., 2010. Current gene therapy, 10(6), pp.423-436, Merten, O.W., et al.,2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017;and Nadeau,I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489.

[0191] The viral vector of the present invention is preferably capable of transducing renal cells. Appropriately, the viral vector of the present invention is capable of specifically transducing renal cells. The viral vector of the present invention is preferably capable of transducing glomerular cells. Appropriately, the viral vector of the present invention is capable of specifically transducing glomerular cells. The viral vector of the present invention is preferably capable of transducing podocytes. Preferably, the viral vector of the present invention is capable of specifically transducing podocytes.

[0192] Adeno-associated virus (AAV) vector

[0193] The viral vector of the present invention is preferably an adeno-associated virus (AAV) vector particle.

[0194] AAV genome

[0195] The AAV vector particle may contain an AAV genome or a fragment or derivative thereof.

[0196] The AAV genome is a polynucleotide sequence that can encode the functions required to produce AAV particles. These functions include functions that operate during the replication and packaging cycles of AAV in host cells, including encapsidating the AAV genome into AAV particles. Naturally occurring AAV is replication-defective and depends on trans-provided helper functions to complete the replication and packaging cycles. Thus, the AAV genome of the AAV vector of the present invention is typically replication-defective.

[0197] The AAV genome can be in single-stranded form (ssAAV), either sense or antisense, or in double-stranded form (dsAAV). The use of the double-stranded form allows bypassing the DNA replication step in the target cell, and thus can accelerate transgene expression. The maximum packaging capacity of the single-stranded form is greater than that of the double-stranded form. Suitably, the AAV genome is in single-stranded form.

[0198] Naturally occurring AAV can be classified according to a variety of biological systems. The AAV genome can be from any naturally derived serotype, isolate, or clade of AAV.

[0199] AAV can be referred to by its serotype. Serotypes correspond to variant subspecies of AAV that have a unique reactivity due to the expression profile of their capsid surface antigens that can be used to distinguish them from other variant subspecies. Generally, AAV vector particles having a particular AAV serotype do not cross-react effectively with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV vector of the invention can be of AAV3B, LK03, AAV9, or AAV8 serotype. In some embodiments, the AAV vector of the invention can be of AAV3B, LK03, or AAV9 serotype.

[0200] AAV can also be referred to by clade or clone. This refers to the phylogenetic relationship of naturally derived AAV and generally refers to a phylogenetic group of AAV that can be traced back to a common ancestor and includes all of its descendants. Additionally, AAV can be referred to by a specific isolate, i.e., a genetic isolate of a particular AAV found in nature. The term genetic isolate describes a population of AAV that has undergone limited genetic mixing with other naturally occurring AAV, thus defining a distinct group that is recognizable at the genetic level.

[0201] Generally, the AAV genome of a naturally derived serotype, isolate, or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequences act in cis to provide a functional origin of replication and allow integration and excision of the vector from the cellular genome. The ITR may be the only sequence that needs to be in cis arrangement next to the therapeutic gene.

[0202] The AAV genome can also contain packaging genes, such as the rep and / or cap genes encoding the packaging functions of AAV particles. A promoter can be operably linked to each packaging gene. Specific examples of such promoters include the p5, p19, and p40 promoters. For example, the p5 and p19 promoters are typically used to express the rep gene, while the p40 promoter is typically 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. These proteins constitute the capsid of the AAV particle, and the capsid determines the AAV serotype. VP1, VP2, and VP3 can be generated by alternative mRNA splicing (Trempe, J.P. and Carter, B.J., 1988. Journal of virology, 62(9), pp. 3356-3363). Thus, VP1, VP2, and VP3 can have the same sequence, but VP2 is truncated at the N-terminus relative to VP1, and VP3 is truncated at the N-terminus relative to VP2.

[0203] The AAV genome can be the complete genome of a naturally occurring AAV. For example, a vector containing the complete AAV genome can be used to prepare an AAV vector.

[0204] Preferably, the AAV genome is derivatized for the purpose of administration to a patient. Such derivatization is standard in the art, and the present invention encompasses any known derivatives of the AAV genome and the use of derivatives that can be generated by applying techniques known in the art. The AAV genome can be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Suitably, the AAV genome is a derivative of AAV2.

[0205] Derivatives of the AAV genome include any truncated or modified form of the AAV genome that permits in vivo expression of the transgene of the AAV vector of the present invention. Generally, it is possible to significantly truncate the AAV genome to include minimal viral sequences while retaining the above functions. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus and to also avoid triggering a cellular immune response due to the presence of viral gene proteins in the target cells.

[0206] Typically, the derivative will include at least one inverted terminal repeat (ITR), preferably more than one ITR, such as two ITRs or more. One or more ITRs can be derived from AAV genomes having different serotypes, or can be chimeric or mutant ITRs. A preferred mutant ITR is a mutant ITR having a trs (terminal resolution site) deletion. This deletion allows the genome to continue to replicate to generate a single-stranded genome containing both coding and complementary sequences, i.e., a self-complementary AAV (scAAV) genome. This allows bypassing DNA replication in the target cell, and thus enables accelerated transgene expression. However, the maximum packaging capacity of scAAV is reduced. Appropriately, the AAV genome is not a scAAV genome.

[0207] The AAV genome can contain one or more ITR sequences from any naturally-derived serotype, isolate, or clade of AAV or its variants. The AAV genome can contain at least one, such as two AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs or their variants. Appropriately, the AAV genome can contain at least one, such as two AAV2 ITRs.

[0208] Preferably includes one or more ITRs to assist in the formation of concatemers of the AAV vector in the host cell nucleus, for example, after the single-stranded vector DNA is converted to double-stranded DNA by the action of the host cell DNA polymerase. The formation of such episomal concatemers protects the AAV vector during the life of the host cell, thus allowing for extended transgene expression in vivo.

[0209] Appropriately, the ITR element is the only sequence retained from the native AAV genome in the derivative. The derivative preferably does 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 stated above and reduces the likelihood of vector integration 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) in addition to the transgene into the vector.

[0210] Accordingly, the following portions can be removed in the derivatives of the present invention: one inverted terminal repeat (ITR) sequence, replication (rep), and capsid (cap) genes. However, the derivative can additionally include one or more rep and / or cap genes of the AAV genome or other viral sequences. Naturally-occurring AAV integrates at a specific locus on human chromosome 19 at a high frequency and shows a negligible random integration frequency, such that retaining the integration ability of the AAV vector in a therapeutic setting can be tolerated.

[0211] The present invention further encompasses sequences that provide the AAV genome in an order and configuration different from the native AAV genome. The present invention also encompasses replacing one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes can be composed of sequences of two or more related viral proteins from different viral species.

[0212] AAV serotype and capsid protein

[0213] AAV vector particles can be encapsidated by capsid proteins. The serotype can facilitate transduction of glomerular cells (such as podocytes), for example, specific transduction of glomerular cells (such as podocytes).

[0214] The AAV vector particles can be kidney-specific vector particles. Preferably, the AAV vector particles are glomerulus-specific (such as podocyte-specific) vector particles. The AAV vector particles can be encapsidated by a glomerulus-specific (such as podocyte-specific) capsid. The AAV vector particles can contain a glomerulus-specific (such as podocyte-specific) capsid protein.

[0215] Suitably, the AAV vector particles can be in a transcapsidated form, in which the AAV genome or derivative with ITRs of one serotype is packaged in a capsid of a different serotype. The AAV vector particles also include a mosaic form, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. The AAV vector particles also include a chemically modified form carrying ligands adsorbed to the capsid surface. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0216] When the derivative contains capsid proteins, namely VP1, VP2, and / or VP3, the derivative can be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses providing sequences of capsid proteins from different serotypes, clades, clones, or isolates within the same vector (i.e., pseudotyped vector). The AAV vector can be in the form of a pseudotyped AAV vector particle.

[0217] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide one or more desired functions to AAV vectors. Thus, compared to AAV vectors containing a naturally occurring AAV genome, these derivatives can exhibit increased gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism range, and / or improved podocyte targeting. Increased gene delivery efficiency can be affected by improved cell surface receptor or coreceptor binding, improved internalization, improved intracellular and nuclear transport, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Increased efficiency can also involve an altered tropism range or podocyte targeting such that the vector dose is not diluted by administration to tissues that do not require it.

[0218] Chimeric capsid proteins include those generated by recombination between two or more capsid-encoding sequences of naturally occurring AAV serotypes. This can be performed, for example, by marker rescue methods, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select for capsid sequences with desired properties. The capsid sequences of different serotypes can be altered by homologous recombination within the cell to generate novel chimeric capsid proteins.

[0219] Chimeric capsid proteins also include those generated by engineering capsid protein sequences to transfer specific capsid protein domains, surface loops, or specific amino acid residues between two or more capsid proteins, for example, between two or more capsid proteins of different serotypes.

[0220] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be produced by randomly fragmenting the sequences of related AAV genes, such as those encoding capsid proteins of multiple different serotypes, and then subsequently recombining the fragments in a self-priming polymerase reaction, which can also cause crossovers in regions of sequence homology. A library of hybrid AAV genes generated in this way by shuffling the capsid genes of multiple serotypes can be screened to identify viral clones with desired functions. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to generate a diverse library of variants, which can then be screened for desired properties.

[0221] The sequence of the capsid gene can also be genetically modified to introduce specific deletions, substitutions or insertions relative to the native wild-type sequence. In particular, the capsid gene can be modified by inserting the sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide can advantageously be a protein or peptide that acts as a ligand for a specific cell type, thereby conferring improved binding to target cells or improving the specificity of the vector for targeting a specific cell population. The unrelated protein can also be a protein that aids in the purification of the viral particles as part of the production process, i.e., an epitope or affinity tag. The insertion site is typically chosen so as not to interfere with other functions of the viral particle, such as internalization and trafficking of the viral particle.

[0222] The capsid protein can be an artificial or mutant capsid protein. As used herein, the term "artificial capsid" means that the capsid particle contains an amino acid sequence that does not exist in nature or an amino acid sequence that contains an amino acid sequence that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, when the artificial capsid amino acid sequence is aligned with the parental capsid amino acid sequence, the artificial capsid protein contains a mutation or variation in the amino acid sequence compared to the sequence of the parental capsid from which it is derived.

[0223] The capsid protein can contain mutations or modifications relative to the wild-type capsid protein that improve its ability to transduce podocytes compared to an unmodified or wild-type viral particle. The improved ability to transduce podocytes can be measured, for example, by measuring the expression of a transgene (e.g., GFP) carried by the AAV vector particle, wherein the expression of the transgene in podocytes is related to the ability of the AAV vector particle to transduce podocytes.

[0224] The AAV vector particle can be an AAV3B, LK03, AAV9 or AAV8 vector particle. The inventors have shown that AAV vector particles with AAV3B, LK03, AAV9 and AAV8 serotypes can transduce podocytes. Preferably, the AAV vector particle is an AAV3B vector particle or an LK03 vector particle. More preferably, the AAV vector particle is an LK03 vector particle.

[0225] The AAV vector particle can contain an AAV3B, LK03, AAV9 or AAV8 capsid protein. Preferably, the AAV vector particle contains an AAV3B capsid protein or an LK03 capsid protein. More preferably, the AAV vector particle contains an LK03 capsid protein.

[0226] AAV vector particles may comprise AAV3B, LK03, AAV9 or AAV8 capsid proteins VP1, VP2 and VP3. Preferably, the AAV vector particles comprise AAV3B or LK03 capsid proteins VP1, VP2 and VP3. More preferably, the AAV vector particles comprise LK03 capsid proteins VP1, VP2 and VP3.

[0227] AAV vector particles may comprise one or more AAV2 ITR sequences and AAV3B capsid protein, LK03 capsid protein, AAV9 capsid protein or AAV8 capsid protein. Preferably, the AAV vector particles comprise one or more AAV2 ITR sequences and AAV3B or LK03 capsid protein. More preferably, the AAV vector particles comprise one or more AAV2 ITR sequences and LK03 capsid protein.

[0228] AAV vector particles may have an AAV2 genome and AAV3B capsid protein (AAV2 / 3B), AAV2 genome and LK03 capsid protein, AAV2 genome and AAV9 capsid protein (AAV2 / 9) or AAV2 genome and AAV8 capsid protein (AAV2 / 8). Preferably, the AAV vector particles comprise an AAV2 genome and AAV3B or LK03 capsid protein. More preferably, the AAV vector particles comprise an AAV2 genome and LK03 capsid protein.

[0229] The nomenclature AAVX / Y may denote a pseudotyped AAV, for example where the ITR sequences are from AAVX and flank a cassette carrying a payload that is encapsidated as serotype AAVY (i.e., has AAVY capsid proteins).

[0230] AAV3B serotype

[0231] AAV vector particles may comprise AAV3B capsid protein. Suitably, the AAV vector particles may be encapsidated by AAV3B capsid protein.

[0232] Two different AAV3 isolates (AAV3A and AAV3B) have been cloned. Compared to vectors based on other AAV serotypes, AAV3 vectors are thought not to transduce most cell types efficiently. However, AAV3B can transduce podocytes efficiently. AA3B has been described in Rutledge, E.A., et al., 1998. Journal of virology, 72(1), pp.309-319.

[0233] The AAV vector particles may comprise an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and / or an AAV3B VP3 capsid protein. Suitably, the AAV vector particles may be encapsidated by an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and / or an AAV3B VP3 capsid protein. Suitably, the AAV vector particles may be encapsidated by AAV3B VP1, VP2, and VP3 capsid proteins.

[0234] Suitably, the AAV3B VP1 capsid protein may comprise the amino acid sequence shown in SEQ ID NO: 1 or a variant that is at least 90% identical to SEQ ID NO: 1 or consist thereof.

[0235] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN

[0236] Example AAV3B VP1 capsid protein (SEQ ID NO: 1)

[0237] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.

[0238] Suitably, the AAV3B VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 1, or N-terminal truncations of variants that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.

[0239] Serotype LK03

[0240] The AAV vector particles may comprise the LK03 capsid protein. Suitably, the AAV vector particles may be encapsidated by the LK03 capsid protein.

[0241] The AAV-LK03 cap sequence is composed of fragments from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, 9) and is described in Lisowski, L., et al., 2014. Nature, 506(7488), pp.382-386. The inventors have demonstrated that the AAV-LK03 vector can achieve nearly 100% high transduction in human podocytes in vitro.

[0242] The AAV vector particles may comprise the LK03 VP1 capsid protein, the LK03 VP2 capsid protein, and / or the LK03 VP3 capsid protein. Suitably, the AAV vector particles may be encapsidated by the LK03 VP1 capsid protein, the LK03 VP2 capsid protein, and / or the LK03 VP3 capsid protein. Suitably, the AAV vector particles may be encapsidated by the LK03 VP1, VP2, and VP3 capsid proteins.

[0243] Suitably, the LK03 VP1 capsid protein may comprise the amino acid sequence shown in SEQ ID NO: 2 or a variant that is at least 90% identical to SEQ ID NO: 2 or consists thereof.

[0244] MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0245] Example LK03 VP1 capsid protein (SEQ ID NO: 2)

[0246] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2.

[0247] Suitably, the LK03 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 2, or N-terminal truncations of variants that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2.

[0248] AAV9 serotype

[0249] The AAV vector particle may comprise an AAV9 capsid protein. Suitably, the AAV vector particle may be encapsidated by the AAV9 capsid protein.

[0250] The present inventors have demonstrated that the AAV9 vector can achieve high transduction in human podocytes in vitro.

[0251] The AAV vector particle may comprise an AAV9 VP1 capsid protein, an AAV9 VP2 capsid protein, and / or an AAV9 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by the AAV9 VP1 capsid protein, the AAV9 VP2 capsid protein, and / or the AAV9 VP3 capsid protein. Suitably, the AAV vector particle may be encapsidated by the AAV9 VP1, VP2, and VP3 capsid proteins.

[0252] Suitably, the AAV9 VP1 capsid protein may comprise the amino acid sequence shown in SEQ ID NO: 3 or a variant that is at least 90% identical to SEQ ID NO: 3 or consists thereof.

[0253] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0254] Exemplary AAV9 VP1 capsid protein (SEQ ID NO: 3)

[0255] Suitably, the variant may be at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3.

[0256] Suitably, the AAV9 VP2 and VP3 capsid proteins may be N-terminal truncations of SEQ ID NO: 3, or N-terminal truncations of variants that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 3.

[0257] Other viral vectors

[0258] Retroviral and lentiviral vectors

[0259] The viral vector of the present invention can be a retroviral vector or a lentiviral vector.

[0260] Retroviral vectors can be derived from or may be capable of being derived from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV).

[0261] Retroviruses can be roughly divided into two categories: "simple" and "complex". Retroviruses can even be further divided into seven groups. Five of these groups represent retroviruses with carcinogenic potential. The remaining two groups are lentiviruses and foamy viruses.

[0262] The basic structures of retroviral and lentiviral genomes share many common features, such as 5' LTR and 3' LTR. The following elements are located between or within these: a packaging signal that enables the genome to be packaged, a primer binding site, an integration site that enables integration into the host cell genome, and the gag, pol, and env genes that encode the packaging components - the packaging components are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as the rev and RRE sequences in HIV, which enable the RNA transcript of the integrated provirus to be effectively exported from the nucleus of the infected target cell to the cytoplasm.

[0263] In the provirus, both ends of these genes are flanked by regions called long terminal repeats (LTRs). The LTR is responsible for proviral integration and transcription. The LTR also acts as an enhancer-promoter sequence and can control the expression of viral genes.

[0264] The LTR itself is the same sequence, which can be divided into three elements: U3, R, and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from the sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of these three elements vary greatly among different retroviruses.

[0265] In the defective retroviral vector genome, gag, pol, and env may be absent or non-functional.

[0266] In a typical retroviral vector, at least a portion of one or more protein-coding regions necessary for replication can be removed from the virus. This renders the viral vector replication-defective. A portion of the viral genome can also be replaced with a library encoding candidate regulatory portions that are operably linked to regulatory control regions and reporter portions in the vector genome to generate a vector containing the candidate regulatory portions that is capable of transducing a target host cell and / or integrating its genome into the host genome.

[0267] Lentiviral vectors are part of a larger group of retroviral vectors. Briefly, lentiviruses can be divided into primate and non-primate. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "lentivirus" visna / maedi virus (VMV), as well as related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0268] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, cannot infect non-dividing or slowly dividing cells, such as those that make up, for example, muscle, brain, lung, and liver tissues.

[0269] As used herein, a lentiviral vector is a vector that contains at least one component that can be derived from a lentivirus. Preferably, the component relates to the biological mechanisms by which the vector infects cells, expresses genes, or replicates.

[0270] A lentiviral vector can be a "primate" vector. A lentiviral vector can be a "non-primate" vector (i.e., derived from a virus that does not primarily infect primates, especially humans). Examples of non-primate lentiviruses can be any member of the lentivirus family that does not naturally infect primates.

[0271] As examples of lentivirus-based vectors, vectors based on HIV-1 and HIV-2 are described below.

[0272] HIV-1 vectors contain cis-acting elements also found in simple retroviruses. Sequences extending into the gag open reading frame have been shown to be important for packaging HIV-1. Thus, HIV-1 vectors typically contain the relevant portion of gag in which the translation initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to the RRE, which permits the export of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and / or the RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to alleviate the need for Rev and the RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0273] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient export of full-length or singly spliced viral RNAs.

[0274] Preferably, the viral vectors used in the present invention have a minimal viral genome.

[0275] "Minimal viral genome" should be understood to mean that the viral vector has been engineered to remove non-essential elements and retain essential elements in order to provide the functions required for infecting, transducing and delivering a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998 / 017815.

[0276] Preferably, the plasmid vector used to generate the viral genome within a host cell / packaging cell will have sufficient lentiviral genetic information to permit, in the presence of packaging components, the packaging of the RNA genome into viral particles capable of infecting target cells but not capable of independent replication to produce infectious virus particles within the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes necessary for replication.

[0277] However, the plasmid vector used to generate the viral genome within a host cell / packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome within the host cell / packaging cell. These regulatory sequences can be native sequences associated with the transcribed viral sequences (i.e., the 5'U3 region), or they can be heterologous promoters such as another viral promoter (e.g., the CMV promoter).

[0278] The vector can be a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with similar efficacy as wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilization of replication-competent virus. This should also be able to regulate the expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

[0279] The vector can be integration-deficient. Integration-deficient lentiviral vectors (IDLVs) can be generated, for example, by packaging the vector with a catalytically inactivated integrase (such as HIV integrase carrying the D64V mutation at the catalytic site), or by modifying or deleting the essential att sequences from the vector LTR, or by a combination of the above.

[0280] Adenoviral vector

[0281] The viral vector of the present invention can be an adenovirus vector.

[0282] Adenoviruses are double-stranded linear DNA viruses that do not go through an RNA intermediate. There are over 50 different human adenovirus serotypes, which are divided into 6 subgroups based on gene sequence homology. The natural targets of adenoviruses are the respiratory and gastrointestinal epithelia and usually cause only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenovirus vector systems and are usually associated with upper respiratory tract infections in young people.

[0283] Adenoviruses have been used as vectors for gene therapy and heterologous gene expression. The large (36 kb) genome can accommodate up to 8 kb of foreign inserted DNA and is capable of replicating efficiently in complementary cell lines to produce very high titers of up to 10 12 . Thus, adenoviruses are one of the best systems for studying gene expression in primary non-replicating cells.

[0284] Expression of viral or foreign genes from the adenovirus genome does not require replicating cells. Adenovirus vectors enter cells by receptor-mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function as linear genomes as episomes (independent of the host genome) in the host nucleus. Thus, the use of recombinant adenoviruses alleviates problems associated with random integration into the host genome.

[0285] Herpes simplex virus vector

[0286] The viral vector of the present invention can be a herpes simplex virus vector.

[0287] Herpes simplex virus (HSV) is a neurotropic DNA virus with favorable properties as a gene delivery vector. HSV is highly infectious, and thus HSV vectors are effective mediators for delivering foreign genetic material into cells. Viral replication can be readily blocked by null mutations in immediate early genes, which can be trans-complemented in vitro, enabling the direct production of high-titer pure preparations of non-pathogenic vectors. The genome is large (152 kb), and many viral genes are unnecessary for replication in vitro, allowing them to be replaced with large or multiple transgenes. Latent infection with wild-type virus results in the persistence of episomal virus in the nuclei of sensory neurons throughout the host's life cycle. The vectors are non-pathogenic, cannot be reactivated, and persist long-term. Latency-active promoter complexes can be used in vector design to achieve long-term stable transgene expression in the nervous system. Due to the broad expression pattern of cell receptors recognized by the virus, HSV vectors transduce a wide range of tissues. Increased understanding of the processes involved in cell entry allows targeting of the tropism of HSV vectors.

[0288] Other viral vectors

[0289] Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p.42.

[0290] The viral vector of the present invention can be an alphavirus vector. The viral vector of the present invention can be a flavivirus vector.

[0291] Self-amplifying ssRNA viruses include alphaviruses (such as Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, and M1) and flaviviruses (such as Kunjin virus, West Nile virus, and dengue virus) with positive-sense genomes. Alphaviruses are mainly applied in preclinical gene therapy research for cancer treatment. Alphavirus vectors can be delivered in the form of naked RNA, layered plasmid DNA vectors, and recombinant replication-deficient or replication-competent particles.

[0292] The viral vector of the present invention can be a rhabdovirus vector. The viral vector of the present invention can be a measles virus vector.

[0293] Rhabdoviruses (such as rabies and vesicular stomatitis virus) and measles virus carry negative-sense genomes. Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied in preclinical gene therapy research. Many gene therapy applications have been found for measles virus (such as MV-Edm).

[0294] The viral vector of the present invention can be a Newcastle disease virus vector.

[0295] The ssRNA paramyxovirus Newcastle disease virus (NDV) replicates specifically in tumor cells and is therefore often used in cancer gene therapy.

[0296] The viral vector of the present invention can be a poxvirus vector.

[0297] Poxviruses are characterized by their dsDNA genomes, which can easily accommodate foreign DNA of more than 30 kb. Poxviruses have found various applications as gene therapy vectors. For example, vaccinia virus vectors have demonstrated potential for treating cancer. Vaccinia virus is a large enveloped poxvirus with a linear double-stranded DNA genome of approximately 190 kb. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it suitable for the delivery of large genes. Many attenuated vaccinia virus strains suitable for gene therapy applications are known in the art, such as the MVA and NYVAC strains.

[0298] The viral vector of the present invention can be a picornavirus vector.

[0299] Picornaviruses are non-enveloped ssRNA viruses. Coxsackieviruses belonging to the Picornaviridae family have been used as oncolytic vectors.

[0300] Protein coding sequence

[0301] The viral vector may contain a protein coding sequence.

[0302] The protein coding sequence can encode any polypeptide of interest, such as a therapeutic protein. For example, the protein coding sequence can encode any polypeptide associated with glomerular diseases. The protein coding sequence can encode a polypeptide involved in GBM-related hereditary glomerular diseases, such as Alport syndrome. The protein coding sequence can encode a polypeptide involved in podocyte-related hereditary glomerular diseases.

[0303] Suitably, the protein coding sequence may encode a COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3 or VEGFC polypeptide.

[0304] Suitably, the protein coding sequence encodes a polypeptide having a length of 1450 amino acids or more, 1500 amino acids or more, 1550 amino acids or more, 1600 amino acids or more or 1650 amino acids or more.

[0305] For polynucleotides encoding proteins, those skilled in the art will understand that due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. In addition, it should be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is to be expressed.

[0306] The protein coding sequence may be codon-optimized. Different cells have different usages of their specific codons. This codon bias corresponds to a bias in the relative abundance of specific tRNAs in the cell type. By changing the codons in the sequence to be customized to match the relative abundance of the corresponding tRNA, it is possible to increase expression. For the same reason, by intentionally selecting codons known to have a sparse corresponding tRNA in a particular cell type, it is possible to reduce expression. Thus, an additional degree of translational control is available. Codon usage tables for mammalian cells (e.g., human) as well as a variety of other organisms are known in the art.

[0307] The protein-coding nucleotide sequences disclosed herein may contain or lack a stop codon at their 3' end. Accordingly, the present disclosure encompasses the SEQ ID NOs. disclosed herein with or without a stop codon.

[0308] COL4A3, COL4A4, and COL4A5 polypeptides

[0309] The protein-coding sequence may encode a COL4A3, COL4A4, or COL4A5 polypeptide, or a fragment or derivative thereof.

[0310] The COL4A3, COL4A4, and COL4A5 proteins are homologous polypeptides of approximately 170 - 185 kDa, which contain collagen Gly-X-Y repeats that are frequently interrupted by non-collagen sequences and form triple-helical repeats. Each polypeptide also contains a large globular non-collagen domain at the carboxyl terminus.

[0311] Alport syndrome (AS) is caused by pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes, which result in abnormalities in the collagen IV α345 network of the basement membrane.

[0312] The COL4A3, COL4A4, or COL4A5 polypeptide, or a fragment or derivative thereof, may be capable of forming a collagen IV α345 network.

[0313] Approximately 200 - 300 amino acids may be removed from each of the COL4A3, COL4A4, and COL4A5 polypeptides to produce a truncated transgene suitable for a minigene approach. The amino acids may be removed from the triple-helical repeats. Preferably, amino acids are not removed from the non-collagen regions.

[0314] In some embodiments, the COL4A3, COL4A4, and COL4A5 polypeptides are full-length polypeptides.

[0315] Preferably, the COL4A3, COL4A4, or COL4A5 polypeptide is human. Exemplary human COL4A3 is COL4A3 with UniProtKB accession number Q01955. Exemplary human COL4A4 is COL4A3 with UniProtKB accession number P53420. Exemplary human COL4A5 is COL4A5 with UniProtKB accession number P29400.

[0316] Suitably, the COL4A3 polypeptide may comprise the polypeptide sequence shown in SEQ ID NO: 4 or a variant that is at least 70% identical to SEQ ID NO: 4 or consists thereof. 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: 4.

[0317] Suitably, the COL4A4 polypeptide may comprise the polypeptide sequence shown in SEQ ID NO: 5 or a variant that is at least 70% identical to SEQ ID NO: 5 or consist thereof. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 5.

[0318] Suitably, the COL4A5 polypeptide may comprise the polypeptide sequence shown in SEQ ID NO: 6 or a variant that is at least 70% identical to SEQ ID NO: 6 or consist thereof. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 6.

[0319]

[0320] Example COL4A3 amino acid sequence - Uniprot reference number Q01955 (SEQ ID NO: 4)

[0321]

[0322] Example COL4A4 amino acid sequence - Uniprot reference number P53420 (SEQ ID NO: 5)

[0323]

[0324] Example COL4A5 amino acid sequence - Uniprot reference number P29400 (SEQ ID NO: 6)

[0325] The protein coding sequence may comprise or consist of a COL4A3, COL4A4, or COL4A5 transgene.

[0326] An example transgene encoding COL4A3 is provided in NM_000091.5. An example transgene encoding COL4A4 is provided in NM_000092.5. An example transgene encoding COL4A5 is provided in NM_000495.5.

[0327] Suitably, the COL4A3 transgene may comprise or consist of the polynucleotide sequence shown in SEQ ID NO: 7 or a variant that is at least 70% identical to SEQ ID NO:7. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 7.

[0328] Suitably, the COL4A4 transgene may comprise or consist of the polynucleotide sequence shown in SEQ ID NO: 8 or a variant that is at least 70% identical to SEQ ID NO:8. 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: 8.

[0329] Suitably, the COL4A5 transgene may comprise or consist of the polynucleotide sequence shown in SEQ ID NO: 9 or a variant that is at least 70% identical to SEQ ID NO:9. 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: 9.

[0330]

[0331] Example COL4A3 transgenic sequence (SEQ ID NO: 7)

[0332]

[0333] Example COL4A4 transgenic sequence (SEQ ID NO: 8)

[0334]

[0335] Example COL4A5 transgenic sequence (SEQ ID NO: 9)

[0336] The COL4A3, COL4A4, or COL4A5 transgene may contain introns or intron sequences, which can be used to improve gene expression. The COL4A3, COL4A4, or COL4A5 transgene may contain a protein tag, such as a hemagglutinin (HA) tag. The HA can be used as an epitope tag and has been shown not to interfere with the biological activity or biodistribution of the protein to which HA has been added. Protein tags can facilitate the detection, isolation, and purification of the transgene. Other suitable protein tags may include Myc tags, polyhistidine tags, and flag tags.

[0337] Nephrotic syndrome (NS)-related transgene

[0338] The protein coding sequence may contain or consist of an NS-related transgene.

[0339] Nephrotic syndrome (NS) is a chronic kidney disease characterized by marked proteinuria, hypoalbuminemia, edema, and hyperlipidemia. An NS-related transgene can be a gene associated with the monogenic form of NS and expressed in podocytes.

[0340] Suitable NS-related transgenes include NPHS2, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAFB, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, and NLRP3.

[0341] NPHS2

[0342] The protein coding sequence may encode NPHS2, or a fragment and / or variant thereof.

[0343] "NPHS2" is the abbreviated name of the polypeptide encoded by the NPHS2 gene, also known as podocin. NPHS2 is a 42 kDa hairpin-like membrane-associated podocyte-specific protein, which is a key component of the protein complex at the slit diaphragm; the cell-cell junction between adjacent podocyte foot processes. It is localized to lipid rafts and interacts with other important slit diaphragm proteins such as nephrin, CD2AP, and TRPC6. It is crucial for maintaining the integrity of the slit diaphragm and the glomerular filtration barrier.

[0344] Fragments and / or variants of NPHS2 can retain NPHS2 activity or function. For example, fragments and / or variants of podocin can regulate glomerular permeability. Appropriately, fragments and / or variants of NPHS2 can have the same or similar activity or function as NPHS2, such as having 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 or function of NPHS2.

[0345] Based on the known structural and functional characteristics of NPHS2 (see, for example, Tabassum, A., et al., 2014. Interdisciplinary Sciences: Computational Life Sciences, 6(1), pp.32-39) and / or based on known variants (see, for example, NCBI Gene ID: 7827 and NCBI HomoloGene: 22826), those skilled in the art will be able to generate fragments and / or variants using conservative substitutions. Appropriately, fragments and / or variants of NPHS2 contain a transmembrane domain with two cytoplasmic domains at the N-terminus and C-terminus.

[0346] The NPHS2 gene is conserved in chimpanzee, rhesus macaque, dog, cow, mouse, and rat. NPHS2 can be human NPHS2. Appropriately, NPHS2 can contain or consist of the polypeptide sequence of UniProtKB accession number Q9NP85 or its fragments and / or variants.

[0347] In some embodiments, NPHS2 contains an amino acid sequence that is at least 70% identical to SEQ ID NO: 10 or a fragment thereof or consists of the same. Appropriately, NPHS2 contains an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10 or a fragment thereof or consists of the same.

[0348] In some embodiments, NPHS2 contains SEQ ID NO: 10 or a fragment thereof or consists of the same.

[0349] MERRARSSSRESRGRGGRTPHKENKRAKAERSGGGRGRQEAGPEPSGSGRAGTPGEPRAPAATVVDVDEVRGSGEEGTEVVALLESERPEEGTKSSGLGACEWLLVLISLLFIIMTFPFSIWFCVKVVQEYERVIIFRLGHLLPGRAKGPGLFFFLPCLDTYHKVDLRLQTLEIPFHEIVTKDMFIMEIDAICYYRMENASLLLSSLAHVSKAVQFLVQTTMKRLLAHRSLTEILLERKSIAQDAKVALDSVTCIWGIKVERIEIKDVRLPAGLQHSLAVEAEAQRQAKVRMIAAEAEKAASESLRMAAEILSGTPAAVQLRYLHTLQSLSTEKPSTVVLPLPFDLLNCLSSPSNRTQGSLPFPSPSKPVEPLNPKKKDSPML

[0350] Example NPHS2 amino acid sequence (SEQ ID NO: 10)

[0351] In some embodiments, the NPHS2 transgene comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 11, or a fragment thereof, or consists of the same. Suitably, the NPHS2 transgene comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 11, or a fragment thereof, or consists of the same.

[0352] In some embodiments, the NPHS2 transgene comprises the nucleotide sequence SEQ ID NO: 11, or a fragment thereof, or consists of the same.

[0353]

[0354] Example NPHS2 transgenic sequence (SEQ ID NO: 11)

[0355] In some embodiments, the NPHS2 transgenic comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 12 or a fragment thereof or consists of the same. Suitably, the NPHS2 transgenic comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 12 or a fragment thereof or consists of the same.

[0356] In some embodiments, the NPHS2 transgenic comprises the nucleotide sequence SEQ ID NO: 12 or a fragment thereof or consists of the same.

[0357]

[0358] Example NPHS2 transgenic sequence (SEQ ID NO: 12)

[0359] Vascular endothelial growth factor (VEGF)C transgene

[0360] The protein coding sequence may comprise or consist of a vascular endothelial growth factor (VEGF)C transgene.

[0361] VEGFC is a lymphangiogenic growth factor known to signal via two receptors, VEGFR-3 (Flt4) and VEGFR-2 (Flk4). VEGFC is produced by cells in a propeptide precursor form that dimerizes before being cleaved into a tetramer.

[0362] The VEGFC transgene may comprise a polynucleotide encoding any form of VEGFC, such as the propeptide precursor form, the tetramer form, an intermediate form, or fully processed mature VEGFC.

[0363] If desired, polynucleotides encoding different forms of VEGFC polypeptides may be used in any combination. Preferably, the VEGFC transgene comprises a polynucleotide encoding one or more polypeptides having VEGFC biological activity (i.e., a peptide that can bind to and activate VEGFR-2 and / or VEGRF-3). More preferably, the VEGFC transgene comprises a polynucleotide encoding a polypeptide that contains a VEGFC homology domain and has 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.

[0364] The VEGFC polynucleotide may comprise the VEGFC open reading frame (ORF) sequence of SEQ ID NO: 13. The VEGFC polynucleotide may comprise a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VEGFC ORF sequence of SEQ ID NO: 13. Variant sequences may encode a VEGFC polypeptide that retains the ability to bind to and activate VEGFR-2 and VEGFR-3.

[0365]

[0366] Example VEGFC polynucleotide (SEQ ID NO: 13)

[0367] Complement proteins and complement inhibitors

[0368] The protein coding sequence may encode a complement protein, or a fragment and / or variant thereof.

[0369] As used herein, "complement protein" is a protein that is part of the complement system. The complement system, also known as the complement cascade, is a central part of innate immunity and serves as the first line of defense against foreign and altered host cells. The complement system consists of plasma proteins produced mainly by the liver or membrane proteins expressed on the cell surface. Complement acts in plasma, tissues, or intracellularly. Complement proteins act in concert as a cascade to opsonize pathogens and induce a series of inflammatory responses, helping immune cells to combat infection and maintain homeostasis (Merle, N.S., et al., 2015. Frontiers in immunology, 6, 262).

[0370] There are three pathways of complement activation: the classical pathway, the alternative pathway, and the lectin pathway. The target recognition mechanisms of the three complement pathways are different, but converge in activating the central complement C3. After this activation, C5 is cleaved and the assembly of the membrane attack complex (MAC) is initiated. The enzymatic cleavage of C3 and C5 results in the production and release of the anaphylatoxins C3a and C5a.

[0371] Suitably, the complement protein is selected from the following table: CFI, CFH, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, and clusterin, or a fragment and / or variant thereof.

[0372] The protein coding sequence may encode an inhibitor of the complement system, or a fragment and / or variant thereof.

[0373] As used herein, "inhibitor of the complement system" or "complement inhibitor" is a protein that prevents the activation of the complement system. Complement is tightly controlled by these inhibitors, which naturally protect self-cells and tissues from unwanted complement activation. Complement inhibitors can regulate complement activation at different stages of the classical pathway, lectin pathway, and alternative pathway. Suitably, the complement inhibitor is a naturally occurring complement inhibitor, or a fragment and / or variant thereof. Preferably, the inhibitor of the complement system is a human complement system inhibitor.

[0374] Complement inhibitors are divided into two categories: soluble inhibitors and membrane-bound inhibitors. Preferably, the complement system inhibitor is a soluble complement inhibitor. Soluble complement inhibitors include C1 inhibitor (C1INH), complement factor I (CFI), complement factor H (CFH), complement factor H-like protein 1 (FHL-1), C4-binding protein (C4BP), clusterin, and vitronectin. Membrane-bound regulators include CD46, CD55, CD59, CD35, and CUB and sushi multiple domain 1 (CSMD1).

[0375] Inhibitors of the complement system may be selected from: CFI, CFH, FHL-1, C1INH, C4BP, CD46, CD55, CD59, CD35, vitronectin, clusterin, and CSMD1, or fragments and / or variants thereof.

[0376] Preferably, inhibitors of the complement system are selected from: CFI, CFH, and FHL-1, or fragments and / or variants thereof.

[0377] CFI

[0378] The protein coding sequence may encode CFI, or a fragment and / or variant thereof.

[0379] Complement factor I (CFI) is a trypsin-like serine protease that inhibits the complement system by cleaving three peptide bonds in the α-chain of C3b and two bonds in the α-chain of C4b, thereby inactivating these proteins.

[0380] CFI is a glycoprotein heterodimer composed of a heavy chain and a light chain linked by a disulfide bond. The heavy chain has four domains: the FI membrane attack complex (FIMAC) domain, the CD5 domain, and the low density lipoprotein receptor 1 and 2 (LDLr1 and LDLr2) domains. The heavy chain plays an inhibitory role in maintaining the enzyme in an inactive state until it encounters a complex formed by the substrate (C3b or C4b) and a cofactor protein (factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein). When the enzyme binds to the substrate:cofactor complex, the heavy chain:light chain interface is disrupted, and the enzyme is activated allosterically. 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 the specific cleavage of C3b and C4b.

[0381] CFI or a fragment and / or variant thereof may be able to cleave C3b into iC3b and / or may be able to cleave iC3b into C3d, g.

[0382] Fragments and / or variants of CFI can retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the C3b inactivating and iC3b degrading activities of native CFI. The C3b inactivating and iC3b degrading activities of CFI and fragments and / or variants of native CFI can be determined using any suitable method known to those skilled in the art. For example, a proteolytic assay can be used.

[0383] Preferably, CFI is human CFI. An exemplary human CFI is the CFI having the UniProtKB accession number P05156.

[0384] Suitably, CFI can comprise the polynucleotide sequence shown in SEQ ID NO: 14, or a variant that is at least 70% identical to SEQ ID NO: 14 or consists thereof.

[0385] MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSCDKVFCQPWQRCIEGTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLECLHPGTKFLNNGTCTAEGKFSVSLKHGNTDSEGIVEVKLVDQDKTMFICKSSWSMREANVACLDLGFQQGADTQRRFKLSDLSINSTECLHVHCRGLETSLAECTFTKRRTMGYQDFADVVCYTQKADSPMDDFFQCVNGKYISQMKACDGINDCGDQSDELCCKACQGKGFHCKSGVCIPSQYQCNGEVDCITGEDEVGCAGFASVTQEETEILTADMDAERRRIKSLLPKLSCGVKNRMHIRRKRIVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTVVDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLISNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVSWGENCGKPEFPGVYTKVANYFDWISYHVGRPFISQYNV

[0386] Exemplary CFI polypeptide sequence (SEQ ID NO: 14)

[0387] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 14.

[0388] An exemplary nucleotide sequence encoding CFI is NM_000204.5. Suitably, the protein coding sequence encoding CFI may comprise the polynucleotide sequence set forth in SEQ ID NO: 15, or a variant that is at least 70% identical to SEQ ID NO: 15 or consists thereof.

[0389]

[0390] Exemplary CFI polynucleotide sequence (SEQ ID NO: 15)

[0391] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 15.

[0392] CFH

[0393] The protein coding sequence may encode CFH, or a fragment and / or variant thereof.

[0394] Complement factor H (CFH) regulates complement activation on self cells and surfaces. CFH competes with complement factor B (CFB) for binding to C3b, serves as a cofactor for the CFI-catalyzed proteolytic cleavage of C3b, and accelerates the irreversible dissociation of C3bBb and C3b2Bb into their individual components. Thus, CFH not only inhibits the formation of convertases, but also shortens the lifespan of any formed convertase complexes.

[0395] CFH is a large (155 kDa) soluble glycoprotein. CFH is composed of a total of 20 domains, each domain containing approximately 60 amino acid residues, called complement control protein modules (CCP) or short consensus repeats, which are linked by short linkers consisting of 3 - 8 residues. The CCP modules are numbered 1 - 20 (from the N-terminus of the protein): CCP 1 - 4 and CCP 19 - 20 bind to C3b, while CCP 7 and CCP 19 - 20 bind to GAG and sialic acid.

[0396] CFH or a fragment and / or variant thereof may be capable of binding C3b and / or C3d; and / or serving as a cofactor for the CFI-catalyzed proteolytic cleavage of C3b; and / or increasing the irreversible dissociation of C3bBb and C3b2Bb into their individual 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 CFH and fragments and / or variants of native CFH can be determined using any suitable method known to those skilled in the art.

[0397] Preferably, CFH is human CFH. An exemplary human CFH is the CFH with UniProtKB accession number P08603.

[0398] Suitably, CFH may comprise the polynucleotide sequence shown in SEQ ID NO: 16, or a variant at least 70% identical to SEQ ID NO: 16 or consisting thereof.

[0399]

[0400] Exemplary CFH polypeptide sequence (SEQ ID NO: 16)

[0401] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 16.

[0402] An exemplary nucleotide sequence encoding CFH is NM_000186.4. Suitably, the protein-coding sequence encoding CFH may comprise the polynucleotide sequence shown in SEQ ID NO: 17, or a variant that is at least 70% identical to SEQ ID NO: 17 or consists thereof.

[0403]

[0404] Exemplary CFH polynucleotide sequence (SEQ ID NO: 17)

[0405] 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: 17.

[0406] The CFH fragment can be a splice variant. For example, complement factor H-like protein 1 (FHL-1) is a splice variant of the CFH gene and is nearly identical to the N-terminal 7 domains (CCP 1-7) of CFH.

[0407] FHL-1

[0408] The protein coding sequence can encode FHL-1, or a fragment and / or variant thereof.

[0409] FHL-1 or a fragment and / or variant thereof may be able to bind C3b and / or C3d. The fragment and / or variant of FHL-1 may retain at least 50%, 60%, 70%, 80%, 90%, 95% or 100% of the activity of native FHL-1. The activity of FHL-1 and the fragment and / or variant of native FHL-1 can be determined using any suitable method known to those skilled in the art.

[0410] Preferably, FHL-1 is human FHL-1. An exemplary human FHL-1 is the FHL-1 having the NCBI reference sequence: NP_001014975.1.

[0411] Suitably, FHL-1 may comprise the polypeptide sequence shown in SEQ ID NO: 18, or a variant at least 70% identical to SEQ ID NO: 18 or consisting thereof.

[0412] MRLLAKIICLMLWAICVAEDCNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPLRKCQKRPCGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPICEVVKCLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKEKPKCVEISCKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSCEEKSCDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRCTLKPCDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGWSPAVPCLRKCYFPYLENGYNQNHGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRCIRVSFTL

[0413] Example FHL-1 polypeptide sequence (SEQ ID NO: 18)

[0414] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 18.

[0415] An example nucleotide sequence encoding FHL-1 is NM_001014975.2. Suitably, the protein-coding sequence encoding FHL-1 may comprise the polynucleotide sequence shown in SEQ ID NO: 19, or a variant at least 70% identical thereto or consisting thereof.

[0416]

[0417] Example FHL-1 polynucleotide sequence (SEQ ID NO: 19)

[0418] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to SEQ ID NO: 19.

[0419] Gene editing agent

[0420] In a preferred embodiment, the protein-coding sequence does not encode a gene editing agent.

[0421] As used herein, "gene editing agent" may refer to a protein that can edit genomic sequences (see, e.g., Raguram, A., Banskota, S. and Liu, D.R., 2022. Therapeutic in vivo delivery of gene editing agents. Cell). Gene editing reagents may be rare-cutting endonucleases, including meganucleases, zinc finger nucleases, TAL effector nucleases or CRISPR endonucleases.

[0422] In some embodiments, the protein-coding sequence does not encode a nuclease. A "nuclease" is an enzyme that can cleave the phosphodiester bonds present within a polynucleotide chain. Suitably, the nuclease is an endonuclease. An endonuclease is capable of breaking the bond from the middle of the chain.

[0423] In some embodiments, the protein-coding sequence does not encode an RNA-guided nuclease. An "RNA-guided nuclease" is a nuclease that can be guided to a specific locus by a guide RNA (see, e.g., Murugan, K., et al., 2017. Molecular cell, 68(1), pp.15-25). RNA-guided nucleases include, but are not limited to, type II CRISPR nucleases such as Cas9, and type V CRISPR nucleases such as Cas12a and Cas12b, and other nucleases derived therefrom. Broadly, RNA-guided nucleases can be defined according to their PAM specificity and cleavage activity.

[0424] In some embodiments, the protein-coding sequence does not encode a type II CRISPR nuclease. In some embodiments, the protein-coding sequence does not encode a Cas9 nuclease. Cas9 is a dual RNA-guided endonuclease associated with the clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system. Cas9 nucleases include well-characterized orthologs from Streptococcus pyogenes (SpCas9). SpCas9 and other orthologs (including SaCas9, FnCa9, and AnaCas9) have been reviewed by Jiang, F. and Doudna, J.A., 2017. Annual review of biophysics, 46, pp.505-529.

[0425] The term "gene editing agent" can also include guide RNAs. A "guide RNA" (gRNA) confers target sequence specificity to an RNA-guided nuclease. A guide RNA is a non-coding short RNA sequence that binds to a complementary target DNA sequence. For example, in the CRISPR / Cas9 system, the guide RNA first binds to the Cas9 enzyme, and the gRNA sequence directs the resulting complex to a specific location on the DNA via base pairing, where Cas9 performs its nuclease activity by cleaving the target DNA strand. The term "guide RNA" encompasses any suitable gRNA that can be used with any RNA-guided nuclease, not just those that are compatible with a specific nuclease such as Cas9.

[0426] Regulatory element

[0427] The viral vectors of the present invention can comprise one or more regulatory sequences that can act either pre-transcriptionally or post-transcriptionally. Suitably, the protein-coding sequence can be operably linked to one or more regulatory sequences. The one or more regulatory sequences can promote the expression of the protein in podocytes.

[0428] A "regulatory sequence" is any sequence that promotes the expression of a polypeptide, such as by acting to increase the expression of a transcript or enhance mRNA stability. Suitable regulatory sequences include, for example, promoters, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites.

[0429] Promoter

[0430] The viral vectors of the present invention can comprise a promoter. Suitably, the promoter can be operably linked to the protein-coding sequence. The promoter can promote the expression of the protein in podocytes.

[0431] A "promoter" is a DNA region that causes the initiation of gene transcription. The promoter is located near the transcription start site of the gene, upstream of the DNA (towards the 5' region of the sense strand). Any suitable promoter can be used and can be readily selected by a person skilled in the art.

[0432] The promoter can be a constitutive promoter or a tissue-specific promoter.

[0433] Suitable constitutive promoters are known to those skilled in the art. For example, in one embodiment, the promoter is the CMV promoter.

[0434] Preferably, the viral vector of the present invention comprises a podocyte-specific promoter. Suitably, the protein-coding sequence is operably linked to the podocyte-specific promoter.

[0435] As used herein, a "podocyte-specific promoter" is a promoter that preferably promotes gene expression in podocytes. Suitably, compared to other cell types, the podocyte-specific promoter can promote higher gene expression in podocytes. Higher expression in podocytes can be measured, for example, by measuring the expression of a transgene such as GFP that is operably linked to the promoter, where the expression of the transgene in podocytes is related to the ability of the promoter to promote gene expression in podocytes. For example, a podocyte-specific promoter can be a promoter that promotes gene expression levels at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500% or at least 1000% higher in podocytes compared to the expression levels in other cell types.

[0436] Suitable podocyte-specific promoters will be well known to those skilled in the art.

[0437] Suitably, the podocyte-specific promoter can be or can be derived from a promoter associated with a gene that is selectively expressed in human podocytes. Genes that are selectively expressed in podocytes will be known to those skilled in the art, and selective gene expression in podocytes can be readily determined by methods known to those skilled in the art, such as using microarrays. Genes that are selectively expressed in podocytes include NPHS1, NPHS2, WT1, FOXC2, ABCA9, ACPP, ACTN4, ADM, ANGPTL2, ANXA1, ASB15, ATP8B1, B3GALT2, BB014433, BMP7, C1QTNF1, CAR13, CD2AP, CD55, CD59A, CD59B, CDC14A, CDH3, CDKN1B, CDKN1C, CEP85L, CLIC3, CLIC5, COL4A1, COL4A2, COL4A3, COL4A4, COL4A5, COLEC12, CRIM1, CST12, DEGS1, DOCK4, DOCK5, EGF, ENPEP, EPHX1, FAM81A, FAT1, FGFBP1, FOXD1, FRYL, GABRB1, GALC, GM10554, H2-D1, H2-Q7, H2BC4, H3C15, HS3ST3A1, HTRA1, IFNGR1, IL18, ILDR2, ITGB5, ITGB8, KIRREL, LAMA1, LAMA5, LAMB1, LAMB2, LMX1B, MAFB, MAGI2, MELA, MERTK, MGAT4A, MYO1D, MYO1E, MYOM2, MYZAP, NEBL, NES, NOD1, NPR3, NR2F2, NUPR1, OPTN, P3H2, PAK1, PARD3B, PDPN, PLAT, PLCE1, PLSCR2, PODXL, PROS1, PTPRO, RAB3B, RDH1, RDH9, SDC4, SEMA3E, SERPINB6B, SH3BGRL2, SLC41A2, SLCO2A1, ST3GAL6, SYNPO, TDRD5, THSD7A, TIMP3, TJP1, TLR7, TM4SF1, TMEM108, TMEM54, TMTC1, TOP1MT, TRAV10, TRAV10N, TRAV5-4, TSHB, UACA, UBA1Y, UPRT, VEGFA, VTCN1, ZBTB20 and 5730407I07RIK.

[0438] Methods for identifying promoter regions associated with genes will be well known to those skilled in the art. Promoters are typically located near or coincide with the transcription start site and contain multiple sequence motifs with which transcription factors (TFs) interact in a sequence-specific manner.

[0439] Suitably, the podocyte-specific promoter is selected from the NPHS1 promoter, NPHS2 promoter, WT1 promoter, FOXC2 promoter, ABCA9 promoter, ACPP promoter, ACTN4 promoter, ADM promoter, ANGPTL2 promoter, ANXA1 promoter, ASB15 promoter, ATP8B1 promoter, B3GALT2 promoter, BB014433 promoter, BMP7 promoter, C1QTNF1 promoter, CAR13 promoter, CD2AP promoter, CD55 promoter, CD59A promoter, CD59B promoter, CDC14A promoter, CDH3 promoter, CDKN1B promoter, CDKN1C promoter, CEP85L promoter, CLIC3 promoter, CLIC5 promoter, COL4A1 promoter, COL4A2 promoter, COL4A3 promoter, COL4A4 promoter, COL4A5 promoter, COLEC12 promoter, CRIM1 promoter, CST12 promoter, DEGS1 promoter, DOCK4 promoter, DOCK5 promoter, EGF promoter, ENPEP promoter, EPHX1 promoter, FAM81A promoter, FAT1 promoter, FGFBP1 promoter, FOXD1 promoter, FRYL promoter, GABRB1 promoter, GALC promoter, GM10554 promoter, H2-D1 promoter, H2-Q7 promoter, H2BC4 promoter, H3C15 promoter, HS3ST3A1 promoter, HTRA1 promoter, IFNGR1 promoter, IL18 promoter, ILDR2 promoter, ITGB5 promoter, ITGB8 promoter, KIRREL promoter, LAMA1 promoter, LAMA5 promoter, LAMB1 promoter, LAMB2 promoter, LMX1B promoter, MAFB promoter, MAGI2 promoter, MELA promoter, MERTK promoter, MGAT4A promoter, MYO1D promoter, MYO1E promoter, MYOM2 promoter, MYZAP promoter, NEBL promoter, NES promoter, NOD1 promoter, NPR3 promoter, NR2F2 promoter, NUPR1 promoter, OPTN promoter, P3H2 promoter, PAK1 promoter, PARD3B promoter, PDPN promoter, PLAT promoter, PLCE1 promoter, PLSCR2 promoter, PODXL promoter, PROS1 promoter, PTPRO promoter, RAB3B promoter, RDH1 promoter, RDH9 promoter, SDC4 promoter, SEMA3E promoter, SERPINB6B promoter, SH3BGRL2 promoter, SLC41A2 promoter, SLCO2A1 promoter, ST3GAL6 promoter, SYNPO promoter, TDRD5 promoter, THSD7A promoter, TIMP3 promoter,The TJP1 promoter, TLR7 promoter, TM4SF1 promoter, TMEM108 promoter, TMEM54 promoter, TMTC1 promoter, TOP1MT promoter, TRAV10 promoter, TRAV10N promoter, TRAV5-4 promoter, TSHB promoter, UACA promoter, UBA1Y promoter, UPRT promoter, VEGFA promoter, VTCN1 promoter, ZBTB20 promoter, and 5730407I07RIK promoter, or a fragment or derivative thereof.

[0440] Suitably, the podocyte-specific promoter is selected from the NPHS1 promoter, NPHS2 promoter, WT1 promoter, FOXC2 promoter, ACTN4 promoter, BMP7 promoter, CD2AP promoter, CDH3 promoter, CDKN1B promoter, CDKN1C promoter, COL4A1 promoter, COL4A2 promoter, COL4A3 promoter, COL4A4 promoter, COL4A5 promoter, CRIM1 promoter, FAT1 promoter, FOXD1 promoter, KIRREL promoter, LAMA1 promoter, LAMA5 promoter, LAMB1 promoter, LAMB2 promoter, LMX1B promoter, MAFB promoter, NES promoter, NR2F2 promoter, PODXL promoter, PTPRO promoter, SYNPO promoter, TJP1 promoter, and VEGFA promoter, or a fragment or derivative thereof.

[0441] Suitably, the podocyte-specific promoter is the NPHS1 promoter, NPHS2 promoter, WT1 promoter, or FOXC2 promoter, or a fragment or derivative thereof.

[0442] Preferably, the podocyte-specific promoter is the NPHS1 promoter or NPHS2 promoter, or a fragment or derivative thereof. More preferably, the podocyte-specific promoter is the NPHS1 promoter, or a fragment or derivative thereof.

[0443] The podocyte-specific promoter can be a minimal podocyte-specific promoter. As used herein, "minimal podocyte-specific promoter" means the smallest sequence that can act as a podocyte-specific promoter.

[0444] Preferably, the podocyte-specific promoter is the minimal NPHS1 promoter or minimal NPHS2 promoter, or a fragment or derivative thereof. More preferably, the podocyte-specific promoter is the minimal NPHS1 promoter, or a fragment or derivative thereof.

[0445] Preferably, the promoter is a human promoter, such as the minimal human NPHS1 promoter.

[0446] NPHS1 promoter

[0447] The viral vector of the present invention may comprise the NPHS1 promoter, or a fragment or derivative thereof. Suitably, the NPHS1 promoter, or a fragment or derivative thereof, may be operably linked to a protein coding sequence.

[0448] The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes.

[0449] The NPHS1 promoter may be a minimal NPHS1 promoter. For example, the NPHS1 promoter may have a length of 1.2 kb or less.

[0450] The minimal 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 minimal NPHS1 is a 1.2 kb fragment and appears to be podocyte-specific. The 1.2 kb promoter region lacks a TATA box but has recognition motifs for other transcription factors, such as the PAX-2 binding element, E-box, and GATA consensus sequences.

[0451] Suitably, the NPHS1 promoter may comprise the nucleotide sequence shown in SEQ ID NO: 20 or a variant that is at least 70% identical to SEQ ID NO:20 or consist thereof.

[0452]

[0453] Exemplary minimal NPHS1 promoter (SEQ ID NO: 20)

[0454] Optionally, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 20.

[0455] In some embodiments, the NPHS1 promoter may comprise the nucleotide sequence shown in SEQ ID NO: 21 or a variant that is at least 70% identical to SEQ ID NO: 21 or consist thereof.

[0456] GGCCCTGGGGTCACGGAGGCTGGGGAGGCACCGAGGAACGCGCCTGGCATGTGCTGACAGGGGATTTTATGCTCCAGGAGCAAGACAGAGAGAGATACTCACAGGGAAGAGGGGAAGAGGAAAACGAGAAAGGGAGGAGAGTAACGGAAAGAGATAAAAAAGAAAAGCAGGTGGCAGAGACACACAGAGAGGGACCCAGAGAAAGCCAGACAGACGCAGGTGGCTGGCAGCGGGCGCTGTGGGGGTCACAGTAGGGGGACCTGTG

[0457] Exemplary minimal NPHS1 promoter - 265 bp (SEQ ID NO: 21)

[0458] Optionally, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 21. The NPHS1 promoter may comprise or consist of a variant of SEQ ID NO: 21 as shown in SEQ ID NO: 22 or SEQ ID NO: 23.

[0459] GGCCCTGGGGTCACGGAGGCTGGGGAGGCACCGAGGAACGCGCCTGGCATGTGCTGACAGGGAATTTTATGCTCCAGGAGCAAGACAGAGAGAGACACTCACAGGGAAGAGGGGAAGAGGAAAACGAGAAAGGGAGGAGAGTAACGGAAAGAGATAAAAAAGAAAAGCAGGTGGCAGAGACACAGAGAGAGGGACCCAGAGAAAGCCAGACAGACGCAGGTGGCTGGCAGCGGGCGCTGTGGGGGTCACAGTAGGGGGACCTGTC

[0460] Exemplary minimal nephrin promoter - 265 bp (SEQ ID NO: 22)

[0461] GGCCCTGGGGTCACGGAGGCTGGGGAGGCACCGAGGAACGCGCCTGGCATGTGCTGACAGGGGATTTTATGCTCCAGGAGCAAGACAGAGAGAGATACTCACAGGGAAGAGGGGAAGAGGAAAACGAGAAAGGGAGGAGAGTAACGGAAAGAGATAAAAAAGAAAAGCAGGTGGCAGAGACACAGAGAGAGGGACCCAGAGAAAGCCAGACAGACGCAGGTGGCTGGCAGCGGGCGCTGTGGGGGTCACAGTAGGGGGACCTGTC

[0462] Exemplary minimal nephrin promoter variant - 265 bp (SEQ ID NO: 23)

[0463] In some embodiments, the NPHS1 promoter may comprise the nucleotide sequence shown in SEQ ID NO: 22 or 23, or a variant that is at least 70% identical to SEQ ID NO: 22 or 23, or consist thereof. Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 22 or 23.

[0464] NPHS2 promoter

[0465] The viral vector of the present invention may comprise the NPHS2 promoter, or a fragment or derivative thereof. Suitably, the NPHS2 promoter, or a fragment or derivative thereof, may be operably linked to a protein coding sequence.

[0466] The NPHS2 gene encodes podocin, which is selectively expressed in podocytes.

[0467] The NPHS2 promoter can be a minimal NPHS2 promoter. For example, the NPHS1 promoter can have a length of 0.6 kb or shorter.

[0468] The minimal human NPHS2 promoter has been described in Oleggini R, et al., 2006. Gene Expr. 13(1):59–66. This minimal NPHS2 is a 630 bp fragment, which has been shown to be expressed in podocytes in vitro.

[0469] Suitably, the NPHS2 promoter may comprise the nucleotide sequence shown in SEQ ID NO: 24 or a variant that is at least 70% identical to SEQ ID NO:24 or consist thereof.

[0470] ggaaagttggggatgaggcgaaatttctgattttaccttaaagtgaccctaattcgatgaccttttgtggtttttttcttttttcttttttcttttttacttggccctgcccaagcaggacctaaaaacaaacagacaaaaaaggttactaacaactgttcctctccacgaaaatctgcagtaaaaggtaaaagatgtattcgttttgaagagaaaccagagcttgcgatgagcttctgtatctccgtcagccctctagcatgacattaggaaccctccaggagatgagtcttcacagcccgggttggcacctgcagacacgcacttttcaacgcccgcaccctgcccggggccggctctcccacccaggcctctctctgcttcagcgccgccccggccgtgggagtcggcgggcgcagtccacagctccaccaagacacagctgtcggggttccgggtgcgccccgcccgcggccccggtgtcccgcccctcgccctcagcccccacccgacggtctttagggtcccccgggcacgccacgcggacccgcagcgactccacagggactgcgctcccgtgcccctagcgctcccgcgctgctgctccagccgcccggcagctctgacc

[0471] Exemplary minimal NPHS2 promoter (SEQ ID NO: 24)

[0472] Suitably, the variant can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 24.

[0473] Enhancer

[0474] The viral vector of the present invention can comprise an enhancer. Suitably, the enhancer can be operably linked to the protein coding sequence. The enhancer can promote the expression of the protein in podocytes.

[0475] An "enhancer" is a DNA region that can be bound by a protein (activator) to increase the likelihood of transcription of a particular gene occurring. Enhancers are cis-acting. They can be located upstream or downstream of the start site, up to 1 Mbp (1,000,000 bp) from the gene. Any suitable enhancer can be used and can be readily selected by a person skilled in the art.

[0476] The viral vector of the present invention can comprise a podocyte-specific enhancer. Suitably, the enhancer can be operably linked to the protein coding sequence.

[0477] As used herein, a "podocyte-specific enhancer" is an enhancer that preferentially promotes the expression of a gene in podocytes. Suitably, compared to other cell types, a podocyte-specific enhancer can promote higher expression of a gene in podocytes. Higher expression in podocytes can be measured, for example, by measuring the expression of a transgene such as GFP that is operably linked to the enhancer, wherein the expression of the transgene in podocytes is related to the ability of the enhancer to promote the expression of the gene in podocytes. For example, a podocyte-specific enhancer can be an enhancer that promotes a gene expression level in podocytes that is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher or at least 1000% higher compared to the expression level in other cell types.

[0478] Suitable podocyte-specific enhancers will be well known to those skilled in the art.

[0479] Suitably, the podocyte-specific enhancer can be or can be derived from an enhancer associated with a gene that has selective expression in human podocytes. Methods for identifying enhancer regions associated with genes will be well known to those skilled in the art.

[0480] Preferably, the podocyte-specific enhancer is the NPHS1 enhancer or the NPHS2 enhancer, or a fragment or derivative thereof. More preferably, the podocyte-specific enhancer is the NPHS1 enhancer, or a fragment or derivative thereof.

[0481] Preferably, the enhancer is a human enhancer, such as the human NPHS1 enhancer.

[0482] The enhancer can be used together with the corresponding promoter. For example, the NPHS1 enhancer can be used together with the NPHS1 promoter. Alternatively, the enhancer can be used together with a different promoter, such as a promoter that is not podocyte-specific, such as the hsp promoter.

[0483] The viral vector of the present invention can comprise a promoter-enhancer. Suitably, the promoter-enhancer can be operably linked to a protein coding sequence. The promoter-enhancer can promote the expression of the protein in podocytes. The promoter-enhancer can be a podocyte-specific promoter-enhancer. The promoter-enhancer can be the NPHS1 promoter-enhancer or the NPHS2 promoter-enhancer, or a fragment or derivative thereof.

[0484] NPHS1 enhancer

[0485] The NPHS1 enhancer has been described in Guo, G., et al., 2004. Journal of the American Society of Nephrology, 15(11), pp.2851-2856. When an 186-bp fragment from the human NPHS1 promoter is placed in front of a heterologous minimal promoter in transgenic mice, it is capable of directing podocyte-specific expression of the β-galactosidase transgene.

[0486] Suitably, the NPHS1 enhancer can comprise the nucleotide sequence shown in SEQ ID NO: 25 or a variant that is at least 70% identical to SEQ ID NO: 25 or consist thereof.

[0487] ctgctgagctgggagaccaccttgatctgacttctcccatcttcccagcctaagccaggccctggggtcacggaggctggggaggcaccgaggaacgcgcctggcatgtgctgacaggggattttatgctccagctgggccagctgggaggagcctgctgggcagaggccagagctgggggctctg

[0488] Exemplary NPHS1 enhancer (SEQ ID NO: 25)

[0489] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 25.

[0490] Kozak sequence

[0491] The viral vector of the present invention may comprise a Kozak sequence. Suitably, the Kozak sequence may be operably linked to the protein coding sequence. The Kozak sequence may be inserted before the start codon of the protein to enhance the initiation of translation.

[0492] Suitable Kozak sequences will be well known to those skilled in the art.

[0493] Suitably, the Kozak sequence may comprise the nucleotide sequence shown in SEQ ID NO: 26 or a variant that is at least 65% identical to SEQ ID NO: 26 or consists thereof.

[0494] GCCGCCACCAUGG

[0495] Exemplary Kozak sequence (SEQ ID NO: 26)

[0496] Suitably, the variant may be at least 75%, at least 85% or at least 90% identical to SEQ ID NO: 26.

[0497] Post-transcriptional regulatory element

[0498] The viral vector of the present invention may comprise a post-transcriptional regulatory element. Suitably, the post-transcriptional regulatory element may be operably linked to the protein coding sequence. The post-transcriptional regulatory element may enhance gene expression.

[0499] The viral vector may comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Suitably, the WPRE may be operably linked to the protein coding sequence.

[0500] The WPRE sequence may have mutations within the X-antigen promoter and / or the start codon of the X-antigen. This may prevent the production of functional X-antigen.

[0501] Suitably, the WPRE may comprise the nucleotide sequence shown in SEQ ID NO: 27, or a variant that is at least 70% identical to SEQ ID NO: 27 or consists thereof.

[0502] aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgc

[0503] Example WPRE (SEQ ID NO: 27)

[0504] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO: 27.

[0505] Polyadenylation signal

[0506] The viral vector of the present invention may comprise a polyadenylation signal. Suitably, the polyadenylation signal may be operably linked to the protein coding sequence. The polyadenylation signal may enhance gene expression.

[0507] Suitable polyadenylation signals include the early SV40 polyadenylation signal (SV40pA), the bovine growth hormone polyadenylation signal (bGH), or the soluble neuropilin-1 polyadenylation signal. Preferably, the polyadenylation signal is the bGH polyadenylation signal or the soluble neuropilin-1 polyadenylation signal.

[0508] Suitably, the polyadenylation signal may comprise the nucleotide sequence shown in SEQ ID NO: 28, or a variant that is at least 70% identical to SEQ ID NO: 28 or consists thereof.

[0509] ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatgg

[0510] Example bGH poly(A) signal sequence (SEQ ID NO: 28)

[0511] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO: 28.

[0512] Suitably, the polyadenylation signal may comprise the nucleotide sequence shown in SEQ ID NO: 29, or a variant that is at least 70% identical to SEQ ID NO: 29 or consists thereof.

[0513] aaataaaatacgaaatg

[0514] Example soluble neuropilin-1 polyadenylation signal (SEQ ID NO: 29)

[0515] Suitably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical to SEQ ID NO: 29.

[0516] Methods of treatment

[0517] In one aspect, the present invention provides a method of treatment, which comprises delivering a therapeutically effective amount of a viral vector according to the method of the present invention.

[0518] In one aspect, the present invention provides a method of treating and / or preventing a kidney disease in a subject in need thereof, which comprises delivering a therapeutically effective amount of a viral vector according to the method of the present invention.

[0519] In one aspect, the present invention provides a viral vector for therapy, wherein the viral vector is delivered by a method according to the present invention.

[0520] In one aspect, the present invention provides a viral vector for treating and / or preventing kidney diseases in a subject, wherein the viral vector is delivered by a method according to the present invention.

[0521] In one aspect, the present invention provides the use of a viral vector for the preparation of a medicament, wherein the medicament is delivered by a method according to the present invention.

[0522] In one aspect, the present invention provides the use of a viral vector for the preparation of a medicament for treating and / or preventing kidney diseases in a subject, wherein the medicament is delivered by a method according to the present invention.

[0523] The subject can be any subject described herein. For example, the kidney disease can be a glomerular disease. For example, the kidney disease can be a podocyte-related glomerular disease. For example, the kidney disease can be a GBM-related glomerular disease.

[0524] The kidney disease can be a genetic kidney disease (see, e.g., Hildebrandt, F., 2010. The Lancet, 375(9722), pp.1287-1295). The viral vector can correspond to the kidney disease to be treated and / or prevented. For example, when the kidney disease is Alport syndrome, the viral vector can encode COL4A3, COL4A4 or COL4A5. For example, when the kidney disease is nephrotic syndrome, the viral vector can encode an NS-related transgene. For example, when the kidney disease is complement-related, the viral vector can encode a complement protein.

[0525] In some embodiments, the kidney disease is a genetic glomerular disease. Genetic glomerular diseases include podocyte-related genetic glomerular diseases such as nephrotic syndrome and GBM-related glomerular diseases such as Alport syndrome.

[0526] In some embodiments, the kidney disease is a podocyte-related genetic glomerular disease. Podocyte-related genetic glomerular diseases include Finnish congenital nephrotic syndrome, type 2 congenital nephrotic syndrome, type 3 familial nephrotic syndrome, Frasier syndrome and Denys-Drash syndrome, Schimke immuno-osseous dysplasia, nephrotic syndrome caused by CD2AP mutations, nephrotic syndrome caused by actin 4 mutations, nephrotic syndrome caused by TRPC6 mutations, and Epstein and Fechtner syndromes. Suitably, the glomerular disease is nephrotic syndrome.

[0527] In some embodiments, the kidney disease is a GBM-related hereditary glomerular disease. GBM-related hereditary glomerular diseases include X-linked Alport syndrome, autosomal recessive Alport syndrome, autosomal dominant Alport syndrome, thin basement membrane disease, 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.

[0528] 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), stx-associated HUS, lupus nephritis, cryoglobulinemia, anti-GBM disease, ANCA-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated renal transplant rejection, membranous nephropathy, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

[0529] In some embodiments, the kidney disease is diabetic nephropathy.

[0530] In some embodiments, the kidney disease is Fabry disease.

[0531] Variants, derivatives, analogs, homologs, and fragments

[0532] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses variants, derivatives, homologs, and fragments thereof.

[0533] In the context of the present invention, a "variant" of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a way that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement, and / or variation of at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0534] As used herein, the term "derivative" with respect to a protein or polypeptide of the present invention includes any substitution, variation, modification, replacement, deletion, and / or addition to one (or more) amino acid residues of the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0535] Generally, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

[0536] The proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues, which produce silent changes and result in functionally equivalent proteins. Intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the residues, so 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; amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0537] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column and preferably in the same row in the third column can be substituted for each other:

[0538]

[0539] As used herein, the term "homolog" refers to a variant having a certain degree of homology to a wild-type amino acid sequence or wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[0540] In the present context, homologous sequences are considered to include amino acid sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, preferably at least 95%, 96% or 97% or 98% or 99% identical. Generally, homologs will contain the same active sites and the like as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0541] In the present context, homologous sequences are considered to include nucleotide sequences that are at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, preferably at least 95%, 96% or 97% or 98% or 99% identical. Although homology can also be considered in terms of similarity, in the context of the present invention, homology is preferably expressed in terms of sequence identity.

[0542] Preferably, a sequence referred to as having a percent identity to any of the SEQ ID NOs detailed herein refers to a sequence having the said percent identity over the full length of the SEQ ID NO mentioned.

[0543] Homology comparisons can be made visually or, more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0544] The percent homology can be calculated for contiguous sequences by aligning one sequence with another and directly comparing each amino acid or nucleotide in one sequence with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "gapless" alignment. Usually, such gapless alignments are carried out only on a relatively small number of residues.

[0545] While this is a very simple and consistent method, it does not take into account, for example, that in pairs of sequences that are otherwise identical, an insertion or deletion in the amino acid or nucleotide sequence may cause subsequent residues or codons to be misaligned, and thus potentially lead to a substantial reduction in the percent homology when a global alignment is carried out. Thus, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0546] However, these more complex methods assign a "gap penalty" to each gap that appears in the alignment, such that for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) will receive a higher score than a sequence alignment with many gaps. An "affine gap penalty" is commonly used, which charges a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is: -12 for the gap and -4 for each extension.

[0547] Thus, calculation of the maximum percent homology first requires generation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid – Ch.18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641), and the GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequence, can also be used to compare protein and nucleotide sequences (FEMS Microbiol. Lett. (1999)174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0548] Although the final percent homology can be measured based on identity, the alignment process itself is generally not based on an all-or-none pairing comparison. Instead, a weighted similarity scoring matrix is typically used, assigning scores for each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix that is commonly used is the BLOSUM62 matrix (the default matrix for the BLAST program suite). The GCG Wisconsin program typically uses either the common default values or a custom symbol comparison table (if provided) (for more details, see the user manual). For some applications, it is preferred to use the common default values of the GCG package, or in the case of other software, the default matrix such as BLOSUM62.

[0549] Once the software has generated an optimal alignment, the percent homology, preferably the percent sequence identity, can be calculated. The software generally does this as part of the sequence comparison and generates a numerical result. The percent sequence identity can be calculated as the percentage of the number of identical residues to the total number of residues of the SEQ ID NO mentioned.

[0550] "Fragment" is also a variant, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of functional interest or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is part of a full-length polypeptide or polynucleotide.

[0551] Such variants, derivatives, homologs, and fragments can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When an insertion is to be made, synthetic DNA encoding the insert and 5' and 3' flanking regions corresponding to the naturally occurring sequences on either side of the insertion site can be prepared. The flanking regions will contain convenient restriction sites corresponding to the sites in the naturally occurring sequences such that the sequences can be cleaved with appropriate enzymes and the synthetic DNA ligated into the incision. The DNA is then expressed according to the invention to prepare the encoded protein. These methods illustrate only many of the standard techniques known in the art for manipulating DNA sequences, and other known techniques can also be used.

[0552] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the capabilities of those of ordinary skill in the art. These 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 hereby incorporated by reference. Examples

[0553] The present invention will now be further described by way of examples, which are intended to assist those of ordinary skill in the art in practicing the invention and are not intended to limit the scope of the invention in any way.

[0554] Example 1 - Direct renal artery injection of an AAV vector encoding podocin under the control of the hNPHS1 promoter

[0555] This study was designed to explore the renal transduction efficiency of rAAV products in healthy pigs when administered via direct renal artery injection (dRAi) with renal artery occlusion, while also testing the biodistribution of the injected rAAV in key organs and tissues.

[0556] Materials and methods

[0557] A schematic diagram of the AAV vector expressing human HA-tagged podocin under the human full-length nephrin (hNPHS1) promoter is shown in Figure 1 and the cassette was encapsulated into the AAV-LK03 serotype for use in the study.

[0558] rAAV was prepared via triple plasmid transfection in HEK293T cells. The virus was incubated for 72 hours after transfection. Virus particles were purified using density gradient separation, which means eradicating / removing most of the empty capsids from the final preparation. The virus was sterile filtered through a 0.2 micron filter. The rAAV product was stored at -80 o °C until use. The vehicle was phosphate buffered saline (PBS).

[0559] The doses of the rAAV products administered to four male pigs are summarized in Table 1. The viral vector was dosed according to body weight (target body weight at dosing was 10 kg).

[0560] Table 1 Dosing schedule and treatment

[0561]

[0562] Before Day 0, all animals were given prophylactic antibiotics twice daily in the feed, and 2 animals were also pre-treated with 1 mg / kg prednisolone. On the day of treatment, the test product was thawed and then formulated in PBS at ambient room temperature to a maximum volume of 5 mL / kg. Standard procedures for surgery and anesthesia were followed, which included Hartmann’s IV fluid therapy.

[0563] Surgery for dRAi with balloon occlusion involved accessing the left renal artery via a percutaneous approach and inflating a balloon to occlude the renal artery before injecting the rAAV test product. After confirming arterial occlusion, the study treatment was administered distal to the balloon for approximately 15 - 20 minutes without using an infusion pump.

[0564] The steps involved in the final surgery for the rAAV test product via dRAi are summarized as follows:

[0565] 1. Carotid artery catheterization

[0566] 2. Introducing a sheath into the carotid artery

[0567] 3. Introduce the guide wire into the renal artery

[0568] 4. Introduce the occlusion balloon catheter into the renal artery

[0569] 5. Balloon-occlude the renal artery by inflating the balloon (for 15 - 20 minutes)

[0570] 6. Use contrast injection and imaging to check the occlusion (angiography from the caudal vein with added contrast to show blood flow and recirculation)

[0571] 7. Flush the contrast agent

[0572] 8. Inject the rAAV test article directly into the renal artery

[0573] 9. Flush with saline to ensure no test article residue remains in the catheter

[0574] 10. Deflate the balloon catheter

[0575] 11. Slowly withdraw the balloon catheter to avoid damaging the blood vessel

[0576] 12. Subsequently withdraw the sheath and close

[0577] Then allow the animal to recover and return to a single enclosure for 24 hours, then return to the group enclosure after removing its carotid artery catheter. Animals numbered 3 to 6 were given post - anesthesia analgesia, IV buprenorphine (20 mg / kg). Throughout the study, all animals were given prophylactic antibiotics in the feed twice a day. Two animals were given prednisolone (10 mg) once a day until termination, and the remaining 2 animals did not receive the drug. Approximately 4 weeks after treatment, euthanasia was performed with sodium pentobarbital (10 - 15 mL) at the surgical anesthesia level.

[0578] Observe the general health status / mortality and moribund state of the animals daily. Record clinical observations and body weight upon arrival at the test facility, then at Day - 1, Day 0, and Weeks 1 - 4. Record pain assessment scores from Day 1 to Day 5 after treatment.

[0579] Collect kidney (cortex and medulla) and other tissue samples (liver, spleen, pancreas) for the following evaluations: immunohistochemistry and histology; western blot; RNA; DNA; and immunofluorescence.

[0580] Results

[0581] This study shows that rAAV AAVLK03 - hPodocin can be successfully delivered to glomeruli, and more specifically to podocytes, via dRAi + occlusion.

[0582] Transcriptomic analysis

[0583] qPCR analysis was performed to find bGH expression in 2 control animals and 4 animals treated with AAVdRAi expressing HA-tagged podocin via the left renal artery to evaluate transduction efficiency in the kidney (by comparing the injected left kidney with the untreated right kidney) and biodistribution (pancreas, liver, spleen). The bGH mRNA expression of each organ was normalized to that of the same organ in the control animals.

[0584] Figure 2 Transcriptomic analysis is shown, where mRNA bGH detection in the left kidney cortex (LKC) of dRAi-treated animals was increased compared to the right kidney cortex (RKC). No significant changes were detected in the expression levels in other organs except the pancreas, where 2 dRAi-treated animals showed an approximately 40 - 45-fold increase in bGH mRNA levels. When LKC was normalized to the left kidney medulla (LKM) of the same animal, all 4 dRAi-treated pigs showed relatively high levels of bGH expression compared to their wild-type counterparts (2 control animals).

[0585] Based on the data generated from the second round of transcriptomic analysis, mRNA bGH expression in the LKC of injected animals was increased compared to RKC or LKM / RKM or other organs. Kidney transcriptomic analysis demonstrated the following trend: LKC > LKM > RKC > RKM, as expected.

[0586] Immunofluorescence study

[0587] To visualize the local penetration and persistence of the virus in the expected regions, glomerular immunofluorescence studies were performed to detect the levels of HA-tagged podocin in the glomeruli of 2 control animals and 4 rAAV-treated animals at the time of sacrifice.

[0588] Figure 3 Successful delivery of dRAi by rAAV was demonstrated by detecting HA-tagged podocin in the LKC and more specifically delivery to the target cells, i.e., podocytes, as shown by the co-localization of nephrin (podocyte-specific protein) and HA-tagged podocin in the middle panel (animal number 6 LKC) compared to untreated (no dRAi) control animal number 2 or animal number 6 RKC. Other animals also underwent immunofluorescence analysis; however, only animal number 2 LKC (control), rAAV-treated animal number 6 (LKC and RKC) are highlighted as representative images.

[0589] Immunofluorescence studies further confirmed that HA-tagged podocin was localized to podocytes in the glomeruli of injected animals compared to non-injected or control pigs.

[0590] In vivo observation / measurement

[0591] There were no deaths during the study. During the study, no AAVLK03-hPodocin-related effects were observed in any of the treated animals by clinical observation or monitoring. During the study, no AAVLK03-hPodocin-related effects on body weight were observed in any of the treated animals.

[0592] Example 2 - Direct renal artery injection of an AAV vector encoding eGFP under the control of the CMV promoter

[0593] This study was designed to compare the renal transduction efficiency of using an rAAV test article in healthy Göttingen pigs when administered via dRAi (with renal artery occlusion) versus IV systemic administration, while also testing the biodistribution of the injected rAAV in key organs and tissues.

[0594] Materials and methods

[0595] A schematic diagram of the AAV vector expressing eGFP under the CMV promoter is shown in Figure 4 . eGFP is widely used as a reporter molecule for gene expression due to its ability to be detected after blue light excitation. The expression of the eGFP protein in this study was driven by the human CMV major promoter, which is known for its ability to produce high levels of recombinant protein in mammalian cells. The cassette was encapsulated into the AAV-LK03 serotype for use in the study.

[0596] rAAV was prepared by triple plasmid transfection in HEK293T cells. The virus was incubated for 72 hours after transfection. The virus particles were purified using density gradient separation, which means eradicating / removing most of the empty capsids from the final preparation. The virus was sterile filtered through a 0.2 micron filter. The rAAV test article was stored at -80 o °C until use. The vehicle was phosphate buffered saline (PBS) buffer (pH 7.4) supplemented with 200 mM NaCl and 0.001% Pluronic F-68, formulated to a maximum volume of 15 mL.

[0597] The doses of the rAAV test article administered in 3 groups are summarized in Table 2, where each group had 3 animals and 1 animal was used as a control animal that did not receive any rAAV test article. The viral vector was dosed according to body weight, and the same fixed volume (15 mL) was used for all experimental animals. The total ischemia time included the flushing step as the flushing was also performed under occlusion in the dRAi+O arm.

[0598] Table 2 Dosing regimens and treatments

[0599]

[0600] The "high" dose was calculated as 5.7×10 12 vg / swine, and the "low" dose was calculated as 5.7×10 11 vg / swine.

[0601] Differences in renal artery anatomy and animal size were anticipated, which would alter the total amount of AAV required. Therefore, to increase consistency, Göttingen minipigs were selected for this study because these pigs are derived from a closed colony and are inbred, and are also known to show low levels of neutralizing antibodies against AAV. All animals were of 4.5 months of age of a single sex (female) and were fed a weight management diet prior to study treatment to limit growth to a body weight of 10 - 12 kg.

[0602] Prior to Day 0, animals were pre - treated prophylactically (3 or 5 days) with amoxicillin (an antibiotic). Prior to Day 0 and surgery, animals were pre - anesthetized. On the day of treatment, the test article was thawed and then formulated in PBS at ambient room temperature to a maximum volume of 15 mL. Standard procedures for surgery and anesthesia were followed, which included Hartmann’s IV fluid therapy.

[0603] Surgery for dRAi with balloon occlusion involved accessing the renal artery of the first kidney via a percutaneous approach and inflating the occlusion balloon catheter to occlude the renal artery for 15 - 20 minutes prior to injection of the rAAV test article. After confirmation of arterial occlusion, the study treatment was administered distal to the balloon via an infusion pump for approximately 15 - 20 minutes.

[0604] The steps involved in surgery for the rAAV test article via dRAi+O are summarized as follows:

[0605] 1. Catheterization of the common femoral artery (CFA) was performed according to standard procedures

[0606] 2. A sheath was introduced into the CFA

[0607] 3. A guide wire was introduced into the renal artery

[0608] 4. An occlusion balloon catheter was introduced into the renal artery

[0609] 5. The renal artery was balloon - occluded by inflating the balloon (15 - 20 minutes)

[0610] 6. Contrast agent injection and imaging were used to check for complete occlusion (angiography from the caudal vein, and contrast agent was added to visualize blood flow and recirculation)

[0611] 7. Flushing with Plasma - Lyte and heparin

[0612] 8. Under non-flow conditions, the rAAV test article was directly injected / infused into the true artery using an infusion pump.

[0613] 9. Flush with saline / PBS to ensure no residual rAAV test article in the catheter.

[0614] 10. Deflate the balloon catheter.

[0615] 11. Slowly withdraw the balloon catheter to avoid damaging the blood vessel.

[0616] 12. Subsequently, withdraw the sheath and close.

[0617] Due to surgical complications (see Table 2), two animals (pigs No. 2 and No. 5) in the low rAAV dose dRAi group were injected into the right kidney instead of the left kidney.

[0618] For the IV of the rAAV test article, the total dose was injected via the jugular vein within 15 minutes. Control animals were not treated with the rAAV test article. However, they were anesthetized to add a line to allow blood sample collection.

[0619] All animals were given post-anesthetic analgesia, IV buprenorphine (20 mg / kg). Pig No. 2 received additional post-operative analgesia (day 0) with acetaminophen (10 mg / kg IV), fentanyl patch (50 mg for 3 days), and then oral acetaminophen 10 mg / kg on day 1. Approximately 4 weeks after treatment, euthanasia was performed with sodium pentobarbital (10 - 15 mL) at the surgical anesthesia level.

[0620] The general health status / mortality and moribund state of the animals were observed. Clinical observations and body weights were recorded upon arrival at the test facility, and then on days -1, 0, and 1 - 4 weeks. Pain assessment scores were recorded from day 1 to day 5 after surgery. Blood samples were collected at the following 3 time points for hematological and biochemical analysis: before intervention (0 hr), day 1 (24 hr), and at termination.

[0621] Kidney (cortex and medulla) and other tissue samples (liver, pancreas, colon) were collected for the following evaluations: immunohistochemistry and histology; RNA (ribonucleic acid); DNA (deoxyribonucleic acid); and immunofluorescence. Brain, lung, heart, and spleen samples were collected and stored for possible future analysis.

[0622] Results

[0623] This study showed that direct renal artery injection (dRAi) with occlusion led to rAAV-mediated transgene transduction and subsequent GFP expression, and GFP localization in glomeruli in the injected kidney was detected by immunofluorescence.

[0624] No safety issues associated with the surgery of blocked dRAi were observed in this study. In all animals administered via this route of administration, no observable injury, fibrosis, necrosis, or sclerosis was found after histological evaluation of all animal samples.

[0625] Immunofluorescence analysis showed consistent and clear GFP expression in the injected left kidney cortex (LKC) of animals that underwent high rAAV dose dRAi. Additionally, moderate GFP expression was observed on the injected side in the cortex of low dose dRAi animals (Pig No. 2 and Pig No. 5 - injected into the right kidney, Pig No. 8 - left kidney). In contrast, after IV administration, no GFP expression was observed in the LKC or right kidney cortex (RKC), except for Pig No. 6 in the high rAAV dose IV group.

[0626] Review of the data from the qPCR results further supported the conclusions of the immunofluorescence images. These results indicated elevated expression in the LKC (injected) of the high rAAV dose dRAi group and slight expression in the RKC of the low rAAV dose dRAi group. This analysis also demonstrated no GFP mRNA expression in the pancreas and colon.

[0627] Importantly, in any of the animals in the high and low rAAV dose dRAi groups, there was no evidence of liver GFP expression at the protein or mRNA levels. In contrast, expression at the protein and mRNA levels was observed in the livers of high rAAV dose IV animals.

[0628] Transcriptomic analysis

[0629] qPCR analysis was performed to analyze mRNA GFP expression in 1 control animal and 6 animals (dRAi - high dose and low dose) injected via the renal artery with rAAV (serotype LK03) expressing GFP driven by the CMV promoter to evaluate the transduction efficiency in the animal kidneys (by comparing the rAAV - injected kidney with the contralateral non - injected kidney) and biodistribution (liver, colon, pancreas). There was an approximately 80 - fold average relative change in GFP in the high rAAV dose (dRAi vs. IV) and an approximately 20 - fold change in the low rAAV dose (dRAi vs. IV) in the injected renal cortex, as Figure 5 shown.

[0630] qPCR analysis was also performed to analyze mRNA GFP expression in 3 animals administered the rAAV test article via IV injection to compare the expression profiles with the dRAi group. The bGH mRNA expression in each organ was normalized to the bGH expression in the same organ of the control animal ( Figure 6)。Therefore, the resulting presentation compares the injected kidney with the contralateral non-injected kidney. For completeness, transduction efficiency data via GFP from the left and right kidneys are presented.

[0631] Immunofluorescence study

[0632] After organ sectioning, 5 - 6 images were taken per sample. The adrenal cortex and medulla were identified by visual inspection of the bisected whole kidney, and the section area was randomly selected (1 section per kidney). The sections shown here are representative samples of all examined sections.

[0633] In animals injected via the left renal artery, most GFP protein expression was found in glomeruli. In some images, GFP was observed to co - localize with the podocyte - specific marker nephrin in a focal linear distribution (linear staining in focal areas indicates co - localization) (using pigs No. 1 and No. 8 as examples) ( Figure 7 ). In contrast, the high - dose rAAV IV injection group demonstrated no kidney GFP protein expression in 2 animals, although one animal (pig No. 6) showed some expression in glomeruli, which seemed much less than in other groups. Due to surgical complications, pigs No. 2 and No. 5 were injected via the right kidney. Therefore, as expected, the animals were injected in the right kidney, and no GFP expression was observed in the left renal cortex and medulla.

[0634] To confirm the widespread expression of GFP in the left renal cortex of animals injected with rAAV via the left renal artery, immunofluorescence analysis was performed in the contralateral kidney, more specifically in the right renal cortex and medulla. Almost no expression was observed in the right cortex of animals injected via the left renal artery ( Figure 8 ), indicating that most GFP expression was localized to the injected left renal cortex. In 2 animals injected with the low - dose rAAV test article via the right artery, GFP signals were observed in podocytes and other cells within glomeruli in the injected kidney. In contrast, the IV injection group showed no GFP protein expression in either kidney of 2 animals, and one animal showed some expression in glomeruli, but far less than in the low - dose group.

[0635] To further confirm kidney localization, images from the high rAAV dose dRAi group were further magnified to show key regions carrying GFP expression within glomeruli ( Figure 9 ). GFP signals were shown to be localized deep within glomeruli where podocytes are located.

[0636] Immunofluorescence analysis of porcine livers was performed after administration of rAAV test articles expressing GFP protein under the CMV promoter by intravenous injection (high dose) and renal artery injection (dRAi - high and low doses). All porcine liver sections imaged after administration of high and low dose dRAi were GFP negative( Figure 10 ). In contrast, GFP was observed in porcine liver sections after rAAV IV injection. The results showed that GFP expression was observed only in the livers of IV animals when compared to animals injected with rAAV via dRAi (high and low doses).

[0637] In vivo observation / measurement

[0638] There were no deaths during the study. No relevant effects of AAV-LK03-eGFP on clinical observations or monitoring were observed in any treatment group during the study. No relevant effects of AAV-LK03-eGFP on body weight were observed in any treatment group during the study.

[0639] Clinical pathology

[0640] All clinical pathology results obtained were within the expected normal range for the same breed of Göttingen minipig. All controls were within range, the analysis was valid, and correct results were obtained.

[0641] Immunohistochemistry and histology

[0642] Sections stained with Masson's trichrome from control animals and all 3 rAAV treatment groups (high dose IV, high dose dRAi, and low dose dRAi) showed normal amounts and distribution of collagen fibers in glomeruli and between renal tubules, with no abnormal accumulation of collagen or fibrin.

[0643] Sections stained with PAS from control animals and rAAV-treated animals showed normal glomeruli, without any significant thickening of the GBM or mesangial cell deposition. The Bowman's capsule cavity and proximal tubules were clearly demarcated. In glomeruli and renal tubules, all other conditions showed normal histology. No rAAV-treated animals developed focal segmental glomerulosclerosis, and no glomeruli were affected by sclerosis.

[0644] Example 3 - Direct renal artery injection of AAV-LK03 vector encoding GFP under the control of the hNPHS1 promoter

[0645] This study was designed to explore the localization of gene expression within glomeruli of the renal cortex using rAAV vectors in healthy pigs when administered by direct renal artery injection with (dRAi+O) or without (dRAi) renal artery occlusion compared to an IV control.

[0646] Materials and methods

[0647] A schematic diagram of an AAV vector expressing GFP under the human full-length nephrin (hNPHS1) promoter is shown in Figure 11 A (hereafter referred to as PS0528). The cassette was encapsulated into the AAV-LK03 serotype for research use.

[0648] rAAV was prepared by triple plasmid transfection in HEK293T cells. After transfection, the virus was incubated for 72 hours. Virus particles were purified using density gradient separation. The virus was sterile filtered through a 0.2 micron filter. The rAAV vector was stored at -80 o °C until use.

[0649] Female Göttingen minipigs were acclimated to the environment for 3 weeks before the start of the study, were between 4.5 and 6.5 months of age at the time of dosing, and weighed between 10 and 15 kg. Water was freely available, while food was provided twice daily.

[0650] Female Göttingen pigs were dosed as shown in Table 3 below, and the study design is also presented as a Figure 11 schematic diagram in B.

[0651] Table 3. Dosing Regimen and Treatments

[0652]

[0653] *Due to an error in dose administration, one pig in Group 2 received a double dose (2x10 13 vg). This pig was excluded from all analyses.

[0654] Each pig received a single kidney. This kidney was designated as the left kidney, unless there were challenges in assessing the renal artery architecture by angiography, in which case the right kidney was dosed. After induction of general anesthesia, the interventional procedures for dRAi (renal artery delivery) and dRAi+O (renal artery delivery with occlusion) were performed as detailed below. Before administration, the vector was diluted in saline (0.9% sodium chloride) to the volume detailed in Table 3 above.

[0655] Local delivery (dRAi / dRAi+O):

[0656] · Catheterization of the common femoral artery (CFA) was performed according to standard procedures

[0657] · A short sheath was introduced into the CFA

[0658] · A guide wire was advanced into the renal artery

[0659] · An occlusion balloon catheter (for dRAi+O) or a diagnostic catheter (for dRAi) was introduced into the renal artery

[0660] ·For dRAi+O only: Balloon occlude the renal artery by inflating the balloon (target total occlusion time <20 minutes)

[0661] ·For dRAi+O only: Use contrast injection and imaging to check for complete occlusion

[0662] ·Administer heparinized saline flush

[0663] ·Use an infusion pump to directly infuse the rAAV test article into the renal artery, set the delivery to 1 mL / min for 15 minutes (dRAi+O) or 2 mL / min for 4 minutes (dRAi)

[0664] ·Administer heparinized saline flush

[0665] ·For dRAi+O only: Deflate the balloon catheter

[0666] ·Withdraw the balloon (dRAi+O) catheter or diagnostic (dRAi) catheter

[0667] ·Withdraw the short sheath

[0668] ·Apply pressure at the access site until hemostasis is achieved

[0669] Then allow the animal to recover and observe daily until the end of the study. Pigs are sacrificed at 28 - 30 days after dosing for tissue collection. At necropsy, kidney (cortex and medulla) sections are excised and fixed in formalin, frozen, or embedded in OCT blocks for the following evaluations: histology, immunohistochemistry, immunofluorescence, RNAscope, ELISA, qPCR, and RTqPCR.

[0670] Results

[0671] Biodistribution

[0672] Thaw frozen renal cortex from treated and untreated kidney samples, extract DNA and analyze by qPCR using primers and probes targeting WPRE.

[0673] Figure 12 A - B shows the AAV genomes detected per milligram (mg) of tissue. In treated kidneys ( Figure 12 A), at a dose level of 1x10 13 the amount of AAV genomes detected using dRAi is more than 7 - fold higher for local delivery compared to IV, and more than 23 - fold higher using dRAi+O. The amount of genomes detected in untreated kidneys increases with increasing administered dose ( Figure 12 B).

[0674] This data demonstrates that local delivery increases the retention of the vector within the dosed kidney.

[0675] RNAscope in situ hybridization (ISH)

[0676] Using a set of probes specific for WPRE, the local presence of AAV mRNA in renal cortical samples of paraffin-embedded formalin-fixed tissues was evaluated by RNAscope in situ hybridization. Stained sections were scanned and images were analyzed visually to determine the proportion of glomeruli with detectable WPRE mRNA. The amount of mRNA-positive areas was quantified and plotted as a frequency distribution.

[0677] Figure 13 A - D show the amount of mRNA-positive areas present in glomeruli of treated kidneys from pigs administered via dRAi (direct renal artery injection without occlusion) or IV. Seventy to 120 glomeruli were quantified per pig. Compared to 1x10 13 vg delivered via IV administration, dRAi delivery of 5x10 12 vg and 1x10 13 vg resulted in a significant increase in the amount of mRNA area per glomerulus.

[0678] This data indicates that local delivery of AAV to the kidney significantly increases glomerular transduction and gene expression.

[0679] Figure 14 A - C show the amount of mRNA-positive areas present in glomeruli of treated kidneys from pigs administered via dRAi + O (direct renal artery injection with occlusion) or IV. Seventy to 120 glomeruli were quantified per pig. Compared to 1x10 13 vg delivered via IV administration, dRAi + O delivery of 1x10 13 vg and 2x10 13 vg resulted in a significant increase in the amount of mRNA area per glomerulus.

[0680] Overall, Figure 13 and Figure 14 show that local delivery to the kidney, with or without blood flow interruption, results in significantly enhanced glomerular transduction and gene expression compared to systemic administration. This result is consistent across a variety of different delivery volumes, infusion rates, and AAV vector concentrations.

[0681] Example 4 - Direct renal artery injection of AAV-LK03 vector encoding podocin under the control of the hNPHS1 promoter

[0682] This study was designed to explore the localization of glomerular gene expression in the renal cortex using rAAV vectors in healthy pigs when administered via direct renal artery injection with renal artery occlusion (dRAi+O), and the selective transduction of podocytes within glomeruli.

[0683] Materials and methods

[0684] A schematic diagram of the AAV vector expressing human HA-tagged podocin under the human full-length nephrin (hNPHS1) promoter is shown in Figure 15 A (hereafter referred to as PS0438). The cassette was encapsulated into the AAV-LK03 serotype for use in the study.

[0685] rAAV was prepared by triple plasmid transfection in HEK293T cells. After transfection, the virus was incubated for 72 hours. Virus particles were purified using density gradient separation. The virus was sterile filtered through a 0.2 micron filter. The rAAV vector was stored at -80 o °C until use.

[0686] Female Göttingen minipigs were acclimated for 2 weeks prior to the start of the study, were between 23 and 25 weeks of age at the time of dosing, and weighed between 11 and 15 kg. Water was freely available, while food was provided twice daily.

[0687] Female Göttingen pigs were dosed as shown in Table 4 below, and the study design is also represented as a schematic diagram in Figure 15 B.

[0688] Table 4 Dosing regimens and treatments

[0689]

[0690] In this study, each pig was dosed with a single kidney. This kidney was designated as the left kidney, unless there was a challenge to the renal artery architecture as assessed by angiography, in which case the right kidney was dosed. After induction of general anesthesia, the intervention procedure for dRAi+O was performed as follows. Prior to administration, the vector was diluted in saline (0.9% sodium chloride) to the volume detailed in Table 4 above.

[0691] Local delivery (dRAi+O):

[0692] · Catheterization of the common femoral artery (CFA) was performed according to standard procedures

[0693] · A short sheath was introduced into the CFA

[0694] · A guide wire was advanced into the renal artery

[0695] · An occlusion balloon catheter was advanced into the renal artery

[0696] ·Occlude the renal artery by balloon inflation (target total occlusion time <20 minutes)

[0697] ·Use contrast agent injection and imaging to check for complete occlusion

[0698] ·Administer heparinized saline flush

[0699] ·Directly infuse the rAAV test article into the renal artery using an infusion pump, set the delivery to 1 mL / min for 15 minutes

[0700] ·Administer heparinized saline flush

[0701] ·Deflate the balloon catheter

[0702] ·Withdraw the balloon catheter

[0703] ·Withdraw the short sheath

[0704] ·Apply pressure at the entry site until hemostasis is achieved

[0705] Then allow the animal to recover and observe daily until the end of the study. The pigs were sacrificed 27 days after dosing. At autopsy, the kidneys (cortex and medulla), liver, and spleen were excised and sections were fixed in formalin, frozen, or embedded in OCT blocks for the following evaluations: histology, immunohistochemistry, immunofluorescence, RNAscope, ELISA, qPCR, and RTqPCR.

[0706] Results

[0707] Biodistribution

[0708] Thaw the frozen renal cortex, spleen, and liver samples from treated and untreated kidneys, extract DNA, and analyze by qPCR using primers and probes targeting WPRE.

[0709] Figure 16 A - D show the AAV genomes detected per milligram (mg) of tissue. Per mg of tissue, the number of genomes detected in the treated kidneys of pigs dosed with PS0438 was 50 to 150 - fold higher than in untreated kidneys, 75 to 430 - fold higher than in the liver, and more than 580 - fold higher than in the spleen.

[0710] This data demonstrates that local delivery to the kidney increases the retention of the vector in the dosed kidney.

[0711] RNAscope in situ hybridization (ISH)

[0712] The local presence of AAV mRNA in renal cortical samples of paraffin-embedded formalin-fixed tissues was evaluated by RNAscope in situ hybridization using a set of WPRE-specific probes. Stained sections were scanned and images were analyzed visually to determine the proportion of glomeruli with detectable WPRE mRNA. The amount of mRNA-positive area was quantified and plotted as a frequency distribution.

[0713] Figure 17 A - B show the amount of mRNA-positive area present in glomeruli of treated and untreated kidneys from pigs administered PS0438 via dRAi+O. 77 to 120 glomeruli were quantified per pig. dRAi+O delivery of 1x10 13 vg resulted in significantly higher mRNA expression in treated kidneys compared to untreated kidneys, localized within glomeruli.

[0714] This data indicates that local delivery significantly increased glomerular transduction and gene expression in treated kidneys.

[0715] Immunofluorescence

[0716] Immunofluorescence (IF) analysis was performed on sections obtained from OCT blocks of the renal cortex. Blocks were sectioned from kidneys locally administered PS0438 in pigs. To evaluate the localization of expression of the HA-tagged podocin therapeutic gene encoded by the AAV vector, multiple markers were utilized. Sections were stained with DAPI and antibodies targeting WT-1 (a podocyte-specific transcription factor), HA (a protein tag present on AAV-encoded podocin), and nephrin (a podocyte-specific slit diaphragm protein that co-localizes with podocin in podocytes).

[0717] Figure 18 A shows a composite IF image of two glomeruli and single-channel images showing HA-tagged podocin ( Figure 18 B) and nephrin ( Figure 18 C). Figure 18 D shows a composite IF image of one glomerulus and single-channel images showing HA-tagged podocin ( Figure 18 E) and nephrin ( Figure 18 F). Co-localization of podocin and nephrin, as well as podocyte-specific WT-1 nuclear staining, was observed, demonstrating podocyte-specific expression of the podocin therapeutic gene.

[0718] Overall, this data demonstrates that local delivery of AAV to the kidney results in successful transduction of podocytes and production of the therapeutic gene.

[0719] Podocin ELISA

[0720] Cryopreserved sections of renal cortex from kidneys harvested from pigs treated with PS0438 were thawed, homogenized and the lysates were analyzed by ELISA using a commercially available kit to quantify podocin. Total protein was also quantified by BCA assay.

[0721] Figure 19 Podocin detected by ELISA in renal cortex samples is shown in ng / mg total protein. Untreated control pigs (UTC) exhibited endogenous levels of podocin, however, levels of podocin 1.7 - 3.8 fold higher were detectable in the cortex of kidneys from pigs administered PS0438 locally.

[0722] Overall, Figure 18 and Figure 19 show that local delivery of an AAV vector expressing podocin (PS0438) to the kidney results in podocyte-specific expression of the therapeutic transgene at levels higher than the endogenous expression level.

[0723] Example 5 - Direct renal artery injection of an AAV9 vector encoding GFP under the control of the CMV promoter

[0724] Three female nude mice were injected with AAV9-CMV-GFP via IV injection at a dose of 1.5x10 12 vg or by direct injection into the renal artery (dRAi) at a dose of 7.5x10 11 vg, or were injected with PBS as a negative control.

[0725] GFP expression in the renal cortex was determined using immunofluorescence and the results are shown in Figure 20 . Direct renal artery injection (dRAi) resulted in higher transduction of glomerular cells than IV administration ( Figure 20 A), even when a larger dose was administered via IV, confirming what was observed in the previous pig study. GFP co-localized with NPHS1 and PDGFb, which confirmed localization in podocytes and mesangial cells respectively ( Figure 20 B).

[0726] GFP expression in the liver was determined by western blot analysis and densitometry and the results are shown in Figure 21 A - B. Quantification of the band density of the western blot ( Figure 21 A) demonstrated that higher GFP expression was observed in the liver following IV administration of the AAV vector compared to direct renal artery injection (dRAi) of the AAV vector ( Figure 21 B).

[0727] This data shows that local delivery of the AAV9 vector to the kidney results in kidney-specific expression in podocytes and mesangial cells.

[0728] Embodiment

[0729] The various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (para).

[0730] 1. A method of delivering a viral vector to a subject's kidney, the method comprising:

[0731] (A) inserting a catheter into the renal artery;

[0732] (b) optionally inflating a balloon to occlude the renal artery; and

[0733] (c) injecting or infusing the viral vector into the renal artery via the catheter.

[0734] 2. The method according to paragraph 1, wherein the method is a minimally invasive procedure.

[0735] 3. The method according to paragraph 1 or 2, wherein the method does not include the step of occluding the renal vein.

[0736] 4. The method according to any one of the preceding paragraphs, wherein the method does not include the step of inserting a catheter into the renal vein and / or does not include the step of directly inserting a catheter into the aorta.

[0737] 5. The method according to any one of the preceding paragraphs, wherein the method does not include the step of clamping the renal artery, renal vein or aorta.

[0738] 6. The method according to any one of the preceding paragraphs, wherein the catheter is an occlusion balloon catheter.

[0739] 7. The method according to any one of the preceding paragraphs, wherein the catheter is inserted into the renal artery via a percutaneous approach.

[0740] 8. The method according to paragraph 7, wherein the percutaneous approach is via the carotid artery or via the femoral artery.

[0741] 9. The method according to paragraph 7 or 8, wherein the insertion of the catheter via the percutaneous approach is facilitated using a sheath.

[0742] 10. The method according to any one of the preceding paragraphs, wherein the catheter is inserted into the renal artery via a guide wire.

[0743] 11. The method according to any one of the preceding paragraphs, wherein the renal artery is occluded for about 1 minute to about 25 minutes.

[0744] 12. The method according to paragraph 11, wherein the renal artery is occluded for about 2 minutes to about 10 minutes, optionally about 5 minutes.

[0745] 13. The method according to paragraph 11, wherein the renal artery is occluded for about 15 minutes to about 25 minutes or about 15 minutes to about 20 minutes, optionally about 20 minutes.

[0746] 14. The method according to any one of the preceding paragraphs, wherein the viral vector is infused into the renal artery under no-flow conditions using an infusion pump.

[0747] 15. The method according to any one of the preceding paragraphs, wherein the viral vector is injected or infused into the renal artery within about 1 minute to about 25 minutes.

[0748] 16. The method according to paragraph 15, wherein the viral vector is injected or infused into the renal artery within about 1 minute to about 5 minutes, optionally about 2 minutes.

[0749] 17. The method according to paragraph 15, wherein the viral vector is injected or infused into the renal artery within about 15 minutes to about 20 minutes, optionally about 17 minutes.

[0750] 18. The method according to any one of the preceding paragraphs, wherein the method results in the delivery of the viral vector to the renal cortex and / or renal medulla, preferably the method results in the delivery of the viral vector to the renal cortex.

[0751] 19. The method according to any one of the preceding paragraphs, wherein the method results in the delivery of the viral vector to the glomeruli, preferably wherein the method results in the delivery of the viral vector to renal podocytes.

[0752] 20. The method according to any one of the preceding paragraphs, wherein the method results in the kidney-specific delivery of the viral vector.

[0753] 21. The method according to any one of the preceding paragraphs, wherein the subject is a human subject.

[0754] 22. The method according to any one of the preceding paragraphs, wherein the subject has a kidney disease or is at risk of kidney disease, optionally wherein the subject has a glomerular disease or is at risk of glomerular disease.

[0755] 23. The method according to any one of the preceding paragraphs, wherein the subject has a hereditary glomerular disease or is at risk of hereditary glomerular disease, optionally wherein the subject has a podocyte-related hereditary glomerular disease or is at risk of podocyte-related hereditary glomerular disease.

[0756] 24. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 1x106 vg / kg to about 1x10 14 vg / kg or about 1x10 6 vg / kg to about 1x10 13 vg / kg of dose delivery.

[0757] 25. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 1x10 9 vg / kg to about 1x10 12 vg / kg of dose delivery.

[0758] 26. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 3x10 9 vg / kg to about 3x10 11 vg / kg of dose delivery.

[0759] 27. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 1x10 8 vg to about 1x10 15 vg or about 1x10 8 vg to about 5x10 14 vg of dose delivery.

[0760] 28. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 1x10 11 vg to about 1x10 14 vg / kg of dose delivery.

[0761] 29. The method according to any one of the preceding paragraphs, wherein the viral vector is at about 2x10 11 vg to about 2x10 13 vg / kg of dose delivery.

[0762] 30. The method according to any one of the preceding paragraphs, wherein the viral vector is capable of transducing renal cells, optionally wherein the vector is capable of specifically transducing renal cells.

[0763] 31. The method according to any one of the preceding paragraphs, wherein the viral vector is capable of transducing glomerular cells, optionally wherein the vector is capable of specifically transducing glomerular cells.

[0764] 32. The method according to any one of the preceding paragraphs, wherein the viral vector is capable of transducing podocytes, optionally wherein the vector is capable of specifically transducing glomerular podocytes.

[0765] 33. The method according to any one of the preceding paragraphs, wherein the viral vector is selected from adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

[0766] 34. The method according to any one of the preceding paragraphs, wherein the viral vector is an adeno-associated virus (AAV) vector particle.

[0767] 35. The method according to paragraph 34, wherein the viral vector is in the form of an AAV vector particle encapsidated by an LK03, AAV3B, or AAV9 capsid protein.

[0768] 36. The method according to paragraph 34 or 35, wherein the viral vector is in the form of an AAV vector particle encapsidated by an LK03 capsid protein.

[0769] 37. The method according to any one of the preceding paragraphs, wherein the viral vector comprises a protein-coding sequence.

[0770] 38. The method according to paragraph 37, wherein the protein-coding sequence encodes a polypeptide associated with a hereditary glomerular disease, optionally a polypeptide associated with a podocyte-related hereditary glomerular disease.

[0771] 39. The method according to paragraph 37 or 38, wherein the protein coding sequence encodes COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3 or VEGFC polypeptide.

[0772] 40. The method according to any one of paragraphs 37 to 39, wherein the protein coding sequence encodes NPHS2 or a fragment and / or variant thereof; a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof; or CFI, CFH or FHL-1, or a fragment and / or variant thereof.

[0773] 41. The method according to any one of paragraphs 37 to 40, wherein the protein coding sequence does not encode a gene editing agent.

[0774] 42. The method according to any one of paragraphs 37 to 41, wherein the protein coding sequence does not encode a nuclease.

[0775] 43. The method according to any one of paragraphs 37 to 42, wherein the protein coding sequence does not encode Cas9.

[0776] 44. The method according to any one of paragraphs 37 to 43, wherein the protein coding sequence is operably linked to a kidney-specific promoter, preferably wherein the protein coding sequence is operably linked to a podocyte-specific promoter.

[0777] 45. The method according to any one of paragraphs 37 to 44, wherein the protein coding sequence is operably linked to an NPHS1 promoter or an NPHS2 promoter.

[0778] 46. The method according to any one of paragraphs 37 to 45, wherein the protein coding sequence is operably linked to a minimal NPHS1 promoter.

[0779] 47. The method according to any one of paragraphs 37 to 46, wherein the protein coding sequence is operably linked to a constitutive promoter.

[0780] 48. The method according to paragraph 47, wherein the protein coding sequence is operably linked to a CMV promoter.

[0781] 49. The method according to any one of paragraphs 37 to 48, wherein the protein coding sequence is operably linked to one or more additional regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences.

[0782] 50. The method according to any one of paragraphs 37 to 49, wherein the protein coding sequence is operably linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0783] 51. The method according to any one of paragraphs 37 to 50, wherein the protein coding sequence is operably linked to a polyadenylation signal, such as the bovine growth hormone polyadenylation signal (bGH).

[0784] 52. The method according to any one of the preceding paragraphs, wherein the viral vector is injected or infused into the renal artery in the form of a viral vector preparation.

[0785] 53. The method according to paragraph 52, wherein the viral vector preparation contains from about 1x10 7 vg / ml to about 1x10 14 vg / ml or from about 1x10 7 vg / ml to about 5x10 13 vg / ml of the viral vector.

[0786] 54. The method according to paragraph 52 or 53, wherein the viral vector preparation contains from about 1x10 10 vg / ml to about 1x10 13 vg / ml of the viral vector.

[0787] 55. The method according to any one of paragraphs 52 to 54, wherein the viral vector preparation contains from about 1x10 10 vg / ml to about 1x1012 the viral vector in an amount of vg / ml.

[0788] 56. The method according to any one of paragraphs 52 to 55, wherein the viral vector preparation comprises an isotonic buffer, such as phosphate buffered saline (PBS) buffer or plasmalyte.

[0789] 57. The method according to any one of paragraphs 52 to 56, wherein the viral vector preparation comprises about 0.001% poloxamer 188.

[0790] 58. The method according to any one of paragraphs 52 to 57, wherein the viral vector preparation has a volume of about 5 mL to about 50 mL, about 5 mL to about 25 mL or about 10 mL to about 25 mL.

[0791] 59. The method according to any one of the preceding paragraphs, wherein the total renal ischemia time is about 60 minutes or less, about 50 minutes or less, about 40 minutes or less or about 30 minutes or less, preferably wherein the total renal ischemia time is about 10 minutes to about 30 minutes or about 15 to about 25 minutes.

[0792] 60. The method according to any one of the preceding paragraphs, wherein the method according to any one of paragraphs 1 to 60 is performed once to deliver the viral vector to a single kidney of the subject, or wherein the method according to any one of paragraphs 1 to 60 is performed twice to deliver the viral vector to two kidneys of the subject.

[0793] 61. A viral vector for therapy, wherein the viral vector is delivered by the method according to any one of paragraphs 1 to 60.

[0794] 62. A viral vector for treating or preventing kidney diseases, wherein the viral vector is delivered by the method according to any one of paragraphs 1 to 60.

[0795] 63. Use of a viral vector for the preparation of a medicament, wherein the medicament is delivered by the method according to any one of paragraphs 1 to 60.

[0796] 64. Use of a viral vector for the preparation of a medicament for treating or preventing kidney diseases, wherein the medicament is delivered by the method according to any one of paragraphs 1 to 60.

Claims

1. A method of delivering a viral vector to a subject's kidney, the method comprising: (a) inserting a catheter into the renal artery of the kidney; (b) optionally inflating a balloon to occlude the renal artery; and (c) injecting or infusing the viral vector into the renal artery via the catheter, wherein the method is a minimally invasive procedure and wherein the method does not include the step of inserting a catheter into the renal vein of the kidney.

2. The method according to claim 1, wherein the method does not include the step of occluding the renal vein of the kidney.

3. The method according to any one of the preceding claims, wherein the method does not include the step of directly inserting a catheter into the aorta.

4. The method according to any one of the preceding claims, wherein the method does not include forming a closed circuit through the kidney.

5. The method according to any one of the preceding claims, wherein the method does not include the step of clamping the renal artery of the kidney, the renal vein of the kidney or the aorta.

6. The method according to any one of the preceding claims, wherein the catheter is an occlusion balloon catheter.

7. The method according to any one of the preceding claims, wherein the renal artery is occluded for about 1 minute to about 25 minutes.

8. The method according to claim 7, wherein the renal artery is occluded for about 2 minutes to about 10 minutes, optionally about 5 minutes.

9. The method according to claim 7, wherein the renal artery is occluded for about 15 minutes to about 25 minutes or about 15 minutes to about 20 minutes, optionally about 20 minutes.

10. A method of delivering a viral vector to a subject's kidney, the method comprising: Inserting a catheter into the renal artery of the kidney and injecting or infusing the viral vector into the renal artery via the catheter, wherein the method does not include the step of occluding the renal artery of the kidney or the renal vein of the kidney and wherein the method does not include the step of clamping the aorta.

11. The method according to claim 10, wherein the method is a minimally invasive procedure.

12. The method according to claim 10 or 11, wherein the method does not include the step of inserting a catheter into the renal vein and / or does not include the step of directly inserting a catheter into the aorta.

13. The method according to any one of claims 10 to 12, wherein the method does not include forming a closed circuit through the kidney.

14. The method according to any one of the preceding claims, wherein the catheter is inserted into the renal artery via a percutaneous approach, optionally wherein the percutaneous approach is via the carotid artery or via the femoral artery and / or wherein the insertion of the catheter via the percutaneous approach is facilitated using a sheath.

15. The method according to any one of the preceding claims, wherein the catheter is inserted into the renal artery via a guide wire.

16. The method according to any one of the preceding claims, wherein the viral vector is infused into the renal artery under no-flow conditions using an infusion pump.

17. The method according to any one of the preceding claims, wherein the viral vector is injected or infused into the renal artery within about 1 minute to about 30 minutes, preferably (a) wherein the viral vector is injected or infused into the renal artery within about 1 minute to about 5 minutes, optionally about 2 minutes or 4 minutes; or (b) wherein the viral vector is injected or infused into the renal artery within about 5 minutes to about 30 minutes or about 15 minutes to about 20 minutes, optionally about 17 minutes.

18. The method according to any one of the preceding claims, wherein the method results in delivery of the viral vector to the renal cortex and / or renal medulla, preferably the method results in delivery of the viral vector to the renal cortex.

19. The method according to any one of the preceding claims, wherein the method results in delivery of the viral vector to glomeruli, preferably wherein the method results in delivery of the viral vector to renal podocytes.

20. The method according to any one of the preceding claims, wherein the method results in kidney-specific delivery of the viral vector.

21. The method according to any one of the preceding claims, wherein the subject is a human subject.

22. The method according to any one of the preceding claims, wherein the subject has a kidney disease or is at risk of a kidney disease, optionally wherein the subject has a glomerular disease or is at risk of a glomerular disease.

23. The method according to any one of the preceding claims, wherein the subject has a hereditary glomerular disease or is at risk of a hereditary glomerular disease, optionally wherein the subject has a podocyte-related hereditary glomerular disease or is at risk of a podocyte-related hereditary glomerular disease.

24. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of from about 1x10 8 vg to about 1x10 15 vg or from about 1x10 8 vg to about 5x10 14 vg.

25. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of from about 1x10 11 vg to about 1x10 14 vg.

26. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of from about 2x10 11 vg to about 2x10 13 vg.

27. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of from about 5x10 11 vg to about 2x10 13 vg.

28. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of from about 1x10 12 vg to about 2x10 13 vg.

29. The method according to any one of the preceding claims, wherein the viral vector is delivered at a dose of about 1x10 13 vg.

30. The method according to any one of the preceding claims, wherein the viral vector is capable of transducing renal cells, optionally wherein the vector is capable of specifically transducing renal cells.

31. The method according to any one of the preceding claims, wherein the viral vector is capable of transducing glomerular cells, optionally wherein the vector is capable of specifically transducing glomerular cells.

32. The method according to any one of the preceding claims, wherein the viral vector is capable of transducing podocytes, optionally wherein the vector is capable of specifically transducing glomerular podocytes.

33. The method according to any one of the preceding claims, wherein the viral vector is selected from adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, adenoviral vectors, herpes simplex virus vectors, alphavirus vectors, flavivirus vectors, rhabdovirus vectors, measles virus vectors, Newcastle disease virus vectors, poxvirus vectors, and picornavirus vectors.

34. The method according to any one of the preceding claims, wherein the viral vector is an adeno-associated virus (AAV) vector particle.

35. The method according to claim 34, wherein the viral vector is in the form of an AAV vector particle encapsidated by the LK03, AAV3B, or AAV9 capsid protein.

36. The method according to claim 34 or 35, wherein the viral vector is in the form of AAV vector particles capsidated by the LK03 capsid protein.

37. The method according to any one of the preceding claims, wherein the viral vector comprises a protein coding sequence.

38. The method according to claim 37, wherein the protein coding sequence encodes a therapeutic protein, preferably wherein the protein coding sequence encodes a polypeptide associated with a hereditary glomerular disease, optionally a polypeptide associated with a podocyte-related hereditary glomerular disease.

39. The method according to claim 37 or 38, wherein the protein coding sequence encodes COL4A3, COL4A4, COL4A5, NPHS2, CFH, CFL, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, clusterin, ADCK4, ALG1, ARHGAP24, ARGHDIA, CD151, CD2AP, COQ2, COQ6, DGKE, E2F3, EMP2, KANK2, LAGE3, LMNA, LMX1B, MAF B, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, PMM2, PODXL, SCARB2, SGPL1, Smad7, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3 or VEGFC polypeptide.

40. The method according to any one of claims 37 to 39, wherein the protein coding sequence encodes NPHS2 or a fragment and / or variant thereof; a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof; or CFI, CFH or FHL-1, or a fragment and / or variant thereof.

41. The method according to any one of claims 37 to 40, wherein the protein coding sequence does not encode a gene editing agent.

42. The method according to any one of claims 37 to 41, wherein the protein coding sequence does not encode a nuclease.

43. The method according to any one of claims 37 to 42, wherein the protein coding sequence does not encode Cas9.

44. The method according to any one of claims 37 to 43, wherein the protein coding sequence is operably linked to a kidney-specific promoter, preferably wherein the protein coding sequence is operably linked to a podocyte-specific promoter.

45. The method according to any one of claims 37 to 44, wherein the protein coding sequence is operably linked to an NPHS1 promoter or an NPHS2 promoter.

46. The method according to any one of claims 37 to 45, wherein the protein coding sequence is operably linked to a minimal NPHS1 promoter.

47. The method according to any one of claims 37 to 46, wherein the protein coding sequence is operably linked to a constitutive promoter.

48. The method according to claim 47, wherein the protein coding sequence is operably linked to a CMV promoter.

49. The method according to any one of claims 37 to 48, wherein the protein coding sequence is operably linked to one or more additional regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences.

50. The method according to any one of claims 37 to 49, wherein the protein coding sequence is operably linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

51. The method according to any one of claims 37 to 50, wherein the protein coding sequence is operably linked to a polyadenylation signal, such as the bovine growth hormone polyadenylation signal.

52. The method according to any one of the preceding claims, wherein the viral vector is injected or infused into the renal artery in the form of a viral vector preparation.

53. The method according to claim 52, wherein the viral vector preparation comprises from about 1x10 7 vg / ml to about 1x10 14 vg / ml or from about 1x10 7 vg / ml to about 5x10 13 vg / ml of the viral vector.

54. The method according to claim 52 or 53, wherein the viral vector preparation comprises the viral vector in an amount of from about 1x10 10 vg / ml to about 1x10 13 vg / ml.

55. The method according to any one of claims 52 to 54, wherein the viral vector preparation comprises the viral vector in an amount of from about 1x10 10 vg / ml to about 1x10 12 vg / ml.

56. The method according to any one of claims 52 to 55, wherein the viral vector preparation comprises an isotonic buffer, such as phosphate buffered saline (PBS) buffer or plasmalyte.

57. The method according to any one of claims 52 to 56, wherein the viral vector preparation comprises about 0.001% poloxamer 188.

58. The method according to any one of claims 52 to 57, wherein the viral vector preparation has a volume of about 5 mL to about 50 mL, about 5 mL to about 25 mL or about 10 mL to about 25 mL.

59. The method according to any one of the preceding claims, wherein the total renal ischemia time is about 60 minutes or less, about 50 minutes or less, about 40 minutes or less or about 30 minutes or less, preferably wherein the total renal ischemia time is about 10 minutes to about 30 minutes or about 15 to about 25 minutes.

60. The method according to any one of the preceding claims, wherein the method according to any one of claims 1 to 60 is performed once to deliver the viral vector to a single kidney of the subject, or wherein the method according to any one of claims 1 to 60 is performed twice to deliver the viral vector to two kidneys of the subject.

61. A viral vector for therapy, wherein the viral vector is delivered by the method according to any one of claims 1 to 60.

62. A viral vector for treating or preventing kidney diseases, wherein the viral vector is delivered by the method according to any one of claims 1 to 60.

63. Use of a viral vector for the preparation of a medicament, wherein the medicament is delivered by the method according to any one of claims 1 to 60.

64. Use of a viral vector for the preparation of a medicament for the treatment or prevention of kidney diseases, wherein the medicament is delivered by the method according to any one of claims 1 to 60.

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