Methods for delivering viral vectors to the kidney

Abstract

The present invention relates to a method for delivering a viral vector to the kidney of a subject, the method comprising the steps of: (a) inserting a catheter into the renal artery; (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. The present invention also relates to a viral vector for use in therapy, wherein the viral vector is delivered by the method.

Description

[Technical Field]

[0001] The present invention relates to a method for delivering a viral vector to the kidney. The present invention also relates to a viral vector for use in therapy, wherein the viral vector is delivered by said method. [Background technology]

[0002] Many diseases affect kidney function by attacking the glomerulus. Glomeruli filter approximately 180 liters of plasma each day, and a healthy glomerular filtration barrier has the remarkable ability to retain approximately 99.9% of large proteins, including albumin, without clogging over a lifetime. The glomerular filtration barrier (GFB) is composed of three major layers: glomerular endothelial cells, glomerular basement membrane (GBM), and podocytes.

[0003] The GBM is composed of a highly cross-linked polymeric network of type IV collagen, proteoglycans, and laminin. Inherited glomerular diseases can be caused by genetic defects in these molecules. For example, Alport syndrome is caused by pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes, resulting in abnormalities in the collagen IVα345 network of the basement membrane. Alport syndrome occurs in approximately 1 in 5,000–10,000 individuals in continental Europe and the United States. The disease usually develops in childhood and is associated with a variety of phenotypes, including progressive renal decline, hearing loss, and eye abnormalities. Other GBM-related disorders include Pearson syndrome and Nail-Patera syndrome (Chiang, CK and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p. 539).

[0004] Podocytes have also been implicated as key cells in the progression of glomerular diseases. Podocytes are mesodermally derived, highly specialized cells found only in renal glomeruli. Podocytes exhibit unique features, such as foot processes and slit diaphragms, which are important for glomerular filtration. Podocyte-related inherited glomerular diseases include nephrotic syndrome, Fraser syndrome, Dennis-Drash syndrome, Schimke immuno-osseous dysplasia, Epstein syndrome, and Fechtner syndrome. (Chiang, CK and Inagi, R., 2010. Nature Reviews Nephrology, 6(9), p.539)

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

[0006] To maximize the potential of gene therapy, optimal methods for delivering viral vectors to glomerular cells, such as podocytes, are needed. However, the kidney is a challenging target for traditional intravenous administration because the selective permeability of the glomerulus prevents most molecular therapeutics from entering the kidney from the blood. Furthermore, various direct kidney injection methods have been tested using viral vectors, such as adeno-associated virus (AAV) vectors, but gene transfer efficiencies have been inconsistent and generally remain too low to effectively treat disease (see, for example, Rubin, JD and Barry, MA, 2020 Molecular Diagnosis & Therapy, 24(4), pp. 375-396).

[0007] Therefore, there is a need for improved methods for delivering viral vectors to glomerular cells such as podocytes. Summary of the Invention

[0008] The present inventors have developed an improved method for delivering viral vectors to the kidney, particularly to glomerular cells such as podocytes, by direct renal artery injection, which can be performed minimally invasively and has improved safety compared to prior art direct kidney injection methods.

[0009] Furthermore, the present inventors surprisingly demonstrated that direct injection of a viral vector into the renal artery can result in significantly increased transgene expression and viral copy number in the kidney, particularly in glomeruli and podocytes, compared with intravenous injection. Furthermore, direct injection of a viral vector into the renal artery can result in transgene expression that is localized to the kidney with minimal expression in other tissues (e.g., the liver).

[0010] In one embodiment, the present invention provides a method for delivering a viral vector to a kidney, the method comprising inserting a catheter into a renal artery and delivering a viral vector into the renal artery via the catheter.

[0011] The method may be a minimally invasive procedure. Preferably, the method does not include occluding the renal vein. Preferably, the method does not include inserting a catheter into the renal vein and / or inserting a catheter directly into the aorta. Preferably, the method does not include forming a closed circuit through the kidney. Preferably, the method does not include clamping the renal artery, renal vein, or aorta. Preferably, 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 minutes to about 25 minutes, or about 5 minutes.

[0012] The catheter can be inserted into the renal artery in any suitable manner. In a preferred embodiment, the catheter is inserted into the renal artery via a percutaneous route. Preferably, the percutaneous route is via the carotid artery or the femoral artery. Preferably, insertion of the catheter via a percutaneous route is facilitated by the use of a sheath. Preferably, the catheter is inserted into the renal artery over a guidewire.

[0013] The viral vector can be introduced into the renal artery by any suitable method. Preferably, the viral vector is injected or infused into the renal artery. In some embodiments, the viral vector is infused into the renal artery under no-flow conditions using an infusion pump. Preferably, the viral vector is delivered to the renal artery over a period of about 1 minute to about 30 minutes. In some embodiments, the viral vector is delivered to the renal artery over a period of 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 over a period of about 15 minutes to about 30 minutes, or about 15 minutes to about 20 minutes, optionally about 17 minutes.

[0014] In some embodiments, the catheter is an occlusion balloon catheter. In some embodiments, the method includes inflating the balloon to occlude the renal artery. Preferably, 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.

[0015] In one embodiment, the present invention is a method of delivering a viral vector to the kidney of a subject, the method comprising: (a) inserting a catheter into a renal artery of a kidney; (b) optionally inflating the balloon to occlude the renal artery; and (c) injecting or infusing a viral vector into the renal artery via the catheter. wherein the method is a minimally invasive procedure and does not include inserting a catheter into the renal vein of the kidney.

[0016] In one embodiment, the present invention provides a method of delivering a viral vector to the kidney of a subject, the method comprising inserting a catheter into a renal artery of the kidney and injecting or infusing a viral vector into the renal artery via the catheter, wherein the method does not include occluding the renal artery of the kidney or the renal vein of the kidney and does not include clamping the aorta.

[0017] The method of the present invention can result in delivery of a viral vector to the kidney. For example, the method can result in delivery of a viral vector to the renal cortex and / or renal medulla, particularly the renal cortex. The method can result in delivery of a viral vector to the renal glomerulus. The method can result in delivery of a viral vector to renal podocytes. The method can result in kidney-specific delivery of a viral vector.

[0018] The subject may be any suitable subject. The subject may be a human. The human subject may be an adult, adolescent, or child. The subject may have or be at risk of having kidney disease. The subject may have or be at risk of having glomerular disease. The subject may have or be at risk of having a genetic glomerular disease, and optionally, the subject may have or be at risk of having a podocyte-associated genetic glomerular disease.

[0019] The viral vector can be delivered at any suitable dose. Preferably, the viral vector is delivered at a dose of about 1 x 10 6 vg / kg ~ approx. 1x10 14 vg / kg, or approximately 1x10 6 vg / kg ~ approx. 1x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg / kg. 9 vg / kg ~ approx. 1x10 12 In some embodiments, the viral vector is delivered at a dose of about 3x10 vg / kg. 9 vg / kg~approx.3x10 11 Preferably, the viral vector is delivered at a dose of about 1 x 10 vg / kg. 8 vg~approx. 1x10 15 vg, or approximately 1x10 8 vg~approx.5x10 14 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 11 vg~approx. 1x10 14 In some embodiments, the viral vector is delivered at a dose of about 2x10 vg. 11vg~approx. 2x10 13 In some embodiments, the viral vector is delivered at a dose of about 5x10 vg. 11 vg~approx. 2x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 12 vg~approx. 2x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 13 vg dose.

[0020] The viral vector may be any suitable viral vector. Preferably, the viral vector is capable of transducing kidney cells, and optionally, the vector can specifically transduce kidney cells. Preferably, the viral vector is capable of transducing glomerular cells, and optionally, the vector can specifically transduce glomerular cells. Preferably, the viral vector is capable of transducing podocytes, and optionally, the vector can specifically transduce glomerular podocytes. Preferably, the viral vector is selected from an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpes simplex virus vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles virus vector, a Newcastle disease virus vector, a poxvirus vector, and a picornavirus vector.

[0021] In a preferred embodiment, the viral vector is an adeno-associated viral (AAV) vector particle. In some embodiments, the viral vector is in the form of an AAV vector particle encapsidated by LK03, AAV3B, or AAV9 capsid protein. In some embodiments, the viral vector is in the form of an AAV vector particle encapsidated by LK03 capsid protein.

[0022] The viral vector may comprise any suitable protein coding sequence. Preferably, the protein coding sequence encodes a therapeutic protein, and preferably, the protein coding sequence encodes a polypeptide involved in a genetic glomerular disease, optionally a polypeptide involved in a podocyte-associated genetic 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.

[0023] 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.

[0024] The protein coding sequence may be operably linked to one or more additional regulatory elements. Preferably, 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 a woodchuck hepatitis post-transcriptional regulatory element (WPRE). In some embodiments, the protein coding sequence is operably linked to a polyadenylation signal, such as a bovine growth hormone polyadenylation signal.

[0025] The viral vector may be in the form of a viral vector formulation. The viral vector formulation may contain any suitable amount of viral vector and may be formulated in any suitable manner. Preferably, the viral vector formulation contains about 1x10 7 vg / ml ~ approx. 1x10 14 vg / ml, or approximately 1x10 7 vg / ml ~ approx. 5x10 13 In some embodiments, the viral vector formulation comprises about 1 x 10 vg / ml of viral vector. 10 vg / ml ~ approx. 1x10 13 In some embodiments, the viral vector formulation comprises about 1 x 10 vg / ml of viral vector. 10 vg / ml ~ approx. 1x10 12The viral vector formulation contains a viral vector in an amount of about 1000 mg / ml. Preferably, the viral vector formulation contains an isotonic buffer solution such as phosphate-buffered saline (PBS) or Plasmalyte. In some embodiments, the viral vector formulation contains about 0.001% poloxamer 188. Preferably, the viral vector formulation 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.

[0026] The viral vector can be delivered to a single kidney or both kidneys of a subject. In some embodiments, the method of the present invention is performed once to deliver the viral vector to a single kidney of a subject. In some embodiments, the method of the present invention is performed twice to deliver the viral vector to both kidneys of a subject.

[0027] In one embodiment, the invention provides a viral vector for use in therapy, wherein the viral vector is delivered by a method according to the invention.

[0028] In one aspect, the invention provides a viral vector for use in the treatment or prevention of kidney disease, wherein the viral vector is delivered by a method according to the invention.

[0029] In one embodiment, the present invention provides the use of a viral vector for the manufacture of a medicament, wherein the medicament is delivered by a method according to the present invention.

[0030] In one aspect, the present invention provides the use of a viral vector for the manufacture of a medicament delivered by a method according to the present invention for treating or preventing kidney disease. [Brief explanation of the drawings]

[0031] [Figure 1]Schematic diagram of the AAV vector encoding podocin under the control of the hNPHS1 promoter. ITR: inverted terminal repeat; hNPHS1 promoter: human full-length nephrin promoter; HA: hemagglutinin; WPRE: woodchuck posttranscriptional regulatory element; bGH: bovine growth hormone polyadenylation signal. [Figure 2] Transcriptome analysis for evaluation of transduction efficiency. Pigs 3-6 were dRAi-treated pigs. Pig 1 and pig 2 were controls. AAV: adeno-associated virus; bGH: bovine growth hormone; LKC: left renal cortex; LKM: left renal medulla; RKC: right renal cortex; RKM: right renal medulla. [Figure 3] Colocalization experiment of nephrin and HA-tagged podocin (LKC and RKC). (A) Single-channel images showing nephrin (left panel) and HA-tagged podocin (middle panel), and the corresponding merged image (right panel) showing the colocalization of nephrin and HA-tagged podocin. (B) Magnified panels of the indicated areas showing the expression of HA-podocin in podocytes, as seen by colocalization with the podocyte marker nephrin. HA: hemagglutinin; LKC: left kidney cortex; RKC: right kidney cortex. [Figure 4] Schematic diagram of an AAV vector encoding eGFP under the control of the CMV promoter. CMV: cytomegalovirus; bGH: bovine growth hormone; eGFP: enhanced green fluorescent protein; ITR: inverted terminal repeat; WPRE: woodchuck posttranscriptional regulatory element. [Figure 5] Transcriptome analysis of mRNA GFP expression (comparison of rAAV-injected and non-injected kidneys) and biodistribution (colon, liver, and pancreas). GFP: green fluorescent protein; IV: intravenous injection; ra: direct injection into the renal artery. [Figure 6] Transcriptome analysis and biodistribution of GFP mRNA in the porcine kidney (colon, liver, and pancreas). GFP: green fluorescent protein; IV: intravenous injection; LKC: left renal cortex; LKM: left renal medulla; ra: direct renal artery injection; RKC: right renal cortex; RKM: right renal medulla. [Figure 7]Double immunofluorescence (anti-GFP) of the left porcine kidney cortex. Merged image (63x magnification) showing colocalization of GFP (green fluorescent protein) and nephrin in the renal cortex. dRAi: direct injection into the renal artery; IV: intravenous injection. [Figure 8] Double immunofluorescence (anti-GFP and anti-nephrin) of the right porcine kidney cortex. Merged image (magnification 63x) showing colocalization of GFP (green fluorescent protein) and nephrin in the kidney cortex. dRAi: direct injection into the renal artery; IV: intravenous injection. [Figure 9] High magnification image of expression within glomerular ultrastructure. Merged image showing co-localization of GFP (green fluorescent protein), nephrin, and DAPI (4',6-diamidino-2-phenylindole) within glomerular ultrastructure. [Figure 10] Double immunofluorescence (anti-GFP) of pig liver. Merged image showing colocalization of GFP (green fluorescent protein) and DAPI (4',6-diamidino-2-phenylindole) in liver tissue. Images from the high-dose and low-dose dRAi groups were all GFP-negative, so representative samples from each animal were randomly taken. dRAi: direct injection into the renal artery; IV: intravenous injection. [Figure 11] Schematic diagram of the AAV vector and experimental design. (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 posttranscriptional regulatory element; bGH: bovine growth hormone poly(A) signal. (B) Schematic diagram of the experimental design. [Figure 12] Biodistribution by qPCR in tissue samples. AAV genomes detected per milligram (mg) of tissue after direct infusion into the renal artery or intravenous infusion with (dRAi+O) or without (dRAi) renal artery occlusion. (A) Treated kidney; (B) Untreated kidney. [Figure 13]RNAscope in-situ hybridization (dRAi vs. IV). To examine AAV mRNA localization in renal cortical samples after direct renal artery infusion (dRAi) without renal artery occlusion or intravenous injection, paraffin-embedded, formalin-fixed tissues were evaluated by RNAscope in-situ hybridization. Pigs received (A) 1x1012 vg dRAi, (B) 5x1012 vg dRAi, (C) 1x1013 vg dRAi, or (D) 1x1013 vg intravenously. [Figure 14] RNAscope in-situ hybridization (dRAi+O vs. IV). To examine AAV mRNA localization in renal cortex samples after direct renal artery infusion with renal artery occlusion (dRAi+O) or intravenous injection, paraffin-embedded, formalin-fixed tissues were evaluated by RNAscope in-situ hybridization. Pigs received (A) 1x1013vg via dRAi+O, (B) 2x1013vg via dRAi+O, or (C) 1x1013vg via intravenous injection. [Figure 15] Schematic diagram of the AAV vector and experimental design. (A) Schematic diagram of the 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 transgene with a hemagglutinin tag; WPRE: woodchuck posttranscriptional regulatory element; bGH: bovine growth hormone poly(A) signal. (B) Schematic diagram of the experimental design. [Figure 16] Biodistribution by qPCR in tissue samples. AAV genomes detected per milligram (mg) of tissue from untreated control pigs (UTC) and pigs treated with PS0438: (A) treated kidney; (B) untreated kidney; (C) liver; and (D) spleen. [Figure 17]RNAscope in-situ hybridization (dRAi+O). To examine the localization of AAV mRNA in renal cortical samples after direct renal artery infusion with renal artery occlusion (dRAi+O), paraffin-embedded, formalin-fixed tissues were evaluated by RNAscope in-situ hybridization. (A) Treated kidney and (B) untreated kidney. [Figure 18] Immunofluorescence of HA-tagged podocin and nephrin. Immunofluorescence (IF) analysis was performed on sections from OCT blocks of kidney cortex. Sections were stained with DAPI and antibodies targeting WT-1, HA, and nephrin. (A) Merged image of two glomeruli and corresponding single-channel images showing (B) HA-tagged podocin (green) and (C) nephrin. (D) Merged image of one glomerulus and corresponding single-channel images showing (E) HA-tagged podocin (green) and (F) nephrin. [Figure 19] Kidney Cortex Podocin ELISA. Podocin protein, expressed in nanograms per milligram of total protein, detected by ELISA in kidney cortex samples from untreated control pigs (UTC) and pigs treated with PS0438. [Figure 20] Double immunofluorescence of glomeruli. (A) Merged images of glomeruli stained with DAPI and antibodies targeting nephrin are shown. GFP expression was not observed in the negative control (PBS, left panel) and was higher after direct intrarenal artery injection (dRAi, right panel) compared with intravenous injection (iv, center panel). (B) Merged images of glomeruli stained with DAPI and antibodies targeting nephrin (left panel) or PDGFb (right panel). GFP expression colocalized with nephrin (NPHS1) and PDGFb, confirming their localization in podocytes and mesangial cells, respectively. [Figure 21] Western blot analysis. (A) Western blot analysis and (B) densitometry of mouse liver samples demonstrated higher GFP expression following intravenous administration of AAV vectors compared to AAV vectors administered via direct renal artery infusion (dRAi). DETAILED DESCRIPTION OF THE INVENTION

[0032] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example. The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Those skilled in the art will appreciate that all features of the invention disclosed herein can be combined without departing from the scope of the disclosed invention.

[0033] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0034] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," and "contains" and are inclusive or open-ended and do not exclude additional non-limiting factors, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0035] Numerical ranges are inclusive of the numbers defining the range. As used herein, the term "about" means approximately, within, roughly, or around. When the term "about" is used in conjunction with a numerical value or range, it modifies that numerical value or range by extending the boundaries above and below the stated numerical value. In general, the terms "about" and "approximately" can be used herein to modify a numerical value by 10% above and below the stated numerical value.

[0036] Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0037] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto. All publications mentioned herein are incorporated herein by reference.

[0038] Glomerular and podocyte gene therapy The glomerulus is the kidney's filtering apparatus. Approximately 180 liters of plasma are filtered daily, and a healthy glomerular filtration barrier has the remarkable ability to retain approximately 99.9% of large proteins, including albumin, without clogging over a lifetime. The afferent arteriole enters the glomerular capillary bed, where filtration occurs, and blood leaves the glomerulus through the efferent arteriole. The glomerular filtration barrier (GFB) contains three main layers: glomerular endothelial cells, the glomerular basement membrane (GBM), and podocytes.

[0039] The third layer of the GFB, the podocyte, plays a crucial role in maintaining the GFB. Podocytes are highly specialized cells that contain a cell body, primary and secondary processes, and foot processes, which interdigitate with the foot processes of neighboring podocytes to form slit membranes. Podocytes form an effective dynamic sieve, which is thought to be primarily due to the integrity of the slit membrane.

[0040] Targeting gene therapy to the glomerulus is challenging. For example, lentiviruses can be useful for transducing renal tubules, but they have not yet been shown to transduce glomeruli in vivo. Furthermore, early attempts to deliver adenovirus via the renal artery or retrogradely through the ureter showed that delivery was primarily to the tubules or interstitium. Early studies in murine kidneys using AAV2 demonstrated that delivery was mostly to the renal tubules and not to the glomerulus.

[0041] Method of delivery In one aspect, the present invention provides a method for delivering a viral vector to the kidney via direct injection into the renal artery. The method may include 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 "delivering" may be used interchangeably with the term "administering."

[0042] The method is preferably an in vivo method (i.e., not an ex vivo method). The method may be a minimally invasive procedure. That is, the method may be a minimally invasive method. As used herein, "minimally invasive procedure" or "minimally invasive surgery" may refer to a surgical technique that limits the size of the required incision, thereby reducing wound healing time, associated pain, and risk of infection (see, e.g., Jaffray, B., 2005. Archives of disease in childhood, 90(5), pp.537-542). In contrast, "open surgery" incisions can sometimes leave large scars, be painful, and take a long time to heal. Many medical procedures, including percutaneous surgery, are referred to as minimally invasive. In a preferred embodiment, the method does not involve open surgery.

[0043] In some embodiments, the method does not include inserting two or more catheters. In some embodiments, the method does not include inserting a catheter into a renal vein. In some embodiments, the method does not include inserting a catheter directly into an aorta (e.g., an infrarenal aorta).

[0044] In preferred embodiments, the method does not include any clamping steps (e.g., clamping of the renal artery, renal vein, or aorta). In some embodiments, the method does not include clamping the renal artery. In some embodiments, the method does not include clamping the renal vein. In some embodiments, the method does not include clamping the aorta (e.g., suprarenal aorta).

[0045] In some embodiments, the method has a total renal ischemia time of about 1 hour or less. As used herein, "renal ischemia time" may refer to the time from when blood supply to the kidney is cut off or reduced until normal blood supply to the kidney is restored. Reduction of renal ischemia may result in reduced kidney damage or reduced risk of kidney damage.

[0046] 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, hi some embodiments, the total renal ischemia time is about 0 minutes.

[0047] 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. In some embodiments, the total renal ischemia time is about 5 minutes.

[0048] Step (a): Insertion of the catheter In the method of the present invention, a catheter is inserted into the renal artery. As used herein, a "catheter" may refer to a thin tube made of medical-grade material that can be inserted into the body to perform a surgical procedure. For most applications, catheters are thin, flexible tubes (soft catheters), although catheters with various levels of stiffness are available depending on the application. Preferably, the catheter is a percutaneous catheter. In some embodiments, the catheter is an occlusion balloon catheter.

[0049] As used herein, insertion "into the renal artery" means that after insertion, the tip of the catheter is positioned within the renal artery. This method is distinct from a method in which the catheter is positioned below the renal artery. The renal artery typically arises from the left medial side of the abdominal aorta, just below the superior mesenteric artery, at a 90° angle, with a radius of approximately 0.25 cm. The renal artery divides into four or five branches before reaching the renal hilum. The term "renal artery" can refer to any of these branches.

[0050] The catheter may be inserted into the renal artery via any suitable route. In a preferred embodiment, the catheter is inserted into the renal artery via a percutaneous route. Such a route is compatible with minimally invasive procedures. The percutaneous route may 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. Standard procedures for inserting a catheter via a percutaneous route are known to those skilled in the art. For example, the carotid artery or the (common) femoral artery may be catheterized, a sheath may be introduced, and a guidewire may be introduced to facilitate the introduction of the catheter into the renal artery.

[0051] In some embodiments, the method includes inserting a catheter into the carotid artery or the (common) femoral artery. In some embodiments, insertion of the catheter via a percutaneous route is assisted by a sheath. In some embodiments, the method includes introducing a sheath into the carotid artery or the (common) femoral artery. As used herein, a "sheath" may refer to a short, hollow tube that may be introduced into a blood vessel to assist in catheter placement. The sheath may remain in place until the procedure is completed and the catheter is removed. In some embodiments, the catheter is inserted into the renal artery over a guidewire. In some embodiments, the method includes introducing the guidewire into the renal artery. As used herein, a "guidewire" may refer to a thin wire used to guide the placement of a catheter into a blood vessel.

[0052] In some embodiments, the step (a) of inserting a catheter into a renal artery comprises the steps of: (a1) Inserting a catheter into the carotid artery or the (common) femoral artery (a2) inserting a sheath into the carotid artery or the (common) femoral artery; (a3) inserting a guidewire into the renal artery; and (a4) Inserting a catheter into the renal artery Includes:

[0053] Step (b): Occlusion of blood vessels The method may optionally include occluding the renal arteries, which may increase the residence time of the viral vector in the kidney and increase viral transduction.

[0054] As used herein, "occlusion" of a blood vessel may refer to the complete blocking of blood flow through the blood vessel. The occlusion can be confirmed by any suitable method, for example, by injecting or injecting a contrast agent and imaging the blood flow by angiography. The occlusion can be achieved by any suitable method, preferably by inflating a balloon. Such methods are compatible with minimally invasive procedures.

[0055] In some embodiments, the method comprises the steps of: (a) inserting a balloon catheter into a renal artery; (b) inflating the balloon to occlude the renal artery; and (c) injecting or infusing the viral vector into the renal artery via a catheter. Includes:

[0056] In some embodiments, the renal artery is occluded for about 1 minute or more, about 2 minutes or more, about 3 minutes or more, about 4 minutes or more, about 5 minutes or more, about 6 minutes or more, about 7 minutes or more, about 8 minutes or more, about 9 minutes or more, about 10 minutes or more, about 11 minutes or more, about 12 minutes or more, about 13 minutes or more, about 14 minutes or more, about 15 minutes or more, about 16 minutes or more, about 17 minutes or more, about 18 minutes or more, about 19 minutes or more, or about 20 minutes or more.

[0057] In some embodiments, the renal artery is occluded for about 30 minutes or less, about 25 minutes or less, about 20 minutes or less, about 19 minutes or less, about 18 minutes or less, about 17 minutes or less, about 16 minutes or less, about 15 minutes or less, about 14 minutes or less, about 13 minutes or less, about 12 minutes or less, about 11 minutes or less, about 10 minutes or less, about 9 minutes or less, about 8 minutes or less, about 7 minutes or less, about 6 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.

[0058] In some embodiments, the renal artery is occluded for about 1 minute to about 30 minutes, or about 1 minute to about 25 minutes, hi 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.

[0059] 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.

[0060] 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.

[0061] In other embodiments, the method does not include occluding the renal arteries.

[0062] In a preferred embodiment, the method does not include occluding the renal vein. Occlusion of the renal vein can increase the risk of renal vein thrombosis. In subjects with kidney disease, the kidneys can be at increased risk of thrombosis.

[0063] In a preferred embodiment, the method does not include the step of occluding the aorta. In some embodiments, the method does not include occluding the renal vein or the aorta.

[0064] In some embodiments, the method does not include occluding the renal artery, renal vein, or aorta.

[0065] In some embodiments, the method does not include occluding any blood vessels in the subject.

[0066] Step (c): Injection or infusion of the viral vector In the methods of the present invention, the viral vector is delivered to the renal artery via a catheter. The viral vector can be delivered by any suitable method, for example, by injection or infusion.

[0067] Preferably, the viral vector is delivered to the renal artery over a period of about 1 minute to about 30 minutes. In some embodiments, the viral vector is delivered to the renal artery over a period of about 1 minute to about 25 minutes. In some embodiments, the viral vector is delivered to the renal artery over a period of 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. Preferably, the viral vector is delivered at a substantially constant rate.

[0068] In some embodiments, the viral vector is injected into the renal artery via a catheter. As used herein, "injection" can refer to delivery under pressure or under flow (e.g., by forcing a liquid into the body with a syringe). In some embodiments, the viral vector is injected into the renal artery without the use of an infusion pump. In some embodiments, the viral vector is injected into the renal artery over 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.

[0069] In some embodiments, the viral vector is infused into the renal artery via a catheter. As used herein, "infusion" can refer to delivery under atmospheric pressure or under no-flow conditions (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 over 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 over 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 over 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.

[0070] Other steps The method of the present invention may include any other suitable steps, such as, for example, flushing the catheter, withdrawing the catheter, withdrawing the sheath, and / or closing any openings.

[0071] In some embodiments, the method further includes 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 can be flushed with saline (e.g., heparinized saline) or phosphate-buffered saline (PBS). In some embodiments, the catheter can be flushed with heparinized saline.

[0072] In some embodiments, the method includes flushing the catheter before injecting or infusing the viral vector. In some embodiments, the method includes flushing the catheter after injecting or infusing the viral vector. In some embodiments, the method includes flushing the catheter before and after injecting or infusing the viral vector.

[0073] In some embodiments, the method further comprises deflating the balloon. In some embodiments, the method further comprises withdrawing the catheter. In some embodiments, the method further comprises withdrawing the sheath. Preferably, the catheter and sheath are withdrawn slowly to avoid damaging the blood vessel. In some embodiments, the method further comprises closing the opening.

[0074] In some embodiments, the method comprises the steps of: (a) Inserting a balloon catheter into a renal artery (b) optionally, inflating the balloon to occlude the renal artery. (c) injecting or infusing the viral vector into the renal artery via a catheter; and (d) flushing the catheter, optionally deflating the balloon, and withdrawing the catheter; Includes:

[0075] 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 separate the viral vector formulation from the subject's systemic circulation. In some embodiments, less than about 20% v / v of the viral vector formulation circulated through the closed circuit leaks outside the closed circuit. For example, the 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 the flow of fluid into the kidney via the renal artery catheter and out of the kidney via the renal vein catheter.

[0076] In some embodiments, the method does not include the use of a membrane oxygenation device. In some embodiments, the viral vector formulation does not pass through a membrane oxygenation device. In some embodiments, the closed circuit further comprises a membrane oxygenation device.

[0077] In some embodiments, the method does not include perfusing the kidney with saline before injecting or infusing the viral vector into the renal artery.

[0078] Kidney-specific delivery The methods of the present invention result in delivery of viral vectors to the kidney. As used herein, "delivery" of a viral vector may refer to transduction by the viral vector, which may result in expression of RNA and / or protein encoded by the viral vector. Delivery of a viral vector to a specific organ, tissue, or cell type may be determined by any suitable method, including transcriptome analysis and / or immunofluorescence.

[0079] The method of the present invention results in transduction of the kidney with a viral vector, followed by expression of RNA encoded by the viral vector in the kidney and / or expression of a protein encoded by the viral vector in the kidney. Transduction of a specific organ, tissue, or cell type can be determined by viral load or viral copy number, which can be determined by any appropriate method (see, for example, Dobnik, D., et al., 2019. Frontiers in Microbiology, 10, p. 1570). RNA expression in a specific 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. Protein expression in a specific organ, tissue, or cell type can be determined by immunofluorescence. For example, proteins can be labeled with fluorescent markers and quantified by fluorescence microscopy.

[0080] The method may result in delivery of the viral vector to the renal cortex and / or renal medulla. In a preferred embodiment, the method results in delivery of the viral vector to the renal cortex. In a more preferred embodiment, the method results in delivery of the viral vector to the renal glomerulus. In an even more preferred embodiment, the method results in delivery of the viral vector to renal podocytes.

[0081] The methods of the present invention can result in kidney-specific delivery of a viral vector. As used herein, "kidney-specific delivery" can mean that the viral vector primarily transduces the kidney, such that the RNA and / or protein encoded by the viral vector is primarily expressed in the kidney.

[0082] Preferably, the method results in 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 delivery of the viral vector to the kidney, but does not result in significant delivery of the viral vector to the liver.

[0083] The viral vector can be delivered to a single kidney or both kidneys of a subject. In some embodiments, the method delivers a viral vector to a single kidney of a subject. In some embodiments, the method is repeated to deliver a viral vector to both kidneys of a subject.

[0084] In one embodiment, the present invention provides a method for delivering a viral vector to a subject, the method comprising: (1) delivering a viral vector to the right kidney of the subject using a method of the present invention; and / or (2) delivering a viral vector to the left kidney of the subject using a method of the present invention.

[0085] In one embodiment, the present invention provides a method for delivering a viral vector to a subject, the method comprising: (1) delivering a viral vector to the right kidney of the subject by a method according to the present invention; and (2) delivering a viral vector to the left kidney of the subject by a method according to the present invention.

[0086] Steps (1) and (2) can be performed in any order and for any period of time. In some embodiments, steps (1) and (2) are performed within one day. In some embodiments, steps (1) and (2) are performed as part of the same procedure. In some embodiments, step (2) directly follows step (1), or step (1) directly follows step (2).

[0087] The method of the present invention can be performed on a subject once or multiple times. The method can be repeated for any suitable period of time. Preferably, the method of the present invention is performed 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 performed once to deliver the viral vector to a single kidney of a subject. In some embodiments, the method is performed twice to deliver the viral vector to both kidneys of a subject.

[0088] subject The subject may be any suitable subject in need of the present invention. The subject may be a mammal. In a preferred embodiment, the subject is a human. The subject may be an adult, an adolescent, or a child.

[0089] The subject has kidney disease or is at risk of suffering from it.For example, the subject has glomerular disease or is at risk of suffering from it.For example, the subject has podocyte-related glomerular disease or is at risk of suffering from it.For example, the subject may have GBM-related glomerular disease or is at risk of suffering from it.

[0090] In some embodiments, the subject has or is at risk of suffering from a genetic glomerular disease, i.e., an inherited glomerular disease, including podocyte-associated inherited glomerular diseases such as nephrotic syndrome and GBM-associated glomerular diseases such as Alport syndrome.

[0091] In some embodiments, the subject has or is at risk of suffering from a podocyte-associated hereditary glomerular disease.Podocyte-associated hereditary glomerular diseases include Finnish congenital nephrotic syndrome, type 2 congenital nephrotic syndrome, type 3 familial nephrotic syndrome, Fraser syndrome, and Dennis-Drash syndrome, Schimke immuno-osseous dysplasia, nephrotic syndrome caused by a mutation in CD2AP, nephrotic syndrome caused by a mutation in actinin-4, nephrotic syndrome caused by a mutation in TRPC6, Epstein syndrome, and Fechtner syndrome.Preferably, the glomerular disease is nephrotic syndrome.

[0092] In some embodiments, the subject has or is at risk of suffering from GBM-related hereditary glomerular disease.GBM-related hereditary glomerular disease includes X-linked Alport syndrome, autosomal recessive Alport syndrome, autosomal dominant Alport syndrome, thin basement membrane disease, Pearson syndrome, and Nail-Patera syndrome.Preferably, the glomerular disease is Alport syndrome (AS).AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease, and thin basement membrane nephropathy.

[0093] In some embodiments, the subject has or is at risk of suffering from a complement-mediated kidney disease. As used herein, "complement-mediated kidney disease" refers to a kidney disease caused by dysregulation of the complement system. The complement system can cause kidney damage in a variety of different diseases. Preferably, the complement-mediated kidney disease is caused by excessive activation of the complement system. Exemplary complement-mediated kidney diseases include IgA nephropathy, C3 glomerulopathy, atypical hemolytic uremic syndrome (aHUS), stx-associated HUS, lupus nephritis, cryoglobulinemia, anti-GBM disease, ANCA-associated vasculitis, bacterial endocarditis, post-infectious glomerulonephritis, antibody-mediated rejection of kidney transplants, membranous nephropathy, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

[0094] In some embodiments, the subject has or is at risk for diabetic nephropathy, also known as diabetic kidney disease, which is the chronic loss of kidney function that occurs in people with diabetes.

[0095] In some embodiments, the subject has or is at risk of developing Fabry disease, a genetic disorder caused by the accumulation of globotriaosylceramide in lysosomes. Renal complications are a common and serious consequence of the disease.

[0096] Viral vector dose The viral vector can be delivered at any suitable dosage (e.g., measured in vector genome (vg) or vg per kg). The dosage can be determined by factors such as the subject's condition, age, weight, and type and severity of the subject's disease, and the appropriate dosage can be determined by a physician. The viral vector can be prescribed accordingly.

[0097] The methods of the present invention may allow for the use of lower doses of viral vectors compared to other delivery methods (e.g., systemic administration such as intravenous administration), which results in fewer risks of immune responses and fewer non-specific effects associated with low or no liver expression of proteins encoded by the viral vector compared to systemic administration such as intravenous administration.

[0098] Preferably, the viral vector is administered at a concentration of about 1 x 10 6 More than vg / kg, about 1x10 7 More than vg / kg, about 1x10 8 More than vg / kg, about 1x10 9 More than vg / kg, about 1x10 10 More than vg / kg, about 1x10 11 vg / kg or more, or about 1x10 12 Preferably, the viral vector is delivered at a dose of about 1 x 10 vg / kg or more. 14 vg / kg or less or about 1x10 13 Preferably, the viral vector is delivered at a dose of about 1 x 10 vg / kg or less. 6 vg / kg ~ approx. 1x10 14 Preferably, the viral vector is delivered at a dose of about 1 x 10 vg / kg. 6 vg / kg ~ approx. 1x10 13 Preferably, the viral vector is delivered at a dose of about 1 x 10 vg / kg. 6 vg / kg~about 10x10 6 vg / kg, approx. 1x10 7 vg / kg~about 10x10 7 vg / kg, approx. 1x10 8 vg / kg~about 10x10 8 vg / kg, approx. 1x10 9 vg / kg~about 10x10 9 vg / kg, approx. 1x10 10 vg / kg~about 10x10 10 vg / kg, approx. 1x10 11 vg / kg~about 10x10 11 vg / kg, or approximately 1x10 12 vg / kg~about 10x10 12vg / kg.

[0099] In some embodiments, the viral vector is about 1 x 10 9 vg / kg ~ approx. 1x10 12 In some embodiments, the viral vector is delivered at a dose of about 3x10 vg / kg. 9 vg / kg~approx.3x10 11 vg / kg.

[0100] Preferably, the viral vector is administered at a concentration of about 1 x 10 8 vg or more, about 1x10 9 vg or more, about 1x10 10 vg or more, about 1x10 11 vg or more, about 1x10 12 vg or more, about 1x10 13 vg or more, or about 1x10 14 Preferably, the viral vector is delivered at a dose of about 1 x 10 15 vg or less or about 1x10 14 Preferably, the viral vector is delivered at a dose of about 1 x 10 8 vg~approx. 1x10 15 Preferably, the viral vector is delivered at a dose of about 1 x 10 8 vg~approx.5x10 14 Preferably, the viral vector is delivered at a dose of about 1 x 10 8 vg~approx. 1x10 14 vg, approx. 1x10 9 vg~approx. 1x10 14 vg, approx. 1x10 10 vg~approx. 1x10 14 vg, approx. 1x10 11 vg~approx. 1x10 14 vg, or approximately 1x10 12 vg~approx. 1x10 14 Preferably, the viral vector is delivered at a dose of about 1 x 10 8 vg~approx. 10x10 8 vg, approx. 1x10 9 vg~approx. 10x10 9 vg, approx. 1x10 10vg~approx. 10x10 10 vg, approx. 1x10 11 vg~approx. 10x10 11 vg, approx. 1x10 12 vg~approx. 10x10 12 vg, approx. 1x10 13 vg~approx. 10x10 13 vg, or approximately 1x10 14 vg~approx. 10x10 14 vg dose.

[0101] In some embodiments, the viral vector is about 1 x 10 11 vg~approx. 1x10 14 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 11 vg, approx. 2x10 11 vg, approx. 3x10 11 vg, approx. 4x10 11 vg, approx. 5x10 11 vg, approx. 6x10 11 vg, approx. 7x10 11 vg, approx. 8x10 11 vg, approx. 9x10 11 vg, approx. 1x10 12 vg, approx. 2x10 12 vg, approx. 3x10 12 vg, approx. 4x10 12 vg, approx. 5x10 12 vg, approx. 6x10 12 vg, approx. 7x10 12 vg, approx. 8x10 12 vg, approx. 9x10 12 vg, approx. 1x10 13 vg, approx. 2x10 13 vg, approx. 3x10 13 vg, approx. 4x10 13 vg, approx. 5x10 13 vg, approx. 6x10 13 vg, approx. 7x10 13 vg, approx. 8x10 13 vg, approx. 9x10 13 vg, or approximately 1x10 14 vg dose.

[0102] In some embodiments, the viral vector is administered at a dose of about 2x10, for example, for a 70 kg subject. 11 vg~approx. 2x10 13 vg dose.

[0103] In some embodiments, the viral vector is about 5x10 11 vg~approx.5x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 12 vg~approx.5x10 13 In some embodiments, the viral vector is delivered at a dose of about 5x10 vg. 12 vg~approx.5x10 13 vg dose.

[0104] In some embodiments, the viral vector is about 5x10 11 vg~approx. 2x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 12 vg~approx. 2x10 13 In some embodiments, the viral vector is delivered at a dose of about 5x10 vg. 12 vg~approx. 2x10 13 vg dose.

[0105] In some embodiments, the viral vector is about 5x10 11 vg~approx. 1x10 13 In some embodiments, the viral vector is delivered at a dose of about 1 x 10 vg. 12 vg~approx. 1x10 13 In some embodiments, the viral vector is delivered at a dose of about 5x10 vg. 12 vg~approx. 1x10 13 vg dose. In some embodiments, the viral vector is about 1 x 10 13 vg dose.

[0106] Viral vector preparations The viral vector can be delivered to the renal artery in the form of a viral vector formulation. As used herein, a "viral vector formulation" may refer to a composition comprising or consisting of a therapeutically effective amount of a viral vector. Preferably, it contains a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). "Pharmaceutically acceptable" includes that the formulation be sterile and pyrogen-free. The carrier, diluent, and / or excipient must be "acceptable" in the sense of being compatible with the viral vector and not harmful to the recipient thereof. Typically, the carrier, diluent, or excipient is sterile, pyrogen-free saline or infusion media, although other acceptable carriers, diluents, and excipients may be used.

[0107] The viral vector may be administered into the renal artery in the form of a sterile aqueous solution which may contain other substances, for example, enough salts and glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0108] Preferably, the viral vector formulation comprises an isotonic buffer (e.g., about pH 7.4). In some embodiments, the viral vector formulation comprises 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 formulation comprises Plasmalyte. In some embodiments, the viral vector formulation comprises about 0.001% Poloxamer 188 (also known as Pluronic® F-68).

[0109] The viral vector formulation can contain any suitable amount of viral vector. Preferably, the viral vector formulation contains about 1x10 7 vg / ml or more, approximately 1x10 8 vg / ml or more, approximately 1x10 9 vg / ml or more, approximately 1x10 10vg / ml or more, approximately 1x10 11 vg / ml or more, approximately 1x10 12 vg / ml or greater, or approximately 1x10 13 Preferably, the viral vector formulation contains about 1 x 10 14 vg / ml or less or about 1x10 13 Preferably, the viral vector formulation contains about 1 x 10 7 vg / ml ~ approx. 1x10 14 Preferably, the viral vector formulation contains about 1 x 10 vg / ml of viral vector. 7 vg / ml ~ approx. 5x10 13 Preferably, the viral vector formulation contains about 1 x 10 vg / ml of viral vector. 7 vg / ml ~ approx. 10x10 7 vg / ml, approx. 1x10 8 vg / ml ~ approx. 10x10 8 vg / ml, approx. 1x10 9 vg / ml ~ approx. 10x10 9 vg / ml, approx. 1x10 10 vg / ml ~ approx. 10x10 10 vg / ml, approx. 1x10 11 vg / ml ~ approx. 10x10 11 vg / ml, approx. 1x10 12 vg / ml ~ approx. 10x10 12 vg / ml, or approximately 1x10 13 vg / ml ~ approx. 10x10 13 Contains a viral vector in an amount of vg / ml.

[0110] In some embodiments, the viral vector formulation comprises about 1 x 10 10 vg / ml ~ approx. 1x10 13 In some embodiments, the viral vector formulation comprises about 1 x 10 vg / ml of viral vector. 10 vg / ml ~ approx. 1x10 12 Contains a viral vector in an amount of vg / ml.

[0111] The viral vector formulation may have any appropriate volume. Preferably, the viral vector formulation 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. In some embodiments, the viral vector formulation 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. The viral vector formulation may further comprise one or more other therapeutic agents.

[0112] viral vectors The viral vector of the present invention is preferably an adeno-associated virus (AAV) vector, but it is considered that other viral vectors can be used.Other suitable viral vectors can include lentivirus vector, retrovirus vector, adenovirus vector, herpes simplex virus vector, alphavirus vector, flavivirus vector, rhabdovirus vector, measles virus vector, Newcastle disease virus vector, poxvirus vector and picornavirus vector.

[0113] The viral vector of the present invention may be in the form of a viral vector particle. Methods for preparing and modifying viral vectors and viral vector particles derived from AAV are well known in the art. Suitable methods are described in Ayuso, E., et al., 2010 Current gene therapy, 10(6), pp.423-436; Merten, OW, et al., 2016 Molecular Therapy-Methods & Clinical Development, 3, p.16017; and Nadeau, I. and Kamen, A., 2003 Biotechnology advances, 20(7-8), pp.475-489.

[0114] The viral vector of the present invention is preferably capable of transducing kidney cells. Suitably, the viral vector of the present invention is capable of specifically transducing kidney cells. Preferably, the viral vector of the present invention is capable of transducing glomerular cells. Suitably, the viral vector of the present invention is capable of specifically transducing glomerular cells. Preferably, the viral vector of the present invention is capable of transducing podocytes. Suitably, the viral vector of the present invention is capable of specifically transducing podocytes.

[0115] Adeno-associated virus (AAV) vectors The viral vectors of the present invention are preferably adeno-associated viral (AAV) vector particles.

[0116] AAV genome An AAV vector particle may comprise an AAV genome or a fragment or derivative thereof. An AAV genome is a polynucleotide sequence that can encode functions necessary for the production of AAV particles. These functions include those that operate in the AAV replication and packaging cycle in a host cell, including encapsidation of the AAV genome into AAV particles. Naturally occurring AAV is replication-deficient and relies on the provision of helper functions in trans to complete the replication and packaging cycle. Thus, the AAV genome of the AAV vector of the present invention is typically replication-deficient.

[0117] The AAV genome may be single-stranded (ssAAV), positive or negative sense, or double-stranded (dsAAV). The use of double-stranded AAV can eliminate the DNA replication step in target cells and promote transgene expression. The maximum packaging capacity of a single-stranded AAV is greater than that of a double-stranded AAV. Preferably, the AAV genome is single-stranded.

[0118] Naturally occurring AAVs can be classified according to various biological systems, and AAV genomes can be derived from any naturally occurring serotype, isolate, or clade of AAV.

[0119] AAV can be designated by serotype. Serotypes correspond to AAV variants with characteristic reactivities that can be used to distinguish them from other variants based on the expression profile of capsid surface antigens. Typically, AAV vector particles having a specific AAV serotype do not efficiently cross-react with neutralizing antibodies specific for other AAV serotypes. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. In some embodiments, the AAV vectors of the present invention can be of the AAV3B, LK03, AAV9, or AAV8 serotype. In some embodiments, the AAV vectors of the present invention can be of the AAV3B, LK03, or AAV9 serotype.

[0120] AAVs can also be referred to by clade or clone. This refers to the phylogenetic relationship of AAVs derived from nature, typically a phylogenetic group of AAVs that can be traced back to a common ancestor and includes all of its descendants. Furthermore, AAVs can be referred to as specific isolates, i.e., genetic isolates of specific AAVs present in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other AAVs present in nature, thereby defining a distinct population that can be recognized at the genetic level.

[0121] Typically, the AAV genome of a naturally occurring serotype, isolate, or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional origin of replication, allowing for the integration and excision of the vector from the cellular genome. The ITR may be the only sequence required in cis adjacent to the therapeutic gene.

[0122] The AAV genome may also contain packaging genes, such as the rep gene and / or the cap gene, which encode packaging functions for AAV particles. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19, and p40 promoters. For example, the p5 and p19 promoters are commonly used to express the rep gene, and the p40 promoter is commonly used to express the cap gene. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52, and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins, such as VP1, VP2, VP3, or variants thereof. These proteins constitute the capsid of the AAV particle, which determines the AAV serotype. VP1, VP2, and VP3 can be produced by alternative mRNA splicing (Trempe, JP and Carter, BJ, 1988, Journal of Virology, 62(9), pp. 3356-3363). Thus, VP1, VP2 and VP3 may have the same sequence, but VP2 is N-terminally truncated relative to VP1 and VP3 is N-terminally truncated relative to VP2.

[0123] The AAV genome can be the entire genome of a naturally occurring AAV. For example, an AAV vector can be prepared using a vector containing the entire AAV genome. Preferably, the AAV genome is derivatized for administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome, as well as derivatives that can be produced by applying techniques known in the art. The AAV genome can be a derivative of any naturally occurring AAV. Preferably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Preferably, the AAV genome is a derivative of AAV2.

[0124] Derivatives of the AAV genome include truncated or modified forms of the AAV genome that allow transgene expression from the AAV vector of the present invention in vivo. Typically, the AAV genome can be significantly truncated to contain minimal viral sequences while retaining the above functions. This is preferred for safety reasons, reducing the risk of recombination between the vector and wild-type virus, and to avoid the induction of cellular immune responses due to the presence of viral gene proteins in target cells.

[0125] Typically, a derivative contains at least one inverted terminal repeat (ITR), preferably two or more ITRs. One or more of the ITRs may be derived from an AAV genome of a different serotype, and may be chimeric or mutant ITRs. A preferred mutant ITR is one that has a trs (terminal resolution site) deletion. This deletion allows genome replication to continue and generate a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV (scAAV) genome. This bypasses DNA replication in the target cell and accelerates transgene expression. However, the maximum packaging capacity of the scAAV is reduced. Preferably, the AAV genome is not a scAAV genome.

[0126] The AAV genome may comprise one or more ITR sequences from any naturally occurring serotype, isolate, clade, or variant of AAV. The AAV genome may comprise at least one, for example, two, of the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11, or variants thereof. Preferably, the AAV genome may comprise at least one, for example, two, of the AAV2 ITRs.

[0127] Preferably, one or more ITRs are included to aid in the formation of concatamers of the AAV vector in the host cell nucleus, e.g., after the single-stranded vector DNA is converted to double-stranded DNA by the action of host cell DNA polymerases. Formation of such episomal concatamers protects the AAV vector for the life of the host cell and allows for long-term transgene expression in vivo.

[0128] Preferably, the ITR elements are the only sequences 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 other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the likelihood of the vector integrating into the host cell genome. Furthermore, the reduced size of the AAV genome allows for greater flexibility in incorporating other sequence elements (such as regulatory elements) into the vector in addition to the transgene.

[0129] Thus, in the derivatives of the present invention, the following portions may be removed: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, the derivatives may further include one or more of the rep and / or cap genes of the AAV genome, or other viral sequences. Because naturally occurring AAV integrates at a high frequency into a specific site on human chromosome 19, with only a negligible frequency of random integration, retaining the integration capacity of AAV vectors may be acceptable in a therapeutic setting.

[0130] The invention further encompasses providing sequences of the AAV genome in an order and arrangement that differs from that of the native AAV genome. The invention also encompasses replacing one or more AAV sequences or genes with sequences from another virus, or with chimeric genes comprised of sequences from two or more viruses. Such chimeric genes can be comprised of sequences from two or more related viral proteins from different viral species.

[0131] AAV serotypes and capsid proteins AAV vector particles can be encapsidated by capsid proteins. Serotypes can promote transduction of glomerular cells (e.g., podocytes), for example, specific transduction of glomerular cells (e.g., podocytes).

[0132] The AAV vector particle can be a kidney-specific vector particle. Preferably, the AAV vector particle is a glomerulus-specific (e.g., podocyte-specific) vector particle. The AAV vector particle can be encapsidated by a glomerulus-specific (e.g., podocyte-specific) capsid. The AAV vector particle can comprise a glomerulus-specific (e.g., podocyte-specific) capsid protein.

[0133] Preferably, the AAV vector particle can be in a transcapsid form, in which an AAV genome or derivative having ITRs of one serotype is packaged into a capsid of a different serotype. AAV vector particles also include mosaic forms in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV vector particles also include chemically modified forms having ligands adsorbed to the capsid surface. For example, such ligands include antibodies that target specific cell surface receptors.

[0134] Where the derivative comprises capsid proteins, i.e., VP1, VP2, and / or VP3, the derivative may be a chimeric, shuffled, or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the present invention encompasses providing capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e., pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle.

[0135] Chimeric, shuffled, or capsid-modified derivatives are typically selected to provide one or more desired functionalities to the AAV vector. Therefore, compared to AAV vectors containing naturally occurring AAV genomes, these derivatives may exhibit improved gene delivery efficiency, reduced immunogenicity (humoral or cellular), altered tropism, and / or improved podocyte targeting. Improved gene delivery efficiency may be achieved by improved receptor or co-receptor binding on the cell surface, improved internalization, improved intracellular and nuclear transport, improved uncoating of viral particles, and improved conversion of single-stranded genomes to double-stranded forms. Improved efficiency may also be related to altered podocyte tropism or targeting, so that the vector dose is not diluted by administration to tissues where it is not needed.

[0136] Chimeric capsid proteins include those generated by recombination between two or more capsid-encoding sequences of naturally occurring AAV serotypes. This can be achieved, for example, by marker rescue, 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 performed to select for capsid sequences with desired properties. Capsid sequences of different serotypes can be modified in cells by homologous recombination to produce novel chimeric capsid proteins.

[0137] 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.

[0138] Shuffled or chimeric capsid proteins can also be generated by DNA shuffling or error-prone PCR. Hybrid AAV capsid genes can be created by randomly fragmenting the sequences of related AAV genes, such as genes encoding capsid proteins of multiple different serotypes, and then reassembling the fragments in a self-priming polymerase reaction, which may result in crossovers in regions of sequence homology. By shuffling the capsid genes of multiple serotypes, a library of hybrid AAV genes created in this way 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 library of diverse variants, which can then be selected for desired properties.

[0139] 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 act as a ligand for a specific cell type, thereby conferring improved binding to target cells or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein can also be an epitope or affinity tag that aids in the purification of viral particles as part of the production process. The insertion site is usually selected so as not to interfere with other functions of the viral particle (e.g., internalization, transport of viral particles).

[0140] 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 occur in nature or that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein contains mutations or changes in its amino acid sequence compared to the sequence of the parent capsid from which it is derived when the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned.

[0141] The capsid protein can contain mutations or modifications to the wild-type capsid protein that improve its ability to transduce podocytes compared to unmodified or wild-type viral particles. The improved ability to transduce podocytes can be measured, for example, by measuring the expression of a transgene, such as GFP, carried by the AAV vector particle, and the expression of the transgene in the podocyte correlates with the ability of the AAV vector particle to transduce podocytes.

[0142] The AAV vector particles can be AAV3B, LK03, AAV9, or AAV8 vector particles. The present inventors have shown that AAV vector particles having AAV3B, LK03, AAV9, and AAV8 serotypes can transduce podocytes. Preferably, the AAV vector particles are AAV3B or LK03 vector particles. More preferably, the AAV vector particles are LK03 vector particles.

[0143] The AAV vector particle may comprise an AAV3B, LK03, AAV9, or AAV8 capsid protein. Preferably, the AAV vector particle comprises an AAV3B capsid protein or an LK03 capsid protein. More preferably, the AAV vector particle comprises an LK03 capsid protein.

[0144] The AAV vector particle may comprise AAV3B, LK03, AAV9, or AAV8 capsid proteins VP1, VP2, and VP3. Preferably, the AAV vector particle comprises AAV3B or LK03 capsid proteins VP1, VP2, and VP3. More preferably, the AAV vector particle comprises LK03 capsid proteins VP1, VP2, and VP3.

[0145] The AAV vector particle may comprise one or more AAV2 ITR sequences and an AAV3B capsid protein, an LK03 capsid protein, an AAV9 capsid protein, or an AAV8 capsid protein. Preferably, the AAV vector particle comprises one or more AAV2 ITR sequences and an AAV3B or LK03 capsid protein. More preferably, the AAV vector particle comprises one or more AAV2 ITR sequences and an LK03 capsid protein.

[0146] The AAV vector particle may have an AAV2 genome and an AAV3B capsid protein (AAV2 / 3B), an AAV2 genome and an LK03 capsid protein, an AAV2 genome and an AAV9 capsid protein (AAV2 / 9), or an AAV2 genome and an AAV8 capsid protein (AAV2 / 8). Preferably, the AAV vector particle comprises an AAV2 genome and an AAV3B or LK03 capsid protein. More preferably, the AAV vector particle comprises an AAV2 genome and an LK03 capsid protein.

[0147] The designation AAVX / Y can indicate a pseudotyped AAV, e.g., one in which the ITR sequences are from AAVX and flank a cassette carrying a payload encapsidated (i.e., having AAVY capsid proteins) in serotype AAVY.

[0148] AAV3B serotype The AAV vector particles may comprise AAV3B capsid proteins. Suitably, the AAV vector particles may be encapsidated by AAV3B capsid proteins.

[0149] Two different AAV3 isolates (AAV3A and AAV3B) have been cloned. Compared with vectors based on other AAV serotypes, AAV3 vectors are thought to transduce most cell types inefficiently. However, AAV3B can efficiently transduce podocytes. AAV3B is described in Rutledge, EA, et al., 1998, Journal of Virology, 72(1), pp. 309-319.

[0150] The AAV vector particle may comprise an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and / or an AAV3B VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an AAV3B VP1 capsid protein, an AAV3B VP2 capsid protein, and / or an AAV3B VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an AAV3B VP1, VP2, and VP3 capsid protein.

[0151] Suitably, the AAV3B VP1 capsid protein comprises or consists of the amino acid sequence shown as SEQ ID NO:1, or a variant that is at least 90% identical to SEQ ID NO:1.

[0152] TIFF2025539119000001.tif51163 Exemplary AAV3B VP1 Capsid Protein (SEQ ID NO: 1)

[0153] 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. Suitably, the AAV3B VP2 and VP3 capsid proteins may be N-terminally truncated versions of SEQ ID NO:1 or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:1.

[0154] LK03 serotype AAV vector particles may comprise LK03 capsid protein. Suitably, AAV vector particles may be encapsidated by LK03 capsid protein.

[0155] The AAV-LK03 cap sequence consists of fragments derived from seven different wild-type serotypes (AAV1, 2, 3B, 4, 6, 8, and 9) and is described in Lisowski, L., et al., 2014. Nature, 506(7488), pp. 382-386. The present inventors have demonstrated that the AAV-LK03 vector can achieve high transduction rates, approaching 100%, in human podocytes in vitro.

[0156] The AAV vector particle may comprise an LK03 VP1 capsid protein, an LK03 VP2 capsid protein, and / or an LK03 VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an LK03 VP1 capsid protein, an LK03 VP2 capsid protein, and / or an LK03 VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an LK03 VP1, VP2, and VP3 capsid protein.

[0157] Suitably, the LK03 VP1 capsid protein may comprise or consist of the amino acid sequence shown as SEQ ID NO:2, or a variant which is at least 90% identical to SEQ ID NO:2.

[0158] TIFF2025539119000002.tif52162 Exemplary LK03 VP1 Capsid Protein (SEQ ID NO: 2)

[0159] 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. Suitably, the LK03 VP2 and VP3 capsid proteins may be N-terminally truncated versions of SEQ ID NO:2, or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:2.

[0160] AAV9 serotype The AAV vector particles may comprise AAV9 capsid proteins. Suitably, the AAV vector particles may be encapsidated by AAV9 capsid proteins.

[0161] The present inventors have demonstrated that AAV9 vectors can achieve high transduction rates in human podocytes in vitro.

[0162] The AAV vector particle may comprise an AAV9 VP1 capsid protein, an AAV9 VP2 capsid protein, and / or an AAV9 VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an AAV9 VP1 capsid protein, an AAV9 VP2 capsid protein, and / or an AAV9 VP3 capsid protein. Preferably, the AAV vector particle may be encapsidated by an AAV9 VP1, VP2, and VP3 capsid protein.

[0163] Suitably, the AAV9 VP1 capsid protein comprises or consists of the amino acid sequence shown as SEQ ID NO:3, or a variant that is at least 90% identical to SEQ ID NO:3.

[0164] TIFF2025539119000003.tif50162 Exemplary AAV9 VP1 Capsid Protein (SEQ ID NO: 3)

[0165] 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.

[0166] Suitably, the AAV9 VP2 and VP3 capsid proteins may be N-terminally truncated versions of SEQ ID NO:3, or variants that are at least 90% identical, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:3.

[0167] Other viral vectors Retroviral and lentiviral vectors The viral vector of the present invention can be a retroviral vector or a lentiviral vector.

[0168] Retroviral vectors can be derived from or be derivable from any suitable retrovirus. Many different retroviruses have been identified, including 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).

[0169] Retroviruses can be broadly divided into two categories: "simple" and "complex." Retroviruses can be further divided into seven groups. Five of these groups are oncogenic retroviruses. The remaining two groups are lentiviruses and spumaviruses.

[0170] The basic genome structure of retroviruses and lentiviruses shares many common features, including 5'LTR and 3'LTR. These include a packaging signal that enables genome packaging, a primer binding site, an integration site that enables integration into the host cell genome, and the gag, pol, and env genes that encode packaging components (polypeptides required for viral particle assembly). Lentiviruses possess additional features, such as the HIV rev and RRE sequences, that allow efficient transport of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells.

[0171] In the provirus, these genes are flanked by regions called long terminal repeats (LTRs) at both ends. LTRs are responsible for the integration and transcription of the provirus. LTRs also function as enhancer-promoter sequences, allowing them to control viral gene expression.

[0172] The LTRs themselves are identical sequences divided into three elements: U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements can vary greatly depending on the retrovirus. In defective retroviral vector genomes, gag, pol and env may be absent or non-functional.

[0173] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication can be removed from the virus, rendering the viral vector replication-deficient. Portions of the viral genome can also be replaced with a library encoding potential regulatory sites operably linked to regulatory control regions and reporter sites in the vector genome to generate vectors containing potential regulatory sites capable of transducing target host cells and / or integrating into the host genome.

[0174] Lentiviral vectors are part of a larger group of retroviral vectors. For convenience, 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 prototypic "slow virus" Visna / Maedi virus (VMV), the related Caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0175] The lentivirus family differs from retroviruses in that it has the ability to infect both dividing and non-dividing cells, whereas other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as those that make up muscle, brain, lung, and liver tissue.

[0176] As used herein, a lentiviral vector is a vector that contains at least one component derivable from a lentivirus, preferably that component is involved in the biological machinery by which the vector infects cells, expresses genes, or replicates.

[0177] The lentiviral vector can be a "primate" vector. The lentiviral vector can be a "non-primate" vector (i.e., derived from a virus that does not primarily infect primates, especially humans). An example of a non-primate lentivirus can be any member of the lentiviridae family that does not naturally infect primates.

[0178] As examples of lentiviral-based vectors, HIV-1 and HIV-2 based vectors are described below.

[0179] 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 HIV-1 packaging. Therefore, HIV-1 vectors often contain a relevant portion of the gag gene with a mutated translation initiation codon. Furthermore, most HIV-1 vectors also contain a portion of the env gene, including the RRE. Rev binds to the RRE, enabling the transport of full-length or single-spliced ​​mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses, such as the Mason-Pfizer monkey virus, can be used to alleviate the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0180] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient delivery of full-length or single-spliced ​​viral RNA.

[0181] Preferably, the viral vectors used in the present invention have a minimal viral genome. By "minimal viral genome" is meant that the viral vector has been engineered to remove non-essential elements and retain essential elements to provide the functions necessary to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell. Details of this method are described in WO 1998 / 017815.

[0182] Preferably, the plasmid vector used to produce the viral genome in the host / packaging cell contains sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles that, in the presence of packaging components, are capable of infecting target cells but are incapable of independent replication to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0183] However, the plasmid vectors used to produce the viral genome in the host cell / packaging cell also contain transcriptional control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These control sequences may be native sequences associated with the transcribed viral sequence (i.e., the 5' U3 region) or may be heterologous promoters such as other viral promoters (e.g., the CMV promoter).

[0184] The vector can be a self-inactivating (SIN) vector, in which the viral enhancer and promoter sequences have been deleted. Once engineered, the SIN vector can transduce non-dividing cells in vivo with efficacy comparable to that of wild-type vectors. Transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent recruitment by replication-competent virus. This should also allow for controlled expression of genes from internal promoters by eliminating the cis-acting effects of the LTR.

[0185] The vector can be integration-deficient. Integration-deficient lentiviral vectors (IDLVs) can be generated, for example, by packaging a catalytically inactive integrase (such as HIV integrase with a D64V mutation in the catalytic site) into the vector, by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.

[0186] Adenovirus vectors The viral vector of the present invention can be an adenoviral vector. Adenoviruses are double-stranded, linear DNA viruses that do not pass through an RNA intermediate. There are over 50 human adenovirus serotypes, divided into six subgroups based on genetic sequence homology. Adenoviruses naturally target respiratory and gastrointestinal epithelia and generally cause only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are the most commonly used adenovirus vector systems and are typically associated with upper respiratory tract infections in young people.

[0187] Adenoviruses have been used as vectors for gene therapy and expression of heterologous genes. Their large (36 kb) genome can accommodate up to 8 kb of foreign DNA insert, replicate efficiently in complementing cell lines, and produce up to 10 12 Adenoviruses can produce very high titers of HIV-1, HIV-1, and HIV-2. Therefore, adenoviruses are one of the best systems for studying gene expression in primary non-replicating cells.

[0188] Expression of viral and 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 host chromosomes. Instead, they function as linear genomes within the host nucleus as episomes (independent of the host genome). Therefore, the use of recombinant adenoviruses can alleviate the problems associated with random integration into the host genome.

[0189] Herpes simplex virus vector The viral vector of the present invention may be a herpes simplex viral vector.

[0190] Herpes simplex virus (HSV) is a neurotropic DNA virus with advantageous properties as a gene transfer vector. Because HSV is highly infectious, HSV vectors are an efficient vehicle for delivering exogenous genetic material into cells. Viral replication is easily disrupted in vitro by null mutations in immediate-early genes that can be complemented in trans, facilitating the production of high-titer, pure preparations of nonpathogenic vectors. The large genome (152 kb) and the majority of viral genes are dispensable for in vitro replication, allowing for replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal persistence in sensory neuronal nuclei for the life of the host. The vector is nonpathogenic, cannot reactivate, and persists for long periods. Latently active promoter complexes can be utilized in vector design to achieve long-term, stable transgene expression in the nervous system. HSV vectors can be delivered to a wide range of tissues due to the broad expression pattern of the cellular receptors recognized by the virus. Advances in understanding the processes involved in cell entry have made it possible to target the tropism of HSV vectors.

[0191] Other viral vectors Other suitable viral vectors include those described in Lundstrom, K., 2018. Diseases, 6(2), p. 42.

[0192] The viral vector of the present invention may be an alphavirus vector. The viral vector of the present invention may be a flavivirus vector.

[0193] Self-amplifying ssRNA viruses include alphaviruses (e.g., Semliki Forest virus, Sindbis virus, Venezuelan equine encephalitis virus, M1) and flaviviruses (e.g., Kunjin virus, West Nile virus, and Dengue virus) with positive-polarity genomes. Alphaviruses are primarily used 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 -deficient particles.

[0194] The viral vector of the present invention may be a rhabdovirus vector.The viral vector of the present invention may be a measles virus vector.

[0195] Rhabdoviruses (e.g., rabies and vesicular stomatitis viruses) and measles virus have negative-strand genomes. Among rhabdoviruses, recombinant vesicular stomatitis virus (VSV) has been applied in preclinical gene therapy studies. Measles viruses (e.g., MV-Edm) have found many gene therapy applications.

[0196] The viral vector of the present invention may be a Newcastle disease virus vector. Newcastle disease virus (NDV), an ssRNA paramyxovirus, is frequently used in cancer gene therapy because it replicates specifically in tumor cells.

[0197] The viral vector of the present invention may be a poxvirus vector. Poxviruses are characterized by their dsDNA genomes, which can easily accommodate foreign DNA of 30 kb or more. Poxviruses have found some use as gene therapy vectors. For example, vaccinia virus vectors have shown potential for cancer therapy. Vaccinia virus is a large, enveloped poxvirus with a linear, double-stranded DNA genome of approximately 190 kb. Vaccinia virus can incorporate foreign DNA up to approximately 25 kb, making it useful for delivering large genes. Numerous attenuated vaccinia virus strains suitable for gene therapy applications are known in the art, such as the MVA and NYVAC strains.

[0198] The viral vector of the present invention may be a picornavirus vector. Picornaviruses are non-enveloped ssRNA viruses. Coxsackieviruses, which belong to the Picornaviridae family, have been used as oncolytic vectors.

[0199] Protein coding sequence The viral vector may comprise a protein coding sequence. 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 related to glomerular disease.The protein coding sequence can encode a polypeptide involved in GBM-related hereditary glomerular disease, such as Alport syndrome.The protein coding sequence can encode a polypeptide involved in podocyte-related hereditary glomerular disease.

[0200] Suitably, the protein coding sequence is selected from the group consisting of 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, The gene may encode a 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.

[0201] Suitably, the protein coding sequence encodes a polypeptide having a length of at least 1450 amino acids, at least 1500 amino acids, at least 1550 amino acids, at least 1600 amino acids, or at least 1650 amino acids.

[0202] With respect to polynucleotides that encode proteins, those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. It should further be understood that those skilled in the art can, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by a polynucleotide of the invention to reflect the codon usage of the particular host organism in which the polypeptide of the invention is expressed.

[0203] Protein-coding sequences can be codon-optimized. Different cells use specific codons differently. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in that cell type. Altering the codons in a sequence to match the relative abundance of the corresponding tRNA can increase expression. Similarly, deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type can decrease expression, thus allowing for even greater translational control. Codon usage tables are known in the art for mammalian cells (e.g., humans) as well as for a variety of other organisms.

[0204] Nucleotide sequences encoding the proteins disclosed herein may or may not contain a stop codon at their 3' terminus. Thus, the present disclosure encompasses the SEQ ID NOs disclosed herein with or without a stop codon.

[0205] COL4A3, COL4A4 and COL4A5 polypeptides The protein coding sequence may encode a COL4A3, COL4A4 or COL4A5 polypeptide, or a fragment or derivative thereof.

[0206] The COL4A3, COL4A4, and COL4A5 proteins are homologous polypeptides of approximately 170–185 kDa containing collagenous Gly-XY repeats, frequently interrupted by non-collagenous sequences, forming triple-helical repeats. Each polypeptide also contains a large globular non-collagenous domain at the carboxyl terminus.

[0207] Alport syndrome (AS) is caused by pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes, resulting in abnormalities in the collagen IVα345 network of the basement membrane. The COL4A3, COL4A4 or COL4A5 polypeptide or a fragment or derivative thereof is capable of forming a collagen IVα345 network.

[0208] Approximately 200-300 amino acids can be removed from each of the COL4A3, COL4A4, and COL4A5 polypeptides to generate truncated transgenes suitable for minigene approaches. Amino acids can be removed from the triple helix repeat. Preferably, no amino acids are removed from non-collagenous regions.

[0209] In some embodiments, the COL4A3, COL4A4 and COL4A5 polypeptides are full-length polypeptides.

[0210] Preferably, the COL4A3, COL4A4, or COL4A5 polypeptide is of human origin. An exemplary human COL4A3 is COL4A3 having UniProtKB accession number Q01955. An exemplary human COL4A4 is COL4A3 having UniProtKB accession number P53420. An exemplary human COL4A5 is COL4A5 having UniProtKB accession number P29400.

[0211] Suitably, the COL4A3 peptide comprises or consists of the polypeptide sequence shown as SEQ ID NO: 4, or a variant that is at least 70% identical to SEQ ID NO: 4. 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.

[0212] Suitably, the COL4A4 peptide comprises or consists of the polypeptide sequence shown as SEQ ID NO: 5, or a variant that is at least 70% identical to SEQ ID NO: 5. 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.

[0213] Suitably, the COL4A5 peptide comprises or consists of the polypeptide sequence shown as SEQ ID NO: 6, or a variant that is at least 70% identical to SEQ ID NO: 6. 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.

[0214] TIFF2025539119000004.tif113164 Exemplary COL4A3 amino acid sequence - Uniprot reference number Q01955 (SEQ ID NO: 4)

[0215] TIFF2025539119000005.tif29162TIFF2025539119000006.tif86162Exemplary COL4A4 amino acid sequence - Uniprot reference number P53420 (SEQ ID NO: 5)

[0216] TIFF2025539119000007.tif113163 Exemplary COL4A5 amino acid sequence - Uniprot reference number P29400 (SEQ ID NO: 6)

[0217] The protein coding sequence comprises or consists of a COL4A3, COL4A4 or COL4A5 transgene. Examples of transgenes encoding COL4A3 are listed in NM_000091.5, examples of transgenes encoding COL4A4 are listed in NM_000092.5, and examples of transgenes encoding COL4A5 are listed in NM_000495.5.

[0218] Suitably, the COL4A3 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 7, or a variant which 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.

[0219] Suitably, the COL4A4 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 8, or a variant which 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.

[0220] Suitably, the COL4A5 transgene may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 9, or a variant which 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.

[0221] TIFF2025539119000008.tif130163TIFF2025539119000009.tif200162Exemplary COL4A3 transgene sequence (SEQ ID NO: 7)

[0222] TIFF2025539119000010.tif38162TIFF2025539119000011.tif249163TIFF2025539119000012.tif51163Exemplary COL4A4 transgene sequence (SEQ ID NO: 8)

[0223] TIFF2025539119000013.tif187163TIFF2025539119000014.tif148163Exemplary COL4A5 transgene sequence (SEQ ID NO: 9)

[0224] The COL4A3, COL4A4, or COL4A5 transgene may contain introns or intronic sequences that can be used to improve gene expression. The COL4A3, COL4A4, or COL4A5 transgene may contain a protein tag, such as a hemagglutinin (HA) tag. HA can be used as an epitope tag and has been shown not to interfere with the biological activity or biodistribution of the attached protein. Protein tags can facilitate detection, isolation, and purification of the transgene. Other suitable protein tags include Myc tags, polyhistidine tags, and Flag tags.

[0225] Nephrotic syndrome (NS)-associated transgene The protein coding sequence may comprise or consist of a NS-associated transgene. Nephrotic syndrome (NS) is a chronic kidney disease characterized by significant proteinuria, hypoalbuminemia, edema, and dyslipidemia. NS-associated transgenes can be genes associated with isolated forms of NS and expressed in podocytes.

[0226] 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, TP53RK, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, ACTN4, INF2, ANKFY1, These include ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, and NLRP3.

[0227] NPHS2 The protein coding sequence may encode NPHS2, or a fragment and / or variant thereof. "NPHS2" is the abbreviation for the polypeptide encoded by the NPHS2 gene, also known as podocin. NPHS2 is a 42-kDa hairpin-like membrane-associated podocyte-specific protein and a key component of the protein complex of the slit diaphragm, the intercellular junction between adjacent podocyte foot processes. It localizes to lipid rafts and interacts with other key slit diaphragm proteins, such as nephrin, CD2AP, and TRPC6. It is essential for maintaining the integrity of the slit diaphragm and, therefore, the glomerular filtration barrier.

[0228] The fragment and / or variant of NPHS2 may retain the activity or function of NPHS2. For example, the fragment and / or variant of podocin may regulate glomerular permeability. Preferably, the fragment and / or variant of NPHS2 may have the same or similar activity or function as NPHS2, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity or function of NPHS2.

[0229] Those skilled in the art can generate fragments and / or variants using conservative substitutions 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 known variants (see, for example, NCBI Gene ID: 7827 and NCBI HomoloGene: 22826). Preferably, the fragment and / or variant of NPHS2 comprises a transmembrane domain having two cytoplasmic domains at the N- and C-termini.

[0230] The NPHS2 gene is conserved in chimpanzees, rhesus monkeys, dogs, cows, mice, and rats. NPHS2 can be human NPHS2. Suitably, NPHS2 can comprise or consist of the polypeptide sequence of UniProtKB accession Q9NP85, or a fragment and / or variant thereof.

[0231] In some embodiments, NPHS2 comprises or consists of an amino acid sequence, or a fragment thereof, that is at least 70% identical to SEQ ID NO: 10. Suitably, NPHS2 comprises or consists of an amino acid sequence, or a fragment thereof, 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.

[0232] In some embodiments, NPHS2 comprises or consists of SEQ ID NO: 10, or a fragment thereof.

[0233] TIFF2025539119000015.tif27151 Exemplary NPHS2 amino acid sequence (SEQ ID NO: 10)

[0234] In some embodiments, the NPHS2 transgene comprises or consists of a nucleotide sequence, or a fragment thereof, that is at least 70% identical to SEQ ID NO: 11. Suitably, the NPHS2 transgene comprises or consists of a nucleotide sequence, or a fragment thereof, 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.

[0235] In some embodiments, the NPHS2 transgene comprises or consists of the nucleotide sequence of SEQ ID NO: 11, or a fragment thereof.

[0236] TIFF2025539119000016.tif70152 Exemplary NPHS2 transgene sequence (SEQ ID NO: 11)

[0237] In some embodiments, the NPHS2 transgene comprises or consists of a nucleotide sequence, or a fragment thereof, that is at least 70% identical to SEQ ID NO: 12. Suitably, the NPHS2 transgene comprises or consists of a nucleotide sequence, or a fragment thereof, 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.

[0238] In some embodiments, the NPHS2 transgene comprises or consists of the nucleotide sequence of SEQ ID NO: 12, or a fragment thereof.

[0239] TIFF2025539119000017.tif71151 Exemplary NPHS2 transgene sequence (SEQ ID NO: 12)

[0240] Vascular endothelial growth factor (VEGF) C transgene The protein coding sequence may comprise or consist of a vascular endothelial growth factor (VEGF) C transgene.

[0241] VEGFC is a lymphangiogenic growth factor known to signal through two receptors, VEGFR-3 (Flt4) and VEGFR-2 (Flk4). VEGFC is produced in cells as a prepropeptide and dimerizes before being cleaved into a tetramer.

[0242] The VEGFC transgene can comprise a polynucleotide encoding any form of VEGFC, such as the prepropeptide form, the tetramer form, an intermediate form, or the fully processed mature VEGFC.

[0243] Polynucleotides encoding different forms of VEGFC polypeptides can be used in any combination, as desired. Preferably, the VEGFC transgene comprises a polynucleotide encoding one or more polypeptides having VEGFC biological activity, i.e., peptides capable of binding to and activating VEGFR-2 and / or VEGRF-3. More preferably, the VEGFC transgene comprises a polynucleotide encoding a polypeptide comprising a VEGFC homology domain and having VEGFC biological activity, i.e., a polypeptide capable of binding to and activating VEGFR-2 and / or VEGRF-3. Further details of suitable VEGFC polynucleotides and polypeptides include those described in WO2015 / 022447 and US2014 / 0087002.

[0244] The VEGFC polynucleotide can comprise the VEGFC open reading frame (ORF) sequence of SEQ ID NO: 13. The VEGFC polynucleotide can 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 can encode VEGFC polypeptides that retain the ability to bind to and activate VEGFR-2 and VEGFR-3.

[0245] TIFF2025539119000018.tif86163 Exemplary VEGFC Polynucleotide (SEQ ID NO: 13)

[0246] Complement Proteins and Complement Inhibitors The protein coding sequence may encode a complement protein, or a fragment and / or variant thereof.

[0247] As used herein, "complement proteins" refer to proteins that are part of the complement system. The complement system, also known as the complement cascade, is a central part of innate immunity, serving as a first line of defense against foreign and altered host cells. The complement system is composed of plasma proteins primarily produced in the liver and membrane proteins expressed on cell surfaces. Complement functions in plasma, tissues, and intracellularly. Complement proteins work together as a cascade to opsonize pathogens and induce a series of inflammatory responses that help immune cells fight infection and maintain homeostasis (Merle, NS, et al., 2015. Frontiers in Immunology, 6, 262).

[0248] There are three pathways of complement activation: the classical pathway, the alternative pathway, and the lectin pathway. These three complement pathways differ in their target recognition mechanisms but converge in the activation of the central component C3. After this activation, C5 is cleaved, initiating the assembly of the membrane attack complex (MAC). Enzymatic cleavage of C3 and C5 leads to the production and release of the anaphylotoxins C3a and C5a.

[0249] Preferably, the complement proteins are selected from the list consisting of CFI, CFH, FHL-1, C1INH, C4BP, MASP2, C3, C5aR1, C5, C5a, CD55, CD35, CD46, CD59, vitronectin, and clusterin, or fragments and / or variants thereof. The protein coding sequence may encode an inhibitor of the complement system, or a fragment and / or variant thereof.

[0250] As used herein, "inhibitors of the complement system" or "complement inhibitors" refer to proteins that prevent activation of the complement system. Complement is tightly regulated 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, lectin, and alternative pathways. Preferably, the complement inhibitor is a naturally occurring complement inhibitor, or a fragment and / or variant thereof. Preferably, the complement inhibitor is an inhibitor of the human complement system.

[0251] Complement inhibitors are classified into two categories: soluble inhibitors and membrane-bound inhibitors. Preferably, the inhibitor of the complement system 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, vitronectin, etc. Membrane-bound regulatory factors include CD46, CD55, CD59, CD35, CUB and Sushi multiple domain 1 (CUB and Sushi multiple domain 1, CSMD1), etc.

[0252] 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.

[0253] Preferably, the inhibitor of the complement system is selected from CFI, CFH, and FHL-1, or fragments and / or variants thereof.

[0254] CFI The protein coding sequence may encode CFI, or a fragment and / or variant thereof.

[0255] Complement factor I (CFI) is a trypsin-like serine protease that inactivates these proteins by cleaving three peptide bonds in the α-chain of C3b and two bonds in the α-chain of C4b, thereby inhibiting the complement system.

[0256] CFI is a glycoprotein heterodimer consisting of a disulfide-linked heavy chain and a light chain. The heavy chain contains 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, keeping the enzyme inactive until it contacts a complex formed by its substrate (C3b or C4b) and cofactor proteins (factor H, C4b-binding protein, complement receptor 1, and membrane cofactor protein). Upon binding of the enzyme to the substrate:cofactor complex, the heavy chain:light chain interface is disrupted, and the enzyme is activated by allostery. The light chain contains only a serine protease domain. This domain contains the catalytic triad of His-362, Asp-411, and Ser-507, responsible for the specific cleavage of C3b and C4b.

[0257] CFI or a fragment and / or variant thereof is capable of cleaving C3b into iC3b and / or cleaving iC3b into C3d,g.

[0258] Fragments and / or variants of CFI may retain at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the C3b inactivating activity and iC3b degrading activity of native CFI. The C3b inactivating activity and iC3b degrading activity of CFI fragments and / or variants relative to native CFI can be determined using any suitable method known to those skilled in the art, for example, using a proteolysis assay.

[0259] Preferably, the CFI is a human CFI. An exemplary human CFI is the CFI having UniProtKB accession number P05156.

[0260] Suitably, the CFI may comprise or consist of the polypeptide sequence shown as SEQ ID NO:14, or a variant that is at least 70% identical to SEQ ID NO:14.

[0261] TIFF2025539119000019.tif43163 Exemplary CFI Polypeptide Sequence (SEQ ID NO: 14)

[0262] 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.

[0263] An example of a nucleotide sequence encoding CFI is NM_000204.5. Suitably, the protein coding sequence encoding CFI may comprise or consist of the polynucleotide sequence set forth as SEQ ID NO: 15, or a variant that is at least 70% identical to SEQ ID NO: 15.

[0264] TIFF2025539119000020.tif117164 Exemplary CFI Polynucleotide Sequence (SEQ ID NO: 15)

[0265] 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.

[0266] CFH The protein coding sequence may encode CFH, or a fragment and / or variant thereof.

[0267] Complement factor H (CFH) regulates complement activation on self-cells and surfaces. CFH competes with complement factor B (CFB) for binding to C3b and serves as a cofactor for the CFI-catalyzed proteolytic cleavage of C3b, promoting its irreversible dissociation into the separate components C3bBb and C3b2Bb. Thus, CFH not only inhibits the formation of convertases but also shortens the lifetime of the formed convertase complexes.

[0268] CFH is a large (155 kDa) soluble glycoprotein. CFH is composed of a total of 20 domains, each containing approximately 60 amino acid residues, called complement control protein modules (CCPs) or short consensus repeats connected by short linkers of 3–8 residues. The CCP modules are numbered 1–20 (from the N-terminus of the protein): CCP1–4 and CCP19–20 bind C3b, while CCP7 and CCP19–20 bind GAG and sialic acid.

[0269] CFH or its fragments and / or variants may bind to C3b and / or C3d; and / or act as a cofactor for CFI-catalyzed proteolytic cleavage of C3b; and / or increase the irreversible dissociation of C3bBb and C3b2Bb into their separate components. CFH fragments and / or variants may retain at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the activity of native CFH. The activity of CFH fragments and / or variants, as well as native CFH, may be determined using any suitable method known to those skilled in the art.

[0270] Preferably, the CFH is human CFH. An example of a human CFH is the CFH having UniProtKB accession number P08603.

[0271] Suitably, CFH may comprise or consist of the polypeptide sequence shown as SEQ ID NO:16, or a variant which is at least 70% identical to SEQ ID NO:16.

[0272] TIFF2025539119000021.tif86163 Exemplary CFH Polypeptide Sequence (SEQ ID NO: 16)

[0273] 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.

[0274] An example of a nucleotide sequence encoding CFH is NM_000186.4. Suitably, the protein coding sequence encoding CFH may comprise or consist of the polynucleotide sequence shown as SEQ ID NO: 17, or a variant that is at least 70% identical to SEQ ID NO: 17.

[0275] TIFF2025539119000022.tif222163TIFF2025539119000023.tif24163Exemplary CFH Polynucleotide Sequence (SEQ ID NO: 17)

[0276] 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. The CFH fragment may 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 of CFH (CCP1-7).

[0277] FHL-1 The protein coding sequence may encode FHL-1, or a fragment and / or variant thereof.

[0278] FHL-1 or its fragments and / or variants may bind C3b and / or C3d. FHL-1 fragments and / or variants may retain at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the activity of native FHL-1. The activity of FHL-1 fragments and / or variants and native FHL-1 may be determined using any suitable method known to those of skill in the art.

[0279] Preferably, the FHL-1 is human FHL-1. An exemplary human FHL-1 is the FHL-1 having the NCBI reference sequence: NP_001014975.1.

[0280] Suitably, FHL-1 may comprise or consist of the polypeptide sequence shown as SEQ ID NO:18, or a variant which is at least 70% identical to SEQ ID NO:18.

[0281] TIFF2025539119000024.tif33163 Exemplary FHL-1 Polypeptide Sequence (SEQ ID NO: 18)

[0282] 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.

[0283] An example of a nucleotide sequence encoding FHL-1 is NM_001014975.2. Suitably, the protein coding sequence encoding FHL-1 may comprise or consist of the polynucleotide sequence set forth as SEQ ID NO: 19, or a variant that is at least 70% identical to SEQ ID NO: 19.

[0284] TIFF2025539119000025.tif91164 Exemplary FHL-1 Polynucleotide Sequence (SEQ ID NO: 19)

[0285] 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.

[0286] gene editing agents In a preferred embodiment, the protein coding sequence does not encode a gene editing agent. As used herein, a "gene editing agent" may refer to a protein that can edit a sequence in a genome (see, e.g., Raguram, A., Banskota, S. and Liu, DR, 2022. Therapeutic in vivo delivery of gene editing agents. in Cell). A gene editing agent may be a meganuclease, a zinc finger nuclease, a TAL effector endonuclease, or a rare-cutting endonuclease, including a CRISPR endonuclease.

[0287] In some embodiments, the protein coding sequence does not encode a nuclease. A "nuclease" is an enzyme that can cleave phosphodiester bonds present in a polynucleotide chain. Preferably, the nuclease is an endonuclease. An endonuclease can cleave bonds in the middle of the chain.

[0288] 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 site by a guide RNA (see, for example, 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, type V CRISPR nucleases such as Cas12a and Cas12b, and other nucleases derived therefrom. RNA-guided nucleases can be broadly defined by their PAM specificity and cleavage activity.

[0289] 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 enzyme associated with the clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system. Cas9 nucleases include a well-characterized ortholog (SpCas9) from Streptococcus pyogenes. SpCas9 and other orthologs (including SaCas9, FnCa9, and AnaCas9) are reviewed by Jiang, F. and Doudna, JA, 2017 Annual review of biophysics, 46, pp. 505-529.

[0290] The term "gene editing agent" can also include guide RNA. "Guide RNA" (gRNA) confers target sequence specificity to RNA-guided nucleases. Guide RNAs are non-coding short RNA sequences that bind to complementary target DNA sequences. For example, in the CRISPR / Cas9 system, the guide RNA first binds to the Cas9 enzyme, and the gRNA sequence guides the resulting complex to a specific location on the DNA through base pairing, where Cas9 exerts its nuclease activity by cleaving the target DNA strand. The term "guide RNA" encompasses not only gRNAs compatible with specific nucleases such as Cas9, but also any suitable gRNA that can be used with any RNA-guided nuclease.

[0291] Control Elements The viral vector of the present invention may contain one or more regulatory sequences that act pre- or post-transcriptionally. Preferably, the protein-coding sequence may be operably linked to one or more regulatory sequences. The one or more regulatory sequences may promote the expression of the protein in podocytes.

[0292] A "regulatory sequence" is any sequence that facilitates expression of a polypeptide, for example, by increasing the expression of a transcript or by increasing the stability of mRNA. Suitable regulatory sequences include, for example, promoters, enhancer elements, post-transcriptional regulatory elements, polyadenylation sites, and the like.

[0293] promoter The viral vector of the present invention may contain a promoter. Preferably, the promoter may be operably linked to a protein-coding sequence. The promoter may promote expression of the protein in podocytes.

[0294] A "promoter" is a region of DNA that initiates transcription of a gene. A promoter is located upstream of the DNA (towards the 5' region of the sense strand) near the transcription start site of the gene. Any suitable promoter can be used, and its selection can be easily made by one skilled in the art. The promoter can be a constitutive promoter or a tissue-specific promoter.

[0295] Suitable constitutive promoters are known to those of skill in the art. For example, in one embodiment, the promoter is a CMV promoter. 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.

[0296] As used herein, a "podocyte-specific promoter" refers to a promoter that preferentially promotes the expression of a gene in podocyte cells. Preferably, a podocyte-specific promoter can promote high expression of a gene in podocytes compared to other cell types. Higher expression in podocytes can be measured, for example, by measuring the expression of a transgene, such as GFP, operably linked to the promoter, and the expression of the transgene in podocytes correlates with the promoter's ability to promote gene expression in podocytes. For example, a podocyte-specific promoter can be a promoter that promotes gene expression levels that are at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in podocytes compared to expression levels in other cell types. Suitable podocyte-specific promoters will be known to those skilled in the art.

[0297] Preferably, the podocyte-specific promoter is a promoter associated with or derived from a gene selectively expressed in human podocytes. Genes selectively expressed in podocytes are known to those skilled in the art, and selective gene expression in podocytes can be easily determined by methods well known to those skilled in the art, such as using microarrays. Genes 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, and COL4. A1, COL4A2, COL4A3, COL4A4, COL4A5, COLEC12, CRIM1, CST12, DEGS1, DOCK4, DOCK5, EGF, ENPEP, EPHX1, FAM81A, FAT1, FGFBP 1, FOXD1, FRYL, GABRB1, GALC, GM10554, H2-D1, H2-Q7, H2BC4, H3C15, HS3ST3A1, HTRA1, IFNGR1, IL18, ILDR2, ITGB5, ITGB8, K IRREL, 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, These include 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.

[0298] Methods for identifying promoter regions associated with genes are well known to those skilled in the art. Promoters are usually located in close proximity to or overlapping the transcription start site and contain several sequence motifs with which transcription factors (TFs) interact in a sequence-specific manner.

[0299] Preferably, the podocyte-specific promoter is an NPHS1 promoter, an NPHS2 promoter, a WT1 promoter, a FOXC2 promoter, an ABCA9 promoter, an ACPP promoter, an ACTN4 promoter, an ADM promoter, an ANGPTL2 promoter, an ANXA1 promoter, an ASB15 promoter, an ATP8B1 promoter, a B3GALT2 promoter, a BB014433 promoter, a BMP7 promoter, a C1QTNF1 promoter, a CAR13 promoter, a CD2AP promoter, a CD55 promoter, a CD59A promoter, a CD59B promoter, a CDC14A promoter, a CDH3 promoter, a CDKN1B promoter, a CDKN1C promoter, a CEP85L promoter, a CLIC3 promoter, a CLIC5 promoter, a COL4A1 promoter, a COL4A2 promoter, a COL4A3 promoter, a COL4A4 promoter, a COL4A5 promoter, a COLEC12 promoter, a CRIM1 promoter, a CST12 promoter, a DEGS1 promoter, a DOCK4 promoter, a DOCK5 promoter, a 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 promoterNR2F2 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, The promoter is selected from the group consisting of a TDRD5 promoter, a THSD7A promoter, a TIMP3 promoter, a TJP1 promoter, a TLR7 promoter, a TM4SF1 promoter, a TMEM108 promoter, a TMEM54 promoter, a TMTC1 promoter, a TOP1MT promoter, a TRAV10 promoter, a TRAV10N promoter, a TRAV5-4 promoter, a TSHB promoter, a UACA promoter, a UBA1Y promoter, a UPRT promoter, a VEGFA promoter, a VTCN1 promoter, a ZBTB20 promoter, and a 5730407I07RIK promoter, or a fragment or derivative thereof.

[0300] Preferably, the podocyte-specific promoter is selected from the group consisting of 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.

[0301] Preferably, the podocyte-specific promoter is the NPHS1 promoter, the NPHS2 promoter, the WT1 promoter, or the FOXC2 promoter, or a fragment or derivative thereof.

[0302] Preferably, the podocyte-specific promoter is the NPHS1 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.

[0303] The podocyte-specific promoter may be a minimal podocyte-specific promoter. As used herein, "minimal podocyte-specific promoter" refers to a minimal sequence that can act as a podocyte-specific promoter.

[0304] Preferably, the podocyte-specific promoter is a minimal NPHS1 or minimal NPHS2 promoter, or a fragment or derivative thereof. More preferably, the podocyte-specific promoter is a minimal NPHS1 promoter, or a fragment or derivative thereof.

[0305] Preferably, the promoter is a human promoter, such as the minimal human NPHS1 promoter.

[0306] NPHS1 promoter The viral vector of the present invention may comprise the NPHS1 promoter, or a fragment or derivative thereof. Preferably, the NPHS1 promoter, or a fragment or derivative thereof, may be operably linked to a protein-coding sequence.

[0307] The NPHS1 gene encodes nephrin, which is selectively expressed in podocytes. The NPHS1 promoter can be a minimal NPHS1 promoter. For example, the NPHS1 promoter can have a length of 1.2 kb or less.

[0308] The minimal human NPHS1 promoter has been described by Moeller et al. 2002 J Am Soc Nephrol, 13(6):1561-7 and Wong MA et al. 2000 Am J Physiol Renal Physiol, 279(6):F1027-32. This minimal NPHS1 is a 1.2 kb fragment that is thought to be podocyte-specific. The 1.2 kb promoter region lacks a TATA box but contains recognition motifs for other transcription factors, such as a PAX-2 binding element, an E-box, and a GATA consensus sequence.

[0309] Suitably, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO:20, or a variant which is at least 70% identical to SEQ ID NO:20.

[0310] TIFF2025539119000026.tif82162 Exemplary Minimal NPHS1 Promoter (SEQ ID NO: 20)

[0311] 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:20.

[0312] In some embodiments, the NPHS1 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO:21, or a variant that is at least 70% identical to SEQ ID NO:21.

[0313] TIFF2025539119000027.tif20163 Exemplary minimal NPHS1 promoter - 265 bp (SEQ ID NO: 21)

[0314] 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: 21. The NPHS1 promoter may comprise or consist of a variant of SEQ ID NO: 21 shown as SEQ ID NO: 22 or SEQ ID NO: 23.

[0315] TIFF2025539119000028.tif20163 Exemplary minimal nephrin promoter - 265 bp (SEQ ID NO: 22)

[0316] TIFF2025539119000029.tif20163 Exemplary minimal nephrin promoter variant - 265 bp (SEQ ID NO: 23)

[0317] In some embodiments, the NPHS1 promoter may comprise or consist of the nucleotide sequence set forth as SEQ ID NO: 22 or 23, or a variant that is at least 70% identical to SEQ ID NO: 22 or 23. 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.

[0318] NPHS2 promoter The viral vector of the present invention may comprise the NPHS2 promoter, or a fragment or derivative thereof. Preferably, the NPHS2 promoter, or a fragment or derivative thereof, may be operably linked to a protein-coding sequence.

[0319] The NPHS2 gene encodes podocin, which is selectively expressed in podocytes. The NPHS2 promoter can be a minimal NPHS2 promoter, for example, the NPHS1 promoter can have a length of 0.6 kb or less.

[0320] The minimal human NPHS2 promoter is described in Oleggini R, et al., 2006. Gene Expr. 13(1):59-66. This minimal NPHS2 is a 630 bp fragment and has been shown to express in podocytes in vitro.

[0321] Suitably, the NPHS2 promoter may comprise or consist of the nucleotide sequence shown as SEQ ID NO:24, or a variant which is at least 70% identical to SEQ ID NO:24.

[0322] TIFF2025539119000030.tif46162 Exemplary Minimal NPHS2 Promoter (SEQ ID NO: 24)

[0323] Suitably, variants 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:24.

[0324] Enhancer The viral vector of the present invention may contain an enhancer. Preferably, the enhancer is operably linked to the protein-coding sequence. The enhancer can promote expression of the protein in podocytes.

[0325] An "enhancer" is a region of DNA to which a protein (activator) can bind, increasing the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. Enhancers can be located up to 1 Mbp (1,000,000 bp) from the gene, upstream or downstream from the start point. Any suitable enhancer can be used, and its selection can be readily made by one of ordinary skill in the art.

[0326] The viral vector of the present invention may comprise a podocyte-specific enhancer. Preferably, the enhancer may be operably linked to the protein-coding sequence.

[0327] As used herein, a "podocyte-specific enhancer" refers to an enhancer that preferentially promotes the expression of a gene in podocyte cells. Preferably, a podocyte-specific enhancer can promote higher expression of a gene in podocytes compared to other cell types. Higher expression in podocytes can be measured, for example, by measuring the expression of a transgene, such as GFP, operably linked to the enhancer, and the expression of the transgene in podocytes correlates with 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 gene expression levels that are at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher, or at least 1000% higher in podocytes compared to expression levels in other cell types.

[0328] Suitable podocyte-specific enhancers are well known to those skilled in the art. Preferably, the podocyte-specific enhancer may be an enhancer associated with or derived from a gene selectively expressed in human podocytes. Methods for identifying enhancer regions associated with genes are well known to those skilled in the art.

[0329] Preferably, the podocyte-specific enhancer is the NPHS1 or NPHS2 enhancer, or a fragment or derivative thereof. More preferably, the podocyte-specific enhancer is the NPHS1 enhancer, or a fragment or derivative thereof.

[0330] Preferably, the enhancer is a human enhancer, such as the human NPHS1 enhancer. An enhancer can be used with a corresponding promoter, for example, the NPHS1 enhancer can be used with the NPHS1 promoter, or an enhancer can be used with a different promoter, for example, a promoter that is not podocyte-specific, such as an hsp promoter.

[0331] The viral vector of the present invention may comprise a promoter-enhancer. Preferably, the promoter-enhancer may be operably linked to a protein-coding sequence. The promoter-enhancer may promote protein expression in podocytes. The promoter-enhancer may be a podocyte-specific promoter-enhancer. The promoter-enhancer may be an NPHS1 promoter-enhancer or an NPHS2 promoter-enhancer, or a fragment or derivative thereof.

[0332] NPHS1 enhancer The NPHS1 enhancer has been described in Guo, G., et al., 2004. Journal of the American Society of Nephrology, 15(11), pp.2851-2856. A 186-bp fragment from the human NPHS1 promoter was able to induce podocyte-specific expression of a β-galactosidase transgene when placed in front of a heterologous minimal promoter in transgenic mice.

[0333] Suitably, the NPHS1 enhancer may comprise or consist of the nucleotide sequence shown as SEQ ID NO:25, or a variant which is at least 70% identical to SEQ ID NO:25.

[0334] TIFF2025539119000031.tif16162 Exemplary NPHS1 enhancer (SEQ ID NO: 25)

[0335] 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.

[0336] Kozak sequence The viral vector of the present invention may contain a Kozak sequence. Preferably, the Kozak sequence may be operably linked to a protein-coding sequence. The Kozak sequence may be inserted before the start codon of a protein to improve translation initiation. Suitable Kozak sequences are well known to those skilled in the art.

[0337] Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence shown as SEQ ID NO:26, or a variant which is at least 65% identical to SEQ ID NO:26.

[0338] TIFF2025539119000032.tif7140 Exemplary Kozak sequence (SEQ ID NO: 26)

[0339] Suitably, the variant may be at least 75%, at least 85%, or at least 90% identical to SEQ ID NO:26.

[0340] Post-transcriptional regulatory elements The viral vector of the present invention may comprise a post-transcriptional regulatory element. Preferably, the post-transcriptional regulatory element is operably linked to a protein-coding sequence. The post-transcriptional regulatory element may improve gene expression.

[0341] The viral vector may include a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). Preferably, the WPRE may be operably linked to a protein coding sequence.

[0342] The WPRE sequence may have mutations within the X antigen promoter and / or the start codon of the X antigen, which may prevent the production of functional X antigen.

[0343] Suitably, the WPRE may comprise or consist of the nucleotide sequence shown as SEQ ID NO:27, or a variant which is at least 70% identical to SEQ ID NO:27.

[0344] TIFF2025539119000033.tif42162 Exemplary WPRE (SEQ ID NO: 27)

[0345] Suitably, variants 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.

[0346] Polyadenylation signal The viral vector of the present invention may contain a polyadenylation signal. Preferably, the polyadenylation signal is operably linked to a protein-coding sequence. The polyadenylation signal can improve gene expression.

[0347] 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.

[0348] Suitably, the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO:28, or a variant which is at least 70% identical to SEQ ID NO:28.

[0349] TIFF2025539119000034.tif20162 Exemplary bGH poly(A) signal sequence (SEQ ID NO: 28)

[0350] 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.

[0351] Suitably, the polyadenylation signal may comprise or consist of the nucleotide sequence shown as SEQ ID NO:29, or a variant which is at least 70% identical to SEQ ID NO:29.

[0352] TIFF2025539119000035.tif8146 Exemplary soluble neuropilin-1 polyadenylation signal (SEQ ID NO: 29)

[0353] Suitably, variants 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.

[0354] Treatment method In one aspect, the invention provides a method of treatment, the method comprising delivering a therapeutically effective amount of a viral vector according to the methods of the invention.

[0355] In one aspect, the present invention provides a method of treating and / or preventing kidney disease in a subject in need thereof, the method comprising delivering a therapeutically effective amount of a viral vector according to the methods of the present invention.

[0356] In one embodiment, the invention provides a viral vector for use in therapy, wherein the viral vector is delivered by a method according to the invention.

[0357] In one aspect, the present invention provides a viral vector for use in treating and / or preventing kidney disease in a subject, said viral vector being delivered by a method according to the present invention.

[0358] In one embodiment, the present invention provides the use of a viral vector for the manufacture of a medicament, said medicament being delivered by a method according to the present invention.

[0359] In one aspect, the present invention provides the use of a viral vector for the manufacture of a medicament for treating and / or preventing kidney disease in a subject, said medicament being delivered by a method according to the present invention.

[0360] Subject can be any subject described herein.For example, kidney disease can be glomerular disease.For example, kidney disease can be podocyte-related glomerular disease.For example, kidney disease can be GBM-related glomerular disease.

[0361] The kidney disease may be a genetic kidney disease (see, for example, Hildebrandt, F., 2010. The Lancet, 375(9722), pp.1287-1295). The viral vector may correspond to the kidney disease intended to be treated and / or prevented. For example, if the kidney disease is Alport syndrome, the viral vector may encode COL4A3, COL4A4, or COL4A5. For example, if the kidney disease is nephrotic syndrome, the viral vector may encode an NS-related transgene. For example, if the kidney disease is complement-related, the viral vector may encode a complement protein.

[0362] In some embodiments, the kidney disease is a genetic glomerular disease, including podocyte-associated genetic glomerular diseases such as nephrotic syndrome and GBM-associated glomerular diseases such as Alport syndrome.

[0363] In some embodiments, the kidney disease is a podocyte-associated hereditary glomerular disease.Podocyte-associated hereditary glomerular disease includes Finnish congenital nephrotic syndrome, type 2 congenital nephrotic syndrome, type 3 familial nephrotic syndrome, Fraser syndrome, as well as Dennis-Drash syndrome, Schimke immuno-osseous dysplasia, nephrotic syndrome caused by CD2AP mutation, nephrotic syndrome caused by actinin-4 mutation, nephrotic syndrome caused by TRPC6 mutation, Epstein syndrome, and Fechtner syndrome.Preferably, the glomerular disease is nephrotic syndrome.

[0364] In some embodiments, the kidney disease is a GBM-related hereditary glomerular disease.GBM-related hereditary glomerular disease includes X-linked Alport syndrome, autosomal recessive Alport syndrome, autosomal dominant Alport syndrome, thin basement membrane disease, Pearson syndrome, and Nail-Patera syndrome.Preferably, the glomerular disease is Alport syndrome (AS).AS is also known as familial nephritis, hereditary nephritis, thin basement membrane disease, and thin basement membrane nephropathy.

[0365] 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 rejection of kidney transplants, membranous nephropathy, membranoproliferative glomerulonephritis I, or membranoproliferative glomerulonephritis III.

[0366] In some embodiments, the kidney disease is diabetic nephropathy. In some embodiments, the kidney disease is Fabry disease.

[0367] Variants, derivatives, analogs, homologs and fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses variants, derivatives, homologues and fragments thereof. In the context of the present invention, a "variant" of any sequence is a sequence in which a specific sequence of residues (whether amino acid or nucleic acid residues) has been altered 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 mutation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0368] The term "derivative" as used herein in relation to a protein or polypeptide of the invention includes substitution, mutation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0369] Typically, amino acid substitutions may be made, for example, from 1, 2 or 3 to 10 or 20 substitutions, provided that the altered sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0370] The proteins used in the present invention may have deletions, insertions, or substitutions of amino acid residues that produce silent mutations and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity 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, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0371] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: TIFF2025539119000036.tif49144

[0372] The term "homologue" as used herein means a variant that has a certain homology to the wild-type amino acid sequence or the wild-type nucleotide sequence. The term "homology" can be equivalent to "identity".

[0373] As used herein, a homologous sequence is intended to include an amino acid sequence that may be at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 96%, 97%, 98%, or 99% identical. Typically, a homolog contains the same active site, etc., as the subject amino acid sequence. Homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), although in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0374] As used herein, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96%, 97%, 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0375] Preferably, reference to a sequence having a certain percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having the stated percent identity over the entire length of the referenced SEQ ID NO.

[0376] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0377] Percent homology can be calculated for consecutive sequences, i.e., one sequence is aligned with the other and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0378] While this is a very simple and consistent method, it does not take into account that, for example, a single insertion or deletion in an amino acid or nucleotide sequence can cause the following residue or codon in an otherwise identical pair of sequences to fall out of alignment, potentially resulting in a significant decrease in percent homology when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0379] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that sequence alignments with as few gaps as possible achieve a higher score than those with more gaps, reflecting a higher relatedness between the two compared sequences for the same number of identical amino acids or nucleotides. The "affine gap cost" method, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap, is commonly used. This is the most commonly used gap scoring system. Higher gap penalties, of course, produce optimized alignments with fewer gaps. Most alignment programs allow you to change the gap penalty; however, it is preferable to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0380] Therefore, to calculate the maximum homology percentage, it is necessary to first create an optimized alignment that takes into account gap penalties.A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387).Other examples of software that can perform sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid.-Chapter 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 series of comparison tools. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid., pp. 7-58 to 7-60). However, for some applications, the GCG Bestfit program may be preferred. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0381] Although the final percent homology can be measured in terms of identity, the alignment process itself is not usually based on absolute pairwise comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the publicly available default values ​​or a custom symbol comparison table if one is provided (see the user manual for details). Depending on the application, it may be preferable to use the public default values ​​of the GCG package, or a default matrix such as BLOSUM62 for other software.

[0382] Once the software has produced an optimized alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. Percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues of the referenced SEQ ID NO.

[0383] "Fragment" is also a variant, and the term typically refers to a selected region of a polypeptide or polynucleotide that is functional or of interest, for example, in an assay. Thus, a "fragment" refers to an amino acid sequence or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0384] Such variants, derivatives, homologs, and fragments can be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where an insertion is to be made, synthetic DNA encoding the insertion site can be generated, along with 5' and 3' flanking regions corresponding to the native sequence on either side of the insertion site. The flanking regions contain restriction sites corresponding to those in the naturally occurring sequence, allowing the sequence to be cleaved with appropriate enzymes and the synthetic DNA to be ligated to the cleavage site. The DNA can then be expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences; other known techniques may also be used.

[0385] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology which are within the capabilities of those skilled in the art. Such techniques are explained in the literature. For example, Skoog, DA, et al. (2013) Fundamentals of Analytical Chemistry, 9th edition, Cengage learning; Walker JM (2009) The Protein Protocols Handbook, 3rd edition, Springer Nature; Green, MR and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM, et al. (2003) Current Protocols in Molecular Biology, John Wiley & Sons; Hill, AJ (2013) DNA Sequencing Protocols, Humana Press; Nielsen, BS 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 See, e.g., J.M., 1999, pp. 111-114, 1999. Each of these general texts is incorporated herein by reference. [Example]

[0386] The present invention will now be further described by way of examples, which are intended to aid those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention.

[0387] Example 1 - Direct renal artery injection of an AAV vector encoding podocin under the control of the hNPHS1 promoter This study was designed to explore the kidney transduction efficiency with rAAV products in healthy pigs when administered via direct renal artery injection (dRAi) with renal artery occlusion, while simultaneously examining the biodistribution of injected rAAV in major organs and tissues.

[0388] Materials and Methods A schematic diagram of the AAV vector expressing human HA-tagged podocin under the control of the human full-length nephrin (hNPHS1) promoter is shown in Figure 1. This cassette was packaged into the AAV-LK03 serotype for testing.

[0389] rAAV was generated by transfecting HEK293T cells with three plasmids. Virus was cultured for 72 hours after transfection. Viral particles were purified using density gradient separation to eliminate / remove the majority of empty capsids from the final preparation. Virus was sterile filtered through a 0.2 micron filter. rAAV preparations were stored at -80°C until use. The vehicle was phosphate-buffered saline (PBS).

[0390] The doses of rAAV formulations administered to four male pigs are summarized in Table 1. The viral vectors were administered according to body weight (target body weight at the time of administration was 10 kg). [Table 1]

[0391] Prior to day 0, all animals received prophylactic antibiotics twice daily in the feed, and two animals were also premedicated with 1 mg / kg prednisolone. On the day of administration, test articles were thawed and added to a maximum of 5 mL / kg in PBS at room temperature. Standard surgical and anesthesia procedures, including Hartmann intravenous fluid therapy, were followed.

[0392] The surgical procedure for dRAi with balloon occlusion involved percutaneous access to the left renal artery and inflating the balloon to occlude the renal artery prior to infusion of the rAAV test formulation. After confirming arterial occlusion, the test procedure was performed distal to the balloon for approximately 15–20 minutes without the use of an infusion pump.

[0393] The steps involved in the final surgical procedure for dRAi-mediated rAAV test preparation are summarized below: 1. Carotid artery catheterization 2. Insertion of the sheath into the carotid artery 3. Guidewire insertion into the renal artery 4. Insertion of the occlusion balloon catheter into the renal artery 5. Renal artery occlusion by balloon dilation (15–20 min) 6. Confirmation of occlusion by contrast injection and imaging (angiography was performed through the tail vein, with contrast added to demonstrate flow and recirculation) 7. Contrast Flushing 8. Direct Infusion of rAAV Test Formulations into the Renal Artery 9. Flush with saline to ensure that no test agent remains in the catheter. 10. Deflation of the balloon catheter 11. Slowly remove the balloon catheter to avoid damaging the blood vessel. 12. The sheath is then withdrawn and closed.

[0394] The animals were then recovered and returned to individual pens for 24 hours. After the carotid artery catheter was removed, they were returned to group pens. Buprenorphine (20 mg / kg) was administered intravenously to animals 3–6 as an analgesic after anesthesia. During the study, all animals received prophylactic antibiotics twice daily during feeding. Two animals received prednisolone (10 mg) once daily until the end of the study, while the remaining two did not. Euthanasia was performed approximately 4 weeks later under anesthesia with sodium pentobarbital (10–15 mL).

[0395] Animals were observed daily for general health / mortality and morbidity. Clinical observations and body weights were recorded upon arrival at the study facility, on day -1, day 0, and weeks 1-4. Pain assessment scores were recorded on days 1-5 after treatment.

[0396] Kidneys (cortex and medulla) and other tissue samples (liver, spleen, pancreas) were collected and evaluated for the following: immunohistochemistry and histology; protein Western blot; RNA; DNA; and immunofluorescence.

[0397] result This study demonstrated that rAAV AAVLK03-hPodocin could be successfully delivered to renal glomeruli, more specifically to podocytes, via dRAi+occlusion.

[0398] Transcriptome analysis qPCR analysis was performed to examine bGH expression in two control animals and four animals treated with dRAi via the left renal artery with AAV expressing HA-tagged podocin to assess transduction efficiency in the kidney (by comparing the injected left kidney with the untreated right kidney) and biodistribution (pancreas, liver, and spleen). Each bGH mRNA expression in an organ was normalized to the bGH expression in the same organ in control animals.

[0399] Figure 2 shows transcriptome analysis of dRAi-treated animals, revealing increased levels of bGH mRNA in the left renal cortex (LKC) compared with the right renal cortex (RKC). While there were no significant changes in expression levels detected in other organs, bGH mRNA levels were increased approximately 40-45-fold in the pancreas in two of the dRAi-treated animals. When LKC was normalized to the left renal medulla (LKM) of the same animals, all four dRAi-treated pigs showed relatively high levels of bGH expression compared with the wild-type (two controls).

[0400] Data from a second round of transcriptome analysis showed increased mRNA bGH expression in LKCs of injected animals compared with RKCs or LKM / RKMs or other organs. Transcriptome analysis of the kidney showed the expected trend of LKC>LKM>RKC>RKM.

[0401] Immunofluorescence experiments To visualize the local penetration and persistence of the virus at the target site, we performed immunofluorescence experiments in glomeruli to detect the amount of HA-tagged podocin protein in the glomeruli of two control animals and four rAAV-treated animals at the time of sacrifice.

[0402] Figure 3 demonstrates successful delivery of rAAV dRAi, as HA-tagged podocin is detected in LKCs and specifically by the target cells, podocytes, as evidenced by the colocalization of nephrin (a podocyte-specific protein) with HA-tagged podocin in the middle panel (LKCs from animal 6) compared with RKCs from untreated (no dRAi) control animal animal 2 or animal 6. While other animals were also subjected to immunofluorescence analysis, LKCs from animal 2 (control) and rAAV-treated animal 6 (LKCs and RKCs) are simply highlighted as representative images.

[0403] Immunofluorescence experiments further confirmed that HA-tagged podocin was localized to podocytes in the glomeruli of injected animals compared with uninjected and control pigs.

[0404] Real-life observations / measurements No deaths occurred during the study period. No AAVLK03-hPodocin-related effects were noted by clinical observation or monitoring in any of the treated animals during the study period. No AAVLK03-hPodocin-related effects on body weight were noted in any of the treated animals during the study period.

[0405] Example 2 - Direct renal artery injection of an AAV vector encoding eGFP under the control of a CMV promoter This experiment was designed to compare the renal transduction efficiency of rAAV test formulations administered via dRAi (with renal artery occlusion) versus intravenous systemic administration in healthy Göttingen pigs, as well as to examine the biodistribution of injected rAAV in major organs and tissues.

[0406] Materials and Methods A schematic diagram of the AAV vector expressing eGFP under the control of the CMV promoter is shown in Figure 4. eGFP is widely used as a reporter molecule for gene expression because it can be detected after blue light excitation. In this study, eGFP protein expression was driven by the human CMV major promoter, which is known for its ability to produce large amounts of recombinant protein in mammalian cells. This cassette was encapsidated into the AAV-LK03 serotype for testing.

[0407] rAAV was produced by transfecting HEK293T cells with three plasmids. Virus was incubated for 72 hours post-transfection. Virus particles were purified using density gradient separation, which meant that the majority of empty capsids were excluded / removed from the final preparation. Virus was sterile-filtered using a 0.2 micron filter. rAAV preparations were stored at -80°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, with a maximum volume of 15 mL.

[0408] The doses of rAAV test formulation administered to the three groups are summarized in Table 2. Each group consisted of three animals, with one animal serving as a control animal not receiving the rAAV test formulation. The viral vector was administered according to body weight, and the same fixed volume (15 mL) was used for all experimental animals. In the dRAi+O group, flushing was also performed under occlusion, so the total ischemic time includes the flush step. [Table 2]

[0409] A "high dose" is 5.7 x 10 per pig. 12 vg, and the "low dose" is 5.7 × 10 per pig. 11 It was calculated as vg. Because of expected variability in renal artery anatomy and animal size, the total amount of AAV required will vary. Therefore, for this study, we selected Göttingen minipigs, derived from a closed colony and known to have low levels of neutralizing antibodies to AAV, for consistency. All animals were 4.5 months old, single-sex (female), and fed a weight-control diet, restricting their body weight to 10-12 kg before study treatment.

[0410] Prior to day 0, animals were prophylactically pretreated (for 3 or 5 days) with amoxicillin (antibiotic). On day 0 and before surgery, animals underwent preanesthesia. On the day of procedure, test articles were thawed and brought to a maximum volume of 15 mL in PBS at room temperature. Standard surgical and anesthesia procedures (including Hartmann fluid therapy) were followed.

[0411] The surgical procedure for balloon occlusion dRAi involved percutaneous access to the renal artery of the first kidney and inflating an occlusion balloon catheter to occlude the renal artery 15–20 minutes before infusion of the rAAV test formulation. After confirming artery occlusion, the test procedure was administered distal to the balloon via an infusion pump for approximately 15–20 minutes.

[0412] The steps of the surgical procedure for rAAV test preparation via dRAi+O are summarized below: 1. Catheterization of the common femoral artery (CFA) according to standard procedures 2. Inserting the sheath into the CFA 3. Guidewire insertion into the renal artery 4. Insertion of an occlusion balloon catheter into the renal artery 5. Renal artery occlusion by balloon dilation (15-20 minutes) 6. Confirm complete occlusion by contrast injection and imaging (angiography is performed via the tail vein, with contrast added to demonstrate flow and recirculation) 7. Plasmalyte and Heparin Flushing 8. Inject / infuse the rAAV test formulation directly into the renal artery under no-flow conditions using an infusion pump. 9. Saline / PBS flushing to ensure no rAAV test formulation remains in the catheter 10. Deflation of the balloon catheter 11. Withdraw the balloon catheter slowly to avoid damaging the blood vessel. 12. Subsequent sheath withdrawal and closure Due to surgical complications (see Table 2), two animals in the low rAAV dose dRAi group (Pigs No. 2 and No. 5) were injected into the right kidney instead of the left kidney.

[0413] For intravenous administration of rAAV test formulations, the total dose was injected via the jugular vein over 15 minutes. Control animals did not receive rAAV test formulations but were anesthetized to allow for the insertion of additional lines for blood sampling.

[0414] Post-anesthetic analgesia buprenorphine (20 mg / kg) was administered intravenously to all animals. Pig 2 also received postoperative analgesia (day 0) with paracetamol (10 mg / kg intravenously), a fentanyl patch (50 mg for 3 days), and oral paracetamol 10 mg / kg on day 1. Euthanasia was performed approximately 4 weeks after the procedure under surgical anesthesia with sodium pentobarbital (10–15 mL).

[0415] Animals were observed for general health / mortality and morbidity. Clinical findings and body weights were recorded upon arrival at the study facility, on day -1, day 0, and weeks 1-4. Pain assessment scores were recorded on postoperative days 1-5. Blood samples were collected for hematology and biochemistry analysis at three time points: pre-intervention (0 hours), day 1 (24 hours), and termination.

[0416] 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 future analysis.

[0417] result This study demonstrated rAAV-mediated gene transfer by direct renal artery infusion (dRAi) with occlusion, followed by immunofluorescence detection of GFP expression localized to the glomeruli of the injected kidney.

[0418] No safety concerns related to the surgical procedure of dRAi with occlusion were observed in this study, and histological evaluation of all animal samples showed no observable damage, fibrosis, necrosis, or sclerosis in all animals treated using this route of administration.

[0419] Immunofluorescence analysis showed consistent and clear GFP expression in the injected left kidney cortex (LKC) of animals receiving high rAAV doses of dRAi. Furthermore, moderate GFP expression was observed in the cortex of animals receiving low-dose dRAi (pig 2 and pig 5, injected in the right kidney; pig 8, injected in the left kidney) on the injected side. In contrast, no GFP expression was observed in the LKC or right kidney cortex (RKC) after intravenous administration, except for pig 6 in the high rAAV dose intravenous administration group.

[0420] The conclusions drawn from the immunofluorescence images were further supported by examination of the data obtained from the qPCR results. These results showed elevated expression in LKCs from the rAAV high-dose dRAi-treated group, and faint expression in RKCs from the rAAV low-dose dRAi-treated group. This analysis also showed the absence of GFP mRNA expression in the pancreas and colon.

[0421] Importantly, GFP expression was not observed in the liver at either the protein or mRNA levels in either the high- or low-rAAV-dose dRAi group, whereas expression was observed at both the protein and mRNA levels in the liver of the high-rAAV-dose IV animals.

[0422] Transcriptome analysis qPCR analysis was performed to analyze mRNA GFP expression in one control animal and six animals injected via the renal artery (dRAi - high and low doses) with rAAV (serotype LK03) expressing GFP driven by a CMV promoter. The transduction efficiency in the animals' kidneys (by comparing the rAAV-injected kidney with the contralateral uninjected kidney) and biodistribution (liver, colon, and pancreas) were assessed. As shown in Figure 5, a mean relative change in GFP expression in the injected kidney cortex was approximately 80-fold with the high rAAV dose (dRAi vs. IV) and approximately 20-fold with the low rAAV dose (dRAi vs. IV).

[0423] qPCR analysis was also performed to analyze mRNA GFP expression in three animals administered the rAAV test formulation intravenously and compare the expression profiles with those in the dRAi group. GFP mRNA expression in each organ was normalized to GFP expression in the same organ in control animals (Figure 6). Therefore, the results presented compare the injected kidney with the contralateral uninjected kidney. For completeness, GFP-mediated transduction efficiency data from the left and right kidneys are shown.

[0424] Immunofluorescence experiments After organ sectioning, 5–6 images were acquired per sample. The adrenal cortex and medulla were identified visually throughout the bisected kidney, and sections were randomly selected (one section per kidney). The sections shown here are a representative sample of all sections examined.

[0425] In animals injected via the left renal artery, the majority of GFP protein expression was observed in the glomeruli. In some images, GFP colocalized with the podocyte-specific marker nephrin in a focal linear distribution (focal linear staining suggesting colocalization) (e.g., pigs 1 and 8) (Figure 7). In contrast, in the high-dose rAAV IV injection group, two animals showed no kidney GFP protein expression, although one (pig 6) showed some glomerular expression (much less than in the other groups). Pigs 2 and 5 were injected via the right kidney due to surgical complications. As expected, GFP expression was not observed in the left cortex or medulla due to the right kidney injection.

[0426] To confirm widespread GFP expression in the left renal cortex of animals injected with rAAV via the left renal artery, we performed immunofluorescence analysis on the contralateral kidney, more specifically, the right renal cortex and medulla. Little expression was observed in the right cortex of animals injected via the left renal artery (Figure 8), suggesting that the majority of GFP expression was localized to the injected left renal cortex. In two animals injected with low doses of rAAV test formulation via the right artery, GFP signal was observed in podocytes in the injected kidney, along with other cells within the glomeruli. In contrast, in the intravenous injection group, two animals showed no GFP protein expression in either kidney, and one animal showed some expression in the glomeruli, but it was much less than in the low-dose group.

[0427] To further confirm the localization in the kidney, we further enlarged the image of the rAAV high-dose dRAi-treated group and showed the main area of ​​GFP expression in the glomerulus (Figure 9). The GFP signal was localized deep within the glomerulus, where podocytes likely reside.

[0428] Immunofluorescence analysis was performed on pig livers after intravenous (high dose) and renal artery injection (dRAi - high and low doses) of rAAV test formulations expressing GFP protein under the control of a CMV promoter. All liver sections of pigs imaged after high- and low-dose dRAi administration were GFP-negative (Figure 10). In contrast, GFP was observed in liver sections of pigs injected intravenously with rAAV. This result indicates that GFP expression was only observed in the livers of IV animals, compared with animals injected with rAAV via dRAi (both high and low doses).

[0429] Real-life observations / measurements No deaths occurred during the study period. No AAV-LK03-eGFP-related effects on clinical observations or monitoring were observed in any treatment group during the study period. No AAV-LK03-eGFP-related effects on body weight were observed in any treatment group during the study period.

[0430] clinical pathology All clinical pathological results obtained were within the normal range expected for the same species of Göttingen minipig. All controls were within range, and the analysis was valid and correct.

[0431] Immunohistochemistry and histology Masson's trichrome-stained sections from control animals and all three rAAV-treated groups (high-dose IV, high-dose dRAi, and low-dose dRAi) showed normal amounts and distribution of collagen fibers in the renal corpuscles and between the renal tubules, with no abnormal accumulation of collagen or fibrin.

[0432] PAS-stained sections from control and rAAV-treated animals show normal glomeruli without obvious GBM thickening or mesangial cell deposition. Bowman's space and proximal tubules are distinct. All other conditions showed normal histology in both glomeruli and tubules. None of the rAAV-treated animals developed focal segmental glomerulosclerosis, and none of the glomeruli were affected by sclerosis.

[0433] Example 3 - Direct renal artery injection of AAV-LK03 vector encoding GFP under the control of the hNPHS1 promoter This study was designed to explore the localization of gene expression within the glomeruli of the renal cortex using rAAV vectors administered by direct renal artery infusion with (dRAi+O) or without renal artery occlusion (dRAi) compared with intravenous controls in healthy pigs.

[0434] Materials and Methods A schematic diagram of the AAV vector expressing GFP under the control of the human full-length nephrin (hNPHS1) promoter (referred to herein as PS0528) is shown in Figure 11A. This cassette was encapsidated into the AAV-LK03 serotype for testing.

[0435] rAAV was produced by transfection of the three plasmids into HEK293T cells. Virus was cultured for 72 hours after transfection. Viral particles were purified using density gradient separation. Virus was sterile filtered through a 0.2 micron filter. rAAV vectors were stored at -80°C until use.

[0436] Female Göttingen minipigs were acclimated for 3 weeks before the start of the study and were 4.5-6.5 months old and weighed 10-15 kg at the time of treatment. Water was available ad libitum and food was provided twice daily.

[0437] Female Gottingen pigs were dosed as shown in Table 3 below, and the experimental design is shown diagrammatically in Figure 11B. [Table 3]

[0438] A single kidney per pig was administered. This kidney was the left kidney, but if the renal artery anatomy was difficult based on angiographic evaluation, the right kidney was administered. The dRAi (renal artery delivery) and dRAi+O (renal artery delivery with occlusion) interventional procedures were performed after induction of general anesthesia as detailed below. The vector was diluted with saline (0.9% sodium chloride) prior to administration to the volumes detailed in Table 3.

[0439] Local delivery (dRAi / dRAi+O): -CFA catheterization using standard techniques -Introduction of short sheath to CFA - Guidewire insertion into the renal artery - Introduction of an occlusion balloon catheter (in the case of dRAi+O) or a diagnostic catheter (in the case of dRAi) into the renal artery -dRAi+O only: Renal artery occlusion by balloon dilation (aiming for total occlusion time of less than 20 minutes) -dRAi+O only: Confirm complete occlusion by contrast injection and imaging -Administration of heparinized saline Direct infusion of the rAAV test formulation into the renal artery using an infusion pump configured to deliver 1 mL / min for 15 minutes (dRAi+O) or 2 mL / min for 4 minutes (dRAi) -Administration of heparinized saline -dRAi+O only: Deflate balloon catheter -Removal of balloon catheter (dRAi+O) or diagnostic catheter (dRAi) -Removal of short sheath -Apply pressure to the access site until hemostasis is achieved.

[0440] Animals were then recovered and observed daily until the end of the study. Pigs were sacrificed 28-30 days after treatment. At necropsy, sections of kidneys (cortex and medulla) were removed and formalin-fixed, frozen, or embedded in OCT blocks for the following evaluations: histology, immunohistochemistry, immunofluorescence, RNAscope, ELISA, qPCR, and RTqPCR.

[0441] result Biodistribution Frozen kidney cortex from treated and untreated kidney samples was thawed, and DNA was extracted and analyzed by qPCR using primers and probes targeted to WPRE. Figures 12A-B show the AAV genomes detected per milligram (mg) of tissue. In treated kidneys (Figure 12A), 1 x 10 13 Comparing the dose levels of topical and intravenous administration, the number of detected AAV genomes was 7-fold higher with dRAi and 23-fold higher with dRAi+O. The number of genomes detected in the untreated kidney increased with dose (Fig. 12B). This data indicates that local administration increases vector retention within the administered kidney.

[0442] RNAscope in-situ hybridization (ISH) Paraffin-embedded, formalin-fixed tissues were evaluated by RNAscope in situ hybridization to examine the localization of AAV mRNA in renal cortical samples using a set of probes specific for WPRE. Stained sections were scanned, and the percentage of glomeruli with detectable WPRE mRNA was visually analyzed. The number of mRNA-positive areas was quantified and plotted as a frequency distribution.

[0443] Figures 13A-D show the number of mRNA-positive areas present in the glomeruli of treated kidneys of pigs administered either via dRAi (direct renal artery injection without occlusion) or intravenous injection. Between 70 and 120 glomeruli were quantified per pig. 5x10 12 vg and 1x10 13 In both vg dRAi delivery, 1x10 delivered by i.v. 13 Administration of α-glucan resulted in a significant increase in the number of mRNA regions per glomerulus compared with administration of α-glucan.

[0444] The data demonstrate that local delivery of AAV to the kidney significantly increases glomerular transduction and gene expression.

[0445] Figures 14A-C show the number of mRNA-positive areas present in glomeruli in treated kidneys of pigs administered either via dRAi+O (direct renal artery injection with occlusion) or intravenous injection. Between 70 and 120 glomeruli were quantified per pig. 1 x 10 13 vg and 2x10 13 In both vg dRAi+O delivery, 1x10 delivered by i.v. 13 vg administration resulted in a significant increase in the number of mRNA regions per glomerulus.

[0446] Taken together, Figures 13 and 14 demonstrate that local delivery to the kidney significantly improves glomerular transduction and gene expression compared to systemic administration, regardless of the presence or absence of blood flow impairment, and this result is consistent across multiple different delivery doses, infusion rates, and AAV vector concentrations.

[0447] Example 4 - Direct renal artery injection of AAV-LK03 vector encoding podocin under the control of the hNPHS1 promoter This study was designed to explore the localization of gene expression within the glomeruli of the renal cortex and the selective transduction of podocytes within the glomerulus using rAAV vectors in healthy pigs when administered by direct renal artery injection with renal artery occlusion (dRAi+O).

[0448] Materials and Methods A schematic diagram of the AAV vector expressing human HA-tagged podocin under the control of the human full-length nephrin (hNPHS1) promoter (referred to herein as PS0438) is shown in Figure 15A. This cassette was encapsidated into the AAV-LK03 serotype for testing.

[0449] rAAV was produced by transfection of the three plasmids into HEK293T cells. Virus was cultured for 72 hours post-transfection. Viral particles were purified using density gradient separation. Virus was sterile filtered through a 0.2 micron filter. rAAV vectors were stored at -80°C until use.

[0450] Female Göttingen minipigs were acclimated for 2 weeks before the start of the study, weighed 11 to 15 kg, and were 23 to 25 weeks old at the time of treatment. Water was available ad libitum and food was provided twice daily.

[0451] Female Gottingen pigs were dosed as shown in Table 4 below, and the experimental design is shown diagrammatically in Figure 15B. [Table 4]

[0452] In this experiment, a single kidney was administered per pig. This kidney was the left kidney unless angiographic assessment indicated a problem with the structure of the renal artery; in such cases, the right kidney was administered. The dRAi+O interventional procedure was performed after general anesthesia as follows: The vector was diluted with saline (0.9% sodium chloride) to the volume detailed in Table 4 above before administration.

[0453] Local administration (dRAi+O): - Catheterization of the common femoral artery (CFA) according to standard procedures - Introduction of short sheath to CFA - Guidewire introduction into the renal artery - Introduction of an occlusion balloon catheter into the renal artery - Renal artery occlusion by balloon dilation (aiming for total occlusion time of less than 20 minutes) - Confirm complete occlusion by contrast injection and diagnostic imaging - Heparinized saline flush - Direct infusion of the rAAV test formulation into the renal artery using an infusion pump set to deliver 1 mL / min for 15 minutes - Heparinized saline flush - Deflation of the balloon catheter - Removal of the balloon catheter - Removal of short sheath - Apply pressure to the access site until hemostasis is achieved

[0454] The animals were then recovered and observed daily until the end of the study. Pigs were sacrificed 27 days after dosing. At necropsy, sections of kidney (cortex and medulla), liver, and spleen were removed and formalin-fixed, frozen, or embedded in OCT blocks for the following evaluations: histology, immunohistochemistry, immunofluorescence, RNAscope, ELISA, qPCR, and RTqPCR.

[0455] result Biodistribution Frozen renal cortex, spleen, and liver samples from treated and untreated kidneys were thawed, and DNA was extracted and analyzed by qPCR using primers and probes targeted to WPRE. Figures 16A-D show the AAV genomes detected per milligram (mg) of tissue. The number of genomes detected per mg of tissue in treated kidneys from pigs treated with PS0438 was 50-150 times higher than in untreated kidneys, 75-430 times higher than in livers, and over 580 times higher than in spleens. This data indicates that local administration to the kidney increases retention of the vector in the administered kidney.

[0456] RNAscope in-situ hybridization (ISH) Paraffin-embedded, formalin-fixed tissues were evaluated by RNAscope in situ hybridization to examine the localization of AAV mRNA in renal cortical samples using a set of probes specific for WPRE. Stained sections were scanned, and the percentage of glomeruli with detectable WPRE mRNA was visually analyzed. The number of mRNA-positive areas was quantified and plotted as a frequency distribution.

[0457] Figures 17A-B show the number of mRNA-positive areas present in glomeruli in treated and untreated kidneys of pigs administered PS0438 via dRAi+O. Between 77 and 120 glomeruli were quantified per pig. 1x10 13 vg dRAi+O delivery resulted in significantly greater expression of glomerular-localized mRNA in treated kidneys than in untreated kidneys. The data demonstrate that local delivery significantly increases glomerular transduction and gene expression in treated kidneys.

[0458] Immunofluorescence Immunofluorescence (IF) analysis was performed on sections obtained from optical computed tomography (OCT) blocks of renal cortex. Sections were cut from blocks obtained from locally administered kidneys of pigs treated with PS0438. Multiple markers were used to assess the expression localization of the HA-tagged podocin therapeutic gene encoded by the AAV vector. Sections were stained with DAPI and antibodies targeting WT-1, a podocyte-specific transcription factor; HA, a protein tag present in the AAV-encoded podocin protein; and nephrin, a podocyte-specific slit membrane protein that colocalizes with podocin in podocytes.

[0459] Figure 18A shows a composite IF image of two glomeruli, side-by-side with single-channel images showing HA-tagged podocin (Figure 18B) and nephrin (Figure 18C). Figure 18D shows a composite IF image of one glomerulus, side-by-side with single-channel images showing HA-tagged podocin (Figure 18E) and nephrin (Figure 18F). Colocalization of podocin and nephrin was observed, along with podocyte-specific WT-1 nuclear staining, indicating podocyte-specific expression of the podocin therapeutic gene.

[0460] Overall, the data demonstrate that local delivery of AAV to the kidney results in podocyte transduction and production of a therapeutic gene.

[0461] Podocin ELISA Cryopreserved sections of renal cortex from kidneys of PS0438-treated pigs were thawed and homogenized, and lysates were analyzed by ELISA using a commercially available kit to quantify podocin protein. Total protein was also quantified by BCA assay.

[0462] Figure 19 shows the podocin protein detected in kidney cortex samples by ELISA in nanograms per milligram of total protein. While untreated control pigs (UTC) exhibited endogenous levels of podocin, 1.7- to 3.8-fold higher levels of podocin protein were detectable in the cortex of locally treated kidneys from pigs treated with PS0438.

[0463] Figures 18 and 19 taken together demonstrate that local delivery of podocin-expressing AAV (PS0438) to the kidney results in podocyte-specific expression of the therapeutic transgene at levels higher than those expressed endogenously.

[0464] Example 5 – Direct renal artery injection of an AAV9 vector encoding GFP under the control of a CMV promoter Three female nude mice were inoculated with 1.5 x 10 12 vg dose or 7.5x10 11 AAV9-CMV-GFP was injected by direct injection into the renal artery (dRAi) at a dose of vg, or PBS was injected as a negative control.

[0465] GFP expression in the renal cortex was measured using immunofluorescence, and the results are shown in Figure 20. Despite the higher doses administered intravenously, direct renal artery infusion (dRAi) resulted in a higher transduction rate into glomerular cells than intravenous administration (Figure 20A), confirming previous observations in pig studies. GFP colocalized with NPHS1 and PDGFb, confirming their localization in podocytes and mesangial cells, respectively (Figure 20B).

[0466] GFP expression in the liver was determined by Western blot analysis and densitometry, and the results are shown in Figures 21A-B. Quantification of band density in the Western blot (Figure 21A) shows that higher GFP expression in the liver was observed after intravenous administration of AAV vectors compared to administration of AAV vectors by direct renal artery infusion (dRAi) (Figure 21B). These data demonstrate that local administration of AAV9 vectors to the kidney results in kidney-specific expression in podocytes and mesangial cells.

[0467] Embodiment Various preferred features and embodiments of the present invention will now be described with reference to the following numbered items:

[0468] 1. A method of delivering a viral vector to the kidney of a subject, 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 a catheter. A method comprising:

[0469] 2. The method according to item 1, wherein the method is a minimally invasive procedure. 3. The method according to item 1 or 2, which does not include a step of occluding the renal vein.

[0470] 4. The method of any preceding item, wherein the method does not include inserting a catheter into the renal vein and / or does not include inserting a catheter directly into the aorta. 5. The method of any preceding item, which does not include clamping the renal artery, renal vein, or aorta.

[0471] 6. The method of any preceding item, wherein the catheter is an occlusion balloon catheter. 7. The method of any preceding item, wherein the catheter is inserted into the renal artery via a percutaneous route.

[0472] 8. The method according to item 7, wherein the percutaneous route is via the carotid artery or via the femoral artery. 9. The method according to item 7 or 8, wherein the insertion of the catheter via the percutaneous route is facilitated by using a sheath.

[0473] 10. The method of any preceding item, wherein the catheter is inserted into the renal artery over a guidewire.

[0474] 11. The method of any preceding item, wherein the renal artery is occluded for about 1 minute to about 25 minutes. 12. The method of item 11, wherein the renal artery is occluded for about 2 minutes to about 10 minutes, optionally about 5 minutes.

[0475] 13. The method according to item 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. 14. The method of any preceding item, wherein the viral vector is infused into the renal artery under no-flow conditions using an infusion pump.

[0476] 15. The method of any preceding item, wherein the viral vector is injected or infused into the renal artery over a period of about 1 minute to about 25 minutes. 16. The method of item 15, wherein the viral vector is injected or infused into the renal artery over a period of about 1 minute to about 5 minutes, optionally about 2 minutes.

[0477] 17. The method of item 15, wherein the viral vector is injected or infused into the renal artery over a period of about 15 minutes to about 20 minutes, optionally about 17 minutes.

[0478] 18. The method of any preceding item, wherein the method results in delivery of the viral vector to the renal cortex and / or renal medulla, preferably to the renal cortex.

[0479] 19. The method of any preceding item, wherein the method results in delivery of the viral vector to renal glomeruli, preferably to renal podocytes.

[0480] 20. The method of any preceding item, wherein the method results in kidney-specific delivery of the viral vector. 21. The method of any preceding item, wherein the subject is a human subject.

[0481] 22. The method of any preceding item, wherein the subject has or is at risk of developing kidney disease, and optionally, wherein the subject has or is at risk of developing glomerular disease.

[0482] 23. The method of any preceding item, wherein the subject has or is at risk of having a genetic glomerular disease, and optionally, wherein the subject has or is at risk of having a podocyte-associated genetic glomerular disease.

[0483] 24. The viral vector is approximately 1x10 6 vg / kg ~ approx. 1x10 14 vg / kg, or approximately 1x10 6 vg / kg ~ approx. 1x10 13 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.05 mg / kg. 25. The viral vector is approximately 1x10 9 vg / kg ~ approx. 1x10 12 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.05 mg / kg.

[0484] 26. The viral vector is approximately 3x10 9 vg / kg~approx.3x10 11 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.05 mg / kg. 27. The viral vector is approximately 1x10 8 vg~approx. 1x10 15 vg, or approximately 1x10 8 vg~approx.5x10 14 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.1 mg / kg.

[0485] 28. The viral vector is approximately 1x10 11 vg~approx. 1x10 14 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.1 mg / kg. 29. The viral vector is approximately 2x1011 vg~approx. 2x10 13 10. The method of any preceding item, wherein the medicament is delivered at a dose of 0.1 mg / kg.

[0486] 30. The method of any preceding item, wherein the viral vector is capable of transducing kidney cells, and optionally, the vector is capable of specifically transducing kidney cells.

[0487] 31. The method of any preceding item, wherein the viral vector is capable of transducing glomerular cells, and optionally, the vector is capable of specifically transducing glomerular cells.

[0488] 32. The method of any preceding item, wherein the viral vector is capable of transducing podocytes, and optionally, the vector is capable of specifically transducing glomerular podocytes.

[0489] 33. The method of any preceding item, wherein the viral vector is selected from an adeno-associated viral (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, a herpes simplex viral vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles viral vector, a Newcastle disease viral vector, a poxvirus vector, and a picornavirus vector.

[0490] 34. The method of any preceding item, wherein the viral vector is an adeno-associated viral (AAV) vector particle. 35. The method of item 34, wherein the viral vector is in the form of an AAV vector particle encapsidated by LK03, AAV3B, or AAV9 capsid protein.

[0491] 36. The method of item 34 or 35, wherein the viral vector is in the form of an AAV vector particle encapsidated by LK03 capsid protein. 37. The method of any preceding item, wherein the viral vector comprises a protein coding sequence.

[0492] 38. The method of item 37, wherein the protein-coding sequence encodes a polypeptide associated with a hereditary glomerular disease, optionally a polypeptide involved in a podocyte-associated hereditary glomerular disease.

[0493] 39. Protein coding sequences include 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, MAFB, NUP85, NUP93, NXF5, OSGEP, PAX2, PDSS2, 39. The method of item 37 or 38, wherein the polypeptide encodes a 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.

[0494] 40. The method of any of items 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.

[0495] 41. The method of any of items 37 to 40, wherein the protein-coding sequence does not encode a gene editing agent. 42. The method of any of items 37 to 41, wherein the protein-coding sequence does not encode a nuclease. 43. The method of any of items 37 to 42, wherein the protein coding sequence does not encode Cas9.

[0496] 44. The method of any of items 37 to 43, wherein the protein coding sequence is operably linked to a kidney-specific promoter, preferably the protein coding sequence is operably linked to a podocyte-specific promoter.

[0497] 45. The method of any of items 37 to 44, wherein the protein-coding sequence is operably linked to the NPHS1 promoter or the NPHS2 promoter. 46. ​​The method of any of items 37 to 45, wherein the protein-coding sequence is operably linked to a minimal NPHS1 promoter.

[0498] 47. The method of any of items 37 to 46, wherein the protein-coding sequence is operably linked to a constitutive promoter. 48. The method of item 47, wherein the protein coding sequence is operably linked to a CMV promoter.

[0499] 49. The method of any of items 37 to 48, wherein the protein-coding sequence is operably linked to one or more further regulatory elements, such as post-transcriptional regulatory elements and / or polyadenylation sequences.

[0500] 50. The method of any of items 37 to 49, wherein the protein coding sequence is operably linked to a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0501] 51. The method of any of items 37 to 50, wherein the protein coding sequence is operably linked to a polyadenylation signal, such as the bovine growth hormone polyadenylation signal (bGH).

[0502] 52. The method of any preceding item, wherein the viral vector is injected or infused into the renal artery in the form of a viral vector formulation. 53. The viral vector preparation is approximately 1x10 7 vg / ml ~ approx. 1x10 14 vg / ml, or approximately 1x10 7 vg / ml ~ approx. 5x10 13 53. The method of claim 52, comprising administering the viral vector in an amount of 0.15 mg / ml.

[0503] 54. The viral vector preparation is approximately 1x10 10 vg / ml ~ approx. 1x10 13 54. The method of item 52 or 53, comprising administering the viral vector in an amount of 0.15 mg / ml. 55. The viral vector preparation is approximately 1x10 10 vg / ml ~ approx. 1x10 12 55. The method of any of items 52 to 54, comprising the viral vector in an amount of 0.15 vg / ml.

[0504] 56. The method of any one of items 52 to 55, wherein the viral vector formulation comprises an isotonic buffer such as phosphate-buffered saline (PBS) buffer or Plasmalyte. 57. The method of any of items 52 to 56, wherein the viral vector formulation comprises about 0.001% poloxamer 188.

[0505] 58. The method according to any one of items 52 to 57, wherein the viral vector formulation 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.

[0506] 59. The method according to any of the preceding items, 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, and preferably, the total renal ischemia time is about 10 minutes to about 30 minutes, or about 15 minutes to about 25 minutes.

[0507] 60. The method of any of the preceding items, wherein the method of any of items 1 to 60 is performed once to deliver the viral vector to a single kidney of the subject, or the method of any of items 1 to 60 is performed twice to deliver the viral vector to both kidneys of the subject.

[0508] 61. A viral vector delivered by the method of any one of items 1 to 60 for use in therapy. 62. A viral vector delivered by the method described in any one of items 1 to 60, for use in treating or preventing kidney disease.

[0509] 63. Use of a viral vector for the manufacture of a medicament, wherein the medicament is delivered by the method described in any one of items 1 to 60. 64. Use of a viral vector for the manufacture of a medicament for treating or preventing kidney disease, wherein the medicament is delivered by the method described in any of items 1 to 60.

Claims

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

2. 10. The method of claim 1, which does not include the step of occluding a renal vein of the kidney.

3. 3. The method of claim 1, wherein the method does not include inserting a catheter directly into the aorta.

4. The method according to any one of claims 1 to 3, which does not include a step of forming a closed circuit through the kidney.

5. The method of any one of claims 1 to 4, which does not include clamping the renal artery of the kidney, the renal vein of the kidney, or the aorta.

6. The method of any one of claims 1 to 5, wherein the catheter is an occlusion balloon catheter.

7. 7. The method of any one of claims 1 to 6, wherein the renal artery is occluded for about 1 minute to about 25 minutes.

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

9. 8. The method of 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 for delivering a viral vector to a subject's kidney, comprising inserting a catheter into a renal artery of the kidney and injecting or infusing the viral vector into the renal artery via the catheter, the method not including occluding the renal artery or renal vein of the kidney and not including clamping the aorta.

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

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

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

14. 14. The method according to any one of claims 1 to 13, wherein the catheter is inserted into the renal artery via a percutaneous route, optionally the percutaneous route being via the carotid artery or via the femoral artery, and / or the insertion of the catheter via the percutaneous route is assisted by a sheath.

15. The method of any one of claims 1 to 14, wherein the catheter is inserted into the renal artery over a guidewire.

16. The method according to any one of claims 1 to 15, wherein the viral vector is infused into the renal artery under no-flow conditions using an infusion pump.

17. 17. The method of any one of claims 1 to 16, wherein the viral vector is injected or infused into the renal artery over a period of about 1 minute to about 30 minutes, preferably (a) the viral vector is injected or infused into the renal artery over a period of about 1 minute to about 5 minutes, optionally about 2 minutes or 4 minutes, or (b) the viral vector is injected or infused into the renal artery over a period of about 5 minutes to about 30 minutes, or about 15 minutes to about 20 minutes, optionally about 17 minutes.

18. 18. The method of any one of claims 1 to 17, 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. 19. The method of any one of claims 1 to 18, wherein the method results in delivery of the viral vector to the renal glomerulus, preferably the method results in delivery of the viral vector to the renal podocyte.

20. 20. The method of any one of claims 1 to 19, wherein the method results in kidney-specific delivery of the viral vector.

21. The method of any one of claims 1 to 20, wherein the subject is a human subject.

22. 22. The method of any one of claims 1 to 21, wherein the subject has or is at risk of developing kidney disease, and optionally the subject has or is at risk of developing glomerular disease.

23. 23. The method of any one of claims 1 to 22, wherein the subject has or is at risk of suffering from a genetic glomerular disease, and optionally, the subject has or is at risk of suffering from a podocyte-associated genetic glomerular disease.

24. The viral vector is approximately 1 × 10 ８ vg ~ approx. 1×10 １５ vg, or approximately 1 x 10 ８ vg ~ approx. 5 x 10 １４ 24. The method of any one of claims 1 to 23, wherein the dose is delivered in a dose of 0.05 vg.

25. The viral vector is approximately 1 × 10 １１ vg ~ approx. 1×10 １４ 25. The method of any one of claims 1 to 24, wherein the dose is delivered in a dose of 0.05 vg.

26. The viral vector is approximately 2 × 10 １１ vg ~ approx. 2 x 10 １３ 26. The method of any one of claims 1 to 25, wherein the dose is delivered in a dose of 0.05 vg.

27. The viral vector is approximately 5 × 10 １１ vg ~ approx. 2 x 10 １３ 27. The method of any one of claims 1 to 26, wherein the dose is delivered in a dose of 0.05 vg.

28. The viral vector is approximately 1 × 10 １２ vg ~ approx. 2 x 10 １３ 28. The method of any one of claims 1 to 27, wherein the dose is delivered in a dose of 0.05 vg.

29. The viral vector is approximately 1 × 10 １３ 29. The method of any one of claims 1 to 28, wherein the dose is delivered in a dose of 0.05 vg.

30. 30. The method of any one of claims 1 to 29, wherein the viral vector is capable of transducing kidney cells, and optionally the vector is capable of specifically transducing kidney cells.

31. 31. The method of any one of claims 1 to 30, wherein the viral vector is capable of transducing glomerular cells, and optionally the vector is capable of specifically transducing glomerular cells.

32. 32. The method of any one of claims 1 to 31, wherein the viral vector is capable of transducing podocytes, and optionally the vector is capable of specifically transducing glomerular podocytes.

33. 33. The method of any one of claims 1 to 32, wherein the viral vector is selected from an adeno-associated viral (AAV) vector, a lentiviral vector, a retroviral vector, an adenoviral vector, a herpes simplex viral vector, an alphavirus vector, a flavivirus vector, a rhabdovirus vector, a measles viral vector, a Newcastle disease viral vector, a poxvirus vector, and a picornavirus vector.

34. 34. The method of any one of claims 1 to 33, wherein the viral vector is an adeno-associated viral (AAV) vector particle.

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

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

37. The method of any one of claims 1 to 36, wherein the viral vector comprises a protein coding sequence.

38. 38. The method of 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 involved in a podocyte-associated hereditary glomerular disease.

39. Protein coding sequences include 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, TP53R K, TPRKB, VDR, WDR73, WT1, ZMPSTE24, APOL1, NPHS1, TRPC6, NUP107, NUP133, NUP160, A 39. The method of claim 37 or 38, wherein the polypeptide encodes a CTN4, INF2, ANKFY1, ANLN, CRB2, ITGA3, KANK1, KANK4, MAGI2, MYO1E, OCRL, PTPRO, SMARCAL1, SYNPO, TBC1D8B, XPO5, TNS2, NLRP3, or VEGFC polypeptide.

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

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

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

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

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

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

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

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

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

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

52. 52. The method of any one of claims 1 to 51, wherein the viral vector is injected or infused into the renal artery in the form of a viral vector formulation.

53. The viral vector preparation is about 1 x 10 ７ vg / ml ~ approx. 1×10 １４ vg / ml, or approximately 1 x 10 ７ vg / ml ~ approx. 5 x 10 １３ 53. The method of claim 52, comprising administering the viral vector in an amount of 1000 mg / ml.

54. The viral vector preparation is about 1 x 10 １０ vg / ml ~ approx. 1×10 １３ 54. The method of claim 52 or 53, comprising the viral vector in an amount of 1000 mg / ml.

55. The viral vector preparation is about 1 x 10 １０ vg / ml ~ approx. 1×10 １２ 55. The method of any one of claims 52 to 54, comprising the viral vector in an amount of 1000 mg / ml.

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

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

58. 58. The method of any one of claims 52 to 57, wherein the viral vector formulation 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. 59. The method of any one of claims 1 to 58, 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 the total renal ischemia time is about 10 minutes to about 30 minutes, or about 15 minutes to about 25 minutes.

60. 60. The method of any one of claims 1-59, wherein the method of any one of claims 1-60 is performed once to deliver the viral vector to a single kidney of the subject, or the method of any one of claims 1-60 is performed twice to deliver the viral vector to both kidneys of the subject.

61. A viral vector for use in therapy delivered by the method of any one of claims 1 to 60.

62. A viral vector for use in treating or preventing kidney disease, delivered by the method of any one of claims 1 to 60.

63. 61. Use of a viral vector for the manufacture of a medicament delivered by the method of any one of claims 1 to 60.

64. 61. Use of a viral vector for the manufacture of a medicament for treating or preventing kidney disease, wherein the medicament is delivered by the method of any one of claims 1 to 60.

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