Locoregional perfusion of the kidney for local gene therapy

Abstract

A method for treating kidney disease by local-regional perfusion of one or both kidneys of a patient is disclosed. A closed circuit can be formed by a perfusion catheter placed in the renal artery of the kidney, a collection catheter placed in the renal vein of the kidney, and an extracorporeal membrane oxygenator positioned therebetween. A perfusion fluid containing, for example, a gene therapy drug, can be circulated through the closed circuit while isolating the closed circuit from the patient's systemic circulation.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATION(S) This application claims priority to U.S. Provisional Patent Application No. 63 / 412,796, filed October 3, 2022, the disclosure of which is incorporated by reference herein in its entirety.

[0002] The present invention relates to the treatment of kidney disease, and in particular to the localized delivery of therapeutic agents to the kidneys of a patient. [Background technology]

[0003] Gene therapy and cell therapy technologies for the treatment of various kidney diseases, such as chronic kidney disease, have attracted increasing attention due to their potential to be uniquely tailored and effective in addressing the underlying pathogenic mechanisms of various kidney diseases. Nevertheless, issues regarding delivery, including vector efficiency, dosage, specificity, and safety, remain. Therefore, further research is needed to achieve more targeted and uniform delivery of drugs to the kidney that are effective, well-tolerated, and minimally invasive, and suitable for the treatment of various kidney diseases. Summary of the Invention

[0004] The following presents a simplified summary of various aspects of the present disclosure to provide a basic understanding of such aspects. This summary is not an extensive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure, nor to delineate the scope of any particular embodiments of the disclosure or the scope of the claims. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0005] In one aspect, a gene therapy vector adapted for transduction of kidney cells in a human subject comprises an adeno-associated virus (AAV) vector and a polynucleotide sequence packaged in the AAV vector, wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0006] In at least one embodiment, the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:3.

[0007] In at least one embodiment, the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:5.

[0008] In at least one embodiment, the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:7.

[0009] In at least one embodiment, the polynucleotide sequence further comprises a promoter sequence operably linked to the polynucleotide sequence encoding the therapeutic protein.

[0010] In at least one embodiment, the promoter sequence is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0011] In at least one embodiment, the promoter sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0012] In at least one embodiment, the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV 13. In at least one embodiment, the serotype of the AAV vector is AAV5.

[0013] In another aspect, a gene therapy agent comprises the gene therapy vector of any of the previous embodiments and a pharmaceutically acceptable carrier.

[0014] In another aspect, a method of treating a kidney-related disease comprises administering a therapeutic dose of the gene therapy agent of the foregoing embodiments to a patient in need thereof.

[0015] In another aspect, a method for effecting gene replacement of a mutated gene comprises administering a therapeutic dose of the gene therapy agent of the foregoing embodiments to a patient in need thereof.

[0016] In another aspect, a method of treating autosomal dominant polycystic kidney disease (ADPKD) in a subject comprises administering to the subject a therapeutic dose of a drug comprising a gene therapy vector described in any of the previous embodiments and a pharmaceutically acceptable carrier.

[0017] In another aspect, a method for performing localized delivery of polynucleotide sequences to renal cells within a kidney of a mammalian subject comprises placing a perfusion catheter in a renal artery of the kidney and placing a recovery catheter in a renal vein of the kidney, wherein the perfusion catheter and the recovery catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney, and further comprising flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid comprises a gene therapy agent as described in the previous embodiment, and wherein the closed circuit substantially isolates the perfusion through the kidney from the subject's systemic circulation.

[0018] In at least one embodiment, the kidney cells comprise tubular cells.

[0019] In at least one embodiment, a dose of AAV vector is delivered via a closed circuit and is at least about 5×10 per milliliter (mL) of plasma during perfusion. 9 The concentration of vector genomes was maintained at approximately 5 x 10 per mL of plasma during perfusion, and the vector exhibiting leakage into the subject's systemic circulation was maintained at approximately 5 x 10 per mL of plasma. 7 The vector genome remains intact and perfusion is maintained for a total of about 30 to about 90 minutes.

[0020] In at least one embodiment, placing a perfusion catheter in the renal artery includes placing a perfusion catheter via the femoral artery.

[0021] In at least one embodiment, placing a collection catheter in the renal vein includes placing a perfusion catheter via percutaneous access through the femoral vein or via the jugular vein.

[0022] In at least one embodiment, placing a retrieval catheter in the renal vein includes placing a perfusion catheter via non-percutaneous cut-down access.

[0023] In at least one embodiment, causing the perfusion fluid to flow through a closed circuit includes passing the perfusion fluid through a membrane oxygenator before entering the renal artery via the perfusion catheter.

[0024] In at least one embodiment, the method further comprises adding additional perfusion fluid to the closed circuit or diluting the perfusion fluid with about 5% to about 50% v / v saline to account for a proportion of the bladder output.

[0025] In at least one embodiment, the closed circuit provides a perfusate flow rate of about 500 mL / min / 1.73 m of body surface area per kidney for a period of about 15 minutes to about 4 hours. 2 ~approximately 650 mL / min per kidney per 1.73 m of body surface area 2 Maintain it.

[0026] In at least one embodiment, the closed circuit provides a perfusate flow rate of about 150 mL / min / 1.73 m of body surface area per kidney for a period of about 15 minutes to about 4 hours. 2 ~ 700 mL / min per kidney per 1.73 m of body surface area 2 Maintain it.

[0027] In at least one embodiment, the method further comprises applying a negative pressure to the retrieval catheter, wherein the negative pressure is in the range of about -100 mmHg to 120 mmHg.

[0028] In at least one embodiment, one or more perfusion and recovery catheters are introduced percutaneously or non-percutaneously.

[0029] In at least one embodiment, less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v of the perfusion fluid circulating through the closed circuit leaks outside the closed circuit, or substantially none (0% v / v) leaks.

[0030] In at least one embodiment, one or more of the perfusion or recovery catheters is a balloon catheter.

[0031] In another aspect, a method of delivering a therapeutic composition to a subject in need thereof comprises locally delivering a therapeutic composition comprising a gene therapy agent as described in the preceding embodiments to the subject's kidney, while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs.

[0032] The above and other features of the present disclosure, its nature and various advantages will become apparent from a consideration of the following detailed description taken in connection with the accompanying drawings. [Brief explanation of the drawings]

[0033] [Figure 1]1 shows a schematic diagram of a first exemplary retrieval catheter having a single balloon configuration in accordance with at least one embodiment. [Figure 2] 1 is a photograph of a retrieval catheter manufactured in accordance with a first exemplary retrieval catheter embodiment. [Figure 3] 1 illustrates deployment of a first exemplary retrieval catheter in accordance with at least one embodiment. [Figure 4] 10 illustrates the deployment of a second exemplary retrieval catheter having a single balloon configuration in accordance with at least one embodiment. [Figure 5] 10 illustrates the deployment of a third exemplary retrieval catheter and a fourth exemplary retrieval catheter, each having a single balloon structure according to at least one embodiment. [Figure 6] 10 illustrates the deployment of a fifth exemplary retrieval catheter having a single balloon structure and a sixth exemplary retrieval catheter without a balloon structure, in accordance with at least one embodiment. [Figure 7] 10 illustrates the deployment of a seventh exemplary retrieval catheter having multiple balloon structures according to at least one embodiment. [Figure 8] 10 illustrates the deployment of an eighth exemplary retrieval catheter having a partially covered, recaptureable stent structure in accordance with at least one embodiment. [Figure 9] 10 illustrates the deployment of a ninth exemplary retrieval catheter having a deployable and retractable stent structure and a balloon structure in accordance with at least one embodiment. [Figure 10] 10 illustrates the deployment of a tenth exemplary retrieval catheter having a covered disc-shaped stent structure in accordance with at least one embodiment. [Figure 11A] FIG. 1 shows a schematic diagram of a first exemplary perfusion catheter having a single balloon configuration in accordance with at least one embodiment. [Figure 11B] FIG. 1 is a schematic diagram of a balloon structure of a first exemplary perfusion catheter in an inflated state in accordance with at least one embodiment. [Figure 11C]FIG. 1 is a schematic diagram of a balloon configuration of a first exemplary perfusion catheter in a retracted state in accordance with at least one embodiment. [Figure 12A] FIG. 10 is a schematic diagram of a second exemplary perfusion catheter having a distal plug in accordance with at least one embodiment. [Figure 12B] FIG. 10 is a schematic diagram of a plug of a second exemplary perfusion catheter in accordance with at least one embodiment. [Figure 12C] FIG. 10 is a schematic diagram of a plug of a second exemplary perfusion catheter in an expanded state in accordance with at least one embodiment. [Figure 13A] FIG. 10 is a schematic diagram of a third exemplary perfusion catheter having a distal wedge in accordance with at least one embodiment. [Figure 13B] FIG. 10 is a schematic diagram of a third exemplary perfusion catheter wedge in accordance with at least one embodiment. [Figure 13C] FIG. 10 is a further schematic diagram of a distal end of a third exemplary perfusion catheter in an expanded state in accordance with at least one embodiment. [Figure 14A] 10 illustrates deployment of a fourth exemplary perfusion catheter having a partially covered, recaptureable stent structure in accordance with at least one embodiment. [Figure 14B] 10 illustrates a fourth exemplary perfusion catheter stent structure in a retracted state in accordance with at least one embodiment. [Figure 14C] 10 illustrates a fourth exemplary perfusion catheter stent structure in a deployed state in accordance with at least one embodiment. [Figure 15A] 10 illustrates the deployment of a fifth exemplary perfusion catheter having a releasable coated braided disc according to at least one embodiment. [Figure 15B] 10 illustrates a braided disc of a fifth exemplary perfusion catheter in a deployed state in accordance with at least one embodiment. [Figure 16A] FIG. 10 is a schematic diagram of a sixth exemplary perfusion catheter having a tapered lumen shaft in accordance with at least one embodiment. [Figure 16B]10 illustrates deployment of a sixth exemplary perfusion catheter in accordance with at least one embodiment. [Figure 16C] 10 illustrates a pre-shaped lumen shaft of a sixth exemplary perfusion catheter in accordance with at least one embodiment. [Figure 17] 1 illustrates an exemplary pre-shaped lumen shaft for an exemplary catheter according to various embodiments. [Figure 18] 1 illustrates an exemplary locoregional perfusion system according to an embodiment of the present disclosure. [Figure 19] 1 is a schematic diagram of an exemplary locoregional perfusion device according to an embodiment of the present disclosure. [Figure 20] Included are radiographs showing the placement of arterial and venous catheters in the renal artery and vein, respectively, of a pig kidney before (top image) and after (bottom image) intravenous injection of contrast agent. [Figure 21] 1 is a plot showing renal transduction and biodistribution following a 60 minute renal LRP performed in accordance with an embodiment of the present disclosure. [Figure 22A] 1 shows vector genomes per mL of plasma measured at various time points during a 60 minute renal LRP treatment at a high vector genome dose. [Figure 22B] 1 shows vector genomes per mL of plasma measured at various time points during a 45 minute renal LRP treatment at low vector genome doses. [Figure 23A] 1 is a plot of C3a levels over several days following renal LRP treatment for two different animals. [Figure 23B] 1 is a plot of % transduction inhibition for various sample dilutions. [Figure 24A] 1 is a plot of flow velocity during renal LRP. [Figure 24B] 1 is a plot of pump rate during renal LRP. [Figure 25] 10 is a plot showing vector genomes per mL of plasma measured at various time points during a 60-minute renal LRP procedure, with the LRP closed circuit to the systemic circulation, for perfusates containing multiple AAV serotypes. [Figure 26] 1 is a plot showing the biodistribution of multiple AAV serotypes in kidney tissue after simultaneous 60 min perfusion. [Figure 27] 1 is a plot showing the biodistribution within the kidney compared to the rest of the liver after 60 minutes of perfusion with multiple AAV serotypes. [Figure 28] 1 is a plot showing relative quantification of transgene mRNA levels in kidney sections treated with AAV5 versus cumulative AAV in the kidney. [Figure 29] 10 is a plot showing vector genomes per mL of plasma measured at various time points during a 60 minute renal LRP procedure, with the LRP closed circuit to the systemic circulation, for delivery of AAV5. [Figure 30] 1 is a plot showing the biodistribution in treated kidneys compared to liver and untreated kidneys after 60 minutes of perfusion of AAV5 in the treated kidneys. [Figure 31] 10 is a plot showing vector genomes per mL of plasma measured at various time points during a 52 minute renal LRP procedure, LRP closed circuit to systemic circulation, for delivery of AAV5. [Figure 32] 1 is a plot showing the biodistribution in treated kidneys compared to the liver, untreated kidneys, and other regions after 52 minutes of perfusion of AAV5 within the treated kidneys. [Figure 33A] FIG. 1 is a schematic diagram of an exemplary perfusion catheter having a balloon in a deflated state, in accordance with at least one embodiment. [Figure 33B] FIG. 1 is a schematic diagram of an exemplary perfusion catheter having a balloon in a deployed state, according to at least one embodiment. [Figure 33C] 1 is a photograph of an exemplary perfusion catheter and its deployed balloon, according to at least one embodiment. [Figure 34A] FIG. 1 is a schematic diagram of an exemplary retrieval catheter with a balloon in a deflated state, in accordance with at least one embodiment. [Figure 34B] FIG. 1 is a schematic diagram of an exemplary retrieval catheter with a balloon in a deployed state, according to at least one embodiment. [Figure 34C] 1 is a photograph of an exemplary retrieval catheter and its deployed balloon, according to at least one embodiment. [Figure 35A] 1 illustrates the deployment of a single perfusion catheter placed in the renal artery of the left kidney, according to at least one embodiment. [Figure 35B] 1 illustrates the deployment of a pair of perfusion catheters positioned within the renal arteries of the left kidney, according to at least one embodiment. [Figure 36A] 1 illustrates the deployment of a single retrieval catheter placed in the renal vein of the left kidney, according to at least one embodiment. [Figure 36B] 1 illustrates the deployment of a pair of retrieval catheters positioned within the renal veins of the left kidney, according to at least one embodiment. [Figure 37] 1 is a plot showing shedding analysis (vector genomes per mL of plasma or urine) detected in the LRP circulation, systemic circulation, and urine of LRP-treated animals. [Figure 38] 1 is a plot showing total vector genomes detected in the urine of LRP-treated animals. [Figure 39] 1 is a plot showing biodistribution analysis of LRP-treated kidneys compared to untreated kidneys and livers separated by kidney section. [Figure 40] 1 is a plot showing GFP protein in LRP-treated kidneys compared to untreated kidneys and livers. [Figure 41] 1 is a plot showing shedding analysis detected in the LRP circuit, systemic circulation, and urine of LRP-treated animals. [Figure 42] 1 is a plot showing shedding analysis detected in the systemic circulation and urine of animals treated by systemic administration. [Figure 43] 1 is a plot showing total vector genomes present in the urine of LRP-treated animals. [Figure 44] 1 is a plot showing the total vector genomes present in the urine of animals treated by systemic administration. [Figure 45]1 is a plot of modeled concentration versus time for AAV5 within the LRP circuit of LRP-treated kidneys. [Figure 46] 1 is a plot of modeled concentration versus time for AAV5 in the systemic circulation of animals treated by systemic administration. [Figure 47] 1 is a plot showing an analysis of the biodistribution measured in kidney sections from LRP-treated animals compared to other organs. [Figure 48] 1 is a plot showing an analysis of biodistribution measured in kidney sections of animals treated by systemic administration compared to other organs. [Figure 49] 1 shows a first exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 50] 1 shows a second exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 51] 1 shows a third exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 52] 1 shows a fourth exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 53] 1 shows a fifth exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 54] 10 depicts a sixth exemplary construct for testing gene expression in kidney cells according to at least one embodiment. [Figure 55] 10 depicts a seventh exemplary construct for testing gene expression in kidney cells according to at least one embodiment.

[0034] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "drug" includes not only a single drug but also a mixture of two or more different drugs, reference to a "viral vector" includes not only a single viral vector but also a mixture of two or more different viral vectors, etc.

[0035] Also, as used herein, "about," when used in connection with a measurand, refers to the normal variation in the measurand that would be expected by one of ordinary skill in the art making the measurement using a level of care commensurate with the purpose of the measurement and the precision of the measuring device. In at least one embodiment, the term "about" includes the stated value plus or minus 10%, such that "about 10" includes 9 to 11.

[0036] Also, as used herein, the term "polynucleotide" has its normal and accustomed meaning in the art and includes any polymeric nucleic acid, such as a DNA molecule or an RNA molecule, as well as chemical derivatives known to those of skill in the art. Polynucleotides include not only those encoding therapeutic proteins, but also sequences that can be used to decrease the expression of a target nucleic acid sequence using techniques known in the art (e.g., antisense nucleic acids, interfering nucleic acids, or small interfering nucleic acids). Polynucleotides can also be used to initiate or increase the expression of a target nucleic acid sequence or the production of a target protein in cells of the cardiovascular system. Target nucleic acids and proteins include, but are not limited to, nucleic acids and proteins normally found in the target tissue, derivatives of such naturally occurring nucleic acids or proteins, naturally occurring nucleic acids or proteins not normally found in the target tissue, or synthetic nucleic acids or proteins. One or more polynucleotides can be used in combination and administered simultaneously and / or sequentially to increase and / or decrease one or more target nucleic acid sequences or proteins.

[0037] Also, as used herein, "perfusion," "perfused," and "perfusing" have their normal and accustomed meaning in the art and refer to administration over a period of time (usually 1 minute or longer) that is substantially longer than the art-recognized terms "injection" or "bolus administration" (usually less than 1 minute). The perfusion flow rate depends at least in part on the amount administered.

[0038] Also, as used herein, a "foreign" nucleic acid or gene is one that is not naturally present in the vector utilized for nucleic acid transfer, e.g., is not naturally found in a viral vector, although the term is not intended to exclude nucleic acids encoding proteins or polypeptides that are naturally present in the patient or host.

[0039] Also, as used herein, "renal cell" includes any cell of the kidney that is involved in maintaining the structure or providing function of the kidney. Examples of renal cells include, but are not limited to, renal tubular epithelial cells (or "renal tubular cells") and podocytes.

[0040] Also, as used herein, "isolated," "substantially isolated," "largely isolated," and variations thereof are terms that do not require complete or absolute isolation of the renal or systemic circulation; rather, they are intended to mean that the majority, preferably the majority or substantially all, of the designated circulation is isolated. Also, as used herein, "partially isolated" refers to isolation of any significant portion of the designated circulation.

[0041] Also, as used herein, "non-naturally restricted" includes any method of restricting fluid flow through a blood vessel, e.g., balloon catheters, sutures, etc., but does not include naturally occurring restrictions, e.g., plaque buildup (stenosis). Non-natural restrictions include, for example, substantial or complete isolation of the renal circulation.

[0042] Also, as used herein, "minimally invasive" is intended to include any procedure that does not require open surgical access to the kidney or blood vessels closely associated with the kidney. Such procedures include the use of endoscopic means to access the kidney, as well as catheter-based means that rely on access via the aorta and vena cava.

[0043] Also, as used herein, the term "adeno-associated virus" or "AAV" encompasses all subtypes, serotypes, and pseudotypes, as well as naturally occurring and recombinant forms. Various AAV serotypes and strains are known in the art and are publicly available from sources such as the ATCC and academic or commercial sources. Alternatively, sequences from AAV serotypes and strains that are publicly available and / or available from various databases can be synthesized using known techniques.

[0044] Also, as used herein, "serotype" refers to an AAV that is identified and distinguished from other AAVs based on the reactivity of its capsid protein with a defined antiserum. There are at least 12 known serotypes of human AAV, including AAV1 to AAV13, but additional serotypes continue to be discovered, and the use of newly discovered serotypes is contemplated.

[0045] Also, as used herein, "pseudotyped" AAV refers to an AAV that contains capsid proteins of one serotype and a viral genome containing 5' and 3' inverted terminal repeats (ITRs) of a different or heterologous serotype. Pseudotyped recombinant AAV (rAAV) would be expected to have the cell surface binding characteristics of the capsid serotype and genetic characteristics consistent with the ITR serotype. Pseudotyped rAAV can contain AAV capsid proteins, including VP1, VP2, and VP3 capsid proteins, and ITRs from any AAV serotype, including any primate AAV serotype from AAV1 to AAV13, so long as the capsid proteins are of a heterologous serotype to the serotype(s) of the ITRs. In pseudotyped rAAV, the 5' and 3' ITRs can be the same or heterologous. Pseudotyped rAAV is generated using standard techniques described in the art.

[0046] As used herein, "chimeric" rAAV vectors also encompass AAV vectors containing heterologous capsid proteins. That is, rAAV vectors can be chimeric with respect to their capsid proteins VP1, VP2, and VP3, such that VP1, VP2, and VP3 are not all from AAVs of the same serotype. As used herein, chimeric AAVs encompass AAVs in which the capsid proteins VP1, VP2, and VP3 vary in serotype, are a mixture of capsid proteins from other parvoviruses, or contain other viral proteins, such as proteins that target the delivery of AAV to desired cells or tissues. As used herein, chimeric rAAVs also encompass rAAVs containing chimeric 5' and 3' ITRs. As used herein, chimeric rAAVs can also include capsids generated from non-AAV sequences, such as those obtained through peptide display screening.

[0047] Also, as used herein, "pharmaceutically acceptable excipient or carrier" or "pharmaceutically acceptable carrier" refers to an inactive ingredient in a composition that is combined with an active drug in a formulation. Pharmaceutically acceptable excipients / carriers may include, but are not limited to, carbohydrates (such as glucose, sucrose, or dextran), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, high molecular weight polymers, gel-forming agents, or other stabilizers and additives. Other examples of pharmaceutically acceptable excipients / carriers include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

[0048] Also, as used herein, "patient" refers to a subject, particularly a human (but will also include non-humans), who is exhibiting clinical signs of a particular condition or symptoms indicating the need for treatment, is being treated prophylactically for a condition, or has been diagnosed with a condition to be treated.

[0049] Also, as used herein, "subject" encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0050] Also, as used herein, "treatment of" and "treating" include the administration of a drug with the intent to reduce the severity of or prevent a condition, e.g., kidney disease or renal failure.

[0051] Also, as used herein, "prevention of" and "preventing" include avoiding the onset of a condition, for example, kidney disease or renal failure.

[0052] Also, as used herein, "condition" or "conditions" refers to those medical conditions, such as kidney disease, that can be treated, alleviated, or prevented by administration of an effective amount of a drug to a subject.

[0053] Also, as used herein, "effective amount" refers to an amount of a drug sufficient to produce a beneficial or desired effect at a level readily detectable by methods commonly used to detect such effects. In some embodiments, such an effect results in at least a 10% change from basal levels when the drug is not administered. In other embodiments, the change is at least 20%, 50%, 80%, or even higher from basal levels. As explained below, the effective amount of a drug may vary from subject to subject, depending on the subject's age, general condition, the severity of the disease being treated, the particular drug being administered, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art by reference to relevant texts or literature and / or by routine experimentation.

[0054] Also, as used herein, "active drug" refers to any substance intended to produce a therapeutic, prophylactic, or other intended effect, regardless of whether it has been approved by a government agency for that purpose.

[0055] The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") presented herein is intended merely to facilitate understanding of certain materials and methods and does not impose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods. DETAILED DESCRIPTION OF THE INVENTION

[0056] Certain embodiments of the present disclosure are directed to systems and methods for minimally invasively treating kidney disease. Certain other embodiments of the present disclosure relate to organ-selective gene delivery to the kidney using a minimally invasive percutaneous delivery system. An exemplary method may include isolating a patient's renal circulation from the patient's systemic circulation and perfusing a fluid, such as a drug-containing fluid, through the patient's isolated or substantially isolated renal circulation. The perfusion may be performed on one or both kidneys and may be used to deliver one or more drugs, including, but not limited to, gene therapy vectors, exosomes, nanoparticles, antibodies, chemotherapy, genetic medicines (e.g., mRNA, siRNA, antisense RNA), etc., without exposing the systemic circulation, and therefore other organs, to the selected drug(s). The method may also be used to isolate the renal circulation to allow, for example, the administration of nephrotoxic drugs into the patient's systemic circulation, in order to protect the patient's kidneys from adverse effects. Isolation of a patient's renal circulation is described in more detail below with reference to Figures 18 and 19.

[0057] Kidney diseases or kidney disorders that can be treated by the methods disclosed herein include, but are not limited to, pyelonephritis, which is caused by an autosomal recessive mutation, particularly in the NPHP1 gene, and autosomal overt polycystic kidney disease (ADPKD), which is caused by haploinsufficiency of the PKD1 and PKD2 genes, particularly. For example, the methods can be used to treat hereditary and acquired glomerulonephritis and polycystic kidney disease.

[0058] Pyelonephritis is an autosomal recessive kidney disease that leads to end-stage renal failure. The most common form is caused by mutations in NPHP1, most commonly biallelic deletions (Hildebrandt, F. et al., Nature Genetics, vol. 17, pp. 149-153, 1997; Saunier, S. et al., Human Molecular Genetics, vol. 6, no. 13, pp. 2317-2323, 1997). The NPHP1 gene encodes nephrocystin-1 (a 2199-base DNA fragment)—a 733-amino acid protein located in the adherens junctions and focal adhesions of renal epithelial cells—which can be vectorized by AAV. It is contemplated that the replacement of nephrocystin-1 in target tissues can alleviate or correct type 1 pyelitis.

[0059] ADPKD, which affects 1 in 400 to 1 in 1,000 people, is characterized by the progressive development of multiple bilateral cysts in the kidneys, leading to the destruction of kidney tissue and ultimately kidney failure. ADPKD accounts for 4 to 10% of kidney failure cases worldwide. ADPKD also has several systemic manifestations, including liver and pancreatic cysts, cerebral aneurysms, and cardiovascular abnormalities. Current management is limited to lifestyle modifications, dialysis, and kidney transplantation. Treatment with tolvaptan can slow disease progression but is associated with significant side effects, such as liver dysfunction and aquaresis.

[0060] Alport syndrome (AS) is a glomerular disease usually associated with sensorineural hearing loss and ocular abnormalities. It has an incidence of 1 in 5,000 to 1 in 10,000 individuals, with 85% of cases associated with mutations in the COL4A5 gene. AS can present with proteinuria and hematuria, early-onset hypertension, sensorineural hearing loss, and progressive loss of kidney function accompanied by ocular abnormalities. Current management approaches are limited to dialysis and kidney transplantation. In AS, pathogenic variants in the genes COL4A3, COL4A4, and COL4A5 result in defective type IV collagen chains, thereby preventing assembly of the glomerular basement membrane (GMB). COL4A3 and COL4A4 are located on chromosome 2, whereas COL4A5 is located on the X chromosome. AS is referred to as X-linked AS when the abnormality results from a mutation in COL4A5. Defects in one of the three collagen genes are directly related to AS but are thought to result in indications other than AS.

[0061] APOL1 nephropathy is a disease associated with APOL1 risk variant expression in podocytes. Patients with two risk alleles have been found to exhibit a more rapid decline in estimated glomerular filtration rate (eGFR). The disease manifests as focal segmental glomerulosclerosis and hypertensive kidney disease. Current supportive care is suboptimal and includes aggressive hypertension control, renin-angiotensin system blockade, steroids, interferon blockade, and conventional immunosuppressants.

[0062] Embodiments of the present disclosure provide therapeutic compositions for the treatment and prevention of one or more of these diseases, as discussed in more detail below. Embodiments further provide methods for localized transduction of kidneys with such therapeutic compositions. Transduction of solid organs by systemic administration of recombinant AAV vectors has been challenging because high doses are required, leading to severe adverse events (SAEs), particularly hepatotoxicity and thrombotic microangiopathy. Certain embodiments relate to locoregional delivery and perfusion systems that enable selective perfusion of solid organs. Embodiments demonstrate the feasibility of targeted delivery of AAV vectors to one or both kidneys without associated excretion into the systemic circulation.

[0063] In an exemplary procedure, to demonstrate the efficacy of the embodiments described herein, the left renal artery and vein of an AAV-seronegative adult pig (approximately 90 kg) were percutaneously catheterized via internal jugular and femoral access. To isolate the kidney from the systemic circulation, a closed loop was established using each animal's own heparinized blood priming (perfusate), and locoregional perfusion (LRP) was initiated using an extracorporeal membrane oxygenation (ECMO) system. An AAV vector carrying a CMV-EGFP transgene cassette was infused into the closed-loop LRP system, and locoregional perfusion of the kidney was performed for up to 2 hours. Blood samples were collected longitudinally for safety assessment, and vector titration and immunological evaluation (e.g., complement activation, anti-AAV antibodies) were performed before, during, and after treatment. After completion of treatment, the vector-containing perfusate was withdrawn and the catheter was removed. The animals were evaluated for 2 weeks and then euthanized and collected for tissue processing. The presence of the vector genome was detected using quantitative PCR (qPCR), and transgene expression was assessed by qPCR, Western blotting, and immunohistochemistry. The procedure was successful in all animals, and no immediate postprocedural complications occurred. Animals recovered rapidly without any clinical signs of renal injury or dysfunction. Vector concentrations remained high and stable in the closed-loop perfusate throughout the procedure, with no associated leakage into the systemic circulation or urine. AAV particles were uniformly distributed within the treated kidney tissue. Green fluorescent protein (GFP) was uniformly expressed within the perfused kidney. No vector was detected in the untreated contralateral kidney, liver, or other organs. Anti-AAV neutralizing antibodies increased only modestly compared to baseline, and complement activation was not detected. Further testing is described in more detail below.

[0064] In some embodiments, the system is inserted, for example, via the femoral artery, and typically delivers 500-600 mL / min of renal blood flow to a 70 kg adult (i.e., 1000-1200 mL / 1.73 m). 2) and an arterial access catheter that can be sealed within the renal artery at a flow rate suitable for perfusing and oxygenating the kidney for the duration of the treatment. In some embodiments, the system includes a venous withdrawal catheter that can be inserted, for example, via the femoral vein, and sealed within the renal vein at a flow rate suitable for withdrawal of venous flow. In some embodiments, the system includes an extracorporeal membrane oxygenation system that fluidly connects venous blood flow from the kidney to the renal arterial blood flow and is capable of oxygenating the venous blood.

[0065] In some embodiments, the system includes one or more additional access lines to allow for drug administration and fluid addition. In some embodiments, a balloon catheter can be inserted into the patient's bladder to measure urinary output during the procedure. In other embodiments, individual urinary catheters are placed in each of the ureters to differentially measure the output of both kidneys. In some embodiments, the system is adapted to replace the volume of perfusion fluid lost due to bladder output. For example, in some embodiments, additional perfusion fluid (e.g., blood) and / or other physiologically acceptable solutions (e.g., plasma or saline) can be used to replace about 5% v / v to about 50% v / v of the lost perfusion fluid volume to account for bladder output.

[0066] In some embodiments, the systems and methods allow for localized regional perfusion of one kidney with a targeted drug for durations of, for example, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or any range defined therein. In some embodiments, the systems and methods allow for selective drug targeting of one or both kidneys with zero or minimal exposure of the systemic circulation and other organs to the drug. In some embodiments, gene therapy drugs, which may utilize viral vectors (e.g., adeno-associated viruses), naked or encapsulated DNA or RNA molecules, or synthetic DNA or RNA analogs (e.g., antisense), may be used to treat kidney disease. In some embodiments, chemotherapy may be used to target renal tumors. In some embodiments, other drugs or biologics / antibodies may be used. In some embodiments, combinations of the above drugs may be used.

[0067] There are several advantages to isolating a patient's renal circulation from their systemic circulation when treating kidney disease. These advantages include, but are not limited to, (1) local-regional delivery of drugs, minimal leakage of drugs into other organs, and reduced total drug dose, (2) increased targeted drug dose, (3) reduced risks and side effects, and (4) the possibility of re-administering to selected patients or to patient populations who were not appropriate candidates for a particular therapy (e.g., viral vector-based gene therapy for patients who had antibodies to the viral vector).

[0068] Other advantages will be readily apparent to those skilled in the art. Certain embodiments relate to methods for perfusing drugs in a minimally invasive manner within one or both kidneys of a patient. Certain embodiments provide methods for circulating perfusion fluid (which may contain one or more of blood or drugs) through one or both kidneys of a patient such that the perfusion fluid is isolated from the patient's systemic circulation. Certain embodiments provide locoregional delivery of drug gene therapy. Certain embodiments may be used to reduce the total dosage of drugs delivered to a patient to treat kidney disease. Certain embodiments may be used to reduce risk and / or adverse immune responses to the administration of drugs suitable for treating kidney disease. Certain embodiments allow for the readministration and / or administration of pharmacological gene therapy drugs to patients who would otherwise be inappropriate candidates for receiving pharmacological gene therapy drugs, for example, patients with neutralizing antibodies to a gene therapy vector. Certain embodiments may be used to circulate perfusion fluid through the kidneys and isolate the renal circulation from the patient's systemic circulation, allowing potentially nephrotoxic drugs to be introduced into the systemic circulation while preventing or reducing drug exposure to the kidney. Certain embodiments may be used to treat kidney diseases such as autosomal dominant polycystic kidney disease and pyelitis.

[0069] Certain embodiments may be used to provide locoregional delivery of pharmacological gene therapy to treat genetic mutations, such as mutations in the PKD1 and PKD2 genes.

[0070] Exemplary Catheter Embodiments Exemplary retrieval and perfusion catheters are described herein. The catheters can be configured for the anatomy of any target organ (e.g., kidney) in which LRP must be performed, as would be understood by one of ordinary skill in the art. Furthermore, it should be understood that any catheter described as a "retrieval catheter" can also be used as a "perfusion catheter," and vice versa. The embodiments described herein are not limited to renal LRP, but can be used to isolate the renal circulation from the systemic circulation, for example, to reduce or prevent kidney exposure to drugs or other substances introduced into the systemic circulation that may have deleterious effects on the kidney. Those skilled in the art will appreciate other uses for the catheter embodiments described herein, such as in applications in which sealing of a blood vessel is desired.

[0071] Exemplary catheter embodiments for use as retrieval catheters in LRP systems are now described. In at least one embodiment, the retrieval catheter is designed to support a fluid aspiration flow rate of about 400 mL / min or greater (e.g., about 700 mL / min or greater). For example, in at least one embodiment, an exemplary catheter can support an in vitro aspiration flow rate of about 800 mL / min at about -80 mmHg.

[0072] 1-10 illustrate various catheter embodiments suitable for fluid withdrawal in an LRP system. Any of the catheters shown in FIGS. 1-10 can be configured to support fluid flow rates (aspiration or irrigation) of at least about 400 mL / min, at least about 450 mL / min, at least about 500 mL / min, at least about 550 mL / min, at least about 600 mL / min, at least about 650 mL / min, at least about 700 mL / min, at least about 750 mL / min, at least about 800 mL / min, at least about 850 mL / min, at least about 900 mL / min, at least about 950 mL / min, or at least about 1000 mL / min. Each catheter can be compatible with a steerable introducer sheath that provides stability and guides the distal end of the catheter and allows the catheter to generate a guided pushing force. Each catheter may also have a pull wire integrated into its shaft assembly, allowing the section proximal to the occlusive structure to bend at angles of up to 120° to achieve better tracking and centering of the occlusive structure.

[0073] In at least one embodiment, one or more catheters may be multi-lumen catheters, such as double-lumen catheters. In at least one embodiment, the multi-lumen catheter allows for fluid flow (e.g., perfusion fluid) and allows for inflation of one or more balloons. In at least one embodiment, one or more catheters may be multi-balloon catheters having two or more balloons. In at least one embodiment, one or more balloons may be independently deployable or deflated.

[0074] FIG. 1 illustrates an exemplary catheter 100 having a lumen shaft 104 / 106 having a proximal end 101 and a distal end 102. The lumen shaft 104 / 106 can be formed from an outer lumen shaft 104 that at least partially surrounds the inner lumen shaft 106 and exposes a distal portion of the inner lumen shaft 106 near the distal end 102. The proximal end 101 includes an outlet structure that can be fluidly coupled to an LRP system. One or more of the outer lumen shaft 104 or the inner lumen shaft 106 can be formed from a durable polymeric material, such as a polyether block amide (PEBA) material (e.g., commercially available as PEBAX®). In at least one embodiment, the innermost diameter ("inner diameter") of the inner lumen shaft 106 is at least about 4 mm to provide a fluid flow path. In at least one embodiment, the catheter 100 can be designed to include additional lumen shafts.

[0075] The catheter 100 includes a tip section 108 at the distal end 102 and an expandable balloon structure 110 disposed along a portion 112 of the inner lumen shaft 106. In at least one embodiment, the tip section 108 includes an elongate shaft extending from the balloon structure 110 to the distal end 102. In at least one embodiment, the length of the elongate shaft of the tip section is between about 2 mm and about 35 mm, between about 5 mm and about 30 mm, between about 10 mm and about 25 mm, between about 15 mm and about 25 mm, or any subrange defined therebetween (e.g., between about 2 mm and about 5 mm). In at least one embodiment, the tip section 108 includes an opening at the distal end 102 and one or more perforations along the elongate shaft. In at least one embodiment, the tip section is formed from a pliable material that is more flexible than the material of the inner lumen shaft 106.

[0076] In at least one embodiment, inner lumen shaft 106 includes a concentric inner flow passage surrounding the liquid flow passage. The concentric inner flow passage provides a path for gas flow from balloon structure 110 to port 114, which can be used to inflate or deflate the balloon in response to pressure applied at port 114. In at least one embodiment, the outermost surface of inner lumen shaft 106 at section 112 is removed such that section 112 is sealed by balloon structure 110, isolating gas flow from the concentric inner flow passage to balloon structure 110. In at least one embodiment, the inflated diameter of the balloon structure is between about 15 mm and about 30 mm, between about 15 mm and about 20 mm, between about 20 mm and about 25 mm, between about 24 mm and about 28 mm, or between about 25 mm and about 30 mm.

[0077] FIG. 2 is an image of a catheter with a similar structure to catheter 100 with the balloon in a deployed state. The catheter dimensions include a 19 Fr (6.3 mm) cross profile, a 12 Fr (4.0 mm) innermost diameter, a usable length of 80 cm, a 25 mm balloon diameter (deployed), and a 20 mm tip length. The lumen shaft may be formed from a polymeric material such as PEBAX® 63 supported by a strong stainless steel braid. The balloon may be formed from a flexible thermoplastic / elastomeric material such as ChronoPrene™ 25A. The tip may be formed from a polymeric material such as PEBAX® 35 and may be filled with a radiopaque filler composition such as a radiomarker or BaSO4.

[0078] FIG. 3 illustrates the insertion of an exemplary catheter 300 into a blood vessel 352 through a larger vessel or chamber 350 (referred to herein as a "vessel") according to at least one embodiment. In the anatomy shown, blood flow from vessels 352 and 354 exits into vessel 350. Catheter 300 may be the same as or similar to catheter 100 and has a proximal end 301, a distal end 302, an inner lumen shaft 304, an outer lumen shaft 306, a tip portion 308, and a balloon structure 310 disposed on portion 312 of inner lumen shaft 304. Balloon structure 310 is flexible enough, upon deployment, to conform to the anatomy of vessel 352 and occlude blood flow through vessel 352 to vessel 350 without creating excessive tissue force. As shown in FIG. 3, catheter 300 is inserted beyond vessel 354 to avoid occluding flow from vessel 354 to vessel 350.

[0079] It should be noted that vessel or chamber 350, vessel 352, and vessel 354 represent the anatomical structures of the right atrium, coronary sinus, and central vein of the heart, respectively, to illustrate the various types of occlusion techniques that may utilize the exemplary catheters. However, these are referred to herein as general vessels, with the understanding that deployment of any of the catheters described herein may be adapted to the specific anatomical structure of the target organ (e.g., kidney) where LRP or occlusion is to be performed. For example, vessel 350 and vessel 352 may correspond to the inferior vena cava and renal vein of the kidney, respectively (if vessel 354 is not present).

[0080] Figures 4-10 illustrate other occlusion techniques according to various embodiments of the present disclosure. The catheters shown in Figures 4-10 may, in certain aspects, be similar to the catheters shown in Figures 1-3, for example, in terms of dimensions, materials, or construction.

[0081] 4 shows a catheter 400 according to at least one embodiment only partially inserted into a blood vessel 352 so as to abut an ostium of the blood vessel 352. The catheter 400 includes a proximal end 401, a distal end 402, an inner lumen shaft 404, an outer lumen shaft 406, a tip section 408, and a balloon structure 410 disposed on a section 412 of the inner lumen shaft 404. In at least one embodiment, the diameter of the balloon structure 410 when deployed is greater than about 15 mm, greater than about 20 mm, greater than about 25 mm, or greater than about 30 mm. The tip section 408 may include one or more perforations in addition to the openings at the distal end 402 to facilitate blood flow from the blood vessel 352 and the blood vessel 354 into the catheter 400.

[0082] In at least one embodiment, during deployment, the outer lumen shaft 406 can move distally and abut the deployed balloon structure 410, resulting in additional pressure by the balloon structure 410 against the ostium of the blood vessel 352 and further stabilizing the position of the catheter 400. In at least another embodiment, a wire structure may be utilized to apply pressure to the balloon structure 410. The wire structure may have, for example, a sinusoidal shape that is deployable into an expanded, flower-like structure extending radially from the outer lumen shaft 406 or the inner lumen shaft 404. Upon contacting the balloon structure 410, the wire structure may create a more uniform pressure profile across the surface of the balloon structure 410. Prior to deployment, the wire structure may be covered by the outer lumen shaft 406 or may be covered by an additional lumen outside of the outer lumen shaft 406.

[0083] 5 illustrates the use of a first catheter 500 and a second catheter 550 to separately occlude and drain blood vessel 352 and blood vessel 354, respectively, according to at least one embodiment. First catheter 500 includes a proximal end 501, a distal end 502, a lumen shaft 504, a tip portion 508, and a balloon structure 510 disposed on portion 512 of lumen shaft 504. Similarly, second catheter 550 includes a proximal end 551, a distal end 552, a lumen shaft 554, a tip portion 558, and a balloon structure 560 disposed on portion 562 of lumen shaft 554. In this configuration, first catheter 500 is inserted into blood vessel 352, while second catheter 550 is inserted directly into blood vessel 354, such that balloon structure 510 does not occlude blood vessel 354. The dimensions of first catheter 500 and second catheter 550 may be selected to provide safe and effective occlusion of blood vessel 352 and blood vessel 354, respectively.

[0084] Figure 6 shows a variation of Figure 5 using two catheters, only one of which has a balloon structure according to at least one embodiment. A first catheter 600 includes a proximal end 601, a distal end 602, a lumen shaft 604, a tip portion 608, and a balloon structure 610 disposed on a portion 612 of the lumen shaft 604. A second catheter 650 includes a proximal end 651, a distal end 652, a lumen shaft 654, and a tip portion 658, but does not include a balloon structure. The first catheter 600 is inserted into the blood vessel 352 such that a portion of the balloon structure 610 occludes the blood vessel 354 and is partially within the blood vessel 350 and the blood vessel 352. The second catheter 650 is inserted directly into the blood vessel 354 and positioned between the blood vessel wall and the balloon structure 610, thereby at least partially occluding the blood vessel 354.

[0085] 7 illustrates the use of a single catheter 700 including multiple balloons according to at least one embodiment. The catheter 700 includes a proximal end 701, a distal end 702, a lumen shaft 704, a tip section 708, a first balloon structure 710 disposed on a first portion 712 of the lumen shaft 704, and a second balloon structure 720 disposed on a second portion 722 of the lumen shaft 704. In at least one embodiment, the catheter 700 is designed for insertion into a blood vessel 352 such that the first balloon structure 710 occludes the blood vessel 352 and the second balloon structure 720 abuts an ostium of the blood vessel 352 to occlude (and further occlude) the blood vessel 352. An intermediate portion 724 of the lumen shaft 704 between the first balloon structure 710 and the second balloon structure 720 includes one or more perforations to allow drainage of the blood vessel 354. In at least one embodiment, the inflated diameter of the second balloon structure 720 is larger than the inflated diameter of the first balloon structure 710. In at least one embodiment, the catheter 700 is a multi-lumen catheter designed to allow each balloon to be deployed and deflated independently of each other.

[0086] 8 illustrates a catheter 800 including a partially covered, recaptureable stent structure 810 according to at least one embodiment. The catheter 800 includes a proximal end 801 and a distal end 802, an inner lumen shaft 804 coupled to the stent structure 810, and an outer lumen shaft 806. A portion of the outer lumen shaft 806 is depicted as a cutaway to show the interior of the inner lumen shaft 804. The stent structure 810 is shown in a deployed state, but can be housed within the outer lumen shaft 806 prior to deployment. The stent structure 810 is further depicted as having a proximal covered portion 810A and a distal uncovered portion 810B, which can be formed from a flexible and durable polymeric material. When inserted into blood vessel 352, coated portion 810A, as shown, occludes blood flow out of blood vessel 352, while uncoated portion 810B provides structural support within blood vessel 352 while allowing blood flow directly into catheter 800 from both blood vessel 352 and blood vessel 354. In at least one embodiment, catheter 800 can be used as a perfusion catheter connected to a supply line.

[0087] 9 illustrates a catheter 900 including a deployable and contractible stent structure 920 according to at least one embodiment. The catheter 900 further includes a proximal end 901, a distal end 902, a lumen shaft 906, a tip section 908, and a balloon structure 910 disposed in a portion 912 of the lumen shaft 906. The catheter 900 may further include an outer lumen shaft (not shown) that substantially encloses the stent structure 920 and the balloon structure 910 prior to deployment. Deployment of the stent structure 920 may be performed by moving the outer lumen shaft in a proximal direction, and contraction of the stent structure 920 may be performed by moving the outer lumen shaft in a distal direction. The stent structure 920 may be formed, for example, from stainless steel and is disposed between the balloon structure 910 and the tip section 908. In at least one embodiment, the lumen shaft 906 includes at least one perforation along a portion 922 between the balloon structure 910 and the stent structure 920 to allow drainage of the blood vessel 354 into the catheter 900. When inserted into the blood vessel 352, the balloon structure 910 abuts the ostium of the blood vessel 352.

[0088] FIG. 10 illustrates a catheter 1000 including a disk-shaped covered stent structure 1010 according to at least one embodiment. The catheter 1000 further includes a proximal end 1001, a distal end 1002, an outer lumen shaft 1006, an inner lumen shaft 1004, and a tip section 1008. The stent structure 1010 may be formed, for example, from a stainless steel stent having a durable polymer coating. The outer lumen shaft 1006 may cover the stent structure 1010 prior to deployment. Once the catheter 1000 is properly positioned, the outer lumen shaft 1006 may be moved proximally to allow deployment of the stent structure 1010. In at least one embodiment, the stent structure 1010 may be partially contained within the inner lumen shaft 1004 and coupled to a tip section 1008 that is actuatable (using a wire) to deploy the stent structure 1010 when moved proximally and to retract the stent structure 1010 when moved distally. In at least one embodiment, stent structure 1010 is large enough that, when deployed, it occludes blood vessel 352 and blood vessel 354 when it abuts the ostium of blood vessel 352. In at least one embodiment, stent structure 1010 has a diameter of about 10 mm to about 30 mm.

[0089] Exemplary catheter embodiments for use as perfusion catheters in an LRP system will now be described. In at least one embodiment, the perfusion catheter is designed to support a liquid perfusion flow rate of about 400 mL / min or greater (e.g., about 700 mL / min or greater). In embodiments utilizing multiple perfusion catheters, a combined flow rate of 700 mL / min or greater can be supported.

[0090] Figures 11-16 illustrate various catheter embodiments suitable for fluid perfusion in an LRP system. Any of the catheters shown in Figures 11-16 can be configured to support fluid flow rates (aspiration or perfusion) of at least about 400 mL / min, at least about 450 mL / min, at least about 500 mL / min, at least about 550 mL / min, at least about 600 mL / min, at least about 650 mL / min, at least about 700 mL / min, at least about 750 mL / min, at least about 800 mL / min, at least about 850 mL / min, at least about 900 mL / min, at least about 950 mL / min, or at least about 1000 mL / min. Each catheter can be designed to have a smooth profile from the proximal catheter body to a low distal profile, for example, using one or more concentric lumen shafts. Additionally, the catheters can be designed with lumen shafts that are pre-shaped according to the anatomy where the LRP procedure will be performed, which can improve overall stability during use.

[0091] In at least one embodiment, one or more catheters may be multi-lumen catheters, such as double-lumen catheters. In at least one embodiment, the multi-lumen catheter allows for fluid flow (e.g., perfusion fluid) and allows for inflation of one or more balloons. In at least one embodiment, one or more catheters may be multi-balloon catheters having two or more balloons. In at least one embodiment, one or more balloons may be independently deployable or deflated.

[0092] 11A-11C show an exemplary catheter 1100 having a lumen shaft 1104 / 1106 with a proximal end 1101 and a distal end 1102 with an opening through which perfusion fluid can flow. The lumen shaft 1104 / 1106 can be formed from an outer lumen shaft 1104 that at least partially surrounds the inner lumen shaft 1106 and exposes a distal portion of the inner lumen shaft 1106 near the distal end 1102. The proximal end 1101 includes an outlet structure that can be fluidly coupled to an LRP system. One or more of the outer lumen shaft 1104 or the inner lumen shaft 1106 can be formed from a durable polymeric material, such as a polyether block amide (PEBA) material (e.g., commercially available as PEBAX®). In at least one embodiment, the innermost diameter of the inner lumen shaft 1106 is at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, or at least about 5 mm to provide a liquid flow path.

[0093] The catheter 1100 includes an expandable balloon structure 1110 disposed along a portion 1112 corresponding to the inner lumen shaft 1106, and a distal end formed by an additional lumen. In at least one embodiment, the inner lumen shaft 1106 includes a concentric inner flow passage surrounding a liquid flow passage. The concentric inner flow passage provides a path for gas flow from the balloon structure 1110 to a port 1114, which can be used to inflate or deflate the balloon structure 1110 in response to pressure applied at the port 1114. In at least one embodiment, the outermost surface of the inner lumen shaft 1106 at the portion 1112 is removed such that the portion 1112 is sealed by the balloon structure 1110, isolating gas flow from the concentric inner flow passage to the balloon structure 1110. In at least one embodiment, the inflated diameter of the balloon structure 1110 is about 15 mm to about 30 mm, about 15 mm to about 20 mm, about 20 mm to about 25 mm, about 24 mm to about 28 mm, or about 25 mm to about 30 mm, or any subrange defined therebetween (e.g., about 20 mm and about 28 mm). Figures 11B and 11C show the balloon structure 1110 in its deployed and deflated states.

[0094] 12 and 13 show catheters including plug and wedge occlusion structures, respectively, that advantageously conform their shape to the vessel or ostium, are formed from highly compressible and atraumatic materials for safe introduction and deployment, are short in length compared to balloon structures, and do not require an additional lumen for inflation as balloon structures do.

[0095] 12A-12C show an exemplary catheter 1200 having a lumen shaft 1204 / 1206 with a proximal end 1201 and a distal end 1202 with an opening through which perfusion fluid can flow. The lumen shaft 1204 / 1206 can be formed from an outer lumen shaft 1204 that at least partially surrounds the inner lumen shaft 1206 and exposes a distal portion of the inner lumen shaft 1206 near the distal end 1202. The proximal end 1201 includes an outlet structure that can be fluidly coupled to an LRP system. One or more of the outer lumen shaft 1204 or inner lumen shaft 1206 can be formed from a durable polymeric material, such as a polyether block amide (PEBA) material (e.g., commercially available as PEBAX®). In at least one embodiment, the innermost diameter of the inner lumen shaft 1206 is at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, or at least about 5 mm to provide a liquid flow path.

[0096] The catheter 1200 further includes a plug 1210 near the distal end 1202. In at least one embodiment, the plug 1210 is formed from a flexible material such as silicone or a foam material. In at least one embodiment, the plug 1210 includes an inner portion 1210A that fits over the inner lumen shaft 1206 and a flexible outer portion 1210B that is shaped to be configurable between a contracted state ( FIG. 12A ) and an expanded state ( FIG. 12C ) in which the outer portion 1210B extends distally from the distal end 1202. The plug 1210 in FIG. 12A is shown as tapering distally. In at least one embodiment, the plug 1210 can be inverted so that it tapers proximally. In at least one embodiment, the outer lumen shaft 1204 can be configured to cover the plug 1210 prior to deployment. When utilized as a perfusion catheter, the pressure of the arterial blood flow into the hollow space between the inner portion 1210A and the outer portion 1210B of the plug 1210 may help improve the sealing of the catheter 1200 within the blood vessel in which it is deployed.

[0097] 13A-13C show an exemplary catheter 1300 having a lumen shaft 1304 / 1306 with a proximal end 1301 and a distal end 1302 with an opening through which perfusion fluid can flow. The lumen shaft 1304 / 1306 can be formed from an outer lumen shaft 1304 that at least partially surrounds the inner lumen shaft 1306 and exposes a distal portion of the inner lumen shaft 1306 near the distal end 1302. The proximal end 1301 includes an outlet structure that can be fluidly coupled to an LRP system. One or more of the outer lumen shaft 1304 or inner lumen shaft 1306 can be formed from a durable polymeric material, such as a polyether block amide (PEBA) material (e.g., commercially available as PEBAX®). In at least one embodiment, the innermost diameter of the inner lumen shaft 1306 is at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, or at least about 5 mm to provide a liquid flow path.

[0098] The catheter 1300 further includes a wedge 1310 near the distal end 1302, which can be shaped to fit the vessel or ostium. In at least one embodiment, the wedge 1310 is formed from a flexible material, such as silicone or a foam material. In at least one embodiment, the outer lumen shaft 1304 can be configured to cover the wedge 1310 prior to deployment. When deployed within a vessel, the shape of the wedge can utilize backup forces from the vessel to further enhance stability during vessel occlusion and perfusion.

[0099] 14A-14C illustrate an exemplary catheter 1400 including a partially covered, recaptureable stent structure 1406 according to at least one embodiment, similar to the catheter 800 described with respect to FIG. 8 . The catheter 1400 is shown inserted into an arterial vessel 1452 via a blood vessel or chamber 1450. The catheter 1400 includes an outer lumen shaft 1402 and an inner lumen shaft 1404 that, in at least one embodiment, is coupled to the stent structure 1406. The stent structure 1406 is further depicted as having a proximal covered portion and a distal uncovered portion, which may be formed from a flexible and durable polymeric material. FIGS. 14B and 14C illustrate the positioning and deployment, respectively, of the stent structure 1406 upon insertion into the vessel 1452. Deployment of the stent structure 1406 is performed by moving the outer lumen shaft 1402 in a proximal direction.

[0100] 15A and 15B show an exemplary catheter 1500 including a releasable covered braided disc 1510, according to at least one embodiment. The catheter 1500 includes an outer lumen shaft 1506 and an inner lumen shaft 1504. The braided disc 1510 is housed within the outer lumen shaft 1506 during placement of the catheter 1500 and can be deployed by moving the outer lumen shaft 1506 in a proximal direction. In at least one embodiment, upon deployment, the braided disc 1510 does not extend beyond the distal end 1502, allowing the distal end 1502 to extend into the blood vessel 1452 while being used to stabilize the catheter 1500 against the ostium of the blood vessel 1452 to reduce the risk of stenosis during occlusion of the blood vessel 1452.

[0101] 16A-16C illustrate an exemplary catheter 1600 having a lumen shaft 1606 with a proximal end 1601 and a distal end 1602 with an opening through which perfusion fluid can flow. The proximal end 1601 includes an outlet structure that can be fluidly coupled to an LRP system. The lumen shaft 1604 can be formed from a durable polymeric material, such as a polyether block amide (PEBA) material (e.g., commercially available as PEBAX®). In at least one embodiment, the innermost diameter of the lumen shaft 1606 is at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, or at least about 5 mm to provide a fluid flow path. In at least one embodiment, the proximal portion 1606A of the lumen shaft 1606 can have a larger diameter than the distal portion 1606B of the lumen shaft 1606 and may gradually taper over the length of the lumen shaft 1606. FIG. 16C shows the lumen shaft in a pre-formed configuration to facilitate introduction and placement of the vessel within the target organ.

[0102] An example of a pre-shaped catheter lumen is shown in Figure 17. The catheter lumen can be shaped to abut against a site in the anatomy upon deployment to utilize backup forces from the vessel wall to further enhance stability during occlusion and perfusion of the target organ.

[0103] Exemplary LRP System Embodiments 18 illustrates an exemplary LRP system 1800 according to an embodiment of the present disclosure. The LRP system 1800 is shown in a closed circuit configuration with a kidney 1810. The LRP system 1800 includes a membrane oxygenator 1820, a blood gas analysis (BGA) monitor 1830, a fluid source 1840, a flow measurement device 1842, an ECMO pump console 1846 for monitoring and controlling fluid flow, and pressure wires and console 1844 for measuring pressure within the closed circuit. In at least one embodiment, a vacuum pump 1848 may also be utilized. The LRP system 1800 may be assembled by placing a first catheter 1822 (sometimes referred to herein as a "perfusion catheter") into the renal artery of the kidney 1810 and a second catheter 1824 (sometimes referred to herein as a "withdrawal catheter," "collection catheter," or "suction catheter") into the renal vein of the kidney 1810. The first catheter 1822 and the second catheter 1824 form a closed circuit with the vasculature of the kidney 1810, the membrane oxygenator 1820, and one or more optional additional components. This closed circuit may isolate or substantially isolate the patient's renal circulation from the patient's systemic circulation.

[0104] The first catheter 1822 and the second catheter 1824 may be introduced percutaneously and in a minimally invasive manner. In some embodiments, the first catheter 1822 and / or the second catheter 1824 may be introduced via antegrade intubation. In other embodiments, the first catheter 1822 and / or the second catheter 1824 may be introduced via retrograde intubation. The first catheter 1822 may be referred to herein as a "drug delivery catheter," and the second catheter 1824 may be referred to as a "drug collection catheter" when the catheter is used for drug delivery to one kidney or multiple kidneys.

[0105] The first catheter 1822 may be a standard infusion catheter, which may optionally include a standard guidewire and infusion pump, for example, capable of delivering perfusion fluid to the kidney 1810, which may include drugs to be delivered to the kidney 1810 during local-regional perfusion. In some embodiments, the first catheter 1822 is placed into the renal artery via the femoral artery. In some embodiments, the second catheter 1824 is placed into the renal vein via the femoral vein. In some embodiments, the second catheter 1824 may be a balloon catheter such that a balloon can be inflated within the renal vein to ensure that all blood circulating through the closed circuit flows through the second catheter 1824. The balloon catheter may be a Fogarty® catheter or any other catheter suitable for the purposes described herein, as will be understood by one of ordinary skill in the art. In some embodiments, the first catheter 1822 and the second catheter 1824 may each be a balloon catheter to help reduce leakage. In some embodiments, either of the catheters may be selected from one or more of the catheters described with respect to FIGS. 1-17 .

[0106] LRP system 1800 may further include one or more additional components, such as, without limitation, one or more pumps (e.g., vacuum pump 1848), one or more suction mechanisms, one or more perfusion fluids, and combinations thereof. For example, LRP system 1800, in some embodiments, may include a pressure wire and console 1844 operably coupled to or part of membrane oxygenator 1820. Pressure wire and console 1844 and ECMO pump console 1846 may collectively be used to control perfusion rate (i.e., flow rate) and ensure safety by continuously monitoring renal artery pressure. First and second pressure sensors may be co-inserted with first catheter 1822 and second catheter 1824, for example, to measure pressure in the renal artery and renal vein, respectively. LRP system 1800 is further depicted as including a BGA monitor 1830 operably coupled to membrane oxygenator 1820 to measure gas concentrations in the perfusate, for example, prior to perfusion via first catheter 1822 and / or after collection of the perfusate (e.g., if the perfusate contains blood) by second catheter 1824. Membrane oxygenator 1820 and one or more additional components may be disposed between first catheter 1822 and second catheter 1824.

[0107] In some embodiments, the LRP system 1800 includes a third catheter 1826 for draining fluid from the bladder 1812. In some embodiments, the third catheter 1826 is a balloon catheter for blocking fluid leakage from the bladder 1812. A flow measurement device 1842 may be used to measure urine output from the bladder 1812 during the LRP procedure. In some embodiments, the fluid source 1840 may be used to replace the amount of drained fluid lost from the perfusion fluid by infusing fluid into a closed circuit via fluid line 1841. In some embodiments, the fluid is the same as the perfusion fluid or has fewer components than all of the components of the perfusion fluid (e.g., does not contain additional drugs). In some embodiments, the fluid is a physiologically acceptable solution (e.g., saline).

[0108] In some embodiments, the LRP system 1800 may be modified to simultaneously establish a closed circuit in each of the patient's kidneys. In some embodiments, two separate LRP systems may be used for each of the patient's kidneys.

[0109] In at least one embodiment, the LRP system 1800 can be modified to include multiple perfusion or retrieval catheters. For example, the first catheter 1822 can be supplemented with one or more additional perfusion catheters that are fluidly coupled to the ECMO pump console 1846 (e.g., directly or via a separate supply line), so that each catheter can be positioned within a different location in the vasculature of the kidney 1810. Similarly, the second catheter 1824 can be supplemented with one or more additional retrieval catheters that are fluidly coupled to the ECMO pump console 1846.

[0110] In some embodiments, while the closed circuit is established, one or more drugs can be perfused through the patient's systemic circulation. For example, if a drug is nephrotoxic or potentially toxic to the kidneys, but systemic delivery is desired, establishing a closed circuit to isolate renal perfusion from the systemic circulation is advantageous in preventing or reducing exposure of the drug to the kidney.

[0111] 19 is a schematic diagram of a membrane oxygenator 1820 that may be used to oxygenate the perfusate, mix the perfusate with other components (e.g., drugs), remove carbon dioxide from the perfusate, and / or push the perfusate through a first catheter 1822. The membrane oxygenator 1820 may be any commercially available ECMO device for exchanging oxygen for carbon dioxide contained in the blood.

[0112] 19 , membrane oxygenator 1820 includes various components, including a heat exchanger 1856 (through which perfusate leaves outlet 1852 before entering first catheter 1822), a delivery pump 1858, a reservoir 1860 (for adding components such as blood and / or drugs to the perfusate returning through second catheter 1824 via inlet 1854), sensors 1862 and 1864 at various stages of the closed circuit (e.g., for measuring pressure and / or blood gas content), and a membrane oxygenator 1866. In some embodiments, deoxygenated blood enters membrane oxygenator 1866 and is mixed with oxygen-rich gas. The oxygen-rich gas may be supplied by a gas blender 1868, which may mix oxygen with carbon dioxide and nitrogen gases in various ratios, and is regulated by a gas flow regulator 1870.

[0113] The perfusion fluid may include one or more of blood (or components thereof, such as plasma or serum) and / or medications suitable for treating kidney disease, and / or excipients, such as saline or dextrose solution. A delivery pump 1858 may deliver the perfusion fluid into the first catheter 1822. In some embodiments, the perfusion fluid may be contained in an IV bag or syringe and administered directly into the first catheter 1822 with or without the use of the delivery pump 1858.

[0114] The suction mechanism can be used to apply a negative suction pressure to the second catheter 1824 to minimize blood and / or drug leakage outside the closed circuit. The negative suction pressure can be about −150 mmHg, about −100 mmHg, about −50 mmHg, about −20 mmHg, about −15 mmHg, about −10 mmHg, about −5 mmHg, about 0 mmHg, or within a subrange defined by any of these points.

[0115] The blood circulating through the closed circuit may be autologous blood, matched blood from a donor, or a combination thereof. In some embodiments, blood components such as serum or plasma are selected according to one or more parameters. One parameter may be the presence or absence of a selected antibody. For example, if the drug is one or more viral vectors containing therapeutic nucleic acid sequences, the patient's autologous blood may be screened to determine whether antibodies against the one or more viral vectors are present. The presence of antibodies in the patient's autologous blood may reduce and / or completely negate the effectiveness of the treatment and / or may result in an undesirable immune response. Therefore, it may be possible to dilute or replace the patient's autologous blood with seronegative matched blood from a donor, thereby reducing the patient's immune response to the drug and increasing its effectiveness.

[0116] While the various components shown in FIG. 19 are shown as being part of membrane oxygenator 1820 or as being separate from membrane oxygenator 1820, it should be understood that this schematic diagram is merely illustrative, as one or more of the components may be included in membrane oxygenator 1820 or may be separate (external) from membrane oxygenator 1820.

[0117] LRP system 1800 may be set up and operated as follows: (1) a collection catheter (e.g., second catheter 1824) is carefully placed and tightly sealed within the renal vein to allow collection of deoxygenated venous blood, (2) a perfusion catheter (e.g., first catheter 1822) is placed in a sealing manner within the renal artery, (3) an additional collection catheter (e.g., third catheter 1826) is inserted in a sealing manner within the bladder, ureter, or both, and (4) the perfusion and collection catheters are then connected to the arterial and venous lines of membrane oxygenator 1820 using standard tubing. (5) Operation of LRP system 1800 is initiated, and the renal artery is perfused antegrade with oxygenated blood while returning deoxygenated blood is collected from the renal vein via a collection catheter using gentle negative pressure, (6) the blood is directed to reservoir 1860, then oxygenated by membrane oxygenator 1866 and antegradely reinfused into the kidney via first catheter 1822 (driven by delivery pump 1858), and (7) the flow rate drained through the bladder is then measured using flow measurement device 1842 and replaced within the perfusate by fluid source 1840. If a drug (e.g., a vector) is to be administered, it can be added to the perfusate via reservoir 1860 after priming with blood or plasma, blood samples can be taken, or drugs can be applied via reservoir 1860 during the entire perfusion process.

[0118] In some embodiments, diluting or replacing a patient's antibody-containing autologous blood with seronegative, matched blood from a donor (e.g., removing venous blood and flushing with antibody-free blood to replace the amount circulating in the system by reducing the amount of circulating antibodies specific to the viral vector being used) can reduce adverse immune responses and / or enhance drug efficacy. For example, when autologous blood is diluted or replaced with seronegative, matched blood from a donor, the adverse immune response of the patient can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or can be completely alleviated, compared to the patient's immune response without dilution or replacement of the autologous blood. When autologous blood is diluted or replaced with seronegative compatible blood from a donor, the efficacy of the administered drug may be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% compared to the efficacy of the drug in a patient without autologous blood dilution or replacement.

[0119] In some embodiments, the blood portion of the perfusate may range from about 5 mL to about 5000 mL, about 50 mL to about 2500 mL, about 100 mL to about 1000 mL, about 150 mL to about 500 mL, about 50 mL, about 75 mL, about 100 mL, about 125 mL, about 150 mL, about 175 mL, about 200 mL, about 225 mL, about 250 mL, about 275 mL, about 300 mL, about 325 mL, about 350 mL, about 375 mL, about 400 mL, about 425 mL, about 450 mL, about 475 mL, about 500 mL, about 550 mL, about 600 mL, about 650 mL, about 700 mL, about 750 mL, about 800 mL, about 850 mL, about 900 mL, about 950 mL, or about 1000 mL.

[0120] The ratio of autologous blood to compatible donor blood in the blood circulating through the closed circuit can be adjusted as needed to obtain a blood mixture that is most tolerant to drugs and least likely to elicit an immune response upon drug introduction. In some embodiments, the ratio can range from about 1:100 to about 100:1, about 1:80 to about 80:1, about 1:50 to about 50:1, about 1:30 to about 30:1, about 1:20 to about 20:1, about 1:10 to about 10:1, about 1:8 to about 8:1, about 1:5 to about 5:1, about 1:3 to about 3:1, or about 1:2 to about 2:1 (volume of autologous blood:volume of compatible donor blood).

[0121] The flow rate of perfusate through the closed circuit can be adjusted to match the patient's blood flow rate. As will be appreciated by those skilled in the art, blood flow rates vary from patient to patient and for any given patient, varying throughout the day. Thus, the flow rate of perfusate circulating through the closed circuit can be adjusted in situ. The flow rate can be measured across the closed circuit. In at least one embodiment, the flow rate can be measured with a transonic probe (such as a clamp on the tubing). In some embodiments, the flow rate of perfusate at a given time during perfusion can be within about 20%, about 15%, about 10%, about 8%, about 5%, about 3%, about 2%, about 1%, or about 0.5% of the patient's blood flow rate, based on mL / min. To avoid ischemia and / or underperfusion, it is important that the flow rate of perfusate circulating through the closed circuit does not deviate significantly from the flow rate of the patient's own blood.

[0122] Exemplary flow rates of the perfusate circulating through the closed circuit can range, without limitation, from about 75 mL / min to about 750 mL / min, from about 100 mL / min to about 650 mL / min, from about 125 mL / min to about 600 mL / min, from about 150 mL / min to about 500 mL / min, from about 175 mL / min to about 400 mL / min, from about 200 mL / min to about 300 mL / min, about 150 mL / min, about 175 mL / min, about 200 mL / min, about 225 mL / min, about 250 mL / min, about 275 mL / min, about 300 mL / min, about 325 mL / min, or about 350 mL / min. In some embodiments, the system regulates the flow rate of perfusate in the closed circuit to about 500 mL / min per 1.73 m of body surface area per kidney for a period of about 15 minutes to about 4 hours. 2 to approximately 650 mL / min per kidney per 1.73 m body surface area 2 Maintain it.

[0123] The perfusion solution may be circulated through the closed circuit for a duration ranging, without limitation, from about 5 minutes to about 5 hours, from about 15 minutes to about 4 hours, from about 30 minutes to about 3 hours, or from about 1 hour to about 2 hours. In some embodiments, the treatment duration may occur over a number of days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.

[0124] According to the systems disclosed herein, in some embodiments, a higher dose of drug may be administered directly and only to one or more kidneys than would otherwise be safely administered by systemic delivery. In some embodiments, a lower total dose of drug may be required to achieve the same therapeutic effect (as achieved with a higher dose to the systemic circulation or with only partial isolation of the renal circulation) because there may be substantially no leakage of perfusate outside the kidney or kidneys.

[0125] In some embodiments, less than about 50% v / v, less than about 40% v / v, less than about 30% v / v, less than about 20%, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v, or substantially none (0% v / v) of the perfusion fluid (e.g., blood and / or drug) circulating through the closed circuit leaks outside the closed circuit during the perfusion process.

[0126] The reduction in perfusate leakage outside the closed circuit (compared to other methods disclosed in the art) may be due to the seals formed within the closed circuit and each individual component utilized within the closed circuit.

[0127] In at least one embodiment, some perfusate leakage from the closed circuit may remain. For example, up to about 0.5% v / v, about 1% v / v, about 2% v / v, about 3% v / v, about 4% v / v, about 5% v / v, about 10% v / v, about 15% v / v, about 20% v / v, about 30% v / v, about 40% v / v, or about 50% v / v of the perfusate circulating through the closed circuit may leak outside the closed circuit. Any drug volume lost due to perfusate leakage may be replaced in the perfusate to maintain consistent drug exposure to the kidney over the calculated exposure time. In at least one embodiment, the calculated exposure time may be about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, about 30 minutes to about 3 hours, about 1 hour to about 2 hours, or any subrange therebetween.

[0128] therapeutic composition Drugs suitable for treating kidney disease (i.e., drugs contained in the perfusate) may comprise a therapeutic polynucleotide sequence. In some embodiments, the therapeutic polynucleotide sequence may encode a protein for treating a kidney condition. The protein for treating kidney disease may be of human origin or may be derived from a different species (e.g., without limitation, murine, feline, porcine, or simian). In some embodiments, the protein encoded by the therapeutic polynucleotide sequence may correspond to a gene expressed in the human kidney. Exemplary proteins and their mechanisms of action are described below.

[0129] Polycystin-1 (PC1) is the product of the PKD1 gene, the most commonly mutated gene in ADPKD. PC1 is a 4,303-amino acid glycoprotein composed of multiple domains, a large amino-terminal (N-terminal) extracellular region containing multiple protein-protein interaction motifs, 11 transmembrane (TM) domains, and an approximately 200-amino acid carboxy-terminal (C-terminal) cytoplasmic tail capable of activating multiple signaling pathways. The N-terminal extracellular region is separated from the 11-TM domain by a G protein-coupled receptor (GPCR) autoproteolysis-inducing (GAIN) domain containing a GPCR proteolytic site (GPS). Cis-autoproteolytic cleavage occurs at the GPS, approximately 20 amino acids before the first TM domain, resulting in an approximately 370-kDa N-terminal fragment (NTF) and an approximately 150-kDa C-terminal fragment (CTF). SEQ ID NO: 1 is the complete amino acid sequence of PC1. SEQ ID NO: 2 is the nucleic acid sequence encoding PC1. SEQ ID NO: 3 is the amino acid sequence of the CTF. SEQ ID NO: 4 is the nucleic acid sequence encoding the CTF.

[0130] Expression of polycystin-2 (PC2), discussed in more detail below, is required for cis-autoproteolytic cleavage to occur. Furthermore, mutations in PC1 that prevent cleavage at GPS prevent proper maturation of PC1, resulting in a protein that is unable to exit the endoplasmic reticulum. Upon cleavage at GPS, the NTF and CTF remain noncovalently associated until dissociated by ligand binding to the NTF. PC1 undergoes an additional cleavage event at the CTF, releasing the approximately 200 amino acid C-terminal cytoplasmic tail (CTT). SEQ ID NO: 5 is the amino acid sequence of CTT. SEQ ID NO: 6 is the nucleic acid sequence encoding CTT.

[0131] PC1 is expressed in epithelial cells of the renal tubules and in various other somatic tissues, including the liver, heart, bone, and endocrine glands. Within epithelial cells, PC1 is found in cilia, lateral domains of the plasma membrane, and adhesion complexes of polarized epithelial cells. PC1 can also be shed from the apical or ciliary membrane in urinary exosome vesicles.

[0132] As a large, complex protein, PC1 has multiple functions. The extracellular N-terminal domain contains multiple PKD repeat motifs, a leucine-rich repeat motif, and a C-type lectin domain, each of which plays an established role in protein-protein and protein-matrix interactions. The motifs present within the N-terminal domain, along with the intracellular localization of PC1, support a role for PC1 in cell-cell and cell-matrix interactions. PC1 may also be involved in fluid flow and pressure sensing in the kidney.

[0133] PC1 possesses structural features consistent with a member of the atypical adhesion G-protein-coupled receptor (aGPCR) family, including the presence of a GPS-containing GAIN domain, a large N-terminal extracellular domain, a potential tethering agonist stalk peptide exposed upon GPS cleavage and NTF dissociation, and a G-protein binding domain within the intracellular C-terminal region (Maser and Calvet, Cellular Signaling, 2020, Vol. 72). Cleavage of PC1 at GPS generates the PC1 CTF, which remains noncovalently bound to the NTF. In the context of the CTF that functions as an aGPCR, cleavage at GPS is not sufficient to activate GPCR signaling. Rather, binding of a ligand (potentially a Wnt ligand) to the NTF (reviewed in Padovano et al., Cellular Signaling, 2020, Vol. 72) translocates the NTF, exposing a stalk peptide, which then functions as a tethering ligand and inserts into an extracellular loop present within the CTF, thereby stimulating G protein-mediated signaling. The G protein α-subunit activated by PC1 regulates the activity of c-Jun N-terminal kinase (JNK) and AP-1 transcription factors, which control differentiation, apoptosis, and cell proliferation. Dysregulation of these functions is consistent with the hallmarks of cyst formation in ADPKD, including loss of epithelial cell polarization and uncontrolled cell proliferation. Importantly, mutations in PC1 that prevent GPS cleavage or that disrupt G-protein coupling by the CTF lead to cyst formation and progression of ADPKD (Parnell et al., Human Molecular Genetics, 2018, Vol. 27; Zhang et al., Development, 2018, Vol. 145). Thus, gene therapy agents that provide production of the PC1 CTF are expected to restore PC1-mediated GPCR signaling and thus prevent, delay, or reverse cyst formation, potentially providing an effective treatment for ADPKD caused by mutations in PKD1. This function of the PC1 CTF is independent of, or may be antagonized by, PC2.Furthermore, because the role of PC2 in PC1 function is to promote the cleavage of PC1 at GPS, providing a gene therapy agent that generates CTFs independently of PC1 autoproteolysis could also overcome the loss or reduction of PC2 function and could therefore be used to effectively treat ADPKD caused by mutations in the PKD1 or PKD2 genes.

[0134] The CTT of PC1 is an approximately 200-amino acid subdomain of the CTF, generated by cleavage of intact PC1 or the CTF via an unidentified proteolytic mechanism. CTT is involved in multiple signaling pathways and appears to contain both nuclear localization and mitochondrial targeting sequences, allowing it to localize to both the nucleus and mitochondria (reviewed in Padovano et al., Cellular Signaling, 2020, Vol. 72). CTT translocates to the nucleus along with components of the Wnt pathway, STAT6 / p100, and other STAT family members, and may regulate transcriptional pathways that control cell proliferation and apoptosis. This nuclear translocation may be part of the mechanosensing function of PC1. CTT can also translocate to mitochondria. In Drosophila, translocation of CTT to mitochondria results in reduced endurance exercise capacity and increased CO2 production, demonstrating that CTT can regulate mitochondrial function. Considering the role of dysregulation of mitochondrial function and metabolism in ADPKD, regulation of mitochondrial function by CTT may play an important role in renal epithelial homeostasis, and loss of this function may play a key role in cyst formation and growth in ADPKD. In PKD1 knockout mice, introduction of a gene encoding CTT prevents or reduces disease progression through a mechanism that may in part involve regulation of mitochondrial function (Onuchic et al., Nature Communications, 2023, Vol. 14). Similarly, in zebrafish or Xenopus oocytes lacking PC1, introduction of mRNA encoding CTT reduces cell proliferation and promotes apoptosis, thereby reversing cyst formation and other features associated with ADPKD (Zhang et al., Development, 2018, Vol. 145; Merrick et al., Developmental Cell 2012, Vol. 22).Thus, gene therapy agents that provide PC1 CTT production are expected to restore PC1-mediated function and thus prevent, delay, or reverse cyst formation, providing an effective treatment for ADPKD caused by mutations in PKD1. This function of PC1 CTT is independent of or can be antagonized by PC2. Furthermore, because the presence of PC2 can stimulate PC1 or CTF cleavage, which results in CTT release, providing gene therapy agents that generate CTT independently of PC2 may also overcome loss or reduction of PC2 function and be useful in effectively treating ADPKD caused by mutations in the PKD1 or PKD2 genes.

[0135] Polycystin-2 (PC2) is the protein product of the PKD2 gene, mutations in which also cause ADPKD. PC2, also known as TRP2, is a 968-amino acid protein that contains six transmembrane domains and intracellular N- and C-termini. PC2 is a Ca channel homologous to the transient receptor potential family of cation channels. 2+ While a portion of the cellular pool colocalizes with PC1 in cilia, the majority of the cellular pool of PC2 appears to reside in intracellular compartments, where it transports Ca from intracellular stores. 2+The channel activity of the ciliary pool of PC1 / PC2 complexes may function to mediate the role of cilia in responding to ciliary bending and transmitting mechanical or chemical stimuli necessary for kidney function. PC2 is also required for proper processing and localization of PC1. Loss or reduction of PC2 leads to cyst formation and growth. In mice, expression of PC2 in PC2 conditional knockout mice reverses ADPKD (Dong et al., Nature Genetics, 2021, Vol. 53). Gene therapy agents that provide PC2 production are expected to restore PC2 function and thus prevent, delay, or reverse cyst formation, providing an effective treatment for ADPKD caused by mutations in PKD2. Restoration of PC2 function may also enhance the Ca2+ receptor agonism of PC2. 2+ This may involve providing a regulatory function, or may involve allowing processing and proper localization of PC1. SEQ ID NO: 7 is the complete amino acid sequence of PC2. SEQ ID NO: 8 is the nucleic acid sequence encoding PC1.

[0136] Exemplary proteins for use in accordance with the embodiments described herein may include, but are not limited to, PC1 (SEQ ID NO: 1), PC2 (SEQ ID NO: 7), COL4A5, COL3A4, COL4A4, APOL1, NPHP1, MUC1, UMON, REN, HNF1B, CD2AP, MYO1E, CFH, CFI, CD46, C3, a functional fragment thereof, a functional subdomain thereof (e.g., SEQ ID NO: 3, SEQ ID NO: 5), a functional variant thereof, or a combination thereof. The one or more proteins used may also be functional variants of the proteins referred to herein and may exhibit significant amino acid sequence identity compared to the original protein. For example, the amino acid identity may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In this context, the term "functional variant" means that a variant of a protein can partially or completely exhibit the function of the corresponding naturally occurring protein. A functional variant of a protein may include a protein that differs from its natural counterpart by, for example, one or more amino acid substitutions, deletions, or additions.

[0137] Amino acid substitutions can be conservative or non-conservative. Preferably, the substitutions are conservative, i.e., substitution of an amino acid residue with an amino acid of similar polarity that acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same amino acid group as the amino acid residue being substituted. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally homologous amino acids that can be used for conservative substitutions include nonpolar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0138] Also considered variants are proteins that differ from their native counterparts by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids. These additional amino acids may be present within the amino acid sequence of the original protein (i.e., as an insertion) or may be added to one or both termini of the protein. Essentially, insertions can be made at any position, provided that the addition of amino acids does not inhibit the function of the polypeptide in the treated subject to perform the function of the native protein. Furthermore, protein variants also include proteins lacking one or more amino acids compared to the original polypeptide. Such deletions may affect any amino acid position, provided that they do not impair the protein's ability to perform its normal function.

[0139] Finally, a variant of a target protein also refers to a protein that differs from the native protein by structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified either by natural processes, such as processing or post-translational modifications, or by chemical modification processes known in the art. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosamination, glutathionylation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent attachment to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids.

[0140] Therapeutic polynucleotide sequences encoding target proteins can be administered to the subject to be treated in the form of a gene therapy vector, i.e., a nucleic acid construct that contains a coding sequence including translation and termination codons, next to other sequences required to provide expression of the exogenous nucleic acid, such as a promoter, a Kozak sequence, a polyA signal, etc.

[0141] For example, gene therapy vectors can be part of a mammalian expression system. Useful mammalian expression systems and expression constructs are commercially available. Also, several mammalian expression systems, such as plasmid or viral vector-based systems, such as LENTI-Smart™ (InvivoGen), GenScript™ Expression vector, pAdVAntage™ (Promega), ViraPower™ Lentiviral, Adenoviral Expression Systems (Invitrogen), and adeno-associated virus expression system (Cell Biolabs), are sold by different manufacturers and can be used in the present invention.

[0142] Gene therapy vectors for expressing exogenous therapeutic polynucleotide sequences of the present invention can be viral or non-viral expression vectors suitable for introducing exogenous therapeutic polynucleotide sequences into cells, for example, for subsequent expression of proteins encoded by the nucleic acid. Expression vectors can be episomal vectors, i.e., vectors capable of autonomous self-replication within host cells, or integrative vectors, i.e., vectors that are stably integrated into the genome of cells. Expression in host cells can be constitutive or regulated (e.g., inducible).

[0143] In certain embodiments, the gene therapy vector is a viral expression vector. Viral vectors for use in the present invention may contain a viral genome with a deletion of a portion of the native sequence to allow for the introduction of a heterologous polynucleotide without destroying the virus's infectivity. Due to the specific interaction between viral components and host cell receptors, viral vectors are highly suitable for efficient gene transfer into target cells. Viral vectors suitable for facilitating gene transfer into mammalian cells can be derived from different types of viruses, such as AAV, adenovirus, retrovirus, herpes simplex virus, bovine papillomavirus, lentivirus, vaccinia virus, polyomavirus, Sendai virus, orthomyxovirus, paramyxovirus, papovavirus, picornavirus, poxvirus, alphavirus, or any other viral shuttle suitable for gene therapy, their variants, and combinations thereof.

[0144] "Adenoviral expression vector" or "adenovirus" is intended to include those constructs that contain sufficient adenoviral sequences to (a) support packaging of a therapeutic polynucleotide sequence construct and / or (b) ultimately express a tissue- and / or cell-specific construct that has been cloned therein. In one embodiment of the present invention, the expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kilobase (kb) linear double-stranded DNA virus, allows for the replacement of large segments of adenoviral DNA with foreign sequences up to 7 kb.

[0145] Adenovirus growth and manipulation is known to those skilled in the art and exhibits a broad host range in vitro and in vivo. This group of viruses can be produced at high titers, e.g., 10 per mL. 9 ~10 11 Adenoviruses can be obtained in plaque-forming units (PFUs) and are highly infectious. The adenovirus life cycle does not require integration into the host cell genome. Foreign genes delivered by adenovirus vectors are episomal, resulting in low genotoxicity to host cells. No adverse reactions have been reported in vaccination studies with wild-type adenovirus, demonstrating its safety and / or therapeutic potential as an in vivo gene transfer vector.

[0146] Retroviruses (also called "retroviral vectors") may be chosen as gene delivery vectors due to their ability to integrate genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in specialized cell lines.

[0147] The retroviral genome contains three genes, gag, pol, and env, which code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genes. These contain strong promoter and enhancer sequences and are also required for integration into the host cell genome.

[0148] To construct a retroviral vector, a nucleic acid encoding a gene of interest (GOI) is inserted into the viral genome in place of a specific viral sequence, generating a replication-deficient virus. To produce virions, a packaging cell line is constructed containing the gag, pol, and / or env genes, but not the long-term repeat (LTR) and / or packaging components. When a recombinant plasmid containing a cDNA, along with retroviral long-term repeat (LTR) and packaging sequences, is introduced into this cell line (e.g., by calcium phosphate acid precipitation), the packaging sequences enable packaging of RNA transcripts of the recombinant plasmid into viral particles that are subsequently secreted into the culture medium. The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are capable of infecting a wide variety of cell types. However, integration and stable expression require host cell division.

[0149] Retroviruses can be derived from any subfamily. For example, vectors derived from murine sarcoma virus, bovine leukemia virus, Rous sarcoma virus, murine leukemia virus, mink cell focus-forming virus, reticuloendotheliosis virus, or avian leukemia virus can be used. Those skilled in the art can combine parts from different retroviruses, such as LTRs, tRNA binding sites, and packaging signals, to produce recombinant retroviruses. These retroviruses are then typically used to produce transduction-competent retroviral vector particles. For this purpose, the vector is introduced into an appropriate packaging cell line. Retroviruses can also be constructed for site-specific integration into the DNA of host cells by incorporating a chimeric integrase enzyme into the retroviral particle.

[0150] Herpes simplex virus (HSV) is of great interest for the treatment of neurological diseases because of its neurotropic nature. Furthermore, the ability of HSV to establish latent infection in non-dividing neural cells without integrating into the host cell chromosome or otherwise altering its metabolism, as well as the presence of promoters that remain active during latency, make HSV an attractive vector. While much attention has focused on the neurotropic applications of HSV, its broad host range allows for its use in other tissues.

[0151] Another factor that makes HSV an attractive vector is the size and organization of its genome. Because HSV is large, the integration of multiple genes or expression cassettes is less of a problem than with other smaller viral systems. Furthermore, the ability to use different viral regulatory sequences with varying performance (time, strength, etc.) allows for greater control of expression than with other systems. Another advantage is that the virus has relatively few spliced ​​messages, further facilitating genetic manipulation.

[0152] HSV is also relatively easy to manipulate and can be grown to high titers. Thus, delivery is less of a problem, both in terms of the amount needed to achieve a sufficient multiplicity of infection (MOI) and in terms of reducing the need for repeated administration. Nonvirulent variants of HSV have been developed and are readily available for use in the context of gene therapy.

[0153] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, as well as other genes with regulatory or structural functions. This increased complexity allows the virus to regulate its life cycle, such as during latent infection. Examples of lentiviruses include human immunodeficiency viruses (HIV-1, HIV-2) or simian immunodeficiency viruses (SIV). Lentiviral vectors have been generated by multiple attenuation of HIV toxic genes; for example, genes env, vif, vpr, vpu, and nef have been deleted, making the vector biologically safe.

[0154] Lentiviral vectors are plasmid-based or virus-based and are designed to carry the necessary sequences for the selection and introduction of foreign nucleic acids into host cells. The gag, pol, and env genes of interest are also known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cells of interest.

[0155] Vaccinia virus vectors have been widely used due to their ease of construction, relatively high levels of expression, broad host range, and large DNA carrying capacity. Vaccinia contains a linear, double-stranded DNA genome of approximately 186 kb that exhibits a pronounced "AT" preference. Approximately 10.5 kb of inverted terminal sequences flank the genome. The majority of essential genes appear to map within the central region, which is the most highly conserved region among poxviruses. Vaccinia virus has an estimated number of open reading frames ranging from 150 to 200. While both strands are coding, extensive overlapping of reading frames is uncommon.

[0156] The vaccinia virus genome can accommodate insertions of at least 25 kb. Prototypical vaccinia vectors contain transgenes inserted into the viral thymidine kinase gene via homologous recombination. Vectors are selected based on the tk phenotype. Inclusion of the nontranslated leader sequence from the encephalomyocarditis virus results in expression levels higher than those of conventional vectors, with the transgene accumulating to more than 10% of the protein in infected cells within 24 hours.

[0157] Empty capsids of papovaviruses, such as mouse polyomavirus, have attracted attention as potential vectors for gene transfer. The use of empty polyomas was first reported when polyoma DNA and purified empty capsids were incubated in a cell-free system. The DNA in the new particles was protected from the action of pancreatic DNase. The reconstituted particles were used to introduce transforming polyoma DNA fragments into rat FIII cells. The empty capsids and reconstituted particles consist of all three polyoma capsid antigens: VP1, VP2, and VP3.

[0158] AAV is a parvovirus belonging to the Dependovirus genus. They are small, non-enveloped, single-stranded DNA viruses that require a helper virus for replication. Superinfection with a helper virus (such as adenovirus, herpesvirus, or vaccinia virus) is required to form functionally intact AAV virions. In vitro, in the absence of superinfection with a helper virus, AAV establishes a latent state in which the viral genome exists in an episomal form but no infectious virions are produced. Subsequent infection with a helper virus "rescues" the genome, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. Recent data indicate that in vivo, both wild-type and recombinant AAV exist primarily as large episomal concatemers. In one embodiment, the gene therapy vector used herein is an AAV vector. The AAV vector may be a purified, replication-incompetent, pseudotyped rAAV particle.

[0159] AAV is not associated with any known human diseases, is generally not considered pathogenic, and does not appear to alter the physiological properties of host cells upon integration. AAV can infect a wide range of host cells, including non-dividing cells, and can infect cells of different species. In contrast to some vectors that are rapidly eliminated or inactivated by both cellular and humoral responses, AAV vectors have been shown to induce sustained transgene expression in various tissues in vivo. The persistence of recombinant AAV-mediated transgenes in non-dividing cells in vivo may be due to the lack of native AAV viral genes and the vector's ability to form ITR-associated episome concatemers.

[0160] AAV is an attractive vector system for use in cell transduction according to the present invention because it has a high frequency of persistence as episomal concatemers, can infect non-dividing cells, including cardiomyocytes, and is therefore useful, for example, for gene delivery to mammalian cells in tissue culture and in vivo.

[0161] Typically, rAAV is produced by cotransfecting a plasmid containing a GOI flanked by two AAV terminal repeats, such as pIM45, and / or an expression plasmid containing a wild-type AAV coding sequence without the terminal repeats. Cells are also infected and / or transfected with a plasmid carrying adenovirus and / or adenovirus genes required for AAV helper function. rAAV stocks produced in this manner are contaminated with adenovirus and must be physically separated from the rAAV particles (e.g., by cesium chloride density centrifugation or column chromatography). Alternatively, adenovirus containing the AAV coding region and / or cell lines containing some or all of the AAV coding region and / or adenovirus helper genes could be used. Cell lines carrying rAAV DNA as an integrated provirus could also be used.

[0162] Multiple serotypes of AAV exist in nature, with at least 12 serotypes (AAV1–AAV13). Despite their high homology, different serotypes have different tissue tropisms. Upon transfection, AAV induces only a minor (if any) immune response in the host. Therefore, AAV is well suited for gene therapy approaches.

[0163] In some embodiments, the present disclosure may be directed to a drug comprising an AAV vector that is one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV13, ANC AAV, chimeric AAVs derived therefrom, variants thereof, and combinations thereof, which are further suitable for high-efficiency transduction in a tissue of interest. In at least one embodiment, the gene therapy vector is an AAV serotype 1 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 2 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 3 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 4 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 5 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 6 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 7 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 8 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 9 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 10 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 11 vector. In at least one embodiment, the gene therapy vector is an AAV serotype 12 vector.

[0164] The appropriate dose of AAV for humans is approximately 1 x 10 per kilogram of body weight. 8 Vector genome (vg / kg) ~ approx. 3 × 10 14 vg / kg, approx. 1×10 8 vg / kg, approx. 1×10 9 vg / kg, approx. 1×10 10 vg / kg, approx. 1×10 11 vg / kg, approx. 1×10 12 vg / kg, approx. 1×10 13 vg / kg, or approximately 1 × 10 14vg / kg DRP, 5×10 15 vg / kg、4×10 15 vg / kg、3×10 15 vg / kg、2×10 15 vg / kg、1×10 15 vg / kg、9×10 14 vg / kg、8×10 14 vg / kg、7×10 14 vg / kg、6×10 14 vg / kg、5×10 14 vg / kg、4×10 14 vg / kg、3×10 14 vg / kg、2×10 14 vg / kg、1×10 14 vg / kg、9×10 13 vg / kg、8×10 13 vg / kg、7×10 13 vg / kg、6×10 13 vg / kg、5×10 13 vg / kg、4×10 13 vg / kg、3×10 13 vg / kg、2×10 13 vg / kg、1×10 13 vg / kg、9×10 12 vg / kg、8×10 12 vg / kg、7×10 12 vg / kg、6×10 12 vg / kg、5×10 12 vg / kg、4×10 12 vg / kg、3×10 12 vg / kg、2×10 12 vg / kg、1×10 12 vg / kg、9×10 11 vg / kg、8×10 11 vg / kg、7×10 11 vg / kg、6×10 11 vg / kg、5×10 11 vg / kg、4×10 11 vg / kg、3×10 11 vg / kg、2×10 11 vg / kg、1×10 11 vg / kg、9×10 10vg / kg, 8 × 10 10 vg / kg, 7 × 10 10 vg / kg, 6 × 10 10 vg / kg, 5 × 10 10 vg / kg, 4 × 10 10 vg / kg, 3 × 10 10 vg / kg, 2 × 10 10 vg / kg, 1 × 10 10 vg / kg, 9 × 10 9 vg / kg, 8 × 10 9 vg / kg, 7 × 10 9 vg / kg, 6 × 10 9 vg / kg, 5 × 10 9 vg / kg, 4 × 10 9 vg / kg, 3 × 10 9 vg / kg, 2 × 10 9 vg / kg, 1 × 10 9 vg / kg, 9 × 10 8 vg / kg, 8 × 10 8 vg / kg, 7 × 10 8 vg / kg, 6 × 10 8 vg / kg, 5 × 10 8 vg / kg, 4 × 10 8 vg / kg, 3 × 10 8 vg / kg, 2 × 10 8 vg / kg, or 1 × 10 8 vg / kg, or about these, or at least these, or at least about these, or less than these, or less than these, or within a range defined by any two of these values. The above indicated doses in vg / kg kidney tissue units.

[0165] According to the systems and methods disclosed herein, in some embodiments, there may be substantially no leakage of perfusate outside the kidney, allowing higher doses of drug to be administered directly and exclusively to the kidney than would otherwise be safely administered by systemic delivery. While not intended to be limiting, it is believed that AAV toxicity may be due to systemic effects such as liver toxicity, platelet activation and loss, and complement activation and loss. These toxicities, and others, may be reduced, minimized, or entirely avoided by the application of locoregional perfusate as described in the methods and systems disclosed herein. Thus, up to about 1×10 15 Doses of 100 to 200 vg / kg kidney tissue or greater may be well tolerated. In at least one embodiment, the AAV dose to the kidney, expressed in vg / kg kidney tissue, may exceed the highest systemically administered dose by about 2 to about 200 fold, about 5 to about 150 fold, about 10 to about 100 fold, or any subrange therebetween.

[0166] Apart from viral vectors, non-viral expression constructs can also be used to introduce genes encoding target proteins or their functional variants or fragments into patient cells. Non-viral expression vectors that enable in vivo protein expression in target cells include, for example, plasmids, modified RNA, mRNA, cDNA, antisense oligomers, DNA-lipid complexes, nanoparticles, exosomes, any other non-viral shuttle suitable for gene therapy, their variants, and combinations thereof.

[0167] In addition to viral and non-viral vectors, nuclease systems may be used in conjunction with vectors and / or electroporation systems to enter patient cells and introduce genes encoding target proteins or functional variants or fragments thereof. Exemplary nuclease systems may include, but are not limited to, clustered regularly interspaced short palindromic repeats (CRISPR), DNA cleavage enzymes (e.g., Cas9), meganucleases, TALENs, zinc finger nucleases, any other nuclease system suitable for gene therapy, variations thereof, and combinations thereof. For example, in one embodiment, one viral vector (e.g., AAV) may be used for the nuclease (e.g., CRISPR), and another viral vector (e.g., AAV) may be used for the DNA cleavage enzyme (e.g., Cas9), and both (nuclease and DNA cleavage enzyme) may be introduced into target cells.

[0168] Another vector delivery system that can be used to deliver therapeutic polynucleotide sequences encoding therapeutic genes into cells is receptor-mediated delivery vehicles. These utilize the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Due to the cell-type-specific distribution of various receptors, delivery can be highly specific. Receptor-mediated gene targeting vehicles can contain two components: a ligand specific for a cellular receptor and a DNA-binding agent.

[0169] Suitable methods for introducing non-viral vectors into target cells include lipofection, calcium phosphate coprecipitation, DEAE-dextran, and direct DNA transfer using glass microtubes, ultrasound, electroporation, etc. Prior to vector transfer, kidney cells can be treated with permeabilizing agents such as phosphatidylcholine, streptolysin, sodium caprate, decanoylcarnitine, tartaric acid, lysolecithin, and Triton X-100. Exosomes can also be used to transfer naked DNA or DNA encapsidated by AAV.

[0170] The gene therapy vectors of the present invention may include a promoter operably linked to a nucleic acid sequence encoding a target protein. The promoter sequence should be compact and ensure strong expression. Preferably, the promoter provides expression of the target protein in the kidney of a patient treated with the gene therapy vector. In some embodiments, the gene therapy vector includes a nephron-specific promoter operably linked to a nucleic acid sequence encoding the target protein. As used herein, "nephron-specific promoter" refers to a promoter whose activity in kidney cells is at least two-fold higher than in any other non-renal cell type. Preferably, nephron-specific promoters suitable for use in the vectors of the present invention have at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, or at least 50-fold higher activity in kidney cells compared to non-renal cell types. Furthermore, nephron-specific promoters may be specific for a particular subunit of the nephron (e.g., proximal tubule, distal tubule, loop of Henle, collecting duct, glomerulus, etc.) and provide higher or exclusive expression in that particular subunit.

[0171] The nephron-specific promoter can be a selected human promoter or a promoter comprising a functionally equivalent sequence having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the selected human promoter. Exemplary, non-limiting promoters include kidney-specific cadherin (KSPC), Na + These may include podocyte-specific promoters such as glucose cotransporter 2 (SGLT2), sodium-potassium chloride cotransporter 2 (NKCC2), and E-cadherin (ECAD), or the podocin promoter NPHS2.

[0172] Vectors useful in the present invention may have various transduction efficiencies. As a result, viral or non-viral vectors transduce greater than, equal to, or at least about about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of cells in the target vascular region. Multiple vectors (viral or non-viral, or a combination thereof) can be used simultaneously or sequentially. Multiple vectors can be used to introduce multiple polynucleotides and / or target multiple cell types. When multiple vectors or multiple drugs are used, multiple transduction / transfection efficiencies may occur.

[0173] Pharmaceutical compositions containing gene therapy vectors can be prepared as either liquid solutions or suspensions. Pharmaceutical compositions of the present invention may contain commonly used pharmaceutically acceptable excipients, such as diluents and carriers. In particular, the compositions contain pharmaceutically acceptable carriers, such as water, saline, Ringer's solution, or dextrose solution. In addition to carriers, pharmaceutical compositions may also contain emulsifiers, pH buffers, stabilizers, dyes, etc.

[0174] In at least one embodiment, the pharmaceutical composition comprises a therapeutically effective gene dose, which is not toxic to the subject and can prevent or treat the subject's kidney disease.Prevention or treatment of kidney disease can be evaluated as the change in the phenotypic characteristics associated with kidney disease, and this change is effective for preventing or treating kidney disease.Therefore, a therapeutically effective gene dose is usually a dose that is sufficient to improve or prevent the pathogenic kidney phenotype of the subject being treated when administered in a physiologically acceptable composition.

[0175] The following exemplary embodiments will now be described.

[0176] Embodiment 1: A gene therapy vector adapted for transduction of kidney cells in a human subject, said gene therapy vector comprising an adeno-associated virus (AAV) vector and a polynucleotide sequence packaged within said AAV vector, said polynucleotide sequence encoding a therapeutic protein having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7.

[0177] Embodiment 2: The gene therapy vector of embodiment 1, wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:3.

[0178] Embodiment 3: The gene therapy vector of embodiment 1, wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:5.

[0179] Embodiment 4: The gene therapy vector of embodiment 1, wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:7.

[0180] Embodiment 5: The gene therapy vector of any of the preceding embodiments, wherein said polynucleotide sequence further comprises a promoter sequence operably linked to said polynucleotide sequence encoding said therapeutic protein.

[0181] Embodiment 6: The gene therapy vector of embodiment 5, wherein the promoter sequence is selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0182] Embodiment 7: The gene therapy vector of embodiment 5, wherein the promoter sequence is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

[0183] Embodiment 8: The gene therapy vector of any of the preceding embodiments, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

[0184] Embodiment 9: The gene therapy vector of any of the preceding embodiments, wherein the serotype of the AAV vector is AAV5.

[0185] Embodiment 10: A gene therapy drug comprising the gene therapy vector of any of the preceding embodiments and a pharmaceutically acceptable carrier.

[0186] Embodiment 11: A method of treating a kidney-related disease, comprising administering a therapeutic dose of the gene therapy agent of embodiment 10 to a patient in need thereof.

[0187] Embodiment 12: A method of performing gene replacement of a mutated gene, comprising administering a therapeutic dose of the gene therapy drug of embodiment 10 to a patient in need thereof.

[0188] Embodiment 13: A method of treating autosomal dominant polycystic kidney disease (ADPKD) in a subject, the method comprising administering to the subject a therapeutic dose of a drug comprising a gene therapy vector of any of embodiments 1-9 and a pharmaceutically acceptable carrier.

[0189] Embodiment 14: A method of performing localized delivery of polynucleotide sequences to renal cells within a kidney of a mammalian subject, the method comprising placing a perfusion catheter in a renal artery of the kidney and placing a withdrawal catheter in a renal vein of the kidney, the perfusion catheter and the withdrawal catheter, together with a membrane oxygenator, forming a closed perfusion circuit through the kidney, and further comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising the gene therapy agent of embodiment 10, the closed circuit substantially isolating perfusion through the kidney from the subject's systemic circulation.

[0190] Embodiment 15: The method of embodiment 14, wherein the kidney cells comprise tubular cells.

[0191] Embodiment 16: A dose of the AAV vector is delivered via the closed circuit and is at least about 5 x 10 per milliliter (mL) of plasma during perfusion. 9 maintain a concentration of vector genomes in the subject's blood at approximately 5 x 10 per mL of plasma during perfusion, and the vector exhibits leakage into the subject's systemic circulation. 7 16. The method of any one of embodiments 14-15, wherein the vector genome remains less than 100% and the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0192] Embodiment 17: The method of any of embodiments 14-16, wherein placing the perfusion catheter in the renal artery comprises placing the perfusion catheter via the femoral artery.

[0193] Embodiment 19: The method of any of embodiments 14-17, wherein placing the collection catheter in the renal vein comprises placing the perfusion catheter via percutaneous access through the femoral vein or via the jugular vein.

[0194] Embodiment 19: The method of any of embodiments 14-18, wherein placing the retrieval catheter in the renal vein comprises placing the perfusion catheter via non-percutaneous cut-down access.

[0195] Embodiment 20: The method of any one of embodiments 14-19, wherein flowing the perfusion fluid through the closed circuit comprises passing the perfusion fluid through the membrane oxygenator before entering the renal artery via the perfusion catheter.

[0196] Embodiment 21: The method of any of embodiments 14-20, further comprising adding additional perfusion fluid to the closed circuit or diluting the perfusion fluid with about 5% to about 50% v / v saline to account for a proportion of bladder output.

[0197] Embodiment 22: The closed circuit maintains the perfusion fluid flow rate at about 500 mL / min / 1.73 m of body surface area per kidney for about 15 minutes to about 4 hours. 2 ~approximately 650 mL / min per kidney per 1.73 m of body surface area 2 22. The method of any one of embodiments 14 to 21, wherein the

[0198] Embodiment 23: The closed circuit maintains the perfusion fluid flow rate at about 150 mL / min / 1.73 m of body surface area per kidney for about 15 minutes to about 4 hours. 2 ~ 700 mL / min per kidney per 1.73 m of body surface area 2 23. The method of any one of embodiments 14 to 22, wherein the

[0199] Embodiment 24: The method of any one of embodiments 14 to 23, further comprising applying a negative pressure to the recovery catheter, wherein the negative pressure is in the range of about -100 mmHg to 120 mmHg.

[0200] Embodiment 25: The method of any of embodiments 14-24, wherein one or more of the perfusion catheter and the recovery catheter are introduced percutaneously or non-percutaneously.

[0201] Embodiment 26: The method of any of embodiments 14 to 25, wherein less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v of the perfusion fluid circulating through the closed circuit leaks outside the closed circuit, or substantially none (0% v / v) leaks.

[0202] Embodiment 27: The method of any of embodiments 14 to 26, wherein one or more of the perfusion catheter or the recovery catheter is a balloon catheter.

[0203] Embodiment 28: A method of delivering a therapeutic composition to a subject in need thereof, the method comprising locally delivering a therapeutic composition comprising the gene therapy agent of embodiment 10 to the kidney of the subject, while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs. [Example]

[0204] Illustrative Examples The following examples are provided to aid in the understanding of the present disclosure and should, of course, not be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments, including the substitution of all currently known or later developed equivalents that would be within the skill of a person of ordinary skill in the art, and changes in formulation or minor variations in experimental design, should be construed as being within the scope of the embodiments incorporated herein.

[0205] The LRP system described below includes the following components: a percutaneous arterial catheter for occlusion antegrade perfusion of the renal artery (accessed via the femoral artery), a percutaneous venous catheter for occlusion of the renal vein and return of venous blood to the LRP system (accessed via the jugular vein), and an ECMO device with a reservoir and associated tubing for providing oxygen and removing carbon dioxide from the blood within the LRP system. The LRP procedure begins when the artery is antegrade perfused with oxygenated blood, while returning deoxygenated blood is collected from the venous system via the venous catheter. The blood is then collected in the reservoir, oxygenated, and antegradely reinfused into the organ via the arterial catheter. Fluid samples can be withdrawn or drugs can be introduced via the reservoir throughout the procedure.

[0206] Example 1: LRP Procedure LRP was performed on pigs utilizing the LRP system 1800 shown and described with respect to Figure 18. The accessory devices used in these examples are listed in Table 1, including their intended use and use in an LRP system according to an embodiment of the present disclosure. [Table 1]

[0207] The custom catheter was used as a venous retrieval catheter and included the following dimensions: a cross profile of 19 French (6.3 mm), an inner diameter of 12 French (4.0 mm), a usable length of 80 cm, a balloon diameter of 25 mm, and a tip length of 20 mm (similar to the exemplary custom catheter shown in and described with respect to Figures 1-3). Materials included Pebax 63 supported by a strong stainless steel braid as the shaft, flexible Chronoprene 25A as the balloon, and Pebax 35 filled with BaSO4 for radiopacity within the tip. The custom catheter was designed to support an aspiration flow rate of approximately 800 mL / min at -80 mmHg.

[0208] Figure 20 includes an x-ray showing the successful placement of arterial and venous catheters in the renal artery and vein, respectively, of a pig kidney. In the bottom image, contrast has been injected intravenously, revealing the overall tightness of the renal vasculature and closure system.

[0209] The detailed protocol of the LRP treatment followed in this example is now described. (1) The research animal is placed in a supine position. (2) Prepare the research animal for intravascular catheter insertion. (3) Angiographically assess the renal vein angle from both jugular and inguinal access, utilizing the least acute angle. (4) Stryker FlowGate from the femoral artery 2 Use the catheter to access the renal arterial circulation (side is determined based on the angle). (5) Access the venous circulation using the custom venous catheter described above (side and access point will be determined on an individual animal basis). (6) Inject contrast and place the catheter in its final position in the open configuration (i.e., balloon down) to visualize the renal circulation. (7) Catheters are placed in the aorta and vena cava until the procedure begins. (8) Flowgate 2 A PressureWire X is placed through the catheter into one of the renal arteries. (9) Prepare the ECMO system by degassing and priming with saline. Connect the venous and arterial lines to the ECMO system by clamping them to avoid air introduction. (10) Turn on the ECMO pump. (11) Remove the clamp from the intravenous line. (12) Begin exchanging saline with blood. If everything is stable, remove the clamp on the arterial line and establish an LRP loop. The venous suction is variable and can be adjusted as needed (e.g., -50 mmHg to 0 mmHg). (13) Place a venous catheter in place in the renal vein. (14) Inflate the balloon. (15) Inject contrast to confirm catheter tightness and position. (16) If the animal is stable: a.Flowgate 2 The renal artery is sealed with a catheter. b. Verify: catheter tightness and position with contrast injection, intrarenal pressure, renal-to-systemic pressure ratio (target ≥ 1), reservoir volume, ECMO pump RPM, and catheter flow rate. (17) If everything is stable for 5 minutes: a. Begin an infusion of glyceryl trinitrate through the arterial line at a rate of 2 μg / kg body weight / min. b. Check the following: intrarenal pressure, renal-to-systemic pressure ratio (target ≥ 1), reservoir volume, ECMO pump RPM, and catheter flow rate. (18) If everything is stable for 5 minutes: a. Treatment is initiated with a gene therapy drug injected into the reservoir. b. For the first group of animals (Group B1): Administer a dose of 5.0E+13 vg (prepared by diluting 1.8 mL of vector solution with a titer of 2.8E+13 vg / mL with 2.2 mL of excipient). c. For the second group of animals (Group B2): Administer 6.0E+14 vg (equivalent to 21.4 mL of vector solution with a titer of 2.8E+13 vg / mL). (19) Continue renal LRP for 60 minutes. (20) Check the following every 5 minutes: intrarenal pressure, renal-to-systemic pressure ratio (targeting a value of 1 or greater), reservoir volume, ECMO pump revolutions per minute, catheter flow rate, and all hemodynamic and cardiovascular parameters (pressure, HR). (21) Check urine output at t = 0, 15, 30, 45, and 60 minutes after the start of treatment. (22) Pay attention to the volume of the LRP reservoir, as there may be overfilling by the phrenic, gonadal, and adrenal veins, or volume loss due to urine production. These volume deviations can be dynamically managed. (23) At t = 0 min, 5 min, 15 min, 30 min, 45 min, and 60 min: Collect blood samples. a. From peripheral blood for shedding analysis. b. From the LRP system for vector infectivity analysis. c. From the LRP system for shedding analysis. (24) At the end of the 60-minute renal LRP: a. Discontinue glyceryl trinitrate. b. Deflate the balloon. c. Remove the catheter. (25) Dispose of the entire LRP circuit, reservoir, blood pump, and catheter in an appropriate biosafety container. (26) Provide immediate postoperative care, including but not limited to compression and administration of protamine.

[0210] The procedure described above demonstrated the feasibility of closed-circuit renal LRP for at least 60 minutes. No acute sequelae were observed, and indigo carmine testing immediately after LRP indicated that renal function was normal / unaffected by the procedure.

[0211] Factors that may be useful in optimizing LRP procedures to reduce leakage of perfusate into the systemic circulation and avoid transduction of proximal and distal organs include, but are not limited to, perfusion time, drug dose, AAV serotype used, presence of neutralizing factors, endothelial permeability, flow rate, and perfusion pressure.

[0212] Example 2: Biodistribution studies Figure 21 shows the renal transduction and biodistribution of 0.05-0.25 vg / dg (vector genome copies per diploid genome) after 60 min of LRP at a high dose of 6.2E+14 vg / kg. No significant contamination of the intact kidney, liver, or other organs was detected, demonstrating the tightness of the LRP closed circuit.

[0213] Intravenous control animals were also tested. Renal LRP resulted in a more uniform transduction profile across the different sections measured, whereas the IV control group showed preferential transduction in the cortical portion of the kidney. Renal LRP resulted in significantly less transduction in the liver compared to the IV control group; 17.2 vg / dg was detected in the liver of the IV control group, while virtually no transduction was observed in the liver of the kidney LRP group.

[0214] Example 3: Vector quantification Figures 22A and 22B show that a high dose of renal LRP (6.2 x 10 14 vg / kg, Figure 22A) and low dose (5.6 × 10 13 Figure 22B shows plasma genomes per mL of plasma measured at various time points during treatment (vg / kg, Figure 22B). Results revealed high retention within the LRP circuit (low vector shedding) over 60 minutes, low exposure of vector to the systemic circulation, and very low leakage of vector into the urine (Figure 22A). Throughout treatment, vector exposure to the kidney appears to be maximized.

[0215] Figure 23A is a plot of C3a levels over several days after renal LRP treatment for two different animals (LRP-1 and LRP2). Figure 23B is a plot of % transduction inhibition for various sample dilutions. Both demonstrate that anti-AAV neutralizing factors remained low for both animals and that there was no complement activation after renal LRP.

[0216] Figures 24A and 24B are plots of the respective flow rates and pump speeds during renal LRP, revealing a substantially constant flow rate of approximately 310 mL / min throughout the procedure.

[0217] These examples demonstrate that targeted AAV delivery to the kidney using clinically relevant animal models results in uniform biodistribution of the transgene. The embodiments described and illustrated herein enable the development of next-generation advanced therapies for the kidney by minimizing systemic adverse effects, significantly reducing the required vector dose, overcoming immunological limitations, and with the potential for repeat treatment. The use of the LRP system and methods is contemplated in combination with other therapeutic agents and treatment strategies.

[0218] In renal LRP experiments for GFP expression, GFP is localized to kidney glomeruli, particularly in podocytes.

[0219] It is contemplated that the methodology described herein can be similarly applied to other organs by applying the LRP procedure to those organs. Exemplary organs may include, but are not limited to, the heart, liver, or pancreas. Cardiac LRP is discussed in International Application No. PCT / IB2020 / 000692, filed August 26, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0220] Example 4: Serotype screening Next, we describe the screening of serotypes to identify highly efficient serotypes for use in local gene delivery to the kidney. Several AAV vector candidates were initially identified. In this study, AAV1, AAV2, AAV5, AAV6, and AAV9 serotypes were selected as candidates. AAV vectors of each serotype, each containing a CMV-GFP transgene cassette, were produced. A total of 4.9 × 10 AAV vectors were generated for each of the five AAV types. 14 vg (5.9 × 10 per serotype) 13 A perfusion solution containing 100 mg of vector (vg) was prepared. Using the same protocol as above and the custom catheter, LRP was performed on the porcine kidney. The total amount of vector injected into the closed loop was 6.4 × 10 12 vg / kg, and closed-circuit perfusion was maintained for 60 min.

[0221] Figure 25 is a plot showing vector genomes per mL of plasma measured at various time points during a 60-minute renal LRP procedure, with a closed LRP circuit to the systemic circulation, for perfusates containing five different AAV serotypes. Results show high retention within the LRP circuit (low vector shedding) over the 60-minute period, and low exposure of vector to the systemic circulation (5 x 10 per mL of plasma). 7 (less than) was revealed.

[0222] Figure 26 is a plot showing the biodistribution of multiple AAV serotypes in the kidney based on measurements of vector genome copy number per diploid genome in various sections of LRP-treated kidneys, demonstrating that AAV5 has significantly higher efficiency than other serotypes (39.3-fold higher efficiency compared to AAV9). Figure 28 is a plot showing relative quantification of transgene mRNA levels in kidney sections treated with AAV5 relative to cumulative AAV in the kidney. Figure 27 is a plot showing biodistribution within the kidney compared to the liver and additional kidney-associated tissues after 60 minutes of perfusion with multiple AAV serotypes, demonstrating that transduction was limited to the treated kidney sections and the renal artery and vein, with minimal transduction in the liver or untreated kidney. Without wishing to be bound by theory, it is believed that the high efficiency of AAV5 may be due to a synergistic effect resulting from maintaining a high concentration of AAV5 in a closed circuit for a period of time (30 minutes to 1 hour) to enable transduction. Without localization, it is believed that the majority of AAV5 in the systemic circulation ends up in the liver or other organs, with little transduction of AAV5 in the kidney.

[0223] AAV5-CMV-GFP was further evaluated alone without other serotypes present in the pig kidneys of two different animals: one with 1.2 × 10 13 LRP treatment for 60 minutes at a dose of 1.3 × 10 13The LRP treatment was performed at a dose of 1000 ng / kg for 52 minutes (Figures 31 and 32). Both studies further demonstrated relatively constant vector concentrations within the LRP circuit, relatively little leakage of vector into the systemic circulation, and localized biodistribution in the treated kidney, with few vector genomes quantified in the untreated kidney and liver.

[0224] Example 5: Single Kidney LRP Treatment for Biodistribution Studies 5.1. Materials and Components Next, we describe the LRP system and protocol for single-kidney LRP used to perform the biodistribution studies described below in Examples 6-8.

[0225] LRP was performed on domestic pigs utilizing the LRP system 1800 shown and described in Figure 18. The accessory devices / components used in the procedures described below, including their intended use and use in an LRP system according to an embodiment of the present disclosure, are listed in Table 2. [Table 2]

[0226] The supply line catheter assembly includes a perfusion catheter, supply dilator, and accessories. The perfusion catheter includes a reinforced inner shaft and an unreinforced outer shaft. The inner shaft, with an inner diameter of 2.7 mm (although diameters between 2 and 3.5 mm are expected), is large enough to establish physiological perfusion. The catheter is compatible with a 14 Fr introducer. The distal section of the shaft is more flexible, allowing for smooth, atraumatic tracking of the catheter into the renal arteries. Flexibility is controlled by the hardness of the Pebax polymer: 72D in the proximal section, 55D in the transition zone, and 35D in the distal section.

[0227] The tip of the perfusion catheter is short (2-3 mm), allowing the balloon to be placed close to the bifurcation when the main stem of the renal artery is short. To ensure atraumatic progression through the vasculature, the tip is flexible (Pebax 35D) and rounded. Furthermore, the inner diameter is slightly reduced to minimize the gap between the dilator and the tip.

[0228] Marker bands are implanted under the balloon near the tip to visualize these landmarks under fluoroscopy.

[0229] The compliant occlusion balloon is attached to the outer shaft and is made of a polyblend or polyurethane material. The soft balloon gently conforms to the shape of the tightly sealed container. The balloon measures 3-5 mm in length and has a disk-like inflated shape with a maximum outer diameter of approximately 11 mm. The balloon diameter is linearly dependent on the inflation volume, ranging from approximately 4 mm in an inflated diameter at approximately 0 mL of inflation volume to approximately 11 mm in an inflated diameter at approximately 0.5 mL of inflation volume. The gap between the inner and outer shafts is used to inflate the balloon, and the outer shaft has multiple inflation holes underneath the balloon.

[0230] A hub is connected to the proximal shaft end and serves as the user interface, allowing the attachment of the following accessories: a luer connector for a syringe to inflate the balloon, a barbed connector to allow attachment of irrigation tubing, and a hemostatic valve with a luer connector for flushing, which can be used to exchange the dilator for guidewires and pressure wires with minimal blood loss.

[0231] The supply line dilator is placed in the inner lumen of the perfusion catheter prior to tracking the device. The dilator fills the large inner lumen of the perfusion catheter, allowing the catheter to be advanced atraumatically through the vasculature. The outer diameter of the dilator is slightly smaller than the inner shaft of the perfusion catheter, allowing for movement of the dilator within the catheter with minimal friction.

[0232] The dilator tip is tapered and rounded to gently dilate the vessel for catheter passage, making it atraumatic. The tip inner diameter is approximately 0.5 mm, minimizing guidewire clearance. The dilator is compatible with guidewires 0.018 inches and smaller. The proximal section of the dilator has an enlarged inner diameter for optimal flexibility. The dilator is made from Pebax 35D and barium sulfate (BaSO4). Barium sulfate is added to make the catheter radiopaque. Additionally, radiopaque markers may be embedded in the shaft to indicate the tapered section. Colored markings may be added to the proximal dilator section to indicate how the dilator should be positioned within the perfusion catheter. A hub is attached to the proximal end of the dilator shaft to facilitate guidewire insertion. Additionally, a Luer connector allows for the attachment of a syringe for flushing.

[0233] A schematic of the perfusion catheter is shown in Figure 33A, where the balloon is in a deflated state, and Figure 33B shows the balloon in a deployed state. Figure 33C is a photograph of the perfusion catheter with the balloon in a deployed state.

[0234] Next, other accessories included with the perfusion catheter will be described. (1) An extension line with a four-way stopcock is delivered along with a catheter that attaches to the balloon inflation port on the hub. (2) A 1 / 4-inch tubing assembly is attached to the barbed connector on the hub. The tubing is approximately 10 cm long. A red on / off tubing clamp is placed on the tubing and used to clamp the perfusion. The red color indicates to the user that this line carries arterial blood. A T-piece is attached to the other end of the tubing. The T-piece is a straight connector with two 1 / 4-inch barbs on the short side and a Luer connector on the long side. The perfusion is directed straight from one 1 / 4-inch barb to the other. A four-way stopcock is attached to the Luer on the side. This side port is used for degassing, contrast injection, pharmaceutical composition injection, and sample collection. (3) After manufacturing, a thin-walled plastic sleeve may be placed over the distal end and balloon to protect the balloon and its connections during shipping and shelf life. The protective sleeve is removed before using the catheter.

[0235] The reflux line catheter assembly includes a retrieval catheter, reflux dilator, and accessories. The reflux catheter includes a reinforced inner shaft and an outer shaft. The inner shaft, with an inner diameter of 4.06 mm (although diameters of 3-4.5 mm are assumed), is large enough to establish a physiological drainage flow. The catheter is compatible with a 22 Fr introducer. The distal shaft section is more flexible, allowing for smooth, atraumatic tracking of the catheter into the renal vein. Shaft flexibility is controlled by the hardness of the Pebax polymer: 72D in the proximal section, 55D in the transition zone, and 35D in the distal section.

[0236] The catheter tip has lateral holes / perforations (4-8 holes, 0.5-2 mm diameter) for drainage. The tip can be positioned close to the vessel wall, and the side holes help to avoid impaired return flow if the distal opening is completely or partially occluded. The tip is short (4-8 mm long), allowing the balloon to be placed close to the bifurcation when the main stem of the renal vein is short. The tip is manufactured from a non-reinforced, soft, radiopaque polymer blend (Pebax 35D + BaSO4). The distal edge of the tip is rounded to ensure atraumatic progression through the vasculature. Additionally, the tip inner diameter is slightly reduced to minimize the gap between the dilator and the tip.

[0237] Marker bands are implanted beneath the balloon to visualize these landmarks under fluoroscopy.

[0238] The compliant occlusion balloon is attached to the outer shaft and is made of a polyblend or polyurethane material. The soft balloon gently conforms to the shape of the tightly sealed container. The balloon measures 8 to 15 mm in length and has a disk-like inflated shape with a maximum outer diameter of approximately 25 mm. The balloon diameter is linearly dependent on the inflation volume, ranging from approximately 6 mm at an inflation volume of approximately 0 mL to approximately 25 mm at an inflation volume of approximately 5 mL. The gap between the inner and outer shafts is used to inflate the balloon, and the outer shaft has multiple inflation holes underneath the balloon.

[0239] A hub is connected to the proximal shaft end and serves as the user interface, allowing the attachment of the following accessories: a luer connector for a syringe to inflate the balloon, a barbed connector to allow attachment of an evacuation tube, and a hemostatic valve with a luer connector for flushing, which can be used to exchange the dilator for a guidewire and pressure wire with minimal blood loss.

[0240] The reflux line dilator is placed in the inner lumen of the retrieval catheter prior to tracking the device. The dilator fills the large inner lumen of the retrieval catheter, allowing the catheter to be advanced atraumatically through the vasculature. The outer diameter of the dilator is slightly smaller than the inner shaft of the retrieval catheter, allowing the dilator to move within the catheter with minimal friction.

[0241] The dilator tip is tapered and rounded to gently dilate the vessel for catheter passage, making it atraumatic. The tip inner diameter is approximately 0.95 mm, minimizing guidewire clearance. The dilator is compatible with guidewires 0.035 inches and smaller. The proximal section of the dilator has an expanded inner diameter to optimize flexibility. The dilator is made from Pebax 35D and BaSO4 (added to make the catheter radiopaque). Additionally, radiopaque markers may be embedded in the shaft to indicate the tapered section. Colored markings may be added to the proximal dilator section to indicate how the dilator should be positioned within the retrieval catheter. A hub is attached to the proximal end of the dilator shaft to facilitate guidewire insertion. Additionally, a Luer connector allows for the attachment of a syringe for flushing.

[0242] A schematic of the retrieval catheter is shown in Figure 34A, where the balloon is in a deflated state, and Figure 34B shows the balloon in a deployed state. Figure 34C is a photograph of the retrieval catheter with the balloon in a deployed state.

[0243] Next, other accessories that may be included with the retrieval catheter will be described. (1) An extension line with a four-way stopcock is delivered along with a catheter that attaches to the balloon inflation port on the hub. (2) A 1 / 4 inch tubing assembly is attached to the barb connector on the hub. The tubing is approximately 10 cm long. A blue on / off tubing clamp is placed on the tubing and used to clamp the outlet flow. The blue color indicates to the user that this line carries venous blood. A T-piece is attached to the other end of the tubing. The T-piece is a straight connector with two 1 / 4 inch barbs on the short side and a Luer connector on the long side. The return flow is directed straight from one 1 / 4 inch barb to the other. A four-way stopcock is attached to the Luer on the side. This side port is used for degassing, contrast injection, pharmaceutical composition injection, and sample collection. (3) After manufacture, a thin-walled plastic sleeve may be placed over the distal end of the catheter and the balloon to protect the balloon and its connections during shipping and shelf life. The protective sleeve is removed before use of the catheter.

[0244] The irrigation and withdrawal catheters, along with all accessories, are secured onto a carton plate using straps, clamps, and protective tubing. The carton plate with the attached device is placed into a Tyvek pouch and heat-sealed. The pouch protects the product from contamination but allows ethylene oxide (EtO) gas to permeate due to the sterilization process. The pouch is then placed into a rigid outer box for transport and storage.

[0245] 5.2 Protocol The entire LRP procedure can be divided into four phases: (1) an initiation phase during which the LRP circuit has not yet been established, (2) a stabilization phase during which the LRP circuit is established, (3) a treatment delivery phase during which the LRP circuit is established, and (4) a removal phase during which the LRP circuit is discontinued. Each phase is described below.

[0246] Initiation stage: interventional placement of arterial and venous catheters in the appropriate positions (i.e., intervention into the left renal main artery and vein, balloon inflation, and assessment of the quality of the seal).

[0247] Stabilization Phase: Initiation of LRP Perfusion Without Payload. The stabilization phase begins when the renal artery is perfused antegrade with oxygenated blood, while returning deoxygenated blood is collected from the renal vein via the collection catheter. LRP perfusion is considered stable when the operator confirms the following criteria: stable reservoir volume, sufficient flow rate through the catheter, physiological renal artery pressure in the absence of pulsation, stable vacuum pressure, a stable rate of glyceryl trinitrate infusion or other vasoactive substance, and complete volume exchange of saline with blood in the reservoir. At this point, payload delivery via the LRP system can begin.

[0248] Therapy Delivery Phase: Once the stabilization phase is complete, the therapy phase begins, during which the therapy payload is introduced into the LRP system. The duration of this phase depends on the therapy delivery protocol. The total time the therapy payload recirculates is referred to as the "LRP duration."

[0249] Removal Phase: After the treatment delivery phase is completed according to a predefined protocol, the LRP perfusion system is stopped and the arterial (perfusion) and venous (withdrawal) catheters are removed from the body according to specific procedures.

[0250] While the protocols discussed below refer to a single perfusion catheter and a single retrieval catheter, it should be noted that multiple catheters may be used to optimize sealing of the LRP circuit based on the patient's anatomy. For example, Figures 35A and 35B illustrate the deployment of a single perfusion catheter and a pair of perfusion catheters, respectively, within the renal artery of the left kidney. Similarly, Figures 36A and 36B illustrate the deployment of a single retrieval catheter and a pair of retrieval catheters, respectively, within the renal vein of the left kidney.

[0251] The detailed protocol for LRP treatment followed in this and subsequent examples is now described. Initiation Phase (1) The research animal is placed in a supine position. (2) Prepare a jugular central venous catheter aseptically. (3) As part of the sterile access site preparation, position the surgical drape and additional drapes to completely cover the animal (with the jugular and thigh regions exposed). (4) An additional set of drapes is placed for use in removing the "contaminated" catheter at the end of the procedure. Femoral artery: (5) Identify the target vessel under ultrasound guidance in the groin region. (6) Introduce an appropriate femoral introducer sheath (14-FR diameter). (7) Flush the catheter with heparinized saline. Jugular vein: (8) Repeat the previous steps (steps 5-7), but for the left jugular vein with a 22-FR introducer sheath. Catheter Placement: (9) A guidewire (0.014 inch or 0.018 inch, e.g., Boston Scientific V18 or Abbott High-Torque, at the discretion of the interventionalist) is placed into the distal renal artery through the multipurpose catheter (5-FR). (10) Place at least one perfusion catheter in the main stem of the renal artery to ensure uniform blood distribution to all parts of the kidney and ensure stable catheter position. (11) A PressureWire X (Abbott) is threaded into the supply line and placed into one of the renal artery branches. (12) Visualize the renal arterial anatomy by antegrade contrast injection through the perfusion catheter to confirm that the catheter is in the desired position and that only renal structures are perfused by this artery. (13) Place a Lunderquist guidewire (or equivalent, 0.035 inches) into the distal renal vein through the 5-FR multipurpose catheter. (14) To ensure uniform blood drainage from all parts of the kidney and to ensure a stable catheter position, at least one retrieval catheter is placed in its position in the main stem of the left renal vein over the Lunderquist guidewire. (15) A second PresureWire X (Abbott) is placed through the return line to measure venous pressure in the renal vein. (16) After confirming proper placement of the venous catheter, inflate the balloon on the retrieval catheter. (17) Evaluate renal vein anatomy by retrograde contrast injection through a diagnostic catheter placed in the lumen of the retrieval catheter. (18) Under fluoroscopy, the tightness of the balloon occlusion is tested by retrograde injection of contrast. The criteria for acceptance of the seal quality are (i) no evidence of paraballoon flow or contrast, (ii) no filling of other venous structures connected to the vena cava, and (iii) no drainage of major extrarenal vessels. (19) Deflate the retrieval catheter balloon to allow the injected contrast agent to be naturally expelled. (20) The perfusion team prepares the LRP system together with the operator. (21) Prepare the LRP system for the LRP procedure. a. Prime with 250 mL of heparinized saline. b. Degassing c. Turn on the LRP pump and operate the circuit within the shunt. d. Use tubing to connect the collection catheter to the reservoir. e. Connect the perfusion catheter to the arterial outflow of the oxygenator via tubing, and completely deflate the tubing and catheter. f. Release the clamp on the venous line and close the shunt. g. Begin filling the reservoir by adding blood. h. When the system is stable, unclamp the arterial line and completely exchange the saline in the reservoir with blood (complete exchange may be confirmed by hematocrit analysis). i. Venous suction and blood pump speed are variable and adapted to physiological needs on a case-by-case basis (typically, vacuum is between -80mmHg and 0mmHg, and blood pump speed is between 2500 and 4500 rpm). Stabilization Phase (22) The LRP circulation begins with the retrieval catheter balloon deployed and the perfusion catheter balloon deflated. a. Deploy the retrieval catheter balloon (optionally reconfirm final position with retrograde contrast injection). b. Check: tightness and position of catheter with contrast injection, intrarenal pressure in both the renal artery and renal vein (acceptable range is 60 mmHg to 140 mmHg in the artery and below central venous pressure in the renal vein), ratio of mean arterial renal pressure to mean systemic pressure (ratio should be slightly below 1 to prevent leakage), stability of reservoir volume (slight volume increases are acceptable, provided the rate of increase does not result in reservoir saturation within the allotted LRP time or hemodynamic instability due to removing too much blood volume from the animal), RPM of the LRP pump, and flow rate through the catheter (a flow rate of 150 mL / min to 800 mL / min is preferred). (23) After the circuit is stabilized and total volume exchange is confirmed by hematocrit analysis, the renal artery is sealed with a perfusion catheter under fluoroscopic guidance. (24) Once both renal vessels are sealed, complete LRP circulation is established. (25) 5 minutes later: a. If necessary (i.e., in the case of hypertension or renal artery spasm), begin an infusion of glyceryl trinitrate (GTN) through the arterial line at a rate of 0 µg / kg body weight / min to 4 µg / kg body weight / min. b. Check: tightness and position of catheter with contrast injection, intrarenal pressure in both the renal artery and renal vein (in the artery, acceptable range is 60 mmHg to 140 mmHg; in the renal vein, acceptable range is equal to or less than central venous pressure), ratio of mean arterial renal pressure to mean systemic pressure (to prevent leakage, the ratio should be slightly below 1), stability of reservoir volume (slight volume increases are acceptable, provided the rate of increase does not result in reservoir saturation within the allotted LRP time or hemodynamic instability due to removing too much blood volume from the animal), RPM of the LRP pump, and flow rate through the catheter (a flow rate of 150 mL / min to 800 mL / min is preferred). (26) Check all the above parameters every 5 minutes for at least 20 consecutive minutes until all parameters are within acceptable limits. Therapeutic Delivery Phase (27) Once all major LRP system components have been stable for at least 20 minutes, the payload can be delivered. The payload is provided via a bolus injection in the venous return line via a three-way stopcock. Alternative injection methods would be continuous injection, a bolus injection into the supply line, or several boluses via either the return line or the supply line. After the payload is injected, a flush is provided from the same location. (28) Perform a single kidney LRP procedure over a predefined period of time. (29) Every 5 minutes, check: catheter tightness and position with contrast injection; intrarenal pressure in both the renal artery and renal vein (in the artery, acceptable range is 60 mmHg to 140 mmHg; in the renal vein, acceptable range is equal to or less than central venous pressure); mean arterial renal pressure to mean systemic pressure ratio (to prevent leakage, the ratio should be slightly below 1); reservoir volume stability (slight volume increases are acceptable, provided the rate of increase does not result in reservoir saturation within the allotted LRP time or hemodynamic instability due to removing too much blood volume from the animal); LRP pump RPM; and flow rate through the catheter (a flow rate of 150 mL / min to 800 mL / min is preferred). (30) At T+5, +10, +15, +20, +25, +30, +45, and 60 minutes (or other predefined time points of interest): a. Obtain peripheral blood samples for blood gas and shedding analysis. b. Collect blood samples from the LRP system for shedding analysis, vector infectivity analysis, ACT, and blood gas analysis. c. Perform urine biochemistry and shedding analysis. Removal Phase (31) At the end of the LRP: a. Discontinue glyceryl trinitrate if used. b. Prepare a 20 mL syringe, fill it with systemic arterial blood, and attach the syringe to the side port of the perfusion catheter. c. Clamp the arterial line and flush the perfusion catheter lumen with a prefilled syringe through the side port. d. Immediately after flushing, deflate the perfusion catheter balloon. e.Extract the arterial line from the body to the exterior. f. During this time, the renal vein balloon should remain engaged and under suction to fill the ECMO reservoir with the remaining payload containing blood. g. After approximately 150 mL has been expelled, clamp the IV line and deflate / retract the recovery catheter balloon. Extract the IV line externally from the body. Completely detach the catheter, taking special care not to spill the blood-containing payload. (32) Dispose of the entire LRP circuit, reservoir, blood pump, and catheter in an appropriate biosafety container. (33) Provide immediate postoperative care.

[0252] Example 6: Biodistribution of AAV5 after administration to the left kidney of domestic pigs via LRP 6.1 Treatment Description and Administration Based on the protocol described in Example 5, 9.8 x 10 AAV5-CMV-eGFP 14 A dose of 1000 mg / kg was used to treat domestic pigs with renal LRP (1-h administration time) from 12 pigs selected after immunological screening for AAV5 pre-existing antibodies and renal computed tomography (CT) scans to ensure that the pigs were physiologically compatible with LRP treatment.

[0253] 6.2 Vector Shedding Analysis To confirm the efficacy of the LRP procedure, multiple blood samples were collected from the LRP system and peripheral blood (systemic circulation) at different time points. Urine samples were also collected to estimate viral shedding during the procedure. Total viral genomes per mL in the animal's blood and urine samples were measured using qPCR with a probe targeting the AAV GFP DNA sequence and titrated against a standard curve using linearized plasmid DNA. All samples were measured in duplicate. Although this pig had challenging anatomy due to a branched left renal vein, which required additional catheterization, the procedure was successful. There was no leakage from the LRP into the systemic circulation.

[0254] Figure 37 is a plot showing shedding analysis (vector genomes per mL of plasma or urine) detected in the LRP circulation, systemic circulation, and urine of treated domestic pigs (1.2 x 10 13 vg / kg, 60 min LRP treatment). Figure 38 is a plot showing the total vector genomes detected in the urine of treated domestic pigs (1.2 x 10 13 (vg / kg, 60-minute LRP treatment). Viral genome levels remained stable over time until the end of treatment, as shown in Figure 37. The vector was also found in the urine early after LRP administration, as shown in Figure 38. In this experiment, urine was collected at the specified time points, and urine volume was measured before emptying the urine bag. Notably, there was fluctuation in urine volume during LRP treatment. Therefore, urine volume was used to calculate the total viral genome in the urine at each time point (Figure 38). Viral shedding was more pronounced 10 minutes after AAV administration to the LRP.

[0255] 6.3 Biodistribution by kidney region (cortex, cone, and papilla) A small piece of each tissue was homogenized using metal beads in a TissueLyser II (Qiagen) and extracted using AllPrep DNA / RNA (Qiagen). DNA concentration and purity were measured using a NanoDrop spectrophotometer. Quantification of viral genomes within cells of tissue samples (vg / dg) was performed using droplet digital PCR (ddPCR). A GFP probe was used to detect the viral genome in animals, whereas a β-actin probe was used for housekeeping genes.

[0256] Figure 39 shows the LRP-treated kidney (1.2 x 10 13 Figure 39 shows the biodistribution analysis of AAV5 (vg / kg, 60-minute LRP treatment) in vg / dg compared with untreated kidney and liver. This pig showed a significant increase in AAV5 viral genomes in the treated kidney (Figure 39, mean 11.5) compared with pigs treated with the same dose of AAV9 (highest observed mean value 0.5 vg / dg). This also confirmed the results of a previous study comparing these two serotypes co-administered at a lower dose (10-fold, see Figure 26) in the same animals. Untreated kidney and liver remained highly detargeted. The observation of comparable levels of transduction throughout the cortical, outer medullary, and inner medullary regions (i.e., pyramidal and papillary regions) indicates that AAV5 delivered via LRP can broadly transduce cells of the nephron as well as other cells in the kidney. Considering that AAV5 delivered by LRP can transduce cells present in both high-perfusion areas (e.g., the cortex) and low-perfusion areas (e.g., the petrous and papillary regions), and that AAV5 can be transported into the urine stream within the renal tubules and excreted, AAV5 may be able to access kidney cells not only via the vascular circulation but also from within the renal tubules. These properties make AAV5 a desirable vector for use in delivering gene medicines to the kidney using LRP.

[0257] 6.4 GFP pg / mg tissue by ELISA The amount of reporter protein in sections of LRP-treated kidneys (AAV5-CMV-GFP) was quantified using a GFP SimpleStep ELISA kit (Abcam #ab171581). The assay was performed according to the manufacturer's instructions. Briefly, a spoonful (30–50 mg) of tissue powder was homogenized in cold extraction buffer. After centrifugation, the supernatant was collected in a clean tube for further processing. Standards were freshly prepared, and samples were run in duplicate. After incubation with the antibody cocktail and extensive washing, substrate was added, and the reaction was stopped before saturation. Optical density (OD) readings were recorded at 450 nm and converted to pg / mg after normalization to total protein concentration.

[0258] Figure 40 shows the LRP-treated kidney (1.2 x 10 13 Figure 1 shows plots of GFP protein levels in pg / mg in treated kidneys (vg / kg, 60-minute LRP treatment) compared to untreated kidneys and livers. Consistent with the observation of high levels of transduction throughout the treated kidneys, high levels of GFP expression were detected throughout the cortical and outer and inner medullary regions of the treated kidneys. No GFP expression was detected in untreated kidneys or livers, further demonstrating effective transduction of only the LRP-treated kidneys. The observation of comparable levels of GFP expression throughout the cortical and outer and inner medullary regions (i.e., the pyramidal and papillary regions) indicates that AAV5 delivered via LRP can broadly transduce and express transgenes in cells of the nephron as well as other cells in the kidney. These properties further make AAV5 a desirable vector for use in delivering gene medicines to the kidney using LRP.

[0259] 6.5 Description of transduced cells observed in RNAScope The in situ hybridization technology assay (ACD, BioTechne brand) allows spatial visualization of single mRNA or episomal DNA molecules. To improve the signal-to-noise ratio, RNAScope uses target-specific probes combined with multiple signal amplifiers. This results in discrete dots that can be visualized by light microscopy. Here, a specific GFP probe was used in combination with other probes specific for different cell types within the kidney (CDH2 - proximal tubule, CDH1 - distal tubule, NPHS1 - podocyte, SLC12A1 - macula densa, and PECAM1 - endothelial cell marker). This allowed the identification of transduced kidney cells after LRP treatment. Briefly, cryosections from freshly frozen kidney sections were fixed in 4% paraformaldehyde and subjected to a cascade of hybridization events, including several washes in between. After mounting, slides were imaged with an inverted Axio Observer microscope (Zeiss). Gross photographs of cortical sections showed uniform staining of glomeruli in treated kidneys. No signal was observed in untreated kidneys or livers. Additional spots were observed in other tubular structures within the cortical and medullary regions, confirming the ability of AAV5 to transduce various cells in the kidney.

[0260] Example 7: Comparison of AAV5 biodistribution after LRP delivery to the left kidney and IV administration 7.1 Description of Treatment and Administration The purpose of this example was to compare two routes of administration of AAV5 to the kidney: 1-hour administration via the renal LRP and intravenous injection (IV), and to evaluate the advantages of AAV administration via the renal LRP compared with IV.

[0261] As in Example 6, the two domestic pigs used in this study were selected based on immunological screening for AAV5 pre-existing antibodies and CT scans of the kidneys to confirm that the pigs were physiologically compatible with LRP treatment.

[0262] For each animal, a total of 9.9 × 10 14vg of AAV5-CAG-eGFP was administered to Animal 1 via LRP as described in Example 5. For Animal 2, AAV5-CAG-eGFP was administered as a bolus dose via a central venous access line. The methodology was identical for Animal 1 and Animal 2, except that for Animal 2, only peripheral blood and urine samples were collected for shedding analysis because the LRP circuit had not been established.

[0263] 7.2 Vector Shedding Analysis To confirm the tightness of the LRP treatment, multiple blood samples were collected at different time points from the LRP system of Animal 1 and peripheral blood (systemic circulation) from both animals. Urine samples were also collected to estimate viral shedding during LRP treatment. Total viral genomes per mL in blood and urine samples were measured using qPCR with a probe targeting the AAV GFP DNA sequence and titrated based on a standard curve using linearized plasmid DNA. All samples were measured in duplicate.

[0264] Figure 41 shows the shedding analysis (vector genomes per mL of plasma or urine) detected in the LRP circuit, systemic circulation, and urine of Animal 1. LRP treatment was successful in Animal 1, with nominal signs of leakage from the LRP into the systemic circulation during the latter part of treatment. Viral genome levels in the circuit remained relatively stable over time until the end of treatment. As shown in Figures 41 and 43, vector was also found in the urine early after LRP administration. In this experiment, urine was collected at the indicated time points, and urine volume was measured before emptying the urine bag. Notably, there was fluctuation in urine volume during LRP treatment. Therefore, urine volume was used to calculate the total viral genomes in the urine at each time point. Viral shedding into the urine was significant, beginning within 10 minutes after AAV administration into the LRP circuit and remaining stable throughout treatment.

[0265] Figure 42 is a plot showing shedding analysis (vector genomes per mL of plasma or urine) detected in the systemic circulation and urine of Animal 2. As shown, IV administration of AAV5 to Animal 2 resulted in a significantly different exposure profile. The AAV5 concentration achieved in the systemic circulation during the first hour post-administration was approximately 100-fold higher than that observed in Animal 1 during LRP treatment, indicating a much higher systemic exposure after IV administration versus LRP administration. Viral genomes in Animal 2 persisted in the circulation at all measured time points up to day 5 post-treatment. Viral genome concentrations declined rapidly in the systemic circulation, with a 1-log decrease from the earliest time point between 5 and 60 minutes post-injection, reaching 1.94 x 10 10 vg / mL ~ 1.93 × 10 9 vg / mL, and on the 5th day, 3.43 × 10 6 Assuming that AAV5 exposure to the kidney is expressed as the systemic AAV concentration in Animal 2, the maximum concentration of AAV5 exposure to the kidney was approximately 20-fold lower than that of Animal 1 receiving the same dose in the LRP (1.94 × 10 10 vg / mL vs. 4.23 × 10 11 Given the potential for AAV5 to transduce cells of the nephron via exposure from the circulation, the substantial difference in the maximum concentration of AAV5 viral genome exposed to the kidney after LRP versus IV administration may have important implications for the delivery of gene therapy to the kidney via AAV5-mediated delivery.

[0266] Similarly, the concentration profile of AAV5 viral genomes in urine was substantially different after IV administration to Animal 2 compared to direct administration to the kidney via the LRP in Animal 1. In Animal 2, viral genomes were evident only at 45 minutes post-injection (8.84 x 10 8Given the possibility that AAV5 transduces tubular epithelial cells of the nephron via the apical (urine-facing) side of these cells, the substantial difference in viral genomes in urine after LRP and IV administration may have even more important implications for the delivery of gene therapy to the kidney via AAV5-mediated delivery.

[0267] Using the LRP shedding data obtained from Animal 1 and the systemic concentration data obtained from Animal 2, we further investigated the differences in AAV5 exposure to the kidney after LRP and after IV administration. Figure 45 shows a plot of modeled AAV5 concentration versus time in the LRP circuit of the treated kidney of Animal 1, derived from the data shown in Figure 41, which is used to model kidney exposure to viral genomes by considering the area under the curve (AUC). For LRP modeling, the approximate AAV5C observed in the LRP circuit of Animal 1 was used. max Plots were derived using the IV model, assuming no leakage and constant AAV5 concentrations throughout treatment. For IV modeling, we assumed that the circulating concentration of AAV5 in peripheral blood represents exposure to the kidney after IV administration. Systemic AAV5 concentration data through day 5 were fitted using a monophasic exponential decay equation (GraphPad Prism) to obtain the decay constant of AAV5 after IV administration to pigs (k = 0.4 / h).

[0268] Figure 46 is a plot of modeled AAV5 concentration versus time in the systemic circulation of Animal 2 after IV administration, derived from the data shown in Figure 42, which is used to model kidney exposure to viral genomes by considering the area under the AUC. The approximate systemic C observed in Animal 2 max Using the decay constant obtained using a monophasic exponential decay model, the decay curve was used to model the concentration versus time profile of AAV5 for a period corresponding to LRP treatment after IV administration.

[0269] AUC analysis (GraphPad Prism) of each model plot showed that AAV5 exposure to the treated kidney during the LRP treatment phase was approximately 200-fold higher when LRP administration was used compared to IV administration during the same period. In principle, the greater AAV5 exposure to the kidney obtained by LRP delivery would result in substantially greater kidney transduction than with IV administration. Furthermore, the large difference in systemic exposure when using these respective administration routes is expected to minimize transduction of other organs (e.g., liver or spleen) after direct administration to the kidney via LRP compared to IV administration. Given the known safety issues associated with AAV transduction of the liver, reducing transduction of non-target organs would have substantial safety implications.

[0270] 7.3 Biodistribution by kidney region (cortex, cone, and papilla) A small piece of each tissue was homogenized using metal beads in a TissueLyser II (Qiagen) and extracted using AllPrep DNA / RNA (Qiagen). DNA concentration and purity were measured using a NanoDrop spectrophotometer. Quantification of viral genomes within cells of tissues (vg / dg) was performed using ddPCR. A GFP probe was used to detect the viral genome in animals, whereas a β-actin probe was used for housekeeping genes.

[0271] Figure 47 is a plot showing the biodistribution analysis (vg / dg) measured in kidney sections compared to other organs in Animal 1, and Figure 48 is a plot showing the biodistribution analysis (vg / dg) measured in kidney sections compared to other organs in Animal 2 (where Tr = treated, Untr = untreated). In Animal 1, where AAV5 was administered to the left kidney via LRP, biodistribution analysis confirmed the robustness of the LRP system. Viral genomes were primarily detected in the treated kidney sections (left kidney), averaging 3.5 vg / dg (Figure 47). Untreated kidney, liver, and spleen remained highly detargeted (untreated kidney averaged 0.01 vg / dg, average of three liver samples was 0.05 vg / dg, and spleen was 0.76 vg / dg). In comparison, the biodistribution of AAV5 after IV administration in Animal 2 was substantially different. As expected from the significantly reduced AAV5 exposure to the kidney, kidney transduction after IV administration was extremely inefficient, with both the left and right kidneys exhibiting a similarly low mean viral genome of 0.2 vg / dg (Figure 48). Furthermore, as expected from the significantly increased systemic exposure after IV administration of AAV5, viral genomes in Animal 2 were significantly higher in the liver and spleen (1.4 vg / dg and 12 vg / dg, respectively).

[0272] To quantify the specificity of targeting kidney relative to transduction in non-target organs achieved by LRP administration of AAV5 compared with IV administration, the kidney-to-liver transduction ratios in Animal 1 and Animal 2 were calculated and are summarized in Table 3. Targeting specificity was calculated by comparing the kidney-to-liver ratios, calculated on a vg / dg basis, between Animal 1 and Animal 2. Targeting specificity, which takes into account differences in cell number per organ, was calculated using organ size as an estimate of the cell number in the corresponding organ.

[0273] In Animal 1, the kidney-to-liver transduction ratio was 63.7, while in Animal 2, the ratio was 0.14. Comparing these ratios indicates that LRP administration provides approximately 450-fold greater specificity for non-renal targeting compared with IV administration. Notably, because the liver is substantially larger in size than the kidney, this analysis provides an average transduction specificity at the cellular level without taking into account the substantially greater number of cells in the liver compared with the kidney. In humans, the volume of a non-diseased liver is approximately 12 times the volume of a non-diseased kidney (Nawaratne et al., "Relationships among liver and kidney volumes, lean body mass, and drug clearance," Br. J. Clin Pharmacol, 1998, Vol. 46, No. 5, 447-452). When comparing the relative volume of the human liver with the human kidney as an approximation of relative cell number, LRP administration of AAV5 provides approximately 5,400-fold greater specificity for kidney targeting compared with non-renal targeting compared with IV administration.

[0274] These findings demonstrate that AAV5 administration via the LRP provides the unexpected advantage of substantial transduction of the treated kidney, which is not achieved by administering the same dose via IV. Furthermore, AAV5 administration via the LRP results in substantial detargeting of the liver and spleen, providing potential safety benefits in addition to the potential therapeutic benefit of effective kidney transduction. [Table 3]

[0275] Example 8: Neutralizing AAV antibody production after tight versus leaky AAV administration via LRP to the kidneys of domestic pigs 8.1 Treatment Description and Administration The purpose of this analysis was to compare systemic AAV-neutralizing antibody formation after AAV administration via the renal LRP when a closed circuit was maintained compared to when substantial leakage was observed during LRP treatment. To make this comparison, animals (referred to as animals 3 and 4 in this example) were treated with AAV9, since systemic exposure to AAV9 is known to induce a relatively strong immune response. Animal 3 received 6.2 x 10 14 vg dose of AAV9-CMV-eGFP (administration time 1 h), and animal 2 received 1.7 × 10 15 Both animals underwent renal LRP treatment with a dose of AAV9-CMV-eGFP (vg) over a 2-hour administration period. Both animals were selected after immunological screening for pre-existing AAV9 antibodies and renal CT scans to confirm that the pigs were physiologically compatible with LRP treatment.

[0276] 8.2 Vector Shedding Analysis To confirm the efficacy of the LRP treatment, multiple blood samples were taken at different time points from the LRP circuit and peripheral blood (systemic circulation). Total viral genomes per mL of blood were measured using qPCR with a probe targeting the AAV GFP DNA sequence and titrated against a standard curve using linearized plasmid DNA. All samples were measured in duplicate.

[0277] To measure neutralizing antibodies against AAV9, 2V6.11 cells were seeded in 96-well white plates and incubated overnight with ponasterone A. The following day, serum samples from AAV9-treated pigs were serially diluted and incubated with AAV9 vectors containing a reporter gene for 1 hour, and the serum sample / vector mix was then added to 2V6.11 cells. After 24 hours of incubation, AAV9 transduction rates were assessed on lysed cells by adding a reporter substrate. Results are reported as the titer required to produce 50% inhibition of transduction. Positive and negative controls were included in the assay, and background signal was assessed using cells treated with medium alone.

[0278] The LRP circuit remained tight during treatment in Animal 1, with no systemic leakage and a maximum concentration of 1.7 × 10 in peripheral blood samples (systemic circulation). 8 In comparison, significant leakage of AAV9 into the peripheral blood was observed during LRP treatment in animal 2, with concentrations reaching 6.6 × 10 at 5 min of treatment. 9 vg / mL to a maximum of 1.2 × 10 during treatment 11 The blood glucose level rose to 1000 mg / mL.

[0279] Anti-AAV9 neutralizing antibody titers were measured on the day of AAV9 administration via kidney LRP (day 0) and on the day of sacrifice (day 15), and the results are summarized in Table 4. Both animals exhibited low levels of anti-AAV9 neutralizing antibodies before administration of AAV9 via kidney LRP. By day 15, the anti-AAV9 neutralizing antibody titer of Animal 4 had significantly increased to over 1 / 4096, the highest serum dilution evaluated. In contrast, the anti-AAV9 neutralizing antibody titer for Animal 3 remained low (1 / 32). Considering safety concerns related to immune responses to AAV, this analysis demonstrates the potential benefits of tight, closed-loop AAV administration via kidney LRP. Furthermore, the low systemic neutralizing antibody response observed in Animal 3 may allow for future rechallenge with the same AAV if administration is required. [Table 4]

[0280] Example 9: Promoters for use in therapeutic constructs 9.1 Exemplary Promoters Therapeutic constructs according to various embodiments of the present disclosure advantageously incorporate promoter sequences to increase expression activity in renal cells. Such promoters contemplated for use in combination with a GOI include, but are not limited to, the CMV promoter (508 bp, SEQ ID NO: 9), the CAG promoter (1733 bp, SEQ ID NO: 10), the NPHS2 promoter (2589 bp, SEQ ID NO: 11), the miniature NPHS2 promoter (265 bp, SEQ ID NO: 12), and KSP-derived promoters, such as the mKSP0.3 promoter (324 bp, SEQ ID NO: 13) and the mKSP1.3 promoter (1341 bp, SEQ ID NO: 14).

[0281] 9.2 Identification of the miniature human kidney-specific cadherin (Ksp-cadherin, cadherin 16) promoter, taking into account DNA sequence predictions for transcription factor binding sites In this example, transcription factor binding sites (TFBSs) were predicted based on the UCSC Genome Browser, which shows genome-wide predicted binding sites for transcription factor binding profiles in the JASPAR CORE database. This open-source database contains a curated, non-redundant set of binding profiles derived from a public collection of experimentally defined TFBSs for eukaryotes.

[0282] JASPAR 2018 used the TFBS Perl module (Lenhard and Wasserman, Bioinformatics, 2002, Vol. 18) and FIMO (Grant, Bailey, and Noble, Bioinformatics, 2011, Vol. 27) as distributed within the MEME suite (version 4.11.2) (Bailey et al., Nucleic Acids Res., 2009, Vol. 37). To scan the genome with the BioPerl TFBS module, profiles were converted to position weight matrices (PWMs) and matches with a relative score ≥ 0.8 were retained. For FIMO scans, profiles were reformatted into MEME motifs and matches with a p-value < 0.05 were retained. TFBS predictions that were inconsistent between the two methods (TFBS Perl module and FIMO) were removed. The remaining TFBS predictions were colored according to their FIMO p-values ​​to allow comparison of prediction reliability between different profiles. For JASPAR2020, DNA sequences were scanned with the JASPAR CORE TF binding profiles independently for each taxon using PWMScan. TFBS predictions with a PWM relative score ≥ 0.8 and a p-value < 0.05 were selected. For genome track coloring and to allow comparison of prediction reliability between different profiles, p-values ​​were scaled from 0 (corresponding to a p-value of 1) to 1000 (p-value ≤ 10-10). JASPAR2022 contains updated transcription factor binding sites along with additional transcription factor profiles.

[0283] The analysis considered the following transcription factors in the promoter design, which are known to regulate renal tubular cells. Examples include: (1) HNF-1β (hepatocyte nuclear factor 1β): HNF-1β is essential for the development and maintenance of renal tubular cells, especially the proximal tubules. Mutations in the HNF-1β gene are associated with renal cysts and diabetes syndrome (RCAD). (2) HNF-4α (hepatocyte nuclear factor 4α): HNF-4α is another important transcription factor involved in the regulation of renal tubular cells. It is expressed in the proximal tubules and plays a role in the differentiation and function of these cells. (3) Pax2 (paired box gene 2): Pax2 is a transcription factor important for the development of various kidney structures, including the renal tubule system. It is expressed early in kidney development and helps form and pattern different tubule segments. (4) Foxi1 (Forkhead box I1): Foxi1 is a transcription factor important for the development and function of the distal tubule and collecting duct in the kidney. It regulates the expression of various genes involved in electrolyte balance and acid-base regulation. (5) HNF-1α (Hepatocyte Nuclear Factor 1α): HNF-1α is expressed in both the proximal and distal tubules and plays a role in maintaining tubular integrity and function. Mutations in the HNF-1α gene are associated with a form of maturity-onset diabetes of the young (MODY), which can lead to renal insufficiency. (6) KLF5 (Kruppel-like factor 5): KLF5 is a transcription factor involved in the regulation of cell proliferation and differentiation in various tissues, including the kidney. It is expressed in renal tubular cells and is involved in kidney injury and repair processes. (7) Pax8 (paired box gene 8): Pax8 is a transcription factor expressed in the developing and mature kidney. It plays a role in the specification and differentiation of different tubule segments, including the proximal and distal tubules. (8) Eya1 (eye absent homolog 1): Eya1 is a transcription factor that forms a complex with Pax proteins, including Pax2 and Pax8. It is essential for metanephros development and regulates the expression of genes involved in renal tubule development and differentiation. (9) AQP2 (aquaporin 2): AQP2 is primarily known as a water channel protein, but its expression is regulated by transcription factors. Several transcription factors, including HNF-1β, CREB (cAMP response element binding protein), and NFAT (nuclear factor of activated T cells), are involved in the regulation of AQP2 expression in renal tubular cells. (10) Sp1 (specificity protein 1): Sp1 is a transcription factor that regulates the expression of numerous genes involved in various cellular processes. It is expressed in renal tubular cells and has been shown to regulate the expression of genes related to renal tubular function, such as aquaporins and transporters. (11) PPARγ (peroxisome proliferator-activated receptor gamma): PPARγ is a transcription factor that belongs to the nuclear hormone receptor superfamily. It is expressed in renal tubular cells and is involved in the regulation of lipid metabolism and inflammation. PPARγ activation has been shown to protect against kidney injury and fibrosis. (12) STAT3 (Signal Transducer and Activator of Transcription 3): STAT3 is a transcription factor activated in response to various signaling pathways, including cytokines and growth factors. It plays a role in renal tubular cell survival, regeneration, and inflammation. Activation of STAT3 is associated with protection against kidney injury. (13) FOXC1 (Forkhead box C1): FOXC1 is primarily expressed in the developing kidney during embryogenesis and continues to be expressed in various kidney structures, including renal tubular cells, throughout development. Its functions in renal tubular cells include epithelial cell differentiation, epithelial-mesenchymal transition (EMT), and ciliary development and function. (14) FOXC2 (Forkhead box C2): FOXC2 is also expressed in the developing kidney and continues to be expressed in various renal compartments, including renal tubular cells, where its functions include epithelial cell differentiation, cell migration and adhesion, epithelial polarity and tight junction formation, regulation of Wnt signaling, and lymphatic vessel development.

[0284] Based on the foregoing analysis, the following sequences were identified in the human genome (CDH16 candidate promoter region (GRCh38 / hg38 reference genome)): (Sequence A) chr16:66920195 to 66920275 (81 bp, SEQ ID NO: 15) (upstream promoter region with TFBS), (Sequence B) chr16:66920085 to 66920275 (191 bp, SEQ ID NO: 16) (extended upstream promoter region with TFBS to include conserved regions in vertebrates and mammals), (Sequence 1) chr16:66918886 to 66919090 (SEQ ID NO: 17, 205 bp) (minimal promoter starting at TSS containing mouse KSP blasting sequence), (Sequence 2) chr16:66918850 to 66919090 (SEQ ID NO: 18, 241 bp) (a minimal promoter extended downstream of the TSS to include a conserved region in vertebrates and particularly mammals), (Sequence 3) chr16:66918850 to 66919173 (SEQ ID NO: 19, 324 bp) (a minimal promoter extended upstream and downstream of the TSS to include conserved regions in vertebrates and particularly mammals), (Sequence 4) chr16: 66918850 to 66919344 (SEQ ID NO: 20, 495 bp) (a minimal promoter extended upstream and downstream from the TSS to include conserved regions in vertebrates and mammals (particularly the upstream extension)),

[0285] Table 5 summarizes the mini-KSP promoter candidates based on the identified sequences and their combinations: [Table 5]

[0286] Example 10: Construct Design Exemplary gene construct designs contemplated for expressing functional PC1 or PC2 protein subunits via local gene replacement to treat ADPKD are described below. Each construct, shown in Table 6 below, contains a gene cassette comprising a promoter operably linked to a GOI (encoding tdTomato or zsGreen fluorescent protein), an optimized post-transcriptional regulatory element (oPRE), and a terminator sequence (SEQ ID NO: 29), flanked by 5' and 3' ITR sequences.

[0287] Testing of the construct can be carried out using tdTomato or zsGreen fluorescent protein as a reporter. Once efficient protein expression is verified, the reporter gene can be exchanged with GOI (e.g., SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8), as will be readily understood by those skilled in the art. For further testing, the gene of interest can further encode an HA tag having the amino acid sequence of SEQ ID NO: 30, and the corresponding nucleic acid sequence is SEQ ID NO: 31. For example, one or more HA tag sequences can be included at the N-terminus of the therapeutic protein. [Table 6-1] [Table 6-2]

[0288] Example 11 (Prophetic): Cell-based screening to identify therapeutic constructs for ADPKD caused by mutations in PKD1 Cell-based assays can be used to identify constructs encoding the PC1 CTF or CTT subdomains that can provide sufficient levels of each PC1 subdomain to reduce or reverse the cellular phenotype observed in PC1-deficient cells. Initial screens can provide a rapid, cost-effective approach to identifying potential therapeutic constructs using transient transfection of a plasmid encoding a therapeutic GOI and an immortalized cell line. Subsequent experiments can be performed to vectorize the GOI in an AAV format, preferably AAV5. However, those skilled in the art will recognize the potential need to use other native AAV serotypes or AAVs within engineered capsids for in vitro experiments. GOI constructs that effectively generate PC1 subdomains that correct PC1 deficiency in immortalized cell lines can be evaluated in PC1-deficient organoids derived from hiPSCs.

[0289] 11.1 Screening in Immortalized Cell Lines In illustrative examples of in vitro systems, immortalized human, porcine, and canine kidney tubular epithelial cells (HK, LLC-PK1, and MDCK, respectively) can be used to identify constructs encoding functional PC1-CTF and CTT. Because ADPKD is a disease of renal tubular epithelial cells, those skilled in the art will recognize that many cell lines representing tubular regions of the nephron, including the proximal tubule, loop of Henle, distal tubule, connecting tubule, or collecting duct (e.g., inner medullary collecting duct cells), can be used to screen for constructs encoding PC1. Furthermore, because the function of polycystic cysts is evolutionarily conserved, cell lines derived from various species, including humans, mice, dogs, pigs, zebrafish, and the like, are suitable for screening for constructs encoding PC1. In parallel, PKD1 disease models can be generated by genome editing using CRISPR / Cas9 technology. Two genotypes will be obtained: (1) homozygous with the complete absence of PC1 protein (PKD1 ); - / - ), and (2) heterozygous with reduced amounts of PC1 protein (PKD1 + / - Homozygous PKD1 with sufficient depletion of PC1 protein - / - The cells can facilitate screening of therapeutic constructs based on evaluation of PKD1 CTF and CTT mRNA and PC1 CTF and CTT protein expression and target engagement. Heterozygous PKD1 cells harbor one PKD1 mutation, thus mimicking the situation of the human ADPKD gene. + / - The cell line can be used for further validation. Plasmids carrying the desired GOI can be designed as described in Example 10. GOIs carrying tags such as HA or FLAG can be prepared to easily distinguish them from endogenous PKD1 mRNA / PC1 protein.

[0290] Transient transfection experiments can be performed in wild-type and genome-edited cell lines using various DNA amounts and transfection techniques appropriate for the cell line of interest. Those skilled in the art will understand that each cell line may require the use of specific transfection reagents or approaches to achieve sufficient transfection levels for experimental evaluation. PKD1 CTF and CTT mRNA levels can be assessed by RT-qPCR using target-specific primer / probe sets and compared to wild-type levels (using PKD1 species-specific probes, if necessary). Protein levels can be assessed by Western blot with antibodies directed against PC1 CTF or CTT or peptide tags and compared to wild-type levels. Constructs providing expression levels ±50% of wild-type levels can be considered for subsequent functional analysis. If desired, experiments can be repeated using constructs encoding the CTF and CTT packaged in AAV capsids. In such experiments, multiplicities of infection (MOIs) ranging from 10,000 to 1,000,000 can be tested.

[0291] PC1 regulates proliferation, apoptosis, and cyst formation, as well as Ca 2+ Because they are involved in signal transduction and metabolic pathways, target engagement in 2D models can be defined by the influence / modulation of the GOI on these pathways. The effect of constructs encoding PC1 CTF and CTT on proliferation can be assessed in proliferation assays based on BrdU incorporation (or other more sensitive compounds, e.g., IdU / CldU, EdU) (Abcam / Thermofisher). Expression of sufficient amounts of PC1 CTF or CTT introduced by transfection or AAV-mediated transduction will reduce the level of proliferation compared to that observed in comparable PC1-deficient cells.

[0292] PC1 LoF causes increased apoptosis. The effect of constructs encoding PC1 CTF and CTT on apoptosis can be assessed using an apoptosis assay, such as a tunneling assay, or by measuring the level of cleaved caspase-3. Expression of sufficient amounts of PC1 CTF or CTT introduced by transfection or AAV-mediated transduction will reduce the level of cleaved caspase-3 or signal in the tunneling assay compared to the signal observed in control PC1-deficient cells.

[0293] Nitric oxide (NO) levels have been shown to be reduced in ADPKD cell lines in vitro, so the effect of constructs encoding PC1 CTF and CTT on NO production can be assessed by flow cytometry using a fluorescent NO indicator (DAF-2 / DA). Upon expression of sufficient amounts of PC1 CTF or CTT introduced by transfection or AAV-mediated transduction, NO levels will be increased compared to those observed in comparable PC1-deficient cells.

[0294] Because PC1 has been shown to regulate mitochondrial function and loss or reduction of PC1 levels leads to metabolic dysregulation as indicated by decreased oxidative phosphorylation and increased glycolysis, the effect of constructs encoding PC1 CTF and CTT on oxidative phosphorylation can be assessed in a seahorse assay. Expression of sufficient amounts of PC1 CTF or CTT introduced by transfection or AAV-mediated transduction will increase the level of oxidative phosphorylation and decrease the level of glycolysis when compared to that observed in control PC1-deficient cells.

[0295] MDCK cells are naturally deficient in PC1 and spontaneously form cysts under appropriate culture conditions (Boletta et al., Molecular Cell, 2000, Vol. 6). MDCK cells can be used to investigate the ability of constructs encoding PC1 CTF and CTT to reverse / prevent cyst growth as described by Boletta et al. Expression of sufficient amounts of PC1 CTF or CTT introduced by transfection or AAV-mediated transduction will reduce or eliminate the level of spontaneous cyst formation by MDCK cells.

[0296] 11.2 Screening in hiPSC-derived ADPKD kidney organoids Constructs encoding PC1 CTF and CTT that effectively reduce or reverse cellular phenotypes associated with loss or reduction of PC1 levels in the above-described assays can be further evaluated in PC1-deficient hiPSC-derived kidney organoids (Freedman et al., Nature Communications, 2015, Vol. 6). These hiPSC-derived kidney organoids provide a 3D model of human tissue composed of podocytes, proximal and distal tubules, and associated endothelium and mesenchyme. PC1-deficient hiPSC-derived organoids mimic some of the phenotypes seen in ADPKD patients, including metabolic dysregulation and cyst formation and growth.

[0297] Transient transfection experiments can be performed in wild-type and genome-edited hiPSCs using various DNA amounts and transfection techniques appropriate for organoids. PKD1 CTF and CTT mRNA levels can be assessed by RT-qPCR using target-specific primer / probe sets and compared to wild-type levels. Protein levels can be assessed by Western blot with antibodies directed against PC1 CTF or CTT or peptide tags and compared to wild-type levels. Constructs providing expression levels ±50% of wild-type levels can be considered for subsequent functional analysis. If desired, experiments can be repeated using constructs encoding the CTF and CTT packaged in AAV capsids. In such experiments, MOIs ranging from 10,000 to 1,000,000 can be tested.

[0298] Because polycystic leukemia has been shown to regulate mitochondrial function and loss or reduction of PC1 levels leads to metabolic dysregulation, as indicated by decreased oxidative phosphorylation and increased glycolysis, the effects of constructs encoding PC1-CTF and CTT on oxidative phosphorylation can be assessed using a seahorse assay. Expression of sufficient amounts of PC1 CTF or CTT in PC1-deficient hiPSC-derived kidney organoids, introduced by transfection or AAV-mediated transduction, will increase the level of oxidative phosphorylation and decrease the level of glycolysis compared to that observed in control PC1-deficient organoids.

[0299] PC1-deficient hiPSC-derived kidney organoids spontaneously form cysts, which grow over time in culture (Freedman et al., Nature Communications, 2015, Vol. 6; Cruz et al., Nature Materials, 2017, Vol. 16). The effect of constructs encoding PC1-CTF and CTT on spontaneous cyst formation will be assessed by scoring the number, volume, and other metrics of cyst formation. Expression of sufficient amounts of PC1 CTF or CTT in PC1-deficient hiPSC-derived kidney organoids by transfection or AAV-mediated transduction will reduce the number and volume of formed cysts compared to the levels observed in control PC1-deficient cells. Depending on the efficiency of transfection or transduction, markers can be introduced into the PC1-encoding construct to identify transfected / transduced cells for analysis. In this case, a comparison of the number and volume of cysts in transfected / transduced versus non-transfected / transduced cells can also be assessed.

[0300] Example 12 (Prophetic): Cell-based screening to identify therapeutic constructs for ADPKD caused by mutations in PKD2 Cell-based assays can be used to identify constructs encoding PC2 that can provide sufficient levels of PC2 to reduce or reverse the cellular phenotypes observed in PC2-deficient cells. Initial screens can provide a rapid, cost-effective means of identifying potential therapeutic constructs using transient transfection of a plasmid encoding a therapeutic GOI and an immortalized cell line. Subsequent experiments will involve vectorizing the GOI in an AAV format, preferably AAV5. However, those skilled in the art will recognize the potential need to use other native AAV serotypes or AAV in engineered capsids for in vitro experiments. GOI constructs that effectively generate PC2 to correct PC2 deficiency in immortalized cell lines can be evaluated in PC1-deficient organoids derived from hiPSCs.

[0301] 12.1 Screening in Immortalized Cell Lines In illustrative examples of in vitro systems, immortalized human, porcine, and canine kidney tubular epithelial cells (HK, LLC-PK1, and MDCK, respectively) can be used to identify constructs encoding functional PC2. Because ADPKD is a disease of renal tubular epithelial cells, those skilled in the art will recognize that many cell lines representing tubular regions of the nephron, including the proximal tubule, loop of Henle, distal tubule, connecting tubule, or collecting duct (e.g., inner medullary collecting duct cells), can be used to screen for constructs encoding PC2. Furthermore, because the function of polycystic cysts is evolutionarily conserved, cell lines derived from various species, including humans, mice, dogs, pigs, zebrafish, and the like, are suitable for screening for constructs encoding PC2. In parallel, PKD2 disease models can be generated by genome editing using CRISPR / Cas9 technology. Two genotypes will be obtained: (1) homozygous with the complete absence of PC2 protein (PKD2 ); - / - ), and (2) heterozygous with reduced amounts of PC2 protein (PKD2 + / - Homozygous PKD2 with sufficient PC2 protein depletion - / -The cells can facilitate screening of therapeutic constructs based on evaluation of PKD2 mRNA and PC2 protein expression and target engagement. Heterozygous PKD2 cells harbor one PKD2 mutation, thus mimicking the situation of the human ADPKD gene. + / - The cell line can be used for further validation. Plasmids carrying the desired GOI will be designed as described in Example 10. GOIs carrying tags such as HA or FLAG can be prepared to easily distinguish them from endogenous PKD2 mRNA / PC2 protein.

[0302] Transient transfection experiments can be performed in wild-type and genome-edited cell lines using various DNA amounts and transfection techniques appropriate for the cell line of interest. Those skilled in the art will understand that each cell line may require the use of specific transfection reagents or approaches to achieve sufficient transfection levels for experimental evaluation. PKD2 mRNA levels can be assessed by RT-qPCR using a target-specific set of primers / probes and compared to wild-type levels (using a PKD2 species-specific probe, if necessary). Protein levels can be assessed by Western blot with antibodies directed against PC2 (sc-28331) or tags (H3663, F7425) and compared to wild-type levels. Constructs that provide expression levels ±50% of wild-type levels can be considered for subsequent functional analysis. If desired, experiments can be repeated using a construct encoding PC2 packaged in an AAV capsid. In such experiments, MOIs ranging from 10,000 to 1,000,000 can be tested.

[0303] PC2 interacts with PC1 and mediates PC1 maturation, apoptosis, metabolic control, cyst formation, and Ca 2+Because PC1 is involved in signal transduction, target engagement in 2D models can be defined by the influence / modification of the GOI on these pathways. In the absence of PC2, PC1 processing (e.g., cleavage at GPS) and export from the ER are impaired. The effect of constructs encoding PC2 on PC1 maturation and export can be assessed by Western blot to measure PC1 expression and IF to monitor PC1 cellular localization. Expression of sufficient amounts of PC2, introduced by transfection or AAV-mediated transduction, will increase PC1 levels compared to those observed in control PC2-deficient cells. The PC1 present will include full-length PC1 as well as PC1 cleaved at GPS to generate the NTF and CTF subdomains of PC1. Furthermore, in cells expressing sufficient amounts of PC2, PC1 is present at the plasma membrane and, if present, in cilia. PC1 cleavage products, such as CTT, may also be present in the nucleus or mitochondria.

[0304] PC2 Loss of Function (LOF) leads to increased apoptosis. The effect of a construct encoding PC2 on apoptosis can be assessed using an apoptosis assay, such as a tunneling assay, or by measuring the level of cleaved caspase-3. Expression of sufficient amounts of PC2, introduced by transfection or AAV-mediated transduction, will reduce the level of cleaved caspase-3 or signal in the tunneling assay compared to the signal observed in control PC2-deficient cells.

[0305] Because PC2 has been shown to regulate mitochondrial function and loss or reduction of PC2 levels leads to metabolic dysregulation as indicated by decreased oxidative phosphorylation and increased glycolysis, the effect of a construct encoding PC2 on oxidative phosphorylation can be assessed in a seahorse assay. Upon expression of sufficient amounts of PC2 introduced by transfection or AAV-mediated transduction, levels of oxidative phosphorylation will increase and levels of glycolysis will decrease when compared to those observed in control PC2-deficient cells.

[0306] PC2 is a Ca channel homologous to the transient receptor potential family of cation channels. 2+ It functions as a permeable, nonselective cation channel. Loss or reduction of PC2 levels results in Ca 2+ Ca in the ER, resulting in dysregulation of Ca 2+ Ca in the ER 2+ The effect of constructs encoding PC2 on calcium can be assessed in a fluorometric assay using a calcium indicator. Expression of sufficient amounts of PC2, introduced by transfection or AAV-mediated transduction, can increase Ca in the ER. 2+ The levels of will be reduced when compared to those observed in comparable PC2-deficient cells.

[0307] 12.2 Screening in hiPSC-derived ADPKD kidney organoids Constructs encoding PC2 that effectively reduce or reverse cellular phenotypes associated with loss or reduction of PC2 levels in the assays described above can be further evaluated in PC2-deficient hiPSC-derived kidney organoids (Freedman et al.). These hiPSC-derived kidney organoids provide a 3D model of human tissue composed of podocytes, proximal and distal tubules, and associated endothelium and mesenchyme. PC2-deficient hiPSC-derived organoids mimic some of the phenotypes seen in ADPKD patients, including metabolic dysregulation and cyst formation and growth.

[0308] Transient transfection experiments can be performed in wild-type and genome-edited hiPSCs using various DNA amounts and transfection techniques appropriate for organoids. PKD2 mRNA levels can be assessed by RT-qPCR using a target-specific primer / probe set and compared to wild-type levels. Protein levels can be assessed by Western blot with antibodies directed against PC2 or its peptide tag and compared to wild-type levels. Constructs providing expression levels ±50% of wild-type levels can be considered for subsequent functional analysis. If desired, experiments can be repeated using a construct encoding PC2 packaged in an AAV capsid. In such experiments, MOIs ranging from 10,000 to 1,000,000 can be tested.

[0309] Because PC2 interacts with PC1 and is involved in PC1 maturation, the effect of constructs encoding PC2 on PC1 maturation and trafficking can be assessed by Western blot to measure PC1 expression and IF to monitor PC1 cellular localization. Expression of sufficient amounts of PC2 in PC2-deficient hiPSC-derived kidney organoids, introduced by transfection or AAV-mediated transduction, will result in increased PC1 levels compared to those observed in control PC2-deficient organoids. The PC1 present will include full-length PC1 as well as PC1 cleaved by GPS to generate the PC1 NTF and CTF. Furthermore, in cells expressing sufficient amounts of PC2, PC1 is present in the plasma membrane and, if present, in cilia. PC1 cleavage products, such as CTT, may also be present in the nucleus or mitochondria.

[0310] Because polycystic leukemia has been shown to regulate mitochondrial function and loss or reduction of PC2 levels leads to metabolic dysregulation as indicated by decreased oxidative phosphorylation and increased glycolysis, the effect of a construct encoding PC2 on oxidative phosphorylation can be assessed using a seahorse assay. Upon sufficient expression of PC2 in PC2-deficient hiPSC-derived kidney organoids introduced by transfection or AAV-mediated transduction, the level of oxidative phosphorylation will increase and the level of glycolysis will decrease compared to the levels observed in control PC2-deficient organoids.

[0311] Loss or reduction of PC2 levels is associated with Ca 2+ Ca in the ER, resulting in dysregulation of Ca 2+ This leads to an imbalance of Ca in the ER. 2+ The effect of constructs encoding PC2 on calcium levels can be assessed in a fluorometric assay using calcium indicators. Expression of sufficient amounts of PC2 in PC2-deficient hiPSC-derived kidney organoids, introduced by transfection or AAV-mediated transduction, allows for the expression of calcium in the ER. 2+ The levels of will be reduced when compared to those observed in control PC2-deficient organoids.

[0312] PC2-deficient hiPSC-derived kidney organoids spontaneously form cysts, which grow over time in culture (Freedman et al., Nature Communications, 2015, Vol. 6; Cruz et al., Nature Materials, 2017, Vol. 16). The effect of a PC2-encoding construct on spontaneous cyst formation can be assessed by scoring the number, volume, and other metrics of cyst formation. Expression of sufficient amounts of PC2 in PC2-deficient hiPSC-derived kidney organoids, introduced by transfection or AAV-mediated transduction, will reduce the number and volume of formed cysts compared to the levels observed in control PC2-deficient cells. Depending on the efficiency of transfection or transduction, a marker can be introduced into the PC2-encoding construct to identify transfected / transduced cells for analysis. In this case, a comparison of the number and volume of cysts in transfected / transduced cells versus untransfected / transduced cells can also be assessed.

[0313] Example 13 (Prophetic): Evaluation of ADPKD Gene Therapy in a Mouse Model of ADPKD Constructs that demonstrate endogenous PC1 or PC2 expression levels ±50%, molecular evidence of pathway engagement, and evidence of reversal or prevention of cellular phenotypes consistent with ADPKD pathology in immortalized 2D and 3D and iPSC-derived 2D cell and 3D organoid models as described in Examples 11 and 12 can be further evaluated in mouse models of ADPKD as follows.

[0314] 13.1 AAV Serotypes and Doses Multiple AAVs can be used to determine the most potent AAV for kidney targeting. This includes AAV peptide display libraries of all major AAV serotypes from AAV1 to AAV13 and the parent serotypes mentioned above, which can be used here to determine lead AAV candidates in terms of kidney tissue specificity and transduction efficiency. Preferably, AAV5 will be considered as one of the major serotypes used based on the study described in Example 4. High and low doses of vectors containing therapeutic constructs and reporter genes can be administered at a dose of 1 × 10 per mouse. 10 ~2×10 14 A range of vector genomes (vg) can be administered in mouse models. It is contemplated that the preferred AAV for kidney transduction may differ depending on the route of administration, and that the use of surrogate serotypes (i.e., serotypes different from the preferred serotypes for human therapeutics delivered via LRP) may be necessary to evaluate therapeutic AAV constructs in mice.

[0315] 13.2 Route of AAV Administration Mice will be followed up from 1 to 24 weeks post-administration. Phenotypic analysis and molecular characterization of the therapeutic construct will be assessed at each time point during the study. Multiple administration routes, including systemic and direct kidney delivery, can be investigated for kidney tissue and kidney cell transduction. Tail vein, facial vein, femoral vein, retro-orbital, jugular vein, and intraperitoneal injections can be used to administer AAV systemically. For direct delivery to the kidney, renal artery, renal vein, intraparenchymal, and ureteral injections (i.e., retroureteral route) can be performed. Clamping for 5 to 45 minutes after renal vein injection can be used to enhance transduction.

[0316] The pathways for AAV delivery to the kidney are summarized in Davis and Park et al., “Gene therapy research for kidney diseases,” Physiol.Genomics 2019 Vol.51(9), 449-461.

[0317] Example 14 (Prophetic): Evaluation of Therapeutic AAV Constructs for Treatment of PKD1 ADPKD This example shows the conditional PKD1-KO mouse model of ADPKD (Pkd1 fl / fl; Pax8 rtTA We used a conditional knockout of PC1 (Tet-Cre) gene (Ma et al., Nature Genetics 2013, Vol. 45). Inactivation of PC1 expression was induced with 2 mg / ml doxycycline in drinking water supplemented with 3% sucrose for 2 weeks from P28 to P42. This resulted in complete systemic loss of PC1 expression in renal epithelial cells and the development of polycystic kidney disease (PKD) as assessed by MRI measurements of kidney weight / body weight (kw / bw%), blood urea nitrogen level (BUN), serum creatinine, kidney volume, and histological evaluation of the kidneys after sacrifice. Disease onset is progressive, with substantially enlarged kidneys and concomitant loss of kidney function observed by 13 weeks after PC1 conditional knockout, and progression continues through 16 and 24 weeks. For these experiments, group sizes were 12–15 mice per group, and the effects of gene therapy treatments were evaluated at various time points.

[0318] 14.1 Evaluation in Prevention Models of ADPKD For evaluation of the constructs in preventive models of disease, AAV constructs encoding PC1 CTF or CTT were administered at 1 × 10 per mouse. 10 ~2×10 14A total dose ranging from 0.01 to 0.01 vg may be administered to P1 pups via retroorbital injection. Littermates not treated with doxycycline may serve as a positive control (e.g., wild-type mice), and doxycycline-induced, non-AAV-treated littermates may serve as comparators. Multiple individuals from each group may be included in the study to allow for evaluation at 10, 13, 16, and 19 weeks of age. In-life evaluations include measurement of BUN, serum creatinine, and kidney volume by MRI. Post-sacrifice evaluations include kw / bw% and histological evaluation of the kidneys. Expression of CTF or CTT in the kidney by transduction with AAV encoding the respective transgenes is expected to effectively delay or prevent the progression of ADPKD after conditional knockout of PKD1, as demonstrated by reduced kw / bw%, BUN levels, serum creatinine levels, and kidney volume in treated versus untreated mice. The effect is dose-dependent, with results at the highest dose of AAV administered approaching those observed in wild-type mice. Differences between treatment and control groups are expected to be negligible at 10 weeks and evident at 13, 16, and 19 weeks. Kidneys from treated mice are also expected to contain substantially fewer and smaller cysts than untreated mice, as assessed by histology.

[0319] 14.2 Evaluation in Treatment Models of ADPKD For evaluation of the constructs in treatment models of disease, AAV constructs encoding PC1 CTF or CTT were administered at 1 x 10 per mouse. 10 ~2×10 14The total dose may be administered at 10 weeks, preferably by systemic route or by direct delivery to the kidney, in the range of 0.5 mg / kg. It is contemplated that the route of administration may vary depending on the AAV serotype used, and that administration at 10 weeks will allow expression of the CTF or CTT protein by 13 weeks. Littermates not treated with doxycycline may serve as positive controls (e.g., wild-type mice), and doxycycline-induced, non-AAV-treated littermates may serve as comparators. Multiple individuals from each group may be included in the study to allow evaluation at 13, 16, and 19 weeks of age. In-life assessments include measurement of BUN, serum creatinine, and kidney volume by MRI. Post-sacrifice assessments include kw / bw% and histological evaluation of the kidneys. Expression of CTF or CTT in the kidney by transduction with AAV encoding the respective transgenes is expected to effectively delay or reverse the progression of PKD after conditional knockout of PKD1, as demonstrated by reduced kw / bw%, BUN levels, serum creatinine levels, and kidney volume in treated versus untreated mice. The effect is dose-dependent, with results at the highest dose of AAV administered approaching those observed in wild-type mice. Differences between the treated and control groups are expected to be evident at 13, 16, and 19 weeks. Kidneys from treated mice are also expected to contain substantially fewer and smaller cysts than untreated mice, as assessed by histology.

[0320] In both preventative and treatment models, transduction and biodistribution assessment of transgene expression can be performed as follows.

[0321] 14.3 Vector genome quantification and biodistribution Total genomic DNA can be extracted from mouse tissue samples after sacrifice. The number of vector genomes in cells can be assessed using ddPCR. Vector genomes can be detected using designed primer-probe sets for CTT, CTF, or AAV ITRs and measured against a housekeeping gene (i.e., a primer-probe set for beta-actin).

[0322] Quantification of vector genomes in the kidney in combination with phenotypic analysis may allow determination of the dose range required to achieve therapeutic efficacy.

[0323] 14.4 Expression analysis by relative mRNA quantification and immunoblotting Total RNA can be isolated from kidney tissue, and RT-qPCR can be used to determine the relative concentration of mRNA encoding CTT or CTF relative to housekeeping genes (PolR2A or GAPDH) using specific primer probe sets.

[0324] Total protein can be isolated from homogenized and lysed tissues, and Western blots can be performed using recommended dilutions of anti-CTT, anti-CTF, and anti-HA monoclonal antibodies.

[0325] An increase in the specific mRNA expressed from the corresponding construct is expected. The full size of the CTT or CTF protein, with or without the HA tag, is also expected in the treated tissue samples.

[0326] Example 15 (Prophetic): Evaluation of Therapeutic AAV Constructs for Treatment of PKD2 ADPKD This example shows the conditional pkd2-KO mouse model of ADPKD (Pkd2 fl / fl; Pax8 rtTA; Tet-Cre) was used (Ma et al., Nature Genetics 2013, Vol. 45). Inactivation of PC2 expression was induced with 2 mg / ml doxycycline in drinking water supplemented with 3% sucrose for 2 weeks from P28 to P42. This resulted in complete systemic loss of PC2 expression in renal epithelial cells and the development of polycystic kidney disease, as assessed by MRI measurements of kidney weight / body weight % (kw / bw), blood urea nitrogen level (BUN), serum creatinine, kidney volume, and histological evaluation of the kidneys after sacrifice. Disease development was progressive, with substantially enlarged kidneys and concomitant loss of kidney function observed by 13 weeks after PC2 conditional knockout, and progression continued through 16 and 24 weeks. For these experiments, group sizes were 12–15 mice per group, and the effects of gene therapy treatment were evaluated at various time points.

[0327] 15.1 Evaluation in Prevention Models of ADPKD For evaluation of constructs in preventive models of disease, AAV constructs encoding PC2 or PC1 CTF or CTT were administered at 1 × 10 per mouse. 10 ~2×10 14A total dose ranging from 0.01 to 0.01 vg may be administered to P1 pups via retroorbital injection. Littermates not treated with doxycycline may serve as a positive control (e.g., wild-type mice), and doxycycline-induced, non-AAV-treated littermates may serve as comparators. Multiple individuals from each group may be included in the study to allow for evaluation at 10, 13, 16, and 19 weeks of age. In-life evaluations include measurement of BUN, serum creatinine, and kidney volume by MRI. Post-sacrifice evaluations include kw / bw% and histological evaluation of the kidneys. Expression of PC2 or PC1 CTF or CTT in the kidney by transduction with AAV encoding the respective transgenes is expected to effectively delay or prevent the progression of ADPKD after conditional knockout of PKD2, as demonstrated by reduced kw / bw%, BUN levels, serum creatinine levels, and kidney volume in treated versus untreated mice. The effect is dose-dependent, with results at the highest dose of AAV administered approaching those observed in wild-type mice. Differences between treated and control groups are expected to be negligible at 10 weeks and evident at 13, 16, and 19 weeks. Kidneys from treated mice are also expected to contain substantially fewer and smaller cysts than untreated mice, as assessed by histology.

[0328] 15.2 Evaluation in Treatment Models of ADPKD For evaluation of the constructs in treatment models of disease, AAV constructs encoding PC2 or PC1 CTF or CTT were administered at 1 x 10 per mouse. 10 ~2×10 14Total doses in the range of 10 ... Expression of PC2 or PC1 CTF or CTT in the kidney by transduction with AAV encoding the respective transgene is expected to effectively delay or reverse the progression of PKD after conditional knockout of PKD2, as demonstrated by reduced kw / bw%, BUN levels, serum creatinine levels, and kidney volume in treated versus untreated mice. The effect is dose-dependent, with results at the highest dose of AAV administered approaching those observed in wild-type mice. Differences between treated and control groups are expected to be evident at 13, 16, and 19 weeks. Kidneys from treated mice are also expected to contain substantially fewer and smaller cysts than untreated mice, as assessed by histology.

[0329] In both preventative and treatment models, transduction and biodistribution assessment of transgene expression can be performed as follows.

[0330] 15.3 Vector genome quantification and biodistribution Total genomic DNA can be extracted from mouse tissue samples after sacrifice. The number of vector genomes in cells can be assessed using ddPCR. Vector genomes can be detected using designed primer-probe sets for PKD2, PKD1 CTT, PKD1 CTF, or AAV ITR, and can be measured against a housekeeping gene (i.e., a primer-probe set for beta-actin).

[0331] Quantification of vector genomes in the kidney in combination with phenotypic analysis may allow determination of the dose range required to achieve therapeutic efficacy.

[0332] 15.4 Expression analysis by relative mRNA quantification and immunoblotting Total RNA can be isolated from kidney tissue, and RT-qPCR can be used to determine the relative concentrations of mRNA encoding PC2 or PC1 CTT or CTF relative to housekeeping genes (PolR2A or GAPDH) using specific primer probe sets.

[0333] Total protein can be isolated from homogenized and lysed tissues, and Western blots can be performed using recommended dilutions of anti-PC2, anti-CTT, anti-CTF, and anti-HA monoclonal antibodies.

[0334] An increase in the specific mRNA expressed from the corresponding construct is expected. The full size of the PC2 or PC1 CTT or CTF protein, with or without the HA tag, is also expected in the treated tissue samples.

[0335] Example 16 (Prophetic): Evaluation of AAV constructs for ADPKD gene therapy in healthy and ADPKD-affected pigs Constructs that demonstrate endogenous PC1 or PC2 expression levels ±50%, molecular evidence of pathway engagement, or evidence of restoration or prevention of a cellular phenotype consistent with ADPKD pathology as described in Examples 11 and 12, and that can prevent, slow, or reverse disease progression in a mouse model of ADPKD as described in Examples 13-15, can be further evaluated in pigs as follows.

[0336] Pigs can be used to evaluate the safety of LRP in combination with ADPKD gene therapy to support evaluation of exemplary embodiments in first-in-human clinical studies. Potential therapeutic constructs can therefore be evaluated in pigs to identify those suitable for IND-enabling trials and further development for ADPKD caused by mutations in the PKD1 or PKD2 genes.

[0337] For constructs intended to treat ADPKD caused by mutations in the PKD1 gene, additional studies can be performed in a PKD1-deficient pig model to evaluate the efficacy of the combination product in a large animal model of the disease.

[0338] 16.1 Evaluation of PKD1 and PKD2 AAV constructs in healthy pigs For a construct intended to treat ADPKD caused by mutations in PKD1, the purpose of this example is to evaluate AAV gene therapy dose versus transduction efficiency and expression when delivered via the renal LRP to (1) aid in the selection of development candidates, (2) determine a dose range for potential evaluation in nonclinical safety studies, (3) determine a dose range for potential evaluation in a porcine model of PKD1-deficient ADPKD, and (4) determine a dose range for potential evaluation in humans.

[0339] For a construct intended to treat ADPKD caused by mutations in PKD2, the purpose of this example is to evaluate AAV gene therapy dose versus transduction efficiency and expression when delivered via the renal LRP to (1) aid in the selection of development candidates, (2) determine a dose range for potential evaluation nonclinical safety studies, and (3) determine a dose range for potential evaluation in humans.

[0340] Healthy pigs, such as Sus scrofa domesticus, adult Yucatan minipigs, or other pigs weighing 50-90 kg to allow for the use of LRP catheter components sized appropriately for use in humans, may be used in these studies (i.e., particularly the arterial supply and venous return perfusion catheters).

[0341] 16.2 AAV Serotypes and Doses The various AAV doses for the constructs encoding PKD1 CTF and CTT and for PKD2 were determined to be 1×10 10 vg~2×10 15 A range of 1000 mg / kg of AAV can be administered to healthy pigs. Multiple AAV variants can be tested, including the major serotypes AAV1-AAV13, preferably AAV5, because it has been experimentally demonstrated to be efficient for LRP-mediated gene delivery to the kidney in pigs (see Example 4) and to provide transduction of various cells in which cysts develop in ADPKD. Additionally, other synthetic AAVs engineered for kidney delivery or demonstrated by us and others to be efficient vectors in the kidney can be tested for efficient kidney transduction.

[0342] It is contemplated that this set of experiments will allow for the determination of highly or most efficient constructs and AAV variants based on transduction and expression data, which can be addressed by the studies described below. Effective vector, dose, and construct combinations are expected to provide sufficient expression of PC1 CTF or CTT or PC2 and, in the case of PC1-CTF or CTT, alleviate symptoms and achieve therapeutic benefit in the PKD1 pig model.

[0343] 16.3 Treatment Pigs for this study may be preselected based on (1) immunological screening for the AAV variant to be used in the study and (2) CT scans to determine optimal physiological compatibility with the LRP procedure. Constructs encoding PKD1 CTF or CTT and PKD2, encapsulated in AAV5, as described in Example 10, or alternative AAV serotypes suitable for administration via renal LRP, may be administered to one or more pigs as described in Example 5. Animals will be followed for 2 to 16 weeks after administration. In-life assessments, which may include blood chemistry parameters, indicators of kidney injury biomarkers, and expression of PC1-CTF, CTT, or PC2 in urinary exosomes, may be periodically assessed as described below. Post-sacrifice assessments, which may include biodistribution quantification, histological assessment for safety and cell-specific transgenic protein expression, and single-cell RNASeq and ATAC-seq to quantify levels of expression and percentage of transduced target cells, may be assessed as described below.

[0344] 16.4 Blood Chemistry Parameters Blood samples may be collected to assess concentrations of blood creatinine (CRE), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase, among other parameters.

[0345] When the constructs are administered to healthy pigs, general blood parameters can be evaluated and compared to untreated animals. When the PKD1 CTF and CTT constructs are administered to PKD1 diseased pigs, general blood parameters can be evaluated compared to healthy wild-type animals, and improvements in kidney function measures (e.g., BUN, serum creatinine) are contemplated for treated versus untreated animals.

[0346] 16.5 Markers of Kidney Injury in Serum and Urine Samples Kidney injury markers such as kidney injury molecule-1 (KIM-1), cystatin C (CysC), and neutrophil gelatinase-associated lipocalin (NGAL) can be measured using ELISA kits.

[0347] No changes in these biomarkers are expected with administration of a PKD1 or PKD2 therapeutic construct to healthy pigs. When a PKD1 CTF or CTT therapeutic construct is administered to a PKD1 diseased pig, lower amounts of the markers can be expected in treated versus untreated diseased animals.

[0348] 16.6 Monitoring PC1 and PC2 Urinary Exosomes as Biomarkers for Transgene Expression Hogan et al., “Identification of Biomarkers for PKD1 Using Urinary Exosomes,” J. Am. Soc. Nephrol. 2015, 26(7), 1661-70, described a test for measuring the urinary exosomal PC1 / TMEM2 or PC2 / TMEM2 ratio for diagnosing and monitoring polycystic kidney disease.

[0349] Urine samples can be collected from all pigs before and after payload administration and tracked until slaughter. Crude exosomes can be isolated by one-step ultracentrifugation. Specific urinary exosome-like vesicles (ELVs), known to contain PC1 or PC2, can be isolated by running a 5-30% sucrose-DO gradient. SDS-PAGE, section recovery, and MS / MS label-free proteomics can be used to determine peptide intensities in collected samples, in addition to quantitative Western blots using specific probes for PC1, PC2, and TMEM2, as described in the previously mentioned studies.

[0350] Therapeutic constructs can be identified based on their ability to provide a dose-dependent increase in the PC1 and PC2 / TMEM2 ratios in healthy pigs. In PKD1-affected pigs, it is contemplated that effective constructs will demonstrate an increase in PC1 / TMEM and / or PC2 / TMEM when compared to untreated animals.

[0351] 16.7 Vector Genome Quantification and Biodistribution Total genomic DNA can be extracted from pig tissue samples after slaughter. The number of vector genomes in cells can be assessed using ddPCR. Vector genomes can be detected using designed primer-probe sets for PKD1 CTF or CTT, PKD2, or AAV ITR, and measured against a housekeeping gene (i.e., a primer-probe set for beta-actin).

[0352] Quantification of vector genomes in pig kidneys is expected to allow determination of appropriate doses to achieve therapeutic benefit.

[0353] 16.8 Expression analysis by relative mRNA quantification and immunoblotting Total RNA can be isolated from kidney tissue, and RT-qPCR can be used to determine the relative concentrations of PKD1 mRNA, encoding the CTF or CTT, or PKD2 mRNA relative to housekeeping genes (PolR2A or GAPDH) using specific primer-probe sets.

[0354] Total protein can be isolated from homogenized and lysed tissues, and Western blot or ELISA assays can be performed using recommended dilutions of anti-PC1 CTF, anti-PC1 CTT, anti-PC2, and anti-HA monoclonal antibodies.

[0355] An increase in the specific mRNA expressed from the corresponding construct is expected. It is contemplated that full-sized PC1 CTT or CTF protein, or PC2 protein, with or without the HA tag, can be expected in treated tissue samples. An increase in PC1 CTT and CTF protein is expected in the PKD1 model.

[0356] 16.9 scRNAs-seq and scATAC-seq Analysis Single-cell RNA sequencing (scRNAseq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) can be performed on both treated and untreated porcine kidney tissue. Nuclei isolation can be performed on freshly flash-frozen kidney tissue. Here, cryopreserved tissue is dissociated into a single-cell suspension, followed by cell count and viability determination to ensure a sufficient number of cells / nuclei with acceptable cell viability (preferably ≥70%). Dead cells are removed before library preparation. Library generation and dead cell removal for each sequencing method can be performed using specific kits, such as those recommended by 10x Genomics® and others. Data can be processed and analyzed using 10x Genomics® Cell Ranger™ and Loupe™ Cell Browser.

[0357] The generated data set may allow for comparable clustering of treated and untreated kidney tissues. Each cluster may be determined based on differential gene expression of a subset of cells. This may then allow for the identification of the precise cellular targets transduced by the exemplary vectors and follow-up of any transcriptomic changes caused by the exemplary constructs.

[0358] It is contemplated that these analyses will demonstrate expression of PKD1 mRNA or PC2 mRNA encoding a CTF or CTT in ≥25% of renal tubular epithelial cells, which would be sufficient to significantly delay cyst progression in humans or animals lacking PC1 or PC2. It is also anticipated that minimal or no expression of these transgenes will be detectable by these methodologies in samples from untreated kidneys.

[0359] 16.10 Histological Evaluation After euthanasia of the anesthetized pigs, kidneys from treated and untreated pigs were collected. The kidneys were cut to cover all major sections, including the cortical, pyramidal, and ampullary sections. Kidney tissues were fixed in paraformaldehyde and embedded in paraffin or OCT. PC1 CTT or CTF was detected using anti-CTT, anti-CTF, and anti-HA antibodies, and PC2 protein was detected using anti-PC2 or anti-HA antibodies for immunohistochemistry (IHC) of paraffin sections or immunofluorescence of OCT samples.

[0360] For IHC, paraffin sections can be subjected to Masson's Trichrome staining and then incubated overnight at 4° C. with the recommended dilution of antibody. Sections can be stained with Histofine Max PO and DAB chromogen after washing off excess antibody. After staining with a counterstain such as hematoxylin, slides can be visualized by microscopy.

[0361] For IF, other markers may be included to co-stain for specific types of cells shown in Table 6 that will be visualized by fluorescence microscopy. [Table 7]

[0362] It is contemplated that these methods will be able to detect increases in PC1 CTT, CTF, and PC2 in treated renal tubular epithelial cells, as well as total spotted proteins of PC1 CTT, CTF, and PC2 compared to untreated groups.

[0363] 16.11 Evaluation of PKD1 AAV constructs in the PKD1 porcine model of ADPKD The goal of this study was to evaluate constructs encoding PKD1 CTF and CTT in a large animal model of ADPKD caused by mutations in PKD1 suitable for treatment with LRP-mediated delivery of AAV therapeutic constructs.

[0364] This study was conducted using the PKD1 pig animal model (i.e., PKD1) described by Watanabe et al., “Generation of heterozygous PKD1 mutant pigs exhibiting early-onset renal cyst formation,” Lab Invest. 2022, 102(5), 560-569. insG / + ), or other porcine models of ADPKD when they become available.

[0365] The dose of the construct encoding the PKD1 CTF or CTT to be administered will be determined based on the above studies in healthy pigs. The AAV therapeutic construct can be administered into the left kidney as described in the previous examples.

[0366] Survival and post-slaughter evaluations may be performed as described above, and specific evaluations may be performed periodically to monitor cyst growth in treated versus untreated pigs as follows:

[0367] Cyst formation can be imaged using an ultrasound device. The kidneys of anesthetized pigs can be examined for cyst formation by a team of experts, which will include measuring the longitudinal and transverse diameters of the kidneys. Pigs will be followed up until 8-10 months of age. Additionally, kidney weight and body weight % after sacrifice can be assessed.

[0368] Animals receiving therapeutically effective doses of AAV-delivered PKD1 CTF or CTT are expected to advantageously exhibit (1) reduced serum and urinary creatine, (2) reduced BUN, (3) increased urinary exosomal PC1 CTF or CTT / TMEM2, (4) reduced kidney volume, and (5) reduced cyst size and number when compared to untreated PKD1 mutant pigs. Additionally, reduced interstitial fibrosis is expected in treated kidneys compared to untreated diseased kidneys.

[0369] In the above description, numerous specific details are set forth, such as particular materials, dimensions, process parameters, etc., to provide a thorough understanding of the present invention. Particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” should not necessarily be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is merely intended to present a concept in a concrete manner. As used in this application, “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless otherwise specified or clear from the context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A, if X includes B, or if X includes both A and B, then “X includes A or B” is satisfied under any of the above illustrative examples. References throughout this specification to "an embodiment," "certain embodiment," or "one embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "embodiment," "particular embodiment," or "one embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0370] The present invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are accordingly to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to be included within the scope of the appended claims.

Claims

1. 1. A gene therapy vector adapted for transduction of kidney cells in a human subject, said gene therapy vector comprising: adeno-associated virus (AAV) vectors, and a polynucleotide sequence packaged in the AAV vector, wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:

7. The gene therapy vector comprising:

2. The gene therapy vector of claim 1 , wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity with SEQ ID NO:

3.

3. The gene therapy vector of claim 1 , wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity with SEQ ID NO:

5.

4. The gene therapy vector of claim 1 , wherein the polynucleotide sequence encodes a therapeutic protein having at least 80% sequence identity to SEQ ID NO:

7.

5. The gene therapy vector of claim 1 , wherein the polynucleotide sequence further comprises a promoter sequence operably linked to the polynucleotide sequence encoding the therapeutic protein.

6. The promoter sequence is SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14 The gene therapy vector of claim 5, selected from the group consisting of:

7. The promoter sequence is SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO:28 The gene therapy vector of claim 5, selected from the group consisting of:

8. 2. The gene therapy vector of claim 1, wherein the serotype of the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13.

9. The gene therapy vector of claim 1 , wherein the serotype of the AAV vector is AAV5.

10. A gene therapy vector according to claim 1, and Pharmaceutically acceptable carrier Gene therapy drugs, including

11. 11. A method for treating a kidney-related disease, comprising administering a therapeutic dose of the gene therapy drug of claim 10 to a patient in need thereof.

12. 11. A method for performing gene replacement of a mutated gene, comprising administering a therapeutic dose of the gene therapy drug of claim 10 to a patient in need thereof.

13. 1. A method of treating autosomal dominant polycystic kidney disease (ADPKD) in a subject, the method comprising: Administering to the subject a therapeutic dose of a drug comprising the gene therapy vector of claim 1 and a pharmaceutically acceptable carrier. The method comprising:

14. 1. A method for performing localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject, said method comprising: placing a perfusion catheter in a renal artery of the kidney; placing a collection catheter into a renal vein of the kidney, the perfusion catheter and the collection catheter, together with a membrane oxygenator, forming a closed perfusion circuit through the kidney; and 11. The method of claim 10, further comprising flowing a perfusion solution through the closed circuit, the perfusion solution comprising the gene therapy agent of claim 10, wherein the closed circuit substantially isolates perfusion via the kidney from the subject's systemic circulation.

15. The method of claim 14, wherein the kidney cells comprise tubular cells.

16. A dose of the AAV vector is delivered through the closed circuit and is at least about 5 x 10 per milliliter (mL) of plasma during perfusion. ９ maintain a concentration of vector genomes in the blood of the subject, and the vector exhibits leakage into the subject's systemic circulation at a concentration of about 5 x 10 per mL of plasma during perfusion. ７ 15. The method of claim 14, wherein the vector genome remains less than 100% and the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

17. 15. The method of claim 14, wherein placing the perfusion catheter in the renal artery comprises placing the perfusion catheter via a femoral artery.

18. 15. The method of claim 14, wherein placing the retrieval catheter in the renal vein comprises placing the perfusion catheter via percutaneous access through the femoral vein or via the jugular vein.

19. 15. The method of claim 14, wherein placing the retrieval catheter in the renal vein comprises placing the perfusion catheter via non-percutaneous cut-down access.

20. flowing the perfusion fluid through the closed circuit, 15. The method of claim 14, comprising passing the perfusion fluid through the membrane oxygenator before entering the renal artery via the perfusion catheter.

21. 15. The method of claim 14, further comprising adding additional perfusion fluid to the closed circuit or diluting the perfusion fluid with about 5% to about 50% v / v saline to account for a proportion of bladder output.

22. The closed circuit maintains the perfusion fluid flow rate at about 500 mL / min / 1.73 m of body surface area per kidney for about 15 minutes to about 4 hours. ２ ~ 650 mL / min / kidney per 1.73 m body surface area ２ 15. The method of claim 14, wherein the temperature is maintained at 0.5°C.

23. The closed circuit maintains the perfusate flow rate at about 150 mL / min / 1.73 m of body surface area per kidney for about 15 minutes to about 4 hours. ２ ~ 700 mL / min / kidney per 1.73 m body surface area ２ 15. The method of claim 14, wherein the temperature is maintained at 0.5°C.

24. 15. The method of claim 14, further comprising applying a negative pressure to the recovery catheter, the negative pressure being in the range of about -100 mmHg to 120 mmHg.

25. 15. The method of claim 14, wherein one or more of the perfusion catheter and the recovery catheter are introduced percutaneously or non-percutaneously.

26. The method of claim 14, wherein less than about 20% v / v, less than about 15% v / v, less than about 10% v / v, less than about 5% v / v, less than about 4% v / v, less than about 3% v / v, less than about 2% v / v, less than about 1% v / v, less than about 0.5% v / v of the perfusion fluid circulating through the closed circuit leaks outside the closed circuit, or substantially none leaks (0% v / v).

27. 15. The method of claim 14, wherein one or more of the perfusion catheter or the recovery catheter is a balloon catheter.

28. 11. A method of delivering a therapeutic composition to a subject in need thereof, the method comprising locally delivering a therapeutic composition comprising the gene therapy agent of claim 10 to the kidney of the subject, while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs.

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