Local regional perfusion of the kidney

Abstract

A method for treating kidney disease by local-regional perfusion of one or both kidneys of a patient is disclosed. A closed circuit may 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 disposed therebetween. A perfusion solution containing, for example, a drug, may 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 APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 399,713, filed August 21, 2022, the disclosure of which is incorporated herein by reference 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 method of performing localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject comprises 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 withdrawal catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney), and flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid comprises the polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, and the AAV vector comprises AAV5 capsid protein, and wherein the closed circuit substantially isolates the perfusion through the kidney from the subject's systemic circulation.

[0006] In at least one embodiment, a dose of AAV vector is delivered via a closed circuit and is maintained at a high concentration (e.g., at least about 5×10 per milliliter (mL) of plasma) during perfusion. 7 The vector genome) is maintained in the blood, and leakage of the vector into the subject's systemic circulation remains extremely low during perfusion (e.g., approximately 5 x 10 per mL of plasma). 7 (Vector genome below). In at least one embodiment, perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0007] In at least one embodiment, the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least 30-fold higher than the same AAV vector that instead comprises a different capsid protein. In at least one embodiment, the number of vector genome copies per diploid genome after perfusion is between about 1 vg / dg and about 25 vg / dg (e.g., about 1 vg / dg, about 2 vg / dg, about 3 vg / dg, about 4 vg / dg, about 5 vg / dg, about 6 vg / dg, about 7 vg / dg, about 8 vg / dg, about 9 vg / dg, about 10 vg / dg, about 11 vg / dg, 12 vg / dg). g, about 14vg / dg, about 15vg / dg, about 16vg / dg, about 17vg / dg, about 18vg / dg, about 19vg / dg, about 20vg / dg, about 21vg / dg, about 22vg / dg, about 23vg / dg, about 24vg / dg, about 25vg / dg, or any range defined between any of the aforementioned points (e.g., about 5vg / dg to about 20vg / dg).

[0008] In another aspect, a method for performing localized delivery of a polynucleotide sequence to renal cells within a kidney of a mammalian subject includes 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 withdrawal catheter, together with a membrane oxygenator, forming a closed perfusion circuit through the kidney, and flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid comprises a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, and wherein a dose of the AAV vector is delivered through the closed circuit and is concentrated at a high concentration (e.g., at least about 5×10 per milliliter (mL) of plasma) during perfusion. 7 The vector genome) is maintained in the blood and leakage into the subject's systemic circulation remains extremely low during perfusion (e.g., approximately 5 x 10 per mL of plasma). 7 Vector genome (hereinafter), perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0009] In another aspect, a method for localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject includes placing a perfusion catheter in a renal artery of the kidney and a withdrawal catheter in a renal vein of the kidney (the perfusion catheter and withdrawal catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney), and flowing a perfusion solution through the closed circuit, wherein the perfusion solution comprises a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector comprising a non-AAV5 capsid protein, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, wherein the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher than the same AAV vector comprising an AAV5 capsid protein instead. In at least one embodiment, the number of vector genome copies per diploid genome after perfusion is between about 2 vg / dg and about 25 vg / dg.

[0010] In another aspect, a method for localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject includes placing a perfusion catheter in a renal artery of the kidney and a collection catheter in a renal vein of the kidney (the perfusion catheter and collection catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney), and flowing a perfusion solution through the closed circuit, wherein the perfusion solution comprises a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector comprising a capsid protein, the closed circuit substantially isolating the perfusion through the kidney from the patient's systemic circulation, wherein the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher than the same AAV vector instead comprising a different capsid protein. In at least one embodiment, the number of vector genome copies per diploid genome after perfusion is between about 2 vg / dg and about 25 vg / dg.

[0011] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

[0012] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-2 or a functional variant thereof.

[0013] In at least one embodiment, the therapeutic polynucleotide sequence comprises a nephron-specific promoter.

[0014] In at least one embodiment, placing a perfusion catheter in the renal artery includes placing a perfusion catheter via the femoral artery.

[0015] In at least one embodiment, placing a retrieval catheter in the renal vein comprises placing the retrieval catheter via percutaneous access (eg, via the femoral vein or via the jugular vein).

[0016] In at least one embodiment, placing the retrieval catheter in the renal vein includes placing the retrieval catheter via non-percutaneous access (eg, cut-down access).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] In at least one embodiment, one or more perfusion and recovery catheters are introduced percutaneously or non-percutaneously.

[0023] 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 blood circulating through the closed circuit leaks outside the closed circuit, or substantially none (0% v / v) leaks.

[0024] 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.

[0025] In at least one embodiment, one or more of the perfusion or recovery catheters is a balloon catheter.

[0026] In another aspect, a system for performing locoregional perfusion of a kidney when fluidly coupled to a patient's kidney includes a perfusion catheter adapted for insertion into a renal artery of the kidney, a collection catheter adapted for insertion into a renal vein of the kidney, the perfusion catheter, the collection catheter, and a membrane oxygenator fluidly coupled to a source of oxygen (when the perfusion catheter is inserted into the renal artery and the collection catheter is inserted into the renal vein, the perfusion catheter, the collection catheter, and the membrane oxygenator together form a closed circuit through the kidney isolated from the patient's systemic circulation), a reservoir containing perfusate (the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector), and a pump configured to drive the flow of perfusion solution through the perfusion catheter and the collection catheter.

[0027] In at least one embodiment, the AAV vector comprises an AAV5 capsid protein.

[0028] In another aspect, a system for performing locoregional perfusion of a patient's kidney includes a perfusion catheter inserted into a renal artery of the kidney, a collection catheter inserted into a renal vein of the kidney, a membrane oxygenator fluidly coupled to the perfusion catheter, the collection catheter, and a source of oxygen (the perfusion catheter, the collection catheter, and the membrane oxygenator, together with the kidney, form a closed circuit through the kidney isolated from the patient's systemic circulation), a reservoir containing perfusate (the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector), and a pump configured to drive flow of perfusate into the kidney via the perfusion catheter and out of the kidney via the collection catheter.

[0029] In at least one embodiment, the AAV vector comprises an AAV5 capsid protein.

[0030] In another aspect, the system of any of the above-described embodiments is configured to perform the method of any of the above-described embodiments.

[0031] In another embodiment, a gene therapy vector adapted for transducing kidney cells in a human subject comprises an adeno-associated virus (AAV) vector comprising AAV5 capsid proteins and a polynucleotide sequence packaged in the AAV vector.

[0032] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

[0033] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-2 or a functional variant thereof.

[0034] In at least one embodiment, the therapeutic polynucleotide sequence comprises a nephron-specific promoter.

[0035] 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 vector described in any of the preceding embodiments to the subject's kidney, while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs.

[0036] In another aspect, a method for screening AAV serotypes to identify a lead serotype for use in local gene delivery to a certain type of organ includes identifying a plurality of AAV vector candidates (each AAV vector candidate corresponding to a different AAV serotype), generating a perfusate composition containing each of the plurality of AAV vector candidates (each AAV vector candidate having a polynucleotide packaged therein), performing local transduction of tissue within an organ of a certain organ type in an animal by creating a closed circuit in the vascular system of the target organ through which the perfusate composition circulates, and selecting as the lead serotype the AAV vector candidate serotype that has the highest vector genome biodistribution and / or the highest RNA / protein expression in the organ compared to the remaining AAV vector candidates.

[0037] In at least one embodiment, the organ type is kidney, heart, pancreas, or liver.

[0038] 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]

[0039] [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 11] 1A shows a schematic diagram of a first exemplary perfusion catheter having a single balloon structure in accordance with at least one embodiment, FIG. 1B shows a schematic diagram of a balloon structure of the first exemplary perfusion catheter in an inflated state in accordance with at least one embodiment, and FIG. 1C shows a schematic diagram of a balloon structure of the first exemplary perfusion catheter in a retracted state in accordance with at least one embodiment. [Figure 12] 1A, 1B, and 1C are schematic illustrations of a second exemplary perfusion catheter having a distal plug according to at least one embodiment, a plug of the second exemplary perfusion catheter according to at least one embodiment, and a plug of the second exemplary perfusion catheter in an expanded state according to at least one embodiment. [Figure 13] 1A is a schematic illustration of a third exemplary perfusion catheter having a distal wedge in accordance with at least one embodiment, FIG. 1B is a schematic illustration of a wedge of the third exemplary perfusion catheter in accordance with at least one embodiment, and FIG. 1C is a further schematic illustration of the distal end of the third exemplary perfusion catheter in an expanded state in accordance with at least one embodiment. [Figure 14] 1A illustrates a fourth exemplary perfusion catheter having a partially covered, recaptureable stent structure in a deployed state according to at least one embodiment; FIG. 1B illustrates a fourth exemplary perfusion catheter stent structure in a stored state according to at least one embodiment; and FIG. 1C illustrates a fourth exemplary perfusion catheter stent structure in a deployed state according to at least one embodiment. [Figure 15] 1A and 1B illustrate a fifth exemplary perfusion catheter having a releasable covered braided disc in a deployed state according to 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 23] A is a plot of C3a levels over several days after renal LRP treatment for two different animals, and B is a plot of % transduction inhibition for various sample dilutions. [Figure 24] A. Plot of flow velocity during renal LRP. B. Plot of pump speed 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 33] 1A is a schematic diagram of an exemplary perfusion catheter with a balloon in a deflated state, according to at least one embodiment; 1B is a schematic diagram of an exemplary perfusion catheter with a balloon in a deployed state, according to at least one embodiment; and 1C is a photograph of an exemplary perfusion catheter and its balloon in a deployed state, according to at least one embodiment. [Figure 34] 1A is a schematic diagram of an exemplary retrieval catheter with a balloon in a deflated state, according to at least one embodiment; 1B is a schematic diagram of an exemplary retrieval catheter with a balloon in a deployed state, according to at least one embodiment; and 1C is a photograph of an exemplary retrieval catheter and its balloon in a deployed state, according to at least one embodiment. [Figure 35]1A and 1B illustrate the deployment of a single perfusion catheter placed in the renal artery of the left kidney, according to at least one embodiment; and 1B illustrates the deployment of a pair of perfusion catheters placed in the renal artery of the left kidney, according to at least one embodiment. [Figure 36] 1A and 1B illustrate the deployment of a single and a pair of retrieval catheters placed in the renal vein 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 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] Also, as used herein, "kidney cell" includes any cell of the kidney that is involved in maintaining the structure or providing function of the kidney.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] Also, as used herein, "pharmaceutically acceptable excipient or carrier" refers to an inactive ingredient in a composition that is combined with an active drug in a formulation. Pharmaceutically acceptable excipients 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 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).

[0054] 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.

[0055] Also, as used herein, "subject" encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0056] 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.

[0057] Also, as used herein, "prevention of" and "preventing" include avoiding the onset of a condition, for example, kidney disease or renal failure.

[0058] 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.

[0059] Also, as used herein, "effective amount" refers to an amount of drug sufficient to produce a beneficial or desired effect at a level readily detectable by methods commonly used to detect such effects. In at least one embodiment, such 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.

[0060] 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.

[0061] 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 clearly contradicted otherwise 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 particular 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

[0062] 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.

[0063] The kidney disease or renal disease that can be treated by the method disclosed herein can include, but is not limited to, pyelonephritis, which is caused by autosomal recessive mutations in NPHP1 gene, and autosomal dominant polycystic kidney disease, which is caused by haploinsufficiency of PKD2 gene.For example, the method can be used to treat hereditary and acquired glomerulonephritis and polycystic kidney disease.

[0064] Pyelonephritis is an autosomal recessive kidney disease leading 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, 149-153, 1997; Saunier, S. et al., Human Molecular Genetics, vol. 6, no. 13, 2317-2323, 1997). The NPHP1 gene encodes the protein nephrocystin-1, which is located in the adherens junctions and focal adhesions of renal epithelial cells and can be delivered to AAV vectors. Targeted delivery of nephrocystin-1 to target tissues may alleviate or correct type 1 pyelitis. Table 1 contains the amino acid sequences of various isoforms of nephrocystin-1. [Table 1-1] [Table 1-2]

[0065] Autosomal dominant polycystic kidney disease (ADPKD) has an incidence of 1 in 1,000 individuals. Approximately 85% of these cases are due to mutations in the PKD1 gene (encoding the polycystin-1 protein, or PC1), and approximately 15% are due to mutations in the PKD2 gene (encoding the polycystin-2 protein, or PC2). PC1 (4,322 amino acids) and PKD2 (968 amino acid polypeptides) are membrane proteins expressed on cilia. The primary pathological mechanism of ADPKD is haploinsufficiency (Veldhuisen, B. et al., American Journal of Human Genetics, vol. 61, 547-555, 1997). Supplementing PKD1 and PKD2 protein levels through AAV-mediated gene therapy is thought to alleviate ADPKD. The amino acid sequence of PC2 is shown in Table 2, and the amino acid sequences of various subdomains of PC1 are shown in Table 3. [Table 2] [Table 3]

[0066] Transduction of solid organs by systemic administration of recombinant AAV vectors has been challenging due to the high doses required, which can lead 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 ability to target delivery of AAV vectors to one or both kidneys without associated excretion into the systemic circulation.

[0067] 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.

[0068] In at least one embodiment, the system is inserted, for example, via the femoral artery and typically delivers 500-600 mL / min renal fluid in a 70 kg adult (i.e., 1000-1200 mL / 1.73 m). 2) and includes 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 at least one embodiment, 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 at least one embodiment, 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.

[0069] In at least one embodiment, the system includes one or more additional access lines to allow for drug administration or fluid addition. In at least one embodiment, 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 at least one embodiment, the system is adapted to replace the volume of perfusion fluid lost due to bladder drainage. For example, in at least one embodiment, 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 drainage.

[0070] In at least one embodiment, the system and method enable localized regional perfusion of one kidney with a targeted drug for a duration of time, e.g., 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or any range defined therein. In at least one embodiment, the system and method enable selective drug targeting of one or both kidneys with zero or minimal exposure of the systemic circulation and other organs to the drug. In at least one embodiment, 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 at least one embodiment, chemotherapy may be used to target kidney tumors. In at least one embodiment, other drugs or biologics / antibodies may be used. In at least one embodiment, combinations of the above drugs may be used.

[0071] There are several advantages to isolating a patient's renal circulation from their systemic circulation when treating kidney disease, including, but 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).

[0072] 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.

[0073] Certain embodiments may be used to provide locoregional delivery of pharmacological gene therapy to treat genetic mutations such as mutations in the PKD2 and NPHP1 genes. Exemplary Catheter Embodiments

[0074] 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.

[0075] Exemplary catheter embodiments for use as retrieval catheters in LRP systems are described herein. 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.

[0076] 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 portion proximal to the occlusive structure to bend at an angle of up to 120° to achieve better tracking and centering of the occlusive structure.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 expanded 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.

[0081] 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) inner 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 polymer material such as PEBAX® 63 supported by a strong stainless steel braid. The balloon may be formed from a flexible thermoplastic / elastomer material such as ChronoPrene™ 25A. The tip section may be formed from a polymer material such as PEBAX® 35 and may be filled with a radiopaque filler composition such as a radiomarker or BaSO4.

[0082] 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.

[0083] 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).

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Exemplary catheter embodiments for use as perfusion catheters in LRP systems are described herein. 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.

[0094] Figures 11-16 illustrate various catheter embodiments suitable for fluid perfusion within 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] The first catheter 1822 and the second catheter 1824 may be introduced percutaneously and in a minimally invasive manner. In at least one embodiment, 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.

[0109] 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 at least one embodiment, the first catheter 1822 is placed into the renal artery via the femoral artery. In at least one embodiment, the second catheter 1824 is placed into the renal vein via the femoral vein. In at least one embodiment, the second catheter 1824 may be a balloon catheter such that a balloon can be inflated within the renal vein to ensure 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 at least one embodiment, the first catheter 1822 and the second catheter 1824 may each be balloon catheters to help reduce leakage. In at least one embodiment, any of the catheters may be selected from one or more of the catheters described with respect to Figures 1-17.

[0110] 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 at least one embodiment, 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.

[0111] In at least one embodiment, the LRP system 1800 includes a third catheter 1826 for draining fluid from the bladder 1812. In at least one embodiment, 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 at least one embodiment, a fluid source 1840 may be used to replace the amount of drained fluid lost from the perfusion solution by injecting fluid into a closed circuit via a fluid line 1841. In at least one embodiment, the fluid is the same as the perfusion solution or has fewer components than all of the components of the perfusion solution (e.g., does not contain additional drugs). In at least one embodiment, the fluid is a physiologically acceptable solution (e.g., saline).

[0112] In at least one embodiment, the LRP system 1800 can be modified to simultaneously establish a closed circuit in each of a patient's kidneys. In at least one embodiment, two separate LRP systems can be used for each of a patient's kidneys.

[0113] 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.

[0114] In at least one embodiment, one or more drugs may be perfused through the patient's systemic circulation while the closed circuit is established. For example, if a drug is potentially nephrotoxic or toxic to the kidneys, but systemic delivery is desired, establishing a closed circuit to isolate renal perfusion from the systemic circulation is advantageous to prevent or reduce exposure of the drug to the kidney.

[0115] 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.

[0116] 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 at least one embodiment, 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.

[0117] 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 at least one embodiment, 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.

[0118] The suction mechanism may 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 may 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.

[0119] The blood circulating through the closed circuit may be autologous blood, matched blood from a donor, or a combination thereof. In at least one embodiment, blood components such as serum or plasma are selected according to one or more parameters. One of the parameters may be the presence or absence of selected antibodies. 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.

[0120] While the various components shown in FIG. 19 are shown as being part of or separate from the 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 the membrane oxygenator 1820 or may be separate (external) from it.

[0121] The 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 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, where it is then oxygenated by membrane oxygenator 1866 and antegradely reinfused into the kidney via first catheter 1822 (driven by delivery pump 1858); (7) the flow rate drained through the bladder is then measured using flow measuring device 1842 and replaced in 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, and blood samples can be taken, or drugs can be applied via reservoir 1860 during the entire perfusion process.

[0122] In at least one embodiment, 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 patient's immune response adversity 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.

[0123] In at least one embodiment, 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.

[0124] 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 at least one embodiment, the ratio can be in the range of 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).

[0125] The flow rate of the perfusate through the closed circuit can be adjusted to match the flow rate of the patient's blood. As will be appreciated by those skilled in the art, blood flow rate varies from patient to patient and for any given patient, varies throughout the day. Thus, the flow rate of the 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 at least one embodiment, the flow rate of the 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 flow rate of the patient's blood, based on mL / min. To avoid ischemia and / or underperfusion, it is important that the flow rate of the perfusate circulating through the closed circuit does not deviate significantly from the flow rate of the patient's own blood.

[0126] 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 at least one embodiment, the system regulates the flow rate of the 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 ~approximately 650 mL / min per kidney per 1.73 m of body surface area 2 Maintain it.

[0127] 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 at least one embodiment, the treatment duration may occur over a number of days, such as, for example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.

[0128] According to the systems disclosed herein, in at least one embodiment, a higher dose of drug may be administered directly and only to one kidney or kidneys than would otherwise be safely administered by systemic delivery. In at least one embodiment, 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.

[0129] In at least one embodiment, 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.

[0130] 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.

[0131] 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. therapeutic composition

[0132] A drug suitable for treating kidney disease (i.e., a drug contained in the perfusate) may comprise a therapeutic polynucleotide sequence. In at least one embodiment, the therapeutic polynucleotide sequence may encode a protein for treating kidney disease. 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 at least one embodiment, the protein encoded by the therapeutic polynucleotide sequence may correspond to a gene expressed in the human kidney.

[0133] Exemplary proteins may include, without limitation, NPHP1 (SEQ ID NOs: 1-4), PC2 (SEQ ID NO: 5), PC1 or its various subdomains (SEQ ID NOs: 6-7), variants thereof, or combinations thereof. The protein(s) 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 reach 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 protein variant can partially or fully exhibit the function of the corresponding naturally occurring protein. A functional variant of a protein can include, for example, a protein that differs from its native counterpart by one or more amino acid substitutions, deletions, or additions.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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).

[0140] 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.

[0141] "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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] To construct a retroviral vector, a nucleic acid encoding a gene of interest 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 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 can infect a wide variety of cell types. However, integration and stable expression require host cell division.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] Typically, rAAV is produced by cotransfecting a plasmid containing a gene of interest 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.

[0159] 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.

[0160] In at least one embodiment, 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, AAV12, 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.

[0161] 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.

[0162] According to the systems and methods disclosed herein, in at least one embodiment, 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 16 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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 + / It may include a podocyte-specific promoter such as glucose cotransporter 2 (SGLT2), sodium-potassium chloride cotransporter 2 (NKCC2), E-cadherin (ECAD), or the podocin promoter NPHS2.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] The following exemplary embodiments will now be described.

[0173] Embodiment 1: A method of performing localized delivery of a polynucleotide sequence to renal cells within a kidney of a mammalian subject, the method comprising placing at least one perfusion catheter in a renal artery of the kidney and placing at least one recovery catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one recovery catheter together with a membrane oxygenator forming a closed perfusion circuit through the kidney, the method further comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising a polynucleotide sequence packaged in an AAV vector, the AAV vector comprising an AAV5 capsid protein, and the closed circuit substantially isolating perfusion through the kidney from the subject's systemic circulation.

[0174] Embodiment 2: A dose of the AAV vector is delivered via the closed circuit and is at least about 5 x 10 per milliliter of plasma during perfusion. 7 and maintaining a concentration of vector genomes in the subject's systemic circulation at about 5 x 10 per milliliter of plasma during perfusion. 7 2. The method of embodiment 1, wherein the vector genome remains below the perfusion limit and said perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0175] Embodiment 3: The method of any preceding embodiment, wherein the number of vector genome copies per diploid genome (vg / dg) after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least 30-fold, or between about 2 vg / dg and about 25 vg / dg, compared to the same AAV vector instead comprising a different capsid protein.

[0176] Embodiment 4: A method of performing localized delivery of polynucleotide sequences to renal cells within a kidney of a mammalian subject, the method comprising placing at least one perfusion catheter in a renal artery of the kidney and placing at least one recovery catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one recovery catheter together with a membrane oxygenator forming a closed perfusion circuit through the kidney, the method further comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, a dose of the AAV vector delivered through the closed circuit, and a dose of at least about 5 x 10 per milliliter of plasma during perfusion. 7 and maintaining a concentration of vector genomes in the subject that exhibit leakage into the subject's systemic circulation at a concentration of about 5 x 10 per milliliter of plasma during perfusion. 7 the vector genome remains below the perfusion limit, and the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0177] Embodiment 5: A method of performing localized delivery of polynucleotide sequences to renal cells within a kidney of a mammalian subject, the method comprising placing at least one perfusion catheter in a renal artery of the kidney and placing at least one recovery catheter in a renal vein of the kidney, wherein the at least one perfusion catheter and the at least one recovery catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney, the method further comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising a polynucleotide sequence packaged in an AAV vector comprising a non-AAV5 capsid protein, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, wherein the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher than the same AAV vector comprising an AAV5 capsid protein instead.

[0178] Embodiment 6: A method of performing localized delivery of a polynucleotide sequence to renal cells within a kidney of a mammalian subject, the method comprising placing at least one perfusion catheter in a renal artery of the kidney and placing at least one recovery catheter in a renal vein of the kidney, wherein the at least one perfusion catheter and the at least one recovery catheter, together with a membrane oxygenator, form a closed perfusion circuit through the kidney, the method further comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising a polynucleotide sequence packaged in an AAV vector comprising a capsid protein, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, wherein the number of vector genome copies per diploid genome after the perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher compared to the same AAV vector instead comprising a different capsid protein.

[0179] Embodiment 7: The method of any preceding embodiment, wherein the polynucleotide sequence encodes a nephrocystin-1 protein or a functional variant thereof.

[0180] Embodiment 8: The method of any preceding embodiment, wherein the polynucleotide sequence encodes a nephrocystin-2 protein or a functional variant thereof.

[0181] Embodiment 9: The method of any preceding embodiment, wherein the therapeutic polynucleotide sequence comprises a nephron-specific promoter.

[0182] Embodiment 10: The method of any preceding embodiment, wherein placing the at least one perfusion catheter in a renal artery comprises placing the at least one perfusion catheter via a femoral artery.

[0183] Embodiment 11: The method of any preceding embodiment, wherein placing the at least one retrieval catheter in the renal vein comprises placing the at least one retrieval catheter percutaneously via a femoral or jugular vein.

[0184] Embodiment 12: The method of any one of embodiments 1 to 10, wherein placing the at least one retrieval catheter in the renal vein comprises non-percutaneously placing the at least one retrieval catheter via cut-down access.

[0185] Embodiment 13: The method of any preceding embodiment, 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 one or more perfusion catheters.

[0186] Embodiment 14: The method of any preceding embodiment, 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.

[0187] Embodiment 15: 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 10. The method of any preceding embodiment, wherein the

[0188] Embodiment 16: 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 10. The method of any preceding embodiment, wherein the

[0189] Embodiment 17: The method of any preceding embodiment, further comprising applying a negative pressure to the at least one retrieval catheter, wherein the negative pressure is in the range of about -100 mmHg to 120 mmHg.

[0190] Embodiment 18: The method of any one of the preceding embodiments, wherein one or more of the at least one perfusion catheter or the at least one recovery catheter is introduced percutaneously or non-percutaneously.

[0191] Embodiment 19: The method of any one of the preceding embodiments, 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 blood circulating through the closed circuit leaks outside the closed circuit, or substantially none leaks.

[0192] Embodiment 20: The method of any one of the preceding embodiments, 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 perfusate circulating through the closed circuit leaks outside the closed circuit, or substantially none leaks.

[0193] Embodiment 21: The method of any one of the preceding embodiments, wherein one or more of the at least one perfusion catheter or the at least one retrieval catheter is a balloon catheter.

[0194] Embodiment 22: A system for performing locoregional perfusion of a patient's kidney when fluidly coupled to the kidney, the system comprising: at least one perfusion catheter adapted for insertion into a renal artery of the kidney; at least one recovery catheter adapted for insertion into a renal vein of the kidney; a membrane oxygenator adapted for fluidly coupling the at least one perfusion catheter, the at least one recovery catheter, and an oxygen source, wherein the at least one perfusion catheter, the at least one recovery catheter, and the membrane oxygenator are adapted together to form a closed circuit through the kidney isolated from the patient's systemic circulation when the at least one perfusion catheter is inserted into the renal artery and the at least one recovery catheter is inserted into the renal vein; a reservoir containing perfusate, the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and a pump configured to drive flow of the perfusion solution through the at least one perfusion catheter and the at least one recovery catheter.

[0195] Embodiment 23: A system for performing locoregional perfusion of a patient's kidney, the system comprising: at least one perfusion catheter inserted into a renal artery of the kidney; at least one recovery catheter inserted into a renal vein of the kidney; a membrane oxygenator fluidly coupled to the at least one perfusion catheter, the at least one recovery catheter, and a source of oxygen, wherein the at least one perfusion catheter, the at least one recovery catheter, and the membrane oxygenator, together with the kidney, form a closed circuit through the kidney isolated from the patient's systemic circulation; a reservoir containing perfusate, the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and a pump configured to drive flow of the perfusate into the kidney via the at least one perfusion catheter and out of the kidney via the at least one recovery catheter.

[0196] Embodiment 24: The system of either embodiment 22 or embodiment 23, wherein the AAV vector comprises an AAV5 capsid protein.

[0197] Embodiment 25: The system of any of embodiments 22 to 24, wherein the polynucleotide sequence encodes a nephrocystin-1 protein or a functional variant thereof.

[0198] Embodiment 26: The system of any of embodiments 22 to 24, wherein the polynucleotide sequence encodes a nephrocystin-2 protein or a functional variant thereof.

[0199] Embodiment 27: A system described in any one of embodiments 22 to 26, configured to perform the method described in any one of embodiments 1 to 21.

[0200] Embodiment 28: A gene therapy vector adapted for transducing kidney cells in a human subject, the gene therapy vector comprising an adeno-associated virus (AAV) vector comprising AAV5 capsid proteins and a polynucleotide sequence packaged within the AAV vector.

[0201] Embodiment 29: The gene therapy vector of embodiment 28, wherein the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

[0202] Embodiment 30: The gene therapy vector of embodiment 28, wherein the polynucleotide sequence encodes nephrocystin-2 or a functional variant thereof.

[0203] Embodiment 31: A gene therapy vector described in any of embodiments 28 to 30, wherein the therapeutic polynucleotide sequence comprises a nephron-specific promoter.

[0204] Embodiment 32: A method of delivering a therapeutic composition to a subject in need thereof, the method comprising locally delivering a therapeutic composition comprising a gene therapy vector of any of embodiments 28-31 to the kidney of the subject, while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs.

[0205] Embodiment 33: A method for screening AAV serotypes to identify a lead serotype for use in local gene delivery to a certain type of organ, the method comprising: identifying a plurality of AAV vector candidates, each AAV vector candidate corresponding to a different AAV serotype; generating a perfusate composition comprising each of the plurality of AAV vector candidates, each AAV vector candidate having a polynucleotide packaged therein; performing local transduction of tissue within an organ of a certain organ type in an animal by creating a closed circuit in the vascular system of the target organ through which the perfusate composition circulates; and selecting the serotype of the AAV vector candidate that has the highest vector genome biodistribution and / or the highest RNA / protein expression in the organ compared to the remaining AAV vector candidates as the lead serotype.

[0206] Embodiment 34: The method of embodiment 33, wherein the organ type is kidney, heart, pancreas, or liver.

[0207] Embodiment 35: The method according to embodiment 32 or embodiment 33, wherein said local transduction of said tissue within said organ is carried out using a system according to either embodiment 22 or embodiment 23. [Example]

[0208] 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.

[0209] 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.

[0210] Example 1: LRP treatment LRP was performed on pigs utilizing the LRP system 1800 shown and described with respect to Figure 18. The accessory devices / components used in these examples, including their intended use and use in an LRP system according to an embodiment of the present disclosure, are listed in Table 4. [Table 4]

[0211] 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.

[0212] 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.

[0213] The detailed protocol of the LRP treatment followed in this example is now described. (1) Place the research animal in a supine position. (2) Preparing research animals 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) Flowgate2 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 introducing air. (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 the tightness and position of the catheter. (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 the gene therapy drug injected into the reservoir. b. For the first group of animals (Group B1): 5.0 x 10 13 Administer a dose of 2.8 x 10 vg (titer 2.8 x 10 13 Prepare by diluting 1.8 mL of vector solution at 1000 mg / mL with 2.2 mL of excipient). c. For the second group of animals (group B2): 6.0 x 10 14 vg (titer 2.8 × 10 13 Administer 100 mg / mL of vector solution (equivalent to 21.4 mL of 100 mg / mL vector solution). (19) Continue renal LRP for 60 minutes. (20) Check the following every 5 minutes: intrarenal pressure, renal-to-systemic pressure ratio (target ≥ 1), 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 from 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.

[0214] 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.

[0215] 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.

[0216] Example 2: Biodistribution studies Figure 21 shows the 6.2 x 10 14 Figure 1 shows kidney transduction and biodistribution of 0.05-0.25 vg / dg (vector genome copies per diploid genome) after 60 min of LRP at high doses of 0.05 vg / kg. No significant contamination of the intact kidney, liver, or other organs was detected, demonstrating the tightness of the LRP closed circuit.

[0217] 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.

[0218] 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] It is envisioned that this method can be applied not only to these organs by applying LRP treatment, but also to other organs.Exemplary organs can 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.

[0226] 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 13 The 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.

[0227] 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.

[0228] 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 5. [Table 5]

[0229] 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.

[0230] 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.

[0231] Marker bands are implanted under the balloon near the tip to visualize these landmarks under fluoroscopy.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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 barb connector on the hub. The tubing is approximately 10 cm long. A red on / off tubing clamp is placed over 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 use of the catheter.

[0238] 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.

[0239] 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.

[0240] Marker bands are implanted beneath the balloon to visualize these landmarks under fluoroscopy.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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).

[0250] 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.

[0251] 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."

[0252] 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.

[0253] 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.

[0254] The detailed protocol for LRP treatment followed in this and subsequent examples is now described. Initiation Phase (1) Place the research animal 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 to ensure stable catheter position. (11) A PressureWire X (Abbott) is threaded through the supply line and placed into one of the renal artery branches. (12) Antegrade contrast injection through the perfusion catheter visualizes the renal arterial anatomy 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 over the Lunderquist guidewire in its position in the main stem of the left renal vein. (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) To evaluate the renal venous 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 material. The criteria for acceptance of the seal quality are (i) no evidence of paraballoon flow or contrast material, (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 the 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. Unclamp 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 fluoroscopy. (24) Once both renal vessels are sealed, a 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 (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). (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) When 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) A single kidney LRP procedure is performed 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 (the ratio should be slightly below 1 to prevent leakage); 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 retrieval catheter balloon. Extract the IV line externally from the body. Fully detach the catheter, taking special care to avoid spilling 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.

[0255] 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.

[0256] 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.

[0257] 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.

[0258] 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.

[0259] Figure 39 shows the LRP-treated kidney (1.2 x 10 13Figure 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.

[0260] 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.

[0261] 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.

[0262] 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.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] 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.

[0270] 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).

[0271] 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.

[0272] 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.

[0273] 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.

[0274] 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).

[0275] To quantify the specificity of targeting kidney relative to transduction in non-target organs achieved by LRP administration of AAV5 compared with IV administration, kidney-to-liver transduction ratios were calculated for Animal 1 and Animal 2 and are summarized in Table 6. 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.

[0276] 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, S. et al., "Relationships among liver and kidney volumes, lean body mass, and drug clearance," Br. J. Clin Pharmacol, vol. 46, no. 5, 447-452, 1998). 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.

[0277] 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 6]

[0278] 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.

[0279] 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.

[0280] 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.

[0281] 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 serum creatinine concentration rose to 1000 mg / mL.

[0282] 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 7. Both animals exhibited low levels of anti-AAV9 neutralizing antibodies before administration of AAV9 via kidney LRP. On 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 7]

[0283] 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 word “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.

[0284] The present invention has been described with reference to specific exemplary embodiments thereof. Accordingly, the specification and drawings are 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 fall within the scope of the appended claims.

Claims

1. 1. A method for effecting localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject, said method comprising: placing at least one perfusion catheter within a renal artery of said kidney; and placing at least one retrieval catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one retrieval catheter forming a closed perfusion circuit through the kidney together with a membrane oxygenator; the method further comprising: the method comprising flowing a perfusion fluid through the closed circuit, the perfusion fluid comprising the polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, the AAV vector comprising an AAV5 capsid protein, and the closed circuit substantially isolating perfusion via the kidney from the subject's systemic circulation.

2. A dose of the AAV vector is delivered through the closed circuit and is at least about 5 x 10 per milliliter of plasma during perfusion. ７ maintain a concentration of vector genomes in the subject's blood at about 5 x 10 per milliliter of plasma during perfusion, and the vector exhibits leakage into the subject's systemic circulation. ７ 10. The method of claim 1, wherein the vector genome remains subcutaneously within the perfusion chamber and the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

3. 2. The method of claim 1, wherein the number of vector genome copies per diploid genome (vg / dg) after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least 30-fold, or between about 2 vg / dg and about 25 vg / dg, compared to the same AAV vector instead comprising a different capsid protein.

4. 1. A method for effecting localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject, said method comprising: placing at least one perfusion catheter within a renal artery of said kidney; and placing at least one retrieval catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one retrieval catheter forming a closed perfusion circuit through the kidney together with a membrane oxygenator; the method further comprising: and flowing a perfusion solution through the closed circuit, the perfusion solution comprising the polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, the closed circuit substantially isolating perfusion through the kidney from the patient's systemic circulation, a dose of the AAV vector delivered through the closed circuit, and a dose of at least about 5 x 10 per milliliter of plasma during perfusion. ７ and the vector is maintained at a concentration of about 5 x 10 per milliliter of plasma during perfusion, and exhibits leakage into the subject's systemic circulation. ７ wherein the vector genome remains below the perfusion threshold and the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

5. 1. A method for effecting localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject, said method comprising: placing at least one perfusion catheter within a renal artery of said kidney; and placing at least one retrieval catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one retrieval catheter forming a closed perfusion circuit through the kidney together with a membrane oxygenator; the method further comprising: a perfusion solution comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector comprising a non-AAV5 capsid protein, the closed circuit substantially isolating perfusion through the kidney from the systemic circulation of the mammalian subject, and wherein the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher than the same AAV vector comprising an AAV5 capsid protein instead.

6. 1. A method for effecting localized delivery of a polynucleotide sequence to renal cells within the kidney of a mammalian subject, said method comprising: placing at least one perfusion catheter within a renal artery of said kidney; and placing at least one retrieval catheter in a renal vein of the kidney, the at least one perfusion catheter and the at least one retrieval catheter forming a closed perfusion circuit through the kidney together with a membrane oxygenator; the method further comprising: a perfusion solution comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector comprising a capsid protein, the closed circuit substantially isolating perfusion through the kidney from the systemic circulation of the mammalian subject, and wherein the number of vector genome copies per diploid genome after perfusion is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold higher than the same AAV vector instead comprising a different capsid protein.

7. 2. The method of claim 1, wherein the polynucleotide sequence encodes a nephrocystin-1 protein or a functional variant thereof.

8. 2. The method of claim 1, wherein the polynucleotide sequence encodes a polycystin-2 protein or a functional variant thereof.

9. The method of claim 1 , wherein the polynucleotide sequence comprises a nephron-specific promoter.

10. 10. The method of claim 1, wherein placing the at least one perfusion catheter in the renal artery comprises placing the at least one perfusion catheter via a femoral artery.

11. 10. The method of claim 1, wherein placing the at least one retrieval catheter in the renal vein comprises placing the at least one retrieval catheter percutaneously via a femoral or jugular vein.

12. The method of claim 1 , wherein placing the at least one retrieval catheter in the renal vein comprises placing the at least one retrieval catheter non-percutaneously via cut-down access.

13. flowing the perfusion fluid through the closed circuit, 10. The method of claim 1, comprising passing the perfusion fluid through the membrane oxygenator before entering the renal arteries via the one or more perfusion catheters.

14. 10. The method of claim 1, 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.

15. 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 The method of claim 1 , wherein the temperature is maintained at 100° C.

16. 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 The method of claim 1 , wherein the temperature is maintained at 100° C.

17. The method of claim 1, further comprising applying a negative pressure to the at least one retrieval catheter, the negative pressure being in the range of about -100 mmHg to 120 mmHg.

18. The method of claim 1 , wherein one or more of the at least one perfusion catheter or the at least one recovery catheter is introduced percutaneously or non-percutaneously.

19. 2. The method of claim 1, 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 blood circulating through the closed circuit leaks outside the closed circuit, or substantially none leaks.

20. The method of claim 1, 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.

21. The method of claim 1 , wherein one or more of the at least one perfusion catheter or the at least one recovery catheter is a balloon catheter.

22. 1. A system for performing locoregional perfusion of a kidney of a patient when fluidly coupled to the kidney, the system comprising: at least one perfusion catheter adapted for insertion into a renal artery of the kidney; at least one retrieval catheter adapted for insertion into a renal vein of the kidney; a membrane oxygenator adapted to be fluidly coupled to the at least one perfusion catheter, the at least one recovery catheter, and an oxygen source, wherein when the at least one perfusion catheter is inserted into the renal artery and the at least one recovery catheter is inserted into the renal vein, the at least one perfusion catheter, the at least one recovery catheter, and the membrane oxygenator are adapted to together form a closed circuit through the kidney that is isolated from the patient's systemic circulation; a reservoir containing a perfusion solution, the perfusion solution comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; a pump configured to drive the flow of the perfusion fluid through the at least one perfusion catheter and the at least one recovery catheter.

23. 1. A system for performing local-regional perfusion of a kidney of a patient, comprising: at least one perfusion catheter inserted into a renal artery of the kidney; at least one retrieval catheter inserted into a renal vein of the kidney; a membrane oxygenator fluidly coupled to the at least one perfusion catheter, the at least one recovery catheter, and an oxygen source, wherein the at least one perfusion catheter, the at least one recovery catheter, and the membrane oxygenator, together with the kidney, form a closed circuit through the kidney that is isolated from the patient's systemic circulation; a reservoir containing a perfusion solution, the perfusion solution comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; a pump configured to drive the flow of the perfusion fluid into the kidney through the at least one perfusion catheter and out of the kidney through the at least one recovery catheter.

24. 23. The system of claim 22, wherein the AAV vector comprises an AAV5 capsid protein.

25. 23. The system of claim 22, wherein the polynucleotide sequence encodes a nephrocystin-1 protein or a functional variant thereof.

26. 23. The system of claim 22, wherein the polynucleotide sequence encodes a polycystin-2 protein or a functional variant thereof.

27. 23. The system of claim 22 configured to perform the method of claim 1.

28. 1. A gene therapy vector adapted for transducing kidney cells in a human subject, said gene therapy vector comprising: an adeno-associated virus (AAV) vector comprising the AAV5 capsid protein; and The polynucleotide sequence packaged in the AAV vector The gene therapy vector comprising:

29. The gene therapy vector of claim 28, wherein the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

30. 29. The gene therapy vector of claim 28, wherein the polynucleotide sequence encodes polycystin-2 or a functional variant thereof.

31. 29. The gene therapy vector of claim 28, wherein the polynucleotide sequence comprises a nephron-specific promoter.

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

33. 1. A method for screening AAV serotypes to identify lead serotypes for use in localized gene delivery to an organ type, said method comprising: identifying a plurality of AAV vector candidates, each AAV vector candidate corresponding to a different AAV serotype; generating a perfusate composition comprising each of said plurality of AAV vector candidates, each of which comprises a polynucleotide packaged therein; performing local transduction of tissue within an organ of said organ type within an animal by creating a closed circuit within the vasculature of said target organ through which said perfusate composition circulates; and selecting the serotype of the AAV vector candidate that has the highest biodistribution of the vector genome and / or the highest RNA / protein expression in the organ compared to the remaining AAV vector candidates as the lead serotype.

34. 34. The method of claim 33, wherein the organ type is kidney, heart, pancreas, or liver.

35. 34. The method of claim 33, wherein the local transduction of the tissue within the organ is performed using the system of claim 22.

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