Local region perfusion of kidney

Abstract

A method of treating a renal condition by local region perfusion of one or both of the kidneys of a patient is disclosed. A closed circuit may be formed with an irrigation catheter positioned in a renal artery of the kidney, a recovery catheter positioned in a renal vein of the kidney, and an extracorporeal membrane oxygenator disposed therebetween. Perfusate containing, for example, a drug may circulate through the closed circuit while isolating the closed circuit from the systemic circulation of the patient.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 399,713, filed on August 21, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Technical field

[0003] The present invention relates to the treatment of kidney diseases, and in particular, to the local delivery of therapeutic agents to the kidneys of a patient. Background art

[0004] Gene therapy and cell therapy technologies have drawn increasing attention in the treatment of various renal disorders such as chronic kidney disease, as these technologies may be uniquely targeted and can effectively address the underlying pathogenic mechanisms of various renal disorders. Nevertheless, there are still delivery - related issues, including vector efficiency, dose, specificity, and safety. Therefore, further research is needed to achieve a more targeted and uniform delivery of drugs suitable for treating various renal disorders to the kidneys, while also being effective, well - tolerated, and minimally invasive. Summary of the invention

[0005] A simplified overview of various aspects of the present disclosure is presented below to provide a basic understanding of such aspects. This overview is not an extensive review of the present disclosure. It is neither intended to identify key or decisive elements of the present disclosure nor to delineate any scope of the specific embodiments of the present disclosure or any scope of the claims. Its sole purpose is to present some concepts of the present disclosure in a simplified form as a prelude to the more detailed description that follows.

[0006] In one aspect, a method of locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject includes: positioning a perfusion catheter in the renal artery of the kidney; positioning a retrieval catheter in the renal vein of the kidney, wherein the perfusion catheter and the retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid contains a polynucleotide sequence packaged in an adeno - associated virus (AAV) vector, wherein the AAV vector contains an AAV5 capsid protein, and wherein the closed circuit substantially isolates the perfusion through the kidney from the systemic circulation of the subject.

[0007] In at least one embodiment, one dose of the AAV vector is delivered via the closed circuit and maintained at a high concentration during perfusion (e.g., at least about 5×10 7 vector genomes per milliliter (mL) of plasma), and the leakage of the vector into the systemic circulation of the subject that is present remains extremely low during perfusion (e.g., not more than about 5×10 per mL of plasma)7 (perfusion of a carrier genome). In at least one embodiment, the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0008] In at least one embodiment, compared to the same AAV vector actually containing different capsid proteins, the vector genome copy number per diploid genome is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold greater after perfusion. In at least one embodiment, the vector genome copy number per diploid genome after perfusion is from about 1 vg / dg to 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, about 12 vg / dg, about 14 vg / dg, about 15 vg / dg, about 16 vg / dg, about 17 vg / dg, about 18 vg / dg, about 19 vg / dg, about 20 vg / dg, about 21 vg / dg, about 22 vg / dg, about 23 vg / dg, about 24 vg / dg, about 25 vg / dg), or within any range defined between any of the foregoing points (e.g., about 5 vg / dg to about 20 vg / dg).

[0009] In another aspect, a method of locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject includes: positioning a perfusion catheter in the renal artery of the kidney; positioning a retrieval catheter in the renal vein of the kidney, wherein the perfusion catheter and the retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid contains a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, wherein the closed circuit substantially isolates the perfusion through the kidney from the patient's systemic circulation, and wherein a dose of the AAV vector is delivered via the closed circuit and maintained at a high concentration during perfusion (e.g., at least about 5×10 7 (carrier genomes) per milliliter (mL) of plasma), and wherein the leakage of the vector into the patient's systemic circulation that is present remains extremely low during perfusion (e.g., not more than about 5×10 7 (carrier genomes) per mL of plasma), wherein the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0010] In another aspect, a method of locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject includes: positioning a perfusion catheter in the renal artery of the kidney; positioning a retrieval catheter in the renal vein of the kidney, wherein the perfusion catheter and the retrieval catheter together with a membrane oxygenation device form 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 vector comprising a non-AAV5 capsid protein, wherein the closed circuit substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein the vector genome copy number per diploid genome is at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater, or at least about 30-fold greater after perfusion compared to the same AAV vector actually comprising an AAV5 capsid protein. In at least one embodiment, the vector genome copy number per diploid genome after perfusion is from about 2 vg / dg to about 25 vg / dg.

[0011] In another aspect, a method of locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject includes: positioning a perfusion catheter in the renal artery of the kidney; positioning a retrieval catheter in the renal vein of the kidney, wherein the perfusion catheter and the retrieval catheter together with a membrane oxygenation device form 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 vector comprising a capsid protein, wherein the closed circuit substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein the vector genome copy number per diploid genome is at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater, or at least about 30-fold greater after perfusion compared to the same AAV vector actually comprising a different capsid protein. In at least one embodiment, the vector genome copy number per diploid genome after perfusion is from about 2 vg / dg to about 25 vg / dg.

[0012] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

[0013] In at least one embodiment, the polynucleotide sequence encodes polycystin-2 or a functional variant thereof.

[0014] In at least one embodiment, the therapeutic polynucleotide sequence comprises a nephron-specific promoter.

[0015] In at least one embodiment, positioning the perfusion catheter in the renal artery includes positioning the perfusion catheter via the femoral artery.

[0016] In at least one embodiment, positioning a retrieval catheter in the renal vein includes positioning the retrieval catheter via a percutaneous access (e.g., via the femoral vein or via the jugular vein).

[0017] In at least one embodiment, positioning a retrieval catheter in the renal vein includes positioning the retrieval catheter via a non-percutaneous access (e.g., a cut-down access).

[0018] In at least one embodiment, flowing a perfusion fluid through a closed loop includes flowing the perfusion fluid through a membrane oxygenation device before entering the renal artery via a perfusion catheter.

[0019] In at least one embodiment, the method further includes adding additional perfusion fluid to the closed loop or diluting the perfusion fluid with a saline solution of about 5% to about 50% v / v to calculate the bladder excretion volume.

[0020] In at least one embodiment, the closed loop maintains the flow rate of the perfusion fluid at about 500 mL / min / 1.73 m 2 body surface area to about 650 mL / min / 1.73 m 2 body surface area per kidney for about 15 minutes to about 4 hours.

[0021] In at least one embodiment, the closed loop maintains the flow rate of the perfusion fluid at about 150 mL / min / 1.73 m 2 body surface area to about 700 mL / min / 1.73 m 2 body surface area per kidney for about 15 minutes to about 4 hours.

[0022] In at least one embodiment, the method further includes applying a negative pressure at the retrieval catheter, wherein the negative pressure is in the range of about -100 mmHg to 120 mmHg.

[0023] In at least one embodiment, one or more of the perfusion catheter and the retrieval catheter are introduced percutaneously or non-percutaneously.

[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, or substantially none (0% v / v) of the blood leaking out of the closed loop circulates through the closed loop.

[0025] 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 or substantially none (0% v / v) of the perfusion fluid circulating via the closed loop leaks outside the closed loop.

[0026] In at least one embodiment, one or more of the perfusion catheter or the retrieval catheter is a balloon catheter.

[0027] In another aspect, a system for performing regional perfusion of a local area of a patient's kidney when fluidly coupled to the patient's kidney includes: a perfusion catheter adapted to be inserted into the renal artery of the kidney; a retrieval catheter adapted to be inserted into the renal vein of the kidney; a membrane oxygenation device fluidly coupled to the perfusion catheter, the retrieval catheter, and an oxygen source, wherein when the perfusion catheter is inserted into the renal artery and the retrieval catheter is inserted into the renal vein, the perfusion catheter, the retrieval catheter, and the membrane oxygenation device together form a closed loop through the kidney, the closed loop being isolated from the patient's systemic circulation; a reservoir containing a perfusion fluid, the perfusion fluid containing a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and a pump configured to drive the perfusion fluid through the perfusion catheter and the retrieval catheter.

[0028] In at least one embodiment, the AAV vector comprises an AAV5 capsid protein.

[0029] In another aspect, a system for performing regional perfusion of a patient's kidney includes: a perfusion catheter inserted into the renal artery of the kidney; a retrieval catheter inserted into the renal vein of the kidney; and a membrane oxygenation device fluidly coupled to the perfusion catheter, the retrieval catheter, and an oxygen source, wherein the perfusion catheter, the retrieval catheter, and the membrane oxygenation device together with the kidney form a closed loop through the kidney, the closed loop being isolated from the patient's systemic circulation; a reservoir containing a perfusion fluid, the perfusion fluid containing a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and a pump configured to drive the perfusion fluid into the kidney via the perfusion catheter and out of the kidney via the retrieval catheter.

[0030] In at least one embodiment, the AAV vector comprises an AAV5 capsid protein.

[0031] In another aspect, the system of any of the foregoing embodiments is configured to perform the method of any of the foregoing embodiments.

[0032] In another aspect, a gene therapy vector suitable for transducing renal cells of a human subject comprises: an adeno-associated virus (AAV) vector comprising an AAV5 capsid protein; and a polynucleotide sequence packaged in the AAV vector.

[0033] In at least one embodiment, the polynucleotide sequence encodes nephrocystin-1 or a functional variant thereof.

[0034] In at least one embodiment, the polynucleotide sequence encodes polycystin-2 or a functional variant thereof.

[0035] In at least one embodiment, the therapeutic polynucleotide sequence comprises a renal-specific promoter.

[0036] In another aspect, a method of delivering a therapeutic composition to a subject in need thereof comprises locally delivering the therapeutic composition to the kidney of the subject while substantially avoiding introduction of the therapeutic composition into the systemic circulation or other organs, the therapeutic composition comprising a gene therapy vector of any of the foregoing embodiments.

[0037] In another aspect, a method of screening AAV serotypes to identify a dominant serotype for local gene delivery to an organ type comprises: identifying a plurality of AAV vector candidates, each AAV vector candidate corresponding to a different AAV serotype; generating an infusion fluid composition comprising each of the plurality of AAV vector candidates, each of the plurality of AAV vector candidates comprising a polynucleotide packaged therein; and performing local transduction of tissue in an organ of an organ type of an animal by creating a closed loop in the vasculature of the target organ, the infusion fluid composition being circulated via the closed loop; and selecting the serotype of the AAV vector candidate having the highest vector genome biodistribution and / or the greatest RNA / protein expression in the organ compared to the remaining AAV vector candidates as the dominant serotype.

[0038] In at least one embodiment, the organ type is kidney, heart, pancreas, or liver. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The foregoing and other features, its nature, and various advantages of the present disclosure will become more apparent upon consideration of the following detailed description in conjunction with the accompanying drawings, in which:

[0040] Figure 1 FIG. shows a schematic diagram of a first exemplary retrieval catheter having a single balloon structure according to at least one embodiment;

[0041] Figure 2 is a photograph of a retrieval catheter manufactured according to one embodiment of the first exemplary retrieval catheter;

[0042] Figure 3 Illustrates the deployment of a first exemplary retrieval catheter according to at least one embodiment;

[0043] Figure 4 Illustrates the deployment of a second exemplary retrieval catheter with a single balloon structure according to at least one embodiment;

[0044] Figure 5 Illustrates the deployment of a third exemplary retrieval catheter and a fourth exemplary retrieval catheter each with a single balloon structure according to at least one embodiment;

[0045] Figure 6 Illustrates the deployment of a fifth exemplary retrieval catheter with a single balloon structure and a sixth exemplary retrieval catheter without a balloon structure according to at least one embodiment;

[0046] Figure 7 Illustrates the deployment of a seventh exemplary retrieval catheter with multiple balloon structures according to at least one embodiment;

[0047] Figure 8 Illustrates the deployment of an eighth exemplary retrieval catheter with a partially covered and retractable stent structure according to at least one embodiment;

[0048] Figure 9 Illustrates the deployment of a ninth exemplary retrieval catheter with a deployable and retractable stent structure and a balloon structure according to at least one embodiment;

[0049] Figure 10 Illustrates the deployment of a tenth exemplary retrieval catheter with a covered disc-shaped stent structure according to at least one embodiment;

[0050] Figure 11A Is a schematic diagram of a first exemplary infusion catheter with a single balloon structure according to at least one embodiment;

[0051] Figure 11B Is a schematic diagram of the balloon structure of the first exemplary infusion catheter in an expanded state according to at least one embodiment;

[0052] Figure 11C Is a schematic diagram of the balloon structure of the first exemplary infusion catheter in a retracted state according to at least one embodiment;

[0053] Figure 12A Is a schematic diagram of a second exemplary infusion catheter with a distal plug according to at least one embodiment;

[0054] Figure 12B Is a schematic diagram of the plug of the second exemplary infusion catheter according to at least one embodiment;

[0055] Figure 12C Schematic diagram of the plug of a second exemplary perfusion catheter according to at least one embodiment in an extended state;

[0056] Figure 13A Schematic diagram of a third exemplary perfusion catheter with a distal wedge according to at least one embodiment;

[0057] Figure 13B Schematic diagram of the wedge of a third exemplary perfusion catheter according to at least one embodiment;

[0058] Figure 13C Another schematic diagram of the distal end of a third exemplary perfusion catheter according to at least one embodiment in an extended state;

[0059] Figure 14A Illustrates the deployment of a fourth exemplary perfusion catheter with a partially covered and retractable stent structure according to at least one embodiment;

[0060] Figure 14B Illustrates the stent structure of a fourth exemplary perfusion catheter according to at least one embodiment in a retracted state;

[0061] Figure 14C Illustrates the stent structure of a fourth exemplary perfusion catheter according to at least one embodiment in a deployed state;

[0062] Figure 15A Illustrates the deployment of a fifth exemplary perfusion catheter with a peelable covered braided disk according to at least one embodiment;

[0063] Figure 15B Illustrates the braided disk of a fifth exemplary perfusion catheter according to at least one embodiment in a deployed state;

[0064] Figure 16A Schematic diagram of a sixth exemplary perfusion catheter with a gradually narrowing lumen axis according to at least one embodiment;

[0065] Figure 16B Illustrates the deployment of a sixth exemplary perfusion catheter according to at least one embodiment;

[0066] Figure 16C Illustrates the preformed lumen axis of a sixth exemplary perfusion catheter according to at least one embodiment;

[0067] Figure 17 Illustrates the exemplary preformed lumen axis of an exemplary catheter according to multiple embodiments;

[0068] Figure 18 Depicts an exemplary local area perfusion system according to an embodiment of the present disclosure;

[0069] Figure 19 is a schematic diagram of an exemplary regional perfusion device according to an embodiment of the present disclosure;

[0070] Figure 20 includes radiographs showing the placement of arterial and venous catheters in the renal artery and renal vein of a porcine kidney before (upper image) and after (lower image) intravenous injection of a contrast agent;

[0071] Figure 21 is a graph showing kidney transduction and biodistribution after 60 minutes of kidney LRP according to an embodiment of the present disclosure;

[0072] Figure 22A shows the vector genome per mL of plasma measured at various time points during a 60-minute kidney LRP procedure with a high vector genome dose;

[0073] Figure 22B shows the vector genome per mL of plasma measured at various time points during a 45-minute kidney LRP procedure with a low vector genome dose;

[0074] Figure 23A is a graph of C3a levels on several days after kidney LRP treatment of two different animals;

[0075] Figure 23B is a graph of percent transduction inhibition at various sample dilutions;

[0076] Figure 24A is a graph of the flow rate during kidney LRP;

[0077] Figure 24B is a graph of the pump speed during kidney LRP;

[0078] Figure 25 is a graph showing the vector genome per mL of plasma measured at various time points during a 60-minute kidney LRP procedure of a perfusion fluid containing multiple AAV serotypes in the LRP closed loop relative to the systemic circulation;

[0079] Figure 26 is a graph showing the biodistribution of multiple AAV serotypes in kidney tissue after 60 minutes of simultaneous perfusion;

[0080] Figure 27 is a graph showing the biodistribution in the kidney compared to the liver after 60 minutes of perfusion with multiple AAV serotypes;

[0081] Figure 28It is a graph showing the relative quantification of transgenic mRNA levels of AAV5 in treated kidney sections relative to cumulative AAV in the kidney;

[0082] Figure 29 It is a graph showing AAV5 delivery of vector genomes per mL of plasma measured at various time points during a 60 - minute kidney LRP procedure in the LRP closed - loop relative to the systemic circulation;

[0083] Figure 30 It is a graph showing the biodistribution in the treated kidney after 60 minutes of perfusion with AAV5 compared to the liver and untreated kidney.

[0084] Figure 31 It is a graph showing AAV5 delivery of vector genomes per mL of plasma measured at various time points during a 52 - minute kidney LRP procedure in the LRP closed - loop relative to the systemic circulation;

[0085] Figure 32 It is a graph showing the biodistribution in the treated kidney after 52 minutes of perfusion with AAV5 compared to the liver, untreated kidney, and other regions;

[0086] Figure 33A It is a schematic diagram of an exemplary perfusion catheter having a balloon in a retracted state according to at least one embodiment;

[0087] Figure 33B It is a schematic diagram of an exemplary perfusion catheter having a balloon in a deployed state according to at least one embodiment;

[0088] Figure 33C It is a photograph of an exemplary perfusion catheter having a balloon in a deployed state according to at least one embodiment;

[0089] Figure 34A It is a schematic diagram of an exemplary retrieval catheter having a balloon in a retracted state according to at least one embodiment;

[0090] Figure 34B It is a schematic diagram of an exemplary retrieval catheter having a balloon in a deployed state according to at least one embodiment;

[0091] Figure 34C It is a photograph of an exemplary retrieval catheter having a balloon in a deployed state according to at least one embodiment;

[0092] Figure 35A It depicts the deployment of a single perfusion catheter deployed within the renal artery of the left kidney according to at least one embodiment;

[0093] Figure 35BIllustrates the deployment of a pair of perfusion catheters within the renal artery of the left kidney according to at least one embodiment;

[0094] Figure 36A Illustrates the deployment of a single retrieval catheter within the renal vein of the left kidney according to at least one embodiment;

[0095] Figure 36B Illustrates the deployment of a pair of retrieval catheters within the renal vein of the left kidney according to at least one embodiment;

[0096] Figure 37 Is a graph showing the excretion analysis (vector genome per mL of plasma or urine) detected in the LRP circulation, systemic circulation, and urine of animals treated with LRP;

[0097] Figure 38 Is a graph showing the total vector genome detected in the urine of animals treated with LRP;

[0098] Figure 39 Is a graph showing the biodistribution analysis of the LRP-treated kidney divided by kidney sections compared to untreated kidneys and livers;

[0099] Figure 40 Is a graph showing the GFP protein in the LRP-treated kidney compared to untreated kidneys and livers;

[0100] Figure 41 Is a graph showing the excretion analysis detected in the LRP circuit, systemic circulation, and urine of animals treated with LRP;

[0101] Figure 42 Is a graph showing the excretion analysis detected in the systemic circulation and urine of animals treated via systemic administration;

[0102] Figure 43 Is a graph showing the total vector genome present in the urine of animals treated with LRP;

[0103] Figure 44 Is a graph showing the total vector genome present in the urine of animals treated via systemic administration;

[0104] Figure 45 Is a graph modeling the concentration and time of AAV5 in the LRP circuit of the LRP-treated kidney;

[0105] Figure 46 Is a graph modeling the concentration and time of AAV5 in the systemic circulation of animals treated via systemic administration;

[0106] Figure 47is a graph showing biodistribution analysis measured in kidney sections compared to other organs from animals treated by LRP; and

[0107] Figure 48 is a graph showing biodistribution analysis measured in kidney sections compared to other organs from animals treated by systemic administration.

[0108] Definition

[0109] As used herein, unless the context otherwise clearly indicates, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a drug" includes a single drug as well as a mixture of two or more different drugs; and reference to "a viral vector" includes a single viral vector as well as a mixture of two or more different viral vectors, and so forth.

[0110] Furthermore, as used herein, when used in connection with a measured quantity, "about" refers to the normal variations of the measured quantity that are expected by one of ordinary skill in the art to make the measurement and operation match the precision of the measurement goal and the measurement device at the level of concern. In at least one embodiment, the term "about" includes ±10% of the recited value, such that "about 10" would include 9 to 11.

[0111] Furthermore, as used herein, "polynucleotide" has its ordinary and customary meaning in the art and includes any polymeric nucleic acid such as a DNA or RNA molecule, as well as chemical derivatives known to those skilled in the art. Polynucleotides include not only those polynucleotides encoding therapeutic proteins, but also sequences that can be used to reduce the expression of a targeted nucleic acid sequence (e.g., antisense, interfering, or small interfering nucleic acids) using techniques known in the art. Polynucleotides can also be used to initiate or increase the expression of a targeted nucleic acid sequence or the production of a targeted protein within cells of the cardiovascular system. The targeted nucleic acids and proteins include (but are not limited to) nucleic acids and proteins normally found in the targeted tissue, derivatives of such naturally occurring nucleic acids or proteins, naturally occurring nucleic acids or proteins not normally found in the targeted tissue, or synthetic nucleic acids or proteins. One or more polynucleotides can be used in combination, administered simultaneously and / or sequentially, to increase and / or decrease one or more targeted nucleic acid sequences or proteins.

[0112] In addition, as used herein, "perfusion", "perfused", and "perfusion" have their ordinary and customary meanings in the art and refer to the administration over a period of time (usually one minute or longer) that is substantially longer than the terms "injection" or "bolus injection" (usually less than one minute) that are recognized in the art. The flow rate of perfusion will depend at least in part on the volume being administered.

[0113] In addition, as used herein, an "exogenous" nucleic acid or gene is a nucleic acid or gene that does not exist in nature but is in a vector used for nucleic acid transfer; for example, a nucleic acid that is not naturally present in a viral vector, but the term is not intended to exclude a nucleic acid encoding a protein or polypeptide that is naturally present in a patient or host.

[0114] In addition, as used herein, "renal cell" includes any kidney cell involved in maintaining structure or providing kidney function.

[0115] In addition, as used herein, "isolated", "substantially isolated", "largely isolated", and variations thereof are terms that do not require complete or absolute isolation from the renal or systemic circulation; indeed, they are intended to mean that most, preferably the major part, or even substantially all of the designated circulation is isolated. In addition, as used herein, "partially isolated" means that any significant part of the designated circulation is isolated.

[0116] In addition, as used herein, "unnatural restriction" includes any method of restricting fluid flow through a blood vessel, such as a balloon catheter, suture, etc., but does not include naturally occurring restrictions such as plaque buildup (stenosis). Unnatural restrictions include, for example, substantial or complete isolation of the renal circulation.

[0117] In addition, 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 using endoscopic means to access the kidney and catheter-based means that rely on access via the large arteries and veins.

[0118] In addition, as used herein, "adeno-associated virus" or "AAV" encompasses all subtypes, serotypes, and pseudotypes, as well as natural and recombinant forms. Various AAV serotypes and strains are known in the art and are publicly available from sources such as ATCC and academic or commercial sources. Alternatively, sequences of AAV serotypes and strains can be synthesized using known techniques from publicly available and / or commercially available databases.

[0119] In addition, 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 twelve known serotypes of human AAV, including AAV1 to AAV13. However, additional serotypes continue to be discovered and consideration is given to using newly discovered serotypes.

[0120] In addition, as used herein, "pseudotyped" AAV refers to an AAV that contains a capsid protein from one serotype and a viral genome that includes 5' and 3' inverted terminal repeats (ITRs) of a different or heterologous serotype. It would be expected that pseudotyped recombinant AAV (rAAV) would have the cell surface binding properties of the capsid serotype and the genetic properties consistent with the ITR serotype. Pseudotyped rAAV can contain AAV capsid proteins, including VP1, VP2, and VP3 capsid proteins, and ITRs from any serotype of AAV, including any primate AAV serotype from AAV1 to AAV13, so long as the capsid protein has a serotype heterologous to the serotype of the ITR. In pseudotyped rAAV, the 5' and 3' ITRs can be the same or heterologous. Pseudotyped rAAV is produced using standard techniques described in the art.

[0121] In addition, as used herein, "chimeric" rAAV vectors encompass AAV vectors that contain heterologous capsid proteins; that is, the rAAV vector can be chimeric with respect to its capsid proteins VP1, VP2, and VP3 such that VP1, VP2, and VP3 are not all of the same serotype of AAV. As used herein, chimeric AAV encompasses AAVs in which the serotypes of the capsid proteins VP1, VP2, and VP3 are different, including, for example (but not limited to), capsid proteins from AAV1 and AAV2; mixtures of capsid proteins from other parvoviruses or containing other viral proteins or other proteins, for example, proteins that target AAV to a desired cell or tissue. As used herein, chimeric rAAV also encompasses rAAV that contains chimeric 5' and 3' ITRs. As used herein, chimeric rAAV can also contain a capsid generated from non-AAV sequences, such as a capsid obtained via peptide display screening.

[0122] In addition, as used herein, "pharmaceutically acceptable excipient or carrier" refers to any inert ingredient combined with the active agent in a formulation in a composition. Pharmaceutically acceptable excipients can 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, gelling agents, or other stabilizers and additives. Examples of other pharmaceutically acceptable carriers include wetting agents, emulsifying agents, dispersing agents, or preservatives particularly suitable for preventing the growth or activity of microorganisms. A variety of preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers, or adjuvants can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition (1985).

[0123] In addition, as used herein, "patient" refers to a subject who has presented clinical manifestations of one or more specific symptoms indicating a need for treatment, is being prophylactically treated for a disorder, or has been diagnosed with a disorder to be treated, particularly a human (but can also encompass non-humans).

[0124] In addition, as used herein, "subject" encompasses the definition of the term "patient" and does not exclude individuals who are otherwise healthy.

[0125] In addition, as used herein, "treatment of" and "treating" include administering a drug with the intention of reducing the severity of a disorder (such as a renal disorder or nephropathy), or preventing the disorder (such as a renal disorder or nephropathy).

[0126] In addition, as used herein, "prevention of" and "preventing" include avoiding the onset of a disorder (such as a renal disorder or nephropathy).

[0127] In addition, as used herein, "condition" or "conditions" refers to a medical disorder that can be treated, alleviated, or prevented by administering an effective amount of a drug to a subject, such as nephropathy.

[0128] In addition, as used herein, an "effective amount" refers to an amount of a drug sufficient to produce a beneficial or desired effect at a level that can be readily detected by methods commonly used to detect such effects. In at least one embodiment, such an effect causes a change in value of at least 10% relative to a baseline level in the absence of the administered drug. In other embodiments, the change is at least 20%, 50%, 80% or even a higher percentage relative to the baseline level. As will be described below, the effective amount of a drug can vary from subject to subject, depending on the subject's age, general condition, severity of the disorder being treated, the particular drug being administered, etc. The appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art with reference to the relevant texts and literature and / or by using routine experimentation.

[0129] In addition, as used herein, an "active agent" refers to any substance intended to produce a therapeutic, prophylactic or other desired effect, whether or not it has been approved by a government agency for such purpose.

[0130] Unless otherwise indicated herein, the recitation of numerical ranges herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated herein or otherwise clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is merely intended to illustrate certain materials and methods and is not intended to impose a limitation on the scope. The language in the specification should not be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods. Detailed Description

[0131] Certain embodiments of the present disclosure relate to systems and methods for treating renal disorders in a minimally invasive manner. Certain other embodiments of the present disclosure relate to the organ-selective delivery of genes to the kidney using a minimally invasive percutaneous delivery system. Exemplary methods can include isolating the patient's renal circulation from the patient's systemic circulation and perfusing a fluid (e.g., a drug-containing fluid) into the patient's isolated or substantially isolated renal circulation. The perfusion can be performed in one or both kidneys and can be used to deliver one or more drugs, including (but not limited to) gene therapy vectors, exosomes, nanoparticles, antibodies, chemotherapeutics, gene drugs (e.g., mRNA, siRNA, antisense RNA), etc., while keeping the systemic circulation and other organs unexposed to the selected drug. The methods can also be used to isolate the renal circulation to allow the administration of, for example, nephrotoxic drugs to the patient's systemic circulation in order to protect the kidneys from adverse effects. The isolation of the patient's renal circulation will be described in more detail below with reference to Figure 18 and Figure 19 More detailed description of the isolation of the patient's renal circulation.

[0132] Renal disorders or diseases treatable by the methods disclosed herein can include (but are not limited to) nephronophthisis, particularly nephronophthisis caused by autosomal recessive mutations in the NPHP1 gene, and autosomal dominant polycystic kidney disease, particularly autosomal dominant polycystic kidney disease caused by haploinsufficiency of the PKD2 gene. For example, the methods can be used to treat hereditary and acquired glomerulonephritis and polycystic kidney disease.

[0133] Nephronophthisis is an autosomal recessive kidney disease that leads to end-stage renal failure. The most common form is caused by mutations in NPHP1 (most commonly biallelic deletions) (Hildebrandt, F. et al., Nature Genetics, Vol. 17, 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 at adherens junctions and focal adhesions in renal epithelial cells and can be vectorized in an AAV vector. It is contemplated that replacement of nephrocystin-1 into the target tissue may alleviate or correct type 1 nephronophthisis. Table 1 includes the amino acid sequences of various isoforms of nephrocystin-1.

[0134] Table 1: Nephrocystin-1 Isoforms

[0135]

[0136]

[0137] Autosomal dominant polycystic kidney disease (ADPKD) has an incidence of 1 / 1000 in the population, with approximately 85% of these cases resulting from mutations in the PKD1 gene (encoding polycystin-1 protein or PC1) and approximately 15% resulting from mutations in the PKD2 gene (encoding polycystin-2 protein or PC2). PC1 (4,322 amino acids) and PKD2 (968 amino acid polypeptide) are membrane proteins expressed post-ciliogenesis. The main pathological mechanism of ADPKD is haploinsufficiency (Veldhuisen, B. et al., American Journal of Human Genetics, Vol. 61, 547 - 555, 1997). It is contemplated that AAV-mediated gene therapy to supplement PKD1 and PKD2 protein levels may 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.

[0138] Table 2: Polycystin-2

[0139]

[0140] Table 3: Polycystin-1 Subdomains

[0141]

[0142]

[0143] Transducing solid organs by systemic administration of recombinant AAV vectors is challenging because it requires high doses and causes serious adverse events (SAEs), particularly hepatotoxicity and thrombotic microangiopathy. Certain embodiments relate to local delivery and perfusion systems capable of selectively perfusing local regions of solid organs. The embodiments demonstrate that it is possible to target delivery of AAV vectors to one or both kidneys without relevant excreta entering the systemic circulation.

[0144] In an exemplary procedure demonstrating the efficacy of the embodiments described herein, the left renal artery and vein of AAV seronegative adult domestic pigs (approximately 90 kg) were percutaneously cannulated via internal jugular vein and femoral artery access. To isolate the kidneys from the systemic circulation, a closed circuit was established using each animal's own heparinized blood perfusion (perfusate), and regional perfusion (LRP) was initiated using an extracorporeal membrane oxygenation (ECMO) system. An AAV vector with a CMV-EGFP transgene cassette was injected into the closed circuit LRP system and regional perfusion of the kidneys was performed for up to 2 hours. Blood samples were collected longitudinally for safety evaluation, and vector titration and immune assessments (e.g., complement activation, anti-AAV antibodies) were performed before, during, and after the procedure. After completion of the procedure, the perfusate containing the vector was withdrawn and the catheters were removed. The animals were evaluated for 2 weeks, then euthanized and harvested for tissue processing. Quantitative PCR (qPCR) was used to detect the presence of the vector genome, and transgene expression was evaluated by qPCR, Western blot, and immunohistochemistry. The surgeries were successful in all animals and there were no perioperative complications. The animals recovered rapidly and there were no clinical signs of renal injury or impairment. The vector concentration remained high and stable in the perfusate of the closed circuit throughout the procedure, and there was no leakage associated with the systemic circulation or urine. AAV particles were evenly distributed in the treated renal tissue. Green fluorescent protein (GFP) was uniformly expressed in the perfused kidneys. No vector was detected in the untreated contralateral kidneys, liver, or other organs. Anti-AAV neutralizing antibodies increased only slightly compared to baseline and no complement activation was detected. Other tests will be discussed in more detail below.

[0145] In at least one embodiment, the system includes an arterial access catheter that can be inserted, for example, via the femoral artery and sealed within the renal artery at a flow rate suitable for perfusing and oxygenating the kidney for the duration of the procedure, the flow rate typically being 500 - 600 mL / min per kidney (or 1000 - 1200 mL / 1.73m in a 70 kg adult). 2 ) In at least one embodiment, the system includes a venous return catheter that can be inserted, for example, via the femoral vein and sealed within the renal vein at a flow rate suitable for venous flow recovery. In at least one embodiment, the system includes an extracorporeal membrane oxygenator system that fluidly connects the venous blood flow from the kidney to the arterial blood flow of the kidney and is capable of oxygenating the venous blood.

[0146] In at least one embodiment, the system includes one or more additional access lines that 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 urine excretion during the procedure. In other embodiments, individual ureteral catheters are placed in each of the two ureters to differentially measure the excretion of the two kidneys. In at least one embodiment, the system is adapted to replace the fluid volume of the perfusate lost due to bladder excretion. For example, in at least one embodiment, additional perfusate (such as blood) and / or other physiologically acceptable solutions (such as plasma or saline solution) can be used to replace approximately 5% v / v to approximately 50% v / v of the lost perfusate volume to calculate bladder excretion.

[0147] In at least one embodiment, the system and method allow for regional perfusion of one kidney with a target drug for a duration, for example, of 15 minutes, 30 minutes, 45 minutes, one hour, 2 hours, 3 hours, 4 hours, or any range defined therebetween. In at least one embodiment, the system and method allow for selective drug targeting of one or both kidneys with minimal or no exposure of the systemic circulation and other organs to the drug. In at least one embodiment, gene therapy drugs can be used to treat renal disorders, and the gene therapy drugs can utilize viral vectors (such as adeno-associated virus), naked or encapsulated DNA or RNA molecules, synthetic DNA or RNA analogs (such as antisense). In at least one embodiment, chemotherapy can be used to target renal tumors. In at least one embodiment, other drugs or biologics / antibodies can be used. In at least one embodiment, combinations of the aforementioned drugs can be used.

[0148] When treating renal disorders, isolating the patient's renal circulation from the patient's systemic circulation has many advantages. These advantages include (but are not limited to): (1) local regional delivery of drugs, minimal leakage of drugs to other organs, and reduced overall drug dosage; (2) increased targeted drug dosage; (3) reduced risks and side effects; and (4) the possibility of readministering to selected patients or administering to patient populations that are not suitable candidates for certain therapies (e.g., using gene therapy with a viral vector in patients having antibodies against the viral vector).

[0149] Other advantages should be apparent to those of ordinary skill in the art. Certain embodiments relate to methods of perfusing a drug into one or both kidneys of a patient in a minimally invasive manner. Certain embodiments provide methods for circulating a perfusion fluid (which may contain one or more of blood or a drug) through one or both kidneys of a patient such that the perfusion fluid is isolated from the patient's systemic circulation. Certain embodiments provide local regional delivery of pharmaco-gene therapy. Certain embodiments can be used to reduce the total dosage of a drug delivered to a patient for treating a renal disorder. Certain embodiments can be used to reduce the risks and / or adverse immune responses associated with administering a drug suitable for treating a renal disorder. Certain embodiments allow readministering to a patient and / or administering a pharmaco-gene therapy drug to a patient who has, for example, neutralizing antibodies against a gene therapy vector and who would otherwise not be a suitable candidate for receiving such a drug. Certain embodiments can be used to circulate a perfusion fluid through the kidneys and isolate the renal circulation from the patient's systemic circulation in order to allow introduction of a potentially nephrotoxic drug into the systemic circulation while preventing or reducing drug exposure to the kidneys. Certain embodiments can be used to treat renal disorders such as autosomal dominant polycystic kidney disease and nephronophthisis.

[0150] Certain embodiments can be used to provide local regional delivery of pharmaco-gene therapy to treat gene mutations, such as mutations in the PKD2 and NPHP1 genes.

[0151] Exemplary catheter embodiments

[0152] Exemplary retrieval catheters and perfusion catheters are now described. Those of ordinary skill in the art will understand that the catheters can be configured for the anatomy of any target organ (e.g., the kidney) for which LRP is performed. Further, it should be understood that any of the catheters described as "retrieval catheters" can also be used as "perfusion catheters" and vice versa. The embodiments described herein are not limited to LRP of the kidney and can also be used to isolate the circulation of the kidney from the systemic circulation, e.g., to reduce or prevent kidney exposure to drugs or other agents introduced into the systemic circulation that may have a harmful effect on the kidney. Those of ordinary skill in the art will appreciate other uses for the catheter embodiments described herein, e.g., for applications that require sealing blood vessels.

[0153] An embodiment of an exemplary catheter used as a retrieval catheter in an LRP system is now described. In at least one embodiment, the retrieval catheter is designed to support a liquid aspiration flow rate of about 400 mL / min or higher (e.g., about 700 mL / min or higher). For example, in at least one embodiment, the exemplary catheter can support an in vitro aspiration flow rate of about 800 mL / min at about -80 mmHg.

[0154] Figures 1 to 10 Depicts various catheter embodiments suitable for fluid retrieval in an LRP system. Figures 1 to 10 Any of the catheters depicted therein can be configured to support a liquid flow rate (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 compatible with a controllable introducer sheath that provides stability and guides the distal end of the catheter and allows the catheter to generate a directional thrust. Each catheter can also have a pull wire integrated into its shaft assembly to allow the section proximal to the occlusive structure to bend at an angle of up to 120° and achieve better tracking and central adjustment of the occlusive structure.

[0155] In at least one embodiment, one or more of the catheters can be a multi-lumen catheter, such as a dual-lumen catheter. In at least one embodiment, the multi-lumen catheter allows liquid flow (e.g., perfusion fluid) and is capable of inflating one or more balloons. In at least one embodiment, one or more of the catheters can be a multi-balloon catheter having two or more balloons. In at least one embodiment, one or more of the balloons can be deployed or deflated independently.

[0156] Figure 1 An exemplary catheter 100 having a lumen shaft 104 / 106 is depicted, the catheter having a proximal end 101 and a distal end 102. The lumen shaft 104 / 106 can be formed by an outer lumen shaft 104 that at least partially surrounds an inner lumen shaft 106 to expose the distal portion of the inner lumen shaft 106 proximal to the distal end 102. The proximal end 101 includes an outlet structure that can be fluidly coupled to the LRP system. One or more of the outer lumen shaft 104 or the inner lumen shaft 106 can be made of a durable polymer material such as a polyether block amide (PEBA) material (e.g., can be formed by commercial purchase). In at least one embodiment, the innermost diameter (“inner diameter”) of the inner lumen axis 106 is at least about 4 mm to provide a liquid flow path. In at least one embodiment, the catheter 100 can be designed to include additional lumen axes.

[0157] The catheter 100 includes a tip portion 108 at the distal end 102 and an expandable balloon structure 110 disposed along a portion 112 of the inner lumen axis 106. In at least one embodiment, the tip portion 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 portion is from about 2 mm to about 35 mm, from about 5 mm to about 30 mm, from about 10 mm to about 25 mm, from about 15 mm to 25 mm, or any sub-range defined therebetween (e.g., from about 2 mm to about 5 mm). In at least one embodiment, the tip portion 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 portion is formed of a flexible material that is more flexible than the material of the inner lumen axis 106.

[0158] In at least one embodiment, the inner lumen axis 106 includes a concentric inner flow path surrounding the liquid flow path. The concentric inner flow path provides a path for air flow from the balloon structure 110 to the orifice 114, which can be used to inflate or deflate the balloon depending on the pressure applied at the orifice 114. In at least one embodiment, the outermost surface of the inner lumen axis 106 at the portion 112 is removed such that the portion 112 is sealed by the balloon structure 110 to isolate the air flow from the concentric inner flow path to the balloon structure 110. In at least one embodiment, the expanded diameter of the balloon structure is from about 15 mm to about 30 mm, from about 15 mm to about 20 mm, from about 20 mm to about 25 mm, from about 24 mm to about 28 mm, or from about 25 mm to about 30 mm.

[0159] Figure 2 is an image of a catheter having a similar structure to the catheter 100 with a balloon in a deployed state. The dimensions of the catheter include: an outer diameter (crossing profile) of 19Fr (6.3 mm); an innermost diameter of 12Fr (4.0 mm); an available length of 80 cm; a balloon diameter of 25 mm (when deployed); and a tip portion length of 20 mm. The lumen axis can be formed of a polymeric material (e.g., 63) supported by a braided stainless steel. The balloon can be formed of a flexible thermoplastic / elastomeric material such as ChronoPrene TM 25A. The tip portion can be formed of a polymeric material such as 35 and can be loaded with a radioactive marker or a radiopaque filler composition such as BaSO4.

[0160] Figure 3 Illustrated in accordance with at least one embodiment, exemplary catheter 300 is inserted into blood vessel 352 via a larger blood vessel or chamber 350 (referred to herein as a "vessel"). In the depicted anatomy, blood flow from blood vessels 352 and 354 is diverted into vessel 350. Catheter 300 may be the same as or similar to catheter 100, having a proximal end 301, a distal end 302, an inner lumen axis 304, an outer lumen axis 306, a tip portion 308, and a balloon structure 310 disposed on a portion 312 of the inner lumen axis 304. When deployed, balloon structure 310 is flexible enough to conform to the anatomy of blood vessel 352 and occlude blood flow into vessel 350 through blood vessel 352 without exerting excessive force on the tissue. As Figure 3 illustrated, catheter 300 is inserted through blood vessel 354 to avoid occlusion of flow into vessel 350 from blood vessel 354.

[0161] It should be noted that vessel or chamber 350, blood vessel 352, and blood vessel 354 illustrate the anatomy of the right atrium of the heart, the coronary sinus, and the middle cardiac vein, respectively, to illustrate various types of occlusion techniques that may utilize the exemplary catheter. However, they are referred to herein as common vessels, and it should be understood that the deployment of any of the catheters described herein may be adapted to the specific anatomy of the target organ (e.g., the kidney) for which LRP or occlusion is to be performed. For example, vessels 350 and 352 may correspond to the inferior vena cava and the renal vein of the kidney (in the absence of blood vessel 354), respectively.

[0162] Figures 4 to 10 Illustrated are other occlusion techniques in accordance with various embodiments of the present disclosure. In some aspects, Figures 4 to 10 the catheter depicted in Figures 1 to 3 may be similar to the catheter depicted in

[0163] Figure 4 Illustrated is catheter 400 in accordance with at least one embodiment, which is inserted only partially into blood vessel 352 such that it abuts the opening of blood vessel 352. Catheter 400 includes a proximal end 401, a distal end 402, an inner lumen axis 404, an outer lumen axis 406, a tip portion 408, and a balloon structure 410 disposed on a portion 412 of the inner lumen axis 404. In at least one embodiment, when deployed, the diameter of balloon structure 410 is greater than about 15 mm, greater than about 20 mm, greater than about 25 mm, or greater than about 30 mm. In addition to the opening at the distal end 402, tip portion 408 may also include one or more perforations to facilitate blood flow from blood vessels 352 and 354 into catheter 400.

[0164] In at least one embodiment, during deployment, the outer lumen shaft 406 can move distally to abut the deployed balloon structure 410, thereby creating additional pressure by the balloon structure 410 against the opening of the blood vessel 352 to further stabilize the position of the catheter 400. In at least another embodiment, a wire structure can be utilized to apply pressure to the balloon structure 410. For example, the wire structure can have a sinusoidal shape that can be deployed into an expanded flower-like structure that radially extends from the outer lumen shaft 406 or the inner lumen shaft 404. When in contact with the balloon structure 410, the wire structure can create a more uniform pressure distribution across the surface of the entire balloon structure 410. Prior to deployment, the wire structure can be covered by the outer lumen shaft 406 or by an additional lumen outside the outer lumen shaft 406.

[0165] Figure 5 Illustrated is the use of a first catheter 500 and a second catheter 550 to separately occlude and drain the blood vessels 352 and 354, respectively, according to at least one embodiment. The 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 a portion 512 of the lumen shaft 504. Similarly, the 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 a portion 562 of the lumen shaft 554. In this configuration, the first catheter 500 is inserted into the blood vessel 352 such that the balloon structure 510 does not occlude the blood vessel 354, while the second catheter 550 is inserted directly into the blood vessel 354. The sizes of the first catheter 500 and the second catheter 550 can be selected to provide safe and effective occlusion of the blood vessels 352 and 354, respectively.

[0166] Figure 6 Illustrated Figure 5 is a variation of, which uses two catheters, where only one has a balloon structure according to at least one embodiment. The 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. The second catheter 650 includes a proximal end 651, a distal end 652, a lumen shaft 654, and a tip portion 658, and 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 vessels 350 and 352. The second catheter 650 is inserted directly into the blood vessel 354 and is disposed between the vessel wall and the balloon structure 610, which at least partially occludes the blood vessel 354.

[0167] Figure 7Illustrated is a single catheter 700 according to at least one embodiment, the catheter including a plurality of balloons. Catheter 700 includes a proximal end 701, a distal end 702, a lumen axis 704, a tip portion 708, a first balloon structure 710 disposed on a first portion 712 of the lumen axis 704, and a second balloon structure 720 disposed on a second portion 722 of the lumen axis 704. In at least one embodiment, 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 opening of the blood vessel 352 to occlude the blood vessel 354 (and further occlude the blood vessel 352). An intermediate portion 724 of the lumen axis 704 between the first balloon structure 710 and the second balloon structure 720 includes one or more perforations that allow drainage of the blood vessel 354. In at least one embodiment, the expanded diameter of the second balloon structure 720 is greater than the expanded diameter of the first balloon structure 710. In at least one embodiment, catheter 700 is a multi-lumen catheter designed to allow each balloon to be deployed and deflated independently of one another.

[0168] Figure 8 Illustrated is a catheter 800 according to at least one embodiment, the catheter including a partially covered and retractable stent structure 810. Catheter 800 includes a proximal end 801 and a distal end 802, an inner lumen axis 804 coupled to the stent structure 810, and an outer lumen axis 806. A portion of the outer lumen axis 806 is depicted in cross-section to illustrate the inner lumen axis 804 therein. The stent structure 810 is depicted in its deployed state but may be contained within the outer lumen axis 806 prior to deployment. The stent structure 810 is further depicted as having a proximally covered portion 810A that may be formed from a flexible and durable polymeric material, and a distally uncovered portion 810B. As shown, when inserted into the blood vessel 352, the covered portion 810A occludes blood flow exiting the blood vessel 352, while the uncovered portion 810B provides structural support within the blood vessel 352 while allowing blood flow from both the blood vessel 352 and the blood vessel 354 to flow directly into the catheter 800. In at least one embodiment, catheter 800 may be used as a perfusion catheter connected to a supply line.

[0169] Figure 9Illustrated is a catheter 900 according to at least one embodiment, the catheter including a deployable and retractable stent structure 920. The catheter 900 also includes a proximal end 901, a distal end 902, a lumen axis 906, a tip portion 908, and a balloon structure 910 disposed on a portion 912 of the lumen axis 906. The catheter 900 may also include an outer lumen axis (not shown) that substantially encapsulates the stent structure 920 and the balloon structure 910 prior to deployment. Deployment of the stent structure 920 may be effected by moving the outer lumen axis in the proximal direction, and retraction of the stent structure 920 may be effected by moving the outer lumen axis in the distal direction. The stent structure 920 may be formed of, for example, stainless steel and is disposed between the balloon structure 910 and the tip portion 908. In at least one embodiment, the lumen axis 906 includes at least one perforation along a portion 922 between the balloon structure 910 and the stent structure 920 to permit drainage of blood vessel 354 into the catheter 900. When inserted into the blood vessel 352, the balloon structure 910 abuts the opening of the blood vessel 352.

[0170] Figure 10 Illustrated is a catheter 1000 according to at least one embodiment, the catheter including a covered, disk-shaped stent structure 1010. The catheter 1000 also includes a proximal end 1001, a distal end 1002, an outer lumen axis 1006, an inner lumen axis 1004, and a tip portion 1008. The stent structure 1010 may be formed of, for example, a stainless steel stent with a durable polymer covering. The outer lumen axis 1006 may cover the stent structure 1010 prior to deployment. After the catheter 1000 is properly positioned, the outer lumen axis 1006 may be moved in the proximal direction to enable deployment of the stent structure 1010. In at least one embodiment, the stent structure 1010 is coupled to the tip portion 1008, which may be partially contained within the inner lumen axis 1004, and is actuatable (using a wire) to deploy the stent structure 1010 when moved in the proximal direction and retract the stent structure 1010 when moved in the distal direction. In at least one embodiment, when deployed, the stent structure 1010 is large enough to occlude the blood vessels 352 and 354 when abutting the opening of the blood vessel 352. In at least one embodiment, the diameter of the stent structure 1010 is from about 10 mm to about 30 mm.

[0171] Embodiments of an exemplary catheter used as a perfusion catheter in an LRP system are now described. In at least one embodiment, the perfusion catheter is designed to support a liquid perfusion flow rate of about 400 mL / min or higher (e.g., about 700 mL / min or higher). In embodiments utilizing multiple perfusion catheters, a combined flow rate of 700 mL / min or higher may be supported.

[0172] Figures 11 through 16 depict various catheter embodiments suitable for fluid perfusion in an LRP system. Any of the catheters depicted in Figures 11 through 16 can be configured to support a liquid flow rate (suction 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 the lower distal profile, such as using one or more concentric lumen axes. Additionally, the catheter can be designed to have a lumen axis that is preformed according to the anatomy of the LRP procedure being performed, which can improve overall stability during use.

[0173] In at least one embodiment, one or more of the catheters can be a multi-lumen catheter, such as a dual-lumen catheter. In at least one embodiment, the multi-lumen catheter allows liquid flow (e.g., perfusion fluid) and is capable of inflating one or more balloons. In at least one embodiment, one or more of the catheters can be a multi-balloon catheter having two or more balloons. In at least one embodiment, one or more of the balloons can be deployed or deflated independently.

[0174] Figures 11A to 11C An exemplary catheter 1100 is shown having lumen axes 1104 / 1106, the catheter having a proximal end 1101 and a distal end 1102 having an opening through which perfusion fluid can flow out. The lumen axes 1104 / 1106 can be formed by an outer lumen axis 1104 that at least partially surrounds an inner lumen axis 1106 to expose a distal portion of the inner lumen axis 1106 proximate the distal end 1102. The proximal end 1101 includes an outlet structure that can be fluidly coupled to the LRP system. One or more of the outer lumen axis 1104 or the inner lumen axis 1106 can be formed of a durable polymer material such as a polyether block amide (PEBA) material (e.g., available commercially). In at least one embodiment, the innermost diameter of the inner lumen axis 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.

[0175] The catheter 1100 includes an expandable balloon structure 1110 disposed along a portion 1112 corresponding to an inner lumen axis 1106 and a tip portion formed by an additional lumen. In at least one embodiment, the inner lumen axis 1106 includes a concentric inner flow path surrounding a liquid flow path. The concentric inner flow path provides a path for air flow to flow from the balloon structure 1110 to an orifice 1114, which can be used to inflate or deflate the balloon structure 1110 according to the pressure applied at the orifice 1114. In at least one embodiment, the outermost surface of the inner lumen axis 1106 at the portion 1112 is removed such that the portion 1112 is sealed by the balloon structure 1110 to isolate the air flow from the concentric inner flow path to the balloon structure 1110. In at least one embodiment, the expanded 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, about 25 mm to about 30 mm, or any sub-range defined therebetween (e.g., about 20 mm to about 28 mm). Figure 11B and Figure 11C illustrates the balloon structure 1110 in a deployed and deflated state.

[0176] Figures 12 and 13 illustrate catheters including a plug and wedge occlusion structure, which advantageously conforms its shape to a blood vessel or opening, is formed of a highly compressible and non-traumatic material for safe introduction and deployment, has a shorter length than the balloon structure, and does not generally require an additional lumen for inflation like the balloon structure.

[0177] Figures 12A to 12C Illustrates an exemplary catheter 1200 having a lumen axis 1204 / 1206, the catheter having a proximal end 1201 and a distal end 1202 having an opening through which a perfusion fluid can flow out. The lumen axis 1204 / 1206 can be formed by an outer lumen axis 1204 that at least partially surrounds an inner lumen axis 1206 to expose a distal portion of the inner lumen axis 1206 proximate 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 axis 1204 or the inner lumen axis 1206 can be formed of a durable polymeric material such as a polyether block amide (PEBA) material (e.g., available commercially). In at least one embodiment, the innermost diameter of the inner lumen axis 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.

[0178] The catheter 1200 also includes a plug 1210 near the distal end 1202. In at least one embodiment, the plug 1210 is formed of a flexible material such as silicone or a foamed material. In at least one embodiment, the plug 1210 includes an internal portion 1210A fitted onto the inner lumen shaft 1206 and a flexible outer portion 1210B shaped to be configurable between a retracted state ( Figure 12A ) and an extended state ( Figure 12C ), wherein the outer portion 1210B extends distally from the distal end 1202. Figure 12A The plug 1210 in

[0179] Figures 13A to 13C is shown as tapering in the distal direction. In at least one embodiment, the plug 1210 can be reversed to taper in the proximal direction. In at least one embodiment, the outer lumen shaft 1204 can be configured to cover the plug 1210 prior to deployment. When used as an infusion catheter, the pressure of arterial blood flowing into the hollow space between the internal portion 1210A and the outer portion 1210B of the plug 1210 can help improve the sealing of the catheter 1200 within the blood vessel in which it is deployed. commercially available) formed. 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.

[0180] The catheter 1300 also includes a wedge 1310 near the distal end 1302, which can be shaped to fit a blood vessel or an opening. In at least one embodiment, the wedge 1310 is formed of a flexible material such as silicone or a foamed material. In at least one embodiment, the outer lumen shaft 1304 can be configured to cover the wedge 1310 prior to deployment. When deployed in a blood vessel, the shape of the wedge can utilize the retrograde force from the blood vessel wall to further enhance stability during occlusion and perfusion of the blood vessel.

[0181] Figures 14A to 14C Shown in connection with Figure 8The described catheter 800 is similar to the exemplary catheter 1400 according to at least one embodiment, which includes a partially covered and retractable stent structure 1406. The catheter 1400 is depicted as inserted into the arterial vessel 1452 via a blood vessel or chamber 1450. In at least one embodiment, the catheter 1400 includes an outer lumen shaft 1402 and an inner lumen shaft 1404 coupled to the stent structure 1406. The stent structure 1406 is further depicted as having a proximally covered portion formed of a flexible and durable polymeric material and a distally uncovered portion. Figure 14B and Figure 14C respectively illustrate the placement and deployment of the stent structure 1406 when inserted into the blood vessel 1452. The deployment of the stent structure 1406 is performed by moving the outer lumen shaft 1402 in the proximal direction.

[0182] Figure 15A and Figure 15B Illustrates an exemplary catheter 1500 according to at least one embodiment, which includes a peelable covered braided disk 1510. The catheter 1500 includes an outer lumen shaft 1506 and an inner lumen shaft 1504. The braided disk 1510 is contained within the outer lumen shaft 1506 during placement of the catheter 1500 and can be deployed by moving the outer lumen shaft 1506 in the proximal direction. In at least one embodiment, when deployed, the braided disk 1510 does not extend beyond the distal end 1502 and is used to stabilize the catheter 1500 against the opening of the blood vessel 1452 to reduce the risk of stenosis during occlusion of the blood vessel 1452 while allowing the distal end 1502 to extend into the blood vessel 1452.

[0183] Figures 16A to 16C Illustrates an exemplary catheter 1600 having a lumen shaft 1606, the catheter having a proximal end 1601 and a distal end 1602 with an opening from which perfusion fluid can flow out. The proximal end 1601 includes an outlet structure that can be fluidly coupled to an LRP system. The lumen shaft 1604 can be formed of a durable polymeric material such as a polyether block amide (PEBA) material (e.g., commercially available). 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 liquid 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 can taper within the length of the lumen shaft 1606. commercially available). Figure 16C Illustrates the lumen shaft in a preformed form for introduction and placement into the blood vessels of a target organ.

[0184] Examples of preformed catheter lumens are illustrated in Figure 17The lumen of the catheter can be shaped to abut an area of the anatomical structure upon deployment, utilizing retrograde forces from the vessel wall to further enhance stability during occlusion and perfusion of the target organ.

[0185] Exemplary LRP system embodiments

[0186] Figure 18 Depicts an exemplary LRP system 1800 according to an embodiment of the present disclosure. The LRP system 1800 is shown in a closed-loop configuration with the kidney 1810. The LRP system 1800 includes a membrane oxygenation device 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 a pressure line and console 1844 for measuring the pressure within the closed loop. In at least one embodiment, a vacuum pump 1848 may also be utilized. The LRP system 1800 can be assembled by positioning a first catheter 1822 (referred to herein as the "perfusion catheter") within the renal artery of the kidney 1810 and a second catheter 1824 (referred to herein as the "recovery catheter", "collection catheter", or "suction catheter") within the renal vein of the kidney 1810. The first catheter 1822 and the second catheter 1824, together with the vasculature of the kidney 1810, the membrane oxygenation device 1820, and one or more optional additional components, form a closed loop. This closed loop can isolate or substantially isolate the patient's renal circulation from the patient's systemic circulation.

[0187] The first catheter 1822 and the second catheter 1824 can be introduced percutaneously in a minimally invasive manner. In at least one embodiment, the first catheter 1822 and / or the second catheter 1824 can be introduced via antegrade cannulation. In other embodiments, the first catheter 1822 and / or the second catheter 1824 can be introduced via retrograde cannulation. When using a catheter to deliver a drug to one or more kidneys, the first catheter 1822 can be referred to herein as the "drug delivery catheter" and the second catheter 1824 can be referred to herein as the "drug collection catheter".

[0188] The first catheter 1822 can be a standard infusion catheter, which can optionally include a standard guidewire and an infusion pump, and is capable of delivering a perfusion fluid to the kidney 1810, which can contain, for example, a drug to be delivered to the kidney 1810 during local regional perfusion. In at least one embodiment, the first catheter 1822 is positioned within the renal artery via the femoral artery. In at least one embodiment, the second catheter 1824 is positioned within the renal vein via the femoral vein. In at least one embodiment, the second catheter 1824 is a balloon catheter, such that the balloon can be inflated within the renal vein to ensure that all blood flowing through the closed loop passes through the second catheter 1824. Those of ordinary skill in the art will appreciate that the balloon catheter can be a catheter, or any other catheter suitable for the purposes discussed herein. In at least one embodiment, the first catheter 1822 and the second catheter 1824 may each be a balloon catheter to help reduce leakage. In at least one embodiment, either of the catheters may be selected from one or more of the catheters described with respect to Figures 1 to 17 those described.

[0189] The LRP system 1800 may also include one or more additional components such as, but not limited to, one or more pumps (such as the vacuum pump 1848), one or more suction mechanisms, one or more perfusion fluids, and combinations thereof. For example, the LRP system 1800 may include a pressure line and a console 1844, which in at least one embodiment is operably coupled to or is part of the membrane oxygenation device 1820. The pressure line and console 1844 and the ECMO pump console 1846 may be used together to control the perfusion rate (i.e., the flow rate) by continuously monitoring the renal artery pressure and to ensure safety. The first pressure sensor and the second pressure sensor may be inserted together with the first catheter 1822 and the second catheter 1824, respectively, to measure the pressures within the renal artery and the renal vein, respectively. The LRP system 1800 is further depicted as including a BGA monitor 1830, which is operably coupled to the membrane oxygenation device 1820 to measure, for example, the gas concentration in the perfusion fluid (such as when the perfusion fluid contains blood) before perfusion via the first catheter 1822 and / or after collecting the perfusion fluid through the second catheter 1824. The membrane oxygenation device 1820 and one or more additional components may be placed between the first catheter 1822 and the second catheter 1824.

[0190] In at least one embodiment, the LRP system 1800 includes a third catheter 1826 for draining the bladder 1812. In at least one embodiment, the third catheter 1826 is a balloon catheter for preventing fluid leakage from the bladder 1812. A flow measurement device 1842 may be used to measure the volume of urine excreted from the bladder 1812 during the LRP procedure. In at least one embodiment, a fluid source 1840 may be used to replace the volume of excreted fluid lost in the perfusion fluid by injecting fluid via a fluid line 1841 into a closed loop. In at least one embodiment, the fluid is the same as the perfusion fluid or less than all components of the perfusion fluid (such as without additional drugs). In at least one embodiment, the fluid is a physiologically acceptable solution (such as a saline solution).

[0191] In at least one embodiment, the LRP system 1800 may be modified to simultaneously establish a closed loop within each of the patient's kidneys. In at least one embodiment, two independent LRP systems may be used for each of the patient's kidneys.

[0192] In at least one embodiment, the LRP system 1800 can be modified to include multiple perfusion or recovery 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 shunt supply line) such that each catheter can be placed in a different location within the vasculature of the kidney 1810. Similarly, the second catheter 1824 can be supplemented with one or more additional recovery catheters that are fluidly coupled to the ECMO pump console 1846.

[0193] In at least one embodiment, when establishing a closed loop, one or more drugs can be perfused via the patient's systemic circulation. For example, if a drug is nephrotoxic or potentially harmful to the kidneys but systemic delivery is required, establishing a closed loop through the kidneys to isolate renal perfusion from systemic perfusion would be beneficial to prevent or reduce drug exposure to the kidneys.

[0194] Figure 19 is a schematic diagram of a membrane oxygenation device 1820 that can be used to oxygenate perfusate, mix the perfusate with other components (such as drugs), remove carbon dioxide from the perfusate, and / or push the perfusate into the first catheter 1822. The membrane oxygenation device 1820 can be any commercially available ECMO device that exchanges carbon dioxide contained in the blood with oxygen.

[0195] As Figure 19 depicted, the membrane oxygenation device 1820 includes various components, including a heat exchanger 1856 through which the perfusate passes before leaving the outlet 1852 and entering the first catheter 1822, a delivery pump 1858, a reservoir 1860 (for adding components such as blood and / or drugs to the perfusate returning via the second catheter 1824 through the inlet 1854), sensors 1862 and 1864 at various stages of the closed loop (e.g., for measuring pressure and / or blood gas content), and a membrane oxygenator 1866. In at least one embodiment, deoxygenated blood enters the membrane oxygenator 1866 and is mixed with oxygen-rich gas. The oxygen-rich gas can be supplied by a gas blender 1868 that can mix oxygen with carbon dioxide and nitrogen in various ratios and is regulated by a gas regulator 1870.

[0196] The perfusate can include one or more of blood (or its components such as plasma or serum) and / or drugs suitable for treating renal disorders and / or a vehicle such as saline or a dextran solution. The delivery pump 1858 can deliver the perfusate into the first catheter 1822. In at least one embodiment, the perfusate can be contained in an IV bag or syringe and can be administered directly to the first catheter 1822 with or without the delivery pump 1858.

[0197] A suction mechanism can be used to apply a negative suction pressure to the second conduit 1824 to minimize blood and / or drug leakage outside the closed loop. The negative suction pressure can be about -150 mmHg, about -100 mmHg, about -50 mmHg, about -20 mmHg, about -15 mmHg, about -10 mmHg, about -5 mmHg, 0 mmHg, or within a sub-range defined by any one of these points.

[0198] The blood circulated via the closed loop can 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 based on one or more parameters. One of the parameters can be the presence or absence of a selected antibody. For example, when the drug is one or more viral vectors encompassing therapeutic nucleic acid sequences, the patient's autologous blood can be screened to determine the presence of antibodies against one or more viral vectors. The presence of antibodies in the patient's autologous blood can reduce and / or completely offset the efficacy of the treatment and / or can cause an unwanted immune response. Thus, it is possible to dilute or replace the patient's autologous blood with serum-negative matched blood from a donor, thereby reducing the patient's immune response to the drug and enhancing the efficacy of the drug.

[0199] Although Figure 19 the various components depicted in are shown as parts of or separate from the membrane oxygenation device 1820, it should be understood that this schematic is illustrative only, as one or more components can be included in or separate from (outside of) the membrane oxygenation device 1820.

[0200] The LRP system 1800 can be set up and operated as follows: (1) Place the retrieval catheter (e.g., the second catheter 1824) carefully and seal it tightly in the renal vein to enable collection of deoxygenated venous blood; (2) Place the perfusion catheter (e.g., the first catheter 1822) in the renal artery in a sealed manner; (3) Insert an additional retrieval catheter (e.g., the third catheter 1826) in a sealed manner into the bladder, ureter, or both; (4) Then connect the perfusion and retrieval catheters to the arterial and venous lines of the membrane oxygenation device 1820 using standard cannulas; (5) Start the operation of the LRP system 1800 and perfuse the renal artery with oxygenated blood in a retrograde manner while collecting the returned deoxygenated blood from the renal vein via the retrieval catheter using gentle negative pressure; (6) Then direct the blood into the reservoir 1860 and subsequently oxygenate it through the membrane oxygenator 1866 and reinfuse it (driven by the delivery pump 1858) into the kidney via the first catheter 1822 in a retrograde manner; and (7) Then use the flow measurement device 1842 to measure the volume of fluid excreted via the bladder and replace the volume of fluid in the perfusion fluid with fluid from the fluid source 1840. If a drug (e.g., a vector) is administered, this drug can be added to the perfusion fluid via the reservoir 1860 after perfusion of blood or plasma, and blood samples can be taken, or the drug can be administered via the reservoir 1860 during the entire perfusion process.

[0201] In at least one embodiment, diluting or replacing the patient's antibody-containing autologous blood with serum-reaction-negative matched blood from a donor (e.g., by removing venous blood and flushing with antibody-free blood to exchange the volume circulating in the system to reduce the amount of circulating antibodies specific to the viral vector used) can result in a reduction in adverse immune responses and / or an improvement in drug efficacy. For example, compared to the immune response of a patient who has not undergone autologous blood dilution or replacement, after diluting or replacing autologous blood with serum-reaction-negative matched blood from a donor, the patient's adverse immune response can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or completely resolved. Compared to the efficacy of a drug in a patient who has not undergone autologous blood dilution or replacement, after diluting or replacing autologous blood with serum-reaction-negative matched blood from a donor, the efficacy of the administered drug can 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%.

[0202] In at least one embodiment, the blood portion of the perfusion fluid can be in the range of 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.

[0203] In the blood circulated via a closed loop, the ratio of autologous blood to matched blood from a donor can be adjusted as needed to obtain a blood mixture that is most receptive to the drug and will produce the least immune response after the introduction of the drug. In at least one embodiment, the ratio of (autologous blood volume):(matched blood volume from a donor) 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.

[0204] The flow rate of the perfusion fluid through the closed loop can be adjusted to match the patient's blood flow rate. Those of ordinary skill in the art will appreciate that blood flow rates vary from patient to patient and, for any given patient, the blood flow rate will vary throughout the day. Accordingly, the flow rate of the perfusion fluid circulated via the closed loop can be adjusted in situ. The flow rate can be measured within the closed loop. In at least one embodiment, the flow rate can be measured using a transonic probe (e.g., a clamp on the tubing). In at least one embodiment, at any given time during perfusion, the flow rate of the perfusion fluid can be within about 20%, about 15%, about 10%, about 8%, about 5%, about 3%, about 2%, about 1%, or about 0.5% of the patient's blood flow rate (in mL / min). Importantly, the flow rate of the perfusion fluid circulated via the closed loop does not deviate significantly from the patient's own blood flow rate to avoid ischemia and / or underperfusion.

[0205] Exemplary flow rates of the perfusate circulating through the closed loop can be in (but are not limited to) the following ranges: about 75 mL / min to about 750 mL / min, about 100 mL / min to about 650 mL / min, about 125 mL / min to about 600 mL / min, about 150 mL / min to about 500 mL / min, about 175 mL / min to about 400 mL / min, 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 maintains the flow rate of the perfusate in the closed loop at about 500 mL / min / 1.73 m 2 body surface area to about 650 mL / min / 1.73 m 2 body surface area for about 15 minutes to about 4 hours.

[0206] The perfusate can circulate through the closed loop for a duration in (but not limited to) the range of about 5 minutes to about 5 hours, about 15 minutes to about 4 hours, about 30 minutes to about 3 hours, or about 1 hour to about 2 hours. In at least one embodiment, the treatment duration can occur over several days, such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, etc.

[0207] In at least one embodiment, using the system disclosed herein, a higher drug dose can be directly and only administered to one or more kidneys than can be safely administered systemically in other ways. In at least one embodiment, because the perfusate does not substantially leak outside of one or more kidneys, a lower total drug dose may be required to achieve the same therapeutic effect (the same as the therapeutic effect achieved with a larger dose that undergoes systemic circulation or only partial isolation of the renal circulation).

[0208] In at least one embodiment, during perfusion, less than about 50% v / v, less than about 40% v / v, less than about 30% v / v, 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, or substantially none (0% v / v) of the perfusate (such as blood and / or drug) circulating through the closed loop leaks outside of the closed loop.

[0209] Reduced leakage of the perfusion fluid outside the closed loop (compared to other methods disclosed in the art) can be attributed to the tight seal formed within the closed loop and each individual component utilized within the closed loop.

[0210] In at least one embodiment, there is still some leakage of the perfusion fluid from the closed loop. 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 perfusion fluid circulating through the closed loop can leak outside the closed loop. Any amount of drug lost due to leakage of the perfusion fluid can be replaced in the perfusion fluid so as to keep the drug exposure of the kidney constant over the calculated exposure time. In at least one embodiment, the calculated exposure time can be in the range of: 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 sub-range therebetween.

[0211] Therapeutic composition

[0212] A drug suitable for treating a renal disorder (i.e., the drug included in the perfusion fluid) can include a therapeutic polynucleotide sequence. In at least one embodiment, the therapeutic polynucleotide sequence can encode a protein for treating a renal disorder. The protein for treating a renal disorder can be of human origin or can be derived from different species (such as but not limited to mouse, cat, pig, or monkey). In at least one embodiment, the protein encoded by the therapeutic polynucleotide sequence can correspond to a gene expressed in the human kidney.

[0213] Exemplary proteins can include (but are not limited to) NPHP1 (SEQ ID NO: 1-4), PC2 (SEQ ID NO: 5), PC1 or various subdomains thereof (SEQ ID NO: 6-7), variants thereof, or combinations thereof. One or more proteins used can also be functional variants of the proteins mentioned herein and can exhibit significant amino acid sequence identity compared to the original protein. For example, the amino acid identity can total 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 a variant of a protein that is capable of partially or fully performing the function of the corresponding naturally occurring protein. Functional variants of a protein can include, for example, proteins that differ from their naturally occurring counterparts by one or more amino acid substitutions, deletions, or additions.

[0214] The amino acid substitution can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, i.e., the amino acid residue is substituted with an amino acid having a similar polarity that serves 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 can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids that can be used for conservative substitution include, for example, non-polar 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.

[0215] In addition, a protein regarded as a variant is a protein that differs from its naturally occurring counterpart by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids. These additional amino acids can be present within the amino acid sequence of the original protein (i.e., as an insertion), or they can be added to one or both ends of the protein. Basically, the insertion can be made at any position provided that the addition of the amino acid does not impair the ability of the polypeptide to perform the function of the naturally occurring protein in the subject being treated. In addition, variants of the protein also include proteins that lack one or more amino acids compared to the original polypeptide. Such deletions can affect any amino acid position provided that they do not impair the ability to perform the normal function of the protein.

[0216] Finally, variants of the target protein also refer to proteins that are different from the naturally occurring protein due to structural modifications, such as modified amino acids. Modified amino acids are amino acids that have been modified by natural processes, such as processing or post-translational modification, or by chemical modification methods known in the art. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, glutathionylation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent binding to phosphatidylinositol, flavine derivatives, lipoteichonic acid, fatty acids, or lipids.

[0217] The therapeutic polynucleotide sequence encoding the target protein can be administered to the subject being treated in the form of a gene therapy vector, i.e., a nucleic acid construct, which contains a coding sequence flanked by other sequences required for the expression of the foreign nucleic acid (such as a promoter, kozak sequence, polyA signal, etc.), including translation and termination codons.

[0218] For example, the gene therapy vector can be part of a mammalian expression system. Suitable mammalian expression systems and expression constructs are commercially available. In addition, several mammalian expression systems are distributed by different manufacturers and can be used in the present invention, such as plasmid- or virus vector-based systems, such as LENTI-Smart TM (InvivoGen), GenScript TM expression vector, pAdVAntage TM (Promega), ViraPower TM lentivirus, adenovirus expression system (Invitrogen) and adeno-associated virus expression system (Cell Biolabs).

[0219] The gene therapy vector for expressing the exogenous therapeutic polynucleotide sequence of the present invention can be, for example, a viral or non-viral expression vector, which is suitable for introducing the exogenous therapeutic polynucleotide sequence into cells for subsequent expression of the protein encoded by the nucleic acid. The expression vector can be an episomal vector, i.e., a vector capable of autonomously replicating itself in a host cell; or an integrating vector, i.e., a vector stably incorporated into the cell genome. Expression in the host cell can be constitutive or regulatable (e.g., inducible).

[0220] In one embodiment, the gene therapy vector is a viral expression vector. The viral vector used in the present invention can contain a viral genome in which a part of the natural sequence has been deleted to introduce a heterologous polynucleotide without destroying the infectivity of the virus. Due to the specific interaction between the viral components and the host cell receptors, viral vectors are highly suitable for efficiently transferring genes into target cells. Viral vectors suitable for promoting 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 virus shuttle suitable for gene therapy, its variants and its combinations.

[0221] "Adenovirus expression vector" or "adenovirus" is intended to include these constructs containing adenovirus sequences that are sufficient to (a) support the packaging of the therapeutic polynucleotide sequence construct, and / or (b) ultimately express the tissue- and / or cell-specific construct cloned therein. In one embodiment of the present invention, the expression vector contains a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kilobase (kb) linear double-stranded DNA virus, allows large segments of adenovirus DNA to be replaced with exogenous sequences of up to 7 kb.

[0222] Adenovirus growth and manipulation are known to those skilled in the art and exhibit a broad host range both in vitro and in vivo. This group of viruses can be obtained at high titers, for example, 10 9 to 10 11 plaque-forming units / ml, and they are highly infectious. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and thus have low genotoxicity to the host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus, indicating its safety and / or therapeutic potential as an in vivo gene transfer vector.

[0223] Retroviruses (also referred to as "retroviral vectors") can be selected as gene delivery vectors because they are able to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a wide range of species and cell types, and be packaged in special cell lines.

[0224] The retroviral genome contains three genes, gag, pol, and env, which encode the capsid protein, polymerase, and envelope components, respectively. Sequences found upstream of the gag gene contain signals for packaging the genome into viral particles. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These sequences contain strong promoter and enhancer sequences and are also required for integration into the host cell genome.

[0225] To construct a retroviral vector, a nucleic acid encoding the gene of interest is inserted into the viral genome in place of certain viral sequences, thereby generating a replication-defective virus. To produce viral particles, a packaging cell line containing the gag, pol, and / or env genes but lacking the LTR and / or packaging components is constructed. When a recombinant plasmid containing cDNA together with the retroviral LTR and packaging sequences is introduced into this cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture medium. Then, the culture medium containing the recombinant retrovirus is collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a wide variety of cell types. However, integration and stable expression require dividing host cells.

[0226] Retroviruses can be derived from any of the subfamilies. For example, vectors from murine sarcoma virus, bovine leukemia virus, Rous Sarcoma Virus, murine leukemia virus, mink cell focus-inducing virus, reticuloendotheliosis virus or avian leukosis virus can be used. Those skilled in the art will be able to combine portions derived from different retroviruses, such as LTRs, tRNA binding sites and packaging signals to provide recombinant retroviruses. These retroviruses are then typically used to generate transduction-competent retroviral vector particles. For this purpose, the vector is introduced into a suitable packaging cell line. Retroviruses can also be engineered to integrate site-specifically into the DNA of the host cell by incorporating a chimeric integrase into the retroviral particle.

[0227] Because herpes simplex virus (HSV) is neurotropic, it has attracted a great deal of attention in the treatment of neurological disorders. In addition, the ability of HSV to establish a latent infection in non-dividing neuronal cells without integrating into the host cell chromosome or otherwise altering the metabolism of the host cell, and the presence of promoters that are active during latency make HSV an attractive vector. And although much attention has been focused on the neurological applications of HSV, this vector can also be used in other tissues given its broad host range.

[0228] Another factor that makes HSV an attractive vector is the size and organization of the genome. Because HSV is large, it is less of a problem to incorporate multiple genes or expression cassettes compared to other smaller viral systems. Additionally, the availability of different viral control sequences with different properties (time, intensity, etc.) allows for greater control of expression compared to other systems. There is also the advantage that the virus has relatively little splicing information, further facilitating genetic manipulation.

[0229] HSV is also relatively easy to manipulate and can be grown to high titers. Thus, delivery is not a problem either in terms of the volume required to obtain a sufficient multiplicity of infection (MOI) or in terms of reducing the need for repeated dosing. Non-toxic variants of HSV have been developed and can be readily used in gene therapy situations.

[0230] Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. This higher complexity allows the virus to regulate its life cycle, as in the case of latent infection. Some examples of lentiviruses include human immunodeficiency virus (HIV-1, HIV-2) and simian immunodeficiency virus (SIV). Lentiviral vectors have been generated by multiple attenuation of the HIV virulence genes, for example, the deletion of the genes env, vif, vpr, vpu, and nef renders the vector biologically safe.

[0231] Lentiviral vectors are plasmid-based or virus-based and are constructed to carry the necessary sequences for incorporating foreign nucleic acids, for selecting nucleic acids, and for transferring nucleic acids into host cells. The gag, pol, and env genes of the vector 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.

[0232] Vaccinia virus vectors have been widely used because they are easy to construct, the expression levels obtained are relatively high, the host range is wide, and the capacity to carry DNA is large. Vaccinia contains a linear double-stranded DNA genome of approximately 186 kb, which exhibits a significant "A-T" bias. The genome is flanked by inverted terminal repeats of approximately 10.5 kb. Most essential genes appear to be located within the central region, which is the most highly conserved in poxviruses. The estimated number of open reading frames in vaccinia virus is 150 to 200. Although both strands are coding, extensive overlap of reading frames is not common.

[0233] At least 25 kb can be inserted into the vaccinia virus genome. The prototype vaccinia vector contains a transgene inserted into the viral thymidine kinase gene via homologous recombination. The vector is selected based on the tk phenotype. Inclusion of the untranslated leader sequence of encephalomyocarditis virus results in higher expression levels than conventional vectors, where the transgene accumulates to 10% or more of the proteins in infected cells within 24 hours.

[0234] Empty capsids of papovaviruses, such as murine polyomavirus, have received attention as possible vectors for gene transfer. The use of empty polyomavirus was first described when polyomavirus DNA and purified empty capsids were incubated in a cell-free system. The DNA of the new particles is protected from the action of pancreatic DNase. The reconstituted particles are used to transfer a transforming polyomavirus DNA fragment into rat FIII cells. The empty capsids and reconstituted particles consist of all three polyomavirus capsid antigens, VP1, VP2, and VP3.

[0235] AAVs are parvoviruses belonging to the Dependovirus genus. They are small non-enveloped single-stranded DNA viruses that require a helper virus for replication. Coinfection with a helper virus (e.g., adenovirus, herpesvirus, or vaccinia virus) is required to form a functionally intact AAV virion. In vitro, in the absence of coinfection with a helper virus, AAV establishes a latent state in which the viral genome exists in an episomal form but does not produce infectious virions. Subsequently, the genome is "rescued" by infection with a helper virus, allowing it to replicate and be packaged into viral capsids, thereby reconstituting infectious virions. Recent data indicate that both wild-type AAV and recombinant AAV exist primarily as large episomal concatemers in vivo. In one embodiment, the gene therapy vector used herein is an AAV vector. The AAV vector can be a purified replication-incompetent pseudotyped rAAV particle.

[0236] AAV is not associated with any known human disease, 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 from different species. Compared to some vectors that are rapidly cleared or inactivated by both cellular and humoral responses, AAV vectors have been shown to induce persistent transgene expression in various tissues in vivo. The persistence of recombinant AAV-mediated transgenes in non-dividing cells in vivo can be attributed to the lack of native AAV viral genes and the ability of the vector to form episomal concatemers via ITR ligation.

[0237] AAV is an attractive vector system for cell transduction in the present invention because it has high-frequency persistence as an episomal concatemer and it can infect non-dividing cells, including cardiomyocytes, thus making it suitable for delivering genes to mammalian cells, for example, in tissue culture and in vivo.

[0238] Typically, rAAV is prepared by co-transfecting a plasmid containing the gene of interest and / or an expression plasmid (e.g., pIM45) containing the wild-type AAV coding sequence without terminal repeats, with the gene of interest flanked by two AAV terminal repeats. The cells are also infected and / or transfected with adenovirus and / or a plasmid carrying the adenovirus genes required for AAV helper functions. Stocks of rAAV prepared in this manner are contaminated with adenovirus, which must be physically separated from the rAAV particles (e.g., by cesium chloride density centrifugation or column chromatography). Alternatively, an adenovirus vector containing the AAV coding region and / or a cell line containing the AAV coding region and / or some or all of the adenovirus helper genes can be used. A cell line carrying rAAV DNA as an integrated provirus can also be used.

[0239] There are multiple serotypes of AAV in nature, with at least twelve serotypes (AAV1 - AAV13). Despite high homology, different serotypes have tropisms for different tissues. After transfection, AAV elicits only a mild immune response (if any) in the host. Thus, AAV is highly suitable for gene therapy methods.

[0240] In at least one embodiment, the present disclosure can be directed to a medicament comprising an AAV vector, wherein the AAV vector is one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, ANCA AAV, chimeric AAVs derived therefrom, variants thereof, and combinations thereof, which would be even more suitable for efficient transduction in the 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.

[0241] A suitable dose of AAV for humans can be in the range of about 1×10 8 vector genomes / kg body weight (vg / kg) to about 3×10 14 vg / kg, about 1×10 8 vg / kg, about 1×10 9 vg / kg, about 1×10 10 vg / kg, about 1×10 11 vg / kg, about 1×10 12 vg / kg, about 1×10 13 vg / kg, or about 1×10 14 vg / kg. The total amount of viral particles or DRP is, is about, is at least, is at least about, does not exceed, or does not exceed about 5×10 15 vg / kg, 4×10 15 vg / kg, 3×1015 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 10 vg / kg, 8×10 10 vg / kg, 7×10 10 vg / kg, 6×10 10 vg / kg, 5×10 10vg / 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 within the range defined by any two of these values. The doses listed above are in units of vg / kg of kidney tissue.

[0242] In at least one embodiment, using the systems and methods disclosed herein, a higher drug dose can be directly and solely administered to the kidney than may be safely administered systemically otherwise because the perfusion fluid essentially does not leak outside the kidney. Without being construed as limiting, it is believed that AAV toxicity can be attributed to systemic effects such as hepatotoxicity, platelet activation and loss, and complement activation and loss. All these toxicities and other effects can be reduced, minimized or completely avoided by applying the local regional perfusion fluid described in the methods and systems disclosed herein. Thus, a dose of up to about 1×10 16 vg / kg of kidney tissue or higher can be well tolerated. In at least one embodiment, the kidney AAV dose expressed as vg / kg of kidney tissue can exceed the highest systemic administration dose by about 2 to about 200-fold, about 5 to about 150-fold, about 10 to about 100-fold, or any sub-range thereof.

[0243] In addition to 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 permit in vivo expression of proteins in target cells include, for example, plasmids, modified RNAs, mRNAs, cDNAs, antisense oligomers, DNA-lipid complexes, nanoparticles, exosomes, any other non-viral shuttle suitable for gene therapy, their variants, and their combinations.

[0244] In addition to viral vectors and non-viral expression vectors, nuclease systems can also be used in combination with vectors and / or electroporation systems to enter patient cells and introduce genes encoding target proteins or their functional variants or fragments therein. Exemplary nuclease systems can include (but are not limited to) clustered regularly interspaced short palindromic repeats (CRISPR), DNA-cleaving enzymes (such as Cas9), meganucleases, TALENs, zinc finger nucleases, any other nuclease system suitable for gene therapy, their variants, and their combinations. For example, in one embodiment, one viral vector (e.g., AAV) can be used for a nuclease (e.g., CRISPR) and another viral vector (e.g., AAV) can be used for a DNA-cleaving enzyme (e.g., Cas9) to introduce both (the nuclease and the DNA-cleaving enzyme) into target cells.

[0245] Other vector delivery systems that can be used to deliver therapeutic polynucleotide sequences encoding therapeutic genes to cells are receptor-mediated delivery vehicles. They 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, the delivery can be highly specific. Receptor-mediated gene targeting vehicles can include two components: a cell receptor-specific ligand and a DNA binder.

[0246] Methods suitable for transferring non-viral vectors into target cells are, for example, liposome transfection, calcium phosphate co-precipitation, DEAE-dextran methods, and direct DNA introduction methods using micropipettes, ultrasound, electroporation, etc. Before introducing the vector, renal cells can be treated with permeabilizing agents such as phosphatidylcholine, streptolysin, sodium caprate, decanoyl carnitine, tartaric acid, lysophosphatidylcholine, Triton X-100, etc. Exosomes can also be used to transfer naked DNA or DNA encapsulated in an AAV capsid.

[0247] The gene therapy vector of the present invention may comprise a promoter functionally linked to a nucleic acid sequence encoding a target protein. The promoter sequence should be tight and ensure strong expression. Preferably, the promoter provides expression of the target protein in the kidneys of a patient who has been treated with the gene therapy vector. In some embodiments, the gene therapy vector comprises a nephron-specific promoter operably linked to a nucleic acid sequence encoding a target protein. As used herein, a "nephron-specific promoter" refers to a promoter that has at least 2-fold higher activity in renal cells than in any other non-renal cell type. Preferably, the nephron-specific promoter suitable for use in the vectors of the present invention has 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 renal cells than in non-renal cell types. In addition, the nephron-specific promoter may be specific for a particular nephron subunit (e.g., proximal tubule, distal tubule, loop of Henle, collecting duct, glomerulus, etc.) to provide higher or exclusive expression in the particular subunit.

[0248] The nephron-specific promoter may 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 may include kidney-specific cadherin (KSPC), Na + / glucose cotransporter 2 (SGLT2), sodium-potassium-chloride 2 cotransporter 2 (NKCC2), E-cadherin (ECAD) or a podocyte-specific promoter such as the podocin promoter NPHS2.

[0249] The vectors suitable for the present invention may have different transduction efficiencies. Thus, a viral vector or a non-viral vector transduces more than, equal to or at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or 100% of the cells at the targeted vascular site. More than one vector (viral or non-viral, or a combination thereof) may be used simultaneously or sequentially. This can be used to transfer more than one polynucleotide, and / or target more than one cell type. When multiple vectors or multiple agents are used, more than one transduction / transfection efficiency may be produced.

[0250] A pharmaceutical composition containing a gene therapy vector can be prepared as a liquid solution or suspension. The pharmaceutical composition of the present invention may include commonly used pharmaceutically acceptable excipients, such as diluents and carriers. Specifically, the composition contains a pharmaceutically acceptable carrier, such as water, physiological saline, Ringer's solution, or dextran solution. In addition to the carrier, the pharmaceutical composition may also contain emulsifiers, pH buffers, stabilizers, dyes, etc.

[0251] In at least one embodiment, the pharmaceutical composition will contain a therapeutically effective gene dose, which is a dose that is non-toxic to the subject and capable of preventing or treating renal disorders in the subject. The prevention or treatment of renal disorders can be evaluated by changes in phenotypic characteristics associated with the renal disorder, where such changes can effectively prevent or treat the renal disorder. Thus, a therapeutically effective gene dose is generally a gene dose that is sufficient to improve or prevent the pathogenic renal phenotype of the treated subject when administered in a physiologically tolerable composition.

[0252] The following exemplary embodiments are now described:

[0253] Embodiment 1: A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising:

[0254] Positioning at least one perfusion catheter in the renal artery of the kidney;

[0255] Positioning at least one recovery catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one recovery catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and

[0256] Flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid contains a polynucleotide sequence packaged in an AAV vector, wherein the AAV vector contains an AAV5 capsid protein, and wherein the closed circuit substantially isolates the perfusion through the kidney from the systemic circulation of the subject.

[0257] Embodiment 2: The method according to Embodiment 1, wherein a dose of the AAV vector is delivered via the closed circuit and maintained at a concentration of at least about 5×10 7 vector genomes per milliliter of plasma during perfusion, and wherein the leakage of the vector into the systemic circulation of the subject present is maintained at less than or equal to about 5×10 7 vector genomes per milliliter of plasma during perfusion, and wherein the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0258] Embodiment 3: The method according to any one of the preceding embodiments, wherein the vector genome copy number (vg / dg) per diploid genome after perfusion is at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater or at least 30-fold greater compared to the same AAV vector actually containing different capsid proteins, or is from about 2 vg / dg to about 25 vg / dg.

[0259] Embodiment 4: A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising:

[0260] Positioning at least one perfusion catheter in the renal artery of the kidney;

[0261] Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and

[0262] Flowing a perfusion fluid through the closed circuit, wherein the perfusion fluid comprises a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, wherein the closed circuit substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein a dose of the AAV vector is delivered via the closed circuit and maintained at a concentration of at least about 5×10 7 vector genomes per milliliter of plasma during perfusion, and wherein the vector leaking into the patient's systemic circulation that is present remains less than or equal to about 5×10 7 vector genomes per milliliter of plasma during perfusion, wherein the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

[0263] Embodiment 5: A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising:

[0264] Positioning at least one perfusion catheter in the renal artery of the kidney;

[0265] Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and

[0266] Flowing a perfusion fluid through the closed loop, wherein the perfusion fluid comprises a polynucleotide sequence packaged in an AAV vector, the AAV vector comprising a non-AAV5 capsid protein, wherein the closed loop substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein the vector genome copy number per diploid genome is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold greater after perfusion compared to the same AAV vector actually comprising an AAV5 capsid protein.

[0267] Embodiment 6: A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising:

[0268] Positioning at least one perfusion catheter in the renal artery of the kidney;

[0269] Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device form a closed perfusion loop through the kidney; and

[0270] Flowing a perfusion fluid through the closed loop, wherein the perfusion fluid comprises a polynucleotide sequence packaged in an AAV vector, the AAV vector comprising a capsid protein, wherein the closed loop substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein the vector genome copy number per diploid genome is at least about 5-fold, at least about 10-fold, at least about 20-fold, or at least about 30-fold greater after perfusion compared to the same AAV vector actually comprising a different capsid protein.

[0271] Embodiment 7: The method according to any one of the preceding embodiments, wherein the polynucleotide sequence encodes nephrocystin-1 protein or a functional variant thereof.

[0272] Embodiment 8: The method according to any one of the preceding embodiments, wherein the polynucleotide sequence encodes polycystin-2 protein or a functional variant thereof.

[0273] Embodiment 9: The method according to any one of the preceding embodiments, wherein the therapeutic polynucleotide sequence comprises a renal-specific promoter.

[0274] Embodiment 10: The method according to any one of the preceding embodiments, wherein positioning the at least one perfusion catheter in the renal artery comprises positioning the at least one perfusion catheter via the femoral artery.

[0275] Embodiment 11: The method according to any one of the preceding embodiments, wherein positioning the at least one retrieval catheter in the renal vein comprises percutaneously positioning the at least one retrieval catheter via the femoral vein or the jugular vein.

[0276] Embodiment 12: The method according to any one of Embodiments 1-10, wherein positioning the at least one recovery catheter in the renal vein includes positioning the at least one recovery catheter percutaneously via an open access.

[0277] Embodiment 13: The method according to any one of the preceding embodiments, wherein flowing the perfusion fluid through the closed loop includes:

[0278] Flowing the perfusion fluid through the membrane oxygenation device before entering the renal artery via the one or more perfusion catheters.

[0279] Embodiment 14: The method according to any one of the preceding embodiments, the method further comprising:

[0280] Adding additional perfusion fluid to the closed loop or diluting the perfusion fluid with a saline solution of about 5% to about 50% v / v to calculate the bladder excretion volume.

[0281] Embodiment 15: The method according to any one of the preceding embodiments, wherein the closed loop maintains the flow rate of the perfusion fluid at about 500 mL / min / 1.73 m 2 body surface area per kidney to about 650 mL / min / 1.73 m 2 body surface area for about 15 minutes to about 4 hours.

[0282] Embodiment 16: The method according to any one of the preceding embodiments, wherein the closed loop maintains the flow rate of the perfusion fluid at about 150 mL / min / 1.73 m 2 body surface area per kidney to about 700 mL / min / 1.73 m 2 body surface area for about 15 minutes to about 4 hours.

[0283] Embodiment 17: The method according to any one of the preceding embodiments, the method further comprising applying negative pressure at the at least one recovery catheter, wherein the negative pressure is in the range of about -100 mmHg to 120 mmHg.

[0284] Embodiment 18: The method according to any one of the preceding embodiments, wherein one or more of the at least one perfusion catheter or the at least one recovery catheter are introduced percutaneously or non-percutaneously.

[0285] Embodiment 19: The method according to any one of the foregoing 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 or substantially no blood circulating through the closed circuit leaks out of the closed circuit.

[0286] Embodiment 20: The method according to any one of the foregoing 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 or substantially no perfusate circulating through the closed circuit leaks out of the closed circuit.

[0287] Embodiment 21: The method according to any one of the foregoing embodiments, wherein one or more of the at least one perfusion catheter or the at least one recovery catheter is a balloon catheter.

[0288] Embodiment 22: A system for performing regional perfusion of a local area of a patient's kidney when fluidly coupled to the kidney, the system comprising:

[0289] At least one perfusion catheter adapted to be inserted into the renal artery of the kidney;

[0290] At least one recovery catheter adapted to be inserted into the renal vein of the kidney;

[0291] A membrane oxygenation device 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 oxygenation device together are adapted to form a closed circuit through the kidney, the closed circuit being isolated from the patient's systemic circulation;

[0292] A reservoir containing a perfusate, the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and

[0293] A pump configured to drive the perfusate through the at least one perfusion catheter and the at least one recovery catheter.

[0294] Embodiment 23: A system for performing regional perfusion of a patient's kidney, the system comprising:

[0295] At least one perfusion catheter inserted into the renal artery of the kidney;

[0296] at least one retrieval catheter inserted into the renal vein of the kidney; and

[0297] a membrane oxygenation device fluidly coupled to the at least one perfusion catheter, the at least one retrieval catheter, and an oxygen source, wherein the at least one perfusion catheter, the at least one retrieval catheter, and the membrane oxygenation device together with the kidney form a closed loop through the kidney, the closed loop being isolated from the systemic circulation of the patient;

[0298] a reservoir containing a perfusion fluid, the perfusion fluid containing a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and

[0299] a pump configured to drive the perfusion fluid into the kidney via the at least one perfusion catheter and out of the kidney via the at least one retrieval catheter.

[0300] Embodiment 24: The system according to Embodiment 22 or Embodiment 23, wherein the AAV vector comprises an AAV5 capsid protein.

[0301] Embodiment 25: The system according to any one of Embodiments 22-24, wherein the polynucleotide sequence encodes polycystin-1 protein or a functional variant thereof.

[0302] Embodiment 26: The system according to any one of Embodiments 22-24, wherein the polynucleotide sequence encodes polycystin-2 protein or a functional variant thereof.

[0303] Embodiment 27: The system according to any one of Embodiments 22-26, the system being configured to perform the method according to any one of Embodiments 1-21.

[0304] Embodiment 28: A gene therapy vector adapted to transduce renal cells of a human subject, the gene therapy vector comprising:

[0305] an adeno-associated virus (AAV) vector comprising an AAV5 capsid protein; and

[0306] a polynucleotide sequence packaged in the AAV vector.

[0307] Embodiment 29: The gene therapy vector according to Embodiment 28, wherein the polynucleotide sequence encodes polycystin-1 or a functional variant thereof.

[0308] Embodiment 30: The gene therapy vector according to Embodiment 28, wherein the polynucleotide sequence encodes polycystin-2 or a functional variant thereof.

[0309] Embodiment 31: The gene therapy vector according to any one of Embodiments 28 - 30, wherein the therapeutic polynucleotide sequence comprises a nephron - specific promoter.

[0310] Embodiment 32: A method of delivering a therapeutic composition to a subject in need thereof, the method comprising locally delivering the therapeutic composition to the kidney of the subject while substantially avoiding introducing the therapeutic composition into the systemic circulation or other organs, the therapeutic composition comprising the gene therapy vector according to any one of Embodiments 28 - 31.

[0311] Embodiment 33: A method of screening AAV serotypes to identify a major serotype for local gene delivery to an organ type, the method comprising:

[0312] identifying a plurality of AAV vector candidates, each AAV vector candidate corresponding to a different AAV serotype;

[0313] generating a perfusion fluid composition comprising each of the plurality of AAV vector candidates, each of the plurality of AAV vector candidates comprising a polynucleotide packaged therein; and

[0314] performing local transduction of tissues in an organ of an organ type of an animal by creating a closed loop in the vasculature of the target organ, the perfusion fluid composition circulating via the closed loop; and

[0315] selecting the serotype of the AAV vector candidate having the highest vector genome biodistribution and / or the greatest RNA / protein expression in the organ compared to the remaining AAV vector candidates as the major serotype.

[0316] Embodiment 34: The method according to Embodiment 33, wherein the organ type is kidney, heart, pancreas or liver.

[0317] Embodiment 35: The method according to Embodiment 32 or Embodiment 33, wherein local transduction of the tissues in the organ is performed using the system according to Embodiment 22 or Embodiment 23.

[0318] Illustrative Examples

[0319] The following examples are set forth to aid in understanding the disclosure and should of course not be regarded as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments include substitution of all equivalents known or developed subsequently, which will be within the scope of those skilled in the art, and minor variations in formulation or experimental design will be considered to fall within the scope of the embodiments incorporated herein.

[0320] The LRP system described below includes the following components: a percutaneous arterial catheter for antegrade perfusion (entering via the femoral artery) of the occluded renal artery; a percutaneous venous catheter for occluding the renal vein and returning venous blood to the LRP system (entering via the jugular vein); and an ECMO device with a reservoir and connected tubing for providing oxygen and removing carbon dioxide from the blood in the LRP system. The LRP procedure begins when oxygenated blood is perfused antegrade into the artery while deoxygenated blood returned is collected from the venous system via the venous catheter. Then, the blood is collected in the reservoir, oxygenated, and reinfused antegrade into the organ via the arterial catheter. During the entire procedure, blood samples can be obtained via the reservoir, or drugs can be introduced.

[0321] Example 1: LRP Procedure

[0322] Using Figure 18 the LRP system 1800 depicted in and described with respect to the accompanying drawings, LRP was performed on pigs. The accessory devices / components used in these examples are listed in Table 4, including their intended uses and uses in the LRP system according to embodiments of the present disclosure.

[0323] Table 4: Components for the LRP Procedure

[0324]

[0325]

[0326] A custom catheter was used as the venous return catheter and it included the following dimensions: an outer diameter of 19Fr (6.3 mm); an inner diameter of 12Fr (4.0 mm); an available length of 80 cm; a balloon diameter of 25 mm; and a tip length of 20 mm (similar to Figures 1 to 3 the exemplary custom catheter shown in and described with respect to the accompanying drawings). The materials included: Pebax 63 supported by a strong stainless steel braid as the shaft; flexible Chronoprene 25A as the balloon; and Pebax 35 loaded with BaSO4 in the tip for radiopacity. The custom catheter was designed to support a suction flow rate of approximately 800 mL / min at -80 mmHg.

[0327] Figure 20 Include radiographs showing the successful placement of the arterial and venous catheters in the renal artery and renal vein of the pig kidney, respectively. In the bottom image, contrast agent was injected intravenously, revealing the overall seal of the renal vasculature and the closure system.

[0328] Now describe the detailed protocol of the LRP procedure followed in this example:

[0329] (1) Place the research animal in the dorsal recumbent position;

[0330] (2) Prepare the animal for endovascular catheterization;

[0331] (3) Using angiography, evaluate the angles of the renal vein with the jugular and femoral accesses using the smallest acute angle;

[0332] (4) Enter the arterial circulation of the kidney from the femoral artery with a Stryker FlowGate 2 catheter (determine the side based on the angle);

[0333] (5) Use the customized venous catheter described above to enter the venous circulation (side and entry point: determined based on the individual animal);

[0334] (6) Place the catheter in its final position in an open configuration (i.e., balloon down) to inject some contrast fluid and observe the renal circulation;

[0335] (7) Place the catheter in the aorta and vena cava until the procedure begins;

[0336] (8) Place a PressureWire X through the Flowgate 2 catheter and place it into one of the renal arteries;

[0337] (9) Prepare the ECMO system by degassing and perfusing with saline; connect the venous and arterial lines to the ECMO while clamping to avoid introducing air;

[0338] (10) Turn on the ECMO pump;

[0339] (11) Release the venous line;

[0340] (12) Begin exchanging blood with saline; if everything is stable, release the arterial line and establish the LRP circuit; the suction on the venous side is variable and adjusted as needed (e.g., from -50 mmHg to 0);

[0341] (13) Place the venous catheter in the appropriate position in the renal vein;

[0342] (14) Inflate the balloon;

[0343] (15) Check the integrity and positioning of the catheter for injecting contrast;

[0344] (16) If the animal is stable, then:

[0345] a. Seal the renal artery with the Flowgate 2 catheter;

[0346] b. Checks: Sealing and position of the catheter for injecting contrast agent; pressure in the kidneys; pressure ratio of the kidneys to the whole body (target > 1); reservoir volume; RPM of the ECMO pump; and flow rate of the catheter;

[0347] (17) If everything is stable for 5 minutes, then:

[0348] a. Start the infusion of nitroglycerin at a rate of 2 micrograms per kilogram of body weight per minute via the arterial line;

[0349] b. Checks: Pressure in the kidneys; pressure ratio of the kidneys to the whole body (target > 1); reservoir volume; RPM of the ECMO pump; and flow rate of the catheter;

[0350] (18) If everything is stable for 5 minutes, then:

[0351] a. Start the treatment by injecting the gene therapy drug into the reservoir;

[0352] b. For the first animal group (Group B1): Administer a dose of 5.0×10 13 (prepared by diluting 1.8 mL of a vector solution with a titer of 2.8×10 13 vg / mL with 2.2 mL of vehicle);

[0353] c. For the second animal group (Group B2): Administer a dose of 6.0×10 14 vg (equivalent to 21.4 mL of a vector solution with a titer of 2.8×10 13 vg / mL);

[0354] (19) Continue renal LRP for 60 minutes;

[0355] (20) Check every 5 minutes: Pressure in the kidneys; pressure ratio of the kidneys to the whole body (target > 1); reservoir volume; RPM of the ECMO pump; flow rate of the catheter; all hemodynamic and cardiovascular parameters (pressure, HR);

[0356] (21) Check the urine output at t = 0, 15, 30, 45, and 60 minutes after the start of the procedure;

[0357] (22) Pay attention to the LRP reservoir volume, as there may be overfilling caused by the phrenic, gonadal, and adrenal veins or volume loss caused by urine production; these volume deviations can be dynamically managed;

[0358] (23) At t = 0, 5, 15, 30, 45, and 60 minutes: Collect blood samples from the following:

[0359] a. For excretion analysis, peripheral blood;

[0360] b. For vector infectivity analysis, the LRP system; and

[0361] c. For excretion analysis, the LRP system;

[0362] (24) At the end of 60 minutes of renal LRP:

[0363] a. Stop the infusion of nitroglycerin;

[0364] b. Deflate the balloon; and

[0365] c. Disengage the catheter;

[0366] (25) Discard the complete LRP circuit, reservoir, blood pump, and catheter in an appropriate biosafety cabinet;

[0367] (26) Immediately perform postoperative care, including (but not limited to) compression and administration of protamine;

[0368] The above procedures show that renal LRP with a sealed closed circuit may last at least 60 minutes. No acute sequelae were observed, and an indigocarmine test performed immediately after the LRP procedure showed normal renal function / was not affected by the procedure.

[0369] Factors that can be applied to optimize the LRP procedure to reduce the leakage of perfusate into the systemic circulation and avoid proximal and distal organ transduction include (but not limited to): perfusion time, drug dose, AAV serotype used, presence of neutralizing factors, endothelial permeability, flow rate, and perfusion pressure.

[0370] Example 2: Biodistribution study

[0371] Figure 21 is a graph showing renal transduction and biodistribution at 0.05 - 0.25 vg / dg (vector genome copies per diploid genome) after 60 minutes of LRP at a higher dose of 6.2×10 14 vg / kg. No significant contamination of untreated kidneys, livers, or other organs was detected, which confirmed the tightness of the LRP closed circuit.

[0372] Intravenous control animals were also tested. It has been found that renal LRP induces a more uniform transduction profile in the different parts measured, while the IV control shows preferential transduction in the cortical part of the kidney. Relative to the IV control, transduction in the liver by renal LRP is less obvious, where for the IV control, 17.2 vg / dg was detected in the liver, while for renal LRP, almost no transduction was observed in the liver.

[0373] Example 3: Vector quantification

[0374] Figure 22A and Figure 22B displayed at high dose (6.2×10 14 vg / kg, Figure 22A ) and low dose (5.6×10 13 vg / kg, Figure 22B ) at various time points during renal LRP of the vector genome per milliliter of plasma. The results revealed that holding for 60 minutes within the LRP circuit had a high retention rate (low vector clearance), less vector exposure to the systemic circulation, and very little vector leakage into the urine ( Figure 22A ). Vector exposure to the kidney appeared to be greatest throughout the procedure.

[0375] Figure 23A is a graph of C3a levels on several days after renal LRP treatment (LRP-1 and LRP2) of two different animals. Figure 23B is a graph of percent transduction inhibition at various sample dilutions. Both revealed that anti-AAV neutralizing factors were low in both animals and that there was no complement activation after renal LRP.

[0376] Figure 24A and Figure 24B are graphs of flow rate and pump speed, respectively, during renal LRP, which revealed a substantially constant flow rate of approximately 310 mL / min throughout the procedure.

[0377] These examples demonstrate the use of a clinically relevant animal model to target delivery of AAV to the kidney, resulting in a homogeneous transgene biodistribution. The embodiments described and exemplified herein are capable of developing a new generation of advanced renal therapies by minimizing systemic adverse reactions, significantly reducing the required vector dose, overcoming immune limitations, and having the potential for repeat treatment. Upon consideration, the use of the LRP system and method can be used in conjunction with other therapeutic agents and strategies.

[0378] Example 4: Serotype Screening

[0379] Serotype screening to identify efficient serotypes for local gene delivery to the kidney is now described as follows. First, multiple AAV vector candidates were identified. In this study, AAV1, AAV2, AAV5, AAV6, and AAV9 serotypes were selected as candidates. AAV vectors of each serotype were generated, each containing a CMV-GFP transgene cassette. Perfusates containing each of the five AAV types were prepared, totaling 4.9×10 14 vg (where 5.9×10 13vg). The LRP of porcine kidneys was performed using a similar protocol as described above and a customized catheter as described above. The total amount of vector injected into the closed circuit was 6.4×10 12 vg / kg, and the perfusion in the closed circuit was maintained for 60 minutes.

[0380] Figure 25 is a graph showing the vector genomes per mL of plasma measured at various time points during a 60-minute kidney LRP procedure in the LRP closed circuit relative to the systemic circulation for perfusates containing five different serotypes. The results revealed that retention within the LRP circuit for 60 minutes had a high retention rate (less vector excretion) and less vector exposure to the systemic circulation (less than 5×10 7 vg per mL of plasma).

[0381] Figure 26 is a graph showing the biodistribution of multiple AAV serotypes in the kidney based on the vector genome copy number per diploid genome measured in various sections of the LRP-treated kidney, which revealed that AAV5 had significantly higher efficiency than other serotypes (39.3-fold higher efficiency than AAV9). Figure 28 is a graph showing the relative quantification of transgenic mRNA levels of AAV5 in the treated kidney sections relative to the cumulative AAV in the kidney. Figure 27 is a graph showing the biodistribution in the kidney compared to the liver and other kidney-related tissues after perfusion with multiple AAV serotypes for 60 minutes, which revealed that transduction was restricted to the treated kidney sections and the renal artery and renal vein, with very little transduction occurring in the liver or the untreated kidney. Without wishing to be bound by theory, it is believed that the high efficiency of AAV5 can be attributed to the synergy resulting from maintaining a high concentration of AAV5 in the closed circuit for a fixed period (30 minutes to 1 hour) to allow transduction. In the absence of localization, it is believed that the systemic circulation would result in low transduction of AAV5 in the kidney, with a large amount of AAV5 ultimately located in the liver or other organs.

[0382] Upon consideration, by applying the LRP procedure to these organs, this method can also be applied to other organs. Exemplary organs can include (but are not limited to) the heart, liver, or pancreas. The LRP of the heart is described in International Application No. PCT / IB2020 / 000692, filed on August 26, 2020, the disclosure of which is hereby incorporated by reference in its entirety.

[0383] AAV5-CMV-GFP was further evaluated independently in the porcine kidneys of two different animals in the absence of other serotypes: one that underwent a 60-minute LRP procedure at a dose of 1.2×10 13 vg / kg ( Figure 29 andFigure 30 );and a LRP procedure that lasts for 52 minutes at a dose of 1.3×10 13 vg / kg ( Figure 31 and Figure 32 ). Two tests further demonstrate the relatively constant concentration of the vector in the LRP circuit and the relatively low amount of vector leakage into the systemic circulation, as well as the local biodistribution in the treated kidney, while the quantitative vector genomes in the untreated kidney and liver are not significant.

[0384] Example 5: Single-Kidney LRP Procedure for Biodistribution Studies

[0385] 5.1 Materials and Components

[0386] The LRP system and protocol for performing single-kidney LRP, which are used for the biodistribution studies described in Examples 6-8 below, are now described.

[0387] Using Figure 18 the LRP system 1800 depicted in

[0388] Table 5: Components for Single-Kidney LRP Procedure

[0389]

[0390]

[0391] The supply line catheter assembly includes a perfusion catheter, a 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 a diameter of 2-3.5 mm is considered) is large enough to establish a physiological perfusion flow. The catheter is compatible with a 14Fr introducer. The distal portion of the shaft is more flexible to allow the catheter to smoothly and non-invasively enter the renal artery. The flexibility is controlled by the hardness of the Pebax polymer: 72D proximal, 55D transition zone, and 35D distal portion.

[0392] The tip of the perfusion catheter is short (2-3 mm) to allow placement of the balloon near the branch in the case of a short main trunk of the renal artery. The tip is soft (Pebax 35D) and rounded to ensure non-invasive advancement in the vasculature. In addition, the inner diameter is slightly reduced to minimize the gap between the dilator and the tip.

[0393] The marker band is embedded below the balloon and near the tip to observe these landmarks under fluoroscopy.

[0394] The flexible occlusion balloon is mounted on the outer shaft and made of a polymer blend or polyurethane material. The soft balloon is gently adapted to the shape of the blood vessel to achieve a tight seal. The balloon is 3 - 5 mm long and has a disc-shaped inflated form, with a maximum outer diameter of approximately 11 mm. The balloon diameter depends on the inflation volume and has a linear relationship from an inflation diameter of approximately 4 mm at an inflation volume of about 0 mL to a diameter of approximately 11 mm at an inflation volume of about 0.5 mL. The gap between the inner shaft and the outer shaft is used to inflate the balloon, and the outer shaft has multiple inflation holes below the balloon.

[0395] The hub is connected to the proximal shaft end to serve as a user interface and allow connection of the following accessories: a luer connector for inflating the balloon with a syringe; a barbed connector for allowing connection of a perfusion catheter; and a hemostatic valve with a luer connector for flushing, where the valve can be used to replace a dilator with a guide wire and a pressure line, thus minimizing blood loss.

[0396] Before the tracking device, a supply line dilator is placed in the inner lumen of the perfusion catheter. The dilator fills the large inner lumen of the perfusion catheter to allow the catheter to advance through the vasculature without trauma. The outer diameter of the dilator is slightly smaller than the inner shaft of the perfusion catheter so that the dilator can move within the catheter with minimal friction.

[0397] The dilator tip tapers gradually to gently dilate the blood vessel for catheter passage and is rounded and atraumatic. The inner diameter of the tip is approximately 0.5 mm to minimize the gap with the guide wire. The dilator is compatible with guide wires of 0.018" and smaller. In the proximal portion of the dilator, its inner diameter gradually increases to optimize flexibility. The dilator is made of Pebax 35D and barium sulfate (BaSO4). Barium sulfate is added to make the catheter radiopaque. Additionally, radiopaque markers can be embedded in the shaft to indicate the tapered portion. Colored markers can be added to the proximal dilator portion to indicate how the dilator should be placed in the perfusion catheter. The hub is connected to the proximal end of the dilator shaft to facilitate guide wire insertion. In addition, a luer connector allows connection of a syringe for flushing.

[0398] A schematic diagram of the perfusion catheter is shown in Figure 33A where the balloon is in a retracted 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.

[0399] Other accessories included with the perfusion catheter are now described.

[0400] (1) An extension line with a 4-way stopcock is delivered with the catheter to connect it to the balloon inflation orifice at the hub.

[0401] (2) The 1 / 4" tubing assembly is connected to the barb connector of the hub. The tubing is approximately 10 cm long. A red on / off tubing clamp is placed on the tubing, which is used to clamp the perfusion flow. The red color indicates to the user that this line carries arterial blood. A T-piece is connected to the other end of the tubing. The T-piece is a straight connector with two 1 / 4" barbs on its short side and one Luer connector on its long side. The perfusion flow is directed directly from one 1 / 4" barb to the other. A 4-way stopcock is connected to the Luer connector on the side. This side port is used for degassing, injecting contrast agent, injecting pharmaceutical compositions and for obtaining samples.

[0402] (3) After production, a thin-walled plastic sleeve can be positioned on the distal end and the balloon to protect the balloon and its bond during transportation and storage. Remove the protective sleeve before using the catheter.

[0403] The return line catheter assembly includes a return catheter, a return dilator and accessories. The return catheter includes a reinforced inner shaft and an outer shaft. The inner shaft with an inner diameter of 4.06 mm (although considering diameters from 3 - 4.5 mm) is large enough to establish the flow of physiological drainage. The catheter is compatible with a 22Fr introducer. The distal shaft portion of the shaft is more flexible to enable the catheter to smoothly and non-invasively enter the renal vein. The flexibility of the shaft is controlled by the hardness of the Pebax polymer: 72D proximal, 55D transition zone and 35D distal portion.

[0404] The tip of the catheter has side holes / perforations (4 - 8 holes, diameter 0.5 - 2 mm) for drainage through it. The tip can be placed close to the vessel wall, and in the case where the distal opening is completely or partially occluded, the side holes help to avoid affecting the backflow. The tip length is short (4 - 8 mm long) to allow the balloon to be placed near the branch in the case where the main trunk of the renal vein is short. The tip is made of a soft and radiopaque polymer blend (Pebax 35D + BaSO4) and is not reinforced. The distal edge of the tip is rounded to ensure non-invasive advancement through the vasculature. In addition, the inner diameter of the tip is slightly reduced to minimize the gap between the dilator and the tip.

[0405] Marker bands are embedded below the balloon to observe these landmarks under fluoroscopy.

[0406] The flexible occlusion balloon is mounted on the outer shaft and is made of a polymer blend or polyurethane material. The soft balloon is gently adapted to the shape of the blood vessel to achieve a tight seal. The balloon size is 8 - 15 mm long, in a disk-shaped inflated form, with a maximum outer diameter of approximately 25 mm. The balloon diameter depends on the inflation volume, having a linear relationship from an inflation diameter of approximately 6 mm at an inflation volume of about 0 mL to a diameter of approximately 25 mm at an inflation volume of about 5 mL. The gap between the inner shaft and the outer shaft is used to inflate the balloon, where the outer shaft has multiple inflation holes below the balloon.

[0407] The hub is connected to the proximal end to serve as a user interface and allow connection of the following accessories: a luer connector for inflating the balloon with a syringe; a barbed connector allowing connection of a drainage catheter; and a hemostatic valve with a luer connector for irrigation, wherein the valve is for replacing a dilator having a guide wire and a pressure line, thereby minimizing blood loss.

[0408] Prior to the tracking device, a return line dilator is placed within the inner lumen of the retrieval catheter. The dilator fills the large inner lumen of the retrieval catheter to allow the catheter to advance through the vasculature without trauma. The outer diameter of the dilator is slightly smaller than the inner shaft of the retrieval catheter such that the dilator can move within the catheter with minimal friction.

[0409] The dilator tip tapers to gently dilate the blood vessel for catheter passage and is rounded and atraumatic. The inner diameter of the tip is approximately 0.95 mm to minimize the gap with the guide wire. The dilator is compatible with guide wires of 0.035" and smaller. In the proximal portion of the dilator, its inner diameter gradually increases to optimize flexibility. The dilator is made of Pebax 35D and BaSO4 added to make the catheter radiopaque. Additionally, radiopaque markers may be embedded in the shaft to indicate the tapered portion. Colored markers may be added to the proximal dilator portion to indicate how the dilator should be placed within the retrieval catheter. The hub is connected to the proximal end of the dilator shaft to facilitate guide wire insertion. Further, a luer connector allows connection of a syringe for irrigation.

[0410] A schematic illustration of the retrieval catheter is shown in Figure 34A wherein the balloon is in a retracted 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.

[0411] Other accessories included with the retrieval catheter are now described.

[0412] (1) An extension line with a 4-way stopcock is delivered with the catheter to connect it to the balloon inflation orifice at the hub.

[0413] (2) A 1 / 4" tubing assembly is connected to the barbed connector of the hub. The tubing is approximately 10 cm long. A blue on / off tubing clamp is placed on the tubing, which is for clamping the flow of drainage. The blue color indicates to the user that this line carries venous blood. A T-piece is connected to the other end of the tubing. The T-piece is a straight connector with two 1 / 4" barbs on the short side and a luer connector on the long side. The return flow is directed directly from one 1 / 4" barb to the other. A 4-way stopcock is connected to the luer connector on the side. This side orifice is for degassing, injecting contrast agent, injecting a pharmaceutical composition, and for obtaining samples.

[0414] (3) After production, a thin-walled plastic sleeve can be positioned over the distal end of the catheter and the balloon to protect the balloon and its bond during shipping and storage. Remove the protective sleeve before using the catheter.

[0415] The perfusion and retrieval catheter with all its accessories is secured to a cardboard sheet with straps, clamps, and a protective sleeve. The cardboard sheet with the connecting device is placed in a Tyvek bag and heat-sealed. The bag protects the product from contamination but allows ethylene oxide (EtO) gas to permeate for sterilization. The bag is then placed in a sturdy outer cardboard box for shipping and storage.

[0416] 5.2 Protocol

[0417] The entire LRP procedure can be divided into four phases: (1) the initiation phase, during which the LRP circuit is not yet established; (2) the stabilization phase, during which the LRP circuit is established; (3) the treatment delivery phase, during which the LRP circuit is established; and (4) the removal phase, during which the LRP circuit is interrupted. Each phase is now described.

[0418] Initiation phase: The arterial and venous catheters are interventionaly placed in their appropriate positions (i.e., into the left renal aorta and vein, the balloon is inflated, and the quality of the seal is evaluated).

[0419] Stabilization phase: LRP perfusion is started without any payload. The stabilization phase begins with the anterograde perfusion of the renal artery with oxygenated blood while deoxygenated blood returning is collected from the renal vein via the retrieval catheter. LRP perfusion is considered stable when the operator confirms the following metrics: a stable reservoir volume, a sufficient flow rate through the catheter, a physiological renal artery pressure in the absence of pulsation, a stable vacuum pressure, a stable rate of nitroglycerin infusion or other vasoactive substances, and the possible completion of the volume exchange of saline and blood in the reservoir. At this point, payload delivery via the LRP system can be initiated.

[0420] Treatment delivery phase: Once the stabilization phase ends, the treatment phase is started, and the therapeutic payload is introduced into the LRP system. The duration of this phase depends on the treatment delivery protocol. The total time of therapeutic payload recirculation is referred to as the "LRP duration".

[0421] Removal phase: Once the treatment delivery phase is completed according to the predetermined protocol, the LRP perfusion system is stopped, and the arterial (perfusion) and venous (retrieval) catheters are removed from the body following a specific procedure.

[0422] It should be noted that although the protocol discussed below refers to a single perfusion catheter and a single retrieval catheter, multiple catheters can be used to optimize the seal of the LRP circuit based on the patient's anatomy. For example, Figure 35A and Figure 35BThe deployment of a single perfusion catheter and a pair of perfusion catheters within the renal artery of the left kidney is shown respectively. Similarly, Figure 36A and Figure 36B the deployment of a single retrieval catheter and a pair of retrieval catheters within the renal vein of the left kidney are shown respectively.

[0423] Now, the detailed protocol of the LRP procedure followed in this and the following examples is described:

[0424] Initial phase

[0425] (1) Place the study animal in the dorsal recumbent position;

[0426] (2) Prepare a jugular central venous catheter in a sterile manner;

[0427] (3) As part of the preparation of the sterile access site, place a surgical drape and additional drapes to completely cover the animal (the jugular region and the femoral region are exposed);

[0428] (4) Place a set of additional drapes for use at the end of the procedure after removing the "contaminated" catheters;

[0429] Femoral artery:

[0430] (5) Identify the target vessel in the inguinal region under ultrasound guidance;

[0431] (6) Introduce an appropriate femoral introducer sheath (14-FR in diameter);

[0432] (7) Flush the catheter with heparinized saline solution;

[0433] Jugular vein:

[0434] (8) Repeat the previous steps (steps 5 to 7), but for the left jugular vein, use a 22-FR introducer sheath;

[0435] Catheter placement:

[0436] (9) Place a guide wire (0.014" or 0.018", e.g., Boston Scientific V18 or Abbott High-Torque, as judged by the intervenor) into the distal renal artery via a multi-purpose catheter (5-FR);

[0437] (10) Place at least one perfusion catheter in the main trunk of the renal artery to ensure homogeneous blood distribution to all parts of the kidney and ensure stable catheter position;

[0438] (11) Pass a PressureWire X (Abbott) through the supply line and place it in one of the renal artery branches;

[0439] (12) Inject contrast agent in an antegrade manner via the perfusion catheter, and observe the renal artery anatomical structure to confirm whether the catheter is in the desired position and only the renal structure is perfused through this artery;

[0440] (13) Place the Lunderquist guide wire (or similar 0.035") in the distal renal vein via a 5-FR multipurpose catheter;

[0441] (14) Place at least one retrieval catheter on the Lunderquist guide wire at its position in the main trunk of the left renal vein to ensure homogeneous blood drainage of all parts of the kidney and ensure the stability of the catheter position;

[0442] (15) Place a second PressureWire X (Abbott) via the return line for venous pressure measurement in the renal vein;

[0443] (16) After determining the proper positioning of the venous catheter, inflate the balloon of the retrieval catheter;

[0444] (17) Evaluate the renal vein anatomical structure by retrograde injection of contrast agent via a diagnostic catheter placed in the lumen of the retrieval catheter;

[0445] (18) Under fluoroscopy, test the tightness of the balloon occlusion by retrograde injection of contrast agent. The acceptance criteria for the sealing quality are: (i) no relative balloon flow or contrast agent trace; (ii) no filling of other venous structures connected to the vena cava; and (iii) no drainage of the main extra-renal vessels;

[0446] (19) Retract the balloon of the retrieval catheter to allow the injected contrast agent to drain and discharge naturally;

[0447] (20) Prepare the LRP system by the perfusion team together with the operator;

[0448] (21) Prepare the LRP system for the LRP procedure:

[0449] a. Perfuse with 250 mL of heparinized saline

[0450] b. Degas

[0451] c. Turn on the LRP pump and run the circuit in a shunt manner;

[0452] d. Connect the retrieval catheter to the reservoir using tubing;

[0453] e. Connect the perfusion catheter to the arterial outlet of the oxygenator via tubing, and completely degas the tubing and catheter;

[0454] f. Loosen the venous line and close the shunt;

[0455] g. Start filling the reservoir by adding blood;

[0456] h. If the system is stable, release the arterial line and completely exchange the saline in the reservoir with blood (complete exchange of volume can be confirmed by hematocrit analysis);

[0457] i. The aspiration on the venous side and the blood pump speed are variable and adjusted to physiological needs according to each situation (usually -80 mmHg to 0 mmHg for vacuum and 2500 - 4500 rpm for blood pump speed);

[0458] Stable phase

[0459] (22) The LRP cycle starts with the deployment of the retrieval catheter balloon and the retraction of the perfusion catheter balloon;

[0460] a. Deploy the retrieval catheter balloon (re - confirm the final position by injecting retrograde contrast agent as needed);

[0461] b. Check: The tightness and position of the catheter for injecting contrast agent; the pressures in the kidneys (both renal artery and renal vein) (the acceptable range is 60 mmHg to 140 mmHg for the artery and the renal vein is equal to or less than the central venous pressure); the ratio of the mean renal artery pressure to the mean systemic pressure (the ratio should be slightly less than 1 to prevent leakage); the stability of the reservoir volume (a slight increase in volume is acceptable provided that the rate of increase does not cause the reservoir to over - fill within the allotted LRP time or does not cause hemodynamic instability due to removal of excessive blood from the animal); the RPM of the LRP pump; and the flow rate through the catheter (the flow rate is preferably between 150 mL / min and 800 mL / min);

[0462] (23) When the circuit is stable and complete exchange of volume is confirmed by hematocrit analysis, seal the renal artery with the perfusion catheter under fluoroscopy;

[0463] (24) After sealing both renal vessels, establish a complete LRP cycle;

[0464] (25) After 5 minutes:

[0465] a. As needed (i.e., in cases of high arterial pressure or renal artery spasm), start infusing nitroglycerin (GTN) via the arterial line at a rate of 0 μg / kg body weight / min to 4 μg / kg body weight / min;

[0466] b. Checks: tightness and position of the catheter for injecting contrast agent; pressures in the kidneys (both renal artery and renal vein) (the acceptable range for the artery is 60 mmHg to 140 mmHg, and the acceptable range for the renal vein is equal to or less than the central venous pressure); the ratio of mean renal artery pressure to mean systemic pressure (the ratio should be slightly less than 1 to prevent leakage); stability of the reservoir volume (a slight increase in volume is acceptable provided that the rate of increase does not cause the reservoir to overfill within the allotted LRP time or does not cause hemodynamic instability due to removal of excessive blood from the animal); RPM of the LRP pump; and flow rate through the catheter (a flow rate between 150 mL / min and 800 mL / min is preferred);

[0467] (26) Check all the parameters listed above every 5 minutes until all parameters remain within the acceptable range continuously for at least 20 minutes;

[0468] Therapeutic delivery phase

[0469] (27) When all major LRP system components have been stable for at least 20 minutes, the payload can be delivered. The payload is provided by rapid injection via a 3-way stopcock in the venous return line. Alternative injection methods would be: continuous injection, rapid injection into the supply line, or multiple injections via the return or supply line. After injecting the payload, flushing is provided at the same location;

[0470] (28) Perform a single-kidney LRP procedure for a predetermined period;

[0471] (29) Check every 5 minutes: tightness and position of the catheter for injecting contrast agent; pressures in the kidneys (both renal artery and renal vein) (the acceptable range for the artery is 60 mmHg to 140 mmHg, and the acceptable range for the renal vein is equal to or less than the central venous pressure); the ratio of mean renal artery pressure to mean systemic pressure (the ratio should be slightly less than 1 to prevent leakage); stability of the reservoir volume (a slight increase in volume is acceptable provided that the rate of increase does not cause the reservoir to overfill within the allotted LRP time or does not cause hemodynamic instability due to removal of excessive blood from the animal); RPM of the LRP pump; and flow rate through the catheter (a flow rate between 150 mL / min and 800 mL / min is preferred);

[0472] (30) At T + 5 minutes, +10 minutes, +15 minutes, +20 minutes, +25 minutes, +30 minutes, +45 minutes, and 60 minutes (or other predetermined relevant time points):

[0473] a. Collect blood samples from peripheral blood for blood gas and excretion analysis;

[0474] b. Collect blood samples from the LRP system for drainage, vector infectivity analysis, ACT, and blood gas analysis;

[0475] c. Perform urine biochemistry and drainage analysis;

[0476] Removal phase

[0477] (31) At the end of LRP:

[0478] a. If used, discontinue nitroglycerin trinitrate;

[0479] b. Prepare a 20 mL syringe, fill the syringe with systemic arterial blood, and connect the syringe to the side port of the perfusion catheter;

[0480] c. Clamp the arterial line and flush the lumen of the perfusion catheter via the side port with the pre-filled syringe;

[0481] d. Immediately after flushing, deflate the perfusion catheter balloon;

[0482] e. Remove the arterial line from the body;

[0483] f. During this time, the renal vein balloon must remain engaged and in aspiration, and fill the ECMO reservoir with the remaining blood-containing payload;

[0484] g. After approximately 150 mL has been drained, clamp the venous line and retract / deflate the retrieval catheter balloon. Remove the venous line from the body. Completely loosen the catheter, taking special care to avoid spillage of the blood-containing payload;

[0485] (32) Discard the entire LRP circuit, reservoir, blood pump, and catheter in an appropriate biosafety cabinet; and

[0486] (33) Immediately perform postoperative care.

[0487] Example 6: Biodistribution of AAV5 after administration to the left kidney of a farm pig via LRP

[0488] 6.1 Procedure description and dosing

[0489] Based on the protocol described in Example 5, a farm pig received kidney LRP (1-hour dosing time) treatment, in which the dose of AAV5-CMV-eGFP was 9.8×10 14 vg. This pig was selected from 12 pigs after immunological screening for pre-existing antibodies to AAV5 and computerized tomography (CT) scanning of the kidney to ensure physiological compatibility of the pig with the LRP procedure.

[0490] 6.2 Vector drainage analysis

[0491] To confirm the tightness of the LRP procedure, multiple blood samples were collected at different time points from the LRP system and peripheral blood (systemic circulation). Urine samples were also collected to estimate viral excretion during the procedure. Quantitative polymerase chain reaction (qPCR) and a probe targeting the AAV GFP DNA sequence were used to measure the total viral genomes per milliliter in the blood and urine samples of this animal, and they were titrated using linearized plasmid DNA based on a standard curve. All samples were measured in duplicate. Despite the challenging porcine anatomy due to the need for additional catheterization for the left renal vein branch, the procedure was still successful. There was no leakage from the LRP to the systemic circulation.

[0492] Figure 37 Is a graph showing the excretion analysis (vector genomes per mL of plasma or urine) detected in the LRP circulation, systemic circulation, and urine of treated farm pigs (1.2 × 10 13 vg / kg, 60-minute LRP procedure). Figure 38 Is a graph showing the total vector genomes detected in the urine of treated farm pigs (1.2 × 10 13 vg / kg, 60-minute LRP procedure). As Figure 37 revealed, the viral genome levels remained stable over time until the end of the procedure. As Figure 38 revealed, vectors were also found in the urine early after LRP administration. In this experiment, urine was collected at selected time points, and the urine volume was measured before emptying the urine bag. Notably, there were fluctuations in urine volume during the LRP procedure. Therefore, the urine volume was used to calculate the total viral genomes in the urine at each time point ( Figure 38 ). Viral excretion was more significant 10 minutes after AAV administration into the LRP.

[0493] 6.3 Biodistribution in the renal regions (cortex, medulla, and papilla)

[0494] A small piece of each tissue was homogenized in a TissueLyser II (Qiagen) using metal beads and extracted using AllPrep DNA / RNA (Qiagen). DNA concentration and purity were measured using a NanoDrop spectrophotometer. Droplet digital PCR (ddPCR) was used to quantify the viral genomes in the cells of the tissue samples (vg / dg). GFP probes were used to detect the viral genomes against the housekeeping gene of the animal using β-actin probes.

[0495] Figure 39 Is a graph showing the LRP-treated kidney (1.2 × 10 13Graph of biodistribution analysis (in vg / dg) following a 60-minute LRP procedure at 1 × 10¹² vg / kg. The pigs demonstrated a significant increase in AAV5 viral genomes in the treated kidneys (mean 11.5) compared to pigs treated with the same dose of AAV9 (highest mean observed 0.5 vg / dg). This also confirmed the results from a previous study that compared these two serotypes co-administered at a lower dose (10¹⁰ vg / kg) in the same animals (see Figure 39 , mean 11.5). This also confirmed the results from a previous study that compared these two serotypes co-administered at a lower dose (10¹⁰ vg / kg) in the same animals (see Figure 26 ). The untreated kidneys and livers remained highly off-target. The comparable transduction levels observed throughout the cortex as well as the outer and inner medullary regions (i.e., the pyramids and papilla) indicate that AAV5 delivered via LRP can widely transduce the cells of the nephrons as well as other cells of the kidney. Given that AAV5 delivered via LRP can transduce cells present in regions of high blood flow (e.g., the cortex) and low blood flow (e.g., the pyramids and papilla), and given that AAV5 can be transported into the urine flow within the renal tubules and secreted, AAV5 may be able to enter kidney cells via the vascular circulation as well as from within the renal tubules. These properties make AAV5 a desirable vector for delivering gene therapeutics to the kidney using LRP.

[0496] 6.4 GFP pg / mg of tissue by ELISA

[0497] The GFP SimpleStep ELISA kit (Abcam #ab171581) was used to quantify the amount of the reporter protein in sections of LRP-treated kidneys (AAV5-CMV-GFP). The assay was performed according to the manufacturer's instructions. Briefly, one scoop (30 - 50 mg) of tissue powder was homogenized in cold extraction buffer. After centrifugation, the supernatant was collected into a clean tube and further processed. Standards were freshly prepared and samples were run in duplicate. After incubation with the antibody mixture and thorough washing, the substrate was added and the reaction was stopped before saturation. The optical density (OD) measurements at 450 nm were recorded and converted to pg / mg after normalization relative to the total protein concentration.

[0498] Figure 40 Shows the GFP expression in LRP-treated kidneys (1.2 × 10 13Graph of GFP protein levels (in pg / mg) in the 60-minute LRP procedure at 10 vg / kg. Consistent with the high levels of transduction observed in the entire treated kidney, high GFP expression levels were detected in the cortex of the entire treated kidney as well as in the outer and inner medullary regions. No GFP expression was detected in the untreated kidney or liver, further demonstrating the effective transduction of only the LRP-treated kidney. The comparable GFP expression levels observed in the entire cortex as well as in the outer and inner medullary regions (i.e., the pyramids and papilla) indicate that AAV5 delivered via LRP can be widely transduced in the cells of nephrons as well as in other cells of the kidney and provide transgene expression. These properties further make AAV5 the desired vector for delivering gene drugs to the kidney using LRP.

[0499] 6.5 Description of transduced cells observed via RNAScope

[0500] In situ hybridization technology assays (ACD, brand of BioTechne) allow for the spatial visualization of individual mRNA or free DNA molecules. To improve the signal-to-noise ratio, RNAscope uses target-specific probes combined with a multiplex signal amplifier. This produces puncta that can be observed with an optical microscope. Here, GFP-specific probes are used in combination with other probes specific for different cell types in the kidney (CDH2 - proximal tubule; CDH1 - distal tubule; NPHS1 - podocyte; SLC12A1 - macula densa; PECAM1 - endothelial cell marker). This allows for the identification of transduced renal cells after the LRP procedure. Briefly, cryosections from freshly frozen kidney fragments are fixed in 4% paraformaldehyde and undergo a cascade of hybridization events, with several washes in between. After mounting, the slides are imaged with an inverted Axio Observer microscope (Zeiss). Overview images of cortical sections show homogeneous staining of glomeruli in the treated kidney. No signal was observed in the untreated kidney or liver. Additional puncta were observed in other tubular structures within the cortical and medullary regions, confirming the ability of AAV5 to transduce a range of kidney cells.

[0501] Example 7: Comparison of the biodistribution of AAV5 after LRP delivery to the left kidney versus IV administration

[0502] 7.1 Procedure description and dosing

[0503] The purpose of this example is to compare two routes of AAV5 administration to the kidney, namely 1-hour kidney LRP dosing and intravenous injection (IV), to evaluate the benefits of AAV administration via kidney LRP versus IV.

[0504] As in Example 6, two farm pigs were selected for this study based on pre-existing antibodies to AAV5 and CT scans of the kidneys to ensure physiological compatibility of the pigs with the LRP procedure.

[0505] Each animal was administered a total of 9.9×10 14 vg of AAV5-CAG-eGFP. AAV5-CAG-eGFP was administered to Animal 1 via LRP as described in Example 5. For Animal 2, AAV5-CAG-eGFP was administered in a bolus dose via a central venous access line. For both Animal 1 and Animal 2, the methods were consistent, except that for Animal 2, only peripheral blood and urine samples were collected for excretion analysis because the LRP circuit was not established in this animal.

[0506] 7.2 Vector excretion analysis

[0507] To confirm the tightness of the LRP procedure, multiple blood samples were collected at different time points from the LRP system of Animal 1 and the peripheral blood (systemic circulation) of both animals. Urine samples were also collected to estimate viral excretion during the LRP procedure. Total viral genomes per milliliter in the blood and urine samples were measured using qPCR and a probe targeting the AAV GFP DNA sequence, and titrated using linearized plasmid DNA based on a standard curve. All samples were measured in duplicate.

[0508] Figure 41 is a graph showing the excretion analysis (vector genomes per mL of plasma or urine) detected in the LRP circuit, systemic circulation, and urine of Animal 1. The LRP procedure was successful in Animal 1, with signs of nominal leakage of the LRP into the systemic circulation in the second half of the procedure. The level of viral genomes within the circuit remained relatively stable over time until the end of the procedure. As Figure 41 and Figure 43 shown, vector was also found in the urine early after LRP administration. In this experiment, urine was collected at selected time points, and the urine volume was measured before emptying the urine bag. Notably, there were fluctuations in urine volume during the LRP procedure. Therefore, urine volume was used to calculate the total viral genomes in the urine at each time point. Viruses were excreted in large amounts into the urine starting within 10 minutes of AAV administration into the LRP circuit and remained stable throughout the procedure.

[0509] Figure 42This is a graph showing the excretion analysis (vector genome 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 provides a very different exposure profile. In the systemic circulation, the AAV5 concentration reached during the first hour after administration is approximately 100-fold higher than that observed in Animal 1 during the LRP procedure, indicating that systemic exposure after IV administration is much higher relative to LRP administration. The viral genome in Animal 2 remained in circulation at all time points measured up to 5 days after the procedure. The concentration of the viral genome decreased rapidly in the systemic circulation, dropping by 1 log from the earliest time point of 5 minutes to 60 minutes after injection, from 1.94×10 10 vg / mL to 1.93×10 9 vg / mL, and continued to decline to day 5, reaching 3.43×10 6 vg / mL. Assuming that the AAV5 exposure to the kidney is represented by the systemic AAV concentration in Animal 2, the maximum concentration of AAV5 exposed to the kidney is approximately 20-fold lower than that after administering the same dose to Animal 1 via LRP (1.94×10 10 vg / mL versus 4.23×10 11 vg / mL, respectively). Given the potential of AAV5 to transduce cells of the renal tubules via exposure to the circulation, the substantial difference in the maximum concentration of the AAV5 viral genome exposed to the kidney relative to IV administration after LRP may have important implications for delivering gene therapy to the kidney via AAV5-mediated delivery.

[0510] Similarly, the concentration profile of the AAV5 viral genome in urine is substantially different after IV administration to Animal 2 compared to direct administration to the kidney via LRP in Animal 1. As Figure 42 and Figure 44 shown, in Animal 2, the viral genome was only evident at 45 minutes after injection (8.84×10 8 vg). Given the potential of AAV5 to transduce the tubular epithelial cells of the renal tubules via the apical (urine-facing side) of these cells, the substantial difference in the viral genome in urine relative to IV administration after LRP may further have important implications for delivering gene therapy to the kidney via AAV5-mediated delivery.

[0511] LRP excretion data obtained from Animal 1 and systemic concentration data obtained from Animal 2 were used to further investigate the differences in AAV5 exposure to the kidney relative to IV administration after LRP. Figure 45 This is a graph of the modeled concentration of AAV5 in the LRP circuit of the treated kidney of Animal 1 versus time, which is derived from Figure 41The data provided in [reference] and used to model the kidney exposure to the viral genome by calculating the area under the curve (AUC). For LRP modeling, the approximate AAV5 C observed in the LRP circuit of animal 1 was used. max And assuming no leakage, so the AAV5 concentration was constant throughout the procedure, the figure was obtained. For IV modeling, it was assumed that the circulating concentration of AAV5 in peripheral blood represented the exposure to the kidney after IV administration. The one-phase exponential decay equation (GraphPad Prism) was used to fit the systemic AAV5 concentration data up to day 5 to obtain the decay constant of AAV5 (k = 0.4 / hr) after IV administration to pigs.

[0512] Figure 46 is a plot of the modeled concentration of AAV5 in the systemic circulation of animal 2 over time after IV administration, which was derived from Figure 42 the data provided in [reference] and used to model the kidney exposure to the viral genome by calculating the area under the curve (AUC). The approximate systemic C observed in animal 2 was used. max And the decay constant obtained using the one-phase exponential decay model, the decay curve was used to model the curve of the concentration of AAV5 over time equivalent to the period of LRP treatment after IV administration.

[0513] AUC analysis of the corresponding model plots (GraphPad Prism) showed that compared to IV administration during the same period, when using LRP administration, the exposure of AAV5 to the treated kidney during the LRP treatment period was approximately 200 times greater. In principle, compared to IV administration, the greater exposure of AAV5 to the kidney obtained via LRP delivery would result in substantially greater transduction of the kidney. Additionally, compared to IV administration, after direct administration of AAV5 to the kidney via LRP, the greater difference in systemic exposure when using these corresponding administration routes was expected to provide minimal transduction of other organs (such as the liver or spleen). Given the known safety issues associated with AAV transduction in the liver, reducing transduction of non-target organs has significant safety implications.

[0514] 7.3 Biodistribution in the kidney regions (cortex, medulla, and papilla)

[0515] Small pieces of each tissue were homogenized in a TissueLyser II (Qiagen) using metal beads and extracted using AllPrep DNA / RNA (Qiagen). The DNA concentration and purity were measured using a NanoDrop spectrophotometer. The viral genome in tissue cells was quantified (vg / dg) using ddPCR. GFP probes were used to detect the viral genome of the animal against the housekeeping gene using β-actin probes.

[0516] Figure 47is a graph showing the biodistribution analysis (vg / dg) measured in kidney sections compared to other organs from animal 1, and Figure 48 is a graph showing the biodistribution analysis (vg / dg) measured in kidney sections compared to other organs from animal 2 (where Tr = treated and Untr = untreated). For animal 1 administered AAV5 to the left kidney via LRP, the biodistribution analysis confirmed the tightness of the LRP system. The viral genome was detected mainly in the treated kidney sections (left kidney), with an average of 3.5 vg / dg( Figure 47 ). The untreated kidney, liver, and spleen remained highly off-target (average of 0.01 vg / dg for the untreated kidney; average of 0.05 vg / dg for three liver samples; spleen at 0.76 vg / dg). In contrast, after IV administration to animal 2, the biodistribution of AAV5 was substantially different. As expected with greatly reduced exposure of the kidneys to AAV5, kidney transduction after IV administration was extremely inefficient, with both the left and right kidneys showing similar low average viral genomes of 0.2 vg / dg( Figure 48 ). Additionally, as expected with substantially greater systemic exposure after IV administration of AAV5, the viral genomes in the liver and spleen were significantly higher in animal 2 (1.4 vg / dg and 12 vg / dg, respectively).

[0517] To quantify the specificity of target kidney transduction relative to non-target organ transduction obtained by LRP administration of AAV5 compared to IV administration, the ratios of kidney to liver transduction in animals 1 and 2 were calculated and summarized in Table 6. The targeting specificity was calculated by comparing the ratios of kidney to liver in animal 1 relative to animal 2 calculated based on vg / dg. The targeting specificity taking into account the difference in cell numbers of each organ was calculated using organ size as an estimate of the number of cells in the corresponding organ.

[0518] For animal 1, the ratio of kidney to liver transduction was 63.7, while for animal 2, this ratio was 0.14. The comparison of these ratios confirmed that LRP administration provides approximately 450-fold greater kidney targeting specificity than non-kidney targeting specificity compared to IV administration. Notably, given that the liver is substantially larger than the kidney, this analysis provides an average of the transduction specificity at the cellular level without considering that the number of cells within the liver is substantially greater than that of the kidney. In humans, the volume of the non-diseased liver is approximately 12-fold that of the 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). Using the relative volume of the human liver compared to the human kidney as an approximation of the relative cell number results in approximately 5400-fold greater kidney targeting specificity than non-kidney targeting specificity when AAV5 is administered via LRP compared to IV administration.

[0519] These findings confirm that administration of AAV5 via LRP confers the unexpected benefit of substantial transduction of the treated kidney, which is not achievable by administering the same dose via IV. In addition, administration of AAV5 via LRP results in significantly reduced targeting of the liver and spleen, conferring potential safety benefits in addition to the potential therapeutic benefits obtained by effective transduction of the kidney.

[0520] Table 6: Overview of the main parameters and conclusions of AAV5 administration to two farm pigs using LRP relative to IV.

[0521]

[0522]

[0523] Example 8: Neutralizing AAV antibody production after tight versus leaky AAV administration to the kidneys of farm pigs via LRP

[0524] 8.1 Procedure description and dosing

[0525] The purpose of this analysis was to compare systemic AAV neutralizing antibody formation after AAV administration via renal LRP when a closed circuit was successfully maintained versus when substantial leakage was observed during the LRP procedure. For this comparison, animals (referred to as animals 3 and 4 in this example) were treated with AAV9 because AAV9 systemic exposure is known to trigger a relatively strong immune response. Animal 3 received 6.2×10 14Treatment of the kidneys with AAV9-CMV-eGFP at a vg dose (1-hour administration time), while animal 2 received treatment of the kidneys with AAV9-CMV-eGFP at a dose of 1.7×10 15 vg (2-hour administration time). Two animals were selected after immunoselection for pre-existing antibodies to AAV9 and CT scanning of the kidneys to ensure physiological compatibility of the pigs with the LRP procedure.

[0526] 8.2 Vector excretion analysis

[0527] To confirm the tightness of the LRP procedure, multiple blood samples were collected at different time points from the LRP circuit and peripheral blood (systemic circulation). Total viral genomes per milliliter in the blood were measured using qPCR and a probe targeting the AAV GFP DNA sequence, and titrated using linearized plasmid DNA based on a standard curve. All samples were measured in duplicate.

[0528] To measure neutralizing antibodies against AAV9, 2V6.11 cells were seeded in a 96-well white plate and incubated overnight with Ponasterone A. The next day, serum samples from AAV9-treated pigs were serially diluted and incubated with an AAV9 vector containing a reporter gene for 1 hour, and then the serum sample / vector mixture was added to 2V6.11 cells. After incubation for 24 hours, the percentage of AAV9 transduction of the lysed cells was evaluated by adding a reporter substrate. Results were reported as the titer required to provide 50% transduction inhibition. Positive and negative controls were included in the assay, and only cells receiving medium were used to evaluate the background signal.

[0529] During the procedure in animal 1, the LRP circuit remained tight, with no signs of systemic leakage, and the maximum concentration in peripheral blood samples (systemic circulation) was 1.7×10 8 vg / mL. In contrast, significant leakage of AAV9 into peripheral blood was observed during the LRP procedure in animal 2, where the concentration rose from 6.6×10 9 vg / mL at 5 minutes into the procedure to a maximum value of 1.2×10 11 vg / mL during the procedure.

[0530] Anti-AAV9 neutralizing antibody titers were measured on the day of AAV9 administration via LRP to the kidney (day 0) and on the day of sacrifice (day 15), and the results are summarized in Table 7. Prior to AAV9 administration via renal LRP, both animals showed low levels of anti-AAV9 neutralizing antibodies. On day 15, the anti-AAV9 neutralizing antibody titer in animal 4 increased significantly to >1 / 4096, the highest dilution of the evaluated serum. In contrast, the anti-AAV9 neutralizing antibody titer in animal 3 remained low (1 / 32). Given the safety concerns associated with the immune response against AAV, this analysis confirms the potential benefits of the closed-loop delivery of AAV to the kidney via LRP. Additionally, the low systemic neutralizing antibody response observed in animal 3 opens the possibility of re-dosing with the same AAV in the future when dosing is required.

[0531] Table 7: Summary of neutralizing AAV9 antibody titers observed in animals 3 and 4

[0532]

[0533] In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, process parameters, etc., to provide a thorough understanding of the present invention. In one or more embodiments, the specific features, structures, materials, or characteristics may be combined in any suitable manner. The terms "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 superior to other aspects or designs. In fact, the use of the terms "example" or "exemplary" is merely intended to present concepts in a concrete fashion. As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or". That is, unless otherwise specified or apparent from the context, "X includes A or B" is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then "X includes A or B" is satisfied in any of the foregoing instances. Throughout this specification, the terms "embodiment", "certain embodiments", or "one embodiment" are used to mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment. Thus, the appearances of the phrases "embodiment", "certain embodiments", or "one embodiment" throughout this specification are not necessarily all referring to the same embodiment.

[0534] 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 present invention will become apparent to those skilled in the art in addition to those shown and described herein and are intended to fall within the scope of the appended claims.

Claims

1. A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising: Positioning at least one perfusion catheter in the renal artery of the kidney; Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device 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, wherein the AAV vector comprises an AAV5 capsid protein, and wherein the closed circuit substantially isolates the perfusion through the kidney from the systemic circulation of the subject.

2. The method according to claim 1, wherein a dose of the AAV vector is delivered via the closed loop and maintained at a concentration of at least about 5×10 7 vector genomes per milliliter of plasma during perfusion, and wherein the vector leaking into the systemic circulation of the subject is maintained at less than or equal to about 5×10 7 vector genomes per milliliter of plasma during perfusion, wherein the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

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

4. A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising: Positioning at least one perfusion catheter in the renal artery of the kidney; Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and Flowing a perfusion fluid through the closed loop, wherein the perfusion fluid contains the polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, wherein the closed loop substantially isolates perfusion through the kidney from the patient's systemic circulation, and wherein a dose of the AAV vector is delivered via the closed loop and maintained at a concentration of at least about 5×10 7 vector genomes per milliliter of plasma during perfusion, and wherein the vector leaking into the patient's systemic circulation that is present remains at less than or equal to about 5×10 7 vector genomes per milliliter of plasma during perfusion, wherein the perfusion is maintained for a total of about 30 minutes to about 90 minutes.

5. A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising: Positioning at least one perfusion catheter in the renal artery of the kidney; Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device 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, the vector comprising a non-AAV5 capsid protein, wherein the closed circuit substantially isolates the perfusion through the kidney from the systemic circulation of the mammalian subject, and wherein the vector genome copy number per diploid genome after the perfusion is at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater or at least about 30-fold greater compared to the same AAV vector actually comprising an AAV5 capsid protein.

6. A method for locally delivering a polynucleotide sequence to renal cells in the kidney of a mammalian subject, the method comprising: Positioning at least one perfusion catheter in the renal artery of the kidney; Positioning at least one retrieval catheter in the renal vein of the kidney, wherein the at least one perfusion catheter and the at least one retrieval catheter together with a membrane oxygenation device form a closed perfusion circuit through the kidney; and Perfuse the perfusion fluid through the closed loop, wherein the perfusion fluid comprises the polynucleotide sequence packaged in an adeno-associated virus (AAV) vector, the vector comprising a capsid protein, wherein the closed loop substantially isolates perfusion through the kidney from the systemic circulation of the mammalian subject, and wherein the vector genome copy number per diploid genome is at least about 5-fold greater, at least about 10-fold greater, at least about 20-fold greater, or at least about 30-fold greater after the perfusion compared to the same AAV vector actually comprising a different capsid protein.

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

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

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

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

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

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

13. The method of claim 1, wherein perfusing the perfusion fluid through the closed loop comprises: passing the perfusion fluid through the membrane oxygenation device before entering the renal artery via the one or more perfusion catheters.

14. The method of claim 1, the method further comprising: adding additional perfusion fluid to the closed loop or diluting the perfusion fluid with a saline solution of about 5% to about 50% v / v to calculate the bladder excretion volume.

15. The method according to claim 1, wherein the closed loop maintains the flow rate of the perfusion fluid at about 500 mL / min / 1.73 m² body surface area to about 650 mL / min / 1.73 m² body surface area per kidney for about 15 minutes to about 4 hours. 2 per kidney for about 15 minutes to about 4 hours. 2 per kidney for about 15 minutes to about 4 hours.

16. The method according to claim 1, wherein the closed loop maintains the flow rate of the perfusion fluid at about 150 mL / min / 1.73 m 2 body surface area to about 700 mL / min / 1.73 m 2 body surface area for about 15 minutes to about 4 hours.

17. The method of claim 1, the method further comprising applying negative pressure at the at least one retrieval catheter, wherein the negative pressure is 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 retrieval catheter are introduced percutaneously or non-percutaneously.

19. 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, or substantially no blood leakage through the closed loop circulates outside the closed loop.

20. The method according to 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 or substantially no perfusate circulating through the closed loop leaks out of the closed loop.

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

22. A system for performing regional perfusion of a local area of a patient's kidney when fluidly coupled to the kidney, the system comprising: At least one perfusion catheter adapted to be inserted into the renal artery of the kidney; At least one retrieval catheter adapted to be inserted into the renal vein of the kidney; A membrane oxygenation device adapted to be fluidly coupled to the at least one perfusion catheter, the at least one retrieval catheter, and an oxygen source, wherein when the at least one perfusion catheter is inserted into the renal artery and the at least one retrieval catheter is inserted into the renal vein, the at least one perfusion catheter, the at least one retrieval catheter, and the membrane oxygenation device together are adapted to form a closed loop through the kidney, the closed loop being isolated from the patient's systemic circulation; A reservoir containing a perfusate, the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; and A pump configured to drive the perfusate through the at least one perfusion catheter and the at least one retrieval catheter.

23. A system for performing regional perfusion of a patient's kidney, the system comprising: At least one perfusion catheter inserted into the renal artery of the kidney; At least one retrieval catheter inserted into the renal vein of the kidney; And A membrane oxygenation device fluidly coupled to the at least one perfusion catheter, the at least one retrieval catheter, and an oxygen source, wherein the at least one perfusion catheter, the at least one retrieval catheter, and the membrane oxygenation device together with the kidney form a closed loop through the kidney, the closed loop being isolated from the patient's systemic circulation; A reservoir containing a perfusate, the perfusate comprising a polynucleotide sequence packaged in an adeno-associated virus (AAV) vector; And A pump configured to drive the perfusate into the kidney via the at least one perfusion catheter and out of the kidney via the at least one retrieval catheter.

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

25. The system according to claim 22, wherein the polynucleotide sequence encodes polycystin-1 protein or a functional variant thereof.

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

27. The system according to claim 22, the system being configured to perform the method according to claim 1.

28. A gene therapy vector adapted to transduce renal cells of a human subject, the gene therapy vector comprising: An adeno-associated virus (AAV) vector comprising an AAV5 capsid protein; and A polynucleotide sequence packaged in the AAV vector.

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

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

31. The gene therapy vector according to claim 28, wherein the polynucleotide sequence comprises a renal-specific promoter.

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

33. A method of screening AAV serotypes to identify a major serotype for local gene delivery to an organ type, the method comprising: Identifying a plurality of AAV vector candidates, each AAV vector candidate corresponding to a different AAV serotype; Generating a perfusion fluid composition comprising each of the plurality of AAV vector candidates, each of the plurality of AAV vector candidates comprising a polynucleotide packaged therein; and Locally transducing tissues in an organ of the organ type of an animal by creating a closed loop in the vasculature of the target organ, the perfusion fluid composition being circulated via the closed loop; and Selecting the serotype of the AAV vector candidate having the highest vector genome biodistribution and / or the greatest RNA / protein expression in the organ compared to the remaining AAV vector candidates as the major serotype.

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

35. The method according to claim 33, wherein the local transduction of the tissues in the organ is performed using the system according to claim 22.

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