Methods and compositions for hydrodynamic gene delivery

Abstract

The present disclosure relates to methods of pressure guided hydrodynamic injection into tissues such as kidney, pancreas, liver, common bile duct, and in certain embodiments, delivery of nucleic acids or viral vectors. This includes an optimal pressure that mediates gene delivery and expression in the subject during the hydrodynamic injection. Described herein are methods of eliminating individual differences during hydrodynamics to achieve a target pressure while avoiding tissue damage, rupture, tearing, reducing toxicity of hydrodynamic injections, and other considerations associated with therapy delivery.

Description

[0001] Related Applications

[0002] This application is related to U.S. Provisional Patent Application Nos. 63 / 374,228, 63 / 374,231, 63 / 374,234, 63 / 374,058, 63 / 374,070, 63 / 374,073, and 63 / 374,216, filed on August 31, 2022, and claims priority under 35 U.S.C. § 119(e). The entire contents of the above patent applications are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to compositions and methods for hydrodynamic gene delivery. Background Art

[0004] Given that the potential disease conditions of each patient may vary, the technical problem lies in understanding which parameters to inject into the patient's body for gene delivery. Gene delivery to specific tissue types has therapeutic prospects.

[0005] The kidney is an organ in the human body that plays an important role in removing toxins from the body and controlling the volume, osmotic pressure, acid-base balance, electrolyte concentration, etc. of various body fluids. Kidney dysfunction can lead to a variety of rare genetic kidney diseases, such as cystinuria, adult polycystic kidney disease, nephrogenic diabetes insipidus, Gitelman syndrome, Fabry disease, thin basement membrane nephropathy, Lowe syndrome, hereditary interstitial nephritis, tuberous sclerosis, renal tuberculosis, and Alport syndrome. In addition to rare kidney diseases, common diseases such as chronic kidney disease, end-stage kidney disease, and immune-mediated glomerular diseases are also very important. Kidney diseases (also known as nephropathy) generally manifest as kidney damage. For example, nephritis is an inflammatory kidney disease that can be classified into several types according to the location of inflammation. Another example is nephrosis, a non-inflammatory kidney disease that can lead to nephritis or nephrotic syndrome. Kidney diseases usually result in the loss of kidney function. Complete loss of kidney function is called end-stage kidney disease and can only be treated by dialysis or kidney transplantation. Gene therapy methods can be used in the treatment algorithms for autoimmune, inflammatory, metabolic, toxic, pre-tumor or cancer of the kidney.

[0006] One strategy for treating kidney diseases is gene therapy, which can provide genes that are missing or insufficient in kidney cells. In addition, adding genes as a treatment method may be beneficial for some clinical prevention strategies or diseases. However, to date, no gene therapy strategy for treating or preventing genetic kidney diseases has been successfully implemented. Therefore, there is an urgent need for gene therapy regimens that can be implemented in the kidney to treat and / or prevent genetic, autoimmune, inflammatory, fibrotic, metabolic, toxic, pre-tumor or malignant diseases of the kidney.

[0007] The pancreas is an important exocrine and endocrine organ in the human body, responsible for food digestion and energy storage. Malfunctions of the pancreas can lead to various diseases, such as cystic fibrosis, hereditary pancreatitis, autoimmune pancreatitis, and various different types of diabetes, including but not limited to type 1 and type 2, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes in adults (LADA), neonatal diabetes, Wolfram syndrome, and Alstrom syndrome. For example, the pancreas plays an important role in the pathogenesis of many different diseases, such as type 1 and type 2 diabetes caused by interrupted or dysregulated insulin secretion. Gene therapy can also be used to deliver proteins against genetic diseases, early-stage tumors, cancers, pancreatic pain, pancreatic inflammation, or autoimmune diseases. Target cells of interest in the pancreas include but are not limited to: pancreatic acinar cells, pancreatic duct cells, islet cells, endothelial cells, and neuronal cells.

[0008] Multiple strategies have been attempted for gene therapy of the pancreas. For example, viral strategies have employed systemic or targeted therapy via the vascular route, while other studies have shown the feasibility of injecting viruses through the pancreatic duct. The drawback is that viral vectors such as AAV have significant size limitations (∼4.8 kb) relative to the nucleotide sequences they can package. Non-viral strategies using hydrodynamic injection via the rat pancreatic artery have also been employed. Unfortunately, these non-viral strategies have not been translated to large animal models. Therefore, there is an urgent need for gene therapy protocols that can be implemented in the pancreas to treat and / or prevent such pancreatic genetic diseases.

[0009] The biliary system is a promising route for delivering genes to the liver. Hydrodynamic gene delivery can be achieved through the biliary system, with high efficiency achievable through the common hepatic duct. Unfortunately, delivery from other locations in the biliary system has not been successful. Therefore, additional administration routes are needed to achieve hydrodynamic gene delivery.

[0010] Gene therapy is a treatment option for cancer. Gene therapy can directly deliver various different types of therapeutic methods into tumors to facilitate their clearance, but current delivery systems are unable to deliver effectively. The efficacy of non-viral delivery for tumors is particularly limited.

[0011] The liver is the main organ that performs many important biological functions, such as detoxification and the synthesis of proteins and biochemical substances. The liver also plays an important role in metabolism, including regulating glycogen storage, the breakdown of red blood cells, glucose and lipid metabolism, and the production of hormones. Liver dysfunction can lead to a variety of rare genetic diseases, such as Wilson's disease, phenylketonuria, hemophilia A, hemophilia B, progressive familial intrahepatic cholestasis, etc. Liver dysfunction can also lead to a variety of autoimmune diseases, such as autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cirrhosis, etc. In addition, infiltrative diseases can also affect the liver, such as amyloidosis and malignancies such as hepatocellular carcinoma or metastatic colorectal cancer. Also, a variety of metabolic diseases can affect the liver, such as non-alcoholic fatty liver and non-alcoholic steatohepatitis. Many liver diseases can be treated by gene therapy, including: hemophilia A, hemophilia B, α-1 antitrypsin deficiency, Wilson's disease, hereditary tyrosinemia, PFIC (progressive familial intrahepatic cholestasis), hereditary hemochromatosis, Crigler-Najjar syndrome, familial hypercholesterolemia, acute hepatic porphyria, acute intermittent porphyria, von Willebrand disease, primary hyperoxaluria, atypical HUS, phenylketonuria, maple syrup urine disease, methylmalonic acidemia, propionic acidemia, NAGS deficiency, CPS I deficiency, OTC deficiency, argininosuccinic aciduria, arginase deficiency, citrullinemia, Fabry disease, MPS, Pompe disease, GSDla, and cystathionine β-synthase deficiency. To describe two examples in more detail, two common genetic liver diseases are hemochromatosis and α-1 antitrypsin deficiency.

[0012] Hemochromatosis is a disease in which iron accumulates in the liver and other organs. The primary form of this disease is one of the most common genetic diseases in the United States, with an estimated incidence of up to 1 in 200 people in the US population. Unfortunately, many people who carry genetic risk factors for hemochromatosis are not aware that they are carriers.

[0013] α-1 antitrypsin deficiency is a genetic liver disease that affects an important liver protein called α-1 antitrypsin, which is either completely absent or present in greatly reduced amounts in affected individuals. People with α-1 antitrypsin deficiency are usually able to produce this protein, but the protein cannot enter the bloodstream and instead accumulates in the liver. The α-1 antitrypsin protein is crucial for protecting the lungs from enzyme damage. In addition, people with this disease are at risk of cirrhosis.

[0014] Multiple strategies have been attempted for gene therapy of the liver. For example, viral strategies employ systemic or targeted therapy via the vascular route, while other studies have shown the feasibility of injecting viruses through the bile duct. The drawback is that viral vectors such as AAV have significant limitations (~4.8 kb) with respect to the size of the nucleotide sequence that can be packaged. Non-viral strategies such as hydrodynamic injection through the rat bile duct have also been employed. Unfortunately, these non-viral strategies have not been effectively translated to large animal models.

[0015] One strategy for treating liver diseases is gene therapy, which can deliver genes that are missing or in insufficient amounts in hepatocytes. However, to date, no gene therapy strategy for treating or preventing liver genetic diseases has been successfully implemented. Therefore, there is an urgent need for gene therapy protocols that can be implemented in the liver to treat and / or prevent such inherited liver diseases. Summary of the Invention

[0016] In some aspects, the present disclosure provides methods of pressure-guided hydrodynamic injection. The methods include mediating gene delivery and expression into the human body at an optimal pressure during biliary hydrodynamic injection. Methods for eliminating individual differences during the hydrodynamic process to achieve the target pressure are described.

[0017] The present disclosure also relates to compositions and methods for treating kidney diseases. More specifically, the present disclosure relates to compositions and methods for treating kidney diseases by gene therapy. As described in detail below, at least a portion of the present disclosure is based on the surprising finding that ureteral hydrodynamic injection can mediate renal cortex rupture at various different flow rates and volumes, and thus only limited parameters are safe to observe.

[0018] The present disclosure also relates to compositions and methods for treating pancreatic diseases. More specifically, the present disclosure relates to compositions and methods for treating pancreatic diseases by gene therapy. As described in detail below, at least a portion of the present disclosure is based on the surprising finding that the volume of liquid required for sufficient gene expression is less than 20 mL, as previously disclosed to be sufficient.

[0019] The present disclosure also provides a method of hydrodynamic gene injection from the common bile duct. The methods herein improve previous strategies of injection through the common bile duct by providing an optimal flow rate and pressure that mediate gene expression within hepatocytes of the liver.

[0020] The present disclosure also provides methods of delivering genes to tumors of the liver and pancreas. These methods include using hydrodynamic injection through the biliary tract as a method of delivering genes to the tumor microenvironment. Methods for injecting parameters and effective doses of DNA into tumors are disclosed.

[0021] The present disclosure also provides methods for mediating the efficient delivery of genes to the liver of a primate. The method includes the step of hydrodynamically delivering a gene to the biliary system of the liver of a primate. Optimal injection parameters for mediating the efficient delivery of genes to the liver of a primate while reducing side effects are described. The pressure for mediating the delivery of genes to the liver of a primate and dosing paradigms are also described.

[0022] The present disclosure also relates to compositions and methods for hydrodynamically delivering genes to the liver through the biliary system. More specifically, the present disclosure relates to compositions and methods for enhancing the efficiency of hydrodynamically delivering genes through the biliary system. As described in detail below, at least a portion of the present disclosure is based on the surprising discovery that by modifying the DNA vector to substantially reduce the bacterial sequence on the plasmid DNA to less than 500 base pairs, the observed hepatocyte transfection area is significantly increased, reaching more than 70% of the hepatocytes, while the transfection area using a conventional plasmid is less than 50%.

[0023] In one aspect, the present disclosure provides a method for determining the flow rate of a hydrodynamic injection into an organ, comprising the steps of:

[0024] Performing a test injection;

[0025] Measuring the pressure during the test injection; and

[0026] Empirically evaluating the differences in stiffness and resistance.

[0027] In an exemplary embodiment, the organ is selected from the liver, pancreas, or kidney.

[0028] In an exemplary embodiment, the liver is injected through the biliary system.

[0029] In an exemplary embodiment, the pancreas is injected through a catheter system.

[0030] In an exemplary embodiment, the kidney is injected through the ureteral system.

[0031] In an exemplary embodiment, a pressure sensor is inserted into the catheter through a dedicated lumen.

[0032] In an exemplary embodiment, the pressure sensor is already present in the catheter.

[0033] In an exemplary embodiment, the pressure sensor is a sensor connected to a fluid-filled lumen for pressure sensing.

[0034] In an exemplary embodiment, the method further comprises the steps of: reading a baseline of the biliary system pressure with the balloon uninflated; measuring the pressure in the biliary system with the balloon inflated; injecting a test solution or nucleic acid into a target duct or blood vessel without plasmid DNA with the balloon inflated; and monitoring the pressure during the injection of the test solution.

[0035] In an exemplary embodiment, the test solution has the same osmolarity, osmolality, and viscosity as the DNA injection solution.

[0036] In an exemplary embodiment, the test solution also does not contain any other active pharmaceutical substances contained in the therapeutic DNA solution.

[0037] In an exemplary embodiment, the pressure reached during hydrodynamic injection is at least 50 mmHg, at least 80 mmHg, or at least 120 mmHg.

[0038] In an exemplary embodiment, the initial test flow rate into the liver is at least 2 mL / second, at least 3 mL / second, or at least 4 mL / second.

[0039] In an exemplary embodiment, the total test volume is a volume of at most 15 mL, at most 10 mL, at most 7 mL, or at most 5 mL.

[0040] In an exemplary embodiment, the total test volume is sufficient to measure the total fluid resistance column in the entire circuit and to evaluate whether sufficient pressure is achieved.

[0041] In an exemplary embodiment, if sufficient pressure is not achieved, increase the flow rate and repeat the test.

[0042] In an exemplary embodiment, if the pressure reaches more than 250 mmHg, decrease the flow rate and repeat the test.

[0043] In an exemplary embodiment, for the second test, the flow rate is increased or decreased by 1 mL / second or increased or decreased by 0.5 mL / second.

[0044] In an exemplary embodiment, the DNA solution is injected at a set flow rate to generate an appropriate pressure.

[0045] In an exemplary embodiment, a series of flow rates are tested during a single test injection, wherein multiple flow rates are tested and the pressure is measured during a single test injection.

[0046] In an exemplary embodiment, at least two or more test flow rates are tested within a single test injection.

[0047] In an exemplary embodiment, the pressure may be related to the increasing flow rate.

[0048] In an exemplary embodiment, the total injection volume is increased to up to 40 mL, 30 mL, or 20 mL, and all injection parameters are tested.

[0049] In an exemplary embodiment, the minimum pressure for effective hydrodynamic gene delivery is greater than 50 mmHg, greater than 80 mmHg, or greater than 100 mmHg.

[0050] In an exemplary embodiment, the maximum pressure for effective hydrodynamic gene delivery is less than 200 mmHg or less than 250 mmHg.

[0051] In one aspect, the present disclosure provides a method for hydrodynamic retrograde ureteral delivery of nucleic acids or viral vectors, comprising the steps of:

[0052] (i) Inserting a cystoscope into the bladder through the urethra,

[0053] (ii) Inserting a balloon catheter into the bladder through the cystoscope,

[0054] (iii) Inserting the balloon catheter into the ureteral orifice,

[0055] (iv) Inflating the balloon catheter at the distal ureter near the entrance of the ureteral orifice,

[0056] (v) Using a power injector to mediate hydrodynamic injection into the kidney,

[0057] wherein the method does not require the use of fluoroscopy for catheter placement.

[0058] In an exemplary embodiment, renal rupture or laceration is avoided by using a total volume equal to or less than 20 mL and an injection parameter of 2 ml / sec.

[0059] In an exemplary embodiment, a flow rate equal to or between 0.5 mL / sec and 2 mL / sec is optimal for achieving rupture-free gene delivery.

[0060] In an exemplary embodiment, the optimal injection volume is between 10 mL and 20 mL to mediate effective gene delivery without rupture.

[0061] In an exemplary embodiment, the balloon is positioned in the bladder muscular wall of the ureter and can be visualized through a cystoscope camera without fluoroscopy.

[0062] a. The method according to claim 5, wherein during injection, the cystoscope camera can be monitored for any ureteral outflow, and if there is no outflow at all, it indicates effective sealing during injection.

[0063] In an exemplary embodiment, for a single kidney of a subject weighing 30 kg or more, the minimum dose of nucleic acid is 1 mg, 2 mg, 3 mg, 4 mg or a higher mass.

[0064] In an exemplary embodiment, the hydrodynamic parameters are sufficient to achieve protein expression within kidney cells after delivery of DNA:

[0065] a. The method according to claim 7, wherein the cell expression is achieved within glomeruli, tubules or endothelial cells inside the kidney.

[0066] b. The method according to claim 7, wherein the difference in cell expression between glomeruli, tubules or endothelial cells is related to the use of different promoters in plasmid DNA.

[0067] In one aspect, the present disclosure provides a method for hydrodynamic retrograde ureteral delivery of nucleic acids or viral vectors, comprising the steps of:

[0068] (i) Inserting a cystoscope into the bladder through the urethra,

[0069] (ii) Inserting a guide wire into the bladder through the cystoscope,

[0070] (iii) Inserting the guide wire into the ureteral orifice,

[0071] (iv) Advancing the guide wire to the kidney,

[0072] (v) Removing the cystoscope and exchanging the guide wire with a balloon catheter,

[0073] (vi) Inflating the balloon catheter in the ureter at a proximal position near the kidney,

[0074] (vii) Using a power injector to mediate hydrodynamic injection into the kidney, wherein the method can utilize a balloon catheter that does not fit into the working channel of the cystoscope.

[0075] In an exemplary embodiment, kidney rupture or tear is avoided by using injection parameters equal to or less than 12 mL and 2 mL / second.

[0076] In an exemplary embodiment, the optimal flow rate for achieving rupture-free gene delivery is equal to or between 1 0.5 and 2 mL / second.

[0077] In an exemplary embodiment, the optimal injection volume is between 7 mL and 12 mL to mediate effective gene delivery without rupture.

[0078] In an exemplary embodiment, the balloon is positioned at least 1 cm, 2 cm or 3 cm away from the renal pelvis and enters the ureter to ensure proper sealing.

[0079] In an exemplary embodiment, a radioactive contrast agent injection is utilized to confirm the positioning of the catheter and the sealing of the balloon prior to injection.

[0080] In an exemplary embodiment, for a single kidney of a subject weighing 30 kg or more, the minimum dose of nucleic acid is 1 mg, 2 mg, 3 mg, 4 mg or a higher quality.

[0081] In an exemplary embodiment, the hydrodynamic parameters are sufficient to achieve protein expression within kidney cells after delivering DNA:

[0082] a. The method of claim 15, wherein the cellular expression is achieved within glomeruli, tubules, or endothelial cells within the kidney.

[0083] b. The method of claim 15, wherein the difference in cellular expression between glomeruli, tubules, or endothelial cells is related to the use of different promoters in the plasmid DNA.

[0084] In an exemplary embodiment, the nucleic acid consists of plasmid DNA, minicircle DNA, mRNA, siRNA, or antisense oligonucleotides.

[0085] In an exemplary embodiment, the viral vector is selected from adenovirus, adeno-associated virus, lentivirus, baculovirus, circovirus, or Sindbis virus.

[0086] In an exemplary embodiment, hydrodynamic injection facilitates better penetration of the viral vector into tissues, binding to cells, and entry into cells compared to injecting the viral vector at a non-hydrodynamic flow rate.

[0087] In an exemplary embodiment, the efficiency of renal cell transduction by retrograde ureteral injection using hydrodynamic injection is higher than that using non-hydrodynamic injection (flow rate < 0.15 mL / second).

[0088] In an exemplary embodiment, a pressure sensor can be further utilized to monitor hydrodynamic injection to ensure a pressure of at least 50 mmHg, 60 mmHg, 70 mmHg, or 80 mmHg is achieved.

[0089] In an exemplary embodiment, distal injection into the ureter is sufficient to generate a pressure of at least 80 mmHg when the flow rate is equal to or greater than 0.5 mL / second.

[0090] In an exemplary embodiment, proximal injection into the ureter is sufficient to generate a pressure of at least 100 mmHg when the flow rate is equal to or greater than 1 mL / second.

[0091] In one aspect, the present disclosure provides a method for hydrodynamic gene delivery to the pancreas via a catheter system, comprising the following steps:

[0092] (a) Insert a catheter into the pancreatic duct by endoscopic retrograde cholangiopancreatography,

[0093] (b) Inflate during injection to seal and increase pressure,

[0094] (c) The injection flow rate is equal to or between 1 and 2 mL / second,

[0095] (d) The injection volume is equal to or less than 0.20 mL per gram of pancreatic weight,

[0096] (e) The DNA dose injected is at least 10 micrograms per gram of pancreatic weight,

[0097] wherein the method is sufficient to mediate gene expression in all pancreatic lobes and results in a decrease in elevated pancreatic enzymes and tissue necrosis.

[0098] In an exemplary embodiment, the injection parameters achieve gene expression in duct cells, islet cells, acinar cells, endothelial cells, and neurons.

[0099] In an exemplary embodiment, for a pancreas weighing more than 60 grams, the total injection volume is not more than 15 mL.

[0100] In an exemplary embodiment, if a flow rate of 2 mL / second is used, the maximum volume used is 0.15 mL per gram of pancreatic weight.

[0101] In an exemplary embodiment, the DNA dose is preferably greater than 20 or 30 micrograms per gram of pancreatic weight.

[0102] In an exemplary embodiment, the amylase or lipase level increases by a maximum of 4-fold on the first day after injection.

[0103] In an exemplary embodiment, the catheter can be placed at:

[0104] (i) Enter the main pancreatic duct through the major duodenal papilla, distal to the confluence of the pancreatic duct and the bile duct, or

[0105] (ii) Enter the accessory pancreatic duct or the dorsal pancreatic duct through the minor duodenal papilla and choose to further advance to the main pancreatic duct.

[0106] In an exemplary embodiment, inflate the balloon passing through the common bile duct in the catheter near the entrance of the pancreatic duct to prevent liquid backflow.

[0107] In an exemplary embodiment, the maximum balloon size for sealing the pancreatic duct is 9 mm to avoid injury.

[0108] In an exemplary embodiment, two or more flow rates are used during hydrodynamic injection to further minimize pancreatic tissue damage while maintaining gene delivery.

[0109] In an exemplary embodiment, for the first 50% of the injection volume, the flow rate is initially 1 mL / second, and then for the remaining injection volume, the flow rate increases to 2 mL / second.

[0110] In an exemplary embodiment, for the first 50% of the injection volume, the flow rate is initially 0.5 mL / second, and then for the remaining injection volume, the flow rate increases to 1.5 mL / second.

[0111] In an exemplary embodiment, a sidewall injection catheter is not used to avoid damage to the tube wall and prevent pancreatitis.

[0112] In one aspect, the present disclosure provides a method for hydrodynamic injection into the gallbladder or liver, comprising the steps of: a) placing a catheter in the common bile duct; b) inflating a balloon in the common bile duct to prevent antegrade flow; c) injecting a DNA solution into the biliary system at a high pressure target and / or flow rate, wherein gene expression can be observed in hepatocytes in the liver and cells in the gallbladder by immunostaining.

[0113] In an exemplary embodiment, the high pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

[0114] In an exemplary embodiment, the flow rate is greater than 2 mL / second, greater than 5 mL / second, or greater than 10 mL / second.

[0115] In an exemplary embodiment, the amount injected is greater than 50 mL per kg of liver weight, greater than 75 mL per kg of liver weight, or greater than 100 mL per kg of liver weight.

[0116] In an exemplary embodiment, before injection, bile is removed from the biliary system, and the biliary system is rinsed and perfused with saline.

[0117] In an exemplary embodiment, a saline solution can optionally be used to fill the gallbladder before injection to reduce the pressure difference during injection.

[0118] In an exemplary embodiment, the preferred DNA dose is at least 20 mg per kg of liver weight, or more.

[0119] In an exemplary embodiment, the preferred DNA concentration of the injection solution is at least 0.5 mg / mL, or at least 2 mg / mL, or at least 5 mg / mL DNA or more.

[0120] In one aspect, the present disclosure provides a method for hydrodynamic injection through the biliary system, wherein the injection is performed in the common bile duct with the assistance of a biliary stent.

[0121] In an exemplary embodiment, the biliary stent is placed before hydrodynamic injection.

[0122] In an exemplary embodiment, a bile duct stent is placed above the cystic duct to prevent fluid from entering the cystic duct.

[0123] In an exemplary embodiment, the biliary stent is at least larger than the diameter of the bile duct to provide sufficient sealing for the duct wall and the stent during injection.

[0124] In an exemplary embodiment, the length of the biliary stent is variable and can reach upstream of the cystic duct from the duodenum.

[0125] In an exemplary embodiment, the balloon catheter is inserted into the stent after placement.

[0126] In an exemplary embodiment, the balloon catheter can be located at any position within the stent, including the common bile duct.

[0127] In an exemplary embodiment, before injection, contrast agent is injected through the stent to confirm that the cystic duct and the gallbladder are not covered and to opacify the upstream biliary system.

[0128] In an exemplary embodiment, the DNA solution is injected according to set parameters to mediate gene delivery into different cells within the liver.

[0129] In an exemplary embodiment, the preferred flow rate during injection is at least 1 mL / second, or at least 2 mL / second.

[0130] In an exemplary embodiment, the preferred injection pressure is at least 50 mmHg, or at least 80 mmHg.

[0131] In an exemplary embodiment, the preferred injection volume is at least 30, 40, 50, or 60 mL per kg of liver weight.

[0132] In an exemplary embodiment, the preferred DNA dose is at least 10 mg per kg of liver weight, or more.

[0133] In an exemplary embodiment, the preferred DNA concentration of the injection solution is at least 0.2 mg / mL DNA or more.

[0134] In an exemplary embodiment, the stent is made of a solid, continuous, and non-porous material such that fluid cannot pass through the wall of the stent.

[0135] In one aspect, the present disclosure provides a method for delivering a non-viral DNA vector into a liver tumor, comprising the steps of: placing a catheter into the biliary system, preferably the common hepatic duct; inflating a balloon in the common hepatic duct to prevent forward flow; and injecting a DNA solution into the biliary system under hydrodynamic pressure, wherein the injection enables expression of the non-viral DNA vector within tumor cells regardless of the location of the tumor within the liver.

[0136] In an exemplary embodiment, the tumor is close to the biliary system to achieve effective delivery.

[0137] In an exemplary embodiment, to effectively deliver tumor genes, the target pressure is at least 50 mmHg, 70 mmHg, or at least 120 mmHg.

[0138] In an exemplary embodiment, a flow rate of at least 2 mL / sec, 4 mL / sec, 7 mL / sec, or at least 10 mL / sec is utilized to achieve effective tumor gene delivery.

[0139] In an exemplary embodiment, the volume injected is at least 30 mL per kg of liver weight. Among them, the non-viral DNA dose injected is at least 10 mg per kg of liver weight, or at least 20 mg per kg of liver weight.

[0140] In an exemplary embodiment, gene expression in tumor cells is highest at the margin of the tumor.

[0141] In one aspect, the present disclosure provides a method for delivering a non-viral DNA vector into a pancreatic tumor, comprising the steps of: a) placing a catheter into the pancreatic duct system upstream of the tumor; b) inflating a balloon in the pancreatic duct to prevent forward flow; c) injecting a DNA solution into the pancreatic duct system under hydrodynamic pressure; wherein the injection enables the expression of the non-viral DNA vector within tumor cells regardless of the location of the tumor within the pancreas.

[0142] In an exemplary embodiment, the tumor is close to the duct system to achieve effective delivery.

[0143] In an exemplary embodiment, a pressure of at least 50 mmHg, 70 mmHg, or at least 120 mmHg is controlled to achieve effective tumor gene delivery.

[0144] In an exemplary embodiment, a flow rate of at least 1 mL / sec is controlled to achieve effective tumor delivery.

[0145] In an exemplary embodiment, a volume of at least 8 mL is injected into the pancreas of an adult.

[0146] In an exemplary embodiment, a non-viral DNA dose of at least 1 mg is injected into the pancreas of an adult.

[0147] In an exemplary embodiment, gene expression in tumor cells is highest at the margin of the pancreatic tumor.

[0148] In an exemplary embodiment, tumor delivery is most effective for pancreatic ductal adenocarcinoma.

[0149] In one aspect, the present disclosure provides a method for delivering a gene into the liver of a primate, comprising the steps of: inserting a catheter into the common hepatic duct of the primate; inflating a balloon in the common hepatic duct to prevent antegrade flow; and injecting a DNA solution into the primate liver under hydrodynamic pressure, wherein the injection achieves > 30% of hepatocytes expressing the gene of interest in the primate liver.

[0150] In an exemplary embodiment, the common hepatic duct is accessed by endoscopic retrograde cholangiopancreatography (ERCP).

[0151] In an exemplary embodiment, the ampulla can be incised to increase the size of the opening for easy insertion of the common hepatic duct during ERCP.

[0152] In an exemplary embodiment, a radiopaque contrast agent injection is used to locate the catheter within the common hepatic duct such that it passes beyond the cystic duct to avoid injection into the gallbladder.

[0153] In an exemplary embodiment, the radiopaque contrast agent injection verifies that the balloon seals the common hepatic duct during injection and visualizes the right and left hepatic ducts.

[0154] In an exemplary embodiment, the DNA solution is a physiological saline solution in which pure recombinant DNA is dissolved.

[0155] In an exemplary embodiment, the DNA in the DNA solution can be plasmid DNA, minicircle DNA or linear closed-ended DNA.

[0156] In an exemplary embodiment, the amount of injection is at least 30 mL per kg of liver weight, or at least 40 / mL / kg or more.

[0157] In an exemplary embodiment, the flow rate is at least 1 mL / second, at least 2 mL / second or 3 mL / second or faster.

[0158] In an exemplary embodiment, the pressure parameter during pressure-guided injection is at least 50 mmHg, at least 80 mmHg or greater than 120 mmHg.

[0159] In an exemplary embodiment, the DNA dose will be at least 10, 20, 30, 40 or 50 mg per kg of liver weight in certain embodiments.

[0160] In an exemplary embodiment, the DNA solution is a DNA vector composition encoding a hepatocyte-specific promoter.

[0161] In an exemplary embodiment, the hepatocyte-specific promoter further comprises one or more hepatocyte-specific enhancers to drive higher levels of transcription.

[0162] In an exemplary embodiment, the gene of interest is codon-optimized using codons selected based on abundance in hepatocytes.

[0163] In an exemplary embodiment, a DNA vector composition and protocol are provided for treating hemophilia B in primates.

[0164] In an exemplary embodiment, the DNA vector composition encodes the human factor IX (hFIX) gene.

[0165] In an exemplary embodiment, the DNA vector composition is a nanoplasmid with a bacterial backbone of less than 500 base pairs.

[0166] In an exemplary embodiment, the total DNA vector composition of hFIX is less than 3 kb.

[0167] In an exemplary embodiment, a dose of 20 mg of the DNA vector composition per kg of primate liver is sufficient to produce 1000 ng / mL of hFIX in primate plasma.

[0168] In an exemplary embodiment, the DNA vector composition can be readministered to ensure further expression.

[0169] In an exemplary embodiment, the process can be repeated a second time in primates, where the expression of two different genes is achieved.

[0170] In one aspect, the present disclosure provides a method for hydrodynamic gene delivery through the biliary system of a subject's liver, comprising the steps of:

[0171] (a) Inserting a catheter into the common hepatic duct,

[0172] (b) Inflating a balloon to seal the catheter and increase pressure during injection,

[0173] (c) Injecting at a flow rate of at least 2 mL / second or a minimum pressure of 50 mmHg,

[0174] (d) Delivering DNA encoding a hepatocyte-specific promoter to drive transgene expression,

[0175] (e) A DNA vector containing a large number of non-mammalian sequence elements,

[0176] (f) Injecting a DNA dose of at least 10 mg DNA per kg of liver weight,

[0177] wherein more than 50% of the hepatocytes express the gene of interest.

[0178] In an exemplary embodiment, in addition, the DNA vector lacks specific modifications, but the optimal injection amount is at least 20 mg DNA per kg of liver weight to achieve hepatocyte expression of the gene of interest in more than 50%.

[0179] In an exemplary embodiment, the selective use of transposons can be utilized to facilitate integration into the host chromosome.

[0180] In an exemplary embodiment, a significant reduction means that the total amount of bacterial or phage DNA sequence is less than 1000 bp.

[0181] In an exemplary embodiment, the DNA is a plasmid DNA vector with a vector bacterial backbone less than 1 kb, or more preferably less than 500 bp.

[0182] In an exemplary embodiment, the plasmid DNA is a nanoplasmid, pFAR or pCOR vector.

[0183] In an exemplary embodiment, the DNA is circular and is microcircular DNA.

[0184] In an exemplary embodiment, the DNA is linear DNA from closed-ended DNA, minicircle DNA or dumbbell DNA.

[0185] In an exemplary embodiment, conversion from a plasmid backbone containing a bacterial sequence greater than 1 kb to the DNA vector of claims 5 and 6 increases the total transfected area of hepatocytes observed by more than 20%.

[0186] In an exemplary embodiment, integration into the host genome using a transposon system can achieve gene expression in at least 40% of hepatocytes for at least 3 months.

[0187] In an exemplary embodiment, the expression duration of the non-integrating DNA of claims 5 and 6 is at least 4 months after injection.

[0188] In an exemplary embodiment, the delivery method expresses for at least 4 months and is capable of generating immune tolerance to foreign transgenes in the liver.

[0189] In an exemplary embodiment, DNA vectors larger than 12 kb, 15 kb or 20 kb in size can be delivered to multiple cell types in the liver, including hepatocytes, endothelial cells and cholangiocytes, thereby generating protein expression.

[0190] In an exemplary embodiment, using larger plasmid DNA of at least 12 kb in size can maintain transfection efficiency.

[0191] In an exemplary embodiment, the DNA dose can be adjusted according to the DNA dose (mg) per kilobase DNA per liver weight (kg) to adjust the DNA size, thereby maintaining equivalent transfection efficiency.

[0192] In an exemplary embodiment, the DNA dose can be predicted using the formula 1 mg / kg / kb to achieve transfection of approximately 50% of the hepatocytes in the liver.

[0193] In an exemplary embodiment, the formula of 2.5 to 5 mg / kg / kb can be used to predict the DNA dose to achieve transfection of approximately 70% of the hepatocytes in the liver.

[0194] In an exemplary embodiment, the process can be repeated with different DNAs expressing the same or different genes during the same injection process such that the expression of two DNAs is now achieved and the expression of the first DNA injection is not eliminated.

[0195] In an exemplary embodiment, the second injection achieves a transfection efficiency similar to that of the first injection and can target the same cells.

[0196] In an exemplary embodiment, the same cell expressing the gene can be observed after injection.

[0197] In an exemplary embodiment, the promoter can be changed to utilize the second injection to achieve expression in different cell types, and the second injection does not change the expression of the first gene.

[0198] In an exemplary embodiment, wherein, the process can be repeated with different DNAs expressing the same or different genes during the same injection process so as to now achieve the expression of two DNAs and the expression of the first DNA injection is not eliminated.

[0199] In an exemplary embodiment, the second injection achieves a transfection efficiency similar to that of the first injection and can target the same cells.

[0200] In an exemplary embodiment, the promoter can be changed to achieve expression in different cell types by the second injection, and the second injection does change the expression of the first gene.

[0201] In an exemplary embodiment, two different DNA molecules can be mixed and delivered during a single injection process such that both DNA molecules enter the same hepatocytes.

[0202] In an exemplary embodiment, DNA doses from 20 mg per kg liver weight to 40 mg per kg liver weight can achieve a similar transfection area.

[0203] In an exemplary embodiment, a DNA dose of up to 40 mg per kg liver weight can be injected without causing significant hepatotoxicity or physiological distress.

[0204] In an exemplary embodiment, a flow rate below 1 mL / second does not produce gene expression.

[0205] In an exemplary embodiment, a flow rate between 1 mL / sec and 2 mL / sec exhibits reduced gene expression compared to a flow rate greater than 2 mL / sec.

[0206] In an exemplary embodiment, a flow rate above 4 mL / sec results in a gradual decrease in hepatocyte delivery efficiency.

[0207] In an exemplary embodiment, a flow rate greater than or equal to 7 mL / sec enables effective bile duct delivery.

[0208] In an exemplary embodiment, a preferred injection volume is between 30 mL / kg and 60 mL / kg per liver tissue.

[0209] In an exemplary embodiment, an injection volume greater than or equal to 70 mL / kg of liver tissue is associated with a decrease in gene delivery efficiency.

[0210] In an exemplary embodiment, the gene injection procedure is well-tolerated in subjects weighing 25 kg, 15 kg, or 5 kg and produces a gene delivery efficiency similar to that of larger mammals.

[0211] In an exemplary embodiment, when combining two or more flow rates during injection, the transfected hepatocyte area by bile hydrodynamics delivery can further increase by at least 10% of the total hepatocytes.

[0212] In an exemplary embodiment, first inject 50% to 66% of the total injection volume at a flow rate of 2 mL / sec, and then inject the remaining volume at a flow rate of 4 mL / sec.

[0213] In an exemplary embodiment, first inject 33% of the volume at a flow rate of 2 mL / sec, then inject 33% of the volume at a flow rate of 3 mL / sec, and then inject the remaining volume at a flow rate of 4 mL / sec.

[0214] In an exemplary embodiment, the catheter is inserted into the common hepatic duct via ERCP, EUS, or image-guided percutaneous approach.

[0215] In an exemplary embodiment, the DNA concentration of the injection solution is at least 0.30 mg / mL, and more preferably a concentration greater than 0.40 mg / mL, 0.50 mg / mL, or 0.60 mg / mL.

[0216] In one aspect, the present disclosure provides a method for achieving expression within liver sinusoidal endothelial cells (LSEC), comprising the step of performing biliary hydrodynamic injection according to claim 1, except for targeted expression in LSEC using a cell-specific promoter.

[0217] In an exemplary embodiment, LSEC can be targeted for expression using a CD36 promoter or an FVIII promoter.

[0218] In an exemplary embodiment, an injection pressure of 80 mmHg can produce more effective expression than an injection pressure of 50 mmHg.

[0219] In an exemplary embodiment, a pressure of 150 to 200 mmHg can produce effective gene expression.

[0220] In an exemplary embodiment, a pressure above 200 mmHg will gradually reduce the gene expression produced.

[0221] In one aspect, a more effective balloon seal can be obtained by advancing the catheter and inflating the balloon into the intrahepatic duct.

[0222] In one aspect, placing the balloon in the extrahepatic duct will result in leakage of the fluid around the balloon.

[0223] In one aspect, the balloon size is at least 2 times, 3 times or 4 times the diameter of the catheter.

[0224] In one aspect, when the balloon is placed in the extrahepatic bile duct, the balloon can be inflated to a maximum size of 3 times the diameter of the catheter.

[0225] In one aspect, when the balloon is placed in the intrahepatic bile duct, the balloon can be inflated to a minimum size of 4 times the diameter of the catheter.

[0226] In one aspect, the balloon of this size will not fully expand within the intrahepatic duct, but will generate additional pressure within the balloon.

[0227] In one aspect, whether intrahepatic or extrahepatic, leakage will occur around the balloon when the balloon size is 8.5 mm or smaller, so these sizes should be avoided.

[0228] In one aspect, to avoid rupture, the balloon is inflated to less than 15 mm in the common hepatic duct.

[0229] In one aspect, the balloon is placed in the right hepatic duct or the left hepatic duct, followed by injection at that location, and then the injection is repeated again in the contralateral hepatic duct to ensure equal injection of both lobes of the liver.

[0230] In one aspect, the balloon seal can be monitored by measuring the pressure within the bile duct lumen.

[0231] In one aspect, the disappearance of the plateau waveform (defined as a decrease of more than 20 mmHg from the start to the end of the plateau) indicates leakage around the balloon.

[0232] In one aspect, the balloon seal can be verified by filling the bile ducts above and below the balloon with a radiopaque contrast agent solution before injection.

[0233] In one aspect, the loss of fluid seal during injection is demonstrated by clearing the contrast agent below the balloon (into the cystic duct and gallbladder, or into the common bile duct).

[0234] In one aspect, the contrast agent above the balloon successfully enters the liver, indicating successful injection.

[0235] In one aspect, hydrodynamic injection can be repeated multiple times in a single procedure to enhance DNA delivery.

[0236] In one aspect, using two or more injections can increase the final gene expression level.

[0237] In one aspect, this strategy allows overcoming the inherent limitations of angiographic or kinetic injection volumes by using multiple injections.

[0238] In one aspect, the liver tissue of primates is more elastic than that of pigs, so different catheter characteristics require changing the balloon size and injection parameters to mediate gene delivery in primates.

[0239] In one aspect, in a pig model, the flow rate must be increased to achieve a given pressure and injection volume.

[0240] In one aspect, a flow rate of at least 4 mL / sec is required to achieve a plateau pressure of at least 80 mmHg.

[0241] In one aspect, primates can tolerate a pDNA dose of at least 80 mg without any significant physiological side effects.

[0242] In one aspect, at least 120 mL of injection volume can be injected into primates per 400 g of liver without any significant interference with vital signs.

[0243] In one aspect, the primate liver can tolerate an injection speed of up to 12 mL / sec without causing tissue damage, changes in vital signs, or bile duct rupture.

[0244] In one aspect, a flow rate greater than 4 mL / sec but less than 8 mL / sec should be used because there is a lack of improved gene delivery at higher flow rates.

[0245] In one aspect, a volume of 30 mL per 400 g can be used, or up to 150 mL per 400 g can be used at most.

[0246] In one aspect, the volume used does not affect the efficiency of gene delivery at a given DNA dose.

[0247] In one aspect, the carrier composition should be dosed according to the copy number of the transgenic expression cassette, and different pDNA doses are required if the DNA vector contains additional foreign DNA.

[0248] In one aspect, using DNA molecules with reduced backbone allows the use of relatively small DNA doses.

[0249] In one aspect, increasing the pDNA dose and / or the carrier expression cassette dose per animal can lead to a quantitatively equivalent increase in the expression of the protein therapeutic of interest.

[0250] In one aspect, a method for hydrodynamic gene delivery through the biliary system of the liver of a subject is provided. In one aspect, the method includes:

[0251] (a) Inserting a catheter into the bile duct;

[0252] (b) Inflating a balloon to seal within the bile duct and prevent forward flow of the solution.

[0253] i. The inflated size of the balloon is at least 2 times, 3 times, or 4 times the diameter of the bile duct to overcome the elasticity of the bile duct in primates;

[0254] ii. Among them, the method for verifying the balloon seal during injection includes placing radioactive contrast agent solutions above and below the balloon to examine the forward movement of the liquid by fluoroscopy.

[0255] (c) Injecting at a flow rate of at least 2 mL / sec, or injecting at a pressure of at least 50 mmHg. i. More preferably, the minimum value is 4 mL / sec, which can generate a minimum of 80 mmHg in the liver of primates at the plateau pressure; among them, the flow rate can be further minimized to less than 12 mL / sec, less than 10 mL / sec, or less than 8 mL / sec without causing loss of total gene expression;

[0256] ii. The injection volume is preferably less than 250 mL / kg liver tissue, less than 150 mL / kg liver tissue, or less than 50 mL / kg liver tissue;

[0257] iii. Among them, the plateau pressure obtained during hydrodynamic injection changes less than 10% mmHg during injection, indicating sufficient seal; and

[0258] iv. Among them, optionally, multiple flow rates can be adopted in a single injection to change and vary the pressure obtained; and

[0259] (d) Delivering a micro DNA vector, in which non-mammalian sequence elements are significantly reduced or absent.

[0260] i. Among them, the miniaturized DNA vector provides a longer expression duration of at least 4 months;

[0261] ii. Among them, the miniaturized DNA vector provides high potency in primates at a given DNA dose, allowing a smaller dose to be used compared to conventional plasmid DNA;

[0262] iii. Among them, the DNA vector preferably contains a hepatocyte-specific promoter to enhance expression in the liver.

[0263] In one aspect, substantially reduced means that the total amount of bacterial or phage DNA sequence is less than 1000 bp.

[0264] In one aspect, the DNA is a vector bacterial backbone or a plasmid DNA vector with a sequence less than 1 kb, or more preferably less than 500 bp.

[0265] In one aspect, the plasmid DNA is a nanoplasmid, GenCircle, pFAR or pCOR vector. In one aspect, the DNA is circular and is microcircular DNA or microvector DNA.

[0266] In one aspect, the DNA is linear DNA from closed-ended DNA, miniDNA or dogbone DNA.

[0267] In one aspect, the linear DNA has only small foreign sequences at either end, each sequence being less than 100 bp in size, and the mammalian expression sequence of interest is the rest of the vector.

[0268] In one aspect, sphincterotomy of the biliary tract is performed during the first surgery of the subject to reduce or eliminate the risk of post-ERCP pancreatitis when re-injecting genetic drugs during subsequent ERCP procedures.

[0269] In one aspect, two injections can be repeated within one treatment course to increase protein expression.

[0270] In one aspect, repeated injection with the same DNA can promote the expression of a single protein, or two different DNA solutions can be used to produce the expression of two different proteins.

[0271] In one aspect, the total sum of the DNA doses for repeated injection is similar to or equivalent to the expression obtained from a single DNA injection.

[0272] In one aspect, the injection procedure can be repeated again after a single administration, such that the transfection efficiency is the same and the peak protein expression is equivalent between injections, without observing immunogenicity.

[0273] In one aspect, repeated injection procedures can be performed at intervals of at least one month, at least three months, at least six months, or at least one year.

[0274] In one aspect, a pressure sensor is used to detect a liquid-filled column in a pressure catheter to monitor pressure, or alternatively, a pressure sensor screwed into the catheter lumen is used to monitor pressure.

[0275] In one aspect, pressure sensor readings are recorded in real time, and the pressure curve is interpreted after injection to determine whether successful sealing and peak expression are achieved.

[0276] In one aspect, optimal hydrodynamic injection can cause the levels of liver enzymes such as ALT and AST to increase by at least 2-fold on the first day after injection compared to the pre-treatment value.

[0277] In one aspect, the liver enzymes return to the normal range within 7 days after injection.

[0278] In one aspect, gene delivery occurs within and around the tumor, in malignant and normal cells, scattered around the tumor.

[0279] In another aspect, the present disclosure provides a method for hydrodynamic injection into the liver through the biliary system, using a partially expanded full-coverage metal or plastic stent, wherein: a) the stent is deployed from the distal end to the proximal end (relative to the stent deployment catheter); b) the released or opened part of the stent is located within the common hepatic duct, and the tip of the stent remains in the delivery catheter that houses the stent, such that it is located around the common hepatic duct, common bile duct, or ampulla of Vater; c) wherein the cystic duct orifice is blocked and / or bypassed by the covered part of the stent, such that the injected fluid solution cannot enter the cystic duct or gallbladder; d) the guide wire is removed from the stent delivery system; e) a DNA solution with a high-pressure target and / or flow rate is injected into the biliary system through the guide wire lumen; wherein, due to the partial deployment and continuous connection of the stent, a closed system is formed with the catheter, thereby preventing antegrade flow in the biliary system, and / or wherein gene expression in hepatocytes within the liver can be observed during immunostaining.

[0280] In one embodiment, the high-pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

[0281] In one embodiment, the flow rate is greater than 2 mL / second, greater than 5 mL / second, or greater than 10 mL / second.

[0282] In one embodiment, the injected volume is greater than 50 mL per kg of liver weight, or greater than 75 mL per kg of liver weight, or greater than 100 mL per kg of liver weight.

[0283] In one embodiment, prior to injection, bile is removed from the biliary system and the biliary system is rinsed and cleaned with saline.

[0284] In one embodiment, a preferred DNA dose is at least 20 mg per kg of liver weight, or more.

[0285] In one embodiment, the DNA concentration of the preferred injection solution is at least 0.5 mg / mL of DNA or more.

[0286] In one embodiment, the diameter of the bile duct stent is at least that of the bile duct to provide sufficient sealing for the tube wall and the stent during injection.

[0287] In one embodiment, the diameter of the bile duct stent is at least 150% of the bile duct diameter, or at least 200% of the bile duct diameter.

[0288] In one embodiment, the length of the bile duct stent is variable and can extend from outside the ampulla to upstream of the bile duct.

[0289] In one embodiment, injection is performed from the tip of the "olive" surface of the stent. During injection, the "olive" may be located between the hepatic end of the stent and the porta hepatis.

[0290] In one embodiment, injection occurs at an opening on the catheter, which is located at the proximal position of the catheter through which the stent is inserted, so that the fluid fills the stent during retrograde flow, and the cone of the stent entering the catheter prevents any antegrade flow.

[0291] In one embodiment, prior to injection, contrast agent is injected through the stent to confirm that the cystic duct and the gallbladder are not covered and the biliary system becomes opaque.

[0292] In one embodiment, the stent is made of a solid material such that fluid cannot pass through the stent wall.

[0293] In one embodiment, the hepatic end of the stent opens into the left or right main hepatic duct rather than the common hepatic duct.

[0294] In one embodiment, once hydrodynamic injection is completed from the left or right main hepatic duct, the same technique is used to inject the replacement catheter.

[0295] In one embodiment, the stent is partially deployed, which means that at most 95% of its length, 75% of its length, 50% of its length, or 25% of its length is deployed outside the catheter in some embodiments, while the remaining length of the stent remains inside the catheter or attached to the catheter.

[0296] In one embodiment, the partially deployed stent forms a funnel or cone shape at its proximal end where it is connected to the catheter, thus forming a closed system.

[0297] Definition

[0298] For ease of understanding the present disclosure, the following terms and phrases are defined:

[0299] Unless otherwise specified or obvious from the context, the term "about" as used herein shall be understood to be within the normal tolerances in the art, such as within 2 standard deviations of the mean. About can be understood to be within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless the context clearly dictates otherwise, all numerical values provided herein are modified by the term about.

[0300] "Agent" refers to any small molecule compound, antibody, nucleic acid molecule, polypeptide or fragment thereof.

[0301] "Improve" means to reduce, inhibit, attenuate, decrease, prevent or stabilize the development or progression of a disease (e.g., a genetic disease of the phenotype of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)).

[0302] "Alter" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide detected by methods known in the standard art (e.g., the methods described herein). Alterations as used herein include a 10% change in the expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change.

[0303] The term "combination therapy" includes the administration of a gene therapy regimen and one or more other therapeutic agents (e.g., erythropoietin, corticosteroids, angiotensin converting enzyme (ACE) inhibitors) as part of a specific treatment regimen, aimed at producing a beneficial (additive or synergistic) effect through the combined action of these therapeutic agents. The beneficial effects of combination therapy include, but are not limited to, pharmacokinetic or pharmacodynamic interactions resulting from the combination of therapeutic agents. The combined administration of these therapeutic agents is typically carried out over a defined period of time (usually minutes, hours, days or weeks, depending on the combination selected). "Combination therapy" is intended to include the administration of these therapeutic agents in a sequential manner, i.e., where each therapeutic agent is administered at a different time, and in a substantially simultaneous or overlapping manner, i.e., the administration of these therapeutic agents or at least two of them. Substantially simultaneous administration can be achieved, for example, by administering to a subject one or more copper chelating compounds while administering a gene therapy regimen as disclosed herein. The sequential or substantially simultaneous administration of each therapeutic agent can be carried out by any suitable route, including but not limited to the oral route, intravenous route, subcutaneous route, intramuscular route, direct absorption through mucosal tissues (e.g., nasal, oral, vaginal and rectal) and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or different routes. For example, one component of a particular combination can be administered by intravenous injection (e.g., a gene therapy regimen), while other components of the combination (e.g., one or more copper chelates) can be administered orally. These components can be administered in any therapeutically effective sequence.

[0304] The phrase "combination" encompasses groups of compounds and / or non-pharmacological gene therapies that can be used as part of the combination therapies disclosed herein.

[0305] In the present disclosure, terms such as "comprises", "comprising", "contains" and "having" can have the meanings ascribed to them under United States patent law and can mean "includes", "including", etc.; "consisting essentially of" or "consisting essentially of" likewise has the meaning ascribed under United States patent law and the term is open-ended, allowing for elements in addition to those recited, provided that the basic or novel characteristics of the recited elements are not changed by the presence of the recited elements, but excluding prior art embodiments.

[0306] "Control" refers to a standard or reference condition.

[0307] "Disease" refers to any condition or disorder that impairs or interferes with the normal function of cells, tissues or organs (e.g., genetic diseases of target tissues such as the kidney, pancreas, bile duct, tumor, liver, etc.).

[0308] "Effective amount" means the amount required to improve the symptoms of a disease (e.g., symptoms of a neurological or other genetic disease in a target tissue such as the kidney, pancreas, common bile duct, tumor, liver, etc.) relative to an untreated patient. For the practice of the present disclosure, the effective amount of an active compound for treating a disease depends on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate dosage and dosing regimen. Such amount is referred to as an "effective" amount. The effective amount may also refer to the level of gene expression (e.g., ATP7B mRNA or protein expression) in the appropriate tissue of a patient.

[0309] "Fragment" means a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can comprise 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more nucleotides or amino acids.

[0310] "Gene therapy composition" should be understood as a DNA composition (e.g., including the full-length PKD1, PKD2, SLC3A1, SLC7A9, NPHP1-NPHP9, MKS1, SLC12A3, OLRL1, CSNCU1, CFTR, IGF-1, Reg3g, ATP7B, UGT1A1, HFE, OTC, LDLR, ABCB4, PBGD, or VWF nucleotide sequences or portions thereof) for preventing and / or treating genetic diseases in a target tissue (such as the kidney, pancreas, common bile duct, tumor, liver, etc.). Thus, in certain aspects, the gene therapy composition is a medicament comprising the full-length PKD1 nucleotide sequence or a portion thereof and is intended for use in humans or animals to prevent and / or treat genetic diseases of the kidney. In certain aspects, the gene therapy composition is a medicament comprising the full-length CFTR nucleotide sequence or a portion thereof and is intended for use in humans or animals to prevent and / or treat genetic diseases of the pancreas. In certain aspects, the gene therapy composition is a medicament comprising the full-length ATP7B, UGT1A1, HFE, OTC, LDLR, ABCB4, PBGD, or VWF nucleotide sequence or a portion thereof and is intended for use in humans or animals to prevent and / or treat genetic diseases of the liver.

[0311] "Hybridization" refers to hydrogen bonding between complementary bases, which can be Watson-Crick hydrogen bonds, Hoogsteen hydrogen bonds, or reverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary bases that pair by forming hydrogen bonds.

[0312] "Isolated polynucleotide" refers to a nucleic acid molecule (e.g., DNA, mRNA, cDNA, etc.) that does not contain a gene in the genome naturally present in an organism, and to which the nucleic acid molecules of the present disclosure are typically related or from which they are derived. This term thus includes, for example, recombinant DNA that can be incorporated (e.g., genomic DNA or cDNA encoding the ATP7B gene, along with associated regulatory elements such as enhancers, promoters, 5' and / or 3' untranslated regions (UTRs), etc.): vectors, autonomously replicating plasmids or viruses, genomic DNA of prokaryotes or eukaryotes, or polynucleotides that exist as separate molecules independent of other sequences (e.g., cDNA or genomic or cDNA fragments or naked DNA constructs such as plasmids or cosmids or linear DNA generated by PCR or restriction endonuclease digestion). In addition, this term includes RNA molecules transcribed from DNA molecules, and recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0313] "Isolated polypeptide" means that the polypeptides of the present disclosure have been separated from their natural accompanying components. Generally, a polypeptide is isolated when it is at least 60% (by weight) free of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75% (by weight), more preferably at least 90% (by weight), and most preferably at least 99% (by weight) the polypeptide of the present disclosure. The isolated polypeptides of the present disclosure can be obtained, for example, by extraction from natural sources, by expressing recombinant nucleic acids encoding such polypeptides, or by chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0314] For the purposes of the present disclosure, "mutation" refers to a DNA sequence found in a patient's gene that is not related to the established wild-type gene sequence, and such a mutation may be due to one or more single nucleotide polymorphisms, one or more deletions or insertions of one or more nucleotides, and deletions or insertions at splice site junctions. "Mutation" can also refer to a pattern in a patient's RNA sequence that cannot be attributed to an expected variation based on known gene information and should reasonably be regarded as a new variation, such as the splicing pattern of a patient's gene.

[0315] Unless explicitly stated or obvious from the context, the term "or" as used herein should be understood as inclusive. Unless explicitly stated or obvious from the context, the terms "a", "an", and "the" as used herein should be understood as singular or plural.

[0316] The term "patient" or "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example only, subjects include, but are not limited to, mammals, including but not limited to humans or non-human mammals, such as non-human primates, cattle, horses, dogs, sheep, or cats.

[0317] "Pharmaceutically acceptable" means approved or recognized by a federal or state government regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopeias, for use in animals (including humans). "Pharmaceutically acceptable excipients, carriers or diluents" means excipients, carriers or diluents that can be administered with a drug to a subject and that, when administered in a dose sufficient to deliver a therapeutically effective amount of the drug, do not destroy the pharmacological activity of the drug and are non-toxic.

[0318] The "pharmaceutically acceptable salts" of the pooled tumor-specific neoantigens described herein can be acid salts or base salts that are generally considered in the art to be suitable for contact with human or animal tissues without undue toxicity, irritation, allergic response or other problems or complications. Such salts include mineral salts and organic acid salts of basic residues (such as ammonia), and alkali metal salts or organic salts of acidic residues (such as carboxylic acids). Specific pharmaceutical salts include, but are not limited to, for example, salts of hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, p-aminobenzenesulfonic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, acids such as HOOC-(CH2)n-COOH (where n is from 0 to 4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium. Those of ordinary skill in the art will recognize further pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those listed in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from the parent compound containing a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0319] As used herein, the terms "prevent", "preventive", "preventive treatment" and the like refer to reducing the probability that a subject will develop a disease or disorder (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)), where the subject does not have the disease or disorder (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)), but is at risk of developing the disease or disorder or is predisposed to developing the disease or disorder (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)).

[0320] An "oligonucleotide set" refers to a set of oligonucleotides that can be used, for example, in PCR. The oligonucleotide set will consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600 or more oligonucleotides.

[0321] The ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or sub-ranges within the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, as well as all intermediate decimal values between the above integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. For sub-ranges, "nested sub-ranges" extending from either endpoint of the range are specifically contemplated. For example, nested sub-ranges of the exemplary range of 1 to 50 can include 1 to 10, 1 to 20, 1 to 30 and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20 and 50 to 10 in the other direction.

[0322] "Reduce" means a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0323] "Reference" means a standard or control condition.

[0324] "Reference sequence" is a defined sequence (e.g., wild-type ATP7B gene sequence) used as a basis for sequence comparison. The reference sequence can be a subset of the designated sequence or the entire sequence; for example, a fragment of a full-length cDNA or genomic sequence, or a complete cDNA or genomic sequence. For polypeptides, the length of the reference polypeptide sequence is typically at least about 10 to 5,000 amino acids, 10 to 4,000 amino acids, 10 to 3,000 amino acids, 10 to 2,000 amino acids, 10 to 1,500 amino acids, 10 to 1,000 amino acids, 10 to 500 amino acids, or 10 to 100 amino acids. Preferably, the length of the reference polypeptide sequence can be at least about 10 to 50 amino acids, more preferably at least about 10 to 40 amino acids, even more preferably about 10 to 30 amino acids, about 10 to 20 amino acids, about 15 to 25 amino acids, or about 20 amino acids. For nucleic acids, the length of the reference nucleic acid sequence is typically at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or in between.

[0325] Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule encoding a polypeptide of the present disclosure (e.g., an ATP7B polypeptide) or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but will generally exhibit substantial identity (e.g., 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%). Polynucleotides having "substantial identity" to an endogenous sequence are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to the pairing of complementary polynucleotide sequences (e.g., the ATP7B gene described herein) or portions thereof to form double-stranded molecules under various stringent conditions (see, e.g., Wahl, G.M. and S.L.Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

[0326] For example, stringent salt concentration conditions are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvents (such as formamide), while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30 °C, more preferably at least about 37 °C, and most preferably at least about 42 °C. Those skilled in the art are familiar with the fact that other parameters can be varied, such as hybridization time, detergent concentration (such as sodium dodecyl sulfate (SDS)), and the addition or exclusion of carrier DNA. Depending on the need, different stringencies can be achieved by combining these different conditions. In a preferred embodiment, hybridization will be carried out at 30 °C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will be carried out at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will be carried out at 42 °C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Those skilled in the art can readily understand the available variations of these conditions.

[0327] For most applications, the stringency of the wash step following hybridization also varies. The stringent conditions for washing can be defined by salt concentration and temperature. As described above, the stringency of washing can be increased by decreasing the salt concentration or increasing the temperature. For example, the stringent salt concentration conditions for the wash step are preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the wash step generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the wash step will be carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will be carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In an even more preferred embodiment, the wash step will be carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations of these conditions will be readily understood by those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0328] "Substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% identity to a reference amino acid or nucleotide sequence (e.g., any one of the amino acid or nucleotide sequences described herein). Preferably, such a sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence or nucleic acid sequence used for comparison (e.g., wild-type ATP7B).

[0329] Sequence identity is typically measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, and a probability score between e-3 and e-100 indicates closely related sequences.

[0330] As used herein, the term "treatment" and the like refer to alleviating or ameliorating a disease and / or its associated symptoms (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)). It should be understood that although not excluded, treating a disease or disorder does not require complete elimination of the disease, disorder, or its associated symptoms.

[0331] The term "treatment effect" refers to a certain degree of alleviation of one or more symptoms (e.g., neurological, kidney-related, liver-related, etc.) of a genetic disease or its associated pathology of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.). As used herein, a "therapeutically effective amount" refers to the amount of an agent or combination therapy that, when administered to a cell or subject in a single dose or multiple doses, is capable of effectively prolonging the survival rate of a patient with a genetic disease of the target tissue, alleviating one or more signs or symptoms of the genetic disease of the target tissue, preventing or delaying the onset of symptoms of the genetic disease of the target tissue, etc., beyond what would be expected in the absence of such treatment. A "therapeutically effective amount" is intended to define the amount required to achieve a treatment effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the required "therapeutically effective amount" of an agent or combination therapy.

[0332] A pharmaceutical composition should generally provide a dose of about 0.0001 mg to about 200 mg of the compound per kg of body weight per day. For example, the dose range for systemic administration to a human patient can be 0.01 to 10 μg / kg, 20 to 80 μg / kg, 5 - 50 μg / kg, 75 - 150 μg / kg, 100 - 500 μg / kg, 250 - 750 μg / kg, 500 - 1000 μg / kg, 1 - 10 mg / kg, 5 - 50 mg / kg, 25 - 75 mg / kg, 50 - 100 mg / kg, 100 - 250 mg / kg, 50 - 100 mg / kg, 250 - 500 mg / kg, 500 - 750 mg / kg, 750 - 1000 mg / kg, 1000 - 1500 mg / kg, 1500 - 2000 mg / kg, 5 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg or 200 mg / kg. Pharmaceutical dosage unit forms are prepared to provide from about 0.001 mg to about 5000 mg, for example from about 100 to about 2500 mg of the compound or combination of essential ingredients per dosage unit form.

[0333] The recitation of a list of chemical groups in any variable definition herein includes defining the variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes taking that embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0334] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0335] In cases where applicable or not explicitly stated, any one embodiment described herein is considered capable of being combined with any other one or more embodiments, even if those embodiments are described under different aspects of the present disclosure.

[0336] These and other embodiments are disclosed and / or encompassed by the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0337] The above and other features and advantages of the present disclosure will be better understood when the following detailed description is read in conjunction with the accompanying drawings, in which:

[0338] Figures 1A to 1D A cartoon and three images are depicted, showing the technique of hydrodynamic injection through the bladder into the ureter, so that pDNA can be delivered to the pig kidney by injecting into the ureter. Figure 1A A cartoon showing catheter positioning. The blue circle indicates the positioning and placement of the catheter balloon. Figure 1B Is a cytoscopic image of the bladder, showing the ureteral orifice close to the urethra. Figure 1CShows a bulge on the bladder wall, confirming inflation after inserting the balloon into the ureteral orifice. Figure 1D Is a fluoroscopic image of the entire ureter and renal pelvis, in which the radioactive contrast agent is filled to near total volume.

[0339] Figures 2A to 2D Images and four photos showing that hydrodynamic injection through the ureter may cause renal rupture. Hydrodynamic injection through the ureter was explored by testing contrast agent injection and saline injection. Figure 2A Shows the escape of contrast agent into the subcapsular space after hydrodynamic injection. The time point of the left image is earlier than that of the right image, only a few seconds later. Gross examination of the kidney after injection ( Figure 2B ) shows blood accumulation and a large hematoma under the renal capsule. Subsequently, the renal capsule was removed ( Figure 2C ) showing a ~1.5 cm tear on the renal surface at the upper pole of the kidney. Figure 2D Anatomy of the kidney in shows that the tear starts from the bottom of the medulla and cuts through the entire cortex.

[0340] Figures 3A to 3C Shows photos and images of GFP staining, indicating that gene delivery can be achieved by hydrodynamic injection through the ureter. The plasmid pCLucf encoding firefly luciferase and GFP was injected into the kidney with different injection parameters. Figure 3A Is a photo showing the upper, middle, and lower pole sections of the kidney, indicated by black circles. Figure 3B Is an image with a higher magnification, showing GFP staining in glomeruli of dispersed cells. Figure 3C Depicts representative photos of luciferase staining at the upper, middle, and lower poles of the cortex / medulla interface. These pictures depict kidneys with an injection rate of 20 BmL and 2 mL / C second, resulting in the delivery of 1.2 mg pDNA. The picture in the lower right corner shows an uninjected kidney as a staining control. Pigs injected with other injection parameters (27 mL, 2 mL / second; 30 mL, 1 mL / second) showed a similar immunohistochemical (IHC) staining pattern (data not shown).

[0341] Figures 4A to 4C Depicts four images showing that hydrodynamic injection achieves expression in multiple cell types in the kidney. GFP staining after injecting pCLucf in different cell types of the kidney is shown. Figure 4A Shows GFP staining in the cortical tubules. Figure 4B Shows that GFP staining occurs in endothelial cells, Figure 4C Shows that GFP staining can be found in small tubules within the renal medulla.

[0342] Figure 5Five photographs are shown demonstrating that hydrodynamic injection at lower parameters enables safer injection. The hydrodynamic injection parameters for distal ureteral injection were a flow rate ranging from 0.5 mL / sec to 2 mL / sec. In these injections, the total volume ranged from 16 mL to 20 mL, as shown. The kidneys were grossly dissected after injection to look for ruptures. In the upper left corner, the kidney injected with 20 mL at 1 mL / sec showed signs of rupture, and the right figure shows bruising when injected with 20 mL at 1.5 mL / sec. The lower right figure shows rupture of the kidney when injected with 20 mL at 0.5 mL / sec, but the lower right figure shows no damage when injected with 16 mL at 2 mL / sec. The fifth photograph is a control group with no injection. The dissected kidneys of different groups shown in the figure did not show signs of rupture.

[0343] Figure 6 Six hydrodynamic injection images are shown demonstrating that gene delivery can still be achieved at lower parameters. The hydrodynamic injection parameters for distal ureteral injection were a flow rate ranging from 0.5 mL / sec to 2 mL / sec. As shown, the volume ranged from 16 mL to 20 mL, and the injected pDNA resulted in the delivery of 2 mg pCLucf. Representative photographs of firefly luciferase staining in the upper pole of the kidney at each parameter are shown below. Two uninjected control tissues are also shown at the bottom. Similar expression patterns were observed in the mid and lower poles of the kidney and when stained with GFP (data not shown).

[0344] Figure 7A and 7B are hydrodynamic injection images at the lowest flow rate, and the expression effect is comparable to that of higher flow rates. The figure shows hydrodynamic injection of a kidney tissue sample at the lowest tested flow rate of 0.5 mL / C sec, indicating that protein expression can still be achieved. Figure 7A shows that GFP staining shows positive cells in the glomeruli, while the control shows no staining. Figure 7B shows the cortex at low magnification, where numerous tubules depicting firefly luciferase staining are shown.

[0345] Figures 8A to 8D are images and photographs showing that proximal ureteral injection can cause kidney injury. Hydrodynamic injection was performed within the ureter at the proximal position of the renal pelvis. Then, injections were performed at 15 mL and 1 mL / sec, using a contrast agent as the injection solution. Figure 8A The fluoroscopy in shows that the balloon located in the renal pelvis could not prevent the contrast agent from flowing anterogradely into the ureter. Figure 8B The post-injection fluoroscopy in shows rupture of the renal capsule, resulting in the escape of the contrast agent into the subcapsular space. After gross examination of the kidneys at autopsy, Figure 8C the photograph in confirms bleeding and rupture. Figure 8DIn the kidney with a proximal injection of 15 mL at 1 mL / sec, no GFP was detected by immunohistochemical staining.

[0346] Figure 9 Two images are depicted, revealing that proximal ureteral injection can cause kidney injury. Hydrodynamic injection at a rate of 15 mL and 1 mL / sec near the kidney can cause severe kidney injury. The top image is a tissue section where extensive tissue damage and lymphocyte infiltration start from the medulla and extend to the cortex. The bottom image shows a high magnification of the necrotic area and areas of neutrophil and lymphocyte infiltration.

[0347] Figure 10 Eight images are presented, showing gene expression of proximal ureteral injection under different injection parameters. To test safety and gene expression, hydrodynamic injection was evaluated from the proximal ureteral position under various injection parameters. The plasmid DNA dose was 10 mg pCLucf, and the injection parameters tested were 11 mL at an injection rate of 2 mL / sec, 12 mL at an injection rate of 1.5 mL / sec, and 10 mL at an injection rate of 1 mL / sec. Pigs were harvested 3 days after injection, and the kidneys were cut into upper, middle, and lower poles. GFP IHC staining was performed on the upper, middle, and lower poles of the renal cortex (left panel), and GFP IHC staining was performed on the upper, middle, and lower poles of the medulla (right panel). Representative images of IHC staining for the different parameters evaluated are shown.

[0348] Figure 11A and 11B Two images and a graph show the correlation between flow rate and pressure during proximal ureteral hydrodynamic injection. Pressure was monitored during distal ureteral hydrodynamic injection. For the experiment, a four-lumen catheter consisting of a guide wire, balloon, pressure sensor, and injection port was obtained. The catheter tip was placed in a proximal position near the kidney. Figure 11A An example of contrast agent filling the ureter and renal pelvis for localization is depicted. A series of flow rates were tested, and pressure was monitored simultaneously. For example, 10 mL was injected at a flow rate of 1 mL / C sec. In Figure 11B it, the pressure trace shows a peak pressure of 105 mmHg. The pressure drops rapidly, possibly due to kidney rupture or balloon failure.

[0349] Figures 12A to 12C Two photos of the kidney and a graph correlating flow rate with pressure during hydrodynamic injection in the distal ureter are shown. Pressure was monitored during hydrodynamic injection in the distal ureter. For the experiment, laparotomy was performed, and the bladder wall was excised. The ureteral orifice was visualized, and the catheter was inserted into the ureter. The catheter itself was connected to a power injector to deliver a saline solution. The pressure sensor was advanced with the catheter into the ureter and could not pass through the lumen due to its large size. Both the pressure sensor and the catheter were advanced to the distal ureter.Figure 12A Shows a surgical ligation made to prevent antegrade flow during injection into the ureter. A series of different flow rates were tested to evaluate the pressure for a fixed volume of 10 mL. Figure 12B Depicts the results of the pressure tracing, with the arrow indicating the injection. It was found that the pressure depends on the flow rate. When the flow rate was 0.5 to 1 mL / C second, the pressure was 70 to 80 mmHg. When the flow rate was 1.5 to 2 mL / C second, the pressure was 120 to 140 mmHg. Additional tests with injecting more volume at a constant flow rate did not further increase the pressure (data not shown). The ureteral baseline was observed to be 5 to 15 mmHg before injection. After a series of injections, the kidney was further dissected. Figure 12C Shows the fluid accumulated in the space between the observed capsule and the kidney surface. Several tears were noted, which occurred during injection at a speed greater than 3 mL / second.

[0350] Figures 13A to 13D Depicts a cartoon and three images evaluating gene delivery to the pancreatic duct of pigs by endoscopic retrograde cholangiopancreatography (ERCP). The pancreatic duct is the conduit for gene delivery by hydrodynamic injection. Pigs are excellent models for ERCP because their organs are similar in size to humans, although the anatomical structure of the pig pancreas differs in the lobular structure between the two species. Figure 13A Shows a cartoon model of inserting a catheter into the pig pancreas, depicting two separate openings for the bile duct and the pancreatic duct. Figure 13B Demonstrates the intubation of the pig pancreatic duct, which is significantly different from that in humans because pigs do not have an ampulla. In pigs, the opening at the pancreatoduodenal junction and the opening at the choledochoduodenal junction are separate. Figure 13C Shows a representative fluoroscopic image of the pancreatic duct before hydrodynamic injection. Figure 13D Shows a representative fluoroscopic image of the pancreatic duct after hydrodynamic injection, confirming no signs of rupture after injection.

[0351] Figures 14A to 14C Shows images and two charts, which demonstrate the safety of hydrodynamic injection into the pancreas. Hydrodynamic injection was performed into the pig pancreas at an injection parameter of 22 mLB and a speed of 2 mL / C second to deliver 1.2 mg pDNA. Figure 14A Shows abdominal CT scans of pancreatic organs from two different pigs obtained 1 day after injection. To measure pancreatic injury, Figure 14B Shows amylase levels at the time points before injection, after injection, 1 day, and 3 days. To measure the inflammation caused by the injection procedure, Figure 14C Shows the white blood cell count (WBC) levels at the time points before injection, after injection, and 1 day.

[0352] Figures 15A to 15DPhotographs, Western blots, and images showing the pancreatic tissue samples, which evaluate gene expression generated by hydrodynamic gene delivery of plasmid DNA into the pancreas. Figure 15A Photographs of the pancreas obtained from the autopsy of the injected pigs are shown. The pancreas was grossly examined to look for signs of liver injury. The gross specimens were evaluated and no gross pancreatic duct or parenchymal injury was observed. Figure 15B Shows the PCR performed in the first step to detect the presence of plasmid DNA in the porcine duodenal lobe, splenic lobe, and connecting lobe. Figure 15C Images of pancreatic tissue of three lobes analyzed by immunohistochemistry (IHC) to detect the presence of firefly luciferase are shown. Figure 15D Images of pancreatic tissue at low magnification analyzed by IHC to detect the presence of firefly luciferase are shown.

[0353] Figures 16A to 16D Images showing reporter gene expression in different pancreatic cell types after hydrodynamic injection are shown. The effect of hydrodynamic gene delivery of plasmid DNA into the pancreas on gene expression in different cell types in the pancreas was evaluated. In Figure 16A , high magnification images of pancreatic duct cells show specific staining of GFP in pancreatic duct cells (left panel) relative to the saline control (middle panel). Cytokeratin staining confirmed the identity of the duct cells. Figure 16B High magnification images in also show specific staining of luciferase in pancreatic islet cells (left panel) relative to the saline control (middle panel). Synaptophysin staining confirmed the identity of the islet cells. In addition to these cell types, expression of the firefly luciferase reporter gene was also observed in endothelial cells ( Figure 16C ) and neurons ( Figure 16D ) in the pancreatic tissue.

[0354] Figure 17 The images shown indicate that hydrodynamic injection into the pancreas produces areas of tissue damage and results in immune infiltration. The pancreatic tissue was evaluated for signs of tissue damage. In the upper two panels, 4 to 5 areas of immune infiltration were visible in each tissue section, among which there were neutrophils and lymphocytes. The lower panel shows an area without cell necrosis, surrounded by an area infiltrated with neutrophils and lymphocytes.

[0355] Figure 18A and 18B Show an image and four photographs depicting the development of duct injury and caseous necrosis of the pancreas after hydrodynamic injection.

[0356] Hydrodynamic injection of the porcine pancreas was performed through the duct system. The 11 mm balloon was inflated before injection to seal the duct. The pigs were injected with an injection parameter of 22 mL @ 2 mL / second, resulting in the delivery of 2 mg pDNA of pCLucf.Figure 18A Fluorescence examination after injection was shown. The contrast agent was observed to be reddish, indicating leakage of the contrast agent injection from the catheter system and rupture of the wall. On the 3rd day after injection, the pig was sacrificed and the pancreatic tissue was analyzed. Figure 18B Gross examination and dissection of the pancreaticoduodenal lobe shown in the photograph in [reference] revealed large areas of pale yellow tissue visible in the photographs on the left, top, and bottom, consistent with necrosis and injury caused by the injection. The photograph on the right shows a magnified view of the necrotic and injured tissue. Histological staining of the remaining organs did not show any gene delivery (data not shown).

[0357] Figures 19A to 19C Images showing gene delivery to the pancreas using minimal injection parameters. Hydrodynamic injection was performed through the catheter system in a pig model. Reduced injection parameters were tested to evaluate whether they could still mediate reduced gene delivery. Figure 19A Representative pictures of IHC staining for firefly luciferase showing expression within islet and duct cells, with injection parameters of 20 mL, 1 mL / sec, resulting in delivery of 4 mg pDNA (pCLucf). Amylase levels were reported before and after the procedure. Gross dissection of the pancreas did not show any abnormalities. Figure 19B Representative pictures of IHC staining for firefly luciferase showing expression within islet and duct cells, after injection at a rate of 15 mL, 2 mL / sec in the first pig, resulting in delivery of 4 mg pDNA (pCLucf). Amylase levels were reported before and after the procedure. Gross dissection of the pancreas did not show any abnormalities. Figure 19C Comparison of the immune infiltration area in pigs injected with 1 mg pDNA at injection parameters of 22 mL, 2 mL / sec and 15 mL, 4 mg pDNA at injection parameters of 2 mL / sec showed a reduction in immune infiltration at the lower injection parameters.

[0358] Figure 20 A traditional straight plastic bile duct stent is shown, which passes through the papilla and cystic duct. This stent is designed to optimize bile drainage and has an internal anti-displacement flared end. This is an example of a stent that, due to the relatively narrow stent diameter and two additional side holes at the bile duct stent end facing the liver stent end, causes the injected fluid to not be sufficiently transferred away from the cystic duct or the area between the stent and the bile duct wall, and thus the stent cannot achieve the purpose of bypassing the cystic duct orifice.

[0359] Figure 21 A relatively magnified view of the placement of a traditional straight plastic bile duct stent passing through the papilla and cystic duct is shown. As described above, this is an example of a stent that, due to the relatively narrow stent diameter and two additional side holes at the bile duct stent end facing the liver stent end, causes the injected fluid to not be sufficiently transferred away from the cystic duct or the area between the stent and the bile duct wall, and thus the stent cannot achieve the purpose of bypassing the cystic duct orifice.

[0360] Figure 22 Show an inflatable occlusion catheter placed within a transpapillary straight plastic biliary stent passing through the cystic duct. As described above, this is an example of a stent type which, due to the relatively narrow stent diameter and additional side holes in the biliary stent end facing the liver stent end, fails to achieve the purpose of bypassing the cystic duct orifice as the injected fluid cannot be adequately transferred out of the cystic duct despite the presence of the occlusion balloon.

[0361] Figure 23 Show a large-diameter fully covered metal biliary stent which is deployed through the papilla and passes through the biliary orifice. The stent will expand almost immediately against the biliary wall to form a seal. An inflatable occlusion catheter with an injection function will be placed within the stent, the balloon inflated against the stent wall to form a seal, and then plasmid DNA or a medical fluid will be injected from within the stent to the common bile duct level.

[0362] Figure 24 Show a large-diameter fully covered metal biliary stent which will be deployed within the bile duct, covering the cystic duct orifice but not passing through the papilla. The stent will expand almost immediately against the biliary wall to form a seal. An inflatable occlusion catheter with an injection function can be placed within the stent or in the common bile duct, below the distal level of the stent. The balloon is inflated against the common bile duct wall to form a seal, and then plasmid DNA or a medical fluid will be injected from within the stent to the common bile duct level. A short tether will be connected from the distal end of the stent and enter the duodenum.

[0363] Figure 25 Show a method of injecting fluid into the liver by placing an inflatable occlusion catheter in the common bile duct and occluding the cystic duct with an inflatable balloon placed percutaneously in the cystic duct.

[0364] Figure 26 Show a method of injecting fluid into the liver by placing an inflatable occlusion catheter in the common bile duct and occluding the cystic duct with an umbrella placed percutaneously in the cystic duct, at the neck of the gallbladder or at the junction where the cystic duct enters the bile duct. The figure also shows how to place the balloon within the stent to achieve an effective seal on the stent so that the contrast agent does not leak along the side of the stent. The figure depicts balloons in different regions of the stent (common hepatic duct and common bile duct) and effectively seals the contrast agent, demonstrating the flexibility of the method.

[0365] Figures 27A to 27C Show an application implementation including a stent, a balloon, and a contrast agent. Figure 27A Show an image obtained of a stent with an internal balloon. Figure 27B Show an image obtained of a stent with an internal balloon, located at approximately the common hepatic duct level, with the contrast agent above the balloon. Figure 27C Show an image obtained of a stent with an internal balloon, located at approximately the common bile duct level, with the contrast agent above the balloon.

[0366] Figures 28A to 28D Show the efficiency of regulating gene expression during biliary hydrodynamic delivery with different flow rates. Four different flow rates (1 mL / sec, 4 mL / sec, 7 mL / sec, 10 mL / sec) were tested at a fixed volume of 40 mL and a fixed DNA dose of 10 mg of plasmid DNA, i.e., pCLucf. The pigs used in the study weighed from 37.1 to 45.9 kg. Immunohistochemical analysis of firefly luciferase protein was performed on liver tissue sections harvested on day 1 after injection. Figure 28A Show that a flow rate of 1 mL / C sec shows minimal staining of bile ducts or hepatocytes. Figure 28B Show that a flow rate of 4 mL / C sec shows the highest transfection rate of hepatocyte staining and also shows delivery to bile ducts and endothelial cells. Figure 28C Show that Figure 28D Flow rates of 7 mL / sec and 10 mL / sec in have a positive cell transfection pattern similar to that of the 4 mL / sec flow rate, with strong expression in bile ducts and endothelial cells. The intensity of protein staining in hepatocytes gradually decreases, and the transfected area slightly decreases. The most prominent staining in hepatocytes appears near the lobular boundary and / or large blood vessels.

[0367] Figures 29A to 29C Show that different volumes regulate the efficiency of gene expression during biliary hydrodynamic delivery. Three different volumes (40 mL, 60 mL, 80 mL) were injected at a fixed flow rate of 2 mL / C sec and a pDNA dose of 10 mg of pCLucf. All pigs weighed 40 kg. Immunohistochemical detection of firefly luciferase protein was performed on liver tissue sections harvested on day 1 after injection. Figure 29A Show that a volume of 40 mL exhibits efficient hepatocyte transfection and expression in bile ducts and endothelial cells. Figure 29B Show that the hepatocyte delivery of a 60 mL volume is relatively similar to that of a 40 mL volume, while maintaining a similar delivery to bile ducts and endothelial cells. Figure 29C The 80 mL volume in seems to reduce the intensity of protein staining in hepatocytes and slightly decrease the transfection efficiency. The intensity of bile duct delivery remains unchanged.

[0368] Figure 30Depict the improvement of gene delivery by nanoplasmid compared with conventional plasmid in terms of biliary hydrodynamic injection efficiency. Test a novel DNA vector platform against a conventional plasmid DNA vector with a traditional bacterial backbone to understand its impact on gene delivery efficiency during biliary hydrodynamic injection. To test this, 10 mg of the conventional plasmid DNA vector and 10 mg of the nanoplasmid were injected into pigs at a rate of 40 mL and 2 mL / second. The nanoplasmid platform contains a vector backbone (<500 bp) smaller than the conventional plasmid DNA (>2 kb). The same LP1-ATP7B, C9 expression cassette was injected into the pigs, and the C9 tag in the porcine liver tissue was stained. Comparing the equivalent pDNA dose normalized by DNA size, it was observed that the transfection area of the nanoplasmid was significantly higher than that of the conventional plasmid (p = 0.0002). Representative photographs of the IHC staining areas of the nanoplasmid and the conventional plasmid are shown in the figure. The parametric t-test was used (significance P < 0.05).

[0369] Figure 31 Show that the nanoplasmid mediates long-term expression in pigs after biliary hydrodynamic injection. Investigate the duration of expression of the free DNA injected into pigs after biliary hydrodynamic injection. The nanoplasmid was hypothesized to mediate long-term expression in the liver compared with the conventional plasmid. Four pigs were injected with 20 mg of the nanoplasmid LP1-ATP7B, C9, and the C9 tag on the liver was stained to verify the efficiency. The pigs were euthanized monthly to evaluate the duration and efficiency of expression. Representative IHC staining images of each porcine liver are shown in the figure, indicating successful expression within four months. The conventional plasmid injected as a control did not show any expression one month after injection (data not shown).

[0370] Figure 32Show that hydrodynamic injection of bile can mediate efficient expression of transposase and maintain it in pigs for a long time. Evaluate the duration of protein expression after hydrodynamic injection of bile using an integration system. The piggyBac transposon system was used to integrate the transgenic cassette of plasmid DNA injected through the hydrodynamic procedure. Three pigs were injected with 15 mg of transposon and 5 mg of transposase. The pigs were injected with the same LP1-ATP7B and C9 expression cassettes, and the C9 tag in the liver tissue of the pigs was stained to evaluate the presence and delivery efficiency of the gene-derived protein. Core and wedge biopsies were performed on the animals halfway through the experiment (1.5 months after injection), and it was found that there was a large amount of protein expression in the canine and cytoplasmic patterns (data not shown). When the animals were euthanized 3 months after injection, abundant ATP7B and C9 staining were present in the hepatocytes of all three animals. Representative IHC images and uninjected control tissues are shown in the figure. The transfection efficiency was estimated to be 41.83% to 44.50%. This was more favorable compared to the delivery efficiency of a group of pigs injected with the same pDNA combination 1 month after injection (42.62% to 48.44%; the difference was not significant). There was no significant difference between these two groups and the pigs sampled on the 3rd day after injection. This indicates that the transposon can mediate stable expression over time, and the relative transfection efficiency does not change. The parametric t-test was used (significance P < 0.05).

[0371] Figure 33 Show that hydrodynamic injection of bile can be repeated, and the efficiency of each gene is similar. An unknown factor in hydrodynamic injection of bile is whether pDNA can be successfully redosed. This was evaluated by a series of injections performed at 4-week intervals. Briefly, three pigs were injected with 15 mg of transposon and 5 mg of transposase, encoding the hepatocyte-specific LP1-ATP7B and C9 expression cassettes. Four weeks later, the pigs were injected with plasmid pCMV-GFP-ATP7B, which carried different protein tags and a ubiquitous promoter. The pigs were harvested three days later, and the C9 tag and GFP tag in the liver tissue of the pigs were stained to evaluate the presence and delivery efficiency of the gene-derived protein. Representative serial sections of a single lobule are depicted on the left, showing that ATP7B, C9, and GFP-ATP7B can be detected in the same lobule and the same cell. GFP staining also indicates that during the second injection, other cell types (bile ducts, endothelial cells) are also likely to be targeted (lower left). Immunofluorescence staining provides further evidence of co-localization in the same hepatocyte (date not shown). The transfection efficiency of the first (C9) and second (GFP) injections was compared by staining the lobular area, and the results showed that they were similar (46.2% vs 50.7%; not significant). The parametric t-test was used (significance P < 0.05).

[0372] Figure 34It was shown that after hydrodynamic injection of bile, an increase in the dose of plasmid DNA could mediate higher transfection efficiency. The effect and ability of gradually increasing pDNA dose on improving transfection efficiency have not been tested. To achieve this goal, we studied four different pDNA doses (10 mg, 20 mg, 30 mg, and 40 mg). All studies were standardized using plasmid pT-LPl-ATP7B, C9. The porcine liver was harvested on the 3rd day after injection and stained for the C9 tag with 1D4 antibody. Representative images of porcine liver showing the 10 mg, 20 mg, 30 mg, and 40 mg doses are presented. The figure shows the quantification of the individual lobule staining area of each pDNA dose in randomly counted lobules per pig, indicating a significant increase in the efficiency of hydrodynamic injection of bile with increasing pDNA dose. The parametric t-test was used (significance P < 0.05).

[0373] Figure 35 Shows the limit of the plasmid DNA size that can be delivered by evaluating hydrodynamic injection of bile. Hydrodynamic injection of bile can mediate the delivery of intracellular naked plasmid DNA, but it is not clear whether there is an upper limit of plasmid DNA for this method. To address this issue, plasmid DNA of 12 kb and 17 kb sizes was injected into porcine liver with injection parameters of 40 mL B and 2 mL / sec. The plasmid DNA doses were 10 mg pDNA and 15 mg pDNA, respectively. The pigs were harvested on the 3rd day after injection. Representative regions of immunostaining of the 12 kB plasmid pCDNA4 / full-length FVIII were depicted at low and high magnifications. The images showed that at low magnification, the gene was delivered to every lobule of the stained section. At high magnification, the staining was deepest around the central vein, reflecting increased local pDNA delivery. Quantification of the staining area showed that 49.48% of hepatocytes expressed the 12 kb gene, with no significant difference from the previously injected 5.5 kb and 8.6 kb plasmids. Testing of the 17 kb plasmid showed similar protein expression ability and relative efficiency (data not shown).

[0374] Figure 36It was shown that hydrodynamic injection of bile could achieve expression in vascular endothelial cells and liver sinusoidal endothelial cells. In previous studies, it was observed that hydrodynamic injection of bile achieved gene expression in endothelial cells and cholangiocytes using a ubiquitous promoter. However, immunostaining near blood vessels or ducts may sometimes be unreliable. To verify the specific delivery of plasmid DNA to these cell types, two different endothelial cell-specific promoters were used, which produced specific GFP expression only within vascular or liver sinusoidal endothelial cells, respectively. Synthetic plasmids pICAM2-GFP and pCD36p-Luc were injected into the livers of pigs at a rate of 2 mL / sec, a volume of 40 mL, and a dose of 10 mg pDNA. Pigs were harvested on the 3rd day after injection. Representative IHC staining for GFP and luciferase was depicted for each plasmid. For the ICAM-2 promoter, the results showed strong and clear GFP staining in each arterial endothelial blood vessel observed throughout the section. Larger venous blood vessels did not seem to have any staining, and there was no staining along the liver sinusoids. For the CD36 promoter, the results showed strong and clear staining along the liver sinusoids, while less staining was retained in the portal tracts.

[0375] Figure 37 Depicted is the verification of the volume-to-liver weight dose for hydrodynamic injection of porcine bile. The conversion of volume injection parameters for individuals of different body sizes remains uncertain. To address this issue, different injection parameters were tested based on volume-to-liver weight dose. These tests included 20 mL / kg, 30 mL / kg, 40 mL / kg, and 55 mL / kg. All injections were performed at the same flow rate of 2 mL / sec with a constant dose of 10 mg pCLucf pDNA. The results of immunohistochemical staining of the injections were demonstrated by IHC staining against firefly luciferase. Example results showed that pigs weighing 25 kg and 27 kg could successfully express proteins at different volume-to-liver weight doses, with test doses of 40 mL / kg and 55 mL / kg, respectively. Pigs weighing 5 kg and 15 kg were tested separately, and the results were similar (data not shown).

[0376] Figure 38Show the evaluation of the pressure threshold for delivering biliary hydrodynamic genes into pigs. The minimum pressure threshold for gene delivery mediated by biliary hydrodynamic injection remains uncertain. To address this issue, pigs were injected with a constant-pressure injection device. The pressure threshold was set at 50 mmHg and maintained throughout the injection. The total injection volume was 30 mL, while the pDNA dose was 10 mg pCLucf. IHC staining for firefly luciferase was performed on liver tissue sections. Control tissue samples injected with pCLucf at 2 mL / C second were provided for comparison. The results showed that a pressure threshold of 50 mmHg was insufficient for injection, and no IHC staining for GFP or luciferase was observed. Another experiment showed that 80 mmHg was sufficient for gene delivery.

[0377] Figures 39A to 39F Show that ERCP-mediated hydrodynamic injection is feasible in non-human primates. The baboon liver anatomy is as Figure 39A shown, and it has a short common hepatic duct (CHD) of approximately 1.5 cm before the bile duct enters the interior of the liver ( Figure 39B ). The duodenoscope used in humans can be advanced into the small intestine and the baboon's bile duct orifice can be identified ( Figure 39C ). Subsequently, the catheter can be successfully inserted into the orifice and into the biliary system ( Figure 39D ). Fluoroscopy before injection shows different branches of the baboon biliary system ( Figure 39E ). The bile duct branches reappear after hydrodynamic injection, showing that they are intact ( Figure 39F ).

[0378] Figures 40A to 40C Show the evaluation of the safety of biliary hydrodynamic injection in non-human primates. Different common clinical laboratory tests were performed before injection and several days after hydrodynamic injection to monitor the potential toxicity of the procedure. The study included four baboons, which were injected with similar parameters. Each baboon ( Figure 40A ) had a set of liver function tests, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), γ-glutamyl transferase (GGT), albumin, and alkaline phosphatase. ( Figure 40B ) had a set of hematology tests, including white blood cell count (WBC), granulocyte count, and hemoglobin. ( Figure 40C ) Pancreatitis after ERCP was evaluated by monitoring amylase and lipase (biomarkers of pancreatic injury).

[0379] Figure 41The elastic increase in the bile duct wall of primates is shown, resulting in leakage around the balloon. A series of fluoroscopic images of three different baboons are shown in the figure. The injection parameters are similar (3 to 4 mL / second and 30 to 40 mL volume). Each time an injection is made, an 8.5-mm-sized balloon is placed in the common hepatic duct (CHD). To help illustrate the potential leakage, contrast agent is placed below the balloon. The pre-injection and post-injection fluoroscopic images of all three baboons show an increase in gallbladder size after injection, reflecting the escape of fluid from around the balloon in the CHD and entering the gallbladder through the cystic duct. The black arrow points to the common bile duct, and it can be seen that the contrast agent in the common bile duct has been cleared after injection, reflecting that the saline has flowed around the balloon in the forward direction. The post-injection fluoroscopic image of baboon #3 also shows significant dilation of all bile ducts compared to before injection, and this finding has also been observed in other baboons tested.

[0380] Figure 42 It is shown that an oversized catheter balloon in the extrahepatic bile duct may have a risk of rupture. A larger balloon size can ensure pressure sealing and avoid leakage, but it will impose greater pressure on the bile duct wall. To test this, a series of time-lapse fluoroscopic images are presented during hydrodynamic injection in a single baboon. The injection parameters are 4 mL / second and the volume of normal saline is 40 mL. Before injection, the diameter of the common hepatic duct is approximately 3 mm, and the balloon is inflated to 15 mm. After hydrodynamic injection, the bile duct wall that has been maximally stretched ruptures around the balloon, resulting in leakage of contrast agent into the surrounding tissue. In the extrahepatic system, similar findings can be replicated in pigs when the ratio of the balloon to the bile duct size is 4 to 5 times.

[0381] Figure 43A and 43B The successful occlusion of the primate liver after adjusting the position and size of the catheter balloon is shown. A series of fluoroscopic images of two different baboons are shown in the figure. The injection parameters for the two injections are similar. In both baboons, the position of the catheter balloon is in the intrahepatic location of the left branch of the liver. The size of the balloon after inflation is 11.5 mm. ( Figure 43A ) The pre-injection and post-injection fluoroscopic images of baboon #1 and baboon #2 are shown. As shown, the injected saline cleared the intrahepatic ducts through hydrodynamic injection, as expected. Importantly, the gallbladder size did not increase in either animal after injection, and the black arrow indicates that the common bile duct did not show clearance of the contrast agent solution. ( Figure 43B ) This is an image of a baboon showing the contrast solution being injected hydrodynamically into the baboon liver through the biliary tract. The entry of the contrast agent into the hepatic parenchyma (acinar formation) is observed.

[0382] Figures 44A to 44DDisplay pressure monitoring can be used to detect fluid leakage due to poor sealing during biliary hydrodynamic injection in primates. A group of baboons received a series of biliary hydrodynamic injections. A pressure sensor (EchoTip Insight, Cook Medical) was connected to a Multi-3V catheter (Olympus), which could sense the liquid-filled column in the injection port. The injection parameters were used as shown in the figure above. The balloon size in the common hepatic duct during injection was 8.5 mm( Figure 44A ), and the pressure gradually decreased during the injection. Faster flow rates and larger volumes were used to test whether this pressure loss could be overcome( Figure 44B ), but the result was the same, with a gradual loss of pressure.( Figure 44C ) An alternative strategy was adopted to change the flow rate in real time during injection. This was effective in stabilizing the pressure and, in some cases, could also increase the pressure, but the total amplitude of the pressure remained low.( Figure 44D ) Adjusting the position of the balloon (intrahepatic position, left branch) and the balloon size to 11.5 mm resulted in stable pressure during injection, without the previously observed decrease. Notably, these pressures were achieved at flow rates significantly lower than those tested in other cases without good sealing.

[0383] Figures 45A to 45D Biliary hydrodynamic injection was shown to mediate gene delivery in non-human primates. A group of baboons was obtained to test the expression of the delivered gene. The balloon size, position, and parameters were similar in all groups.( Figure 45A ) Two baboons received repeated injections of a DNA vector encoding human FIX (hFIX). The first injection dose was 20 mg pDNA, while the second injection dose was 60 mg pDNA, three times that of the former. Both animals showed a dose-dependent response, with hFIX expression three times higher in individual baboons.( Figure 45B ) Repeating the gene delivery procedure monthly in individual baboons achieved similar peak hFIX expression at similar doses and parameters on the first day after injection.( Figure 45C ) Comparing the alternative DNA molecule nanoplasmid DNA with conventional plasmid DNA with a large bacterial backbone (such as an antibiotic resistance gene), the latter was twice as high. As shown in the figure, nanoplasmid allowed a lower DNA dose but could still produce an hFIX expression level comparable to that of conventional pDNA at much higher DNA doses.( Figure 45D ) In another experiment, baboons were injected with GFP / luciferase reporter DNA by biliary hydrodynamic injection and euthanized 24 hours after injection. GFP expression was detected by immunohistochemical staining, and positive gene expression was found around the central veins of the baboon liver lobules.

[0384] Figure 46Mechanical damage caused by the guide wire can lead to bile leakage within the liver. The guide wire is first used to assist in the insertion of the catheter and entry into the biliary system, and then the catheter is driven through the guide wire into the bile duct. As shown in the fluoroscopic images, it is common for the guide wire to be advanced into the intrahepatic system, where the guide wire protrudes into the deep left duct system. However, after the catheter is placed in the bile duct and contrast agent is injected, a small amount of contrast agent can be observed at the tip of the guide wire. During subsequent hydrodynamic injections, the size of the contrast agent / bile leakage increases, emphasizing its communication with the injected fluid and serving as a sieve and limiting factor for pressure generation.

[0385] Figure 47 A to 47D show that biliary hydrodynamic injection can be performed using a stent-based method. The figure depicts a series of fluoroscopic images demonstrating the utility of injecting contrast fluid into the liver using a stent catheter without a balloon. ( Figure 47 A) The catheter with the unexpanded stent is advanced through the cystic duct into the common hepatic duct. ( Figure 47 B) The stent within the catheter is expanded by 50%, and the stent opens distally, as indicated by the black arrow. ( Figure 47 C) Contrast agent is injected through the guide wire channel of the catheter and exits the olive tip of the catheter. No contrast agent is detected within the stent, nor does any contrast agent extend from around the stent into the cystic duct. ( Figure 47 D) The stent and catheter are retracted into the common bile duct and contrast agent is injected through the catheter, now showing that the cystic duct has become opaque. At the same time, stent deployment is able to prevent fluid from entering the cystic duct and has the potential to deliver bile hydrodynamic injection.

[0386] Figures 48A to 48D Show that biliary hydrodynamic injection enables the delivery of genes to liver tumors. The key tumor suppressor gene and oncogene TP53 R167H and KRAS 31212 are mutated in the Oncopig model, and these mutations can be activated by introducing Cre recombinase. After Cre is introduced into cells by a virus, large tumors can grow in multiple different tissue types within 1 to 2 weeks. ( Figure 48A ) The figure shows liver tumors (1 to 2 cm in size) induced in a pig liver, which histologically show large, highly necrotic tumors. GFP / luciferase reporter DNA was injected into the tumor-bearing pig by biliary hydrodynamic injection, and GFP immunohistochemical staining was performed to detect positive cell types. ( Figure 48B ) GFP-positive cells were detected in the peritumoral area at the tumor boundary, where normal tissue is invaded by tumor tissue. ( Figure 48C ) GFP-positive cells were also observed within the tumor tissue itself. ( Figure 48D ) Positive expression of GFP in normal hepatocytes and normal lobules could still be observed in the same liver section, indicating the ability to target both cell types.

[0387] Figures 49A to 49C Demonstrating that hydrodynamic injection of the catheter enables gene delivery to pancreatic tumors. Key tumor suppressor genes and oncogenes TP53 R167H and KRAS° 12D are mutated in the Oncopig model, and these mutations can be activated by introducing Cre recombinase. After Cre is introduced into cells by virus, large tumors can grow in multiple different tissue types within 1 to 2 weeks.( Figure 49A ) Inducing pancreatic tumors in porcine pancreases. Tumors can be seen histologically in pancreatic sections (black arrows). GFP / luciferase reporter DNA was injected into pigs with tumors by hydrodynamic injection through the pancreatic duct, and GFP immunohistochemical staining was performed to detect positive cell types.( Figure 49B ) Obtaining pancreatic tumor sections and performing GFP staining, showing scattered positive cells of different intensities.( Figure 49C ) Sections of normal pancreatic tissue on the same slide also showed that the gene was mainly delivered to ductal cells, as detected by GFP staining. Therefore, DNA delivery can target both tumors and normal tissues simultaneously by a single injection. Detailed Description

[0388] Examples of hydrodynamic injection of the biliary tract have been established in the prior art, with a focus on flow rate as a key parameter for successful gene delivery to liver tissue. The prior art gives suggestions on the optimal flow rate for achieving gene delivery from hydrodynamic injection of the biliary tract, as well as suggestions on targeting specific regions in the liver using specific flow rates.

[0389] The present disclosure relates to improved hydrodynamic injection and delivery methods that are more advanced than prior art methods. In certain aspects, the present disclosure relates to compositions and methods for treating kidney diseases. More specifically, the present disclosure relates to compositions and methods for treating kidney diseases by gene therapy. As described in detail below, at least a part of the present disclosure is based on the surprising finding that hydrodynamic injection of the ureter can mediate rupture of the renal cortex at different flow rates and volumes, such that only limited parameters are observed to be safe. It is very likely that there is no escape of blood vessels or lymphatic vessels, which has never been detected before. It has been found that volume is the key parameter leading to rupture, because the smaller the volume, the better the tolerance regardless of the flow rate. However, we have found that the injection parameters are safe and can mediate gene delivery, which has never been described before. In fact, it was doubted whether any injection parameters could be found. Equally surprising is that the procedure can be performed without the need for fluoroscopy, which has previously limited all other gene therapy procedures. An additional surprise is the relative sensitivity of the kidney to injury during proximal injection, and that relatively small flow rates can cause a significant increase in pressure during injection.

[0390] Overview

[0391] The kidney is an attractive target for gene therapy. Current treatment strategies are ineffective for many patients with chronic kidney disease (CKD) who require regular dialysis. In terms of the medical burden, CKD is one of the largest drivers of healthcare costs in the United States. Any approach to treating CKD, whether reversing it or halting its progression, would be extremely valuable. Chronic dialysis is a demanding and debilitating procedure for patients. Kidney transplantation can cure the disease, but many patients are unable to receive a transplant due to limited donors. Kidney transplantation also has many significant risk factors, particularly rejection and chronic immunosuppression complications.

[0392] In addition to chronic kidney disease, there are several other important kidney diseases. One relatively common rare disease is adult polycystic kidney disease (PKD), which affects approximately 500,000 people in the United States. PKD is caused by mutations in the PKD1 or PKD2 genes. Pathogenic mutations in these genes lead to hyperactive tubules, which in turn cause cysts to form throughout the kidney. Over time, these cysts can lead to chronic kidney disease, requiring kidney transplantation. Current treatment methods are poor and ineffective. Many people with PKD ultimately require kidney transplantation. Other major diseases are various glomerular diseases, including anti-glomerular basement membrane disease and IgA nephropathy. These are typically characterized by immune processes that affect these organs, leading to the destruction of glomeruli through inflammation or fibrosis, along with thickening of the glomerular basement membrane.

[0393] Currently, treatments for all of these different kidney diseases are ineffective. Gene therapy is a new treatment approach that could help fill this clinical gap. However, kidney gene therapy is limited by several factors. Many researchers have utilized systemic infusion of viral vectors, including adeno-associated virus (AAV). AAV is efficient and can deliver genes to many organs, such as the liver. Unfortunately, AAV has poor transduction ability in the kidney. Poor transduction ability is thought to be caused by multiple factors, but the size exclusion of glomeruli during blood filtration is the most problematic. There is no traditional pathway for viral vectors to migrate from the systemic circulation to the kidney. Since systemic injection of viral vectors is ineffective, other researchers have begun to attempt local injection of AAV into the kidney. Examples of local routes include injection into the kidney via the renal artery or vein. These administration routes have shown promise for achieving delivery in mouse models, but the efficiency remains relatively low. Another approach is to inject the vector through the ureter. This method is a relatively non-invasive route that does not require any skin incisions. However, most viral vectors injected through the ureter quickly escape from the kidney and return to the systemic circulation (Hum Gene Ther. December 2019;30(12):1559 - 1571). In these cases, liver transduction is more efficient than kidney transduction.

[0394] Non-viral methods are attractive for kidney gene delivery. There are more than 30 million patients with chronic kidney disease in the United States, which means that the total number of patients far exceeds the capacity to manufacture viral vectors. Treatment of any kidney disease may require the ability to repeat dosing because the half-life of kidney cells is shorter than that of more stable cells such as hepatocytes. Non-viral vectors can compensate for this deficiency because they do not trigger an immune response. Unfortunately, the most common non-viral vector delivery strategies also have the same limitations as viral vectors. Methods for delivering nucleic acids use delivery vehicles such as lipid nanoparticles, which can range in size from 100 to 200 nanometers. Lipid nanoparticles are very effective in delivering siRNA or mRNA to different cell types, including hepatocytes and macrophages. Lipid nanoparticles are particularly useful when bound to messenger RNA for vaccine applications. Lipid nanoparticles are less efficient for DNA because the delivery they mediate is insufficient to enter the nucleus.

[0395] If lipid nanoparticles (LNPs) can be obtained and administered systemically via the vascular system, they will face the same size-limiting barrier as viral vectors in the glomeruli. Therefore, LNPs cannot effectively enter the kidney to transfect renal cells. Local administration of nanoparticles to the kidney has not been described in previous studies.

[0396] As an alternative method, hydrodynamic gene delivery has great promise. It uses hydrodynamic pressure to physically deliver DNA into different types of cells. This is achieved by transiently creating pores in the cells, which causes naked DNA to enter these types of cells. This strategy is most well-known in mouse models, where hydrodynamic tail vein injection can effectively introduce plasmid DNA directly into the mouse liver. The same hydrodynamic tail vein technique can also deliver to other organs at a much lower percentage. Researchers have attempted to apply local hydrodynamic pressure to these organs to drive DNA into various other organs. Ultimately, local administration is the only method that can be translated to large animal models.

[0397] A study on hydrodynamic injection into the kidney via the renal vein (Mol Ther. Jun 2008;16(6):1098 - 104). The renal vein was exposed surgically in a rat model. The reported peak injection pressure was 100 mmHg and the DNA concentration injected was 100 ug / mL. To deliver genes to the kidneys of a larger porcine model, a catheter was inserted into the right renal vein using an interventional radiology - guided fluoroscopy procedure. The junction of the renal vein and the inferior vena cava (IVC) was blocked using an IVC occlusion balloon so that the fluid would not leak back into the IVC during the hydrodynamic injection procedure. Using this strategy, the researchers were able to obtain gene expression in the target kidneys of porcine kidneys, which could be measured by firefly luciferase luminescence activity. The procedure seemed to be safe with no elevation of creatinine, but no immunohistochemical staining data were reported.

[0398] Although the vascular strategy is promising, another hydrodynamic approach is injection via the urinary system. The proof - of - concept of this method has been studied in a mouse model, in which the hydrodynamic injection procedure was directly into the renal pelvis (SciRep. Mar 20, 2017;7:44904). Before injection, the mouse kidney was incised and exposed via a retroperitoneal surgery, a needle was inserted into the renal pelvis, and injection was performed under high pressure. The injected solution contained naked plasmid DNA. Scattered positive cells of various cell types were observed in the kidney.

[0399] Previous studies have provided various different routes for applying hydrodynamic pressure to the kidney. A third route, which has not been explored in the published literature, is hydrodynamic injection via the ureter. The ureter is a one - way vessel from the kidney to the bladder, so retrograde injection may be more likely to generate higher fluid pressure. In addition, the ureter enters the bladder easily via simple cystoscopy.

[0400] Hydrodynamic retrograde ureteral injection

[0401] A previous study focusing on retrograde ureteral injection proposed the idea of a hydrodynamic procedure via the ureter. This idea has not been verified by any experimental evidence, so the designated procedure may or may not be successful in gene delivery. The previously reported procedures for hydrodynamic gene delivery to the kidney are summarized as follows:

[0402] The first step is to obtain a cystoscope and advance it through the urethra into the bladder. Subsequently, a catheter is advanced through the cystoscope into the right or left ureter. Then, the catheter is advanced distally into the ureter and into the renal pelvis, preferably using injection of contrast agent and fluoroscopy to confirm the position of the catheter. Optionally, once hydrodynamic injection is initiated, fluid can be aspirated through the catheter to drain urine from the renal pelvis, thereby reducing potential toxicity. An inflatable balloon is opened to seal the ureter to prevent antegrade flow of the solution during hydrodynamic injection. The next step is to inject contrast agent and image with fluoroscopy to confirm balloon sealing, and then the contrast agent can be optionally removed. Then, a DNA solution is perfused through the catheter circuit from a power injector to the distal tip. Subsequently, hydrodynamic injection of a fluid containing nucleic acid and / or protein is performed.

[0403] After the injection is complete, the balloon is deflated. Injection of contrast agent and fluoroscopy can be repeated. The catheter and guidewire can be removed from the ureter, and the procedure can be repeated on the non-injected kidney if needed. After the procedure is completed, the catheter is removed from the ureter, bladder, and urethra outside the patient's body along with the cystoscope. Optionally, creatinine, blood urea nitrogen, and post-injection glomerular filtration rate can be monitored after injection to monitor damage caused by hydrodynamic injection.

[0404] To simplify the procedure, some additional steps can be added. A guidewire can be used to facilitate insertion into the ureteral orifice and then the catheter. A pressure catheter can be used to monitor injection of a fluid containing nucleic acid and / or protein at high speed and high pressure using a power injector to ensure the quality of reaching the injection target. Another option is to chase the fluid containing macromolecules with a solution without macromolecules (such as normal saline) to ensure complete delivery of the solution to the kidney.

[0405] Several hypothetical injection parameters are outlined in the study, although no data are provided to support their effectiveness rather than their biological principles. The recommended flow rate is at least 1 mL / second, or at least 2 mL / second, or at least 3 mL / second. The total volume of injection recommended is up to 10 mL, 20 mL, 30 mL, 40 mL, or 50 mL, with the result that the volume escapes from the renal pelvis into the parenchymal tissue. It is not clear how changes in the size of the pig would affect this volume. The pressure recommended to be achieved during kidney injection is at least 50 mmHg, or at least 75 mmHg, or at least 100 mmHg. It is not clear whether these pressures are effective for gene delivery to the kidney.

[0406] In other aspects of this procedure, methods of using various different drug solutions in combination are also introduced, which can reduce inflammation and / or potential infection during injection. The optimal drugs to be used at appropriate doses and their efficacy at these doses are not described. Methods for cell-specific targeting are described, i.e., targeting the expression of specific cell types by injecting DNA molecules containing cell type-specific promoters, but no data are confirmed.

[0407] In summary, previous studies have not determined the precise injection parameters that can effectively deliver DNA into kidney cells. This includes the effective flow rate, volume, and pressure during injection. The relationship between the injection volume and the size of the mammal or kidney was not provided. It is not clear what volume is suitable for applying this technique to human patients.

[0408] Previous studies have not proposed how much DNA dose is required to achieve gene expression when placed from the proximal ureteral kidney into the kidney. In addition, there is no information on the safety of the injection technique regarding the proposed injection parameters. The studies to date have not proposed a gene therapy method that is safe, effective, and can produce the desired gene therapy results.

[0409] Detailed description:

[0410] The present disclosure also details an effective method for hydrodynamic gene delivery through the ureter to target gene expression to the kidney. The present disclosure also employs a new method of performing this procedure to provide higher efficacy and safety.

[0411] The first procedural method for gene delivery

[0412] Previous studies have disclosed a method of hydrodynamic injection through the ureter, where the catheter balloon is located in the ureter near the kidney. This method is difficult to replicate. Placing the balloon near the kidney makes it impossible for the catheter not to slip into the renal pelvis. It was found that placing the balloon inside the renal pelvis would cause liquid to escape during the hydrodynamic injection process due to poor sealing (see, for example Figure 8A )

[0413] To develop a more robust and easier-to-perform method for doctors, we designed a new procedure for hydrodynamic injection through the ureter. The first step of this procedure is to insert a cystoscope through the urethra into the bladder. The catheter can be advanced into the bladder through the working channel of the cystoscope. The bladder wall can be examined using the camera of the cystoscope, and the ureteral orifice (UO) can be seen. Then the catheter is advanced into the ureteral orifice for calculation.

[0414] After the catheter enters the ureteral orifice, it is advanced approximately 1, 2, or 3 cm, at which point the balloon on the catheter is inflated so that it is exactly at the opening where the ureteral orifice enters the bladder. The inflated balloon will appear as a bulge on the bladder wall with the help of the cystoscope camera, thus confirming the position of the balloon. The advantage of this method is that the position of the balloon can be verified through the visualization of the cystoscope camera. Therefore, the position of the catheter can be determined without fluoroscopy. This brings numerous safety advantages, including no need for radiation safety checks and no need to use expensive equipment (including C-arm).

[0415] Another advantage is that the bladder wall provides additional reinforcement to the balloon through the muscular wall during inflation, which helps prevent ureteral rupture. In addition, during infection, the ureteral orifice can be visually monitored for fluid leakage, providing real-time feedback on the success of hydrodynamic injection. The main advantage of this procedure and method is that it can be performed in facilities without fluoroscopic imaging, but rather in a routine outpatient setting. In addition, patients and clinicians are exposed to less radiation. This new procedure improves the availability of the technique because bedside cystoscopy is relatively routine and can be performed in any outpatient setting.

[0416] A series of tests were performed to evaluate hydrodynamic injection after distal ureteral balloon placement. In all studies, the balloon was able to successfully occlude the ureter and block all antegrade fluid flow into the bladder.

[0417] An important aspect of hydrodynamic gene delivery is that the optimal injection parameters change with minor modifications to the procedure. Given that the balloon is located in the distal ureter, a direct consideration for catheter placement is how much volume is needed to fill the ureter and renal pelvis, with the ultimate goal of pushing fluid into the renal parenchyma during hydrodynamic injection. In empirical tests performed prior to injection, the ureteral volume was estimated to be between 4 and 5 mL. When slowly filled with a radiopaque contrast solution, the volume of the entire ureter and renal pelvis was between 6 and 9 mL.

[0418] The optimal injection parameters for the distal ureteral location were determined empirically. The first step in the testing was to determine how much volume and flow rate could be safely injected into the kidney without causing severe damage. The third parameter was to test how much volume of the injection solution could mediate the entry of radiopaque contrast into the renal parenchyma, which was an alternative method for DNA entry into tissue in previous liver studies. To achieve this, a series of injections were performed to verify the safe and effective parameters of the injection.

[0419] The present disclosure also presents the injection volume that avoids renal rupture during hydrodynamic injection. Renal rupture is associated with a physical tear of the renal medulla extending into the renal cortex. As shown by the data in the present disclosure, the tear typically occurs in only one location in the kidney and is approximately 1 cm in size. Renal rupture results in a small to large amount of bleeding within the fibrous capsule surrounding the kidney. Repeated hydrodynamic injection tests on different kidneys showed that the induced tear bleeding eventually stopped, and thus no pigs were severely harmed due to kidney injury. However, it is uncertain whether this would lead to more serious problems if it occurred in a human patient. Therefore, hydrodynamic injection should be optimized to avoid renal rupture.

[0420] In the best case scenario, the injection parameters should be less than 20 mL in volume and the flow rate should be less than or equal to 2 mL / second to avoid renal rupture when injecting near the ureteral orifice. When the volume exceeds 20 mL, even at a low flow rate such as 0.5 mL / second, the incidence of renal rupture is high. At these volumes, flow rates greater than 2 mL / second should also be avoided as they have been found to cause rupture. Flow rates between 0.5 mL / second and 2 mL / second intermittently cause renal rupture in some animals. Larger volumes greater than 20 mL can be considered, but lower flow rates in the range of 1 mL / second to 1.5 mL / second are required and there is still a risk of causing renal rupture in some animals.

[0421] Considering the effectiveness of gene delivery from these parameters, the optimal injection parameters for mediating gene entry into renal cells are preferably to inject a volume between 10 and 20 mL into a kidney with a mass of 70 to 80 g. To achieve gene expression while avoiding harmful renal trauma and injury, this volume should be injected at a rate of 0.5 to 2 mL / second. Gene expression can also be seen at higher injection flow rates, but this injection carries the above-mentioned risk of renal rupture, which is not desirable.

[0422] An unexpected result is that relatively low flow rates in the ureter generate significant pressure. It has been observed that flow rates of 0.5 to 1 mL / second generate a pressure of 80 mmHg. Flow rates of 1.5 to 2 mL / second generate a pressure of 120 to 140 mmHg. Thus, the flow rate required to generate pressure is small, which explains why even 0.5 mL / second can achieve gene expression and why relatively slow flow rates are still associated with the risk of renal rupture. In the best case scenario, the pressure during injection should be at least 80 mmHg in order to achieve gene expression in different cell types of the kidney.

[0423] Ideally, the balloon will be deflated after hydrodynamic injection, which can be confirmed by visual inspection with the camera of the cystoscope. The catheter can be withdrawn from the ureteral orifice and into the bladder. Then the catheter is withdrawn through the cystoscope and the cystoscope is withdrawn from the patient. Any damage to the kidney can be monitored by measuring serum creatinine and other biomarkers.

[0424] The second procedural method for gene delivery

[0425] In another example, the procedure is carried out by another method. The cystoscope will be advanced through the urethra into the bladder. Then, a guide wire will be advanced through the cystoscope into one of the ureteral orifices. The guide wire will be advanced all the way to the renal pelvis, where it will start to loop back, indicating that it has entered the kidney, or resistance will be felt when advancing the guide wire. The cystoscope will be withdrawn from the bladder. Subsequently, a catheter will be inserted through the guide wire and advanced to the kidney. Fluoroscopic imaging using a C-arm monitors the advancement of the catheter. Preferably, the catheter has radio-opaque markers to verify its position during advancement.

[0426] In this example, the catheter will be placed near the renal pelvis. The catheter remains within the ureter. The ureter can be successfully occluded by testing for obstruction of the antegrade contrast agent flow. In one embodiment, the guide wire remains in the ureter even after balloon inflation and injection to assist in verifying its position. The guide wire can extend above or below the balloon. In another example, the guide wire will be removed and the position of the catheter can be determined by injecting contrast agent only.

[0427] Preferably, the catheter balloon is located at least 1 cm, 2 cm, or 3 cm below the renal pelvis to ensure that the balloon can completely occlude the ureter (the ureter is cylindrical while the renal pelvis is conical). A balloon with an optimal size of 11, 12, 13, 14, or 15 mm can successfully seal and occlude the ureter.

[0428] For proximal injection near the renal pelvis point, the optimal injection parameters disclosed are as follows:

[0429] For a volume less than or equal to 15 mL / sec, the flow rate should be less than or equal to 2 mL / sec. Optimally, the total volume injected is equal to or less than 10 mL. Due to the small volume of the upstream renal pelvis and ureter (estimated to be 3 to 4 mL in total), a smaller injection volume is required. Additionally, a smaller injection volume is used to avoid kidney rupture. Preferably, the flow rate is between 0.5 and 1 mL / sec (inclusive) to mediate gene expression of plasmid DNA within the kidney.

[0430] In another example, a constant-pressure injection device can be used for injection. This will bring the pressure to a specific setting, thus reducing the risk of kidney rupture. Additionally, considering that many patients' kidneys have chronic kidney disease, the relative stiffness of the tissue will be different from that of a normal kidney. Stiffness affects the relative resistance of the tissue, thus increasing the pressure. For the proximal position, the pressure generated is evaluated under given parameters. For example, 10 mL is injected at a flow rate of 1 mL / sec. Pressure tracing shows a peak pressure of 105 mmHg is reached. In the optimal embodiment of the present disclosure, a pressure of at least 100 mmHg can be injected from the proximal position. In other embodiments, a pressure of at least 80 mmHg can be injected from the proximal position.

[0431] After successful injection under these parameters, the balloon will be deflated and the catheter will be removed from the ureter, bladder, and urethra. Subsequently, the guide wire will also be removed from the ureter, bladder, and urethra. The patient will be monitored for any toxicity via subsequent urine analysis and serum chemistry tests, including creatinine levels. Hydrodynamic, retrograde ureteral injection of viral vectors.

[0432] Fluid injection has traditionally been used to deliver plasmid DNA into cells. As such, it has traditionally been considered a non-viral method. However, fluid injection is a non-specific process that can mediate the entry of any number of DNA, RNA, proteins, and even viruses into cells (J Gene Med. July 2006; 8(7):852-73). During this process, hydrodynamic injection can have a variety of different mechanisms, from more entry into cells to more penetration of macromolecules into tissues.

[0433] Viral vectors are the traditional mainstay of gene delivery. Through millions of years of evolution, viruses have acquired the properties of targeting, internalizing into cells, and DNA viruses entering the nucleus. However, viruses used for gene therapy have not been able to adapt to the required high level of delivery efficiency. For example, viruses only need to target a small fraction of cells in a given tissue to replicate. Viruses have not easily acquired the ability to penetrate tissues required for gene therapy, such as replacing genes within each cell.

[0434] There is limited research on the potential synergy between hydrodynamic gene delivery and traditional viral vectors. In one study, intravenous injection was used to increase the delivery of the SV40 viral vector into the liver (Hum Gene Ther. March 2005; 16(3):361-71). Notably, the expression level was much higher than that of the viral factor alone. In this case, the SV40 vector did not target only the liver with its natural tropism, so hydrodynamic delivery helped to concentrate the delivery of all viral vectors into tissues.

[0435] Even for therapeutic modalities with a natural tropism for the liver, such as adenoviruses, hydrodynamic delivery can play a beneficial role. Since hydrodynamic injection is a local procedure that forces the delivery of substances to a specific site, its benefit is that it greatly reduces the viral vector dose required for treatment. Early mouse model studies have found this phenomenon, in which hydrodynamic injection of an adenoviral vector increases liver transduction while reducing the level of inflammation normally caused by adenoviruses (Mol Ther. July 2005;12(1):99-106). This study has also been applied to non-human primate models, in which injection at a higher efficiency also helps reduce the vector dose required, resulting in longer-lasting expression than other modalities (Mol Ther. April 2007;15(4):732-40). The disease-related capabilities of this method have been demonstrated in a hyperbilirubinemic rat model, in which a lower viral vector dose is used when combined with hydrodynamic gene delivery to the liver (Hum Gene Ther. April 2011;22(4):483-8).

[0436] As described above, there have been a small number of studies on hydrodynamic gene delivery to the kidney, but these studies have only shown insignificant gene delivery effects. Viral vectors have been injected via the renal vein, renal artery, subcapsular space of the kidney, and ureter, and all of these routes have only mediated moderate transduction of the kidney (Hum Gene Ther. Dec 2019;30(12):1559-1571). Currently, there is a large gap between efficacy and delivery efficiency in treating clinically relevant diseases, and depending on the disease, most cells in the glomeruli or renal tubules need to be targeted.

[0437] One study examined the combination of viral vector delivery via the renal vein and hydrodynamic gene delivery in rats (Mol Ther. June 2008;16(6):1098-104). The authors believe that our vector combined with hydrodynamic delivery can improve delivery efficiency compared to vectors delivered at a normal rate. The authors claim that at normal infusion rates, there is little or no adenoviral transduction, and GFP staining only occurs with hydrodynamic delivery in rats. This experiment was conducted in a rat model, so this finding may not occur in large animal models.

[0438] This disclosure also improves on prior studies and describes a method for co-delivering viral vectors through the ureteral system under hydrodynamic pressure. Although viral vectors have been injected through the ureteral system before, they have never been injected under hydrodynamic pressure. This disclosure combines these two methods to make up for the low efficiency of the minimal gene delivery previously achieved through the ureter. Another goal is how to extend any of these methods to large animals in order to translate these methods into treatments for human subjects.

[0439] The present disclosure also describes the optimal hydrodynamic parameters that can be used to deliver viral vectors. These hydrodynamic parameters are slightly lower than the maximum pressure that causes tissue damage when the kidney ruptures, as described elsewhere in the present disclosure. The present disclosure teaches that, compared with the conventional method of injecting viral vectors into the ureter at a flow rate of less than 0.1 mL / second, the higher flow rate and volume used for injecting the virus in this method more effectively increase the penetration of the viral vector into tissues and result in viral transduction. The mechanism of higher transduction is caused by higher fluid pressure during injection, which leads to higher permeability between tissues.

[0440] Viral vectors that can be used in the present disclosure include adeno-associated virus, adenovirus, lentivirus, retrovirus, baculovirus, anellovirus, and Sindbis virus. The present disclosure is not specific to the virus type or viral serotype because the hydrodynamic gene delivery process is particle-nonspecific, and thus all viral vector particles can benefit from this combination.

[0441] The present disclosure also relates to reusable viral vector technology. The hydrodynamic injection procedure can be modified to first push a non-viral vector solution, which will clear any antibodies present. Subsequently, a solution containing the viral vector is rapidly injected such that the fluid pressure immediately pushes the viral vector into the tissue and target cells. This can eliminate any antibodies present, so that the viral vector can be stably injected into the kidney.

[0442] Previous studies have not described the synergistic effect of these two methods, so it is still uncertain whether retrograde ureteral hydrodynamic injection of viral vectors will improve transduction. In particular, previous evidence has shown that viral vectors can be easily expelled from the kidney by retrograde ureteral injection, so it is still uncertain whether additional pressure will promote this process rather than result in enhanced gene delivery (Hum Gene Ther. December 2019; 30(12): 1559-1571).

[0443] The ultimate goal of hydrodynamic injection is to generate pressure, which helps to form pores in the cell membrane, thus enabling gene delivery. A previous paper found that the flow rate is a key parameter determining the pressure in the biliary system during hydrodynamic injection (Huang, PLOS One 2021). However, a challenge in the prior art is that the flow rate may not be the optimal injection parameter for hydrodynamic injection. The actual pressure achieved during hydrodynamic injection depends on many different factors, including bile duct diameter, liver stiffness, liver volume, liver size, and the viscosity of the injected substance. The injection parameters in the prior art are all formulated around liver size, which is roughly equal within a narrow range (800 to 1000 grams), and the viscosity of the injection solution is also similar. In addition, in non-diseased animals, all livers are healthy. More importantly, at the cellular level, the degree of fibrosis in porcine livers is significantly higher than that in human livers at baseline. Specifically, this is manifested in the significant fibrotic tissue between each porcine liver lobule. Different components of the porcine liver may affect the injection resistance of the system, which will ultimately affect the pressure achieved and the injection flow rate. These differences together make it a challenge to translate porcine injection parameters to humans, despite the similar organ sizes of humans and pigs. Therefore, the prior art may face challenges in achieving the precise gene delivery efficiency achieved in porcine studies.

[0444] Another challenge is that the flow rate achieved during biliary hydrodynamic injection may not be easily convertible to the expected pressure. More specifically, according to the prior art, the challenge in converting the flow rate during biliary hydrodynamic injection to pressure is that it does not seem to produce a reliable conversion. For example, both 1 mL / second and 2 mL / second seem to generate a pressure of 80 mmHg during injection (Huang, PLOS One 2021). Therefore, if a given pressure threshold is used as a reference, doctors will not be able to determine how to program the power injector for gene delivery.

[0445] Reference will now be made in detail to the exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with the exemplary embodiments, it should be understood that it is not intended to limit the present disclosure to these embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0446] The present disclosure describes and teaches a solution to this challenge. The present disclosure teaches a method of injection during biliary hydrodynamics injection at a defined pressure perimeter rather than a defined flow rate parameter. This is because due to the above factors, the flow rate required for gene delivery may vary from patient to patient. A prominent example is patients with cirrhosis or non-alcoholic steatohepatitis. The pressure parameters defined in the present disclosure make these thresholds sufficient to achieve gene delivery into the porcine liver or another mammalian liver. In addition, the present disclosure teaches a pressure threshold that should not be exceeded due to loss of gene delivery efficiency. The present disclosure also teaches that a given pressure threshold will result in tissue damage and injury.

[0447] To achieve pressure-directed biliary hydrodynamics injection, the first step is to obtain a balloon catheter that contains a lumen that can mount a pressure sensor or be connected to a sensor. The pressure sensor should be able to monitor and reflect the pressure within the biliary system. The pressure sensor will be connected to a device that can provide real-time pressure results and / or real-time plot the pressure reached during the injection.

[0448] The biliary hydrodynamics injection procedure will start in a manner similar to the established protocols in the prior art, advancing the balloon catheter to the common hepatic duct. In other embodiments, the catheter can be placed within the common bile duct. After the catheter is in the common hepatic duct, the pressure sensor will be connected or inserted into the catheter through a dedicated lumen. In some embodiments, the pressure sensor is already present in the catheter. A baseline reading of the pressure in the biliary system will be taken with the balloon deflated and then a pressure measurement will be taken with the balloon inflated. A test solution without plasmid DNA or other nucleic acids will be injected into the bile duct. The test solution should have the same osmolality, weight molality, and viscosity as the DNA injection solution. It should not contain any other active pharmaceutical substances that may be contained in the therapeutic DNA solution, except for DNA or other nucleic acids. An electric syringe will be loaded with the test solution substance.

[0449] Next, the balloon on the catheter will be inflated. Subsequently, the test solution will be injected into the biliary system while monitoring the pressure. The target pressure to be achieved is greater than 80 mmHg. The initial injection parameter for the test injection should be 3 mL / second. The total volume of the test injection should be at most 15 mL, 10 mL, or 5 mL. In other cases, the total volume of the test injection should be at most 40 mL, 30 mL, or 20 mL. This volume should be sufficient to measure the total fluid resistance column in the entire circuit and determine whether sufficient pressure will be reached. Previous publications have indicated that a steady-state pressure can be achieved within the first second of injection, which supports that in the test injection, only a short injection time is required to complete the total injection volume (Huang et al., PLOS One 2021).

[0450] Based on the results of the test injection, the flow rate will be adjusted. If the pressure generated by the flow rate is higher than 80 mmHg, the flow rate at this threshold will be selected for injection, and the DNA solution injection will continue. If the pressure exceeds 250 mmHg, the flow rate will be reduced to below 3 mL / second. If the flow rate does not exceed 80 mmHg, the flow rate will be adjusted to a higher speed. For any possible adjustments, a second test injection will be performed. Generally speaking, whether it is to increase or decrease the pressure experience, the flow rate can be increased or decreased by 1 mL / second to achieve the desired result. In other embodiments, shorter increments of 0.5 mL / second can be used to adjust the flow rate. Once the correct pressure level is reached in the test injection, the DNA solution injection will begin. The DNA solution injection will be programmed at the established flow rate to generate the required pressure.

[0451] In other embodiments of the present disclosure, a series of flow rates is performed during a single test injection, in which multiple flow rates are tested. In this embodiment, at least two or more test flow rates will be tested during a single test injection. For example, the injection can start at 1 mL / second and then progress to 2 mL / second, 3 mL / second, and 4 mL / second. During this series of test injections, the pressure will be continuously monitored to create a curve of multiple different pressures. This pressure curve can have a variety of different forms, including a step function. Through this test injection, it can be accurately determined which is the best within the tested flow rate range and reaches the pressure required for injection. To perform the test series, it is envisioned that the total test volume needs to be greater than a single test injection. In these embodiments, the total injection volume can be at most 40 mL, 30 mL, or 20 mL in order to test all different injection parameters.

[0452] In a preferred embodiment of the present disclosure, the minimum pressure for effective hydrodynamic gene delivery will be greater than 50 mmHg, or greater than 80 mmHg, or greater than 100 mmHg. The maximum pressure for effective hydrodynamic gene delivery will be less than 200 mmHg or less than 250 mmHg.

[0453] The prior art teaches that "during hydrodynamic gene delivery, the pressure within the bile duct will be equal to or greater than 40 mmHg, 50 mmHg or higher. In at least some aspects, the upper limit can be 200 mmHg, although higher pressures can be employed in certain systems." However, the prior art only teaches the pressure levels that a power injector can achieve based on its technical specifications. In addition, although a power injector can theoretically achieve these different pressures, the prior art does not link these results to actual gene delivery. Therefore, it is not clear which actual gene delivery parameters are sufficient for gene injection. The present disclosure remedies the deficiencies of the prior art by providing data on the injection pressures for achieving given gene delivery parameters.

[0454] For example, the present disclosure teaches that hydrodynamic pressures below 50 mmHg enable only minimal gene delivery through the biliary system, as measured by immunohistochemical staining (<10%) of reporter genes, firefly luciferase, and green fluorescent protein. Effective immunohistochemical staining (>20%) of reporter genes was only observed at pressures of 80 mmHg or above. Additionally, within the pressure range of 100 to 150 mmHg, the transfection area increased more compared to lower pressures.

[0455] An advantage of the present disclosure is that it is not restricted to any particular species, allowing for the empirical measurement and customization of injection parameters for that species during the process. Another advantage of the present disclosure is that it can account for genetic differences among subjects within the same species, thus enabling the customization of injection parameters.

[0456] This disclosure is achieved through the rapid accumulation of pressure within the biliary system, such that only a minimal amount of fluid needs to be injected to determine what flow rate results in what pressure level.

[0457] The present disclosure relates to compositions and methods for treating pancreatic diseases. More specifically, the present disclosure relates to compositions and methods for treating pancreatic diseases by gene therapy. As described in detail below, the present disclosure is at least partially based on the surprising finding that the amount of fluid required for sufficient gene expression is less than 20 mL, such as the amount of fluid previously disclosed as sufficient for expression. The injection volume can be as low as 5 to 15 mL and still achieve effective gene delivery. The flow rate required to transfect pancreatic cells is lower than the previously reported 2 mL / s. The flow rate sufficient to mediate gene delivery is at least 1 mL / second. In other studies, the flow rate was at least 0.5 mL / second. These two findings are related to the finding that if these lower injection parameters are used, tissue damage caused by pancreatic hydrodynamic injection can be eliminated, with significant differences in immune infiltration and necrosis compared to the parameters outlined in the prior art. The present disclosure also outlines an optimal occlusion balloon size smaller than those listed in the prior art, finding that larger balloon sizes may cause pancreatic duct rupture, but smaller balloon sizes do not cause this and can still have sufficient sealing for injection. Specifically, an 8 mm balloon is sufficient. When translated to patients, one may inflate the balloon to a diameter 1, 2, 3, 4, 5 mm or up to 5 mm larger than the pancreatic duct diameter. The present disclosure also corrects a gap in the prior art by teaching that the pressure required to transfect pancreatic cells is below 75 mmHg above the baseline pancreatic duct pressure. It may be between 30 and 75 mmHg.

[0458] Overview

[0459] Pancreatic gene therapy is a potential approach for treating a variety of diseases, from autoimmune diabetes to hereditary chronic pancreatitis to pancreatic cancer. Proof-of-concept studies using viral vectors have achieved modest efficiency in rodents. Importantly, delivery of genes into the pancreas of large animal models of human size has not been achieved, representing a major gap in the field. Here we demonstrate proof-of-concept that an endoscopic procedure accessing the pancreatic duct via endoscopic retrograde cholangiopancreatography (ERCP) can achieve non-viral hydrodynamic delivery of naked plasmid DNA solution, mediating efficient delivery into multiple cell types in the pancreas of adult pigs, including almost 100% transfection of islet cells, paving the way for future therapeutic development.

[0460] The pancreas is an important endocrine and exocrine organ in the human body and plays an important role in food digestion and energy storage. Disorders of pancreatic function can lead to a variety of diseases. Pancreatic insufficiency results from the secretion of pancreatic digestive enzymes through the pancreatic duct. This chronic enzyme secretion occurs in patients with cystic fibrosis. Chronic pancreatitis is characterized by inflammation, pancreatic scarring, and can also lead to pancreatic insufficiency, i.e., the pancreas is unable to produce enough digestive enzymes to break down food and absorb nutrients. Hereditary pancreatitis is a genetic disease mutation where patients develop pancreatic insufficiency and / or chronic damage at a young age.

[0461] The pancreas is an important organ in the pathogenesis of a variety of diseases, such as type 1 and type 2 diabetes caused by interruption or dysregulation of insulin secretion. The β islet cells of the pancreas secrete and regulate insulin, which regulates the total storage of fat and protein in the body. In type 1 diabetes, the insulin-producing β islet cells are destroyed by autoreactive T cells, resulting in absolute insulin deficiency and hyperglycemia. Type 2 diabetes leads to systemic insulin resistance and relative insulin deficiency. Pancreatic cancer is another devastating disease with low survival rates. One strategy for treating pancreatic diseases is gene therapy, which can deliver defective genes found in pancreatic cells or by transforming pancreatic cells. Gene therapy can also be used to deliver proteins that can counter genetic diseases, cancer, or autoimmune diseases.

[0462] People have attempted various pancreatic gene therapy strategies. Viral strategies employ systemic or targeted therapy via the vascular route (see, for example, Griffin, M A et al., "A novel gene delivery method transduces porcine pancreatic duct epithelial cells." Gene Therapy 21, 2 (2014): 123 - 30. doi:10.1038 / gt.2013.62), while other studies have shown that injecting virus through the pancreatic duct is feasible (see, for example, Wang, Yuhan et al., "Long - Term Correction of Diabetes in Mice by In Vivo Reprogramming of Pancreatic Ducts." Molecular Therapy: The Journal of the American Society of Gene Therapy 26, 5 (2018): 1327 - 1342).

[0463] Non - viral strategies have also been employed, using hydrodynamic injection through the rat pancreatic artery (see, for example, Ogawa, Kohei et al., "Efficacy and Safety of Pancreas - Targeted Hydrodynamic Gene Delivery in Rats." Molecular Therapy - Nucleic Acids 9 (2017): 80 - 88.) and by Yamada Y et al. through the mouse pancreatic duct. In vivo transgene expression in the pancreas by intraductal injection of naked plasmid DNA. Journal of Pharmaceutical Sciences.

[0464] In February 2018, 107(2):647 - 653). However, these non - viral strategies have not been translated to large animal models, and there is only one published report on a viral vector in neonatal pigs (about 1.5 kg), in which AAV was delivered through the celiac artery of the pig, spreading the vector into the left gastric artery and splenic artery that act on the pancreas (see, for example, Griffin, M A et al.). If a translatable pancreatic gene therapy strategy can be achieved in adult animals, other important factors will be to figure out how to effectively deliver genes to the pancreas and which cell types should be effectively targeted.

[0465] In the present disclosure, endoscopic retrograde cholangiopancreatography (ERCP) is used to mediate non-viral hydrodynamic gene delivery into the porcine pancreatic duct. Non-viral gene therapy is used because of its significantly lower cost, which may allow for its routine use in metabolic diseases such as diabetes. Previously, ERCP has been used to effectively deliver plasmid DNA into hepatocytes (see, e.g., Kumbhari V et al. Successful hepatic-directed gene delivery by ERCP-guided hydrodynamic injection61 (with video). Gastrointest Endosc 2018;88:755 - 763.e5).

[0466] Although gene therapy has great potential for pancreatic diseases, very little research has been done on pancreatic diseases. One approach to gene therapy is the use of viral vectors or nanoparticles, but they do not accumulate to a sufficient extent in the pancreas. A more successful approach is the local administration of viral vectors or nanoparticles to the pancreas, such as by injection into the pancreatic artery or vein. Intraductal delivery is another commonly used technique. A drawback of early studies was the use of mouse and rat models because their response to treatment was very inefficient. More research is needed to determine whether viral vectors are an effective method for delivering gene therapy.

[0467] Hydrodynamic injection is a technique for mediating the delivery of non-viral nucleic acids (including DNA and RNA). Studies conducted by different research groups on mouse models have shown that hydrodynamic injection can mediate limited gene delivery via the intravenous or catheter route. The data from rodent studies are not reliable, and there have been few follow-up studies.

[0468] Hydrodynamic delivery in large animal models is a new technique that enables more effective and robust gene therapy delivery. The catheter system was chosen for hydrodynamic injection because it can directly reach the entire pancreatic tissue through a network (Figure 13).

[0469] Complex, the catheter system is unidirectional, so the high-pressure liquid injected retrogradely can directly enter the pancreatic tissue.

[0470] Previous studies have found that catheter hydrodynamic injection can effectively mediate efficient delivery into a variety of different types of pancreatic cells. These cells include islet cells, duct cells, endothelial cells, and pancreatic neurons (Figures 15, 16). This is achieved by injecting a ubiquitous promoter that can label multiple cell types.

[0471] Techniques previously used for catheter hydrodynamic gene delivery include:

[0472] (1) Insert a catheter through the major duodenal papilla into the main pancreatic duct, distally to the part of the pancreatic duct that merges with the common bile duct, or into the minor duodenal papilla in the accessory pancreatic duct or the dorsal pancreatic duct, and optionally further advance the catheter into the main pancreatic duct;

[0473] (2) Optionally remove the fluid in the pancreatic duct to remove digestive enzymes from the lumen;

[0474] (3) Inject a contrast agent into the pancreatic duct to confirm correct catheter placement;

[0475] (4) Inflate a balloon in the catheter at the entrance of the pancreatic duct near the common bile duct to prevent backflow of fluid;

[0476] (5) Inject a solution containing at least 1 mg

[0477] DNA at a flow rate of at least 2 mL / second for a volume of at least 20 mL,

[0478] (6) wherein the flow rate, volume, and DNA dose are sufficient to mediate gene expression in all pancreatic lobes and multiple pancreatic cell types.

[0479] Monitoring of pancreatic tissue damage is performed by detecting the levels of amylase and lipase in the serum. To reduce pancreatic damage, previous studies have suggested using different drugs during or after hydrodynamic injection, although the optimal drug combinations and concentrations to be used have not been described.

[0480] The specified injection parameters described above are:

[0481] (1) The amount injected into the pancreas is at least 20 mL, or can exceed 30 or 40 mL, so that the amount can escape from the pancreatic duct into the parenchymal tissue.

[0482] (2) The flow rate of the procedure exceeds 2 mL / second, and in other embodiments is 3 mL / second or 4 mL / second.

[0483] (3) The optimal catheter pressure for pancreatic gene delivery is greater than 50 mmHg, greater than 75 mmHg, greater than 100 mmHg, greater than 150 mmHg, or greater than 200 mmHg.

[0484] Other methods described in previous studies include strategies to alter promoter specificity in order to target specific cell types in the pancreas. For example, this would allow exclusive expression in alpha-islet cells, beta-islet cells, acinar cells, or duct cells. Gene delivery to the pancreas can only be successful when using gene delivery parameters of 2 mL / second and 20 mL or greater. It is uncertain whether other injection parameters can successfully perform gene delivery.

[0485] Delivery of genes to the pancreas of large animals such as pigs by hydrodynamic injection is a major advancement, but there are still some challenges and limitations to this technique.

[0486] The first drawback is that the injection itself seems to cause severe tissue damage. Although most of the tissue appears normal, there are many areas of necrosis on each tissue section ( Figure 17 ). The corresponding histological images show a large infiltration of lymphocytes and neutrophils in small areas. These areas are not seen in the non-injected pig pancreas. It is uncertain whether the necrotic areas will develop into full-blown pancreatitis at longer time points. During the short time interval of this study, the amylase levels did seem to return to normal (Figure 14), but necrotic areas were still observable.

[0487] Although the pig pancreas may be tolerant to pancreatic injury, it is well known that the pancreas of primates, including humans, is extremely sensitive to any perturbation. The introduction of necrotic areas may be risky or unacceptable for treating human patients. Therefore, methods and strategies to reduce pancreatic injury are important advancements and improvements to this technique.

[0488] One potential strategy to reduce pancreatic injury is to adjust the parameters in order to reduce the pressure on the pancreas. One way to change the parameters is to lower the flow rate, thus reducing the peak pressure experienced by the pancreas. A second way is to reduce the volume, thus shortening the duration of pancreatic compression.

[0489] An important factor in pancreatic gene delivery is that the total volume of the pancreatic duct is only 2 to 3 mL. Previous studies injected drugs that were 4 to 6 times larger than the normal volume into the pancreatic duct. This additional volume did not cause rupture of the pancreatic tissue, indicating that the additional volume can be absorbed by blood vessels or the surrounding retroperitoneal cavity. However, lower injection volumes can be tested, which are sufficient to achieve effective gene delivery.

[0490] Given the small volume of the pancreatic duct, lower flow rates may generate a similar threshold pressure within the lumen, thus pushing plasmid DNA directly into the surrounding tissue. In previous studies, the lowest tested flow rate to produce gene expression was 2 mL / second. Lower flow rates have the potential to achieve gene expression.

[0491] Previous studies did not define the target volume for pancreatic injection. The parameters were defined for pigs of a specific body weight (40 to 54 kg) and were not adjusted for pigs of different sizes. This is important because volume parameters are an important factor for clinical translation to humans of different sizes. One assumption of previous studies was that the adult human pancreas is similar in size to the tested pig pancreas, so parameters including DNA dose could be directly translated.

[0492] Hydrodynamic injection of the pancreas

[0493] Hydrodynamic injection into the main pancreatic duct or accessory pancreatic duct using endoscopic retrograde cholangiopancreatography (ERCP) can improve the efficiency and safety of the procedure. The catheter can be placed at the head or tail of the pancreas. Other modifications of the new method and improved gene delivery techniques are described below:

[0494] Balloon size

[0495] The first improvement to the method is to determine the maximum balloon inflation size for hydrodynamic injection to avoid damage to the pancreatic duct and surrounding pancreatic tissue. It has been observed that balloon inflation to a diameter of 11 mm or greater may cause damage to the pancreatic wall (Figure 18), which may result in leakage of fluid into the surrounding tissue at the start of hydrodynamic injection.

[0496] Generally, the pancreatic duct is slightly smaller than the bile duct. The diameter of the bile duct in the human body is generally 4 mm, and in some individuals it can dilate to 6 to 8 mm. However, in some patients, the diameter of the bile duct is only 2 to 3 mm. The diameter of the pancreatic duct at the head of the pancreas is 3.5 mm, the diameter of the pancreatic duct at the tail of the pancreas is 2.5 mm, and the diameter of the ureter is 6 to 8 mm.

[0497] When the balloon in the catheter is inflated, the bile duct has greater plasticity. It can accommodate a larger dilation and remains intact at the maximum balloon size. In contrast, the pancreatic duct does not have enough plasticity to accommodate a larger balloon size. Pancreatic tissue is generally more sensitive and fragile, making it prone to injury.

[0498] The Multi-3 V Plus triple-lumen disposable stone extraction balloon catheter (Olympus Medical) has been used in liver and pancreas research. The balloon on the catheter can be inflated to three different sizes (8.5, 11, and 15 mm) and can withstand hydrodynamic pressure. It has been observed that the 11 mm balloon size causes damage, so future tests will use balloon sizes smaller than 11 mm, preferably less than 10 mm or 9 mm.

[0499] Injection volume calculation

[0500] Second improvement of the injection procedure, which defines and extends the exact dose injected into the pancreas in hydrodynamic injection. Previously, to successfully deliver genes, a dose of more than 20 mL needed to be injected into the pancreas. However, this specification did not take into account differences in individual size, nor how to adjust the volume for human patients. For example, the porcine pancreas has two different lobes, consisting of three different lobules, while the human pancreas is a single lobe or mass consisting of two main lobes. The relative mass of the porcine and human pancreases is comparable (discussed in detail below). In theory, the parameter data of pigs should be applicable to adult human patients. Previous studies only disclosed the injection strategy in pigs weighing approximately 40 to 50 kg, so it was uncertain how the injection volume would change for human patients with significantly larger or smaller body weights. Previous studies provided guidance on dosing based on pancreatic mass, but this did not explain how to use this information to translate into a volume suitable for hydrodynamic injection of the pancreas.

[0501] The current disclosure remedies this deficiency by detailing how to calculate a safe and effective injection volume into the pancreas. As shown in Table 1, the body weights of four pigs and their respective pancreatic weights are shown below, as well as the injection parameters for their injections. It should be noted that in Study 1, some of the early porcine pancreatic dissections included adipose tissue around the pancreatic organ, making these measurements less accurate when interpreting dose recommendations based on body weight.

[0502] Table 1: Gene delivery by injection

[0503]

[0504] Although the body weight distribution of pigs is different from that of humans, the sizes of the visceral organs may be similar. The pancreatic weight data of pigs in these studies are similar to those of the human pancreas, as shown in Table 2.

[0505] Table 2: DNA not injected

[0506] Animal ID# Animal weight Pancreas weight Pig #5 34.7 kg 73.59g Pig #6 40 kg 80.1g Pig #7 36.4 kg 78.2g Pig #8 37.7 kg 64.62g

[0507] One way to address this difference is to weigh the pancreas before injection. The weight determines the injection volume. This can avoid injecting too much fluid into the pancreas, thus risking damage to the organ. Although the data of the porcine pancreas are similar to those of adult humans, it is more accurate to calculate the pancreas based on weight.

[0508] There are some examples in the literature that can determine how to estimate the pancreatic weight of human individuals. An early study in the Journal of The Anatomical Record was published in 1926, summarizing the autopsy data of pancreatic weights of adult men and women known at that time (The normal weight of the pancreas in the adult human being: A biometric study - https: / / doi.org / 10.1002 / ar.1090320204).

[0509] The studies summarized in this article indicate that the normal weight of the pancreas in adult men is 60 to 100 grams. The average weight is 80 grams, and the extreme values are 60 to 100 grams, 70 to 108 grams, or 70 to 90 grams. Other studies have reported that the average weight of the normal pancreas in men is 70 grams, while that in women is 66 grams.

[0510] In another study, 30 cadaveric pancreatic specimens were dissected and carefully measured. The recorded weights averaged 91.8 grams (range: 40.9 to 182 grams). (Am J Surg. February 1994;167(2):261 - 3)

[0511] Another study showed that the average age of the patients was 47.9 ± 17.8 years (between 25 and 88 years), height was 172.2 ± 7.5 cm (between 145 and 190 cm), weight was 78.1 ± 15.2 kg (between 42 and 120 kg), the average BMI was 26.2 ± 4.7 kg / m² (between 17 and 38 kg / m²), and the BSA was 1.9 ± 0.2 m² (Forensic and Anatomical Research, Volume 02, Issue 03 (2014)). The average pancreatic weight was reported to be 87.3 ± 30.6 grams.

[0512] Another study on cadaveric pancreatic weights found a significant difference between normal individuals and patients with type 1 diabetes, with the latter having an atrophied pancreatic volume (JAMA. 2012;308(22):2337 - 2339). This is an important consideration when applying hydrodynamic gene delivery doses in this patient population. The researchers found that the average pancreatic weight of non - diabetic patients (control group) was 81.4 grams (95% CI, 73.0 to 89.8 grams), while that of the group positive for only a single autoantibody was 61.3 grams (95% CI, 46.8 grams to 75.8 grams; P =.02), and that of the type 1 diabetes group was 44.9 grams (95% CI, 36.0 grams to 53.9 grams; P <.001).

[0513] Another method of administration is to calculate the pancreatic volume directly through imaging studies or calculations. The density of the pancreas is estimated to be 1.1 g / mL (Cellular Transplantation, 2007). Therefore, the volume can be converted to mass according to the guidelines here to determine the appropriate dose. Similarly, those skilled in the art can use conventional dimensional analysis to convert the mass-based guidelines here into volume-based guidelines. An example method for calculating the pancreatic volume by MRI is provided here as an example of one of many studies on this technique (PLoS One. 2014;9(3):e92263).

[0514] Although average anatomical studies of the pancreas are useful, for the clinical translation of pancreatic gene delivery, it is very important to more accurately estimate the pancreatic weight. To achieve this goal, a previous study provided a solution for how to calculate the pancreatic weight before hydrodynamic injection.

[0515] This study analyzed the pancreatic weights of 354 cadaveric donors, including gender, age, weight, height, body mass index (BMI), and body surface area (BSA) (Cell Transplant. 2006;15(2):181 to 5). The researchers created a mathematical formula to predict pancreatic weight based on patient-specific factors.

[0516] "For young donors (<40 years old), weight and age are the main predictors of pancreatic weight [pancreatic weight (g) = 4.355 + 0.742 x weight (kg) + 0.837 x age (years) (R2 = 0.564, p < 0.001)]."

[0517] "The pancreatic weight of older donors (>40 years old) is best predicted by BSA and gender [pancreatic weight (g) = -17.624 + 60.036 x BSA (m2) - 7.152 x gender (R2 = 0.372, p < 0.001; "gender": 1 = female, 0 = male)]."

[0518] Using the mass of the pancreas, the current technology describes how to determine the injection dose based on the pancreatic weight.

[0519] In one embodiment of the present disclosure, the volume of hydrodynamic injection can be calculated by multiplying the pancreatic weight by 0.30 mL / g, 0.35 mL / g, or 0.40 mL / g or more, where the mass in the denominator is the pancreatic weight. The ratio multiplier will reflect a volume greater than 22 mL.

[0520] Reduce the injection volume

[0521] In the present disclosure, new volume targets are disclosed to reduce the toxicity of hydrodynamic injection into the pancreas duct. These volume targets address the drawbacks of previous methods, where microscopic lesions of necrosis and damage caused by elevated amylase were observed. These new volume targets are a way to find the minimum but most effective hydrodynamic gene delivery parameters.

[0522] In one example, the volume of hydrodynamic injection should be 0.15 mL / g or 0.20 mL / g of pancreatic tissue weight to reduce the amount of tissue damage observed. These calculations are intended to represent that for a pig or an average adult with a body weight of about 40 kg, the total injection volume is less than 20 mL (as in previous tests). In a more preferred embodiment, the injection volume is 15 mL, or the total volume is 10 mL.

[0523] New data supporting the present disclosure show that when combined with a flow rate of 2 mL / sec, reducing the volume to 15 mL can still result in sufficient hydrodynamic gene delivery. Gene expression was observed in a variety of different pancreatic cell types, and its efficiency was similar to that of the prior art disclosed using larger injection volumes ( Figure 19A 、 Figure 19B ). The purpose of reducing the volume is to reduce tissue damage during the operation. This is verified by the reduced area of necrosis observed histologically and the reduced increase in amylase after injection ( Figure 19C ). The total volume of the pancreatic duct is only 4 to 5 mL, indicating that a smaller injection volume can allow the DNA solution to enter the pancreatic tissue.

[0524] Table 3 below provides a summary of the pancreatic injection experiments, recording the weights of different pigs and pancreases, and the calculated parameters used for these injections.

[0525] The dose guidelines in the present disclosure are based on a synthesis of best practices from previous experiments, taking into account the observed gene delivery efficiency and safety.

[0526] Table 3: Pancreatic injection experiments

[0527]

[0528] Reduced flow rate

[0529] Regarding other injection parameters, the current technology's consideration of the flow rate is also different from previous methods. It has been determined that at a flow rate of 2 mL / sec, a volume greater than 20 mL is sufficient to produce gene expression in the pancreas and a variety of cell types.

[0530] In the present disclosure, the preferred total injection volume is less than 20 mL, which raises the question: what flow rate is optimal or sufficient for gene delivery. Previously, a flow rate of 2 mL / sec or lower was determined to be preferred and sufficient for gene expression.

[0531] In a study with a total volume of 20 mL, a flow rate of 1 mL / second was found to be sufficient for gene expression. Thus, the preferred flow rate range of the present disclosure is equal to or between 1 and 2 mL / second. Higher flow rates above 2 mL / second can be considered, but their association with pancreatic tissue damage is increasing, and their use should be avoided.

[0532] Using multiple flow rates during injection

[0533] By incorporating different flow rates during injection, the flow rate can be further adjusted to further reduce pancreatic damage while maintaining gene delivery and expression. This strategy requires the use of a powered syringe that can be programmed with multiple injection parameters during a given injection procedure.

[0534] For less than 50% of the injection volume, the initial flow rate is 1 to 1.5 mL / second, and for the remaining injection volume, the flow rate is 1.5 to 2 mL / second. This shortens the time at which the peak fluid pressure occurs, such that the peak fluid pressure occurs when the DNA solution completely fills the catheter system.

[0535] Another technique is to use an initial flow rate between 0.5 and 1 mL / second for less than 50% of the injection volume, and set the flow rate to 1 to 2 mL / second for the remaining injection volume. This shortens the time of the peak fluid pressure such that it occurs when the DNA solution completely fills the catheter system.

[0536] This strategy successfully delivers genes efficiently into all relevant cell types (data not shown). In addition, the number of tissue necrosis areas is reduced, and the peak amylase elevation is also decreased.

[0537] Hydrodynamic injection through the liver is an exciting new gene therapy method. Its advantage is that it is non-viral, and thus more scalable and safer than viral methods. Hydrodynamic injection can deliver naked DNA alone, which is the simplest gene therapy method. It is well known that this technique is very effective in mouse models, but traditionally it has not scaled well to large animal models.

[0538] Over the years, multiple research groups have studied the ability to extend hydrodynamic injection to large animal models. An inherent challenge in developing new hydrodynamic injection techniques is the translation of the technique between animals. New methods must be adjusted and optimized for each animal and organ, and it must be empirically tested which strategies are effective. Pigs are a convenient animal test model because depending on the age of the pig, its liver size can be equivalent to that of an adult human. Thus, hydrodynamic techniques established in pigs are likely to be translatable to humans. However, there are still several considerations for this translation, including the presence of lobular fibrosis between the liver lobes in pigs, which may introduce some uncertainty.

[0539] In this context, hydrodynamic gene delivery work in mouse, rat, and rabbit models is undoubtedly exciting proof-of-concept work, but unfortunately, it does not show how to perform this process in human patients and how to optimize gene delivery efficiency. A simple example is that many research groups isolate specific lobes of the liver and inject through the hepatic vein located in that lobe. This technique was first published in a rabbit paper in Human Gene Therapy 2002 Nov 20; 13(17):2065-77. Subsequently, a very similar technique was used in pigs, with the same strategy of isolating a single lung lobe with a balloon-occluded catheter and then injecting hydrodynamically into that lobe (Mol Ther. Mar 2009; 17(3):491-9). However, the flow rates and volumes used to mediate gene delivery in these later studies in pigs could not be predicted from the rabbit studies because, unlike traditional drugs or biologics, there is no simple body weight-based scaling for the hydrodynamic injection procedure. This finding can even be seen in the simple hydrodynamic tail vein injection in mice and rats, where the volumes and flow rates used in mice cannot be simply translated into rates after adjustment according to their larger size.

[0540] Another example of variability is that the choice of blood vessel or catheter for hydrodynamic injection can greatly alter the nature of the injection and the ultimate gene delivery outcome. Such examples have been seen in pig models, where single-lobe injection seems to be more effective than the strategy of reaching the entire liver by injecting through the hepatic vein, and the strategy of clamping the inferior vena cava and portal vein to isolate the blood flow there is even more effective. The subtle differences between these different blood vessels only become apparent after conducting experiments.

[0541] Similarly, researchers have also investigated the biliary system as another route for hydrodynamic gene delivery. The biliary system is unique in that its total volume is much smaller than that of the blood vessels supplying the liver. In contrast, the volume of the biliary system is approximately 30 mL, while the volume of adult blood is approximately 600 mL. The biliary system also has a unique unidirectionality, so the injected liquid does not flow out from the unsealed end simultaneously. This non-directionality of the biliary system is particularly beneficial for maintaining appropriate fluid pressure during injection and ensuring that the high-pressure DNA solution is ejected into the surrounding liver tissue rather than being ejected outward through another hepatic blood vessel.

[0542] Although the hypothesis of liver-directed gene delivery has existed for more than 20 years, the practicality of how to perform this procedure effectively and what the outcome of this procedure will be in large animal models remains uncertain.

[0543] Regarding the development history of biliary gene delivery, the first description of hydrodynamic gene delivery via the bile duct was based on experiments in dogs. The group used surgery, injection rate (∼1 mL / sec), and sutures to prevent antegrade flow within the biliary system (Hum Gene Ther. October 10, 1997; 8(15):1763 - 72). Importantly, the researchers injected the liquid into the bile duct such that the liquid was injected into the gallbladder in addition to the liver. The researchers were only able to detect a small amount of luciferase activity, but no protein detection was observed by histochemical analysis of the liver, so it is likely that an undetectable percentage of pDNA entered hepatocytes. Additionally, no protein was detected on Western blot.

[0544] Parameters:

[0545] · Plasmid DNA dose: 10 to 20 mg

[0546] · Occlusion: Use sutures to occlude the bile duct to prevent antegrade flow

[0547] · Volume: 200 to 400 mL

[0548] · Dog weight: 4.2 kg to 10.8 kg

[0549] · Flow rate: Up to 1.66 mL / sec

[0550] · Pressure: Applicable to mice, not applicable to dogs

[0551] · Others: Optionally occlude the IVC to enhance the effect

[0552] To improve this result, another research group described using ERCP to deliver genes into the livers of dogs and pigs (GIE 2005, T1249 abstract). The ERCP aspect represents an improvement of this strategy over surgery. Other innovations in the procedural method used by the researchers were the use of a balloon to prevent antegrade flow during hydrodynamic injection, thereby increasing the liquid fraction of the DNA-containing solution entering the liver. The group used an injection rate of 5 mL / min in the procedure and continued to inject through the common bile duct, thus injecting into the liver and gallbladder simultaneously. The researchers did not report any efficiency of gene delivery to the liver, as reflected by detectable protein expression on IHC, IF, or Western blot. However, it was reported that a protein was detected in the systemic circulation. The efficiency and utility of this procedure when applied to liver diseases remain uncertain.

[0553] Parameters:

[0554] · Volume: 3 to 10 ml / kg (body weight)

[0555] · Flow rate: 5 ml / min

[0556] · Pressure: 40 to 47 mmHg

[0557] · Weight: Not provided

[0558] · DNA dosage: Not provided

[0559] · Occlusion: Balloon catheter

[0560] Meanwhile, another research group reported that hydrodynamic injection of pDNA into the bile ducts of rats via a surgical approach could mediate gene delivery (Gut. October 2005; 54(10): 1473-1479), but as mentioned above, the hydrodynamic parameters in rodent models are inherently not scalable to large mammals such as dogs, pigs, and humans. For example, the flow rate used by this research group was 0.54 mL / min, and a surgical tape was used to help occlude the bile duct. The relative efficiency of delivering pDNA to hepatocytes via histochemical staining was only up to 1% of hepatocytes, which was much lower than that of the similar hydrodynamic tail vein method.

[0561] Reference will now be made in detail to the exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with the exemplary embodiments, it should be understood that it is not intended to limit the present disclosure to these embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0562] Previous studies have investigated hydrodynamic injection through the biliary system. The initial studies were injections through the common bile duct. The common bile duct is connected to the gallbladder through the cystic duct and to the liver through the intrahepatic ducts, respectively. Researchers tested hydrodynamic injection through the common bile duct in dog and pig models. The researchers reported that protein expression and systemic protein expression could be achieved through luciferase assays, although immunostaining did not show delivery to liver tissue.

[0563] The present disclosure aims to improve the strategy of hydrodynamic injection through the common bile duct. The strategy for common bile duct injection includes inflating a balloon within the common bile duct to prevent the forward flow of fluid into the small intestine. The balloon can be inflated anywhere within the common bile duct. In an alternative strategy, the balloon will be inflated such that the balloon is inflated at the junction of the common bile duct and the common hepatic duct, thereby occluding the opening of the cystic duct.

[0564] The diameter or maximum width of the balloon can be 8 mm (depending on the shape of the balloon) or greater, depending on the estimated lumen diameter. To achieve sufficient sealing during injection, the width / diameter of the balloon at this position needs to be at least 1.5 times the width / diameter of the bile duct, but more likely 2 times, 3 times or more the width / diameter of the bile duct to ensure sufficient sealing. The width / diameter can be fixed at least 4 mm larger, or can be at least 50% larger. Alternatively, the width / diameter can be fixed at least 5 mm, 6 mm, 7 mm or 8 mm larger, or can be at least 60%, 70%, 80%, 90%, 100% or more larger. An overly large width / diameter may cause the bile duct to stretch and subsequently cause bile duct injury or perforation, most likely during hydrodynamic injection.

[0565] The adequacy of bile duct sealing can be confirmed by measuring the pressure in the bile duct upstream of the balloon (during rest or injection testing). If the pressure reading indicates insufficient sealing, the balloon is further inflated. Another method to confirm sufficient sealing is to inject a contrast agent through a catheter upstream or downstream of the balloon to confirm that the position is distal to the porta hepatis and occluded at or upstream of the level of the cystic duct outlet. The pressure can also be measured from a lumen having an opening proximal / downstream of the balloon.

[0566] Parameters studied previously included an injection volume range from 200 mL to 400 mL. In another study, the injection volume range was 3 to 10 mL per kg of subject body weight.

[0567] The required DNA dose range is 10 to 20 mg, injected into a 5 to 10 kg dog. Another study did not report the DNA dose employed. Regarding the flow rate, in one study, the goal was a flow rate of up to 1 mL per second. The flow rate in another study was 5 mL per minute. One prior art reference teaches that the optimal injection pressure is between 40 and 50 mmHg.

[0568] The present disclosure aims to improve the prior art and teaches a method of hydrodynamic injection through the common bile duct to simultaneously deliver genes to the gallbladder and the liver. In other embodiments, only the liver is targeted. The present disclosure teaches the appropriate injection parameters and DNA dose required for hydrodynamic injection through the common bile duct.

[0569] Intra-bile duct injection

[0570] The present disclosure includes several steps to achieve gene delivery in the gallbladder and / or liver. The first step is to access the common bile duct. The common bile duct can be accessed by endoscopic retrograde cholangiopancreatography (ERCP), which is a routine clinical procedure where a catheter is inserted from the small intestine into the biliary system, usually through the major papilla. Alternatively, the common bile duct can be accessed by endoscopic ultrasound guidance (EUS), where a needle is inserted into the duodenal wall and directly into the common bile duct.

[0571] Once the catheter is in place within the bile duct column, the catheter preferably has a balloon that can be inflated to completely seal the common bile duct. The diameter of the common bile duct is relatively wide, so a relatively large balloon size, such as 8 mm, 11 mm, 13 mm, 15 mm or larger, may be required to completely seal the common bile duct. In some embodiments, after sealing the common bile duct, bile can be withdrawn through the catheter by aspiration in order to remove as much bile as possible from the common bile duct, the common hepatic duct, and the gallbladder. In other embodiments, the catheter can be advanced through the cystic duct and bile can be withdrawn from the gallbladder in a similar manner in order to remove it prior to injection. In addition to aspirating bile alone, the bile duct can be flushed with a neutral fluid (such as saline) so that a portion of the bile volume in the bile duct is replaced by this neutral fluid.

[0572] In other embodiments of the present disclosure, the bile can remain in situ prior to injection because the substance fills the gallbladder prior to injection. In other strategies of the present disclosure, the gallbladder is filled with a liquid or gel-like substance prior to hydrodynamic injection of the DNA solution. The purpose of these strategies is to fill the gallbladder such that there is no remaining volume for additional fluid to enter during injection. In certain embodiments of the present disclosure, a salt solution can be pre-loaded into the gallbladder for this purpose. The total volume of the pre-loaded salt solution may be between 40, 50 or 60 mL, or greater. In other embodiments, the salt solution can contain a contrast agent such that the filling of the gallbladder can be monitored in real time.

[0573] In other embodiments of the present disclosure, a gel-like substance can be injected into the gallbladder to fill the gallbladder space. The polymeric substance will then degrade within a few hours. In some examples of the present disclosure, poly(lactic-co-glycolic acid) (PLGA) powder can be injected into the gallbladder in order to empty the gallbladder space prior to injection.

[0574] In the preferred embodiments of the present disclosure, the injection volume is 50 mL / kg or 100 mL / kg of liver weight. This volume accounts for the total volume of the gallbladder, biliary system, and intrahepatic duct system. In other embodiments, the injection volume is at least 150 mL / kg of liver weight, or at least 200 mL / kg of liver weight.

[0575] In certain embodiments of the present disclosure, the DNA dose is at least 20 mg per kg of liver weight. In other embodiments, the DNA dose is at least 40 mg per kg of liver weight.

[0576] In other embodiments of the present disclosure, the minimum DNA concentration injected is at least 0.2 mg / mL of volume. In other embodiments, the minimum DNA concentration is at least 0.5 mg / mL of injection volume.

[0577] The optimal flow rate taught by the current disclosure is significantly different from the prior art. The current disclosure teaches that a flow rate of at least 2 mL / second or 5 mL / second is required. In other embodiments, a flow rate of at least 10 mL / second is required during any injection.

[0578] The present disclosure also determines the optimal pressure during injection. The present disclosure teaches that a minimum pressure of at least 50 mmHg is necessary for effective gene delivery. In other embodiments, the minimum pressure is at least 80 mmHg, or 120 mmHg for injection. In some examples, the pressure during gene delivery can be achieved by a constant pressure injection device that will monitor the pressure and adjust it in real time.

[0579] As an example of the effectiveness of this gene delivery strategy, effective gene expression is achieved within the bile duct wall, common bile duct, and common hepatic duct. This also demonstrates the expression of the reporter gene within the gallbladder wall. The injection strategy at the specified injection parameters also shows an application in mediating gene delivery into hepatocytes.

[0580] In some embodiments of the present disclosure, antibiotics will be mixed with the DNA injection solution because intestinal bacteria are more likely to appear during injection at lower positions in the biliary system, which can cause cholangitis. In another embodiment, antibiotics will be mixed with the fluid used to irrigate the biliary system after aspirating bile, rather than with the DNA solution itself.

[0581] In summary, a newly discovered method for effective gene delivery through the common bile duct is presented herein. The current strategy remedies previous attempts by injecting at higher flow rate and pressure levels to drive more expression within host hepatocytes. The present disclosure also teaches gene delivery into the extrahepatic biliary system, which has not been reported in previous studies. The present disclosure expands the versatility of using the biliary system as a route for gene therapy strategies.

[0582] Strategy of injection through the common bile duct and / or the junction of the common bile duct and cystic duct

[0583] The present disclosure describes a method of hydrodynamic injection through the biliary system. The injection describes a method of injecting through the common bile duct while bypassing the cystic duct, allowing all fluid to enter the common hepatic duct and subsequently the liver.

[0584] The method describes a new strategy of placing a stent within the extrahepatic bile duct, with the stent extending to the bile duct orifice and entering the common hepatic duct. The diameter of the stent is preferably larger than the bile duct to ensure its stability and to ensure that no fluid flows around the bile duct, but rather all solutions flow through the interior of the stent. This will effectively prevent any injected solution from entering the cystic duct or the gallbladder. The optimal stent should be made of solid material or have a non-perforated covering such that fluid cannot penetrate the walls of the stent itself. The stent can be deployed such that it passes through the major papilla and into the duodenum for easy removal. The stent can also be deployed such that it is entirely within the biliary system, but a cord or tether is attached to the stent so that it can be easily grasped and removed when deployed.

[0585] In the preferred embodiment of the present disclosure, the catheter will be placed at several different locations. In one embodiment, the injection can be performed downstream of the common bile duct stent. The common bile duct will be inflated to prevent antegrade flow into the intestine. The injection solution containing DNA will flow retrograde through the stent into the common hepatic duct and then into the liver.

[0586] In another embodiment of the present disclosure, the catheter is located within the stent in the bile duct. Inflating the balloon within the stent will allow the balloon to be safely inflated such that its shape will change from spherical to cylindrical within the stent without damaging the biliary system because the stent will bear the force of the balloon rather than the bile duct wall directly ( Figure 27A ).

[0587] The exact location of the catheter can be the common hepatic duct portion of the stent ( Figure 27B ), the common bile duct portion of the stent ( Figure 27C ) or the cystic duct junction of the stent. The balloon will be inflated within the stent to adequately seal and prevent forward flow of the solution. Additionally, it is expected that the pressure generated by the fluid injection will firmly press the stent against the bile duct wall, thereby further improving the seal. Finally, it is expected that with an inflated balloon inside, the catheter can be used to keep the stent secure during hydrodynamic injection.

[0588] After the injection is complete, regardless of the catheter location, the balloon will be deflated and the stent will be removed along with the catheter and the bile duct.

[0589] The optimal injection parameters for injection through the common bile duct and the stent will follow the injection parameters of the previously described hydrodynamic injection of the common hepatic duct. The main difference is that an additional volume of 5 to 10 mL is added to the calculated volume to account for the additional volume of the common bile duct and the common hepatic duct lumen. The calculated volume will utilize the published common hepatic duct strategy.

[0590] The preferred embodiment of this strategy is to inject a contrast agent into the biliary system to verify whether the gallbladder is opaque. This will effectively confirm that the stent blocks the cystic duct, thereby preventing the DNA solution from entering during the injection process.

[0591] This method is applicable to cases where the common hepatic duct is extremely short or damaged. This method is also applicable to smaller subjects, such as neonates, because in such cases the common hepatic duct itself is very short.

[0592] In the best embodiment of the present disclosure, the following parameters can be used for injection. These parameters are similar to the strategy adopted for common hepatic duct injection, except that the additional volume involved is limited.

[0593] · The preferred flow rate during injection is at least 1 mL / second or 2 mL / second.

[0594] · The preferred injection pressure is at least 50 mmHg, and at least 80 mmHg in other embodiments.

[0595] · The preferred injection volume is at least 30, 40, 50 or 60 mL per kg of liver weight.

[0596] Alternatively, it is recommended to additionally inject 5 mL of solution to address the additional lumen within the biliary system.

[0597] · The preferred DNA dose is at least 20 mg or greater per kg of liver weight.

[0598] · The preferred DNA concentration is at least 0.5 mg per mL of DNA or higher.

[0599] · It is preferred to use a hepatocyte-specific promoter to increase the hepatocyte transfection area.

[0600] Blocking the cystic duct by a secondary approach combined with a bile duct strategy

[0601] One strategy to avoid plasmid DNA solution loss is to block the cystic duct. It can be temporary or permanent, and allows injection at the level of the common bile duct through a balloon catheter. An example is to have the patient undergo a cholecystectomy. Another example is to have the patient undergo a choledochoenterostomy (cystic duct to small intestine anastomosis) (through surgery, percutaneous interventional radiology or endoscopic ultrasound endoscopy) and place a catheter with a balloon from the gallbladder to block the cystic duct. In the above examples, as an alternative to using a balloon to block the cystic duct, a catheter can be used that allows the release of an umbrella-shaped cover from its tip, and the catheter can be placed above or within the cystic duct of the bile duct.

[0602] Intended use of the bile duct strategy

[0603] The current disclosure may have various uses. Unconventional anatomical structures of the biliary system, where the outlet of the cystic duct is not downstream of the porta hepatis, are not uncommon during biliary imaging (Sarawagi et al., Pol J Radiol. 2016;81:250-255). Sometimes, the cystic duct orifice is located upstream of the porta hepatis (i.e., emerging from the left or right intrahepatic bile duct). This would make injection strategies that bypass the cystic duct and gallbladder very complex. In such cases, injection from the common bile duct is preferred.

[0604] This strategy can also be used in a veterinary setting, as the biliary anatomy of veterinarians is different from that of humans. In some animals, there is no common hepatic duct or traditional cystic duct. This strategy can also be used to treat extrahepatic biliary diseases, such as treating cholangiocarcinoma or fibrotic strictures of the bile duct. This strategy can also be used to treat certain gallbladder diseases that can be treated by gene intervention. This strategy can also be used to deliver genes to the liver, where the common hepatic duct may be too small to be adequately positioned. In these cases, injection from the common bile duct would be the only viable solution for delivering genes to the liver. An example is injecting certain injectables into neonates.

[0605] Gene therapy against cancer is an attractive idea. There are many ways to use gene therapy against cancer. Among them, an attractive approach is to try to directly deliver genes into the tumor microenvironment. These genes can encode a range of therapeutic proteins, including proteins that regulate and reconstitute the immune system. In other cases, genes can be directly delivered into tumor cells. In this case, suicide genes can be encoded to immediately kill the tumor.

[0606] Several gene therapy strategies have been published previously. Previous tumor delivery strategies have included using viral vectors, oncolytic viruses with the ability to replicate in tumors, and different non-viral strategies. Non-viral strategies include using local delivery strategies to directly inject DNA or RNA into tumors. Another common strategy is to use lipids or polymers to directly deliver plasmid DNA into tumors. For these strategies, tumor-specific uptake can be enhanced by having specific tumor-binding ligands on the surface of the nanoparticles, or by using polymers or lipids that have a natural affinity for specific types of tumors.

[0607] The drawback of all these strategies is that it is difficult for gene therapy vectors to penetrate into the tumor microenvironment of the cancer cells themselves. Most strategies rely on the systemic administration of gene therapy vectors. In these cases, the gene therapy vectors must cross the endothelial barrier and penetrate deep into the tissue. Many types of tumors are rich in blood vessels around the tumor margin, but have fewer blood vessels inside the tumor. Therefore, vascular administration is difficult to penetrate deep into the tumor. Other types of tumors have a large amount of fibrosis within the tumor, so it is difficult for any nanoparticles or viral vectors to penetrate the tumor.

[0608] A potential strategy for non-viral gene delivery is hydrodynamic delivery. The hydrodynamic delivery leverages fluid pressure to form pores in the cell membrane, enabling DNA to enter the interior of the cell. When delivered via the mouse tail vein, hydrodynamic delivery is a highly effective gene delivery method. During tail vein injection, within a time span of 5 to 7 seconds, the liquid is rapidly injected into the mouse liver, accounting for approximately 10% of the body weight. The liquid injected into the tail vein and inferior vena cava returns from the right heart to the mouse liver, resulting in severe fluid congestion. The fluid pressure forms pores in the mouse liver, thereby enabling efficient gene expression.

[0609] Although hydrodynamic tail vein injection is a very common technique, whether hydrodynamic injection can effectively deliver drugs to tumors within the liver has largely remained unexplored. Even when testing this hypothesis, the biggest obstacle is the attempt to generate local tumors within the mouse liver. Most mouse models form multifocal large tumors over time through germline mutations.

[0610] To date, the only study evaluating the delivery of genes to hepatocellular carcinoma (HCC) by hydrodynamic injection was conducted in rats (J Gene Med. August 2006; 8(8): 1018 - 26). Rats can also undergo hydrodynamic injection. The rats in this study were treated with a chemical, diethylnitrosamine, which causes mutations in the rat liver. Over time, these mutations eventually lead to the development of cancer. The researchers found that hydrodynamic tail vein injection was very ineffective, with almost no expression detectable, and was unable to deliver genes to the tumor. Expression could only be detected in the tumor when the DNA carrier was administered via the hepatic artery. Therefore, the delivery efficiency seems to be highly dependent on the route of administration.

[0611] A core challenge of hydrodynamic injection is the uncertainty and unpredictability of scaling up this technology from rodents to large animals. When considering how to scale up hydrodynamic injection, the volume and flow rate increase by an order of magnitude compared to the parameters used in rodents. Additionally, the access routes and the methods of accessing these routes in large animal models are significantly different from those in rodent models.

[0612] Since there is no data to support the actual delivery of genes to the liver and pancreatic tissues of large animals, it is currently impossible to predict whether gene delivery by hydrodynamic injection is effective. In particular, it is impossible to predict which areas of the liver, if any, will express the injected genes. Another important unknown is whether different types of tumors in the liver, each with a different type of tissue structure, will absorb any DNA carriers in different ways when delivered by hydrodynamic injection. Therefore, it is uncertain whether hydrodynamic injection can reach the tumors.

[0613] Reference will now be made in detail to the exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with the exemplary embodiments, it should be understood that it is not intended to limit the present disclosure to these embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0614] The present disclosure describes a method for delivering genes to liver tumors by hydrodynamic injection. The method utilizes the biliary tract pathway to deliver fluid pressure to the tumor microenvironment. The biliary system is a dynamic network, a branched structure that contacts all hepatocytes. However, prior studies before the present disclosure have shown that the biliary network does not contact the tumor microenvironment. This includes primary liver tumors and metastatic liver tumors. Therefore, the prior art of this study speculated that this method would fail. The present disclosure shows that the biliary tract pathway can effectively mediate gene delivery to different aspects of tumors. The present disclosure also shows that multiple tumor types, not just hepatocytes, can be gene-delivered through the biliary system.

[0615] The first step of the method is to perform an endoscopy. ERCP is used to access the common hepatic duct in the biliary system. Once in the common hepatic duct, an inflatable balloon is used to seal the catheter. Meanwhile, a DNA solution will be prepared, where the DNA encodes a therapeutic protein for treating the tumor. In other embodiments, the DNA of interest may encode a diagnostic protein to help identify the tumor. The DNA solution will be loaded into a power injector. In a preferred embodiment, the volume of the loaded solution will be at least 30 mL per kg of liver weight. In other embodiments, the volume of the loaded solution will be at least 40 mL per kg of liver weight. In a preferred embodiment, the flow rate will be specified as at least 2 mL per second. In other embodiments, the flow rate is at least 4 mL per second. This technique has been optimized for delivery to normal hepatocytes before. It has been taught in the prior art to deliver to normal hepatocytes at a flow rate of at least 2 mL per second. For intratumoral delivery, the inventors teach that a higher flow rate is more desirable to allow deeper penetration of the tumor microenvironment. In a preferred embodiment, a flow rate of up to 10 mL per second can be utilized to increase DNA expression within the tumor.

[0616] The minimum DNA dose effectively delivered into the tumor is at least 1 mg DNA per kg of liver weight. In other embodiments, the minimum DNA dose is 5 mg per kg of liver weight. In other preferred embodiments, a DNA dose of 10 mg per kg of liver weight may be more desirable. Generally, this specific tumor targeting technique requires a higher DNA dose because the hydrodynamic method delivers DNA to the entire liver, and thus only a small amount of DNA will ultimately enter the tumor microenvironment. The amount of DNA in the tumor affects the degree of transfection.

[0617] In some embodiments of the present disclosure, a way to address this limitation is to position the catheter in the right or left hepatic duct of the biliary system, rather than in the common hepatic duct. This will facilitate the delivery of DNA only in the right or left liver. If the tumor is located only in the right or left hepatic duct, this strategy can be used to increase the amount of DNA delivered to that specific tumor. In other embodiments, where there are multiple hepatic tumors or existing and / or metastases throughout the liver, injection is preferably from the common hepatic duct.

[0618] In some embodiments of the present disclosure, the DNA molecule can be further formulated. The DNA molecule can be formulated with a variety of polymers, peptides, or lipids, which are reported to help the tumor absorb more. It is conceivable that the hydrodynamic injection method can act synergistically with these delivery reagents to promote the more effective delivery of these nanoparticles into the tumor. Hydrodynamic injection will particularly help them penetrate into the interior of the tumor. Given that tumor cells are dividing, the nuclear membrane breaks down more frequently, allowing more non-viral DNA to enter the nucleus for expression.

[0619] The process of gene delivery to the pancreas is similar, but ERCP accesses the pancreatic duct and the dose is calculated based on the estimated pancreatic weight. Most tumors are located in the head of the pancreas, so the tumor should be downstream of the head of the pancreas to deliver DNA to the tumor under pressure.

[0620] Single-gene hereditary liver diseases cover a range of diseases from metabolic disorders to coagulation disorders, including hemophilia A and B, α-1 antitrypsin deficiency, familial hypercholesterolemia, Wilson's disease, Crigler-Najjar syndrome, methylmalonic acidemia, ornithine transcarbamylase deficiency, etc.

[0621] These diseases cause severe distress to patients even with modern therapies. The cure for these diseases is liver transplantation, but the available livers are limited. In addition, transplantation itself requires lifelong immunosuppression, leading to the risk of infection and drug side effects.

[0622] As an alternative to liver transplantation, gene therapy, which modifies the tissues of a patient's own genes that are missing or malfunctioning, has been explored. The success of adeno-associated virus (AAV) in preclinical models has translated into clinical trials, but has also revealed some limitations, including the presence of neutralizing antibodies against AAV in many patients; high levels of AAV input trigger an immune response, and subsequently AAV is cleared; the gene packaging size is limited, about 4.8 kb, and so on.

[0623] To address these issues, the inventors developed a non-viral gene therapy with hydrodynamic injection via an endoscopic approach. In a preliminary study using endoscopic retrograde cholangiopancreatography (ERCP), a gene was successfully delivered into the porcine liver, achieving a transfection efficiency of 30 to 50%, which may meet the needs for treating many monogenic liver diseases. Previous work on ERCP gene therapy for hemophilia B received R21 funding from NHLBI / NIH, and this gene therapy research will be explored in non-human primates in the future.

[0624] This study will explore the optimal injection method for injecting into the primate liver and apply it to human patients. This study also describes the optimal methods and components for gene therapy of hemophilia B using hydrodynamic injection.

[0625] Mutations in the genes of a small number of people disrupt basic liver processes such as metabolism or blood clotting. Disruptions in metabolism or blood clotting lead to various problems, including thickening of blood vessels due to cholesterol, accumulation of metals such as copper in tissues to toxic levels, or continuous bleeding.

[0626] A possible treatment for these methods is to insert a functional copy of the gene into a person's liver to reverse these processes. This study will explore this gene therapy using a common medical procedure of inserting a tube through the throat, through the stomach into the intestine. Then a thin wire can be guided to the liver and directly target DNA delivery. This study will explore a method that does not use a virus as a delivery vector, which will improve safety. The potential impact may be to cure these patients, saving them from frequent infusions of other drugs and hundreds of thousands of dollars in drug costs per year.

[0627] Reference will now be made in detail to the exemplary embodiments of the present disclosure. While the present disclosure will be described in conjunction with exemplary embodiments, it should be understood that it is not intended to limit the present disclosure to these embodiments. Instead, it is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0628] The present disclosure describes a series of steps for effectively delivering DNA to the primate liver for gene therapy.

[0629] The first step is to enter the biliary system for gene delivery and injection. In a preferred embodiment, an endoscopic retrograde cholangiopancreatography (ERCP) procedure will be performed to place a balloon catheter into the common hepatic duct of the baboon. In a preferred embodiment, the ampulla can be incised to increase the size of the opening and the convenience of catheter insertion.

[0630] Using a radiopaque contrast agent solution, the catheter is placed in the common hepatic duct, passing over the cystic duct to avoid injection into the gallbladder. Contrast agent injection will also be used to verify whether the balloon seals the catheter during injection and whether the left and right hepatic ducts are visible.

[0631] Subsequently, the injection procedure begins. The injection is performed using a power injector filled with the target DNA solution. The catheter connects the end of the power injector to the bile duct catheter to allow the DNA solution to be injected through one of its lumens.

[0632] The DNA solution in the preferred embodiment is saline, in which pure recombinant DNA is dissolved. The DNA can be of various types, including plasmid DNA or minicircle DNA. In other embodiments, linear closed-ended DNA can be used.

[0633] The injection should follow the specified parameters to mediate effective gene delivery. The volume parameter will be set to 30 mL per kg of liver weight. The volume can also be 40 / mL / kg or greater.

[0634] The flow rate parameter in the preferred embodiment should be at least 2 mL per second. In other embodiments, the flow rate can be at least 3 mL per second or greater.

[0635] The pressure parameter, when using a dedicated constant pressure injection device, is at least 50 mmHg. In other embodiments, higher pressures will be employed, including greater than 80 mmHg, or greater than 120 mmHg.

[0636] In certain embodiments, the DNA dose is at least 10, 20, 30, 40, or 50 mg per kg of liver weight. The purpose of a higher DNA dose is to produce a high enough serum or plasma concentration to generate an effective level of human factor IX for the treatment of hemophilia B.

[0637] The DNA vector composition will employ a hepatocyte-specific promoter. The vector in the preferred embodiment should also have one or more hepatocyte-specific enhancers to drive higher levels of transcription. A codon-optimized gene cassette will be selected, where the codons are selected to be abundant in hepatocytes. This can be applied to the expression of human factor IX in the preferred embodiment. A strong polyadenylation sequence will also be used.

[0638] The injection of the primate liver will also seek to monitor signs of liver injury, which can be done through common liver chemistry tests including transaminase function.

[0639] After injection, deflate the balloon and then withdraw the catheter from the bile duct. Injection can be repeated during the same procedure or on different operative days as indicated.

[0640] The present disclosure relates to compositions and methods for hydrodynamic gene delivery to the liver via the biliary system. More specifically, the present disclosure relates to compositions and methods for enhancing the efficiency of hydrodynamic gene delivery via the biliary system. As described in detail below, the present disclosure is at least in part based on the following surprising findings: Modifying the DNA vector to substantially reduce the bacterial sequences on the plasmid DNA to less than 500 base pairs significantly increases the observed hepatocyte transfection area to more than 70% of the hepatocytes, while the transfection area of conventional plasmids is less than 50%. The present disclosure also elucidates the following surprising finding: When the dose is equal to or higher than 20 mg DNA per kg liver weight, the gene delivery efficiency is greatly increased and then reaches saturation. This non-linear relationship is unexpected but very useful in designing therapeutic dosing regimens. The present disclosure also yields the surprising finding that more than 40% of the high-level transfection of the integrated reporter gene shows no loss of level within three months, contrary to previous reports on transposons, and for the first time shows immunotolerance at this threshold of antigen expression. The present disclosure also yields the surprising finding that free vectors delivered by hydrodynamic injection can be expressed for 4 months, the longest expression duration observed to date, and they can survive after substantial dilution loss in the liver, i.e., with a volume increase of up to 50%. The present disclosure also reports the surprising finding that very large DNA vector sizes can be delivered into large animals, exceeding 12 kb. More surprisingly, although the DNA vector size gradually increases, the transfection efficiency does not seem to decrease. This is contrary to every published report on non-viral DNA vectors. The present disclosure also finds that gene injection can be repeated and the gene can be successfully expressed with the same efficiency as the first injection, such that both proteins can be detected in the same cell. It is not certain whether the first hydrodynamic injection disrupts the liver structure to prevent a second injection. The present disclosure also finds that a high DNA vector dose of up to 40 mg per kg liver weight is not toxic, which has never been tested before, and more importantly, the DNA concentration in the injection solution is the main determinant of the observed delivery efficiency. The present disclosure also elucidates new findings regarding the gene expression efficiency related to the flow rate and how to optimize the injection with an appropriate volume dose. These two aspects have never been reported before. Finally, the present disclosure reports that liver sinusoidal endothelial cells can be targeted for gene delivery, which has not been shown in previous publications.

[0641] Overview

[0642] Hydrodynamic injection via the liver is an exciting new gene therapy method. Its advantage is that it is non-viral, so it is highly scalable and safer than viral methods. Hydrodynamic injection can deliver naked DNA alone, which is the simplest gene therapy method. As is well known, this technique is very effective in mouse models, but traditionally it has not been well scalable to large animal models.

[0643] For many years, several research groups have studied the ability to scale up hydrodynamic injection to large animal models. The inherent challenge in developing new hydrodynamic injection techniques is the translation of the technique between animals. New methods must be adjusted and optimized for each animal and organ, and which strategies are effective must be tested empirically. Pigs are a convenient animal test model because, depending on the age of the pig, its liver size can be equivalent to that of an adult human. Therefore, hydrodynamic techniques established in pigs are likely to be translatable to humans. There are still several considerations for this translation. For example, the pig liver has lobular fibrosis between the lobes, which may cause some uncertainties.

[0644] The work on hydrodynamic gene delivery carried out in mouse, rat, and rabbit models is a strong proof of concept, but unfortunately, it does not show how to perform this process in human patients and how to optimize gene delivery efficiency. One example is that many research groups isolate specific lobes of the liver and inject through the hepatic vein located in that lobe. This technique was first described in a paper using a rabbit model, published in Human Gene Therapy 2002 Nov 20;13(17):2065-77.

[0645] Subsequently, similar techniques have also been used in pigs, applying the same strategy, that is, using a balloon-occluded catheter to isolate a single lung lobe and then injecting it into the lung lobe via hydrodynamic injection (Mol Ther. Mar 2009;17(3):491-9). However, the flow rate and volume of pig gene delivery used in these later studies cannot be predicted from rabbit studies because, unlike traditional drugs or biologics, there is no simple weight-based scaling for hydrodynamic injection procedures. This finding was confirmed in simple hydrodynamic tail vein injection in mice and rats, where the volume and flow rate used in mice are not easily translated into predictable rates when adjusting for their larger size.

[0646] Another example of variability is that the choice of blood vessel or catheter for hydrodynamic injection can vary the injection parameters, as well as the way gene delivery works and its outcome. An example of this was observed in a porcine model, where a single-lobe injection was more effective than an injection through the hepatic vein in reaching the entire liver, compared to a strategy of clamping the inferior vena cava and portal vein to isolate the blood flow there. These subtle differences between these different blood vessels could not be predicted prior to conducting the experiment.

[0647] Similarly, the biliary system has also been studied as another route for hydrodynamic gene delivery. The biliary system is unique in that its overall volume is much smaller than that of the blood vessels supplying the liver. In contrast, the volume of the biliary system is approximately 15 to 30 mL, while the blood volume of an adult is approximately 600 mL. The biliary system is also unidirectional, so the injected liquid does not flow out of the unsealed end simultaneously. This unidirectional property of the biliary system is beneficial for maintaining an appropriate fluid pressure during injection and ensuring that the high-pressure DNA solution is ejected into the surrounding liver tissue rather than being ejected out through another hepatic blood vessel.

[0648] Although the hypothesis of liver-directed gene delivery has been around for more than 20 years, researchers still do not have a good grasp of how to effectively implement the procedure in large animal models and the specific details of the possible outcomes. The first description of hydrodynamic gene delivery through the bile duct was based on experiments on dogs. The research group used surgery, an injection rate (∼1 mL / second), and sutures to prevent antegrade flow within the biliary system (Hum Gene Ther. October 10, 1997; 8(l5):1763 - 72). The researchers injected the liquid into the common bile duct, so that the liquid was injected into the gallbladder in addition to the liver. The researchers were only able to detect a small amount of luciferase activity, but no protein detection was observed by liver histochemical analysis, so it is likely that an undetectable percentage of pDNA entered the hepatocytes.

[0649] Another group applied ERCP to deliver genes to the livers of dogs and pigs (GIE 2005, T 1249 abstract). ERCP is a better technique than surgery. Other improvements the researchers made to this method were to use a balloon to prevent antegrade flow during hydrodynamic injection, thus increasing the amount of liquid containing the DNA solution that enters the liver. This group used an injection rate of 5 mL / min in this method and continued to inject through the common bile duct, thus injecting into the liver and gallbladder simultaneously. The researchers did not report any efficiency of gene delivery to the liver, as reflected by detectable protein expression on IHC, IF, or Western blot. However, it was reported that a protein was detected in the systemic circulation. The efficiency and practicality of applying this procedure to liver diseases remain uncertain.

[0650] Meanwhile, another research group reported that surgical injection of pDNA into the bile duct of rats could mediate gene delivery (Gut. October 2005; 54(10): 1473-1479), but as mentioned above, the hydrodynamic parameters in rodent models cannot be extended to large mammals such as dogs, pigs, and humans. For example, the flow rate used by this research group was 0.54 mL / min, and the surgical tape helped seal the bile duct. The relative efficiency of delivering pDNA to hepatocytes by histochemical staining was only about 1% of hepatocytes, which was much less efficient than the similar hydrodynamic tail vein method.

[0651] Explore improved techniques for hydrodynamic injection into the biliary tract. In the first study, the research mediated gene delivery into pigs by hydrodynamic injection and detected it by immunostaining observation (Kumbhari, GIE 2018). Explore the new injection parameters that the porcine biliary system can tolerate. The porcine bile duct ruptured at parameters above 2 mL / second and 30 mL / second, so these parameters were selected for subsequent work. Enter the porcine biliary system through ERCP, and place a balloon in the common hepatic duct to avoid cystic duct and gallbladder injection. Compared with previous studies, this is a key innovation because avoiding gallbladder injection can generate greater pressure in the liver during injection, thus delivering genes more effectively. Keep the balloon inflated for 1 minute after injection to increase more fluid delivery.

[0652] As a result, for the first time, experimental data verified the presence of genes expressed by pDNA in the liver (Kumbhari, GIE 2018). PCR showed the presence of pDNA in all lobes and parts of the liver. Western blot confirmed that the delivered protein was expressed in all liver tissues. Immunofluorescent staining of the delivered protein was also observed, although the delivery efficiency was less than 1% of hepatocytes. The expression of the protein seemed to be stable, and the Sleeping Beauty transposon effect lasted up to 60 days.

[0653] Although this previous study was a major advancement, its delivery efficiency was insufficient to treat any clinical diseases because clinical diseases require more than 10 to 20% of hepatocytes in the liver to express the target transgene. Compared with previous studies, improvements are still needed, and the published data discuss this in detail (Kruse, GIE 2021).

[0654] The first area of improvement focuses on a better composition of the vector injected into the liver. Using a hepatocyte-specific promoter to drive expression, along with codon optimization of the transgene and stabilization of the 3'UTR. Higher doses of plasmid were introduced, and a more active transposase system was introduced in piggyBac. These changes to the composition, pDNA dose, and transposase together increased the transfection efficiency by an order of magnitude, with over 30% of hepatocytes expressing the transgene. These improvements are all described in Kruse, GIE 2021.

[0655] To improve the technique, the researchers also targeted multiple cell types in the liver via a ubiquitous promoter. This included cholangiocytes, endothelial cells, and neurons. The improved vector composition demonstrated the ability of cell-specific delivery, showing that when using the corresponding hepatocyte or vascular endothelial cell promoter, only hepatocytes or endothelial cells could express the desired transgene. Other new findings included the ability to change the flow rate to zone 3 rather than zone 1 of the hepatic lobule. Determining the flow rate that causes elevated transaminases and liver injury. The first description of an alternative strategy for biliary hydrodynamic injection into the biliary tract system other than ERCP.

[0656] A more comprehensive understanding of the tolerance of the bile duct to higher flow rates and volumes without rupture. Determining the relationship between pressure and injection parameters and determining the pressure achieved during biliary hydrodynamic injection sufficient to mediate gene delivery. These findings are described in Huang's PLOS One 2021.

[0657] Subsequently, another research group contributed to biliary hydrodynamic gene delivery (Mol Ther Methods Clin Dev. January 19, 2022; 24:268 - 279). This group studied the hydrodynamic gene delivery technique in young pigs weighing between 4 and 6 kg. The methods and techniques employed by this group were different from the results published by Kumbhari and Kruse; thus their efficacy was different. For their protocol, they accessed the biliary tract system surgically rather than via ERCP. Instead of using a balloon catheter, they used different surgical clips on the cystic duct and multiple different veins. A single-lumen catheter was used for injection. No clips were placed on the bile duct itself, presumably to avoid catheter breakage, or if the catheter was in place, the surgical clips would never seal. The injection itself occurred in the common hepatic duct, but there was no antegrade flow into the common bile duct, resulting in reduced pressure and leakage of the DNA solution into the intestine.

[0658] The injection parameters were significantly higher than those used in previous studies by Kruse and Kumbhari, with a target of 10 mL / sec and a 100 mL injection volume. The team was able to detect the presence of DNA in all lobes of the porcine liver by PCR. Similarly, luciferase activity was also detected in all lobes. Unfortunately, very little protein expression was detected after liver immunostaining, with only sparse cells detected. Another important factor is that although protein expression could initially be detected, it almost completely disappeared within 10 days after injection. The researchers used free microcircles and nano-carrier / nanoplasmid carriers as DNA constructs. These carriers were reported to be superior to regulatory plasmid DNA, but they were not sufficient to increase the expression level on IHC or mediate long-term expression. In summary, the results indicate limited effectiveness of their method / technique, and it is doubtful whether these alternative carriers can improve the expression of biliary hydrodynamic techniques.

[0659] This disclosure aims to provide improved technologies, methods, and compositions to enhance gene delivery efficiency after hydrodynamic injection of bile in large animals.

[0660] Other improvements include new safety strategies and methods of gene expression targeting specific cell types. Other improvements include re-administration of gene carriers and strategies for achieving long-term expression. The possible size limitations of biliary hydrodynamic injection are also described.

[0661] Increase the transfection area

[0662] A key goal of gene therapy technology is to deliver DNA into as many target cells as possible and have the DNA reach the cell nucleus so that protein expression can be detected in as many target cells as possible. This is crucial for both viral vectors and non-viral strategies. Previously, expression was achieved in 30% to 50% of porcine hepatocytes, but the DNA doses were different. However, there may be significant differences in expression in each lobe. In addition, for this result, only one plasmid DNA composition and one transgene were described, which may affect its efficiency when applying this technology to other vector sizes and proteins.

[0663] To deliver DNA to more hepatocytes, further modifications were made to the hydrodynamic injection vector composition. The modified vector composition can act in synergy with the program parameters to produce better results. Modifying plasmid DNA by reducing or removing the bacterial backbone can greatly increase the transfection area observed in porcine liver slices, reaching more than 50% of hepatocytes. In some slices of the transfected area, 60%, 70%, 80%, or even 90% of the hepatocytes within the lobule expressed the gene of interest. On average, 72% of the hepatocytes stained positive for the gene of interest. The stained area was significantly darker, indicating higher intracellular endogenous protein expression.

[0664] This improved transfection efficiency is achieved by injecting a vector that is a circular DNA molecule lacking most bacterial sequences. The bacterial sequences that exist are less than a thousand bases in length, and in some cases even less than 500 base pairs. An example is a nanovector or nanoplasmid with a reduced origin of replication and bacterial selection region. The nanoplasmid has an R6K conditional origin of replication and requires pir+ E. coli host cells for reproduction. Selection is done by the RNA-OUT system, which downregulates the SacB gene. In another example, the vector is pF AR, which has a smaller bacterial backbone of less than 1000 base pairs. The pCOR backbone consists of three bacterial elements: a 0.4kb R6K y conditional origin of replication (ori y), which requires a functional R6K71 promoter protein, a 0.2kb optional tRNA suppressor gene (sup Phe), and a 0.4kb cer (ColEl resolution) fragment to resolve the pCOR oligomer, totaling less than 1000 base pairs. Any bacterial backbone of varying structure that is less than 1000 base pairs, preferably less than 500 bp, is suitable for this approach. Another option is to use a minicircle vector, which is a circular DNA molecule that does not contain bacterial elements. Minicircle vectors are created by recombination of two different sites on plasmid DNA. Recombining these sequences removes the bacterial sequence on the plasmid DNA, leaving only a small remnant of the mammalian sequence and the recombination site.

[0665] Another DNA vector composition that produces a higher transfection area after biliary hydrodynamic injection is a linear DNA molecule. In a preferred embodiment, the linear DNA vector is a linear DNA molecule with closed ends (PLoS One. August 1, 2013; 8 (8): e69879), also known as a mini DNA molecule (Mol Ther Nucleic Acids. June 2014; 3 (6): e165) or dog bone DNA (Hum Vaccin Immunother. August 2015; 11 (8): 1972-1982). These linear DNA molecules lack any bacterial sequences and have covalently closed ends. Therefore, they will not trigger the DNA damage response and will not be integrated into the genome. These linear DNA molecules move more freely in the solution, which helps them to translocate during hydrodynamic injection into the nucleus. Linear DNA molecules have smaller residual sequences (up to 50bp at each end), which are residues of viral or bacterial production excision sites, but do not contain other bacterial sequences in addition.

[0666] These vector compositions have a synergistic effect with hydrodynamic injection into the biliary tract because the lack of bacterial elements gives them a better expression profile. Another hypothesis is that the smaller size may facilitate translocation to the nucleus during hydrodynamic injection. The results were unexpected because these types of DNA molecules did not significantly improve the transfected area in mouse studies, and most of their effects were thought to be cell-specific expression. It is currently unclear whether these molecules will improve the transfected area during hydrodynamic injection. In a study of hydrodynamic injection into the biliary tract, reduced bacterial scaffolds (minicircles, nanoplasmids) failed to improve the process (Mol Ther Methods Clin Dev. January 19, 2022; 24:268-279). However, for gene delivery using hydrodynamic injection into the biliary tract, the above vector compositions should be used to maximize protein expression in hepatocytes.

[0667] Improved expression longevity

[0668] An important aspect of gene therapy is the length of expression. The length of expression is affected by a variety of different factors, including the intrinsic silencing of gene vectors, dilution loss of episomal DNA constructs, death of the cells receiving the gene, and adaptive immune responses against the gene product. These factors can be greatly influenced by the delivery vector and / or the procedure used to deliver the gene.

[0669] For hydrodynamic gene delivery, the procedure itself may cause trauma by injecting a high pressure of liquid into the tissue. The tissue will swell temporarily due to the liquid, which may lead to cell death of a variety of different cell types. This is particularly evident in mouse models, where after hydrodynamic injection, some tissues will necrose, accompanied by a transient inflammatory response with immune infiltration. In a canine study using vascular hydrodynamic gene delivery, it was observed that the specified hydrodynamic injection parameters were associated with an early loss of transgene expression. It is thought that this is because they trigger an immune response against the transgene product. Transgene products expressed in the inflammatory environment of hydrodynamic injection can have a vaccine-like effect. Therefore, it is uncertain whether any hydrodynamic procedure will result in long-term expression, either due to intrinsic DNA reasons or due to an immune response against the transgene product.

[0670] Hydrodynamic injection can result in short-term expression of the delivered transgene, which is consistent with observations in mouse models where hydrodynamic injection of a plasmid encoding hepatitis B virus antigen results in acute clearance. In contrast, adeno-associated virus (AAV)-mediated introduction of hepatitis B virus antigen can result in long-term expression of the viral protein without causing inflammation. Therefore, it is uncertain whether hydrodynamic injection will result in short-term or long-term expression, especially compared to other gene delivery methods.

[0671] It is not clear how hydrodynamic injection of bile duct fluid will modulate or interact with the host immune system to achieve long-term expression, as this technique is inherently different from other hydrodynamic strategies. Published data suggest that the transposon strategy of hydrodynamic injection of bile duct can express genes for up to two months (Kumbhari, GIE 2018). However, the amount of protein produced in this study was minimal, and only a very small number of cells (<1%) expressed the delivered gene. The amount of protein produced by clinically relevant transfection rates was insufficient for the immune system to recognize and respond. Based on previous studies, it is not possible to predict whether the immune system will initiate an adaptive immune response after high-level transfection. Considering subsequent improvements in bile duct hydrodynamic technology, it is not possible to predict whether additional proteins will be more clearly recognized by the immune system and thus ultimately eliminated. Published data suggest that expression is maintained for three weeks, but adaptive immunity may take up to two months to develop, so three weeks is a very early time point to consider. A longer expression process is needed to know whether the integrated transgene will express genes beneficial to the life of the animal.

[0672] This disclosure shows that the combination of an optimized expression cassette with a hepatocyte-specific promoter and the piggyBac transposon was able to successfully mediate transgene expression in porcine hepatocytes for up to three months. The relative transfected area of hepatocytes expressing the protein was not significantly different compared to the transfection percentages at day 3 and 1 month. This confirmed stable expression and no immune response occurred. The specified parametric technique and the vector composition are the best choices to achieve the expression goal of three months or longer.

[0673] A major challenge in gene therapy is that DNA integration into the host chromosome can be genotoxic and thus lead to carcinogenesis. Therefore, many regulatory agencies and physicians prefer strategies that deliver episomal DNA into cells. Episomal DNA does not integrate into the host genome and is expected to have minimal or no impact on host cell viability. The challenge with episomal DNA strategies is that they typically lose their expression capacity over time due to various reasons, including silencing of episomal genes and dilution loss of plasmid DNA during cell division. Improvements are still needed to develop best practices, methods, and vector compositions to achieve long-term expression of episomal DNA after hydrodynamic injection of bile.

[0674] Previous studies of hydrodynamic injection via the porcine vascular route have observed that extrachromosomal pDNA expression persists for up to two months after injection, although the transfected area decreased by approximately 50% during this period (Mol Ther Nucleic Acids. October 2013;2(10):e28).

[0675] Mouse studies have shown that most of the injected plasmid DNA molecules are silenced after one month, so it is unclear how long the expression of non-viral constructs will last. Even for viral vectors such as AAV, the expression of free DNA is significantly reduced within a few weeks to 2 to 3 months after injection (bioRxiv 2022.03.24.485675). Although previous studies on hydrodynamic injection into blood vessels have been exciting, it is unclear whether the biliary approach can achieve a similar or even longer duration of expression, especially in growing pigs with progressive pDNA silencing. No study has described the expression of pDNA by biliary injection for more than 1 week in large mammals.

[0676] The present disclosure addresses these limitations by describing a method that achieves up to four months of free DNA expression using a vector composition lacking large bacterial DNA backbone sequences. In previous hydrodynamic studies in blood vessels, this method was either not employed or could not achieve long-term expression using a reduced bacterial backbone vector. This modified vector DNA backbone must be delivered to cells using the hydrodynamic biliary method under set parameters so as not to trigger an immune response and so that the pDNA can be correctly translocated into the cell nucleus. The present disclosure is an improvement because it achieves the longest duration of biliary hydrodynamic injection pDNA expression to date. In addition, the techniques herein also overcome previous barriers in free-type vector gene delivery, where during the current experiment, the size of the pig liver increased by 50% from the time of DNA administration to when the liver was harvested at the end of the four-month experiment. According to the literature on free-type vectors, this typically results in a significant dilution loss of the vector genome, thereby reducing the observed hepatocyte transfection area (Hum Gene Ther. May 2012;23(5):533-539). In contrast, the hepatocyte transfection area in our study remained relatively similar throughout the study, indicating that the biliary hydrodynamic gene delivery method and the vector composition with a reduced bacterial backbone size have special and unexpected characteristics.

[0677] Repetition of gene expression in the operating room

[0678] One limitation of all current gene therapy methods is the inability to reuse gene therapy vectors. This is mainly because viral vectors generate an immune response against the viral capsid, so a strong immune response prevents the free use of the vector during a second administration. Hydrodynamic injection may be a way to address this limitation because it is non-viral and lacks any protein components that may generate an immune response. In a mouse model, hydrodynamic injection can be repeatedly administered, and the result is that the protein expression produced by the second injection is similar to that of the original injection. However, hydrodynamic injection in mice has not been shown to have an additive effect on the original injection, possibly because pDNA gene silencing makes it difficult to demonstrate this in the experiment.

[0679] Although hydrodynamic injection has been shown to be reproducible in some murine models, studies in large animal models have not been successful. A study of repeated hydrodynamic injection in a dog model showed that despite the use of immunosuppressive drugs, no manifestation occurred after the second injection (Hum Gene Ther. July 2017;28(7):551-564). This is thought to be due to a strong immune response to the transgene during hydrodynamic injection, which can be re-stimulated with each subsequent injection. In another study of pigs, readministration was achieved by a second injection of human α-1 antitrypsin, but the contribution of the first and second doses to the observed expression was difficult to determine. Thus, previous studies have not revealed whether hydrodynamic injection in large animal models can be repeatedly dosed, or whether two different genes can be continuously and repeatedly dosed without suppressing the expression of the first-administered gene, and whether the second administration can target the same cells within the liver for expression. Previous studies have not determined whether repeated gene therapy is feasible from the perspective of gene expression and immune response in the biliary system. There is no data from the biliary system to support the possibility of repeated injection. It has been reported previously that the procedure itself can be repeated a second time in pigs without causing bile duct rupture or severe damage to the liver, but this does not address whether gene expression using the procedure is feasible. More importantly, it does not determine whether the second injection has the same gene delivery efficiency as the first injection.

[0680] To address the challenges of repeated dosing, the present disclosure describes a method for achieving repeated non-viral gene therapy dosing via the biliary system. One method is to perform a first injection using a vector composition having a cell-specific or ubiquitous promoter, using established optimized biliary injection parameters. This second injection procedure can be completed at least one week, two weeks, three weeks, four weeks or later after the first injection, thereby achieving significant gene expression. This result can be achieved without changing the injection procedure technique. The second injection does not result in loss of expression from the first injection, and individual cells in the liver can express DNA from both injections, so the second injection does not result in translocation of DNA from the first injection or gene expression silencing. The transfection efficiency is not reduced or altered by the second injection. Depending on the promoter used for the second injection, the second injection can target DNA to all cell types, including cholangiocytes and endothelial cells, which are not disturbed during the first injection.

[0681] Repeating gene expression within a single procedure

[0682] Previous reports have indicated that multiple fluid injections into the bile duct cannula of a pig are technically feasible and that pigs tolerate fluid injections well. What is not clear is whether DNA can be injected multiple times during a pig's surgery to produce the expression of different plasmid DNA molecules. There is reason to suspect that this may not be technically feasible and may have harmful effects on the pig liver. Previous studies have shown that liquid fdl vesicles accumulate within the cytoplasm of pigs within 15 minutes after high-flow bile injection (Huang, PLOS One 2021). If these vesicles accumulate, it is uncertain whether hepatocytes can tolerate more vesicles and / or whether the vesicles will disrupt the transport of a second DNA through the cytoplasm into the nucleus. In addition, while multiple injections can be tolerated within a day, it is not clear whether the long-term effects on the pig will be toxic to the liver and whether the potential liver toxicity at longer time points after injection will increase, leading to protein expression ablation.

[0683] There are two main clinical reasons for multiple injections during a single injection procedure. In one example, a patient requires different DNA constructs encoding different proteins to treat their disease. The cost and logistics of mixing these DNA constructs into a single volume may be impractical or may alter the total fluid volume, thereby endangering the efficacy of hydrodynamic delivery. More specifically, combining volumes and / or increasing the DNA concentration into a single injection may alter the optimized parameters of the gene delivery protocol. Therefore, as an alternative to this approach, a second injection can be performed on the same pig to deliver the gene to the liver again.

[0684] The second reason for multiple hydrodynamic injections during a single ERCP procedure is that if technical difficulties and / or technical failures occur, the injection may fail due to the balloon slipping out of place, the balloon deflating, a power injector failure, or a failure of the luer lock connection between the catheter and the power injector tubing. In such a case, a second injection of the full dose of DNA can be immediately repeated. The important reason for attempting a second injection at that time rather than rescheduling is that there is some risk of pancreatitis with each ERCP procedure, so the total number of ERCP procedures should be reduced. In addition, if there is a second DNA solution, it is relatively easy to reload the power injector and continue the injection with the catheter already in place.

[0685] Previous studies have not demonstrated that repeated injection of genes during a single biliary surgery is technically feasible. Current technical methods indicate that this is feasible and practical in human patients. In the present disclosure, the second gene injection can be performed within 5 minutes after the first injection. In other examples, the gene injection can be performed 10 minutes, 15 minutes, or 20 minutes or more after the first injection. Current technical methods indicate that after the second DNA treatment, two proteins expressed from these DNA molecules can be detected simultaneously within the same liver despite different injection times. Current technical methods also indicate that proteins from the delivered DNA can co-localize within the same hepatocyte. For this method, the present invention indicates that injection parameters do not have to be adjusted for its intended use as a single injection. However, a flow rate exceeding 5 mL / sec should be avoided during the first injection to prevent the development of a large number of vacuoles that would impede the second injection.

[0686] Mixing pDNA vectors during a single injection for co-delivery into the same cell

[0687] In some cases, it is desirable to co-deliver multiple different genes into a cell simultaneously. In previous studies, this could be achieved by placing multiple gene cassettes on the same plasmid DNA. However, a drawback of this method is the large size of the plasmid DNA, which may reduce the translocation of the plasmid DNA into the cell nucleus. Additionally, for manufacturing protocols where plasmid DNA molecules have been produced separately, in many cases, it is desirable to combine the plasmid DNA molecules into a single injection rather than two different injections. In these cases, cloning the plasmid DNA constructs into a single plasmid would become infeasible and costly. Additionally, larger plasmid DNA may reduce the delivery efficiency.

[0688] Previous studies have never demonstrated that two different plasmid DNAs can be co-delivered to achieve gene expression of both plasmid DNAs. It is currently uncertain whether the two plasmid DNAs will be equally expressed in different cells, or whether one plasmid will be superior to the other in terms of gene delivery efficiency. Additionally, it is unclear whether the two plasmids will co-localize within certain cell types, which is necessary for the efficacy of some diseases.

[0689] Current technical methods indicate that it is feasible to co-deliver two different plasmid DNAs simultaneously via biliary hydrodynamics, and gene expression can co-localize within the same cell. By the biliary hydrodynamics method, after gene delivery, co-localization of the two proteins appears to be present in almost all cells. This indicates that the same hepatocytes receive the same amount of fluid pressure simultaneously, resulting in co-delivery of the plasmid DNA.

[0690] DNA size limitation

[0691] When determining the potential applications of gene therapy, the DNA vector size limitations of specific gene therapy methods are crucial. Current vector systems, including adeno-associated virus (AAV, 4.8 kb genome size), are highly restricted in terms of DNA payload size, which limits their ability to deliver complete genes for several rare diseases (VWD, hemophilia A) and other therapeutic possibilities. Viral vectors are inherently limited in packaging size because they must encapsulate their entire genome into the geometry defined by the capsid. Non-viral strategies generally do not have this limitation because they are not packaged into a viral capsid. However, it has been observed that the transfection efficiency of all non-viral strategies varies depending on the plasmid DNA size used. For example, some transfection reagents do not transfect larger pDNA constructs and smaller pDNA sizes. In mouse models, hydrodynamic injection is known to be able to deliver a relatively large amount of DNA intracellularly. However, it has never been studied whether this fact applies to hydrodynamic strategies in large animals. In addition, it has never been demonstrated whether hydrodynamic injection into the bile duct can deliver relatively large plasmid DNA molecules with sizes greater than 8 kb, 9 kb, or 10 kb.

[0692] Previous studies have shown that plasmid DNA molecules with a size of 5.5 KB can be efficiently delivered into the porcine liver by hydrodynamic injection into the bile duct. No further data on plasmids or larger sizes have been published. In the present disclosure, strategies for delivering DNA molecules with sizes exceeding 12 kilobases, and strategies for delivering DNA molecules with sizes greater than 15 kilobases and 20 kilobases via the bile duct are described. These strategies include delivering DNA into multiple cell types, including hepatocytes, cholangiocytes, and endothelial cells. Strategies for obtaining plasmid DNA with sizes exceeding 10 kb and using higher DNA mass doses to maintain an equivalent DNA dose per molecule are described.

[0693] Hydrodynamic injection into the bile duct uses injection parameters of volume >30 mL / kg and greater than 2 mL / second, which are sufficient to deliver large DNA constructs into cell types. Expression can be achieved using ubiquitous promoters, such as the cytomegalovirus promoter. Specific promoters that can be used to increase expression can also be used to target cells.

[0694] The newly discovered ability to deliver larger DNA helps to determine guidelines for appropriate dosages. If the ratio of molecule to vector is the same, then transfection efficiency can be maintained even if the plasmid is larger. The first step is to standardize all DNA weight-based dosages (mg DNA / kg liver) by the size of the indicator plasmid to reach the transfection threshold of the indicator plasmid. For example, in this case, the size of the indicator plasmid used to determine transfection efficiency is 8.6 kb, and the known goal is that 50% of the dosage is ~10 mg / kg and 70% of the dosage is ~20 mg / kg. Within these parameter ranges, multiply the body weight-based dosage by the plasmid DNA size (in kb) divided by the 8.6 kb indicator plasmid size to obtain a plasmid DNA dosage that should produce similar injection results.

[0695] To simplify clinical application, the DNA dosage for any given liver weight (kg) and plasmid DNA size (kb) is calculated using the following formula, as shown in Table 4.

[0696] Table 4: DNA Dosage Formula

[0697] DNA dose (mg) / liver (kg) / plasmid DNA (kb) % Transfection rate 1 mg / kg / kb 50％ 2.5 mg / kg / kb >70％ 3.5 mg / kg / kb >70％ 5 mg / kg / kb >70％

[0698] Minor deviations within 0.5 mg / kg / kb in this formula do not have a significant impact on the percentage and produce similar dosage efficacy.

[0699] This calculation does not apply to nanoplasmids or other vectors with reduced bacterial backbone sizes because, as observed in the above experiments, nanoplasmids or other vectors with reduced backbone sizes have inherently better properties in mediating higher transfection efficiency even when considering different plasmid DNA sizes. Therefore, when using this formula, only similar vector types should be compared. Similar calculations can be performed in nanoplasmid dosage experiments to clarify appropriate dosages.

[0700] Summarizing the above experiments, it was found that a 5.8 kb nanoplasmid could achieve a 70% transfection rate. The 70% formula would calculate 14.5 mg pDNA, while in fact only 10 mg pDNA is needed. Therefore, nanoplasmids themselves have higher intrinsic activity. To account for this activity, the DNA dosage can be reduced by 30% to account for the higher nanoplasmid activity. Alternatively, a similar series of empirical dosages can be calculated, or the current dosage formula can be used, but it should be noted that dosages above 2.5 mg / kg / kb are unnecessary.

[0701] Increasing DNA Dosage

[0702] Another research approach is to determine whether increasing the plasmid DNA dose can improve hydrodynamic injection into the biliary tract. A previously published report only tested pDNA doses of 3 mg and 5.5 mg, and the results showed that the higher the pDNA dose, the larger the transfection area (Kruse, GZE 2021). It would be useful to know whether gradually increasing the dose can continue to increase the transfection area and whether different pDNA molecules are effective. This information will be very valuable for guiding the appropriate amount of clinical dose for gene therapy procedures. Although the purpose of increasing the pDNA dose is to ensure that more genes are delivered into hepatocytes, thereby increasing the observed transfection area, a secondary issue with increasing the pDNA dose is whether any toxicity will occur. Naked plasmid DNA can be recognized by intracellular innate immune receptors, thereby causing inflammation. Generally speaking, dose-dependent toxicity is a major problem faced by all gene therapy vectors today, which has been observed in AAV and adenovirus gene therapy trials. Dose-limiting toxicity can also be observed in clinical trials using lipid nanoparticle vectors delivering mRNA or siRNA or naked oligonucleotide conjugates. There is a possibility that a certain concentration or amount of DNA can be hydrodynamically injected into the liver of large mammals, resulting in severe toxicity. Toxicity may also manifest as an abnormal decrease in transgene expression due to cell death or triggering of innate immunity, thereby reducing the expression of pDNA. Such knowledge will provide practitioners with information on the appropriate dose level for biliary hydrodynamic delivery technology.

[0703] The toxicity issue was empirically studied to improve the technical method. Four different concentrations of plasmid pT-LPl-ATP7B, C9 were injected into pigs weighing from 36 kg to 41 kg. The concentrations were ~10 mg, ~20 mg, ~30 mg, and ~40 mg pDNA. The flow rate was kept at 2 mL / second in all studies. It was observed that the pDNA continued to increase in the transfection area as the dose increased up to 20 mg pDNA. There was no significant difference between the higher doses of 30 mg or 40 mg pDNA and 20 mg pDNA. This finding avoids waste of pDNA dose, thereby allowing more patients to be treated. There was no difference in potential toxicity compared to injecting a higher dose of pDNA, as there were no changes in vital signs and no elevation of transaminases after injection.

[0704] Higher doses of pDNA can be added to achieve a higher transfection area, exceeding the previously described 5 mg of pDNA. Doses up to 20 mg, 30 mg, or 40 mg are well tolerated. The present disclosure shows that DNA doses equal to or greater than 20 mg of pDNA can increase the transfection area to 70% of the total hepatocytes. Considering that 5.5 mg and 10 mg of pDNA only account for 50%, this represents an unexpected increase in efficiency. Additionally, it is unexpectedly found that the effect saturates at around 20 mg / kg, and for a given plasmid DNA size, larger plasmid DNA doses are not necessary as they do not increase the transfection area. Furthermore, DNA doses of at least up to 40 mg of pDNA do not cause cell-mediated toxicity that would reduce transgene expression. Higher doses can be safely used by bile hydrodynamic injection and can be considered when applying this technique to a variety of different treatments.

[0705] Injection parameters

[0706] Previous studies have revealed that the combination of vector composition and procedural techniques can achieve efficient gene delivery, thereby enabling efficient expression in the transfected area of tissues. The parts not defined in this technique are how different flow rates and volumes affect gene expression and how to improve gene delivery.

[0707] Although all components of this technical method are important, determining the optimal injection force is crucial for delivering more pDNA into cells while avoiding potential toxicity.

[0708] Previous studies have described the injection parameters used in gene delivery strategies as 2 mL / sec to 4 mL / sec. The total volume tested was 30 mL to 40 mL. The studies also described that the biliary system can tolerate various injection flow rates, and the highest injection flow rate tested was 10 mL / sec. The highest injection volume tested was 140 mL into the liver. However, although it has been shown that these parameters are tolerable from a technical perspective and do not cause bile duct rupture or severe liver injury, it does not show how they will affect DNA delivery and which parameters are optimal.

[0709] A series of studies were conducted to understand how higher and lower flow rates affect the gene delivery efficiency of biliary surgery, as evaluated by the transfection area through immunohistochemical staining. Another set of studies tested the effect of volume on these parameters. All studies kept other variables the same, including the plasmid DNA construct and the plasmid DNA dose.

[0710] Low flow rate

[0711] Lower flow rates are attractive because they can reduce injection-induced tissue damage and inflammation and may be safer for the patient. However, the techniques provided herein show that a flow rate of 1 mL / sec, in combination with the procedural techniques employed, results in little or no gene expression in the liver. The vector compositions in this experiment have the ubiquitous promoters of CMV and SV40 that drive reporter gene expression. Thus, flow rates equal to or below this threshold should be avoided, unless the goal is to load the DNA solution into the liver prior to a faster injection.

[0712] High flow rate

[0713] Regarding the effect of increasing the flow rate, the hypothesis was that higher flow rates might mediate more pDNA entry into cells, thus transfecting more hepatocytes. However, the present disclosure shows that using significantly higher flow rates is not optimal. A flow rate of 4 mL / sec reflects the results of previous studies, where staining of hepatocytes and non-hepatocyte cell types (such as bile ducts) was similar. A flow rate of 4 mL / sec may bias more expression towards the peripheral regions of the lobule, although central delivery was still observable. Surprisingly, higher flow rates of 7 mL / sec and 10 mL / sec decreased the efficiency of transfection into hepatocytes, with a progressive decrease in the number of hepatocytes expressing the gene. At higher flow rates, delivery to non-hepatocyte cell types remained robust, with significant bile duct staining.

[0714] Current techniques indicate that the flow rate for efficient gene delivery is preferably greater than 1 mL / sec and less than 7 mL / sec. Optimally, the flow rate for hepatocyte-targeted delivery is at least 2 mL / sec and does not exceed 4 mL / sec. Alternatively, by increasing the flow rate to greater than 4 mL / sec, bile duct cells can be preferentially targeted. In other embodiments, the flow rate for targeted gene delivery to bile duct cells is greater than 7 mL / sec or greater than 10 mL / sec to drive more DNA into this cell type.

[0715] Volume

[0716] Regarding the effect of increasing the volume, it is unclear whether delivering a larger volume of a liquid with a certain concentration of plasmid DNA (pDNA) to the liver actually increases the observed expression. One hypothesis is that a larger pDNA volume would increase the saturation of the DNA solution into the bile ducts and ensure that more pDNA volume enters the cells. On the other hand, a larger volume might also act to dilate the biliary system and its surrounding tight junctions, allowing more fluid to escape into the vasculature. The pDNA would not enter the hepatocytes. A related consideration is that only a certain proportion of the DNA solution volume is "active", so a lower net concentration of pDNA in this active portion could actually be detrimental. Additionally, a more diluted DNA solution might also mean less effective DNA enters individual cells, which would reduce the transfection efficiency.

[0717] In the experiment, 10 mg of pDNA was injected, diluted in 40 mL, 60 mL, and 80 mL of physiological saline, and the flow rate for each injection was 2 mL / second. The pDNA liquid solution was pushed into the hepatic lobules.

[0718] The results of the drug delivery technology study found that under the conditions of a constant flow rate and a certain concentration of pDNA, the larger the injection volume, the lower the gene expression. There was little IHC staining among porcine hepatocytes injected with a dose of 80 mL, while the efficiencies of injecting 40 mL and 60 mL doses were relatively close.

[0719] Preferably, the volume of the pDNA solution should be less than 80 mL. In other embodiments, the pDNA solution should be less than 60 mL. These volumes should be adjusted according to the weight of the target liver, which was approximately 1 kg in these studies.

[0720] Converting this information into a liver weight-based drug delivery strategy, the optimal flow rates are 20 mL / kg, 30 mL / kg, 40 mL / kg, 50 mL / kg, or 60 mL / kg, where kg represents the liver weight and can be determined by various different methods in the literature.

[0721] DNA Dosage Guidelines

[0722] Previous studies have indicated that nucleic acids include DNA administered in an amount of at least 1 mg of DNA per kg of the total liver tissue weight of the subject. However, there is no information on the optimal concentration for preparing the DNA solution. The current study shows that high doses may have a harmful effect on transfection efficiency, but this can be offset by increasing the concentration of DNA injected at high doses.

[0723] Here, the DNA concentration (mg / mL) is preferably greater than 0.30, 0.40, 0.50, or preferably greater than 0.60 mg / mL. For simplicity, the DNA dosage per kg of liver weight can be used as an example. In this case, doses of 10 mg / kg, 20 mg / kg, 30 mg / kg, or 40 mg / kg or higher can be used. Whichever DNA dosage is chosen, the volume should be adjusted to achieve these parameters.

[0724] Improving Transfection Efficiency by Adjusting the Flow Rate

[0725] Injection parameters are a crucial part of successfully achieving gene delivery. Regardless of the hydrodynamic drug delivery route, it has been observed in all animal models that minor changes in injection parameters can greatly affect the observed gene expression results.

[0726] Previous studies have shown that hydrodynamic injection of bile duct fluid uses a set flow rate to achieve gene expression. All experiments were carried out at a single flow rate, which is recommended to mediate efficient expression. In addition, as the flow rate increases, the distribution of expression can be slightly adjusted from zone 3 to zone 1 of the hepatic lobule.

[0727] Previous studies have not proposed a method of using multiple different flow rates during the operation. Although this idea has been considered before, it is not clear which combination of flow rates can achieve the highest level of expression. The currently proposed technical method recommends modifying the gene delivery method, thereby generating a larger hepatocyte transfection area than the current test paradigm.

[0728] This disclosure shows that by using at least two different flow rates during injection, the transfection area in the liver can be further increased. In other cases, at least three different flow rates are used. Previous studies have shown that this is technically feasible, but have not specifically stated which combination of flow rates and times is most suitable for increasing the transfection area.

[0729] This disclosure shows that the initial hydrodynamic injection is preferably started at a slower injection flow rate to avoid premature dilation of the bile ducts and canaliculi due to higher pressure, thereby allowing more vascular fluid to escape. In addition, the injection protocol teaches that it is best to first fill the biliary system with liquid and then gradually increase the pressure.

[0730] For example, the initial injection speed is 2 mL / second, and then the injection speed is increased to 4 mL / second. This first targets gene expression to zone 3 in the center of the hepatic lobule, and then targets gene expression to the periphery of the hepatic lobule at a faster flow rate. The final result of designing the injection parameters in this way is to more effectively cover the entire hepatic lobule, thereby increasing the number of hepatocytes expressing the target gene. In the best case, the slow phase will inject half of the liquid volume. In another example, the slow phase will continue for two-thirds of the injection volume. The fast phase is kept to a minimum because the increased force can be immediately delivered to the distal fluid and does not need to experience a longer time. It is best to avoid premature dilation of the catheter and sinusoid by a faster flow rate so that a smaller pressure can be achieved by later flow rates.

[0731] In another example, the first fluid phase is carried out at a speed of 1 mL / second until the total volume reaches the estimated volume of the large bile ducts in the liver, and then one or more faster volumes are carried out to increase the expression of pDNA. Alternatively, a fast flow rate occurs first, followed by a slower flow rate, to introduce plasmid DNA into the center of the lobule. In this example, the initial rapid injection will account for the first half of the total injection volume, followed by a slow injection volume, thereby shortening the injection time. The best method is to use the total injection volume to calculate how much time is required for each injection phase. Time is not the most important factor because the injection volume may vary for different procedures, depending on the size of the large mammal being injected.

[0732] In another example, the flow rate increases in a step function such that three or more flow rates can be used. In one example, a flow rate of 2 mL / sec is first used to inject 33% of the volume, then a flow rate of 3 mL / sec is used to inject 33% of the volume, and then the remaining volume is injected at 4 mL / sec.

[0733] Scaling the procedure to smaller and larger livers

[0734] Previous studies have shown that pigs weighing 40 kg and above can tolerate injection into the biliary system using the set parameters and balloon inflation. However, these studies did not show whether smaller pigs could tolerate the procedure, or whether their bile ducts would rupture, or whether their livers would be severely damaged.

[0735] One previous study reported that pigs weighing approximately 5 kg could tolerate injection through the biliary system at parameters of 100 mL and 10 mL / sec. This study did not use a balloon catheter to ensure pressure during injection, so it was not determined what the effective pressure within the biliary system was. Additionally, there were several other details of the procedure that differed, including the use of surgery.

[0736] Thus, although the study showed that the procedure was tolerated in small animals, it did not address the problems that occur when performing the procedure on large animals. This means that the method used in the study cannot be transferred to large animals without further testing.

[0737] To ensure that hydrodynamic injection into the biliary system can be scaled down to smaller animals, current technical methods show that pigs weighing at least 25 kg and at least 15 kg can tolerate hydrodynamic injection well without causing rupture of the biliary system. Additionally, injection of a smaller dose of a drug into these pigs still results in robust gene expression as measured by immunohistochemistry.

[0738] This disclosure presents a method of scaling the injection volume to a particular animal. Previous studies have specified volume parameters for hydrodynamic injection and proposed the goal of dosing by liver weight, but the exact details were limited to 30 mL / kg liver weight and 100 mL / kg liver weight. This disclosure remedies these deficiencies by providing additional recommendations for weight-based dosing.

[0739] In the best case, the volume injected during hydrodynamic injection through the biliary system is 40 mL / kg of liver tissue weight. In another example, the volume is 50 mL / kg of liver tissue weight. In other cases, the volume is 60 mL / kg of liver tissue weight. Before biliary hydrodynamic injection, the liver tissue weight is calculated either through computations available in the liver or by direct imaging methods. The present disclosure indicates that the total volume is preferably not more than 70 mL / kg, as this is associated with lower efficiency. In the best case, the injection volume is between 30 and 70 mL / kg, or more preferably between 40 and 60 mL / kg.

[0740] These new doses per kg of liver tissue volume are supported by additional porcine experimental data, indicating that they can effectively mediate gene delivery, resulting in the expression of a reporter gene in the liver.

[0741] Pressure-mediated injection

[0742] Previous studies have disclosed that the pressure for effective gene delivery is between 50 mmHg and 150 mmHg. The present disclosure attempts to further describe which pressure within this range and in higher pressure ranges can result in the most effective gene delivery. A series of tests are conducted using a constant pressure injection device that employs real-time pressure monitoring and uses it to adjust the injection in real-time to maintain a given pressure.

[0743] Using this device, a constant pressure injection of approximately 50 mmHg is tested. It is found that the injection efficiency is very low at this pressure threshold, with limited hepatocyte transfection observed. Using a higher pressure threshold of 80 mmHg, a higher proportion of hepatocyte transfection expressing the target gene is observed. Therefore, for hydrodynamic injection, a pressure of at least 80 mmHg is preferred. Pressures above 150 mmHg are further tested to determine whether they are suitable for hydrodynamic gene delivery. It is observed that pressures of 175 mmHg and 200 mmHg can still result in effective gene delivery, with an efficiency similar to that of injections at lower pressures below 150 mmHg. Compared with these lower pressure thresholds, injections at pressures above 200 mmHg begin to produce lower transfection efficiencies.

[0744] Targeting liver sinusoidal endothelial cells ...

Claims

1. A method for determining the flow rate of hydrodynamic injection into an organ, comprising the following steps: Perform a test injection; Measure the pressure during the test injection; and Empirically evaluate the differences in stiffness and resistance.

2. The method according to claim 1, wherein The organ is selected from the liver, pancreas or kidney.

3. The method according to claim 2, wherein Inject the liver through the biliary system.

4. The method according to claim 2, wherein Inject the pancreas through the catheter system.

5. The method according to claim 2, wherein Inject the kidney through the ureter system.

6. The method according to claim 1, wherein Insert a pressure sensor into the catheter through a dedicated lumen.

7. The method according to claim 1, wherein The pressure sensor already exists in the catheter.

8. The method according to claim 1, further comprising the following steps: Read the baseline pressure of the biliary system with the balloon uninflated; Measure the pressure of the biliary system with the balloon inflated; Inject a test solution or nucleic acid into the target catheter or blood vessel without plasmid DNA with the balloon uninflated; and Monitor the pressure during the injection of the test solution 9. The method according to claim 8, wherein The test solution has the same osmolality, weight - molality and viscosity as the DNA injection solution.

10. The method according to claim 8, wherein The test solution also does not contain any other active pharmaceutical substances contained in the therapeutic DNA solution.

11. The method according to claim 1 or 8, wherein The pressure reached during hydrodynamic injection is at least 50 mmHg, at least 80 mmHg or at least 120 mmHg.

12. The method according to claim 8, wherein The initial test flow rate into the liver is at least 2 mL / sec or at least 3 mL / sec.

13. The method according to claim 8, wherein The total test volume is a volume of at most 40 mL, at most 30 mL, at most 20 mL, at most 10 mL or at most 5 mL.

14. The method according to claim 13, wherein The total test volume is sufficient to measure the total fluid resistance column in the entire circuit and evaluate whether sufficient pressure is reached.

15. The method according to claim 8, wherein If the appropriate pressure is not reached, increase the flow rate and repeat the test.

16. The method according to claim 8, wherein If the pressure above 250 mmHg is reached, reduce the flow rate and repeat the test.

17. The method according to claim 15 or 16, wherein For the second test, the flow rate is increased or decreased by 1 mL / sec or increased or decreased by 0.5 mL / sec.

18. The method according to claims 14 to 17, wherein The DNA solution injection is performed at a set flow rate to generate an appropriate pressure.

19. The method according to claim 8, wherein Test a series of flow rates during a single test injection, where multiple flow rates are tested and the pressure is measured throughout the single test injection.

20. The method according to claim 19, wherein Test at least two or more test flow rates within a single test injection.

21. The method according to claim 19 or 20, wherein The pressure is related to the increasing flow rate.

22. The method according to claim 19, wherein Increase the total injection volume to a total volume of at most 40 mL, 35 mL or 20 mL and test all injection parameters.

23. The method according to claim 1, wherein The minimum pressure for effective hydrodynamic gene delivery is greater than 50 mmHg, greater than 80 mmHg or greater than 100 mmHg.

24. The method according to claim 1, wherein The maximum pressure for effective hydrodynamic gene delivery is less than 200 mmHg or less than 250 mmHg.

25. A method for hydrodynamic retrograde ureteral delivery of nucleic acids or viral vectors, comprising the steps of: (i) Insert a cystoscope into the bladder via the urethra (ii) Insert a balloon catheter into the bladder through the cystoscope (iii) Use the balloon catheter to insert into the ureteral orifice (iv) Inflate the balloon catheter at the distal ureter near the entrance of the ureteral orifice (v) Use a power injector to mediate hydrodynamic injection into the kidney, where the method does not require the use of fluoroscopy to place the catheter, 26. The method according to claim 25, wherein By using injection parameters with a total volume equal to or less than 20 mL to avoid kidney rupture or tear. And 2 ml / sec 27. The method according to claim 25, wherein A flow rate equal to or between 0.5 mL / sec and 2 mL / sec is optimal for achieving rupture - free gene delivery.

28. The method according to claim 25, wherein The optimal injection volume is between 10 mL and 20 mL to mediate effective gene delivery without rupture.

29. The method according to claim 25, wherein The balloon is located in the bladder muscular wall of the ureter and can be visualized through a cystoscope camera without the need for fluoroscopy.

30. The method according to claim 29, wherein During injection, any ureteral outflow can be monitored through the cystoscope camera. If there is no outflow at all, it indicates effective sealing during injection.

31. The method according to claim 25, wherein For a single kidney of a subject weighing 30 kg or more, the minimum dose of the nucleic acid is 1 mg, 2 mg, 3 mg, 4 mg or higher quality.

32. The method according to claims 25 to 31, wherein The hydrodynamic parameters are sufficient to achieve protein expression within renal cells after delivering DNA.

33. The method according to claim 32, wherein Cell expression is achieved within glomeruli, tubules or endothelial cells inside the kidney.

34. The method according to claim 32, wherein The difference in cell expression among glomeruli, tubules or endothelial cells is related to the use of different promoters in plasmid DNA.

35. A method for hydrodynamic retrograde ureteral delivery of nucleic acids or viral vectors, comprising the steps of: (i) Insert the cystoscope through the urethra into the bladder (ii) Insert a guide wire into the bladder through the cystoscope (iii) Insert the guide wire into the ureteral orifice (iv) Advance the guide wire towards the kidney (v) Remove the cystoscope and replace it with a balloon catheter through the guide wire. (vi) Inflate the balloon catheter within the ureter at the proximal position of the kidney (vii) Use a power injector to inject liquid into the kidney Among them, this method can utilize a balloon catheter that is not suitable for the working channel of the cystoscope.

36. The method according to claim 35, wherein, By using injection parameters equal to or less than 12 mL and 2 mL / second to avoid renal rupture or tear.

37. The method according to claim 35, wherein, A flow rate equal to or between 0.5 mL / second and 2 mL / second is optimal for achieving rupture-free gene delivery.

38. The method according to claim 35, wherein, The optimal volume for injection is 7 mL to 12 mL to mediate effective gene delivery without rupture.

39. The method according to claim 35, wherein, The balloon is positioned at least 1 cm, 2 cm or 3 cm away from the renal pelvis and inserted into the ureter to ensure proper sealing.

40. The method according to claim 35, wherein, Before injection, a radiopaque agent is injected to confirm the positioning of the catheter and the sealing of the balloon.

41. The method according to claim 35, wherein, For a single kidney of a subject weighing 30 kg or more, the minimum dose of the nucleic acid is 1 mg, 2 mg, 3 mg, 4 mg or higher quality.

42. The method according to claims 35 to 41, wherein, The hydrodynamic parameters are sufficient to achieve protein expression within renal cells after delivering DNA.

43. The method according to claim 42, wherein, Cell expression is achieved within glomeruli, tubules or endothelial cells inside the kidney.

44. The method according to claim 42, wherein, The difference in cell expression among glomeruli, tubules or endothelial cells is related to the use of different promoters in plasmid DNA.

45. The nucleic acid composition according to claims 25 and 35, wherein, The nucleic acid consists of plasmid DNA, minicircle DNA, mRNA, siRNA or antisense oligonucleotide.

46. The viral vector composition according to claims 1 and 8, wherein, The viral vector is selected from adenovirus, adeno-associated virus, lentivirus, baculovirus, anellovirus or Sindbis virus.

47. The method according to claims 25 and 35, wherein, Compared with a viral vector injected at a non-hydrodynamic flow rate, hydrodynamic injection is used to promote better penetration of the viral vector into tissues, binding to cells and entry into cells.

48. The method according to claim 47, wherein, The transduction efficiency of renal cells by retrograde ureteral injection is higher with hydrodynamic injection than with non-hydrodynamic injection (flow rate < 0.15 mL / second).

49. The method according to claims 25 and 35, wherein, The hydrodynamic injection can also be monitored with a pressure sensor to ensure a pressure of at least 50 mmHg, 60 mmHg, 70 mmHg or 80 mmHg is achieved.

50. The method according to claim 49, wherein, Inject distally into the ureter at a pressure of at least 80 mmHg at a flow rate equal to or greater than 0.5 mL / second.

51. The method according to claim 49, wherein, Inject proximally into the ureter at a pressure of at least 100 mmHg at a flow rate equal to or greater than 1 mL / second.

52. A method for hydrodynamic gene delivery to the pancreas via a catheter system, comprising: (a) Insert a catheter into the pancreatic duct by endoscopic retrograde cholangiopancreatography (b) Inflate the balloon during injection to seal and increase the pressure (c) Inject at a flow rate equal to or between 1 and 2 mL / second (d) The injection volume is equal to or less than 0.20 mL per gram of pancreatic weight (e) The DNA dose injected is at least 10 micrograms per gram of pancreatic weight Wherein this method is sufficient to mediate gene expression in all pancreatic enzyme-elevating-reducing and tissue necrotic lobes.

53. The method according to claim 52, wherein, The injection parameters achieve gene expression in duct cells, islet cells, acinar cells, endothelial cells, and neurons.

54. The method according to claim 52, wherein, For a pancreas weighing more than 60 grams, the total injection volume is not more than 15 mL.

55. The method according to claim 52, wherein, If a flow rate of 2 mL / second is used, the maximum volume used is 0.15 mL per unit pancreatic weight.

56. The method according to claim 52, wherein, The DNA dose is preferably greater than 20 or 30 micrograms per gram of pancreatic weight.

57. The method according to claim 52, wherein, The amylase or lipase content increases by a maximum of 4-fold on the first day after injection.

58. The method according to claim 52, wherein, The catheter can be placed: (i) Through the major duodenal papilla into the main pancreatic duct, distal to the confluence of the pancreatic duct and the bile duct, or (ii) Through the minor duodenal papilla into the accessory pancreatic duct or the dorsal pancreatic duct, and optionally further advanced into the main pancreatic duct.

59. The method according to claim 52, wherein, Inflate the balloon in the catheter near the entrance of the pancreatic duct that crosses the common bile duct to prevent liquid backflow.

60. The method according to claim 57, wherein, The maximum balloon size for sealing the pancreatic duct is 9 mm to avoid injury.

61. The method according to claim 52, wherein, Optionally use two or more flow rates during hydrodynamic injection to further minimize pancreatic tissue damage while maintaining gene delivery.

62. The method according to claim 52, wherein, For the first 50% of the injection volume, the flow rate is initially 1 mL / second, and then for the remaining injection volume, the flow rate is increased to 2 mL / second.

63. The method according to claim 52, wherein, For the first 50% of the injection volume, the flow rate is initially 0.5 mL / second, and then for the remaining injection volume, the flow rate is increased to 1.5 mL / second.

64. The method according to claim 52, wherein, Do not use a sidewall injection catheter to avoid wall damage and prevent pancreatitis.

65. A method for hydrodynamic injection into the gallbladder or liver, comprising: a) Place the catheter in the common bile duct; b) Inflate the balloon in the common bile duct to prevent forward blood flow; c) Inject the DNA solution into the biliary system at a high pressure target and / or flow rate, where gene expression immunostaining can be observed in hepatocytes in the liver and cells in the gallbladder.

66. The method according to claim 65, wherein, The high pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

67. The method according to claim 65, wherein, The flow rate is greater than 2 mL / second, greater than 5 mL / second, or greater than 10 mL / second.

68. The method according to claim 65, wherein, The injection volume is greater than 50 mL per kg of liver weight, or greater than 75 mL per kg of liver weight, or greater than 100 mL per kg of liver weight.

69. The method according to claim 65, wherein, Before injection, remove bile from the biliary system and rinse and wash the biliary system with saline.

70. The method according to claim 65, wherein, Optionally, a saline solution can be used to fill the gallbladder before injection to reduce the pressure difference during injection.

71. The method according to claim 65, wherein, The preferred DNA dose is at least 20 mg or more per kg of mouse body weight.

72. The method according to claim 65, wherein, The preferred DNA concentration of the injection solution is at least 0.5 mg / mL DNA or more.

73. A method for hydrodynamic injection through the biliary system, wherein, The injection is performed in the common bile duct with the assistance of a biliary stent.

74. The method according to claim 73, wherein, The biliary stent is placed prior to hydrodynamic injection.

75. The method according to claim 73, wherein, The bile duct stent is placed over the cystic duct to prevent fluid from entering the cystic duct.

76. The method according to claim 73, wherein, The diameter of the biliary stent is at least that of the bile duct to provide sufficient sealing between the wall of the duct and the stent during injection.

77. The method according to claim 73, wherein, The biliary stent has a variable length and can reach from the ampulla to the cystic duct.

78. The method according to claim 73, wherein, The balloon catheter is inserted into the stent after placement.

79. The method according to claim 78, wherein, The balloon catheter can be located at any position within the stent, including the common bile duct or the common hepatic duct.

80. The method according to claim 79, wherein, Prior to injection, a contrast agent is injected through the stent to confirm that the cystic duct and gallbladder are not visualized and that the biliary system becomes visualized.

81. The method according to claim 73, wherein, A DNA solution is injected with set parameters to mediate gene delivery to different cells within the liver.

82. The method according to claim 73, wherein, The preferred flow rate during injection is at least 1 mL / sec or at least 2 mL / sec.

83. The method according to claim 73, wherein, The preferred injection pressure is at least 50 mmHg or at least 80 mmHg.

84. The method according to claim 73, wherein, The preferred injection volume is at least 30, 40, 50, or 60 mL per kg of liver weight.

85. The method according to claim 73, wherein, The preferred DNA dose is at least 20 mg or more per kg of liver weight.

86. The method according to claim 73, wherein, The preferred DNA concentration of the injection solution is at least 0.5 mg / mL DNA or more.

87. The method according to claim 73, wherein, The stent is made of a solid material such that fluid cannot pass through the wall of the stent.

88. A method for delivering a non-viral DNA vector to a liver tumor, comprising the following steps: A catheter is placed into the biliary system, preferably the common bile duct; the balloon is inflated within the common hepatic duct to prevent forward blood flow; and the DNA solution is injected into the biliary system with hydrodynamic pressure wherein the injection enables the expression of a non-viral DNA vector within tumor cells regardless of the location of the tumor within the liver.

89. The method according to claim 88, wherein, The tumor is close to the biliary system to enable effective delivery.

90. The method according to claim 88, wherein, A pressure of at least 50 mmHg, 70 mmHg, or at least 120 mmHg is the target for effective tumor gene delivery.

91. The method according to claim 88, wherein, A flow rate of at least 2 mL / sec, 4 mL / sec, 7 mL / sec, or at least 10 mL / sec is utilized to achieve effective tumor gene delivery.

92. The method according to claim 88, wherein, The volume of injection is at least 30 mL per kg of liver weight.

93. The method according to claim 88, wherein, The non-viral DNA dose for injection is at least 10 mg per kg of liver weight or at least 20 mg per kg of liver weight.

94. The method according to claim 88, wherein, Gene expression in tumor cells is highest along the margin of the tumor.

95. A method for delivering a non-viral DNA vector to a pancreatic tumor, comprising the steps of: a) Place a catheter into the pancreatic duct system, upstream of the tumor; b) Inflate the balloon within the pancreatic duct to prevent forward flow; and c) Inject the DNA solution into the pancreatic duct system with hydrodynamic pressure, wherein the injection enables the expression of non-viral DNA vectors within tumor cells regardless of the location of the tumor within the pancreas.

96. The method according to claim 95, wherein, The tumor is close to the duct system to enable effective delivery.

97. The method according to claim 95, wherein, Control a pressure of at least 50 mmHg, 70 mmHg, or at least 120 mmHg to achieve effective tumor gene delivery.

98. The method according to claim 95, wherein, Control a flow rate of at least 1 mL / sec to achieve effective tumor delivery.

99. The method according to claim 95, wherein, Inject a volume of at least 8 mL into the pancreas of an adult.

100. The method according to claim 95, wherein, Inject a non-viral DNA dose of at least 1 mg into the pancreas of an adult.

101. The method according to claim 95, wherein, Gene expression in tumor cells is highest along the margin of the pancreatic tumor.

102. The method according to claim 95, wherein, This tumor delivery is most effective for pancreatic ductal adenocarcinoma.

103. A method for delivering a gene to the liver of a primate, comprising the steps of: Insert a catheter into the common hepatic duct of a primate; Inflate the balloon within the common hepatic duct to prevent forward blood flow; and Inject a DNA solution into the liver of a primate under hydrodynamic pressure, wherein the injection enables hepatocytes expressing a gene of interest in the primate liver to reach >30%.

104. The method according to claim 103, wherein, The common hepatic duct is accessed by endoscopic retrograde cholangiopancreatography (ERCP).

105. The method according to claim 104, wherein, The ampulla of Vater can be incised to increase the size of the opening, thereby facilitating the insertion of a common hepatic duct cannula during ERCP.

106. The method according to claim 104, wherein, Use a radiopaque agent injection to locate the catheter located in the common hepatic duct so that it crosses the cystic duct to avoid injection into the gallbladder.

107. The method according to claim 104, wherein, The radiopaque agent injection confirms that the balloon seals the common hepatic duct during injection, and the right and left hepatic ducts are visualized.

108. The method according to claim 103, wherein, The DNA solution is a physiological saline solution, wherein pure recombinant DNA is dissolved in the solution.

109. The method according to claim 103, wherein, The DNA in the DNA solution can be plasmid DNA, minicircle DNA or linear closed-ended DNA.

110. The method according to claim 103, wherein, The amount of the injection is at least 30 mL per kg of liver weight or at least 40 / mL / kg or more.

111. The method according to claim 103, wherein, The flow rate is at least 1 mL / second, at least 2 mL / second or 3 mL / second or faster.

112. The method according to claim 103, wherein, During pressure-guided injection, the pressure parameter is at least 50 mmHg, at least 80 mmHg or greater than 120 mmHg.

113. The method according to claim 103, wherein, In certain embodiments, the DNA dose will be at least 10, 20, 30, 40 or 50 mg per kg of liver weight.

114. The method according to claim 103, wherein, The DNA solution is a DNA vector composition encoding a hepatocyte-specific promoter.

115. The method according to claim 114, wherein, The hepatocyte-specific promoter further contains one or more hepatocyte-specific enhancers to drive higher levels of transcription.

116. The method according to claim 115, wherein, The gene of interest is optimized using codons selected for abundance in hepatocytes.

117. The method according to claims 103 to 116, wherein, Provide a DNA vector composition and protocol for treating hemophilia B in primates.

118. The method according to claim 117, wherein, The DNA vector composition encodes the human factor IX (hFIX) gene.

119. The method according to claim 117, wherein, The DNA vector composition is a nanoplasmid with a bacterial backbone of less than 500 base pairs.

120. The method according to claim 117, wherein, For hFIX, the total DNA vector composition size is less than 3 kb.

121. The method according to claim 117, wherein, A DNA vector composition dose of 20 mg per kg of primate liver is sufficient to produce 1000 ng / mL of hFIX in primate plasma.

122. The method according to claim 117, wherein, The DNA vector composition can be reused to ensure further expression.

123. The method according to claims 103 to 122, wherein, The procedure can be repeated a second time in primates, wherein the expression of two different genes is achieved.

124. A method for hydrodynamic gene delivery through the biliary system of a subject's liver, comprising: (a) Insert a catheter into the common hepatic duct (b) Inflate the balloon to seal the catheter and increase the pressure during injection (c) The injection flow rate is at least 2 mL / second or the minimum pressure is 50 mmHg (d) Deliver DNA encoding a hepatocyte-specific promoter to drive transgene expression (e) A DNA vector with significantly reduced or absent non-mammalian sequence elements (f) The injected DNA dose is at least 10 mg DNA per kg of liver weight, wherein more than 50% of the hepatocytes express the gene of interest.

125. The method according to claim 124, wherein, Optionally, the DNA vector lacks specific modifications, but the optimal injection amount is at least 20 mg DNA per kg of liver weight to achieve more than 50% of the hepatocytes expressing the gene of interest.

126. The method according to claim 124, wherein, The optional use of transposons can be utilized to facilitate integration into the host chromosome.

127. The method according to claim 124, wherein, The average total amount of bacterial or phage DNA sequences less than 1000 bp is significantly reduced.

128. The method according to claim 124, wherein The DNA is a plasmid DNA vector having a carrier bacterial backbone or sequence, and the size of the carrier bacterial backbone or sequence is less than 1 kb or more preferably less than 500 bp.

129. The method according to claim 128, wherein The plasmid DNA is a nanoplasmid, pFAR or pCOR vector.

130. The method according to claim 124, wherein The DNA is circular and is microcircular DNA.

131. The method according to claim 124, wherein The DNA is linear DNA from closed-ended DNA, minicircle DNA or dumbbell DNA.

132. The method according to claim 124, wherein Conversion from a plasmid backbone comprising a bacterial sequence greater than 1 kb to the DNA vectors of claims 5 and 6 results in an increase in the total transfected area of hepatocytes observed by more than 20%.

133. The method according to claim 124, wherein Integration into the host genome using a transposon system enables gene expression in at least 40% of hepatocytes for at least 3 months.

134. The method according to claim 124, wherein The expression duration of the non-integrating DNA of claims 5 and 6 is at least 4 months after injection.

135. The method according to claim 124, wherein The delivery method expresses for at least 4 months and is capable of generating immune tolerance to foreign transgenes in the liver.

136. The method according to claim 124, wherein DNA vectors greater than 12 kb, 15 kb or 20 kb in size can be delivered to multiple cell types in the liver, including hepatocytes, endothelial cells and cholangiocytes, resulting in protein expression.

137. The method according to claim 136, wherein Larger plasmid DNA with a size of at least 12 kb is used to maintain transfection efficiency.

138. The method according to claim 136, wherein The DNA dose can be adjusted according to the DNA dose (mg) per kilobase DNA per liver weight (kg) to adjust the DNA size, thereby maintaining equivalent transfection efficiency.

139. The method according to claim 136, wherein The formula 1 mg / kg / kb can be used to predict the DNA dose to achieve transfection of approximately 50% of hepatocytes in the liver.

140. The method according to claim 136, wherein The formula of 2.5 to 5 mg / kg / kb can be used to predict the DNA dose to achieve transfection of approximately 70% of hepatocytes in the liver.

141. The method according to claim 124, wherein This procedure can be repeated with different DNA expressing the same or different genes a second time, such that the expression of the first gene is not eliminated and now the expression of two genes is achieved.

142. The method according to claim 132, wherein The second injection achieves a transfection efficiency similar to the first injection and can target the same cells.

143. The method according to claim 132, wherein Gene expression in the same cells can be observed after injection.

144. The method according to claim 132, wherein The promoter can be altered to utilize the second injection to achieve expression in different cell types, and the second injection does not alter the expression of the first gene.

145. The method according to claim 124, wherein This process can be repeated with different DNA expressing the same or different genes during the same injection procedure, such that the expression of two DNAs is now achieved and the expression of the first DNA injection is not eliminated.

146. The method according to claim 136, wherein The second injection achieves a transfection efficiency similar to the first injection and can target the same cells.

147. The method according to claim 136, wherein The promoter can be altered to utilize the second injection to achieve expression in different cell types, and the second injection does alter the expression of the first gene.

148. The method according to claim 124, wherein Two different DNA molecules can be mixed and delivered during a single injection procedure such that the two DNA molecules enter the same hepatocytes.

149. The method according to claim 124, wherein, DNA doses from 20 mg per kg liver weight to 40 mg per kg liver weight achieve similar transfected areas.

150. The method according to claim 124, wherein, DNA can be injected at a dose of up to 40 mg per kg liver weight without causing significant hepatotoxicity or physiological distress.

151. The method according to claim 124, wherein, Flow rates below 1 mL / sec do not result in gene expression.

152. The method according to claim 124, wherein, Flow rates between 1 mL / sec and 2 mL / sec exhibit reduced gene expression compared to flow rates greater than 2 mL / sec.

153. The method according to claim 124, wherein, Flow rates above 4 mL / sec result in a gradual decrease in hepatocyte delivery efficiency.

154. The method according to claim 124, wherein, Flow rates greater than or equal to 7 mL / sec enable effective gene delivery into cholangiocytes.

155. The method according to claim 124, wherein, The preferred injection volume is between 30 mL / kg and 60 mL / kg per liver tissue.

156. The method according to claim 124, wherein, Injection volumes greater than or equal to 70 mL / k liver tissue are associated with reduced gene delivery efficiency.

157. The method according to claim 124, wherein, This gene injection procedure is well-tolerated in subjects weighing 25 kg or 15 kg or 5 kg and produces gene delivery efficiency similar to that of larger mammals.

158. The method according to claim 124, wherein, When combining two or more flow rates during injection, the transfected hepatocyte area of bile hydrodynamic delivery can further increase by at least 10% of the total hepatocytes.

159. The method according to claim 149, wherein, First, inject 50% to 66% of the total injection volume at a flow rate of 2 mL / sec, and then inject the remaining volume at a flow rate of 4 mL / sec.

160. The method according to any one of the above claims, wherein, First, inject 33% of the volume at a flow rate of 2 mL / sec, inject the second 33% of the volume at a flow rate of 3 mL / sec, and then inject the remaining volume at a flow rate of 4 mL / sec.

161. The method according to claim 124, wherein, The catheter is inserted into the common hepatic duct via ERCP, EUS, or an image-guided percutaneous approach.

162. The method according to claim 124, wherein, The DNA concentration of the injection solution is at least 0.30 mg / mL, and a more preferred concentration is greater than 0.40 mg / mL, 0.50 mg / mL, or 0.60 mg / mL.

163. A method for achieving expression in liver sinusoidal endothelial cells (LSECs), comprising hydrodynamic injection into the biliary tract according to claim 1, except for targeted expression in LSECs using a cell-specific promoter.

164. The method according to claim 163, wherein, LSEC can be expressed using a CD36 promoter or an FVIII promoter.

165. The method according to claim 124, wherein, An injection pressure of 80 mmHg produces more effective expression than an injection pressure of 50 mmHg.

166. The method according to claim 124, wherein, Pressures between 150 and 200 mmHg produce effective gene expression.

167. The method according to claim 124, wherein, Pressures above 200 mmHg result in a gradual decrease in gene expression.

168. A method for hydrodynamic gene delivery through the biliary system of a primate liver, comprising: (a) Insert the catheter into the common hepatic duct, right hepatic duct, or left hepatic duct (b) Inflate the balloon to seal the catheter and increase pressure during injection (c) Inject at a flow rate of at least 2 mL / sec or inject at a minimum pressure of 50 mmHg (d) Deliver DNA encoding a hepatocyte-specific promoter to drive transgene expression (e) A DNA vector with significantly reduced or absent non-mammalian sequence elements (f) The injected DNA dose is at least 10 mg DNA per kg liver weight, where more than 50% of the hepatocytes express the gene of interest.

169. The method according to claim 168, wherein, More effective balloon sealing is obtained by advancing the catheter and inflating the balloon into the intrahepatic duct.

170. The method according to claims 168 and 169, wherein, Placing the balloon in the extrahepatic duct results in leakage of the fluid around the balloon.

171. The method according to claims 168 to 170, wherein, The balloon size is at least 2 times, at least 3 times, or at least 4 times the diameter of the catheter.

172. The method according to claim 171, wherein, When the balloon is placed in the extrahepatic bile duct, the balloon can be inflated to a maximum size of 3 times the diameter of the catheter.

173. The method according to claim 171, wherein, When the balloon is placed in the intrahepatic bile duct, the balloon can be inflated to a minimum size of 4 times the diameter of the catheter.

174. The method according to claim 173, wherein, The balloon size does not fully inflate within the intrahepatic duct, but generates additional pressure within the balloon.

175. The method according to claims 168 to 170, wherein, Leakage around the balloon occurs when the size is 8.5 mm or smaller, whether intrahepatic or extrahepatic, so these sizes should be avoided.

176. The method according to claims 168 to 170, wherein, The balloon is inflated in the common hepatic duct to a size less than 15 mm to avoid rupture.

177. The method according to claim 168, wherein, If the balloon is placed in the right hepatic duct or the left hepatic duct and subsequent injection is performed at that location, injection is repeated again in the opposite duct to ensure equal injection of both lobes of the liver.

178. The method according to claim 168, wherein, Balloon sealing can be monitored by measuring the intraluminal bile duct pressure.

179. The method according to claim 168, wherein, Disappearance of the plateau waveform (defined as a decrease of more than 20 mmHg from the start to the end of the plateau) indicates a leak around the balloon.

180. The method according to claim 168, wherein, Balloon sealing can be verified by filling the bile ducts above and below the balloon with a radiopaque agent solution prior to injection.

181. The method according to claim 180, wherein, Loss of fluid seal during injection is demonstrated by clearance of the agent below the balloon, which enters the cystic duct and gallbladder or enters the common bile duct.

182. The method according to claims 180 to 181, wherein, Clearance of the agent above the balloon into the liver indicates successful injection.

183. The method according to claim 168, wherein, Hydrodynamic injection can be repeated multiple times in a single procedure to enhance DNA delivery.

184. The method according to claim 183, wherein, Use of two or more injections can be cumulative to achieve the final gene expression level.

185. The method according to claims 183 to 184, wherein, This strategy allows overcoming the inherent limitations of angiography or kinetic injection volumes by using multiple injections.

186. The method according to claim 168, wherein, Primate liver tissue is more elastic than porcine tissue, so different catheter characteristics require changing balloon size and injection parameters to mediate gene delivery in primates.

187. The method according to claim 186, wherein The flow rate must be increased to achieve a given pressure and injection in the porcine model.

188. The method according to claim 187, wherein A flow rate of at least 4 mL / second is required to achieve a plateau pressure of at least 80 mmHg.

189. The method according to claim 168, wherein Primates can tolerate a pDNA dose of at least 80 mg without any significant physiological side effects.

190. The method according to claim 168, wherein An injection volume of at least 120 mL per 400 grams of liver can be injected into primates without any significant perturbation of vital signs.

191. The method according to claim 168, wherein The primate liver can tolerate an injection rate of up to 12 mL / second without causing tissue damage, change in vital signs, or bile duct rupture.

192. The method according to claim 168, wherein A flow rate greater than 4 mL / second but less than 8 mL / second should be used because there is a lack of improved gene delivery at higher flow rates.

193. The method according to claim 168, wherein A volume of 30 mL per 400 g or up to 150 mL per 400 g volume can be used.

194. The method according to claims 192 and 193, wherein The volume used does not affect the gene delivery efficiency of a given DNA dose.

195. The method according to claim 168, wherein The vector composition should be dosed by the copy number of the transgene expression cassette, so that if additional foreign DNA is included in the DNA vector, different pDNA doses are required.

196. The method according to claim 195, wherein Using DNA molecules with a reduced backbone allows for relatively small DNA doses.

197. The method according to claim 168, wherein Increasing the pDNA dose and / or the vector expression cassette dose per animal results in a quantitatively equivalent increase in the expression of the protein therapeutic of interest.

198. A method for hydrodynamic gene delivery through the biliary system of a subject's liver, comprising: (a) Insert the catheter into the bile duct: (b) Inflate the balloon to seal the bile duct and prevent antegrade flow of the solution i. The size of the balloon after inflation is at least 2 times, 3 times, or 4 times the diameter of the bile duct to overcome the elasticity of the primate bile duct; ii. Wherein, Methods for verifying balloon seal during injection include placing a radiopaque agent solution above and below the balloon and detecting antegrade movement of the fluid by fluoroscopy (c) Inject at a flow rate of at least 2 mL / second or at a pressure of at least 50 mmHg i. More preferably, the minimum is 4 mL / sec, which can generate at least 80 mmHg of pressure in the primate liver under the plateau pressure; wherein, the flow rate can be further minimized to less than 12 mL / sec, less than 10 mL / sec, or less than 8 mL / sec without loss of total gene expression; ii. The injection volume is preferably less than 250 mL / kg of liver tissue, less than 150 mL / kg of liver tissue, or less than 50 mL / kg of liver tissue; iii. wherein, the plateau pressure obtained during the hydrodynamic injection process changes by less than 10% mmHg during the injection and gives a signal of sufficient sealing; and iv. wherein, multiple flow rates can be arbitrarily adopted in a single injection to change the obtained pressure; and (d) Delivering a micro DNA vector, wherein non-mammalian sequence elements are significantly reduced or absent i. wherein, the miniaturized DNA vector can provide a longer expression persistence, at least lasting for 4 months; ii. wherein, the micro DNA vector provides high potency on a given DNA For primates, compared with conventional plasmid DNA, a smaller dose can be used; iii. wherein, the DNA vector preferably contains a hepatocyte-specific promoter to enhance the expression in the liver.

199. The method according to claim 198, wherein The average total amount of bacterial or phage DNA sequences less than 1000 bp is significantly reduced.

200. The method according to claim 198, wherein, The DNA is a plasmid DNA vector with a vector bacterial backbone or sequence, and the size of the vector bacterial backbone or sequence is less than 1 kb or more preferably less than 500 bp.

201. The method according to claim 200, wherein, The plasmid DNA is a nano plasmid, GenCircle, pFAR or pCOR vector.

202. The method according to claim 198, wherein, The DNA is circular and is a microcircular DNA or a microvector DNA.

203. The method according to claim 198, wherein, The DNA is linear DNA from closed-end DNA, small-chain DNA or dogbone DNA.

204. The method according to claim 203, wherein, The linear DNA has only small foreign sequences at either end, each sequence being less than 100 bp in size, and the mammalian expression sequence of interest is the remaining part of the vector.

205. The method according to claim 198, wherein, During the first surgery of the subject, sphincterotomy of the biliary tract is performed to reduce or eliminate the risk of post-ERCP pancreatitis when re-administering genetic drugs during subsequent ERCP procedures.

206. The method according to claim 198, wherein, The injection procedure can be repeated twice within a course of treatment to increase protein expression.

207. The method according to claim 198, wherein, Repeated injections can use the same DNA to promote the expression of a single protein, or can use two different DNA solutions to produce the expression of two different proteins.

208. The method according to claim 198, wherein, The total sum of the DNA doses for repeated injections is similar or equal to the expression obtained from a single DNA injection.

209. The method according to claim 198, wherein, After a single administration, the injection procedure can be repeated again such that the transfection efficiency is the same, and the peak protein expression is equivalent between injections, and no immunogenicity is observed.

210. The method according to claim 209, wherein, The repeated injection procedure can be performed at intervals of at least one month, at least 3 months, at least 6 months, or at least one year.

211. The method according to claim 198, wherein, Use a pressure sensor to detect the liquid-filled column in the pressure catheter to monitor the pressure, or use a pressure sensor screwed into the catheter lumen to monitor the pressure.

212. The method according to claim 198, wherein, Immediately record the pressure sensor readings and interpret the pressure curve after injection to determine whether successful sealing and peak expression are achieved.

213. The method according to claim 198, wherein, Optimal hydrodynamic injection results in at least a two-fold increase in hepatic enzymes (e.g., ALT and AST levels) on day 1 post-injection compared to pre-treatment values.

214. The method according to claim 198, wherein, Hepatic enzymes return to the normal range within 7 days post-injection.

215. The method according to claim 198, wherein, Gene delivery occurs within and around the tumor, in malignant and normal cells, scattered between and around the tumors.

216. A method for hydrodynamic injection into the liver through the biliary system, using a partially deployed full-coverage metal or plastic stent, wherein: a) The stent is deployed from the distal end to the proximal end (relative to the stent deployment catheter) b) The released or opened part of the stent is located within the common hepatic duct, and the tip of the stent remains within the delivery catheter that houses the stent, and thus is located within the common hepatic duct, the common bile duct, or the ampulla of Vater c) wherein, The cystic duct orifice is blocked and / or bypassed by the covered portion of the stent such that the injected fluid solution cannot enter the cystic duct or the gallbladder. d) Remove the guide wire from the stent delivery system. e) Inject a DNA solution at a high pressure target and / or flow rate through the guide wire lumen into the biliary system. f) wherein Due to the partial deployment and continuous connection of the stent, a closed system is formed with the catheter, thereby preventing antegrade flow in the biliary system. wherein gene expression can be observed in hepatocytes within the liver on immunostaining.

217. The method according to claim 216, wherein, The high pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

218. The method according to claim 216, wherein, The flow rate is greater than 2 mL / sec, greater than 5 mL / sec, or greater than 10 mL / sec.

219. The method according to claim 216, wherein, The injected volume is greater than 50 mL per kg of liver weight, greater than 75 mL per kg of liver weight, or greater than 100 mL per kg of liver weight.

220. The method according to claim 216, wherein, Prior to injection, bile is removed from the biliary system and the biliary system is flushed and cleaned with saline.

221. The method according to claim 216, wherein, The preferred DNA dose is at least 20 mg or more per kg of liver weight.

222. The method according to claim 216, wherein, The preferred DNA concentration of the injection solution is at least 0.5 mg / mL DNA or more.

223. The method according to claim 216, wherein, The diameter of the biliary stent is at least the diameter of the bile duct to provide sufficient sealing for the tube wall and the stent during injection.

224. The method according to claim 223, wherein, The diameter of the biliary stent is at least 150% of the diameter of the bile duct or at least 200% of the diameter of the bile duct.

225. The method according to claim 216, wherein, The biliary stent has a variable length and can reach upstream of the cystic duct from outside the ampulla.

226. The method according to claim 216, wherein, Injection is performed from the olive tip of the stent, and during injection, the "olive body" may be located between the hepatic end of the stent and the porta hepatis.

227. The method according to claim 216, wherein, Injection occurs at the opening on the catheter, which is located at the proximal position of the catheter into which the stent is inserted, such that the fluid fills the stent during retrograde flow, and the cone of the stent entering the catheter prevents any forward flow.

228. The method according to claim 216, wherein, Prior to injection, a contrast agent is injected through the stent to confirm that the cystic duct and the gallbladder are not visualized and the biliary system becomes opaque.

229. The method according to claim 216, wherein, The stent is made of a solid material such that fluid cannot pass through the wall of the stent.

230. The method according to claim 216, wherein, The hepatic end of the stent opens in the left or right main hepatic duct, rather than in the common hepatic duct.

231. The method according to claim 230, wherein, Once hydrodynamic injection is completed from the left or right main hepatic duct, an alternative catheter is injected using the same technique.

232. The method according to claim 216, wherein, The stent is partially deployed, which means that in some embodiments, at most 95% of its length, 75% of its length, 50% of its length, or 25% of its length is deployed outside the catheter, while the remaining length of the stent remains inside the catheter or attached to the catheter.

233. The method according to claim 216, wherein, The partially deployed stent forms a funnel or conical shape at its proximal end where it is connected to the catheter, thereby forming a closed system.

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