Methods and compositions for hydrodynamic gene delivery

Abstract

In certain embodiments, the present disclosure relates to a method for pressure-induced hydrodynamic injection into tissues such as the kidney, pancreas, liver, and common bile duct for the delivery of nucleic acids or viral vectors, including optimal pressures that mediate gene delivery and expression in a subject during such hydrodynamic injection. A method is described that overcomes interindividual variability to achieve target pressures during the hydrodynamic procedure while avoiding tissue damage, rupture, and tearing, while reducing the toxicity of hydrodynamic injection and other issues associated with therapeutic delivery.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Applications Nos. 63 / 374,228, 63 / 374,231, 63 / 374,234, 63 / 374,058, 63 / 374,070, 63 / 374,073, and 63 / 374,216, filed August 31, 2022. The entire contents of the aforementioned patent applications are incorporated herein by this reference.

[0002] Field The present disclosure relates to compositions and methods for hydrodynamic gene delivery. [Background technology]

[0003] The technical challenge concerns how to know what parameters to inject into a patient for gene delivery, given that every patient may have differences in their underlying disease state. Gene delivery to specific tissue types holds therapeutic promise.

[0004] The kidneys are bodily organs that play a vital role in removing toxins from the body and regulating the volume, osmolality, acid-base balance, and electrolyte concentrations of various bodily fluids. Disruptions in renal function lead to a wide variety of rare genetic kidney diseases, including cystinuria, adult-onset polycystic kidney disease, nephrogenic diabetes insipidus, Gitelman syndrome, Fabry disease, thin basement membrane disease, Lowe syndrome, hereditary interstitial kidney disease, tuberous sclerosis, nephronophthisis, and Alport disease. In addition to rare kidney diseases, common disorders such as chronic kidney disease, end-stage renal disease, and immune-mediated glomerular disorders are also of great importance. Kidney disease (also known as renal disease) generally manifests as damage to the kidney. For example, nephritis is an inflammatory kidney disease that can be divided into several categories depending on the location of inflammation. Another example is nephrosis, a non-inflammatory kidney disease that can result in either nephritis or nephrotic syndrome. Kidney disease usually leads to a decline in kidney function. Complete loss of kidney function is known as end-stage kidney disease and can only be treated by dialysis or kidney transplantation. Gene therapy approaches could be used in the treatment of kidney autoimmune, inflammatory, metabolic, toxic, precancerous, or cancerous conditions.

[0005] One strategy for treating kidney disorders is gene therapy, which can deliver the gene that is found to be missing or present at insufficient levels in kidney cells.In addition, some clinical prevention strategies or disorders can benefit from the addition of gene as a therapeutic approach.However, to date, the gene therapy strategy for treating or preventing kidney genetic diseases has not been successfully implemented.Therefore, there is an urgent need for a gene therapy protocol that can be implemented in the kidney to treat and / or prevent kidney genetic diseases, autoimmune diseases, inflammatory diseases, fibrotic diseases, metabolic diseases, toxic diseases, precancerous diseases or malignant diseases.

[0006] The pancreas is an important exocrine and endocrine organ of the body that plays a role in food digestion and energy storage. Disruptions in pancreatic function lead to a wide variety of disorders, such as cystic fibrosis, hereditary pancreatitis, autoimmune pancreatitis, and several different types of diabetes, including, but not limited to, type I and type II diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes in adults (LADA), neonatal diabetes, Wolfram syndrome, and Alström syndrome. For example, the pancreas plays an important role in the pathogenesis of a wide range of diseases, such as type I and type II diabetes, which are caused by disrupted or dysregulated insulin production. One strategy for treating pancreatic disorders is gene therapy, which may deliver genes found to be defective in pancreatic cells or by transforming pancreatic cells. Gene therapy can also be used to deliver proteins that can combat genetic diseases, early neoplasia, cancer, pancreatic pain, pancreatitis, or autoimmune diseases. Target cells of interest in the pancreas include, but are not limited to, pancreatic acinar cells, pancreatic ductal cells, pancreatic islet cells, pancreatic endothelial cells, and pancreatic neural cells.

[0007] Various strategies have been attempted for pancreatic gene therapy. For example, viral strategies have used systemic or targeted therapy via vascular routes, while other studies have demonstrated the feasibility of injecting viruses via the pancreatic duct. Unfortunately, viral vectors, such as AAV, have a significant size limit (approximately 4.8 kb) for the nucleotide sequences they can package. Non-viral strategies have also been used by utilizing hydrodynamic injection through the pancreatic artery in rats. Unfortunately, these non-viral strategies have not been translated into 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 this genetic disease of the pancreas.

[0008] The biliary system is a promising route for gene delivery to the liver. Hydrodynamic gene delivery can be achieved through the biliary system, with high efficiency achieved from the common hepatic duct. Unfortunately, delivery from other locations in the biliary system has not been successful. Therefore, additional administration routes for hydrodynamic gene delivery are needed.

[0009] Gene therapy is one of the therapeutic options for treating cancer. Gene therapy can deliver many different types of treatments directly to tumors to help eliminate them, but current delivery systems fail to deliver them efficiently. Non-viral delivery is particularly limited in its effectiveness within tumors.

[0010] The liver is a major organ that performs many essential biological functions, such as detoxification and the synthesis of proteins and biochemicals. It also plays an important role in metabolism, including regulating glycogen storage, red blood cell breakdown, glucose and lipid metabolism, and hormone production. Disturbances in liver function can lead to a variety of rare genetic disorders, such as Wilson's disease, phenylketonuria, hemophilia A, hemophilia B, and progressive familial intrahepatic cholestasis. Disturbances in liver function can also lead to various autoimmune disorders, such as autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis. Furthermore, invasive diseases can affect the liver, including amyloidosis and malignant tumors such as hepatocellular carcinoma or metastatic colorectal cancer. In addition, various metabolic diseases, such as nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, can also affect the liver. There are numerous liver diseases that could potentially be treated by gene therapy. These include: hemophilia A, hemophilia B, alpha-1 antitrypsin deficiency, Wilson's disease, hereditary tyrosinemia, PFIC (progressive familial intrahepatic cholestasis), hereditary hemochromatosis, Crigler-Najjar disease, familial hypercholesterolemia, acute hepatic porphyria, acute intermittent porphyria, von Willebrand's disease, primary hyperoxaluria, atypical HUS, phenylketonuria, maple syrup urine disease, methylmalonic acidemia, propionic acidemia, NAGS deficiency, CPS I deficiency, OTC deficiency, argininosuccinic aciduria, argininemia, citrullinemia, Fabry disease, MPS, Pompe disease, GSD1a, and cystathionine beta-synthase deficiency. To describe two examples in more detail, two common inherited liver diseases are hemochromatosis and alpha-1 antitrypsin deficiency.

[0011] Hemochromatosis is a disease in which iron deposits accumulate in the liver and other organs. The primary form of the disease is one of the most common genetic disorders in the United States, with an estimated prevalence of the disease in the U.S. population of as high as 1 in 200. Unfortunately, many people who carry genetic risk factors for hemochromatosis are unaware that they are carriers.

[0012] Alpha-1 antitrypsin deficiency is a genetic liver disease that affects a critical liver protein known as alpha-1 antitrypsin, which is either completely absent or present at very low levels in affected individuals. Although people with alpha-1 antitrypsin deficiency are often able to produce the protein, it is unable to enter the bloodstream and instead accumulates in the liver. The alpha-1 antitrypsin protein plays an important role in protecting the lungs from enzymatic damage. Furthermore, people with this disease are at risk of developing cirrhosis.

[0013] Various strategies have been attempted for liver gene therapy. For example, viral strategies have used systemic or targeted therapy via the vascular route, while other studies have demonstrated the feasibility of injecting viruses via the bile duct. Unfortunately, viral vectors, such as AAV, have a significant size limit (approximately 4.8 kb) for the nucleotide sequence they can package. Non-viral strategies have also been used by utilizing hydrodynamic injection via the bile duct in rats. Unfortunately, these non-viral strategies have not been effectively translated into large animal models.

[0014] One strategy for treating liver damage is gene therapy, which can be thought of as being able to deliver the gene that is found to be missing or present at insufficient levels in liver cells.However, to date, the gene therapy strategy for treating or preventing liver genetic disease has not been successfully implemented.Therefore, there is an urgent need for a gene therapy protocol that can be implemented in the liver to treat and / or prevent this gene-based liver disease. Summary of the Invention

[0015] In certain aspects, the present disclosure provides methods for pressure-induced hydrodynamic infusion, including optimal pressures to mediate gene delivery and expression in humans during biliary hydrodynamic infusion. Methods are described that eliminate individual variability in achieving target pressures during hydrodynamic procedures.

[0016] The present disclosure also relates to compositions and methods for treating kidney disease. More particularly, the present disclosure relates to compositions and methods for treating kidney disease by gene therapy. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that ureteral hydrodynamic injection can mediate renal cortical rupture at a variety of different flow rates and volumes, and as a result, only limited parameters are observed to be safe.

[0017] The present disclosure further relates to compositions and methods for treating diseases of the pancreas. More particularly, the present disclosure relates to compositions and methods for treating diseases of the pancreas by gene therapy. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that the volume of fluid required for sufficient gene expression is less than the 20 mL previously published as sufficient.

[0018] The present disclosure also provides a method of hydrodynamic gene injection proceeding from the common bile duct, which improves upon previous strategies of injecting via the common bile duct by providing optimal flow rates and pressures to mediate gene expression within hepatocytes in the liver.

[0019] The present disclosure also provides methods for delivery to liver and pancreatic tumors.These methods include the use of hydrodynamic injection through the bile duct as a method for gene delivery into tumor microenvironment.Methods are disclosed regarding the procedure parameters and effective DNA dose for tumor injection.

[0020] The present disclosure further provides a method for mediating efficient gene delivery to the liver of a primate. The method comprises the step of performing hydrodynamic delivery to the biliary system of the liver of a primate. Optimal injection parameters are described that mediate efficient gene delivery to the liver of a primate while reducing side effects. Pressures and administration paradigms that mediate gene delivery to the liver of a primate are also described.

[0021] The present disclosure also relates to compositions and methods for hydrodynamic gene delivery to the liver via the biliary system. More particularly, the present disclosure relates to compositions and methods for increasing the efficiency of hydrodynamic gene delivery via the biliary system. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that by modifying a DNA vector so that the bacterial sequence on the plasmid DNA is significantly reduced to less than 500 base pairs, the observed transfected area of ​​hepatocytes is significantly increased to more than 70% of hepatocytes, an increase from less than 50% when using conventional plasmids.

[0022] In one aspect, the present disclosure provides a method for determining a flow rate for hydrodynamic injection into an organ, the method comprising: conducting a test injection; measuring the pressure during the test injection; and Empirically assessing stiffness and resistance differences The present invention provides a method comprising:

[0023] In an exemplary embodiment, the organ is selected from the liver, pancreas, or kidney.

[0024] In an exemplary embodiment, the liver is infused via the biliary system.

[0025] In an exemplary embodiment, the pancreas is infused via the ductal system.

[0026] In an exemplary embodiment, the kidney is infused via the urinary system.

[0027] In an exemplary embodiment, the pressure sensor would be inserted into the catheter through a dedicated lumen.

[0028] In an exemplary embodiment, the pressure sensor is already present in the catheter.

[0029] In an exemplary embodiment, the pressure sensor is a transducer attached to the fluid-filled lumen for pressure sensing.

[0030] In an exemplary embodiment, the method further includes the steps of taking a baseline reading of pressure in the biliary system without inflating the balloon; inflating the balloon and taking a pressure measurement in the biliary system; inflating the balloon and injecting a test solution or nucleic acid into the duct or vessel of interest that does not contain plasmid DNA; and monitoring the pressure during injection of the test solution.

[0031] In an exemplary embodiment, the test solution has the same osmolarity, osmolality, and viscosity as the DNA injection solution.

[0032] In an exemplary embodiment, the test solution does not further contain any other active drug substances contained in the therapeutic DNA solution.

[0033] In exemplary embodiments, the pressure achieved during hydrodynamic injection is at least 50 mmHg, at least 80 mmHg, or at least 120 mmHg.

[0034] In exemplary embodiments, the test flow rate to the liver is initially at least 2 mL / sec, or at least 3 mL / sec, or at least 4 mL / sec.

[0035] In exemplary embodiments, the total test volume is at most 15 mL, at most 10 mL, at most 7 mL, or at most 5 mL.

[0036] In an exemplary embodiment, the total test volume is sufficient to measure the column of total fluid resistance in the entire circuit to estimate whether sufficient pressure has been achieved.

[0037] In an exemplary embodiment, if sufficient pressure is not achieved, the flow rate is increased and the test is repeated.

[0038] In an exemplary embodiment, if a pressure above 250 mmHg is achieved, the flow rate is reduced and the test is repeated.

[0039] In an exemplary embodiment, the flow rate is increased or decreased by 1 mL / sec, or increased or decreased by 0.5 mL / sec for the second test.

[0040] In an exemplary embodiment, injection of the DNA solution proceeds at an established programmed flow rate that will produce the appropriate pressure.

[0041] In an exemplary embodiment, a series of flow rates are performed during one single test injection, and multiple flow rates are tested and pressures are measured throughout the single test injection.

[0042] In an exemplary embodiment, at least two or more test flow rates are tested in a single test injection.

[0043] In an exemplary embodiment, the pressure can be correlated to which flow rate caused the increase.

[0044] In exemplary embodiments, the total infusion volume is increased up to a total volume of 40 mL, 30 mL, or 20 mL to test all of the infusion parameters.

[0045] In exemplary embodiments, the minimum pressure for efficient hydrodynamic gene delivery is greater than 50 mmHg, or greater than 80 mmHg, or greater than 100 mmHg.

[0046] In exemplary embodiments, the maximum pressure for efficient hydrodynamic gene delivery is less than 200 mmHg or less than 250 mmHg.

[0047] In one aspect, the present disclosure provides a method for hydrodynamic retrograde ureteral delivery of a nucleic acid or viral vector, comprising: (i) inserting a cystoscope through the urethra into the bladder; (ii) inserting a balloon catheter into the bladder through a cystoscope; (iii) cannulating the ureteral orifice with a balloon catheter; (iv) inflating a balloon catheter in the distal ureter near the ureteral orifice entrance; (v) using an autoinjector to mediate hydrodynamic injection into the kidney; wherein the method does not require the use of fluoroscopy for catheter placement.

[0048] In an exemplary embodiment, kidney rupture or laceration is avoided by using a total volume equal to or less than 20 mL and injection parameters of 2 ml / sec.

[0049] In an exemplary embodiment, a flow rate equal to or between 0.5 mL / sec and 2 mL / sec is optimal to achieve gene delivery without manifesting bursting.

[0050] In an exemplary embodiment, the optimal volume for injection is 10 mL to 20 mL to mediate effective gene delivery without bursting.

[0051] In an exemplary embodiment, the balloon is placed in the muscle wall of the bladder within the ureter and can be visualized by a cystoscope camera without the need for fluoroscopy.

[0052] a. The method of claim 5, wherein a cystoscope camera can monitor for outflow from the ureter during injection, with a complete absence of outflow indicating an effective seal during injection.

[0053] In exemplary embodiments, the nucleic acid is administered at a minimum dose of 1 mg, 2 mg, 3 mg, 4 mg or more by mass to each kidney in a subject weighing 30 kg or more.

[0054] In an exemplary embodiment, the hydrodynamic parameters are sufficient to achieve protein expression within the cells of the kidney following delivery of the DNA.

[0055] a. The method of claim 7, wherein cellular expression is achieved within glomerular, tubular, or endothelial cells within the kidney.

[0056] b. The method of claim 7, wherein differences in cellular expression between glomeruli, tubules, or endothelium correlate with the use of multiple different promoters in the plasmid DNA.

[0057] In one aspect, the present disclosure provides a method for hydrodynamic retrograde ureteral delivery of a nucleic acid or viral vector, comprising: (i) inserting a cystoscope through the urethra into the bladder; (ii) inserting a guidewire into the bladder through the cystoscope; (iii) cannulating the ureteral orifice with a guidewire; (iv) advancing a guidewire toward the kidney; (v) removing the cystoscope and replacing it with a balloon catheter over the guidewire; (vi) inflating a balloon catheter within the ureter at a proximal location near the kidney; (vii) using an automatic 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 a cystoscope.

[0058] In an exemplary embodiment, kidney rupture or laceration is avoided by using injection parameters equal to or less than 12 mL and 2 ml / sec.

[0059] In an exemplary embodiment, a flow rate equal to or between 0.5 and 2 mL / sec is optimal to achieve gene delivery without bursting.

[0060] In an exemplary embodiment, the optimal volume for injection is 7 mL to 12 mL to mediate effective gene delivery without bursting.

[0061] In an exemplary embodiment, the balloon is placed within the ureter at least 1 cm, 2 cm, or 3 cm from the renal pelvis to ensure a proper seal.

[0062] In an exemplary embodiment, radiocontrast injection is utilized to verify catheter alignment and balloon seal prior to injection.

[0063] In exemplary embodiments, the nucleic acid is administered at a minimum dose of 1 mg, 2 mg, 3 mg, 4 mg or more by mass to each kidney in a subject weighing 30 kg or more.

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

[0065] a. The method of claim 15, wherein cellular expression is achieved within glomerular, tubular, or endothelial cells within the kidney.

[0066] b. The method of claim 15, wherein differences in cellular expression between glomeruli, tubules, or endothelium correlate with the use of multiple different promoters in the plasmid DNA.

[0067] In exemplary embodiments, the nucleic acid consists of plasmid DNA, minicircle DNA, mRNA, siRNA, or antisense oligonucleotides.

[0068] In exemplary embodiments, the viral vector is selected from an adenovirus, an adeno-associated virus, a lentivirus, a baculovirus, anellovirus, or a Sindbis virus.

[0069] In an exemplary embodiment, hydrodynamic injection is used to facilitate better penetration of viral vectors into tissues, cell binding, and cellular internalization compared to viral vectors injected at non-hydrodynamic flow rates.

[0070] In an exemplary embodiment, the transduction efficiency of kidney cells through retrograde ureteral injection is higher using hydrodynamic injection than using non-hydrodynamic injection (flow rate less than 0.15 mL / sec).

[0071] In an exemplary embodiment, the hydrodynamic infusion can be further monitored using a pressure sensor to ensure that a pressure of at least 50 mmHg, 60 mmHg, 70 mmHg, or 80 mmHg is reached.

[0072] In an exemplary embodiment, distal injection into the ureter is sufficient to achieve a pressure of at least 80 mmHg at a flow rate equal to or greater than 0.5 mL / sec.

[0073] In an exemplary embodiment, proximal injection into the ureter is sufficient to achieve a pressure of at least 100 mmHg with a flow rate equal to or greater than 1 mL / sec.

[0074] In one aspect, the present disclosure provides a method of hydrodynamic gene delivery to the pancreas via the ductal system, comprising: (a) inserting a catheter into the pancreatic duct via endoscopic retrograde cholangiopancreatography; (b) inflating and sealing the balloon to increase pressure during injection; (c) injecting at a flow rate equal to or between 1 and 2 mL / sec; (d) injecting a volume equal to or less than 0.20 mL per gram of pancreatic weight. Including, (e) the injected DNA dose is at least 10 micrograms per gram of pancreas weight; The method is provided wherein the method is sufficient to mediate gene expression in all lobes, accompanied by a reduction in pancreatic enzyme elevation and tissue necrosis.

[0075] In an exemplary embodiment, the injection parameters achieve gene expression in duct cells, islet cells, acinar cells, endothelial cells, and neurons.

[0076] Alternatively, in an exemplary embodiment, the total volume injected does not exceed 15 mL for a pancreas weighing more than 60 grams.

[0077] In an exemplary embodiment, a maximum volume of 0.15 mL per gram of pancreas weight is used when a flow rate of 2 mL / sec is utilized.

[0078] In exemplary embodiments, the DNA dose is preferably greater than 20 or 30 micrograms per gram of pancreas weight.

[0079] In an exemplary embodiment, amylase or lipase levels increase up to four-fold on the first day after injection.

[0080] In an exemplary embodiment, the catheter comprises: (i) Through the major duodenal papilla into the main pancreatic duct distal to where it fuses with the common bile duct; or (ii) through the accessory duodenal papilla, into the accessory or dorsal pancreatic duct, and optionally further into the main pancreatic duct; can be placed.

[0081] In an exemplary embodiment, a balloon within the catheter is inflated through the common bile duct near the entrance to the pancreatic duct to prevent backflow of fluid.

[0082] In an exemplary embodiment, the maximum balloon size used to seal the pancreatic duct is 9 mm to avoid injury.

[0083] In an exemplary embodiment, alternatively, two or more flow rates are used during hydrodynamic injection to further minimize pancreatic tissue damage while preserving gene delivery.

[0084] In an exemplary embodiment, the flow rate is initially 1 mL / sec for the first 50% of the injection volume, and then increases to 2 mL / sec for the remaining injection volume.

[0085] In an exemplary embodiment, the flow rate is initially 0.5 mL / sec for the first 50% of the injection volume, and then increases to 1.5 mL / sec for the remaining injection volume.

[0086] In an exemplary embodiment, a sidewall injection catheter is not used to avoid ductal wall injury and prevent pancreatitis.

[0087] In one aspect, the present disclosure provides a method of 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; and 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.

[0088] In exemplary embodiments, the high pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

[0089] In exemplary embodiments, the flow rate is greater than 2 mL / sec, greater than 5 mL / sec, or greater than 10 mL / sec.

[0090] In exemplary embodiments, the volume injected is greater than 50 mL / kg of liver weight, or greater than 75 mL / kg of liver weight, or greater than 100 mL / kg of liver weight.

[0091] In an exemplary embodiment, prior to infusion, bile is removed from the biliary system and saline solution is used to flush and prime the biliary system.

[0092] In an exemplary embodiment, saline solution can optionally be used to fill the gallbladder prior to injection to reduce pressure differentials during injection.

[0093] In an exemplary embodiment, the preferred DNA dose is at least or greater than 20 mg / kg liver weight.

[0094] In exemplary embodiments, the preferred DNA concentration of the injection solution is or exceeds at least 0.5 mg / mL, or at least 2 mg / mL, or at least 5 mg / mL DNA.

[0095] In one aspect, the present disclosure provides a method of hydrodynamic injection through the biliary tree, where the injection is performed within the common bile duct with the aid of a biliary stent.

[0096] In an exemplary embodiment, a biliary stent is placed prior to hydrodynamic injection.

[0097] In an exemplary embodiment, a biliary stent is placed across the cystic duct to prevent fluid from entering the cystic duct.

[0098] In an exemplary embodiment, the biliary stent will at least exceed the diameter of the bile duct to ensure an adequate seal between the duct wall and the stent during injection.

[0099] In an exemplary embodiment, the biliary stent is of variable length and can reach from the ampulla to upstream of the cystic duct.

[0100] In an exemplary embodiment, a balloon catheter is inserted through the stent after it has been deployed.

[0101] In an exemplary embodiment, the balloon catheter may be positioned anywhere within the stent, including the common bile duct.

[0102] In an exemplary embodiment, prior to injection, contrast is injected through the stent to ensure that the cystic duct and gallbladder are not opacified and that the upstream biliary tree is opacified.

[0103] In an exemplary embodiment, a DNA solution is injected with set parameters to mediate gene delivery to different cells within the liver.

[0104] In an exemplary embodiment, the preferred flow rate during injection is a minimum of 1 mL / sec, or at least 2 mL / sec.

[0105] In exemplary embodiments, the preferred infusion pressure is at least 50 mmHg, or at least 80 mmHg.

[0106] In exemplary embodiments, the preferred injection volume is at least 30, 40, 50, or 60 mL per kilogram of liver weight.

[0107] In an exemplary embodiment, the preferred DNA dose is at least or greater than 10 mg / kg liver weight.

[0108] In an exemplary embodiment, the preferred DNA concentration of the injection solution is at least or greater than 0.2 mg / mL DNA.

[0109] In an exemplary embodiment, the stent is made from a solid, continuous, non-fenestrated material so that fluid cannot pass through the wall of the stent.

[0110] In one aspect, the present disclosure provides a method for delivering a non-viral DNA vector into a tumor in the liver, comprising the steps of: placing a catheter into the biliary system, preferably into the common hepatic duct; inflating a balloon in the common hepatic duct to prevent antegrade flow; and injecting a DNA solution into the biliary system with hydrodynamic pressure, wherein the injection achieves expression of the non-viral DNA vector in tumor cells, regardless of the location of the tumor within the liver.

[0111] In an exemplary embodiment, the tumor is in close proximity to the biliary tree to achieve efficient delivery.

[0112] In exemplary embodiments, pressures of at least 50 mmHg, 70 mmHg, or at least 120 mmHg are targeted for efficient tumor gene delivery.

[0113] In exemplary embodiments, flow rates of at least 2 mL / sec, 4 mL / sec, 7 mL / sec, or at least 10 mL / sec are utilized to achieve efficient tumor gene delivery.

[0114] In an exemplary embodiment, a volume of at least 30 mL per kg of liver weight is utilized for infusion. 6. The method of claim 1, wherein a non-viral DNA dose of at least 10 mg per kg of liver weight, or at least 20 mg per kg of liver weight, is used for infusion.

[0115] In an exemplary embodiment, gene expression in tumor cells is highest along the tumor margin.

[0116] In one aspect, the present disclosure provides a method for delivery of a non-viral DNA vector to a pancreatic tumor, comprising the steps of: a) placing a catheter within the pancreatic duct system upstream of the tumor; b) inflating a balloon within the pancreatic duct to prevent antegrade flow; and c) injecting a DNA solution into the pancreatic duct system with hydrodynamic pressure, wherein the injection achieves expression of the non-viral DNA vector in tumor cells regardless of tumor location within the pancreas.

[0117] In an exemplary embodiment, the tumor is in close proximity to the ductal system to achieve efficient delivery.

[0118] In exemplary embodiments, a pressure of at least 50 mmHg, 70 mmHg, or at least 120 mmHg is controlled for efficient tumor gene delivery.

[0119] In an exemplary embodiment, a flow rate of at least 1 mL / sec is controlled to achieve efficient tumor delivery.

[0120] In an exemplary embodiment, a volume of at least 8 mL is injected into the pancreas of an adult human.

[0121] In an exemplary embodiment, a non-viral DNA dose of at least 1 mg is injected into the pancreas of an adult human.

[0122] In an exemplary embodiment, gene expression in tumor cells is highest along the periphery of a pancreatic tumor.

[0123] In an exemplary embodiment, tumor delivery is most efficient for pancreatic ductal adenocarcinoma.

[0124] In one aspect, the present disclosure provides a method for gene delivery into the liver of a primate, comprising the steps of inserting a catheter into the common hepatic duct of the primate; inflating a balloon within the common hepatic duct to prevent antegrade flow; and injecting a DNA solution into the liver of the primate with hydrodynamic pressure, wherein the injection results in more than 30% of the hepatocytes in the liver of the primate expressing the gene of interest.

[0125] In an exemplary embodiment, the common hepatic duct is accessed by endoscopic retrograde cholangiopancreatography (ERCP).

[0126] In an exemplary embodiment, the ampulla of Vater may be cut to increase the size of the opening to facilitate cannulation of the common hepatic duct during ERCP.

[0127] In an exemplary embodiment, radiocontrast injection is used to localize a catheter placed through the cystic duct into the common hepatic duct to avoid injection into the gallbladder.

[0128] In an exemplary embodiment, radiocontrast injection verifies that the balloon seals the common hepatic duct during injection and that the right and left hepatic ducts are visualized.

[0129] In an exemplary embodiment, the DNA solution is a saline solution having pure recombinant DNA dissolved in the solution.

[0130] In exemplary embodiments, the DNA in the DNA solution can be plasmid DNA, minicircle DNA, or linear closed-end DNA.

[0131] In an exemplary embodiment, the volume injected is at least 30 milliliters per kilogram of liver weight, or at least 40 / mL / kg or more.

[0132] In exemplary embodiments, the flow rate is at least 1 mL / sec, at least 2 mL / sec, or 3 mL / sec or greater.

[0133] In exemplary embodiments, the pressure parameter is at least 50 mmHg, at least 80 mmHg, or greater than 120 mmHg during pressure-guided infusion.

[0134] In exemplary embodiments, the DNA dose is, in certain embodiments, at least 10, 20, 30, 40, or 50 milligrams per kilogram of liver weight.

[0135] In an exemplary embodiment, the DNA solution is a DNA vector composition encoding a hepatocyte-specific promoter.

[0136] In exemplary embodiments, the hepatocyte-specific promoter also contains one or more hepatocyte-specific enhancers to drive higher levels of transcription.

[0137] In an exemplary embodiment, the gene of interest is codon optimized with codons selected for abundance in selected hepatocytes.

[0138] In an exemplary embodiment, DNA vector compositions and protocols are provided for the treatment of hemophilia B in primates.

[0139] In an exemplary embodiment, the DNA vector composition encodes the human Factor IX (hFIX) gene.

[0140] In an exemplary embodiment, the DNA vector composition is a nanoplasmid with a bacterial backbone of less than 500 base pairs.

[0141] In an exemplary embodiment, the overall size of the DNA vector composition is less than 3 kb for hFIX.

[0142] In an exemplary embodiment, a dose of 20 mg of DNA vector composition per kg of primate liver is sufficient to produce 1000 ng / mL of hFIX in the plasma of the primate.

[0143] In an exemplary embodiment, the DNA vector composition can be readministered to ensure further increased expression.

[0144] In an exemplary embodiment, the procedure can be repeated again in a primate to achieve expression of two different genes.

[0145] In one aspect, the present disclosure provides a method of hydrodynamic gene delivery via the biliary system of the liver of a subject, comprising: (a) inserting a catheter into the common hepatic duct; (b) inflating the balloon to seal the conduit and increase pressure during injection; (c) injecting at a minimum flow rate of 2 mL / sec or a minimum pressure of 50 mmHg; (d) delivering DNA encoding a hepatocyte-specific promoter to drive transgene expression; Includes; (e) the DNA vector has significantly reduced or no non-mammalian sequence elements; (f) the injected DNA dose is a minimum of 10 mg of DNA per kilogram of liver weight; A method is provided in which greater than 50% of the hepatocytes express the gene of interest.

[0146] In an exemplary embodiment, alternatively, the DNA vector lacks specific modifications, but optimally, a minimum of 20 mg of DNA is injected per kilogram of liver weight to achieve greater than 50% of hepatocytes expressing the gene of interest.

[0147] In exemplary embodiments, the optional use of transposons may be utilized to facilitate integration into the host chromosome.

[0148] In an exemplary embodiment, significantly reduced means that the total amount of bacterial or phage DNA sequence is less than 1000 bp.

[0149] In an exemplary embodiment, the DNA is a plasmid DNA vector with a vector bacterial backbone that is less than 1 kb in size, or more preferably less than 500 bp in size.

[0150] In exemplary embodiments, the plasmid DNA is a nanoplasmid, pFAR, or pCOR vector.

[0151] In an exemplary embodiment, the DNA is circular and is minicircle DNA.

[0152] In exemplary embodiments, the DNA is linear DNA derived from closed-end DNA, ministring DNA, or dogbone DNA.

[0153] In an exemplary embodiment, switching from a plasmid backbone containing more than 1 kb of bacterial sequences to a DNA vector as described in claims 5 and 6 increases the observed total transfected area of ​​hepatocytes by more than 20%.

[0154] In an exemplary embodiment, gene expression in at least 40% of the hepatocytes for at least three months can be achieved through the use of a transposon system for integration into the host genome.

[0155] In an exemplary embodiment, the duration of expression of the non-integrated DNA of claims 5 and 6 is at least 4 months after injection.

[0156] In an exemplary embodiment, the delivery method results in expression for at least four months and is capable of generating immune tolerance to the exogenous transgene in the liver.

[0157] In exemplary embodiments, DNA vectors greater than 12 kb, 15 kb, or 20 kb in size can be delivered through the liver to multiple cell types, including hepatocytes, endothelial cells, and cholangiocytes, resulting in protein expression.

[0158] In an exemplary embodiment, transfection efficiency is maintained with larger plasmid DNA sizes of at least 12 kb in size.

[0159] In an exemplary embodiment, DNA dose can be adjusted according to DNA dose (mg) per liver weight (kg) per kilobase (kb) of DNA to adjust for DNA size in order to maintain comparable transfection efficiency.

[0160] In an exemplary embodiment, a formulation of 1 mg / kg / kb may be utilized, with the DNA dose planned to achieve transfection of approximately 50% of the hepatocytes in the liver.

[0161] In an exemplary embodiment, a formulation of 2.5-5 mg / kg / kb can be utilized to schedule the DNA dose to achieve transfection of approximately 70% of hepatocytes in the liver.

[0162] In an exemplary embodiment, the procedure can be repeated on a second date with different DNA expressing the same or a different gene, such that expression of the first gene is not disabled and expression of both genes is then achieved.

[0163] In an exemplary embodiment, the second injection can achieve the same transfection efficiency as the first injection and target the same cells.

[0164] In an exemplary embodiment, the same cells can be observed to express the gene after injection.

[0165] In an exemplary embodiment, the promoter can be altered to achieve expression in a different cell type using a second injection, which does not alter expression of the first gene.

[0166] In an exemplary embodiment, the procedure can be repeated in the same injection procedure with different DNAs expressing the same or different genes, so that expression of both DNAs is achieved therein and expression of the first DNA injection is not abolished.

[0167] In an exemplary embodiment, the second injection can achieve the same transfection efficiency as the first injection and target the same cells.

[0168] In an exemplary embodiment, the promoter can be altered to achieve expression in a different cell type by a second injection, which alters expression of the first gene.

[0169] In an exemplary embodiment, two different DNA molecules can be delivered mixed together during a single injection, resulting in both DNA molecules entering the same liver cells.

[0170] In exemplary embodiments, DNA doses of 20 mg / kg liver weight to 40 mg / kg liver weight achieve similar transfected areas.

[0171] In an exemplary embodiment, DNA doses of up to 40 mg / kg liver weight can be injected without causing significant hepatotoxicity or physiological distress.

[0172] In an exemplary embodiment, flow rates below 1 mL / sec do not result in expression of the gene.

[0173] In an exemplary embodiment, flow rates between 1 mL / sec and 2 mL / sec show a decrease in gene expression compared to those above 2 mL / sec.

[0174] In an exemplary embodiment, flow rates above 4 mL / sec result in progressively less efficient hepatocyte delivery.

[0175] In an exemplary embodiment, a flow rate of greater than or equal to 7 mL / sec achieves efficient delivery to the bile duct.

[0176] In an exemplary embodiment, the preferred injection volume is 30 mL / kg to 60 mL / kg of liver tissue.

[0177] In an exemplary embodiment, injection volumes greater than or equal to 70 mL / kg of liver tissue are associated with decreased efficiency of gene delivery.

[0178] In exemplary embodiments, the gene injection procedure is well tolerated by subjects 25 kg or 15 kg or 5 kg in size, resulting in gene delivery as efficient as in larger mammals.

[0179] In an exemplary embodiment, the transfected hepatocyte area by biliary hydrodynamic delivery can be further increased by at least 10% of the total hepatocyte area when two or more flow rates are incorporated during infusion.

[0180] In an exemplary embodiment, a flow rate of 2 mL / sec is first used for 50% to 66% of the total injection volume, followed by 4 mL / sec for the remaining volume.

[0181] In an exemplary embodiment, a flow rate of 2 mL / sec is used for the first 33% of the volume injection, 3 mL / sec is used for the second 33% of the volume, and then 4 mL / sec is used for the remaining injection volume.

[0182] In an exemplary embodiment, the catheter is inserted into the common hepatic duct via ERCP, EUS, or an imaging-guided percutaneous route.

[0183] In exemplary embodiments, the DNA concentration of the injected solution is at least 0.30 mg / mL, and more preferably greater than 0.40 mg / mL, 0.50 mg / mL, or 0.60 mg / mL.

[0184] In one aspect, the present disclosure provides a method for achieving expression in liver sinusoidal endothelial cells (LSECs), comprising performing biliary hydrodynamic injection as described in claim 1, with the exception of using a cell-specific promoter to target expression in LSECs.

[0185] In exemplary embodiments, LSECs can be targeted for expression by the CD36 promoter or the FVIII promoter.

[0186] In an exemplary embodiment, an injection pressure of 80 mmHg produces a more efficient ejection than an injection pressure of 50 mmHg.

[0187] In an exemplary embodiment, a pressure of 150-200 mmHg results in efficient gene expression.

[0188] In an exemplary embodiment, pressures above 200 mmHg result in progressively less gene expression.

[0189] In certain aspects, a more efficient balloon seal is obtained by advancing the catheter and inflating the balloon within the intrahepatic duct.

[0190] In certain aspects, placement of the balloon within the extrahepatic duct results in fluid leakage around the balloon.

[0191] In certain embodiments, the balloon size is at least 2 times, at least 3 times, or at least 4 times the diameter of the conduit.

[0192] In certain embodiments, when the balloon is placed within the extrahepatic bile duct, the balloon can be inflated to a maximum size of three times the diameter of the duct.

[0193] In certain embodiments, when the balloon is placed in the intrahepatic bile duct, the balloon can be inflated to a minimum size of four times the diameter of the duct.

[0194] In certain aspects, the balloon size is not fully inflated within the intrahepatic duct, but rather additional pressure is created within the balloon.

[0195] In certain embodiments, leakage around the balloon, whether intrahepatic or extrahepatic, occurs at sizes of 8.5 mm or less, and therefore these sizes should be avoided.

[0196] In certain embodiments, the balloon is inflated to a size of less than 15 mm within the common hepatic duct to avoid rupture.

[0197] In certain embodiments, if a balloon is placed in the right or left hepatic duct and an injection is subsequently performed at that location, the injection is repeated again in the contralateral duct to ensure that both lobes of the liver are injected equally.

[0198] In certain embodiments, the balloon seal can be monitored by measuring intraluminal biliary pressure.

[0199] In certain embodiments, loss of the plateau waveform, as defined by a drop of more than 20 mmHg from the beginning to the end of the plateau, indicates leakage around the balloon.

[0200] In certain aspects, balloon sealing may be verified by filling the bile duct above and below the balloon with radiocontrast solution prior to injection.

[0201] In certain embodiments, loss of fluid-tight seal during injection is evidenced by the escape of contrast agent under the balloon into either the cystic duct and gallbladder or the common bile duct.

[0202] In certain embodiments, the contrast agent above the balloon flows into the liver, indicating a successful injection.

[0203] In certain embodiments, hydrodynamic injection can be repeated multiple times in a single procedure to enhance DNA delivery.

[0204] In certain embodiments, the use of two or more injections is additive towards achieving the final gene expression level.

[0205] In certain aspects, this strategy allows for overcoming inherent limitations of vascular contrast or power injection volume through the use of multiple injections.

[0206] In certain aspects, primate liver tissue is more elastic than porcine tissue, and as a result, different ductal properties necessitate changes in balloon size and injection parameters to mediate gene delivery in primates.

[0207] In certain aspects, the flow rate must be increased to achieve a given pressure relative to infusion in the porcine model.

[0208] In certain embodiments, a flow rate of at least 4 mL / sec is required to achieve a pressure plateau of at least 80 mmHg.

[0209] In certain embodiments, the primate can tolerate a dose of at least 80 mg of pDNA without any significant physiological side effects.

[0210] In certain embodiments, an injection volume of at least 120 mL per 400 grams of liver can be injected into a primate without any significant disturbance of vital signs.

[0211] In certain embodiments, infusion rates of up to 12 mL / sec can be tolerated by the liver of a primate without tissue damage, changes in vital signs, and bile duct rupture.

[0212] In certain embodiments, if no improvement in gene delivery is seen at higher flow rates, a flow rate greater than 4 mL / sec but less than 8 mL / sec should be used.

[0213] In certain embodiments, a volume of 30 mL per 400 g may be used, or alternatively, a volume of up to 150 mL per 400 g may be used.

[0214] In certain embodiments, the volume utilized does not affect the efficiency of gene delivery at a given DNA dosage.

[0215] In certain embodiments, the vector composition should be administered with copies of the transgene expression cassette, and consequently, different pDNA doses are required if additional exogenous DNA is included in the DNA vector.

[0216] In certain embodiments, the use of backbone-reduced DNA molecules allows for relatively smaller DNA doses.

[0217] In certain embodiments, increasing the pDNA dose and / or vector expression cassette dose per animal leads to a quantitatively equivalent increase in expression of the protein therapeutic of interest.

[0218] In certain aspects, methods are provided for hydrodynamic gene delivery via the biliary system of the liver of a subject. In certain aspects, the methods include: (a) inserting a catheter into the bile duct; (b) inflating the balloon to seal within the bile duct and prevent antegrade flow of the solution; i. the balloon inflation size is at least two times, at least three times, or at least four times the diameter of the bile duct to overcome the elasticity of the primate's duct; ii. The method for verifying balloon sealing during injection consists of placing radiocontrast solution above and below the balloon and detecting antegrade fluid movement by fluoroscopy; Steps; (c) injecting at a flow rate of at least 2 mL / sec or at a pressure of at least 50 mmHg; i. more preferably, a minimum of 4 mL / sec capable of producing a plateau pressure of at least 80 mmHg in the primate liver, and 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 global gene expression; ii. the volume injected 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. A plateau pressure is obtained during hydrodynamic injection that fluctuates by less than 10% mmHg over the course of the injection, indicating an adequate seal; iv. Optionally, multiple flow rates can be used in a single injection to modify and vary the resulting pressure; Steps; and (d) delivering a miniaturized DNA vector having significantly reduced or no non-mammalian sequence elements, i. The miniaturized DNA vector results in a more persistent expression lasting at least 4 months; ii. Miniaturized DNA vectors provide high efficacy at a given DNA dose in primates, allowing for the use of smaller doses compared to conventional plasmid DNA; iii. The DNA vector optimally contains a hepatocyte-specific promoter to enhance expression in the liver; Steps; Includes.

[0219] In certain embodiments, significantly reduced means that the total amount of bacterial or phage DNA sequence is less than 1000 bp.

[0220] In certain embodiments, the DNA is a plasmid DNA vector with a vector bacterial backbone or sequence that is less than 1 kb in size, or more preferably less than 500 bp in size.

[0221] In certain embodiments, the plasmid DNA is a nanoplasmid, GenCircle, pFAR, or pCOR vector.

[0222] In some embodiments, the DNA is circular and is a minicircle DNA or a minivector DNA.

[0223] In certain embodiments, the DNA is linear DNA derived from closed-end DNA, ministring DNA, or dogbone DNA.

[0224] In certain embodiments, the linear DNA has only small exogenous sequences at either end, each less than 100 bp in size, with the mammalian expression sequence of interest being the remainder of the vector.

[0225] In certain embodiments, a choledocholic sphincterotomy is performed in a subject during a first procedure to reduce or eliminate the risk of post-ERCP pancreatitis during a subsequent ERCP procedure to readminister the genetic medicine.

[0226] In certain embodiments, the injection procedure can be repeated twice within one session to increase protein expression.

[0227] In certain embodiments, the repeated injections may use the same DNA to boost individual protein expression, or may use two different DNA solutions to result in two different proteins being expressed.

[0228] In certain embodiments, the total DNA dose from repeated injections is similar or equivalent to the expression obtained from a single DNA injection.

[0229] In certain aspects, the injection procedure can be repeated again after a single administration, resulting in the same transfection efficiency, comparable peak protein expression between injections, and a lack of observed immunogenicity.

[0230] In certain embodiments, repeated injection procedures may be spaced apart by at least one month, at least three months, at least six months, or at least one year.

[0231] In certain embodiments, pressure is monitored using a pressure transducer that senses a fluid-filled column within the pressure catheter, or alternatively, using a pressure sensor threaded into the catheter lumen.

[0232] In certain embodiments, pressure sensor readings are recorded in real time and the pressure curve is interpreted after injection to determine whether a successful seal and peak expression was achieved.

[0233] In certain embodiments, optimal hydrodynamic infusion achieves at least a two-fold increase in liver enzymes, such as ALT and AST levels, compared to pre-treatment values ​​by day 1 post-infusion.

[0234] In certain embodiments, liver enzymes return to normal limits within seven days of infusion.

[0235] In certain embodiments, gene delivery occurs intratumorally and peritumorally, in both malignant and normal cells, and disseminatedly surrounding the tumor.

[0236] In another aspect, the present disclosure provides a method of hydrodynamic injection into the liver via the biliary system using a partially deployed, fully covered metal or plastic stent, comprising: a) deploying the stent from distal to proximal (relative to the stent placement catheter); b) retaining the stent in a delivery catheter containing the stent such that the position of the released or open component of the stent is within the common hepatic duct and the tip of the stent is located either in the common hepatic duct, the common bile duct, or around the duodenal ampulla; c) blocking and / or bypassing the cystic duct opening with the covered portion of the stent so that the injected fluid solution cannot enter the cystic duct or gallbladder; d) removing the guidewire from the stent delivery system; and e) injecting a DNA solution into the biliary system through the guidewire lumen at a high pressure target and / or flow rate; the stent, which forms a closed system with the catheter by partial deployment and continuous connection, prevents antegrade flow within the biliary system and / or allows gene expression to be observed in hepatocytes within the liver by immunostaining.

[0237] In one embodiment, the high pressure target is greater than 50 mmHg, greater than 80 mmHg, or greater than 120 mmHg.

[0238] In one embodiment, the flow rate is greater than 2 mL / sec, greater than 5 mL / sec, or greater than 10 mL / sec.

[0239] In one embodiment, the volume injected is greater than 50 mL / kg liver weight, or greater than 75 mL / kg liver weight, or greater than 100 mL / kg liver weight.

[0240] In one embodiment, prior to infusion, bile is removed from the biliary system and saline solution is used to flush and cleanse the biliary system.

[0241] In one embodiment, the preferred DNA dose is at least or greater than 20 mg / kg liver weight.

[0242] In one embodiment, the preferred DNA concentration of the injection solution is at least or greater than 0.5 mg / mL DNA.

[0243] In one embodiment, the biliary stent is at least the diameter of the bile duct to ensure an adequate seal between the duct wall and the stent during injection.

[0244] In one embodiment, the biliary stent diameter is at least 150% of the bile duct diameter, or at least 200% of the bile duct diameter.

[0245] In one embodiment, the biliary stent is of variable length and can reach from outside the ampulla to upstream of the cystic duct.

[0246] In one embodiment, injection is performed from the tip of the "olive" aspect of the stent, which may be located between the liver end of the stent and the porta hepatis during injection.

[0247] In one embodiment, the injection occurs at an opening on the catheter proximal to where the stent is fed into the catheter, so that the fluid fills the stent as it advances retrograde, the cone of the stent within the catheter preventing antegrade flow.

[0248] In one embodiment, prior to injection, contrast is injected through the stent to ensure that the cystic duct and gallbladder are not opacified and that the biliary tree is opacified.

[0249] In one embodiment, the stent is made of a solid material so that fluid cannot pass through the wall of the stent.

[0250] In one embodiment, the liver end of the stent opens into the left main hepatic duct or the right main hepatic duct, but not into the common hepatic duct.

[0251] In one embodiment, once hydrodynamic injection is completed through either the left or right main hepatic duct, the other duct is injected using the same technique.

[0252] In one embodiment, the stent is partially deployed, i.e., up to 95% of its length, 75% of its length, 50% of its length, or in some embodiments only 25% of its length is deployed outside the catheter, with the remainder of the stent length remaining within or attached to the catheter.

[0253] In one embodiment, the partially deployed stent forms a funnel or cone shape at the proximal end where the stent is attached to the catheter, thereby forming a closed system.

[0254] definition To facilitate understanding of this disclosure, several terms and phrases are defined below.

[0255] As used herein, unless specifically stated otherwise or clear from the context, the term "about" is understood to mean within a normal range of tolerance in the art, for example, within 2 standard deviations of the mean value. About can be understood as 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 otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0256] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, polypeptide, or fragment thereof.

[0257] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest, or stabilize the occurrence or progression of a disease (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.) phenotype).

[0258] "Alteration" refers to a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

[0259] The phrase "combination therapy" encompasses the administration of a gene therapy protocol and one or more additional therapeutic agents (e.g., erythropoietin, corticosteroids, ACE inhibitors) as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the interaction of these therapeutic agents. Beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The administration of these therapeutic agents in combination typically occurs over a defined period of time (usually minutes, hours, days, or weeks, depending on the combination selected). "Combination therapy" is intended to encompass the administration of these therapeutic agents in a sequential manner, i.e., each therapeutic agent is administered at a different time, as well as the administration of these therapeutic agents or at least two of the therapeutic agents in a substantially simultaneous or overlapping manner. Substantially simultaneous administration can be achieved, for example, by administering one or more copper chelating compounds to a subject while administering a gene therapy protocol as disclosed herein. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissue (e.g., nasal, oral, vaginal, and rectal), and ocular (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same or different routes. For example, one component of a particular combination can be administered by intravenous infusion (e.g., in a gene therapy protocol), while the other component of the combination (e.g., one or more copper chelating compounds) can be administered orally. The components can be administered in any therapeutically effective order.

[0260] The term "combination" encompasses groups of compounds and / or non-drug gene therapies useful as part of a combination therapy as disclosed herein.

[0261] In this disclosure, "comprises," "comprising," "containing," "having," and the like can have the meaning ascribed to them in U.S. patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially of" likewise have the meaning ascribed to them in U.S. patent law, and the term is open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of the recited items are not changed by the presence of more than what is recited.

[0262] "Control" means a standard or reference condition.

[0263] "Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)).

[0264] "Effective amount" refers to the amount required to improve disease symptoms (e.g., neurological or other symptoms of a genetic disease in a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)) compared to an untreated patient. The effective amount of an active compound used to practice the present disclosure for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and administration regimen. Such an amount is referred to as an "effective" amount. An effective amount can also refer to the level of gene expression (e.g., ATP7B mRNA or protein expression) in the appropriate tissue of a patient.

[0265] "Fragment" refers to 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 entire length of the reference nucleic acid molecule or polypeptide. A fragment may 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.

[0266] A "gene therapy composition" should be understood to mean a DNA composition (e.g., comprising a 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 sequence, or a portion thereof) for preventing and / or treating a genetic disease in a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.). Thus, in certain embodiments, the gene therapy composition is a pharmaceutical comprising the full-length PKD1 nucleotide sequence or a portion thereof, intended for use in humans or animals for preventing and / or treating a genetic disease of the kidney. Thus, in certain embodiments, the gene therapy composition is a pharmaceutical comprising the full-length CFTR nucleotide sequence or a portion thereof, intended for use in humans or animals for preventing and / or treating a genetic disease of the pancreas. Thus, in certain embodiments, the gene therapy composition is a pharmaceutical comprising a full-length ATP7B, UGT1A1, HFE, OTC, LDLR, ABCB4, PBGD or VWF nucleotide sequence, or a portion thereof, intended for use in humans or animals to effect prevention and / or treatment of genetic diseases of the liver.

[0267] "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0268] An "isolated polynucleotide" refers to a nucleic acid molecule (e.g., DNA, mRNA, cDNA, etc.) that does not contain the gene with which the nucleic acid molecule of the present disclosure is normally associated or derived in the naturally occurring genome of an organism. Thus, this term includes, for example, recombinant DNA (e.g., genomic DNA or cDNA encoding the ATP7B gene and associated regulatory elements, such as enhancers, promoters, 5' and / or 3' untranslated regions (UTRs)) that can be incorporated into a vector, or an autonomously replicating plasmid or virus, or genomic DNA of a prokaryotic or eukaryotic organism, or a polynucleotide that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction enzyme digestion, or naked DNA constructs such as plasmids, cosmids, or linear DNA). Furthermore, this term also includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0269] "Isolated polypeptide" refers to a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60% free, by weight, from 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%, and most preferably at least 99% polypeptide of the present disclosure. Isolated polypeptides of the present disclosure can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0270] For purposes of this disclosure, "mutation" refers to a DNA sequence found in a patient's gene that does not correlate with the established wild-type gene sequence; such mutations may result from one or more single nucleotide polymorphisms, one or more deletions or insertions of one or more nucleotides, and deletions or insertions of splice site junctions. "Mutation" may also refer to a pattern in the sequence of RNA from a patient that is not attributable to variations predicted based on known information about the gene and is reasonably believed to be, for example, a novel variation in the splicing pattern of the patient's gene.

[0271] As used herein, unless specifically stated otherwise or clear from context, the term "or" is understood to be inclusive. As used herein, unless specifically stated otherwise or clear from context, the terms "a," "an," and "the" are understood to be singular or plural.

[0272] The term "patient" or "subject" refers to an animal that has been the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including a human or a non-human mammal, such as, but not limited to, a non-human primate, cow, horse, dog, sheep, or cat.

[0273] "Pharmaceutically acceptable" refers to approved or appropriable by a regulatory agency of the federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, including humans.

[0274] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug and that is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug without impairing its pharmacological activity.

[0275] A "pharmaceutically acceptable salt" of a pooled tumor-specific neo-antigen as recited herein can be a salt of an acid or base generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include mineral and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic 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 (e.g., acetic acid, HOOC-(CH)-COOH, where n is 0 to 4), and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize additional pharmaceutically acceptable salts for the pooled tumor-specific neoantigens provided herein, including those listed by 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 a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base forms of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0276] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disease or condition (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)) in a subject who does not have the disease or condition (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)), but who is at risk of or susceptible to developing the disease or condition (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)).

[0277] "Primer set" refers to a set of oligonucleotides that can be used, for example, for PCR. A primer set may 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 primers.

[0278] Ranges provided herein are understood to be shorthand for all values ​​within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from 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 intervening decimal values ​​between the aforementioned integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either endpoint of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could 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.

[0279] By "decreasing" is meant a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0280] "Reference" means a standard or control condition.

[0281] A "reference sequence" is a defined sequence used as a basis for sequence comparison (e.g., a wild-type ATP7B gene sequence). A reference sequence can be a subset or the entirety of a specified sequence; for example, a segment of a full-length cDNA or genomic sequence, or the entire cDNA or genomic sequence. For polypeptides, the length of a reference polypeptide sequence is generally 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 a reference polypeptide sequence can be at least about 10 to 50 amino acids, more preferably at least about 10 to 40 amino acids, and 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 a reference nucleic acid sequence is generally at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, even more preferably about 100 nucleotides or about 300 nucleotides, or any integer therebetween or thereabout.

[0282] 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 do not need to be 100% identical to an endogenous nucleic acid sequence, but typically exhibit substantial identity (e.g., 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%). A polynucleotide having "substantial identity" to an endogenous sequence can typically hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridizing" refers to the formation of a double-stranded molecule between complementary polynucleotide sequences (e.g., the ATP7B gene described herein) or portions thereof under various stringency conditions. (See, e.g., Wahl, GM and SL Berger (1987) Methods Enzymol. 152:399; Kimmel, AR (1987) Methods Enzymol. 152:507).

[0283] For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, preferably less than about 500 mM NaCl and less than 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low stringency hybridization can be achieved in the absence of organic solvents, such as formamide, while high stringency hybridization can be achieved 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. Various additional parameters, such as hybridization time, detergent (e.g., sodium dodecyl sulfate (SDS)) concentration, and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency can be achieved by combining these various conditions as needed. In a preferred embodiment, hybridization is carried out in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS at 30° C. In a more preferred embodiment, hybridization is carried out in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization is carried out in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be readily apparent to those of skill in the art.

[0284] For most applications, post-hybridization washing steps also vary in stringency. Stringency conditions for washing can be defined by salt concentration and temperature. As described above, washing stringency can be increased by decreasing salt concentration or increasing temperature. For example, stringent salt concentrations for washing steps are preferably less than about 30 mM NaCl and less than 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and less than 1.5 mM trisodium citrate. Stringent temperature conditions for washing steps typically include temperatures 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 washing step is performed at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 42° C. In a more preferred embodiment, the wash steps are performed in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. Further variations of these conditions will be readily apparent to one of skill 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.

[0285] "Substantially identical" refers to a polypeptide or nucleic acid molecule that 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 or nucleic acid sequence (e.g., wild-type ATP7B) used for comparison.

[0286] Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX program, from Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). 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 approach to 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.

[0287] As used herein, the terms "treat," "treated," "treating," "treatment," and the like refer to alleviating or ameliorating a disorder and / or its associated symptoms (e.g., a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.)). Although not excluded, it is understood that treating a disorder or condition does not require that the disorder, condition, or its associated symptoms be completely eliminated.

[0288] The term "therapeutic effect" refers to some degree of alleviation of one or more symptoms (e.g., neurological, kidney-related, liver-related, etc.) of a genetic disease of a target tissue (e.g., kidney, pancreas, common bile duct, tumor, liver, etc.) or its associated pathology. As used herein, a "therapeutically effective amount" refers to an amount of a drug or combination therapy that, upon administration to a cell or subject in a single dose or multiple doses, is effective in extending the survival of a patient with a genetic disease of the target tissue, alleviating one or more signs or symptoms of a genetic disease of the target tissue, preventing or delaying the onset of symptoms of a 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 limit the amount required to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the required "therapeutically effective amount" of a drug or combination therapy.

[0289] Pharmaceutical compositions should typically provide a dosage of from about 0.0001 mg to about 200 mg of compound per kilogram of body weight per day. For example, dosages for systemic administration to human patients can be in the range of 0.01-10 μg / kg, 20-80 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 a combination of essential ingredients per dosage unit form.

[0290] The recitation of a list of chemical groups in any definition of a variable herein includes definition of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0291] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0292] Where applicable, or unless specifically stated otherwise, it is contemplated that any one of the embodiments described herein may be combined with any other one or more embodiments, even if the embodiments are described under different aspects of the disclosure.

[0293] These and other embodiments are disclosed and / or encompassed by the following detailed description.

[0294] The above and other features and advantages of the present disclosure will be better understood from the following detailed description read in conjunction with the following drawings. [Brief explanation of the drawings]

[0295] [Figure 1-1] Figures 1A-1D show a schematic diagram and three images demonstrating the technique of hydrodynamic injection by accessing the ureter through the bladder. Injection into the ureter can be used to deliver pDNA to the porcine kidney. Figure 1A shows a schematic diagram of the catheter position. The blue circle represents where the catheter balloon is positioned and placed. Figure 1B is a cystoscopic image of the bladder revealing the ureteral orifice adjacent to the urethra. Figure 1C shows the bulge in the bladder wall, confirming inflation of the balloon after insertion into the ureteral entrance into the bladder. Figure 1D is a fluoroscopic image showing the entire ureter and renal pelvis filled with radiocontrast agent, approximately depicting the total volume. [Figure 1-2] (As mentioned above.) [Figure 1-3](As mentioned above.) [Figure 1-4] (As mentioned above.) [Figure 2-1] Figures 2A-2D show images and four photographs of hydrodynamic injection through the ureter, which may be a cause of kidney rupture. Hydrodynamic injection of fluid through the ureter was explored by testing both contrast injection and saline injection. Figure 2A shows the outflow of contrast into the subcapsular space after hydrodynamic injection. The image on the left is from an earlier time point than the image on the right, just a few seconds later. Macroscopic examination of the kidney after injection (Figure 2B) reveals blood pooling under the renal capsule and a large hematoma. Subsequent removal of the renal capsule (Figure 2C) reveals an approximately 1.5 cm tear on the kidney surface at the upper pole of the kidney. Dissection of the kidney in Figure 2D shows that the tear originates at the base of the medulla and tears through the entire cortex. [Figure 2-2] (As mentioned above.) [Figure 3-1] Figures 3A-3C show photographs and GFP staining images demonstrating that hydrodynamic injection via the ureter can result in gene delivery. The pCLucf plasmid, encoding firefly luciferase and GFP, was injected into kidneys using different injection parameters. Figure 3A shows a photograph of a kidney sectioned at the upper, middle, and lower poles, indicated by black circles. Figure 3B is a higher magnification image showing GFP staining in glomeruli among scattered cells. Figure 3C shows representative photographs of luciferase staining at the upper, middle, and lower poles at the cortex / medulla interface. These images are for kidneys injected at 20 mL and 2 mL / sec, resulting in the delivery of 1.2 mg of pDNA. The image on the bottom right shows an uninjected kidney as a control for staining. Pigs injected with other injection parameters (27 mL, 2 mL / sec; 30 mL, 1 mL / sec) also showed similar immunohistochemical (IHC) staining patterns (data not shown). [Figure 3-2] (As mentioned above.) [Figure 4-1]Figures 4A-4C depict four images demonstrating that hydrodynamic injection achieves expression in multiple cell types in the kidney. GFP staining after pCLucf injection in various cell types in the kidney is shown. Figure 4A shows GFP staining in renal tubules within the cortex. Figure 4B shows that GFP staining occurs in endothelial cells, and Figure 4C shows that GFP staining is seen in a small population of renal tubules within the renal medulla. [Figure 4-2] (As mentioned above.) [Figure 5] Figure 5 shows five photographs demonstrating that hydrodynamic injection at lower parameters results in safer injections. Hydrodynamic injection parameters for distal ureteral injections were performed at flow rates ranging from 0.5 mL / sec to 2 mL / sec. Total volumes ranged from 16 mL to 20 mL for these injections. After injection, the kidneys were grossly dissected and examined for rupture. The upper left photograph shows signs of rupture in the kidney injected with 20 mL at 1 mL / sec, while the right photograph shows bruising after injection of 20 mL at 1.5 mL / sec. The lower right photograph shows rupture in the kidney injected with 20 mL at 0.5 mL / sec, but no injury was observed after injection of 16 mL at 2 mL / sec. The fifth photograph is a control, with no injection. Dissected kidneys from different groups are depicted, none of which show signs of rupture. [Figure 6] Figure 6 shows six images of hydrodynamic injections at lower parameters, still allowing gene delivery. Hydrodynamic injection parameters for distal ureteral injection were performed at flow rates ranging from 0.5 mL / sec to 2 mL / sec. Injection volumes ranged from 16 mL to 20 mL, resulting in the delivery of 2 mg of pCLucf. For each parameter, representative images of firefly luciferase staining within the upper pole of the kidney are shown. Two uninjected control tissues are also shown at the bottom. Similar expression patterns were observed in the middle and lower poles of the kidney, as well as when stained with GFP (data not shown). [Figure 7]Figures 7A and 7B are images of hydrodynamic injection at the lowest flow rate, which resulted in expression comparable to higher flow rates. Hydrodynamic injection of tissue samples from kidneys at the lowest flow rate tested, 0.5 mL / sec, demonstrates that protein expression can still be achieved. Figure 7A shows GFP staining positive cells in glomeruli, while the control showed no staining. Figure 7B shows a low magnification of the cortex, depicting abundant tubules stained with firefly luciferase. [Figure 8-1] Figures 8A-8D show images and photographs demonstrating that proximal ureteral injection can cause kidney injury. Hydrodynamic injection was performed within the ureter proximal to the renal pelvis. Next, contrast medium was used as the injection fluid, with injections at 15 mL and 1 mL / sec. Fluoroscopy in Figure 8A shows that the balloon placed in the renal pelvis was unable to prevent antegrade flow of contrast medium into the ureter. Fluoroscopy after injection in Figure 8B shows rupture of the renal capsule, with contrast medium spilling into the subcapsular space. Macroscopic examination of the kidney at necropsy confirmed bleeding and rupture in the photograph in Figure 8C. Figure 8D shows the disappearance of GFP by immunohistochemical staining from a kidney injected proximally with 15 mL at 1 mL / sec. [Figure 8-2] (As mentioned above.) [Figure 8-3] (As mentioned above.) [Figure 9] Figure 9 depicts two images demonstrating that proximal ureteral injection can cause kidney injury. Hydrodynamic injections of 15 mL and 1 mL / sec close to the kidney can cause significant kidney injury. The top image is a tissue section showing extensive areas of tissue injury and lymphocytic infiltration from the medulla to the cortex. The bottom image shows a high magnification of the necrotic area along with areas of neutrophil and lymphocytic infiltration. [Figure 10]Figure 10 shows eight images demonstrating gene expression following proximal ureteral injection at various low injection parameters. To test safety and gene expression, hydrodynamic injection was evaluated from the proximal ureteral location at various low injection parameters. A 10 mg pCLucf plasmid DNA dose was delivered, and the injection parameters tested were 11 mL at 2 mL / sec, 12 mL at 1.5 mL / sec, and 10 mL at 1 mL / sec. Three days after injection, the pigs were harvested, and the upper, middle, and lower poles of the kidney were dissected. IHC staining for GFP was performed on the upper, middle, and lower poles of the kidney in the renal cortex in the left image and in the medulla in the right image. Representative images of IHC staining for the various parameters evaluated are shown. [Figure 11] Figures 11A and 11B show two images and a graph showing the correlation between flow rate and pressure during hydrodynamic injection in the proximal ureter. Pressure was monitored during hydrodynamic injection in the distal ureter. For the experiment, a quadruple-lumen catheter consisting of a guidewire, balloon, pressure sensor, and injection port was obtained. The catheter tip was placed proximally near the kidney. Figure 11A shows an example of contrast agent filling the ureter and renal pelvis for localization. A series of flow rates was tested, and pressure was simultaneously monitored. As an example, 10 mL was injected at a flow rate of 1 mL / sec. In Figure 11B, the pressure trace graph shows a peak pressure of 105 mmHg. The rapid drop in pressure was likely due to kidney rupture or balloon damage. [Figure 12-1]Figures 12A-12C show two photographs of the kidney and a correlation graph of flow rate and pressure during hydrodynamic infusion into the distal ureter. Pressure was monitored during hydrodynamic infusion into the distal ureter. For the experiment, a laparotomy was performed and the bladder wall was resected. The ureteral orifice was visualized, and a catheter was inserted into the ureter. The catheter itself was connected to a power injector for injecting saline solution. A pressure sensor was inserted into the ureter along with the catheter, but it was too large to fit into the lumen. Both the pressure sensor and catheter were advanced distally into the ureter. Figure 12A shows a surgical tie performed to prevent antegrade flow during ureteral infusion. A series of different flow rates were tested to assess pressure at a fixed volume of 10 mL. Figure 12B shows pressure tracings with arrows indicating the infusion. In this study, pressure was observed to be flow rate dependent. Flow rates of 0.5-1 mL / s resulted in pressures of 70-80 mmHg. At flow rates of 1.5 to 2 mL / s, the pressure was 120 to 140 mmHg. Additional tests were performed with increasing volumes injected at a constant flow rate, but the pressure did not increase further (data not shown). The baseline ureter pressure before injection was 5 to 15 mmHg. After a series of injections, the kidney was further dissected. Figure 12C shows the observed pooling of fluid in the space between the capsule and the kidney surface. Several tears were noted during injections above 3 mL / s. [Figure 12-2] (As mentioned above.) [Figure 13-1]Figures 13A-13D depict a schematic diagram and three images depicting the evaluation of the pancreatic duct by endoscopic retrograde cholangiopancreatography (ERCP) for gene delivery in pigs. The pancreatic duct is the conduit for gene delivery via hydrodynamic injection. Because the pig organ is similar in size to humans, pigs are an excellent model for ERCP; however, the anatomy of the pig pancreas in terms of lobule structure differs between the two species. Figure 13A shows a schematic model of catheter placement in the pig pancreas, depicting two separate openings for the bile duct and pancreatic duct. Figure 13B shows cannulation of the pig pancreatic duct, which is significantly different from humans because the pancreatic duct does not have the ampulla of Vater. In pigs, the openings at the pancreaticoduodenal junction and the biliary duodenal junction are separate. Figure 13C shows a representative fluoroscopy image of the pancreatic duct before hydrodynamic injection. Figure 13D shows a representative fluoroscopic image of the pancreatic duct after hydrodynamic injection, confirming that there are no signs of rupture after injection. [Figure 13-2] (As mentioned above.) [Figure 13-3] (As mentioned above.) [Figure 14-1] Figures 14A-14C show images and two graphs demonstrating the safety of pancreatic hydrodynamic injection. Hydrodynamic injection into pig pancreases was performed with injection parameters of 22 mL at 2 mL / sec, resulting in the delivery of 1.2 mg of pDNA. Figure 14A shows abdominal CT scans of the pancreases of two different pigs obtained one day after injection. To measure pancreatic injury, Figure 14B shows amylase levels before injection, after injection, on day 1, and on day 3. To measure inflammation induced by the injection procedure, Figure 14C shows white blood cell (WBC) levels before injection, after injection, and on day 1. [Figure 14-2] (As mentioned above.) [Figure 15-1]Figures 15A-15D show photographs, Western blots, and images of pancreatic tissue samples evaluating gene expression following hydrodynamic gene delivery of plasmid DNA to the pancreas. Figure 15A shows a photograph of the pancreas obtained from necropsy of an injected pig. A macroscopic examination of the pancreas is performed to look for signs of liver injury. Gross specimens were evaluated, and no gross pancreatic duct or parenchymal injury was observed. Figure 15B shows PCR performed as a first step to detect the presence of plasmid DNA in the duodenal, splenic, and connecting lobes of the pig. Figure 15C shows images of pancreatic tissue from three lobes analyzed by immunohistochemistry (IHC) for the presence of firefly luciferase. Figure 15D shows an image of pancreatic tissue analyzed by IHC for the presence of firefly luciferase at low magnification. [Figure 15-2] (As mentioned above.) [Figure 16-1] Figures 16A-16D show images of reporter gene expression observed in different pancreatic cell types after hydrodynamic injection. Hydrodynamic gene delivery of plasmid DNA into the pancreas was evaluated for gene expression in different pancreatic cell types. In Figure 16A, a high-magnification image of pancreatic duct cells shows specific GFP staining of pancreatic duct cells (left panel) relative to the saline control (center panel). Cytokeratin staining confirmed the identity of pancreatic duct cells. In Figure 16B, a high-magnification image also shows specific luciferase staining of pancreatic islet cells (left panel) relative to the saline control (center panel). Synaptophysin staining confirmed the identity of pancreatic islet cells. In addition to these cell types, firefly luciferase reporter gene expression was also observed in pancreatic tissue endothelial cells (Figure 16C) and neurons (Figure 16D). [Figure 16-2] (As mentioned above.) [Figure 17-1] Figure 17 shows images demonstrating that hydrodynamic injection into the pancreas can result in areas of tissue injury and potentially immune infiltration. Pancreatic tissue was evaluated for signs of tissue damage. In the top two images, areas of immune infiltration with neutrophils and lymphocytes were observed 4-5 times per tissue section. In the bottom image, acellular areas of necrosis are observed surrounded by areas of neutrophil and lymphocyte infiltration. [Figure 17-2](As mentioned above.) [Figure 18-1] Figures 18A and 18B show an image and four photographs demonstrating the development of ductal injury and pancreatic caseous necrosis after hydrodynamic injection. Hydrodynamic injection was performed into a pig's pancreas through the ductal system. An 11 mm balloon was inflated prior to injection to seal the duct. The pig was injected with 22 mL @ 2 mL / sec injection parameters, resulting in the delivery of 2 mg of pDNA in pCLucf. Figure 18A shows fluoroscopy after injection. Redness of the contrast agent was observed, revealing that the contrast injection had leaked from the ductal system and caused wall rupture. Three days after injection, the pig was removed, and the pancreatic tissue was analyzed. Gross examination and dissection of the duodenal lobe of the pancreas depicted in the photograph in Figure 18B revealed large areas of pale yellow tissue consistent with injection-induced necrosis and injury, as seen in the left, top, and bottom photographs. The photograph on the right shows a magnification of the necrotic and injured tissue. Histological staining of the remainder of the organ did not reveal any gene delivery (data not shown). [Figure 18-2] (As mentioned above.) [Figure 19-1]Figures 19A-19C show images of gene delivery to the pancreas with minimal injection parameters. Hydrodynamic injection through the ductal system was performed in a pig model. Reduced injection parameters were tested to assess whether reduced injection parameters could still mediate reduced gene delivery. Figure 19A depicts representative photographs of IHC staining for firefly luciferase, showing expression within pancreatic islets and ductal cells, after the first pig was injected with 20 mL at 1 mL / sec, resulting in the delivery of 4 mg of pDNA (pCLucf). Amylase levels are reported before and after the procedure. Gross dissection of the pancreas revealed no abnormalities. Figure 19B depicts representative photographs of IHC staining for firefly luciferase, showing expression within pancreatic islets and ductal cells, after the first pig was injected with 15 mL at 2 mL / sec, resulting in the delivery of 4 mg of pDNA (pCLucf). Amylase levels are reported before and after the procedure. Gross dissection of the pancreas revealed no abnormalities. Figure 19C compares the area of ​​immune infiltration in pigs infused with injection parameters of 22 mL of 1 mg pDNA at 2 mL / sec and 15 mL of 4 mg pDNA at 2 mL / sec, demonstrating reduced immune infiltration with lower injection parameters. [Figure 19-2] (As mentioned above.) [Figure 20] Figure 20 shows a conventional straight-type plastic biliary stent placed across the papilla and cystic duct. This type of stent is designed for optimal bile drainage and has an internal anti-migration flare. This is an example of a type of stent that does not facilitate the goal of bypassing the cystic duct opening because the relatively narrow caliber of the stent and two additional side holes in the bile duct side of the stent toward the liver end of the stent do not adequately direct injected fluid away from the bile duct or between the stent and the bile duct wall. [Figure 21]Figure 21 shows a relatively close-up view of a conventional straight plastic biliary stent positioned to cross the papilla and cystic duct. As noted above, this is an example of a type of stent that, due to its relatively narrow caliber and two additional side holes in the bile duct side of the stent toward the liver end of the stent, does not adequately direct injected fluid away from the bile duct or between the stent and the bile duct wall, thereby failing to facilitate the goal of bypassing the cystic duct orifice. [Figure 22] Figure 22 shows a balloon occlusion catheter placed within a transcapillary straight plastic biliary stent crossing the cystic duct. As noted above, this is an example of a type of stent that, due to its relatively narrow caliber and additional side holes in the bile duct side of the stent toward the liver end of the stent, is unable to sufficiently direct injected fluid away from the cystic duct orifice, despite the presence of an occlusion balloon, thereby failing to facilitate the goal of bypassing the cystic duct orifice. [Figure 23] Figure 23 shows a large-diameter, fully covered metallic biliary stent placed transcapillary and positioned across the cystic duct orifice. The stent quickly expands to adhere to the bile duct wall and form a tight seal. A balloon occlusion catheter with an infusion function is placed within the stent, and the balloon is inflated to adhere to the stent wall and form a tight seal. Plasmid DNA or medical fluids are then injected through the stent at the common bile duct level. [Figure 24] Figure 24 shows a large-diameter, fully covered metallic biliary stent placed within the bile duct, covering the cystic duct orifice but not across the papilla. The stent quickly expands to seal tightly against the bile duct wall. A balloon occlusion catheter with infusion capabilities can be placed within the stent or within the common bile duct below the level of the distal aspect of the stent. The balloon is inflated to seal tightly against the common bile duct wall, and plasmid DNA or medical fluids are injected through the stent at the level of the common bile duct. A short tether is attached to the distal end of the stent and delivered to the duodenum. [Figure 25] FIG. 25 illustrates a method for placing a balloon occlusion catheter in the common bile duct and infusing fluid into the liver while the cystic duct is occluded by a balloon placed percutaneously in the cystic duct. [Figure 26] Figure 26 shows how a balloon occlusion catheter can be placed in the common bile duct to infuse fluid into the liver while occluding the cystic duct with an umbrella placed percutaneously at the junction of the cystic duct, gallbladder neck, or cystic duct entrance to the bile duct. This figure also shows how the balloon can be positioned within the stent so that it effectively seals the entire stent and prevents contrast leakage along the sides of the stent. The flexibility of this approach is demonstrated by the placement of balloons in different regions of the stent (common hepatic duct vs. common bile duct) to effectively encapsulate contrast. [Figure 27-1] Figures 27A-27C show application embodiments including a stent, a balloon, and contrast media. Figure 27A shows an image obtained with a stent with a balloon placed inside. Figure 27B shows an image of a stent with a balloon placed inside at approximately the level of the common hepatic duct, with contrast media placed above the balloon. Figure 27C shows an image of a stent with a balloon placed inside at approximately the level of the common bile duct, with contrast media above the balloon. [Figure 27-2] (As mentioned above.) [Figure 28-1]Figures 28A-28D show that different flow rates modulate gene expression efficiency during biliary hydrodynamic delivery. Four separate flow rates (1 mL / s, 4 mL / s, 7 mL / s, and 10 mL / s) were tested at a fixed volume of 40 mL and a fixed DNA dose of 10 milligrams of plasmid DNA, pCLucf. The pigs used in the study ranged in size from 37.1 to 45.9 kg. Immunohistochemistry for firefly luciferase protein was performed on pig liver tissue sections collected 1 day after injection. Figure 28A shows that a flow rate of 1 mL / s resulted in little staining of bile ducts or hepatocytes. Figure 28B shows that a flow rate of 4 mL / s resulted in the highest transfection rate of hepatocyte staining, while also demonstrating delivery to bile ducts and endothelial cells. Figure 28C shows that flow rates of 7 mL / sec and 10 mL / sec in Figure 28D show a similar transfection pattern of positive cells as the 4 mL / sec flow rate, with strong expression observed in bile ducts and endothelial cells. The amount of protein staining intensity in hepatocytes gradually decreased with a slight decrease in the transfection area. The most prominent staining in hepatocytes was observed near the lobule borders and / or large blood vessels. [Figure 28-2] (As mentioned above.) [Figure 29-1] Figures 29A-29C show that different volumes modulate gene expression efficiency during biliary hydrodynamic delivery. Three separate volumes (40 mL, 60 mL, and 80 mL) were injected at a fixed flow rate of 2 mL / sec and a pDNA dose of 10 mg pCLucf. All pigs weighed 40 kg. Immunohistochemistry for firefly luciferase protein was performed on pig liver tissue sections harvested 1 day after injection. Figure 29A shows that the 40 mL volume demonstrated efficient hepatocyte transfection, with expression in bile ducts and endothelial cells. Figure 29B shows that the 60 mL volume demonstrated relatively similar hepatocyte delivery to the 40 mL volume, while maintaining similar delivery to bile ducts and endothelial cells. Figure 29C shows that the 80 mL volume resulted in reduced protein staining intensity in hepatocytes and a mild reduction in transfection efficiency. However, delivery intensity to the bile duct was maintained. [Figure 29-2] (As mentioned above.) [Figure 30] Figure 30 illustrates the improvement in gene delivery efficiency during biliary hydrodynamic injection by comparing Nanoplasmid versus regular plasmid. The novel DNA vector platform was tested against regular plasmid DNA vectors with a conventional bacterial backbone to understand its effect on gene delivery efficiency during biliary hydrodynamic injection. To test this, 10 mg of regular plasmid DNA vector and 10 mg of Nanoplasmid were infused into pigs at 40 mL and 2 mL / s. The Nanoplasmid platform contains a smaller vector backbone (less than 500 bp) compared to regular plasmid DNA (greater than 2 kb). Pigs were injected with the same LP1-ATP7B,C9 expression cassette, and pig liver tissue was stained for C9 tags. Comparing equivalent pDNA doses normalized by DNA size, we observed that the transfection area with Nanoplasmid was significantly larger than that with regular plasmid (p=0.0002). Representative photographs of IHC stained areas with Nanoplasmid and regular plasmid are shown. A parametric t-test was used (significant p<0.05). [Figure 31] Figure 31 shows that nanoplasmid mediates long-term expression in pigs after biliary hydrodynamic injection. The duration of expression from episomal DNA injected into pigs after biliary hydrodynamic injection was examined. It was hypothesized that nanoplasmid mediates long-term expression in the liver compared to regular plasmid. Four pigs were injected with 20 mg of nanoplasmid LP1-ATP7B,C9, and efficiency was verified using C9-tag staining on the liver. Pigs were euthanized monthly to evaluate the duration and efficiency of expression. Representative images of IHC staining of each pig's liver are shown, demonstrating successful expression up to 4 months. Regular plasmid injected as a control showed no expression at 1 month after injection (data not shown). [Figure 32]Figure 32 demonstrates that biliary hydrodynamic injection mediates efficient, long-term transposase expression in pigs. The duration of protein expression after biliary hydrodynamic injection was evaluated using an integration system. The piggyBac transposon system was used to integrate a transgene cassette from plasmid DNA injected via the hydrodynamic procedure. Three pigs were injected with 15 mg of transposon and 5 mg of transposase. Pigs were injected with the same LP1-ATP7B,C9 expression cassette, and liver tissue from the pigs was stained for the C9 tag to assess the presence and delivery efficiency of the gene-derived protein. Midway through the experiment, 1.5 months after injection, core and wedge biopsies were taken from the animals, revealing abundant protein expression in the neck and in a cytoplasmic pattern (data not shown). Upon euthanasia of the animals 3 months after injection, abundant staining for ATP7B,C9 was observed between hepatocytes in all three animals. Representative IHC images are shown alongside those of uninjected control tissue. The estimated transfection efficiency was calculated to be 41.83%-44.50%. This compares favorably with the transfection efficiency (42.62%-48.44%; not significant) one month after injection in a cohort of pigs injected with the same pDNA combination. Neither cohort was significantly different from pigs sampled three days after injection. This demonstrates that the transposon can mediate stable expression over time without any change in relative transfection efficiency. Using a parametric t-test (significant p<0.05), [Figure 33]Figure 33 shows that biliary hydrodynamic injection can be re-administered with similar efficiency achieved for each gene. A key unknown with biliary hydrodynamic injection is whether pDNA can be successfully re-administered. This was assessed through a series of injections spaced 4 weeks apart. Briefly, three pigs were injected with 15 mg of transposon and 5 mg of transposase encoding the hepatocyte-specific LP1-ATP7B,C9 expression cassette. Four weeks later, the pigs were injected with the plasmid pCMV-GFP-ATP7B, which carries a different protein tag and a ubiquitous promoter. Three days later, the pigs were harvested, and the C9 and GFP tags in the pig liver tissue were stained to assess the presence and delivery efficiency of the gene-derived proteins. Representative serial sections of a single lobule are depicted on the left, demonstrating that ATP7B,C9, and GFP-ATP7B can be detected in the same lobule and the same cells. GFP staining also reveals that other cell types (bile ducts, endothelial cells) can be targeted during the second injection (bottom left). Immunofluorescence staining provided further evidence of colocalization within the same hepatocyte (data not shown). Transfection efficiency, as measured by the stained lobule area, was compared between the first (C9) and second (GFP) injections and showed similar results (46.2% vs. 50.7%; not significant) using a parametric t-test (significant P<0.05). [Figure 34]Figure 34 shows that increasing the plasmid DNA dose mediates higher transfection efficiency after biliary hydrodynamic injection. The effect and ability of gradually increasing the pDNA dose to increase transfection efficiency was not tested. For this purpose, four different pDNA doses (10 mg, 20 mg, 30 mg, and 40 mg) were examined. The plasmid pT-LP1-ATP7B,C9 was used in all studies for standardization. Pig livers were harvested three days after injection and stained for the C9 tag with the 1D4 antibody. Representative images of pig livers showing the 10 mg, 20 mg, 30 mg, and 40 mg doses are shown. Quantification of the stained area of ​​individual lobules for each pDNA dose in randomly counted lobules from each pig is shown, demonstrating a significant increase in the efficiency of biliary hydrodynamic injection with increasing pDNA dose. A parametric t-test was used (significant p<0.05). [Figure 35] Figure 35 shows an evaluation of the size limit of plasmid DNA that can be delivered by biliary hydrodynamic injection. While biliary hydrodynamic injection can mediate the delivery of naked plasmid DNA into cells, it is unclear whether there is an upper limit to the size of plasmid DNA for this approach. To address this, 12 kb and 17 kb plasmid DNA were injected into pig livers at 40 mL and 2 mL / sec injection parameters. The plasmid DNA doses were 10 mg pDNA and 15 mg pDNA, respectively. Pigs were harvested 3 days after injection. Representative areas of immunostaining for the 12 kb plasmid, pCDNA4 / full-length FVIII, are depicted at low and high magnification. At low magnification, the image shows gene delivery to all lobules of the stained section. At high magnification, the most intense staining is seen around the central vein, reflecting increased localized pDNA delivery. Quantitation of stained area revealed that 49.48% of hepatocytes expressed the 12 kb gene, which was not significantly different from the previously injected 5.5 kb and 8.6 kb plasmids. Tests of the 17 kb plasmid showed similar potency and relative efficiency of protein expression (data not shown). [Figure 36]Figure 36 demonstrates that biliary hydrodynamic injection can achieve expression in vascular endothelial cells and hepatic sinusoidal endothelial cells. Previous studies have observed that biliary hydrodynamic injection achieves gene expression in endothelial cells and bile duct cells using ubiquitous promoters. However, immunostaining near blood vessels and bile ducts can sometimes be unreliable. To verify specific delivery of plasmid DNA to these cell types, two different endothelial cell-specific promoters were employed to achieve GFP expression specifically in vascular endothelial cells or hepatic sinusoidal endothelial cells, respectively. Plasmids pICAM2-GFP and pCD36p-Luc were synthesized and injected into pig livers at 2 mL / s in a 40 mL volume at a 10 mg pDNA dose. Pigs were harvested 3 days after injection. Representative IHC staining for GFP and luciferase for each plasmid is shown. The ICAM-2 promoter showed strong and distinct GFP staining in all arterial endothelial vessels observed throughout the entire section. No staining was observed in large venous vessels or along the hepatic sinusoids. The CD36 promoter showed strong and clear staining along the hepatic sinusoids, but little staining in the portal vein region. [Figure 37] Figure 37 shows the validation of volume-to-liver weight dosing for biliary hydrodynamic infusion in pigs. The applicability of volume-to-liver weight dosing parameters to animals of different sizes remains uncertain. To address this question, different infusion parameters were tested based on volume-to-liver weight dosing. These included 20 mL / kg, 30 mL / kg, 40 mL / kg, and 55 mL / kg. All infusions were performed at the same flow rate of 2 mL / sec with a fixed dose of 10 mg pDNA in pCLucf. Immunohistochemical staining results of the infusions are shown by IHC staining for firefly luciferase. The results of the example demonstrate that pigs weighing 25 kg and 27 kg can successfully express proteins across different volume-to-liver weight doses, as tested at 40 mL / kg and 55 mL / kg, respectively. Smaller pigs weighing 5 kg and 15 kg were also tested separately, with similar results (data not shown). [Figure 38] Figure 38 shows the evaluation of the pressure threshold for biliary hydrodynamic gene delivery to pigs. The minimum pressure threshold for gene delivery via biliary hydrodynamic infusion remains unknown. To address this, pigs were infused using a constant-pressure infusion device. The pressure threshold was set at 50 mmHg and maintained throughout the infusion. The total injection volume was 30 mL, and the pDNA dose was 10 mg pCLucf. IHC staining of firefly luciferase was performed on liver tissue sections. For comparison, a control tissue sample infused with pCLucf at 2 mL / sec is provided. Results showed that a pressure threshold of 50 mmHg was insufficient for infusion, and no IHC staining for GFP or luciferase was observed. A separate experiment demonstrated that 80 mmHg was sufficient for gene delivery. [Figure 39] Figures 39A-39F demonstrate that hydrodynamic injection via ERCP is feasible in nonhuman primates. The anatomy of the baboon liver is depicted (Figure 39A), with a short common hepatic duct (CHD) of approximately 1.5 cm before the bile duct enters the liver (Figure 39B). A duodenoscope used in humans can be advanced into the small intestine to confirm the biliary opening in the baboon (Figure 39C). The opening can then be successfully cannulated and advanced into the biliary system (Figure 39D). Pre-injection fluoroscopy demonstrates the distinct branches of the baboon's biliary system (Figure 39E). After hydrodynamic injection, the bile duct branches are again visualized and shown to be intact (Figure 39F). [Figure 40]Figures 40A-40C show the safety evaluation of biliary hydrodynamic infusion in nonhuman primates. To monitor potential toxicity of the procedure, various common clinical tests were tested before and several days after hydrodynamic infusion. The study consisted of four baboons infused with similar parameters. Each baboon underwent (Figure 40A) a panel of liver function tests consisting of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), gamma-glutamyltransferase (GGT), albumin, and alkaline phosphatase. (Figure 40B) a panel of hematological tests consisting of white blood cell count (WBC), granulocyte count, and hemoglobin. (Figure 40C) Post-ERCP pancreatitis was assessed by monitoring amylase and lipase enzymes, biomarkers of pancreatic injury. [Figure 41] Figure 41 shows that the bile duct wall in primates becomes more elastic, resulting in leakage around the balloon. A series of fluoroscopic images from three baboon species are shown. Injection parameters were similar (3–4 mL / sec, 30–40 mL volume). The balloon was positioned 8.5 mm into the common hepatic duct (CHD) for each injection. Contrast medium was placed under the balloon to visualize potential leaks. Pre- and post-injection fluoroscopic images are shown for all three baboons. The increase in gallbladder size after injection reflects fluid flowing out from around the balloon in the CHD and into the gallbladder through the cystic duct. The black arrow points to the common bile duct, and the removal of contrast medium after injection reflects antegrade flow of saline solution around the balloon. Post-injection fluoroscopic images from Baboon #3 show significant dilation of all bile ducts compared to pre-injection, a finding observed in the other baboons tested. [Figure 42]Figure 42 demonstrates the risk of rupture if the catheter balloon is too large in the extrahepatic bile duct. Increasing the balloon size ensures a pressure seal and prevents leakage, but increases the strain on the bile duct wall. To test this, we present a time-lapse series of fluoroscopic images of a single baboon during hydrodynamic injection. Injection parameters were 4 mL / s with a 40 mL volume of saline solution. The common hepatic duct diameter before injection was approximately 3 mm, and the balloon was inflated to 15 mm. Upon hydrodynamic injection, the already maximally stretched bile duct wall was observed to rupture around the balloon, resulting in the spillage of contrast agent into the surrounding tissue. Similar findings could be reproduced in pigs with a balloon-to-extrahepatic bile duct size ratio of 4-5 times. [Figure 43] Figures 43A and 43B demonstrate successful liver sealing in primates by adjusting the catheter balloon position and size. Serial fluoroscopic images from two different baboons are shown. Injection parameters were similar between the two injections. In both baboons, the catheter balloon was positioned intrahepatically in the left hepatic branch. The balloon was inflated to a size of 11.5 mm. (Figure 43A) Pre- and post-injection fluoroscopic images are shown for Baboon #1 and Baboon #2. As shown, the injected saline passed through the intrahepatic ducts during hydrodynamic injection, as expected. Importantly, the gallbladder size in both animals did not increase after injection, and the black arrows indicate that the common bile duct did not clear the contrast solution. (Figure 43B) Images from one baboon are shown demonstrating hydrodynamic injection of the contrast solution into the baboon's liver via the biliary tract. The contrast is observed to penetrate the liver parenchyma (acinar contrast). [Figure 44]Figures 44A–44D demonstrate that pressure monitoring can be used to detect fluid leaks due to poor sealing during biliary hydrodynamic infusion in primates. A series of biliary hydrodynamic infusions was performed in a cohort of baboons. A pressure transducer (EchoTip Insight, Cook Medical) sensing the fluid-filled column in the infusion port was connected to a Multi-3V catheter (Olympus). Infusion parameters were as shown above the graphs. For the infusion in Figure 44A, the balloon size was 8.5 mm within the common hepatic duct, demonstrating a gradual drop in pressure during the infusion. For Figure 44B, a faster flow rate and a larger volume were used to test whether this pressure drop could be overcome, but the results were the same with a gradual drop in pressure. For Figure 44C, an alternative strategy was employed, varying the flow rate in real time during the infusion. This was effective in stabilizing and, in some cases, increasing the pressure, but the overall pressure amplitude remained low. (Figure 44D) Adjusting the balloon placement (intrahepatic location, left branch) and size to 11.5 mm resulted in stable plateau pressures throughout the infusion period without the previously observed losses. Notably, these pressures were achieved at flow rates significantly lower than the other flow rates tested without a good seal. [Figure 45]Figures 45A-45D demonstrate that biliary hydrodynamic injection can mediate gene delivery in nonhuman primates. Cohorts of baboons were obtained to test for expression of the delivered gene. Balloon size, position, and parameters were similar in all groups. (Figure 45A) Two baboons were repeatedly injected with a DNA vector encoding human FIX (hFIX). The first injection was a 20 mg pDNA dose, while the second injection was a threefold higher 60 mg pDNA dose. Both animals demonstrated a dose-dependent response, with threefold higher hFIX expression within each individual baboon. (Figure 45B) Monthly repeated gene delivery procedures in each individual baboon achieved similar peak expression of hFIX at day 1 postinjection with similar doses and parameters. (Figure 45C) An alternative DNA molecule, nanoplasmid DNA, was compared to regular plasmid DNA with a twofold higher expression of a large bacterial backbone (e.g., antibiotic resistance genes). As shown, nanoplasmid allows for lower DNA doses that can still result in hFIX expression levels comparable to regular pDNA at much higher DNA doses (Figure 45D). In another experiment, baboons were injected with GFP / luciferase reporter DNA via biliary hydrodynamic injection and euthanized 24 hours after injection. Detection of GFP expression by immunohistochemical staining revealed positive gene expression around the central vein of the baboon's liver lobule. [Figure 46] Figure 46 demonstrates that mechanical injury caused by a guidewire can lead to an intrahepatic biliary leak. A guidewire is initially used to cannulate and aid in accessing the biliary system, after which a catheter is inserted over the guidewire into the bile duct. As shown in the fluoroscopic image, it is common for a guidewire to enter the intrahepatic system, protruding deep into the left duct system. However, once the catheter was placed in the bile duct and contrast was injected, a small contrast reddening was observed at the location of the guidewire tip. During the subsequent hydrodynamic injection, the size of the contrast / bile leak increased, highlighting its potential to communicate with the injected fluid and act as a sieve and restrictor for pressure generation. [Figure 47]Figures 47A-47D demonstrate that a stent-based approach can be used for biliary hydrodynamic injection. A series of fluoroscopic images demonstrate the utility of a balloon-less stent catheter for the injection of contrast fluid into the liver. (Figure 47A) An unstented catheter is advanced beyond the cystic duct into the common hepatic duct. (Figure 47B) The stent within the catheter is 50% deployed, and the stent opens at its distal end, as indicated by the black arrow. (Figure 47C) Contrast was injected through the catheter's guidewire channel, leaving the olive-colored tip of the catheter. No contrast was detected within the stent, nor was there any extension of contrast around the stent into the cystic duct. (Figure 47D) The stent and catheter were retracted into the common bile duct, and contrast was injected through the catheter, showing the cystic duct becoming opacified. Together, stent placement can block fluid flow into the cystic duct, enhancing the feasibility of delivering biliary hydrodynamic injection. [Figure 48] Figures 48A-48D demonstrate that biliary hydrodynamic injection can achieve gene delivery to liver tumors. The Oncopig model harbors mutations in TP53R167H and KRASG12D, key tumor suppressors and oncogenes, which can be activated by the introduction of Cre recombinase. After virally introducing Cre into cells, large tumors can grow within 1-2 weeks in multiple different tissue types. (Figure 48A) Liver tumors induced in the pig liver are depicted (1-2 cm in size), showing histologically large, highly necrotic tumors. Tumor-bearing pigs were injected with GFP / luciferase reporter DNA by biliary hydrodynamic injection, and immunohistochemical staining for GFP was performed to detect positive cell types. (Figure 48B) GFP-positive cells were detected in the peritumoral area at the tumor border, where tumor tissue invaded normal 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 was still observed in the same liver section, demonstrating the ability to target both cell types. [Figure 49]Figures 49A-49C demonstrate that ductal hydrodynamic injection can achieve gene delivery to pancreatic tumors. The Oncopig model harbors mutations in TP53R167H and KRASG12D, key tumor suppressors and oncogenes, which can be activated by the introduction of Cre recombinase. After virally introducing Cre into cells, large tumors grow within 1-2 weeks in multiple different tissue types. (Figure 49A) Pancreatic tumors were induced in the pancreas of a pig. Tumors are visible histologically in pancreatic sections (black arrows). Tumor-bearing pigs were injected with GFP / luciferase reporter DNA via ductal hydrodynamic injection, and immunohistochemical staining for GFP was performed to detect positive cell types. (Figure 49B) Pancreatic tumor sections were obtained and stained for GFP, revealing scattered positive cells of varying intensity. (Figure 49C) Normal pancreatic tissue sections on the same slide also demonstrate gene delivery primarily to ductal cells, as detected by GFP staining. In this way, both tumor and normal tissues can be targeted simultaneously for DNA delivery in a single injection. DETAILED DESCRIPTION OF THE INVENTION

[0296] The prior art has established embodiments of biliary hydrodynamic injection that focus on flow rate as a key parameter for successfully programming gene delivery to liver tissue. The prior art provides recommendations regarding optimal flow rates for achieving gene delivery by biliary hydrodynamic injection, along with recommendations regarding the use of specific flow rates to target specific regions in the liver.

[0297] The present disclosure relates to improved hydrodynamic injection and delivery methods that offer advances over prior art approaches. In certain aspects, the present disclosure relates to compositions and methods for treating kidney disease. More particularly, the present disclosure relates to compositions and methods for treating kidney disease via gene therapy. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that ureteral hydrodynamic injection can mediate renal cortical rupture at a variety of different flow rates and volumes, resulting in the observation that only limited parameters are safe. There is likely no vascular or lymphatic escape route, which has not been previously tested. What was discovered was that volume was the critical parameter that led to rupture, as lower volumes were better tolerated, regardless of flow rate. Nevertheless, injection parameters that appear to be safe and capable of mediating gene delivery have been discovered, none of which have been previously described, and indeed, it was doubtful whether any would ever be discovered. It was also surprising that the procedure could be performed without the need for fluoroscopy, a limitation of all other previous gene therapy procedures. Even more surprising was the relative susceptibility of the kidney to injury during proximal infusion, and furthermore, the relatively low flow rates allowed for large increases in pressure during infusion.

[0298] Overview The kidney is an attractive target for gene therapy. Current treatment strategies are ineffective for many patients who require regular dialysis for chronic kidney disease (CKD). In terms of healthcare burden, CKD is one of the largest contributors to rising healthcare costs in the United States. Any solution directed at treating CKD, whether by reversing CKD or preventing its onset, would be highly beneficial. Chronic dialysis is a burdensome and debilitating procedure for patients. While kidney transplantation is potentially curative, many patients cannot undergo kidney transplantation due to limited donor availability. Kidney transplantation also has many significant risk factors, particularly with the complications of rejection and chronic immunosuppression.

[0299] In addition to chronic kidney disease, there are several other important kidney diseases. A 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. Causative mutations in these genes lead to overactivation of renal tubules, which results in the formation of cysts throughout the kidney. The accumulation of these cysts over time can later lead to chronic kidney disease and the need for a transplant. Current treatments are inadequate and lack significant efficacy. Many individuals ultimately require a kidney transplant due to PKD. Other major diseases include various glomerular disorders, including anti-GBM disease and IgA nephropathy. These are typically characterized by immune processes affecting these organs, leading to the destruction of glomeruli through either inflammation or fibrosis with thickening of the glomerular basement membrane.

[0300] Current treatments are ineffective for all of these indications of various kidney diseases. Gene therapy is an emerging treatment that may help address this clinical gap. However, kidney gene therapy is limited by several factors. Many researchers have utilized systemic injection of viral vectors, including adeno-associated viruses (AAVs). AAVs are efficient and deliver genes to numerous organs, including the liver. Unfortunately, AAVs exhibit very poor transduction in the kidney. While this poor transduction can be attributed to many factors, the most significant problem is glomerular size exclusion when filtering blood. There is no conventional route for viral vectors to travel from the systemic circulation to the kidney. Because systemic injection of viral vectors is ineffective, some researchers have pursued local administration of AAVs to the kidney. Examples of local routes include injection into the kidney via the renal artery or renal vein. While these administration routes have shown promise in achieving delivery in mouse models, they remain relatively inefficient. Another approach is to inject the vector via the ureter. This strategy is relatively non-invasive and does not require any skin incision. However, most viral vectors injected via the ureter rapidly leak from the kidney and return to the systemic circulation (Hum Gene Ther. 2019 Dec;30(12):1559-1571). In these cases, transduction of the liver is more effective than transduction of the kidney.

[0301] Nonviral approaches appear attractive for kidney gene delivery. Chronic kidney disease affects 30 million or more people in the United States, meaning the total number of patients far exceeds the manufacturing capacity of viral vectors. Renal cells have a shorter half-life than more stable cells such as hepatocytes, so any kidney damage treatment likely requires the ability to be re-administered. Nonviral vectors potentially address this weakness because they do not initiate an immune response. Unfortunately, the most common strategy for delivering nonviral vectors also suffers from the same limitations as viral vectors. Approaches for delivering nucleic acids use delivery vehicles such as lipid nanoparticles, which range in size from 100 to 200 nanometers. Lipid nanoparticles are highly efficient at delivering siRNA or mRNA to various cell types, including hepatocytes and macrophages, among others. Lipid nanoparticles are particularly useful for vaccine applications in combination with messenger RNA. Lipid nanoparticles are less efficient for DNA delivery due to their poor nuclear delivery.

[0302] If lipid nanoparticles (LNPs) were obtained and administered systemically via the vascular system, they would face the same size-limiting barrier as viral vectors in the renal glomerulus. Therefore, LNPs cannot be efficiently internalized into the kidney to transfect renal cells. Previous studies have not described the local administration of nanoparticles to the kidney.

[0303] As an alternative approach, hydrodynamic gene delivery shows great promise. It utilizes fluid pressure to physically deliver DNA into various cell types. This occurs via the transient generation of pores in the cells, which leads to the internalization of naked DNA into these cell types. This strategy is best known in mouse models, where hydrodynamic tail vein injection can efficiently introduce plasmid DNA directly into the mouse liver. The same hydrodynamic tail vein technique can also be used to deliver DNA to other organs, although at a much lower rate. Researchers have attempted to apply localized fluid pressure to various other organs to drive DNA internalization in these organs. Ultimately, local administration is the only method that can be adapted to large animal models.

[0304] One study of hydrodynamic injection into the kidney was performed via the renal vein (Mol Ther. 2008 Jun;16(6):1098-104). In a rat model, the renal vein was exposed through a surgical procedure. The reported peak pressure during injection was 100 mmHg, and a DNA concentration of 100 μg / mL was injected. For gene delivery to the kidney of a larger pig model, catheterization of the right renal vein was performed using an interventional radiology-guided fluoroscopic procedure. An inferior vena cava (IVC) occlusion balloon was utilized to block the junction where the renal vein returns to the IVC, preventing fluid leakage back into the IVC during hydrodynamic injection. Using this strategy, researchers were able to obtain gene expression in the targeted pig kidney, as measured by firefly luciferase activity. The procedure appeared safe with no increase in creatinine, although immunohistochemical staining data were not reported.

[0305] While vascular strategies are promising, an alternative hydrodynamic approach is injection via the urinary system. Proof-of-concept for this approach was studied in a mouse model, where the hydrodynamic injection procedure was directed toward the renal pelvis (Sci Rep. 2017 Mar 20;7:44904). Prior to injection, the mouse kidney was accessed through a surgical incision and exposure of the kidney through the retroperitoneum. A needle was inserted into the renal pelvis, and the injection proceeded with high fluid pressure. The injected solution contained naked plasmid DNA. As a result, scattered positive cells of multiple cell types were observed within the kidney.

[0306] Previous studies have demonstrated several different routes to applying hydrodynamic pressure to the kidney. A third route, not explored in the published literature, is hydrodynamic infusion via the ureter. Because the ureter is a unidirectional vessel that runs from the kidney to the bladder, it may be easier to apply higher fluid pressures during retrograde infusion. Furthermore, the ureter is easily accessible through simple cystoscopy into the bladder.

[0307] Hydrodynamic retrograde ureteral injection Previous studies focusing on retrograde ureteral injection have presented ideas about how hydrodynamic procedures through the ureter might be performed. These ideas have not been validated by any experimental evidence, and therefore, the specified procedures may or may not be successful in gene delivery. Previously disclosed procedures for hydrodynamic gene delivery to the kidney are summarized as follows:

[0308] The first step is to obtain a cystoscope and advance it through the urethra into the bladder. A catheter is then advanced through the cystoscope into the right or left ureter. The catheter is then advanced to the distal end of the ureter and its entrance into the renal pelvis, preferably using contrast injection and fluoroscopy to confirm catheter position. Optionally, fluid can be aspirated through the catheter to drain urine from the renal pelvis and reduce potential toxicity when hydrodynamic injection begins. The balloon is opened to seal the ureter, preventing antegrade flow of solution during hydrodynamic injection. The next step is to inject contrast and image using fluoroscopy to confirm balloon sealing, optionally followed by removal of the contrast. The catheter circuit is then primed with DNA solution from the power injector to the distal tip. Hydrodynamic injection of the nucleic acid and / or protein-containing fluid then proceeds.

[0309] The balloon is then deflated after the injection has stopped. Contrast injection and fluoroscopy may be repeated. The catheter and guidewire are removed from the ureter, and the procedure may be repeated on the uninjected kidney, if desired. Upon complete completion of the procedure, the catheter, along with the cystoscope, is removed from the patient's ureter, bladder, and urethra. Following injection, there is an optional step of monitoring post-injection creatinine, blood urea nitrogen, and glomerular filtration rate to monitor for damage from the hydrodynamic injection.

[0310] Several additional steps can be incorporated to help facilitate the procedure. The use of a guidewire can facilitate cannulation of the ureteral orifice and subsequent catheter insertion. Injection of nucleic acid and / or protein-containing fluids using an automatic injector at high speed and pressure can be monitored with a pressure catheter to ensure the quality of the injection target is reached. Another option is to follow up the macromolecule-containing fluid with a macromolecule-free solution, such as saline solution, to ensure complete delivery of the solution into the kidney.

[0311] This study outlined several hypothetical injection parameters, but did not provide data supporting their effectiveness or provide a biological basis for them. Flow rates of at least 1 mL / sec, at least 2 mL / sec, or at least 3 mL / sec were suggested. Total injection volumes of 10 mL, 20 mL, 30 mL, 40 mL, or up to 50 mL were suggested, but the results indicated that the volume leaked from the renal pelvis into the parenchyma. It is unclear how this volume would be affected by variations in pig size. Pressures suggested to be achieved during kidney injection are a minimum of 50 mmHg, at least 75 mmHg, or at least 100 mmHg. It is unclear whether any of these pressures would be effective in delivering genes to the kidney.

[0312] In other aspects of the procedure, the method of simultaneous administration of various different drug solutions was also presented to reduce inflammation and / or potential infection due to the injection procedure.The best drugs to be used at appropriate doses and their effectiveness at those doses were not described.A method of cell-specific targeting was described by injecting DNA molecules containing cell-type specific promoters that target the expression of specific cell types, but there was no confirmation data.

[0313] In summary, previous studies have not established the precise injection parameters for effective delivery of DNA into kidney cells. This ranges from the effective flow rate, volume, and pressure achieved during injection. The relationship between mammalian size or kidney size and injected volume has not been demonstrated. It is unclear what volume would be appropriate for translating this technique to human patients.

[0314] Previous studies have not demonstrated what DNA dose is required to achieve gene expression in the kidney from ureteral proximal kidney placement. Furthermore, no information has been reported regarding the safety of injection procedures for the proposed injection parameters. Previous studies have not demonstrated a procedure for gene therapy that is safe, effective, and produces the desired gene therapy results.

[0315] Detailed Description of the Invention This disclosure also provides details regarding an effective method for hydrodynamic gene delivery via the ureter to target gene expression to the kidney. This disclosure also describes novel methods for performing the procedure to provide greater efficacy and safety.

[0316] First Procedure Method for Gene Delivery Previous studies have disclosed a method of hydrodynamic injection via the ureter, in which a catheter balloon was placed in the ureter in close proximity to the kidney. This method has been difficult to reproduce. It was not possible to place the balloon in close proximity to the kidney without sliding the catheter into the renal pelvis. It was found that placing the balloon inside the renal pelvis resulted in rapid fluid leakage during hydrodynamic injection due to an ineffective seal (see, e.g., Figure 8A).

[0317] To develop a new method that is more robust and easier for physicians to perform, a novel procedure for hydrodynamic insufflation through the ureter was designed. The first step in this procedure is to insert a cystoscope into the bladder through the urethra. A catheter can be advanced into the bladder through the working channel of the cystoscope. The ureteral orifice (UO) can be visualized by examining the bladder wall using the cystoscope's camera. The catheter is then advanced toward the ureteral orifice so that infusion can be achieved.

[0318] Once the catheter enters the ureteral orifice, it is advanced approximately 1, 2, or 3 cm into the ureter. At this point, the balloon on the catheter is inflated so that it is just at the opening of the ureteral orifice in the bladder. The inflated balloon is visible as a bulge in the bladder wall through the cystoscope camera, thereby confirming its location. An advantage of this strategy is that balloon placement can be verified by visualization through the cystoscope camera. Therefore, fluoroscopy is not required to localize catheter placement. This offers immense safety benefits, including the elimination of radiation safety precautions and the elimination of expensive equipment, including a C-arm.

[0319] Another advantage is that the bladder wall, along with its muscular wall, provides reinforcement around the balloon as it expands, thereby helping to prevent the problem of ureteral rupture. Furthermore, the ureteral orifice can be visually monitored during injection to determine if there is fluid leakage, providing real-time feedback that the hydrodynamic injection was successful. A major advantage of this procedure and approach is that it can be performed in facilities without fluoroscopic imaging, rather than in a routine outpatient clinic setting. Furthermore, patients and clinical providers are exposed to less radiation. This new procedure increases the availability of this technique because bedside cystoscopy is relatively routine and can be performed in any outpatient facility.

[0320] A series of tests were performed to evaluate hydrodynamic infusion with distal ureteral balloon placement. In all tests, the balloon successfully sealed the ureter and blocked all antegrade hydrodynamic outflow to the bladder.

[0321] An important aspect of hydrodynamic gene delivery is that optimal injection parameters vary with minor procedural modifications. Given the distal ureteral placement of the balloon, an immediate consideration for this catheter position is what volume is required to fill the ureter and renal pelvis, with the ultimate goal of forcing fluid into the renal parenchyma during hydrodynamic injection. The ureteral volume was estimated to be 4–5 mL in empirical testing performed prior to injection. The total volume of the ureter and renal pelvis ranged from 6–9 mL when slowly filled with radiocontrast solution.

[0322] Optimal injection parameters from the distal ureteral location were empirically derived. The first step in these studies was to determine what volume and flow rate could be safely injected into the kidney without significant injury. The third parameter was to test what volume of injected fluid could mediate internalization of radiocontrast injection into the renal parenchyma, which in previous studies in the liver has been used as a surrogate for DNA internalization into the tissue. To accomplish this, a series of injections were performed to verify safe and effective parameters for injection.

[0323] This disclosure also presents an injection volume that avoids kidney rupture during hydrodynamic injection. Kidney rupture involves a physical tear extending from the renal medulla to the renal cortex. As shown in the data in this disclosure, the tear usually occurs in only one location (approximately 1 cm in size) in the kidney. Kidney rupture causes minor to major bleeding into the fibrous capsule surrounding the kidney. Repeated hydrodynamic injection tests into different kidneys demonstrated that bleeding from the induced tear eventually stopped, and as a result, none of the pigs suffered significant kidney damage. However, it is unclear whether this would be a more serious problem if it occurred in human patients. Therefore, hydrodynamic injection should be optimized to prevent kidney rupture.

[0324] Optimally, when injections are performed near the opening of the ureteral orifice, injection parameters of less than 20 mL in volume and less than 2 mL / sec in flow rate are utilized to avoid kidney rupture. Renal rupture frequently occurred at volumes greater than 20 mL, even when flow rates as low as 0.5 mL / sec were used. Flow rates greater than 2 mL / sec have also been found to cause rupture at these volumes and should be avoided. Flow rates of 0.5 mL / sec to 2 mL / sec have intermittently caused kidney rupture in some animals. Larger volumes greater than 20 mL can be envisioned, but require the use of reduced flow rates in the 1 mL / sec to 1.5 mL / sec range, which still poses a risk of kidney rupture in some animals.

[0325] Regarding the efficacy of gene delivery according to these parameters, the optimal injection parameters for mediating gene internalization into renal cells are a volume of 10-20 mL injected into a kidney weighing 70-80 g. This volume is injected at a rate of 0.5-2 mL / sec to achieve gene expression while avoiding harmful kidney trauma and damage. Gene expression has also been observed at higher injection flow rates, but this injection carries the aforementioned risk of unwanted kidney rupture.

[0326] One unexpected result was that even relatively low flow rates within the ureter could generate significant pressure. Flow rates of 0.5–1 mL / s were observed to generate pressures of 80 mmHg. Flow rates of 1.5–2 mL / s generated pressures of 120–140 mmHg. Thus, only a small flow rate is required to generate pressure, explaining why even 0.5 mL / s was able to produce gene expression and why relatively slow flow rates still carry the risk of kidney rupture. Optimally, a pressure of at least 80 mmHg can be targeted during infusion to achieve gene expression in various kidney cell types.

[0327] Optimally, the balloon is deflated after hydrodynamic injection, which can be confirmed by visual camera examination through the cystoscope. The catheter can be withdrawn from the ureteral orifice into the bladder. The catheter is then withdrawn through the cystoscope, which is then withdrawn from the patient. Kidney damage could be monitored by serum testing for creatinine and other biomarkers.

[0328] Second Procedure Method for Gene Delivery In a different example, the procedure proceeds via an alternative method: A cystoscope is advanced through the urethra into the bladder. A guidewire is then advanced through the cystoscope into one ureteral orifice. The guidewire is advanced completely into the renal pelvis, where it begins to loop, indicating its presence in the kidney, or alternatively, resistance is felt as the guidewire is advanced. The cystoscope is withdrawn from the bladder. A catheter is then inserted over the guidewire and advanced toward the kidney. The advancement of the catheter is monitored with fluoroscopic imaging using a C-arm. Preferably, the catheter has radiopaque markings to confirm its placement while it is advanced.

[0329] In this example, the catheter is positioned adjacent to the renal pelvis. The catheter is still positioned within the ureter. Successful ureteral occlusion is confirmed by examining the occlusion of antegrade radiocontrast flow. In one embodiment, a guidewire remains within the ureter after balloon inflation and injection to help verify its position. The guidewire can extend above or below the balloon. In another example, the guidewire is removed and the catheter position is confirmed solely by contrast injection.

[0330] Preferably, the catheter balloon is at least 1, 2, or 3 cm below the renal pelvis to ensure that the balloon can completely occlude the ureter, which is cylindrical in shape as opposed to the renal pelvis, which has a more conical shape. Optimally, balloon sizes of 11, 12, 13, 14, or 15 millimeters are used to successfully seal and block the ureter.

[0331] For this proximal injection near the renal pelvic point, the optimal injection parameters disclosed are as follows: For volumes 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 less than or equal to 10 mL. Small injection volumes are required because the upstream renal pelvis and ureter have small volumes, estimated at 3-4 mL total. Additionally, small injection volumes are used to avoid kidney rupture. Preferably, the flow rate is 0.5-1 mL / sec, inclusive, to mediate gene expression of the plasmid DNA within the kidney.

[0332] In another example, a constant pressure infusion device can be used for infusion. This can be tailored to a specific pressure target, reducing the risk of kidney rupture. Furthermore, given that many patients' kidneys have chronic kidney disease, the relative stiffness of the tissue differs from that of normal kidneys. Stiffness affects the relative resistance of the tissue, thereby increasing the pressure. The resulting pressure was evaluated for predetermined parameters at the proximal location. In one example, 10 mL was infused at a flow rate of 1 mL / sec. Pressure tracing revealed that a peak pressure of 105 mmHg was achieved. In an optimal embodiment of the present invention, a pressure of at least 100 mmHg can be targeted for infusion from the proximal location. In other embodiments, a pressure of at least 80 mmHg can be targeted for infusion from the proximal location.

[0333] After successful injection at these parameters, the balloon is deflated and the catheter is withdrawn from the ureter, bladder, and urethra. The guidewire is then withdrawn from the ureter, bladder, and urethra. Patients are monitored for any toxicity with follow-up urinalysis and serum chemistry, including creatinine levels.

[0334] Hydrodynamic retrograde ureteral injection of viral vectors Hydrodynamic injection has traditionally been used to deliver plasmid DNA into cells. As a result, it is traditionally considered a non-viral modality. However, hydrodynamic injection is a non-specific process that mediates the delivery and internalization of any number of DNA / RNA proteins, or even viruses, into cells (J Gene Med. 2006 Jul;8(7):852-73). Hydrodynamic injection can have multiple different mechanisms in this process, ranging from the expansion of cell internalization to the expansion of macromolecular penetration into tissues.

[0335] Viral vectors are the traditional backbone of gene delivery. Over a long period of evolution, viruses have acquired the ability to target and internalize cells, and in the case of DNA viruses, to translocate into the nucleus. However, viruses repurposed for gene therapy have not been adapted to the high level of delivery efficiency required. For example, viruses only need to target a portion of cells in a given tissue to replicate. Viruses have not immediately acquired the ability to penetrate tissues at the level desired for gene therapy, for example, to replace genes in all cells.

[0336] There has been limited research into the potential synergy between hydrodynamic gene delivery and its use with conventional viral vectors. One study used intravenous infusion to increase delivery of SV40 viral vectors to the liver (Hum Gene Ther. 2005 Mar;16(3):361-71). Expression levels were found to be much higher than with viral vectors alone. In this case, the SV40 vector did not target only the liver, as it has a natural tropism, so hydrodynamic delivery helped concentrate all viral vector delivery in the tissue.

[0337] Even with modalities that have a natural tropism for the liver, such as adenovirus, hydrodynamic delivery can play a beneficial role. Because hydrodynamic injection is a localized procedure that forces delivery of a substance to a specific tissue, it has the advantage of significantly reducing the viral vector dose required for treatment. This was seen in an early study in a mouse model, where hydrodynamic injection of adenovirus vectors increased liver transduction while reducing the level of inflammation normally induced by adenovirus (Mol Ther. 2005 Jul;12(1):99-106). This study was also transferred to a non-human primate model, where more efficient injection also helped reduce the amount of vector dose required, leading to longer expression than would normally be required (Mol Ther. 2007 Apr;15(4):732-40). The disease efficacy of this approach was demonstrated in a rat model of hyperbilirubinemia, where lower viral vector doses were used when combined with hydrodynamic gene delivery to the liver (Hum Gene Ther. 2011 Apr;22(4):483-8).

[0338] As discussed above, there are few studies on hydrodynamic gene delivery to the kidney, but these have shown only modest efficacy. Viral vectors have been injected via the renal vein, renal artery, intercapsular space, and ureter, all of which mediated only modest transduction into the kidney (Hum Gene Ther. 2019 Dec;30(12):1559-1571). Currently, to treat clinically important diseases, there is a significant gap in efficacy in terms of delivery efficiency, and depending on the disorder, a large proportion of glomerular or tubular cells must be targeted.

[0339] One study examined viral vector delivery in combination with hydrodynamic gene delivery via the renal vein in rats (Mol Ther. 2008 Jun;16(6):1098-104). The authors suggested that the combination of their vector with hydrodynamic delivery increased delivery efficiency compared to vectors delivered at a normal rate. The authors claimed that in rats, normal infusion rates resulted in minimal or no adenoviral transduction, while hydrodynamic delivery resulted in only GFP staining. This experiment was performed in a rat model, so this finding may not occur in larger animal models.

[0340] The present disclosure also describes a method for synergistically delivering viral vectors through the urinary system with hydrodynamic pressure, improving upon previous work. Viral vectors have been injected through the urinary system before, but not with hydrodynamic pressure. The present disclosure combines these two modalities to improve upon the previously low efficiency of minimal gene delivery through the ureter. Another objective is to scale up either of these methods to larger animals in order to translate these methods into the treatment of human subjects.

[0341] The present disclosure also describes optimal hydrodynamic parameters that can be used for viral vector delivery. These hydrodynamic parameters are slightly below the maximum pressure that causes tissue damage in kidney rupture, as described elsewhere in this invention. The present invention teaches that, compared with conventional viral vector injection into the ureter at a flow rate of less than 0.1 mL / sec, the higher flow rates and volumes used to inject the virus in this procedure are more effective in increasing viral vector penetration into tissues and resulting in viral transduction. The mechanism for higher transduction is caused by increased permeability between tissues via higher fluid pressure during injection.

[0342] Exemplary viral vectors that can be used in the present invention include adeno-associated viruses, adenoviruses, lentiviruses, retroviruses, baculoviruses, anelloviruses, and Sindbis viruses. Because the hydrodynamic gene delivery process is particle-nonspecific, the present invention is not specific to virus type or virus serotype, and as a result, all viral vector particles can benefit from this combination.

[0343] The present disclosure also relates to a re-administrable viral vector approach. The hydrodynamic injection procedure can be modified to first push in a non-viral vector solution to remove any antibodies present. The viral vector-containing solution is then rapidly injected, so that hydrodynamic fluid pressure immediately pushes the viral vector into the tissue and target cells. This eliminates any antibodies present, allowing the viral vector to be reliably administered to the kidney.

[0344] The synergistic effect of these two modalities has not been described in previous studies, and therefore it is unclear whether retrograde ureteral hydrodynamic injection of viral vectors improves transduction. In particular, previous evidence suggests that viral vectors can be easily expelled from the kidney by retrograde ureteral injection, so it is unclear whether the additional pressure facilitates the process rather than leading to enhanced gene delivery (Hum Gene Ther. 2019 Dec;30(12):1559-1571).

[0345] The ultimate goal of hydrodynamic injection is to generate pressure, which helps create pores in the cell membrane to enable gene delivery. Previous publications have found that flow rate is a key parameter determining the pressure within the biliary system during hydrodynamic injection (Huang, PLOS One 2021). However, a challenge with prior art methods is that 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 injectate formed. All prior art injection parameters were formulated around approximately equal liver sizes within a narrow range (800-1000 grams) and using injection solutions of similar viscosity. Furthermore, all livers were healthy in animals without any disease. More importantly, at the cellular level, pig livers have significantly more fibrosis at baseline compared to human livers. Specifically, this is seen in the prominent fibrous tissue between all pig lobules. The different composition of pig livers may affect the infusion resistance in this system, ultimately affecting the pressure achieved depending on the infusion flow rate. Despite the similar organ sizes of humans and pigs, these differences combine to make it difficult to translate infusion parameters from pigs to humans. Therefore, prior art techniques may be difficult to achieve the precise gene delivery efficiency achieved in pig studies.

[0346] Another challenge is that the flow rate achieved during biliary hydrodynamic injection may not immediately translate to the expected pressure. More specifically, however, the challenge of converting flow rate to pressure during biliary hydrodynamic injection is that, according to prior art, the conversion does not appear to be reliable. For example, both 1 mL / sec and 2 mL / sec appear to produce a pressure of 80 mmHg during injection (Huang, PLOS One 2021). This leaves physicians uncertain about how to program an autoinjector for gene delivery when guided by a predetermined pressure threshold.

[0347] 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 will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the intention is to cover alternatives, modifications, and equivalents which may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0348] The present disclosure describes and teaches a solution to this problem. Instead of the prescribed flow rate parameters implemented during biliary hydrodynamic injection, the present disclosure teaches a method of injecting at a prescribed pressure boundary. This is because the flow rate required for gene delivery may vary between patients due to the factors specified above. A notable example of this is patients with cirrhosis or nonalcoholic steatohepatitis. The pressure parameters specified in the present disclosure are such that these thresholds are sufficient to achieve gene delivery to pig livers or other mammalian livers. Furthermore, the present disclosure teaches a pressure threshold that should not be exceeded in order to prevent efficient gene delivery. The present disclosure also teaches that a predetermined pressure threshold causes tissue damage and injury.

[0349] To achieve pressure-directed biliary hydrodynamic infusion, the first step is to obtain a balloon catheter with a lumen that can accommodate a pressure sensor or be connected to a transducer. This pressure sensor should be able to monitor and reflect the pressure within the biliary system. The pressure sensor is connected to a device that can give real-time pressure results and / or graph the pressure achieved during the infusion in real time.

[0350] The biliary hydrodynamic injection procedure begins with advancing a balloon catheter into the common hepatic duct, similar to established protocols in the prior art. In other embodiments, the catheter can be placed within the common bile duct. After the catheter is positioned within the common hepatic duct, a pressure sensor is attached to or inserted into the catheter through a dedicated lumen. In some embodiments, the pressure sensor is already present within the catheter. A baseline reading of pressure in the biliary system is taken without inflating the balloon, followed by pressure measurements upon inflation of the balloon. A test solution containing no plasmid DNA or other nucleic acids is injected into the bile duct. This test solution should have the same osmolarity, osmolality, and viscosity as the DNA injection solution. Besides DNA or other nucleic acids, it should not contain any other active pharmaceutical ingredients that may be included in the therapeutic DNA solution. The test solution material is loaded into an automatic injector.

[0351] The balloon on the catheter is then inflated. The test solution is then infused into the biliary system while the pressure is monitored. The target pressure to be achieved is greater than 80 mmHg. The initial infusion parameters for the test infusion should be 3 mL / sec. The total volume for the test infusion should be a maximum of 15 mL, 10 mL, or 5 mL. In other cases, the total volume for the test infusion should be a maximum of 40 mL, 30 mL, or 20 mL. This volume should be sufficient to measure the total fluid resistance column throughout the circuit and gauge when sufficient pressure is achieved. Previous literature has shown that steady-state pressure is achieved within the first second of the infusion, supporting the requirement for a short infusion time for the total infusion volume for the test infusion (Huang, et al. PLOS One 2021).

[0352] Depending on the results of the test injection, the flow rate is adjusted. If the flow rate produces a pressure above 80 mmHg, that flow rate is selected for injection at that threshold, and injection of the DNA solution proceeds. If the pressure exceeds 250 mmHg, the flow rate is reduced to less than 3 mL / sec. If the flow rate does not exceed 80 mmHg, the flow rate is adjusted to a higher rate. If an adjustment is made, a second test injection is performed. Generally, the flow rate can be adjusted up or down by 1 mL / sec to achieve the desired result, whether increasing or decreasing the applied pressure. In other embodiments, smaller increments of 0.5 mL / sec can be used to adjust the flow rate. If the correct pressure level is achieved during the test injection, injection of the DNA solution is initiated. Injection of the DNA solution is programmed at the established flow rate that will produce the desired pressure.

[0353] In other embodiments of the present invention, a series of flow rates is performed during a single test injection, testing multiple flow rates. In this embodiment, at least two or more test flow rates are tested within a single test injection. For example, the injection can begin at 1 mL / sec and then progress to 2 mL / sec, 3 mL / sec, and 4 mL / sec. During this series of test injections, pressure is monitored throughout, and multiple different pressure curves are generated. These pressure curves can take many different forms, including a stepped function. From these test injections, it is possible to accurately determine which, within the range of tested flow rates, is optimal and achieves the desired pressure for the injection. It is anticipated that to perform a series of tests, the total test volume will need to be larger than a single test injection. In these embodiments, the total injection volume may be up to a total volume of 40 mL, 30 mL, or 20 mL to test all of the different injection parameters.

[0354] In preferred embodiments of the present invention, the minimum pressure for efficient hydrodynamic gene delivery is greater than 50 mmHg, or greater than 80 mmHg, or greater than 100 mmHg, and the maximum pressure for efficient hydrodynamic gene delivery is less than 200 mmHg or less than 250 mmHg.

[0355] The prior art teaches that "the pressure in the biliary tract during hydrodynamic gene delivery is 40 mmHg or greater, 50 mmHg or greater, or greater. In at least some embodiments, the upper limit may be 200 mmHg, although higher pressures may be used in certain systems." However, the prior art only teaches pressure levels achievable by an autoinjector based on its technical specifications. Furthermore, while an autoinjector can theoretically achieve these different pressures, the prior art does not link any of these results to actual gene delivery. Therefore, it is unclear which actual gene delivery parameters are sufficient for gene injection. The present disclosure improves upon the prior art by providing data regarding injection pressures that achieve predetermined gene delivery parameters.

[0356] For example, the present disclosure teaches that hydrodynamic pressures below 50 mmHg achieve minimal gene delivery through the biliary system (less than 10%), as measured by immunohistochemical staining for the reporter genes firefly luciferase and green fluorescent protein. Only at pressures of 80 mmHg or greater could efficient immunohistochemical staining of the reporter genes be observed (greater than 20%). Furthermore, at pressures in the 100-150 mmHg range, the transfection area was further increased compared to lower pressures.

[0357] The present disclosure is advantageous in that it is not tied to any given species, allowing for empirical measurement and adjustment of infusion parameters for that species during the procedure. The present disclosure is also advantageous in that it allows for accounting for inherent differences between subjects within a species and for accounting for customized infusion parameters.

[0358] The present disclosure is made possible by the rapid buildup of pressure within the biliary tree, such that only minimal fluid volumes are required for injection to determine what flow rates cause what pressure levels.

[0359] The present disclosure relates to compositions and methods for treating pancreatic diseases. More particularly, the present disclosure relates to compositions and methods for treating pancreatic diseases through gene therapy. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that the volume of fluid required for sufficient gene expression is less than the 20 mL previously reported to be sufficient. The volume injected can be as little as 5-15 mL and still achieve effective gene delivery. The flow rate required to transfect pancreatic cells is less than the previously reported 2 mL / sec. A flow rate of at least 1 mL / sec is sufficient to mediate gene delivery. Other studies have found flow rates of at least 0.5 mL / sec. These two findings were coupled with the discovery that tissue damage from hydrodynamic injection into the pancreas can be eliminated when these lower injection parameters are utilized, resulting in significant differences in immune infiltration and necrosis compared to parameters outlined in the prior art. The present disclosure also outlines an optimal occlusion balloon size that is smaller than the cited prior art, finding that larger balloon sizes may rupture the pancreatic duct, while smaller balloon sizes do not, and still provide a sufficient seal for infusion. Specifically, an 8 mm balloon is sufficient. Applying this to a patient, the balloon can likely be inflated to a diameter 1, 2, 3, 4, 5, or even up to 5 mm larger than the diameter of the pancreatic duct. The present disclosure also addresses a gap in the prior art, teaching that the pressure required to transfect pancreatic cells is less than 75 mmHg above baseline pancreatic duct pressure, which is likely to be between 30 and 75 mmHg.

[0360] Overview Pancreatic gene therapy is a promising modality for a variety of diseases, ranging from autoimmune diabetes to hereditary chronic pancreatitis and pancreatic cancer. Proof-of-concept studies using viral vectors have achieved some efficacy in rodents. Importantly, gene delivery to the pancreas has not been achieved in large, human-sized animal models, representing a major gap in the field. Herein, we demonstrate proof-of-concept that an endoscopic procedure accessing the pancreatic duct by endoscopic retrograde cholangiopancreatography (ERCP) enables nonviral hydrodynamic delivery of naked plasmid DNA solutions and mediates efficient delivery to various cell types within the pancreas of adult pigs, including nearly 100% transfection of islet cells, paving the way for future therapeutic development.

[0361] The pancreas is an important exocrine and endocrine organ of the body that plays a key role in food digestion and energy storage. Disruptions in pancreatic function can lead to a wide variety of disorders. Secretion of pancreatic digestive enzymes through the pancreatic duct leads to the disorder pancreatic insufficiency. This type of chronic enzyme secretion is seen in cystic fibrosis. Chronic pancreatitis is characterized by inflammation and scarring of the pancreas and can also lead to pancreatic insufficiency, a condition that occurs when the pancreas does not produce enough digestive enzymes to break down food and absorb nutrients. Hereditary pancreatitis is a genetic disease mutation that causes patients to suffer from pancreatic insufficiency and / or chronic damage at a very young age.

[0362] The pancreas is a key organ in the pathogenesis of a wide variety of diseases, including type 1 and type 2 diabetes, caused by disrupted or dysregulated insulin production. Pancreatic beta islet cells secrete and regulate insulin, modulating systemic fat and protein storage. In type 1 diabetes, insulin-producing beta islet cells are destroyed by autoreactive T cells, leading to absolute insulin deficiency and hyperglycemia. Type 2 diabetes causes systemic insulin resistance and relative insulin deficiency. Pancreatic cancer is another devastating disease with a low survival rate. One strategy for treating pancreatic disorders is gene therapy, which can deliver genes found to be defective in pancreatic cells or by transforming pancreatic cells. Gene therapy can also be used to deliver proteins that can combat genetic disorders, cancer, or autoimmune diseases.

[0363] Various strategies for pancreatic gene therapy have been attempted. Viral strategies have used systemic or targeted delivery via vascular routes (see, e.g., Griffin, MA et al. “A novel gene delivery method transduces porcine pancreatic duct epithelial cells.” Gene therapy vol. 21,2 (2014): 123-30. doi:10.1038 / gt.2013.62), while other studies have demonstrated the feasibility of injecting viruses via the pancreatic duct (see, e.g., 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 vol. 26,5 (2018): 1327-1342). Non-viral strategies have also been used by utilizing hydrodynamic injection via the pancreatic artery in rats (e.g., Ogawa, Kohei et al. "Efficacy and Safety of Pancreas-Targeted Hydrodynamic Gene Delivery in Rats." Molecular therapy. Nucleic acids vol. 9 (2017): 80-88.) and via the pancreatic duct in mice (Yamada Y, et al. In Vivo Transgene Expression in the Pancreas by the Intraductal Injection of Naked Plasmid DNA. Journal of Pharmaceutical Sciences. 2018 Feb;107(2):647-653).However, these non-viral strategies have not been translated into large animal models, with only one published report of viral vectors in newborn pigs (approximately 1.5 kg) at birth, where AAV was delivered via the pig's celiac artery, resulting in vector dissemination to the left gastric vein and splenic artery, which supply the pancreas (see, e.g., Griffin, MA et al.). If a pancreatic gene therapy strategy transferable to adult animals can be achieved, other important factors are how efficiently gene delivery can be delivered to the pancreas and which cell types should be efficiently targeted.

[0364] In this study, we used endoscopic retrograde cholangiopancreatography (ERCP) to mediate nonviral hydrodynamic gene delivery through the pancreatic duct of pigs. Nonviral gene therapy was used because its significantly lower cost allows for routine use in metabolic conditions such as diabetes. ERCP has previously been used to efficiently deliver plasmid DNA to hepatocytes (see, for example, Kumbhari V, et al. Successful liver-directed gene delivery by ERCP-guided hydrodynamic injection (with videos). Gastrointest Endosc 2018;88:755-763.e5).

[0365] Although gene therapy has considerable potential to have a meaningful impact on pancreatic disease, little research has been conducted in this area. One approach to gene therapy is the administration of viral vectors or nanoparticles, but these do not accumulate to the required extent in the pancreas. A more successful approach targets local administration of nanoparticle viral vectors to the pancreas, such as through injection through the pancreatic artery or vein. Intraductal delivery is another technique used. One drawback of early studies is the use of mouse and rat models, as these models respond very inefficiently to treatment. Further research is needed to confirm whether viral vectors are an efficient method for delivering gene therapy.

[0366] Hydrodynamic injection is a technique that mediates the nonviral delivery of nucleic acids, including DNA and RNA. Studies in mouse models conducted by various groups have shown that hydrodynamic injection can mediate limited amounts of gene delivery via intravenous or ductal routes. Data obtained from rodent studies are not robust, and few follow-up studies have been conducted.

[0367] Hydrodynamic delivery in large animal models is a novel technique that produces more efficient and reliable delivery of gene therapy. The ductal system was selected for hydrodynamic injection because it directly reaches the entire pancreatic tissue through a single network (Figure 13). Furthermore, the ductal system is unidirectional, allowing retrograde injection of fluid at high pressure directly into the pancreatic tissue.

[0368] Previous studies have found that ductal hydrodynamic injection is effective in mediating efficient delivery to multiple different pancreatic cell types, including islet cells, ductal cells, endothelial cells, and neurons in the pancreas (Figures 15 and 16). This was achieved by injecting a ubiquitous promoter capable of tagging multiple cell types.

[0369] Previous techniques used for ductal hydrodynamic gene delivery include: (1) placing a catheter through the major duodenal papilla into the main pancreatic duct distal to where the pancreatic duct merges with the common bile duct, or through the minor duodenal papilla into the accessory or dorsal pancreatic duct, and optionally advancing the catheter further into the main pancreatic duct; (2) optionally removing fluid present within the pancreatic duct to remove digestive enzymes from the pancreatic duct lumen; (3) injecting contrast into the pancreatic duct to confirm correct placement of the catheter; (4) inflating a balloon within the catheter through the common bile duct near its entrance to the pancreatic duct to prevent backflow of fluid; (5) injecting a solution containing at least 1 mg of DNA at a flow rate of at least 2 mL / sec and a volume of at least 20 mL; are listed; (6) Here, the flow rate, volume, and DNA dose are sufficient to mediate gene expression in all pancreatic lobes and multiple pancreatic cell types.

[0370] Pancreatic tissue damage is monitored by serum tests for amylase and lipase levels. To reduce pancreatic damage, previous studies have suggested administering various drugs during or after hydrodynamic infusion, but the best combinations and concentrations of drugs to use have not been described.

[0371] The previously specified injection parameters are as follows: (1) The volume injected into the pancreas is a minimum of 20 mL, or volumes can exceed 30 or 40 mL, resulting in leakage of the volume from the pancreatic duct into the parenchyma. (2) The flow rate for the procedure is greater than 2 mL / sec, and in other embodiments, greater than 3 mL / sec or 4 mL / sec. (3) The optimal ductal 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.

[0372] Additional methods described in previous studies include strategies to alter promoter specificity to target specific cell types in the pancreas. This allows for targeting exclusive expression in, for example, alpha islet cells, beta islet cells, acinar cells, or ductal cells. Gene delivery to the pancreas was only successful when using parameters of 2 mL / sec and a volume of 20 mL or greater for gene delivery. It is uncertain whether other injection parameters will result in successful gene delivery.

[0373] Although the successful method of gene delivery to the pancreas by hydrodynamic injection in large animals such as pigs represents a major advance, several challenges and limitations remain with this approach.

[0374] The first drawback is that the injection itself appears to cause significant tissue damage. While most tissue appeared normal, numerous areas of necrosis were present on all tissue sections (Figure 17). Corresponding histological images revealed small areas with significant lymphocyte and neutrophil infiltration. These areas were not seen in uninjected pig pancreases. It is uncertain whether the necrotic areas will progress to full-blown pancreatitis at longer time points. Over the short time intervals in this study, amylase levels appeared to normalize (Figure 14), but necrotic areas were still observed.

[0375] Although porcine pancreases can tolerate pancreatic injury, it is well known that pancreases from primates, including humans, are highly susceptible to any injury. The introduction of necrotic regions may be risky or unacceptable for treating human patients. Therefore, methods and strategies for reducing pancreatic injury would be important advances and improvements for this procedure.

[0376] A promising strategy for reducing pancreatic injury appears to be modulating parameters to apply less force and stress to the lesion. One way to change parameters is by decreasing flow rate and imposing less peak pressure. A second way is by reducing volume so that pressure is exerted on the pancreas for less time.

[0377] A key factor in pancreatic gene delivery is that the total volume of the pancreatic duct is only 2–3 mL. Previous studies have injected volumes 4–6 times the normal volume into the pancreatic duct. This additional volume did not rupture the pancreatic tissue, suggesting that this additional volume could be absorbed by the vasculature or the surrounding retroperitoneal space. However, injection of smaller volumes could be tested and may be sufficient for efficient gene delivery.

[0378] Given the small size of the pancreatic duct volume, a lower flow rate could create a similar threshold pressure in the duct lumen to push the plasmid DNA directly into the surrounding tissue. The lowest flow rate tested to produce gene expression in previous studies was 2 mL / sec. Gene expression can be achieved at lower flow rates.

[0379] Previous studies have not defined target volumes for pancreatic infusion. Parameters have been defined for pigs of a specific weight (40–54 kg) but have not been adjusted for pigs of different sizes. This is important because volume parameters are essential factors for clinical translation to humans, which vary in size. Previous studies have assumed that the adult and porcine pancreases being tested are of similar size, and as a result, parameters, including DNA dose, can be directly translated.

[0380] Hydrodynamic injection in the pancreas The use of endoscopic retrograde cholangiopancreatography (ERCP) to access either the main or accessory pancreatic duct for hydrodynamic injection improves the efficiency and safety of the procedure. Catheters can be placed in either the head or tail of the pancreas. Further modifications to novel and improved methods of gene delivery are described below:

[0381] Safe balloon size The first modification to the procedure is to define a maximum balloon inflation size for hydrodynamic injection that avoids pancreatic duct injury and damage to the surrounding pancreatic tissue. It has been observed that inflation of the balloon to a diameter of 11 mm or greater can result in pancreatic wall injury (Figure 18), which can lead to fluid leakage into the surrounding tissue when hydrodynamic injection is initiated.

[0382] In general, the pancreatic duct is slightly smaller than the bile duct. The average diameter of the bile duct in humans is 4 mm, and in some individuals it can dilate to 6–8 mm. However, in some patients, the bile duct is only 2–3 mm in diameter. In comparison, the pancreatic duct is 3.5 mm in the head of the pancreas, 2.5 mm in the tail of the pancreas, and the ureter is 6–8 mm.

[0383] When inflating a balloon within a catheter, the bile duct has greater flexibility, allowing it to accommodate more dilation and remain intact with the largest balloon size. In comparison, the pancreatic duct does not have sufficient flexibility to accommodate larger balloon sizes. Pancreatic tissue is generally more sensitive, fragile, and susceptible to damage.

[0384] The Multi-3 V Plus, a disposable triple-lumen balloon catheter for stone removal (Olympus Medical) has been utilized in liver and pancreatic studies. The balloon on the catheter can be inflated to three different sizes (8.5, 11, and 15 mm) and withstands hydrodynamic pressure. Because the 11 mm balloon size was observed to cause injury, subsequent studies will use balloons smaller than 11 mm, preferably 10 mm or 9 mm.

[0385] Injection volume calculation The second modification to the injection procedure clarifies and expands the precise volume administered to the pancreas for hydrodynamic injection. Previously, volumes greater than 20 mL have been injected into the pancreas for successful gene delivery. However, this specification does not consider differences in individual size or how to adjust the volume for transition to human patients. For example, the porcine pancreas has two distinct wings composed of three distinct lobes, whereas the human pancreas is a single wing or tissue mass composed of two main lobes. The relative mass of the porcine and human pancreas is comparable (discussed in further detail below). Theoretically, data regarding parameters in porcine samples should be translatable to adult patients. Previous studies have only disclosed injection strategies in pigs approximately 40–50 kilograms in size, making it uncertain how injection volumes would change for significantly larger or smaller individuals in human patients. Previous studies have provided guidance on administration by pancreatic mass, but this does not demonstrate how this information can be used to convert to an appropriate volume for hydrodynamic injection into the pancreas.

[0386] The present disclosure remedies that shortcoming by detailing how to calculate a safe and effective injection volume to be administered to the pancreas. As shown in Table 1, the body weights of each of the four pigs and their respective pancreatic weights are listed below, along with the injection parameters for those injections. Of note, some of the pancreatic dissections in the early pigs tested included fatty tissue surrounding the pancreatic organ, making these measurements inaccurate when interpreting weight-based dosage recommendations.

[0387] [Table 1]

[0388] Although the weight distribution in pigs is different from that in humans, organ size may be comparable. The porcine pancreatic mass from these studies is comparable to the human pancreatic mass data, as shown in Table 2.

[0389] [Table 2]

[0390] One way to address this discrepancy is to weigh the pancreas before injection. The weight determines the appropriate injection volume. This avoids injecting too much fluid into the pancreas, which risks damaging the organ. While data from porcine pancreases are comparable to adult humans, it is more accurate to calculate the pancreas based on its weight.

[0391] Examples exist in the literature for determining how to estimate pancreatic weight in individual humans. An early study in the journal The Anatomical Record, published in 1926, summarized known data from autopsies at the time regarding the weight of the pancreas in adult men and women (The normal weight of the pancreas in the adult human being: A biometric study - https: / / doi.org / 10.1002 / ar.1090320204).

[0392] The research summarized in this paper states that the normal weight of the pancreas in adult men is 60 to 100 grams. The average weight is 80 grams, with extreme values ​​ranging from 60 to 100 grams, 70 to 108 grams, or 70 to 90 grams. Further studies have reported that the average weight of a normal pancreas in men is 70 grams, while the average weight in women is 66 grams.

[0393] In another study, 30 cadaveric pancreatic specimens were dissected and carefully weighed. Weights were recorded, with a mean of 91.8 g (range: 40.9–182 g) (Am J Surg. 1994 Feb;167(2):261–3).

[0394] Another study showed that the mean age of patients was 47.9 ± 17.8 years (range 25-88 years), height was 172.2 ± 7.5 cm (range 145-190 cm), weight was 78.1 ± 15.2 kg (range 42-120 kg), mean BMI was 26.2 ± 4.7 kg / m² (range 17-38 kg / m²), and BSA was 1.9 ± 0.2 m² (Forensic Medicine and Anatomy Research, Vol. 02 No. 03 (2014)). The mean pancreatic weight was reported to be 87.3 ± 30.6 grams.

[0395] Another study of pancreatic weight in cadavers found a significant difference between normal individuals and those with type 1 diabetes, with the latter having reduced pancreatic size (JAMA. 2012;308(22):2337-2339). This is an important consideration when administering hydrodynamic gene delivery in this patient population. The researchers found that the mean weight of pancreases from nondiabetic pancreases (controls) was 81.4 g (95% CI, 73.0-89.8 g), compared with 61.3 g (95% CI, 46.8 g-75.8 g; P = .02) in the group positive for only a single autoantibody and 44.9 g (95% CI, 36.0 g-53.9 g; P < .001) in the T1D group.

[0396] An alternative method for administration is to calculate only the pancreas volume, either directly from imaging or by calculation. The density of the pancreas is estimated to be 1.1 grams / mL (Cellular Transplantation, 2007). Therefore, volume can be converted to the appropriate mass for administration according to the guidelines herein. Similarly, the mass-based guidelines herein can be converted to volume-based guidelines by those skilled in the art using standard dimensional analysis. An example of a method for calculating pancreas volume based on MRI is provided herein as one example of many studies that have investigated this technique (PLoS One. 2014; 9(3): e92263).

[0397] While average anatomical studies of the pancreas are useful, obtaining a more accurate estimate of pancreatic weight is important for clinical translation of pancreatic gene delivery. Toward this goal, one previous study provided a solution on how to calculate pancreatic weight before hydrodynamic injection.

[0398] The study examined pancreatic weight from 354 cadaveric donors, taking into account sex, age, weight, height, body mass index (BMI), and body surface area (BSA) (Cell Transplant. 2006;15(2):181-5). The researchers developed a mathematical formula to predict pancreatic weight from patient-specific factors.

[0399] "In younger donors (<40 years), body weight and age were the major predictors of pancreatic weight [pancreatic weight (g) = 4.355 + 0.742 × body weight (kg) + 0.837 × age (years) (R2 = 0.564, p < 0.001)]."

[0400] Pancreatic weight in older donors (>40 years) was best predicted by BSA and gender [pancreatic weight (g) = -17.624 + 60.036 × BSA (m²) - 7.152 × gender (R² = 0.372, p < 0.001; 'gender': 1 = female, 0 = male)].

[0401] Current technology uses pancreatic mass to describe how to administer an infused volume based on pancreatic weight.

[0402] In one embodiment of the present invention, the volume for hydrodynamic infusion can be calculated by multiplying the pancreas weight by 0.30 mL / g, or 0.35 mL / g, or 0.40 mL / g or more, where the mass in the denominator is the pancreas weight. The ratio multiplier reflects that volumes greater than 22 mL can be obtained.

[0403] Reduced volume for injection This disclosure describes new volume targets for reducing toxicity from pancreatic duct hydrodynamic infusion. These volume targets address the shortcomings of previous methods, where damage was observed with microscopic lesions of necrosis along with elevated amylase levels. These new volume targets are a way to find minimal yet effective gene delivery parameters for hydrodynamic gene delivery.

[0404] In one case, the volume for 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 a total volume injected of less than 20 mL for an approximately 40 kg pig or an average adult, as previously discussed. In more preferred embodiments, the volume injected is 15 mL or 10 mL total volume.

[0405] New data supporting this disclosure demonstrate that a reduced volume of 15 mL still results in sufficient hydrodynamic gene delivery when combined with a 2 mL / sec flow rate. Gene expression was observed in multiple different pancreatic cell types with similar efficiency to prior art techniques disclosed with larger injection volumes (Figures 19A and 19B). The purpose of reducing the volume is to reduce tissue damage during the procedure, as verified by a reduction in the area of ​​necrosis observed histologically, as well as a reduced increase in amylase activity after injection (Figure 19C). The total volume of the pancreatic duct was only 4-5 mL in total, suggesting that even smaller injection volumes can result in internalization of the DNA solution into the pancreatic tissue.

[0406] A summary of the pancreatic infusion experiments is presented below in Table 3, recording several different pigs and pancreatic weights along with the calculated parameters used for these infusions. Guidelines for administration in the present invention are based on a synthesis of best practices from previous experiments that describe the observed gene delivery efficiency and safety.

[0407] [Table 3]

[0408] Decrease in flow rate As for other injection parameters, flow rate considerations for the current technique also differ from previous methods. It was established that volumes of over 20 mL at a flow rate of 2 mL / sec were sufficient to produce gene expression in the pancreas and multiple cell types.

[0409] In the present disclosure, the preferred total volume injected is less than 20 mL, which raises the question of what flow rate is best or sufficient for gene delivery. Previously, flow rates of 2 mL / sec or less were found to be preferred and sufficient for gene expression.

[0410] A flow rate of 1 mL / sec was found to be sufficient for gene expression in tests with a total volume of 20 mL. Therefore, preferred flow rates within the scope of the present invention are equal to or between 1 and 2 mL / sec. Higher flow rates above 2 mL / sec may be considered, but they are associated with more pancreatic tissue damage and progressive injury and should be avoided.

[0411] Use of multiple flow rates during injection To further reduce pancreatic injury while preserving gene delivery and expression, flow rate can be further modulated by incorporating multiple different flow rates during the infusion. This strategy requires the use of an automated injector that can be programmed with multiple injection parameters during a given infusion.

[0412] Use an initial flow rate of 1-1.5 mL / s for less than 50% of the injection volume, and then increase the flow rate to 1.5-2 mL / s for the remaining injection volume. This reduces the duration of the main part of the peak fluid pressure so that the peak fluid pressure occurs when the DNA solution completely fills the conduit system.

[0413] Another approach is to use an initial flow rate of 0.5-1 mL / s for less than 50% of the injection volume, and then set the flow rate to 1-2 mL / s for the remaining injection volume. This reduces the time to peak fluid pressure, so that peak fluid pressure occurs when the DNA solution completely fills the conduit system.

[0414] This strategy successfully maintained efficient gene delivery to all relevant cell types (data not shown). Furthermore, the number of tissue necrotic areas was reduced, as were amylase peaks.

[0415] Hydrodynamic injection through the liver is an exciting new gene therapy modality. It has the advantage of being non-viral, and therefore scalable and safer than viral approaches. Hydrodynamic injection can deliver naked DNA alone, making it the simplest gene therapy modality available. This technique has been shown to be highly efficient in mouse models, but has not traditionally been well-extended to larger animal models.

[0416] Several groups have been investigating for many years whether hydrodynamic injection can be extended to large animal models. An inherent challenge in developing new techniques for hydrodynamic injection is the challenge of translating the technique between animals. New methods must be adapted and optimized for each animal and organ, empirically testing which strategies work. Pigs are a convenient animal model for testing because their livers can be comparable in size to adults, depending on the pig's age. Therefore, hydrodynamic techniques established in pigs are likely to be translatable to humans. However, several caveats remain, including the presence of interlobular fibrosis between the lobes of the porcine liver, which can create some uncertainty.

[0417] In this regard, while studies of hydrodynamic gene delivery in mouse, rat, and rabbit models are certainly interesting proof-of-concept studies, they unfortunately do not teach how the procedure would be performed in human patients and how gene delivery efficiency might be optimized. One simple example of this is that many groups have isolated specific lobes of the liver and infused them through the hepatic vein that supplies that lobe. This technique was first described in rabbits in Human Gene Therapy 2002 Nov 20;13(17):2065-77. Since then, a very similar technique has been used in pigs, where the same strategy of isolating individual lobes using a balloon occlusion catheter is applied to those lobes using hydrodynamic infusion (Mol Ther. 2009 Mar;17(3):491-9). However, unlike traditional pharmaceuticals or biologics, the flow rates and volumes used in these later studies to mediate gene delivery in pigs could not be predicted from rabbit studies, as there is no simple weight-based scaling for the hydrodynamic infusion procedure. This finding is also seen with simple hydrodynamic tail vein infusions in mice and rats, where the volumes and flow rates used in mice do not simply translate into rates when adjusted for larger size.

[0418] Another example of variability is that the vessel or conduit chosen for hydrodynamic injection can significantly alter the characteristics of the injection, along with the outcome of gene delivery. One example of this has been seen in a porcine model, where single-lobe injection appears to be more efficient compared to clamping the inferior vena cava and portal vein to isolate blood flow there, rather than injecting through the hepatic vein to reach the entire liver. The subtle differences for these different vessels are not clear until experiments are performed.

[0419] Similarly, researchers have also explored the biliary system as a different route for hydrodynamic gene delivery. The biliary system is unique in that it has a much smaller total volume than the vascular system that supplies the liver. This comparison compares the biliary system volume of approximately 30 mL to the blood volume of an adult human, which is approximately 600 mL. The biliary system is also unique in that it is unidirectional, preventing the injected fluid from simultaneously leaking out through unsealed ends. This unidirectional nature of the biliary system is particularly advantageous for maintaining adequate fluid pressure during injection, ensuring that the high-pressure DNA solution is expelled into the surrounding liver tissue rather than spurting outward through other hepatic vessels.

[0420] Although the hypothesis of liver-directed gene delivery has been around for over 20 years now, the practicality of how to perform this procedure efficiently and what the outcomes of such a procedure would be in large animal models have remained uncertain.

[0421] Regarding the history of biliary gene delivery, the first description of hydrodynamic gene delivery via the bile duct was based on experiments in dogs. This group used surgical procedures, an injection rate of approximately 1 mL / sec, and sutures to prevent antegrade flow within the biliary system (Hum Gene Ther. 1997 Oct 10;8(15):1763-72). Importantly, the researchers injected into the common bile duct, ensuring that the fluid was delivered to the gallbladder in addition to the liver. Although the researchers were only able to detect low levels of luciferase activity, no protein was detected by histochemical analysis of the liver, suggesting that an undetectable proportion of pDNA was incorporated into hepatocytes. Furthermore, no protein was detected by Western blot.

[0422] parameter: Plasmid DNA dosage: 10-20mg Obstruction: Alternatively, sutures were used in the bile duct to obstruct it and prevent antegrade flow. ·Capacity: 200~400mL Dog weight: 4.2kg~10.8kg Flow rate: up to 1.66 mL / sec Pressure: Provided to mice, not dogs Other: Optional IVC occlusion to enhance efficacy

[0423] To improve upon these results, another group described the use of ERCP to mediate gene delivery to the liver of dogs and pigs (GIE 2005, T1249 Abstract). This aspect of ERCP represents a transitional improvement over surgery. Another innovation in the procedural methodology used by the researchers was the use of a balloon to prevent antegrade flow during hydrodynamic injection, thereby increasing the portion of fluid containing the DNA solution that entered the liver. This group used a 5 mL / min infusion rate for the procedure and continued the infusion through the common bile duct, thus simultaneously infusing the liver and gallbladder. The researchers did not report any gene delivery efficiency to the liver, as reflected by detectable protein expression on IHC, IF, or Western blot. However, there were reports of protein detection in the systemic circulation. The efficiency and usefulness of this procedure, when applied to liver disease, remain uncertain.

[0424] parameter: ·Capacity: 3~10ml / kg (weight) ·Flow rate: 5ml / min Pressure: 40~47mmHg Weight: Not provided DNA dose: Not provided Occlusion: Balloon catheter

[0425] Concurrently, another group reported that injection of pDNA through the bile duct via a surgical approach in rats could mediate gene delivery (Gut. 2005 Oct; 54(10): 1473-1479). However, as discussed above, the hydrodynamic parameters in rodent models are inherently infeasible for larger mammals (e.g., dogs, pigs, and humans). For example, the flow rate used by this group was 0.54 mL / min, and surgical ligation served to seal the bile duct. The relative efficiency of pDNA delivery to hepatocytes was only approximately 1% of hepatocytes based on histochemical staining, which is significantly less efficient than the equivalent hydrodynamic tail vein approach.

[0426] 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 will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the intention is to cover alternatives, modifications, and equivalents which may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0427] Previous studies have investigated hydrodynamic injection through the biliary tree. The first studies involved injection through the common bile duct, which connects to the gallbladder via the cystic duct and to the liver via the intrahepatic duct. Researchers tested hydrodynamic injection through the common bile duct in canine and porcine models. They reported that protein expression could be achieved by luciferase assay and systemic protein expression, but did not demonstrate liver tissue delivery as demonstrated by immunostaining.

[0428] The present disclosure seeks to improve upon strategies for hydrodynamic injection through the common bile duct. The strategy for common bile duct injection involves inflating a balloon within the common bile duct to block antegrade flow into the small intestine. The balloon can be inflated anywhere along the common bile duct. An alternative strategy involves inflating the balloon at the junction of the common bile duct and the common hepatic duct to occlude the opening of the cystic duct.

[0429] The balloon may be 8 mm in diameter or maximum width (depending on the balloon shape) or larger depending on the assessed luminal diameter. To achieve a proper seal during injection, the balloon needs to be a minimum of 1.5 times the width / diameter of the bile duct at this location, but more likely 2 or 3 times or more the width / diameter of the bile duct to provide a reliable seal. The width / diameter can be fixed at least 4 mm larger, or at least 50% larger. Alternatively, the width / diameter can be fixed at least 5 mm, 6 mm, 7 mm, or 8 mm larger, or at least 60%, 70%, 80%, 90%, 100%, or more. If the width / diameter is too large, the bile duct may be stretched, potentially causing subsequent bile duct injury or perforation, which is most likely to occur during the hydrodynamic injection process.

[0430] Verification of adequate bile duct sealing is confirmed by measuring the bile duct pressure (at rest or during a test injection) upstream of the balloon. If the pressure reading suggests an inadequate seal, the balloon is further inflated. Another method to confirm adequate sealing is to inject contrast through the catheter upstream or downstream of the balloon to confirm that the location is distal to the porta hepatis and occluded at or upstream of the cystic duct bifurcation. Pressure can also be measured from the lumen with an opening proximal / downstream of the balloon.

[0431] Previous parameters investigated include volume-based infusions ranging from 200 mL to 400 milliliters. In different studies, infusion volumes ranged from 3 to 10 mL per kilogram of subject body weight.

[0432] The required DNA dose ranges from 10 to 20 milligrams of DNA for a 5-10 kg dog. Other studies did not report the DNA dose used. Regarding flow rate, one study targeted a maximum flow rate of 1 mL / sec. Another study proceeded with a flow rate of 5 mL / min. One prior art reference teaches that the optimal injection pressure for injection is 40-50 mmHg.

[0433] The present disclosure aims to improve the prior art, and teaches the method of hydrodynamic injection through the common bile duct to simultaneously deliver gene to the gallbladder and liver.In other embodiments, only the liver is targeted.The present disclosure teaches the appropriate injection parameters and DNA dosage required to achieve hydrodynamic injection through the common bile duct.

[0434] Injection into the common bile duct The present disclosure includes several steps to achieve gene delivery in the gallbladder and / or liver. The first is accessing the common bile duct. The common bile duct can be accessed by endoscopic retrograde cholangiopancreatography (ERCP), a routine clinical procedure in which a catheter is inserted from the small intestine into the biliary tree, typically via the major papilla. Alternatively, the common bile duct can be accessed via endoscopic ultrasound guidance (EUS), in which a needle is inserted directly into the common bile duct through the duodenal wall.

[0435] Once the catheter is in place within the common bile duct, it preferably has a balloon that can be inflated to completely seal the common bile duct. The common bile duct is simply relatively large in diameter, so that a relatively large balloon size of 8, 11, 13, or 15 mm or more may be necessary to completely seal the common bile duct. After sealing the common bile duct, in some embodiments, bile can be withdrawn through the catheter by suction to remove as much volume as possible from the common bile duct, common hepatic duct, and gallbladder. In other embodiments, the catheter can also be advanced through the cystic duct, and bile can be withdrawn from the gallbladder to remove it before infusion. In addition to aspiration of bile alone, irrigation of the bile duct with a neutral fluid, such as saline solution, can be performed to replace a portion of the bile fluid volume in the bile duct with this neutral fluid.

[0436] In other embodiments of the present disclosure, bile may be left in place prior to injection, allowing the material to fill the gallbladder prior to injection. In other strategies of the present disclosure, the gallbladder is filled with a fluid or gel-like material prior to hydrodynamic injection of the DNA solution. The goal of these strategies is to fill the gallbladder so that no remaining volume is available for additional fluid during injection. In certain embodiments of the present disclosure, saline solution may be pre-filled into the gallbladder for this purpose. The pre-filled saline solution may have a total volume of 40-50 or 60 mL or more. In other embodiments, the saline solution may contain a contrast agent so that gallbladder filling can be monitored in real time.

[0437] In other embodiments of the present disclosure, a gelatinous material can be injected into the gallbladder to fill the gallbladder space. The polymeric material then degrades within a few hours. In some examples of the present disclosure, poly(lactic-co-glycolic acid) (PLGA) powder can be injected into the gallbladder to fill the space prior to injection.

[0438] In optimal embodiments of the present disclosure, the injection is performed at a volume of 50 mL / kg or 100 mL / kg liver weight, which corresponds to the total volume of the gallbladder, biliary system, and intrahepatic duct system. In other embodiments, the injection volume is at least 150 mL / kg liver weight, or at least 200 mL / kg liver weight.

[0439] In certain embodiments of the present disclosure, the DNA dose is at least 20 milligrams per kilogram of liver weight, hi other embodiments, the DNA dose is at least 40 milligrams per kilogram of liver weight.

[0440] In other embodiments of the present disclosure, the minimum DNA concentration for injection is at least 0.2 mg / mL volume. In other embodiments, the minimum DNA concentration is at least 0.5 mg / mL injection volume.

[0441] The optimal flow rates taught by the present disclosure differ significantly from the prior art. The present disclosure teaches that a flow rate of at least 2 mL / sec, or 5 mL / sec is required. In other embodiments, a flow rate of at least 10 mL / sec is required during any injection.

[0442] The optimal pressure during injection is also established by the present disclosure. The present disclosure teaches that a minimum pressure of at least 50 mmHg is necessary for efficient gene delivery. In other embodiments, the minimum pressure is at least 80 mmHg or 120 mmHg for injection. In some cases, the pressure during gene delivery can be achieved by a constant pressure injection device that monitors and adjusts the pressure in real time.

[0443] As an example of the effectiveness of this gene delivery strategy, efficient gene expression is achieved within the bile duct wall, common bile duct, and common hepatic duct. This also demonstrates reporter gene expression within the gallbladder wall. The injection strategy, with the specified injection parameters, also demonstrates its applicability for mediating gene delivery to hepatocytes.

[0444] In some embodiments of the present disclosure, antibiotics are mixed with the DNA injection solution because injection into the lower biliary tree is associated with a higher propensity for the presence of enteric bacteria that can lead to cholangitis. In another embodiment, antibiotics are mixed with the fluid used to flush the biliary tree after bile aspiration has been performed, but not with the DNA solution itself.

[0445] In summary, presented herein is the novel discovery of efficient gene delivery through the common bile duct. This strategy improves upon previous attempts by performing injections at higher flow rates and pressure levels to drive greater expression within host hepatocytes. This disclosure also teaches gene delivery to the extrahepatic biliary system, which has not been reported in previous studies. This disclosure expands the versatility of using the biliary system as a route for gene therapy strategies.

[0446] Strategies for injection via the common bile duct and / or the common bile duct-cystic duct junction The present disclosure describes a method for performing hydrodynamic infusion through the biliary system, via the common bile duct while bypassing the cystic duct to allow all fluid to enter the common hepatic duct and subsequently the liver.

[0447] This method describes a novel strategy in which a stent is placed within the extrahepatic bile duct, extending beyond the cystic duct orifice into the common hepatic duct. The stent optimally has a larger diameter than the bile duct to ensure its stability and ensure that fluid does not flow around the duct, but rather that all solutions flow through the interior of the stent. This effectively blocks infusion solutions from entering the cystic duct or gallbladder. An optimal stent is made of a solid material or has a nonporous coating on the wall to prevent fluid from penetrating the stent's wall. The stent can be deployed across the major papilla and into the duodenum for easy removal. Stents can also be deployed so that they are entirely within the biliary tree, but with a string or tether attached to them for easy grasping and removal when deployed.

[0448] In one preferred embodiment of the present disclosure, the catheter can be placed in several different locations. In one embodiment, the injection can occur downstream of the stent in the common bile duct. A balloon is inflated in the common bile duct to prevent antegrade flow into the intestine. The injection solution containing DNA passes retrograde through the stent and into the common hepatic duct, then into the liver.

[0449] In another embodiment of the present disclosure, a catheter is placed within a stent in the bile duct. Inflating a balloon within the stent allows the balloon to safely inflate so that its shape changes from spherical to cylindrical within the stent without damaging the biliary tree, since the stent is directly subjected to the force of the balloon, as opposed to the bile duct wall (FIG. 27A). The exact location of the catheter can be either on the common hepatic duct side of the stent (FIG. 27B), within the common bile duct side of the stent (FIG. 27C), or at the cystic duct junction of the stent. The balloon is believed to be inflated inside the stent to allow for a proper seal and prevent antegrade flow of solution. Furthermore, pressure from the fluid injection is expected to press the stent firmly against the bile duct wall, further improving the seal. Finally, with the inflated balloon inside, the catheter is expected to be used to hold the stent in place during hydrodynamic injection.

[0450] After the injection is complete, regardless of the position of the catheter, the balloon is deflated and the stent is removed along with the catheter and bile duct.

[0451] The optimal injection parameters for injection through the common bile duct and stent are believed to follow those previously described for hydrodynamic injection in the common hepatic duct. The main difference is that an additional 5–10 mL is added to the calculated volume to account for the additional volume of the common bile duct and common hepatic duct lumen. The volume calculation utilizes the published strategy for the common hepatic duct.

[0452] In an optimal embodiment of this strategy, contrast is injected into the biliary tree to verify that the gallbladder is not opacified, effectively confirming that the stent is blocking the cystic duct and preventing internalization of the DNA solution during injection.

[0453] This strategy is used in situations where the common hepatic duct is extremely short or damaged. This strategy can also be used in smaller subjects, such as neonates, where the common hepatic duct itself is extremely short.

[0454] In an optimal embodiment of the present disclosure, the following parameters can be used for injection: These parameters are similar to the strategy used by common hepatic duct injection, considering that only limited additional volume is involved. The preferred flow rate during injection is a minimum of 1 ml / sec, or 2 ml / sec. Preferred infusion pressures are at least 50 mmHg, and in other embodiments at least 80 mmHg. The preferred infusion volume is at least 30, 40, 50, or 60 mL per kilogram of liver weight. Optionally, an additional 5 mL of infusion solution is recommended to account for additional lumens within the biliary tree. The preferred dose of DNA is at least 20 mg / kg liver weight or more. The preferred DNA concentration is at least 0.5 mg / mL DNA or more. · The preferred use of hepatocyte-specific promoters to increase the transfected area of ​​hepatocytes.

[0455] Cystic duct obstruction by a secondary approach combined with a common bile duct strategy One strategy to counteract the loss of the plasmid DNA solution is to occlude the cystic duct. This can be done temporarily or permanently, allowing infusion through a balloon catheter to occur at the level of the common bile duct. One example is a patient undergoing a cholecystectomy. Another example is a patient who has a biliary-enteric anastomosis (anastomosis from the gallbladder to the small intestine) created (surgically, percutaneously via interventional radiology, or endoscopically via EUS) in which a catheter equipped with an occlusion balloon is placed through the gallbladder to occlude the cystic duct. In the above example, instead of using a balloon to occlude the cystic duct, a catheter can be used that can release an umbrella-shaped sheath from its tip and be placed over the cystic duct branch from the bile duct or within the cystic duct itself.

[0456] Intended Use of the Common Bile Duct Strategy The present disclosure may have several applications. Unconventional anatomy of the biliary tree, in which the cystic duct branch is not downstream from the hilum of the liver, is frequently encountered in biliary imaging (Sarawagi et al., Pol J Radiol. 2016; 81: 250-255). Sometimes, the cystic duct opening is upstream of the hilum of the liver (i.e., away from the left or right intrahepatic bile duct). This would significantly complicate injection strategies that bypass the cystic duct and gallbladder. In this case, injection through the common bile duct is advantageous.

[0457] The strategy also has applications in veterinary settings where the biliary anatomy differs from that of humans. Some animals lack a common hepatic duct or a conventional cystic duct. This strategy is also useful in situations where extrahepatic bile duct disease is being treated, such as treating cholangiocarcinoma or fibrous strictures of the bile duct. The strategy may also be useful in treating certain gallbladder disorders that can be treated by genetic intervention. This strategy is also useful for gene delivery to the liver, where the common hepatic duct is too small to be properly localized. In these cases, injection through the common bile duct is the only viable solution for gene delivery to the liver. One example would be certain injections into newborns.

[0458] Gene therapy for cancer is an attractive idea. There are many ways to fight cancer with gene therapy. One attractive approach is to try to deliver genes directly into the tumor microenvironment. These genes can encode a host of therapeutic proteins, including those that modulate and remodel the immune system. In other cases, genes can be delivered directly into tumor cells. In this case, they can encode suicide genes that would kill the tumor immediately.

[0459] Several gene therapy strategies have been previously published. Previous tumor delivery strategies include the use of viral vectors, oncolytic viruses that replicate and function in tumors, and different non-viral strategies. Non-viral strategies include the direct administration of DNA or RNA to tumors via local delivery strategies. Another common strategy is the use of lipids or polymers to deliver plasmid DNA directly to tumors. For these strategies, tumor-specific uptake can be enhanced by incorporating specific tumor-binding ligands on the nanoparticle surface, or alternatively by using polymers or lipids that have a natural affinity for specific tumor types.

[0460] The drawback of all these strategies is the inherent difficulty of penetrating gene therapy vectors into the tumor microenvironment of cancer cells themselves. Most strategies rely on systemic administration of gene therapy vectors. In these cases, gene therapy vectors must cross the endothelial barrier to penetrate deep into the tissue. Many tumor types are well vascularized around the tumor periphery but lack vascularization toward the interior of the tumor. This makes intratumor penetration by vascular administration extremely difficult. Other tumor types have a lot of fibrosis within the tumor, making it difficult to penetrate the tumor with nanoparticles or viral vectors.

[0461] One promising strategy for non-viral gene delivery is hydrodynamic delivery. The efficacy of hydrodynamic delivery is due to fluid pressure, which creates pores in the cell membrane to allow DNA to enter the cell. Hydrodynamic delivery is a highly efficient gene delivery modality to the mouse liver when delivered via the mouse tail vein. During tail vein injection, approximately 10% of body weight of fluid is rapidly injected into the mouse liver over a period of 5–7 seconds. Fluid injection into the tail vein and IVC causes backflow from the right heart into the mouse liver, causing severe fluid stasis. The fluid pressure creates pores in the mouse liver, resulting in efficient gene expression.

[0462] Although hydrodynamic tail vein injection is a very common technique, little research has been done into whether hydrodynamic injection can mediate efficient delivery to tumors within the liver. A major obstacle to even testing this hypothesis is the generation of localized tumors within the mouse liver. Most mouse models develop large, multifocal tumors over time via germline mutations.

[0463] The only study to date evaluating hydrodynamic injection for gene delivery to hepatocellular carcinoma (HCC) was conducted in rats (J Gene Med. 2006 Aug;8(8):1018-26). Rats can also be subjected to hydrodynamic injection. The rats in this study were treated with diethylnitrosamine, a chemical that induces mutations in the rat liver. Over time, these mutations can ultimately lead to the development of cancer-like diseases. The researchers found that hydrodynamic tail vein injection was highly inefficient, did not mediate delivery to tumors, and little expression was observed. Expression could only be detected in tumors when the DNA vector was administered via the hepatic artery. Therefore, the efficiency of delivery appears to be highly dependent on the route of administration.

[0464] A central challenge of hydrodynamic injection is the uncertainty and unpredictability of scaling up the technique from rodents to large animals. When determining how to scale hydrodynamic injection, volumes and flow rates increase by an order of magnitude compared to parameters used in rodents. Furthermore, the internalization pathways and methods for accessing those pathways differ significantly in large animal models compared to those implemented in rodent models.

[0465] Because there is no data supporting actual delivery to liver and pancreatic tissues in large animals, it is currently impossible to predict whether gene delivery by hydrodynamic injection will be effective. In particular, it is impossible to predict which areas of the liver, if any, will express the injected gene. Another important unknown is whether various tumor types in the liver, all of which have different types of tissue structure, will differentially take up any DNA vector when delivered by hydrodynamic injection. Therefore, it is uncertain whether tumors can be reached by hydrodynamic injection.

[0466] 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 will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the intention is to cover alternatives, modifications, and equivalents which may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0467] This disclosure describes a method for gene delivery to liver tumors via hydrodynamic injection. This method utilizes the biliary tract to deliver fluid pressure within the tumor microenvironment. The biliary system is a dynamic network of branching structures that contacts all liver cells. However, previous studies prior to this disclosure taught that the biliary network does not contact the tumor microenvironment, including primary and metastatic liver tumors. Therefore, prior art to this study assumed that this method would fail. This disclosure teaches that the biliary tract is effective in mediating gene delivery to different aspects of tumors. This disclosure also teaches that multiple tumor types, not just liver cells, can be engaged via the biliary system for gene delivery.

[0468] The first step in this method is endoscopy. ERCP is used to access the common hepatic duct within the biliary tree. Once inside the common hepatic duct, a balloon is inflated to seal the duct. Simultaneously, a DNA solution is prepared, with the DNA encoding a therapeutic protein of interest for tumor treatment. In other embodiments, the DNA of interest may encode a diagnostic protein to aid in tumor identification. The DNA solution is loaded into an autoinjector. In a preferred embodiment, the volume of the solution loaded is at least 30 mL per kilogram of liver weight. In other embodiments, the volume of the solution loaded is at least 40 mL per kilogram of liver weight. In a preferred embodiment, the flow rate is specified to be at least 2 mL / sec. In other embodiments, the flow rate is at least 4 mL / sec. This technique has previously been optimized for delivery to normal hepatocytes. Delivery to normal hepatocytes has been taught in the prior art at a minimum flow rate of 2 mL / sec. For intratumoral delivery, the inventors teach that higher flow rates are preferable to allow deeper penetration into the tumor microenvironment. In a preferred embodiment, flow rates of up to 10 mL per second can be utilized to increase expression of DNA within the tumor.

[0469] The minimum DNA dose for efficient delivery into tumors is at least 1 milligram of DNA per kg of liver weight.In another embodiment, the minimum DNA dose is 5 mg per kg of liver weight.In another preferred embodiment, a DNA dose of 10 milligrams per kg of liver weight may be preferred.Generally, for this particular method of tumor targeting, a higher DNA dose is required, because the hydrodynamic approach delivers DNA to the entire liver, and as a result, only a small amount of DNA reaches the tumor microenvironment.The amount of DNA in tumors affects the degree of transfection evaluated.

[0470] In some embodiments of the present disclosure, a method to address this limitation is to position the catheter in the right or left hepatic duct of the biliary system rather than the common hepatic duct. This facilitates DNA delivery to only the right or left side of the liver. If a tumor is localized 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 multiple liver tumors are present and / or metastases are present throughout the liver, injection through the common hepatic duct is preferred.

[0471] In some embodiments of the present disclosure, DNA molecules can be further formulated. DNA molecules can be formulated with various polymers, peptides, or lipids, which have been reported to promote greater uptake into tumors. It is anticipated that hydrodynamic injection can work synergistically with these delivery reagents to promote even more efficient delivery of these nanoparticles to tumors. Hydrodynamic injection particularly helps them penetrate and reach the interior of tumors. Considering that tumor cells are dividing, the nuclear membrane breaks down more frequently, allowing more non-viral DNA to access the nucleus for expression.

[0472] Gene delivery to the pancreas follows similar parameters, except that ERCP accesses the pancreatic duct and the dose is based on estimated pancreatic weight. Most tumors are located in the head of the pancreas, and therefore the tumor must be downstream to deliver DNA under tumor pressure.

[0473] Monogenic inherited liver diseases encompass a spectrum of disorders ranging from metabolic disorders to coagulation disorders, including hemophilia A and B, alpha-1 antitrypsin deficiency, familial hypercholesterolemia, Wilson's disease, Crigler-Najjar syndrome, methylmalonic acidemia, and ornithine transcarboxylase deficiency. These diseases cause significant morbidity in patients, even with today's modern therapies. The curative treatment for these disorders is liver transplantation, but liver availability is limited. Furthermore, transplantation itself requires lifelong immunosuppression, leading to the risk of infection and adverse drug reactions.

[0474] As an alternative to liver transplantation, gene therapy, which involves modifying a patient's own tissue with a missing or dysfunctional gene, is being investigated. Following the success of adeno-associated virus (AAV) in preclinical models, it has progressed to clinical trials. However, several limitations have become apparent, including the fact that many patients have pre-existing neutralizing antibodies to AAV; high levels of AAV injection induce an immune response followed by clearance of AAV; and the gene packaging size is limited to approximately 4.8 kb.

[0475] To address these issues, we developed a non-viral gene therapy method using hydrodynamic injection via an endoscopic route. In a pilot study using endoscopic retrograde cholangiopancreatography (ERCP), we successfully delivered genes to pig livers, achieving transfection efficiencies of 30-50%, potentially meeting the need for cures for many monogenic liver diseases. Our previous work on ERCP gene therapy for hemophilia B has received R21 funding from NHLBI / NIH to further explore this gene therapy research in non-human primates.

[0476] This study will explore optimal methods of infusion into the liver of primates that can be translated to human patients. This study also describes optimal methods and compositions for hydrodynamic gene therapy of hemophilia B.

[0477] Rare individuals have mutations in these genes that can disrupt fundamental processes in the liver, such as metabolism or clotting, which can lead to a variety of problems, including thickening of blood vessels with cholesterol, metals such as copper building up to toxic levels in tissues, or a person bleeding continuously and without stopping.

[0478] A potential cure for these approaches would be to insert a functional copy of a gene into a person's liver, thereby reversing these processes. This study will explore this gene therapy using a common medical procedure in which a tube is inserted down the throat, through the stomach, and into the intestine. A thin wire can then be guided directly to the liver for targeted DNA delivery. The study will explore approaches that do not use viruses as delivery vehicles, improving safety. The potential impact is a cure for these patients, sparing them from frequent infusions of other drugs and drug costs that can amount to hundreds of thousands of dollars per year.

[0479] 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 will be understood that they are not intended to limit the disclosure to those embodiments. On the contrary, the intention is to cover alternatives, modifications, and equivalents which may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[0480] This disclosure describes a series of steps toward the effective delivery of DNA to the liver of primates for the purpose of gene therapy.

[0481] The first step involves accessing the biliary system for gene delivery and infusion. In a preferred embodiment, an endoscopic retrograde cholangiopancreatography (ERCP) procedure is performed to place a balloon catheter inside the baboon's common hepatic duct. In a preferred embodiment, the ampulla of Vater may be cut to increase the size of the opening to facilitate cannulation of the duct.

[0482] To avoid injection into the gallbladder, a radiocontrast solution is used to localize the catheter placed through the cystic duct into the common hepatic duct. Contrast injection is also used to verify that the balloon seals the duct during injection and that the right and left hepatic ducts are visualized.

[0483] The injection protocol is then initiated. The injection proceeds using an autoinjector loaded with the desired DNA solution. Tubing connects the end of the autoinjector to the bile duct catheter to inject the DNA solution through one of the lumens.

[0484] In a preferred embodiment, the DNA solution is a saline solution with pure recombinant DNA dissolved therein. The DNA can be of many types, including plasmid DNA or minicircle DNA. In other embodiments, linear, closed-end DNA can be utilized.

[0485] The injection should proceed at specified parameters that mediate efficient gene delivery. The volume parameter is set at 30 milliliters per kilogram of liver weight. The volume can also be 40 mL / kg or higher.

[0486] In preferred embodiments, the flow rate parameter shall be at least 2 mL / sec. In other embodiments, the flow rate may be at least 3 mL / sec or more.

[0487] Pressure parameters are at least 50 mmHg when using specialized constant pressure infusion devices. In other embodiments, higher pressures are used, including greater than 80 mmHg or greater than 120 mmHg.

[0488] The DNA dose, in certain embodiments, is at least 10, 20, 30, 40, or 50 milligrams per kilogram of liver weight. The purpose of the higher DNA dose is to obtain a sufficiently high serum or plasma concentration to obtain an effective level of human factor IX to treat hemophilia B.

[0489] It is considered that the DNA vector composition uses a hepatocyte-specific promoter. In a preferred embodiment, the vector should also have one or more hepatocyte-specific enhancers to drive even higher levels of transcription. A codon-optimized gene cassette is selected with codons selected for abundance in hepatocytes. This can be applied to a preferred embodiment for the expression of human factor IX. A strong polyadenylation sequence is also utilized.

[0490] Infusion into the primate liver is also intended to monitor for signs of liver damage, which can be accomplished by a general liver panel of chemistries including transaminase function.

[0491] The balloon is deflated after injection, after which the catheter is withdrawn from the bile duct. Injections can be repeated within the same procedure or on different procedure days, as indicated.

[0492] 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 increasing the efficiency of hydrodynamic gene delivery via the biliary system. As described in detail below, the present disclosure is based, at least in part, on the surprising discovery that by modifying a DNA vector so that the bacterial sequence on the plasmid DNA was significantly reduced to less than 500 base pairs, the observed transfected area of ​​hepatocytes was significantly increased to more than 70% of hepatocytes, an increase from less than 50% when using conventional plasmids. The present disclosure also reveals the surprising finding that the efficiency of gene delivery significantly increases at doses of DNA equal to or greater than 20 mg per kg of liver weight, followed by saturation thereafter. Such a nonlinear relationship was unexpected but is highly useful in planning therapeutic administration schemes. The present invention also surprisingly found that high-level transfection of over 40% of the integrated reporter gene showed no loss of levels even after three months, contradicting previous reports using transposons and the first report of immune tolerance at this threshold of antigen expression. The present disclosure also surprisingly found that episomal vectors delivered by hydrodynamic injection could be expressed for up to four months, the longest period of expression observed to date, and that they survived extensive dilutional loss in the liver, increasing in size by up to 50%. The present disclosure also reports the surprising finding that very large DNA vectors, exceeding 12 kb in size, can be delivered to large animals. Even more surprisingly, transfection efficiency did not appear to decrease despite the progressive increase in DNA vector size, contradicting all published reports using non-viral DNA vectors. The present disclosure also demonstrates that repeat gene injections successfully achieved gene expression with efficiency comparable to the first injection, resulting in both proteins being detected and found in the same cells. It was uncertain whether the hydrodynamic injection from the first injection would perturb the liver structure and prevent the second injection.This disclosure also makes the previously untested discovery that high DNA vector doses up to 40 mg / kg liver weight do not exhibit toxicity, and more importantly, that the DNA concentration in the infusion fluid is a major determinant of the observed delivery efficiency. This disclosure also reveals new findings related to the efficiency of gene expression relative to flow rate and how to optimize infusion for appropriate volumetric doses, both of which have not been reported previously. Finally, this disclosure reports that hepatic sinusoidal endothelial cells can be targeted for gene delivery, which has not been shown in previous publications.

[0493] Overview Hydrodynamic injection through the liver is an interesting new gene therapy modality. It is advantageous because it is non-viral, therefore highly scalable, and safer than viral approaches. Hydrodynamic injection can deliver only naked DNA, making it the simplest possible gene therapy modality. This technique is known to be highly efficient in mouse models, but has not traditionally been well-scaled to larger animal models.

[0494] Several groups have been investigating for many years whether hydrodynamic injection can be extended to large animal models. An inherent challenge in developing new techniques for hydrodynamic injection is the challenge of translating the technique between animals. New methods must be adapted and optimized for each animal and organ, empirically testing which strategies work. Pigs are a convenient animal model for testing because their livers can be comparable in size to adults, depending on the pig's age. Therefore, hydrodynamic techniques established in pigs are likely to be transferable to humans. However, some caveats remain. For example, pig livers have interlobular fibrosis between lobes, which can create some uncertainty.

[0495] Although the research on hydrodynamic gene delivery in mouse, rat and rabbit models is a solid proof-of-concept study, unfortunately, it does not teach how to perform the procedure in human patients and how to optimize gene delivery efficiency.One example of this is that many groups have isolated specific lobes of the liver and injected them through the hepatic vein that supplies that lobe.This technique was first described in a paper using rabbits in Human Gene Therapy 2002 Nov20;13(17):2065-77.

[0496] A similar approach has since been used in pigs, applying the same strategy of isolating individual lobes with a balloon occlusion catheter to the lobes via hydrodynamic infusion (Mol Ther. 2009 Mar;17(3):491-9). However, unlike traditional pharmaceuticals or biologics, there is no simple weight-based scaling for hydrodynamic infusion procedures, so the flow rates and volumes used in these later studies to mediate gene delivery in pigs could not be predicted from rabbit studies. This finding is also seen with simple hydrodynamic tail vein infusions in mice and rats; the volumes and flow rates used in mice do not easily translate to rates when adjusted for larger sizes.

[0497] Another example of variability is that the vessel or conduit chosen for hydrodynamic injection alters the parameters of the injection, along with how gene delivery works and its outcome. One example of this has been observed in a porcine model, where single-lobe injection is more efficient than a strategy of injecting through the hepatic vein to reach the entire liver, compared to clamping the inferior vena cava and portal vein to isolate blood flow there. The subtle differences between these different vessels are not clear until experiments are performed.

[0498] Similarly, the biliary system has been investigated as a different route for hydrodynamic gene delivery. The biliary system is unique in that it has a much smaller total volume than the vascular system that supplies the liver. This comparison compares the biliary system volume of approximately 15–30 mL to the blood volume of an adult human, which is approximately 600 mL. The biliary system is also unidirectional, preventing the injected fluid from simultaneously leaking out through unsealed ends. This unidirectional nature of the biliary system is advantageous for maintaining adequate fluid pressure during injection, ensuring that the high-pressure DNA solution is expelled into the surrounding liver tissue rather than spurting outward through another hepatic vessel.

[0499] Although the hypothesis of liver-directed gene delivery has existed for over 20 years, specific details regarding how to efficiently perform this procedure in large animal models and the resulting outcomes have eluded researchers. The first description of hydrodynamic gene delivery via the bile duct was based on experiments in dogs. This group used surgical techniques, an injection rate of approximately 1 mL / sec, and sutures to prevent antegrade flow within the biliary tree (Hum Gene Ther. 1997 Oct 10;8(15):1763-72). The researchers injected into the common bile duct, ensuring that fluid was delivered to the gallbladder in addition to the liver. Although the researchers were only able to detect low levels of luciferase activity, histochemical analysis of the liver did not reveal any protein, suggesting that an undetectable proportion of pDNA was internalized by hepatocytes.

[0500] Another group applied the use of ERCP to mediate gene delivery to the liver of dogs and pigs (GIE 2005, T1249 Abstract). ERCP is an improvement over surgery. Another modification to the procedure used by the researchers was the use of a balloon to prevent antegrade flow during hydrodynamic injection, thereby increasing the volume of fluid containing the DNA solution entering the liver. The group used an infusion rate of 5 mL / min for their procedure and continued the infusion through the common bile duct, thus simultaneously infusing the liver and gallbladder. The researchers did not report any gene delivery efficiency to the liver, as reflected by detectable protein expression on IHC, IF, or Western blot. However, there were reports of protein detection in the systemic circulation. The efficiency and usefulness of this procedure, when applied to liver disease, remain uncertain.

[0501] Concurrently, another group reported that injection of pDNA through the bile duct via a surgical approach in rats could mediate gene delivery (Gut. 2005 Oct; 54(10): 1473-1479). However, as discussed above, the hydrodynamic parameters in rodent models cannot be scaled up to larger mammals (e.g., dogs, pigs, and humans). As an example, the flow rate used by this group was 0.54 mL / min, and surgical ligation served to seal the bile duct. The relative efficiency of pDNA delivery to hepatocytes was only approximately 1% of hepatocytes based on histochemical staining, which is significantly less efficient than the equivalent hydrodynamic tail vein approach.

[0502] Improved techniques for biliary hydrodynamic injection have been investigated. The first study investigated the mediation of gene delivery to pigs via hydrodynamic injection and the observation of detection by immunostaining (Kumbhari, GIE 2018). New injection parameters that could be tolerated by the pig biliary system were tested. Because pig bile ducts burst at parameters higher than 2 mL / s and 30 mL / s, these parameters were selected for subsequent testing. The pig biliary system was accessed via ERCP, and a balloon was placed in the common hepatic duct to avoid injection into the cystic duct and gallbladder. This was a significant innovation compared to previous studies, as avoiding gallbladder injection generates more pressure on the liver during injection and therefore allows for more efficient gene delivery. The balloon was left inflated for 1 minute after injection to increase fluid delivery.

[0503] The results demonstrated for the first time experimental data verifying the presence of genes expressed from pDNA in the liver (Kumbhari, GIE 2018). PCR demonstrated the presence of pDNA in all lobes and sides of the liver. Western blot analysis confirmed that the delivered protein was expressed in all liver tissues. Immunofluorescent staining of the delivered protein was also observed, although delivery efficiency was less than 1% of hepatocytes. Protein expression appeared to be stable for up to 60 days due to the Sleeping Beauty transposon effect.

[0504] While this previous work was a major advance, its delivery efficiency was not sufficient to treat any clinical disease that requires a large proportion of the liver (>10-20% of hepatocytes) to express the transgene of interest. Improvements beyond this previous work are needed, and are discussed in detail in published data (Kruse, GIE 2021).

[0505] The first improvements focused on a better vector composition injected into the liver. A hepatocyte-specific promoter was used to drive expression, along with codon optimization of the transgene and stabilization of the 3'UTR. A higher dose of plasmid was delivered, and a more active transposase system was introduced into piggyBac. Taken together, these changes to composition, pDNA dose, and transposase resulted in the largest increase in transfection efficiency, with over 30% of hepatocytes expressing the transgene. All of these improvements are described in Kruse, GIE 2021.

[0506] Further efforts to improve this technique included targeting multiple cell types in the liver via ubiquitous promoters, including cholangiocytes, endothelial cells, and neurons. Improved vector composition demonstrated the ability for cell-specific delivery, demonstrating that only hepatocytes or endothelial cells could express the desired transgene when the respective hepatocyte or endothelial cell promoter was utilized. Further novel findings included the ability to target zone 3 of the liver lobule instead of zone 1 by altering the flow rate. The flow rate that induces transaminase elevation and liver injury was clarified. For the first time, an alternative strategy for biliary hydrodynamic infusion beyond ERCP to access the biliary system was described.

[0507] The ability of the bile duct to tolerate higher flow rates and volumes without rupture was better characterized. The pressure relationship with infusion parameters was clarified, and the pressure reached during biliary hydrodynamic infusion sufficient to mediate gene delivery was also identified. These findings were described by Huang, PLOS One 2021.

[0508] Another group subsequently contributed to the development of biliary hydrodynamic gene delivery (Mol Ther Methods Clin Dev. 2022 Jan 19;24:268-279). This group studied hydrodynamic gene delivery techniques in young pigs weighing 4-6 kg. The methods and techniques employed by this group differed from those published by Kumbhari and Kruse, and consequently, their effectiveness differed. In their protocol, they accessed the biliary system via a surgical procedure rather than ERCP. They did not use a balloon catheter; instead, they utilized multiple different surgical clamps across the cystic duct and several different veins. A single-lumen catheter was used for infusion. No clamps were placed on the bile duct itself, likely to avoid catheter breakage or because surgical clamps would not seal with the catheter in place. The infusion itself was performed in the common hepatic duct, but antegrade flow into the common bile duct was not blocked, resulting in a pressure drop and leakage of the DNA solution into the intestine.

[0509] The injection parameters were significantly higher than those employed in the previous Kruse and Kumbhari study, which targeted a 10 mL / sec injection rate and a 100 mL injection volume. This group was able to detect the presence of DNA by PCR in all lobes of the porcine liver. Similarly, luciferase enzyme activity was also detected in all lobes. Unfortunately, immunostaining of the liver was largely undetectable, with only rare cells detected. Another important factor was that although protein expression was initially detectable, it decreased to the point of almost complete disappearance by 10 days postinjection. The researchers used episomal minicircle vectors and nanovectors / nanoplasmid vectors as DNA constructs. Although these vectors have been reported to be superior to control plasmid DNA, they were insufficient to increase expression levels in IHC or mediate long-term expression. Taken together, these results suggest the limited effectiveness of their method / technique, and it is questionable whether these alternative vectors could improve expression by biliary hydrodynamic techniques.

[0510] The present disclosure seeks to provide improved techniques, methods, and compositions for increasing the efficiency of gene delivery after biliary hydrodynamic injection in large animals. Further improvements include new safety strategies and methods for targeting gene expression to specific cell types. Other improvements include re-administration of gene vectors and strategies for achieving long-term expression. The size limitations possible with biliary hydrodynamic injection have also been described.

[0511] Increased transfection area The key goals of gene therapy techniques are to deliver DNA into as many target cells as possible and to ensure that the DNA reaches the nucleus so that protein expression can be detected in as many target cells as possible. This is important for both viral vector and non-viral strategies. To date, expression has been achieved in 30%–50% of pig hepatocytes, albeit with varying DNA doses. However, there can be significant differences in expression in each lobe. Furthermore, these results describe only one plasmid DNA composition and one transgene, which may bias the efficiency of this technique when applied to other vector sizes and proteins.

[0512] To target the current delivery of DNA to more hepatocytes, further modifications to the hydrodynamically injected vector composition were initiated. The modified vector composition is believed to produce better results through synergistic interaction with procedural parameters. Plasmid DNA modification by reducing or eliminating the bacterial backbone significantly increased the transfected area observed in pig liver sections to over 50% of hepatocytes. In specific sections of the transfected region, 60%, 70%, 80%, or even 90% of hepatocytes within the lobule expressed the gene of interest. On average, 72% of hepatocytes stained positive for the gene of interest. The stained area was significantly denser, indicating higher intrinsic protein expression within the cells.

[0513] This improved transfection efficiency was achieved by injecting vectors that are circular DNA molecules lacking most bacterial sequences. The bacterial sequences present are less than 1 kilobase long, or in some cases, less than 500 base pairs. One example is a nanovector or nanoplasmid with a reduced origin of replication and bacterial selection region. This nanoplasmid has an R6K conditional origin of replication that requires pir+ E. coli host cells for growth. Selection is achieved by an RNA-OUT system that downregulates the SacB gene. In another example, the vector is pFAR, which has a smaller bacterial backbone of less than 1,000 base pairs. The pCOR backbone consists of three bacterial elements: a 0.4 kb R6Kγ conditional origin of replication (oriγ) that requires a functional R6Kπ initiator protein, a 0.2 kb selectable tRNA suppressor gene (sup Phe), and a 0.4 kb cer (ColE1 degradation) fragment for degrading pCOR oligomers, totaling less than 1000 base pairs. Any bacterial backbone of varying composition and less than 1000 base pairs, preferably less than 500 bp, is considered suitable for this approach. Another option is the use of minicircle vectors, which are circular DNA molecules lacking bacterial elements. Minicircle vectors are generated by recombination of two distinct sites on the plasmid DNA. Recombination of these sequences removes the bacterial sequences on the plasmid DNA, leaving only short remnants of mammalian sequences and recombination sites.

[0514] 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 closed-end linear DNA molecule (PLoS One. 2013 Aug 1;8(8):e69879), also known as a ministring DNA molecule (Mol Ther Nucleic Acids. 2014 Jun;3(6):e165) or dogbone DNA (Hum Vaccin Immunother. 2015 Aug;11(8):1972-1982). These linear DNA molecules lack any bacterial sequences and have covalently closed ends. As a result, they do not induce a DNA damage response and are not integrated into the genome. These linear DNA molecules also move more freely in solution, which aids their hydrodynamic movement to the cell nucleus. The linear DNA molecule has small residual sequences (up to 50 bp at each end) that are remnants of the viral or bacterial excision sites from which it was generated, but otherwise contains no other bacterial sequences.

[0515] These vector compositions work synergistically with biliary hydrodynamic injection, as the lack of bacterial elements provides a better expression profile. Another hypothesis is that their smaller size may aid in migration to the nucleus during hydrodynamic injection. These types of DNA molecules did not significantly improve the transfected area in mouse studies, a result unexpected because most of their effects were thought to be cell-specific with respect to expression. It is unclear whether these molecules improve the transfection area during hydrodynamic injection. In one study of biliary hydrodynamic injection, the reduction of bacterial scaffolds (minicircles, nanoplasmids) failed to improve the procedure (Mol Ther Methods Clin Dev. 2022 Jan 19;24:268-279). However, for gene delivery using biliary hydrodynamic injection, the above vector compositions should be used to maximize the amount of protein expression in hepatocytes.

[0516] Improved expression lifespan An important aspect of gene therapy is the length of expression.The length of expression is affected by several different factors, including the inherent silencing of gene vectors, the dilution loss of episomal DNA constructs, the death of cells that receive genes, and the adaptive immune response to gene products.These can be greatly affected by the delivery vehicle and / or the procedure used to deliver genes.

[0517] In the case of hydrodynamic gene delivery, the procedure itself can cause traumatic injury through the high-pressure injection of fluid into tissues. Tissues transiently swell with fluid, which can cause cell death in several different cell types. This is particularly evident in mouse models after hydrodynamic injection, where some tissue necrosis and a transient inflammatory response accompanied by immune infiltration are observed. In a study in dogs using vascular hydrodynamic gene delivery, it was observed that specified hydrodynamic injection parameters were associated with early loss of transgene expression. This was thought to occur because they elicited an immune response against the transgene product. Transgene products expressed in the inflammatory environment of hydrodynamic injection may have a vaccine-like effect. Therefore, it is unclear whether any hydrodynamic procedure will have long-term expression, whether due to DNA-specific reasons or an immune response to the transgene product.

[0518] The possibility that hydrodynamic injection may lead to short-term expression of the delivered transgene is consistent with observations in mouse models in which hydrodynamic injection of a plasmid encoding a hepatitis B virus antigen led to acute elimination. In contrast, adeno-associated virus (AAV)-mediated delivery of hepatitis B virus antigen led to long-term expression of viral proteins without inflammation. Therefore, it may be uncertain whether hydrodynamic injection leads to short-term or long-term expression, especially compared with other modes of gene delivery.

[0519] It is unclear how the biliary hydrodynamic injection method modulates or interacts with the host immune system to enable long-term expression, as this method is inherently different from other hydrodynamic strategies. Published data indicate that a transposon strategy using biliary hydrodynamic injection can express genes for up to two months (Kumbhari, GIE 2018). However, the amount of protein produced in that study was very low, and only a small percentage of cells (less than 1%) expressed the delivered gene. There was no protein at a clinically relevant transfection rate sufficient for the immune system to recognize and respond. Based on previous studies, it was unpredictable whether the immune system would mount an adaptive immune response after high levels of transfection. Given subsequent improvements to the biliary hydrodynamic method, it is unpredictable whether additional proteins will become more recognizable to the immune system, leading to eventual elimination. Although published data show that expression was maintained for 3 weeks, this is a very early time point to examine, as adaptive immunity can take up to 2 months to develop. A longer time course of expression is needed to determine whether the integrated transgene expresses the gene throughout the life of the animal.

[0520] This disclosure demonstrates that the combination of an optimized expression cassette with a hepatocyte-specific promoter and piggyBac transposon successfully mediates transgene expression in pig hepatocytes for up to three months. No significant difference was observed in the relative transfection area of ​​protein-expressing hepatocytes when comparing the transfection rates at day 3 and month 1. This demonstrates stable expression and the absence of immune responses. The specified parameter approach and this vector composition are the best options for achieving the goal of an expression duration of three months or longer.

[0521] A major challenge with gene therapy is the potential for genotoxicity due to DNA integration into host chromosomes, which can lead to oncogenic transformation. As a result, many regulatory agencies and physicians prefer strategies in which episomal DNA is delivered to cells. Episomal DNA does not integrate into the host genome and is expected to have minimal or no effect on host cell viability. A challenge with episomal DNA strategies is that they often lose their expression potential over time due to a variety of reasons, including episomal gene silencing and dilutional loss of plasmid DNA with cell division. Improvements are still needed to develop best practices, methods, and vector compositions for long-term expression of episomal DNA after biliary hydrodynamic injection.

[0522] Previous studies of hydrodynamic injection via the vascular route in pigs have observed episomal pDNA expression for up to two months after injection, although the transfected area decreased by approximately 50% during that time (Mol Ther Nucleic Acids. 2013 Oct;2(10):e128). Literature from mouse studies has shown that the majority of injected plasmid DNA molecules silence expression after one month, so it is unclear how long expression will last with nonviral constructs. Even for viral vectors such as AAV, substantial loss of expression from episomal DNA occurs between several weeks and two to three months after injection (bioRxiv 2022.03.24.485675). While previous studies of vascular hydrodynamic injection are promising, it is unclear whether the biliary approach can achieve similar or even longer expression periods, especially in growing pigs with gradual pDNA silencing. No studies have described expression of pDNA from bile infusion for more than one week in large mammals.

[0523] The present disclosure addresses these limitations by describing a method for achieving episomal DNA expression for up to four months using a vector composition lacking large bacterial DNA backbone sequences. Previous vascular hydrodynamic studies have not pursued this approach or have failed to achieve long-term expression using reduced bacterial backbone vectors. This modified vector DNA backbone must be delivered into cells using a hydrodynamic biliary approach with set parameters to avoid eliciting an immune response and ensure proper pDNA transport to the nucleus. This disclosure represents an improvement in achieving pDNA expression from biliary hydrodynamic injection for the longest period of time to date. Furthermore, the approach described herein also overcomes previous obstacles to episomal vector gene delivery; in this experiment, pig livers increased in size by 50% from the time of DNA administration to the time of liver harvest at the end of the four-month experiment. Based on the literature for episomal vectors, this would normally result in significant dilutional loss of the vector genome, reducing the observed transfected area of ​​hepatocytes (Hum Gene Ther. 2012 May; 23(5): 533-539). In contrast, the transfected area of ​​hepatocytes in our study remained relatively consistent throughout the study period, suggesting a unique and unexpected feature of the biliary hydrodynamic gene delivery approach and vector composition with reduced bacterial backbone size.

[0524] Re-administration of gene expression between procedures A current limitation of all gene therapy approaches is the inability to re-administer gene therapy vectors. This is primarily due to the fact that viral vectors elicit an immune response against the viral capsid, which prevents the free administration of the vector for a second dose. Hydrodynamic injection, which is non-viral and lacks any protein components that could elicit an immune response, may offer a solution to this limitation. Re-administration of hydrodynamic injection in mouse models has been shown to result in comparable levels of protein expression after the second injection. However, hydrodynamic injection in mice has not been shown to be additive to the original injection, likely due to pDNA gene silencing, which makes this experimentally difficult to demonstrate.

[0525] Although hydrodynamic injection has been shown to be successful in certain mouse models, studies in large animal models have not been successful. A study of hydrodynamic injection in a dog model failed to achieve any expression with the second administration, despite the administration of immunosuppressive drugs (Hum Gene Ther. 2017 Jul;28(7):551-564). This is thought to be caused by a significant immune response to the transgene during hydrodynamic injection, which may be re-stimulated with each subsequent injection. In another study in pigs, readministration was achieved with a second administration of human alpha-1 antitrypsin, but it is difficult to determine the contribution of the first and second doses to the observed expression. As a result, previous studies have not demonstrated whether hydrodynamic injection readministration can be performed in large animal models, or whether two different genes can be sequentially readministered without inhibiting expression of the first administered gene, and whether the same cells in the liver can be targeted for expression with the second administration. Previous studies have not clarified whether re-administration of gene therapy is feasible in terms of gene expression and immune responses in the biliary system. No data from the biliary system support the feasibility of re-administration. While it has previously been shown that the procedure can be repeated a second time in pigs without rupturing the bile duct or substantial damage to the liver, this does not address whether gene expression is possible using this procedure. More importantly, it is unclear whether the second injection has the same gene delivery efficiency as the first injection.

[0526] To address the challenge of re-administration, this disclosure describes an approach for achieving non-viral gene therapy re-administration via the biliary system. One approach is to use a vector composition with a cell-specific or ubiquitous promoter for the first infusion using optimized, established bile infusion parameters. This second infusion procedure can be performed at least one, two, three, four, or more weeks after the first infusion, thereby achieving significant gene expression. The injection procedure does not need to be modified to achieve this result. The second infusion does not result in a loss of expression from the first infusion; individual cells within the liver can express DNA from both infusions. As a result, the second infusion does not shift or silence gene expression from the DNA from the first infusion. Transfection efficiency is neither reduced nor altered by the second infusion. Depending on the promoter used during the second infusion, the second infusion can contain DNA targeted to all cell types, including bile duct cells and endothelial cells targeted in the first infusion, without interference.

[0527] Re-administration of gene expression within a single procedure It has previously been shown that multiple hydrodynamic injections through the bile duct in a single bile duct cannulation in a single pig are technically feasible and well tolerated. What remains unclear is whether multiple injections of DNA can be performed during a single pig procedure to generate expression from multiple different plasmid DNA molecules. There is reason to suspect that this would not technically work and could have deleterious effects on the pig liver. It has previously been demonstrated that within 15 minutes of bile injection at high flow rates, fluid-filled vesicles can accumulate in the pig's cytoplasm (Huang, PLOS One 2021). If these vesicles accumulate, it is uncertain whether the hepatocytes can tolerate additional vesicles and / or whether the vesicles interfere with the transport of a second DNA molecule through the cytoplasm to the nucleus. Furthermore, although multiple injections per day were tolerated, it is unclear whether the long-term effects on the pig would be toxic to the liver and whether potential hepatotoxicity would increase at longer time points after injection, leading to loss of protein expression.

[0528] There are two primary clinical reasons for performing multiple injections in a single procedure. In one example, a patient requires treatment with different DNA constructs encoding multiple distinct proteins to treat their disease. The cost and logistics of combining these DNA constructs into a single volume may be impractical or may alter the total fluid volume, compromising the effectiveness of hydrodynamic delivery. More specifically, combining volumes and / or increasing DNA concentrations into a single injection may alter the optimized parameters of the gene delivery procedure. Therefore, an alternative to this approach is to perform a second injection in the same pig to retarget gene delivery to the liver.

[0529] A second reason for performing multiple hydrodynamic injections within a single ERCP procedure is in the event of technical difficulty and / or technical failure, where the injection fails due to the balloon moving out of position, the balloon deflation, the autoinjector malfunctioning, or the failure of the Luer-lock connection in the catheter to the autoinjector tubing. In these situations, a second injection can be immediately repeated using the entire DNA dose. An important reason for attempting a second injection at the time, rather than rescheduling it for a different day, is that each ERCP procedure carries some risk of pancreatitis, and therefore the total number of ERCP procedures should be reduced. Furthermore, if a second DNA solution is available, it may be relatively easy to reload the autoinjector with the catheter already in place and proceed with the injection.

[0530] Previous studies have not demonstrated the technical feasibility of gene re-administration within a single bile duct procedure. The present technology demonstrates its feasibility and practicality in human patients. In this disclosure, a second gene infusion can proceed within 5 minutes of the first infusion. In other examples, gene infusion can proceed within 10, 15, or 20 minutes, or even longer, after the first infusion. The present technology demonstrates that after a second DNA treatment, two proteins expressed from these DNA molecules can be detected alongside each other in the same liver, despite being injected at different times. The present technology also demonstrates that proteins derived from the delivered DNA can be found coexisting within the same hepatocyte. With this method, the present invention demonstrates that injection parameters do not need to be modified from their intended use as a single infusion. However, flow rates above 5 mL / sec are avoided during the first infusion to prevent significant fluid vesicles, which would inhibit the second infusion.

[0531] Mixing pDNA vectors in a single injection for simultaneous delivery to the same cells In some cases, it is preferable to simultaneously deliver multiple different genes into cells. Previous studies have shown this can be achieved by placing multiple gene cassettes on the same plasmid DNA. However, a drawback of this approach is that larger plasmid DNA sizes can reduce the transport of the plasmid DNA into the nucleus. Furthermore, for manufacturing schemes in which both plasmid DNA molecules have already been created separately, it is often preferable to combine the plasmid DNA molecules into a single injection volume rather than two separate injections. In these cases, cloning the plasmid DNA constructs into a single plasmid can be impractical and costly. Furthermore, larger plasmid DNAs can reduce delivery efficacy.

[0532] Previous studies have not demonstrated that two different plasmid DNAs can be delivered simultaneously, resulting in gene expression from both plasmid DNAs. It is uncertain whether both plasmid DNAs are expressed equally in different cells or whether one plasmid predominates over the other in terms of gene delivery efficiency. Furthermore, it is unclear whether both plasmids coexist within a particular cell type, as is necessary for efficacy in certain disease indications.

[0533] This technique demonstrates that simultaneous biliary hydrodynamic delivery of two different plasmid DNAs is feasible and that gene expression can be achieved within the same cells. Through the biliary hydrodynamic approach, almost all cells appear to have both proteins present within them after gene delivery. This suggests that the same hepatocytes are experiencing the same amount of fluid pressure simultaneously, thus leading to simultaneous delivery of the plasmid DNAs.

[0534] DNA size restriction The size limitations of DNA vectors for a given gene therapy approach are important when defining potential applications for gene therapy. Current vector systems, including adeno-associated viruses (AAVs, genome size 4.8 kb), are severely limited in their DNA cargo size, limiting their ability to fully deliver genes for several rare diseases (VWD, hemophilia A), among other therapeutic possibilities. Viral vectors must encapsulate their entire genome within a defined capsid, inherently limiting their packaging size. Nonviral strategies generally do not share this limitation because they are not packaged into viral capsids. However, all nonviral strategies have been observed to exhibit varying transfection efficiencies depending on the size of the plasmid DNA used. For example, some transfection reagents transfect neither larger pDNA constructs nor smaller pDNA sizes. It is known that large DNA fragments can be delivered into cells in mouse models by hydrodynamic injection. However, whether this extends to hydrodynamic strategies in larger animals has not been investigated. Furthermore, it has not been demonstrated whether biliary hydrodynamic injection can deliver large plasmid DNA molecules of greater than 8 kb, 9 kb, or 10 kb.

[0535] Previous studies have shown that plasmid DNA molecules up to 5.5 kb in size can be delivered to pig livers with high efficiency via biliary hydrodynamic injection. No further data has been published on what happens with larger plasmid sizes. In this disclosure, strategies for delivering DNA molecules greater than 12 kb in size are described, along with the delivery of DNA molecules greater than 15 and 20 kb in size via the biliary approach. These strategies include the delivery of DNA to multiple cell types, including hepatocytes, cholangiocytes, and endothelial cells. Obtaining plasmid DNA greater than 10 kb in size and utilizing higher DNA mass doses to maintain equivalent per molecular DNA dose are described. Biliary hydrodynamic injection is described using volumes greater than 30 mL / kg and injection parameters greater than 2 mL / sec, which are sufficient for the delivery of large DNA constructs to these cell types. Expression can be achieved using ubiquitous promoters, such as the cytomegalovirus promoter. Cell targeting can also be used to increase expression using specific promoters.

[0536] The newly discovered ability to deliver larger DNA sizes helps define guidelines for appropriate dosage. Transfection efficiency can be maintained despite larger plasmid sizes when the molecule-to-vector ratio is the same. The first step is to normalize all DNA weight-based dosages (mg DNA / kg liver) by the index plasmid size to achieve the transfection threshold of the index plasmid. For example, in this case, the index plasmid used to establish transfection efficiency has a size of 8.6 kb, with known targets of approximately 10 mg / kg dose for 50% and approximately 20 mg / kg dose for 70%. Within these parameters, multiplying the weight-based dose by the plasmid DNA size (kb) and dividing by the index plasmid size of 8.6 kb yields a plasmid DNA dose that should produce similar injection results.

[0537] To simplify clinical application, the following formula is used to calculate the DNA dose for any given liver weight (kg) and plasmid DNA size (kb), as summarized in Table 4.

[0538] [Table 4]

[0539] Small deviations in this formulation within 0.5 mg / kg / kb do not significantly affect the percentages and result in similar dose effectiveness.

[0540] This calculation should not be used for nanoplasmids or other vectors with reduced bacterial backbone size, as it has been observed in the above experiments that nanoplasmids or other vectors with reduced backbone size have inherently better properties for mediating higher transfection efficiency, even when differences in plasmid DNA size are taken into account. Therefore, only similar vector types should be compared when using this formulation. Similar calculations can be performed in nanoplasmid administration experiments to elucidate the appropriate dose.

[0541] Summarizing the above experiments, it was found that the 5.8 kb Nanoplasmid achieved approximately 70% transfection. The 70% formulation was calculated as 14.5 mg of pDNA when only 10 mg of pDNA was required. Therefore, the Nanoplasmid itself possesses more intrinsic activity. To account for this activity, a 30% reduction in DNA dose was applied, which could account for the higher Nanoplasmid activity. Alternatively, a similar empirical dose series could be calculated, or the current dosing formulation could be used, with the caveat that doses higher than 2.5 mg / kg / kb are unnecessary.

[0542] DNA dose escalation Another avenue of research is to determine whether biliary hydrodynamic infusion can be improved by increasing the dose of plasmid DNA. Previously published reports have only tested pDNA doses of 3 mg and 5.5 mg, demonstrating higher transfected areas with higher doses (Kruse, GIE 2021). It would be useful to determine whether the transfected area continues to increase with incremental dose increases and whether different pDNA molecules function. This information would be invaluable for guiding the appropriate clinical dosage for gene therapy procedures. While the intent of higher pDNA doses is to ensure greater gene delivery to hepatocytes and thereby increase the observed transfected area, a secondary concern with incrementally increasing pDNA doses is whether any toxicity will occur. Naked plasmid DNA can be recognized by innate immune receptors within cells, leading to inflammation. Generally, dose-dependent toxicity is a major issue with all gene therapy vectors today, and this has been observed in gene therapy trials using AAV and adenovirus. Dose-limiting toxicity in clinical trials can also be observed using lipid nanoparticle vectors delivering mRNA or siRNA, or using naked oligonucleotide conjugates. It is possible that a given concentration or amount of DNA hydrodynamically injected into the liver of a large mammal can cause significant toxicity. Toxicity can also manifest itself as a paradoxical reduction in transgene expression due to cell-based death or by triggering innate immunity that reduces expression from pDNA. Such findings inform clinicians about the appropriate dosage level for this biliary hydrodynamic delivery technique.

[0543] Toxicity issues have been empirically investigated to improve the technical approach. Four different concentrations of the plasmid, pT-LP1-ATP7B, C9, were infused into pigs ranging in weight from 36 kg to 41 kg. The concentrations were approximately 10 mg, 20 mg, 30 mg, and 40 mg of pDNA. The flow rate was kept constant at 2 mL / s in all studies. It was observed that pDNA continued to increase the transfected area with increasing doses up to 20 mg. Higher doses of 30 mg and 40 mg of pDNA showed no significant difference from 20 mg pDNA. This finding avoids wasting pDNA doses and allows more patients to be treated. The absence of changes in vital signs and transaminase elevations after infusion indicated no fundamental difference in toxicity between infusions using higher pDNA doses.

[0544] Higher doses of pDNA beyond the previously described 5 mg pDNA can be incorporated to achieve higher transfection areas. Doses of up to 20 mg, 30 mg, or 40 mg are well tolerated. This disclosure demonstrates that DNA doses of 20 mg pDNA or greater can increase the transfection area up to 70% of total hepatocytes. This represents an unexpected increase in efficiency, given that only 5.5 mg and 10 mg pDNA reached 50%. Furthermore, it was unexpected that the effect saturates at approximately 20 mg / kg, and that higher plasmid DNA doses for a given plasmid DNA size are unnecessary because they do not increase the transfected area. Furthermore, DNA doses up to at least 40 mg pDNA do not cause cell-mediated toxicity, which reduces transgene expression. Higher doses can be safely utilized via biliary hydrodynamic injection and may be considered when applying the technique for multiple different therapeutic applications.

[0545] Injection parameters Previous studies have demonstrated combinations of vector compositions and procedural techniques that result in efficient gene delivery, achieving highly efficient transfected areas of tissue expression. What remains unclear is how different flow rates and volumes affect gene expression and how to improve gene delivery.

[0546] Although all components of the technique are important, determining the optimal injection pressure is crucial for delivering more pDNA into cells while avoiding potential toxicity.

[0547] Previous studies have described injection parameters of 2 mL / s to 4 mL / s used in gene delivery strategies. The volumes tested ranged from 30 mL to 40 mL in total. This study also demonstrates that the biliary system tolerates a wide range of injection flow rates, up to a maximum of 10 mL / s. The largest volume tested was 140 mL, which entered the liver. However, while these parameters have been shown to be tolerable from a technical standpoint without bile duct rupture or severe liver injury, it has not been shown how they affect DNA delivery and which parameters are optimal for use.

[0548] A series of studies was conducted to elucidate how higher and lower flow rates affect the gene delivery efficiency of the bile procedure, as assessed by immunohistochemical staining of the transfected area. Another series of studies examined the effect of volume on these parameters. All studies kept other variables, including the plasmid DNA construct and the plasmid DNA dose, the same.

[0549] Low flow rate Lower flow rates are attractive because they may induce less tissue damage and inflammation during injection, making them safer for patients. However, the methodology herein demonstrates that a flow rate of 1 mL / sec, combined with the procedure employed, results in little or no gene expression in the liver. The vector composition in this experiment has the ubiquitous promoters of CMV and SV40, which drive reporter gene expression. Therefore, flow rates at or below this threshold should be avoided unless the goal is to load the liver with DNA solution prior to faster infusion.

[0550] High flow rate Regarding the effect of increasing flow rate, the hypothesis was that a higher flow rate would mediate greater pDNA internalization into cells, resulting in more transfected hepatocytes. However, this disclosure demonstrates that using significantly higher flow rates is not optimal. A flow rate of 4 mL / s mirrored previous studies using similar staining of hepatocytes and non-hepatocytes, such as bile ducts. A 4 mL / s flow rate biased expression more toward the peripheral regions of the lobule, but central delivery was still observed. Higher flow rates of 7 mL / s and 10 mL / s surprisingly reduced transfection efficiency into hepatocytes, resulting in progressively fewer hepatocytes expressing the gene. Delivery to non-hepatocyte types remained strong at higher flow rates, accompanied by prominent biliary staining.

[0551] The present technology demonstrates that the optimal flow rate for efficient gene delivery is greater than 1 mL / sec and less than 7 mL / sec. Optimally, the flow rate for targeted delivery to hepatocytes is at least 2 mL / sec and no more than 4 mL / sec. Alternatively, by increasing the flow rate beyond 4 mL / sec, cholangiocytes can be preferentially targeted. In other embodiments, the flow rate for targeted gene delivery to cholangiocytes is greater than 7 mL / sec or greater than 10 mL / sec to direct more DNA into this cell type.

[0552] capacity Regarding the effect of increasing volume, it is unclear whether delivering a larger fluid volume containing a set plasmid DNA (pDNA) concentration to the liver actually increases the observed expression. One hypothesis is that a larger pDNA volume increases saturation of the DNA solution in the bile duct, ensuring that more pDNA enters the cells. On the other hand, a larger volume also acts to dilate the biliary system and the tight junctions surrounding it, thereby allowing more fluid to leak into the vasculature. It is believed that pDNA delivery to hepatocytes does not occur. A related consideration is that only a certain portion of the DNA solution volume is "active," and a low net concentration of pDNA in that active portion may actually be harmful. Furthermore, a more dilute DNA solution also means that less effective DNA enters individual cells, which is thought to reduce transfection efficiency.

[0553] Studies were conducted by injecting 10 mg of pDNA and diluting it into 40 mL, 60 mL, or 80 mL of saline solution. For all injections, the flow rate was maintained at 2 mL / sec, which is believed to force the pDNA fluid solution into the liver lobule.

[0554] Testing of this delivery technique found that higher volumes at a constant flow rate and set pDNA concentration reduced gene expression. Little immunohistochemical staining was observed in hepatocytes in pigs injected with an 80 mL volume, while doses of 40 mL and 60 mL were similar in efficacy.

[0555] Preferably, the volume of the pDNA solution should be less than 80 mL. In other embodiments, the volume of the pDNA solution should be less than 60 mL. These volumes should be adjusted based on the weight of the target liver, which in these studies was approximately 1 kg.

[0556] Translating this information into a liver weight dosing strategy optimally applies flow rates of 20 mL / kg, 30 mL / kg, 40 mL / kg, 50 mL / kg or 60 mL / kg, where kg represents liver weight, which can be determined by a variety of different methods in the literature.

[0557] DNA administration guidelines Previous studies have identified nucleic acids containing DNA administered in amounts of at least 1 mg of DNA per kilogram of total liver tissue weight. However, there was no information regarding the optimal concentration of the prepared DNA solution. This study demonstrates that large volumes can have a detrimental effect on transfection efficiency, but this can be counteracted by increasing the concentration of DNA injected in large volumes.

[0558] In this specification, DNA concentration (mg / mL) should be optimally more than 0.30, 0.40, 0.50mg / mL, or preferably more than 0.60mg / mL can be used.For simplicity, the DNA dose per kg liver weight can be used as a standard, and in this case, 10mg / kg, 20mg / kg, 30mg / kg or 40mg / kg or more doses can be used.The amount should be adjusted according to the DNA dose selected so that these parameters are achieved.

[0559] Increased transfection rate through flow rate modulation Injection parameters are a critical part of achieving successful gene delivery. Regardless of the route of hydrodynamic administration, it has been observed that in all animal models, small modifications to injection parameters can greatly affect the observed gene expression outcome.

[0560] Previous studies have shown that biliary hydrodynamic infusion uses a set flow rate to achieve gene expression. All experiments are performed at a single flow rate, which is recommended to mediate effective expression. Furthermore, the distribution of expression can be slightly modulated from zone 3 to zone 1 of the liver lobule with increasing flow rate.

[0561] Previous studies have not demonstrated the use of multiple different flow rates during the procedure. While this concept has been explored previously, there are no details regarding which combination of flow rates achieves the highest levels of expression. The presented technique suggests a modification of gene delivery methods that results in even higher transfected areas of hepatocytes beyond those achieved with current testing paradigms.

[0562] This disclosure demonstrates that the transfection area in the liver can be further increased by using at least two different flow rates during injection. In other examples, at least three different flow rates are used. Previous studies have described this as technically possible, but have not provided details on which combination of flow rate and time is optimal for increasing the transfection area.

[0563] This disclosure demonstrates that it is optimal to begin the initial hydrodynamic injection with a slower injection volume to avoid prematurely dilating the bile ducts and canaliculi at higher pressures, thereby causing more vascular fluid leakage. Furthermore, the injection protocol teaches that it is best to first fill the biliary system with fluid before increasing to higher pressures.

[0564] For example, the initial injection proceeds at 2 mL / sec, and then the injection rate is increased to 4 mL / sec. This serves to initially target gene expression to zone 3 in the center of the liver lobule, followed by targeting gene expression to the periphery of the lobule at a faster flow rate. The net result of designing injection parameters in this manner is more efficient coverage of the entire lobule, thereby increasing the number of hepatocytes expressing the gene of interest. Optimally, the slow phase of the injection proceeds over half the fluid volume. In another example, the slow phase proceeds over two-thirds of the injection volume. Fast phases are kept to a minimum because increased forces can be transmitted to the distal fluid instantly and do not need to be imposed for a longer period of time. It is preferable to avoid premature dilation of the ducts and sinusoids by faster flow rates, so that later flow rates achieve lower pressures.

[0565] In another example, the first fluid injection step proceeds at 1 mL / sec until the total volume reaches the estimated volume of the large ducts in the liver, followed by one or more faster volumes to increase pDNA expression. Alternatively, a fast flow rate is used first, followed by a slower flow rate to introduce the plasmid DNA into the center of the lobule. In this example, the initial fast injection is performed for the first half of the total volume, followed by a slower fluid volume for a shorter period. Optimally, the total volume is used to calculate how long each fluid injection step requires. Time is not the most important factor, as the volume injected can vary between many different procedures and depends on the size of the large mammal being injected.

[0566] In another example, the flow rate is increased in a stepped function such that three or more flow rates are used. In one example, a flow rate of 2 mL / sec is first used for 33% of the volume injection, then 3 mL / sec is used for 33% of the volume, followed by 4 mL / sec for the remaining injection volume.

[0567] Scaling the procedure to small and large livers Previous studies have shown that pigs 40 kilograms or larger can tolerate infusion through the biliary tree using set parameters and balloon inflation, but they have not shown whether smaller pigs can tolerate such a procedure or whether their smaller bile ducts would rupture or their livers would suffer significant damage.

[0568] One study previously reported that pigs weighing approximately 5 kg could tolerate infusions through the biliary system at 100 mL and 10 mL / sec. Because this study did not use a balloon catheter to ensure pressure during infusion, it is unclear what the effective pressure inside the biliary system was. Furthermore, there are several other details about the procedure that differ, including the use of surgery. Consequently, while this study demonstrates that the procedure is tolerated in small animals, it does not address concerns that arise when this procedure is performed on larger animals. This means that the method used in this study cannot be transferred to larger animals without further testing.

[0569] To confirm that biliary hydrodynamic infusion can be scaled down to smaller animals, the present technique demonstrates that smaller sized pigs, up to at least 25 kilograms and at least 15 kilograms, tolerate hydrodynamic infusion well without rupture in the biliary system. Furthermore, the smaller volumes infused into these pigs still resulted in strong gene expression as measured by immunohistochemistry.

[0570] This disclosure demonstrates a method for scaling down injection volumes for specific animals. Previous studies have identified volume parameters for hydrodynamic injection and described volumetric dose targets relative to liver weight, but precise details are limited to 30 mL / kg liver weight and 100 mL / kg liver weight. This disclosure improves on these shortcomings by providing additional recommendations for weight-based dosing.

[0571] Optimally, the volume injected during hydrodynamic infusion via the biliary system is 40 mL / kg liver tissue weight. In another example, the volume is 50 mL / kg liver tissue weight. In another example, the volume is 60 mL / kg liver tissue weight. The liver tissue weight was calculated by calculation of available liver tissue or by direct imaging modality prior to biliary hydrodynamic infusion. This disclosure indicates that it is best not to exceed a total volume of 70 mL / kg, as this is associated with a decrease in efficiency. In optimal cases, the injection volume is 30-70 mL / kg, or more preferably 40-60 mL / kg.

[0572] These new dosages in terms of volume per kilogram of liver tissue are supported by data from additional porcine studies, showing that they are effective in mediating gene delivery leading to reporter gene expression in the liver.

[0573] pressure-mediated injection Previous studies have disclosed pressures for effective gene delivery of 50 mmHg to 150 mmHg. The present disclosure seeks to further delineate which pressures within this range and higher pressure ranges result in the most efficient gene delivery. A series of tests was conducted using a constant pressure infusion device that provides real-time pressure monitoring and uses it to adjust the infusion in real time to maintain a predetermined pressure.

[0574] Using this device, constant pressure injection at approximately 50 mmHg was tested. It was found that injection at this pressure threshold was largely inefficient, with limited hepatocyte transfection observed. When a higher pressure threshold of 80 mmHg was used, a more highly transfected portion of the hepatocytes was observed to express the gene of interest. Therefore, pressures of at least 80 mmHg are preferred for hydrodynamic injection. Pressures above 150 mmHg were further tested to determine whether they were effective in hydrodynamic gene delivery. It was observed that pressures of 175 mmHg and 200 mmHg were as efficient as injections at lower pressures below 150 mmHg and could still result in effective gene delivery. Injections at pressures higher than 200 mmHg resulted in less efficient transfection compared to these lower pressure thresholds.

[0575] Targeting liver sinusoidal endothelial cells Previous studies have demonstrated that biliary hydrodynamic injection can mediate gene delivery to multiple different cell types in the liver. However, they have not demonstrated whether hepatic sinusoidal endothelial cells (LSECs) can be targeted. This cell type is adjacent to the sinusoids and space of Disse, which help regulate access to hepatocytes and have highly specialized functions. However, given that the sinusoids are adjacent to these endothelial cells, it is possible that pressure buildup along these cells is not sufficient to enable transfection. Alternatively, it is possible that the injection technique could reach these cells as well. To confirm whether biliary hydrodynamic injection can reach LSECs, we developed a cell-specific promoter for hepatic sinusoidal endothelial cells. This cell-specific promoter, CD36, is expected to be expressed in all LSECs throughout the entire hepatic lobule.

[0576] Hydrodynamic injection into the biliary tree can efficiently induce expression in LSECs, with expression most concentrated around zone 3 but also spreading outward toward zone 1. Therefore, we demonstrate a method for targeting the LSEC population. The injection parameters used for LSEC delivery are similar to those for gene delivery to other liver cell types. Established volume and flow rate parameters can then be utilized for LSECs. Different DNA doses can be envisioned, starting with a DNA dose of 5 milligrams / kilogram (liver weight) or higher. [Example]

[0577] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all ...

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[Claim 1] The invention described in the specification.

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