Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime

Definitions

• the invention generally relates to techniques for transferring genes into mammalian parenchymal cells in vivo. More particularly, a method is provided for transfecting parenchymal cells with viral encapsulated polynucleotides and viral vectors delivered intravascularly.
• hepatocytes have been genetically modified by retroviral vectors [Wolff et al. 1987, Ledley et al. 1987] and re-implanted into the livers in animals and in people [Chowdhury et al. 1991, Grossman et al. 1994]. Retroviral vectors have also been delivered directly to livers in which hepatocyte division was induced by partial hepatectomy [Kay et al. 1992, Ferry et al. PNAS; 1991, Kaleko et al. 1991].
• the present invention provides for the transfer of polynucleotides into parenchymal cells within tissues in situ and in vivo.
• An intravascular route of administration enables a viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide to be more evenly distributed to the parenchymal cells and expressed more efficiently than direct parenchymal injections.
• the efficiency of polynucleotide and viral vector delivery and expression is increased by increasing the permeability of vessels to the delivery vector.
• a volume is injected into the lumen of a vessel at an appropriate rate thereby increasing movement of the molecule or complex out of vessels and into the extravascular space.
• Increasing vessel permeability may further comprise blocking the flow of fluid through vessels into and/or out of a target tissue or area, increasing the intravascular hydrostatic (physical) pressure, and/or increasing the osmotic pressure.
• a method for delivering a polynucleotide to an organ or tissue cell comprising: injecting a viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide in a solution into the lumen of an afferent or efferent vessel of the organ or tissue.
• the method may further comprise occluding one or more afferent and/or efferent vessels of the organ or tissue. Occlusion of vessels facilitates increasing the volume of fluid in the target tissue or organ when the solution is injected.
• a method for delivering a polynucleotide to a muscle cell comprising: inserting the polynucleotide into an efferent or afferent vessel of the tissue communicating with the muscle cell of the mammal such that the polynucleotide is transfected into the parenchymal cell.
• a process for delivering a polynucleotide to a parenchymal cell of a mammal for expression of a gene, comprising, transporting the polynucleotide to a vessel containing a fluid; and, increasing the hydrostatic and/or osmotic pressure against the vessel wall for a time sufficient to complete delivery of the polynucleotide.
• a process for increasing the transit of a viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide out of a vessel and into a surcounding tissue in a mammal in vivo comprising: injecting a sufficient volume of injection solution containing the viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide into an afferent or efferent vessel of the target tissue, thus forcing fluid out of the vasculature into the extravascular space.
• the target tissue is the tissue that the artery supplies with blood.
• the target tissue is the tissue from which the vein drains blood.
• the target tissue is the liver.
• the injection solution may further contain a compound or compounds which may aid in delivery and may or may not associate with the molecule or complex.
• the permeability of the vessel may be further increased by delivering to the mammal a compound which is known in the art to increase vessel permeability.
• a compound which is known in the art to increase vessel permeability may be selected from the list comprising: histamine, vascular permeability factor, calcium channel blockers, beta-blockers, phorbol esters, ethylene- diaminetetraacetic acid, adenosine, papaverine, atropine, nifedipine, and hypertonic solutions.
• the described devices and processes can be used to deliver a viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide to a mammalian cell for the purpose of altering the endogenous properties of the cell.
• Altering the endogenous properties of the cell may be for therapeutic purposes, for facilitating pharmaceutical drug discovery, for facilitating drug target validation or for biological research.
• the mammal can be selected from the group comprising: mouse, rat, rabbit, guinea pig, dog, pig, goat, sheep, cow, primate and human.
• the cell may be selected from the group comprising parenchymal cell, liver cell, spleen cell, heart cell, kidney cell, lung cell, skeletal muscle cell, diaphragm cell, prostate cell, skin cell, testis cell, fat cell, bladder cell, brain cell, pancreas cell, and thymus cell.
• FIG. 1 Cross section of rat muscle (upper leg medial) following intra-arterial injection of adenovirus containing the ⁇ -galactosidase gene and ⁇ -galactosidase staining (lOOx magnification).
• FIG. 2 Cross section of rat muscle (lower leg posterior) following intra-arterial injection of adenovirus containing the ⁇ -galactosidase gene and ⁇ -galactosidase staining, lower expression area (1 OOx magnification).
• Vessel permeability is increased by one or more of the following: inserting a sufficient volume of an appropriate injection solution containing the molecule into the vessel, inserting the solution into the vessel at an appropriate rate, impeding fluid flow into and out of the target tissue during the process, and increasing permeability of the vessel wall.
• deliver means that the molecule or complex becomes associated with the cell thereby altering the endogenous properties of the cell.
• the molecule or complex can be on the membrane of the cell or inside the cytoplasm, nucleus, or other organelle of the cell.
• Other terms sometimes used interchangeably with deliver include transfect, transfer, or transform.
• In vivo delivery of a molecule or complex means to transfer the molecule or complex from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal.
• the delivery of a biologically active compound is commonly known as "drug delivery".
• a delivery system is the means by which a biologically active compound becomes delivered. The tenn encompasses all compounds, including the biologically active compound itself, and all processes required for delivery including the form and method of administration.
• the described delivery system comprises an intravascular administration route.
• Vessels comprise internal hollow tubular structures connected to a tissue or organ within the body of an animal, including a mammal. Bodily fluid flows to or from the body part within the lumen of the tubular structure. Examples of bodily fluid include blood, lymphatic fluid, and bile. Vessels may be selected from the group comprising: arteries, arterioles, capillaries, venules, sinusoids, veins (including peripheral veins), lymphatics, and bile ducts. Afferent vessels are directed towards the organ or tissue and through which fluid flows towards the organ or tissue under normal physiological conditions. Conversely, efferent vessels are directed away from the organ or tissue and through which fluid flows away from the organ or tissue under normal physiological conditions.
• the hepatic vein is an efferent blood vessel since it normally carries blood away from the liver into the inferior vena cava.
• the portal vein and hepatic arteries are afferent blood vessels in relation to the liver since they normally carry blood towards the liver.
• a vascular network consists of the directly connecting vessels supplying and/or draining fluid in a target organ or tissue.
• An injector such as a needle or catheter, is used to inject the viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide into the vascular system. The injection can be performed under direct observation following an incision and visualization of the tissues blood vessels.
• a catheter can be inserted at a distant site and threaded so that it resides in the vascular system that connects with the target tissue.
• the injection can also be performed using a needle that traverses the intact skin and enters the lumen of a vessel.
• the viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide can be injected into a blood vessel at a distal or proximal point.
• the viral vector, virally encapsulated polynucleotide or a virally associated polynucleotide can also be injected into a peripheral vein.
• the injection solution can be injected into either an afferent vessel or an efferent vessel.
• the injection solution can be inserted into the hepatic artery or the portal vein or via retrograde injection into the hepatic vein.
• the injection solution can be inserted into either arteries or veins.
• the solution is injected in the direction of normal (antegrade) flow rather than in a retrograde direction.
• Efficient delivery via intravascular administration primarily depends on the volume of the injection solution and the injection rate. Vessel occlusion is also an important factor for delivery to many tissues.
• the composition of the injection solution can depend on the nature of the molecule or complex that is to be delivered. We have observed that certain complexes maybe delivered more efficiently using low salt injection solutions. The use or hypertonic or hypotonic injection solutions or the use of vasodilators in the injection solution may further enhance delivery.
• injection volume and rate are dependent upon: the size of the animal, the size of the vessel into which the solution is injected, the size and or volume of the target tissue, the bed volume of the target tissue vasculature, and the nature of the target tissue or vessels supplying the target tissue.
• delivery to liver may require less volume because of the porous nature of the liver vasculature.
• the precise volume and rate of injection into a particular vessel, for delivery to a particular target tissue may be determined empirically. Larger injection volumes and/or higher injection rates are typically required for a larger vessels, target sizes, etc.
• efficient delivery to mouse liver may require injection of as little as 1 ml or less (animal weight ⁇ 25 g).
• Injection rates can vary from 0.5 ml/sec or lower to 4 ml/sec or higher, depending on animal size, vessel size, etc. Occlusion of vessels, by balloon catheters, clamps, cuffs, natural occlusion, etc, can limit or define the vascular network size or target area.
• vasculature may not be identical from one individual to another, methods may be employed to predict or control appropriate injection volume and rate. Injection of iodinated contrast dye detected by fluoroscopy can aid in determining vascular bed size. Also, an automatic injection system can be used such that the injection solution is delivered at a preset pressure. For such a system, pressure may be measured in the injection apparatus, in the vessel into which the solution is injected, in a branch vessel within the target tissue, or within an efferent or afferent vessel within the target tissue.
• Injecting into a vessel an appropriate volume at an appropriate rate increases the volume of fluid in the tissue while increasing permeability of the vessel to the injection solution and the molecules or complexes therein. Permeability can be further increased by occluding outflow of fluid (both bodily fluid and injection solution) from the tissue or local vascular network.
• fluid both bodily fluid and injection solution
• a solution is rapidly injected into an afferent vessel supplying an organ while the efferent vessel(s) draining the tissue is transiently occluded.
• Branching vessels may also be occluded. Natural occlusions may also he used.
• the afferent vessel may also be transiently occluded proximal to the injection site.
• the vessels are partially or totally occluded for a period of time sufficient to allow delivery of a molecule or complex present in the injection solution.
• the occlusion may be released immediately after injection or may be released only after a determined length of time which does not result in tissue damage due to ischemia.
• Permeability is defined herein as the propensity for macromolecules to move out of a vessel and enter the extravascular space.
• One measure of permeability is the rate at which macromolecules move through the vessel wall and out of the vessel.
• Another measure of permeability is the lack of force that resists the movement of fluid or macromolecules through the vessel wall and out of the vessel.
• Endothelial cells lining the interior of blood vessels and connective material both function to limit permeability of blood vessels to macromolecules.
• Increasing the size of the tissue is defined herein as increasing extracellular volume and/or cell volume in the specific tissue.
• cuff means an device for impeding fluid flow through mammalian vessels, particularly blood vessels. More particularly, a cuff refers specifically to a device applied exterior to the mammal's skin that touches the skin in a non-invasive manner. The cuff applies external compression to the mammalian skin such that vessel walls, in an area underneath the cuff, are forced to constrict an amount sufficient to impede fluid from flowing through the vessels at a normal rate. Impeding fluid flow into and out of an area such as a limb, combined with injection of a solution, causes increased vessel permeability to increase the size and volume of the tissue.
• a cuff is a sphygmomanometer which is normally used to measure blood pressure.
• Another example is a tourniquet.
• An exterior cuff may be applied prior to insertion of the injection solution, subsequent to insertion, or concurrent with insertion.
• the described intra-arterial and intravenous processes require that blood flow be impeded for substantially less time than is required to cause tissue damage by ischemia.
• a common anesthesia for human limb surgery e.g., carpal tunnel repair
• the minimal elevations of muscle-derived enzymes found in serum provide significant evidence against any consequential muscle damage.
• vasodilators known in the art for increasing vascular permeability, including drugs or chemicals and hypertonic solutions.
• Drugs or chemicals can increase the permeability of the vessel by causing a change in function, activity, or shape of cells within the vessel wall; typically interacting with a specific receptor, enzyme or protein of the vascular cell.
• Other agents can increase permeability by changing the extracellular connective material.
• Examples of drugs or chemicals that may be used to increase vessel permeability include histamine, vascular permeability factor (VPF, which is also known as vascular endothelial growth factor, VEGF), calcium channel blockers (e.g., verapamil, nicardipine, diltiazem), beta-blockers (e.g., lisinopril), phorbol esters (e.g., PKC), ethylenediaminetetraacetic acid (EDTA), adenosine, papaverine, atropine, and nifedipine.
• Hypertonic solutions have increased osmolarity compared to the osmolality of blood thus increasing osmotic pressure and causing cells to shrink.
• hypertonic solutions containing salts such as NaCl or sugars or polyols such as mannitol are used.
• Molecules and complexes can be efficiently delivered to skeletal muscle cells in vivo via intravascular delivery.
• up to 21% of all muscle cells in rat hind limbs express ⁇ -galactosidase after injection of 500 ⁇ g pCI-LacZ plasmid DNA in 10 ml saline into the iliac artery [Zhang et al. 2001].
• Similar experiments in pig heart demonstrated that cardiac tissue can be efficiently transfected following injection of 1.5 mg plasmid DNA in 30 ml saline. Delivery of plasmid DNA to heart muscle cells, as determined by luciferase expression, is equally efficient following injection into coronary arteries or veins.
• PTCA percutaneous transluminal coronary angioplasty
• injections can be directed into the inferior cava which is occluded both proximally and distally to the entry of the hepatic vein into the inferior vena cava.
• the downstream inferior vena cava occlusion is placed between the diaphragm and the entry point of the hepatic vein.
• the upstream inferior vena cava occlusion is placed just upstream of the entry point of the renal veins.
• the hepatic artery, mesenteric artery, renal vein and portal vein can also be occluded.
• injections solutions may be inserted into both the bile duct and the portal vein. It is envisioned that the described processes may be used repetitively in a single mammal. Multiple injections may be used to provide delivery to additional tissues, to increase delivery to a single tissue, or where multiple treatments are indicated, or to facilitate longer term expression.
• mice, rats, dogs, pig, and non-human primates The processes are shown to be effective in mice, rats, dogs, pig, and non-human primates. That delivery is observed in each of these animals strongly suggests that the processes are generally applicable to all mammals. In particular, the effectiveness of the processes in delivering molecules and complexes to nonhuman primates indicates that the processes will also be successful in humans.
• delivery vehicles or vectors or other delivery enhancing groups comprise: transfection reagents, "naked" plasmid DNA, siRNA, non-viral vectors, lipids, polymers, polycations, amphipathic compounds, targeting signals, nuclear targeting signals, and membrane active compounds.
• Delivery may also be improved by the use of tissue specific cellular targeting signals; enhance binding to receptors, cytoplasmic transport to the nucleus and nuclear entry (nuclear localizing signals) or release from endosomes or other intracellular vesicles.
• Cellular receptor signals are any signal that enhances the association of the gene with a cell, including ligands and non-specific cell binding.
• a targeting signal can be a protein, peptide, lipid, steroid, sugar, carbohydrate, nucleic acid or synthetic compound.
• polynucleotide is a term of art that refers to a string of at least two (nucleotide) base-sugar-phosphate combinations. Nucleotides are the monomeric units of nucleic acid polymers. The term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the form of an oligonucleotide messenger RNA, anti-sense, plasmid DNA, parts of a plasmid DNA or genetic material derived from a virus. Anti-sense is a polynucleotide that interferes with the function of DNA and/or RNA.
• DNA deoxyribonucleic acid
• RNA ribonucleic acid
• Anti-sense is a polynucleotide that interferes with the function of DNA and/or RNA.
• a polynucleotide can be delivered to a cell in order to produce a cellular change that is therapeutic or furthers other research purposes.
• the delivery of polynucleotides or other genetic material for therapeutic(the art of improving health in an animal including treatment or prevention of disease) and/or research purposes is gene therapy.
• the polynucleotides are coded to express a whole or partial protein, or may be or encode anti-sense or other functional nucleic acid (i.e. siRNA),.
• the protein can be missing or defective in an organism as a result of genetic, inherited or acquired defect in its genome.
• a polynucleotide maybe coded to express the protein dystrophin that is missing or defective in Duchenne muscular dystrophy.
• the coded polynucleotide is delivered to a selected group or groups of cells and incorporated into those cell's genome or remain apart from the cell's genome. Subsequently, protein expressed from the polynucleotide is produced by the formerly deficient cells.
• Other examples of imperfect protein production that can be treated with gene therapy include the addition of the protein clotting factors that are missing in the hemophilias and enzymes that are defective in inborn errors of metabolism such as phenylalanine hydroxylase.
• a delivered polynucleotide can also be therapeutic in acquired disorders such as neurodegenerative disorders, cancer, heart disease, and infections. The polynucleotide has its therapeutic effect by entering the cell.
• Duchenne Delivery of a polynucleotide means to transfer a polynucleotide from a container outside a mammal to within or near the outer cell membrane of a cell in the mammal.
• the term transfection is used herein, in general, as a substitute for the term delivery, or, more specifically, the transfer of a polynucleotide from outside a cell membrane to within the cell membrane. If the polynucleotide is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm.
• the polynucleotide is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein.
• the polynucleotide may contain sequences that are required for transcription and translation. These sequences may include promoter and enhancer sequences that are required for initiation. DNA and thus the corresponding messenger RNA (transcribed from the DNA) may contain introns, poly A sequences, and sequences required for the initiation and termination of its translation into protein. Therefore if a polynucleotide expresses its cognate protein, then it must have entered a cell.
• a therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane or being secreted and dissociating from the cell where it can enter the general circulation and blood.
• Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g. alpha-antitrypsin) and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein.
• the low density lipoprotein (LDL) receptor could be expressed in hepatocytes and lower blood cholesterol levels and thereby prevent atherosclerotic lesions that can cause strokes or myocardial infarction.
• Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic). A protein within a cell could also interfere with the replication of a virus.
• a therapeutic effect of an siRNA or oligonucleotide in attenuating or preventing an unwanted cellular state can be accomplished by the siRNA or oligonucleotide entering the cell and acting on messenger RNA in the cytoplasm or nucleus, or by an oligonucleotide acting on genomic DNA or precursor RNAs within the nucleus of a cell.
• the delivered polynucleotide can stay within the cytoplasm or nucleus apart from the endogenous genetic material.
• the polynucleotide could recombine (become a part of) with the endogenous genetic material.
• DNA can insert into chromosomal DNA by either homologous or non-homologous recombination.
• Parenchymal cells are the distinguishing cells of a gland or organ contained in and supported by the connective tissue framework.
• the parenchymal cells perform a function that is unique to the particular organ.
• the term "parenchymal” excludes cells that are common to many organs and tissues such as fibroblasts and endothelial cells within the blood vessels.
• the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules.
• liver parenchyma The major constituent of the liver parenchyma are polyhedral hepatocytes (also known as hepatic cells) that presents at least one side to an hepatic sinusoid and apposed sides to a bile canaliculus.
• Liver cells that are not parenchymal cells include cells within the blood vessels such as the endothelial cells or fibroblast cells.
• the parenchymal cells include myoblasts, satellite cells, myotubules, and myofibers.
• the parenchymal cells include the myocardium also known as cardiac muscle fibers or cardiac muscle cells and the cells of the impulse connecting system such as those that constitute the sinoatrial node, atrioventricular node, and atrioventricular bundle.
• the parenchymal cells include cells within the acini such as zymogenic cells, centroacinar cells, and basal or basket cells and cells within the islets of Langerhans such as alpha and beta cells.
• the parenchymal cells include reticular cells and blood cells (or precursors to blood cells) such as lymphocytes, monocytes, plasma cells and macrophages.
• the parenchymal cells include neurons, glial cells, microglial cells, oligodendrocytes, Schwann cells, and epithelial cells of the choroid plexus.
• parenchymal cells include cells of collecting tubules and the proximal and distal tubular cells.
• the parenchyma includes epithelial cells.
• the parenchymal cells include cells that produce hormones.
• the parenchymal cells include the principal cells (chief cells) and oxyphilic cells.
• the parenchymal cells include follicular epithelial cells and parafoUicular cells.
• the parenchymal cells include the epithelial cells within the adrenal cortex and the polyhedral cells within the adrenal medulla.
• the parenchymal cells include epithelial cells, glandular cells, basal, and goblet cells.
• the parenchymal cells include the epithelial cells, mucus cells, goblet cells, and alveolar cells.
• the parenchymal cells include adipose cells or adipocytes.
• the parenchymal cells include the epithelial cells of the epidermis, melanocytes, cells of the sweat glands, and cells of the hair root.
• the parenchyma includes chondrocytes.
• the parenchyma includes osteoblasts, osteocytes, and osteoclasts.
• Polypeptide refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxy group of contiguous amino acid residues.
• Vectors include polynucleic molecules originating from a virus, a plasmid, or the cell of an organism into which another nucleic fragment of appropriate size can be integrated without loss of the vectors capacity for self- replication; vectors introduce foreign DNA into host cells, where it can be reproduced. Examples are plasmids, cosmids, yeast artificial chromosomes and viruses. Vectors are often recombinant molecules containing DNA sequences from several sources.
• a vector includes a viral vector selected from the list comprising: adeno-associated virus (Parvoviridae), adenovirus (icosahedral virus that contains DNA; there are over 40 different adenovirus varieties, some of which cause the common cold), herpes simplex virus (HSV), vaccinia virus (Poxviridae), retrovirus
• adeno-associated virus Parvoviridae
• adenovirus icosahedral virus that contains DNA; there are over 40 different adenovirus varieties, some of which cause the common cold
• HSV herpes simplex virus
• PV vaccinia virus
• retrovirus retrovirus
• Afferent blood vessels of organs are defined as vessels which are directed towards the organ or tissue and in which blood flows towards the organ or tissue under normal physiologic conditions.
• the efferent blood vessels of organs are defined as vessels which are directed away from the organ or tissue and in which blood flows away from the organ or tissue under normal physiologic conditions.
• the hepatic vein is an efferent blood vessel since it normally carries blood away from the liver into the inferior vena cava.
• the portal vein and hepatic arteries are afferent blood vessels in relation to the liver since they normally carry blood towards the liver.
• a liver blood vessel includes the portal venous system which transports blood from the gastrointestinal tract and other internal organs (e.g. spleen, pancreas and gall bladder) to the liver.
• Another liver blood vessel is the hepatic vein.
• the hepatic vein may also be reached via the inferior vena cava or another blood vessel that ultimately connects to the liver.
• Adenoviral vectors can be delivered to muscle parenchymal cells by an intravascular route.
• Adult Sprague-Dawley rats 120-140 g were anesthetized with isoflorane and the surgical field was shaved and prepped with an antiseptic. The animals were placed on a heating pad to prevent loss of body heat during the surgical procedure.
• a 4 cm long abdominal midline incision was made after which skin flaps were folded away and held with clamps to expose the target area.
• a moist gauze was applied to prevent excessive drying of internal organs.
• Intestines were moved to visualize the iliac veins and arteries.
• Microvessel clips were placed on the external iliac, caudal epigastric, internal iliac, deferent duct, and gluteal arteries and veins as well as on the inferior vena cava near the bifurcation to block both outflow and inflow of the blood to the leg.
• An efflux enhancer solution e.g., 0.5 mg of papaverine and 40 ng of collagenase in 3 ml saline
• the adenoviral vector CMVLuc expresses the luciferase gene from the immediate early promoter of the human cytomegalovirus [Yang T et al. 1996].
• the microvessel clips were removed 2 minutes after the injection and bleeding was controlled with pressure and gel foam.
• the abdominal muscles and skin were closed with 4-0 dexon suture. Two days after injection the leg muscle were assayed for luciferase as above.
• Luciferase Assays Results of the rat AdV-Luc injections are provided in relative light units (RLU) and/or micrograms ( ⁇ g) of luciferase produced. To determine RLU, 10 ⁇ l of cell lysate were assayed using a EG&G Berthold LB9507 luminometer and total muscle RLU were determined by multiplying by the appropriate dilution factor.
• RLU relative light units
• ⁇ g micrograms
• Table 1 Distribution of luciferase activity following the intraarterial injection of adenovirus CMVLuc. Muscle Group Luciferase (ng) Upper Leg Anterior 59.04 Upper Leg Posterior 18.33 Upper Leg Medial 4.44 Lower Leg Posterior 11.04 Lower Leg Anterior 5.33 Foot 0.22 Total 98.40 The increased permeability of vessels resulting form the injection procedure enabled delivery of adenovirus to muscles cells in the leg and expression of the adenovirus encoded luciferase gene.
• Recombinant AAV viral particles containing reporter genes were delivered to muscles in the leg of a rat via a single intra-arterial injection and the resulting gene expression was determined.
• rAAV recombinant AAV
• Recombinant AAV particles (AAV-Luciferase, AAV-LacZ, AAV-AAT) were injected into rat leg muscle by either a single intra-arterial injection into the external iliac as indicated above for delivery of adenovirus or by direct injections into each of 5 major muscle groups of the leg [see Wolff et al. 1990].
• lxlO 12 rAAV particles were split and equal amounts were injected into each of 5 muscle groups: upper leg anterior, upper leg posterior, upper leg medial, lower leg anterior, lower leg posterior. All rats used were female and approximately 150 grams and each received a total of 1 x 10 rAAV particles via injection. Luciferase of ⁇ -galactosidase expression in muscle cells was determined at various times after injection.
• Luciferase Assays Results of the rat AdV-Luc injections are provided in relative light units (RLU) and/or micrograms ( ⁇ g) of luciferase produced. To determine RLU, 10 ⁇ l of cell lysate were assayed using a EG&G Berthold LB9507 luminometer and total muscle RLU were determined by multiplying by the appropriate dilution factor.
• RLU relative light units
• ⁇ g micrograms
• rAAV CMV-LacZ Delivery To confirm that AAV mediated gene expression was occurring within muscle parenchyma, injections were performed using an AAV vector containing the reporter gene ⁇ -galactosidase.
• Recombinant AAV CMV-LacZ (lxlO 12 particles) was injected into 7 animals (4 intra-arterial, 3 direct muscle injection) as described above. All animals tolerated the procedure well and muscle was harvested at 2, 4, 6 and 8 weeks for the intra-arterial delivery and 2, 4 and 8 weeks for the direct injections.
• ⁇ -galactosidase Assays Animals were euthanized at the indicated times. Following excision, muscles were cut into two pieces and frozen in liquid nitrogen cooled isopentane. Frozen muscles were embedded, sliced into thin sections on a cryostat (6- 10 microns thick) and mounted onto glass slides. Mounted sections were fixed in glutaraldehyde and stained for ⁇ -galactosidase activity.
• ⁇ -galactosidase staining of thin sections indicated that the LacZ reporter gene was efficiently expressed in myofibers (FIG. 1, upper leg medial, high expression area; FIG. 2, lower leg posterior, lower expression area). All muscles excised (from both intra-arterial and direct injection) displayed ⁇ -galactosidase expression within myofibers with the major difference being the distribution of the staining. In the direct muscle injection samples, ⁇ -galactosidase staining was localized to areas near the injection site as expected. In the rats receiving intra- arterial injections, myofiber expression was much more widespread with LacZ positive stained cells being found in all parts of the excised leg muscles. Expression of ⁇ -galactosidase in the rat muscle following intra-arterial injection was much more widespread than following direct injection.
• rAAV CMV-LacZ results in high levels of myofiber gene expression using both intra-arterial and direct muscle injection.
• a single intra-arterial (external iliac) injection of rAAV resulted in ⁇ -galactosidase expression in all major muscle groups in the rat leg.
• Expression patterns were qualitatively different between the two injection procedures with more widespread myofiber expression occurring in muscle groups receiving intra-arterial injections.
• Percent myofiber staining ranged from ⁇ 2 - 5% (low expressing areas) to ⁇ 100% blue cells (high expressing areas) for intra-arterial delivery and ⁇ 0% (away from injection site) to -100% (near injection site) for direct muscle injection depending on location.
• Example 4 Example 4.
• Adenovirus to Limb Skeletal Muscle via venous injection Delivery of Adenovirus to Limb Skeletal Muscle via venous vein injection: 120-140 g adult Sprague-Dawley rats were anesthetized with ⁇ soflurane and the surgical field was shaved and prepped with an antiseptic. The animals were placed on a heating pad to prevent loss of body heat during the surgical procedure. A latex tourniquet was wrapped around the upper limb and secured with a hemostat. A 1.5 cm incision was made on the inside of the limb to expose the medial saphenous vein. A 25-gauge needle catheter was inserted into the distal great saphenous vein and secured with a microvascular clip.
• the needle catheter was connected to a two-way connector for delivering both papaverine and pDNA and fluid was injected in the direction of normal blood flow. All animals were injected with 1.5 ml of papaverine (0.25mg in saline) over 6 seconds using a syringe pump. After 5 min, 5 ml normal saline containing 2 l0 9 Adenovirus particles encoding firefly Luciferase was injected at varying flow rates. Some injections also contained 5 ⁇ g of the siRNA targeted against firefly luc + (siRNA-luc + ). 2 minutes after injection, the tourniquet and catheter were removed and the skin was closed with 4-0 Vicryl.
• Adenovirus and siRNA Delivery of Adenovirus and siRNA to limb muscle cells via direct muscular injection: 120-140 g adult Sprague-Dawley rat was anesthetized with isoflurane. The animals were placed on a heating pad to prevent loss of body heat during the procedure. 1x10 Adenovirus particles encoding firefly Luciferase in 2.5 ml of saline was injected into each hind gastrocnemius muscle group of the animal.
• Example 5 Delivery to Rat Skeletal Muscle cells In Vivo Using Intra-iliac Iniection. 250 ⁇ g pCI-Luc plasmid DNA in 10 ml Ringer's injection solution was injected into iliac artery of rats using a Harvard Apparatus PHD 2000 programmable syringe pump. Varying injection rates were used. Specifically, animals were anesthetized and the surgical field shaved and prepped with an antiseptic. The animals were placed on a heating pad to prevent loss of body heat during the surgical procedure. A midline abdominal incision was be made after which skin flaps were folded away and held with clamps to expose the target area. A moist gauze was applied to prevent excessive drying of internal organs.
• Intestines were moved to visualize the iliac veins and arteries.
• Microvessel clips were placed on the external iliac, caudal epigastric, internal iliac, deferent duct, and gluteal arteries and veins to block both outflow and inflow of the blood to the leg.
• An efflux enhancer solution e.g., 0.5 mg papaverine in 3 ml saline
• An efflux enhancer solution e.g., 0.5 mg papaverine in 3 ml saline
• 12 mL injection solution containing the indicated complexes was injected in approximately 10 seconds.
• the microvessel clips were removed 2 minutes after the injection and bleeding was controlled with pressure and gel foam.
• the abdominal muscles and skin were closed with 4-0 dexon suture. Seven days after injection, the animals were sacrificed, and a luciferase assays were conducted on leg muscles. Results show that efficiency of delivery is affected by the rate of solution injection.
• Luciferase expression after delivery of plasmid DNA to muscle via iliac administration route.
• Example 6 Delivery of polynucleotides to liver in mouse: Comparison of Ringer's and low- salt glucose injection solutions for delivery by peripheral vein (tail vein) injections. Two solutions were used in this experiment. Solution A was prepared consisting of 290 mM glucose, 5 mM Hepes, adjusted to pH 7.5. Solution B was Ringer's.
• pDNA (45 ⁇ g, 22.5 ⁇ L of a 2 ⁇ g/ ⁇ L solution in water) was added to 11.25 mL of Solution A. To this solution was added Histone HI (36 ⁇ g, 3.6 ⁇ L of a 10 mg/mL solution in water), and the sample was mixed.
• Complex V. pDNA (45 ⁇ g, 22.5 ⁇ L of a 2 ⁇ g/ ⁇ L solution in water) was added to 11.25 mL of Solution B.
• Complex VI. pDNA (45 ⁇ g, 22.5 ⁇ L of a 2 ⁇ g/ ⁇ L solution in water) was added to 11.25 mL of Solution B.
• pDNA (45 ⁇ g, 22.5 ⁇ L of a 2 ⁇ g/ ⁇ L solution in water) was added to the mixture, and the sample was mixed for 5 min on a vortexer.
• Complex X The lipid DOTAP-Chloride (225 ⁇ g, 9 ⁇ L of a 25 mg/mL solution in chloroform, Avanti Polar Lipids) and the lipid DOPE (225 ⁇ g, 9 ⁇ L of a 25 mg/mL solution in chloroform, Avanti Polar Lipids) were added to 500 ⁇ L of chloroform. The solution was concentrated under a stream of N 2 into a film, and dried for 16 hrs under vacuum.
• the film was hydrated with 11.25 mL of Solution B for 5 min, and sonicated for 20 min.
• pDNA 45 ⁇ g, 22.5 ⁇ L of a 2 ⁇ g/ ⁇ L solution in water
• the sample was mixed for 5 min on a vortexer.
• livers were harvested and homogenized in lysis buffer (0.1% Triton X-100, 0.1 M K-phosphate, 1 mM DTT, pH 7.8). Insoluble material was cleared by centrifugation and 10 ⁇ l of the cellular extract or extract diluted 10x was analyzed for luciferase activity as previously reported [Wolff et al 1990].
• the results show that cationic polymer/DNA complexes were more efficiently delivered to liver cells when the complexes are injected in Solution A, relative to Solution B.
• anionic polymer/pDNA complexes were more efficiently delivered to liver cells when the complexes are injected in Solution B, relative to Solution A.
• Cationic liposomes with pDNA were more efficiently delivered to liver cells when injected with Solution A relative to Solution B (Table 4).
• Example 7 Delivery of plasmid DNA to liver cells via iniection into the bile duct vessel: Retrograde injection was used to deliver nucleic acid expression cassettes to hepatocytes in mouse, rat, and dog. Repetitive injections of a therapeutic gene into the bile duct were also accomplished.
• the pCILuc plasmid expresses a cytoplasmic luciferase from the human CMV immediately early (hCMV ID) promoter.
• pCILux expresses peroxisomal luciferase under control of the hCMV IE promoter.
• pCILacZ plasmid expressed the ⁇ -galactosidase gene.
• the pCMVGH expresses human growth hormone..
• Plasmid delivery into the hepatic vessels was performed in 6 week old ICR mice, 2.5 6.25 month old, 200-300 gram Sprague Dawley rats, and beagle dogs. Ventral midline incisions were performed to expose the liver and associated vessels.
• the mice were anesthetized with intramuscular injections of 1000 ⁇ g of ketamine HC1 (Parke Davis, Morris Plains, NJ) and by inhalation of methoxyflurane (Pitman Moore, Mudelein, IL) as needed.
• the rats were anesthetized with ether and the dogs were anesthetized with halothane by inhalation. Plasmids were injected in solutions containing 2.5 units/ml or heparin (Qian et al.
• Bile duct injections in mice were performed using manual injections with a 30-gauge, 1/2 inch needle and 1 ml syringe. A 5 1 mm, Kleinert Kutz microvessel clip was used to occlude the bile duct downstream from the point of injection in order to prevent flow to the duodenum and away from the liver. The gallbladder inlet was not occluded. In some of the bile duct injections, the junction of the hepatic vein and caudal vena cava clamped as above.
• the portal vein and hepatic artery were clamped in addition to the occlusion of the hepatic vein.
• Repetitive injections into the bile duct were done by placing a polyethylene tube (I.D. 0.28 mm, O.D. 0.61 mm; Intramedic Clay Adams Brand, Becton Dickinson Co., Sparks, MD, USA) catheter into the bile duct after making a hole with a 27 gauge needle.
• the tubing was secured by a suture around the bile duct and tubing; thereby occluding the bite duct.
• the other end of the tubing was placed outside the skin of the animal's back so that surgery was not required for repeat injections.
• Mouse liver sections were added to 0.7 ml lysis buffer (0.1% Triton X-100, 0.1 M potassium phosphate, 1 mM DTF pH 7.8). For rats, liver sections were added to 4 ml lysis buffer. For the dog livers, approximately 10% of each lobe was divided into 5-20 pieces and placed into 2 ml lysis buffer. The samples were homogenized using a PRO 200 homogenizer (PRO Scientific Inc., Monroe, CT) and centrifuged at 4,000 rpm for 10 min at 4°C. 20 ⁇ l supernatant was analyzed for luciferase activity.
• PRO 200 homogenizer PRO Scientific Inc., Monroe, CT
• pg 5.1xl0 5 x RLU + 3.683
• Serum ALT and GGT assays were performed on mice one and eight days after each of the above injections with pCILuc (4 mice for each condition). No increases in GOT were observed. Serum ALT levels increased to 200-400 U/L one day after bile duct injections. Eight days after injection, serum ALT levels decreased to baseline levels in all animals.
• the liver in one of the four mice was grossly yellow and scarred as a result of the bile duct ligation and did not express any luciferase.
• the decrease in hGH expression following repeat procedures is presumed to result from immune response since the same animals expressed luciferase following pCTLuc delivery.
• Example 8 Delivery of DNA/polycation complexes to prostate and testis via iniection into dorsal vein of penis: DNA and L-cystine-l,4-bis(3-aminopropyl)piperazine cationic copolymer were mixed at a 1 : 1.7 wtwt ratio in water, diluted to 2.5 ml with Ringers solution and injected rapidly into the dorsal vein of the penis (within 7 seconds).
• clamps were applied to the inferior vena cava and the anastomotic veins just prior to the injection and removed just after the injection (within 5 - 10 seconds). Mice were sacrificed 24 h after injection and various organs were assayed for luciferase expression.
• Table 4 show efficient and functional delivery of DNA containing complexes to prostate, testis and other tissues.
• Example 9 Plasmid DNA delivery to heart muscle cells via catheter mediate coronary vein injection: 30-50 kg Sale domestic swine (Sus scrofa) were sedated with telezol (20- 30mg IM), induced with pentobarbitol (250-500 mg TV), and endotracheally intubated. Anesthesia was maintained with inhaled isoflurane (0.5 - 3%). The right carotid artery and internal jugular vein were exposed by surgical cutdown and coronary angiography was performed. Heparin (100 U/kg, IV) was administered.
• a 10 Fr guiding catheter was advanced to the coronary sinus, and a 7 Fr balloon-tipped triple lumen catheter was advanced over a 0.014 inch guidewire into the cardiac vein draining the left anterior descending (great cardiac vein) or right posterior descending (middle cardiac vein) territories. Injections of diluted iodinated contrast were used, in conjunction with the coronary angiogram, to delineate the myocardial territory drained by each vein.
• the larger lumen of the balloon-tipped triple lumen catheter was used for fluid injection, while the smaller lumen was used to monitor cardiac vein pressures during plasmid DNA infusion.
• the third lumen was used to inflate and deflate the balloon.
• the balloon was inflated, and 6 ml saline or 6 ml saline with 3 mg papaverine was instilled through the large lumen (which opened distal to the balloon).
• the installation required 3-20 seconds and resulted in slightly increased venous pressure (10 - 350 mm Hg).
• the balloon was deflated for 20-30 seconds and then inflated again followed by injection solution delivery.
• a saline solution containing 100 ⁇ g/ml pCI-Luc + was rapidly delivered through the main lumen. 25-30 ml injection solution was injected in 8-20 seconds. Intravenous pressure increased (120-500 mmHg).
• Example 10 Delivery of polynucleotide to the diaphragm in monkey: The monkey was anesthetized with ketamine followed by halothane inhalation. A 2 cm long incision was made in the upper thigh close to the inguinal ligament just in front of the femoral artery. Two clamps were placed around the femoral vein after separating the femoral vein from smrounding tissue. At an upstream location, the femoral vein was ligated by the clamp and a guide tube was inserted into the femoral vein anterogradely.
• a French 5 balloon catheter (D 1.66mm) with guide wire was inserted into the inferior vena cava through the guide tube and an X-ray monitor was used for instructing the direction of guide wire.
• the guide wire was directed into the inferior phrenic vein.
• the catheter position in the inferior phrenic vein was checked by injecting iodine.
• the balloon was inflated to block blood flow through the inferior phrenic vein.
• 20 ml 0.017% papaverine in normal saline was injected.
• 40 ml of DNA solution (3 mg) was injected in 65 sec (0.615 ml/sec). 2 minutes after DNA injection, the balloon was released and the catheter was removed.
• the animal was sacrificed and the diaphragm was taken for luciferase assay 7 days after the procedure. The results indicate successful delivery of plasmid DNA to the portion of the diaphragm supplied by the injected vessel.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Pharmacology & Pharmacy (AREA)
• Epidemiology (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=34711827

Family Applications (1)

Country Status (2)

Families Citing this family (4)

Citations (2)

Family Cites Families (2)

• 2004
  • 2004-02-06 EP EP04709076A patent/EP1691609A4/de not_active Withdrawn
  • 2004-02-06 WO PCT/US2004/003506 patent/WO2005060746A1/en active Application Filing

Patent Citations (2)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events