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Inhibition of gene expression by delivery of polynucleotides to animal cells in vivoRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Whole Live Micro-organism, Cell, Or Virus Containing, Genetically Modified Micro-organism, Cell, Or Virus (e.g., Transformed, Fused, Hybrid, Etc.)Inhibition of gene expression by delivery of polynucleotides to animal cells in vivo description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128169, Inhibition of gene expression by delivery of polynucleotides to animal cells in vivo. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is continuation-in-part of application Ser. No. 10/186,757, filed Jul. 1, 2002, application Ser. No. 10/012,804, filed Nov. 6, 2001, application Ser. No. 09/707,117, filed Nov. 6, 2000, application Ser. No. 10/855,175, filed May 27, 2004, and application Ser. No. 10/600,098, filed Jun. 20, 2003, application Ser. No. 10/600,098 is a continuation of application Ser. No. 09/447,966, filed Nov. 23, 1999, issued as U.S. Pat. No. 6,627,616, which is a continuation-in-part of application Ser. No. 09/391,260, filed Sep. 7, 1999, which is a divisional of application Ser. No. 08/975,573, filed Nov. 21, 1997, issued as U.S. Pat. No. 6,265,387, which is a continuation of application Ser. No. 08/571,536, filed Dec. 13, 1995, abandoned, and application Ser. No. 10/186,757 claims the benefit of U.S. Provisional Application Nos. 60/315,394, Aug. 27, 2001 and 60/324,155, filed Nov. 20, 2001. BACKGROUND OF THE INVENTION [0002] Gene therapy is the purposeful delivery of genetic material to cells for the purpose of (a) treating disease, or (b) biomedical investigation and research. Gene therapy includes the delivery of a polynucleotide to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to produce a specific physiological characteristic not naturally associated with the cell. In some cases, the polynucleotide itself, when delivered to a cell, can alter expression of a gene in the cell. If Appropriate delivery of genetic material has the potential to enhance a patient's health and, in some instances, lead to a cure. Delivery of genetic material to cells in vivo is also beneficial in basic research into gene function as well as for drug development and target validation for traditional small molecule drugs. [0003] Gene or polynucleotide transfer to cells is an important technique for biological and medical research as well as potentially therapeutic applications. The route of cellular entry for most conventional drugs is diffusion across the biological membrane. For this reason, drugs tend to be small (MW<500) and amphipathic, containing both hydrophobic and hydrophilic functionalities. These characteristics engender molecules with water solubility, while allowing them to cross the nonpolar lipid bilayer of the cell membrane. In contrast, the drugs used in antisense and gene therapies are relatively large hydrophilic polymers and are frequently highly negatively charged as well. Both of these physical characteristics preclude their direct diffusion across the cell membrane. [0004] It was first observed that injection of naked plasmid DNA directly into muscle in vivo enabled expression of foreign genes in the muscle (Wolff et al. 1990). This discovery led to successful direct injection of naked plasmid DNA into other organs, including liver and heart. [0005] An early attempt to deliver viruses to limb tumors utilized an isolated limb perfusion technique (Milas et al.). This method used a tourniquet to isolate the limb circulation from the rest of the circulation and then re-circulated a solution containing adenovirus through the isolated limb. Solution containing adenovirus was circulated through the limb vasculature through a loop connecting both the femoral artery--for fluid inflow to the limb--and the femoral vein--for fluid outflow. An external pump circulated fluid through this loop. Bridges et al. improved on this method to enable delivery of the virus to muscle cells in the limb by administering the vascular permeability-enhancing agent histamine. Mann et al. show naked DNA could be delivered to tissues by placing the tissue in a sealed inelastic enclosure and establishing a isotropic incubation pressure around the cells. [0006] The method described herein provides an improved method of delivering nucleic acids to extravascular cells. The described method is faster to perform, requires access to only an artery or a vein-not both, does not require an inelastic enclosure be placed around the target tissue, does not require an external re-circulation pump, does not require extended perfusion times, delivers naked nucleic acids and non-viral nucleic acid particles as well a viruses, and is effective without the requirement for vascular permeability-enhancing agents. [0007] Double-stranded RNA (dsRNA) of sequence that is identical or highly similar to a target gene results in the inhibition of expression of the gene in a natural process termed RNA interference (RNAi). Inhibition can result from degradation or inhibition of translation of messenger RNA (mRNA) (Sharp 2001). RNAi is mediated by short interfering RNAs (siRNA) or microRNAs (miRNA) of approximately 21-25 nucleotides in length. [0008] The ability to inhibit specific target; gene expression in vivo by RNAi has obvious benefits. For example, inhibition of gene expression can be used to study gene function. RNAi also enables the generation animals that mimic genetic "knockout" animals. In addition, many diseases arise from the abnormal expression of a particular gene or group of genes or expression of an dominant mutant gene. RNAi can be used to inhibit the expression of the genes and therefore alleviate disease symptoms. For example, genes contributing to a cancerous state can be inhibited. In addition, viral genes can be inhibited. Inhibiting genes such as cyclooxygenase or cytokines can be used to reduce inflammation to treat diseases such as arthritis. The ability to safely and efficiently deliver siRNA to mammalian cells in vivo has potential for the treatment of infections and diseases as well as drug discovery and pharmaceutical target validation. SUMMARY OF THE INVENTION [0009] In one embodiment, processes are described for delivering a polynucleotide to a mammalian extravascular cell in vivo comprising, injecting the polynucleotide in a solution into an efferent or afferent vessel of a target tissue wherein the volume of the solution and rate of the injection results in increasing permeability of vessels in the target tissue and increasing the volume of extravascular fluid in the target tissue. [0010] In a preferred embodiment, the polynucleotide consists of a naked polynucleotide. In another preferred embodiment, the polynucleotide comprises a polynucleotide complex. A polynucleotide complex comprises the polynucleotide in association with one or more molecules that aid in delivery or function of the polynucleotide. The polynucleotide complex can be a non-viral complex or a viral complex. The polynucleotide can be selected from the list comprising: DNA, RNA, double strand polynucleotide, partially double strand polynucleotide, and single strand polynucleotide. The polynucleotide can be delivered to the mammalian cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to produce a specific physiological characteristic not naturally associated with the cell. [0011] In a preferred embodiment, increasing permeability of vessels in the target tissue and increasing the volume of extravascular fluid in the target tissue is enhanced by occluding fluid flow out of, or away from, the target tissue. Occluding, or impeding, fluid flow out of, or away from, the target tissue may be performed before, during or after the injection. Preferably, fluid flow out of, or away from, the target tissue is occluded prior to and during injecting the polynucleotide. The occlusion may remain until immediately after the injection or may remain for a period of time such that the target tissue is not at risk of damage from ischemia. In a preferred embodiment, occluding fluid flow out of, or away from, the target tissue comprises blocking the flow of fluid through one or more afferent of efferent vessels of the target tissue. Fluid flow though a vessel may be occluded by applying compressive pressure against the vessel. Compressive pressure may be applied by a clamp placed directly on the vessel or may be applied indirectly, such as by externally applying pressure against the vessel. Externally applying pressure to occlude blood, or fluid, flow through a vessel is well known in the art and includes, but is not limited to, applying a cuff over the skin, such as a sphygmomanometer (or other device with a bladder than is inflated) or a tourniquet. The use of an external cuff to occlude fluid flow enables polynucleotides to be delivered to limb tissue without the invasive placement of clamps directly on vessels. Fluid flow though a vessel may also be occluded using a balloon catheter which is inserted into the lumen of the vessel. In a preferred embodiment, increasing permeability of vessels in the target tissue may further comprise injecting a vascular permeability enhancing factor. [0012] In a preferred embodiment, the process further comprises administration of at least one anesthetic or analgesic drug or adjuvant. Administration of anesthetics or analgesic lessens potential discomfort or pain experienced by the mammal during or after the procedure. Anesthetics and analgesics are well known in the art and examples include lidocaine, NSAIDs, clonidine, ketamine, neuromuscular blockers, and immunsuppressants. [0013] In a preferred embodiment, a polynucleotide is delivered to a mammalian cell for the purpose of facilitating pharmaceutical drug discovery or target validation. The mammalian cell may be in vitro or in vivo. Specific inhibition of a target gene can aid in determining whether an inhibition of a protein or gene has a significant phenotypic effect. Specific inhibition of a target gene can also be used to study the target gene's effect on the cell. [0014] Further objects, features, and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES [0015] FIGS. 1A-1D. Schematic diagram of catheter-mediated intravenous injection of nucleic acids into mammalian limb A) IV delivery to distal hind limb of rats. B) IV delivery to distal hind limb of dog. C) IV delivery to distal hind limb of primate. D) IV delivery to distal leg of human. Right panel in each illustrates major veins of the limb. Occlusion sites and injection sites shown in the diagrams are for illustrative purposes. Different occlusion and injection sites are possible as indicated in the description and examples. [0016] FIG. 2. Delivery of fluorescently labeled PMOs to liver via increased pressure tail vein injection. Fluorescently-labeled PMO is shown in white. Cell nuclei and peripheral actin cytoskeleton are shown in gray. [0017] FIG. 3. Inhibition of CD26 expression by mCD26-1 PMO after increased pressure tail vein injection in mice. Liver sections (10 .mu.m) are shown. Histochemical staining for CD26 activity appears dark and is localized at bile canaliculi of hepatocytes. [0018] FIG. 4. Inhibition of firefly luciferase expression by luc-1 PMO. 2 .mu.g of plasmid pGL3 (firefly luciferase) and 0.2 .mu.g of plasmid pRL-SV40 (Renilla luciferase) were co-injected into mice by high pressure tail vein injection with increasing amounts of luc-1 PMO or a standard control PMO (std ctrl). One day after injection, livers were harvested and the homogenate assayed for both luciferase activities. The ratio of firefly to Renilla luciferase activities was calculated and then normalized to the ratio from mice receiving plasmids only. SD bars are shown, n=3. [0019] FIG. 5. Photomicrographs of rat limb gastrocnemius (A) and shin (B) muscles stained for .beta.-galactosidase following repeat (triple) intravenous injections of 500 .mu.g of pDNA (pCI-LacZ). [0020] FIG. 6. Intravascular delivery of siRNA inhibits EGFP expression in the liver of transgenic mice. EGFP (green), phalloidin (red). 10 week old mice (strain C57BL/6-TgN(ACTbEGFP)10sb) expressing EGFP were injected with 50 .mu.g siRNA (mice #1 and 2), 50 .mu.g control siRNA (mice #3 and 4) or were not injected (mouse #5). Livers were harvested 30 h post-injection, sectioned, fixed, and counterstained with Alexa 568 phalloidin in order to visualize cell outlines. Images were acquired using a Zeiss Axioplan fluorescence microscope outfitted with a Zeiss AxioCam digital camera. Continue reading about Inhibition of gene expression by delivery of polynucleotides to animal cells in vivo... 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