US20070128169A1

US20070128169A1 - Inhibition of gene expression by delivery of polynucleotides to animal cells in vivo

Info

Publication number: US20070128169A1
Application number: US11/649,502
Authority: US
Inventors: David Lewis; Jon Wolff; Vladimir Budker; Hans Herweijer; James Hagstrom
Original assignee: Individual
Current assignee: Arrowhead Madison Inc
Priority date: 1995-12-13
Filing date: 2007-01-04
Publication date: 2007-06-07
Also published as: US20030125281A1; EP1427450A4; EP1427450A2; US7101995B2; WO2003080794A3; JP2005519976A; WO2003080794A2

Abstract

Described is a process for intravascular delivery of a polynucleotide to an extravascular cell of a mammal to inhibit gene expression. A polynucleotide containing sequence that is similar to a sequence in the gene to be expressed is made and inserted into a vessel in the mammal. The polynucleotide is delivered to a cell wherein expression of the gene is inhibited.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

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

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.
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.
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.
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.
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.
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.
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

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.
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.
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.
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.
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.
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

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.
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.
FIG. 3. Inhibition of CD26 expression by mCD26-1 PMO after increased pressure tail vein injection in mice. Liver sections (10 μm) are shown. Histochemical staining for CD26 activity appears dark and is localized at bile canaliculi of hepatocytes.
FIG. 4. Inhibition of firefly luciferase expression by luc-1 PMO. 2 μg of plasmid pGL3 (firefly luciferase) and 0.2 μg 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.
FIG. 5. Photomicrographs of rat limb gastrocnemius (A) and shin (B) muscles stained for β-galactosidase following repeat (triple) intravenous injections of 500 μg of pDNA (pCI-LacZ).
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 μg siRNA (mice #1 and 2), 50 μg 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.
FIG. 7. Graph illustrating reduction in PPAR levels following delivery of PPAR-siRNA expression cassettes in vivo.
FIG. 8A-8B. A. Graph illustrating levels of HMG-CoA reductase mRNA in mice treated with 50 mg/kg atorvastatin. B. Graph illustrating prevention of atorvastatin-induced upregulation of HMGCR levels in vitro by co-delivery of HMGCR siRNA.
FIG. 9. Relative levels of PPARα mRNA in groups of mice injected with siRNAs. mRNA levels are shown relative to total input RNA. Black bar=Experimental group; Grey bars=control group.
FIGS. 10A-10C. A. Graph illustrating effect of statin treatment on LDLR mRNA in primary hepatocytes. B. Graph illustrating relative levels of LDLR mRNA in hepatocytes treated with statins and siRNAs. Dark bars=HMGCR siRNA-treated cells; Light bars=GL3-treated cells. C. Graph illustrating lower doses of atorvastatin necessary to get comparable statin/no statin ratios in cells treated with HMGCR siRNAs.
FIG. 11. Graph Illustrating in vivo inhibition of Hepatitis B surface antigen (HBsAg) following delivery of HepB-sAG specific siRNA via intravascular injection.
FIG. 12. Graph Illustrating in vivo inhibition of Hepatitis B surface antigen (HBsAg) following delivery of HepB-sAG specific siRNA via intravascular injection.
FIG. 13A-13D. A) Delivery of siRNA-Luc+. Maximal inhibition is achieved at 10 nM siRNA-Luc+. B) Delivery of morpholino-Luc+. Maximal specific inhibition is achieved at 100 nM morpholino-Luc+. C) Comparison of inhibitory power of siRNA-Luc+ (1.0 nM) alone, morpholino-Luc+ (100 NM) alone and siRNA-Luc+ (1.0 nM) plus morpholino (100 nM) together. When siRNA and morpholino are added together at these concentrations, the degree of inhibition is greater than either siRNA or morphlino alone. D) Comparison of inhibitory power of siRNA-Luc+ (10 nM) alone, morpholino-Luc+ (100 nM) alone and siRNA-Luc+ (10 nM) plus morpholino (100 nM) together. When siRNA and morpholino are added together at these concentrations, the degree of inhibition is greater than either siRNA or morphlino alone.
FIGS. 14A-14B. Photomicrographs of mouse hind limb gastrocnemius muscle stained for β-galactosidase following single intravenous injection of 0.19×10⁹transducing units of adeno-associated virus in 0.8 ml saline at a rate of 3 ml per minute.
FIG. 15 Bar graph illustrating effective knockdown of gene expression in vivo using PCR generated hairpin siRNA expression cassettes. Expression in each sample (n=4) was measured as the firefly luc+/Renilla ratio and normalized to that ratio of reporter plasmids only samples.

DETAILED DESCRIPTION

The described invention provides methods for delivery of polynucleotides to extravascular cells in a mammal. The invention comprises rapidly injecting the polynucleotides, in a relatively large volume of a pharmaceutically acceptable solution, into a vessel in the mammal. The injection volume, combined with the injection rate, results in increased permeability of the target tissue vasculature, movement of the injection solution and the polynucleotides contained therein out of the vasculature and into the extravascular space, an increase in the volume of extravascular fluid, and entry of the polynucleotides into the surrounding extravascular cells. An intravascular route of administration allows polynucleotides to be delivered to an increased number of cells in a more even distribution than direct parenchymal injections.

Vessels

Vessels comprise internal hollow tubular structures connected to a tissue or organ within a mammal. Fluid flows to or from an organ, tissue, or body part within the cavity of the tubular structure. Examples of fluids normally found in the mammal include blood, lymphatic fluid, and bile. Examples of vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts. Afferent blood vessels of a target tissue are defined as vessels which are directed towards the organ or tissue and in which blood flows towards the tissue under normal physiological conditions. Conversely, efferent blood vessels of a target tissue are defined as vessels which are directed away from the tissue and in which blood flows away from the organ or tissue under normal physiological conditions. In the liver, the hepatic vein is an efferent blood vessel since it normally carries blood away from the liver into the inferior vena cava. Also in the liver, the portal vein and hepatic arteries are afferent blood vessels in relation to the liver since they normally carry blood towards the liver. By inserting the polynucleotide into a vessel in a relatively large volume and at a relatively high rate, the polynucleotide is forced out of the vasculature of the target tissue and into extravascular cells of the target tissue. The polynucleotide may be injected into a vessel in an antegrade or retrograde direction.
For delivery to some target tissues, particularly limb muscle, retrograde injection into a vein is limited by the presence of valves. Valves normally prevent the retrograde flow of blood in limb veins. However, described herein is a means to deliver polynucleotides throughout a limb through the limb venous system via injection of a solution such that permeability of the vessels in the limb is increased but without damaging venous valves or being impeded by venous valves (FIG. 1). After occluding the limb (proximal to the injection site; i.e., nearer to the attachment point of the limb to the body), a sufficiently large volume can be injected into a vein in a direction that does not cause damage to the valves but still results in delivery of polynucleotides throughout the limb. The solution travels in the direction of normal fluid flow (antegrade) in the injected valved vein (thus not damaging the valves), but is free to enter, in a retrograde direction, branches of the large vein which do not have valves. This injection method allows polynucleotides to be delivered to muscle cells throughout the limb.

Volume and Rate of Injection

The polynucleotide is injected into a vessel in a relatively large injection volume. A volume that is relatively large means that the volume is sufficiently large to force the polynucleotides out of the particular target tissue vasculature and into the surrounding extravascular cells. The injection volumes used for the present invention are greater than the volume of fluid that is normally present in the target tissue vasculature. Thus, the injection volume exceeds the capacity of the target tissue vasculature. The injection solution is not simultaneously withdrawn as is done during perfusion procedures.
The injection volume and injection rate are dependent upon: the size of the animal, the size of the vessel into which the solution is injected, and the size and/or volume of the target tissue. Larger injection volumes and/or higher injection rates are required for larger target tissues. For delivery to larger animals, injection of larger volumes is expected. One method of determining target tissue size is through volume displacement measurement, where possible such as with a limb or appendage, or through MRI scan, which can be used to determine tissue mass. The precise volume and rate of injection into a particular vessel, for delivery to a particular target tissue of a given mammal species, may be determined empirically.
The injection volume is dependent on the size of the animal. The injection volume can be from 1.0 to 3.0 ml or greater for small animals (e.g. tail vein injections into mice, ave. wt. about 25 g). The injection volume for slightly larger animals, such as rats (ave. weight 150 g), can be from 6 to 35 ml or greater. The injection volume for Rhesus macaque primates can be 40 to 200 ml or greater, depending on the target tissue. The injection volumes in terms of ml/body weight can be 0.03 ml/g to 0.1 ml/g or greater.
The injection volume is also dependent on the target tissue. For example, for delivery of a polynucleotide to a limb, an injection volume of 5 ml or greater may be used for a rat hind limb while a volume of 40 ml of greater may be used for a Rhesus macaque primate limb. In another example delivering polynucleotides to a limb of a primate (rhesus monkey), the polynucleotide can be in an injection volume of about 0.6 to about 1.8 ml/g of limb target muscle
Intravascular delivery of a polynucleotide to mammalian extravascular cells also requires that the injection volume be injected into the vessel relatively rapidly. A volume that is injected relatively rapidly means that the volume is injected sufficiently rapidly to force the polynucleotides out of the particular target tissue vasculature and into the surrounding extravascular cells. The rate of the injection is partially dependent on the volume to be injected, the size of the vessel into which the volume is injected, the size of the target tissue, and the size of the animal. In one embodiment the total injection volume (1-3 ml) can be injected in about 4 to about 15 seconds into the vascular system of mice. In another embodiment the total injection volume (6-35 ml) can be injected into the vascular system of rats in about 7 to about 20 seconds. In another embodiment the total injection volume (40-200 ml) can be injected into the vascular system of monkeys in about 120 seconds or less.
The injection volume and injection rate can be varied to optimize delivery for a particular application. For a given volume, the efficiency of delivery may be altered by altering the injection rate. Conversely, at a given rate of injection, altering the injection volume can affect delivery efficiency.
The described method is effective for delivering polynucleotides to extravascular cells in mouse, rat, rabbit, dog, and nonhuman primate. Because the method is readily adapted to use in rats, dogs, and nonhuman primates, it is expected that the method is also readily adapted to use in other mammals, including humans. By increasing the amount of polynucleotide injected and the volume of injection, the method described for intravascular delivery of polynucleotides to extravascular cells in small mammals, such as rats, is readily adapted to use in larger animals. Injection rate may also be increased for delivery to larger mammals. Conversely, for delivery to smaller animals or tissues, the injection volume and/or rate is reduced. For example, for delivery to rat hind-limb (150 g animal total weight), injection of 0.2-3 ml injection solution at a rate of 0.5-25 ml/min into the saphenous vein results in delivery of polynucleotides to multiple muscle cells throughout the limb. For delivery to beagle dog (˜9.5 kg total weight) forelimb, injection of 36-40 ml injection solution at a rate of 2 ml/sec into a limb vein results in delivery of polynucleotides to multiple muscle cells throughout the limb. For delivery to rhesus monkey limb, injection of 40-100 ml injection solution at a rate of 1.7-2 ml/sec into a limb vein results in delivery of polynucleotides to multiple muscle cells throughout the limb. This volume corresponds to from about 0.2 to about 0.6 ml of injection solution per ml of displaced target limb volume in rhesus monkey. Target limb volume is the volume of the limb or portion of the limb distal to the vessel occlusion or isolated by the vessel occlusion.

Occlusion of Vessels

Occluding (also impeding or obstructing) the outflow of fluid from the target tissue during the injection further enhances delivery of polynucleotides to extravascular mammalian cells. By occluding outflow of fluid from the target tissue, the injection volume is retained in the target tissue which leads to increased pressure and vascular permeability and enhanced polynucleotide delivery. As used herein, to occlude is to block or inhibit the flow of fluid through a vessel. Fluid flow though a vessel may be occluded by applying compressive pressure against the vessel. Compressive pressure my 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. Use of an external device to occlude fluid flow through vessels is particularly useful because it does not require an invasive procedure. As used herein, the term invasive refers to a medical procedure involving the making of an incision in the skin or other tissues, or the insertion of an instrument or device into the body. Fluid flow though a vessel may also be occluded using a balloon catheter which is inserted into the lumen of the vessel. Vessels are partially or totally occluded for a period of time sufficient for delivery of polynucleotides 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. An occlusion may be released immediately after the injection, within 2 min, within 5 min, within 10 min, or within 20 min after the injection.
One method for occluding fluid flow is the application of an external cuff. The term cuff means an externally applied device for impeding fluid flow to and from a mammalian limb. The cuff applies compression around the limb such that vessels, in an area underneath the cuff, are constricted in an amount sufficient to impede fluid from flowing through the vessels at a normal rate. One example of a cuff is a sphygmomanometer, which is normally used to measure blood pressure. Another example is a tourniquet. A third example is a modified sphygmomanometer cuff containing two air bladders such as is used for intravenous regional anesthesia (i.e. Bier Block). Double tourniquet, double cuff tourniquet, oscillotonometer, oscillometer, and haemotonometer are also examples of cuffs. A sphygmomanometer can be inflated to a pressure above the systolic blood pressure, above 500 mm Hg or above 700 mm Hg or greater than the intravascular pressure generated by the injection.
A vessel may also be occluded using a balloon catheter. A catheter can be inserted at a distant site and threaded through the lumen of a vessel so that it resides in or near a target tissue. Catheters are available in many different sizes (diameter and length), to accommodate applications in different sized and localized vessels. A balloon catheter is a type of catheter with an inflatable balloon somewhere along its length, typically near the tip. The deflated balloon catheter is positioned, then inflated to perform the necessary procedure, and deflated again in order to be removed. Balloon catheters generally include a lumen that provides for controlling inflation of the balloon. In the inflated condition, the balloon will fix the catheter in place in the targeted vessel and prevent flow of fluid through the vessel. Multi-lumen catheters provide for delivery of fluid and other devices (such as a pressure transducer for measuring real time pressure) in addition to controlling the balloon. Single and multi-balloon catheters may also be used. The fluid injection port in a balloon catheter can be located proximal to the balloon, distil to the balloon, or between balloons in a multi-balloon catheter.
The vessel into with the polynucleotide is injected may be occluded. One or more vessels other than the injected vessel-may also be occluded. For example, an afferent vessel supplying a target tissue may be the injected vessel and an efferent vessel draining the target tissue may be occluded. Conversely, an efferent vessel of a target tissue may be the injected vessel and an afferent vessel may be occluded. In some tissues, such as the heart and liver, capillary beds within the target tissue provide sufficient resistance to outflow of fluid from the target tissue to enable polynucleotide delivery. The occlusion point is chosen such that the injection solution is able to reach the target tissue through the target tissue vasculature and such that the injection solution is sufficiently retained in the target tissue during the injection.
As used herein, with respect to vessel occlusion, proximal generally refers to a location that is closer to the center of the body, and more specifically the heart, than another part. More particularly, the term proximal is used in reference to a location in a vessel relative to the target tissue. For an afferent vessel, a proximal location is upstream with respect to the direction of normal fluid flow. For an efferent blood vessel, a proximal location is downstream with respect to the direction of normal fluid flow. With respect to a limb, proximal refers to a location nearer the point of attachment of the limb to the body. The hip or thigh is proximal to the knee, which is proximal to the foot. Similarly, the shoulder is proximal to the elbow, which is proximal to the hand. By occluding a vessel or limb proximal to the site of injection, the injection solution is directed towards or retained in the target tissue.
Because 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. MRI can also be used to determine bed size. Also, an automatic injection system can be used such that the injection solution is delivered at a preset pressure or rate. 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 a vein or artery within the target tissue.

Vascular Permeability Enhancing Factors

Permeability of a vessel can be further increased by administration of a vascular permeability enhancing factor or vasodilator. The vascular permeability enhancing factor or vasodilator can be administered prior to injecting the polynucleotide or simultaneously with the polynucleotide. Vascular permeability enhancing factors are proteins or molecules that increase the permeability of the vessel by causing a change in function, activity, or shape of cells, such as the endothelial or smooth muscle cells, within the vessel wall. Vascular permeability enhancing molecules and vasodilators include, not are not limited to, histamine, vascular permeability factor (VPF, which is also known as vascular endothelial growth factor VEGF), calcium channel blockers, adenosine, papaverine, beta adrenergic blockers, angiotensin converting enzyme (ACE) inhibitors, and angiotensin II receptor antagonists,
Vessel permeability can also be further increased by increasing the osmotic pressure within the vessel. To increase the osmotic pressure within a vessel, hypertonic solutions containing salts such as NaCl, sugars or polyols such as mannitol are typically used. Hypertonic means that the osmolarity of the injection solution is greater than physiological osmolarity. Isotonic means that the osmolarity of the injection solution is the same as the physiological osmolarity (the tonicity or osmotic pressure of the solution is similar to that of blood). Hypertonic solutions have increased tonicity and osmotic pressure relative to the osmotic pressure of blood and cause cells to shrink.

Target Tissue

As used herein, the term target tissue can encompass one or more organs or tissue types or may include part of an organ or tissue. For delivery to skeletal muscle, a target tissue can be an individual muscle or a plurality of muscles. For example, the target tissue can be all the muscles of a limb proximal to a tourniquet. The target tissue is determined by the injection location and can be further delimited by vessel occlusions and/or capillary beds. In other words, the target tissue consists of the cells supplied by the injected vessel and may optionally be limited by vessel occlusions. For injection into an artery, the target tissue is the cells that the arteries supply with blood. For injection into a vein, the target tissue is the cells from which the vein drains blood. Arteries undergo enormous ramification in their course throughout the body, and end in very minute vessels, called arterioles, which in turn open into a close-meshed network of microscopic vessels termed capillaries. These capillaries then join to form larger venules which in turn join to form larger veins. Within a mammal, the injected solution is able to flow through connecting vessels, including arterial and venous vessels. Tissue to which a sufficiently large volume of the injected solution is able to reach and cause increased vessel permeability constitutes the target tissue. Thus, for injection into a hepatic vein, hepatic artery, portal vein, or bile duct, the target tissue is the liver. For injection into the femoral artery or saphenous vein distal to a tourniquet placed around the limb, the target tissue includes the limb skeletal muscle distal to the tourniquet. The injection volume typically causes visible swelling in the target tissue.
The extravascular cell may be selected from the group comprising: cardiac muscle cells, liver cells (including hepatocytes), kidney cells, prostate cells, diaphragm cells, skeletal muscle cells (myofiber, myocytes) bone cells (osteocytes, osteoclasts, osteoblasts), bone marrow cells, stroma cells, joint cells (synovial and cartilage cells), connective tissue cells (fibroblasts, fibrocytes, chondrocytes, mesenchyme cells, mast cells, macrophages, histiocytes), cells in tendons cells in the skin and cells in the lymph nodes, lung cells, fat cells, pancreatic cells, spleen cells, thymus cells, and gastrointestinal cells.
Parenchymal cells are the distinguishing cells of a gland or organ contained in and supported by the connective tissue framework. The parenchymal cells typically 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 blood vessels.
For example, in a liver organ, the parenchymal cells include hepatocytes, Kupffer cells and the epithelial cells that line the biliary tract and bile ductules. 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 opposed 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. In one preferred embodiment hepatocytes are targeted by injecting the polynucleotide or polynucleotide complex into the portal vein or bile duct of a mammal.
In striated muscle, the parenchymal cells include myoblasts, satellite cells, myotubules, and myofibers. In cardiac muscle, 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.
In a pancreas, 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.
In spleen, thymus, lymph nodes and bone marrow, the parenchymal cells include reticular cells and blood cells (or precursors to blood cells) such as lymphocytes, monocytes, plasma cells and macrophages.
In the kidney, parenchymal cells include cells of collecting tubules and the proximal and distal tubular cells. In the prostate, the parenchyma includes epithelial cells.
In glandular tissues and organs, the parenchymal cells include cells that produce hormones. In the parathyroid glands, the parenchymal cells include the principal cells (chief cells) and oxyphilic cells. In the thyroid gland, the parenchymal cells include follicular epithelial cells and parafollicular cells. In the adrenal glands, the parenchymal cells include the epithelial cells within the adrenal cortex and the polyhedral cells within the adrenal medulla.
In the parenchyma of the gastrointestinal tract such as the esophagus, stomach, and intestines, the parenchymal cells include epithelial cells, glandular cells, basal, and goblet cells.
In the parenchyma of lung, the parenchymal cells include the epithelial cells, mucus cells, goblet cells, and alveolar cells.
In fat tissue, the parenchymal cells include adipose cells or adipocytes. In the skin, the parenchymal cells include the epithelial cells of the epidermis, melanocytes, cells of the sweat glands, and cells of the hair root.

Multiple/Repeat Injections

The described process may be used repetitively in a single animal. Multiple injections may be used to provide delivery to additional tissues, to increase delivery to a single tissue, or where multiple treatments are indicated. Multiple injections may be performed in different vessels or tissues of the same animal or within the same vessel or tissue of the animal. The site of vessel occlusion may also be the same or different for multiple injections in the same animal.

Injection Device

A syringe needle, cannula, catheter or other injection device may be used to inject the polynucleotide into a vessel. Single and multi-port injectors may be used, as well as single or multi-balloon catheters and single and multi-lumen injection devices. Catheters are available in many different sizes (diameter and length), to accommodate applications in different sized and localized vessels. A catheter can be inserted at a distant site and threaded through the lumen of a vessel so that it resides in or near a target tissue. The injection can also be performed using a needle that enters the lumen of a vessel.

Injection Solution

The polynucleotide is injected in a pharmaceutically acceptable solution. Pharmaceutically acceptable refers to those properties and/or substances which are acceptable to the mammal from a pharmacological/toxicological point of view. The phrase pharmaceutically acceptable refers to molecular entities, compositions and properties that are physiologically tolerable and do not typically produce an allergic or other untoward or toxic reaction when administered to a mammal. Preferably, as used herein, the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
In addition to the polynucleotide, the injection solution may contain one or more molecules selected from the list comprising: anesthetics, analgesics, vascular permeability enhancing agents, salts, sugars, and components of polynucleotide complexes. The composition of the injection solution can depend on the nature of the polynucleotide or polynucleotide complex that is to be delivered. Certain complexes may be delivered more efficiently using low salt injection solutions.

Polynucleotides

The term polynucleotide, or nucleic acid or polynucleic acid, is a term of art that refers to a polymer containing at least two nucleotides. Nucleotides are the monomeric units of polynucleotide polymers. Polynucleotides with less than 120 monomeric units are often called oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-phosphate backbone. An artificial or synthetic polynucleotide is any polynucleotide that is polymerized in vitro or in a cell free system and contains the same or similar bases but may contain a backbone of a type other than the natural ribose-phosphate backbone. These backbones include, but are not limited to: PNAs (peptide nucleic acids), phosphorothioates, phosphorodiamidates, morpholinos, and other variants of the phosphate backbone of native nucleic acids. Bases include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs. Synthetic derivatives of purines and pyrimidines include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides. The term base encompasses any of the known base analogs of DNA and RNA. A polynucleotide may comprise modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. The term polynucleotide includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations of DNA, RNA and other natural and synthetic nucleotides. DNA may be in form of cDNA, in vitro polymerized DNA, plasmid DNA, parts of a plasmid DNA, genetic material derived from a virus, linear DNA, chromosomal DNA, an oligonucleotide, antisense DNA, or derivatives of these groups. RNA may be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, siRNA (small interfering RNA), dsRNA (double stranded RNA), RNAi, ribozymes, in vitro polymerized RNA, or derivatives of these groups. The polynucleotide can be single strand, double strand or triple strand. A double strand polynucleotide may comprise two separate complementary strands. Alternatively, a double strand polynucleotide may comprise a single strand which is self-complementary. A self-complementary strand may comprise two complementary sequences which are connected by a non-nucleotide linker. A self-complementary strand may also comprise two complementary sequences connected by a nucleotide linker, referred to in the art as a loop. A double strand polynucleotide may be completely double stranded, mostly double stranded or partially double stranded. A double strand oligonucleotide, including double strand RNA oligonucleotide is double strand polynucleotide in which the region of complementarity is less than 50 bases.
The term expression cassette refers to a natural or recombinantly produced nucleic acid molecule that is capable of expressing a gene or genetic sequence in a cell. An expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins or RNAs. Optionally, the expression cassette may include transcriptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals. An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins. Optionally, the expression cassette may include translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non-coding sequences. Optionally, the expression cassette may include a gene or partial gene sequence that is not translated into a protein. The nucleic acid can effect a change in the DNA or RNA sequence of the target cell. This can be achieved by hybridization, multi-strand nucleic acid formation, homologous recombination, gene conversion, RNA interference or other yet to be described mechanisms.
The term gene generally refers to a nucleic acid sequence that comprises coding sequences necessary for the production of a nucleic acid (e.g., siRNA) or a polypeptide or precursor. A polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction) of the full-length polypeptide or fragment are retained.
The term also encompasses the coding region of a gene and the including sequences located adjacent to the coding region on both the 5′ and 3′ ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5′ of the coding region and which are present on the mRNA are referred to as 5′ untranslated sequences. The sequences that are located 3′ or downstream of the coding region and which are present on the mRNA are referred to as 3′ untranslated sequences. The term gene encompasses synthetic, recombinant, cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed introns, intervening regions or intervening sequences. Introns are segments of a gene which are transcribed into nuclear RNA. Introns may contain regulatory elements such as enhancers. Introns are removed or spliced out from the nuclear or primary transcript; introns therefore are absent in the mature RNA transcript. Components of a gene also include, but are not limited to, promoters, enhancers, transcription factor binding sites, polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, matrix attachment regions. Non-coding sequences influence the level or rate of transcription and/or translation of the gene. Covalent modification of a gene may influence the rate of transcription (e.g., methylation of genomic DNA), the stability of mRNA (e.g., length of the 3′ polyadenosine tail), rate of translation (e.g., 5′ cap), nucleic acid repair, nuclear transport, and immunogenicity. Gene expression can be regulated at many stages in the process. Up-regulation or activation refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while down-regulation or repression refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called activators and repressors, respectively. Episomal vectors are vectors that are replicated during the eukaryotic cell division (e.g., plasmid DNA vectors containing a papilloma virus origin of replication, artificial chromosomes).
Different promoter, enhancers, and even introns can affect the level or duration of expression of a delivered polynucleotide. It may be desirable to regulate expression of the delivered polynucleotide using regulated promoters. Regulated promoters may be inducible or repressible. Regulated gene expression systems may be selected from the list comprising: drug-dependent gene regulation, tetracycline/doxycycline-inducible, tetracycline/doxycycline-repressible, rapamycin-inducible, β-galactoside, streptogramin-regulated, bacterial repressor protein, antiprogestin-inducible GeneSwitch®(Valentis, Inc., induced by mifepristone), nuclear hormone receptor ligand binding domain (antiprogestin-, antiestrogen-, ecdysteroid-, glucocorticoid-responsive), heterodimeric protein, metabolic regulated, hypoxia responsive, and glucose responsive systems. Some of these systems are regulated by proteins naturally occurring in mammalian cells while others require co-delivery of a gene encoding a transcription activator or repressor.
It may also be desirable for the delivered polynucleotide to be expressed from a tissue or cell-type specific promoter. Muscle specific promoters may be selected from the list comprising: muscle creatine kinase (MCK), myosin light chain, myosin light chain 3F, desmin, alpha-actin, enolase, utrophin, dystrophin, sarcoglycan and other dystrophin-associated glycoprotein promoters. Still other transcription elements that function in muscle include: actin and δ-actin promoters, E-box elements, MEF-2 elements, TEF-1 elements, SRE sites, myogenin enhancer sequences, and viral promoters such as CMV and SV40.
A delivered polynucleotide can stay within the cytoplasm or enter the nucleus. A polynucleotide can be delivered to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to affect a specific physiological characteristic not naturally associated with the cell. Altering gene expression may comprise: altering splicing of an RNA, affecting mRNA levels, and altering gene expression through binding to transcription factors. A polynucleotides can also alter the sequence of an endogenous or exogenous polynucleotide in a cell. Altering the sequence of a polynucleotide in a cell includes altering the sequence through gene conversion or recombination. Chimeroplasts (hybrid molecules of RNA and DNA) and single stranded polynucleotides have been used to alter chromosomal DNA sequences.
The described method can be used to deliver a polynucleotide to a mammalian cell for the purpose of altering the endogenous properties of the cell, for example altering the endogenous properties of the cell for therapeutic purposes, for augmenting function, for facilitating pharmaceutical drug discovery, for facilitating drug target validation or for investigating gene function (i.e., research). The polynucleotide can express in the cell a protein or nucleic acid that is not normally present in the cell. The polynucleotide can also inhibit expression of an expressed gene in the cell. The polynucleotide can express a gene product (e.g. protein) that is retained in or on the cell or is secreted from the cell. The polynucleotide can be delivered to a mammalian cell in vivo for the treatment of a disease or infection. Multiple polynucleotides or polynucleotides containing more that one gene may be delivered using the described process.
Certain polynucleotides known in the art can inhibit gene expression or function through degradation or inhibition of translation or function of a specific cellular RNA, usually a mRNA, in a sequence-specific manner. Such polynucleotides are selected from the group comprising: small interfering RNA (siRNA), microRNA (miRNA), dsRNA, ribozymes, and antisense polynucleotides. Antisense polynucleotides may be RNA, DNA, or artificial polynucleotides. An siRNA comprises a double stranded RNA molecule capable of inducing RNA interference. SiRNA comprises a double stranded structure typically containing 15-50 base pairs and preferably 19-25 base pairs and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA sequence within the cell. An siRNA may be composed of two annealed polynucleotides or a single polynucleotide that forms a hairpin structure. MicroRNAs (miRNAs) are small noncoding polynucleotides, about 22 nucleotides long, that direct destruction or translational repression of their mRNA targets. SiRNA and microRNA can be generated in the cell from longer dsRNAs or may be transcribed from expression vectors. We demonstrate that delivery of siRNA and antisense inhibitors to cells of post-embryonic animals interferes with specific gene expression in those cells. The inhibition is gene specific and does not cause general translational arrest. Thus RNAi can be effective in post-embryonic mammalian cells in vivo.
We have disclosed expression or inhibition of reporter genes in multiple target tissues following delivery of polynucleotides. The disclosed delivery method is independent of the sequence of the polynucleotide. Therefore, delivery of polynucleotides encoding or inhibiting reporter genes indicates that delivery of other polynucleotide would be equally efficient.
A therapeutic effect of the protein in attenuating or preventing a 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 may be selected from the list comprising: hormones, cytokines, interferons, enzymes (e.g. lysosomal enzymes), growth factors, clotting factors, anti-protease proteins (e.g., alphal-antitrypsin), angiogenic proteins (e.g., vascular endothelial growth factor, fibroblast growth factors), anti-angiogenic proteins (e.g., endostatin, angiostatin), and other proteins that are present in the blood. Proteins on the membrane can provide a receptor on the cell surface. For example, 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. Expressed proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite, as in phenylketonuria, cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic), or interfere with replication of a virus. Intracellular proteins can be part of the cytoskeleton (e.g., actin, dystrophin, myosins, sarcoglycans, dystroglycans) and thus have a therapeutic effect in cardiomyopathies and musculoskeletal diseases (e.g., Duchenne muscular dystrophy, limb-girdle disease). Other proteins of particular interest to treating disease include polypeptides affecting cardiac contractility (e.g., calcium and sodium channels), inhibitors of restenosis (e.g., nitric oxide synthetase), angiogenic factors, and anti-angiogenic factors.
Levels of treatment considered beneficial by a person having ordinary skill in the art of gene therapy differ from disease to disease, for example: Hemophilia A and B are caused by deficiencies of the X-linked clotting factors VIII and IX, respectively. Their clinical course is greatly influenced by the percentage of normal serum levels of factor VIII or IX: <2%, severe; 2-5%, moderate; and 5-30% mild. This indicates that in severe patients an increase from 1% to 2% of the normal level can be considered beneficial. Levels greater than 6% prevent spontaneous bleeds but not those secondary to surgery or injury. A person having ordinary skill in the art of gene therapy would reasonably anticipate beneficial levels of expression of a gene specific for a disease based upon sufficient levels of marker gene results. In the hemophilia example, if marker genes were expressed to yield a protein at a level comparable in volume to 2% of the normal level of factor VIII, it can be reasonably expected that the gene coding for factor VIII would also be expressed at similar levels.
A polynucleotide can be delivered to a cell to prevent or treat an injection. Because viruses inject their genomes into the host cell, delivery of siRNA and miRNA to a mammalian cell infected by a virus can be effective in inhibition expression of viral genes. In fact, research suggests that RNAi is a natural protective response to viral infection. The genes involved in viral reproduction and virulence are well known in the art. In addition the design of siRNAs and microRNAs that inhibit genes is straightforward. The polynucleotide may reduce or block microbe production, virulence, or both. Delivery of the polynucleotide may delay progression of disease until endogenous immune protection can be acquired. Viral genes involved in transcription, replication, virion assembly, immature viral membrane formation, extracellular enveloped virus formation, early genes, intermediate genes, late genes, and virulence genes may be targeted. Combinations of polynucleotide targeted to the same or different viral genes or classes of genes (e.g., transcription, replication, virulence, etc) are delivered to an infected mammalian cell in vivo.
Alternatively, instead of inhibiting a pathogen gene, a polynucleotide may to reduce virulence of the pathogen, such as by decreasing expression of an endogenous host gene. The polynucleotide may be delivered to a cell in a mammal to reduce expression of a cellular receptor. For example, the lethality of the bacterial pathogen, Anthrax, is primarily mediated by a secreted tripartite toxin which requires the mammalian anthrax toxin receptor (ATR) for cellular entry (Bradley 2001). Reducing expression of ATR may decrease Anthrax toxicity. Receptors to which other pathogens bind may also be targeted.
RNAi technology has an advantage over traditional drugs in that it is more readily adapted to new, mutated, or engineered infectious agents. A new infectious agent can be quickly sequenced and RNAi molecules synthesized to combat the new pathogen. Delivery of the polynucleotide would remain the same regardless of the specific sequence.
Viral genes for transcription, replication, or virulence may be targeted to decrease the contagiousness or to delay onset of major disease. Delaying onset or reducing symptoms of infection can boost survival of infected individuals. Since host immune response is responsible for the toxicity of some infectious agents, reducing this response may increase the survival of an infected mammal. Also, inhibition of immune response is beneficial for a number of other therapeutic purposes, including gene therapy, where immune reaction often greatly limits transgene expression, organ transplantation, and autoimmune disorders.
Pathogen infection or replication does not need to be completely eliminated for a therapy to provide a therapeutic benefit. Reducing toxicity, reducing replication, or reducing a negative host response can provide a benefit by delaying onset of symptoms or lessening symptoms. Delaying or lessening symptoms can provide additional time for an animal's natural immune system to react to the pathogen, or enhance the effectiveness of other pharmaceutical drugs.
Several aspects of current pharmaceutical research and therapeutic treatment are candidates for in vivo polynucleotide delivery. For the purposes of target validation, gene inactivation allows the investigator to assess the potential therapeutic effect of inhibiting a specific gene product. Expression arrays can be used to determine the responsive effect of inhibition on the expression of genes other than the targeted gene or pathway. Other methods of gene inactivation, generation of mutant cell lines or knockout mice suffer from serious deficiencies including embryonic lethality, expense, and inflexibility. Also, these methods frequently do not adequately model larger animals.
A polynucleotide may express a peptide or protein antigen, thereby resulting in induction of an immune response in the animal. Induction of an immune response can be used to produce antibodies, provide a vaccine or immunization, or provide a therapeutic response, such as to cancer or infection.
Delivery of a gene to a cell that expresses a protein not previously expressed in the animal can result in the induction of an immune response directed against the newly expressed protein. Also, the polynucleotide itself, or other potential components of the injection solution, may illicit an immune response. In some instances, therefore, it may be beneficial to provide immunosuppressive drugs to the mammal. Suppression of immune response to an expressed gene can prolong expression of the gene. Immunosuppressive drugs can be given before, during, or after injection of the polynucleotide. Immunosuppression can be of immediate duration (within 2 days of injection of the polynucleotide), short term duration (less than 3 months) or long term duration (longer than 3 months).

Naked Polynucleotides and Polynucleotide Complexes

The invention is meant to encompass the delivery to cells of naked polynucleotides, modified polynucleotides, and polynucleotide complexes. A polynucleotide complex may be a viral complex or a non-viral complex.
As used herein, a naked polynucleotide is not physically associated with a transfection reagent or other delivery vehicle that is required for the polynucleotide to be delivered to the parenchymal cell.
A modified polynucleotide comprises a polynucleotide to which one or more functional groups is covalently attached. The functional group may be selected from the group comprising: lipophilic compounds, targeting signals, detectable labels, fluorescent molecules, fluorescence and chemiluminescence quenchers, steric stabilizers, interaction modifiers, reactive groups, and membrane active compounds. The functional groups may be linked to the 5′ or 3′ end of the polynucleotide, to the backbone, to a sugar moiety or to a base moiety. The functional group can alter the stability, hybridization, solubility, or cellular uptake of the polynucleotide.
A viral complex, or viral vector, comprises a nucleic acid encapsulated by a intact viral particle. Viral vectors are typically assembled in a eukaryotic cell. Viral vectors may be selected from the list comprising: adeno-associated virus, adenovirus, herpes simplex virus, vaccinia virus, retrovirus, murine leukaemia virus, lentivirus, human immunodeficiency virus, syndbis virus, and vesicular stomatitis virus. Viruses used for nucleic acid delivery are typically recombinant viruses in which the nucleic acid to be delivered is integrated into the viral genome, either in addition to the viral genes or place or one or more viral genes or sequences. In some viral vectors, most or the entire viral genome is replaced by the desired nucleic acid sequences.
A non-viral complex, or non-viral vector, comprises a polynucleotide that is not encapsulated by an intact viral particle but is in association with one or more compounds that aid in delivery of the polynucleotide to a cell. Compounds associated with polynucleotides to aid in delivery of the polynucleotide to a cell comprise: transfection reagents, hydrophobic or lipophilic groups, lipids, surfactants, cationic lipids and surfactants, polymers, proteins, viral proteins, peptides, viral peptides, polycations, polyampholytes, amphipathic compounds and polymers, targeting signals, membrane active compounds, cell penetrating compounds, targeting signals, interaction modifiers and steric stabilizers. The compounds may be associated with the polynucleotide through covalent or non-covalent interactions. Non-covalent interactions include ionic or electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions. One or more functional groups may be covalently or non-covalently associated with a polynucleotide complex. The functional groups may be attached to one or more of the components prior to complex formation. Alternatively, the functional group(s) may be attached to the complex after formation of the complex. The functional group may be selected from the list comprising: hydrophobic groups, membrane active compounds, cell penetrating compounds, targeting signals, interaction modifiers and steric stabilizers. Non-viral vectors include protein and polymer complexes (polyplexes), lipids and liposomes. (lipoplexes), combinations of polymers and lipids (lipopolyplexes), and multilayered and recharged particles. Polycations and cationic lipid may condense nucleic acids.
A transfection reagent or delivery vehicle is a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and enhances or mediates their entry into cells. Examples of transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, polylysine, and polyampholyte complexes. It has been shown that cationic proteins like histones and protamines, or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents. Typically, the transfection reagent has a component with a net positive charge that binds to the oligonucleotide's or polynucleotide's negative charge.
One or more components of a non-viral polynucleotide complex may be covalently linked, or crosslinked, to another component of the non-viral complex. Crosslinking can stabilize the non-viral complex.
A polyelectrolyte, or polyion, is a polymer possessing more than one charge, i.e. the polymer contains groups that have either gained or lost one or more electrons. A polycation is a polyelectrolyte possessing net positive charge. The polycation can contain monomer units that are charge positive, charge neutral, or charge negative, however, the net charge of the polymer must be positive. A polycation also can mean a non-polymeric molecule that contains two or more positive charges. A polyanion is a polyelectrolyte containing a net negative charge. The polyanion can contain monomer units that are charge negative, charge neutral, or charge positive, however, the net charge on the polymer must be negative. A polyanion can also mean a non-polymeric molecule that contains two or more negative charges. The term polyelectrolyte includes polycation, polyanion, zwitterionic polymers, and neutral polymers. The term zwitterionic refers to the product (salt) of the reaction between an acidic group and a basic group that are part of the same molecule.
As used herein, functional groups include targeting groups, membrane active compounds, cell penetrating compounds, hydrophobic groups, and interaction modifiers.
Targeting groups direct a molecule or complex to a cell or cellular organelle by interacting with or binding to components of the cell or cellular organelle. Targeting groups enhance the association of molecules with a cell. The targeting group can be, but is not limited to, a protein, peptide, lipid, steroid, sugar, carbohydrate, or synthetic compound. Examples of targeting groups include ligands that bind to receptor proteins on a cell surface. An examples of a receptor is the asialoglycoprotein receptor which binds asialoglycoproteins or galactose molecules. Other ligands, such as insulin, EGF, RDG-containing peptides, transferrin, and certain viral coat proteins, can be used for targeting. Folate and other vitamins can also be used for targeting. Targeting groups can also include molecules that interact with membranes, such as fatty acids, cholesterol, dansyl compounds, and amphotericin derivatives. A variety of ligands have been used to target drugs and genes to cells and to specific cellular receptors. The ligand may seek a target within the cell membrane, on the cell membrane or near a cell. Binding of a ligand to a receptor may initiate endocytosis.
After a polynucleotide complex has been directed to a cell, other targeting groups can be used to increase the delivery of the polynucleotide to certain organelles, such as the nucleus. Nuclear localization signals are examples of targeting groups that enhance localization of molecules to a cellular organelle. Nuclear localizing signals enhance the targeting of the pharmaceutical into proximity of the nucleus and/or its entry into the nucleus. Such nuclear transport signals can be a protein or a peptide such as the SV40 large T antigen NLS or the nucleoplasmin NLS. These nuclear localizing signals interact with a variety of nuclear transport factors such as the NLS receptor (karyopherin alpha) which then interacts with karyopherin beta. The nuclear transport proteins themselves could also function as NLS's since they are targeted to the nuclear pore and nucleus. For example, karyopherin beta itself could target the nuclear pore complex. Several peptides have been derived from the SV40 T antigen. Other NLS peptides have been derived from M9 protein, nucleoplasmin, and c-myc.
Membrane active compounds or polymers are molecules that are able to inducing one or more of the following effects upon a biological membrane: an alteration or disruption that allows small molecule permeability, pore formation in the membrane, a fusion and/or fission of membranes, an alteration or disruption that allows large molecule permeability, or a dissolving of the membrane. This alteration can be functionally defined by the compound's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis and release of endosomal contents. More specifically membrane active compounds allow for the transport of molecules with molecular weight greater than 50 atomic mass units to cross a membrane. This transport may be accomplished by either the loss of membrane structure or the formation of holes or pores in the membrane. Membrane active polymers may be selected from the list comprising: membrane active toxins such as pardaxin, melittin, cecropin, magainin, PGLa, indolicidin, dermaseptin, and their derivative; viral fusogenic peptides such as the influenza virus hemagglutinin subunit HA-2 peptide; certain synthetic amphipathic peptides; and amphipathic polymers such as butyl polyvinyl ether. Because there exists little sequence homology or predictable structural similarity between the different membrane active peptides, they are defined by their membrane activity.
Cell penetrating compounds, which include cationic import peptides (also called peptide translocation domains, membrane translocation peptides, arginine-rich motifs, cell-penetrating peptides, and peptoid molecular transporters) are typically rich in arginine and lysine residues and are capable of crossing biological membranes. In addition, they are capable of transporting molecules to which they are attached across membranes. Examples include TAT (GRKKRRQRRR, SEQ ID NO. 1), VP22 peptide, and an ANTp peptide (RQIKIWFQNRRMKWKK, SEQ ID NO. 2). Cell penetrating compounds are not strictly peptides. Short, non-peptide polymers that are rich in amines or guanidinium groups are also capable of carrying molecules crossing biological membranes. Like membrane active peptides, cationic import peptides are defined by their activity rather than by strict amino acid sequence requirements.
An interaction modifier changes the way that a molecule interacts with itself or other molecules relative to molecule containing no interaction modifier. The result of this modification is that self-interactions or interactions with other molecules are either increased or decreased. Polyethylene glycol is an interaction modifier that decreases interactions between molecules and themselves and with other molecules. One class of interaction modifier is also termed a steric stabilizer. A steric stabilizer is a long chain hydrophilic group that prevents aggregation by sterically hindering particle to particle or polymer to polymer electrostatic interactions. Examples include: alkyl groups, PEG chains, polysaccharides, alkyl amines. Electrostatic interactions are the non-covalent association of two or more substances due to attractive forces between positive and negative charges.

Labile Bonds and Polymers

A labile bond is a covalent bond that is capable of being selectively broken. More specifically, a labile bond is a chemical covalent bond may be selectively broken in the presence of other covalent bonds without the breakage of other covalent bonds in the molecule. More specifically, as used herein, a labile bond is a covalent bond that can be selectively broken without the breakage of other covalent bonds in the molecule under typical mammalian physiological conditions. For example, a disulfide bond is capable of being broken in the presence of thiols or glutathione without cleavage of any other bonds, such as carbon-carbon, carbon-oxygen, carbon-sulfur, carbon-nitrogen bonds, which may also be present in the molecule. Labile also means cleavable. As used herein, the phrase physiological conditions relates to the range of chemical (e.g., pH, ionic strength) and biochemical (e.g., enzyme concentrations) conditions likely to be encountered in the intracellular and extracellular fluids of mammalian tissues.
In one embodiment, peptides and polypeptides (both referred to as peptides) are modified by an anhydride. The amine (lysine), alcohol (serine, threonine, tyrosine), and thiol (cysteine) groups of the peptides are modified by the anhydride to produce an amide, ester or thioester acid. In the acidic environment of the internal vesicles (pH less than 6.5, greater than 4.5) (early endosomes, late endosomes, or lysosome) the amide, ester, or thioester is cleaved displaying the original amine, alcohol, or thiol group and the anhydride.
A variety of endosomolytic and amphipathic peptides can be used in this embodiment. A positively-charged amphipathic/endosomolytic peptide is converted to a negatively-charged peptide by reaction with the anhydrides to form the amide acids and this compound is then complexed with a polycation-condensed nucleic acid. After entry into the endosomes, the amide acid is cleaved and the peptide becomes positively charged and is no longer complexed with the polycation-condensed nucleic acid and becomes amphipathic and endosomolytic. In one embodiment the peptides contains tyrosines and lysines. In yet another embodiment, the hydrophobic part of the peptide (after cleavage of the ester acid) is at one end of the peptide and the hydrophilic part (e.g. negatively charged after cleavage) is at another end. The hydrophobic part could be modified with a dimethylmaleic anhydride and the hydrophilic part could be modified with a citraconyl anhydride. Since the dimethylmaleyl group is cleaved more rapidly than the citraconyl group, the hydrophobic part forms first. In another embodiment the hydrophilic part forms alpha helixes or coil-coil structures.
pH-labile refers to the selective breakage of a covalent bond under acidic conditions (pH<7). That is, the pH-labile bond may be broken under acidic conditions in the presence of other covalent bonds without breakage of the other covalent bonds.
pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over a defined range of pH. A more narrow definition demands significant changes in the polymer in a physiologically tolerated pH range (usually pH 5.5-8). Polyions can be divided into three categories based on their ability to donate or accept protons in aqueous solutions: polyacids, polybases and polyampholytes. Use of pH-sensitive polyacids in drug delivery applications usually relies on their ability to become soluble with increased pH (acid/salt conversion), to form complexes with other polymers over a change of pH, or to undergo significant change in hydrophobicity/hydrophilicity balance. Combinations of all three above factors are also possible.
For polynucleotide complexes, the polynucleotide must be dissociated from components of the complex in the cell in order for the polynucleotide to be active. This dissociation may occur outside the cell, within cytoplasmic vesicles or organelles (i.e. endosomes), in the cytoplasm, or in the nucleus. We have developed bulk polymers prepared from disulfide bond containing co-monomers and cationic co-monomers to better facilitate this process. These polymers have been shown to condense polynucleotides, and to release the nucleotides after reduction of the disulfide bond. These polymers can be used to effectively complex with nucleic acids and can also protect the nucleic acid from nucleases during delivery to the liver and other organs. After delivery to the cells the polymers are reduced to monomers, effectively releasing the nucleic acid. For instance, the disulfide bonds may be reduced by glutathione which is present in higher concentrations inside the cell. Negatively charged polymers can be fashioned in a similar manner, allowing the condensed nucleic acid particle to be “recharged” with a cleavable anionic polymer resulting in a particle with a net negative charge that after reduction of disulfide bonds will release the nucleic acid. The reduction potential of the disulfide bond in the reducible co-monomer can be adjusted by chemically altering the disulfide bonds environment. Therefore one can construct particles whose release characteristics can be tailored so that the nucleic acid is released at the proper point in the delivery process.

EXAMPLES

The following examples are intended to illustrate, but not limit, the present invention.

Example 1

Functional delivery of siRNA to liver cells in vivo for inhibition of gene expression. Single-stranded, gene-specific sense and antisense RNA oligomers with overhanging 3′ deoxyribonucleotides were prepared and purified by PAGE. The two oligomers, 40 μM each, were annealed in 250 μl buffer containing 50 mM Tris-HCl, pH 8.0 and 100 mM NaCl, by heating to 94° C. for 2 minutes, cooling to 90° C. for 1 minute, then cooling to 20° C. at a rate of 1° C. per minute. The resulting siRNA was stored at −20° C. prior to use.
The sense and antisense oligomers with identity to positions 155-173 of the luc+ reading frame had the sequence: 5′-rCrUrUrArCrGrCrUrGrArGrUrArCrUrUrCrGrATT-3′ (SEQ ID NO. 3), and 5′-rUrCrGrArArGrUrArCrUrCrArGrCrGrUrArArGTT-3′ (SEQ ID NO. 4), respectively. The annealed oligomers containing luc+ coding sequence are referred to as siRNA-luc+. The sense and antisense oligomers with identity to the ColE1 origin of replication had the sequence: 5′-rGrCrGrArUrArArGrUrCrGrUrGrUrCrUrUrArCTT-3′ (SEQ ID NO. 5), and 5′-rGrUrArArGrArCrArCrGrArCrUrUrArUrCrGrCTT-3′ (SEQ ID NO. 6), respectively. The annealed oligomers containing ColEl sequence are referred to as siRNA-ori. The letter “r” preceding a nucleotide indicates that nucleotide is a ribonucleotide.
Plasmid pMIR48 (10 μg), containing the luc+ coding region (Promega Corp.) and a chimeric intron downstream of the cytomegalovirus major immediate-early enhancer/promoter, was mixed with 0.5 or 5 μg siRNA-luc+, diluted in 1-3 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) and injected into the tail vein of ICR mice over 7-12 seconds.
Volume equaled 1 ml per 10 g body weight. The tail vein can be considered to be an afferent vessel of the liver in rodents. Under the conditions used, a significant portion of the injected volume is directed to the liver. The tail vein injection will also delivery the solution and polynucleotides to the spleen, heart, and kidneys. One day after injection, the livers were harvested and homogenized in lysis buffer (0.1% Triton X-100, 0.1 M KPO₄, 1 mM DTT, pH 7.8). Insoluble material was cleared by centrifugation. 10 μl of the cellular extract or extract diluted 10× was analyzed for luciferase activity using the Enhanced Luciferase Assay kit (Mirus Bio Corporation, Madison, Wis.).
Co-injection of 10 μg pMIR48 and 0.5 μg siRNA-luc+ resulted in 69% inhibition of Luc+ activity as compared to injection of 10 μg pMIR48 alone. Co-injection of 5 μg siRNA-luc+ with 10 μg pMIR48 resulted in 93% inhibition of Luc+ activity.

Example 2

Inhibition of gene expression by siRNA in vivo is gene specific. Two plasmids were injected simultaneously either with or without siRNA-luc+ as described in Example 1. The first plasmid, pGL3 control (Promega Corp, Madison, Wis.), contained the luc+ coding region and a chimeric intron under transcriptional control of the simian virus 40 enhancer and early promoter region. The second, pRL-SV40, contained the coding region for the Renilla reniformis luciferase under transcriptional control of the Simian virus 40 enhancer and early promoter region.
10 μg pGL3 control and 1 μg pRL-SV40 was injected as described in Example 1 with 0, 0.5 or 5.0 μg siRNA-luc+. One day after injection, the livers were harvested and homogenized as described in Example 1. Luc+ and Renilla Luc activities were assayed using the Dual Luciferase Reporter Assay System (Promega). Ratios of Luc+ to Renilla Luc were normalized to the no siRNA-Luc+ control. siRNA-luc+ specifically inhibited the target Luc+ expression 73% at 0.5 μg co-injected siRNA-luc+ and 82% at 5.0 μg co-injected siRNA-luc+.

Example 3

Inhibition of gene expression in vivo by siRNA is gene and siRNA specific. 10 μg pGL3 control and 1 μg pRL-SV40 were injected as described in Example 1 with either 5.0 μg siRNA-luc+ or 5.0 μg control siRNA-ori. One day after injection, the livers were harvested and homogenized as described in Example 1. Luc+ and Renilla Luc activities were assayed using the Dual Luciferase Reporter Assay System (Promega). Ratios of Luc+ to Renilla Luc were normalized to the siRNA-ori control. siRNA-Luc+ inhibited Luc+ expression in liver by 93% compared to siRNA-ori indicating inhibition by siRNAs is sequence specific.

Example 4

Functional delivery of siRNA to liver cells via bile duct injection. 10 μg pGL3 control and 1 μg pRL-SV40 with 5.0 μg siRNA-luc+ or 5.0 μg siRNA-ori were injected into the bile duct of mice. A total volume of 1 ml in Ringer's buffer was delivered at 6 ml/min. The inferior vena cava was clamped above and below the liver before injection and clamps were left on for two minutes after injection. One day after injection, the liver was harvested and homogenized as described in Example 1. Luc+ and Renilla Luc activities were assayed using the Dual Luciferase Reporter Assay System (Promega). Ratios of Luc+ to Renilla Luc were normalized to the siRNA-ori control. siRNA-Luc+ inhibited Luc+ expression in liver by 88% compared to the control siRNA-ori.

Example 5

Delivery of siRNA to liver cells in vivo for inhibition of SEAP gene expression. Single-stranded, SEAP-specific sense and antisense RNA oligomers with overhanging 3′ deoxyribonucleotides were prepared and purified by PAGE. The two oligomers, 40 μM each, were annealed in 250 μl buffer containing 50 mM Tris-HCl, pH 8.0 and 100 mM NaCl, by heating to 94° C. for 2 min, cooling to 90° C. for 1 min, then cooling to 20° C. at a rate of 1° C. per min. The resulting siRNA was stored at −20° C. prior to use.
The sense and antisense oligomer with identity to positions 362-380 of the SEAP reading frame had the sequence: 5′-rArGrGrGrCrArArCrUrUrCrCrArGrArCrCrArUTT-3′ (SEQ ID NO. 7) and 5′-rArUrGrGrUrCrUrGrGrArArGrUrUrGrCrCrCrUTT-3′(SEQ ID NO. 8), respectively. The letter “r” preceding a nucleotide indicates that nucleotide is a ribonucleotide. The annealed oligomers containing SEAP coding sequence are referred to as siRNA-SEAP.
Plasmid pMIR141 (10 μg), containing the SEAP coding region under transcriptional control of the human ubiquitin C promoter and the human hepatic control region of the apolipoprotein E gene cluster, was mixed with 0.5 or 5 μg siRNA-SEAP or 5 μg siRNA-ori, diluted in 1-3 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂), and injected into the tail vein over 7-12 seconds. Control mice also included those injected with pMIR141 alone. Each mouse was bled from the retro-orbital sinus one day after injection. Cells and clotting factors were pelleted from the blood to obtain serum. The serum was then evaluated for the presence of SEAP by a chemiluminescence assay using the Tropix Phospha-Light kit. Results showed that SEAP expression was inhibited by 59% when 0.5 μg siRNA-SEAP was delivered and 83% when 5.0 μg siRNA-SEAP was delivered (Table 1). No decrease in SEAP expression was observed when 5.0 μg siRNA-ori was delivered indicating the decrease in SEAP expression by siRNA-SEAP was gene specific.

TABLE 1

Inhibition of SEAP expression in vivo following delivery by tail

vain injection of SEAP expression plasmid and siRNA-SEAP.

injection Ave. SEAP (ng/ml) St. Dev.

plasmid only 2239 1400

siRNA-ori (5.0 μg) 2897 1384

siRNA-SEAP (0.5 μg) 918 650

siRNA-SEAP (5.0 μg) 384 160

Example 6

Intravascular delivery of naked PMOs to hepatocytes by increased pressure tail vein injection. Injection of naked plasmid DNA into the tail vein of mice has been shown to be an efficient method for hepatocyte transfection in vivo [Zhang et al. 1999; Liu et al. 1999]. We tested this method for the ability to deliver PMOs to the liver. Briefly, 30 μg of fluorescein labeled PMO or PMO:ODN hybrid duplexes in 1-2.5 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) were injected into the tail vein of mice in 4-5 sec using a 27 gauge needle. 24 h after injection, the animals were sacrificed and the livers harvested. Tissue was immediately placed in embedding compound and snap frozen in liquid nitrogen. Cryosections (10 μm) were fixed for 15 min in 3.6% formaldehyde in PBS, pH 7.4, rinsed 3×5 minutes in PBS and then counterstained for 20 minutes with a 1:40,000 dilution of the nucleic acid stain, ToPro-3 (Molecular Probes). After rinsing with PBS, the sections were mounted in VectaShield (Vector Laboratories, Inc.) and examined using a Zeiss LSM 510 confocal microscope.
Using this technique, approximately 10% of hepatocytes were observed to contain PMO (FIG. 2). In contrast, we observed no PMO uptake by liver cells following tail vein injection using 250 μl volume (data not shown). No difference in the extent of PMO uptake was observed when the PMO was delivered alone or in PMO:DNA hybrid duplexes. The intracellular distribution was diffuse cytoplasmic accompanied by accumulation in the nucleus. These results indicate that tail vein injections can be used to deliver PMOs to hepatocytes when a sufficient volume is injected at a sufficient rate. More importantly, using this method, PMOs are delivered to the cytoplasmic and nuclear compartments, the cellular locations of PMO action.

Example 7

Inhibition of gene expression in liver hepatocytes following intravascular delivery of PMOs. The PMO, mCD26-1, was designed to base pair to positions −3 to +20 of the mouse CD26 coding sequence. Mice were injected into the tail vein as above with a 2.5 ml solution containing either 100 μg mCD26-1 or no oligonucleotide. Using histochemical staining, an overall decrease in CD26 activity could be observed in animals receiving the mCD26-1 antisense PMO compared (FIG. 3). Some areas of each section appeared to contain higher levels of CD26 gene expression inhibition than others and may reflect the uneven distribution of the PMOs after injection using this method.
Animals were sacrificed and their livers harvested. Tissues were immediately placed in embedding compound and snap frozen in liquid nitrogen. Cryosections (10 μm) were fixed for 15 min in 3.6% formaldehyde in PBS, pH 7.4 at RT and then rinsed 3×5 minutes in PBS. The fixed cryosections will be placed in blocking buffer (PBS, 1% BSA) for 1 h at RT. The cryosections were be incubated in a 1:1000 dilution of the goat anti-CD26 primary antibody (Research Diagnostics) in blocking buffer at 40° C. overnight. After 3×20 min washes in blocking buffer, the cryosections were incubated in a 1:400 dilution of Cy3™ conjugated donkey anti-goat F(ab′)2 fragments (Jackson Laboratories) and 1:40,000 dilution of the nuclear stain ToPro-3 (Molecular Probes) in blocking buffer for 2 h at RT. After rinsing with PBS, the sections were mounted in VectaShield (Vector Laboratories, Inc.) and examined using a Zeiss LSM 510 confocal microscope.
Similarly, intravascular tail vein injection was used to co-deliver a firefly luciferase expression plasmid (pGL3) and an expression plasmid containing the unrelated Renilla luciferase (pRL-SV40) along with a PMO designed to base pair to positions −3 to +22 of the firefly luciferase coding region (Luc-1 PMO), a control PMO or no PMO. The Renilla luciferase served as an internal control to normalize for plasmid delivery efficiency. 24 h after injection, the activities of both luciferase enzymes in liver homogenates were assayed. The ratio of firefly luciferase activity to Renilla luciferase activity was then compared with the ration obtained in mice injected with solution containing plasmids only. Results are shown in FIG. 4. In mice are co-injected with increasing amounts of luc-1 PMO, increasing amounts of firefly luciferase expression inhibition was observed. The greatest inhibition, 83%, was observed in mice receiving 50 μg luc-1 PMO. No inhibition was observed in mice receiving the control PMO. We have thus demonstrated gene-specific inhibition of expression following intravascular delivery of PMOs using the describe technique.

Example 8

Functional delivery of siRNA to limb skeletal muscle cells in vivo via hydrodynamic intra-iliac injection. 10 μg pGL3 control and 1 μg pRL-SV40 with 5.0 μg siRNA-luc+ or 5.0 siRNA-ori were injected into iliac artery of rats under increased pressure. The iliac artery is an afferent vessel of the limb skeletal muscle. Specifically, animals were anesthetized and the surgical field shaved and prepped with an antiseptic. Animals were placed on a heating pad to prevent loss of body heat during the surgical procedure. A midline abdominal incision will 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) was injected into the external iliac artery though a 25 g needle, followed by the plasmid DNA and siRNA containing solution (in 10 ml saline) 1-10 minutes later. The solution 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.
Four days after injection, rats were sacrificed and the quadriceps and gastrocnemius muscles were harvested and homogenized as described in Example 1. Luc+ and Renilla Luc activities were assayed using the Dual Luciferase Reporter Assay System (Promega). Ratios of Luc+ to Renilla Luc were normalized to the siRNA-ori control. siRNA-Luc+ inhibited Luc+ expression in quadriceps and gastrocnemius by 85% and 92%, respectively, compared to the control siRNA-ori. Thus siRNA was effectively delivered to muscle cells in the leg using the delivery procedure. The placement of a tourniquet or cuff around the limb to prevent injection fluid from rabidly escaping the limb can be used in place of the clamps on individual vessels. Further, effective delivery is also attained without the presence of the vascular permeability agent, papaverine.

Example 9

Hydrodynamic limb vein delivery of siRNAs to limb skeletal muscle cells in rat and primate. To deliver siRNA to extravascular limb cells to achieve RNA interference in myofibers in vivo, siRNAs (targeted against firefly luciferase) were co-injected with pDNA encoding firefly luciferase (pCI-Luc-K) into the great saphenous vein of C57B1/6 mice, Sprague-Dawley rats and a rhesus macaque. At 2 days post-injection, greater than 95% inhibition of the targeted gene was achieved in the limbs that received the siRNA encoding the firefly luciferase in both species.
For delivery of siRNA to rat limb muscle cells, 150 g Sprague Dawley rats were co-injected into the great saphenous vein with 250 μg of a pDNA encoding firefly luciferase (pSP-luc+, Promega) and 25 μg of a pDNA (pRL-SV40, Promega) encoding Renilla reniformis luciferase. Injections were performed using 3 mls injection volume as described above. One group of animals (n=5) received plasmids alone, one group (n=5) received plasmids plus 12.5 jig of a siRNA targeted against firefly luciferase (siRNA-luc+) and a control group (n=5) received plasmids plus 12.5 μg of a siRNA targeted against enhanced green fluorescent protein (siRNA-EGFP, Clontech). Muscle was harvested 72 hours after injection.

Expression levels were measured by preparing homogenates and measuring activity of the firefly luciferase and the renilla luciferase using the dual luciferase assay kit (Promega). The mean expression levels (from all harvested muscle groups) in animals receiving the siRNA targeted against firefly luciferase was normalized to those animals receiving the control siRNA (EGFP). Animal receiving siRNA against firefly luciferase showed ˜60 fold reduction in firefly luciferase expression relative to Renilla luciferase expression (Table 2).

TABLE 2


Inhibition of gene expression by siRNA delivered to muscle cells

Muscle Group

	quad	biceps	hamstring	gastroc.	shin	total

no siRNA
average firefly	2,331,015	2,197,626	5,701,719	6,368,653	648,859	17,247,871
luciferase expression
average Renilla	102,322	98,349	242,450	319,224	31,129	793,474
luciferase expression
average ratio	23.4 ± 4.5	22.3 ± 4.0	23.8 ± 3.4	22.2 ± 4.7	21.3 ± 1.7	22.8 ± 3.1
(firefly/Renilla)
control siRNA
average firefly	692,220	2,317,722	4,767,100	5,296,748	514,189	12,425,792
luciferase expression
average Renilla	25,566	105,572	188,049	252,630	24,196	540,647
luciferase expression
average ratio	25.6 ± 6.5	24.1 ± 3.7	26.3 ± 5.2	22.2 ± 3.5	21.3 ± 0.9	24.3 ± 3.9
(firefly/Renilla)
siRNA
average firefly	44,754	103,421	105,719	223,126	54,779	531,799
luciferase expression
average Renilla	115,517	292,509	300,648	521,484	104,106	1,334,265
luciferase expression
average ratio	0.46 ± 0.20	0.37 ± 0.04	0.35 ± 0.05	0.44 ± 0.04	0.49 ± 0.09	0.40 ± 0.03
(firefly/Renilla)

Delivery of siRNA to primate limb_muscle cells. One front limb of a rhesus macaque was injected via the cephalic vein with 40 ml of saline containing 10 mg of a pDNA encoding firefly luciferase (pCI-Luc-K), 2.2 mg of a pCMV-Renilla encoding Renilla reniformis (sea pansy) luciferase and 750 μg of a siRNA targeted against firefly luciferase (siRNA-luc+). The opposite lower -hind limb was injected on the same day via the great saphenous vein with 50 ml of saline containing the same plasmids plus 750 μg of a siRNA targeted against enhanced green fluorescent protein (siRNA-EGFP). 96 hours after injection, animals were euthanized and muscles were harvested. Expression levels were measured with the same technique described in the rat studies. Data was normalized to values obtained for the control siRNA (EGFP). Co-delivery of a plasmid containing an expressible reporter gene was used as a convenient method to quantitatively assay delivery of the siRNA. The invention does not require co-delivery of a plasmid for delivery of siRNA and absence of plasmid DNA in the injection solution will not effect siRNA delivery. For all muscle groups of the forearm (palmaris longus, pronator teres, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profuindus, pronator quadratus, brachioradialis, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, anconeus, extensor carpi ulnanis, supinator, abductor pollicis longus, ext. digiti secund et teriti, extensor digiti quart et minimi, muscles of the thumb, interosseus, other, muscles of the hand), the ratio of firefly luciferase espression to Renilla luciferase expression was 0.019±0.015. For all muscle groups of the lower hind limb (gastrocnemius medial, gastrocnemius lateral, soleus, popliteus, flexor digitorum longus, flexor hallucis longus, tibialis posterior, tibialis anterior, extensor hallucis longus, extensor digitorum longus, abductor hal lucis longus, peronaus longus, peronaus brevis, extensor di gitorum brevis, extensor hallucis brevis, other muscles of the foot), the ratio of firefly luciferase espression to Renilla luciferase expression was 0.448±0.155. Muscles receiving the firefly specific siRNA showed 23.6 fold lower expression of firefly luciferase relative to Renilla luciferase.

Example 10

Multiple (repeat) injections: A Sprague-Dawley rat was injected intravenously three-times with 500 μg of pCI-LacZ on days 0, 4, and 8 and muscles were harvested on day 10. Injections were performed, via catheterization, on days 0, 4, and 8 at different sites: lateral plantar vein, small saphenous, and great saphenous respectively. For each injection, all volumes and amounts injected were as described as above. β-galactosidase staining was performed as described above. Additional injections resulted in significantly higher percentages of cells expressing the transgene (FIG. 5). In the gastrocnemius of the rat limb that was thrice injected, β-galactosidase expression was observed in about 60-80% of the cells in high-expressing areas (FIG. 5). β-galactosidase enzyme assays on the individual muscle groups correlated the histochemical analyses, 52,959,500 RLUs in the gastrocnemius muscle and 11,894,700 RLUs in the shin muscle.

Example 11

Functional delivery of siRNA to cardiac muscle cells in vivo by hydrodynamic intravascular injection to inhibit gene expression. Animal #1 and #2—Pigs were injected with plasmids and siRNA known to inhibit expression of the gene expressed from the plasmid. The injection solution was prepared by adding 100 μg/ml each of plasmids encoding either Firefly luciferase or Renilla luciferase genes as well as 45 μg/ml of Firefly-specific siRNA, siRNA-luc+. Control animals were injected with plasmid only (no siRNA present). The injection solution was saline with 2.5 mg/ml lidocaine. The injection volume was 20 ml and the rate of injection was 5.0 ml/second. Injection was done by retrograde instillation into the left anterior descending vein using a balloon catheter. The animals were sacrificed 48 h after injection and the heart was excised. Tissue specimens (approximately 1 gram each) were obtained from the heart tissue perfused by the left anterior descending vessel vasculature (i.e. from the muscle surrounding the left anterior descending artery and vein). Specimens were frozen in liquid nitrogen and stored at −80° C. Expression levels were measured by preparing homogenates and measuring activity of the firefly luciferase⁺ and the renilla luciferase using a commercial available assay kit (Promega). Data is expressed as a ratio fireflyluc⁺ /renillaluc.
Animal #3 and #4—This animal was injected with plasmids only. The injection solution was prepared by adding 100 μg/ml each of Fireflyluc⁺ and Renillaluc to a saline solution which also contained 2.5 mg/ml of lidocaine. The injection volume was 12.5-16 ml and the rate of injection was 4.5-6 ml/second. Injection was done by retrograde instillation into the left anterior descending vein using a balloon catheter. The animal was sacrificed 48 h after injection and the heart was excised. Tissue specimens (approximately 1 gram each) were obtained from the heart tissue perfuised by the left anterior descending vessel vasculature (i.e. from the muscle surrounding the left anterior descending artery and vein). Specimens were frozen in liquid nitrogen and stored at −80° C. Expression levels were measured by preparing homogenates and measuring activity of firefly luciferase⁺ and the renilla luciferase using a commercial available assay kit (Promega). Data is expressed as a ratio fireflyluc⁺ /renillaluc.

The data, shown in Table 3 and Table 4, show that siRNA was efficiently delivered to heart muscle cells near the left anterior descending artery and vein vasculature following retrograde injection of a solution containing the siRNA into the left anterior descending vein. In animals receiving siRNA, firefly luciferase expression was reduced 70-90% (ratio of firefly luciferase to Renilla luciferase in control vs. siRNA animals). Co-delivery of siRNA with a plasmid containing an expressible reporter gene was used as a convenient method to quantitatively assay delivery of the siRNA. The invention does not require co-delivery of a plasmid for delivery of siRNA. The absence of plasmid DNA in the injection solution does not effect siRNA delivery. Each value for animal #4 represents the average of two samples.

TABLE 3


Delivery of siRNA to cardiac muscle cells via retrograde
instillation into the left anterior descending vein

Luciferase Activity (RLUs)

Firefly/

	Renilla	Firefly	Renilla

animal #

1	2,606,023	45,255	0.017
		1,865,868	41,435	0.022
		1,009,623	25,600	0.025
		123,958	6,385	0.052
		293,653	755	0.003
		23,148	320	0.014
		76,803	945	0.012
	average	857,010	17,242	0.020
	total	5,999,073	120,695
	animal #2	183,465	21,350	0.12
		990,065	38,630	0.039
		120,695	9,580	0.079
		7,505	2,265	0.30
		39,650	11,380	0.29
		108,645	5,325	0.049
		15,635	1,465	0.094
		15,155	1,055	0.070
		12,585	2,145	0.17
		16,175	1,275	0.079
		170,635	8,630	0.051
		13,840	2,580	0.19
	average	141,171	8,807	0.062
	total	1,694,050	105,680

TABLE 4


Renilla and Firefly luciferase expression in control
animals not receiving firefly-specific siRNA.

Luciferase Activity (RLUs)

Firefly/

	Renilla	Firefly	Renilla

animal #3	75,923	304,450	4.01
no siRNA control	501,513	1,033,850	2.06
	199,222	472,570	2.37
	9,283	19,580	2.11
	12,202	7,460	0.61
	316,663	603,570	1.91
	90,412	103,980	1.15
	628,963	1,150,540	1.83
	118,152	211,310	1.79
	73,843	83,680	1.13
	9,133	10,220	1.12
	16,682	16,980	1.02
	22,112	40,930	1.85
	142,813	226,130	1.58
	120,072	263,610	2.20
	19,272	39,420	2.05
	41,033	72,440	1.77
average	141,017	274,160	1.94
total	2,397,293	4,660,720
animal #4	15656	33905	2.21
no siRNA control	20501	55155	2.69
	34148	104293	3.04
	50838	154645	2.99
	979421	3005915	2.89
	4127548	13391003	3.23
	5773616	18548940	3.20
	7232438	25413183	3.50
	9061823	32462075	3.59
	17620838	68046103	3.88
average	4491683	16121522	3.12
total	44916825	161215215

Example 12

In vivo luciferase expression from naked plasmid DNA transfected to a pig heart: Solutions of pCI-Luc⁺ were injected into coronary arteries and veins in pig heart. pCI-Luc⁺ is a plasmid DNA expression vector in which an optimized version of the firefly luciferase gene (Promega, Madison, Wis.) is expressed under transcriptional control of the
CMV promoter (basic expression vector is pCI, Promega, Madison, Wis.). The hearts of 30-40 kg domestic pigs were accessed via a limited left thoracotomy through the fifth intercostal space. A 27-gauge needle was inserted into a left anterior descending (LAD, great cardiac) or right posterior descending (middle cardiac) vein or artery, and ligated in place. The ligation serves to keep the needle in place and to direct flow distal from the needle. The corresponding artery or vein was transiently occluded during the injection. A pre-injection into the coronary artery or vein of 6 ml papaverine solution (0.5 mg/ml) was given in 15-20 seconds. After 5 minutes, a 30 ml solution of 50 μg/ml plasmid DNA in saline with 15% mannitol (w/v) was injected in ˜20-30 seconds. Following injection, the ligation and needle were removed, bleeding stopped, and the pericardium and chest closed. In most pigs, the LAD bed and a site in the circumflex were injected. In two pigs, direct interstitial injections were also performed for comparison.
48 h after injection, the animals were sacrificed and sections from the injection site were excised and assayed for reporter gene expression. Sections from the heart (ca. 1.5 gram each) were homogenized in a Triton X-100 lysis buffer. Luciferase activity was measured with an Analytical Luminescence Laboratories luminometer. Activity levels are expressed as the amount of luciferase protein per gram of heart tissue. Plasmid DNA was obtained from BayouBioLabs (Harahan, La.) and was supercoiled purified and endotoxin free.
Luciferase expression in the area around the injection site averaged 26.2 ng/g tissue (range 2.3-61.8; n=5). Both arterial and venous delivery resulted in efficient luciferase expression. In one animal, we compared intravenous delivery while transiently occluding the corresponding artery with leaving arterial flow open. Luciferase expression levels were 7.22 vs. 7.76 ng/g, respectively. This suggests that the capillary bed itself accounts for sufficient resistance to retrograde flow to increase vascular permeability above the required threshold for efficient plasmid DNA extravasation.
Direct interstitial injection of 500 μg plasmid DNA in 500 μl saline resulted in an average expression level of 70.3 ng luciferase per gram tissue (range 9.6-115.2; n=3). Expression appeared far more limited to the area of injection. Analysis of tissues around the injected bed after intravascular delivery, showed lower levels of expression extending to relatively distant sites. All of the pigs injected recovered well from the procedure, and little damage was observed. Tissue sections from the injection site and stained with hematoxylin-eosin did not show any major histological abnormalities.

Example 13

Retrograde cardiac venous gene delivery. Pigs (30-50 kg) of either sex were anesthetized with Telazol (tiletamine HCl and zolazepam HCl, 4-5 mg/kg IM) and thiopental (7-9 mg/kg IV), intubated and mechanically ventilated. Anesthesia was maintained with inhaled isoflurane (0.5%-1.5%). The right carotid artery was exposed via a midline incision and cannulated with an 8-9 Fr introducer sheath. A 10.5-11 Fr sheath was placed in the right jugular vein. The left main coronary artery was engaged with a coronary angiographic catheter and a left coronary artery angiogram was obtained. A modified 10 Fr guiding catheter was advanced through the jugular sheath into the coronary sinus. The delivery catheter (modified triple-lumen 7 Fr balloon-tipped pulmonary artery catheter with an infusion lumen, a balloon inflation/deflation lumen and an end-hole lumen for intravascular pressure measurements) was advanced over a guidewire into the proximal great (anterior) cardiac vein. The vein was occluded by inflating the latex balloon on the distal tip of the catheter. Low pressure injections of diluted iodinated contrast were used, in conjunction with the left coronary angiogram, to delineate the myocardial territory drained by the vein. Retrograde infusions of 15-30 ml of pDNA solution were performed in 17 pigs using an injection pump (MedRad, Inc., Pittsburgh, Pa.) at constant flow rates (3-7 mf/sec). In injections resulted in a rapid rise in coronary intravenous pressure to a mean injection pressure of 804±242 mmHg (range 515-1225 mm Hg). No ventricular tachycardia was observed during the 11 experiments were the mean injection pressure was less than 1000 mm Hg. The occlusion balloon was left inflated for 2 min following the injection. Following gene delivery, both vessels were ligated and the neck incision was closed. In some animals, 6 ml of a 0.5 mg/ml papaverine solutions was injected 5 minutes prior to plasmid DNA delivery.
Total luciferase expression in the heart 48 hours after transfection with this method was 673±587 ng (range 18 to 2,124 ng), with peak expression of luciferase in the anterior wall of 118±113 ng luciferase per gram heart tissue (range 4.4-455 ng/g). Luciferase expression was typically observed in the entire anterior and anteroseptal walls of the left ventricle. For the 8 experiments where the mean intravenous injection pressure was between 500 and 700 mm Hg, total luciferase expression was 624±663 ng (range 18-2024 ng).
For comparison, direct intramyocardial injections were performed. For these injections, the heart was exposed via a fourth interspace left thoracotomy and 0.5 ml of pDNA solution was directly injected in the lateral wall of the left ventricle using a 25 gauge needle. Following pDNA injections, the pericardium was closed, the ribs approximated, and the muscles and skin were closed by individual layers. Direct intramyocardial injection of 500 μg pCI-Luc⁺ at 5 sites in 3 separate pigs resulted in a total luciferase expression of 16.8±8.4 ng in the myocardium at each injection site, with peak expression of 9.6±8.4 ng/g myocardium. Luciferase expression was limited to a relatively small area of myocardium immediately adjacent to the injection site.

Example 14

Inhibition of green fluorescent protein expression in transgenic mice. The commercially available mouse strain C57BL/6-TgN(ACTbEGFP)10sb (The Jackson Laboratory) has been reported to express enhanced green fluorescent protein (EGFP) in all cell types except erythrocytes and hair. These mice were injected with siRNA targeted against EGFP (siRNA-EGFP) or a control siRNA (siRNA-control) as in example 1.30 h post-injection, the animals were sacrificed and sections of the liver were prepared for fluorescence microscopy. Liver sections from animals injected with 50 μg siRNA-EGFP displayed a substantial decrease in the number of cells expressing EGFP compared to animals injected with siRNA-control or mock injected (FIG. 6). Thus, the described delivery procedure is effective for delivery of oligonucleotides to extravascular cells in vivo to inhibit expression of endogenously expressed genes.

Example 15

Inhibition of a naturally occurring endogenous gene, cytosolic alanine aminotransferase (ALT). Single-stranded, cytosolic alanine aminotransferase-specific sense and antisense RNA oligomers with overhanging 3′ deoxyribonucleotides were prepared and purified by PAGE. The two oligomers, 40 μM each, were annealed in 250 μl buffer containing 50 mM Tris-HCl, pH 8.0 and 100 mM NaCl, by heating to 94° C. for 2 minutes, cooling to 90° C. for 1 minute, then cooling to 20° C. at a rate of 1° C. per minute. The resulting siRNA was stored at −20° C. prior to use. The sense and antisense oligomers with identity to positions 928-946 of the mouse and rat cytosolic alanine aminotransferase reading frame had the sequence: 5′-rCrArCrUrCrArGrUrCrUrCrUrArArGrGrGrCrUTT-3′ (SEQ ID NO. 9), and 5′-rArGrCrCrCrUrUrArGrArGrArCrUrGrArGrUrGTT-3′ (SEQ ID NO. 10), respectively. The letter “r” preceding a nucleotide indicates that nucleotide is a ribonucleotide. The annealed oligomers containing cytosolic alanine aminotransferase coding sequence are referred to as siRNA-ALT
Mice were injected into the tail vein as described in example 1. Control mice were injected with Ringer's solution without siRNA. Two days after injection, the livers were harvested and homogenized in 0.25 M sucrose. ALT activity was assayed using the Sigma diagnostics INFINITY ALT reagent according to the manufacturers instructions. Total protein was determined using the BioRad Protein Assay. Mice injected with 40 μg siRNA-ALT had an average decrease in ALT specific activity of 32% compared to mice injected with Ringer's solution alone.

Example. 16

Reduction of PPAR levels in vivo following hydrodynamic delivery of siRNA expression cassettes. PPARα, peroxisome proliferator-activated receptor α, is a transcription factor and a member of the nuclear hormone receptor superfamily. The gene, found in both mice and humans, plays an important role in the regulation of mammalian metabolism. In particular, PPARα is required for the normal maintenance of metabolic pathways whose misregulation can facilitate the development metabolic disorders such as hyperlipidemia and diabetes. When bound to its ligand, PPARα binds to the retinoid X receptor (RXR) and activates the transcription of genes implicated in maintaining homeostatic levels of serum lipids and glucose. The manipulation of PPARα levels using RNA interference may be a safe and effective way to modulate mammalian metabolism and treat pathogenic hyperlipidemia and diabetes. We used a tail vein injection procedure to delivery plasmid DNA encoding an siRNA expression cassette to modulate endogenous PPARα levels using RNA interference in mice. Our results provide a model for the therapeutic delivery of siRNAs synthesized in vivo from delivered plasmid DNA. This method, or variations thereof, will be generally useful in the modulation of the levels of an endogenous gene using RNA interference.
siRNA hairpin sequences: Initially, we identified a series of plasmid DNA-based siRNA hairpins that exhibited RNA activity against PPARα in primary cultured hepatocytes. The general hairpin structure consists of a polynucleotide sequence with sense and antisense target sequences flanking a micro-RNA hairpin loop structure. Transcription of the siRNA hairpin constructs was driven by the promoter from the human U6 gene. In addition, the end of the hairpin construct contains five T's to serve as an RNA Polymerase III termination sequence. The siRNA hairpin directed against PPARα had the sequence 5′-GGAGCTTTGGGAAGAGGAAGGTGTCATCcttcctgtcaGATGGCATCTTCCTCTTCCCGAAGCTCCTTTTT-3′ (SEQ ID NO. 11). Lower-case letters indicate the sequence of the hairpin loop motif. The entire hairpin construct encoding the PPARα siRNA (consisting of the U6 promoter, the PPARα siRNA hairpin, and the termination sequence) is referred to as pMIR303. The negative control siRNA hairpin directed against GL3 had the sequence 5′-GGATTCCAATTCAGCGGGAGCCACCTGATgaagcttgATCGGGTGGCTCTCGCTGAGTTGGAATCCATTTTT-3′ (SEQ ID NO. 12). The entire hairpin construct encoding the GL3 siRNA (consisting of the U6 promoter, the GL3 siRNA hairpin, and the termination sequence) is referred to as pMIR277.
Injections of mice: Ten mice in each experimental group were injected three times each with 40 μg/injection of either pMIR277 (GL3 siRNA construct) or pMIR303 (PPARα siRNA construct) using a tail vein injection procedure. Volumes of Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) corresponding to 10% of each animal's body weight and containing the 40 μg of pMIR277 or pMIR303 were injected into mice over a period of 10 seconds with each injection. For each animal, injection 1 was performed on Day 0, injection 2 was performed on Day 2, and injection 3 was performed on Day 4. Seven days after Injection 3 (Day 11), livers from all mice were harvested and total RNA was isolated using the Tri-Reagent protocol.
Isolation of total RNA and cDNA synthesis: Total mRNA from injected mouse livers was isolated using Tri-Reagent. 500 ng of ethanol precipitated, total RNA suspended in RNase-free water was used to synthesize the first strand cDNA using SuperScript III reverse transcriptase. cDNAs were then diluted 1:50 and analyzed by quantitative, real-time qPCR. Quantitative, real-time PCR: Bio-Rad's iCycler quantitative qPCR system was used to analyze the amplification of PPARα and GAPDH amplicons in real time. The intercalating agent SYBR Green was used to monitor the levels of the amplicons. Primer sequences used to amplify PPARα sequences were 5′-TCGGGATGTCACACAATGC-3′ (SEQ ID NO. 13) and 5′-AGGCTTCGTGGATTCTCTTG-3′ (SEQ ID NO. 14). Primer sequences used to amplify GAPDH sequences were 5′-CCTCTATATCCGTTTCCAGTC-3′ (SEQ ID NO. 15) and 5′-TTGTCGGTGCAATAGTTCC-3′ (SEQ ID NO. 16). Serial dilutions (1:20, 1:100 and 1:500) of cDNA made from Ringer's control samples were used to create the standard curve from which mRNA levels were determined. PPARα levels were quantitated relative to both GAPDH mRNA and total input RNA.
Results: Mouse livers injected with the PPARα hairpin constructs contained 50% or 35% less PPARα mRNA than those injected with GL3 siRNA control hairpins when compared to GAPDH mRNA or total input RNA, respectively. FIG. 7 shows the relative levels of PPARα mRNA as compared to GAPDH mRNA or total input RNA in each 10-mouse group. The experimental error is expressed as the total standard deviation among all samples. That this delivery procedure is able to achieve up to 50% knockdown of an endogenous target transcript demonstrates its general utility for in vivo modulation of gene expression.

Example 17

Reduction of serum triglyceride levels in vivo following delivery of HMG CoA reductase siRNA. We have demonstrated a reduction of serum triglyceride levels in mice upon treatment with siRNA directed against HMG CoA reductase. Group A (series2) mice (5 mice) were each injected with 50 μg of an siRNA directed against mouse HMG CoA reductase mRNA. Group B (Seriesl) mice (5 mice) were an uninjected control group. Group A and Group B animals were bled 7 days before, 2 days after, 4 days after, and 7 days after the injection. Serum samples were stored at −20° C. until all time points had been collected. Each group's serum samples from a given time-point were pooled prior to the triglyceride assays. Triglyceride assays were performed in quintuplicate.
Mice: Experiments were performed in Apoetm1Unc mice obtained from The Jackson Laboratories (Bar Harbor, Me.). Mice homozygous for the Apoetm1Unc mutation show a marked increase in total plasma cholesterol levels that is unaffected by age or sex. Fatty streaks in the proximal aorta are found at 3 months of age. The lesions increase with age and progress to lesions with less lipid but more elongated cells, typical of a more advanced stage of pre-atherosclerotic lesion. Moderately increased triglyceride levels have been reported in mice with this mutation on a mixed C57BL/6×129 genetic background.
siRNA reagents: Single-stranded, HMG CoA reductase-specific sense and antisense RNA oligomers with overhanging 3′ deoxyribonucleotides were ordered from Dharmacon, Inc. The annealed RNA duplex was resuspended in Buffer A (20 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl₂) and stored at −20° C. prior to use. Prior to injection, siRNAs were diluted to the desired concentration (50 μg/2.2 ml) in Ringer's solution.
Oligonucleotide sequences: The sense and antisense oligomers with identity to positions 2324-2344 of the HMG CoA reductase reading frame had the sequence: 5′-rArCrArUrUrGrUrCrArCrUrGrCrUrArUrCrUrATT-3′ (SEQ ID NO. 17) and 5′-rUrArGrArUrArGrCrArGrUrGrArCrArArUrGrUTT-3′ (SEQ ID NO. 18), respectively. The letter “r” preceding a nucleotide indicates that nucleotide is a ribonucleotide. The annealed oligomers are referred to as siRNA-HMGCR.
A total of 50 μg of siRNA-HMGCR was dissolved in 2.2 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂), and injected into the tail vein of ApoE (−/−) mice over 7-12 seconds. Control mice were not injected and are referred to here as naive. Each mouse was bled from the retro-orbital sinus at various times prior to and after injection. Cells and clotting factors were pelleted from the blood to obtain serum. The serum triglyceride levels were then assayed by a enzymatic, colorimetric assay using the Infinity Triglyceride Reagent (Sigma Co.). Results showed that triglyceride levels in siRNA-HMGCR treated mice (Series2) were reduced 62% after two days, 56% after two days, and returned to normal levels after 7 days. No decrease in serum triglyceride levels was observed in uninjected mice (Series1).

Triglyceride assays: Serum samples were diluted 1:100 in the Infinity Triglyceride Reagent (2 μl in 200 μl) in a clear, 96-well plate. Each assay plate was then incubated at 37° C. for five minutes, removed and allowed to cool to room temperature. Absorbance was measured at 520 nm using a SpectraMax Plus plate reader (Molecular Devices, Inc). Background absorbance (no serum added) was subtracted from each reading and the resulted data was plotted versus timepoint (Table 5).

TABLE 5


Triglyceride levels in animals following
delivery of HMGCR-specific siRNA

Triglyceride levels

		siRNA-HMGCR
day	No Injection Control	Treated Animals

−7	0.181 ± 0.010	0.218 ± 0.008
+2	0.194 ± 0.011	0.082 ± 0.006
+4	0.175 ± 0.012	0.095 ± 0.005
+7	0.284 ± 0.021	0.189 ± 0.012

Example. 18

Delivery of siRNA to cells in vivo enhances statin effectiveness. Treatment with inhibitors of HMG CoA reductase, commonly known as statins, has been shown to markedly reduce the serum lipid levels of hyperlipidemia patients. Statins inhibit the activity of HMG-CoA reductase. In turn, this inhibition triggers a feedback mechanism through which the cellular levels of HMG-CoA reductase mRNA is markedly upregulated. Here, we present work that demonstrates a significant reduction in the levels of HMGCR mRNA in cells treated with atorvastatin. Addition of bioavailable siRNAs to the treatment regiments of patients on statins will lower the required statin dose, thereby reducing the required dosage of stains and cutting deleterious side effects.
Single-stranded, HMG CoA reductase-specific sense and antisense RNA oligomers (positions 2324-2344 of the HMG CoA reductase reading frame) with overhanging 3′ deoxyribonucleotides were ordered from Dharmacon, Inc (SEQ ID NO. 17 and SEQ ID NO. 18). The annealed RNA duplex, siRNA-HMGCR, was resuspended in Buffer A (20 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl₂) and stored at −20° C. prior to use. Prior to injection or transfection, siRNAs were diluted to the desired concentration (50 μg/2.2 ml) in Ringer's solution or (25 nM) in OPTI-MEM/TRANSIT-TKOTM (Mirus Bio Corp., Madison, Wis.), respectively. A total of 50 μg of siRNA-HMGCR was dissolved in 2.2 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂), and injected into the tail vein of mice over ˜7 seconds. Control mice were not injected and are referred to here as naive.
C57B6 mice treated for 48 hours with a 50 mg/kg dose of atorvastatin showed an expected and marked increase in HMG-CoA reductase mRNA levels as measured by quantitative, real-time PCR (FIG. 8A). Livers from groups of 10 mice were harvested 48 hours after treatment was commenced and pooled mRNA populations (10 mice/pool) were assayed for HMGCR levels. Mice treated with atorvastatin had, on average, an 800% increase in HMGCR mRNA.
Primary hepatocytes were isolated from C57B6 mice and cultured for 24 hours in the presence or absence of anti-HGMCR siRNAs and 10 μm atorvastatin. Total RNA from these cells was isolated and transcribed into cDNA using an oligo-dT primer and reverse transcriptase. Subsequently, HMGCR levels were assayed using quantitative, real-time PCR using the Bio-Rad iCycler system and iCycler reagents and primers (SEQ ID NO. 15 and SEQ ID NO. 16) to amplify HMGCR sequences. HMGCR mRNA levels were induced 400% relative to vehicle-treated cells after 24 hours of exposure to atorvastatin (FIG. 8B). Simultaneous administration of the anti-HMGCR siRNA along with the statin in vivo held HMGCR levels to those seen in vehicle-treated controls. In addition, treatment of hepatocytes with the HMGCR-directed siRNA alone resulted in the knockdown of HMGCR mRNA to approximately 20% of that seen in control cells. These results show that the simultaneous delivery of an siRNA against HMGCR to cells treated with an HMGCR inhibitor can reduce the relative level of HMGCR mRNA to wild type levels seen in control cells. This strategy should reduce the amount of drug needed to inhibit cellular HMGCR and potentially lower the dose of drug needed in target validation or therapeutic applications in this and other protein families.

Example 19

Combination therapy using statins and siRNA delivery for the treatment of hyperlipidemia. Initially, we identified a series of siRNAs that exhibited RNAi activity against PPARA in primary cultured hepatocytes. Having identified several highly active siRNAs, we selected one to use in our in vivo demonstration of siRNA delivery.
RNA oligonucleotides were ordered from Dharmacon, Inc. The siRNA duplex directed against PPARα contained the target sequence:

- 5′-rGrArTrCrGrGrArGrCrTrGrCrArArGrArTrTrC-3′ (SEQ ID NO. 19).

A control GL3 siRNA duplex contained the target sequence

- 5′-rArArCrUrUrArCrGrCrUrGrArGrUrArCrUrUrCrGrA-3′ (SEQ ID NO. 20).

An “r” before a base indicate that that oligonucleotides was a ribonucleotide. All siRNAs contained dTdT overhangs.
Four mice in each experimental group were injected with 50 μg of siRNA using the high-pressure tail vein procedure. A volume of Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) corresponding to 10% of each animal's body weight and containing 50 μg of PPARα siRNA (or control) were injected into mice over a period of ˜7-10 seconds. After 48 h, livers from injected mice were harvested and total RNA was isolated. Total mRNA from injected mouse livers was isolated using Tri-Reagent. 500 ng of ethanol precipitated, total RNA suspended in RNase-free water was used to synthesize the first strand cDNA using SuperScript III reverse transcriptase. cDNAs were then diluted 1:50 and analyzed by quantitative real-time qPCR using a Bio-Rad iCycler to determine PPARα and GAPDH levels. The intercalating agent SYBR Green was used to monitor the levels of the amplicons. Primer sequences used to amplify PPARα sequences were SEQ ID NO. 13 and SEQ ID NO. 14. Primer sequences used to amplify GAPDH sequences were SEQ ID NO. 15 and SEQ ID NO. 16. Primers used to amplify PTEN sequences were 5′-GGGAAGTAAGGACCAGAGAC-3′ (SEQ ID NO. 21) and 5′-ATCATCTTGTGAAACAGCAGTG-3′ (SEQ ID NO. 22). Serial dilutions (1:20, 1:100 and 1:500) of cDNA made from Ringer's control samples were used to create the standard curve from which mRNA levels were determined. Mouse livers injected with siRNAs directed against PPARα contained 17% or 37% less PPARα mRNA than Ringer's control or GL3 siRNA control animals, respectively. FIG. 9 shows the relative levels of PPARα mRNA as compared to total input RNA in each four-mouse group. The experimental error is expressed as the standard deviation of the mean.

Example 20

Combination treatment to reduce LDL-cholesterol levels in liver cells. Treatment with inhibitors of HMG-CoA Reductase, commonly known as statins, has been shown to markedly reduce serum LDL-cholesterol levels in hyperlipidemia patients. Statins inhibit the enzymatic activity of HMG-CoA Reductase. Inhibition of HMG-CoA Reductase causes decreased levels of cholesterol biosynthesis. To compensate for the reduced levels of cholesterol synthesis occurring in cells treated with statins, the low density lipoprotein receptor (LDLR) is upregulated through a specific, SREBP-dependent mechanism that senses the effective levels of cholesterol in cellular membranes. This upregulation of the LDLR results in increased cellular uptake of LDL-cholesterol and is one mechanism through which statins may exert their lipid-lowering effects. However, inhibition of cholesterol biosynthesis also triggers a feedback mechanism through which the cellular levels of HMG-CoA Reductase mRNA is markedly upregulated.
When HMG-CoA reductase activity drops below a certain threshold, the cell compensates by upregulating the LDL receptor, bringing cholesterol into the cell to replace the depleted endogenous stores. LDL receptor upregulation can be used as an indicator that HMG-CoA reductase activity had dropped below this threshold. We demonstrate that the levels of HMG-CoA reductase activity can be reduced by cotreatment with both statins and siRNA.
siRNA reagents. Single-stranded, HMG CoA reductase-specific sense and antisense RNA oligomers with overhanging 3′ deoxyribonucleotides were synthesized (Dharmacon, Inc). These single-stranded oligomers were annealed by stepwise cooling of a solution of the oligos from 96° C. to 15° C. The annealed RNA duplex was resuspended in Buffer A (20 mM KCl, 6 mM HEPES-KOH pH 7.5, 0.2 mM MgCl₂) and stored at −20° C. prior to use. Prior to transfection, siRNAs were diluted to the desired concentration (25 nM) in OPTI-MEM/TransIT-TKO (Mirus, Inc).
Oligonucleotide sequences. The antisense oligomer with identity to the murine HMG CoA reductase gene has the sequence: 5′-rCrCrArCrArArArUrGrArArGrArCrUrUrArUrATT-3′ (SEQ ID NO. 23), which corresponds to positions 2793-2812 of the HMG CoA reductase reading frame in the sense direction. The antisense oligomer with identity to the murine HMG CoA reductase gene has the sequence: 5′-rUrArUrArArGrUrCrUrUrCrArUrUrUrGrUrGrGTT-3′ (SEQ ID NO. 24), which corresponds to positions 2793-2812 of the HMG CoA reductase reading frame in the sense direction. The letter “r” preceding a nucleotide indicates that the nucleotide is a ribonucleotide. The annealed oligomers containing HMG CoA reductase coding sequence are referred to as siRNA-HMGCR.
Transfection and atorvastatin treatment of hepatocytes. Just prior to the addition of siRNA transfection cocktails (see below), fresh hepatocyte maintenance media supplemented with various concentrations of atorvastatin was added to each well in a 12-well, collagen coated plate that had been seeded with primary hepatocytes 24 hours previously. Then 100 μl of the siRNA transfection cocktail was added to each well. Hepatocyte maintenance media was a 1:1 mixture of DMEM-F 12/0.1% BSA/0.1% galactose.
siRNA transfection cocktail. Each 100 μl aliquot of siRNA transfection cocktail contained 3.8 μl TransIT-TKO, 275 nM siRNA, and the remaining volume of OPTI-MEM transfection media. The 100 μl aliquots were added to cells in 1 ml of media such that the final siRNA concentration was 25 nM.
RNA isolation. After 24 hours of siRNA transfection and atorvastatin treatment, cells were harvested in Tri-Reagent. RNA was isolated, quantitated, and corresponding cDNAs from an oligo-dT primer were synthesized with reverse transcriptase.
qPCR assays. Quantitative, real-time PCR was performed using the Bio-Rad iCycler system and icycler reagents as recommended by the manufacturer. The primers used to amplify LDLR sequences were 5′-GCATCAGCTTGGACAAGGTGT-3′ (SEQ ID NO. 25) and 5′-GGGAACAGCCACCATTGTTG-3′ (SEQ ID NO. 26).
Primary hepatocytes were isolated from C57BL6 mice and plated on collagen-coated 12-well plates. After allowing them to adhere to the plates for 24 hours, one of two different procedures was followed. In the first, cells were treated with 200 nM atorvastatin in DMSO or DMSO alone for 24 hours. In the second, cells were covered with 1 ml of hepatocyte maintenance media. Next, 100 μl of an siRNA (HMGCR or GL3 control) cocktail (see above) was added to each well such that the final concentration of atorvastatin was 200 nM, 100 nM, 50 nM, 25 nM, or 0 nM and the final concentration of siRNA was 25 nM. Cells were incubated in the atorvastatin/siRNA mixture for 24 hours. Following all 24-hour incubations, cells were harvested in Tri-Reagent and processed for qPCR as described above.
Induction of the LDL receptor in primary murine hepatocytes. Primary hepatocytes isolated by perfusion of C57BL6 mice and treated with 200 nM atorvastatin for 24 hours showed a marked increase in LDL receptor mRNA levels as measured by quantitative, real-time PCR (FIG. 10A). This result shows the expected upregulation of LDLR mRNA upon treatment with atorvastatin. Next, we treated isolated hepatocytes with a range of atorvastatin concentrations and measured the amount of LDLR mRNA in each sample. In addition, cells were transfected with siRNAs against HMG-CoA reductase or control siRNA against luciferase (GL3). In each case, atorvastatin triggered a dose-dependent increase in LDLR mRNA levels (FIG. 10B). Furthermore, addition of siRNA to the cells further increased LDLR levels. The data in FIG. 10B indicate that cells treated with HMGCR siRNAs required lower doses of atorvastatin to achieve a corresponding level of LDLR upregulation. For example, one can compare HMGCR siRNA-treated cells exposed to 25 nm or 50 nM statin with GL3 siRNA-treated cells exposed to 200 nM statin and see a similar level of LDLR mRNA was present in those cells. In addition, FIG. 10B indicates that simply reducing the amount of HMGCR in the cell results in an approximately 3-fold upregulation of LDLR mRNA (0 nM atorvastatin lanes). This shows that the HMGCR siRNA alone is effective in reducing cellular HMG-CoA reductase activity and thus increasing LDLR levels.
We used cells treated with GL3 siRNA and 0 nM atorvastatin as a baseline to compare the upregulation of LDLR mRNA in the other samples. The relative starting quantity of LDLR mRNA in each sample was plotted relative to the “baseline” LDLR mRNA level seen in GL3/no statin cells (FIG. 10C). The plot in FIG. 10C clearly shows that lower doses of atorvastatin were necessary to get comparable statin/no statin ratios in cells treated with HMGCR siRNAs.
In summary, we have demonstrated that siRNAs can be used to lower the effective dose of a small molecule inhibitor directed against the product of a gene targeted by the siRNA. This technology has applications in small molecule combination therapies as well as in drug discovery and research applications. For example, using siRNAs to decrease the gene dosage in cells being screened with small molecule libraries can sensitize cell-based assays and make otherwise difficult to detect cellular phenotypes apparent. The principle demonstrated here can be applied to situations in which the target of the small molecule and the siRNA are not the same. For example, a small molecule inhibitor of a protein required for the efflux of cellular cholesterol (e.g., ABCA1), coupled with an siRNA against HMGCR mRNA, could work together to lower the levels of total serum cholesterol. This would be expected to result in the upregulation of the LDL receptor and a corresponding increase in LDL-C uptake. In addition, G-protein coupled receptor (GPCR) mediated signaling pathways could be modulated by simultaneously treating cells with GPCR antagonists and siRNAs targeting the second messenger pathways within cells.

Example 21

Inhibition of Hepatitis B surface antigen (HBsAg) gene expression by delivery of siRNA in liver cells in vivo. The siRNAs used in this example were obtained from Dharmacon (Lafeyette, Colo.) and consisted of 21-nucleotide sense and antisense oligonucleotides each containing a two deoxynucleotide overhang at the 3′ end. The control siRNA targeted positions 155-173 of the luc+ coding sequence:

sense

(SEQ ID NO. 3)

5′-rCrUrUrArCrGrCrUrGrArGrUrArCrUrUrCrGrATT-3′,

antisense

(SEQ ID NO. 4)

5′-rUrCrGrArArGrUrArCrUrCrArGrCrGrUrArArGTT-3′.
HBsAg siRNA-1 targeted positions 177-195 of the HBsAg coding sequence:

sense

(SEQ ID NO. 27)

5′-rUrCrArCrUrCrArCrCrArArCrCrUrCrUrUrGrUTT-3′,

antisense

(SEQ ID NO. 28)

5′-rArCrArArGrArGrGrUrUrGrGrUrGrArGrUrGrATT-3′.
HBsAg siRNA-2 targets positions 392-410 of the HBsAg coding sequence:

sense:

(SEQ ID NO. 29)

5′-rCrCrUrCrUrArUrGrUrArUrCrCrCrUrCrCrUrGTT-3′;

antisense:

(SEQ ID NO. 30)

5′-rCrArGrGrArGrGrGrArUrArCrArUrArGrArGrGTT-3′.
The letter “r” preceding a nucleotide indicates that nucleotide is a ribonucleotide. Sense and antisense strands for each siRNA, 40 μM each, were annealed in 250 μl of buffer containing 50 mM Tris-HCl, pH 8.0 and 100 mM NaCl, by heating to 94° C. for 2 minutes, cooling to 90° C. for 1 minute, then cooling to 20° C. at a rate of 1° C. per minute. The resulting siRNA was stored at −20° C. prior to use.
Plasmid containing the HBsAg gene under the transcriptional control of the CMV enhancer promoter (pRc/CMV-HBs(S)) was obtained from Aldevron (Fargo, N.Dak.). Plasmid (pRc/CMV-HBs(S), 10 μg) was mixed with no siRNA, control siRNA (5 μg), 0.5 or 5 μg of HepB sAg siRNA-1, or 5 μg of HBsAg siRNA-2 and then diluted in 2 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) and injected into the tail vein of ICR mice (N=3) over 5-7 seconds. Serum was collected one day after injection. The amount of HBsAg in the serum was assayed by ELISA according to the manufacturer's instructions (Ortho) using purified HBsAg protein as the standard (Aldevron).
Co-injection of 10 μg pRc/CMV-HBs(S) and 0.5 μg HepB sAg siRNA-1 resulted in 22% inhibition of HBsAg expression compared to co-injection of 5 μg of the control siRNA (FIG. 11). Co-injection of 5 82 g of HepBsAg siRNA-1 resulted in 43% inhibition of HBsAg expression as compared to co-injection of 5 82 g of the control siRNA. Co-injection of 10 82 g pRc/CMV-HBs(S) and 5 82 g HBsAg siRNA-2 resulted in 65% inhibition of HBsAg expression compared to injection of the plasmid DNA alone (FIG. 12). These results indicate delivery of HBsAG-specific siRNA to cells in vivo can inhibit expression of HBsAg in the liver.

Example 22

siRNA -mediated inhibition of a virally expressed gene in mammalian cells. HeLa cells in culture were first infected with adenovirus containing the luciferase gene under control of the phosphoglycerol kinase (PGK) enhancer/promoter (Ad2PGKluciferase). Infection of HeLa cells with Ad2PGKluciferase resulted in expression of luciferase in these cells. After infection, siRNA targeted to the luciferase coding region or control siRNAs were delivered to the cells and the amount of luciferase activity was determined 24 h later.
HeLa cells were seeded to 50% confluency in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a T25 flask and incubated in a 5% CO₂humidified incubator at 37° C. 16 h later, cells were washed with PBS, trypsinized, harvested and resuspended in 13 ml DMEM/10% FBS. 500 μl of the cell suspension was distributed to each well in a 24 well plate. After 16 h incubation, the media in each well was replaced with 100 μl DMEM/10% FBS containing 5 μl Ad2PGKluciferase (2.5×10¹⁰particles/ml stock). After incubation for 2 h, 400 μl DMEM/10% FBS was added to each well followed by the addition of siRNA complexed with TRANSIT™-TKO (Mirus Bio Corporation, Madison, Wis.). For preparation of the siRNA complexes 7.5 μg TRANSIT-TKO was diluted in 50 μl serum-free Opti-MEM and incubated at room temperature for 5 minutes. siRNA was added to give a final concentration of siRNA per well of 0, 1, 10 or 100 nM and incubated for 5 minutes at RT. Complexes were then added directly to the wells. SiRNAs targeted to the either luciferase gene, the Luc⁺ gene, or an unrelated gene product were used (siRNA-Luc, siRNA-Luc⁺, and siRNA-c respectively). Only siRNA-Luc contained sequence identical to Ad2PGKluciferase. All assay points were performed in duplicate wells.
24 hours after delivery of siRNA, cells were lysed and luciferase activity was assayed. Results indicate that luciferase activity was inhibited 35% at 1 nM siRNA-Luc and 53% at 10 nM siRNA-Luc (Table 6). No inhibition was observed using either siRNA-Luc⁺, which contains three base pair mismatches relative to siRNA-luc or siRNA-c.

TABLE 6

SiRNA-mediated inhibition of virally encoded luciferase.

% Luciferase activity

siRNA conc. siRNA-Luc siRNA-Luc⁺ siRNA-c

0 nM 100 NA NA

1 nM 65 101 91

10 nM 47 117 129
These results demonstrate that siRNA can be used to inhibit expression of a virally encoded gene. In addition, the fact that siRNA-luc⁺ was unable to inhibit luciferase expression demonstrates that siRNA-mediated RNAi exhibits high sequence specificity. Thus, whether the gene is expressed from a viral genome, from a delivered extrachromosomal expression cassette, or from an endogenous gene, delivery of the proper siRNA to an in vivo or in vitro cell in which the target gene is expressed results in inhibition of expression of the target gene.

Example 23

Simultaneous delivery of siRNA and morpholino antisense oligonucleotide to mammalian cells. HeLa cells were maintained in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum. All cultures were maintained in a humidified atmosphere containing 5% CO₂at 37° C. Approximately 24 hours prior to transfection, cells were plated at an appropriate density in a T75 flask and incubated overnight. At 50% confluency, cells were initially transfected with pGL3 control (firefly luciferase, Promega, Madison Wis.) and pRL-SV40 (sea pansy luciferase, Promega, Madison, Wis.) using TRANSIT-LT1® transfection reagent (Mirus Corporation, Madison, Wis.) according to the manufacturer's recommendations. 15 μg pGL3 control and 50 ng pRL-SV40 were added to 45 μl TRANSIT-LT1® in 500 μl Opti-MEM (Invitrogen) and incubated 5 min at RT. DNA complexes were then added to cells in the T75 flask and incubated 2 h at 37° C. Cells were washed with PBS, harvested with trypsin/EDTA, suspended in media, plated into a 24-well plate with 250 μl DMEM+10% serum and incubated 2 h at 37° C. After incubation for 2 h, 400 μl DMEM/10% FBS was added to each well followed by the addition of siRNA and morpholino complexed with TRANSIT-TKO ® (Mirus Corporation). For preparation of the siRNA and morpholino-containing complexes, 2 μl TRANSIT-TKO® was diluted in 50 μl serum-free Opti-MEM and incubated at RT for 5 minutes. siRNA was added in order to give a final concentration of siRNA per well of 0, 0.1, or 10 nM and morpholino added to give a final concentration of morpholino per well of 0, 10, 100 or 1000 nM and incubated for 5 min at RT. Complexes were then added directly to the wells. All assay points were performed in duplicate wells.
Morpholino antisense molecule and siRNAs used in this example were as follows:

- Morpholino-Luc (GeneTools Philomath, Oreg.), 5′-TTATGTTTTTGGCGTCTTCCATGGT-3′ (SEQ ID NO. 31) (Luc+−3 to +22 of pGL3 Control Vector), was designed to base pair to the region surrounding the Luc+ start codon in order to inhibit translation of mRNA. Sequence of the start codon in the antisense orientation is underlined.
- Standard control morpholino, 5′-CCTCTTACCTCAGTTACAATTTATA-3′ (SEQ ID NO. 32), contains no significant sequence identity to Luc+ sequence or other sequences in pGL3 Control Vector.
- GL3 siRNA-Luc+ (see example 1):

Single-stranded, gene-specific sense and antisense RNA oligomers with overhanging 3′ deoxynucleotides were prepared and purified by PAGE (Dharmacon, LaFayette, Colo.). The two complementary oligonucleotides, 40 μM each, are annealed in 250 μl 100 mM NaCl/50 mM Tris-HCl, pH 8.0 buffer by heating to 94° C. for 2 minutes, cooling to 90° C. for 1 minute, then cooling to 20° C. at a rate of 1° C. per minute. The resulting siRNA was stored at −20° C. prior to use.
In order to deliver the morpholino to cells in culture using the cationic transfection reagent, TRANSIT-TKOO the morpholino was first annealed to a DNA oligonucleotide of complementary sequence. The sequence of the DNA strand is as follows: 5′-GCCAAAAACATAAACCATGGAAGACT-3′ (SEQ ID NO. 33). The morpholino and complementary DNA oligonucleotide, 0.5 mM each, are annealed in 5 mM Hepes, pH 8.0 buffer by heating to 94° C. for 2 minutes, cooling to 90° C. for 1 minute, then cooling to 20° C. a rate of 1° C. per minute. The resulting morpholino/DNA complex was stored at −20° C. prior to use.
Cells were harvested after 24 h and assayed for luciferase activity using the Promega Dual Luciferase Kit (Promega). A Lumat LB 9507 (EG&G Berthold, Bad-Wildbad, Germany) luminometer was used. The amount of luciferase expression was recorded in relative light units. Numbers were then adjusted for control sea pansy luciferase expression and are expressed as the percentage of firefly luciferase expression in the absence of siRNA (FIG. 13) Numbers are the average for at least two separate wells of cells. These data demonstrate that when siRNA and morpholino are delivery simultaneously, the degree of inhibition is greater than with delivery of either siRNA or morpholino alone.

Example 24

Enhanced inhibition of gene expression by delivery of antisense morpholino and siRNA in vivo. Morpholino antisense molecule and siRNAs used in this example were as follows:

Morpholino-Luc: SEQ ID NO. 31
Standard control morpholino: SEQ ID NO. 32
GL3 siRNA-Luc+: SEQ ID NO. 3 and SEQ ID NO. 4.

DL88:DL88C siRNA (targets EGFP 477-495, nt765-783):

(SEQ ID NO. 34)

5′-rGrArArCrGrGrCrArUrCrArArGrGrUrGrArArCdTdT-3′,

(SEQ ID NO. 35)

5′-rGrUrUrCrArCrCrUrUrGrArUrCrCrCrGrUrUrCdTdT-3′
Two plasmid DNAs±siRNA and ±antisense morpholino in 1-3 ml Ringer's solution (147 mM NaCl, 4 mM KCl, 1.13 mM CaCl₂) were injected, in 7712 seconds, into the tail vein of mice for delivery to liver. The plasmids were pGL3 control, containing the luc+ coding region under transcriptional control of the simian virus 40 enhancer and early promoter region, and pRL-SV40, containing the coding region for the Renilla reniformis luciferase under transcriptional control of the Simian virus 40 enhancer and early promoter region. 2 μg pGL3 control and 0.2 μg pRL-SV40 were injected with or without 5.0 μg siRNA and with or without 50 μg morpholino-Luc. One day after injection, the 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 were cleared by centrifugation. The homogenate was diluted 10-fold in lysis buffer and 5 μl was assayed for Luc+ and Renilla luciferase activities using the Dual Luciferase Reporter Assay System (Promega Corp.). Ratios of Luc+ to Renilla Luc were normalized to the 0 μg siRNA-Luc+ control.

These experiments demonstrate the near complete inhibition of gene expression in vivo when antisense morpholino is delivered together with siRNA (Table 7). This level if inhibition was greater than that for either morpholino of siRNA individually.

TABLE 7


Inhibition of luciferase expression from pGL3 control
plasmid in mouse liver after delivery of 50 μg
antisense morpholino, 5 μg siRNA or both.

		percent inhibition of
Antisense morpholino	siRNA	luciferase expression

—	—	0
Standard	DL88:DL88C	0
DL94	DL88:DL88C	85.4 ± 2.7
Standard	GL3 siRNA-Luc+	92.0 ± 1.9
DL94	GL3 siRNA-Luc+	98.6 ± 0.5

Example 25

Delivery of siRNA-containing non-viral particles to liver cells in vivo via hydrodynamic intravascular injection. 10 μg pGL3 control plasmid and 1 μg pRL-SV40 were complexed with 11 μl TRANSIT™ In Vivo in 2.5 ml total volume according the manufacturer's recommendation (Mirus Corporation, Madison, Wis.). For siRNA delivery, 10 μg pGL3 control, 1 μg pRL-SV40, and either 5 μg siRNA-Luc+ or 5 μg control siRNA were complexed with the 16 μl of the polycationic TRANSIT™ In vivo transfection reagent in 2.5 ml total volume. Particles were injected into the tail vein of 25-30 g ICR mice as described in Example 1, with an injection time of about 7 seconds. One day after injection, the livers were harvested and homogenized as described in Example 1. Luc+ and Renilla Luc activities were assayed using the Dual Luciferase Reporter Assay System (Promega). Ratios of Luc+ to Renilla Luc were normalized to the no siRNA control. siRNA-luc+ specifically inhibited the target Luc+ expression 96% (Table 8).

TABLE 8


Functional delivery of siRNA to mouse liver cells
via hydrodynamic injection of siRNA/TRANSIT ™ In
Vivo labile polycation complexes.

			relative	% inhibition
		expression	LUC+	of Luc+
complex	gene	(RLUs)	expression	expression

Plasmid	Luciferase	31973057	5.1855	0.0
alone	Renilla	6165839
Plasmid +	Luciferase	853332	0.2069	96.0
siRNA-Luc+	Renilla	4124726
Plasmid +	Luciferase	5152933	2.1987	57.5
control	Renilla	2343673
SiRNA

These data show that siRNA-containing particles, in this case siRNA/polycation complexes, can be delivered to cells in target tissues via the described hydrodynamic intravascular injection method.

Example 26

Delivery of Adenovirus to Limb Skeletal Muscle via venous injection. Delivery of Adenovirus and siRNA to limb muscle cells via saphenous vein injection: 120-140 g adult Sprague-Dawley rats were anesthetized with isoflurane 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 (0.25 mg in saline) over 6 seconds using a syringe pump. After 5 min, 5 ml normal saline containing 2×10⁹Adenovirus particles encoding firefly Luciferase was injected at varying flow rates. Some injections also contained 5 μg 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.
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. 1×10⁹Adenovirus particles encoding firefly Luciferase in 2.5 ml of saline was injected into each hind gastrocnemius muscle group of the animal.

Conclusion: Injection of adenovirus into a leg vein, using the describe delivery process, resulted in efficient delivery of the virus to muscle cells in the leg and expression of a virally encoded reporter gene (Table 9). Delivery was much more efficient that direct muscle injections. Co-injection of siRNA specific to the luciferase gene resulted in efficient inhibition of luciferase expression.

TABLE 9


Delivery of adenovirus to skeletal muscle:
intravenous vs. intramuscular injection.

injection

luciferase activity

nucleic acid

Volume

rate

ng per

Adenovirus	siRNA	Route	(ml)	(ml/min)	total RLUs	limb

2.0 × 10⁹		saphenous	5.0	16.6	1,613,367	101.8
2.0 × 10⁹		saphenous	5.0	12.5	656,435	48.2
2.0 × 10⁹		saphenous	5.0	12.5	222,172	16.2
2.0 × 10⁹	5 μg	saphenous	5.0	16.6	112,325	7.3
2.0 × 10⁹	5 μg	saphenous	5.0	16.6	101,824	5.4
1.0 × 10⁹		IM (gastroc)	2.5		3,262	0.2
1.0 × 10⁹		IM (gastroc)	2.5		2,724	0.2

Example 27

Delivery of adeno-associated virus (AAV) to limb muscle cells via increased pressure IV injection. Mice were anesthetized with 1-2% isoflurane throughout each procedure. A small latex tourniquet was wrapped tightly around the upper hind limb (above quadriceps) and held in place with a hemostat. A small incision (˜1 cm) was made in the skin to expose a segment of the distal great saphenous vein. A 30 gauge needle catheter was inserted into the distal great saphenous vein, advanced about 0.5 cm and held in place during the injection. The catheter was connected to a Harvard PHD 2000 syringe pump and the AAV (0.19×10⁹transducing units, CMV-LacZ) saline solution (0.8 ml) injected (antegrade) at a rate of 3 ml/min. The tourniquet was removed 2 minutes after injection and the skin was closed with 5-0 suture. Two weeks after injection, the limb muscles were harvested and frozen in cold isopentane and stored at −80° C. 10 μm thick cryosections were made and fixed in 1.25% gluteraldehyde. The sections were then incubated in X-gal staining solution (Mirus Bio Corporation) for 1 hour at 37° C. FIG. 14 shows photomicrographs of mouse hind limb gastrocnemius muscle stained for β-galactosidase, showing that AAV was efficiently delivered to muscle cells.

Example 28

rAAV CMV-Luciferase Delivery Protocol. 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. We compared the efficiency of single-site intravascular gene delivery system to that of multi-site direct intramuscular injection for the delivery of recombinant AAV (rAAV) to muscle. 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 or by direct injections into each of 5 major muscle groups of the leg. For direct intramuscular injections, 1×10¹²rAAV 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 1×10¹²rAAV particles via injection. Luciferase of β-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 (μg) of luciferase produced. To determine RLU, 10 μl 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.

Delivery of rAAV CMV-Luc into rat muscle via the described intravascular delivery procedure resulted in high levels of transgene expression. Average total luciferase levels were higher for the single-site intra-arterial (10.07 μg per animal; n=8) method than for the multi-site direct muscle injections (6.70 μg per animal; n=7) (Table 10a). High level luciferase expression is stable out to at least 8 weeks in rats with an intact immune system (Table 10b).

TABLE 10a


Comparison of luciferase expression in skeletal
muscle cells following delivery of AAV by intravascular
delivery or intramuscular injection.

muscle
group	relative light units (RLU)

Intravascular (IV) Delivery - 2 week time point

upper leg	1.80 × 10¹⁰	6.10 × 10¹⁰	6.37 × 10¹⁰	1.03 × 10¹⁰
anterior
upper leg	2.38 × 10¹⁰	5.17 × 10¹⁰	8.27 × 10⁹	4.62 × 10⁹
posterior
upper leg	1.12 × 10¹⁰	3.50 × 10¹⁰	9.29 × 10⁹	5.79 × 10⁹
medial
lower leg	1.30 × 10¹⁰	1.09 × 10¹¹	2.01 × 10¹⁰	8.32 × 10⁹
posterior
lower leg	4.32 × 10⁹	5.01 × 10⁹	1.29 × 10¹⁰	5.04 × 10⁹
anterior
foot	5.08 × 10⁸	9.51 × 10⁹	1.52 × 10⁸	8.20 × 10⁷
total	7.08 × 10¹⁰	2.71 × 10¹¹	1.14 × 10¹¹	3.41 × 10¹⁰
RLU
μg	3.61	13.85	5.84	1.75
luciferase

average

6.26 μg luciferase per leg

Direct Intramuscular (IM) Injection - 2 week time point

upper leg	1.61 × 10¹⁰	1.21 × 10¹⁰	1.41 × 10¹⁰	3.41 × 10¹⁰
anterior
upper leg	3.34 × 10⁹	3.62 × 10⁹	4.49 × 10⁹	3.26 × 10⁹
posterior
upper leg	8.88 × 10⁹	1.75 × 10¹⁰	6.49 × 10⁹	1.41 × 10¹⁰
medial
lower leg	8.12 × 10⁹	1.03 × 10¹⁰	1.71 × 10¹⁰	7.73 × 10⁹
posterior
lower leg	0.23 × 10⁹	1.47 × 10¹⁰	3.57 × 10¹⁰	4.09 × 10¹⁰
anterior
foot	6.01 × 10⁶	5.23 × 10⁷	2.35 × 10⁶	2.26 × 10⁶
total	3.67 × 10¹⁰	5.82 × 10¹⁰	7.79 × 10¹⁰	1.00 × 10¹¹
RLU
μg	1.86	2.97	3.97	5.11
luciferase

average	3.48 μg luciferase per leg

TABLE 10b


Comparison of luciferase expression in skeletal
muscle cells following delivery of AAV by intravascular
delivery or intramuscular injection.

	IV delivery - 4 weeks	IM injection - 4 weeks

upper leg	1.03 × 10¹¹	1.69 × 10¹⁰	5.71 × 10¹⁰	4.21 × 10¹⁰
anterior
upper leg	6.65 × 10¹⁰	1.73 × 10¹⁰	3.96 × 10¹⁰	1.36 × 10¹⁰
posterior
upper leg	1.17 × 10¹¹	4.23 × 10¹⁰	1.86 × 10¹⁰	3.30 × 10¹⁰
medial
lower leg	2.15 × 10¹¹	6.09 × 10¹⁰	3.19 × 10¹⁰	2.46 × 10¹⁰
posterior
lower leg	1.62 × 10¹⁰	2.00 × 10¹⁰	3.62 × 10¹⁰	2.36 × 10¹⁰
anterior
foot	7.50 × 10⁹	0.36 × 10⁹	0.09 × 10⁹	0.05 × 10⁸
total	5.25 × 10¹¹	1.58 × 10¹¹	1.84 × 10¹¹	1.37 × 10¹¹
RLU
μg	26.82	8.05	9.36	6.97
luciferase

average	17.44 μg luciferase per leg	8.17 μg luciferase per leg

	IV delivery - 8 weeks	IM injection - 8 weeks

upper leg	6.82 × 10¹⁰	4.03 × 10¹⁰	9.93 × 10¹⁰
anterior
upper leg	2.22 × 10¹⁰	4.40 × 10⁹	3.13 × 10¹⁰
posterior
upper leg	4.98 × 10¹⁰	3.57 × 10¹⁰	1.89 × 10¹⁰
medial
lower leg	5.87 × 10¹⁰	7.56 × 10¹⁰	9.39 × 10¹⁰
posterior
lower leg	2.29 × 10¹⁰	2.77 × 10¹⁰	8.35 × 10¹⁰
anterior
foot	0.7 × 10⁹	0.28 × 10⁹	0.10 × 10⁹
total	2.23 × 10¹¹	1.84 × 10¹¹	3.27 × 10¹¹
RLU
μg	11.27	9.38	16.68
luciferase

average	10.32 μg luciferase per leg	16.68 μg luciferase per leg

Example 29

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 surrounding 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 1.66 mm) 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. 5 min after papaverine injection, 40 ml of DNA solution (3 mg) was injected in 65 sec (0.615 ml/sec). 2 min 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 (Table 11).

TABLE 11


Luciferase expression in diaphragm from monkey sacrificed
7 days after injection of pCI-Luc⁺.

	total luciferase	ng luciferase/
diaphragm section	(ng)	gram if tissue

anterior part of left side	0	0
posterior part of left side	0	0
left conjunction area	0	0
anterior part of right side	221.94	27.88
posterior part of right side	15.98	2.12
right conjunction area	34.21	17.82

Example 30

Delivery of DNA/polycation complexes to prostate and testis via injection into dorsal vein of penis. DNA and L-cystine-1,4-bis(3-aminopropyl)piperazine cationic copolymer were mixed at a 1:1.7 wt:wt ratio in water, diluted to 2.5 ml with Ringers solution and injected rapidly into the dorsal vein of the penis (within 7 seconds). For directed delivery to the prostate, 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. The results, Table 12, show efficient and functional delivery of DNA containing complexes to prostate, testis and other tissues.

TABLE 12

Delivery of DNA containing plasmid to prostate

and testis via injection in dorsal vein.

Organ Luciferase (RLUs)

Prostate 129,982,450

Testis 4,229,000

fat (around bladder) 730,300

bladder 618,000

Example 31

Delivery of polynucleotides to primate liver. Cynomolgus monkeys (n=6) weighing 2.5 to 3.3 kg and were sedated with ketamine (10-15 mg/kg IM). After sedation, animals were intubated and anesthesia was maintained with 1.0 to 2.0% isoflurane. An intravenous catheter was inserted into the cephalic vein for administering fluids and EKG electrodes were attached to the limbs for monitoring heart rate. The surgical area was prepped and draped for surgery using aseptic technique. A femoral cutdown was performed and a 5 cm segment of the femoral vein was dissected free of connective tissue. The vein was ligated distally and a 6F introducer was inserted into the vein and secured with a vessel tourniquet. A 4F injection catheter was inserted through the introducer and advanced into the inferior vena cava (IVC). An abdominal incision was made extending from just below the xyphoid to the pubis. A retractor was placed inside the abdominal cavity and moist sponges were used to retract and hold the intestines. The infra hepatic IVC was exposed and the exact placement of the injection catheter within the IVC was adjusted so that the tip of the catheter was adjacent to the hepatic vein. A vessel tourniquet was placed loosely around the infra hepatic IVC to prevent backflow during the nucleic acid injection. The supra hepatic IVC was dissected free of connective tissue and ligament attachments. In some animals (n=3), a catheter (20 gauge) was inserted into the portal vein and attached to a pressure transducer to measure pressure changes during the injection. Immediately before the injection, a vascular clamp was placed on supra hepatic IVC and the vessel tourniquet was tightened around the infra hepatic IVC, thus directing injection solution to the liver. The IVC remained occluded during the injection and for 2 minutes post injection. After the injection, the clamps were removed and the portal vein pressure catheter was pulled. The abdominal cavity was closed in 3 layers with 3-0 PDS suture. The catheter and introducer were pulled from the femoral vein, the vessel was ligated and the incision was closed in 2 layers with 3-0 PDS suture. Prior to the completion of the surgery, the animal was given buprenorphine (0.005-0.01 mg/kg IM) as an analgesic. Blood samples were collected on days 0, 1, 4, 7, 14 and 21 for measuring reporter gene expression (secreted alkaline phosphatase), liver enzymes and a complete blood count.
Two 60 ml syringes were filled with a total volume of 120 ml of injection solution containing 0.9% NaCl, 7.5% mannitol and 10 mg of CMV-SEAP. Syringes were attached to a syringe pump (Harvard Instrument) and connected to the injection catheter via an extension line with a 3-way stopcock. The injection flow rate was set at either 60, 120 or 160 ml/min. In addition to using the syringe pump, hand injections were performed on two animals. The flow rate was continuous during the injection except for one animal that had a preset pause (5 seconds) half way through the injection. During the injection, the entire liver swelled and patchy areas were blanched from the injection solution. In five of the six animals, it was noted that the liver swelled to the point where fluid would begin to leak out the exterior of the liver capsule by the end of the injection.
The portal vein pressure during injection was measured at two injection flow rates. An injection rate of 120 ml/min produced a transient elevation in portal vein pressure to 45 mm Hg while a flow rate of 160 ml/min resulted in a pressure increase to 105 mm Hg. This pressure increase lasted for the length of the injection and rapidly returned to preinjection levels.
All animals recovered and regained normal activity within 24 hours after surgery. Liver enzymes were elevated in all animals after surgery with the highest levels on day 1. Enzyme levels gradually declined thereafter and returned to normal levels around day 14. Day one levels of alanine aminotransferase (ALT) varied from 4960-338 U/L. The animals with the highest ALT levels were also the animals with the highest level of gene expression. One of the animals that showed both high reporter gene expression and elevated liver enzymes was sacrificed at 21 days to harvest tissue for histology. H+E slides of the right and left lobes showed normal tissue with no significant pathology.

In all the animals in this study, reporter gene expression peaked at day 2 (Table 13). The first animal (animal #1) in this study was a hand injection and resulted in the highest gene expression (day 2=2414 ng/ml SEAP) in this study. This hand injection delivered ˜120 ml of solution/minute. Using a syringe pump we found that 60 ml/min (animal #3) and 120 ml/min (animal #2) resulted in a low level of gene expression on day 2 (99 and 71 ng/ml SEAP respectively) but a flow rate of 160 ml/min (animal #5) dramatically improved expression (day 2=1991 ng/ml SEAP). This injection rate was repeated in a second animal (animal #6) but with a preset pause (5 seconds) halfway through the injection to determine if continuous flow was important for high gene expression. The expression in this animal was also high (day 2=985 ng/ml) but lower than the continuous syringe pump injection. In one additional animal, the pump failed during the injection and had to be helped by hand (animal #4). This injection delivered 120 ml in approximately one minute and resulted in a SEAP level of 350 ng/ml on day 2.

TABLE 13


Hepatic Vein Delivery of Nucleic Acid to Primate Liver. Expression
of plasmid CMV-SEAP in liver and analysis of toxicity.

day

0

1

2

4

7

14

21

animal	SEAP expression (ng/ml)

1	2300.7	2414.7	1605.1	1144.1	504.2	0.7
2	70.9	70.8	43.4	18.0	3.4	1.6
3	63.9	98.8	85.5	49.5	13.4	9.2
4	206.7	350.1	184.9	59.3	55.7	2.5
5	1686.1	1990.7	1737.8	831.9	371.7	155.8
6	740.9	985.1	864.4	634.6	232.5	106.7

Example 32

SiRNA expression cassettes are effective in inhibiting gene expression in vivo. ICR mice (n=4 each group) were co-injected via hydrodynamic tail vein injection as in example 1 with two reporter plasmids, firefly luc+ (pGL3-Control) and Renilla luciferase (pRL-SV40). Each mouse was injected with 15 μg pGL3-Control and 0.1 tg pRL-SV40. Co-delivered with the reporter plasmids were 10 μg of control hU6 vector plasmid, Luc⁺-siRNA (targeting GL3 luciferase) expression plasmid, 383 bp Luc⁺-siRNA linear expression cassette, 422 bp Luc⁺-siRNA linear expression cassette, 1065 bp Luc⁺-siRNA linear expression cassette, or synthetic Luc⁺-siRNA Mice were sacrificed 54 h after injection. The livers were harvested and homogenized in LUX lysis buffer. The Dual Luciferase assays were performed as described above. The averaged ratios of firefly/Renilla expression in each group of mice was normalized to the ratio from the mice that were injected with reporter plasmids only. Results are shown in FIG. 15. Firefly luciferase was inhibited 89% from expression plasmid, 90% from the 383 bp pMIR225 fragment, 88% from the 422 bp pMIR225 fragment, 85% from the 1065 bp pMIR225 fragment, and 92% from the synthetic GL3 siRNA. The PCR fragments mediated RNAi in the mice to the same degree as the siRNA expression plasmid and the synthetic siRNA indicated their utility in vivo.

Example 33

Human H1 promoter driving siRNA from small fragments of DNA in vivo. The hH1 promoter (100 bp) is much smaller than the hU6 promoter (264 bp). We sought to determine if a small fragment of DNA bearing the hH1 promoter in an siRNA expression cassette could mediate knockdown in vivo. ICR mice were injected in the tail vein with 1 μg firefly expression plasmid and 20 μg Renilla luciferase expression plasmid. Co-delivered with the reporter plasmid were 10 μg of either Luc⁺-siRNA H1 promoter expression plasmid, 221 bp Luc⁺-siRNA H1 promoter expression cassette, 434 bp Luc⁺-siRNA H1 promoter expression cassette, or 478 bp Luc⁺-siRNA H1 promoter expression cassette. On day 3 the mice were sacrificed and livers were harvested, homogenized, and assayed for Dual Luciferase activity. The firefly/Renilla ratios were normalized to reporter plasmids only. Firefly luciferase expression was knocked down 42% by the Luc⁺-siRNA expression plasmid, 50% by the 221 bp expression cassette, 56% by the 434 bp expression cassette, and 32% by the expression cassette.
The foregoing is considered as illustrative only of the principles of the invention. Furthermore, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described. Therefore, all suitable modifications and equivalents fall within the scope of the invention.

Claims

1. An in vivo process for delivering a polynucleotide containing sequence similar to a sequence present in a gene that is expressed in an extravascular cell in an animal, comprising:

a) making the polynucleotide consisting of a double strand or partially double strand polynucleotide containing a sequence that is essentially complementary to a sequence in the gene;

b) injecting the polynucleotide, in a single injection, into an afferent or efferent vessel of a target tissue in which the extravascular cell is located in a sufficient volume to increase permeability of vessels in the tissue and deliver the polynucleotide to the extravascular cell.

2. The process of claim 1 wherein the polynucleotide inhibits expression of the gene.

3. The process of claim 2 wherein the gene consists of a viral gene.

4. The process of claim 3 wherein inhibiting the viral gene decreases viral toxicity.

5. The process of claim 3 wherein inhibiting the viral gene decreases viral replication.

6. The process of claim 3 wherein inhibiting the viral gene decreases viral virulence.

7. The process of claim 3 wherein the viral gene consists of a variola virus gene.

8. The process of claim 2 wherein inhibiting gene expression alters expression of an endogenous gene.

9. The process of claim 8 wherein inhibiting the endogenous gene alters a host response to a pathogen infection.

10. The process of claim 1 wherein the polynucleotide consists of a naked polynucleotide.

11. The process of claim 1 wherein the polynucleotide is associated with a compound.