Method for genetic immunization and introduction of molecules into skeletal muscle and immune cells

Abstract

A method is disclosed for enhanced vaccination and genetic vaccination of mammals. The vaccination is accomplished by delivering molecules such as proteins and nucleic acids into skeletal muscle and other cells residing in the skeletal muscle in vivo. The protein or nucleic acid is first injected into the muscle at one or multiple sites. Immediately or shortly after injection, electrodes are placed flanking the injection site and a specific amount of electrical current is passed through the muscle. The electrical current makes the muscle permeable, thus allowing the pharmaceutical drug or nucleic acid to enter the cell. The efficiency of transfer permits robust immune responses using DNA vaccines and produces sufficient secreted proteins for systemic biological activity to be observed.

Description

[0001] This application is a continuation of copending U.S. patent application Ser. No. 09 / 565,140 of Iacob Mathiesen and Stig Tollefsen filed May 5, 2000 and entitled "Method for Genetic Immunization and Introduction of Molecules into Skeletal Muscle and Immune Cells," which is related to and claims the benefit of U.S. Pat. No. 6,110,161 of Iacob Mathiesen and Terje L.o slashed.mo filed Apr. 3, 1998 and entitled "Method for Introducing Pharmaceutical Drugs and Nucleic Acids Into Skeletal Muscle," which is related to and claims the benefit of U.S. Provisional Application Ser. No. 60 / 042,594 of Iacob Mathiesen and Terje L.o slashed.mo filed Apr. 3, 1997 and entitled "Apparatus and Method for Introducing Pharmaceutical Drugs and Genetic Material Into Skeletal Muscle." These applications and this patent are hereby incorporated by reference.[0002] The present invention is related to a method for immunizing an animal by making the skeletal muscle semipermeable to nucleic acids and other ...

AI Technical Summary

Benefits of technology

[0047] FIG. 28--is a bar graph illustrating mean luciferace activity in mouse muscles after a second immunization with 85B and luciferace cDNA. A low value indicates a strong cellular immune response and efficient killing of transfected cells.

[0052] As illustrated in FIG. 1, generally, skeletal muscle is exposed and a predetermined amount of a molecule is injected into the muscle. In one embodiment the DNA is dissolved in 0.9% sodium chloride (NaCl). The exact solvent, however, is not critical to the invention. For example, it is well known in the art that other solvents such as sucrose are capable of increasing DNA uptake in skeletal muscle. Other substances may also be co-transfected with the molecule of interest for a variety of beneficial reasons. For example, P188 (Lee, et al. PNAS., 4524-8, 10, 89 (1992)), which is known to seal electropermeabilized membranes, may beneficially affect transfection efficiencies by increasing the survival rate of transfected fibers.

[0058] As illustrated in FIGS. 3 and 5-8, the method of the present invention dramatically increases the efficiency of drug and DNA delivery into skeletal muscle. In one embodiment, rat soleus or EDL muscles were injected with DNA plasmid containing the .beta.-galactosidase gene (lac Z). The .beta.-galactosidase gene yields a protein capable of converting a colorless substrate into a blue substrate that can be visually analyzed or measured spectrophotometrically. FIG. 3 depicts representative soleus and EDL muscles that have been transfected with .beta.-galactosidase gene using various stimulation parameters.

[0059] FIG. 3a illustrates the improved DNA delivery efficiency of soleus and EDL muscles that have been transfected according to the method of the present invention. Soleus and EDL muscles (n=3) were first denervated by transecting the sciatic nerve. This was done to eliminate any influence of nerve-induced activity that arguably could contribute to the increased transfection efficiency observed. Three days post-denervation, the muscles were injected with the .beta.-galactosidase gene as described above. After the DNA injection, the muscles were either untreated or, immediately after the DNA injection, the muscles were stimulated according to the method of the present invention.

[0070] As illustrated in the examples below, molecules other than nucleic acids can be delivered to the muscle using the technique of the present invention. In one embodiment, rhodamin conjugated dextran injected into the muscles and stimulated according to the method of the present invention was able to enter muscle cells. In addition, nucleic acid and proteins can be simultaneously introduced into an electroporated muscle. In one embodiment, the large T-antigen nuclear localization signal was mixed with a plasmid containing the DNA coding region for Lac Z. The large T-antigen nuclear localization signal is a protein that binds DNA and facilitates its transport into the nucleus of a cell. In other systems, large T-antigen nuclear localization signal has been shown to increase transfection efficiency. Using the method of the present invention, large T-antigen nuclear localization signal also increased the transfection efficiency of Lac Z indicating that the protein was able to bind the DNA and enter the muscle cell.

Problems solved by technology

Despite these new discoveries, a major obstacle facing the medical profession is how to safely deliver effective quantities of these agents to patients to treat disease or for genetic immunization.

First, a large percent of orally or intravenously delivered drugs are degraded by the body before arriving at the target organ or cells.

Second, oral and intravenous drug and gene delivery is non-specific.

Currently, however, there is no non-viral method for effectively delivering pharmaceutical drugs, proteins, and DNA into skeletal muscle in vivo.

The clinical applicability of direct muscle injection, however, is limited mainly because of low transfection efficiency, typically less than 1% transfection efficiency.

While injection in regenerating muscles induced by Bivucain show higher efficiency, the method has limited applicability in humans because of the severe damage caused to the muscle.

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