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

United States Patent:  6,261,281

Assignee:  Electrofect AS (NO)

Filed:  May 5, 2000

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.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a method for delivering or transfecting pharmaceutical drugs, proteins, and DNA into skeletal muscle and other cells residing within the skeletal muscle. Without being bound by theory, the method is thought to be similar to electroporation. Electroporation works on the principle that a cell acts as an electrical capacitor and is generally unable to pass current. Subjecting cells to a high-voltage electric field, therefore, creates transient permeable structures or micropores in the cell membrane. These pores are large enough to allow pharmaceutical drugs, DNA, and other polar compounds to gain access to the interior of the cell. With time, the pores in the cell membrane close and the cell once again becomes impermeable.

Conventional electroporation, however, employs high field strengths from 0.4 to several kV/cm. In contrast to conventional electroporation, the field strength used in the present invention ranges from about 10 V/cm to about 300 V/cm. These lower field strengths are thought to cause less muscle damage without sacrificing, and indeed increasing, transfection efficiencies. Furthermore, using the method of the present invention, transfection efficiencies can be tightly regulated by altering such parameters as frequency, pulse duration and pulse number.

The increase in DNA transfection efficiency is observed only if the muscle is electrically stimulated immediately, or shortly after the DNA injection. Thus, the semipermeable quality of the tissue induced by the stimulation is reversible. Moreover, it is dependent on current through the muscle; activity induced through the nerve does not affect transfection efficiency.

Once transfected, the muscle cells are able to express the proteins coded by the nucleic acid. Therefore, the transfection method of the present invention can be used, for example, to transfect expression vectors for genetic immunization (i.e., DNA vaccines). In one embodiment, rabbits were transfected with a plasmid containing the cDNA for rat agrin. The transfected muscles produced and secreted agrin protein. Nineteen days post-transfection, rabbit serum contained significant antibodies against rat agrin.

In a second embodiment, mice and rats were transfected using the method of the present invention with one or more of three different eukaryotic expression vectors containing the coding sequences for DH-CNTF, an agonistic variant of human ciliary neurotrophic factor, AADH-CNTF, an antagonistic variant of human ciliary neurotrophic factor and sec-DHCNTF, a secreted form of DH-CNTF. The muscles were either not electrically stimulated or stimulated immediately after DNA injection. Blood was collected at various time points and the antibody titers determined. In both rats and mice, electrical stimulation immediately after DNA injection led to approximately 5 to 10-fold higher antibody titers than simple DNA injection.

The transfection method of the present invention can also be used to systemically deliver proteins to treat diseases. In one preferred embodiment, a DNA plasmid harboring the erythropoietin (EPO) gene was injected into skeletal muscle and stimulated according to the method of the present invention. Controls were either not stimulated or transfected with a control vector not harboring the EPO gene. After 14 days, only the mice transfected with EPO according to the method of the present invention displayed an increased hematocrit indicating the transfected muscles were able to produce and secrete into the blood stream substantial amounts of EPO.

Non-nucleic acids may also be transfected by the method of the present invention. In one embodiment, rhodamin conjugated dextran was injected into the muscle followed by electrical stimulation. Three to five days later the muscles were frozen in liquid nitrogen and sectioned on a cryostat. Fluorescence was observed in cells injected and stimulated, indicating the rhodamin conjugated dextran was able to enter and remain in the muscle cells.

In order to reduce pain that may be associated with the method of the present invention, a local anesthetic can be injected at the site of treatment prior to or in conjunction with the injection of DNA. For example, in one embodiment of the current invention, DNA may be mixed with Marcain, a local anesthetic, followed by electroporation.

Claim 1 of 34 Claims

We claim:

1. A method of delivering a DNA molecule to the immune system of a mammal in vivo comprising:

mixing a first solution comprising the DNA molecule with a second solution comprising a local anesthetic to produce a DNA-anesthetic mixture;

injecting the DNA-anesthetic mixture into an injection site in a skeletal muscle of the mammal;

positioning electrodes near the injection site such that current traveling through the electrodes passes through the injection site; and

electrically stimulating the muscle with an electrical current having a field strength of from about 10 V/cm to about 300 V/cm.

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