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

• US 6,261,281 B1
• Filed: 05/05/2000
• Issued: 07/17/2001
• Est. Priority Date: 04/03/1997
• Status: Expired due to Term

• Current Assignee: Inovio AS (Inovio Pharmaceuticals, Inc.)
• Original Assignee: Electrofect AS
• Inventors: Tollefsen, Stig, Mathiesen, Iacob
• Primary Examiner(s): Bockelman, Mark
• Application Number:
  US09/565,140
• Time in Patent Office: 438 Days
• Field of Search: 604/20-21, 604/500, 435/173.6, 435/285.2
• US Class Current: 604/500
• CPC Class Codes: A61K 2039/53 DNA (RNA) vaccination
• CPC Class Codes: A61K 2039/54 characterised by the route of administration
• CPC Class Codes: A61K 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2039/57 characterised by the type of response, e.g. Th1, Th2
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/04 Mycobacterium, e.g. Mycobacterium tuberculosis
• CPC Class Codes: A61K 41/00 Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• CPC Class Codes: A61N 1/325 for iontophoresis, i.e. transfer of media in ionic state by an electromotoric force into the body electrodes for external use A61N1/0428
• CPC Class Codes: A61N 1/36 for stimulation
• CPC Class Codes: A61N 1/36003 of motor muscles, e.g. for walking assistance
• CPC Class Codes: A61N 1/36017 with leads or electrodes penetrating the skin
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

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.

Claims

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.

2. The method of claim 1, wherein the local anesthetic is Marcain.

3. The method of claim 2, wherein the electrical current has a field strength of from about 10 V/cm to about 233 V/cm.

4. The method of claim 2, wherein the electrical current has a field strength of from about 12 V/cm to about 175 V/cm.

5. The method of claim 2, wherein the electrical current has a field strength of from about 125 V/cm to about 233 V/cm.

6. The method of claim 2, wherein the electrical current is delivered as one or more square bipolar pulses.

7. A method of immunizing a mammal with an antigen comprising:

injecting the antigen into an injection site in the 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.

8. The method of claim 7, wherein the electrical current is delivered in the form of a single square bipolar pulse.

9. The method of claim 7, wherein the electrical current is delivered in the form of between about 2 to about 30,000 square bipolar pulses.

10. The method of claim 7, wherein the antigen is a protein.

11. The method of claim 7, the electrical current has a field strength of from about 10 V/cm to about 233 V/cm.

12. The method of claim 7, wherein the electrical current has a field strength of from about 12 V/cm to about 175 V/cm.

13. The method of claim 7, wherein the electrical current has a field strength of from about 125 V/cm to about 233 V/cm.

14. A method of inducing a cellular immune response in a mammal comprising:

injecting DNA encoding an antigen into an injection site in the 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 about 10 V/cm to about 233 V/cm.

15. The method of claim 14, wherein the electrical current has a field strength of from about 12 V/cm to about 175 V/cm.

16. The method of claim 14, wherein the electrical current has a field strength of from about 125 V/cm to about 233 V/cm.

17. The method of claim 14, wherein the electrical current is delivered as one or more square bipolar pulses.

18. A method of inducing a humoral immune response in a mammal comprising:

injecting an antigen into an injection site in the 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 about 10 V/cm to about 233 V/cm.

19. The method of claim 18, wherein the antigen is a protein.

20. The method of claim 18, wherein the electrical current has a field strength of from about 12 V/cm to about 175 V/cm.

21. The method of claim 18, wherein the electrical current has a field strength of from about 125 V/cm to about 233 V/cm.

22. The method of claim 18, wherein the electrical current is delivered as one or more square bipolar pulses.

23. A method of genetically immunizing a mammal comprising:

injecting DNA encoding an antigen 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 less than about 100 V/cm.

24. The method of claim 23, wherein the electrical current has a field strength of at least about 10 V/cm.

25. The method of claim 23, wherein the electrical current has a field strength of from about 10 V/cm to about 25 V/cm.

26. A method of delivering a molecule to cells residing within the skeletal muscle of a mammal in vivo comprising:

injecting the molecule into an injection site in the 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 233 V/cm.

27. The method of claim 26 wherein, the current has a field strength of from about 125 to about 233 V/cm.

28. The method of claim 26, wherein the molecule is a nucleic acid molecule.

29. The method of claim 28, wherein the nucleic acid molecule is a DNA molecule.

30. A method of enhancing an immune response of a mammal comprising:

injecting an antigen into a first injection site in the skeletal muscle of the mammal;

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

electrically stimulating the muscle with a first electrical current having a field strength of from about 10 V/cm to about 233 V/cm;

injecting a DNA molecule coding for the antigen into a second injection site in the skeletal muscle of the mammal;

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

electrically stimulating the muscle with a second electrical current having a field strength of from about 10 V/cm to about 233 V/cm;

wherein the DNA molecule is injected at a time of from about one week to about several years after the injection of the antigen.

31. The method of claim 30, wherein the antigen is a protein.

32. The method of claim 30, wherein the DNA molecule is injected at a time of from about two weeks to about four months after injection of the antigen.

33. The method of claim 30, wherein the DNA molecule is injected at a time of from about one month to about two months after injection of the antigen.

34. The method of claim 30, wherein the first and second electrical currents have field strengths of from about 125 V/cm to about 233 V/cm.