SKIN AND MUSCLE-TARGETED GENE THERAPY BY PULSED ELECTRICAL FIELD

• US 20020099323A1
• Filed: 07/13/1999
• Published: 07/25/2002
• Est. Priority Date: 07/13/1998
• Status: Active Grant

• Current Assignee: Genetronics, Inc. (Inovio Pharmaceuticals, Inc.)
• Original Assignee: Genetronics, Inc. (Inovio Pharmaceuticals, Inc.)
• Inventors: ZHANG, LEI, NOLAN, EDWARD, RABUSSAY, DIETMAR P., HOFMANN, GUNTER A., WIDERA, GEORG, DEV, NAGENDU B., TONNESSEN, ARNT
• Granted Patent: US 6,697,669 B2
• Time in Patent Office: Days
• US Class Current: 604/21
• CPC Class Codes: A61N 1/0424 Shape of the electrode
• CPC Class Codes: A61N 1/0452 Specially adapted for transcutaneous muscle stimulation [TMS]
• CPC Class Codes: A61N 1/306 Arrangements where at least part of the apparatus is introduced into the body
• 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 1/327 for enhancing the absorption properties of tissue, e.g. by electroporation

Abstract

The present invention describes an in vivo method, using pulsed electric field to deliver therapeutic agents into cells of the skin and muscle for local and systemic treatments. In particular, therapeutic agents include naked or formulated nucleic acid, polypeptides and chemotherapeutic agents.

Claims

1. An in vivo method for introducing a therapeutic agent into skin or muscle cells of a subject, said method comprising applying a pulsed electric field to said skin or muscle cells substantially contemporaneously with the application of said therapeutic agent to said skin or muscle cells, such that said therapeutic agent is introduced into said skin or muscle cells.

2. A method according to claim 1, wherein said electric pulse is of sufficient voltage and duration to cause electroporation.

3. A method according to claim 2, wherein, substantially contemporaneously with said application of said electric pulse, a second electric pulse is applied to said skin or muscle cells, said second electric pulse being of sufficient voltage and time to cause transmembrane permealization.

4. A method according to claim 1, wherein said therapeutic agent is selected from a chemotherapeutic agent, a nucleic acid sequence, a polypeptide, and combinations thereof.

5. A method according to claim 4, wherein said nucleic acid sequence is selected from the group consisting of double stranded DNA, single-stranded DNA, complexed DNA, encapsulated DNA, naked RNA, encapsulated RNA, and combinations thereof.

6. A method according to claim 4, said nucleic acid encoding a therapeutic polypeptide, and said nucleic acid sequence being operably associated with a regulatory sequence such that said therapeutic polypeptide is expressed in said skin or muscle cells.

7. A method according to claim 6, wherein said polypeptide comprises an antigen.

8. A method according to claim 6, wherein said polypeptide comprises a pathogen-derived protein, or a peptide hormone.

9. A method according to claim 8, wherein said peptide hormone is selected from the group consisting of calcitonin (CT), parathyroid hormone (PTH), erythropoietin (Epo), insulin, a cytokine, a growth hormone or a growth factor.

10. A method according to claim 6, wherein said polypeptide is a blood coagulation factor or a lymphokine.

11. A method according to claim 10, wherein said lymphokine is selected from the group consisting of tumor necrosis factor, interleukins 1, 2, and 3, lymphotoxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, alpha-interferon, beta-interferon, gamma-interferon and subtypes thereof.

12. A method according to claim 6, wherein said blood coagulation factor is Factor VIII or Factor IX.

13. A method according to claim 6, wherein said nucleic acid sequence further encodes a selectable marker polypeptide.

14. A method according to claim 6, wherein said regulatory sequence comprises a promoter.

15. A method according to claim 14, wherein said promoter is skin or muscle specific.

16. A method according to claim 14, wherein said promoter is selected from the group consisting of CMV, RSV LTR, MPSV LTR, SV40, and keratin specific promoters.

18. A method according to claim 17, wherein said electric pulse is of sufficient voltage and duration to cause electroporation.

19. A method according to claim 18, wherein, substantially contemporaneously with said application of said electric pulse, a second electric pulse is applied to said skin or muscle cells, said second electric pulse being of sufficient voltage and time to cause iontophoresis.

20. A method according to claim 17, wherein said immune response-inducing agent is selected from a nucleic acid sequence, a polypeptide, and combinations thereof.

21. A method according to claim 20, said nucleic acid encoding a polypeptide, and said nucleic acid sequence being operably associated with a regulatory sequence.

22. A method according to claim 21, wherein said regulatory sequence comprises a promoter.

23. A method according to claim 22, wherein said polypeptide comprises an antigen.

24. A method according to claim 17, wherein said immune response is a humoral response.

25. A method according to claim 17, wherein said immune response is a cellular response.

26. A method according to claim 17, wherein said immune response includes a cellular and a humoral response.

27. A method according to claim 1, wherein said electric pulse comprises a square wave pulse of at least 50 V for about 10 up to 20 ms.

28. A method according to claim 1, wherein said electric pulse is monopolar or bipolar.

29. A method according to claim 17, wherein said electric pulse comprises an exponential decay pulse of 120 V for about 10 up to 20 ms.

30. A method according to claim 17, wherein said electric pulse is monopolar or bipolar.

31. A method according to claim 27, wherein said electric pulse is applied through an electrode selected from the group consisting of a caliper electrode, a meander electrode, a needle electrode, a microneedle array electrode, a micropatch electrode, a ring electrode, and combinations thereof.

32. A method according to claim 31, wherein each electrode of said caliper electrode has an area of about 1 cm².

33. A method according to claim 31, wherein said caliper electrode is applied to a skin fold having a thickness of about 1 mm to about 6 mm.

34. A method according to claim 29, wherein said electric pulse is applied through an electrode selected from the group consisting of a caliper electrode, a meander electrode, a needle electrode, a microneedle array electrode, a micropatch electrode, a ring electrode, and combinations thereof.

35. A method according to claim 34, wherein each electrode of said caliper electrode has an area of about 1 cm².

36. A method according to claim 34, wherein said caliper electrode is applied to a skin fold having a thickness of about 1 mm to about 6 mm.

37. A method according to claim 17, wherein said immune response is protective.

40. A micropatch electrode according to claim 39, wherein said patch elements are separated by electrically insulating material.

41. A micropatch electrode according to claim 40, wherein said electrically insulating material can be pierced by an injection needle.

42. An electrode kit for use in conjunction with electroporation therapy, said kit comprising:

a) a micropatch electrode according to claim 39, and

b) an injection needle, optionally comprising one or more holes disposed along its length and proximal to the needle tip,

wherein said holes are in fluid communication with the hollow interior of said injection needle.

17. A method for inducing an immune response in a subject, said method comprising applying a pulsed electric field to skin or muscle cells of said subject substantially contemporaneously with the application of an immune response-inducing agent to said skin cells or muscle, such that said immune response-inducing agent is introduced into said skin or muscle cells thereby inducing in said subject an immune response.

38. A method for the therapeutic application of electroporation to skin or muscle cells of a subject for introducing topically applied molecules into said cells, said method comprising:

a) providing an array of electrodes, at least one of the electrodes having a needle configuration for penetrating tissue;

b) inserting the needle electrode into selected tissue;

c) positioning a second electrode of the array of electrodes in conductive relation to the selected tissue; and

d) applying pulses of high amplitude electric signals to the electrodes, proportionate to the distance between the electrodes, for electroporation of said tissue;

such that said topically applied molecules are introduced into said skin or muscle cells.

39. A micropatch electrode for use with an electroporation apparatus, said micropatch electrode comprising a substantially planar array of patch elements, each patch element comprising two sets of electrodes, wherein each set of electrodes comprises a first electrode and a second electrode electrically insulated from one another, such that when different electric potentials are applied to said first and second electrodes, a voltage is produced therebetween.

43. An electrode for use with an electroporation apparatus, said electrode comprising:

a) a ring-shaped electrode having an electrically insulating shield, wherein said electrically insulating shield provides support to said ring-shaped electrode and electrically insulates a tissue under treatment employing said electrode, and

b) an electrically conducting injection needle, optionally comprising one or more holes disposed along its length and proximal to the needle tip, wherein said holes are in fluid communication with the hollow interior of said injection needle, wherein a potential difference applied to said ring-shaped electrode and said needle electrode creates a voltage therebetween.

44. An electrode for use with an electroporation apparatus, said electrode comprising an array of a plurality of paired electrode needles and, at least one injection needle, wherein a potential difference applied said paired electrode needles creates a voltage therebetween.

45. An electrode according to claim 44, said injection needle comprising one or more holes disposed along its length and proximal to the needle tip, wherein said holes are in fluid communication with the hollow interior of said needle.

47. An electrode according to claim 46, wherein said suction generating device is a syringe comprising a piston slidably engaged with, and sealingly disposed about said injection needle electrode.

50. A needle electrode according to claim 48, wherein said substance-releasing material is a porous material.

46. An electrode for use with an electroporation apparatus, said electrode comprising a suction generating device comprising a ring electrode disposed about an injection needle electrode, such that when said ring electrode is contacted with skin of a subject and suction is generated by said suction generating device, said skin is pulled up around the injection needle electrode, causing the injection needle electrode to pierce the skin, and

wherein a potential difference applied to said ring-shaped electrode and said injection needle electrode creates a voltage therebetween.

48. A needle electrode for use with an electroporation apparatus, said needle electrode comprising an injection needle and paired electrically conducting wires disposed within the hollow core of said injection needle and protruding from the tip thereof, said electrically conducting wires having an electrically insulated portion and an exposed electrically conducting portion, said exposed electrically conducting portion being at the end of said wires protruding from the tip of said injection needle.

49. A needle electrode for use with an electroporation apparatus, said needle electrode having disposed around a portion of its length a substance-releasing material.

51. A needle electrode for use with an electroporation apparatus, said needle electrode comprising:

a) a hollow central core,

b) at least four holes along its length, wherein said four holes comprise two paired holes, each of said two paired holes comprising a hole proximal to the tip of said needle and a hole distal to said tip of said needle, and

c) a pair of electrically conducting wires having an electrically insulated portion and an exposed electrically conducting portion,

wherein said pair of electrically conducting wires are located in said hollow core, except for said exposed electrically conducting portion which runs outside of said needle, extending outward from said distal paired hole to said proximal paired hole where it re-enters said hollow core.

52. A needle electrode for use with an electroporation apparatus, said electrode comprising a plurality of electrically conducting needles disposed within a depth guide, wherein said tip of said needles extend for a predetermined distance beyond said depth guide.