Ocular gene therapy using avalanche-mediated transfection
View Patent ↗The present invention provides a method of treating an ocular disease in a subject. In a first step, a nucleic acid is introduced into cells or a tissue. The nucleic acid is introduced by electron avalanche transfection. With this technique, a high electric field induces a vapor bubble and plasma discharge between an electrode and the surrounding medium. The formation of a vapor bubble generates mechanical stress. Plasma discharge through the ionized vapor in the bubble enables connectivity between the electrode and the surrounding medium, so that mechanical stress and electric field are applied simultaneously, which results in permeabilization of the cells or tissue. This permeabilization in turn allows the nucleic acid to enter the cell or tissue. Cells or tissue containing the nucleic acid are then transplanted into an ocular region of the subject.
1. A method of delivering a nucleic acid to an ocular region of a subject, comprising:
providing a nucleic acid and target cells or target tissue, the target cells being an autologous or allogeneic cell line or being isolated from an autologous or allogeneic individual and the target tissue being from an autologous or allogeneic individual;
applying a voltage of about 100 V to about 10 kV for a duration of about 20 μsec to about 100 msec to an electrode to generate both an electric field and a mechanical stress wave, the electrode positioned to provide both the electric field and the mechanical stress wave to the target cells or target tissue sufficient to transfect the nucleic acid into the cells or the tissue; and
transplanting the transfected cells or the transfected tissue into an ocular region of the subject,
wherein the nucleic acid is expressed in the ocular region.
2. The method of claim 1 , wherein generating both the electric field and the mechanical stress wave comprises forming a plasma discharge.
3. The method of claim 1 , further comprising isolating the cells or the tissue from the subject before transfection.
4. The method of claim 1 , further comprising culturing the cells or the tissue prior to transplanting the transfected cells or the transfected tissue.
5. A method of delivering a nucleic acid to an ocular region of a subject, comprising:
placing an electrode at a target tissue within the ocular region of the subject;
providing a nucleic acid to the target tissue; and
applying voltage of about 100 V to about 10 kV for a duration of about 20 μsec to about 100 msec to the electrode to form a plasma discharge,
the electrode providing both an electric field and a mechanical stress wave to the tissue sufficient to deliver the nucleic acid into the tissue.
6. The method of claim 1 , wherein the cells or the tissue originate from the ocular region of the individual.
7. The method of claim 6 , wherein the cells or the tissue is conjunctival fibroblasts, scleral cells, epithelial cells, or cells of the ciliary body or ciliary muscle.
8. The method of claim 1 , wherein the subject has a condition selected from the group consisting of age-related macular degeneration, choroidal neovascularization, retinal degeneration, glaucoma, diabetic retinopathy, and retinal dystrophies.
9. The method of claim 1 , wherein the cells or the tissue are transplanted by placing the cells or the tissue in an implantable device, and implanting the device within an ocular region of the subject.
10. The method of claim 1 , wherein the nucleic acid is DNA.
11. The method of claim 1 , wherein the nucleic acid encodes a therapeutic protein or shRNA.
12. The method of claim 5 , wherein the target tissue comprises conjunctival fibroblasts, scleral cells, epithelial cells, or cells of the ciliary body or ciliary muscle.
13. The method of claim 5 , wherein the subject has a condition selected from the group consisting of age-related macular degeneration, choroidal neovascularization, retinal degeneration, glaucoma, diabetic retinopathy, and retinal dystrophies.
14. The method of claim 5 , wherein the nucleic acid is expressed in the ocular region.
15. The method of claim 5 , wherein the nucleic acid is DNA.
16. The method of claim 5 , wherein the nucleic acid encodes a therapeutic protein or shRNA.
17. The method of claim 1 , wherein the voltage is about 100 V to about 1 kV.
18. The method of claim 1 , wherein the duration of the voltage pulse is about 20 μsec to about 1 msec
19. The method of claim 5 , wherein the voltage is about 100 V to about 1 kV.
20. The method of claim 5 , wherein the duration of the voltage pulse is about 20 μsec to about 1 msec.