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Apparatus for generating electrical pulses and methods of using the same

a technology of electrical pulses and apparatus, applied in the direction of antibody medical ingredients, drug compositions, therapy, etc., can solve the problems of cell type, especially those that grow in suspension, that the cell type cannot be effectively transfected by electropermeabilization, so as to enhance gene expression in a cell

Inactive Publication Date: 2006-11-30
EASTERN VIRGINIA MEDICAL SCHOOL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] In accordance with at least one embodiment of the invention, a method of enhancing gene expression includes providing a preparation comprising the cell and the nucleotide sequence to be delivered into the cell, and applying nanosecond pulse electric fields to said preparation, wherein said application facilitates the entry of the agent into the nucleus.
[0023] In accordance with one or more embodiments of the invention, a method is provided for enhancing gene expression in a cell using a multi-pulse generator. The method comprises the steps: charging a capacitor; triggering a high voltage, high current transistor to initiate discharge of the charge accumulated in the capacitor into at least one cell to cause electroporation at the plasma membrane of the at least one cell; triggering the high voltage, high current transistor to stop the discharge of the capacitor after a predetermined long duration; actuating a switch to decouple the capacitor from the at least one cell; charging a transmission line; triggering a high voltage switch to initiate discharge of the charge accumulated in the transmission line into the at lest one cell to cause electroporation at the nuclear membrane of the at least one cell; and triggering the high voltage switch to stop discharge of the transmission line after a predetermined short duration.

Problems solved by technology

Alternatively, using different electrical or cellular conditions, electroporation can cause rupture / death of cells.
Some cell types, especially those that grow in suspension, can only be effectively transfected by electropermeabilization.

Method used

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  • Apparatus for generating electrical pulses and methods of using the same
  • Apparatus for generating electrical pulses and methods of using the same
  • Apparatus for generating electrical pulses and methods of using the same

Examples

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example 1

[0098] As stated above, experiments were performed in which electropermeabilization of cells was followed by exposure of these cells to nsPEFs. These experiments resulted in increased expression of a green fluorescent protein (GFP) reporter gene in HL-60 cells. FIGS. 1-6 show the effects of different pulses on HL-60 cells.

Materials and Methods

[0099] HL-60 cells were removed from growth media, washed, and re-suspended in low conductivity buffer (LCB) containing 0.85 mM K2HPO4, 0.3 mM K2HPO4 (pH 7.2), and 10 mM KCl (conductivity 1.5 mS / cm at 22° C.). Osmolality was adjusted to 290 mOsm by the addition of inositol. Cell suspensions (106 cells / ml) were loaded into the BioRad gene Pulser® cuvettes (Bio-Rad Laboratories, Hercules, Calif.) prior to nsPEF pulsing. A cable pulse generator was use to deliver the NsPEF pulses. NsPEFs were delievered by means of a cable pulse generator to cells suspended in a cuvette with parallel plate electrodes separated by 0.1, 0.2, or 0.4 cm. Briefly, t...

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Abstract

A method and apparatus are provided for delivering an agent into a cell through the application of nanosecond pulse electric fields (“nsPEF's”). The method includes circuitry for delivery of an agent into a cell via known methods followed by the application of nanosecond pulse electric fields to said cell in order to facilitate entry of the agent into the nucleus of the cell. In a preferred embodiment, the present invention is directed to a method of enhancing gene expression in a cell comprising the application of nanosecond pulse electric fields to said cell. An apparatus for generating long and short pulses according to the present invention is also provided. The apparatus includes a pulse generator capable of producing a first pulse having a long duration and low voltage amplitude and a second pulse having a short duration and high voltage amplitude.

Description

REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 487,932, filed Jul. 18, 2003, U.S. Provisional Application No. 60 / 499,921, filed Sep. 4, 2003, and U.S. Provisional Application No. 60 / 526,585, filed Dec. 4, 2003, the disclosures of which are incorporated herein by reference.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] The present invention was made with Government support under AFOSR MURI Grant No. F49620-02-1-0320 awarded by the United States Air Force Office of Scientific Research. The Government has certain rights in the invention.BACKGROUND OF THE INVENTION [0003] Both the detection and use of electricity / electric fields in medicine and biology are widespread and well accepted. Electrocardiography (EKG) and electroencephalography (EEG) are used to detect electrical activity in the heart and brain, respectively. Cardioversion, the application of a pulsed elect...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K39/395C12N15/87A61NA61N1/32C12M3/00
CPCA61K48/00A61N1/0412C12N15/87C12M35/02C12N13/00A61N1/327A61P21/00A61P35/00A61P35/02
Inventor BEEBE, STEPHEN J.SCHOENBACH, KARL H.
Owner EASTERN VIRGINIA MEDICAL SCHOOL
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