Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2001-04-05
2003-09-30
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S173100, C435S173600, C435S461000, C204S450000, C204S157620, C204S600000
Reexamination Certificate
active
06627421
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the application of energy to biological samples for the purpose of inducing transfection and cell transformation. More particularly, the invention relates to methods and systems, including a robotic system, for applying multi-mode energy, including sonic, optical and electromagnetic energy, and combinations thereof, to biological samples.
BACKGROUND OF THE INVENTION
Cells are the basic structural and functional units of all living organisms. All cells contain cytoplasm surrounded by a plasma membrane. Most bacterial and plant cells are enclosed in a rigid or semi-rigid cell wall. The cells contain DNA that may be (1) arranged in a nuclear membrane or (2) free in cells lacking a nucleus. While the cell membrane is known to contain naturally occurring ion channels, compounds that are therapeutically advantageous to cells are usually too large to pass through the naturally occurring ion channels. Conventional interventional methods for delivering compounds to cells have proved difficult in view of the need for the compounds to pass through the cell membrane, cell wall and nuclear membrane.
Molecular biology has resulted in mapping the genomes of many plants and animals including the mapping of much of the human genome. The potential for advances in the understanding of the genetic basis of diseases is great. A variety of methods have been used to insert genes into plant and animal cells. Calcium phosphate DNA precipitation has been used to deliver genetic material into cells in cell culture but efficiency of transfection and gene expression has been very low. Improved transfection has been attained with viral vectors, e.g., adenovirus and retrovirus, but difficulties with gene expression persist. In addition, substantial concerns regarding antigenicity and the potential mutagenic effects of mutant viruses exist. Liposomes, manufactured more easily than viral vectors, have shown promise as gene delivery agents. Liposomes have less biological concerns (e.g., non-antigenic) but the efficiency of transfection and gene expression using liposomes has generally been lower than with viruses.
Electroporation and sonoporation involve the application of energy to enhance transfection of genetic material into cells. For example, electroporation concerns the formation of surface “pores” to allow permeation of macromolecules into cells in the presence of an electric field. Sonoporation, on the other hand, involves the application of ultrasound to enhance transfection of genetic material into cells. There are numerous exemplary practical applications of such techniques, including screening, experimental, pharmaceutical manufacturing, and the like. Further background on sonoporation can be found in U.S. patent application Ser. No. 08/841,169, filed Apr. 29, 1997, “Methods for Delivering Compounds Into a Cell,” the content of which is hereby incorporated by reference in its entirety.
Electroporation has been studied extensively and reviewed in a number of publications. See, for example, Weaver J C, Journal of Cellular Biochemistry, (1993 April) 51 (4) 426-35. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for: introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; selective biochemical loading of one size cell in the presence of many smaller cells; introduction of virus and other particles; cell killing under nontoxic conditions; and insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including: enhanced cancer tumor chemotherapy, gene therapy, transdermal drug delivery, and noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cells and artificial planar bilayer membranes. For short pulses (microsecond to millisecond), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5 to 1.5 V. In the case of isolated cells, the pulse magnitude is 10
3
to 10
4
V/cm. These pulses cause reversible electrical breakdown (REB) accompanied by a tremendous increase in molecular transport across the membrane. Reversible electrical breakdown results in a rapid membrane discharge, with the elevated voltage U(t) returning to low values within a few microseconds of the pulse. Membrane recovery can, however, be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death.
As mentioned, sonoporation involves the use of ultrasound to enhance transfection of genetic material into cells. This phenomenon is believed to be due to mechanisms involving facilitation of uptake and molecular rearrangement of DNA to promote transcription and translation. To make more efficient use of this discovery, it would be beneficial to adapt the sonoporation technique to large scale and/or miniaturized and/or high-throughput screening of samples. One aspect of the invention described herein has utility for genomics library screening, large-scale transfection of cells, and other specialized operations where the ability to achieve high levels of transfection in a short time and in high quantity is desirable.
SUMMARY OF THE INVENTION
Accordingly, the present invention addresses the need for improved methods and systems for applying energy of various types to biological materials so as to promote the formation of “pores” and thus transfection and/or cell transformation. The invention also encompasses the use of dual function transducers allowing for either electroporation or ultrasound-mediated transfection, or a combination of both (“sonoelectroporation”), to significantly increase the potential for successful cell transformation.
In one embodiment, the present invention provides a robotic system for applying energy to cells so as to elicit the formation of pores, transfection, and/or cell transformation. The inventive system includes a computer, a plurality of acoustic probes coupled to the computer for controllably applying acoustic energy to batches of cells, and a robot coupled to the computer for effecting relative movement between the probes and the batches of cells. The present invention also encompasses an automated method for applying energy to cells, comprising the use of a computer, a robot and a plurality of acoustic probes to controllably apply acoustic energy to batches of cells and to effect relative movement between the probes and the batches of cells.
In another embodiment, the present invention provides a sonoelectroporation method comprising the application of ultrasonic energy in combination with electrical energy to cells so as to enhance cell uptake of a desired material and to also enhance subsequent gene expression.
In yet another embodiment the present invention provides a method of applying energy to cells to elicit formation of pores and to enhance transfection. The inventive method comprises the use of a first energy source to elicit pore formation and the use of a second energy source to enhance transfection. The first energy source may be any one of the group consisting of ultrasound, electricity, optical and magnetic energy, and the second energy source may be any one of the group consisting of ultrasound, electricity, optical and magnetic energy, provided the first and second energy sources are not identical.
Other aspects of the present invention are disclosed below.
REFERENCES:
patent: 5019034 (1991-05-01), Weaver et al.
patent: 5246437 (1993-09-01), Abela
patent: 5445611 (1995-08-01), Eppstein et al.
patent: 5795755 (1998-08-01), Lemelson
patent: 5830430 (1998-11-01), Unger et al.
patent: 5885211 (1999-03-01), Eppstein et al.
patent: 5977538 (1999-11-01), Unger et al.
patent: 6041253 (2000-03-01), Kost et al.
patent: WO 9740679 (1997-11-01), None
Palumbo et al., “Targeted gene transfer in euca
McCreery Thomas
Unger Evan C.
Wu Yunqiu
IMARX Therapeutics, Inc.
Weber Jon P.
Woodcock & Washburn LLP
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