Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses
Reexamination Certificate
1995-05-05
2002-02-19
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Modification of viruses
C435S173300
Reexamination Certificate
active
06348338
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method whereby living cells modified by a biological substance, introduced into them by ballistic transfer, are efficiently separated from remaining unmodified cells.
BACKGROUND OF THE INVENTION
Many methods of modern cell biology require the transfer of matter, mainly nucleic acids, into living cells (hereafter referred to as transfection). Traditionally, this transfer of matter has been important to both the fields of biological and medical research. Recent progress, however, in the understanding of the body's functions as regarded to molecular mechanisms has led to the idea of treating human desease by using molecular approaches (colloquially referred to as “gene therapy”). Many of the biological methods suggested in this approach require the transfection of somatic cells. A number of techniques have been developed to achieve this aim: microinjection; electroporation; transfection by viral vectors or liposomes; and direct bombardment of cells with particles (“gene gun”). For a review on methods see
Methods in Enzymology
217, (1993), pp. 461-655, (Academic Press, San Diego, Calif.).
Apart from microinjection, in which a single cell is injected directly with the transfecting matter, these methods suffer from a rather low and unreliable efficiency, efficiency being measured as percentage of successfully transfected cells out of total of treated cells. Microinjection's efficiency is very high; however, the number of treated cells is generally too low for this technique to be clinically valuable.
If the object of the transfection is to insert genetic information into the cell, then successful transfection requires the passage of the transfecting nucleic acid not only into the cell cytoplasm, but into the nucleus. The nuclear membrane is a barrier more difficult to cross than the cytoplasmic membrane. Many of the cells transfected by means of electroporation or lipofection that have incorporated the transfecting matter into their cytoplasm, will not express any genetic message transferred into them. For expression of any genetic message to happen, the genetic message has to pass into the nucleus. The transfection of cells with DNA by electroporation is most likely successful only when it happens during cell division, because the division process momentarily renders the nucleus permeable for the transfecting DNA.
In contrast, the ballistic transfection method achieves transport into the nucleus by the kinetic energy of the passing particle. The probability of nuclear passage of the microcarrier particle is governed by the ratio of nucleus diameter to cell diameter, which for many cells, is very favourable for nuclear passage. Thus, it can be expected that any clinical approach to transfection of cells with DNA would increase efficiency, employing the ballistic transfection method.
A current estimate of the number of transfected cells needed in a clinical protocol is in the order of 10
7
-10
8
cells. For the reasons given above, we believe that of the transfection methods mentioned, the ballistic transfer, i.e. directly bombarding cells with particles that carry the transfecting matter into the cells, has the greatest potential to achieve this aim.
Various embodiments of the idea of bombarding cells in order to achieve transfection have been published. They differ in the propulsion of the particles, the nature of the particles and various other aspects. A number of patents have been filed describing these embodiments (see: Jones, Frey, Gleason, Chee, Slightom: Gas driven microprojectile accelerator and method of use U.S. Pat. No. 5,066,587; Jones, Frey, Gleason, Chee, Slightom: Gas driven microprojectile accelerator WO 9111526, U.S. Pat. No. 471,216; Sanford, Wolf, Allen: Apparatus for delivering substances into cells and tissues in a non-lethal manner EP 0 331 855; Tome: improved particle gun EP 0 397 413; Brill, McCabe, Yang: Particle-mediated transformation of animal somatic cells WO 91/00359; Mets: Aerosol beam injector WO 91/00915; WO 91/02071; Johnston, Williams, Sanford, McElligott: Particle-mediated transformation of animal tissue cells WO 91/07487; Bruner, deVit, Johnston, Sanford: Improved method and apparatus for introducing biological substances into living cells WO 91/18991; Bellhouse, Sarphie: Ballistic apparatus. WO 9204439, GB 9018892.1). However, only one embodiment to our knowledge, is commercially manufactured. This embodyment is the “Biolistic” apparatus invented by John C Sanford and manufactured under licence from Cornell University and DuPont by Bio-Rad (Hercules, Calif.). The propulsion of the microcarriers is achieved by adsorbing the microcarriers to a macrocarrier polymer sheet, which is accelerated towards the cells by a cold gas shock wave. After retaining the macrocarrier, the microcarrier sheaf continues towards the target cell layer, eventually impacting and unloading the adsorbed transfecting matter into the cells.
The method of ballistic transfection implies that only a (sometimes large) fraction of the target cells is transfected successfully. The microcarrier sheaf is rarely homogeneous, and has to be of sufficiently small density in order not to kill too many of the target cells, which invariably suffer from stress exerted on them by both the shock wave and the impacting microprojectiles. A balance must be found between a high survival rate and a high transfection rate, which leaves part of the target cells untransfected.
In many of the plausible clinical uses, a separation of transfected cells from untransfected cells is desirable, if not strictly required. Ex vivo transfection of tumor cells for cancer gene therapy is only one example. The current state of the art employs time-consuming separation protocols based on expression of markers. The genetic information for these markers is introduced into the cell with the transfecting DNA. This procedure requires cell culture of the transfected cells ex vivo for a prolonged period of time, raising the risk of both contamination and alteration of cell characteristics. A method enabling a quick, simple separation of transfected cells would clearly be of great value.
Magnetic separation techniques have been in use in biology for years. These methods primarily employ paraferromagnetic beads, the size of which ranges in micrometers. Paramagnetic particles of such size retain some residual magnetic orientation after removal of an external magnetic field, leading to aggregation in solution. Recently, a new separation technique has been introduced (Miltenyi: Methods and materials for high gradient magnetic separation of biological materials WO 90/07380; DE 3720 844) that is based upon the coupling of biological material onto submicroscopic-size magnetic particles. These particles have the property of being “super-paramagnetic,” meaning their magnetic core is smaller than the the size of a Weiss domain: the area of similar magnetic dipole orientation within a paraferromagnetic solid. In the absence of an external magnetic field, these particles do not retain any macroscopic magnetic orientation; thus do not attract each other, making them ideal for suspension in fluids. Their retainment requires a very strong “high gradient” magnetic field, since the magnetic force exerted on the particle is small due to its minimal size. A typical separation apparatus employs a mesh of iron wool embedded in polymer, through which the suspension is passed. In the presence of a strong magnetic field, the local field inside the wool mesh is strong enough to retain the particles. After removal of the external field, the particles can be washed out.
BRIEF DESCRIPTION OF THE DRAWINGS
The cytograms show the results of fluorescence measurements. The abscissa represents fluorescence in log scale, the ordinate cell number. U represents unsorted cells after ballistic transfer; M and N represent the magnetic and nonmagnetic fractions, respectively.
BRIEF SUMMARY OF THE INVENTION
The invention refers to a method by which cells that have been transfected by ballistic transf
Schroff Matthias
Wittig Burghardt
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