Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-12-23
2002-11-05
Nguyen, Dave T. (Department: 1633)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S091400, C435S320100, C435S455000, C424S486000, C424S497000
Reexamination Certificate
active
06475994
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to compositions of matter and methods which are useful in delivering genetic materials to the interior of plant and animal cells, and more particularly to gene transfection particles and particle bombardment methods for gene transfection.
BACKGROUND OF THE INVENTION
Particle bombardment, also know as the gene gun method, provides a potentially effective method for introducing genetic materials into plant and animal cells in the treatment and control of a variety of genetic, neoplastic and infectious diseases, and for creating transgenic species. Particle bombardment methods of introducing genetic materials into plant and animal cells involves accelerating microscopic particles coated with a genetic material through the wall of the cell to deliver the genetic material to the interior of the cell. The motive force used to accelerate the particles can be generated by high-voltage electric discharge, helium pressure discharge or other means. The microscopic projectile transfection particles generally comprise a gold particle having a diameter of from about 1 to 15 micrometers, and genetic materials attached thereto. Gold particles are preferred because they are chemically inert, have no cytotoxic effects in the cells, and have a high density which permits greater momentum and penetration into cells. Binding agents such as spermidine (N-3-aminopropyl-1-4-butanediamine) have been used to attach genetic materials to gold particles.
A disadvantage with known particle bombardment methods is that they may not provide a sufficient quantity of genetic material to achieve a desired therapeutic effect without causing unacceptably high levels of cell damage. Ning-Sun Yong et al., in Gene Therapeutics: Methods and Applications of Direct Gene Transfer, have reported that for confluent monolayer cell cultures with cells 15-20 micrometers in size, a particle bombardment density of 0.1 particles per square centimeter of 0.9 micrometer particles, which delivers about two particles per cell, results in more than 90% of monolayer cells and 75% of suspension cells being viable and healthy after particle bombardment, but that for most tissue samples, higher particle bombardment densities cause excessive cell and tissue damage. Because of the relatively low binding capacity of the linear polyamines used to attach genetic materials to gold particles, and the relatively low levels of particle bombardment which can be tolerated without excessive cell damage, known transfection particles may not be capable of introducing sufficient quantities of genetic materials to cells to achieve a desired therapeutic or other effect while avoiding excessive cell damage.
Accordingly, there is a need for transfection particles which are capable of delivering higher amounts of genetic materials into cells using particle bombardment methods while minimizing cell damage.
SUMMARY OF THE INVENTION
The invention provides highly efficient transfection particles and bombardment methods which are capable of delivering higher amounts of genetic materials to cells while minimizing cell damage. The gene transfection particles of this invention comprise a composite material including a polymer, a support particle conjugated with the polymer, and genetic material conjugated with the polymer.
The bombardment method of this invention involves the steps of forming a gene transfection particle including a polymer, and genetic material conjugate, with or without a support particle, and accelerating the gene transfection particle toward a cell with sufficient motive force to cause the gene transfection particle to penetrate and enter the cell.
An advantage of the invention is that it provides a method whereby the density of the gene transfection particles can be adjusted as required depending on the robustness of the cell membrane which must be penetrated.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The gene transfection particles of the preferred embodiment of the invention preferably include genetic material which is conjugated to polymer, and a metal, in the form of one or more atoms, one or more ion complexes, clusters, or particles, or other support which is also conjugated to the polymer. The metal component provides the gene transfection particles with the density needed to achieve the momentum which is necessary to penetrate the cell wall and/or membrane when the particles are accelerated toward the cell so that the carried genetic material can be delivered to the interior of the cell. The metal used in preparing the gene transfection particles must be chemically inert in the cell environment, have essentially no, or at least very low, cytotoxic effects in the cells, and be capable of being conjugated to a dendritic polymer. The metal preferably has a relatively high density to allow greater momentum and, hence, adequate penetration of the cell wall and/or membrane. Gold is presently preferred because of its established acceptance for use in gene therapy. However, other metals may be employed in certain applications. Examples of metals which may be suitable for use in particle bombardment gene therapy methods include gold, tungsten, silver, copper, magnesium, calcium and combinations thereof.
The metal component may be conjugated to the dendritic polymer in the form of an individual atom, ion, or complex, or in the form of clusters of atoms or microscopic size particles. In addition to metal particles, other suitable supports includes silica particles, alumina particles, and other solid supports having Lewis acid surface functionality. Also, it has been determined that dendritic polymers conjugated to genetic materials, without any metals or other support materials conjugated thereto may also be usefully employed as gene transfection particles in particle bombardment methods.
The gene transfection particles prepared by contacting a dendritic polymer with a metal atom or a metal atom-containing entity, and a genetic material may have a diameter or maximum dimension of from about 1 nm to about 15 nm. Particles comprised of dendritic clusters or aggregates can have a maximum dimension of from about 2 nm up to at least several micrometers. However, the gene transfection particles of this invention preferably have a particle size of from about 1 nm to about 1000 nm, and more preferably from about 1 nm to about 100 nm.
The genetic materials which may be used in preparing the gene transfection particles of this invention include biological response modifiers, such as interleukins, interferons, and viruses, viral fragments and other genetic materials. The term “genetic material” as used herein refers to nucleotide based materials, including without limitation, viruses and viral fragments, deoxyribonucleic acid (DNA), plasmids, ribonucleic acid (RNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), catalytic RNA (cRNA), smaller nuclear RNA (snRNA), exons, introns, codons, and anti-sense oligonucleotides. Genetic material, especially viruses and viral fragments, may incidentally include some protein.
The polymers are preferably dendritic, but linear and other non-dendritic polymers may be suitable. The dendritic polymers used in preparing the gene transfection particles of the preferred embodiment include generally any dendritic polymers which include functional groups, on the interior and/or exterior, which are capable of binding or conjugating the metal with the dendritic polymer. The dendritic polymers used to coat the metal particles have surface functional groups which have an affinity for (i.e., will stick to) the surface of the support particles, with the preferred dendritic polymers being those having amine functional groups e.g., such as PAMAM, POPAM and PEI dendritic polymers. The support particles and the dendritic polymers are contacted under conditions sufficient to cause the dendritic polymer molecules to adhere to the surface of the support particles. Thereafter, the dendritic polymer-support particle conjugate is contacted with genetic material to form a highly effic
Balogh Lajos
Tomalia Donald A.
Kimble Karen L.
Nguyen Dave T.
Tomalia Donald A.
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