Synthetic transfection vectors

Details Synthetic transfection vectors Synthetic transfection vectors

2001-10-04

2004-10-26

Priebe, Scott D. (Department: 1635)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C435S320100, C424S489000, C424S490000, C424S493000, C424S497000, C424S491000

Reexamination Certificate

active

06809082

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to synthetic transfection vectors.
This invention is in the field of gene therapy and concerns the design and use of an entirely novel means of safely introducing therapeutic genes into mammalian and human cells to achieve various useful effects.
Steady progress over the past twenty years in the field of genetic engineering, nucleic acids research, chromosome mapping, DNA cloning, and other related fields has brought modern medicine to the threshold of gene therapy. It may be possible to treat some diseases by constructing pieces of DNA or RNA which code information which could correct the physiological malfunction causing the disease. It may also be possible in agriculture to make certain beneficial changes in the protein product of some animals or plants.
A crucial limiting factor in progress in this field is the difficulty of causing such new genes to enter the cells of intact organisms where they can commence doing their work. For some small animals and plants, genes have been introduced into the small number of cells involved in the early embryo and so caused to replicate and ultimately appear in many or all of the cells of the adult organism. However, this approach is not viable for treating diseases in human children or adults where the disease is discovered after conception or more commonly after birth. Further, in agriculture, this method proves to be extremely expensive and difficult to carry out when large numbers of large animals such as cattle are meant to be so treated.
A variety of methods have been attempted for introducing genes into adult animals. These methods include direct injection of naked DNA plasmids into individual cells, attempts at reapplying calcium phosphate transfection techniques, inclusion of DNA into liposomes, and construction of simulated viruses which can carry the new DNA as a sort of infection. The greatest progress has been made with a group of techniques in which DNA is coated onto gold colloid particles and the particles then subject to powerful electromagnetic fields in order to accelerate them to high speeds and so to hurl them against the cell walls of tissues. The particles plunge through the tissue surface and many viable DNA chains arrive inside the cell along with their non-degradable gold carrier. To reach tissues other than skin, a surgical operation is performed, and e.g. the tip of the liver is exposed and then a bombardment is carried out. This permits access only to surface layers of exposed tissues, is obviously injurious (since petechial hemorrhages immediately appear on the tissue surface), and deposits substantial amounts of non-degradable degradable gold in the tissues.
In the method of this invention, the new DNA, RNA, plasmids, ribosomal particles, nucleic acid binding proteins and any other necessary molecules are caused to adhere to the outer surface of any one of a variety of metal oxide or mixed metal crystals of coated or uncoated type or to He attached to the surface of or included in the body of a variety of other types of biodegradable particles of appropriate size and capable of surface attachment to a cell adhesion molecule. These particles are in the size range of 5 to 100 nm in diameter including all attached coatings and other surface molecules. Included on the surface is one of a variety of nerve adhesion molecules or muscle adhesion molecules which bind to the surface of nerve and muscle cells, but preferrably to muscle cells.
When such particles are constructed and then administered by routine percutaneous intramuscular injection, an exceedingly safe and efficient transfection process is initiated. The particles adhere to the outer surfaces of muscle cells and to the outer surfaces of the axon termini of motor nerve cells or preferably to the dendritic or sensory process of sensory axons within the muscle. After adherence, the particles are ingested into the nerve and muscle cells by a natural process termed adsorptive endocytosis.
Experiments carried out by the inventor have demonstrated a surprising efficiency for the uptake of such particles after intramuscular injection. Further, particularly when such particles are made of iron salts, the particles are completely biodegradable. Normally, particulate material injected into muscle is rapidly cleared by the lymphatic system and the particles are taken into lysosomal vesicles where they are subject immediately to degradative enzymes. However, the inventor has shown that when the process of adsorptive endocytosis by muscle cells is entrained, the bulk of the injected material is carried into protected compartments within neural and muscular cells.
Many cells have means of destroying any foreign DNA or RNA which appears in their cytoplasmic compartments, however muscle cells are uniquely ineffective at destroying incoming nucleic acids. In this fashion, and using intramuscular injection, the agents can be caused to enter the very large intracellular volume provided by the cells of muscles. Upon uptake by neurons, it is also possible to take advantage of the natural ability of the dendritic processes of neurons to carry out protein synthesis from RNA at great distance from the controlling influence of the neuron cell body. Use of sensory specific nerve adhesion molecules such as Nerve Growth Factor is helpful at efficiently selecting sensory rather than motor neurons where this is useful. In some situations, it may be useful to inject the agent into or near and dorsal root ganglion so that the agent can be carried by axonal transport to reach all of the tissues innervated by sensory processes from cells in the ganglion.
Treatment of muscle cells or treatments where gene therapy products are dumped into the neuromuscular synapse after production in the nerve process terminus are particularly helpful for treating disorders such as muscular dystrophy or other diseases which particularly affect muscle or for treating diseases which affect neuromuscular transmission.
It must be noted, however, that such agents are sufficiently small that they can be safely injected intravenously. Because of their potentially hydrophilic coatings with e.g. dextran, the inventor has shown extended plasma half life for such agents with up to 25% of the initial injectate remaining in circulation for over four 10 hours. This provides targeting access to a wide variety of cells in the blood marrow, circulating blood, and various glands and tissues. In all these cases, selection of appropriate targeting molecules for these particles will cause preferential adsorption to various useful cell types. While efficiency of phagocytosis of selectively adsorbed particles varies among tissues, there are a very wide variety of accessible intracellular sites. When the metal oxide core is constructed in such a way as to demonstrate superparamagnetism, then external magnetic fields (as from U.S. Pat. No 4,869,247) can be used to aid in targeting the agents.
In one example of synthesis of such compounds, the nucleic acid attachment to the particle is effected by specific nucleic acid binding proteins. A DNA plasmid or strand is constructed to include both the desired treatment gene and a segment with very high affinity for a selected nucleic acid binding the attachment DNA segment to immobile latex particles using a cyanogen bromide immobilisation technique. Various nucleic acid binding proteins and other cell constituents are then passed through an affinity column made up to such DNA tagged latex particles. The specific fraction of nucleic acid binding protein is the eluted for use in making the particle.
A mixture of ferrous and ferric chloride salts is dissolved in a saturated dextran solution after the fashion of U.S. Pat. No. 4,452,773 and precipitated by addition or 7.55 ammonia solution. The product is then moved into 0.1 M acetate buffer pH 6.4 by Sephadex 150 column filtration, concentrated with Amicon Centriprep 30 ultrafilters, and passed through a 2.5 cm by 20 cm column of Sephacryl 200 to clear gelatinous hydrous oxides and excess dextr

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