Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2002-07-24
2003-04-29
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C536S023100
Reexamination Certificate
active
06555376
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a process of entrapping genetic materials in nanoparticles of inorganic compounds of size below 100 nm diameter to form non-viral carriers suitable for delivery of genes including those of therapeutic interest in appropriate cells.
BACKGROUND OF THE INVENTION
As it is known, the ability to safely and efficiently transfer foreign DNA into cells is a fundamental goal in biotechnology. In recent years, with the advent of recombinant DNA technology, a surge in research activity has occurred in the field of DNA transfer across cell lines. This activity, which has taken the shape of what is popularly known as gene therapy, is a medical/surgical intervention technique which is being developed as a ‘molecular medicine’ and requires genes to be introduced into cells in order to treat a wide variety of till now incurable human diseases. Potential applications are numerous, given the diversity of the genes to be used as well as the possible target cells.
Today's gene therapy research may be seen as pursuing intelligent drug design through a logical extension of results of fundamental biomedical research on the molecular basis of disease. The term gene therapy applies to approaches to disease treatment based on the insertion of genetic material (DNA and RNA) into a cell's genetic pool either to correct an underlying defect or to modify the characteristics of a cell via expression of the newly inserted gene. In order to successfully implement this technique, effective means of delivering the therapeutic gene to the target cell is required, in such a way that the gene can be expressed at the appropriate level and for a sufficient duration. Two broad approaches have been used to deliver DNA and RNA to cells, namely viral and non-viral vectors, which have different advantages as regards efficiency, ease of production and safety. One of the most powerful methods for gene-transfer is the use of viral vectors. A viral vector is genetically engineered from ‘wild-type’ virus, and consists of a modified viral genome and virion structure. By retaining the protein coat of the original virus, the vector is able to bind and penetrate the cell more effectively while protecting the genome from endogenous enzymes. As for the original viral genome (wild-type), only the essential viral sequence necessary for transcription is retained. There are a number of viral vectors that are currently being used for transfecting cells. Of interest are retroviruses (enveloped single strand RNA), adenoviruses (non-enveloped double stranded DNA) and adeno-associated viruses (linear single stranded DNA). Due to their inherent nature of penetrating and inserting their genetic material (genome) into the target cell, viral vectors result in very high transfection rates. In addition to escaping the target cell's endonucleases, viral genes also possess promoters and enhancers that increase the probability of genetic expression.
Although viral vectors are attractive in terms of the scientific strategy of exploiting natural mechanism, there are some major drawbacks associated with them. They suffer from inherent difficulties of effective pharmaceutical processing, immunogenicity, difficulty in targeting to specific cell types, scale up and the possibility of reversion of an engineered virus to the wild type The safety risks include ‘Insertional Mutagenesis’ and toxicity problems. Ever since the death of Jesse Gelsinger in September 2000, scientists have began to severely question the safety aspects related to viral vector mediated gene delivery. Consequently, a major focus is now being given at the development and use of alternative vectors based on synthetic, non-viral systems for safe and efficient gene delivery.
The problems associated with viral vectors have led to a growing interest in non-viral gene delivery systems. Non-viral vectors are techniques of introducing a coding DNA sequence without the means of a virus. The self-assembly of artificial plasmid (pDNA) containing vectors is required for the development of such vectors. These methods of gene transfer require only a small number of gene, have a virtually infinite capacity, have no infectious or mutagenic capability and large scale production is possible using pharmaceutical techniques. DNA itself is negatively charged, as is the cell membrane and therefore the entry of naked DNA is restricted due to electrical repulsion forces. To reduce this repulsion, many researchers have encased the polynucleotide with a cationic membrane so as to alter the electrical distribution and charge of the complex. These include lipid-based carriers, polycationic lipids, polylysine, polyornithine, histones and other chromosomal proteins, hydrogel polymers and precipitated calcium phosphate (CaPi). One of the major drawbacks of the use of these non-viral vectors is their low transfection efficiency which is caused due to exposure of DNA in the hostile DNAse environment due to simple electrostatic compaction of DNA with the polymeric materials. Among these, the technique of calcium phosphate co-precipitation for in vitro transfection is used as a routine laboratory procedure. This procedure involves a reaction of calcium chloride with sodium phosphate to form a water insoluble calcium phosphate precipitate, which can bind to pDNA. This method heavily relies on the fact that divalent metal cations, such as Ca
2+
, Mg
2+
, Mn
2+
and Ba
2+
can form ionic complexes with the helical phosphates of DNA. Calcium phosphate, therefore, forms complexes with the nucleic acid backbone and thus may impart a stabilizing function to certain DNA structures. When added to a cell monolayer, the cells take up the water insoluble calcium phosphate-pDNA complex (Ca Pi-pDNA) by transportation across the membrane through Ca
2+
ion mediated channel formation. This process is an example of ion channel mediated endocytosis. Once inside the cell, the CaPi-pDNA complex is broken down inside the endosome, thereby releasing the pDNA into the cytosol, which, under suitable circumstances, can be incorporated into the host cell genome. In addition, being inorganic particles, calcium phosphate is highly stable, non-toxic, non-antigenic and non-carcinogenic.
Although extremely safe, the major shortcoming of this process is the poor transfection efficiency as compared to that of viral vectors. The general belief is that the transfection with CaPi-DNA is a low efficiency procedure partly because most of the endocytosed DNA is quickly degraded and excreted to the cytosol. A small fraction of the remaining DNA macromolecules important for gene transfer may be delivered from the endosomal compartment through membrane bound organelles to the nucleus without traversing the cytosol. Moreover, although calcium phosphate precipitation method is simple, effective and still widely used in laboratory for in vitro transfection, the method is hampered by the difficulty of applying to in vivo studies, especially delivery of DNA to any particular cell types. Due to bulk precipitation of calcium phosphate, the method also suffers from variation in calcium phosphate-DNA particle size, which causes variation among experiments.
Process for production of inorganic nanoparticles has been described in U.S. Pat. Nos. 5,460,831 and 5,879,715. Although the process has described the method of preparation of particles of size as small as 10 nm diameter the preparative method does not describe anything about the encapsulation of biologically active materials inside the matrices of these nanoparticles. Calcium Phosphate nanoparticles of size 300 nm and above have been reported in U.S. Pat. No. 6,355,271, which have been, used as carriers and as controlled release matrices for biologically active materials. Virus-like-size particles i.e. particles of size below 100 nm diameter encapsulating genetic materials, which are biologically safe and cost effective, are the main criteria of a non-viral vector for effective delivery of genes. We have described in this invention o
Maitra Amarnath
Mitra Susmita
Mozumdar Subho
Roy Indrajit
Ketter James
Sidley Austin Brown & Wood LLP
University of Delhi, Department of Chemistry
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