SYNTHETIC DNA PROCESS
Scientists at CytoGenix have developed a novel method for producing large amounts of high quality therapeutic circular DNA with significant reductions in residual contaminants compared to traditional fermentation methods. Specifically, we have identified and optimized a method for in vitro DNA synthesis and amplification for the production of GMP drug substances.
Cell-free amplification of c-DNA has many important benefits beginning with the size and composition of the plasmid. Under this system, there is no need for bacterial replication genes or selection markers such as antibiotic resistant genes. In most cases, this will reduce the size and weight of the therapeutic c-DNA by at least 3,000 base pairs or a molecular weight of approximately 2,000 kilo Daltons. The total absence of bacteria and growth media assures that there is no need to employ mechanical or chemical purification methods to extract cell or animal proteins, RNA, genomic DNA and backbone molecules. This feature allows the designer more control of coding for non-specific and specific immune responses.
Greater biological activity
Our experiments have shown that the biological response to c-DNAs with no vector backbone is approximately one and one-half times higher than traditional plasmids. Other investigators have reported greater activity levels of in vivo studies using "DNA mini circles". At the molecular level, 100ng of
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traditional plasmid DNA is equal to 60ng of bacteria-free c-DNA. This may explain the result of our transfection experiments and suggest that less in vitro synthesized DNA may be required to generate the same biological effect as its plasmid counterpart. In terms of cost, this would translate in substantial savings.
Low risk, low cost and fast cycle time
Our novel process is bench-scale and requires little equipment, space or human intervention in comparison to bioprocess manufacturing facilities. No cell banks are needed and no yield optimization steps that can add weeks or months to the production process. This process lends itself to liquid-handling automation and one technician can synthesize gram quantities in a matter of days working in custom manufacturing suites. The capital costs for physical plant and on-going fixed and variable operating expenses are a fraction of the costs of large-scale bioprocess methods. We believe that this synthetic process will be more consistent, yield material of higher purity, and will enable delivery of higher concentrations of API if needed.
Improved regulatory profile
Benefits provided by the cell-free synthesis from a Regulatory Agency review and compliance perspective are significant. By beginning with a well-characterized synthetic master construct, there is no need for cell bank systems (master or working cell banks), thus reducing the risk and amount of documentation, space and cost. Similarly, product cGMP manufacturing procedures detailing methods for cell collection, processing and culture conditions would no longer be necessary and would substantially reduce QA/QC and compliance overhead costs.
Cytogenix is carrying out a multi-pronged strategy with the objective of commercializing this technology within the next year. The following briefly describes our plans:
o PROCESS OPTIMIZATION AND SCALE-UP, continued experimentation to achieve yield optimization, reduced reagent usage and reduced costs. Manufacture milligram quantities consistently, conduct several animal studies with various plasmids to verify bioactivity equal to or greater than plasmids produced via traditional methods. Scale-up to produce gram quantities.
o DEVELOP REAGENT SUPPLIERS AND SOURCING, establish relationships with manufacturers of enzymes. Develop quality parameters.
o CONSTRUCT CGMP PRODUCTION SUITE, build and equip a side-by-side manufacturing suite for simultaneous production of batches cGMP pDNA.
o BUSINESS DEVELOPMENT, we are currently evaluating options for industry partnership for continued development of the process including contract manufacturing
All primary research and development at CytoGenix is conducted in the on-site laboratory located adjacent to the executive offices at the same address. The Company's primary research and development experiments are being conducted in human lung cancer cells (A549 cells) and human liver cells (HepG2 cells) to determine the expression levels of single-stranded catalytic DNA and single-stranded Antisense DNA targeting c-raf kinase mRNA transcripts, bcl-2mRNA transcripts, and mouse double minute oncogene 2 (MDM2) MRNA transcripts.
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The Company is currently supporting two (2) Sponsored Research Agreements (SRA):
Dr. Cy Stein's lab at Albert Einstein College of Medicine is using Cytogenix's proprietary gene silencing DNA technology against a gene that is expressed in melanoma cells that produces a protein known to counteract the effect of several chemotherapeutic agents in difficult to treat cancers.
Dr. Charles Densmore's lab at Baylor College of Medicine is conducting animal trials to determine the effect of CytoGenix's gene silencing technology on cancer in the lungs of mice using aerosol gene delivery system.
Other research is contemplated to explore various plasmid delivery systems.
Yin Chen, the Company's Vice President of Research and Development, was invited to give presentations at 1) the 10th International Conference on Gene Therapy in Cancer held from December 13 to 15, 2001 in San Diego, CA; 2) 3rd Annual Conference: RNA in Drug Development - RNA as Tool and Target, San Diego, CA, November 10-13, 2003 and 3) A Novel Single-Stranded DNA Expression Vector. Louisiana at the Crossroads: Moving Biotechnology from Research to Commercialization, Shreveport, LA, June 24, 2004.
The first phase of a Small Business Technology Transfer Program (STTR) Grant entitled, "ssDNA Expression of Triplex-Forming Oligonucleotides" was approved for funding by the National Institute of Child Health/Human Development (NICHD) of the National Institutes of Health and has been completed.
The second SBIR grant entitled, "PEI aerosol delivery of ssDNA expression vector" was awarded by the National Cancer Institute, NIH in 2004.
The Company and its cooperating university scientists have published a number of scientific papers and presented at scientific meetings. These publications include:
1. Tan, X. & Chen, Y., A novel genomic approach identifies bacterial DNA-dependent RNA polymerase as the target of an antibacterial oligodeoxynucleotide, RBL-1 Biochemistry, 2005 (in press).
2. Tan, X., Actor, J.K., & Chen, Y. PNA antisense oligomer as a therapeutic strategy against bacterial infection: proof of principle using mouse peritonitis model, Antimicrobial Agent and Chemotherapy, (submitted), 2005.
3. Tan, X, & Chen, Y. Discovery of novel antibiotics using cell-based screening (Review), Current Drug Discovery, pp. 21-23, April, 2004.
4. Tan, X., Knesha, R., Margolin, W. and Chen, Y. DNA enzyme generated by a novel single-stranded DNA expression vector inhibits expression of the essential bacterial cell division gene ftsZ, Biochemistry, 43:
1111-1117, 2004.
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5. McMicken, H., Bates, P. and Chen, Y. Antiproliferative activity of G-quartet-containing oligonucleotides generated by a novel single-stranded DNA expression system, Cancer Gene Therapy, 10(12):867-869, 2003.
6. Chen, Y. and McMicken, H. Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector, Gene Therapy, 10:1776-1780, 2003.
7. Chen, Y. , Ji, Y., and Conrad, C. Expression of single-stranded DNA in mammalian cells, Biotechniques, 34:167-171, 2003.
8. Chen, Y., Novel Technologies for target validation, Genetic Engineering News, 23(11):7-9, 2003. 9. Chen, Y. A novel single-stranded DNA (ssDNA) expression vector (Review), Expert Opinion on Biological Therapy, 2:735-740, 2002.
10. Chen, Y. Meeting highlights, 10th International conference on gene therapy of cancer, Expert Opinion on Biological Therapy, 2:443-445, 2002.
11. Chen, Y., Growth of oligo-based drugs, Genomics & Proteomics, October, 2002.
12. Datta, H., and Glazer, P. Intracelullar generation of single-stranded DNA for chromosomal triplex formation and induced recombination, Nucleic Acid Research, 29:5140-5147, 2001. A marvel of biochemical engineering means cells can produce DNA enzyme to attach cancer, New Scientist, January, 2001.
13. Chen, Y. , Ji, Y., Roxby, R., and Conrad, C. In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to c-raf, Antisense & Nucleic Acid Drug Development, 10:
