The administration of the naked DNA of gene expression cassettes into animals is increasingly being used as a research tool to study the role of genes and their cognate proteins in the pathogenesis of disease in animal models (Herweijer and Wolff, Gene Therapy 2003; 10:453-458). This in vivo gene delivery technology is also being used in human clinical trials for genetic vaccines, Duchenne muscular dystrophy, peripheral limb ischemia, and cardiac ischemia. Naked DNA is an attractive non-viral vector because of its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It is particularly useful in studying the functions of secreted proteins, which are present in blood circulation, regardless where the proteins are made.
A basic expression cassette is a fragment of double stranded DNA that contains a target gene of interest, a promoter, an expression terminator, and a mRNA stabilization element. Naked DNA can be readministered multiple times into mammals including primates without inducing an antibody response against the DNA itself (Jiao et al. Gene Therapy 1992; 3:21-33). Naked DNA can exist in episomal form for a long duration, without chromosome integration. With the advent of intravascular and intramuscular injection coupled with electroporation techniques, the target protein expression can reach significant levels that yield a pharmacological response and therapeutic efficacy (Herweijer et al. J Gene Med. 2001; 3:280-291; Miao et al. Molecular Therapy2001; 3:947-957).
Direct in vivo gene transfer with naked DNA was first demonstrated in skeletal muscles (Wolff et al. Science 1990; 247:1465-1468). Subsequent studies found foreign gene expression in other tissues such as heart, thyroid, skin, and liver after direct injection (Li et al. J Mol Cel Cardiol 1997; 29:1499-1504). In the case of intramuscular delivery, when plasmid DNA is injected directly into skeletal muscles, target proteins such as luciferase can be detected in the injected tissues (Wolff et al. Science 1990; 247:1465-1468). In the case of intravascular delivery, plasmid DNA is injected into the tail vein, and target gene expression is generally found in the liver (Liu et al. Gene Therapy 1999; 6:1258-1266). When the target gene is a secreted protein, the protein is secreted out of cells and can be detected in blood circulation. A technique called hydrodynamic tail vein (HTV) injection is a tail vein injection method in which a large volume of liquid (typically saline around 10% of animal body weight) is injected within a few seconds, and is known to be effective in producing significant levels of target gene expression in rodents, sometimes in the range of microgram per milliliter (reviewed by Suda and Liu Molecular Therapy 2007; 15:2063-2069).
LakePharma has developed advanced expression vectors and formulation procedures to significantly enhance in vivo gene expression level and duration of injected target gene DNA. These vectors can be used for both intramuscular injection or hydrodynamic tail vein injection. Target protein can be detected as high as 200 mg/L and as long as 8 weeks for quite a number of targets. Below is sample data of in vivo delivery of DNA expressing either leptin or a protein that neutralizes leptin. Note that the leptin level remained elevated six weeks after in vivo DNA delivery.
DNA immunization is a technique for producing an immunological response by injecting hosts with genetically engineered DNA. When DNA is injected into the cells of the host, the inner machinery of the the host cells reads the DNA and converts it into pathogenic proteins. These proteins are recognized as foreign and trigger an antibody response. LakePharma has developed highly efficient DNA immunization vectors and procedures where immune response can be induced against not only foreign proteins, (for infectious diseases) but also native host proteins (for therapeutic vaccines).