Therapeutic gene modulation

Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene (perhaps through the introduction of a gene encoding a novel modulatory protein) whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly. Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene (perhaps through the introduction of a gene encoding a novel modulatory protein) whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly. Modulation of gene expression can be mediated at the level of transcription by DNA-binding agents (which may be artificial transcription factors), small molecules, or synthetic oligonucleotides. It may also be mediated post-transcriptionally through RNA interference. An approach to therapeutic modulation utilizes agents that modulate endogenous transcription by specifically targeting those genes at the gDNA level. The advantage to this approach over modulation at the mRNA or protein level is that every cell contains only a single gDNA copy. Thus the target copy number is significantly lower allowing the drugs to theoretically be administered at much lower doses. This approach also offers several advantages over traditional gene therapy. Directly targeting endogenous transcription should yield correct relative expression of splice variants. In contrast, traditional gene therapy typically introduces a gene which can express only one transcript, rather than a set of stoichiometrically-expressed spliced transcript variants. Additionally, virally-introduced genes can be targeted for gene silencing by methylation which can counteract the effect of traditional gene therapy. This is not anticipated to be a problem for transcriptional modulation as it acts on endogenous DNA. There are three major categories of agents that act as transcriptional gene modulators: triplex-forming oligonucleotides (TFOs), synthetic polyamides (SPAs), and DNA binding proteins. Triplex-forming oligonucleotides (TFO) are one potential method to achieve therapeutic gene modulation. TFOs are approximately 10-40 base pairs long and can bind in the major groove in duplex DNA which creates a third strand or a triple helix. The binding occurs at polypurine or polypyrimidine regions via Hoogsteen hydrogen bonds to the purine (A / G) bases on the double stranded DNA that is already in the form of the Watson-Crick helix. TFOs can be either polypurine or polypyrimidine molecules and bind to one of the two strands in the double helix in either parallel or antiparallel orientation to target polypurine or polypyrimidine regions. Since the DNA-recognition codes are different for the parallel and the anti-parallel fashion of TFO binding, TFOs composed of pyrimidines (C / T) bind to the purine-rich strand of the target double helix via Hoogsteen hydrogen bonds in a parallel fashion. TFOs composed of purines (A / G), or mixed purine and pyrimidine bind to the same purine-rich strand via reverse Hoogsteen bonds in an anti-parallel fashion. TFO's can recognize purine-rich target strands for duplex DNA. In order for TFO motifs to bind in a parallel fashion and create hydrogen bonds, the nitrogen atom at position 3 on the cytosine residue needs to be protonated, but at physiological pH levels it is not, which could prevent parallel binding. Another limitation is that TFOs can only bind to purine-rich target strands and this would limit the choice of endogenous gene target sites to polypurine-polypyrimidine stretches in duplex DNA. If a method to also allow TFOs to bind to pyrimidine bases was generated, this would enable TFOs to target any part of the genome. Also the human genome is rich in polypurine and polypyrimidine sequences which could affect the specificity of TFO to bind to a target DNA region. An approach to overcome this limitation is to develop TFOs with modified nucleotides that act as locked nucleic acids to increase the affinity of the TFO for specific target sequences.