Lessons from the First Decade and a Half of Gene Therapy
Fund for Inherited Disease Research Newtown, PA, United States
The prospect of treating diseases by using the vast store of information in the genomes of living things has excited the imagination of clinical investigators everywhere. Often overlooked in this excitement is the magnitude of the challenge to effectively harness genes as medicines. Because of the success of transgenic technology in microbes and rodents, the initial human gene therapy trials also used transgenes to substitute for a disease-causing mutant or to give targeted production of a gene product. Unfortunately many ex vivo protocols floundered at the interface of vector biology and cell biology when the targeted cell lacked the receptor for the vector or the cell’s position in the growth cycle was not receptive to transgene/vector integration or expression. The challenge of in vivo transgene delivery also rapidly became apparent as the laws of physics (diffusion inversely correlates with molecular mass) collided with the need to give DNA complexes of huge size. Despite these problems, slow /steady progress has been made in several areas with cures now reported for X-SCID and ADA-SCID as well as treatment successes in patients with vascular insufficiency, cancer, AIDS and other indications. Although therapy with transgenes will continue to improve, systemic problems with random vector integration, widely variable transgene dose from cell to cell, lack of physiologic regulation of transgene expression (and loss of alternatively splicing with cDNA) and the inability to treat dominant disorders highlights the need for new approaches for many genetic disorders. Homologous recombination has not been used because it has a high frequency of illegitimate recombination events and because its efficiency in mammalian cells is so low as to require the addition of selectable genes. During the past decade techniques that employ synthetic DNA / RNA-DNA polymers have been reported that seemed to offer an alternative to both transgene addition and HR. Many of the most spectacular of these reports have not been reproducible and the entire field has suffered from over interpretation of data and a lack of scientific rigor. Finally reliable / reproducible data has begun to appear from several sources showing that it is possible to edit the genome in living cells, a process active in bacteria, plants, mammalian and even human cells. In some systems we have tested it was possible to reliably substitute, add &/or delete specific bases to a genomic DNA section spanning up to 8 nucleotides and to isolate and expand the modified cells for extensive analysis. Genome editing still does not have the efficiency to be used for in vivo human treatment – but the power to specifically modify the genome in living cells - to change SNPs or a few bases or codons – without introducing transgenes or foreign DNA – has great potential for future medical and commercial applications. The generation of cattle that are rendered genetically resistant to “mad cow disease” will be discussed as an example of a currently practical application of genome editing.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in plenary session 901 (Immunobiology).