Treatment Of Human Genetic Diseases Research Articles

Rapid Detection of Genetic Mutations Using the Chemiluminescent Hybridization Protection Assay (HPA): Overview and Comparison with Other Methods

The detection of genetic mutations is of paramount importance for the study, diagnosis, and treatment of human genetic disease. Methods of detection generally fall into one of two categories: those to scan for unknown mutations and those to detect known mutations. This review focuses on methods for the detection of known mutations. The hybridization protection assay (HPA) is described in detail. The HPA method utilizes short oligonucleotide probes covalently labeled with a highly chemiluminescent acridinium ester (AE). The assay format is completely homogeneous, requiring no physical separation steps, and can rapidly and sensitively detect all single-base mismatches as well as multiple mismatches, insertions, deletions, and genetic translocations. When very low copy number targets are assayed, HPA is coupled with transcription-mediated amplification (TMA), an isothermal method that amplifies DNA or RNA targets. Other methods that are described for the detection of known mutations include hybridization with sequence-specific oligonucleotides, hybridization to oligonucleotide arrays, allele-specific amplification, ligase-mediated detection, primer extension, and restriction fragment analysis. The advantages and limitations of each of these methods are discussed. Methods to scan for unknown mutations are briefly described.

Human gene therapy

Somatic cell gene therapy for the correction of many human genetic diseases is now technically possible. We review several methods of gene transfer that have been successfully used in animal studies, and discuss the promise and potential limitations of these methods in the treatment of human genetic diseases.

Human gene expression in murine hemopoietic cells in vivo.

Somatic gene transfer offers the possibility of a new approach in the treatment of human genetic disease. Defects affecting the blood and blood forming tissues are candidates for therapies involving transfer of new genetic information into hemopoietic stem cells. One such defect, adenosine deaminase (ADA) deficiency, is being used as a model in which hemopoietic gene transfer techniques can be developed and evaluated. In this model, gene transfer is mediated by a retroviral vector. Retroviral vectors have been used extensively to deliver information to hemopoietic cells1–14. We have previously reported delivery and expression of human ADA (hADA) sequences in murine hemopoietic progenitors in vitro15 and in vivo16, but were not able to demonstrate long term stability of expression. We describe here the construction and testing of four new vectors, representing the first demonstration of efficient transfer and long term in vivo expression of hADA in murine hemopoietic cells.