Nine structural genes (furA, katG, inhA, kasA, Rv0340, iniB, iniA, iniC, and efpA) and two regulatory regions (the oxyR-ahpC intergenic region and the promoter of mabA-inhA) in 87 isoniazid (INH)-monoresistant and 50 INH-susceptible Mycobacterium tuberculosis isolates collected from five provinces of China were analyzed by sequencing. Eighty-two (94.3%) INH-resistant isolates had mutations in the katG gene, with the katG Ser315Thr mutation predominant (55.2%). No mutation at codon 463 of katG was detected among the 50 INH-susceptible isolates with different IS6110 fingerprints. In addition, there were 35 (40.2%) INH-resistant isolates that had a mutation at codon 463 of katG. Of the INH-resistant strains, 20 (23.0%) isolates harbored double mutations at two separate loci of katG. Mutations in the inhA promoter region occurred in 13 (14.9%) isolates; 4.6% of the isolates had inhA structural gene mutations, and 11.5% harbored mutations in the oxyR-ahpC intergenic region. Drug resistance-associated mutations were detected in the iniBAC region and efpA.Tuberculosis (TB) represents one of the world's greatest sources of mortality and morbidity, with approximately 8 million new infections and 2.5 million to 3 million deaths per year. China has one of the highest burdens of TB in the world, with the second-largest total number of TB cases globally (9, 34, 41). Currently, throughout the world, isoniazid (INH) and rifampin (RIF) together represent the backbone of short-course chemotherapy treatment for Mycobacterium tuberculosis infections. The rise of multidrug-resistant tuberculosis (MDR-TB), defined as TB showing resistance to at least INH and RIF, is a serious threat to TB control. The World Health Organization estimates that 50 million people worldwide are infected with MDR-TB. The highest percentages of MDR-TB cases that are newly contracted have been found in China (11%) and eastern Europe (7 to 14%) (5,10,11).RIF resistance appears to be mediated by mutations of the ␤ subunit of RNA polymerase, which is encoded by the rpoB gene. Previous work indicates that more than 95% of RIFresistant strains are associated with mutations within an 81-bp region of the rpoB gene. Specific mutations, insertions, and deletions have been detected, and this 81-bp region has been termed the rifampin resistance determinant region (7,13,42). In contrast, INH resistance is apparently controlled by a more complex genetic system that involves several genes (15, 31). INH, a first-line antituberculosis drug, has a simple chemical structure consisting of a pyridine ring and a hydrazide group. INH is a prodrug that enters actively growing tubercle bacilli by passive diffusion (4). The bifunctional bacterial enzyme catalase-peroxidase (KatG) converts INH to a range of oxygenated and organic toxic radicals that attack multiple targets in the mycobacterial cell (32, 33). The best-characterized target of these radicals is the cell wall mycolic acid, but DNA, carbohydrates, lipids, and DNA metabolism may be targeted as well (6). Reports suggest th...

Murine leukemia viruses (MuLV) have been adapted for use as gene transfer vectors for experimental and human gene therapy applications. Their utility for these purposes has been circumscribed by the limited host range and relatively low titer of available producer clones. Pseudotyping of MuLV particles with the vesicular stomatitis virus envelope protein (VSV-G), expressed transiently in cells producing MuLV Gag and Pol proteins, has yielded vector preparations with a broader host range that can be concentrated by ultracentrifugation. We have explored the use of steroid-inducible and tetracycline-modulated promoter systems (necessary because the VSV-G protein is toxic to cells when constitutively expressed) to derive stable producer cell lines capable of substantial production of VSV-G pseudotyped MuLV particles. A packaging cell line and producer clones capable of expressing a chimeric transcription factor, composed of the tetracycline repressor (tetR) and the VP16 trans-activating sequences of herpes simplex virus VP16 gene and containing the VSV-G coding sequences linked to a minimal promoter having seven tandem copies of the tetracycline responsive operator (tetO), exhibited high levels of VSV-G protein expression when cultured in the absence of tetracycline. Vector particles, produced at titers of 10(5)-10(6) infectious colony forming units per ml (cfu/ml), could be concentrated effectively by ultracentrifugation yielding vector preparations having a titer of 10(9) cfu/ml. These cell lines grew normally when VSV-G protein expression was repressed by tetracycline. Such producer clones hold promise for future human gene therapy applications.

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RNA repair or reprogramming is a new avenue for human gene therapy. Unlike conventional gene therapy, in which exogenous cDNAs are introduced into cells, RNA repair approaches, which are based on spliceosome-mediated pre-mRNA trans-splicing, trans-splicing ribozymes, and tRNA-splicing endonuclease, allow the correction of endogenous RNA species. Recently published accounts that in vivo phenotypic correction of a variety of inherited diseases can be achieved by RNA repair are encouraging. Nevertheless, the science of RNA repair for treatment of human diseases is just beginning and faces several scientific and technical challenges that must be addressed and surmounted. In this review, we summarize recent advances in spliceosome-mediated pre-mRNA trans-splicing. We also provide an update on the progress of this emerging technology toward the development of molecular therapy and diagnosis for human diseases and discuss the outstanding issues and challenges confronting RNA therapeutics.

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