A gene, NMB1638, with homology to the recently characterized gene encoding a phosphoethanolamine transferase, lpt-3, has been identified from the Neisseria meningitidis genome sequence and was found to be present in all meningococcal strains examined. Homology comparison with other database sequences would suggest that NMB1638 and lpt-3 represent genes coding for members of a family of proteins of related function identified in a wide range of gram-negative species of bacteria. When grown and isolated under appropriate conditions, N. meningitidis elaborated lipopolysaccharide (LPS) containing a lipid A that was characteristically phosphorylated with multiple phosphate and phosphoethanolamine residues. In all meningococcal strains examined, each lipid A species contained the basal diphosphorylated species, wherein a phosphate group is attached to each glucosamine residue. Also elaborated within the population of LPS molecules are a variety of "phosphoforms" that contain either an additional phosphate residue, an additional phosphoethanolamine residue, additional phosphate and phosphoethanolamine residues, or an additional phosphate and two phosphoethanolamine residues in the lipid A. Mass spectroscopic analyses of LPS from three strains in which NMB1638 had been inactivated by a specific mutation indicated that there were no phosphoethanolamine residues included in the lipid A region of the LPS and that there was no further phosphorylation of lipid A beyond one additional phosphate species. We propose that NMB1638 encodes a phosphoethanolamine transferase specific for lipid A and propose naming the gene "lptA," for "LPS phosphoethenolamine transferase for lipid A."Lipopolysaccharide (LPS) is found in all gram-negative bacteria and is usually composed of three regions: lipid A, core oligosaccharide (OS), and a polysaccharide molecule often referred to as the "O-antigen." LPS from Neisseria meningitidis lacks the polysaccharide and is sometimes referred to as "lipooligosaccharide" (LOS) to reflect this. The core OS unit of N. meningitidis LPS comprises an inner core diheptose-N-acetylglucosamine backbone, wherein the two L-glycero-D-mannoheptose (Hep) residues can provide a point of attachment for outer core OS residues (8). Meningococcal LPS has been classified into 12 distinct LPS immunotypes (L1 to L12), originally defined by monoclonal antibody (MAb) reactivities (21) but further defined by structural analyses (4,5,7,9,14,16,26). The structural basis of the immunotyping scheme is primarily governed by the location of a phosphoethanolamine (PEtn) moiety on the distal heptose residue (HepII) at either the 3-or 6-position or absent, but is also dictated by the length and nature of OS extension from the proximal heptose residue (HepI) and the presence or absence of a glucose at HepII (17 [ Fig. 1]). The lipid A region of the LPS is responsible for much of the toxicity of the LPS molecule, and LPS is indeed sometimes referred to as "endotoxin." The lipid A region consists of a disaccharide of pyranosyl glucosamine r...

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.

SummaryWe have identified a gene, lpt-3, that is required for the addition of phosphoethanolamine to the 3-position (PEtn-3) on the b-chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn-3 substituent is characteristic of the LPS of a majority (ª 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface-accessible epitope. All strains of Nm that have PEtn-3 possess the lpt-3 gene. In some lpt-3-containing strains, the 3-position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt-3 resulted in loss of PEtn-3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt-3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a L9 (Jennings et al., 1983) have been elucidated. There has been significant success in identifying genes involved in LPS biosynthesis, including lgtA, lgtB, lgtE (Jennings et al., 1995a), lgtC (Gotschlich, 1994), rfaC (Stojiljkovic et al., 1997), lgtF, rfaK (Kahler et al., 1996a,b), rfaF (Jennings et al., 1995b) and lgtG (Banerjee et al., 1998). A characteristic of meningococcal LPS is reversible, highfrequency phase variation of its outer core structures mediated by slippage-like mechanisms in homopolymeric DNA tracts present in LPS biosynthetic genes . These homopolymeric tracts are absent in inner core LPS biosynthetic genes such as rfaC, rfaK, rfaF and lgtF, making this structure relatively stable and an attractive candidate for incorporation into a vaccine.In an attempt to investigate LPS epitopes for potential inclusion into a vaccine against serogroup B meningococcal disease, we have previously identified an inner core LPS epitope defined by a monoclonal antibody designated B5 (mAb B5). The cognate epitope of mAb B5 was found to be present on 70% of all Nm strains (Plested et al., 1999). mAb B5 recognises LPS inner core structures that contain phosphoethanolamine (PEtn) attached specifically at the 3-position (PEtn-3) on the b-chain heptose (HepII). L1, L3, L7, L8 and L9 LPS immunotypes of Nm containing PEtn-3 were mAb B5 reactive (B5+), whereas those containing PEtn in an exocyclic position (L2, L4 and L6 immunotypes) or glycoforms that completely lack PEtn (L5 immunotype) were mAb B5 nonreactive (B5-).Recent studies have revealed that antibodies specific for the mAb B5+ epitope are present in sera from infants recovering from invasive meningococcal disease (Plested et al., 2000). These antibodies exhibited functional activity towards meningococci in an in vitro opsonophagocytic (OP) assay . Taken together, these findings indicate that the mAb B5+ epitope is a target for protective antibodies, and that the inner core...