Single Systemic Administration of a Gene Therapy Leading to Disease Treatment in Metachromatic Leukodystrophy Arsa Knock-Out Mice.

Metachromatic leukodystrophy (MLD) is a rare, inherited, demyelinating lysosomal storage disorder caused by mutations in the arylsulfatase-A gene (ARSA). In patients, levels of functional ARSA enzyme are diminished and lead to deleterious accumulation of sulfatides. Herein, we demonstrate that intravenous administration of HSC15/ARSA restored the endogenous murine biodistribution of the corresponding enzyme, and overexpression of ARSA corrected disease biomarkers and ameliorated motor deficits in Arsa KO mice of either sex. In treated Arsa KO mice, when compared with intravenously administered AAV9/ARSA, significant increases in brain ARSA activity, transcript levels, and vector genomes were observed with HSC15/ARSA Durability of transgene expression was established in neonate and adult mice out to 12 and 52 weeks, respectively. Levels and correlation between changes in biomarkers and ARSA activity required to achieve functional motor benefit was also defined. Finally, we demonstrated blood-nerve, blood-spinal and blood-brain barrier crossing as well as the presence of circulating ARSA enzyme activity in the serum of healthy nonhuman primates of either sex. Together, these findings support the use of intravenous delivery of HSC15/ARSA-mediated gene therapy for the treatment of MLD.SIGNIFICANCE STATEMENT Herein, we describe the method of gene therapy adeno-associated virus (AAV) capsid and route of administration selection leading to an efficacious gene therapy in a mouse model of metachromatic leukodystrophy. We demonstrate the therapeutic outcome of a new naturally derived clade F AAV capsid (AAVHSC15) in a disease model and the importance of triangulating multiple end points to increase the translation into higher species via ARSA enzyme activity and biodistribution profile (with a focus on the CNS) with that of a key clinically relevant biomarker.

Modifications and applications of the Acinetobacter venetianus RAG-1 exopolysaccharide, the emulsan complex and its components.

Since its discovery in the late 1970s, emulsan has been the subject of significant interest for fundamental biosynthesis and structure-function relationships as well as for its potential industrial applications. These studies initially examined the emulsification properties of the compound, while more recent efforts have focused on potential biomedical applications. As a result of this change of focus, it became necessary to more completely characterize the structure of the emulsan molecule and to develop a more reproducible purification process. We review previous studies with emulsan and explain how prior notions were recently shown to be incorrect through the development of a new purification process. More recent genetic modification of the relevant operon is also reviewed. Finally, the potential applications for the new purified polymer will be discussed.

Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component.

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.

Influence of deletions in the apoemulsan gene cluster on A. venetianus RAG-1 polysaccharide biosynthesis.

Apoemulsan is a biopolymer with potent emulsification activity, produced by Acinetobacter venetianus RAG-1 (RAG-1). The wee gene cluster is responsible for apoemulsan biosynthesis. The analysis of (i) a putative polysaccharide copolymerase mutant (wzc), (ii) a putative polymerase mutant (wzy), and (iii) an apoemulsan-deficient variant (2) indicated that the wee gene cluster controls the synthesis of two polysaccharides: high molecular weight (HMW) and low molecular weight (LMW). LMW polysaccharide of wee origin was present in LPS isolated from RAG-1 cells, suggesting a link to the Lipid A-core of LPS molecules. SDSPAGE analysis indicated that apoemulsan is copurified with LPS polysaccharide, with implications in the emulsification activity of RAG-1 polymer.