Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of "virtual family shuffling" to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.

The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.

A novel alkaliphilic, sulfate-reducing bacterium, strain MLF1 T , was isolated from sediments of soda Mono Lake, California. Gram-negative vibrio-shaped cells were observed, which were 0?6-0?761?2-2?7 mm in size, motile by a single polar flagellum and occurred singly, in pairs or as short spirilla. Growth was observed at 15-48 6C (optimum, 37 6C), >1-7 % NaCl, w/v (optimum, 3 %) and pH 8?0-10?0 (optimum, 9?5). The novel isolate is strictly alkaliphilic, requires a high concentration of carbonate in the growth medium and is obligately anaerobic and catalasenegative. As electron donors, strain MLF1 T uses hydrogen, formate and ethanol. Sulfate, sulfite and thiosulfate (but not sulfur or nitrate) can be used as electron acceptors. The novel isolate is a lithoheterotroph and a facultative lithoautotroph that is able to grow on hydrogen without an organic source of carbon. Strain MLF1 T is resistant to kanamycin and gentamicin, but sensitive to chloramphenicol and tetracycline. The DNA G+C content is 63?0 mol% (HPLC). DNA-DNA hybridization with the most closely related species, Desulfonatronum lacustre Z-7951 T , exhibited 51 % homology. Also, the genome size (1?6610 9 Da) and T m value of the genomic DNA (71±2 6C) for strain MLF1 T were significantly different from the genome size (2?1610 9 Da) and T m value (63±2 6C) for Desulfonatronum lacustre Z-7951 T . On the basis of physiological and molecular properties, the isolate was considered to be a novel species of the genus Desulfonatronum, for which the name Desulfonatronum thiodismutans sp. nov. is proposed (the type strain is MLF1 T

A novel, obligately anaerobic, mesophilic, haloalkaliphilic spirochaete, strain ASpG1 T , was isolated from sediments of the alkaline, hypersaline Mono Lake in California, USA. Cells of the Gramnegative strain were motile and spirochaete-shaped with sizes of 0?2-0?2268-18 mm. Growth of the strain was observed between 10 and 44˚C (optimum 37˚C), in 2-12 % (w/v) NaCl (optimum 3 % NaCl) and between pH 8 and 10?5 (optimum pH 9?5). The novel strain was strictly alkaliphilic, required high concentrations of carbonates in the medium and was capable of utilizing D-glucose, fructose, maltose, sucrose, starch and D-mannitol. End products of glucose fermentation were H 2 , acetate, ethanol and formate. Strain ASpG1 T was resistant to kanamycin and rifampicin, but sensitive to gentamicin, tetracycline and chloramphenicol. The G+C content of its DNA was 58?5 mol%. DNA-DNA hybridization analysis of strain ASpG1 T with its most closely related species, Spirochaeta alkalica Z-7491 T , revealed a hybridization value of only 48?7 %. On the basis of its physiological and molecular properties, strain ASpG1 T appears to represent a novel species of the genus Spirochaeta, for which the name Spirochaeta americana is proposed (type strain ASpG1 T =ATCC BAA-392 T =DSM 14872 T ).The genus Spirochaeta contains a group of free-living, saccharolytic, obligately or facultatively anaerobic, helixshaped bacteria (Canale-Parola, 1977, 1984. Analysis of 16S rRNA gene sequences has shown that the spirochaetes represent a monophyletic phylum within the Bacteria (Paster et al., 1991). At the time of writing, the genus Spirochaeta contained 13 species of bacteria. These organisms occur in a wide variety of freshwater and marine environments (Canale-Parola, 1992), such as aquatic habitats (Spirochaeta aurantia) (Breznak & Canale-Parola, 1975), freshwater mud (Spirochaeta zuelzerae, Spirochaeta stenostrepta) (Canale-Parola, 1980), marine mud (Spirochaeta isovalerica, Spirochaeta litoralis) (Hespell & Canale-Parola, 1970;Harwood & Canale-Parola, 1983), oilfields (Spirochaeta smaragdinae) (Magot et al., 1997) and microbial mats (Teal et al., 1996). Some members of the genus Spirochaeta are microbial extremophiles that inhabit environments with extremes of salinity, alkalinity, pressure and/or temperature. The spirochaete extremophiles may harbour important enzymes and proteins and, consequently, are of particular interest to biotechnologists (Bermudes et al., 1987;Barth et al., 1991;Munson et al., 1993).Prior studies have shown that some species of the genus Spirochaeta are naturally resistant to sulfide concentrations at levels that inhibit the growth of many other microorganisms. The mesophilic halophile Spirochaeta halophila was isolated from black mud (smelling strongly of H 2 S) of Solar Lake, a high-salinity pond on the Sinai shore of the Gulf of Aqaba (Greenberg & Canale-Parola, 1976). The obligate anaerobe strain GS-2 is a mesophilic barophile that was isolated from sulfide-rich mud samples collected at a depth of 2550 m near deep-sea hydrotherm...