Characterization and quality control of recombinant adenovirus vectors for gene therapy.

Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.

Investigation of the first step of biotin biosynthesis in Bacillus sphaericus. Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate.

The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli. The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein. Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP. Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme. The pH profile under Vmax. conditions showed a maximum around 8.5. Apparent Km values for pimelate, CoASH, ATP.Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM. The enzyme was inhibited by Mg2+ above 10 mM. This acid-CoA ligase exhibited a very sharp substrate specificity, e.g. neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed. The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+. The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA. The synthase was a homodimer with a 28,000-M(r) subunit. N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene. A careful study of pimelate uptake by B. sphaericus, E. coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.

The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.

Biotin formation by recombinant strains of Escherichia coli: influence of the host physiology.

Strains of Escherichia coli were transformed with different plasmids bearing the gene clusters bioXWF and bioDAYB isolated from the Gram positive bacterium Bacillus sphaericus. These genes encode for the enzymes involved in the metabolic pathway which synthesizes biotin from the precursor pimelic acid. Transformed E. coli strains were grown in bioreactors to reach a biomass of 18 g l-1 cell dry weight in 1 litre batch culture with substrate feeding and approximately 50 g l-1 in 10 l fed batch culture. Improved yields of total vitamers and biotin formed in these processes were achieved after a comparative analysis of different culture conditions, medium compositions, host strains and expression systems. Production of 27 mg l-1 of biotin and 200 mg l-1 of vitamers was achieved in 1 litre batch culture. Using a 10-1 fed batch process, biotin and vitamer concentrations reached maximum values of 45 mg l-1 and 350 mg l-1, respectively.

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli.

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.

Cloning, sequencing and analysis of the yeast S. uvarum ERG10 gene encoding acetoacetyl CoA thiolase.

The ERG10 gene specific to S. uvarum, a brewing yeast, has been cloned by complementation of an S. cerevisiae erg10 mutant. S. uvarum contains two different ERG10 genes. One of these is similar to the S. cerevisiae ERG10 gene; they are structurally different, but functionally homologous. The cloned ERG10 gene has been located on chromosome XVI, and we have shown that it is allelic to the previously isolated tsm0115 mutants. Northern blot and sequence analysis indicate that the ERG10 gene is highly expressed, and biochemical and genetic evidence show that it encodes the cytoplasmic acetoacetyl CoA thiolase.

Comparison of immunosorbent electron microscopy, enzyme immunoassay and counterimmunoelectrophoresis for detection of human rotavirus in stools.

The detection of human rotaviruses by routine electron microscopy examination of stool specimens has been compared with the sensitivity of detection obtainable by three different immunoassays. These assays are: 1) immunosorbent electron microscopy (ISEM), which consists of the serological trapping of viruses on electron microscopy grids coated with protein A and specific viral antiserum; 2) an enzyme-linked immunosorbent assay (ELISA), in which the primary antibody is rabbit anti-rotavirus immunoglobulin, the secondary antibody is chicken anti-rotavirus immunoglobulin extracted from egg yolk of immunized hens, and the indicator antibody is alkaline phosphatase-conjugated rabbit anti-chicken immunoglobulin; 3) counterimmunoelectrophoresis (CIE). A total of 63 stool specimens from infants with gastroenteritis were examined. Of these, 23 and 24 specimens were found to contain rotavirus by electron microscopy and CIE, respectively. When scored by ELISA and ISEM, 37 and 39 were found to be positive, respectively. Confirmatory inhibition assays were necessary to eliminate some false positive reactions in ELISA. Detection of human rotaviruses in stools by ISEM is as sensitive as by ELISA, but in weakly positive specimens, ISEM offers the additional advantage of a direct visual demonstration of the presence of the aetiological agent.

The nucleotide sequence of spinach chloroplast tryptophan transfer RNA.

Spinach chloroplast tRNATrp, purified by column chromatography and two-dimensional gel electrophoresis, has been sequenced using in vitro labeling techniques. The sequence is : pG-C-G-C-U-C-U-U-A-G-U-U-C-A-G-U-U-C-Gm-G-D-A-G-A-A-C-m2G-psi-G-G-G-psi-C-U-C-A-A*-A-A-C-C-C-G-A-U-G-N-C-G-U-A-G-G-T-psi-C-A-A-G-U-C-C-U-A-C-A-G-A-G-C-G-U-G -C-C-AOH. Like the E. coli suppressor tRNA psu+UGA which translates both the opal terminator codon U-G-A and the tryptophan codon U-G-G, spinach chloroplast tRNATrp has C-C-A as an anticodon and contains an A-U pair in the D-stem.