Imaging and tissue biodistribution of 99mTc-labeled adenovirus knob (serotype 5).

Hepatic sequestration of systemically administered adenoviral vectors reduces the number of viral particles available for delivery to other tissues. The biological basis of this phenomenon was investigated using a new in vivo technique which permitted imaging in real time. Recombinant adenovirus serotype 5 knob (Ad5K) was radiolabeled with the gamma-emitter 99mTc (half-life = 6 h). Scatchard analysis of the 99mTc-Ad5K showed specific, high-affinity binding to U293 cells (Kd = 1.4 +/- 0.5 nM), demonstrating that the radiolabeling process had no effect on receptor binding. In vivo dynamic imaging with an Anger gamma camera revealed that the liver binding followed an exponential rise to maximum, with a measured 100% extraction efficiency. Initially, the liver binding capacity was 3.1 +/- 0.4 micrograms Ad5K, equivalent to approximately 17,000 Ad5K molecules per liver cell. Liver binding was blocked by preincubation of Ad5K with neutralizing anti-Ad5K antibody; a 50% reduction in liver uptake was demonstrated by imaging. Unlabeled Ad5K was more effective in blocking liver uptake of 99mTc-Ad5K, whereas irrelevant unlabeled Ad3K had no effect. Imaging data for the liver uptake studies were in agreement with biodistribution determined by removing and measuring tissues. These data demonstrated that in vivo imaging is a sensitive tool for measuring changes to liver tropism. Similar imaging techniques can be applied to adenovirus vectors to measure specific targeting for gene therapy.

Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.

To expand the utility of recombinant adenovirus vectors for gene therapy applications, methods to alter native viral tropism to achieve cell-specific transduction would be beneficial. To this end, we are pursuing genetic methods to alter the cell recognition domain of the adenovirus fiber. To incorporate these modified fibers into mature virions, we have developed a method based on homologous DNA recombination between two plasmids. A fiber-deleted, propagation-defective rescue plasmid has been designed for recombination with a shuttle plasmid encoding a variant fiber gene. Recombination between the two plasmids results in the derivation of recombinant viruses containing the variant fiber gene. To establish the utility of this method, we constructed a recombinant adenovirus containing a fiber gene with a silent mutation. In addition, we generated an adenovirus vector containing chimeric fibers composed of the tail and shaft domains of adenovirus serotype 5 and the knob domain of serotype 3. This modification was shown to alter the receptor recognition profile of the virus containing the fiber chimera. Thus, this two-plasmid system allows for the generation of adenovirus vectors containing variant fibers. This method provides a rapid and facile means of generating fiber-modified recombinant adenoviruses. In addition, it should be possible to use this system in the development of adenovirus vectors with modified tropism to allow cell-specific targeting.

[Study of the structure-function organization of the variola virus genome. IV. Sequencing and analysis of the nucleotide sequence of the right terminus of the India-1967 strain genome]. / Izuchenie strukturno-funktsional'noi organizatsii genoma virusa natural'noi ospy. IV. Sekvenirovanie i analiz posledovatel'nosti nukleotidov pravogo kontsa genoma shtamma Indiia-1967.

Sequencing and computer analysis of the variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region have been carried out. Fifty nine potential open reading frames (ORFs) of over 60 amino acid residues have been identified. Structure-function organization of VAR-IND DNA segment under study was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple distinctions in the genetic map of VAR-IND from VAC-COP and VAC-WR have been revealed along with the high similarity to the corresponding VAR-HAR segment. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

Biochemical characterization of VspI methyltransferase.

The gene (vspIM) encoding VspI methyltransferase (MTase) has previously been cloned and sequenced, and shown to belong to the gamma class of m6-adenine MTases [Degtyarev et al., Nucleic Acids Res. 21 (1993) 2015]. Here it is shown that the MTase modifies the third adenine within the recognition sequence 5'-ATTAAT-3'.

[Expression in Escherichia coli cells of the nucleotide sequence of DNA coding for the bovine lipotropic hormone]. / Ekspressiia v kletkakh Escherichia coli nukleotidnoi posledovatel'nosti DNK, kodiruiushchei lipotropnyi gormon byka.

A cDNA fragment of bovine proopiomelanocortin coding for beta-lipotropic hormone was joined with a promoter and ribosome binding site of B. amyloliquefaciens and cloned in E. coli in pBR 327 plasmid. The level of beta-lipotropin synthesis in bacterial cells transformed by the obtained plasmid was estimated immunochemically. The level of beta-lipotropin production was shown to be 5 mg per liter of bacterial culture.

[Chemico-enzymatic synthesis of genetic elements for expression of synthetic genes in Bacillus subtilis cells]. / Khimiko-fermentativnyi sintez geneticheskikh élementov dlia ékspressii iskusstvennykh genov v kletkakh Bacillus subtilis.

In order to obtain the recombinant Bacillus subtilis strain, a transcriptional-translational control unit of the alpha-amylase gene of B. amyloliquefaciens was synthesized. The oligodeoxyribonucleotides were prepared by the modified triester method in solution and by the solid-phase approach. Then these oligonucleotides were joined by DNA ligase into two fragments which were cloned in the phage M13mp9 DNA and the plasmid pBR327. A plasmid harboring the site regulating the transcription of the alpha-amylase gene may be employed as vector for cloning the promoter-containing fragments in E. coli cells.