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The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

ABSTRACT: Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comprising five early transcription units, E1A, E1B, E2, E3, and E4, two delayed early units IX and IVa2, and the major late unit, in total 39 putative and 7 hypothetical open reading frames. A recombinant E1-deleted Ad3 was generated on a bacterial artificial chromosome. This prototypic virus efficiently transduced CD46-positive rodent and human cells. Our results will help in clarifying the biology and pathology of adenoviruses and enhance therapeutic applications of viral vectors in clinical settings.

SUBMITTER: Sirena D

PROVIDER: S-EPMC7172737 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

Sirena Dominique D Ruzsics Zsolt Z Schaffner Walter W Greber Urs F UF Hemmi Silvio S

Virology 20050916 2

Human adenovirus (Ad) serotype 3 causes respiratory infections. It is considered highly virulent, accounting for about 13% of all Ad isolates. We report here the complete Ad3 DNA sequence of 35,343 base pairs (GenBank accession DQ086466). Ad3 shares 96.43% nucleotide identity with Ad7, another virulent subspecies B1 serotype, and 82.56 and 62.75% identity with the less virulent species B2 Ad11 and species C Ad5, respectively. The genomic organization of Ad3 is similar to the other human Ads comp ...[more]

PMID: 16169033

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Project description:BackgroundHemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.MethodsWe infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months.ResultsAAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values.ConclusionsPeripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).

Project description:Efficient infection with adenovirus (Ad) vectors based on serotype 5 (Ad5) requires the presence of coxsackievirus-adenovirus receptors (CAR) and alpha(v) integrins on cells. The paucity of these cellular receptors is thought to be a limiting factor for Ad gene transfer into hematopoietic stem cells. In a systematic approach, we screened different Ad serotypes for interaction with noncycling human CD34(+) cells and K562 cells on the level of virus attachment, internalization, and replication. From these studies, serotype 35 emerged as the variant with the highest tropism for CD34(+) cells. A chimeric vector (Ad5GFP/F35) was generated which contained the short-shafted Ad35 fiber incorporated into an Ad5 capsid. This substitution was sufficient to transplant all infection properties from Ad35 to the chimeric vector. The retargeted, chimeric vector attached to a receptor different from CAR and entered cells by an alpha(v) integrin-independent pathway. In transduction studies, Ad5GFP/F35 expressed green fluorescent protein (GFP) in 54% of CD34(+) cells. In comparison, the standard Ad5GFP vector conferred GFP expression to only 25% of CD34(+) cells. Importantly, Ad5GFP transduction, but not Ad5GFP/F35, was restricted to a specific subset of CD34(+) cells expressing alpha(v) integrins. The actual transduction efficiency was even higher than 50% because Ad5GFP/F35 viral genomes were found in GFP-negative CD34(+) cell fractions, indicating that the cytomegalovirus promoter used for transgene expression was not active in all transduced cells. The chimeric vector allowed for gene transfer into a broader spectrum of CD34(+) cells, including subsets with potential stem cell capacity. Fifty-five percent of CD34(+) c-Kit(+) cells expressed GFP after infection with Ad5GFP/F35, whereas only 13% of CD34(+) c-Kit(+) cells were GFP positive after infection with Ad5GFP. These findings represent the basis for studies aimed toward stable gene transfer into hematopoietic stem cells.

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The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

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The nucleotide sequence and a first generation gene transfer vector of species B human adenovirus serotype 3.

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