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495. Adenovirus Serotype 35: Genome Sequence and Gene Transfer Vector Development
NOVEL AD VECTORS AND APPLICATIONS 495. Adenovirus Serotype 35: Genome Sequence and Gene Transfer Vector Development Wentao Gao,1 Huijie Sun,1 Paul D. Robbins,2 Andrea Gambotto.1,2 1 Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; 2Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States; 3United States. Adenoviral vectors are widely used as gene delivery vehicles. The most adenoviral vectors are currently developed from adenoviral serotype 5. However, the prevalence of pre-existing immunity to adenovirus type 5 may become an obstacle for its use in humans as gene transfer vector. To date, 51 serotypes of adenovirus have been identified in humans. To find out adenoviruses among the known serotypes that have lower pre-existing immunity will benefit the adenoviral vector application in humans. We report here the complete sequence of adenovirus type 35(Ad35) strain Holden genome. 47 open reading frames (ORF) were identified which include early (E1, 2, 3, 4) and late (L1, 2, 3, 4, 5) genes of known adenoviruses. The gene layout is similar to that of other human adenoviruses. The viral genome shows a very compact organization. Overlapping genes were found on both strands. Additional 14 ORFs were identified which do not encode for known adenoviral conserved genes. The predicated translational features of those conserved genes were compared with other adenoviruses. The result showed that Ad35 shares high homology with Ad 11, Ad7, Ad3, Ad21, Ad17 and simian Ads25. Based upon the Ad35 DNA sequence, we have developed an E1/E3 region deleted recombinant adenoviral 35 vector encoding an enhanced yellow fluorescence protein (Ad35-EYFP) and functionally analyzed the pre-existing neutralizing antibody by using serum samples from South Africa, Gambia and USA on A549 cell. The assay results from flow cytometry showed that about 3%22% of serum samples have different level of neutralizing antibody against Ad35 comparing to 42-90% serum samples existing neutralizing antibody against Adenovirus type 5. Transduction efficiency of ad35-EYFP was evaluated in various tumor cell lines as well as human and monkey dendritic cells. The results showed high transduction efficiency for most of tumor cell lines tested, as well as human and monkey dendritic cells. Our study suggested that Ad35 may become a useful adenoviral vector for both cancer gene therapy and vaccine development.
496. Optimization of the FLPe/frt Recombination System for the Production of HighCapacity, Helper-Dependent Adenoviral (HD) Vector II: Detailed Molecular Analysis of the Stability of the Flpe/frt System over Repeated Passaging Xiangpeng Yuan,1 Jinwei Hu,1 Pedro R. Lowenstein,1,2 Maria Castro.1,2 1 Gene Therapeutics Research Institute, Davis Building, Cedars Sinai Medical Center, Los Angeles, CA ; 2Department of Medicine, David Geffen School of Medicine, UCLA. HD vectors have many advantages, such as, increased cloning capacity (~30kb), reduced toxicity, and prolonged transgene expression in vivo. The present study was designed to investigate the propagation of HD vectors, and the stability and genome structure surrounding the packaging site (Ψ) of helper virus contaminating high titer HD vectors, produced with our novel Flpe/frt packaging system [1]. We analyzed helper virus genomes present in passages 1-10 of HD vector preparations. To analyze the sequences surrounding Ψ of the helper virus, following passage 5 and 10, crude HD vector preparations were used to infect 293FLPe cells at an MOI = 10 of helper virus present in such preparations. Viral DNA S194
from infected cells was extracted and subjected to PCR analysis. PCR primers were designed to amplify the sequences around Ψ within the helper virus genome to assess the efficiency of Ψ excision by Flpe recombination. One primer was labeled with fluorescein to facilitate quantification of the PCR product. Analysis of passage 5 showed a very faint 493bp fragment, indicative of sequences surrounding Ψ in the absence of excision, and a strong signal from a 288bp fragment, a result of the amplification of packaging signalexcised sequences from helper virus genomes. Relative quantification of the fragments, using Fluochem analysis showed that the 493bp fragment (containing Ψ) is approximately 3% of the excised 288bp fragment, and thus 97% of helper virus present at passage 5 had undergone Ψ excision by Flpe. Similar results were obtained following the analysis of virus obtained at passage 10. Thus, we found no significant increment in the number of helper virus from which Ψ had not been excised from passage 5 to passage 10. This indicated that there was no overgrowth of helper virus potentially containing a mutated packaging site that could not be excised anymore by Flpe mediated recombination of the frt sites flanking Ψ, even after 10 serial passages. Such a mutated virus would increase in titer upon passaging, and this would be detrimental to HD vector titers obtained. To further our analysis, the 493bp and 288bp fragments of PCR were subcloned and sequenced. The sequences showed that the 493bp fragment contained 2 intact frt sites flanking Ψ, while the 288bp fragment contained only one, and no Ψ sequences, confirming the absence of a Flpe-resistant mutation within the helper viral genome, even at passage 10. Thus, helper virus within HD vector preparations maintains its sensitivity to Flpe-mediated recombination. Passage 10 was further purified by CsCl gradient centrifugation. Titration of HD vectors’ transducing genome number was performed using a novel quantitative TaqMan PCR method [2]. Passage 10 HD vectors had higher than 1011 transducing genome copies per milliliter, with unexcised helper virus contamination of 0.1% [2]. In conclusion, high titer HD vectors can be produced by extended passaging without helper viral overgrowth, even after 10 passages of rescue/amplification using the Flpe/frt recombination packaging system. [1] Nat.Biotech.19:582-5, 2001. [2] Hu et al., Am. Soc. Gene Therapy Abs., 2003.
497. Construction of a Novel Vector Plasmid for the Production of High-Capacity, HelperDependent Adenoviral (HD) Vector Using the FLPe/frt Recombination Maximiliano J. Jimenez-Dalmaroni,1 Pablo Umana,2 Pedro R. Lowenstein,1 Maria G. Castro.1 1 Department of Medicine, David Geffen School of Medicine, UCLA, Gene Therapeutics Research Institute, Davis Building, Cedars Sinai Medical Center, Los Angeles, CA, United States; 2 Glycart Biotechnology AG, Glycart Biotechnology AG, Zurich, Switzerland. First generation adenoviral vectors can be produced at high titer, and transduce dividing as well as non-dividing cells. However, antiadenoviral immune responses are an important limitation to long term expression, since they eliminate transgene expression, and cause extended inflammation. To reduce immune responses HD vectors have been developed. HD vectors only contain the cis-acting elements required for virus replication and packaging while the respective proteins are provided in trans by helper virus. We recently described a novel HD packaging system, in which helper virus is eliminated by Flpe induced recombination between two frt sites flanking Y. Thus, only HD genomes can be packaged. To achieve HD packaging without recombination or rearrangements, the vector plasmid needs to have a length of at least 27 Kb, most of which is made up of non-coding sequences (called ‘stuffer’ DNA), together Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
496. Optimization of the FLPe/frt Recombination System for the Production of HighCapacity, Helper-Dependent Adenoviral (HD) Vector II: Detailed Molecular Analysis of the Stability of the Flpe/frt System over Repeated Passaging Xiangpeng Yuan,1 Jinwei Hu,1 Pedro R. Lowenstein,1,2 Maria Castro.1,2 1 Gene Therapeutics Research Institute, Davis Building, Cedars Sinai Medical Center, Los Angeles, CA ; 2Department of Medicine, David Geffen School of Medicine, UCLA. HD vectors have many advantages, such as, increased cloning capacity (~30kb), reduced toxicity, and prolonged transgene expression in vivo. The present study was designed to investigate the propagation of HD vectors, and the stability and genome structure surrounding the packaging site (Ψ) of helper virus contaminating high titer HD vectors, produced with our novel Flpe/frt packaging system [1]. We analyzed helper virus genomes present in passages 1-10 of HD vector preparations. To analyze the sequences surrounding Ψ of the helper virus, following passage 5 and 10, crude HD vector preparations were used to infect 293FLPe cells at an MOI = 10 of helper virus present in such preparations. Viral DNA S194
from infected cells was extracted and subjected to PCR analysis. PCR primers were designed to amplify the sequences around Ψ within the helper virus genome to assess the efficiency of Ψ excision by Flpe recombination. One primer was labeled with fluorescein to facilitate quantification of the PCR product. Analysis of passage 5 showed a very faint 493bp fragment, indicative of sequences surrounding Ψ in the absence of excision, and a strong signal from a 288bp fragment, a result of the amplification of packaging signalexcised sequences from helper virus genomes. Relative quantification of the fragments, using Fluochem analysis showed that the 493bp fragment (containing Ψ) is approximately 3% of the excised 288bp fragment, and thus 97% of helper virus present at passage 5 had undergone Ψ excision by Flpe. Similar results were obtained following the analysis of virus obtained at passage 10. Thus, we found no significant increment in the number of helper virus from which Ψ had not been excised from passage 5 to passage 10. This indicated that there was no overgrowth of helper virus potentially containing a mutated packaging site that could not be excised anymore by Flpe mediated recombination of the frt sites flanking Ψ, even after 10 serial passages. Such a mutated virus would increase in titer upon passaging, and this would be detrimental to HD vector titers obtained. To further our analysis, the 493bp and 288bp fragments of PCR were subcloned and sequenced. The sequences showed that the 493bp fragment contained 2 intact frt sites flanking Ψ, while the 288bp fragment contained only one, and no Ψ sequences, confirming the absence of a Flpe-resistant mutation within the helper viral genome, even at passage 10. Thus, helper virus within HD vector preparations maintains its sensitivity to Flpe-mediated recombination. Passage 10 was further purified by CsCl gradient centrifugation. Titration of HD vectors’ transducing genome number was performed using a novel quantitative TaqMan PCR method [2]. Passage 10 HD vectors had higher than 1011 transducing genome copies per milliliter, with unexcised helper virus contamination of 0.1% [2]. In conclusion, high titer HD vectors can be produced by extended passaging without helper viral overgrowth, even after 10 passages of rescue/amplification using the Flpe/frt recombination packaging system. [1] Nat.Biotech.19:582-5, 2001. [2] Hu et al., Am. Soc. Gene Therapy Abs., 2003.
497. Construction of a Novel Vector Plasmid for the Production of High-Capacity, HelperDependent Adenoviral (HD) Vector Using the FLPe/frt Recombination Maximiliano J. Jimenez-Dalmaroni,1 Pablo Umana,2 Pedro R. Lowenstein,1 Maria G. Castro.1 1 Department of Medicine, David Geffen School of Medicine, UCLA, Gene Therapeutics Research Institute, Davis Building, Cedars Sinai Medical Center, Los Angeles, CA, United States; 2 Glycart Biotechnology AG, Glycart Biotechnology AG, Zurich, Switzerland. First generation adenoviral vectors can be produced at high titer, and transduce dividing as well as non-dividing cells. However, antiadenoviral immune responses are an important limitation to long term expression, since they eliminate transgene expression, and cause extended inflammation. To reduce immune responses HD vectors have been developed. HD vectors only contain the cis-acting elements required for virus replication and packaging while the respective proteins are provided in trans by helper virus. We recently described a novel HD packaging system, in which helper virus is eliminated by Flpe induced recombination between two frt sites flanking Y. Thus, only HD genomes can be packaged. To achieve HD packaging without recombination or rearrangements, the vector plasmid needs to have a length of at least 27 Kb, most of which is made up of non-coding sequences (called ‘stuffer’ DNA), together Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy