WO2003031633A2 - Adenovirales vektorsystem - Google Patents
Adenovirales vektorsystem Download PDFInfo
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- WO2003031633A2 WO2003031633A2 PCT/DE2002/003846 DE0203846W WO03031633A2 WO 2003031633 A2 WO2003031633 A2 WO 2003031633A2 DE 0203846 W DE0203846 W DE 0203846W WO 03031633 A2 WO03031633 A2 WO 03031633A2
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10351—Methods of production or purification of viral material
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
Definitions
- the invention relates to a special adenoviral vector based on human adenoviruses of group B, in particular of subtype 11, of the heterologous elements according to the invention, inverted terminal repeats (ITRs) in combination with the corresponding pack signal of a virus of another serotype, preferably a virus of the Type B contains.
- a heterologous promoter preferably the SV40 promoter, is contained in the viral vector and positioned between the pack signal and the natural position for protein IX. This vector can additionally be deleted in reading frames of the regions E1, E2, E3 or E4.
- the invention also describes the use of this viral vector for the production of a high-capacity vector based on the adenovirus 11, which has only ITRs and pack signals on adenoviral sequences and contains human genomic filling sequences.
- These heterologous elements in the viral vector on the one hand enable stable reproducibility in complementing cell lines and on the other hand enable use as a helper virus to multiply a viral vector with filling sequences (high-capacity vector). It is possible to select the high-capacity vector against the helper virus on the basis of the heterologous elements. This selection principle can be combined with other selection principles. Cell lines for the amplification of these viral vectors and the use of the vectors in medicine are also described
- the success of viral gene therapies largely depends on the availability of suitable vectors.
- the vector was to transport and express the therapeutic gene with high efficiency in the cell nucleus of the target cell, stabilize it there and, moreover, neither have a direct toxic effect nor trigger an immune response.
- the vector must also be able to be produced on an industrial scale.
- Adenoviral vectors have a special position among viral systems. They are characterized by a broad host spectrum, the ability to infect resting cells and extremely high titers. The infection effectiveness, based on the required amounts of nucleic acid, exceeds that of all other viral systems and exceeds that of bare DNA (in application) by 100,000 times. Types 4 and 7 adenoviruses have been used on a large scale as live vaccines and have a good safety profile.
- the extremely low tendency to integrate adenoviruses is also advantageous because it minimizes the risk of insertion mutagenesis and oncogene activation.
- 52 different serotypes of human adenoviruses offer a selection of different virus envelopes with very different tropism and are therefore particularly infectious for liver or muscle (group C), cells of the central nervous system (representative of group D) or cells of the hemopoietic system (group B).
- adenovirus vectors from the frequently used serotypes 2 and 5 (group C) stands in the way of their widespread use and a generally high antibody titre in substantial parts of the population. This can reduce the effectiveness of the application up to ineffectiveness.
- the infection of the population with viruses of different serotypes varies greatly. As a result, viruses can be found whose envelopes are only rarely inactivated by antibodies and which are particularly suitable for certain target tissues.
- Type 11 adenoviruses a rare serotype in the western hemisphere, are effective in infecting hemopoietic stem cells, dentritic cells and certain tumors. So far, however, there is no vector system of this type. Despite these key advantages, the uses of adenovirus vectors are limited.
- adenovirus vectors which are free of viral genes have been developed in recent years (Hardy et al. 1997; Kochanek et al. 1996; Kumar-Singh and Chamberlain 1996; Mitani et al. 1995; Parks et al 1996). These helper-dependent or high-capacity vectors show a significantly changed behavior in the animal. The otherwise typical inflammation symptoms after infection of the liver with medium doses of adenoviruses are completely absent, and the expression per virus increases up to 100 times, which makes a significant dose reduction possible.
- Production begins with the transfection of a helper-dependent vector, the ITRs of which are present either terminally after restriction cleavage or as head-to-head fusion (Parks et al. 1996) into the production cell line (293cre).
- the cells are also infected with the adenovirus helper. After achieving a cytopathic effect, the cells are harvested and the vectors are freed by freeze-thaw steps or mild detergents.
- the cell extract is used to infect further passages, which must also be infected with the helper virus, insofar as the inoculum comes from cells that exert a selection pressure on the helper. This process continues for 5-6 passages until enough vector is available for large scale infection.
- the amplification factor moves by 20 and is therefore lower than with 1st generation vectors (30-80).
- helper viruses are created with great frequency during the selection, which escape the selection pressure by mutation and are accordingly amplified. These make a vector preparation unusable.
- the object of the invention is to establish a viral vector based on human adenovirus 11. This vector should be stably reproducible and safe to use. There is still the task of finding robust production processes for such vectors, which enable a minimization of the helper combination.
- Such a vector is established with the aim of restricting the neutralization of the vector by pre-existing antibodies and of being able to infect new target cells or tissues and thus to expand the host spectrum.
- reading frames from different regions or all viral reading frames must be removed.
- heterologous elements are inverted terminal repeats (ITRs) in combination with the corresponding pack signal which originate from a virus of another serotype, preferably a virus of type B. These elements allow amplification of the virus, but also allow use as a helper to produce an adenovirual vector with filling sequences (high-capacity vector). They also include a heterologous promoter, preferably the SV40 promoter, which is positioned between the pack signal and the natural position for protein IX , The object is further achieved in that special filling sequences for a high-capacity vector are provided which allow the stable amplification of this vector.
- ITRs inverted terminal repeats
- This virus uses a variety of infection pathways from the adenovirus subtypes used to date, thus expanding the range of applications.
- the vectors are used as therapeutics or vaccines.
- Vector system based on the human adenovirus type 1
- the following describes the establishment of a vector system for a serotype, adenovirus type 1 1, which is rare in the western hemisphere.
- group C type 2 and 5 adenoviruses the serotypes common in gene therapy developments and trials, causes high titers of neutralizing antibodies in large parts of the population and thus reduces the effectiveness of gene therapy use.
- the low prevalence of the Adl 1 serotype thus represents a great advantage over the use of previously conventional vectors, which are based, for example, on Ad5.
- the nucleic acid sequence of this virus is hitherto unknown.
- Adl 1 differs significantly in the sequence of the terminal 22bp from other representatives of group B, eg Ad7, but is completely correct match the sequence of group C viruses (Ad2 and 5).
- Adl 1's ITR has significantly fewer target sequences for transcription factors than Ad5. The lack of the NF3 binding sequence is particularly striking.
- group C adenoviruses this cellular factor is involved in the initiation of replication, the covalent activation of the terminal protein (pTP) by reaction with deoxycytidine triphosphate (dCTP).
- Adl 1 replicates more slowly and limits the range of permissive cell types beyond capsid-receptor interaction.
- Adl 1 needs a different, possibly less ubiquitous and efficient support from the host cell.
- El proteins are removed from vectors because of their transforming properties and the transactivating effect on all other viral promoters from the genome of vectors. These proteins or homologs with identical function must then be provided in a helper cell. The partial remaining of El sequences leads to an overlap of sequences between the helper cell line and the vector and is the cause of the development of wild-type viruses by homologous recombination. Therefore, El sequences are completely removed from the vectors. This leaves only 64bp in front of the start codon of protein IX, a protein necessary for efficient virus packaging, which has no binding sites for known transcription factors or a start region typical for group C viruses. The mechanism of expression of the pIX factor is unclear.
- this protein is encoded by a specific m-RNA that is controlled by an independent promoter.
- Adl l Protein IX elements in the E1A or B region could be responsible.
- the expression of this open reading frame should therefore be supported by the insertion of heterologous known promoter elements, namely the early SV40 promoter.
- the insertion leads to a dramatic increase in virus replication. This increase unexpectedly also occurs when the promoter is prevented by a directly connected poly A signal from driving the expression of PXI. It is concluded that specific transcription factor binding sequences found in the SV40 promoter region generally support the amplification of adenovirus 11 as representatives of group B2.
- a stabilized Ad 11 vector is generated by the insertion of SV40 promoter sequences.
- the packaging of adenovirus DNA requires the specific interaction of ITR and pack signal with viral and cellular proteins. The interaction with viral proteins is described as subtype-specific (Zhang, 2001).
- An Ad 5 virus with a mutant protein L152 / 55k cannot pack its DNA and this defect is not complemented by the corresponding protein from other serotypes. It suggests itself that the specificity is mediated through an interaction with the pack signal.
- a viral Adl 1 vector with a heterologous ITR but autologous pack signal should therefore be capable of replication due to the end sequences in the ITR being identical to Ad5, and should also be packable due to the Adl 1 pack signal.
- Such a vector is unexpectedly not viable. All the more, a vector with Adl 1 genome and ITR and pack signal from Ad5 should not be viable. However, such a vector, which is described in accordance with the invention, can be effectively propagated without additional helper function in the absence of Adl 1 packing sequences. It generates a clear excess of empty virus envelopes and is therefore used in a special application as a helper virus for a high-capacity vector.
- This packaging lock can be combined with other methods aimed at deleting ice active sequences for packaging.
- the heterologous pack signal is flanked by the target sequences of the recombinases and cut or inverted in cells which express the corresponding recombinases.
- the modifications of the Adl 1-based vector can be used separately from one another. In a preferred application, they are used together in the vector.
- a deletion is introduced in a further region of the genome in order to generate a 2nd generation Adl 1 virus. The possibilities for such deletions are known to the person skilled in the art. In particular, the deletion is carried out in the E2B area.
- Production then takes place in a cell which carries the DNA polymerase gene of a group C adenovirus.
- the adenovirus type 1 1 polymerase gene can be expressed in the cell.
- the gene of the preterminal protein of a C-type virus can also be expressed.
- the increased oncogenic potential is likely to be located in the E4 region.
- the E4 region of the virus is therefore partially deleted.
- the El region is deleted from Ad11 and thus a virus is generated which, in its replication, is dependent on the trans-complementation of the El proteins by the host cell.
- the most effective complement is expected to be achieved through the El Region of Adl 1.
- the regions EIA and E1B can either be as separate cassettes, each fused with heterologous transcription signals (promoter, poly A) or as a unit, connected with only one heterologous promoter before EIA and one heterologous poly A following the reading frame of E1B 55k in the Production cell to be established. It must be ensured that there are no or only short overlaps between the sequences in the cell line and vector that do not allow homologous recombination. Such overlaps can be between 1 and 6bp. The establishment in separate cassettes offers further protection against homologous recombination.
- EIA region brings about a change in the cell cycle combined with a stimulation of virus replication. They also have a transcription-activating effect, but do not bind to DNA on their own, but interact with cellular factors.
- the skilled worker is therefore furthermore aware that EIA of one serotype can complement the vector of another serotype. As is known, this does not apply to E1B because it interacts with other viral proteins, in particular with E4orf6. It is therefore not possible to amplify the vector according to the invention in the cell line 293 which expresses the El region of Ad5.
- the E1B region encodes two independent proteins of 19k and 55k, which are encoded by a common mRNA.
- the E1B promoter is located in the EIA region.
- E1B region As a unit either from the autologous promoter or from a heterologous promoter.
- region As a unit of 55k and 19K potein only insufficiently complements the vector, regardless of whether the expression is from a heterologous or an autologous promoter.
- effective complementation is only possible if E1B 55k is controlled separately from E1B 19k by a separate promoter.
- the ineffectiveness of the common expression could be due to the fact that the 55k reading frame is the second reading frame of this mRNA and the mechanism for its initiation is ineffective.
- Packaging are absolutely necessary. These occupy less than 2% of the genome size. However, effective packaging requires a linear DNA molecule of 75-103% of the wild-type genome size, the ends of which are flanked by viral terminal repeats (ITR) and contain a packing signal near one of the ITRs.
- ITR viral terminal repeats
- the replication mechanism of adenovirus is based on the synthesis of linear DNA molecules starting from a protein priming by terminal protein (TP) and stabilized single-stranded DNA as an intermediate. This condition requires the juxtaposition of different molecules in homologous areas with the result of a high recombination frequency. Recombination at homologous sections within the filler DNA results in heterogeneous populations of vectors, possibly with loss of the transgene. Frequent repetitions of the sequence must therefore be avoided in the filling DNA become. This places an additional strain on the use of human DNA
- the DNA used should also be free of additional, potentially expressible
- Introns of cellular genes are used. For safety reasons, only DNA sections that do not contain any endogenous retroviruses or elements of these viruses are selected. For a large number of applications, it is desirable that this DNA is not recognized as foreign in the cell and does not trigger any defense reactions. This condition is best achieved when using human DNA.
- sequences consist of introns of the CXorf ⁇ gene and exons of less than 200 bp, which do not result in independent open reading frames. They do not contain retroviral LTRs. Repeats are limited to short caca, gaga and gtttgttt simple repeats.
- the GC content of the vector is 47.1%, which is far from that of adenovirus 5.
- the replication ability of a vector depends on the properties of its DNA. The underlying mechanisms have not been examined in detail.
- GC is far more effective than that of type 5 adenovirus (55.2%).
- the sequences contain DNA from the 1st intron of the FMR2 gene. They are also free of retroviral elements and only contain phylogenetically old shine, line and simple repets that are already far away from the respective consensus sequences.
- the GC portion in the vector is 38.1%. The vector is therefore particularly suitable for GC-poor people
- Adenovirus types e.g. of the AT adenovirus genus.
- sequences were extracted from genomic DNA, whole blood from a healthy one
- Subjects obtained by PCR and in vectors containing both ITR and packing sequences from
- the DsRedl gene was subsequently introduced as a foreign gene.
- the replication of the vector was with the existing one
- Vectors A and B were examined by co-replication using the Cre-Lox helper system.
- the replication ability of a vector depends on the properties of its DNA.
- GC is far more effective than that of type 5 adenovirus (55.2%). According to published hypotheses, reduced replication of the vector was expected. Contrary to expectations, it was found experimentally that a vector with these sequences is superior in replication.
- An example here is a fusion with the pack signal and the ITRs of adenovirus type 5 nt
- the invention has a viral vector based on the sequence of the human adenovirus serotype 11, which contains inverted terminal repeats and the pack signal from a virus of another serotype, ITRs and pack signal together from a group C adenovirus, preferably from type 5 adenovirus , comes from.
- the vector is deleted in genome areas in such a way that independent replication without trans-complementation by factors whose genes have been inactivated by the deletion is impossible and the recombinant viruses in one or more reading frames of the EI region and preferably further reading frames of the E2 and / or E4 region are deleted.
- the vector according to the invention is based on the sequence of the human adenovirus serotype 11, in particular on a viral vector which contains a heterologous promoter.
- the heterologous promoter is preferably an SV40 promoter, the promoter being located between the pack signal and the natural position of the protein IX.
- the vector according to the invention is further characterized in that filling sequences are inserted as replacements in the recombinant viruses instead of certain genome areas or in the entire genome, with the exclusion of the left and right ITR and the pack signal.
- the invention further relates to vector constructs which contain components of the viral vector or are suitable for the production thereof.
- the invention relates to a cell line, characterized in that it can be infected by human adenovirus serotype 11 and complements deletions in the viral vector according to claims 1-7. It is characterized in that it forms adenovirus 11 E1B 55k or a functional homologue thereof as an independent expression unit, separate from ElB19k, with its own promoter.
- the cell line is derived from HEK 293.
- the viral vector according to the invention is further characterized in that, as a helper virus, it enables the multiplication of a viral vector, the helper virus being characterized in that the pack signal of the virus is flanked by sequences of site-specific recombinases and that in a cell line complementing adenovirus functions, which the corresponding recombinase carries, is inactivated.
- Human sequences are used as foreign sequences, which are continuous, interrupted or inverted and are used as filler sequences to more than 80% intron sequences. there filling sequences are taken completely or partially from the region of the X chromosome of X152941900- X152976000 and / or chrX149493805-149526200.
- the invention further relates to a therapeutic or a vaccine containing a viral vector according to one of claims 1-6 or 14-16.
- the vaccination method according to the invention comprises administering a viral vector according to one of claims 1-6 or 14-16. to the person to be vaccinated.
- the features of the invention go from the elements of claims and. from the description, both individual features and several in the form of combinations representing advantageous versions for which protection is sought with this document.
- the combination consists of known (viral vectors, adenoviruses deleted in individual or all reading frames) and new elements (hererologists ITRs, promoters and filling sequences), which mutually influence one another and whose overall effect enables the advantageous extraction of recombinant viruses.
- the invention results in deleted adenoviral vectors of human subtype 11, including adenoviral vectors deleted (completely) in all viral reading frames.
- the vectors according to the invention contain the sequence adenovirus type 11 sequence in recombinant form, combined with specific heterologous sequences. These viruses are deleted in genome areas in such a way that independent replication without trans complementation by suitable factors is impossible - they are deleted in the reading frame of the El Region
- Human sequences that are continuous, interrupted or inverted are used as foreign sequences. More than 80% of intron sequences are used as filler sequences, the filler sequences being wholly or partly the region of the X chromosome - from X 152941900- X 152976000 or
- the viral vector according to the invention is amplified in a cell line which complements adenoviral gene functions.
- the cell line contains genes from the El region of Ad11 as a continuous sequence with a heterologous promoter before EIA and a heterologous poly A signal after the EIB 55K stop codon.
- the cell line is characterized by the fact that it forms the Ad 11 EIB 55k or a functional homologue thereof as an independent expression unit with its own promoter, separate from ElB19k.
- the helper virus is a deleted adenovirus in areas essential for replication, such as El, for example. It carries a heterologous ITR and pack signal.
- the virus pack signal can be flanked by sequences of site-specific recombinases. In this case it is additionally inactivated by the cell line which complements an adenovirus function and which carries the corresponding recombinase.
- the hallmark of the helper virus is that
- Adl 1 acquired from American Type Culture Collection (VR-12), was amplified on Hep-2 cells and purified using a Cs gradient. Virus DNA was isolated using pronase treatment, phenol extraction and ethanol precipitation. The DNA was subjected to a treatment with 4N NaOH for 60 min at 37 ° C., renatured with 0.4N NaCl, 3M NaCl, 0.5M Tris 7.5 for 6 h at 65 ° C. and 12 h at room temperature, and re-precipitated. This DNA is completely deproteinated. It was treated with Klenow polymerase, digested with Hind III, and the fragment mixture was cloned between the Hinc II and Hind III sites of pUC 18.
- a 1.3 kb fragment was found in several clones. This fragment is homologous to known sequences of ITR and pack signal of adenovirus 11. It is completely identical to that of adenovirus type 5 in the terminal 22nt CATCATCAATAATATACCTTAT. To obtain the 3 'end, primer with the terminal sequence was flanked by a Pacl site AdlIITRF CTTAATTAACATCATCAATAATATC and primer 11-S2 with the sequence GCTCCGTGCGACTGCTGTTT, selected from the fiber region of the virus gb L08232, were used. Adl 1ITRF contains a single base deletion TAATATAC compared to the wt sequence, caused by a sequencing error.
- a 2.8 kb fragment was amplified and cloned. It contains the 3 'end of the virus. The sequence is identical to the 5 'end of nt 1- 132, the ITR is therefore 132 bp long. A 472bp fragment of the 5 'end flanked by Pacl and the internal SnaBI site, with which the pack signal is completely included, the 5' end was obtained and cloned in such a way that a shuttle vector for the homologous recombination of the virus DNA into a plasmid results. As a result of the recombination in E.
- plasmid of approximately 15 kb was formed, which contains the left end of the virus in duplicate. This could be due to the presence of an internal, naturally occurring reverted sequence.
- a plasmid (pAdl lwt) of 37.5 kb was generated, the internal Hind III fragments of approx. 14 kb, 5.7 kb 5, lkb 3.3 kb, 2.5 kb 2.2 kb, l, 3 kb 0.7 kb 0, 3kb releases. Only the 35 kb plasmid is capable of producing a cytopathic effect after 16 days after cleavage with Pacl and Ca transfection in 293 cells.
- Plasmid pAdl lwt was therefore digested with Ncol and a 4600bp fragment was religated.
- Adl IITR-F-Pme sequence ACCGGTTTAAACATCATCAATAATATACCTTAT
- Adl l ITR end flanked by a Pmel site a 2000 bp frame was amplified and cloned into a KanR minimal vector (pAdl lNco).
- PvuII cleavage a fragment encoding gfp, containing a unique Notl site, was cloned into pAdl lNco and the resulting vector after Ncol cleavage was recombined with pAdl lwt.
- the resulting plasmid pAdl 1 wtgfp contains a complete Ad 11 genome flanked by Pmel sites with a gfp gene cloned in position E at position 459 from the left end of the virus in El orientation.
- This plasmid served as the supplier of the Adl 1 sequences.
- Embodiment 2 Cloning of vectors with heterologous ITR and or pack signals
- Adl 1 wt was cleaved with EcorV in the plasmid vector and Afel in the virus, the resulting plasmid was religated and inserted between SnaBi at the end of the pack signal and Bglll gfp (pAdl 1 Afegfp). After recombination with pAdl 1 wtgfp split with Notl, p Adl 1 deltaElgfp was formed. With the help of the shuttle vector pi5pl lgfpPme, Ad5 5 'ITR and flanking sequences nt 1 -190, the Adl 1 pack signal nt 198-440, the Ad53 TTR (103bp) as well
- the resulting vector pHip51 lfrt contains the Adl 1 sequence deleted in E1, ITRs and pack signal from Ad5, the pack signal being flanked by frt sites, and the SV40 promoter before the reading frame of PXI. Likewise, the HindIII fragment from pAdl 1FRT (1-5) SV40 downstream from gfp in
- a tk polyadenylation signal was taken from pgfpNl as PvuII BspHI and cloned in StuI from pshAdip51 1 frt.
- This poly A signal is located directly after the SV40 promoter and blocks its direct effect as a promoter, but not an indirect effect as an enhancer on other genome sections.
- Embodiment 4 Production of a complementing cell line for deleted vectors of Adl 1
- the coding sequence of Adl 1E1 B55k without promoter and poly A was determined with the aid of the primers Ad 1155kF-XhoI AACTCGAGAATGGATCCCGC AGACT and Ad 11 E 1 R-Kpnl AATGGTACCTTAGTCCTCTTTC template amplified and cloned into the vector phPGKLgfp after Kpnl and Xhol digestion.
- EI A is under the control of the human PGK promoter and the EIB polyadenylation signal is replaced by the early poly A signal from SV40.
- This plasmid was transfected into HEK293 cells using Polyfect (Qiagen) and selected for 3 weeks with 400 ⁇ g / ml G418. Clones were separated by dilution sowing in 96 well plates and subsequently checked for the presence of the gene of Adl lElB55k using the primers Adl 155kF-XhoI and Adl lElR-Kpnl. Positive clones were transfected with the plasmid Adl delta El gfp after linearization and clones with visible CPE after 9 days were used for reamplification of the virus in comparison. The clone with the maximum number of gfp -inducing units D7 formed was used as a cryopreserved and as a helper cell line.
- Embodiment 5 Embodiment 5
- Clone D7 (expressing Adl 1 55k) or transfected in 293 cells in 2 wells of a 6 well plate. The cells were examined daily for the presence of a cytopathic effect. For vectors with the gfp gene, the well was examined simultaneously for an expansion of the gfp expression.
- Hip51 lfrt and Adl ldelgfp were each cleaned from 20 15 cm cell culture dishes, 293 clone D7 via a CsCl step gradient and a continuous gradient. It was found that for Hip51 lfrt a 20 times stronger upper band (empty virus envelopes) and a weak lower band (complete virus) formed. In contrast, a clear lower band and a weak upper band appeared in Adl ldelgfp. This confirms the hypothesis that Hip51 lfrt is easily amplifiable in 293Klon D7, but produces an excess of empty shells due to ineffective packaging.
- Embodiment 6 Cloning of the vector pHCA
- Genomic DNA was isolated from 15 ml whole blood of a subject by SDS lysis followed by saline precipitation and ethanol precipitation. In each case 100 ng DNA were used as
- Fragment 3 was first cloned into the vector pPCR4blunttopo (Invitrogene).
- a shuttle vector was created for the construction of a high-capacity vector with ITR and pack signals of the adenovirus type 5.
- the adenovirus 5 sequence of ntl -nt 443 was flanked by Pmel at the 5 'and Ecor52I and Hind III at the 3' end, and the sequence from 35520-35935 flanked by Hind III and Ecor52I at the 5 ' end and Pmel at the 3' End amplified by PCR (expand high fidelity PCR kit, Röche).
- Both fragments were then co-amplified with outer primers by annealing in the overlap region and cloned into the EcoRV site of the vector pAC.
- a polylinker with the sequence GGCCGGATATCGATATCTTCGAACGGTTAATTA was subsequently inserted between Hind III and Eco 521.
- the resulting vector was linearized with Notl and used for homologous recombination with the PCR fragment lin Ecoli RecA + RecBC-sbcBC-. Subsequently, fragment 2 was inserted in the same way after linearization with Sall.
- the resulting vector has unique BstBI and Clal locations at the transitions from fragments 1 and 2, and 2 and 3, respectively.
- Shuttle vectors were created for both locations, which contain 300 bp on both sides of the unique locations.
- a ⁇ -galactosidase gene controlled by the RSV promoter and DsRedl (red fluorescent protein, ClonTech) under the control of the CMV promoter were cloned into the shuttle vector for BstBI and inserted into pHCA by means of recombination.
- PHCAredfp and a control vector A which carries the gfp gene under the control of the CMV promoter, were separated from the plasmid backbone by restriction cleavage and in each case 2 ⁇ g together with 2 ⁇ g of helper virus genome released from the plasmid in 293 cells Calcium phosphate precipitation co-transfected. After the culture was completely infected, the lysate was harvested and again 293 cells were infected. The proportion of cells with green and red fluorescence was evaluated in the following passages. It was found that in passage 3 about 15% of the cells fluoresce red, but only 7% green. The vector pHCA is thus superior to the control vector in the amplification rate as a result of replication and packaging.
- the vector system based on the human adenovirus type 1 is also explained: It is known that the proteins of the EIA region cause a change in the cell cycle combined with a stimulation of virus replication. They also have a transcription-activating effect, but do not bind to DNA on their own, but interact with cellular factors. The skilled worker is therefore furthermore aware that EIA of one serotype can complement the vector of another serotype. As is known, this does not apply to EIB because it interacts with other viral proteins, in particular with E4orf6. It is therefore not possible to amplify the vector according to the invention in cell line 293, which expresses the E1 region of Ad5.
- the EIB region codes for 2 independent proteins of 19k and 55k, which are encoded by a common mRNA.
- the EIB promoter is located in the EIA region. Since 19k of Ad5 is present and formed in 293 cells and no serotype specificity is expected due to the apoptosis-inhibiting function, EIB 55k is expressed as a single gene, controlled by heterologous poly A and promoters in HEK293 cells.
- the cell line according to claim 9 is further characterized in that it expresses adenovirus 11 EIB 55k.
- Sequence no.1 sequence between Adl 1 ElB55k stop codon and pIX start codon
- Ad2 Ad2, 5, or 1 1 adenovirus serotype bp base pairs cDNA to mRNA complementary DNA
- a new adenoviral vector Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.
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Application Number | Priority Date | Filing Date | Title |
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EP02779157A EP1434868A2 (de) | 2001-10-04 | 2002-10-04 | Adenovirales vektorsystem |
DE10294594T DE10294594D2 (de) | 2001-10-04 | 2002-10-04 | Adenovirales Vektorsystem |
AU2002342536A AU2002342536A1 (en) | 2001-10-04 | 2002-10-04 | Adenoviral vector system |
US10/491,936 US20050054105A1 (en) | 2001-10-04 | 2002-10-04 | Adenoviral vector system |
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DE10150940.5 | 2001-10-04 |
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WO2003031633A2 true WO2003031633A2 (de) | 2003-04-17 |
WO2003031633A3 WO2003031633A3 (de) | 2003-10-30 |
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PCT/DE2002/003846 WO2003031633A2 (de) | 2001-10-04 | 2002-10-04 | Adenovirales vektorsystem |
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US (1) | US20050054105A1 (de) |
EP (1) | EP1434868A2 (de) |
AU (1) | AU2002342536A1 (de) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005033320A1 (en) | 2003-10-02 | 2005-04-14 | Crucell Holland B.V. | Packaging cells for recombinant adenovirus |
WO2005071084A1 (fr) * | 2003-12-24 | 2005-08-04 | Qijun Qian | Construction d'un adenovirus recombinant vide exprimant efficacement des anticorps comprenant la region constante humaine et utilisations correspondantes |
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US5643567A (en) * | 1990-12-04 | 1997-07-01 | Board Of Regents, The University Of Texas System | Methods for the suppression of neu mediated tumors by adenoviral E1A and SV40 large T antigen |
US6410013B1 (en) * | 1999-01-25 | 2002-06-25 | Musc Foundation For Research Development | Viral vectors for use in monitoring HIV drug resistance |
US20030215423A1 (en) * | 1999-04-01 | 2003-11-20 | Merck & Co., Inc. | Gene therapy for obesity |
US6913922B1 (en) * | 1999-05-18 | 2005-07-05 | Crucell Holland B.V. | Serotype of adenovirus and uses thereof |
US6541245B1 (en) * | 1999-09-23 | 2003-04-01 | Genzyme Corporation | Adenoviral helper vectors |
-
2002
- 2002-10-04 DE DE10247403A patent/DE10247403A1/de not_active Ceased
- 2002-10-04 US US10/491,936 patent/US20050054105A1/en not_active Abandoned
- 2002-10-04 AU AU2002342536A patent/AU2002342536A1/en not_active Abandoned
- 2002-10-04 DE DE10294594T patent/DE10294594D2/de not_active Expired - Fee Related
- 2002-10-04 EP EP02779157A patent/EP1434868A2/de not_active Withdrawn
- 2002-10-04 WO PCT/DE2002/003846 patent/WO2003031633A2/de not_active Application Discontinuation
Non-Patent Citations (6)
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SKOG JOHAN ET AL: "Human adenovirus serotypes 4p and 11p are efficiently expressed in cell lines of neural tumour origin." JOURNAL OF GENERAL VIROLOGY, Bd. 83, Nr. 6, Juni 2002 (2002-06), Seiten 1299-1309, XP002249516 June, 2002 ISSN: 0022-1317 * |
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STONE DANIEL ET AL: "The complete nucleotide sequence, genome organization, and origin of human adenovirus type 11." VIROLOGY, Bd. 309, Nr. 1, 25. April 2003 (2003-04-25), Seiten 152-165, XP002249518 ISSN: 0042-6822 -& DATABASE GENBANK [Online] retrieved from NCBI Database accession no. AY163756 XP002249519 * |
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005033320A1 (en) | 2003-10-02 | 2005-04-14 | Crucell Holland B.V. | Packaging cells for recombinant adenovirus |
JP2007507216A (ja) * | 2003-10-02 | 2007-03-29 | クルセル ホランド ベー ヴェー | 組換えアデノウイルス用パッケージング細胞 |
EA010924B1 (ru) * | 2003-10-02 | 2008-12-30 | Круселл Холланд Б.В. | Способ получения партий рекомбинантного аденовируса и используемые для этого пакующие клетки |
AU2004278517B2 (en) * | 2003-10-02 | 2010-04-01 | Crucell Holland B.V. | Packaging cells for recombinant adenovirus |
US7816104B2 (en) | 2003-10-02 | 2010-10-19 | Crucell Holland B.V. | Packaging cells for recombinant adenovirus |
CN1863918B (zh) * | 2003-10-02 | 2011-03-30 | 克鲁塞尔荷兰公司 | 用于重组腺病毒的包装细胞 |
JP4749334B2 (ja) * | 2003-10-02 | 2011-08-17 | クルセル ホランド ベー ヴェー | 組換えアデノウイルス用パッケージング細胞 |
CN102174536A (zh) * | 2003-10-02 | 2011-09-07 | 克鲁塞尔荷兰公司 | 用于重组腺病毒的包装细胞 |
US8114637B2 (en) | 2003-10-02 | 2012-02-14 | Crucell Holland B.V. | Packaging cells for recombinant adenovirus |
KR101203817B1 (ko) * | 2003-10-02 | 2012-11-23 | 크루셀 홀란드 비.브이. | 재조합 아데노바이러스를 위한 포장 세포 |
CN102174536B (zh) * | 2003-10-02 | 2013-02-13 | 克鲁塞尔荷兰公司 | 用于重组腺病毒的包装细胞 |
WO2005071084A1 (fr) * | 2003-12-24 | 2005-08-04 | Qijun Qian | Construction d'un adenovirus recombinant vide exprimant efficacement des anticorps comprenant la region constante humaine et utilisations correspondantes |
Also Published As
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DE10294594D2 (de) | 2004-08-19 |
DE10247403A1 (de) | 2003-09-25 |
WO2003031633A3 (de) | 2003-10-30 |
EP1434868A2 (de) | 2004-07-07 |
US20050054105A1 (en) | 2005-03-10 |
AU2002342536A1 (en) | 2003-04-22 |
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