Nucleic acid construct and use of the same
09534233 ยท 2017-01-03
Assignee
Inventors
Cpc classification
C12N2840/44
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention is related to a nucleic acid construct comprising an expression unit for the expression of E1B, wherein the expression unit comprises a promoter, a nucleotide sequence coding for E1B, and a 3UTR, wherein the promoter is operatively linked to the nucleotide sequence coding for E1B, wherein the 3UTR comprises 30 or less than 30 Exonic Enhancer Elements (ESEs), preferably 20 or less than 20 Exonic Enhancer Elements (ESEs), and wherein the 3 UTR is a non-viral 3 UTR.
Claims
1. A nucleic acid construct comprising (i) an expression unit for the expression of E1 B, wherein the expression unit comprises a promoter, a nucleotide sequence coding for E1 B, and a 3 UTR, wherein the promoter is operatively linked to the nucleotide sequence coding for E1 B, wherein the 3 UTR comprises 30 or fewer than 30 Exonic Splicing Enhancer elements (ESEs), and wherein the 3 UTR is a non-viral 3 UTR, and (ii) an expression unit for the expression of E1A, wherein the expression unit comprises a promoter, a nucleotide sequence coding for E1A, and a 3 UTR, wherein the promoter is operatively linked to the nucleotide sequence coding for E1A, wherein the expression unit for the expression of E1A and the expression unit for the expression of EIB form a combined expression unit, wherein the combined expression unit comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 15.
2. The nucleic acid construct of claim 1, wherein the 3 UTR comprises 20 or fewer than 20 Exonic Splicing Enhancer elements (ESEs).
3. The nucleic acid construct of claim 1, wherein the nucleic acid construct is a single nucleic acid molecule comprising both the expression unit for the expression of E1A and the expression unit for the expression of E1 B.
4. The nucleic acid construct according to claim 1, wherein the Exonic Splicing Enhancer elements (ESEs) are contained within a stretch of nucleotides of the 3 UTR of the expression unit for the expression of E1 B, and wherein said stretch of nucleotides comprises the 200 nucleotides of the 5 end of the 3 UTR of the expression unit for the expression of E1 B.
5. The nucleic acid construct according to claim 1, wherein the 3 UTR of the expression unit for the expression of E1 B is a mammalian 3 UTR.
6. The nucleic acid construct according to claim 1, wherein the expression unit for the expression of E1A and the expression unit for the expression of E1 B are arranged in a 5->3 direction in the nucleic acid construct as follows: the promoter of the expression unit for the expression of E1A, the nucleotide sequence coding for E1A and the 3 UTR, the promoter of the expression unit for the expression of E1 B, the nucleotide sequence coding for E1 B, a splice donor site, an intron, a splice acceptor site and the 3 UTR.
7. The nucleic acid construct according to claim 1, wherein the nucleic acid construct is coding for and capable of expressing E1A, E1 B 55K, E1 B 19K and/or E1 B 84R.
8. The nucleic acid construct according to claim 7, wherein the nucleic acid construct is capable of expressing E1A, E1 B 55K, and E1 B 19K.
9. A vector comprising the nucleic acid construct according to claim 1.
10. A cell comprising a the vector according to claim 9.
11. The cell according to claim 10, wherein the cell is an amniocytic cell line.
12. The nucleic acid construct according to claim 7, wherein the nucleic acid construct is capable of expressing E1A, E1 B 55 K, E1 B 19 K and E1 B 84R.
Description
(1) The present invention is further illustrated by the figures, examples and the sequence listing from which further features, embodiments and advantages may be taken, wherein
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C. EXAMPLES
Example 1
Cloning of the Ad5 E1 Expressing Construct pSTK146UBE2I
(8) Cloning Strategy
(9) According to previous results human primary amniocytes can be transformed by E1 proteins of hAd5 (Schiedner et al., 2000). The E1 expressing construct pSTK146 of the prior art used in these experiments contains non-coding SV40 sequence elements including intron and 3 UTR that are often found in many transcription cassettes enhancing gene expression. An intron at the 3 end of the E1B 55K coding sequence including a splice acceptor is necessary for splicing of the MB mRNAs and efficient expression of E1B 55K protein. An embodiment of the nucleic acid construct of the present invention which his referred to herein as pSTK146 UBE2I, was generated, replacing the SV40 intron and 3 UTR by a short intron, including splice donor and splice acceptor and a 3 UTR of the human gene UBE2I (NCBI Reference Sequence: NT_010393.16, SEQ IC NO: 25. Additionally, a short sequence of the pIX gene was inserted which allows for expression of the minor E1B 84R protein. For an enhanced expression of E1B 84R and a reduced homology to corresponding Ad5 sequences, the E1B 84R-encoding sequence was codon-optimized. The latter measure is particularly useful in case the cell line containing the nucleic acid construct of the present invention is used for production of E1Ad vectors. To further reduce sequence overlaps between transgene expression cassettes of the E1 Ad vectors and the Ad5 sequences of E1-transformed cell lines the 3 UTR of SV40 including the polyadenylation site was replaced by a human 3 UTR of the UBE2I gene.
(10) Actual Cloning
(11) Starting from plasmid pBKSII E1B containing the E1B promoter, E1B coding sequence and SV40 sequences (intron and 3 UTR) a site-directed mutagenesis was performed to remove the splice donor site at nt. 3510 of Ad5 and to introduce a NdeI restriction site within the plasmid. The resulting plasmid pBKSII E1B QC NdeI was digested with BamHI and NdeI to release a 1 kb fragment thereby removing all SV40 sequences. The human UBE2I intron was obtained by polymerase chain reaction (PCR) using genomic DNA isolated from low passage human N52.E6 cells (Schiedner et al., 2000) and oligonucleotides #73 (5-gttcagCATATGcaggtacggggcctccgcctctg-3 (SEQ ID NO: 26) and #74 (5-TCAAGGTGGGGGAGGGTtctgtgccagagacaaaaacacaagac-3(SEQ ID NO: 27). The PCR product called PCR intron is flanked by a NdeI site (underlined) and codon-optimized Ad5 sequences (nt. 3595 to nt. 3612) encoding for the C-terminal part of E1B 84R at the 5 or 3 end, respectively. The 3 UTR of UBE2I was isolated using oligonucleotides #75 (5-gaACCCTCCTCCACCTTGAATTGCCCGTTTCCATACAGGGTC-3 (SEQ ID NO: 28) and #76 (5-ctggatccGCGGTGGGGCTGCAGGTG-3(SEQ ID NO: 29)) resulting in the PCR product PCR 3 UTR which is flanked by the same Ad5 sequences (nt. 3595 to nt. 3612) as mentioned above and a BamHI restriction site (underlined) at the 5 or 3 end, respectively. The overlapping Ad5 sequences at the 3 end of PCR intron and at the 5 end PCR 3 UTR allowed to fuse these two PCR products thereby using oligonucleotides #73 and #76. The resulting fusion PCR fragment flanked by NdeI and BamHI was then inserted between the NdeI and BamHI sites of pBSK E1B QC NdeI resulting in pBSK E1B UBE2I. To generate pSTK146 UBE2I the BglII/BamHI fragment from pBSK E1B UBE2I containing the UBE2I intron, codon-optimized C-terminal part of E1B 84R and UBE2I 3 UTR was subcloned between the BglII and BamHI sites of pSTK146. The resulting plasmid was named pSTK146UBE2I (Sequence ID NO 9) and is also depicted in
Example 2
Western Blot Analysis to Detect Steady-State Levels of E1 Proteins
(12) To determine expression levels of the E1B 55K protein after transient transfection using various E1B 55K expressing nucleic acid constructs 110.sup.6 Hela cells were seeded in 6 cm dishes. The next day the cells were washed with phosphate-buffered saline (PBS) and fresh medium was added. The cells were transfected with 3 g of plasmid pSTK146 (expressing both E1A and E1B) and plasmid pBSKII E1B (expressing only E1B from the natural E1B promoter), pBSKII E1B UBE2I (expressing E1B from the natural E1B promoter and containing the UBE2I elements) and plasmid pSTK146 UBE2I using polyethylenimine (PEI) as transfection reagent. After 48 hours the cells were washed with PBS, detached with 50 mM EDTA in PBS and pelleted by centrifugation. Cell pellets were lysed with 200 l RIPA lysis buffer (40 mM Tris/HCL, pH 8, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, 0.5% (w/v) sodium desoxycholate) for 30 minutes on ice. After repeated freezing and thawing the cell debris was removed by centrifugation, and the protein concentration was determined (Bio-Rad protein assay). Fifty g of whole cell extract was analysed by 10% SDS-PAGE and immunoblotting. The E1B 55K protein was detected using the E1B 55K-specific 2A6 antibody.
(13) Expression of the E1B 55K protein after transfection of pBSKII E1B UBE2I and pSTK146 UBE2I was approximately 10-fold higher than after transfection of pBSKII E1B and of pSTK146 as shown in
Example 3
Determination of Aberrant Splicing of E1B mRNA Transcripts in pSTK146-Transfected Cells
(14) Western blot analysis of pSTK146-transfected cells by the N-terminal binding E1B-specific antibody 2A6 showedin addition to E1B 55Ka faster migrating E1B protein named E1B 37K. To test at the mRNA level, if the E1B 37K protein resulted from aberrant splicing events, total RNA from pSTK146-transfected Hela cells was extracted with Trizol Reagent and further purified using Phase Lock Gel tubes (PLG, Eppendorf) and RNeasy Mini Kit (Qiagen) including DNAse treatment according to the manufacturers' instructions. Complementary DNA (cDNA) was synthesised with the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, USA) as described by manufacturer's protocol and RNA was reverse transcribed using an oligo-dT primer. PCR amplification of cDNA was performed with Taq Polymerase (NEB) using the forward oligonudeoxycleotide #59 (5-CTGAACTGTATCCAGAACTGAG-3(SEQ ID NO: 30)), which binds 3 to the splice donor (SD)1 that is normally used (located at nucleotide (nt.) sequence 2,255 of Ad5 or nt. 4,503 of pSTK146) and the SV40-specific, reverse oligonucleotide #60 (5-ACTGCTCCCATTCATCAGTTC-3(SEQ ID NO: 31)), which binds 3 to SV40 splice acceptor (SA). The amplified cDNAs were gel-purified and their 3 overhangs were removed by T4-DNA Polymerase (NEB). The cDNAs were then inserted into the EcoRV site of the cloning vector pBluescript II SK, and sequenced (Entelechon GmbH, Regensburg, Germany).
(15) Sequence analysis of E1B mRNA transcripts revealed aberrant splicing using an SD (nt. 2,324 of Ad5 or nt. 4,572 of pSTK146) 69 nt. downstream of SD1 and the SA of the SV40 3 UTR (nt. 5,832). The usage of the splice donor SD2, which is usually used for splicing of E1B 55K encoding mRNA transcripts, could not be detected. The resulting E1B 37K protein only shares the first 102 amino acids with E1B 55K. For efficient transformation, however, various motifs in the central part and C-terminus of E1B 55K are required. By introducing an intron and an 3UTR region from UBE2I gene, cryptic splicing may be inhibited leading to the expression of a full length E1B 55K protein harbouring all sequence motifs contributing to transformation (Blackford et al., 2009, Endter et al., 2001, Schreiner et al., 2011). Taken together, following cell transfection with plasmid pSTK146, only a very small amount of the E1B 55K protein was detected, rather an abberrant E1B 37 K protein was found that results from aberrant splicing as shown by sequence analysis following RT-PCR.
Example 4
Transfection of Human Amniocytes with Plasmids pSTK146 and pSTK146 UBE2I
(16) Transfection of human amniocytes essentially followed the procedure as described in Schiedner et al., 2000, and in EP00979539 with some modifications as detailed below.
(17) Culture of Amniocytes
(18) Samples of amniotic fluid containing primary cells obtained by diagnostic amniocenteses, were added to cell culture medium in plastic culture dishes Amniotic fluid cells generally began to attach and proliferate within 2-4 days after seeding. Primary cell populations were cultured in adherent culture in plastic cell culture dishes initially in Ham's F10 medium supplemented with 10% fetal bovine serum, 4 mM glutamine and 2% Ultroser. Later, when the cells had been expanded to two 15 cm cell culture dishes, they were adapted during two passaging steps to OptiPro medium (Gibco) supplemented with 2% Ultroser (Cytogen) and 2% Glutamax (Gibco), in a first step to 50% OptiPro medium and in a second step to 100% OptiPro medium. Culture medium was changed every 3-4 days. At a visual confluency of 70-90%, primary amniocytes were detached with TrypLE Select (Gibco) and expanded to larger vessels or split by a factor of four. Starting in the fourth passage after seeding, several vials of cells were frozen in every passage (freezing procedure described below). The culture was maintained until over 50% of cells had acquired the senecent phenotype, characterized by enlargement and flattening of cells as well as arrested cell division. This change was generally observed between passage 7 and 11, corresponding to 30-38 population doublings.
(19) Freezing and Storage of Cell Stocks
(20) For long-term storage of primary cells, cells were detached with TrypLE Select, collected and separated from culture medium by centrifugation. They were resuspended in fresh culture medium containing 5% cell culture grade dimethylsulfoxid (DMSO, Sigma) at a cell density of 110.sup.6 to 110.sup.7 cells per milliliter. The suspension was filled in vials for storage in liquid nitrogen (Nalgene). The tubes were placed in a Nalgene freezing device containing isopropanol as a cooling agent; the device was stored overnight at 80 C., resulting in a cooling rate of about 1K per minute. The frozen vials were then placed in the gaseous phase of a liquid nitrogen container for long-term storage.
(21) The same freezing procedure was used also for cell clones and cell lines derived from primary amniocytes.
(22) Preparation of Transfection Complexes, Transfection of Amniocytes
(23) Transfection was performed on primary amniocytes between passage 7 and 9, corresponding to PD 30 to 35, shortly before the onset of senescence.
(24) Materials used: Plasmid pSTK146 UBE2I DNA in Tris-EDTA buffer, pH 7.5, following linearization with restriction enzyme BspHI according to standard procedures. BspHI cleaves in the plasmid backbone, not within the E1A/E1B expression cassettes. Solution of linear polyethyleneimine (PEI), 7.5 mM (0.32 g/l; PEI nitrogen molarity: 43 g/mol), pH 7.0, sterile filtered (0.2 m) Sodium chloride (NaCl) solution, 150 mM, sterile filtered (0.2 m)
(25) For each culture dish to be transfected, 2 g of linearized plasmid DNA and 36 l of PEI solution were separately diluted ad 250 l with NaCl solution. Each PEI dilution was added to one DNA dilution, resulting in an N/P (nitrogen/phosphorous) ratio of 45. Preparations were mixed and incubated at room temperature for 15 to 20 minutes to allow for the formation of PEI-DNA complexes. Primary amniocytes, seeded in 6 cm culture dishes on the previous day at a visual density of 50-70%, were washed with PBS and supplied with fresh culture medium. Each transfection complex was added to one prepared dish.
(26) Culture after Transfection
(27) Twentyfour hours after transfection, cells were detached from culture dishes with TrypLE Select (Gibco) and transferred to 14 cm dishes. Over a period of 3 to 6 weeks, medium was changed every 3 to 4 days, or cells on one culture dish were passaged to two dishes, if cells reached a visual confluency over 90%. During this period, dishes were observed daily under 2.5-fold magnification to screen for emerging foci of transformed cells.
(28) Harvesting and Expansion of Transformed Cell Clones
(29) Three to six weeks after transfection, foci of transformed cells became visible among the primary amniocytes. Transformants were recognized by their distinctive morphology, small cell size and rapid growth among very large and non-dividing senescent primary cells. The round foci were removed mechanically from the culture surface by scraping and aspiration with a sterile pipette tip and seeded in a culture well. Each harvested clone was expanded to larger culture vessels for three passages before a first cell stock was frozen.
(30) For clarity, the term clone or cell clone and its plural forms are preferably used herein to describe proliferating cells derived from isolated single cell foci that are generated after transfection with the E1-expressing plasmids. These single foci, as described above are removed from the cell culture dish by aspiration and seeded in individual cell culture dishes. At this stage they are assumed to be polyclonal, since multiple clones are derived from the same cell culture dish and it cannot be excluded that a clone consists of cells derived from more than one integration and immortalization event. The term cell line and its plural form are preferably used herein to describe immortalized and permanently proliferating cells obtained following single cell cloning so that they can be considered monoclonal.
(31) Use of Different Ad5 E1 Expressing Plasmids pSTK146 and pSTK146UBE2I for Transfection and Generation of Immortalized Cell Lines
(32) The described transfection procedure was performed using two different E1-expressing constructs: pSTK146 and the pSTK146 UBE2I. Both transfections resulted in successful generation of foci consisting of small and rapidly proliferating cells. However, significant differences were observed in the long-term stability of clones in culture.
(33) During the passages following isolation, a portion of clones underwent crisis characterized by morphological changes including strong increase in size and flattening, slow cell division and ultimately cessation of growth, and in part signs of cell death. The clones transformed with pSTK146 were much more susceptible to these changes: At least 60% of each clone batch (78% over all experiments) ceased to grow during the early phase of culture (polyclonal passage 1 to 4, PD<55). Subsequently, only seven of 14 clones selected for good growth and adenovirus vector productivity kept proliferating beyond polyclonal passage 10 (corresponding to a total of approximately 65 PDs after seeding of the primary cells), and none survived beyond polyclonal passage 13 (75 PDs).
(34) Clones transformed with the pSTK146 UBE2I construct, however, survived the early passages at a much higher rate (average loss 29% up to passage 4), and only few clones entered crisis at a later point. Eight clones selected for high productivity were kept in culture up to polyclonal passage 23 or higher (100 PDs) and were considered for the generation of monoclonal cell lines.
(35) Further details of the transformation experiments are summarized in the following Tables 1 and 2.
(36) TABLE-US-00001 TABLE 1 Results following transfection of primary amniocytes with pSTK146 until polyclonal passage 4 Passage no. of proliferating % of isolated cell no. Culture vessel cell clones clones 1 24-well dish 473 2 6-well dish 226 48 3 9.2 cm dish 156 33 4 cryovial (frozen stock) 103 22
(37) TABLE-US-00002 TABLE 2 Results following transfection of primary amniocytes with pSTK146 UBE2I until polyclonal passage 4 Passage no. of proliferating % of isolated cell no. Culture vessel cell clones clones 1 24-well dish 221 2 6-well dish 185 84 3 9.2 cm dish 164 74 4 cryovial (frozen stock) 157 71
(38) Comparable results were obtained in amniocytes from two different amniocenteses.
Example 5
Single Cell Cloning of Amniocyte Cell Lines after Transformation with pSTK146 UBE2I
(39) Due to the mechanical method of isolation, described above, and the fact that multiple clones are isolated from each transfected cell culture dish, an isolated cell clone cannot be considered to be monoclonal, i.e. derived from a single cell. They are rather considered polyclonal at this stage. The following procedure was performed to obtain monoclonal cell lines from well-growing transformants.
(40) Transformed cells in stable growing culture (polyclonal passage P 20, approx. 90 PDs) were detached, resuspended in culture medium and counted in a haemocytometer. Three dilutions were prepared of each cell suspension, containing 10, 20 or 50 viable cells/ml. Each of these dilutions was used to seed one flat-bottom 96-well dish with 100 l of this cell suspension per well, resulting in a seeding density of 1, 2 or 5 viable cells per well.
(41) For one week after seeding, each well was observed closely under 10-fold magnification to screen for attaching cells. Only wells with a single colony of cells, growing from a single attached cell, were chosen for further culture. From the point of seeding, each well was treated as a separate cell line, taking precautions against cross-contamination. The cell lines were expanded to larger culture vessels up to 9.2 cm dishes, at which point a first cell stock was frozen as described above. Cultures based on this stock were tested for the ability to produce E1 Ad vectors, and highly productive cell lines were expanded for further cell banking. Two monoclonal cell lines with both good growth properties and good Ad vector production capability were named SGT11 1T3.1D9 and SGT11 1T3.1G3. These cell lines were deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstrasse 7B, 38124 Braunschweig and received the number DSMZ ACC3134 (cell line SGT11 1T3.1D9) and DSM ACC3135 (cell line SGT11 1T3.1G3).
Example 6
Investigation of Senescence-Associated Beta-Galactosidase Expression
(42) The changes in cellular morphology and the stop in proliferation of primary human amniocytes approaching passage 10 suggested that cells entered senescence at this point. Senescent cells express senescence-associated (SA) -galactosidase at a much higher level than actively dividing immortal or tumor cells.
(43) SA beta-galactosidase expression was evaluated in primary amniocytes, amniocytes at the polyclonal stage (clones) following transfection with plasmid pSTK146 UBE2I, immortalized monoclonal amniocyte cell lines and in 293 cells. The following samples were investigated:
(44) a) primary human amniocytes of donor number 11 at passage 9 (SGT11 P9); at this stage the size of cells had already increased and the growth rate was reduced, i.e. the cells started to show a senescent phenotype;
(45) b) polyclonal amniocyte cell clone of the same donor obtained from one focus after transfection with pSTK146 UBE2I at passages 8 and 24 (SGT11 1T3 P8 and SGT11 1T3 P24); and
(46) c) immortalized amniocyte cell line established from the above polyclonal cell clone after single cell cloning at passage 12 (1T3.1D9 P12).
(47) The staining procedure was performed using the Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, Saint Louis, Mo., USA) following standard procedures and the manufacturer's recommendations. Following staining, evaluation was performed using phase contrast with an inverted microscope. 10 images were randomly taken from each sample and SA -galactosidase-positive cells were counted.
(48) As the result 98.5% of SGT11 P9 were positive for SA -galactosidase expression, 40.41% of SGT11 1T3 P8, 18.73% of SGT11 1T3 P24 and 13.29% of 1T3.1D9 P12. Results of these experiments are shown in
(49) This data indicated, that the change in morphology and growth arrest of primary amniocytes at around passage 10 was, indeed, senescence-associated and that this state was overcome by transfection of the primary cells with plasmid pSTK146 UBE2I.
Example 7
Determination of Telomere Length in Primary Amniocytes and Cell Lines
(50) Human telomeric DNA is usually about 10 kb in length on average in primary cells. To investigate whether primary human amniocytes enter replicative senescence due to a telomere erosion-mediated DNA damage response, the length of the telomeric DNA of primary amniocytes and of established cell lines after transfection with pSTK146 UBE2I was determined. The N52.E6 cell lines that had been established by plasmid pST146 (Schiedner et al., 2000) served as control.
(51) The length of telomeres is conveniently measured by a standard method, determining the telomere restriction fraction (TRF) (Harley et al., 1990). Genomic DNA is treated with restriction enzymes, which do not cleave within the repeating hexanucleotide 5-TTAGGG-3 sequence constituting telomeric DNA. The cleaved DNA is separated by gel electrophoresis and the length of the TRFs is determined by hybridization with a probe that recognizes this hexanucleotide. As telomeres within one cell differ from chromosome to chromosome, and shortening may differ from cell to cell, the distribution of telomeric DNA in each sample is quite heterogeneous. The TRFs of the DNA samples from the following primary cells and cell lines in different passages were analysed:
(52) a) primary human amniocytes of donor SGT11 at passages 6 and 9: SGT11 P6 and SGT11 P9;
(53) b) polyclonal cell clones of the same donor after transfection with plasmid pSTK146 UBE2I at polyclonal passage 5 and 21: SGT11 1T3 P5 and SGT11 1T3 P21;
(54) c) monoclonal cell lines of the same donor at monoclonal passages 9 and 23: 1T3.D9 P9 and 1T3.1D9P23; and d) as control, N52.E6 cells (transfected with pSKT146) were used.
(55) The mean TRF of SGT11P6 and SGT11P9 was determined to be 10.3 and 9.8, respectively. The mean TRF of SGT11 1T3 P5 and SGT11 1T3 P21 was determined to be 7.8 and 8.4 kb, respectively. The mean TRF of SGT11 1T3.D9 P9 and SGT11 1T3.1D9 P23 was determined to be 7.9 and 7.9 kb, respectively. The mean TRF of N52.E6 cells was determined to be 4.6 kb. The TRFs from N52.E6 cells was determined from cells in a cell passage that corresponded to SGT11 1T3.1D9P23 cells. Results of these experiments are shown in
(56) From this experiment, two conclusions can be drawn: first, the senescence phenotype of primary human amniocytes at late passage (P11) is not caused by obvious telomere erosion. Rather it is likely due to so-called stress-induced premature senescence (SIPS), which is thought to be caused by accumulation of stresses in culture cells (Toussaint et al., 2000; Weinberg, R. A., 2007 supra). Second, the mean TRFs of N52.E6 cells, generated by transformation with pSTK146, are very short compared to cells that have been immortalized with pSTK146 UBE2I. This data, together with the observation that primary amniocytes do not enter crisis when immortalized with pSTK146 UBE2I, but do undergo crisis when transfected with pSTK146, indicates that replicative senescence is prevented by transformation of primary amniocytes with plasmid pSTK146 UBE2I.
Example 8
Production of E1 Adenovirus Vectors in Aminocyte-Derived Clones and Cell Lines
(57) The following screening protocol was used to compare and quantify the vector production capability of isolated and expanded clones. The results of these screenings, in addition to the stability of cell growth, were the basis for selection of clones for further culture and single-cell cloning.
(58) Following standard procedures, one day after seeding at a defined density, cells were infected with a E1 Ad vector carrying a GFP expression cassette (Ad1stGFP) at an infectious multiplicity of infection (MOI) of 5 to 20. Cells were harvested mechanically using a cell scraper 48 hours after infection, separated from culture medium by centrifugation, resuspended in buffer and lysed by three rounds of freezing in liquid nitrogen and thawing in a 37 C. water bath. The resulting lysate, containing the produced vector particles, was cleared of cell debris by centrifugation.
(59) Dilutions of the clarified lysates were used to infect A549 cells (in which E1 Ad vectors cannot replicate), also seeded at a defined density. Further 48 hours after infection, A549 were harvested and analyzed by flow cytometry, using the mean fluorescence intensity (corresponding to the level of intracellular GFP expression) as a measure for the number of infectious vector particles received per A549 cell. For a certain range of infectious MOI, the correlation between infectious dose and mean fluorescence intensity in A549 is linear. Therefore, by infection of A549 with defined infectious MOI to establish a standard curve, the average number of infectious particles produced per cell can be calculated.
(60) It was found that the monoclonal cell lines SGT11 1T3.1D9 and SGT11 1T3.1G3 allowed production of Ad1stGFP at high levels with production of more than 2500 infectious Ad1stGFP particles per cell in SGT11 1T3.1D9 cells and more than 1000 infectious Ad1stGFP particles per cells in SGT11 1T3.1G3 cells as may also be taken from
Example 9
Generation of RCA During Vector Production
(61) The possible risk of RCA generation during vector production in the new cell lines was assessed by serial passage of a E1Ad vector in two different permanent amniocyte cell lines SGT11 1T3.1D9 and SGT11 1T3.1G3. Since it is known that E1Ad vectors, when produced in HEK293 cells, frequently result in the generation of RCA HEK293 cells were used as a control. RCA is generated by DNA recombination due to sequence overlap between DNA of the E1Ad vector and the chromosomally integrated adenoviral DNA. The assay was performed in two different formats.
(62) In a first format, 10 wells of each cell line (1.510.sup.6 cells/well in 6-well cell culture dishes) were infected with an MOI of 10 infectious particles per cell of the E1 Ad vector Ad1stGFP and harvested after 48 h. Cells were lysed by three times freezing and thawing. 10% of each cell lysate (high infection format) was used to infect cells of the same cell line for another cycle of 48 h. This procedure was repeated for a total 15 passages.
(63) In a second format of this assay, cells were infected with 0.1% of cell lysate from the previous passage (low infection format) and harvested when a cytopathic effect (CPE) became visible after 5-8 days. This procedure was also repeated for 15 passages.
(64) RCA detection was performed essentially as described previously (Fallaux et al. Hum Gene Ther 1998, 9, 1909-17). The assay is performed on human cell lines (A549 cells and HeLa cells) that do not allow replication of a E1 Ad vector. Only in the case of RCA generation and the presence of RCA, a full infectious cycle can occur resulting in a classical CPE. Thus, the final lysate was incubated on HeLa cells for 4 days. Then, HeLa cells were lysed by freezing and thawing and the lysate was added to A549 cells for 10 days. A visible CPE on A549 indicated the presence of RCA, as a first-generation vector could not replicate in either HeLa or A549. To test the detection limit of this assay, control HeLa dishes were infected with lysates spiked with Ad5 wild-type particles at very low multiplicity of infection. The assay has been found sensitive enough to detect 6 RCA particles per infected HeLa dish.
(65) After 15 virus passages, no RCA was detected in any lysate of amniocyte-based cell lines (40 lysates tested), while three in 20 final lysates of HEK293 cells were found to contain RCA. Also, there was no evidence for the generation of HDEPs in SGT11 1T3.1D9 and in SGT11 1T3.1G3 cells. HDEPs would have become apparent as CPE, when A549 or HeLa cells were exposed to the cell lysates obtained from the serial passages.
Example 10
Karyotype Analyses
(66) Karyotype analyses were performed from metaphases following standard procedures as they are routinely used in cytogenetic laboratories. Metaphases were analysed using the METAFER 4 equipment of MetaSystems GmbH, Altlussheim, Germany. Images were edited with the IKAROS software in order to obtain the following karyogramms Primary amniocytes, polyclonal cell clones at 2 different passages (Passages 10 and 20) and monoclonal cell lines (passages 14 and 23) were analysed.
(67) The following results were obtained:
(68) a) Primary amniocytes from individual SGT11 in passage 8 (SGT11 P8), corresponding to an estimated total PD of 30.
(69) Karyotype: 46,XX (normal female karyotype)
(70) b) Polyclonal clone established from the same individual in polyclonal passage 10 (SGT11 1T3 P10), corresponding to an estimated total PD of 68 to 70.
(71) Karyotype: 71,XXX, +mar(del(8)t(X,8)(q;p)
(72) c) Polyclonal clone established from the same individual in polyclonal passage 20 (SGT11 1T3 P20), corresponding to an estimated total PD of 90.
(73) Karyotype: 75, XXX, +mar(del(8)t(X,8)(q;p)+elongation of 1q
(74) d) Monoclonal cell line established from the same individual in monoclonal passage 14 (SGT11 1T3.1D9 P14), corresponding to an estimated total PD of 140.
(75) Karyotype: 55, XX, +mar(del(8)t(X,8)(q;p)
(76) e) Monoclonal cell line established from the same individual in monoclonal passage 23 (SGT11 1T3.1D9 P23), corresponding to an estimated total PD of 160.
(77) Karyotype: 61,XX, +mar(del(8)t(X,8)(q;p)+homologous stained region (HSR) on 1p
(78) Starting from a normal female karyotype (46,XX) in primary amniocytes, a polyploid karyotype was observed with chromosome numbers between 75 and 55 and one consistent translocation (t(X;8(q;p) observed in the polyclonal cell clone in passage 10 (total PD of 68 to 70) and in passage 20 (total PD of 90), and in the monoclonal cell line in passage 14 (total PD of 140) and in passage 23 (total PD of 160). Only an elongation of 1q visible in passage 20 in the polyclonal status, and one HSR on 1p visible in passage 23 in the single cell cloned status of the cell line. No additional structural abnormalities were detected despite long-term cultivation, indicating a remarkable stability of the karyotype.
Example 11
Structural Characteristics of the E1 Region of Ad 5
(79) The E1 region of Ad5 like is characterized by a complex structure with overlapping reading frames encoding for several E1A and E1B proteins. Within the E1B sequence two SD and three SA site are present enabling alternative splicing of the E1B mRNA transcript. In addition to the consensus splice sites, the use of cryptic splice sites in this region may give rise to unwanted E1B protein products and/or may result in a lower expression of the major E1B protein E1B 55K. When analysing different genomic 3UTR sequences for the presence of ESEs using the 238 ESEs shown in Table 3 it was found that these sequences exhibited a decreased number of ESEs compared to the SV40 sequences present in pSTK146.
(80) TABLE-US-00003 TABLE3 Listof238candidateESEsaspredicted byFairbrotheretal.,2002 1 AAAACC 2 AAAAGA 3 AAAAGC 4 AAACAG 5 AAACCA 6 AAACCT 7 AAACGA 8 AAAGAA 9 AAAGAC 10 AAAGAG 11 AAAGAT 12 AAAGCA 13 AAAGCT 14 AAAGGA 15 AAATCC 16 AACAAC 17 AACAAG 18 AACAGA 19 AACCAA 20 AACGAA 21 AACTGG 22 AACTTC 23 AAGAAA 24 AAGAAC 25 AAGAAG 26 AAGAAT 27 AAGACA 28 AAGACT 29 AAGAGA 30 AAGAGG 31 AAGATC 32 AAGATG 33 AAGCAA 34 AAGCAG 35 AAGCCA 36 AAGCTA 37 AAGGAA 38 AAGGAC 39 AAGGAT 40 AATCAA 41 AATCCA 42 AATGAC 43 AATGGA 44 ACAAAG 45 ACAACG 46 ACAACT 47 ACAAGA 48 ACAGAA 49 ACCTGA 50 ACGAAA 51 ACGAAG 52 ACGACT 53 ACTGAA 54 ACTTCA 55 ACTTCG 56 AGAAAA 57 AGAAAC 58 AGAAAG 59 AGAACA 60 AGAACT 61 AGAAGA 62 AGAAGC 63 AGAAGG 64 AGAAGT 65 AGAATT 66 AGACAA 67 AGACAT 68 AGACGA 69 AGAGAA 70 AGAGAT 71 AGAGGA 72 AGATGA 73 AGATGC 74 AGATGT 75 AGCAAA 76 AGCAGA 77 AGGAAA 78 AGGAAC 79 AGGAAG 80 AGGACA 81 AGGAGA 82 AGTGAA 83 ATCAAA 84 ATCAAG 85 ATCAAT 86 ATCAGA 87 ATCCAA 88 ATGAAG 89 ATGAGA 90 ATGATG 91 ATGCAA 92 ATGGAA 93 ATGGCG 94 ATTCAG 95 ATTGGA 96 CAAAAC 97 CAAAAG 98 CAAAGA 99 CAACTT 100 CAAGAA 101 CAAGAT 102 CAAGTA 103 CAATCA 104 CAGAAA 105 CAGAAG 106 CAGAAT 107 CAGAGG 108 CAGGAA 109 CCTGAA 110 CGAAAA 111 CGAACA 112 CGAAGA 113 CGACGA 114 CGTATG 115 CTGAAA 116 CTGAAG 117 CTTCAG 118 GAAAAA 119 GAAAAC 120 GAAAAG 121 GAAACA 122 GAAACC 123 GAAACG 124 GAAACT 125 GAAAGA 126 GAAAGC 127 GAAATC 128 GAACAA 129 GAACAT 130 GAACTG 131 GAACTT 132 GAAGAA 133 GAAGAC 134 GAAGAG 135 GAAGAT 136 GAAGCA 137 GAAGGA 138 GAAGTA 139 GAAGTT 140 GAATCA 141 GACAAA 142 GACAAT 143 GACGAA 144 GACGAC 145 GAGAAA 146 GAGAAG 147 GAGAGA 148 GAGATG 149 GAGGAA 150 GAGGAG 151 GAGGAT 152 GATATC 153 GATATG 154 GATCAA 155 GATCAT 156 GATGAA 157 GATGAG 158 GATGAT 159 GATGCA 160 GATGGA 161 GATTCA 162 GCAAAA 163 GCAAGA 164 GCAGAA 165 GGAAAA 166 GGAAAC 167 GGAAGA 168 GGAGAA 169 GGAGGA 170 GGATCA 171 GTCAAG 172 GTGAAG 173 TACAAG 174 TACAGA 175 TATGGA 176 TCAAGA 177 TCAGAA 178 TCAGGA 179 TGAAAC 180 TGAAAG 181 TGAAGA 182 TGAAGC 183 TGAAGG 184 TGAAGT 185 TGAOAA 186 TGATGA 187 TGCAAC 188 TGGAAA 189 TGGAAG 190 TGGAAT 191 TGGATC 192 TTCAGA 193 TTCGAA 194 TTGAAG 195 TTGCGA 196 TTGGAA 197 TTGGAT 198 TTTGGA 199 AAAAAG 200 AAACTC 201 AACATG 202 AACCAG 203 AACTAC 204 AAGGAG 205 AATACG 206 AATCAG 207 AATGAA 208 ACATGA 209 ACGCAA 210 ACTACA 211 ACTGGA 212 AGTGAC 213 ATCTTC 214 ATGAAA 215 ATGGAT 216 ATGGTC 217 CAAACA 218 CAGATC 219 CATCAG 220 CGAATG 221 CGTCGC 222 CTACAT 223 CTCCAT 224 GAAAAT 225 GAACCA 226 GCGAAT 227 GGAGAT 228 GTCGAC 229 GTGTCG 230 GTTGGA 231 TATGAA 232 TCAACG 233 TCATCA 234 TCGTCG 235 TCTTCA 236 TGACTG 237 TGGAAC 238 TGTGGA
(81) A comparison of this analysis for pSTK146 and for pSTK146 UBE2I is shown in Table 4. In this table also the results of the ESE analysis for other suitable 3UTRs, derived from the ARF5, the DAXX, the HPRT and RING1 locus are shown.
(82) TABLE-US-00004 TABLE 4 ESEs present in pSTK146, in pSTK146BE2I and in additional sequences Number of ESEs in 200 nucleotide length of Origin of Intron Origin of 3 UTR DNA 3UTR SV40 poly late SV40 poly late pSTK146 40 gi|9628421|ncbi|NC_001669.1 gi|9628421|ncbi|NC_001669.1 UBE2I UBE2I pSTK146 1 gi|224589807: 1359180-1375390| gi|224589807: 1359180-1375390| UBE2I ncbi|NC_000016.9| ncbi|NC_000016.9| ARF5 ARF5 1 gi|224589819: 127228406-127231759| gi|224589819: 127228406-127231759| ncbi|NC_000007.13 ncbi|NC_000007.13 DAXX DAXX 24 gi|224589818: c33290793-33286335| gi|224589818: c33290793-33286335| ncbi|NC_000006.11 ncbi| NC_000006.11 HPRT HPRT 13 gi|224589822: 133594175-133634698| gi|224589822: 133594175-133634698| ncbi|NC_000023.10 ncbi|NC_000023.10 RING1 RING1 15 gi|224589818: 33176286-33180499| gi|224589818: 33176286-33180499| ncbi|NC_000006.11 ncbi|NC_000006.11
Example 12
Generation of Immortalized Amniocyte Cell Lines with a Nucleic Acid Construct where the E1A Genes are Under Regulatable Promoter Control
(83) In the nucleic acid construct of the present invention used in this example, the E1A cDNA of hAd5 was placed under the control of a Tetracycline (Tet)-inducible promoter.
(84) The Tet-On Advanced System (Clontech) was used to generate a nucleic acid construct, in which the E1A is tetracycline-inducible. This system is based on expression of the Tet-On Advanced transactivator, which is a fusion protein derived from a mutant version of the E. coli Tet repressor protein, rTetR, which is joined to three minimal transcription activation domains from the HSV VP16 protein. In the presence of doxycycline (Dox), Tet-On Advanced binds to the tetracycline response element (TREMod) in PTight, which is placed in front of a coding sequence of choice, resulting in activation of gene expression.
(85) A nucleic construct was generated containing both the transactivator and the transgene sequence on one nucleic acid molecule named plasmid pTL13 (
(86) a) the expression cassette coding for the Tet-On Advanced transactivator (rTET M2-VP16),
(87) b) the E1A cDNA under the control of the Tet-inducible promoter (PTight), and
(88) c) the E1B cDNA operatively linked to the natural E1B promoter and followed by the UBE2I intron, part of the pIX5UTR (to allow for expression of the E1B84R protein) and the UBE2I 3 UTR.
(89) For construction of pTL13, the plasmid pSTK146 UBE2I was digested with EcoRV and NotI to release a 3.7 kb fragment containing the Ad5 E1A coding sequence, the Ad5 E1B cDNA operatively linked to the natural E1B promoter and followed by the UBE2I intron, the C-terminus of E1B 84R and the UBE2I 3 UTR. This fragment was then cloned into the pTRE-Tight (Clontech) vector's multiple cloning site (Sural, NotI) located downstream of the Tet-inducible promoter Ptight to obtain a construct named pTL12. To generate pTL13, the 4.4. kb BamHI fragmentcontaining the transactivator sequenceobtained from the plasmid pTet-On-Advanced (Clontech) was then subcloned into the BamHI site of pTL12.
(90) For generation of immortalized amniocyte cell lines plasmid pTL13 is transfected into primary amniocytes as described above for the pSTK146 UBE2I plasmid, with doxycycline added to the cell culture medium at a concentration following the manufacturer's recommendation and in general being at a range between 0.01 and 2 g/ml.
Example 13
Immortalization of Primary Human Amniocytes by Transfection with Two Plasmids Expressing E1A and E1B Independently
(91) This example illustrates the nucleic acid construct of the present invention, whereby the nucleic acid construct is a two-piece nucleic acid construct.
(92) This two-step transfection procedure essentially follows the one-step transfection described above in example 4, with minor modifications. The two nucleic acid constructs, one expressing E1A, the second E1B, are transfected into primary human amniocytic cells, either at the same time (e.g. by mixing the two plasmid DNAs) or in two consecutive transfections. The latter procedure (i.e. two transfections) increases the chance of integration of the two nucleic acid constructs at different chromosomal sites, which may further reduce the risk of RCA generation if the resulting cell line is used for production of E1 Ad vectors. The amount of PEI as transfection reagent is lowered in the separate transfection steps to minimize its cytotoxic effects.
(93) Materials Used:
(94) Plasmid DNA in Tris-EDTA buffer, pH 7.5, linearized with restriction enzyme BspHI pBSK E1B UBE2I pmPGK E1A, expressing E1A from the murine PGK promoter Solution of linear PEI, 7.5 mM, pH 7.0, sterile filtered NaCl solution, 150 mM, sterile filtered
(95) The sequence of the functional sequence elements contained in plasmid pBSK E1B UBE2I is provided as SEQ ID NO 7: it contains the E1B promoter, the E1B 19K and 55K coding region, the UBE2I intron and comprising part of the 5-UTR of the pIX gene, and the UBE2I 3UTR. Thus, in this example plasmid pBSK E1B UBE2I expresses E1B under control of its natural promoter. The 3 UTR region is identical to that of the pSTK146 UBE2I plasmid described above. The second nucleic construct required for immortalization of primary amniocytes comprises an expression unit coding for the E1A functions. The sequence of an expression unit coding for E1A is provided in SEQ ID NO 8. It contains as a constitutive promoter the murine pgk promoter, the E1A coding region and the 3UTR from the UBE2I gene.
(96) If transfection of the two plasmids is performed at the same time, the two plasmid DNAs can be mixed and transfection is performed as described above for plasmids expressing both the E1A and E1B functions.
(97) If transfection of the two plasmids is performed consecutively, the procedure is performed as follows:
(98) For each culture dish to be transfected, 2 g of linearized pmPGK E1A expressing E1A and 18 l of PEI solution are separately diluted ad 250 l with NaCl solution. Each PEI dilution is added to one DNA dilution. Preparations are mixed and incubated at room temperature for 15 to 20 minutes. Primary human amniocytes, seeded in 6 cm culture dishes on the previous day, are washed with PBS and supplied with fresh culture medium. Each transfection complex is added to one prepared dish. Two days later, the same procedure is performed with the E1B expressing plasmid pBSK E1B UBE2I. The day after the second transfection, cells are passaged to 14 cm culture dishes, and subsequently treated according to the same protocols as the single-plasmid transfected cultures.
(99) The procedure can also be performed vice versa, i.e. transfecting first the E1B expressing plasmid, followed by the E1A expressing plasmid. The procedure can also be performed by using other transfection reagents then PEI or by using retroviral or lentiviral vectors for delivery of both nucleic acid constructs coding for E1A and E1B, respectively, into primary amniocytic cells.
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(101) The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.