Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons - PubMed (original) (raw)
Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons
M Sena-Esteves et al. J Virol. 1999 Dec.
Abstract
We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV-Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 10(6) and 10(7) transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.
Figures
FIG. 1
Amplicon constructs. (A) Schematic representation of the HSV-EBV-retrovirus hybrid amplicons which code for the MMLV gag-pol and env genes. These genes are under the control of the CMV promoter and are followed by the bovine growth hormone polyadenylation signal (BGHpA). The HERElacZ A1 and A7 amplicons code for the ecotropic env gene, while the HERAlacZ B3 and B7 amplicons code for the amphotropic env gene derived from the 4070A genome. (B) Schematic representation of the HERlacZ amplicons C1 and C5 which are missing the MMLV gag-pol-env genes. Amplicon clones A7, B3, and C5 bear the retrovirus lacZ cassette in opposite orientation to the GPE cassette, whereas clones A1, B7, and C1 have it in the same orientation. Abbreviations: S.D., splicing donor; S.A., splicing acceptor; ψ, retrovirus packaging signal; lacZ, E. coli β-galactosidase gene; SV40, simian virus 40 promoter; pac, HSV packaging signal; p15A, E. coli origin of plasmid replication; Ampr, ampicillin resistance gene; RSV, RSV promoter; EBNA-1 (del), EBNA-1 with most of the internal Gly-Ala repetitive sequence deleted (67); oriP, EBV latent origin of replication which contains two elements, the family of repeats (FR) and dyad symmetry (DS) element; oriS, HSV origin of DNA replication; IE4/5; HSV immediate/early 4/5 promoter; EGFP, enhanced green fluorescent protein gene; SV40polyA, SV40 polyadenylation signal.
FIG. 2
Retrovirus production in 293T/17 cells after transfection with amplicon constructs. Two million cells were transfected by calcium phosphate coprecipitation with HERElacZ A1 (1), HERElacZ A7 (2), HERAlacZ B3 (3), HERAlacZ B7 (4), HERlacZ C1 (5), and HERlacZ C5 (6) amplicon plasmids. Cells were also cotransfected with the BABE LacZ and pcDNA3.1MOV12 plasmids (7) and the BABE LacZ and pcDNA3.1MOVAmpho plasmids (8). Retrovirus titers assessed 48 h posttransfection represent the average of two experiments repeated in triplicate, and the error bars represent standard deviations.
FIG. 3
Retrovirus vector production in 293T/17 cells 48 h after infection with amplicon vectors. (A) To test whether the orientation of the retrovirus vector cassette in relation to the CMV-GPE cassette had any effect on retrovirus titers, cells were infected at an MOI of 2 with amplicon vectors HERElacZ A1 (1), HERElacZ A7 (2), HERAlacZ B3 (3), and HERAlacZ B7 (4), and 48 h later the supernatants were harvested for titering. A neutralization experiment was performed to demonstrate that retrovirus production is dependent on amplicon transduction. HERAlacZ B7 amplicon vector stocks (MOI of 2) were incubated for 10 min with a rabbit anti-HSV-1 antibody (5), normal rabbit serum (6), and an unrelated rabbit antibody (7) before infection of 293T/17 cells. Media were harvested 48 h later for determination of titers. (B) Relation between MOI and retrovirus titers generated 48 h postinfection. Cells were infected at different MOIs with HERElacZ A1 (□) and HERAlacZ B7 (⧫). The relation between MOI and transduction efficiency was also evaluated (▵). When supernatants were harvested for retrovirus titering, cells were analyzed by FACS to determine the percentage of cells expressing GFP as a measure of the amplicon transduction efficiency.
FIG. 4
Retrovirus production over time for J3T and Gli-36 cells infected with HERAlacZ B7 amplicon vector at different MOIs. One day prior to infection 5 × 105 cells were seeded on 60-mm plates. (A) J3T cells were infected at MOIs of 1 (□), 2 (⧫), and 5 (■). (B) Gli-36 cells were infected at MOIs of 1 (□), 2 (⧫), and 5 (■). Media were harvested at 2, 5, and 8 days postinfection and used for retrovirus titering. At 2 and 5 days postinfection cells were counted and replated at the same density as on day 0. (C) The percentage of GFP-positive cells was determined at 2, 5, and 8 days postinfection for J3T (MOI = 2, □; MOI = 5, ○) and Gli-36 cells (MOI = 2, ●; MOI = 5, ▴).
FIG. 5
Western blot analysis of Pr65_gag_ and gp70 expression in J3T and Gli-36 cells at 2 and 8 days postinfection with HERAlacZ B7 and HERlacZ C1 amplicon vectors at an MOI of 2. (A) Pr65_gag_ expression. (B) gp70 expression. Lanes 1 and 4, lysates of naive J3T cells at 2 and 8 days; lanes 2 and 5, lysates of J3T cells at 2 and 8 days postinfection with HERAlacZ B7; lanes 3 and 6, lysates of J3T cells at 2 and 8 days postinfection with HERlacZ C1; lanes 7 and 10, lysates of naive Gli-36 cells at 2 and 8 days; lanes 8 and 11, lysates of Gli-36 cells at 2 and 8 days postinfection with HERAlacZ B7; lanes 9 and 12, lysates of Gli-36 cells at 2 and 8 days postinfection with HERlacZ C1. Goat anti-p30 and anti-gp70 antibodies were used at a 1:3,000 dilution. Anti-goat peroxidase-conjugated IgG was used as secondary antibody at a 1:5,000 dilution. Blots were developed with ECL reagents and exposed to film for 1 min.
FIG. 6
β-Galactosidase activity in amplicon-infected J3T and Gli-36 cells over time. (A) J3T (□) and Gli-36 cells (⧫) were infected with HERAlacZ B7 amplicon vector at an MOI of 2, and the LacZ activity in the population was measured at different time points for up to 1 month. LacZ activities at 48 h for J3T and Gli-36 cells were 317.2 and 79.7 mU/mg of protein, respectively. (B) J3T (□) and Gli-36 cells (⧫) were infected with HERlacZ C1 amplicon vector at an MOI of 2, and the LacZ activity in the population was assayed at different time points of up to 1 month. (C) Gli-36 cells were infected with HERAlacZ B7 amplicon vector at MOIs of 2 (◊), 1 (■), 0.5 (⧫), and 0.1 (□), and LacZ activity in the population was measured at different time points for up to 15 days. LacZ activities at 48 h postinfection for MOIs of 2, 1, 0.5, and 0.1 were 147.5, 94.6, 62.3, and 9.6 mU/mg of protein, respectively. LacZ activities for all experiments were represented as the percentage of LacZ activity at 48 h. Results represent the average of two experiments repeated in triplicate for each cell line.
FIG. 7
Spreading of transgene expression in glioma cells. J3T (A to C) and Gli-36 (D to F) glioma cells were infected at MOIs of 2 and 0.1, respectively, with HERAlacZ B7 (A, D, B, and E) and HERlacZ C1 (C and F) amplicon vectors achieving initial percentages of transduction (GFP positive) of 20%. Cells were fixed at 2 (A and B) and 5 (B, C, E, and F) days postinfection. LacZ expression (red) was detected by immunofluorescence by using a rhodamine-conjugated secondary antibody, and GFP was detected by its intrinsic fluorescence.
FIG. 8
Model of cumulative increase in transgene expression in cell populations infected with the tribrid vector. The hypothesized dynamics of transgene expression are shown for two different transduction efficiencies immediately after infection with the tribrid amplicon vector and 2 weeks later, assuming a dividing cell population. Right after infection, uninfected cells (indicated as open circles) do not express the LacZ transgene, and cells infected with the amplicon vector (solid circles with “lollipop”) express LacZ and retroviral proteins, which interfere with their ability to be infected with retrovirus vectors. After 2 weeks a number of parallel phenomena have occurred. Some initially uninfected cells are now infected once by a retrovirus vector (LacZ+, solid circles) or multiple times with retrovirus vectors (higher LacZ expression, solid circles with halo). Over the same period, some cells initially infected with the amplicon vector have lost the episomal amplicon and hence their ability to produce LacZ or retroviral proteins (open circle with “×”), while some of these have subsequently become infected by retrovirus vectors produced by amplicon-infected cells that retain the episomal tribrid (LacZ+, solid circles with “×”). Therefore, at low transduction efficiency, when only a small fraction of cells are initially infected with the amplicon vector, there is a larger increase in the total LacZ activity of the population than in populations with high levels of initial transduction. Eventually the episome tribrid is lost from all cells, and then the population should reach a steady-state level of LacZ expression, reflecting the number of successful infections with retrovirus vectors.
References
- Aboody, K. S., N. Rainov, J. E. Small, S. Liu, K. A. Bower, U. Herrlinger, P. Black, X. O. Breakefield, and E. Y. Snyder. Neural stem cells migrate throughout and express foreign genes within experimental gliomas: a potential gene therapy approach to brain tumors. Submitted for publication.
- Berens M E, Bjotvedt G, Levesque D C, Rief M D, Shapiro J R, Coons S W. Tumorigenic, invasive, karyotypic, and immunocytochemical characteristics of clonal cell lines derived from a spontaneous canine anaplastic astrocytoma. In Vitro Cell Dev Biol Anim. 1993;29A:310–318. - PubMed
- Brown, A. B., E. Marecos, R. Weissleder, and X. O. Breakefield. Vascular targeting of therapeutic cells to tumors. Submitted for publication.
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