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. 2014 May;25(5):428-42.
doi: 10.1089/hum.2013.172. Epub 2014 Apr 10.

Long-term Episomal Transgene Expression From Mitotically Stable Integration-Deficient Lentiviral Vectors

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Free PMC article

Long-term Episomal Transgene Expression From Mitotically Stable Integration-Deficient Lentiviral Vectors

Hanna Kymäläinen et al. Hum Gene Ther. .
Free PMC article

Abstract

Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection. The use of a scaffold/matrix attachment region did not result in improved episomal retention in this system, and episomes did not form after transduction with adeno-associated viral or minicircle vectors under the same conditions. Investigations into the episomal status of the vector genome using (1) linear amplification-mediated polymerase chain reaction followed by deep sequencing of vector-genome junctions, (2) Southern blotting, and (3) fluorescent in situ hybridization strongly suggest that the vector is not integrated in the vast majority of cells. In conclusion, we have developed an IDLV procedure generating mitotically stable episomes capable of long-term transgene expression. The application of this approach to stem cell populations could significantly improve the safety profile of a range of stem and progenitor cell gene therapies.

Figures

<b>FIG. 1.</b>
FIG. 1.
Cell cycle phase analysis of IDLV-transduced CHO cells with and without induction of cell cycle arrest by methionine and serum depletion. CHO cells were plated, transduced with IDLV vectors [IDLV-SG (IDLV-SV40-GFP); IDLV-SGm (IDLV-SV40-GFP-mMAR)] or mock control after 1 day, and then treated with MSD medium from day 2 to day 7. At day 7, cultures were returned to full growth medium and allowed to proliferate as normal with routine passaging as required. To confirm induction of cell cycle arrest, cell cycle phase analyses were performed by propidium iodide staining and flow cytometry at day 7. The nonarrested controls were analyzed at the same time point. The colored sections indicate the average percentages of cells in each cell cycle phase. The proportion of cells in different cell cycle phases that were significantly different between treatments was determined by Student's t-test (*p<0.05, **p<0.01, ***p<0.001). CHO, Chinese hamster ovary; IDLV, integration-deficient lentiviral vector; MSD medium, methionine- and serum-depleted medium.
<b>FIG. 2.</b>
FIG. 2.
Transient induction of cell cycle arrest leads to substantial levels of stable eGFP expression in IDLV-transduced CHO cells. CHO cells were seeded and transduced at MOI 0.5 with vectors IDLV-SG or IDLV-SGm. (a) Cells were induced to undergo transient cell cycle arrest by methionine and serum depletion of the culture medium at day 2 or (b) allowed to proliferate freely. From day 7, all cells were allowed to proliferate normally in full growth medium with routine passaging for over 2 months (up to day 72). Levels of eGFP expression were determined by flow cytometry at a range of time points (mean±SEM; n=3). (c) Stabilized transduction levels at day 72. Transduction in cultures subject to an initial cell cycle arrest were substantial, and very significantly higher than for the cell cultures allowed to proliferate continuously (***p<0.001; one-way ANOVA with Tukey's post hoc test). MOI, multiplicity of infection.
<b>FIG. 3.</b>
FIG. 3.
The establishment of stable episomes is specific to IDLV-transduced cells. CHO cells were plated into culture, and after 1 day transduced at an MOI of 105 with single-stranded AAV vectors (a) and self-complementary AAV vectors (b) or transfected with minicircle and plasmid vectors at 2 μg per well (c). Cells were induced to undergo transient cell cycle arrest by methionine and serum depletion of the culture media at day 2, and from day 7 all cells were allowed to proliferate normally in full growth medium with routine passaging for up to 42 days. In all of the transduced cells, the average proportion of the population expressing GFP dropped to below 1.5% by 30 days posttransduction or transfection. The final percentages of cells expressing GFP after 42 days (AAV vectors) or 30 days (minicircle and plasmid vectors) of continuously proliferating culture are shown in (d). AAV, adeno-associated viral.
<b>FIG. 4.</b>
FIG. 4.
Clonal stability of eGFP expression during long-term proliferation of CHO cell lines derived by IDLV transduction with transient cell cycle arrest. CHO cell lines derived by dilution cloning from parent populations transduced with IDLV-SG and IDLV-SGm and subjected to transient cell cycle arrest were maintained in continuous proliferating culture for up to 60 days with routine passaging approximately 3 times weekly at a 1:5 split. (a and c) eGFP flow cytometry and fluorescence micrographs at culture day 100 for the parental (nonclonal) populations of CHO cells transduced with IDLV-SG and IDLV-SGm, respectively. (b and d) eGFP flow cytometry (days 16 and 50), and eGFP fluorescence micrographs (day 40) for three examples of clonal cell populations transduced with IDLV-SG and IDLV-SGm, respectively.
<b>FIG. 5.</b>
FIG. 5.
Episomal status of vector sequences in CHO cell clones derived by IDLV transduction and transient cell cycle arrest is confirmed by LAM-PCR analyses and high-throughput deep sequencing. After 60 days of continuous proliferation, genomic DNA was prepared from the stable GFP-expressing CHO clones derived from IDLV transduction and transient cell cycle arrest. DNAs were subject to LAM-PCR, and the amplicons were analyzed by high-throughput deep sequencing. (a) Diagrammatic representation of potential LAM-PCR amplicons. (b) Distribution of sequencing results according to the vector sequence content for the clones analyzed. Amplicons containing exclusively vector sequence are shown in blue and any other amplicons shown in red. (c) The analysis of LAM-PCR amplicons by alignment against the Chinese hamster genomic sequence. The table shows the number of successful sequencing reads per sample (Verified reads), number of reads containing only vector sequences (Vector), number of reads containing sequences verified by QuickMap alignment against the hamster genome (Host), and the number of hamster loci indicated (Host locus). Vector sequences in CHO cell clones derived by IDLV transduction and transient cell cycle arrest contained mostly vector sequences indicative of circular episomes. As expected in the control CHO cells transduced with an IPLV, LAM-PCR amplicons generated high amounts of nonvector sequence reads characteristic of extensive genomic integration. IPLV, integration-proficient lentiviral vector; LAM-PCR, linear amplification-mediated polymerase chain reaction.
<b>FIG. 6.</b>
FIG. 6.
Evaluation by Southern blot hybridization of the episomal status of vector sequences in CHO cell clones derived by IDLV transduction and transient cell cycle arrest. Genomic DNA was prepared after 60 days of continuous proliferation from the stable GFP-expressing CHO clones derived from IDLV transduction and transient cell cycle arrest, as well as 24 hr after transduction for IDLV and IPLV control populations. Genomic DNAs were digested with EcoRI (IDLV-SG and IPLV-SG) or XhoI (IDLV-SGm) and 10 μg subjected to agarose gel electrophoresis, Southern blot transfer, and hybridization with a 32P-labeled probe corresponding to GFP transgene sequences. A diagrammatic linear representation of the expected episomes indicating the location of the probe and the restriction enzyme sites is shown in (a). The resulting blot image is shown in (b), and the expected band sizes for each of the two episomal conformations are shown in (c). Ticks indicate the presence of bands corresponding to the expected sizes.
<b>FIG. 7.</b>
FIG. 7.
Cytogenetic FISH analysis of the episomal status of vector sequences in CHO cell clones derived by IDLV transduction and transient cell cycle arrest. Stable GFP-expressing CHO clones derived from IDLV transduction and transient cell cycle arrest [IDLV-SG (clone 4) and IDLV-SGm (clone 11)], and clonal CHO cells stably transduced with an integrating lentivector (IPLV-SG), along with nontransduced cells were prepared for FISH and hybridized simultaneously to a mixture of two probes labeled with either SpectrumOrange or SpectrumGreen fluorochromes. Slides were counterstained with DAPI, and at least 20 metaphases were located on each slide and examined by epifluorescence microscopy, here shown at ×100 magnification (a–l). In nontransduced negative control cells (a–c), rare background spots positive for only one probe color were observed. In the clone transduced with integrating lentivector (d–f ), several doublet signals labeled with both probes and indicative of integrated vector are shown. In clone 4 (g–i) and clone 11 (j–l), obtained by IDLV transduction and transient cell cycle arrest, strong individual signals labeled with both probes and indicative of episomal DNAs are shown. (m) Signal specificity was evaluated by counting the number of FISH signals associated with nuclear profiles and the number of total nuclei in 20 fields for each population. Statistical analysis shows a significant difference between the lentivector transduced clones and the negative control cells (t-test, p<0.0001). (n) Singlet and doublet signals in the IDLV-SG and IDLV-SGm and the control integrating LV populations were counted. In the IPLV-SG clonal population, 23% of the signals observed were doublets (8 out of 35), whereas in the IDLV-SG (clone 4) and IDLV-SGm (clone 11) cells in total only 2% of the signals observed were doublets (1 out of 52). FISH, fluorescent in situ hybridization.
<b>FIG. 8.</b>
FIG. 8.
Potential origins of replication in circular episomes. Schematic representations of 1-LTR episomes expected to arise during cell transduction with (a) IDLV-SG or (b) IDLV-SGm vectors are aligned with their corresponding profiles of strand separation potential from SIDD analyses. The SIDD plots show G(x), the average free energy of strand separation for each individual base pair in kcal/mol, plotted against the episome base sequence number where position 1 is set at the start of the 5′LTR. The red arrows indicate the destabilization troughs generated by the cPPT present in each vector. cPPT, central polypurine tract; LTR, long terminal repeat; SIDD, stress-induced DNA duplex destabilization.

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