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. 2022 May 18:28:877-891.
doi: 10.1016/j.omtn.2022.05.029. eCollection 2022 Jun 14.

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells

Taekyu Ha  1 , Michael DiPrima  1 , Vishal Koparde  2   3 , Parthav Jailwala  2   3 , Hidetaka Ohnuki  1 , Jing-Xin Feng  1 , Murali Palangat  4 , Daniel Larson  4 , Giovanna Tosato  1
Affiliations

Antisense transcription from lentiviral gene targeting linked to an integrated stress response in colorectal cancer cells

Taekyu Ha et al. Mol Ther Nucleic Acids. .

Abstract

Advances in gene therapy research have resulted in the successful development of new therapies for clinical use. Here, we explored a gene targeting approach to deplete ephrinB2 from colorectal cancer cells using an inducible lentiviral vector. EphrinB2, a transmembrane ephrin ligand, promotes colorectal cancer cell growth and viability and predicts poor patient survival when expressed at high levels in colorectal cancer tissues. We discovered that lentiviral vector integration and expression in the host DNA frequently drive divergent host gene transcription, generating antisense reads coupled with splicing events and generation of chimeric vector/host transcripts. Antisense transcription of host DNA was linked to development of an integrated stress response and cell death. Despite recent successes, off-target effects remain a concern in genetic medicine. Our results provide evidence that divergent gene transcription is a previously unrecognized off-target effect of lentiviral vector integration with built-in properties for regulation of gene expression.

Keywords: MT: Oligonucleotides; Therapies and Applications; antisense reads; colorectal cancer; ephrinB2; gene therapy; integrated stress response; lentiviral vector; shRNA; vector integration.

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Conflict of interest statement

All authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
EFNB2 knockdown in colorectal cancer cells (A) pTRIPZ-CMV-shRNAmiR vector and DNA sequences for EFNB2 targeting and non-silencing control. 5′ LTR, 5′ long terminal repeat; Ψ, Psi packaging sequence; RRE, Rev response element; cPPT, central polypurine tract; Puro, puromycin resistance gene; 3′ LTR, 3′ self-inactivating long terminal repeat; CMV, tetracycline-inducible minimal CMV promoter; RFP, TurboRFP reporter; shRNAmiR, microRNA (miR-30)-adapted shRNA; UBC, human ubiquitin C promoter; rtTA3, reverse tetracycline-transactivator 3; WPRE, woodchuck hepatitis posttranscriptional regulatory element. (B) EphrinB2 and EphA2 protein, representative of six immunoblots. (C) Cell death and RFP (red fluorescent protein) by differential interference contrast (DIC) and fluorescence; scale bar: 200 μm. (D) Cleaved caspase-3 in ephrinB2-depleted 596-7 clone, representative of three immunoblots. NT, non-silencing inducible vector; Empty, empty inducible vector. (E) Proliferation (mean ± SD, triplicate cultures; ∗∗p < 0.01 by unpaired Student’s t test), representative of three experiments. (F) Growth curves; clone 596-7 with or without Dox. (G) Tumor volume in NOD-SCID mice injected subcutaneously with clone 596-7. On day 7, groups of 10 mice were randomized to chow with or without Dox. (H) Relative EFNB2 and (I) NNMT mRNA levels in clone 596-7 (mean ± SD of triplicate measurements; representative of three to five experiments). Clone 596-7 was superinfected with three constitutive NNMT shRNAs or control (I). See also Figure S1.
Figure 2
Figure 2
Defective mRNA translation and altered gene expression after EFNB2 silencing (A) Experimental design. Linear sucrose gradient profile of polysomes isolated from clone 596-7 with or without Dox treatment. The positions of the small (S) and large (L) ribosomal subunits, monosomes, and polysomes are indicated; one of two experiments. (B) Time-dependent activation of phospho (p)-eIF2α (S51) in clone 596-7 treated with Dox; representative immunoblot of three experiments. (C) Magnitude (fold change) and statistical significance of changes in RNA expression induced by Dox in clone 596-7 over 24 h compared with uninduced 596-7 cells. RNA expression by total RNA-seq. Each dot in the volcano plot represents an annotated RNA; significantly induced RNAs are shown as red dots and significantly repressed RNAs are shown as blue dots. Some RNAs are identified as gene products. Vertical and horizontal dotted lines reflect cutoffs for the parameters fold change and significance. (D) Genes expressed at significantly different levels (p < 0.005) in control and Dox-treated 596-7 cells (n = 4/group) displayed by row-wide Z score (color bar) ordered alphabetically. The gene categories are inclusive of pathways “endoplasmic reticulum stress,” “unfolded protein response,” “role of PKR in interferon induction,” and “pattern recognition receptors” (Ingenuity Pathway Analysis). (E) Differentially expressed (p < 0.005) “cell death and survival” genes (Ingenuity Pathway Analysis) in control and Dox-treated 596-7 cells (n = 4/group) displayed by row-wide Z score (color bar) ordered alphabetically. (F) Time-dependent PKR phosphorylation (T446) and eIF2α phosphorylation (S51) in 596-7 cells treated with Dox; PERK (T980) and GCN2 (T899) are not phosphorylated. Representative immunoblotting from three experiments. See also Figure S2.
Figure 3
Figure 3
Antisense transcription from the YTHDC2 and PURB genes and chimeric vector/host RNA reads (A and B) Vector integration site in the host DNA and the junction between host RNA and vector RNA are marked by the blue vertical lines for the YTHDC2 (A) and PURB (B) genes. The red arrow below the schematic gene structure indicates the sense (5′ to 3′) direction of gene transcription. Sense (red) and antisense (green) reads by stranded RNA-seq were derived from clone 596-7 treated or not treated with Dox. Each read is representative of four replicate samples. The read scale is indicated on the read panels. The bar graphs reflect the quantification of chimeric antisense reads from cells treated with (orange bars) or without (blue bars) Dox. The results reflect the means of four samples. The vector-derived sequences within the chimeric reads are mapped to the vector sequence. A simplified schematic of the annotated shRNA vector is shown above the bar graphs joining to the host antisense DNA strand. The hybrid vector-host reads reveal splicing from the splice vector donor (3′ LTR and UBC) into the host splice acceptor site; the dotted lines indicate the shRNA-host splicing junctions.
Figure 4
Figure 4
Antisense transcription from the VPS52 and ABCC4 genes and chimeric vector/host RNA reads (A and B) Vector integration site in the host DNA and the junction between host RNA and vector RNA are marked by the blue vertical lines for the VPS52 (A) and ABCC4 (B) genes. The red arrow indicates the sense (5′ to 3′) direction of gene transcription. Sense (red) and antisense (green) reads from the VPS52 (A) and ABCC4 (B) genes were derived from clone 596-7 treated or not treated with Dox. Each read is representative of four replicate samples. The bar graphs in (A) and (B) reflect the quantification of chimeric antisense reads from cells treated (orange bars) or not (blue bars) with Dox. The results reflect the means of four samples. The vector-derived sequences within the chimeric reads are mapped to the vector sequence joining to the host antisense DNA strand. The hybrid vector-host reads reveal splicing from the splice vector donor (3′ LTR and UBC) into the host splice acceptor site; the dotted lines indicate the shRNA-host splicing junctions. (C) Quantification of all chimeric sense (host and vector-derived reads are in the sense orientation) and antisense (host-derived reads are in the antisense orientation and vector reads are in the sense orientation) reads coming from all 15 sites of vector integration in the host DNA. The vector component of the hybrid reads is mapped to the vector sequence.
Figure 5
Figure 5
Characterization of HT29 cell clones infected with ephrinB2 shRNA 596 (A) Relative mRNA levels of EFNB2, shRNA, IFNB1, and IFNL1 in each of the indicated clones infected with shRNA 596, with or without Dox, for 24 h. Values are normalized to mRNA levels in clone 7 without Dox. The results reflect the means of triplicate measurements. Error bars show the standard deviation. (B) Protein levels of ephrinB2, cleaved caspase-3, and GAPDH in HT29 clones infected with EFNB2 shRNA 596, with or without treatment with Dox for 24 h. Immunoblotting results are shown. (C) Number and location of vector integration sites in each of the HT29 clones infected with EFNB2 shRNA 596-7. (D) Shared DNA integration sites among 10 HT29 clones infected with EFNB2 shRNA 596.
Figure 6
Figure 6
Antisense reads detected in distinct HT29 cell clones (A–C) Sense and antisense reads from the SETD1B gene in clone 596-6 (A), from the SCFD1 gene in clone 596-1 (B), and from the JPH1 gene in clone 596-11 (C). Vector integration site in the host DNA and the chimeric host-vector RNA junction are marked by the blue vertical lines. The direction of sense gene transcription is indicated by the red arrow below the schematic gene structure. The read scale is 0–200 for all SCFD1 and JPH1 reads and 0–250 for all SETD1B reads. Quantification of chimeric antisense reads is displayed in the bar graphs (orange bars reflect reads from cells treated with Dox and blue bars reflect reads from cells not treated with Dox). The results reflect the means of duplicate samples. The vector-derived sequences within the chimeric reads are mapped to the shRNA sequence. Splicing events from the vector splice donor (3′ LTR, 5′ LTR, or RFP) into the host splice acceptor site are identified; the dotted lines indicate the shRNA-host splice junctions. See also Figure S4.
Figure 7
Figure 7
Antisense gene transcription in HT29 cell clones (A–D) Quantification of all chimeric sense and antisense reads from all sites of vector integration in clones 596-1 (A), 596-6 (B), 596-7 (C), and 596-11 (D) with or without Dox. The vector hybrid reads are mapped to the vector sequence. (E) Quantification of all chimeric antisense reads from all DNA sites of vector integration in each of the clones 596-1, 596-6, 596-7, and 596-11 with or without Dox. The results reflect the means of two separate sequencing results and are displayed as normalized counts/106 reads. (F) Correlation between integrated vector copy number and relative antisense read number (antisense reads/106 reads) in clones 596-1, 596-6, 596-7, and 596-11. (G) Correlation between relative shRNA expression (shRNA/RLP30 × 103) and relative antisense read number (antisense reads/106 reads) in clones 596-1, 596-6, 596-7, and 596-11. Correlation between two variables was calculated by Spearman’s rank correlation coefficient.
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