Long-Distance Effects of Insertional Mutagenesis

Most common systems of genetic engineering of mammalian cells are associated with insertional mutagenesis of the modified cells. Insertional mutagenesis is also a popular approach to generate random alterations for gene discovery projects. A better understanding of the interaction of the structural elements within an insertional mutagen and the ability of such elements to influence host genes at various distances away from the insertion site is a matter of considerable practical importance.We observed that, in the context of a lentiviral construct, a transcript, which is initiated at an internal CMV promoter/enhancer region and incorporates a splice donor site, is able to extend past a collinear viral LTR and trap exons of host genes, while the polyadenylation signal, which is naturally present in the LTR, is spliced out. Unexpectedly, when a vector, which utilizes this phenomenon, was used to produce mutants with elevated activity of NF-κB, we found mutants, which owed their phenotype to the effect of the insert on a gene located tens or even hundreds of kilobases away from the insertion site. This effect did not result from a CMV-driven transcript, but was sensitive to functional suppression of the insert. Interestingly, despite the long-distance effect, expression of loci most closely positioned to the insert appeared unaffected.We concluded that a polyadenylation signal in a retroviral LTR, when occurring within an intron, is an inefficient barrier against the formation of a hybrid transcript, and that a vector containing a strong enhancer may selectively affect the function of genes far away from its insertion site. These phenomena have to be considered when experimental or therapeutic transduction is performed. In particular, the long-distance effects of insertional mutagenesis bring into question the relevance of the lists of disease-associated retroviral integration targets, which did not undergo functional validation

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Examples of regulated expression of RIPK1 in mutant cells.

<p>(A) Elevated expression of RIK1 in the mutant clones. RNA was isolated from mutant clones 2B2.1, 2B1.4 and 2B3, and from the parental HEK293ZeoTK cells, and analyzed by RT-PCR with RIPK1-specific primers (upper panel). The bottom panel presents results of RT-PCR on the same samples with primers for a housekeeping gene (GAPDH). (B) Dependence of RIPK1 expression in the mutant clones on the presence of the insert. Mutant clones 2B1.2 and 2B1.4 were infected with a Cre-expressing construct or the respective empty vector control. The cell lysates were compared by Western blotting with polyclonal anti-RIPK1. (C) Dependence of RIPK1 expression in the mutant clones on the function of the inserted promoter. Mutant clone 2B1.4 infected with a tTS-expressing construct was cultured with or without doxycycline. The cell lysates were analyzed by Western blotting with polyclonal anti-RIPK1 and compared to those of the parental HEK293ZeoTK cells.</p

Functional organization of pAIM.

<p>The vector is shown in the form of a provirus collinearly integrated in a host gene. The vector is flanked by promoter-deficient LTRs (“dLTR”), which harbor Cre-recombinase recognition sites. The immediate early promoter and enhancer region from human cytomegalovirus (“CMV”) is preceded by a cluster of tet-operators (“TO”), which permit suppression of this promoter in the presence of a tetracycline-controlled transcription silencer (tTS). The coding region of copGFP (“copGFP”) ends with the 2A sequence and an unpaired splice donor (“2A” and “SD” respectively). In the scenario shown above the vector, CMV-driven transcript is polyadenylated on the LTR-derived sequence and encodes only copGFP. In the scenario shown below the vector, transcription continues into the host DNA, and a mature fusion product is produced by splicing, which removes the LTR sequences. If the open reading frames of copGFP and the host gene coincide, both copGFP and a host protein could be produced when the RNA is translated.</p

Activity of NF-κB upon overexpression of genes in the vicinity of inserts compared to parental HEK293ZeoTK cells.

<p>NF-êB dependent luciferase reporter activity was measured after cells were co-transfected with respective expression constructs. Only RIPK1 showed elevated NF-κB activity.</p

Location of inserts in the three mutant clones.

<p>A fragment of p25 region of chromosome 6 is shown, indicating the relative positions of known genes. The inserts are shown as arrows pointing in the direction of the CMV promoter. Approximate distances from the first annotated exon of RIPK1 are indicated. The drawing in not to scale.</p

Identification of the fusion transcripts generated by trans-LTR splicing in two independent clones.

<p>The products of nested ligation-mediated PCR on MboI-digested cDNA were sequenced. As predicted, the clones contain host sequence, followed by an MboI site (GATC) and the adapter. The 5′-end of the transcript is derived from the vector. The junction point corresponds to host intron/exon boundary.</p

A example of the dependence of NF-κB activity in mutant cells on the function of RIPK1.

<p>Mutant 2B2.1 and parental HEK293ZeoTK cells were assayed for NF-κB activity using transient transfection of a luciferase reporter. A decline in the reporter activity (indicated by the red arrow) resulted from adding an shRNA against RIPK1 to the transfection mix. An shRNA against p53 (a protein that is expressed, but is inactivated in these cells) was used as the control.</p