Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay

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The main reason why tumours are not controlled by the immune system is that, unlike pathogens, they do not express potent tumour rejection antigens (TRAs). Tumour vaccination aims at stimulating a systemic immune response targeted to, mostly weak, antigens expressed in the disseminated tumour lesions. Main challenges in developing effective vaccination protocols are the identification of potent and broadly expressed TRAs1, 2, 3 and effective adjuvants to stimulate a robust and durable immune response4, 5, 6. Here we describe an alternative approach in which the expression of new, and thereby potent, antigens are induced in tumour cells by inhibiting nonsense-mediated messenger RNA decay (NMD)7, 8, 9, 10. Small interfering RNA (siRNA)-mediated inhibition of NMD in tumour cells led to the expression of new antigenic determinants and their immune-mediated rejection. In subcutaneous and metastatic tumour models, tumour-targeted delivery of NMD factor-specific siRNAs conjugated to oligonucleotide aptamer ligands led to significant inhibition of tumour growth that was superior to that of vaccination with granulocyte–macrophage colony-stimulating factor (GM-CSF)-expressing irradiated tumour cells11, and could be further enhanced by co-stimulation. Tumour-targeted NMD inhibition forms the basis of a simple, broadly useful, and clinically feasible approach to enhance the antigenicity of disseminated tumours leading to their immune recognition and rejection. The cell-free chemically synthesized oligonucleotide backbone of aptamer–siRNAs reduces the risk of immunogenicity and enhances the feasibility of generating reagents suitable for clinical use.

At a glance


  1. Expression of Upf2 or Smg1 shRNA in CT26 tumour cells leads to immune-mediated inhibition of tumour growth.
    Figure 1: Expression of Upf2 or Smg1 shRNA in CT26 tumour cells leads to immune-mediated inhibition of tumour growth.

    a, Intratumoral accumulation of OVA-specific OT-I T cells in response to NMD inhibition. B16/F10 tumour cells transduced with shRNA-encoding lentiviral vectors (described in Supplementary Fig. 1a) were stably transfected with an NMD reporter plasmid (described in Supplementary Fig. 1b) containing the class I-restricted epitope of chicken ovalbumin (OVA). Mice were implanted subcutaneously with parental tumour cells (wild-type (WT) B16) or with the lentivirus-transduced tumour cells, and either received or did not receive doxycycline in their drinking water. When tumours became palpable, mice were injected with either OT-I or Pmel-1 transgenic CD8+ T cells (three mice per group). Six days later, tumours were excised and analysed for OT-I and Pmel-1 T-cell content by flow cytometry. Ctrl, control. n = 2 b, Balb/c mice were implanted subcutaneously with CT26 tumour cells stably transduced with the shRNA inducible lentiviral vector encoding Smg1, Upf2 and control shRNA (ten mice per group). Each group was divided into two subgroups receiving (filled circles) or not receiving (open circles) doxycycline in the drinking water. n = 2. c, Same as b except that tumour cells were injected into immune-deficient nude mice. n = 1.

  2. Inhibition of tumour growth in mice treated with PSMA aptamer targeted Upf2 and Smg1 siRNAs.
    Figure 2: Inhibition of tumour growth in mice treated with PSMA aptamer targeted Upf2 and Smg1 siRNAs.

    a, Balb/c mice were implanted subcutaneously with PSMA-CT26 tumour cells and 3 days later injected via the tail vein with PBS (filled circles) or with PSMA aptamer–siRNA conjugates (open circles, control siRNA; open squares, Upf2 siRNA; filled squares, Smg1 siRNA) (5 mice per group). n = 2. b, C57BL/6 mice were implanted with PSMA-B16/F10 tumour cells by tail vein injection, and 5days later were injected with PSMA aptamer–siRNA conjugates (ten mice per group). Metastatic load was determined by measuring lung weight at the time of euthanization. n = 2. c, Combination immunotherapy using NMD inhibition and 4-1BB co-stimulation. PSMA-CT26 tumour-bearing mice (five mice per group) were treated with various combinations of PSMA aptamer conjugated to Smg1 or control siRNA and an agonistic or co-stimulation-deficient 4-1BB aptamer dimer26 (mut4-1BB) and monitored for tumour growth. n = 1.

  3. PSMA aptamer-Smg1 siRNA rejection of PSMA-expressing, but not parental, CT26 tumour cells.
    Figure 3: PSMA aptamer–Smg1 siRNA rejection of PSMA-expressing, but not parental, CT26 tumour cells.

    a, Mice were co-implanted subcutaneously with PSMA-expressing (left flank) and parental (right flank) CT26 tumour cells and injected with PSMA aptamer–Smg1 siRNA via the tail vein. b, Fifteen days after tumour inoculation, 32P-labelled aptamer–siRNA was injected, and 3 or 24h later tumours were excised and the 32P content determined. n = 3. c, Three days after tumour inoculation, mice were injected with aptamer–siRNA conjugate (eight mice per group) as described in Fig. 2a and tumour growth was monitored. Open circles, parental CT26; filled circles, PSMA-CT26. n = 2.

  4. Comparison of PSMA aptamer-Smg1 siRNA treatment to vaccination with GM-CSF expressing irradiated tumour cells.
    Figure 4: Comparison of PSMA aptamer–Smg1 siRNA treatment to vaccination with GM-CSF expressing irradiated tumour cells.

    C57BL/6 mice were injected intravenously with B16/F10 tumour cells and treated with PSMA aptamer–siRNA conjugates starting at day 5 as described in Fig. 2b, or vaccinated with GM-CSF-expressing irradiated B16/F10 tumour cells (GVAX) starting at days (D) 1 or 5 using the protocol described previously29. n = 1.


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Author information


  1. Department of Microbiology & Immunology, Dodson Interdisciplinary Immunotherapy Institute, University of Miami Miller School of Medicine Miami, Florida 33134, USA

    • Fernando Pastor,
    • Despina Kolonias &
    • Eli Gilboa
  2. Department of Internal Medicine and Department of Radiation Oncology, Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA

    • Paloma H. Giangrande


F.P. suggested the approach and was responsible for designing the aptamer–siRNA conjugates and interpreting the results, D.K. was responsible for the mouse studies, P.H.G. helped design the aptamer–siRNA conjugates, and E.G. oversaw experimental design, data analysis, and wrote the manuscript.

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The authors declare no competing financial interests.

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