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

Vol 465 13 May 2010 Induction of tumour immunity by targeted inhibitionof nonsense-mediated mRNA decay Fernando Pastor1, Despina Kolonias1, Paloma H. Giangrande2 & Eli Gilboa1 The main reason why tumours are not controlled by the immune antigenicity of disseminated tumours leading to their immune system is that, unlike pathogens, they do not express potent recognition and rejection. The cell-free chemically synthesized tumour rejection antigens (TRAs). Tumour vaccination aims at oligonucleotide backbone of aptamer–siRNAs reduces the risk of stimulating a systemic immune response targeted to, mostly weak, immunogenicity and enhances the feasibility of generating antigens expressed in the disseminated tumour lesions. Main chal- reagents suitable for clinical use.
lenges in developing effective vaccination protocols are the iden- Disseminated metastatic disease is the primary cause of death tification of potent and broadly expressed TRAs1–3 and effective among cancer patients. Cancer vaccination stimulates a systemic adjuvants to stimulate a robust and durable immune response4–6.
immune response against judiciously chosen tumour antigens Here we describe an alternative approach in which the expression expressed in the tumour cells that seeks out and destroys the disse- of new, and thereby potent, antigens are induced in tumour minated tumour lesions. The development of effective cancer vac- cells by inhibiting nonsense-mediated messenger RNA decay cines will require the identification of potent and broadly expressed (NMD)7–10. Small interfering RNA (siRNA)-mediated inhibition TRAs1–3 as well as effective adjuvants to stimulate a robust and dur- of NMD in tumour cells led to the expression of new antigenic able immune response4–6. An alternative approach to vaccination is determinants and their immune-mediated rejection. In subcutan- to express new, and hence potent, antigens in tumour cells in situ.
eous and metastatic tumour models, tumour-targeted delivery of How to express new antigens in the disseminated tumour lesions, but NMD factor-specific siRNAs conjugated to oligonucleotide apta- not in normal tissue, have precluded the development of such strat- mer ligands led to significant inhibition of tumour growth that egies so far. NMD is an evolutionarily conserved surveillance mech- was superior to that of vaccination with granulocyte–macrophage anism in eukaryotic cells that prevents the expression of mRNAs containing a premature termination codon (PTC)8–10. Inhibition of tumour cells11, and could be further enhanced by co-stimulation.
NMD in cultured human cell lines using siRNAs targeted to any of its Tumour-targeted NMD inhibition forms the basis of a simple, factors, SMG1, UPF1, UPF2 or UPF3, results in the upregulation of broadly useful, and clinically feasible approach to enhance the several products encoded by the PTC-containing mRNAs (see, for Upf2 shRNA Smg1 shRNA umour volume (mmT c )3 1,500
Upf2 shRNA Smg1 shRNA centage Pmel-1 per shRNA Smg1 shRNA Smg1 Figure 1 Expression of Upf2 or Smg1 shRNA in CT26 tumour cells leads to were injected with either OT-I or Pmel-1 transgenic CD81 T cells (three mice immune-mediated inhibition of tumour growth. a, Intratumoral per group). Six days later, tumours were excised and analysed for OT-I and accumulation of OVA-specific OT-I T cells in response to NMD inhibition.
Pmel-1 T-cell content by flow cytometry. Ctrl, control. n 5 2 b, Balb/c mice B16/F10 tumour cells transduced with shRNA-encoding lentiviral vectors were implanted subcutaneously with CT26 tumour cells stably transduced (described in Supplementary Fig. 1a) were stably transfected with an NMD with the shRNA inducible lentiviral vector encoding Smg1, Upf2 and control reporter plasmid (described in Supplementary Fig. 1b) containing the class shRNA (ten mice per group). Each group was divided into two subgroups I-restricted epitope of chicken ovalbumin (OVA). Mice were implanted receiving (filled circles) or not receiving (open circles) doxycycline in the subcutaneously with parental tumour cells (wild-type (WT) B16) or with the drinking water. n 5 2. c, Same as b except that tumour cells were injected lentivirus-transduced tumour cells, and either received or did not receive into immune-deficient nude mice. n 5 1.
doxycycline in their drinking water. When tumours became palpable, mice 1Department of Microbiology & Immunology, Dodson Interdisciplinary Immunotherapy Institute, University of Miami Miller School of Medicine Miami, Florida 33134, USA.
2Department of Internal Medicine and Department of Radiation Oncology, Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa 52242, USA.
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NATURE Vol 465 13 May 2010 example, refs 12–15). Many of these products, resulting from aber- No T-cell responses were detected against tumour cells that did not rant splicing or NMD-dependent autoregulated alternative splic- express Smg1 shRNA or against normal tissues including liver, colon ing7,8,16, encode new peptides that have not induced tolerance (see and prostate (Supplementary Fig. 3). This is consistent with the Supplementary Discussion). We proposed that the upregulation of hypothesis that tumour rejection was mediated by the induction of such products when NMD is inhibited in tumour cells will elicit an immune responses against NMD-controlled products that were immune response against (some of) the new products, and that the upregulated when NMD was inhibited in the tumour cells.
immune response will inhibit tumour growth. Moreover, there is In the experiment shown in Fig. 1b, tumour growth was comple- evidence that frameshift mutations in cancer cells exhibiting DNA tely prevented when NMD was inhibited in all tumour cells from the mismatch repair generate PTC-containing transcripts that are nega- time of tumour implantation. Simulating a more relevant clinical tively controlled by NMD17. Inhibiting NMD could, therefore, fur- model, we tested whether inhibition of NMD in pre-existing tumours ther augment the production of such tumour-specific antigens (see can induce therapeutically useful tumour immunity. To preclude NMD inhibition in normal cells, the NMD factor siRNAs were tar- To determine whether NMD inhibition in tumour cells can geted to tumour cells using oligonucleotide aptamer ligands21,22.
stimulate protective anti-tumour immunity, we tested whether Smg1 and Upf2 siRNA were conjugated to an oligonucleotide apta- the stable expression of NMD factor short hairpin RNAs mer that binds to prostate-specific membrane antigen (PSMA)23 as (shRNAs) in tumour cells inhibits their growth potential in mice.
shown in Supplementary Fig. 4. PSMA-expressing CT26 and B16 CT26 colon carcinoma tumour cells were transduced with a lenti- tumour cell lines were generated by transduction with a PSMA- viral vector (PTIG-U6tetOshRNA) encoding Smg1 or Upf2 shRNAs encoding expression vector, and PSMA expression was confirmed expressed from a tet-regulated U6 promoter18. shRNA expression by flow cytometry (not shown). The PSMA-conjugated siRNAs can be upregulated in vitro by adding doxycycline to the culturemedium, and in vivo by providing doxycycline in the drinkingwater. Doxycycline-induced Smg1 and Upf2 shRNA expression in cultured CT26 cells results in downregulation of the corresponding mRNA (Supplementary Fig. 1a) and inhibition of NMD (Supplementary Fig. 1b). Long-term inhibition of NMD, or other functions controlled by SMG1 or UPF2, had no measurable effects Normal lung weight on the viability or proliferative capacity of the CT26 cells in vitro (data not shown).
To determine whether siRNA inhibition of NMD in the tumour- Lung weight (g) 0.5 bearing mice can stimulate immune responses against products that umour volume (mmT are normally under NMD control, we measured the intratumoral accumulation of T cells recognizing a model tumour antigen that is suppressed as a result of NMD. B16/F10 tumour cells containing thedoxycycline-inducible Smg1, Upf2 or control shRNA were stably PSMA-ctrl + 4-1BB transfected with an NMD reporter plasmid encoding the dominantmajor histocompatibility complex (MHC) class I epitope of the chicken ovalbumin gene (OVA) upstream of a PTC (diagrams in Fig. 1a and Supplementary Fig. 1a). Tumour-bearing mice wereinfused with OT-I transgenic CD81 T cells that recognize the OVA MHC class I-restricted epitope20, or with Pmel-1 transgenic CD81 T cells that recognize an MHC class I-restricted epitope in the endo-genous gp100 tumour antigen expressed in B16 tumour cells19. gp100 expression is not under NMD control. As shown in Fig. 1a, unlikePmel-1 T cells, the OT-I T cells failed to accumulate to significant PSMA-Smg1 + mut4-1BB PSMA-Smg1 + 4-1BB levels in the OVA-negative B16/F10 tumours or in tumours trans- umour volume (mmT fected with the PTC-containing b-globin-OVA construct encoding but not expressing Smg1 or Upf2 shRNA. However, upregulation ofSmg1 or Upf2 shRNA, but not control shRNA (doxycycline in the drinking water) resulted in a significant accumulation of OT-I T cells in the tumours. This experiment shows that siRNA inhibition of NMD in tumour cells can induce an immune response in vivo againstan antigen that is under NMD control.
To determine whether siRNA-mediated inhibition of NMD affects tumour growth, the lentiviral-transduced CT26 cells expressing a Figure 2 Inhibition of tumour growth in mice treated with PSMA aptamer control, Smg1 or Upf2 shRNA were implanted subcutaneously into targeted Upf2 and Smg1 siRNAs. a, Balb/c mice were implanted mice and tumour growth was monitored in the presence or absence subcutaneously with PSMA-CT26 tumour cells and 3 days later injected via of doxycycline administered in the drinking water. Figure 1b shows the tail vein with PBS (filled circles) or with PSMA aptamer–siRNA that tumour cells expressing Smg1 or Upf2 shRNA, but not control conjugates (open circles, control siRNA; open squares, Upf2 siRNA; filled shRNA, grew initially but failed to progress. Tumour inhibition was squares, Smg1 siRNA) (5 mice per group). n 5 2. b, C57BL/6 mice were immune-mediated because the tumours grew in nude mice (Fig. 1c), implanted with PSMA-B16/F10 tumour cells by tail vein injection, and and mice that rejected the tumours shown in Fig. 1b, but not age- 5 days later were injected with PSMA aptamer–siRNA conjugates (ten miceper group). Metastatic load was determined by measuring lung weight at the matched control mice, resisted a second challenge with parental time of euthanization. n 5 2. c, Combination immunotherapy using NMD tumour cells (not shown). Delaying doxycycline treatment of mice inhibition and 4-1BB co-stimulation. PSMA-CT26 tumour-bearing mice expressing Smg1 shRNA diminished the tumour inhibitory effect that (five mice per group) were treated with various combinations of PSMA was completely lost when drug treatment was delayed for 6 days aptamer conjugated to Smg1 or control siRNA and an agonistic or co- (Supplementary Fig. 2). Tumour rejection correlated with the induc- stimulation-deficient 4-1BB aptamer dimer26 (mut4-1BB) and monitored tion of T-cell responses against tumour cells expressing Smg1 shRNA.
for tumour growth. n 5 1.
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NATURE Vol 465 13 May 2010 Figure 3 PSMA aptamer–Smg1 siRNA rejectionof PSMA-expressing, but not parental, CT26tumour cells. a , Mice were co-implanted subcutaneously with PSMA-expressing (leftflank) 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-labelledaptamer–siRNA was injected, and 3 or 24 h later tumours were excised and the 32P content determined. n 5 3. c, Three days after tumour PSMA-Smg1 siRNA inoculation, mice were injected with aptamer–siRNA conjugate (eight mice per group) as described in Fig. 2a and tumour growth was Smg1 siRNA monitored. Open circles, parental CT26; filled circles, PSMA-CT26. n 5 2.
umour volume (mmT bound to and were taken up by PSMA-expressing, but not parental, 6. As shown in Fig. 2c, combination therapy with PSMA aptamer– tumour cells (Supplementary Fig. 5), leading to the downregulation Smg1 siRNA and 4-1BB aptamer was more than additive.
of their target RNAs (Supplementary Fig. 6).
To determine whether tumour inhibition shown in Fig. 2 is a result We next tested whether systemic administration of PSMA apta- of aptamer targeting of siRNA to PSMA-expressing tumour cells, mice mer–siRNA conjugates by tail vein injection can inhibit tumour were implanted in opposite flanks with PSMA-expressing and parental growth. As shown in Fig. 2a, treatment of day 3 subcutaneously CT26 tumour cells and PSMA aptamer conjugated to control or Smg1 implanted PSMA-CT26 tumour cells with PSMA-conjugated Smg1 siRNA was administered systemically by tail vein injection (Fig. 3a).
siRNA, and to a lesser extent Upf2 siRNA, significantly inhibited Figure 3b shows that 32P-labelled PSMA aptamer–Smg1 siRNA con- tumour growth. Two out of seven mice treated with the PSMA apta- jugate accumulated preferentially in PSMA-expressing tumour cells.
mer–Smg1 siRNA conjugate rejected the implanted tumours and Figure 3c shows that systemic administration of PSMA aptamer-con- remained tumour-free (Supplementary Fig. 7). When treatment jugated Smg1, but not control, siRNA inhibited the growth of PSMA- intensity was increased by doubling the dose of the aptamer– expressing CT26 tumour cells but not the contralaterally implanted siRNA conjugate and extending treatment to seven injections, six parental CT26 tumour cells. Supplementary Fig. 8 shows a snapshot of out of seven of mice rejected the tumour long term. Treatment with the tumour-bearing mice at the day of euthanization.
PSMA aptamer conjugated to control siRNA had a small inhibitory To assess the potency of tumour-targeted NMD inhibition, we effect that could have resulted from the binding of the PSMA apta- compared the anti-tumour effects of treating tumour-bearing mice mer–siRNA to the tumour cells, or be due to non-specific immune with PSMA aptamer–Smg1 siRNA conjugate and vaccination with stimulatory effects of the oligonucleotide24,25. We found no increase GM-CSF-expressing irradiated syngeneic tumour cells (GVAX)11,27.
in IFNa levels in the serum of mice treated with PSMA aptamer– In therapeutic protocols when vaccination is initiated 2–4 days control or Smg1 siRNA conjugates (data not shown). As shown in after tumour inoculation, the anti-tumour impact of GVAX is Fig. 2b, the treatment of day 5 PSMA-B16/F10 tumour-implanted limited, unless combined with other treatments such as CTLA-4 mice with PSMA aptamer-conjugated Upf2 or Smg1 siRNA inhibited blockade28 or T-regulatory cell depletion29. As shown in Fig. 4, in the development of lung metastasis that was more profound in the the B16 lung metastasis model described in Fig. 2b, GVAX treat- SMG1 group. To determine whether the anti-tumour response eli- ment of day 1 tumour bearing mice significantly inhibited meta- cited by NMD inhibition can be further enhanced by co-stimulation, stasis, whereas treatment of day 5 tumour bearing mice had a PSMA-CT26 tumour-bearing mice were treated with PSMA apta- limited anti-metastatic effect that barely reached statistical signifi- mer–Smg1 siRNA and an agonistic 4-1BB aptamer dimer26. The cance. By comparison, treatment of day-5 tumour-bearing mice stringency of NMD inhibition and 4-1BB co-stimulation was with PSMA aptamer–Smg1 siRNAs inhibited metastasis to an adjusted to elicit a limited anti-tumour effect when applied separately extent comparable to that of administering GVAX at day 1.
by delaying treatment with PSMA aptamer–siRNA conjugates from Given that these are first generation aptamer–siRNA conjugates days 3 to 5 and administering a single dose of 4-1BB aptamer on day and the dose and schedule of aptamer–siRNA treatment have not P < 0.0001 Figure 4 Comparison of PSMA aptamer–Smg1siRNA treatment to vaccination with GM-CSF P = 0.0442 expressing irradiated tumour cells. C57BL/6mice were injected intravenously with B16/F10 P = 0.0012 tumour cells and treated with PSMAaptamer–siRNA conjugates starting at day 5 as described in Fig. 2b, or vaccinated with GM-CSF- expressing irradiated B16/F10 tumour cells PSMA-Smg1 (D5) (GVAX) starting at days (D) 1 or 5 using the protocol described previously29. n 5 1.
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NATURE Vol 465 13 May 2010 been optimized, these results indicate that tumour-targeted siRNA- ¨hlemann, O., Eberle, A. B., Stalder, L. & Zamudio Orozco, R. Recognition and mediated NMD inhibition is more effective than a commonly used elimination of nonsense mRNA. Biochim. Biophys. Acta 1779, 538–549 (2008).
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tion protocols—NMD inhibition generates new antigenic determi- Nonsense surveillance regulates expression of diverse classes of mammalian nants in situ in the disseminated tumour lesions. It should be noted transcripts and mutes genomic noise. Nature Genet. 36, 1073–1078 (2004).
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Supplementary Information is linked to the online version of the paper at Received 21 October 2009; accepted 2 March 2010.
Acknowledgements We thank J. Zhang for assistance in the mouse studies, A.-M.
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Jegg for technical assistance in characterizing Smg1 siRNAs, J. Rossi for advising in Novellino, L., Castelli, C. & Parmiani, G. A listing of human tumor antigens the design of aptamer–siRNA conjugates, and S. Nair and D. Boczkowski for advice in recognized by T cells. Cancer Immunol. Immunother. 54, 187–207 (2005).
performing T-cell assays. This work was supported by the Dodson foundation and Schietinger, A., Philip, M. & Schreiber, H. Specificity in cancer immunotherapy.
the Sylvester Comprehensive Cancer Center (Medical School, University of Miami).
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Author Contributions F.P. suggested the approach and was responsible for Melief, C. J. Cancer immunotherapy by dendritic cells. Immunity 29, 372–383 (2008).
designing the aptamer–siRNA conjugates and interpreting the results, D.K. was Pardoll, D. M. Spinning molecular immunology into successful immunotherapy.
responsible for the mouse studies, P.H.G. helped design the aptamer–siRNA Nature Rev. Immunol. 2, 227–238 (2002).
conjugates, and E.G. oversaw experimental design, data analysis, and wrote the Frischmeyer, P. A. & Dietz, H. C. Nonsense-mediated mRNA decay in health and disease. Hum. Mol. Genet. 8, 1893–1900 (1999).
Behm-Ansmant, I. et al. mRNA quality control: an ancient machinery recognizes Author Information Reprints and permissions information is available at and degrades mRNAs with nonsense codons. FEBS Lett. 581, 2845–2853 (2007).
The authors declare no competing financial interests.
Maquat, L. E. Nonsense-mediated mRNA decay: splicing, translation and mRNP Correspondence and requests for materials should be addressed to E.G.
dynamics. Nature Rev. Mol. Cell Biol. 5, 89–99 (2004).
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PSMA aptamer–siRNA conjugates. The PSMA aptamer, 59-GGGAGG CTGAGGAGAAG-39 and reverse primer 59-GGGTGTTGGCGGGTGTC-39, cloned in the pcDNA3.1 plasmid (Invitrogen) and used to transfect parental CGGCAGACGACUCGCCCGA-39 was cloned into pUC57 between KpnI and pTIG-U6tetOshRNA transduced B16/F10 tumour cells.
and BamHI restriction sites. siRNAs were screened using the psiCHECK RT–PCR. RNA was isolated using RNAsy columns (Qiagen) from cells grown in system (Promega) from candidates generated by the HPCdispatcher and the presence or absence of 1 mg ml21 doxycycline (Sigma) for 5 days were OpenBiosystem algorithms. The DNA template for the aptamer–siRNA guide reverse-transcribed and PCR-amplified using the following primers. CT26 and strand was generated by PCR amplification using forward primer 59- pTIG-U6tetOshRNA transduced CT26 tumour cells: actin: forward, 59- TAATACGACTCACTATAGGGAGGACGATGCGG-39 and reverse primers GC-39. Smg1: forward, 59-GCCCATCGTGTTTGCTTTGG-39; reverse, 59- TCTCGTTCCCAGTGGTGTTACAG-39. Upf2: forward, 59-ACCCGGGGCUA AUGUUGAC-39; reverse, 59-CUUGGUAAUGUUAGGCGUUUUCUC-39. BG, for Smg1 siRNA. The PCR products were purified using the QIAprep Spin col- BGPTC and OVA-BGPTC transduced cells: b-globin: forward, 59-ACCACC umns (Qiagen) RNA was transcribed using the T7(Y639F) polymerase and hybri- dized to the corresponding passenger strands (control siRNA sequence: 59- Transfection of cells with aptamer–siRNA conjugates. CT26 and PSMA-CT26 AAUUCUCCGAACGUGUCACdTdT-39; Upf2 siRNA sequence: 59-GCGUUA tumour cells were incubated with 400 nM siRNA or PSMA aptamer–siRNA UGUUUGGUGGAAGdTdT-39; Smg1 siRNA sequence: 59-GCCAUGACUAA conjugate in the presence of absence of Lipofectamine 2000 (Invitrogen) for 2 days and analysed for RNA expression or NMD inhibition.
Derivation of PSMA-expressing CT26 tumour cell lines. The PSMA comple- Tumour infiltration of OT-1 and Pmel-1 T cells. C57BL/6 mice (CD45.2; mentary DNA, provided by V. Ponomarev, was PCR-amplified using forward Thy1.2) were implanted subcutaneously with 5 3 104 B16 tumour cells and 8 days after tumour inoculation 5 3 106 peptide-activated OT-I (CD45.1) or Pmel-1 CD81 T cells were injected intravenously via the tail vein. At the same ACTTCACTC-39, and cloned into the SalI and Not1 restriction sites of the retro- day the drinking water was supplemented with 10% sucrose (Sigma) and with or viral vector pBMN (Addgene). Plasmid was transiently transfected into the without 2 mg ml21 doxycycline (Sigma). At day 14 after tumour implantation Phoenix-AMPHO 293 packaging cell lines and viral supernatant was used to mice were euthanized, tumours removed and mechanically disaggregated by transduce CT26 colon carcinoma (H-2d) and B16/F10 melanoma (H-2b) tumour collagenase treatment (400 U ml21). Cells were ficolled and stained with cell lines. PSMA-expressing cells were isolated by cell sorting using PSMA-PE- FITC-labelled anti-CD45.1 antibody and allophycocyanin (APC)-labelled anti- labelled anti-PSMA antibody from MBL.
CD8 antibody for OT-1 T cells or with phycoerythrin (PE)-labelled anti-Thy1.1 Confocal microscopy. The passenger strand of the siRNAs was labelled with Cy3 antibody and APC-labelled anti-CD8 antibody for Pmel-1 T cells and analysed before hybridization to the PSMA-aptamer guide strand using the Silencer RNA by flow cytometry. All antibodies used were from BD Bioscience.
labelling kit (Ambion). Tumour cells were plated on glass plates, washed with Tumour homing or 32P-labelled atpamer–siRNA conjugates. The PSMA apta- PBS and incubated with 40 nM of Cy3-labelled aptamer–siRNA or with mer was transcribed in vitro in the presence of 1/1,000 parts of a32P-ATP 10 mg ml21 anti-PSMA antibody (MBL) and Alexa Fluor 488 goat anti-mouse (3000 Ci mmol21) (PerkinElmer) and annealed to Smg1 siRNA as described IgG (Molecular Probes). Coverslips were mounted with Prolong Gold-DAPI earlier. Balb/c mice were co-implanted with CT26 and PSMA-CT26 tumour cells (Molecular Probes).
in the opposite flanks, and 15 days later injected via the tail vein with Generation of stably transduced shRNA-expressing CT26 and B16/F10 5 3 105 c.p.m. 32P-labelled aptamer–siRNA. After aptamer–siRNA injection, tumour cell lines. Double-stranded oligonucleotides corresponding to the guide tumours were surgically removed, cells dispersed by incubation with and passenger strands of Smg1, Upf2 or control siRNA modified to contain 400 U ml21 of collagenase, washed three times with PBS, and cell-associated32 overhangs compatible with BglII and KpnI restriction sites were cloned into P was measured in a scintillation counter.
the BglII and KpnI sites of pFRT-U6tetO plasmid30. The U6tetO-shRNA cas- Tumour immunotherapy studies. Three-hundred-thousand parental or pTIG- settes from the pFRT plasmids were isolated by PCR (forward primer: 59- U6tetOshRNA transduced CT26 tumour cells were implanted subcutaneously in Balb/c or Nude mice. At the day of tumour implantation mice started receiving 59-GTTAAGCATGCCCACACTGGACTAGTGGATC-39) and cloned into the water supplemented with 10% sucrose with or without 2 mg ml21 doxycycline NotI/SphI restriction sites of PTIG lentiviral vector to generate pTIG- U6tetOshRNA plasmids30. pTIG-U6tetOshRNA DNA was cotransfected into To evaluate the anti-tumour effects of PSMA aptamer–siRNAs, mice were 293T cells with lentiviral packaging plasmids pCHPG-2, pCMV-rev and implanted with 1 3 106 PSMA-CT26 tumour cells and injected with 400 pmoles PCMV-gag and lentivirus-containing supernatant was collected and concen- of aptamer–siRNA in 100 ml PBS via the tail vein at days 3, 5, 7, 9, 11 and 13. In trated by centrifugation31. CT26 colon carcinoma (H-2d) and B16/F10 mela- combination therapy, treatment with PSMA aptamer–siRNA was administered noma (H-2b) tumour cell lines were infected with lentiviral vectors and stably at days 5, 7, 9, 11 and 13, and a single dose of 500 pmoles of 4-1BB aptamer dimer transduced GFP-expressing cells were isolated by sorting.
was administered on day 6.
shRNA oliognuclotides used were as follows. Control shRNAs: 59- To monitor metastasis, C57BL/6 mice were implanted with 105 B16-PSMA transduced cells via the tail vein and injected with 400 pmoles of aptamer–siRNA conjugates at days 5, 8, 11, 14 and 17. When about half of the mice in the control AGGAAGTGACACGTTCGGAGAATT-39. Upf2 shRNA: 59-GATCGCGTTATG groups had shown signs of morbidity (approximately days 25–28), the mice were euthanized and their lungs were weighed. GM-CSF-expressing B16/F10 tumour cells, provided by G. Dranoff, were irradiated (50 Gy) and 5 3 105 cells were TCCACCAAACATAACGC-39. Smg1 shRNA: 59-GATCGCCACCAAAGACA injected subcutaneously at days 1, 4 and 7, or days 5, 8 and 11, as described For statistical analysis P values were calculated using a Student's t-test.
30. Aagaard, L. et al. A facile lentiviral vector system for expression of doxycycline- CT26 and B16/F10 tumour cell lines containing BG, BGPTC and OVA-BGPTC.
inducible shRNAs: knockdown of the pre-miRNA processing enzyme Drosha.
The SIINFEKL peptide was cloned into the first exon of the b-globin gene Mol. Ther. 15, 938–945 (2007).
between second (valine) and third (histidine) amino-terminal amino acids of 31. Li, M. J. & Rossi, J. J. Lentiviral vector delivery of recombinant small interfering the BG and BGPTC plasmids3, provided by L. Maquat, by PCR using the forward RNA expression cassettes. Methods Enzymol. 392, 218–226 (2005).
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