Preclinical Justification of pbi-shRNA EWS/FLI1 Lipoplex (LPX) Treatment for Ewing's Sarcoma
Donald D Rao, Christopher Jay, Zhaohui Wang, Xiuquan Luo, Padmasini Kumar, Hilary Eysenbach, Maurizio Ghisoli, Neil Senzer, John Nemunaitis, Donald D Rao, Christopher Jay, Zhaohui Wang, Xiuquan Luo, Padmasini Kumar, Hilary Eysenbach, Maurizio Ghisoli, Neil Senzer, John Nemunaitis
Abstract
The EWS/FLI1 fusion gene is well characterized as a driver of Ewing's sarcoma. Bi-shRNA EWS/FLI1 is a functional plasmid DNA construct that transcribes both siRNA and miRNA-like effectors each of which targets the identical type 1 translocation junction region of the EWS/FLI1 transcribed mRNA sequence. Previous preclinical and clinical studies confirm the safety of this RNA interference platform technology and consistently demonstrate designated mRNA and protein target knockdown at greater than 90% efficiency. We initiated development of pbi-shRNA EWS/FLI1 lipoplex (LPX) for the treatment of type 1 Ewing's sarcoma. Clinical-grade plasmid was manufactured and both sequence and activity verified. Target protein and RNA knockdown of 85-92% was demonstrated in vitro in type 1 human Ewing's sarcoma tumor cell lines with the optimal bi-shRNA EWS/FLI1 plasmid. This functional plasmid was placed in a clinically tested, liposomal (LP) delivery vehicle followed by in vivo verification of activity. Type 1 Ewing's sarcoma xenograft modeling confirmed dose related safety and tumor response to pbi-shRNA EWS/FLI1 LPX. Toxicology studies in mini-pigs with doses comparable to the demonstrated in vivo efficacy dose resulted in transient fever, occasional limited hypertension at low- and high-dose assessment and transient liver enzyme elevation at high dose. These results provide the justification to initiate clinical testing.
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References
- Leavey, PJ and Collier, AB (2008). Ewing sarcoma: prognostic criteria, outcomes and future treatment. Expert Rev Anticancer Ther 8: 617–624.
- Esiashvili, N, Goodman, M and Marcus, RB Jr (2008). Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: Surveillance Epidemiology and End Results data. J Pediatr Hematol Oncol 30: 425–430.
- Gamberi, G, Cocchi, S, Benini, S, Magagnoli, G, Morandi, L, Kreshak, J et al. (2011). Molecular diagnosis in Ewing family tumors: the Rizzoli experience–222 consecutive cases in four years. J Mol Diagn 13: 313–324.
- Arvand, A and Denny, CT (2001). Biology of EWS/ETS fusions in Ewing's family tumors. Oncogene 20: 5747–5754.
- Tirode, F, Laud-Duval, K, Prieur, A, Delorme, B, Charbord, P and Delattre, O (2007). Mesenchymal stem cell features of Ewing tumors. Cancer Cell 11: 421–429.
- Riggi, N, Suvà, ML, Suvà, D, Cironi, L, Provero, P, Tercier, S et al. (2008). EWS-FLI-1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells. Cancer Res 68: 2176–2185.
- Meltzer, PS (2007). Is Ewing's sarcoma a stem cell tumor? Cell Stem Cell 1: 13–15.
- Kovar, H, Ban, J and Pospisilova, S (2003). Potentials for RNAi in sarcoma research and therapy: Ewing's sarcoma as a model. Semin Cancer Biol 13: 275–281.
- Oikawa, T (2004). ETS transcription factors: possible targets for cancer therapy. Cancer Sci 95: 626–633.
- Toretsky, JA, Connell, Y, Neckers, L and Bhat, NK (1997). Inhibition of EWS-FLI-1 fusion protein with antisense oligodeoxynucleotides. J Neurooncol 31: 9–16.
- Tanaka, K, Iwakuma, T, Harimaya, K, Sato, H and Iwamoto, Y (1997). EWS-Fli1 antisense oligodeoxynucleotide inhibits proliferation of human Ewing's sarcoma and primitive neuroectodermal tumor cells. J Clin Invest 99: 239–247.
- Mateo-Lozano, S, Gokhale, PC, Soldatenkov, VA, Dritschilo, A, Tirado, OM and Notario, V (2006). Combined transcriptional and translational targeting of EWS/FLI-1 in Ewing's sarcoma. Clin Cancer Res 12: 6781–6790.
- Dohjima, T, Lee, NS, Li, H, Ohno, T and Rossi, JJ (2003). Small interfering RNAs expressed from a Pol III promoter suppress the EWS/Fli-1 transcript in an Ewing sarcoma cell line. Mol Ther 7: 811–816.
- Prieur, A, Tirode, F, Cohen, P and Delattre, O (2004). EWS/FLI-1 silencing and gene profiling of Ewing cells reveal downstream oncogenic pathways and a crucial role for repression of insulin-like growth factor binding protein 3. Mol Cell Biol 24: 7275–7283.
- Nozawa, S, Ohno, T, Banno, Y, Dohjima, T, Wakahara, K, Fan, DG et al. (2005). Inhibition of platelet-derived growth factor-induced cell growth signaling by a short interfering RNA for EWS-Fli1 via down-regulation of phospholipase D2 in Ewing sarcoma cells. J Biol Chem 280: 27544–27551.
- Kinsey, M, Smith, R and Lessnick, SL (2006). NR0B1 is required for the oncogenic phenotype mediated by EWS/FLI in Ewing's sarcoma. Mol Cancer Res 4: 851–859.
- Herrero-Martín, D, Osuna, D, Ordóñez, JL, Sevillano, V, Martins, AS, Mackintosh, C et al. (2009). Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target. Br J Cancer 101: 80–90.
- Hu-Lieskovan, S, Heidel, JD, Bartlett, DW, Davis, ME and Triche, TJ (2005). Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma. Cancer Res 65: 8984–8992.
- Toub, N, Bertrand, JR, Tamaddon, A, Elhamess, H, Hillaireau, H, Maksimenko, A et al. (2006). Efficacy of siRNA nanocapsules targeted against the EWS-Fli1 oncogene in Ewing sarcoma. Pharm Res 23: 892–900.
- Ramon, AL, Bertrand, JR, de Martimprey, H, Bernard, G, Ponchel, G, Malvy, C et al. (2013). siRNA associated with immunonanoparticles directed against cd99 antigen improves gene expression inhibition in vivo in Ewing's sarcoma. J Mol Recognit 26: 318–329.
- Stahl, M, Ranft, A, Paulussen, M, Bölling, T, Vieth, V, Bielack, S et al. (2011). Risk of recurrence and survival after relapse in patients with Ewing sarcoma. Pediatr Blood Cancer 57: 549–553.
- Ozaki, T, Hillmann, A, Hoffmann, C, Rübe, C, Blasius, S, Dunst, J et al. (1996). Significance of surgical margin on the prognosis of patients with Ewing's sarcoma. A report from the Cooperative Ewing's Sarcoma Study. Cancer 78: 892–900.
- Bacci, G, Picci, P, Ferrari, S, Mercuri, M, Brach del Prever, A, Rosito, P et al. (1998). Neoadjuvant chemotherapy for Ewing's sarcoma of bone: no benefit observed after adding ifosfamide and etoposide to vincristine, actinomycin, cyclophosphamide, and doxorubicin in the maintenance phase–results of two sequential studies. Cancer 82: 1174–1183.
- Klingebiel, T, Pertl, U, Hess, CF, Jürgens, H, Koscielniak, E, Pötter, R et al. (1998). Treatment of children with relapsed soft tissue sarcoma: report of the German CESS/CWS REZ 91 trial. Med Pediatr Oncol 30: 269–275.
- Rodriguez-Galindo, C, Billups, CA, Kun, LE, Rao, BN, Pratt, CB, Merchant, TE et al. (2002). Survival after recurrence of Ewing tumors: the St Jude Children's Research Hospital experience, 1979-1999. Cancer 94: 561–569.
- Shankar, AG, Ashley, S, Craft, AW and Pinkerton, CR (2003). Outcome after relapse in an unselected cohort of children and adolescents with Ewing sarcoma. Med Pediatr Oncol 40: 141–147.
- Barker, LM, Pendergrass, TW, Sanders, JE and Hawkins, DS (2005). Survival after recurrence of Ewing's sarcoma family of tumors. J Clin Oncol 23: 4354–4362.
- McTiernan, AM, Cassoni, AM, Driver, D, Michelagnoli, MP, Kilby, AM and Whelan, JS (2006). Improving Outcomes After Relapse in Ewing's Sarcoma: Analysis of 114 Patients From a Single Institution. Sarcoma 2006: 83548.
- Bacci, G, Ferrari, S, Longhi, A, Donati, D, De Paolis, M, Forni, C et al. (2003). Therapy and survival after recurrence of Ewing's tumors: the Rizzoli experience in 195 patients treated with adjuvant and neoadjuvant chemotherapy from 1979 to 1997. Ann Oncol 14: 1654–1659.
- Rasper, M, Jabar, S, Ranft, A, Jürgens, H, Amler, S and Dirksen, U (2014). The value of high-dose chemotherapy in patients with first relapsed Ewing sarcoma. Pediatr Blood Cancer 61: 1382–1386.
- Navid, F, Billups, C, Liu, T, Krasin, MJ and Rodriguez-Galindo, C (2008). Second cancers in patients with the Ewing sarcoma family of tumours. Eur J Cancer 44: 983–991.
- Merchant, TE, Kushner, BH, Sheldon, JM, LaQuaglia, M and Healey, JH (1999). Effect of low-dose radiation therapy when combined with surgical resection for Ewing sarcoma. Med Pediatr Oncol 33: 65–70.
- Saylors, RL 3rd, Stine, KC, Sullivan, J, Kepner, JL, Wall, DA, Bernstein, ML et al.; Pediatric Oncology Group. (2001). Cyclophosphamide plus topotecan in children with recurrent or refractory solid tumors: a Pediatric Oncology Group phase II study. J Clin Oncol 19: 3463–3469.
- Wagner, LM, McAllister, N, Goldsby, RE, Rausen, AR, McNall-Knapp, RY, McCarville, MB et al. (2007). Temozolomide and intravenous irinotecan for treatment of advanced Ewing sarcoma. Pediatr Blood Cancer 48: 132–139.
- Wagner, LM, Crews, KR, Iacono, LC, Houghton, PJ, Fuller, CE, McCarville, MB et al. (2004). Phase I trial of temozolomide and protracted irinotecan in pediatric patients with refractory solid tumors. Clin Cancer Res 10: 840–848.
- Wagner, LM (2010). Oral irinotecan for treatment of pediatric solid tumors: ready for prime time? Pediatr Blood Cancer 54: 661–662.
- Wagner, LM, Perentesis, JP, Reid, JM, Ames, MM, Safgren, SL, Nelson, MD Jr et al. (2010). Phase I trial of two schedules of vincristine, oral irinotecan, and temozolomide (VOIT) for children with relapsed or refractory solid tumors: a Children's Oncology Group phase I consortium study. Pediatr Blood Cancer 54: 538–545.
- Wagner, L, Turpin, B, Nagarajan, R, Weiss, B, Cripe, T and Geller, J (2013). Pilot study of vincristine, oral irinotecan, and temozolomide (VOIT regimen) combined with bevacizumab in pediatric patients with recurrent solid tumors or brain tumors. Pediatr Blood Cancer 60: 1447–1451.
- Casey, DA, Wexler, LH, Merchant, MS, Chou, AJ, Merola, PR, Price, AP et al. (2009). Irinotecan and temozolomide for Ewing sarcoma: the Memorial Sloan-Kettering experience. Pediatr Blood Cancer 53: 1029–1034.
- Raciborska, A, Bilska, K, Drabko, K, Chaber, R, Pogorzala, M, Wyrobek, E et al. (2013). Vincristine, irinotecan, and temozolomide in patients with relapsed and refractory Ewing sarcoma. Pediatr Blood Cancer 60: 1621–1625.
- McGregor, LM, Stewart, CF, Crews, KR, Tagen, M, Wozniak, A, Wu, J et al. (2012). Dose escalation of intravenous irinotecan using oral cefpodoxime: a phase I study in pediatric patients with refractory solid tumors. Pediatr Blood Cancer 58: 372–379.
- Navid, F, Willert, JR, McCarville, MB, Furman, W, Watkins, A, Roberts, W et al. (2008). Combination of gemcitabine and docetaxel in the treatment of children and young adults with refractory bone sarcoma. Cancer 113: 419–425.
- Mora, J, Cruz, CO, Parareda, A and de Torres, C (2009). Treatment of relapsed/refractory pediatric sarcomas with gemcitabine and docetaxel. J Pediatr Hematol Oncol 31: 723–729.
- Rapkin, L, Qayed, M, Brill, P, Martin, M, Clark, D, George, BA et al. (2012). Gemcitabine and docetaxel (GEMDOX) for the treatment of relapsed and refractory pediatric sarcomas. Pediatr Blood Cancer 59: 854–858.
- Rao, DD, Maples, PB, Senzer, N, Kumar, P, Wang, Z, Pappen, BO et al. (2010). Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference. Cancer Gene Ther 17: 780–791.
- Pizzorno, MC, O'Hare, P, Sha, L, LaFemina, RL and Hayward, GS (1988). trans-activation and autoregulation of gene expression by the immediate-early region 2 gene products of human cytomegalovirus. J Virol 62: 1167–1179.
- Zeng, Y, Cai, X and Cullen, BR (2005). Use of RNA polymerase II to transcribe artificial microRNAs. Methods Enzymol 392: 371–380.
- Matranga, C, Tomari, Y, Shin, C, Bartel, DP and Zamore, PD (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123: 607–620.
- Lu, C, Stewart, DJ, Lee, JJ, Ji, L, Ramesh, R, Jayachandran, G et al. (2012). Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans. PLoS One 7: e34833.
- Bernstein, M, Kovar, H, Paulussen, M, Randall, RL, Schuck, A, Teot, LA et al. (2006). Ewing's sarcoma family of tumors: current management. Oncologist 11: 503–519.
- Rocchi, A, Manara, MC, Sciandra, M, Zambelli, D, Nardi, F, Nicoletti, G et al. (2010). CD99 inhibits neural differentiation of human Ewing sarcoma cells and thereby contributes to oncogenesis. J Clin Invest 120: 668–680.
- Zhang, PJ, Barcos, M, Stewart, CC, Block, AW, Sait, S and Brooks, JJ (2000). Immunoreactivity of MIC2 (CD99) in acute myelogenous leukemia and related diseases. Mod Pathol 13: 452–458.
- Waheed Roomi, M, Kalinovsky, T, Roomi, NW, Niedzwiecki, A and Rath, M (2013). Inhibition of the SK-N-MC human neuroblastoma cell line in vivo and in vitro by a novel nutrient mixture. Oncol Rep 29: 1714–1720.
- Franzetti, GA, Laud-Duval, K, Bellanger, D, Stern, MH, Sastre-Garau, X and Delattre, O (2013). MiR-30a-5p connects EWS-FLI1 and CD99, two major therapeutic targets in Ewing tumor. Oncogene 32: 3915–3921.
- Barve, M, Wang, Z, Kumar, P, Jay, CM, Luo, X, Bedell, C et al. (2015). Phase 1 trial of Bi-shRNA STMN1 BIV in refractory cancer. Mol Ther 23: 1123–1130.
- Senzer, N, Barve, M, Kuhn, J, Melnyk, A, Beitsch, P, Lazar, M et al. (2012). Phase I trial of “bi-shRNAi(furin)/GMCSF DNA/autologous tumor cell” vaccine (FANG) in advanced cancer. Mol Ther 20: 679–686.
- Senzer, N, Nemunaitis, J, Barve, M, Orr, D, Kuhn, J, Magee, M et al. (2013) Long term follow up: phase I trial of “bi-shRNA furin/GMCSF DNA/Autologous Tumor Cell” immunotherapy (FANG™) in advanced cancer J Vaccines and Vaccination 4:209.
- Rao, DD, Senzer, N, Cleary, MA and Nemunaitis, J (2009). Comparative assessment of siRNA and shRNA off target effects: what is slowing clinical development. Cancer Gene Ther 16: 807–809.
- Liu, SH, Rao, DD, Nemunaitis, J, Senzer, N, Zhou, G, Dawson, D et al. (2012). PDX-1 is a therapeutic target for pancreatic cancer, insulinoma and islet neoplasia using a novel RNA interference platform. PLoS One 7: e40452.
- Ghisoli, M, Barve, M, Schneider, R, Mennel, R, Lenarsky, C, Wallraven, G et al. (2015). Pilot Trial of FANG Immunotherapy in Ewing's Sarcoma. Mol Ther 23: 1103–1109.
- Nemunaitis, J, Barve, M, Orr, D, Kuhn, J, Magee, M, Lamont, J et al. (2014). Summary of bi-shRNA/GM-CSF augmented autologous tumor cell immunotherapy (FANG™) in advanced cancer of the liver. Oncology 87: 21–29.
- Phadke, AP, Jay, CM, Wang, Z, Chen, S, Liu, S, Haddock, C et al. (2011). In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein. DNA Cell Biol 30: 715–726.
- Templeton, NS, Lasic, DD, Frederik, PM, Strey, HH, Roberts, DD and Pavlakis, GN (1997). Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat Biotechnol 15: 647–652.
- Templeton, NS, Kumar, P, Sanchez,R, Templeton, N, Senzer, N Maples, P et al. Non-viral vectors for the treatment of disease. in Keystone Symposia on Molecular and Cellular Biology of Gene Therapy. Salt Lake City, Utah, 1999.
- Jay, CM, Ruoff, C, Kumar, P, Maass, H, Spanhel, B, Miller, M et al. (2013). Assessment of intravenous pbi-shRNA PDX1 nanoparticle (OFHIRNA-PDX1) in yucatan swine. Cancer Gene Ther 20: 683–689.
- Jay, C, Nemunaitis, G, Nemunaitis, J, Senzer, N, Hinderlich, S, Darvish, D et al. (2008). Preclinical assessment of wt GNE gene plasmid for management of hereditary inclusion body myopathy 2 (HIBM2). Gene Regul Syst Bio 2: 243–252.
- Wu, J, Liu, S, Yu, J, Zhou, G, Rao, D, Jay, CM et al. (2014). Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform. Cancer Gene Ther 21: 48–53.
- Liu, SH, Zhou, G, Yu, J, Wu, J, Nemunaitis, J, Senzer, N et al. (2013). Notch1 activation up-regulates pancreatic and duodenal homeobox-1. Genes (Basel) 4: 358–374.
- Ramesh, R, Saeki, T, Templeton, NS, Ji, L, Stephens, LC, Ito, I et al. (2001). Successful treatment of primary and disseminated human lung cancers by systemic delivery of tumor suppressor genes using an improved liposome vector. Mol Ther 3: 337–350.
- Judge, AD, Sood, V, Shaw, JR, Fang, D, McClintock, K and MacLachlan, I (2005). Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 23: 457–462.
- Robbins, M, Judge, A and MacLachlan, I (2009). siRNA and innate immunity. Oligonucleotides 19: 89–102.
- Senzer, N, Nemunaitis, J, Nemunaitis, D, Bedell, C, Edelman, G, Barve, M et al. (2013). Phase I study of a systemically delivered p53 nanoparticle in advanced solid tumors. Mol Ther 21: 1096–1103.
- Nemunaitis, G, Jay, CM, Maples, PB, Gahl, WA, Huizing, M, Yardeni, T et al. (2011). Hereditary inclusion body myopathy: single patient response to intravenous dosing of GNE gene lipoplex. Hum Gene Ther 22: 1331–1341.
- Nemunaitis, G, Maples, PB, Jay, C, Gahl, WA, Huizing, M, Poling, J et al. (2010). Hereditary inclusion body myopathy: single patient response to GNE gene Lipoplex therapy. J Gene Med 12: 403–412.
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