Targeted Cellular Delivery of CRISPR/Cas9 with RNA Aptamer-Cationic Liposome As A Potential Therapy For Metastatic Prostate Cancer

Main Article Content

Kelvin Theandro Gotama
Alessa Fahira
Bashar Adi Wahyu Pandhita


Prostate cancer is the sixth deadliest cancer worldwide due to its metastatic ability, rendering conventional treatment futile. Editing of target oncogenes with the help of CRISPR/Cas9 is gaining recognition as promising treatment modality. However, implementation of CRISPR/Cas9 in clinical setting remains difficult due to challenges in delivery to target tissue. Therefore, this literature review focuses on exploring nanocarrier suitable for CRISPR/Cas9 delivery. The method used to assemble this literature review is by performing a comprehensive literature search with corresponding keywords. Results shows that targeted delivery system of CRISPR/Cas9 using cationic liposome conjugated with RNA aptamer is an excellent candidate. Modification of cationic liposome, a well-established nanocarrier, with PEG and RNA aptamer as targeting ligand increases specificity, efficiency, stability, and circulation time in bloodstream. However, further research is still needed to confirm these findings for future implementation of this technology as prostate cancer therapy.

Keywords: Aptamer, Cancer, CRISPR/Cas9, Liposome, Prostate.

Article Details

How to Cite
Gotama, K. T., Fahira, A. and Pandhita, B. A. W. (2019) “Targeted Cellular Delivery of CRISPR/Cas9 with RNA Aptamer-Cationic Liposome As A Potential Therapy For Metastatic Prostate Cancer”, Journal of Asian Medical Students’ Association. Kuala Lumpur, Malaysia, 7(1). Available at: (Accessed: 18April2021).
Original Papers


Ito K. Prostate cancer in Asian men. Nat Rev Urol. 2014 March 4; 11: 197-212.

Stephenson AJ. Epidemiology, etiology, and prevention of prostate cancer. In: Wein AJ, Kavoussi LR, Partin AW, Peters CA. Campbell-Walsh urology. 11th ed. 2016; Canada: Saunders Elsevier. p.2543-2564.

Nelson WG, Carter HB, DeWeese TL, Antonarakis ES, Eisenberger MA. Prostate Cancer. In: Niederhuber JE, Armitage JO, Doroshow JH, Kastan MB, Tepper JE. Abeloff’s clinical oncology. 5th ed. 2014; China: Churchill Livingstone. p.1463-1496.

Flint PW, Cummings CW, editors. Cummings otolaryngology: head and neck surgery. 6th ed. Philadelphia, Pa: Elsevier, Saunders; 2015. Chapter 73, Fundamentals of molecular biology and gene therapy; p.1053-69

Hart T, et al. High-resolution CRISPR screens reveal fitness genes and genotype-specific cancer liabilities. Cell. 2015 Dec 3;163(6):1515-26.

Munoz DM, et al. CRISPR screens provide a comprehensive assessment of cancer vulnerabilities but generate false-positive hits for highly amplified genomic regions. Cancer discovery. 2016 Aug 1;6(8):900-13.

Zhen S, Hua L, Takahashi Y, Narita S, Liu YH, Li Y. In vitro and in vivo growth suppression of human papillomavirus 16-positive cervical cancer cells by CRISPR/Cas9. Biochem Biophys Res Commun. 2014; 450:1422–6.

Zhen S, et al. Harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR- associated Cas9system to disrupt the hepatitis B virus. Gene Ther. 2015; 22:404–12.

Kawamura N, Nimura K, Nagano H, Yamaguchi S, Nonomura N, Kaneda Y. CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 2015 Sep 8;6(26):22361.

Ye R, Pi M, Cox JV, Nishimoto SK, Quarles LD. CRISPR/Cas9 targeting of GPRC6A suppresses prostate cancer tumorigenesis in a human xenograft model. Journal of Experimental & Clinical Cancer Research. 2017 Jun 28;36(1):90.

Zhen S, Takahashi Y, Narita S, Yang YC, Li X. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome. Oncotarget. 2017 Feb 7;8(6):9375.

Li L, He ZY, Wei XW, Gao GP, Wei YQ. Challenges in CRISPR/CAS9 delivery: potential roles of nonviral vectors. Human gene therapy. 2015 Jun 2;26(7):452-62.

Epstein JI, Lotan TT. The lower urinary tract and male genital system. In: Kumar V, Abbas AK, Aster JC. Robbins and Cotran pathologic basis of disease. 9th ed. 2015; Canada: Saunders Elsevier. p.959-90.

Schelling LA, Williamson SR, Zhang S, Yao JL, Wang M, Huang J, et al. Frequent TMPRSS2-ERG rearrangement in prostatic small cell carcinoma detected by fluorescence in situ hybridization: the superiority of fluorescence in situ hybridization over ERG immunohistochemistry. Human Pathol. 2013; 44(10): 2227-33.

Gonzalgo ML, Sfanos KS, Meeker AK. Molecular genetics and cancer biology. In: Wein AJ, Kavoussi LR, Partin AW, Peters CA. Campbell-Welsh urology. 11th ed. 2016; Philadelphia: Saunders Elsevier. p.459-81.

Biagioni A, Chillà A, Andreucci E, Laurenzana A, Margheri F, Peppicelli S et al. Type II CRISPR/Cas9 approach in the oncological therapy. Journal of Experimental & Clinical Cancer Research. 2017;36(1).

Redman M, King A, Watson C, King D. What is CRISPR/Cas9?. Archives of disease in childhood - Education & practice edition. 2016;101(4):213-215.

Amitai G, Sorek R. CRISPR–Cas adaptation: insights into the mechanism of action. Nature Reviews Microbiology. 2016;14(2):67-76.

Hsu P, Lander E, Zhang F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell. 2014;157(6):1262-1278.

Brouns S, Jore M, Lundgren M, Westra E, Slijkhuis R, Snijders A et al. Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science. 2008;321(5891):960-964.

Rouillon C, Zhou M, Zhang J, Politis A, Beilsten-Edmands V, Cannone G et al. Structure of the CRISPR Interference Complex CSM Reveals Key Similarities with Cascade. Molecular Cell. 2013;52(1):124-134.

Spilman M, Cocozaki A, Hale C, Shao Y, Ramia N, Terns R et al. Structure of an RNA Silencing Complex of the CRISPR-Cas Immune System. Molecular Cell. 2013;52(1):146-152.

Shmakov S, Abudayyeh O, Makarova K, Wolf Y, Gootenberg J, Semenova E et al. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Molecular Cell. 2015;60(3):385-397.

Jiang F, Doudna J. CRISPR–Cas9 Structures and Mechanisms. Annual Review of Biophysics. 2017;46(1):505-529.

Vasiliou S, Diamandis E, Church G, Greely H, Baylis F, Thompson C et al. CRISPR-Cas9 System: Opportunities and Concerns. Clinical Chemistry. 2016;62(10):1304-1311.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J, Charpentier E. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. 2012;337(6096):816-821.

Ran F, Hsu P, Wright J, Agarwala V, Scott D, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nature Protocols. 2013;8(11):2281-2308.

Travis J. Breakthrough of the Year: CRISPR makes the cut. Science Magazine. 2015. Available from: http://www.sciencemag. org/news/2015/12/and-science-s-breakthrough-year.

Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

Gaj T, Gersbach C, Barbas C. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology. 2013;31(7):397-405.

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science. 2007;315(5819):1709-1712.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. 2013;339(6121):819-823.

Hsu P, Scott D, Weinstein J, Ran F, Konermann S, Agarwala V et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nature Biotechnology. 2013;31(9):827-832.

Wright A, Nuñez J, Doudna J. Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering. Cell. 2016;164(1-2):29-44.

Barrangou R, Horvath P. A decade of discovery: CRISPR functions and applications. Nature Microbiology. 2017;2:17092.

Vogelstein B, Papadopoulos N, Velculescu VE, et al. Cancer genome landscapes. Science. 2013;339(6127):1546–1558.

Kandoth C, McLellan MD, Vandin F, et al. Mutational landscape and significance across 12 major cancer types. Nature. 2013;502(7471):333–339.

Ahmed Khan F, Pandupuspitasari N, Chun-Jie H, Ao Z, Jamal M, Zohaib A et al. CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases. Oncotarget. 2016;7(32):52541-52552.

Cong L, Ran FA, CoxD, Lin S, Barretto R, etal. 2013. Multiplex genome engineering using CRISPR/Cas9 systems. Science 339(6121):819–23

Silva J, et al. NANOG is the gateway to the pluripotent ground state. Cell. 2009; 138:722–37.

Lee M, Nam EJ, Kim SW, Kim S, Kim JH, Kim YT. Prognostic impact of the cancer stem cell–related marker NANOG in ovarian serous carcinoma. International Journal of Gynecological Cancer. 2012 Nov 1;22(9):1489-96.

Zhang J, et al. NANOG modulates stemness in human colorectal cancer. Oncogene. 2013 Sep 12;32(37):4397.

Han J, et al. RNA interference-mediated silencing of NANOG reduces cell proliferation and induces G0/G1 cell cycle arrest in breast cancer cells. Cancer letters. 2012 Aug 1;321(1):80-8.

Moon JH, et al. NANOG-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells. Biochemical and biophysical research communications. 2011 Aug 19;412(1):175-81.

Jeter CR, et al. NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011 Sep 8;30(36):3833.

Mathieu J, et al. HIF induces human embryonic stem cell markers in cancer cells. Cancer research. 2011 Jul 1;71(13):4640-52.

Zhang J, et al. The human pluripotency gene NANOG/NANOGP8 is expressed in gastric cancer and associated with tumor development. Oncology letters. 2010 May 1;1(3):457-63.

Du Y, Shi L, Wang T, Liu Z, Wang Z. Nanog siRNA plus Cisplatin may enhance the sensitivity of chemotherapy in esophageal cancer. J Cancer Res Clin Oncol. 2012; 138:1759–67.

Pi M, Quarles LD. GPRC6A regulates prostate cancer progression. The Prostate. 2012 Mar 1;72(4):399-409.

Pi M, Quarles LD. Multiligand specificity and wide tissue expression of GPRC6A reveals new endocrine networks. Endocrinology. 2012 Feb 28;153(5):2062-9.

Nimptsch K, Rohrmann S, Nieters A, Linseisen J. Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European prospective investigation into cancer and nutrition. Cancer Epidemiol Prev Biomarkers. 2009;18:49–56.

Pi M, Kapoor K, Wu Y, Ye R, Senogles SE, Nishimoto SK, Hwang DJ, Miller DD, Narayanan R, Smith JC, Baudry J. Structural and functional evidence for testosterone activation of GPRC6A in peripheral tissues. Molecular Endocrinology. 2015 Dec 1;29(12):1759-73.

Hayashi Y, Kawakubo-Yasukochi T, Mizokami A, Takeuchi H, Nakamura S, Hirata M. Differential roles of carboxylated and uncarboxylated osteocalcin in prostate cancer growth. J Cancer. 2016;7:1605.

Li L, He ZY, Wei XW, Gao GP, Wei YQ. Challenges in CRISPR/CAS9 delivery: potential roles of nonviral vectors. Human gene therapy. 2015 Jun 2;26(7):452-62.

Fu Y, Foden JA, Khayter C, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013;31:822–826.

Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science 2013;339:823–826.

Liu Q, Muruve D. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 2003;10:935–940.

Sun J, Anand-Jawa V, Chatterjee S, et al. Immune responses to adeno-associated virus and its recombinant vectors. Gene Ther 2003;10:964–976.

Schiener M, Hossann M, Viola JR, et al. Nanomedicine-based strategies for treatment of atherosclerosis. Trends in molecular medicine. 2014 May 31;20(5):271-81.

Lonez C, Vandenbranden M, Ruysschaert JM. Cationic liposomal lipids: from gene carriers to cell signaling. Prog Lipid Res 2008;47:340–347.

Lakhin AV, Tarantul VZ, Gening LV. Aptamers: problems, solutions and prospects. Acta Naturae (англоязычная версия). 2013;5(4 (19)).

Chercia L, Giangrande PH, McNamara JO, de Franciscis V. Cell-specific aptamers for targeted therapies, Methods Mol boil. 2009; 535:59–78.

Zhou J, Li H, Li S, Zaia J, Rossi JJ. Novel dual inhibitory function aptamer-siRNA delivery system for HIV-1 therapy. Mol Ther. 2008; 16:1481–1489.

Rockey WM, Hernandez FJ, Huang SY, et al. Rational truncation of an RNA aptamer to prostate-specific membrane antigen using computational structural modeling. Nucleic acid therapeutics. 2011 Oct 1;21(5):299-314.

Zhou J, Rossi JJ. Aptamer-targeted RNAi for HIV-1 therapy. Methods Mol Biol. 2011;721:366–371.

Lupold SE, Hicke BJ, Lin Y, Coffey DS. Identi cation and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-speci c membrane antigen, Cancer Res. 2002; 62:4029–4033.

Wullner U, Neef I, Eller A, Kleines M, Tur MK, Barth S. Cell-speci c induction of apoptosis by rationally designed bivalent aptamer-siRNA transcripts silencing eukaryotic elongation factor 2. Curr Cancer Drug Targets. 2008; 8:554–565.

Baek SE, Lee KH, Park YS, et al. RNA aptamer-conjugated liposome as an efficient anticancer drug delivery vehicle targeting cancer cells in vivo. Journal of Controlled Release. 2014 Dec 28;196:234-42.

Troyer JK, Beckett ML, Wright GL. Location of prostate-specific membrane antigen in the LNCaP prostate carcinoma cell line. Prostate. 1997; 30:232–242.

Weiß L, Efferth T. Polo-like kinase 1 as target for cancer therapy. Experimental hematology & oncology. 2012 Dec 10;1(1):38.

Degenhardt Y, Lampkin T. Targeting Polo-like kinase in cancer therapy. Clinical Cancer Research. 2010 Jan 15;16(2):384-9.

Maire V, et al. Polo-like kinase 1: a potential therapeutic option in combination with conventional chemotherapy for the management of patients with triple-negative breast cancer. Cancer research. 2013 Jan 15;73(2):813-23.

Allera-Moreau C, et al. DNA replication stress response involving PLK1, CDC6, POLQ, RAD51 and CLASPIN upregulation prognoses the outcome of early/mid-stage non-small cell lung cancer patients. Oncogenesis. 2012 Oct;1(10):e30.

Rödel F, et al. Polo-like kinase 1 as predictive marker and therapeutic target for radiotherapy in rectal cancer. The American journal of pathology. 2010 Aug 31;177(2):918-29.

Zhang Z, et al. Plk1 inhibition enhances the efficacy of androgen signaling blockade in castration-resistant prostate cancer. Cancer research. 2014 Nov 15;74(22):6635-47.

Steegmaier M, Hoffmann M, Baum A, et al. BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo. Curr Biol 2007;17:316–22.

Nogawa M, Yuasa T, Kimura S, et al. Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer. J Clin Invest 2005;115:978–85.

Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, et al. 2013. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31(9):822–26

Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK. 2014. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32(3):279–84