Sains Malaysiana 51(2)(2022): 567-576

http://doi.org/10.17576/jsm-2022-5102-20

TAT Kappa (TATK): A Novel Cell Penetrating Peptide for Delivery of Pluripotent Proteins into Target Cells

(TAT Kappa (TATK): Sel Baharu Menembusi Peptida untuk Penghantaran Protein Pluripoten kepada Sel Sasaran)

UBASHINI VIJAKUMARAN¹, FAZLINA NORDIN¹*, ZARIYANTEY ABD HAMID², MAHA ABDULLAH³ & GEE JUN TYE⁴

¹Centre for Tissue Engineering and Regenerative Medicine (CTERM), Hospital Canselor Tuanku Mukhriz UKM (HCTM), Faculty of Medicine, Jalan Yaacob Latiff, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Biomedical Science Programme & Centre for Diagnostic, Therapeutic and Investigative Science (CODTIS), Faculty of Health Sciences, Universiti Kebangsaan Malaysia (UKM), Kuala Lumpur Campus

Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Federal Territory, Malaysia

³Immunology Unit, Department of Pathology, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

⁴Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800 USM, Pulau Pinang, Malaysia

Received: 8 March 2021/Accepted: 8 June 2021

ABSTRACT

Induced pluripotent stem cell (iPSC) holds a magnificent place in the medical revolution. Its emergence is expected to instigate development of novel therapies for regenerative medicine and treatment of malignant diseases. Moreover, iPSC usage also resolved a long-time ethical controversy on the usage of the embryo as a pluripotent stem cells source. Since Yamanaka’s iPSC discovery in 2006, several pieces of research have proven that the enforced expression of transcription factors Oct-3/4, KLF4, and Sox2 can induce the reprogramming of previously differentiated cells, to generate iPSC. However, the conventional method using viral vectors leads to genetic modification due to exogene integration and subsequently tumorigenicity, which is unsafe for clinical application. Therefore, our study utilised an improved novel protein transduction domain, trans-activator of transcription kappa (TAT), a synthetic TAT-HIV to deliver these transcription factors gene as an alternative method for iPSC generation via non-viral reprogramming. With this new strategy, we have established a stable clone of 293T cells expressing TATk fusion proteins (TATκ-GFP, TATκ-KLF4, TATκ-Sox2, and TATκ-Oct-3/4) that expresses and secretes their respective cloned reprogramming proteins. These stable clones successfully transduced our target cell (U937) monocyte cell line. TATκ-GFP, a marker protein and fusion proteins TATκ-KLF4, TATκ-Sox2, and TATκ-Oct-3/4 transduced the targeted (U937) monocyte cell line, proving that this novel TATκ possesses an ability to translocate across the cell membrane. Morphological changes were successfully observed in U937 cells after 20 days of transduction, however the presence of bonifide iPSC colonies were unable to be elicited. This might be due to the incomplete reprogramming or insufficient duration of protein transduction to generate iPSC cells.

Keywords: Cell-penetrating peptide; induced pluripotent stem cells; pluripotent proteins; reprogramming; transduction

ABSTRAK

Sel induk pluripoten terjana (iPSC) memainkan peranan penting dalam revolusi perubatan. Kemunculannya dijangka akan membuka ruang untuk pembangunan terapi baharu untuk perubatan regeneratif dan rawatan penyakit kanser malignan. Selain itu, penggunaan iPSC berupaya menyelesaikan kontroversi berhubung penggunaan embrio sebagai sumber sel induk pluripoten. Sejak penemuan iPSC pada tahun 2006 oleh Yamanaka, beberapa siri penyelidikan telah membuktikan bahawa pengekspresan gen secara paksa faktor transkripsi seperti Oct-3/4, KLF4 dan Sox2 berupaya memprogram-semula sel yang telah membeza untuk menghasilkan sel induk pluripoten. Namun, penggunaan kaedah konvensional vektor virus yang boleh mengubah genetik sel melalui integrasi atau penggabungan dengan eksogen berupaya mengakibatkan tumor dan ini menjadikan penggunaannya tidak selamat untuk aplikasi klinikal. Oleh itu, kajian ini menggunakan domain transduksi protein yang baharu dan diperbaharui yang dikenali sebagai trans-activator of transcription kappa (TATκ), iaitu TAT-HIV yang disintesis untuk menghantar gen faktor transkripsi sebagai kaedah alternatif penghasilan iPSC tanpa melalui proses program-semula menggunakan virus. Melalui kaedah baharu ini, klon stabil sel 293T yang merembeskan dan mengekspresikan protein program-semula tergabung TATκ (TATκ-GFP, TATκ-KLF4, TATκ-Sox2, dan TATκ-Oct-3/4) telah dihasilkan. Klon stabil ini telah berjaya mentransduksikan sel monosit sasaran (U937). TATκ-GFP yang memainkan peranan sebagai protein penanda serta protein tergabung TATκ-KLF4, TATκ-Sox2 dan TATκ-Oct-3/4 telah berjaya ditransduksikan ke dalam sel (U937), seterusnya membuktikan bahawa kaedah baharu yang mengaplikasikan TATκ ini berupaya untuk melalui proses translokasi merentasi membran sel. Perubahan morfologi sel U937 berjaya diperhatikan selepas 20 hari proses transduksi namun sel koloni iPSC tidak dapat dijana. Ini mungkin disebabkan oleh proses program-semula yang tidak lengkap atau tempoh transduksi yang tidak mencukupi untuk penjanaan sel iPSC.

Kata kunci: Domain transduksi protein; faktor transkripsi; program-semula; sel induk pluripoten terjana; transduksi

REFERENCES

Bechara, C. & Sagan, S. 2013. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Letters 587(12): 1693-1702.

Denault, J.B. & Leduc, R. 1996. Furin/PACE/SPC1: A convertase involved in exocytic and endocytic processing of precursor proteins. FEBS Letters 379(2): 113-116.

Deng, X.Y., Wang, H., Wang, T., Fang, X.T., Zou, L.L., Li, Z.Y. & Liu, C.B. 2015. Non-viral methods for generating integration-free, induced pluripotent stem cells. Current Stem Cell Research & Therapy 10(2): 153-158.

Dimos, J.T., Rodolfa, K.T., Niakan, K.K., Weisenthal, L.M., Mitsumoto, H., Chung, W., Croft, G.F., Saphier, G., Leibel, R., Goland, R., Wichterle, H., Henderson, C.E. & Eggan, K. 2008. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321(5893): 1218-1221.

Flinterman, M., Farzaneh, F., Habib, N., Malik, F., Gäken, J. & Tavassoli, M. 2009. Delivery of therapeutic proteins as secretable TAT fusion products. Molecular Therapy 17(2): 334-342.

Frankel, A.D. & Pabo, C.O. 1988. Cellular uptake of the TAT protein from human immunodeficiency virus. Cell 55(6): 1189-1193.

Green, M. & Loewenstein, P.M. 1988. Autonomous functional domains of chemically synthesized human immunodeficiency virus TAT trans-activator protein. Cell 55(6): 1179-1188.

Jiang, C., Zeng, X., Xue, B., Campbell, D., Wang, Y., Sun, H., Xu, Y. & Wen, X. 2019. Screening of pure synthetic coating substrates for induced pluripotent stem cells and IPSC-derived neuroepithelial progenitors with short peptide based integrin array. Experimental Cell Research 380(1): 90-99.

Kaji, K., Norrby, K., Paca, A., Mileikovsky, M., Mohseni, P. & Woltjen, K. 2009. Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458(7239): 771-775.

Kim, D., Kim, C.H., Moon, J.I., Chung, Y.G., Chang, M.Y., Han, B.S., Ko, S., Yang, E., Cha, K.Y., Lanza, R. & Kim, K.S. 2010. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4(6): 472-476.

Kingston, R.E., Chen, C.A. & Rose, J.K. 2003. Calcium phosphate transfection. Current Protocols in Molecular Biology 63(1): Chapter 9: Unit 9.1.

Mi, Z., Mai, J., Lu, X. & Robbins, P.D. 2000. Characterization of a class of cationic peptides able to facilitate efficient protein transduction in vitro and in vivo. Molecular Therapy 2(4): 339-347.

Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N. & Yamanaka, S. 2008. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nature Biotechnology 26(1): 101-106.

Nemes, C., Varga, E., Polgar, Z., Klincumhom, N., Pirity, M.K. & Dinnyes, A. 2014. Generation of mouse induced pluripotent stem cells by protein transduction. Tissue Engineering - Part C: Methods 20(5): 383-392.

Nordin, F., Tye, G.J., Gaken, J. & Farzaneh, F. 2014. TATκ fusion protein of OCT-3/4 and KLF-4: Stable mixed population cell lines capable of delivering fusion proteins to target cells. Journal of Cell Science & Therapy 5(2): 5-11.

Okita, K., Ichisaka, T. & Yamanaka, S. 2007. Generation of germline-competent induced pluripotent stem cells. Nature 448(7151): 313-317.

Rana, T.M. & Jeang, K.T. 1999. Biochemical and functional interactions between HIV-1 tat protein and TAR RNA. Archives of Biochemistry and Biophysics 365(2): 175-185.

Takahashi, K. & Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663-676.

Tikhonov, I., Ruckwardt, T.J., Berg, S., Hatfield, G.S. & Pauza, C.D. 2004. Furin cleavage of the HIV-1 Tat protein. FEBS Letters 565(1-3): 89-92.

Wernig, M., Meissner, A., Foreman, R., Brambrink, T., Ku, M., Hochedlinger, K., Bernstein, B.E. & Jaenisch, R. 2007. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448(7151): 318-324.

Woltjen, K., Michael, I.P., Mohseni, P., Desai, R., Mileikovsky, M., Hämäläinen, R., Cowling, R., Wang, W., Liu, P., Gertsenstein, M., Kaji, K., Sung, H.K. & Nagy, A. 2009. PiggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458(7239): 766-770.

Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D. & Carpenter, M.K. 2001. Feeder-free growth of undifferentiated human embryonic stem cells. Nature Biotechnology 19(10): 971-974.

Yamanaka, S. 2009. A fresh look at IPS cells. Cell 137(1): 13-17.

Zhang, H., Ma, Y., Gu, J., Liao, B., Li, J., Wong, J. & Jin, Y. 2012. Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors. Biomaterials 33(20): 5047-5055.

Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak, G., Schöler, H.R., Duan, L. & Ding, S. 2009. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell 4(5): 381-384.

*Corresponding author; email: nordinf@ppukm.ukm.edu.my