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

 

Diserahkan: 8 Mac 2021/Diterima: 8 Jun 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

 

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*Pengarang untuk surat-menyurat; email: nordinf@ppukm.ukm.edu.my

 

 

   

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