Sains Malaysiana 53(3)(2024): 549-559

http://doi.org/10.17576/jsm-2024-5303-06

Evaluation of Lentiviral Based Gene Delivery System in Adherent and Suspension in vitro Cell Models

(Penilaian Sistem Angkut Serang Gen Berasaskan Lentivirus dalam Model Sel Adheren dan Ampaian secara in vitro)

IZYAN MOHD IDRIS^1,2, FAZLINA NORDIN^1,*, NUR JANNAIM MUHAMAD², SITI ZULEHA IDRIS³, GEE JUN TYE⁴, WAN SAFWANI WAN KAMARUL ZAMAN⁵ & MIN HWEI NG¹

¹Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), 56000 Cheras, Kuala Lumpur, Malaysia

²Institute for Medical Research (IMR), National Institutes of Health (NIH), Ministry of Health Malaysia, Jalan Setia Murni U13/52, Seksyen U13 Setia Alam, 40170 Shah Alam, Selangor, Malaysia

³Immunology Unit, Pathology Department, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

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

⁵Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, 50603, Kuala Lumpur, Malaysia

Diserahkan: 6 April 2023/Diterima: 9 Februari2024

Abstract

Lentiviruses are a highly robust gene delivery system capable of in vitro and in vivo gene transfer into multiple cell types. Recent fourth-generation lentiviral systems have been designed for enhanced safety, however, the increased recombination events required to produce infective lentiviral particles may reduce production efficiency. A set protocol for all types of target cells is not recommended and optimization of conditions for gene transfer into different target cells is required. In this study, we aim to evaluate the efficiency and reproducibility of lentiviral production using a fourth-generation lentiviral packaging system and identify optimal parameters for successful transduction in two different cell models, adherent and suspension cells. Lentiviral production, effect of viral volume, sustained gene expression and transduction adjuvants on adherent and suspension gene- cell models were evaluated. Transfection and transduction efficiency of lentiviruses was evaluated by fluorescence microscopy and flow cytometry. This study demonstrates that production of green fluorescent protein (GFP)- lentiviruses using the fourth-generation lentiviral packaging is consistent and reproducible. Optimal transduction of adherent cell types is achieved at lower multiplicity of infection (MOI) compared to suspension cells and produces GFP-expressing cells with higher intensity. Expression of GFP is sustained in all cell types over multiple passages. Polycation DEAE-dextran was determined to improve transduction in suspension cells, however, provides similar transduction efficiency as polybrene in adherent cells. In conclusion, fourth generation lentiviral system reproducibly generates high titre lentiviruses capable of infecting multiple cell types, however transduction protocols for different cell types require further optimization.

Keywords: Gene transfer; in vitro model; lentivirus

Abstrak

Lentivirus merupakan kaedah pemindahan gen yang sangat teguh yang mampu memindahkan gen secara in vitro dan in vivo kepada pelbagai jenis sel. Sistem lentivirus generasi keempat yang terbaharu, direka untuk mempertingkatkan keselamatan, walau bagaimanapun, bilangan keadaan rekombinasi yang diperlukan bagi menghasilkan zarah lentivirus berjangkit boleh mengurangkan kecekapan pengeluaran. Protokol yang ditetapkan untuk semua jenis sel tidak digalakkan dan pengoptimuman keadaan bagi pemindahan gen ke dalam sel sasaran yang berbeza diperlukan. Dalam penyelidikan ini, kami berhasrat untuk menilai kecekapan dan kebolehulangan pengeluaran lentivirus menggunakan sistem lentiviral generasi keempat dan mengenal pasti parameter optimum bagi transduksi yang berjaya dalam dua model sel yang berbeza iaitu sel adheren dan sel ampaian. Penghasilan lentivirus, kesan isi padu virus, pengekspresan gen yang berterusan dan adjuvan transduksi pada model sel adheren atau ampaian telah dinilai.  Kecekapan pemindahan dan transduksi lentivirus dinilai oleh mikroskop pendarfluor dan sitometri aliran. Kajian ini menunjukkan bahawa pengeluaran lentivirus ‘green fluorescent protein’ (GFP) menggunakan pembungkusan lentiviral generasi keempat adalah tekal dan boleh dihasilkan semula. Transduksi optimum jenis sel adheren dicapai pada kegandaan jangkitan (MOI) yang lebih rendah berbanding sel ampaian dan menghasilkan sel yang mengekspresi GFP dengan keamatan yang tinggi. Ekspresi GFP dikekalkan dalam semua jenis sel yang melalui pemindahan. Polycation DEAE-dextran didapati telah meningkatkan transduksi dalam sel ampaian, walau bagaimanapun, ia menghasilkan kecekapan yang sama seperti polibren dalam sel adheren. Kesimpulannya, sistem lentivirus generasi keempat mempunyai kebolehulangan dalam menghasilkan titer lentivirus tinggi yang mampu menjangkiti pelbagai jenis sel, namun protokol bagi transduki bagi jenis sel berbeza memerlukan pengoptimuman selanjutnya. 

Kata kunci: in vitro; lentivirus; pemindahan gen

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