Evaluation of lentiviral based gene delivery system in adherent and suspension in vitro cell models

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 fourthgeneration 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

Intestinal stem cells and gut microbiota therapeutics: hype or hope?

The vital role of the intestines as the main site for the digestion and absorption of nutrients for the body continues subconsciously throughout one’s lifetime, but underneath all the complex processes lie the intestinal stem cells and the gut microbiota that work together to maintain the intestinal epithelium. Intestinal stem cells (ISC) are multipotent stem cells from which all intestinal epithelial cells originate, and the gut microbiota refers to the abundant collection of various microorganisms that reside in the gastrointestinal tract. Both reside in the intestines and have many mechanisms and pathways in place with the ultimate goal of co-managing human gastrointestinal tract homeostasis. Based on the abundance of research that is focused on either of these two topics, this suggests that there are many methods by which both players affect one another. Therefore, this review aims to address the relationship between ISC and the gut microbiota in the context of regenerative medicine. Understanding the principles behind both aspects is therefore essential in further studies in the field of regenerative medicine by making use of the underlying designed mechanisms

The establishment of in vitro human induced pluripotent stem cell-derived neurons

Induced pluripotent stem cells (iPSCs) have been generated using different reprogramming strategies. Lentiviruses remain a strategic method for cell reprogramming as it is highly efficient in gene transfer. The latest fourth-generation lentiviral packaging systems claimed to be efficient and safe. However, modifications made to enhance safety of lentiviral vectors have been shown to affect vector performance. In this study, we established that the fourth-generation lentiviral packaging system can produce high-titre lentiviruses with high-transduction efficiencies. Subsequently, the robustness and reproducibility of generating iPSCs from adult human dermal fibroblasts were tested using these lentiviruses. The use of fourth-generation lentiviruses consistently generates iPSCs with similar efficiency and quality in different primary cell lines. This study demonstrated that the human-derived iPSCs can be maintained using mitomycin-C inactivated feeder cells. The iPSCs clones highly expressed key pluripotency markers and can spontaneously differentiate into cells from the three embryonic germ layers. The iPSCs generated were able to differentiate into neural stem cell lineages, producing cells expressing Nestin and Sox2 as well as able to further differentiate into neurons with more than 70% efficiency. The data demonstrated that the use of the fourth-generation lentiviral packaging to produce lentiviruses for iPSCs generation is robust and reproducible as it can generate iPSCs from different adult dermal fibroblasts with the potential to differentiate into neural stem cells and neurons. The use of safer lentiviral packaging systems combined with established vector plasmids will help to expedite the generation of iPSCs for clinical applications

CD74 and HLA-DRA in Cervical Carcinogenesis: Potential Targets for Antitumour Therapy

Background and Objectives: Abnormal expressions of CD74 and human leukocyte antigen-DR alpha (HLA-DRA) have been reported in various cancers, though their roles in cervical cancer remain unclear. This study aimed to evaluate the gene and protein expressions of CD74 and HLA-DRA in the progression from normal cervix to precancerous cervical intraepithelial neoplasia (CIN) and finally to squamous cell carcinoma (SCC). Materials and Methods: The gene expression profiles of CD74 and HLA-DRA were determined in formalin-fixed paraffin-embedded tissues, with three samples each from normal cervixes, human papillomavirus type 16/18-positive, low-grade CIN (LGCIN), high-grade CIN (HGCIN), and squamous cell carcinoma (SCC) using Human Transcriptome Array 2.0. Immunohistochemical expression of the proteins was semi-quantitatively assessed in another cohort of tissue microarray samples comprising 7 normal cervix cases, 10 LGCIN, 10 HGCIN, and 95 SCC. Results: The transcriptomics profile and proteins’ expression demonstrated similar trends of upregulation of CD74 and HLA-DRA from normal cervix to CIN and highest in SCC. There was a significant difference in both proteins’ expression between the histological groups (p = 0.0001). CD74 and HLA-DRA expressions were significantly associated with CIN grade (p = 0.001 and p = 0.030, respectively) but not with the subjects’ age or SCC stage. Further analysis revealed a positive correlation between CD74 and HLA-DRA proteins. Conclusions: CD74 appears to promote cervical carcinogenesis via oncogenic signalling mechanisms and may serve as a potential antitumour target. Additionally, the upregulation of HLA-DRA, often associated with stronger immunogenicity, could be a promising biomarker for developing immunotherapies

The potential applications of T cell receptor (TCR)-like antibody in cervical cancer immunotherapy

Cervical cancer is ranked as the fourth most common cancer in women worldwide. Monoclonal antibody has created a new dimension in the immunotherapy of many diseases, including cervical cancer. The antibody’s ability to target various aspects of cervical cancer (oncoviruses, oncoproteins, and signaling pathways) delivers a promising future for efficient immunotherapy. Besides, technologies such as hybridoma and phage display provide a fundamental platform for monoclonal antibody generation and create the opportunity to generate novel antibody classes including, T cell receptor (TCR)-like antibody. In this review, the current immunotherapy strategies for cervical cancer are presented. We have also proposed a novel concept of T cell receptor (TCR)-like antibody and its potential applications for enhancing cervical cancer therapeutics. Finally, the possible challenges in TCR-like antibody application for cervical cancer therapeutics have been addressed, and strategies to overcome the challenges have been highlighted to maximize the therapeutic benefits

TAT Kappa (TATK) : a novel cell penetrating peptide for delivery of pluripotent proteins into target cells

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 TATκ 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

Development of cell penetrating peptides for effective delivery of recombinant factors into target cells

The cell membrane is a protective layer that strictly controls the passage of molecules restricting the delivery of biomolecules such as drugs, oligonucleotides, peptides, and siRNA into the cells. This shortcoming has been overcome by the discovery of Cell-Penetrating Peptides (CPPs) that has undergone 30 years of evolution. To date, CPPs are largely modified to improve its efficacy and to suit the different delivery applications. The modes of CPPs penetration are still an unresolved mystery and requires further investigations to increase its effectiveness and to diversify its use. Despite having huge potential as a biomolecule carrier, CPPs also have some drawbacks. In this review, the natural and synthetic CPPs, the modifications that have been conducted on CPPs to improve its efficacy, its extended applications, modes of penetration and limitation as well as challenges will be discussed

Non-Integrating Lentiviral Vectors in Clinical Applications: A Glance Through

Lentiviral vectors (LVs) play an important role in gene therapy and have proven successful in clinical trials. LVs are capable of integrating specific genetic materials into the target cells and allow for long-term expression of the cDNA of interest. The use of non-integrating LVs (NILVs) reduces insertional mutagenesis and the risk of malignant cell transformation over integrating lentiviral vectors. NILVs enable transient expression or sustained episomal expression, especially in non-dividing cells. Important modifications have been made to the basic human immunodeficiency virus (HIV) structures to improve the safety and efficacy of LVs. NILV-aided transient expression has led to more pre-clinical studies on primary immunodeficiencies, cytotoxic cancer therapies, and hemoglobinopathies. Recently, the third generation of self-inactivating LVs was applied in clinical trials for recombinant protein production, vaccines, gene therapy, cell imaging, and induced pluripotent stem cell (iPSC) generation. This review discusses the basic lentiviral biology and the four systems used for generating NILV designs. Mutations or modifications in LVs and their safety are addressed with reference to pre-clinical studies. The detailed application of NILVs in promising pre-clinical studies is also discussed