Progress and pitfalls of gene editing technology in CAR-T cell therapy: a state-of-the-art review.

CAR-T cell therapy has shown remarkable promise in treating B-cell malignancies, which has sparked optimism about its potential to treat other types of cancer as well. Nevertheless, the Expectations of CAR-T cell therapy in solid tumors and non-B cell hematologic malignancies have not been met. Furthermore, safety concerns regarding the use of viral vectors and the current personalized production process are other bottlenecks that limit its widespread use. In recent years the use of gene editing technology in CAR-T cell therapy has opened a new way to unleash the latent potentials of CAR-T cell therapy and lessen its associated challenges. Moreover, gene editing tools have paved the way to manufacturing CAR-T cells in a fully non-viral approach as well as providing a universal, off-the-shelf product. Despite all the advantages of gene editing strategies, the off-target activity of classical gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) remains a major concern. Accordingly, several efforts have been made in recent years to reduce their off-target activity and genotoxicity, leading to the introduction of advanced gene editing tools with an improved safety profile. In this review, we begin by examining advanced gene editing tools, providing an overview of how these technologies are currently being applied in clinical trials of CAR-T cell therapies. Following this, we explore various gene editing strategies aimed at enhancing the safety and efficacy of CAR-T cell therapy.

A review of animal models utilized in preclinical studies of approved gene therapy products: trends and insights.

Scientific progress heavily relies on rigorous research, adherence to scientific standards, and transparent reporting. Animal models play a crucial role in advancing biomedical research, especially in the field of gene therapy. Animal models are vital tools in preclinical research, allowing scientists to predict outcomes and understand complex biological processes. The selection of appropriate animal models is critical, considering factors such as physiological and pathophysiological similarities, availability, and ethical considerations. Animal models continue to be indispensable tools in preclinical gene therapy research. Advancements in genetic engineering and model selection have improved the fidelity and relevance of these models. As gene therapy research progresses, careful consideration of animal models and transparent reporting will contribute to the development of effective therapies for various genetic disorders and diseases. This comprehensive review explores the use of animal models in preclinical gene therapy studies for approved products up to September 2023. The study encompasses 47 approved gene therapy products, with a focus on preclinical trials. This comprehensive analysis serves as a valuable reference for researchers in the gene therapy field, aiding in the selection of suitable animal models for their preclinical investigations.

Gene therapy in glioblastoma multiforme: Can it be a role changer?

Glioblastoma multiforme (GBM) is one of the most lethal cancers with a poor prognosis. Over the past century since its initial discovery and medical description, the development of effective treatments for this condition has seen limited progress. Despite numerous efforts, only a handful of drugs have gained approval for its treatment. However, these treatments have not yielded substantial improvements in both overall survival and progression-free survival rates. One reason for this is its unique features such as heterogeneity and difficulty of drug delivery because of two formidable barriers, namely the blood-brain barrier and the tumor-blood barrier. Over the past few years, significant developments in therapeutic approaches have given rise to promising novel and advanced therapies. Target-specific therapies, such as monoclonal antibodies (mAbs) and small molecules, stand as two important examples; however, they have not yielded a significant improvement in survival among GBM patients. Gene therapy, a relatively nascent advanced approach, holds promise as a potential treatment for cancer, particularly GBM. It possesses the potential to address the limitations of previous treatments and even newer advanced therapies like mAbs, owing to its distinct properties. This review aims to elucidate the current status and advancements in gene therapy for GBM treatment, while also presenting its future prospects.

The paths and challenges of "off-the-shelf" CAR-T cell therapy: An overview of clinical trials.

The advent of chimeric antigen receptor T cells (CAR-T cells) has made a tremendous revolution in the era of cancer immunotherapy, so that since 2017 eight CAR-T cell products have been granted marketing authorization. All of these approved products are generated from autologous sources, but this strategy faces several challenges such as time-consuming and expensive manufacturing process and reduced anti-tumor potency of patients' T cells due to the disease or previous therapies. The use of an allogeneic source can overcome these issues and provide an industrial, scalable, and standardized manufacturing process that reduces costs and provides faster treatment for patients. Nevertheless, for using allogeneic CAR-T cells, we are faced with the challenge of overcoming two formidable impediments: severe life-threatening graft-versus-host-disease (GvHD) caused by allogeneic CAR-T cells, and allorejection of allogeneic CAR-T cells by host immune cells which is called "host versus graft" (HvG). In this study, we reviewed recent registered clinical trials of allogeneic CAR-T cell therapy to analyze different approaches to achieve a safe and efficacious "off-the-shelf" source for chimeric antigen receptor (CAR) based immunotherapy.

Engineering chimeric autoantibody receptor T cells for targeted B cell depletion in multiple sclerosis model: An in-vitro study.

Background: Recent evidence suggests that B cells and autoantibodies have a substantial role in the pathogenesis of Multiple sclerosis. T cells could be engineered to express chimeric autoantibody receptors (CAARs), which have an epitope of autoantigens in their extracellular domain acting as bait for trapping autoreactive B cells. This study aims to assess the function of designed CAAR T cells against B cell clones reactive to the myelin basic protein (MBP) autoantigen. Methods: T cells were transduced to express a CAAR consisting of MBP as the extracellular domain. experimental autoimmune encephalomyelitis (EAE) was induced by injecting MBP into mice. The cytotoxicity, proliferation, and cytokine production of the MBP-CAAR T cells were investigated in co-culture with B cells. Results: MBP-CAAR T cells showed higher cytotoxic activity against autoreactive B cells in all effector-to-target ratios compared to Mock T cell (empty vector-transduced T cell) and Un-T cells (un-transduced T cell). In co-cultures containing CAAR T cells, there was more proliferation and inflammatory cytokine release as compared to Un-T and Mock T cell groups. Conclusion: Based on these findings, CAAR T cells are promising for curing or modulating autoimmunity and can be served as a new approach for clone-specific B cell depletion therapy in multiple sclerosis.

The effect of serum origin on cytokines induced killer cell expansion and function.

BACKGROUND: Cytokine-induced killer (CIK) cells have shown promising results in adoptive immunotherapy. However, serum may play a determining role in the large-scale expansion of these cells for clinical applications. According to Good Manufacturing Practice (GMP) guidelines to reduce the use of animal products in cell-based therapies; therefore, this study sought to investigate the impact of serum origin and the reduced serum concentration on the pattern of cell expansion and function. METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from a healthy donor were expanded based on the CIK cell expansion protocol. The cell culture medium was supplemented with three types of sera comprising fetal bovine serum (FBS), human serum (HS), or human-derived platelet lysate (hPL) at different concentrations (10%, 5%, and 2.5%). The proliferation kinetics for each group were investigated for 30 days of cell culture. RESULTS: Cell proliferation in 10% concentration of all sera (hPL, FBS, HS) was higher than their lower concentrations. Moreover, hPL was significantly associated with higher expansion rates than FBS and HS in all three concentrations. Furthermore, cells cultured in hPL showed higher viability, cytotoxicity effect, and CIK CD markers expression. CONCLUSION: hPL at a concentration of 10% showed the best effect on CIK cell proliferation and function.

Cytotoxicity of WT1-reactive T cells against Wilms tumor: An implication for antigen-specific adoptive immunotherapy.

Introduction: T cells that recognize WT1 peptides have been shown to efficiently eliminate WT1-expressing tumor cells. This study was designed to investigate the feasibility of isolating WT1-reactive T cells from peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with Wilms tumor, and to assess the cytotoxicity mediated by these cells against Wilms tumor cells (WiTu cells). Methods: WT1-reactive T cells were enriched and isolated by stimulating PBMCs with a WT1 peptide pool and interferon-γ capture-based immunomagnetic separation (IMS). Using the lactate dehydrogenase release assay, the in vitro cytotoxicity of the isolated cells and standard chemotherapy was evaluated on WiTu cells. Results: Higher proportions of WT1-reactive T cells were isolated from patients with Wilms tumor compared to those isolated from HDs. WT1-reactive T cells produced > 50% specific lysis when co-cultured with WT1+ WiTu cells at the highest effector-to-target (E:T) ratio in this study (i.e., 5:1), compared to <23% when co-cultured with WT1- WiTu cells at the same ratio. WT1-reactive T cells showed anti-tumoral activity in a dose-dependent manner and mediated significantly greater cytotoxicity than the non-WT1-reactive fraction of PBMCs on WT1+ WiTu cells. The cytotoxicity of standard chemotherapy was significantly lower than that of WT1-reactive T cells when co-cultured with WT1+ WiTu cells at E:T ratios of 2:1 and 5:1. Conclusion: WT1-reactive T cells can be effectively enriched from the PBMCs of patients with Wilms tumor. Ex vivo generated WT1-reactive T cells might be considered an adoptive immunotherapeutic option for WT1+ Wilms tumors.

Regenerative potential of multinucleated cells: bone marrow adiponectin-positive multinucleated cells take the lead.

BACKGROUND: Polyploid cells can be found in a wide evolutionary spectrum of organisms. These cells are assumed to be involved in tissue regeneration and resistance to stressors. Although the appearance of large multinucleated cells (LMCs) in long-term culture of bone marrow (BM) mesenchymal cells has been reported, the presence and characteristics of such cells in native BM and their putative role in BM reconstitution following injury have not been fully investigated. METHODS: BM-derived LMCs were explored by time-lapse microscopy from the first hours post-isolation to assess their colony formation and plasticity. In addition, sub-lethally irradiated mice were killed every other day for four weeks to investigate the histopathological processes during BM regeneration. Moreover, LMCs from GFP transgenic mice were transplanted to BM-ablated recipients to evaluate their contribution to tissue reconstruction. RESULTS: BM-isolated LMCs produced mononucleated cells with characteristics of mesenchymal stromal cells. Time-series inspections of BM sections following irradiation revealed that LMCs are highly resistant to injury and originate mononucleated cells which reconstitute the tissue. The regeneration process was synchronized with a transient augmentation of adipocytes suggesting their contribution to tissue repair. Additionally, LMCs were found to be adiponectin positive linking the observations on multinucleation and adipogenesis to BM regeneration. Notably, transplantation of LMCs to myeloablated recipients could reconstitute both the hematopoietic system and BM stroma. CONCLUSIONS: A population of resistant multinucleated cells reside in the BM that serves as the common origin of stromal and hematopoietic lineages with a key role in tissue regeneration. Furthermore, this study underscores the contribution of adipocytes in BM reconstruction.

Challenges in Mesenchymal Stromal Cell-based Therapies.

Over 50 years have passed since discovering mesenchymal stromal cells (MSCs). Initially, despite gaps in the knowledge of the identity of these cells, their therapeutic aspects were recognized. Consequently, MSCs became candidates for treating a wide range of diseases. However, the therapeutic effects of MSCs are not stable in the long term, and there are inconsistent data on their clinical efficacy. Even though more than 1000 MSC-based clinical trials have been registered, and the safety of MSCbased cell therapies has been proven, data on the clinical efficacy of MSCs have not been enough to warrant FDA approval for clinical treatment and marketing purposes. The available information on MSCs still contains some controversies, perhaps owing to little progress in understanding their in vivo identity. MSCs have been used for therapeutic purposes despite poor knowledge of their in vivo origin or functions. Hence, perhaps we need to go back to the basics of MSCs and spend more time understanding the biology of these cells. An improved understanding of MSCs' location and function within tissues may improve their therapeutic efficacy and, consequently, their establishment as a cell therapy product.

Efficacy of adoptively transferred allogeneic CIK cells on colorectal cancer: Augmentative antitumoral effects of GvHD.

OBJECTIVE: A preclinical study was designed to evaluate the effects of adoptively transferred cytokine-induced killer (CIK) cells on colorectal adenocarcinoma. METHODS: Forty NOG mice bearing HT-29 xenograft tumors were developed and equally divided into 2 groups of treatment and control. The mice in the treatment group received cumulatively 40-60 × 106 CIK cells in four divided doses. RESULTS: Median tumor doubling times for HT-29 xenograft tumors in the treatment and control groups were found to be 8.98 and 4.32 days; respectively. The treatment resulted in tumor growth delay (TGD) of 52.5 %. CIK cell-induced log cell kill (LCK) was found to be 0.67, which implies reduction of 78.6 % of neoplastic colorectal cells. Median length of survival in the treated mice was significantly longer than controls (57 (41-63) vs 41 (31-57) days, P < 0.001). Mice in the treatment group experienced graft-versus-host disease (GvHD) from median of day 13th after the cell therapy. LCK and TGD significantly increased after emergence of GvHD. After necropsy, tumors of the treatment group contained high levels of human-originated CD3+, CD4+ and CD8+ cells and showed significantly lower mitotic counts (P < 0.001) and residual tumor scores (P = 0.005) than the controls (entirely negative for the mentioned CD markers). Ninety percent of the treated mice were found to be responding. CONCLUSIONS: Adoptive transfer of allogeneic CIK cells may be an efficient antitumoral therapy for colorectal cancer. Allogeneic CIK cell-mediated GvHD may contribute to amplification of graft-versus-tumor effects of the cellular therapy.

Retinal and Choroidal Neovascularization Antivascular Endothelial Growth Factor Treatments: The Role of Gene Therapy.

Neovascularization in ocular vessels causes a major disease burden. The most common causes of choroidal neovascularization (CNV) are age-related macular degeneration and diabetic retinopathy, which are the leading causes of irreversible vision loss in the adult population. Vascular endothelial growth factor (VEGF) is critical for the formation of new vessels and is the main regulator in ocular angiogenesis and vascular permeability through its receptors. Laser therapy and antiangiogenic factors have been used for CNV treatment. Bevacizumab, ranibizumab, and aflibercept are commonly used anti-VEGF agents; however, high costs and the need for frequent intraocular injections are major drawbacks of anti-VEGF drugs. Gene therapy, given the potency of one-time treatment and no need for frequent injections offers the real possibility of such a lasting treatment, with fewer adverse effects and higher patient quality of life. Herein, we reviewed the role of gene therapy in the CNV treatment. In addition, we discuss the advantages and challenges of current treatments compared with gene therapy.

Gene therapy clinical trials, where do we go? An overview.

There have been many ups and downs since the introduction of gene therapy as a therapeutic modality for diseases. However, the journey of gene therapy has reached a fundamental milestone, as evidenced by the increasing number of gene therapy products on the market. Looking at the currently approved and under-approval products, as well as the numerous clinical trials in this field, gene therapy has a promising future. Trend of changes in gene therapy strategies, vectors, and targets could be insightful for pharmaceutical companies, policymakers, and researchers. In this paper, following a brief history of gene therapy, we reviewed current gene therapy products as well as gene therapies that may be approved in the near future. We also looked at ten-year changes in gene therapy clinical trials strategies, such as the use of vectors, target cells, transferred genes, and ex-vivo/in-vivo methods, as well as the major fields that gene therapy has entered. Although gene therapy was initially used to treat genetic diseases, cancer now has the greatest number of gene therapy clinical trials. Changes in gene therapy strategies, particularly in pioneering countries in this field, may point to the direction of future clinical products.

In vivo study of the angiogenesis potential of bone marrow-derived mesenchymal stem cell aggregates in their niche like environment.

BACKGROUND: Sufficient blood vessel formation in bioengineered tissues is essential in order to keep the viability of the organs. Impaired development of blood vasculatures results in failure of the implanted tissue. The cellular source which is seeded in the scaffold is one of the crucial factors involved in tissue engineering methods. MATERIALS AND METHODS: Considering the notable competence of Bone Marrow derived Mesenchymal Stem Cell aggregates for tissue engineering purposes, in this study BM-aggregates and expanded BM-MSCs were applied without any inductive agent or co-cultured cells, in order to investigate their own angiogenesis potency in vivo. BM-aggregates and BM-MSC were seeded in Poly-L Lactic acid (PLLA) scaffold and implanted in the peritoneal cavity of mice. RESULT: Immunohistochemistry results indicated that there was a significant difference (p < 0.050) in CD31+ cells between PLLA scaffolds contained cultured BM-MSC; PLLA scaffolds contained BM-aggregates and empty PLLA. According to morphological evidence, obvious connections with recipient vasculature and acceptable integration with surroundings were established in MSC and aggregate-seeded scaffolds. CONCLUSION: Our findings revealed cultured BM-MSC and BM-aggregates, capacity in order to develop numerous connections between PLLA scaffold and recipient's vasculature which is crucial to the survival of tissues, and considerable tendency to develop constructs containing CD31+ endothelial cells which can contribute in vessel's tube formation.

An outlook on antigen-specific adoptive immunotherapy for viral infections with a focus on COVID-19.

Although not a standard-of-care yet, adoptive immunotherapeutic approaches have gradually earned a place within the list of antiviral therapies for some of fatal and hard-to-treat viral diseases. To maintain robust antiviral immunity and to effectively target the viral particles and virally-infected cells, immune cells capable of recognizing the viral antigens are required. While conventional vaccination can induce these cells in vivo; another option is to prime and generate antigen-specific immune cells ex vivo. This approach has been successfully trialed for virulent opportunistic viral infections after bone marrow transplantation. Amid the crisis of SARS-CoV2 pandemic, which has been followed by the success of certain early-authorized vaccines; some institutions and companies have explored the effects of viral-specific adoptive cell transfers (ACTs) in trials, as alternative treatments. Aimed at outlining a perspective on antigen-specific adoptive immunotherapy for viral infections, this review article specifically provides an appraisal of ACT-based studies/trials on SARS-CoV2 infection.

Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy.

BACKGROUND: The successful ex vivo expansion of T-cells in great numbers is the cornerstone of adoptive cell therapy. We aimed to achieve the most optimal T-cell expansion condition by comparing the expansion of T-cells at various seeding densities, IL-2 concentrations, and bead-to-cell ratios. we first expanded the peripheral blood mononuclear cells (PBMCs) of a healthy donor at a range of 20 to 500 IU/mL IL-2 concentrations, 125 × 103 to 1.5 × 106 cell/mL, and 1:10 to 10:1 B:C (Bead-to-cell) ratios and compared the results. We then expanded the PBMC of three healthy donors using the optimized conditions and examined the growth kinetics. On day 28, CD3, CD4, and CD8 expression of the cell populations were analyzed by flow cytometry. RESULTS: T-cells of the first donor showed greater expansion results in IL-2 concentrations higher than 50 IU/mL compared to 20 IU/mL (P = 0.02). A seeding density of 250 × 103 cell/mL was superior to higher or lower densities in expanding T-cells (P = 0.025). Also, we witnessed a direct correlation between the B:C ratio and T-cell expansion, in which, in 5:1 and 10:1 B:C ratios T-cell significantly expanded more than lower B:C ratios. The results of PBMC expansions of three healthy donors were similar in growth kinetics. In the optimized condition, 96-98% of the lymphocyte population expressed CD3. While the majority of these cells expressed CD8, the mean expression of CD4 in the donors was 19.3, 16.5, and 20.4%. CONCLUSIONS: Our methodology demonstrates an optimized culture condition for the production of large quantities of polyclonal T-cells, which could be useful for future clinical and research studies.

Effects of human placenta-derived mesenchymal stem cells with NK4 gene expression on glioblastoma multiforme cell lines.

Poor prognosis and low survival are commonly seen in patients with glioblastoma multiforme (GBM). Due to the specific nature of solid tumors such as GBM, delivery of therapeutic agents to the tumor sites is difficult. So, one of the major challenges in the treatment of these tumors is a selection of appropriate method for drug delivery. Mesenchymal stem cells (MSCs) have a unique characteristic in migration toward the tumor tissue. In this regard, the present study examined the antitumor effects of manipulating human placenta-derived mesenchymal stem cells (PDMSCs) with NK4 expression (PDMSC-NK4) on GBM cells. After separation and characterization of PDMSCs, these cells were transduced with NK4 which was known as the antagonist of hepatocyte growth factor (HGF). The results indicated that engineered PDMSCs preferably migrate into GBM cells by transwell coculture system. In addition, the proliferation of the GBM cells significantly reduced after coculture with these cells. In fact, manipulated PDMSCs inhibited growth of tumor cells by induction of apoptosis. Our findings suggested that besides having antitumor effects, PDMSCs can also be applied as an ideal cellular vehicle to target the glioblastoma multiforme.

Antigen-independent killer cells prepared for adoptive immunotherapy: One source, divergent protocols, diverse nomenclature.

Adoptive cell therapy (ACT) using tumor antigen-independent killer cells has been widely used in clinical trials of cancer treatment. Circumventing the need for identification of a particular tumor-associated antigen on tumor cells, the approach has opened possibilities for the extension of ACT immunotherapy to patients with a wide variety of cancer types. Namely, Natural Killer (NK), Lymphokine-activated Killer (LAK) cells and Cytokine-induced killer (CIK) cells are the most commonly used cell types in antigen-independent adoptive immunotherapy of cancer. They all originate from peripheral blood mononuclear cells and share several common features in their killing mechanisms. However, despite broad application in clinical settings, the boundaries between these cell types are not very clearly defined. The current study aims to review different aspects of these cell populations in terms of phenotypical characteristic and preparation media, to clarify how the boundaries are set.

ANTXR1 (TEM8) overexpression in gastric adenocarcinoma makes the protein a potential target of immunotherapy.

BACKGROUND: Despite the promise of immunotherapy for gastric adenocarcinoma, choices for the selection of effective antigenic targets are very limited. Previously published data and our own in-house computational analysis have suggested that ANTXR1 is a potential target, simultaneously expressed in malignant tumor cells and the endothelial cells of the tumors. However, the expression pattern of ANTXR1 protein in clinical samples of gastric adenocarcinoma has not been fully evaluated. METHODS: Using immunohistochemistry (IHC), we recorded the percentage of ANTXR1 positive cells separately in tumor cells and endothelial cells in the primary tumor, non-tumor gastric tissue adjacent to the primary tumor, and tumor in metastatic sites of 140 gastric adenocarcinoma patients. We also evaluated the association of ANTXR1 expression with the Lauren histological classification of the primary tumors, the patient's history of neoadjuvant chemotherapy and/or radiotherapy, and the patient's overall survival. RESULTS: ANTXR1 was expressed in a mean of 73.89 ± 30.12% of tumor cells and 13.55 ± 20.53% of endothelial cells in the primary tumors. Intestinal adenocarcinomas had lower ANTXR1 expression in the tumor cells and higher ANTXR1 expression in the endothelial cells of the tumor regions, and a history of neoadjuvant therapy was associated with increased ANTXR1 expression in the endothelial cells of the tumor regions. Finally, above median expression of ANTXR1 in the tumor cells of the tumor regions was associated with significantly lower overall patient survival. CONCLUSIONS: Our findings suggest that ANTXR1 is a promising candidate for preclinical and clinical evaluation for gastric adenocarcinoma immunotherapy.

Impact of various culture conditions on ex vivo expansion of polyclonal T cells for adoptive immunotherapy.

Currently, adoptive immunotherapy is considered as one of the leading treatments in cancer. Successful adoptive immunotherapy depends on producing large numbers of desired T cells ex vivo for infusion. This requires an effective protocol for maximum functional T-cell expansion while keeping the time and costs to a minimum. Current T-cell expansion protocols are diverse in their methodology, and a universal protocol of expansion is wanting. Also, new findings regarding T-cell biology, signaling, and activation have reshaped the strategies of T-cell propagation over the years, introducing new ways to expand T cells. Here, we reviewed different conditions for blood-derived polyclonal T-cell expansion so as to elucidate the influential factors of T-cell expansion and their efficacy.

Strategies for Prevention and Treatment of Peritoneal Fibrosis: A Scientometric Study.

BACKGROUND: Interest in using peritoneal dialysis (PD) shows global and national increase. However, it remains a challenge to prevent the progression of PD-associated fibrosis in clinical practice. Here, we assessed the status of scientific publications in prevention and management of PD-associated fibrosis in a scientometric study. METHODS: We retrieved the bibliometric data by search terms "encapsulating peritoneal fibrosis," "treatment or prevention," and their synonyms in the Scopus databases until December 2, 2017. Data were analyzed using Scopus analysis tools, SPSS version 15 and Visualizing Scientific Landscapes viewer version 1.6.5. RESULTS: Number of publications showed a steady significant increase (P < 0.001) reaching to 390 documents. Japan had the highest share (21.3%) followed by United Kingdom. Coauthorship network assessment assigned "Ikeda M." from Japan as the top author. The top source of documents was "Peritoneal Dialysis International." Most of documents were original articles focusing on prevention and management of malignant fibrosis of peritoneum (72.6%). The documents were cited totally 5636 times with average citations per article of 14.45, and relatively high H-index of 38. CONCLUSIONS: Despite the global increasing trend in scientific output in this field, contribution of our country is very small. Perhaps more national and international collaboration is required to encourage our researchers for producing more scientific products.