Biology subjects

Tiryaki, N. N.

Publications and source records attributed to Tiryaki, N. N..

3 recordsLinked to original sources

bioRxiv2022

CAR-T Cells with Phytohemagglutinin (PHA) Provide Anti-Cancer Capacity with Better Proliferation, Rejuvenated Effector Memory, and Reduced Exhausted T Cell Frequencies

The development of genetic modification techniques has led to the opening of a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. Since not only cancerous cells but also healthy cells are damaged by the drugs, intensive efforts are made to develop cancer-targeted techniques. The most promising approach is genetically modified CAR-T cell therapy. The high central memory T cell (Tcm) and stem cell-like memory T cell (Tscm) ratios in the CAR-T cell population increase the effectiveness of immunotherapy. Therefore, it is important to increase the populations of CAR-expressing Tcm and Tscm cells to ensure that CAR-T cells remain long-term and have cytotoxic (anti-tumor) efficacy. In this study, we aimed to improve CAR-T cell therapys time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the subpopulation of Tcm and Tscm in CAR-T cells, we investigated to produce a long-term stable and cytotoxic efficient CAR-T cell by modifications in the cell activation-dependent production method using Phytohemagglutinin. Phytohemagglutinin (PHA), a lectin that binds to the membranes of T cells and increases metabolic activity and cell division, is studied to increase the Tcm and Tscm population. Although it is known that PHA significantly increases Tcm cells, B-lymphocyte antigen CD19 specific CAR-T cell expansion, its anti-cancer and memory capacity has not yet been tested compared to aCD3/aCD28. Two different types of CAR (aCD19 scFv CD8- (CD28 or 41BB)-CD3z-EGFRt) expressing T cells were generated and their immunogenic phenotype, exhausted phenotype, Tcm-Tscm populations, and cytotoxic activities were determined in this study. The proportion of T cell memory phenotype in the CAR-T cell populations generated by PHA was observed to be higher than that of aCD3/aCD28-amplified CAR-T cells with similar cytotoxic (anti-tumor) and higher proliferation capacity. Here, we show that PHA provides long-term and efficient CAR-T cell production, suggesting a potential alternative to aCD3/aCD28-amplified CAR-T cells.

cancer biology↗

bioRxiv2022

Autonomous Remotely Controlled Closed System Transgenic Cell Technologies Robot: CRISPR.BOT

In manually advancing experimental processes, the stages may be long-term and need to be repeated. Human errors with the repetition of the steps turn into a time-consuming and high-cost for the experiment processes. For this reason, autonomous liquid processing systems are promising technologies. However, in addition to the high cost of fully automatic systems, their maintenance is also quite expensive. Furthermore, conventional systems usually require system-specific protocols and laboratory equipment. Here, we aimed to show that the autonomous robotic systems may provide a closed and error-free molecular biology bench to perform genetic engineering automatically, quickly, and practically 7-24. In this way, researchers can save time from repetitive experiment processes and perform BSL3 experiments including pathogens without human contact. In this study, we built CRISPR.BOT robotic systems to perform Green Fluorescent Protein (GFP) encoding plasmid DNA transfer into bacteria, lentiviral transduction of the gene-of-interests including GFP encoding gene and CRISPR-Cas9 with gRNAs genetic editing system to a human cell line. Furthermore, we showed that CRISPR.BOT system achieved to accomplish single-cell subcloning of GFP+ CRISPR-gRNA+ cells with 90-100% purity. This study suggests that CRISPR.BOT-like approaches may reduce manpower in a safely closed bench in which molecular biology and genetic engineering can be done by robots in a closed system without touching pathogenic microorganisms (virus or bacteria, for example, SARS CoV-2 virus). Furthermore, LEGO Mindstorms robots showed to have the potential to be used in daily laboratory routines with their cost-effectiveness reduced by up to 50 times compared to normal commercial robots.

molecular biology↗

bioRxiv2022

Exon7 Targeted CRISPR-Prime Editing Approaches for SMN2 Gene Editing in Spinal Muscular Atrophy (SMA) Disease Can Increase In Vitro SMN Expression

Spinal Muscular Atrophy (SMA) is a fatal neuromuscular disease characterized by motor neuron loss and advanced muscle weakness, which occurs in functional SMN (Survival Motor Neuron) protein deficiency with SMN1 gene-induced deletions and mutations. The incidence of SMA, which is an autosomal recessive disease, is 1/10,000 in the world. The SMN protein acts as a molecular chaperone in the formation of the spliceosome complex, which catalyzes the splicing of pre-mRNA, enabling mRNAs and non-coding RNAs to mature. Since the current SMN1-encoding Adeno-associated virus (AAV) or SMN2 gene targeting antisense oligonucleotide-based strategies cannot provide long-term stable SMN expression in neuron cells, more effective methods need to be developed. CRISPR technology, which adds a new dimension to genetic engineering and gene therapies, makes it possible to treat many genetic diseases. In terms of SMA, some previous studies in the literature prove that it is possible to treat SMA with the CRISPR strategy. Homology Directed Repair (HDR)-based CRISPR technology, which results in a high rate of in-del (insertion-deletion) mutations rather than editing, was shown unsuitable for therapeutic applications. CRISPR-Prime editing (PE) technology is a new generation of gene editing approach that precisely provides various genomic modifications without the need for double-strand breakage or donor DNA sequences. CRISPR-Prime Editing method has also been used in rare diseases such as sickle cell anemia and Tay-Sachs, and their efficiency in editing various pathogenic mutations has been demonstrated. However, CRISPR Prime Editing-mediated gene editing for Spinal Muscular Atrophy (SMA) have not yet been investigated. The c.840 T-C transition and c.859 G-C transformations in the SMN2 gene and the correction of these point mutations with a single pegRNA at the same time were investigated for the first time in this study. Here, we showed that CRISPR-PE systems could increase SMN2 gene activity and SMN protein expression by ensuring exon 7 participation by editing c.840 T-C transition and c.859 G-C transformations. The fact that Prime Editing method showed the efficacy and stability of modifications in SMN2 genes that were investigated in SMN-low Jurkat cells as a proof-of-concept. This study enabled the next step with the CRISPR-Prime Editing approach to be tested ex vivo in primary cell lines from SMA patients and SMN-low neuronal cells.

genetics↗