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.

The development of genetic modification techniques has led to a new era in cancer treatments that have been limited to conventional treatments such as chemotherapy. intensive efforts are being performed to develop cancer-targeted therapies to avoid the elimination of non-cancerous cells. One of the most promising approaches 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 therapy’s time-dependent efficacy and stability, increasing the survival time and reducing the probability of cancer cell growth. To increase the sub-population of Tcm and Tscm in CAR-T cells, we investigated the production of a long-term stable and efficient cytotoxic CAR-T cell by modifications in the cell activation-dependent production using Phytohemagglutinin (PHA). 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 with aCD3/aCD28-amplified CAR-T cells. Two different types of CARs (aCD19 scFv CD8-(CD28 or 4-1BB)-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 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.

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Every year, millions of drugs are thrown away by consumers, even before their expiration date, assuming they have deteriorated. We need to see the fact that this has negative repercussions for our country and the world. In addition, when the data of the World Health Organization is examined, when it is considered that approximately 50% of the drugs are used incorrectly or unnecessarily or they are thrown away without even opening the box, we can see that the waste is large. We should not forget that the waste of drugs also brings environmental problems. It is crucial to maintain proper medicine storage conditions and keep out anything that could cause them to deteriorate. The most important factors that play a role in the deterioration of drugs are light, temperature and humidity. If drugs are not stored under appropriate conditions, their chemical structures may change and their effectiveness may decrease or disappear. One of the main factors affecting the deterioration of drugs is that drugs are not kept in the cold chain except for heat, light and humidity. These factors affecting degradation also affect the degradation of DNA. Therefore, it is anticipated that a DNA-based ink will be developed and used in the packaging of drugs, and drug degradation can be followed easily. As a groundbreaking project in the field of health, it is aimed that the person using the drug will be able to notice the deterioration in drugs over time with the DNAINK method. With DNAINK, it is aimed to understand whether the products still maintain their stability effectively, since the text on the drug boxes will be written with DNA ink. The DNA will be fluorescently marked, placed in ink, and injected into the writing on the medicine box. The target is that if the double-stranded DNA is left in conditions such as ultraviolet, light, temperature and humidity for a long time or if it is dependent on time, breaks will occur in the ink containing DNA, and it is aimed to determine the rate of degradation by following these breaks. Against counterfeit drug production, which has become a major health threat in the market, the DNAINK project will additionally develop barcoding technology, an important solution to this problem. Aims to bring Barcoding on the purchased medicine box will help to understand that the medicine came out of the right production place, and to bring a great solution to the health problems experienced due to counterfeit medicine production. With the barcoding technology, it will be possible to monitor whether the purchased drug is fake or not and whether the material deteriorates over time with the DNAINK method. Restriction enzyme analysis will be performed in plasmid DNA design and pHIV-EGFP plasmid will be used at this stage. Then the DNA will be mixed with the ink, and this ink will be subjected to certain tests to measure its stability and effectiveness. Afterwards, it is aimed to reach the desired stage of the project by performing DNA barcode sequencing and analysis. Within the scope of the realization of this p...

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