ACTIVE GENE CARRIER SYSTEM FOR USE IN TUMOR TREATMENTS.

Abstract

The invention relates to an active gene delivery system based on CRISPR / Cas9 technology for the treatment of various tumor types, particularly glioblastoma (GBM). This gene delivery system comprises a nanostructured lipid transporter (NLC) platform, a 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) lipid matrix containing cinnamon oil, and a pMGS7 plasmid containing the genetic sequence for Cas9 protein expression. This plasmid also contains a guide RNA (gRNA) sequence that varies depending on the target gene. With all these components, the invention enables the treatment of various cancer tumors, primarily glioblastoma, at both the molecular and genetic levels.

Description

1 TARIFF ACTIVE GENE CARRIER SYSTEM FOR USE IN TUMOR TREATMENTS. Technical Field to Which the Invention Relates 5 The invention is suitable for the treatment of different tumor types, particularly glioblastoma (GBM). It is an active gene delivery system for cinnamon oil and... N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) lipid matrix containing nanostructured lipid transporter (NLC) platform, and CRISPR / Cas9-supported double-stranded circular It contains a DNA vector. The NLC platform in question has cinnamon 10 in its formulation. With the use of the oil, the gene transfer system that is the subject of the invention becomes an active carrier. Furthermore, the aforementioned double-stranded circular DNA vector targets the Cas9 protein. target gene-specific genes that direct to the cell and vary depending on the target cell. It contains a guide RNA (gRNA) sequence. Thanks to this discovery, glioblastoma (GBM) in particular including the removal of tumors of various types of cancer from the area and those that have grown and become 15 It prevents its spread (metastasis). State of the Art Glial brain tumors are tumors that affect 20 neurons in the brain and spinal cord that provide structural and functional support to neurons. Originating from glial cells and ranging from low-grade types to high-grade types such as glioblastoma. It is a group of tumors ranging from graded aggressive types [1]. The formation of these tumors In short, genetic and epigenetic factors accumulate in glial lineage cells or progenitor cells. changes affect cell proliferation, differentiation, invasion, and treatment resistance. It is summarized as activating disruptive signal networks [2]. Especially high 25 In grade 1 gliomas, widespread infiltration, marked cellular heterogeneity, and tumor stem are characteristic. cell-like subpopulations, complete removal of the entire mass by surgery This makes it more difficult and increases the risk of relapse [3]. Therefore, treatment is of great importance. This is because these tumors not only shorten life expectancy, but also Over time, it also has a serious impact on neurological functions, cognitive status, and quality of life. This leads to losses [4]. In current techniques for treating glial brain tumors, the primary goal is to first establish safe margins. to perform maximum surgical resection to reduce the tumor burden, then 2 The goal is to apply radiotherapy to suppress residual disease and improve local control. In glioblastoma in particular, the standard approach is concomitant radiotherapy after surgery and temozolomide administration, followed by adjuvant temozolomide treatment of remaining microscopic cells. The aim is to suppress the disease and prolong survival. Surgery is recommended in selected cases. During carmustine-impregnated polymer implant placement, the drug is delivered locally to the tumor. 5 Administering it to the patient and reducing systemic exposure while increasing regional antitumor efficacy. It is used to increase [5]. The most important advantage of surgical resection is, It rapidly reduces tumor burden, lowers intracranial pressure, and provides diagnostic tissue. This is the case, but due to the diffuse and infiltrative nature of glial tumors, normal brain tissue is not affected. Since cells that spread to the tissue often cannot be completely removed, they are not permanent on their own. It is unable to provide control. On the other hand, radiotherapy targets residual tumor cells. It is a fundamental method in that it strengthens local control by suppressing it, however risk of damage to surrounding healthy tissue, neurocognitive effects, and radioresistance developing over time. It has certain limitations. Chemotherapeutic agents such as temozolomide, however, are standard. It can prolong survival as part of treatment, but the drug crosses the blood-brain barrier 15 efficacy is restricted due to its ability to pass through, systemic toxicity, and especially MGMT-related resistance. It remains limited in many patients. In some patients, these are known as tumor treatment areas. The electric field-based TTFields approach studies mitotic processes in dividing tumor cells. It is used to provide additional benefit to standard treatment by disrupting [6]. Molecular Targeted therapies are also used in selected glial tumors; for example, 20 In grade 2 astrocytomas and oligodendrogliomas carrying IDH1 or IDH2 mutations Vorasidenib slows tumor growth and reduces the need for more toxic additional therapies. It is implemented to delay [7]. The advantage of the TTFields approach is that the standard a drug that can be added to treatment and is generally well tolerated, requiring no administration to the body It is a method, however, the device requires long-term and regular use, patient 25 It creates problems such as dependence on compatibility and accessibility. Gene carrier In approaches using systems, the aim is to identify therapeutic genes, RNA payloads, or genes. By delivering regulatory components to tumor cells, tumor suppressor pathways are reactivated. to activate, silence oncogenic signals, reduce resistance to chemotherapy and radiotherapy The aim is to reduce or strengthen the immune response. In this context, adenovirus and AAV 30 In addition to viral vectors, lipid, polymer and nanoparticle-based nonviral vectors are also used. carriers also specifically aim to overcome the blood-brain barrier and increase safety. is being developed [8]. 3 Gene delivery systems stand out among these existing techniques because... The fundamental problem is not just reducing the tumor mass, but also the tumor itself. the cell's genetic program, treatment resistance, and immune evasion mechanisms It considers this as direct targeting. It also targets viral and non-viral gene carriers. platforms, therapeutic genes, small interfering RNA (siRNA) payloads, or gene 5 more selectively deliver regulatory components to the tumor site and blood-brain pathways. its potential to overcome the barrier, makes this approach superior to classical surgery, radiotherapy and systemic This makes it a more flexible and future-oriented strategy compared to chemotherapy. Gen In the simplest terms, delivery systems safely deliver the therapeutic genetic payload to tumor cells. and are transport platforms designed to transport in a functional manner. Glioblastoma 10 The importance of these systems lies in the fact that the tumor is very aggressive, highly infiltrative, genetically heterogeneous and prone to developing resistance to classical treatments Therefore, it not only kills cells but also alters tumor biology at the molecular level. It also allows for modification. In glioblastoma, this is done with gene delivery systems. Among the main goals of the studies is the recovery of tumor suppressor genes, 15 silencing oncogenes, reducing resistance to chemotherapy and radiotherapy, tumor This includes targeting stem cells and enhancing antitumor immunity. [8]. Therefore, the systems used in this field are not only “carriers” but also Tumor selectivity, crossing the blood-brain barrier, intracellular entry, endosomal escape, and Multi-component therapeutic platforms that attempt to solve functions such as controlled release together 20 has become [9]. Delivery systems developed for glioblastoma are generally classified as viral and non-viral. They are divided into two main groups [8]. Viral systems include adenoviruses, lentiviruses and AAVs. Vectors are used; these usually contain a therapeutic DNA cassette, this A promoter and the necessary regulatory sequences are inserted to execute it, so that 25 After entering the carrier cell, a missing or suppressed function is restored. can be created [9]. In this context, tumor suppressors such as p53 can be created in glioblastoma [9]. Gene delivery, suicide gene systems such as HSV-tk, and ganciclovir-like precursors the conversion of drugs into toxic forms within cells or the activation of immune-stimulating genes in tumors It is attempted to transfer it to the bed [8]. In non-viral systems, liposomes, lipid 30 nanoparticles, polymeric nanoparticles, dendrimers, polymeric micelles, gold Nanoparticles and exosomes stand out. Among these systems are plasmid DNA, mRNA, siRNA, microRNA, antisense oligonucleotides, or CRISPR components 4 It can be transported. For example, the lysosomal system called SGT-53 has wild-type p53 in its nucleus. It carries plasmid DNA and, thanks to the transferrin on its surface, interacts with both blood and brain. transferrin receptors in both the barrier and glioblastoma cells It targets Bcl2L12 in the gold nanoparticle-based system called NU-0129. By loading siRNA against the oncogene, the tumor cell's 5 The survival advantage is being targeted. Arginine-glycine-aspartic acid (RGD), Tumor necrosis in polymeric systems such as polyethylene glycol (PEG) and polyethyleneimine (PEI). factor-associated apoptosis-inducing ligand (TRAIL) gene transported integrins Targeting rich glioblastoma tissue is being increased, while miR-145 is used on other platforms. Proliferation using anti-HIF-1α siRNA or STAT3-suppressing RNA payloads, 10 Invasion, stem cell characteristics, and resistance mechanisms are weakened. Exosome In carriers based on natural vesicle structure, proteins, lipids and RNA are transported. The cargoes are transported to the tumor in a biocompatible manner, and these systems are particularly effective in the blood-brain pathway. They are noteworthy because they can overcome the barrier [8]. Because glioblastoma is one of the most aggressive tumors, it has been studied extensively in recent years. CRISPR / Cas9-based transport systems are also attracting particular attention. Here, the carriers It mostly contains Cas9 mRNA or Cas9 protein along with target-specific sgRNA. It is involved and the aim is to interrupt, silence, or reduce tumor resistance by targeting a specific gene. This involves disabling the pathways that play a role. In experimental studies, O6- methylguanine-DNA methyltransferase (MGMT), aldehyde dehydrogenase 1-A3 (ALDH1A3), 20 Temozolomide or mucin 1 (MUC1) and other resistance-associated genes are targeted. Reducing radiotherapy resistance, as well as playing a role in tumor microenvironment and immune evasion. The aim is to regulate the pathways that play

[10] . Cas9 with lipid nanoparticles. mRNA and sgRNA can be transported together, and this is particularly evident in glioblastoma stem cells. It has also been shown that gene editing can be performed in vivo. This means that treatment targets can be directly targeted. It is important for verification within the tumor. However, this area... There are significant shortcomings that need to be addressed. The first major problem is the carrier's systemic... when administered, it does not reach the tumor in sufficient quantities and crosses the blood-brain barrier, affecting the brain and tumor. Due to the barrier and irregular intratumoral vascularization, the therapeutic load is not homogeneous. The first problem is that it does not disperse. The second problem is that the polymeric systems used (e.g., PEI) have a high 30°C. This leads to cytotoxicity. The third problem, especially with CRISPR and some viral... Off-target effects in systems, immunogenicity, difficulty with repeat dosing, and long-term risks. These are security uncertainties. One of the prominent studies conducted within this scope is that of Küçüktürkmen and Bozkır. It is a cationic solid lipid nanoparticle (cSLN) system that he developed for glioblastoma. This The carrier used in the study contained both pemetrexed and miR-21 antisense oligonucleotide. (anti-miR-21) is loaded into the system. The aim of this system is to treat glioblastoma with a chemotherapeutic agent. The goal is to enhance the antitumor effect by combining gene silencing approaches within the same transporter. 5 The main components of this carrier are a solid lipid core, cationic lipid / surface, and surfactant. These consist of a stabilization layer and therapeutic loads. Solid materials are used in SLN / cSLN platforms. The lipid core provides physical stability to the carrier by maintaining structural integrity and is hydrophobic. It helps in the retention of drugs. The cationic surface, on the other hand, is associated with negatively charged oligonucleotides. It facilitates nucleic acid charging by establishing electrostatic interactions. In addition, 10 Surfactants contribute to the preservation of particle size and dispersion stability. It contributes. In this example, pemetrexed, the first therapeutic element placed inside the carrier, It was used to provide a classic cytotoxic / antimetabolite effect, the second element being anti- miR-21, which is associated with oncogenic behavior in glioblastoma, suppression of miR-21. It aims to achieve this. The summary of the study shows that cSLNs can encapsulate these two payloads together and 15 It states that this is being evaluated in vitro for the treatment of glioblastoma. Furthermore, this... The biological rationale behind the approach is that miR-21 suppression is used in chemotherapy. Its ability to increase sensitivity and weaken resistance mechanisms. This study The subject is cationic lipid nanoparticles that primarily target anti-miR-21 without being degraded intracellularly. to protect, to facilitate its approach to the cell membrane, and to allow it to be released alone 20 It aims to increase the entry of oligonucleotides into the cell. At the same time... Having pemetrexed on the same platform allows two different therapeutic approaches to be delivered to the same cell. The aim is to provide a more coordinated delivery of these types of lipid nanoparticles for glioma / GBM. The reason for its preference in this field is generally its biocompatibility, high drug / nuclear compatibility. acid transport potential and increased tumor accumulation via BBB passage in brain tumors 25 They show potential

[11] . However, the biological carrier of this study Its effectiveness is largely due to miR-21 suppression combined with pemetrexedin administration. Because of its reliance on heterogeneous subpopulations within the tumor, this system, in particular To what extent are stem cell-like resistant glioblastoma cells and invasive cells present? It is unclear what it encompasses. The system in this study does not permanently edit the genome. 30 and primarily provides temporary gene silencing and drug-based biosuppression. In contrast, CRISPR / Cas9 systems, when properly designed, can directly target a specific gene. more permanent molecular by cutting, disabling or regulating It can produce results. 6 Another example of the most concrete gene delivery systems developed against glioblastoma is: This is a 2014 study by Kim and colleagues. The main hypothesis of this study is: wild-type transported within a lysosomal nanocomplex targeting the transferrin receptor p53 plasmid DNA disrupts the p53 axis in temozolomide-resistant glioblastoma cells. by reactivating it, it can specifically break down MGMT-mediated chemotherapy resistance and 5 Thus, it may increase sensitivity to temozolomide. In the study, the targeting surface The p53 plasmid was loaded into a cationic lysosomal nanocomplex carrying a fragment, first MGMT levels, apoptosis, and in vitro in T98G and LN-18 glioblastoma cells were determined. Temozolomide susceptibility was investigated, followed by subcutaneous and orthotopic intracranial injections in mice. Tumor penetration after intravascular application in xenograft models, tumor detection by MRI 10 Volume and survival were evaluated. The findings suggest that this system bypasses the blood-brain barrier. It can selectively reach the tumor region by bypassing the barrier, and approximately increase MGMT expression. It can reduce apoptosis by 90%, and when given in combination with temozolomide, it significantly reduces apoptosis. and significantly extended survival in TMZ-resistant orthotopic models. This has been shown. For example, in two cycles of combination therapy, some mice showed 60, 77, 137, 15 It has even been reported that it can live for up to 177 days. However, the important aspect of this study is... It has shortcomings. Firstly, the study is at the preclinical level and efficacy data are predominantly... xenograft models that do not fully represent the immune system with specific cell lines This is based on, therefore, the complete heterogeneity and immunological abnormalities in human glioblastoma. The microsystem reflectivity is limited. Secondly, the data shows that the p53 load alone is 20 It did not create a significant survival advantage; the main efficacy was with temozolomide. This shows that it emerged in combination. This also indicates that the platform alone is curatorial. This suggests that it acts more like a sensitizing tool than a sensitizing tool

[12] . Glioblastoma is one of the most aggressive types of glial brain tumors. The main reason why gene delivery systems are coming to the forefront in the treatment of this type of tumor is... The reason is that they provide not only symptomatic or cytotoxic suppression, but also tumor suppression. They have the potential to redirect biology at the molecular level. However, polymers commonly used in gene delivery systems in current technology Its high cytotoxicity poses a significant risk. In addition, the current To achieve a more lasting effect from gene delivery systems in this technique, CRISPR / Cas9 with 30 Although there are studies that support it, it is not directly related to glioblastoma. On human cell lines, a homogeneous distribution that can cross the blood-brain barrier, 7 It has low cytotoxicity and remains stable in the blood for a long time, maintaining its effect. There is a need to develop new gene delivery systems. Brief Description and Objectives of the Invention The invention analyzes the genetic makeup of various tumor types, particularly glioblastoma (GBM). It is a gene delivery system used in treatment. The gene delivery system in question... The system uses a nanostructured lipid carrier (NLC) platform, N-[1-(2,3-dioleyloxy)propyl]-N,N,N- Trimethylammonium (DOTMA) and CRISPR / Cas9-supported double-stranded circular DNA vector It includes cinnamon oil added to the NLC platform structure, to the aforementioned plasmid. In this way, a guide RNA (gRNA) is inserted into the target gene. Thus, the invention, 10 By specifically targeting tumor cells, it inhibits tumor development and proliferation. It prevents. One aim of the invention is to treat various types of tumors, particularly glioblastoma (GBM). The aim is to provide a gene delivery system for genetic therapy. Within this scope, the invention... Cationic 15-bit nanostructured lipid carrier (NLC) platform formulation using DOTMA The plasmid is loaded and forms a complex with DNA. In this way, the plasmid DNA and the gRNA it contains are protected from degradation within the cell, preventing endosomal escape. This facilitates high-rate transfection of the relevant tumor cells. The carrier system, which is taken up into target cells, contains genes carried by its plasmid. It synthesizes the Cas9 protein and gRNA molecule. This Cas9 protein, 20 Thanks to gRNA, it finds the target gene sequence and creates cuts in the oncogene. These incisions trigger the death of tumor cells, causing the tumor to be removed from the area. It cleans the area and prevents it from growing and spreading (metastasis). Therefore... The invention is particularly useful for targeting glial brain tumor cells at the genetic level, and It enables treatment. 25 Another aim of the invention is to develop gene carriers for genetic therapy of various tumor types. The aim is to ensure that the system is an active system. Located within the carrier system that is the subject of the invention. The NLC platform is formulated with cinnamon oil. Cinnamon oil has powerful anti-cancer properties. and its anti-inflammatory properties mean that the system described in the invention is not merely a passive carrier, but also This ensures that the treatment has a strengthening effect over time. Also, 30 With its antioxidant properties, it also protects the CRISPR / Cas9 plasmid DNA it carries from oxidative stress. By protecting it, it increases the targeting potential of genetic material. The gene that is the subject of the invention 8 The invention, due to the cinnamon oil present in the NLC formulation of the carrier system, It goes beyond being a passive transport device and offers a synergistic contribution to the treatment process. Another aim of the invention is to remove the carried gene from the blood and in the serum. to prevent fragmentation and thus maintain the security profile of the systemic application. The aim is to improve it. This invention, aimed at achieving this goal, physically processes DNA using a lipid matrix formulation. It acts as a shield surrounding the gene carrier that is the subject of the invention. The system is protected against serum nucleases and intracellular lysosomal degradation, and The biological half-life is extended. In addition, the cinnamon oil contained in the invention... Its antioxidant effect prevents DNA damage caused by oxidative stress. This invention... Thanks to these components, plasmid DNA remains stable in serum for 24 hours without degradation. It remains in this form. In short, the invention involves degrading the plasmid DNA it contains in the serum. It increases the safety of dose adjustment by exhibiting a protective effect against enzymes. Another aim of the invention is to achieve high transfection while simultaneously reducing cytotoxicity. The aim is to reduce. The formulation of the NLC platform of the carrier system subject to the invention. It is optimized with biocompatible and low immunogenic ingredients. Additionally, 15 The DOTMA polymer it contains, like other polymers, has a free and dense positive charge. It does not create. In addition, thanks to the lipid structure of the formulation, it is passive. Targeting enhances the targeted distribution of the inoculation within the tumor microenvironment. This allows the gene carrier system, which is the subject of the invention, to accumulate only around the tumor. Exposure to healthy tissues is being increased and reduced. 20 treated with this invention While cell morphology and safety are preserved, survival rates are also increased. is kept at a high level. Therefore, the invention is low in its repeated applications. It provides safe use through cytotoxicity and tumor-specific targeting. Another aim of the invention is to create a gene delivery system suitable for combination therapies. The invention concerns a gene delivery system that can deliver chemotherapeutic agents or RNA-based 25. Like other therapies, it can be used in combination with different types of treatment agents. It is a suitable platform for loading. Thus, the proliferation of the target tumor cell and Simultaneous intervention in the different molecular pathways it follows for metastasis This is provided. In addition, combination therapy shows that gene therapy alone is insufficient. In cases where it remains in use, it increases effectiveness, reduces the development of resistance, and lowers the level of resistance. It allows therapeutic effects to be achieved with doses and minimizes systemic side effects. It contributes to its limitation. 9 The gene delivery system described in the invention has a CRISPR / Cas9-supported structure and formulation. Thanks to the therapeutic cinnamon oil it contains, it increases the efficiency of gene transfer. This... In addition, in challenging pathologies such as glioblastoma, the treatment has a synergistic effect. It strengthens the gene delivery system that is the subject of the invention, by carrying the DNA intracellularly and... By protecting it from degrading enzymes located outside, it passively targets its specific target cells. Targeting increases transfection efficiency and, thanks to its biocompatible structure, allows for repeated transfection. It offers a safe system option even at low doses. Explanation of the Figures Figure 1. Triangular phase diagram developed for NLC-DOTMA. Figure 2. Morphological image of the NLC-DOTMA formulation. 10 Figure 3. Agarose gel images showing the binding efficiency of the NLC-DOTMA formulation. Figure 4. Protection of NLC-DOTMA-plasmid formulation against benzoase enzymes. electrophoresis image showing its effectiveness Figure 5. Serum stability of the NLC-DOTMA-plasmid formulation. electrophoresis image 15 Figure 6. Efficiency of NLC-DOTMA-plasmid formulation in U87 transfection. microscopic image showing Figure 7. MDA-MB-231 (breast cancer), U87-MG (glioblastoma), and LnCap (prostate cancer) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in cancer cell lines Graph 20 showing the test result Figure 8. 3-(4,5-dimethylthiazol- in MDA-MB-231, U87-MG and LnCap cancer cell lines. Microscope showing the test result of 2-yl)-2,5-diphenyltetrazolium bromide (MTT). images Explanation of References in Figures 25 4. M: Molecular weight marker, gRNA+Cas9+PuroR: gRNA of the TRAIL-R2 gene, The plasmid containing the Cas9 enzyme and the gene for resistance to the antibiotic Puromycin, AAVS1 gRNA+ Cas9+ eGFP: gRNA of the AVVS1 gene, Cas9 enzyme, and green fluorescent protein. Plasmid containing the (GFP) gene. 5. M: Molecular weight indicator 30 6. GFP: Green Fluorescent Protein, pDNA: plasmid DNA 7. A1: Control application on MDA-MB-231 cell line, B1: MDA-MB-231 Cell DOTMA application in the line, Lipofectamine® in C1: MDA-MB-231 cell line. Application A2: Control application in U87-MG cell line, B2: U87-MG cell DOTMA application in the line, Lipofectamine® 5 in the C2: U87-MG cell line. Application, A3: Control application in LnCap Cell line, B3: LnCap Cell DOTMA application in the line, Lipofectamine® in the C3:LnCap Cell line application Detailed Description of the Invention 10 The invention offers potential applications in the treatment of various tumor types, particularly glioblastoma (GBM). It relates to a gene delivery system intended for use. This gene delivery system,  cinnamon oil,  N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) lipid matrix, 15  cholesterol,  lipophilic base,  at least one short circuit that functions as a solvent, co-solvent and / or co-surfactant chain monohydric alcohol,  solvent or co-solvent, 20  surfactant,  emulsifier,  water phase  Nanostructured lipid carrier (NLC) platform containing and 25  at least one DNA molecule encoding at least one CRISPR-related nuclease protein polynucleotide,  Guide RNA (gRNA) specific to the target gene region to be cut  a double-stranded circular DNA vector containing It includes. The gene delivery system subject to the invention has a final product volume of 30. NLC platform contains 0.63% cinnamon oil and 0.65% DOTMA lipid by weight / volume. matrix, 0.31% cholesterol, 1.56% lipophilic base, 15.83% solvent, co-solvent and / or co- 11 At least one short-chain monohydric alcohol acting as a surfactant, 7.92% solvent or co-solvent, 7.92% surfactant, 15.83% emulsifier, 49.35% DNase / Rnase. It contains an ultrafiltered sterile water phase that does not contain glyceride as the lipophilic base. hard oil base (e.g., Suppocire® AM), polyoxyethylated castor oil as an emulsifier. oil derivative emulsifier (e.g., Cremopohor® EL), sorbitan 5 as surfactant. monolaurate, at least one short-acting compound that functions as a solvent, co-solvent and / or co-surfactant. Ethanol as a chain-based monohydric alcohol, polyethylene glycol as a solvent or co-solvent. It contains 400 (PEG400). Additionally, it contains SpCas9 within the double-stranded circular DNA vector. or SpCas9, which is one of the Cas9 isoforms such as SaCas9 or Cas12. The invention pMGS7 10 contains the genetic sequence of the SpCas9 isoform used in one of its applications. The accession number for the plasmid scaffold is Addgene, 48138. It is used in the treatment of glioblastoma. For use within a plasmid, the adeno-associated virus integration region 1 (AAVS1) or tumor necrosis factor-associated apoptosis-inducing ligand receptor 2 (TRAIL-R2) There is a guide RNA (gRNA) specific to the target gene region. The invention is one of them. In the application, the nucleotide sequence with SEQUENCE NO: 1 targeting the AAVS1 DNA locus is 15. It contains gRNA. The plasmid described in this invention has a glyceride-based hard oil base with a low melting point. This prevents the NLC formulation from being heated to very high temperatures, Cinnamon oil has a strong antioxidant effect, which contributes to the low toxicity of the NLC formulation. This is ensured by the fact that the cholesterol NLC formulation resembles the cell membrane. 20 It is used for the purpose of increasing the transfection efficiency of the invention, polyoxyethylated castor oil derivative emulsifier and sorbitan monolaurate surfactant It serves the purpose of NLC with ethanol and PEG400 as auxiliary surfactants. It enables the reduction of particle size in the formulation, while DOTMA is cationic. It is used for the purpose of applying load. In this way, plasmid 25 is placed within the carrier system. by preventing degradation within the cell and facilitating endosomal escape, the relevant tumor High levels of transfection are achieved into the cells. The method for producing the gene delivery system described in this invention is as follows: i. lipophilic base, cinnamon oil, cholesterol, functioning as co-solvent and / or co-surfactant. Seen with at least one short-chain monohydric alcohol, emulsifier, surfactant, 30 by mixing and melting the solvent or co-solvent and DOTMA substances Obtaining an oil-surfactant-co-surfactant mixture, 12 ii. the resulting oil-surfactant-co-surfactant mixture, drop by drop Emulsification by adding DNase / RNase-free ultrafiltered sterile water, iii. (ii). The emulsion obtained at the end of the process step is coated with 4oC DNase / Rnaz. Emulsion of ultrafiltered sterile water: water ratio will be 1:10 by weight / volume. added drop by drop, 5 iv. Restriction of the double-stranded circular DNA vector scaffold with a restriction enzyme, v. Inactivation of the restriction enzyme after cutting the double-stranded circular DNA vector being done, vi. double-stranded circular DNA vector cut by standard purification protocol purification, 10 vii. Two pairs of DNA that will form the target gene region-specific gRNA sequence Phosphorylation of complementary oligonucleotides and complementary regions cloning of double-stranded circular DNA vector by hybridization obtaining a suitable gRNA sequence, viii. The resulting gRNA sequence and the purified double-stranded circular DNA vector 15 ensuring ligation, ix. Transformation of ligation products into competent bacteria, x. transformed bacteria in agar petri dishes containing ampicillin being indoctrinated, xi. Selection of colonies growing on agar petri dishes and colony formation with appropriate primers. Positive colonies are identified by performing polymerase chain reaction (PCR). determination, xii. Isolation of double-stranded circular DNA vector from positive colonies, xiii. NLC of double-stranded circular DNA vector isolated from positive colonies After being added to the platform and incubated, the gene carrier 25 obtaining the system It includes the steps involved in the process. In the production method described in the invention, the materials in step (i) are based on the volume of the final product. taken as 1.56% lipophilic base by weight / volume, glyceride-based hard fat base, 0.63% Cinnamon oil, 0.31% cholesterol, 7.92% sorbitan monolaurate as a surfactant, 30 15.83% polyoxyethylated castor oil derivative emulsifier, 15.83% ethanol. It will contain 7.92% PEG400 as solvent or co-solvent and 0.65% DOTMA lipid matrix. weighed and mixed as follows and melted at 60°C, DNase / Rnaz in step (ii). 13 Ultrafiltered sterile water, which does not contain water, is added into the NLC platform in step (iii). Emulsion: Cold distilled water is added in such a way that the ratio of distilled water to emulsion is 1:10 by volume, and While adding water, the mixture is stirred continuously at 1000 rpm for 15 minutes (iv). In this step, the pMGS7 plasmid scaffold (Access No: Addgene, 48138) is mixed with the BbsI enzyme. is cut, in step (v), BbsI enzyme is inactivated at 65oC for 20 minutes, 5 (vii) In step AAVS1, nucleotide SEQUENCE NO: 2 and SEQUENCE NO: 3 specific to the target gene region gRNA oligonucleotides with specific sequences are phosphorylated by T4 polynucleotide kinase, Complementary regions are hybridized with the T4 DNA ligase enzyme and SEQUENCE NO: 1 Obtaining a gRNA sequence suitable for cloning into the pMGS7 plasmid containing the nucleotide sequence. (viii) step, the pMGS7 plasmid and gRNA sequence are ligated with T4 DNA ligase 10 ligation is achieved with the enzyme, in step (xi) SEQUENCE NO: 4 nucleotide sequence PCR using a forward primer and a back primer with a nucleotide sequence numbered 5. being carried out, step (xiii) will contain 1.85 µL NLC for 150 ng pMGS7 plasmid. A gene carrier system is obtained by incubating the mixture while shaking it at a temperature of 37°C. is being done. 15 The gene delivery system described in this invention is a delivery system based on CRISPR / Cas9 technology. Within the scope of the invention, the aforementioned double-stranded circular DNA vector The term 'vector' refers to the organ that contains the gene intended to be transferred and... Double-stranded circular DNA that allows the gene to be delivered to the target cell. It refers to molecules and is not limited to a specific plasmid. Plasmids are 20 and other double-stranded circular DNA vectors that can perform the same function It includes. In one application of the invention, as a double-stranded circular DNA vector. The pMGS7 plasmid (Access No: Addgene, 48138) is being used, and this plasmid is compatible with Cas9. SpCas9 protein, an isoform of the protein, enables intracellular expression of the protein. The sequence number contains a DNA polynucleotide sequence with 6 nucleotide sequences. The aforementioned 25 This Cas9 protein creates cuts in the target gene to form segments in the relevant oncogene. The gRNA sequence is also added to the same plasmid for target cell orientation. after being transfected into it and the carrier system, which is the subject of this invention, enters the cell nucleus Once it reaches the source, the expression of the plasmid DNA begins. The plasmid DNA... Both Cas9 protein and gRNA molecules are synthesized through expression. Cell 30 This gRNA molecule, synthesized within it, binds the Cas9 protein to the target oncogene. It directs. When the Cas9 protein reaches the target oncogene, it acts on the relevant gene. By creating cuts, it causes irreversible damage to the gene. This 14 As a result of the damage, the affected gene is deactivated, causing apoptosis in the cell. The tumor is either starting to spread or its growth (metastasis) is prevented. Figure 1 shows the requirements for the NLC formulation developed by the microemulsion method. It represents the triangular phase diagram. This triangular phase diagram is equilateral 60°. It is a diagram consisting of 5 triangles. To prepare this diagram, 5 different triangles are needed. surfactant:cosurfactant ratios: varying amounts of fat, surfactant / cosurfactant The amount of water the mixture will absorb has been determined. Then, these 6 different amounts of oil Triangular phase diagram with surfactant / co-surfactant and water-containing mixture percentages. is drawn. The midpoint of the area shown in the triangular phase diagram represents the ideal emulsion. It gives the ratio. When evaluating the midpoint, 10 parallel to each side of the triangle. Lines are drawn. Depending on the reading direction, ideal lines are drawn from the points where the parallel lines intersect. The percentage of fat, surfactant / surfactant percentage, and water percentage were determined. This diagram... the white circle corresponding to the ideal ratio of the formulation at its midpoint Point weight / volume: 1.56% glyceride-based hard oil base, 0.63% cinnamon oil, 0.31% Cholesterol, 15.83% polyoxyethylated castor oil derivative emulsifier, 7.92% sorbitan 15 monolaurate, containing 15.83% ethanol, 7.92% PEG400, 0.65% DOTMA, and 49.35% water phase. Figure 2 represents the mixture. Figure 2 shows the electron configuration of this prepared NLC formulation. This is its morphological appearance under the microscope. Accordingly, the formulation is spherical in shape and Therefore, this supports the idea that the NLC formulation was developed correctly. Figure 3, The different values ​​for the NLC platform and plasmid ratio to be combined will result in gene binding 20 It demonstrates its capacity. As can be seen from the relevant figure, the optimum plasmid is: NLC. The ratio used in the invention is 8:1. Figure 4 shows the plasmid in a medium containing the DNase enzyme. The results of the study examining how well the bound NLC-DOTMA protects the plasmid. This is shown. The TRAIL-R2 gRNA+Cas9+PuroR plasmid shown in the relevant figure is TRAIL- The gRNA of the R2 gene, the Cas9 enzyme, and the puromycin antibiotic resistance gene were analyzed in 25 cases. while the AAVS1 gRNA+ Cas9+ eGFP plasmid contains the gRNA of the AVVS1 gene, The gene that enables Cas9 enzyme and green fluorescent protein (GFP) expression. It includes. Accordingly, the 0:1 ratio is the ratio where only the plasmid is present, and DNase In the presence of the enzyme, the plasmid in question was degraded (no band). However, in the medium When the NLC platform containing DOTMA is added, 30 different formulations of the plasmid are available. It was protected from degradation in the presence of DNase at concentrations of 25%. Figure 5, 25% In the presence of fetal bovine serum (FBS), how much serum is added to the plasmid on the NLC platform? This shows that it provides stability. As a result of the obtained findings, the NLC plasmid It has been proven to provide protection. Figure 6 shows the most commonly used in current technology. Developed with transfection agents polyethylene imine (PEI) and Lipofectamamine 2000 Transfection of the NLC platform developed within the scope of the invention with the NLC platform. Their activities were compared. In this context, both platforms used plasmids and complexes. were formed and then the U87 cell line was transfected with these complexes. 5 The images obtained with a confocal microscope are used in the current technique. Transfection agents disrupt cell morphology and are even toxic to cells. It has been observed to have an effect. However, the NLC-plasmid gene developed within the scope of the invention More cells are transfected via the carrier system, and cell morphology is improved. It appears that it is not affected. In this study, the gene transport system was monitored in cell 10 a protein that will synthesize a green fluorescent protein (GFP) when it enters the nucleus It was made using a plasmid. Figures 7-8, 3-(4,5-dimethylthiazol-2-yl)-2,5- This shows the result of the diphenyltetrazolium bromide (MTT) test. MDA-MB-231 (breast in cancer cell lines), U87-MG (glioblastoma) and LnCap (prostate cancer) The invention states that the NLC platform maintains cell viability at a rate above 70%. This is shown. The same analysis results also show the toxic effects of Lipofectamamine 2000. This demonstrates that these results are consistent with the agents currently used in the existing technique. compared, the gene delivery system that is the subject of the invention has higher transfection and cell transmission rates. This demonstrates low toxicity effects. Furthermore, these experiments... As a result, the gene delivery system that is the subject of the invention can be used not only under laboratory conditions, but also under GBM 20 This has been shown to be effective even in aggressive types of cancer. In general, the CRISPR / Cas9 plasmid is anionic, meaning it is negatively charged. Electrostatic repulsion across the cell membrane makes it difficult for the cell to take uptake into the cell. In addition, the plasmid is rapidly degraded by endonucleases present in the serum, This disrupts its stability in circulation. To prevent this, the invention 25 Within this scope, a plasmid with an NLC platform formulation containing a DOTMA lipid matrix. a complex is formed. Thanks to this lipid complex, the plasmid enzymatically Its stability is ensured by protecting it from degradation, and it is protected by a negatively charged cell membrane. It provides imaging. In addition, the gene delivery system that is the subject of the invention is an NLC platform. The antioxidant property of cinnamon oil, which is included in the formulation, also affects the oxidative plasmid 30. It contributes to its stability by protecting it from stress. Serum stability test results According to the invention, the gene delivery system in question can show its effectiveness in serum for up to 24 hours. It continues. 16 As lyoprotectants in gene-loaded and unloaded NLC-DOTMA nanoparticles. Trehalose, sucrose, and mannitol are added at percentages of 30, 40, and 50% w / v, respectively. It has been processed and optimized. The lyophilized dry powders are nuclease-free. Redispersed with extra pure water and diluted at a ratio of 1:100, particle size (PS), Particle size distribution (PDI) and zeta potential values ​​at 15 seconds, 30 seconds, and 60 seconds. The nanoparticles were analyzed at mixing times of seconds. The nanoparticles were prepared in the systems. Expected properties for lyophilized products include cake appearance and short-term hydration. These include processing times. In addition, redispersed dispersions that cannot be lyophilized The liquid formulation is also expected to retain all its properties. Hydration The duration is 10, and it should be fast, reliable, and consistent in the hands of the user in the clinic. Ideally, the products should be added immediately after the hydration medium is added. It needs to be rehydrated and disintegrate spontaneously. A lyophilized product. If it does not dissolve and disperse completely, the excipients that are in an undissolved state It needs to be filtered before application, which results in filtered nanoparticles. This substance causes dose loss. 15 Table 1. Results of using 30-40-50% Trehalose as a lyoprotectant. Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 30% Trehalose NLC-DOTMA- pDNA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 second 210.40 ± 16.91 0.577 ± 0.012 18.3 ± 1.05 second 265.30 ± 20.27 0.670 ± 0.074 14.20 ± 0.577 1 minute 286.20 ± 27.45 0.699 ± 0.150 10.10 ± 0.837 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 40% Trehalose NLC-DOTMA- pDNA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 165.40 ± 6.79 0.285 ± 0.011 32.10 ± 2.20 seconds 147.50 ± 8.54 0.242 ± 0.010 30.70 ± 0.82 1 minute 159.60 ± 3.69 0.242 ± 0.057 31.70 ± 0.91 17 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 50% Trehalose NLC-DOTMA- pDNA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 second 164.70 ± 12.85 0.359 ± 0.070 31.50 ± 0.83 seconds 159.00 ± 2.40 0.342 ± 0.065 30.60 ± 0.49 1 minute 167.70 ± 1.80 0.248 ± 0.023 31.20 ± 0.90 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 40% Trehalose Gen-NLC-DOTMA- PDNA Beginning 127.00 ± 14.03 0.455 ± 0.175 13.00 ± 0.32 Lyophilization post- 215.70 ± 14.31 0.549 ± 0.043 -9.28 ± 0.25 Table 2. Results of using 30-40-50% sucrose as a lyoprotectant. Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 30% Sucrose NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 102.90 ± 2.38 0.305 ± 0.021 25.50 ± 1.47 seconds 105.90 ± 3.46 0.358 ± 0.050 23.40 ± 0.32 1 minute 97.24 ± 4.95 0.334 ± 0.057 18.70 ± 3.61 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 40% Sucrose NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 94.07 ± 1.49 0.309 ± 0.008 23.90 ± 0.47 seconds 87.92 ± 2.63 0.324 ± 0.033 22.60 ± 0.87 1 minute 96.91 ± 3.18 0.323 ± 0.106 21.00 ± 0.36 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 50% Sucrose NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 95.62 ± 1.47 0.470 ± 0.087 23.30 ± 0.12 18 seconds 95.91 ± 2.38 0.494 ± 0.013 22.50 ± 0.57 1 minute 93.71 ± 1.90 0.454 ± 0.013 20.00 ± 0.72 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 40% Sucrose Gen-NLC- DOTMA Beginning 127.00 ± 14.03 0.455 ± 0.175 13.00 ± 0.32 Lyophilization post- 153.40 ± 4.51 0.491 ± 0.044 4.03 ± 0.98 Table 3. Results of using 30-40-50% Mannitol as a lyoprotectant. Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 30% Mannitol NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 193.30 ± 9.56 0.479 ± 0.068 23.40 ± 0.346 seconds 180.50 ± 5.20 0.578 ± 0.029 24.50 ± 0.67 1 minute 190.40 ± 6.79 0.682 ± 0.112 22.90 ± 0.71 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 40% Mannitol NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 second 183.50 ± 12.19 0.561 ± 0.035 32.40 ± 1.38 seconds 191.50 ± 8.03 0.512 ± 0.084 31.40 ± 1.04 1 minute 166.20 ± 5.29 0.473 ± 0.019 30.70 ± 0.10 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 50% Mannitol NLC-DOTMA Initial 123.9 ± 1.552 0.305 ± 0.023 35.70 ± 1.18 seconds 189.00 ± 6.78 0.400 ± 0.031 26.30 ± 0.15 seconds 196.90 ± 9.06 0.429 ± 0.040 23.70 ± 0.25 19 1 minute 194.00 ± 9.10 0.441 ± 0.065 20.90 ± 1.25 Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 30% Mannitol Gen-NLC- DOTMA Beginning 127.00 ± 14.03 0.455 ± 0.175 13.00 ± 0.32 Lyophilization post- 317.00 ± 32.76 0.576 ± 0.016 4.19 ± 1.32 Table 4. Results of using 20% ​​Trehalose and 20% Sucrose as lyoprotectants. Formulation Time PB (nm) ± SS PDI ± SS ZP (mV) ± SS 20% Trehalose- 20% Sucrose Gen-NLC- DOTMA Beginning 127.00 ± 14.03 0.455 ± 0.175 13.00 ± 0.32 Lyophilization post- 271.20 ± 4.30 0.511 ± 0.047 -2.99 ± 0.51 When the findings in Tables 1-4 are evaluated, with 30%, 40% and 50% (w / v) trehalose Lyophilized NLC-DOTMA nanoparticle formulations, within themselves compared. Zeta potential values ​​to the baseline value (35.70 ± 1.18 mV) Except for formulations that remain close and those containing 30% trehalose, zeta is used in other formulations. Its potential was determined to vary between 30.70 ± 0.82 and 35.70 ± 1.18 mV. Also, 10 Significant changes in particle size and PDI values ​​in time-dependent measurements. It has been observed that formulations containing 40% trehalose do not show PDI. their values ​​are lower compared to formulations containing 30% and 50% trehalose Therefore, this formulation was chosen as the ideal cryoprotectant concentration. NLC-DOTMA nanoparticles 15 prepared using 30%, 40% and 50% sucrose When the formulations were compared, the zeta potential values ​​in all formulations were different. It decreased from the initial value (35.70 ± 1.18 mV) to 18.70 ± 3.61 and 23.90 ± 0.47 It has been determined that it varies between mV. In time-dependent measurements, 40% sucrose-containing PDI values ​​of lyophilized nanoparticles compared to formulations containing 50% sucrose It has been observed that formulations containing 30% and 40% sucrose remain more stable. Particle size, PDI, and zeta potential showed similar results. However, In formulations containing 30% sucrose, zeta 5 is obtained after 60 seconds of mixing. Due to its potential dropping to 18.70 ± 3.61 mV, containing 40% sucrose NLC-DOTMA nanoparticle formulations were selected as the ideal formulation. 30%, NLC-DOTMA nanoparticles prepared using 40% and 50% mannitol. When the formulations are evaluated, the zeta potential values ​​are compared to the initial value. (35.70 ± 1.18 mV) decreased and varied between 20.90 ± 1.25 and 32.40 ± 1.38 mV. 10 It has been observed that the highest zeta potential values ​​were obtained in formulations containing 40% mannitol. This has been achieved. However, time-dependent measurements show that these formulations contain particles. Because its size tends to decrease, it is considered an ideal formulation. Formulations containing 30% and 50% mannitol were not evaluated in terms of particle size. They exhibited similar profiles in terms of PDI and zeta potential. However, 50% 15 Zeta potential values ​​of formulations containing mannitol with 30% mannitol Due to its lower content compared to other formulations, NLC- containing 30% mannitol. DOTMA lyophilized nanoparticles were selected as the most suitable formulation. This After this stage, ideal NLC-DOTMA formulations are prepared by gene loading. The selected compositions were 40% trehalose, 40% sucrose, 30% mannitol, 20% trehalose, and 20% 20% The mixture was lyophilized by adding sucrose w / v. Subsequently, particle size, PDI, and zeta were determined. Potential value and gene integrity were analyzed using gel electrophoresis. Gene integrity analysis performed after lyophilization revealed 40%. The lyophilization process performed using sucrose at a certain ratio best preserves the gene. It has been determined that it provides protection. 25 Table 5. Particle size of NLC-DOTMA and NLC-DOTMA-plasmid formulations. size distribution, zeta potential, and conductivity Formulation Particle Size (nm) PDI Zeta Potential (mV) Conductivity (mS / cm) NLC-DOTMA 132.65±9.83 0.47±0.05 33.65±8.84 0.08±0.02 21 NLC-DOTMA- plasmid 117.73±1.99 0.50±0.01 12.97±0.32 0.01±0.00 Table 5 shows the particle size and poldispersity index of the developed NLC formulations. (PDI), zeta potential and conductivity were investigated. Both plasmid-charged NLCs. The particle size of both the formulation and the plasmid-free formulation is greater than 200 nm. It was found to be small. This is important for the particle to cross the blood-brain barrier. 5 The PDI value of the formulations was found to be less than 0.5. This indicates that the developed formulations... It indicates that NLCs exhibit a homogeneous distribution. The zeta potential of the particles... It is considered as surface charge. The surface charge decreases with the binding of the plasmid. It has been observed. Conductivity provides information about the external phase. It contains lipids. It is a formulation whose external phase is water, despite containing a substance. 10 In addition to stability, the aforementioned NLC platform does not cause toxicity in the cell. The absence of any polymers or chemicals makes the invention chemically safe. It creates a profile. The gene delivery system that is the subject of the invention contains an NLC platform formulation. Cinnamon oil synergistically enhances the therapeutic properties of the invention. This 15 This is because the gene delivery system discussed in the invention is not merely a passive delivery system. This carrier system itself, when it enters the cell, has anti-cancer and anti-inflammatory properties. Cinnamon oil, which possesses certain properties, exerts a therapeutic effect through its release. So, the discovery involves inducing apoptosis not only at the genetic level but also at the molecular level. It includes elements that support treatment. Therefore, the gene delivery system that is the subject of the invention is 20 This provides treatment at both the genetic and molecular levels. In summary, the invention relates to various types of cancer, primarily glioblastoma (GBM). Enabling the treatment of tumors at both the molecular and genetic levels, A safe, active gene carrier with high transfection potential in terms of toxicity. The system offers 25 22 REFERENCES [1] The 2021 WHO Classification of Tumors of the Central Nervous System: A summary Neuro-Oncology, 23(8), 1231-1251. NCI Dictionary of Cancer Terms. (n.d.). Definitions of glioma and glioblastoma. National Cancer Institute. [2] Choate, KA, Pratt, EPS, Jennings, MJ, Winn, RJ, & Mann, PB (2024). 5 IDH Mutations in Glioma: Molecular, Cellular, Diagnostic, and Clinical Implications. Biology, 13(11), 885. https: / / doi.org / 10.3390 / biology13110885 [3] Pećina-Šlaus, N., & Hrašćan, R. (2024). Glioma Stem Cells-Features for New Therapy Design. Cancers, 16(8), 1557. https: / / doi.org / 10.3390 / cancers16081557 [4] Ng, A. T., Steve, T., Jamouss, K. T., Arham, A., Kawtharani, S., & Assi, H. I. (2024). 10 The challenges and clinical landscape of glioblastoma immunotherapy. CNS oncology, 13(1), 2415878. https: / / doi.org / 10.1080 / 20450907.2024.2415878 [5] National Cancer Institute. (2025, March 28). Central Nervous System Tumors Treatment (PDQ®)–Health Professional Version. [6] Serventi, J. N., & Newton, H. B. (2025). Tumor Treating Fields: An Innovative 15 Therapy for Glioblastoma and Other Solid Tumors. Journal of the advanced practitioner in oncology, 16(5), 181–191. https: / / doi.org / 10.6004 / jadpro.2025.16.5.3 [7] U.S. Food and Drug Administration. (2024, August 6). FDA approves vorasidenib for Grade 2 astrocytoma or oligodendroglioma with a susceptible IDH1 or IDH2 mutation. 20 [8] Shah, S., Green, J., Graff, SA, Li, Q., & Heiss, JD (2025). Gene Therapy for Glioblastoma Multiforme. Viruses, 17(1), 118. https: / / doi.org / 10.3390 / v17010118 [9] Taghdiri, M., & Mussolino, C. (2024). Viral and Non-Viral Systems to Deliver Genes Therapeutics to Clinical Targets. International journal of molecular sciences, 25(13), 7333. https: / / doi.org / 10.3390 / ijms25137333 25

[10] Begagić, E., Beculić, H., Đuzić, N., Džidić-Krivić, A., Pugonja, R., Muharemović, A., Jaganjac, B., Salković, N., Sefo, H., & Pojskić, M. (2024). CRISPR / Cas9-Mediated Gene Therapy for Glioblastoma: A Scoping Review. Biomedicines, 12(1), 238. https: / / doi.org / 10.3390 / biomedicines12010238 23

[11] Küçüktürkmen, B., Devrim, B., Saka, OM, Yılmaz, Ş., Arsoy, T., & Bozkır, A. (2017). Co-delivery of pemetrexed and miR-21 antisense oligonucleotide by lipid- polymer hybrid nanoparticles and effects on glioblastoma cells. Drug Development and Industrial Pharmacy, 43(1), 12-21.

[12] Kim, S. S., Rait, A., Kim, E., Pirollo, K. F., Nishida, M., Farkas, N., Dagata, J. A., 5 & Chang, E. H. (2014). A nanoparticle carrying the p53 gene targets tumors including cancer stem cells, sensitizes glioblastoma to chemotherapy and improves survival. ACS nano, 8(6), 5494–5514. https: / / doi.org / 10.1021 / nn5014484

Claims

28 xi. Selection of colonies growing on agar petri dishes and SEQUENCE NO: 4 nucleotide forward primer with sequence NO: 5 and reverse primer with sequence NO: 5 nucleotide sequence Colony polymerase chain reaction (PCR) using primer by identifying positive colonies, xii. Isolation of pMGS7 plasmids from positive colonies, 5 xiii. For every 150 ng of pMGS7 plasmid isolated from positive colonies, 1.85 After the plasmid is added to the NLC platform, which will be a µL NLC then after being shaken and incubated at 37°C, the gene obtaining the carrier system It includes the steps of the process. 10 27 14. It is a method according to claim 13, and its characteristic is; i. as 1.56% lipophilic base by weight / volume, based on final product volume. Glyceride-based hard fat base, 0.63% cinnamon oil, 0.31% cholesterol, 7.92% surface oil. active ingredient: sorbitan monolaurate, 15.83%, emulsifier. Polyoxyethylated castor oil derivative emulsifier, 15.83% solvent, co-solvent 5 and / or at least one short-chain monohydric alcohol functioning as a co-surfactant as ethanol, 7.92% solvent or co-solvent, PEG400 and 0.65%. DOTMA lipid matrix should be weighed, mixed, and heated to 60°C. Obtaining an oil-surfactant-co-surfactant mixture by melting, ii. The resulting oil-surfactant-co-surfactant mixture is then drizzled with 10 drops. Ultrafiltered sterile water, free of DNase / RNase, into the NLC platform. emulsifying by adding, iii. (ii). The emulsion obtained at the end of the process step is coated with 4oC Emulsion of ultrafiltered sterile water free of DNase / RNase: water ratio Adding drop by drop at a weight / volume ratio of 1:10, and the same 15 the mixture is continuously heated at 1000 rpm for 15 minutes. The result of mixing them is the NLC platform. iv. pMGS7 plasmid scaffold (Access No: Addgene, 48138) with BbsI enzyme cutting, v. After the pMGS7 plasmid was discontinued, the BbsI enzyme was induced at 65°C for 20 seconds. inactivation in minutes, vi. pMGS7 plasmid cut by standard purification protocol purification, vii. AAVS1 target gene region-specific nucleotide sequences 2 and 3. gRNA oligonucleotides with sequences 25 are mixed with T4 polynucleotide kinase. phosphorylation and complement of the oligonucleotides in question pMGS7 is hybridized with T4 DNA ligase enzyme from its regions. gRNA containing nucleotide sequence NO: 1 suitable for cloning into plasmid obtaining the sequence, viii. The pMGS7 plasmid purified with the obtained gRNA sequence was ligated by T4 DNA ligase 30 ligation with enzyme, ix. Transformation of ligation products into competent bacteria, x. transformed bacteria on ampicillin-containing agar culturing in petri dishes, 26 i. lipophilic base, cinnamon oil, cholesterol, solvent, co-solvent and / or co- At least one short-chain monohydric alcohol that functions as a surfactant, emulsifier, surfactant, solvent or co-solvent and DOTMA oil-surfactant-co-surfactant by mixing and melting the substances obtaining the mixture, 5 ii. the resulting oil-surfactant-co-surfactant mixture, with drops Emulsifying by adding water that does not contain DNase / RNase droplets, iii. (ii). Cold distillate is added to the emulsion obtained at the end of the process step water is added drop by drop while the mixture is continuously stirred The mixing ultimately results in the NLC platform, 10 iv. restriction of the double-stranded circular DNA vector scaffold with restriction enzyme cutting, v. restriction enzyme after the double-stranded circular DNA vector is cut inactivation, vi. Double-stranded circular DNA cut by standard purification protocol 15 purification of the vector, vii. Two pairs of DNA that will form the target gene region-specific gRNA sequence Phosphorylation of complementary oligonucleotides and complementary regions hybridized via a double-stranded circular DNA vector Obtaining a gRNA sequence suitable for cloning, 20 viii. Purified double-stranded circular DNA with the obtained gRNA sequence ensuring the ligation of the vector, ix. Transformation of ligation products into competent bacteria, x. transformed bacteria on ampicillin-containing agar Culturing in petri dishes, 25 xi. Selection of colonies growing on agar petri dishes and preparation with appropriate primers. A positive result was obtained by performing colony polymerase chain reaction (PCR). identification of colonies, xii. Isolation of double-stranded circular DNA vector from positive colonies, xiii. NLC 30 of double-stranded circular DNA vector obtained from positive colonies After being added to the platform and incubated, the gene isolation of the carrier system It includes the steps of the process. - 15.83% emulsifier, - 49.35% water phase It includes.

3. A gene delivery system according to Claim 1 or Claim 2, characterized by being surfactant. The substance is sorbitan monolaurate. 5 4. A gene delivery system according to any of claims 1-3, and its characteristic is: The emulsifier is a polyoxyethylated castor oil derivative emulsifier.

5. A gene delivery system that, according to any of claims 1-4, has the characteristic of being lipophilic. The base is a glyceride-based, hard oil base.

6. A gene delivery system with the characteristic of being a solvent, according to any of claims 1-5; 10 at least one short-chain solvent that functions as a co-solvent and / or co-surfactant The monohydric alcohol is ethanol.

7. A gene delivery system with the characteristic of being a solvent, according to any of claims 1-6. or the co-solvent is polyethylene glycol 400 (PEG400).

8. A gene delivery system according to any of claims 1-7, characterized by having a double-stranded 15 circular DNA vector, CRISPR-associated SpCas9 or SaCas9 or Cas12 at least one DNA polynucleotide that codes for at least one of the nuclease proteins It includes.

9. According to claim 8, it is a gene delivery system characterized by its double-stranded circular DNA. The vector contains the sequence number 6 nucleotide 20 encoding the CRISPR-related SpCas9 protein. DNA contains a polynucleotide sequence with the following sequence.

10. A gene delivery system according to any of claims 1-9, characterized by its double-stranded nature. Circular DNA vector, adeno-associated virus integration region 1 (AAVS1) or tumor necrosis factor-associated apoptosis-inducing ligand receptor 2 (TRAIL-R2) contains guide RNA (gRNA) specific to the target gene region. 25 11. According to claim 10, it is a gene delivery system characterized by its double-stranded circular DNA. The vector has a nucleotide sequence with SEQUENCE NO:1 specific to the AAVS1 target gene region. It contains gRNA.

12. A gene carrier system according to any of claims 1-11, characterized by its paired nature. 30-strand circular DNA vector with pMGS7 plasmid (Access No: Addgene, 48138) It is the fact that.

13. In the treatment of various tumor types, especially glioblastoma (GBM). It is a method of producing a gene delivery system for use in; its characteristic feature is: 24 REQUESTS 1. In the treatment of various tumor types, especially glioblastoma (GBM). It is a gene delivery system intended for use; its characteristic is:  cinnamon oil, 5  N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTMA) lipid matrix,  cholesterol,  lipophilic base,  at least one short circuit acting as a solvent, co-solvent and / or co-surfactant 10 chain monohydric alcohol,  solvent or co-solvent,  surfactant,  emulsifier,  water phase 15 nanostructured lipid carrier (NLC) platform containing And  at least one DNA molecule encoding at least one CRISPR-related nuclease protein polynucleotide,  Guide RNA (gRNA) specific to the target gene region to be cut 20 double-stranded circular DNA vector containing It includes.

2. According to Claim 1, various tumor types, primarily glioblastoma It is a gene delivery system intended for use in treatment; its characteristic is; word 25 the subject of the NLC platform, - 0.63% cinnamon oil, - 0.65% DOTMA lipid matrix, - 0.31% cholesterol, - 1.56% lipophilic base, 30 - 15.83% solvent, at least one acting as a co-solvent and / or co-surfactant short-chain monohydric alcohol, - 7.92% solvent or co-solvent, - 7.92% surfactant

Images