Cancer: From Genomics to Pharmaceutics

192 NEW THERAPEUTIC APPROACHES IN GENOME1. IntroductionGene therapy is the transfer of specific genetic material to alter the encoding of a geneproduct or tissues’ biological characteristics to treat various illnesses (1, 2). After discoveringthe DNA helix structure, the application of cutting-edge technology to the research enlightenedmany aspects of the functional genome, many of which are now being applied in therapeuticsettings. Several molecular approaches that support the editing of the DNA and alteringmRNA through post-transcriptional modifications have been developed over the past fewdecades. And now, the term Gene Therapy combines varying delicate techniques in whicheach should be intensively worked out and accomplished for successful treatment.Gene therapy could be reviewed in two sections:I. Gene Delivery SystemsII. Genome EditingWe will initially discuss the gene transfer techniques and delivery systems for gene therapyand then focus on genome editing.Genes are physical and functional subunits carried on chromosomes. With the specificcodes on the genes, they encode the necessary information from the cellular structure of thecells to their components and turn them into proteins. Changes in genes for various reasonscause genetic diseases by causing changes in RNA and especially protein. Gene therapy treatsdisease by transferring RNA or DNA into patients’ cells. The transfer of genetic materialcould be performed in two ways: ex vivo or in vivo.In vivo, gene therapy involves injecting the genetic material directly into the target organ,while ex vivo gene therapy involves modifying host cells before reinjecting them to the siteof action (1, 3). Both somatic and germline cells can be applied for gene therapy. The maindifference between the target cells is; in somatic cell gene therapy, only modified cells will beimpacted by such treatment. In germline cell gene therapy, genetic modifications are expectedto pass on to the progeny. Therefore, there are yet, no human germline gene therapy clinicaltrials that are approved by ethical committees (1, 4).Three goals of gene therapy: i) enabling the expression of the transferred gene in placefor the damaged gene(s), ii) inhibiting or silencing the activity of a damaged gene, or iii)modifying the target gene (1, 5, 6).To achieve such goals, initially, we will discuss the gene delivery systems for gene therapy.2. Gene Delivery Systems for Gene TherapyIn situ, the transfer of the editing machinery into individual cells and tissues involvesthe transfer of foreign genomic material into the host tissue to alter an expression of a geneproduct or alter a cell’s biological makeup for therapeutic purposes. Strategies for genetransfer could be either biological, physical, or chemical applications (Figure 1). In biologicalmethods, traditionally incompetent viruses were applied as vectors, including adenovirus,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 193adeno-associated virus, retrovirus, herpes virus, lentivirus, and baculo viruses. In recentyears, the technology has enabled the use of non-viral gene delivery methods advancingin more efficiency and less complication in transfer techniques. Such procedures could bedivided into chemical and physical approaches. Physical methods make use of physicalproperties and forces. To transport vectors that carry gene editing machinery into the cell,such as electro-, hydro-, sono- poration, enables the cell membrane to enlarge and open a pore.Gene guns and direct injection of genetic material into the cells are other physical options.Chemical methods involve specific polymers and lipids. Instead of a single method for all celltypes and treatments, strategies should be selected according to the types of target cells andexperimental requirements. In addition, the criteria for deciding the optimal method shouldinclude; easy to operate and repeat with high transfection efficiency, low cell toxicity, andminimal impact on normal physiology.Figure 1: The Strategies for Gene Delivery Systems.2.1. Biological Methods2.1.1. Viral VectorsIn gene delivery systems mediated by viruses, the main objective is the virus’s ability toinject its DNA into host cells. Their natural viral structures enable effective gene transmissionand prevent DNA degradation (7, 8). Gene delivery systems generally use retrovirus,adenovirus, adeno-associated viruses, retroviruses, and herpes simplex viruses. Viruses canbe used in germline gene delivery and somatic gene delivery. Although germline gene deliveryCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

194 NEW THERAPEUTIC APPROACHES IN GENOMEsystems have great potential, as discussed earlier, germline gene therapy could not be utilizedethically (9, 10). In studies, human gene delivery systems have been limited to only somaticcells. The viruses applied for gene transmission could be DNA- or RNA-based vectors. Viralvectors based on DNA for gene delivery are generally longer lived and integrate into genomes.DNA-based viral vectors include the vaccinia virus, adenovirus, adeno-associated virus, andherpes simplex virus (11). RNA-based viral vectors for gene delivery have been developedto transcribe infectious RNA transcripts directly. RNA-based gene delivery is not permanentand is usually temporary. In RNA-based gene delivery systems, the human foamy virus ismade with oncoretro-viral vectors and lentiviral viruses (12). Studies using viral vectors haveshown that expression in cancer cells using adenovirus causes cancer cell death via activationof p53 and mitochondrial apoptosis (13). It has also elicited antitumor immunity via oncolyticadenovirus, IFN-7- and TNF-?-Co-producing T cells expressing IL-23 and p35. Thus, it hasbeen revealed that cytokine-mediated immune gene therapy with viral vectors can be used incancer treatments (14).2.1.2. Physical MethodsPhysical methods use physical force to increase the cell membrane’s permeability and allowthe gene to enter the cell. Physical methods of gene delivery have revolutionized the efficiencyof non-viral gene transfer and, in some cases, reached the efficiency of viral vectors. Theprimary advantage of physical methods is that they are easy and reliable to use (15). However,they also have the disadvantage of causing tissue damage in some applications. Injection,electroporation, gene gun, sonoporation-mediated methods, and hydrodynamic systems arethe most commonly used physical methods.The injection: The injection of genetic material into the tissue via a syringe or a vein into thesystem. It is the simplest and safest method of gene transfer without any carrier. The mostsuitable tissues for injection are muscle, skin, liver, heart muscle, and solid tumors. Althoughit is the simplest method, the efficiency is low due to rapid degradation by nucleases in serumand clearance by the mononuclear phagocyte system (16, 17)Electroporation: Electroporation is a gene transfer system based on the principle ofopening nanometer-sized temporary pores in the cell membrane by applying a short-termand high-power electrical field to cells or tissues (18). When using this system, attentionshould be paid to the current intensity. Pore formation takes place in about ten nanoseconds.Excess current can cause a complete rupture of the cell membrane (19).Gene gun: Particle bombardment, also known as gene gunning, is a physical method ofintroducing nucleic acids into cells, where gene delivery is done directly (20). It is used totransfect cells with foreign DNA by bombarding target cells with DNA-coated microparticles.In this method, nucleic acids or other biological molecules are coated on high-density gold ortungsten microparticles, followed by a high velocity with a helium pulse to propagate throughCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 195the cell walls and membranes toward the target (21). Microparticles of ∼ 0.5-1?m in size,usually made of gold, are used in the gene gun (15, 18).Sonoporation: It is a non-viral gene delivery system for sonoporation to improve plasmidDNA transfer across biological cell membranes. It is a method that allows therapeutic DNAto enter cells through the transient permeabilization of the cell membrane and the applicationof ultrasound energy throughout the tissue (22).Hydroporation: Hydrodynamic pressure is the method used to penetrate the cell membrane.Hydrodynamic pressure is created by injecting a large volume of DNA solution quickly.This increases the permeability of the capillary endothelium and creates pores in the plasmamembrane surrounding the parenchyma cells. The relevant therapeutic gene can reach the cellthrough these pores, and these membrane pores are then closed, allowing the genetic materialto remain inside the cell. This technique is most commonly used for gene therapy research inhepatic cells (23, 24).2.2. Chemical MethodsChemical-based gene delivery methods use natural or artificial compounds to createparticles that improve the transfer of target genes into cells. Chemical vectors usually entercells via endocytosis, protecting the genetic material from degradation. The synthetic vectorcan electrostatically interact with RNA or DNA and bind to compress genetic informationin inserting larger genetic transfers (25). Generally, there are two non-viral vectors, such asliposomes and polymers (26, 27). Liposome-based non-viral vectors use liposomes to facilitategene delivery by forming lipoplexes. When negatively charged DNA comes into contact withpositively charged liposomes, lipoplexes are formed spontaneously. Polymer-based non-viralvectors use polymers to interact with DNA to create polyplexes.Liposomes: synthetic lipid spheres containing fatty acids with one or more bilayer membranestructures surrounding an aqueous core that can be used to encapsulate small molecules (28).Polymers: Polymers are long chain structures of small attached molecules called monomers.Polymers consisting of a repeating monomer are called homopolymers, and those composedof two monomers are called copolymers. Polymeric gene carriers provide genetic materialfor gene therapy via electrostatic interactions with nano-sized polyplexes (29).Liposome and polymer structures used in synthetic gene delivery should protect the negativelycharged phosphate-DNA skeleton from charge repulsion against the anionic cell surface. Inaddition, DNA, which has a macromolecular structure while being taken into the cellularsystem, must be reduced to micrometer size for endocytosis or phagocytosis and must protectthe DNA from all extracellular and intracellular nuclease degradation.3. Genome EditingGenome editing is a technique that uses nucleases to cause double-stranded breaks (DSB)at particular chromosomal locations. The nuclease-induced DSBs, which stimulate veryCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

196 NEW THERAPEUTIC APPROACHES IN GENOMEeffective cellular DNA recombination mechanisms in mammalian cells, are the precursorof targeted DNA changes by triggering cellular endogenous DNA repair and starting thegenome editing process (30-33). (Figures 2 and 3). Once DSB is formed, one of two repairmechanisms is activated in all cell types and species, which are in the cell– non-homologousend joining (NHEJ) and homology-dependent repair (HDR), both of which produce changesin DNA by resulting in either targeted integration or gene disruptions, respectively (34).During NHEJ, DNA end breaks are brought together (35, 36), and mutations create smalldeletions and insertions (37). It is used in NHEJ to disable gene function with loss-of-functionmutations. NHEJ is error-prone because it may result in nucleotide deletions or insertions inthe damaged locus. HDR requires donor DNA, which requires homology on both sides ofthe break. Since HDR uses a homologous template to repair DNA damage, it differs fromNHEJ by the double-strand break creation feature of the CRISPR/Cas9 system (38) and lesschance of causing error (39-42). It processes DSB ends to leave 3’ overhangs that occupydonor DNA at homologous regions, using it as a template for DNA synthesis in HDR. (43).For this purpose, varying types of nucleases are used to induce the DSB required for genomeediting which all be explained in the following section (44, 45).3.1. The Structure and Mechanism of Tools for Genome EditingThe development of genome editing techniques has made it possible to directly target andalter the genomic sequences in practically all eukaryotic cells. These techniques are based onbacterial or manufactured nucleases. The transition of gene editing from theory to clinicalapplication has been substantially accelerated by recent developments in programmablenucleases; based on their structural differences, there are four distinct gene-editing nucleases;Meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases(TALENs), and CRISPR-associated nucleases (34). (Figure 2). Research has centered ondeveloping various zinc finger nucleases or meganucleases to generate desired DSBs at eachDNA target site during the early stages of genome editing (34, 46, 47). These nucleasesystems provided unique tools for generating artificial proteins consisting of customizablesequence-specific DNA binding domains that bind to a non-specific nuclease for targetcleavage (34, 48). The TALENs, ZFNs, and meganuclease systems used before the discoveryof CRISPR were created by combining the DNA binding site with the non-specific nucleaseregion of the restriction enzyme whose catalytic domain is produced from bacterial proteinscalled Flavobacterium okeanokoites (FokI), have been successfully used in many differentorganisms, including plants for genome editing (49). Subsequently, transcription activator-likeeffectors were developed for precision genome editing (34, 50). Meganuclease, ZNFs, andTALENs nucleases depend on Protein-DNA interaction to cut the DNA. However, theseprotein engineering techniques are expensive, and the enzymes sometimes cut non-targetsequences, causing off-target and toxic effects. In contrast, the CRISPR- related nucleases,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 197including Cas9 apply RNA-DNA interaction, CRISPR RNA-targeted nucleases do not requireprotein engineering and rely on simply known Watson-Crick base pairing rules, making thesystem more suitable for use in genome engineering (51).Figure 2: Platforms frequently used for gene editing. Zinc Finger protein; Meganucleases; TALE;CRISPR/ CAS9.Figure 3: Genome editing with double-stranded breaks (DSB) and cellular endogenous DNA repairprocesses homology-directed repair (HDR) and non-homologous end-joining (NHEJ).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

198 NEW THERAPEUTIC APPROACHES IN GENOME3.2. Meganucleases (MNs)The era of genome editing began with the advent of meganucleases (MNs), the firstbiological molecules to be utilized for precise gene modification (52, 53). MNs can carryout therapeutic tasks, including gene editing and therapeutic gene splicing, thanks to theircapacity to detect and cleave target genes. MNs are another name for homing endonucleases.It is natively expressed by specific homing endonuclease genes found in microorganisms anddetects lengthy, asymmetric DNA sequences (20–30 bp). At the target site (14–40 bp), targetsites of interest are recognized by sequence-specific endonucleases and the encoded enzymeproduces DSBs that HDR or NHEJ can repair. Thus, the KO gene mutation results whenexogenous DNA is added (53-55). Because of its minimal cytotoxicity, it is dependable forgenome editing. The synthesis of fusion proteins using existing MN domains and the directmodification of protein residues in the DNA-binding domain are two engineering strategiesnow in use.On the other hand, numerous drawbacks prevent using meganucleases in individualizedanti-cancer therapy (52, 56, 57). Firstly, specific oncological mutations that cause cancer andsuch genetic variations vary from patient to patient. Using natural MNs to fix cancer-relatedmutations is challenging since they can cause DSBs at specific recognition sites that are notjust present in oncological genes (52, 56, 57). The usage of MNs is constrained by thedifficulty of restructuring and poor regulatory effectiveness (1, 58). However, since theycan target sequences outside of their natural recognition sites with a high significance level,designed MNs can get around this issue. MN engineering approaches will therefore increasethe viability of individualized anti-cancer treatments. The efficiency of gene targeting couldbe higher. MN reportedly had a targeting efficiency as high as 66% in the human 293H cellline (52, 59), despite most research having only seen 1%–20% efficiency in human cells (52,60, 61). In addition, off-target issues hinder the use of mega-nuclease-based applications forpersonalized anti-cancer therapies, mainly correcting cancer-causing somatic mutations (52,62, 63).3.3. Zinc Finger Nucleases (ZFNs)The first custom DNA nucleases, ZFNs, are programmable synthetic proteins that attachto particular DNA regions and use DNA endonucleases to produce DSBs on DNA, whichare repaired through HDR and NHEJ, making genome engineering possible (45, 55, 64).(Figure 3). These enzymes are fusions of two domains: zinc finger repeats consisting of sixor more fingers that can recognize approximately 9-18 bp, and restriction FokI endonuclease,which dimerizes to target and cleave regions of DNA naturally found in bacteria. HDR orNHEJ mechanisms can fix these breaks (55, 65-67). The zinc finger protein with site-specificbinding to DNA was discovered in Xenopus oocytes as part of transcription factor IIIa (68).The zinc-finger domain contains a series of Cys2-His2-ZFs; It is the DNA recognition domainCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 199and is considered the most common type of DNA binding motif in eukaryotic transcriptionfactors (69, 70). Each zinc finger unit recognizes three base pairs in DNA. FokI endonucleasemust be dimerized to cleave DNA, requiring two ZFN molecules to bind. After DNA splicingby ZFNs, DSBs in a specific part of the genome and desired changes are created in NHEJor HDR repair systems (34, 68) and created synthetically by combining a site-specific zincfinger protein with the FokI restriction endonuclease’s non-specific cleavage domain. Tocreate a three-fingered sequence that binds to the nine base pair target site and non-specificcleavage site, ZFN has three zinc fingers, each defining three base pairs of DNA sequence.(58, 71). Off-target breakage may occur if ZFNs are not specific at the target site. Suchoff-target breakdown may lead to DBS, which can cause cell death. An off-target break mayaid the random integration of donor DNA. (1, 71, 72) Since ZFPs are eukaryotic tiny proteinmotifs that may bind DNA in a sequence-specific way and are controlled by zinc ions, theirdiscovery fundamentally altered the scenario of genome editing. They control the expressionof endogenous genes when fused to ZFP-TFs, which are transcriptional activators or repressors(50, 73-75).3.4. Transcription Activator-Like Effector Nucleases (TALENs)The subsequent development is the programmable nuclease, TALEN, made possibleby the transcription activator-like effector (TALE) proteins secreted from Xanthomonasbacteria. These proteins precisely recognize one base instead of three bases (50, 73-75).The DNA-binding domain of TALE effectors has a conserved 33- 35 amino acid repeatsequence with 12th and 13th amino acids (1). They are created by joining a DNA cleavagedomain to a TAL effector DNA binding domain. Such fusion enzymes contain two essentialand distinct domains, capable of splicing specific DNA sequences (14-20 bp); when used witha nuclease, TALEs can be created to attach to virtually any desired DNA sequence and causeprecise DNA splicing (76). The catalytic site for DNA binding and DNA cleavage is similarto ZFNs. DNA regions of interest can be targeted and cut for the knockout or knock in genesusing TALENs. (55, 77). ZFN and TALEN both require an addition of plasmids transfectedinto cells. It is an engineered nuclease strain that is more specific and exhibits better activitythan ZFNs. To obtain high activity, it appears that the number of bases between two differentTALEN binding sites and the number of amino acid residues between the FokI cleavage siteand the TALE DNA binding site are critical factors (78, 79). After the TALEN components arecombined, they are added to the plasmids, and the target cells are transfected with the plasmidsto ensure the expression of gene products. TALEN constructs can also be delivered to cellsas mRNA, thus eliminating the possibility of genomic integration of the TALEN-expressingprotein. Using the mRNA vector can also significantly increase the success of gene editing byhomology-directed repair (79). ZFN/TALEN is copied and translated into proteins that enterthe nucleus and bind to DNA to cleave the target sequence. Non-homologous end joiningCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

200 NEW THERAPEUTIC APPROACHES IN GENOMEbinds directly to DNA from both sides of the DSB with little or no sequence overlap forannealing. This repair mechanism can render the encoded gene products dysfunctional bycausing errors in the genome through indels. The off-target activity of an active nuclease canlead to unwanted double-strand breaks, resulting in chromosomal rearrangements and/or celldeath (79). Since homology-directed repair transfected double-stranded sequences are usedas templates, foreign DNA can be inserted into the DSB. Repair enzymes can produce the KOgene, or synthetic DNA can be incorporated to create the KI gene. (45, 55, 80).3.5. CRISPR-Cas SystemThe clustered, regularly spaced short palindromic repeats (CRISPR) andCRISPR-associated protein 9 (Cas9) system (CRISPR-Cas9) is an adaptive defense systemseen in bacteria and archaea. This system has many innate immune-like systems, such asreceptor mutation, restriction modification in bacteria, and features that provide acquiredimmunity against specific and exogenous genetic elements (81).CRISPR repeat sequencesbegan in 1987 when Ishino and his team first noticed unique and repetitive CRISPR sequencesin the genome of Escherichia coli during their study of genes involved in phosphate metabolism(82). In those years, the functions of these sequences remained mysterious due to the need forgenome projects and insufficient data. In 1993, when the identical CRISPR sequences weredetected in Haloferax mediterranei archaea, he started studies on the human genome (83).As the investigations progressed, it was realized that bacteria and archaea had regions withthe same genomic sequence as the bacteriophages and viruses that infect them (83). Withthese developments, studies on the status of CRISPR sequences of different groups began in2005, and in 2012 Jennifer Doudna and her team discovered that some groups of clusters inbacteria were identical to the sequence in the viruses they infect (84). The study developed amechanism using the type II Cas9 protein obtained from Streptococcus pyogenes (SpCas9),which can cut and regulate the double-stranded DNA in the cell (85). In this mechanism,Cas9 created double-strand breaks at genomic loci under the guidance of programmable guideRNA, that is, Cas9 was shown to cut precisely like DNA endonuclease (86). In two separatestudies conducted in 2013, the use of CRISPR systems in genome editing engineering with Casproteins obtained from Streptococcus thermophilus and Streptococcus pyogenes for genomeediting in mammalian cells was successfully carried out (87, 88). Under the leadership ofthese studies, many researchers used the CRISPR technique for gene editing.3.5.1. CRISPR ClassificationCRISPR systems have many variable features in various categories, such as proteincomposition, pre-crRNA processing and interference, genome locus architecture, adaptationmechanisms and complex effector structure, etc. The main reason for the variability andrapid evolution of CRISPR/Cas systems has been the rapid evolution of Cas genes’ constantwarfare with viruses(89). Comparative sequence analysis, experimental data, and structuralCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 201studies show that evolutionarily all CRISPR/Cas variants exhibit common architectural andfunctional principles, and the basic building blocks are descended from common ancestry.Cas proteins interact with nucleic acids such as nucleases, helicases, and RNA-bindingproteins in the CRISPR system. Cas1 and Cas2 proteins play a role in adaptation, and theseproteins are present in all CRISPR-Cas systems (90). Other Cas proteins are associated onlywith certain types of CRISPR-Cas systems. According to the organization of the CRISPRregion, the content of Cas genes is divided into two classes; Class 1 and Class 2, and gatheredunder 6 types (91) (Table 1). The main difference between the two classes is the effectormolecules (92). Class 1 effectors contain more than one subunit, while class 2 effectorsconsist of single large proteins (93). The specific types in each class depend on the particularCas endonuclease responsible for cleavage and its mechanism of action. The molecularmechanism of each CRISPR type is unique, as Cas proteins are responsible for crRNAbiogenesis and the recognition and degradation of invasive nucleic acids (94).Table 1: CRISPR Classification # of subtypes Cas endonuclease Target Requires tracrRNA? type 1 7 cas3 DNA no Class l type III 4 cas10 DNA/RNA no type IV* 1 -- -- -- type II 3 cas9 DNA yes Class 2 type V 3 cas12 DNA yes (1 subtype) type VI 3 cas13 RNA no *putative subtype 3.5.2. CRISPR/Cas9 MechanismIn the CRISPR/Cas9 system, initially detected in prokaryotic cells, the DNA fragmentsformed due to the fragmentation of the foreign DNA entering the cell for the first timeare included as intermediate sections between the repeat sequences in the bacterial/archaealgenome (84). The CRISPR sequences thus formed in case another virus or plasmid containingthe identical sequences in its genome enters the cell, cleaves the foreign DNA through theCas9 enzyme, which is an adaptive immune response mechanism, and renders it ineffective(95). The system briefly synthesizes foreign precursor CRISPR RNA (pre-crRNA) from theCRISPR sequence. It processes it into crRNA, the complementary region of the pre-crRNAto the relevant foreign DNA. After synthesizing a complementary trans-CRISPR RNA(tracrRNA) to crRNA, crRNA, and tracrRNA form a complex and bind to the region wherecrRNA is complementary to foreign DNA, and Cas9 endonuclease creates a double-strandbreak in DNA in the relevant region (96). Applying the CRISPR/Cas9 method in eukaryotesonly requires a guide RNA (gRNA) that mimics the crRNA tracrRNA complex and a Cas9Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

202 NEW THERAPEUTIC APPROACHES IN GENOMEprotein complex with nuclease activity (97). (Figure 4).Targeting specificity depends mainly on the gRNA sequence of about 20 bases; changesof a few bases in this sequence can affect specificity. For Cas9 to function, the enzymerequires several specific sequences of bases, known as protospacer adjacent motifs (PAMs)(98). The Cas9-sgRNA (Cas9-single guide RNA) complex searches the genome’s PAMsequence, unwinds the dsDNA, and DNA-RNA base pairing occurs. During double-strandbreak formation, two nuclease subunits (HNH and RuvC) come into play and cut DNAstrands independently of each other (94). For the CRISPR construct to be active, thepresence of CRISPR-related (Cas) genes adjacent to CRISPR sequences and encoding proteinsfor immune response is required. These characteristic CRISPR sequences are formed byinserting non-repeating sequences derived from short segments of foreign genetic materialinto repetitive sequences (99). In other words, certain parts of the virus DNA that infectthe bacteria are inserted into the CRISPR region with their repeat genes. Another featureassociated with CRISPR regions is the presence of conserved sequences, called leaders, thatlie behind CRISPR according to the transcription direction. The most crucial point in editingthe genome with CRISPR/Cas9 is the selective targeting of a particular DNA sequence. Forthis, Cas9 protein and guide RNA form a complex to identify target sequences with highselectivity. The Cas9 protein is responsible for finding and fragmenting target DNA in bothnatural and artificial CRISPR/Cas systems. It consists of six units of the Cas9 protein, namelyREC I, REC II, Bridge Helix, PAM Interacting, HNH, and RuvC (100). Rec I is the largestunit responsible for binding guide RNA. The existence of the REC II area has been identified,but its role still needs to be fully understood. The arginine-rich bridge Helix helix is crucialfor initiating cleavage activity by binding of target DNA (99). The PAM-Interaction domainconfers PAM specificity and is therefore responsible for starting binding to target DNA (101,102). The HNH and RuvC domains are nuclease domains that cut single-stranded DNA. Theyare similar to other proteins’ HNH and RuvC domains (103) (Figure 4).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 203Figure 4: Activation of Cas9 Protein Complex. Adopted from (100)In the absence of guide RNA, Cas9 protein remains inactive (100). In engineered CRISPRsystems, guide RNA consists of a single strand of RNA with a T-shape consisting of a tetraloopand two or three stem-loops (99). The 5 end of the guide RNA is designed to be the targetcomplementary region that is complementary to the target DNA sequence. This artificialguide RNA binds to the Cas9 protein, causing a conformational change in the protein uponbinding. The conformational change converts the inactive protein into its active form. Themechanism of conformational change is not fully understood. Still, Jinek et al. hypothesizethat sterile interactions or weak binding between protein side chains and RNA bases maytrigger the difference (101). Following the Cas9 protein activation, the search for targetDNA is initiated by binding with sequences that match the adjacent protospacer motif (PAM)sequence (104). The PAM sequence is a two- or three-base sequence located one nucleotidedownstream of the complementary region of the guide RNA (104). The PAM sequence inStreptococcus pyogenes is 5’-NGG-3 (105). When the Cas9 protein finds the target sequencewith the appropriate PAM, it will dissolve the bases just upstream of the PAM and pair themwith the complementary region in the guide RNA (102). If the complement and target sitesmatch correctly, the RuvC and HNH nuclease domains will cut the target DNA after the thirdnucleotide base upstream of the PAM sequence (106).CRISPR sequences and Cas genes have a universal anatomy, although they may differbetween species. When we look at the CRISPR universal anatomy, palindromic repeatsequences are found first. The length of the repeat sequences varies between 4–47 base pairs(bp) and often contains short 5–7 nt palindromic repeats. Palindromic sequences are stable,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

204 NEW THERAPEUTIC APPROACHES IN GENOMEhighly conserved sequences that contribute to forming the RNA stemloop secondary structure.The repeat regions of the CRISPR genome can be several or hundreds. CRISPR repeats areseparated from each other by specific unique sequences called Spacer DNA regions. Thelength of SPACER DNAs with high variability of the CRISPR locus varies between 26-72bp and can be found more than once in the genome. Spacer DNA is specific DNA sequencescontaining fragments of nucleic acids of plasmids or viruses. Spacer DNA sequences, whichfunction as recognition sequences, are responsible for preventing viruses from infecting thehost by acting as memory. The regions called the leader sequence are rich in Adenine andThymine nucleotides located at the 5’ end of the CRISPR locus containing approximately 500nucleotides, which are not encoded, and are the starting point of transcription. The leadersequence has no open reading frame and is not conserved across species. Spacer DNAs tobe newly added to the CRISPR locus are inserted from the proximal end where the leadersequence is located (Figure 5).Figure 5: CRISPR Sequence3.5.3. CRISPR in Genome EditingThe CRISPR/Cas system is an RNA-directed DNA targeting system. The role of theCRISPR/Cas system in gene editing can be explained by the use of CRISPR to alter thegenetic sequence of nucleotides in DNA strands rather than cutting the DNA chain. Theprimary purpose of the method is to make changes in the targeted DNA sequences by usingNHEJ and HR DNA repair mechanisms that repair double-strand breaks. Double-strandbreaks in the genome are repaired by a recombinational repair mechanism that combinesdouble-strand breaks, since DNA is not a complementary DNA sequence across the damagedregion. Which method to repair double chain breaks depends on the cell cycle stage. Sincethe synthesis phase has not yet occurred and sister chromatids have not formed in the G0/G1 phase, HR is not activated, and NHEJ is used. Homologous recombination is usedduring the S and G2 phases of the cell cycle, where sister chromatids are readily accessible.Since sister chromatids are exact copies of each other, it is a suitable method for homologousrecombination. The basic working principle of NHEJ is that ligases directly link two brokenDNA strands. During repair, the two broken ends are randomly joined, and permanent changesin the original DNA sequence may occur as a result of the loss of some nucleotides. Becausethe nucleotides lost as a result of the deletion cannot be controlled, NHEJ is prone to error andCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 205can create mutations in the genome. Unlike NHEJ, which is simple, plain and random, HR isthe more complex but most accurate method of arranging its genetic sequence by the presenceof sister chromatids. In this repair mechanism, the DSB is resynthesized according to theprepared template sequence. Thus, the target region is translated into the desired nucleotidesequence. The error-prone NHEJ system, in which the two ends are randomly joined in doublechain breaks created by Cas9 enzyme, causes insertion, deletion or indel mutations. If indelmutations occur within the coding region of a gene, they cause the silencing of genes bycausing a frameshift or creating an early stop code (Figure 6).Studies using double-strand breaks as a gene editing tool use NHEJ to generate knockoutmutations, while gene editing and knock-in studies use the more sensitive HR. With CRISPRgenome editing technology, NHEJ or HR DNA repair mechanisms should be given in thecomplex form of sgRNA and cas9 vectors explicitly designed for the region to be edited,using different transfection methods such as viral, non-viral and plasmid, in order to makearrangements such as knock out and knock in in the desired region.Figure 6: CRISPR - Cas9 System and DSB Repair.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

206 NEW THERAPEUTIC APPROACHES IN GENOME3.5.4. CRISPR - Cas9 System Versus ZFN, TALENPrevious DNA editing techniques have been achieved through homologous recombinationwith a DNA template with 5’- and 3’- homology in the target genomic region. This approachhas proven inefficient in various mammalian cells as it is time-consuming and the engineeredconstruct requires a long DNA template (107). Genome editing has advanced with theemergence of potent bioengineering methods like ZFNs, TALENs, and CRISPR-Cas thatenable long-lasting modifications in a specific genomic area on the target organism (45). WhileZFN and TALEN are tools that enhance the ability to edit genes, both have disadvantagesthat the Crispr-Cas system aims to overcome. The design of the zinc finger domains of ZFNsis challenging and time-consuming, and the target specificity is unpredictable. TALENs arebased on protein-DNA interactions that affect the specificity of the technique. Comparedto ZFN, it is more flexible and straightforward because each TALEN domain recognizesonly one nucleotide with well-defined target specificities (55, 108, 109). In vivo or in vitrodelivery of ZFN and TALEN is toxic and lethal due to external target binding that causesbreaks at unwanted sites. Among the gene editing techniques, the most up-to-date Crispr-Castechnology has become one of the most promising tools with several advantages over ZFNsand TALENs. With a more straightforward design for any DNA target, ease of usage, highereffectiveness, fewer off-target areas, high specificity and efficiency, low cost, and the abilityto change many genomic regions at once by adding numerous gRNAs, and ease of applicationmade the system more preferred (54, 55, 108, 109). Thus, this method has been generallyused in studies since 2013(110).3.5.5. Application of CRISPR /Cas9 System in TherapyToday, the CRISPR/Cas9 technique is the most popular tool in gene therapy. Knockoutor knockdown, gene silencing or suppression of gene expression, knock-in transgenic studies,gene therapy, chromosomal deletions and insertions, transcription control, modification ofepigenetic imprints and creation of 3D organoid cell lines can be performed with this technique(111) (Table 2). There has been a significant increase in studies with CRISPR/Cas9 followingthe identification of how the technique can be used successfully to target a specific genomiclocus (110) (Figure 7).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 207Figure 7: Application of CRISPR /Cas9 System in Therapy3.5.5.1 Immunotherapy ApplicationsIn the human body’s homeostasis, the cells that might be dangerous to the nature of thebody that escapes from checkpoints are identified and eliminated by the immune system.If this system is disrupted or somehow inactive, then oncogenic cells survive and thrive,leading to cancer formation. Immunotherapy is a scientific field that seeks to understand theimmune system-cancer cell relationship to use and strengthen natural immune mechanismsto fight disease. CRISPR editing on genes known to have a role in immune system functionand cancer prevention has been among the subjects that have been studied extensively inrecent years. With higher efficiency than traditional transfection and transduction methods,CRISPR can produce specially designed cells, such as CAR-T cells and can be used directlyin immunotherapeutic approaches.3.5.5.2 Chimeric Antigen Receptor (CAR) T-cell therapyChimeric Antigen Receptor (CAR) T-cell therapy is a treatment that involves geneticmodification of the patient’s autologous T-cells to express tumor antigen-specific CAR,followed by ex vivo amplification and reinfusion into the patient (112). CAR-T is afusion protein with an antigen recognition moiety and T cell signaling domains. T-cellgenetic modification can occur through the direct transfer of mRNA by viral-based genetransfer methods, DNA-based transposons, or non-viral methods such as CRISPR/Cas9and electroporation (113). Recently, genetically engineered T cells expressing CAR T-cellshave shown unprecedented efficacy in hematological malignancies (114). One of the mostsuccessful clinical trials is the application of anti-CD19 CAR-T cells in B-cell malignancyCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

208 NEW THERAPEUTIC APPROACHES IN GENOME(115). In 2017, the clinical efficacy of CAR-T cells Kymriah® and Yescarta® in the treatmentof pediatric/young adult B lymphoblastic leukemia, Tecartus® in the treatment of adult mantlecell lymphoma (MCL) patients and in February 2021, Breyanzi® in the treatment of recurrentresistant B-cell lymphoma in adults were approved by the US Food and Drug Administration(FDA) (116-118).On the other hand, the approach to producing personalized T-cells is very time-consumingand costly, preventing the best use of immunotherapy, especially in rapidly progressingdiseases. In addition, the inability to generate enough high-quality T-cells from lymphopenicpatients with poor prognosis during the manufacturing process limits its clinical applicability.Finally, heterogeneity among autologous CAR-T products results in unpredictable and variableclinical activity.3.5.5.3 Production of Universal Allogeneic CAR-T Cells with CRISPR/Cas9In some cases, T cells may not be isolated in patients who are decided to be treated withCAR-T. Failure to isolate enough T cells is the biggest obstacle to treatment. Numerousstrategies have been developed to overcome the barriers to this limitation. One of the mostviable approaches is to generate allogeneic universal CAR-T cells from healthy donors (119).In this method, standardization of CAR-T cells is ensured. Compared to autologous CAR-Tcells, ”off-the-shelf” allogeneic CAR-T cells can be transfused into the patient immediatelyfrom cryopreserved CAR-T cells for patients in urgent need (120). The presence ofendogenous HLA and TCR in T lymphocytes of donors causes complications such as potentialalloreactivity and vaccine versus host disease (Graft versus Host). Complications, which arethe most significant problems encountered, can be prevented by advances in gene editingtechnology and destruction of endogenous HLA and TCR. In previous studies, CD19-specificCAR-T cells were generated by disruption of the endogenous TCR via ZFNs to reduce hostresponses to the vaccine. In another study using TALENs, universal CAR-T cells wereproduced by deactivating the ?? chains of the TCR (121). Simultaneous deactivation ofTCR and HLA molecules was planned to have fully allogeneic CAR-T cells by the samemethods, but it was seen that an efficient and sensitive gene editing technique was needed.The ability of the CRISPR/Cas9 system to knock out multiple gene loci simultaneously andefficiently was thought to be an easy way to obtain TCR HLA classes with lentiviral vectorsthrough the CRISPR protocol incorporating various guide RNAs (122). Based on this, heused CRISPR/Cas9 to generate CAR-T cells in which endogenous TCR, HLA-I, and PD-1were simultaneously suppressed, which showed strong antitumor activity in vitro and inanimal models (123). The edited cells were found to exhibit enhanced antitumor activityover conventional CAR-T cells in vitro and in mouse models of acute lymphoblastic leukemia(122).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 2093.5.5.4 Inhibition of the Immune Checkpoint Signaling PathwayAlthough it is effective in cancers, especially in the treatment of hematologicalmalignancies, CAR-T adoptive cell therapy has yet to achieve the desired effect in manypatients due to immunosuppressive tumor microenvironment and T cell depletion (124).Gene profiling and phenotypic studies in humans and mice have shown that depletedtumor-infiltrating T lymphocytes typically express highly inhibitory receptors, includingmany other inhibitory molecules such as PD-1, cytotoxic T lymphocyte antigen-4 (CTLA-4),lymphocyte activation gene 3 (LAG-3), and T cell immunoglobulin domain (TIM-3) in Tcells which might all have in tumorigenesis (125). Using the CRISPR/Cas9 system, in recentyears, these genes have been inhibited and have aroused broad interest in cancer researchdue to their critical role in antitumor immunity. CRISPR/Cas9-mediated knockdown of PD-1expression has been shown to increase the ability of CAR-T cells to kill tumor cells in vitro andto clear PD-L1+ tumor xenografts in vivo (126). Destruction of the granulocyte-macrophagecolony-stimulating factor (GM-CSF) gene has been shown to increase the function of CAR-Tcells as well as reduce the risk of cytokine release syndrome (CRS) and inflammation (127).Studies have also confirmed that the knockdown of endogenous TGF-? receptor II (TGFBR2)on CAR-T cells with CRISPR/Cas9 technology can reduce the depletion of CAR-T cells andincrease solid tumor killing efficacy both in vitro and in vivo (128). Also, CRISPR knockdownof CD7 and TRAC in CAR T cells appeared to increase effectiveness in treating T-cell acutelymphoblastic leukemia (T-ALL) (129).CRISPR/Cas9 has also been applied to disrupt programmed death-1 (PD-1) expressionon CAR-T cells and antigen-specific cytotoxic T lymphocytes (CTLs) (130-132). In thestudy, it was reported that PD-1 inactivation is possible by electroporation of plasmidsencoding the CRISPR/Cas9 system, and it does not affect T cell viability in vitro. In contrast,modified T cells increase IFN-? secretion and antitumor cytotoxicity. A recently completedclinical trial showed that PD-1 cleavage of T cells by CRISPR/Cas9 is safe and feasible butineffective in patients with non-small cell lung cancer (133). In addition to PD-1 knockdown,disruption of PD-L1 in tumor cells has also been shown to increase the efficacy of cancerimmunotherapy. In a study, Tu et al. He designed a new type of nanoparticle sensitive toweak acidity, featured by the CRISPR/Cas9-Cdk5 plasmid (Cas9-Cdk5) and paclitaxel (PTX).PTX and CRISPR/Cas9 plasmids encapsulated in nanoparticles were delivered by targetingthe cyclin-dependent kinase 5 (CDK5) gene to mediate PD-L1 attenuation on tumor cellsto enhance the antitumor immune response. As a result, it was observed that immunogeniccells died and immune suppressor cells decreased (134). Also, Zhao et al. (2020) createda photo-switched CRISPR/Cas9 system to target the PD-L1 gene. It has been shown in thestudy that this system can effectively disable the PD-L1 gene not only in cancer cells but alsoin cancer stem cell-like cells. In addition to using antibodies to block CTLA-4, CRISPR/Cas9Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

210 NEW THERAPEUTIC APPROACHES IN GENOMEwas applied to disable CTLA-4 (135).3.5.5.5 Application of CRISPR/Cas9 Technology in Tumor CellsCRISPR-Cas9 gene editing technology has been started to be used in tumor cells to knockout tumor suppressors and oncogenes, to prevent the initiation and progression of cancer bycorrecting mutations and unbalanced expressions in chemoresistant genes, metabolism-relatedgenes and cancer stem cell-related genes. In addition, epigenetic modeling studies withCRISPR and studies on monitoring and screening of cancer cells promise developments incancer treatment.Table 2: Applications On Crispr/Cas and Tumor CellsCRISPR/Cas System in AntiTUMOR Therapy Applications Type ReferencesImmunotherapy CRISPR Immunotherapy of CART cells (112-117) Improving CAR-T Cell Function with CRISPR/Cas9 (124-129) Inhibition of the Immune Checkpoint Signaling Pathway (130-135) Production of Universal Allogeneic CAR-T Cells with CRISPR/Cas9 (119-123) Tumor CellsDNA-based knockout/in Oncogenes (136-142) DNA-based knockout/in Tumor Suppressor Genes (143, 144) Chemotherapy Resistance Genes (145-147) Metabolism Related Genes (148-152) Cancer Stem Cell Related Genes (153-158) 3D Organoid (159-161) • DNA-based knockout/in OncogenesThe ability of CRISPR/Cas9 to target a desired genomic locus based on the sequencecomplementarity of the gRNA is a viable method for the knockout of oncogenes locatedat that specific locus. As it is known, the off-target nuclease activity of Cas9 raisessignificant concerns about the safe use of this gene editing technology for clinicalapplications, so it has developed a mutated wild-type dCas9 (136). dCas9 cannotcleave DNA, but can bind to target genes with the same specificity when guided bysgRNA. Successful inhibition of cell growth and migration and induction of apoptosishas been demonstrated in the bladder cancer cell line SW780 using this inactive formCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 211of Cas9 and a gRNA that binds the promoter region and enables the expressionof the oncogenic lncRNA SNGH3 (137). CRISPR/Cas9-mediated knockdown ofD133 in colon cancer cells has been shown to downregulate vimentin expression,significantly reduce cell proliferation and colony formation, and significantly inhibitcell migration and invasion (138). Knockdown of miR-3064 by CRISPR/Cas9 wasshown to significantly inhibit the proliferation, invasion and tumorigenic capacity ofpancreatic cancer (PC) cells (139). In addition, oncogenic mutant EGFR alleles weredemonstrated using CRISPR/Cas9 to inhibit the growth and proliferation of lung cancercell lines H1975, A549 and H1650 and to cause tumor size reduction in xenograft miceimplanted with H1975 and A549 cells (140). KRAS mutation Non-Small Cell LungCancer (NSCLC) cells have been shown to affect drug resistance by knocking downthe FAK gene using CRISPR/Cas9, causing detectable DNA damage and increasedsensitivity to radiotherapy (141). Furthermore, CRISPR/Cas9-mediated deletion of theE3 ubiquitin ligase UBR5 has been shown to significantly inhibit tumor growth andmetastasis in vivo in an experimental mouse model of triple-negative breast cancers(TNBC) (142).• DNA-based knockout/in Tumor Suppressor GenesInactivation of tumor suppressor genes is essential in cancer initiation and progression(143). The tumor suppressor gene silencing, deficiency, or mutation activatesoncogenes, leading to tumor initiation and progression. CRISPR/Cas9 technologyhas been used to demonstrate therapeutic cancer benefits by repairing inactivated tumorsuppressor genes. In studies, it was combined with the CRISPR/dCas9 trans-activatorto activate PTEN expression in cancer cells with low-level PTEN expression (144).The results showed that the dCas9-VPR system increased the expression level of PTENin melanoma and TNBC cell lines and that PTEN activation inhibited downstreamoncogenic pathways (144).• Chemotherapy Resistance GenesOne of the biggest obstacles in cancer treatment is chemotherapy drug resistance. Themechanism called chemoresistance arises from the dysregulation of genes related tochemoresistance and occupies an important place in therapy by regulating the expressionof these genes.In a study, it was shown that CRISPR/Cas9-mediated suppression of NRF2 expressionin a lung cancer xenograft mouse model (145) and suppression of SKA3 expressionin laryngeal cancer cells reduced the developed resistance to cisplatin and carboplatinin patients (146). Knockdown of aurora B (AURKB) by CRISPR/Cas9 in NSCLCcell lines restored the expression of the tumor suppressor gene TP53 and increased itsCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

212 NEW THERAPEUTIC APPROACHES IN GENOMEsensitivity to cisplatin and paclitaxel (147).• Metabolism Related GenesCancer cells rearrange their growth, survival, reproduction, and metabolism for theirlong needs. The common feature of this altered metabolism is increased glucoseuptake and fermentation of glucose to lactate (148). This process is known asthe Warburg Effect, which is observed even in the presence of fully functioningmitochondria. In cancer cells, genes for glucose metabolism disorders and genes inabnormal lipid metabolism, amino acid metabolism, mitochondrial biogenesis, andother energy metabolism pathways are very important (149). Common markersexpressed at high levels in cellular hypoxia, glucose transporter-1 (GLUT-1) andhypoxia-inducible factor-1? (HIF-1?), and glutaminase (GLS), the primary enzyme inglutamine metabolism, have been shown to be associated with the biological behaviorof cancer (150, 151). Therefore, understanding the mechanism of energy metabolismis essential in the understanding and treatment of cancer biogenesis.It has been shown that GLUT-1 and hif1a proteins are important for glucose uptakeand glycolysis steps in cells under hypoxic stress conditions in laryngeal cancer cells.From here, it performed HIF-1?, resulting in decreased proliferation, migration, andinvasion. HIF-la and GLUT-1 gene knockdown appeared to cause a significant reductionin glucose uptake and lactic acid of HEp-2 cells (152).• Cancer Stem Cell Related GenesCancer stem cells (CSCs) have the characteristic features of normal stem cells. Theycan form tumors, advance, and relapse by using their ability to self-renew and transforminto many different cell types (153). The power of these cells to differentiate into allcell types is more prone to form tumors than other cancer cells. For these reasons, itis predicted that the identification of the CSC-associated gene will create new cancertherapy targets.Ovarian cancer stem cells (OCSC) are associated with a poor ovarian cancer prognosis.Nanog has been identified as the essential gene that maintains CSC pluripotency andself-renewal ability (154). Androgen receptors (AR) are involved in the malignantbehavior of other tumors (155). In a study, a green fluorescent protein (GFP)-taggedcell model was created in ovarian cell lines A2780 and SKOV3 using the CRISPR/Cas9system, and it was shown that by suppressing transcriptional activation of Nanog, itsinteraction with AR signaling can cause or contribute to the regulation of OCSC (156).Another study used CRISPR/Cas9 technology to knock down the transcription factorYB-1 gene in cancer stem cells and observed that the absence of, leading to cell cyclearrest, apoptosis, and irreversible differentiation (157). In colorectal cancer, abnormalCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 213Wnt signaling is critical for developing and maintaining cancer stem cells. Hwang etal. used CRISPR/Cas9 to suppress REG4 in colorectal cancer spheres containing bothAPC and KRAS mutations and showed that knockdown of REG4 inhibited Wnt/?-linkedprotein signaling, thereby effectively suppressing CSC properties (158). These findingsprovided new insights for investigating the regulatory mechanisms of cancer stem cellsin many aspects and for CSC targeting cancer treatment.3.5.6. 3D Organoid CRISPRThree-dimensional (3D) cell culture methods are widely used in several cell types,including stem cells, to fully modulate the cellular biophysical and biochemicalmicroenvironment and control various cell signaling cues (159). More in vivo-likemicroenvironments in 3D cultures are epitomized, particularly by forming multicellularspheroids and organoids, which may provide more valid disease mechanisms. Recently,genome engineering tools such as CRISPR-Cas9 have been used to control gene expression,complementing external signaling cues with intracellular control elements. The combinationof CRISPR-Cas9 and 3D cell culture methods is expected to advance the understanding of themolecular mechanisms underlying several disease phenotypes and pave the way for developingnew therapeutics that can more rapidly and effectively advance to clinical candidates. Genomemodification to multipotent stem cell types, including neural stem cells (NSCs), has been usedin studies to model diseases such as cancer in cerebral organoids (160). Direct introduction ofCRISPR-Cas9 to mutate several tumor suppressor genes in 3D embryoid bodies was performedat the NSC stage to identify critical genes that, when mutated, result in abnormal growthassociated with various cancers. Mutations in specific genes resulted in the rapid proliferationof organoids in culture. Further investigation revealed mRNA changes in the expressionof several genes between mutated cancerous organoids and non-cancerous organoids. Suchdisease models have provided a wealth of information on how cells change with tumorigenesisand how this knowledge can be used to create better potential drug candidates to reducetumorigenesis (161).3.6. Gene Editing with Noncoding RNAMutant tumor suppressor genes and oncogenes offer excellent possibilities for applyinggenome editing techniques (34, 162). ZFN-induced targeting was first used to specificallyblock the expression of the human BCR-ABL oncogene, which was transformed into amouse cell line in 1994 (163). Therapeutic ZFN targeted the promoter function of thehuman T cell leukemia virus type 1 (HTLV-1) long terminal repeat, which selectively killedHTLV-1-infected cells in an in vivo adult T cell leukemia model. (164). In addition, theBCR-ABL fusion gene cleavage by the specific ZFN has been reported to terminate BCR-ABLprotein translation and induce apoptosis in imatinib-resistant CML cells (165). In addition,to restore wild-type p53 function, as the tumor suppressor gene p53 plays an essential role inCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

214 NEW THERAPEUTIC APPROACHES IN GENOMEcancer development, a yeast hybrid (Y1H) four-fingered ZFN was designed to replace mutantp53 with wild-type p53 in several cancer lines via ZFN-induced HR. Modifications of the p53loci provided a framework for the study, despite the fact that HR events were not especiallyeffective in this case (166). In 2003, Gendicine®, the first anti-cancer gene therapy, wasauthorized in China for treating head and neck cancer in patients with p53 tumor suppressormutations (167). Oncorine® therapy uses a genetically modified adenovirus that selectivelyreplicates in p53-deficient tumor cells but does not replicate in normal cells (168).RNA interference (RNAi): In conventional gene therapy, a normal copy of a particulardefective gene is delivered to target cells and restores the defective gene’s function. This”gene addition” approach has been changed to employ RNA interference (RNAi), whichprevents the defective gene from being expressed (169, 170). RNAi is the most widelyused reverse genetics approach to study gene function in mammalian cells. In RNAi genetherapy, chemically synthesized small inhibitory RNAs (siRNA) are delivered directly to cells.Alternatively, the gene encoding short hairpin RNA (shRNA), which will produce siRNA, isdelivered to target cells using the viral vector. In the target cell, siRNAs base pairs the defectivegene with mRNAs and promote degradation. As a result, it inhibits defective gene production.In 2018, Onpattro® became the first RNAi-based gene therapy for inherited transthyretinamyloidosis to receive approval. Antisense oligonucleotides, which influence the expressionor splicing of target genes, are used comparably. Oligonucleotide-based gene therapies havebeen authorized for the treatment of Duchenne muscular dystrophy (Exondys 51®), familialhypercholesterolemia (Kynamro®), spinal muscular atrophy (Spinraza®) (171, 172). SmallRNAs regulate gene expression, and by the endogenous RNAi mechanism, synthetic smallRNAs such as siRNA and shRNA are introduced into the cell (173). After small RNAs entercells, they are screened by the dsRNA reagents DICER.The added RNA is loaded into the RNA-induced silencing complex (RISC) and degradesthe target mRNA (174). In this way, target protein levels after target mRNA can be reducedpost-transcriptionally (108). Considering that the RNAi mechanism is active primarily inthe cytoplasm, nuclear transcripts e.g. long noncoding RNAs (lncRNAs) can be challengingto target. lncRNAs may not elicit complete loss-of-function phenotypes via RNAi (175).The most crucial advantage of RNAi is that the silencing mechanism is present in almostevery mammalian somatic cell (108). Limited sequence complementarity non-target mRNAscan also be silenced by siRNAs, frequently through contact with the 3’UTR (176). Itcauses translational repression and/or degradation in this manner. One siRNA can suppresshundreds of transcripts, depending on the sequence. May have off-target impacts that are notsequence-specific (177). RNAi technology differs from nuclease-dependent genome editingstrategies. Although genome editors facilitate DSBs in the genomic region, RNAi performspost-transcriptional gene silencing by cleaving mRNA molecules. (77, 178). The mechanismCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 215was first described in 1998 by exposure of the model organism Caenorhabditis elegans todsRNA resulting in gene silencing (179). RNAi constitutes a conserved process for directlycontrolling genes in eukaryotes. miRNAs and siRNAs represent a group of noncoding RNAscommonly used for cancer gene therapy (Figure 8). Both use the RNAi mechanism.siRNAs are derived from longer dsRNAs, while miRNAs are produced by double-strandedprecursor miRNAs (55, 180). Endogenous small noncoding RNAs known as miRNAs, have18–25 nucleotides, control gene expression in a sequence-specific way by either inhibiting thetranslation of proteins or degrading their target mRNA. RNA poI II transcribes the miRNAgenes to create pri-miRNAs with the 3’ poly(A) end and 5’ cap (181, 182). Pri-miRNAsare cleaved with the Drosha complex in the nucleus, resulting in pre-miRNA containing 70nucleotide hairpin structures and flanking sequences. Exportin-5 transports the pre-miRNAto the cytosol, where DICER (Rnase III enzyme) and its partners TRBP (TAR-RNA bindingprotein) are cleaved into the 22 nucleotide miRNA:miRNA duplex (183). RISC is coupledto the Argonaute 2 (ago2) protein. The miRNA sequence (guide strand) is selectivelyincorporated into the RISC complex, and the complex is specifically directed to mRNA targetsthrough complementary base-pairing interactions between the seed sequence (2-8 bases at the5’ end of the mature miRNA) and the target mRNA binding site (184, 185). RNAi exerts itssilencing function through this mechanism via mRNA degradation or translation inhibition.Similarly, shRNA with root loop structure is transcribed by RNA pol III. The siRNA guidestrand is assembled into RISC for RNAi mechanism target cleavage and gene silencing (181)(Figure 8).3.6.1. Delivery Systems of Noncoding RNAA crucial factor that improves the effectiveness of noncoding RNAs for cancer genetherapy is the in vivo delivery mechanism, in addition to the chemical structures of RNA(RNA sequence and end modification). Several barriers exist, such as defense against immunedetection, avoidance of endogenous nuclease digestion, and promotion of extravasation fromblood vessels to target tissues and cells (181).Deliveries of naked noncoding RNA molecules can occur at a surprisingly high rate. (Table3). For instance, the oncomir miR-10b inhibitor was effectively used to suppress breast cancermetastasis after being injected into a mouse tail vein (186). Non-coding RNA is transportedusing lipid-based carriers, polymersomes, cell-penetrating peptides, and inorganic particlesto prevent degradation and improve distribution (187-189). Viral vectors are an additionaltechnique for in vivo transfer (Figure 1). For instance, the administration of miR-26a caused aregression in the development of cancer cells and the induction of apoptosis in a mouse modelof liver cancer (181, 190).3.6.2. Cancer and RNAiCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

216 NEW THERAPEUTIC APPROACHES IN GENOMEFigure 8: The siRNA enters the cytoplasm and binds to the RISC. The antisense strand is loaded ontothe RISC, and the sense strands are discarded. The antisense strand then causes specific splicing anddegradation of the target mRNA.Table 3: Noncoding RNA delivery methods * Method RNA species delivered Advantages Disadvantages Non-viral vectors Naked delivery miRNA, siRNA No carriers needed High dosage required Lipid-based carriers miRNA, siRNA Robust, effective, and selective delivery Sophisticated preparation needed Polymersomes siRNA Robust, effective, and selective delivery Sophisticated preparation needed Cell-penetrating peptides miRNA (e.g., pHLIP) Effective and selective delivery Expensive, sophisticated preparation Inorganic nanoparticles siRNA Easy preparation Limited efficiency, sometimes toxic Viral vectors miRNA, shRNA Effective delivery, stable expression Biosafety risk, immunogenic Transgene miRNA Stable expression, nonimmunogenic Research purpose only *Adopted from (181)RNAi has resulted in the treatment of incurable illnesses to enhance treatment outcomes.Cancer is one of the most fatal illnesses with a poor prognosis (191). Application of RNAiCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 217targets the inhibition of tumor-apoptosis genes, control of tumor signal transduction pathway,inhibition of tumor angiogenesis-related factors, affect oncogenes, tumor suppressor genes,and reduction of tumor drug resistance in cancer (192). With its gene expression regulatoryeffect, RNAi has been accepted as the ideal strategy for cancer. Furthermore, it increasesthe effectiveness of cancer treatment (193). The accumulation of different gene mutationsand the interaction of these genes lead to tumors. Gene editing is, therefore, the primarymode of treatment. RNAi knocks down the expression of target genes with low side effectsand low risk. It also accelerates the development of effective and targeted drugs to controltumor growth. Therefore, cancer is the primary target of RNAi-based therapy, with high geneexpression and cell proliferation (194). Administration of siRNA in lung cancer resulted inan effective reduction of overexpressed PLK1 expression, which is the main regulatory factorof mitosis, in tumor cells, and an increase in the percentage of cell death caused by in vivo/invitro apoptosis (195).Additionally, RNAi drugs have been proposed as a possible method for overcoming thelimitations of chemotherapy through the selective silencing of oncogenes and genes linkedto multidrug resistance (196). Pancreatic cancer is a malignant tumor usually asymptomaticin its early stages and is fatal with delayed diagnosis (197). Radiotherapy, chemotherapydrug resistance, and a poor prognosis are all decreased when combined with RNAi therapies(192). Target gene knockdown treatment with RNAi has shown high therapeutic potential.Recently, a miRNA-based therapeutic agent has also been developed for pancreatic cancer.Some preclinical studies in breast cancer have investigated RNAi-based strategies for humanepidermal growth factor 2+ (HER2+) breast cancer (198). In breast cancer, using miRNAas a functional marker to determine cell properties and siRNA to overcome chemoresistancein cancer are at the forefront (199). Overexpression of the epidermal growth factor receptor(EGFR) family in colorectal cancer predisposes to tumor formation. Dimerized EGFR sendsmitotic signals to tumor cells while binding to particular ligands, causing cell proliferation andresistance to apoptosis. As a result, siRNA-mediated EGFR inactivation is being consideredas a possible therapeutic approach (200). In addition to these cancers, RNAi has extensiveuse in other cancer types, including ovarian, hepatocellular, stomach, and cervical cancer.Transcription Factor Decoys; Transcription Factor Decoys (TFDs) areoligodeoxynucleotides (ODN) with double-stranded ends that are intended to blockparticular regulatory pathways (201). ODNs are short double-stranded DNA moleculesthat compete with the unique binding sites of transcription factors by containing thetranscription factor sequence of a particular gene or the consensus DNA recognition motif ofthe transcription factor. In cancer therapy, TFDs are targeting STAT3 to induce apoptosisand cell cycle arrest in ovarian, lung, and neck cancers and NF-KB for metastasis inhibition.(202, 203).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

218 NEW THERAPEUTIC APPROACHES IN GENOME3.7. CRISPRiThe enzymatically dormant form of Cas9 (deadCas9, dCas9) is fused to a repressor domain(such as KRAB) and initiates heterochromatin-mediated gene silencing in transcription startsite (TSS), just like TALE transcriptional repressors (204). This technique, called CRISPRi,causes transcriptional suppression of the target RNA, much like TALE. CRISPRi exhibits moresevere loss-of-function phenotypes than RNAi (205). Chromatin accessibility is necessaryfor Cas9 to reach its target site in CRISPRi. Therefore, genomic areas with closed chromatinstates might prevent Cas9 from functioning by preventing binding (206). The -500 bpwindow downstream from the TSS is the site the KRAB domain addresses to effectivelyinhibit transcription (207). It is crucial to understand the transcripts’ TSS realm. Numerousgenes may have TSSs that are very far apart from one another, alternative transcripts, or maydiffer between different kinds of cells (108, 208).3.7.1. RNAi or CRISPRiCompared to RNAi, CRISPRi operates more effectively. The CRISPR mechanism’sof-targeting rate is reduced. RNAi application does not require the addition of additionalgenes to cells, so it is faster to administer and saves time and money (207). When itcomes to transcriptome editing, the CRISPR-Cas system uses sgRNAs that can remove alltranscript variants of a gene, decreasing off-target effects, in contrast to the conventionalpost-transcriptional gene silencing approach, RNAi (54, 108). Following the completion ofthe human genome project, it was necessary to disrupt regular gene expression and examinethe resulting phenotypes to identify genes of unknown function (209). The discovery of RNAibroke new ground with the injection of double-stranded RNA into C.elegans by powerfullysilencing a gene sequence and creating phenotypes that reveal gene function(210). Soon after,RNAi mechanisms began inhibiting specific genes in human cells (211).Furthermore, RNAi produces hypomorphic phenotypes that do not always reflect thecomplete loss of function that would occur with a genetic mutation. In this way, new toolsfor reverse genetics began to be developed (108). RNAi, in contrast to CRISPRi, does nottarget TSSs, allowing it to be applied to species for which genome sequencing data are notyet accessible but the transcriptome is present (212). RNAi can target transcript variantsand conserved regions among members of the same gene family, just like CAS9 nuclease.Therefore, regardless of where the TSS originates, si/shRNA can target all transcripts. Thisis in contrast with CRISPRi and emphasizes RNAi (108). Finally, RNAi does not targetchromosomal DNA in the nucleus; instead, it targets RNA transcripts in the cytoplasm.Compared to CRISPR-based methods, accessibility does not seem to be linked to chromatinstate (206, 213).Gene knockout: A homogeneous knockout phenotype should be the outcome of a trueknockout, which is defined as total dysfunction of the target gene. It should be emphasizedCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 219that the randomness of the genomic mutations caused by DSB NHEJ repair (214). As a result,it is anticipated that some of the mutations caused by Cas9 nuclease will occur in-frame,preventing ORF disruption. However, amino acids added or removed can change activitybased on the position within a protein, altering the phenotypic impact for each mutation andeach gene (108). In other words, genetic heterogeneity can cause phenotypic heterogeneity.Gene knockdown: While knockdown does not permanently eliminate gene function, it hassome benefits over knockouts. RNAi and CRISPRi can imitate the effects of inhibitory drugs.A complete knockout may reflect an idealized loss-of-function phenotype for the most potentinhibitory drugs. Genetic heterogeneity problems are reduced and gene knockdowns arereversible because RNAi and CRISPRi-induced knockdowns are not frameshift-dependent.Studying the function of essential genes whose deactivation would be deadly is made possibleby partial knockdown (108, 215).3.8. Gene AdditionGene addition is one of the most practical approaches in gene therapy. It is inserting orsimply adding a gene by non-homologous recombination. The adding gene may be an activecopy of a defective innate gene. The gene addition technique requires a vector system. Viralor non-viral-based vectors are successfully used for this scenario. Retroviruses are the mostsuccessful vectors tested up to now for gene addition techniques due to their adaptive naturefor delivering genes into cells. Moreover, in comparison to gene replacement, gene additionis more successful. However, there are several advantages as well. One disadvantage is thedisordered insertion of genes into the genome. Because of this, the inserted genes may beexpressed erroneously or inappropriately. The introduction of novel genes into human cellshas traditionally been referred to as gene therapy. Thus, the idea of gene therapy emerged,in which defective DNA is replaced with exogenous DNA. (216). By inserting a therapeuticgene into the cells, it restores the function of the deleterious or absent gene (217). The newgene may be a regular version of the defective gene or a gene that improves how it works.Therapeutic gene integration can have unpredictable consequences for gene expression andunwanted results for nearby genes (218).In vivo, gene editing has attracted attention for targeted gene insertion into tissues wherecell transplantation is challenging or unfeasible, in addition to ex vivo gene editing of bloodand immune cells. The first demonstration of high-throughput, nuclease-mediated geneediting in vivo, using AAV vectors to deliver ZFNs and a factor IX cDNA without a promoterto the liver of a mouse model of hemophilia. Cleavage of the first intron of the mutatedhuman factor IX gene by ZFNs catalyzed the efficient integration of factor IX cDNA intothe locus, leading to the correction of the hemophilic phenotype (219) (Figure 9). This isthe first study that hepatocytes are actively dividing, and hence HDR pathways are active.It has been carried out in newborns. A later study showed efficacy in adult mice, whereCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

220 NEW THERAPEUTIC APPROACHES IN GENOMEhepatocytes likely exited the cell cycle, although the integrations were due to a combinationof HDR and NHEJ-mediated events. It provides high-throughput gene editing by generatinga predetermined region-specific DSB in the genome. Its repair by NHEJ or HDR pathwaysresults in targeted gene disruption or splicing (216, 220).The diversity and resilience of cancer motivate gene treatments to create novel therapeuticmodalities (221). Two different gene-splicing strategies will be discussed here; the firstcontains herpes simplex viral vector with viral gene deletions that would replicate only intumor cells and cause oncolysis. It provides the gene encoding an immune-stimulatingcytokine known as a granulocyte-macrophage colony-stimulating factor (GM-CSF), whichhelps immune effector cells attack tumor cells (222). In a phase III clinical trial for melanoma,the crucial function of GM-CSF gene addition in initiating the systemic antitumor responsewas revealed. The second method involves implanting T cells ex vivo with a gene encodingthe chimeric antigen receptor that recognizes CD19 which are then administered again topatients in order to cure B-cell malignancies (222, 223). T cells equipped with anti-CD19CAR were shown to reverse advanced lymphoma in one patient. This strategy is being studiedin treating B cell malignancies (223, 224).Insertional mutagenesis is when retroviral vectors used in gene therapies randomlyintegrate into the genome of their target cells. Cancer results if insertional mutagenesisaccidentally triggers or alters a gene’s expression (225). Using non-integrated vectors, suchas AAV vectors, is unlikely to integrate into the genome of target cells. Another solution issite-specific integration, where genome editing is used to insert curative genes into the ”safe”genomic locus, thus avoiding intervening mutagenesis. (226). The most effective method maybe DNA-free genome modification. This approach uses lipids or nanoparticles to transportnuclease proteins or mRNAs to target cells. (219).4. ConclusionGene therapies treat a disease in various ways, such as replacing malfunctioning genes withtherapeutic genes, gene knockdown, deactivating genes, and inserting a new gene. The use ofgene transfers to treat human diseases has been successful in several diseases. Compared withallogeneic bone marrow transplantation, ex vivo gene therapy using autologous cells eliminatesthe need to search for an appropriate donor, removes the risk of graft-versus-host disease,and sometimes reduces the intensity of the required preparative regimen before transplant,which also reduces toxicity. Thus, this therapeutic approach can now be considered analternative to standard therapy in some diseases. Insertional mutagenesis, which has resultedin severe adverse events in several trials, has stimulated the rapid development of putativesafer vector systems being tested in human trials but remains challenging. Rapid progress inmolecular technology, such as high-throughput sequencing, high-efficiency gene editing, andthe development of new sources of expandable stem cell sources, offers significant potentialCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSFigure 9: Types of therapeutic genomic modifications (219). a. Silencing a pathogenic gene throughgene disruption b. Correction of the NHEJ gene: removal of a pathogenic insertion c. HDR gene repaireliminates a harmful mutation. d. Adding a therapeutic gene to the HDR gene.for ongoing development of gene transfer in regenerative biology for a wide range of humanconditions.REFERENCES1. Belete TM. The current status of gene therapy for the treatment of cancer. Biologics:targets & therapy. 2021;15:67.2. Wirth T, Parker N, Yla-Herttuala S. History of gene therapy. Gene. 2013;525(2):162-9. ¨3. Templeton NS. Gene and cell therapy: therapeutic mechanisms and strategies: CrcPress; 2008.4. Vermezovic J, Stergiou L, Hengartner M, D’Adda Di Fagagna F. Differential regulation ofDNA damage response activation between somatic and germline cells in Caenorhabditiselegans. Cell Death & Differentiation. 2012;19(11):1847-55.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

222 NEW THERAPEUTIC APPROACHES IN GENOME5. Colella P, Ronzitti G, Mingozzi F. Emerging issues in AAV-mediated in vivo genetherapy. Molecular Therapy-Methods Clinical Development. 2018;8:87-104.6. Tang R, Xu Z. Gene therapy: a double-edged sword with great powers. Molecular andcellular biochemistry. 2020;474(1-2):73-81.7. Singh BN, Prateeksha, Gupta VK, Chen J, Atanasov AG. Organic Nanoparticle-BasedCombinatory Approaches for Gene Therapy. Trends Biotechnol. 2017;35(12):1121-4.8. Nayerossadat N, Maedeh T, Ali PA. Viral and nonviral delivery systems for gene delivery.Adv Biomed Res. 2012;1:27.9. Resnik DB, Langer PJ. Human germline gene therapy reconsidered. Hum Gene Ther.2001;12(11):1449-58.10. McDonough PG. The ethics of somatic and germline gene therapy. Ann N Y Acad Sci.1997;816:378-82.11. Goncalves GAR, Paiva RMA. Gene therapy: advances, challenges and perspectives.Einstein (Sao Paulo). 2017;15(3):369-75.12. Howells A, Marelli G, Lemoine NR, Wang Y. Oncolytic Viruses—Interaction of Virusand Tumor Cells in the Battle to Eliminate Cancer. Frontiers in Oncology. 2017;7.13. Yoon AR, Hong J, Yun C-O. Adenovirus-mediated decorin expression inducescancer cell death through activation of p53 and mitochondrial apoptosis. Oncotarget.2017;8(44):76666-85.14. Choi I-K, Li Y, Oh E, Kim J, Yun C-O. Oncolytic Adenovirus Expressing IL-23 andp35 Elicits IFN-- and TNF--Co-Producing T Cell-Mediated Antitumor Immunity. PLoSONE. 2013;8(7):e67512.15. Gao X, Kim K-S, Liu D. Nonviral gene delivery: what we know and what is next. TheAAPS journal. 2007;9:E92-E104.16. Gascon AR, del Pozo-Rodr ´ ´ıguez A, Solin´ıs MA. Non-viral delivery systems in gene ´therapy. Gene therapy-tools and potential applications: IntechOpen; 2013.17. Herweijer H, Wolff J. Progress and prospects: naked DNA gene transfer and therapy.Gene therapy. 2003;10(6):453-8.18. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ, et al.Theory and in Vivo Application of Electroporative Gene Delivery. Molecular Therapy.2000;2(3):178-87.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 22319. Heller R, Gilbert R, Jaroszeski MJ. Clinical trials for solid tumors usingelectrochemotherapy. Methods Mol Med. 2000;37:137-56.20. Lin MT, Pulkkinen L, Uitto J, Yoon K. The gene gun: current applications in cutaneousgene therapy. International journal of dermatology. 2000;39(3):161-70.21. Mhashilkar A, Chada S, Roth JA, Ramesh R. Gene therapy: therapeutic approaches andimplications. Biotechnology advances. 2001;19(4):279-97.22. Miller DL, Pislaru SV, Greenleaf JE. Sonoporation: mechanical DNA delivery byultrasonic cavitation. Somat Cell Mol Genet. 2002;27(1-6):115-34.23. Suda T, Liu D. Hydrodynamic gene delivery: its principles and applications. Mol Ther.2007;15(12):2063-9.24. Yudin MA, Bykov VN, Nikiforov AS, Al-Shekhadat RI, Ivanov IM, Ustinova TM. Studyof the Efficiency of the Hydroporation for Delivery of Plasmid DNA to the Cells on theModel of Toxic Neuropathy. Bull Exp Biol Med. 2018;164(6):798-802.25. Jin L, Zeng X, Liu M, Deng Y, He N. Current progress in gene delivery technologybased on chemical methods and nano-carriers. Theranostics. 2014;4(3):240-55.26. Zhi D, Zhang S, Wang B, Zhao Y, Yang B, Yu S. Transfection efficiency ofcationic lipids with different hydrophobic domains in gene delivery. Bioconjug Chem.2010;21(4):563-77.27. Schaffert D, Troiber C, Wagner E. New sequence-defined polyaminoamides withtailored endosomolytic properties for plasmid DNA delivery. Bioconjug Chem.2012;23(6):1157-65.28. Pezzoli D, Kajaste-Rudnitski A, Chiesa R, Candiani G. Lipid-based nanoparticles asnonviral gene delivery vectors. Nanomaterial Interfaces in Biology: Methods andProtocols. 2013:269-79.29. Choi JS, Nam K, Park JY, Kim JB, Lee JK, Park JS. Enhanced transfection efficiencyof PAMAM dendrimer by surface modification with L-arginine. J Control Release.2004;99(3):445-56.30. Carroll D. Genome engineering with targetable nucleases. Annual review ofbiochemistry. 2014;83:409-39.31. Porteus M. Genome editing: a new approach to human therapeutics. Annual review ofpharmacology and toxicology. 2016;56:163-90.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

224 NEW THERAPEUTIC APPROACHES IN GENOME32. Kosicki M, Tomberg K, Bradley A. Repair of double-strand breaks inducedby CRISPR–Cas9 leads to large deletions and complex rearrangements. Naturebiotechnology. 2018;36(8):765-71.33. Rouet P, Smih F, Jasin M. Expression of a site-specific endonuclease stimulateshomologous recombination in mammalian cells. Proceedings of the National Academyof Sciences. 1994;91(13):6064-8.34. Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editingtechnology in the targeted therapy of human diseases: mechanisms, advances andprospects. Signal transduction and targeted therapy. 2020;5(1):1-23.35. Wyman C, Kanaar R. DNA double-strand break repair: all’s well that ends well. AnnuRev Genet. 2006;40:363-83.36. Jasin M, Haber JE. The democratization of gene editing: Insights from site-specificcleavage and double-strand break repair. DNA repair. 2016;44:6-16.37. Porteus MH. A new class of medicines through DNA editing. New England Journal ofMedicine. 2019;380(10):947-59.38. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, et al. Doublenicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell.2013;154(6):1380-9.39. Ernst MP, Broeders M, Herrero-Hernandez P, Oussoren E, van der Ploeg AT, PijnappelWP. Ready for repair? Gene editing enters the clinic for the treatment of human disease.Molecular Therapy-Methods & Clinical Development. 2020;18:532-57.40. Sallmyr A, Tomkinson AE. Repair of DNA double-strand breaks bymammalian alternative end-joining pathways. Journal of Biological Chemistry.2018;293(27):10536-46.41. Suleiman AA, Saedi WY, Muhaidi MJ. Widely used gene editing strategies in cancertreatment a systematic review. Gene Reports. 2021;22:100983.42. Zhang H-X, Zhang Y, Yin H. Genome editing with mRNA encoding ZFN, TALEN, andCas9. Molecular Therapy. 2019;27(4):735-46.43. Sun W, Liu H, Yin W, Qiao J, Zhao X, Liu Y. Strategies for enhancing thehomology-directed repair efficiency of CRISPR-cas systems. The CRISPR journal.2022;5(1):7-18.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 22544. Breier D, Peer D. Genome editing in cancer: Challenges and potential opportunities.Bioactive Materials. 2023;21:394-402.45. Gaj T, Gersbach CA, Barbas CF. ZFN, TALEN, and CRISPR/Cas-based methods forgenome engineering. Trends in biotechnology. 2013;31(7):397-405.46. Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, et al. Meganucleasesand other tools for targeted genome engineering: perspectives and challenges for genetherapy. Current gene therapy. 2011;11(1):11-27.47. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing withengineered zinc finger nucleases. Nature Reviews Genetics. 2010;11(9):636-46.48. Cathomen T, Joung JK. Zinc-finger nucleases: the next generation emerges. MolecularTherapy. 2008;16(7):1200-7.49. Bortesi L, Fischer R. The CRISPR/Cas9 system for plant genome editing and beyond.Biotechnol Adv. 2015;33(1):41-52.50. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, et al. Breaking the code ofDNA binding specificity of TAL-type III effectors. Science. 2009;326(5959):1509-12.51. Wilkinson R, Wiedenheft B. A CRISPR method for genome engineering. F1000PrimeRep. 2014;6:3.52. Lan T, Que H, Luo M, Zhao X, Wei X. Genome editing via non-viral delivery platforms:Current progress in personalized cancer therapy. Molecular Cancer. 2022;21(1):1-15.53. Paques F, Duchateau P. Meganucleases and DNA double-strand break-induced ˆrecombination: perspectives for gene therapy. Current gene therapy. 2007;7(1):49-66.54. Khalil AM. The genome editing revolution. Journal of genetic engineering andbiotechnology. 2020;18(1):1-16.55. Boti MA, Athanasopoulou K, Adamopoulos PG, Sideris DC, Scorilas A. RecentAdvances in Genome-Engineering Strategies. Genes. 2023;14(1):129.56. Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer.Cancer research. 2012;72(10):2457-67.57. Lin Y, Wu Z, Guo W, Li J. Gene mutations in gastric cancer: a review of recentnext-generation sequencing studies. Tumor Biology. 2015;36:7385-94.58. Zaman QU, Li C, Cheng H, Hu Q. Genome editing opens a new era of geneticimprovement in polyploid crops. The Crop Journal. 2019;7(2):141-50.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

226 NEW THERAPEUTIC APPROACHES IN GENOME59. Munoz IG, Prieto J, Subramanian S, Coloma J, Redondo P, Villate M, et al. Molecularbasis of engineered meganuclease targeting of the endogenous human RAG1 locus.Nucleic acids research. 2011;39(2):729-43.60. Gouble A, Smith J, Bruneau S, Perez C, Guyot V, Cabaniols JP, et al. Efficient in tototargeted recombination in mouse liver by meganuclease-induced double-strand break.The Journal of Gene Medicine: A cross-disciplinary journal for research on the scienceof gene transfer and its clinical applications. 2006;8(5):616-22.61. Szczepek M, Brondani V, B¨uchel J, Serrano L, Segal DJ, Cathomen T. Structure-basedredesign of the dimerization interface reduces the toxicity of zinc-finger nucleases.Nature biotechnology. 2007;25(7):786-93.62. Gambardella V, Tarazona N, Cejalvo JM, Lombardi P, Huerta M, Rosello S, et al. ´Personalized medicine: recent progress in cancer therapy. Cancers. 2020;12(4):1009.63. Yee JK. Off-target effects of engineered nucleases. The FEBS journal.2016;283(17):3239-48.64. Carroll D. Genome engineering with zinc-finger nucleases. Genetics.2011;188(4):773-82.65. Schierling B, Dannemann N, Gabsalilow L, Wende W, Cathomen T, Pingoud A. A novelzinc-finger nuclease platform with a sequence-specific cleavage module. Nucleic acidsresearch. 2012;40(6):2623-38.66. Gupta RM, Musunuru K. Expanding the genetic editing tool kit: ZFNs, TALENs, andCRISPR-Cas9. The Journal of clinical investigation. 2014;124(10):4154-61.67. Lanigan TM, Kopera HC, Saunders TL. Principles of genetic engineering. Genes.2020;11(3):291.68. Kim Y-G, Cha J, Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusionsto Fok I cleavage domain. Proceedings of the National Academy of Sciences.1996;93(3):1156-60.69. Beerli RR, Barbas III CF. Engineering polydactyl zinc-finger transcription factors.Nature biotechnology. 2002;20(2):135-41.70. Beerli RR, Schopfer U, Dreier B, Barbas CF. Chemically regulated zinc fingertranscription factors. Journal of Biological Chemistry. 2000;275(42):32617-27.71. Palpant N, Dudzinski D. Zinc finger nucleases: looking toward translation. Gene therapy.2013;20(2):121-7.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 22772. Cassandri M, Smirnov A, Novelli F, Pitolli C, Agostini M, Malewicz M, et al. Zinc-fingerproteins in health and disease. Cell death discovery. 2017;3(1):1-12.73. Li T, Huang S, Zhao X, Wright DA, Carpenter S, Spalding MH, et al. Modularlyassembled designer TAL effector nucleases for targeted gene knockout and genereplacement in eukaryotes. Nucleic acids research. 2011;39(14):6315-25.74. Lubroth P, Colasante G, Lignani G. In vivo genome editing therapeutic approachesfor neurological disorders: where are we in the translational pipeline? Frontiers inNeuroscience. 2021:142.75. Rebar EJ, Huang Y, Hickey R, Nath AK, Meoli D, Nath S, et al. Induction ofangiogenesis in a mouse model using engineered transcription factors. Nature medicine.2002;8(12):1427-32.76. Boch J. TALEs of genome targeting. Nature biotechnology. 2011;29(2):135-6.77. Setten RL, Rossi JJ, Han S-p. The current state and future directions of RNAi-basedtherapeutics. Nature reviews Drug discovery. 2019;18(6):421-46.78. Mussolino C, Morbitzer R, L¨utge F, Dannemann N, Lahaye T, Cathomen T. A novelTALE nuclease scaffold enables high genome editing activity in combination with lowtoxicity. Nucleic acids research. 2011;39(21):9283-93.79. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, et al. A TALE nucleasearchitecture for efficient genome editing. Nature biotechnology. 2011;29(2):143-8.80. Ain QU, Chung JY, Kim Y-H. Current and future delivery systems for engineerednucleases: ZFN, TALEN and RGEN. Journal of Controlled Release. 2015;205:120-7.81. Kamruzzaman M, Yan A, Castro-Escarpulli G. Editorial: CRISPR-Cas Systems inBacteria and Archaea. Front Microbiol. 2022;13:887778.82. Ishino Y, Krupovic M, Forterre P. History of CRISPR-Cas from Encounter witha Mysterious Repeated Sequence to Genome Editing Technology. J Bacteriol.2018;200(7).83. Makarova KS, Aravind L, Grishin NV, Rogozin IB, Koonin EV. A DNA repair systemspecific for thermophilic Archaea and bacteria predicted by genomic context analysis.Nucleic Acids Res. 2002;30(2):482-96.84. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmabledual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science.2012;337(6096):816-21.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

228 NEW THERAPEUTIC APPROACHES IN GENOME85. Nowak CM, Lawson S, Zerez M, Bleris L. Guide RNA engineering for versatile Cas9functionality. Nucleic Acids Res. 2016;44(20):9555-64.86. Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced shortpalindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology(Reading). 2005;151(Pt 8):2551-61.87. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineeringusing CRISPR/Cas systems. Science. 2013;339(6121):819-23.88. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineeringwith CRISPR-Cas9. Science. 2014;346(6213):1258096.89. Makarova KS, Wolf YI, Koonin EV. The basic building blocks and evolution ofCRISPR-CAS systems. Biochem Soc Trans. 2013;41(6):1392-400.90. Shmakov S, Smargon A, Scott D, Cox D, Pyzocha N, Yan W, et al. Diversity andevolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017;15(3):169-82.91. Shmakov S, Abudayyeh OO, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, etal. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.Mol Cell. 2015;60(3):385-97.92. Makarova KS, Wolf YI, Alkhnbashi OS, Costa F, Shah SA, Saunders SJ, et al.An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol.2015;13(11):722-36.93. Liu TY, Doudna JA. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, andregulation. J Biol Chem. 2020;295(42):14473-87.94. Makarova KS, Aravind L, Wolf YI, Koonin EV. Unification of Cas protein families anda simple scenario for the origin and evolution of CRISPR-Cas systems. Biol Direct.2011;6:38.95. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, etal. CRISPR provides acquired resistance against viruses in prokaryotes. Science.2007;315(5819):1709-12.96. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided humangenome engineering via Cas9. Science. 2013;339(6121):823-6.97. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, et al. TargetingDNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186(2):757-61.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

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230 NEW THERAPEUTIC APPROACHES IN GENOME112. Miliotou AN, Papadopoulou LC. CAR T-cell Therapy: A New Era in CancerImmunotherapy. Curr Pharm Biotechnol. 2018;19(1):5-18.113. Azangou-Khyavy M, Ghasemi M, Khanali J, Boroomand-Saboor M, Jamalkhah M,Soleimani M, et al. CRISPR/Cas: From Tumor Gene Editing to T Cell-BasedImmunotherapy of Cancer. Front Immunol. 2020;11:2062.114. Hu KJ, Yin ETS, Hu YX, Huang H. Combination of CRISPR/Cas9 System and CAR-TCell Therapy: A New Era for Refractory and Relapsed Hematological Malignancies.Curr Med Sci. 2021;41(3):420-30.115. Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, et al. Chimericantigen receptor T cells for sustained remissions in leukemia. N Engl J Med.2014;371(16):1507-17.116. Braendstrup P, Levine BL, Ruella M. The long road to the first FDA-approvedgene therapy: chimeric antigen receptor T cells targeting CD19. Cytotherapy.2020;22(2):57-69.117. Zheng PP, Kros JM, Li J. Approved CAR T cell therapies: ice bucket challenges onglaring safety risks and long-term impacts. Drug Discov Today. 2018;23(6):1175-82.118. Riviere I, Sadelain M. Chimeric antigen receptors: a cell and gene therapy perspective. `Molecular therapy. 2017;25(5):1117-24.119. Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. ’Off-the-shelf’ allogeneic CAR Tcells: development and challenges. Nat Rev Drug Discov. 2020;19(3):185-99.120. Zhang Y, Tacheva-Grigorova SK, Sutton J, Melton Z, Mak YSL, Lay C, et al. AllogeneicCAR T Cells Targeting DLL3 Are Efficacious and Safe in Preclinical Models of SmallCell Lung Cancer. Clin Cancer Res. 2023:OF1-OF15.121. Poirot L, Philip B, Schiffer-Mannioui C, Le Clerre D, Chion-Sotinel I, Derniame S, et al.Multiplex Genome-Edited T-cell Manufacturing Platform for ”Off-the-Shelf” AdoptiveT-cell Immunotherapies. Cancer Res. 2015;75(18):3853-64.122. Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJ, Hamieh M, Cunanan KM, etal. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017;543(7643):113-7.123. Ren J, Zhang X, Liu X, Fang C, Jiang S, June CH, et al. A versatile system for rapidmultiplex genome-edited CAR T cell generation. Oncotarget. 2017;8(10):17002-11.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 231124. Haslauer T, Greil R, Zaborsky N, Geisberger R. CAR T-Cell Therapy in HematologicalMalignancies. Int J Mol Sci. 2021;22(16).125. Jiang W, He Y, He W, Wu G, Zhou X, Sheng Q, et al. Exhausted CD8+T Cells inthe Tumor Immune Microenvironment: New Pathways to Therapy. Front Immunol.2020;11:622509.126. Rupp LJ, Schumann K, Roybal KT, Gate RE, Ye CJ, Lim WA, et al.CRISPR/Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of humanchimeric antigen receptor T cells. Sci Rep. 2017;7(1):737.127. Sterner RM, Sakemura R, Cox MJ, Yang N, Khadka RH, Forsman CL, et al. GM-CSFinhibition reduces cytokine release syndrome and neuroinflammation but enhancesCAR-T cell function in xenografts. Blood. 2019;133(7):697-709.128. Tang N, Cheng C, Zhang X, Qiao M, Li N, Mu W, et al. TGF-beta inhibition viaCRISPR promotes the long-term efficacy of CAR T cells against solid tumors. JCIInsight. 2020;5(4).129. Cooper SL, Brown PA. Treatment of pediatric acute lymphoblastic leukemia. PediatrClin North Am. 2015;62(1):61-73.130. Chamberlain CA, Bennett EP, Kverneland AH, Svane IM, Donia M, Met O. Highlyefficient PD-1-targeted CRISPR-Cas9 for tumor-infiltrating lymphocyte-based adoptiveT cell therapy. Mol Ther Oncolytics. 2022;24:417-28.131. Nakazawa T, Natsume A, Nishimura F, Morimoto T, Matsuda R, Nakamura M, et al.Effect of CRISPR/Cas9-Mediated PD-1-Disrupted Primary Human Third-GenerationCAR-T Cells Targeting EGFRvIII on In Vitro Human Glioblastoma Cell Growth. Cells.2020;9(4):998.132. Guo X, Jiang H, Shi B, Zhou M, Zhang H, Shi Z, et al. Disruption of PD-1 Enhancedthe Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against HepatocellularCarcinoma. Front Pharmacol. 2018;9:1118.133. Liu M, Wang X, Li W, Yu X, Flores-Villanueva P, Xu-Monette ZY, et al. TargetingPD-L1 in non-small cell lung cancer using CAR T cells. Oncogenesis. 2020;9(8):72.134. Deng H, Tan S, Gao X, Zou C, Xu C, Tu K, et al. Cdk5 knocking out mediated byCRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity.Acta Pharm Sin B. 2020;10(2):358-73.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

232 NEW THERAPEUTIC APPROACHES IN GENOME135. Wang H, Hu S, Chen X, Shi H, Chen C, Sun L, et al. cGAS is essential for theantitumor effect of immune checkpoint blockade. Proceedings of the National Academyof Sciences. 2017;114(7):1637-42.136. Rahman MM, Tollefsbol TO. Targeting cancer epigenetics with CRISPR-dCAS9:Principles and prospects. Methods. 2021;187:77-91.137. Cao Y, Hu Q, Zhang R, Li L, Guo M, Wei H, et al. Knockdown of Long Non-codingRNA SNGH3 by CRISPR-dCas9 Inhibits the Progression of Bladder Cancer. Front MolBiosci. 2021;8:657145.138. Li W, Cho M-Y, Lee S, Jang M, Park J, Park R. CRISPR-Cas9 mediated CD133 knockoutinhibits colon cancer invasion through reduced epithelial-mesenchymal transition. PLOSONE. 2019;14(8):e0220860.139. Yan J, Jia Y, Chen H, Chen W, Zhou X. Long non-coding RNA PXN-AS1 suppressespancreatic cancer progression by acting as a competing endogenous RNA of miR-3064 toupregulate PIP4K2B expression. Journal of Experimental & Clinical Cancer Research.2019;38(1).140. Koo T, Yoon AR, Cho HY, Bae S, Yun CO, Kim JS. Selective disruption of an oncogenicmutant allele by CRISPR/Cas9 induces efficient tumor regression. Nucleic Acids Res.2017;45(13):7897-908.141. Tang KJ, Constanzo JD, Venkateswaran N, Melegari M, Ilcheva M, Morales JC, etal. Focal Adhesion Kinase Regulates the DNA Damage Response and Its InhibitionRadiosensitizes Mutant KRAS Lung Cancer. Clin Cancer Res. 2016;22(23):5851-63.142. Wu B, Song M, Dong Q, Xiang G, Li J, Ma X, et al. UBR5 promotes tumor immuneevasion through enhancing IFN-?-induced ≺i≻PDL1≺/i≻ transcription in triple negativebreast cancer. Theranostics. 2022;12(11):5086-102.143. Liu Y, Hu X, Han C, Wang L, Zhang X, He X, et al. Targeting tumor suppressor genesfor cancer therapy. Bioessays. 2015;37(12):1277-86.144. Moses C, Nugent F, Waryah CB, Garcia-Bloj B, Harvey AR, Blancafort P. ActivatingPTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. Mol Ther NucleicAcids. 2019;14:287-300.145. Banas K, Rivera-Torres N, Bialk P, Yoo B-C, Kmiec EB. Temporal analyses ofCRISPR-directed gene editing on NRF2, a clinically relevant human gene involvedin chemoresistance. 2019.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

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234 NEW THERAPEUTIC APPROACHES IN GENOME158. Hwang JH, Yoon J, Cho YH, Cha PH, Park JC, Choi KY. A mutant KRAS-inducedfactor REG4 promotes cancer stem cell properties via Wnt/beta-catenin signaling. Int JCancer. 2020;146(10):2877-90.159. Suarez-Martinez E, Suazo-Sanchez I, Celis-Romero M, Carnero A. 3D and organoidculture in research: physiology, hereditary genetic diseases and cancer. Cell Biosci.2022;12(1):39.160. Bian S, Repic M, Guo Z, Kavirayani A, Burkard T, Bagley JA, et al.Genetically engineered cerebral organoids model brain tumor formation. Nat Methods.2018;15(8):631-9.161. Luo C, Lancaster MA, Castanon R, Nery JR, Knoblich JA, Ecker JR. CerebralOrganoids Recapitulate Epigenomic Signatures of the Human Fetal Brain. Cell Rep.2016;17(12):3369-84.162. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz Jr LA, Kinzler KW. Cancergenome landscapes. science. 2013;339(6127):1546-58.163. Choo Y, Sanchez-Garc ´ ´ıa I, Klug A. In vivo repression by a site-specific DNA-bindingprotein designed against an oncogenic sequence. Nature. 1994;372(6507):642-5.164. Tanaka A, Takeda S, Kariya R, Matsuda K, Urano E, Okada S, et al. A novel therapeuticmolecule against HTLV-1 infection targeting provirus. Leukemia. 2013;27(8):1621-7.165. Huang N, Huang Z, Gao M, Luo Z, Zhou F, Liu L, et al. Induction of apoptosis inimatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption ofbcr-abl oncogene with zinc finger nucleases. Journal of Experimental & Clinical CancerResearch. 2018;37:1-14.166. Herrmann F, Garriga-Canut M, Baumstark R, Fajardo-Sanchez E, Cotterell J, MinocheA, et al. p53 Gene repair with zinc finger nucleases optimised by yeast 1-hybrid andvalidated by Solexa sequencing. PloS one. 2011;6(6):e20913.167. Zhang W-W, Li L, Li D, Liu J, Li X, Li W, et al. The first approved gene therapyproduct for cancer Ad-p53 (Gendicine): 12 years in the clinic. Human gene therapy.2018;29(2):160-79.168. Liang M. Oncorine, the world first oncolytic virus medicine and its update in China.Current cancer drug targets. 2018;18(2):171-6.169. Vaishnaw AK, Gollob J, Gamba-Vitalo C, Hutabarat R, Sah D, Meyers R, et al. A statusreport on RNAi therapeutics. Silence. 2010;1:1-13.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

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˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 237197. Rawat M, Kadian K, Gupta Y, Kumar A, Chain PS, Kovbasnjuk O, et al. MicroRNA inpancreatic cancer: from biology to therapeutic potential. Genes. 2019;10(10):752.198. Ngamcherdtrakul W, Castro DJ, Gu S, Morry J, Reda M, Gray JW, et al. Currentdevelopment of targeted oligonucleotide-based cancer therapies: perspective onHER2-positive breast cancer treatment. Cancer treatment reviews. 2016;45:19-29.199. Ma L, Liang Z, Zhou H, Qu L. Applications of RNA indexes for precision oncology inbreast cancer. Genomics, Proteomics Bioinformatics. 2018;16(2):108-19.200. Binkhathlan Z, Alshamsan A. Emerging nanodelivery strategies of RNAimolecules for colon cancer therapy: preclinical developments. Therapeutic delivery.2012;3(9):1117-30.201. Hecker M, Wagner AH. Transcription factor decoy technology: A therapeutic update.Biochemical Pharmacology. 2017;144:29-34.202. Roma-Rodrigues C, Rivas-Garc´ıa L, Baptista PV, Fernandes AR. Gene therapy in cancertreatment: why go nano? Pharmaceutics. 2020;12(3):233.203. Rad SMAH, Langroudi L, Kouhkan F, Yazdani L, Koupaee AN, Asgharpour S, etal. Transcription factor decoy: a pre-transcriptional approach for gene downregulationpurpose in cancer. Tumor Biology. 2015;36:4871-81.204. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. RepurposingCRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013;152(5):1173-83.205. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, et al.CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell.2013;154(2):442-51.206. Kuscu C, Arslan S, Singh R, Thorpe J, Adli M. Genome-wide analysis revealscharacteristics of off-target sites bound by the Cas9 endonuclease. Nature biotechnology.2014;32(7):677-83.207. Gilbert LA, Horlbeck MA, Adamson B, Villalta JE, Chen Y, Whitehead EH, etal. Genome-scale CRISPR-mediated control of gene repression and activation. Cell.2014;159(3):647-61.208. Georgakilas G, Vlachos IS, Paraskevopoulou MD, Yang P, Zhang Y, Economides AN,et al. microTSS: accurate microRNA transcription start site identification reveals asignificant number of divergent pri-miRNAs. Nature communications. 2014;5(1):5700.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

238 NEW THERAPEUTIC APPROACHES IN GENOME209. 52 MGLLAWKAGPJFEAGMSCFS, Center* BCoMHGS, Center* WUGS, Institute*B, Institute* CsHOR. Identification and analysis of functional elements in 1% of thehuman genome by the ENCODE pilot project. nature. 2007;447(7146):799-816.210. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent andspecific genetic interference by double-stranded RNA in Caenorhabditis elegans. nature.1998;391(6669):806-11.211. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. nature.2001;411(6836):494-8.212. Kodzius R, Kojima M, Nishiyori H, Nakamura M, Fukuda S, Tagami M, et al. CAGE:cap analysis of gene expression. Nature methods. 2006;3(3):211-22.213. Wu X, Scott DA, Kriz AJ, Chiu AC, Hsu PD, Dadon DB, et al. Genome-widebinding of the CRISPR endonuclease Cas9 in mammalian cells. Nature biotechnology.2014;32(7):670-6.214. Valerie K, Povirk LF. Regulation and mechanisms of mammalian double-strand breakrepair. Oncogene. 2003;22(37):5792-812.215. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al.Senescence and tumour clearance is triggered by p53 restoration in murine livercarcinomas. Nature. 2007;445(7128):656-60.216. Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy.Molecular Therapy. 2016;24(3):430-46.217. Naldini L. Gene therapy returns to centre stage. Nature. 2015;526(7573):351-60.218. Baum C, Von Kalle C, Staal FJ, Li Z, Fehse B, Schmidt M, et al. Chance or necessity?Insertional mutagenesis in gene therapy and its consequences. Molecular Therapy.2004;9(1):5-13.219. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges.Nature medicine. 2015;21(2):121-31.220. Anguela XM, Sharma R, Doyon Y, Miller JC, Li H, Haurigot V, et al. RobustZFN-mediated genome editing in adult hemophilic mice. Blood, The Journal of theAmerican Society of Hematology. 2013;122(19):3283-7.221. Brenner MK, Gottschalk S, Leen AM, Vera JF. Is cancer gene therapy an empty suit?The lancet oncology. 2013;14(11):e447-e56.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 239222. Goins WF, Huang S, Cohen JB, Glorioso JC. Engineering HSV-1 vectors for genetherapy. Herpes Simplex Virus: Methods and Protocols. 2014:63-79.223. Wang D, Gao G. State-of-the-art human gene therapy: part Ii. gene therapy strategiesand applications. Discovery medicine. 2014;18(98):151.224. Kochenderfer JN, Rosenberg SA. Treating B-cell cancer with T cells expressinganti-CD19 chimeric antigen receptors. Nature reviews Clinical oncology.2013;10(5):267-76.225. Baum C, Modlich U, Gohring G, Schlegelberger B. Concise review: managing ¨genotoxicity in the therapeutic modification of stem cells. Stem Cells.2011;29(10):1479-84.226. Ramamoorth M, Narvekar A. Non viral vectors in gene therapy-an overview. Journal ofclinical and diagnostic research: JCDR. 2015;9(1):GE01.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCHAPTER 9TARGETING STRATEGIES WITH NEW DRUGDELIVERY SYSTEMS IN CANCER THERAPYYavuz Selim C¸ EL˙IK1,2, Burcu MESUT3, Yıldız OZSOY ¨ 41PhD candidate, ˙Istanbul University Institute of Graduate Studies in Health Sciences, ˙Istanbul, T¨urkiye2˙Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Technology, ˙Istanbul, T¨urkiyeE-mail: [email protected]., ˙Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Technology, ˙Istanbul,T¨urkiyeE-mail: [email protected]., ˙Istanbul University, Faculty of Pharmacy, Department of Pharmaceutical Technology, ˙Istanbul, T¨urkiyeE-mail: [email protected]: 10.26650/B/LSB28LSB48LSB56.2024.019.009ABSTRACTNanotechnology has been researched extensively for the treatment of cancer and used for it, since nanoparticlescan be an overall effective drug delivery technique. Nanoparticle-based drug delivery provides distinct benefits overmore conventional and traditional drug delivery methods, such as greater biocompatibility and stability, increasedretention effect and permeability, as well as precision targeting. Many nanotechnology applications have been created,and numerous medications based on nanotechnology are already available on the market, therefore the impact ofnanotechnology on healthcare is already being seen. The worldwide pharmaceutical market and healthcare systemare expected to be significantly impacted by pharmaceutical nanomedicine products. Researchers working on findingbiomarkers and diagnosing cancer are interested in nanomaterials as well. In fact, preclinical and clinical trials arenow being conducted on a number of anticancer nanodrugs, which have shown promise in therapeutic and othersituations. In this chapter, nano based drug carriers such as liposomes, polymeric nanoparticles, polymeric micelles,dendrimers, inorganic nanomaterials and also passive and active targeting issues are highlighted.Keywords: Cancer therapy, active targeting, passive targetingCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Yavuz Selim C¸ EL˙IK, Burcu MESUT, Yıldız OZSOY ¨ 2411. IntroductionAs drug carriers, nanomaterials provide a variety of benefits and as a result, thepharmacological and pharmacokinetic properties of the drugs are improved. (1) Nanocarrierscan also deliver drugs to specific tissues or cells, enhancing therapeutic efficacy and limitingaccumulation of drugs in non-targeted organs like kidneys, spleen, liver and other tissues.Additionally, they can deliver a combination of imaging and therapeutic agents for real-timemonitoring of therapeutic effects (2,3).There have been a lot more authorized nano-based medicinal products and uses since 1989(4). Over 80 nanomedicine products have received commercial approval from the Food andDrug Administration (FDA) and the European Medicines Agency (EMA) in the past 25 years.A list of approved nano-based products for use in cancer treatment is given in Table 1.Table 1: Nano-based products approved for cancer treatment by FDA and EMA21.IntroductionAs drug carriers, nanomaterials provide a variety of benefits and as a result, the pharmacological andpharmacokinetic properties of the drugs are improved. (1) Nanocarriers can also deliver drugsto specifictissues or cells, enhancing therapeutic efficacy and limiting accumulation of drugsin non targeted organslike kidneys, spleen, liver and other tissues. Additionally, they can deliver a combination of imagingand therapeutic agents for real-time monitoring of therapeutic effects (2,3).There have been a lot more authorized nano-based medicinal products and uses since 1989 (4). Over80 nanomedicine products have received commercial approval from the Food and Drug Administration(FDA) and the European Medicines Agency (EMA) in the past 25 years. A list of approved nano-basedproducts for use in cancer treatment is given in Table 1.Table 1. Nano-based products approved for cancer treatment by FDA and EMAName Form Approval Active Ingredient(s) CompanyAbraxane®Albumin-boundNanoparticleFDA (2005, 2012,2013), EMA (2008)PaclitaxelCelgene Pharmaceutical Co.Ltd.Apealea® Micellar EMA (2018) Paclitaxel Oasmia Pharmaceutical ABCaelyx® Liposomal EMA (1996) Doxorubicin Janssen PharmaceuticalsDaunoXome® LiposomalEMA (1996)FDA (1996)Daunorubicin Galen Ltd.Doxil® LiposomalFDA (1995)EMA (1996)Doxorubicin(adriamycin)Johnson & JohnsonGenexolPM® Micellar FDA (2007) Paclitaxel Lupin Ltd.Lipodox® Liposomal FDA (2013)DoxorubicinhydrochlorideSun Pharma Global FZELipusu® Liposomal FDA (2016) Paclitaxel Luye PharmaMarqibo® Liposomal FDA (2012) Vincristine Talon TherapeuticsMepact® Liposomal EMA (2009) Mifamurtide Takeda France SASMyocet® Liposomal EMA (2000)DoxorubicinhydrochlorideTeva PharmaceuticalIndustries Ltd.Neulasta®Polymer-ProteinConjugateFDA (2002) Pegfilgrastim Amgen, Inc.Onivyde® Liposomal FDA (2015) Irinotecan Merrimack PharmaceuticalsOnpattro® LiposomalFDA (2018)EMA (2018)Patisiran AlnylamPazenir®Albumin-boundNanoparticleEMA (2019) Paclitaxel Ratiopharm GmbHVyxeos® LiposomalFDA (2017)EMA (2018)Daunorubicin andcytarabineJazz PharmaceuticsA new phase of cancer therapy has been made possible by nanotechnology in medicine, and theintersection of these two domains merits further investigation (5). This review highlights the existingobstacles, discusses the future areas of research, and defines the fundamental ideas underlying the useof the nano-carrier system in cancer therapy.A new phase of cancer therapy has been made possible by nanotechnology in medicine, andthe intersection of these two domains merits further investigation (5). This review highlightsthe existing obstacles, discusses future areas of research, and defines the fundamental ideasunderlying the use of the nano-carrier system in cancer therapy.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.