Recent Advancements in Gene Therapy in the Treatment of Triple-Negative Breast Cancer Using Nanotechnology

Abstract

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer. Due to the deficiency in estrogen, progesterone, and ERBB2 receptor expression, these TNBC cells are unmanageable in nature. Till now the specific treatment for this TNBC cancer is not present which leads to the death of the patient. So to overcome this at present scientists are trying to use gene therapy to treat TNBC. RNA-based therapeutic strategies are used in the treatment of TNBC via gene therapy. Currently, two methods are under investigation- (i) RNA interference (RNAi), in this method, RNAi uses gene silencing mechanism, and (ii) RNA nano therapy where chemotherapeutics is inserted into the host body or target cell by using RNA-derived nanoparticles. Additionally small interfering RNA (siRNA) or microRNA (miRNA) are used as vectors to perform RNA-based gene therapy in the treatment of TNBC. Both of these methods are very complex processes. Different types of nanoparticles such as polymer-based, lipid-based, nanoparticles, beside this the amorphous drug–polyelectrolyte nanoparticle complexes are also been used for RNA-based therapeutics of gene therapy. Besides this, there is CRISPR/Cas-based therapeutics in the treatment of TNBC is also under investigation. In this method, CRISPR-based gene therapy DNA binding domain-dependent orthodox techniques, specifically, zinc finger nuclease (ZFNs) and transcriptional activator-like effector nuclease (TALENs) have gained exceptional importance in genetically engineered breast tissue organoids, cellular carcinoma models, and gene therapy investigations. In the delivery of CRISPR/Cas 9, several organic and inorganic nanoparticles, lipid and protein-based nanoparticles are used to deliver dynamic DNA, RNA, and mRNA.

Keywords

Introduction

Figure 1: Advancement, limitation, and future scope of CRISPR/Cas9 based gene therapy in breast cancer treatment

Gene therapy for TNBC through CRISPR/Cas (CRISPR)

 For the CRISPR-based gene therapy, zinc finger nuclease (ZFNs) and transcriptional activator-like effector nuclease (TALENs) gets bind to the DNA by domain dependent orthodox techniques and it has achieved a significant importance in performing genetical modification in breast tissue organoids, cellular carcinoma models, and gene therapy investigations [13]. Its complexity and incompetence, is the main drawback of this comprehensive application of this therapeutic approach . Where this therapy is bounded, on the other hand, for therapeutic treatment of cancer, for expressing the gene RNAi has shown scientific and medicinal importance in administration [11]. It has a transitory silencing impact but despite this to gain a specific level of gene knockout, it must be constantly administrated [14]. At present, adaptive immunity that has been collected or derived from invented archaea, that therapeutic method is based on the principle of double-stranded RNA endonuclease, called CRISPR-associated protein-9 (Cas9) and has earned an important role in this therapeutic model of treating cancer [14].

Editing and screening of genome in TNBC through advanced CRISPR therapeutics

A powerful method, genome screening is efficient to spot proteomes or genes which is the regions of origin of tumour which is occurred through continuous mutation and proliferation, and the advanced therapeutic technology has made it easier to tackle via the CRISPR-Cas9 gene therapy system that develops new inflects for targeting noncoding and coding genes [15]. This screening is very much useful to find out either the entered gene sequence has expressed in the target cell or not and to know if it is effective for loss of function or overexpression of a particular gene sequence or a complete gene present in the target cell by gene silencing method and to identify if it affects in the resistance or tolerance of genes, immunosuppression, proliferation, invasion, and metastasis in the treatment of TNBC [16]. In occasion to this, CRISPR knockout gene-editing tool likeCRISPR/pSpCas9(BB)-2A-puro v2.0 plasmid, CRISPR/Cas13a polylysine black phosphorus nanosheets NPs helps in gene manipulation. For example, CRISPR/pSpCas9(BB)-2A-puro v2.0 plasmid tool targets the BAG3 molecule,Negatively regulates cyclogenesis and induces resistance to the therapy through the in vitro study which shows effective results in silencing the BG3 molecule, significantly decreased invasion, expression of ZEB1 and SNAI1 (induce EMT), AURKA (cilium disassemble) and CDK1 (phosphorylate PLK1 to disassemble cilia) and cell migration [16]. The AKT/mTOR/MAPK oncogenic signaling cascades have been reduced by the activation of PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) activation in TNBC via dead (d)-Cas9 merged with VP64-p65-Rta. Moreover, tRNA-based multiplex CRISPR/dCas9 also used in identification of HER2+ breast cancer cells [17].

There are some other regulatory elements that are successfully able to knock out gene sequences by utilizing CRISPR gene therapeutics. For example, inhibition of BET as a candidate is targeted by BBDIs (Bromodomain inhibitors) for therapeutic aid of TNBC relapse, while it shows intrinsic and acquired tolerance against BBDIs frontier clinical potential. Additionally, the BET signaling pathway of activation, SRC, AXL, YAP signaling, systemic regulation, and chemo-resistance in TNBC cell lines SUM149, and SUM159 has been revealed by a small-molecule inhibitor such as JQ1 and by silencing the gene sequence with CRISPR [18].  The CRISPR-based dual 10-base pair of the gene cut the first and second exon of transcriptional factor Snail1, and it opens up the partial involvement of the protein in the EMT phenotype in the TNBC mutant gene [17, 14]. Further, during the genome editing and screening, CRISPR has immensely exploited in the loss-of-function screen of 2240 genes [18], 2500 novel super-enhancer, including TGF-β pseudo receptor dependencies on BAMBI [19], and ATAC profiling at 104,592 ?cells [20] for the identification of specific phenotypic susceptibilities in TNBC. It is also seen that, mTOR/Hippo pathway inhibits YAP protein in CRISPR screening [20].

Transcriptomic analysis using CRISPR/Cas

Formerly lncRNA, miRNA, shRNA, and siRNA-dependent interference were used for silencing but these techniques were not always perfect, and to make the complete deactivation of mRNA, the functional part of the residual mRNA is also performed in the target site and thus it resist the identification of the target site and silent the [19, 15]. This problem of screening analysis due to those techniques can be overcome by using CRISPR/Cas9 or CRISPR/dCas9 [15]. In this occasion, better analysis of synergistic drug matrix, drug target efficacy, and identification of any mischaracterization can now be analysed through the modern CRISPR/siRNA-based silencing method of gene therapy, which is able to explain that the capable or applicable inhibitor of CDK11 in TNBC is an orally active, small antagonist, high affinity and selective T-LAK cell originated protein kinase (TOPK) inhibitor hydrocarbon which is known as OTS964 hydrocarbon, and it shows multiple carcinoma addiction over CDK11.

Biomarkers for TNBC and discovery of drug using CRISPR aids

To identify which biomarker should be used in the early TNBC proliferation, the wide array screening for genome using CRISPR is an efficient technique that shows the significant result and helps in diagnosis [19]. For example, RING finger protein 208 (RNF208) is an estrogen-inducible E3 ligase, overexpression of this protein is engineered by the therapeutic treatment of CRISPR/Cas9, which induces the proteasomal degradation of Vimentin protein encoded by the VIM gene suppress the proliferation of  TNBC cell. Activation or overexpression of RNF208, due to CRISPR/Cas9-based gene alteration interacts with a serine residue (Ser39) of phosphorylated Vimentin protein, and from the head domain of Vimentin protein the lysine residue(Lys97) leads toward degradation of the proteasome, which helps to reduce the  lung metastasis and invasion in TNBC cells. On the other hand in TNBC drug development using nanoparticles, the negative feedback of RNF208 protein or inhibitors serves as an important and efficient biomarker [21]. But till now, in case of development of drug for TNBC using the proteins and the process of incorporating them in nanoparticles is a multi-step, complex, and challenging process in which scientists and pharmacists have to face several of genetic complexity such as drug target structure correlation, tissue exposure, and disease selectivity, drug target conformation and authentication, clinical dose, drug efficacy [21], prolong testing of about 12 years and huge expense that may exceed $ 1 billion are involved [20, 21]. This advanced therapeutic model is also very efficient to produce multiple insertion/mutations through which the proliferation of the TNBC cell could be reduced, it helped in knockout, knock-in screening to diagnose TNBC [21], and it is also responsible for the complete activation and suppression of the desired targets [15]. As proof, patient-derived xenograft model by CRISPR/Cas-based genome-wide screening has able to build up a new combinatorial Verteporfin-Torin1 TNBC therapy. Dual druggable targets which is blocked by CRISPR, is able to generate a better anti-tumour effect in the TNBC in in vivo model, is revealed by the CRISPR/Cas system [20].

Chemotherapy using CRISPR/Cas-based therapeutic

The efficiency of chemotherapy is increased by CRISPR system as TNBC shows hybridism, it includes evaluation in breast cancer stem cells and tumour microenvironments (TME) [22, 23]. For example,  Paclitaxel and Doxorubicin sensitivity in TNBC cell is enhanced by the knockdown of MALAT1 lncRNA via altering several lncRNA (LINC-PINT, NEAT1, and USP3-AS1) and suppressing STAT1, NUPR1, SREBF1, RELA, interferon regulatory factor 1 (IRF1), ERAP1, aminopeptidase regulator, histone-associated proteins such as

H3C12, H1-5, and H2AC4, insulin-like growth factor-binding protein 1 (IGFBP1), granulin precursor (GRN), angiogenin, and oxidative phosphorylation reputable pathway. Similar way,  in MDA-MB-436 and MDA-MB-231 ?cell lines which is facing PARP1(Poly(ADP-Ribose) Polymerase 1), a Protein Coding gene-generated deficiency, prominently decrease the Doxorubicin, Gemcitabine and Docetaxel doses according to its requirements to understand the efficiency of therapeutic in even 3D tumour-on-a-chip model [24, 25]. Thus, induced by TGF-β/Smad signalling the profoundly expressed transmembrane prostate androgen-induced protein (TMEPAI), propel TGF-β act as a tumorigenic carcinoma in TNBC patients. Complete silencing of exon 4 of TMEPAI by sgRNA does not shows resistance against Paclitaxel, Doxorubicin [19] but it has a very less tolerance for Bicalutamide and Cisplatin, which indicates the partial potency of TMEPAI knockout for chemotherapeutic usage. In the advancement technology, BCL-2 family antagonist BH3 mimetics like S63845 is now versatile application as an efficient therapeutic method in chemo-immuno combo-therapy.  Tumour cells generate drug resistance or chemoresistance after repeated cycles of chemotherapy as a common result, and this is the most critical problem and due to this problem, most of the single or complex-therapies have not been successful in the treatment of TNBC. For instance, due to complete BAG3 silencing via CRISPR/Cas9, it decreases the expression of ZEB1 and SNAI1 that induce EMT in TNBC, which cut down the TNBC cell migration and penetration in tissues as well as shows some efficient results in therapy resistance and thus CRISR/Cas9 gene therapy offers advanced therapeutic approach to overcome chemoresistance in tumour cells [19, 20]. 

Immunotherapy via CRISPR/Cas-based therapeutic

TNBC is a complex and immuno-based heterogeneous form of gene in nature and this gene makes its profiling complicated due to the involvement of various signalling molecules, intra and extra cellular matric, specialized T-cells along with diverse kind of mutations, and genomic instability [23]. To enhance response during TNBC treatment several exogenous inductions of PD-L1 by PI3K/AKT/mTOR, JAK/STAT1/IRF1, NF-κB/JAK/STAT3, ECM, IFN-γ, EGFR, interleukins (ILs), TNF-α, NPM1 or B23, vascular endothelial growth factor receptor-2 (VEGFR2), and PARP1 were being used [21]. The meticulous transcriptional control of PD-L1 in TNBC remains contentious and several ongoing clinical research or pre-clinical trials combinatorial, targeted, ligand-specific, NAC or chemo-immunotherapies are being examined to identify and classify foreseeable biomarkers that would be helpful in the advance treatment consequences among TNBC patients,

which may be due to the use of the immuno exclusion process, and several agents used for treatment and immune checkpoint markers or inhibitors which have been used to address the developed tumour which have been causing this issue most of TNBC patients do not retort to anti-PD-1/PD-L1 [26, 27]. TNBC destroys the immunityin tumorigenesis progression [24, 25], carcinoma cells deter immune effector cells through excretion of extrinsic aspects by disturbing the (TME) but the immunity reaction can be elevated by jam-packed blocking of CD47 protein [27]. An innovative effort induced by ECM to understand the immuno exclusion study shows, the discoidin domain receptor 1 (Ddr1) gene was entirely silenced by All-in-One Lentivector CRISPR/Cas9 system in E0771, M-Wnt and AT-3 mouse mammary tissues, which revealed the potent involvement of untethered-extra cellular domain (ECD) of DDR1 protein [28]. The discoidin domain receptors (DDRs) belong to the non-integrin collagen receptors family which have tyrosine kinase activity. The DDR1-ECD interacts with collagen, disturbs fibre alignment, induces immuno exclusion, deter immunity, and uses collagen fibre in tumour defence. Additionally, its membrane-bound intracellular kinase domain assists DDR1-ECD against antitumor T-cells [21]. This CRISPR-based finding of anti-PD-1/PD-L1 immunotherapy onlyworks on fewer TNBC patients . A new antibody against DDR1 needs to be developed to reduce metastasis of TNBC cells and enhance the life span of the TNBC patients.

Further, during the CRISPR base gene therapy treatment during immune checkpoint inhibitors documentation the proposed prime target are the carcinoma-precise polyclonal memory CD4⁺, and CD8⁺T [29]. The T cell-dependent immunotherapy is credited to the application of ex vivo exploited T lymphocytes with tumour-associated antigen (TAA) or CAR-T cells, and T cell receptors (TCR)-engineered T lymphocytes stops the growth of the tumour and help to abolish tumour [20,26]. In CAR-T cell therapy, the T cells are possibly originated from an allogeneic (cells derived from another person) or autologous (cells taken from the same person) donor but autologous T cells utilization is hypothetically is an inefficient process and it mostly depends on the quantity and quality of autologous T cells harvested from the subjected patient. On the other hand, using allogeneic grafted T cells challenging due to the TCR on donor's T lymphocytes, and endogenous MHC class I complex works as allergen on the host body means it possess alloreactivity and graft-versus-host disease (GVHD). Therefore, the CAR gene is inserted by the CRISPR/Cas9 gene-editing technology and it arrests the TCR effectively. For instance, the highly expressed receptors on TNBC cells is EGFR, which is successfully engineered to expressby using third-generation CAR-T targeting EGFR in TNBC both in vivo xenograft mouse model and in vitro, and it has revealed limited cytotoxicity, retard tumour cell growth, enhanced IFN-γ, activate PARP, and Fas-associated death domain (FADD), and caspase signalling. The same way, 95% in TNBC cases, CRISPR-engineered CAR-T cells with scFv of grafted monoclonal TAB004 antibody coupled to CD3 and CD28 for mutant glycosylated tumour of MUC1 (MUC28z), significantly victimized cytotoxicity over an array of human TNBC cells, upon identification of mutant MUC1, MUC28z CAR-T cells elevate synthesis of IFN-γ, granzyme B, Th1 and other cyto- and chemokines [25]. This theory helps to understand that a single dose of CRISPR/Cas9 engineered CAR-T cells can significantly reduce tumour growth in NOD-Prkdc^scid IL2rg^null (NSG) mice xenograft model and may be this model of therapy would be helpful for human too [29].

Clinical studies of CRISPR theraputics

In pre-clinical research for tumor CRISPR therapeutics has played several significant importance, and due to this from pre-clinical research it went to advanced stage or went for clinical studies [30]. Till present, according to the National Library of Medicine (https://www.clinicaltrials.gov) there are registration of almost 18 cases which are going through clinical trials. Among those most of the CRISPR clinical trial is dependent on the PD-1 based immunotherapy, on the other hand to identify the efficiency and safety of using this CRISPR technique this clinical trial need to be performed and continuously assess. Additionally, use of the autologous CAR-T cells prepared by the genetic modification using CRISPR till present is only applicable for lung cancer or non-small-cell lung cancer so till now this limitation has not overcome till now for which clinical trial is going on [30]. With the advanced CRISPR-based allogeneic T-cell therapy it is more time consuming and this therapeutics has more ability, less costly, and rapid than autologous CAR-T cell therapy [29].

Components of CRISPR/cas

The principle of CRISPR-Cas gene therapy or gene editing technology is based on the immuno-adaptive mechanism of prokaryotes against an invasion of foreign DNA/RNA [29] Till present, gene editing technique of CRISPR-Casis mainly clasified into two major classes and further sub-divided into six types and those subtypes are again branched into 33 sub-types [29]. The CRISPR/Cas system which has two parts Class-I and Class-II is mainly based on site-specific Cas endonuclease and CRISPR-RNA [29]. The Class-1 CRISPR-Cas is consists of multiple effector protein complexes, while Class-2 CRISPR-Cas systems have a single effector protein complex [30]. Further, Class-1 consists of Type I (CRISPR-Cas3), III (CRISPR-Cas10), and IV (CRISPR-Cas6), while Class-2 comprises of Type II (CRISPR-Cas9), V (CRISPR-Cas12 and CRISPR-Cas14), and VI (CRISPR-Cas13).

CRISPR-based gene editing and targeting in TNBC

CRISPR/Cas system could be synthesized from plasmid, ribonucleoprotein (RNP), and mRNA-based methods and could be used to screen, treat, and engineer in vitro and in vivo TNBC genome at their desired location. This genome editing through CRISPR-Cas could be done by several techniques, one of the technique is Plasmid based genome editing where pLKO-based plasmid [30], and dual vector bearing-plasmid system is a modest and lucrative alternative approach for RNP, which signifies tractability in scheme owing to the affluence of DNA sequences integration into plasmids retaining unpretentious molecular cloning systems. The second technique of genome editing is Cas9-RNA ribonucleoprotein-based genome editing scheme also declines the cell type quality since transcriptional directing cannot be engaged in the targeted tumour cells [30]. It is also an important fleeting practical genome-engineering method which i9s responsible for the lesser off-target impact, immuno stimulation, and cytotoxicity and it is a pragmatic approach. The third one is mRNA-based genome editing where the mRNA-based technique is used to reduce genome excision deferral where mRNA indoctrination sgRNA and Cas9is transferred in the target carcinoma cell or tumour cell.

Figure 2: Several mechanisms for delivering CRISPR/Cas 9 system targeting TNBC cells

Delivery of CRISPR/Cas system through viruses

Delivery vector for CRISPR/Cas based targeting in TNBC

Three distinct methods to achieve the CRISPR-mediated genome editing are (i) physical method for transporting CRISPR/Cas9 system, (ii) lentiviral/adenoviral-based delivery system, and (iii) nanocarriers-based delivery In CRISPR/Cas based gene therapy to insert the system some viruses like Lentivirus, adenovirus (Ad), adeno-associated virus (AAV) is used and retrovirus-based CRISPR delivery are widely used among all the delivery method through viruses, to manage redox, pH, enzymes, and

Other multiple stimuli-responsive elements/environments [12]. For example, screening of the TNBC cell can be performed to demonstrate or identify the clonogenic growth, impairment of tumour spheroid, and other significant distinctive nature of TNBC cells through the lentivirus-CRISPR complex screening.

Delivery of CRISPR/Cas9 system through physical method

The physical methods for expressing the CRISPR/Cas9 complex in the host cell is highly used recently due to its high accuracy and non-aggressive or non-attacking strategies like optical, temperature, magnetic field, pH, ultrasound-responsive elements and this physical methods includes electroporation, and microinjection to deliver CRISPR and it has also observed that during this delivery, it depends on the time of release and an extended space remain present . Thus, physical method of CRISPR/Cas system insertion offers the high transfection competence, but in case of in vivo drives many unsuccessful deliveries has been recorded and have shown series uptake on this approach, but identifies the specific weak and viable cell. In in vivo experiments the hydrodynamic mode of delivering CRISPR/Cas plays a significant role [29].

Delivery of CRISPR/Cas9 system through nanoparticles

Dynamic DNA, RNA or mRNA, and proteome could be non-toxic and effectually be transferred at the targeted cells or tissues, unsolicited genetic aberrations, inadequate packaging size, and immunogenicity are the principal problems in translational gene therapeutics and genome editing [30].Now a days, for nanoparticles (NPs) are getting considerable attention in delivering CRISPR/Cas cargo to the targeted sites due to the dynamic small sizes, exceptional loading capacity, comfortable penetration capability through the phospholipid bilayer, and other intracellular barriers that help in amplified pervasion and delivery. Nanocarriers like cargo-encumbered organic (exosome, dendrimers, micelles, nucleic acid, and liposomes) and inorganic (generally metal and magnetic-based) components are used in present to insert and maintain the stability, biodegradation and to accomplish meritorious biocompatibility by engaging it with NPs (Nanoparticles) in clinical translation and the unique features of NPs convey the competency to transport the captured CRISPR cargo/complexes into the cell or even within the nucleus's targeted sits. Depending on the shape, size, and surface chemistry of NPs some most important factors such as cellular uptake, biodistribution and rapid clearance can be regulated or systematically maintained [26].

Protein-based nanocarriers 

For protein based nano carriers a protein compound named albumin is vividly utilized as nanoparticles that offers safety, biocompatibility, and suitable surface adjustment which is  able to get attached to the existing amino and carboxylic groups which could be proved as an innovation of medical science [29]. This protein could be enormously found from blood of Homo sapiens, this protein is 67kDa (kilodalton) andit has mostly 19 days half-life. It could be used as a carrier for several compounds, such as copper and zinc metal ions and bilirubin, which shows positive result in the transportation and solubilization of long hydrophobic fatty acid tail. Beside this, the albumin NPs have some significant binding sites that easily perform proliferation of hydrophobic and hydrophilic drugs with the particle medium.

 Lipid-based nanocarriers 

For batter insertion of the CRISPR/Cas complex into the target tumour cells and to fight against the ejection of the system and to get solution of the multidrug confrontation problem since from 2017 the lipid-based CRISPR/Cas delivery vector has deduced to use. To use lipid based nano carriers the lipid molecules has swallowing amphiphilic characteristics is attached along with the hydrophobic tail and polar head that are allied between the two domains, systematically categorized into ionisable, cationic, and neutral lipids [30]. At the least pH value, the ionizable lipids easily get protonated but residual amphipathic or impartial at biological pH and besides, the ionised lipid uses the pH-responsive character to transfer complimentary sgRNA through in vivo technique, as the negatively charged membranes of blood cells does not very commonly interact with the neutral lipids, the biological compatibility of lipid nanoparticles (LNPs) get increased [26, 27]. 

Polymer-based nanocarriers 

The nanoparticles which act as nanocarrier and could be produced from synthetic or natural biocompatible and decomposable monomers or polymers is known as Polymer-based NPs are nanosized materials or polymer based nanocarriers. The polymer based nanocarriers could be synthesized by using several process such as solvent displacement or diffusion emulsification [26], ionic gelation nanoprecipitation and microfluidics. Inside the NPs core, several types of CRISPR system such as multiple, dual, or single could be engineered depending on the structure and polymer characteristics of the nano carrier and then it is chemically conjugated, entrapped in the matrix of polymer, or NPs-bound surface polymer and then it could be transferred and released payload via bulk or superficial erosion, diffusion and inflammation [29]. Polymer-based polymeric NPs, micelles, and dendrimers have been very commonly and effectively be used for pDNA, mRNA, oligonucleotides, and nucleic acid delivery and signifies prodigious packaging capability and noteworthy synthetic properties in alleviating desired gene encoding Cas9-sgRNA complex counter to serum-tempted accumulation in TNBC.

Inorganic nanocarriers 

Because of the strong applicability for CRISPR/Cas safe delivery, drug distribution and imaging implication several types of rigid nanoparticles or nano sized inorganic materials like black phosphorus, crbon (C) nanotubes, calcium carbonate (CaCO₃), gold (Au), graphene, silica (SiO₂), and iron oxide (Fe₂O₃) NPs are used for nanoformulation [29]. To be capable to carry various types of the nanostructure, surface morphology, sizes, and geometries these inorganic NPs are specifically engineered and these specifically modified inorganic nanocarriers have essential significance quality of the material and due to this characteristic it has inimitable magnetic, physical, optical, and electrical characteristics. For example eventually the gold nanocarriers was prepared which has its own free electrons at their outermost shell that constantly oscillate in a frequency-dependent manner according to the shape, size, and magnitude, benevolent the photothermal character fascinated significant consideration to emerging pioneering CRISPR/Cas9 carrier structures for targeting tumours and it is a well-researched prepared NP nanostructures that are exquisitely exploited in innumerable forms, including nanostars, nanospheres, nanocages, nanorods, and nanoshells   and widely used in effective breast therapy. Gold NPs also has an different characteristics property to modify the surface and due to that it is significantly incorporated in the advance treatment of breast carcinoma diagnosis based on free-DNA which is caused due to effective site-specialized primer design and false-positive signals limitations and permanently could be overcome via CRISPR/Cas-dependent fluorescent biosensing system. Carbon is an another inorganic nanocarrier, which has showed effective result in the expansion of improved carrier delivery systems due to the governable topographies such as wider surface area concerning volume ratio, simple surface alteration to upsurge uptake, tuneable size, immunologically inactive surface, and excellent stability in an internal biological setting. Similarly, an inorganic nanocarrier iron oxide is an element which is very preciously examined for production of nanomedicine, and most of those elements are acknowledged by FDA (Food and Drug Administration) and has gained the permission to be used as nanodrugs or vehicles for gene delivery [30].

DISCUSSION

Here, we have discussed that still there is no specific and scientifically established treatment for the carcinoma of TNBC as the reduction of complete triple negative carcinoma mass and

distinct organ metastasis is not achieved through clinical trial. Because of the specific microenvironment for tumour, heterogeneity of multiple cellular and oncogenic complexities TNBC are more challenging and the radiotherapy, chemotherapy and monotherapy is not able to provide prime success in the treatment of TNBC. So various research is still going on in order to establish a successful treatment against TNBC to provide new life to the patients. That’s why RNA-based therapies and CRISPR/Cas based therapeutics treatment has ensure a very interesting place in past few years and use of this process has been increased in the treatment of several types of disease, among those TNBC has ensure the more interesting area of research using this therapeutics. But till date, very limited amount of clinical trial has been registered and investigated for the treatment of TNBC using RNA-based therapeutics. Despite of several potentials of progress it has some challenges and to get the solution of these hurdles is very important before clinically this therapeutic treatment method get permission. One of the major challenge of this therapy is that RNAi is the lack of miRNA and because of this the RNAi is not always able to bind perfectly to the target mRNA and due to this another set of mRNA could be targeted and get degraded by a single miRNA. When the molecules are injected systemically an another concern of nuclease-mediated degradation of naked siRNAs and miRNAs could be observed, beside this as in the naked siRNA and miRNA has negative charge at the neutral pH level, repulsion at the cell membrane level could be experienced and penetration in the weak tissues and due to this non-specific immune stimulation can also be observed. This challenges need to be overcome and could be overcome by the use of distinct delivery vehicles, such as liposomes and cationic lipids but these vectors have another challenges which the scientists are trying to overcome for successful treatment of TNBC. Beside this RNAs therapeutic the CRISPR/Cas cutting-edge gene technology has shown reconstituted result in cancer therapeutics; but its insertion process is difficult and can arrive several significant problems, the impact occurred by missing the specific target and genotoxicity due to virus-based transfection creates threatful impact in biotechnology. The main critical concern related to CRISPR/Cas 9 based gene therapy for treating TNBC is, the targeting is not error free and prolonged aggregation of payload in the genome and related immunogenicity could be easily observed and these creates limitations in the clinical trial of TNBC. These limitations could include, firstly severe health related issue during CRISPR based genome –editing induces permanent alteration. Secondly till now it is not successfully established gene therapy to eliminate the breast and ovarian carcinoma bearing cells of the patients until modern technology is obtained. If these criterias could be overcome then in future it would have the most diverse platform in the treatment of TNBC carcinoma. As the scientists are trying to use the nano particles to prepare nano medicine to deliver the drug for gene therapy, it is also not a very flawless method to use. The nanoparticles may have the high clearance rate from the body for the proper use in diagnosis or drug delivery. An augmented chemical reactivity of these particles could be observed which leads to pressing uncertainty and react under different condition and then the nano particle enters the cell and the increased chemical reactivity of nanoparticles could create imbalance of reactive oxygen species (ROS), which may cause oxidative stress, inflammation, and damage to DNA, proteins and membranes, ultimately leading to toxicity. If these major drawbacks could be overcome then it would have a high potential to prepare nano medicine in the

treatment of nervous system, cancer, vascular thrombosis etc.

REFERENCE

1. Deo SVS, Sharma J, Kumar S. GLOBOCAN 2020 Report on Global Cancer Burden: Challenges and Opportunities for Surgical Oncologists. Ann Surg Oncol. 2022 Oct;29(11):6497-6500. doi: 10.1245/s10434-022-12151-6. Epub 2022 Jul 15. PMID: 35838905.
2. Ensenyat-Mendez M, Llinàs-Arias P, Orozco JIJ, Íñiguez-Muñoz S, Salomon MP, Sesé B, DiNome ML, Marzese DM. Current Triple-Negative Breast Cancer Subtypes: Dissecting the Most Aggressive Form of Breast Cancer. Front Oncol. 2021 Jun 16;11:681476. doi: 10.3389/fonc.2021.681476. PMID: 34221999; PMCID: PMC8242253.
3. Sporikova Z, Koudelakova V, Trojanec R, Hajduch M. Genetic Markers in Triple-Negative Breast Cancer. Clin Breast Cancer. 2018 Oct;18(5):e841-e850. doi: 10.1016/j.clbc.2018.07.023. Epub 2018 Aug 4. PMID: 30146351.
4. Sandhu GS, Erqou S, Patterson H, Mathew A. Prevalence of Triple-Negative Breast Cancer in India: Systematic Review and Meta-Analysis. J Glob Oncol. 2016 Jun 29;2(6):412-421. doi: 10.1200/JGO.2016.005397. PMID: 28717728; PMCID: PMC5493252.
5. Medina MA, Oza G, Sharma A, Arriaga LG, Hernández Hernández JM, Rotello VM, Ramirez JT. Triple-Negative Breast Cancer: A Review of Conventional and Advanced Therapeutic Strategies. Int J Environ Res Public Health. 2020 Mar 20;17(6):2078. doi: 10.3390/ijerph17062078. PMID: 32245065; PMCID: PMC7143295.
6. Kwapisz D. Pembrolizumab and atezolizumab in triple-negative breast cancer. Cancer Immunol Immunother. 2021 Mar;70(3):607-617. doi: 10.1007/s00262-020-02736-z. Epub 2020 Oct 5. PMID: 33015734; PMCID: PMC10992894.
7. Kim C, Gao R, Sei E, Brandt R, Hartman J, Hatschek T, Crosetto N, Foukakis T, Navin NE. Chemoresistance Evolution in Triple-Negative Breast Cancer Delineated by Single-Cell Sequencing. Cell. 2018 May 3;173(4):879-893.e13. doi: 10.1016/j.cell.2018.03.041. Epub 2018 Apr 19. PMID: 29681456; PMCID: PMC6132060.
8. Haque S, Cook K, Sahay G, Sun C. RNA-Based Therapeutics: Current Developments in Targeted Molecular Therapy of Triple-Negative Breast Cancer. Pharmaceutics. 2021 Oct 15;13(10):1694. doi: 10.3390/pharmaceutics13101694. PMID: 34683988; PMCID: PMC8537780.
9. Menbari MN, Rahimi K, Ahmadi A, Mohammadi-Yeganeh S, Elyasi A, Darvishi N, Hosseini V, Abdi M. miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8>oncogene. J Cell Physiol. 2020 Mar;235(3):2631-2642. doi: 10.1002/jcp.29167. Epub 2019 Sep 11. PMID: 31508813.
10. Adams D, Gonzalez-Duarte A, O'Riordan WD, Yang CC, Ueda M, Kristen AV, Tournev I, Schmidt HH, Coelho T, Berk JL, Lin KP, Vita G, Attarian S, Planté-Bordeneuve V, Mezei MM, Campistol JM, Buades J, Brannagan TH 3rd, Kim BJ, Oh J, Parman Y, Sekijima Y, Hawkins PN, Solomon SD, Polydefkis M, Dyck PJ, Gandhi PJ, Goyal S, Chen J, Strahs AL, Nochur SV, Sweetser MT, Garg PP, Vaishnaw AK, Gollob JA, Suhr OB. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med. 2018 Jul 5;379(1):11-21. doi: 10.1056/NEJMoa1716153. PMID: 29972753.
11. Zhu H, Li C, Gao C. Applications of CRISPR-Cas in agriculture and plant biotechnology. Nat Rev Mol Cell Biol. 2020 Nov;21(11):661-677. doi: 10.1038/s41580-020-00288-9. Epub 2020 Sep 24. Erratum in: Nat Rev Mol Cell Biol. 2020 Nov;21(11):712. doi: 10.1038/s41580-020-00304-y. Erratum in: Nat Rev Mol Cell Biol. 2020 Dec;21(12):782. doi: 10.1038/s41580-020-00312-y. PMID: 32973356.
12. Lafond M, Yoshizawa S, Umemura SI. Sonodynamic Therapy: Advances and Challenges in Clinical Translation. J Ultrasound Med. 2019 Mar;38(3):567-580. doi: 10.1002/jum.14733. Epub 2018 Oct 19. PMID: 30338863.
13. Pickar-Oliver A, Gersbach CA. The next generation of CRISPR-Cas technologies and applications. Nat Rev Mol Cell Biol. 2019 Aug;20(8):490-507. doi: 10.1038/s41580-019-0131-5. PMID: 31147612; PMCID: PMC7079207.
14. Kaminski MM, Abudayyeh OO, Gootenberg JS, Zhang F, Collins JJ. CRISPR-based diagnostics. Nat Biomed Eng. 2021 Jul;5(7):643-656. doi: 10.1038/s41551-021-00760-7. Epub 2021 Jul 16. PMID: 34272525.
15. Winkle M, El-Daly SM, Fabbri M, Calin GA. Noncoding RNA therapeutics - challenges and potential solutions. Nat Rev Drug Discov. 2021 Aug;20(8):629-651. doi: 10.1038/s41573-021-00219-z. Epub 2021 Jun 18. PMID: 34145432; PMCID: PMC8212082.
16. Moses C, Nugent F, Waryah CB, Garcia-Bloj B, Harvey AR, Blancafort P. Activating PTEN Tumor Suppressor Expression with the CRISPR/dCas9 System. Mol Ther Nucleic Acids. 2019 Mar 1; 14:287-300. doi: 10.1016/j.omtn.2018.12.003. Epub 2018 Dec 14. PMID: 30654190; PMCID: PMC6348769.
17. Mei Y, Cai D, Dai X. Modulating cancer stemness provides luminal a breast cancer cell with HER2 positive-like features. J Cancer. 2020 Jan 1;11(5):1162-1169. doi: 10.7150/jca.37117. PMID: 31956362; PMCID: PMC6959057.
18. Subramaniyan B, Sridharan S, M Howard C, M C Tilley A, Basuroy T, de la Serna I, Butt E, Raman D. Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer. Cancers (Basel). 2020 Aug 21;12(9):2372. doi: 10.3390/cancers12092372. PMID: 32825729; PMCID: PMC7563118.
19. Linder B, Klein C, Hoffmann ME, Bonn F, Dikic I, Kögel D. BAG3 is a negative regulator of ciliogenesis in glioblastoma and triple-negative breast cancer cells. J Cell Biochem. 2022 Jan;123(1):77-90. doi: 10.1002/jcb.30073. Epub 2021 Jun 27. PMID: 34180073.
20. Ogbu SC, Rojas S, Weaver J, Musich PR, Zhang J, Yao ZQ, Jiang Y. DSTYK Enhances Chemoresistance in Triple-Negative Breast Cancer Cells. Cells. 2021 Dec 29;11(1):97. doi: 10.3390/cells11010097. PMID: 35011659; PMCID: PMC8750327.
21. Qin G, Wang X, Ye S, Li Y, Chen M, Wang S, Qin T, Zhang C, Li Y, Long Q, Hu H, Shi D, Li J, Zhang K, Zhai Q, Tang Y, Kang T, Lan P, Xie F, Lu J, Deng W. NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. Nat Commun. 2020 Apr 3;11(1):1669. doi: 10.1038/s41467-020-15364-z. PMID: 32245950; PMCID: PMC7125142.
22. Qiao J, Li W, Lin S, Sun W, Ma L, Liu Y. Erratum: Author Correction: Co-expression of Cas9 and single-guided RNAs in Escherichia coli streamlines production of Cas9 ribonucleoproteins. Commun Biol. 2019 Nov 27; 2:447. doi: 10.1038/s42003-019-0695-9. Erratum for: Commun Biol. 2019 May 03; 2:161. doi: 10.1038/s42003-019-0402-x. PMID: 31799441; PMCID: PMC6881436.
23. Tatiparti K, Rauf MA, Sau S, Iyer AK. Carbonic Anhydrase-IX Guided Albumin Nanoparticles for Hypoxia-mediated Triple-Negative Breast Cancer Cell Killing and Imaging of Patient-derived Tumor. Molecules. 2020 May 19;25(10):2362. doi: 10.3390/molecules25102362. PMID: 32438691; PMCID: PMC7287925.
24. Wang P, Zhang L, Zheng W, Cong L, Guo Z, Xie Y, Wang L, Tang R, Feng Q, Hamada Y, Gonda K, Hu Z, Wu X, Jiang X. Thermo-triggered Release of CRISPR-Cas9 System by Lipid-Encapsulated Gold Nanoparticles for Tumor Therapy. Angew Chem Int Ed Engl. 2018 Feb 5;57(6):1491-1496. doi: 10.1002/anie.201708689. Epub 2018 Jan 15. PMID: 29282854.
25. Iida K, Tsuchiya A, Tamura M, Yamamoto K, Kawata S, Ishihara-Sugano M, Kato M, Kitamura T, Goyama S. RUNX1 Inhibition Using Lipid Nanoparticle-Mediated Silencing RNA Delivery as an Effective Treatment for Acute Leukemias. Exp Hematol. 2022 Aug-Sep;112-113:1-8. doi: 10.1016/j.exphem.2022.05.001. Epub 2022 May 26. PMID: 35644277.
26. Weng Y, Huang Q, Li C, Yang Y, Wang X, Yu J, Huang Y, Liang XJ. Improved Nucleic Acid Therapy with Advanced Nanoscale Biotechnology. Mol Ther Nucleic Acids. 2020 Mar 6;19:581-601. doi: 10.1016/j.omtn.2019.12.004. Epub 2019 Dec 17. PMID: 31927331; PMCID: PMC6957827.
27. Yang B, Song BP, Shankar S, Guller A, Deng W. Recent advances in liposome formulations for breast cancer therapeutics. Cell Mol Life Sci. 2021 Jul;78(13):5225-5243. doi: 10.1007/s00018-021-03850-6. Epub 2021 May 11. PMID: 33974093; PMCID: PMC11071878.
28. Brown SB, Wang L, Jungels RR, Sharma B. Effects of cartilage-targeting moieties on nanoparticle biodistribution in healthy and osteoarthritic joints. Acta Biomater. 2020 Jan 1; 101:469-483. doi: 10.1016/j.actbio.2019.10.003. Epub 2019 Oct 4. PMID: 31586725; PMCID: PMC7025912.
29. Ghosh S, Javia A, Shetty S, Bardoliwala D, Maiti K, Banerjee S, Khopade A, Misra A, Sawant K, Bhowmick S. Triple negative breast cancer and non-small cell lung cancer: Clinical challenges and nano-formulation approaches. J Control Release. 2021 Sep 10;337:27-58. doi: 10.1016/j.jconrel.2021.07.014. Epub 2021 Jul 14. PMID: 34273417.
30. Xiong R, Hua D, Van Hoeck J, Berdecka D, Léger L, De Munter S, Fraire JC, Raes L, Harizaj A, Sauvage F, Goetgeluk G, Pille M, Aalders J, Belza J, Van Acker T, Bolea-Fernandez E, Si T, Vanhaecke F, De Vos WH, Vandekerckhove B, van Hengel J, Raemdonck K, Huang C, De Smedt SC, Braeckmans K. Photothermal nanofibres enable safe engineering of therapeutic cells. Nat Nanotechnol. 2021 Nov;16(11):1281-1291. doi: 10.1038/s41565-021-00976-3. Epub 2021 Oct 21. PMID: 34675410; PMCID: PMC7612007