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

Breast cancer is the second most diagnosed cancer globally as it is reported. Due to uncontrolled proliferation of breast tissues inside different parts of breast like milk ducts and lobules is the main reason of breast cancer. Ductal carcinoma occurs when uncontrolled growth seen in milk ducts and it is reported that almost 80% cases of breast cancer happen due to ductal carcinoma, while 10% of breast cancer cases are reported as lobular carcinoma which is observed due to the unnatural proliferation in lobules. Moreover, when the cells do not have estrogen and progesterone receptors and do not prepare sufficient HER2 protein, that carcinoma is defined as Triple-negative breast cancer (TNBC). There are some frequent subtypes of TNBC which are also found in patients with high mortality rates. According to a statistical report published by GLOBCON in 2020, approximately 20 lakh women were diagnosed with breast cancer; amongst them, 6.85 lac patients have died from this disease [1].

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer [2]. Due to the deficiency in estrogen, progesterone, and ERBB2 receptor expression, these TNBC cells are unmanageable in nature. TNBC has the ability to adapt the area where it starts proliferation so it is highly aggressive and it is not easy to perform any therapeutic treatment because it shows very less response as it creates a metastatic resistance. There is no specific treatment for TNBC, mostly in advance stages and sometimes shows systemic relapse in the patients . Treatment options for TNBC are limited since the hormonal receptor and HER-2 antagonists typically used for other breast cancers are ineffective. In the initial stages of TNBC, chemotherapy could be helpful but with the progression of the disease, the effectivity of chemotherapy gets reduced and shows almost no effect in later or advanced stages of. Due to this, TNBC patients frequently develop resistance and metastasis, and most of the hormonal therapies also failed because of that metastatic resistance and the peculiar triple-negative nature. Due to the overexploitation of proteinaceous transporters in cell cytoplasm, amplified drug biotransformation and modification occurred in the cell due to this make the cell able to resist against chemotherapy. So to overcome these problems occurred from the regular treatment of cancer and to get an effective treatment for TNBC, several new mechanisms has been introduced in this field and those methods are nanotechnology-based approaches. For the treatment of TNBC, the developed drug delivery mechanism using nanotechnology includes liposomes, dendrimers, polymeric micelles, polymeric nanoparticles, carbon nanotubes, metallic nanoparticles, Nanoemulsions, solid lipid nanoparticles (SLN), and nanostructured lipid carriers (NLC). Gene therapy is a therapeutic treatment where the abnormal genes or missing genes are replaced with the new gene. It focuses on the genetic modification of cells to produce a therapeutic effect or the treatment of disease by repairing or reconstructing defective genetic material. Gene vectors are the specific part of nucleic acid or gene that has the capability of editing the gene with foreign genetic material. The therapeutic protein or drug is attached to these prepared gene vectors and then it is inserted into the target cell so that it could produce the required therapeutic proteins in the diseased cells. The nucleic acid is inserted into the target cell by modifying the secreted organelles, and exosomes, which naturally ruminate the communications between cells [3].  The main aim of research regarding the field of cancer is to develop more treatment method and to make those treatment method more advanced and to find out a permanent solution of this aggressive and dangerous subtype of cancer. It has been reported that the researchers or scientists have shown more interest on immunotherapy, gene therapy where pharmaceutical conjugation is observed with the vector and the drug delivery mechanism is done using nanoparticles for the treatment of cancer. When these nanoparticles and gene therapy are combined together for medical imaging and various treatments then that is known as theranostics. The success of this technique involves binding a vector carrying the genetic information with a nanoparticle which will provide the signal for magnetic resonance imaging. This binding assay shows the specific uptake of the used nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. The nanoparticle is attached to the vector or the probe and then it is inserted so that it could stop and demonstrate the high specificity of the targeting and high efficacy for tumor growth inhibition in TNBC tumors. Now the oncologist and the nanotechnology scientist are focusing to enhance the availability and to achieve the targeted cellular uptake with minimal toxicity by nano-carriers.  Here, in this review, we will explore different nanotechnology-based technologies that can be utilized to treat TNBC in the current future such as gene-loaded nanocarriers along with the possible conjugations to achieve a higher therapeutic profile with reduced side effects via target-specific gene delivery [4]. The deficiency in estrogen, progesterone, and ERBB2 receptor expression leads TNBC cells unmanageable. Nowadays to treat TNBC several methods like RNA-based therapeutics, gene therapy, and nanoparticles have been used.

RNA-based therapeutics in the treatment of TNBC

RNA-based gene therapeutics can change the internal cancer pathway in the patient making the improvement in the patient and shows a ray of hope to those patients and because of that RNA based therapeutics is represented as a novel tool in oncology. In recent advancement techniques RNA-based therapeutics exist as two major areas of investigation- (i) RNA interference (RNAi), where 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, RNAi can be again classified into small interfering RNA (siRNA) and microRNA (miRNA) are used as therapeutic vectors to perform RNA-based gene therapy in the treatment of TNBC [5].

siRNA-based Therapeutics

In the broader area of gene therapy, one of the most interesting and important area of investigation is siRNA-based therapeutics and while performing this method of gene therapy, genetic material is synthesized synthetically and inserted into the targeted cancerous cells of the host to treat the disease [6]. siRNA specifically silences those genes which are directly involved with pathogenesis and thus shows a positive therapeutic result, producing a therapeutic effect; for example, oncogenic mRNAs get suppressed and blocks the translation by the use of miRNAs and thus it prevents the proliferation of cancerous cell and stops spreading cancer into the host’s body and this method is performed based on the principle of gene silencing mechanism [5]. The first siRNA-based therapeutics which get approval was, Onpattro^® (patisiran), this drug is used to treat hereditary transthyretin amyloidosis (hATTR),  mutations in the gene encoding transthyretin and abnormal deposits (amyloids) of transthyretin protein, causing polyneuropathy and cardiomyopathy is the main reason of this hereditary disease. RNA interference (RNAi), an endogenous mechanism for controlling gene expression, shows results in the cleavage of target messenger RNA (mRNA) by small interfering RNAs bound to the RNA-induced silencing complex. This helps to reduce the TTR protein serum level, which helps to cut down the amount of stored amyloid that accumulate in different tissues. Givlaari^® (givosiran), a under trial nanomedicine which is also an another approved siRNA-based therapeutics that is used to treat acute hepatic porphyria (AHP), an inherited metabolic disorder that leads to the accumulation of “neurotoxic precursors δ-aminolevulinic acid (ALA) and porphobilinogen (PBG)” [7, 8].

miRNA-based therapeutics

In the treatment of TNBC miRNA-based therapeutics also has an expanded area of research in cancer inside RNAi therapeutics. But still, till date no drug has gotten approval on miRNA based theraputic. Due to its high versatility, the technology is very much capable in cancer therapeutics in gene therapy for the treatment of cancer and for both suppression and improving or enhancing functions, miRNA therapeutics can show very effective result. In a constraint function when miRNAs are used through Anti-miRNA therapy, Prometastatic miRNAs get suppressed where miRNA functions as a suppressor [7]. The miRNA mimics is responsible for expantion of miRNAs where the inserted synthetic miRNA works as the endogenous miRNAs and functions in the degradation of mRNA and silence the gene using gene silencing mechanism. Miravirsen (SPC3649) a prime example of anti-miRNA therapy, which is an under trial drug which researchers are trying to use in the treatment of hepatitis C virus (HCV) infections [9].

RNA-based therapeutics using nanoparticles

Evidently, gene therapy based on both miRNA and siRNA therapeutics is a complicated process and requires specificity in their delivery as it is a targeted treatment [10]. In the advanced treatment, delivery systems of RNAi are based on three principles, which are used for RNAi therapeutics: polymer-based, lipid-based, and using the amorphous drug–polyelectrolyte nanoparticle complexes. For RNAi-based gene therapy treatment, RNAi delivery also can be done by using cell-penetrating peptides, and several types of nanoparticles (NPs), including liposomes and micelles. For insertion of RNAi in the RNAi therapeutic three different types of nanoparticles are mostly used which are inorganic nanoparticles, polymeric nanoparticles, and lipid nanoparticles (LNPs) [11].  There are polymeric NPs that are mostly cationic polymer-based, among all the cationic polymer, for the insertion of the siRNA complex into the cancerous cells the most commonly used cationic polymer is the branched polyethyleneimine (PEI). Both siRNA and RNAi-based gene therapy use short duplex RNA molecules that exert gene silencing effects at the post-transcriptional level by targeting messenger RNA (mRNA), still, their mechanisms of action and clinical applications are very different because siRNA. Is highly specific with only one mRNA target, whereas RNAi therapy has multiple targets [11].

CRISPR/Cas-based therapeutics in the treatment of TNBC

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity mechanism of archaea and bacteria that is combined with nanotechnology, to prepare a dynamic gene editing tool which would play an extended role  in the treatment of cancer where the cancerous genome will be edited using this tool to treat the cancer and this mechanism is very commonly used or investigated further and has grown an area of interest to the researchers due to its quite comfortable and easy process of using and low cost [12].  The efficiency of this technique was improved but however the effectiveness of the CRISPR/Cas@nano complex also consists some problems such as biodegradability and toxicity remains to render in the cell which causes disturbance in another medical implications while delivering the CRISPR/Cas complex into the target TNBC cells [12].