MICRO-RNA BASED THERAPEUTICS

INTRODUCTION:

MicroRNAs (miRNAs) are a type of non-coding RNA molecules that regulate cell differentiation, proliferation, and survival by binding to complementary target mRNAs and inhibiting or degrading their translation.

miRNAs' capacity to target multiple mRNAs that are altered during disease conditions makes them promising candidates for therapeutics or therapeutic targets. Advances in the delivery of RNA molecules in vivo have made miRNA-based therapeutics a possibility.

MicroRNA (miRNA)-based therapeutics are of two types - miRNA mimics and inhibitors of miRNAs known as antimiRs.

MiRNA mimics are synthetic double-stranded small RNA molecules that are designed to match the sequence of the relevant miRNA and, as a result, attempt to replace lost miRNA expression in diseases.

AntimiRs, on the other hand, are single-stranded antisense oligonucleotides (ASOs) intended to target mRNAs or modified with locked nucleic acids (LNAs).

MicroRNA BIOGENESIS:

RNA polymerase II (Pol II) transcribes the primary transcripts (pri-miRNA). Then gets cleaved by the RNAse III Drosha into precursor miRNAs (pre-miRNAs).

Exportin-5 (Exp5) transports the pre-miRNAs out of the nucleus to cytoplasm where Dicer cleaves them into a mature single stranded miRNA.

The mature miRNA is subsequently attached onto the RNA-induced silencing complex (RISC), which causes the target mRNA to degrade or suppress the translation of mRNA targets.

MicroRNA TRANSLATION TO CLINICAL SETTINGS:

However, several obstacles must be overcome before miRNAs may be used in clinical settings. The delivery system is the biggest stumbling block.

Chemical alterations, as well as the use of viral vectors or nanoparticles, may be able to assist in overcoming this obstacle.

Despite these flaws in delivery, it is possible that miRNAs will play a significant role in cancer treatment in the future.

MiRNA therapies, in combination with standard chemotherapeutic techniques and drug targets, offer a novel approach to cancer treatment, though more research is needed before this promising paradigm can be implemented in the clinical settings.

ECONOMICAL PROSPECT:

RNA Therapeutics is a new field of biotherapeutics that is rapidly getting momentum. These treatments are based on a strong and adaptable platform with practically limitless potential for addressing unmet clinical needs.

For many disorders, RNA Therapeutics are intended to transform the standard of care. The number of RNA medications being developed and tested in clinical trials is continuously increasing. The capacity to solve the difficulties of stability, delivery, and immunogenicity has fueled the rapid expansion of RNA therapies.

While there is always opportunity for development and innovation in each of these areas, the technology has progressed to the point where RNA Therapeutics are now a viable option.

Despite the emergence of several dominant businesses in the RNA biopharma market, new tiny biotech startups and research organisations with game-changing concepts are emerging.

Furthermore, hospital-based RNA therapeutics initiatives will speed the discovery of RNA-based drugs and the translation of revolutionary cures from the lab bench to the patient's bedside.

COMPARISON OF miRNA WITH siRNA:

RNAi therapeutics have been growing. Patisiran and givosiran, two siRNA-based drugs, were approved by the Food and Drug Administration in 2018 and 2019, respectively. However, there is rare news on the advance of miRNA drugs (another therapeutic similar to siRNA drug). Here the existing obstacles of miRNA therapeutics by analyses for resources available in a drug target perspective, despite being appreciated when it began. Only 10 obtainable miRNA drugs have been in clinical trials with none undergoing phase III, while over 60 siRNA drugs are in complete clinical trial progression including two approvals. Two types of drug are compared and found that their major distinction lay in the huge discrepancy of the target number of two RNA molecules, which was caused by different complementary ratios. One miRNA generally targets tens and even hundreds of genes. Further, two adverse events from the discontinuation of two miRNA therapeutics were exactly answered by TMTME.

SPECIFICITY AND EFFECTIVENESS OF MicroRNA TREATMENT:

miRNAs are involved in onset and progression of a varied amount of biological abnormality.It has been a challenge to deliver the miRNA or their mimics to the target tissue safely and efficiently.

There are few factors that limit delivery of miRNAs, which include:

- Susceptibility to degradation of nucleases

- Rapid clearance from blood

- Immunotoxicity

- Low tissue permeability

For specificity and effectiveness of microRNA treatment it can be done by following ways:

- By modifying the miRNAs chemically their stability is increased and also this leads to better protection against nucleases.

- Use of oligonucleotide carriers also improve their stability and tissue penetration.

- As they are nearly similar to siRNAs so that same pharmaceutical formulations like encompassing liposomes, polymeric nanoparticles and viral systems can increase the stability as well enhance miRNA oligonucleotides pharmacokinetic behaviour.

- There are different viral and non-viral delivery methods which can be used as strategy to evade the multitude of the disease.

VIRUS-BASED miRNA and ANTI-miRNA OLIGONUCLEOTIDE:

Retroviral vectors

- They are derived from the Moloney murine leukaemia viruses (MoMLVs).

- By binding to the target cell, the viral RNA enters the cytoplasm and then they are reverse transcribed into dsDNA and they randomly integrate into the host chromosome.

- This imparts “Janus-faced” character to RVs. They have promising ability to deliver miRNA in regenerative medicine.

Lentiviral vectors

- With the help of nuclear pores, they can translocate across the nuclear membrane which makes the able to target quiescent and non-quiescent cells.

- They can actively integrate with transcribing units which can reduce the chances of triggering oncogenesis.

- In a study in a mouse model, lentiviral delivery with high level of miRNAs (miR-15a, and miR-16) for chronic lymphocytic leukaemia resulted in depletion of malignant B cells and also weakened the disease.

Adeno-associated vectors

- Key feature: Can infect resting and dividing cells.

- Disadvantage: immune activation against AAV by human cells. Several factors like properties of the transgene, vector dose and serotype, administration route, host species, and the presence of pre-existing neutralizing antibodies influences the immunogenicity against AAV.

- Recently in a study the tissue-specific miRNA target sequences were incorporated into 3′-UTR of an AAV vector cassette which can help prevent unintentional transgene expression in liver without affecting the expression in other tissues.

Bacteriophage based VLP vectors

- To avoid challenges like high cytotoxicity, carcinogenic potential, and strong immunogenicity of eukaryotic virus-based system this system was developed.

- An example from a study miRNA-23b delivery to the targeted tissue by a bacteriophage PP7 VLPs to hepatoma cells resulted in migration of these cells and reducing the risk of getting associated cancer.

Non-viral based miRNA AND Anti-miRNA OLIGONUCLEOTIDE:

Inorganic-compound based nanoparticle

- Primarily: Gold, Fe3O4-based, and silica-based nanoparticles.

- These particles fused with functional thiol and amino can increase interaction with miRNAs.

- An example: gold nanoparticles conjugated to PEG for delivery of miR-1 cancer cells. Resulted in high transfection and low cytotoxicity.

Extracellular-vesicle (EVs) based

- They are involved in intercellular communication which helps in the transport of biomolecules.Surface modification can result in targeted delivery of biomolecules to specific tissues.

- Classification: exosomes, microvesicles and apoptotic bodies.

- Exosomes: low cytotoxicity and antigenicity therefore highly efficient. Recently in a study exosome co-delivered anticancer drug along with miR-21 inhibitor in colorectal cancer cell lines for avoiding drug resistance and improving the efficacy of treatment.

- Microvesicles: In a study microvesicles with miRNA-29a/c was delivered which was capable of suppressing tumor development in gastric cancer.

Lipid-based

- Most popular and classical approach, by optimizing the formulations by changing their structure and multi-component composition there can be increase in the loading capacity and delivery efficiency.

- Cationic lipoplexes have low efficiency. Cationic lipoplexes made up of protamine, DOTAP, cholesterol and PEG-DPSE was used to deliver miRNA to lung metastases and the result showed inhibition of tumour growth in mice. This resulted accumulation in liver tissue.

- Neutral lipoplexes- neutral lipid emulsion with synthetic miRNA-34a and let-7 was tested for efficiency of NSCLC in mouse model. These particles showed less accumulation compared to cationic lipoplexes.

- Concern: Non-specific and systemic accumulation of miRNA

- Recent strategy: use of aptamers

Polymeric

- Primarily used: polyethyleneimines (PEIs). Lower molecular weight PEIs are less cytotoxicity but their low transfection efficiency and cytotoxicity makes it unfavourable for clinical applications.

- Alternative can be done by fusing PEG or poly L-Lysine (PLL) covalently so that it improves the biocompatibility (less toxic to cells).

- An example: PEG/PEI nanocomplex polymeric vectors were proved stable and effective in transfecting miR-150 in human leukaemia cells.

- Addition to synthetic polymers less toxic natural cell-penetrating peptides (CPPs) is also used in delivering miRNA. Chitosan another example for biocompatible natural polysaccharide that is used for delivery.

EMERGING METHODS:

- Argininocalix arene 1 – synthetic cationic surfactant with basic amino acids

- Tetraargininocalix arene – multivalent macrocyclic carrier

- To overcome restricted efficiency and specificity of non-viral oligonucleotide carriers, studies showed that engineered nanobody-functionalized nucleic acid nanogel can be used for the targeted delivery of miRNAs for tumor cells and prevent the growth of tumor.

- Example: In the study an engineered multipronged DNA star motif which can carry three miRNA molecules forms a Shuriken-like shape upon miRNA loading. This nanostructure can be used for delivery of tumor suppressive miRNA to human colorectal cancer cells.

Since micro-RNA based treatments involve manipulation at a genetic level, can they potentially harm an individual via mutations or improper gene expression?

MicroRNA based treatment involves controlling gene expression mainly by binding to messenger RNA (mRNA) in the cell cytoplasm. This microRNA will either destroy and recycle components of the mRNA, or silence it, or it will be preserved and translated later. This prevents the rapid translation of mRNA into protein. MicroRNA based treatment must be successful in term of efficacy and should not cause any harm to the individual receiving the treatment

However, there are chances of errors sliding in. If the level of a particular microRNA is underexpressed / abnormally low, overexpression of the protein it normally regulates would take place. Whereas, if the microRNA is overexpressed (its level is unusually high), its protein will be underexpressed. Thus, the varying levels of mRNA cause improper gene expression. Abnormal gene expression has been implicated in many human cancers.

Mutation in a microRNA genes itself will make it non- functional and cause the cell to be devoid of that particular microRNA ergo reducing it to a low level in the cell. These unusually low levels of a microRNA can lead to overexpression of genes which can lead to cancer development and progression. Hence, manipulation at a genetic level, can potentially harm an individual either via mutations or by improper gene expression.

CONCLUSION:

New therapeutic strategies are urgently needed, as drug resistance remains a serious setback in the clinical setting, causing to relapse and metastatic spread in many cancer types. The discovery of miRNAs has paved the way for a better understanding of the molecular processes behind cancer, allowing for the development of novel and more effective treatment options. Many researchers are looking at microRNAs as possible therapeutic agent. Because miRNAs influence various signalling pathways and regulatory networks, even small changes in miRNA expression can have a big impact on disease progression and cancer prognosis.

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