Role of miRNAs in Neurodegeneration In disease causing mechanisms and tools of biomarker discovery and therapeutics

Building a Biomedical Diagnostic Research Hub in Asia

Globally, there remains an unmet need for advanced research in healthcare biotechnology and diagnostics. Singapore’s reputation for quality research and development (R&D), supportive operating environment, and talent pool made it an easy decision when deciding where to build our first diagnostics research arm in the world.

2. Why the need to launch a biomarker research arm?

Menarini Biomarkers (A. Menarini Biomarkers Singapore Pte Ltd) was founded with the mission of

MAURIZIO LUONGO

CEO, A. Menarini Asia-Pacific Holdings Pte Ltd. (Menarini Asia-Pacific)

1. Tell us more about Menarini Biomarkers, Singapore. Why set up an R&D operation in Asia instead of Europe or North America?

Menarini Asia-Pacific is just over a decade old, having established its regional headquarters in Singapore back in November 2011. Over the past decade, the island-nation’s positioning has changed from just a pharmaceutical manufacturing outpost to an international biomedical hub that encompasses the entire innovation and manufacturing value chain.

identifying, developing, and validating new biomarkers on circulating human cells for applications in diagnostics. There is a current healthcare gap in precision medicine surrounding the fields of early disease prediction and disease control, particularly related to precision rare-cell isolation, analysis, and disease diagnosis. Menarini Biomarkers uses single-cell analysis to provide clinicians with new tools for early detection, tracking disease progression, and the measurement of real-time responses to innovative therapies. The R&D team specialises in pathogenic single-cell isolation that enables scientists to pinpoint specific disease markers, also known as biomarkers. The identification and selection of pathogenic single cells from blood samples is incredibly useful in disease treatment, as it helps healthcare practitioners narrow therapies down to target cells.

3. Are there any current use applications you can share to contextualise the work underway?

Pregnant women face complex healthcare journeys, undergoing different tests at various gestational stages and this does not necessarily include genetic testing. At the same time, an increasingly educated segment of the population wish to understand the potential health risks carried in their babies’ genes without undergoing invasive tests.

Last October, Menarini Biomarkers signed a Memorandum of Understanding with the SingHealth Duke-NUS Maternal and Child Health Research Institute (MCHRI) to develop the first non-European based international medical hub for non-invasive testing in prenatal care.[1] The Biomarkers team have developed a technology that enables the isolation of rare cells taken from the blood of pregnant women. These rare cells are released from the unborn foetus and can provide the entire genome of a baby from the isolated foetal cells alone. This patented technology can surpass any prenatal testing technology currently available in the market. It is a next-generation non-invasive clinical prenatal test (CB-NIPT) that can detect pathogenic microdeletions and microduplications in the foetal genome. The test can be conducted as early as the first trimester (10 weeks) to give an indication of potential diseases that can be treated in utero or immediately after birth. This avoids the crucial delay between disease discovery and treatment. Previously, in some cases, diseases were only formally diagnosed well into early childhood. In the years to come, the team anticipates increased demand for CB-NIPT, owing to the rising average maternal age, as women choose to give birth later in life. Increased literacy rates, financial stability, and awareness of genome sequencing are also empowering women to demand access to world-class healthcare.

4. Looking ahead, what are your plans for Biomarkers in the next five years?

Beyond the SingHealth partnership, Menarini Biomarkers is also collaborating with the KK Women's and Children's Hospital, Singapore Eye Research Institute, and Singapore General Hospital to develop singlecell based diagnostic platforms for applications in the immuno-oncology field.

The team will continue to place strong emphasis on advancing highly specialised and scientifically valuable fields through close collaborations with academic and medical institutions to elevate the standard of healthcare not only in Asia, but globally.

AUTHOR BIO

In his role as Chief Executive Officer, Maurizio ensures the smooth operations of Menarini in the region. A veteran marketing professional, he was previously appointed as Corporate Consumer Health Business Unit Director of Menarini Consumer Health, where he drove the distribution expansion of the portfolio. Prior to joining Menarini, Maurizio worked in various industries with companies including Angelini, Tigi Italia Healthcare, L’Oreal and Saritel SpA.

Role of miRNAs in Neurodegeneration

In disease causing mechanisms and tools of biomarker discovery and therapeutics

This article highlights the relevance of studying brain-enriched miRNAs, the mechanisms underlying their regulation of target gene expression, their dysregulation leading to progressive neurodegeneration, and their potential for biomarker marker and therapeutic intervention. This article has been written to emphasize ways for the effective diagnosis and prevention of these neurodegenerative disorders in the near future.

Bidisha Roy, Department of Biological Sciences, Rutgers University

Neurodegenerative diseases

Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic lateral sclerosis (ALS) are a group of age-related progressive disorders initiated by the neuronal loss that eventually leads to cognitive and movement disorders. These diseases are thought to be caused by alterations to protein-coding genes. Non-coding RNAs participate in translational regulation and comprise 95 per cent of total human cellular RNAs (Figure 1)

PostmortemContol and AD brain - (A, B) represents post mortem formalin fixed human brain whole (A) or hemi segments of the human brain from control patients (B). (C, D) represents post mortem formalin fixed human brain whole (C) or degenerated shrunk hemi segments of the brain (D) from AD patients.

MicroRNAs

MicroRNAs are small, non-coding RNA molecules transcribed from RNA polymerase II and III. MicroRNAs are 22 nucleotides long and fixed in the 3' untranslated section. Mature miRNA are formed in the effector complex to act as a post-transcriptional regulator with its target mRNA. Each miRNA comprises of a seed region that is highly conserved and comprises of an area between 2 to 8 nucleotides. This region spans from the 5’ to 3’ end of the miRNA and has a perfect complementarity match with the 3’UTR of the target mRNA.

Figure 1: PostmortemContol and AD brain - (A, B) represents post mortem formalin fixed human brain whole (A) or hemi segments of the human brain from control patients (B). (C, D) represents post mortem formalin fixed human brain whole (C) or degenerated shrunk hemi segments of the brain (D) from AD patients.

Extensive research, led to the understanding that small noncoding miRNAs played an important role in fine-tuning the genome. Complex networks in the brain are formed by the usage of transcriptome in wide range of combinations. Modulation of expression of thousands of genes is achieved by specific miRNAs controlling the target mRNA expression, leading to various physiological processes. Primary transcripts of miRNA are synthesised by RNA polymerase from miRNA genes. These transcripts are processed in the nucleus by the Drosha enzyme to produce a hairpin-like precursor miRNA (premiRNA). The pre-miRNA is transported from the nucleus to the cytoplasm by exportin. In the cytoplasm, the premiRNA undergoes shearing by dicer to form the mature miRNA. Ago 1 and Ago2 (Argonaute-1 and Argonaute-2) proteins, mature miRNA containing RISC (RNA induced silencing complex) binds to the 3'UTR of target mRNA. This leads to post-transcriptional inhibition or degradation of the target mRNA. Neuronal degeneration onset begins due to dysregulation in Dicer, Drosha, and RISC complexes leading to disruption of miRNA biogenesis and defective cellular processes (Figure 2)

Biogenesis of microRNA -Various steps of mature microRNA formation and mode of gene regulation in the various cellular compartments of the neuron.

Role of microRNAs in neurodegenerative diseases

Alzheimer’s Disease affects about 60 per cent of age dependent dementia cases amongst elderly people. This neurodegenerative disorder is associated with loss in neuronal tissue, memory, impaired cognitive functioning, and impaired learning. The exact cause of AD is still unknown but prior research shows that there are two biomarkers strongly associated with AD. The first biomarker, tau, is a microtubule-associated protein that promotes vesicle transportation. In AD, hyper phosphorylation of tau causes it to lose its affinity to other molecules. Consequently, this hyper phosphorylated tau develops a stronger affinity for other tau molecules and cause them to adhere together to form Tau aggregates. Increased levels of Tau aggregates lead to a decrease in neuronal communication, due to microtubule instability. Tau is a microtubule binding protein and phosphorylation of Tau lead to its dissociation from the microtubules, thereby destabilizing the microtubule assembly. This eventually lead to defective axonal transport and impaired synaptic transmission across neurons in a neuronal circuit. The second biomarker is amyloid- , which is a product of the APP (amyloid- precursor protein), and is known to form amyloid- plaques in AD patients. Research studies show that there is another biomarker in addition to hyper-phosphorylated Tau and amyloidplaques, known as miRNAs. Several research investigations discuss the presence of miRNAs circulating within the blood and cerebrospinal fluid. Most of these miRNAs have experimentally validated targets, known to be important in regulating various pathological processes in the neurons leading to AD (Figure 3). On the other hand, certain miRNAs have been shown to have altered levels in blood plasma, cerebrospinal fluid, or post- mortem brain tissues in AD patients. Additionally, there are various molecular players modulated by miRNAs in other neurodegenerative diseases like Parkinson’s

Disease (PD), Huntington's Disease (HD) and Amyotrophic Lateral Sclerosis (ALS). The miRNA target gene schematic diagram (Figure 4) highlights some of the important genes whose levels post-transcriptionally modified miRNAs, leading to alteration of various vital cellular functions of the neurons. This eventually leads to their degeneration (Figure 3).

Gene Regulatory network of miRNAs in AD - Network depicting the modulation of various genes affecting function of Tau and Amyloid beta by different microRNAs leading to Alzheimer’s disease. [Adapted from Roy et al., Genes (Basel), 2022, (6)] (Figure 4).

Gene Regulatory network of miRNAs in other neurodegenerative diseases - Network depicting the modulation of various genes by different microRNAs, affecting neuronal function in PD, HD and ALS. [Adapted from Roy et al., Genes (Basel), 2022]

MicroRNAs as biomarkers

Molecular, genetic and biochemical components that aid in the identification and analysis of pathological processes in the human body are referred to as biomarkers. They can be used as tools to assess the different stages of diseases, especially the early or preclinical stages. Detection of biomarkers

Figure 2: Biogenesis of microRNA - Various steps of mature microRNA formation and mode of gene regulation in the various cellular compartments of the neuron.

Figure 3: Gene Regulatory network of miRNAs in AD - Network depicting the modulation of various genes affecting function of Tau and Amyloid beta by different microRNAs leading to Alzheimer’s disease. [Adapted from Roy et al., Genes (Basel), 2022, (6)]

Figure 4: Gene Regulatory network of miRNAs in other neurodegenerative diseases - Network depicting the modulation of various genes by different microRNAs, affecting neuronal function in PD, HD and ALS. [Adapted from Roy et al., Genes (Basel), 2022]

in the early stage of any disease may enable patients to receive early therapy. MicroRNA has been advocated as a possible biomarker for different types of diseases regarding diagnosis and treatment. These diseases include, neurodegenerative diseases such as AD and different types of cancer (lung carcinoma, gastric cancer, oesophageal cancer, breast cancer, colorectal cancer). A widespread methodology used in miRNA biomarker study is the utilisation of their levels in the cerebrospinal fluid (CSF) as a minimally invasive detection tool for preclinical markers for central nervous system diseases like AD, lymphomas and gliomas. Circulating miRNAs in the body fluids, showing statistically significant alteration levels, have been widely accepted as an important diagnostic factor in miRNA biomarker research in many diseases, including AD. Several research groups used different human patient samples (serum, peripheral blood mononuclear cells, cerebrospinal fluid) and different techniques (to measure circulating microRNAs) (Figure 5).

Table listing the miRNAs found as potential biomarkers in Alzheimer’s disease– The table lists the various techniques and tissue samples used for measuring miRNA levels in AD patient samples and can serve as potential strategies for biomarker studies in clinical diagnostics.

Usage of CSF samples can be a useful methodology for detecting circulating disease specific or associated miRNAs. Despite few ongoing studies, further research and more easily viable, cost effective and user friendly applications or methodologies are needed for implementing miRNAs as biomarkers for AD. The application of CSF to detect miRNAs is highly recommended in comparison to blood. Blood samples are easily obtained at a low-cost and low risk. However, the molecular profile generated from blood and CSF samples can vary, as there is a possibility of additional factors being found in the blood (which are absent from the brain), due to blood’s systemic circulation properties. Cerebrospinal fluid, on the other hand might be a more reliable biomarker, because it is protected by the blood brain barrier, which reduces unreliable biological factors that may influence the miRNA composition in AD or any other brain disorder. Internal and external factors influencing miRNA levels, include genetic variation, age, gender, race, inflammatory status, lifestyle, methodologies or techniques used to process samples and measure miRNA levels. MicroRNA concentration levels may vary in serum and plasma samples, within the same person. Research study have shown that different types and concentrations of miRNA were found in blood, plasma, serum and exosome samples. Thus, a careful analysis of data from these various samples should be done as a downstream processing protocol in every biomarker study. Additionally, precaution should be taken in the process of analysing the downstream data, for understanding the statistical significance and the relevance of the data in biomarker studies. The variation of miRNA measurement requires a more standardised protocol, such as consistent sample preparation, statistical calculations, and systematic analysis of unvarying miRNA levels. In summary, miRNA biomarker research has the potential in understanding the pathogenesis of these age related neurodegenerative disease and deciphering the underlying regulatory molecular mechanisms leading to their clinical manifestations. As a step forward, miRNA biomarker research in a more detailed and comprehensive manner might help us to monitor and diagnose these diseases at the earlier pre-clinical stage.

MicroRNAs as therapeutics

Overview: Delivery of miRNA into the central nervous system, is very challeng-

TECHNIQUE USED ALTERED MIRNA IDENTIFIED NEURODEGENERATIVE DISEASE CAUSED REFERENCE

Microarray Analysis miR-12

RNA Sequencing and MiR-98-5p, miR-885-5p, miR-4833p, miR-342-3p, miR-191-5p and miR-let-7d-5p

miR-128/miR-491-5p, miR- 132/miR-491-5p, and miR- 874/miR-491-5p, miR-134/miR- 370, miR-323-3p/miR-370, and miR-382/miR-370

Nanostring Technology let-7d-5p, let-7g-5p, miR-15b-5p, miR-142-3p, miR-191-5p, miR- 301a-3p, and miR-545-3p

let-7b Alzheimer's Disease

Mild Cognitive Impairment

Alzheimer's Disease

Alzheimer's Disease

Alzheimer's Disease 9, 151

152

153

155

156

qRT-PCR

Microarray + qRT-PCR miR-27a-3p

miR-15a Alzheimer's Disease

Alzheimer's Disease 157

158

NONVIRAL DELIVERY SYSTEMS

Liposomes

STRENGTHS

1) Reduce the efflux of drugs out of the BBB. 2) Entrap both hydrophilic and lipophilic drugs. 3) W eakly immunogenic and biodegradable. 4) Protects the encapsulated therapeutic agent against rapid enzymatic degradation. 5) High versatility and flexibility in the surface modification with target recognition molecules. 6) Minimizing unwanted inactivating effects of the body and improving the biodistribution of the encapsulated drug to specific cells. 7) Low elimination by the liver and spleen, increases the circulation time of therapeutic agents in the bloodstream and improves the bioavailability of encapsulated molecules for therapeutic action.

Polymeric Nanoparticles 1) High biodegradability, biocompatibility, nonallergic, low immunogenicity, and lack of or low cytotoxicity, higher stability in biological fluids and protection of the RNA against degradation by RNases, reduced nonspecific biodistribution, encapsulate large amounts of genetic material (high drug-binding capacity), and high delivery efficacy, facilitate the cellular uptake via endocytosis.

WEAKNESSES

1) Traditional liposomes have low transfection efficiency into cells due to their lack of surface charges. 2) Nonspecific uptake, and unwanted immune response. 3) Usually heterogeneous in size owing to interactions between water molecules and the hydrophobic groups of lipids, and sometimes the large size of the liposomes produces microembolisms giving a false impression of brain uptake. 4) Conventional liposomes, composed of cholesterol and phospholipids, suffer from high plasma clearance and low transport

1) High cellular toxicity.

Lipoplexes: Formed by cationic liposomes that self- assemble in the presence of RNA due to the electrostatic interaction between the positively charged lipids and the negatively charged RNA molecules.

Exosomes

Dendrimers (Composed by repetitive units of branched molecules; ability to control their structure)

Cyclodextrins

Polymeric micelles : Amphiphilic copolymers composed by a hydrophobic core and hydrophilic surface.

1) Efficient internalization of RNA via membrane fusion with the host cell, and high rate of endosomal release of RNA after entering the cell.

1) Derived from intraluminal vesicles and are released from the plasma membrane; contain proteins, lipids, and miRNAs that can mediate various signaling functions; CNS-derived exosomes are released into physiological biofluids such as CSF and blood. 2) Exosomes can be used as diagnostic tools and have reduced immunogenicity and toxicity. 1) Possible effects of nucleic acids and proteins derived from dendritic cells and carried with the exosomes on the target cell need to be further explored.

1) High versatility to incorporate multiple molecules in the peripheral end groups. 2) Improve solubility, pharmacokinetics, and biodistribution of the therapeutic agents. 3) High loading capacity and transfection efficiency. 4) Low toxicity and immunogenicity; triggering endosomal escape and release RNA into the cytoplasm. They are cleared rapidly by the bloodstream, preventing ‘long-term’ accumulation in nontargeted organs, such as kidneys, lungs, and liver, reducing potential side 1) Controlled drug release and high drug loading still remain challenges with dendrimers. 2) Their cytotoxicity increases proportionally with the generation number.

1) Naturally derived materials with the ability to deliver therapeutic agents across the BBB. 2) Cyclodextrins have been investigated intensely in the targeted delivery of small therapeutic molecules due to their nontoxicity and not producing immune stimulation

1) Easy to formulate, incorporated at different sites in micelles. 2) Small particle size that allows escaping from the reticuloendothelial system. 3) Enhanced drug solubility, drug pharmacokinetics, and biodistribution; high physical stability. 1) Enhanced penetration for a number of useful drugs, using this non-viral delivery system would also open the BBB to potentially toxic substances.

Figure 6: Non-viral methods of delivery – Various methods of non-viral mode of delivery in the central nervous system are listed in this table, along with their strengths and weaknesses, aiding in understanding the best strategy for miRNA delivery to the brain for effective neuro-therapy.

1) It can induce inflammatory effects and unwanted interaction with negatively charged serum proteins, which can lead to opsonization and clearance of the lipoplex