The Promise and Challenges of Developing miRNA-Based Therapeutics for Parkinson's Disease.

MicroRNAs (miRNAs) are small double-stranded RNAs that exert a fine-tuning sequence-specific regulation of cell transcriptome. While one unique miRNA regulates hundreds of mRNAs, each mRNA molecule is commonly regulated by various miRNAs that bind to complementary sequences at 3'-untranslated regions for triggering the mechanism of RNA interference. Unfortunately, dysregulated miRNAs play critical roles in many disorders, including Parkinson's disease (PD), the second most prevalent neurodegenerative disease in the world. Treatment of this slowly, progressive, and yet incurable pathology challenges neurologists. In addition to L-DOPA that restores dopaminergic transmission and ameliorate motor signs (i.e., bradykinesia, rigidity, tremors), patients commonly receive medication for mood disorders and autonomic dysfunctions. However, the effectiveness of L-DOPA declines over time, and the L-DOPA-induced dyskinesias commonly appear and become highly disabling. The discovery of more effective therapies capable of slowing disease progression -a neuroprotective agent-remains a critical need in PD. The present review focus on miRNAs as promising drug targets for PD, examining their role in underlying mechanisms of the disease, the strategies for controlling aberrant expressions, and, finally, the current technologies for translating these small molecules from bench to clinics.


miRNA mimics
A microRNA imitator, composed by short double-stranded RNA nucleotides identical to the endogenous miRNA. Synthetic miRNA mimics are used in experimental assays to generate a condition in which the endogenous microRNA is present at the physiological level or overexpressed.

AntimiR
AntimiRs are microRNA inhibitors, formed by a single-stranded chain of antisense nucleotides. The antimiR will bind to a specific endogenous mature miRNA containing a complementary sequence, thus blocking the miRNA function in the cell. Box 2. FDA guidelines for drug development process applied to miRNA-based drugs.
The following information describes how the conventional process of drug discovery and testing could be applied for miRNA-based drugs, as shown in figure 1. In the discovery stage, Step 1, researchers will test if a candidate miRNA target has the potential to produce therapeutic effects. In PD, for example, a synthetic oligonucleotide that inhibits or, in contrast, imitates a specific microRNA must show efficacy to protect neurons exposed to a damaging agent. Researchers must employ experimental preparations that recapitulate changes found in the parkinsonian brainthe models of PD. Preliminary analysis in silico may inform if the candidate microRNA is involved in underlying mechanisms of the disease, for example, regulating pathogenic proteins like α-Syn. Finally, promising targets are those microRNAs with aberrant patterns of expression in PD patients and animal models. In the subsequent 'development' phase of Step 1, the study may incorporate technologies in the candidate miRNA-based drug. Which is the best nanoparticle for brain delivery? Should the microRNA nucleotides receive chemical modifications to improve specificity and durability? How to inject the preparation for reaching affected brain areas? Which is the lowest dose of oligonucleotides that protect neurons without causing toxicity?
The candidate miRNA-based drug discovered in Step 1 will progress to Step 2 for preclinical testing of efficacy and safety, before entering the clinical research. A microRNA inhibitor for hepatitis C named miravirsen is a good example of drug discovery/development and also preclinical testing in non-human primates, as described by Lindow & Kaupinen et al. (2012) [1]. Pre-clinical testing of anti-Parkinson drugs requires a model that best recapitulates critical features of PD (i.e., the loss of nigral cells, alpha-synuclein accumulation, motor deficit) and that provides reliable endpoints of neuroprotection and motor improvement, as reviewed elsewhere [2].
Clinical research, Step 3, encompasses three phases that explore how the candidate drug acts in the human body. The number of individuals, cost, and duration of the study increase progressively as the drug reaches endpoints and evolves to the subsequent phase. Phase I trials addresses safety and dosage. The study normally recruits healthy volunteers (N=20 -100) but may also involve patients with the disease/condition to which the drug was developed. Phase I studies commonly execute a dose escalation analysis to find the maximum tolerated dose and explore pharmacodynamic and pharmacokinetic properties. In Phase II, efficacy and side effects are examined in a higher number of individuals (N=50 -500) that present the disease/condition. Finally, Phase III clinical research is a multicentric and multi-country trial aimed to test if the drug shows efficacy in an even larger sample of individuals who have the disease (N=300 -3,000) and causes no significant adverse effects. A previous review illustrated the road of selected RNAi-based drugs across Phases I -III that finally led to the first FDA-approved small-interfering RNAs (siRNAs), patisiran and givosiran in 2018 and 2019, respectively [3,4]. Brief definitions of RNAi, siRNA, miRNA mimics, and AntimiR are present in Box 1 of supplementary material.

In
Step IV, all data have been produced across clinical trials, especially the Phase III, and FDA will examine if the drug is safe and effective for the intended disease. A critical decision on whether to approve or not the drug is based on a document named the 'new drug application' (NDA) that contains all results generated across the development process. Prescribing information is commonly refined at this stage, referred as the 'labeling' process. FDA decision at Step 4 is the final part of product development, and the approved drug is allowed to entering the market. The FDA-approved drug is now ready for commercialization and use for the intended disease. Currently, two RNAi-based drugs received FDA approval: patisiran [4], and givosiran [5].
Post-marketing safety monitoring represent the Step 5 of drug development, commonly refereed as the pharmacovigilance study or Phase IV. FDA keeps monitoring drugs in the marketplace, to check if some undesirable effect has emerged and deserves some action. If safety issues appear when drug usage reaches a large number of individuals, FDA will adopt appropriate measures regarding cautions on dosage or usage information, or, indeed, some more restrictive actions. Figure 1. Multi-step development of a biotechnological product. The process begins at the stage of discovery and development (step 1), with posterior preclinical testing in animals (step 2), and the subsequent clinical trials (Phase I -III). FDA review and approval in Step IV allows the drug to enter the market. A subsequent Step 5 represents the pharmacovigilance phase for FDA-approved drugs.