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The miRNA biogenesis pathway begins with processing of the primary miRNA (pri-miRNA) transcript in the nucleus by the microprocessor complex (DGCR8 and DROSHA), which trims off the 5' and 3' regions flanking the characteristic stem-loop structure. The newly formed precursor miRNA (pre-miRNA) is then exported to the cytoplasm where it is a substrate for DICER1, an RNA endonuclease that cleaves the terminal loop leaving behind a miRNA duplex. With the help of Argonaute (AGO) the miRNA is loaded with one strand being retained in the miRNA Induced Silencing Complex (miRISC). The mature miRNA brings miRISC to a target mRNA through imperfect base pairing between the miRNA and the target to promote gene silencing. Image adapted from Nature Reviews Cancer, Kasinski and Slack 2012.
Background
MiRNAs, small RNAs of approximately 22 nucleotides in length, were originally identified in the nematode, Caenorhabditis elegans in 1993. It was not until 2000 that the first human-encoded miRNA was identified, which followed with a seminal study that determined that loss of two miRNAs can predispose individuals to chronic lymphocytic leukemia. This work, published in 2002, generated an appreciation for the fact that these small RNAs are not only present in the human genome, but that their misregulation contributes to disease, and that perhaps their restoration may represent a valid therapeutic option. The reason that loss of these small RNAs has such profound effects stems from mechanistic insight into the way that miRNAs function. During their biogenesis, mature miRNAs are loaded into the miRNA Induced Silencing Complex (miRISC) where the miRNA then brings RISC to a target RNA based on imperfect-complementary between the miRNA and the target, which promotes target gene silencing. The imperfect-complementary between the miRNAs and its’ targets is a key aspect of miRNA biology that supports why loss (or gain) of a single miRNA can lead to profound cellular consequences contributing to various diseases. Binding of a miRNA to a target is typically dictated by nucleotides 2-8 of the miRNA (numbering from the 5' end of the miRNA), which is referred to as the seed sequence. Additional nucleotides outside of the seed sequence enhance affinity and contribute to other functions, but it is the seed that is critical for driving the interaction. This low degree of complementarity between a miRNA and its targets results in numerous targets that can be regulated by a single miRNA. Thus, as would be expected, depletion of a single miRNA results in upregulation of a cohort of genes, many of which work in the same or complementary pathways. Restoring this balance is the basis for miRNA-based therapeutics.
Proposed mechanism of action of endosomal escape of RNA cargo mediated by ligand-targeted delivery of nigericin. (1) The nascent endosome is rich in extracellular fluid with a high concentration of Na+ (blue shading), whereas the intracellular space contains high levels of K+ (yellow shading). (2) Upon ligand-targeted delivery, nigericin, a K+/H+ anti-porter, translocates to the endosomal membrane causing an influx of K+ (green shading) in exchange for H+. (3) The exchange of K+ for the osmotically inactive H+ leads to buildup of osmotic pressure, which causes endosomal swelling and release of RNA cargo into the cytosol. Image from Molecular Therapy – Nucleic Acids, Orellana,… and Kasinski 2019.
Research Areas
Delivery:
One of Kasinski labs biggest accomplishments and what the lab is international recognized for includes the use of ligands to deliver therapeutic microRNAs in vivo. Historically, delivery of RNAs has been a challenge, especially to sites other than the liver. Previously, the Kasinski group, and others, relied on encapsulating miRNAs inside of protective vehicles with the assumption that naked RNAs would be degraded rapidly in circulation. Unfortunately, many of these vehicles are associated with toxic side effects or are too bulky to penetrate the dense architecture of the tumor. Working with a diverse team of experts, spanning chemistry and the life sciences, the Kasinski Lab led a first-in-class study that found that miRNAs can be delivered virtually unprotected, in the absence of toxic delivery vehicles. This mechanism successfully delivers miRNAs in therapeutic quantities, specifically to tumor cells, through direct conjugation of the miRNAs to relevant ligands, including folate, 5-methyltetrahydrofolate (5-MTHF), and 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA). These ligand-conjugated miRNAs are quickly and specifically taken up by tumor cells, both in culture and in vivo through their interaction with their respective high affinity cell surface receptor (Kd 7-10 nM) that is overexpressed specifically on tumor cells (>1x106 copies/cell). In light of being conjugated to the ligands, the miRNAs retain their targeting efficiency, producing a sustained effect that ultimately reduces tumor cell progression at doses as low as 0.07 mg/kg [(0.1 nmol), Science Translational Medicine, 2017; Molecular Therapy-Nucleic Acids, 2019; Oncogene, 2023; and Molecular Therapy-Nucleic Acids, 2024]. While studies using these ligands are highly encouraging, there are still challenges that remain with ligand-mediated delivery of small RNAs, including sequestration of the delivered RNAs inside of the endosome and poor RNA stability.
Indeed, ligands and their bound conjugates are internalized via endocytosis. Thus, the cargo, in this case the miRNA, is sequestered in the endosome with only a small amount (<1%) reaching the cytoplasm where it is bioactive. If the RNA is degraded rapidly, there is limited opportunity for a substantial amount of the RNA to act on its targets. Thus, the Kasinski lab determined that inclusion of a small molecule ionophore can promote release of the ligand-delivered miRNAs from the endosome and into the cytosol where they are biologically active (Bioconjugate Chemistry, 2018; Molecular Therapy - Nucleic Acids, 2019; and Molecular Therapy-Nucleic Acids, 2024). Inclusion of the ionophore, nigericin, results in more diffuse cytoplasmic localization of the delivered miRNA, increases engagement of the miRNA in the RNA Induced Silencing Complex (RISC), reduces targeting latency (targeting occurs as soon as 18 hours post-delivery, opposed to >72 hours without nigericin), and increases targeting efficiency (from ~50% to ~85% downregulation of a miRNA reporter). Addition of the ionophore is expected to allow for reduced dosing and greater efficacy than first-generation ligand-conjugates.
Chemical composition and stability of partially modified (PM) and fully modified (FM) miR-34a. A) Chemical modification pattern of PM-miR-34a and FM-miR-34a. B) Structure of the various chemical modifications used in (A). C) Representative gel-Red-stained poly-acrylamide gel of PM- and FM-miR-34a highlighting successful annealing of miRNA duplexes as indicated by mobility shifts on the gel (n > 5). D) Representative gel-Red-stained poly-acrylamide gel of unmodified (UM-), PM- and FM-miR-34a following exposure to 50% serum over a time course (n = 3). Figure from Oncogene, Abdelaal,... and Kasinski 2023.
RNA modifications:
The Kasinski lab demonstrated that in vivo delivery of ligand-conjugated-miR-34a reduces tumor latency in vivo; however, dosing every 3 days was needed due to poor stability of partially modified (PM) miR-34a. To overcome this need for frequent dosing, therapeutic RNAs can be modified to increase in vivo stability. The increased stability leads to reduced frequency of dosing and increased targeting. Indeed, short interfering RNAs (siRNAs) approved by the Food and Drug Administration (FDA) include stabilizing modifications on the ribose and the phosphodiester backbone, both of which, in the correct combination, increase stability from minutes to days. Despite the value of increasing RNA stability, identifying modifications that do not alter targeting, especially for miRNAs that have multiple targets, is challenging. Each RNA requires testing modifications across all positions of the duplex to identify the combination that increases stability, maintains targeting, and prevents off-targeting. Recently the Kasinski lab identified a versions of miR-34a with >400-fold increased stability and outstanding in vivo efficacy. Because miR-34a targets are relevant to various malignancies, this stabilized version of miR-34a will have overarching clinical utility. In brief, the lab determined that delivery of folate-conjugated fully-modified miR-34a (FM-miR-34a) into mice results in downregulation of a synthetic target (miR-34a Renilla reporter) that is sustained for at least 92 hours. However, they found that endogenous targets are repressed for >120 hours following a single systemic injection of folate-FM-miR-34a. Using the same folate-FM-miR-34a in a longitudinal study the lab was able to irradicate tumors in two of six mice completely and reduce tumor latency is the remaining four.
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