Noncoding-RNA-Based Therapeutics with an Emphasis on Prostatic Carcinoma: History
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Subjects: Allergy
Contributor:
VICTOR NAVA

Noncoding RNAs (ncRNAs) defy the central dogma by representing a family of RNA molecules that are not translated into protein but can convey information encoded in their DNA.  The importance of understanding ncRNA is apparent since these molecules regulate gene expression at the transcriptional and post-transcriptional level exerting pleiotropic effects critical in development, oncogenesis, and immunity. Multiple therapeutic modalities are available for advanced PC, include hormone therapy, chemotherapy, immunotherapy, radiation and salvage prostatectomy, or various combinations of the aforementioned strategies. Despite significant therapeutic progress, metastatic Prostatic Carcinoma (PC)  remains incurable, and therefore, new treatments, including ncRNA therapeutics (which may be used alone or in combination with current options) are necessary. 

  • noncoding RNA
  • ncRNA therapeutics
  • ncRNA “vaccines”
  • prostatic carcinoma

1. Introduction

Noncoding RNAs (ncRNAs) represent a family of RNA molecules that are not translated into protein but execute a multitude of biological functions informing significant therapeutic potential. It is possible that some ncRNAs are nonfunctional products of spurious transcription, sometimes referred to as “junk RNA”. However, the exact number and function of ncRNAs are still a matter of debate and the focus of active research. It has been estimated that ncRNAs represent approximately 97% of the transcriptome, widely surpassing the amount of coding messenger RNAs (mRNAs). Approximately 30,000 molecules of ncRNAs have been identified, which may be functionally as important as proteins. Interestingly, it has been recognized that approximately 22% of ncRNAs have been misclassified and do encode small polypeptides, which may muddle translational research [1][2]. Overall, ncRNAs can be divided according to their length and function. Molecules longer than 200 nucleotides are referred to as long-intervening ncRNAs (lncRNAs or lincRNAs). They can be intronic or intergenic (lincRNAs) and play multifunctional roles regulating gene expression. Similar to mRNAs, lncRNAs are transcribed by RNA polymerase II, have a capped 5′ end, a polyadenylated 3′ end, and are processed by splicing. The nucleotide sequence of lncRNAs is not well conserved among species. However, their function is evolutionarily conserved, because lncRNAs with diverse sequences share a similar tertiary structure (hairpin) and bind to analogous proteins in different organisms [3]. In addition to proteins, lncRNAs can also directly bind to nucleic acids with bidirectionally (sense or antisense) base paring ability.
In contrast, the smaller ncRNA transcripts are usually about 20 nucleotides in length and represent about 2000 to 5000 molecules with evolutionarily well-conserved sequences that are processed by endonucleases. In common with lncRNAs, the location of smaller ncRNAs can be intergenic or intronic.
Functionally, ncRNAs are divided into homeostatic/housekeeping and regulatory types, although some molecules defy categorization. For example, circular RNAs (covalently closed molecules), which are usually lncRNAs that can, on occasion, encode proteins and be structurally shorter than 200 nucleotides long [4].
Well-known homeostatic ncRNAs include transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). tRNAs are short (~80 nucleotides) ncRNAs that ferry individual amino acids to ribosomes allowing protein translation. In contrast, rRNAs represent a specialized type of long ncRNAs (1500 to 3000 nucleotides in length) that constitute ~60% of the mass of the ribosomes and bind to riboproteins, contributing to the formation of the catalytic sites necessary for protein translation from mRNAs.
Regulatory ncRNAs have been functionally grouped into at least four main categories, sometimes displaying overlapping features: (1) splicing RNAs, (2) self-modifying RNAs, (3) transcriptional regulatory/gene silencing RNAs (all three usually belonging to the small ncRNA subclass), and (4) multifunctional gene-regulatory long RNAs, exemplified by lncRNAs. Splicing RNAs comprise small nuclear RNAs (snRNAs) that bind proteins in the spliceosome. Self-modifying RNAs represent small nucleolar RNAs (snoRNAs), which form part of the ribonuclear protein complex of the nucleolus and have functional similarity to guiding RNAs, serving to chemically modify other RNA molecules. The transcriptional regulatory/gene-silencing RNAs denote at least three types of molecules—namely, (a) small interfering RNAs (siRNAs), involved in gene silencing; (b) microRNAs (miRNAs), which represses mRNA translation by binding to the 3′ untranslated regions of mRNA, promoting its degradation; (c) PIWI-interacting RNAs (piRNAs), which interact with the piwi-subfamily of Argonaute proteins (a highly conserved family of RNA-binding proteins abbreviated as PIWI for the “P-element induced wimpy testis in Drosophila”), which predominantly silence transposable elements through both epigenetic, transcriptional and post-transcriptional mechanisms.
This overwhelming variety of ncRNA species, together with their abundance and rich overlapping functionality, suggests regulatory effects on almost every gene. For example, it has been estimated that in humans, one molecule of miRNA is able to bind with perfect or imperfect complementary base pairing to hundreds (100 to 500) of different mRNA molecules. Therefore, because more than 2000 mature miRNAs have been deposited in the miRNABase V22 [5], this implies that potentially every one of the ~20,000 genes in the human genome could be under miRNA regulation. Consequently, a tremendous effort to unravel the precise involvement of ncRNAs in biology is underway, to understand their crucial roles regulating development, immunity, and oncogenesis, and to explore novel therapeutic applications [6].
In this regard, ncRNA agents could act as immunomodulators or even therapeutic “vaccines”, when defined as medicines that contribute to eliminating or controlling disease, recognizing that so far the immunogenic potential of ncRNAs has not been exploited. Therefore, ncRNA therapeutics cannot be currently considered bona fide vaccines, and quotation marks will be used for clarification in the following sections. ncRNA “vaccines” can be mechanistically diverse, acting directly at a genetic or epigenetic level, or indirectly by modulating innate and adaptive immune responses to find and destroy harmful infectious agents and cancer cells. Modulating ncRNA represents an attractive strategy in oncologic therapy, as well as many other disciplines, and although RNA-based therapeutics have experienced significant prominence in the last 20 years, employing predominantly antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), important hurdles need to be overcome. Several products have gained regulatory approval by the US Food and Drug Administration (FDA) or the European Medicines Agencies (EMA). However, trial results have been disappointing so far due to a combination of factors that include limited efficacy or significant toxicity. Alternative RNA products, such as lncRNAs and anti-miRNAs, are currently undergoing clinical evaluation, but importantly, other ncRNA species have not been extensively targeted to develop therapeutics yet.

2. Noncoding RNA Therapeutics Approved by Regulatory Agencies

Several excellent reviews have recently covered a variety of strategies to manipulate the medicinal potential of ncRNAs by using small interfering RNAs (siRNAs), antisense oligonucleotides (ASOs), short hairpin RNAs (shRNAs), anti-microRNAs (anti-miRNAs), miRNA mimics, miRNA sponges, therapeutic circular RNAs (cirRNAs), and CRISPR-Cas9-based gene editing [6][7][8]. The value of developing therapeutic targeting of ncRNA, while obvious, faces significant obstacles associated with specificity, delivery, and tolerability. Some clinical trials have been stopped, mainly due to lack of efficacy, and it remains uncertain if proper delivery was achieved. In addition, intolerable side effects related to activation of the immune system have promoted the termination of clinical trials. Toxicity is commonly related to the recognition of RNA as a foreign antigen by TLRs, which activate the transcription factor NFkB, leading to cytokine storm [9][10]. Nevertheless, this intrinsic ability of foreign ncRNA to elicit immune responses could be harnessed to avert tumoral immune escape and increase tolerability of ncRNA “vaccines”. Promising advances in this direction have been achieved with other RNA therapeutics, based on breakthroughs in immunology research [11][12]. Therefore, ncRNA could be deployed as a double-edged sword to modulate immune activation, allowing only desirable immunotherapeutic effects while simultaneously synergizing conventional anti-oncogenic chemotherapy. For instance, in the last decade, several studies have shown that intercellular communication can be mediated by nanosized extracellular vesicles (exosomes), which are present in all human body fluids and are composed of bilayer lipid membranes containing diverse cargo molecules (proteins, lipids, ncRNAs, and DNAs) [13]. The release of exosomal content into the cytoplasm of recipient cells after cell membrane fusion has been shown to alter protein expression in the recipient cells, regulating adaptive immune responses to microbes and tumors [14][15]. In addition, it has been specifically shown that ncRNA cargo in the exomes may alter the tumor microenvironment and macrophage function [16]. Recent studies have also addressed the role of exosomal ncRNAs in PC cell lines. Exosomal delivery of miR-26a inhibited metastasis and tumor growth in a PC mouse xenograft model [17]. Another report found that exosomal long lncRNA HOXD-AS1 was upregulated in the serum of patients with metastatic PC, suggesting that it may promote tumorigenesis by acting on the miR-361-5P/FOXM1 axis [18]. Collectively, these data reaffirm the possibility of developing tailored ncRNA therapeutics with reduced toxicity and improved specificity by exploiting recent innovations in chemical engineering.
Despite the rapidly evolving refinements in precision medicine, ncRNA “vaccine” development remains daunting. Many different ncRNA therapeutic products are in the pipeline, but only a few have been approved by the FDA and/or the EMA. Most of them were designed to treat monogenic inherited diseases by using siRNAs or ASOs that would specifically downregulate wild-type genes or alter pathological splicing of mutated genes restoring functionality. However, none of these tools have been approved to treat carcinomas due, in part, to the complex multigenic nature of malignancy. Nevertheless, several ncRNA therapeutics are in phase II or III clinical development, which include different molecules such as miRNA mimics and anti-miRNAs [6]. Of these, at least a few are intended for carcinomas such as pancreatic, nonsmall-cell lung, and colorectal.
Notably, three products have been deployed in clinical trials for advanced PC (Table 1).
Table 1. Clinical trials for advanced prostatic carcinoma using ncRNA therapeutics.
Trial Title Therapeutic (Type) and MOA Characteristics Regimen Major Outcomes
A Randomized Phase II Study of OGX-427 (a Second-Generation Antisense Oligonucleotide to Heat Shock Protein-27) in Patients with Castration-Resistant Prostate Cancer Who Have Not Previously Received Chemotherapy for Metastatic Disease.
(NCT01120470)		 Apatorsen/OGX-427 (a second-generation ASO) targets cytoprotective Hsp27. Downregulation of Hsp27 is expected to enhance sensitivity to cytotoxic agents. 74 patients were randomized to receive apatorsen + prednisone (n  =  36) or prednisone alone (n  =  38). The primary endpoint was disease progression at 12 weeks. Three loading doses at 600 mg IV within the first 10 days of initiating treatment, followed by weekly doses of 1000 mg IV
up to 12 weeks.		 Apatorsen  +  prednisone produced a significant PSA decline but did not change the proportion of CRPC patients without disease progression at 12 weeks, compared with prednisone alone.
A Randomized Phase III Study Comparing Cabazitaxel/Prednisone in Combination with Custirsen (OGX-011) to Cabazitaxel/Prednisone for Second-Line Chemotherapy in Men with Metastatic Castrate-Resistant Prostate Cancer (AFFINITY)
(NCT01578655)		 Custirsen (ASO) downregulates Clusterin. Clusterin (a cytoprotective heat shock protein) regulates apoptosis and is upregulated by chemotherapy. 635 patients were randomized. Co-primary objectives were to evaluate overall survival (OS) in patients receiving Cbz/P/C (n = 317) versus Cbz/P (n = 318) alone. 21-day cycles of 25 mg/m2 IV Cbz on day 1 with 10 mg oral P daily with or without 640 mg IV of C on days 1, 8, and 15 (plus 3 prior loading doses) until disease progression, unacceptable toxicity, or 10 cycles obtained. No significant survival benefits were demonstrated.
Randomized Phase II Trial of Docetaxel (Taxotere) and Oblimersen (Antisense Oligonucleotide Directed to BCL-2) versus Taxotere Alone in Patients with Hormone-Refractory Prostate
Cancer (NCT00085228)		 Oblimersen (ASO) selectively downregulates Bcl-2 (anti-apoptotic proto-oncogene) expression. 115 Chemotherapy naive patients were randomized to receive docetaxel + oblimersen (n = 58) or docetaxel (n = 57) alone. Biologic anti-tumor activity (based on PSA response: Bubley Criteria) Docetaxel 75 mg/m2 on day 1 or oblimersen 7 mg/kg/day continuous IV infusion on days 1–7 with docetaxel 75 mg/m2 on day 5 every 3 weeks for ≤12 cycles. Patients in the docetaxel group received a median of eight cycles and those in the docetaxel + oblimersen group received a median of six cycles The selected endpoint (reduction of PSA > 30%) was not achieved in any arm of the study, indicating that oblimersen was not beneficial in this selected cohort, but Bcl-2 expression was not analyzed.
MOA = mechanism of action, Cbz = cabazitaxel, P = prednisone, C = custirsen.

3. Conclusions

In conclusion, herein concluded exciting and growing applications of ncRNA therapeutics, focusing on clinical trials and future prospects for PC. The results of the trials in PC have been so far contradictory and plagued with issues related to toxicity and suboptimal delivery. However, substantial scientific advances reaffirm the potential of ncRNA therapeutics for the treatment of a multitude of diseases, including PC. The fact that one ncRNA molecule controls numerous different genes in an amplifying effect reinforces the therapeutic value of these agents.
Third-generation chemical modification of ncRNA “vaccines” has achieved a level of sophistication that ensures acceptable tolerability and pharmacokinetic profiles. New delivery systems, including conjugates with nanopolymers and/or specific antibodies, highlight the promise of precise targeting. To date, most of the clinical trials have used anti-miRNA ASO (antagomiRs) and siRNA, which leaves the field wide open for development and experimentation with other ncRNA therapeutics in the future. Ample opportunities to explore ncRNA species exist that have not been used in the clinic, such as miRNA sponges (linear or circular artificial RNA molecules able to simultaneously inhibit more than one species of miRNA) [19][20] or miRNA-masking ASOs (able to block the access of native miRNA to the target mRNA by annealing to the latter in a sequence-specific manner) [21]. Furthermore, some types of lncRNAs, such as circular RNAs or natural antisense transcripts, may represent innovative therapeutic opportunities, which have only recently gained attention for clinical trials [6]. The realization of the potential of ncRNA therapeutics will require a multidisciplinary approach combining scientific advances in the fields of molecular biology, immunology, chemistry (nanotechnology), and pharmacology, coupled with translational research in various clinical disciplines including oncology. The ideal ncRNA product should act specifically on one or various genetic pathways in the appropriate tissue type without eliciting intolerable toxicity related to an exaggerated immune response. Encouraging creative solutions are emerging in ncRNA therapeutics, which allow cautious optimism and certainly will foster additional multidisciplinary research.
In summary, the field of ncRNA therapeutics is reaching maturity and offers tremendous hope to deliver a revolutionary change in the treatment of carcinoma and many other maladies.

This entry is adapted from the peer-reviewed paper 10.3390/vaccines10020276

References

  1. Palazzo, A.F.; Lee, E.S. Non-coding RNA: What is functional and what is junk? Front. Genet. 2015, 6, 2.
  2. Hao, Y.; Zhang, L.; Niu, Y.; Cai, T.; Luo, J.; He, S.; Zhang, B.; Zhang, D.; Qin, Y.; Yang, F.; et al. SmProt: A database of small proteins encoded by annotated coding and non-coding RNA loci. Brief. Bioinform. 2018, 19, 636–643.
  3. Field, A.R.; Jacobs, F.; Fiddes, I.T.; Phillips, A.; Reyes-Ortiz, A.M.; LaMontagne, E.; Whitehead, L.; Meng, V.; Rosenkrantz, J.L.; Olsen, M.; et al. Structurally conserved primate LncRNAs are transiently expressed during human cortical differentiation and influence cell-type-specific genes. Stem Cell Rep. 2019, 12, 245–257.
  4. Lasda, E.; Parker, R. Circular RNAs: Diversity of form and function. RNA 2014, 20, 1829–1842.
  5. Alles, J.; Fehlmann, T.; Fischer, U.; Backes, C.; Galata, V.; Minet, M.; Hart, M.; Abu-Halima, M.; Grässer, F.A.; Lenhof, H.P.; et al. An estimate of the total number of true human miRNAs. Nucleic Acids Res. 2019, 47, 3353–3364.
  6. Winkle, M.; El-Daly, S.M.; Fabbri, M.; Calin, G.A. Noncoding RNA therapeutics—Challenges and potential solutions. Nat. Rev. Drug Discov. 2021, 20, 629–651.
  7. Rupaimoole, R.; Slack, F.J. MicroRNA therapeutics: Towards a new era for the management of cancer and other diseases. Nat. Rev. Drug Discov. 2017, 16, 203–222.
  8. Harper, K.L.; Mottram, T.J.; Whitehouse, A. Insights into the evolving roles of circular RNAs in cancer. Cancers 2021, 13, 4180.
  9. Schlee, M.; Hartmann, G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 2016, 16, 566–580.
  10. Uehata, T.; Takeuchi, O. RNA recognition and immunity-innate immune sensing and its posttranscriptional regulation mechanisms. Cells 2020, 9, 1701.
  11. Wardell, C.M.; Levings, M.K. mRNA vaccines take on immune tolerance. Nat. Biotechnol. 2021, 39, 419–421.
  12. Zhang, C.; Maruggi, G.; Shan, H.; Li, J. Advances in mRNA vaccines for infectious diseases. Front. Immunol. 2019, 10, 594.
  13. Samuelson, I.; Vidal-Puig, A.J. Fed-EXosome: Extracellular vesicles and cell-cell communication in metabolic regulation. Essays Biochem. 2018, 62, 165–175.
  14. Li, X.B.; Zhang, Z.R.; Schluesener, H.J.; Xu, S.Q. Role of exosomes in immune regulation. J. Cell. Mol. Med. 2006, 10, 364–375.
  15. Pucci, F.; Garris, C.; Lai, C.P.; Newton, A.; Pfirschke, C.; Engblom, C.; Alvarez, D.; Sprachman, M.; Evavold, C.; Magnuson, A.; et al. SCS macrophages suppress melanoma by restricting tumor-derived vesicle-B cell interactions. Science 2016, 352, 242–246.
  16. Li, W.; Wang, X.; Li, C.; Chen, T.; Yang, Q. Exosomal non-coding RNAs: Emerging roles in bilateral communication between cancer cells and macrophages. Mol. Ther. 2021, S1525-0016, 638–639.
  17. Wang, X.; Wang, X.; Zhu, Z.; Li, W.; Yu, G.; Jia, Z.; Wang, X. Prostate carcinoma cell-derived exosomal MicroRNA-26a modulates the metastasis and tumor growth of prostate carcinoma. Biomed. Pharmacother. 2019, 117, 109109.
  18. Jiang, Y.; Zhao, H.; Chen, Y.; Li, K.; Li, T.; Chen, J.; Zhang, B.; Guo, C.; Qing, L.; Shen, J.; et al. Exosomal long noncoding RNA HOXD-AS1 promotes prostate cancer metastasis via miR-361-5p/FOXM1 axis. Cell Death Dis. 2021, 12, 1129.
  19. Ebert, M.S.; Neilson, J.R.; Sharp, P.A. MicroRNA sponges: Competitive inhibitors of small RNAs in mammalian cells. Nat. Methods 2007, 4, 721–726.
  20. Lima, J.F.; Cerqueira, L.; Figueiredo, C.; Oliveira, C.; Azevedo, N.F. Anti-miRNA oligonucleotides: A comprehensive guide for design. RNA Biol. 2018, 15, 338–352.
  21. Murakami, K.; Miyagishi, M. Tiny masking locked nucleic acids effectively bind to mRNA and inhibit binding of microRNAs in relation to thermodynamic stability. Biomed. Rep. 2014, 2, 509–512.
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