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Treating Disease at the RNA Level
with Oligonucleotides (siRNA) - Review
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Arthur A. Levin, Ph.D.
NEJM Jan 3 2019
Inclisiran is an experimental therapeutic agent that induces cleavage of the mRNA encoding proprotein convertase subtilisin–kexin type 9 (PCSK9), an enzyme that negatively regulates levels of the LDL receptor (LDLR).2,4,28 Persons with naturally occurring genetic variants that reduce the activity of PCSK9 have increased LDLR levels, reduced LDL cholesterol levels, and reduced cardiovascular risk as compared with persons who do not have these variants.29 Inclisiran cleaves and inactivates PCSK9 mRNA, which has the effect of decreasing levels of the PCSK9 and therefore increasing both LDLR levels and the clearance of LDL cholesterol and reducing circulating levels of LDL cholesterol. Inclisiran is being tested in late-stage clinical trials (e.g., NCT03397121 opens in new tab, NCT03400800. opens in new tab, NCT03705234. opens in new tab, and NCT03399370. opens in new tab).
Challenges for Oligonucleotide Therapeutic Agents
Similar to other new technological developments in medicine, including monoclonal antibody therapy and gene therapy, the field of oligonucleotide-based treatment has overcome a number of challenges during a period of maturation. Challenges related to chemistry and manufacturing have been addressed. Synthetic sources of oligonucleotide precursors have reduced the costs of manufacture, and chemical modifications to oligonucleotides have improved resistance to metabolism by nucleases and produced more favorable pharmacokinetic profiles.
Despite considerable progress, two major hurdles stand in the way of widespread application of oligonucleotide therapeutics: drug safety and delivery. The administration of oligonucleotides has been associated with the activation of innate immunity through interactions with toll-like receptors (TLRs). Some oligonucleotides bind to TLRs and induce immune responses similar to those induced by viral and bacterial RNA and DNA. Different members of the TLR family are activated by single-stranded, DNA-like oligonucleotides (e.g., RNase H–dependent and splice-skipping oligonucleotides), and different sequence motifs have been identified as agonists of TLR family members. These immunostimulatory effects can be minimized by avoiding these sequence motifs and using chemical modifications.34-38
The proinflammatory nature of single-stranded phosphorothioate oligonucleotides stymied some early therapeutic programs. Injection-site reactions were commonly observed after subcutaneous injections of antisense drugs, including mipomersen and the splice-skipping drug candidate drisapersen.39,40 Constitutional symptoms such as fever, chills, and rigors have been associated with high doses of phosphorothioate oligonucleotides.41,42 The response of adaptive immunity has been more muted. Treatment with some sequences of single-stranded phosphorothioate oligonucleotides has been associated with weak titers of antidrug antibodies.40
The use of some siRNA therapeutics in clinical trials may be associated with another liability: inflammatory responses to the lipid nanoparticle formulations used to promote the uptake of siRNAs.
Lipid nanoparticles are known to induce a complex antiviral-like response of innate immunity.43,44 To diminish the immunostimulatory effects of the formulations, siRNAs in lipid nanoparticles have been administered in combination with antihistamines, nonsteroidal antiinflammatory drugs, and glucocorticoids.3,4,31
Other challenges for single-stranded phosphorothioate oligonucleotides include renal accumulation and a rare but notable reduction in platelet count.30,40 Single-stranded, phosphorothioate-modified oligonucleotides are generally protected from glomerular filtration because they are bound to plasma proteins. However, the small unbound fraction is readily reabsorbed by renal proximal tubular cells. Mild, low-molecular-weight proteinuria and, in rarer instances, glomerular nephritis have been reported in some patients after treatment with some phosphorothioate oligonucleotides.30,40,45 An analysis of data on renal function from a database of approximately 2400 patients suggested no clinically meaningful changes in levels of protein, creatinine, or plasma urea across multiple sequences.46 In a phase 1 trial, the administration of a single-stranded phosphorothioate oligonucleotide at a dose of 5 mg per kilogram of body weight per week, modified with locked nucleic acids, produced acute tubular necrosis.47,48 This toxic effect has not been observed with other oligonucleotide drugs and appears to be sequence-related but nevertheless has led regulatory authorities to recommend increased surveillance for renal toxicity in clinical trials involving phosphorothioate-modified oligonucleotides.
At least three phosphorothioate oligonucleotides have produced marked thrombocytopenia in small subgroups of patients in clinical trials. These events occurred in three unrelated indications with no overlap in oligonucleotide sequences. Platelets dropped precipitously to class 4 thrombocytopenia in 3% of patients receiving long-term treatment (14 to 26 months) with drisapersen at a dose of 6 mg per kilogram per week40,45 but the condition has also been observed in patients receiving treatment with phosphorothioate-modified sequences for triglyceridemias and transthyretin amyloidosis.30,49
All observed toxic effects are dose-related. Some adverse effects may be minimized with newer versions of oligonucleotide drugs in which the chemistry has been improved or the delivery made more efficient in an effort to reduce doses.
Delivery remains one of the greatest challenges to more widespread application of oligonucleotide therapeutics. Because oligonucleotide drugs have molecular weights in the range of 5 to 15 kDa and are hydrophilic in nature, their ability to penetrate cell membranes is limited, thus diminishing access to their site of activity in cytoplasmic or nuclear compartments. The first successful delivery strategy for an oligonucleotide agent was intravitreal administration of fomiversen; this was approved in 1998 for the treatment of cytomegalovirus retinitis, which was followed more recently by nusinersen, which is locally administered through intrathecal injection for spinal muscle atrophy.
Systemic delivery to most organs and tissues, with the exception of the liver, has proved to be challenging. After intravenous or subcutaneous delivery, phosphorothioate oligonucleotides and siRNAs in lipid nanoparticles are taken up primarily by nonparenchymal cells and to a lesser extent by hepatocytes. N-acetylgalactosamine (GalNAc) conjugated to oligonucleotides binds to the asialoglycoprotein receptors (ASGPR) on hepatocytes to transport and release the oligonucleotides into the intracellular compartment.50 This receptor-mediated uptake allows for dosing that is lower than that required for the therapeutic delivery of unconjugated oligonucleotides.8 Both single-stranded and double-stranded oligonucleotides can be delivered to hepatocytes with the use of GalNAc conjugates. ASGPR-directed delivery to hepatocytes is being used in the experimental treatment of diseases related to hepatocyte-derived protein products and liver diseases. Delivery to other cell types, such as muscle cells, can be accomplished by targeting antibodies or antibody fragments against cell-surface proteins known to be involved in intracellular transport.51
Polymeric nanoparticles have been used to deliver oligonucleotides, but the efficiency with which the liver clears plasma of nanoparticles makes it difficult to direct delivery to other tissues and organs. The technology has not progressed beyond early clinical studies with an siRNA payload.52
In nature, the transfer of genetic information between cells is mediated by membrane-bound nanovesicles or exosomes, which bud from some cell types. Exosomes range from 20 to 200 nm in diameter and are known to move between cells, delivering mRNA, microRNA (miRNA), and proteins that modulate neighboring cell function.53-55 Early studies involving harvested exosomes loaded with synthetic miRNAs exploit exosome membrane proteins and sugars for delivery to distal sites.56-59 The growing body of information on naturally circulating exosomes, their membrane composition, and the membrane-associated proteins will lead to better delivery systems.
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Mechanisms of Action
Therapeutic oligonucleotides are generally 15 to 30 nucleotides. opens in new tab in length and are designed to be complementary to a specific region of a messenger RNA (mRNA). opens in new tab encoding a disease-related protein or a regulatory RNA. After parenteral administration, the oligonucleotide enters a cell and binds to any complementary RNA. When designing a therapeutic candidate, the goal is to identify sequences that are highly specific for the target RNA and to avoid sequences that hybridize to unintended but homologous "bystander" RNAs. With careful design guided by bioinformatics, specific sequences can be identified such that even single members of closely related gene families can be targeted selectively.
Once the oligonucleotide drug has bound to its complementary mRNA or pre-mRNA, a series of events ensues. The outcomes depend partly on the nature of the targeted sequence and include destruction of the mRNA by means of enzymatic cleavage (which is helpful when the mRNA is mutated and encodes a pathogenic protein), a change in the pre-mRNA splicing pattern (which is helpful when the "default" splicing pattern produces a pathogenic product), or a change in the function of a regulatory RNA. The choice of strategy depends on the disease mechanism and on whether the intended outcome is gain or loss of RNA function.
Currently approved oligonucleotide drugs induce cleavage of a target mRNA or alter the splicing pattern. Cleavage-inducing oligonucleotides have structural and chemical features that recruit endogenous enzymes to the site on the target mRNA where the drug hybridizes. Oligonucleotides that alter splicing hybridize with pre-mRNA near a site that controls splicing; the hybridized oligonucleotide effects a change, through steric hindrance, in the action of enzymes that edit pre-mRNA.

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