Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates

In the paper

“For liver targets, lipid nanoparticles (LNPs) and N-acetylgalactosamine (GalNAc) conjugates are two clinically validated delivery strategies, with multiple regulatory approvals that include Onpattro® (patisiran) 2,3 , Givlaari® (givosiran) 4 , Oxlumo® (lumaisran) 5 , and Leqvio® (inclisiran) [6][7][8] , and numerous additional programs in various stages of clinical development across several thera”

What the sources say

• Partly supported

  The paper’s introduction describes patisiran as a hepatically directed RNAi therapeutic, and the Results report benefit in a phase 3 trial, supporting its use as a clinical example for a liver target. But our copy calls patisiran investigational and does not identify its delivery system as a lipid nanoparticle or report regulatory approval. The citation is attached to Onpattro (patisiran), not to the other named drugs. (LLM, conf=0.84)

  cites [2] Adams, D. et al. Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N. Engl. J. Med. 379, 11–21 (2018). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [3] Kristen, A. V. et al. Patisiran, an RNAi therapeutic for the treatment of hereditary transthyretin-mediated amyloidosis. Neurodegener. Dis. Manag. 9, 5–23 (2019). source
• Supported

  This citation is attached to Givlaari (givosiran), not to the other listed drugs. The paper says givosiran uses a GalNAc conjugate for targeted delivery to hepatocytes and reports its FDA and EMA approvals (Discussion). (LLM, conf=0.93)

  cites [4] Balwani, M. et al. Phase 3 trial of RNAi therapeutic givosiran for acute intermittent porphyria. N. Engl. J. Med. 382, 2289–2301 (2020). source
• Partly supported

  The Results identify lumasiran as a liver-directed RNAi therapeutic, and the Discussion reports substantial oxalate reductions in its phase 3 trial, supporting its use as a clinically studied example. This citation is attached to the lumasiran example, not the other named drugs. Our copy does not identify lumasiran as a GalNAc conjugate or state that Oxlumo received regulatory approval; it calls lumasiran investigational. (LLM, conf=0.84)

  cites [5] Garrelfs, S. F. et al. Lumasiran, an RNAi therapeutic for primary hyperoxaluria type 1. N. Engl. J. Med. 384, 1216–1226 (2021). sourceExperimental — accuracy not yet measured

In the paper

“The naturally occurring RNAi pathway can also be harnessed in extrahepatic organs by building on nearly two decades of efforts on liver siRNA design optimization [9][10][11][12] ; however, novel delivery approaches are needed.”

What the sources say

• Partly supported

  The Introduction states that synthetic siRNAs harness the endogenous RNAi pathway and describes targeted delivery to hepatocytes; the Discussion describes further optimization of siRNA designs. The paper does not establish that these designs can be used in extrahepatic organs or state that novel delivery approaches are needed for them. No page numbers are available in our copy. (LLM, conf=0.85)

  cites [9] Foster, D. J. et al. Advanced siRNA designs further improve in vivo performance of GalNAc-siRNA conjugates. Mol. Ther. 26, 708–717 (2018). sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction (page unavailable) says exogenous siRNAs can harness the natural RNAi pathway and describes chemical modifications and GalNAc-mediated delivery to hepatocytes. It does not address harnessing RNAi in extrahepatic organs, establish nearly two decades of liver siRNA design optimization, or say that novel delivery approaches are needed for those organs. (LLM, conf=0.85)

  cites [10] Janas, M. M. et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat. Commun. 9, 723 (2018). sourceExperimental — accuracy not yet measured
• Partly supported

  The Introduction describes advances in siRNA design and chemistry and explains how GalNAc conjugation enables delivery to the liver. It does not establish a nearly two-decade period of liver optimization or address whether those designs can be applied to extrahepatic organs. The statement that novel delivery approaches are needed follows the citation rather than being directly attributed to it. (LLM, conf=0.82)

  cites [11] Nair, J. K. et al. Impact of enhanced metabolic stability on pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates. Nucleic Acids Res. 45, 10969–10977 (2017). sourceExperimental — accuracy not yet measured

In the paper

“2'-O-C16 was well tolerated across both strands, with potency similar to the unconjugated siRNA, except for a few positions known to be sensitive to sterically demanding 2'-modi cations 9 .”

What the sources say

• Partly supported

  The Introduction and Results describe how sterically demanding 2′ modifications can reduce RNAi activity at particular positions in the sense and antisense strands. But the paper examines 2′-OMe and 2′-F, not 2′-O-C16; it does not establish that 2′-O-C16 was tolerated across both strands or had potency similar to unconjugated siRNA. Page numbers are unavailable in our copy. (LLM, conf=0.79)

  cites [9] Foster, D. J. et al. Advanced siRNA designs further improve in vivo performance of GalNAc-siRNA conjugates. Mol. Ther. 26, 708–717 (2018). sourceExperimental — accuracy not yet measured

In the paper

“Additionally, off-target mitigation strategies involving placing a thermally destabilizing modi cation such as GNA in the antisense strand seed region 10 also performed well, thus allowing for translation of safety improvements with the ESC+ platform beyond liver.”

What the sources say

• Partly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  cites [10] Janas, M. M. et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat. Commun. 9, 723 (2018). sourceExperimental — accuracy not yet measured

In the paper

“Dose dependent silencing in the frontal cortex corresponded with drug exposure through the 0.07 mg, 0.3 mg, and 0.9 mg groups (Fig. 2f), with the siRNA half-life for the 0.9 mg group calculated to be three to four months.”

In the paper

“APP siRNAs, with or without 5'-VP on the antisense strand and/or sense N6 2'-O-C16 conjugation, were well-tolerated and there were no test item-related microscopic ndings in the examined brain, spinal cord, and dorsal root ganglia sections.”

In the paper

“Nadir of knockdown was observed as early as one week post-dose (earliest time point tested), with sustained >75% APP silencing for three months post-dose.”

In the paper

“One month after a single 0.9 mg IT dose, most tested positions provided activity equivalent to sense strand N6 (Fig. 1c), con rming tolerance of 2'-O-C16 modi cation at multiple positions of the siRNA sense strand.”

In the paper

“Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route.”

• Also stated in: § Introduction · hedged

  “Our results indicate that C16-siRNAs administered via a clinically feasible intrathecal (IT) route of administration have durable activity across CNS regions and cell types, with a favorable nonclinical safety pro le to date.”

In the paper

“Consistent with the rat data, siRNA containing both 5'-VP and 2'-O-C16 had the best activity across CNS regions, with up to 70% APP knockdown in the spinal cord and up to 80% in the brain, including 25% knockdown in deep brain (striatum).”

In the paper

“Silencing appeared to be additive with 88%, 85%, and 81% silencing observed in frontal cortex, cerebellum, and thoracic spinal cord, respectively, at 1 month after three 0.3 mg doses, compared to 85%, 82%, and 82% in those regions at the same time point following a single 0.9 mg dose (Fig. 2d).”

In the paper

“APP knockdown recovered to 50% at 4.5 months, with full recovery at nine months in most animals as assessed by CSF protein biomarkers.”

In the paper

“In the lower spinal cord, the maximal level of target knockdown was observed as early as one week post-dose (earliest time point tested), with sustained silencing out to six months.”

In the paper

“Following a single IT dose of 60 mg, an unconjugated siRNA lacking 2'-O-C16 as well as 5'-VP had minimal activity in the spinal cord and no activity in the brain at three months post-dose (Fig. 3a).”

In the paper

“The siRNA combining both 5'-VP and 2'-O-C16 had the best activity across CNS regions, with up to 90% SOD1 knockdown in the spinal cord and up to 75% in the brain.”

In the paper

“IHC also revealed siRNA in vascular endothelium and perivascular macrophages (data not shown), which was shown to be nonfunctional by RT-qPCR (Fig. 2b).”

In the paper

“Addition of 5'-VP to the antisense strand or internal 2'-O-C16 conjugation at N6 of the sense strand produced knockdown in the spinal cord (up to 60% APP lowering), but only modest activity in the brain (up to 30% APP lowering) at three months post-dose.”

In the paper

“RNAi activity in the CNS generally increased with lipid chain length, with 2'-O-C16 providing optimal activity in the rat spinal cord and brain at two weeks after a single IT dose of 0.9 mg (Fig. 1a).”

In the paper

“Fortuitously, the variability in delivery allowed us to observe a robust exposure response relationship, which was conserved across these tissues.”

In the paper

“Notably, higher concentrations of C16-siRNA and greater silencing were observed in prefrontal cortex and thoracic spinal cord than in the deep brain region of the striatum.”

In the paper

“Evidence of accumulation was observed in this tissue following repeat doses, with the siRNA exposure following the third monthly 0.3 mg dose similar to a single 0.9 mg dose, and additive accumulation observed following the fourth and fth doses.”

In the paper

“Brain regions showed >75% silencing for at least three months with partial recovery out to six months, except for temporal cortex where SOD1 knockdown started to recover at two months post-dose.”

In the paper

“The addition of either 5'-VP to the antisense strand or internal 2'-O-C16 conjugation at sense strand N6 produced robust knockdown in the spinal cord (up to 80% SOD1 mRNA lowering), but only modest knockdown in the brain (up to 50% SOD1 mRNA lowering).”

In the paper

“Doses of 0.3, 1, 3, or 10 mg/kg by IN injection resulted in SOD1 mRNA reductions of 57%, 53%, 71%, and 68%, respectively, while a single intravenous (IV) injection at 30 mg/kg reduced SOD1 mRNA by 42% at the same day ten timepoint (Fig. 5e).”

In the paper

“Here we report for the rst time a systematic evaluation of lipophiles of varying lengths and siRNA design features to identify an optimal conjugate with 2'-O-C16 that enables widespread siRNA distribution in the spinal cord and brain following administration into rodent and NHP CSF via a clinically feasible IT dosing route, with no safety signals observed in our studies.”

In the paper

“SOD1 mRNA in lung was reduced by 62%-78% in the rst month, with sustained activity out to two months post-dose with 57% mRNA lowering.”

In the paper

“Within the affected areas, there were minimal decreases in Luxol Fast Blue (LFB) staining intensity and a minimal increase in the number of IBA-1 positive microglia.”

In the paper

“The same conjugation strategy also enables broad siRNA distribution and durable activity in the eye and lung after local delivery.”

In the paper

“At one month, aqueous humor TTR protein levels in animals treated with the unconjugated siRNA showed no knockdown at the 10 μg/eye dose and a modest 33% knockdown at the 30 μg/eye dose, compared to 81% knockdown observed for the 2'-O-C16 conjugate at 10 μg/eye (Fig. 4d).”

In the paper

“RNA interference (RNAi) therapeutics utilize an endogenous mechanism whereby short interfering RNAs (siRNAs) direct the RNA-induced silencing complex (RISC) to target gene transcripts, resulting in a sequence-speci c and catalytic RNA cleavage and subsequent lowering of the target protein 1 .”

What the sources say

• ?Not checked — no full text of the sourcecites [1] Setten, R. L., Rossi, J. J. & Han, S. P. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 18, 421–446 (2019). source

In the paper

“Proper placement of the needle was con rmed via CSF back ow in the hub of the needle.”

In the paper

“Intravitreal injections were performed by a board-certi ed veterinary ophthalmologist.”

In the paper

“Standards and samples were added to the plate, 100 µL/well, in duplicate and incubated for 2 hours at room temperature with shaking at 600 rpm.”

In the paper

“H&E based microscopic ndings were recorded in the Pristima v7.0.0 System (Xybion, NJ) and graded on a scale of 1-5, (1=minimal, 2= mild, 3=moderate, 4=marked, and 5=severe) by a board-certi ed veterinary pathologist.”

In the paper

“All studies were conducted using protocols consistent with local, state and federal regulations, as applicable, and approved by the Institutional Animal Care and Use Committee (IACUC) at Alnylam Pharmaceuticals, Charles River Laboratories (CRL), or LabCorp (formerly Covance, Inc.), as applicable.”

In the paper

“Proper placement of the needle was con rmed via CSF back ow in the hub of the needle.”

In the paper

“The identities and purities of all oligonucleotides were con rmed using ESI-LC/MS and IEX HPLC, respectively.”