A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system

Classified as probably not a substantial claim. May combine two claims.

The most advanced CNS-active ASO scaffold utilizes a mixed backbone configuration with at least 70-80% PS content, maintaining efficacy and distribution with minimal risk of toxic phenotypes.

“The most advanced CNS-active ASO scaffold utilizes a mixed backbone configuration with at least 70–80% PS content. This modification maintains efficacy and distribution with minimal risk of toxic phenotypes 13, 14 via mechanisms that are not fully understood15.”

• cites [13] Flierl, U. et al. Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators. J. Exp. Med. 212, 129–137 (2015). sourceDisputed

  The Results and Discussion sections on PS backbone modification and therapeutic relevance report platelet activation by PS-modified oligonucleotides and warn that these effects should be assessed in clinical trials. That is contrary to the claim of minimal toxic risk. The paper does not evaluate a CNS-active scaffold with 70–80% PS content, so its findings do not establish the risk of that specific mixed backbone. (LLM, conf=0.76)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured
• cites [14] Sewing, S. et al. Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia. PLoS ONE 12, e0187574 (2017). sourcePartly supported

  The Discussion (page not provided) reports that reducing PS content may reduce platelet activation and thrombocytopenia risk, and that LNA modifications improve binding to RNA targets. But the Methods describe the tested oligonucleotides as fully phosphorothioated; the paper does not establish a CNS-active mixed-backbone scaffold with 70–80% PS content or show maintained efficacy and distribution. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

PS modifications are essential for clinically-advanced siRNA configurations.

“PS modifications are also essential for clinically-advanced siRNA configurations1, 16.”

• cites [1] Khvorova, A. & Watts, J. K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 35, 238–248 (2017). sourcePartly supported

  In “Chemical evolution of siRNAs > Metabolic stabilization” (page unavailable), the paper says phosphorothioate modifications are incorporated into many leading clinical siRNA candidates and improve nuclease resistance and in-vivo accumulation. It does not say they are essential to clinically advanced siRNA configurations; “many” does not establish necessity. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [16] Hassler, M. R. et al. Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo. Nucleic Acids Res. 46, 2185–2196 (2018). sourcePartly supported

  The Results (page not specified) say phosphorothioate (PS) linkages were included in the tested siRNAs to improve stability and resistance to exonucleases. The Introduction (page not specified) says additional PS modifications can enhance long-term efficacy of conjugated siRNAs. Neither passage establishes that PS modifications are essential for clinically advanced siRNA configurations. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

May combine two claims.

siRNAs are large, negatively-charged, double-stranded molecules that require formulation or chemical conjugation for delivery to tissues, especially the CNS.

“siRNAs are large, negatively-charged, double-stranded molecules that require formulation6 or chemical conjugation7–10 for delivery to tissues (especially CNS).”

• cites [6] Coelho, T. et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N. Engl. J. Med. 369, 819–829 (2013). sourcePartly supported

  Citation 6 is attached to formulation. The paper describes lipid-nanoparticle formulations that deliver siRNAs to hepatocytes, supporting that part of the sentence. But it says these systemically administered formulations do not penetrate the CNS, so it does not support the CNS qualifier. The provided copy gives no page number for the relevant passage. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [7] Alterman, J. F. et al. Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids 4, e266 (2015). sourceSupported
• cites [8] Nikan, M. et al. Docosahexaenoic acid conjugation enhances distribution and safety of siRNA upon local administration in mouse brain. Mol. Ther. Nucleic Acids 5, e344 (2016). sourceSupported
• cites [9] Osborn, M. F. & Khvorova, A. Improving small interfering RNA delivery in vivo through lipid conjugation. Nucleic Acid Ther. 28, 128–136 (2018). sourceSupported
• cites [10] Nikan, M. et al. Synthesis and evaluation of parenchymal retention and efficacy of a metabolically stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA conjugate in mouse brain. Bioconjug. Chem. 28, 1758–1766 (2017). sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

IBA-1 is located within a major histocompatibility complex and increases as a result of microglial activation.

“IBA-1 is located within a major histocompatibility complex and increases as a result of microglial activation7.”

• cites [7] Alterman, J. F. et al. Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids 4, e266 (2015). sourceSupported

May combine two claims.

Increasing the PS content of individual siRNA duplexes to 80% can limit RISC efficacy and increase toxicity.

“However, increasing the PS content of individual siRNA duplexes to 80% can limit RNA-induced silencing complex (RISC) efficacy 17, 18 and increase toxicity19, 20.”

• cites [17] Behlke, M. A. Progress towards in vivo use of siRNAs. Mol. Ther. 13, 644–670 (2006). sourcePartly supported

  The Chemical modification section says extensively PS-modified siRNA duplexes show reduced potency and/or toxicity. It does not specify 80% PS content or establish that the reduced potency results from impaired RISC efficacy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [18] Winkler, J., Stessl, M., Amartey, J. & Noe, C. R. Off-target effects related to the phosphorothioate modification of nucleic acids. Chem. Med. Chem. 5, 1344–1352 (2010). sourceNot checked — no full text of the source
• cites [19] Amarzguioui, M., Holen, T., Babaie, E. & Prydz, H. Tolerance for mutations and chemical modifications in a siRNA. Nucl. Acids Res. 31, 589–595 (2003). sourcePartly supported

  The Chemical modification section reports about 70% cell death with the most extensively phosphorothioated siRNA, supporting the toxicity part. The Discussion says shorter phosphorothioate stretches were well tolerated; our copy does not specify 80% PS content or show that PS modification limits RISC efficacy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [20] Harborth, J. et al. Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucl. Acid Drug Devel. 13, 83–105 (2003). sourceNot checked — no full text of the source

May combine two claims.

The 1C2 antibody detects polyQ expansions and is specific to the mutant protein.

“The 1C2 antibody detects polyQ expansions33; and thus, is specific to the mutant protein.”

• cites [33] Herndon, E. S. et al. Neuroanatomic profile of polyglutamine immunoreactivity in Huntington disease brains. J. Neuropathol. Exp. Neurol. 68, 250–261 (2009). sourcePartly supported

  The Introduction says 1C2 binds preferentially to long-glutamine-stretch-containing mutant huntingtin, supporting its use to detect polyQ expansions. It does not support specificity to the mutant protein: the antibody was raised against normal TATA-binding protein, and the Abstract reports 1C2 immunoreactivity in controls. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

IBA-1 and GFAP are well-established markers of immune stimulation.

“To evaluate the impact of Di-siRNA injection on microglia activation and gliosis, we measured two well-established markers of immune stimulation, IBA-1 and GFAP.”

• cites [7] Alterman, J. F. et al. Hydrophobically modified siRNAs silence huntingtin mRNA in primary neurons and mouse brain. Mol. Ther. Nucleic Acids 4, e266 (2015). sourcePartly supported

  The Results section (page not provided) uses IBA-1-positive microglia to assess innate immune activation and says IBA-1 is upregulated after brain injury. The cited paper’s passages do not identify GFAP as a marker or use it to assess gliosis, so they support only the IBA-1 part of the claim. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [30] Achuta, V. S. et al. Tissue plasminogen activator contributes to alterations of neuronal migration and activity-dependent responses in fragile X mice. J. Neurosci. 34, 1916–1923 (2014). sourcePartly supported

  The section on increased tPA expression identifies GFAP as an astrocytic marker, supporting its use to identify glial cells. Our copy does not identify GFAP as a marker of immune stimulation or discuss IBA-1; the claimed role of both markers in assessing immune stimulation is therefore not established by this source. No page number is available in the indexed copy. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

A single subcutaneous injection of trivalent GalNAc-conjugated siRNA can achieve efficacy for >12 months.

“A single subcutaneous injection of trivalent GalNAc-conjugated siRNA can achieve efficacy for >12 months22, 2324.”

• cites [22] Fitzgerald, K. et al. A highly durable RNAi therapeutic inhibitor of PCSK9. N. Engl. J. Med. 376, 41–51 (2017). sourcePartly supported

  The Methods and Results describe a single subcutaneous dose of inclisiran, a triantennary GalNAc-conjugated siRNA, with reductions in PCSK9 and LDL cholesterol maintained at day 180. The paper reports follow-up only to 180 days and discusses potential dosing every 3 or 6 months; it does not establish efficacy beyond 12 months. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [23] Ray, K. K. et al. Inclisiran in patients at high cardiovascular risk with elevated LDL cholesterol. N. Engl. J. Med. 376, 1430–1440 (2017). sourcePartly supported

  The Methods describe a single subcutaneous dose of inclisiran, an siRNA, and the LDL Cholesterol Levels and Other Laboratory Measures section reports LDL-cholesterol reductions after a single dose through day 240. That is less than 12 months; our copy does not establish efficacy beyond 12 months or identify inclisiran as trivalent GalNAc-conjugated. (LLM, conf=0.89)

  Experimental — accuracy not yet measured
• cites [24] Lee, Y. C. et al. Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features. J. Biol. Chem. 258, 199–202 (1983). sourceNot checked — no full text of the source

Classified as probably not a substantial claim. May combine two claims.

GFAP is an intermediate filament protein and increases as a result of astrocyte proliferation.

“GFAP is an intermediate filament protein and increases as a result of astrocyte proliferation30.”

• cites [30] Achuta, V. S. et al. Tissue plasminogen activator contributes to alterations of neuronal migration and activity-dependent responses in fragile X mice. J. Neurosci. 34, 1916–1923 (2014). sourcePartly supported

  The Materials and Methods section identifies GFAP as glial fibrillary acidic protein, and the section on increased tPA expression calls it an astrocytic marker. The paper does not describe GFAP as an intermediate filament protein or say that GFAP increases because of astrocyte proliferation; the reported increase is in tPA, not GFAP. No page numbers are available in our copy. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The most clinically-advanced siRNA conjugate is the multi-valent GalNAc, which drives delivery to and silencing in the liver.

“Indeed, the most clinically-advanced siRNA conjugate is the multi-valent N-acetylgalactosamine (GalNAc)21, which drives delivery to and silencing in the liver.”

• cites [21] 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). sourcePartly supported

  The Introduction (page unavailable) describes triantennary GalNAc as enabling efficient siRNA delivery to the liver, and the Results and Discussion report liver gene silencing. The Conclusion mentions programs progressing into clinical development, but the paper does not establish that GalNAc is the most clinically advanced siRNA conjugate compared with others. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

NeuN and GFAP antibodies are neuron-specific and glial cell-specific, respectively.

“To evaluate any potential preference for specific cell-types, we performed immunofluorescence on sections of Di-siRNA-treated NHP brains using neuron-specific (NeuN)34 and glial cell-specific (GFAP)35 antibodies.”

• cites [34] Weyer, A. & Schilling, K. Developmental and cell type-specific expression of the neuronal marker NeuN in the murine cerebellum. J. Neurosci. Res. 73, 400–409 (2003). sourceNot checked — no full text of the source
• cites [35] Takala, R. S. et al. Glial fibrillary acidic protein and ubiquitin C-Terminal hydrolase-L1 as outcome predictors in traumatic brain injury. World Neurosurg. 87, 8–20 (2016). sourceSupported

May combine two claims.

Increasing the percentage of PS modifications in the siRNA, either uniformly or strategically, may interfere with RISC and reduce tolerability.

“Since increasing the percentage of PS modifications in the siRNA, either uniformly (i.e. every other nucleotide linkage) or strategically, may interfere with RISC 17, 18 and reduce tolerability, a multi-valency approach may be preferable for fine-tuning siRNA uptake and toxicity.”

• cites [17] Behlke, M. A. Progress towards in vivo use of siRNAs. Mol. Ther. 13, 644–670 (2006). sourcePartly supported

  The Chemical modification section (page unavailable) says limited PS modification preserves siRNA potency, while extensive modification can reduce potency and/or cause toxicity. It does not say that PS modification interferes with RISC or assess an every-other-linkage pattern, so those specifics are unsupported. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [18] Winkler, J., Stessl, M., Amartey, J. & Noe, C. R. Off-target effects related to the phosphorothioate modification of nucleic acids. Chem. Med. Chem. 5, 1344–1352 (2010). sourceNot checked — no full text of the source

May combine two claims.

ASO delivery to deep brain structures is limited, with no significant HTT silencing observed in NHP caudate after multiple injections.

“However, ASO delivery to deep brain structures is limited4, with no significant HTT silencing observed in NHP caudate after multiple injections42.”

• cites [4] Kordasiewicz, H. B. et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of huntingtin synthesis. Neuron. 74, 1031–1044 (2012). sourcePartly supported

  The Results section (‘Suppression of huntingtin levels in the CNS after ASO infusion into non-human primates’; page not provided) reports that caudate huntingtin mRNA trended toward a 25% reduction, but the result was not statistically significant (p=0.23). The study used continuous infusion into the cerebrospinal fluid for 21 days, not multiple injections. The separate claim about limited delivery to deep brain structures is attached to citation 4, not this source. (LLM, conf=0.90)

  Experimental — accuracy not yet measured
• cites [42] Southwell, A. L. et al. Huntingtin suppression restores cognitive function in a mouse model of Huntington’s disease. Sci. Transl. Med. 10, eaar3959 (2018). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

siRNA off-target effects are a major concern in development of siRNA-based therapeutics.

“siRNA off-target effects are a major concern in development of siRNA-based therapeutics38.”

• cites [38] Janas, M. M. et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat. Commun. 9, 723 (2018). sourceSupported

Classified as probably not a substantial claim.

Thrombocytopenia (low platelet count) has been reported as a PS-mediated rare adverse event.

“In addition, we found no measurable changes in blood chemistry or cell counts (Supplementary Fig. 20), which is particularly important because thrombocytopenia (low platelet count) has been reported as a PS-mediated rare adverse event13, 14.”

• cites [13] Flierl, U. et al. Phosphorothioate backbone modifications of nucleotide-based drugs are potent platelet activators. J. Exp. Med. 212, 129–137 (2015). sourcePartly supported

  The Results and Discussion section, under “Relevance for PS-modified, nucleotide-based therapeutics” (page unavailable), identifies thrombocytopenia as a reported side effect of the PS-modified drug oblimersen. It does not say the event is rare or establish that the PS modification causes thrombocytopenia. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [14] Sewing, S. et al. Assessing single-stranded oligonucleotide drug-induced effects in vitro reveals key risk factors for thrombocytopenia. PLoS ONE 12, e0187574 (2017). sourcePartly supported

  The Introduction (page unavailable) reports rare platelet-count reductions and cases of severe thrombocytopenia in trials of phosphorothioate oligonucleotides. But the Discussion (page unavailable) says the clinical mechanism remains unknown and may be multifactorial, so the paper does not establish that PS itself mediated those events. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

A non-selective strategy would allow for treatment of all HD patients with a single entity.

“a non-selective strategy would allow for treatment of all HD patients with a single entity49”

• cites [49] Pfister, E. L. et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr. Biol. 19, 774–778 (2009). sourceCould not verify

  The Discussion (page unavailable) addresses allele-selective siRNAs: one could target nearly half the studied patients, while five could cover about three-quarters. A search for a non-selective, single-entity strategy found no passage assessing whether it could treat all HD patients. The paper's limited coverage estimates for selective siRNAs do not settle that different proposal. (LLM, conf=0.74)

  Experimental — accuracy not yet measured

May combine two claims.

In Cynomolgus macaques, a bolus injection of Di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects.

“In Cynomolgus macaques, a bolus injection of Di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects.”

May combine two claims.

Silencing in mice persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels.

“Silencing persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels.”

May combine two claims.

Guide strand accumulation in different brain regions ranged between 2-7 μg/g at one month, 0.8-2.5 μg/g at four months, and 0.2-1.8 μg/g at six months, suggesting relatively slow clearance.

“Guide strand accumulation in different brain regions ranged between 2–7 µg/g at one month, 0.8–2.5 µg/g at four months, and 0.2– 1.8 µg/g at six months, suggesting relatively slow clearance (Fig. 2b).”

May combine two claims.

At six months, the degree of silencing differed between brain regions: > 90% in hippocampus, ~50% in thalamus and striatum, and high variability in the cortex.

“At six months, the degree of silencing differed between brain regions: > 90% in hippocampus, ~50% in thalamus and striatum, and high variability in the cortex.”

May combine two claims.

The higher dose (475 μg) resulted in approximately a two-fold maximum increase in guide strand tissue retention and a wider distribution compared to the lower dose (60 μg).

“The higher dose resulted in approximately a two-fold maximum increase in guide strand tissue retention and a wider distribution compared to the lower dose (0.1 to 4 µg/g at 60 µg; 2–8 µg/g at 475 µg dose, Supplementary Fig. 6b).”

May combine two claims.

In the cortical regions, initial accumulation at one month was 3-4 μg/g and fell below the 0.5 μg/g threshold by six months, resulting in loss of silencing in some animals.

“In the cortical regions, initial accumulation at one month was 3–4 µg/g and fell below the 0.5 µg/g threshold by six months, resulting in loss of silencing in some animals.”

May combine two claims.

Forty-eight hours after ICV injection of 475 μg Cy3-Di-siRNA, the mouse brain was uniformly pink to the naked eye, indicating broad distribution.

“Forty-eight hours after the injection, the mouse brain was uniformly pink to the naked eye (Fig. 1e), indicating broad distribution.”

May combine two claims.

The protein silencing IC50 value is estimated to be ~0.5 μg/g and the IC90 value to be ~2 μg/g of guide strand accumulation in tissue.

“We estimate the protein silencing IC50 value to be ~0.5 µg/g and the IC90 value to be ~2 µg/g of guide strand accumulation in tissue.”

May combine two claims.

For genes differentially expressed at a 1% FDR, there were no significantly over-represented 6-mers and the HTT siRNA seed complement, AGAUUA, was present in the middle of the distribution.

“For genes differentially expressed at a 1% FDR, there were no significantly over-represented 6-mers and the HTT siRNA seed complement, AGAUUA, was present in the middle of the distribution (Fig. 6b).”

May combine two claims.

There was no detectable difference between pre-operative and one-month post-injection MRIs, indicating that Di-siRNA injection did not cause a major inflammatory response.

“There was no detectable difference between these scans, indicating that Di-siRNA injection did not cause a major inflammatory response (Fig. 5a).”

May combine two claims.

The level of guide strand accumulation was sufficient for potent HTT mRNA silencing in all regions tested, with the exception of a single outlier in cervical white matter.

“Again, the level of guide strand accumulation was sufficient for potent HTT mRNA silencing in all regions tested (p < 0.01), with the exception of a single outlier in cervical white matter (Fig. 4e).”

May combine two claims.

There was no major difference in silencing between the ipsilateral and contralateral sides of the brain, consistent with uniform distribution.

“There was no major difference in silencing between the ipsilateral and contralateral sides of the brain, which is consistent with uniform distribution.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Slightly higher accumulation was seen in the lumbar compared to the cervical region.

“Slightly higher accumulation was seen in the lumbar compared to the cervical region.”

May combine two claims.

Fluorescent microscopy confirmed the visual distribution, with significant delivery to the cortex, caudate, and hippocampus.

“Fluorescent microscopy confirmed the visual distribution, with significant delivery to the cortex, caudate, and hippocampus.”

May combine two claims.

A single injection of Di-siRNAHTT showed potent down-regulation of both mutant and wild type HTT protein, with a silencing efficiency similar to that in wild type mice at four months post injection.

“A single injection of Di-siRNAHTT showed potent down-regulation of both mutant and wild type HTT protein (Supplementary Fig. 15), with a silencing efficiency similar to that in wild type mice at four months post injection (Fig. 2d).”

May combine two claims.

Upon sectioning, uniform cortical distribution with evident delivery to deep brain structures, including striatum and hippocampus, was observed.

“Upon sectioning of the brain, we observed uniform cortical distribution with evident delivery to deep brain structures, including the striatum (caudate and putamen) and hippocampus (Fig. 3c).”

May combine two claims.

Forty-eight hours after unilateral injection, uniform distribution was observed throughout the NHP brain, similar to mice.

“Forty-eight hours after unilateral injection, we observed uniform distribution throughout the non-human primate (NHP) brain, similar to mice.”

May combine two claims.

Robust distribution to all spinal cord regions was observed, with guide strand accumulation ranging from 4-7 μg/g.

“We observed robust distribution to all spinal cord regions, with guide strand accumulation ranging from 4–7 µg/g (Fig. 4d).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Animals woke up within 15 minutes after anesthesia was discontinued, with no observable adverse events.

“Animals woke up within 15 minutes after anesthesia was discontinued, with no observable adverse events.”

May combine two claims.

With a 1% FDR, only the "immune system processes" category was significantly enriched among differentially expressed genes, with 12 out of 576 expressed genes in this category showing differential expression.

“With a 1% FDR, only the “immune system processes” category was significantly enriched among differentially expressed genes, with 12 out of 576 expressed genes in this category showing differential expression.”

May combine two claims.

Efficient distribution of Di-siRNA to both neurons and glial cells in the hippocampus and cortex was observed, with co-localization of Di-siRNA with both neurons and glial cells.

“We observed efficient distribution of Di-siRNA to both neurons and glial cells in the hippocampus and cortex, and observed co-localization of Di-siRNA (red) with both neurons (green) and glial cells (purple) (Fig. 3d).”

May combine two claims.

Relaxing the differential expression threshold to 10% FDR, there were several overrepresented 6-mers, but none of them matched AGAUUA.

“Relaxing the differential expression threshold to 10% FDR, there were several overrepresented 6-mers, but none of them matched AGAUUA (Supplementary Fig. 21).”

May combine two claims.

The brain appeared visibly pink with no obvious difference in the degree of oligonucleotide distribution in injected vs non-injected sides.

“The brain appeared visibly pink with no obvious difference in the degree of oligonucleotide distribution in injected vs non-injected sides (Fig. 3b).”

May combine two claims.

For protein silencing, >90% silencing in cortex, >80% in hippocampus, ~50-85% in caudate, and ~40-70% in putamen were observed.

“For protein silencing (Fig. 3f, Supplementary Fig. 18 for raw westerns), we observed >90% silencing in cortex (p < 0.001), >80% silencing in hippocampus (p < 0.01), ~50–85% silencing in the caudate (p < 0.001), and ~40–70% silencing in putamen (p < 0.05).”

May combine two claims.

At one-month post injection, guide strand accumulation was 1-9 μg/g, with highest in cortex (9 μg/g), hippocampus (6 μg/g), and thalamus (6 μg/g), and lowest in caudate (2 μg/g) and putamen (1 μg/g).

“At one-month post injection (Fig. 3e), we observed 1–9 µg/g guide strand accumulation, with the highest accumulation detected in the cortex (9 µg/g), hippocampus (6 µg/g), and thalamus (6 µg/g), and the lowest accumulation in the caudate (2 µg/g) and putamen (1 µg/g).”

May combine two claims.

Downregulated transcripts were slightly enriched for perfect complementarity to the guide strand seed region (P = 0.04) while no enrichment was observed in up-regulated genes (P = 0.59).

“Downregulated transcripts were slightly enriched for perfect complementarity to the guide strand seed region (P = 0.04) while no enrichment was observed in up-regulated genes (P = 0.59).”

May combine two claims.

Animals with a single treatment of Di-siRNA targeting HTT showed significant silencing of HTT with minimal transcriptome-wide changes.

“Animals with a single treatment of Di-siRNA targeting HTT showed significant silencing of HTT with minimal transcriptome-wide changes.”

May combine two claims.

Di-siRNA is delivered to most cortical neurons, resulting in non-selective, potent silencing of wild type and mutant HTT in the CNS, independent of brain size.

“Di-siRNA is delivered to most cortical neurons, resulting in non-selective, potent silencing of wild type and mutant HTT in the CNS, independent of brain size.”

Classified as probably not a substantial claim.

Broad distribution of ASOs, systemically and in the CNS, has been achieved primarily using phosphorothioate (PS) backbone modifications.

“Broad distribution of ASOs, systemically and in the CNS, has been achieved primarily using phosphorothioate (PS) backbone modifications12.”

• cites [12] Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 24, 374–387 (2014). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The PNA hybridization assay provides quantitative accumulation data and does not depend on the presence of a fluorescent label.

“To quantitatively evaluate guide strand accumulation, we used a PNA hybridization assay, which provides quantitative accumulation data and does not depend on the presence of a fluorescent label25.”

• cites [25] Roehl, I., Schuster, M. & Seiffert, S. US Patent 20110201006 A1 (2011).Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Observed APOE silencing is sequence specific and not caused by the Di-siRNA chemical scaffold.

“NTC had no impact on APOE expression, indicating that observed silencing is sequence specific and not caused by the Di-siRNA chemical scaffold.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Multiple cell surface receptors have been recently identified that may be involved in phosphorothioate-mediated oligonucleotide uptake.

“The underlying mechanism is not fully understood, but multiple cell surface receptors have been recently identified that may be involved in phosphorothioate-mediated oligonucleotide uptake15.”

• cites [15] Crooke, S. T., Wang, S., Vickers, T. A., Shen, W. & Liang, X. H. Cellular uptake and trafficking of antisense oligonucleotides. Nat. Biotechnol. 35, 230–237 (2017). sourceNot checked — no full text of the source

May combine two claims.

RNA-seq analyses of Di-siRNAHTT treated NHP brains reveal no major transcriptional changes (aside from HTT silencing) and minimal, if any, off-targeting activity.

“In general, RNA-seq analyses of Di-siRNAHTT treated NHP brains reveal no major transcriptional changes (aside from HTT silencing) and minimal, if any, off-targeting activity.”

Classified as probably not a substantial claim. May combine two claims.

We delivered Di-siRNAHTT via ICV injection, using MRI and CT to confirm needle placement in the lateral ventricle.

“We delivered Di-siRNAHTT via ICV injection, using Magnetic resonance Imaging (MRI) and Computerized Tomography (CT) to confirm needle placement in the lateral ventricle.”

Classified as probably not a substantial claim. May combine two claims.

NeuN and BF-1 are markers for neuronal viability.

“To determine whether there was any effect on neuronal viability, we examined two additional markers by western blot, NeuN36 and BF-137.”

• cites [36] Lind, D., Franken, S., Kappler, J., Jankowski, J. & Schilling, K. Characterization of the neuronal marker NeuN as a multiply phosphorylated antigen with discrete subcellular localization. J. Neurosci. Res. 79, 295–302 (2005). sourceNot checked — no full text of the source
• cites [37] Dou, C. L., Li, S. & Lai, E. Dual role of brain factor-1 in regulating growth and patterning of the cerebral hemispheres. Cereb. Cortex 9, 543–550 (1999). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

HTT is involved in several cellular processes.

“HTT is involved in several cellular processes40, raising the potential for transcriptional changes downstream of HTT silencing (on-target vs off-target effects).”

• cites [40] Zuccato, C., Valenza, M. & Cattaneo, E. Molecular mechanisms and potential therapeutical targets in Huntington’s disease. Physiol. Rev. 90, 905–981 (2010). sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

siRNA off-target effects are predominantly due to seed (position 2-7 of the guide strand) complementarity in the 3' UTR of other targets.

“siRNA off-target effects are predominantly due to seed (position 2–7 of the guide strand) complementarity in the 3’ UTR of other targets39.”

• cites [39] Birmingham, A. et al. 3’ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat. Methods 3, 199–204 (2006). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Linking of two ASOs via their 5' ends resulted in better systemic efficacy compared to monomeric ASO variants.

“Linking of two ASOs via their 5’ ends resulted in better systemic efficacy compared to monomeric ASO variants46.”

• cites [46] Bhagat, L. et al. Novel oligonucleotides containing two 3’-ends complementary to target mRNA show optimal gene-silencing activity. J. Med. Chem. 54, 3027–3036 (2011). sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Our findings are consistent with recent efforts to fine-tune ASO potency and toxicity profiles via optimization of PS content.

“Our findings are consistent with recent efforts to fine-tune ASO potency and toxicity profiles via optimization of PS content (Moazami et al., in review).”

• cites Moazami et al., in reviewNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Given the level of accumulation in cortex and hippocampus, silencing is expected to last 6-9 months in these regions, and as long as 2-4 months in the caudate and putamen.

“Given the level of accumulation in cortex and hippocampus, silencing is expected to last 6–9 months in these regions, and as long as 2–4 months in the caudate and putamen.”

• AssumptionRests on an assumption: The relationship between tissue accumulation and duration of silencing observed in mice holds in NHPs.

Probably not a substantial claim: may not pass S3 (load-bearing).

The RNA-seq data from non-human primate samples have been deposited in GEO under accession code GSE130132, including processed transcriptome read counts.

“The RNA-seq data from non-human primate samples have been deposited in GEO under accession code GSE130132, including processed transcriptome read counts.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

All experimental studies involving animals were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC Protocol #A-2411 and #A-2515) and performed according to the guidelines and regulations therein described.

“All experimental studies involving animals were approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC Protocol #A-2411 and #A-2515) and performed according to the guidelines and regulations therein described.”