Lipid-siRNA conjugate accesses a perivascular transport mechanism and achieves widespread and durable knockdown in the central nervous system

Systemic administration of siRNA-based therapeutics does not reach the CNS in meaningful concentrations due to the blood-brain barrier.

“Unfortunately, at present, systemic administration of siRNA-based therapeutics do not reach the CNS in meaningful concentrations due to the blood-brain barrier (BBB).3”

• cites [3] Wu D. The blood–brain barrier: structure, regulation, and drug delivery. Signal Transduct. Target. Ther. 8, 1– 27 (2023). 37231000 PMC10212980 sourcePartly supported

  The Introduction (page unavailable) says the intact BBB excludes macromolecular therapeutics from the brain, supporting the general delivery barrier. It does not establish that systemically administered siRNA therapeutics, as a class, fail to reach meaningful CNS concentrations; the “Other non-invasive strategies” section describes an injected siRNA nanoparticle formulation that improved BBB crossing. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

The striatum receives the most perivascular CSF flow.

“Intriguingly, multiple orthogonal studies suggest that the striatum receives the most perivascular CSF flow, highlighting the potential of this transport pathway for accessing deep brain structures.15,16”

• cites [15] Rey J. A. Perivascular network segmentations derived from high-field MRI and their implications for perivascular and parenchymal mass transport in the rat brain. Sci. Rep. 13, 9205 (2023). 37280246 PMC10244386 sourcePartly supported

  The Results report that the striatum had the highest average percentage of segmented perivascular volume after contrast-agent infusion. That measures tracer uptake, not CSF flow; the authors also note that infusion into periventricular tissue in one rat likely increased the striatal measurement. The Discussion says their MRI data do not establish the direction of transport, so the paper does not show that the striatum receives the most perivascular CSF flow. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [16] Albargothy N. J. Convective influx/glymphatic system: tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. Acta Neuropathol. (Berl.) 136, 139– 152 (2018). 29754206 PMC6015107 sourcePartly supported

  The Results (page unavailable) report deep periarterial tracer penetration in the caudoputamen, a striatal region, and the greatest penetration there at 30 minutes in older mice. But the pons had the greatest penetration at 5 minutes, and the study measured tracer penetration distance, not which region receives the most CSF flow. The unqualified claim that the striatum receives the most perivascular CSF flow therefore overstates the finding. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

High local concentrations of cholesterol are toxic.

“In addition to reducing penetration and delivery homogeneity, high local concentrations of cholesterol are toxic.26,27”

• cites [26] Nikan M. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain. Mol. Ther. Nucleic Acids 5, e344 (2016). 27504598 PMC5023396 sourcePartly supported

  The Results section reports severe toxicity with cholesterol-conjugated, fully modified siRNA above 25 µg and links it to accumulation near the injection site. The Discussion likewise attributes the observed toxicity to local retention of the conjugate. Neither passage establishes that high local concentrations of cholesterol itself are toxic. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [27] Sarli S. L. Quantifying the activity profile of ASO and siRNA conjugates in glioblastoma xenograft tumors in vivo. Nucleic Acids Res. gkae260 (2024) doi: 10.1093/nar/gkae260. PMC11109979 38613388 sourcePartly supported

  The Results section reports acute neurotoxicity in mice after intratumoral injection of cholesterol-conjugated siRNA, and the Discussion describes that conjugate as more neurotoxic than less hydrophobic alternatives (page unavailable). The paper does not establish that high local concentrations of cholesterol itself are toxic or test a concentration-dependent effect. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Perivascular transport has been investigated for antibody-based therapies and ASOs but not for siRNA.

“While perivascular transport has been investigated for antibody-based therapies8 and ASOs,17 it has not been examined for siRNA.”

• cites [8] Pizzo M. E. Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J. Physiol. 596, 445– 475 (2018). 29023798 PMC5792566 sourceNot checked — no full text of the source
• cites [17] Mazur C. Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging. JCI Insight 4, e129240. PMC6824309 31619586 sourceSupported

C16-siRNA did not achieve knockdown in the striatum of rats.

“Notably, C16-siRNA did not achieve knockdown in the striatum of rats, and the divalent siRNA was not effective throughout the striatum of Dorset sheep.22,49”

• cites [22] Brown K. M. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat. Biotechnol. 40, 1500– 1508 (2022). 35654979 sourceSupported
• cites [49] Ferguson C. M. Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep. JCI Insight 6, e152203 (2021). 34935646 PMC8783676 sourceCould not verify

  The Results section reports di-siRNA silencing in Dorset sheep, including variable effects in the caudate and putamen. Our copy does not report whether C16-siRNA achieved striatal knockdown in rats; searches for C16-siRNA and rat striatal knockdown found no passage addressing that claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Chol-siRNA cell-association prevents effective transport across large distances.

“While cholesterol conjugation has been used to promote cell uptake, our findings mirror prior studies showing that Chol-siRNA cell-association prevents effective transport across large distances.26”

• cites [26] Nikan M. Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain. Mol. Ther. Nucleic Acids 5, e344 (2016). 27504598 PMC5023396 sourcePartly supported

  The Results report that Chol-hsiRNA remains concentrated near the injection site in mouse brain and associates with myelin sheaths and nerve bundles. They say this association *may* explain its limited spread; the claim presents that proposed mechanism as established. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Divalent siRNA was not effective throughout the striatum of Dorset sheep.

“Notably, C16-siRNA did not achieve knockdown in the striatum of rats, and the divalent siRNA was not effective throughout the striatum of Dorset sheep.22,49”

• cites [22] Brown K. M. Expanding RNAi therapeutics to extrahepatic tissues with lipophilic conjugates. Nat. Biotechnol. 40, 1500– 1508 (2022). 35654979 sourceCould not verify

  The CNS optimization section reports no activity in the rat striatum, but it does not report a divalent-siRNA study in Dorset sheep. Searches for “Dorset sheep divalent siRNA striatum” and “divalent siRNA sheep striatum” found no passage addressing the attributed sheep finding. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [49] Ferguson C. M. Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep. JCI Insight 6, e152203 (2021). 34935646 PMC8783676 sourcePartly supported

  The Results section on HTT mRNA silencing reports incomplete striatal efficacy for some routes: intrathecal-catheter delivery had no detectable activity in the putamen, and intrastriatal injection produced no silencing on the contralateral side. But it also reports substantial silencing in the caudate and putamen with other routes, so the unqualified claim that divalent siRNA was not effective throughout the striatum overstates the finding. No page number is available in our copy. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Chol-siRNA has the highest cellular uptake among L2-siRNA, Chol-siRNA, and unconjugated siRNA in serum-free media.

“More L2-siRNA was taken up than the parent siRNA, while Chol-siRNA, a benchmark chosen for its known cell penetration capacity,24 had the highest level of uptake in serum-free media (Supplementary Fig. 1A).”

Chol-siRNA is characterized by steep concentration gradients away from the CSF-brain interface in the brain.

“In contrast, Chol-siRNA exhibited high brain retention at both timepoints, but it was characterized by steep concentration gradients away from the CSF-brain interface (Fig. 1C).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

L2-siRNA appears within all CSF interfaces in the parenchyma: ventricular (trans-ependymal), subarachnoid (trans-pial, including cisternal), and perivascular.

“In contrast, L2-siRNA appeared within all CSF interfaces in the parenchyma (Fig. 1C): ventricular (trans-ependymal), subarachnoid (trans-pial, including cisternal; Fig. 1E), and perivascular.”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

L2-siRNA achieves greater perivascular delivery than Chol-siRNA.

“Measuring Cy5 intensity between these markers confirmed that L2-siRNA achieves greater perivascular delivery than Chol-siRNA (Fig. 1H,I).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

Unconjugated siRNA is minimally retained in the CNS at 48 hours after ICV administration.

“We observed that unconjugated siRNA rapidly disperses through the mouse brain and spinal cord by 2 hours post-administration, but that the siRNA was minimally retained in the CNS by 48-hours (Fig. 1C,D).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

L2-siRNA carrier-free gene silencing activity is equivalent to Chol-siRNA at 250 nM.

“Despite uptake differences, L2-siRNA carrier-free gene silencing activity was equivalent to Chol-siRNA at 250 nM (Supplementary Fig. 1B).”

More L2-siRNA is taken up than the parent unconjugated siRNA in neuroblastoma cells in serum-free media.

“More L2-siRNA was taken up than the parent siRNA, while Chol-siRNA, a benchmark chosen for its known cell penetration capacity,24 had the highest level of uptake in serum-free media (Supplementary Fig. 1A).”

Chol-siRNA exhibits high brain retention at 2 and 48 hours after ICV administration.

“In contrast, Chol-siRNA exhibited high brain retention at both timepoints, but it was characterized by steep concentration gradients away from the CSF-brain interface (Fig. 1C).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

At 1 month, L2-siRNAHtt induces robust HTT protein knockdown in the striatum.

“Similarly, at the protein level, siRNAHtt mediated some silencing in the cortex and hippocampus, but less than L2-siRNAHtt, which also induced robust knockdown in the striatum (Fig. 2C and Supplementary Fig. 6).”

At 1 month, siRNAHtt mediates some HTT protein silencing in the cortex and hippocampus.

“Similarly, at the protein level, siRNAHtt mediated some silencing in the cortex and hippocampus, but less than L2-siRNAHtt, which also induced robust knockdown in the striatum (Fig. 2C and Supplementary Fig. 6).”

L2-siRNA localizes within the perivascular space between endothelial cells (Glut1+) and the astrocytic parenchymal glia limitans (Aquaporin-4+).

“Localization within the perivascular space was confirmed by the presence of L2-siRNA between endothelial cells (Glut1+) and the astrocytic parenchymal glia limitans (Aquaporin-4+) (Fig. 1G).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

Unconjugated siRNA accumulates in kidneys at 2 hours after ICV administration.

“Coupled with the observation of accumulation of siRNA in kidneys at 2 hours (Supplementary Fig. 2), these results suggest that unconjugated siRNA is not efficiently internalized by CNS cells causing it to be eliminated quickly from the CNS.”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

scRNA-seq shows L2-siRNA gene silencing activity across diverse cell populations in the CNS parenchyma and at brain borders.

“Single-cell RNA sequencing further demonstrates gene silencing activity across diverse cell populations in the parenchyma and at brain borders, which may provide new avenues for neurological disease-modifying therapies.”

• AssumptionRests on an assumption: The perivascular CSF transport pathway and its accessibility to L2-siRNA observed in rodents is representative of humans.

Chol-siRNA exhibits steep gradients from the subarachnoid space toward the central canal in the spinal cord.

“This delivery pattern was also present in the spinal cord, where Chol-siRNA exhibited steep gradients from the subarachnoid space toward the central canal (Fig. 1D).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

At 1 month, unconjugated siRNAHtt exhibits Htt mRNA silencing only in the cortex and brainstem.

“At 1 month after injection, Htt-targeting L2-siRNA (referred to as L2-siRNAHtt) demonstrated potent mRNA knockdown (>50%) in all brain regions tested, whereas unconjugated siRNA (referred to as siRNAHtt) only exhibited silencing in the cortex and brainstem, highlighting the benefit of L2 conjugation (Fig. 2B).”

Unconjugated siRNA rapidly disperses through the mouse brain and spinal cord by 2 hours after ICV administration.

“We observed that unconjugated siRNA rapidly disperses through the mouse brain and spinal cord by 2 hours post-administration, but that the siRNA was minimally retained in the CNS by 48-hours (Fig. 1C,D).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

Cy5 intensity profile across the midbrain to the interpeduncular cistern shows peaks of L2-siRNA consistent with perivascular transport to deep brain structures.

“First, we measured the Cy5 intensity profile across the midbrain to the interpeduncular cistern (far from injection site) and detected peaks of L2-siRNA consistent with perivascular transport to deep brain structures (Fig. 1F).”

• AssumptionRests on an assumption: The Cy5 fluorescent label does not substantially alter the biodistribution of the siRNA conjugates.

L2-siRNA associates with albumin in ex vivo human CSF.

“In addition, we confirmed L2-siRNA association with albumin in ex vivo human CSF, as demonstrated by fast protein liquid chromatography (FPLC).”

At 5 months, L2-siRNAHtt still achieves Htt mRNA silencing in all examined parenchymal regions.

“Impressively, at a prolonged 5-month timepoint, L2-siRNAHtt still achieved mRNA silencing in all examined parenchymal regions.”

At 3 months post-injection, siRNAHtt no longer exhibits significant HTT protein knockdown in any brain region.

“At 3 months post-injection, mice receiving siRNAHtt no longer exhibited significant protein knockdown in any regions, and only the hippocampus exhibited a reduction in the target mRNA.”

Microglia showed the highest L2-siRNA uptake among examined CNS cell types.

“Microglia, which are resident CNS phagocytes, showed the highest L2-siRNA uptake (Supplementary Fig. 9).”

siRNA levels detected by PNA correlated with gene silencing after intrathecal L2-siRNAHtt in rats.

“The PNA assay revealed detectable levels of siRNA in every tested region, which correlated with gene silencing (Fig. 7D).”

• AssumptionRests on an assumption: Intrathecal administration into lumbar CSF in rats closely mimics the clinical standard for CSF drug delivery.

GFAP immunohistochemical staining 1 month after ICV injection showed no differences between L2-siRNA groups (NTC, Htt) and vehicle.

“Immunohistochemical staining of GFAP 1 month after ICV injection did not show any differences between L2-siRNA groups (NTC, Htt) and vehicle (Fig. 6A,B).”

L2-siRNA shows widespread uptake in MHCII+ macrophages.

“MHCII+ macrophages are most abundant in the choroid plexus and meninges,43 and we similarly observe widespread uptake of L2-siRNA in this population (Fig. 5F).”

siRNA accesses the entire perivascular network 2 hours after ICV delivery.

“This suggests that relatively small nucleic acid therapies are well-suited for this transport, and indeed we observe siRNA (which are small, negatively charged linear molecules) readily accessing the entire perivascular network 2 hours after ICV delivery.”

L2-siRNA is transported along penetrating vessels at the surface and in deep regions of the rat brain.

“To further confirm perivascular localization, we stained vessels with rat endothelial cell antibody (RECA1), which revealed considerable transport along penetrating vessels both at the surface and in deep regions of the brain (Fig. 7C).”

• AssumptionRests on an assumption: Intrathecal administration into lumbar CSF in rats closely mimics the clinical standard for CSF drug delivery.

L2-siRNA shows widespread perivascular transport throughout the brain and spinal cord after intrathecal delivery in rats.

“Widespread perivascular transport throughout the brain and spinal cord was also apparent as bright red puncta.”

• Also stated in: § Discussion: · plain

  “These transport mechanisms were further substantiated in rats following intrathecal injection, where we observed considerable perivascular delivery throughout the spinal cord and parenchyma.”

• AssumptionRests on an assumption: Intrathecal administration into lumbar CSF in rats closely mimics the clinical standard for CSF drug delivery.

L2-siRNAPpib potentiates ~50% Ppib silencing in activated microglia.

“Compared to the non-targeting control, L2-siRNAPpib potentiated gene silencing in homeostatic (~40%) and activated microglia (~50%), as well as resident CNS macrophages (~70%) (Fig. 4E and Supplementary Fig. 10D).”

L2-siRNA shows robust distribution throughout all regions of the spinal cord after intrathecal delivery in rats.

“Robust distribution throughout all regions of the spinal cord was observed, which is expected for intrathecal delivery based on proximity to the injection site (Fig. 7B).”

• AssumptionRests on an assumption: Intrathecal administration into lumbar CSF in rats closely mimics the clinical standard for CSF drug delivery.

L2-siRNAHtt gene silencing activity remains high in all segments of the spinal cord at both mRNA and protein levels at 1, 3, and 5 months.

“These levels are lower than the parenchyma, however, L2-siRNAHtt gene silencing activity remained high in all segments of the spinal cord at both the mRNA and protein levels, whereas unconjugated siRNA did not mediate knockdown of HTT protein (Fig. 3A,B).”

The PNA assay detected siRNA in every tested region after intrathecal L2-siRNAHtt in rats.

“The PNA assay revealed detectable levels of siRNA in every tested region, which correlated with gene silencing (Fig. 7D).”

• AssumptionRests on an assumption: Intrathecal administration into lumbar CSF in rats closely mimics the clinical standard for CSF drug delivery.

L2-siRNAPpib causes robust gene silencing in MHCII+ macrophages.

“While the Lyve1+ population was too sparse to evaluate, we found robust gene silencing in the MHCII+ macrophages (Fig. 5G).”

L2-siRNAPpib potentiates considerable Ppib knockdown in astrocytes and oligodendrocytes.

“For example, glial cells such as astrocytes and oligodendrocytes exhibit considerable Ppib knockdown, as well as ependymal cells, which line the ventricles and directly interface with CSF (Fig. 4G).”

Regions with the highest L2-siRNA delivery (striatum, hippocampus, and cortex) also had the most gene silencing at all timepoints.

“Consistently, regions with the highest delivery (striatum, hippocampus, and cortex) also had the most gene silencing at all timepoints.”

Western blot quantification of GFAP in the hippocampus was consistent with no difference between L2-siRNA and vehicle.

“These results are consistent with Western blot quantification of GFAP in the hippocampus (Fig. 6C), collectively suggesting that L2-siRNA does not induce astrogliosis.”

Every mouse injected with an L2-siRNA conjugate survived to its predetermined endpoint.

“Overall, we did not observe visible signs of toxicity after surgery and every mouse injected with an L2-siRNA conjugate (Htt, Ppib, NTC) survived to their predetermined endpoint.”

L2-siRNA is extensively delivered to perivascular Lyve1+ macrophages.

“We observe extensive delivery to perivascular Lyve1+ macrophages, as evidenced by localization of bright L2-siRNA puncta (Fig. 5E).”

L2-siRNA levels in the spinal cord decrease linearly over time from ~3 ng/mg to ~1 ng/mg.

“Evaluation of the cervical, thoracic, and lumbar spinal cord regions at 1-, 3-, and 5-months post-injection by the PNA assay showed that L2-siRNA levels in the spinal cord decrease linearly over time from ~3 ng/mg to ~1 ng/mg (Fig. 3C,D).”

L2 conjugation increases total and cell-specific uptake of siRNA in mouse CNS cells after ICV administration.

“We found that total and cell-specific uptake were increased by L2 conjugation (Supplementary Fig. 9), in agreement with our prior in vitro data (Supplementary Fig. 1A).”

L2-siRNAPpib potentiates ~40% Ppib silencing in homeostatic microglia.

“Compared to the non-targeting control, L2-siRNAPpib potentiated gene silencing in homeostatic (~40%) and activated microglia (~50%), as well as resident CNS macrophages (~70%) (Fig. 4E and Supplementary Fig. 10D).”

L2-siRNA is highly concentrated within perivascular cells throughout the gray matter parenchyma.

“We visualized the vascular network in gray matter in three dimensions using CLARITY and light sheet microscopy and observed a high concentration of L2-siRNA within perivascular cells throughout the parenchyma (Fig. 5D).”

L2-siRNAPpib produces slightly higher Ppib silencing in venous/capillary endothelial cells than in arterial endothelial cells.

“Endothelial cells were subclustered into venous/capillary and arterial populations (Fig. 4F); there was low but significant knockdown in arterial endothelial cells, and slightly higher silencing in the venous/capillary cells (Fig. 4G).”

qRT-PCR confirmed Ppib gene silencing in Cd11bpos and Cd11bneg populations with an average of 45% and 65% knockdown, respectively.

“To verify gene silencing with orthogonal methodology, cells not utilized for scRNA-seq were analyzed in parallel using qRT-PCR, which confirmed gene silencing in both Cd11bpos and Cd11bneg populations with an average of 45% and 65% Ppib knockdown, respectively (Fig. 4B).”

No changes in serum markers were observed after ICV injection of L2-siRNA.

“Consistent with prior studies showing no evidence of systemic toxicity induced by L2-siRNA after intravenous injection,30,46 we similarly did not observe changes in serum markers after ICV injection (Supplementary Fig. 14).”

Unconjugated siRNA does not mediate knockdown of HTT protein in the spinal cord.

“These levels are lower than the parenchyma, however, L2-siRNAHtt gene silencing activity remained high in all segments of the spinal cord at both the mRNA and protein levels, whereas unconjugated siRNA did not mediate knockdown of HTT protein (Fig. 3A,B).”

L2-siRNA causes robust gene silencing in choroid plexus epithelial cells.

“Lastly, we examined delivery to other cells in the choroid plexus and noted a high level of delivery to choroid plexus epithelial cells corresponding to robust gene silencing (Fig. 5H,I).”

At 3 months, L2-siRNAHtt exhibits significant reduction in HTT protein in the striatum, hippocampus, cortex, and cerebellum.

“In contrast, mice receiving L2-siRNAHtt exhibited sustained gene and protein knockdown; we note significant reduction in HTT protein at 3 months in the striatum, hippocampus, cortex, and cerebellum.”

Cholesterol conjugation limits penetration into perivascular spaces, with minimal perivascular signal beyond superficial vessels.

“Yet, the conjugation of cholesterol limits penetration into these spaces, with minimal perivascular signal beyond superficial vessels likely due to extensive interactions with cells and potentially other tissue components.”

Neurons exhibit modest Ppib gene silencing after L2-siRNAPpib.

“Neurons exhibit modest gene silencing, and due to the low capture of this population, we could not subcluster knockdown into neuron subtypes.”

L2-siRNAPpib potentiates considerable Ppib knockdown in ependymal cells.

“For example, glial cells such as astrocytes and oligodendrocytes exhibit considerable Ppib knockdown, as well as ependymal cells, which line the ventricles and directly interface with CSF (Fig. 4G).”

Iba1 protein levels were unchanged after L2-siRNA injection compared to vehicle.

“Iba1 protein levels were unchanged after L2-siRNA injection compared to vehicle (Fig. 6D,E).”

No difference in pro-inflammatory cytokine, chemokine, or growth factor levels was observed between vehicle and L2-siRNAHtt in cortical and striatal tissue two weeks after ICV injection at 15 nmol or 5 nmol.

“Notably, no difference in pro-inflammatory cytokine, chemokine, or growth factor levels were observed between vehicle and L2-siRNAHtt (Fig. 6F and Supplementary Fig. 13).”

L2-siRNAHtt is more effective in the striatum (~80% reduction) than ASOHtt (~25% reduction).

“Of particular note, L2-siRNAHtt was more effective in the striatum (~80% reduction) than ASOHtt (~25% reduction), which is highly relevant considering the striatum is the region predominantly affected by Huntington’s disease.”

• AssumptionRests on an assumption: The striatum is the brain region predominantly affected by Huntington's disease.

At 3 months, siRNAHtt exhibits a reduction in Htt mRNA only in the hippocampus.

“At 3 months post-injection, mice receiving siRNAHtt no longer exhibited significant protein knockdown in any regions, and only the hippocampus exhibited a reduction in the target mRNA.”

L2-siRNAPpib produces low but significant Ppib knockdown in arterial endothelial cells.

“Endothelial cells were subclustered into venous/capillary and arterial populations (Fig. 4F); there was low but significant knockdown in arterial endothelial cells, and slightly higher silencing in the venous/capillary cells (Fig. 4G).”

L2-siRNA delivers at high levels to choroid plexus epithelial cells.

“Lastly, we examined delivery to other cells in the choroid plexus and noted a high level of delivery to choroid plexus epithelial cells corresponding to robust gene silencing (Fig. 5H,I).”

No visible signs of toxicity were observed after surgery in mice injected with L2-siRNA conjugates.

“Overall, we did not observe visible signs of toxicity after surgery and every mouse injected with an L2-siRNA conjugate (Htt, Ppib, NTC) survived to their predetermined endpoint.”

L2-siRNAPpib potentiates ~70% Ppib silencing in resident CNS macrophages.

“Compared to the non-targeting control, L2-siRNAPpib potentiated gene silencing in homeostatic (~40%) and activated microglia (~50%), as well as resident CNS macrophages (~70%) (Fig. 4E and Supplementary Fig. 10D).”

The greatest fibroblast knockdown potency is observed in cells directly contacting CSF, such as arachnoid barrier cells and pial fibroblasts.

“The greatest fibroblast knockdown potency was observed in cells directly contacting CSF, such as arachnoid barrier cells, pial fibroblasts, and arachnoid barrier cells (Fig. 5C).”

L2-siRNA uptake into neurons was the lowest among the examined CNS cell types.

“L2-siRNA uptake into neurons was the lowest out of the cells examined, but this may have been due to poor neuron recovery in our dissociation protocol (<2% of total population).”

More knockdown was observed in inner arachnoid cells and arachnoid barrier cells (~75%) than dural border cells (30%) after L2-siRNA.

“Interestingly, L2-siRNA mediated gene silencing in fibroblasts spanning the arachnoid membrane, where knockdown correlated with distance from CSF; more knockdown was observed in inner arachnoid cells and arachnoid barrier cells (~75%) than dural border cells (30%), suggesting that L2-siRNA can either cross or bypass the arachnoid barrier.”

L2-siRNAHtt was more potent than dose-matched ASOHtt across multiple brain regions, including the striatum.

“Further, both L2-siRNA and ASO silence a wide variety of cell types, but in a direct comparison we demonstrated that L2-siRNAHtt was more potent than dose matched ASOHtt across multiple brain regions, including the striatum where HD pathology manifests.”

L2-siRNA accumulates in both Lyve1+ and MHCII+ macrophages.

“L2-siRNA accumulates in both type of macrophages, and we observe robust gene silencing in MHCII+ macrophages.”

L2-siRNANTC treatment does not change Htt expression appreciably compared to vehicle in mouse brain regions.

“We determined that treatment with L2-siRNANTC did not change Htt expression appreciably compared to vehicle (Supplementary Fig. 5) and therefore proceeded with L2-siRNANTC as the primary negative control for gene silencing studies.”

The Lyve1+ macrophage population was too sparse to evaluate for knockdown.

“While the Lyve1+ population was too sparse to evaluate, we found robust gene silencing in the MHCII+ macrophages (Fig. 5G).”

L2-siRNA did not exhibit any detectable toxicities at the doses utilized to achieve potent and durable target gene silencing.

“Collectively, L2-siRNA did not exhibit any detectable toxicities at the doses utilized to achieve potent and durable target gene silencing, suggesting that L2-siRNA has a practical therapeutic index.”

Ppib knockdown measured by scRNA-seq closely matched RT-qPCR bulk silencing for both Cd11bpos and Cd11bneg populations.

“We compared RT-qPCR bulk silencing to Ppib knockdown measured by scRNA-seq and these outputs closely matched for both CD11bpos and CD11bneg populations (Fig. 4C), providing confidence in the accuracy of examining cell-specific gene silencing with scRNA-seq.”

L2-siRNA can achieve delivery in larger species using intrathecal administration.

“Collectively this evidence showcases the potential of using L2-siRNA to achieve delivery in larger species using intrathecal administration.”

• AssumptionRests on an assumption: Findings in rats after intrathecal delivery predict delivery in larger mammalian species.

L2-siRNA has a practical therapeutic index.

“Collectively, L2-siRNA did not exhibit any detectable toxicities at the doses utilized to achieve potent and durable target gene silencing, suggesting that L2-siRNA has a practical therapeutic index.”

L2-siRNA does not induce astrogliosis.

“These results are consistent with Western blot quantification of GFAP in the hippocampus (Fig. 6C), collectively suggesting that L2-siRNA does not induce astrogliosis.”

Microglia are notoriously refractory to gene silencing.

“Microglia are notoriously refractory to gene silencing, highlighting the significance of this result.38”

• cites [38] Ralvenius W. T. Nanoparticle-Mediated Delivery of Anti-PU.1 siRNA via Localized Intracisternal Administration Reduces Neuroinflammation. Adv. Mater. 36, 2309225 (2024). 38018280 sourceNot checked — no full text of the source

CSF to brain delivery is driven by a combination of diffusion from subarachnoid/ventricular CSF and convective movement along CSF-filled perivascular spaces.

“CSF to brain delivery is driven by a combination of diffusion from subarachnoid/ventricular CSF and convective movement along CSF-filled perivascular spaces.8”

• cites [8] Pizzo M. E. Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery. J. Physiol. 596, 445– 475 (2018). 29023798 PMC5792566 sourceNot checked — no full text of the source

Ppib is abundantly and ubiquitously expressed across CNS cell types.

“Here, we used an siRNA sequence targeting Ppib because this gene is abundantly and ubiquitously expressed across CNS cell types.34”

• cites [34] Vanlandewijck M. A molecular atlas of cell types and zonation in the brain vasculature. Nature 554, 475– 480 (2018). 29443965 sourceNot checked — no full text of the source

Once delivered, the L2-siRNA complex remains active in tissue for a prolonged period.

“This evidence suggests that, once delivered, this siRNA complex remains active in tissue for a prolonged period, a feature that is advantageous for minimizing treatment frequency in chronic diseases.”

Relatively small nucleic acid therapies are well-suited for perivascular transport.

“This suggests that relatively small nucleic acid therapies are well-suited for this transport, and indeed we observe siRNA (which are small, negatively charged linear molecules) readily accessing the entire perivascular network 2 hours after ICV delivery.”

L2-siRNAPpib exhibits broad gene silencing activity across diverse CNS cell types.

“Overall, L2-siRNAPpib exhibits broad gene silencing activity across diverse CNS cell types, suggesting that it can be leveraged as a platform technology for modulation of neurological diseases.”

• AssumptionRests on an assumption: The perivascular CSF transport pathway and its accessibility to L2-siRNA observed in rodents is representative of humans.

L2-siRNA achieves potent gene inhibition comparable to other siRNA conjugates developed for CSF delivery.

“L2-siRNA achieves potent gene inhibition comparable to other siRNA conjugates developed for CSF delivery.”

L2-siRNA yields a promising platform technology for silencing genes implicated in CNS disorders.

“Collectively, these data suggest that L2-siRNA possesses unique properties that enhance its transport and brain-wide distribution relative to other conjugates, thus yielding a promising platform technology for silencing genes implicated in CNS disorders.”

MHCII+ macrophages are most abundant in the choroid plexus and meninges.

“MHCII+ macrophages are most abundant in the choroid plexus and meninges,43 and we similarly observe widespread uptake of L2-siRNA in this population (Fig. 5F).”

• cites [43] Van Hove H. A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment. Nat. Neurosci. 22, 1021– 1035 (2019). 31061494 sourceNot checked — no full text of the source

Oligodendrocytes were not effectively silenced with C16.

“Furthermore, not all cell types examined achieve effective gene silencing; for example, oligodendrocytes were not effectively silenced with C16.”

L2-siRNA possesses unique properties that enhance its transport and brain-wide distribution relative to other conjugates.

“Collectively, these data suggest that L2-siRNA possesses unique properties that enhance its transport and brain-wide distribution relative to other conjugates, thus yielding a promising platform technology for silencing genes implicated in CNS disorders.”