Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain

In the paper

“These results indicate that LNPs are much more efficient and less toxic than current methods used to deliver siRNA to cultured neurons such as electroporation, calcium phosphate, or lipofection, which usually result in transfection rates of 1–10%.13”

What the sources say

• Disputed

  The Lipofection section reports siRNA delivery into postmitotic neurons at up to 83% efficiency, contrary to the cited sentence’s characterization of siRNA-delivery methods as usually achieving 1–10%. Its low lipofection rates of typically 1–5% are explicitly for plasmid-based vectors, not siRNA. The paper does not compare LNP efficiency or toxicity with these methods. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [13] sourceExperimental — accuracy not yet measured

In the paper

“By contrast, current nonviral vectors exhibit transfection efficiencies of 10% or less and are often accompanied by cell damage.13”

What the sources say

• Disputed

  The Electrical transfection methods section directly contradicts the claim’s 10% ceiling: it reports nucleofection rates averaging 60–80% after optimization, with up to 95% in embryonic rat neurons and better cell survival than conventional electroporation. The paper does report cell damage with some nonviral techniques, including biolistics, but does not characterize nonviral methods as uniformly low-efficiency and damaging. Page numbers were not available in our copy. (LLM, conf=0.90)

  Checked by a reasoning model — may be wrong.

  cites [13] sourceExperimental — accuracy not yet measured

In the paper

“Furthermore, a number of clinical trials are now in progress for treatments of serious neurological diseases that rely on continuous infusion of ASOs into the CSF.27,28”

What the sources say

• ?Not checked — no full text of the sourcecites [27] source
• Disputed

  The Discussion (page unavailable) describes a Phase I trial in human familial ALS patients as planned, not in progress, and says safety studies were underway in animals. The Results describe continuous ASO infusion in rats and a nonhuman primate, but the paper does not report multiple ongoing clinical trials. (LLM, conf=0.88)

  Checked by a reasoning model — may be wrong.

  cites [28] sourceExperimental — accuracy not yet measured

In the paper

“Previous work has shown that LNP systems with this lipid composition can silence target genes in hepatocytes following IV injection at dose levels as low as 0.01 mg siRNA/kg body weight8,9 in an apolipoprotein E (ApoE)–dependent fashion.10”

What the sources say

• Supported

  The Results (page not supplied) report 50% factor VII silencing at 0.01 mg siRNA/kg in mice, and Materials and Methods (page not supplied) specify intravenous administration. The ApoE-dependent mechanism is attached to citation 10 in the citing sentence, not to this source. (LLM, conf=0.91)

  cites [8] source
• Supported

  The Figure 7 results report hepatic gene silencing in mice with an ED50 of 0.005 mg siRNA/kg—below the claimed 0.01 mg/kg—and the paper describes the experiments as using intravenous administration. Its mechanism discussion states that ApoE mediates hepatocyte uptake of these LNPs. Page numbers are not available in our copy. (LLM, conf=0.90)

  cites [9] source
• ?Not checked — no full text of the sourcecites [10] source

In the paper

“The NMDAR ion channel is heteromeric, composed of two obligatory GluN1 subunits and two GluN2 or GluN3 subunits.”

What the sources say

• Supported

  In “C. Synaptic and Extrasynaptic N-Methyl-D-aspartate Receptors” (page not supplied), the review describes NMDA receptors as heterotetramers with two GluN1 subunits and two GluN2 or GluN3 subunits. The same passage calls GluN1 an obligate subunit for channel function. (LLM, conf=0.94)

  cites [19] source

In the paper

“Injection of ASOs into the CSF represents another promising method to silence neuronal gene expression in vivo.25”

What the sources say

• Supported

  The Results section reports that ASOs infused into the cerebrospinal fluid of rhesus monkeys reached neurons and produced sustained reductions in huntingtin mRNA across brain and spinal cord regions. The Discussion describes the approach as promising for human therapy. The paper used continuous infusion rather than a single injection, but this does not change the claim's point about CSF delivery of ASOs in vivo. (LLM, conf=0.93)

  cites [25] source

In the paper

“With respect to the mechanism of action, the LNP delivery systems used here contain the ionizable cationic lipid DMAP-BLP, a lipid that exhibits optimized bilayer destabilizing and pKa properties leading to highly potent gene silencing in hepatocytes following IV administration (Supplementary Data) that is similar to “gold standard” lipids such as DLinMC3-DMA.8”

What the sources say

• Partly supported

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

  cites [8] sourceExperimental — accuracy not yet measured

In the paper

“In addition, there are now well-established methods to reduce or eliminate siRNA immunogenicity by adjusting siRNA chemistry.29”

What the sources say

• Supported

  The section “Abrogating TLR7/8 activation by chemical modification” describes chemical substitutions that reduce siRNA immunogenicity and says some effectively abrogate TLR interaction. The “Quick guide to chemical siRNA design” adds that 2′-OMe modification may fully abrogate TLR7/8 responses. (LLM, conf=0.92)

  cites [29] source

In the paper

“However, two major problems associated with ASOs are the high doses required to achieve gene silencing effects and immunostimulatory effects.26,27”

What the sources say

• Partly supported

  The Cellular Delivery and Associated Safety section says phosphorothioate antisense oligonucleotides (AONs) can have immunostimulatory effects, while noting that appropriate modifications can suppress them. Neither that section nor the AON Mechanisms section says high doses are required to achieve gene silencing; the paper instead discusses dosage control and possible dose-dependent toxicity. Our copy provides no page numbers for these passages. (LLM, conf=0.87)

  cites [26] sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [27] source

In the paper

“In vivo, intracortical or intracerebroventricular (ICV) siRNA-LNP injections resulted in knockdown of target genes either in discrete regions around the injection site or in more widespread areas following ICV injections with no apparent toxicity or immune reactions from the LNPs.”

In the paper

“LNPs were nontoxic at the concentrations used, with no observable differences in cell death relative to controls measured either by comparing nuclear morphology or by lactate dehydrogenase (LDH) release (Figure 2c).”

In the paper

“In this article, we show a method for using small interfering RNA (siRNA) in lipid nanoparticles (LNPs) to efficiently silence neuronal gene expression in cell culture and in the brain in vivo through intracranial injection.”

• Also stated in: § Introduction · boosted

  “In this article, we report the conditions under which LNP delivery of siRNA is a remarkably efficient method for silencing neuronal gene expression in both primary neuronal culture and following intracranial injection in vivo.”

In the paper

“With no added ApoE, there was no evidence of neuronal LNP uptake, whereas graded uptake was observed when ApoE concentrations were increased above 0.1μg/ml to 1 μg/ml and 5 µg/ml ApoE.”

In the paper

“Western blot analysis, which revealed that PTEN protein levels were significantly reduced at distances within 1 mm of the injection tract but not 1–3 mm away (Figure 5b).”

In the paper

“As a positive control, cultures treated with 1% Triton X-100 showed a large increase in LDH release (90.21 ± 0.62%).”

In the paper

“In this article, we demonstrate that a single LNP-siRNA injection of 2.5 µg of siRNA per rat by intracortical administration results in pronounced (up to 91%) gene silencing that is sustained for at least 15 days after injection.”

In the paper

“In experiments in vivo, localized injection of siRNA-LNPs within the cortex or ICV injections to cause more diffuse and widespread LNP uptake led to consistent decreases in both mRNA and protein expression again with no indication of cell loss or damage from the uptake of LNPs.”

In the paper

“however, sequence number 1 resulted in the most robust knockdown, and it was therefore used for the subsequent experiments in vivo.”

In the paper

“The results suggest that there was no immunostimulatory effect when luc siRNA-LNPs (246 nmol/l) were applied to brain slices. TNF-α levels in the luc siRNA-LNP–treated group were not significantly different from the control group (Figure 4f, P > 0.05).”

In the paper

“To get the NMDA component, we measured the amplitude of the outward current at Vh + 40 mV 50 ms after the stimulation artifact, a time point when the AMPAR current had returned to baseline (this was verified by subtracting the trace in the presence of the NMDAR antagonist, DL-2-amino-5-phosphonopentanoic acid (APV) to show the outward INMDAR (blue) and IAMPAR (red) components separately).”

In the paper

“In ApoE knockout mice, LNP-siRNA gene silencing activity is inhibited in hepatocytes and can be reestablished by incubating the LNPs with ApoE before IV administration.”

What the sources say

• ?Not checked — no full text of the sourcecites [10] source

In the paper

“NMDARs are typically arranged in clusters at synaptic and extrasynaptic sites.20,21”

What the sources say

• ?Not checked — no full text of the sourcecites [20] source
• ?Not checked — no full text of the sourcecites [21] source

In the paper

“The AMPA component was measured as the inward current when the cell was held at −70 mV (Figure 8d).”

In the paper

“Therefore, a common protocol to test the NMDAR/AMPAR ratio in response to the synaptic release of glutamate is to measure the ratio of the evoked excitatory postsynaptic current when the NMDAR is blocked by holding the cell at −70 mV to obtain a pure AMPAR component versus when the cell is held at +40 mV to get a mixed AMPAR + NMDAR component (Figure 8d).22,23”

What the sources say

• ?Not checked — no full text of the sourcecites [22] Sah P, 1991, J Physiol (Lond) source
• ?Not checked — no full text of the sourcecites [23] source

In the paper

“These results demonstrate that when administered directly into the brain, LNPs are capable of diffusing through the extracellular milieu to deliver siRNA to neurons and induce gene silencing at sites distant from the site of injection.”

In the paper

“Neurons that were chosen for experiments were within 500 µm of the injection site and were verified to have taken up LNPs based on punctate intracellular DiI fluorescence (Figure 8c), and the ratio of NMDAR/AMPAR was measured.”

In the paper

“The use of LNPs can circumvent these issues, and provides a method to rapidly test various siRNA constructs against the expression of different target proteins, and then examine the protein's function.”

In the paper

“This is a region with robust LNP uptake and gene knockdown but not damaged from the injection needle itself, suggesting that LNPs themselves are nontoxic in vivo.”

In the paper

“The results presented in this article show that LNP-siRNA systems can exhibit efficient gene silencing properties in neurons both in vitro and in vivo that lead to functional consequences without inducing significant toxicity.”

In the paper

“Clinical studies suggest that the immunostimulatory effects of LNP-siRNA injections are infrequent and readily managed, see http://www.alnylam.com/capella/wp-content/uploads/2012/07/Alnylam-ALN-TTR02-PhaseI-Results-120716.pdf.”

What the sources say

• ?Not matched

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

  cites http://www.alnylam.com/capella/wp-content/uploads/2012/07/Alnylam-ALN-TTR02-PhaseI-Results-120716.pdf

In the paper

“Cell density was measured in 10 µm z-stacks of 200 × 200 µm, and all cells including partial cells were counted, therefore resulting in an overestimate of cell density/mm3.”

In the paper

“The extracellular solutions were supplemented with 50 µmol/l picrotoxin (Sigma) and 8 mmol/l of each MgCl2 and CaCl2 to block GABAA synaptic potentials, block epileptiform activity, and minimize polysynaptic responses.”

In the paper

“Cells were allowed to dialyze for at least 15 minutes before starting recordings.”

In the paper

“The LNP platform is highly flexible, potentially allowing delivery of other materials to neurons such as drugs, fluorescent dyes and plasmids.”

In the paper

“A lipid-based formulation of cytarabine has gained clinical acceptance for treatment of brain cancer following direct administration into CSF.34”

What the sources say

• ?Not checked — no full text of the sourcecites [34] source

In the paper

“The therapeutic utility of direct intracranial administration of LNP-siRNA for treatment of neurological disorders remains to be established; however, there are now a number of clinically accepted therapies relying on direct pumping of therapeutics into the CSF.32,33”

In the paper

“Vhold was then returned to −70 mV at the end of the experiment to verify that there was no change in the baseline.”

In the paper

“Access resistance was continuously monitored during the experiments.”

In the paper

“Media containing Triton X-100 (1%) was used as a positive control for cell death.”

In the paper

“As a negative control experiment, primary antibody was omitted during the immunostaining.”