Identifying an Optimal Neuroinflammation Treatment Using a Nanoligomer Discovery Engine

Abstract

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leading to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and it is causally linked to a range of neurodegenerative diseases such as Alzheimer's diseases (AD), Multiple Sclerosis (MS), Parkinson's disease (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier, making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer Discovery Engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1β or IL-1β, tumor necrosis factor-alpha or TNFα, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-κβ) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and artificial intelligence-based ranking method to design sequence-specific peptide molecules to up-or downregulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-κβ (SB.201.17D.8_NF-κβ1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low dose (5 mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

Safety and Biodistribution of Nanoligomers Targeting the SARS-CoV-2 Genome for the Treatment of COVID-19

“…While a longer-term study would be necessary to confirm a lack of T-cell response and antibody production, there is thus far no indication that Nanoligomers cause activity in the immune system (Figures and S5–S9). Recent studies have shown that human primary astrocytes treated with missense Nanoligomers show the cytokine profile as measured in a 65-plex panel similar to untreated cells, indicating the effects observed are due to sequence-specific action of Nanoligomers . This is in contrast with siRNA therapies, PNA conjugated to CPP, and lipid-encapsulated ASOs. ,− Nanoligomers are readily available to key organs such as the lungs, kidneys, liver, and spleen.…”

“…Another major advantage of Nanoligomers discovery engine is the ability to target gene(s) of interest on demand to up- or downregulate expression. Multiple studies have shown the use of Nanoligomers to target the library of immunological genes including interleukin-1β, TNF receptor 1, inflammasome (NLRP1), granulocyte-macrophage colony-stimulating factor (CSF2), and nuclear factor kappa-B (NF-κβ) to identify the effective therapeutic interventions. , Recently, a low dose of 5 mg/kg Nanoligomer SB_NI_111, a combination treatment targeting NF-κβ and TNFR1, has been shown to significantly reduce inflammation in mouse hippocampus in an LPS-induced neuroinflammation …”

“…Nanoligomers were designed and synthesized (Sachi Bio Inc.) according to the published methods. , The Nanoligomer is composed of a nanobiohybrid molecule based on an antisense peptide nucleic acid (PNA) − ,,, moiety conjugated to nanoparticles − for improved delivery and membrane transport. The PNA moiety was chosen to be 15 bases long (to maximize both solubility and specificity) antisense to the SARS-CoV-2 viral genome (NC_045512) .…”

“…Recent studies have shown that human primary astrocytes treated with missense Nanoligomers show the cytokine profile as measured in a 65-plex panel similar to untreated cells, indicating the effects observed are due to sequence-specific action of Nanoligomers. 31 This is in contrast with siRNA therapies, PNA conjugated to CPP, and lipid-encapsulated ASOs. 41,43−45 Nanoligomers are readily available to key organs such as the lungs, kidneys, liver, and spleen.…”

“…The antiviral compound presented here, called SBCoV202 (Sachi Bio), relies on antisense technology called Nanoligomer, a nanobiohybrid molecule that comprises of a short 15-mer peptide nucleic acid (PNA) sequence, conjugated to a gold nanoparticle, which can specifically and tightly bind a target sequence of SARS-CoV2 RNA (Figure A,B). − PNAs are a third generation of antisense oligomers (ASO) comprised of a backbone of N -(2-aminoethyl) glycine units, which are linked to bases as side groups. , This creates a nucleic acid sequence with a rigid, stable amine backbone and a nucleotide sequence capable of highly specific binding to a target with single-base-pair specificity. − PNAs have been shown to function in a RNAseH-independent manner and can modulate processing of RNA by interfering with splicing machinery or arrest of translation by masking the AUG translation initiation codon. ,, The PNAs used in these Nanoligomers were rationally designed to target the translation initiation site of the RNA-dependent RNA polymerase within the SARS-CoV-2 genome while minimizing off-target binding within the host genome. A PNA molecule developed by Li et al, designed to target the translation initiation site with the use of a cell-penetrating peptide (CPP) to aid transport, showed efficacy in reducing SARS-CoV-2 infection .…”