bims-limsir 2023-02-05 papers

RNA. 2023 Feb 01. pii: rna.079526.122. [Epub ahead of print]

Acyclic (S)-Glycol Nucleic Acid (S-GNA) Modification of siRNAs Improves the Safety of RNAi Therapeutics While Maintaining Potency.

Martin Egli, Mark K Schlegel, Muthiah Manoharan.

Glycol nucleic acid (GNA) is an acyclic nucleic acid analogue connected via phosphodiester bonds. Crystal structures of RNA-GNA chimeric duplexes indicated that nucleotides of the right-handed (S)-GNA were better accommodated in the right-handed RNA duplex than were the left-handed (R)-isomers. GNA nucleotides adopt a rotated nucleobase orientation within all duplex contexts, pairing with complementary RNA in a reverse Watson-Crick mode, which explains the inabilities of GNA C and G to form strong base pairs with complementary nucleotides. Transposition of the hydrogen bond donor and acceptor pairs using novel (S)-GNA isocytidine and isoguanosine nucleotides resulted in stable base pairing with the complementary G and C ribonucleotides, respectively. GNA nucleotide or dinucleotide incorporation into an oligonucleotide increased resistance against 3'-exonuclease-mediated degradation. Consistent with the structural observations, small interfering RNAs (siRNAs) modified with (S)-GNA had greater in vitro potencies than identical sequences containing (R)-GNA. (S)-GNA is well tolerated in the seed regions of antisense and sense strands of a GalNAc-conjugated siRNA in vitro. The siRNAs containing a GNA base pair in the seed region had in vivo potency when subcutaneously injected into mice. Importantly, seed pairing destabilization resulting from a single GNA nucleotide at position 7 of the antisense strand mitigated RNAi-mediated off-target effects in a rodent model. Two GNA-modified siRNAs have shown an improved safety profile in humans compared with their non-GNA-modified counterparts, and several additional siRNAs containing the GNA modification are currently in clinical development.

Keywords: Glycol Nucleic Acids; Oligonucleotide Therapeutics; RNA Therapeutics; RNAi Therapeutics; siRNAs, GNA

DOI: https://doi.org/10.1261/rna.079526.122

Nucleic Acids Res. 2023 Feb 02. pii: gkad023. [Epub ahead of print]

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Brendan T Finicle, Kazumi H Eckenstein, Alexey S Revenko, Brooke A Anderson, W Brad Wan, Alison N McCracken, Daniel Gil, David A Fruman, Stephen Hanessian, Punit P Seth, Aimee L Edinger.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

DOI: https://doi.org/10.1093/nar/gkad023

Methods Mol Biol. 2023 ;2616 419-425

Gene Silencing in the Brain with siRNA to Promote Long-Term Post-Stroke Recovery.

Bharath Chelluboina, Raghu Vemuganti.

RNA interference is a promising strategy to degrade target RNAs of interest after stroke using small interfering RNA (siRNA). An optimized targeting such as combining a siRNA with a nontoxic transfection reagent that facilitates the effective delivery of siRNAs to the brain and subsequent cellular uptake after stroke is needed. Furthermore, an appropriate route of administration such as intravenous (tail vein or retro-orbital sinus) or cerebroventricular injection has to be used. Using siRNAs tagged with fluorescent probes shows the cellular uptake of siRNA. Efficacy and window of opportunity for a siRNA needs to be determined by testing multiple doses and time frame that alters the long-term functional outcomes. Real-time PCR/western blots can be used to determine the siRNA efficiency by evaluating the knockdown of the RNA/protein of interest. In siRNA studies, it is also essential to identify a proper dose (efficacious, but not toxic) by histopathologic testing to identify any toxicity in the peripheral organs and CNS. This chapter describes the strategies to deliver siRNAs to treat stroke and to facilitate post-stroke long-term recovery.

Keywords: Cerebral ischemia; In vivo delivery; Recovery; Toxicity; Transfection; siRNA

DOI: https://doi.org/10.1007/978-1-0716-2926-0_29

Nat Biotechnol. 2023 Feb 02.

Synapse-tuned CARs enhance immune cell anti-tumor activity.

Peter J Chockley, Jorge Ibanez-Vega, Giedre Krenciute, Lindsay J Talbot, Stephen Gottschalk.

Chimeric antigen receptor (CAR) technologies have been clinically implemented for the treatment of hematological malignancies; however, solid tumors remain resilient to CAR therapeutics. Natural killer (NK) cells may provide an optimal class of immune cells for CAR-based approaches due to their inherent anti-tumor functionality. In this study, we sought to tune CAR immune synapses by adding an intracellular scaffolding protein binding site to the CAR. We employ a PDZ binding motif (PDZbm) that enables additional scaffolding crosslinks that enhance synapse formation and NK CAR cell polarization. Combined effects of this CAR design result in increased effector cell functionality in vitro and in vivo. Additionally, we used T cells and observed similar global enhancements in effector function. Synapse-tuned CAR immune cells exhibit amplified synaptic strength, number and abundance of secreted cytokines, enhanced killing of tumor cells and prolonged survival in numerous different tumor models, including solid tumors.

DOI: https://doi.org/10.1038/s41587-022-01650-2