Lipid Nanoparticle Delivery of Chemically Modified NGF^R100W mRNA Alleviates Peripheral Neuropathy

Abstract

Messenger RNA (mRNA) carries genetic instructions to the cell machinery for the transient production of antigens or therapeutic proteins and shows enormous potential in vaccine development, cancer immunotherapy, protein replacement therapy, and genome engineering. Here, the synthesis of chemically modified nerve growth factor mutant (NGF R100W ) mRNA through in vitro transcription is described. After the replacement of the original signal peptide sequence with the Ig Kappa leader sequence, codon-optimized NGF R100W mRNA yielded high secretion of mature NGF R100W , which promotes axon growth in PC12 cells. Using lipid nanoparticle (LNP)-delivery of N1-methylpseudouridine-modified mRNA in mice, NGF R100W -mRNA-LNPs result in the successful expression of NGF R100W protein, which significantly reduces nociceptive activity compared to that of NGF WT . This indicates that NGF R100W derived from exogenous mRNA elicited "painless" neuroprotective activity. Additionally, the therapeutic value of NGF R100W mRNA is established in a paclitaxel-induced peripheral neuropathy model by demonstrating the rapid recovery of intraepidermal nerve fibers. The results show that in vitro-transcribed mRNA has significant flexibility in sequence design and fast in vivo functional validation of target proteins. Furthermore, the results highlight the therapeutic potential of mRNA as a supplement to beneficial proteins for preventing or reversing some chronic medical conditions, such as peripheral neuropathy.

Nanoparticle-Facilitated Therapy: Advancing Tools in Peripheral Nerve Regeneration

“…The beneficial role of nanoparticles acting in peripheral nerve repair transcends the confines of structural reinforcement, deeply exploring the orchestration of neuroprotective cues. 48 …”

“…Within the sphere of encapsulation, therapeutic interventions undergo a profound metamorphosis, evolving from transient actions into orchestrated and sustained endeavors. 10 , 48 This transformative shift gives rise to a regenerative milieu adeptly fostering and guiding the complicated processes of healing.…”

Off-target effects in CRISPR/Cas9 gene editing

“…RNP electroporation exhibits higher on-target editing efficiency and lower off-target mutations than the other delivery methods ( Kim et al, 2014 ; Ramakrishna et al, 2014 ). Similar to RNP electroporation, Cas9 mRNA and sgRNA can also be delivered into cells via electroporation or liposome-based vectors ( Evers et al, 2022 ; Yu et al, 2023 ) to improved genome editing fidelity. The rationale behind all these methods is to achieve a transient peak expression of CRISPR/Cas9 followed by quick turnover of these editors to avoid off-target effects due to prolonged editor expression.…”

Lipid Nanoparticle (LNP) Enables mRNA Delivery for Cancer Therapy

“…developed nerve growth factor mutant (NGF R100W ) mRNA‐LNPs and injected them locally at the hind paw to treat chemotherapy‐induced peripheral neuropathy. [ 142 ] Riley et al. utilized LNPs to achieve in utero mRNA delivery by directly injecting them through the vitelline vein.…”

“…[141] In addition, Yu et al developed nerve growth factor mutant (NGF R100W ) mRNA-LNPs and injected them locally at the hind paw to treat chemotherapy-induced peripheral neuropathy. [142] Riley et al utilized LNPs to achieve in utero mRNA delivery by directly injecting them through the vitelline vein. [143] Of note, Melamed et al were the first to achieve pancreatic mRNA delivery by intraperitoneal injection of LNPs containing cationic lipids.…”