Unifying in vitro and in vivo IVT mRNA expression discrepancies in skeletal muscle via mechanotransduction

Abstract

The translational efficiency of an in vitro transcribed (IVT) mRNA was measured upon delivery to primary skeletal muscle cells and to a mouse model system, towards the development of a predictive in vitro assay for the screening and validation of intramuscular mRNA-based vaccines. When IVT mRNA was delivered either naked or complexed with novel aminoglycoside-based delivery vehicles, significant differences in protein expression in vitro and in vivo were observed. We hypothesized that this previously anticipated discrepancy was due to differences in the mechanism of IVT mRNA endosomal entry and release following delivery. To address this, IVT mRNA was fluorescently labeled prior to delivery, to visualize its distribution. Colocalization with endosomal markers indicated that different entry pathways were utilized in vivo and in vitro, depending on the delivery vehicle, resulting in variations in protein expression levels. Since extracellular matrix stiffness (ECM) influences mRNA entry, trafficking and release, the effect of mechanotransduction on mRNA expression was investigated in vitro upon delivery of IVT mRNA alone, and complexed with delivery vehicles to skeletal muscle cells grown on ∼10 kPa hydrogels. This in vitro hydrogel model more accurately recapitulated the results obtained in vivo upon IM injection, indicating that this approach may assist in the characterization of mRNA based vaccines.

Proximity Ligation Assays for In Situ Detection of Innate Immune Activation: Focus on In Vitro-Transcribed mRNA

“…Furthermore, mRNA complexed with CholK was retained in the muscle longer than free mRNA, inducing a stronger RIG-I response at 6 hr and a stronger MDA5 activation at 16 hr. We hypothesize that CholK enhanced cytoplasmic delivery of the mRNAs in tissue, causing the stronger cytoplasmic PRR responses of complexed mRNA over naked mRNA, as supported by our prior work 40 . The extended mRNA retention in the muscle tissue is likely the main cause of MDA5 activation at 16 hr post-injection.…”

“…IVT mRNA was injected intramuscularly (i.m.) either naked or formulated with the nanoparticle CholK, for improved delivery, especially to lymph nodes over naked delivery 40 . At 1.5 hr following injection, IVT mRNA delivered alone or complexed to CholK was evenly distributed throughout the muscle, primarily between skeletal muscle fibers.…”

“…IVT mRNA encodes for the antigenic protein(s) and is concurrently detected by anti-viral PRRs, which activate innate immune responses, facilitating the development of protective immune responses 17, 18, 19, 34, 35, 36, 37, 38, 39. By pre-labeling IVT mRNA with multiply labeled tetravalent RNA imaging probes (MTRIPs), its distribution can be assessed by confocal microscopy without affecting metabolism, localization, or translatability 30, 40. The colocalization of mRNA and PLA signals allows the correlation of immune responses in cells and tissue with downstream immune function.…”

mRNA as a Transformative Technology for Vaccine Development to Control Infectious Diseases

“…injection. 137 Although not as clinically advanced as lipid-based systems for mRNA delivery, polymers have shown considerable potential in mRNA vaccine development. One example is the dendrimer-based nanoparticle system developed by Chahal et al, 138,139 which comprises molecules of high amine density with organizing branching structures condensing around mRNA to form monodisperse spheres.…”

Tetrafunctional Block Copolymers Promote Lung Gene Transfer in Newborn Piglets

“…Possible explanations for these differences are sparse, 47 but some new concepts are starting to emerge. For example, in vitro and in vivo discrepancies in gene transfer in skeletal muscle can be unified by mechano-transduction 50 . Further understanding these differences is likely to lead to the design of better non-viral gene transfer vectors in vivo .…”