RG7800/RG7619 binds to the SMN2 pre-mRNA sequence and promotes a conformational change that increases e7 splicing activator affinity, whereas LMI070 stabilizes U1 small nuclear RNA interactions at the e7 5′ splice site 8, 11.Ī, Schematic depicting the SMN2-on cassette. 1c), possibly reflecting their different mechanisms. LMI070 showed greater induction activity than did RG7800 (Extended Data Fig.
SPLICE 2 IZLE PLUS
For indSMN2 plus LMI070, there was an approximately 20-fold induction (Fig. Both drugs induced e7 splicing, with a complete splicing switch evident at concentrations greater than 1 μM (Extended Data Fig. IndSMN2 was further evaluated in response to various doses of LMI070 or RG7800. For this, the original minigene was modified at the e7 acceptor splice site to reduce the approximately 10% background splicing in the native condition (indSMN2 Extended Data Fig. Our first derivation of a splicing switch for controlled translation used a modified SMN2 minigene, such that exon 7 (e7) exclusion and premature termination would occur in the absence of drug, but e7 inclusion and in-frame gene expression would occur in the presence of splice modifiers (Fig. Notably, these drugs are in later-stage clinical testing (LMI070) 9 or have been approved for use in humans (RG7800/RG7619) 10. Two drugs, LMI070 and RG7800/RG7619, can improve the proportion of correct SMN2 splicing, and both show efficacy in animal models of spinal muscular atrophy 7, 8.
The rationale is as follows: SMN2, a pseudogene of SMN1, is correctly spliced only around 10% of the time, resulting in an SMN2 protein that is functionally equivalent to SMN1 6. Initially, alternative splicing for the regulated control of protein translation was achieved using drugs developed for the treatment of spinal muscular atrophy, a disorder that is caused by reduced levels of SMN1. As such, the X on system can be applied to any genetic element of interest in cells or in animals. Notably, the X on switch does not require any foreign elements for regulation, but rather takes advantage of drugs that are orally bioavailable and in human use to induce the rapid inclusion of an exon containing a start codon. By controlling alternative splicing, we can regulate which exons are spliced in (or out). The X on system takes advantage of alternative RNA splicing 5, a mechanism that provides for RNA and protein diversity through the inclusion or exclusion of different protein-coding exons, parts of exons, and different 5′ and 3′ noncoding exons. To address this gap, here we develop a method to finely control protein translation via a drug-inducible switch. However, the cargo itself-and more importantly the elements that control the expression from that cargo-have not received the same attention, although engineered promoters, riboswitches or other 3′ regulatory elements that restrict expression to certain cell types have advanced 1, 2, 3, 4. Additionally, lipid nanoparticles have been refined for improved uptake. For example, viral capsid evolution and engineering has improved the cell and tissue targeting of adeno-associated viruses (AAVs), and the landscape of cell targeting for lentiviruses has been expanded by using different envelopes in viral production. Additionally, owing to the oral bioavailability and safety of the drugs used, the X on switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.Īlthough viral and non-viral approaches for gene therapies have undergone substantial advancement over the past twenty years, the major focus has been on the cargo-delivery system. The ability of X on to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Using X on, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. Moreover, the switch system, which we denote X on, does not require the co-expression of any regulatory proteins.
The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. So far, gene therapies have relied on complex constructs that cannot be finely controlled 1, 2.
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