SMaRT-Based Repair of NF1 Pre-mRNA
Abstract
The proposed research project investigates the applicability and suitability of a method called spliceosome- mediated pre-mRNA trans-splicing (SMaRT) for correcting faulty pre-mature NF1 messenger RNA (pre- mRNAs) in cells. If successful, this method could be used to transiently correct a patient’s NF1 mutation – not in the NF1 gene (DNA) but in NF1 transcripts (RNA) before these are translated into NF1 protein (neurofibromin). The technique utilizes the cell’s own splicing machinery, the spliceosome, which by default removes non-coding regions, so called introns, from premature messenger RNAs and combines the coding sequences, so-called exons, into mature mRNAs. SMaRT requires the delivery of an engineered RNA molecule, called the pre-trans-splicing molecule (PTM), to the cell’s nucleus. The PTM contains the replacement sequence for one or more NF1 exons in the pre-mRNA that harbor a pathogenic mutation and will be designed to specifically bind to intronic NF1 pre-mRNA. Interaction between spliceosome, PTM, and mutant NF1 pre-mRNA will then result in the insertion of the PTM’s replacement exon(s) into the NF1 pre-mRNA instead of the corresponding disease-causing exon(s), as well as generation of a correct transcript, allowing translation into functional neurofibromin. SMaRT has been studied for over two decades and applied to several target pre-mRNAs and disease contexts with varying degrees of successful repair. In recent years, several improvements in the PTM design have increased the efficiency and specificity of trans-splicing. For example, trans-splicing efficiencies of more than 40% have been achieved in human cells following optimization of the PTM targeting pre-mRNA from a gene, called rho-dopsin. The level of NF1 repair that is ultimately achieved depends on many factors, including the sequence of the targeted NF1 intron, the position and length of the intronic NF1 pre-mRNA binding domain, and other PTM design elements. Recently published research by us and collaborators suggests that less than 50% NF1 repair is necessary to observe at least partial reconstitution of normal cellular behavior. Moreover, a genetically engineered mouse that produces only approximately 50% of functional neurofibromin in its cells does not show any Neurofibromatosis Type I (NF1) phenotype. Together, this suggests that, thanks to improved PTM designs, SMaRT-based repair efficiencies of mutant transcripts are now reaching levels that could potentially normalize NF1 downstream signaling and be therapeutic for NF1 patients. This is the underlying hypothesis of our proposal. Of note, our approach does not correct mutant DNA, which would be permanent, but instead it corrects faulty RNA. Consequently, PTMs (RNA) would likely have to be repeatedly delivered to the patient’s cells to be therapeutic. On the other hand, safety issues of gene replacement and gene editing therapies such as insertional mutagenesis, overexpression of the target gene, or off-target genome editing are of no concern with SMaRT. If successful, our approach could help patients with pathogenic NF1 variants, where the mutation is in an exon that can be efficiently replaced using a designed PTM. We envision the use of therapeutic PTMs that systemically correct a patient’s NF1 germline mutation (first hit) in the transcript to reduce the overall risk of NF1-associated tumors. In addition, PTMs correcting NF1 germline and/or somatic exon mutations may also be therapeutic against NF1-related tumors. While off-target effects such as binding of the PTM to pre-mRNA other than NF1 are, in principle, possible, they are likely not harmful if the product of such an event is non-functional or readily degraded. Moreover, it has been shown that such specificity issues can be reduced by improving the PTM design. The likeliest major contribution of this project will be a proof of concept showing that SMaRT-based correction of mutant NF1 transcripts is possible in
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 04, 2024
- Source ID
- HT94252310185
Entities
People
- Andre Leier
Organizations
- United States Army
- University of Alabama at Birmingham