Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing

Abstract

Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by transsplicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects.mRNA repair ͉ gene correction ͉ reporter I n the postgenomic era, a great impetus has been generated toward designing therapeutic and diagnostic agents that are able to capitalize on the wealth of genetic information now available. Although proteins are the ultimate effectors of genetic programming, there are several preceding steps in the cascade of gene expression where interventions for therapy or diagnosis are possible. Because of the complex tertiary folding and singular structure of each individual protein, it is unfeasible (at present) to design molecules specific for an arbitrary protein based on sequence alone. However, given that the principles behind targeting arbitrary nucleic acid sequences have been well established (1) and our profound knowledge of the human genomic sequence, it is practicable to design molecules that interact with specific genes at the nucleotide level. By exploiting the WatsonCrick base-pairing nature of nucleic acids, researchers have been able to design sequence-specific molecules for purposes ranging from in vivo antisense therapeutics to in vitro detection (2, 3).The concept of therapeutic intervention at the level of nucleic acids has been advanced recently by spliceosome-mediated RNA trans-splicing (SMaRT) (4, 5). SMaRT has effectively repaired disease-causing mutant genes at the level of RNA splicing for several disorders including hemophilia and cystic fibrosis (6, 7). The principle behind SMaRT is centered on the use of engineered pre-trans-splicing molecules (PTMs), which can replace the mutated portion of a disease-causing gene with the wild-type sequence. SMaRT can also be used to regulate the trans-splicing and expression of almost any desired gene sequence, such as those encoding reporter or toxic molecules (4,8). Embedded within each PTM are active splicing elements that are recognized by the cell's splicing machinery. These promote the formation of spliceosome complexes that trans-splice the PTM encoded exon(s) into the target transcript rather than allowing cis-splicing within the target pre-mRNA to occur. The specificity of the trans-splicing reaction is conferred primarily by the bindin...

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies

“…The most significant applications of SMaRT have been the functional repair of the CFTR⌬F508 mutation of the cystic fibrosis transmembrane conductance regulator in human airway epithelial cells (23) and the phenotypic correction of hemophilia A in mice (24). SMaRT is also a promising technology for imaging gene expression in vivo (25).Here, we show that tau RNA can be reprogrammed by trans-splicing and that exon 10 Ϫ to exon 10 ϩ tau RNA converConflict of interest statement: M.A.G.-B. is a founder of and consultant for Intronn, which owns and commercializes the SMaRT technology.…”

“…The most significant applications of SMaRT have been the functional repair of the CFTR⌬F508 mutation of the cystic fibrosis transmembrane conductance regulator in human airway epithelial cells (23) and the phenotypic correction of hemophilia A in mice (24). SMaRT is also a promising technology for imaging gene expression in vivo (25).…”

Preparation and Evaluation of 99mTc-Epidermal Growth Factor Receptor (EGFR)-Peptide Nucleic Acid for Visualization of EGFR Messenger RNA Expression in Malignant Tumors

“…Direct imaging of messenger RNA (mRNA) of overexpressed oncogenes may provide information on cellular gene expression patterns and reveal molecular changes in disease states at relatively early stages (23). The successful use of antisense peptide nucleic acid (PNA) radiolabeled with 99m Tc has been reported for targeting and imaging tumor-associated mRNA (24)(25)(26)(27).…”

Molecular imaging of endogenous mRNA expression in a mouse tumor model by adenovirus harboring trans‐splicing ribozyme

“…While Bhaumik and colleague did show splicing-dependent bioluminescence imaging in living animals using spliceosomemediated RNA trans-splicing (SMaRT), they targeted an exogenous gene which was driven by a strong constitutive promoter, and not an endogenous mRNA [15]. Our method, which visualizes endogenous mRNA in infused cancer cells, potentially offers an attractive and comprehensive approach for imaging mRNA in vivo.…”