The Cellular Processing Capacity Limits the Amounts of Chimeric U7 snRNA Available for Antisense Delivery

Abstract

Many genetic diseases are induced by mutations disturbing the maturation of pre-mRNAs, often affecting splicing. Antisense oligoribonucleotides (AONs) have been used to modulate splicing thereby circumventing the deleterious effects of mutations. Stable delivery of antisense sequences is achieved by linking them to small nuclear RNA (snRNAs) delivered by viral vectors, as illustrated by studies where therapeutic exon skipping was obtained in animal models of Duchenne muscular dystrophy (DMD). Yet, clinical translation of these approaches is limited by the amounts of vector to be administered. In this respect, maximizing the amount of snRNA antisense shuttle delivered by the vector is essential. Here, we have used a muscle- and heart-specific enhancer (MHCK) to drive the expression of U7 snRNA shuttles carrying antisense sequences against the human or murine DMD pre-mRNAs. Although antisense delivery and subsequent exon skipping were improved both in tissue culture and in vivo, we observed the formation of additional U7 snRNA by-products following gene transfer. These included aberrantly 3′ processed as well as unprocessed species that may arise because of the saturation of the cellular processing capacity. Future efforts to increase the amounts of functional U7 shuttles delivered into a cell will have to take this limitation into account.

<p>Nucleic Acid Therapy for β-Thalassemia</p>

“…Unfortunately, several studies showed a modification of U7 snRNA resulting in two isoforms, full length and truncated, of U7 snRNA that might interfere with the antisenses function to block the splicing machinery in certain animal models. 46 , 53 A similar finding also found in the erythroid progenitor cells of thalassemic mice (unpublished data). Therefore finding the best model to test the SSOs would prove very important before its further application.…”

“… 38 Not only in β-thalassemia, but the modified U7 snRNAs has also been extensively explored in several RNA mis-splicing diseases including DMD and SMA. 46 – 52 …”

Pathological mechanism and antisense oligonucleotide-mediated rescue of a non-coding variant suppressing factor 9 RNA biogenesis leading to hemophilia B

“…In essence, both antisense strategies lead to a rescue of F9 mRNA expression and an increase in coagulation activity, which may be able to revert the phenotype into a mild or subclinical hemophilia B. Interestingly, the RNA duplex formation and association with U7snRNP did not hamper translation in the cytosol, which suggests detachment of the complex at the nuclear pore or during the pioneer round of translation. However, we will have to determine if the partial rescue was due to limitation of the cellular processing capacity of snRNP particles [40].…”

Enhancement of β-Globin Gene Expression in Thalassemic IVS2-654 Induced Pluripotent Stem Cell-Derived Erythroid Cells by Modified U7 snRNA

“…Although a complete restoration of correctly spliced b-globin mRNA was not achieved, it is estimated that only 10% level of correction would already lead to a clinically applicable result [60]. This incomplete correction may be the consequence of a lower production of functional U7 snRNA by the host cells due to their limited processing capacity, as reported previously [61,62]. Although no significant downregulation of HBA and HBE genes was found in the corrected cells, they were prone to express these genes at a lower level than patient or healthy cells.…”