RNA‐based therapeutic approaches for coagulation factor deficiencies

Abstract

To cite this article: Pinotti M, Bernardi F, Dal Mas A, Pagani F. RNA-based therapeutic approaches for coagulation factor deficiencies. J Thromb Haemost 2011; 9: 2143-52.Summary. Substitutive therapy has significantly ameliorated the quality of life of patients with coagulation factor deficiencies. However, there are some limitations that support research towards alternative therapeutic approaches. Here we focus on the rescue of coagulation factor biosynthesis by targeting the RNA processing and translation, which would permit restoration of the altered gene expression while maintaining the gene regulation in the physiological tissues. The essential prerequisite of the three reported RNA-based correction approaches (i-iii), which rely on mutation types and are applicable even to large size mRNAs, is the presence in cells of the precursor (pre-mRNA) or mature mRNA forms. (i) In the F7 gene, modification of the small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, re-directs correct usage of a mutated exonintron junction, triggering synthesis of correct mRNA and secretion of functional factor (F)VII. (ii) Spliceosome-mediated RNA trans-splicing (SMaRT) between mutated and engineered pre-mRNAs produces normal FVIII mRNA and secretion of functional protein. (iii) Aminoglycoside drugs induce ribosome readthrough and suppress premature translation termination caused by nonsense mutations in FVII, VIII and IX. The rescued expression levels ranged from very low (aminoglycosides) to moderate (U1 snRNA and SMaRT), which could result in amelioration of the disease phenotypes. These findings prompt further studies aimed at demonstrating the clinical translatability of RNA-based strategies, which might open new avenues in the treatment of coagulation factor deficiencies.

Activation of a cryptic splice site in a potentially lethal coagulation defect accounts for a functional protein variant

“…It has been demonstrated that U1-snRNA variants engineered to restore the complementarity with defective donor splice sites can re-direct splicing to the correct junction [17,21,23]. Although mutations that disrupt the invariant GT dinucleotide of 5'ss are usually not rescued [23], the c.165 + 1G > T mutation in the FANCC gene was successfully treated [8]. It must be noticed that in the latter case, the + 1G > T change does not completely abrogate splicing and it is compatible with synthesis of correct transcripts.…”

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

“…18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models. However, these U1snRNAs are often tailored on the disease-causing mutation and have the intrinsic risk of off-target effects by recognizing the partially conserved donor splice site 22 in other splicing units.…”

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation

“…25 Among all coagulation factor deficiencies, FV deficiency represents a particularly suitable target for RNA therapy, because (1) no FV concentrate or recombinant FV preparation is available for substitutive therapy; (2) the FV requirement for adequate hemostasis is extremely low, making a few percentages of FV sufficient to prevent life-threatening bleeding 19,[35][36][37] ; and (3) the large size of the F5 cDNA makes conventional gene therapy particularly challenging. Here, we present the first application of RNA therapy to severe FV deficiency and we provide in vitro and ex vivo evidence that mutation-specific antisense molecules can effectively rescue a F5 splicing mutation.…”

“…Accordingly, F5 splicing mutations are usually associated with severe bleeding symptoms. 14,[16][17][18][19][20][21] Several studies on various genetic disorders have shown that splicing defects are amenable to "RNA therapy" with antisense molecules specifically designed to anneal to the mutant pre-mRNA (without triggering its degradation) and to direct its maturation into the correct transcript (reviewed in Cooper et al, 22 Du and Gatti, 23 Hammond and Wood, 24 and Pinotti et al 25 ). In particular, mutations that disrupt an existing donor splice site can be corrected with U1 small nuclear RNA (snRNA) specifically modified to recognize the mutated splice site, 26 whereas mutations that create a new splice site can be targeted with short antisense oligonucleotides or snRNAs that mask the aberrant splice site and prevent its interaction with the spliceosome.…”