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Review
. 2019 Jun 21;20(12):3036.
doi: 10.3390/ijms20123036.

Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies

Dario Balestra  1 , Alessio Branchini  2
Affiliations
Review

Molecular Mechanisms and Determinants of Innovative Correction Approaches in Coagulation Factor Deficiencies

Dario Balestra et al. Int J Mol Sci. .

Abstract

Molecular strategies tailored to promote/correct the expression and/or processing of defective coagulation factors would represent innovative therapeutic approaches beyond standard substitutive therapy. Here, we focus on the molecular mechanisms and determinants underlying innovative approaches acting at DNA, mRNA and protein levels in inherited coagulation factor deficiencies, and in particular on: (i) gene editing approaches, which have permitted intervention at the DNA level through the specific recognition, cleavage, repair/correction or activation of target sequences, even in mutated gene contexts; (ii) the rescue of altered pre-mRNA processing through the engineering of key spliceosome components able to promote correct exon recognition and, in turn, the synthesis and secretion of functional factors, as well as the effects on the splicing of missense changes affecting exonic splicing elements; this section includes antisense oligonucleotide- or siRNA-mediated approaches to down-regulate target genes; (iii) the rescue of protein synthesis/function through the induction of ribosome readthrough targeting nonsense variants or the correction of folding defects caused by amino acid substitutions. Overall, these approaches have shown the ability to rescue the expression and/or function of potentially therapeutic levels of coagulation factors in different disease models, thus supporting further studies in the future aimed at evaluating the clinical translatability of these new strategies.

Keywords: CRISPR activation; RNA-based correction approaches; TALEs; chaperone-like compounds; coagulation factor deficiencies; gene therapy; modified U1 snRNA; ribosome readthrough.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The series of enzymatic reactions in the coagulation cascade. Schematic representation of the coagulation cascade, showing the several direct (black rows) or feedback (dotted rows) reactions that can be subdivided into initiation (red), amplification (green) and propagation (blue) phases, ultimately leading to the fibrin clot. Boxed items indicate the interaction of active enzymes (FVIIa, FXa, FIXa) with their cofactors (TF, FVa, FVIIIa). F, factor; a, activated form; PT, prothrombin; TF, tissue factor.
Figure 2
Figure 2
Overview of the interventions at the DNA, RNA and protein levels. Schematic representation of the DNA–RNA–protein flow (left) as well as of the corresponding molecular approaches with the indicated level at which each strategy works (right). In the pre-mRNA scheme, exons and introns are indicated in cyan and grey, respectively.
Figure 3
Figure 3
Gene editing and transcription modulation-based approaches for coagulation factor deficiencies. Approaches aimed at correcting and/or modulating the expression of coagulation factor genes through ZFNs (A), cleavage by CRISPR/Cas9 (B), engineering of the albumin locus to drive F9 gene expression (C), correction of intron 22 inversion in F8 gene (D), and TALE-TF or the CRISPRa systems leading to an increase in luciferase (reporter constructs) or endogenous activity due to F7 promoter transactivation (E). Asterisks represent the c.-94C>G (*) and c.-61T>G (**) nucleotide changes.
Figure 4
Figure 4
Elements regulating the splicing of pre-messenger RNA (mRNA). Major regulatory elements of pre-mRNA splicing, namely donor/acceptor splice sites and polypyrimidine tract, are shown with the corresponding trans-acting factors (U1, U2 snRNPs and U2AF65-35). The Exonic Splicing Enhancer (ESE), Silencer (ESS), Intron Splicing Enhancer (ISE) or Intron Splicing Silencer (ISS) sequence(s) positively or negatively contribute to exon recognition through interaction with serine–arginine-rich (SR) proteins and heterogeneous ribonuclear particles (hnRNPs), respectively.
Figure 5
Figure 5
U1-mediated rescue in coagulation FVII deficiency. Coagulation FVII deficiency caused by c.859+5G>A (A) and c.681+1G>T (B) nucleotide changes in the F7 gene. Schematic representation of the genomic context (left panel), splicing transcripts (middle panel) and protein isoforms (right panel) is reported. Sequences of the splice sites and position of mutations are indicated. Frameshift of the coding sequence and premature stop codons are reported by asterisks and red X letter, respectively. Percentages of transcripts, antigen and relative coagulation activity are reported. Values in rounded brackets indicate experiments in mouse model of the disease.
Figure 6
Figure 6
U1-mediated rescue in hemophilia B. Hemophilia B models caused by multiple nucleotide changes at the 3’ss (exon 5, A), at the 5’ss (exons 2, 3 and 5, B, C and D) or within exon (exon 5, panel D) of various exons of the F9 gene. Schematic representation of the genomic context (left panel), splicing transcripts (middle panel) and protein isoforms (right panel) is reported. Sequences of the splice sites and position of mutations (black arrows) are indicated. Frameshift of the coding sequence and premature stop codons are reported by asterisks and red X letter, respectively. Percentages of transcripts, antigen and relative coagulation activity are reported. Values in rounded brackets indicate experiments in mouse model of the disease.
Figure 7
Figure 7
U1-mediated rescue in hemophilia A. Hemophilia A model caused by multiple nucleotide changes within the F8 exon 19. Schematic representation of the genomic context (left panel), splicing transcripts (middle panel) and protein isoforms (right panel) is reported. Frameshift of the coding sequence and premature stop codons are reported by asterisks and red X letter, respectively. Percentages of transcripts, antigen and relative coagulation activity are shown on the right.
Figure 8
Figure 8
Translation termination and readthrough-mediated PTC suppression. (A) Normal translation termination involving eRF1 and eRF3-GTP at 3’ natural stop codons. (B) Aberrant translation termination at PTCs and possible outputs resulting from nonsense changes. (C) Mechanism of ribosome readthrough resulting in PTC suppression, either spontaneous or induced by compounds, and synthesis of the full-length protein.
Figure 9
Figure 9
Amino acid insertions and productive protein outputs arising from readthrough. Type of amino acids inserted at PTCs as a function of the type of stop codon (left panel) and molecular determinants influencing the productive output of readthrough (right panel).
Figure 10
Figure 10
Impact of missense changes on protein folding and correction approaches through chaperone-like compounds. (A) Protein folding in normal conditions assisted by molecular chaperones, with a final production of the native protein conformation. (B) Aberrant folding and the consequent aggregation/degradation of misfolded proteins, unable to reach the native conformation, due to amino acid substitutions caused by missense changes. (C) Rescue of folding of misfolded proteins mediated by chaperone-like compounds.
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