Therapeutic Manipulation of mtDNA Heteroplasmy: A Shifting Perspective
Authors: Christopher B. Jackson, Doug M. Turnbull, Michal Minczuk, Payam A. Gammage
Journal: Trends in Molecular Medicine (2020)

Organelle-targeted nucleases allow specific destruction of mutant mtDNA.In combination with adeno-associated viruses, these nucleases allow organ-specific changes in mtDNA heteroplasmy.Recent data have provided early proof of principle for gene therapy of incurable mtDNA disorders using these engineered enzymes. Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease. Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease. In the past 30 years the medical relevance of mtDNA (see Glossary) has brought this unusual genome to the fore [1.Lightowlers R.N. et al.Mutations causing mitochondrial disease: what is new and what challenges remain?.Science. 2015; 349: 1494-1499Crossref PubMed Scopus (176) Google Scholar]. This is, in the most part, owing to the increasing recognition of mitochondria as a major biochemical hub, the confirmation of mitochondrial dysfunction and mtDNA mutations in common diseases [1.Lightowlers R.N. et al.Mutations causing mitochondrial disease: what is new and what challenges remain?.Science. 2015; 349: 1494-1499Crossref PubMed Scopus (176) Google Scholar,2.Gammage P.A. Frezza C. Mitochondrial DNA: the overlooked oncogenome?.BMC Biol. 2019; 17 (53–10)Crossref PubMed Scopus (42) Google Scholar], and the role of mtDNA in disease-relevant cell signalling pathways [3.Bock F.J. Tait S.W.G. Mitochondria as multifaceted regulators of cell death.Nat. Rev. Mol. Cell Biol. 2019; 17: 608Google Scholar,4.Suomalainen A. Battersby B.J. Mitochondrial diseases: the contribution of organelle stress responses to pathology.Nat. Rev. Mol. Cell Biol. 2018; 19: 77-92Crossref PubMed Scopus (169) Google Scholar], in concert with a number of high-profile attempts at preventing untreatable, hereditary mitochondrial disease using in vitro fertilisation techniques [known as mitochondrial replacement therapy or mitochondrial donation] [5.Herbert M. Turnbull D. Progress in mitochondrial replacement therapies.Nat. Rev. Mol. Cell Biol. 2018; 19: 71-72Crossref PubMed Scopus (31) Google Scholar]. With such an upsurge of interest in mtDNA, effort towards generating methods for robust manipulation of mtDNA, for both clinical and experimental means, has been galvanised [6.Patananan A.N. et al.Modifying the mitochondrial genome.Cell Metab. 2016; 23: 785-796Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar,7.Gammage P.A. et al.Mitochondrial genome engineering: the revolution may not be CRISPR-ized.Trends Genet. 2018; 34: 101-110Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar]. Mitochondrial diseases are one of the most common forms of inherited metabolic disease, with asymptomatic carriers of pathogenic mtDNA variants estimated at one in 200 adults, and approximately one in 5000 adults affected by resultant mitochondrial disease [8.Gorman G.S. et al.Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.Ann. Neurol. 2015; 77: 753-759Crossref PubMed Scopus (422) Google Scholar]. The presentation of patients bearing identical mtDNA mutations is often highly variable, with mostly unpredictable penetrance and severity. When combined with the current lack of reliable biomarkers of mitochondrial disease, diagnosis of these conditions is a major challenge [4.Suomalainen A. Battersby B.J. Mitochondrial diseases: the contribution of organelle stress responses to pathology.Nat. Rev. Mol. Cell Biol. 2018; 19: 77-92Crossref PubMed Scopus (169) Google Scholar]. Despite being clinically and genetically described for several decades, there are still no curative therapeutics for patients with mtDNA disease. As a branch of medicine still lacking such treatment, the clinical potential of gene therapy for mitochondrial disease is considerable [1.Lightowlers R.N. et al.Mutations causing mitochondrial disease: what is new and what challenges remain?.Science. 2015; 349: 1494-1499Crossref PubMed Scopus (176) Google Scholar]. Human mtDNA is a small, multicopy genome encoding 13 protein subunits of the respiratory chain complexes I, III, and IV and ATP synthase, and all RNA molecules necessary for their translation by mitochondrial ribosomes [9.Gustafsson C.M. et al.Maintenance and expression of mammalian mitochondrial DNA.Annu. Rev. Biochem. 2016; 85: 133-160Crossref PubMed Scopus (264) Google Scholar]. As with any genetic material, mtDNA is susceptible to the introduction of point mutations and rearrangements by a combination of endogenous and exogenous factors, and these genetic lesions are observed in human mitochondrial disease [8.Gorman G.S. et al.Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease.Ann. Neurol. 2015; 77: 753-759Crossref PubMed Scopus (422) Google Scholar,10.Gorman G.S. et al.Mitochondrial diseases.Nat. Rev. Dis. Primers. 2016; 2: 16080Crossref PubMed Scopus (468) Google Scholar]. However, as mtDNA is multicopy, mutated genomes can coexist with wild-type genomes, a state termed heteroplasmy (Figure 1A ). For the impact of heteroplasmic mtDNA mutations to biochemically and clinically manifest, the ratio of mutated to nonmutated mtDNA has to exceed a certain upper limit, a phenomenon known as the threshold effect (Figure 1B) [10.Gorman G.S. et al.Mitochondrial diseases.Nat. Rev. Dis. Primers. 2016; 2: 16080Crossref PubMed Scopus (468) Google Scholar]. This effect is mutation specific, with large deletions of mtDNA that encompass several genes generally manifesting at lower heteroplasmic levels (~60%) than single point mutations. These thresholds also vary between individuals with the same mutation as well as between tissues in a single patient (Figure 1A,B). While there is general agreement on the maternal inheritance of human mtDNA, the exact mechanisms of the inheritance and segregation of mtDNA, from tissues to populations, remain a matter of debate. A genetic bottleneck during female gamete development has been identified as the major source of variance between generations, often resulting in unaffected mothers bearing children with severe mitochondrial disease (Figure 1A) [11.Floros V.I. et al.Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos.Nat. Cell Biol. 2018; 20: 144-151Crossref PubMed Scopus (80) Google Scholar]. Further complexity is added through mutation-specific variance, where the same mutation can be of variable distribution and pathogenicity within an individual as well as between individuals [10.Gorman G.S. et al.Mitochondrial diseases.Nat. Rev. Dis. Primers. 2016; 2: 16080Crossref PubMed Scopus (468) Google Scholar]. While the stochastic transmission of mtDNA continues to be a major clinical complication, the threshold effect has been the source of inspiration for numerous therapeutic strategies, aiming to reduce the heteroplasmy of deleterious variants to alleviate disease through both direct and indirect targeting of pathogenic mitochondrial genomes [12.Viscomi C. et al.Emerging concepts in the therapy of mitochondrial disease.Biochim. Biophys. Acta. 2015; 1847: 544-557Crossref PubMed Scopus (75) Google Scholar], an approach often referred to as heteroplasmy shifting. This approach takes advantage of the multicopy nature of mtDNA and the functionally recessive nature of virtually all pathogenic mutations in mtDNA [13.Naeem M.M. Sondheimer N. Heteroplasmy shifting as therapy for mitochondrial disorders.Adv. Exp. Med. Biol. 2019; 1158: 257-267Crossref PubMed Scopus (9) Google Scholar]. As heteroplasmic mtDNA mutations coexist with wild-type mtDNA, shifting mtDNA heteroplasmy below the pathogenic threshold in somatic tissues offers a rational approach to the treatment of primary mitochondrial disease [13.Naeem M.M. Sondheimer N. Heteroplasmy shifting as therapy for mitochondrial disorders.Adv. Exp. Med. Biol. 2019; 1158: 257-267Crossref PubMed Scopus (9) Google Scholar] (Figure 2A ). Many avenues to the manipulation of mtDNA heteroplasmy have been explored, including: upregulation of mitochondrial turnover [14.Suen D.-F. et al.Parkin overexpression selects against a deleterious mtDNA mutation in heteroplasmic cybrid cells.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 11835-11840Crossref PubMed Scopus (215) Google Scholar,15.Kandul N.P. et al.Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila.Nat. 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The first reported heteroplasmy shifting approaches made use of peptide nucleic acid (PNA) oligomers [16.Taylor R.W. et al.Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.Nat. Genet. 1997; 15: 212-215Crossref PubMed Scopus (206) Google Scholar,23.Chinnery P.F. et al.Peptide nucleic acid delivery to human mitochondria.Gene Ther. 1999; 6: 1919-1928Crossref PubMed Scopus (143) Google Scholar]. PNAs are synthetic polymers comprising canonical DNA bases linked in series through a repeating N-(2-aminoethyl)-glycine backbone by peptide bonds. The absence of negatively charged phosphates in PNA, compared with DNA, was seen as advantageous with regard to mitochondrial import competence, as the electrochemical potential across the inner mitochondrial membrane (IMM) sits at ~−180 mV, effectively precluding the import of DNA and RNA molecules. Once within mitochondria, the binding of a PNA molecule to a specific mutant mtDNA sequence, but not wild type, was predicted to preferentially block the replication of mutant mtDNA, thereby shifting mtDNA heteroplasmy in a replication-dependent fashion. Initial work reported encouraging results, with specific stalling of DNA replication through PNA binding to mutant mtDNA templates in vitro [16.Taylor R.W. et al.Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids.Nat. Genet. 1997; 15: 212-215Crossref PubMed Scopus (206) Google Scholar]. However, later efforts that conjugated PNA oligomers to mitochondrial targeting sequences (MTS) or positively charged triphenylphosphonium moieties that facilitate the mitochondrial uptake of small molecules [23.Chinnery P.F. et al.Peptide nucleic acid delivery to human mitochondria.Gene Ther. 1999; 6: 1919-1928Crossref PubMed Scopus (143) Google Scholar, 24.Flierl A. et al.Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid.Mol. Ther. 2003; 7: 550-557Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25.Muratovska A. et al.Targeting peptide nucleic acid (PNA) oligomers to mitochondria within cells by conjugation to lipophilic cations: implications for mitochondrial DNA replication, expression and disease.Nucleic Acids Res. 2001; 29: 1852-1863Crossref PubMed Scopus (143) Google Scholar] failed to demonstrate mtDNA heteroplasmy shifts in cultured cells. More recently, MTS-mediated delivery of PNA 4-mers to the mitochondrial matrix of living cells has been achieved [26.Hoogewijs K. et al.Assessing the delivery of molecules to the mitochondrial matrix using click chemistry.Chembiochem. 2016; 17: 1312-1316Crossref PubMed Scopus (10) Google Scholar]. However, PNAs of at least 7-mer length would be required for a unique site in mtDNA to be targeted, and whether single-stranded mtDNA at the replication fork is readily bound by PNA remains to be determined. A similar approach has also emerged attempting to achieve mtDNA mutation-specific antireplicative effects using imported RNA molecules [27.Loutre R. et al.Can mitochondrial DNA be CRISPR-ized: pro and contra.IUBMB Life. 2018; 70: 1233-1239Crossref PubMed Scopus (23) Google Scholar]. However, in addition to the question of single-stranded mtDNA accessibility at the replication fork, the import of RNA into mammalian mitochondria is contentious, with strong evidence against the existence of any efficiently operating pathway [7.Gammage P.A. et al.Mitochondrial genome engineering: the revolution may not be CRISPR-ized.Trends Genet. 2018; 34: 101-110Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar]. The earliest experiments in the field of nuclease-mediated heteroplasmy shifting used mitochondrially targeted restriction endonucleases (mitoREs), highly specific bacterial endonucleases that target and cleave specific foreign sequences in the 'innate immunity' restriction-modification system of bacteria [28.Srivastava S. Moraes C.T. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease.Hum. Mol. Genet. 2001; 10: 3093-3099Crossref PubMed Scopus (118) Google Scholar]. This approach was first explored using a mitochondrially targeted form of the restriction endonuclease PstI, which was capable of depleting mtDNA copy number when expressed in mouse cells containing mtDNA with a PstI recognition site [28.Srivastava S. Moraes C.T. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease.Hum. Mol. Genet. 2001; 10: 3093-3099Crossref PubMed Scopus (118) Google Scholar]. Heteroplasmy shifting activity was also demonstrated when a hybrid cell line containing both rat mtDNA (bearing two PstI sites) and an alternative mouse mtDNA (without any PstI sites) was transfected with the same mitoPstI construct, resulting in a specific shift towards the mouse haplotype [28.Srivastava S. Moraes C.T. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease.Hum. Mol. Genet. 2001; 10: 3093-3099Crossref PubMed Scopus (118) Google Scholar]. 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Besides an unidentified form of copy number control, which both maintains stable cellular mtDNA copy number over time and ensures the recovery of copy number following nucleolytic cleavage of mutant mtDNAs [31.Moraes C.T. What regulates mitochondrial DNA copy number in animal cells?.Trends Genet. 2001; 17: 199-205Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar], the precise mechanism underlying heteroplasmy shifts in these experiments was unclear. Initially, a lack of DNA repair in mitochondria was the speculated mechanism underpinning heteroplasmic shifts after the introduction of DNA double-strand breaks (DSBs) [31.Moraes C.T. What regulates mitochondrial DNA copy number in animal cells?.Trends Genet. 2001; 17: 199-205Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar]. 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Nevertheless, while the mechanism underlying heteroplasmy shifts remained unclear, these early studies offered proof of principle for nuclease-mediated heteroplasmy shifting approaches, tempered by the reality that m.8993T>G is one of very few pathogenic mutations known to introduce a unique mitoRE site into mtDNA. Limited clinical application notwithstanding, mitoRE approaches developed further [35.Bacman S.R. et al.Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a "differential multiple cleavage-site" model.Gene Ther. 2007; 14: 1309-1318Crossref PubMed Scopus (65) Google Scholar, 36.Bayona-Bafaluy M.P. et al.Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease.Proc. Natl. Acad. Sci. U. S. 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However, mitoRE approaches are profoundly limited by the availability of REs for any given site produced by mtDNA mutations, and the refractory nature of REs to the engineering of alternate DNA binding specificity rendered this avenue unsuitable for further development. With longstanding efforts in nuclear genome manipulation yielding zinc-finger protein (ZFP) and transcription activator-like effector (TALE) technologies [40.Gaj T. et al.ZFN, TALEN and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (2175) Google Scholar], early forms of both mitochondrially targeted zinc-finger nucleases (mtZFNs) and mitochondrially targeted TALE nucleases (mitoTALENs) were soon to follow [21.Gammage P.A. et al.Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.EMBO Mol. Med. 2014; 6: 458-466Crossref PubMed Scopus (146) Google Scholar,22.Bacman S.R. et al.Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs.Nat. Med. 2013; 19: 1111-1113Crossref PubMed Scopus (233) Google Scholar,41.Minczuk M. et al.Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.Nucleic Acids Res. 2008; 36: 3926-3938Crossref PubMed Scopus (128) Google Scholar]. The principle of both technologies lies in sequence specific DNA-binding domains, delivered to mitochondria using MTS peptides, coupled to the functionally dimeric, sequence nonspecific endonuclease domain from the RE FokI (Figure 2B). This approach creates a DSB only when both monomers are bound to the target DNA, limiting off-target activity as the nucleolytic reagent is assembled and active only at the target site [40.Gaj T. et al.ZFN, TALEN and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol. 2013; 31: 397-405Abstract Full Text Full Text PDF PubMed Scopus (2175) Google Scholar]. For both mtZFNs and mitoTALENs, sequence specificity is conveyed via protein–DNA interactions through tandemly linked repetitive modules, varying at key nucleic acid-contacting residues that are derived from either eukaryotic (ZFP) or bacterial (TALE) DNA-binding proteins. Two studies describing the use of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in mitochondria have been published in recent years [42.Jo A. et al.Efficient mitochondrial genome editing by CRISPR/Cas9.Biomed. Res. Int. 2015; 2015: 305716Crossref PubMed Scopus (104) Google Scholar,43.Bian W.-P. et al.Knock-in strategy for editing human and zebrafish mitochondrial DNA using mito-CRISPR/Cas9 System.ACS Synth. Biol. 2019; 8: 621-632Crossref PubMed Scopus (20) Google Scholar]. However, the major hurdle to this approach – RNA import of guide RNA-containing molecules into mitochondria, an essential component of this programmable nuclease system – is not adequately addressed by the authors and these reports are replete with unconvincing data [7.Gammage P.A. et al.Mitochondrial genome engineering: the revolution may not be CRISPR-ized.Trends Genet. 2018; 34: 101-110Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar]. Further, Cas9 endonucleases are subject to target site limitations in terms of the length of site that may be efficiently targeted (typically ~15–22 bp) and the proscribed protospacer-adjacent motif (PAM). PAM sites are 2–6 bp sequences that are essential for the introduction of a DSB into DNA, varying between the Cas9 species used. The limitations in target site preference presented by even the most permissive PAM-bearing Cas9 variants would render a mitochondrial CRISPR/Cas9 approach unable to target the majority of pathogenic mtDNA mutations. Therefore, even if an efficient mitochondrial CRISPR/Cas9 platform were to be created, it would be of limited use in the context of therapeutic heteroplasmy shifting (see Table 1 for a summary).Table 1Comparison of Engineered Mitochondrial NucleasesFeaturemtZFNmitoTALENCRISPR/Cas9MotifββαRVDCas9/gRNAInteractionProtein–DNAProtein–DNARNA–DNABinding1 ZFP:3 (+1) bp1 TALE:1 bp1 gRNA:18–22 bpSpecificity1 bp1 bpUnknownOff target mtDNAAt high expressionAt high expressionUnknown NuclearNot detectedUnknownUnknownAAV compatibleYesTwo capsids neededYesProsSmall, highly specific, several in-body clinical trialsEasy to designEasy to designConsDifficult to designLarge, limited target sitesMitochondrial RNA import, limited target sites Open table in a new tab Zinc-finger nucleic acid-binding motifs are among the most abundant protein folds in eukaryotic biology. The capacity for DNA sequence-specific binding of Cys2His2 ZFPs is mediated by repeating ~25-amino-acid (aa) ZFP modules, or fingers, that each bind a 3-bp sequence. This binding is coordinated 5′–3′ in a C-terminal to N-terminal orientation through hydrogen bonding in the major groove, with additional cross-strand contacts to the final nucleotide of the preceding triplet and nonspecific phosphate interactions [44.Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu. Rev. Biochem. 2010; 79: 213-231Crossref PubMed Scopus (461) Google Scholar] (Figure 2B,C). These zinc-finger domains, originally characterised in the murine transcription factor zif268, comprise antiparallel β-strands and a zinc ion-stabilised α-helix formed through the invariant cysteine and histidine residues for which the fold is named, arranged in a ββα configuration. DNA binding is conveyed by the α-helical residues 1, 2, 3, and 6 of the ZFP [44.Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu. Rev. Biochem. 2010; 79: 213-231Crossref PubMed Scopus (461) Google Scholar] (Figure 2C), which when arranged in tandem using short flexible linkers, form an engineered, sequence-specific DNA-binding ZFP. In the case of engineered ZFPs, typically four to six fingers (12–18 bp target site) are used per monomer, providing a total binding site of 24–36 bp. The rational construction of ZFPs from preselected libraries is challenging, as DNA binding of consecutive fingers is highly DNA/ZFP context dependent. As such, substantial libraries of candidate ZFPs and linkers were created using phage display [44.Klug A. The discovery of zinc fingers and their applications in gene regulation and genome manipulation.Annu. Rev. Biochem. 2010; 79: 213-231Crossref PubMed Scopus (461) Google Scholar], which have been further iterated to provide targeting depth that allows the generation of ZFPs to virtually any DNA sequence [45.Paschon D.E. et al.Diversifying the structure of zinc finger nucleases for high-precision genome editing.Nat. Commun. 2019; 10: 1133Crossref PubMed Scopus (33) Google Scholar]. The potential of zinc-finger-based approaches to mtDNA manipulation was first seen in the demonstration of site-specific methylase activity by a three-finger, monomeric, mitochondrially targeted ZFP specific to m.8993T>G, fused to DNA methyltransferase 3a [46.Minczuk M. et al.Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19689-19694Crossref PubMed Scopus (109) Google Scholar]. The use of a nuclear export signal (NES) was essential for mitochondrial localisation of ZFPs in these experiments, as cryptic internal nuclear localisation signals form an intrinsic part of the ZFP motif. This work was further built on with a rudimentary mtZFN design for heteroplasmy shifting, where two FokI domains separated by a long, flexible linker are attached to a single ZFP monomer specific to m.8993T>G [41.Minczuk M. et al.Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA.Nucleic Acids Res. 2008; 36: 3926-3938Crossref PubMed Scopus (128) Google Scholar]. This early mtZFN design was capable of shifting heteroplasmy in cybrid cells but was limited to a single ZFP binding site (four fingers, 12 bp) and was a constitutively active nuclease, leading to undesired mtDNA copy number depletions that limited further use. Early attempts at generating a dimeric mtZFN failed to produce efficacious rea