New approaches to the treatment of orphan genetic disorders: Mitigating molecular pathologies using chemicals

VELHO, RENATA V.; SPERB-LUDWIG, FERNANDA; SCHWARTZ, IDA V.D.

doi:10.1590/0001-3765201520140711

RENATA V. VELHO

Gene Therapy Center, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, 90035-903 Porto Alegre, RS, Brasil

Post-Graduation Program on Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43323M, Caixa Postal 15053, 91501-970 Porto Alegre, RS, Brasil

* These authors contributed equally

FERNANDA SPERB-LUDWIG

Post-Graduation Program in Medical Science, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2400, 90035-903 Porto Alegre, RS, Brasil

BRAIN Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, 90035-903 Porto Alegre, RS, Brasil

* These authors contributed equally

IDA V.D. SCHWARTZ

Post-Graduation Program on Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43323M, Caixa Postal 15053, 91501-970 Porto Alegre, RS, Brasil

Post-Graduation Program in Medical Science, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2400, 90035-903 Porto Alegre, RS, Brasil

BRAIN Laboratory, Experimental Research Center, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, 90035-903 Porto Alegre, RS, Brasil

Department of Genetics and Molecular Biology, Universidade Federal do Rio Grande do Sul, Avenida Bento Gonçalves, 9500, Prédio 43433, Sala 207, 91501-970 Porto Alegre, RS, Brasil

Correspondence to: Ida Vanessa Doederlein Schwartz E-mail: ischwartz@hcpa.edu.br

*
These authors contributed equally

Figures (3)
Tables (1)

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Figure 1
- Mutation-targeted therapies reviewed in this paper.

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Figure 2
- Schematic diagram of gene regulation and readthrough strategy. The relative positions of caps, polyadenylation sequences, and initiation and termination codons within mRNAs are shown. (a) In wild type cells, normal mRNAs encode complete proteins. (b) In mutant cells, the mRNAs contain a premature stop codon, and truncated proteins are translated in the absence of aminoglycoside or the transcript is degraded by the NMD pathway. The presence of aminoglycoside allows the incorporation of a random amino acid at the terminal stop codon of the mutant mRNA. Full-length proteins result from the aminoglycoside-induced readthrough.

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Figure 3
- Antisense oligonucleotides (AONs) therapies are used to correct pre-mRNA splicing. (a) Exon exclusion: Large-scale deletions can result in out-of-frame mRNA transcripts. AONs directed toward an exon splice enhancer element within the mutated exon result in exclusion of the exon and, thus, an alternative in-frame transcript. (b) Exon inclusion: A mRNA transcript can lose one exon owing to negative splicing regulatory elements within and surrounding this exon and produce a less functional protein. Treatment with AON directed toward an intron splice silencer (ISS) can increase the inclusion of the exon from 20% to approximately 95%, as seen in the SMN2 gene (Hua et al. 2010HUA Y, SAHASHI K, HUNG G, RIGO F, PASSINI MA, BENNETT CF AND KRAINER AR. 2010. Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model. Genes Dev 24: 1634-1644.). (c) Cryptic splice sites: This can result in inclusion of introns or partial deletions of exons from mRNA. The hypothetical intronic mutation illustrated creates an intronic 5' splice site (ss) and activates a cryptic 3' ss within intron 2. AONs targeting the mutated intron 5' ss blocks recognition by the splicing machinery and generates transcripts without the aberrant intron element. Similarly, a mutation within exon 2 creates a cryptic 5' ss preferentially utilized over the natural splice site. AONs directed to the mutant cryptic site cause steric blockage of the splicing machinery and redirect splicing to the natural 5' ss. (d) Pseudoexon: A C-A mutation within intron 1 strengthened the 50ss of a pseudoexon (dashed box), which maintains a strong 3' ss. AONs directed to the pseudoexon 5' ss and inclusion of C-A mutation restored the natural transcript (Adapted from Hammond and Wood 2011HAMMOND SM AND WOOD MJ. 2011. Genetic therapies for RNA mis-splicing diseases. Trends Genet 27: 196-205.).

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TABLE I
Examples of mutation-targeted therapies.

Therapy	Target*	Diseases tested	References
Aminoglycosides	Stop codon mutations	cystic fibrosis (CF), Duchene muscular dystrophy (DMD), Hurler’s syndrome (MPS I), and diabetes, among others	Diop et al. 2007, Nudelman et al. 2009, Goldmann et al. 2010
Oligonucleotides (peptide nucleic acids – PNAs; alternating locked nucleic acids - LNAs and; phosphoradiamidate morpholino-based oligomers - PMOs)	Splicing mutations	CF, β-thalassemia, Hutchinson-Gilford progeria syndrome, DMD and spinal muscular atrophy	Friedman et al. 1999, Suwanmanee et al. 2002, Scaffidi and Misteli 2005, van Deutekom et al. 2007, Kinali et al. 2009, Cirak et al. 2011, Hua et al. 2010, Nlend Nlend et al. 2010, Taylor et al. 1999, Giles et al. 1999
Single-stranded oligonucleotides (ssODNs)	Missense mutations	–	Falgowski et al. 2011, Bertoni et al. 2009
Chimeric RNA/DNA oligonucleotides (RDOs)	Missense mutations	–	Sargent et al. 2011
Small DNA fragments (SDFs)	Missense mutations	–
Triplex-forming oligonucleotides (TFOs)	Missense mutations	–	Falgowski et al. 2011, Bertoni et al. 2009
Adeno-associated virus vectors (AAVs)	Missense mutations	CF, Pompe disease, α-1 antitrypsin deficiency, muscular dystrophy, Batten’s disease, Parkinson’s disease, Leber’s congenital amaurosis, hemophilia, choroideremia, Lipoprotein Lipase Deficiency	Mittermeyer et al. 2012, Zhong et al. 2012, Smith et al. 2013, MacLaren et al. 2014, Mendell et al. 2015, Mingozzi and High 2011
Zinc-finger nucleases (ZFNs)	Missense and Frameshift mutations	X-linked severe combined immune deficiency (SCID)	Granja et al. 2014, Aarts and Riele 2011, Urnov et al. 2005, Rahman et al. 2011
Single-stranded DNA (ssDNA)	Frameshift mutations	–	Morita et al. 2011
Cell therapy combined with gene therapy	Frameshift mutations	β-thalassemia	Chang et al. 2006
iPS cells	Frameshift mutations	sickle cell anemia and β-thalassemia	Hanna et al. 2007, Ye et al. 2009

[1] Correspondence to: Ida Vanessa Doederlein Schwartz E-mail: ischwartz@hcpa.edu.br