Genetic therapies for RNA mis-splicing diseases

Hammond, Suzan M.; Wood, Matthew J.A.

doi:10.1016/j.tig.2011.02.004

Cited by 147 publications

(121 citation statements)

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“…Here, we report that a highly active peptide, Pip6a, directly conjugated to a morpholino PMO permits highly efficient systemic delivery, which enhances bodywide SMN expression, including in brain and spinal cord; rescues the phenotype; and dramatically prolongs the life span of severe SMA mice. These data demonstrate powerful SMA disease modification by peptide-PMO therapy, a benefit that could be extended to many other neurodegenerative disorders (8,31,32). …”

mentioning

confidence: 75%

“…A rational, gene therapy-based approach for SMA uses singlestranded antisense splice-switching oligonucleotides (SSOs) to enhance SMN2 pre-mRNA exon 7 inclusion via steric block of splice regulatory pre-mRNA elements (8). Targeting the intron splice silencer N1 (ISS-N1) site within intron 7, by deletion or SSOmediated splice switching, improves exon 7 inclusion (9, 10).…”

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confidence: 99%

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Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

Hammond

Hazell

Shabanpoor

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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166

129

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The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA.spinal muscular atrophy | survival motor neuron | antisense oligonucleotide | splice switching oligonucleotide | cell-penetrating peptide S pinal muscular atrophy (SMA), a leading genetic cause of infant mortality primarily due to lower motor neuron degeneration and progressive muscle weakness, results from loss of the ubiquitous survival motor neuron 1 gene (SMN1) (1, 2). Humans have a second nearly identical copy, SMN2, that differs from SMN1 by a crucial nucleotide transition within exon 7 leading to the predominant generation of an alternative exon 7-excluded transcript and only marginally functional protein (3-7). SMN2 therefore fails to compensate for loss of SMN1 unless sufficient copies are present to generate functional levels of full-length SMN protein (2).A rational, gene therapy-based approach for SMA uses singlestranded antisense splice-switching oligonucleotides (SSOs) to enhance SMN2 pre-mRNA exon 7 inclusion via steric block of splice regulatory pre-mRNA elements (8). Targeting the intron splice silencer N1 (ISS-N1) site within intron 7, by deletion or SSOmediated splice switching, improves exon 7 inclusion (9, 10). ISS-N1-targeted SSOs used to treat presymptomatic severely affected neonatal SMA mice, via systemic or intracerebroventricular administration, extend survival from 10 to >100 d (11, 12). Although SSO targeting to the CNS is essential, there is also evidence for a peripheral role for the SMN in SMA (13-24).Although SSO therapy is currently at an advanced stage of de...

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confidence: 75%

mentioning

confidence: 99%

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

Hammond

Hazell

Shabanpoor

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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166

129

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“…Furthermore, promising approaches are being developed to rescue splicing defects, including 59ss mutations not affecting positions +1 or +2. Such technologies are based on antisense oligonucleotides or larger RNA molecules that can affect splicing (Hammond and Wood 2011). These few examples illustrate that a better understanding of the mechanisms of 59ss selection will likely improve the molecular diagnosis of 59ss mutations and facilitate therapeutics development.…”

Section: Implications For Genetic Diseasesmentioning

confidence: 99%

Pick one, but be quick: 5′ splice sites and the problems of too many choices

2013

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Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 59 Splice sites (59ss) are the critical elements at the 59 end of introns and are extremely diverse, as thousands of different sequences act as bona fide 59ss in the human transcriptome. Most 59ss are recognized by base-pairing with the 59 end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 59ss selection, highlighting the difficulties of establishing how basepairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:59ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 59ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 59ss might lead to selection of a single 59ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 59ss selection.Questions about the mechanisms by which 59 splice sites (59ss) are selected are deeply rooted in the history of research on pre-mRNA splicing. Identification of the sequences associated with 59ss triggered the first key insights into splicing mechanisms, efforts that are reflected now in the widespread use of genomic methods to quantify the contributions of other sequences and their cognate factors. The first factors shown to modulate alternative splicing affected 59ss selection, and the difficulties of working out the molecular mechanisms involved provided a foretaste of the complexities awaiting investigations into other regulatory proteins. Despite the many insights resulting from such studies over the years, it is clear that our conceptual frameworks are not yet adequate. New ideas and models are needed for studies on splice site selection. One purpose of this review is to emphasize that developing new ideas may involve first the challenge of uprooting commonsense but unsubstantiated preconceptions hidden in established models.The 59ss is involved in both steps of splicing. In the first step, the 29-hydroxyl group of the branchpoint adenosine attacks the phosphodiester bond at the 59ss and displaces the 59 exon; in the second step, the 39-hydroxyl group of the 59 exon attacks the phosphodiester bond at the 39 splice site (39ss) and displaces the lariat intron. Splicing was discovered just before new, gel-based DNA-sequencing methods transformed molecular biology. Thus, although the original discoveries were made without the benefit of sequence information (Berget et al. 1977;Chow et al. 1977), sequences emerged rapidly thereafter and revealed clear similarities among 59ss. Moreover, the ''consensus'' sequence (comprising, at each position, the nucleotide most commonly found there) was complementary to the sequence at the 59 end of U1 small nuclear RNA (snRNA), which immediately suggested a mechanism for recognition of t...

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“…Their role is to restrict the binding of regulatory splicing proteins that mediate exons inclusion into the mature mRNA by concealing exonic splicing enhancer motifs. 175 Despite the small internal deletion, the protein's functionality is unaltered. AAV9 administration in myosin-binding protein C 3 -knock in mice (MYBPC3-KI mice) produced a stable functional protein which temporarily prevented the disease phenotype.…”

Section: Gene Therapy For Hcmmentioning

confidence: 99%