U7 snRNA,a Small RNA with a Big Impact in Gene Therapy

Abstract

The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3′ end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.

Introduction

Many human diseases are caused by mutations in the genomic sequence that alter RNA splicing, impairing the production of a functional protein. The ability to target aberrant RNA provides an opportunity to modulate splicing and potentially treat numerous genetic disorders. Early strategies for modulating splicing focused on the use of antisense oligonucleotides (AONs) to interfere with important splice sites or exon regulatory elements.^1

–6 The effectiveness of AONs in altering splicing patterns was first demonstrated by Dominski and Kole.⁷ They showed that AONs targeted against β-globin premessenger RNA (pre-mRNA) could correct the aberrant splicing patterns which cause β-thalassemia. Since this landmark publication, AONs for splicing modulation have been studied and used to treat patients with several genetic disorders.

AONs efficiently compete with the spliceosome for recognition of splicing motifs, which often results in exclusion of the targeted exon from the mRNA. However, the effects of AONs are transient due to degradation by endo and exonucleases, meaning that therapies would require regular life-long readministration.^7,8 As an alternative to standard AONs, derivatives of uridine-rich 7 (U7) small nuclear RNA (snRNA) containing optimized Sm protein-binding sites and a modified antisense sequence (termed U7 Sm OPT snRNA) has proved to be promising modulators of pre-mRNA splicing. Expression of U7 Sm OPT snRNA allows for the continuous nuclear accumulation of therapeutic antisense sequences embedded in a stable small nuclear ribonucleoprotein (snRNP) complex.^8,9 This long-term expression of antisense prevents or significantly reduces the need for readministration of the therapy.

Delivery of U7 Sm OPT snRNA via a viral vector such as adeno-associated virus (AAV) enables the selective inclusion or exclusion of a targeted exon in tissues of interest. This technology is also referred to as vectorized exon skipping/inclusion (VES/VEI) and can be used to ameliorate the effects of genetic errors. Although VES/VEI has been explored in many animal models across several diseases, in 2020, this approach has finally entered its first clinical trial using systemic AAV delivery in patients with Duchenne muscular dystrophy (DMD; NCT04240314), one of the most common and severe neuromuscular disorders. This review covers the natural function of U7 snRNA in histone pre-mRNA processing, its development as a splicing modulation tool, and current advancements in VES/VEI, underlining its promise as therapeutic tool for a broad range of diseases.

U7 snRNA Transcription and Assembly

U snRNAs are a family of RNAs with diverse and complex functions. The best-known U snRNAs are those of the spliceosome, which are involved in removing introns from pre-mRNAs during transcription (U1, U2, U4, U5, and U6). The U7 snRNA was first discovered in sea urchins, where it is crucial for histone pre-mRNA 3′ end processing.^10,11 Later, U7 snRNA was identified to have the same role in mice and humans.^12

–15 It is now known that U7 snRNA exists in nearly all animals.^16,17 Like almost all other U snRNAs, the U7 snRNA has its own promoter and is transcribed by RNA polymerase II (pol II). The U7 promoter contains U snRNA-specific elements not found in other genes transcribed by pol II that are essential for its transcription: a distal sequence element responsible for the enhancement of transcription and a proximal sequence element important for specifying the site of transcription initiation.^18,19

U snRNAs do not contain a polyadenylation signal and their 3′ untranslated region is processed through cleavage only.^20
–22 Efficient formation of the 3′ ends of the pre-snRNA transcript requires a 3′ end signal called the 3′ box.^20,22 This signal is only recognized if transcription is initiated from a U snRNA promoter, and replacement with a noncompatible pol II snRNA promoter results in inefficient 3′ end formation.^22,23

Maturation of U snRNAs begins in the nucleus, continues in the cytoplasm, and completes in the nucleus following nuclear reentry. In the nucleus, the U snRNA transcript initially acquires a standard m7GpppG 5′ cap structure for export to the cytoplasm. In the cytoplasm, U7 snRNA associates with five Sm proteins (B/B′, D3, E, F, and G) and two U7-specific Sm-like proteins (Lsm10 and Lsm11), which are important for histone pre-mRNA processing.²⁴ In contrast, the spliceosomal snRNAs associate with seven Sm proteins (B/B′, D1, D2, D3, E, F, and G).

Assembly of the Sm core stabilizes and protects the snRNA from nuclease degradation and is required for downstream processing of the snRNA. Following heptamer formation, the snRNA 3′ end is trimmed, and the cap structure is hypermethylated to a 2,2,7-trimethyl guanosine (3mG) cap, which functions as a nuclear localization signal.²⁵ Once in the nucleus, the snRNP reenters cajal bodies for further RNA modification and maturation.^26,27 After maturation, the U7 snRNP localizes to nuclear structures involved in the assembly of the machinery needed for histone pre-mRNA processing (reviewed in Nizami et al. ²⁸).

U7 snRNP Structure and Function

The U7 snRNP involved in histone RNA 3′ end processing has three major components: (1) a histone downstream element (HDE)-binding sequence, (2) a Sm/Sm-like binding site, and (3) a 3′ stem-loop that stabilizes the RNA¹⁰ (Fig. 1A). U7 snRNP functions by recognizing histone pre-mRNAs through base pairing to the HDE that follows the histone pre-mRNA 3′ processing site.¹² Once bound, U7 recruits the appropriate cleavage factors essential for histone pre-mRNA processing.^11,29,30 This process relies on both Lsm11 and Lsm10 (Fig. 1A). If these two proteins are exchanged with other Sm proteins, the resulting U7 snRNP molecule can still bind to histone pre-mRNAs, but is unable to process their 3′ end.³¹

Figure 1.

Structure of wild-type U7 snRNP and processing of histone pre-mRNA 3′ end processing. (A) Mechanism and additional factors involved in the 3′ end processing reaction. The SLBP binds to the stem loop located near the 3′ end of histone pre-mRNA and alters the histone pre-mRNA structure allowing U7 snRNP to bind to the HDE sequence (via recruitment of the Lsm11- FLASH protein complex). Once bound, ZFP100 helps to coordinate and stabilize the cleavage complex (composed of endonucleases 64 kDa) subunit of the cleavage stimulation factor [CstF64]), 73-kDa and 100-kDa subunit of the cleavage and polyadenylation specificity factors (CPSF73 and CPSF100), and Symplekin on histone pre-mRNA. (B) U7 snRNP contains three major components: (i) a HDE binding sequence (Salmon rectangle), (ii) a Sm/Sm-like binding site that binds five Sm proteins (dark green circles) and two Lsm proteins (Lsm10 and Lsm11; light green circles), and (iii) a 3′ stem-loop that stabilizes the RNA (gray loop). Modification of the wild-type U7 Sm-binding site with the consensus sequence derived from the binding site of spliceosomal snRNAs (D1 and D2 instead of Lsm 10 and Lsm11; dark green spheres; referred as U7 sm OPT) allows modified U7 snRNA to assemble with the seven Sm proteins found in the spliceosomal snRNAs. FLASH, FLICE-associated huge; HDE, histone downstream element; mRNA, messenger RNA; SLBP, stem-loop binding protein; snRNP, small nuclear ribonucleoprotein; U7, uridine-rich 7; ZFP100, 100 kDa zinc finger protein.

U7 Sm OPT snRNA as a Splicing Modulation Tool

The ability to target aberrant pre-mRNA splicing could provide treatments for a variety of genetic disorders caused by alteration of typical splicing patterns. The spliceosome carries out pre-mRNA splicing by recognizing important conserved sequence elements (also referred to as exon definition elements) located at intron-exon junctions: the 5′ donor (GT), the 3′ acceptor (AG) splice sites, and the branch point sequence near the 3′ splice site of the intron (Fig. 2A).

Figure 2.

The classical spliceosome machinery and its interaction with U7 sm OPT. (A) The spliceosome is composed of five major U snRNPs: U1, U2, U4/U6, and U5. This complex recognizes among other factors, the 5′ (GU) and 3′ splice sites (AG) and the branch point (A+(Y)_n). Early in splicing, base pairing between U1 snRNP with the 5′ splice site occurs. Subsequently, non-snRNP-associated factors such as SF1 recognize the branch point sequence and U2AFs recognize the polypyrimidine tract. Interaction between U2 snRNP and U1 snRNP brings the 5′ splice site, branch point adenosine, and 3′ splice site into proximity. Several steps follow (not shown) that result in the joining of exons/spliced mRNA and the release of intron lariats. (B) Introduction of a U7 sm OPT with an antisense sequence complementary to the 3′ acceptor splice site competes with the SF1/U2AF for recognition resulting in the skipping of exon 2. (C) Additional regulatory elements promote exon recognition and selection of splice sites. These can be found within exons: ESEs and ESSs; and within introns: ISEs and ISSs. The ESE and ESS sequences function as binding sites for regulatory factors such as the SR protein family, which promote the association of the snRNP subunits of the splicing machinery to adjacent splice sites. In contrast, ISE and ISS sequences bind factors from the hnRNP family and prevent the association of the snRNP subunits to nearby splice sites. Depicted is a U7 snRNP modified to bind an ESE, competing with SR. The displayed U7 snRNP promotes the exclusion of exon 2. ESE, exonic splicing enhancer; ESS, exonic splicing silencer; hnRNP, heterogeneous nuclear ribonucleoprotein; ISE, intronic splicing enhancer; ISS, intronic splicing silencer; SF1, splicing factor 1; SR, serine- and arginine-rich; U2AF, U2 auxiliary factor.

In addition, the spliceosome's ability to recognize a specific exon depends on the exon's own definition sequences and the relative strength of surrounding sequences/elements known as splicing regulatory elements (SREs). SREs promote exon recognition and selection of splice sites (reviewed in Cartegni et al. ³²). Binding of regulatory proteins and the added effects of positive and negative elements determines whether the spliceosome will recognize and include/exclude an exon (reviewed in Cartegni et al. ³², Matera and Wang³³, Smith and Valcárcel³⁴). Positive elements are exonic splicing enhancers (ESEs) and intronic splicing enhancers (ISEs). Negative elements are exonic splicing silencers (ESSs) and intronic splicing silencers (ISSs) (Fig. 2C).

Replacement of the wild-type U7 Sm-binding sequence with the consensus Sm-binding sequence found in the major snRNAs creates the U7 Sm OPT snRNA, which can associate with all the Sm proteins of spliceosomal snRNAs.³¹ The resulting modified snRNP is more efficient at accumulating in the nucleus than its wild-type counterpart and is no longer able to induce cleavage of histone pre-mRNAs.^8,9,31,35 Further, by replacing the intrinsic HDE-binding sequence of the U7snRNA with a custom antisense sequence (Figs. 1B and 2B), the splicing pattern of a target pre-mRNA can be modified.

U7 Sm OPT snRNAs usually contain one antisense sequence and thus are termed “single-target” constructs (Fig. 3A). However, improved efficiency can be obtained with the “double-target” U7 Sm OPT snRNA constructs, which carry two tandem antisense sequences targeting two different splice sites (3′ acceptor splice site and 5′ donor splice site)³⁶ (Fig. 3B). Double-target constructs induce removal of the targeted exon (exon skipping). Additionally, a 5′ or 3′ tail can be added to the antisense sequence to form a “bifunctional” U7 Sm OPT snRNA (Fig. 3C). Such tails carry established ESE, ESS, ISE, or ISS sequences to provide binding sites for splicing enhancer or silencer factors to improve the likelihood of the desired splicing event occurring (for reviews see Meyer and Schümperli³⁷, Benchaouir and Goyenvalle³⁸). Taken together, the ability to exchange sequence elements as desired makes U7 Sm OPT snRNAs a stable, antisense-carrying molecule that can be used as a therapeutic for a variety of human diseases. Importantly, the small size of U7 expression cassettes (U7 promoter and U7 Sm OPT snRNA sequence) allows the use of AAV vectors for delivery to target cells or organs. Using this system, the U7 snRNA will be continuously expressed in the nucleus of transduced cells and does not have to be readministered for an extended period.

Figure 3.

Modified U7 snRNA as a splicing modulator and its derivatives. (A) Additional replacement of U7's HDE-binding sequence with one complementary to a specific target (e.g., one that interferes with splice sites or regulatory elements within the exon or intron of the target pre-mRNA; yellow rectangle) renders it capable of modulating splicing (referred as “single-target construct”). (B) “Double-target constructs” contain two target sequences, one complementary to the 5′ splice site (purple rectangle), and the other complementary to the 3′ splice site (yellow rectangle) of the exon of interest. (C) “Bifunctional targets” contain complementary sequences targeting a specific sequence (yellow rectangle) in combination with an extra “tail” sequence (gray rectangle) containing binding sites for transacting regulatory factors (ESE or ESS) that, respectively, either promote or prevent exon inclusion/exclusion.

Other modified U snRNAs have also been explored to mediate exon skipping/inclusion.^39
–41 Technically, the antisense sequence can be incorporated into any of the U snRNAs. However, this may lead to competition with the wild-type counterparts, thus interfering with natural function. Therefore, the major U snRNAs may be less suited for therapeutic modification. Since the Sm-binding site of the U7 Sm OPT snRNA has been modified to eliminate U7 snRNA's natural function, and due to the lower expression levels of the U7 snRNA compared to the major spliceosomal U snRNAs, U7 snRNA has a more appealing safety profile than other U snRNAs.

Among the other U snRNAs, the one that has been most tested for splicing modulation is U1 snRNA due to its higher levels of expression; for example, U1 has a sixfold higher natural expression than wild-type U7.³¹ Despite this, modified U1 constructs are often less efficient than U7 Sm OPT snRNAs, and replacing the U7 promoter with the U1 does not significantly increase the levels of snRNA or splicing.^39,40 Modified U6 snRNA constructs have also been used; however, they are not as efficient possibly because U6 snRNA alone is unable to access splice sites as it relies on U4 snRNA for snRNP formation.³⁹ To more efficiently reach target cells, these modified U snRNAs tools have been delivered using both lentiviral^42,43 and AAV vectors⁴⁴ (reviewed in Benchaouir and Goyenvalle³⁸).

Use of the U7 snRNA Sm OPT Antisense System to Treat Human Disease

As demonstrated in Table 1, U7 snRNAs have been used in several disorders.

Table 1.

Overview of uridine-rich 7 small nuclear RNA studies to treat various diseases ^a

Disease	U7 approach	Targeted region	In vitro	Skipping/inclusion %	Protein %	In vivo	Skipping/inclusion %	Protein %	Authors	PMID
Exon skipping
DMD	Exon skipping	DMD exon 23 skipping	C2C12 and mdx myoblasts	NA	NA	NA	NA	NA	Brun et al. (2003)⁴⁹	12737315
DMD	Exon skipping	DMD exon 23 skipping	NA	NA	NA	mdx mice	NA	50–80%	Goyenvalle et al. (2004)⁴⁴	15528407
DMD	Exon skipping+ α sarcoglycan expression	DMD exon 23 skipping	NA	NA	NA	mdx and Sgca -null mice	NA	NA	Bartoli et al. (2006)⁵⁰	16107863
DMD	Exon skipping	DMD exon 51 skipping	T antigen immortalized DMD myoblasts	30%	NA	SCID mice	NA	NA	Quenneville et al. (2007)⁵¹	17235323
DMD	Exon skipping	DMD exon 51 skipping	DMD myoblasts	NA	Near normal	hDMD mice	NA	NA	Goyenvalle et al. (2009)⁵²	19455105
DMD	Exon skipping	DMD exon 23 skipping	NA	NA	NA	mdx mice (brain injection)	NA	5–60% (SR of hippocampus)	Vaillend et al. (2010)⁵³	20588257
Dysferlinopathy	Exon skipping	Dysf exon 32 skipping	Patient derived fibromyoblast	NA	NA	NA	NA	NA	Wein et al. (2010)⁵⁴	19953532
DMD	Exon skipping	Multi exon skipping; DMD exons 45–55	DMD primary myoblasts and normal myoblasts	NA	Near normal	hDMD mice	18–95%	NA	Goyenvalle et al. (2012)⁵⁵	22354379
DMD	Exon skipping	DMD exons 6–8	NA	NA	NA	GRMD dogs	NA	15–20% of normal canine	Bish et al. (2012)⁵⁶	22146342
DMD	Exon skipping	DMD exons 6–8	NA	NA	NA	GRMD dogs	NA	16–80%	Vulin (2012)⁵⁷	22968479
DMD	Exon skipping	DMD exons 6–8	NA	NA	NA	GRMD dogs	NA	NA	Barbash et al. (2013)⁵⁸	22551778
Factor V deficiency	Exon skipping	Factor V cryptic donor splice site inactivation	COS-1, HepG2 cells and patient megakaryocytes	1–2 orders of magnitude	NA	NA	NA	NA	Nuzzo et al. (2013)⁵⁹	24085767
Central core disease	Exon skipping	RyR1 pseudo-exon skipping	Primary muscle cells	80%	1.5-fold increase	NA	NA	NA	Rendu et al. (2013)⁶⁰	23805838
DMD	Exon skipping	DMD exons 6–8	NA	NA	NA	GRMD dogs	NA	18–76%	Le Guiner et al. (2014)⁶¹	25200009
Lowe syndrome	Exon skipping	OCRL 1 pseudo-exon skipping	Primary patient fibroblasts	79%	30%	NA	NA	NA	Rendu et al. (2017)⁶²	27790796
Dysferlinopathy	Exon skipping	Dysf exons 37–38 skipping	C2C12	12.90%	NA	MMex38 mice	6.60%	20%	Malcher et al. (2018)⁶³	30292141
Out of frame exon skipping
DMD	Exon skipping	DMD exon 2 skipping and IRES activation	Patient derived fibromyoblast	88.60%	NA	dup2 mice	Near complete	18.7–43.2%	Wein et al. (2014)⁴⁸	25108525
ALS	Exon skipping	SOD1 mutant exon 2 skipping to generate PTC	HEK293T	40–80%	NA	OD1G93A mice	<80%	70%	Biferi et al. (2017)⁶⁴	28663100
Exon inclusion/intron skipping
β-thalassemia	Exon inclusion	β-globin gene skipping of aberrant and cryptic splicing sites	HeLa cells	65%	NA	NA	NA	NA	Gorman et al. (1998)⁸	9560205
β-thalassemia	Exon inclusion	β-globin gene skipping of aberrant and cryptic splicing sites	HeLa cells	25–95%	NA	NA	NA	NA	Suter et al. (1999)³⁶	10556289
Cancers and congenital adrenal insufficiency	Intron skipping/Exon inclusion	PTCH1, BRCA1 and CYP11 mutations skipping	HeLa cells	NA	NA	NA	NA	NA	Uchikawa et al. (2007)⁶⁵	17851636
SMA	Exon inclusion	SMN2 exon 7 inclusion	HeLa cells and patient primary cells from fibroblasts	up to 97%	2–2.7 fold	NA	NA	NA	Marquis et al. (2007)⁶⁶	17505471
SMA	Exon inclusion	SMN2 exon 7 inclusion	NA	NA	NA	SMA mice	26–52%	NA	Meyer et al. (2009)⁴³	19010792
SMA	Exon inclusion	SMN2 exon 7 inclusion	NA	NA	NA	SMN▵7 mice	NA	NA	Odermatt et al. (2016)⁶⁷	27456062
Pompe disease	Exon inclusion	GAA exon 2 inclusion	Primary patient fibroblasts	NA	NA	NA	NA	NA	van der Wal et al. (2017)⁶⁸	28624228
Nasu-Hakola disease	Exon inclusion (U7 and U1)	TREM 2 exon 3 inclusion	HEK293 cells and THP 1 cells	NA	NA	NA	NA	NA	Yanaizu et al. (2018)⁶⁹	29720600
β-thalassemia	Exon inclusion	βE-globin gene skipping of aberrant splicing sites	Patient erythroid cells; HeLa cells	90–100%	NA	NA	NA	NA	Preedagasamzin et al. (2018)⁷⁰	29550480
β-thalassemia	Intron skipping	β-globin skipping of aberrant splicing site and BP	Patient erythroid cells; HeLa cells	up to 75%	NA	NA	NA	NA	Nualkaew et al. (2019)⁷¹	31113996
Nonexon skipping transcript silencing
DM1	UTR repeats skipping	DMPK 3′ UTR removal of expanded CUG repeats	DM1 primary myoblasts	NA	NA	NA	NA	NA	François et al. (2011)⁷²	21186365

Nonexhaustive list.

ALS, amyotrophic lateral sclerosis; BP, branching point; DM1, myotonic dystrophy type 1; DMD, Duchenne muscular dystrophy; DMPK, dystrophia myotonica protein kinase; dup2, duplication of exon 2 of DMD; dysf, dysferlin; GAA, acid α-glucosidase; GRMD, golden retriever muscular dystrophy; hDMD, humanized DMD; ICV, intracebroventricular; IRES, internal ribosome entry site; IV, intravenous; LGMD 2D, limb girdle muscular dystrophy α sarcoglycan; mdx, muscular dystrophy x linked; NA, not available; OCRL 1, inositol polyphosphate 5-phosphatase OCRL gene; PTC, premature termination codon; RyR1, type 1 ryanodine receptor; SMA, spinal muscular atrophy; SMN2, survival of motoneurons 2; SOD1, Cu/Zn superoxide dismutase 1; SR, stratum radiatum; TREM 2, triggering receptor expressed on myeloid cells 2; U7, uridine-rich 7; UTR, untranslated region.

β-thalassemia

Gorman et al. were the first to prove that U7 snRNA's natural function of complementary base-pairing to histone pre-mRNA could be reprogrammed to function in an AON-like manner.⁸ They used U7 Sm OPT snRNA constructs to promote exon exclusion in β-thalassemia. β-thalassemia occurs when a point mutation in the β-globin gene creates a novel 5′ splice site and activates a cryptic 3′ splice site in intron 2. This results in the inclusion of an aberrant “exon” (an intron fragment containing an in-frame stop codon) in the β-globin mRNA and consequently leads to a loss of β-globin protein expression. Different U7 Sm OPT snRNAs containing single-target sequences complementary to aberrant 5′ or 3′ splice sites were able to induce exon skipping. This led to the restoration of correct β-globin mRNA splicing and the corresponding protein in patient-derived cell lines.⁸

Human immunodeficiency virus 1

Acquired immunodeficiency syndrome (AIDS) is a life-threatening condition caused by infection with the human immunodeficiency virus 1 (HIV-1). HIV works by damaging the immune system, leaving the body defenseless against organisms that cause disease. The efficient replication of HIV-1 virus relies on the integration of protein cyclophilin A (CyPA) into HIV-1 virions.⁴² A double-target U7 Sm OPT snRNA system was used by Liu et al. to target the 3′ and 5′ splice sites of CyPA exons 3 or 4 to promote their exclusion in the final transcript of CyPA.⁴² Exclusion of these exons would result in a truncated protein unable to integrate into viral particles; hence delaying HIV-1 infection.

Efficient skipping of these exons and a reduction in CyPA protein was observed after lentiviral transduction of a human T cell line alongside delayed HIV-1 replication.⁴² While this approach showed high rates of reduction of HIV-1 expression, it was significantly less efficient than small interfering RNA (siRNA) targeting the same regions, although there are still some questions regarding the clinical applications of siRNA given their reliance on the saturable RNA-induced silencing complex and Drosha complexes.^45
–47 Moreover, siRNAs and miRNAs can only reduce mRNA or protein levels produced and are not suited for changing of mRNA structure (exon inclusion/exclusion). Thus, using a modified U7 snRNA to induce gene knockdown by generating an out-of-frame transcript may prove superior as it has never been shown to saturate any cellular processes. To increase efficacy of the U7 snRNA approach, several U7 Sm OPT snRNA cassettes can be added into the same viral vector.⁴⁸

Spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder that affects 1:6,000–10,000 patients and causes progressive muscle loss and premature death (reviewed in Kolb and Kissel⁷³). SMA is caused by homozygous mutations in the survival motor neuron 1 (SMN1) gene leading to a loss of expression of the SMN1 protein. In addition to SMN1, another similar gene (99% homology) named survival motor neuron 2 (SMN2) exists. This gene encodes the SMN2 protein, which in theory can compensate for the lack of SMN1. However, SMN2 contains a single-nucleotide polymorphism (C > T transition) in exon 7 compared to SMN1. This nucleotide variation disrupts an ESE site and creates an ESS site. In addition, exon 7 contains an inhibitory terminal stem loop and ISSs upstream and downstream of its vicinity, which causes it to be poorly recognized by the spliceosome machinery. Furthermore, SMN2 contains a weak 3′ splice site that competes with the strong 3′ splice site found in exon 8 for recognition by the spliceosome (reviewed in Nlend Nlend et al. ⁷⁴). Altogether, this leads to an aberrantly spliced transcript that lacks exon 7.

Complete absence of the SMN protein is embryonic lethal and all SMA patients produce reduced levels of the protein from the SMN2 gene. The number of SMN2 genes and other factors influencing the efficiency of correct SMN2 mRNA splicing affect disease severity. Hence, increasing the correct splicing of the SMN2 pre-mRNA is an attractive therapeutic approach for SMA patients. Among the drugs investigated, Nusinersen, an AON, was the first approved drug used in treating SMA.^75
–77

Single-target U7 Sm OPT snRNA constructs with complementary sequences to the strong 3′ splice site found in exon 8 allowed the spliceosome to recognize the weak 3′ splice site found in exon 7, leading to its incorporation into the final mRNA and producing a full length SMN2 protein in vitro.⁷⁸ Alternatively, another group has used bifunctional U7 Sm OPT targeting the 3′ end of exon 7 with an ESE tail that attracts splicing enhancer proteins to force exon 7 inclusion. This strategy allowed generation of 90% full-length SMN2 transcript in a HeLa cell minigene screening system and in fibroblasts from SMA patients, leading to a 50% increase in SMN protein.⁷⁹

Treatment of a mouse model of SMA using this construct demonstrated extended lifespan, improved muscle performance, and extended motoneuron survival. When compared in the same mouse model to the gene replacement strategy, in which the SMN coding sequence is delivered via AAV, the splicing correction approach was slightly less efficacious.⁸⁰ However, this result may be attributable to the use of a strong ubiquitous promoter for the expression of the SMN cDNA. Due to their varied mechanisms of action, a combination of the two approaches might warrant further investigation and could lead to improved patient outcomes. The transgene replacement gene therapy (Zolgensma) was recently approved by the US Food and Drug Administration (FDA), European Medicines Agency (EMA), and in Japan for treatment of SMA patients.⁴³

Duchenne muscular dystrophy

The potential of the U7 snRNA system has been best demonstrated in DMD (reviewed in Benchaouir and Goyenvalle³⁸), the most common form of muscular dystrophy. DMD is a lethal, X-linked, genetic disorder that affects roughly 1:5,000 boys born every year.^81,82 DMD is characterized by a loss of ambulation before 12 years of age. DMD can be caused by a wide variety of mutations in the DMD gene, which usually lead to an absence of the dystrophin protein. Exon skipping approaches for DMD seek to restore the DMD reading frame. Skipping of exons surrounding a deleteriously removed exon or direct skipping of an exon containing a nonsense mutation results in a partially functional but truncated dystrophin protein. Such proteins are characteristic of Becker muscular dystrophy (BMD), in which patients have a significantly milder phenotype (loss of ambulation past the age of 12 and increased life expectancy). In essence, exon skipping can convert a DMD patient's phenotype to that of BMD.

Exon skipping strategies in DMD are well-documented and AONs targeting various exons of DMD have been in preclinical and clinical trials (for reviews see Refs.^83

–88). However, as mentioned previously, a major challenge posed by AON-mediated exon skipping is the requirement to continuous delivery of the compound for long-term protein restoration, as such compounds get degraded by endogenous nucleases (for reviews see Refs.^89
–91). In addition, while such AONs can enter many muscles, their penetration in the heart and diaphragm is inefficient.^92
–94

To overcome these limitations, several groups have taken advantage of VES (reviewed in Benchaouir and Goyenvalle³⁸) to induce exon skipping and long-term dystrophin expression. They used a wide range of animal models of DMD such as the mdx mouse (nonsense mutation in exon 23 of DMD); the Dup2 mouse (out-of-frame duplication of DMD exon 2), and the golden retriever muscular dystrophy (GRMD) dog (out-of-frame natural skipping of DMD exon 7). In all these models, as described below, U7 snRNA demonstrated efficient exon skipping, long-term dystrophin expression, and functional muscle improvement.

One of the first in vivo proof of concepts of VES was performed by Goyenvalle et al., for DMD. This group demonstrated that VES induced persistent exon skipping and restored dystrophin expression in the mdx mouse.⁴⁴ Specifically, they used a double-target U7 Sm OPT snRNA targeting both the DMD exon 22 branch point and the DMD exon 23 splicing donor site. This engineered U7 Sm OPT snRNA was inserted into an AAV1 viral vector and injected into the tibialis anterior and extensor digitorum longus of the mice. Four weeks postinjection, dystrophin was being expressed in muscles along with improvements in muscle force generation.⁴⁴ Other studies using exon 51 cell lines showed that bifunctional U7 Sm OPT snRNAs carrying an antisense targeting exon 51 and a splicing silencer sequence induce complete skipping of exon 51 in vitro and allow restoration of near wild-type levels of dystrophin in DMD patient cells.⁵²

As all these studies were conducted in vitro or in mice, a looming question was whether VES would retain its functionality in larger animals. This was answered in a series of studies, in which VES proved both effective and safe in GRMD. This model carries a natural point mutation in intron 6 that leads to an out-of-frame deletion of exon 7. Skipping of exon 6 and 8 can restore the reading frame of the mRNA and leads to expression of a slightly truncated form of the protein. Thus, a U7 Sm OPT snRNA carrying two antisense sequences (U7 Sm OPT snRNA6/8) was used for these studies.⁵⁶ Percutaneous transendocardial injection of rAAV6–U7 snRNA6/8 resulted in corrected transcript and cardiac dystrophin expression measured up to 13 months postinjection in GRMD dogs.⁵⁶

Another group validated the longevity of these VES results using an rAAV1–U7 snRNA6/8 and measured protein expression up to 5 years postinjection.⁹⁵ Beyond establishing the efficacy of VES in a larger animal model, this cohort study concluded that VES was safe and the restored dystrophin did not induce an immune response, a promising sign for VES. While initial rates of dystrophin expression and exon skipping were high, a large decline in the treatment's effects over the course of the 5 years was observed, with an especially steep decline over years 1 and 2. These data were challenged by a later large-cohort study conducted in 18 GRMD dogs injected intramuscularly with VES.⁶¹ Although this study did not look 1-year postinjection, they did not observe lower rates of dystrophin restoration in their 3.5- and 7-month follow-ups. They hypothesized that this difference could be attributed to their earlier injection age of GRMD dogs (3–4 months vs. 5–6 months).⁶¹

Earlier injection may provide earlier dystrophin restoration, preventing muscle degradation and resulting in long-lasting effects by preventing leakage of AAV episomes encoding the therapeutic U7 snRNA.⁹⁶ Based on these data, it will likely be beneficial for patients to receive therapy as early as possible, supporting the effort to include DMD in new-born screening panels.

Often overlooked is the role that dystrophin plays in nonmuscle tissues such as the brain. While the molecular mechanisms remain poorly understood, many DMD patients present with cognitive defects.^97
–99 Therefore, to create the greatest benefit for patients, it is important to consider targeting every tissue expressing dystrophin when designing treatments for DMD. Evidence of U7 Sm OPT snRNA's ability to function in the brain was demonstrated in the mdx mouse model. Groups found that a single intrahippocampal injection of rAAV1–U7 rescued dystrophin expression, restored gamma aminobutyric acid (GABA) receptor clusters in the hippocampus, and improved levels of long-term potentiation (LTP) measured at 2 months.^53,100 It has been shown that the lack of certain dystrophin isoforms in the brain reduces type A GABA receptor clusters and enhances LTP.¹⁰⁰ This demonstrated the potential of VES therapies to provide benefit to both muscular and neurological phenotypes.¹⁰¹

The first clinical trial using VES recently began in the United States (NCT04240315). The therapy, developed by Wein et al., targets mutations in exon 2 of DMD.^48,102,103 Preclinical studies were conducted in a mouse model carrying an exon 2 duplication (Dup2 mouse) and using an AAV1-U7 to skip the duplicated exon 2. Treatment of the Dup2 mouse resulted either in single exon 2 skipping, which leads to the production of full-length dystrophin, or skipping of both copies of exon 2, which results in an out-of-frame transcript. However, this mRNA lacking both copies of exon 2 still produces a highly functional truncated protein, thanks to a downstream internal ribosome entry site.⁴⁸ This study further supported the ability of VES to induce efficient exon skipping, protein restoration, and force improvement. A follow-up dose escalation study was also performed in Dup2 mice using the same VES approach to determine the minimal efficacious dose (MED).¹⁰² MED was found to be 3e13 vg/kg.

Finally, two additional studies were performed to assess potential off targets of this VES, one in the Dup2 mouse and one in wild-type nonhuman primates (NHPs). No off-target or other adverse events were found in NHPs.¹⁰³ Based on these results, a phase I/IIa clinical trial (NCT04240315) using this exon 2 VES approach began in 2020. Astellas Pharma, the sponsor of this trial, recently presented initial results of increased dystrophin expression and muscle strength in one patient. While these results are promising, the number of patients in the clinical trial is very small, and the data have not yet undergone peer review and thus must be interpreted with care (LBO3, Waldrop et al., 2020 WMS).¹⁰⁴

For DMD, several other gene therapy strategies are currently being explored in clinical trials. Since AAV packaging limits make delivery of the full-length dystrophin transcript impossible, microdystrophin (microdys) has emerged as a promising alternative. Various forms of microdys are currently being explored in three different clinical trials in the United States.

A major advantage to microdys is its potential to treat DMD patients regardless of their mutation. However, VES offers distinct advantages. Namely, exon skipping restores a near full-length dystrophin, while microdys only represents about 30% of the full-length protein. Thus, the VES induced protein is expected to have a higher functionality compared to microdys. Additionally, exon skipping utilizes the natural dystrophin promoter, ensuring that expression of the restored protein is properly regulated. Finally, the engineered microdys is more likely to be immunogenic as it contains several unnatural epitopes. Due to its several advantages, VES may be a superior choice when compared to microdys for patients with mutations correctable by VES approaches.

Beyond the described studies, U7 Sm OPT snRNA has also been explored as a possible treatment for Myotonic Dystrophy Type 1 (DM1),⁷² Dysferlinopathies,⁶³ Pompe disease,⁶⁸ amyotrophic lateral sclerosis,⁶⁴ Hemophilia, and several other genetic disorders (Table 1).

Conclusion: Advantages/Disadvantages and Future Steps for U7 snRNA VES/VEI

U7 snRNA is a versatile platform for creating therapies that overcomes obstacles faced by AONs. However, important questions remain regarding U7 snRNAs. Since AONs have been more widely used than U7 snRNA for modulating splicing, the ability of AONs to compete with endogenous proteins for regulatory element binding sites is better characterized.¹⁰⁵ For example, results from DeepCLIP,¹⁰⁶ a tool which accelerates antisense design by providing predicted sites of splicing regulatory element binding to RNA, were validated using AONs. It will be of interest to perform similar studies that characterize U7 snRNA antisense's ability to compete with regulatory element binding sites. Such research will be valuable as an AON or U7 snRNA carrying the same antisense sequence may vary in their ability to bind to a particular RNA regulatory element.

While AAV delivered splicing modulation is significantly longer lasting when compared to regular AONs, the precise longevity of the treatment remains unknown and is unlikely to be permanent.⁹⁶ This issue may be partially circumvented in some disorders by starting treatment early, but a form of readministration might eventually be necessary. Unfortunately, the potential immune response elicited by the AAV capsid complicates VES reinjection. However, in the future, immunosuppressants, alterations of the AAV capsid, or nonviral vectors may make readministration of U7 snRNA-based therapies possible.

Another alternative could also be to first treat with VES and later administer an AON treatment if necessary. This combined approach was recently tested and proved to be highly promising in a study where VES was used in combination with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO).¹⁰⁷ Results showed significant improvements in skeletal muscle, diaphragm, and heart function that resulted in significant life extension beyond PPMO or VES alone. Alternatively, if long-term expression of VES/VEI is found to be toxic, strategies to remove the therapeutic AAV episomes will have to be developed. This may be done through designing an AAV that forms a less stable episome or introducing molecules to knockdown the introduced AAV episomes. A similar idea has been explored in the development of “self-deleting” AAV encoding CRISPR-Cas9.¹⁰⁸

An additional potential challenge for U7 Sm OPT snRNA approaches is that although they do not interfere with natural U7 snRNA pathways, the formation of U7 Sm OPT snRNPs and other U snRNPs could be saturated. When expressed at high levels, a fraction of U7 Sm OPT molecules are truncated at the 3′ end. It is expected that these misprocessed sequences are not assembled into snRNPs and lose therapeutic function.¹⁰⁹ Thus, reaching sufficiently high levels of intracellular antisense sequence may require a further optimized U7 Sm OPT that does not undergo such misprocessing. Although not detected in safety studies in mice, dogs, and nonhuman primates, it is possible that inducing high levels of snRNP formation poses a safety concern. This concern is somewhat mitigated by the finding that U7 Sm OPT misprocessing is a result of the Sm OPT sequence itself.²⁴ Nevertheless, additional study regarding the effects of expressing high levels of therapeutic U7 snRNPs on natural snRNP processing might be needed.

Recently, genome editing tools based on modified bacterial proteins have gained popularity for treating genetic disorders. While the advantages of modifying genomic DNA rather than RNA are clear and may one day prove superior, in vivo delivery of these DNA-editing technologies carries the risk of permanent off-target effects and immune responses triggered by the introduction of foreign proteins. Meanwhile, VES has been shown to have negligible off-target effects and its targeting of RNA makes for an inherently safer choice.¹¹⁰

In some cases, DNA-editing may lead to undesired mutations inducing improper splicing. If predicted ahead of time, VES/VEI may be administered alongside the DNA-editing vector to correct the unintended alterations in splicing caused by DNA-editing off-targets or improper repairs. In addition, using DNA-editing and VES/VEI in tandem may be beneficial if a single gene harbors more than one deleterious mutation (e.g., an improperly included exon and downstream nonsense mutation). This may greatly reduce off-target risks when compared to using two DNA-editing vectors.

Altogether, VES/VEI have shown to be a highly adaptable and effective tools for modulating splicing in many genetic disorders and have the potential to play a prominent role in developing personalized treatments. Genetic disorders for which VES/VEI may be used as a treatment will typically fall into one of three categories: (1) diseases caused by the improper inclusion or exclusion of exons, (2) diseases in which the effects of a mutation in one gene can be alleviated by altering the splicing of a second gene, and (3) diseases caused by a nonsense mutation located within an exon whose removal is in-frame and does not affect overall protein function. For diseases that fall into these categories and more, VES/VEI is an emerging technology that has the potential to play a key role in treating patients with genetic disorders.

Footnotes

Authors' Contributions

D.L., Y.R., D.R., and N.W. wrote the article. Y.R. compiled and created the figures.

Author Disclosure

Nationwide Children's Hospital optioned a program to Astellas Pharma. N.W. received royalty payments from this option.

Funding Information

This review was written while D.L., Y.R., and N.W were supported by Nationwide Children's internal and D.R. and N.W. by a patient donation (Nicholoff Family).

References

Skordis

, Dunckley

, Yue

, et al. Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci U S A, 2003; 100:4114–4119.

Aartsma-Rus

, Janson

AAM

, Kaman

, et al. Antisense-induced multiexon skipping for Duchenne muscular dystrophy makes more sense. Am J Hum Genet, 2004; 74:83–92.

Van Deutekom

, Janson

, Ginjaar

, et al. Local dystrophin restoration with antisense oligonucleotide PRO051. N Engl J Med, 2007; 357:2677–2686.

Aoki

, Nakamura

, Yokota

, et al. In-frame dystrophin following exon 51-skipping improves muscle pathology and function in the exon 52-deficient mdx mouse. Mol Ther, 2010; 18:1995–2005.

Hammond

, Wood

MJA

. Genetic therapies for RNA mis-splicing diseases. Trends Genet, 2011; 27:196–205.

Wilton

, Lloyd

, Carville

, et al. Specific removal of the nonsense mutation from the mdx dystrophin mRNA using antisense oligonucleotides. Neuromuscul Disord, 1999; 9:330–338.

Dominski

, Kole

. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci, 1993; 90:8673–8677.

Gorman

, Suter

, Emerick

, et al. Stable alteration of pre-mRNA splicing patterns by modified U7 small nuclear RNAs. Proc Natl Acad Sci U S A, 1998; 95:4929–4934.

, Yokota

, Takeda

, et al. The status of exon skipping as a therapeutic approach to Duchenne muscular dystrophy. Mol Ther, 2011; 19:9–15.

10.

De Lorenzi

, Rohrer

, Birnstiel

. Analysis of a sea urchin gene cluster coding for the small nuclear U7 RNA, a rare RNA species implicated in the 3' editing of histone precursor mRNAs. Proc Natl Acad Sci U S A, 1986; 83:3243–3247.

11.

Schaufele

, Gilmartin

, Bannwarth

, et al. Compensatory mutations suggest that base-pairing with a small nuclear RNA is required to form the 3' end of H3 messenger RNA. Nature, 1986; 323:777–781.

12.

Soldati

, Schümperli

. Structural and functional characterization of mouse U7 small nuclear RNA active in 3' processing of histone pre-mRNA. Mol Cell Biol, 1988; 8:1518–1524.

13.

Gruber

, Soldati

, Burri

, et al. Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear RNA. BBA Gene Struct Expr, 1991; 1088:151–154.

14.

Massa

, Longo

, Zuo

, et al. Cloning of rat grp75, an hsp70-family member, and its expression in normal and ischemic brain. J Neurosci Res, 1995; 40:807–819.

15.

Mowry

, Steitz

. Identification of the human U7 snRNP as one of several factors involved in the 3′ end maturation of histone premessenger RNA's. Science (80-), 1987; 238:1682–1687.

16.

Dominski

, Yang

, Purdy

, et al. Cloning and characterization of the Drosophila U7 small nuclear RNA. Proc Natl Acad Sci U S A, 2003; 100:9422–9427.

17.

Marz

, Mosig

, Stadler

BMR

, et al. U7 snRNAs: a computational survey. Genomics Proteomics Bioinformatics, 2007; 5:187–195.

18.

Parry

, Scherly

, Mattaj

. “Snurpogenesis”: the transcription and assembly of U snRNP components. Trends Biochem Sci, 1989; 14:15–19.

19.

Southgate

, Busslinger

. In vivo and in vitro expression of U7 snRNA genes: cis- and trans-acting elements required for RNA polymerase II-directed transcription. EMBO J, 1989; 8:539–549.

20.

Hernandez

. Formation of the 3′ end of U1 snRNA is directed by a conserved sequence located downstream of the coding region. EMBO J, 1985; 4:1827–1837.

21.

Cuello

, Boyd

, Dye

, et al. Transcription of the human U2 snRNA genes continues beyond the 3' box in vivo . EMBO J, 1999; 18:2867–2877.

22.

Uguen

, Murphy

. The 3′ ends of human pre-snRNAs are produced by RNA polymerase II CTD-dependent RNA processing. EMBO J, 2003; 22:4544–4554.

23.

Hernandez

, Weiner

. Formation of the 3′ end of U1 snRNA requires compatible snRNA promoter elements. Cell, 1986; 47:249–258.

24.

Pillai

, Will

, Lührmann

, et al. Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein. EMBO J, 2001; 20:5470–5479.

25.

Fischer

, Lührmann

. An essential signaling role for the m3G cap in the transport of U1 snRNP to the nucleus. Science (80-), 1990; 249:786–790.

26.

Jády

, Darzacq

, Tucker

, et al. Modification of Sm small nuclear RNAs occurs in the nucleoplasmic Cajal body following import from the cytoplasm. EMBO J, 2003; 22:1878–1888.

27.

CHH

, Murphy

, Gall

. The Sm binding site targets U7 snRNA to coiled bodies (spheres) of amphibian oocytes. RNA, 1996; 2:811–823.

28.

Nizami

, Deryusheva

, Gall

. The Cajal body and histone locus body. Cold Spring Harb Perspect Biol, 2010; 2:1–12.

29.

Gick

, Krämer

, Keller

, et al. Generation of histone mRNA 3′ ends by endonucleolytic cleavage of the pre-mRNA in a snRNP-dependent in vitro reaction. EMBO J, 1986; 5:1319–1326.

30.

Sun

, Zhang

, Aik

, et al. Structure of an active human histone pre-mRNA 3′-end processing machinery. Science (80-), 2020; 367:700–703.

31.

Grimm

, Stefanovic

, Schümperli

. The low abundance of U7 snRNA is partly determined by its Sm binding site. EMBO J, 1993; 12:1229–1238.

32.

Cartegni

, Chew

, Krainer

. Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet, 2002; 3:285–298.

33.

Matera

, Wang

. A day in the life of the spliceosome. Nat Rev Mol Cell Biol, 2014; 15:108–121.

34.

Smith

CWJ

, Valcárcel

. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci, 2000; 25:381–388.

35.

Stefanovic

, Hacki

, Luhrmanni

, et al. Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes. Nucleic Acids Res, 1995; 23:3141–3151.

36.

Suter

, Tomasini

, Reber

, et al. Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human β-thalassemic mutations. Hum Mol Genet, 1999; 8:2415–2423.

37.

Meyer

, Schümperli

. Antisense derivatives of U7 small nuclear RNA as modulators of pre-mRNA splicing. Altern pre-mRNA Splicing Theory Protoc, 2012:481–494.

38.

Benchaouir

, Goyenvalle

. AAV-mediated exon skipping for Duchenne muscular dystrophy. Muscle Gene Ther, 2019:355–370.

39.

Gorman

, Mercatante

, Kole

. Restoration of correct splicing of thalassemic β-globin pre-mRNA by modified U1 snRNAs. J Biol Chem, 2000; 275:35914–35919.

40.

De Angelis

, Sthandier

, Berarducci

, et al. Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48–50 DMD cells. Proc Natl Acad Sci U S A, 2002; 99:9456–9461.

41.

Yanaizu

, Sakai

, Tosaki

, et al. Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-Transcriptional regulation. Sci Rep, 2018:8:1–11.

42.

Liu

, Asparuhova

, Brondani

, et al. Inhibition of HIV-1 multiplication by antisense U7 snRNAs and siRNAs targeting cyclophilin A. Nucleic Acids Res, 2004; 32:3752–3759.

43.

Meyer

, Marquis

, Trü

, et al. Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation. Hum Mol Genet, 2009; 18:546–555.

44.

Goyenvalle

, Vulin

, Fougerousse

, et al. Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science (80-), 2004; 306:1796–1799.

45.

Sellier

, Freyermuth

, Tabet

, et al. Sequestration of DROSHA and DGCR8 by expanded CGG RNA Repeats Alters microRNA processing in fragile X-associated tremor/ataxia syndrome. Cell Rep, 2013; 3:869–880.

46.

Klironomos

, Berg

. Quantitative analysis of competition in posttranscriptional regulation reveals a novel signature in target expression variation. Biophys J, 2013; 104:951–958.

47.

Curtis

, Seow

, Wood

MJA

, et al. Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7. Nucleic Acids Res, 2017; 45:7870–7885.

48.

Wein

, Vulin

, Falzarano

, et al. Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med, 2014; 20:992–1000.

49.

Brun

, Suter

, Pauli

, et al. U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping. Cell Mol Life Sci, 2003; 60:557–566.

50.

Bartoli

, Poupiot

, Goyenvalle

, et al. Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies. Gene Ther, 2006; 13:20–28.

51.

Quenneville

, Chapdelaine

, Skuk

, et al. Autologous transplantation of muscle precursor cells modified with a lentivirus for muscular dystrophy: human cells and primate models. Mol Ther, 2007; 15:431–438.

52.

Goyenvalle

, Babbs

, van Ommen

, et al. Enhanced exon-skipping induced by U7 snRNA carrying a splicing silencer sequence: Promising tool for DMD therapy. Mol Ther, 2009; 17:1234–1240.

53.

Vaillend

, Perronnet

, Ros

, et al. Rescue of a dystrophin-like protein by exon skipping in vivo restores GABAA-receptor clustering in the hippocampus of the mdx mouse. Mol Ther, 2010; 18:1683–1688.

54.

Wein

, Avril

, Bartoli

, et al. Efficient bypass of mutations in dysferlin deficient patient cells by antisense-induced exon skipping. Hum Mutat, 2010; 31:136–142.

55.

Goyenvalle

, Wright

, Babbs

, et al. Engineering multiple U7snRNA constructs to induce single and multiexon-skipping for Duchenne muscular dystrophy. Mol Ther, 2012; 20:1212–1221.

56.

Bish

, Sleeper

, Forbes

, et al. Long-term restoration of cardiac dystrophin expression in golden retriever muscular dystrophy following rAAV6-mediated exon skipping. Mol Ther, 2012; 20:580–589.

57.

Vulin

, Barthelemy

, Goyenvalle

, et al. Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping. Mol Ther, 2012; 20:2120–2133.

58.

Barbash

, Cecchini

, Faranesh

, et al. MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Gene Ther, 2013; 20:274–282.

59.

Nuzzo

, Radu

, Baralle

, et al. Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation. Blood, 2013; 122:3825–3831.

60.

Rendu

, Brocard

, Denarier

, et al. Exon skipping as a therapeutic strategy applied to an RYR1 mutation with pseudo-exon inclusion causing a severe core myopathy. Hum Gene Ther, 2013; 24:702–713.

61.

Le Guiner

, Montus

, Servais

, et al. Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients. Mol Ther, 2014; 22:1923–1935.

62.

Rendu

, Montjean

, Coutton

, et al. Functional characterization and rescue of a deep intronic mutation in OCRL gene responsible for lowe syndrome. Hum Mutat, 2017; 38:152–159.

63.

Malcher

, Heidt

, Goyenvalle

, et al. Exon skipping in a dysf-missense mutant mouse model. Mol Ther Nucleic Acids, 2018; 13:198–207.

64.

Biferi

, Cohen-Tannoudji

, Cappelletto

, et al. A New AAV10-U7-mediated gene therapy prolongs survival and restores function in an ALS mouse model. Mol Ther, 2017; 25:2038–2052.

65.

Uchikawa

, Fujii

, Kohno

, et al. U7 snRNA-mediated correction of aberrant splicing caused by activation of cryptic splice sites. J Hum Genet, 2007; 52:891–897.

66.

Marquis

, Meyer

, Angehrn

, et al. Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. Mol Ther, 2007; 15:1479–1486.

67.

Odermatt

, Trub

, Furrer

, et al. Somatic therapy of a mouse SMA model with a U7 snRNA gene correcting SMN2 splicing. Mol Ther, 2016; 24:1797–1805.

68.

van der Wal

, Bergsma

, Pijnenburg

, et al. Antisense oligonucleotides promote exon inclusion and correct the common c.-32-13T>G GAA splicing variant in pompe disease. Mol Ther Nucleic Acids, 2017; 7:90–100.

69.

Yanaizu

, Sakai

, Tosaki

, et al. Small nuclear RNA-mediated modulation of splicing reveals a therapeutic strategy for a TREM2 mutation and its post-transcriptional regulation. Sci Rep, 2018; 8:6937.

70.

Preedagasamzin

, Nualkaew

, Pongrujikorn

, et al. Engineered U7 snRNA mediates sustained splicing correction in erythroid cells from beta-thalassemia/HbE patients. Biochem Biophys Res Commun, 2018; 499:86–92.

71.

Nualkaew

, Jearawiriyapaisarn

, Hongeng

, et al. Restoration of correct beta(IVS2-654)-globin mRNA splicing and HbA production by engineered U7 snRNA in beta-thalassaemia/HbE erythroid cells. Sci Rep, 2019; 9:7672.

72.

Francois

, Klein

, Beley

, et al. Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs. Nat Struct Mol Biol, 2011; 18:85–87.

73.

Kolb

, Kissel

. Spinal muscular atrophy: a timely review. Arch Neurol, 2011; 68:979–984.

74.

Nlend Nlend

, Meyer

, Schümperli

. Repair of pre-mRNA splicing: prospects for a therapy for spinal muscular atrophy. RNA Biol, 2010; 7:430–440.

75.

Finkel

, Chiriboga

, Vajsar

, et al. Treatment of infantile-onset spinal muscular atrophy with nusinersen: a phase 2, open-label, dose-escalation study. Lancet, 2016; 388:3017–3026.

76.

Kolb

, Ph

, Coffey

, et al. Natural history of infantile-onset spinal muscular atrophy. Ann Neurol, 2017; 82:883–891.

77.

Finkel

, Mercuri

, Darras

, et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. New, 2017; 377:1723–1732.

78.

Madocsai

, Lim

, Geib

, et al. Correction of SMN2 Pre-mRNA splicing by antisense U7 small nuclear RNAs. Mol Ther, 2005; 12:1013–1022.

79.

Marquis

, Meyer

, Angehrn

, et al. Spinal muscular atrophy: SMN2 Pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence. Mol Ther, 2007; 15:1479–1486.

80.

Dabbous

, Maru

, Jansen

, et al. Survival, motor function, and motor milestones: comparison of AVXS-101 relative to nusinersen for the treatment of infants with spinal muscular atrophy type 1. Adv Ther, 2019; 36:1164–1176.

81.

Rodino-Klapac

, Mendell

, Sahenk

. Update on the treatment of Duchenne muscular dystrophy. Curr Neurol Neurosci Rep, 2013; 13:332.

82.

Mendell

, Shilling

, Leslie

, et al. Evidence-based path to newborn screening for Duchenne muscular dystrophy. Ann Neurol, 2012; 71:304–313.

83.

Aartsma-Rus

, Van Ommen

GJB

. Progress in therapeutic antisense applications for neuromuscular disorders. Eur J Hum Genet, 2010; 18:146–153.

84.

Koo

, Wood

. Clinical trials using antisense oligonucleotides in Duchenne muscular dystrophy. Hum Gene Ther, 2013; 24:479–488.

85.

Stein

, Castanotto

. FDA-approved oligonucleotide therapies in 2017. Mol Ther, 2017; 25:1069–1075.

86.

Scoto

, Finkel

, Mercuri

, et al. Genetic therapies for inherited neuromuscular disorders. Lancet Child Adolesc Heal, 2018; 2:600–609.

87.

Bennett

. Therapeutic antisense oligonucleotides are coming of age. Annu Rev Med, 2019; 70:307–321.

88.

Syed

. Eteplirsen: first global approval. Drugs, 2016; 76:1699–1704.

89.

Bennett

, Swayze

. RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol, 2010; 50:259–293.

90.

Scoles

, Minikel

, Pulst

. Antisense oligonucleotides: a primer. Neurol Genet, 2019; 5:1–8.

91.

Bennett

, Baker

, Pham

, et al. Pharmacology of antisense drugs. Annu Rev Pharmacol Toxicol, 2017; 57:81–105.

92.

, Rabinowitz

, Yun

, et al. Systemic delivery of antisense oligoribonucleotide restorers dystrophin expression in body-wide skeletal muscles. Proc Natl Acad Sci U S A, 2005; 102:198–203.

93.

Alter

, Lou

, Rabinowitz

, et al. Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med, 2006; 12:175–177.

94.

Heemskerk

, de Winter

, de Kimpe

, et al. In vivo comparison of 2'-O-methyl phosphorothioate and morpholino antisense oligonucleotides for Duchenne muscular dystrophy exon skipping. J Gene Med, 2009; 11:257–266.

95.

Vulin

, Barthélémy

, Goyenvalle

, et al. Muscle function recovery in golden retriever muscular dystrophy after AAV1–U7 exon skipping. Mol Ther, 2012; 20:2120–2133.

96.

Le Hir

, Goyenvalle

, Peccate

, et al. AAV genome loss from dystrophic mouse muscles during AAV-U7 snRNA-mediated exon-skipping therapy. Mol Ther, 2013; 21:1551–1558.

97.

Taylor

, Betts

, Maroulis

, et al. Dystrophin gene mutation location and the risk of cognitive impairment in Duchenne muscular dystrophy. PLoS One, 2010; 5:e8803.

98.

Bresolin

, Castelli

, Comi

, et al. Cognitive impairment in Duchenne muscular dystrophy. Neuromuscul Disord, 1994; 4:359–369.

99.

Anderson

, Head

, Rae

, et al. Brain function in Duchenne muscular dystrophy. Brain, 2002; 125:4–13.

100.

Dallérac

, Perronnet

, Chagneau

, et al. Rescue of a dystrophin-like protein by exon skipping normalizes synaptic plasticity in the hippocampus of the mdx mouse. Neurobiol Dis, 2011; 43:635–641.

101.

Goyenvalle

, Leumann

, Garcia

. Therapeutic Potential of Tricyclo-DNA antisense oligonucleotides. J Neuromuscul Dis, 2016; 3:157–167.

102.

Simmons

, Vetter

, Huang

, et al. Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping. Mol Ther Methods Clin Dev, 2021; 21:325–340.

103.

Gushchina

L V

, Frair

, Rohan

, et al. Lack of toxicity in non-human primates receiving clinically relevant doses of an AAV9.U7snRNA vector designed to induce DMD exon 2 skipping. Hum Gene Ther 2021, DOI: 10.1089/hum.2020.286.

104.

Waldrop

, Lawlor

, Vetter

, et al. Expression of apparent full-length dystrophin in-human gene therapy trial using the scAAV9.U7-ACCA vector. Neuromuscul Disord, 2020; 30:S166–S167.

105.

Bruun

, Doktor

, Borch-Jensen

, et al. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation. BMC Biol, 2016; 14:54.

106.

Grønning

AGB

, Doktor

, Larsen

, et al. DeepCLIP: predicting the effect of mutations on protein–RNA binding with deep learning. Nucleic Acids Res, 2020; 48:7099–7118.

107.

Forand

, Muchir

, Mougenot

, et al. Combined treatment with peptide-conjugated phosphorodiamidate morpholino oligomer-PPMO and AAV-U7 rescues the severe DMD phenotype in mice. Mol Ther Methods Clin Dev, 2020; 17:695–708.

108.

, Lee

, Hurley

, et al. A self-deleting AAV-CRISPR system for in vivo genome editing. Mol Ther Methods Clin Dev, 2019; 12:111–122.

109.

Eckenfelder

, Tordo

, Babbs

, et al. The cellular processing capacity limits the amounts of chimeric U7 snRNA available for antisense delivery. Mol Ther Nucleic Acids, 2012; 1:e31.

110.

Domenger

, Allais

, François

, et al. RNA-seq analysis of an antisense sequence optimized for exon skipping in Duchenne patients reveals no off-target effect. Mol Ther Nucleic Acids, 2018; 10:277–291.