Restoration of SMN function: delivery of a trans-splicing RNA re-directs SMN2 pre-mRNA splicing.
ABSTRACT Spinal muscular atrophy (SMA) is caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene called SMN2 is present in all SMA patients; however SMN2 produces low levels of functional protein due to alternative splicing. Recently a therapeutic approach has been developed referred to as trans-splicing. Conceptually, this strategy relies upon pre-messenger RNA (pre-mRNA) splicing occurring between two separate molecules: (i) the endogenous target RNA and (ii) the therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. SMN trans-splicing RNAs were initially examined and expressed from a plasmid-backbone and shown to re-direct splicing from a SMN2 mini-gene as well as from endogenous transcripts. Subsequently, recombinant adeno-associated viral vectors were developed that expressed and delivered trans-splicing RNAs to SMA patient fibroblasts. In the severe SMA patient fibroblasts, SMN2 splicing was redirected via trans-splicing to produce increased levels of full-length SMN mRNA and total SMN protein levels. Finally, small nuclear ribonucleoprotein (snRNP) assembly, a critical function of SMN, was restored to SMN-deficient SMA fibroblasts following treatment with the trans-splicing vector. Together these results demonstrate that the alternatively spliced SMN2 exon 7 is a tractable target for replacement by trans-splicing.
- SourceAvailable from: Stéphanie Lorain[Show abstract] [Hide abstract]
ABSTRACT: GNE myopathy is a rare neuromuscular autosomal recessive disease, resulting from mutations in the gene UDP N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). The most frequent mutation is the single homozygous missense mutation, M712T-the Middle Eastern mutation-located ten amino acids before the end of the protein. We have used an adeno-associated virus (AAV)-based trans-splicing (TS) vector as a gene therapy tool to overcome this mutation by replacing the mutated last exon of GNE by the wild-type exon while preserving the natural endogenous regulatory machinery. We have designed relevant plasmids directed either to mouse or to human GNE. Following transfection of C2C12 murine muscle cells with the mouse TS vectors, we have been able to detect by nested RT-PCR trans-spliced molecules carrying the wild-type exon 12 of GNE. Similarly, transfection of HEK293 human cells with the human-directed TS vectors resulted in the generation of trans-spliced human GNE RNA molecules. Furthermore, infection of primary muscle cells from a GNE myopathy patient carrying the homozygous M712T mutation, with an AAV8-based viral vector carrying a human-directed TS construct, resulted in the generation of wild-type GNE transcripts in addition to the mutated ones. These studies provide a proof of concept that the TS approach could be used to partially correct the Middle Eastern mutation in GNE myopathy patients. These results provide the basis for in vivo research in animal models using the AAV platform with TS plasmids as a potential genetic therapy for GNE myopathy.Neuromolecular medicine 11/2013; 16(2). · 5.00 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is a debilitating neuromuscular disease caused by the loss of survival of motor neuron (SMN) protein. Previously we demonstrated that ISIS 396443, an antisense oligonucleotide (ASO) targeted to the SMN2 pre-mRNA, is a potent inducer of SMN2 exon 7 inclusion and SMN protein expression, and improves function and survival of mild and severe SMA mouse models. Here we demonstrate that ISIS 396443 is the most potent ASO in central nervous system (CNS) tissues of adult mice, compared to several other chemically modified ASOs. We evaluated methods of ISIS 396443 delivery to the CNS and characterized its pharmacokinetics and pharmacodynamics in rodents and non-human primates (NHPs). Intracerebroventricular (ICV) bolus injection is a more efficient method of delivering ISIS 396443 to the CNS of rodents, compared to ICV infusion. For both methods of delivery, the duration of ISIS-396443-mediated SMN2 splicing correction is long-lasting, with maximal effects still observed 6 months after treatment discontinuation. Administration of ISIS 396443 to the CNS of NHPs by a single intrathecal bolus injection results in widespread distribution throughout the spinal cord. Based upon these preclinical studies, we have advanced ISIS 396443 into clinical development.Journal of Pharmacology and Experimental Therapeutics 04/2014; · 3.89 Impact Factor
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ABSTRACT: Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease, which causes death of motor neurons in the anterior horn of spinal cord. Genetic cause of SMA is the deletion or mutation of SMN1 gene, which encodes the SMN protein. Although SMA patients include SMN2 gene, a duplicate of SMN1 gene, predominant production of exon 7 skipped isoform from SMN2 pre-mRNA fails to rescue SMA patients. Here we show that hnRNP M, a member of hnRNPs protein family, when knocked down, promote exon 7 skipping of both SMN2 and SMN1 pre-mRNA. By contrast, overexpression of hnRNP M promotes exon 7 inclusion of both SMN2 and SMN1 pre-mRNA. Significantly, hnRNP M promotes exon 7 inclusion in SMA patient cells. Thus, we conclude that hnRNP M promotes exon 7 inclusion of both SMN1 and SMN2 pre-mRNA. . we also demonstrate that hnRNP M contacts an enhancer on exon 7, which was previously shown to provide binding site for tra2β. We present evidence that hnRNP M and tra2β contact overlapped sequence on exon 7 but with slightly different RNA sequence requirements. In addition, hnRNP M promotes U2AF65 recruitment on the flanking intron of exon 7. We conclude that hnRNP M promotes exon 7 inclusion of SMN1 and SMN2 pre-mRNA through targeting an enhancer on exon 7 through recruiting U2AF65. Our results provide a clue that hnRNP M is a potential therapeutic target for SMA.Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms 04/2014; · 5.44 Impact Factor
Restoration of SMN Function: Delivery of a
Trans-splicing RNA Re-directs SMN2
Tristan H Coady1, Monir Shababi1, Gregory E Tullis2 and Christian L Lorson1
1Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA; 2Department of Ophthalmology,
Boston University Medical School, Boston, Massachusetts, USA
Spinal muscular atrophy (SMA) is caused by loss of survival
motor neuron-1 (SMN1). A nearly identical copy gene
called SMN2 is present in all SMA patients; however SMN2
produces low levels of functional protein due to alternative
splicing. Recently a therapeutic approach has been devel-
oped referred to as trans-splicing. Conceptually, this strategy
relies upon pre-messenger RNA (pre-mRNA) splicing occur-
ring between two separate molecules: (i) the endogenous
target RNA and (ii) the therapeutic RNA that provides the
correct RNA sequence via a trans-splicing event. SMN trans-
splicing RNAs were initially examined and expressed from
a plasmid-backbone and shown to re-direct splicing from a
SMN2 mini-gene as well as from endogenous transcripts.
Subsequently, recombinant adeno-associated viral vectors
were developed that expressed and delivered trans-splicing
RNAs to SMA patient fibroblasts. In the severe SMA patient
fibroblasts, SMN2 splicing was redirected via trans-splicing
to produce increased levels of full-length SMN mRNA and
total SMN protein levels. Finally, small nuclear ribonucleo-
protein (snRNP) assembly, a critical function of SMN, was
restored to SMN-deficient SMA fibroblasts following treat-
ment with the trans-splicing vector. Together these results
demonstrate that the alternatively spliced SMN2 exon 7 is
a tractable target for replacement by trans-splicing.
Received 15 February 2007; accepted 25 April 2007; advance online
publication 5 June 2007. doi:10.1038/sj.mt.6300222
Spinal muscular atrophy (SMA) is an autosomal recessive neuro-
muscular disorder and the leading genetic cause of death in chil-
dren under the age of two.1 SMA is caused by the homozygous
loss of survival motor neuron 1 (SMN1).2 In humans, two nearly
identical copies of the SMN gene exist in chromosome 5, desig-
nated SMN1 and SMN2.2 SMN2 is distinguished from SMN1 by
a small number of non-polymorphic nucleotide differences; how-
ever, the critical distinction between the two genes is at the RNA
processing level: SMN1 produces exclusively full-length tran-
scripts, while SMN2 primarily produces an alternatively spliced
transcript lacking the final coding exon (exon 7).2,3 A single silent
nucleotide difference disrupts an exonic splice enhancer in SMN2
exon 7 and dictates the splicing patterns for these closely related
genes.4–6 Since the non-polymorphic nucleotide differences do
not alter the coding capacity of SMN2, this gene has been envi-
sioned as an ideal target for a variety of therapeutic strategies.7
SMN exon 7 pre-messenger RNA (pre-mRNA) splicing is
extensively regulated through a complex interplay between posi-
tively and negatively acting RNA splicing factors. Exonic splice
enhancers have been identified that serve as binding substrates,
directly or indirectly, for regulatory proteins including SF2/ASF,
hTra2β1, SRp30c, RBMY, and hnRNP-G.4,8–10 SF2/ASF, a serine/
arginine-rich protein, binds an enhancer that overlaps the criti-
cal C/T transition, and binds only in the SMN1 context.4 In the
SMN2 context, the SF2/ASF site is disrupted, and a novel hnRNP-
A1 dependent splicing silencer is created by the C/T transition.11
Additional RNA sequences influence SMN alternative splicing,
such as an extended inhibitory context and novel intronic repres-
sors located in flanking introns (11–15).
Nucleic acid-based therapies have emerged that are designed
to overcome the alternative splicing of SMN2 by promoting the
inclusion of SMN exon 7.12–14 For example, oligonucleotides that
disable the SMN exon 8 splice site increase the usage of the exon
7 splice site.12,13 Additionally, peptide nucleic acids comprised
an SMN exon 7 anti-sense domain, and a minimalistic splicing-
activation-domain peptide were shown to efficiently increase
SMN2 exon 7 inclusion.15 Similarly, a novel synthetic RNA class
called bi-functional RNAs were developed that contained an exon
7 targeting domain and a non-complementary tail region that
acts to recruit splicing factors to the SMN2 exon 7 splice site.16,17
Recently a novel therapeutic approach has been developed,
based upon the principle of trans-splicing.18,19 Conceptually, this
strategy relies upon pre-mRNA splicing occurring between two dif-
ferent molecules: (i) the mutant endogenous RNA and (ii) the exog-
enous therapeutic RNA that provides the correct RNA sequence
via a trans-splicing event. Trans-splicing has been shown to effi-
ciently replace constitutively spliced as well as alternatively spliced
exons.20,21 This technology has been applied to several disease con-
texts including cystic fibrosis in which the common CFTR∆F508
The first two authors contributed equally to this work.
Correspondence: Christian L. Lorson, Department of Veterinary Pathobiology, Bond Life Sciences Center, 1201 Rollins Road, Room 471G,
University of Missouri, Columbia, Missouri 65211-7310, USA, E-mail: firstname.lastname@example.org
© The American Society of Gene Therapy
www.moleculartherapy.org? ? ?
© The American Society of Gene Therapy© The American Society of Gene Therapy
Restoration of SMN Function
mutation was efficiently replaced with the wild-type sequence via a
trans-splicing reaction.20,22,23 Similarly, tau exon 10, an alternatively
spliced exon associated with Alzheimer’s disease, can be redirected
such that exon 10 is included at a high frequency.21
In this report, the goal was to develop a trans-splicing system
that results in an increase in full-length SMN protein expres-
sion from the SMN2 gene. Since SMN2-derived transcripts are
inherently stable and the SMA patient population universally
expresses high levels of SMN∆7 transcript, we sought to deter-
mine whether SMN exon 7 was a tractable target for the develop-
ment of an exon 7 trans-splicing system.
Development of a trans-splicing system for sMN2
Trans-splicing has previously been implemented to overcome
genetic defects by re-directing pathogenic RNA splicing defects.18
To determine whether SMN2 exon 7 was a tractable target for
trans-splicing, a trans-splicing molecule was developed that con-
sists of the following: a SMN intron 6 annealing sequence com-
prising approximately 130 nucleotides; an optimized heterologous
splice site; the SMN1 exon 7 sequence; and two tandem copies
of the Hemagglutinin (HA) motif downstream of SMN exon 7 to
allow for straightforward monitoring of SMN RNA and protein
expression derived from trans-splicing (Figure 1). A translation
termination codon exists at the 3′ end of the endogenous SMN
exon 7; therefore, the native stop codon has been disrupted by a
single nucleotide substitution to allow translation of the HA epit-
ope. The initial analysis was performed using a SMN2 mini-gene,
pSMN2, and the trans-spliced RNA (tsRNA)-expressing plas-
mid, pMU2-tsRNAHA (Figure 1). The SMN2 mini-gene contains
the genomic sequences from SMN exon 6 through SMN exon 8
(including intronic sequences) and recapitulates the endogenous
SMN2 pre-mRNA splicing pattern.5 In the reverse transcription
polymerase chain reaction (RT-PCR) analysis, different primer
pairs can be utilized to differentiate transcripts derived exclusively
from pSMN2 (pCI-Fwd and pCI-Rev) versus trans-spliced tran-
scripts (pCI-Fwd and HA-Rev) (Figure 1).
sMN2 is a tractable target for trans-splicing
To determine whether trans-splicing was occurring between
the tsRNA and SMN2 transcripts, HeLa cells were transiently
co-transfected with the tsRNA expressing plasmid, pMU2-
tsRNAHA, and pSMN2. When expressed alone, pSMN2 pro-
duced predominately the SMN∆7 transcript and undetectable
levels of the trans-spliced product, as expected (Figure 2a, lanes
2–5). However, co-transfection of pSMN2 and pMU2-tsRNAHA
resulted specifically in the detection of the trans-spliced product
(Figure 2a, lane 8). A pMU2 plasmid containing a scrambled RNA
lacking tsRNAHA sequences did not result in the production of
the trans-spliced SMN2 RNA (Figure 2a, lanes 9, 10). A low level
of trans-splicing was detected when a pMU2-tsRNAHA derivative
was used that lacked the promoter for tsRNA (Figure 2a, lanes
11, 12). This low level of trans-splicing is likely due to the tran-
scriptional activity of the adeno-associated virus-2 (AAV2) ter-
minal repeats located upstream of the tsRNA start site and have
previously been shown to mediate transcriptional activity.24,25
To confirm that trans-splicing had occurred, the predicted
trans-spliced RT-PCR product (Figure 2a) was excised and
Predicted cis/trans-splice products
Exon 6 Exon 7Exon 8
Figure 1 Development of a survival motor neuron (sMN) trans-splicing
system. Schematic of trans-splicing in the context of SMN2 pre-mRNA splic-
ing. The trans-splicing RNA (tsRNAHA) binds to endogenous SMN pre-mRNA
(exons 6 through 8) at the intron 6 region by complementary base-pairing.
The SMN1 exon 7 is contained within the trans-splicing RNA and precedes
two tandem hemagglutinin (HA) epitopes followed by a poly-adenylation
signal (pA). Primers for reverse transcription polymerase chain reaction are
represented by arrows (pCI-Fwd; pCI-Rev; HA-Rev), and the branch point
(BP) and poly-pyrimidine tract (pPy) are indicated. The proposed mRNA
products represent the combination of primers pairs and splicing events
(lower panel). Fwd, forward; mRNA, messenger RNA; Rev, reverse.
tsRNA Exon 7HA
Figure 2 Identification and characterization of the survival motor
neuron (sMN) trans-spliced messenger RNA. (a) HeLa cells were co-
transfected with 1.25 µg of pSMN2 and 2.0 µg of pMU2-tsRNAHA. Total
cellular RNA was harvested 48 hours later and reverse transcription poly-
merase chain reaction was performed using the indicated primers: pCI-
Fwd, pCI-Rev, and hemagglutinin (HA)-Rev. Products derived exclusively
from pSMN1 and pSMN2 (“FL”, full-length; “∆7”, SMN∆7) and the trans-
spliced product (Trans*) are indicated. Primers that were used in reactions
are indicated by the “+” below the gel. (b) Sequence of SMN trans-spliced
product using Chromo-mat imaging software. Sequences derived from
SMN exon 6, SMN exon 7 and the HA tag are indicated. cDNA, comple-
mentary DNA; Fwd, forward; Rev, reverse; tsRNA, trans-splicing RNA.
© The American Society of Gene Therapy© The American Society of Gene Therapy
Restoration of SMN Function
sequenced. Sequence analysis identified proper SMN splicing
and that a trans-splicing event had occurred in the presence of:
the HA-tag, SMN1 exon 7 sequences, SMN exon 6 sequences
and pCI-derived (the pSMN2 plasmid backbone) sequences
(Figure 2b). An additional band was noted 130 nucleotides above
the predicted trans-spliced product; sequencing determined that
this minor species results from un-spliced vector-derived intron
upstream of the pSMN exon 6.
Additional tsRNA vectors were developed to identify molecu-
lar constituents that influenced the efficiency of the SMN trans-
splicing reaction. The U6 or cytomegalovirus (CMV) promoters
were used to drive expression of the tsRNA. In addition, tsRNAs
were engineered to include a modified U7 stem-loop struc-
ture cloned in front of or internal to the Intron 6 SMN binding
domain. The stem-loop has previously been proposed to facilitate
the inclusion of RNAs into the cellular splicing machinery due to
the presence of a modified Smith core binding motif and increase
stability of the RNA.26,27 We compared the efficiency of each plas-
mid in generating the trans-spliced product by RT-PCR (data not
shown). Our results demonstrated that the original tsRNA driven
by the CMV promoter, regardless of additional modifications
such as the U7 stem loop, was the most efficient construct and was
therefore used in all subsequent experiments. Transcript analysis
by RT-PCR demonstrated that the CMV-derived tsRNAHA effi-
ciently generated a trans-spliced product in a dose-dependent
manner (Figure 3). Interestingly, low but detectable levels of
trans-splicing occurred even at the lowest concentration tested
(0.05 µg) (Figure 3). Taken together, these results demonstrate
that the SMN exon 7 is amenable to a trans-splicing replacement
strategy and that the CMV-driven vector results in high levels of
endogenous sMN transcripts can be re-directed
To determine whether trans-splicing occurred between the tsRNA
and endogenous SMN transcripts, HeLa cells were transiently
transfected with increasing concentrations of pMU2-tsRNAHA. In
these reactions, SMN primer pairs specific to SMN exon 6 and
exon 8 were used to amplify endogenous RNA species (Figure 4a),
and primer pairs specific to SMN exon 6 and the HA sequences
were used to specifically amplify the trans-spliced product
(Figure 4a). Cells transfected with pMU2-tsRNAHA generated
a trans-spliced product with all three concentrations tested and
a modest dose-response was observed (Figure 4a). Importantly,
untransfected cells, cells transfected with the promoter-less vector
(pMU2KO-tsRNAHA), or the parent vector expressing non-specific
RNA sequences (pMU2) did not produce a SMN trans-spliced
product (Figure 4b). Since HeLa cells contain SMN1 and SMN2,
these analyses cannot distinguish between SMN1 and SMN2 tran-
scripts that had been trans-spliced.
To confirm the trans-splicing event at the protein level,
extracts from HeLa cells transfected with pMU2-tsRNAHA were
immunoprecipitated with an anti-HA antibody, and bound frac-
tions were analyzed by Western blot using an anti-SMN antibody.
The HA epitope is derived specifically from a trans-splicing event,
and if expressed, HA-SMN and endogenous SMN would likely
be in a complex since SMN contains two potent self-association
domains.28,29 Consistent with this, HA-immunoprecipitated com-
plexes were enriched for SMN (Figure 4c). In this assay, SMN and
the HA-tagged SMN species could not be specifically resolved.
However, these results demonstrate that endogenous SMN tran-
scripts are suitable targets for trans-splicing and trans-spliced
RNA is translated to protein.
Figure 3 Cytomegalovirus-driven trans-splicing RNA (tsRNA) mediates
survival motor neuron (sMN) trans-splicing in a dose-dependent man-
ner. Total RNA was isolated from HeLa cells co-transfected with 1.25 µg of
pSMN2 and 0.05, 0.1, 0.5, 0.75, 1.0, and 2.0 µg of pMU2-tsRNAHA. Reverse
transcription polymerase chain reaction analysis was performed with primer
pairs to identify trans-spliced levels (Trans*; pCI Fwd/HA Rev primers) and a
HPRT control (bottom panel: HPRT forward/reverse). HA, hemagglutinin.
Figure 4 pMU2-tsRNAHA trans-splices endogenous survival motor neu-
ron (sMN) transcript. HeLa cells were transfected with (a) 3.0, 6.0 or
8.0 µg of pMU2-tsRNAHA or 6.0 µg of control plasmids (pMU2KO-tsRNA;
pMU2), (b) and total RNA was harvested 48 hours post-transfection.
Reverse transcription polymerase chain reaction was performed with SMN
exon 6 (forward) and exon 8 (reverse) primers (top panel), exon 6 (for-
ward) and hemagglutinin (HA)-reverse primers (middle panel), or HPRT
primers (bottom panel). (c) SMN within HA-immunoprecipitated (IP)
complex. HeLa cell extracts derived from transfection with pMU2-tsRNAHA
were IP with anti-HA antibody. Bound fractions were resolved by sodium
dodecyl sulfate polyacrylamide gel electrophoresis and visualized with a
anti-SMN (4B7) antibody. SMN, cross-reactive heavy and light chains from
the immunoprecipitation (“HC” and “LC”), and size markers (“48” and
“36”) are indicated. A low level of SMN can be observed non-specifically
associated with the HA-resin. ∆7, SMN∆7; FL, full-length SMN; tsRNA,
www.moleculartherapy.org? ? ?
© The American Society of Gene Therapy
Restoration of SMN Function
Viral delivery of tsRNAs stimulates the levels of
full-length sMN in sMA fibroblasts
The development of recombinant AAV vectors expressing the SMN
tsRNA would provide a gene therapy approach directly applicable
to SMA, since recombinant AAV serotypes have been reported
to be efficiently delivered to the central nervous system through
retrograde transport.30 The tsRNA plasmids (pMU2 derivatives)
function in many respects like a typical plasmid, although when
expressed in the appropriate packaging cell line, tsRNA-expressing
recombinant AAV vectors can be readily developed.17 The flanking
AAV2 terminal repeats are present and facilitate the excision and
subsequent replication of the recombinant AAV (rAAV) genome.
The AAV coding sequences have been removed and replaced with
heterologous sequences including enhanced green fluorescent
protein (enhanced GFP), which facilitates the selection of the
infected cells and separate promoters controlling the expression
of the tsRNA cassette and enhanced GFP.
Type I (severe) SMA patient primary fibroblasts (3813 cells)
were chosen as a cell-based model to test the rAAV-derived
tsRNA activity. These cells lack SMN1 and contain two intact
copies of SMN2 and therefore provide an ideal genetic context
for testing SMA therapeutics. One of the hallmarks of the SMN
protein in fibroblasts is its presence in discrete nuclear foci called
“gems.”31 Gems in SMA patient fibroblasts are reduced in number
compared to unaffected carrier-derived fibroblasts.32 Since gems
are an established measure of SMN protein levels, and the num-
bers of gems correlate with disease severity, an increase in SMN-
positive gems would indicate an amelioration of a hallmark of
To determine whether virally derived tsRNAs can re-direct
SMN2 splicing in SMA patient cells, transcripts were analyzed
in 3813 cells following transduction with vMU2-tsRNAHA. Two
separate experiments are shown in which an multiplicity of
infection of 100 for the rAAV vectors was used to infect 3813
cells (Figure 5). In both instances, trans-splicing was detected
between the tsRNAHA and the endogenous SMN2 transcripts
(Figure 5, lanes 1, 2). Consistent with the production of the
trans-spliced product, the relative abundance of the endoge-
nous SMN∆7 transcript decreases following rAAV transduction
(Figure 5, compare lanes 3, 4 and 6), suggesting that the decrease
in SMN∆7 is due to the accumulation of the trans-spliced
product. As expected, promoter-less and empty vectors did not
alter expression patterns of endogenous SMN2 in 3813 cells
(data not shown). Transduction with vMU2-tsRNAHA resulted
in an increase in cytoplasmic SMN staining in GFP-positive cells
as well as an increase in SMN-positive gems, although not at lev-
els correlative with the efficiency of the RT-PCR data (data not
shown). Taken together these results demonstrate that endoge-
nous SMN2 transcripts can be targeted for trans-splicing, result-
ing in the production of full-length SMN from the SMN2 gene.
second-generation tsRNA: restoration
of sMN function
During the analysis of the initial panel of tsRNA molecules, it
became apparent that levels of SMN RNAs that were trans-spliced
did not translate into comparable levels of SMN-HA protein.
We speculated that the C-terminal HA-epitope tag decreased
the stability of the SMN protein, consistent with our analysis of
C-terminal SMN-GFP fusions (C.L. Lorson and E.J. Androphy,
unpublished results). While the epitope tag provided a convenient
method of detection at RNA and protein levels, a more suitable
vector for potential SMA therapeutics would lack extraneous epit-
opes such as HA. Therefore, a tsRNA was developed that encoded
only the SMN exon 7 peptide. However, to allow detection of the
trans-splicing event, the M13 primer sequence was engineered
immediately downstream from the normal SMN stop codon
within the tsRNAM13.
Consistent with the analysis of the tsRNAHA vector, the
tsRNAM13 vector specifically generated a SMN trans-spliced RNA
species that was detected by RT-PCR using primer pairs SMN
exon 5 Fwd and M13-Rev (Figure 6a). To determine whether
tsRNAM13 resulted in increased SMN protein levels, SMA fibro-
blasts were transduced with the recombinant AAV2 vector vMU2-
tsRNAM13. Fifteen days post-infection, SMN levels were analyzed
by immunofluorescence and Western blot. Treatment with the
vMU2-tsRNAM13 resulted in an increase of gems to approximately
45 per 100 nuclei, significantly above untreated fibroblasts and
cells infected with vMU2 or vMU2KO-tsRNAM13 (Figure 6b and c).
Additionally, in vMU2-tsRNAM13-treated cells, SMN co-localized
with members of the Gemin complex in more than 90% of gems
(Figure 6b and data not shown). Western blot analysis of extracts
from SMA fibroblasts 15 days post-infection also demonstrated
a significant increase in SMN levels as evidenced by the sixfold
to eightfold increase in total SMN protein in rAAV2-tsRNAM13
treated extracts compared to untreated SMA patient fibroblasts
(Figure 6d). In comparison, fibroblast extracts from untreated
and the rAAV2KO-tsRNAM13 vector contained expectedly lower
levels of SMN protein (Figure 6d).
Although the specific SMN function associated with the
development of SMA is still unknown, the SMN protein performs
a well-described role in small nuclear ribonucleoprotein (snRNP)
assembly.33 To determine whether snRNP assembly activity can
Exon 6 Fwd
Exon 8 Rev
Figure 5 endogenous survival motor neuron-2 (sMN2) transcripts
are trans-spliced following vMU2-tsRNAHA transduction in primary
sMA fibroblasts. 3813 cells transduced at a multiplicity of infection
of 100 with vMU2-tsRNAHA were harvested 8 days post-infection and
total RNA was isolated. Reverse transcription polymerase chain reaction
analysis was performed with the indicated primers (as described above)
and expected products are indicated (FL, full-length SMN; ∆7, SMN∆7;
trans-spliced product: Trans*). Two independent experiments are shown
(“A” and “B” lanes). HA, hemagglutinin; tsRNA, trans-splicing RNA.
© The American Society of Gene Therapy
Restoration of SMN Function
be increased by SMN trans-splicing in the SMN-deficient cellu-
lar extracts derived from SMA fibroblasts, cells were transfected
with the pMU2-tsRNAM13 plasmid or the negative control plasmid
(pMU2KO-tsRNAM13). As expected, HeLa cell extracts that contain
high levels of endogenous SMN supported high levels of snRNP
assembly and failed to appreciably assemble on the negative
control snRNA that lacks that Sm assembly site (Figure 7a). In
extracts from either untransfected SMA fibroblasts, or fibroblasts
transfected with pMU2KO-tsRNAM13, or salmon sperm DNA, Sm
assembly was at expectedly low levels. In contrast, transfection
of the pMU2-tsRNAM13 plasmid resulted in a marked increase
in snRNP assembly, demonstrating the trans-splicing results in
full-length SMN transcripts, but more importantly, resulted in
functional SMN protein. Importantly, snRNP assembly was simi-
larly elevated in extracts of 3813 cells that were infected with the
vMU2-tsRNAM13 viral vector (Figure 7b), demonstrating the
potential usefulness of a gene therapy approach using the trans-
splicing mechanism. This was a highly specific reaction as cold
100 bp ladder
Gems per 100 GFP+ cells
Figure 6 second-generation of trans-splicing RNAs efficiently redi-
rect pre-mRNA splicing and promote functional full-length survival
motor neuron (sMN) expression. (a) Total RNA was isolated from HeLa
cells transfected with 2 µg pMU2-tsRNAM13 48 hours post-transfection.
Reverse transcription polymerase chain reaction was performed with
SMN exon 5 forward and M13 reverse, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) specific primers. (b) Spinal muscular atrophy
fibroblasts (3813 cells) were transduced at an multiplicity of infection
(MOI) of 100 with vMU2KO-tsRNAM13 and vMU2-tsRNAM13; immunofluo-
rescence was used to identify SMN (green), Gemin2 (red), and green fluo-
rescent protein (GFP) (grayscale). GFP was shown in grayscale and SMN/
Gemin2 were pseudo-colored (SMN: AMCA) to allow for easier visual-
ization of the merged and overlapping images. (c) Semi-quantitative
analysis of gem distribution following trans-splicing. Gem numbers were
analyzed in untreated 3813 cells or cells infected with vMU2, vMU2KO-
tsRNAM13, or vMU2-tsRNAM13. Values represent the average from three
independent experiments in which 100 GFP-positive (indicative of infec-
tion) cells were analyzed by immunofluorescence for SMN staining
in gems. (d) Western blot analysis demonstrates an increase in SMN
protein in 3813 cells following transduction with recombinant adeno-
associated virus (rAAV) vectors. Extracts from 3814 (untreated) and
3813 cells (untreated) or infected with rAAV vectors (vMU2KO-tsRNAM13,
vMU2-tsRNAM13) at an MOI of 100 were harvested 15 days post-infection
and resolved by Western blotting. Monoclonal antibodies were used to
identify SMN and actin. bp, base pair; tsRNA, trans-spliced RNA.
SMA fibroblasts (3813)
HeLa SMA patient fibroblasts
b a ba b a b a b
Figure 7 Trans-splicing derived survival motor neuron (sMN) pro-
tein functions in small nuclear ribonucleoprotein (snRNP) assembly.
(a) Stimulation of SMN function in snRNP assembly assays. Radio-labeled
small nuclear RNA (snRNA) templates (WT, “a” lanes; and ∆Sm, “b” lanes)
were incubated with cytoplasmic fractions from HeLa or 3813 cells that
were untreated or transfected for 48 hours with 2 µg of salmon sperm DNA
(ssDNA), pMU2KO-tsRNAM13, pMU2-tsRNAM13. Complexes were immunopre-
cipitated with an anti-Sm antibody (Y12) and resolved on an 8% sequenc-
ing gel; (b) Virally derived tsRNAs stimulate snRNP assembly. Extracts from
uninfected HeLa cells, or 3813 cells infected with vMU2KO-tsRNAM13, vMU2-
tsRNAM13 vectors at 14 days post-infection. snRNP assembly was performed
as described above; (c) snRNP assembly is highly specific. In extracts from
3813 cells infected with vMU2-tsRNAM13 at 14 days post-infection, snRNP
assembly assays were performed in the presence of increasing concentra-
tions of unlabeled U1snRNA (U1snRNA*) or an amount of transfer RNA
(tRNA) equivalent to the highest concentration of U1snRNA. SMA, spinal
muscular atrophy; tsRNA, trans-splicing RNA.
www.moleculartherapy.org? ? ?
© The American Society of Gene Therapy
Restoration of SMN Function
U1snRNA efficiently completed the SMN activity in extracts
from vMU2-tsRNAM13 infected cells, while high levels of transfer
RNA failed to inhibit snRNP assembly (Figure 7c). Overall, these
results demonstrate that trans-splicing can not only increase SMN
protein levels from the endogenous SMN2 gene but the protein
derived from the trans-splicing event restores a critical SMN func-
tion in patient fibroblasts.
Recently trans-splicing has been envisioned as a potential thera-
peutic intervention for a variety of genetic disease states.18,19 The
potential effectiveness of this strategy has been demonstrated in
disease contexts including cystic fibrosis, hyper-IgM X-linked
immunodeficiency, and hemophilia A.20,34,35 While the majority of
these studies have been performed using plasmid-derived tsRNAs
in tissue culture or in bronchial xenografts,20,23,36–38 one report
has demonstrated that rAAV can be used to efficiently deliver a
trans-splicing molecule in a cystic fibrosis model.22 Additionally,
an adenovirus-based delivery system for trans-splicing RNAs effi-
ciently corrected the hemophilia A phenotype in a mouse model
of disease.34 Since SMN2-derived transcripts are inherently stable
and the SMA patient population universally express high levels of
SMN∆7 transcript, strategies designed to elevate full-length SMN
protein expression from SMN2, such as a trans-splicing approach,
hold promise within the context of SMA.
The genetic context of SMA represents an exciting prospect for
a number of therapeutic approaches including trans-splicing. In
other examples of trans-splicing that have been shown to be effec-
tive, the exons that were targeted were: (i) constitutively expressed
or (ii) wild-type alternatively spliced in which all of the native
endogenous splice signals were intact and fully functional. In the
SMN2 gene, the intrinsic quality of the exon 7 splice acceptor site
is reduced due to the C/T transition. Therefore, the competition
between SMN2 cis-splicing and trans-splicing is likely reduced,
providing a potential advantage to trans-splicing in the SMN con-
text compared to other alternatively regulated exons that retain
fully functional splice sites.
This is the first series of SMN trans-splicing RNAs; however,
it was clear that the expression from a strong CMV promoter
resulted in significantly higher levels of SMN trans-splicing than
the pMU2 U6 promoter-driven RNAs. Additionally, the inclusion
of the previously described modified U7 stem-loop and consensus
Sm core binding motif did not markedly increase the efficiency of
the trans-splicing reaction.26,27 This sequence has been reported to
stabilize RNAs, such as anti-sense RNAs, and promote the incor-
poration of these artificial RNAs into the spliceosome, thereby
increasing their potency as RNA splicing modifiers. Since SMN
exon 7 splicing is dependent upon splice enhancer sequences, one
possible means to increase trans-splicing efficiency would be to
improve the relative strength of the exonic splice enhancers within
the trans SMN exon 7 sequence without altering the overlapping
SMN coding sequence.
The use of viral vectors as a means to deliver various fac-
tors with the ultimate goal of developing a gene therapy is an
ever-expanding and accelerating field. We chose the AAV
vector as our mode of delivery for many reasons but not with-
out understanding certain potential drawbacks. Wild-type AAV
can integrate into chromosomal DNA at a specific location on
chromosome 19; however, recombinant AAV can only integrate
randomly and does so at such a low incidence that this activ-
ity is negligible.39 The majority of the rAAV genomes exist as
episomal concatemers within dividing or non-dividing cells.39
Importantly, from an SMA perspective, rAAV2 has a high tro-
pism for neurons and muscle cells and can be retrogradely trans-
ported to neurons in vivo. The characterization of serotypes
with greater neuronal specificity or pseudo-typed vectors with
neuronal-specific peptides are promising areas of gene therapy
investigation.40,41 Efficient retrograde delivery of the full-length
SMN complementary DNA has been reported using a rabies-G
pseudo-typed Lentiviral vector in a mouse model of SMA.42 One
of the benefits of trans-splicing is that the levels of SMN would
be regulated based upon the endogenous SMN promoter since
the trans-splicing RNA does not encode a functional protein
prior to the trans-splicing event with the endogenous transcript.
The introduction of SMN trans-splicing RNAs and the com-
parison between full-length SMN gene rescue experiments is an
important step forward in determining the potential application
of trans-splicing within the context of SMA.
MATeRIAls AND MeTHODs
Plasmids and cloning. The parental pMU2 vector17 was used to create the
series of tsRNA expression vectors. The CMV promoter was cloned upstream
of the tsRNA sequences. The original tsRNA was produced with the Expand
High Fidelity PCR kit (Roche, AG, Basel, Switzerland) by amplifying a region
of SMN Intron 6 (In6 Rev: 5′-GATCAAGCTTGGAAAATAAAGGAA
GTTAAAAAAAATAGC-3′; In6 Fwd: 5′-GATCGGATCCCGCGGCTGA
TCATATTTTGTTGAATAAAATAAG-3′) and annealing DNA oligo-
nucleotides (Spacer Top: 5′-GGAACATTATTATAACGTTGCTCGAAT
ACTAAGTGGTACCC-3′; Spacer Bottom: 5′-TCGAGGGTACCAGTT
AGTATTCGAGCAACGTTATAATAATGTTCCGC-3′; Splice Site Top:
CC-3′; Splice Site Bottom: 5′-TCGAGGGATCCATTTAAGGAATGTGA
GATATCAAAAAAAAAAGAAGAGGTAC-3′; HA Top: 5′-GATCCTA
C C C ATAC G AC G T T C C AG AT TAC G C T TAC C C ATAC
GACGTTCCAGATTACGCTTAAC-3′; HA Bottom: 5′-TCGAGTTA
GTCGTATGGGTAG-3′) and directionally cloning into pMU2 plasmid
intermediates. The tsRNA was cloned into the vector pMU2 using AvrII
and SacI sites.
Modifications to pMU2-tsRNA were created using overlapping PCR
mutagenesis using pMU2-NheI Fwd#1 (5′-GCGCTAGCGGATCTGAC
GGTTCAC-3′) and pMU2-SalI Rev (5′-TTAATTAACAATTGGTCGA
CAGCC-3′). Fragments were re-cloned into the pMU2 and pMU2CMV
using NsiI and SalI sites. The HA tag was deleted using primers M13
TAATTTAAGGAATGTGAG-3′; M13 Reverse 5′-CTCACATTCCTTA
The SMN1 and SMN2 mini-genes (pSMN1, pSMN2) have been previously
Transfections. The transfections were performed using linear polyethyl-
enimine at 50 pmol/l of linear polyethylenimine per 1.0 μg of DNA which
correlates to a nitrogen to phosphorous ratio of 22.3:1. pMU2 clones were
transfected into HeLa or HEK293 cells grown to 80% confluence before
transfection. tsRNA plasmids were diluted in a 150 mmol/l NaCl solution
before adding filtered 7.5 mmol/l polyethylenimine. Cells were incubated
© The American Society of Gene Therapy
Restoration of SMN Function
with plasmid overnight and re-fed with fresh Dulbecco’s modified Eagle’s
medium and harvested 48 hours post-transfection. Controls include
salmon sperm DNA equilibrated for total DNA transfection loads. Trans-
fection of 3813 cells was performed with Lipofectamine 2000 in 3:1 ratio of
lipofectamine to µg of DNA.
RT-PCR. 48 hours post transfection or transduction, total RNA was harvested
from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to
the manufacturer’s instruction. Total RNA concentration was determined
and normalized before complementary DNA synthesis. RT-PCR was per-
formed as previously described.17 The trans-splicing product was amplified
using a reverse primer that overlaps the end of SMN exon 7 and a portion
of the HA tag (5′-AGCGTAATCTGGAACGTCGTATGGGTAGGATCCA
TTTCAAGGAAT-3′), the M13 reverse primer (5′-GTCATAGCTGTTTCC
TGCGAC-3′), and a mini-gene specific primer, pCI-Fwd#1.5 The mini-
gene transcripts were amplified using pCI-Fwd#1 and pCI-Rev.5 The
primers used in endogenous PCR amplifications were an SMN Exon 6-
Forward (5′-CCCCCACCACCTCCCATATGT-3’), SMN exon 5-Forward
(5′-GGACCAGGAAAGCCAGGTCTAAAATTC-3′), and SMN Exon
8-Reverse (5′-AGTGGTGTCATTTAGTGCTGC-3′). Mini-gene-derived
transcripts were amplified using the following conditions: 93 °C for 1:30;
90 °C for 1:00; 58 °C for 1:30; 72 °C for 1:30; repeat steps 2–4 for 25 cycles;
72 °C for 5:00. Endogenous transcripts were amplified using the follow-
ing conditions: 94 °C for 5:00; 94 °C for 0:45; 60 °C for 1:00; 68 °C for 2:00;
repeat steps 2–4 for 30 cycles; 68 °C for 5:00. Representative results are
shown and have been repeated at least three different times.
rAAV Production. Plasmid DNA from each tsRNA clone was triple trans-
fected with the previously described pXX6 (encodes: VA, E2A and E4)
and pAD8 (encodes: AAV2 Rep and Cap proteins) helper plasmids into
HEK 293 XDC cells43,44 using 7.5 mmol/l PEI. Transfections were done in
a 1:1:3 molar ratio of plasmids pMU2 derived clones, pAD8, and pXX6,
respectively. A total concentration of 10 μg DNA was delivered per 100 mm
dish. 293 cells containing vMU2 derivatives were harvested 48 hours later
and subjected to three freeze thaw cycles to extract the virus from living
cells. The cycles were performed using a liquid nitrogen bath followed by
10 minutes at 37 °C, then vortexing for 2 additional minutes. To separate
the virus from the cellular debris, samples were centrifuged twice at 9 K
for 10 minutes at 4 °C. Supernatant was separated from the pellet and
stored at –20 °C in 350 μl aliquots. The multiplicity of infection was deter-
mined using HeLa cells and scoring GFP expression 2 days post infection.
1.5 × 103 HeLa cells were plated and infected 24 hours later with 25, 50, and
100 μl of pellet supernatant. Titer units were averaged across 3 dilutions of
virus and over a total area of 9 sq. mm.
Immunofluorescence microscopy. SMA patient fibroblasts (Coriell Cell
Repositories, Camden, NJ; 3813 cells) were plated at a density of 2 × 103
and infected with vector genomes to produce a multiplicity of infection
of 100. Infections occurred at room temperature for 30 minutes with the
appropriate volume of virus supernatant and the total volume of cells
diluted in complete Dulbecco’s modified Eagle’s medium at 37 °C. Cells
were incubated at 37 °C for additional 7–15 days. Prior to harvest, samples
were washed twice in phosphate-buffered saline pH7.5. Fixation of cells
was performed using cold acetone/methanol (50:50).17 5% bovine serum
albumin was used as a blocking medium for 1 hour; then the samples
were washed in phosphate-buffered saline pH7.0. Primary antibody was
diluted 1:50 in 1.5% phosphate-buffered saline (rabbit anti-HA; Santa
Cruz, CA, sc-Y11; mouse anti-PML; Santa Cruz, CA, sc-966; anti-SMN,
4B7).45 Secondary antibodies (goat anti-rabbit AlexaFlor-350: Molecular
Probes, Eugene, OR; goat anti-mouse Texas Red-594: Jackson Immuno-
Research Laboratories, West Grove, PA; goat anti-rabbit TRITC: Sigma,
St. Louis, MO) were used according to manufacturer’s instructions. Cells
were washed with phosphate-buffered saline and nuclei stained with
4′,6-diamidino-2-phenylindole.45 Microscope images were captured on a
Nikon Eclipse E1000 using Meta-Morph software (Tokyo, Japan). SMN
and gemin co-localization were performed with 4B7 and either Gemin2
or Gemin3 antibodies (BD Biosciences, San Jose, CA). Gem counts were
performed in triplicate counting 100 GFP-positive fibroblasts. Total SMN/
gem per nucleus scores were performed in triplicate counting 100 GFP
Western blot. HeLa cell pellets were prepared in RSB-100 lysis buffer
(100 mmol/l NaCl; 20 mmol/l Tris–HCl pH 7.4; 2.5 mmol/l MgCl; 0.01%
NP40) and centrifuged at 10,000g for 5 minutes at 4 °C. Supernatant was
removed and used for subsequent immunoprecipitation experiments as
previously described.46 Beads and lysate were incubated overnight and
washed 5 times over 1 hour. Samples were boiled in loading dye and resolved
in a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Blots
were probed with a 1:100 dilution of an mouse anti-SMN monoclonal
antibody, 4B7,45 and visualized with a horeseradish peroxidase-conjugated
rabbit anti-mouse antibody. Images were captured and quantitated using a
Fuji Imager and Multi-Gauge V2.3 system (Valhalla, NY). Representative
results are shown and have been repeated at least three different times.
In vitro snRNP assembly reactions. Cloned U1 snRNA (ΔSm) complemen-
tary DNA has the Smith core site (5′-AUUUGUGG-3′) sequence deleted
to inhibit non-specific products.47 The plasmids containing the U1 snRNAs
(pT7-WT or -ΔSm) were linearized with EcoRI (New England Biolabs,
Ipswich, MA). Linear plasmid, T7 polymerase (Promega, Madison, WI),
and ribonucleotide triphosphates supplemented with P32 labeled rUTP
were incubated for 90 minutes at 37 °C. 3 µl RNAsin was added and incu-
bated for an additional 30 minutes. The radio-labeled probe was phenol-
chloroform extracted and ethanol precipitated twice. Cells were harvested
and lysed in RSB-100 buffer, and nuclei were removed by centrifugation
at 1,000 rpm for 15 minutes at 4 °C. The S100 fraction was removed and
(7X) protease inhibitor cocktail (Roche, AG, Basel, Switzerland) added
to preserve the SMN complex. To assemble the Sm core, 20 U of RNA-
sin (Promega, Madison, WI) was added to S100 extract with 2.5 mmol/l
adenosine triphosphate yeast transfer RNA and radio-labeled U1 snRNA
(100,000 cpm) for 20 minutes at 30 °C. Anti-mouse Y12 antibody immuno-
precipitated Sm core proteins (NeoMarkers, Fremont, CA). 500 µl of RSB-
500 salt buffer (500 mmol/l NaCl; 20 mmol/l Tris–HCl pH 7.4; 2.5 mmol/l
MgCl; 0.01% NP40) was used in the wash steps to clean up bead frac-
tions (Protein A/G Sephrose, Oncogene Research Products, Darmstadt,
Germany). RNAs were denatured in (5×) formamide loading dye and run
on an 8% TBE-Urea gel. Quantitative measurements of snRNP products
were obtained after a 2-hour exposure on a phospho-screen. Bands were
imaged using Fuji-Imager and normalized to background.
We would like to thank G. Matera for the generous gift of the U1sn-
RNA plasmid. M.S. is supported by a Developmental Grant from the
Muscular Dystrophy Association. This work was funded by grants from
the MU Research Board grant, MU College of Veterinary Medicine
and the National Institutes of Health (C.L.L., R01 NS41584; G.T., R01
NS444494). No conflicts of interest exist for the authors.
1. Crawford, TO and Pardo, CA (1996). The neurobiology of childhood spinal muscular
atrophy. Neurobiol Dis 3: 97–110.
2. Lefebvre, S, Burglen, L, Reboullet, S, Clermont, O, Burlet, P, Viollet, L et al. (1995).
Identification and characterization of a spinal muscular atrophy-determining gene.
Cell 80: 155–165.
3. Gennarelli, M, Lucarelli, M, Capon, F, Pizzuti, A, Merlini, L, Angelini, C et al. (1995).
Survival motor neuron gene transcript analysis in muscles from spinal muscular
atrophy patients. Biochem Biophys Res Commun 213: 342–348.
4. Cartegni, L and Krainer, AR (2002). Disruption of an SF2/ASF-dependent exonic
splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1.
Nat Genet 30: 377–384.
5. Lorson, CL, Hahnen, E, Androphy, EJ and Wirth, B (1999). A single nucleotide in the
SMN gene regulates splicing and is responsible for spinal muscular atrophy.
Proc Natl Acad Sci U S A 96: 6307–6311.
www.moleculartherapy.org? ? ?
© The American Society of Gene Therapy
Restoration of SMN Function
6. Monani, UR, Lorson, CL, Parsons, DW, Prior, TW, Androphy, EJ, Burghes, AH et al.
(1999). A single nucleotide difference that alters splicing patterns distinguishes the
SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet 8: 1177–1183.
Sumner, CJ (2006). Therapeutics development for spinal muscular atrophy. NeuroRx
Hofmann, Y, Lorson, CL, Stamm, S, Androphy, EJ and Wirth, B (2000). Htra2-beta 1
stimulates an exonic splicing enhancer and can restore full-length SMN expression
to survival motor neuron 2 (SMN2). Proc Natl Acad Sci U S A 97: 9618–9623.
Hofmann, Y and Wirth, B (2002). hnRNP-G promotes exon 7 inclusion of survival
motor neuron (SMN) via direct interaction with Htra2-beta1. Hum Mol Genet
10. Lorson, CL and Androphy, EJ (2000). An exonic enhancer is required for inclusion
of an essential exon in the SMA-determining gene SMN. Hum Mol Genet
11. Kashima, T and Manley, JL (2003). A negative element in SMN2 exon 7 inhibits
splicing in spinal muscular atrophy. Nat Genet 34: 460–463.
12. Madocsai, C, Lim, SR, Geib, T, Lam, BJ and Hertel, KJ (2005). Correction of SMN2
Pre-mRNA splicing by antisense U7 small nuclear RNAs. Mol Ther 12: 1013–1022.
13. Lim, SR and Hertel, KJ (2001). Modulation of survival motor neuron pre-mRNA
splicing by inhibition of alternative 3′ splice site pairing. J Biol Chem
14. Sangiuolo, F, Filareto, A, Spitalieri, P, Scaldaferri, ML, Mango, R, Bruscia, E et al.
(2005). In vitro restoration of functional SMN protein in human trophoblast cells
affected by spinal muscular atrophy by small fragment homologous replacement.
Hum Gene Ther 16: 869–880.
15. Cartegni, L and Krainer, AR (2003). Correction of disease-associated exon skipping by
synthetic exon-specific activators. Nat Struct Biol 10: 120–125.
16. Skordis, LA, Dunckley, MG, Yue, B, Eperon, IC and Muntoni, F (2003).
Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that
stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci U S A
17. Baughan, T, Shababi, M, Coady, TH, Dickson, AM, Tullis, GE and Lorson, CL (2006).
Stimulating full-length SMN2 expression by delivering bifunctional RNAs via a viral
vector. Mol Ther 14: 54–62.
18. Garcia-Blanco, MA (2003). Messenger RNA reprogramming by spliceosome-mediated
RNA trans-splicing. J Clin Invest 112: 474–480.
19. Garcia-Blanco, MA, Baraniak, AP and Lasda, EL (2004). Alternative splicing in disease
and therapy. Nat Biotechnol 22: 535–546.
20. Liu, X, Jiang, Q, Mansfield, SG, Puttaraju, M, Zhang, Y, Zhou, W et al. (2002).
Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway
epithelia by spliceosome-mediated RNA trans-splicing. Nat Biotechnol 20: 47–52.
21. Rodriguez-Martin, T, Garcia-Blanco, MA, Mansfield, SG, Grover, AC, Hutton, M,
Yu, Q et al. (2005). Reprogramming of tau alternative splicing by
spliceosome-mediated RNA trans-splicing: implications for tauopathies.
Proc Natl Acad Sci U S A 102: 15659–15664.
22. Liu, X, Luo, M, Zhang, LN, Yan, Z, Zak, R, Ding, W et al. (2005).
Spliceosome-mediated RNA trans-splicing with recombinant adeno-associated
virus partially restores cystic fibrosis transmembrane conductance regulator
function to polarized human cystic fibrosis airway epithelial cells. Hum Gene Ther
23. Mansfield, SG, Kole, J, Puttaraju, M, Yang, CC, Garcia-Blanco, MA, Cohn, JA et al.
(2000). Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.
Gene Ther 7: 1885–1895.
24. Flotte, TR, Afione, SA, Solow, R, Drumm, ML, Markakis, D, Guggino, WB et al. (1993).
Expression of the cystic fibrosis transmembrane conductance regulator from a novel
adeno-associated virus promoter. J Biol Chem 268: 3781–3790.
25. Haberman, RP, McCown, TJ and Samulski, RJ (2000). Novel transcriptional regulatory
signals in the adeno-associated virus terminal repeat A/D junction element. J Virol
26. De Angelis, FG, Sthandier, O, Berarducci, B, Toso, S, Galluzzi, G, Ricci, E et al. (2002).
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 Delta 48-50 DMD cells. Proc Natl Acad Sci USA
27. Goyenvalle, A, Vulin, A, Fougerousse, F, Leturcq, F, Kaplan, JC, Garcia, L et al. (2004).
Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science
28. Lorson, CL, Strasswimmer, J, Yao, JM, Baleja, JD, Hahnen, E, Wirth, B et al. (1998).
SMN oligomerization defect correlates with spinal muscular atrophy severity.
Nat Genet 19: 63–66.
29. Young, PJ, Man, NT, Lorson, CL, Le, TT, Androphy, EJ, Burghes, AH et al. (2000).
The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in
self-association and SIP1 binding. Hum Mol Genet 9: 2869–2877.
30. Kaspar, BK, Llado, J, Sherkat, N, Rothstein, JD and Gage, FH (2003). Retrograde viral
delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301: 839–842.
31. Liu, Q, Fischer, U, Wang, F and Dreyfuss, G (1997). The spinal muscular atrophy
disease gene product, SMN, and its associated protein SIP1 are in a complex with
spliceosomal snRNP proteins. Cell 90: 1013–1021.
32. Coovert, DD, Le, TT, McAndrew, PE, Strasswimmer, J, Crawford, TO, Mendell, JR et al.
(1997). The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet
33. Yong, J, Wan, L and Dreyfuss, G (2004). Why do cells need an assembly machine for
RNA-protein complexes? Trends Cell Biol 14: 226–232.
34. Chao, H, Mansfield, SG, Bartel, RC, Hiriyanna, S, Mitchell, LG, Garcia-Blanco, MA
et al. (2003). Phenotype correction of hemophilia A mice by spliceosome-mediated
RNA trans-splicing. Nat Med 9: 1015–1019.
35. Tahara, M, Pergolizzi, RG, Kobayashi, H, Krause, A, Luettich, K, Lesser, ML et al.
(2004). Trans-splicing repair of CD40 ligand deficiency results in naturally regulated
correction of a mouse model of hyper-IgM X-linked immunodeficiency. Nat Med
36. Mansfield, SG, Clark, RH, Puttaraju, M, Kole, J, Cohn, JA, Mitchell, LG et al. (2003).
5’ exon replacement and repair by spliceosome-mediated RNA trans-splicing. RNA
37. Puttaraju, M, DiPasquale, J, Baker, CC, Mitchell, LG and Garcia-Blanco, MA (2001).
Messenger RNA repair and restoration of protein function by spliceosome-mediated
RNA trans-splicing. Mol Ther 4: 105–114.
38. Puttaraju, M, Jamison, SF, Mansfield, SG, Garcia-Blanco, MA and Mitchell, LG (1999).
Spliceosome-mediated RNA trans-splicing as a tool for gene therapy. Nat Biotechnol
39. Flotte, TR (2004). Gene therapy progress and prospects: recombinant
adeno-associated virus (rAAV) vectors. Gene Ther 11: 805–810.
40. Liu, JK, Teng, Q, Garrity-Moses, M, Federici, T, Tanase, D, Imperiale, MJ et al. (2005).
A novel peptide defined through phage display for therapeutic protein and vector
neuronal targeting. Neurobiol Dis 19: 407–418.
41. Choi, VW, McCarty, DM and Samulski, RJ (2005). AAV hybrid serotypes: improved
vectors for gene delivery. Curr Gene Ther 5: 299–310.
42. Azzouz, M, Le, T, Ralph, GS, Walmsley, L, Monani, UR, Lee, DC et al. (2004).
Lentivector-mediated SMN replacement in a mouse model of spinal muscular atrophy.
J Clin Invest 114: 1726–1731.
43. Matsushita, T, Elliger, S, Elliger, C, Podsakoff, G, Villarreal, L, Kurtzman, GJ et al.
(1998). Adeno-associated virus vectors can be efficiently produced without helper
virus. Gene Ther 5: 938–945.
44. Xiao, X, Li, J and Samulski, RJ (1998). Production of high-titer recombinant
adeno-associated virus vectors in the absence of helper adenovirus. J Virol
45. Wolstencroft, EC, Mattis, V, Bajer, AA, Young, PJ and Lorson, CL (2005).
A non-sequence-specific requirement for SMN protein activity: the role of
aminoglycosides in inducing elevated SMN protein levels. Hum Mol Genet
46. Young, PJ, Day, PM, Zhou, J, Androphy, EJ, Morris, GE and Lorson, CL (2002). A direct
interaction between the survival motor neuron protein and p53 and its relationship to
spinal muscular atrophy. J Biol Chem 277: 2852–2859.
47. Shpargel, KB and Matera, AG (2005). Gemin proteins are required for efficient
assembly of Sm-class ribonucleoproteins. Proc Natl Acad Sci USA 102: 17372–17377.