Restoration of SMN Function: Delivery of a Trans-splicing RNA Re-directs SMN2 Pre-mRNA Splicing
Authors: Tristan H Coady, Monir Shababi, Gregory E Tullis, Christian L Lorson
Journal: Molecular Therapy (2007)

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. 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. IntroductionSpinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and the leading genetic cause of death in children under the age of two.1Crawford TO Pardo CA The neurobiology of childhood spinal muscular atrophy.Neurobiol Dis. 1996; 3: 97-110Crossref PubMed Scopus (399) Google Scholar SMA is caused by the homozygous loss of survival motor neuron 1 (SMN1).2Lefebvre S Burglen L Reboullet S Clermont O Burlet P Viollet L et al.Identification and characterization of a spinal muscular atrophy-determining gene.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2938) Google Scholar In humans, two nearly identical copies of the SMN gene exist in chromosome 5, designated SMN1 and SMN2.2Lefebvre S Burglen L Reboullet S Clermont O Burlet P Viollet L et al.Identification and characterization of a spinal muscular atrophy-determining gene.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2938) Google Scholar SMN2 is distinguished from SMN1 by a small number of non-polymorphic nucleotide differences; however, the critical distinction between the two genes is at the RNA processing level: SMN1 produces exclusively full-length transcripts, while SMN2 primarily produces an alternatively spliced transcript lacking the final coding exon (exon 7).2Lefebvre S Burglen L Reboullet S Clermont O Burlet P Viollet L et al.Identification and characterization of a spinal muscular atrophy-determining gene.Cell. 1995; 80: 155-165Abstract Full Text PDF PubMed Scopus (2938) Google Scholar,3Gennarelli M Lucarelli M Capon F Pizzuti A Merlini L Angelini C et al.Survival motor neuron gene transcript analysis in muscles from spinal muscular atrophy patients.Biochem Biophys Res Commun. 1995; 213: 342-348Crossref PubMed Scopus (176) Google Scholar A single silent nucleotide difference disrupts an exonic splice enhancer in SMN2 exon 7 and dictates the splicing patterns for these closely related genes.4Cartegni L Krainer AR Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1.Nat Genet. 2002; 30: 377-384Crossref PubMed Scopus (570) Google Scholar,5Lorson CL Hahnen E Androphy EJ Wirth B A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.Proc Natl Acad Sci U S A. 1999; 96: 6307-6311Crossref PubMed Scopus (1136) Google Scholar,6Monani UR Lorson CL Parsons DW Prior TW Androphy EJ Burghes AH et al.A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2.Hum Mol Genet. 1999; 8: 1177-1183Crossref PubMed Scopus (716) Google Scholar Since the non-polymorphic nucleotide differences do not alter the coding capacity of SMN2, this gene has been envisioned as an ideal target for a variety of therapeutic strategies.7Sumner CJ Therapeutics development for spinal muscular atrophy.NeuroRx. 2006; 3: 235-245Crossref PubMed Scopus (83) Google ScholarSMN exon 7 pre-messenger RNA (pre-mRNA) splicing is extensively regulated through a complex interplay between positively 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.4Cartegni L Krainer AR Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1.Nat Genet. 2002; 30: 377-384Crossref PubMed Scopus (570) Google Scholar,8Hofmann Y Lorson CL Stamm S Androphy EJ Wirth B 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. 2000; 97: 9618-9623Crossref PubMed Scopus (278) Google Scholar,9Hofmann Y Wirth B hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1.Hum Mol Genet. 2002; 11: 2037-2049Crossref PubMed Google Scholar,10Lorson CL Androphy EJ An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN.Hum Mol Genet. 2000; 9: 259-265Crossref PubMed Google Scholar SF2/ASF, a serine/arginine-rich protein, binds an enhancer that overlaps the critical C/T transition, and binds only in the SMN1 context.4Cartegni L Krainer AR Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1.Nat Genet. 2002; 30: 377-384Crossref PubMed Scopus (570) Google Scholar 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.11Kashima T Manley JL A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy.Nat Genet. 2003; 34: 460-463Crossref PubMed Scopus (422) Google Scholar Additional RNA sequences influence SMN alternative splicing, such as an extended inhibitory context and novel intronic repressors 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 7.12Madocsai C Lim SR Geib T Lam BJ Hertel KJ Correction of SMN2 Pre-mRNA splicing by antisense U7 small nuclear RNAs.Mol Ther. 2005; 12: 1013-1022Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,13Lim SR Hertel KJ Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3′ splice site pairing.J Biol Chem. 2001; 276: 45476-45483Crossref PubMed Scopus (136) Google Scholar,14Sangiuolo F Filareto A Spitalieri P Scaldaferri ML Mango R Bruscia E et al.In vitro restoration of functional SMN protein in human trophoblast cells affected by spinal muscular atrophy by small fragment homologous replacement.Hum Gene Ther. 2005; 16: 869-880Crossref PubMed Scopus (21) Google Scholar For example, oligonucleotides that disable the SMN exon 8 splice site increase the usage of the exon 7 splice site.12Madocsai C Lim SR Geib T Lam BJ Hertel KJ Correction of SMN2 Pre-mRNA splicing by antisense U7 small nuclear RNAs.Mol Ther. 2005; 12: 1013-1022Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,13Lim SR Hertel KJ Modulation of survival motor neuron pre-mRNA splicing by inhibition of alternative 3′ splice site pairing.J Biol Chem. 2001; 276: 45476-45483Crossref PubMed Scopus (136) Google Scholar 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.15Cartegni L Krainer AR Correction of disease-associated exon skipping by synthetic exon-specific activators.Nat Struct Biol. 2003; 10: 120-125Crossref PubMed Scopus (246) Google Scholar 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.16Skordis LA Dunckley MG Yue B Eperon IC Muntoni F 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: 4114-4119Crossref PubMed Scopus (225) Google Scholar,17Baughan T Shababi M Coady TH Dickson AM Tullis GE Lorson CL Stimulating full-length SMN2 expression by delivering bifunctional RNAs via a viral vector.Mol Ther. 2006; 14: 54-62Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarRecently a novel therapeutic approach has been developed, based upon the principle of trans-splicing.18Garcia-Blanco MA Messenger RNA reprogramming by spliceosome-mediated RNA trans-splicing.J Clin Invest. 2003; 112: 474-480Crossref PubMed Scopus (62) Google Scholar,19Garcia-Blanco MA Baraniak AP Lasda EL Alternative splicing in disease and therapy.Nat Biotechnol. 2004; 22: 535-546Crossref PubMed Scopus (430) Google Scholar Conceptually, this strategy relies upon pre-mRNA splicing occurring between two different molecules: (i) the mutant endogenous RNA and (ii) the exogenous therapeutic RNA that provides the correct RNA sequence via a trans-splicing event. Trans-splicing has been shown to efficiently replace constitutively spliced as well as alternatively spliced exons.20Liu X Jiang Q Mansfield SG Puttaraju M Zhang Y Zhou W et al.Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.Nat Biotechnol. 2002; 20: 47-52Crossref PubMed Scopus (512) Google Scholar,21Rodriguez-Martin T Garcia-Blanco MA Mansfield SG Grover AC Hutton M Yu Q et al.Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.Proc Natl Acad Sci U S A. 2005; 102: 15659-15664Crossref PubMed Scopus (53) Google Scholar This technology has been applied to several disease contexts including cystic fibrosis in which the common CFTRΔF508 mutation was efficiently replaced with the wild-type sequence via a trans-splicing reaction.20Liu X Jiang Q Mansfield SG Puttaraju M Zhang Y Zhou W et al.Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing.Nat Biotechnol. 2002; 20: 47-52Crossref PubMed Scopus (512) Google Scholar,22Liu X Luo M Zhang LN Yan Z Zak R Ding W et al.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. 2005; 16: 1116-1123Crossref PubMed Scopus (60) Google Scholar,23Mansfield SG Kole J Puttaraju M Yang CC Garcia-Blanco MA Cohn JA et al.Repair of CFTR mRNA by spliceosome-mediated RNA trans-splicing.Gene Ther. 2000; 7: 1885-1895Crossref PubMed Scopus (84) Google Scholar 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.21Rodriguez-Martin T Garcia-Blanco MA Mansfield SG Grover AC Hutton M Yu Q et al.Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.Proc Natl Acad Sci U S A. 2005; 102: 15659-15664Crossref PubMed Scopus (53) Google ScholarIn this report, the goal was to develop a trans-splicing system that results in an increase in full-length SMN protein expression from the SMN2 gene. Since SMN2-derived transcripts are inherently stable and the SMA patient population universally expresses high levels of SMNΔ7 transcript, we sought to determine whether SMN exon 7 was a tractable target for the development of an exon 7 trans-splicing system.ResultsDevelopment of a trans-splicing system for SMN2Trans-splicing has previously been implemented to overcome genetic defects by re-directing pathogenic RNA splicing defects.18Garcia-Blanco MA Messenger RNA reprogramming by spliceosome-mediated RNA trans-splicing.J Clin Invest. 2003; 112: 474-480Crossref PubMed Scopus (62) Google Scholar To determine whether SMN2 exon 7 was a tractable target for trans-splicing, a trans-splicing molecule was developed that consists of the following: a SMN intron 6 annealing sequence comprising 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 epitope. The initial analysis was performed using a SMN2 mini-gene, pSMN2, and the trans-spliced RNA (tsRNA)-expressing plasmid, 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.5Lorson CL Hahnen E Androphy EJ Wirth B A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy.Proc Natl Acad Sci U S A. 1999; 96: 6307-6311Crossref PubMed Scopus (1136) Google Scholar 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 transcripts (pCI-Fwd and HA-Rev) (Figure 1).SMN2 is a tractable target for trans-splicingTo 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 produced predominately the SMNΔ7 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 transcriptional activity of the adeno-associated virus-2 (AAV2) terminal repeats located upstream of the tsRNA start site and have previously been shown to mediate transcriptional activity.24Flotte TR Afione SA Solow R Drumm ML Markakis D Guggino WB et al.Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.J Biol Chem. 1993; 268: 3781-3790Abstract Full Text PDF PubMed Google Scholar,25Haberman RP McCown TJ Samulski RJ Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.J Virol. 2000; 74: 8732-8739Crossref PubMed Scopus (96) Google ScholarFigure 2Identification 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 polymerase 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; "Δ7", SMNΔ7) 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, complementary DNA; Fwd, forward; Rev, reverse; tsRNA, trans-splicing RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To confirm that trans-splicing had occurred, the predicted trans-spliced RT-PCR product (Figure 2a) was excised and 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 molecular 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 structure 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.26De Angelis FG Sthandier O Berarducci B Toso S Galluzzi G Ricci E 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 Delta 48-50 DMD cells.Proc Natl Acad Sci USA. 2002; 99: 9456-9461Crossref PubMed Scopus (110) Google Scholar,27Goyenvalle A Vulin A Fougerousse F Leturcq F Kaplan JC Garcia L et al.Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping.Science. 2004; 306: 1796-1799Crossref PubMed Scopus (405) Google Scholar We compared the efficiency of each plasmid 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 efficiently 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 SMN trans-splicing.Figure 3Cytomegalovirus-driven trans-splicing RNA (tsRNA) mediates survival motor neuron (SMN) trans-splicing in a dose-dependent manner. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Endogenous SMN transcripts can be re-directed by trans-splicingTo 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 transcripts that had been trans-spliced.Figure 4pMU2-tsRNAHA trans-splices endogenous survival motor neuron (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 (forward) 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. Δ7, SMNΔ7; FL, full-length SMN; tsRNA, trans-splicing RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 fractions 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.28Lorson CL Strasswimmer J Yao JM Baleja JD Hahnen E Wirth B et al.SMN oligomerization defect correlates with spinal muscular atrophy severity.Nat Genet. 1998; 19: 63-66Crossref PubMed Scopus (410) Google Scholar,29Young PJ Man NT Lorson CL Le TT Androphy EJ Burghes AH et al.The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding.Hum Mol Genet. 2000; 9: 2869-2877Crossref PubMed Scopus (80) Google Scholar Consistent with this, HA-immunoprecipitated complexes 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 transcripts are suitable targets for trans-splicing and trans-spliced RNA is translated to protein.Viral delivery of tsRNAs stimulates the levels of full-length SMN in SMA fibroblastsThe 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.30Kaspar BK Llado J Sherkat N Rothstein JD Gage FH Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model.Science. 2003; 301: 839-842Crossref PubMed Scopus (741) Google Scholar 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.17Baughan T Shababi M Coady TH Dickson AM Tullis GE Lorson CL Stimulating full-length SMN2 expression by delivering bifunctional RNAs via a viral vector.Mol Ther. 2006; 14: 54-62Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar 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."31Liu Q Fischer U Wang F Dreyfuss G The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins.Cell. 1997; 90: 1013-1021Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar Gems in SMA patient fibroblasts are reduced in number compared to unaffected carrier-derived fibroblasts.32Coovert DD Le TT McAndrew PE Strasswimmer J Crawford TO Mendell JR et al.The survival motor neuron protein in spinal muscular atrophy.Hum Mol Genet. 1997; 6: 1205-1214Crossref PubMed Scopus (571) Google Scholar Since gems are an established measure of SMN protein levels, and the numbers of gems correlate with disease severity, an increase in SMN-positive gems would indicate an amelioration of a hallmark of disease severity.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 endogenous SMNΔ7 transcript decreases following rAAV transduction (Figure 5, compare lanes 3, 4 and 6), suggesting that the decrease in SMNΔ7 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 levels correlative with the efficiency of the RT-PCR data (data not shown). Taken together these results demonstrate that endogenous SMN2 transcripts can be targeted for trans-splicing, resulting in the production of full-length SMN from the SMN2 gene.Figure 5Endogenous 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; Δ7, SMNΔ7; trans-spliced product: Trans*). Two independent experiments are shown ("A" and "B" lanes). HA, hemagglutinin; tsRNA, trans-splicing RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Second-generation tsRNA: restoration of SMN functionDuring 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