Development of a single vector system that enhances trans-splicing of SMN2 transcripts - PubMed (original) (raw)

Development of a single vector system that enhances trans-splicing of SMN2 transcripts

Tristan H Coady et al. PLoS One. 2008.

Abstract

RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1. A nearly identical copy gene called SMN2 produces low levels of functional protein due to alternative splicing. We previously reported a trans-splicing RNA (tsRNA) that re-directed SMN2 splicing. Now we show that reducing the competition between endogenous splices sites enhanced the efficiency of trans-splicing. A single vector system was developed that expressed the SMN tsRNA and a splice-site blocking antisense (ASO-tsRNA). The ASO-tsRNA vector significantly elevated SMN levels in primary SMA patient fibroblasts, within the central nervous system of SMA mice and increased SMN-dependent in vitro snRNP assembly. These results demonstrate that the ASO-tsRNA strategy provides insight into the trans-splicing mechanism and a means of significantly enhancing trans-splicing activity in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Development of a single plasmid vector enhances SMN based _trans_-splicing.

(A) Proposed ASO-tsRNA mechanism. SMN2 transcripts alternative splice producing two mRNA products. The alternative splicing pathway is represented by gray dashed lines, SMN2 mRNA products: “Δ7”- Exon 7 skipped, “FL”- full length. Trans_-splicing RNA identifies SMN2 pre-mRNA intron 6 which overlaps the endogenous branch point then incorporated in the final mRNA is catalyzed by the spliceosome. Circles surrounding symbol “BP” identifies known branchpoints in the model. We demonstrate a novel mechanism of disrupting downstream intron 7 splicing elements causes enhancement of trans_-splicing. (Bold “TRANS*” and large black arrow indicate enhanced trans_-splicing pathway). The effect is accomplished via enhancing Antisense Oligonucleotides “ASO In711” targeted to the distal intron and exon boundary. The stop codons “STOP” and SMN2 “C-T” or tsRNA “C*” nucleotide changes are denoted with vertical line marking approximate RNA position. Bracketed objects indicate promoters and gene products produced from pMU3 plasmid pictured below by a black line. (B) Increasing concentrations of transcription mutant RNA polymerase promotes trans_-splicing. HeLa cells were transfected of static amounts mini-gene p_SMN1 (lane a) or p_SMN2 (lanes b–g) 1.25 µg , pM13 (lanes c–g) 0.75 µg, and increasing amounts of mutant Rpb1 RNA Polymerase subunit (pAT7-Rpb1MT) at 0.25, 0.75, 1.0 and 2.0 µg were harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. (C) A genetically disabled SMN2 intron 7 deletion mini-gene displays enhances trans-splicing at reduced tsRNA plasmid concentrations. HeLa cells were cotransfected with p_SMN1 (lane a), p_SMN2 (lanes b,d), p_SMN2_ ΔIn7 (lanes e–h) 1.25 µg each and increasing concentrations of pM13 (lane d) 0.25 µg, (lanes e–h) 0.10, 0.25, 1.0 and 2.0 µg and the RNA harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control.

Figure 2

Figure 2. ASO mediated inhibition of downstream splicing increases _SMN2 trans_-splicing.

(A) HeLa cells transfected with static amounts of p_SMN1_ (lane a), p_SMN2_ 1.25 µg (lanes b–e) and pM13 (lane c–e) 0.75 µg are cotransfected with increasing concentrations of pIn711 (lane d,e) 0.25, 1.0 µg and RNA harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. Graph represents independent triplicate repeats using a quantitative M13-Cy3 labeled primer and linear-range PCR amplification. Inset graph represents the average of triplicate repeats and error bars indicate ±s.d. (B) SMN intron 6 ASO controls contribute to a reduction of trans_-splicing. HeLa cells were triple transfected with static amounts of mini-gene p_SMN1 (lane a) p_SMN2_ (lane b–g) 1.25 µg and pM13 (lane c–g) 0.75 µg with increasing amounts of pIn65 (lane e–g) 0.25, 0.75 and 1.0 µg and RNA harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. pIn711 (lane c) 1.0 µg serves as a positive ASO-tsRNA control. (C) ASOs targeted internally to SMN intron 7 reduce trans_-splicing. HeLa cells were triple transfected with static amounts of mini-gene p_SMN1 (lane a) p_SMN2_ (lanes b–g) 1.25 µg and pM13 (lanes c–g) 1.0 µg with increasing amounts of pIn71 (lanes e–g) 0.25, 0.75 and 1.0 µg and RNA harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. pIn711 (lane c) at 0.25 µg serves as a positive ASO-tsRNA control.

Figure 3

Figure 3. Endogenous enhancement of _trans_-splicing utilizing combined delivery of ASO and tsRNA.

(A) Dose dependant ASO In711 enhancement of endogenous _SMN trans_-splicing. HeLas were cotransfected with pM13 (lanes a–e) 1.0 µg and increasing concentrations of ASO pIn711 (lanes c–e) 0.25, 2.0 and 5.0 µg and RNA harvested at 48 hrs. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. pIn65 (lane a) 2.0 µg serves as a negative control. (B) Dose dependant ASO pIn711 enhancement of pM13 mediated SMN protein induction. 3813 SMA patient fibroblasts were co-transfected with pM13 (lanes d–f) 0.75 µg and increasing concentrations of pIn711 (lanes d–f) 0.50, 1.0 and 2.0 µg and cells harvested at 24 hrs and run on 10% SDS-PAGE. pIn711 alone (lane c) 2.75 µg and ssDNA (lanes a, b) 2.75 µg serve as negative controls. β-actin antibody panel serves as a normalization control.

Figure 4

Figure 4. Development of a single plasmid ASO-tsRNA system (pMU3) enhances endogenous _SMN trans_-splicing.

(A) pMU3 enhances _trans_-splicing capabilities in a single vector. HeLas were transfected with pM13 (lane a) 0.75 µg or pMU3KO (lane b) 0.75 µg or pMU3 (lanes c,d) 0.75 and 1.0 µg and RNA harvested 48 hrs later. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. Inset graph represents triplicate repeats. (B) In SMA relevant contexts the single plasmid ASO-tsRNA system (pMU3) produces the greatest amount of _trans_-splicing. 3813 SMA patient fibroblasts were transfected with pM13 for basal level (lane c) 0.75 µg or cotransfected with pIn711 0.50 µg and pM13 0.50 µg together (lane d) or pMU3 (lane e) 0.75 µg and RNA harvested at 48 hrs. ssDNA (lane a) 0.75 µg and pIn711 alone (lane b) 2.0 µg serve as negative controls. Reverse transcriptase PCR gel is displayed with GAPDH normalization control. Inset graph represents the average of triplicate repeats and error bars indicate ±s.d.

Figure 5

Figure 5. Single vector dosages with enhanced _trans_-splicing produces functional SMN protein in patient fibroblasts.

(A) The pMU3 single plasmid ASO-tsRNA system produces the greatest amount SMN protein induction. 3813 SMA patient fibroblasts were transfected with pM13 for basal level (lane d) 0.75 µg or cotransfected with pIn711 0.50 µg and pM13 0.50 µg together (lane e) or pMU3 (lane f) 0.75 µg cells were harvested at 24 hrs and run on 10% SDS-PAGE. ssDNA (lanes a,b) 0.75 µg and pIn711 alone (lane c) 2.0 µg serve as negative controls. β-actin antibody panel serves as a normalization control. Inset graph represents the average of triplicate repeats and error bars indicate ±s.d. (B) The pMU3 system restores SMA primary fibroblasts in vitro capacity to assemble U1snRNP. 3813 SMA patient fibroblasts were transfected with pM13 (lane e) 0.75 µg for basal level or pMU3 (lane d) 0.75 µg cells were harvested at 24 hrs and S100 extract prepared. ssDNA (lane b) 0.75 µg and pIn711 alone (lane c) 2.0 µg serve as negative controls. Inset graph represents the average of triplicate repeats and error bars indicate ±s.d. (C) The pMU3 system restores the SMA primary fibroblasts in vitro capacity for minor spliceosome U11 snRNP assembly. 3813 SMA patient fibroblasts were transfected with pMU3 (lane d) 0.75 µg or ssDNA (lanes b,c) 0.75 µg. ssDNA. 3813 and 3814 (lanes b and c) serve as negative and positive controls respectively for U1 and U11ATAC assembly. (*) indicates band of interest.

Figure 6

Figure 6. pMU3 transfected SMA primary fibroblasts display increased gem numbers.

(A) 3813 SMA patient fibroblasts were transfected with pMU3 0.75 µg or ssDNA 0.75 µg and fixed at 24 hrs. Immunohistochemistry was performed using αSMN antibody 4B7 and visualized with Texas Red 594, nuclei with DAPI. White arrows indicate examples of gem structures in the red anti-SMN panel. (B) Graphical summary of averaged SMN gem counts in 300 GFP positive 3813 fibroblasts. Error bars represent ±SD. Statistical significance relating pM13 to pMU3 determined by values of p<0.05.

Figure 7

Figure 7. Intracerebroventricular 25 kDa PEI pMU3 transfections in SMA model mice increases SMN protein in the spinal cord.

(A) Heterozygous (m_SMN_ +/+, h_SMN_ +/+, h_Δ7 SMN_ cDNA +/+) (lane a) or homozygous (lanes b–g) (m_SMN_ −/−, h_SMN_ +/+, h_Δ7 SMN_ cDNA +/+) neonatal mice (PND 0-1) were injected with pMU3 plasmid at 10 µg (1.14×1012 plasmid copies) with 25 kDa PEI over two ventricles (lanes c–g). Mock transfection mice are shown in lane a and b. Whole spinal cord homogenates were prepared for protein or RNA extraction 24 hours post injection. In vivo RT-PCR results for trans_-spliced SMN are shown in the upper panel. Lower panel depicts controls for vector expression via northern blot of mock or treatment group spinal cord RNA loaded on nitrocellouse and developed with a radio-labeled GFP probe. (B) SMA mouse spinal cord protein was resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. (C) Intra-ventricular transfections of pM13 in SMA model mice do not alter SMN levels in the spinal cord. Homozygous (m_SMN −/−, h_SMN_ +/+, h_Δ7 SMN_ cDNA +/+) (lanes b,c) neonatal mice (PND 0-1) were injected with 10 µg of plasmid with 25 kDa PEI over both ventricles. Whole spinal cord homogenates were prepared for protein and resolved on 10% SDS-PAGE. β-actin antibody panel serves as a normalization control. Mock treatment is depicted in lane a. (D) Graphical summary of injection outcomes relative to SMN protein induction western blot in mice. Error bars represent ±s.d. HET, KO, pM13 (n = 4). pMU3 (n = 5).

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