Hammerhead ribozyme-mediated destruction of nuclear foci in myotonic dystrophy myoblasts  Marc-André Langlois, Nan Sook Lee, John J Rossi, Jack Puymirat  Molecular Therapy  Volume 7, Issue 5, Pages 670-680 (May 2003) DOI: 10.1016/S1525-0016(03)00068-6 Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Selection of candidate target sites in the 3′ untranslated region of the DMPK mRNA. (A) Schematic representation of antisense ODN target sites in the 3′UTR of DMPK mRNA and primer annealing sites. Normal DMPK mRNA is approximately 2.8 kb in length. Five target sites were selected between the termination codon (UGA) and the beginning of the repeat expansion. (B) ODN and primer sequences used for accessibility site determination. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Identification of accessible ribozyme cleavage sites. (A) Agarose gel showing accessible sites. Whole-cell extracts were prepared from differentiated DM1 myoblasts with 750 repeats. Antisense ODNs were added for 5 min, and then total RNA was isolated from the extracts. RT-PCR was then performed using DMPK and β-actin primers. Amplification of a band at 235 bp indicates an inaccessible target site in the 3′UTR. The β-actin amplicon at 348 bp serves as an internal loading control. (B) Histogram showing quantification by densitometry of band amplification in three unrelated experiments (n = 3). Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 3 DNA construct used for ribozyme expression. (A) tRNAmet-ribozyme expression cassette. Transcripts can be produced in vitro either by T7 RNA polymerase or by RNA polymerase III using the internal tRNA-methionine promoter when transfected into myoblasts. There is a sequence coding for a stem–loop 3′ of the cassette that confers resistance to 3′-to-5′ degradation of the RNA. Deletion of the final 10 nucleotides of the mature tRNAmeti transcript prevents its maturation and export from the nucleus [45,46]. Transcription by Pol III is terminated by a stretch of five thymidines. (B) Hammerhead ribozyme sequence and structure with its DMPK target. The mRNA cleavage site is located after the GUC consensus on the DMPK mRNA. The ribozyme's catalytic activity is dependent on the presence of the guanosine indicated by an asterisk. The mutant ribozyme used in our assays was generated by replacing the G with an A. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 4 In vitro cleavage assays of a transcribed DMPK substrate. A [32P]UTP-labeled DMPK RNA substrate was produced by in vitro transcription of the pRMKΔ-100 vector, which contains a NarI/XhoI deletion of the pRMK-CTG100 vector generating an 806-nt transcript with 100 CUG repeats. Ribozymes were produced by in vitro transcription of the tRNAmet-RBZ construct digested with HindIII. All transcriptions were initiated by a T7 promoter. Ribozyme cleavage of the substrate generates fragments of 214 and 554 nt. (A) Target/RBZ ratio optimization. Two nanomolar labeled substrate was incubated for 1 h with various concentrations of ribozymes in the presence of 10 mM MgCl2. (B) MgCl2 requirement optimization. Two nanomolar labeled substrate was incubated for 1 h in the presence of 100 nM ribozyme and varying concentrations of MgCl2. (C) Cleavage time course. Two nanomolar labeled substrate was incubated for various periods in the presence of 100 nM ribozyme and 20 mM MgCl2. (D) Two nanomolar labeled substrate was incubated for various periods in the presence of 100 nM mutant and nonsense ribozymes in the presence of 20 mM MgCl2. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Ribozyme-mediated cleavage of DMPK mRNA by transient transfection of tRNAmet-RBZ constructs in DM-750 myoblasts. DM-750 myoblasts were grown in a 60-mm dish and transfected with either 10 or 20 μg of total DNA containing wild-type or mutant tRNAmet-RBZ constructs and the CMV-REV-eGFP construct. The transfection ratio of ribozyme to reporter was 3 to 1. Cells were then differentiated for 3 days and mRNA was extracted by proteinase K/poly(A)+ isolation. Normal DMPK transcripts (fewer than 20 CUG repeats) have a length of 2.8 kb and mutant transcripts from DM myoblasts with 750 CUG repeats generate a band at 5.1 kb. One representative experiment of four is shown here. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Destruction of nuclear speckles in ribozyme-transfected DM-750 myoblasts. Normal and DM-750 myoblasts were cotransfected with either wild-type or mutant ribozyme constructs and the CMV-REV-eGFP vector. The transfection ratio of ribozyme to reporter was 3 to 1. The cells were then differentiated for 3 days. After fixation and permeabilization cells were hybridized with a PNA Cy3-(CAG)5 probe. Red speckles show sequestered mutant DMPK transcripts, the REV-eGFP fusion protein is depicted in green within the nucleoli of the myoblasts, and in blue is a DAPI staining of the nucleus. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Restoration of insulin receptor splicing. Normal differentiated myoblasts predominantly express the B isoform of the insulin receptor. In DM1 myoblasts, the insulin receptor A isoform is predominantly expressed. RT-PCR analysis of insulin receptor splicing in normal and DM-750 eGFP-positive myoblasts transfected with either tRNA-RBZmut or tRNA-RBZ-wt construct is shown. Myoblasts were allowed to differentiate for 4 days before total RNA was extracted. Amplification products are of 131 bp for the A isoform and 167 bp for the B isoform of the insulin receptor using primers flanking the boundaries between exons 10 and 12 [21]. One representative experiment of two is shown. Molecular Therapy 2003 7, 670-680DOI: (10.1016/S1525-0016(03)00068-6) Copyright © 2003 The American Society of Gene Therapy Terms and Conditions