Gene Therapy (2003) 10, 2005–2012 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta Y Smicun1, MW Kilpatrick1, J Florer2, I Toudjarska1,GWu3, RJ Wenstrup2 and P Tsipouras1 1Department of Pediatrics, University of Connecticut Health Center, Farmington, CT, USA; 2Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH, USA; and 3Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA Antisense hammerhead ribozymes have the capability to achieved, a function directly related to high steady-state level cleave complementary RNA in a sequence-dependent of intracellular ribozyme. We report significantly enhanced manner. In osteogenesis imperfecta, a genetic disorder of expression of Col1A1Rz547 by vaccinia T7 polymerase connective tissue, mutant collagen type I has been shown to following infection with an attenuated T7-pol vaccinia virus as participate in but not sustain formation of the triple helix. shown both by the intracellular level of the ribozyme and the Selective ablation of mutant collagen gene transcript could cleavage of the mutant Col1A1 gene transcript. We also potentially remove the mutant gene product and reverse the describe the engineering of a multimeric ribozyme construct dominant-negative effect exerted by the abnormal protein. In comprising eight subunits, which can self-cleave to mono- earlier studies we showed that the hammerhead ribozyme mers. These studies suggest the potential use of multimeric Col1A1Rz547 selectively cleaved a mutant Col1A1 gene ribozymes expressed by a vaccinia-based system in the transcript in a murine calvarial osteobleast cell line. In order therapy of a variety of disorders. to test the possible therapeutic efficacy of this approach, a Gene Therapy (2003) 10, 2005–2012. doi:10.1038/ dramatic downregulation of the mutant transcript must be sj.gt.3302108 Keywords: hammerhead ribozyme; osteogenesis imperfecta; collagen type I; vaccinia vector; multimeric ribozyme Introduction mRNA. Additionally, hammerhead ribozymes have the advantage of being able to discriminate between closely The application of nucleic acids as antisense reagents related target RNAs, even when the two RNA species for the downregulation of expression of disease causing differ by as little as a single nucleotide.15 genes has been the focus of a great deal of attention for Selective intracellular ablation is a strategy particu- more than 20 years.1,2 Hammerhead ribozymes are larly suited to conditions like osteogenesis imperfecta small RNA molecules with a catalytic core of less than (OI), a systemic disorder of connective tissue presenting 40 nucleotides (nt) that can be resolved into a substrate with osseous fragility, short stature, hearing loss, dental and a catalytic strand.3–7 It is possible that any RNA defects, and blue sclerae.16,17 Mutations in either of the molecule can be cleaved by hammerhead ribozymes in genes encoding the proa1(I) and proa2(I) chains of trans, provided it contains a putative cleavage site. These collagen type I have been identified in many affected ribozymes have been applied to downregulate a variety individuals, and have been determined to be the under- of genes, and their potential as therapeutic agents in the lying cause of OI.16 management of genetic disorders, particularly those Several studies on the molecular pathology of OI have whose pathogenesis is the result of a dominant-negative identified two mechanisms of collagen type I defects.16 In effect exerted by a mutant gene product, has been widely one, null mutations result in the synthesis of mutant recognized.8–11 collagen chains that cannot be incorporated in the More recently, the discovery of the role of RNA collagen triple helix. This mechanism leads to the interference (RNAi) in post-transcriptional gene silen- deposition of half normal levels of collagen type I in cing has led to the suggestion that a new class of the extracellular matrix and is associated with a mild RNA-based therapeutics might be developed.12–14 Unlike clinical phenotype.18 The second mechanism results from the antisense and RNAi approaches, which both rely on the incorporation of mutant protein into the collagen the action of the cellular machinery for their effect, triple helix, thus disrupting the collagen fibrillar struc- hammerhead ribozymes act directly to cleave their target ture. These dominant-negative mutations are associated with phenotypes ranging in severity from moderate to lethal.16 Correspondence: P Tsipouras, Department of Pediatrics, University of 19–22 Connecticut Health Center, 263 Farmington Avenue, Farmington, CT Several murine models of OI have been described. 23 06030, USA Pereira et al generated mice transgenic for an internally Received 29 March 2003; accepted 30 May 2003 deleted human gene for the proa1(I) chain of collagen Ablation of Collagen 1 target by specific ribozymes Y Smicun et al 2006 type I. The deleted minigene (pMG155) was designed to synthesize shortened proa1(I) chain which could parti- cipate in but not sustain the formation of the triple helix, thus depleting the endogenous procollagen type I through protein suicide.23,24 Wenstrup et al25 developed MC3T3-E1, a murine calvarial osteoblast cell line stably expressing pMG155 that has been used as a model system to study the effect of dominant-negative OI mutations. We previously tested the use of an antisense ribozyme complementary to a particular sequence of the human mutant minigene target, pMG155, abundantly expressed in MC3T3-E1 cells. After intracellular delivery of a ribozyme expression construct, intracellular ribozyme transcript was detected along with a relative reduction of pMG155 mutant collagen transcript level.26 Thus, the ribozyme was shown to be biologically active and its potential value in the therapy of OI was demonstrated. However, the utility of this monomeric ribozyme was limited by the restrictions of the transient expression system used. Previous studies have established that even a small fraction of mutant protein is sufficient to produce a clinically severe OI phenotype.19 Therefore, in order to achieve a therapeutic effect, a dramatic downregula- tion of the mutant transcript might be necessary. This could be accomplished by increasing the level of ribozyme available intracellularly. We report here on two alternate yet complementary modalities aimed at achieving higher intracellular ribozyme availability: firstly, a multimeric ribozyme construct that produces a single transcriptional unit containing multiple copies of the ribozyme motif and capable of self-cleavage;27 secondly, infection with a modified vaccinia virus Ankara (MVA) that produces large quantities of recom- binant T7 polymerase, which acts on T7 promoter bearing ribozyme sequence to achieve rapidly a high level of ribozyme transcript.28 Results The octameric ribozyme cleaves both in cis and trans In vitro transcription of the octameric transcript demon- strated that the transcript efficiently underwent cis- cleavage to produce the monomeric subunit. Transcription was carried out in the presence of 8 mM MgCl2 to minimize cis-cleavage during the transcription reaction, and synthesized transcript was subsequently Figure 1 Structure and function of the octameric ribozyme designed to cleave both in cis and trans. (a) Monomeric Col1A1547Rz subunit incubated in 20 mM MgCl2 to assay for cis-cleavage designed to cleave both in cis and in trans. The ribozyme sequence is shown activity. A ladder of fragments of between 70 and in the secondary structure that facilitates cis-cleavage on the left and is B500 nt, the sizes expected if the octameric transcript bound to its minigene target sequence on the right. The putative CUC were to cis-cleave (Figure 1b), was observed following in cleavage site is shown in bold and the site of cleavage indicated by a vitro transcription. Subsequent incubation in the pre- vertical arrow. (b) Schematic representation of the octameric ribozyme sence of the higher MgCl concentration led to accumu- construct. The sequence of the monomeric subunit is shown on top, with 2 the octameric derivative underneath. Vertical arrows depict the expected lation of the 70 nt monomeric subunit (Figure 1c). sites of cis-cleavage of the octameric transcript. (c) In vitro transcription of 1 Incubation at 50 C resulted in increased cleavage as the octameric ribozyme construct in the presence of 8 mM MgCl2 (lane 1) compared to 371C, as has been observed previously for and incubation of the resultant transcript in 20 mM MgCl2 (lanes 2 and 3) hammerhead ribozymes.8 In addition, the octameric at 37 or 501C, respectively. Lane M contains an RNA marker ladder. Sizes ribozyme was able to cleave its target sequence in trans. of marker fragments are shown on the left and sizes of the fragments Incubation of a 205 nt minigene target transcript with resulting from cis-cleavage of the transcript on the right. (d) In vitro ribozyme cleavage assay of the 205 nt minigene target transcript with octameric ribozyme transcript led to accumulation of octameric ribozyme at 371C. Lanes 1, 30, 60, and 120 show minigene increasing amounts of the 130 and 75 nt subfragments target transcript incubated with ribozyme for 1, 30, 60, and 120 min, expected if the ribozyme cleaved its putative cleavage respectively. The sizes of the full-length minigene transcript and