Molecular Vision 2001; 7:6-13 <>
Received 1 November 2000 | Accepted 15 January 2001 | Published 26 January 2001

An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa

Lynn C. Shaw,1 Anna Skold,2 Fulton Wong,3 Robert Petters,4 William Hauswirth,2,5 Alfred S. Lewin2

1Department of Pharmacology and Therapeutics, 2Department of Molecular Genetics and Microbiology, and 5Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL; 3Departments of Ophthalmology, Neurobiology, and Pathology, Duke University School of Medicine, Durham, NC; 4Department of Animal Science, North Carolina State University, Raleigh, NC

Correspondence to: Alfred S. Lewin, PhD, Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Box 100266, Gainesville, FL, 32610; Phone: (352) 392-0676; FAX: (352) 392-3133; email:


Purpose: To develop a hammerhead ribozyme-based gene therapy for a porcine model of autosomal dominant retinitis pigmentosa (ADRP).

Methods: Hammerhead ribozymes were developed and assayed in vitro against RNA targets homologous to the opsin P347S mutants found in a transgenic porcine model and in humans. Both cloned and synthetic RNA oligonucleotide versions of ribozymes and targets were tested under multiple-turnover conditions using oligonucleotide RNA targets. Digestion of full-length P347S mRNA from porcine retina was performed.

Results: The porcine P347S hammerhead ribozyme was specific for the opsin P347S sequence. Multiple-turnover analysis yielded the following kinetic parameters: Vmax=7.3±0.5 nM/min, Km=2.1±0.6 mM, and kcat=1.5±0.4 min-1. The human P347S hammerhead ribozyme was substantially less active (~10,000 fold).

Conclusions: We have developed a hammerhead ribozyme to use as a model for gene therapy of autosomal dominant retinitis pigmentosa in a transgenic porcine model. Based on kinetic characterization of this ribozyme compared to others used for gene therapy, this should be an effective reagent RNA. The allele specific ribozyme we tested for the human sequence, however, is not likely to be useful for gene therapy indicating that an alternative approach is necessary.


Retinitis pigmentosa (RP) is the most common inherited form of blindness in the United States. RP is a group of genetic disorders that result in the breakdown of the rod and, eventually, cone photoreceptor cells in the retina [1,2]. Symptoms of retinitis pigmentosa usually begin in adolescence or in early adulthood and may result in total blindness by the fifth or sixth decade of life [1]. Initial symptoms of RP include an inability to see in low light conditions (night blindness), impaired adaptation, problems with discriminating colors and a loss of the mid-peripheral visual field resulting in tunnel vision [1]. This process continues throughout life until central vision is lost as well.

There are three basic classes of RP based on mode of inheritance: autosomal dominant, autosomal recessive, and X-linked [3]. However, 70% of RP patients have no family history of RP [4]. There are 36 known or predicted RP genes (RetNet) in humans and over 70 loci linked to photoreceptor dysfunction or degeneration have been found in Drosophila [3,4]. RP affects approximately 1 in 3,500 people worldwide and 50,000 to 100,000 people in the United States [5]. Mutations in the rhodopsin gene account for 40% of the cases of the autosomal dominant form of RP in the United States [3,6], and more than 100 separate mutations in this gene result in RP. The majority of these mutations are missense mutations resulting in a single amino acid substitution [2]. These mutations demonstrate the allelic heterogeneity of RP through the variations in onset and severity of symptoms [2,4]. In addition, clinical heterogeneity can be found with the same mutation causing different symptoms even within members of the same family [2,4].

Mutations resulting in changes in the C-terminal domain of rhodopsin result in some of the more severe forms of the disease, with total blindness occurring in early adulthood [7]. The rhodopsin C-terminal sequence QVS (A)PA is recognized by specific factors in the trans-Golgi network [7,8]. Mutations resulting in changes in this region produce RP due to formation of abnormal post-Golgi membranes and from the aberrant subcellular localization of rhodopsin [7-9]. A transgenic porcine model for the rhodopsin P347L mutation has been produced and shows a disease phenotype similar to that found in humans [10-12]. In addition, parallel to severe rod degeneration, ectopic synapses form early between cone photoreceptors and rod bipolar cells and this introduces a complicating factor into the design of therapy [13]. A P347S transgenic pig model has also been produced that showed a much more gradual rate of rod degeneration (data not shown). This model affords a more extended time period for therapeutic intervention. Accordingly, we have chosen the porcine transgenic P347S model for use in the development of a gene therapy for ADRP. Currently, there is no treatment for RP other than ingestion of high doses of vitamin A [4]. Our gene therapy approach is to use hammerhead ribozymes cloned in recombinant adeno-associated viral (AAV) vectors and packaged into AAV to treat ADRP (Figure 1) [14].

Hammerhead ribozymes are small catalytic RNAs capable of cleaving other RNAs in cis or in trans. This ribozyme was originally found in satellite RNA of tobacco ringspot virus where it cleaves the RNA genome as part of a rolling-circle model of replication [15]. Hammerhead ribozymes cleave RNA at the target sequence NUX where N is any base, and X is any base except G [16]. GUC has been reported to be the optimal sequence for recognition and cleavage by a hammerhead ribozyme [16].

The specific cleavage by ribozymes of mutant mRNAs that code for disease-associated proteins is the basis of ribozyme-based gene therapy [17,18]. Ribozyme gene therapy is being developed for a number of diseases including AIDS [19,20], hemophilia A and B [21], Parkinson's disease [22] and retinitis pigmentosa [23-25]. One hammerhead ribozyme has been designed against the P23H mutation in rhodopsin and has been successfully tested in a transgenic rat model [24]. This ribozyme successfully slowed rod cell apoptosis and maintained rod cell function for a minimum of 8 months after a single subretinal injection. While rod cells continued to be lost over this time period, the maintenance of rod cells and rod cell function was well above that observed in the control eyes that received no ribozyme treatment.

We have designed and tested in vitro hammerhead ribozymes against the transgenic porcine P347S model and the human P347S mutation. The porcine ribozyme will eventually be tested in vivo after packaging into AAV [26]. The porcine model is an ideal precursor to eventual ribozyme gene therapy in the human eye for a number of reasons [10,12]. First, the size and architecture of the porcine eye is similar to the human eye. Second, even though the porcine eye lacks a macula, the rod and cone density of the porcine retina and the human retina are similar. Third, the porcine eye is a good model for testing dose response and potential toxicity problems with ribozyme treatments and for refining a method of ribozyme delivery to the retina. Here we describe the in vitro testing of these hammerhead ribozymes.


Synthetic RNA targets and ribozymes

RNA oligonucleotides for the porcine and human P347S hammerhead ribozymes and targets were purchased from Dharmacon (Boulder, CO) and deprotected following the manufacturer's protocol. RNA oligonucleotides were 5'-end labeled with [g-32P]-dATP (ICN, Irvine, CA) using polynucleotide kinase (Promega, Madison, WI).

Production of the porcine P347S hammerhead ribozyme clone

The porcine P347S hammerhead ribozyme coding sequence was generated by extension of two overlapping synthetic DNA oligonucleotides (Life Technologies, Gaithersburg, MD) flanked by EcoRI and MluI sites (Figure 2). The overlapping DNA oligonucleotides were held at 65 °C for 2 min and annealed by slow cooling to room temperature for 30 min. The annealed oligonucleotides were extended by DNA polymerase (Klenow, New England Biolabs, Beverly, MA) in the presence of 5 mM dNTPs and polymerase buffer (10 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 7.5 mM dithiothreitol) at 37 °C for 60 min. The resulting double-stranded DNA fragment was digested with EcoRI and MluI and ligated into the pHC T7 RNA expression vector [27]. The ligated plasmids were transformed into DH5a cells. The ribozyme clone was verified by sequencing and was designated as p347Rz.

Production of the porcine P347S target clone

The porcine P347S target was cloned into pHC using the same method used to clone the porcine P347S hammerhead ribozyme. The two DNA oligonucleotides used were, 5'-CCG GAA TTC GCC AGG TAG GCG TCA GCT AAG GAT CCG CC-3' and 5'-GGC GGA TCC TTA GCT GAC GCC TAC CTG GCG AAT TCC GG-3'. The target clone was verified by sequencing and was designated as p347Tar.

In vitro transcription to produce the porcine P347S hammerhead ribozyme target

Run-off transcripts were produced using the MluI-digested p347Rz plasmid or the MluI-digested p347Tar plasmid and T7 RNA polymerase as previously described [28].

Time course analysis of ribozyme cleavage of RNA oligonucleotides

Both the cloned version and the RNA oligonucleotide version of the ribozyme were used. Two pmol of ribozyme with a final concentration of 15 nM in 40 mM Tris-HCl, pH 7.5 were held at 65 °C for 2 min then cooled to 25 °C for 10 min. Dithiothreitol (DTT) with a final concentration of 20 mM, 4 units RNasin (Promega), and MgCl2 with a final concentration of 20 mM were added and the mixture was held at 37 °C for 10 min. Cleavage was initiated by the addition of the 32P-end labeled target RNA oligonucleotide, and the reaction proceeded at 37 °C. Aliquots were removed at various times and added to formamide stop buffer (90% formamide, 50 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol) and held on ice. The samples were then heat denatured, cooled and separated on 10% polyacrylamide-8 M urea gels. The gels were analyzed on a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Multiple-turnover kinetic analysis of the hammerhead ribozymes

Multiple-turnover kinetic reactions were performed in a final volume of 20 ml. Ribozyme (cloned or RNA oligonucleotide; 0.3 pmol; 15 nM final concentration) in 40 mM Tris-HCl, pH 7.5 was held at 65 °C for 2 min and then cooled at 25 °C for 10 min. DTT (20 mM final concentration) and MgCl2 (20 mM final concentration) and 4 units of RNasin were added. The reactions were held at 37 °C for 10 min, and cleavage was initiated by the addition of increasing concentrations of the target oligonucleotide (0-300 pmol; 0-1500 nM final concentration). The reactions proceeded at 37 °C for a fixed interval determined in the time course analysis of cleavage. Reactions were terminated by the addition of 20 ml of formamide stop buffer and held on ice. The samples were then heat denatured at 85 °C, cooled on ice, and separated on 10% polyacrylamide-8 M urea gels. The gels were analyzed on a Molecular Dynamics PhosphorImager.

Preparation of total porcine retinal RNA

Total retinal RNA was extracted from freshly dissected porcine eyes using a guanidinium thiocyanate procedure described in RNAgents Total RNA Isolation System (Fisher, Pittsburgh, PA).

Ribozyme cleavage of the full-length rhodopsin mRNA

The oligonucleotide version of the porcine P347S hammerhead incubated with total porcine retinal RNA isolated from a wild type animal and from a transgenic P347S rhodopsin animal model. 0.3 pmol of ribozyme (15 nM final concentration) in 40 mM Tris-HCl, pH 7.5 was held at 65 °C for 2 min and then cooled at 25 °C for 10 min. Reactions were brought to the same incubation conditions as used above (20 mM DTT, 4 units RNasin, 20 mM MgCl2 and held at 37 °C for 10 min). Cleavage was initiated by the addition of 1 mg total retinal RNA and incubation continued at 37 °C for 16 h. The mixture was precipitated by the addition of 0.1 volume of 5 M ammonium acetate and 2.5 volumes of absolute ethanol. After centrifugation, the supernatant was discarded and the sample was dried in a Speed Vac (Savant, Holbrook, NY). At this point, samples were taken directly to RT-PCR.

RT-PCR using total porcine retinal RNA

The dried RNA samples from the ribozyme cleavage reaction and uncleaved RNA samples were directly used to measure intact rhodopsin mRNA by RT-PCR. For reverse transcription, the samples were resuspended in 7 ml of buffer A (14 mM Tris-HCl, pH 7.5, 70 mM KCl, 0.14 mM EDTA) followed by the addition of 1 ml (1 pmol) of the antisense oligonucleotide (5'-GTG ACC TGG TTC TGA CAG GTG-3'). The samples were held at 80 °C for 2 min and then cooled at 25 °C for 15 min. Next, 16.5 ml of buffer B (6.25 mM dNTPs, 250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl2, 15 mM DTT) and 0.5 ml (100 units) of Superscript II RNase H- MMLV reverse transcriptase (Life Technologies) were added. The samples were incubated at 50 °C for 5 min. The reaction was stopped by the addition of 2 ml of 5 M ammonium acetate and precipitated with 90 ml of absolute ethanol. The supernatant was discarded and the samples were dried. For PCR, the samples were resuspended in 5 ml of concentrated PCR buffer supplied by the manufacturer (Sigma, St. Louis, MO), 2 ml 10 mM dNTPs, 1 ml (15 pmol) each of the sense (5'-GCA TGC TCA CCA CGC TCT GC-3') and antisense oligonucleotides and 39 ml water. The PCR reaction was initiated by the addition of 2.5 units of Taq DNA polymerase (Sigma) and placed in a PerkinElmer (Wellesly, MA) thermal cycler for 25 cycles (30 s at 94 °C, 30 s at 60 °C, 3 s at 72 °C). Prior to the last cycle, 1 mCi of [a-32P]-dCTP was added to the reaction and the last cycle was allowed to proceed. The samples were stored at -20 °C until restriction enzyme analysis was performed. Each restriction enzyme digestion used 10 ml of the PCR reaction. The restriction enzymes used were BanI (New England Biolabs) and HgaI (New England Biolabs) and the conditions were as described by the manufacturer. The products of digestion were separated on a non-denaturing 6% polyacrylamide gel. The gel was dried and analyzed on a Molecular Dynamics PhosphorImager.


The porcine P347S hammerhead ribozyme specifically cleaves the P347S mutant RNA target sequence

Figure 3A shows an autoradiograph from a polyacrylamide-urea gel used to separate the products of ribozyme cleavage. The P347S hammerhead ribozyme was incubated with the P347S mutant and P347 wild type RNA target oligonucleotides as described above. Both the mutant and wild type target oligonucleotides are 13 nucleotides in length with molecular weights of 4101.5 and 4100.5, respectively. Due to conformational differences in this incompletely denaturing system, the wild type target oligonucleotide migrated faster than the mutant target on the polyacrylamide-urea gel. This experiment demonstrates that the P347S hammerhead ribozyme (porcine) was specific for the P347S mutant sequence. Total cleavage of the P347S mutant target oligonucleotide occurred in 60 min at a ribozyme to target molar ratio of 1:10 (Figure 3B). No cleavage was observed of the wild type target oligonucleotide under the same conditions. Figure 3C shows the time course of cleavage for the human P347S hammerhead ribozyme. This ribozyme was much slower than the porcine P347S hammerhead ribozyme, giving around 5% cleavage of the human P347S RNA target oligonucleotide in 60 min. The time course reaction in Figure 3B was performed to determine the time point where 15% of the RNA target oligonucleotide was cleaved (6 min with the porcine P347S ribozyme and 6.5 h with the human P347S ribozyme). As an estimate of Vo, all multiple-turnover kinetic reactions were performed at these time points.

Target length affects the rate of ribozyme cleavage

We produced two versions of the porcine P347S RNA target. The first was a 13 nucleotide synthetic RNA oligonucleotide. The second was a 34 nucleotide RNA produced by in vitro transcription from a cloned version of the mutant sequence. The RNA oligonucleotide target annealed to the ribozyme with no 5' or 3' overhanging bases. The cloned target annealed to the ribozyme with a 5' overhang of 15 bases and a 3' overhang of 6 bases. All of these extra bases were vector derived. Our time course analysis (Figure 3B) shows that initially the porcine P347S hammerhead ribozyme cleaved the RNA oligonucleotide target faster than the cloned target. Ultimately, both targets were cleaved to greater than 96% completion by 3 h.

Kinetic parameters for the hammerhead ribozymes were determined using multiple-turnover kinetics

Multiple-turnover kinetic reactions and analysis were done on both the porcine and human P347S hammerhead ribozymes as described in Methods. Figure 4 shows a Lineweaver-Burke double-reciprocal plot of a substrate saturation experiment for the porcine P347S hammerhead ribozyme. Data from six repetitions of the analysis generated the following kinetic parameters for this ribozyme: Vmax=7.3±0.5 nM/min, Km=2.1±0.6 mM, and kcat=1.5±0.4 min-1. These data include experiments using both the oligonuleotide version and the cloned version of the ribozyme. The results obtained from both versions were the same within experimental error. Data from duplicate experiments confirmed that the human P347S hammerhead ribozyme was much slower: Vmax=1.4±0.5 pM/min, Km=42±1.8 mM, and kcat=1.1x10-4±0.4x10-4 min-1. Enzymatic efficiency is often indicated by the ratio of kcat/Km, which is 7.5x105 min-1M-1 for the porcine specific ribozyme and 2.6 min-1M-1 for the human specific ribozyme. The human P347S hammerhead ribozyme was dropped from further analysis because its enzymatic efficiency suggested that it would not be a viable treatment for ADRP in vivo. Other ribozymes used for gene therapy have kcat values of around 1 min-1 [29]. We believe that the large difference we found between the porcine and human ribozymes is attributed to secondary structures in both the ribozyme and the target that prevent adequate binding of these two molecules (Figure 5).

The porcine P347S hammerhead ribozyme cleaves the full-length P347S mRNA

Total porcine retinal RNA from a P347S transgenic animal was incubated with the porcine P347S hammerhead ribozyme. Cleavage of the mutant mRNA was monitored by RT-PCR followed by restriction enzyme digestion of the RT-PCR products (Figure 6A). The products of restriction enzyme digestion were separated on a non-denaturing 6% polyacrylamide gel. The wild type P347 RT-PCR product contains a BanI site, but the mutant P347S RT-PCR product has lost the BanI site and has a unique HgaI site. The percent cleavage of the P347S mRNA was determined using the following formula [30]:

(% total mRNA - % treated mRNA) / (% total mRNA)

Figure 6B is an autoradiograph from a non-denaturing 6% polyacrylamide gel used to separate the restriction enzyme digested RT-PCR products generated after incubation of the porcine P347S hammerhead ribozyme with total mRNA isolated from porcine retinas as described in Methods. Thirty percent of mutant target was cleaved by the ribozyme in this reaction.


The P347S and P347L mutations in rhodopsin cause particularly severe forms of ADRP in humans resulting in total blindness by early adulthood [7,8]. We have designed hammerhead ribozymes that are specific for the P347S mutants of rhodopsin found in both a transgenic porcine model and in humans [10,12]. We produced two versions of the porcine P347S hammerhead ribozyme in order to determine if sequence context affected their behavior in vitro. The cloning of a DNA version of a ribozyme into a transcription vector can be time consuming and in vitro testing might ultimately reveal that the ribozyme is too slow for testing in animals. However, the use of synthetic RNA oligonucleotides for initial in vitro screening of potential ribozymes is fast and ultimately more economical than cloning. The first version was a synthetic RNA oligonucleotide purchased from Dharmacon. This ribozyme was 35 nucleotides in length and results in a secondary structure with 6 bp stems I and III and a 4 bp stem II (Figure 1B). The second version of the ribozyme was transcribed from a T7 RNA polymerase expression vector with a DNA copy of the ribozyme inserted downstream of the T7 RNA polymerase start site. This second version is identical to the first except for an additional eight nucleotides at its 5'-end and an additional single nucleotide at its 3'-end (Figure 2B). None of these additional bases participate in the formation of structures required for ribozyme function. We found no difference in the reaction kinetics between these two versions of the porcine P347S hammerhead ribozyme, suggesting that flanking nucleotides from cloning do not impair cleavage. In contrast, a cloned target of 34 nucleotides was cleaved considerably more slowly than the 13 oligonucleotide target (Figure 3B), indicating that sequences surrounding the cleavage site, some of which are vector derived, can reduce the apparent activity of ribozymes. Our in vitro testing also showed that the porcine P347S hammerhead ribozyme was specific for the porcine P347S mutant sequence (Figure 3A). This specificity was found with both the 13 nucleotide RNA oligonucleotide target and the full-length P347S mutant mRNA from total retinal RNA isolates (data not shown). In vitro experiments with the porcine P347S hammerhead ribozyme suggest that it may be a more potent gene therapy agent than a hammerhead ribozyme designed for another rhodopsin mutant, P23H, which has been tested in vivo in a transgenic rat model [23,24]. The P23H ribozyme exhibited a low Km (28 nM) but also a modest turnover number of 0.55 min-1. The porcine P347S hammerhead has a kcat of 1.5 min-1 (three times faster than the P23H ribozyme) but has a 1000 fold higher Km (2.1 mM). The relatively higher Km for the porcine P347S ribozyme suggests that the ribozyme:target intermediate may be pushed toward cleavage rather than target release more efficiently than the P23H ribozyme [31]. By comparison the human P347S hammerhead ribozyme has a kcat=1.1x10-4 and cleaves its target about 10,000 times slower than the porcine ribozyme. We believe that the poor in vitro performance of the human P347S ribozyme results mainly from the formation of secondary structures within the ribozyme and target that inhibit ribozyme:target binding (Figure 5), but also from the suboptimal NUX target site (CUC) in the human P347S target. This ribozyme has been dropped from further consideration.

The problems with the human P347S ribozyme illustrate one of the major problems in target selection. That is, finding a target site accessible to the ribozyme. By specifically targeting the human P347S mutation we have limited ourselves to a single target site. Inefficient cleavage at this site eliminates the human P347S hammerhead ribozyme as a potential effective gene therapy treatment. Another strategy for the treatment of the human P347S ADRP disease would be to design a hammerhead ribozyme specific for both wild type and mutant rhodopsin mRNAs. This ribozyme, along with an engineered replacement copy of the rhodopsin gene, could be packaged into a gene therapy vector like AAV. The engineered replacement copy of rhodopsin would still produce a wild type rhodopsin protein, but its mRNA would not be a substrate for the hammerhead ribozyme. The replacement RNA would be modified by replacing the NUX target site with a silent mutation (e.g., GUC to GUG which both encode valine). This approach allows the selection of an ideal cleavage site for the hammerhead ribozyme. In addition, this hammerhead ribozyme-rhodopsin gene construct would allow for the treatment of any ADRP stemming from mutations in the rhodopsin gene. Millington-Ward et al. [32] and O'Neill et al. [33] have also proposed this approach for the treatment of ADRP. This method has already been tested for dominant mutations in the gene for a1-antitrypsin [34].

The porcine P347S hammerhead ribozyme has been cloned in a rAAV vector (Figure 7) and packaged into AAV for subretinal injection into P347S transgenic pigs. These in vivo studies are currently underway and will be used to work out the parameters needed to do this type of gene therapy in human-sized eyes (e.g., volume and route of injection). While we have abandoned the human P347S hammerhead ribozyme as a viable agent for treatment of ADRP, in vivo testing of the porcine version should yield important dosage and delivery information in developing this type of treatment in humans.


This work was supported by grants from The Foundation Fighting Blindness, The March of Dimes Foundation, Research to Prevent Blindness, Inc., and The National Institutes of Health.


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