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Fritz Eckstein - One of the best experts on this subject based on the ideXlab platform.
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in vitro selection of a purine nucleotide specific hammerheadlike ribozyme
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Narendra K Vaish, Paul A Heaton, Olga Fedorova, Fritz EcksteinAbstract:The in vitro selection for an intramolecular AUG-cleaving hammerhead-like ribozyme is described. One of the Ribozymes selected was found to cleave after this triplet, both intramolecularly and intermolecularly, with rates comparable to the rate of the native GUC-cleaving hammerhead ribozyme. Although the selection was designed for cleavage 3′ of the AUG triplet, the ribozyme also cleaves 3′ of the AUA triplet. AUU and AUC triplets are, however, not cleaved, and thus the selected ribozyme is purine-specific for the third position in the triplet. In addition, cleavage 3′ of the AAG triplet has been observed, thus the central U is not essential. Nuclease digestion indicates that the selected ribozyme has a secondary structure similar to that of the native hammerhead ribozyme, although with an altered core and stem–loop II sequence. All nucleotides in the core, except one, are essential for activity. The nucleotides in loop II are sensitive to changes and cannot, as in the hammerhead ribozyme, be replaced by other sequences or a nonnucleotide linker. Thus there are differences between these two Ribozymes even though they have similar two-dimensional structures. The new ribozyme enlarges the application of hammerhead Ribozymes for the inhibition of gene expression by extending the range of cleavable triplets.
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the structure function and application of the hammerhead ribozyme
FEBS Journal, 1997Co-Authors: Klara R Birikh, Paul A Heaton, Fritz EcksteinAbstract:The hammerhead ribozyme is one of the smallest Ribozymes known and catalyses the site-specific hydrolysis of a phosphodiester bond. This small ribozyme is of interest for two reasons. It offers a convenient system to study the structure/function relationship of a nucleotide sequence, and is a potential vehicle for the inhibition of gene expression. The first part of the review summarizes the sequence requirements of the hammerhead, its three-dimensional structure and the proposed mechanism, in addition to ribozyme specificity and turnover. The second part of the review focuses on the in vivo application of the ribozyme. The processes involved in designing Ribozymes for efficient cleavage in vivo are described, together with possible delivery strategies.
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mg2 dependent conformational changes in the hammerhead ribozyme
Biochemistry, 1996Co-Authors: Marcus Menger, Fritz Eckstein, Thomas Tuschl, Dietmar PorschkeAbstract:Conformational changes of the hammerhead ribozyme were examined by fluorescence changes of 2-aminopurine riboside incorporated either in the substrate or in the ribozyme. Fluorescence changes could be observed for both the substituted substrate and ribozyme upon complex formation, indicating a different environment for the 2-aminopurine in the complex. Ribozyme-substrate constructs for ciscleavage containing 2-aminopurine at various sites were used for the determination of binding constants of Mg2+ and Ca2+. Depending upon the site of 2-aminopurine substitutions, the fluorescence intensity upon addition of Mg2+ or Ca2+ was reduced by 0-50%. The measurements were performed in high ionic strength buffers such that base pairing in the helical regions is expected to be complete. With three of the Ribozymes, the dependence of the fluorescence emission as a function of Mg2+ concentration could be fitted by single binding processes, whereas for the two remaining Ribozymes a second binding process needed to be included. The binding constants range from 7600 M-1 down to 12 M-1 in 75 mM Tris-HCl (pH 7.5) and indicate the presence of multiple binding sites in the Ribozymes with varying degrees of affinity toward the metal ions. Mg2+ binding constants determined in the same buffer from the Mg2+ dependence of the cleavage rate are of the order of 100 M-1; thus, Mg2+ sites directly involved in catalysis are of intermediate affinity. The ribozyme containing 2-aminopurine in loop III demonstrated the highest binding constant whereas the ribozyme with a 2-aminopurine next to a 2'-deoxy-2'-aminocytosine at the cleavage site exhibited only low metal ion affinity. The data obtained for Ca2+ are very similar to those found for Mg2+. This approach provides a first set of data describing a Mg2+ binding topography to hammerhead RNA molecules and should be useful for the analysis of other RNA molecules.
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high activity and stability of hammerhead Ribozymes containing 2 modified pyrimidine nucleosides and phosphorothioates
Journal of Biological Chemistry, 1994Co-Authors: Olaf Heidenreich, Fritz Benseler, A Fahrenholz, Fritz EcksteinAbstract:The influence of chemical modifications on the catalytic activity and stability of a hammerhead ribozyme directed against the long terminal repeat RNA of the human immunodeficiency virus 1 was examined. Previous studies had shown that substitution of all pyrimidine nucleosides by their 2'-fluoro analogs led to an 8-fold decrease in catalytic efficiency in the cleavage reaction compared to the unmodified ribozyme (Heidenreich, O., and Eckstein, F. (1992) J. Biol. Chem. 267, 1904-1909). It is shown here that replacement of the 2'-fluoro-2'-deoxyuridines in the conserved region of this ribozyme, positions 4 and 7, by 2'-amino-2'-deoxyuridines fully restores catalytic activity of the ribozyme. Ribozymes containing these 2'-modifications show an increased stability against RNases present in fetal calf serum and in cell culture supernatant. The stability is increased further by the incorporation of four terminal phosphorothioates as protection against 3'-exonucleases, the degree of which depends on the secondary structure of the ribozyme. Such Ribozymes are stable in undiluted fetal calf serum for at least 24 h. The results clearly demonstrate the potential to design stable Ribozymes without any loss of catalytic activity.
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function of specific 2 hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2 modifications
Proceedings of the National Academy of Sciences of the United States of America, 1992Co-Authors: David M Williams, Wolfgang Pieken, Fritz EcksteinAbstract:Abstract The importance of the 2'-hydroxyl group of several guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. Five Ribozymes in which single guanosine residues were substituted with 2'-amino-, 2'-fluoro-, or 2'-deoxyguanosine were chemically synthesized. The comparison of the catalytic activity of the three 2' modifications at a specific position allows conclusions about the functional role of the parent 2'-hydroxyl group. Substitutions of nonconserved nucleotides within the ribozyme caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, when either of the guanosines within the single-stranded loop between stem I and stem II of the ribozyme was replaced by 2'-deoxyguanosine or 2'-fluoro-2'-deoxyguanosine, the catalytic activities of the resulting Ribozymes were reduced by factors of at least 150. The catalytic activities of the corresponding Ribozymes containing 2'-amino-2'-deoxyguanosine substitutions at these positions, however, were both reduced by factors of 15. These effects resulted from decreases in the respective kcat values, whereas variations in the Km values were comparatively small. A different pattern of reactivity of the three 2' modifications was observed at the guanosine immediately 3' to stem II of the ribozyme. Whereas both 2'-deoxyguanosine and 2'-amino-2'-deoxyguanosine at this position showed catalytic activity similar to that of the unmodified ribozyme, the activity of the corresponding 2'-fluoro-2'-deoxyguanosine-containing ribozyme was reduced by a factor of 15. The implications of these substitution-specific reactivities on the functional role of the native 2'-hydroxyl groups are discussed.
Alfred S. Lewin - One of the best experts on this subject based on the ideXlab platform.
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Ribozyme Knockdown of the ␥-Subunit of Rod cGMP Phosphodiesterase Alters the ERG and Retinal Morphology in Wild-Type Mice
2020Co-Authors: Jianwen Liu, Alfred S. Lewin, Adrian M Timmers, William W. HauswirthAbstract:PURPOSE. To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the ␥-subunit of the rod cGMP phosphodiesterase (PDE␥) mRNA in the retina of wild-type mice. METHODS. Two hammerhead Ribozymes, HRz35 and HRz42, were designed to target the PDE␥ gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the Ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDE␥ gene were monitored by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblot assay. RESULTS. The Ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K cat of 0.48 minute Ϫ1 and a K m of 980 nM, and HRz42 showed a K cat of 0.17 minute Ϫ1 and a K m of 971 nM. Both Ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and fulllength mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a-and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDE␥ mRNA and protein paralleled the ERG results. CONCLUSIONS. Ribozyme-mediated somatic knockdown of wildtype PDE␥ mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent. (Invest Ophthalmol Vis Sci. 2005;46: 3836 -3844
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An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.
Molecular vision, 2001Co-Authors: Lynn C. Shaw, William W. Hauswirth, Skold A, Fulton Wong, Robert M. Petters, Alfred S. LewinAbstract: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.
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ribozyme uses in retinal gene therapy
Progress in Retinal and Eye Research, 2000Co-Authors: William W. Hauswirth, Alfred S. LewinAbstract:In this chapter we discuss the design, delivery and preclinical testing of mutation-specific Ribozymes for the treatment of dominantly inherited retinal disease. We focus particular attention on the initial screening of Ribozymes in vitro, because the activity of RNA enzymes in cell-free systems can be used to predict their suitability for animal experiments. Current techniques for delivering genes of interest to cells of the retina using viral vectors are then briefly surveyed emphasizing vector properties that best match to the needs of a ribozyme-based therapy. Using these considerations, analysis of ribozyme gene therapy for an autosomal dominant RP-like disease in a rodent model is outlined emphasizing the desirability of combining biochemical, morphological and electrophysiological measures of therapy. Finally, we describe alternative, perhaps more general, ribozyme approaches that have yet to be tested in the context of retinal disease.
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Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.
Nature medicine, 1998Co-Authors: Alfred S. Lewin, Kimberly A. Drenser, William W. Hauswirth, Shimpei Nishikawa, Douglas Yasumura, John G. Flannery, Matthew M. LavailAbstract:Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control Ribozymes had less effect on the retinal degeneration. Intracellular production of Ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.
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ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa
Nature Medicine, 1998Co-Authors: Alfred S. Lewin, Kimberly A. Drenser, William W. Hauswirth, Shimpei Nishikawa, Douglas Yasumura, John G. Flannery, Matthew M. LavailAbstract:Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene1, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP (ref. 2). Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control Ribozymes had less effect on the retinal degeneration. Intracellular production of Ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.
Peter V Coveney - One of the best experts on this subject based on the ideXlab platform.
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structure dynamics and function of the hammerhead ribozyme in bulk water and at a clay mineral surface from replica exchange molecular dynamics
Langmuir, 2015Co-Authors: Jacob B Swadling, David W Wright, James L Suter, Peter V CoveneyAbstract:Compared with proteins, the relationship between structure, dynamics, and function of RNA enzymes (known as Ribozymes) is far less well understood, despite the fact that Ribozymes are found in many organisms and are often conceived as "molecular fossils" of the first self-replicating molecules to have arisen on Earth. To investigate how ribozymal function is governed by structure and dynamics, we study the full hammerhead ribozyme in bulk water and in an aqueous clay mineral environment by computer simulation using replica-exchange molecular dynamics. Through extensive sampling of the major conformational states of the hammerhead ribozyme, we are able to show that the hammerhead manifests a free-energy landscape reminiscent of that which is well known in proteins, exhibiting a "funnel" topology that guides the ribozyme into its globally most stable conformation. The active-site geometry is found to be closely correlated to the tertiary structure of the ribozyme, thereby reconciling conflicts between previously proposed mechanisms for the self-scission of the hammerhead. The conformational analysis also accounts for the differences reported experimentally in the catalytic activity of the hammerhead ribozyme, which is reduced when interacting with clay minerals as compared with bulk water.
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structure dynamics and function of the hammerhead ribozyme in bulk water and at a clay mineral surface from replica exchange molecular dynamics
Langmuir, 2015Co-Authors: Jacob B Swadling, David W Wright, James L Suter, Peter V CoveneyAbstract:Compared with proteins, the relationship between structure, dynamics, and function of RNA enzymes (known as Ribozymes) is far less well understood, despite the fact that Ribozymes are found in many organisms and are often conceived as “molecular fossils” of the first self-replicating molecules to have arisen on Earth. To investigate how ribozymal function is governed by structure and dynamics, we study the full hammerhead ribozyme in bulk water and in an aqueous clay mineral environment by computer simulation using replica-exchange molecular dynamics. Through extensive sampling of the major conformational states of the hammerhead ribozyme, we are able to show that the hammerhead manifests a free-energy landscape reminiscent of that which is well known in proteins, exhibiting a “funnel” topology that guides the ribozyme into its globally most stable conformation. The active-site geometry is found to be closely correlated to the tertiary structure of the ribozyme, thereby reconciling conflicts between previ...
William W. Hauswirth - One of the best experts on this subject based on the ideXlab platform.
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Ribozyme Knockdown of the ␥-Subunit of Rod cGMP Phosphodiesterase Alters the ERG and Retinal Morphology in Wild-Type Mice
2020Co-Authors: Jianwen Liu, Alfred S. Lewin, Adrian M Timmers, William W. HauswirthAbstract:PURPOSE. To generate an animal model of retinal degeneration by using AAV-mediated ribozyme knockdown of the ␥-subunit of the rod cGMP phosphodiesterase (PDE␥) mRNA in the retina of wild-type mice. METHODS. Two hammerhead Ribozymes, HRz35 and HRz42, were designed to target the PDE␥ gene in wild-type C57BL/6 mice. The efficiency and specificity of the ribozyme cleavage was tested in vitro against three different types of target: short synthetic RNA oligomers, longer targets transcribed from clones, and full-length mRNA from total retinal RNA extracts. After in vitro validation, the Ribozymes were cloned and packaged in a recombinant adenoassociated virus (rAAV) containing a proximal 472-bp murine rod opsin promoter (MOPS) to drive ribozyme synthesis. Three-week-old wild-type C57BL/6 mice were injected subretinally with the vectors. For treated versus partner control retinas, responses to light were measured by full-field electroretinography (ERG), and retinal tissues were examined by light microscopy. Messenger RNA and protein levels of PDE␥ gene were monitored by reverse transcription-polymerase chain reaction (RT-PCR) and Western immunoblot assay. RESULTS. The Ribozymes had comparable in vitro kinetic properties in multiple turnover kinetic analyses. Ribozyme HRz35 exhibited a K cat of 0.48 minute Ϫ1 and a K m of 980 nM, and HRz42 showed a K cat of 0.17 minute Ϫ1 and a K m of 971 nM. Both Ribozymes cleaved at accessible sites in the RNA, as they digested long transcripts transcribed from clones and fulllength mRNA from total retinal RNA extracts in vitro. At increasing intervals after subretinal injection with either AAV ribozyme, a 30% to 90% reduction in a-and b-wave amplitudes was observed compared with those in contralateral control eyes that were not injected. Retinal tissue analysis showed that loss of the photoreceptor cells and PDE␥ mRNA and protein paralleled the ERG results. CONCLUSIONS. Ribozyme-mediated somatic knockdown of wildtype PDE␥ mRNA in vivo can efficiently reduce the target RNA leading to a loss in rod photoreceptors and in rod-mediated ERG amplitudes, thus generating an animal model of retinal degeneration resembling human RP in an essentially normal adult retina. This vector ribozyme technique should be applicable to other genes associated with RP and perhaps also to mRNAs of phototransduction genes not yet associated with RP. Application of this approach may be age and species independent. (Invest Ophthalmol Vis Sci. 2005;46: 3836 -3844
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An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.
Molecular vision, 2001Co-Authors: Lynn C. Shaw, William W. Hauswirth, Skold A, Fulton Wong, Robert M. Petters, Alfred S. LewinAbstract: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.
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ribozyme uses in retinal gene therapy
Progress in Retinal and Eye Research, 2000Co-Authors: William W. Hauswirth, Alfred S. LewinAbstract:In this chapter we discuss the design, delivery and preclinical testing of mutation-specific Ribozymes for the treatment of dominantly inherited retinal disease. We focus particular attention on the initial screening of Ribozymes in vitro, because the activity of RNA enzymes in cell-free systems can be used to predict their suitability for animal experiments. Current techniques for delivering genes of interest to cells of the retina using viral vectors are then briefly surveyed emphasizing vector properties that best match to the needs of a ribozyme-based therapy. Using these considerations, analysis of ribozyme gene therapy for an autosomal dominant RP-like disease in a rodent model is outlined emphasizing the desirability of combining biochemical, morphological and electrophysiological measures of therapy. Finally, we describe alternative, perhaps more general, ribozyme approaches that have yet to be tested in the context of retinal disease.
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Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.
Nature medicine, 1998Co-Authors: Alfred S. Lewin, Kimberly A. Drenser, William W. Hauswirth, Shimpei Nishikawa, Douglas Yasumura, John G. Flannery, Matthew M. LavailAbstract:Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control Ribozymes had less effect on the retinal degeneration. Intracellular production of Ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.
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ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa
Nature Medicine, 1998Co-Authors: Alfred S. Lewin, Kimberly A. Drenser, William W. Hauswirth, Shimpei Nishikawa, Douglas Yasumura, John G. Flannery, Matthew M. LavailAbstract:Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene1, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP (ref. 2). Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control Ribozymes had less effect on the retinal degeneration. Intracellular production of Ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.
Olaf Heidenreich - One of the best experts on this subject based on the ideXlab platform.
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high activity and stability of hammerhead Ribozymes containing 2 modified pyrimidine nucleosides and phosphorothioates
Journal of Biological Chemistry, 1994Co-Authors: Olaf Heidenreich, Fritz Benseler, A Fahrenholz, Fritz EcksteinAbstract:The influence of chemical modifications on the catalytic activity and stability of a hammerhead ribozyme directed against the long terminal repeat RNA of the human immunodeficiency virus 1 was examined. Previous studies had shown that substitution of all pyrimidine nucleosides by their 2'-fluoro analogs led to an 8-fold decrease in catalytic efficiency in the cleavage reaction compared to the unmodified ribozyme (Heidenreich, O., and Eckstein, F. (1992) J. Biol. Chem. 267, 1904-1909). It is shown here that replacement of the 2'-fluoro-2'-deoxyuridines in the conserved region of this ribozyme, positions 4 and 7, by 2'-amino-2'-deoxyuridines fully restores catalytic activity of the ribozyme. Ribozymes containing these 2'-modifications show an increased stability against RNases present in fetal calf serum and in cell culture supernatant. The stability is increased further by the incorporation of four terminal phosphorothioates as protection against 3'-exonucleases, the degree of which depends on the secondary structure of the ribozyme. Such Ribozymes are stable in undiluted fetal calf serum for at least 24 h. The results clearly demonstrate the potential to design stable Ribozymes without any loss of catalytic activity.
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hammerhead ribozyme mediated cleavage of the long terminal repeat rna of human immunodeficiency virus type 1
Journal of Biological Chemistry, 1992Co-Authors: Olaf Heidenreich, Fritz EcksteinAbstract:Three Ribozymes targeted against different sites of the long terminal repeat RNA (LTR RNA) of human immunodeficiency virus type 1 cleaved a LTR RNA transcript 1,000 x less efficiently than corresponding short synthetic oligoribonucleotide substrates. Varying the stem lengths of the ribozyme resulted in changes of the catalytic efficiency. Almost no cleavage was observed for a ribozyme forming only 10 base pairs instead of 14 with the LTR RNA. Increasing the base pairs to 16 or elongation of the stem formed within the ribozyme revealed only small changes in kcat/Km. The influence of chemical modifications within the ribozyme on the cleavage of the LTR RNA was also examined. 2'-Fluorocytidine substitutions as well as four terminal phosphorothioate internucleotidic linkages influenced the catalytic efficiency of Ribozymes only negligibly. However, substitution of uridine by 2'-fluorouridine resulted in a 5-fold decrease of kcat/Km. A ribozyme containing all these modifications revealed only a 7-fold lower catalytic efficiency but a markedly increased stability in cell culture supernatant. These results demonstrate that it is possible to increase the stability of Ribozymes toward nucleases without a serious loss in catalytic efficiency.
