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Biomedical subjects

A S Lewin

Publications and source records attributed to A S Lewin.

At least 19 recordsLinked to original sources
PubMed2006

Preservation of photoreceptor morphology and function in P23H rats using an allele independent ribozyme.

To develop an allele independent ribozyme for the treatment of autosomal dominant retinitis pigmentosa (ADRP) associated with mutations in the rhodopsin (RHO) gene, a ribozyme targeting dog, mouse, human but not rat rhodopsin (RHO) mRNA was designed and tested in vitro. Activity of this ribozyme was tested in tissue culture by co-transfection of HEK 293 cells with plasmids expressing opsin mRNA and ribozyme, followed by quantitative RT-PCR to evaluate the level of RHO mRNA. For experiments in vivo, Rz525 driven by the mouse opsin proximal promoter was inserted in plasmids with AAV 2 terminal repeats (TR) and packaged in AAV serotype 5 capsids. AAV-Rz525 was injected subretinally into the right eyes of P23H rat pups. Left eyes were injected with virus expressing GFP from the identical promoter. Animals were analyzed at 4, 8 and 12 weeks post-injection by full field scotopic electroretinography (ERG). After 12 weeks, animals were sacrificed and retinas were dissected, fixed and sectioned. Rz525 had high catalytic activity in vitro and led to a 50% reduction of RHO mRNA in cells. AAV-Rz525 injection into P23H transgenic rats led to significant preservation (about 50%) of scotopic ERG a- and b-wave amplitudes. Histological analysis showed an increased number of ONL nuclei in the central and superior retina of treated eyes relative to control eyes. RT-PCR analysis revealed 46% reduction of transgenic (mouse) RHO mRNA in right eyes relative to left eyes and no change in rat RHO mRNA. AAV5 delivery of Rz525 resulted in a partial rescue of the light response and structural preservation of photoreceptors in transgenic rats. This ribozyme may be a useful component of an RNA replacement gene therapy for ADRP.

Alleles↗
PubMed2006

RNA knockdown as a potential therapeutic strategy in Parkinson's disease.

Parkinson's disease is a prevalent progressive degenerative disorder of the elderly. There is a current need for novel therapeutic strategies because the standard levodopa pharmacotherapy is only temporarily efficacious. Recently, there have been some high-profile successful preclinical results obtained in animal models of neurological disorders using small interfering RNAs delivered by viral vectors. RNA interference can theoretically be applied to Parkinson's disease since over-expression of various proteins is known to kill the dopamine neurons of the substantia nigra in animal models and in familial forms of Parkinson's disease. Potential RNA interfering strategies and caveats are discussed in this review.

Animals↗
PubMed2003

Reduction in preretinal neovascularization by ribozymes that cleave the A2B adenosine receptor mRNA.

Adenosine modulates a variety of cellular functions by interacting with specific cell surface G protein-coupled receptors (A1, A2A, A2B, and A3) and is a potential mediator of angiogenesis through the A2B receptor. The lack of a potent, selective A2B receptor inhibitor has hampered its characterization. Our goal was to design a hammerhead ribozyme that would specifically cleave the A2B receptor mRNA and examine its effect on retinal angiogenesis. Ribozymes specific for the mouse and human A2B receptor mRNAs were designed and cloned in expression plasmids. Human embryonic kidney (HEK) 293 cells were transfected with these plasmids and A2B receptor mRNA levels were determined by quantitative real-time RT-PCR. Human retinal endothelial cells (HRECs) were also transfected and cell migration was examined. The effects of these ribozymes on the levels of preretinal neovascularization were determined using a neonatal mouse model of oxygen-induced retinopathy (OIR). We produced a ribozyme with a Vmax of 515+/-125 pmol/min and a Kcat of 36.1+/-8.3 min(-1) (P< or =1x10(-5)). Transfection of HEK293 cells with the plasmid expressing the ribozyme reduced A2B receptor mRNA levels by 45+/-4.8% (P=5.1x10(-5)). Transfection of HRECs reduced NECA-stimulated migration of cells by 47.3+/-1.2% (P=7x10(-4)). Intraocular injection of the constructs into the mouse model reduced preretinal neovascularization by 53.5+/-8.2% (P=4.5x10(-5)). Our results suggest that the A2B receptor ribozyme will provide a tool for the selective inhibition of this receptor and provide further support for the role of A2B receptor in retinal angiogenesis.

Animals↗
PubMed2001

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

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.

Alleles↗
PubMed2001

Ribozyme gene therapy: applications for molecular medicine.

RNA enzymes--ribozymes--are being developed as treatments for a variety of diseases ranging from inborn metabolic disorders to viral infections and acquired diseases such as cancer. Ribozymes can be used both to downregulate and to repair pathogenic genes. In some instances, short-term exogenous delivery of stabilized RNA is desirable, but many treatments will require viral-mediated delivery to provide long-term expression of the therapeutic catalyst. Current gene therapy applications employ variations on naturally occurring ribozymes, but in vitro selection has provided new RNA and DNA catalysts, and research on trans-splicing and RNase P has suggested ways to harness the endogenous ribozymes of the cell for therapeutic purposes.

Acquired Immunodeficiency Syndrome↗
PubMed2001

Noninvasive imaging by optical coherence tomography to monitor retinal degeneration in the mouse.

PURPOSE: Optical coherence tomography (OCT) is a high-resolution imaging technique that measures the intensity of backscattered light from biological microstructures in living tissue. The objective was to evaluate OCT as a routine, noninvasive technique for quantitative measurements of retinal thickness and detachment in small animal models of retinal degenerative diseases. METHODS: An OCT scanning unit was designed and built to visualize retinal tissue from rodents at high resolution in vivo. Several normal and retinal degeneration (rd) mouse strains with different pigmentation, as well as a transgenic mouse strain that carries a wild-type beta-PDE gene in an rd/rd background, were analyzed at different ages. Retinal detachment was induced by subretinal injection of saline. Retinal function was evaluated by full-field ERG, and then each retina was cross-sectionally scanned by OCT. OCT image analysis and measurements of retinal thickness were performed. Animals were then killed and retinal histology was documented. RESULTS: OCT images of the mouse retina revealed structural landmarks allowing assignment of retinal structures. There was no difference in the OCT pattern between pigmented and nonpigmented mice. Changes in the retinal thickness measured by OCT correlated very well with the loss in function measured by ERG and histology in rd/rd and rd/rd/tg(+) transgenic mice at a variety of ages. In addition, retinal detachment caused by surgery was easily visualized and observed by OCT imaging. CONCLUSIONS: OCT imaging is applicable to the mouse retina. There is excellent agreement between the retinal thickness measured by OCT, ERG amplitude, and retinal histology, thus validating OCT imaging as a sensitive and noninvasive tool for monitoring the structural progression of retinal diseases in rodent models. OCT also appears useful for visualizing retinal detachments in the mouse.

Animals↗
PubMed2000

Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

Animals↗
PubMed2000

Ribozyme uses in retinal gene therapy.

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.

Animals↗
PubMed2000

Ribozyme gene therapy for autosomal dominant retinal disease.

Gene delivery to cells of the retina, particularly to photoreceptor cells, has broad potential both for answering basic questions of retinal biology and for more applied therapeutic purposes. The use of ribozymes as therapy for autosomal dominant retinal diseases is a promising technique, and the theoretical and practical basis for their use is discussed. The process involves designing and testing ribozymes first in vitro and then in animal models of retinal disease. Viral vectors based on the nonpathogenic human adeno-associated virus, when coupled with the strong, rod photoreceptor specific opsin promoter, offer an efficient and nontoxic way to deliver and express ribozymes in photoreceptor cells for long time periods of time. Effective ribozyme-mediated therapy also demands careful in vitro analysis of a ribozyme's ability to efficiently and specifically distinguish between mutant and wild type RNAs. Finally, effective demonstration of therapy in an animal model requires careful analysis of any rescue effect in the retina using multiple criteria, including biochemical, structural and physiological assays. For this purpose, ribozyme therapy in a transgenic rat model of retinitis pigmentosa containing a dominant rod opsin mutation (proline-to-histidine change at position 23) is discussed in detail.

Animals↗
PubMed1999

An RNA binding motif in the Cbp2 protein required for protein-stimulated RNA catalysis.

The fifth and terminal intron of yeast cytochrome b pre-mRNA (a group I intron) requires a protein encoded by the nuclear gene CBP2 for splicing. Because catalysis is intrinsic to the RNA, the protein is believed to promote formation of secondary and tertiary structure of the RNA, resulting in a catalytically competent intron. In vitro, this mitochondrial intron can be made to self-splice or undergo protein-facilitated splicing by varying the Mg(2+) and monovalent salt concentrations. This two-component system, therefore, provides a good model for understanding the role of proteins in RNA folding. A UV cross-linking experiment was initiated to identify RNA binding sites on Cbp2 and gain insights into Cbp2-intron interactions. A 12-amino acid region containing a presumptive contact site near the amino terminus was targeted for mutagenesis, and mutant proteins were characterized for RNA binding and stimulation of splicing. Mutations in this region resulted in partial or complete loss of function, demonstrating the importance of this determinant for stimulation of RNA splicing. Several of the mutations that severely reduced splicing did not significantly shift the overall binding isotherm of Cbp2 for the precursor RNA, suggesting that contacts critical for activity are not necessarily reflected in the dissociation constant. This analysis has identified a unique RNA binding motif of alternating basic and aromatic residues that is essential for protein facilitated splicing.

Amino Acid Sequence↗
PubMed1998

Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

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.

Animals↗
PubMed1998

Ribozyme-targeted destruction of RNA associated with autosomal-dominant retinitis pigmentosa.

PURPOSE: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa. METHODS: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction. RESULTS: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA. CONCLUSIONS: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.

Animals↗
PubMed1997

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.

Apoproteins↗
PubMed1997

Potentiation of antifungal activity of amphotericin B by azithromycin against Aspergillus species.

The potential role of azithromycin in combination with amphotericin B against 25 clinical isolates of Aspergillus was assessed. The MIC of amphotericin B was 1 microg/ml for 44% of the isolates, 0.5 microg/ml for 48%, and 0.25 microg/ml for 8%. All isolates were resistant to azithromycin. Synergism, defined as a > or = twofold reduction in the MIC of both drugs upon combination, was demonstrated between amphotericin B and azithromycin for all 25 isolates. To prove that azithromycin exerts its antifungal effect by inhibiting protein synthesis, we studied [35S]-methionine incorporation into protein in one Aspergillus isolate. Neither amphotericin B at 0.125 microg/ml (fourfold below its MIC) nor azithromycin at 16 microg/ml (> or = 16-fold below its MIC) had any effect on protein synthesis when tested alone. Upon combination, however, a 68% inhibition in protein synthesis was evident by the inhibition of [35S]-methionine incorporation.

Amphotericin B↗
PubMed1996

The Cbp2 protein suppresses splice site mutations in a group I intron.

The Cbp2 protein facilitates the folding of a group I intron in the COB pre-mRNA of yeast mitochondria. Based on its ability to suppress mutations affecting the auto-catalytic reaction, the protein appears to play a role in the selection of splice sites. Adding Cbp2 did not overcome the effects of mutations in P1 whose primary effect was on the first step of splicing. In contrast, most mutations affecting the ligation of exons were suppressed in vitro by Cbp2. These included mutations in P1, P9.0 and P10. In fact, a mutant transcript lacking both P9.0 and P10 ligated efficiently in the presence of Cbp2. P9.0 and P10 mutations also reduced the rate of cleavage at the 5' splice junction, and this effect was only partially mitigated by adding Cbp2. A competitive secondary structure near the 3' splice junction blocked Cbp2-stimulated splicing, but this mutation could be suppressed by co-transcriptional splicing in the presence of Cbp2. Our data underscore the importance of the interaction between the 5' and 3' splice junctions in group I introns and suggest that nucleotide-nucleotide interactions that stabilize the structure of group I introns can be superceded by protein-RNA interactions.

Apoproteins↗
PubMed1995

Protein-induced folding of a group I intron in cytochrome b pre-mRNA.

Some group I introns have been shown to be self-splicing in vitro, but perhaps all require proteins for splicing in vivo. Sequence differences affect the stability of secondary structures and may explain why some group I introns function efficiently without protein cofactors while others require them. The terminal intron of the cytochrome b pre-mRNA from yeast mitochondria needs a nucleus-encoded protein for splicing, even though it splices autocatalytically in high salt in vitro. This system has the advantage that the protein is specific for this intron, and yet the structure of the catalytically active RNA can be studied in its absence. We have modified the intron by chemical and enzymatic treatment in the presence and absence of the protein to determine the impact of the protein on the secondary and tertiary structures of the intron. We found protein-induced formation of secondary and tertiary structures within the intron, and the same structures also form in high salt autocatalytic conditions. We have also studied UV cross-links to determine those bases of the intron that interact directly with the protein and found that the protein contacts the intron most intimately at the structures denoted P1, L2, P4, and P6a.

Base Sequence↗