Hammerhead Ribozyme Cleavage of Apolipoprotein B mRNA Generates a Truncated Protein

Wang, Jian Ping; Enjoji, Munechika; Tiebel, M.; Ochsner, Scott A.; Chan, Lawrence; Teng, Babie

doi:10.1074/jbc.274.34.24161

Cited by 13 publications

(19 citation statements)

References 48 publications

(34 reference statements)

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“…To further validate this view, we examined the effects of a specific hammerhead ribozyme against NBC-1 (RZ-NBC), which was designed to target at the common region of human NBC-1 mRNA. Consistent with the highly efficient ability of hammerhead ribozyme to inhibit gene expression (20), adenovirus-mediated transfer of RZ-NBC into HLE cells quite effectively suppressed the expression of NBC-1 protein as estimated by Western blot analysis ( Figure 6). In cells transfected with RZ-NBC, the pH i recovery from acid load in Cl --free HCO 3 -solution containing amiloride was markedly slowed (Figure 7a), and the residual DIDS-sensitive Na + -HCO 3 -cotransport activity at 48 hours was only 20 ± 3% (n = 12) of the control value (Figure 7b).…”

Section: Specificity Of Anti-nbc-1 Ab'smentioning

confidence: 53%

“…The oligonucleotide, 5′-AACATCCCTGATGAGTC-CGTGAGGACGAAACTTGC-3′, and the complementary oligonucleotide were annealed together and subcloned into the NruI site of this plasmid to construct a NBC-1 mRNA-specific hammerhead ribozyme, RZ-NBC. The RZ sequence consisted of two flanking regions complementary to NBC-1 mRNA, targeting at the NBC-1 common region (GUA 723 in kNBC-1 or GUA 823 in pNBC-1), and a conserved catalytic sequence (20). The plasmid was then subcloned into pAdCMVlink lacking cytomegalovirus enhancer and promoter, linearized with EcoRI, and cotransfected to 293 cells together with adenoviral DNA digested with ClaI to construct the adenovirus vector carrying RZ-NBC.…”

Section: Methodsmentioning

confidence: 99%

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Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Usui¹,

Hara²,

Satoh³

et al. 2001

J. Clin. Invest.

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Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na + -HCO 3 -cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na + -HCO 3 -cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH i ) measurements revealed the presence of Cl --independent, electrogenic Na + -HCO 3 -cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH i recovery from acid load in the presence of HCO 3 -/CO 2 was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO 3 -transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

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Section: Specificity Of Anti-nbc-1 Ab'smentioning

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Usui¹,

Hara²,

Satoh³

et al. 2001

J. Clin. Invest.

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“…One such strategy employs ribozymes, small RNA molecules with specific RNA cleaving capacity. Adenoviral expression of an apoB-specific ribozyme has been shown to decrease full-length apoB mRNA concentration and consequently the production of full-length apoB protein in vitro [Wang et al, 1999] as well as in vivo [Enjoji et al, 2000] (see Fig. 1F).…”

Section: Decreasing Levels Of Apob-100mentioning

confidence: 99%

Gene Therapy in Disorders of Lipoprotein Metabolism

Vaessen

Twisk²,

Kastelein³

et al. 2007

CGT

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Current pharmacologic interventions in lipid metabolism are insufficient in a subset of patients at increased risk of cardiovascular disease. In particular, several monogenetic disorders of lipid metabolism with diverse clinical complications are beyond treatment to date. Somatic gene transfer is a potential approach to treat these disorders. This review describes the efforts made thus far to develop gene therapy for 3 major classes of dyslipidemia: Increased levels of low-density lipoprotein cholesterol, reduced levels of high-density lipoprotein cholesterol and increased plasma triglyceride levels. For many of the genetic causes underlying these conditions, proof-of-principle studies have been performed and in combination with improved vectors some of these strategies may be feasible for clinical use in the future.

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“…Our laboratory [2,3] has previously demonstrated that adenovirus mediated apolipoprotein B (apoB) mRNA-specific hammerhead ribozyme cleaved apoB mRNA efficiently. In the dyslipidemic mouse model, the effect of decreased apoB mRNA results in a marked decrease in plasma cholesterol, triglyceride and apo B levels [3] and these hypolipidemic effects persist to day-21.…”

Section: Introductionmentioning

confidence: 99%

“…To decrease apoB mRNA expression, we have designed a hammerhead ribozyme targeted at GUA 6679 of apoB mRNA (RB15) to cleave apoB mRNA [2,3]. In this study, we constructed the apolipoprotein B mRNA-specific hammerhead ribozyme (RB15) driven by a liver-specific transthyretin (TTR) promoter [6] using the AAV2 serotype virus vector (rAAV2-TTR-RB15).…”

Section: Introductionmentioning

confidence: 99%

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

Zhong

Sun

Teng

2004

Genet Vaccines Ther

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BackgroundIn humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo.MethodsWe constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15.ResultsThe effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene expression in the mice livers, which did not produce the therapeutic effects on alteration the lipid levels or the inhibition of atherosclerosis development. In contrast, the ribozyme RB15 RNA mediated by scAAV2-TTR-RB15 vector was expressed immediately at day-1 after transduction in HepG2 cells. The apoB mRNA levels were decreased 47% (p = 0.001), compared to the control vector scAAV2-TTR-RB15-mutant.ConclusionThis study provided evidence that the rAAV2 single-strand vector mediated a prolonged but not efficient transduction in mouse liver. However, the scAAV2 double-strand vector mediated a rapid and efficient gene expression in liver cells. This strategy using scAAV2 vectors represents a better approach to express small molecules such as ribozyme.

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Hammerhead Ribozyme Cleavage of Apolipoprotein B mRNA Generates a Truncated Protein

Cited by 13 publications

References 48 publications

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Gene Therapy in Disorders of Lipoprotein Metabolism

The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

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