Construction of a functional human suppressor tRNA gene: an approach to gene therapy for β-thalassaemia

Temple, Gary F.; Dozy, Andrée M.; Roy, Kanak; Kan, Yuet Wai

doi:10.1038/296537a0

Cited by 142 publications

(86 citation statements)

References 32 publications

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“…In addition, cell lines carrying the X. laevis amber Su' tRNATYr gene and human amber Su' tRNAser gene have been established (15, 32a). Glutamine-and lysine-inserting amber Su' tRNA genes also have been generated from human tRNA genes (37) and found to be functional in mammalian cells (13).…”

mentioning

confidence: 99%

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Ganguly¹,

Sharp²,

RajBhandary³

1988

Mol. Cell. Biol.

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We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNALU gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNAI"U gene. Extracts from HeLa or CV1 cells transcribed both tRNALeU genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNALu.Surprisingly, when the same tRNAI"U genes were introduced into CV1 cells, only pre-tRNAsLu were produced. The pre-tRNAsI"u made in vivo were of the same size and contained the same 5'-leader and 3'-trailer sequences as did pre-tRNAsLu made in vitro. Furthermore, the pre-tRNAsI"u made in vivo were processed to mature tRNAIeU when incubated with HeLa cell extracts. A tRNALeU gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNALu in CV1 cells is blocked at the level of 5'-and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAslu synthesized in CV1 cells are not transported to the appropriate site.

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mentioning

confidence: 99%

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Ganguly¹,

Sharp²,

RajBhandary³

1988

Mol. Cell. Biol.

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“…The amounts of specific RNA were determined by Northern hybridization as described previously (25,41 . Immunoprecipitation of translated products with an antibody against human x-globin (a gift from S. Boyer) was performed as described previously (40). All protein analyses were performed in 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels (24).…”

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confidence: 99%

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

Lau¹,

Lin²,

Kan³

1984

Mol. Cell. Biol.

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We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human a-globin gene (Na2) and a hybrid gene (MaG) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural aL2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500-to 1,000-fold increase in the number of copies of DHFR and human ot-globin genes. The transcription of the a-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid MaG gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human a-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that a-globin mRNA transcribed from the amplified a-globin genes was functional and directed a-globin chain synthesis. In situ hybridization of 3H-labeled oa-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.

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“…Several studies demonstrated efficient nonsense suppression in mammalian models, although the recovery of functional proteins was rarely shown. 15,20,[22][23][24][25] In this work we wish to: (1) evaluate the potential impact of using nonsense suppression in HDGC; (2) determine the number of tRNAs necessary to treat nonsense mutation-carrying HDGC families; and (3) assess the possibility of recovering expression of a functional protein from an allele carrying a nonsense mutation described in HDGC using a sup-tRNA. To our knowledge, this is the first demonstration of functional recovery of a causative gene in cancer by cognate amino acid replacement with sup-tRNAs.…”

Section: Cdh1mentioning

confidence: 99%

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

et al. 2014

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Hereditary diffuse gastric cancer (HDGC) syndrome, although rare, is highly penetrant at an early age, and is severe and incurable because of ineffective screening tools and therapy. Approximately 45% of HDGC families carry germline CDH1/Ecadherin alterations, 20% of which are nonsense leading to premature protein truncation. Prophylactic gastrectomy is the only recommended approach for all asymptomatic CDH1 mutation carriers. Suppressor-tRNAs can replace premature stop codons (PTCs) with a cognate amino acid, inducing readthrough and generating full-length proteins. The use of suppressor-tRNAs in HDGC patients could therefore constitute a less invasive therapeutic option for nonsense mutation carriers, delaying the development of gastric cancer. Our analysis revealed that 23/108 (21.3%) of E-cadherin-mutant families carried nonsense mutations that could be potentially corrected by eight suppressor-tRNAs, and arginine was the most frequently affected amino acid. Using site-directed mutagenesis, we developed an arginine suppressor-tRNA vector to correct one HDGC nonsense mutation. E-cadherin-deficient cell lines were transfected with plasmids carrying simultaneously the suppressor-tRNA and wild-type or mutant CDH1 mini-genes. RT-PCR, western blot, immunofluorescence, flow cytometry and proximity ligation assay (PLA) were used to establish the model, and monitor mRNA and protein expression and function recovery from CDH1 vectors. Cells expressing a CDH1 mini-gene, carrying a nonsense mutation and the suppressor-tRNA, recovered full-length E-cadherin expression and its correct localization and incorporation into the adhesion complex. This is the first demonstration of functional recovery of a mutated causative gene in hereditary cancer by cognate amino acid replacement with suppressor-tRNAs. Of the HDGC families, 21.3% are candidates for correction with suppressor-tRNAs to potentially delay cancer onset.

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Construction of a functional human suppressor tRNA gene: an approach to gene therapy for β-thalassaemia

Cited by 142 publications

References 32 publications

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

Rescue of wild-type E-cadherin expression from nonsense-mutated cancer cells by a suppressor-tRNA

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