1 Gene Therapy 2003 Vol: 10(22):1910-1916. DOI: 10.1038/sj.gt.3302096

Correction/mutation of acid α-D-glucosidase gene by modified single-stranded oligonucleotides: in vitro and in vivo studies

Deficiency in acid -D-glucosidase results in Pompe's disease. Modified single-stranded oligonucleotide (ODN) was designed to correct the acid -D-glucosidase gene with a C1935 A (Asp Glu) point mutation which causes a complete loss of enzymatic activity for glycogen digestion in the lysosome. The ODN vectors contained a stretch of normal oligonucleotide flanked by phosphorothioated sequences. The 25mer and 35mer ODNs were homologous to the target sequence, except for a mismatched base in the middle. The ODNs caused permanent and inheritable restoration of acid -D-glucosidase activity in skin fibroblast cells carrying this mutation derived from a Pompe's disease patient. Gene correction was confirmed by amplification refractory mutation system-PCR (ARMS-PCR), restriction fragment length polymorphism (RFLP) and direct DNA cloning and sequencing. The increased acid -D-glucosidase activity was detected using 4-MUG as the artificial substrate. The correction efficiency, ranging from 0.5 to 4%, was dependent on the length and polarity of the MSSOV used, the optimal design being a sense-strand 35mer ODNs. Repeated treatment of the mutant fibroblast cells with the ODNs substantially increased correction. We also constructed ODN vectors to trigger specific and in vivo nonsense mutation in the mouse acid -D-glucosidase gene. The ODNs were in complex with YEEE-K18, an asialoglycoprotein-receptor ligand tagged with polylysine and targeted to hepatocytes and renal cells in vivo through intravenous injection. The mutated genotype was detected in the liver and the kidney by ARMS-PCR and glycogen accumulation in the lysosome of the liver cells. The studies demonstrate the utility of single-stranded ODN to direct targeted gene correction or mutation in a human hereditary disease and in an animal model. Our data open the possibility of developing ODN vector as a therapeutic approach for treatment of human hereditary diseases caused by point mutation.

Mentions
Figures
Figure 1: Detection C1935 A mutation in Pompe's disease patients. (a) Nested RT-PCR product (529 bp) was amplified from total RNA of skin fibroblasts of Pompe disease patients by first and second primers for the human acid -D-glucosidase gene (upper panel). The RT-PCR products were digested by AatII. Normal alleles generated the 434 and 95-bp bands. For the C1935 A mutant allele, the RT-PCR product remained unchanged due to the loss of the AatII site (lower panel). M, 1-kb DNA marker (GibcoBRL, Maryland); PC, a normal individual control, P6–P8, patient no. 6–8. (b) Direct sequencing of nested RT-PCR product of the acid -D-glucosidase gene from patient P7 using the Glu-3'-2 primer. The asterisk indicates the site of substitution (C1935 A). Figure 2: ODN vectors of the acid -D-glucosidase gene. A portion of the acid -D-glucosidase gene making the C1935 A mutation site is shown at the top. AatII cutting site is underlined. The ODN sequences are shown below. Upper-case letters indicate normal DNA sequence and lower-case letters indicate phosphorothioate DNA sequence. The mismatched bases are in bold letters. Figure 3: Detection of gene correction in cultured C1935 A mutant skin fibroblasts. (a) ARMS-PCR analysis of mutant skin fibroblasts treated with different ODNs. (b) RFLP analysis of cDNA derived from total RNA isolated from the mutant fibroblast cells treated with ODNs. The arrow designates the 95-bp size marker. PC: fibroblast of normal individual, NC: P7 fibroblasts without transfection; S25, S35, A25 and A35 represent P7 fibroblasts treated with Glu-S25, Glu-S35, Glu-A25 and Glu-A35, respectively. (c) Autoradiograms for colony hybridization are shown, the nested-PCR products were cloned. Colony hybridizations were conducted as described in 'Experimental protocol'. 1: DNA from normal individual, 2: DNA from P7 fibroblasts 3 and 4: DNA from P7 fibroblasts treated with Glu-S35 and Glu-A35, respectively. (d) DNA sequence analysis of the corrected acid -D-glucosidase gene in a positive clone isolated colony hybridization. The asterisk designates the corrected base. Figure 4: Acid -D-glucosidase enzyme activity of C1935 A mutant fibroblasts after ODN treatment. The activity of different ODN-treated fibroblasts and the normal individual (PC). The experiments were triplicated. Numericals indicate the mean values obtained. Figure 5: Restriction analysis of cDNAs generated from total RNA isolated from the skin fibroblasts of a Pompe's disease C1935 A patient after repeated treatment with ODN, see legend of for lane labels. Figure 6: In vivo introduction of a nonsense mutation in the mouse acid -D-glucosidase gene. The translational reading frame is underlined. The stop codon (TGA) created by the G2063 T mutation is shown. The ODN vector, mGlu-S35 and mGlu-A35 used for introducing this mutation are shown below. Bold letters indicate the mismatched bases. Lower-case letters indicate phosphorothioate nucleotides. (b) Structure of YEEE-K18. 'K18' is the 18mer lysine chain. The synthesis of YEEE was described previously.19 Figure 7: Microscopic photos of the tissue biopsies. (a) In vivo delivery of fluorescein-labeled ODN/ YEEE-K18 complex to mice by tail vein injection. Mice were killed 1.5 h postinjection. Frozen sections of the liver (a) and kidney (b) biopsies were inspected by fluorescence microscope. (b) Mice were treated with mGlu-S35/YEEE-K18 (S35), mGlu-A35/ YEEE-K18 (A35), or YEEE-K18 only (NC) three times every second day. At 2 weeks after the last treatment, the mice were killed. The liver biopsies were fixed by formalin and embedded in paraffin. The liver biopsy sections were stained by PAS to detect glycogen accumulation. Pictures shown are views of cell tissues around blood vessels. Figure 8: Detection of in vivo mutation of the acid -D-glucosidase gene in the liver and kidney of treated mice. Mice were treated with mGlu-S35/YEEE-K18 (S35), mGlu-A35/YEEE-K18 (A35), or YEEE-K18 only (NC) by i.v. three times every second day. At 2 weeks after the last treatment, mice were killed. Total RNAs were subjected to ARMS-PCR. The nested RT-PCR products were used as internal controls.
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References
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    • . . . Glycogen storage disease type II (GSD II), also known as Pompe's disease or acid maltase deficiency (AMD),1,2 is genetically transmitted through autosomal recessive inheritance. 3 It is caused by a deficiency of acid -D-glucosidase, a glycogen-degrading lysosomal enzyme . . .
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    • . . . A defect in the enzyme results in lysosomal glycogen accumulation in almost all body tissues, with cardiac and skeletal muscle affected most seriously.4,5 Based on clinical symptoms and the age of onset, such defects could be divided into three clinical types: infantile form, juvenile form and adult form . . .
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    • . . . The infantile form is most severe and the patients die within a year.6,7 There is currently no effective treatment for this fatal disease . . .
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    • . . . The infantile form is most severe and the patients die within a year.6,7 There is currently no effective treatment for this fatal disease . . .
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    • . . . Considerable efforts have been devoted to the development of gene therapy for Pompe's disease using viral vectors with only modest short-term successes due to limitations in transfection-targeting efficiency and immune responses.8 Enzyme replacement therapy (ERT) for GSD II was reported, but ERT suffers from transient effectiveness and high cost.9,10,11 Long-term correction of acid -D-glucosidase deficiency requires a more permanent gene therapy protocol to provide stable expression of acid -D-glucosidase, while circumventing problems associated with vector delivery or ERT. . . .
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    • . . . Considerable efforts have been devoted to the development of gene therapy for Pompe's disease using viral vectors with only modest short-term successes due to limitations in transfection-targeting efficiency and immune responses.8 Enzyme replacement therapy (ERT) for GSD II was reported, but ERT suffers from transient effectiveness and high cost.9,10,11 Long-term correction of acid -D-glucosidase deficiency requires a more permanent gene therapy protocol to provide stable expression of acid -D-glucosidase, while circumventing problems associated with vector delivery or ERT. . . .
  10. Van den Hout JM et al. Enzyme therapy for Pompe disease with recombinant human alphaglucosidase from rabbit milk. J Inherit Metab Dis 2001; 24: 266-274 , .
    • . . . Considerable efforts have been devoted to the development of gene therapy for Pompe's disease using viral vectors with only modest short-term successes due to limitations in transfection-targeting efficiency and immune responses.8 Enzyme replacement therapy (ERT) for GSD II was reported, but ERT suffers from transient effectiveness and high cost.9,10,11 Long-term correction of acid -D-glucosidase deficiency requires a more permanent gene therapy protocol to provide stable expression of acid -D-glucosidase, while circumventing problems associated with vector delivery or ERT. . . .
  11. Amalfitano A et al. Recombinant human acid alpha-glucosidase enzyme therapy for infantile glycogen storage disease type II: results of a phase I/II clinical trial. Genet Med 2001; 3: 132-138 , .
    • . . . Considerable efforts have been devoted to the development of gene therapy for Pompe's disease using viral vectors with only modest short-term successes due to limitations in transfection-targeting efficiency and immune responses.8 Enzyme replacement therapy (ERT) for GSD II was reported, but ERT suffers from transient effectiveness and high cost.9,10,11 Long-term correction of acid -D-glucosidase deficiency requires a more permanent gene therapy protocol to provide stable expression of acid -D-glucosidase, while circumventing problems associated with vector delivery or ERT. . . .
  12. Moerschell RP, Tsunasawa S, Sherman F. Transformation of yeast with synthetic oligonucleotides. Proc Natl Acad Sci USA 1998; 85: 524-528 , .
    • . . . Modified single-stranded oligonucleotide (ODN) vectors have been developed to target and trigger gene correction.12,13,14,15 An ODN vector is a relatively short oligonucleotide containing 25–100 bases homologous to the target sequence except for a single mismatch; an ODN vector also contains a specific number of modified termini linkages . . .
    • . . . ODN vectors direct gene correction via DNA mismatch repair mechanisms.12 Workers have shown that ODNs are effective in gene repair in cell-free extracts, cultured mammalian cells and yeast13,14 with comparable levels of gene repair to chimeraplasty,15,16 an RNA–DNA double-stranded ODN . . .
  13. Yamamoto T et al. Strand-specificity in the transformation of yeast with synthetic oligonucleotides. Genetics 1992; 131: 811-819 , .
    • . . . Modified single-stranded oligonucleotide (ODN) vectors have been developed to target and trigger gene correction.12,13,14,15 An ODN vector is a relatively short oligonucleotide containing 25–100 bases homologous to the target sequence except for a single mismatch; an ODN vector also contains a specific number of modified termini linkages . . .
    • . . . ODN vectors direct gene correction via DNA mismatch repair mechanisms.12 Workers have shown that ODNs are effective in gene repair in cell-free extracts, cultured mammalian cells and yeast13,14 with comparable levels of gene repair to chimeraplasty,15,16 an RNA–DNA double-stranded ODN . . .
    • . . . In contrast, Yamamoto's study13 in transformation of yeast by oligonucleotides showed that the sense strand affected the target sequence 50–100-fold more effectively than antisense oligonucleotides . . .
  14. Igoucheva O, Alexeev V, Yoon K. Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Therapy 2001; 8: 391-399 , .
    • . . . Modified single-stranded oligonucleotide (ODN) vectors have been developed to target and trigger gene correction.12,13,14,15 An ODN vector is a relatively short oligonucleotide containing 25–100 bases homologous to the target sequence except for a single mismatch; an ODN vector also contains a specific number of modified termini linkages . . .
    • . . . Yoon and colleagues have previously addressed strand bias in gene repair using MSSOV and found that antisense (ie ODN complements to the nontranscribed strand of DNA) is 1000-fold more efficient than sense constructs.14 Liu et al15 also showed that antisense exhibited a five- to six-fold increase in correction efficiency . . .
  15. Liu L, Rice MC, Kmiec EB. In vivo gene repair of point and frameshift mutations directed by chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides. Nucleic Acids Res 2001; 29: 4238-4250 , .
    • . . . Modified single-stranded oligonucleotide (ODN) vectors have been developed to target and trigger gene correction.12,13,14,15 An ODN vector is a relatively short oligonucleotide containing 25–100 bases homologous to the target sequence except for a single mismatch; an ODN vector also contains a specific number of modified termini linkages . . .
    • . . . ODN vectors direct gene correction via DNA mismatch repair mechanisms.12 Workers have shown that ODNs are effective in gene repair in cell-free extracts, cultured mammalian cells and yeast13,14 with comparable levels of gene repair to chimeraplasty,15,16 an RNA–DNA double-stranded ODN . . .
    • . . . Yoon and colleagues have previously addressed strand bias in gene repair using MSSOV and found that antisense (ie ODN complements to the nontranscribed strand of DNA) is 1000-fold more efficient than sense constructs.14 Liu et al15 also showed that antisense exhibited a five- to six-fold increase in correction efficiency . . .
  16. Parekh-Olmedo H, Czymmek K, Kmiec EB. Targeted gene repair in mammalian cells using chimeric RNA/DNA oligonucleotides and modified single-stranded vectors. Sci STKE 2001; 2001: PL1 , .
    • . . . ODN vectors direct gene correction via DNA mismatch repair mechanisms.12 Workers have shown that ODNs are effective in gene repair in cell-free extracts, cultured mammalian cells and yeast13,14 with comparable levels of gene repair to chimeraplasty,15,16 an RNA–DNA double-stranded ODN . . .
  17. Lin CY, Hwang B, Hsiao KJ, Jin YR. Study of -D-glucosidase activity in patients with Pompe's disease. J Formosan Med Assoc 1986; 85: 766-770 , .
    • . . . A cytidine to adenosine (C1935 A) transversion causing a substitution of glutamic acid for aspartic acid at position 645 results in a complete loss of acid -D-glucosidase activity.17 This mutation is found in the major infantile form of GSDII prevalent in southern China and Taiwan.18 Here, we investigated the use of modified single-stranded ODN vectors to correct this mutation in skin fibroblast cells derived from a patient . . .
  18. Lin CY, Shieh JJ. Molecular study on the infantile form of Pompe disease in Chinese in Taiwan. Acta Paed Sin 1996; 37: 115-121 , .
    • . . . A cytidine to adenosine (C1935 A) transversion causing a substitution of glutamic acid for aspartic acid at position 645 results in a complete loss of acid -D-glucosidase activity.17 This mutation is found in the major infantile form of GSDII prevalent in southern China and Taiwan.18 Here, we investigated the use of modified single-stranded ODN vectors to correct this mutation in skin fibroblast cells derived from a patient . . .
    • . . . The acid -D-glucosidase gene contains an AatII restriction cleavage site at C1935,18 and the C1935 A point mutation abolishes the site . . .
    • . . . The detection of the C1935 A mutation is consistent with the clinical manifestation and the almost complete loss of acid -D-glucosidase activity18 in this patient . . .
  19. Jiaang WT, Tseng PH, Chen ST. Facile solid phase synthesis of YEE(ah-GalNAc)3, a ligand with known high affinity for the asialoglycoprotein receptor. Synletter 2000; 6: 797-800 , .
  20. Hangeland JJ, Levis TT, Lee YC, Ts'o POP. Cell-type specific and ligand specific enhancement of cellular uptake of oligonucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3. Bioconjugate Chem 1995; 6: 695-701 , .
    • . . . YEEE-K18 is a synthetic compound19 containing a ligand of the asialoglycoprotein receptor20 and a polysine chain for interacting with ODN . . .
  21. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning, A Laboratory Use 2nd edn. 1989, pp. I.90-I.104 , .
    • . . . Colony hybridization was carried out using the standard protocol.21 The membranes were hybridized at 48°C for 24 h with a [32P] end-labeled allele-specific oligonucleotide probe, Glu-p, 5' CCCGGCTGCAGACGC 3', in which the underlined 'G' was the corrected nucleotide . . .
  22. David A et al. Alpha-1,4-glucosidase activity in Pompe's disease. FASEB J 2002; 16: 754-756 , .
    • . . . Recently, Goukassian et al found that small single-stranded DNA fragments activate p53, enhance DNA repair and compensate for age-associated decline in DNA repair capacity in primary cultured fibroblast.22 It may be the cause for the enhancement . . .
  23. Geffen I, Spiess M. Asialoglycoprotein receptor. Int Rev Cytol 1992; 137B: 181-219 , .
    • . . . Asialoglycoprotein receptor of the liver is known to bind and endocytose old serum asialoglycoprotein23 and has been utilized for liver-targeting delivery.24 In the kidney, the occurrence of such an event is more controversial . . .
  24. Plank C et al. Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand. Bioconjugate Chem 1992; 3: 533-539 , .
    • . . . Asialoglycoprotein receptor of the liver is known to bind and endocytose old serum asialoglycoprotein23 and has been utilized for liver-targeting delivery.24 In the kidney, the occurrence of such an event is more controversial . . .
  25. Seow YY, Tan MG, Woo KT. Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells. Nephron 2002; 91: 431-438 , .
    • . . . However, Seow et al25 have recently demonstrated the existence of asialoglycoprotein receptor in human renal tubular epithelial cells . . .
  26. Hu H, Serra D, Amalfitano A. Persistence of an [E1-,polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice. Hum Gene Ther 1999; 10: 355-364 , .
    • . . . Chen and colleagues26,27 have developed a new strategy called hepatic gene therapy for treatment of GSD II using adenovial vectors for transduction of hepatocytes . . .
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    • . . . Chen and colleagues26,27 have developed a new strategy called hepatic gene therapy for treatment of GSD II using adenovial vectors for transduction of hepatocytes . . .
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