- Email: [email protected]
DNA oligonucleotide-based gene therapy
Kidney International, Vol. 61, Symposium 1 (2002), pp. S47–S51
Chimeric RNA/DNA oligonucleotide-based gene therapy LI-WEN LAI and YEONG-HAU H. LIEN Department of Medicine, Sections of Endocrinology and Nephrology, University of Arizona Health Sciences Center, Tucson, Arizona, USA
Chimeric RNA/DNA oligonucleotide-based gene therapy. Background. Chimeric RNA/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models. Methods. Six recent studies of chimeric RNA/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site with the exception of a mismatch in the center, were delivered via different routes and delivery vehicles to target different tissues and organs. Corrections of the mutation at genotypic and phenotypic levels were assessed using various methods, including allele-specific polymerase chain reaction assay, restriction enzyme digestion, colony-lifting assays, sequencing, Northern and Western blot analyses, enzyme activity assay, immunohistochemical staining, and functional studies. Results. The gene correction frequency varied, ranging from less than 1% to more than 40%. This represented several magnitudes higher conversion rate compared with homologous recombination frequency, which is in the range of 10⫺5 to 10⫺6. The resulting phenotype changes lasted longer than one year in some studies. Conclusion. Chimeric RNA/DNA oligonucleotide-based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or tissues. Further efforts to elucidate the mechanisms of chimeric RNA/DNA oligonucleotide-based gene therapy are warranted in order to increase the efficacy and safety of this method.
Most of the current strategies for gene therapy are based on the delivery of therapeutic genes using viral or plasmid vectors in vivo and ex vivo. With a few exceptions, such as severe combined immune deficient syndrome [1], the results of most expression vector-based gene therapy trials for genetic diseases have been disappointing. Severe complications, including one death [2], have led the public to doubt the values of gene therapy and investigators in this field to re-examine their strategies. The immune reactions induced by adenoviral vecKey words: genetic disease, homologous pairing, mismatch repair, carbonic anhydrase II, Fabry disease, tyrosinase, Crigler-Najjar syndrome type I, UDP-glucuronosyltransferase, Duchenne muscular dystrophy, dystrophin.
2002 by the International Society of Nephrology
tors not only prevent long-term expression of therapeutic genes, but also result in serious complications. Retroviral vectors may cause random integration and disrupt the normal genes. Nonviral vectors such as liposomes and polycations are not able to achieve stable long-term gene expression [3–6]. In addition, a full-length cDNA is required to construct the therapeutic genes, which is a problem especially for a gene with large transcripts. Furthermore, the transfected genes may not have the appropriate transcriptional regulatory machinery, which is critical for cell specificity and proper function of the transgenes. Recently, a novel strategy to correct a point mutation using a chimeric RNA/DNA oligonucleotide has been reported [7–9]. This strategy evolved from the finding that RNA/DNA hybrids were more active in homologous pairing reactions in vitro than corresponding DNA [10–12]. Based on this finding, a chimeric RNA/DNA oligonucleotide, containing one DNA strand aligning to 25 residues of genomic DNA flanking the site of mutation with the exception of a single mismatched nucleotide located at the center and one complementary RNA/ DNA hybrid strand with two blocks of 10 2⬘-O-methylated RNA residues flanking both sides of a five-residue stretch of DNA, was created (Fig. 1). The folded doublehairpin structure, containing four T residues in each loop, a 5 bp guanine-cytosine (GC) clamp, and the modified RNA residues, was designed to improve resistance to nuclease degradation. The alignment of the chimeric molecule with the genomic site appears to trigger an endogenous repair system that facilitates homologous conversion. The mechanism of gene correction seems to involve both homologous pairing and mismatch repair activities [13]. Cole-Strauss et al recently reported that chimeric RNA/DNA oligonucleotide-mediated gene repair in mammalian cell-free extracts from the HuH-7 hepatoma cell line depended on the presence of mismatch repair enzyme MSH2 protein [14]. Different levels of endogenous mismatch repair activities among different cell types may explain some of the variation in the efficiency of gene conversion using different cells and tissues. Gamper et al have shown that the DNA strand but not the chimeric strand of the chimeric RNA/DNA oligonucleotide served as a template for high-fidelity
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Lai and Lien: Site-specific gene correction
Fig. 1. Sequence of the normal and CA II (⫺) alleles and chimeric RNA/DNA oligonucleotide. The site of mutation is underlined. The chimeric oligonucleotide contains 25 homologous DNA residues identical to normal allele and only one bp different from CA II (⫺) allele. The antisense strand contains five core DNA residues that is flanked by 10 2⬘-Omethyl RNA residues at each side. A doublehairpin configuration is also included. DNA residues are capitalized, and 2⬘-O-methyl RNA residues are in lowercase.
Table 1. Disease models tested using chimeric RNA/DNA oligonucleotide based gene therapy Disease Fabry disease CA II deficiency Albinism Crigler-Najjar syndrome type I Duchenne muscular dystrophy
Gene ␣-Galatosidase A CA II Tyrosinase UGT1A1 Dystrophin
Tissue/cell type Lymphoblast Renal tubule Melanocyte Liver Muscle
Vehicle
References
Effectene PEI Cytofectin L-PEI and liposome None
[19] [20] [22] [23] [24, 25]
Abbreviations are: PEI, polyethylenimine; UGT1A1, UDP-glucuronosyltransferase; CA II, Carbonic Anhydrase II; L-PEI, Lactosylated PEI.
gene correction when one of its bases was mismatched to the target gene [15]. The role of the chimeric strand appeared to be facilitating the complex formation with the target, thus increasing the frequency of gene conversion. The feasibility of this oligonucleotide-based gene therapy was tested in an in vitro system of sickle cell anemia [16] and human hepatoma cell line HuH-7 [17]. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the S mutation, conversion of the mutant allele to the normal sequence was confirmed by direct sequencing of the polymerase chain reaction (PCR)-amplified fragment [16]. In the hepatoma cell line system, a single base pair mutation in the alkaline phosphatase gene was introduced following polyethylenimine (PEI) transfection with the chimeric molecule [17]. The overall frequency of conversion was up to 11.9%, and when corrected for transfection efficiency, it approached 43%. Subsequently, Kren et al reported the first in vivo study using lactosylated 25 kD PEI as a delivery system specific to the liver and were able to mutate factor IX gene to prolong the activated partial thromboplastin time by 25% and achieve up to 40% conversion in rat liver cells when the rats were injected twice [18]. Since then, several groups have reported a successful chimeric RNA/DNA oligonucleotidebased gene therapy in various genetic disease models. These technologies and respective results are summarized in Table 1 and are reviewed as follows. CORRECTION OF A POINT MUTATION IN ␣-GALACTOSIDASE A GENE IN FABRY LYMPHOBLASTS Fabry disease is an X-linked recessive disease, caused by a defective lysosomal enzyme, ␣-galactosidase A,
characterized by skin lesions and involvement of heart, central nerve system, blood vessels, and kidneys. We have identified a family of patients with Fabry disease. Reverse transcription-polymerase chain reaction (RTPCR) sequencing of the peripheral lymphocyte RNA from all female carriers and a male patient reveals a C to T mutation, resulting in an amino acid substitution of Pro-40 by Ser. The serum ␣-galactosidase A activity from the male patient was undetectable, and the lymphocyte ␣-galactosidase A activity was less than 10% of the normal value. We transfected the chimeric RNA/DNA oligonucleotides with the correct sequence into cultured peripheral lymphocytes and EB virus-transformed lymphoblasts from the male patient [19]. The conversion of mutant T to C was confirmed by allele-specific PCR assay on the genomic DNA. The treatment increased the ␣galactosidase enzymatic activity from baseline 9.2 ⫾ 0.7% and 8.5 ⫾ 1.6% (of the normal value) to 15.0 ⫾ 2.1% and 20.9 ⫾ 4.9% in cultured peripheral lymphocytes and EB virus-transformed lymphoblasts, respectively. Potentially, this gene therapy strategy can be used to treat Fabry disease in this family. IN VIVO CORRECTION OF THE CARBONIC ANHYDRASE II MUTATION IN CARBONIC ANHYDRASE II-DEFICIENT MICE The carbonic anhydrase (CA) II-deficient mouse model was used to test the feasibility of gene therapy using the chimeric RNA/DNA oligonucleotides. The mutation is caused by a point mutation in amino acid codon 154, converting Gln (CAA) to a stop codon (TAA). Previously, we have shown that liposome-mediated gene therapy is sufficient to correct renal tubular
Lai and Lien: Site-specific gene correction
acidosis; however, the gene expression was transient [6]. The chimeric RNA/DNA oligonucleotide (Oligo-CA) was complexed with PEI and injected intravenously in adult CA II-deficient mice. Replacement of the mutant sequence by the exogenous normal sequence at the appropriate genomic locus, and the resulting expression of the wild-type gene product was confirmed by RT-PCR sequencing, allele-specific PCR assay, Northern blot analysis, and Western blot analysis [20]. The mutation conversion lasted for at least six months, and the conversion rate was improved by repeated intravenous injection (unpublished data). In addition, we investigated whether unwanted mutation to the nontargeting sequence would occur after the chimeric RNA/DNA oligonucleotide gene targeting [21]. After BLAST genome database search, we identified a mouse gene, regucalcin, which is highly homologous to the target sequence. We synthesized a 97 bp putative mutation sequence of regucalcin gene containing the homologous region and developed an allele-specific PCR assay to detect the putative mutation sequence. After gene targeting with Oligo-CA in cultured CA II-deficient renal tubular cells, the genomic DNA was isolated and subjected to allele-specific PCR for both CA II and regucalcin. The gene conversion rate for CA II was 1 to 5%, whereas no point mutation in the regucalcin gene was detected. Our data suggest that gene targeting by chimeric RNA/DNA oligonucleotides is feasible to correct the mutation in CA II gene and is highly sequence specific. CORRECTION OF THE TYROSINASE GENE MUTATION IN ALBINO BALB/c MOUSE SKIN Alexeev et al reported that the detection of pigmented hairs in approximately 50% of animals received either topical application or intradermal injection of the RNADNA oligonucleotide designed to correct a point mutation in tyrosinase gene [22]. Active tyrosinase was detected in skin sections three months after the last treatment with chimeric oligonucleotide, which corresponds with more than one hair cycle. The gene correction may also occur in precursor melanocytes, as suggested by the prolonged expression of tyrosinase activity found in some hairs. The frequency of gene correction at the phenotypic level, as demonstrated by the number of dark pigmented hairs and DOPA-positive hair follicles, appeared to be much lower than the frequency of chromosomal DNA sequence correction observed from the same skin biopsy. A likely explanation is that phenotypic change is exclusively caused by melanocytes, which compose less than 1% of total cells in skin, whereas genotypic change is measured in all cells in skin biopsy. It is also possible that gene correction of multiple melanocytes per hair follicle may be required for hair pigmentation.
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If the number of the corrected melanocytes is under the threshold, the phenotypic change may not be observed. CORRECTION OF A SINGLE BASE PAIR DELETION IN THE UDPGLUCURONOSYLTRANSFERASE GENE IN THE GUNN RAT MODEL OF CRIGLER-NAJJAR SYNDROME TYPE I For the first time, Kren et al have successfully shown that chimeric RNA/DNA oligonucleotide-based gene therapy can correct a single base pair deletion in vivo [23]. A single guanosine (G) base deletion in UDP-glucuronosyltransferase gene (UGT1A1) resulted in a frameshift and a premature stop codon, lack of enzyme activity, and hyperbilirubinemia in Gunn rat. A slightly longer chimeric oligonucleotide (28 bp sequence homology to the target gene except the inserted G base) was either complexed with PEI or encapsulated in anionic liposomes, administered intravenously, and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, DNA sequencing, and genomic Southern blot analysis. DNA repair was stable throughout the six-month observation period and was associated with a reduction of serum bilirubin levels. The data indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, resulting in improvement in the metabolic abnormality. The time course for hepatic disappearance of the fluorescein label in rats injected with the liposome or PEI complexed chimeric RNA/DNA oligonucleotides was also characterized. The fluorescence level was stable until 24-hours postinjection and decreased dramatically throughout the liver by 48 hours. By 120 to 168 hours, there was only background fluorescence. Disappearance of the fluorescein label in the other tissues was similar to that observed in the liver. CORRECTION OF DYSTROPHIN GENE MUTATION IN CANINE MODEL OF DUCHENNE MUSCULAR DYSTROPHY A point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA and results in frame shift that causes early termination of translation. Bartlett et al have demonstrated in vivo repair of the dystrophin gene mutation by direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog [24]. Analysis of biopsy and necropsy samples suggested that the repair was sustained for 48 weeks. RT-PCR analysis of exons 5 to 10 demonstrated increasing levels of exon 7 inclusion with time. Synthesis
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Lai and Lien: Site-specific gene correction
of normal-sized dystrophin product and positive localization to the sarcolemma was confirmed using an exon 7-specific dystrophin antibody. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism–PCR and sequencing of the PCR product. This work supports evidence for the long-term repair of a specific dystrophin point mutation in dog muscle using a chimeric oligonucleotide.
tide-based gene therapy, a therapeutic gene will be customer made to target the mutation site specifically. Recently, the mutated genes and their sequences have been identified in a variety of genetic renal diseases. The chimeric RNA/DNA oligonucleotide-based gene therapy may be a promising approach for treating these diseases. ACKNOWLEDGMENTS
RESCUE OF DYSTROPHIN EXPRESSION IN mdx MOUSE MUSCLE In a similar study using mdx mouse model for Duchenne muscular dystrophy disease, Rando et al [25] reported the correction of the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 (the correcting chimeric oligonucleotide with 30 bp sequence homology to the target gene except the center mismatch) into muscles of mdx mice, immunohistochemical analysis showed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1 to 2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after two weeks, suggesting that the expression was stable. Immunohistochemical staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoprecipitation and immunoblot analysis. RT-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1. CONCLUSION These studies provide the base for further studies of chimeric oligonucleotide-mediated gene therapy as a therapeutic modality to genetic diseases caused by a point mutation. The major obstacle—the low correction rate in some disease models—can be improved by (1) optimizing the delivery systems, (2) modifying the chimeric oligonucleotide structure, for example, increasing the length of the homologous oligonucleotides and/or modifying nucleotide analogues to increase the efficiency of homologous pairing and mismatch repair systems, and (3) increasing the mismatch repair efficiency by stimulating the repair enhancing elements or suppressing the inhibitors for the repair system. Chimeric RNA/DNA oligonucleotide-based gene therapy would require mutation analysis for the individual patient with a known genetic disease. Once a mutation is identified and determined as suitable for chimeric RNA/DNA oligonucleo-
This work was supported by NIH Grant RO1DK56660, the Arizona Disease Control Research Commission Grant #9812, and a grant from the Dialysis Clinic, Inc., a nonprofit organization. Reprint requests to Yeong-Hau H. Lien, M.D., Ph.D., Section of Renal Disease Department of Medicine University of Arizona Health Sciences Center Tucson, Arizona 85724, USA. E-mail: [email protected]
REFERENCES 1. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al: Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669–672, 2000 2. Brenner M: Reports of adenovector “death” are greatly exaggerated. Mol Ther 1:205, 2000 3. Lien YH, Lai L: Gene therapy for renal diseases. Kidney Int 52(Suppl 61):S85–S88, 1997 4. Lien YH, Lai L: Liposome-mediated gene transfer into the tubules. Exp Nephrol 5:132–136, 1997 5. Lai L, Moeckel GW, Lien YH: Kidney-targeted liposome-mediated gene transfer in mice. Gene Ther 4:426–431, 1997 6. Lai L, Chan D, Erickson RP, et al: Correction of renal tubular acidosis in carbonic anhydrase II deficient mice with gene therapy. J Clin Invest 101:1320–1325, 1998 7. Kmiec EB: Targeted gene repair. Gene Ther 6:1–3, 1999 8. Lai L, Lien YH: Homologous recombination-based gene therapy: Mini-review. Exp Nephrol 7:11–14, 1999 9. Yoon K: Single-base conversion of mammalian genes by an RNADNA oligonucleotide. Biogenic Amines 15:137–167, 1999 10. Kotani H, Kmiec EB: A role for RNA synthesis in homologous pairing events. Mol Cell Biol 14:6097–6106, 1994 11. Kotani H, Kmiec EB: Transcription activates RecA-promoted homologous pairing of nucleosomal DNA. Mol Cell Biol 14:1949– 1955, 1994 12. Kotani H, Germann MW, Andrus A, et al: RNA facilitates RecAmediated DNA pairing and strand transfer between molecules bearing limited regions of homology. Mol Gen Genet 250:626–634, 1996 13. Yoon K, Cole-Strauss A, Kmiec EB: Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc Natl Acad Sci USA 93:2071– 2076, 1996 14. Cole-Strauss A, Gamper H, Holloman WK, et al: Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract. Nucleic Acids Res 27:1323–1330, 1999 15. Gamper HB, Jr, Cole-Strauss A, Metz R, et al: A plausible mechanism for gene correction by chimeric oligonucleotides. Biochem 39:5808–5816, 2000 16. Cole-Strauss A, Yoon K, Xiang Y, et al: Correction of the mutation responsible for sickle cell anemia by an RNA-DNA Oligonucleotide. Science 273:1386–1389, 1996 17. Kren BT, Cole-Strauss A, Kmiec EB, Steer CJ: Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25:1462–1468, 1997 18. Kren BT, Bandyopadhyay P, Steer CJ: In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides. Nat Med 4:285–290, 1998
Lai and Lien: Site-specific gene correction 19. Lai L, Luo M, Lien YH: Correction of a point mutation in ␣galactosidase gene by chimeric RNA/DNA oligonucleotides in cultured peripheral lymphocytes and lymphoblasts from a patient with Fabry disease. J Am Soc Nephrol 11:409A, 2000 20. Lai L, Chau B, Kahn R, Lien YH: Gene targeting in the kidney of carbonic anhydrase II deficient mice by chimeric RNA/DNA oligonucleotides. J Am Soc Nephrol 10:447A, 1999 21. Luo M, Lien YH, Lai L: Mutation analysis for non-target gene after gene targeting by chimeric RNA/DNA oligonucleotides. Mol Ther 1:S228, 2000 22. Alexeev V, Igoucheva O, Domashenko A, et al: Localized in vivo
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genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotech 18:43–47, 2000 23. Kren BT, Parashar B, Bandyopadhyay P, et al: Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide. Proc Natl Acad Sci USA 96:10349–10354, 1999 24. Bartlett RJ, Stockinger S, Denis MM, et al: In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotech 18:615–622, 2000 25. Rando TA, Disatnik MH, Zhou LZ: Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 97:5363–5368, 2000
Chimeric RNA/DNA oligonucleotide-based gene therapy LI-WEN LAI and YEONG-HAU H. LIEN Department of Medicine, Sections of Endocrinology and Nephrology, University of Arizona Health Sciences Center, Tucson, Arizona, USA
Chimeric RNA/DNA oligonucleotide-based gene therapy. Background. Chimeric RNA/DNA oligonucleotides, emerging as a potential strategy for gene therapy, have been shown to induce site-specific correction of point mutations in several genetic disease models. Methods. Six recent studies of chimeric RNA/DNA oligonucleotide-based gene therapy in genetic disease models are reviewed. Chimeric RNA/DNA oligonucleotides, complementary to 25 to 30 residues of genomic DNA flanking the mutation site with the exception of a mismatch in the center, were delivered via different routes and delivery vehicles to target different tissues and organs. Corrections of the mutation at genotypic and phenotypic levels were assessed using various methods, including allele-specific polymerase chain reaction assay, restriction enzyme digestion, colony-lifting assays, sequencing, Northern and Western blot analyses, enzyme activity assay, immunohistochemical staining, and functional studies. Results. The gene correction frequency varied, ranging from less than 1% to more than 40%. This represented several magnitudes higher conversion rate compared with homologous recombination frequency, which is in the range of 10⫺5 to 10⫺6. The resulting phenotype changes lasted longer than one year in some studies. Conclusion. Chimeric RNA/DNA oligonucleotide-based gene therapy has the potential to develop into powerful therapeutic modality for genetic diseases. It can offer permanent expression and normal regulation of corrected genes in appropriate cells or tissues. Further efforts to elucidate the mechanisms of chimeric RNA/DNA oligonucleotide-based gene therapy are warranted in order to increase the efficacy and safety of this method.
Most of the current strategies for gene therapy are based on the delivery of therapeutic genes using viral or plasmid vectors in vivo and ex vivo. With a few exceptions, such as severe combined immune deficient syndrome [1], the results of most expression vector-based gene therapy trials for genetic diseases have been disappointing. Severe complications, including one death [2], have led the public to doubt the values of gene therapy and investigators in this field to re-examine their strategies. The immune reactions induced by adenoviral vecKey words: genetic disease, homologous pairing, mismatch repair, carbonic anhydrase II, Fabry disease, tyrosinase, Crigler-Najjar syndrome type I, UDP-glucuronosyltransferase, Duchenne muscular dystrophy, dystrophin.
2002 by the International Society of Nephrology
tors not only prevent long-term expression of therapeutic genes, but also result in serious complications. Retroviral vectors may cause random integration and disrupt the normal genes. Nonviral vectors such as liposomes and polycations are not able to achieve stable long-term gene expression [3–6]. In addition, a full-length cDNA is required to construct the therapeutic genes, which is a problem especially for a gene with large transcripts. Furthermore, the transfected genes may not have the appropriate transcriptional regulatory machinery, which is critical for cell specificity and proper function of the transgenes. Recently, a novel strategy to correct a point mutation using a chimeric RNA/DNA oligonucleotide has been reported [7–9]. This strategy evolved from the finding that RNA/DNA hybrids were more active in homologous pairing reactions in vitro than corresponding DNA [10–12]. Based on this finding, a chimeric RNA/DNA oligonucleotide, containing one DNA strand aligning to 25 residues of genomic DNA flanking the site of mutation with the exception of a single mismatched nucleotide located at the center and one complementary RNA/ DNA hybrid strand with two blocks of 10 2⬘-O-methylated RNA residues flanking both sides of a five-residue stretch of DNA, was created (Fig. 1). The folded doublehairpin structure, containing four T residues in each loop, a 5 bp guanine-cytosine (GC) clamp, and the modified RNA residues, was designed to improve resistance to nuclease degradation. The alignment of the chimeric molecule with the genomic site appears to trigger an endogenous repair system that facilitates homologous conversion. The mechanism of gene correction seems to involve both homologous pairing and mismatch repair activities [13]. Cole-Strauss et al recently reported that chimeric RNA/DNA oligonucleotide-mediated gene repair in mammalian cell-free extracts from the HuH-7 hepatoma cell line depended on the presence of mismatch repair enzyme MSH2 protein [14]. Different levels of endogenous mismatch repair activities among different cell types may explain some of the variation in the efficiency of gene conversion using different cells and tissues. Gamper et al have shown that the DNA strand but not the chimeric strand of the chimeric RNA/DNA oligonucleotide served as a template for high-fidelity
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Lai and Lien: Site-specific gene correction
Fig. 1. Sequence of the normal and CA II (⫺) alleles and chimeric RNA/DNA oligonucleotide. The site of mutation is underlined. The chimeric oligonucleotide contains 25 homologous DNA residues identical to normal allele and only one bp different from CA II (⫺) allele. The antisense strand contains five core DNA residues that is flanked by 10 2⬘-Omethyl RNA residues at each side. A doublehairpin configuration is also included. DNA residues are capitalized, and 2⬘-O-methyl RNA residues are in lowercase.
Table 1. Disease models tested using chimeric RNA/DNA oligonucleotide based gene therapy Disease Fabry disease CA II deficiency Albinism Crigler-Najjar syndrome type I Duchenne muscular dystrophy
Gene ␣-Galatosidase A CA II Tyrosinase UGT1A1 Dystrophin
Tissue/cell type Lymphoblast Renal tubule Melanocyte Liver Muscle
Vehicle
References
Effectene PEI Cytofectin L-PEI and liposome None
[19] [20] [22] [23] [24, 25]
Abbreviations are: PEI, polyethylenimine; UGT1A1, UDP-glucuronosyltransferase; CA II, Carbonic Anhydrase II; L-PEI, Lactosylated PEI.
gene correction when one of its bases was mismatched to the target gene [15]. The role of the chimeric strand appeared to be facilitating the complex formation with the target, thus increasing the frequency of gene conversion. The feasibility of this oligonucleotide-based gene therapy was tested in an in vitro system of sickle cell anemia [16] and human hepatoma cell line HuH-7 [17]. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the S mutation, conversion of the mutant allele to the normal sequence was confirmed by direct sequencing of the polymerase chain reaction (PCR)-amplified fragment [16]. In the hepatoma cell line system, a single base pair mutation in the alkaline phosphatase gene was introduced following polyethylenimine (PEI) transfection with the chimeric molecule [17]. The overall frequency of conversion was up to 11.9%, and when corrected for transfection efficiency, it approached 43%. Subsequently, Kren et al reported the first in vivo study using lactosylated 25 kD PEI as a delivery system specific to the liver and were able to mutate factor IX gene to prolong the activated partial thromboplastin time by 25% and achieve up to 40% conversion in rat liver cells when the rats were injected twice [18]. Since then, several groups have reported a successful chimeric RNA/DNA oligonucleotidebased gene therapy in various genetic disease models. These technologies and respective results are summarized in Table 1 and are reviewed as follows. CORRECTION OF A POINT MUTATION IN ␣-GALACTOSIDASE A GENE IN FABRY LYMPHOBLASTS Fabry disease is an X-linked recessive disease, caused by a defective lysosomal enzyme, ␣-galactosidase A,
characterized by skin lesions and involvement of heart, central nerve system, blood vessels, and kidneys. We have identified a family of patients with Fabry disease. Reverse transcription-polymerase chain reaction (RTPCR) sequencing of the peripheral lymphocyte RNA from all female carriers and a male patient reveals a C to T mutation, resulting in an amino acid substitution of Pro-40 by Ser. The serum ␣-galactosidase A activity from the male patient was undetectable, and the lymphocyte ␣-galactosidase A activity was less than 10% of the normal value. We transfected the chimeric RNA/DNA oligonucleotides with the correct sequence into cultured peripheral lymphocytes and EB virus-transformed lymphoblasts from the male patient [19]. The conversion of mutant T to C was confirmed by allele-specific PCR assay on the genomic DNA. The treatment increased the ␣galactosidase enzymatic activity from baseline 9.2 ⫾ 0.7% and 8.5 ⫾ 1.6% (of the normal value) to 15.0 ⫾ 2.1% and 20.9 ⫾ 4.9% in cultured peripheral lymphocytes and EB virus-transformed lymphoblasts, respectively. Potentially, this gene therapy strategy can be used to treat Fabry disease in this family. IN VIVO CORRECTION OF THE CARBONIC ANHYDRASE II MUTATION IN CARBONIC ANHYDRASE II-DEFICIENT MICE The carbonic anhydrase (CA) II-deficient mouse model was used to test the feasibility of gene therapy using the chimeric RNA/DNA oligonucleotides. The mutation is caused by a point mutation in amino acid codon 154, converting Gln (CAA) to a stop codon (TAA). Previously, we have shown that liposome-mediated gene therapy is sufficient to correct renal tubular
Lai and Lien: Site-specific gene correction
acidosis; however, the gene expression was transient [6]. The chimeric RNA/DNA oligonucleotide (Oligo-CA) was complexed with PEI and injected intravenously in adult CA II-deficient mice. Replacement of the mutant sequence by the exogenous normal sequence at the appropriate genomic locus, and the resulting expression of the wild-type gene product was confirmed by RT-PCR sequencing, allele-specific PCR assay, Northern blot analysis, and Western blot analysis [20]. The mutation conversion lasted for at least six months, and the conversion rate was improved by repeated intravenous injection (unpublished data). In addition, we investigated whether unwanted mutation to the nontargeting sequence would occur after the chimeric RNA/DNA oligonucleotide gene targeting [21]. After BLAST genome database search, we identified a mouse gene, regucalcin, which is highly homologous to the target sequence. We synthesized a 97 bp putative mutation sequence of regucalcin gene containing the homologous region and developed an allele-specific PCR assay to detect the putative mutation sequence. After gene targeting with Oligo-CA in cultured CA II-deficient renal tubular cells, the genomic DNA was isolated and subjected to allele-specific PCR for both CA II and regucalcin. The gene conversion rate for CA II was 1 to 5%, whereas no point mutation in the regucalcin gene was detected. Our data suggest that gene targeting by chimeric RNA/DNA oligonucleotides is feasible to correct the mutation in CA II gene and is highly sequence specific. CORRECTION OF THE TYROSINASE GENE MUTATION IN ALBINO BALB/c MOUSE SKIN Alexeev et al reported that the detection of pigmented hairs in approximately 50% of animals received either topical application or intradermal injection of the RNADNA oligonucleotide designed to correct a point mutation in tyrosinase gene [22]. Active tyrosinase was detected in skin sections three months after the last treatment with chimeric oligonucleotide, which corresponds with more than one hair cycle. The gene correction may also occur in precursor melanocytes, as suggested by the prolonged expression of tyrosinase activity found in some hairs. The frequency of gene correction at the phenotypic level, as demonstrated by the number of dark pigmented hairs and DOPA-positive hair follicles, appeared to be much lower than the frequency of chromosomal DNA sequence correction observed from the same skin biopsy. A likely explanation is that phenotypic change is exclusively caused by melanocytes, which compose less than 1% of total cells in skin, whereas genotypic change is measured in all cells in skin biopsy. It is also possible that gene correction of multiple melanocytes per hair follicle may be required for hair pigmentation.
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If the number of the corrected melanocytes is under the threshold, the phenotypic change may not be observed. CORRECTION OF A SINGLE BASE PAIR DELETION IN THE UDPGLUCURONOSYLTRANSFERASE GENE IN THE GUNN RAT MODEL OF CRIGLER-NAJJAR SYNDROME TYPE I For the first time, Kren et al have successfully shown that chimeric RNA/DNA oligonucleotide-based gene therapy can correct a single base pair deletion in vivo [23]. A single guanosine (G) base deletion in UDP-glucuronosyltransferase gene (UGT1A1) resulted in a frameshift and a premature stop codon, lack of enzyme activity, and hyperbilirubinemia in Gunn rat. A slightly longer chimeric oligonucleotide (28 bp sequence homology to the target gene except the inserted G base) was either complexed with PEI or encapsulated in anionic liposomes, administered intravenously, and targeted to the hepatocyte via the asialoglycoprotein receptor. G insertion was determined by PCR amplification, colony lift hybridizations, restriction endonuclease digestion, DNA sequencing, and genomic Southern blot analysis. DNA repair was stable throughout the six-month observation period and was associated with a reduction of serum bilirubin levels. The data indicate that correction of the UGT1A1 genetic lesion in the Gunn rat restores enzyme expression and bilirubin conjugating activity, resulting in improvement in the metabolic abnormality. The time course for hepatic disappearance of the fluorescein label in rats injected with the liposome or PEI complexed chimeric RNA/DNA oligonucleotides was also characterized. The fluorescence level was stable until 24-hours postinjection and decreased dramatically throughout the liver by 48 hours. By 120 to 168 hours, there was only background fluorescence. Disappearance of the fluorescein label in the other tissues was similar to that observed in the liver. CORRECTION OF DYSTROPHIN GENE MUTATION IN CANINE MODEL OF DUCHENNE MUSCULAR DYSTROPHY A point mutation within the splice acceptor site of intron 6 leads to deletion of exon 7 from the dystrophin mRNA and results in frame shift that causes early termination of translation. Bartlett et al have demonstrated in vivo repair of the dystrophin gene mutation by direct skeletal muscle injection of the chimeric oligonucleotide into the cranial tibialis compartment of a six-week-old affected male dog [24]. Analysis of biopsy and necropsy samples suggested that the repair was sustained for 48 weeks. RT-PCR analysis of exons 5 to 10 demonstrated increasing levels of exon 7 inclusion with time. Synthesis
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of normal-sized dystrophin product and positive localization to the sarcolemma was confirmed using an exon 7-specific dystrophin antibody. Chromosomal repair in muscle tissue was confirmed by restriction fragment length polymorphism–PCR and sequencing of the PCR product. This work supports evidence for the long-term repair of a specific dystrophin point mutation in dog muscle using a chimeric oligonucleotide.
tide-based gene therapy, a therapeutic gene will be customer made to target the mutation site specifically. Recently, the mutated genes and their sequences have been identified in a variety of genetic renal diseases. The chimeric RNA/DNA oligonucleotide-based gene therapy may be a promising approach for treating these diseases. ACKNOWLEDGMENTS
RESCUE OF DYSTROPHIN EXPRESSION IN mdx MOUSE MUSCLE In a similar study using mdx mouse model for Duchenne muscular dystrophy disease, Rando et al [25] reported the correction of the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 (the correcting chimeric oligonucleotide with 30 bp sequence homology to the target gene except the center mismatch) into muscles of mdx mice, immunohistochemical analysis showed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1 to 2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after two weeks, suggesting that the expression was stable. Immunohistochemical staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoprecipitation and immunoblot analysis. RT-PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1. CONCLUSION These studies provide the base for further studies of chimeric oligonucleotide-mediated gene therapy as a therapeutic modality to genetic diseases caused by a point mutation. The major obstacle—the low correction rate in some disease models—can be improved by (1) optimizing the delivery systems, (2) modifying the chimeric oligonucleotide structure, for example, increasing the length of the homologous oligonucleotides and/or modifying nucleotide analogues to increase the efficiency of homologous pairing and mismatch repair systems, and (3) increasing the mismatch repair efficiency by stimulating the repair enhancing elements or suppressing the inhibitors for the repair system. Chimeric RNA/DNA oligonucleotide-based gene therapy would require mutation analysis for the individual patient with a known genetic disease. Once a mutation is identified and determined as suitable for chimeric RNA/DNA oligonucleo-
This work was supported by NIH Grant RO1DK56660, the Arizona Disease Control Research Commission Grant #9812, and a grant from the Dialysis Clinic, Inc., a nonprofit organization. Reprint requests to Yeong-Hau H. Lien, M.D., Ph.D., Section of Renal Disease Department of Medicine University of Arizona Health Sciences Center Tucson, Arizona 85724, USA. E-mail: [email protected]
REFERENCES 1. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, et al: Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288:669–672, 2000 2. Brenner M: Reports of adenovector “death” are greatly exaggerated. Mol Ther 1:205, 2000 3. Lien YH, Lai L: Gene therapy for renal diseases. Kidney Int 52(Suppl 61):S85–S88, 1997 4. Lien YH, Lai L: Liposome-mediated gene transfer into the tubules. Exp Nephrol 5:132–136, 1997 5. Lai L, Moeckel GW, Lien YH: Kidney-targeted liposome-mediated gene transfer in mice. Gene Ther 4:426–431, 1997 6. Lai L, Chan D, Erickson RP, et al: Correction of renal tubular acidosis in carbonic anhydrase II deficient mice with gene therapy. J Clin Invest 101:1320–1325, 1998 7. Kmiec EB: Targeted gene repair. Gene Ther 6:1–3, 1999 8. Lai L, Lien YH: Homologous recombination-based gene therapy: Mini-review. Exp Nephrol 7:11–14, 1999 9. Yoon K: Single-base conversion of mammalian genes by an RNADNA oligonucleotide. Biogenic Amines 15:137–167, 1999 10. Kotani H, Kmiec EB: A role for RNA synthesis in homologous pairing events. Mol Cell Biol 14:6097–6106, 1994 11. Kotani H, Kmiec EB: Transcription activates RecA-promoted homologous pairing of nucleosomal DNA. Mol Cell Biol 14:1949– 1955, 1994 12. Kotani H, Germann MW, Andrus A, et al: RNA facilitates RecAmediated DNA pairing and strand transfer between molecules bearing limited regions of homology. Mol Gen Genet 250:626–634, 1996 13. Yoon K, Cole-Strauss A, Kmiec EB: Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide. Proc Natl Acad Sci USA 93:2071– 2076, 1996 14. Cole-Strauss A, Gamper H, Holloman WK, et al: Targeted gene repair directed by the chimeric RNA/DNA oligonucleotide in a mammalian cell-free extract. Nucleic Acids Res 27:1323–1330, 1999 15. Gamper HB, Jr, Cole-Strauss A, Metz R, et al: A plausible mechanism for gene correction by chimeric oligonucleotides. Biochem 39:5808–5816, 2000 16. Cole-Strauss A, Yoon K, Xiang Y, et al: Correction of the mutation responsible for sickle cell anemia by an RNA-DNA Oligonucleotide. Science 273:1386–1389, 1996 17. Kren BT, Cole-Strauss A, Kmiec EB, Steer CJ: Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25:1462–1468, 1997 18. Kren BT, Bandyopadhyay P, Steer CJ: In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides. Nat Med 4:285–290, 1998
Lai and Lien: Site-specific gene correction 19. Lai L, Luo M, Lien YH: Correction of a point mutation in ␣galactosidase gene by chimeric RNA/DNA oligonucleotides in cultured peripheral lymphocytes and lymphoblasts from a patient with Fabry disease. J Am Soc Nephrol 11:409A, 2000 20. Lai L, Chau B, Kahn R, Lien YH: Gene targeting in the kidney of carbonic anhydrase II deficient mice by chimeric RNA/DNA oligonucleotides. J Am Soc Nephrol 10:447A, 1999 21. Luo M, Lien YH, Lai L: Mutation analysis for non-target gene after gene targeting by chimeric RNA/DNA oligonucleotides. Mol Ther 1:S228, 2000 22. Alexeev V, Igoucheva O, Domashenko A, et al: Localized in vivo
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genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotech 18:43–47, 2000 23. Kren BT, Parashar B, Bandyopadhyay P, et al: Correction of the UDP-glucuronosyltransferase gene defect in the Gunn rat model of Crigler-Najjar syndrome type I with a chimeric oligonucleotide. Proc Natl Acad Sci USA 96:10349–10354, 1999 24. Bartlett RJ, Stockinger S, Denis MM, et al: In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nat Biotech 18:615–622, 2000 25. Rando TA, Disatnik MH, Zhou LZ: Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 97:5363–5368, 2000