Familial amyloidotic polyneuropathy (FAP) is the common form of hereditary generalized amyloidosis and is characterized by the accumulation of amyloid fibrils in the peripheral nerves and other organs. Liver transplantation has been utilized as a therapy for FAP, because the variant transthyretin (TTR) is predominantly synthesized by the liver, but this therapy is associated with several problems. Thus, we need to develop a new treatment that prevents the production of the variant TTR in the liver. In this study, we used HepG2 cells to show in vitro conversion of the TTR gene by single-stranded oligonucleotides (SSOs), embedded in atelocollagen, designed to promote endogenous repair of genomic DNA. For the in vivo portion of the study, we used liver from transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. The level of gene conversion was determined by real-time RCR combined with mutant-allele-specific amplification. Our results indicated that the level of gene conversion was approximately 11 and 9% of the total TTR gene in HepG2 cells and liver from transgenic mice, respectively. Gene therapy via this method may therefore be a promising alternative to liver transplantation for treatment of FAP.
Familial amyloidotic polyneuropathy (FAP) is the term for a heterogeneous collection of familial diseases marked by the accumulation of amyloid fibrils in the peripheral nerves and other organs.1,2 The amyloid fibrils in the most common type of FAP consist predominantly of variant transthyretin (TTR) with single or double amino-acid substitutions; more than 80 TTR mutations have been identified in association with FAP.3 Clinically, FAP produces various symptoms: sensory–motor polyneuropathy, autonomic dysfunction, cardiomyopathy, vitreous opacity, carpal tunnel syndrome, renal dysfunction and gastrointestinal tract disorders. Liver transplantation has been used since 1990 as a therapy for FAP,4 because amyloidogenic TTR (ATTR), the pathogenic protein of FAP, is predominantly synthesized by the liver. By the end of 2000, more than 500 patients with FAP had undergone the surgery, with about 80% of the patients surviving.5 Liver transplantation is now considered beneficial for saving the lives of patients with FAP.6,7,8,9,10 However, there are several problems with this therapy: (1) It is extremely expensive. (2) Patients with transplants must continue lifelong administration of immunosuppressants after the surgery. (3) Carriers of the ATTR gene who do not have any clinical symptoms cannot undergo liver transplantation. Thus, we should devise a new treatment that prevents the production of variant TTR in the liver.
Chimeric RNA/DNA oligonucleotides (chimeraplasts) have been developed to facilitate correction of single-base mutations of episomal and chromosomal targets in mammalian cells.11,12 Chimeraplasts consist of short regions of correcting DNA bounded by long stretches of 2′-O-methyl RNA, hairpin loops, and GC clamps. Chimeraplasts were shown to cause site-specific chromosomal correction or mutation in tissue culture cells and in vivo.13,14,15,16,17 As permanent and stable gene correction by chimeraplasts was demonstrated by clonal analysis at the level of the genomic sequence, and as shown by protein and phenotypic changes,16 chimeraplasts-mediated gene repair may be a powerful tool for the treatment of genetic diseases.
The strategy of using single-stranded oligonucleotides (SSOs) for gene repair is another therapeutic option. SSOs were developed in studies using cell-free assays to characterize and improve chimeraplasty.18,19 SSOs containing three phosphorothioate bonds at the 3′- and 5′-termini were more effective than the chimeraplasts in gene repair assays conducted with cell-free extracts19 and a yeast system.20 As SSOs are significantly less expensive than chimeraplasts and are far simpler to synthesize and purify, they may prove to be an invaluable resource for treating a variety of genetic diseases.
To test the feasibility of gene therapy in FAP to prevent the production of variant TTR in the liver by using chimeraplasts and SSOs with several nonviral vectors, we first applied chimeraplasts and SSOs to HepG2 cells secreting human wild-type TTR. We then demonstrated gene repair induced by SSOs in transgenic murine liver,21 in which the intrinsic wild-type TTR was replaced by the murine TTR Val30Met gene.
Design of chimeraplasts and SSOs
We designed the chimeraplasts and SSOs to introduce human ATTR Val30Met and murine wild-type TTR into HepG2 cell and transgenic mice expressing murine TTR Val30Met, respectively (Figure 1). The DNA sequence of the human22 and murine23 served as the basis for the design.
Evaluation of transfection efficiency in HepG2 cells
Nonviral vectors (Fugene 6, ExGen 500, and atelocollagen) were tested for the delivery of oligonucleotides. Fluorescent-labeled oligonucleotides along with fluorescence confocal microscopy were used to measure the cellular uptake and localization of the oligonucleotide in individual cells. Approximately 50% of the cells were fluorescent in the nucleus when the cells were transfected with atelocollagen (Figure 2a). In contrast, approximately 20% of the cells transfected with other nonviral vectors exhibited weak nuclear fluorescence (Figure 2b).
Properties of the oligonucleotide/atelocollagen complex
We examined the properties of several types of oligonucleotide/atelocollagen complex under a fluorescence microscope. As the length of the oligonucleotide increased, the oligonucleotide/atelocollagen complex became smaller (Figure 3a–c). The 74-mer SSOs complex was observed as small particles with a mean diameter of 18.73 μm under the dispersed condition (Figure 3d). However, particles were not observed when the SSOs were used alone (data not shown).
TTR gene conversion assay by real-time RCR combined with mutant-allele-specific amplification (MASA)
Real-time RCR combined with MASA24 was employed to allow discrimination between the normal human TTR gene and the ATTR Val30Met gene or normal murine TTR gene and the TTR Val30Met gene. When PCR was performed with the upstream primer whose 3′-end corresponded to the first nucleotide of the human ATTR Val30Met codon (ATG) or whose 3′-end corresponded to the first nucleotide of normal murine TTR codon (GTG), the human ATTR Val30Met allele and normal murine TTR allele were amplified effectively, whereas the normal human TTR allele and murine TTR Val30Met allele were barely amplified (Figure 4a and c). The fit-point method was used to set the threshold manually between background and significant for data analysis, and the level of gene conversion in the samples was determined by using a standard curve generated from plotting external standards consisting of the 100% human ATTR Val30Met gene, 10% ATTR Val30Met gene, and the 1% ATTR Val30Met gene, or 100% normal murine TTR gene, 10% normal TTR gene, and 1% normal TTR gene against the threshold cycle. To establish the accuracy of the level of TTR gene conversion estimates by the method, a mixing experiment was performed in duplicate with the FAP ATTR Val30Met homozygote and normal subject possessing the wild-type TTR gene in various proportions from 50:50, 25:75, 12.5:87.5, 6.25:93.75, and 3.125:96.875. The R2 statistic for the regression line relating ATTR Val30Met frequencies by this method versus actual ATTR Val30Met frequencies was 0.9956 (Figure 4b). Then, a mixing experiment was performed in duplicate with the normal mouse and transgenic mouse possessing murine TTR Val30Met gene in various proportions from 50:50, 25:75, 12.5:87.5, 6.25:93.75, and 3.125:96.875. The R2 statistic for the regression line relating normal murine TTR frequencies by the method versus actual normal murine TTR frequencies was 0.9975 (Figure 4d).
To examine the possibility whether SSOs act as a template, real-time RCR combined with MASA was performed using SSOs instead of genomic DNA. Higher background fluorescence level and shallower amplification curve were observed in SSOs than in genomic DNA (Figure 5a). Melting curve analysis revealed the different melting peak pattern and melting temperature (Tm) in SSOs in comparison with that in external positive standards (Figure 5b).
TTR gene conversion with chimeraplasts and SSOs in cultured HepG2 cells
Gene conversion induced by chimeraplasts and SSOs was examined in HepG2 cells. To compare gene conversion induced by chimeraplasts with that induced by SSOs, type A or type B of the oligonucleotide/atelocollagen complex (300 μl) (Table 1) was added to the medium. At 5 days after the addition of type B, the level of gene conversion was 0.4% (Figure 6a). No such conversion was observed after type A (chimeraplasts) administration (Figure 6a). As shown in Figure 6b, melting curve peaks and Tm from amplification of the sample after type B administration were consistent with those of external positive standards, suggesting that amplification of the sample after type B administration was specific (Figure 6b).
Determination of optimal conditions for SSOs-induced TTR gene conversion in cultured HepG2 cells
To determine optimal conditions for TTR gene conversion, we tested several different combinations of oligonucleotide/atelocollagen complexes, types C–H (Table 1). The level of gene conversion was evaluated via real-time RCR combined with MASA.24 At 5 days after the administration of complex types C and D, the level of gene conversion was 0 and 0.1%, respectively (Figure 7a). The levels of gene conversion after the administration of types E and F were 0 and 2.7% and after the administration of types G and H were 0.7 and 11.1%, respectively (Figure 7a). No such conversion was observed after SSOs administration without atelocollagen (data not shown). The specificity of PCR amplification in each sample was confirmed by melting curve analysis (Figure 7b).
TTR gene repair in transgenic mice possessing the murine TTR Val30Met gene
The injection of oligonucleotide/atelocollagen complex type I or type J (Table 1) into the peritoneal cavity of transgenic mice produced no gene conversion (data not shown). However, direct injection of 400 μl of the oligonucleotide/atelocollagen type I or type J into the liver of transgenic mice resulted in levels of gene repair of 8.7 and 0.3%, respectively (Figure 8a). The specificity of PCR amplification in each sample was confirmed by melting curve analysis (Figure 8b). The average level of gene repair after type I injection was 7.9±0.9% (n=5). To detect the de novo production of murine wild-type TTR in the serum of the transgenic mice, mass-spectrometric pattern of TTR purified from the serum was compared before and after type I injection by MALDI/TOF-mass spectrometry (MS). Although only one main peak with the molecular sizes of 13 672 kDa that corresponded to the reduced forms of murine TTR Val30Met was observed before type I injection, the peak with the molecular size of 13 640 kDa that corresponded to the reduced forms of murine wild-type TTR was detected in addition to that with the molecular size of 13 672 kDa after type I injection (Figure 9). The ratio of wild-type TTR to total TTR after type I injection was calculated to be 7.7% in comparison with the peak intensities of wild-type TTR and TTR Val30Met.
It has been well documented that liver transplantation is effective for halting the progression of FAP.6,7,8,9,10 However, this procedure is associated with several problems. This situation has stimulated us to attempt suppression of the production of variant TTR in the liver by use of gene therapy. Targeted gene repair by oligonucleotides11,12,13,14,15,16,17,18,19,20 and suppression of TTR expression by antisense25,26 and ribozymes27,28,29 have been studied. However, suppression of TTR expression in mRNA by antisense and ribozyme therapy may not be an essential therapy because TTR is a rapid turnover protein and FAP patients may need to undergo treatment every day or every hour.30 Kren et al reported, however, that genomic and phenotypic changes that were induced by targeted gene repair were stable during an 18-month period,15,17 which suggests that the effect of targeted gene repair could be permanent and that this method might be effective for genomic TTR therapy.
The strategy called targeted gene repair was developed to facilitate the process of gene therapy by using chimeraplasts11,12,13,14,15,16,17 and SSOs.18,19,20 Many reports have demonstrated the feasibility of using chimeraplasts and SSOs to induce point conversion in the targeted genes in vitro and in vivo. However, most of these reports are unsatisfactory, because of poor reproducibility and an inadequate rate of gene conversion. To overcome these problems, we focused on two main objectives. One was to develop an optimized oligonucleotide delivery system. The other was to contrive a more improved structure of the oligonucleotides for gene repair.
Vehicles for gene delivery are divided into viral and nonviral delivery systems.31,32 Although many viral vectors have the potential for effective transfer of a gene to the targeted tissues, they may also cause problems related to immunogenicity of the viral components as well as random integration of the transgene. Even though the efficiency of gene delivery with a nonviral delivery system is less than that with a viral delivery system, the nonviral system has potential advantages of being less immunogenic and allowing for greater ease of production, purity, and standardization of molecules. Recently, atelocollagen, a biocompatible polymer, has attracted attention as a new gene transfer tool that allows prolonged release and expression of plasmid DNA in vivo.33,34 As collagen has been widely used in medicine and cosmetics,35 and atelocollagen has had antigenic telopeptides attached to both ends of the collagen molecules removed by pepsin treatment, atelocollagen is considered to be one of the safest nonviral vectors. Among all the nonviral vectors tested here, atelocollagen showed the highest transfection efficiency and accumulation of fluorescently labeled oligonucleotides in the nucleus of HepG2 cells (Figure 2). These findings suggested that atelocollagen was the most suitable nonviral vector for our gene therapy.
Gene conversion was confirmed by the real-time RCR combined with MASA. To date, fluorescently labeled probes have been used in the majority of studies on real-time PCR quantification. However, we used the SYBR green dye because the dye can be used as an alternative for the detection of amplified PCR products,36,37 and there was a possibility that fluorescently labeled probes could bind the SSOs. As shown in Figure 4, the R2 statistic for the regression line relating ATTR Val30Met frequencies by this method versus actual ATTR Val30Met frequencies or normal murine TTR frequencies by this method versus actual normal murine TTR frequencies was 0.9956 and 0.9975, suggesting that this method actually reflected the level of gene conversion. It has been reported that all different types of PCR products could be distinguished by their specific patterns of melting curve.36,37 We used the QIAamp DNA Mini Kit (QIAGEN, Tokyo, Japan) for DNA extraction to exclude the influence of the SSOs on the PCR amplification, because the QIAamp membrane did not bind up to 100 bp DNA in length. Even if an infinitesimal amount of SSOs was mixed, PCR amplification of SSOs demonstrated high background fluorescence and shallow amplification curve (Figure 5a). In addition, different melting peak pattern and Tm were observed (Figure 5b), indicating that SSOs did not work as a template. Moreover, it did not take time to perform this analyses compared with the conventional methods such as PCR-RFLP12,14,16 and colony lift hybridization assay.11,13,15,17 Thus, the real-time RCR combined with MASA was considered to be a rapid and reliable method for analyzing the level of gene conversion.
We first evaluated whether chimeraplasts or SSOs were more suitable for gene conversion in HepG2 cells, because chimeraplasts and SSOs have quite different structures and different gene conversion mechanisms.19,38 As atelocollagen has the characteristics of prolonged release and expression of plasmid DNA unlike other nonviral delivery systems,33,34 we cultured the HepG2 cells after the administration of chimeraplasts or SSOs for 5 days to raise the level of gene conversion. Our results indicated that SSOs were far superior to chimeraplasts for gene conversion with the atelocollagen delivery system in HepG2 cells (Figure 6). Although the reason why chimeraplasts did not generate significant gene conversion was unknown, the possibility exists that difficulty in synthesis and purity of functional chimeraplasts, cell types, and targeted genomic loci might influence the genetic correction.39
We then determined the optimal conditions for TTR gene conversion in HepG2 cells with oligonucleotide/atelocollagen complex. The level of gene conversion was different depending on the length of SSOs and about 11% after the administration of oligonucleotide/atelocollagen complex type H (Figure 7). During the 5-day culture with oligonucleotide/atelocollagen complex, cell death was hardly seen. As the length of the oligonucleotide increased, SSOs may be folded and become compact in size in the presence of atelocollagen (Figure 3). Tm in each SSOs was 75°C at 25-mer, 90°C at 45-mer, and 96°C at 74-mer, suggesting that 74-mer SSOs may have the highest stability of hybrid of SSOs and target gene. These findings suggested that 74-mer SSOs with atelocollagen was considered to be the most suitable for inducing the highest gene conversion.
On the basis of the report of Tagalakis et al,40 we tried to the convert TTR gene in HepG2 cells with ExGen 500 and 600 nM chimeraplasts or SSOs, but almost no gene conversion was observed (data not shown). As shown in Figure 7, a much greater amount of SSOs than that described by Tagalakis et al40 was needed for gene conversion in our study, and the level of gene conversion increased as the concentration of SSOs with atelocollagen increased. This finding suggested that transport of large amounts of SSOs into the nucleus was indispensable for significant gene conversion.
Transgenic mice possessing the intrinsic murine TTR Val30Met genes21 were thought to be excellent for the analysis of in vivo gene repair, compared with transgenic mice having both the intrinsic murine TTR gene and many copies of the human ATTR Val30Met gene.41 First, the copy number of the murine TTR Val30Met gene in the liver is 1, as is that of the human TTR gene in the human liver, which suggests that this gene conversion result may mimic gene therapy with FAP patients. Second, analysis of gene conversion is easier with these mice because they have no intrinsic TTR gene showing high homology with the human TTR gene. After the direct administration of the oligonucleotide/atelocollagen complex type I into the liver, gene conversion was estimated as 8.7% (Figure 8) and de novo murine wild-type TTR in the serum of transgenic mice was estimated as 7.7% (Figure 9). Several times intrahepatic administration of oligonucleotide/atelocollagen complex via hepatic artery or portal vein may be promising as the gene therapy for FAP.
In summary, gene therapy with SSOs and atelocollagen is thought to be a promising method for gene repair, even though the level of gene conversion was not sufficient for suppression of the production of variant TTR in clinical terms. This method and strategy may be useful for autosomal recessive disorders. Further studies should be performed to determine the optimal delivery system consisting of SSOs with atelocollagen or times of administration.
Materials and methods
Chimeraplasts and oligonucleotides
Chimeraplasts were obtained from MWG-Biotech (Ebersberg, Germany) and SSOs were obtained from Nihon Gene Research Laboratories (Sendai, Japan).
Preparation of the oligonucleotide/atelocollagen formulation
Atelocollagen, a highly purified type I collagen of calf dermis obtained by pepsin treatment,42 was provided by Koken Co., Ltd (Tokyo, Japan). All types of the oligonucleotide/atelocollagen formulation were prepared as follows. Atelocollagen and chimeraplasts or SSOs were diluted with phosphate-buffered saline (PBS), with the concentration adjusted to twice that of the intended formulations. Equal amounts of PBS solutions containing atelocollagen and chimeraplasts or SSOs were mixed vigorously and left overnight to form the oligonucleotide/atelocollagen complexes.43 The characteristics of the oligonucleotide/atelocollagen complexes used in this study are shown in Table 1. All complexes were kept at 4°C until use.
Evaluation of transfection efficiency in HepG2 cells
To evaluate transfection efficiency, 68-mer oligonucleotides were fluorescently labeled and were transfected into HepG2 cells with Fugene 6, ExGen 500, or atelocollagen. After 24 h of transfection, the distribution of oligonucleotides in the cells was determined by using a confocal microscope (IX-FLA, OLYMPUS Co., Ltd, Tokyo, Japan).
Fluorescence microscopic observation of the size of the oligonucleotide/atelocollagen complexes
The properties of the oligonucleotide/atelocollagen complex types D, F, and H (Table 1) were observed via a fluorescence microscope (BX50, OLYMPUS Co., Ltd, Tokyo, Japan). After 8 μl of the complex formulation and 2 μl of five-times-diluted YOYO (single-strand staining reagent; Molecular Probes Inc., Eugene, OR, USA) were mixed, pictures were obtained via a fluorescence microscope equipped with a digital camera system (HC-2500, Fuji Photo Film Co., Ltd, Tokyo, Japan). To measure the size of type H accurately, type H was observed under 10 times dilution with PBS.
Gene conversion assay
Genomic DNA was extracted from HepG2 cells and samples of transgenic murine liver using a QIAamp DNA Mini Kit (QIAGEN, Tokyo, Japan). The level of gene conversion was analyzed by use of real-time RCR combined with MASA.24 Serial samples of genomic DNA from an FAP homozygous for the ATTR Val30Met gene were diluted 10-fold with that from a normal subject possessing the wild-type TTR gene. These samples, 100:0, 90:10, and 1:99 of ATTR Val30Met and wild-type TTR genes, served as external standards for the analyses of gene conversion in the HepG2 cells. Serial samples of genomic DNA from a normal mouse were diluted 10-fold with that from a transgenic mouse expressing murine TTR Val30Met. These samples, 100:0, 90:10, and 1:99 of wild-type murine TTR and murine TTR Val30Met genes, served as external standards for the analyses of gene repair in the transgenic mice. To establish the accuracy of the level of TTR gene conversion estimates by the method, a mixing experiment was performed in duplicate with the FAP ATTR Val30Met homozygote and normal subject possessing the wild-type TTR gene or the normal mouse and transgenic mouse possessing murine TTR Val30Met gene in various proportions from 50:50, 25:75, 12.5:87.5, 6.25:93.75, and 3.125:96.875.
For evaluation of TTR gene conversion in HepG2 cells, the upstream and downstream primer sequences were 5′-IndexTermGCCATCAATGTGGCCA-3′, in which the 3′-end corresponded to the first nucleotide of the mutation codon (ATG), and 5′-IndexTermAGGGGCAAACGGGAAGA TAA-3′, respectively.22 For evaluation of TTR gene conversion in transgenic mice, the upstream and downstream primer sequences were 5′-IndexTermGCTGTAGACGTGGCTG-3′, in which the 3′-end corresponded to the first nucleotide of the normal codon (GTG), and 5′-IndexTermCACGAATAAGAGCAAATGGG-3′, respectively.23 PCR was performed with the QuantiTect SYBR Green PCR kit (QIAGEN, Tokyo, Japan) according to the manufacturer’s instructions. The 20 μl final volume contained 1 × QuantiTect SYBR Green PCR Master Mix, each primer at 0.5 μM, and 30 ng of template DNA. The real-time quantitative PCR program with the LightCycler instrument (Roche Molecular Biochemicals, Tokyo, Japan) (LC-PCR) consisted of an initial 15-min denaturation at 95°C and 40 cycles of 15 s at 94°C, 20 s at 60°C, and 10 s at 72°C. The crossing point values of these standards were used to generate an external standard curve, which provided accurate quantification of samples. To examine the specificity of the PCR amplification, detailed melting curve analysis of the PCR products was performed in the same closed capillary using SYBR green dye after amplification.
Cell culture and transfection
HepG2 cells were maintained in DMEM (Invitrogen Corp.) supplemented 10% FBS (Invitrogen Corp.) and 2 mM L-glutamine (Invitrogen Corp.). For transfection experiments, 2 × 105 HepG2 cells were seeded per well in a 12-well plate 24 h before the transfection. To determine whether chimeraplasts or SSOs were more suitable for gene conversion, 300 μl of oligonucleotide/atelocollagen complex type A or B (Table 1) was added to the culture medium after 300 μl of this medium had been removed. To ascertain the optimal conditions for gene conversion by SSOs, 600 μl of each oligonucleotide/atelocollagen complex types C to H (Table 1) was added to the culture medium after 400 μl of this medium had been removed. To evaluate the efficiency of gene conversion, cells were harvested for DNA isolation 5 days after the transfection.
For the study of gene therapy with the liver, we used 10- to 12-week-old transgenic C57BL/5 mice (five mice) that possessed the murine TTR Val30Met gene.21
In vivo delivery systems
The oligonucleotide/atelocollagen complex type I or J (400 μl) (Table 1) was directly injected into the liver of transgenic mice whose intrinsic wild-type TTR gene was replaced by the murine TTR Val30Met gene. For the gene conversion study, the mice were exsanguinated 2 weeks after the injection of the oligonucleotide/atelocollagen complex, and the liver samples were removed for DNA isolation.
Analysis of TTR forms by MALDI/TOF-MS in the serum of transgenic mouse
Test mice sera (50 μl) were mixed with 10 μl of 2.7 mM dithiothreitol and 20 μl of anti-TTR antibody (Santa Cruz Biotechnology, Inc., CA, USA): The generated precipitate was centrifuged at 9000 g for 5 min and washed with 100 μl saline and 100 μl water twice, respectively, at 4°C. The precipitate was dissolved in 50 μl of 4% acetic acid and 4% acetonitrile in water.
All experiments were performed using a Bruker Reflex mass spectrometer (Bruker Franzen Analytik GmbH, Bremen, Germany) operated at a wavelength of 337 nm. The best spectra of TTR were obtained at an ion accelerating voltage of 27.5 kV and a reflectron voltage of 30 kV. The spectra were calculated using external calibration with (M+H)-ions produced from horse cytochrome C (m/z 12360.08) and horse myoglobin (m/z 16951.46). The matrix was a saturated solution of sinapinic acid in 1:2 acetonitrile:water containing 0.1% TFA. The samples were deposited onto the sample probe assembly.44
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The corresponding author's work was supported by grants from the Amyloidosis Research Committee, and the Pathogenesis and Therapy of Hereditary Neuropathy Research Committee, Surveys and Research on Specific Diseases, the Ministry of Health, Labor and Welfare of Japan, Charitable Trust Clinical Pathology Research Foundation of Japan, and Grants-in-Aid for Scientific Research (C) 13670655 and (B) 15390275 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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Nakamura, M., Ando, Y., Nagahara, S. et al. Targeted conversion of the transthyretin gene in vitro and in vivo. Gene Ther 11, 838–846 (2004) doi:10.1038/sj.gt.3302228
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