Amelioration of β654-thalassemia in mouse model with the knockdown of aberrantly spliced β-globin mRNA

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GENETICS AND GENOMICS J. Genet. Genomics 35 (2008) 595601

www.jgenetgenomics.org

Amelioration of E654-thalassemia in mouse model with the knockdown of aberrantly spliced E-globin mRNA Shuyang Xie 1, Wei Li 1, Zhaorui Ren, Jingzhi Zhang, Xinbin Guo, Shu Wang, Shuzhen Huang, Fanyi Zeng *, Yi-Tao Zeng * Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China. Received for publication 4 March 2008; revised 31 August 2008; accepted 2 September 2008

Abstract Large amounts of aberrantly spliced mRNA from the E654 allele was present in erythroid cells, which might impair the erythropoiesis. A therapeutic strategy for E-thalassemia was explored by knocking down the aberrantly spliced mRNA of E-globin. Lentiviral vector with siRNA fragment targets on the specific portion of E654-globin aberrantly spliced pre-mRNA was constructed. In HeLa E654 cells, the siRNA vector could reduce approximately 60% of aberrantly spliced mRNA, which was assessed by RT-PCR and qRT-PCR. Furthermore, a disease model of E654 thalassemia mice with lentiviral-mediated siRNA was produced by subzonal injection (named HEi-Hbbth-4/Hbb+ transgenic mice). Our results showed that the hemotological parameters were improved in HEi-Hbbth-4/Hbb+ transgenic mice. This study provides a potential way for E654-thalassemia therapy by knocking down the aberrantly spliced E-globin mRNA, whilst supporting that the aberrantly spliced E-globin mRNA may aggravate the disease. Keywords: E-thalassemia; small interfering RNA (siRNA); hemoglobin

Introduction E-thalassemia is a common inherited blood disorder resulting from genetic defects in the E-globin gene expression (Kazazian and Boehm, 1988), in which deficient synthesis of effective E-chain of the molecule leads to imbalanced globin chain synthesis. The excess chain precipitates onto the erythrocytic membrane, resulting in intramedullary destruction of the erythroid precursors, ineffective erythropoiesis, oxidative membrane damage, and eventually apoptosis (Ho, 1999; Cao and Moi, 2000). Patients with severe E-thalassemia require regular blood transfusion to sustain their lives. Many patients die before age of 25, directly resulting from cardiac iron deposition if improperly treated. Present address. Shuyang Xie: Binzhou Medical University, Binzhou, Shandong Province, China. * Corresponding author. Tel: +86-21-6279 0545; Fax: +86-21-6247 5476. E-mail address: [email protected] (Y. T. Zeng); [email protected] (F. Zeng) 1 These authors contributed equally to this work.

CĺT substitution at the position IVS-2 nt 654 (the second intron of E-globin gene 654 CĺT, E654) in the Eglobin gene comprises one of the most common E-thalassemia alleles in the Chinese (Zhang et al., 1988; Liu et al., 1989; Huang et al., 1990). This splicing defective mutation generated a CĺT dinucleotide that, within the context of the surrounding sequences, forms an apparent 5c donor-like splice site and simultaneously activates a crytic acceptor 3c splice site at the position of nt 579 of IVS-2 sequence that is inserted between exon 2 and 3 as an “extra exon” (extra exon 73 bp) (Huang et al., 1994). It can also be found that the synthesis of highly unstable E-globin chain can precipitate with the concomitant excess chain, leading to increasing ineffective erythropoiesis in E-thalassemia patients (the 121 G>T mutation) (Ho et al., 1997). An increased level of aberrantly spliced E-globin mRNA was also found in E654-thalassemia (IVS-654, CĺT mutation) (Ho et al., 1998). The above discovery led us to hypothesize that the aberrantly spliced E-globin mRNA was harmful to patients and the anemia symptom would be

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ameliorated with the reduction of aberrantly spliced E654-globin mRNA. In the present study, we design siRNA vector to investigate whether the aberrantly spliced E654-globin mRNA in HeLa E654 cells could be effectively suppressed by siRNA or the correctly spliced E-globin could be increased by siRNA. The anemia phenotype of E654-thalassemia mice was further observed to estimate the therapy efficiency with the decrease of the aberrantly spliced of E654 -globin mRNA by using this approach. Materials and methods Vectors design The H1 promoter element of RNA polymerase III was rescued from the Tc vector described previously (Xie et al., 2005). Afterwards, the H1 promoter was cut off and cloned into pREP4 vector (Invitrogen, USA), generating PH1 vector. We designed other four pairs of siRNA oligonucleotides for suppressing the aberrantly spliced E654-globin mRNA expression on the basis of Berns’ principle (Berns et al., 2004). Each siRNA oligo sequence was homologous to a specific portion of the “extra exon 73 bp” of E654-globin pre-mRNA. pEi1 and pEi4 were targeted to “nt 579 and 654” of “intron 2” , respectively, and pEi 2 and pEi3 were specific for the site of “nt 623” of “intron 2”. pEi1 (19 nt): 5c-ATCTCTTTCTTTCAGGGCA-3c (5 bp homologous to the “extra exon 73 bp” of E654-globin gene and 14 bp homologous to the intron of E654-globin gene), pEi2 (19 nt): 5c-GTATCATGCCTCTTTGCAC-3c (only homologous to the “extra exon 73 bp”), pEi3 (19 nt): 5c-GCACCATTCTA-

AAGAATAA-3c (only homologous to the “extra exon 73 bp”), and pȕi4 (19 nt): 5c-AGGTAATAGCAATATTTCT- 3c (2 bp homologous to the “extra exon 73 bp” of ȕ654-globin gene and 17 bp homologous to the intron of ȕ654-globin gene). The oligos were then annealed and cloned into the Hind III and Nhe I sites of PH1 vector and the siRNA constructs are named pEi1, pEi2, pEi3, and pEi4, respectively. Then, H1 promoter and the siRNA oligo were cut off by EcoR V and Pvu II from pEi2, and cloned into FUGW vector (kindly given by Dr. Zack Wang, Massachusetts General Hospital, Harvard University, USA) by Pac I site (blunted), constructing Len-pEi2 vector. The sequences and the vectors structure were shown in Fig 1. All the siRNA vectors used in this experiment were blasted for the specificity (http://www.ncbi.nlm.nih.gov/BLAST/). Cell culture and transfection HeLa E654 cell lines (in which human aberrantly spliced E654-globin pre-mRNA stably expressed, kindly given by Professor R. Kole, University of North Carolina, USA) were cultivated in DMEM medium (GIBICO, USA) supplemented with 10% fetal calf serum and penicillin/ streptomycin. The cells were cultured at 37oC with 5% CO2. Transfection: 5 × 105 HeLa E654 cells were transfected with 1 Pg of plasmid and 2.5 PL of lipofectamine (Invitrogen, USA), according to the manufacturer’s instructions. One microgramme of pEi vector was used to treat cells. Two days after transfection, the hygromycin (300–500 Pg/mL, Sigma, USA) were added into the medium to select positive cells. The expressions of the aberrantly spliced E654-globin mRNA were determined after three weeks when the cell clones were formed.

Fig. 1. Structure of the vectors used in this study. Pol III: RNA polymerase III promoter. TTTTT: polyT termination signal. pCMV: cytomegalovirus promoter; pH1: H1 promoter; pEi1, pEi2, pEi3 and pEi4: the segment homologous to aberrantly spliced E-globin mRNA. Control: the random segment control of siRNA segment (pDi or pEi).

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Virus production Virus was produced by transient co-transfection of 293T cells using the Len-pEi 2, packaging and enveloping plasmids (VSV-G). The virus-containing supernatant was collected at 60 h after transfection and concentrated by low-speed filtration using 0.2 Pmol/L polyethersulfone filter (Hyclone, USA) followed by ultracentrifugation (20,000 r/min × 90 min). The virus was concentrated 1,000fold from viral supernatants. Because the FUGW vector contains eGFP gene, viral titers were determined by infecting HeLa cells (ATCC) and analyzing enhanced green fluorescence protein (eGFP) expression by fluorescence-activated cell-sorter scanner (FACS). The titers of the lentivirus were above 5 × 108 U/mL. Production of E654 mice integrated with lentivirus-mediated siRNA vector and identification by PCR/FISH The E654-thalassemia mice (B6;129P2-Hbbtm2Unc) were obtained from the Jackson Laboratory (JAX), which is a heterozygote (Hbbth-4/Hbb+) carrying a human gene with EIVS-2-654 CoT splicing mutation, manifesting a typical sign of a moderate form of E-thalassemia (Lewis et al., 1998) . The E654 mouse zygotes were treated with lentivirus-mediated siRNA (pEi2) virus particles according to the method we described previously (Xie et al., 2007). Each zygote was injected with 5–10 U virus (virus solution of 5 × 108 U/mL). The transgenic mice carrying lentivirusmediated siRNA oligos were identified by PCR analysis three weeks after birth. The E654 mouse zygotes from the same mouse were injected with control virus (FUGW without siRNA segment as a control). The PCR primers were designed on the basis of LTR sequence of lentivirus vector and human E654-thalassemia mutation. The integration of vector was further defined with FISH (Matsuda et al., 1992; Xie et al., 2007). RT-PCR Total RNA was isolated from the cultured cells using a RNA isolation kit (Gentra, Australia) according to the

manufacturer’s instructions, 1.5 Pg of total RNA from the cells was mixed with 2 PL of RT buffer, 4 PL of dNTP (10 mmol/L), 0.5 PL of oligo-dT (0.5 mg/mL), and 1 PL of reverse transcriptase. Diethyl pyrocarbonate-treated water (DEPC H2O) was used to make a total volume of 20 PL. cDNA was then synthesized at 37oC for one hour followed by heating at 70oC for 10 min and left on ice for at least 2 min. The aberrantly spliced E654-globin mRNA and GAPDH (internal control) cDNAs were also amplified for 24 cycles in PCR machine: denaturing at 94oC for 30 s; annealing at 60oC for 30 s; and extension at 72oC for 45 s. The primers used are shown in Table 1. The PCR products were run on 2% agarose electrophoretic gel. Real-time quantitative RT-PCR After reverse transcription reaction of the RNA from HeLa E654 cells or peripheral blood of mice, the levels of aberrantly spliced E-globin mRNA and GAPDH mRNA was quantitatively determined by real-time PCR using the RG3000 system (Corbett Research) with the Quantitect SYBRGreen Kit (Qiagen, Hilden, Germany). The reaction condition was: after an initial denaturation at 95oC for 3 min, 30 cycles of 95oC for 30 s, 60oC for 30 s, and 72oC for 30 s. The primers were shown in Table 1. Each sample was measured in triplicate. Hematologic studies Mouse peripheral blood smears were made from 1 to 2 PL of blood collected in heparinized microhematocrit tubes, air dried, and stained with Wright staining. Whole blood samples from mice at least six weeks old were collected in 40 PL microhematocrit tubes containing 2 PL of 0.5 mol/L EDTA (pH 8.0). The red blood cell (RBC) count, hemoglobin (Hb) level, and reticulocyte count for each sample were determined using a Hematology Analyzer (KX-21, Sysmex, Japan) equipped with software to analyze murine cells. Bone marrow smears were also stained with Wright-Giemsa to calculate the proportion of nucleated cells.

Table 1 Oligonucleotide primers for RT-PCR analysis Primers

Sequences

E-5c E-3c GAPDH1 GAPDH2

5c-CTCGGTGCCTTTAGTGATGG-3c 5c-AGCCTGCACTGGTGGGGTGAA-3c 5c-GTCTTCACCACCATGGAGAAGG-3c 5c-GCCTGCTTCACCACCTTCTTGA-3c 5c-AGGGCCTAGCTTGGACTCAG-3c 5c-AGTGATAATTTCTGGGTTCAGGT-3c 5c-TGGAAGGGCTAATTCACTC-3c 5c-TGCTAGAGATTTTCCACACTG-3c

M654-1 M654-2 LTR1 LTR2

597

Notation E-5c and E-3c are used to detect aberrantly spliced E-globin mRNA (254 bp) GAPDH1 and GAPDH2 are used to amplify GAPDH mRNA (reference, 490 bp) M1 and M2 are used to amplify human E654 thalassemia gene mutation (179 bp) LTR1 and LTR2 are used to amplify LTR sequence of lentivirus (500 bp)

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Results Reduction of E654 aberrantly spliced mRNA in HeLa̓ E654 cells by siRNA RT-PCR analysis showed that the aberrantly spliced E654-globin mRNA was obviously reduced for all the siRNA treated groups (Fig. 2A), and siRNA did not restore correctly spliced E-globin mRNA. Real-time RT-PCR analysis further demonstrated that the amount of aberrantly spliced E654-globin mRNA decreased dramatically to 0.26 ± 0.001 (pEi1), 0.14 ± 0.007 (pEi2), 0.33 ± 0.013 (pEi3), and 0.85 ± 0.033 (pEi4) as compared to the control (1.05 ± 0.019, P<0.05) (Fig. 2B). We did not find that the correctly spliced E-globin mRNA was reduced by the siRNA vectors (data not shown here). We found that pEi2 siRNA vector inhibited the expression of aberrantly spliced E654-glboin mRNA more effectively than other vectors. Therefore, we chose pEi 2 vector to suppress the aberrantly spliced E-globin mRNA expression in mice experiments. Production and identification of Hȕi-Hbbth-4/Hbb+ transgenic mice As pEi2 vector had the most significant inhibition of aberrantly spliced E-globin mRNA expression, lentivirial-

mediated siRNA vector was designed to infect the zygotes of E654 mice by microinjection for generating transgenic mice carrying siRNA (pEi2). PCR analysis showed that two E654 mice were integrated with siRNA (named as HEi-Hbbth-4/Hbb+) (Fig. 3). FISH analysis demonstrated that lentivirus-mediated siRNA fragments were integrated in chromosomes of E654 mice (Fig. 4). We also produced the control transgenic mice (HCo-Hbbth-4/ Hbb+), which were integrated with lentivirus without siRNA segment. Reduction of aberrantly spliced E-globin mRNA and amelioration of anemia in HEi-Hbbth-4/Hbb+ mice Peripheral blood smears and the hematological observations were performed in HEi-Hbbth-4/Hbb+ mice at 4 months after birth. The total quantity of poikilocytosis plus target cells in the peripheral blood smear of HEi-Hbbth-4/ Hbb+ mice was (38.4 ± 2.3)%, less than that of E654-control mice (Hbbth-4/Hbb+, approximately 50%) (P<0.05, Figs. 5 and 6). The hemoglobin level of HEi-Hbbth-4/Hbb+ was about (11.8 ± 0.1) g/dL, higher than that of the Hbbth-4/Hbb+ mice (about 10 g/dL). The RBC count of HEi-Hbbth-4/Hbb+ was also improved as compared to that of Hbbth-4/Hbb+-control. We also found that the reticulocyte count of HEi-Hbbth-4/Hbb+ [(126.2 ± 6.5) ‰] was much

Fig. 2. Analysis of the aberrantly spliced E-globin mRNA. A: RT-PCR analysis of the aberrantly spliced E-globin mRNA on 2% agarose gel electrophoresis. GAPDH mRNA is as an internal control. Lane 1, HeLa E654 aberrantly spliced-control; lanes 25, pEi1, pEi2, pEi3 and pEi4 group (siRNA), respectively. M, 100 bp marker. B: the aberrantly spliced E-globin mRNA expression in HeLa E654 cells by real-time RT-PCR analysis. 1, HeLa-control; 2, pEi1; 3, pEi2; 4, pEi3; 5, pEi4. **P<0.01; *P<0.05 vs. HeLa-control. C: the aberrantly spliced E-globin mRNA expression in blood of HEi-Hbbth-4/Hbb+ mice by real-time PCR analysis. 1, Hbb+ /Hbb+; 2, Hbbth-4/Hbb+; 3, HEi-Hbbth-4/Hbb+-1; 4, HEi-Hbbth-4/Hbb+-2. **P<0.01 vs. Hbbth-4/Hbb+.

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Fig. 3. PCR analysis of lentiviral vector integration in the HEi-Hbbth-4/Hbb+ mice. Lane 1, Hbb+/Hbb+ mouse that was both LTR and E654 negative; lane 2, Hbbth-4/Hbb+ mouse that was E654 positive; lane 3, lentivirus-integrated Hbb+/Hbb+ mouse that was LTR positive; lanes 46 were lentivirus-integrated E654 mice (HEi-Hbbth-4/Hbb+) that were detected both LTR and E654 positive. M, DNA marker.

Fig. 4. FISH analysis of lentivirus integrated E654 mice. Left: FISH results; right: G-banding. The arrows show the integration of the chromosomal localization (green arrow) in the transgenic mice (HEi-Hbbth-4/Hbb+).

Fig. 5. The changes of peripheral blood and bone marrow smears of HEi-Hbbth-4/Hbb+ stained with Wright-Giemsa (detected at four months after birth, 400 ×). A: observation of poikilocytosis plus target cells in blood smears under microscopy. B: observation of the proportion of nucleated cells in bone marrow smears under microscopy.

less than that of Hbbth-4/Hbb+-control (about 150 ‰) (Fig. 6). The proportion of nucleated cells in bone marrow was considerably decreased in HEi-Hbbth-4/Hbb+, indicating the improvement of abnormal bone marrow proliferation. Real-time PCR analysis also showed that the expression of aberrantly spliced E-globin mRNA in HEi-Hbbth-4/Hbb+ was obviously decreased (Fig. 2C). While the control transgenic mouse (HCo-Hbbth-4/Hbb+) still showed marked

anisocytosis, poikilocytosis plus target cells closely resemble human E654-thalassemia phenotype. The spleen size of treatment mice was (0.211 ± 0.05) g, smaller than that of control E654 mice (0.275 ± 0.044) g. The improvement of hematological parameters of HEi-Hbbth-4/Hbb+ might be relevant to reducing the expression of aberrantly spliced E-globin mRNA by siRNA.

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Fig. 6. The counts of poikilocytosis plus target cells and hematologic studies in transgenic mice. A: RBC counts. B: hemoglobin levels. C: percentage of poikilocytosis plus target cells. D: reticulocyte counts. The total quantity of poikilocytosis plus target cells in the peripheral blood smear of HEi-Hbbth-4/Hbb+mice is much less than that of E654-control mice (n = 47 per group, P<0.05). RBC counts, hemoglobin levels, reticulocyte count is shown at four months after birth. All measured parameters show significant increases in transgenic mice as compared with the wild type correspondents (as the negative control, n= 49 per group, P<0.05).

Discussion RNA interference (RNAi) was first discovered in the nematode worm, Caenorhabditis elegans in 1998 (Fire et al., 1998), when double-stranded RNA (dsRNA) was found to be more effective in suppressing the gene expression than the antisense approach. RNAi can silence gene expression at different levels, such as DNA, pre-mRNA, mRNA, and affect the translation. In addition, the RNAi effect can be amplified in vivo (Zamore et al., 2000). Therefore, RNAi has become a powerful tool for the analysis of gene function in many fields (Chen et al., 2007). In this paper, we hypothesized that the aberrantly spliced E-globin mRNA was harmful to patients and the anemia symptom would be ameliorated with the aberrantly spliced E-globin mRNA reduced. Then, four siRNA vectors (pEi1, pEi2, pEi3 and pEi4) were designed and the aberrantly spliced E-globin mRNA was found to be reduced in cultured HeLa E654 by the siRNA, especially in pEi2 group. On the basis of in vitro study that pEi2 vector had significant inhibition of aberrantly spliced Eglobin mRNA expression, we constructed Lenti-pEi2 vector and produced lentiviral-mediated siRNA integrated E654-thalassemia mice (HEi-Hbbth-4/Hbb+) to investigate whether the hematological parameters of HEi-Hbbth-4/Hbb+ was improved when aberrantly spliced E-globin mRNA expression was inhibited. The E654-thalassemia mice used in our experiment is a heterozygote (Hbbth-4/Hbb+) carrying a human gene with

EIVS-2-654 splice mutation and the normal mouse E-globin locus, which represents the prototypic signs of the moderate form of E-thalassemia (Lewis et al., 1998). The Hbbth-4/Hbb+ heterozygous mice have low RBC counts, dedicating ineffective erythropoiesis and increased RBC destruction. The peripheral blood smears from the Hbbth-4/Hbb+ mice showed marked anisocytosis and poikilocytosis plus target cells, which closely resemble human E654-thalassemia phenotype. In in vivo study, we found that hematologic parameters (RBC, Hb, blood smears, the proportion of nucleated cells in bone marrow etc.) of Hbbth-4/Hbb+ mice were ameliorated after treatment with lentivirus-mediated siRNA. We also produced one control transgenic mice, which were integrated with lentivirus without siRNA. No reducing aberrantly spliced E-globin mRNA was found in this transgenic mice in genetic background. The ameliorated effect of Hbbth-4/Hbb+ mice may be due to the reduction of the synthesis of highly unstable E-globin chain mRNA or protein expression by siRNA. In summary, we have successfully used the siRNA to knock down the aberrantly spliced E-globin mRNA, and our results supported that the aberrantly spliced E-globin mRNA might aggravate E654-ҏthalassemia and reduce the aberrantly spliced E-globin mRNA to ameliorate E654-thalassemia. So, combined with other therapy approach, using RNAi approach to knock down the aberrantly spliced E-globin mRNA is another potential way for E654-thalassemia therapy.

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Acknowledgements This work was supported by the National Basic Research Program of China (973 Program) (No. 2004CB518806), the National High-tech R&D Program (863 Program) (No. 2007AA021206), the National Natural Science Foundation of China (No. 30571777), and the Chinese National and Shanghai Leading Academic Discipline Project (No. B204).

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