Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on August 22, 2007
Human Molecular Genetics 2007 16(21):2616-2625; doi:10.1093/hmg/ddm218

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Restoration of the balanced /ß-globin gene expression in ß⁶⁵⁴-thalassemia mice using combined RNAi and antisense RNA approach

Shu-Yang Xie, Zhao-Rui Ren, Jing-Zhi Zhang, Xin-Bin Guo, Qing-Xue Wang, Shu Wang, Dan Lin, Xiu-Li Gong, Wei Li, Shu-Zhen Huang, Fanyi Zeng and Yi-Tao Zeng^*

Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China

^* To whom correspondence should be addressed at: 24/1400 West Beijing Road, Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai 200040, People's Republic of China. Tel: +8621 62790545; Fax: +8621 62475476; Email: ytzeng{at}stn.sh.cn

Received June 5, 2007; Accepted August 4, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
The ß-thalassemia is associated with abnormality in ß-globin gene, leading to imbalanced synthesis of -/ß-globin chains. Consequently, the excessive free -globin chains precipitate to the erythrocyte membrane, resulting in hemolytic anemia. We have explored post-transcriptional strategies aiming at -globin reduction and ß-globin enrichment on ß⁶⁵⁴ (Hbb^th-4/Hbb⁺) mouse, carrying a human splicing-deficient ß-globin allele (Hbb^th-4). Lentiviral vectors of short hairpin RNA (shRNA) targeting -globin and/or antisense RNA facilitating ß-globin correct splicing were microinjected into ß⁶⁵⁴ single-cell embryos. Three transgenic strains were generated, as _i-Hbb^th-4/Hbb⁺(shRNA), ß_a-Hbb^th-4/Hbb⁺(antisense) and _iß_a-Hbb^th-4/Hbb⁺(both shRNA and antisense). Without notable abnormalities, all the founders and their offsprings showed sustained amelioration of hematologic parameters, ineffective erythropoiesis and extramedullary hematopoiesis. Augmented effects appeared in _iß_a-Hbb^th-4/Hbb⁺, which correlated with a better-balanced -/ß-globin mRNA level. Among the transgenic mice integrated with shRNA and antisense RNA, one homozygous mouse (Hbb^th-4/Hbb^th-4) had been viable, and the 3-week survival rate for heterozygotes (Hbb^th-4/Hbb⁺) was 97%, compared with 45.4% for untreated. Our data have demonstrated the feasibility of techniques for ß-thalassemia therapy by balancing the synthesis of -/ß-globin chains.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
The ß-thalassemia, caused by ß-globin gene abnormality, is one of the most common genetic diseases, especially in the Mediterranean region, the Middle East and South East Asia. More than 200 mutations affecting the human ß-globin gene have been described. Among the most frequent types, the C T substitution at nucleotide 654 of intron 2 (ßIVS-2-654), which induces aberrant splicing leading to a nonfunctional polypeptide, accounts for 18–36% of total ß-thalassemia alleles in China (1,2). Deficient synthesis of effective ß-chain leads to an imbalance in the production of - and ß-globin chains (1). As a result, the excess free chains precipitate to the erythrocyte membrane, causing intramedullary destruction of the erythroid precursors, ineffective erythropoiesis and oxidative membrane damage associated with apoptosis of red blood cells (RBC) (3,4). The severity of ß-thalassemia has been suggested to be linked to the degree of imbalance in the production of - and ß-globin chains (5). Despite extensive efforts to develop effective agents, most drugs under development have not yet become clinical options due to severe toxic effects (6,7). At present, most patients with severe ß-thalassemia depend on lifelong blood transfusion combined with iron chelation therapy. Consequently, affected individuals often die before the age of 25 as a result of cardiac iron deposition.

Genetic manipulation aiming to increase functional hemoglobin (Hb) expression in autologous hematopoietic stem cells has been demonstrated to be a feasible therapeutic approach to correct hemoglobinopathies. May et al. (8,9) first demonstrated production of ß-globin, correction of anemia and reduction of extramedullary hematopoiesis (EMH) in ß-thalassemia mice following engraftment with bone marrow cells transduced with a lentiviral vector encoding the human ß-globin gene. By using lentiviral-mediated gene transfer combined with RNA interference (RNAi) technologies, Samakoglu et al. (10) have successfully induced erythroid-specific expression of the -globin transgene and concomitantly suppressed endogenous ß^S transcripts in CD34⁺ cells from patients with sickle cell anemia.

Despite the indisputable success of therapy in cultured cells or in mouse models from the above studies by increasing non--globin, here, we attempted to treat ß-thalassemia by a combined strategy to use RNAi to knock down -globin mRNA levels and antisense RNA to promote correct splicing of the thalassemia allele.

ß⁶⁵⁴-thalassemia mouse (Hbb^th-4/Hbb⁺) is a good model system for our investigation purpose. In this animal model, the two cis murine adult ß-globin genes (i.e. major and minor) (together referred as ‘mß^A’ hereafter) have been replaced with a single copy of the human ßIVS-2-654 gene (Hbb^th-4) (11). As the same aberrant splicing was observed as their human counterparts, heterozygous mice (Hbb^th-4/Hbb⁺) produce reduced amounts of the mouse ß-globin chains and no functional human ß-globin, and have an intermediate form of ß-thalassemia. No homozygous mice (Hbb^th-4/Hbb^th-4) survived postnatally (11). Our group and others have used antisense oligonucleotides or RNAs to block the aberrant splice sites in cultured human thalassemic erythroid cells and/or its progenitors (12–14). In the current study, we would like to investigate whether the antisense RNA strategy was able to correct the splicing defect of Hbb^th-4 mouse in vivo so that the Hbb^th-4 transcripts followed the natural splicing pathway. As a result, stable reduction of the aberrant mRNAs and significant accumulation of normal ß-globin transcripts were observed.

In addition, to the best of our knowledge, until now, gene therapy for ß-thalassemia has been limited on ß-globin engineering. Given the significant impact of free chains on apoptosis of RBC, it is reasonable to hypothesize that suppression of -globin would be an alternative therapeutic option to treat ß-thalassemia. As exogenous target gene-complementary short hairpin RNAs (shRNAs) are capable of down-regulating target gene expression through sequence-specific pre-mRNA degradation (15–17) via a process known as RNAi (18), we also designed -globin-specific shRNA constructs to suppress -globin in ß⁶⁵⁴-thalassemia mice.

Taken together, we sought to evaluate the therapeutic potentials to ß⁶⁵⁴ mice through lessening the imbalance in -/ß-globin production by RNAi-based -globin reduction and/or antisense RNA-mediated ß-globin enhancement. We have designed shRNA and antisense RNA constructs and delivered them into single-cell zygotes of ß⁶⁵⁴ mice (Hbb^th-4/Hbb⁺), utilizing the widely recommended lentiviral vector system. Three sets of transgenic mice, with shRNA, antisense RNA or combined constructs, respectively, were produced, and their phenotypes were analyzed. Our data revealed significant amelioration of ß-thalassemia in all these transgenic mice and their F1 progeny, especially in those receiving combined constructs. Interestingly, among the mice integrated with combined constructs, one homozygous mouse (Hbb^th-4/Hbb^th-4) had been identified, and the 3-week survival rate for the heterozygous mice (Hbb^th-4/Hbb⁺) was 97%, compared with 45.4% for untreated.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
Sequence-specific shRNA knock down -globin gene expression in ß⁶⁵⁴-thalassemia mice
To investigate the effects of abating free -globin accumulation in ß⁶⁵⁴ mice, we injected into the perivitelline space of single-cell embryos of ß⁶⁵⁴ mice (Hbb^th-4/Hbb⁺) with mouse -globin sequence-specific shRNA constructs (Fig. 1). To ensure high efficiency of transgenesis, sustained expression of transgene and less invasive to the embryos, we chose lentiviruses as gene delivery vehicles based on previous study results (19).

View larger version (21K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Structure of hemoglobin mRNAs and recombinant lentiviral vectors. (A) -globin mRNA and the specific shRNA (i). Boxes indicate exons; lines, introns; short bar below RNA, shRNA targeting site. The i sequence was shown as hairpin structure, which was linked to pH1 (RNA polymerase III H1 promoter) and TTTT (poly T termination signal). (B) Diagrammatic representation of recombinant lentiviral vectors (FUGW). Three types of vectors were constructed to correct aberrant splicing of ß-globin (ßa), suppress -globin expression (i) or both (ißa). pßA, ß globin gene promoter. (C) ß-globin mRNA. The dashed lines represent correct and aberrant splicing pathways. Short bar below RNA, antisense RNA (ßa) blocking region.

 
Embryos were implanted into pseudopregnant females and were carried to term. Founder mice with both ß⁶⁵⁴ allele and vector integrations were identified (designated as _i-Hbb^th-4/Hbb⁺) by PCR analysis. Fluorescence in situ hybridization (FISH) analysis performed on the spleen cells visualized the chromosomal insertions of vectors (Supplementary Materials 1). No notable abnormalities were observed with these transgenic mice, as well as the other two sets of transgenic animals described below. Real-time PCR analysis showed that the relative abundance of -globin mRNA in the blood cells of _i-Hbb^th-4/Hbb⁺ mice decreased 20–35%, as compared to that in the blood cells of Hbb^th-4/Hbb⁺ controls (P < 0.01; Fig. 2A). The mRNA abundance of -globin was not affected in the transgenic mice integrated with lentiviral vectors hosting antisense RNA against aberrant splicing of Hbb^th-4, excluding the possibility of non-specific -globin suppression caused by lentiviral vectors (Fig. 2A).

View larger version (32K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Reduction of -globin transcripts and restoration of ß-globin normal splicing in transgenic mice. Lentiviral vectors of i, ßa or ißa were microinjected to one cell zygotes of ß654 heterozygotes (Hbbth-4/Hbb+) mated with wild-type mice (Hbb+/Hbb+). As a result, three types of transgenic mice were produced, namely, i-Hbbth-4/Hbb+, ßa-Hbbth-4/Hbb+ and ißa-Hbbth-4/Hbb+, respectively. (A) The abundance of -globin mRNA from mouse blood cells was determined by real-time PCR, using ß-actin as internal control. Hbb+/Hbb+, wild-type mice (n = 5); Hbbth-4/Hbb+, ß654 heterozygote (n = 5); for the rest, n = 2. In comparison with wild-type mice, -globin mRNA reduction was observed in i-Hbbth-4/Hbb+ and ißa-Hbbth-4/Hbb+ mice (P < 0.01), but not in Hbbth-4/Hbb+ and ßa-Hbbth-4/Hbb+, demonstrating selective suppression of -globin mRNA by i vector. (B) Normal human ß-globin transcript was detected from the blood cells of ßa-integrated mice. Using RT–PCR assay with ß-5' and ß-3' primers (Table 1), correctly spliced pre-mRNA would yield a 181 bp fragment, whereas aberrant splicing gave rise to a 254 bp fragment. M, 100 bp marker (ßa-Hbbth-4/Hbb+: upper figure lane 1 and ißa-Hbbth-4/Hbb+: lower figure lane 2). (C) Human ß-globin protein is detected by western blotting assay. 1, Hbbth-4/Hbb+ mouse; 2, ßa-Hbbth-4/Hbb+ mouse; 3, ißa-Hbbth-4/Hbb+ mouse. Normal human ß-globin protein is detected from the blood cells of ßa-integrated mice.

 

View this table: [in this window] [in a new window]   Table 1. Oligonucleotide primers for PCR analysis

 
Antisense RNA restores the correct splicing pathway of ß-globin pre-mRNA in ß⁶⁵⁴-thalassemia mice
Previous studies indicated that the ß⁶⁵⁴ mutation had created an aberrant splicing site, dominant over the natural one (20). The mutant gene gives rise to a non-functional ß-globin mRNA with the inclusion of a 73 bp intron fragment. We and other groups have demonstrated that the genetic deficiency of ß⁶⁵⁴ can be corrected partially through blocking the aberrant splicing sites by antisense RNA treatments in vitro (13,14). To further evaluate the effects of antisense RNA in vivo, we utilized the similar approaches to make antisense RNA transgenic mice (designated as ß_a-Hbb^th-4/Hbb⁺). Our data revealed that although no detectable normal human ß-globin transcripts and/or proteins were in Hbb^th-4/Hbb⁺ and/or Hbb⁺/Hbb⁺ controls, they were clearly detected in the peripheral blood of the ß_a-Hbb^th-4/Hbb⁺ (Fig. 2B and C). This result indicated that antisense RNA was also able to fix the abnormal splicing to some degree and enforce the production of normal human ß⁶⁵⁴ globin mRNA and protein in vivo.

Both ß-globin enrichment and -globin reduction were observed in those transgenic mice (designated as _iß_a-Hbb^th-4/Hbb⁺) receiving constructed vectors harboring both shRNA and antisense RNA. -globin mRNA was reduced over 30% when compared with that in Hbb^th-4/Hbb⁺ mice (P < 0.01, Fig. 2A), and the normal human ß-globin transcripts and proteins were also partially recovered (Fig. 2B and C). Conceivably, the imbalance of -/ß-globin had been lessened better in these _iß_a-Hbb^th-4/Hbb⁺, in comparison with either _i-Hbb^th-4/Hbb⁺ or ß_a-Hbb^th-4/Hbb⁺, which only received a single correction.

Sustained amelioration of anemia in all three types of transgenic mice
To determine whether the better balanced -/ß-globin level in these transgenic mice would be of therapeutic benefits, we have conducted a series of examinations on the phenotypes of these transgenic mice, Hbb⁺/Hbb⁺ and Hbb^th-4/Hbb⁺ controls for several months. The marked decrease of 30–50% in poikilocytosis and target cells were observed in peripheral blood of all _i-Hbb^th-4/Hbb⁺, ß_a-Hbb^th-4/Hbb⁺ and _iß_a-Hbb^th-4/Hbb⁺ mice (Fig. 3). Such a reduction has been sustained throughout the monitoring period up to 8 months.

View larger version (136K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. The peripheral blood patterns of the transgenic mice (400 x). Blood smears strained with Wright strain. Tests were performed at 2.5 months after birth. Note that the poikilocytosis with target cells in the peripheral blood of all three types of transgenic mice are less than that in ß654 mice (Hbbth-4/Hbb+).

 
The Hb values and RBC counts in the transgenic mice were increased 20–30% in comparison with those in untreated Hbb^th-4/Hbb⁺ controls (P < 0.01; Fig. 4). The reticulocyte counts were also diminished 25–30% (data not shown), suggesting a decrease in abnormal erythropoiesis resulting from prolonged RBC life span.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. Hematologic studies in transgenic mice and their offspring. RBC counts, hemoglobin levels are shown 10 weeks after birth. All measured parameters show significant increases in transgenic mice when compared with wild-type correspondents (n = 4–9 per group, P < 0.05).

 
Amelioration of EMH and improvement of erythropoiesis in _iß_a-Hbb^th-4/Hbb⁺ mice
To determine the impact of the synergy of shRNA and antisense RNA on hematopoiesis, we investigated the extent of splenomegaly and EMH in _iß_a-Hbb^th-4/Hbb⁺ and age-matched Hbb^th-4/Hbb⁺ control mice. Spleen size and weight in Hbb^th-4/Hbb⁺ control mouse was much larger and heavier (about 6-fold) than that of mßA/mßA mouse. However, it reduced by half in _iß_a-Hbb^th-4/Hbb⁺ mouse (Fig. 5). The transgenic spleen also looked reddish, that is, similar to the normal one.

View larger version (109K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. Significant reduced spleen size in ißa-Hbbth-4/Hbb+ mice. The spleens were collected at 6 months old. I, wild-type mouse; II, ß654 heterozygote (Hbbth-4/Hbb+); III, ißa-Hbbth-4/Hbb+.

 
The bone marrow of the _iß_a-Hbb^th-4/Hbb⁺ mouse showed an increase in erythroid elements and the presence of myeloid cells (Fig. 6). Histopathologic analysis on spleen of _iß_a-Hbb^th-4/Hbb⁺ revealed less expansion of the red pulp and morphologically intermediate changes between those of wild type and ß⁶⁵⁴ controls. Hemosiderin was obviously decreased in the red pulp of the _iß_a-Hbb^th-4/Hbb⁺ mouse, indicating that less RBCs were destroyed in treated mice (Fig. 6). Similarly, the amelioration of EMH in liver was also evidenced (Fig. 6).

View larger version (146K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 6. Histopathologic analysis of mouse hematopoietic tissues. The following tissues were examined at 6 months after birth. (A) Bone marrow (original magnification, 100 x). Left: wild type. the marrow shows various stages of normal erythroid maturation. Megakaryocytes as well as a significant numbers of maturing myeloid cells are present. Center: ß654 heterozygote (Hbbth-4/Hbb+). Misshapen erythroid cells and red cell fragments are present in the sinuses. Some of the immature erythroid cells have dysmorphic nuclei. Mature erythroid cells are decreased. Right: ißa-Hbbth-4/Hbb+. The marrow shows a relative increase in erythroid elements; myeloid cells are also present. (B) Spleen (original magnification, 100 x). Left: wild type. The red pulp contains a mixture of erythroid and myeloid cells. The sinuses are open. Center: Hbbth-4/Hbb+. The red pulp consists primarily of immature erythroid cells. Mitotic figures are easily identified. The immature erythroid cells often have abnormal nuclei. Haemosiderin is obviously in red pulp. Right: ißa-Hbbth-4/Hbb+. It showed less expansion of the red pulp and were morphologically intermediate between those of the healthy and ß654 control mice. There is almost no haemosiderin in red pulp. (C) Liver (original magnification, 100 x). Left: wild type. Splenomegaly, EMH is absent. Center: Hbbth-4/Hbb+. Extensive EMH is present. Right: ißa-Hbbth-4/Hbb+. Moderate EMH is present between those of the healthy and ß654 control mice. (D) Immunohistochemistry analysis of CD68 in mouse spleens (original magnification, 200 x). Left: wide type. Almost no macrophages are found. Center: Hbbth-4/Hbb+. Lots of macrophages are present. Right: ißa-Hbbth-4/Hbb+. Fewer macrophase are present.

 
The significantly reduced RBC apoptosis in the spleen tissue of _iß_a-Hbb^th-4/Hbb⁺ mouse has been further demonstrated by immunohistochemistry assay using rat anti-mouse polyclonal antibody against CD68, a marker reflecting the function of macrophages. Much less CD68 positive cells were found in _iß_a-Hbb^th-4/Hbb⁺ mouse than that in the untreated Hbb^th-4/Hbb⁺ mouse (Fig. 6), indicating that less macrophages were required for clearance of destroyed RBC debris.

The similarities in phenotypes between offspring and their parents indicated capabilities of genetic transmission of the experimental constructs
To investigate whether the favorable effects could be passed down to their progeny, we examined the phenotype of F1 generation of the transgenic mice. We noticed that changes of the percentage of poikilocytosis with target cells, and especially, the patterns of all hematologic parameters observed in peripheral blood of F1 generation, were extremely similar to that of their corresponding transgenic parents. The improved hematologic parameters and blood smear appearances also persisted for months in the mice inheriting the experimental constructs. Stable amelioration of anemia was also observed in the F2 generation of _iß_a-Hbb^th-4/Hbb⁺ mouse (Fig. 4).

Enhanced survival of Hbb^th-4/Hbb⁺ mice born of all three sets of transgenic mice and one Hbb^th-4/Hbb^th-4 mouse had been rescued upon combined treatment
To further investigate the effective therapy action to Hbb^th-4/Hbb⁺ by balancing the synthesis of -/ß-globin chains, we analyzed the genotypes and the lentiviral integrations of the F1 progeny of all three types of transgenic mice and the untreated Hbb^th-4/Hbb⁺ mice, mated to Hbb⁺/Hbb⁺ mice, respectively, at three weeks after birth, using listed primers specific for Hbb^th-4/Hbb⁺ alleles, and lentiviral vector long terminal region (LTR) sequences (Table 1). Interestingly, we found only 22.7% of the offspring of untreated mice carrying Hbb^th-4 allele (Table 2), deviating significantly from 50% according to Mendel’s rule. Further investigation throughout the entire pregnancy period and at birth revealed that the frequencies of the Hbb^th-4 allele were complied with Mendel’s rule all the time (Table 2). This result indicated that although Hbb^th-4/Hbb⁺ mice are viable, less than half (i.e. 22.7/50 = 45.4%) could reach 3-week-old when left untreated. However, in the cases of the _i-Hbb^th-4/Hbb⁺, ß_a-Hbb^th-4/Hbb⁺ or _iß_a- Hbb^th-4/Hbb⁺ mice, the proportion was as high as 41.2, 41.9 and 48.5%, respectively, representing the 3-week survival rate of 82.4 (41.2/50), 83.8 (41.9/50) and 97% (48.5/50), respectively.

View this table: [in this window] [in a new window]   Table 2. The survival rate of transgenic mice at 3 weeks after birth

 
Furthermore, although no Hbb^th-4/Hbb^th-4 mouse survived postnatally (11), surprisingly, among the progeny of _iß_a-Hbb^th-4/Hbb⁺ mice, we have identified one Hbb^th-4/Hbb^th-4 homozygote with lentiviral vector integration (Fig. 7). None has been found among the untreated. These data altogether demonstrated that treatments through suppression of -globin and/or enrichment of ß-globin using the aforementioned shRNA or antisense RNA were effective, especially when combined treatments were applied.

View larger version (31K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 7. One Hbbth-4/Hbbth-4 homozygote is detected upon combined treatment. 1, Hbbth-4/Hbbth-4; 2, Hbbth-4/ Hbb+; 3, Hbb+/Hbb+.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
It has been observed that co-inheritance of -thalassemia in ß-thalassemia patients ameliorates thalassemic phenotype due to a more balanced -/ß-globin chain biosynthesis (5). This suggests that suppression of -globin gene expression could be used as an approach to correct ß-thalassemia. In this study, we tested this possibility by reducing -globin gene expression using specific shRNA. Indeed, we observed enhanced erythropoiesis and survival of _i-Hbb^th-4/Hbb⁺ mice, in which the -globin expression had been suppressed by specific shRNA, but leaving the defective ß-globin gene untouched. Also, the enhancements were more profound in mice with both -globin reduction and ß-globin enrichment (_ißa-Hbb^th-4/Hbb⁺) than those receiving ß-globin correction alone (ßa-Hbb^th-4/Hbb⁺). Together, our data provide convincing evidence demonstrating the therapeutic potential of -globin inhibition in the management of ß-thalassemia mice.

Combined manipulation aiming at both ß-globin enrichment and -globin reduction should be of greater value in treating ß-thalassemia, compared with single correction of either ß-globin or -globin, as the data revealed augmented ameliorations of hematologic parameters and EMH in _ißa-Hbb^th-4/Hbb⁺ mice. Top 3-week survival rate (i.e. 97%) of heterozygotes (Hbb^th-4/Hbb⁺) and one viable homozygote (Hbb^th-4/Hbb^th-4) were also observed in this group. Quantitative analysis revealed a better-balanced -/ß-globin mRNA level in these mice, compared with that in _i-Hbb^th-4/Hbb⁺, ßa-Hbb^th-4/Hbb⁺ or untreated Hbb^th-4/Hbb⁺. This result is consistent with clinical observations that the severity of ß-thalassemia correlates with the degree of imbalance in the production of /ß-globin chains (5). Therefore, the observations suggest that the -/ß-globin ratio can be a good indicator in evaluating the effectiveness of a genetic option against ß-thalassemia.

Gene replacement therapy has been utilized in mouse model of ß-thalassemia (8). Such a strategy often requires transfer of large gene cassettes, and associates with transient loss of phenotypes resulting from epigenetic modifications or instabilities of the transgenes. Here we explored post-transcriptional approaches in ß⁶⁵⁴ mouse model, using antisense RNA to repair aberrant splicing of defective ß-globin pre-mRNA and engineered shRNA to induce RNAi-based -globin suppression. Significant amount of normal human ß-globin mRNA and stable reduction of mouse -globin was detected in corresponding transgenic mice and their offsprings, in which sustained ameliorations of anemia was observed during the entire monitoring period up to 8 months. These results have provided in vivo evidences demonstrating effective genomic integration, persistent expression and genetic transmission of exogenous antisense RNA and shRNA, and their abilities in regulating targeted endogenous gene expression. Interestingly, based on our preliminary data, the degree of gene expression reduction/correction or hematologic parameter improvement does not seem to be directly linked to the copy number of the integrated vectors (Supplementary Materials 2). One explanation for the engineered shRNA is that the cellular regulation system would execute its controlling power over the expression of integrated shRNAs, as it does to endogenous miRNAs, so that an oversaturation of cellular RNAi machinery may be prevented. In all the dozens of transgenic mice hosting exogenous shRNAs or antisense RNA, neither visible abnormalities nor toxicity effects such as significant weight loss and/or death have been identified. Elevation in white blood cell counts that implies the activation of the interferon pathway had not occurred, either. These results provide additional evidences supporting the great potentials of shRNA and antisense RNA technologies in gene therapy.

The advantage of post-transcriptional mechanism-based technologies is avoid of problems resulting from gene replacement with large exogenous cassettes, so that the chromosome microenvironment can be retained and overt transcription and gene regulations better preserved. Notably, this is the first report to combine the RNAi and antisense RNA strategies in order to reduce and restore gene expression simultaneously. Our results have demonstrated the feasibility of such a combined approach, providing new insights to possible treatments of various disorders, not ß-thalassemia only, which have been associated with ‘gain-of-function’ mutations as well as aberrant splicing that occurred in > 15% of point mutations contributing to genetic diseases (21).

In this study, we have utilized the lentivirus to deliver transgenes into single-cell embryos. In comparison with pronucleus microinjection, it is more efficient, less invasive to the embryos and has overcome many of the limitations of the former (22). Lentiviral vectors are not developmentally silenced, and therefore allow faithful expression of transgenes (19). Quantitative analysis on transgenic mice revealed that each had carried several copies of transgenes (Supplementary Materials 2) indicating high efficiency of this gene transfer technique. Sustained ameliorations of anemia in founder mice and their progeny confirmed persistent expression and effective therapy action by balancing -/ß-globin production. On the other hand, however, our results also revealed some disadvantages of the technique using the FUGW lentivirus: (i) the position and frequency of insertion of transgenes into host genomes could not be controlled and was poorly understood; (ii) The expression in some F1 mice was not consistent with its founder mice. The reason may be owned to the vector methylated or silenced during gene transmission; (iii) Among the transgenic mice integrated with combined constructs, we have identified only one homozygote (Hbb^th-4/Hbb^th-4) and (iv) Our approach may not be directly used in human patient at present. Nevertheless, the results described in this study provide a direct evidence supporting the importance and potential of therapy to ß-thalassemia by balancing the synthesis of -/ß-globin chains.

In conclusion, we have explored combined post-transcriptional approaches for gene therapy of ß-thalassemia. Through lentiviral vectors, three types of ß⁶⁵⁴ transgenic mice have been produced, namely i-Hbb^th-4/Hbb⁺, ßa-Hbb^th-4/Hbb⁺ and _iß_a-Hbb^th-4/Hbb⁺, integrated with -globin-specific shRNA for suppression, antisense RNA for interfering aberrant splicing of ß-globin pre-mRNA and both, respectively. Our data revealed significant and persistent amelioration of ß-thalassemia and EMH in all transgenic mice and their F1 progeny, especially in _iß_a-Hbb^th-4/Hbb⁺. Interestingly, among the transgenic mice integrated with combined constructs, one homozygous mouse (Hbb^th-4/Hbb^th-4) had been identified, and the 3-week survival rate for the transgenic heterozygous mice (Hbb^th-4/Hbb⁺.) was 97%, compared with 45.4% for untreated. The findings hint at the potential success of shRNA and antisense RNA for gene therapy of ß-thalassemia and other human diseases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
ß⁶⁵⁴-thalassemia mouse
The ß⁶⁵⁴-thalassemia mice (B6; 129P2-Hbb^tm2Unc) were obtained from the Jackson Laboratory (JAX). This heterozygote (Hbb^th-4/Hbb⁺) carries a normal mouse ß-globin allele, and a defective human ßIVS-2-654 allele associated with aberrant splicing due to C T substitution at nt654 of intron 2. Therefore, the ß⁶⁵⁴ mice produce half of the normal mouse ß-globin chains but no functional human ß-globin, manifesting typical signs of a moderate form of ß-thalassemia (anemia, splenomegaly, abnormal hematologic indices) (11). The use of ß⁶⁵⁴ mice for this study was reviewed and approved by the Review Board of Shanghai Children’s Hospital.

ShRNA vectors for RNAi targeting mouse -globin mRNA
The H1 promoter element of Polymerase III was rescued from the Tc vector as described previously (23), and subcloned into pREP4 (Invitrogen), generating pH1 vector. Oligonucleotides aiming at suppression of the -globin gene expression were designed based on the principle described by Berns et al. (24). The 19-mers were complementary to mouse -globin mRNA (Fig. 1). Paired siRNA oligos were annealed and inserted into HindIII/NheI-digested pH1 vector. Among these shRNA constructs, one of the most effective in vitro was used in animal study.

Antisense RNA vector for blocking the aberrant splice site of ß⁶⁵⁴
The antisense RNA for ß⁶⁵⁴-globin gene was prepared according to our previous report (14). The ß-globin promoter (–1649 to +75 bp) and the antisense RNA were then cloned into pcDNA3.1 (+) vector (Invitrogen) at EcoRI and NotI sites to form the pcDNA-antisense vector.

Lentiviral-mediated shRNA or antisense RNA vectors
The fragment harboring H1 promoter and shRNA sequence was cut off by EcoRV and PvuII and cloned into blunt-ended PacI-digested FUGW vector (kindly provided by Dr. Zack Wang, Massachusetts General Hospital, Harvard University), resulting in _i vector. Similarly, NheI–XbaI element containing ß-globin promoter and the antisense RNA from the pcDNA vector were inserted into the XbaI site of FUGW vector to form the ß_a vector. We also used the PacI–XbaI site to construct _iß_a vector (Fig. 1). Virus production, cell culture and transfection were carried out using standard protocols.

RT–PCR
Total RNA was isolated from mouse blood cells using RNA isolation kit (U-gene) according to the manufacturer’s instructions. After RT reaction, the -globin and GAPDH cDNA (internal control) were amplified for 26 cycles in a PCR machine (Eppendorf): denaturing at 94°C for 30 s; annealing at 55°C for 30 s; followed by extension at 72°C for 45 s. The aberrant ß⁶⁵⁴-globin was amplified for 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 30 s. The primers used are shown in Table 1. PCR products were analyzed by gel electrophoresis using 2% agarose.

Quantitative real-time–PCR
To evaluate the relative abundance of each mRNA, total RNAs from mouse blood cells were isolated and subjected to RT reaction, followed by quantitative real-time PCR using the RG3000 system (Corbett Research) with the Quantitect SYBRGreen Kit (Qiagen, Hilden). The primers for each sample and control were shown in Table 1. The reaction condition was as follows: an initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s. Fluorescence was detected at 585 nm at each step of 72°C. Each sample was measured thrice, and GAPDH or ß-actin mRNAs were used as internal controls. A standard dilution curve was obtained. The relative mRNA abundance was determined by the ratio of sample-to-control.

Copy number of transgenes
The copies of transgenes were estimated by the ratio of LTR of lentiviral vectors versus mouse -globin, by quantitive PCR analysis on DNA isolated from mouse tails. The primers were shown in Table 1. Duplicate reactions were performed in a total volume of 20 µl, containing 1 µl of SYBR Green PCR Master Mix (Cambrex Bio Science Rockland), 15 µM of forward and reverse primers and 80 ng DNA template. The samples were heated for 10 min at 95°C and amplified for 40 cycles of 30 s at 95°C followed by 60 s at 60°C. Blank and positive controls were run in parallel to determine amplification efficiency within each experiment. The quantity of each sample was determined by comparing to the standard curve.

FISH analysis
The mouse spleen cells were used for FISH analysis according to the methods described previously (25,26). To detect the ß⁶⁵⁴-allele, a 4.2 kb fragment was prepared as probes, which was homolog to the human ß-globin gene, but not to its mouse counterpart. To visualize LV vector integration, corresponding vectors, i.e. _i, ß_a or _iß_a, were labeled with DIG-Nick or Biotin-Nick Translation Mix (Roche, Germany) according to the manufacturer’s protocol. FISH signals were examined with Leica DM RXA2 fluorescent microscope.

Detection of human ß-globin by western blotting assay
Fresh periphreral blood from mouse tail vein was collected and then lysated. Proteins were separated by 12% SDS–PAGE and then transferred to nylon membrane using electronic transfer method. Primary human ß-globin monoclonal antibody (H00003043-M01, Abnova, 1:1000 diluted) was used to hybridization at 4°C for 2 h. After the primary incubation was completed, the secondary hybridization was performed using peroxidase-conjugated goat anti-mouse IgG (Rockland, 1:1000 diluted) at 4°C for another 2 h. Hb bands were visualized by DAB staining.

Hematologic studies
Peripheral blood smears were prepared using 1–2 µl of blood samples collected in heparinized microhematocrit tubes, air dried and stained with Wright staining. Whole blood samples from mice at least 6 weeks old were collected in 40 µl microhematocrit tubes containing 2 µl of 0.5 M EDTA (pH 8). The RBC count, Hb, mean corpuscular volume, reticulocyte counts and mean corpuscular hemoglobin for each sample were determined using a Hematology Analyzer (KX-21, Sysmex) equipped with software to analyze murine cells.

Generation of transgenic mice via a lentiviral-mediated approach
The ß⁶⁵⁴ male mice were mated to superovulated wild-type females. The zygotes were collected and subjected for the injection of shRNA or antisense RNA virus particles. Each zygote was injected with 5 x 10^–4 µl of virus suspension (10⁸ U/ml). The injected zygotes were placed back in the oviduct of pseudo-pregnant wild-type females, and allowed to develop to full term. The shRNA or antisense RNA ß⁶⁵⁴ transgenic mice were verified by PCR analysis 3 weeks after birth. The PCR primers were designed based on the sequence of the LTR of lentivirus vector and the ß⁶⁵⁴ allele. The chromosomal integration of the transferred genes in the transgenic mice was visualized with FISH assay.

Histopathology analysis
Small pieces of tissue were embedded in paraffin wax, followed by cutting with a LEICA RM 2135, and then mounted onto glass slides. The tissue sections were stained with hematoxylin and eosin or Gomori iron, and examined under light microscopy. Immunohistochemistry analysis was also performed as previously described (26), using rat anti-mouse CD68 polyclonal antibody MCA1957GA (Serotec Ltd, UK).

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
Supplementary Material is available at HMG Online.

	FUNDING

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 
National Basic Research Program (973 Program) of Chinese Ministry of Science and Technology (Grant No. 2004CB518806), National Natural Science Foundation of China (Grant No. 30571777), Shangai Pujiang Program (Grant No. 06PJ14060).

	ACKNOWLEDGEMENTS

 
We appreciate Yi-Wen Zhu, Zhi-Juan Gong for their technical assistance. We also thank Professor C.P. Pang, Chinese University of Hong Kong, and Professor Deming Sun, University of Louisville, for their helpful comments and discussions.

Conflict of Interest statement. None declared.

	FOOTNOTES

 
Present address: Binzhou Medical College, Binzhou, ShanDong Province, People's Republic of China.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL FUNDING REFERENCES

 

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