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【摘要】
Objective- Using intravenous injection of adeno-associated viral (AAV) vectors based on novel serotypes 7 and 8, we examined whether liver-specific expression of human apolipoprotein E (apoE) in apoE-deficient mice would completely prevent atherosclerosis after 1 year of sustained expression.
Methods and Results- Chow-fed apoE -/- mice were injected via the tail vein with vectors based on AAV2 or novel serotypes AAV7 and AAV8 encoding human apoE3 driven by a liver-specific promoter. In contrast to the first-generation AAV2 vector, apoE levels of mice injected with chimeric AAV2/7 and AAV2/8 vectors reached 2-fold greater than normal human plasma levels by week 4 and maintained therapeutic levels up to 1 year. Cholesterol levels of AAV2/7-apoE and AAV2/8-apoE-treated mice were reduced to normal murine wild-type levels and were maintained for 1 year. At termination after 1 year, extensive atherosclerosis was present in the thoracic aortas and aortic roots of control AAV2/8-lacZ and AAV2-apoE-injected mice, but was completely prevented in both the AAV2/7 and AAV2/8-apoE-treated mice.
Conclusion- We demonstrate that intravenous administration of AAV2/7- and AAV2/8-apoE vectors effectively mediated robust and sustained hepatic-specific expression of apoE and completely prevented atherosclerosis at 1 year.
Intravenous administration of AAV2/7- and AAV2/8-apoE vectors effectively mediated robust and sustained hepatic-specific expression of apoE in apoE-deficient mice, resulting in complete prevention of atherosclerosis at 1 year.
【关键词】 adenoassociated virus apolipoprotein E atherosclerosis gene therapy
Introduction
Human apolipoprotein E (apoE) is a 34-kDa multifunctional protein that plays a key role in lipoprotein metabolism 1 and inhibits the development of atherosclerosis. 2 Deficiency of apoE in mice results in hypercholesterolemia and atherosclerosis on a chow diet. 3-6 In our previous studies, somatic gene transfer and expression of apoE in apoE-deficient mice using a second generation adenoviral vector acutely reduced plasma cholesterol 7 and regressed atherosclerosis in apoE deficient mice. 8 In similar studies using low-density lipoprotein (LDL) receptor-deficient mice, apoE overexpression resulted in reduced lesion development 9 and regression of advanced atherosclerosis 10 without reducing cholesterol levels. 9,10 However, these studies did not resolve the important question whether hepatic-specific expression of apoE would be sufficient to completely prevent atherosclerosis in apoE-deficient mice over a long period of time. Vectors that permit longer-term stable hepatic expression of apoE at adequate concentrations are needed to test this hypothesis.
Adeno-associated virus (AAV) is a small, single-stranded DNA, nonenveloped, human parvovirus that has been of interest as a somatic gene transfer vector. 11 Recombinant AAV (rAAV) is attractive for gene transfer, because it does not contain sequences encoding viral proteins and because it has the potential to provide stable transgene expression. 12 The liver is one important target for rAAV-mediated somatic gene transfer. However, first-generation AAV vectors such as AAV2 have generally resulted in low levels of transgene expression, in part because the proportion of hepatocytes stably transduced with AAV2 vectors is only about 5%. 13 This has limited the use of first generation AAV vectors for in vivo gene transfer to genes such as coagulation factor IX (F.IX) that provide therapeutic correction of a deficiency phenotype at very low levels of expression. 14
Gene transfer vectors based on novel AAV serotypes 7 15-17 and 8 15-23 were more recently reported. AAV2/7 and AAV2/8 are chimeric vectors that have the inverted terminal repeats of AAV2 and are packaged with the capsid from AAV7 or AAV8. A comparative study of AAV serotypes delivered intraportally showed that AAV2/8 vectors achieved the highest levels of hepatic transgene expression and ranged from 16- to 110-times greater than that of AAV2; gene transfer from AAV2/7 was intermediate. 15 Intraportal injection of LDL receptor (LDLr)-deficient mice using AAV2/7 and AAV2/8 encoding the human LDLr driven by the liver-specific thyroxine binding globulin (TBG) promoter 24 resulted in sustained LDLr protein expression for at least 20 weeks, nearly complete normalization of lipids, and significantly reduced atherosclerosis. 16 In another instance, transgene expression from intraportally injected AAV2/8 was shown to persist for 1 year. 22 Although the majority of AAV liver expression studies in mice have used intraportal injections, some recent studies in mice demonstrated that intravenous injection via the tail vein achieved significant physiological effects. 18,23 The present study demonstrates that the intravenous injection of AAV7- and AAV8-based vectors results in the hepatic-specific stable expression of apoE in apoE-deficient mice and complete prevention of atherosclerosis after 1 year.
Materials and Methods
Vector Construction
The cis-plasmid pAAV.TBG.hapoE3 contains the human apoE3 cDNA insert followed by SV-40 poly A tail, and is driven by the liver-specific human TBG promoter. 24 This entire region is flanked by 2 AAV2 inverted terminal repeats, which serve as origins of DNA replication and the packaging signal ( Figure 1 ). AAV2/2-apoE3 was produced by triple transfection into 293 cells with an adenovirus helper plasmid and a packaging vector expressing both rep and cap of AAV2. The pseudotyped AAV2/7-apoE3 and AAV2/8-apoE3 were produced similarly using a chimeric packaging construct in which the AAV2 rep gene is fused with the cap gene of serotype 7 or 8. The control vector consisted of the LacZ gene packaged into 2/8 serotype. Genome copy was determined by TaqMan (Applied Biosystems, Foster City, Calif) analysis.
Figure 1. Gene cassette for human apoE3 (A) and LacZ (B). Human apoE3 and LacZ cDNAs were inserted into the pAAV vector driven by the liver-specific human thyroxine binding globulin (TBG) promoter/enhancer fused to a chimeric intron. The cDNA sequences are followed by an SV40 poly A signal and the entire cassette is flanked by inverted terminal repeats of AAV2.
Animals
Female apoE -/- mice on a C57Bl/6 background were purchased from Jackson Labs (Bar Harbor, Me). For virus injection, 2- to 3-month-old mice were slowly infused with 1 x 10 12 genome copies of AAV vectors in phosphate-buffered saline intravenously (tail vein) with a 27-gauge needle. Blood samples were collected at several time points thereafter under anesthesia with isoflurane (Vedeco Inc, St. Joseph, Mo). Mice were euthanized 1 year after injection at which time liver, heart, and aorta were harvested.
Plasma Analysis
Blood samples were collected by retroorbital venous plexus puncture with heparinized capillary tubes (Fisher Scientific, Pittsburgh, Pa). Plasma was separated by low-speed centrifugation. Cholesterol was measured enzymatically on a Cobas Fara II autoanalyzer (Roche Diagnosistic Systems Inc, Nutley, NJ) using Wako Chemicals (Richmond, Va) reagents. Human apoE concentrations in plasma were determined by immunoturbidometric assay using reagents from Wako Chemicals run on the Cobas Fara II autoanalyzer (Roche).
Fast Protein Liquid Chromatography (FPLC) Fractionation of Plasma Lipoproteins
A pool of 120 µL of plasma from AAV2/2-apoE, AAV2/7-apoE, AAV2/8-apoE, or AAV2/8-LacZ (control) injected mice was diluted 2-fold with FPLC column buffer (1 mmol/L EDTA, 154 mmol/L NaCl, pH 8.0); 200 µL of diluted sample was run on a Superose 6 column (Amersham Biosciences Corp, Piscataway, NJ) at a flow rate of 0.4 mL/min and 0.5 mL fractions were collected. The cholesterol content of each fraction was analyzed using an enzymatic microplate assay (Wako Chemicals).
Immunoblotting of Human ApoE in Liver
Liver lysates were homogenized using a Sonifier S-450 (Branson Ultrasonics Corp, Danbury, Conn). Total protein was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, Ill), 25 and 30 µg of pooled protein was used for immunoblotting. The liver lysate was resolved on NuPAGE 4% to 12% Bis-Tris SDS-PAGE (Invitrogen, Carlsbad, Calif) and transferred to ECL Hybond membrane (Amersham). The apoE was detected using a commercial anti-human apoE primary antibody (Wako Chemicals) and horseradish peroxidase-conjugated anti-goat IgG.
Analysis of Human ApoE mRNA Expression by Real-Time Reverse-Transcription Polymerase Chain Reaction
Total RNA from 20 mg of liver was isolated using an RNeasy Mini kit (Qiagen, Valencia, Calif). For reverse transcription (RT), 300 ng of total RNA was used with SuperScript First-Strand Synthesis System (Invitrogen). Each polymerase chain reaction (PCR) reaction contained 0.5 ng of cDNA, 10 µL of 2 x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif), and 900 nM of optimized human apoE-specific primer pair. Primer sequences were 5'-ACC CAG GAA CTG AGG GC-3' (forward) and 5'-CTC CTT GGA CAG CCG TG-3' (reverse). 26 Real-time quantitative PCR was performed using an ABI PRISM 7300 Sequence Detection System (Applied Biosystems) by using the default thermal cycling conditions (10 minutes at 95°C and then 40 cycles of 15 seconds at 95°C plus 1 minute at 60°C). Relative quantitation was performed using the comparative cycle threshold method, as recommended by the manufacturer. All quantitations are normalized to an endogenous control using the 18S rRNA (Applied Biosystems). All samples were run in triplicate.
Atherosclerotic Lesion Analysis
Methods for the quantification of atherosclerotic lesions were the same as previously reported by Tangirala et al 27 In brief, the upper half of the heart containing the aortic root was embedded in OCT and frozen at -80°C. The remainder of the aortas were removed by cutting off minor branching arteries and fixed in 10% buffered formalin phosphate. Aortas were cut open longitudinally, stained with Sudan IV (Sigma Aldrich, St. Louis, Mo). Serial 8-µm-thick cryosections from the aortic sinus to aortic arch were mounted on masked slides and stained with Oil-red O or trichrome (Sigma Aldrich) and counterstained hematoxylin (Sigma Aldrich). Atherosclerosis in the aortas and aortic root sections were quantified using Image Pro Plus version 5.0.1 image analysis software (MediaCybernetics Inc, Silver Spring, Md).
Statistics
Data are presented as means±SEM. Student t test was used to compare the mean values between 2 groups, respectively. Statistical significance for all comparisons was assigned at P <0.05.
Results
Expression of Human ApoE
Plasma human apoE concentrations were determined at multiple time points through 1 year ( Table, A). Plasma levels of apoE approached normal human plasma levels as early as day 14 after injection in the AAV2/7-apoE and AAV2/8-apoE-injected mice, whereas there were undetectable plasma apoE levels in AAV2/2-apoE-treated mice. The peak of expression was at day 28 in the AAV2/7-apoE (87±5 µg/mL) and AAV2/8-apoE-treated mice (97±17 µg/mL). Expression was relatively stable through 128 days, after which apoE levels declined to <30 µg/mL but remained easily measurable. Hepatic apoE was detected by Western blot of liver homogenates ( Figure 2 A) and quantitative PCR of liver cDNA ( Figure 2 B) obtained 1 year after injection. AAV2/7- and AAV2/8-apoE-treated mice had readily detectable hepatic apoE protein, whereas AAV2/2-apoE-treated mice had none. AAV2/7- and AAV2/8-apoE-treated mice also had 370-fold increased apoE mRNA in liver compared with AAV2/2-apoE-treated mice.
Human ApoE Levels and Total Cholesterol Levels in Plasma
Figure 2. Human apoE immunoblot of mouse liver lysate. A, 30 µg of pooled mouse liver lysate protein from each experimental group was loaded. The livers of AAV2/7- and AAV2/8-apoE-injected mice had higher human apoE protein expression than that of AAV2/2-apoE and AAV2/8-LacZ groups. Human apoE mRNA levels of mouse liver. B, 300 ng of liver mRNA from each mouse was used for quantitative PCR. AAV2/7- and AAV2/8-apoE-injected mice had 370-fold expression levels over AAV2/2-apoE.
Plasma Cholesterol and Lipoprotein Reduction
The mean baseline total cholesterol of the normal chow-fed apoE -/- mice before AAV injection was 765±25 mg/dL. Total cholesterol levels were reduced to normal (59±3 mg/dL) by day 14 after intravenous injection in AAV2/7-apoE-injected mice and AAV2/8-apoE-injected mice, whereas AAV2/2-apoE-injected mice had no reduction in cholesterol ( Table, B). Even at the 1 year time point, total cholesterol levels in AAV2/7-apoE and AAV2/8-apoE-injected mice remained normal. The very-low-density lipoprotein and intermediate density lipoprotein/LDL fractions in AAV2/7-apoE and AAV2/8-apoE-injected mice decreased to virtually undetectable levels by day 42 ( Figure 3 A) and remained low through 1 year ( Figure 3 B).
Figure 3. Distribution of cholesterol in female apoE -/- mice, 42 days (A) and 360 days (B) after virus injection with AAV2/8-LacZ ( or diamonds), AAV2/2-apoE ( or squares), AAV2/7-apoE ( or closed triangles), AAV2/8-apoE (X or crosses). Very-low-density lipoprotein and intermediate density lipoprotein/LDL fractions in AAV2/7-apoE and AAV2/8-apoE-injected mice dropped dramatically by day 14 after injection (data not shown) and remained low through day 42 (A) and day 360 (B).
Prevention of Atherosclerosis
Atherosclerosis was quantified using 2 independent methods 1 year after injection. Formalin-fixed thoracic aortas were stained with Sudan IV ( Figure 4 A) and aortic root sections were stained with Oil-red O ( Figure 4 B) and trichrome ( Figure 4 C). The percentage of thoracic aorta covered by lesion in AAV2/8-LacZ-treated mice was 50.4% ± 4.4% and in AAV2/2-apoE-treated mice was 31.5% ± 3.3%. In contrast, the thoracic aorta in AAV2/7-apoE and AAV2/8-apoE-treated mice had absolutely no detectable atherosclerosis ( Figure 5 A). Mean aortic root lesion area in AAV2/8-LacZ-treated mice was 6.1±0.4 µm 2 x 10 5 per section and in AAV2/2-apoE-treated mice was 4.0±1.6 µm 2 x 10 5 per section. In contrast, AAV2/7-apoE and AAV2/8-apoE-treated mice had no atherosclerosis in the aortic root ( Figure 5 B). These data show that atherosclerosis in AAV2/7-apoE and AAV2/8-apoE-treated mice was completely prevented at 1 year after vector injection.
Figure 4. Aortic atherosclerosis 1 year after AAV injection. A, Thoracic aortas were stained with Sudan IV which stains neutral lipid red (arrows); (a) AAV2/8-LacZ and (b) AAV2/2-apoE-treated mice exhibited lesion, whereas (c) AAV2/7-apoE-treated and (d) AAV2/8-apoE-treated mice had no atherosclerosis. B, Aortic roots were stained red with Oil-red-O which stains neutral lipid red (arrows); (a) AAV2/8-LacZ and (b) AAV2/2-apoE showed lesion, whereas (c) AAV2/7-apoE-treated and (d) AAV2/8-apoE-treated mice had none. C, Aortic roots were stained with trichrome which stains collagen blue (arrows); (c) AAV2/7-apoE-treated and (d) AAV2/8-apoE-treated mice showed no atherosclerosis.
Figure 5. Quantification of atherosclerosis in mouse aorta. A, Mouse thoracic aorta. AAV2/7 and AAV2/8-apoE-treated mice showed no lesion; (a) AAV2/8-LacZ: 50.0% ± 4.4% of lesion, (b) AAV2/2-apoE: 31.5% ± 3.3% of lesion, (c) AAV2/7-apoE: 0.0% ± 0.0% of lesion, (d) AAV8-apoE: 0.0±0.0% of lesion. B, Mouse aortic root. AAV2/7- and AAV2/8-apoE -treated mice exhibited no lesion; (a) AAV2/8-LacZ: 6.1±0.4 (µm 2 x 10 5 / section), (b) AAV2/2-apoE: 4.0±1.6 (µm 2 x 10 5 / section), (c) AAV2/7-apoE: 0.0±0.0 (µm 2 x 10 5 per section), (d) AAV2/8-apoE: 0.0±0.0 (µm 2 x 10 5 / section).
Discussion
In this study, we demonstrate that a single tail vein injection of AAV7- and AAV8-based vectors encoding human apoE3 driven by the liver-specific TBG promoter resulted in robust and sustained hepatic expression of apoE and complete prevention of atherosclerosis at 1 year. The present study is the first report of the use of somatic gene transfer and hepatic expression of apoE to completely prevent long-term atherosclerosis in apoE-deficient mice. The AAV7- and AAV8-based vectors were dramatically more effective than the first-generation AAV2-based vector, which failed to generate detectable levels of apoE or reduce atherosclerosis compared with control AAV2/8-LacZ-injected mice. Our results are among the first to demonstrate that AAV7- and AAV8-based vectors can be used to achieve stable hepatic expression of genes at levels exceeding normal endogenous levels with simple tail vein injection and thus fully correct a deficiency phenotype with severe consequences such as the marked atherosclerosis associated with apoE deficiency. These results also prove that atherosclerosis can be completely prevented in apoE-deficient mice by liver-specific apoE expression alone.
It has been recognized for some time that liver-directed gene transfer is an excellent tool for the investigation of lipoprotein metabolism in animal models 7,8,28 and a potential strategy for a therapeutic approach to severe genetic dyslipidemias in humans. 9,28 Indeed, recombinant adenoviral vectors have been used extensively to probe aspects of lipoprotein physiology in animals. 29 Although they generate high levels of expression, they have the major disadvantages of transient expression and induction of an acute inflammatory response. 30,31 Helper-dependent "gutted" adenoviral vectors provide longer-term expression 32 but are difficult to prepare and still have the potential to induce immune/inflammatory responses through the viral capsid proteins. 33 AAV-based vectors have been of interest with regard to liver-directed gene transfer because of a reduced immune response and potential for long-term stable expression. Indeed, first-generation AAV-based vectors have been used to express F.IX in the liver and stably correct F.IX deficiency in mice and dogs 34 and have even been used in a clinical trial for liver-directed gene transfer of F.IX. 35 However, first generation AAV vectors generate only low levels of hepatic expression, which while adequate for correction of a disorder such as F.IX deficiency (which requires 5% of normal levels), 36 is inadequate for many other situations, such as expression of high-abundance apolipoproteins.
The discovery of second generation AAV serotypes such as AAV7 and AAV8 isolated from rhesus monkeys 15 has enabled the testing of the hypothesis that recombinant vectors based on these serotypes would be more efficient in transducing liver and therefore permit higher levels of transgene expression than first generation AAV vectors. Indeed, an initial study using rhesus monkey choriogonadotropic hormone (CG) as a reporter suggested that this is the case, 15 and a few subsequent studies using other transgenes have confirmed these results. 16-18 Here we extend these findings in several ways. First, we used AAV7- and AAV8-based vectors to achieve hepatic-specific expression of apoE in apoE-deficient mice, entirely correcting the hypercholesterolemia and completely preventing the development of atherosclerosis for up to 1 year. Second, we directly demonstrated that the first generation vector AAV2 was markedly inferior to AAV2/7 and AAV2/8. 15-18 Third, in contrast to the other reports, 15,16,18 we find no major difference between AAV2/7 and AAV2/8 with regard to efficiency of liver-directed gene transfer, expression of apoE, and physiological effect. Finally, in contrast to the numerous reports that used intraportal administration of AAV vectors, we show that simple tail vein administration is adequate for robust transgene expression using these vectors. 23
The apoE levels peaked 28 days after injection at levels 2-fold higher than normal human levels. They then declined gradually to about normal human levels by day 128 and to 25% of normal levels by 1 year. Plasma cholesterol levels remained completely normalized even at 1 year, consistent with previous studies that have suggested that 2% to 5% of normal plasma apoE is adequate to normalize plasma lipids. 8 The reason for the decline in apoE levels over time in our studies is unknown, but could be related to an immune response to the human apoE as a foreign protein. Indeed, we were able to detect antibodies to human apoE in the plasma as early as day 42 (data not shown).
In summary, somatic gene transfer and liver-specific expression of apoE in apoE -/- mice using AAV2/7 and AAV2/8 vectors resulted in sustained expression of apoE, reduction of plasma cholesterol, and complete prevention of the development of atherosclerosis. These data indicate that hepatic expression of apoE is sufficient to prevent atherosclerosis and suggest that AAV2/7 and 2/8 vectors are promising tools for achieving high-level stable hepatic expression of genes, necessary conditions for many applications in the field of lipid disorders and atherosclerosis.
Acknowledgments
We are indebted to Anthony Secreto, Aisha Wilson, Anna DiFlorio, and Linda Morrell for excellent technical assistance and Dr Jane Glick and for helpful discussions.
Sources of Funding
This study was supported by P01 HL59407 from the National Heart Lung and Blood Institute. D.J.R. is a recipient of a Clinical-Scientist Award in Translational Research from the Burroughs Wellcome Fund.
Disclosures
None.
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作者单位：From Institution for Translational Medicine and Therapeutics (K.K., G.C.M., D.H.M., D.J.R.) and Gene Therapy Program, Division of Medical Genetics (G.G., J.M.W.), Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pa.