CN105916990B - Adeno-associated virus vector for transduction of adipose tissue - Google Patents
Adeno-associated virus vector for transduction of adipose tissue Download PDFInfo
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- CN105916990B CN105916990B CN201380080085.2A CN201380080085A CN105916990B CN 105916990 B CN105916990 B CN 105916990B CN 201380080085 A CN201380080085 A CN 201380080085A CN 105916990 B CN105916990 B CN 105916990B
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Abstract
本发明涉及用于转导脂肪组织的腺相关病毒载体。本发明还涉及用于产生这种腺相关病毒载体的多核苷酸、质粒、载体和方法。本发明还涉及用于需要调节基因表达水平的疾病的治疗的方法。The present invention relates to adeno-associated viral vectors for transduction of adipose tissue. The present invention also relates to polynucleotides, plasmids, vectors and methods for producing such adeno-associated viral vectors. The present invention also relates to methods for the treatment of diseases requiring modulation of gene expression levels.
Description
技术领域technical field
本发明涉及用于转导脂肪组织的腺相关病毒(AAV)载体。该载体可以以组织特异性的方式转导白色或褐色脂肪组织。The present invention relates to adeno-associated virus (AAV) vectors for transduction of adipose tissue. This vector can transduce white or brown adipose tissue in a tissue-specific manner.
背景技术Background technique
脂肪组织,除了其作为脂肪库以及能量平衡的调节器的已知作用,近来已被认为是主要的新陈代谢和内分泌器官。已经提出,减损的白色脂肪组织(WAT)功能,以及降低的褐色脂肪组织(BAT)活性或BAT质量,是肥胖症(肥胖,obseity)发展的主要因素。在这方面,已经所描述了人类的脂肪细胞和WAT功能障碍。此外,还报告了BAT活性和体重指数(BMI)之间的逆相关。参见Yee J等,Lipids Health Dis.2012,11:19-30、Ichimura A等,Nature2012;483:350-354、Mazzatti D等,Arch.Physiol.Biochem.2012;118(3):112-120、CypessA等,N.Engl.J.Med.2009;360:1509-1517和van Marken Lichtenbelt W等,N.Engl.J.Med.2009;360:1500-1508。Adipose tissue, in addition to its known role as a fat depot and regulator of energy homeostasis, has recently been recognized as a major metabolic and endocrine organ. Impaired white adipose tissue (WAT) function, as well as reduced brown adipose tissue (BAT) activity or BAT mass, have been proposed to be major factors in the development of obesity. In this regard, adipocyte and WAT dysfunction in humans has been described. In addition, an inverse correlation between BAT activity and body mass index (BMI) was also reported. See Yee J et al, Lipids Health Dis. 2012, 11:19-30, Ichimura A et al, Nature 2012;483:350-354, Mazzatti D et al, Arch.Physiol.Biochem.2012;118(3):112-120, Cypess A et al, N. Engl. J. Med. 2009; 360: 1509-1517 and van Marken Lichtenbelt W et al, N. Engl. J. Med. 2009; 360: 1500-1508.
肥胖症的发病在过去几十年之中已经显著增加以致达到流行病的比例。据估计超过5亿人是肥胖的。肥胖症是现今主要的公共卫生问题。参见IASO,“Global Prevalence ofAdult Obesity,Report IOTF 2008”(IASO,London,GB,2009)。肥胖症自身增加死亡的风险并且与胰岛素耐受性和2型糖尿病长期强烈相关。参见Peeters A等,Ann.Intern.Med.2003;138:24-32和Moller D等,N.Engl.J.Med.1991;325:938-948。此外,脂肪细胞的功能障碍和肥胖症对于特定类型的癌症和许多其他严重的疾病,如心脏病、免疫功能障碍、高血压、关节炎和神经退行性疾病而言也是显著的风险因素。见Roberts D等,Annu.Rev.Med.2010;61:301-316、Spiegelman B等,J.Biol.Chem.1993;268(10):6823-6826和Whitmer R等,Curr.Alzheimer Res.2007;4(2):117-122。The incidence of obesity has increased significantly over the past few decades to reach epidemic proportions. It is estimated that more than 500 million people are obese. Obesity is a major public health problem today. See IASO, "Global Prevalence of Adult Obesity, Report IOTF 2008" (IASO, London, GB, 2009). Obesity itself increases the risk of death and is strongly associated with insulin resistance and
饮食和锻炼是肥胖症的主要治疗方法,但是越来越多的患者还需要药物治疗干预以降低并保持体重。然而,药物治疗不会引发非自愿的或显著的体重减轻,并且此外,抗肥胖药物经常由于它们的全身作用表现出严重的副作用。因此,存在对于预防并对抗当前的肥胖症流行的新的和安全的途径的急切的需要。在这方面,阐明肥胖症基础的病理事件对于开发新的抗肥胖症疗法是至关紧要的。候选基因在体内基因转移至白色和褐色脂肪组织可以提供巨大的潜力以了解肥胖症的发病和发作之下的分子机制。此外,目标为脂肪细胞的基因治疗途径可以为未来的肥胖症及其相关的失调的治疗,同时最小化全身的反应开创新的机会。然而,迄今为止转移至白色和褐色脂肪组织的有效的和特定的基因仍然难以确定。Diet and exercise are the main treatments for obesity, but an increasing number of patients also require pharmacological interventions to lose and maintain weight. However, drug treatment does not trigger involuntary or significant weight loss, and in addition, anti-obesity drugs often exhibit severe side effects due to their systemic effects. Therefore, there is an urgent need for new and safe ways to prevent and combat the current obesity epidemic. In this regard, elucidating the pathological events underlying obesity is critical for the development of new anti-obesity therapies. In vivo gene transfer of candidate genes to white and brown adipose tissue may offer great potential to understand the molecular mechanisms underlying the pathogenesis and onset of obesity. Furthermore, gene therapy approaches targeting adipocytes could open up new opportunities for future treatment of obesity and its associated disorders while minimizing systemic responses. However, efficient and specific genes for transfer to white and brown adipose tissue have so far remained elusive.
最近,当血清型1的AAV(AAV1)与非离子表面活性剂或雷公藤红素结合时,示出其适度感染的小鼠体内的WAT。见Mizukami H等,Hum.Gene Ther.2006;17:921-928和Zhang F等,Gene Ther.2011;18:128-134。已经报告其他AAV血清型,如AAV6、AAV7、AAV8或AAV9是高度感染性的,但是它们的脂肪转导效率是不清楚的。参见Gao G等,Proc.Natl.Acad.Sci.USA 2002;99:11854-11859、Nakai H等,J.Virol.2005;79:214-224、Pacak C等,Circ.Res.2006;99:e3-e9、Broekman M等,Neuroscience 2006;138:501-510、Wang Z等,Diabetes 2006;55:875-884、Taymans J等,Hum.Gene Ther.2007;18:195-206、Bish L等,Hum.Gene Ther.2008;19:1359-1368和Lebherz C等,J.Gene Med.2008;10:375-382。因此,在本领域中存在对于开发使脂肪组织特定转导,以及,此外使特定类型的脂肪细胞转导的载体的需要。Recently, AAV of serotype 1 (AAV1) was shown to show WAT in moderately infected mice when it was combined with a nonionic surfactant or triptolide. See Mizukami H et al, Hum. Gene Ther. 2006;17:921-928 and Zhang F et al, Gene Ther. 2011;18:128-134. Other AAV serotypes, such as AAV6, AAV7, AAV8 or AAV9, have been reported to be highly infectious, but their adipotransduction efficiency is unclear. See Gao G et al, Proc. Natl. Acad. Sci. USA 2002; 99: 11854-11859, Nakai H et al, J. Virol. 2005; 79: 214-224, Pacak C et al, Circ. Res. 2006; 99: e3-e9, Broekman M et al. Neuroscience 2006; 138:501-510, Wang Z et al. Diabetes 2006; 55:875-884, Taymans J et al. Hum. Gene Ther. 2007; 18:195-206, Bish L et al. , Hum. Gene Ther. 2008; 19: 1359-1368 and Lebherz C et al, J. Gene Med. 2008; 10: 375-382. Accordingly, there is a need in the art to develop vectors that specifically transduce adipose tissue, and, in addition, specific types of adipocytes.
发明内容SUMMARY OF THE INVENTION
本发明涉及使感兴趣的多核苷酸转移至特定类型的脂肪细胞并且使它们表达的腺相关病毒载体(AAV)。本发明的脂肪组织特异调控元件的使用限制感兴趣的多核苷酸在白色脂肪组织或褐色脂肪组织中的表达。此外,已经证明本发明的载体对于脂肪组织相关的疾病,如,例如2型糖尿病的治疗是有用的。本发明的发明方面在权利要求中公开。The present invention relates to adeno-associated viral vectors (AAV) that transfer polynucleotides of interest to specific types of adipocytes and express them. The use of adipose tissue-specific regulatory elements of the present invention limits the expression of polynucleotides of interest in white or brown adipose tissue. Furthermore, the vectors of the present invention have proven useful for the treatment of adipose tissue-related diseases such as, for example,
微生物保藏Microorganism preservation
质粒pAAV-mini/aP2-null和pAAV-mini/UCP1-null在2012年6月8日分别以登录号DSM 26057和DSM 26058保藏于DSMZ(Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH),Inhoffenstraβe 7 B,D-38124 Braunschweig,德意志联邦共和国。Plasmids pAAV-mini/aP2-null and pAAV-mini/UCP1-null were deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) on June 8, 2012 under accession numbers DSM 26057 and DSM 26058, respectively, Inhoffenstraβe 7 B, D- 38124 Braunschweig, Federal Republic of Germany.
附图说明Description of drawings
图1.通过AAV的eWAT内部给予的白色脂肪细胞的转导。A-B.在用血清型1、2、4和5(A)的AAV-CAG-GFP与普兰尼克F88(Pluronics F88)一起或不与普兰尼克F88(PluronicsF88)一起处理eWAT的部分和用血清型6、7、8和9(B)的AAV-CAG-GFP处理eWAT的部分中针对绿色荧光蛋白(GFP,绿色)的免疫染色。蓝色,细胞核。箭头表示转导的脂肪细胞。初始放大倍数100×(B,左侧图片)和200×(A;B,右侧图片)。C.用血清型1、6、7、8、或9的AAV-CAG-GFP处理的eWAT中的GFP含量(n=5每组)。示出的值是平均值±SEM。RLU,相对光单位(relativelight unit)。D.接收AAV8-CMV-LacZ的eWAT的全部X-gal着色。X-gal着色分布贯穿整个转导的eWAT。相反地,在来自用生理盐水溶液处理的动物的eWAT中没有检测到X-gal着色。E.由用AAV9-CMV-mHKII或AAV0-CMV-null载体处理的动物的eWAT获得的分离细胞中的相对mHKII表达水平。F.由来自用AAV9-CMV-null和AAV-CMV-mHKII注射的小鼠的分离的脂肪细胞摄取的基础的和胰岛素刺激的2[1-3H]脱氧-D-葡萄糖。脂肪细胞获得自至少5小鼠/组。G.在2×1011vg的AAV8或AAV9-CAG-GFP载体的iWAT内部给予两个星期之后在腹股沟白色脂肪组织(iWAT)部分中的针对GFP(棕色)的免疫染色。初始放大倍数×100。H:注射2×1011vg的AAV8或AAV9-CAG-GFP后两个星期iWAT中的GFP表达水平(n=6)。示出的值是平均值±SEM。*p<0.05、**p<0.01并且***p<0.001;在相同的胰岛素浓度下相对于AAV9-CMV-null#p<0.05。所有的分析是在2×1011vg/eWAT的eWAT内部给予之后两个星期进行的。Figure 1. Transduction of white adipocytes by internal administration of eWAT of AAV. AB. Sections of eWAT treated with AAV-CAG-GFP of
图2.通过mini/aP2调控区的AAV的eWAT内部给予之后白色脂肪细胞的特异性转导。A.在接收1012vg/eWAT的AAV8和AAV9-mini/aP2-GFP载体的eWAT中针对GFP(棕色)的免疫染色。分析是在注射后两个星期进行的。初始放大倍数×400。B.循环hSeAP水平。将4×1012vg/鼠的剂量的AAV9-mini/aP2-hSeAP载体双侧注射入eWAT并在注射后的几个时间点进行循环hSeAP水平的测量。RLU,相对光单位。示出的值是平均值±SEM。n=3(生理盐水)以及n=4(AAV9-mini/aP2-SeAP)。C.4×1012vg/鼠的AAV9-mini/aP2-hSeAP的eWAT内部给予一年后,肝脏和eWAT中的相对hSeAP表达水平。D.在AAV9-mini/aP2-null和AAV9-mini/aP2-mHKII载体的eWAT内部给予(1.4×1012vg/鼠)两个星期后评估由eWAT、iBAT和心脏的体内2-[1-3H]脱氧-D-葡萄糖摄取。示出的值是平均值±SEM。n=7只小鼠每组。*p<0.05。Figure 2. Specific transduction of white adipocytes following eWAT internal administration of AAV via the mini/aP2 regulatory region. A. Immunostaining for GFP (brown) in eWAT receiving 10 12 vg/eWAT of AAV8 and AAV9-mini/aP2-GFP vectors. Analysis was performed two weeks after injection. Initial magnification × 400. B. Circulating hSeAP levels. A dose of 4 x 1012 vg/mouse of AAV9-mini/aP2-hSeAP vector was bilaterally injected into eWAT and measurements of circulating hSeAP levels were performed at several time points after injection. RLU, relative light unit. Values shown are mean ± SEM. n=3 (saline) and n=4 (AAV9-mini/aP2-SeAP). C. Relative hSeAP expression levels in liver and eWAT after one year intra-eWAT administration of AAV9-mini/aP2-hSeAP of 4×10 12 vg/mouse. D. Assessment of in vivo 2-[1-2-[1- by eWAT, iBAT and heart after two weeks of intra-eWAT administration of AAV9-mini/aP2-null and AAV9-mini/aP2-mHKII vectors (1.4×10 12 vg/mouse) 3 H]deoxy-D-glucose uptake. Values shown are mean ± SEM. n=7 mice per group. *p<0.05.
图3.通过AAV的iBAT内部给予的棕色脂肪细胞的转导。A.在用2×109vg/鼠的AAV8或AAV9-CAG-GFP处理的iBAT的部分中,针对GFP(棕色)的免疫染色。初始放大倍数×200和×400(小图)。B.接收2×109vg/鼠的AAV8或AAV9-CAG-GFP的iBAT中的相对GFP表达水平。示出的值是平均值±SEM。n=5只小鼠每组。*p<0.05。C.接收2×1010vg/鼠的AAV4或AAV8-CMV-RFP或2×1010vg/鼠的AAV1、AAV2、AAV5、AAV6、AAV7、AAV8、或AAV9-CMV-RFP的iBAT中的相对红色荧光蛋白(RFP)表达水平。转导模式与eWAT相似。参见图1。AAV6、AAV7、AAV8和AAV9是用于转导iBAT最高效的血清型。D.在用1011vg/鼠的AAV9-CMV-RFP处理的iBAT的部分中针对RFP(棕色)的免疫染色。初始放大倍数×200和×400(小图)。分析是在给予AAV后两个星期进行的。Figure 3. Transduction of brown adipocytes given internally by iBAT of AAV. A. Immunostaining for GFP (brown) in sections of iBAT treated with 2 x 109 vg/mouse of AAV8 or AAV9-CAG-GFP. Initial magnifications ×200 and ×400 (panel). B. Relative GFP expression levels in iBAT receiving 2 x 109 vg/mouse of AAV8 or AAV9-CAG-GFP. Values shown are mean ± SEM. n=5 mice per group. *p<0.05. C. Relative in iBAT receiving 2 x 10 10 vg/mouse of AAV4 or AAV8-CMV-RFP or 2 x 10 10 vg/mouse of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, or AAV9-CMV-RFP Red fluorescent protein (RFP) expression levels. The transduction pattern is similar to eWAT. See Figure 1. AAV6, AAV7, AAV8 and AAV9 are the most efficient serotypes for transduction of iBAT. D. Immunostaining for RFP (brown) in sections of iBAT treated with 10 11 vg/mouse of AAV9-CMV-RFP. Initial magnifications ×200 and ×400 (panel). Analysis was performed two weeks after AAV administration.
图4.通过mini/UCP1调控区的AAV的iBAT内部给予之后棕色脂肪细胞的特异性转导。A.通过注射2×1011vg/鼠的AAV8或AAV9-mini/UCP1-GFP载体后两个星期针对GFP(棕色)的免疫染色评估棕色脂肪细胞的转导。初始放大倍数×200和×400(小图)。B.在给予AAV8-mini/UCP1-null和AAV8-mini/UCP1-mHKII载体(7×1010vg/鼠)之后两个星期由iBAT、eWAT和心脏摄取的体内2-[1-3H]脱氧-D-葡萄糖。n=6(AAV8-mini/UCP1-mHKII)以及n=10(AAV8-mini/UCP1-null)只小鼠每组。C-E.递送2×1011vg/鼠的AAV9-mini/UCP1-mVEGF164或AAV9-mini/UCP1-null载体之后两个星期,iBAT中的相对mVEGF164(C)、总mVEGF(D)和mPECAM1(E)表达水平。n=5只小鼠每组。F.注射2×1011vg/鼠的AAV9-mini/UCP1-mVEGF164或AAV9-mini/UCP1-null载体后两个星期,iBAT中针对α-SMA(棕色)的免疫染色。初始放大倍数×400。示出的值是平均值+SEM。*p<0.05。红色箭头指示毛细结构。Figure 4. Specific transduction of brown adipocytes following iBAT internal administration of AAV via the mini/UCP1 regulatory region. A. Transduction of brown adipocytes was assessed by immunostaining for GFP (brown) two weeks after injection of 2×10 11 vg/mouse of AAV8 or AAV9-mini/UCP1-GFP vector. Initial magnifications ×200 and ×400 (panel). B. In vivo 2-[ 1-3H ] uptake by iBAT, eWAT and heart two weeks after administration of AAV8-mini/UCP1-null and AAV8-mini/UCP1-mHKII vectors (7×10 10 vg/mouse) Deoxy-D-glucose. n=6 (AAV8-mini/UCP1-mHKII) and n=10 (AAV8-mini/UCP1-null) mice per group. CE. Relative mVEGF164 (C), total mVEGF (D) and mPECAM1 in iBAT two weeks after delivery of 2 x 10 11 vg/mouse of AAV9-mini/UCP1- mVEGF164 or AAV9-mini/UCP1-null vector (E) Expression levels. n=5 mice per group. F. Immunostaining in iBAT for α-SMA (brown) two weeks after injection of 2×10 11 vg/mouse of AAV9-mini/UCP1-mVEGF 164 or AAV9-mini/UCP1-null vector. Initial magnification × 400. Values shown are mean + SEM. *p<0.05. Red arrows indicate capillary structures.
图5.通过AAV载体全身递送至瘦鼠的白色和棕色脂肪细胞的转导。A.eWAT部分中针对GFP(绿色)的免疫染色。蓝色,细胞核。初始放大倍数×100(左侧图片)和×200(右侧图片)。B.eWAT中的相对GFP表达水平。C.eWAT中的GFP含量。D.iBAT部分中针对GFP(棕色)的免疫染色。初始放大倍数×200和×400(小图)。E.IBAT中的相对GFP表达水平。F.iBAT中的GFP含量。G-H.将载体静脉(iv)给予至ICR小鼠(G)和C57Bl6小鼠(H)(ICR:对AAV8n=3并且对于AAV9n=5;C57Bl6:n=4)之后,腹股沟(iWAT)、腹膜后(rWAT)、肠系膜(mWAT)、eWAT和iBAT中的相对GFP表达水平。所有的分析是在5×1012vg/鼠的AAV8或AAV9-CAG-GFP载体的尾部静脉给予之后两个星期进行的。AU,任意单位。RLU,相对光单位。Figure 5. Transduction of white and brown adipocytes by systemic delivery of AAV vectors to lean mice. A. Immunostaining for GFP (green) in the eWAT section. Blue, nucleus. Initial magnification ×100 (left image) and ×200 (right image). B. Relative GFP expression levels in eWAT. C. GFP content in eWAT. D. Immunostaining for GFP (brown) in the iBAT section. Initial magnifications ×200 and ×400 (panel). E. Relative GFP expression levels in IBAT. F. GFP content in iBAT. GH. After intravenous (iv) administration of vector to ICR mice (G) and C57B16 mice (H) (ICR: n=3 for AAV8 and n=5 for AAV9; C57B16: n=4), inguinal (iWAT), peritoneal Relative GFP expression levels in posterior (rWAT), mesentery (mWAT), eWAT and iBAT. All analyses were performed two weeks after tail vein administration of 5 x 1012 vg/mouse of AAV8 or AAV9-CAG-GFP vectors. AU, arbitrary unit. RLU, relative light unit.
图6.通过全身给予mini/UCP1调控区的AAV之后,棕色脂肪细胞的特异性转导。A.在接收AAV8或AAV9-mini/UCP1-GFP载体的iBAT中,针对GFP(棕色)的免疫染色。初始放大倍数×200和×400(小图)。B-C.在注射两个月后用2×1012vg小鼠的AAV9-mini/UCP1-mVEGF164或AAV9-mini/UCP1-null处理的iBAT中的相对mVEGF164(B)和总mVEGF(C)表达水平。D-E.在静脉给予8×1012vg的AAV9-mini/UCP1-VEGF164或AAV9-mini/UCP1-null(n=5)一个月后,iBAT中的VEGF164(B)和PECAM1(C)表达水平。F-G.在与B-C相同的组中针对CD105(棕色)(D)和α-SMA(棕色)(E)的免疫染色。红色箭头指示血管。初始放大倍数×400和×1000(小图)。示出的值是平均值+SEM。n=5只小鼠每组,*p<0.05。Figure 6. Specific transduction of brown adipocytes following systemic administration of AAV in the regulatory region of mini/UCP1. A. Immunostaining for GFP (brown) in iBAT receiving AAV8 or AAV9-mini/UCP1-GFP vectors. Initial magnifications ×200 and ×400 (panel). BC. Relative mVEGF 164 (B) and total mVEGF (C) in iBAT treated with AAV9-mini/UCP1-mVEGF 164 or AAV9-mini/UCP1-null from 2×10 12 vg mice two months after injection The expression level. DE. VEGF164 (B) and PECAM1 (C) expression levels in iBAT one month after intravenous administration of 8 x 10 12 vg of AAV9-mini/UCP1-VEGF 164 or AAV9-mini/UCP1-null (n=5) . FG. Immunostaining for CD105 (brown) (D) and α-SMA (brown) (E) in the same panel as BC. Red arrows indicate blood vessels. Initial magnifications ×400 and ×1000 (panel). Values shown are mean + SEM. n=5 mice per group, *p<0.05.
图7.AAV的全身给予之后通过mini/aP2调控区和脂肪限制的转基因表达的脂肪细胞的转导。A.在来自接收AAV8或AAV9-mini/aP2-GFP载体的动物的iBAT部分中,通过针对GFP(棕色)的免疫染色评估棕色脂肪细胞的转导。初始放大倍数×200和×400(小图)。B-C.注射两个星期后通过针对GFP(棕色)的免疫染色评估非脂肪组织的转导。GFP表达在用AAV8或AAV9-mini/aP2-GFP(B)和AAV8或AAV9-mini/UCP1-GFP(C)处理的动物的肝脏中是最小的并且不存在于心脏中。初始放大倍数×100和×400(小图)。所有的分析是在2×1012vg/鼠的全身注射两个星期后进行的。Figure 7. Transduction of adipocytes expressing via mini/aP2 regulatory region and fat-restricted transgenes following systemic administration of AAV. A. Transduction of brown adipocytes was assessed by immunostaining for GFP (brown) in iBAT fractions from animals receiving AAV8 or AAV9-mini/aP2-GFP vectors. Initial magnifications ×200 and ×400 (panel). BC. Transduction of non-adipose tissue was assessed by immunostaining for GFP (brown) two weeks after injection. GFP expression was minimal in the liver of animals treated with AAV8 or AAV9-mini/aP2-GFP (B) and AAV8 or AAV9-mini/UCP1-GFP (C) and absent in the heart. Initial magnifications x 100 and x 400 (panel). All analyses were performed two weeks after systemic injection of 2 x 1012 vg/mouse.
图8.AAV的eWAT内部给予后,脱靶(off-target)转基因表达。A.来自接收AAV9-CAG-GFP的动物的iBAT中的针对GFP(棕色)的免疫染色。初始放大倍数×200和×400(小图)。B.在来自用AAV9-CAG-GFP处理的动物的iBAT和eWAT中的相对GFP表达水平。示出的值是平均值±SEM。n=5只小鼠每组。*p<0.05。C.通过针对GFP(绿色)的免疫染色评估非动物脂肪器官的转导。GFP表达在来自用AAV7、AAV8、或AAV9注射的动物的心脏和肝脏中是明显的。初始放大倍数×100。在AAV-CAG-GFP载体(4×1011vg小鼠)的eWAT内部给予两个星期后进行分析。Figure 8. Off-target transgene expression following eWAT internal administration of AAV. A. Immunostaining for GFP (brown) in iBAT from animals receiving AAV9-CAG-GFP. Initial magnifications ×200 and ×400 (panel). B. Relative GFP expression levels in iBAT and eWAT from animals treated with AAV9-CAG-GFP. Values shown are mean ± SEM. n=5 mice per group. *p<0.05. C. Transduction of non-animal fat organs was assessed by immunostaining for GFP (green). GFP expression was evident in hearts and livers from animals injected with AAV7, AAV8, or AAV9. Initial magnification × 100. Analysis was performed two weeks after intra-eWAT administration of the AAV-CAG-GFP vector ( 4 x 1011 vg mice).
图9.通过mini/aP2调控区的脂肪组织限制的AAV-介导的转基因表达。在1012vg的AAV8和AAV9-mini/aP2-GFP载体的局部eWAT内部给予两个星期后,在肝脏和心脏中通过针对GFP的免疫染色没有检测到GFP表达。初始放大倍数×100。Figure 9. AAV-mediated transgene expression restricted by adipose tissue through the mini/aP2 regulatory region. Two weeks after local eWAT administration of 10 12 vg of AAV8 and AAV9-mini/aP2-GFP vectors, no GFP expression was detected in liver and heart by immunostaining for GFP. Initial magnification × 100.
图10.通过AAV载体的iBAT内部给予的非动物脂肪器官的转导。注射两个星期后通过针对GFP(棕色)的免疫染色评估非脂肪器官的转导。在来自用2×109vg/鼠的AAV8和AAV9-CAG-GFP载体iBAT内部注射的动物的心脏和肝脏中GFP的表达是明显的。初始放大倍数×200。Figure 10. Transduction of non-animal adipose organs by internal administration of iBAT of AAV vectors. Transduction of non-adipose organs was assessed by immunostaining for GFP (brown) two weeks after injection. Expression of GFP was evident in the heart and liver from animals injected internally with 2 x 109 vg/mouse of AAV8 and AAV9-CAG-GFP vector iBAT. Initial magnification × 200.
图11.AAV的iBAT内部给予之后借助通过mini/aP2调控区和脂肪限制的转基因表达的棕色脂肪细胞的转导。A.2×1011vg/鼠的AAV9-mini/aP2-GFP载体的iBAT内部递送两个星期后通过针对GFP的免疫染色(棕色)评估棕色脂肪细胞的转导。初始放大倍数×200和×400(小图)。B.将AAV8或AAV9-mini/UCP1-GFP载体(2×1011vg/鼠)局部注射iBAT两个星期后通过针对GFP(棕色)的免疫染色评估非脂肪器官的转导。肝脏中的GFP表达较少。初始放大倍数×100和×400(小图)。Figure 11. Transduction of brown adipocytes by transgene expression via mini/aP2 regulatory region and fat restriction following internal administration of iBAT of AAV. A. Transduction of brown adipocytes was assessed by immunostaining (brown) for GFP two weeks after intra-iBAT delivery of AAV9-mini/aP2-GFP vector at 2 x 1011 vg/mouse. Initial magnifications ×200 and ×400 (panel). B. Transduction of non-adipose organs was assessed by immunostaining for GFP (brown) two weeks after local injection of AAV8 or AAV9-mini/UCP1-GFP vector ( 2 x 1011 vg/mouse) into iBAT. There is less GFP expression in the liver. Initial magnifications x 100 and x 400 (panel).
图12.AAV的尾部静脉递送之后广泛的转基因表达。通过尾部静脉注射5×1012vg/鼠的AAV9-CAG-GFP载体两个星期后,针对GFP(绿色)的免疫染色显示出肝脏、心脏、骨骼肌、睾丸和肾脏的转导。蓝色,细胞核。初始放大倍数×200。Figure 12. Extensive transgene expression following tail vein delivery of AAV. Immunostaining for GFP (green) showed transduction of liver, heart, skeletal muscle, testis and kidney two weeks after 5 x 1012 vg/mouse injection of AAV9-CAG-GFP vector via tail vein. Blue, nucleus. Initial magnification × 200.
图13.AAV载体全身给予至肥胖-糖尿病小鼠之后WAT和BAT的转导。A-B.将3×1012vg的AAV8或AAV9-CAG-GFP载体静脉给予至ob/ob(A)和db/db小鼠(B)之后附睾白色脂肪组织(eWAT)、腹股沟白色脂肪组织(iWAT)和肩胛间褐色脂肪组织(iBAT)部分中针对GFP(棕色)的免疫染色。初始放大倍数×200。(ob/ob:n=4;db/db:n=4)。C-D.在来自ob/ob(C)和db/db小鼠(D)的相同组的腹股沟(iWAT)、腹膜后(rWAT)、肠系膜(mWAT)、eWAT和iBAT库中的GFP表达。AU,任意单位。所有的分析是在载体递送两个星期后进行的。示出的值是平均值±SEM。相对于AAV9*p<0.05。Figure 13. Transduction of WAT and BAT following systemic administration of AAV vectors to obese-diabetic mice. AB. Epididymal white adipose tissue (eWAT), inguinal white adipose tissue (iWAT) after intravenous administration of 3×10 12 vg of AAV8 or AAV9-CAG-GFP vector to ob/ob (A) and db/db mice (B) ) and immunostaining for GFP (brown) in interscapular brown adipose tissue (iBAT) fractions. Initial magnification × 200. (ob/ob: n=4; db/db: n=4). CD. GFP expression in inguinal (iWAT), retroperitoneal (rWAT), mesenteric (mWAT), eWAT and iBAT pools from the same group of ob/ob (C) and db/db mice (D). AU, arbitrary unit. All analyses were performed two weeks after vector delivery. Values shown are mean ± SEM. vs. AAV9*p<0.05.
图14.用mirT序列的肝脏和心脏的高效脂肪细胞转导和转基因表达的去靶(脱靶,de-targeting)。静脉注射1012vg的AAV9-CAG-GFP、AAV9-CAG-GFP-miRT122、AAV9-CAG-GFP-miRT1或AAV9-CAG-GFP-双miRT载体两个星期后附睾白色脂肪组织(eWAT)、腹股沟白色脂肪组织(iWAT)、肩胛间褐色脂肪组织(iBAT)、肝脏和心脏中的GFP免疫染色。初始放大倍数×100(肝脏和心脏)、×200(eWAT和iWAT)和×400(iBAT和小图)。Figure 14. Efficient adipocyte transduction and de-targeting of transgene expression in liver and heart with mirT sequences. Epididymal white adipose tissue ( eWAT ), inguinal white adipose tissue (eWAT), groin GFP immunostaining in white adipose tissue (iWAT), interscapular brown adipose tissue (iBAT), liver and heart. Initial magnifications x 100 (liver and heart), x 200 (eWAT and iWAT) and x 400 (iBAT and panels).
具体实施方式Detailed ways
本发明公开了当局部给予时,能够高效介导基因转移至WAT或BAT的基于AAV6、AAV7、AAV8和AAV9血清型的腺相关病毒载体。参见图1B和1C和图3A-3D。这些载体的全身给予还导致至WAT和BAT二者中高效的基因递送。参见图5A和5D。虽然由AAV8和AAV9载体介导的基因递送是高效的,其不局限于脂肪组织。本发明公开了AAV8和AAV9载体与脂肪组织特异启动子区的组合使脂肪组织中感兴趣的多核苷酸特定表达。特别地,AAV8或AAV9的局部给予包含表达框(表达盒,expression cassette),其中,在mini/aP2调控区控制下的异源基因(例如GFP)导致其在WAT中的表达而没有在肝脏或心脏中的表达。参见图2A和图9。此外,AAV8或AAV9的局部给予包含表达框,其中,在mini/UCP1调控区控制下的异源基因(例如GFP)导致其在BAT中表达而没有在心脏中表达以及仅较少的肝脏表达。参见图4A和11B。因此,通过本发明的载体和启动子形成的组合的局部给予提供了用于治疗多种疾病的基于体内脂肪细胞的转导的安全机制。The present invention discloses adeno-associated virus vectors based on AAV6, AAV7, AAV8 and AAV9 serotypes that can efficiently mediate gene transfer to WAT or BAT when administered locally. See Figures IB and 1C and Figures 3A-3D. Systemic administration of these vectors also resulted in efficient gene delivery into both WAT and BAT. See Figures 5A and 5D. Although gene delivery mediated by AAV8 and AAV9 vectors is efficient, it is not limited to adipose tissue. The present invention discloses that the combination of AAV8 and AAV9 vectors and adipose tissue-specific promoter regions enables specific expression of polynucleotides of interest in adipose tissue. In particular, local administration of AAV8 or AAV9 comprises an expression cassette in which a heterologous gene (eg, GFP) under the control of the mini/aP2 regulatory region results in its expression in WAT but not in liver or expression in the heart. See Figure 2A and Figure 9. In addition, local administration of AAV8 or AAV9 contained an expression cassette in which a heterologous gene (eg, GFP) under the control of the mini/UCP1 regulatory region resulted in its expression in BAT but not in the heart and only to a lesser extent in the liver. See Figures 4A and 11B. Thus, local administration by the combination of the vector and promoter of the present invention provides a safe mechanism for in vivo adipocyte-based transduction for the treatment of various diseases.
此外,本发明的组合的全身给予构成了用于脂肪细胞的转导的替代途径。在这方面,本发明公开了AAV8或AAV9-mini/UCP1和AAV9或AAV9-mini/aP2组合的全身给予对于分别转导BAT和WAT是高效的。参见图6A和7A。无论使用哪两种调控区,本发明的组合的全身给予导致脂肪组织中感兴趣的多核苷酸的高度受限的表达,而没有心脏中的表达且肝脏中仅较少的表达。参见图7B和图7C。Furthermore, systemic administration of the combinations of the present invention constitutes an alternative route for transduction of adipocytes. In this regard, the present invention discloses that systemic administration of AAV8 or AAV9-mini/UCP1 in combination with AAV9 or AAV9-mini/aP2 is highly efficient for transduction of BAT and WAT, respectively. See Figures 6A and 7A. Regardless of which two regulatory regions were used, systemic administration of the combinations of the invention resulted in highly restricted expression of the polynucleotide of interest in adipose tissue, with no expression in the heart and only less expression in the liver. See Figures 7B and 7C.
1.通用术语和表达的定义1. Definitions of common terms and expressions
如在本文中可互换地使用的术语“腺相关病毒”、“AAV病毒”、“AAV病毒粒子”、“AAV病毒颗粒”和“AAV颗粒”是指由至少一种AAV衣壳蛋白(优选地全部由特定AAV血清型的衣壳蛋白)和衣壳化的多核苷酸AAV基因组组成的病毒颗粒。如果该颗粒包含由AAV末端反向重复序列侧接的异源多核苷酸(即非野生型AAV基因组的多核苷酸,如转基因递送至哺乳动物细胞),其通常称作“AAV载体颗粒”或“AAV载体”。AAV是指属于依赖病毒属细小病毒科的病毒。AAV基因组为大约4.7千碱基长,并且由可以是正或负向的单链脱氧核糖核酸(ssDNA)组成。该基因组在DNA链两端包含末端反向重复序列(ITR),和两种开放阅读框(ORF):rep和cap。rep框架由四个编码AAV生命周期所需的Rep蛋白质的重叠基因形成。cap框架含有衣壳蛋白的重叠核苷酸序列:VP1、VP2和VP3,它们共同交互以形成二十面体对称的衣壳。参见Carter B,Adeno-associated virus and adeno-associated virus vectors for genedelivery、Lassic D等,Eds.,“Gene Therapy:Therapeutic Mechanisms and Strategies”(Marcel Dekker,Inc.,New York,NY,US,2000)和Gao G等,J.Virol.2004;78(12):6381-6388。As used interchangeably herein, the terms "adeno-associated virus", "AAV virus", "AAV virion", "AAV virion" and "AAV particle" refer to proteins composed of at least one AAV capsid protein (preferably A virus particle consisting entirely of the capsid protein of a specific AAV serotype) and an encapsidated polynucleotide AAV genome. If the particle comprises a heterologous polynucleotide flanked by AAV terminal inverted repeats (ie, a polynucleotide that is not a wild-type AAV genome, eg, for delivery of a transgene to mammalian cells), it is commonly referred to as an "AAV vector particle" or "AAV vector". AAV refers to a virus belonging to the Parvoviridae family of the genus Dependovirus. The AAV genome is approximately 4.7 kilobases long and consists of single-stranded deoxyribonucleic acid (ssDNA) that can be positive or negative. The genome contains inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The rep framework is formed by four overlapping genes encoding Rep proteins required for the AAV life cycle. The cap framework contains overlapping nucleotide sequences of capsid proteins: VP1, VP2, and VP3, which interact together to form an icosahedral symmetric capsid. See Carter B, Adeno-associated virus and adeno-associated virus vectors for genedelivery, Lassic D et al., Eds., "Gene Therapy: Therapeutic Mechanisms and Strategies" (Marcel Dekker, Inc., New York, NY, US, 2000) and Gao G et al, J. Virol. 2004;78(12):6381-6388.
如本文中使用的术语“腺相关病毒ITR”或“AAV ITR”是指存在于腺相关病毒的基因组的DNA链两端的末端反向重复序列。ITR序列是AAV基因组的高效增殖所需的。这些序列的另一特性是它们形成发夹的能力。此特性有助于其自身引导(self-priming),这允许不依靠引发酶的第二DNA链的合成。还示出ITR对于野生型AAV DNA整合至宿主细胞基因组(即人类的第19染色体)并从其中拯救,以及对于与产生的完全组装的,耐脱氧核糖核酸酶的AAV颗粒结合的AAV DNA的高效衣壳化是必需的。The term "Adeno-Associated Virus ITR" or "AAV ITR" as used herein refers to inverted terminal repeats present at both ends of the DNA strand of the genome of an Adeno-Associated Virus. ITR sequences are required for efficient proliferation of the AAV genome. Another property of these sequences is their ability to form hairpins. This property facilitates its self-priming, which allows the synthesis of a second DNA strand independent of priming enzymes. Also shown is the high efficiency of ITR for integration and rescue of wild-type AAV DNA into the host cell genome (i.e., chromosome 19 in humans), and for AAV DNA bound to the fully assembled, DNase-resistant AAV particles produced Encapsidation is required.
如在本文中使用的术语“AAV2”是指具有在GenBank登录号NC001401中限定的具有基因组序列的腺相关病毒的血清型。The term "AAV2" as used herein refers to the serotype of adeno-associated virus having a genomic sequence as defined in GenBank Accession No. NC001401.
如本文中使用的术语“AAV载体”进一步指包含一种或多种由AAV末端重复序列(ITR)侧接的感兴趣的多核苷酸(或转基因)的载体。当这类AAV载体存在于宿主细胞时,它们可以复制并包装为感染性病毒颗粒,该宿主细胞已经用可以编码并表达rep和cap基因产物(即AAV Rep和Cap蛋白质)的载体转染,并且其中,宿主细胞已经用编码和表达来自腺病毒开放阅读框E4orf6的蛋白质的载体转染。当AAV载体结合至更大的多核苷酸中(例如,染色体或另一载体,如用于克隆或转染的质粒)时,则该AAV载体通常称作“蛋白-载体(pro-vector)”。该蛋白-载体可以通过在AAV包装功能和由E4orf6提供的必要的辅助功能存在下的复制和衣壳化来获得“拯救”。The term "AAV vector" as used herein further refers to a vector comprising one or more polynucleotides (or transgenes) of interest flanked by AAV terminal repeats (ITRs). Such AAV vectors can replicate and package into infectious viral particles when present in host cells that have been transfected with vectors that encode and express the rep and cap gene products (ie, AAV Rep and Cap proteins), and Wherein, the host cell has been transfected with a vector encoding and expressing a protein from the adenovirus open reading frame E4orf6. When an AAV vector is incorporated into a larger polynucleotide (eg, a chromosome or another vector, such as a plasmid used for cloning or transfection), the AAV vector is often referred to as a "pro-vector" . This protein-vector can be "rescue" by replication and encapsidation in the presence of AAV packaging functions and the necessary helper functions provided by E4orf6.
如本文中使用的术语“脂肪组织”是指由成熟的脂肪细胞(即脂细胞(fat cell))以及小血管、神经组织、淋巴结和基质血管成分(SVF)的组合组成的组织。SVF是由内皮细胞、成纤维细胞、脂肪细胞前体细胞(即前脂肪细胞)和免疫细胞,如巨噬细胞和T细胞组成的。在哺乳动物中,两种不同类型的脂肪组织通常区分为:白色脂肪组织(WAT)和褐色脂肪组织(BAT)。脂肪组织主要作用为以脂肪的形式储存能量、通过非战栗产热产生热量以及分泌脂肪因子。The term "adipose tissue" as used herein refers to tissue composed of mature adipocytes (ie, fat cells) and a combination of small blood vessels, neural tissue, lymph nodes, and stromal vascular components (SVF). SVF is composed of endothelial cells, fibroblasts, adipocyte precursor cells (i.e. preadipocytes) and immune cells such as macrophages and T cells. In mammals, two different types of adipose tissue are commonly distinguished: white adipose tissue (WAT) and brown adipose tissue (BAT). The main functions of adipose tissue are to store energy in the form of fat, to generate heat through non-shudder thermogenesis, and to secrete adipokines.
如本文中使用的术语“脂肪组织细胞”或“脂肪细胞”是指包含脂肪组织,并且特异化为作为脂肪储存能量,或通过非战栗产热产生热量,并且分泌脂肪因子的细胞类型。脂肪组织细胞包括白色脂肪细胞和褐色脂肪细胞。The term "adipose tissue cell" or "adipocyte" as used herein refers to a cell type that comprises adipose tissue and is specialized to store energy as fat, or generate heat by non-shudder thermogenesis, and secrete adipokines. Adipose tissue cells include white adipocytes and brown adipocytes.
如本文中使用的术语“脂肪组织特异转录调控区”是指用作启动子(即调节可操作地与启动子连接的选择的核酸序列的表达)的核酸序列,并且其影响选择的核酸序列在特定组织细胞,如脂肪细胞中的表达。脂肪组织特异转录调控区可以是组成性的或可诱导的。The term "adipose tissue-specific transcriptional regulatory region" as used herein refers to a nucleic acid sequence that acts as a promoter (ie, regulates the expression of a selected nucleic acid sequence operably linked to the promoter) and which affects the selected nucleic acid sequence in Expression in specific tissue cells, such as adipocytes. Adipose tissue-specific transcriptional regulatory regions can be constitutive or inducible.
如本文使用的术语“碱性磷酸酶”或“AP”是指催化来自许多类型的分子,包括核苷酸、蛋白质和生物碱的磷酸酯基团的水解的酶(EC 3.1.3.1)。The term "alkaline phosphatase" or "AP" as used herein refers to enzymes that catalyze the hydrolysis of phosphate groups from many types of molecules, including nucleotides, proteins and alkaloids (EC 3.1.3.1).
如本文中使用的术语“血管发生(angiogenesis)”是指由其他预先存在的血管形成新的血管的过程,并且包括血管生成(vasculogenesis)和动脉生成(arteriogenesis)。The term "angiogenesis" as used herein refers to the process of forming new blood vessels from other pre-existing blood vessels, and includes vasculogenesis and arteriogenesis.
如本文中使用的术语“动脉生成”是指具有平滑肌介质层的血管的形成、生长或发育。The term "arteriogenesis" as used herein refers to the formation, growth or development of blood vessels with layers of smooth muscle media.
如本文中使用的术语“褐色脂肪组织细胞”或“褐色脂肪细胞”是指多边形的脂肪细胞的类型,并且其特征在于将脂质积聚为多个较小的“多腔”液滴,以及它们在细胞质内大量的具有层状脊堆叠的大型线粒体。不同于白色脂肪细胞,这些细胞具有相当多的细胞质。细胞核是圆形的,并且虽然其位置偏离中心,但不在细胞的外周。棕色脂肪细胞的众多线粒体和棕色脂肪库丰富的血管分布是BAT为棕色颜色的主要原因。棕色脂肪细胞位于典型的BAT库中并且负责通过非战栗产热产生热量。参见Enerback S,N.Engl.J.Med.2009;360:2021-2023。The term "brown adipose tissue cell" or "brown adipocyte" as used herein refers to the type of fat cell that is polygonal and characterized by the accumulation of lipids into multiple smaller "multi-lumen" droplets, and their Numerous large mitochondria with lamellar ridges packed within the cytoplasm. Unlike white adipocytes, these cells have considerable cytoplasm. The nucleus is round, and although its location is off-center, it is not at the periphery of the cell. The numerous mitochondria of brown adipocytes and the rich vascularity of brown fat depots are the main reasons for the brown color of BAT. Brown adipocytes are located in the typical BAT pool and are responsible for heat production through non-shudder thermogenesis. See Enerback S, N. Engl. J. Med. 2009;360:2021-2023.
如本文中使用的术语“CAG调控区”是指由巨细胞病毒的早期增强子元件和鸡β-肌动蛋白启动子形成的组合。参见Alexopoulou A等,BMC Cell Biology 2008;9(2):1-11。The term "CAG regulatory region" as used herein refers to the combination formed by the early enhancer element of cytomegalovirus and the chicken beta-actin promoter. See Alexopoulou A et al, BMC Cell Biology 2008;9(2):1-11.
如本文中使用的术语“cap基因”或“AAV cap基因”是指编码Cap蛋白的基因。如本文中使用的术语“Cap蛋白”是指具有至少一种天然AAV Cap蛋白(例如,VP1、VP2、VP3)的功能活性的多肽。Cap蛋白(例如,VP1、VP2、VP3)的功能活性的实例包括诱导衣壳的形成的能力、促进单链DNA的积聚、促进AAV DNA包装进入衣壳(即衣壳化)、结合至细胞受体并促进病毒粒子进入宿主。The term "cap gene" or "AAV cap gene" as used herein refers to the gene encoding the Cap protein. The term "Cap protein" as used herein refers to a polypeptide having the functional activity of at least one native AAV Cap protein (eg, VP1, VP2, VP3). Examples of functional activities of Cap proteins (eg, VP1, VP2, VP3) include the ability to induce capsid formation, promote the accumulation of single-stranded DNA, promote AAV DNA packaging into the capsid (ie, encapsidation), bind to cellular receptors body and facilitate the entry of virions into the host.
如在本文中使用的术语“衣壳”是指其中包装病毒基因组的结构。衣壳由多种通过蛋白形成的低聚构造亚单元构成。例如,AAV具有通过三种衣壳蛋白:VP1、VP2、VP3的交互形成的二十面体衣壳。The term "capsid" as used herein refers to the structure in which the viral genome is packaged. The capsid is composed of various oligomeric structural subunits formed by proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins: VP1, VP2, VP3.
如在本文中使用的术语“细胞成分”是指包含本发明的脂肪细胞和至少另一种组分的材料复合物。该成分可以配制为单个制剂或可能呈现为各个组分的单独制剂,其可以结合为联合制剂用于联合使用。该组合物可以是其中每种组分单独配制并包装的套装试剂盒(kit-of-parts)。The term "cellular component" as used herein refers to a composite of material comprising the adipocytes of the present invention and at least one other component. The ingredients may be formulated as a single formulation or may be presented as separate formulations of the individual components, which may be combined into a combined formulation for combined use. The compositions may be kit-of-parts in which each component is formulated and packaged separately.
如本文中使用的术语“组成型启动子”是指其活性在生物体的所有细胞中,或在大部分的发育阶段过程中维持在相对恒定水平的启动子,以及与细胞环境条件关联较少或不相关。The term "constitutive promoter" as used herein refers to a promoter whose activity is maintained at relatively constant levels in all cells of an organism, or during most developmental stages, and is less dependent on cellular environmental conditions or irrelevant.
如本文中使用的术语“增强子”是指转录因子结合至其中以增加基因转录的DNA序列元件。The term "enhancer" as used herein refers to a DNA sequence element into which a transcription factor binds to increase transcription of a gene.
如本文中使用的术语“表达框”是指用一系列特化的核酸元件重组地或合成地生成的核酸构结构,其允许特定的核酸在靶细胞中转录。The term "expression cassette" as used herein refers to a nucleic acid construct, recombinantly or synthetically produced with a series of specialized nucleic acid elements, which allows transcription of a particular nucleic acid in a target cell.
如在本文中使用的术语“提供辅助功能的基因”是指编码进行这样的功能的多肽的基因,AAV依赖于该功能(即“辅助功能”)进行复制。辅助功能包括对于AAV复制,包括,但不限于涉及AAV基因转录的激活、阶段特异性AAV mRNA剪接、AAV DNA复制、cap表达产物的合成和AAV衣壳组装那些部分所必需的那些功能。基于病毒的附属功能可以源自任何已知的辅助病毒,如腺病毒、疱疹病毒(而非1型单纯疱疹病毒)和牛痘病毒。辅助功能包括但不限于,腺病毒El、E2a、VA和E4或疱疹病毒UL5、UL8、UL52和UL29以及疱疹病毒聚合酶。The term "gene that provides a helper function" as used herein refers to a gene encoding a polypeptide that performs the function on which AAV depends (ie, "helper function") for replication. Auxiliary functions include those necessary for AAV replication, including, but not limited to, those involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions can be derived from any known helper virus, such as adenovirus, herpes virus (rather than herpes simplex virus type 1), and vaccinia virus. Helper functions include, but are not limited to, adenovirus E1, E2a, VA and E4 or herpesvirus UL5, UL8, UL52 and UL29 and herpesvirus polymerase.
如本文中使用的术语“己糖激酶”或“HK”是指催化己糖的磷酸化以形成磷酸己糖的酶。在大部分生物体中,葡萄糖是HK的主要底物,并且葡萄糖-6-磷酸酯是最重要的产物。The term "hexokinase" or "HK" as used herein refers to an enzyme that catalyzes the phosphorylation of hexose sugars to form hexose phosphates. In most organisms, glucose is the major substrate for HK, and glucose-6-phosphate is the most important product.
如本文中使用的术语“高血压(high blood pressure)”或“高动脉压(arterialhypertension)”是指其中动脉的血压升高的医学状况。血压包括两种测量,收缩和舒张,其取决于心肌在搏动之间是否收缩(心脏收缩)或放松(心脏舒张)。静止时的正常血压在100-140mmHg收缩(最高读数)以及60-90mmHg舒张(最低读数)的范围内。如果其持续位于140/90mmHg或以上,则称为存在高血压症。高血压(Hypertension)分类为原发性(自发性)高血压和继发性高血压;约90-95%的情况归类为“原发性高血压”,这是指没有明显的潜在医学起因的高血压症。其余5-10%的情况(即继发性高血压)是由其他影响肾脏、动脉、心脏或内分泌系统的条件引起的。肥胖症中常见的胰岛素耐受性也认为促进了高血压。高血压是中风、心肌梗塞(即心脏病发作)、心力衰竭、动脉的动脉瘤(例如,主动脉瘤)、外周动脉疾病的主要风险因素并且是慢性肾病的起因。即使中度的动脉血压升高也与预期寿命的缩短相关。The term "high blood pressure" or "arterial hypertension" as used herein refers to a medical condition in which the blood pressure of the arteries is elevated. Blood pressure includes two measurements, systole and diastole, which depend on whether the heart muscle contracts (systole) or relaxes (diastole) between beats. Normal blood pressure at rest is in the range of 100-140 mmHg systolic (highest reading) and 60-90 mmHg diastolic (lowest reading). Hypertension is said to be present if it is consistently at or above 140/90mmHg. Hypertension is classified as primary (spontaneous) hypertension and secondary hypertension; approximately 90-95% of cases are classified as "essential hypertension", which means there is no apparent underlying medical cause of hypertension. The remaining 5-10% of cases (ie, secondary hypertension) are caused by other conditions affecting the kidneys, arteries, heart, or endocrine system. Insulin resistance, which is common in obesity, is also thought to contribute to high blood pressure. Hypertension is a major risk factor for stroke, myocardial infarction (ie, heart attack), heart failure, arterial aneurysm (eg, aortic aneurysm), peripheral arterial disease and a cause of chronic kidney disease. Even moderate increases in arterial blood pressure are associated with reduced life expectancy.
如本文中使用的术语“高血糖”是指与空腹基准水平相比,其中出现异常的高血糖水平的状态。特别地,高血糖理解为当空腹血液葡萄糖水平始终高于126mg/dL,餐后葡萄糖水平高于140mg/dL时,或给予每公斤体重1.75克的剂量的葡萄糖2小时之后静脉血浆中的葡萄糖水平超过200mg/dL时发生。The term "hyperglycemia" as used herein refers to a state in which abnormally high blood sugar levels occur compared to fasting baseline levels. In particular, hyperglycemia is understood as the glucose level in
如本文中使用的术语“胰岛素耐受性”是指其中细胞不正确地对胰岛素响应的失调。结果是,身体响应于高血液葡萄糖水平而产生更多的胰岛素。具有胰岛素耐受性的患者经常表现出高葡萄糖水平和高循环胰岛素水平。胰岛素耐受性经常与肥胖症、高血压和高血脂相关。此外,胰岛素耐受性经常出现在具有2型糖尿病的患者中。The term "insulin resistance" as used herein refers to a disorder in which cells respond incorrectly to insulin. As a result, the body produces more insulin in response to high blood glucose levels. Patients with insulin resistance often exhibit high glucose levels and high circulating insulin levels. Insulin resistance is frequently associated with obesity, hypertension and hyperlipidemia. In addition, insulin resistance frequently occurs in patients with
如本文中使用的术语“局部给予”是指将本发明的多核苷酸、载体、多肽、或药物组合物在特定的位点或附近给予至受试者。The term "topical administration" as used herein refers to the administration of a polynucleotide, vector, polypeptide, or pharmaceutical composition of the invention to a subject at or near a specific site.
如本发明中使用的术语“肥胖症”是指由WHO提供的,基于体重指数(BMI)(其是由个人的重量(以kg计)和它们的以米计的高度的平方之间的比率构成)的定义。根据此标准,低于18.5kg/m2的BMI认为是重量不足或瘦,18.5-24.9kg/m2的BMI认为是正常重量25.0-29.9kg/m2的BMI认为是1级超重,30.0-39.0kg/m2的BMI认为是2级超重以及大于或等于40.0kg/m2的BMI认为是病态的肥胖症。可替代地,存在用于定义受试者的肥胖症的程度的其他方法,如在肋部下边缘和骨盆上边缘之间的中点测量的腰部直径(以cm计)、皮肤皱襞的厚度、生物阻抗,其基于瘦肉组织(lean mass)比脂肪组织(fatty mass)更好地传输电流的原理。The term "obesity" as used in the present invention refers to the ratio provided by WHO, based on body mass index (BMI), which is the ratio between an individual's weight (in kg) and their height in meters squared composition) definition. According to this standard, a BMI of less than 18.5kg /m2 is considered underweight or thin, a BMI of 18.5-24.9kg /m2 is considered normal weight, a BMI of 25.0-29.9kg/ m2 is considered to be
如本文中使用的术语“可操作地连接”是指启动子序列相对于感兴趣的多核苷酸的功能关系和位置(例如,启动子或增强子可操作地与编码序列连接,如果其影响该序列的转录)。通常,可操作地连接的启动子与感兴趣的序列邻接。然而,增强子无须邻接感兴趣的序列以控制其表达。The term "operably linked" as used herein refers to the functional relationship and location of a promoter sequence relative to a polynucleotide of interest (eg, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence). Typically, an operably linked promoter is contiguous with the sequence of interest. However, the enhancer need not be adjacent to the sequence of interest to control its expression.
在本文中可互换地使用的术语“药学上可接受的运载体(carrier)”、“药学上可接受的稀释剂”、“药学上可接受的赋形剂”、或“药学上可接受的媒介(vehicle)”是指非毒性的固体、半固体、或液体填料,稀释剂,包封材料,或所有常规类型的制剂辅助剂。药学上可接受的运载体基本上在采用的剂量和浓度下对接受者是非毒性的,并且与制剂的其他成分相容。药学上可接受的运载体的数量和性质取决于期望的给予形式。药学上可接受的运载体是已知的并且可以通过本领域已知的方法制备。参见Faulíi Trillo C,“Tratado deFarmacia Galénica”(Ed.Luzán 5,S.A.,Madrid,ES,1993)和Gennaro A,Ed.,“Remington:The Science and Practice of Pharmacy”20版(Lippincott Williams&Wilkins,Philadelphia,PA,US,2003)。The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable diluent", "pharmaceutically acceptable excipient", or "pharmaceutically acceptable excipient" are used interchangeably herein. "Vehicle" means a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation adjuvant of all conventional types. A pharmaceutically acceptable carrier is substantially non-toxic to recipients at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. The amount and nature of the pharmaceutically acceptable carrier will depend on the desired form of administration. Pharmaceutically acceptable carriers are known and can be prepared by methods known in the art. See Faulíi Trillo C, "Tratado de Farmacia Galénica" (Ed.
如本文中使用的术语“启动子”是指位于多核苷酸序列的上游的作用为控制一种或多种多核苷酸的转录的核酸片段,并且通过存在的用于以下各项的结合位点而被结构识别:DNA依赖的RNA聚合酶的结合位点、转录起始位点和任何其他DNA序列,包括(但不限于)转录因子结合位点、抑制子和激活蛋白结合位点,以及任何其他本领域已知的直接或间接作用以调节来自启动子的转录的量的核苷酸的序列。“组织特异”启动子是指仅在特定类型的分化细胞或组织中具有活性的启动子。The term "promoter" as used herein refers to a nucleic acid segment located upstream of a polynucleotide sequence that functions to control the transcription of one or more polynucleotides, and through the presence of binding sites for and are recognized by structures: DNA-dependent RNA polymerase binding sites, transcription initiation sites, and any other DNA sequences, including (but not limited to) transcription factor binding sites, repressor and activator protein binding sites, and any Other sequences of nucleotides known in the art that act directly or indirectly to regulate the amount of transcription from a promoter. A "tissue-specific" promoter refers to a promoter that is active only in a particular type of differentiated cell or tissue.
如本文中使用的术语“多核苷酸”是指含有脱氧核糖核苷酸或核糖核苷酸的核酸分子,DNA或RNA。核酸可以是双链的、单链的、或含有双链或单链序列二者的部分。术语“多核苷酸”包括,但不限于具有编码与细胞或其中治疗的受试者的内源多核苷酸部分或全部互补的多肽和核酸序列,从而遵循其转录的能力的核酸序列,其产生能够杂交和抑制内源多核苷酸的表达的RNA分子(例如,microRNA、shRNA、siRNA)。The term "polynucleotide" as used herein refers to a nucleic acid molecule, DNA or RNA, containing deoxyribonucleotides or ribonucleotides. Nucleic acids can be double-stranded, single-stranded, or contain portions of both double-stranded or single-stranded sequences. The term "polynucleotide" includes, but is not limited to, nucleic acid sequences that have the ability to encode polypeptides and nucleic acid sequences that are complementary to part or all of an endogenous polynucleotide in a cell or a subject treated therein, thereby following its transcription, resulting in RNA molecules (eg, microRNA, shRNA, siRNA) capable of hybridizing and inhibiting the expression of endogenous polynucleotides.
如本文中使用的术语“转录后调控区”是指促进包含于表达框或得到的基因产物中的含有的序列的表达、稳定、或定位的任意多核苷酸。The term "post-transcriptional regulatory region" as used herein refers to any polynucleotide that facilitates the expression, stabilization, or localization of sequences contained in an expression cassette or resulting gene product.
如本文中使用的术语“预防(prevent)”、“预防的(preventing)”和“预防(prevention)”是指抑制受试者中疾病的开始或降低其发生率。预防可以是彻底的(例如,受试者中完全没有病理细胞)或部分的。预防还指减少对临床状态的易感性。The terms "prevent," "preventing," and "prevention" as used herein refer to inhibiting the onset or reducing the incidence of disease in a subject. Prevention can be complete (eg, complete absence of pathological cells in the subject) or partial. Prevention also refers to reducing susceptibility to clinical conditions.
如本文中使用的术语“重组病毒基因组”是指其中至少一种外源表达框多核苷酸插入至自然产生的AAV基因组中的AAV基因组。The term "recombinant viral genome" as used herein refers to an AAV genome in which at least one exogenous expression cassette polynucleotide is inserted into a naturally occurring AAV genome.
如本文使用的术语“rep基因”或“AAV rep基因”是指编码Rep蛋白的基因。如本文中使用的术语“Rep蛋白”是指具有至少一种天然AAV Rep蛋白(例如Rep 40、52、68、78)的功能活性的多肽。Rep蛋白(例如Rep 40、52、68、78)的“功能活性”是与该蛋白的生理功能相关的任何活性,包括通过识别促进DNA的复制,连接和切口(平切,nicking)DNA复制的AAV原点以及DNA解旋酶活动。另外的功能包括调节来自AAV的(或其他异源的)启动子的转录并将AAV DNA位点特异地整合至宿主染色体中。The term "rep gene" or "AAV rep gene" as used herein refers to the gene encoding the Rep protein. The term "Rep protein" as used herein refers to a polypeptide having the functional activity of at least one native AAV Rep protein (eg
如本文中使用的术语“受试者”是指个体、植物、或动物,如人类、非人类灵长类(例如,黑猩猩和其他类人猿和猴子物种)、家畜(例如,禽类、鱼类、牛、绵羊、猪、山羊和马)、或实验动物(例如,啮齿动物,如小鼠(mice)、大鼠(rat)和豚鼠)。该术语不表示特定的年龄或性别。术语“受试者”包含胚胎和胎儿。The term "subject" as used herein refers to an individual, plant, or animal, such as humans, non-human primates (eg, chimpanzees and other ape and monkey species), livestock (eg, birds, fish, cattle , sheep, pigs, goats, and horses), or experimental animals (eg, rodents such as mice, rats, and guinea pigs). The term does not denote a specific age or gender. The term "subject" includes embryos and fetuses.
如本文中使用的术语“全身给予的”和“全身给予”是指将本发明的多核苷酸、载体、多肽、或药物组合物以非局部的方式给予受试者。本发明的多核苷酸、载体、多肽、或药物组合物的全身给予可以达到受试者的整个身体的多个器官或组织,或可以达到受试者的特定器官或组织。例如,本发明的药物组合物的静脉给予可以产生受试者中多于一个组织或器官的转导。As used herein, the terms "administered systemically" and "administered systemically" refer to the non-local administration of a polynucleotide, vector, polypeptide, or pharmaceutical composition of the invention to a subject. Systemic administration of a polynucleotide, vector, polypeptide, or pharmaceutical composition of the invention can reach multiple organs or tissues throughout the body of a subject, or can reach a specific organ or tissue in a subject. For example, intravenous administration of a pharmaceutical composition of the present invention can result in transduction of more than one tissue or organ in a subject.
如本文中使用的术语“转录调控区”是指能够调节一种或多种基因的表达的核酸片段。本发明的多核苷酸的调控区包括启动子和增强子。The term "transcriptional regulatory region" as used herein refers to a nucleic acid segment capable of regulating the expression of one or more genes. The regulatory regions of the polynucleotides of the present invention include promoters and enhancers.
如本文中使用的术语“转导(transduce)”或“转导(transduction)”是指借其将外来的核苷酸序列通过病毒载体引入细胞的过程。The term "transduce" or "transduction" as used herein refers to the process by which a foreign nucleotide sequence is introduced into a cell by a viral vector.
如本文中使用的术语“转染”是指将DNA引入受体真核细胞。The term "transfection" as used herein refers to the introduction of DNA into recipient eukaryotic cells.
如本文中使用的术语“治疗(treat)”或“治疗(treatment)”是指给予本发明的化合物或组合物以在疾病的临床征象出现之后控制其进展。控制疾病进展理解为是指有利的或期望的临床结果,其包括但不限于:症状的减少、疾病持续时间的减少、病理状态的稳定(特别地,避免另外的恶化)、推迟疾病的进展、改善病理状态并缓解(部分和全部二者)。与如果不应用治疗的预期存活相比,疾病的进展的控制还包括延长存活。The term "treat" or "treatment" as used herein refers to the administration of a compound or composition of the present invention to control the progression of a disease after the appearance of clinical signs. Controlling disease progression is understood to mean a favorable or desired clinical outcome including, but not limited to: reduction of symptoms, reduction of disease duration, stabilization of pathological state (in particular, avoidance of further exacerbations), delay of disease progression, Improvement in pathological status and remission (both partial and total). Control of disease progression also includes prolonging survival as compared to expected survival if no treatment is applied.
如本文中使用的术语“2型糖尿病”是指表征为不适当的血糖水平增加的疾病。糖尿病的慢性高血糖与不同器官的长期损伤、功能障碍和失效相关,同时导致各种并发症,如视网膜病、肾病和周围神经病。2型糖尿病是由外围组织(主要是骨骼肌、脂肪组织和肝脏)的胰岛素耐受性和由于降低的β-细胞团和功能的组合导致的不适当的补偿性胰岛素分泌响应所引起的。除了葡萄糖浓度增加,胰岛素作用欠缺经常导致胆甾醇或甘油三酯水平的增加。The term "
如本文中使用的术语“血管生成”是指源自未分化的或分化中的细胞的血管的形成、生长、发育或增殖。The term "angiogenesis" as used herein refers to the formation, growth, development or proliferation of blood vessels derived from undifferentiated or differentiated cells.
如本文中使用的术语“载体(vector)”是指能够将一种或多种感兴趣的多核苷酸递送,并且可选地表达至宿主细胞的结构。载体的实例包括但不限于:病毒载体、裸DNA或RNA表达载体、质粒、粘粒(cosmid)或噬菌体载体、与阳离子缩合剂(condensing agents)关联的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体和某些真核细胞,如生产细胞(producer cell)。载体可以是稳定的并且可以自身复制。关于可以使用的载体的类型没有限制。载体可以是克隆载体,适用于繁殖和获得结合至多种异源生物体的多核苷酸、基因结构或表达载体。适合的载体包括原核表达载体(例如pUC18、pUC19、Bluescript和它们的衍生物)、mp18、mp19、pBR322、pMB9、CoIEl、pCRl、RP4、噬菌体和穿梭载体(例如,pSA3和pAT28),和基于病毒载体的真核表达载体(例如,腺病毒、腺相关病毒以及逆转录病毒和慢病毒),以及非病毒载体,如pSilencer 4.1-CMV(Life Technologies Corp.,Carslbad,CA,US)、pcDNA3、pcDNA3.1/hyg pHCMV/Zeo、pCR3.1、pEFl/His、pIND/GS、pRc/HCMV2、pSV40/Zeo2、pTRACER-HCMV、pUB6/V5-His、pVAXl、pZeoSV2、pCI、pSVL和pKSV-10、pBPV-1、pML2d和pTDTl。The term "vector" as used herein refers to a structure capable of delivering, and optionally expressing, one or more polynucleotides of interest to a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, encapsulation in lipids DNA or RNA expression vectors in vivo and certain eukaryotic cells, such as producer cells. The vector can be stable and self-replicating. There are no restrictions on the type of carrier that can be used. The vector may be a cloning vector, suitable for propagating and obtaining polynucleotides, genetic constructs or expression vectors that bind to various heterologous organisms. Suitable vectors include prokaryotic expression vectors (eg, pUC18, pUC19, Bluescript and their derivatives), mp18, mp19, pBR322, pMB9, CoIE1, pCR1, RP4, phage and shuttle vectors (eg, pSA3 and pAT28), and virus-based Eukaryotic expression vectors of vectors (eg, adenovirus, adeno-associated virus, and retroviruses and lentiviruses), as well as non-viral vectors, such as pSilencer 4.1-CMV ( Life Technologies Corp., Carslbad, CA, US), pcDNA3, pcDNA3.1/hyg pHCMV/Zeo, pCR3.1, pEFl/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5 -His, pVAX1, pZeoSV2, pCI, pSVL and pKSV-10, pBPV-1, pML2d and pTDT1.
如本文中使用的术语“VEGF”是指血管内皮生长因子。“VEGF”包括但不限于VEGF变体A、B、C、D、E和F。参见Hamawy A等,Curr.Opin.Cardiol.1999;14:515-522、Neufeld G等,Prog.Growth Factor Res.1994;5:89-97、Olofsson B等,Proc.Natl.Acad.Sci.USA 1996;93:2576-2581、Chilov D等,J.Biol.Chem.1997;272:25176-25183和Olofsson B等,Curr.Opin.Biotechnol.1999;10:528-535。VEGF变体包括,但不限于异形体VEGF164、VEGF121、VEGF145、VEGF167、VEGF165、VEGF189和VEGF206。参见Tischer E等,J.Biol.Chem.1991;266:11947-11954和Poltorak Z等,J.Biol.Chem.1997;272:7151-7158。术语“VEGF”还包括血管通透因子或血管调理素(促血管生成素,vasculotropin)(VPF)。参见Keck P等,Science 1989;246:1309-1312和Senger D等,Science 1983;219:983-985。VPF目前在本领域中已知为VEGF A。也可以使用VEGF家族的其他成员,包括胎盘生长因子PIGF I和II。适合的VEGF的序列是容易获得的(例如,国家生物技术信息中心(National Center forBiotechnology Information,http://www.ncbi.nlm.nih.gov/June 2012)。例如,人类VEGF家组成员的基因座(loci)包括:VEGF-A-P15692和NP003367;VEGF-B-NP003368、P49765、AAL79001、AAL79000、AAC50721、AAB06274和AAH08818;VEGF-C-NP005420、P49767,S69207、AAB36425和CAA63907;VEGF-D-NP004460、AAH27948、O43915、CAA03942和BAA24264;VEGF-E-AAQ88857;VEGF-F-2VPFF;PIGF-1-NP002623、AAH07789、AAH07255、AAH01422、P49763、CAA38698和CAA70463;PIGF-1的链A-1FZVA和链B-1FZVB的合成结构;以及PIGF-2-AAB25832和AAB30462。优选地,VEGF是人类来源的。然而,根据本发明也可以使用来自其他物种,如小鼠的VEGF。The term "VEGF" as used herein refers to vascular endothelial growth factor. "VEGF" includes, but is not limited to, VEGF variants A, B, C, D, E and F. See Hamawy A et al, Curr. Opin. Cardiol. 1999; 14:515-522, Neufeld G et al, Prog. Growth Factor Res. 1994; 5:89-97, Olofsson B et al, Proc.Natl.Acad.Sci.USA 1996; 93: 2576-2581, Chilov D et al., J. Biol. Chem. 1997; 272: 25176-25183 and Olofsson B et al., Curr. Opin. Biotechnol. 1999; 10: 528-535. VEGF variants include, but are not limited to, isoforms VEGF164 , VEGF121 , VEGF145 , VEGF167 , VEGF165 , VEGF189 , and VEGF206 . See Tischer E et al, J. Biol. Chem. 1991; 266: 11947-11954 and Poltorak Z et al, J. Biol. Chem. 1997; 272: 7151-7158. The term "VEGF" also includes vascular permeability factor or vascular opsonin (vasculotropin) (VPF). See Keck P et al. Science 1989;246:1309-1312 and Senger D et al. Science 1983;219:983-985. VPF is currently known in the art as VEGF A. Other members of the VEGF family can also be used, including placental growth factors PIGF I and II. Sequences of suitable VEGFs are readily available (eg, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/June 2012). For example, genes from members of the human VEGF family Loci include: VEGF-A-P15692 and NP003367; VEGF-B-NP003368, P49765, AAL79001, AAL79000, AAC50721, AAB06274 and AAH08818; VEGF-C-NP005420, P49767, S69207, AAB36425 and CAA6390; NP004460, AAH27948, O43915, CAA03942, and BAA24264; VEGF-E-AAQ88857; VEGF-F-2VPFF; Synthetic structure of B-1FZVB; and PIGF-2-AAB25832 and AAB30462. Preferably, the VEGF is of human origin. However, VEGF from other species, such as mice, can also be used according to the present invention.
如本文中使用的术语“白色脂肪组织相细胞”或“白色脂肪细胞”是指多面体至球形,并且含有较大的,由薄层的细胞质围绕的“单室”脂质液滴的脂肪细胞的类型。所述细胞的细胞核是扁平的并且位于外周。白色脂肪细胞的直径在根据库部位在30和70μm之间的范围内变化。存储的脂肪处于半液体状态,并且主要由甘油三酯和胆固醇酯组成。白色脂肪细胞分泌多种肽和蛋白,统称为脂肪因子,如抵抗素、脂连素和瘦素。The term "white adipose tissue phase cells" or "white adipocytes" as used herein refers to adipocytes that are polyhedral to spherical and contain large, "unicompartmental" lipid droplets surrounded by a thin layer of cytoplasm type. The nuclei of the cells are flat and peripherally located. The diameter of white adipocytes ranged between 30 and 70 μm depending on the depot site. Stored fat is in a semi-liquid state and consists mainly of triglycerides and cholesterol esters. White adipocytes secrete a variety of peptides and proteins collectively referred to as adipokines, such as resistin, adiponectin, and leptin.
如本文中使用的术语“土拨鼠肝炎病毒转录后调控元件”或“WPRE”是指当转录时产生能够增强基因表达的三级结构的DNA序列。参见Lee Y等,Exp.Physiol.2005;90(1):33-37和Donello J等,J.Virol.1998;72(6):5085-5092。The term "woodchuck hepatitis virus post-transcriptional regulatory element" or "WPRE" as used herein refers to a DNA sequence that, when transcribed, produces tertiary structure capable of enhancing gene expression. See Lee Y et al, Exp. Physiol. 2005; 90(1):33-37 and Donello J et al, J. Virol. 1998; 72(6):5085-5092.
如本文中使用的术语“microRNA”或“miRNA”是涉及在转录后水平下RNA介导的基因沉默的小型(~22-nt)的、进化保守的调控RNA。参见Bartel DP.Cell 2004;116:281–297。通过与互补区域(最常在细胞信使RNA(mRNA)的3'未翻译区(3'UTR))的碱基配对,miRNA可以起抑制mRNA翻译,或在高序列同源性时,造成mRNA的催化解聚。由于许多miRNA高度分化的组织表达,可以采用细胞miRNA以介导基因治疗载体的组织特异定位。通过病毒载体内的与组织特异miRNA(miRT)完全互补的靶元件的工程串联拷贝,可以高效抑制在不期望的组织中的转基因表达。The term "microRNA" or "miRNA" as used herein is a small (~22-nt), evolutionarily conserved regulatory RNA involved in RNA-mediated gene silencing at the post-transcriptional level. See Bartel DP. Cell 2004;116:281-297. By base pairing with complementary regions, most often in the 3' untranslated region (3' UTR) of cellular messenger RNA (mRNA), miRNAs can act to inhibit mRNA translation or, in the case of high sequence homology, cause mRNA Catalytic depolymerization. Due to the highly differentiated tissue expression of many miRNAs, cellular miRNAs can be employed to mediate tissue-specific localization of gene therapy vectors. Transgene expression in undesired tissues can be efficiently inhibited by engineered tandem copies of target elements fully complementary to tissue-specific miRNAs (miRTs) within viral vectors.
2.提供脂肪组织特异表达的腺相关病毒载体2. Adeno-associated virus vector providing adipose tissue-specific expression
在第一方面,本发明涉及腺相关病毒(AAV)载体,包含重组病毒基因组,其中,所述重组病毒基因组包含表达框,该表达框包含可操作地连接至感兴趣的多核苷酸的脂肪组织特异的转录调控区。In a first aspect, the present invention relates to an adeno-associated virus (AAV) vector comprising a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette comprising adipose tissue operably linked to a polynucleotide of interest specific transcriptional regulatory regions.
根据本发明的AAV包括已知的AAV的42种血清型中的任何血清型。通常,AAV的血清型具有在氨基酸和核酸水平具有显著同源性的基因组序列,提供等同的系列的遗传功能,产生在物理和功能方面实质相同的病毒粒子,并且通过实际上相同的机制复制和装配。特别地,本发明的AAV可以属于血清型1的AAV(AAV1)、AAV2、AAV3(包括类型3A和3B)、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、鸟类AAV、牛类AAV、犬类AAV、马类AAV、羊类AAV和任何其他的AAV。不同的AAV血清型的基因组序列的实例可以在文献中或在公开数据库,如GenBank中找到。参见GenBank登录号AF028704.1(AAV6)、NC006260(AAV7)、NC006261(AAV8)和AX753250.1(AAV9)。在优选的实施方式中,本发明的腺相关病毒载体选自由AAV6、AAV7、AAV8和AAV9血清型所组成的组中的血清型。AAVs according to the present invention include any of the 42 serotypes of known AAVs. Generally, serotypes of AAV have genomic sequences with significant homology at the amino acid and nucleic acid levels, provide an equivalent set of genetic functions, produce virions that are physically and functionally substantially identical, and replicate and replicate through virtually the same mechanism. assembly. In particular, the AAV of the invention may belong to AAV of serotype 1 (AAV1), AAV2, AAV3 (including types 3A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine Class AAV, canine AAV, equine AAV, ovine AAV and any other AAV. Examples of genomic sequences of different AAV serotypes can be found in the literature or in public databases such as GenBank. See GenBank Accession Nos. AF028704.1 (AAV6), NC006260 (AAV7), NC006261 (AAV8) and AX753250.1 (AAV9). In a preferred embodiment, the adeno-associated viral vector of the invention is a serotype selected from the group consisting of AAV6, AAV7, AAV8 and AAV9 serotypes.
根据本发明的AAV的基因组通常包含顺式作用的5'和3'末端反向重复序列和表达框。参见Tijsser P,Ed.,,“Handbook of Parvoviruses”(CRC Press,Boca Raton,FL,US,1990,pp.155-168)。ITR序列为约145bp长。虽然这些序列一定程度的小部分修改是允许的,优选地将基本整个编码ITR的序列用于分子。用于修改这些ITR序列的方法是在本领域中已知的。参见Brown T,“Gene Cloning”(Chapman&Hall,London,GB,1995)、Watson R等,“Recombinant DNA”,第2版(Scientific American Books,New York,NY,US,1992)、Alberts B等,“Molecular Biology of the Cell”(Garland Publishing Inc.,New York,NY,US,2008)、Innis M等,Eds.,“PCR Protocols.A Guide to Methods andApplications”)Academic Press Inc.,San Diego,CA,US,1990)、Erlich H,Ed.,“PCRTechnology.Principles and Applications for DNA Amplification”(Stockton Press,New York,NY,US,1989)、Sambrook J等,“Molecular Cloning.A Laboratory Manual”(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,US,1989)、Bishop T等,“Nucleic Acid and Protein Sequence.A Practical Approach”(IRL Press,Oxford,GB,1987),Reznikoff W,Ed.,“Maximizing Gene Expression”(ButterworthsPublishers,Stoneham,MA,US,1987),Davis L等,“Basic Methods in MolecularBiology”(Elsevier Science Publishing Co.,New York,NY,US,1986)和Schleef M,Ed.,“Plasmid for Therapy and Vaccination”(Wiley-VCH Verlag GmbH,Weinheim,DE,2001)。在优选的实施方式中,AAV重组基因组包含5'和3'AAV ITR。在另一实施方式中,5'和3'AAV ITR源自AAV2。在更加优选的实施方式中,AAV重组基因组缺少rep开放阅读框和cap开放阅读框。在一种实施方式中,选择AAV2ITR以生成假型AAV(即具有衣壳和源自不同血清型的ITR的AAV)。The genome of an AAV according to the invention generally comprises cis-acting 5' and 3' terminal inverted repeats and an expression cassette. See Tijsser P, Ed., "Handbook of Parvoviruses" (CRC Press, Boca Raton, FL, US, 1990, pp. 155-168). The ITR sequence is about 145 bp long. While some minor modification of these sequences is permissible, it is preferred to use substantially the entire sequence encoding the ITR for the molecule. Methods for modifying these ITR sequences are known in the art. See Brown T, "Gene Cloning" (Chapman & Hall, London, GB, 1995), Watson R et al, "Recombinant DNA", 2nd Ed. (Scientific American Books, New York, NY, US, 1992), Alberts B et al, " Molecular Biology of the Cell" (Garland Publishing Inc., New York, NY, US, 2008), Innis M et al., Eds., "PCR Protocols. A Guide to Methods and Applications") Academic Press Inc., San Diego, CA, US, 1990), Erlich H, Ed., "PCR Technology. Principles and Applications for DNA Amplification" (Stockton Press, New York, NY, US, 1989), Sambrook J et al., "Molecular Cloning. A Laboratory Manual" (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, US, 1989), Bishop T, et al., "Nucleic Acid and Protein Sequence. A Practical Approach" (IRL Press, Oxford, GB, 1987), Reznikoff W, Ed., "Maximizing Gene Expression" (Butterworths Publishers, Stoneham, MA, US, 1987), Davis L et al., "Basic Methods in Molecular Biology" (Elsevier Science Publishing Co., New York, NY, US, 1986) and Schleef M, Ed., "Plasmid for Therapy and Vaccination" (Wiley-VCH Verlag GmbH, Weinheim, DE, 2001). In a preferred embodiment, the AAV recombinant genome comprises 5' and 3' AAV ITRs. In another embodiment, the 5' and 3' AAV ITRs are derived from AAV2. In a more preferred embodiment, the AAV recombinant genome lacks the rep open reading frame and the cap open reading frame. In one embodiment, AAV2 ITRs are selected to generate pseudotyped AAVs (ie AAVs with capsids and ITRs derived from different serotypes).
本发明的多核苷酸可以包含源自任一AAV血清型的ITR。在优选的实施方式中,ITR源自AAV2血清型。The polynucleotides of the present invention may comprise ITRs derived from any AAV serotype. In a preferred embodiment, the ITR is derived from the AAV2 serotype.
本发明的AAV包含来自任何血清型的衣壳。在特定的实施方式中,该衣壳源自由以下所组成的组中的AAV:AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8和AAV9。在优选的实施方式中,本发明的AAV包含源自AAV8或AAV9血清型的衣壳。在另一优选的实施方式中,AAV衣壳的VP1序列具有序列表SEQ ID NO.18。参见GenBank登录号AY530579。The AAVs of the present invention comprise capsids from any serotype. In a specific embodiment, the capsid is derived from an AAV in the group consisting of: AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In a preferred embodiment, the AAV of the invention comprises a capsid derived from AAV8 or AAV9 serotype. In another preferred embodiment, the VP1 sequence of the AAV capsid has SEQ ID NO. 18 of the Sequence Listing. See GenBank Accession No. AY530579.
在一些实施方式中,用于本发明的方法的AAV Cap可以通过突变(即通过插入、删除、或替代)上述AAV Cap或其编码核酸之一产生。在一些实施方式中,AAV Cap与上述的一种或多种AAV Cap至少70%、75%、80%、85%、90%、95%、98%、或99%相似。In some embodiments, AAV Caps for use in the methods of the present invention may be generated by mutation (ie, by insertion, deletion, or substitution) of one of the above-described AAV Caps or their encoding nucleic acids. In some embodiments, the AAV Cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the AAV Caps described above.
在一些实施方式中,AAV Cap是嵌合的,包含来自两种、三种、四种、或更多种前述AAV Cap的结构域。在一些实施方式中,AAV Cap是来源自两种或三种不同的AAV或重组AAV的VP1、VP2和VP3单体的嵌合体。在一些实施方式中,rAAV组合物包含多于一种的前述Cap。In some embodiments, the AAV Cap is chimeric, comprising domains from two, three, four, or more of the aforementioned AAV Caps. In some embodiments, the AAV Cap is a chimera of VP1, VP2, and VP3 monomers derived from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned Caps.
在一些实施方式中,将用于rAAV组合物中的AAV Cap设计为含有异源序列或其他修饰。例如,可以将赋予选择性定位或免疫逃避的肽或蛋白序列引入至Cap蛋白。可替代地或另外地,Cap可以化学改性使得rAAV的表面是聚乙二醇化SEQ ID NO.(即聚乙二醇修饰的),其可以促进免疫逃避。Cap蛋白还可以是诱变的(例如去除其天然受体结合,或掩盖免疫原表位)。In some embodiments, AAV Caps used in rAAV compositions are designed to contain heterologous sequences or other modifications. For example, peptide or protein sequences that confer selective localization or immune evasion can be introduced into the Cap protein. Alternatively or additionally, Cap can be chemically modified such that the surface of the rAAV is PEGylated SEQ ID NO. (ie, polyethylene glycol modified), which can facilitate immune evasion. Cap proteins can also be mutagenized (eg, to remove their native receptor binding, or to mask immunogenic epitopes).
在另一特定的实施方式中,AAV载体是假型AAV载体(即该载体包含来源自至少两种不同AAV血清型的序列或组件)。在特定的实施方式中,假型AAV载体包含源自一种AAV血清型(例如AAV2)的AAV基因组,和至少部分源自不同的AAV血清型的衣壳。这种假型AAV载体的特定的实例包括,但不限于包含源自任何AAV血清型(例如来自AAV1至AAV11)的基因组,在源自AAV6、AAV7、AAV8、或AAV9的衣壳中的载体。In another specific embodiment, the AAV vector is a pseudotyped AAV vector (ie, the vector comprises sequences or components derived from at least two different AAV serotypes). In a specific embodiment, a pseudotyped AAV vector comprises an AAV genome derived from one AAV serotype (eg, AAV2), and a capsid derived at least in part from a different AAV serotype. Specific examples of such pseudotyped AAV vectors include, but are not limited to, vectors comprising a genome derived from any AAV serotype (eg, from AAV1 to AAV11) in a capsid derived from AAV6, AAV7, AAV8, or AAV9.
在一种实施方式中,AAV载体包含一个启动子,其中,加入至少一种miRNA的至少一个靶序列,该miRNA可以选自以下列表:miR122(miRBase数据库登录号MI0000442)、miR152(MI0000462)、miR199(MI0000242)、miR215(MI0000291)、miR192(MI0000234)、miR148a(MI0000253)、miR194(MI0000488)、miR1(MI0000651)、miRT133(MI0000450)、miR206(MI0000490)、miR208(MI0000251)、miR124(MI0000443)、miR125(MI0000469)、miR216(MI0000292)、miR130(MI0000448)。参考序列已经获得自miRBase(根据31/07/2013的版本,http://www.mirbase.org/)。In one embodiment, the AAV vector comprises a promoter into which at least one target sequence of at least one miRNA is added, the miRNA may be selected from the following list: miR122 (miRBase database accession number MI0000442), miR152 (MI0000462), miR199 (MI0000242)、miR215(MI0000291)、miR192(MI0000234)、miR148a(MI0000253)、miR194(MI0000488)、miR1(MI0000651)、miRT133(MI0000450)、miR206(MI0000490)、miR208(MI0000251)、miR124(MI0000443)、miR125 (MI0000469), miR216 (MI0000292), miR130 (MI0000448). Reference sequences have been obtained from miRBase (version according to 31/07/2013, http://www.mirbase.org/).
在一种实施方式中AAV载体含有一个启动子,其中加入至少一种可以选自以下列表的miRNA靶序列:In one embodiment the AAV vector contains a promoter into which is added at least one miRNA target sequence that can be selected from the following list:
列表1
miRT122a(5’CAAACACCATTGTCACACTCCA3’)、miRT122a(5'CAAACACCATTGTCACACTCCA3'),
miRT152(5’AGTCACGTACTGTCTTGAACC3’)、miRT152(5'AGTCACGTACTGTCTTGAACC3'),
miR199a-5p(5’GGGTCACAAGTCTGATGGACAAG3’)、miR199a-5p (5'GGGTCACAAGTCTGATGGACAAG3'),
miR99a-3p(5’TGTCATCAGACGTGTAACCAAT3’)、miR99a-3p (5'TGTCATCAGACGTGTAACCAAT3'),
miRT215(5’TACTGGATACTTAACTGTCTG3’)、miRT215(5'TACTGGATACTTAACTGTCTG3'),
miRT192(5’GGCTGTCAATTCATAGGTCAG3’)、miRT192(5'GGCTGTCAATTCATAGGTCAG3'),
miRT194(5’ACATTGTCGTTGAGGTACACCT3’)、miRT194(5'ACATTGTCGTTGAGGTACACCT3'),
miRT1(5’TTACATACTTCTTTACATTCCA3’)、miRT1(5'TTACATACTTCTTTACATTCCA3'),
mirT148(5’AGTCACGTGATGTCTTGAAACA3’)、mirT148(5'AGTCACGTGATGTCTTGAAACA3'),
mirT133a(5’AAACCAGGGGAAGTTGGTCGAC3’)、mirT133a (5'AAACCAGGGGAAGTTGGTCGAC3'),
miRT206(5’ACCTTACATTCCTTCACACACC3’)、miRT206(5'ACCTTACATTCCTTCACACACC3'),
miRT124(5’ATTCCGTGCGCCACTTACGG3’)、miRT124(5'ATTCCGTGCGCCACTTACGG3'),
miRT125(5’AGGGACTCTGGGAAATTGGACACT3’)、miRT125(5'AGGGACTCTGGGAAATTGGACACT3'),
miRT216(5’ATTAGAGTCGACCGTTGACACT3’)、miRT216(5'ATTAGAGTCGACCGTTGACACT3'),
miRT130(5’GTCACGTTACAATTTTCCCGTA3’)。miRT130 (5'GTCACGTTACAATTTTCCCGTA3').
在一种实施方式中AAV载体含有一个启动子,其中加入至少一个具有与选自以上提及的列表1的miRNA靶序列85%同源的miRNA靶序列。In one embodiment the AAV vector contains a promoter into which at least one miRNA target sequence having 85% homology to a miRNA target sequence selected from
在一种实施方式中AAV载体含有一个启动子,其中加入至少一个具有与选自以上提及的列表1的miRNA靶序列功能等效的miRNA靶序列。在这种情况下,术语功能等效物是指能够与相同的miRNA结合的任何核苷酸序列,该miRNA与原始序列结合。例如,miRT122a的功能等效物是与相同的miRNA杂交的任意序列,该miRNA与mirT122a杂交。In one embodiment the AAV vector contains a promoter into which is added at least one miRNA target sequence that is functionally equivalent to a miRNA target sequence selected from
功能等效的核苷酸序列保持了参考mirT序列的相关生物活性。这是指mirT的功能等效物具有,以与参考mirT序列相同的方式在不期望的组织中抑制转基因表达的能力。A functionally equivalent nucleotide sequence retains the relevant biological activity of the reference mirT sequence. This means that the functional equivalent of mirT has the ability to inhibit transgene expression in undesired tissues in the same manner as the reference mirT sequence.
在另一特定的实施方式中,miRNA靶序列可以选自mirT122a(5'CAAACACCATTGTCACACTCCA3'),参考为序列表SEQ ID NO:19或mirT1(5'TTACATACTTCTTTACATTCCA3'),参考为序列表SEQ ID NO:20。In another specific embodiment, the miRNA target sequence can be selected from mirT122a (5'CAAACACCATTGTCACACTCCA3'), with reference to SEQ ID NO: 19 of the Sequence Listing or mirT1 (5'TTACATACTTCTTTACATTCCA3'), with reference to the SEQ ID NO: 20 of the Sequence Listing .
转录调控区可以包含启动子,以及可选地增强区域。优选地,该启动子是对脂肪组织特异的。增强子对脂肪组织不需要是特异的。可替代地,转录调控区可以包含脂肪组织特异的启动子和脂肪组织特异的增强子。Transcriptional regulatory regions may comprise promoters, and optionally enhancer regions. Preferably, the promoter is specific for adipose tissue. Enhancers need not be specific for adipose tissue. Alternatively, the transcriptional regulatory region may comprise an adipose tissue-specific promoter and an adipose tissue-specific enhancer.
在一种实施方式中,组织特异的启动子是脂肪细胞特异的启动子,例如脂肪细胞蛋白2(aP2,也称作脂肪酸结合蛋白4(FABP4))、PPARy启动子、脂连素启动子、磷酸烯醇丙酮酸羧激酶(PEPCK)启动子,该启动子源自人芳香酶细胞色素p450(p450arom),或Foxa-2启动子。参见Graves R等,Genes Dev.1991;5:428-437、Ross S等,Proc.Natl.Acad.Sci.USA1990;87:9590-9594、Simpson E等,US 5,446,143、Mahendroo M等,J.Biol.Chem.1993;268:19463-19470、Simpson E等,Clin.Chem.1993;39:317-324和Sasaki H等,Cell 1994;76:103-115。在优选的实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。In one embodiment, the tissue-specific promoter is an adipocyte-specific promoter, such as adipocyte protein 2 (aP2, also known as fatty acid binding protein 4 (FABP4)), PPARy promoter, adiponectin promoter, Phosphoenolpyruvate carboxykinase (PEPCK) promoter, derived from the human aromatase cytochrome p450 (p450arom), or the Foxa-2 promoter. See Graves R et al, Genes Dev. 1991; 5:428-437, Ross S et al, Proc. Natl. Acad. Sci. USA 1990; 87: 9590-9594, Simpson E et al, US 5,446,143, Mahendroo M et al, J. Biol Chem. 1993; 268: 19463-19470, Simpson E et al. Clin. Chem. 1993; 39: 317-324 and Sasaki H et al. Cell 1994; 76: 103-115. In a preferred embodiment, the enhancer region is selected from the group consisting of the fat-specific aP2 enhancer and the fat-specific UCP1 enhancer.
在优选的实施方式中,根据本发明的AAV的脂肪组织特异调控区包含脂肪特异aP2增强子和aP2基本启动子(基本aP2启动子,basal ap2 promoter)。参见Rival Y等,J.Pharmacol.Exp.Ther.2004:311(2):467-475。该包含脂肪特异aP2增强子和aP2基本启动子的区域也称为“mini/aP2调控区”,并且是由aP2基因的基本启动子和所述aP2基因的脂肪特异增强子形成的。优选地,aP2启动子是鼠类的。参见Graves R等,Mol.Cell Biol.1992;12(3):1202-1208和Ross S等,Proc.Natl.Acad.Sci.USA 1990;87:9590-9594。在特定的实施方式中,mini/aP2调控区具有序列表SEQ ID NO:2的序列。In a preferred embodiment, the adipose tissue-specific regulatory region of the AAV according to the present invention comprises an adipose-specific aP2 enhancer and an aP2 basal promoter (basal ap2 promoter). See Rival Y et al., J. Pharmacol. Exp. Ther. 2004:311(2):467-475. This region comprising the fat-specific aP2 enhancer and the aP2 basic promoter is also referred to as the "mini/aP2 regulatory region" and is formed by the basic promoter of the aP2 gene and the fat-specific enhancer of the aP2 gene. Preferably, the aP2 promoter is murine. See Graves R et al, Mol. Cell Biol. 1992; 12(3): 1202-1208 and Ross S et al, Proc. Natl. Acad. Sci. USA 1990; 87:9590-9594. In a specific embodiment, the mini/aP2 regulatory region has the sequence of SEQ ID NO:2 of the Sequence Listing.
在另一优选的实施方式中,根据本发明的AAV的脂肪组织特异调控区包含脂肪特异UCP1增强子和UCP1基本启动子(基本UCP1启动子,basal UCP1 promoter)。参见del MarGonzález-Barroso M等,J.Biol.Chem.2000;275(41):31722-31732和Rim J等,J.Biol.Chem.2002;277(37):34589-34600。包含脂肪特异UCP1增强子和UCP1基本启动子的该区域也称为“mini/UCP调控区”并且是指UCP1基因的基本启动子和所述UCP1基因的脂肪特异增强子的组合。优选使用大鼠UCP1启动子。参见Larose M,等,J.Biol.Chem.1996;271(49):31533-31542和Cassard-Doulcier A等,Biochem.J.1998;333:243-246。在特定的实施方式中,mini/UCP1调控区具有序列表SEQ ID NO:3的序列。In another preferred embodiment, the adipose tissue-specific regulatory region of the AAV according to the present invention comprises an adipose-specific UCP1 enhancer and a UCP1 basal promoter (basal UCP1 promoter). See del Mar González-Barroso M et al, J. Biol. Chem. 2000; 275(41):31722-31732 and Rim J et al, J. Biol. Chem. 2002; 277(37):34589-34600. This region comprising the fat-specific UCP1 enhancer and the UCP1 base promoter is also referred to as the "mini/UCP regulatory region" and refers to the combination of the base promoter of the UCP1 gene and the fat-specific enhancer of the UCP1 gene. The rat UCP1 promoter is preferably used. See Larose M, et al, J. Biol. Chem. 1996; 271(49): 31533-31542 and Cassard-Doulcier A et al, Biochem. J. 1998; 333: 243-246. In a specific embodiment, the mini/UCP1 regulatory region has the sequence of SEQ ID NO:3 of the Sequence Listing.
在另一实施方式中,组成本发明的AAV的部分的表达框进一步包含表达控制序列,其包括但不限于适当的转录序列(即起始、终止、启动子和增强子)、有效的RNA加工信号(例如,剪接和聚腺苷酸化(polyA)信号)、稳定细胞质mRNA的序列、增强翻译效率的序列(即Kozak共有序列)、增强蛋白稳定性的序列、以及当需要时,增强编码的产物的分泌的序列。非常多的表达控制序列,包括天然的、组成的、诱导的、或组织特异的启动子是本领域中已知的并且可以根据本发明利用。In another embodiment, the expression cassettes that form part of the AAVs of the invention further comprise expression control sequences including, but not limited to, appropriate transcription sequences (ie initiation, termination, promoters and enhancers), efficient RNA processing Signals (e.g., splicing and polyadenylation (polyA) signals), sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (ie, Kozak consensus sequences), sequences that enhance protein stability, and, when desired, encoded products the secretory sequence. A wide variety of expression control sequences, including native, constitutive, inducible, or tissue-specific promoters, are known in the art and can be utilized in accordance with the present invention.
在另一实施方式中,构成本发明的AAV的部分的表达框进一步包含转录后调控区。在优选的实施方式中,该转录后调控区是土拨鼠肝炎病毒转录后区(WPRE)或其功能变体和片段,以及PPT-CTS或其功能变体和片段。参见Zufferey R等,J.Virol.1999;73:2886-2892和Kappes J等,WO 2001/044481。在特定的实施方式中,该转录后调控区是WPRE。In another embodiment, the expression cassette forming part of the AAV of the invention further comprises a post-transcriptional regulatory region. In preferred embodiments, the post-transcriptional regulatory region is the woodchuck hepatitis virus post-transcriptional region (WPRE) or functional variants and fragments thereof, and PPT-CTS or functional variants and fragments thereof. See Zufferey R et al, J. Virol. 1999;73:2886-2892 and Kappes J et al, WO 2001/044481. In a specific embodiment, the post-transcriptional regulatory region is WPRE.
构成根据本发明的AAV的部分的表达框包含“感兴趣的多核苷酸”。在优选的实施方式中,该感兴趣的多核苷酸编码作用于全身的蛋白。在另一实施方式中,感兴趣的多核苷酸编码作用于脂肪细胞或其附近区域的蛋白。在优选的实施方式中,作用于所述脂肪细胞或其附近区域的蛋白是己糖激酶(HK),包括相对于不同的底物,在亚细胞位置和动力学上不同的任意四种哺乳动物HK同功酶(EC 2.7.1.1)。例如,HK包括HK1(GenBank 登录号NP000179、NP277031、NP277032、NP277033、NP277035)、HK2(GenBank登录号NP000180)、HK3(GenBank登录号NP002106)和HK4或葡糖激酶(GenBank登录号NP000153、NP277042、NP277043)。在另一优选的实施方式中,HK是葡糖激酶,其在本文中与己糖激酶4或HK 4可互换地使用,并且指具有HK1、HK2、或HK3的100倍高的葡萄糖的Km的己糖激酶的异形体。An expression cassette forming part of an AAV according to the invention comprises a "polynucleotide of interest". In a preferred embodiment, the polynucleotide of interest encodes a protein that acts throughout the body. In another embodiment, the polynucleotide of interest encodes a protein that acts on adipocytes or regions near them. In a preferred embodiment, the protein acting on the adipocyte or its vicinity is a hexokinase (HK), including any four mammalian species that differ in subcellular location and kinetics relative to different substrates HK isozyme (EC 2.7.1.1). For example, HK includes HK1 (GenBank Accession No. NP000179, NP277031, NP277032, NP277033, NP277035), HK2 (GenBank Accession No. NP000180), HK3 (GenBank Accession No. NP002106) and HK4 or glucokinase (GenBank Accession No. NP000153, NP277042, NP277043) ). In another preferred embodiment, HK is glucokinase, which is used interchangeably herein with hexokinase 4 or HK 4 and refers to a Km with
在实施方式中,作用于所述脂肪细胞或其附近区域的蛋白是碱性磷酸酶(AP),包括但不限于肠类或IAP(GenBank登录号NP001622)、胎盘类或PLAP(GenBank登录号NP001623)和非组织特异同功酶或ALPL(GenBank登录号NP000469、NP001120973.2和NP001170991.1)。在另一实施方式中,作用于所述脂肪细胞或其附近区域的蛋白是VEGF,包括但不限于VEGF变体A、B、C、D、E和F。In embodiments, the protein acting on the adipocyte or its vicinity is alkaline phosphatase (AP), including but not limited to intestinal or IAP (GenBank Accession No. NP001622), placental or PLAP (GenBank Accession No. NP001623) ) and the non-tissue specific isozyme or ALPL (GenBank accession numbers NP000469, NP001120973.2 and NP001170991.1). In another embodiment, the protein acting on the adipocyte or its vicinity is VEGF, including but not limited to VEGF variants A, B, C, D, E and F.
在另一实施方式中,感兴趣的多核苷酸编码通常产生的并且由脂肪细胞分泌的多肽。在另一实施方式中,由脂肪细胞产生并分泌的多肽是脂肪酶(例如特别地,丝氨酸蛋白酶同系物的脂肪酶)、脂连素、瘦素、抵抗素、或肥胖基因(ob gene)的蛋白产物。In another embodiment, the polynucleotide of interest encodes a polypeptide that is normally produced and secreted by adipocytes. In another embodiment, the polypeptide produced and secreted by an adipocyte is a lipase (eg, in particular, a lipase of a serine protease homolog), adiponectin, leptin, resistin, or of an ob gene protein product.
其他还有用的感兴趣的多核苷酸包括编码激素以及生长和分化因子的那些,包括,但不限于胰岛素、高血糖素、生长激素(GH)、甲状旁腺素(PTH)、生长激素释放因子(GRF)、促卵泡激素(FSH)、促黄体生成激素(LH)、人绒毛膜促性腺激素(hCG)、血管生成素、血管抑制素、粒细胞集落刺激因子(GCSF)、红细胞生成素(EPO)、结缔组织生长因子(CTGF)、碱性成纤维细胞生长因子(bFGF)、酸性成纤维细胞生长因子(aFGF)、表皮生长因子(EGF)、血小板源生长因子(PDGF)、胰岛素生长因子I和II(IGF-I和TGF-II)、转化生长因子超家族中的任一种,包括TGFa、活化素、抑制素、或骨形态发生蛋白(BMP)BMP 1-15中的任一种、生长因子的调节蛋白(heregluin)/神经调节蛋白/ARIA/Neu分化因子(NDF)家族中的任一种、神经生长因子(nerve growth factor)(NGF)、脑源性神经营养因子(BDNF)、神经营养蛋白NT-3和NT-4/5、睫状神经营养因子(CNTF)、胶质细胞源性神经营养因子(GDNF)、神经生长因子(neurturin)、聚集蛋白(agrin)、臂板蛋白(semaphorins)/崩溃蛋白家族的任一种、神经突起导向因子(netrin)-1和神经突起导向因子-3、肝细胞生长因子(HGF)、肝配蛋白(ephrin)、头蛋白(noggin)、音猬因子(sonic hedgehog)和酪氨酸羟基化酶。Other also useful polynucleotides of interest include those encoding hormones and growth and differentiation factors, including, but not limited to, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Human Chorionic Gonadotropin (hCG), Angiopoietin, Angiostatin, Granulocyte Colony Stimulating Factor (GCSF), Erythropoietin ( EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and TGF-II), any of the transforming growth factor superfamily, including any of TGFa, activin, inhibin, or bone morphogenetic protein (BMP) BMPs 1-15 , any of the heregluin/neuregulin/ARIA/Neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) , neurotrophin NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (neurturin), aggregated protein (agrin), arm plate Any of the semaphorins/collapsin family, neurite guidance factor (netrin)-1 and neurite guidance factor-3, hepatocyte growth factor (HGF), ephrin, noggin , sonic hedgehog and tyrosine hydroxylase.
其他有用的感兴趣的多核苷酸包括编码调节免疫系统的蛋白的那些,包括,但不限于细胞因子和淋巴细胞活素,如血小板生成素(TPO)、白细胞介素(IL)IL-1至IL-25(例如IL-2、IL-4、IL-12和IL-18)、单核细胞趋化蛋白、白血病抑制因子、粒细胞-巨噬细胞集落刺激因子、Fas配体、肿瘤坏死因子α和β、干扰素α、β和γ、干细胞因子、flk-2/flt3配体。由免疫系统生产的基因产物对本发明也是有用的。这些包括,但不限于免疫球蛋白IgG、IgM、IgA、IgD和IgE、嵌合免疫球蛋白、人源化抗体、单链抗体、T细胞受体、嵌合T细胞受体、单链T细胞受体、I类和II类MHC和HLA分子,以及工程化免疫球蛋白以及MHC和HLA分子。有用的基因产物还包括补体调节蛋白,如补体调节蛋白、膜辅因子蛋白(MCP)、衰变加速因子(MCP)、CRl、CF2和CD59。Other useful polynucleotides of interest include those encoding proteins that modulate the immune system, including, but not limited to, cytokines and lymphokines such as thrombopoietin (TPO), interleukin (IL), IL-1 to IL-25 (eg IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor Alpha and beta, interferon alpha, beta and gamma, stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cells Receptors, Class I and II MHC and HLA molecules, and engineered immunoglobulins and MHC and HLA molecules. Useful gene products also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (MCP), CR1, CF2, and CD59.
其他有用的感兴趣的多核苷酸包括编码激素、生长因子、细胞因子、淋巴细胞活素、调节蛋白和免疫系统蛋白的受体中任一种的那些。本发明包含用于胆甾醇调节或脂调节的受体,包括低密度脂蛋白(LDL)受体、高密度脂蛋白(HDL)受体、极低密度脂蛋白(VLDL)受体和清道夫受体。本发明号也包含基因产物,如包括糖皮质激素受体和雌激素受体、维生素受体和其他核受体的类固醇激素受体总家族成员。此外,有用的基因产物包括转录因子,如jun、fos、max、mad、血清效应因子(SRF)、AP1、AP2、myb、MyoD和肌细胞生成素、包含蛋白的ETS盒(ETS-box containing protein)、TFE3、E2F、ATF1、ATF2、ATF3、ATF4、ZF5、NFAT、CREB、HNF-4、C/EBP、SP1、CCAAT盒结合蛋白(CCAAT-box binding protein)、干扰素调节因子(IRF-1)、肾胚细胞瘤(Wilms tumor)蛋白、ETS结合蛋白、STAT、GATA盒结合蛋白(例如GATA-3)和翼状螺旋蛋白(winged helix protein)的叉头(forkhead)家族。Other useful polynucleotides of interest include those encoding receptors for any of hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins. The present invention encompasses receptors for cholesterol regulation or lipid regulation, including low density lipoprotein (LDL) receptors, high density lipoprotein (HDL) receptors, very low density lipoprotein (VLDL) receptors and scavenger receptors body. Inventive numbers also encompass gene products, such as members of the general family of steroid hormone receptors that include glucocorticoid and estrogen receptors, vitamin receptors, and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum effector factor (SRF), AP1, AP2, myb, MyoD and myogenin, ETS-box containing protein ), TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding protein, interferon regulatory factor (IRF-1) ), Wilms tumor proteins, ETS binding proteins, STAT, GATA box binding proteins (eg GATA-3) and the forkhead family of winged helix proteins.
其他有用的感兴趣的多核苷酸包括编码以下酶的那些,如氨基甲酰基合成酶I、鸟氨酸转氨甲酰酶、精氨琥珀酸合成酶、精氨琥珀酸裂解酶、精氨酸酶、富马酰乙酰乙酸水解酶、苯丙氨酸羟化酶、α-1抗胰蛋白酶、葡萄糖-6-磷酸酶、胆色素原脱氨基酶、胱硫醚(cystathione)β-合酶、支链酮酸脱羧酶、白蛋白、异戊酰-辅酶A脱氢酶、丙酰辅酶A羧化酶、甲基丙二酰辅酶A变位酶、戊二酰辅酶A脱氢酶、胰岛素、β-葡糖苷酶、丙酮酸盐羧化酶(pyruvate carboxylate)、肝磷酸化酶、磷酸化酶激酶、甘氨酸脱羧酶、H蛋白、T蛋白、囊性纤维化跨膜调节子(CFTR)序列和抗肌营养不良蛋白(肌萎缩蛋白,dystrophin)基因产物(例如,微小肌营养不良蛋白或小肌营养不良蛋白)。还有用的基因产物包括用于代替治疗的酶,如,例如用于治疗溶酶体贮积病的含有甘露糖-6-磷酸酯的酶(例如,编码β-葡糖醛酸酶(GUSB)的适合的基因)。Other useful polynucleotides of interest include those encoding enzymes such as carbamoyl synthase I, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase, arginine Enzymes, fumaric acetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, Branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, insulin, β-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase, glycine decarboxylase, H protein, T protein, cystic fibrosis transmembrane regulator (CFTR) sequence and Dystrophin (dystrophin) gene product (eg, dystrophin mini or dystrophin mini). Also useful gene products include enzymes for replacement therapy, such as, for example, mannose-6-phosphate-containing enzymes (eg, encoding beta-glucuronidase (GUSB) for the treatment of lysosomal storage diseases) suitable genes).
AAV载体的包装尺寸限制限于母体野生型AAV基因组的尺寸,其在基于AAV血清型的尺寸的范围内(即4087至4767)。参见Wu Z等,Mol.Ther.2010;7(l):80-86。例如,野生型AAV02具有4679的基因组尺寸并且野生型AAV-6具有4683的基因组尺寸。在一些实施方式中,重组体RNA载体的克隆容量可能受限并且期望的编码序列可能涉及完全替换病毒的4.8千碱基基因组。因此大的基因在一些情况下可能不适用于标准的重组AAV载体。技术人员应该理解的是用于克服受限的编码容量的选项是在本领域中可获得的。例如,可以将两个基因组的AAV ITR退火以形成头接尾的串连体(concatamer),几乎加倍载体的容量。剪接位点的插入使得ITR从转录物中去除。其他用于克服受限的克隆容量的选项对于技术人员是显而易见的。Packaging size constraints for AAV vectors are limited to the size of the maternal wild-type AAV genome, which is in the range of sizes based on AAV serotypes (ie, 4087 to 4767). See Wu Z et al., Mol. Ther. 2010;7(1):80-86. For example, wild-type AAV02 has a genome size of 4679 and wild-type AAV-6 has a genome size of 4683. In some embodiments, the cloning capacity of the recombinant RNA vector may be limited and the desired coding sequence may involve a complete replacement of the 4.8 kilobase genome of the virus. Therefore large genes may not be suitable for standard recombinant AAV vectors in some cases. The skilled artisan will appreciate that options for overcoming the limited coding capacity are available in the art. For example, AAV ITRs of two genomes can be annealed to form head-to-tail concatamers, nearly doubling the capacity of the vector. The insertion of the splice site allows the removal of the ITR from the transcript. Other options for overcoming limited cloning capacity will be apparent to the skilled person.
3.基于AAV6、AAV7、AAV8和AAV9对脂肪组织的嗜性的治疗方法3. Therapeutic methods based on the tropism of AAV6, AAV7, AAV8 and AAV9 for adipose tissue
在第二方面中,本发明公开了能够高效转导脂肪组织细胞的AAV6、AAV7、AAV8和AAV9血清型的腺相关病毒载体。该特征使得开发用于治疗需要或可以受益于脂肪细胞中感兴趣的聚核苷酸的表达的疾病的方法成为可能。特别地,此发现通过将本发明的AAV载体给予至患者,促进感兴趣的多肽递送至需要其的受试者,从而产生能够表达感兴趣的多核苷酸并在体内编码多肽的脂肪细胞。如果编码的多肽是分泌的多肽,其可以由脂肪细胞分泌,使得以这种方式全身递送该多肽。In a second aspect, the present invention discloses adeno-associated viral vectors of AAV6, AAV7, AAV8 and AAV9 serotypes capable of efficiently transducing adipose tissue cells. This feature enables the development of methods for treating diseases that require or can benefit from the expression of polynucleotides of interest in adipocytes. In particular, this discovery facilitates delivery of a polypeptide of interest to a subject in need thereof by administering an AAV vector of the present invention to a patient, thereby generating adipocytes capable of expressing a polynucleotide of interest and encoding the polypeptide in vivo. If the encoded polypeptide is a secreted polypeptide, it can be secreted by adipocytes, allowing for systemic delivery of the polypeptide in this manner.
因此,在另一实施方式中,本发明提供了包含重组病毒基因组的腺相关病毒载体以用于需要感兴趣的聚核苷酸的表达的疾病的治疗或预防,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的转录调控区,其中,AAV的血清型选自由AAV6、AAV7、AAV8和AAV9组成的组。Accordingly, in another embodiment, the present invention provides an adeno-associated virus vector comprising a recombinant viral genome for the treatment or prevention of a disease requiring expression of a polynucleotide of interest, wherein the recombinant viral genome comprises An expression cassette comprising a transcriptional regulatory region operably linked to the polynucleotide of interest, wherein the AAV serotype is selected from the group consisting of AAV6, AAV7, AAV8 and AAV9.
在另一实施方式中,本发明提供了包括重组病毒基因组的腺相关病毒载体用于需要所述感兴趣的多核苷酸的表达的疾病的治疗或预防,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的脂肪组织特异的转录调控区。In another embodiment, the present invention provides an adeno-associated virus vector comprising a recombinant viral genome comprising an expression cassette for the treatment or prevention of a disease requiring expression of the polynucleotide of interest , the expression cassette comprises an adipose tissue-specific transcriptional regulatory region operably linked to the polynucleotide of interest.
在另一实施方式中,本发明提供了用于需要感兴趣的多核苷酸在受试者中表达的疾病的治疗或预防的方法,其包括将腺相关病毒载体给予所述受试者,该腺相关病毒载体包含重组病毒基因组,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的转录调控区,其中AAV的血清型选自由AAV6、AAV7、AAV8和AAV9组成的组。In another embodiment, the present invention provides a method for the treatment or prevention of a disease requiring expression of a polynucleotide of interest in a subject, comprising administering to the subject an adeno-associated viral vector, which The adeno-associated virus vector comprises a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette comprising a transcriptional regulatory region operably linked to a polynucleotide of interest, wherein the AAV serotype is selected from AAV6, AAV7 , AAV8 and AAV9.
在另一实施方式中,本发明提供用于需要感兴趣的多核苷酸在受试者中表达的疾病的治疗或预防的方法,其包括将腺相关病毒载体给予至所述受试者,该腺相关病毒载体包含重组病毒基因组,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的脂肪组织特异转录调控区。In another embodiment, the present invention provides a method for the treatment or prevention of a disease requiring expression of a polynucleotide of interest in a subject, comprising administering to the subject an adeno-associated viral vector, the The adeno-associated viral vector comprises a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette comprising an adipose tissue-specific transcriptional regulatory region operably linked to a polynucleotide of interest.
用于本发明的治疗方法的AAV包含含有感兴趣的多核苷酸和转录调控区的表达框。转录调控区可以包含启动子,以及可选的增强区域。AAVs for use in the therapeutic methods of the present invention comprise expression cassettes containing the polynucleotide of interest and transcriptional regulatory regions. Transcriptional regulatory regions may comprise promoters, and optionally enhancer regions.
在一种实施方式中,转录调控区允许感兴趣的多核苷酸的组成型表达。组成型启动子的实例包括,但不限于逆转录病毒劳氏肉瘤病毒(RSV)LTR启动子(可选地带有RSV增强子)、巨细胞病毒(CMV)启动子(可选地带有CMV增强子)、SV40启动子、二氢叶酸还原酶启动子、β-肌动蛋白启动子、甘油磷酸激酶(PGK)启动子和EFla启动子。参见Boshart M等,Cell1985;41:521-530。In one embodiment, the transcriptional regulatory region allows for constitutive expression of the polynucleotide of interest. Examples of constitutive promoters include, but are not limited to, retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter (optionally with CMV enhancer) ), SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, glycerophosphokinase (PGK) promoter and EFla promoter. See Boshart M et al. Cell 1985;41:521-530.
在另一实施方式中,转录调控区包含β-肌动蛋白启动子。β-肌动蛋白启动子可以源自任何哺乳动物,包括人和啮齿动物,或禽类,包括鸡。优选使用鸡β-肌动蛋白。In another embodiment, the transcriptional regulatory region comprises a beta-actin promoter. The beta-actin promoter can be derived from any mammal, including humans and rodents, or avian, including chickens. Preferably chicken beta-actin is used.
在又一实施方式中,转录调控区进一步包含增强子区。优选地,增强子区是CMV增强子区。In yet another embodiment, the transcriptional regulatory region further comprises an enhancer region. Preferably, the enhancer region is a CMV enhancer region.
在特定的实施方式中,调控区是CAG调控区。在优选的实施方式中,CAG调控区具有序列表SEQ ID NO:1的序列。In a specific embodiment, the regulatory region is a CAG regulatory region. In a preferred embodiment, the CAG regulatory region has the sequence of SEQ ID NO: 1 of the Sequence Listing.
在另一实施方式中,转录调控区是脂肪组织特异的转录调控区。In another embodiment, the transcriptional regulatory region is an adipose tissue-specific transcriptional regulatory region.
如果启动子对脂肪组织特异,则增强子不需要也对脂肪组织特异。可替代地,转录调控区可以包含脂肪组织特异的启动子和脂肪组织特异的增强子。If the promoter is specific for adipose tissue, the enhancer need not also be specific for adipose tissue. Alternatively, the transcriptional regulatory region may comprise an adipose tissue-specific promoter and an adipose tissue-specific enhancer.
在一种实施方式中,组织特异的启动子是脂肪细胞特异的启动子,如,例如脂肪细胞蛋白2(aP2,也称作脂肪酸结合蛋白4(FABP4))、PPARy启动子、脂连素启动子、磷酸烯醇丙酮酸羧激酶(PEPCK)启动子,该启动子源自人类芳香酶细胞色素p450(p450arom)和Foxa-2启动子。参见上文的Graves(1991)、Ross(1990)、Simpson(US 5,446,143)、Mahendroo(1993)、Simpson(1993)和Sasaki(1994)。In one embodiment, the tissue-specific promoter is an adipocyte-specific promoter, such as, eg, adipocyte protein 2 (aP2, also known as fatty acid binding protein 4 (FABP4)), PPARy promoter, adiponectin promoter promoter, the phosphoenolpyruvate carboxykinase (PEPCK) promoter, which is derived from the human aromatase cytochrome p450 (p450arom) and Foxa-2 promoters. See Graves (1991), Ross (1990), Simpson (US 5,446,143), Mahendroo (1993), Simpson (1993) and Sasaki (1994), supra.
在一种实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。In one embodiment, the enhancer region is selected from the group consisting of a fat-specific aP2 enhancer and a fat-specific UCP1 enhancer.
在优选的实施方式中,根据本发明的AAV的脂肪组织特异调控区包含脂肪特异aP2增强子和鼠类aP2基本启动子(基本鼠类ap2启动子,basal murine aP2 promoter)。在特定的实施方式中,mini/aP2调控区具有序列表SEQ ID NO:2的序列。In a preferred embodiment, the adipose tissue-specific regulatory region of the AAV according to the present invention comprises an adipose-specific aP2 enhancer and a basal murine aP2 promoter (basal murine aP2 promoter). In a specific embodiment, the mini/aP2 regulatory region has the sequence of SEQ ID NO:2 of the Sequence Listing.
在另一优选的实施方式中,根据本发明的AAV的脂肪组织特异调控区包含脂肪特异UCP1增强子和大鼠UCP1基本启动子(基本大鼠UCP1启动子,basal rat UCP1 promoter)。在特定的实施方式中,mini/UCP1调控区具有序列表SEQ ID NO:3的序列。In another preferred embodiment, the adipose tissue-specific regulatory region of the AAV according to the present invention comprises an adipose-specific UCP1 enhancer and a rat UCP1 basal promoter (basal rat UCP1 promoter). In a specific embodiment, the mini/UCP1 regulatory region has the sequence of SEQ ID NO:3 of the Sequence Listing.
在另一实施方式中,表达框进一步包含转录后调控区。在优选的实施方式中,转录后调控区是WPRE或其功能变体和片段,以及PPT-CTS或其功能变体和片段。In another embodiment, the expression cassette further comprises a post-transcriptional regulatory region. In preferred embodiments, the post-transcriptional regulatory regions are WPRE or functional variants and fragments thereof, and PPT-CTS or functional variants and fragments thereof.
其他适合的感兴趣的多核苷酸可以用本发明的AAV载体并且用于疾病的治疗或预防。参见表1。Other suitable polynucleotides of interest can be used with the AAV vectors of the invention and for the treatment or prevention of disease. See Table 1.
表1Table 1
感兴趣的多核苷酸polynucleotide of interest
如本文中使用的术语“需要感兴趣的多核苷酸的表达的疾病”是指任何其中期望感兴趣的多核苷酸的表达的疾病。如本文中描述的感兴趣的多核苷酸可以是编码感兴趣的多肽的基因,或可替代地,在转录时产生能够调节细胞中内源多核苷酸的表达的分子的核酸序列。因此,需要感兴趣的多核苷酸的表达的疾病可以是,其中期望增加或降低基因的表达水平的疾病。The term "disease requiring expression of a polynucleotide of interest" as used herein refers to any disease in which expression of a polynucleotide of interest is desired. A polynucleotide of interest as described herein can be a gene encoding a polypeptide of interest, or alternatively, a nucleic acid sequence that, when transcribed, produces a molecule capable of modulating the expression of an endogenous polynucleotide in a cell. Thus, a disease requiring expression of a polynucleotide of interest may be a disease in which it is desired to increase or decrease the expression level of a gene.
此外,感兴趣的多核苷酸可以编码分泌并作用于全身的蛋白,或作用于脂肪细胞或其附近区域的蛋白。在特定的实施方式中,需要感兴趣的多核苷酸的表达的疾病是需要感兴趣的多核苷酸在脂肪组织中,更优选地,在白色脂肪组织或褐色脂肪组织中的表达的疾病。In addition, the polynucleotide of interest can encode a protein that is secreted and acts throughout the body, or a protein that acts on adipocytes or their vicinity. In a specific embodiment, the disease requiring expression of the polynucleotide of interest is a disease requiring expression of the polynucleotide of interest in adipose tissue, more preferably in white adipose tissue or brown adipose tissue.
需要感兴趣的多核苷酸的表达的疾病的实例包括,但不限于肥胖症、高血糖、胰岛素耐受、2型糖尿病、高血压、癌症、心脏病、免疫性疾病、关节炎、中枢神经系统的疾病和衰老相关的疾病。Examples of diseases requiring expression of the polynucleotide of interest include, but are not limited to, obesity, hyperglycemia, insulin resistance,
已经证明本发明的AAV对脂肪组织相关的疾病的基因治疗是有用的,如在WAT和BAT二者中由本发明的AAV介导的己糖激酶的递送,以增加基础葡萄糖摄取。参见图2D和4B。因此在特定的实施方式中,需要调节感兴趣的多核苷酸的表达的疾病是选自由肥胖症、高血糖、胰岛素耐受性、2型糖尿病和高血压的组成的组中的疾病。The AAVs of the invention have proven useful for gene therapy of adipose tissue-related diseases, such as the delivery of hexokinase mediated by the AAVs of the invention in both WAT and BAT to increase basal glucose uptake. See Figures 2D and 4B. Thus in particular embodiments, the disease requiring modulation of expression of the polynucleotide of interest is a disease selected from the group consisting of obesity, hyperglycemia, insulin resistance,
示例性的基因与相关的疾病状态包括,但不限于用于治疗糖尿病的胰岛素、用于治疗囊肿性纤维化的CFTR、用于治疗乙型血友病(hemophilia B)的IX因子、用于治疗甲型血友病(hemophilia A)的VIII因子、葡萄糖-6-磷酸酶,与肝糖贮积症1A型相关;磷酸烯醇丙酮酸-羧基酶,与Pepck不足相关;半乳糖-1-磷酸尿苷酰转移酶,与半乳糖血症相关;苯丙氨酸羟化酶,与苯丙酮尿症相关;支链α-酮酸脱氢酶,与枫糖尿症相关;富马酰乙酰乙酸水解酶,与1型酪氨酸血症相关;甲基丙二酰-辅酶A变位酶,与甲基丙二酸血症相关;中链酰基辅酶A脱氢酶,与中链酰基辅酶A缺乏症相关;鸟氨酸氨甲酰基转移酶,与鸟氨酸氨甲酰基转移酶缺乏症相关;精氨基琥珀酸合成酶,与瓜氨酸血症相关;低密度脂蛋白受体蛋白,与家族性高胆固醇血症相关;UDP-葡萄糖醛酸转移酶(UDP-glucouronosyltransferase),与克里格勒-纳贾尔症(Crigler-Najjar disease)相关;腺苷酸脱胺酶,与重度联合免疫缺陷疾病相关;次黄嘌呤鸟嘌呤磷酸核苷转移酶,与痛风(Gout)和Lesch-Nyan综合症相关;生物素酶,与生物素酶缺乏症相关;β-葡糖脑苷酯酶,与高歇疾病相关;β-葡糖醛酸糖苷酶与斯赖(Sly)综合症相关;过氧化物酶体膜蛋白70kDa,与Zellweger综合症相关;胆色素原脱氨酶,与急性间歇性卟啉症相关;α-1抗胰蛋白酶,用于治疗α-1抗胰蛋白酶缺乏症(肺气肿);红细胞生成素,用于治疗由于地中海贫血或肾衰竭的贫血;血管内皮生长因子、血管生成素-1和纤维原细胞生长因子,用于治疗缺血性疾病;血栓调节蛋白和组织因子途径抑制物,用于治疗如在,例如动脉粥样硬化、血栓、或栓塞中所见的闭塞的血管;芳香族氨基酸脱羧酶(AADC)和酪氨酸脱羧酶(TH),用于治疗帕金森症;β-肾上腺素能受体,受磷蛋白的反义或突变形式、肌质(内质)网腺苷三磷酸酶-2(SERCA2)和心脏腺苷酸环化酶,用于治疗充血性心力衰竭;肿瘤抑制基因,如p53,用于治疗各种癌症;细胞因子,如各种白细胞介素之一,用于治疗炎性和免疫功能失调以及癌症;肌营养不良蛋白或小肌营养不良蛋白(minidystrophin)和肌营养相关蛋白(utrophin)或小肌营养相关蛋白(miniutrophin)用于治疗肌营养不良;腺苷脱氨酶(ADA),用于治疗腺苷脱氨酶(ADA)缺乏症;亨廷顿蛋白(HTT),用于治疗亨廷顿氏舞蹈症;低密度脂蛋白受体(LDLR)或脱脂蛋白B(APOB),用于治疗家族性高胆甾醇血症;苯基丙氨酸羟化酶(PAH),用于治疗苯丙酮尿症;多囊性肾病变1(PKD1)和多囊性肾病变2(PKD2),用于治疗多囊性肾病变;TNFR:Fc,用于治疗关节炎;AAT,用于治疗遗传性肺气肿;肌聚糖,用于治疗肌肉萎缩症;GAD65或GAD67,用于治疗帕金森氏病,AAC,用于治疗卡纳万氏病(Canava's disease),CLN2,用于治疗巴滕氏病(Batten'sdisease),NGF,用于治疗阿尔茨海默氏病;VEGF拮抗剂,用于治疗黄斑变性;IGF/HGF,用于治疗充血性心力衰竭,NGF,用于治疗中枢神经系统紊乱;以及针对HIV的中和抗体,用于治疗HIV、HIV感染、或AIDS。Exemplary genes and associated disease states include, but are not limited to, insulin for treatment of diabetes, CFTR for treatment of cystic fibrosis, factor IX for treatment of hemophilia B, treatment for Factor VIII, glucose-6-phosphatase in hemophilia A (hemophilia A), associated with hepatoglycososis type 1A; phosphoenolpyruvate-carboxylase, associated with Pepck deficiency; galactose-1-phosphate Uridyltransferase, associated with galactosemia; phenylalanine hydroxylase, associated with phenylketonuria; branched-chain alpha-keto acid dehydrogenase, associated with maple syrup urine disease; hydrolysis of fumarylacetoacetate Enzymes, associated with type 1 tyrosinemia; methylmalonyl-CoA mutase, associated with methylmalonic acidemia; medium-chain acyl-CoA dehydrogenase, associated with medium-chain acyl-CoA deficiency ornithine carbamyltransferase, associated with ornithine carbamyltransferase deficiency; argininosuccinate synthase, associated with citrullinemia; low-density lipoprotein receptor protein, associated with the family associated with hypercholesterolemia; UDP-glucouronosyltransferase, associated with Crigler-Najjar disease; adenylate deaminase, associated with severe combined immunodeficiency Disease-related; hypoxanthine guanine phosphorylated nucleoside transferase, associated with gout (Gout) and Lesch-Nyan syndrome; biotinidase, associated with biotinidase deficiency; β-glucocerebrosidase, associated with high β-glucuronidase associated with Sly syndrome; peroxisomal membrane protein 70 kDa associated with Zellweger syndrome; porphyrinogen deaminase associated with acute intermittent porphyrin alpha-1 antitrypsin, used to treat alpha-1 antitrypsin deficiency (emphysema); erythropoietin, used to treat anemia due to thalassemia or renal failure; vascular endothelial growth factor, angiogenesis Thrombomodulin-1 and fibroblast growth factor for the treatment of ischemic diseases; thrombomodulin and tissue factor pathway inhibitors for the treatment of occlusions as seen in, eg, atherosclerosis, thrombosis, or embolism Vascular; aromatic amino acid decarboxylase (AADC) and tyrosine decarboxylase (TH), used in the treatment of Parkinson's disease; beta-adrenergic receptors, antisense or mutant forms of phosphoproteins, sarcoplasmic (endoplasmic) ) Reticulo-ATPase-2 (SERCA2) and cardiac adenylate cyclase, used to treat congestive heart failure; tumor suppressor genes, such as p53, used to treat various cancers; cytokines, such as various leukocytes One of interleukins used in the treatment of inflammatory and immune dysfunction and cancer; dystrophin or minidystrophin and utrophin or miniutrophin used in treatment muscular dystrophy; adenosine deaminase (ADA), with for the treatment of adenosine deaminase (ADA) deficiency; huntingtin protein (HTT) for the treatment of Huntington's disease; low-density lipoprotein receptor (LDLR) or apolipoprotein B (APOB) for the treatment of familial high blood pressure Cholesterolemia; phenylalanine hydroxylase (PAH), used to treat phenylketonuria; polycystic kidney disease 1 (PKD1) and polycystic kidney disease 2 (PKD2), used to treat polycystic nephropathy; TNFR:Fc, used to treat arthritis; AAT, used to treat hereditary emphysema; sarcoglycan, used to treat muscular dystrophy; GAD65 or GAD67, used to treat Parkinson's disease, AAC, For the treatment of Canava's disease, CLN2, for the treatment of Batten's disease, NGF, for the treatment of Alzheimer's disease; VEGF antagonist, for the treatment of macular degeneration; IGF/HGF, for the treatment of congestive heart failure, NGF, for the treatment of central nervous system disorders; and neutralizing antibodies against HIV, for the treatment of HIV, HIV infection, or AIDS.
可以通过本发明的AAV递送的感兴趣的多核苷酸的实例包括但不限于,己糖激酶、葡糖激酶、UCP2、UCP3、PPAR-α、瘦素、瘦素受体OB-Rb和GLP-1。在特定的实施方式中,感兴趣的基因选自由己糖激酶(HK)、葡糖激酶(GK)、碱性磷酸酶(AP)和血管内皮生长因子(VEGF)组成的组。在另一种实施方式中,将包含表达己糖激酶或葡糖激酶的多核苷酸的本发明的AAV给予需要其的受试者用于治疗或预防2型糖尿病。在另一实施方式中,将包含表达血管内皮生长因子的多核苷酸的本发明的AAV给予需要其的受试者用于治疗或预防肥胖症。Examples of polynucleotides of interest that can be delivered by the AAVs of the present invention include, but are not limited to, hexokinase, glucokinase, UCP2, UCP3, PPAR-alpha, leptin, leptin receptor OB-Rb, and GLP- 1. In particular embodiments, the gene of interest is selected from the group consisting of hexokinase (HK), glucokinase (GK), alkaline phosphatase (AP) and vascular endothelial growth factor (VEGF). In another embodiment, an AAV of the invention comprising a polynucleotide expressing hexokinase or glucokinase is administered to a subject in need thereof for the treatment or prevention of
可以用本发明的方法治疗的疾病的示例性的,但是非限制性的实例包括肥胖症、高血糖、胰岛素耐受性、2型糖尿病、高血压和高动脉压。优选地,2型糖尿病可以通过在脂肪细胞附近区域或全身表达瘦素使其到达下丘脑来治疗。Exemplary, but non-limiting examples of diseases that can be treated with the methods of the present invention include obesity, hyperglycemia, insulin resistance,
此外,已经证明本发明的AAV对BAT的遗传工程是有用的,因为通过AAV 9-mini/UCP1的VEGF164的内部iBAT给予导致总VEGF表达水平的增加和iBAT中血管数量增加。参见图4C-4F。因此,在特定的实施方式中,本发明涉及本发明的AAV或药物组合物用于治疗或预防需要VEGF表达的疾病,例如对该疾病的管理可以受益于诱导血管生成、动脉生成、或血管发生的那种疾病。需要VEGF的表达的疾病的实例包括,但不限于急性外科手术和外伤伤口、烧伤、烫伤、静脉性溃疡、动脉性溃疡、褥疮性溃疡(pressure sore)(又名褥疮(decubitusulcer))、糖尿病性溃疡、辐射后伤口、皮肤移植、混合病因的溃疡和其他慢性的或坏死性的伤口。Furthermore, the AAVs of the present invention have been shown to be useful for genetic engineering of BAT, as internal iBAT administration of VEGF164 via AAV 9-mini/UCP1 resulted in increased levels of total VEGF expression and increased number of blood vessels in iBAT. See Figures 4C-4F. Thus, in certain embodiments, the present invention relates to AAVs or pharmaceutical compositions of the present invention for use in the treatment or prevention of diseases requiring VEGF expression, eg, the management of which may benefit from induction of angiogenesis, arteriogenesis, or angiogenesis of that disease. Examples of diseases requiring expression of VEGF include, but are not limited to, acute surgical and traumatic wounds, burns, scalds, venous ulcers, arterial ulcers, pressure sore (aka decubitusulcer), diabetic Ulcers, post-radiation wounds, skin grafts, ulcers of mixed etiology, and other chronic or necrotizing wounds.
此外,用AAV9-mini/aP2病毒载体的hSeAP基因(即源自人类胎盘的分泌的碱性磷酸酶)的内部eWAT给予导致hSeAP的循环水平的持续增加。因此,在特定的实施方式中,本发明涉及本发明的AAV或药物组合物,以用于治疗或预防需要AP的表达的疾病,LPS(即脂多糖)介导的或恶化的疾病。需要AP的表达的疾病的实例包括,但不限于炎症性肠病、败血症或脓毒性休克、全身炎症反应综合征(SIRS)、脑膜炎球菌血症、创伤失血性休克、人身伤害(hum injury)、心血管外科手术或心肺转流术、肝脏手术或移植、肝病、胰腺炎、坏死性小肠结肠炎、牙周病、肺炎、囊肿性纤维化、哮喘、冠心病、充血性心力衰竭、肾病、溶血性尿毒症、肾脏透析、癌症、阿尔兹海默氏病和自身免疫性疾病,如类风湿性关节炎和系统性红斑狼疮。Furthermore, internal eWAT administration of the hSeAP gene (ie, secreted alkaline phosphatase derived from human placenta) with the AAV9-mini/aP2 viral vector resulted in a sustained increase in circulating levels of hSeAP. Accordingly, in a specific embodiment, the present invention relates to AAVs or pharmaceutical compositions of the present invention for use in the treatment or prevention of diseases requiring expression of AP, LPS (ie lipopolysaccharide) mediated or exacerbated diseases. Examples of diseases requiring expression of AP include, but are not limited to, inflammatory bowel disease, sepsis or septic shock, systemic inflammatory response syndrome (SIRS), meningococcalemia, traumatic hemorrhagic shock, hum injury , cardiovascular surgery or cardiopulmonary bypass, liver surgery or transplantation, liver disease, pancreatitis, necrotizing enterocolitis, periodontal disease, pneumonia, cystic fibrosis, asthma, coronary heart disease, congestive heart failure, kidney disease, Hemolytic uremic disease, renal dialysis, cancer, Alzheimer's disease and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus.
4.用于体外转导细胞的方法4. Methods for transducing cells in vitro
在第三方面,本发明涉及通过使用本发明的AAV载体用于体外转导细胞的方法。因此,本发明还涉及用于体外转导细胞的方法,包括将所述细胞与包含重组病毒基因组的AAV接触,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的脂肪组织特异的转录调控区。In a third aspect, the present invention relates to a method for transducing cells in vitro by using the AAV vector of the present invention. Accordingly, the present invention also relates to a method for transducing cells in vitro, comprising contacting said cells with an AAV comprising a recombinant viral genome comprising an expression cassette comprising a polynucleotide of interest Acid operably linked adipose tissue-specific transcriptional regulatory region.
在优选的实施方式中,用于体外转导细胞的方法的腺相关病毒载体具有选自由AAV6、AAV7、AAV8和AAV9血清型所组成的组的血清型。在另一实施方式中,腺相关病毒ITR是AAV2ITR。In a preferred embodiment, the adeno-associated viral vector used in the method of transducing cells in vitro has a serotype selected from the group consisting of AAV6, AAV7, AAV8 and AAV9 serotypes. In another embodiment, the adeno-associated virus ITR is AAV2 ITR.
在另一实施方式中,腺相关病毒载体包含脂肪组织特异的转录调控区。在又一实施方式中,脂肪组织特异的转录调控区包含选自鼠类aP2基本启动子和大鼠UCP1基本启动子组成的组的启动子区。在又一实施方式中,脂肪组织特异的转录调控区进一步包含与启动子区可操作地连接的增强子区。在又一实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。在更加优选的实施方式中,脂肪组织特异的转录调控区选自由以下组成的组:In another embodiment, the adeno-associated viral vector comprises an adipose tissue-specific transcriptional regulatory region. In yet another embodiment, the adipose tissue-specific transcriptional regulatory region comprises a promoter region selected from the group consisting of a murine aP2 basal promoter and a rat UCP1 basal promoter. In yet another embodiment, the adipose tissue-specific transcriptional regulatory region further comprises an enhancer region operably linked to the promoter region. In yet another embodiment, the enhancer region is selected from the group consisting of a fat-specific aP2 enhancer and a fat-specific UCP1 enhancer. In a more preferred embodiment, the adipose tissue-specific transcriptional regulatory region is selected from the group consisting of:
i)包含脂肪特异aP2增强子和鼠类aP2基本启动子的多核苷酸以及i) a polynucleotide comprising a fat-specific aP2 enhancer and a murine aP2 basic promoter and
ii)包含脂肪特异UCP1增强子和大鼠UCP1基本启动子的多核苷酸。ii) A polynucleotide comprising a fat-specific UCP1 enhancer and a rat UCP1 basal promoter.
在另一实施方式中,表达框进一步包含转录后调控区。在又一实施方式中,该转录后调控区是WPRE。In another embodiment, the expression cassette further comprises a post-transcriptional regulatory region. In yet another embodiment, the post-transcriptional regulatory region is WPRE.
在另一实施方式中,感兴趣的多核苷酸编码选自由作用于全身的分泌蛋白和作用于所述脂肪细胞或其附近区域的蛋白组成的组中的蛋白。在更优选的实施方式中,感兴趣的多核苷酸编码选自由己糖激酶、葡糖激酶、碱性磷酸酶和血管内皮生长因子组成的组的蛋白。In another embodiment, the polynucleotide of interest encodes a protein selected from the group consisting of a secreted protein that acts throughout the body and a protein that acts on the adipocyte or its vicinity. In a more preferred embodiment, the polynucleotide of interest encodes a protein selected from the group consisting of hexokinase, glucokinase, alkaline phosphatase and vascular endothelial growth factor.
在另一实施方式中,本发明涉及用于用包含重组病毒基因组的AAV体外转导细胞的方法,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的转录调控区,其中,AAV的血清型选自由AAV6、AAV7、AAV8和AAV9组成的组。In another embodiment, the present invention relates to a method for in vitro transduction of cells with AAV comprising a recombinant viral genome comprising an expression cassette that is operable with a polynucleotide of interest A tightly linked transcriptional regulatory region, wherein the AAV serotype is selected from the group consisting of AAV6, AAV7, AAV8, and AAV9.
在一种实施方式中,腺相关病毒ITR是AAV2ITR。In one embodiment, the adeno-associated virus ITR is AAV2 ITR.
在另一实施方式中,腺相关病毒载体包含含有转录调控区的重组基因组。在一种实施方式中,该转录调控区是组成型启动子。在优选的实施方式中,该组成型转录调控区包含肌动蛋白启动子。在又一实施方式中,该组成型转录调控区进一步包含与启动子区可操作地连接的增强子区。在更加优选的实施方式中,该增强子区是巨细胞病毒增强子。In another embodiment, the adeno-associated viral vector comprises a recombinant genome containing transcriptional regulatory regions. In one embodiment, the transcriptional regulatory region is a constitutive promoter. In a preferred embodiment, the constitutive transcriptional regulatory region comprises an actin promoter. In yet another embodiment, the constitutive transcriptional regulatory region further comprises an enhancer region operably linked to the promoter region. In a more preferred embodiment, the enhancer region is a cytomegalovirus enhancer.
在一种实施方式中,转录调控区是脂肪组织特异的转录调控区。在又一实施方式中,脂肪组织特异的转录调控区包含选自由鼠类aP2基本启动子和大鼠UCP1基本启动子组成的组的启动子区。在再一实施方式中,脂肪组织特异的转录调控区进一步包含与启动子区可操作地连接的增强子区。在又一实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。在更加优选的实施方式中,脂肪组织特异的转录调控区选自由以下组成的组:In one embodiment, the transcriptional regulatory region is an adipose tissue-specific transcriptional regulatory region. In yet another embodiment, the adipose tissue-specific transcriptional regulatory region comprises a promoter region selected from the group consisting of a murine aP2 basal promoter and a rat UCP1 basal promoter. In yet another embodiment, the adipose tissue-specific transcriptional regulatory region further comprises an enhancer region operably linked to the promoter region. In yet another embodiment, the enhancer region is selected from the group consisting of a fat-specific aP2 enhancer and a fat-specific UCP1 enhancer. In a more preferred embodiment, the adipose tissue-specific transcriptional regulatory region is selected from the group consisting of:
i)包含脂肪特异aP2增强子和鼠类aP2基本启动子的多核苷酸以及i) a polynucleotide comprising a fat-specific aP2 enhancer and a murine aP2 basic promoter and
ii)包含脂肪特异UCP1增强子和大鼠UCP1基本启动子的多核苷酸。ii) A polynucleotide comprising a fat-specific UCP1 enhancer and a rat UCP1 basal promoter.
在另一实施方式中,表达框进一步包含转录后调控区。在又一实施方式中,该转录后调控区是WPRE。In another embodiment, the expression cassette further comprises a post-transcriptional regulatory region. In yet another embodiment, the post-transcriptional regulatory region is WPRE.
在另一实施方式中,感兴趣的多核苷酸编码选自由以下所组成的组的蛋白:作用于全身的分泌蛋白和作用于所述脂肪细胞或其附近区域的蛋白。在更加优选的实施方式中,感兴趣的多核苷酸编码选自由己糖激酶、葡糖激酶、碱性磷酸酶和血管内皮生长因子组成的组的蛋白。In another embodiment, the polynucleotide of interest encodes a protein selected from the group consisting of a secreted protein that acts throughout the body and a protein that acts on the adipocyte or its vicinity. In a more preferred embodiment, the polynucleotide of interest encodes a protein selected from the group consisting of hexokinase, glucokinase, alkaline phosphatase, and vascular endothelial growth factor.
使用本发明的体外方法可以转导任何细胞。在特定的实施方式中,AAV用于转导脂肪组织细胞。在更加优选的实施方式中,脂肪组织细胞是褐色脂肪细胞或白色脂肪细胞。Any cell can be transduced using the in vitro methods of the present invention. In a specific embodiment, AAV is used to transduce adipose tissue cells. In a more preferred embodiment, the adipose tissue cells are brown adipocytes or white adipocytes.
当进行根据本发明的用于转导细胞的体外方法以转导白色脂肪细胞时,AAV内的转录调控区优选地包含mini/aP2调控区。在另一实施方式中,当进行根据本发明的用于转导细胞的体外方法以转导棕色脂肪细胞时,AAV内的转录调控区优选地包含表达框,该表达框包含mini/UCP1调控区。When performing the in vitro method for transducing cells according to the present invention to transduce white adipocytes, the transcriptional regulatory region within the AAV preferably comprises the mini/aP2 regulatory region. In another embodiment, when performing the in vitro method for transducing cells according to the present invention to transduce brown adipocytes, the transcriptional regulatory region within the AAV preferably comprises an expression cassette comprising the mini/UCP1 regulatory region .
在本发明的另一方面,为改善由mini/aP2和mini/UCP1启动子获得的转基因表达,CAG启动子与组织特异的miRNA靶序列结合使用,以求获得脂肪组织中的高表达水平和由脱靶器官的去靶转基因表达。这导致当局部或全身给予时,AAV载体遗传性修改脂肪组织的潜力进一步加强。In another aspect of the invention, to improve transgene expression obtained from the mini/aP2 and mini/UCP1 promoters, the CAG promoter was used in combination with tissue-specific miRNA target sequences to obtain high expression levels in adipose tissue and Off-target transgene expression in off-target organs. This leads to further enhancement of the potential of AAV vectors to genetically modify adipose tissue when administered locally or systemically.
在另外的实施方式,本发明涉及通过使用本发明的AAV载体并将它们在体外培养用于分离体内转导的细胞的方法。在另一实施方式中,本发明涉及所述分离的转导细胞以及该细胞和包含它们的药物组合物。In a further embodiment, the present invention relates to methods for isolating cells transduced in vivo by using the AAV vectors of the present invention and culturing them in vitro. In another embodiment, the present invention relates to the isolated transduced cells as well as the cells and pharmaceutical compositions comprising them.
5.转导的脂肪细胞和脂肪细胞细胞组合物,离体治疗方法5. Transduced adipocytes and adipocyte cell compositions, ex vivo methods of treatment
在第四方面,本发明涉及通过本发明的体外方法获得的脂肪细胞。在另一实施方式中,本发明涉及包含根据本发明的方法获得的脂肪细胞的细胞组合物。此外,本发明还涉及包含根据本发明的AAV的基因组的脂肪细胞或脂肪细胞细胞组合物。优选地,至少50%的细胞组合物包含根据本发明的脂肪细胞。更优选地,至少60%、70%、80%、90%、95%和100%的细胞组合物包含根据本发明的脂肪细胞。In a fourth aspect, the present invention relates to adipocytes obtained by the in vitro method of the present invention. In another embodiment, the present invention relates to a cellular composition comprising adipocytes obtained according to the method of the present invention. Furthermore, the present invention also relates to adipocytes or adipocyte cell compositions comprising the genome of the AAV according to the present invention. Preferably, at least 50% of the cellular composition comprises adipocytes according to the present invention. More preferably, at least 60%, 70%, 80%, 90%, 95% and 100% of the cellular composition comprise adipocytes according to the present invention.
如上所述,本发明的AAV可用于体外转导细胞以将感兴趣的多核苷酸引入所述细胞。接着,可以将转导细胞植入人或动物体内以获得期望的治疗效果。As described above, the AAVs of the present invention can be used to transduce cells in vitro to introduce a polynucleotide of interest into the cells. The transduced cells can then be implanted in humans or animals to achieve the desired therapeutic effect.
因此,在另一实施方式中,本发明涉及包含根据本发明的方法获得的脂肪细胞的脂肪细胞或细胞组合物用于在药物中使用。Thus, in another embodiment, the present invention relates to adipocytes or cell compositions comprising adipocytes obtained according to the method of the present invention for use in medicine.
在另一实施方式中,本发明涉及包含根据本发明的方法获得的脂肪细胞的脂肪细胞或细胞组合物,以用于需要感兴趣的多核苷酸的表达的疾病的治疗。In another embodiment, the present invention relates to adipocytes or cellular compositions comprising adipocytes obtained according to the methods of the present invention for use in the treatment of diseases requiring expression of a polynucleotide of interest.
在另一实施方式中,本发明涉及用于治疗或预防疾病的方法,其包括将根据本发明的方法获得的脂肪细胞或细胞组合物给予至需要其的受试者。可以用此途径解决的疾病的实例已经在以上本发明的AAV的上下文中限定。In another embodiment, the present invention relates to a method for treating or preventing a disease, comprising administering to a subject in need thereof an adipocyte or cellular composition obtained according to the method of the present invention. Examples of diseases that can be addressed by this approach have been defined above in the context of the AAV of the present invention.
6.多核苷酸、载体和质粒6. Polynucleotides, Vectors and Plasmids
在第五方面,本发明涉及对产生根据本发明的AAV有用的多核苷酸。因此,在另一实施方式中,本发明涉及多核苷酸(“本发明的多核苷酸”),其包含通过腺相关病毒ITR侧接的表达框,其中,所述表达框包含与感兴趣的多核苷酸可操作地连接的脂肪组织特异调控区。In a fifth aspect, the present invention relates to polynucleotides useful for producing AAVs according to the present invention. Accordingly, in another embodiment, the present invention relates to a polynucleotide ("polynucleotide of the present invention") comprising an expression cassette flanked by an adeno-associated virus ITR, wherein the expression cassette comprises an An adipose tissue-specific regulatory region to which a polynucleotide is operably linked.
在优选的实施方式中,脂肪组织特异的调控区包含选自由鼠类aP2基本启动子和大鼠UCP1基本启动子的组的启动子区。In a preferred embodiment, the adipose tissue-specific regulatory region comprises a promoter region selected from the group consisting of a murine aP2 basal promoter and a rat UCP1 basal promoter.
在另一实施方式中,脂肪组织特异的调控区进一步包含与启动子区可操作地连接的增强子区。在更加优选的实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。In another embodiment, the adipose tissue-specific regulatory region further comprises an enhancer region operably linked to the promoter region. In a more preferred embodiment, the enhancer region is selected from the group consisting of a fat-specific aP2 enhancer and a fat-specific UCP1 enhancer.
在另一实施方式中,该调控区选自由以下所组成的组:In another embodiment, the regulatory region is selected from the group consisting of:
i)包含脂肪的特异aP2增强子和鼠类aP2基本启动子的多核苷酸以及i) a polynucleotide comprising a fat specific aP2 enhancer and a murine aP2 basic promoter and
ii)包含脂肪特异UCP1增强子和大鼠UCP1基本启动子的多核苷酸。ii) A polynucleotide comprising a fat-specific UCP1 enhancer and a rat UCP1 basal promoter.
在另一实施方式中,本发明的多核苷酸的表达框进一步包含转录后调控元件。在又一实施方式中,转录后调控区是WPRE。In another embodiment, the expression cassette of the polynucleotide of the invention further comprises post-transcriptional regulatory elements. In yet another embodiment, the post-transcriptional regulatory region is a WPRE.
在另一实施方式中,包含在本发明的多核苷酸中的感兴趣的多核苷酸编码选自由己糖激酶、葡糖激酶、碱性磷酸酶和血管内皮生长因子组成的组的蛋白。In another embodiment, the polynucleotide of interest comprised in the polynucleotides of the present invention encodes a protein selected from the group consisting of hexokinase, glucokinase, alkaline phosphatase, and vascular endothelial growth factor.
本发明的多核苷酸可以结合至载体如,例如质粒。因此,在另外的实施方式中,本发明涉及包含本发明的多核苷酸的载体或质粒。根据本发明,术语“载体”和“质粒”是可互换的。The polynucleotides of the present invention can be conjugated to a vector such as, for example, a plasmid. Accordingly, in further embodiments, the present invention relates to vectors or plasmids comprising the polynucleotides of the present invention. According to the present invention, the terms "vector" and "plasmid" are interchangeable.
在特定的实施方式中,本发明的多核苷酸结合至腺相关的病毒载体或质粒。优选地,所有的其他生产腺相关病毒所需的结构和非结构编码序列不存在于病毒载体中,因为可以将它们通过另一载体,如质粒,或通过将序列稳定地结合至包装细胞系,以反式提供。In specific embodiments, the polynucleotides of the invention are conjugated to adeno-associated viral vectors or plasmids. Preferably, all other structural and non-structural coding sequences required for adeno-associated virus production are not present in the viral vector, as they can be passed through another vector, such as a plasmid, or by stably binding the sequences to a packaging cell line, Provided in trans.
本发明的多核苷酸可以使用本领域中已知的分子生物学技术获得。参见上文的Brown(1995)、Watson(1992)、Alberts(2008)、Innis(1990)、Erlich(1989)、Sambrook(1989)、Bishop(1987)、Reznikoff(1987)、Davis(1986)和Schleef(2001)。在另一实施方式中,本发明涉及AAV载体,其中,基因组包含本发明的多核苷酸。The polynucleotides of the present invention can be obtained using molecular biology techniques known in the art. See Brown (1995), Watson (1992), Alberts (2008), Innis (1990), Erlich (1989), Sambrook (1989), Bishop (1987), Reznikoff (1987), Davis (1986) and Schleef, supra (2001). In another embodiment, the present invention relates to an AAV vector, wherein the genome comprises a polynucleotide of the present invention.
7.用于获得AAV的方法7. Methods for obtaining AAV
在第六方面,本发明涉及用于获得本发明的AAV的方法。所述AAV可以通过将本发明的多核苷酸引入至组成型表达rep和cap的细胞来获得。因此,在另一实施方式中,本发明涉及用于获得腺相关病毒载体的方法,包括以下步骤:In a sixth aspect, the present invention relates to a method for obtaining the AAV of the present invention. The AAV can be obtained by introducing a polynucleotide of the invention into cells constitutively expressing rep and cap. Therefore, in another embodiment, the present invention relates to a method for obtaining an adeno-associated viral vector, comprising the steps of:
i)提供包含由AAV ITR侧接的本发明的多核苷酸、AAV cap蛋白、AAV rep蛋白或AAV依赖于其复制的病毒或细胞蛋白的细胞,i) providing a cell comprising a polynucleotide of the invention flanked by an AAV ITR, an AAV cap protein, an AAV rep protein or a viral or cellular protein on which AAV depends for replication,
ii)将该细胞维持在适合AAV组装的条件下,以及ii) maintaining the cell under conditions suitable for AAV assembly, and
iii)纯化由该细胞生产的腺相关病毒载体。iii) purifying the adeno-associated virus vector produced by the cell.
在优选的实施方式中,构成本发明的多核苷酸的部分的脂肪组织特异调控区包含选自鼠类aP2基本启动子和大鼠UCP1基本启动子的组的启动子区。In a preferred embodiment, the adipose tissue-specific regulatory region forming part of the polynucleotide of the present invention comprises a promoter region selected from the group consisting of a murine aP2 basal promoter and a rat UCP1 basal promoter.
在另一实施方式中,脂肪组织特异的调控区进一步包含与启动子区可操作地连接的增强子区。在更加优选的实施方式中,增强子区选自由脂肪特异aP2增强子和脂肪特异UCP1增强子组成的组。In another embodiment, the adipose tissue-specific regulatory region further comprises an enhancer region operably linked to the promoter region. In a more preferred embodiment, the enhancer region is selected from the group consisting of a fat-specific aP2 enhancer and a fat-specific UCP1 enhancer.
在另一实施方式中,该调控区选自由以下所组成的组:In another embodiment, the regulatory region is selected from the group consisting of:
i)包含脂肪的特异aP2增强子和鼠类aP2基本启动子的多核苷酸以及i) a polynucleotide comprising a fat specific aP2 enhancer and a murine aP2 basic promoter and
ii)包含脂肪特异UCP1增强子和大鼠UCP1基本启动子的多核苷酸。ii) A polynucleotide comprising a fat-specific UCP1 enhancer and a rat UCP1 basal promoter.
在另一实施方式中,本发明的多核苷酸的表达框进一步包含转录后调控元件。在又一实施方式中,该转录后调控区是WPRE。In another embodiment, the expression cassette of the polynucleotide of the invention further comprises post-transcriptional regulatory elements. In yet another embodiment, the post-transcriptional regulatory region is WPRE.
在另一实施方式中,包含在本发明的多核苷酸中的感兴趣的多核苷酸编码选自由己糖激酶、葡糖激酶、碱性磷酸酶和血管内皮生长因子组成的组的蛋白。In another embodiment, the polynucleotide of interest comprised in the polynucleotides of the present invention encodes a protein selected from the group consisting of hexokinase, glucokinase, alkaline phosphatase, and vascular endothelial growth factor.
用于载体化转基因的重组AAV(rAAV)的生产已经在前文描述。参见Ayuso E等,Curr.Gene Ther.2010;10:423-436、Okada T等,Hum.Gene Ther.2009;20:1013-1021、Zhang H等,Hum.Gene Ther.2009;20:922-929和Virag T等,Hum.Gene Ther.2009;20:807-817。可以使用或修改这些实验报告以生成本发明的AAV。在一种实施方式中,将生产细胞系用本发明的多核苷酸(包含由ITR侧接的表达框)以及用编码rep和cap蛋白并且提供辅助功能的结构暂时转染。在另一实施方式中,生产细胞系稳定地提供辅助功能,并且用本发明的多核苷酸(包含由ITR侧接的表达框)以及用编码rep和cap蛋白的结构暂时转染。在另一实施方式中,细胞系稳定地提供rep和cap蛋白以及辅助功能,并且用本发明的多核苷酸暂时转染。在另一实施方式中,细胞系稳定地提供rep和cap蛋白,并且用本发明的多核苷酸以及编码辅助功能的多核苷酸暂时转染。在又一实施方式中,细胞系稳定地提供本发明的多核苷酸、rep和cap蛋白以及辅助功能。制备并使用这些的方法和其他AAV生产系统已经在本领域中描述。参见Muzyczka N等,US 5,139,941、Zhou X等,US 5,741,683、Samulski R等,US6,057,152、Samulski R等,US 6,204,059、Samulski R等,US 6,268,213、Rabinowitz J等,US 6,491,907、Zolotukhin S等,US 6,660,514、Shenk T等,US 6,951,753、Snyder R等,US7,094,604、Rabinowitz J等,US 7,172,893、Monahan P等,US 7,201,898、Samulski R等,US 7,229,823和Ferrari F等,US 7,439,065。The production of recombinant AAV (rAAV) for vectorized transgenes has been described above. See Ayuso E et al, Curr. Gene Ther. 2010; 10:423-436, Okada T et al, Hum. Gene Ther. 2009; 20: 1013-1021, Zhang H et al, Hum. Gene Ther. 2009; 20:922- 929 and Virag T et al. Hum. Gene Ther. 2009;20:807-817. These experimental reports can be used or modified to generate the AAVs of the present invention. In one embodiment, a producer cell line is transiently transfected with a polynucleotide of the invention (comprising an expression cassette flanked by ITRs) and with constructs encoding rep and cap proteins and providing helper functions. In another embodiment, a producer cell line stably provides helper functions and is transiently transfected with a polynucleotide of the invention (comprising an expression cassette flanked by an ITR) and with constructs encoding rep and cap proteins. In another embodiment, the cell line stably provides rep and cap proteins and helper functions and is transiently transfected with a polynucleotide of the invention. In another embodiment, the cell line stably provides rep and cap proteins and is transiently transfected with a polynucleotide of the invention and a polynucleotide encoding a helper function. In yet another embodiment, the cell line stably provides the polynucleotides of the invention, rep and cap proteins, and helper functions. Methods of making and using these and other AAV production systems have been described in the art. See Muzyczka N et al, US 5,139,941, Zhou X et al, US 5,741,683, Samulski R et al, US 6,057,152, Samulski R et al, US 6,204,059, Samulski R et al, US 6,268,213, Rabinowitz J et al. , Shenk T et al, US 6,951,753, Snyder R et al, US 7,094,604, Rabinowitz J et al, US 7,172,893, Monahan P et al, US 7,201,898, Samulski R et al, US 7,229,823 and Ferrari F et al, US 7,439,065.
在另一实施方式中,可以通过提供两种可以退火以形成头接尾的串连体的基因组的AAV ITR来增加AAV的转基因递送容量。通常,当AAV进入宿主细胞时,含有转基因的单链DNA由宿主细胞DNA聚合酶复合物转化为双链DNA,其后ITR帮助在细胞核中形成串连体。作为替代,AAV可以设计为自身互补(sc)的AAV,其使得病毒载体可以在进入靶细胞时绕过第二链合成的步骤,提供具有更快的,潜在更高的(例如多达100次折叠)转基因表达的scAAV病毒载体。例如,可以将AAV设计为具有包含两个相连的单链DNA的基因组,该单链DNA分别编码转基因单元及其补体,其可以随着递送至靶细胞而结合,产出编码感兴趣的转基因单元的双链DNA。自身互补的AAV已经在本领域中描述。参见Carter B,US 6,596,535、CarterB,US 7,125,717和Takano H等,US 7,456,683。In another embodiment, the transgene delivery capacity of AAV can be increased by providing two AAV ITRs that can anneal to form a head-to-tail tandem of genomes. Typically, when AAV enters a host cell, the single-stranded DNA containing the transgene is converted to double-stranded DNA by the host cell DNA polymerase complex, after which ITR helps form tandem in the nucleus. Alternatively, AAVs can be designed as self-complementary (sc) AAVs that allow viral vectors to bypass the step of second-strand synthesis upon entry into target cells, providing faster, potentially higher (e.g., up to 100 times) folded) scAAV viral vector for transgene expression. For example, an AAV can be designed to have a genome comprising two linked single-stranded DNA encoding a transgene unit and its complement, respectively, which can bind upon delivery to target cells, yielding a transgenic unit encoding the transgenic unit of interest of double-stranded DNA. Self-complementary AAVs have been described in the art. See Carter B, US 6,596,535, Carter B, US 7,125,717 and Takano H et al. US 7,456,683.
已经报告Cap蛋白对宿主嗜性、细胞、组织、或器官特异性、受体使用、侵染效率和AAV病毒的免疫原性的具有作用。因此,可以考虑以下因素选择用于rAAV的AAV Cap,例如受试者的种族(例如,人类或非人类)、受试者的免疫状态、受试者的对长期或短期治疗的适应性、或特定的治疗应用(例如,特定的疾病或失调的治疗,或递送至特定的细胞、组织、或器官)。在另一实施方式中,rAAV Cap是基于来自两种或三种或更多种的AAV血清型的Cap。在特定的实施方式中,AAV cap基因源自血清型AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8、或AAV9。在优选的实施方式中,AAV cap基因源自血清型AAV6、AAV7、AAV8和AAV9。Cap proteins have been reported to have effects on host tropism, cell, tissue, or organ specificity, receptor usage, infection efficiency, and immunogenicity of AAV viruses. Thus, an AAV Cap for rAAV can be selected taking into account factors such as the race of the subject (eg, human or non-human), the immune status of the subject, the subject's suitability for long-term or short-term treatment, or A specific therapeutic application (eg, treatment of a specific disease or disorder, or delivery to a specific cell, tissue, or organ). In another embodiment, the rAAV Cap is based on Caps from two or three or more AAV serotypes. In specific embodiments, the AAV cap gene is derived from serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In a preferred embodiment, the AAV cap gene is derived from serotypes AAV6, AAV7, AAV8 and AAV9.
在一些实施方式中,用于本发明的方法的AAV Cap可以通过诱变(即通过插入、删除、或替代)前述AAV Cap或其编码核酸之一生成。在一些实施方式中,AAV Cap与前述的一种或多种AAV Cap相似至少70%、75%、80%、85%、90%、95%、98%、或99%。In some embodiments, AAV Caps for use in the methods of the invention can be generated by mutagenesis (ie, by insertion, deletion, or substitution) of one of the aforementioned AAV Caps or their encoding nucleic acids. In some embodiments, the AAV Cap is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% similar to one or more of the aforementioned AAV Caps.
在一些实施方式中,AAV Cap是嵌合的,包含来自至少两种前述AAV Cap的结构域。在一些实施方式中,AAV Cap是来自两种或三种不同的AAV或重组AAV的VP1、VP2和VP3单体的嵌合体。在一些实施方式中,rAAV组合物包含多于一种的前述Cap。In some embodiments, the AAV Cap is chimeric, comprising domains from at least two of the aforementioned AAV Caps. In some embodiments, the AAV Cap is a chimera of VP1, VP2, and VP3 monomers from two or three different AAVs or recombinant AAVs. In some embodiments, the rAAV composition comprises more than one of the aforementioned Caps.
在一些实施方式中,将用于rAAV组合物中的AAV Cap设计为包含异源序列或其他修饰。例如,可以将赋予选择性定位或免疫逃避的肽或蛋白序列设计进入Cap蛋白。可替代地或另外地,Cap可以化学改性,使得rAAV的表面是聚乙二醇化(即聚乙二醇修饰),其可以促进免疫逃避。Cap蛋白还可以是诱变的(例如,去除其天然受体结合,或掩盖免疫原表位)。In some embodiments, AAV Caps for use in rAAV compositions are designed to contain heterologous sequences or other modifications. For example, peptide or protein sequences that confer selective localization or immune evasion can be engineered into Cap proteins. Alternatively or additionally, Cap can be chemically modified such that the surface of the rAAV is pegylated (ie, polyethylene glycol modification), which can facilitate immune evasion. Cap proteins can also be mutagenized (eg, to remove their native receptor binding, or to mask immunogenic epitopes).
在特定的实施方式中,AAV rep基因源自血清型AAV1、AAV2、AAV4、AAV5、AAV6、AAV7、AAV8和AAV9。在优选的实施方式中,AAV rep和cap基因源自血清型AAV6、AAV7、AAV8和AAV9。In specific embodiments, the AAV rep gene is derived from serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In a preferred embodiment, the AAV rep and cap genes are derived from serotypes AAV6, AAV7, AAV8 and AAV9.
基因AAV rep、AAV cap和提供辅助功能的基因可以通过使所述基因结合至载体,例如质粒,并将所述载体引入细胞而引入至细胞。可以将基因结合至相同的质粒或不同的质粒。在优选的实施方式中,将AAV rep和cap基因结合至一种质粒并将提供辅助功能的基因结合至另一种质粒。适用于本发明的方法的包含AAV rep和cap基因的质粒的实例包括pHLP19和pRep6cap载体。参见Colisi P,US 6,001,650和Russell D等,US 6,156,30。在优选的实施方式中,提供辅助功能的基因源自腺病毒。The genes AAV rep, AAV cap, and genes that provide helper functions can be introduced into cells by incorporating the genes into a vector, such as a plasmid, and introducing the vector into the cell. The genes can be bound to the same plasmid or to different plasmids. In a preferred embodiment, the AAV rep and cap genes are incorporated into one plasmid and the genes providing helper functions are incorporated into the other plasmid. Examples of plasmids comprising AAV rep and cap genes suitable for use in the methods of the present invention include pHLP19 and pRep6cap vectors. See Colisi P, US 6,001,650 and Russell D et al. US 6,156,30. In a preferred embodiment, the gene providing the helper function is derived from an adenovirus.
本发明的多核苷酸以及包含AAV rep和cap基因或提供辅助功能的基因的多核苷酸可以通过使用任何适合的本领域中已知的方法引入细胞。参见Ausubel等,Eds.,“ShortProtocols in Molecular Biology”,4th Ed.(John Wiley and Sons,Inc.,New York,NY,US,1997),上文的Brown(1995)、Watson(1992)、Alberts(2008)、Innis(1990)、Erlich(1989)、Sambrook(1989)、Bishop(1987)、Reznikoff(1987)、Davis(1986)和Schleef(2001)。转染方法的实例包括,但不限于用磷酸钙、DEAE-葡聚糖、聚凝胺共沉淀、电穿孔、显微注射、脂质体介导的融合、脂转染、逆转录病毒感染和基因枪(biolistic)转染。在特定的实施方式中,通过用磷酸钙的共沉淀进行转染。当细胞缺少任何AAV rep和cap基因以及提供腺病毒辅助功能的基因的表达时,可以将所述基因与本发明第一方面的多核苷酸同时引入细胞。可替代地,可以将所述基因在引入本发明第一方面的多核苷酸之前或之后引入细胞。在特定的实施方式中,将细胞与以下三种质粒同时转染:The polynucleotides of the present invention, as well as polynucleotides comprising AAV rep and cap genes or genes providing helper functions, can be introduced into cells using any suitable method known in the art. See Ausubel et al., Eds., "Short Protocols in Molecular Biology", 4th Ed. (John Wiley and Sons, Inc., New York, NY, US, 1997), supra Brown (1995), Watson (1992), Alberts (2008), Innis (1990), Erlich (1989), Sambrook (1989), Bishop (1987), Reznikoff (1987), Davis (1986) and Schleef (2001). Examples of transfection methods include, but are not limited to, co-precipitation with calcium phosphate, DEAE-dextran, polybrene, electroporation, microinjection, liposome-mediated fusion, lipofection, retroviral infection, and Biolistic transfection. In a specific embodiment, transfection is performed by co-precipitation with calcium phosphate. When the cell lacks expression of any AAV rep and cap genes and genes that provide adenovirus helper functions, the genes can be introduced into the cell simultaneously with the polynucleotide of the first aspect of the invention. Alternatively, the gene may be introduced into the cell before or after introduction of the polynucleotide of the first aspect of the invention. In a specific embodiment, cells are transfected simultaneously with the following three plasmids:
1)包含本发明的多核苷酸的质粒1) Plasmids comprising the polynucleotides of the present invention
2)包含AAV rep和cap基因的质粒2) Plasmids containing AAV rep and cap genes
3)包含提供辅助功能的基因的质粒3) Plasmids containing genes that provide helper functions
培养包装细胞的方法和促进AAV载体颗粒释放,如产生细胞裂解物的示例性条件,可以根据本文实施例中所描述地进行。生产细胞生长适合的时段以促进病毒载体释放入介质。通常,细胞可以成长约24小时、约36小时、约48小时、约72小时、约4天、约5天、约6天、约7天、约8天、约9天、多达约10天。在约10天后(或更快,取决于培养条件和使用的特定的生产细胞),生产水平通常显著下降。通常,从产生病毒的点测量培养的时间。例如,在AAV的情况下,通常当在如本文中描述的适当的生产细胞中提供辅助病毒功能时开始病毒产生。通常,在辅助病毒侵染之后(或开始产生病毒之后)约48至约100,优选地约48至约96,优选地约72至96,优选地约68至约72小时收获细胞。Exemplary conditions for culturing packaging cells and promoting AAV vector particle release, such as producing cell lysates, can be performed as described in the Examples herein. The producer cells are grown for a suitable period of time to facilitate release of the viral vector into the medium. Typically, cells can grow for about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, up to about 10 days . After about 10 days (or sooner, depending on the culture conditions and the particular producer cells used), production levels typically drop significantly. Typically, the time of incubation is measured from the point of virus production. For example, in the case of AAV, virus production typically begins when helper virus function is provided in an appropriate producer cell as described herein. Typically, cells are harvested about 48 to about 100, preferably about 48 to about 96, preferably about 72 to 96, preferably about 68 to about 72 hours after helper virus infection (or after virus production begins).
本发明的AAV可以由:i)用本发明的多核苷酸转染的细胞和ii)转染后一段时间之后,优选地72小时后的所述细胞的培养基二者获得。可以使用任何用于纯化来自所述细胞或所述培养基的AAV以获得本发明的AAV。在特定的实施方式中,遵循基于聚乙二醇沉淀步骤和两个连续的氯化铯(CsCl)梯度的优化方法纯化本发明的AAV。参见上文的Ayuso,2010。本发明的纯化的AAV可以用PBS渗析,过滤并在-80℃下储存。病毒基因组的滴度可以通过定量PCR测定,该测定遵循对于使用线性化的质粒DNA作为标准曲线的AAV2参考标准材料描述的流程。参见Lock M等,Hum.Gene Ther.2010;21:1273-1285。The AAVs of the invention can be obtained from both: i) cells transfected with the polynucleotides of the invention and ii) the culture medium of said cells some time after transfection, preferably 72 hours later. Any AAV used to purify the cells or the culture medium to obtain the AAV of the present invention can be used. In a specific embodiment, the AAV of the invention is purified following an optimized method based on a polyethylene glycol precipitation step and two consecutive cesium chloride (CsCl) gradients. See Ayuso, 2010, supra. The purified AAV of the present invention can be dialyzed against PBS, filtered and stored at -80°C. The titer of the viral genome can be determined by quantitative PCR following the procedure described for the AAV2 reference standard material using linearized plasmid DNA as the standard curve. See Lock M et al., Hum. Gene Ther. 2010;21:1273-1285.
在一些实施方式中,该方法进一步包括纯化步骤,如用核酸酶处理细胞裂解物,在CsCl梯度上纯化细胞裂解物,或通过硫酸肝素层析纯化细胞裂解物。参见Halbert C等,Methods Mol.Biol.2004;246:201-212。In some embodiments, the method further comprises a purification step, such as treating the cell lysate with a nuclease, purifying the cell lysate on a CsCl gradient, or purifying the cell lysate by heparin sulfate chromatography. See Halbert C et al, Methods Mol. Biol. 2004;246:201-212.
适用于生产AAV的,各种自然发生的和重组的AAV、它们的编码核酸、AAV Cap和Rep蛋白和它们的序列、以及用于分离或产生、繁殖和纯化这种AAV,以及特别是它们的衣壳的方法是本领域中已知的。参见上文的Gao,2004、Russell D等,US 6,156,303、Hildinger M等,US 7,056,502、Gao G等,US 7,198,951、Zolotukhin S,US 7,220,577、Gao G等,US 7,235,393、Gao G等,US 7,282,199、Wilson J等,US 7,319,002、Gao G等,US 7,790,449、GaoG等,US 20030138772、Gao G等,US 20080075740、Hildinger M等,WO 2001/083692、WilsonJ等,WO 2003/014367、Gao G等,WO 2003/042397、Gao G等,WO 2003/052052、Wilson J等,WO 2005/033321、Vandenberghe L等,WO 2006/110689、Vandenberghe L等,WO 2007/127264和Vandenberghe L等,WO 2008/027084。Suitable for the production of AAV, various naturally occurring and recombinant AAVs, their encoding nucleic acids, AAV Cap and Rep proteins and their sequences, and for the isolation or production, propagation and purification of such AAVs, and in particular their Methods of capsid are known in the art. See above, Gao, 2004, Russell D et al, US 6,156,303, Hildinger M et al, US 7,056,502, Gao G et al, US 7,198,951, Zolotukhin S, US 7,220,577, Gao G et al, US 7,235,393, Gao G et al, US 7,282,199, Wilson J et al, US 7,319,002, Gao G et al, US 7,790,449, Gao G et al, US 20030138772, Gao G et al, US 20080075740, Hildinger M et al, WO 2001/083692, Wilson J et al, WO 2003/014367, Gao G et al, WO 2003/ 042397, Gao G et al, WO 2003/052052, Wilson J et al, WO 2005/033321, Vandenberghe L et al, WO 2006/110689, Vandenberghe L et al, WO 2007/127264 and Vandenberghe L et al, WO 2008/027084.
8.药物组合物8. Pharmaceutical compositions
本发明的AAV可以通过传统方法给予至人或动物体,其需要所述载体在药物组合物中的制剂。因此,在第七方面,本发明涉及包含AAV的药物组合物(以下称为“本发明的药物组合物”),其中,所述AAV包含重组病毒基因组,其中,所述重组病毒基因组包含表达框,该表达框包含与感兴趣的多核苷酸可操作地连接的脂肪组织特异转录调控区。可替代地,本发明的药物组合物可以包含本发明的多核苷酸或多肽。The AAVs of the present invention can be administered to the human or animal body by conventional methods, which require formulation of the carrier in a pharmaceutical composition. Accordingly, in a seventh aspect, the present invention relates to a pharmaceutical composition comprising an AAV (hereinafter referred to as "the pharmaceutical composition of the present invention"), wherein the AAV comprises a recombinant viral genome, wherein the recombinant viral genome comprises an expression cassette , the expression cassette comprises an adipose tissue-specific transcriptional regulatory region operably linked to the polynucleotide of interest. Alternatively, the pharmaceutical compositions of the present invention may comprise the polynucleotides or polypeptides of the present invention.
所述药物组合物可以包括在治疗上有效量的本发明的AAV和药学上可接受的载体(carrier)。The pharmaceutical composition may include a therapeutically effective amount of an AAV of the present invention and a pharmaceutically acceptable carrier.
本发明的组合物可以配制为用于递送至动物(例如家畜(牛、猪、其他))用于兽医学目的,以及递送至其他非人类哺乳动物受试者,以及人类受试者。可以用生理学可接受的载体配制AAV以用于基因转移和基因治疗应用。制剂的剂量可以测量或计算为病毒颗粒或基因组拷贝(“GC”)/病毒基因组(“vg”)。The compositions of the present invention can be formulated for delivery to animals (eg, livestock (bovine, porcine, others)) for veterinary purposes, as well as to other non-human mammalian subjects, as well as human subjects. AAVs can be formulated with physiologically acceptable carriers for gene transfer and gene therapy applications. The dosage of a formulation can be measured or calculated as viral particle or genome copies ("GC")/viral genome ("vg").
本领域中已知的任何方法可以用于测定本发明的病毒组合物的基因组拷贝(GC)数。一种用于进行AAV GC数滴定的方法如下:将纯化的AAV载体样品首先用脱氧核糖核酸酶处理以除去未壳体化的AAV基因组DNA或来自生产过程的污染质粒DNA。然后使耐脱氧核糖核酸酶的颗粒经受热处理以从衣壳中释放基因组。随后通过使用靶定病毒基因组的特定区域的引物/探针组的实时PCR测定释放的基因组的数量。Any method known in the art can be used to determine the genome copy (GC) number of the viral compositions of the invention. One method for performing AAV GC number titrations is as follows: Purified AAV vector samples are first treated with deoxyribonuclease to remove unencapsidated AAV genomic DNA or contaminating plasmid DNA from the production process. The DNase-resistant particles are then subjected to heat treatment to release the genome from the capsid. The amount of released genome was then determined by real-time PCR using primer/probe sets targeting specific regions of the viral genome.
另外,可以以剂量单位配制病毒组合物以包含约1.0×109GC至约1.0×1015GC(以治疗平均体重70kg的受试者),并且优选地1.0×1012GC至1.0×1014GC范围内的量的病毒载体用于人类患者。优选地,制剂中病毒的剂量是1.0×109GC、5.0×109GC、1.0×1010GC、5.0×1010GC、1.0×1011GC、5.0×1011GC、1.0×1012GC、5.0×1012GC、1.0×1013GC、5.0×1013GC、1.0×1014GC、5.0×1014GC、或1.0×1015GC。Additionally, the viral composition can be formulated in a dosage unit to contain from about 1.0 x 10 9 GC to about 1.0 x 10 15 GC (to treat a subject with an average body weight of 70 kg), and preferably from 1.0 x 10 12 GC to 1.0 x 10 14 Viral vectors are used in human patients in amounts in the GC range. Preferably, the dose of virus in the formulation is 1.0×10 9 GC, 5.0×10 9 GC, 1.0×10 10 GC, 5.0×10 10 GC, 1.0×10 11 GC, 5.0×10 11 GC, 1.0×10 12 GC , 5.0×10 12 GC, 1.0×10 13 GC, 5.0×10 13 GC, 1.0×10 14 GC, 5.0×10 14 GC, or 1.0×10 15 GC.
病毒载体可以以传统方式使用一种或多种生理学上可接受的运载体或赋形剂配制。AAV可以配置为通过注射(例如,通过弹丸式注射(bolus injection)或持续输注(continuous infusion))进行非消化道给予。用于注射的制剂可以以单位剂量(例如,安瓿瓶或以多剂量容器)的形式存在,其中添加防腐剂。病毒组合物可以采取这些形式,如在油性或水性媒介中的悬浮液、溶液或乳液,并可以包含配制剂,如悬浮剂、稳定剂和/或分散剂。AAV制剂的液体剂型可以通过传统方法,用药学上可接受的添加剂,如悬浮剂(例如,山梨糖醇糖浆、纤维素衍生物或氢化食用脂肪)、乳化剂(例如,卵磷脂或阿拉伯树胶)、非水媒介(例如,扁桃仁油、油性酯、乙醇或分馏的植物油)和防腐剂(例如,甲基或丙基-对羟基苯酸酯或山梨酸)来制备。该剂型还可以含有缓冲盐。可替代地,组合物可以是粉末形式,用于在使用之前与适合的媒介(例如,无菌的无热源的水)组合。Viral vectors can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. AAVs can be configured for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection may be presented in unit dose (eg, in ampoules or in multi-dose containers) with an added preservative. The viral compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Liquid dosage forms of AAV formulations can be prepared by conventional methods with pharmaceutically acceptable additives such as suspending agents (eg, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (eg, lecithin or acacia) , a non-aqueous vehicle (eg, almond oil, oily esters, ethanol or fractionated vegetable oils) and a preservative (eg, methyl or propyl-paraben or sorbic acid). The dosage form may also contain buffer salts. Alternatively, the compositions may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.
还包括了辅剂与本发明的AAV结合或混合使用。预期的辅剂包括,但不限于无机盐辅剂或无机盐凝胶辅剂、颗粒辅剂、微粒辅剂、粘膜辅剂和免疫刺激辅剂。Also included are adjuvants used in combination or in admixture with the AAVs of the present invention. Contemplated adjuvants include, but are not limited to, inorganic salt adjuvants or inorganic salt gel adjuvants, granular adjuvants, particulate adjuvants, mucosal adjuvants, and immunostimulating adjuvants.
可以将辅剂作为与本发明的AAV的混合物,或与AAV结合使用给予受试者。The adjuvant can be administered to a subject as a mixture with the AAV of the invention, or in combination with the AAV.
本发明的药物组合物可以局部或全身给予。在优选的实施方式中,该药物组合物在要转导其细胞的组织或器官附近给予。在特定的实施方式中,本发明的药物组合物通过WAT内部或BAT内部注射局部给予至白色脂肪组织(WAT)或褐色脂肪组织(BAT)中。在另一优选的实施方式中,本发明的药物组合物全身给予。The pharmaceutical compositions of the present invention can be administered locally or systemically. In a preferred embodiment, the pharmaceutical composition is administered near the tissue or organ whose cells are to be transduced. In specific embodiments, the pharmaceutical compositions of the present invention are administered locally into white adipose tissue (WAT) or brown adipose tissue (BAT) by intra-WAT or intra-BAT injection. In another preferred embodiment, the pharmaceutical composition of the present invention is administered systemically.
药物组合物可以根据常规方法配制为适合于静脉注射、皮下、或肌内给予至人类的药物组合物。注射剂可以以常规形式,作为液体溶液或悬浮液,在注射之前适用于液体中的溶液或悬浮液的固体形式,或作为乳液制备。必要时,组合物还可以包括,局部麻醉剂,如利多卡因以便在注射位置止痛。当组合物通过渗透给予时,其可以省去含有药物质量的水或盐溶液的渗透瓶。当通过注射给予组合物时,可以提供用于注射的水瓶或无菌盐溶液从而可以在给予之前混合成分。The pharmaceutical composition can be formulated according to conventional methods as a pharmaceutical composition suitable for intravenous injection, subcutaneous, or intramuscular administration to humans. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The composition may also include, if necessary, a local anesthetic, such as lidocaine, for pain relief at the injection site. When the composition is administered by osmosis, it can dispense with an osmotic bottle containing a drug-quality water or saline solution. When the composition is administered by injection, a water bottle for injection or sterile saline solution can be provided so that the ingredients can be mixed prior to administration.
术语“治疗有效量”是指计算以产生期望的效果的本发明的多核苷酸、载体、多肽、或药物组合物的量,并且总体上连同其它原因,由本发明的多核苷酸、载体、多肽和药物组合物的自身特征以及要获得的疗效决定。通过本文描述的或本领域另外已知的标准临床技术测定在疾病的治疗中有效的本发明的多核苷酸、载体、多肽或药物组合物的量。此外,还可以可选地使用体外测定以帮助确定最佳的剂量范围。在制剂中使用的精确的剂量取决于给予途径,和病情的严重性,并且应该以医生的判断决定并取决于每个患者的情况。有效剂量可以推断自来自于体外试验系统或动物中的模型的一对剂量响应曲线。对于全身给予,治疗有效剂量可以最初由体外试验估计。例如,可以在动物模型中配制剂量以达到包括已经在细胞培养中测定的IC50的循环浓度范围。所述信息可用于精确地测定人类中有用的剂量。首次剂量还可以估计自使用现有技术中已知的技术的体内数据(例如动物模型)。本领域中的普通技术人员可以容易地基于动物中的数据优化对人类的给予。The term "therapeutically effective amount" refers to the amount of a polynucleotide, vector, polypeptide, or pharmaceutical composition of the invention calculated to produce the desired effect, and generally, among other reasons, from the polynucleotide, vector, polypeptide of the invention and the own characteristics of the pharmaceutical composition and the therapeutic effect to be obtained. The amount of a polynucleotide, vector, polypeptide or pharmaceutical composition of the invention effective in the treatment of a disease is determined by standard clinical techniques described herein or otherwise known in the art. In addition, in vitro assays can optionally be used to help determine optimal dosage ranges. The precise dose to be employed in the formulation will depend on the route of administration, and the seriousness of the condition, and should be determined with the judgment of the physician and will depend on each patient's circumstances. Effective doses can be extrapolated from a pair of dose-response curves derived from in vitro test systems or models in animals. For systemic administration, the therapeutically effective dose can be estimated initially from in vitro tests. For example, dosages can be formulated in animal models to achieve a range of circulating concentrations that include IC50s that have been determined in cell culture. Such information can be used to accurately determine useful doses in humans. The first dose can also be estimated from in vivo data (eg, animal models) using techniques known in the art. One of ordinary skill in the art can readily optimize administration to humans based on data in animals.
这种全身给予包括,但不限于任何意味着并不直接注入脂肪组织的给予途径。更特别地,全身给予包括本发明的多核苷酸、载体、多肽和药物组合物的全身注射,如肌内(im)、血管内(ie)、动脉内(ia)、静脉内(iv)、腹膜内(ip)、皮下或经皮注射。外周给予还包括本发明的多核苷酸、载体、多肽和药物组合物的口腔给予,使用植入物递送、或使用喷雾剂、气雾剂或任何其他适当的制剂通过呼吸系统(例如鼻内)滴注给予。优选地,全身给予是通过im、ip、ia或iv注射。最优选地,通过iv注射给予本发明的多核苷酸、载体、多肽和药物组合物。参见During M,WO 1996/040954和Monahan P等,WO 2001/091803。Such systemic administration includes, but is not limited to, any route of administration that is not intended to be injected directly into adipose tissue. More particularly, systemic administration includes systemic injection of the polynucleotides, vectors, polypeptides and pharmaceutical compositions of the invention, such as intramuscular (im), intravascular (ie), intraarterial (ia), intravenous (iv), Intraperitoneal (ip), subcutaneous or transdermal injection. Peripheral administration also includes oral administration of the polynucleotides, carriers, polypeptides and pharmaceutical compositions of the invention, delivered using implants, or via the respiratory system (eg, intranasally) using sprays, aerosols, or any other suitable formulation given by instillation. Preferably, systemic administration is by im, ip, ia or iv injection. Most preferably, the polynucleotides, vectors, polypeptides and pharmaceutical compositions of the present invention are administered by iv injection. See During M, WO 1996/040954 and Monahan P et al, WO 2001/091803.
本发明的药物组合物可以以单一剂量给予,或在本发明的特定的实施方式中,可以采用多次剂量(例如两次、三次、四次、或更多次给予)以达到治疗效果。优选地,当需要多次剂量时,包含在本发明的药物组合物中的AAV来自不同的血清型。The pharmaceutical compositions of the present invention may be administered in a single dose, or in certain embodiments of the present invention, multiple doses (eg, two, three, four, or more administrations) may be employed to achieve a therapeutic effect. Preferably, when multiple doses are required, the AAVs contained in the pharmaceutical compositions of the present invention are from different serotypes.
所有上文提及的公开通过引证将它们的整体结合在此。All publications mentioned above are incorporated herein by reference in their entirety.
虽然已经为了清楚和理解,较详细地描述了前述发明,本领域技术人员通过阅读本公开应该理解的是,可以在形式和细节上做出各种改变而不偏离本发明和所附权利要求的真实范围。Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be understood by those skilled in the art from reading this disclosure that various changes in form and detail may be made therein without departing from the invention and the appended claims true range.
一般方法general method
1.受试者特性1. Subject Characteristics
使用8-12周大的雄性ICR小鼠、9-13周大的C57Bl/6J小鼠和8周大的B6.V-Lepob/OlaHsd(ob/ob)和BKS.Cg-+Leprdb/+Leprdb/OlaHsd(db/db)小鼠。小鼠用标准食物(TekladGlobalHarlan Labs.,Inc.,Madison,WI,US)自由采食,并在光暗周期下(在8:00a.m.光照)保持12h。对于组织采样,将小鼠通过吸入麻醉异氟烷(AbbottLaboratories,Abbott Park,IL,US)麻醉并割去头部。切离感兴趣的组织并在-80℃下或使用甲醛保存直至分析。8-12 week old male ICR mice, 9-13 week old C57Bl/6J mice and 8 week old B6.V-Lep ob /OlaHsd(ob/ob) and BKS.Cg-+Lepr db / +Lepr db /OlaHsd(db/db) mice. Standard chow for mice (TekladGlobal Harlan Labs., Inc., Madison, WI, US) fed ad libitum and maintained for 12 h under a light-dark cycle (light at 8:00 a.m.). For tissue sampling, mice were anesthetized by inhalation of isoflurane ( Abbott Laboratories, Abbott Park, IL, US) anesthetized and decapitated. Tissues of interest were excised and stored at -80°C or with formaldehyde until analysis.
2.重组AAV载体2. Recombinant AAV Vectors
根据标准方法,通过HEK293细胞的三次转染产生载体。参见上文的Ayuso,2010。在10个滚瓶中(850cm2,平面;CorningTM,Sigma-Aldrich Co.,Saint Louis,MO,US)10%FBS的DMEM中培养细胞至80%细胞覆盖,并通过磷酸钙方法,用携带由AAV2ITR侧接的表达框的质粒、携带AAV2rep基因和AAV血清型1、2、4、5、6、7、8或9的cap基因的辅助质粒、以及携带辅助功能腺病毒的质粒共转染。使用的转基因是:a)由以下驱动的eGFP:a1)杂交巨细胞病毒增强子/鸡β-肌动蛋白组成型启动子(CAG)和WPRE调控元件,a2)鼠mini/aP 2调控区或a3)小鼠mini/UCP1调控区;b)由以下驱动的鼠类己糖激酶II(mHKII)cDNA:b1)CMV遍在启动子(ubiquitous promoter)和WPRE,b2)鼠mini/aP2调控区或b3)小鼠mini/UCP1调控区,c)由鼠mini/aP2调控区和WPRE源自人类胎盘的分泌的碱性磷酸酶(hSeAP),d)由小鼠mini/UCP1调控区驱动的鼠类VEGF164,和e)由CMV遍在启动子驱动的RFP。参见上文的Ross,1990、Graves,1992和Cassard-Doulcier,1998。使用携带CMV启动子的非编码质粒(pAAV-MCS,StratageneTM,Agilent Technologies,Inc.,Santa Clara,CA,US)、mini/aP2调控区或mini/UCP1调控区以及多克隆位点产生空颗粒。AAV利用基于聚乙二醇沉淀步骤和两个连续的氯化铯(CsCl)梯度的优化的方法进行纯化。这种第二代的基于CsCl的方法显著地减少了空AAV衣壳以及DNA和蛋白杂质。参见上文的Ayuso,2010。可以将纯化的AAV载体对PBS进行渗析,过滤并在-80℃下保存。病毒基因组的滴度通过定量PCR确定,该定量PCR遵循对于使用线性化的质粒DNA作为标准曲线的AAV2参考标准材料描述的方法。参见Lock M等,Hum.Gene Ther.2010;21:1273-1285。根据本领域中已知的分子生物学技术构造载体。参见上文的Brown(1995)、Watson(1992)、Alberts(2008)、Innis(1990)、Erlich(1989)、Sambrook(1989)、Bishop(1987)、Reznikoff(1987)、Davis(1986)和Schleef(2001)。Vectors were generated by three transfections of HEK293 cells according to standard methods. See Ayuso, 2010, supra. Cells were cultured to 80% cell coverage in 10 roller bottles (850 cm 2 , flat; Corning ™ , Sigma-Aldrich Co., Saint Louis, MO, US) in DMEM with 10% FBS and carried Co-transfection with a plasmid containing the expression cassette flanked by the AAV2 ITR, a helper plasmid carrying the AAV2rep gene and the cap gene of
3.体内AAV载体的eWAT内部给予3. In vivo eWAT administration of AAV vectors
将小鼠用氯胺酮(100mg/kg)和赛拉嗪(10mg/kg)的腹膜内注射麻醉。进行剖腹术以暴露附睾的白色脂肪组织。将AAV载体再悬浮于具有或不具有2%普兰尼克F88(BASFCorp.,Florham Park,NJ,US)的盐溶液中并直接注射入附睾脂垫。每个附睾脂垫用50μL的AAV溶液注射两次(一次注射接近睾丸并且另一次在脂垫中部)。用无菌盐溶液漂洗腹部并且用两层方法闭合。Mice were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg). A laparotomy was performed to expose the white adipose tissue of the epididymis. AAV vectors were resuspended in saline with or without 2% Pluronic F88 (BASF Corp., Florham Park, NJ, US) and injected directly into the epididymal fat pad. Each epididymal fat pad was injected twice with 50 [mu]L of AAV solution (one injection close to the testis and another in the middle of the fat pad). The abdomen was rinsed with sterile saline and closed with a two-layer approach.
4.体内AAV载体的iBAT内部和iWAT内部给予。4. Intra-iBAT and intra-iWAT administration of AAV vectors in vivo.
将小鼠用氯胺酮(100mg/kg)和赛拉嗪(10mg/kg)的腹膜内注射麻醉。在肩胛间或腹股沟区域切开纵向1.5-2cm长的切口,以分别暴露iBAT或iWAT。使用汉密尔顿注射器使每个iBAT或iWAT接受4次10μl的AAV溶液的注射将载体分散至整个库。使用一层方法闭合皮肤。Mice were anesthetized with intraperitoneal injections of ketamine (100 mg/kg) and xylazine (10 mg/kg). A longitudinal 1.5-2 cm long incision was made in the interscapular or inguinal region to expose the iBAT or iWAT, respectively. Each iBAT or iWAT received four injections of 10 μl of AAV solution using a Hamilton syringe to disperse the vector throughout the pool. Close the skin using a one-layer method.
5.AAV载体的全身给予5. Systemic Administration of AAV Vectors
将适量的AAV溶液在200μL的盐溶液中稀释并在递送时手动注射入尾部侧静脉而不施加压力。在注射之前,将动物置于250W的红外加热灯(Philips NV,Amsterdam,NL)下几分钟以扩张血管并便于观察且更易达到尾部静脉。使用塑料限制器(Harvard Apparatus,Holliston,MA,US)固定动物用于注射。由于采用适当的限制装置,不使用麻醉。利用30针径的针对动物注射。The appropriate amount of AAV solution was diluted in 200 μL of saline and manually injected into the lateral caudal vein at the time of delivery without pressure. Prior to injection, animals were placed under a 250W infrared heating lamp (Philips NV, Amsterdam, NL) for several minutes to dilate blood vessels and facilitate visualization and better access to the tail vein. Animals were immobilized for injection using plastic restraints (Harvard Apparatus, Holliston, MA, US). Anesthesia was not used due to the use of appropriate restraint devices. Animals are injected with a 30 gauge needle.
6.免疫组织化学6. Immunohistochemistry
将组织在甲醛中固定12至24小时,嵌入石蜡,并切片用于GFP、RFP和α-SMA的检测。将切片用1:300稀释的羊抗GFP抗体(Abcam plc,Cambridge,MA,US)、用1/400稀释的兔抗RFP抗体(Abcam plc,Cambridge,MA,US)或用1/300稀释的鼠抗-α-SMA抗体(Sigma-AldrichCo.,Saint Louis,MO,US)在4℃下温育过夜。将1:300稀释的生物素化的驴抗山羊抗体(Santa Cruz Biotechnology,Inc.,Santa Cruz,CA,US),或1/300稀释的生物素化的羊抗兔抗体(Pierce Antibodies,Thermo Fisher Scientific Inc.,Rockford,IL,US)或1/300稀释的生物素化的马抗鼠抗体(Vector Laboratories,Burlingame,CA,US)用作第二抗体。将1:300稀释的链霉亲和素Alexa Fluor 488(Streptavidine Alexa Fluor 488)(MolecularLife Technologies Corp.,Carslbad,CA,US)用作荧光染料并且将Hoescht bisbenzimide(Sigma-Aldrich Co.,Saint Louis,MO,US)用于细胞核对比染色。可替代地,使用1:50稀释的ABC过氧物酶试剂盒(Pierce Biotechnology,Inc.,Rockford,IL,US)并将切片在Mayer的苏木精中对比染色。Tissues were fixed in formaldehyde for 12 to 24 hours, embedded in paraffin, and sectioned for detection of GFP, RFP, and α-SMA. Sections were plated with 1:300 diluted goat anti-GFP antibody (Abcam plc, Cambridge, MA, US), 1/400 diluted rabbit anti-RFP antibody (Abcam plc, Cambridge, MA, US) or 1/300 diluted Murine anti-α-SMA antibody (Sigma-Aldrich Co., Saint Louis, MO, US) was incubated overnight at 4°C. Biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, US) at 1:300 dilution, or biotinylated goat anti-rabbit antibody (Pierce Antibodies, Thermo Fisher) at 1/300 dilution Scientific Inc., Rockford, IL, US) or a 1/300 dilution of biotinylated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA, US) were used as secondary antibodies. Streptavidin Alexa Fluor 488 (Molecular Life Technologies Corp., Carslbad, CA, US) was used as a fluorescent dye and Hoescht bisbenzimide (Sigma-Aldrich Co., Saint Louis, MO, US) was used for nuclei contrast staining. Alternatively, a 1:50 dilution of the ABC Peroxidase Kit (Pierce Biotechnology, Inc., Rockford, IL, US) was used and sections were counterstained in Mayer's Hematoxylin.
7.eWAT样品中β-半乳糖苷酶表达的分析7. Analysis of β-galactosidase expression in eWAT samples
为了检测eWAT中存在的所有β-半乳糖苷酶,将组织样品在4%多聚甲醛中固定1h,在PBS溶液中洗涤两次,并且然后在5mM K3Fe(CN)5、5mM K4Fe(CN)6和1mM MgCl2的PBS中的X-Gal(5-溴-4-氯-3-β-D-半乳糖苷(galactopyranoside))中在37℃下在黑暗中温育6-8h。To detect all β-galactosidase present in eWAT, tissue samples were fixed in 4% paraformaldehyde for 1 h, washed twice in PBS solution, and then in 5 mM K 3 Fe(CN) 5 , 5 mM K 4 Fe( CN ) and 1 mM MgCl in X-Gal (5-bromo-4-chloro- 3 -β-D-galactopyranoside) in PBS for 6-8 h at 37 °C in the dark .
8.GFP含量8. GFP content
为测定GFP含量,将组织在1mL的裂解缓冲液(PBS中,50mM/L Tris、1%NonidetP40、0.25%脱氧胆酸钠、150mM/L NaCl、1mM/L EDTA,pH 7.4,无菌过滤)中用型号组织匀浆器机械破碎并在室温下温育10分钟。温育之后,将样品以14000rpm离心10分钟。将上层清液移至新管并且用具有488nm激发波长和512nm发射波长的闪光光谱仪Flx800(Bio-Tek Instruments,Inc,Winooski,VT,US)测量100μL的该溶液中的GFP含量。通过含有蛋白的样品校正总GFP含量值。To determine GFP content, tissues were lysed in 1 mL of lysis buffer (50 mM/L Tris, 1% NonidetP40, 0.25% sodium deoxycholate, 150 mM/L NaCl, 1 mM/L EDTA, pH 7.4, sterile filtered) in PBS. Chinese use Model tissue homogenizers were mechanically disrupted and incubated for 10 min at room temperature. After incubation, samples were centrifuged at 14000 rpm for 10 minutes. The supernatant was transferred to a new tube and 100 μL of this solution was measured for GFP content with a flash spectrometer Flx800 (Bio-Tek Instruments, Inc, Winooski, VT, US) with an excitation wavelength of 488 nm and an emission wavelength of 512 nm. Total GFP content values were corrected for samples containing protein.
9.来自附睾脂垫的脂肪细胞的分离9. Isolation of Adipocytes from Epididymal Fat Pads
使用改进的Rodbell的方法分离AAV转导的脂肪细胞。参见Rodbell M,J.Biol.Chem.1964;239:375-380。通过去除头部处死异氟烷麻醉的小鼠并将附睾WAT切碎,并在35-45分钟中在37℃下在含有4%BSA(不含脂肪酸)、0.5mM/L葡萄糖和0.5mg/mL II型胶原酶(C6885;Sigma-Aldrich Co.,Saint Louis,MO,US)的Krebs-Ringer碳酸氢盐HEPES缓冲液(KRBH)中消化。通过温和的离心分离脂肪细胞并且用新鲜的不含胶原酶的没有葡萄糖的KRBH洗涤三次。将脂肪细胞再悬浮于不含葡萄糖的新鲜的KRBH中,并且如先前描述的估计细胞数。参见DiGirolamo M等,Am.J.Physiol 1971;221:850-858。AAV-transduced adipocytes were isolated using a modified Rodbell's method. See Rodbell M, J. Biol. Chem. 1964;239:375-380. Isoflurane-anaesthetized mice were sacrificed by decapitation and epididymal WAT was minced and incubated at 37°C for 35-45 minutes in 4% BSA (without fatty acids), 0.5mM/L glucose and 0.5mg/L mL of collagenase type II (C6885; Sigma-Aldrich Co., Saint Louis, MO, US) in Krebs-Ringer bicarbonate HEPES buffer (KRBH). Adipocytes were isolated by gentle centrifugation and washed three times with fresh KRBH without collagenase without glucose. Adipocytes were resuspended in fresh KRBH without glucose and cell numbers were estimated as previously described. See DiGirolamo M et al. Am. J. Physiol 1971;221:850-858.
10.RNA分析10. RNA Analysis
总RNA通过分别使用QIAzol裂解试剂(Qiagen NV,Venlo,NL)或tripure分离试剂(Roche Diagnostics Corp.,Indianapolis,IN,US),以及RNeasy脂质组织迷你试剂盒(Lipid Tissue Minikit)由分离的脂肪细胞和脂肪库或肝脏获得。为了除去残留的病毒基因组,用DNAseI(Qiagen NV,Venlo,NL)处理总RNA。对于RT-PCR,使用Superscript VILOcDNA合成试剂组(InvitrogenTM,Life Technologies Corp.,Carslbad,CA,US)逆转录1μg的RNA样品。在使用EXPRESS SYBRGreen qPCR supermix(InvitrogenTM,Life TechnologiesCorp.,Carslbad,CA,US)的(Cepheid,Sunnyvale,USA)中进行实时定量PCR。正义和反义寡核苷酸引物的序列是:Total RNA was isolated from adipose tissue using QIAzol lysis reagent (Qiagen NV, Venlo, NL) or tripure isolation reagent (Roche Diagnostics Corp., Indianapolis, IN, US), and RNeasy Lipid Tissue Minikit, respectively. Cells and fat depots or liver obtained. To remove residual viral genome, total RNA was treated with DNAseI (Qiagen NV, Venlo, NL). For RT-PCR, 1 μg of RNA samples were reverse transcribed using the Superscript VILO cDNA synthesis kit (Invitrogen ™ , Life Technologies Corp., Carslbad, CA, US). After using EXPRESS SYBRGreen qPCR supermix (Invitrogen ™ , Life Technologies Corp., Carslbad, CA, US) (Cepheid, Sunnyvale, USA) for real-time quantitative PCR. The sequences of the sense and antisense oligonucleotide primers are:
数据用36B4值标准化并如先前描述的进行分析。参见Pfaffl M,Nucleic AcidsRes.2001;29(9):e45。Data were normalized with 36B4 values and analyzed as previously described. See Pfaffl M, Nucleic Acids Res. 2001;29(9):e45.
11.分离的脂肪细胞中的离体葡萄糖摄取11. Ex vivo glucose uptake in isolated adipocytes
对于来自小鼠的分离的脂肪细胞,如先前描述的,在不同的胰岛素浓度下测量2-[1-3H]脱氧-D-葡萄糖(2-DG;Amersham Pharmacia Biotech Inc.,Piscataway,NJ,US)摄取。参见Traxinger R等,J.Biol.Chem.1989;264:8156-8163。简要地,分离的脂肪细胞通过来自如之前描述的饲养的小鼠的附睾WAT的胶原酶消化获得。用KRBH+4%BSA(不含脂肪酸)、10mM/L脱氧-葡萄糖、0.4μCi 2-[1-3H]脱氧-D-葡萄糖和不同的胰岛素浓度温育250μL的脂肪细胞悬液5分钟。最终,在聚丙烯管中通过硅油(Sigma-Aldrich Co.,Saint Louis,MO,US)分离脂肪细胞和培养基,并且通过液体闪烁计数评估脂肪细胞样品中的放射性。结果表示为pmol的2-[3H]-DG每106细胞每分钟。For isolated adipocytes from mice, 2-[ 1-3H ]deoxy-D-glucose (2-DG; Amersham Pharmacia Biotech Inc., Piscataway, NJ, US) intake. See Traxinger R et al, J. Biol. Chem. 1989;264:8156-8163. Briefly, isolated adipocytes were obtained by collagenase digestion of epididymal WAT from mice raised as previously described. 250 μL of adipocyte suspensions were incubated with KRBH + 4% BSA (without fatty acids), 10 mM/L deoxy-glucose, 0.4 μCi 2-[1- 3 H]deoxy-D-glucose and various insulin concentrations for 5 minutes. Finally, adipocytes and media were isolated in polypropylene tubes by silicone oil (Sigma-Aldrich Co., Saint Louis, MO, US), and radioactivity in adipocyte samples was assessed by liquid scintillation counting. Results are expressed as pmol of 2-[ 3 H]-DG per 10 6 cells per minute.
12.体内葡萄糖摄取12. Glucose uptake in the body
如先前描述的测定体内基础葡萄糖利用指数。参见Franckhauser S等,Diabetes2002;51:624-630。简要地,将148GBq(4μCi)的不可代谢的葡萄糖类似物,脱氧-D-[1,2-3H]葡萄糖(2-DG;PerkinElmer,Inc.,Waltham,MA,US)混合在BSA-柠檬酸盐缓冲液中。在零时间将放射性同位素标记的混合物的闪烁注射剂(flash injection)给予麻醉的(氯胺酮+赛拉嗪)饲养小鼠的颈静脉中。如先前描述的,用注射后1、15和30分钟获得的25μL血液样品(尾部静脉)测定具体的血液2-DG清除率。参见Somogyi M,J.Biol.Chem.1945;160:69-73。在注射后30分钟移除组织样品。葡萄糖利用指数是通过测量放射性标记化合物的积聚测定的。参见FerréP等,Biochem.J.1985;228:103-110。将每毫克蛋白的2-DG-6磷酸酯的量除以测量的2-DG与未标记的葡萄糖的浓缩比的积分。因为值不是由葡萄糖代谢途径中的2-DG的“判别常数(discrimination constant)”校正的,结果表示为葡萄糖利用的指数,以每分钟皮摩尔每毫克的蛋白计。In vivo basal glucose utilization index was determined as previously described. See Franckhauser S et al. Diabetes 2002;51:624-630. Briefly, 148 GBq (4 μCi) of the non-metabolizable glucose analog, deoxy-D-[ 1,2-3H ]glucose (2-DG; PerkinElmer, Inc., Waltham, MA, US), was mixed in BSA-Lemon in salt buffer. A flash injection of the radiolabeled mixture was administered into the jugular vein of anesthetized (ketamine + xylazine) housed mice at time zero. Specific blood 2-DG clearance was determined using 25 [mu]L blood samples (tail vein) obtained at 1, 15 and 30 minutes post-injection as previously described. See Somogyi M, J. Biol. Chem. 1945;160:69-73. Tissue samples were removed 30 minutes after injection. The Glucose Utilization Index is determined by measuring the accumulation of radiolabeled compounds. See Ferré P et al, Biochem. J. 1985;228:103-110. Divide the amount of 2-DG-6 phosphate per milligram of protein by the integral of the measured concentration ratio of 2-DG to unlabeled glucose. Because the values were not corrected for the "discrimination constant" of 2-DG in the glucose metabolic pathway, the results were expressed as an index of glucose utilization in picomoles per milligram of protein per minute.
13.血液hSeAP水平的测量13. Measurement of blood hSeAP levels
使用Phospha-LightTM系统(Applied BiosystemsTM,Life TechnologiesCorp.,Carslbad,CA,US),由5μL的血清测定循环hSeAP水平。use Circulating hSeAP levels were determined from 5 μL of serum on the Phospha-Light ™ system (Applied Biosystems ™ , Life Technologies Corp., Carslbad, CA, US).
14.统计分析14. Statistical analysis
所有的值表示为平均值±SEM。由学生t检验(Student's t-test)比较组之间的差值。在p<0.05认为差值是显著的。All values are expressed as mean ± SEM. Differences between groups were compared by Student's t-test. Differences were considered significant at p<0.05.
实施例1Example 1
通过AAV的局部给予的白色脂肪细胞的体内转导In vivo transduction of white adipocytes by local administration of AAV
为评估使用AAV载体的白色脂肪组织(WAT)的体内转导效率,将4×1011病毒基因组(vg)/鼠的AAV血清型1、2、4、5、6、7、8和9(在遍在启动子CAG控制下编码标记蛋白GFP(AAV-CAG-GFP)),带有或没有非离子表面活性剂普兰尼克F88,双侧注射入小鼠的附睾白色脂肪组织(eWAT)中。参见Croyle M等,Mol.Ther.2001;4:22-28、Gebhart C等,J.ControlRelease 2001;73:401-416、Mizukami H等,Hum.Gene Ther.2006;17:921-928和Sommer J等,Mol.Ther.2003;7:122-128。如由在eWAT中针对GFP的免疫染色所评估的,不带有普兰尼克F88的AAV1、AAV2、AAV4和AAV5的给予在注射两个星期后导致非常低的白色脂肪细胞转导的百分比。此外,通过添加普兰尼克F88没有实现由任何测试的血清型介导的脂肪转导效率的改善。参见图1A。因此,对于随后的实验弃去该非离子表面活性剂的使用。与普兰尼克F88的添加无关,AAV1比AAV2、AAV4和AAV5在eWAT体内转导中更加高效。参见上文的Mizukami,2006。与由AAV1转导的少量分散的脂肪细胞和极少的脂肪细胞的组相反,用AAV 6和AAV7注射的动物表现出多个较大的GFP+白色脂肪细胞的组。此外,用AAV8和AAV9处理的动物示出了大得多的eWAT的转导,并且每个eWAT区域绝大多数的脂肪细胞被转导。参见图1B。通过荧光分析的给予两个星期后的eWAT中的GFP含量的定量进一步确认血清型6、7、8和9的AAV比AAV1在eWAT体内转导中是更高效的。参见图1C。值得注意的的是用AAV8和AAV9注射的附睾脂垫表现出最高的GFP含量,并且它们之间没有显著的统计差。参见图1C。在单侧eWAT内部给予2×1011vg/鼠的,受遍在CMV启动子控制的编码LacZ基因的AAV8(AA8-CMV-LacZ)两个星期之后,用LacZ底物5-溴-4-氯-3-吲哚基-β-D-半乳糖苷(X-gal)的染色表现出遍及eWAT转导的脂肪细胞的广泛分布。参见图1D。AAV8和AAV9-CAG-GFP载体局部给予至腹股沟WAT(iWAT)介导了该库(depot)中白色和浅褐色脂肪细胞的大量转导。参见图1G和H。总而言之,这些结果表明AAV8和AAV9是体内遗传WAT最适合的载体。To evaluate the in vivo transduction efficiency of white adipose tissue (WAT) using AAV vectors, 4 x 10 11 viral genomes (vg)/mouse of
此外,AAV载体的eWAT内部给予导致实际上局限于eWAT的转导。注射两个星期后,用AAV1、AAV6、AAV7、AAV8、或AAV9-CAG-GFP处理的动物并没有表现出肠系膜、腹膜后和腹股沟库的转导,然而少量的GFP+棕色脂肪细胞存在于用AAV9eWAT内部注射的小鼠的iBAT中。参见图8A。然而,BAT中的转基因表达相比eWAT中检测的极少。参见图8B。关于非脂肪组织的转导,AAV7、AAV8和AAV9-CAG-GFP的eWAT内部给予还导致显著的转移至肝脏和心脏以及至较少数量的胰腺的外分泌细胞的基因。参见图8C。Furthermore, intra-eWAT administration of AAV vectors results in transduction that is virtually restricted to eWAT. Two weeks after injection, animals treated with AAV1, AAV6, AAV7, AAV8, or AAV9-CAG-GFP did not exhibit transduction of mesenteric, retroperitoneal, and inguinal depots, whereas small amounts of GFP + brown adipocytes were present in cells treated with AAV1, AAV6, AAV7, AAV8, or AAV9-CAG-GFP. AAV9eWAT in iBAT of mice injected internally. See Figure 8A. However, transgene expression in BAT was minimal compared to that detected in eWAT. See Figure 8B. Regarding transduction of non-adipose tissue, eWAT administration of AAV7, AAV8 and AAV9-CAG-GFP also resulted in significant gene transfer to the liver and heart and to a smaller number of exocrine cells of the pancreas. See Figure 8C.
作为构思的证明,评估体内AAV转导的脂肪细胞是否可以是研究脂肪功能的可行的模型,将4×1011vg/鼠的,受遍在启动子CMV控制的,编码鼠类的酶的己糖激酶II(mHKII)的AAV9载体(AAV9-CMV-mHKII)或等剂量的AAV9-CMV-null载体双侧注射入健康小鼠的eWAT。注射两个星期后,来自用AAV9-CMV-mHKII处理的动物的分离的脂肪细胞与来自用AAV9-CMV-null注射的小鼠的脂肪细胞相比表现出mHKII表达的3倍增加。参见图1E。为分析脂肪细胞中AAV介导的mHKII的过表达,测定离体分离的脂肪细胞摄取的基础和胰岛素刺激的2-[1-3H]脱氧-D-葡萄糖。在AAV9-CMV-mHKII转导的脂肪细胞中基础2-[1-3H]脱氧-D-葡萄糖摄取相比AAV9-CMV-null转导的脂肪细胞略微增加。相反,相比来自用AAV9-CMV-null载体处理的动物的脂肪细胞,胰岛素刺激导致由AAV9-CMV-mHKII转导的脂肪细胞摄取的2-[1-3H]脱氧-D-葡萄糖的增加较大。参见图1F。As a proof-of-concept to assess whether AAV-transduced adipocytes in vivo could be a viable model for studying adipocyte function, 4 x 1011 vg/mouse, controlled by the ubiquitous promoter CMV, encoding a murine enzyme AAV9 vector of glycokinase II (mHKII) (AAV9-CMV-mHKII) or an equal dose of AAV9-CMV-null vector was injected bilaterally into eWAT of healthy mice. Two weeks after injection, isolated adipocytes from animals treated with AAV9-CMV-mHKII exhibited a 3-fold increase in mHKII expression compared to adipocytes from mice injected with AAV9-CMV-null. See Figure 1E. To analyze AAV-mediated overexpression of mHKII in adipocytes, basal and insulin-stimulated 2-[ 1-3H ]deoxy-D-glucose uptake by ex vivo isolated adipocytes was determined. Basal 2-[ 1-3H ]deoxy-D-glucose uptake was slightly increased in AAV9-CMV-mHKII transduced adipocytes compared to AAV9-CMV-null transduced adipocytes. In contrast, insulin stimulation resulted in increased 2-[ 1-3H ]deoxy-D-glucose uptake by AAV9-CMV-mHKII-transduced adipocytes compared to adipocytes from animals treated with AAV9-CMV-null vector larger. See Figure 1F.
实施例2Example 2
白色脂肪细胞的体内AAV介导的特异遗传工程化In vivo AAV-mediated specific genetic engineering of white adipocytes
评估仅由脂肪细胞特异的增强子组成的短版本的鼠类脂肪细胞蛋白2(mini/aP2)启动子与aP2基本启动子的结合使用。参见上文的Ross,1990和Graves,1992。该试验的目的是将AAV介导的转基因表达式限定于脂肪细胞。单侧eWAT内部给予1012vg/鼠的,受mini/aP2调控区控制的,编码GFP的AAV8和AAV9介导了有限的白色脂肪细胞的转导,并且注射两个星期后在肝脏和心脏中没有可检测的GFP表达。参见图2A和9。The use of a short version of the murine adipocyte protein 2 (mini/aP2) promoter consisting only of adipocyte-specific enhancers in combination with the aP2 basal promoter was evaluated. See Ross, 1990 and Graves, 1992, supra. The purpose of this assay was to restrict AAV-mediated transgene expression to adipocytes. AAV8 and AAV9 encoding GFP mediated limited transduction of white adipocytes in unilateral eWAT administered internally at 10 12 vg/mouse, controlled by the mini/aP2 regulatory region, and in liver and heart two weeks after injection There was no detectable GFP expression. See Figures 2A and 9.
为评估由小鼠中的mini/aP2调控区介导的转基因表达的时间过程,将4×1012vg/鼠剂量的,受mini/aP2调控区控制的,编码源自人类胎盘的分泌的碱性磷酸酶(hSeAP)cDNA的AAV9载体(AAV9-mini/aP2-SeAP)或盐溶液双侧注射入eWAT,并在AAV给予之后不同的时间点测量循环hSeAP水平。直到AAV递送两个星期之后,检测出高水平的hSeAP,并且逐渐增加多达40天。此后,循环hSeAP水平维持注射后至少一年的跟踪期间。通过qPCR定量的肝脏和eWAT中的hSeAP表达水平确认eWAT是负责主要生产hSeAP的组织。参见图2B-2C。To assess the time course of transgene expression mediated by the mini/aP2 regulatory region in mice, a dose of 4 x 10 12 vg/mouse, controlled by the mini/aP2 regulatory region, encoding a secreted base derived from human placenta was administered. AAV9 vector (AAV9-mini/aP2-SeAP) or saline solution of sex phosphatase (hSeAP) cDNA was injected bilaterally into eWAT, and circulating hSeAP levels were measured at various time points after AAV administration. High levels of hSeAP were detected until two weeks after AAV delivery and gradually increased for up to 40 days. Thereafter, circulating hSeAP levels were maintained for a follow-up period of at least one year after injection. Quantification of hSeAP expression levels in liver and eWAT by qPCR confirmed that eWAT is the tissue responsible for the main production of hSeAP. See Figures 2B-2C.
为评估AAV介导的白色脂肪细胞的特异的遗传工程化是否可以构成研究小鼠体内脂肪细胞功能、分化和代谢的新工具,将1.4×1012vg/鼠的,受mini/aP2调控区控制的,编码mHKII的AAV9载体(AAV9-mini/aP2-mHKII)或等剂量的AAV9-mini/aP2-null载体给予eWAT。注射两个星期后,测定体内基础2-[1-3H]脱氧-D-葡萄糖摄取。相比用AAV9-mini/aP2-null载体处理的动物,接收AAV9-mini/aP2-mHKII载体的动物示出了增加的由eWAT摄取的基础2-[1-3H]脱氧-D-葡萄糖。在基础条件下,在iBAT中和在,例如心脏的组织(其中,mini/aP2调控区的使用阻止转基因表达)中的组之间没有发现2-[1-3H]脱氧-D-葡萄糖摄取的差别。参见图2D。To assess whether AAV-mediated specific genetic engineering of white adipocytes could constitute a new tool to study adipocyte function, differentiation, and metabolism in mice, 1.4×10 12 vg/mouse of adipocytes, controlled by the mini/aP2 regulatory region, was added. Yes, an AAV9 vector encoding mHKII (AAV9-mini/aP2-mHKII) or an equal dose of AAV9-mini/aP2-null vector was administered to eWAT. Two weeks after injection, basal in vivo 2-[ 1-3H ]deoxy-D-glucose uptake was determined. Animals receiving AAV9-mini/aP2-mHKII vector showed increased basal 2-[l - 3H]deoxy-D-glucose uptake by eWAT compared to animals treated with AAV9-mini/aP2-null vector. Under basal conditions, no 2-[ 1-3H ]deoxy-D-glucose uptake was found between groups in iBAT and in tissues such as the heart, where the use of the mini/aP2 regulatory region prevents transgene expression difference. See Figure 2D.
实施例3Example 3
体内褐色脂肪细胞通过AAV载体的局部递送的遗传工程化Genetic engineering of in vivo brown adipocytes via localized delivery of AAV vectors
考虑到AAV8和AAV9是最高效的介导白色脂肪细胞的遗传工程载体,评估通过相同的血清型的褐色脂肪细胞的转导。将2×109vg/鼠的AAV8和AAV9-CAG-GFP载体给予至肩胛间褐色脂肪组织(iBAT)两个星期后,检测到多种GFP+褐色脂肪细胞。参见图3A。通过qPCR的GFP表达水平评估表明在2×109vg/鼠的剂量下通过AAV8的转导效率相比AAV9较高。参见图3B。一旦使用AAV8和AAV9载体证明了褐色脂肪细胞的转导,我们表征使用血清型1、2、4、5、6、7、8和9的AAV的褐色脂肪组织(BAT)体内转导效率。为此,将1.2×1010vg/鼠剂量的血清型4和8的AAV或1011vg/鼠剂量的血清型1、2、4、5、6、7、8和9的AAV(AAV-CMV-RFP)(受遍在启动子CMV控制,编码标记蛋白RFP)注射入小鼠的iBAT。iBAT内部给予两个星期后,通过qPCR的RFP表达水平的定量显示出于AAV1、2、4、5和6相比,通过AAV7、AAV8和AAV9的iBAT的转导效率较高。参见图3C。一致的是在接收AAV9载体的动物的iBAT中检测道广泛的RFP+褐色脂肪细胞分布。参见图3D。Considering that AAV8 and AAV9 are the most efficient genetically engineered vectors to mediate white adipocytes, transduction of brown adipocytes by the same serotype was evaluated. Two weeks after administration of 2 x 109 vg/mouse of AAV8 and AAV9-CAG-GFP vectors to interscapular brown adipose tissue (iBAT), multiple GFP + brown adipocytes were detected. See Figure 3A. Evaluation of GFP expression levels by qPCR indicated higher transduction efficiency by AAV8 compared to AAV9 at a dose of 2 x 109 vg/mouse. See Figure 3B. Once transduction of brown adipocytes was demonstrated using AAV8 and AAV9 vectors, we characterized brown adipose tissue (BAT) transduction efficiency using AAV of
此外,AAV的iBAT内部给予导致该库的局限性转导,其中,在附睾、肠系膜、腹膜后和腹股沟库中检测不到的转基因表达。关于非脂肪组织的转导,iBAT内部用AAV7、AAV8和AAV9载体处理的动物表现出心脏和肝脏的转导,而在胰腺、肠、脾脏、肺脏、肾脏、骨骼肌、睾丸、副睾和大脑中检测不到GFP表达。参见图10。Furthermore, intra-iBAT administration of AAV resulted in limited transduction of this pool, with undetectable transgene expression in epididymal, mesenteric, retroperitoneal and inguinal pools. Regarding transduction of non-adipose tissue, animals treated with AAV7, AAV8 and AAV9 vectors inside iBAT exhibited transduction in the heart and liver, while in pancreas, intestine, spleen, lung, kidney, skeletal muscle, testis, epididymis and brain GFP expression was not detectable in . See Figure 10.
实施例4Example 4
体内AAV介导的褐色脂肪细胞的特异遗传工程化AAV-mediated specific genetic engineering of brown adipocytes in vivo
利用由给予棕色脂肪细胞特异表达的增强子和大鼠UCP1基因的基本启动子组成的mini UCP1(mini/UCP1)调控区特异地调节感兴趣的基因在褐色脂肪组织中的表达。参见Boyer B等,Mol.Cell Biol.1991;11:4147-4156、Kozak U等,Mol.Cell Biol.1994;14:59-67、上文的Cassard-Doulcier,1998,和Larose,1996。iBAT内部给予2×1011vg/鼠的AAV8或AAV9-mini/UCP1-GFP载体两个星期后,得到褐色脂肪细胞的高效转导。参见图4A。类似地,iBAT内部递送2×1011vg/鼠的AAV8和AAV9-mini/aP2-GFP也转导褐色脂肪细胞。参见图11A。此外,mini/UCP1调控区实现高度脂肪细胞特异的GFP表达,完全消除了心脏中AAV介导的转基因表达,并且仅介导少量的肝脏转导。参见图11B。The expression of a gene of interest in brown adipose tissue is specifically regulated using a mini UCP1 (mini/UCP1) regulatory region consisting of an enhancer that imparts brown adipocyte-specific expression and a basal promoter of the rat UCP1 gene. See Boyer B et al., Mol. Cell Biol. 1991; 11:4147-4156, Kozak U et al., Mol. Cell Biol. 1994; 14:59-67, Cassard-Doulcier, 1998, supra, and Larose, 1996. High-efficiency transduction of brown adipocytes was obtained two weeks after intra-iBAT administration of 2 x 10 11 vg/mouse of AAV8 or AAV9-mini/UCP1-GFP vector. See Figure 4A. Similarly, AAV8 and AAV9-mini/aP2-GFP delivered internally in iBAT at 2 x 1011 vg/mouse also transduced brown adipocytes. See Figure 11A. Furthermore, the mini/UCP1 regulatory region achieves highly adipocyte-specific GFP expression, completely abolishes AAV-mediated transgene expression in the heart, and mediates only a small amount of liver transduction. See Figure 11B.
为检验AAV介导的iBAT的转导可以是研究褐色脂肪细胞功能的新模型,将7×1010vg/鼠的AAV8-mini/UCP1-mHKII载体给予iBAT。与用AAV8-mini/UCP1-null载体处理的动物相比,接收AAV8-mini/UCP1-mHKII载体的动物表现出增加的通过iBAT摄取的基础2-[1-3H]脱氧-D-葡萄糖,并且在基础条件下,在eWAT和心脏的组之间没有发现2-[1-3H]脱氧-D-葡萄糖摄取的差别。参见图4B。然后,将2×1011vg/小鼠的AAV9-mini/UCP1-VEGF164载体或AAV9-mini/UCP1-null载体递送iBAT内部。注射两个星期后,接收AAV9-mini/UCP1-VEGF164载体的动物表现出VEGF164的过表达和与用AAV9-mini/UCP1-null载体处理的动物相比增加的iBAT中的总VEGF水平。此外,在过表达VEGF164的动物中获得PECAM1(常用的内皮细胞标记物)的过表达。如通过针对iBAT中α-SMA的免疫染色所证明的,用AAV9-mini/UCP1-VEGF164载体处理的动物相比接收AAV9-mini/UCP1-null载体的动物表现出增加的血管的数量。参见图4C-4F。To examine that AAV-mediated transduction of iBAT could be a new model for studying brown adipocyte function, 7×10 10 vg/mouse of AAV8-mini/UCP1-mHKII vector was administered to iBAT. Animals receiving AAV8-mini/UCP1-mHKII vector exhibited increased basal 2-[1- 3 H]deoxy-D-glucose uptake via iBAT compared to animals treated with AAV8-mini/UCP1-null vector, And under basal conditions, no difference in 2-[ 1-3H ]deoxy-D-glucose uptake was found between the eWAT and heart groups. See Figure 4B. Then, 2×10 11 vg/mouse of AAV9-mini/UCP1-VEGF 164 vector or AAV9-mini/UCP1-null vector was delivered inside iBAT. Two weeks after injection, animals receiving AAV9-mini/UCP1-VEGF 164 vector exhibited overexpression of VEGF 164 and increased total VEGF levels in iBAT compared to animals treated with AAV9-mini/UCP1-null vector. In addition, overexpression of PECAM1, a commonly used endothelial cell marker, was obtained in animals overexpressing VEGF 164 . Animals treated with AAV9-mini/UCP1-VEGF 164 vector exhibited an increased number of blood vessels compared to animals receiving AAV9-mini/UCP1-null vector as demonstrated by immunostaining for α-SMA in iBAT. See Figures 4C-4F.
实施例5Example 5
通过AAV8和AAV9的全身给予的白色和褐色脂肪细胞的体内遗传工程化In vivo genetic engineering of white and brown adipocytes by systemic administration of AAV8 and AAV9
将5×1012vg/鼠剂量的AAV8或AAV9-CAG-GFP载体通过尾部静脉给予至瘦鼠。注射两个星期后评估脂肪库的转导。针对eWAT部分的GFP的免疫染色表现出AAV8和AAV9介导的白色脂肪细胞的转导。此外,GFP表达水平和GFP含量的测量证明AAV8和AAV9的转导效率相似。此外,全身递送5×1012vg/鼠的AAV8或AAV9载体高效地介导iBAT的褐色脂肪细胞的转导。通过qPCR的GFP表达水平的测量和通过荧光分析的GFP含量表明AAV8表现出相比AAV9优良的iBAT转导效率的倾向。此外,通过qPCR的GFP表达水平的测量证明基因转移至腹股沟、腹膜后和肠系膜的库。然而,在库之中观察到显著的转导效率的差别。参见图5A-5H。重要地,AAV8或AAV9载体静脉给予至糖尿病-肥胖ob/ob小鼠或db/db小鼠也导致WAT和BAT的遗传工程化,并且效率与在瘦鼠中得到的相似。参见图13。AAV8或AAV9-CAG-GFP载体的全身给予转导多种非脂肪组织。参见图12。A dose of 5 x 1012 vg/mouse AAV8 or AAV9-CAG-GFP vector was administered to lean mice via tail vein. Transduction of fat depots was assessed two weeks after injection. Immunostaining of GFP against eWAT fractions demonstrated AAV8- and AAV9-mediated transduction of white adipocytes. Furthermore, measurements of GFP expression levels and GFP content demonstrated that the transduction efficiencies of AAV8 and AAV9 were similar. Furthermore, systemic delivery of 5 x 10 12 vg/mouse of AAV8 or AAV9 vectors efficiently mediated the transduction of iBAT brown adipocytes. Measurement of GFP expression levels by qPCR and GFP content by fluorescence analysis indicated that AAV8 showed a tendency to exhibit superior iBAT transduction efficiency compared to AAV9. Furthermore, measurement of GFP expression levels by qPCR demonstrated gene transfer to inguinal, retroperitoneal and mesenteric repertoires. However, significant differences in transduction efficiency were observed among the pools. See Figures 5A-5H. Importantly, intravenous administration of AAV8 or AAV9 vectors to diabetic-obese ob/ob mice or db/db mice also resulted in the genetic engineering of WAT and BAT with efficiencies similar to those obtained in lean mice. See Figure 13. Systemic administration of AAV8 or AAV9-CAG-GFP vectors transduced various non-adipose tissues. See Figure 12.
实施例6Example 6
利用AAV-mini/aP2和AAV-mini/UCP1载体的白色和褐色脂肪细胞的体内特异遗传工程化In vivo-specific genetic engineering of white and brown adipocytes using AAV-mini/aP2 and AAV-mini/UCP1 vectors
虽然提供较低水平的转基因表达式,全身递送2×1012vg小鼠的AAV8或AAV9-mini/aP2-GFP导致白色和褐色脂肪细胞的转导。参见图7A。相反地,全身给予2×1012vg小鼠的AAV8或AAV9-mini/UCP1-GFP载体介导显著的褐色脂肪细胞的转导。参见图6A。此外,静脉注射递送的AAV-mini/aP2-GFP和AAV-mini/UCP1-GFP载体实现高度脂肪细胞特异的GFP表达,在心脏中没有可检测的转基因表达以及在肝脏中仅表现出少数分散的GFP+肝细胞的少量表达。参见图7B-7C。Although providing lower levels of transgene expression, systemic delivery of AAV8 or AAV9-mini/aP2-GFP to 2 x 1012 vg mice resulted in transduction of white and brown adipocytes. See Figure 7A. In contrast, systemic administration of AAV8 or AAV9-mini/UCP1-GFP vectors to 2 x 1012 vg mice mediated significant transduction of brown adipocytes. See Figure 6A. In addition, the AAV-mini/aP2-GFP and AAV-mini/UCP1-GFP vectors delivered intravenously achieved highly adipocyte-specific GFP expression, with no detectable transgene expression in the heart and only a few scattered in the liver. Small expression of GFP + hepatocytes. See Figures 7B-7C.
为进一步证明通过AAV全身给予的脂肪细胞的遗传工程化,将2×1012vg的AAV9-mini/UCP1-VEGF164或AAV9-mini/UCP1-null通过尾部静脉递送。注射后两个月,接收AAV9-mini/UCP1-VEGF164载体的动物表现出与用AAV9-mini/UCP1-null载体处理的动物相比增加的VEGF164的表达。参见图6B-6C。此外,将8×1012vg剂量的AAV9-mini/UCP1-VEGF164或AAV9-mini/UCP 1-null载体也通过尾部静脉传递。注射后一个月,接收8×1012vg的AAV9-mini/UCP1-VEGF164载体的动物表现出增加的VEGF164和PECAM1的表达和增加的血管密度。参见图6D-6G。To further demonstrate the genetic engineering of adipocytes administered systemically by AAV, 2 x 1012 vg of AAV9-mini/UCP1-VEGF 164 or AAV9-mini/UCP1-null were delivered via tail vein. Two months after injection, animals receiving AAV9-mini/UCP1-VEGF 164 vector showed increased expression of VEGF 164 compared to animals treated with AAV9-mini/UCP1-null vector. See Figures 6B-6C. In addition, AAV9-mini/UCP1-VEGF164 or AAV9-mini/UCP1-null vector was also delivered via tail vein at a dose of 8×10 12 vg. One month after injection, animals receiving 8 x 1012 vg of AAV9-mini/UCP1-VEGF164 vector exhibited increased expression of VEGF164 and PECAM1 and increased vessel density. See Figures 6D-6G.
实施例7Example 7
使用AAV-mini/aP2-GK的褐色脂肪细胞的体内特异遗传工程化In vivo-specific genetic engineering of brown adipocytes using AAV-mini/aP2-GK
将2×1011vg/鼠剂量的,受mini/aP2调控区控制的,编码小鼠葡糖激酶的AAV9载体(AAV9-mini/aP2-rGK)或等剂量的AAV9-mini/aP2-null载体局部给予至小鼠的iBAT。注射两个星期后/一个月,测定体内基础和胰岛素刺激的2-[1-3H]脱氧-D-葡萄糖摄取,以评估接收AAV9-mini/aP2-rGK载体的动物相比于用AAV9-mini/aP2-null载体处理的动物是否表现出增加的特异的通过iBAT的基础2-[1-3H]脱氧-D-葡萄糖摄取。The AAV9 vector (AAV9-mini/aP2-rGK) encoding mouse glucokinase or an equal dose of AAV9-mini/aP2-null vector controlled by the mini/aP2 regulatory region was administered at a dose of 2 × 10 11 vg/mouse. Topically administered to iBAT of mice. Two weeks/month post-injection, basal and insulin-stimulated 2-[ 1-3H ]deoxy-D-glucose uptake in vivo was determined to evaluate animals receiving AAV9-mini/aP2-rGK vector compared to those treated with AAV9- Whether mini/aP2-null vehicle-treated animals exhibited increased specific basal 2-[1- 3 H]deoxy-D-glucose uptake by iBAT.
实施例8Example 8
AAV载体的全身给予之后来自具有mirT序列的肝脏和心脏的高效脂肪细胞转导和转基因表达的去靶Efficient adipocyte transduction and off-targeting of transgene expression from liver and heart with mirT sequences following systemic administration of AAV vectors
全身给予1012vg/鼠剂量的,在表达框的3'UTR中克隆的,受遍在CAG启动子(AAV9-CAG-GFP)或CAG启动子外加四个肝脏特异miR122a的串连靶位点(AAV9-CAG-GFP-miRT122)、心脏特异miR1(AAV9-CAG-GFP-miRT1)或二者(AAV9-CAG-GFP-双miRT)控制的,编码GFP标记蛋白的AAV9载体。注射两个星期后,在来自接收AAV9-CAG-GFP、AAV9-CAG-GFP-miRT122、AAV9-CAG-GFP-mirT1或AAV9-CAG-GFP-双miRT载体的小鼠的白色和褐色脂肪细胞中观察到较高水平的GFP表达。相反的,在分别用AAV9-CAG-GFP-mirT122或AAV9-CAG-GFP-mirT1载体处理的小鼠中,几乎完全消除了肝脏或心脏中的GFP产生。显然的是在来自用AAV9-CAG-GFP-双miRT处理的小鼠的肝脏和心脏中GFP产生被大幅抑制。参见图14。Systemically administered at a dose of 10 12 vg/mouse, cloned in the 3'UTR of the expression cassette, supported by the ubiquitous CAG promoter (AAV9-CAG-GFP) or the CAG promoter plus four tandem target sites of liver-specific miR122a AAV9 vector encoding a GFP-tagged protein controlled by (AAV9-CAG-GFP-miRT122), heart-specific miR1 (AAV9-CAG-GFP-miRT1), or both (AAV9-CAG-GFP-dual-miRT). Two weeks after injection, in white and brown adipocytes from mice receiving AAV9-CAG-GFP, AAV9-CAG-GFP-miRT122, AAV9-CAG-GFP-mirT1, or AAV9-CAG-GFP-dual-miRT vectors Higher levels of GFP expression were observed. In contrast, GFP production in liver or heart was almost completely abolished in mice treated with AAV9-CAG-GFP-mirT122 or AAV9-CAG-GFP-mirT1 vectors, respectively. It is evident that GFP production is largely inhibited in liver and heart from mice treated with AAV9-CAG-GFP-dual-miRT. See Figure 14.
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