口服重组腺相关病毒基因药物

重组腺相关病毒 (rAAV) 载体介导的口服基因药物引起业界广泛的重视。尽管经口服给药后转基因的有效表达面临许多障碍, 但该技术的有效性已得到大量实验证实。本文总结了口服rAAV基因药物的临床前研究结果, 重点阐述了该类型药物的传递、吸收、分布和基因转导等药动学特点。已证实rAAV基因药物对人体的安全性高, 但口服rAAV基因药物的临床应用仍需对其作用机制和生物药剂学特征进行深入和广泛的研究。     

Recombinant adeno-associated virus-mediated human kallikrein gene therapy protects against hypertensive target organ injuries through inhibiting cell apoptosis

Aim： Overexpression of human tissue kallikrein （HK）, mediated by recombinant adeno-associated virus （rAAV）, decreased blood pressure in spontaneous hypertensive rats （SHRs） and reduced injury to the heart, aorta and kidney. In this study, we used both an in vivo animal model and in vitro cell culture system to investigate whether rAAV-mediated HK gene therapy protects against organ damage by inhibiting cell apoptosis. Methods： rAAV encoding HK （rAAV-HK） or LacZ （rAAV-lacZ） were delivered as a control to spontaneously hypertensive rats （SHRs） and cultured human embryonic kidney （HEK） 293 cells. Results： Treatment with rAAV-HK decreased cell apoptosis in the target organs of SHRs and also inhibited lipopolysaccharide （LPS）-induced HEK 293 apoptosis. The rAAV-HK delivery system also increased the levels of apoptosis-inhibiting proteins bcl-2 and bcl-XL, and decreased the level of Bax and the activity of caspase 3, two promoters of apoptosis. In addition to its role in the inhibition of apoptosis, rAAV-HK also activated the cell survival and proliferation signaling pathways ERK1/2 and PI3K/AKT. Conclusion： rAAV-mediated HK gene delivery has multiple therapeutic possibilities for treating hypertension, not only by decreasing blood pressure, but also by directly inhibiting end-organ damage.     

The potential of adeno-associated viral vectors for gene delivery to muscle tissue

Introduction: Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8⁺ T-cell response against AAV capsid in humans.

Areas covered: In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application.

Expert opinion: Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.     

In vitro and in vivo induction of bone formation based on adeno-associated virus-mediated BMP-7 gene therapy using human adipose-derived mesenchymal stem cells

Aim： To determine whether adeno-associated virus （AAV）-2-mediated, bone morphogenetic protein （BMP）-7-expressing human adipose-derived mesenchymal stem cells （ADMS） cells would induce bone formation in vitro and in vivo. Methods： ADMS cells were harvested from patients undergoing selective suction-assisted lipectomy and transduced with AAV carrying the human BMP-7 gene. Non-trans- duced cells and cells transduced with AAV serotype 2 carrying the enhanced green fluorescence protein gene served as controls. ADMS cells were qualitatively assessed for the production of BMP-7 and osteocalcin, and subjected to alkaline phosphatase （ALP） and Chinalizarin staining. A total of 2.5×10^6 cells mixed with type I collagen were implanted into the hind limb of severe combined immune-deficient （SCID） mice and subjected to a histological analysis 3 weeks post implantation. Results： Transfection of the ADMS cells achieved an efficiency of 99% at d 7. Transduction with AAV2-BMP-7 induced the expression of BMP-7 until d 56, which was markedly increased by d 7. The cells were positively stained for ALP. Osteocalcin production and matrix mineralization further con- firmed that these cells differentiated into osteoblasts and induced bone formation in vitro. A histological examination demonstrated that implantation of BMP-7- expressing ADMS cells could induce new bone formation in vivo. Conclusion： The present in vitro and in vivo study demonstrated that human ADMS cells would be a promising source of autologous mesenchymal stem cells for BMP gene therapy and tissue engineering.     

FKBP52在重组腺相关病毒载体胞内转运中的作用研究

目的探讨细胞质中FKBP52对重组腺相关病毒(AAV)介导的基因转导效率的作用。方法测定AAV在FKBP52-野生型(WT)、FKBP52-杂合型(HE)及FKBP52-基因敲除(KO)鼠胚纤维母细胞(MEFs)中的基因转导效率,观察羟基脲(HU)对不同基因型MEFs中AAV转导效率的影响。采用流式细胞术、EMSA、Southernblot、免疫沉淀、West-ernblot等方法分析FKBP52对AAV在细胞内转运的作用。结果传统的单链AAV不能有效地转导FKBP52-WTMEFs,在FKBP52-HE及FKBP52-KOMEFs中的转导效率也未见明显增加。在这些细胞中,AAV不能有效地转运至细胞核。HU处理后能使AAV在WTMEFs中的转导效率提高25倍,但是在KOMEFs中AAV转导效率仅提高4倍。为避免AAV第二链合成的影响,进一步采用自身互补的AAV(scAAV)载体进行研究,结果发现,HU能使WTMEFsscAAV基因转导效率增强23倍,但在KOMEFs中转导效率仅增加4倍。提示,HU处理后KOMEFs中转导效率未增加的原因并非由于AAV第二链的合成障碍所致。HU处理后,59%的AAV基因组DNA出现在WTMEFs细胞核,KOMEFs细胞核中为28%,提示HU介导的AAV转运通路在KOMEFs中存在缺陷。转染FKBP52表达质粒的KOMEFs经HU处理后,AAV所介导的基因转导效率恢复至WTMEFs的水平,且直接与其转运至细胞核的转运功能改善相吻合。结论作为一种细胞分子伴侣蛋白,FKBP52能促进AAV在细胞内转运,有益于重组AAV载体在人类基因治疗中的应用。     

A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting

Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising.Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson''s disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway.These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described.     

腺相关病毒介导的pdx-1基因表达促进糖尿病大鼠肝脏细胞向胰岛素产生细胞转化的研究

目的：胰十二指肠同源盒基因-l（pancreatic duodenal homeobox-1，pdx-1）是胰岛发育和分化过程中重要的转录因子。本实验旨在研究腺相关病毒（adeno-associated virus，AAV）介导的pdx-1基因的表达是否可以诱导糖尿病大鼠肝脏细胞分化成产生胰岛素的细胞，以进一步为糖尿病的细胞替代疗法提供信息。方法：门静脉途径注射AAV．pdx-1或AAV对照载体到大鼠体内。6周后，用RT-PCR和免疫细胞化学法检测肝脏中胰岛素的基因表达及测定大鼠血糖、体重的变化。结果：AAV-pdx-1治疗组明显提高了糖尿病大鼠肝脏中胰岛素的基因表达和胰岛素阳性细胞数，改善了糖尿病大鼠的高血糖状态及增加了体重。结论：AAV-pdx-1可诱导糖尿病大鼠肝脏细胞分化成产生胰岛素的细胞，缓解了糖尿病状态。其分化的机制及分化的肝脏细胞亚群需进一步实验研究，以提高分化效率。     

Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD)

A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a ～ d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.     

Intramuscular delivery of rAAV-mediated kallikrein gene reduces hypertension and prevents cardiovascular injuries in model rats

Aim： The overexpression of the human tissue kallikrein （HK） gene can reduce blood pressure and ameliorate the secondary syndromes associated with hyper- tension in animal models. The current study was designed to investigate hy- potensive effect of intramuscular delivery of HK gene. Methods： We generated an recombinant adeno-associated virus （rAAV） vector expressing human tissue kallikrein under the control of a cytomegalovirus promoter and administered the rAAV-HK vector to a spontaneously hypertensive rat model at a dose of lx 10^10 virons/rat through intramuscular injection. Results： A persistent, high-level ex- pression of HK post-gene delivery was confirmed by ELISA. The systolic blood pressure in the rats receiving rAAV-LacZ and saline increased from 171.3 mmHg to 182.3 mmHg 28 weeks＇ post injection. In contrast, the delivery of the HK gene by AAV vectors attenuated the increase of the systolic blood pressure in the treated group. The systolic blood pressure was only slightly lowered （from a level of 174 mmHg to 170.5 mmHg） post-vector administration. The difference in blood pres- sure between the treated group and the control groups is statistically significant at 12.6 mmHg. The hypotensive effect of rAAV-HK persisted until the end of the testing period. In addition, a significant amelioration of cardiovascular hypertrophy, renal injury, and collagen depositions in the rAAV-HK-treated ani- mals were also observed. Conclusion： All the effects are comparable with those of intravenous delivery. Therefore, the intramuscular administration of rAAV-HK may be used in gene therapy for hypertension.     

人甲胎蛋白启动子控制HIV-1 Vpr表达的重组腺病毒体内抑瘤效果研究

目的：研究人甲胎蛋白启动子控制表达HIV Vpr基因的重组腺病毒对裸鼠肝癌模型的肿瘤组织生长抑制作用,探讨其应用于肝癌基因治疗的可行性。方法：将人甲胎蛋白启动子控制表达HIV Vpr基因的重组腺病毒（rvAdAFP-Vpr）直接注射到利用BEL-7402细胞建立的肝癌裸鼠模型瘤体内,同时往裸鼠腹腔内注射环磷酰胺（CTX）,经过1个月的治疗,利用肿瘤生长曲线、增殖指数、凋亡指数等指标,观察rvAdAFP-Vpr对肝癌组织生长抑制的效果。结果：人甲胎蛋白启动子控制表达HIV Vpr基因的重组腺病毒在肝癌组织中表达了Vpr蛋白,rvAdAFP-Vpr注射明显诱导肿瘤细胞凋亡,抑制了体内肝癌组织的生长,rvAdAFP-Vpr治疗组和rvAdAFP-Vpr＋CTX治疗组抑瘤率分别达到41％和66．7％,与空白对照组和rvAd-null组相比,具有统计学意义,差异显著（P〈0．05,P〈0．01）,两组的Ki-67指数和凋亡指数相比,对照组和空腺病毒载体（rvAd-null）组同样具有统计学意义,差异显著（P〈0．05,P〈0．01）。结论：人甲胎蛋白启动子控制表达HIV Vpr基因的重组腺病毒rvAdAFP-Vpr通过引起肝癌细胞凋亡,有效抑制了体内肝癌组织的生长。本研究结果为HIV-1 Vpr应用于肝癌治疗提供了依据。     

携带人内皮一氧化氮合酶基因的AAV重组表达载体的构建

目的：构建一个新型的携带有人内皮一氧化氮合酶(eNOS)cDNA的质粒载体并研究其体外表达,以用于基因治疗．方法：eNOS cDNA插入到腺相关病毒质粒pSNAV-1的EcoR I位点．该质粒的启动子为CMV启动子,并携带有腺相关病毒的末端重复序列．构建好的质粒转染到两种哺乳动物细胞BHK和C2C12细胞中,通过PCR和RT—PCR分别检测eNOS cDNA和mRNA．结果：限制性内切酶分析证明,eNOS cDNA以正确的方向插入到pSNAV-1质粒中．PCR检测表明pSNAV—eNOS被转入BHK和C2C12细胞中．RT—PCR检测表明转染有pSNAV—eNOS的细胞可表达eNOS mRNA．结论：成功构建的pSNAV—eNOS可在体外培养的哺乳动物细胞中表达人eNOS mRNA．     

聚乳酸/乙醇酸共聚物纳米基因载体的制备及转染效率考察

目的：为了制备转染效率高的新型基因载体，本实验制备由聚酰胺一胺型树状大分子（PAMAM）与聚乳酸／乙醇酸共聚物（PLGA）复合的新型基因载体，并评价了其转染效率。方法：采用溶剂挥发法制备了PAMAM-PLGA新型基因载体，并且通过考察纳米基因载体粒径，粒子形态，稳定性，zeta电位、pH值等，从而确定了最佳的制备工艺。用电泳实验和绿色荧光蛋白标记方法研究了它的转染效率。结果：新型基因载体能够很好的携带DNA进入细胞。结论：在适合的制备工艺条件下可以制备出粒径大约100nm，分散系数为0.088的新型纳米基因载体，其转染效率要高于单纯的PAMAM。     

超声微泡介导的基因递送系统应用进展

超声波可聚焦于体内的特定部位。含气体微泡既可以作为医学超声显像的造影剂，又可以作为药物或基因载体。超声微泡有望实现基因的靶向递送，因此成为药物递送系统研究的热门领域。本文阐述了超声微泡介导的基因递送系统在心肌、血管、骨骼肌和肿瘤组织等方面的研究进展，讨论其在未来应用中面临的问题。     

Lung epithelial binding peptide-linked high mobility group box-1 A box for lung epithelial cell-specific delivery of DNA

High mobility group box-1 A box (HMGB1A) is an anti-inflammatory peptide originating from HMGB1. A previous report demonstrated that recombinant HMGB1A could deliver DNA into cells. Lung epithelial-specific gene delivery is required for the gene therapy of various lung diseases such as acute lung injury. In this study, a lung epithelial-specific DNA carrier was produced by linking the lung epithelial binding peptide (LEBP) to HMGB1A. An LEBP-linked HMGB1A (LEBP-HMGB1A) expression vector, pET21a-LEBP-HMGB1A, was constructed. LEBP-HMGB1A was expressed in BL21 strain and purified by consecutive applications of nickel affinity chromatography and cationic exchange chromatography. In a gel retardation assay, LEBP-HMGB1A completely retarded DNA at a 5:1 weight ratio (peptide:DNA). LEBP-HMGB1A/DNA complexes were prepared at various weight ratios, to which a fixed amount of polyethylenimine (2?kDa, PEI2k) was added to increase the proton buffering effect of the complex. LEBP-HMGB1A had the highest transfection efficiency to L2 lung epithelial cells at a 20:1 weight ratio (peptide:DNA). At this ratio, LEBP-HMGB1A had a higher transfection efficiency than poly-L-lysine (PLL) as well as HMGB1A without LEBP. A cytotoxicity assay showed that LEBP-HMGB1A was not toxic to L2 cells. Therefore, LEBP-HMGB1A may be useful in developing gene therapies for lung diseases.     

重组人粒细胞集落刺激因子壳聚糖胶囊的制备和体外释药特性

目的 :制备人重组粒细胞集落刺激因子 (rhG CSF)冻干粉剂壳聚糖胶囊 ,并对其体外释药性能进行评价。方法 :将rhG CSF冻干粉剂装入壳聚糖胶囊中 ,再以邻苯二甲酸羟丙基甲基纤维素 (HPMCP)包裹胶囊 ,用氮兰四唑蓝 (MTT)比色法测定其在人工胃液及小肠液中的体外释放性能。将荧光素钠 (FS)作为模型化合物在相同条件下进行实验 ,以激发波长 470nm、发射波长 5 13nm荧光检测FS壳聚糖胶囊在人工大肠液中的体外释放性能。用扫描电镜法评价壳聚糖胶囊在大肠内容物中的降解作用。结果 :在人工大肠液中壳聚糖具有明显降解作用。rhG CSF壳聚糖胶囊在人工胃液 (2h)和人工小肠液 (6h)内累积释药量为 (15 .5± 6 .5 ) % ,n =6。而在人工大肠液中 ,FS壳聚糖胶囊 4h释药基本完全。结论 :用HPMCP包膜的rhG CSF壳聚糖胶囊具有潜在的结肠靶向释药特性。     

Recent advances in the development of polyethylenimine-based gene vectors for safe and efficient gene delivery

Introduction: Despite the great therapeutic potential of gene therapy for treating critical diseases, the clinical application is limited by lack of safe and effective gene delivery vectors. Nonviral gene vectors have attracted tremendous attention due to the favorable loading capacity and facile manufacture. Among them, polyethylenimine-based gene vectors (PEIs) hold great promise for highly efficient gene delivery.

Areas covered: In this review, we outline the multiple biological barriers associated with gene delivery process and point out several challenges exist in the clinical usage of PEIs. We then provide an overview of the most impressive progresses made to overcome such challenges in recent years, including modifications of PEIs (i.e. to enhance biocompatibility, specific targeting effect, and buffering capacity) and stimuli-responsive strategies (i.e. endogenous and exogenous stimuli) for safe and efficient gene delivery.

Expert opinion: Rational modification of PEIs with diverse functionalized segments or the development of stimuli-responsive PEIs is an appealing strategy to meet some requirements involved in gene delivery. Nevertheless, further optimization by combining the two strategies is needed for the maximized transfection efficiency and minimized side effects, shedding new light on the development of nonviral gene delivery for clinical application.     

Optimization of ultrasound and microbubbles targeted gene delivery to cultured primary endothelial cells

Ultrasound and microbubbles targeted gene delivery (UMTGD) is a promising technique for local gene delivery. As the endothelium is a primary target for systemic UMTGD, this study aimed at establishing the optimal parameters of UMTGD to primary endothelial cells. For this, an in vitro ultrasound (US) setup was employed in which individual UMTGD parameters were systematically optimized. The criteria for the final optimized protocol were: (1) relative high reporter gene expression levels, restricted to the US exposed area and (2) induction of not more than 5% cell death. US frequency and timing of medium replacement had a strong effect on UMTGD efficiency. Furthermore, US intensity, DNA concentration and total duration of US all affected UMTGD efficiency. Optimal targeted gene delivery to primary endothelial cells can be accomplished with Sonovue® microbubbles, using 20 μg/ml plasmid DNA, a 1 MHz US exposure of Ispta 0.10 W/cm² for 30 s with immediate medium change after UMTGD. This optimized protocol resulted in both an increase in the number of transfected cells (more than three fold) and increased levels of transgene expression per cell (170%).     

Cardiac adenovirus-associated viral Presenilin 1 gene delivery protects the left ventricular function of the heart via regulating RyR2 function in post-ischaemic heart failure

Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca²⁺ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca²⁺ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca²⁺ leak, thereby restoring the diminished intracellular Ca²⁺ transients and SR Ca²⁺ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.