Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector

Adenovirus (Ad) and adeno-associated virus (AAV) have attractive and complementary properties that can be exploited for gene transfer purposes. Ad vectors are probably the most efficient vehicles to deliver foreign genes both in vitro and in vivo. AAV exhibits the unique ability to establish latency by efficiently integrating at a specific locus of human chromosome 19 (AAVS1). Two viral elements are necessary for the integration at AAVS1: Rep68/78 and the inverted terminal repeats (AAV-ITRs). In this study, we report the development of two helper-dependent adenoviral (HD) vectors, one carrying the Rep78 gene, the other an AAV-ITR-flanked transgene. Although Rep proteins have been demonstrated to interfere with Ad replication, HD Rep78 vector was successfully amplified on serial passages in 293CRE4 cells with a yield of 50-100 transducing units per cell. DNA integration at the AAVS1 site also was demonstrated in hepatoma cells coinfected with the HD-expressing Rep78 and with the second HD vector carrying a transgene flanked by AAV-ITRs. The high transduction efficiency, large cloning capacity, and high titer of the HD, combined with the site-specific integration machinery provided by AAV-derived components, make the Ad/AAV hybrid viruses a promising vehicle for gene therapy.     

Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 10⁴ vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 10¹² VG/larva and 4.6 × 10¹² VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.     

A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal

Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an ≈10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of ≈10¹⁰ transducing virions/ml, with <0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.     

Persistent liver expression of murine apoA-l using vectors based on adeno-associated viral vectors serotypes 5 and 1

Plasma levels of high-density lipoprotein-cholesterol (HDL-C) and apolipoprotein A-l (apoA-l) are inversely related to risk for coronary heart disease. Overexpression of apoA-l inhibits atherosclerosis in animal models. A method of stably expressing apoA-l using somatic gene transfer would be of interest. Pseudotyped adeno-associated virus (AAV) vectors comprised of inverted terminal repeats from AAV serotype 2 have been used for liver-directed gene transfers. We hypothesized that liver-directed gene transfer of apoA-l using vectors based on AAV serotypes 1 and 5 would result in higher-level, prolonged expression of apoA-l and increased HDL-C. To test this hypothesis we injected apoA-l-/- mice via the tail vein with either AAV2, AAV1 or AAV5 vectors encoding the murine apoA-l cDNA driven by the liver-specific thyroxine binding globulin promoter. Plasma levels of murine apoA-l and HDL-C were highest in mice injected with the AAV1-based vector and lowest in mice injected with the AAV2-based vector. Expression of apoA-l was stable up to 1 year after vector injection. These results indicate that AAV5 and AAV1 are more effective vectors for achieving higher levels of stable transgene expression of apoA-l after liver-directed gene transfer than AAV2. Furthermore, AAV1-based vectors generate higher apoA-l levels than AAV5-based vectors. It is possible that the levels of expression achieved using these vectors will be therapeutic in preventing atherosclerosis.     

Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.

Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.     

Prevention and reversal of lupus in NZB/NZW mice by costimulatory blockade with adeno-associated virus-mediated gene transfer

OBJECTIVE: To investigate the potency of costimulatory blockade with adeno-associated virus (AAV)-mediated gene transfer in the prevention and reversal of lupus in a murine model. METHODS: AAV vectors expressing CTLA-4Ig or CD40Ig were injected into NZB/NZW mice. Serum levels of transgene expression and autoantibody titers were determined by enzyme-linked immunosorbent assay. The therapeutic effects on proteinuria, renal pathologic features, and survival rate were evaluated. Splenic T cell phenotypes were analyzed by flow cytometry. The humoral immune response to a foreign antigen was also examined in treated mice. RESULTS: A single injection of AAV serotype 8 (AAV8)-CTLA-4Ig in neonatal NZB/NZW mice before the onset of lupus effectively delayed and inhibited autoantibody production, proteinuria, and kidney damage and prolonged their lifespan. In addition, coinjection of AAV8-CTLA-4Ig and AAV8-CD40Ig vectors into neonatal mice achieved a synergistic effect and the best efficacy. The preventive effects were attributed to suppression of CD4+ T cell activation and the transition from naive to memory T cells. Moreover, coinjection of these 2 vectors in adult mice reversed the existing autoantibody levels, suppressed the development of proteinuria, and prolonged their lifespan. The therapeutic effects were found to be dependent on the vector dose. In addition, AAV-mediated long-term gene expression did not severely suppress the host humoral response to foreign antigen. CONCLUSION: Our findings show that delivery of costimulatory inhibitor transgenes by AAV vectors could prevent and reverse lupus in this murine model, suggesting the potential of AAV-mediated gene transfer as an alternative treatment for lupus.     

Production of high-titer helper-free retroviruses by transient transfection.

The generation of high-titer, helper-free retroviruses by transient transfection has been achieved by using the highly transfectable 293T cell line into which are stably introduced constructs that express retroviral packaging functions. The resulting ecotropic virus packaging cell line BOSC 23 produces infectious retrovirus at > 10(6) infectious units/ml of supernatant within 72 hr after CaPO4-mediated transfection. A stringent assay for replication-competent virus showed that no helper virus was present. The system can produce high titers of retroviral vectors expressing genes that are extremely difficult to propagate at high titer in stable producer lines. This method should facilitate and extend the use of helper-free retroviral gene transfer, as well as be useful for gene therapy.     

A shortened adeno-associated virus expression cassette for CFTR gene transfer to cystic fibrosis airway epithelia

Adeno-associated viruses (AAVs) such as AAV5 that transduce airway epithelia from the apical surface are attractive vectors for gene transfer in cystic fibrosis (CF). However, their utility in CF has been limited because packaging of the insert becomes inefficient when its length exceeds approximately 4,900-5,000 bp. To partially circumvent this size constraint, we previously developed a CF transmembrane conductance regulator (CFTR) transgene that deleted a portion of the R domain (CFTRDeltaR). In this study, we focused on shortening the other elements in the AAV expression cassette. We found that portions of the CMV immediate/early (CMVie) enhancer/promoter could be deleted without abolishing activity. We also tested various intervening sequences, poly(A) signals, and an intron to develop an expression cassette that meets the size restrictions imposed by AAV. We then packaged these shortened elements with the CFTRDeltaR transgene into AAV5 and applied them to the apical surface of differentiated CF airway epithelia. Two to 4 weeks later, the AAV5 vectors partially corrected the CF Cl(-) transport defect. These results demonstrate that a single AAV vector can complement the CF defect in differentiated airway epithelia and thereby further the development of effective CF gene transfer.     

DR-nm23基因慢病毒表达载体的构建及其在人结直肠癌SW620细胞中的表达

目的:构建DR-nm23基因慢病毒表达载体及建立DR-nm23基因稳定表达的人结直肠癌SW620细胞系,为DR-nm23基因功能研究奠定基础,方法:应用逆转录聚合酶链反应方法筛选无内源性DR-nm23基因表达的人结直肠癌细胞系.通过AgeⅠ酶切获得DR-nm23 eDNA片段,并将其交换连接到慢病毒表达载体pGC-FU...     

Efficient expression of the vascular endothelial growth factor gene in vitro and in vivo, using an adeno-associated virus vector

Vascular endothelial growth factor (VEGF) has proven to be one of the most effective growth factors for therapeutic angiogenesis. The biological efficacy of the adeno-associated virus (AAV) vector has recently been demonstrated in muscle tissues, including the heart. Apart from these promising insights into VEGF and the AAV vector, studies on VEGF gene transfer using the AAV vector have been limited. Here, we evaluate AAV-mediated VEGF gene transfer, both in vitro and in vivo, using the AAV-mVEGF vector that contains cDNA for murine VEGF(120) within an HCMV-driven expression cassette. Transient transfection of AAV-mVEGF plasmid significantly increased mVEGF expression in 293T cells. The secreted VEGF in the conditioned medium had strong biological activity, as confirmed by the Miles' vascular permeability assay. Transduction of 293T and HeLa cells with AAV-mVEGF stock of high titer, that is essentially adenovirus-free, showed significantly increased mVEGF expression above that of AAV-eGFP-transduced cells. When human umbilical vein endothelial cells were transduced, a higher level of mVEGF expression, together with higher cell counts, was observed compared to AAV-eGFP-transduced cells. In vivo transduction of mouse tibialis anterior muscle resulted in an increased level of mVEGF expression, and higher capillary-to-myofibre ratio, 8 weeks post-transduction. In a rat hindlimb ischemia model, regional blood flow, as well as the capillary-to-myofibre ratio, was significantly increased at 4 weeks post-transduction. These findings demonstrate the efficient delivery of the VEGF gene using an AAV vector, which has implications for angiogenic gene therapy in ischemic diseases.     

Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector.

Adeno-associated virus (AAV) vectors expressing the normal cystic fibrosis transmembrane conductance regulator (CFTR) cDNA complement the cystic fibrosis (CF) defect in vitro. Unlike other DNA virus vectors, AAV is a stably integrating virus, which could make possible long-term in vivo complementation of the CF defect in the airway epithelium. We report AAV-CFTR gene transfer and expression after infection of primary CF nasal polyp cells and after in vivo delivery of AAV-CFTR vector to one lobe of the rabbit lung through a fiberoptic bronchoscope. In the rabbit, vector DNA could be detected in the infected lobe up to 6 months after administration. A 26-amino acid polypeptide sequence unique to the recombinant AAV-CFTR protein was used to generate both oligonucleotide probes and a polyclonal antibody which allowed the unambiguous identification of vector RNA and CFTR protein expression. With these reagents, CFTR RNA and protein were detected in the airway epithelium of the infected lobe for up to 6 months after vector administration. AAV vectors do, therefore, efficiently promote in vivo gene transfer to the airway epithelium which is stable over several months. These findings indicate that AAV-CFTR vectors could potentially be very useful for gene therapy.     

Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy

Adeno-associated viral (AAV) vectors have demonstrated considerable promise for gene therapy of inherited diseases. However, with a packaging size of <5 kb, applications have been limited to relatively small disease genes. Based on the finding that AAV genomes undergo intermolecular circular concatamerization after transduction in muscle, we have developed a paradigm to increase the size of delivered transgenes with this vector through trans-splicing between two independent vectors coadministered to the same tissue. When two vectors encoding either the 5' or 3' portions of the erythropoietin genomic locus were used, functional erythropoietin protein was expressed in muscle subsequent to the formation of intermolecular circular concatamers in a head-to-tail orientation through trans-splicing between these two independent vector genomes. These findings will allow for the application of AAV technologies to a wider variety of diseases for which therapeutic transgenes exceed the packaging limitation of present AAV vectors.     

腺相关病毒Rep78蛋白抑制乙型肝炎病毒复制的体外研究

目的 研究腺相关病毒(adeno-associated virus,AAV)Rep78蛋白对乙型肝炎病毒(HBV)复制的抑制作用及机制.方法 将土拨鼠肝炎病毒(WHV)全基因组DNA从质粒中酶切回收,线状DNA重新连接呈环状.用脂质体Fugene 6体外转染至HepG2细胞,同时共同转染含有Rep78的质粒AAVdl52-91.5 d后收获细胞DNA,Southern blot检测WHV DNA复制.以含有HBV全长的质粒为模板,PCR法分别扩增出HBV-S、C和X基因全长.以凝胶电泳阻滞实验(EMSA)检测Rep78与HBV-S、C和X的结合.结果 Southern blot结果表明,AAV-Rep78可以明显抑制WHV病毒在HepG2细胞中的复制,并呈剂量依赖关系.EMSA结果显示,Rep78蛋白在体外能够与HBV-S、C和X的DNA结合,其中与HBV-C的结合最强而且有剂量依赖关系.此外,这种Rep78蛋白与HBV-C DNA结合可以被特异性的Rep78抗体阻滞,形成超结合带.结论 AAV-Rep78可以抑制HBV DNA的复制,其机制可能在于Rep78蛋白结合并抑制了HBV-C基因.     

Adeno-associated virus serotypes 7 and 8 outperform serotype 9 in expressing atheroprotective human apoE3 from mouse skeletal muscle

Intramuscular injection of adeno-associated viral (AAV) vectors is potentially a safe, minimally invasive procedure for the long-term gene expression of circulating antiatherogenic proteins. Here, we compare secretion and atheroprotective effects of human apoE3 after injection of 3 pseudotyped AAV vectors (AAV2/7, AAV2/8, or AAV2/9), driven by the CMV enhancer/chicken β-actin (CAG) promoter, into skeletal muscle of hyperlipidemic apolipoprotein E-deficient (apoE^−/−) mice. Vector viabilities were verified by transducing cultured C2C12 mouse myotubes and assessing secretion of human apoE3 protein. Both hind limb tibialis anterior muscles of female C57BL/6 apoE^−/− mice, 2 months old and fed a high-fat diet, were each injected with 1 × 10¹⁰ vector genomes of AAV vector. Identical noninjected mice served as controls; and blood was collected at weeks 0, 1, 2, 4, and 13. At termination (13 weeks), the brachiocephalic artery was excised; and after staining sections, plaque morphometry and fractional lipid content were quantified by computerized image analysis. Intramuscular injection of AAV2/7 and AAV2/8 vectors produced up to 2 μg human apoE3 per milliliter plasma, just below the threshold to reverse dyslipoproteinemia. AAV2/9 was notably less effective, mice having a 3-fold lower level of plasma apoE3 at 13 weeks and a 50% greater burden of atherosclerotic plaque lipid in their brachiocephalic arteries. We conclude that although vector refinement is needed to exploit fully apoE3 atheroprotective functions, AAV2/7 and AAV2/8 are promising gene transfer vectors for muscle-based expression of antiatherogenic circulating proteins.     

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 x 10(11)/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.     

Biodistribution of a low dose of intravenously administered AAV-2, 10, and 11 vectors to cynomolgus monkeys

In gene therapy trials, adeno-associated virus (AAV) vectors are injected directly into target tissues such as muscle and liver. Direct injection can lead to the introduction of a low level of the vector into blood circulation. To determine the systemic effects of the vector released in the blood, we extensively examined the biodistribution of intravenously administered AAV serotype 2 (AAV2) vector in cynomolgus monkeys. Although the vector distribution pattern varied from monkey to monkey, the vector DNA was maintained in the various tissues beyond 7 months post-inoculation (pi). The vector DNA was detected in the lymphoid tissues, particularly in the spleen, more frequently and at a much higher level than in the other tissues tested (i.e., brain, lung, liver, heart, gallbladder, pancreas, colon, kidney, ovary, uterus, etc.). The expression of a transgene was detected in the lymph nodes at 3 months pi. The distribution of two pseudotyped vectors, AAV2/10 and AAV2/11, was similar to that of the AAV2 vector. The present results suggest that when introduced intravenously, the AAV vector DNA persists and may induce transgene expression in various monkey tissues. Thus, the possibility of inadvertent gene transfer to various non-target tissues should be considered in a gene therapy strategy with an AAV vector.     

Suppression of transforming growth factor β1 in lung alveolar epithelium-derived cells using adeno-associated virus type 2/5 vectors to carry short hairpin RNA

Since the discovery of RNA interference, short interfering RNA (siRNA) has become a standard research tool. However, expression of siRNA in lung alveolar epithelial cells has remained a problem. Adeno-associated virus (AAV) vectors are known to have low toxicity, and AAV type 5 vectors transduce these cells efficiently. In this study, LacZ expression was higher using AAV2/5-LacZ and LA-4 cells compared with transfection of plasmid or transduction to 3T12-3 cells. The authors designed 10 different siRNAs against mouse transforming growth factor β1 (Tgfβ1), selected one with the highest knockdown efficiency, and transduced the AAV vectors carrying the short hairpin RNA (shRNA) to target cells. The AAV vectors transduced LA-4 cells 50 times more efficiently than 3T12-3 cells, and suppression of Tgfβ1 protein expression was similar, at approximately 50%. Knockdown of mRNA was only seen in LA-4 cells. Inhibition of Tgfβ1 resulted in higher number of LA-4 cells, lower number of 3T12-3 cells, and decreased procollagen expression in LA-4 cells. Higher transduction was seen in H23 cells than in H1975 cells, and low transduction was seen MH-S cells. This study shows that AAV2/5 can be used to carry shRNA and suppress gene function in lung alveolar epithelium-derived cells.     

In vivo marking of rhesus monkey lymphocytes by adeno-associated viral vectors: direct comparison with retroviral vectors.

We have compared adeno-associated virus (AAV)-based and retrovirus-based vectors for their ability to transduce primary T lymphocytes in vitro and then tracked the persistence of these genetically marked lymphocytes in vivo, using the rhesus monkey model. To avoid the complication of immune rejection of lymphocytes transduced with xenogeneic genes in tracking studies primarily designed to investigate transduction efficiency and in vivo kinetics, the vectors were designed without expressed genes. All vectors contained identically mutated beta-galactosidase gene (beta-gal) and neomycin resistance gene (neo) DNA sequences separated by different length polylinkers, allowing simple differentiation by polymerase chain reaction (PCR). Each of 2 aliquots of peripheral blood lymphocytes from 4 rhesus monkeys were transduced with either AAV or retroviral vectors. The in vitro transduction efficiency (mean vector copy number/cell) after the ex vivo culture was estimated by PCR at 0.015 to 3.0 for AAV, varying depending on the multiplicity of infection (MOI) used for transduction, and 0.13 to 0.19 for the retroviral transductions. Seven days after transduction, Southern blot analysis of AAV-transduced lymphocytes showed double-stranded and head-to-tail concatemer forms but failed to show integration of the AAV vector. AAV and retroviral aliquots were reinfused concurrently into each animal. Although the retrovirally marked lymphocytes could be detected for much longer after infusion, AAV transduction resulted in higher short-term in vivo marking efficiency compared with retroviral vectors, suggesting possible clinical applications of AAV vectors in lymphocyte gene therapy when long-term vector persistence is not required or desired.