Mosaic vectors comprised of modified AAV1 capsid proteins for efficient vector purification and targeting to vascular endothelial cells

Vascular-targeted gene therapies have the potential to treat many of the leading causes of mortality in the western world. Unfortunately, these therapies have been ineffective due to poor vascular gene transfer. The use of alternative virus serotypes and the incorporation of vascular targeting ligands into vectors has resulted in only modest increases in vascular gene transfer. Adeno-associated virus (AAV) 1 has shown the most promise among the AAV vectors for the transduction of vascular endothelial cells. However, no straightforward small-scale purification strategy exists for AAV1 as it does for AAV2 making it difficult to quickly produce AAV1 vector for analysis. Here we have combined two AAV1 capsid protein modifications to enhance vascular gene transfer and allow easy purification of vector particles. Mosaic vector particles have been produced comprised of capsid proteins containing the well-characterized RGD4C modification to target integrins present on the vasculature, and capsid proteins containing a modification that permits metabolic biotinylation and efficient purification of mosaic particles by avidin affinity chromatography. We show that the RGD modification results in a 50-100-fold enhancement in endothelial cell gene transfer that is maintained in biotinylated mosaic AAV1 particles. These results suggest that mosaic virions hold significant promise for targeted gene delivery to the vasculature.     

Avidin-based targeting and purification of a protein IX-modified, metabolically biotinylated adenoviral vector.

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors.     

Metabolic biotinylation of lentiviral pseudotypes for scalable paramagnetic microparticle-dependent manipulation.

Nonviral, host-derived proteins on lentiviral vector surfaces can have a profound effect on the vector's biology as they can both promote infection and provide resistance to complement inactivation. We have exploited this to engineer a specific posttranslational modification of a "nonenvelope," virally associated protein. The bacterial biotin ligase (BirA) and a modified human DeltaLNGFR have been introduced into HEK293T cells and their protein products directed to the lumen of the endoplasmic reticulum. The BirA then couples biotin to an acceptor peptide that has been fused to the DeltaLNGFR. This results in the covalent linkage of biotin to the extracellular domain of the DeltaLNGFR expressed on the cell surface. Lentiviral vectors from these cells are metabolically labeled with biotin in the presence of free biotin. These biotinylated lentiviral vectors have a high affinity for streptavidin paramagnetic particles and, once captured, are easily manipulated in vitro. This is illustrated by the concentration of lentiviral vectors pseudotyped with either the VSV-G or an amphotropic envelope in excess of 4500-fold. This new cell line has the potential for widespread application to envelope pseudotypes compatible with lentiviral vector production.     

An efficient rHSV-based complementation system for the production of multiple rAAV vector serotypes

Recombinant herpes simplex virus type 1 (rHSV)-assisted recombinant adeno-associated virus (rAAV) vector production provides a highly efficient and scalable method for manufacture of clinical grade rAAV vectors. Here, we present an rHSV co-infection system for rAAV production, which uses two ICP27-deficient rHSV constructs, one bearing the rep2 and cap (1, 2 or 9) genes of rAAV, and the second bearing an AAV2 ITR-gene of interest (GOI) cassette. The optimum rAAV production parameters were defined by producing rAAV2/GFP in HEK293 cells, yielding greater than 9000 infectious particles per cell with a 14:1 DNase resistance particle to infectious particle (DRP/ip) ratio. The optimized co-infection parameters were then used to generate large-scale stocks of rAAV1/AAT, which encode the human alpha-1-antitrypsin (hAAT) protein, and purified by column chromatography. The purified vector was extensively characterized by rAAV- and rHSV-specific assays and compared to transfection-made vector for in vivo efficacy in mice through intramuscular injection. The co-infection method was also used to produce rAAV9/AAT for comparison to rAAV1/AAT in vivo. Intramuscular administration of 1 x 10(11) DRP per animal of rHSV-produced rAAV1/AAT and rAAV9/AAT resulted in hAAT protein expression of 5.4 x 10(4) and 9.4 x 10(5) ng ml(-1) serum respectively, the latter being clinically relevant.     

Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.

Aggregation of recombinant AAV2 results in reduced yield during purification and may have deleterious effects on vector transduction efficiency, biodistribution and immunogenicity following in vivo administration. Studies to elucidate the mechanism of vector aggregation and methods to prevent its occurrence are reported. In excipient screening studies, the sugars sorbitol, sucrose, mannitol, trehalose, or glycerol at concentrations of up to 5% (w/v), or surfactants Tween 80 or Pluronic F68, did not prevent aggregation. Aggregation was prevented by the use of various salts at concentrations corresponding to solution ionic strengths of >200 mM. AAV2 vectors purified by double cesium chloride gradient centrifugation, cation-exchange chromatography, or combined chromatography and gradient centrifugation each demonstrated a similar requirement for ionic strength to prevent aggregation. AAV2 vectors concentrated to 6.7 x 10(13) vector genome (vg)/mL in neutral-buffered isotonic saline resulted in 59+/-6.0% recovery of nonaggregated material compared to 96+/-4.4% recovery in an isotonic formulation with elevated ionic strength. The latter showed no aggregation following storage or after 10 freeze-thaw cycles at -20 degrees C. AAV2 vectors stored for an extended period in an elevated ionic strength formulation retained a high infectivity titer (13 vg/infectious unit) and transduction efficiency. Nuclease digestion of purified AAV2 vectors reduced aggregation, implicating trace amounts of vector surface nucleic acids in interparticle binding.     

Simultaneous targeting of HCV replication and viral binding with a single lentiviral vector containing multiple RNA interference expression cassettes.

Chronic hepatitis C virus (HCV) infection has a major medical impact and current treatments are often unsuccessful. RNA interference represents a promising new approach to tackling this problem. The current study details the design and testing of self-inactivating lentiviral vectors (LV) delivering RNA interference to prevent HCV replication and infection. Vectors were constructed with single, double, and triple cassettes expressing short hairpin RNAs (shRNAs) simultaneously targeting two regions of the HCV 1b genome and the host cell receptor, CD81. The shRNAs directed against HCV IRES or NS5b regions were shown to be effective in inhibiting HCV replication in vitro (82 and 98%, respectively). No evidence of shRNA-related interferon production was observed. Vectors containing CD81 shRNA reduced cell surface expression up to 83% and reduced cell binding of HCV surface protein E2 up to 82% while not affecting levels of unrelated surface protein (Ber-EP4) or HCV replication. Double or triple shRNA vectors were independently effective in simultaneously reducing HCV replication, CD81 expression, and E2 binding. This study demonstrates lentiviral delivery of multiple shRNA, inhibiting HCV in a specific, IFN-independent, manner. The targeting of multiple viral and host cell elements simultaneously by RNAi could increase the potency of antiviral gene therapies.     

Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector

The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.     

AAV2/5 vector expressing galactocerebrosidase ameliorates CNS disease in the murine model of globoid-cell leukodystrophy more efficiently than AAV2.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive lysosomal storage disorder caused by mutations in the galactosylceramidase (GALC) gene. Infantile GLD has a lethal course with severe cerebral demyelination that progresses to death by 2 years of age. In the current study twitcher mice, an authentic murine model of infantile GLD, were given intracranial injections of either recombinant adeno-associated virus serotype 2 encoding the murine Galc cDNA (AAV2-GALC) or the same genome pseudotyped with AAV5 capsid proteins (AAV2/5-GALC) on day 3 of age. The group injected intracranially with AAV2/5-GALC had approximately 25-fold greater than normal Galc levels in the brain, while AAV2-GALC-injected animals had 28% normal levels. The average life expectancy of twitcher mice ( approximately 38 days) was significantly (P < 0.0001) increased to 48 and 52 days for the AAV2-GALC- and AAV2/5-GALC-treated groups, respectively. The AAV2/5-GALC group performed significantly better in a battery of behavioral tests compared to untreated, AAV2-GFP-treated, or AAV2-treated twitcher animals. This longitudinal study demonstrated that AAV2/5-GALC-mediated gene therapy resulted in higher levels of Galc expression and slowed the neurologic deterioration more completely than AAV2-GALC in the murine model of globoid-cell leukodystrophy. However, the clinical improvements, as assessed by behavioral tests and life span, were only modest.     

Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates.

Gene therapy using recombinant adeno-associated virus (rAAV2) vectors for cystic fibrosis has shown gene transfer and remarkable safety, yet indeterminate expression. A new construct has been characterized with a powerful exogenous promoter, the cytomegalovirus enhancer/chicken beta-actin promoter, driving a truncated CF transmembrane conductance regulator (CFTR), pseudotyped in an AAV5 viral coat. Our goal is to demonstrate that airway delivery of a pseudotyped rAAV5 vector results in gene transfer as well as expression in non-human primates. Aerosolized pseudotyped rAAV5-DeltaCFTR or rAAV5-GFP (green fluorescent protein) genes were delivered to four and six lungs, respectively. The pseudotyped rAAV5 vector did result in GFP gene transfer (1.005x10(6) copies/mug DNA on average) and quantifiable gene expression. Microscopy confirmed protein expression in airway epithelium. Similarly, the vector also resulted in vector-specific CFTR DNA (1.24x10(5) copies/microg) and mRNA expression. Immunoprecipitation and (32)P phosphoimaging were used to demonstrate CFTR protein expression, as qualitatively enhanced beyond the barely detectable endogenous expression in untreated animals. Based on these promising studies, this CFTR minigene construct is a therapeutic candidate.     

人肿瘤抑素基因真核表达载体的构建及其活性研究

目的构建携带人肿瘤抑素(Tumstatin)基因的重组腺相关病毒载体,研究其对人脐静脉内皮细胞(HUVECs)增殖的影响。方法构建重组载体pAAV-Tum质粒,并建立一株稳定表达Tumstatin基因的HU-VECs细胞株。用逆转录聚合酶链反应检测该细胞系中Tumstatin基因的表达,四甲基唑蓝法检测该细胞增殖情况。结果构建的pAAV-Tum重组腺相关病毒载体能在HUVECs细胞中表达,其表达产物能抑制该细胞增殖(0.335±0.011)。结论成功构建了pAAV-Tum重组载体,构建的重组载体能有效表达,为其后的抗肿瘤血管生成治疗研究奠定了基础。     

Bioadhesive microdevices with multiple reservoirs: a new platform for oral drug delivery.

A variety of delivery systems have been devised, in recent years, to improve the oral bioavailability of drugs including enterically coated tablets, capsules, particles, and liposomes. Microfabrication technology may offer some potential advantages over conventional drug delivery technologies. This technology, combined with appropriate surface chemistry, may permit the highly localized and unidirectional release of drugs, permeation enhancers, and/or promoters. In this study, we demonstrate the fabrication of prototype reservoir-containing microdevices and a surface chemistry protocol that can be used to bind lectin via avidin-biotin interactions to these micromachined drug delivery vehicles. The use of microfabrication allows one to tailor the size, shape, reservoir volume, and surface characteristics of the drug delivery vehicle. In vitro studies show enhanced bioadhesion of these lectin conjugated silicon microdevices. This approach may be used to improve the absorption of pharmacologically active biopolymers such as peptides, proteins and oligonucleotides into circulation at targeted sites in the GI system via the creation of a robust hybrid organic/inorganic delivery system. This paper describes one of the first applications of microfabrication to oral drug delivery.     

Comparison of adeno-associated viral vector serotypes for spinal cord and motor neuron gene delivery

Gene therapy for motor neuron diseases requires efficient gene delivery to motor neurons (MNs) throughout the spinal cord and brainstem. The present study compared adeno-associated viral (AAV) vector serotypes 1, 6, 8, and 9 for spinal cord delivery in adult mice, by the intraparenchymal or intrathecal route of administration. Whereas intraparenchymal injections resulted in local transduction of the lumbar segment of the spinal cord, intrathecal injections led to a broader distribution, transducing cells along the sacral, lumbar, and lower thoracic spinal cord. Overall, AAV6 and AAV9 performed better than the other serotypes. Dramatic differences in cell-specific expression patterns could be observed when constructs bearing the chicken β-actin (Cba) versus cytomegalovirus (CMV) promoter were compared. In summary, intrathecal delivery of AAV6 or AAV9 vectors containing the CMV promoter yielded the strongest levels of biodistribution and MN transduction in the spinal cord.     

New multifunctional molecular conjugate vector for targeting, imaging, and therapy of tumors.

We report the in vitro and in vivo characteristics of a new molecular conjugate vector for targeting and imaging of tumors. Its core is a cyclodecapeptide platform named RAFT, onto which two spatially independent functional domains can be covalently and stereospecifically linked: a cell-targeting domain for tumor targeting and a labeling domain able to carry two drugs and/or labeling agents. To prove the interest of this carrier, we used a well-known cRGD cyclopeptide, a ligand for the alphavbeta3 integrin. We demonstrate that this vector presenting four cRGD motifs very efficiently prevents alphavbeta3-mediated cell adhesion to vitronectin. Furthermore, it is actively endocytosed because of the multivalent cRGD presentation, a major advantage for drug delivery. In vivo experiments in nude mice reveal that repeated intratumoral injections of low doses of RAFT(cRGD)4 reduce tumor growth. Furthermore, RAFT(cRGD)4 significantly improves the targeting specificity of subcutaneous tumor masses as well as that of disseminated metastasis after intravenous injection. Thus, RAFT(cRGD)4 is specific, internalized, and perfectly controlled and can carry multiple biological functions on a single, spatially defined backbone, making it a powerful and versatile synthetic vector for drug delivery, molecular imaging, or both.     

Smad3基因siRNA表达载体的构建及沉默效应

背景:研究提示抑制Smad3可望抑制纤维增生和胶原的大量产生.目的:构建针对Smad3基因的siRNA表达载体,观察RNA干扰对增生性瘢痕成纤维细胞Smad3基因表达的抑制作用.方法:根据siRNA设计原则,设计3条针对Smad3基因的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后与载体pRNAT-U6连接,然后进行酶切鉴定和DNA序列测定.用脂质体包裹转染增生性瘢痕成纤维细胞,荧光定量PCR和Western blot方法检测Smad3基因的表达情况.结果与结论:测序分析结果显示:克隆入pRNAT-U6载体的针对Smad3基因的siRNA的双链寡核苷酸片段插入正确;荧光定量PCR和Western blot检测显示:转染的增生性瘢痕成纤维细胞Smad3基因的表达水平明显降低,尤以AGA CAG ACT GTG ACC AGT A(1156)为靶序列的siRNA沉默作用最强,抑制效率于转染后48 h可达45%.证实实验构建的沉默增生性瘢痕成纤维细胞Smad3基因表达的siRNA载体获得成功.     

Smad3基因siRNA表达载体的构建及沉默效应

背景：研究提示抑制Smad3可望抑制纤维增生和胶原的大量产生。目的：构建针对Smad3基因的siRNA表达载体,观察RNA干扰对增生性瘢痕成纤维细胞Smad3基因表达的抑制作用。方法：根据siRNA设计原则,设计3条针对Smad3基因的siRNA靶序列,分别合成两条互补的寡核苷酸链,退火后与载体pRNAT-U6连接,然后进行酶切鉴定和DNA序列测定。用脂质体包裹转染增生性瘢痕成纤维细胞,荧光定量PCR和Western blot方法检测Smad3基因的表达情况。结果与结论：测序分析结果显示：克隆入pRNAT-U6载体的针对Smad3基因的siRNA的双链寡核苷酸片段插入正确;荧光定量PCR和Western blot检测显示：转染的增生性瘢痕成纤维细胞Smad3基因的表达水平明显降低,尤以AGA CAG ACT GTG ACC AGT A（1156）为靶序列的siRNA沉默作用最强,抑制效率于转染后48h可达45%。证实实验构建的沉默增生性瘢痕成纤维细胞Smad3基因表达的siRNA载体获得成功。     

Inadvertent germline transmission of AAV2 vector: findings in a rabbit model correlate with those in a human clinical trial.

The risk of germline transmission of vector sequences in humans is a major safety concern, because the enrollment of subjects of reproductive age in early-phase clinical trials of gene transfer continues to increase. In a study of adult men with hemophilia B, adeno-associated virus serotype 2 (AAV2) delivered to the liver via the hepatic artery resulted in unexpected transient vector dissemination to the semen. Here we report that intravenous AAV2 injection in rabbits proved a useful model to assess biologic parameters of vector dissemination to the semen. Detectable vector sequences in semen disappeared in a dose-dependent and time-dependent fashion. AAV infectious particles were present only as long as day 4 after injection and were undetectable thereafter. The kinetics of vector clearance was faster in the semen fractions enriched for motile sperm than in the total semen. In addition, increased frequency of semen sampling accelerated the clearance of vector sequences from semen. Long-term follow-up, spanning hundreds of spermatogenesis cycles, showed that there was no recurrence of detectable vector sequences in semen, thus reducing the probability of inadvertent transduction of early spermatogonia not committed to differentiation at the time of vector injection. We conclude that AAV2 presents minimal germline transmission risk for humans.     

沉默Toll样受体4基因表达的RNAi慢病毒载体的构建

背景:Toll样受体4(toll-like receptor4,TLR4)是介导内毒素/脂多糖应答的主要受体,在由内毒素诱导的炎性反应的信号通路中发挥着重要作用。目的:构建人TLR4基因的RNA干扰慢病毒载体,并观察其对人脐静脉内皮细胞TLR4在蛋白水平的沉默效应。方法:利用Invitrogen在线软件设计人TLR4基因shRNA序列,合成、退火形成双链寡核苷酸后克隆到线性载体pENTRTM/H1/TO的黏性末端,并进行DNA测序。得到的阳性重组子再与慢病毒载体进行重组反应,从而获得干扰TLR4基因真核表达的慢病毒载体。在脂质体的介导下将慢病毒包装辅助复合体和TLR4基因的真核表达慢病毒载体导入293FT细胞包装病毒,测定病毒滴度,感染人脐静脉内皮细胞,检验其干扰TLR4基因表达的有效性。结果与结论:实验成功构建TLR4基因真核表达慢病毒干扰载体并获得相应的慢病毒,病毒滴度为8.7×106U/mL。免疫印迹杂交结果表明,所获得的慢病毒感染人脐静脉内皮细胞TLR4基因在蛋白水平的表达显著降低。实验成功构建了人TLR4基因慢病毒RNA干扰表达载体,并验证了其在人脐静脉内皮细胞上的有效性。     

沉默Toll样受体4基因表达的RNAi慢病毒载体的构建

背景：Toll样受体4（toll-like receptor4,TLR4）是介导内毒素/脂多糖应答的主要受体,在由内毒素诱导的炎性反应的信号通路中发挥着重要作用。目的：构建人TLR4基因的RNA干扰慢病毒载体,并观察其对人脐静脉内皮细胞TLR4在蛋白水平的沉默效应。方法：利用Invitrogen在线软件设计人TLR4基因shRNA序列,合成、退火形成双链寡核苷酸后克隆到线性载体pENTRTM/H1/TO的黏性末端,并进行DNA测序。得到的阳性重组子再与慢病毒载体进行重组反应,从而获得干扰TLR4基因真核表达的慢病毒载体。在脂质体的介导下将慢病毒包装辅助复合体和TLR4基因的真核表达慢病毒载体导入293FT细胞包装病毒,测定病毒滴度,感染人脐静脉内皮细胞,检验其干扰TLR4基因表达的有效性。结果与结论：实验成功构建TLR4基因真核表达慢病毒干扰载体并获得相应的慢病毒,病毒滴度为8.7×106U/mL。免疫印迹杂交结果表明,所获得的慢病毒感染人脐静脉内皮细胞TLR4基因在蛋白水平的表达显著降低。实验成功构建了人TLR4基因慢病毒RNA干扰表达载体,并验证了其在人脐静脉内皮细胞上的有效性。