Scalable manufacturing methodologies for improving adeno-associated virus-based pharmaprojects

Adeno-associated virus （AAV） is a promising viral vector and meets most requirements of being a safe biological agent. However, the commercialization of AAV has been hampered due to the limitation of large-scale production, and only a small number of clinical trials have been launched. In recent years, progresses in scalable manufacturing of AAV have dramatically improved AAV- based clinical researches, and have assisted the develop- ment of investigational drug products. An AAVl-based investigational product, Glybera, has been formally approved by European Commission for the treatment of lipoprotein lipase deficiency （LPLD）. Glybera was the first gene therapy product in the western world, and the pro- duction process involves a scalable baculovirus-insect cell system. However, many problems still need to be solved to improve the productivity and quality of AAV. The present review gives critical insights into current state-of-the-art scalable producing methodologies of AAV, such as bacu-lovirus-insect cell system, HSV complementation system, and Ad complementation system, along with a discussion on the problems, solutions, and developmental trends.Novel AAV-producing platforms in Saccharomyces cere- visiae and vaccinia virus complementation system will also be discussed.     

Extension of the Ye and Shreeve group contribution method for density estimation of ionic liquids in a wide range of temperatures and pressures

An extension of the Ye and Shreeve group contribution method [C. Ye, J.M. Shreeve, J. Phys. Chem. A 111 (2007) 1456–1461] for the estimation of densities of ionic liquids (ILs) is here proposed. The new version here presented allows the estimation of densities of ionic liquids in wide ranges of temperature and pressure using the previously proposed parameter table. Coefficients of new density correlation proposed were estimated using experimental densities of nine imidazolium-based ionic liquids. The new density correlation was tested against experimental densities available in literature for ionic liquids based on imidazolium, pyridinium, pyrrolidinium and phosphonium cations. Predicted densities are in good agreement with experimental literature data in a wide range of temperatures (273.15–393.15 K) and pressures (0.10–100 MPa). For imidazolium-based ILs, the mean percent deviation (MPD) is 0.45% and 1.49% for phosphonium-based ILs. A low MPD ranging from 0.41% to 1.57% was also observed for pyridinium and pyrrolidinium-based ILs.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated virus

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cosl4 and cos56, which represents the HSV-1 genome was used for generation of this rHSV.Rep andcap genes of AAV-2 were inserted intoXba I site ofUL2 gene on cod, generating cos6rcΔUL2. After being digested withPac I, cos6-rcΔUL2 and the other 4 cosmids were cotransfected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carryingrep andcap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence ofrep andcap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cos14 and cos56, which represents the HSV-1 genome was used for generation of this rHSV. Rep and cap genes of AAV-2 were inserted into Xba Ⅰ site of UL2 gene on cos6, generating cos6-rcΔUL2. After being digested with Pac Ⅰ , cos6-rcΔUL2 and the other 4 cosmids were cotrans-fected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carrying rep and cap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence of rep and cap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

Application of heparinized cellulose affinity membranes in recombinant adeno-associated virus serotype 2 binding and delivery

The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this research, we tested a novel application of the microporous, heparinized cellulose membranes for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2, which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We conjugated rAAV2 to an epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, human fibroblasts exhibited proliferation kinetics similar to those on the standard polystyrene tissue-culture surface. Using fluorescent proteins as the reporter, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. In addition, we applied the affinity membrane to adsorb unpurified rAAV2 from the crude lysate of packaging cells via the ligand–receptor binding, avoiding the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a new application of affinity cellulose membranes in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.     

利用昆虫病毒系统生产表达胰岛素的腺相关病毒

腺相关病毒(Adeno-associated virus-2,AAV-2)用于构建基因治疗病毒载体,在基因治疗领域受到了普遍重视和关注,而rAAV在应用过程中一个很重要的限制因素就是缺乏简单有效的大规模制备方法.运用昆虫细胞表达系统来提高AAV-2的产量,分别采用绿色荧光蛋白(GFP)和胰岛素(Insulin)基因替代AAV-2的结构基因后,与其他重组病毒共转染293t细胞,3 d后即可检测到高效表达的GFP和Insulin.结果表明,该方法能够比较理想地提高AAV的产量,为临床使用奠定了基础.