Functional and biodegradable dendritic macromolecules with controlled architectures as nontoxic and efficient nanoscale gene vectors

Gene therapy has provided great potential to revolutionize the treatment of many diseases. This therapy is strongly relied on whether a delivery vector efficiently and safely directs the therapeutic genes into the target tissue/cells. Nonviral gene delivery vectors have been emerging as a realistic alternative to the use of viral analogs with the potential of a clinically relevant output. Dendritic polymers were employed as nonviral vectors due to their branched and layered architectures, globular shape and multivalent groups on their surface, showing promise in gene delivery. In the present review, we try to bring out the recent trend of studies on functional and biodegradable dendritic polymers as nontoxic and efficient gene delivery vectors. By regulating dendritic polymer design and preparation, together with recent progress in the design of biodegradable polymers, it is possible to precisely manipulate their architectures, molecular weight and chemical composition, resulting in predictable tuning of their biocompatibility as well as gene transfection activities. The multifunctional and biodegradable dendritic polymers possessing the desirable characteristics are expected to overcome extra- and intracellular obstacles, and as efficient and nontoxic gene delivery vectors to move into the clinical arena.     

Reprint of: The diversity of oomycetes on crayfish: Morphological vs. molecular identification of cultures obtained while isolating the crayfish plague pathogen

Numerous oomycetes colonise the crayfish cuticle, the best known being the crayfish plague pathogen Aphanomyces astaci. Although other oomycetes associated with crayfish complicate the isolation and molecular detection of A. astaci, their diversity is little known. To improve this knowledge, we analysed 95 oomycete isolates obtained during attempts to isolate A. astaci from crayfish presumably infected by this pathogen. We characterized the isolates morphologically and by sequencing of the nuclear internal transcribed spacer (ITS) region. We identified 13 taxa by molecular analysis. Ten of them were assigned to five genera; the remaining three were affiliated with the order Saprolegniales but could not be reliably assigned to any genus. Morphological identification to species level was only possible for 15 % of isolates; all corresponded to Saprolegnia ferax, which was confirmed by ITS sequencing. The most frequently isolated species were S. ferax and Saprolegnia australis. Only seven isolates of A. astaci were obtained, all from one disease outbreak. We show that oomycete cultures obtained as by-products of parasite isolation are valuable for oomycete diversity studies, but morphological identification may uncover only a fraction of their diversity. Further, we show that crayfish may be frequently associated with potentially serious parasites of other organisms.     

本氏烟Ⅰ型启动子的克隆及其转录起始位点分析

启动子是转录水平上一个重要的调控元件,其决定着基因的表达模式和表达强度。Ⅰ型启动子具有高转录活性和种属间特异性等特点。如将其应用于植物RNA病毒载体表达系统,有利于提高表达系统的表达效率和生物安全性。本氏烟(Nicotiana benthaminana)是一种被广泛地应用于植物生物反应器和植物病理学的模式生物,但是现有核酸数据库中尚没有其Ⅰ型启动子的相关信息。因此,克隆本氏烟Ⅰ型启动子并分析其转录起始位点就具有重要的应用价值。通过半巢式PCR获得了514 bp的本氏烟Ⅰ型启动子序列(KC352713);生物信息学分析初步预测其转录起始位点位于其核心序列TATA(G)TA(N)GGGGG中的第3位A处;通过植物RNA病毒表达载体和5'RACE技术在体内验证本氏烟Ⅰ型启动子转录起始位点与生物信息学预测结果一致。研究结果为深入研究Ⅰ型启动子和构建Ⅰ型启动子介导转录的植物RNA病毒载体表达系统奠定了基础。     

非复制型痘苗病毒的生物学性状研究进展

痘病毒被广泛用于针对多种感染性疾病和癌症的疫苗研究,而非复制型痘苗病毒的发展又进一步提高了其安全性。来源于中国天花疫苗株的复制缺陷型痘苗病毒天坛株(NTV)正是一株非复制型痘苗病毒。为了促进未来NTV作为疫苗候选株在临床中的应用,我们对NTV与另外2株非复制型痘苗病毒,即改良痘苗病毒安卡拉株(MVA)和NYVAC的生物学性状做简要综述,主要讨论了它们的基因组结构、体外生长特性、病毒复制和形态发生、组织中的病毒分布和病毒-宿主细胞间的相互作用。     

高光效基因植物表达载体的构建

通过一系列中间载体的构建 ,将来自C4作物 (甘蔗 )光合作用中的关键酶基因 (高光效基因 )RbcS和PEPCase基因插入到植物表达质粒中 ,获得了具有卡那霉素抗性的RbcS植物表达载体 pC2 0SNR和具有PPT除草剂抗性的PEPCase基因的正义 (pC30SNP)和反义 (pC30SNPr)植物表达载体。再将这 3个载体分别转入农杆菌LBA4 4 0 4和A2 81或EHA10 5中 ,获得了适于C3 植物转化的农杆菌重组工程菌株。为进一步利用C4植物高光效基因转化C3 植物 ,以期提高光合效率提供基础。     

Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor

Recombinant retroviruses are now an established tool for gene delivery. Presently they are mainly produced using adherent cells. However, due to the restrictive nature of adherent cell culture, this mode of production is hampered by low cell-specific productivity and small production units. The large-scale production of retroviral vectors could benefit from the adaptation of retrovirus packaging cell lines to suspension culture. Here, we describe the ability of a 293 packaging cell line to produce retroviral vectors in suspension culture at high titer. Adherent 293GPG cells, producing a Moloney Murine Leukemia Virus (MoMLV) retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein, were adapted to suspension culture in calcium-free DMEM. At a cell density similar to adherent cell culture, the suspension culture produced retroviral vector consistently in the range of 1 x 10(7) infectious viral particles/mL (IVP/mL), with a specific productivity threefold higher than adherent culture. Furthermore, at the same medium replacement frequency, the suspension producer cells could be cultured at higher density than their adherent counterparts, which resulted in virus titer of 3-4 x 10(7) IVP/mL at 11.0 x 10(6) cells/mL. This corresponds to a 10-fold increase in viral concentration compared to adherent cells. The capacity to up scale the retroviral vector production was also demonstrated by performing a 2 VVD perfusion culture for 9 days in a 3L Chemap bioreactor. The combination of suspension and perfusion led to a 20-fold increase in maximum virus productivity compared to the adherent culture.     

Capacity of human beta-defensin expression in gene-transduced and cytokine-induced cells

The purpose of this study was to determine the capacity of cells transduced with human beta-defensins (HBDs) to express antimicrobial peptides, since sufficient expression level is required for effective antimicrobial activity. Retroviral vector pBabeNeo and lentiviral vector SIN18cPPTRhMLV (SIN18) carrying HBDs were utilized to transduce non-HBD-expressing cells such as fibroblasts or HBD-producing oral epithelial cells. We found that HBD-3 gene transfer to fibroblasts was possible not via retrovirus but by direct vector transfection. SIN18 had high transduction efficiencies (80.9-99.9%) and transduced cells expressed higher amounts of HBD-2 than those by pBabeNeo. Primary human gingival epithelial cells (HGECs) expressed greater amounts of HBD-2 than primary fibroblasts after lentiviral transduction. Additionally, HBD-2 secretion from transduced HGECs cells was further increased when stimulated with IL-1 or TNFalpha. Our data indicate that while HBD-2 expression is limited in primary fibroblasts, its expression in HGECs may be maximized by gene transduction plus cytokine induction.