One of the most promising gene transfer vectors in human clinical trials is AAV2. The quality of the vector preparations is a key element in obtaining reliable and reproducible data in preclinical studies. However, established protocols either result in impure, low infectious virus (CsCl2 gradient centrifugation) or demand a high level of manual and technical skills (CsCl2 gradient centrifugation, iodixanol/heparin or HPLC purification). In this study, we present an easy-to-do single-step column purification (SSCP) of AAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation. Various vector preparations generated by our method reproducibly showed high titers, infectivity, and purity. In vivo, our single-step column-purified AAV2 vectors mediate significantly higher transduction efficiency compared with conventional protocols. Investigators still unsatisfied with previously published techniques or new to the field of AAV production may find in our method an interesting alternative.

Mutational heterogeneity represents a significant barrier to development of therapies for many dominantly inherited diseases. For example, >100 mutations in the rhodopsin gene (RHO) have been identified in patients with retinitis pigmentosa (RP). The development of therapies for dominant disorders that correct the primary genetic lesion and overcome mutational heterogeneity is challenging. Hence, therapeutics comprising two elements--gene suppression in conjunction with gene replacement--have been investigated. Suppression is targeted to a site independent of the mutation; therefore, both mutant and wild-type alleles are suppressed. In parallel with suppression, a codon-modified replacement gene refractory to suppression is provided. Both in vitro and in vivo validation of suppression and replacement for RHO-linked RP has been undertaken in the current study. RNA interference (RNAi) has been used to achieve ~90% in vivo suppression of RHO in photoreceptors, with use of adeno-associated virus (AAV) for delivery. Demonstration that codon-modifed RHO genes express functional wild-type protein has been explored transgenically, together with in vivo expression of AAV-delivered RHO-replacement genes in the presence of targeting RNAi molecules. Observation of potential therapeutic benefit from AAV-delivered suppression and replacement therapies has been obtained in Pro23His mice. Results provide the first in vivo indication that suppression and replacement can provide a therapeutic solution for dominantly inherited disorders such as RHO-linked RP and can be employed to circumvent mutational heterogeneity.

Vectors based on hybrids consisting of adeno-associated virus types 2 (ITRs and Rep) and 5 (Cap) were evaluated for muscle-directed gene transfer (called AAV2/5). Evaluation in immune-competent mice revealed greater transduction efficacy with AAV2/5 than with AAV2 and no cross-neutralization between AAV2/5 and AAV2. Interestingly, we saw no immunologic evidence of previous exposure to AAV5 capsids in a large population of healthy human subjects.Vectors based on the human parvovirus adeno-associated virus (AAV) are being evaluated in preclinical and clinical models of gene therapy. The majority of experiments have been performed with vectors based on serotype 2 (14). A number of principles have emerged from these studies. A wide spectrum of permissivity for AAV2 vector transduction exists, ranging from skeletal muscle, where transduction rates are high, to hematopoietic stem cells, which require large vector doses for detection of transduction. When achieved, transgene expression is remarkably stable and largely void of destructive T-cell responses to nonsecreted transgene products (11, 13).Six serotypes of AAV have been isolated and fully characterized with respect to nucleotide sequence (8,12,15,16). Serotype 5 was isolated from a human condylomatous skin lesion, while the other serotypes were identified as contaminants of human adenovirus preparations (3, 6, 12). Serotypes 1, 2, 3, and 4 represent distinct molecular isolates with significant homology, particularly in the inverted terminal repeat (ITR) and Rep regions; within this group, Rep proteins can bind to heterologous ITRs and support rescue and replication. Serotype 6 represents a hybrid virus consisting of serotypes 1 and 2. Serotype 5 is quite dissimilar to the others, with a distinct ITR structure and only 67% homology of the rep gene to that of AAV2 (7).Other investigators have generated vectors based on AAV5 by using an AAV5 rep-cap genome for production of vector and a vector with AAV5 ITRs. They have shown enhanced performance in terms of transduction efficiency in the murine lung, central nervous system, and most recently, in skeletal muscle (5,10,17).In this study, an AAV vector based on serotype 5 was generated by transfection of the vector (i.e., ITRs and transgene), a rep-cap-expressing construct, and a plasmid expressing E2a, E4-orf6, and VA from adenovirus (2). The standard AAV5 vector was produced with an AAV5 rep-cap construct together with a vector containing AAV5 ITRs; the recombinant virions were purified by cesium chloride sedimentation (called AAV5).A "pseudotyped" version of AAV2 was created by using rep from AAV2 and cap from AAV5 together with a plasmid containing a vector based on AAV2; this was also purified by cesium chloride sedimentation. The resulting vector is referred to as AAV2/5. In comparing the performance of vectors based on AAV2 versus AAV2/5, we utilized common production protocols but different methods of purification. The AAV2 vector was purified by heparin chromatography, which has been shown by several investig...

Transient transfection allows for fast production of recombinant proteins. However, the current bottlenecks in transient transfection are low titers and low specific productivity compared to stable cell lines. Here, we report an improved transient transfection protocol that yields titers exceeding 1 g/l in HEK293E cells. This was achieved by combining a new highly efficient polyethyleneimine (PEI)-based transfection protocol, optimized gene expression vectors, use of cell cycle regulators p18 and p21, acidic Fibroblast Growth Factor, exposure of cells to valproic acid and consequently the maintenance of cells at high cell densities (4 million cells/ml). This protocol was reproducibly scaled-up to a working volume of 2 l, thus delivering >1 g of purified protein just 2 weeks after transfection. This is the fastest approach to gram quantities of protein ever reported from cultivated mammalian cells and could initiate, upon further scale-up, a paradigm shift in industrial production of such proteins for any application in biotechnology.