A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Abstract

Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and sizeexclusion column chromatography. The purity and infectious activity of adenovectors isolated by either method were comparable. The new method yielded three to four times more adenovectors with RGD-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors, but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3 hours. Therefore, this method is rapid and efficient for production recombination adenovectors, especially those with RGD-modified fibers.Adenoviral vectors (adenovectors) have high in vivo transduction efficiencies and are easy to construct and produce. They are thus frequently used for in vitro and in vivo gene delivery and for gene therapy in clinical trials [1] . Substantial efforts have been made to characterize adenovirus-host cell interactions and to improve gene delivery by adenovectors, including minimizing vector gene expression, reducing vector related toxicity and immunogenicity, increasing their capacity to accommodate large foreign genes, and increasing their transduction efficiency.It is now known that adenovirus interacts with host cells by binding to the coxsackievirus and adenovirus receptor (CAR) [2], a cellular receptor for Ad5 and most other adenovirus serotypes. After attachment, the virion is then internalized by endocytosis through the interaction of its penton base with α v β 3 and α v β 5 integrins on the host cell surface [3]. However, there is growing evidence that the expression of CAR is frequently suppressed in various types of cells, including tumor cells and in primary tumors [4,5], resulting in resistance to adenovirus infection [6][7][8] Unfortunately, technical difficulties may be encountered in production of certain modified adenovectors. We have encountered difficulties when producing certain adenovectors by this method, especially those with RGD-modified fibers or "sticky" virus. For example, we have experienced difficulty in purifying various RGD-modified adenoviral vectors by conventional two-cycle (2x) of CsCl ultracentrifugation because the RGD-modified adenoviral vectors tend to aggregate, especially in the second round of ultracentrifugation, forming floccules without a sharp band. This results in a low yield or even failure of purification. Thus, an alternative method for efficient production of these modified adenov...

Intratumoral Immunization by p19Arf and Interferon-β Gene Transfer in a Heterotopic Mouse Model of Lung Carcinoma

“…Adenovector production was performed by transfection of linearized plasmids into HEK293A cells followed by amplification cycles and purification using iodixanol gradient [44]. Purified adenovectors were stored in PBS 7% glycerol at −80°C.…”

Downstream processing and chromatography based analytical methods for production of vaccines, gene therapy vectors, and bacteriophages

“…Ultracentrifugation of viruses can be carried out in different density gradients like caesium chloride [21], sucrose [22] or iodixanol [23,24]. In general the method enables efficient purification and concentration of virus particles in one step, but requires expensive equipment, process times are long (with subsequent step of density gradient material removal by dialysis or size exclusion chromatography) and sometimes several cycles are needed [21,25] Although new generation chromatography media are very efficient, chromatography based purification of viruses intended for human use still represents only a part of the virus downstream processing (DSP) scheme which usually consists of several purification steps and needs to deliver a virus preparation of high purity and efficacy (e.g.…”

“…For example, high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus[24] leading to loss of virus infectivity and unsatisfactory infectious virus yields[25]. Another problem is extensive virus aggregation during gradient ultracentrifugation which was observed by Peng et al[23] when trying to purify recombinant adeno vectors in CsCl density gradient.Selective precipitation uses different chemical agents like ammonium sulfate and polyethylene glycol[25] to precipitate either the virus or the impurities. However, this method is not very suitable for preparative scale virus purification/production; it is especially challenging to carry out this method in a batch mode under cGMP conditions.…”

Adenovirus-based virotherapy enabled by cellular YB-1 expression in vitro and in vivo

“…19 Ad-Delo3-RGD, dl312 and Ad-WT were produced in HEK 293 cells (Clontech, Mountain View, CA) and purified by two consecutive iodixanol gradient centrifugations and additionally by size-exclusion chromatography (Disposable PD-10 Desalting Columns, GE Healthcare, Freiburg, Germany). 20 To exclude contamination of Ad-Delo3-RGD by Ad-WT and to verify the viral construct PCR against e1a13s, e1a12s, e1b19k and RGD was performed (E1A13Sfw: 5 0 -AATGGCCGCCAGTCTTTT; E1A13Srev: 5 0 -GCCA TGCAAGTTAAAC ATTATC; E1A12Sfw: 5 0 -GCATG TTTGTCTACAGTAAGTG; E1A12Srev: 5 0 -GCCATGC AAGTTAAACATTATC; E1B19fw: 5 0 -GCGTAACTTG CTGGAACAGAG; E1B19rev: 5 0 -TCAGTTCTGGA; RGDfw: 5 0 -CTGCCGCGGAGACTGTTTC; RGDrev: 5 0 -CTGCAATTGAAAAATAAACACGTTGAAAC. Viral titers were determined by AdEasy Viral Titer Kit (Stratagene, La Jolla, CA).…”