Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

Zolotukhin, Sergei; Byrne, Barry; Mason, Emily Grace; Zolotukhin, Irene; Potter, Mark; Chesnut, Kye; Summerford, Candace; Samulski, R. Jude; Muzyczka, Nicholas

doi:10.1038/sj.gt.3300938

Cited by 1,174 publications

(945 citation statements)

References 37 publications

(33 reference statements)

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“…Nevertheless, aggregation of adeno-associated virus (AAV) vectors during CsCl ultracentrifugation was reported previously by others [25]. Use of iodixanol discontinuous gradient centrifugation was reported to solve the aggregation problem of AAV observed during purification by CsCl method and significantly improve infectious titer and yield of AAV vectors [18,22].…”

Section: Discussionmentioning

confidence: 95%

“…We used iodixanol as the ultracentrifugation gradient medium because this agent has been successfully used to isolate cells [12,13], organelles [14,15], macromolecules [16,17], and viruses [18][19][20][21][22]. Using iodixanol discontinuous density ultracentrifugation followed by sizeexclusion chromatography, we successfully isolated and purified both RGD-modified and non-RGD-modified adenovectors with a degree of purity and infectivity comparable to that of adenovectors purified by traditional 2× CsCl ultracentrifugation.…”

mentioning

confidence: 99%

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

Peng¹,

Wu²,

Davis³

et al. 2006

Analytical Biochemistry

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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...

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Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 99%

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

Peng¹,

Wu²,

Davis³

et al. 2006

Analytical Biochemistry

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“…AAV1.SERCA2a was produced using the 2‐plasmids protocol described by Zolotukhin et al10 with the following modifications: HEK293T cells (ATCC) were grown in triple flasks for 24 hours (DMEM, 10% fetal bovine serum). Calcium phosphate precipitate was added for cotransfection of plasmid DNA.…”

Section: Methodsmentioning

confidence: 99%

Primary Effect of SERCA2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction

Motloch

Cacheux

Ishikawa

et al. 2018

JAHA

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Background SERCA2a gene transfer (GT) improves mechano‐electrical function in animal models of nonischemic heart failure Whether SERCA2a GT reverses pre‐established remodeling at an advanced stage of ischemic heart failure is unclear. We sought to uncover the electrophysiological effects of adeno‐associated virus serotype 1.SERCA2a GT following myocardial infarction (MI).Methods and ResultsPigs developed mechanical dysfunction 1 month after anterior MI, at which point they received intracoronary adeno‐associated virus serotype 1.SERCA2a (MI+SERCA2a) or saline (MI) and were maintained for 2 months. Age‐matched naive pigs served as controls (Control). In vivo ECG‐and‐hemodynamic properties were assessed before and after dobutamine stress. The electrophysiological substrate was measured using optical action potential (AP) mapping in controls, MI, and MI+SERCA2a preparations. In vivo ECG measurements revealed comparable QT durations between groups. In contrast, prolonged QRS duration and increased frequency of R′ waves were present in MI but not MI+SERCA2a pigs relative to controls. SERCA2a GT reduced in in vivo arrhythmias in response to dobutamine. Ex vivo preparations from MI but not MI+SERCA2a or control pigs were prone to pacing‐induced ventricular tachycardia and fibrillation. Underlying these arrhythmias was pronounced conduction velocity slowing in MI versus MI+SERCA2a at elevated rates leading to ventricular tachycardia and fibrillation. Reduced susceptibility to ventricular tachycardia and fibrillation in MI+SERCA2a pigs was not related to hemodynamic function, contractile reserve, fibrosis, or the expression of Cx43 and Nav1.5. Rather, SERCA2a GT decreased phosphoactive CAMKII‐delta levels by >50%, leading to improved excitability at fast rates.Conclusions SERCA2a GT increases conduction velocity reserve, likely by preventing CAMKII overactivation. Our findings suggest a primary effect of SERCA2a GT on myocardial excitability, independent of altered mechanical function.

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“…rAAV vectors were produced by a two-plasmid transfection 25 and purification method. 26 Briefly, 293 cells at 70-80% confluence were transfected with the rAAV plasmid plus the helper plasmid ( pDG ) by standard CaCl 2 technique for a period of 72 hours. After harvesting the cells, three freeze / thaw cycles were applied and the sample was treated with DNase (Benzonase ).…”

Section: Raav Construction and Productionmentioning

confidence: 99%

Adeno-associated virus–mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo

et al. 2002

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A variety of approaches has demonstrated that interfering with tumor -induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno -associated viral ( rAAV ) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV -HuEndo ( 1Â10 9 i.u. ) led to a sustained serum endostatin level of $35 -40 ng / mL. This endostatin level was sufficient to inhibit tumor cell -induced angiogenesis and to suppress both the initiation and subsequent growth of a human colorectal cancer model.

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Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield

Cited by 1,174 publications

References 37 publications

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

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

Primary Effect of SERCA2a Gene Transfer on Conduction Reserve in Chronic Myocardial Infarction

Adeno-associated virus–mediated gene transfer of endostatin inhibits angiogenesis and tumor growth in vivo

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