Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

Abstract

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA inert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV.

Description

TECHNICAL FIELD OF THE INVENTION [0001] This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA insert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV. BACKGROUND OF THE INVENTION [0002] A recombinant virus carrying a foreign DNA insert may be used to deliver genes to cells, where the gene may be expressed, if desired, to permit production of recombinant proteins in vitro or in vivo, vaccination of human and non-human mammals, or treatment or amelioration of diseases or genetic defects in humans or in non-human mammals. One may treat or ameliorate diseases or genetic defects by providing some effective level of normal gene products,...

AI Technical Summary

Benefits of technology

[0014] The instant invention provides an alternative production method that results in high yields of rAAV vector and is amenable to large-scale industrial applications. The invention provides a novel adenovirus vector comprising rep and cap genes, thus providing AAV rep and cap and adenovirus helper functions in one component. In the adenovirus vector of the instant invention, the native AAV p5 promoter upstream of rep is removed and replaced with a minimal promoter or with no promoter. This novel vector, when infected into cells containing a nucleic acid sequence comprising a transgene flanked by AAV ITRs, results in the production of high levels of rAAV. The nucleic acid sequence comprising the transgene flanked by AAV ITRs may be established in the host cell by stable integration into the host cell chromosome, secondary infection with an adenovirus or other viral vector carrying the transgene flanked by ITRs (see, e.g, U.S. Pat. No. 5,856,152), infection with an rAAV comprising the transgene, or any other method known in the art, such as transfection, lipofection or microinjection, of plasmid DNA comprising the transgene flanked by ITRs.

[0015] In one embodiment of the invention, rep, operably linked to a minimal promoter or to no promoter, is inserted into either the E1 or E3 regions of an adenovirus. The adenovirus is deleted in E1 or E3 alone, or a combination of both. In another embodiment, the adenovirus vector is further deleted in E4. In this embodiment, rep sequences may be inserted in E4, while upstream of these rep sequences there may be no promoter or a minimal promoter. In a preferred embodiment, cap is inserted along with the rep gene into the adenoviral vector. In another aspect of the invention, the adenoviral vector comprising the minimal promoter or promoterless rep is used in a method to produce rAAV. The advantage of this method is that it is easily scaled for industrial production of rAAV.

Problems solved by technology

However, the disadvantage of the methods taught by U.S. Pat. No. 5,753,500 and Li et al. is that co-transfection of two plasmids along with infection by a helper virus is inefficient, may exhibit poor reproducibility, may result in generation of pseudo-wildtype replication-competent AAV (rcAAV), and cannot be easily scaled up for industrial production of rAAV. rcAAV, comprising rep and cap flanked by ITRs, is produced when the rep and cap genes recombine with the ITRs flanking the transgene which results in deletion of the transgene.

The disadvantages of the first method are shared with this method.

The disadvantage of this method is that it requires making a cell line that expresses sufficient levels of rep and cap, and requires multiple components—including the cell line, the rAAV genome, and an adenovirus—to produce rAAV, which do not lend themselves to easy and convenient downstream manufacturing processes.

In addition, some of these packaging cell lines do not produce high levels of rAAV.

U.S. Pat. No. 5,354,678 does not disclose the levels of rAAV, if any, produced by this method.

As described above, current rAAV production methods are not amenable for production of sufficient rAAV for pharmaceutical applications in a convenient manner.

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