Methods for large-scale production of recombinant rAAV vectors

摘要

Disclosed is a method of serial amplification of recombinant adeno-associated virus (rAAV). The method includes the steps of: (f) infecting a competent host cell line with a rAAV and an ICP27 defective herpes virus comprising an AAV rep gene and an AAV cap gene, each operably linked to a homologous or heterologous promoter; (g) culturing the host cell under conditions effective to produce rAAV; (h) lysing said cell to obtain a lysate; (i) amplifying rAAV from said lysate through serial passaging; and (j) collecting the rAAV so produced. Wherein amplification is at least 200-fold over amount of input rAAV vector at each passage. Suitable promoters include p5, p19, p40, hCMV, HSV-1 tk, CMV40, HIV LTR, HCMV IE and HSV-1 IE 110 promoter. The defective herpes virus may be d27.1rc or a herpes simplex virus. The total amplification may be greater than 106 after 3 cycles of passaging. Said method is useful for multiplying rAAV including a therapeutic transgene. (62) Divided Out of 511171