Adenovirus-AAV hybrid vectors for gene therapy.

摘要

Curative treatment of genetic diseases demands a vehicle that can efficiently deliver therapeutic genes, not elicit an immune response, and engender safe, regulated, long-term therapeutic gene expression. Due to a large DNA-carrying capacity, wide tissue tropism, relatively favorable safety profile, and efficient transduction capability, adenovirus (Ad) has been widely pursued as a vector for gene therapy. However, humoral and cellular immune responses against Ad proteins, transient expression from episomally-maintained therapeutic genes, and difficulties in vector production have restrained the clinical application of Ad vectors for treating genetic diseases. Cellular immune responses against viral proteins have been significantly curtailed by using "gutted" vectors that carry a transgene of interest, but are devoid of all viral genes. There is no Ad producer cell line, and the conventional Cre-lox method for producing gutted Ad is inefficient, difficult to reproduce, and poorly, scalable. One goal of this study was to improve an alternate system for producing gutted Ad that utilizes Ad/AAV hybrid viruses. In these viruses, the complete terminal repeat (TR) of adeno-associated virus (AAV) was inserted into an E1-deleted Ad genome, between a transgene cassette and the rest of the Ad genome. A major advance was the development of an E1-complementing cell line that is able to express AAV Rep proteins that are normally cytotoxic. Upon infection of this cell line with Ad/AAV hybrid virus, Rep catalyzes endonucleolytic cleavage at the TR and separates the left transgene-containing portion of the viral genome from the rest of the genome. Since this released left portion carries the Ad packaging signal, this DNA is selectively encapsidated to yield gutted Ad vectors. The second goal of this study was to develop an Ad vector that can integrate a transgene into a specific locus in human chromosomal DNA. This is important from the standpoint of achieving long-term therapeutic gene expression without resorting to vector re-administration, or incurring a high risk of insertional mutagenesis. Using a cis-acting integration element from AAV, an Ad/AAV hybrid vector was developed that can integrate a transgene with high specificity and efficiency into the human AAVS1 locus in a Rep-dependent manner.