AAV serotype 2 vectors preferentially integrate into active genes in mice

Abstract

Recombinant adeno-associated virus serotype 2 (rAAV2) is a promising vector for gene therapy because it can achieve long-term stable transgene expression in animals and human subjects after direct administration of vectors into various target tissues. In the liver, although stable transgene expression primarily results from extrachromosomal vector genomes, a series of experiments has shown that vector genomes integrate into host chromosomes in hepatocytes at a low frequency. Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice and two human subjects have raised concerns about the potential for rAAV2-mediated insertional mutagenesis. Here we characterize rAAV2-targeted chromosomal integration sites isolated from selected or non-selected hepatocytes in vector-injected mouse livers. We document frequent chromosomal deletions of up to 2 kb at integration sites (14 of 14 integrations, 100%; most of the deletions were <0.3 kb) and preferred integration into genes (21 of 29 integrations, 72%). In addition, all of the targeted genes analyzed (20 of 20 targeted genes, 100%) were expressed in the liver. This is the first report to our knowledge on host chromosomal effects of rAAV2 integration in animals, and it provides insights into the nature of rAAV2 vector integration into chromosomes in quiescent somatic cells in animals and human subjects.

State-of-the-art 2003 on PKU gene therapy

“…Hence, comparing with other viral vectors, rAAV vectors appear to be safer and more efficient, and the existence of different AAV serotypes expands the usefulness of the rAAV vectors, and leads to more specific and strong transgene expression with capsid pseudotypes from alternative serotypes in different organs and tissues [38]. These high promises are somewhat flawed by a recent report indicating that rAAV (type 2) vectors, despite low integration efficiency, may integrate in murine hepatocytes, i.e., quiescent cells, preferentially into actively transcribed genes, and cause small genomic deletions, hence having the potential for dangerous side effects [39,40].…”

Absence of ocular malignant transformation after sub-retinal delivery of rAAV2/2 or integrating lentiviral vectors in p53-deficient mice

“…rAAV integration studies in the mouse liver reported that a high frequency (53-72%) of integration events occurred in transcribed regions. 9,10 A number of integration hot-spots in the mouse genome was identified, the most prominent being rRNA gene repeats. Those studies also identified integrations (3.5%) near or within cancer-related genes.…”

Gene therapy for optic nerve disease

“…Of particular concern is the theoretical risk of insertional oncogenesis, as the recombinant form of the virus mediates random incorporation of transgenes into the host chromosomes. 20 Also, the usefulness of AAV as a vector is limited by the relatively small amount of passenger DNA that can be incorporated. Although genes up to 6.0 kb have been packaged into AAV, these oversized viruses were not infectious, 21 and the usual packaging limit for AAV appears to be 5.1-5.3 kb.…”