Site-specific integration of adeno-associated virus involves partial duplication of the target locus

Abstract

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

“…Transgene integration inito the AAVS1 locus reduces MBS85 expression at the mRNA level, but this reduction can be avoided by modifying the transgene [4]. Pluripotent stem cells with exogenous DNA inserted into the AAVS1 locus show normal phenotype and differentiation potential [5,6]. However, misgivings have arisen because the function of MBS85 is not yet completely understood [1].…”

Optogenetic Approaches to Target Specific Neural Circuits in Post-stroke Recovery

“…These viral vectors are nonreplicating and well tolerated in rodents. AAV is a safer viral vector, as it is less likely to induce insertional mutagenesis and has a low immune response [176]. Thus, AAV2 has been commonly and safely used in a number of human clinical trials, including treatments for Parkinson's and Alzheimer's diseases [177,178].…”

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1

“…Interestingly, most of the AAV serotypes have three contiguous repeats flanked by pseudo-repeats, a feature that is shared with AAVS1 (18). The site-specific integration process is contingent on the presence of the large regulatory proteins Rep78/Rep68, the AAVS1 site, and a cis-acting viral DNA sequence (1,17,19,20). Earlier studies reported that for the AAVS1 region a minimum sequence of 33 bp containing the RBS and trs sequences is essential for site-specific integration (16,17,21).…”

“…The requirement of site-specific nicking of the AAVS1 trs site suggests that some of the steps resemble the terminal resolution reaction during AAV DNA replication. This process is a variation of the mechanism used by DNA relaxases during DNA conjugation (20,24,25). The first step in site-specific integration is the assembly of a Rep78/ Rep68-DNA complex guided by the recognition of GCTC repeats through the N-terminal origin-binding domain (OBD).…”

“…Samples (2,5,10,20, and 40 M) were loaded in the cells, using in all cases size exclusion buffer. Samples were centrifuged in two-sector carbon-filled Epon centerpieces at 20°C.…”