Integration site selection by retroviral vectors: molecular mechanism and clinical consequences

Abstract

Retroviral DNA integration into the host cell genome is an essential feature of the retroviral life cycle. The ability to integrate their DNA into the DNA of infected cells also makes retroviruses attractive vectors for delivery of therapeutic genes into the genome of cells carrying adverse mutations in their cellular DNA. Sequencing of the entire human genome has enabled identification of integration site preferences of both replication-competent retroviruses and retroviral vectors. These results, together with the unfortunate outcome of a gene therapy trial, in which integration of a retroviral vector in the vicinity of a protooncogene was associated with the development of leukemia, have stimulated efforts to elucidate the molecular mechanism underlying integration site selection by retroviral vectors, as well as the development of methods to direct integration to specific DNA sequences and chromosomal regions. This review outlines our current knowledge of the mechanism of integration site selection by retroviruses in vitro, in cultured cells, and in vivo; the outcome of several of the more recent gene therapy trials, which employed these vectors; and the efforts of several laboratories to develop vectors that integrate at predetermined sites in the human genome.

FIG. 1.

Early steps of the retroviral life cycle. After entry, the RNA genome of the retrovirus or retroviral vector undergoes reverse transcription in the cytoplasm of infected cells. Viral DNA, integrase (IN), and certain viral and cellular proteins constitute the preintegration complex, which is imported into the nucleus (Flint et al., 2004). IN then catalyzes the integration of viral DNA into the host cell DNA. As indicated by the asterisk (*), this process applies only to some retroviruses, such as human immunodeficiency virus (HIV)-1 and avian sarcoma virus (ASV; Flint et al., 2004). Murine leukemia virus (MLV) and MLV-based vectors cannot pass readily through the nuclear pore and access the host cell DNA unless the nuclear envelope is dissolved during mitosis (Roe et al., 1993). IN(n), integrase multimer.

FIG. 2.

Retroviral DNA integration. For details see text. IN(n), integrase multimer.

FIG. 3.

LEDGF/p75 role in integration site selection. LEDGF/p75 binds to specific sites, thereby mediating contact between IN and host cell chromatin. This association tethers the HIV-1 preintegration complex near or in genes.

FIG. 4.

A possible role for chromatin structure in HIV-1 DNA integration. HIV-1 DNA integration occurs preferentially in the vicinity of certain epigenetic modifications, such as H3 K4 methylation (H3K4Me) and H3 and H4 acetylation (H3-Ac and H4-Ac, respectively). Integration seems to be disfavored in the vicinity of H3K27 trimethylation (H3K27Me3) and CpG islands in the DNA (see text and Wang et al., 2007).

FIG. 5.

A model for targeting integration by fusion proteins of integrase and a heterologous DNA-binding domain. A DNA-binding domain (DBD) of a cellular DNA-binding protein (DBP) can be fused to either the N terminus or C terminus of IN. The resulting fusion protein can be incorporated into the virion and the preintegration complex, and targets integration toward the DNA sequences recognized by the DBD, the target site (TS).