Site-specific integration of retroviral DNA in human cells using fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc-finger protein E2C

Abstract

During the life cycle of retroviruses, establishment of a productive infection requires stable joining of a DNA copy of the viral RNA genome into host cell chromosomes. Retroviruses are thus promising vectors for the efficient and stable delivery of genes in therapeutic protocols. Integration of retroviral DNA is catalyzed by the viral enzyme integrase (IN), and one salient feature of retroviral DNA integration is its lack of specificity, as many chromosomal sites can serve as targets for integration. Despite the promise for success in the clinic, one major drawback of the retrovirus-based vector is that any unintended insertion events from the therapy can potentially lead to deleterious effects in patients, as demonstrated by the development of malignancies in both animal and human studies. One approach to directing integration into predetermined DNA sites is fusing IN to a sequencespecific DNA-binding protein, which results in a bias of integration near the recognition site of the fusion partner. Encouraging results have been generated in vitro and in vivo using fusion protein constructs of human immunodeficiency virus type 1 IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence. This review focuses on the method for preparing infectious virions containing the IN fusion proteins and on the quantitative PCR assays for determining integration site specificity. Efforts to engineer IN to recognize specific target DNA sequences within the genome may lead to development of effective retroviral vectors that can safely deliver gene-based therapeutics in a clinical setting.

Gene therapy for the neurological manifestations in lysosomal storage disorders

“…Replication-defective lentiviral vectors derived from the human immunodefi ciency virus or equine immunoassociated virus are becoming particularly prominent because of their ability to transduce nonproliferating cells (143)(144)(145). Although insertional mutagenesis resulting from vector integration presents a risk, safety concerns continue to be allayed through the use of modifi ed vectors that harbor self-inactivating mutations in their long terminal repeat to minimize the activation of cellular oncogenes or that impart preferences for integration at specifi c sites, thereby limiting the spectrum of genomic target sites (146)(147)(148)(149).…”

Mechanistic and pharmacological analyses of HIV-1 integration

“…Trans incorporated integrase proteins that harbored heterologous DNA binding elements catalyzed significant levels of HIV-1 integration [69, 70] and moreover imparted some target sequence specificity [70]. Su et al [71] detail in this issue their virological and downstream PCR methods for quantifying site-specific HIV-1 integration in the human genome.…”

Using Lentiviral Vectors as Delivery Vehicles for Gene Therapy

“…The insertion led to uncontrolled proliferation of mature T cells ( 60 ) . Alternatives to avoid random integration include fusing the integrase enzyme to a sequence-specifi c DNA-binding protein, which can direct integration into specifi c safe sites in the gene ( 62 ) . Another strategy that has emerged over the past few years consists of introducing different point mutations that affect the integration process, creating integration-defi cient LVs (IDLVs) that are identical to AAV in that they lead to sustained transgene expression without integration into the host genome ( 63,64 ) .…”