Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Nanotopography-based engineering of retroviral DNA integration patterns.

Controlling the interactions between cells and viruses is critical for treating infected patients, preventing viral infections, and improving virus-based therapeutics. Chemical methods using small molecules and biological methods using proteins and nucleic acids are employed for achieving this control, albeit with limitations. We found, for the first time, that retroviral DNA integration patterns in the human genome, the result of complicated interactions between cells and viruses, can be engineered by adapting cells to the defined nanotopography of silica bead monolayers. Compared with cells on a flat glass surface, cells on beads with the highest curvature harbored retroviral DNAs at genomic sites near transcriptional start sites and CpG islands during infections at more than 50% higher frequencies. Furthermore, cells on the same type of bead layers contained retroviral DNAs in the genomic regions near cis-regulatory elements at frequencies that were 2.6-fold higher than that of cells on flat glass surfaces. Systems-level genetic network analysis showed that for cells on nanobeads with the highest curvature, the genes that would be affected by cis-regulatory elements near the retroviral integration sites perform biological functions related to chromatin structure and antiviral activities. Our unexpected observations suggest that novel engineering approaches based on materials with specific nanotopography can improve control over viral events.

Shifting Retroviral Vector Integrations Away from Transcriptional Start Sites via DNA-Binding Protein Domain Insertion into Integrase.

The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3' end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.