The study investigates the integration site preferences of three retroviruses—ASLV, HIV, and MLV—in human cells. Using 3,127 integration site sequences from various cell types, the researchers found that each virus has distinct integration site preferences. HIV vectors strongly favored integration near active genes, while MLV vectors showed a strong bias towards integration near transcription start sites. ASLV vectors, in contrast, showed only a weak preference for active genes and no preference for transcription start regions. The analysis also revealed that integration sites were influenced by gene activity and tissue-specific transcription, with highly expressed genes and specific transcription patterns in different cell types leading to tissue-specific integration biases. Additionally, the study identified substructures within chromosomal regions, such as CpG islands, that affect integration frequency. The findings suggest that virus-specific binding of integration complexes to chromatin features likely guides site selection, and that these preferences could be exploited for safer gene therapy.The study investigates the integration site preferences of three retroviruses—ASLV, HIV, and MLV—in human cells. Using 3,127 integration site sequences from various cell types, the researchers found that each virus has distinct integration site preferences. HIV vectors strongly favored integration near active genes, while MLV vectors showed a strong bias towards integration near transcription start sites. ASLV vectors, in contrast, showed only a weak preference for active genes and no preference for transcription start regions. The analysis also revealed that integration sites were influenced by gene activity and tissue-specific transcription, with highly expressed genes and specific transcription patterns in different cell types leading to tissue-specific integration biases. Additionally, the study identified substructures within chromosomal regions, such as CpG islands, that affect integration frequency. The findings suggest that virus-specific binding of integration complexes to chromatin features likely guides site selection, and that these preferences could be exploited for safer gene therapy.