-
Human Gene Therapy Nov 2023Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA...
Integration of naturally occurring adeno-associated viruses (AAV; wild-type AAV [wtAAV]) and those used in gene therapy (recombinant AAV [rAAV]) into host genomic DNA has been documented for over two decades. Results from mouse and dog studies have raised concerns of insertional mutagenesis and clonal expansion following AAV exposure, particularly in the context of gene therapy. This study aimed to characterize the genomic location, abundance, and expansion of wtAAV and rAAV integrations in macaque and human genomes. Using an unbiased, next-generation sequencing-based approach, we identified the genome-wide integration loci in tissue samples (primarily liver) in 168 nonhuman primates (NHPs) and 85 humans naïve to rAAV exposure and 86 NHPs treated with rAAV in preclinical studies. Our results suggest that rAAV and wtAAV integrations exhibit similar, broad distribution patterns across species, with a higher frequency in genomic regions highly vulnerable to DNA damage or close to highly transcribed genes. rAAV exhibited a higher abundance of unique integration loci, whereas wtAAV integration loci were associated with greater clonal expansion. This expansive and detailed characterization of AAV integration in NHPs and humans provides key translational insights, with important implications for the safety of rAAV as a gene therapy vector.
Topics: Animals; Humans; Dependovirus; Genetic Therapy; Genetic Vectors; Liver; Macaca; Virus Integration
PubMed: 37930949
DOI: 10.1089/hum.2023.134 -
Current Opinion in Biotechnology Oct 1992Adeno-associated virus is a human parvovirus that integrates its DNA genome into host cell chromosomes with very high efficiency. This suggests that adeno-associated... (Review)
Review
Adeno-associated virus is a human parvovirus that integrates its DNA genome into host cell chromosomes with very high efficiency. This suggests that adeno-associated virus may be a useful vector for human gene therapy. Interest in adeno-associated virus vectors increased greatly in the last year following reports that adeno-associated virus genome integration may be site specific and occur at preferred sites in the human genome. Several genes relevant to the treatment of genetic or infectious diseases have been expressed in adeno-associated virus vectors in vitro.
Topics: Biotechnology; Dependovirus; Genetic Therapy; Genetic Vectors; Genome, Viral; Humans; Safety; Virus Integration
PubMed: 1369403
DOI: 10.1016/0958-1669(92)90082-t -
Microbiology Spectrum Aug 2022Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase...
Moloney murine leukemia virus (MLV) infects BALB/c mice and induces T-cell lymphoma in mice. Retroviral integration is mediated by the interaction of the MLV integrase (IN) with members of the bromodomain and extraterminal motif (BET) protein family (BRD2, BRD3, and BRD4). The introduction of the W390A mutation into MLV IN abolishes the BET interaction. Here, we compared the replication of W390A MLV to that of wild-type (WT) MLV in adult BALB/c mice to study the role of BET proteins in replication, integration, and tumorigenesis . Comparing WT and W390A MLV infections revealed similar viral loads in the blood, thymus, and spleen cells. Interestingly, W390A MLV integration was retargeted away from GC-enriched genomic regions. However, both WT MLV- and W390A MLV-infected mice developed T-cell lymphoma after similar latencies represented by an enlarged thymus and spleen and multiorgan tumor infiltration. Integration site sequencing from splenic tumor cells revealed clonal expansion in all WT MLV- and W390A MLV-infected mice. However, the integration profiles of W390A MLV and WT MLV differed significantly. Integrations were enriched in enhancers and promoters, but compared to the WT, W390A MLV integrated less frequently into enhancers and more frequently into oncogene bodies such as and . We conclude that host factors direct MLV integration site selection. Although BET proteins target WT MLV integration preferentially toward enhancers and promoters, insertional lymphomagenesis can occur independently from BET, likely due to the intrinsically strong enhancer/promoter of the MLV long terminal repeat (LTR). In this study, we have shown that the replication of murine leukemia virus happens independently of BET proteins, which are key host determinants involved in retroviral integration site selection. This finding opens a new research line in the discovery of alternative viral or host factors that may complement the dominant host factor. In addition, our results show that BET-independent murine leukemia virus uncouples insertional mutagenesis from gene enhancers, although lymphomagenesis still occurs despite the lack of an interaction with BET proteins. Our findings also have implications for the engineering of BET-independent MLV-based vectors for gene therapy, which may not be a safe alternative.
Topics: Animals; Genomics; Integrases; Leukemia Virus, Murine; Lymphoma, T-Cell; Mice; Nuclear Proteins; Transcription Factors; Virus Integration
PubMed: 35852337
DOI: 10.1128/spectrum.01478-22 -
Genomics, Proteomics & Bioinformatics Apr 2023Integration of oncogenic DNA viruses into the human genome is a key step in most virus-induced carcinogenesis. Here, we constructed a virus integration site (VIS) Atlas...
Integration of oncogenic DNA viruses into the human genome is a key step in most virus-induced carcinogenesis. Here, we constructed a virus integration site (VIS) Atlas database, an extensive collection of integration breakpoints for three most prevalent oncoviruses, human papillomavirus, hepatitis B virus, and Epstein-Barr virus based on the next-generation sequencing (NGS) data, literature, and experimental data. There are 63,179 breakpoints and 47,411 junctional sequences with full annotations deposited in the VIS Atlas database, comprising 47 virus genotypes and 17 disease types. The VIS Atlas database provides (1) a genome browser for NGS breakpoint quality check, visualization of VISs, and the local genomic context; (2) a novel platform to discover integration patterns; and (3) a statistics interface for a comprehensive investigation of genotype-specific integration features. Data collected in the VIS Atlas aid to provide insights into virus pathogenic mechanisms and the development of novel antitumor drugs. The VIS Atlas database is available at https://www.vis-atlas.tech/.
Topics: Humans; Epstein-Barr Virus Infections; Genome, Human; Herpesvirus 4, Human; Carcinogenesis; High-Throughput Nucleotide Sequencing; Virus Integration
PubMed: 36804047
DOI: 10.1016/j.gpb.2023.02.005 -
Cell Host & Microbe May 2016Advances in technology have made it possible to analyze integration sites in cells from HIV-infected patients. A significant fraction of infected cells in patients on... (Review)
Review
Advances in technology have made it possible to analyze integration sites in cells from HIV-infected patients. A significant fraction of infected cells in patients on long-term therapy are clonally expanded; in some cases the integrated viral DNA contributes to the clonal expansion of the infected cells. Although the large majority (>95%) of the HIV proviruses in treated patients are defective, expanded clones can carry replication-competent proviruses, and cells from these clones can release infectious virus. As discussed in this Perspective, it is likely that cells that produce virus are strongly selected against in vivo, and cells with replication competent proviruses expand and survive because only a small fraction of the cells produce virus. These findings have implications for strategies that are intended to eliminate the reservoir of infected cells that has made it almost impossible to cure HIV-infected patients.
Topics: Animals; HIV Infections; HIV-1; Humans; Proviruses; Virus Integration
PubMed: 27173927
DOI: 10.1016/j.chom.2016.04.010 -
Plant Science : An International... May 2016Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of...
Horizontal gene transfer (HGT) is known to be a major force in genome evolution. The acquisition of genes from viruses by eukaryotic genomes is a well-studied example of HGT, including rare cases of non-retroviral RNA virus integration. The present study describes the integration of cucumber mosaic virus RNA-1 into soybean genome. After an initial metatranscriptomic analysis of small RNAs derived from soybean, the de novo assembly resulted a 3029-nt contig homologous to RNA-1. The integration of this sequence in the soybean genome was confirmed by DNA deep sequencing. The locus where the integration occurred harbors the full RNA-1 sequence followed by the partial sequence of an endogenous mRNA and another sequence of RNA-1 as an inverted repeat and allowing the formation of a hairpin structure. This region recombined into a retrotransposon located inside an exon of a soybean gene. The nucleotide similarity of the integrated sequence compared to other Cucumber mosaic virus sequences indicates that the integration event occurred recently. We described a rare event of non-retroviral RNA virus integration in soybean that leads to the production of a double-stranded RNA in a similar fashion to virus resistance RNAi plants.
Topics: Base Sequence; Cucumovirus; Gene Expression Regulation, Plant; Gene Library; Genome, Plant; Phylogeny; Plant Viruses; RNA, Messenger; RNA, Plant; Glycine max; Virus Integration
PubMed: 26993236
DOI: 10.1016/j.plantsci.2016.01.011 -
Cancer Discovery Apr 2023The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous...
UNLABELLED
The human papillomavirus (HPV) genome is integrated into host DNA in most HPV-positive cancers, but the consequences for chromosomal integrity are unknown. Continuous long-read sequencing of oropharyngeal cancers and cancer cell lines identified a previously undescribed form of structural variation, "heterocateny," characterized by diverse, interrelated, and repetitive patterns of concatemerized virus and host DNA segments within a cancer. Unique breakpoints shared across structural variants facilitated stepwise reconstruction of their evolution from a common molecular ancestor. This analysis revealed that virus and virus-host concatemers are unstable and, upon insertion into and excision from chromosomes, facilitate capture, amplification, and recombination of host DNA and chromosomal rearrangements. Evidence of heterocateny was detected in extrachromosomal and intrachromosomal DNA. These findings indicate that heterocateny is driven by the dynamic, aberrant replication and recombination of an oncogenic DNA virus, thereby extending known consequences of HPV integration to include promotion of intratumoral heterogeneity and clonal evolution.
SIGNIFICANCE
Long-read sequencing of HPV-positive cancers revealed "heterocateny," a previously unreported form of genomic structural variation characterized by heterogeneous, interrelated, and repetitive genomic rearrangements within a tumor. Heterocateny is driven by unstable concatemerized HPV genomes, which facilitate capture, rearrangement, and amplification of host DNA, and promotes intratumoral heterogeneity and clonal evolution. See related commentary by McBride and White, p. 814. This article is highlighted in the In This Issue feature, p. 799.
Topics: Humans; Human Papillomavirus Viruses; Papillomavirus Infections; Gene Rearrangement; Oropharyngeal Neoplasms; Clonal Evolution; Virus Integration; Papillomaviridae
PubMed: 36715691
DOI: 10.1158/2159-8290.CD-22-0900 -
Current Opinion in Immunology Oct 2012Retroviral vectors are unique in their ability to integrate their genome into the host genome of transduced cells. Several members of the retrovirus family show distinct... (Review)
Review
Retroviral vectors are unique in their ability to integrate their genome into the host genome of transduced cells. Several members of the retrovirus family show distinct pattern for preferential integration into the host genome. Despite many years of investigation, precise mechanisms of target site selection and the fundamental interplay of viral integrase and host cell proteins are still unknown. Improved methods to detect retroviral integrations genome-wide as well as recent advances on the retroviral integrase structure and integrase interacting proteins may lead to further uncover the process of retroviral target site selection. A better knowledge of these mechanisms and interactions will allow further improving safety of retroviral vectors for gene therapy by providing an opportunity to retarget retroviral integration into non-harmful genomic positions.
Topics: Animals; DNA, Viral; Gene Products, pol; Genetic Loci; Genetic Therapy; Genetic Vectors; Humans; Proviruses; Retroviridae; Virus Integration
PubMed: 22981243
DOI: 10.1016/j.coi.2012.08.006 -
Cellular and Molecular Life Sciences :... Jul 2018Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to... (Review)
Review
Integration is central to HIV-1 replication and helps mold the reservoir of cells that persists in AIDS patients. HIV-1 interacts with specific cellular factors to target integration to interior regions of transcriptionally active genes within gene-dense regions of chromatin. The viral capsid interacts with several proteins that are additionally implicated in virus nuclear import, including cleavage and polyadenylation specificity factor 6, to suppress integration into heterochromatin. The viral integrase protein interacts with transcriptional co-activator lens epithelium-derived growth factor p75 to principally position integration within gene bodies. The integrase additionally senses target DNA distortion and nucleotide sequence to help fine-tune the specific phosphodiester bonds that are cleaved at integration sites. Research into virus-host interactions that underlie HIV-1 integration targeting has aided the development of a novel class of integrase inhibitors and may help to improve the safety of viral-based gene therapy vectors.
Topics: Gene Targeting; HIV Integrase; HIV-1; Host-Pathogen Interactions; Humans; Virus Integration; Virus Internalization; Virus Latency
PubMed: 29417178
DOI: 10.1007/s00018-018-2772-5 -
International Journal of Molecular... Jul 2022Human papillomavirus (HPV) integration within the host genome may contribute to carcinogenesis through various disruptive mechanisms. With next-generation sequencing...
Human papillomavirus (HPV) integration within the host genome may contribute to carcinogenesis through various disruptive mechanisms. With next-generation sequencing (NGS), identification of viral and host genomic breakpoints and chimeric sequences are now possible. However, a simple, streamlined bioinformatics workflow has been non-existent until recently. Here, we tested two new, automated workflows in CLC Microbial Genomics, i.e., Viral Hybrid Capture (VHC) Data Analysis and Viral Integration Site (VIS) Identification for software performance and efficiency. The workflows embedded with HPV and human reference genomes were used to analyze a publicly available NGS dataset derived from pre- and cancerous HPV+ cervical cytology of 21 Gabonese women. The VHC and VIS workflow median runtimes were 19 and 7 min per sample, respectively. The VIS dynamic graphical outputs included read mappings, virus-host genomic breakpoints, and virus-host integration circular plots. Key findings, including disrupted and nearby genes, were summarized in an auto-generated report. Overall, the VHC and VIS workflows proved to be a rapid and accurate means of localizing viral-host integration site(s) and identifying disrupted and neighboring human genes. Applying HPV VIS-mapping to pre- or invasive tumors will advance our understanding of viral oncogenesis and facilitate the discovery of prognostic biomarkers and therapeutic targets.
Topics: Alphapapillomavirus; DNA, Viral; Female; Genomics; Humans; Papillomaviridae; Papillomavirus Infections; Uterine Cervical Neoplasms; Virus Integration; Workflow
PubMed: 35897706
DOI: 10.3390/ijms23158132