Vector-omics?
Authors: Matthew D. Weitzman
Journal: Molecular Therapy (2004)

Post-genomic tools for molecular biology provide a new window through which to view virus-host interactions. High-density nucleic acid arrays allow us to measure the level of expression of thousands of genes simultaneously. These arrays are thus useful for examining changes in the pattern of gene expression that accompany the characteristic responses of cells or tissues to a particular treatment. Indeed, microarray analysis has become an integral part of the drug development pipeline, contributing to both drug discovery and optimization [1Gerhold D.L. Jensen R.V. Gullans S.R. Better therapeutics through microarrays.Nat. Genet. 2002; 32: S547-S551Crossref PubMed Scopus (115) Google Scholar]. Perhaps it is now time to consider incorporating microarray analysis into the development pipeline for gene delivery vectors. The design and testing of vectors for gene delivery could benefit significantly from microarray analysis of the effects of both wild-type and mutant viruses on cells, as well as of the effects of individual transgenes. Such analyses could facilitate the elimination of poor vectors and the optimization of promising ones, while at the same time providing insight into the mechanism behind the adverse events that have been observed in the clinic and in animal models. During infection with wild-type viruses, there is a complicated interplay between the virus and the host cell. The virus attempts to establish optimal conditions for its replication and the production of progeny, while the host cell attempts to fight the intruder. Viruses have developed ingenious mechanisms by which to inactivate some of these host antiviral defense systems. Some viruses encode multiple regulators of transcription that establish a temporal cascade of viral gene expression. In addition, certain viral transactivators regulate cellular genes to set up the appropriate cellular milieu, while viral-encoded repressors dampen transcriptional elements of some of the host defense responses. Therefore, one might imagine that viral infection could induce alterations in the level of expression of a large number of cellular genes. Cellular genes induced by viral infection might reflect the cellular innate immune response or might be activated for the benefit of the virus itself. Recently, a number of studies have used gene expression profiling to analyze global effects of viral infection on cellular genes. Viruses tested include adenovirus (Ad) [2Zhao H. et al.Strategic attack on host cell gene expression during aden ovirus infection.J. Virol. 2003; 77: 11006-11015Crossref PubMed Scopus (64) Google Scholar, 3Lohr K. et al.Mutual interference of adenovirus infection and myc expression.J. Virol. 2003; 77: 7936-7944Crossref PubMed Scopus (17) Google Scholar], human cytomegalovirus (HCMV) [4Zhu H. et al.Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays.Proc. Natl. Acad. Sci. USA. 1998; 95: 144705Google Scholar, 5Goodrum F.D. et al.Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: a model for latency.Proc. Natl. Acad. Sci. USA. 2002; 99: 16255-16260Crossref PubMed Scopus (201) Google Scholar], herpes simplex virus type 1 (HSV-1) [6Eidson K.M. et al.Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection.J. Virol. 2002; 76: 2180-2191Crossref PubMed Scopus (142) Google Scholar, 7Mossman K.L. Smiley J.R. Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.J. Virol. 2002; 76: 1995-1998Crossref PubMed Scopus (95) Google Scholar, 8Taddeo B. Esclatine A. Roizman B. The patterns of accumulation of cellular RNAs in cells infected with a wild-type and a mutant herpes simplex virus 1 lacking the virion host shutoff gene.Proc. Natl. Acad. Sci. USA. 2002; 99: 17031-17036Crossref PubMed Scopus (82) Google Scholar] and human immunodeficiency virus (HIV) [9Corbeil J. et al.Temporal gene regulation during HIV-1 infection of human CD4+ T cells.Genome Res. 2001; 11: 1198-1204Crossref PubMed Scopus (96) Google Scholar, 10Mitchell R. et al.Global analysis of cellular transcription following infection with an HIV-based vector.Mol. Ther. 2003; 8: 674-687Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 11van ‘t Wout A.B. et al.Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4+-T-cell lines.J. Virol. 2003; 77: 1392-1402Crossref PubMed Scopus (161) Google Scholar]. Surprisingly, the number of cellular genes altered during infection with wild-type adenovirus was fairly small [2Zhao H. et al.Strategic attack on host cell gene expression during aden ovirus infection.J. Virol. 2003; 77: 11006-11015Crossref PubMed Scopus (64) Google Scholar, 3Lohr K. et al.Mutual interference of adenovirus infection and myc expression.J. Virol. 2003; 77: 7936-7944Crossref PubMed Scopus (17) Google Scholar]. Stress responsive genes were upregulated, while some genes involved in the innate immune response were downregulated [2Zhao H. et al.Strategic attack on host cell gene expression during aden ovirus infection.J. Virol. 2003; 77: 11006-11015Crossref PubMed Scopus (64) Google Scholar]. The most dramatic changes in gene expression following HCMV infection involved genes regulated by interferon [4Zhu H. et al.Cellular gene expression altered by human cytomegalovirus: global monitoring with oligonucleotide arrays.Proc. Natl. Acad. Sci. USA. 1998; 95: 144705Google Scholar]. Interferon-responsive genes were also induced by HSV-1 which had been either UV-irradiated or which lacked the ICP0 viral transactivator, but this response appeared to be disarmed during infection with wild-type virus [6Eidson K.M. et al.Expression of herpes simplex virus ICP0 inhibits the induction of interferon-stimulated genes by viral infection.J. Virol. 2002; 76: 2180-2191Crossref PubMed Scopus (142) Google Scholar, 7Mossman K.L. Smiley J.R. Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.J. Virol. 2002; 76: 1995-1998Crossref PubMed Scopus (95) Google Scholar]. Interferon-regulated genes were upregulated during infection with Kaposi-associated herpesvirus (KSHV) [12Mikovits J. et al.Potential cellular signatures of viral infections in human hematopoietic cells.Dis. Markers. 2001; 17: 173-178Crossref PubMed Scopus (16) Google Scholar, 13Poole L.J. et al.Altered patterns of cellular gene expression in dermal microvascular endothelial cells infected with Kaposi's sarcoma-associated herpesvirus.J. Virol. 2002; 76: 3395-3420Crossref PubMed Scopus (105) Google Scholar] and also by expression of the latency-associated nuclear antigen [14Renne R. et al.Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus.J. Virol. 2001; 75: 458-468Crossref PubMed Scopus (177) Google Scholar]. Thus, there are different pathways by which viral proteins at the cell surface or in the nucleus act to modulate the host interferon response. The cellular transcriptional response has been analyzed during HIV infection, although conflicting conclusions from these reports highlight some of the concerns with interpretation of microarray data. One study of infection in cultured human CD4+ lymphoblastoid cells suggested that there was a dramatic effect on cellular gene expression, with changes observed in 30% of the mRNAs, including those encoding factors involved in the interferon response, the DNA damage response and apoptosis [9Corbeil J. et al.Temporal gene regulation during HIV-1 infection of human CD4+ T cells.Genome Res. 2001; 11: 1198-1204Crossref PubMed Scopus (96) Google Scholar]. However, reanalysis of the same data by a different group suggested very few statistically significant changes [10Mitchell R. et al.Global analysis of cellular transcription following infection with an HIV-based vector.Mol. Ther. 2003; 8: 674-687Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar], and a separate study of infection in cultured T cells also found only modest changes [11van ‘t Wout A.B. et al.Cellular gene expression upon human immunodeficiency virus type 1 infection of CD4+-T-cell lines.J. Virol. 2003; 77: 1392-1402Crossref PubMed Scopus (161) Google Scholar]. Two recent studies published in Molecular Therapy have focused specifically on the transcriptional impact of commonly used vector systems. Analysis by Mitchell et al. of HIV vector transduction suggested little change in gene expression with this vector system [10Mitchell R. et al.Global analysis of cellular transcription following infection with an HIV-based vector.Mol. Ther. 2003; 8: 674-687Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. The genes that were affected suggest that the recognition of the virus produces both an immune response and a DNA damage response [10Mitchell R. et al.Global analysis of cellular transcription following infection with an HIV-based vector.Mol. Ther. 2003; 8: 674-687Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar]. In this issue of Molecular Therapy, a study by Stilwell and Samulski interrogated the response of primary human lung cells to both Ad and adeno-associated virus (AAV) [15Stilwell J.L. Samulski R.J. Role of viral vectors and virion shells on cellular gene expression.Mol. Ther. 2004; (this issue.)Google Scholar]. Recombinant Ad vectors (rAd) produced changes in the level of expression of 4.2% of the approximately 7000 genes on the microarray chip used (Affymetrix’ HuGENE-Fl). These included elevation in expression of genes encoding cytokines and chemokines, as well as stress responsive genes. Although wild-type Ad was not analyzed in this study, the number of genes altered with rAd was significantly greater that that observed in two recent reports evaluating Ad2 [2Zhao H. et al.Strategic attack on host cell gene expression during aden ovirus infection.J. Virol. 2003; 77: 11006-11015Crossref PubMed Scopus (64) Google Scholar] and Ad5 [3Lohr K. et al.Mutual interference of adenovirus infection and myc expression.J. Virol. 2003; 77: 7936-7944Crossref PubMed Scopus (17) Google Scholar]. This may be due to different experimental conditions or methods of analysis, or may reflect the fact that viral genes that counteract the cellular response are not expressed in the replication-defective rAd vector. In contrast, AAV affected only 1.1% of the genes, giving rise to similar results for both wild-type and recombinant forms of the virus. AAV infection produced decreases in the levels of mRNA for some genes linked to cellular proliferation and the cell cycle. These microarray data correlate nicely with observations from previous studies that demonstrated an effect of AAV on cellular proliferation. They also provide a molecular basis by which to understand the documented induction of an inflammatory response to rAd vectors, but not to those derived from rAAV. Interactions of viral coat proteins with cell surface receptors may be responsible for many of the changes observed in cellular gene expression by induction of intracellular signaling cascades. For example, in the case of HCMV, viral glycoprotein B is largely responsible for the induction of the interferon response [16Simmen K.A. et al.Global modulation of cellular transcription by human cytomegalovirus is initiated by viral glycoprotein B.Proc. Natl. Acad. Sci. USA. 2001; 98: 7140-7145Crossref PubMed Scopus (183) Google Scholar]. Another example comes from HIV, where the gp120 protein of the envelope can itself induce expression of a large number of cellular genes, including cytokines, chemokines and transcription factors [17Cicala C. et al.HIV envelope induces a cascade of cell signals in nonproliferating target cells that favor virus replication.Proc. Natl. Acad. Sci. USA. 2002; 99: 9380-9385Crossref PubMed Scopus (141) Google Scholar]. Envelope-mediated signaling may help to overcome postentry barriers to viral infection and contribute to productive infection. The impact of the initial events during virus infection and vector transduction is further highlighted by the study from Stilwell and Samulski [15Stilwell J.L. Samulski R.J. Role of viral vectors and virion shells on cellular gene expression.Mol. Ther. 2004; (this issue.)Google Scholar]. They developed elegant methods to generate empty AAV and Ad virions, which enabled them to reveal the impact of the capsid shell on host gene expression during vector transduction. Removal of the Ad genome dramatically reduced (down to 0.7%) the number of cellular genes that were affected, although it did not completely eliminate induction of genes associated with stress and inflammation. In contrast, there was very little difference between the changes in expression observed due to infection with empty AAV virions, rAAV or wild-type AAV. Empty capsids of AAV induced effects on the cell cycle that were similar to those seen with wild-type virus infection. These experiments clearly demonstrate that primary steps in vector transduction can affect cellular gene expression. The results with empty Ad virions support the idea that the incoming vector particle is sufficient to induce an immune response [18Kafri T. et al.Cellular immune response to adenoviral vector infected cells does not require de novo viral gene expression: implications for gene therapy.Proc. Natl. Acad. Sci. USA. 1998; 95: 11377-11382Crossref PubMed Scopus (224) Google Scholar]. As a large number of genes are affected by empty virion shells, presumably through interactions with cellular receptors, it will be interesting to determine the differences with alternative serotypes of AAV and Ad that use different entry pathways. It will also be informative to test the effects of individual coat proteins on cellular gene expression. The difference between rAd and empty Ad also suggests that either the genome itself or expression of viral genes from the vector has a significant impact on cellular gene expression. It will be interesting to test gutted Ad vectors or second-generation variants to determine what factors are responsible for this difference. Comparison of these vector variants with rAd expressing a reporter gene will also allow an assessment of the impact of a specific transgene on cellular gene expression. As with all microarray studies, rigorous statistics must be used when analyzing multiple replicates and when comparing different experimental conditions. Additional insight may come from analysis of datasets by different researchers and from comparisons between datasets. Therefore, these data might be more useful to the gene therapy community if they were collected into a user-friendly database, which is one suggestion to come from these recent papers. There are a number of problems inherent to such public repositories of microarray data [19Stoeckert Jr., C.J. Causton H.C. Ball C.A. Microarray databases: standards and ontologies.Nat. Genet. 2002; 32: S469-S473Crossref PubMed Scopus (132) Google Scholar]. There will need to be a set of standards for data representation and formatting to consolidate and coordinate the data. Microarray data are highly context dependent, and therefore the experimental data would need to be presented in a comprehensible format with sufficient information to allow a separate research group to evaluate the quality of the imported data. To generate meaningful and reproducible findings across multiple data sets would require standardization of the production and purification of the specific vector used, the handling of cells and RNA samples, and the processing of the large amounts of data. If successful, such a database could provide a means to assess the potential toxicity of newly developed or modified vectors during preclinical studies. Generating large amounts of data with microarrays is the easy part. A much greater challenge is to understand the significance of the data at the mechanistic level and to convert the information into improvements in vector performance. Comparing different vectors and iterations thereof may suggest ways by which to diminish negative effects of vector transduction. The datasets in these first few studies using basic vectors may provide reference standards upon which to assess the impact of improvements. An obvious follow-on question is how well changes in RNA levels correlate with changes in protein levels. Therefore, there is a need for profiling technologies that go beyond measuring RNA levels. Numerous alterations occur in proteins that are not reflected in changes of the cognate RNA, and it is already known that viruses can induce many targeted modifications of cellular proteins, such as phosphorylation, ubiquitinylation and even degradation. Proteomic approaches to analyze cellular responses will thus be essential for a complete understanding of changes induced during vector transduction. There have been very few reports that have attempted to extend observations from in vitro microarray studies to determine whether they are reflected in an in vivo model of infection or transduction. Variation in cell types transduced in a specific tissue and the influence of immune cells that are recruited will complicate interpretation of expression profiles for in vivo settings. One study with hybridization of a relatively small number of genes arrayed on a filter demonstrated an increase in RNA levels of some immune response genes [20Haberberger T.C. Kupfer K. Murphy J.E. Profiling of genes which are differentially expressed in mouse liver in response to adenoviral vectors and delivered genes.Gene Ther. 2000; 7: 903-909Crossref PubMed Scopus (29) Google Scholar], but clearly more global analysis in different tissues is required. In vivo studies will be aided by laser microdissection of transduced cells, and by evolving techniques for amplifying small amounts of mRNA for microarray analysis. Expression of viral genes in large and complicated viral genomes can also benefit from viral-specificmicroarrays. Arrays made up of individual viral open reading frames may be useful in assessing residual viral gene expression after mutation of specific gene products in vectors. Although the virus that produces the most benign affect on cellular transcription may seem the most attractive, it will be important to compare gene expression with proteomics and in vivo transduction perfomance. Ultimately the effects on the host of the virus coat, the vector genome and the remaining viral genes, will all be important parameters to be considered when developing viral delivery systems. Global analysis of gene expression changes reveals a fingerprint of the molecular consequences of vector transduction, and hopefully this can be interpreted to make predictions of cellular toxicity that ultimately lead to better vectors.