Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy
Authors: Eirini P Papapetrou, Axel Schambach
Journal: Molecular Therapy (2016)

Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy. Genomic safe harbors (GSHs) are sites in the genome able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements: (i) function predictably and (ii) do not cause alterations of the host genome posing a risk to the host cell or organism. GSHs are thus ideal sites for transgene insertion whose use can empower functional genetics studies in basic research and therapeutic applications in human gene therapy. Currently, no fully validated GSHs exist in the human genome. Here, we review our formerly proposed GSH criteria and discuss additional considerations on extending these criteria, on strategies for the identification and validation of GSHs, as well as future prospects on GSH targeting for therapeutic applications. In view of recent advances in genome biology, gene targeting technologies, and regenerative medicine, gene insertion into GSHs can potentially catalyze nearly all applications in human gene therapy. The majority of gene therapy applications that are currently in the clinic or in advanced stages of preclinical development rely on gene addition. Efficient transgene insertion and expression is predominantly achieved through gamma-retroviral and lentiviral vectors.1Cartier N Hacein-Bey-Abina S Bartholomae CC Veres G Schmidt M Kutschera I et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy.Science. 2009; 326: 818-823Crossref PubMed Scopus (1177) Google Scholar,2Gaspar HB Cooray S Gilmour KC Parsley KL Zhang F Adams S et al.Hematopoietic stem cell gene therapy for adenosine deaminase-deficient severe combined immunodeficiency leads to long-term immunological recovery and metabolic correction.Sci Transl Med. 2011; 3: 97ra80Crossref PubMed Scopus (242) Google Scholar,3Hacein-Bey-Abina S Pai SY Gaspar HB Armant M Berry CC Blanche S et al.A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.N Engl J Med. 2014; 371: 1407-1417Crossref PubMed Scopus (296) Google Scholar,4Biffi A Montini E Lorioli L Cesani M Fumagalli F Plati T et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (906) Google Scholar,5Aiuti A Biasco L Scaramuzza S Ferrua F Cicalese MP Baricordi C et al.Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome.Science. 2013; 341: 1233151Crossref PubMed Scopus (809) Google Scholar However, transgenes newly inserted into random genomic positions interact with the host genome in unpredictable ways. 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While the former effects diminish or abrogate the therapeutic effect, the latter can have catastrophic consequences for the host organism. Dysregulated expression of key genes may alter the cellular behavior in dramatic ways, promoting clonal expansion or malignant transformation of the host cell, as demonstrated in clinical trials of retroviral gene transfer into hematopoietic stem cells, resulting in "preleukemia" or myelodysplastic syndrome in some cases and in overt acute leukemia in others.11Hacein-Bey-Abina S Von Kalle C Schmidt M McCormack MP Wulffraat N Leboulch P et al.LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.Science. 2003; 302: 415-419Crossref PubMed Scopus (3001) Google Scholar,12Ott MG Schmidt M Schwarzwaelder K Stein S Siler U Koehl U et al.Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1.Nat Med. 2006; 12: 401-409Crossref PubMed Scopus (983) Google Scholar,13Stein S Ott MG Schultze-Strasser S Jauch A Burwinkel B Kinner A et al.Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease.Nat Med. 2010; 16: 198-204Crossref PubMed Scopus (631) Google Scholar,14Howe SJ Mansour MR Schwarzwaelder K Bartholomae C Hubank M Kempski H et al.Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.J Clin Invest. 2008; 118: 3143-3150Crossref PubMed Scopus (947) Google Scholar,15Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google Scholar We previously introduced a conceptual framework for genomic safe harbors (GSHs)—genomic sites that can support predictable transgene expression while minimizing the risk of unwanted interactions with the host genome.16Papapetrou EP Lee G Malani N Setty M Riviere I Tirunagari LM et al.Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells.Nat Biotechnol. 2011; 29: 73-78Crossref PubMed Scopus (248) Google Scholar,17Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (148) Google Scholar In order to avoid insertions that can lead to dysregulation of the endogenous gene expression program of the host cell, we proposed five GSH criteria.16Papapetrou EP Lee G Malani N Setty M Riviere I Tirunagari LM et al.Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells.Nat Biotechnol. 2011; 29: 73-78Crossref PubMed Scopus (248) Google Scholar,17Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (148) Google Scholar These were intended to avoid the two types of insertional events that predominantly result in gene dysregulation: activation of adjacent genes and gene disruption. The most common mechanism of insertional oncogenesis is activation of oncogene expression through increase in the rate of transcription by the vector's promoter/enhancer load. In these cases, vector integration sites are typically located just upstream of the dysregulated gene, most commonly within 50 kb of the transcriptional start site. The second most common mechanism of dysregulation of endogenous genes results from insertions within transcription units, mediated by formation of new gene products through fusion of viral and cellular sequences as a result of aberrant splicing or read-through, or by truncation of endogenous transcription units removing negative regulatory sequences.15Cavazzana-Calvo M Payen E Negre O Wang G Hehir K Fusil F et al.Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia.Nature. 2010; 467: 318-322Crossref PubMed Scopus (1035) Google Scholar Our five criteria thus aimed to exclude regions of the genome in close proximity (within 50 kb) to coding and noncoding genes—requiring extra distance (at least 300 kb) from genes known to play a role in cancer in humans or model organisms and microRNA genes.18Li Q Peterson KR Fang X Stamatoyannopoulos G Locus control regions.Blood. 2002; 100: 3077-3086Crossref PubMed Scopus (344) Google Scholar,19Bejerano G Pheasant M Makunin I Stephen S Kent WJ Mattick JS et al.Ultraconserved elements in the human genome.Science. 2004; 304: 1321-1325Crossref PubMed Scopus (1256) Google Scholar,20Fraser P Transcriptional control thrown for a loop.Curr Opin Genet Dev. 2006; 16: 490-495Crossref PubMed Scopus (88) Google Scholar They also aimed at avoiding disruption of endogenous coding genes and conserved genetic elements by exclusion of sites located inside transcription units and ultra-conserved genomic regions. We provided proof of principle in a disease model of β-thalassemia showing that a therapeutic β-globin transgene inserted in sites meeting the five criteria—i.e., extragenic sites distant from endogenous genes—can afford therapeutic levels of expression without dysregulation of expression of endogenous genes.16Papapetrou EP Lee G Malani N Setty M Riviere I Tirunagari LM et al.Genomic safe harbors permit high β-globin transgene expression in thalassemia induced pluripotent stem cells.Nat Biotechnol. 2011; 29: 73-78Crossref PubMed Scopus (248) Google Scholar No chromosomal location in the human genome has yet been demonstrated to qualify as a bona fide GSH—i.e., to be adequate for reliable and safe therapeutic transgene addition—with any level of confidence. Three sites have to date mostly been targeted for transgene addition: (i) the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19; (ii) the chemokine (C-C motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as an HIV-1 coreceptor; and (iii) the human ortholog of the mouse Rosa26 locus, a locus extensively validated in the murine setting for the insertion of ubiquitously expressed transgenes.21Irion S Luche H Gadue P Fehling HJ Kennedy M Keller G Identification and targeting of the ROSA26 locus in human embryonic stem cells.Nat Biotechnol. 2007; 25: 1477-1482Crossref PubMed Scopus (222) Google Scholar,22Kotin RM Linden RM Berns KI Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination.EMBO J. 1992; 11: 5071-5078Crossref PubMed Scopus (409) Google Scholar,23Liu R Paxton WA Choe S Ceradini D Martin SR Horuk R et al.Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2563) Google Scholar,24Perez EE Wang J Miller JC Jouvenot Y Kim KA Liu O et al.Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases.Nat Biotechnol. 2008; 26: 808-816Crossref PubMed Scopus (829) Google Scholar While none of the above sites perfectly match the above criteria, some functional data is available for some of them on robustness of expression of integrated transgenes and their effects on their neighboring genome.25Lombardo A Cesana D Genovese P Di Stefano B Provasi E Colombo DF et al.Site-specific integration and tailoring of cassette design for sustainable gene transfer.Nat Methods. 2011; 8: 861-869Crossref PubMed Scopus (263) Google Scholar The AAVS1 site on chromosome 19, in particular, has gained popularity because of the commercial availability of effective tools for its targeting and its ability to support transgene expression in multiple cell types.26Smith JR Maguire S Davis LA Alexander M Yang F Chandran S et al.Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration.Stem Cells. 2008; 26: 496-504Crossref PubMed Scopus (154) Google Scholar,27Yang L Soonpaa MH Adler ED Roepke TK Kattman SJ Kennedy M et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (1152) Google Scholar,28Zou J Sweeney CL Chou BK Choi U Pan J Wang H et al.Oxidase-deficient neutrophils from X-linked chronic granulomatous disease iPS cells: functional correction by zinc finger nuclease-mediated safe harbor targeting.Blood. 2011; 117: 5561-5572Crossref PubMed Scopus (209) Google Scholar,29Ramachandra CJ Shahbazi M Kwang TW Choudhury Y Bak XY Yang J et al.Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors.Nucleic Acids Res. 2011; 39: e107Crossref PubMed Scopus (35) Google Scholar However, it cannot support faithful transgene expression in at least some cell lineages and transgenes inserted at the AAVS1 locus can be silenced through mechanisms that include DNA methylation.30Ordovás L Boon R Pistoni M Chen Y Wolfs E Guo W et al.Efficient recombinase-mediated cassette exchange in hPSCs to study the hepatocyte lineage reveals AAVS1 locus-mediated transgene inhibition.Stem Cell Reports. 2015; 5: 918-931Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Furthermore, integration in the AAVS1 locus disrupts the gene phosphatase 1 regulatory subunit 12C (PPP1R12C) and the consequences of its haploinsufficiency or complete inactivation in various cell types have not been investigated in depth. The CCR5 gene locus was identified as a putative safe harbor after the discovery that people with a naturally occurring homozygous deletion leading to complete CCR5 gene disruption were resistant to HIV-1 infection and had no signs of overt pathology,23Liu R Paxton WA Choe S Ceradini D Martin SR Horuk R et al.Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2563) Google Scholar although subsequent studies associated CCR5 gene knockout with increased susceptibility to disease caused by infection with the West Nile Virus and the related Japanese Encephalitis Virus.31Glass WG McDermott DH Lim JK Lekhong S Yu SF Frank WA et al.CCR5 deficiency increases risk of symptomatic West Nile virus infection.J Exp Med. 2006; 203: 35-40Crossref PubMed Scopus (402) Google Scholar,32Lim JK Murphy PM Chemokine control of West Nile virus infection.Exp Cell Res. 2011; 317: 569-574Crossref PubMed Scopus (48) Google Scholar,33Larena M Regner M Lobigs M The chemokine receptor CCR5, a therapeutic target for HIV/AIDS antagonists, is critical for recovery in a mouse model of Japanese encephalitis.PLoS One. 2012; 7: e44834Crossref Scopus (27) Google Scholar Therefore, the safety of all of the above sites is at present unknown at best and—taking into account their localization inside coding genes and in gene-dense areas and their proximity to genes implicated in cancer—very uncertain.17Sadelain M Papapetrou EP Bushman FD Safe harbours for the integration of new DNA in the human genome.Nat Rev Cancer. 2012; 12: 51-58Crossref Scopus (148) Google Scholar Although sites like AAVS1, CCR5, and other intragenic regions may be acceptable for research applications, a much higher burden of proof of safety is needed for clinical applications. Given this uncertainty and the growing body of information on functional features of the human genome, we next discuss additional considerations that could be applied to the core GSH criteria, as well as approaches toward the identification and validation of GSHs (Figure 1). The GSH definition presented above is a reflection of lessons learned from side effects related to insertional mutagenesis by LTR-driven retroviruses. The criteria were mostly empirically based on known genome annotations, primarily coding and noncoding genes. With regard to putative regulatory genomic sequences, a conservative approach of excluding only regions exhibiting very high conservation was adopted. Perspectively, GSH criteria will need to be amended to take into account emerging information on genome organization and function, including three-dimensional nuclear organization and the rapid discovery of new genomic functional elements. The distances specified in the original criteria may also be revised as additional information on how the regional, domain-level, and large-scale organization of the genome influences the function of genetic sequences. For example, longer-range interactions exceeding the 300 kb limit that we originally set seem increasingly possible. Data supporting the possibility of oncogene activation by a viral genome integrated ~500 kb upstream has more recently been reported, albeit in HeLa cells which are highly aneuploid.34Adey A Burton JN Kitzman JO Hiatt JB Lewis AP Martin BK et al.The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line.Nature. 2013; 500: 207-211Crossref PubMed Scopus (233) Google Scholar Below, we discuss some putative additions to the original criteria. It should be noted that our GSH definition does not include any toxicity that may result from expression (at physiologic or supra-physiologic levels) of the transgene itself. Recent studies taking advantage of CRISPR and other high-throughput methods to knockout genes have addressed gene essentiality in human cells.35Blomen VA Májek P Jae LT Bigenzahn JW Nieuwenhuis J Staring J et al.Gene essentiality and synthetic lethality in haploid human cells.Science. 2015; 350: 1092-1096Crossref PubMed Scopus (503) Google Scholar,36Wang T Birsoy K Hughes NW Krupczak KM Post Y Wei JJ et al.Identification and characterization of essential genes in the human genome.Science. 2015; 350: 1096-1101Crossref PubMed Scopus (945) Google Scholar These genome-scale systematic approaches identified ~2,000 genes (~10% of the ~20,000 protein-coding genes in the human genome) that are indispensable for cell viability. Although essentiality of a specific gene may be heavily context-dependent and disruption of only one allele may be tolerated, inactivation of this set of genes should probably be avoided. Similarly, essential and nonessential genes can be teased at the organismal level from genome-wide association studies, as well as from rare human knockouts (like the CCR5 case discussed above).37MacArthur DG Balasubramanian S Frankish A Huang N Morris J Walter K 1000 Genomes Project Consortium et al.A systematic survey of loss-of-function variants in human protein-coding genes.Science. 2012; 335: 823-828Crossref PubMed Scopus (863) Google Scholar,38Sulem P Helgason H Oddson A Stefansson H Gudjonsson SA Zink F et al.Identification of a large set of rare complete human knockouts.Nat Genet. 2015; 47: 448-452Crossref PubMed Scopus (154) Google Scholar However, all the above—gene essentiality screens and human genetics—only inform on which genomic regions should not be disrupted and not on potential problems that may arise from aberrant transcriptional upregulation. These include regulatory noncoding RNAs (ncRNAs) other than miRNAs (which are taken into consideration in the original criteria), mainly regulatory small noncoding RNAs and long noncoding RNAs (lncRNAs).39Huang B Zhang R Regulatory non-coding RNAs: revolutionizing the RNA world.Mol Biol Rep. 2014; 41: 3915-3923Crossref Scopus (47) Google Scholar Some lncRNAs have been shown to play important roles in various cellular and physiologic processes, including gene expression and gene regulation, chromatin dynamics, differentiation, and development.40Fatica A Bozzoni I Long non-coding RNAs: new players in cell differentiation and development.Nat Rev Genet. 2014; 15: 7-21Crossref PubMed Scopus (2240) Google Scholar The disruption or dysregulation of ncRNAs can lead to diseases, e.g., cancer and immunological disorders. For example, HOTAIR (HOX antisense intergenic RNA) encodes a lncRNA regulating key epigenetic regulators and silencing and its dysregulation could lead to cancer formation and other diseases.41Bhan A Mandal SS LncRNA HOTAIR: a master regulator of chromatin dynamics and cancer.Biochim Biophys Acta. 2015; 1856: 151-164PubMed Google Scholar Even though the role of most lncRNAs remains at present uncertain, it is probably prudent that a GSH is not located in close proximity to or disrupt a ncRNA, especially if the latter is well characterized. Our original criteria take into consideration exclusively kilobase-scale genomic interactions, involving primarily enhancer–promoter interactions. Indeed, all known to date insertional oncogenesis events have occurred within this scale. As our perception of our genome's dynamic folding and packaging inside the cell nucleus evolves, we may become increasingly aware of the influence of additional levels of genome organization and higher order chromatin structure on transgene expression.42Sexton T Cavalli G The role of chromosome domains in shaping the functional genome.Cell. 2015; 160: 1049-1059Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar Genome-wide interaction studies using chromosome conformation capture-based approaches (such as 3C, 5C, and Hi-C) revealed that the genome is partitioned into megabase-scale topologically associated domains (TADs).43Dixon JR Selvaraj S Yue F Kim A Li Y Shen Y et al.Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3955) Google Scholar,44Nora EP Lajoie BR Schulz EG Giorgetti L Okamoto I Servant N et al.Spatial partitioning of the regulatory landscape of the X-inactivation centre.Nature. 2012; 485: 381-385Crossref PubMed Scopus (1807) Google Scholar Genetic elements frequently interact with each other within a domain but engage very rarely in interdomain interactions. TAD boundary regions (the generally "non-looped" stretch of DNA between two TADs) often contain CTCF-binding sites that block interactions across adjacent TADs. Thus, TADs are believed to represent regulatory genomic units within which enhancers and promoters can interact and which are insulated from each other. There are still more questions than answers on how chromatin topology relates to genome function. Genome folding is not as rigid and stable, as for example protein structure and whether it is a cause or consequence of genome activity is still unclear.45Cavalli G Misteli T Functional implications of genome topology.Nat Struct Mol Biol. 2013; 20: 290-299Crossref PubMed Scopus (305) Google Scholar However, there clearly is a correlation between chromatin topology and gene activity. Furthermore, TADs and their boundaries are largely conserved across different cell types. Thus, the relative location of a genomic site with respect to TADs could inform the choice of GSHs. For example, it might be useful to avoid sites within TADs enriched in cancer genes or to favor a GSH located in a TAD border. An additional layer of complexity is added by epigenetic modifications which register, signal, or perpetuate altered genomic activity states.46Bird A Perceptions of epigenetics.Nature. 2007; 447: 396-398Crossref PubMed Scopus (2048) Google Scholar These include DNA modifications (e.g., 5-methylcytosine, 5-hydroxymethylcytosine), posttranslational histone tail modifications and nucleosomal remodeling.47Boland MJ Nazor KL Loring JF Epigenetic regulation of pluripotency and differentiation.Circ Res. 2014; 115: 311-324Crossref PubMed Scopus (173) Google Scholar Several features can help distinguish accessible and transcriptionally active from repressive chromatin, and these may help predict the capacity of a given genomic site to support adequate transgene expression. Such predictions can be made in a cell type-specific manner using data obtained by various methods and through large-scale initiatives (ENCODE, Roadmap), including ChIP-Seq data on transcription factor binding, genome-wide DNA methylation, promoter/enhancer signatures inferred by histone marks and chromatin accessibility. Nucleosomes in the vicinity of active enhancers typically contain histones with characteristic posttranslational modifications, such as histone H3 lysine 4 monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) at their amino termini, while inactive enhancers show enrichment of the Polycomb protein-associated repressive H3K27me3 mark.48Shlyueva D Stampfel G Stark A Transcriptional enhancers: from properties to genome-wide predictions.Nat Rev Genet. 2014; 15: 272-286Crossref PubMed Scopus (805) Google Scholar Moreover, chromatin accessibility as assessed by DNAse I hypersensitivity or more recently ATACseq identifies DNA sequences that are not bound by nucleosomes and therefore accessible to the transcriptional machinery. Despite the currently rather loose definition of regulatory DNA and much debate surrounding the portion of the genome that is functional, there is likely more to the human genome than is covered by the core criteria that should not be interfered with. In contrast to the linear organization of the genome, which can be easily analyzed by DNA sequencing in cases of insertional mutagenesis in preclinical models and the clinic, the contribution of epigenetic features, if any, to insertional dysregulation of genes is very hard to study, as epigenetic modifications are dynamic, unstable, and cell context dependent. In addition, vector insertion likely reshapes the surrounding chromatin in ways that we still do not adequately understand.25Lombardo A Cesana D Genovese P Di Stefano B Provasi E Colombo DF et al.Site-specific integration and tailoring of cassette design for sustainable gene transfer.Nat Methods. 2011; 8: 861-869Crossref PubMed Scopus (263) Google Scholar It is generally assumed that markers of "active" chromatin would be desirable in a GSH as they are predictive of high transcriptional activity and thus likelihood of transgene expression, while on the contrary integration into an area of "repressive" chromatin will likely result in silencing. Furthermore, as discussed below, "closed" chromatin may also hamper gene targeting. In view of the current state of knowledge, no formal guidelines based on epigenetic information can be added to the DNA sequence-based criteria. However, information on chromatin status may facilitate predictions regarding the likelihood of expression from any given genomic site in any given cell type. For example, a GSH may be a locus with active chromatin in the desired cell type(s), but not in others in which aberrant expression may be detrimental, such as tissue stem cells. Different types of GSHs can be envisioned depending on the specific application, taking into account parameters such as the transgene (levels of expression required), the target cell type (risk of transformation and potential for ex vivo manipulation), and the disease (absolute number and proportion of genetically modified cells required for therapeutic effect). What is an acceptable GSH may vary widely. For example, applications that require transgene overexpression for therapeutic benefit4Biffi A Montini E Lorioli L Cesani M Fumagalli F Plati T et al.Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy.Science. 2013; 341: 1233158Crossref PubMed Scopus (906) Google Scholar will need to utilize sites that can afford these levels at a given tissue. "Conditional" or application-specific GSHs can be envisioned in some cases. For example, a site which disrupts a highly expressed gene and "hijacks" the transcriptional output corresponding to one of its two (or more in some cases) endogenous copies may be a viable option, if the function of the endogenous gene is not dosage dependent and/or if it is expressed at such high levels that its haploinsufficiency phenotype is minimal or inconsequential for the target cell. For example, in a recent study, the liver-specific albumin gene locus was used to insert a human coagulation factor IX via a recombinant adeno-associated virus vector.49Barzel A Paulk NK Shi Y Huang Y Chu K Zhang F et al.Promoterless gene targeting without nucleases ameliorates haemophilia B in mice.Nature. 2015; 517: 360-364Crossref PubMed Scopus (177) Google Scholar An argument could thus be made that genomic loci that have been carefully studied might constitute preferable GSHs than sites meeting the criteria discussed before, which mostly select extragenic regions likely to be less studied. In another study, a human factor IX gene was inserted into ribosomal DNA (rDNA) sequences, which exist in ~400 copies in the human genome.50Lisowski L Lau A Wang Z Zhang Y Zhang F Grompe M et al.Ribosomal DNA integrating rAAV-rDNA vectors allow for stable transgene expression.Mol Ther. 2012; 20: 1912-1923Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Alternatively, one or few "universal" GSHs, sites with robust expression and no side effects across all cell types and all stages of differentiation, if identified and validated, could accommodate most or all