Date Published: October 27, 2016
Publisher: Public Library of Science
Author(s): Lenard S. Vranckx, Jonas Demeulemeester, Zeger Debyser, Rik Gijsbers, Yasuhiro Ikeda.
The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.
The capacity to integrate transgenes into the host cell genome makes retroviral vectors (RV) an interesting tool for gene therapeutic applications as stable insertion of transgenes into the genome ensures long-term expression. Use of RV-mediated gene transfer resulted in successful cure of several monogenic, primary immunodeficiency disorders [1–3]. Yet, stable insertion occasionally altered endogenous gene regulation resulting in insertional mutagenesis. Due to this major setback 5 out of 19 treated patients developed leukemia in otherwise successful clinical gene therapy trials for X-SCID and 2 out of 2 patients treated for X-CGD acquired myelodysplastic syndrome [3–6]. Both trials employed murine leukemia virus (MLV)-based gammaretroviral vectors (γRV) that integrate in close proximity to gene regulatory regions [7–9] and resulted in transcriptional deregulation due to up-regulated LMO2 expression [10–13]. Similar reports on insertional mutagenesis were published after integration of γRV near CCDN2, BMI1 and EVI1 [14,15]. Despite improvements in vector design (e.g. self-inactivating (SIN) vectors) their use is still not risk-free [3,4,6,14–16], which shifted attention from yRV towards HIV-derived lentiviral vectors (LV). Even though LV display a more favorable integration pattern, induction of aberrant splicing [17,18] and insertional mutagenesis remain a major concern, as clonal expansion was observed in a gene therapy trial for β-thalassemia . In addition, two recent independent studies revealed clonal expansion in HIV-1 infected patients on antiretroviral therapy due to HIV-1 virus triggered insertional mutagenesis [20,21]. Retroviral integration is a non-random process which is, depending on the viral genus, associated with specific chromatin marks and genomic features [22–24]. yRV predominantly integrate in the vicinity of gene regulatory regions, whereas LV preferably target the body of active transcription units [10,25]. Integration is catalyzed by the viral integrase (IN), whereas integration site choice bias is attributed to the cellular chromatin readers that are co-opted by the viral IN. Whereas the bromodomain and extra-terminal domain (BET) family of proteins (BRD2, 3 and 4) guide MLV integration [26–28], LV integration is directed by Lens epithelium-derived growth factor p75 (LEDGF/p75) [29,30]. Both function as molecular tethers in the cell, combining a chromatin-binding and a protein-interacting region (reviewed in ). For LEDGF/p75 (Fig 1A), the chromatin-binding part contains an N-terminal Pro-Trp-Trp-Pro (PWWP) epigenetic reader domain (aa 1–93), recognizing H3K36me3 chromatin marks [32–36], and a set of DNA-binding motifs (Fig 1A, [37,38]). Together, these elements allow LEDGF/p75 to explore the chromatin in a dynamic scan-and-lock fashion . Even though its cellular role is not fully understood, it is clear that LEDGF/p75 acts as a molecular hub for a variety of endogenous proteins next to the lentiviral integrase (Fig 1A) [40,41],,,. All these proteins, including the lentiviral integrase, bind the C-terminal Integrase-Binding Domain (IBD, aa 347–429; Fig 1A) of LEDGF/p75. We and others showed that replacement of the N-terminal LEDGF/p75 DNA-binding region (aa 1–325) with alternative DNA-binding domains retargets LV integration towards genomic loci bound by these domains [35,45–47]. Fusion of the heterochromatin binding Chromobox protein homolog 1 (CBX1) to the IN-binding C-terminal end of LEDGF/p75 shifted LV integration into the cognate H3K9mex-marked chromatin environment, pericentric heterochromatin and intergenic regions . Despite integration in regions enriched in epigenetic marks associated with gene silencing, transgene expression remained efficient and resulted in successful phenotypic correction in a cell model for X-CGD .
Integration of retroviral vectors into the host cell genome makes them invaluable tools for gene therapeutic applications where life-long correction is key. Previous reports showed effective gene transfer enabling long-term gene correction (For a review see ). However, severe adverse events in these clinical studies (using full-LTR driven gamma-retrovirus vectors) raised serious concerns regarding the safety of gene therapy when using integrating vectors (derived from the family of retroviruses) [14,15]. The yRV preference for integration into enhancer regions and concomitant activation of proto-oncogenes led to malignant transformation of cells and clonal expansion [10–12]. Therefore, multiple studies have been triggered to increase the safety of the used retroviral vectors, which include the use of other subtypes (lenti or alpha instead of gammaretroviral), SIN-LTR design [72–75], tissue specific promoters , changing integration properties [45–47] and insulator sequences as enhancer and silencer blockers . Meanwhile, lentivirus vectors became the mean of choice when using retroviruses for gene transfer and clinical gene therapy due to their safer integration profile and lower genotoxicity in preclinical models. As such, any successful modification avoiding an increased integration of these vectors into gene coding regions may be relevant for translation into the clinics. Stable integration however will always imply the intrinsic risk of vector-induced genomic perturbation, open reading frame-disruption, leading to loss of function or transcriptional deregulation of neighbouring genes as indicated by the report on SIN-LV affected splicing . In addition, also LV integration may lead to clonal dominance as reported in the beta-thalassemia trial, which could be an indicator of upcoming malignant transformation . Therefore it is important to gain additional mechanistic insights into the molecular mechanism of integration and integration site selection for LVs to be accepted for general therapeutic use. We and others substantially contributed to the elucidation of the role of LEDGF/p75 as a molecular tether of lentiviral vector integration. As a cellular cofactor of lentiviral integration, LEDGF/p75 orchestrates lentiviral integration preference by binding H3K36me3 in the body of active transcription units via its N-terminal PWWP domain, but it is the vector-encoded integrase that catalyzes the integration reaction. Depletion of LEDGF/p75 by knockdown or knockout strategies shifts lentiviral vector integration out of active genes, yet integration is not completely random [67,79], which at least in part can be explained by residual targeting via HRP-2 . Here we set out to study whether different LEDGF-hybrids could be generated to distribute lentiviral integration sites more randomly. This line of vector development is based on the further increasing interest in new vector platforms displaying a close-to-random insertional profile potentially reducing the probability of proto-oncogene activation lowering the genotoxic potential [51,80,81]. In an effort to achieve a more random integration site distribution, we deleted the specific chromatin-binding PWWP module of LEDGF/p75 (aa 1–93), or we replaced it with alternative pan-chromatin binding modules. In case of LEDGF/p75, it is demonstrated that the PWWP domain recognizes H3K36me3, a chromatin mark that is particularly enriched in the body of active transcription units [32–36]. Complementation of LEDGF-depleted cells with a LEDGF/p75-protein that had its PWWP domain deleted (ΔN93-LEDGF) or replaced with alternative chromatin binding modules showed unique subnuclear distributions for each of the constructs, indicating that these deletion of the PWWP domain, or the replacements with any of the other peptides, resulted in a specific redistribution within the nuclear compartment of the artificial LEDGF chimera (Fig 3). The latter phenotype can be attributed to the AT-hook motifs and charged regions present in the N-terminal end of ΔN93-LEDGF, together with the specific peptides that replaced the PWWP domain. After working up integration sites, analysis showed that lentiviral integration preferences for most of the constructs resulted in a more random distribution than under LEDGF depleted conditions (genomic and the epigenetic heat map representations; Fig 6A and 6B), except for PFV Gag534-546-ΔN93-LEDGF. For example, in the latter cells LV integration was still enriched near epigenetic markers for transcriptionally active chromatin, albeit less outspoken than observed with LEDGFWT and LEDGFBC cells (S4B Fig). Interestingly, peptide addition was not required to obtain a more random distribution. Lentiviral integrations in ΔN93-LEDGF expressing cells were redistributed in a fairly random manner, with tile colors shifting to grey and black (for the genomic and the epigenetic heat map representations, respectively) indicating that integration frequencies for these features are not enriched nor depleted compared to the matched random integration site distribution. Comparison with LEDGF KD shows that integration is more randomly distributed than under LEDGF depletion (*** p<0.001, Wald statistics; Fig 6A and 6B). Fusion of short pan-chromatin binding peptides to the truncated ΔN93-LEDGF resulted in similar shifts towards a more randomized integration profile. The fact that all peptide fusions display a unique subnuclear location, suggest that their interaction with chromatin is different. Even though the overall integration frequencies are highly similar (considering the genomic and the epigenetic features analyzed), larger integration site datasets (>10e5 sites) would be required to allow more detailed analysis on the specific subsets. In an effort to estimate the effect of the more randomized distribution on safety, we calculated the frequency of integration relative to a set of safe harbor criteria for the individual integration site datasets . This analysis showed that the more random distributions resulted in a lower genotoxic profile with 18–22% of integrations meeting safe harbor criteria for our LEDGF-chimera compared to only 5.4% for cells carrying wild-type LEDGF/p75, all LEDGF-chimera resulted in a safer distributions over the genome. Fully targeted integration towards safe harbor regions like the AAVS1 or CCR5 locus would be the ultimate solution to circumvent insertional mutagenesis [59,66,82]. Several methods for site-directed gene correction have been developed using genetic scissors based on Zinc-finger nucleases, transcription activator like effector nucleases or more recently RNA-guided nucleases (CRISPR/Cas9) (for a review ). However, site directed integration would no doubt impair transduction efficiencies. Our approach improves the therapeutic potential of lentiviral vectors by decreasing the risk/benefit ratio, still supporting high transduction efficiencies. The fact that integration can be directed to genomic regions that are not targeted under wild-type conditions nor LEDGF-depleted conditions, indicates that integration in these areas is disfavored due to the absence of a tether, rather than the presence of specific obstacles such as steric hindrance resulting from the condensed chromatin structure. As an alternative to the generation of stable cell lines as employed here, we demonstrated earlier that mRNA-electroporation ensures timely, high-level recombinant protein expression that is sufficient to retarget lentiviral vector integration . When combined with IN mutant lentiviral vectors that selectively bind complementary LEDGF/p75 variants , this approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as genetic modification of patient-specific iPS cells and improve safety of lentiviral vectors. With the occurrence of potential adverse effects being of multi-factorial nature  novel therapeutic approaches should be evaluated in relevant functional assays able to predictively assess the cytotoxicity observed in vivo , a continuous effort aiming at abolishing the risk of insertional mutagenesis will be required for gene therapy to become a broadly accepted treatment alternative.