Subcellular localization of LEDGF/p75-hybrids in interphase cells.

<p>LEDGF/p75 depleted cell lines were complemented with the respective LEDGF-fusions. Laser scanning confocal images of HelaP4 cells, stained using an Ab recognizing LEDGF_480-530, are shown in green. Nuclei were stained using Dapi (shown in blue). A merge of green and blue fluorescence is shown. Data depicted are representative for the respective cell lines. PFV, Prototype foamy virus; LANA, Latency associated nuclear antigen; HPV, Human papilloma virus; LEDGF, Lens epithelium-derived growth factor; DAPI, 4',6-Diamidino-2-Phenylindole.</p

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras

<div><p>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.</p></div

Peptide characteristics.

<p>Fig showing the acronyms, aa-sequences and binding characteristics of the peptides used to generate the artificial LEDGF tethers. PFV, Prototype foamy virus; HPV, Human papilloma virus; KSHV, Kaposi’s sarcoma herpes virus; LANA, Latency associated nuclear antigen.</p

LEDGF-hybrids retarget lentiviral integration towards a more randomized pattern.

<p>(a) Genomic heat map comparing integration site data sets obtained from HeLaP4 LEDGF/p75 KD cells overexpressing different artificial LEDGF-hybrids to genomic features. Tile color depicts the correlation for an integration dataset with the respective genomic feature (left) relative to matched random controls, as indicated by the colored receiver operating characteristic (ROC) curve area scale at the bottom of the panel. Statistical significance (asterisks, ***p<0.001 ranked Wald tests) is shown relative to LEDGF KD population (double dash). Columns indicate different data sets, while rows indicate different genomic features analyzed (described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164167#pone.0164167.ref063" target="_blank">63</a>]). LANA, Latency associated nuclear antigen; HPV, Human papilloma virus; PFV, Prototype foamy virus; a; LEDGF, Lens epithelium-derived growth factor. (b) Epigenetic heat map comparing integration site data sets obtained from HeLaP4 LEDGF/p75 KD cells overexpressing different artificial LEDGF-hybrids to epigenetic features. Tile color depicting a positive or negative correlation to the respective epigenetic feature (10kb windows), relative to MRC, as indicated by the receiver operating characteristic (ROC) curve area scale at the bottom of the panel. Statistical significance (asterisks, ***p<0.001, ranked Wald tests) is shown relative to LEDGF KD population (dashed). Columns indicate different data sets while rows indicate different epigenetic features analyzed. Included features were limited to those identified in high-throughput studies HeLaP4 and primary CD4+ T-cells. Detailed information on epigenetic marks and their roles can be found in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164167#pone.0164167.ref087" target="_blank">87</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164167#pone.0164167.ref088" target="_blank">88</a>]. LANA, Latency associated nuclear antigen; HPV, Human papilloma virus; PFV, Prototype foamy virus; a; LEDGF, Lens epithelium-derived growth factor.</p

Rescue of lentiviral vector transduction by artificial LEDGF-hybrids.

<p>LEDGF-fusions were evaluated for their ability to support lentiviral vector transduction. LEDGF-depleted HelaP4-based cell lines stably complemented with LEDGF-hybrids were challenged with a VSV-G pseudo-typed lentiviral reporter vector encoding enhanced Green Fluorescent Protein (eGFP). Fluorescence was measured by fluorescence activated cell sorting and the different variables plotted:. (a) Percentage eGFP positive cells (transduction efficiency) and (b) Mean Fluorescence Intensity (MFI). Data are compiled for a representative experiment and depict averages of 3 replicates for 3 different vector dilutions (mean ± SD). (c) Lentiviral integrated proviral copies were determined by Q-PCR analysis on genomic DNA extracts of cells transduced with an MOI = 1. Data are represent the mean of 3 replicates ± SD. Statistical significance is calculated using a two-tailed t-test relative to LEDGF_KD or ΔN₉₃-LEDGF. PFV, Prototype foamy virus; LANA, Latency associated nuclear antigen; HPV, Human papilloma virus; LEDGF, Lens epithelium-derived growth factor.</p

IN dimerization interface mutants exhibit reduced affinity for LEDGF/p75 <i>in vitro</i>.

<p>An AlphaScreen assay measuring mutant IN interaction with LEDGF/p75, as compared to that of the WT IN-LEDGF/p75 interaction, showed that none of the mutant IN proteins substantially interact with LEDGF/p75. The highest amount of interaction was between K103G and LEDGF/p75, but this protein still only reached about 30% of the WT interaction level.</p

A symmetric region of the HIV-1 dimerization interface is conserved across other lentiviruses.

<p>(A) PyMOL representation of the highly symmetric region at the HIV-1 IN dimeric interface. A four-tiered aromatic interaction between W61 and W108 from each IN monomer is flanked by two salt bridges composed of E85 and R107, and E87 and K103 from each monomer. The four-tiered aromatic interaction donates at least −10 kcal/mol of stabilization energy to the interface. (B) Sequence alignment of relevant lentiviral IN residues, beginning at IN residue 55. Red text denotes highly conserved residues, while blue signifies moderately conserved. W61, E85, E87, K103, R107, and W108 are all completely conserved throughout HIV-1, SHIV, SIV, and HIV-1 viruses. Aromaticity is heavily conserved across most lentiviruses for positions 61 and 108.</p