Hepatocyte-targeted expression by integrase-defective lentiviral vectors induces antigen-specific tolerance in mice with low genotoxic risk

Abstract

Lentiviral vectors are attractive tools for liver-directed gene therapy because of their capacity for stable gene expression and the lack of preexisting immunity in most human subjects. However, the use of integrating vectors may raise some concerns about the potential risk of insertional mutagenesis. Here we investigated liver gene transfer by integrase-defective lentiviral vectors (IDLVs) containing an inactivating mutation in the integrase (D64V). Hepatocyte-targeted expression using IDLVs resulted in the sustained and robust induction of immune tolerance to both intracellular and secreted proteins, despite the reduced transgene expression levels in comparison with their integrase-competent vector counterparts. IDLV-mediated and hepatocyte-targeted coagulation factor IX (FIX) expression prevented the induction of neutralizing antibodies to FIX even after antigen rechallenge in hemophilia B mice and accounted for relatively prolonged therapeutic FIX expression levels. Upon the delivery of intracellular model antigens, hepatocyte-targeted IDLVs induced transgene-specific regulatory T cells that contributed to the observed immune tolerance. Deep sequencing of IDLV-transduced livers showed only rare genomic integrations that had no preference for gene coding regions and occurred mostly by a mechanism inconsistent with residual integrase activity.

Conclusion: IDLVs provide an attractive platform for the tolerogenic expression of intracellular or secreted proteins in the liver with a substantially reduced risk of insertional mutagenesis.

Fig. 1

IDLV performance in hepatocytes culture (A) Schematic representation of the third-generation self-inactivating vector used for these studies. SD: splicing donor site. SA: splicing acceptor site. ψ: packaging signal (including 5' portion of GAG gene (GA)). RRE: Rev responsive element. cPPT: central polypurine tract. Wpre: woodchuck hepatitis virus post-regulatory elements. 142T: miR-142 target sequence made of 4 tandem copies of a sequence perfectly complementary to miR-142. Vectors were produced with integrase-competent (ICLV) or integrase-defective (IDLV) construct. The green fluorescent protein (GFP), coagulation Factor IX (FIX) and ovalbumin (OVA) were driven from the hepatocyte-specific ET promoter composed of synthetic hepatocyte-specific enhancers and transthyretin promoter or the ubiquitously expressed phosphoglycerokinase (PGK) promoter (B) Percentage of GFP⁺ cells and mean fluorescence intensity of GFP (MFI; left axis) and vector copies/diploid genome (vector copy number – VCN; right axis) in Huh7 cells transduced with ICLV or IDLV at the indicated multiplicity of infection (MOI) and analyzed 3 days or 2 weeks after transduction by flow cytometry. Black bars correspond to ICLV-transduced cells, grey bars to IDLV-transduced cells. Circles show VCN. The results are presented as mean ± standard error of the mean (SEM; n = 3). (C) Representative images of human primary hepatocytes transduced as indicated or left untreated (UNT) and analyzed by live fluorescence microscopy 1 week after transduction. Nuclei are stained with Hoechst. (D) Percentage of GFP⁺ cells and MFI of GFP (5 fields per sample; left axis) and VCN (circles, right axis) in cultures from quiescent human primary hepatocytes. The results are presented as mean ± range (n = 2). Abbreviations: MOI, multiplicity of infection; UNT, untreated.

Fig. 2

IDLV performance in the mouse liver (A) Morphometric analysis of GFP-expressing hepatocytes and VCN in the liver of mice injected with the indicated HIV-1 p24 Gag equivalents of IDLV.ET.GFP.142T and analyzed at the indicated time post-injection. GFP⁺ cells were identified either by immunostaining or direct fluorescence. Results are presented as mean ± SEM of n = 3 mice per time point, 5-10 optical fields scored from 5-10 non-consecutive GFP-immunostained (filled bars) or unstained (open bars) liver sections per mouse (left axis). Circles show VCN (right axis), as mean ± SEM. (B) Same analysis performed for IDLV- (grey bars) vs. ICLV- (black bars) injected mice, n = 3-7 mice per IDLV time point from 3 different experiments. (C) Representative images of unstained liver sections from mice reported in (B). Nuclei were stained with TOPRO-3. Abbreviation: UNT, untreated.

Fig. 3

Functional FIX expression in hemophilic mice FIX KO mice were injected with ICLV.ET.FIX.142T (A) or IDLV.ET. FIX.142T (B) and FIX activity was monitored in the mouse plasma collected by retro-orbital bleeding. Partial hepatectomy (PHX) was performed at week 12. In both panels, grey lines correspond to untreated (UNT) FIX KO mice. (C) FIX activity in FIX KO mice injected with IDLV.ET.FIX.142T monitored for 1 year post injection and not subjected to partial hepatectomy. Abbreviations: PHX, partial hepatectomy; UNT, untreated.

Fig. 4

Assessment of residual IDLV integration in vitro and in the liver (see also Supporting Figure 2-4 and Table 1) (A) Distribution of integration sites determined by LAM-PCR that were located in gene coding regions of ICLV- (black bars) or IDLV- (grey bars) transduced mouse SC-1 and C1498 cells (SC-1 cells: 533 IDLV IS, 2419 ICLV IS; C1498: 271 IDLV IS, 898 ICLV IS). (B) LTR deletions (red triangles) and genomic deletions (blue triangles) identified in IDLV and ICLV integrants from which both the 5' and 3' genomic flanking region were identified by LAM-PCR. The terminal CA dinucleotide (underlined, GT at end of 5' strand LTR, complementary to CA at end of 3' strand LTR) is shown if exist. The 5 bp direct repeat of host DNA flanking the provirus is delineated in bold. The number of deleted bp in host DNA is indicated (Del.). LTR: long terminal repeat. LV: lentiviral vector. B2-PCR product gel image for primer set 1 (C) and primer set 2 (D). The arrows indicate the position of the respective PCR bands; densitometric analysis of the B2-PCR bands received with primer set 1 (E) and primer set 2 (F) in the untreated (UNT), IDLV- and ICLV-treated liver samples. Statistically significant differences (*) were found between ICLV and IDLV (t-test: P < 10^-4 and P < 10^-11) as well as between UNT and IDLV (t-test: P < 0.002) with both primer sets 1 and 2, respectively. Abbreviation: UNT, untreated.

Fig. 5

Kinetics of liver lymphocytes infiltrate after IDLV gene transfer Mononuclear cells (MNC) were isolated from the liver of mice injected with matched doses of hepatocyte targeted ET.GFP.142T IDLV (IDLV.142T) or control PGK.GFP IDLV (IDLV) at the indicated time post-injection (IDLV.142T n = 20, IDLV n = 19, untreated n = 9 in 3 indipendent experiment) (A) Frequency of CD8⁺ T, (B) GFP_200-208-pentamer-positive, and (C) CD4⁺CD25⁺Foxp3⁺ Tregs infiltrating cells was evaluated by flow cytometry. Data are expressed as the mean % ± SD (n = 3 per group per time point; a representative experiment out of 3 is shown). (D) Ratio of T regs to CD8+ T cell. Abbreviation: CTL, cytotoxic T lymphocyte.

Fig. 6

Transgene-specific tolerance after IDLV liver gene transfer (A) Secondary immune response to the vector-encoded antigen was assessed by frequency of IFN-γ-producing, GFP-specific CD8⁺ T cells in the spleen of mice subjected to antigen re-challenge (by intramuscular vaccination with GFP-encoding plasmids) 6 weeks after IDLV treatment. Single values are plotted and the mean ± SD number of GFP-specific CD8⁺ T cells per 10⁶ total CD8⁺ T cells is shown (n = 6 per group). Representative wells are shown on top. (B) Quantification of the CD8⁺ GFP_200-208-pentamer-positive T cells infiltrating the liver of mice treated with the indicated IDLV (IDLV.PGK n = 9; IDLV.PGK.142T n = 6; IDLV.ET.142T n = 3) after antigen re-challenge. Single values are plotted and mean ± SD is shown. Mean ± SD VCN in the liver is indicated. (C) Morphometric analysis of GFP⁺ hepatocytes and VCN in the liver of immune-deficient mice previously injected with ET.GFP.142T ICLV and adoptively transferred (AT) with cells derived from naïve, IDLV.PGK- and IDLV.ET.142T-treated mice (from 5A-C). Mean ± SEM of n = 3 mice (left axis). Circles show mean ± SEM VCN (right axis). A representative field is shown on top. Note that residual vector DNA in the liver of mice in which GFP-expressing hepatocytes were completely cleared can be ascribed to the persistence of vector genomes in transduced macrophages and endothelial cells that did not express GFP. (D) Plasma samples from the various groups of animals were screened for anti-FIX neutralizing antibodies using an aPTT mixing assay using a positive control of 2.6 Bethesda Units (BU) and a known negative control. Samples were assayed in a blinded fashion. Groups include FIX KO mice treated with the indicated ET.FIX.142T vector or untreated (UNT) 12 weeks after vector administration, with or without challenge with canine FIX and IFA. Only untreated mice receiving FIX + IFA had detectible neutralizing anti-FIX antibodies and all were greater than 2.6 BU. Abbreviations: Ag, antigen; ns, not significant; UNT, untreated.

Fig. 7

Induction of transgene-specific Tregs by IDLV treatment CD4⁺ cells isolated from OTII Ly5.2 ^Foxp3-GFP transgenic mice were FACS-sorted to remove GFP⁺ cells obtaining an homogeneous population of CD4⁺ non-regulatory T cells with a unique antigen specificity (OVA_323-339 presented in IA^b molecule). (A) Tregs-depleted OTII CD4⁺GFP^- (2.5&times;10⁶/mouse) were adoptively transfer intravenously into naïve C57BL/6 Ly5.1 recipient mice one day before the injection of IDLV.PGK (n = 3) or IDLV.ET.142T (n = 3) encoding for OVA. Three weeks after IDLV administration livers were harvested and infiltrating lymphocytes isolated. OVA-specific induced Tregs (iTregs) were measured as GFP⁺ cells gated on CD4⁺Ly5.2⁺. (B) a representative histogram and (C) mean % induced Tregs ± SEM is reported.