Genome editing for scalable production of alloantigen‐free lentiviral vectors for in vivo gene therapy

Research Article23 August 2017Open Access Source DataTransparent process Genome editing for scalable production of alloantigen-free lentiviral vectors for in vivo gene therapy Michela Milani Michela Milani San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Andrea Annoni Andrea Annoni San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Bartolaccini Sara Bartolaccini San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Mauro Biffi Mauro Biffi San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Fabio Russo Fabio Russo San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Tiziano Di Tomaso Tiziano Di Tomaso San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Raimondi Andrea Raimondi IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Johannes Lengler Johannes Lengler Baxalta (former Baxter) Innovation GmbH, Vienna, Austria Search for more papers by this author Michael C Holmes Michael C Holmes Sangamo Therapeutics, Inc., Richmond, CA, USA Search for more papers by this author Friedrich Scheiflinger Friedrich Scheiflinger Baxalta (former Baxter) Innovation GmbH, Vienna, Austria Search for more papers by this author Angelo Lombardo Angelo Lombardo San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Alessio Cantore Alessio Cantore San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, ItalyThese authors share senior authorship Search for more papers by this author Luigi Naldini Corresponding Author Luigi Naldini [email protected] orcid.org/0000-0002-7835-527X San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, ItalyThese authors share senior authorship Search for more papers by this author Michela Milani Michela Milani San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Andrea Annoni Andrea Annoni San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Sara Bartolaccini Sara Bartolaccini San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Mauro Biffi Mauro Biffi San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Fabio Russo Fabio Russo San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Tiziano Di Tomaso Tiziano Di Tomaso San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Andrea Raimondi Andrea Raimondi IRCCS San Raffaele Scientific Institute, Milan, Italy Search for more papers by this author Johannes Lengler Johannes Lengler Baxalta (former Baxter) Innovation GmbH, Vienna, Austria Search for more papers by this author Michael C Holmes Michael C Holmes Sangamo Therapeutics, Inc., Richmond, CA, USA Search for more papers by this author Friedrich Scheiflinger Friedrich Scheiflinger Baxalta (former Baxter) Innovation GmbH, Vienna, Austria Search for more papers by this author Angelo Lombardo Angelo Lombardo San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy Search for more papers by this author Alessio Cantore Alessio Cantore San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, ItalyThese authors share senior authorship Search for more papers by this author Luigi Naldini Corresponding Author Luigi Naldini [email protected] orcid.org/0000-0002-7835-527X San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, ItalyThese authors share senior authorship Search for more papers by this author Author Information Michela Milani1,2, Andrea Annoni1, Sara Bartolaccini1, Mauro Biffi1, Fabio Russo1, Tiziano Di Tomaso1, Andrea Raimondi3, Johannes Lengler4, Michael C Holmes5, Friedrich Scheiflinger4, Angelo Lombardo1,2, Alessio Cantore1 and Luigi Naldini *,1,2 1San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy 2Vita Salute San Raffaele University, Milan, Italy 3IRCCS San Raffaele Scientific Institute, Milan, Italy 4Baxalta (former Baxter) Innovation GmbH, Vienna, Austria 5Sangamo Therapeutics, Inc., Richmond, CA, USA *Corresponding author. Tel: +39 02 2643 4681; E-mail: [email protected] EMBO Mol Med (2017)9:1558-1573https://doi.org/10.15252/emmm.201708148 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy. Synopsis Lentiviral vectors (LV) lacking major histocompatibility complexes (MHC) and with low vesicular stomatitis virus glycoprotein G (VSV.G) content escape immune recognition by human T cells and complement. Cell line-packaged LV offer scalable and consistent LV production for in vivo gene therapy. Site-specific integration of the LV genome into an inducible packaging cell line allows consistent generation of high-yield producers of the LV of interest. Cell line-produced LV achieve equivalent gene transfer in the mouse liver as LV produced by conventional transfection but are more stable in human sera, due to a lower VSV.G content. Genetic inactivation of beta-2 microglobulin (B2M) in LV producer cells allows production of MHC-free LV with preserved infectivity, devoid of immunogenic alloantigens in human recipients. Introduction Gene therapy has recently shown remarkable progress in clinical trials and promises to provide effective treatment for several genetic and acquired diseases (Naldini, 2015). Underlying this success is the development of improved gene transfer vectors (Kay, 2011; Mingozzi & High, 2011; Nathwani et al, 2014). Among them, lentiviral vectors (LV) are emerging as versatile vehicles of relatively large capacity for stable transgene integration in the genome of target cells. LV are currently exploited both for ex vivo gene therapy, in which target cells (such as hematopoietic stem/progenitors cells, HSPC or T cells) are harvested from the patient, transduced, and then re-infused, and for in vivo gene therapy, in which LV are directly injected into the patient, either into the bloodstream or in situ, such as in the brain (Palfi et al, 2014). Whereas several ongoing clinical trials support the efficacy and safety of LV for ex vivo gene therapy (Cartier et al, 2009; Cavazzana-Calvo et al, 2010; Aiuti et al, 2013; Biffi et al, 2013; Maude et al, 2014), in vivo liver-directed gene therapy with LV remains more challenging. Indeed, LV particles undergo a complex assembly with the outer envelope deriving from the membrane of packaging cells, thus comprising an array of proteins beside the viral antigens that may act as immune triggers upon recognition and phagocytosis by professional antigen presenting cells (APC; Annoni et al, 2013). Moreover, current manufacturing of LV mostly relies on transient plasmid co-transfection, which is labor-intensive and poorly amenable to scale-up and standardization, and leads to substantial amount of impurities, including residual plasmid DNA, which may further aggravate APC activation. In addition, the reported instability of vesicular stomatitis virus glycoprotein G (VSV.G)-pseudotyped LV in human serum remains a concern for in vivo administration (DePolo et al, 2000; Croyle et al, 2004; Trobridge et al, 2010; Hwang & Schaffer, 2013). We have previously reported that, upon intravenous administration, LV allow stable gene transfer to the liver, provided that transgene expression is stringently targeted to hepatocytes, and have shown dose-dependent therapeutic efficacy in a mouse and a canine model of hemophilia B, a coagulation disorder due to mutations in the factor IX (FIX)-encoding gene (Brown et al, 2006; Matrai et al, 2011; Cantore et al, 2012, 2015). Despite the therapeutic potential, some hurdles remain to be addressed before considering clinical translation of in vivo LV administration, such as the manufacturing of sufficiently large, consistent, and highly purified batches for in vivo delivery, the vector stability in the circulation, and the risk of acute toxicity and immunogenicity triggered by particle components or contaminants. Here, we describe an inducible scalable packaging cell line, which supports consistent generation of high-yield producers of LV of interest by a targeted integration strategy. LV produced by these cells achieve equivalent levels of gene transfer in the liver and are stable upon concentration and purification as LV produced by conventional transfection, but are more resistant to inactivation in human sera and lack plasmid DNA contaminants. Moreover, by further editing the genome of LV producer cells, we modified the protein composition of their plasma membrane and in turn of the LV envelope and obtained novel LV with enhanced capacity to escape immune recognition, which are better suited for in vivo applications. Results Reproducible generation of LV producer cell lines by targeted integration In order to avoid toxicity due to stable expression of viral components, we took advantage of a regulated, tetracycline (Tet)-dependent system, in which a Tet-regulated transcriptional repressor (Tet-R) binds to DNA sequences included in a promoter and represses transcription by steric hindrance (Yao et al, 1998; Jones et al, 2005). Upon addition of doxycycline (dox), Tet-R is released, allowing transcription. To generate LV producer cell lines, we thus started from a 293 cell line stably expressing Tet-R. We then stepwise transfected the plasmids expressing third-generation LV components, human immunodeficiency virus type 1 (HIV) Rev, Gag/Pol, and the VSV.G pseudotype (Dull et al, 1998), under the control of Tet-regulated promoters and coupled with antibiotic resistance cassettes (Fig 1A and B). We first introduced Rev, showed inducible Rev expression in 32/33 clones analyzed (Appendix Fig S1A), then pooled > 4,000 clones, and stably transfected Gag/Pol. We then screened clones for inducible LV particle production (Appendix Fig S1B), pooled the six highest expressing clones, and stably transfected VSV.G. We confirmed inducible Rev and VSV.G expression (Appendix Fig S1C) and pooled the highest expressing clones to obtain the packaging cell line. This line yielded 66 ± 14 ng of HIV Gag p24/ml after dox addition, with > 500-fold induction over a very low basal level (Fig 1C and Appendix Fig S1B), a productivity that, although in the lower range of that obtained by transient transfection, prompted us to continue development of this cell line. We found one copy of Rev, Gag, and VSV.G DNA per genome in the packaging cell line (Fig 1D), suggesting that integration site selection rather than copy accumulation played a role in the higher expression. We thus adopted site-specific integration as an efficient and reproducible means to introduce a full-length, self-inactivating (SIN)-LV genome transfer construct (Zufferey et al, 1998; Follenzi et al, 2000), and convert the packaging into a producer cell line (see Fig 1B). We targeted the adeno-associated virus site 1 (AAVS1), previously described as permissive to high-level stable expression and tolerant to integration, exploiting site-specific endonucleases and homology-directed repair (Lombardo et al, 2011). We included a GFP selector preceded by a splice acceptor site and a sequence encoding the self-cleaving 2A peptide after the LV 3′ long terminal repeat (Figs 1E and EV1A). Because integration occurs in the first intron of the PPP1R12C gene, GFP expression originates from the endogenous promoter (Lombardo et al, 2011), allowing selection of targeted cells. We performed three independent targeted integrations (T.I.) of LV constructs expressing GFP or human FIX, either wild-type (wt) or a codon-optimized hyper-functional version (FIX-Padua; Fig EV1A; Cantore et al, 2015), by transiently co-delivering zinc-finger nucleases (ZFN) targeting AAVS1 and the plasmid donor DNA. We achieved between 2 and 5% of GFP-positive cells, then enriched the GFP-positive cells by fluorescence-activated cell sorting (FACS), and obtained bulk and several single-cell-derived clones (n = 51) for each targeting (Fig EV1B). All clones analyzed (43/43 that grew well in culture among the 51 clones) were GFP positive, with some variation in mean fluorescence intensity (MFI) which was lower in those targeting in which GFP expression relied on splicing and 2A-mediated protein release, as expected (Fig EV1C). All clones except for one showed one copy of Rev, Gag, and VSV.G DNA per genome and no integration of ZFN DNA (Fig EV1D and E); the majority of the clones (44/51) presented the two expected AAVS1-LV genome junctions by PCR (Fig 1F), 34 clones (67%) had one LV copy per cell, 10 (20%) had two, four (8%) > 2, and three (6%) had none (Fig 1G and H). Together, these data show a high rate of mono-allelic on-target integration of this strategy, as previously reported for different purposes (Lombardo et al, 2011). Figure 1. Generation and molecular characterization of LV producer cell lines A. Schematic representation of the plasmids expressing third-generation LV packaging components (HIV Rev, Gag/Pol) and the surface glycoprotein of the vesicular stomatitis virus, VSV.G (pseudotype; Dull et al, 1998), coupled with antibiotic resistance cassettes, used to generate the LV packaging cell line. CMV-2xTetO2, immediate/early enhancer/promoter of cytomegalovirus (CMV) with two tetracycline operator elements (TetO2); BGH pA, bovine growth hormone polyadenylation signal; SV40, simian virus 40 promoter; SV40 pA, simian virus 40 polyadenylation signal; SD, splice donor site; SA, splice acceptor site. B. Flowchart of the generation of LV packaging and producer cell lines. Rev, Gag/Pol, and VSV.G-expressing plasmids were introduced into a 293 cell line stably expressing a tetracycline-regulated transcriptional repressor (293 T-REx; Yao et al, 1998) by subsequent rounds of transfection and antibiotic selection, to obtain the packaging cell line. Further genome engineering allows modifying the packaging cell line for the desired features. Targeted integration of a LV genome transfer construct allows consistent generation of producer cell lines of LV of interest. GOI, gene of interest. C. LV physical particle content (ng of HIV Gag p24/ml) in medium collected from the packaging cell line 3 days after dox induction. D. DNA copies of Rev (pink bar), Gag (gray bar) or VSV.G (blue bar) per diploid genome in the packaging cell line. E. Schematic representation of the plasmid used as donor DNA (pLV) for homologous recombination (top) to target the LV genome transfer construct into AAVS1 (bottom), which is found within the first intron of the PPP1R12C gene (see also Fig EV1A). Brown and light blue arrows represent the sequences homologous to the genomic target site. The HIV U3 region of the 5′ long terminal repeat (LTR) is replaced by the CMV promoter/enhancer allowing synthesis of the full-length RNA for packaging (Dull et al, 1998). The HIV enhancer/promoter was deleted from the 3′ LTR (ΔU3), thus obtaining SIN LV (Zufferey et al, 1998). Ψ, packaging signal; Prom, internal promoter; wpre, woodchuck hepatitis virus post-transcriptional regulatory element (Zufferey et al, 1999; Zanta-Boussif et al, 2009); 2A, porcine teschovirus-1 2A sequence. The black arrow shows transcription of the locus, and the brown and light blue arrows represent the primers used to detect the LV genome junctions. F–H. PCR analyses (F) for the 5′ and 3′ LV genome junctions generated by targeted integration (T.I.) of the donor DNA into the locus, or DNA copies of LV genome construct per diploid genome (G, H) in bulk GFP-positive (+) or GFP-negative (−) sorted populations and single-cell clones obtained from three independent T.I. experiments performed with the indicated donor DNA (see also Fig EV1A). (F) Red borders show images taken from different gels. Data information: In (C), data are presented as mean with standard error of the mean, SEM, of three independent inductions. Source data are available online for this figure. Source Data for Figure 1 [emmm201708148-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation of LV producer cell lines A. Schematic representation of the plasmid used as donor DNA (pLV) for homologous recombination to target the LV genome transfer constructs used in this work into the AAVS1 (see also Fig 1E). Brown and light blue arrows represent the sequences homologous to the genomic target site, respectively. PGK, phosphoglycerate kinase promoter; ET, enhanced transthyretin promoter (Cantore et al, 2015); FIX, coagulation factor IX, co-FIX-Padua, codon-optimized hyper-functional FIX transgene (Cantore et al, 2015); 142T, four tandem repeats of microRNA 142 target sequences (Cantore et al, 2015). The brown and light blue arrows represent the primers used to detect the LV genome junctions (see Fig 1F). B. Percentage of GFP-positive cells (mean with range or single value, n = 1–2), in LV packaging cell line transiently transfected with the indicated amount of ZFN-expressing plasmid and 1 μg of the indicated donor plasmid and analyzed by flow cytometry, 2 weeks after transfection. C. Percentage of GFP-positive cells (black bars, left y-axis) and MFI (gray bars, right y-axis) in bulk GFP-positive (+) or GFP-negative (−) sorted populations and single-cell clones obtained from three independent T.I. experiments performed with the indicated donor DNA, analyzed by flow cytometry at least 1 month after sorting. D, E. DNA copies of Rev (pink bars), Gag (gray bars), or VSV.G (blue bars) per diploid genome (D) and ZFN copies (DNA copies of FOK) per diploid genome (E) in untreated 293T cells (UNT), bulk GFP-positive (+) or GFP-negative (−) sorted populations and single-cell clones obtained from three independent T.I. experiments performed with the indicated donor DNA. Download figure Download PowerPoint Robust and scalable LV production by stable producer cell lines We measured infectious titer, physical particles, and specific infectivity of LV-containing supernatant of dox-induced bulk-sorted positive populations and single-cell clones, selected for robust growth rate, for the three T.I. experiments. At peak production, 3 days after induction (Fig EV2A and B), we found on average 1.5 × 106 transducing units (TU)/ml, 56 ng/ml p24 equivalents, 3 × 104 TU/ng p24, reaching up to 4.4 × 106 TU/ml, 222 ng p24/ml, and 8 × 104 TU/ng p24 (Fig 2A). The infectivity of cell line-produced LV was in the lower-bound range of that obtained for LV produced by transient transfection in our standard conditions. We compared the LV output of several different inductions of the bulk-sorted LV-GFP producer cells over the course of 1 year and observed that productivity is maintained also after a freeze/thaw cycle and even without antibiotic selective pressure (Fig 2B). Lentiviral vectors production was scaled in cell factories up to 6 liters with similar or higher output in the collected conditioned medium than measured in small-scale experiments (Table 1), and this medium was processed by our previously reported two-step chromatography purification (Biffi et al, 2013), giving the expected final yield of vector. Specific infectivity was maintained both throughout the purification process and after concentration by ultracentrifugation (Fig EV2C). Concentrated LV was stable at −80°C after 10 months of storage (Fig EV2D). Overall, these data indicate comparable stability of LV particles produced by the cell line or by transient transfection. We found undetectable titer (< 100 TU/ml) and 120 pg p24/ml in medium collected from the LV-GFP producer cell line in the absence of dox (see also Appendix Fig S1B), suggesting that the low amount of LV particles produced are not infectious, possibly because the low levels of Rev in the non-induced condition limit nuclear exports of full-length LV genome for encapsidation. These data are consistent with the reported low leakiness of the Tet-R system and support the long-term stability observed for the cell line, which may be protected from the toxicity of viral proteins and from LV superinfection in the non-induced state. Overall, these data show that single-copy integration into AAVS1 mediates robust transcription of the LV genome and the generation of highly infectious vector particles. Click here to expand this figure. Figure EV2. Yield and stability of cell line-produced LV A, B. LV infectious titer (TU/ml, black line, plotted on left y-axis), physical particles (ng p24/ml, dashed line, plotted on right y-axis) and specific infectivity (TU/ng p24, gray line, plotted on left y-axis) in (A) conditioned medium of LV-GFP producer cell line, collected at the indicated day after dox induction (n = 1 per time point), and in (B) conditioned medium of LV-GFP producer cell line induced at the indicated dox concentration, collected 3 days after induction (n = 3 per dox concentration, mean with SEM). C. Percentage of recovery of infectivity (single values and mean with SEM) upon ultracentrifugation of LV produced by cell lines (circles, n = 18) or of LV produced by transient transfection (squares, n = 17). D. Infectious titer (TU/ml; single values and mean or mean with range, n = 1–2) of two concentrated LV produced by LV-GFP producer cell line, determined at the indicated time (months) upon storage at −80°C. Download figure Download PowerPoint Figure 2. Evaluation of LV produced by the producer cell lines A, B. LV infectious titer (TU/ml, black bars or line, plotted on left y-axis), physical particles (ng p24/ml, dashed bars or line, plotted on right y-axis), and specific infectivity (TU/ng p24, gray bars or line, plotted on left y-axis) in conditioned medium of (A) bulk GFP-positive (+) sorted populations and single-cell clones obtained from three independent T.I. experiments performed with the indicated donor DNA, 3 days after dox induction, or (B) at the indicated time (months) of continuous culture in the absence of antibiotic selective pressure. Axis interruption indicates a freeze/thaw cycle. The n of independent inductions of LV production from bulk-sorted populations (+) or single-cell clones is shown on top of the bars in panel (A), when not 1. C–F. Percentage of GFP-positive cells (C, D) and VCN (E, F) in the CD34-positive cells culture (C, E) or pooled colonies (D, F) from CFC assays (MOI 10 and 100, n = 4 transductions in 2 independent experiments using three different LV batches per production method; MOI 300, n = 2 transductions). HSPC (n = 4 healthy cord blood donors) were transduced with LV produced by transient transfection ("transfection", white squares) or by LV-GFP producer cell line ("cell line", black circles) at the indicated MOI and analyzed at 7 (in C) or 14 (in D–F) days after transduction. G, H. Percentage of GFP-positive cells (G) and VCN (H) in T lymphocytes transduced and analyzed as in (A–D) ("cell line", black circles, n = 4 transductions using two different LV batches; "transfection", white squares, n = 2 transductions). I, J. Human FIX expression (% of normal) in the plasma over time (I) and VCN (J) in liver DNA of hemophilia B mice treated with LV-FIX produced by transient transfection (n = 4, white squares) or from producer cell line (n = 10 using two different LV batches, black circles). No significant differences. Data information: In (A–J), data are presented as mean with SEM (for n ≥ 3), mean with range (for n = 2), and/or single values. Significance was assessed by Mann–Whitney test in (C–F) and (J) or by two-way ANOVA for repeated measures in (I). Download figure Download PowerPoint Table 1. Purification of cell line-produced LV Titer Particles Infectivity Volume Total TU/ml ng p24/ml TU/ng p24 ml TU ng p24 Initial 4.2 × 106 396 1.1 × 104 6,000 2.5 × 1010 2.4 × 106 Final 1.1 × 108 10,207 1.1 × 104 64.5 7.2 × 109 6.6 × 105 Yield – − − − 29% 28% The table shows infectious titer, physical particles, and specific infectivity of (i) LV-containing conditioned medium collected from LV-FIX-Padua producer cells 3 days after dox addition (initial material) and (ii) concentrated purified LV formulated in saline solution at 5% dimethyl sulfoxide (final product), according to our previously reported process (Biffi et al, 2013; Cantore et al, 2015). Note that the yield of the process is in line with results previously reported for LV produced by conventional transient transfection (Aiuti et al, 2013; Biffi et al, 2013) and that the recovery in specific infectivity is 100%. We then transduced human cord blood-derived HSPC with concentrated LV produced by the two most productive clones of the LV-GFP producer cell line, or by transient transfection. We observed a LV dose-dependent increase in the percentage of transduced cells and vector copies per diploid genome (vector copy number, VCN), reaching up to 45% GFP-positive and 1.9 VCN in the cultured cells, 70% GFP-positive cells and 2.8 VCN in CFC assay, at the highest multiplicity of infection (MOI) of the cell line-produced LV, and a two- to fivefold lower dose–response than observed for transient transfection LV (Fig 2C–F). The percentage of erythroid (CD235a-positive) and myeloid (CD33-positive) cells among the total CFC did not change significantly among the different transductions (Fig EV3A–C). The lower transduction efficiency of cell line-derived than transient transfection-derived LV at matched MOI likely reflects the lower specific infectivity of the former vector. However, the cell line-produced LV still allowed reaching clinically relevant VCN in the HSPC (Aiuti et al, 2013; Biffi et al, 2013). We also transduced activated primary human T cells and achieved > 90% transduction and VCN > 5 at the highest MOI of LVs produced by either method (Fig 2G and H). We did not observe any skewing in the CD4/CD8 ratio among the transduced lymphocytes at any tested dose (Fig EV3D–F). We then intravenously administered 4.5 × 108 TU/mouse of LV-FIX derived either from the two most productive cell line clones or from transient transfection to hemophilia B mice and observed stable reconstitution of circulating FIX at 50–100 ng/ml and 1.5 VCN in the liver of treated mice (Fig 2I and J). Importantly, we did not detect any difference in FIX expression or VCN in mice treated with LV produced by either method. Click here to expand this figure. Figure EV3. HSPC and T-cell transduction A. Gating strategy for HSPC and CFC population analysis. FMO: fluorescence minus one; arrows indicate sub-gating from the previously gated population. B, C. Mean with SEM (MOI 10 and 100, n = 4) or range (MOI 300, n = 2) of the CFC composition in (B) erythroid progenitors (CD235a-positive cells) and (C) myeloid progenitors (CD33-positive cells), 2 weeks after