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original article

Lentiviral Vector Gene Therapy Protects XCGD Mice From Acute Staphylococcus aureus Pneumonia and Inflammatory Response Giada Farinelli1, Raisa Jofra Hernandez1, Alice Rossi2, Serena Ranucci2, Francesca Sanvito3, Maddalena Migliavacca1,4, Chiara Brombin5, Aleksandar Pramov5, Clelia Di Serio5, Chiara Bovolenta6, Bernhard Gentner1,7, Alessandra Bragonzi2 and Alessandro Aiuti1,4,8 1 San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), San Raffaele Scientific Institute, Milan, Italy; 2Infection and Cystic Fibrosis Unit, San ­ affaele Scientific Institute, Milan, Italy; 3Pathology Unit, San Raffaele Scientific Institute, Milan, Italy; 4Pediatric Immunohematology and Bone Marrow R ­Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy; 5CUSSB-University Center Center for Statistics in the Biomedical Sciences, Vita-Salute San Raffaele University, Milan, Italy; 6MolMed S.p.A., Milano, Italy; 7Haematology and Bone Marrow Transplantation Unit, San Raffaele Scientific ­Institute, Milan, Italy; 8Vita-Salute San Raffaele University, Milan, Italy, Italy

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. CGD patients are characterized by an increased susceptibility to bacterial and fungal infections, and to granuloma formation due to the excessive inflammatory responses. Several gene therapy approaches with lentiviral vectors have been proposed but there is a lack of in vivo data on the ability to control infections and inflammation. We set up a mouse model of acute infection that closely mimic the airway infection in CGD patients. It involved an intratracheal injection of a methicillin-sensitive reference strain of S. aureus. Gene therapy, with hematopoietic stem cells transduced with regulated lentiviral vectors, restored the functional activity of NADPH oxidase complex (with 20–98% of dihydrorhodamine positive granulocytes and monocytes) and saved mice from death caused by S. aureus, significantly reducing the bacterial load and lung damage, similarly to WT mice even at low vector copy number. When challenged, gene therapytreated XCGD mice showed correction of proinflammatory cytokines and chemokine imbalance at levels that were comparable to WT. Examined together, our results support the clinical development of gene therapy protocols using lentiviral vectors for the protection against infections and inflammation. Received 4 April 2016; accepted 17 July 2016; advance online publication 6 September 2016. doi:10.1038/mt.2016.150

INTRODUCTION

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by defects in one of the subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase

complex, crucial for the reactive oxygen species (ROS)-dependent killing of microorganisms.1–4 XCGD is caused by mutations in the CYBB gene encoding the gp91phox subunit of the NADPH oxidase complex and presents earlier and more severe manifestations than the autosomal recessive form, AR-CGD.5 The disease is characterized by an unusual predisposition to infections, resulting in severe recurrent bacterial and fungal infections and granuloma formation due to an abnormal inflammatory response.2,6 Typical clinical manifestations include pneumonia, inflammation of the gastrointestinal tract, lymphadenitis, liver abscess, and osteomyelitis.7,8 The microorganisms responsible for the majority of infections in CGD are Staphylococcus aureus, Gram-negative enteric bacilli (including Serratia marcescens, Salmonella species, and Burkholderia cepacia), and the Aspergillus species.9–11 Microorganisms are normally phagocytosed, but in the absence of effective elimination they can persist within cells and cause infection. Conservative treatment is based on lifelong antibiotic and antifungal prophylaxis but mortality remains high.8 The only available cure is hematopoietic stem cell transplant (HSCT) that has shown an improved outcome in patients with a human leukocyte antigen (HLA)-compatible donor.12 Nevertheless, HSCT remains associated with the risk of Graft versus Host Disease (GvHD), toxicity and infections.12 Gene therapy with autologous hematopoietic stem and progenitor cells (HSPCs) may represent a definitive cure for patients without a HLA-matched donor or who cannot receive HSCT due to severe chronic infections or steroid-resistant chronic inflammations.13 Previous clinical trials with retroviral vectors (RVs) for CGD showed a transient clinical benefit followed by low or short-term engraftment14,15 or insertional mutagenesis and methylation of the promoter.16–18 Our group and others have developed alternative gene therapy strategies based on lentiviral vectors (LVs) under the control of various myeloidspecific promoters or miRNA 126-based detargeting, resulting in post-transcriptional downregulation of gp91phox expression by miR-126 found in high levels in HSC but not in the myeloid progeny.19–21 With these vectors, gp91phox expression is targeted to the

The last two authors shared equal senior authorship. The study was conducted in Milan, Italy. Correspondence: Alessandro Aiuti, San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. E-mail: [email protected] Molecular Therapy  vol. 24 no. 10, 1873–1880 oct. 2016

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differentiated myeloid cells to reduce the risk of genotoxicity and perturbation of reactive oxygen species levels in HSCs.22–25 Those vectors showed a restored gp91phox in differentiated human or mouse cells; however, the preclinical efficacy of gene therapy, in terms of protection against infections and inflammatory responses, has not been studied yet. We established a model of S. aureus airways infection in XCGD mice to evaluate the in vivo antimicrobial efficacy and beneficial effect on the inflammatory response of gene therapy. After infection with a methicillin-sensitive reference strain of S.  aureus (MSSA), XCGD mice showed acute pneumonia, increased production of inflammatory cytokines, and higher mortality than WT mice. The disease course and the inflammatory response in LV gene therapy-treated XCGD mice was as mild as it was in the WT control mice, indicating that gene therapy effectively controls S. aureus infection. Taken together, our results would support the clinical development of gene therapy protocols using lentiviral vectors to protect against infections and inflammation.

RESULTS Establishment of a model of S. aureus acute airways infection in XCGD mice

To determine suitable conditions for inducing appropriate acute airway infection, we challenged XCGD (n = 8) and age-matched WT (n = 9) untreated mice with two different doses of S. aureus methicillin-sensitive (MSSA), reference strain Newman, via intratracheal injection. After infection, mortality and change in body weights were monitored daily for up to 6 days (Figure 1a). In XCGD mice, infection with the highest dose (2 × 107 colonyforming units (CFU)) (n = 4) led to 100% mortality within 3 days (72 hours) while the lowest dose (1 × 107 CFU) (n = 4) resulted in 50% of the animals affected by a lethal infection within 6 days (Supplementary Figure S1a; Supplementary Table S3). By contrast, all WT mice survived the infection (n = 5 treated with 2 × 107 CFU and n = 4 treated with 1 × 107 CFU). By using a linear mixed effect model, the loss of weight was significantly higher for XCGD 1 × 107 -treated group than for the WT 1 × 107 -treated group, particularly during the first 3 days, and XCGD mice gained less weight compared to the WT group (Supplementary Figure S1b). Six days after the infection, surviving mice were sacrificed and murine lungs were homogenized and plated to quantify bacterial load (Supplementary Figure S1c). We observed a massive increase in bacterial load in the XCGD mice infected with 1 × 107 CFU compared to WT mice at both doses, confirming a severe defect in S. aureus clearance in XCGD mice. These data indicate that S. aureus infection is more severe in the XCGD group than in the WT mice, mimicking hallmarks of the human disease (Supplementary Table S4).

Gene therapy with regulated vectors effectively clears S. aureus infection To test the efficacy of gene therapy in vivo, 8-week-old XCGD mice were initially treated with HSPC transduced with regulated lentiviral vectors in which the transgene gp91phox is driven by a myeloid-specific promoter23,26 (MSP) with (MSP.gp91_126T(2), n = 9) or without (MSP.gp91, n = 5) a miRNA126 detargeting element.23 Consequently, Lineage negative (Lin−) cells harvested from bone marrow (BM) of XCGD mice were transduced with the therapeutic vectors and transplanted into lethally irradiated recipients. 1874

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All gene therapy-treated mice were bled at different time-points and checked for the expression of gp91phox and the correction of the NADPH oxidase complex activity (Figure 1b). Ten weeks after gene therapy, NADPH oxidase activity was restored in a 20– 98% range of Dihydrorhodamine (DHR)-positive granulocytes and monocytes and the correction was correlated to the vector copy number (VCN) ranging between 0.33 and 9.80 copies per cell (Supplementary Figure S2). Gene therapy mice were infected with 1 × 107 CFU of S. aureus MSSA Newman strain via intratracheal administration 13 weeks after gene therapy. As a control, we infected untreated WT (n = 15), XCGD (n = 13) mice and XCGD mice previously mock transplanted with untransduced WT (bone marrow transplanted (BMT) WT, n = 7) or XCGD HSPC (XCGD MOCK, n = 4). After infection, mice were monitored daily for survival and body weights for up to 5 days (Figure 1b). S. aureus infection was lethal in 60% of XCGD and 50% of XCGD MOCK mice within 5 days (Figure 2a). By contrast, all gene therapy-treated mice and WT control mice survived after the infection, indicating that the restoration of gp91phox expression and the correction of the NADPH oxidase activity effectively recovered the ability to control infection (Figure 2a ; Supplementary Table S1). Unlike XCGD mice, all gene therapy-treated, WT and BMT WT mice gained weight from 2 days after infection (Figure  2b; Supplementary Table S2). The fitted Linear Mixed Effects model showed that for all the treatment groups, apart from XCGD MOCK, the estimated loss of weight was significantly lower than the reference group XCGD (Figure 2b). Five days after infection, surviving mice were sacrificed and the bacterial load was assessed in broncho-alveolar lavage (BAL) and in lung homogenate. Bacterial load in BAL and lung homogenate of XCGD and XCGD MOCK mice were similar (range between 104–105 CFU) and significantly higher than in the groups of gene therapy-treated and WT mice (Figure 3a). Consequently, gene therapy effectively restored the ability of XCGD mice to control the infections, even at low VCN. BAL cytology showed an increased neutrophil count in XCGD and XCGD MOCK mice, whereas in gene therapy-treated and in BMT WT mice, the neutrophil count was comparable to WT mice (Figure 3b). This result illustrates the inability of CGD macrophages to clear neutrophils recruited into tissue during inflammation, and the consequent production of inflammatory signals for the recruitment of more neutrophils.

Histopathological analysis of lungs after S. aureus infection To better characterize the histopathological features of infected lungs, microscopic analysis was performed on formalin-fixed, paraffin-embedded specimens 5 days after infection (Figure 4). Lungs of XCGD-untreated mice typically present acidophilic macrophagic pneumonia (AMP), bronchiectasis, and marked and diffused inflammatory cell infiltrates.27 Inflammatory cell infiltrates in XCGD mice, mainly represented by lymphocytes, monocytes and neutrophils, were observed around blood vessels and bronchi; occasionally micro-abscesses were observed in the parenchyma suggesting that these lungs had extensive tissue damage. By contrast, lungs of gene therapy-treated mice and WT mice showed focal and less prominent bronchiectasis and inflammatory cell infiltrates. Inflammatory www.moleculartherapy.org  vol. 24 no. 10 oct. 2016

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Figure 1 Experimental plan. (a) XCGD and WT mice were injected intratracheally at 21 weeks, with S. aureus Newman strain at different doses (1 × 107 CFU and 2 × 107 colony-forming units (CFU)). Survival and weight changes were monitored daily. Surviving mice were euthanized 6 days after the infection and the lungs were harvested, homogenized, and cultured to determine the bacterial load. (b) Eight-week-old XCGD mice were treated with HSPCs transduced with different lentiviral vectors and bled at 14 and 18 weeks for DHR and VCN. At week 21, gene therapy-treated XCGD mice, XCGD and WT were injected intratracheally with S. aureus Newman strain (1 × 107 CFU). Survival and weight changes were monitored daily. Surviving mice were euthanized 5 days after the infection and broncho-alveolar lavage fluid was recovered. The lungs were harvested and either homogenized and cultured to determine the bacterial load and cytokine levels or subjected to histopathological analysis.

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infiltrates were mainly perivascular and peribroncheal and predominantly represented by lymphocytes and macrophages (Figure 4).

Correction of cytokine and chemokine imbalance in gene therapy-treated S. aureus-infected mice To better characterize the airway inflammatory response and the effect of gene therapy treatment, we measured the concentration of a panel of cytokines and chemokines in murine lung homogenates 5 days after infection. Results show that the levels of pro-inflammatory cytokines (IL-1α, IL-1β, and MIP1α) in lung homogenates of XCGD mice were significantly higher than those in WT and BMT WT mice, and a similar trend was observed for XCGD MOCK (Figure 5). By contrast, all gene therapy-treated mice showed a normalization of proinflammatory cytokines to levels comparable to WT mice and consistent with Molecular Therapy  vol. 24 no. 10 oct. 2016

the amelioration observed in these mice after infection. Levels of other cytokines, including IL-6, KC, granulocyte-colony stimulating factor (G-CSF), and MIP-1β were increased in XCGD and in XCGD MOCK mice, but differences did not reach statistical significance when compared to WT mice. Also in this case, gene therapy-treated mice showed lower levels of all proinflammatory cytokines and chemokines analyzed similarly to WT and BMT WT mice, confirming that gene therapy was efficacious in controlling inflammatory responses.

DISCUSSION

Lentiviral vector gene therapy could overcome the hurdles of transient efficacy and insertional mutagenesis observed in previous clinical trials for XCGD based on retroviral vectors. However, preclinical evidence of the ability to control infection and inflammation 1875

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Figure 3 Bacterial load and broncho-alveolar lavage (BAL) cytology after S. aureus Newman acute infection. (a) The graph shows the bacterial load determined in the BAL fluid and in lung homogenate lungs. (b) The graph shows the content of total neutrophils, macrophages and lymphocytes recovered in the BAL fluid. Statistics shown in the figures represent the Kruskal Wallis test (nonparametric test with Dunns multiple comparison test; ***P < 0.001, **P < 0.05, *P < 0.01). WT

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Figure 4 Lung histopathological analysis following S. aureus infection. Images show hematoxylin- and eosin-stained lung sections of infected mice (5×). Scale bar = 200 microns.

is lacking. We developed an in vivo model with XCGD mice that were highly sensitive to S. aureus lung infection, one of the most frequent infection routes in humans. Most of the mice died from pneumonia but those that survived lung infection developed a hyper-inflammatory response mimicking findings typical of the 1876

human phenotype. Previous studies described an enhanced susceptibility to infection of A. fumigatus, B. cepacia, or S. aureus in XCGD mice but to date none of these models has been used to study protection against acute respiratory infection after LVs gene therapy.28–32 Dinauer’s group first showed that XCGD mice treated with retroviral www.moleculartherapy.org  vol. 24 no. 10 oct. 2016

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Figure 5 Cytokine and chemokine responses to S. aureus Newman acute infection. Cytokine and chemokine expression levels were assessed in lung homogenates obtained 5 days from infection. Results are shown as mean ± standard error of the mean. Statistics shown in the figures represent the Kruskal Wallis test (nonparametric test with Dunns multiple comparison test; *P < 0.01).

vectors (RVs) were able to control the acute inflammation and the development of chronic granulomatous lesions of CGD, induced by intradermal injection of Aspergillus fumigatus (AF) hyphae.33 We found that mice treated with gene therapy using regulated LVs were protected from the lethal disease and, similarly to WT mice, had no further consequences. Moreover, gene therapy-treated XCGD mice were able to control lung damage and inflammations induced by S. aureus intratracheal acute infection. Notably, the mouse model of respiratory infection, induced by intratracheal injection and used in our work, differs from the models generated previously that reproduced the systemic infection with S. aureus introduced by an intraperitoneal injection.30 In our model of acute pneumonia, it should be pointed out that mice with DHR activity as low as 20% or with a low vector copy number of 0.3 were protected from death and inflammation. These results would suggest that a reduced-intensity conditioning, such as the one adopted for previous CGD trials or other diseases, could be used.34 Our study included a wide distribution of Molecular Therapy  vol. 24 no. 10 oct. 2016

VCN (ranging from 0.3 to 9.8). While these VCNs are in line with previous studies from our group and others36–38 it is expected that lower VCNs would be observed in the clinical application based on our previous studies in human CD34+ cells,23 and clinical trials in other therapeutic areas.34,39 Unfortunately, no published data is available regarding the protection from the infections in clinical trials with lentiviral vectors encoding for gp91phox. In a previous retroviral gene therapy trial, two patients cleared infections with 4–25% oxidase activity and are clinically well with very low persistent marking level (0.03 and 0.7%) while another succumbed to the infection after losing gene marking.35 The bacterial load quantified in lung homogenates of mice treated with gene therapy lies within the range of observed in the WT infected mice, indicating that the clearance of S. aureus after 5 days from the infection was similar in GT-treated and WT mice. A distinctive component of CGD is “sterile inflammation”, that is often unrelated to infection; this may lead to an exaggerated 1877

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inflammatory response due to a dysregulation of proinflammatory cytokines levels and defects in autophagy.40,41 In a recent paper, Bagaitkar and colleagues identified how NADPH oxidase deficiency in mice can exacerbate neutrophilic inflammation by enhancing the release of IL-1α and promoting an excessive G-CSF regulated neutrophilic response following a peritoneal inflammation elicited by tissue injury.42 This excessive IL-1a/G-CSF response is the major driver of enhanced sterile inflammation in CGD patients and could lead to a dysregulated hematopoietic homeostasis that might contribute significantly to the engraftment failure of modified HSCs.43 In our study, analysis of proinflammatory cytokine and chemokine expression levels showed that in surviving XCGD mice, IL-1α, IL-1β, and IL-6 were substantially increased after the infection and a similar tendency was observed in XCGD mice that were transplanted with nontransduced HSCs (MOCK mice). These cytokines are produced by monocytes and macrophages and they participate in the regulation of immune responses and inflammatory reactions that play a key role in the acute phase responses.44 KC (keratinocyte chemoattractant), a powerful neutrophil chemoattractant, was also increased in X-CGD, in accordance with the high number of neutrophils in the lungs of these mice. G-CSF, MCP-1, MIP-1α, and MIP-1β levels were also increased in XCGD and this might explain the acute inflammatory state in the recruitment and activation of inflammatory cells in these untreated mice (Figure 3b and 4). By contrast, all gene therapytreated mice were able to control the expression of proinflammatory cytokines and chemokines to levels observed in WT and BMT WT mice. This demonstrates the efficacy of gene therapy in controlling inflammation, and restricting proinflammatory cytokine expression levels. A continuous inflammatory status may contribute to alteration of the HSC composition and/or HSC engraftment in gene therapy patients, suggested as a factor that contributed to low HSC engraftment in previous RV gene therapy trials. In this regard, chronic models of inflammation in XCGD mice, similarly to cystic fibrosis, may provide information useful for the design of clinical studies, including preparatory regimens to dampen inflammation at HSC collection or transplantation.45 Finally, our data further validate the use of regulated vectors based on transcriptional or post-transcriptional regulators that allow the adequate expression of a functional transgene while preventing ectopic expression in undifferentiated HSCs.23 Taken together, our results provide a useful and manageable model that mimics the hallmarks of human disease and demonstrates robust preclinical evidence of efficacy and supports the progression to the clinical settings of gene therapy with regulated lentiviral vectors.

MATERIALS AND METHODS

Ethics statement. Animal studies were conducted according to protocols

approved by the San Raffaele Scientific Institute and Institutional Animal Care and Use Committee (IACUC, Number 673) and adhered strictly to the Italian Ministry of Health guidelines for the use and the care of experimental animals. All efforts were made to minimize the number of animals used and their suffering.

Mice. B6.129S6− Cybbtm1Din/J mice, Ly5.2 (XCGD) and C57BL/6N (Wild-

Type) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) or Charles River (Calco, Italy) and maintained in specific pathogen-free conditions at San Raffaele Scientific Institute SPF Animal Facility.

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Isolation and transduction of murine HSPC. Murine BM samples were harvested from the femurs and tibias of B6.129S6- Cybbtm1Din/J mice, Ly5.2 (The Jackson Laboratory) and from C57BL/6-LY5.1 (Charles River, Calco). Lin− progenitors were isolated using the Lineage Cell Depletion Kit (Miltenyi, Germany) following manufacturer’s instructions. Lin− cells were cultured in StemSpan serum-free medium for 12/24 hours (StemCell Technologies, Vancouver, Canada) supplemented with 1% l-glutamine, 1% penicillin/streptomycin and full cytokine cocktail 100 ng/ml murine stem cell factor (SCF) (mu SCF), 50 ng/ml mu TPO, 100 ng/ml hu Flt3L, and 20 ng/ml hu IL-3 (Peprotech, Germany). After prestimulation, cells were transduced at multiplicity of infection 200 for 16 hours and after that 5 × 105 cells were administered in the tail vein (in 200 μl saline solution) of XCGD mice irradiated at 900 RAD.23 Multiplicity of infection is the average number of virus particles infecting each cell and was calculated as follows: multiplicity of infection = plaque forming units (pfu) of virus used for infection/number of cells per ml. Lentiviral vectors production. Vectors used in this study are a third gener-

ation, self-inactivating vesicular stomatitis virus (VSV.G) pseudotyped lentiviral vector constructed on a pCCL backbone, previously described.46,47 The vector lots were produced and purified by MolMed s.p.a. (Milan, Italy) using a large-scale validated process following GMP protocols.

Analyses of vector copy number. Cells were cultured for 14 days after transduction allowing elimination of nonintegrated vector forms. Genomic DNA was extracted by using QIAamp DNA Blood mini kit or micro kit (QIAGEN, Hilden, Germany), according to manufacturer’s instructions. Vector copy numbers per genome (VCN) were quantified by quantitative real-time PCR (Q-PCR) using the following primers for HIV: HIV fwd: 5′-TACTGACGCTCTCGCACC-3′; HIV rev 5′TCTCGACGCAGGACTCG-3′) and probe (FAM 5′-ATCTCTCTCC TTCTAGCCTC-3′) against the primer binding site (PBS) region of LV. Endogenous DNA amount was quantified by a primer/probe set against the murine β-actin gene (Act fwd: 5′-AGAGGGAAATCGTGCGTGAC-3′; Act rev: 5′-CAATAGTGATGACCTGGCCGT-3′; probe: VIC 5′-CAC TGACGCATCCTCTTCCTCCC-3′). Copies per genome were calculated by the formula: (copies LV/copies endogenous DNA)/(n° of housekeeping gene copies in the standard curve). The standard curve was generated by using a CEM cell line (CEMA 301#25) stably carrying one vector integrant (MolMed, Italy). DNA isolated from CEMA 301#37 cell line (MolMed, Italy) carrying four copies of vector integrant was used as a positive control. All reactions were carried out in duplicate in a VIIA7 (Thermo Fisher Scientific, Waltham, MA). Bacterial strain. S. aureus MSSA reference strain Newman was inoculated

in tryptic soy broth (Becton Dickinson, Sparks, MD). Prior to animal experiments, Newman was grown for 3 hours to reach the exponential phase. Next, the bacteria were pelleted by centrifugation (2,700 g, 15 minutes, 4 °C), washed twice with sterile PBS and the OD of the bacterial suspension was adjusted by spectrophotometry at 600 nm.48 The intended number of CFU was extrapolated from a standard growth curve. Appropriate dilutions with sterile PBS were made to prepare the inoculum of 1–2 × 107 CFU/ml.

Mouse model of acute lung infection. To perform acute lung infection in XCGD and WT mice, mice were anesthetized by an intra-peritoneal injection of a solution of 2.5% Avertin (2,2,2-tribromethanol 97%, Sigma Aldrich, St. Louis, MO) in 0.9% NaCl and administered in a volume of 0.015 mlg−1 body weight as described previously.49 Trachea was directly visualized by a ventral midline incision, exposed and intubated with a sterile, flexible 22-g cannula (Becton Dickinson, Italy) into the lung attached to a 1 ml syringe. Infection was performed using 1–2 × 107 CFU of S. aureus Newman strain. After infection, mortality and body weight were monitored in one group of mice over 5 days. In another group of mice, lungs were excised and used for histopathology or excised after BAL collection, homogenized and plated onto TBS-agar plates for CFU counting.50 www.moleculartherapy.org  vol. 24 no. 10 oct. 2016

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BAL collection and analysis. BAL was collected with a 22-g venous catheter by washing the lungs with 3 ml of RPMI-1640 (Euroclone, Milan, Italy) with protease inhibitors (Complete tablets, Roche Diagnostic, Basel, Switzerland) as previously described.50 Total cells present in the BAL were counted, and a differential cell count was performed on cytospins stained with Diff Quick (Dade, Biomap, Italy). BAL was serially diluted and plated on tryptic soy broth-agar plates. Lung homogenization and cytokine analysis. Lungs were removed and homogenized in PBS with Ca2+/Mg2+ containing protease inhibitors. Samples were serially diluted and plated on the above agar media for CFU counts. Lung homogenates were then centrifuged at 14,000 rpm for 30 minutes at 4 °C, and the supernatants were stored at −80 °C for cytokine analysis. Total lung homogenates protein content was quantified with Brad­ ford’s assay (Bio-RAD) at the final concentration of 500 µg/ml. A panel of 23 murine chemokines and cytokines were measured using BioPlex pro Mouse Cytokine Standard 23-Plex, Group I according to the manufacturer’s instructions (Bio-RAD, California, USA). Histologic analysis. Lungs were removed, fixed in 10% buffered forma-

lin for at least 24 hours, and embedded in paraffin. Representative consecutive 3 (micron) µm sections of the five lung lobes were stained by Haematoxylin-Eosin according to standard procedures for histopathological examination.

Statistical analysis

Survival curve estimation. We used the Kaplan-Meier (KM) estimator to estimate the survival curves of the analyzed mice lifetime data. The event considered here is death and there was no informative censoring during the period of the analysis. The KM estimator is nonparametric; in particular, it does not require specific parametric assumptions for the shape of survival curves. For both data sets, we use the nonparametric log-rank test (Mantel-Haenszel) for equality of the survival curves considered here. We use the estimation procedure implanted in the survival package in R 3.2.2 by Thernau.51 For further information, please refer to the Statistical Analysis section in the Supplementary Material. Growth curve modeling. Growth curve data were modeled by applying Linear Mixed Effects model framework52,53, which are suitable for repeated measures, allow to specify linear and nonlinear growth trends over time and accounts for unobservable heterogeneity among experimental subjects. Data from Figures 3 and 4 and Supplementary Figures S1c and S2a,b were analyzed by a Kruskal-Wallis test (nonparametric), followed by Dunn’s post-hoc test.

SUPPLEMENTARY MATERIAL Figure S1.  Survival and weight loss in response to S. aureus Newman acute infection. Figure S2.  Restored NADPH oxidase activity in XCGD mice treated with gene-corrected Lin-negative cells. Figure S3.  Schematic maps of the lentiviral vectors used in this study. Figure S4.  Scatterplot matrix in both vectors groups, where all variables were plotted against each other. Table S1.  Pair-wise log-rank tests for equality of the survival curves in the first experiment. Table S2.  Pair-wise log-rank tests for equality of the survival curves in the second experiment. Table S3.  Fitted growth model in the first experiment. The “:” represents an interaction term. Table S4. Fitted growth model in the second experiment. The “:” represents an interaction term. Supplementary Material

ACKNOWLEDGMENTS This work was supported by grants from European Commission (E-rare project EURO-CGD to A.A., CELL-PID HEALTH-F5-2010–261387 to AA) and Fondazione Telethon (TIGET core grant to A.A.). We thank Erika

Molecular Therapy  vol. 24 no. 10 oct. 2016

S. aureus Infection in XCGD Gene Therapy Mice

Zonari for preliminary studies on XCGD mice and Martina Rocchi for her technical work in histological preparation of specimens. We thank Fiona Johnston, a native English speaker, for revising the manuscript. We thank GSK R&D Rare Disease Unit delegates for reviewing the manuscript and their suggested edits to the text. In 2010, Telethon and OSR, through TIGET, entered a strategic alliance with GSK for the development-up-to-marketing authorization of hematopoietic stemcell gene therapy for various rare diseases including CGD. While TIGET remains responsible for the pre-clinical development and early clinical testing of such therapies, GSK has option rights once clinical proof-ofconcept is achieved. Ch.B. is an employee of MolMed S.p.A.

AUTHOR CONTRIBUTIONS G.F., R.J.H., A.R., and S.R. performed the infection experiments in mice, carried out functional readouts and analyzed the data. F.S. performed the histopathological analysis. M.M. contributed to the mouse studies. C.B., A.P., and Cd.S. contributed to the statistical analysis. Ch.B. participated in the vector production and characterization. B.G. contributed to the design of the lentiviral vectors and supervised the gene therapy part. A.B. and A.A. conceived and supervised the studies, analyzed the data and wrote the paper. All authors critically revised the manuscript.

References

1. Segal, BH, Leto, TL, Gallin, JI, Malech, HL and Holland, SM (2000). Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 79: 170–200. 2. Holland, SM (2010). Chronic granulomatous disease. Clin Rev Allergy Immunol 38: 3–10. 3. Roos, D, Kuhns, DB, Maddalena, A, Bustamante, J, Kannengiesser, C, de Boer, M et al. (2010). Hematologically important mutations: the autosomal recessive forms of chronic granulomatous disease (second update). Blood Cells Mol Dis 44: 291–299. 4. Matute, JD, Arias, AA, Wright, NA, Wrobel, I, Waterhouse, CC, Li, XJ et al. (2009). A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity. Blood 114: 3309–3315. 5. van den Berg, JM, van Koppen, E, Ahlin, A, Belohradsky, BH, Bernatowska, E, Corbeel, L et al. (2009). Chronic granulomatous disease: the European experience. PLoS One 4: e5234. 6. Seger, RA (2010). Chronic granulomatous disease: recent advances in pathophysiology and treatment. Neth J Med 68: 334–340. 7. Winkelstein, JA, Marino, MC, Johnston, RB Jr, Boyle, J, Curnutte, J, Gallin, JI et al. (2000). Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 79: 155–169. 8. Seger, RA (2008). Modern management of chronic granulomatous disease. Br J Haematol 140: 255–266. 9. Song, E, Jaishankar, GB, Saleh, H, Jithpratuck, W, Sahni, R and Krishnaswamy, G (2011). Chronic granulomatous disease: a review of the infectious and inflammatory complications. Clin Mol Allergy 9: 10. 10. Deffert, C, Cachat, J and Krause, KH (2014). Phagocyte NADPH oxidase, chronic granulomatous disease and mycobacterial infections. Cell Microbiol 16: 1168–1178. 11. Grimm, MJ, Vethanayagam, RR, Almyroudis, NG, Lewandowski, D, Rall, N, Blackwell, TS et al. (2011). Role of NADPH oxidase in host defense against aspergillosis. Med Mycol 49 Suppl 1: S144–S149. 12. Güngör, T, Teira, P, Slatter, M, Stussi, G, Stepensky, P, Moshous, D, et al. (2013). Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study. Lancet 6736: 1–13. 13. Grez, M, Reichenbach, J, Schwäble, J, Seger, R, Dinauer, MC and Thrasher, AJ (2011). Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther 19: 28–35. 14. Kang, EM, Choi, U, Theobald, N, Linton, G, Long Priel, DA, Kuhns, D et al. (2010). Retrovirus gene therapy for X-linked chronic granulomatous disease can achieve stable long-term correction of oxidase activity in peripheral blood neutrophils. Blood 115: 783–791. 15. Kang, HJ, Bartholomae, CC, Paruzynski, A, Arens, A, Kim, S, Yu, SS et al. (2011). Retroviral gene therapy for X-linked chronic granulomatous disease: results from phase I/II trial. Mol Ther 19: 2092–2101. 16. Ott, MG, Schmidt, M, Schwarzwaelder, K, Stein, S, Siler, U, Koehl, U et al. (2006). Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12: 401–409. 17. Stein, S, Ott, MG, Schultze-Strasser, S, Jauch, A, Burwinkel, B, Kinner, A et al. (2010). Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med 16: 198–204. 18. Bianchi, M, Hakkim, A, Brinkmann, V, Siler, U, Seger, RA, Zychlinsky, A et al. (2009). Restoration of NET formation by gene therapy in CGD controls aspergillosis. Blood 114: 2619–2622. 19. Brown, BD, Venneri, MA, Zingale, A, Sergi Sergi, L and Naldini, L (2006). Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nat Med 12: 585–591. 20. Gentner, B, Visigalli, I, Hiramatsu, H, Lechman, E, Ungari, S, Giustacchini, A et al. (2010). Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy. Sci Transl Med 2: 58ra84.

1879

S. aureus Infection in XCGD Gene Therapy Mice

21. Gentner, B and Naldini, L (2012). Exploiting microRNA regulation for genetic engineering. Tissue Antigens 80: 393–403. 22. Santilli, G, Almarza, E, Brendel, C, Choi, U, Beilin, C, Blundell, MP et al. (2011). Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of transgene expression in myeloid cells. Mol Ther 19: 122–132. 23. Chiriaco, M, Farinelli, G, Capo, V, Zonari, E, Scaramuzza, S, Di Matteo, G, et al. (2014). Dual-regulated lentiviral vector for gene therapy of X-linked chronic granulomatosis. Mol Ther 22: 1472–1483. 24. Farinelli, G, Capo, V, Scaramuzza, S and Aiuti, A (2014). Lentiviral vectors for the treatment of primary immunodeficiencies. J Inherit Metab Dis 37: 525–533. 25. Ludin, A, Gur-Cohen, S, Golan, K, Kaufmann, KB, Itkin, T, Medaglia, C et al. (2014). Reactive oxygen species regulate hematopoietic stem cell self-renewal, migration and development, as well as their bone marrow microenvironment. Antioxid Redox Signal 21: 1605–1619. 26. Barde, I, Laurenti, E, Verp, S, Wiznerowicz, M, Offner, S, Viornery, A et al. (2011). Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease. Gene Ther 18: 1087–1097. 27. Bingel, SA (2002). Pathology of a mouse model of x-linked chronic granulomatous disease. Contemp Top Lab Anim Sci 41: 33–38. 28. Pollock, JD, Williams, DA, Gifford, MA, Li, LL, Du, X, Fisherman, J et al. (1995). Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat Genet 9: 202–209. 29. Björgvinsdóttir, H, Ding, C, Pech, N, Gifford, MA, Li, LL and Dinauer, MC (1997). Retroviral-mediated gene transfer of gp91phox into bone marrow cells rescues defect in host defense against Aspergillus fumigatus in murine X-linked chronic granulomatous disease. Blood 89: 41–48. 30. Dinauer, MC, Gifford, MA, Pech, N, Li, LL and Emshwiller, P (2001). Variable correction of host defense following gene transfer and bone marrow transplantation in murine X-linked chronic granulomatous disease. Blood 97: 3738–3745. 31. Sousa, SA, Ulrich, M, Bragonzi, A, Burke, M, Worlitzsch, D, Leitão, JH et al. (2007). Virulence of Burkholderia cepacia complex strains in gp91phox-/- mice. Cell Microbiol 9: 2817–2825. 32. Liu, W, Yan, M, Sugui, JA, Li, H, Xu, C, Joo, J et al. (2013). Olfm4 deletion enhances defense against Staphylococcus aureus in chronic granulomatous disease. J Clin Invest 123: 3751–3755. 33. Goebel, WS, Mark, LA, Billings, SD, Meyers, JL, Pech, N, Travers, JB et al. (2005). Gene correction reduces cutaneous inflammation and granuloma formation in murine X-linked chronic granulomatous disease. J Invest Dermatol 125: 705–710. 34. Aiuti, A, Biasco, L, Scaramuzza, S, Ferrua, F, Cicalese, MP, Baricordi, C et al. (2013). Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341: 1233151. 35. Kang, EM and Malech, HL (2012). Gene therapy for chronic granulomatous disease. Methods Enzymol 507: 125–154. 36. Mortellaro, A, Hernandez, RJ, Guerrini, MM, Carlucci, F, Tabucchi, A, Ponzoni, M et al. (2006). Ex vivo gene therapy with lentiviral vectors rescues adenosine deaminase (ADA)-deficient mice and corrects their immune and metabolic defects. Blood 108: 2979–2988. 37. Visigalli, I, Delai, S, Politi, LS, Di Domenico, C, Cerri, F, Mrak, E et al. (2010). Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model. Blood 116: 5130–5139.

1880

© The American Society of Gene & Cell Therapy

38. Leon-Rico, D, Aldea, M, Sanchez-Baltasar, R, Mesa-Nunez, C, Record, J, Burns, SO, et al. (2016). Lentiviral vector mediated correction of a mouse model of leukocyte adhesion deficiency type I. Hum Gene Ther. doi:10.1089/hum.2016.016 (epub ahead of print). 39. Biffi, A, Montini, E, Lorioli, L, Cesani, M, Fumagalli, F, Plati, T et al. (2013). Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science 341: 1233158. 40. Meissner, F, Seger, RA, Moshous, D, Fischer, A, Reichenbach, J and Zychlinsky, A (2010). Inflammasome activation in NADPH oxidase defective mononuclear phagocytes from patients with chronic granulomatous disease. Blood 116: 1570–1573. 41. de Luca, A, Smeekens, SP, Casagrande, A, Iannitti, R, Conway, KL, Gresnigt, MS et al. (2014). IL-1 receptor blockade restores autophagy and reduces inflammation in chronic granulomatous disease in mice and in humans. Proc Natl Acad Sci USA 111: 3526–3531. 42. Bagaitkar, J, Pech, NK, Ivanov, S, Austin, A, Zeng, MY, Pallat, S et al. (2015). NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis. Blood 126: 2724–2733. 43. Weisser, M, Demel, UM, Stein, S, Chen-wichmann, L, Touzot, F, Santilli, G, et al. (2016). Hyperinflammation in patients with chronic granulomatous disease leads to impairment of hematopoietic stem cell functions. S Allergy Clin Immunol 138: 219–228. 44. Gabay, C (2006). Interleukin-6 and chronic inflammation. Arthritis Res Ther 8 Suppl 2: S3. 45. Bragonzi, A (2010). Murine models of acute and chronic lung infection with cystic fibrosis pathogens. Int J Med Microbiol 300: 584–593. 46. Marangoni, F, Bosticardo, M, Charrier, S, Draghici, E, Locci, M, Scaramuzza, S et al. (2009). Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models. Mol Ther 17: 1073–1082. 47. Scaramuzza, S, Biasco, L, Ripamonti, A, Castiello, MC, Loperfido, M, Draghici, E et al. (2013). Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome. Mol Ther 21: 175–184. 48. Baba, T, Bae, T, Schneewind, O, Takeuchi, F and Hiramatsu, K (2008). Genome sequence of Staphylococcus aureus strain Newman and comparative analysis of staphylococcal genomes: polymorphism and evolution of two major pathogenicity islands. J Bacteriol 190: 300–310. 49. Lorè, NI, Cigana, C, De Fino, I, Riva, C, Juhas, M, Schwager, S et al. (2012). Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts. PLoS One 7: e35648. 50. Facchini, M, De Fino, I, Riva, C and Bragonzi, A (2014). Long term chronic Pseudomonas aeruginosa airway infection in mice. J Vis Exp: (85) 1–10. 51. Therneau, TM (2014). A Package for Survival Analysis in S. R package version 2.37.7. Survival (Lond) http://CRAN.R-project.org/package=survival. 52. Laird, NM and Ware, JH (1982). Random-effects models for longitudinal data. Biometrics 38: 963–974. 53. Pinheiro, JC and Bates, DM (2000). Mixed effects models in Sand S-PLUS. New York SpringerVerlag. doi:10.1007/978-1-4419-0318-1. 54. Kaplan, EL and Meier, P (1958). Nonparametric Estimation from Incomplete Observations. J Am Stat Assoc 53: 457–481. 55. Mantel, N (1966). Evaluation of survival data and two new rank order statistics arising in its consideration. Cancer Chemother Rep 50: 163–170.

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Lentiviral Vector Gene Therapy Protects XCGD Mice From Acute Staphylococcus aureus Pneumonia and Inflammatory Response.

Chronic granulomatous disease (CGD) is a primary immunodeficiency due to a deficiency in one of the subunits of the nicotinamide adenine dinucleotide ...
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