Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Study design

Sample size in experiments with mice was choosen according to previous experience with experimental models and assays. Mouse studies were designed to evaluate susceptibility of LV to phagocytosis after i.v. administration. LV-derived transgene expression was measured in the plasma of treated animals and LV transduction of different cell types was determined at end-point. NHP study was designed to evaluate acute toxicity, transduction efficiency and biodistribution of i.v. administered LV, by measuring transgene expression in the plasma, blood chemistry, histological and molecular analyses. Sample size in the NHP study was limited by ethical and feasibility reasons. No sample or animal was excluded from the analyses. Mice and NHP were randomly assigned to each experimental group. Investigators were not blinded. Vector production Large-scale purified LV batches used for the NHP study were produced by MolMed S.p.A., as previously described ( 9 ), and formulated in PBS 5% dimethyl sulfoxide (DMSO). Results of selected quality control assays performed on these batches are reported in Table S1 . A sterile “vehicle” solution (PBS 5% DMSO) was prepared in parallel. Lab-grade VSV.G-pseudotyped third-generation self-inactivating (SIN) LV were produced by calcium phosphate transient transfection into 293T cells, or by LV stable producer cell lines ( 24 ). 293T cells were transfected with a solution containing a mix of the selected LV genome transfer plasmid, the packaging plasmids pMDLg/pRRE and pCMV.REV, pMD2.G and pAdvantage, as previously described ( 9 ). Medium was changed 14-16 hours after transfection and supernatant was collected 30 hours after medium change. Alternatively, LV production was induced when LV producer cells were in a sub-confluent state, by replacing the culture medium with medium containing doxycycline (Sigma) 1 μg/mL and supernatant was collected 3 days after induction. LV-containing supernatants were sterilized through a 0.22 μm filter (Millipore) and, when needed, transferred into sterile poliallomer tubes (Beckman) and centrifuged at 20,000 g for 120 min at 20° C (Beckman Optima XL-100K Ultracentrifuge). LV pellet was dissolved in the appropriate volume of PBS to allow 500-1000X concentration. VSV.G-pseudotyped SIN RV were produced by calcium phosphate transient transfection into 293T cells. 293T cells were transfected with pRT43.3.PGK.CD47, the packaging plasmid pCMV-Gag/Pol (Moloney Leukemia Virus) and pMD2.G, as described ( 44 ). Medium was changed 14-16 hours after transfection and RV-containing supernatant collected 30 hours after medium change and concentrated 1000X by ultracentrifugation as above. Mice experiments Founder C57BL/6 F9 knock out mice were originally obtained from the laboratory of Dr. Inder Verma at the Salk Institute ( 45 ). NOD and wild-type C57BL/6 mice were purchased by Charles River. All mice were maintained in specific pathogen-free conditions. Vector administration was carried out in adult (7-10 week old) mice by tail-vein injection. Mice were bled from the retro-orbital plexus using capillary tubes and blood was collected into 0.38% sodium citrate buffer, pH 7.4. Mice were deeply anesthetized with tribromoethanol (Avertin) and euthanized by CO 2 inhalation at the scheduled times. All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee.

Intravital imaging

C57BL/6 or NOD mice were surgically prepared for liver IV2PM as described ( 28 ). Mice were i.v. injected with PE-conjugated anti-F4/80 Ab (clone BM8, Biolegend) 20 min before imaging. GFP-labeled LV, CD47hi or CD47-free LV were i.v. injected 2 min after the start of video recording. Images (TriMScope II) were obtained with a Nikon Ti-U fluorescence inverted microscope and a 25x objective (NA 0.95). For four-dimensional analysis, 8–12 z-stacks (spacing 4 μm) of 300- to 400-μm 2 xy-sections were acquired every 20 seconds for 40 min. Liver sinusoids were visualized by injecting i.v. non-targeted Quantum Dots 655 (Invitrogen) immediately prior to imaging. Sequences of image stacks were transformed into volume-rendered four-dimensional videos using Imaris software (Bitplane). NHP study Seven adults (3-5 kg body weight) male Macaca Nemestrina were purchased by Bioprim (Baziège, France). Macaques were housed in an enriched environment with access to toys, fresh fruits, and vegetables at the Boisbonne Center (Nantes, France), under protocol APAFIS#4302-2015122314563838 that was approved by the Institutional Animal Care and Use Committee of the Pays De Loire. For LV administration, animals were anesthetized with Demetomidine (Domitor) 30 μg/kg and Ketamine 7 mg/kg and maintained under gas anesthesia, 1-2% Isoflurane (Vetflurane). The LV-containing solution or vehicle solution (PBS 5% DMSO) was administered using a syringe with controlled flow rate fixed at 10 mL/kg/hour via a catheter in the saphenous vein for an approximate time of 2 hours. The administered LV dose was verified by sampling and titering the dosing solution at the end of infusion. Whereas 5 out of 6 animals received the target dose, between 6.9 and 7.7x10 9 TU/kg, one animal (CD47hi-LV2) received a higher dose of 1.0x10 10 TU/kg CD47hi LV, due to a mistake in pooling LV aliquots of different volume. Note, however, that this animal showed the lowest transgene expression among its group, thus suggesting that the slightly higher administered dose was not instrumental in driving the outcome of higher transgene expression for the CD47hi LV group. An anti-inflammatory and antihistamine pre-medication regimen was administered: dexamethasone (Dexadreson, 0.3 mg/kg) i.v. 24 hours before LV and just before LV and Dexchlorpheniramine (Polaramine, 4 mg/kg) i.v. 30 minutes before LV. Blood samples were taken at different time points from the femoral vein upon anesthesia with 10 mg/kg ketamine (Imalgene) intramuscularly. For hematology, 1 mL of total blood samples was collected on EDTA-coated tubes. For clinical biochemistry, 2 mL of total blood samples was collected on heparin-coated tubes and for hemostasis, 1.8 mL of total blood was collected in citrate-coated tubes. Blood tests were performed on fresh samples at the Veterinary School of Nantes (LDHvet, Oniris). Biochemistry parameters were analyzed by automatons and analyzers based on spectrometry, reflectometry, potentiometry and enzyme immunoassays. Hemostasis was analyzed by a hemostasis analyzer based on electro-mechanical clot detection (viscosity-based detection system). Tissue samples were collected following euthanasia, performed by overdose pentobarbital sodium (Dolethal) i.v. injection.

FIX assays

The concentration of human FIX was determined in mouse plasma by an enzyme-linked immunosorbent assay (ELISA) specific for human FIX antigen (Asserachrom IX:Ag, Stago) following manufacturer’s instructions. Absorbance of each sample was determined spectrophotometrically, using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized to antigen standard curves. The concentration of human FIX was determined in NHP plasma by an ELISA specific for human FIX antigen (Haematologic Technologies, AHIX-5041). Human FIX activity was quantified in NHP plasma by a modified FIX chromogenic assay, human FIX in plasma samples was first captured by a human FIX specific Ab immobilized onto a 96-well plate (Haematologic Technologies, AHIX-5041) then chromogenic activity of human FIX was measured using Hyphen Biophen Factor IX kit (Aniara Corp., 221806-RUO) following manufacturer’s instructions. Standard curves were obtained by diluting recombinant human FIX (BENEFIX, Pfizer) into untreated NHP plasma. Total anti-human FIX Abs were quantified in heat-inactivated NHP serum by ELISA, coating recombinant human FIX (BENEFIX, Pfizer) and developing with a HRP-conjugated mouse anti-monkey IgG Ab (Southern Biotech 1:8,000). Anti-human FIX / human FIX complexes were quantified in NHP serum by ELISA, coating with a human-FIX specific Ab (Haematologic Technologies, AHIX-5041) and developing with a HRP-conjugated mouse anti-monkey IgG Ab (Southern Biotech 1:8,000). Absorbance of each sample was determined spectrophotometrically, using a Multiskan GO microplate reader (Thermo Fisher Scientific) and normalized to coated monkey IgG standard curves ( MyBioSource.com ). Neutralizing anti-human FIX Abs were determined by Bethesda assay. The test sample was incubated for 2 hours at 37° C with recombinant human FIX (BENEFIX, Pfizer, 1 U/mL) with FIX activity of 100%. Residual FIX activity was measured using Hyphen Biophen Factor IX kit (Aniara Corp., 221806-RUO) and converted into Bethesda Units (BU)/ml, where one BU is defined as the inverse of the dilution factor of the test sample that yields 50% residual FIX activity.

Statistical analysis

Statistical analyses were performed upon consulting with professional statisticians at the San Raffaele University Center for Statistics in the Biomedical Sciences (CUSSB). When normality assumptions were not met, non-parametric statistical tests were performed. Mann-Whitney or Kruskall-Wallis tests with Dunn’s multiple comparison post-test were performed when comparing 2 or more experimental groups, respectively. For repeated measures over time, two-way ANOVA was performed. Comparison between the proportions of IS retrieved in liver and spleen in NHP was performed by the Fisher’s exact test. When sample size was