stm.sciencemag.org/cgi/content/full/11/493/eaav7325/DC1
Supplementary Materials for
Phagocytosis-shielded lentiviral vectors improve liver gene therapy in
nonhuman primates
Michela Milani, Andrea Annoni, Federica Moalli, Tongyao Liu, Daniela Cesana, Andrea Calabria, Sara Bartolaccini, Mauro Biffi, Fabio Russo, Ilaria Visigalli, Andrea Raimondi, Susannah Patarroyo-White, Douglas Drager, Patrizia Cristofori,
Eduard Ayuso, Eugenio Montini, Robert Peters, Matteo Iannacone, Alessio Cantore*, Luigi Naldini*
*Corresponding author. Email: [email protected] (A.C.); [email protected] (L.N.)
Published 22 May 2019, Sci. Transl. Med. 11, eaav7325 (2019)
DOI: 10.1126/scitranslmed.aav7325
The PDF file includes:
Materials and Methods Fig. S1. Fractionation and sorting of liver cell subpopulations. Fig. S2. LV biodistribution within the liver cell subpopulations. Fig. S3. Generation of CD47-negative cells. Fig. S4. Generation of CD47-overexpressing cells. Fig. S5. Cytokine and chemokine response to LV, CD47hi LV, or CD47-free LV administration in NOD mice. Fig. S6. Cytokine and chemokine response to LV or CD47hi LV administration in NHPs. Fig. S7. LV gene therapy in NHPs. Fig. S8. IS analysis in CD47hi LV– or LV-treated NHP spleen. Table S1. Large-scale LV batches used in NHP study. Table S2. Clinical biochemistry, hematology, and hemostasis of vehicle NHP. Table S3. Clinical biochemistry, hematology, and hemostasis of LV1. Table S4. Clinical biochemistry, hematology, and hemostasis of LV2. Table S5. Clinical biochemistry, hematology, and hemostasis of LV3. Table S6. Clinical biochemistry, hematology, and hemostasis of CD47hi LV1. Table S7. Clinical biochemistry, hematology, and hemostasis of CD47hi LV2. Table S8. Clinical biochemistry, hematology, and hemostasis of CD47hi LV3. Table S9. LV IS in NHPs. Table S10. LV IS in cancer genes. Table S11. LV CIS. Legend for movie S1 References (46–53)
Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/493/eaav7325/DC1)
Movie S1 (.mov format). IV2PM of LVs, CD47hi LVs, or CD47-free LVs upon administration.
Materials and Methods
Plasmid construction
The LV constructs expressing human FIX were previously described (8). The expression cassette
contains an engineered hepatocyte-specific promoter (Enhanced Transthyretin, GenBank
accession number AY661265) and target sequences for the hematopoietic-lineage specific
microRNA 142, abrogating off-target transgene expression in antigen-presenting cells (APC) in
the liver and spleen (mature human microRNA 142 sequence – miR-142-3p:
uguaguguuuccuacuuuaugga). The Cas9 and sgRNA expressing plasmids were previously
described (46). The sequences of the CRISPR used to generate the sgRNA are: CD47 A
(CTACTGAAGTATACGTAAAGTGG), B (CTTGTTTAGAGCTCCATCAAAGG), C
(ATCGAGCTAAAATATCGTGTTGG). To generate SIN RV genome transfer PGK.CD47, the
gene synthesized human codon-optimized version of the CD47 cDNA (Genewiz) was exchanged
with GFP into pRT43.3.PGK.GFP (BamHI-NotI)(44). GFP-pp60Src
fusion construct was
generated by cloning the gene synthesized sequence encoding for the first 15 amino acids of
pp60Src
and the poly-glutamine spacer (26) upstream the GFP encoding sequence into
pMAX.GFP.
LV and RV titration
For LV titration, 1x105
293T cells were transduced with serial LV dilutions in the presence of
polybrene (8 μg/ml). For LV-GFP, cells were analyzed by flow cytometry 3-7 days after
transduction and infectious titer, expressed as transducing units293T (TU)/mL, was calculated
using the formula TU/mL = ((% GFP+ cells/100)x100,000x(1/dilution factor)). For all other LV,
genomic DNA (gDNA) was extracted 14 days after transduction, using Maxwell 16 Cell DNA
Purification Kit (Promega), following manufacturer’s instructions. VCN was determined by
quantitative PCR (qPCR) starting from 100 ng of template gDNA using primers (HIV fw: 5’-T
ACTGACGCTCTCGCACC-3’; HIV rv: 5’-TCTCGACGCAGGACTCG-3’) and a probe (FAM
5’-ATCTCTCTCCTTCTAGCCTC-3’) designed on the primer binding site region of LV. The
amount of endogenous DNA was quantified by a primers/probe set designed on the human
telomerase gene (Telo fw: 5’-GGCACACGTGGCTTTTCG-3’; Telo rv: 5’-
GGTGAACCTCGTAAGTTTATGCAA-3’; Telo probe: VIC 5’-
TCAGGACGTCGAGTGGACACGGTG-3’ TAMRA) or the human GAPDH gene (Applied
Biosystems HS00483111_cm) VCN was calculated by the formula = (ng LV/ng endogenous
DNA)xVCN of sample used for the standard curve. The standard curve was generated, by using
a CEM cell line stably carrying 1 vector integrant, which was previously determined by Southern
blot and fluorescent in situ hybridization (FISH). All reactions were carried out in duplicate or
triplicate in a Viia7 Real Time PCR thermal cycler (Applied Biosystems). Each qPCR run
carried an internal control generated by using a CEM cell line stably carrying 4 vector integrants,
which were previously determined by Southern blot and FISH analysis. Infectious titer,
expressed as TU/mL, was calculated using the formula TU/mL = (VCNx100,000x(1/dilution
factor). LV physical particles were measured by HIV-1 Gag p24 antigen immunocapture assay
(Perkin Elmer) following manufacturer’s instructions. LV specific infectivity was calculated as
the ratio between infectious titer and physical particles. RV titration was performed as for LV,
except that RV VCN was determined by droplet digital PCR (ddPCR) starting from 5-20 ng of
template gDNA using primers (sinRV fw: 5’-ACCTATCTGTGTCTGTCCGA-3’; sinRV rv: 5’-
CAGAACTCGTCAGTTCCACC-3’) and a probe (FAM 5’-
ACTAGCTCTGTATCTGGCGGACCCGT-3’) designed on the packaging signal of RV. The
amount of endogenous DNA was quantified by a primers/probe set designed on the human
telomerase gene, as above. The PCR reaction was performed with each primer (900 nM) and the
probe (250 nM, 500 nM for Telo) following manufacturer’s instructions (Biorad), read with
QX200 reader and analyzed with QuantaSoft software (Biorad).
Fractionation and sorting of liver cell sub-populations
The liver was perfused (2.5 mL/min) via the inferior vena cava with 12.5 mL of the following
solutions at subsequent steps: 1) PBS EDTA (0.5 mM), 2) HBSS (Hank’s balanced salt solution,
Gibco) and HEPES (10 mM), 3) HBSS-HEPES 0.03% Collagenase IV (Sigma). The digested
liver tissue was harvested, passed through a 70 μm cell strainer (BD Biosciences) and processed
into a single-cell suspension. This suspension was subsequently centrifuged three times (30, 25
and 20 g, for 3 minutes, at room temperature) to obtain PC-containing pellets. The nPC-
containing supernatant was centrifuged (650 g, 7 minutes, at room temperature) and recovered
cells were loaded onto a 30/60% Percoll (Sigma) gradient (1800 g, for 20 minutes at room
temperature). nPC interface was collected and washed twice. The nPC were subsequently
incubated with the following monoclonal antibodies: e-fluor 450-conjugated anti-CD45 (30-F11,
e-Bioscience), Allophycocyanin (APC)-conjugated anti-CD31 (MEC13.3, BD Biosciences),
phycoerythrin (PE)-conjugated F4/80 (CI:A3-1, Biorad), PE-Cy5-conjugated anti-CD45R/B220
(from BD Biosciences), PE-Cy7-conjugated anti-CD11c (N418, e-Bioscience), purified anti-
CD16/32 (2.4G2, BD Biosciences). nPC subpopulations (LSEC, KC, pDC) were sorted by
FACS, MOFLO-DAKO-Beckman-Coulter; the nPC contaminating the PC suspension, were
removed by FACS excluding cells labeled by APC-conjugated anti-CD31/anti-CD45 cocktail,
thus obtaining sorted hepatocytes (Hep).
Immune-fluorescence analysis and RNA ISH
For immune-fluorescence analysis, the liver was fixed in 4% paraformaldehyde, embedded in
optimal cutting temperature (OCT) and frozen in liquid nitrogen. Cryostat sections (10 μm thick)
were blocked with 5% FBS (Euroclone), 1% BSA, 0.1% Triton X-100 in PBS, incubated with
rabbit anti-GFP (1:200, Molecular Probes) washed and incubated with (FITC)-conjugated goat
anti-rabbit IgG (1:500, Molecular Probes). Sections were analyzed under a 3-laser confocal
microscope (Radiance 2100, Bio-Rad). Sections form untreated mice were used as negative
controls. Nuclei were stained with TOPRO-3 (1:1000, Molecular Probes). For RNA ISH, the
liver was fixed in formalin for 24 hours then embedded in paraffin and sectioned. A RNAscope
probe was designed to target the WPRE sequence and the signal was detected using BOND RX
system (Leica Biosystem) with RNAscope
2.5 HD Reagent Kit-RED (Advanced Cell
Diagnostics, Inc., 322360) following manufacturer’s instructions. Nuclei were stained with
DAPI.
Cell cultures and in vitro experiments
293T and LV producer cell lines were maintained in Iscove's modified Dulbecco's medium
(IMDM, Sigma) supplemented with 10% fetal bovine serum (FBS, Euroclone), 4 mM glutamine
(Lonza), penicillin and streptomycin 100 IU/mL (Lonza). THP-1 cell line was maintained in
Roswell Park Memorial Institute medium (RPMI, Corning) supplemented with 20% FBS, 4 mM
glutamine, penicillin and streptomycin 100 IU/mL. Primary human macrophages were obtained
from CD14-positive cells isolated by negative selection (Pan Monocyte Isolation Kit, Miltenyi
Biotec), from buffy coats of healthy donors (obtained according to a protocol approved by the
San Raffaele Scientific Institute Ethical Committee) and differentiated in IMDM, supplemented
with 5% human serum, 4 mM glutamine, penicillin and streptomycin 100 IU/mL for 7 days. The
purity of CD14-positive cells was determined by flow cytometry and was >90%. All cells were
maintained in a 5% CO2 humidified atmosphere at 37° C. All cell lines were routinely tested for
mycoplasma contamination. THP-1 cell line was differentiated with PMA (100 ng/mL) for 24
hours, before transduction. Human primary macrophages, differentiated THP-1 and 293T were
transduced for 1 hour with spinoculation (at 1,100 g, at 37° C), then washed with PBS and
cultured for 3 days.
Gene disruption and mismatch-selective endonuclease assay
Gene disruption was performed by calcium phosphate-mediated transient transfection of the
indicated amount of the desired sgRNA-expressing plasmid and the Cas9-expressing plasmid.
The mismatch-selective endonuclease assay was used to measure the extent of mutations
consequent to non-homologous end joining (NHEJ) at the Cas9 target sites, as described (47).
PCR was performed using primers flanking the sgRNA binding site in in the CD47 gene (fw: 5’-
TTCCTTTCCAGGATCAGCTCAGC-3’; rv: 5’-TTGATTCAAAGGAGTACCTATCCC -3’).
The PCR product was denatured, allowed to re-anneal and digested with Surveyor nuclease assay
(Transgenomic). Because this enzyme cuts DNA at sites of duplex distortions, the products of re-
annealing between wild type and mutant alleles (carrying mutations or deletions consequent to
the nuclease activity) are specifically digested. The reaction products were separated on a
Spreadex EL1200 Wide Mini gel (Elchrom Scientific), stained by ethidium bromide or GelRed
(Biotium) and the intensity of the bands was quantified by ImageQuant TL 5 software. The ratio
of the uncleaved parental fragment to the two lower migrating cleaved products was calculated
using the formula (1-(parental fraction)1/2
)x100.
Flow cytometry
Flow cytometry analyses were performed using a FACSCanto analyzer (BD Biosciences),
equipped with DIVA Software. Between 100,000-500,000 cells were harvested, washed with
PBS or MACS buffer (PBS pH 7.2 0.5% BSA, 2Mm EDTA), treated with Fc Receptor-Block
(Miltenyi Biotec) when antibody stained and then re-suspended in the buffer used for washing.
Staining was performed in MACS buffer, incubating cells with antibodies (in the proportion
indicated in the table below) for 20 minutes at 4° C in the dark. Anti-murine IgG beads were
used for single-staining controls (BD Biosciences).
CD14 PE-Vio770 clone TÜK4 (Miltenyi Biotec, 1:20)
CD19 PE clone SJ25C1 (BD Biosciences, 1:50)
CD3 APC clone UCHT1 (BD Biosciences, 1:50)
CD16 Pacific Blue clone 3G8 (BD Biosciences, 1:50)
CD47 Pacific Blue clone B6H12 (BD Biosciences, 1:20)
Electron microscopy
Few microliters of concentrated LV batches were adsorbed on glow discharged carbon coated
formvar copper grids and fixed for 20 minutes with 8% paraforlmadehyde in PBS. After several
washes in 50 mM glycine in PBS grids were blocked in 1% BSA in PBS and incubated with
primary antibodies diluted in blocking buffer for 30-90 minutes (Anti-VSV.G, KeraFAST, 1:50,
Anti-CD47, BD Biosciences, 1:10). After several washes in 0.1% BSA in PBS, samples were
incubated for 30 minutes with Protein A-gold (10 nm), fixed with 1% glutaraldehyde, stained
with 2% uranyl acetate and air-dried. Grids were observed with a Zeiss LEO 512 transmission
electron microscope. Images were acquired by a 2k x 2k bottom-mounted slow-scan Proscan
camera controlled by EsivisionPro 3.2 software. For quantification of labeling density, random
images of viral particles were taken at nominal magnification of 16k and gold particles
associated to virions were manually counted using ImageJ. Virions were defined based on
expected size (approximately 120 nm) and an electron-dense core.
Cytokine and anti-VSV.G Abs ELISA
The concentrations of cytokines and chemokines were determined in mouse or NHP serum by a
magnetic-based multiplex ELISA 23 analytes (Bio-Plex 23-Plex, Group I, Biorad or Milliplex
NHP Cytokine Magnetic Beads Kit, Millipore, respectively), following manufacturer’s
instructions.
The concentrations of anti-VSV.G Abs were determined in NHP serum by ELISA, coating with
recombinant VSV.G (Alpha Diagnostic Intl.) 1 µg/mL and and developing with a HRP-
conjugated mouse anti-monkey IgG Ab (Southern Biotech 1:10,000).
RNA extraction and qRT-PCR
RNA extraction was performed using the RNeasy Plus mini Kit (Qiagen) according to
manufacturer’s instructions and reverse transcribed using the SuperScript Vilo kit (11754250;
Invitrogen). All q-PCR analyses were done using TaqMan probes from Applied Biosystems
(WPRE: primer fw 5’-GGCTGTTGGGCACTGACAAT-3’; primer rv 5’-
ACGTCCCGCGCAGAATC-3’; probe FAM 5’-TTTCCTTGGCTGCTCGCCTGTGT-3’ NGB).
Q-PCR was run for 40 cycles using the Viia 7 instrument and raw data (Ct) were analyzed as
follows: to determine gene expression, the difference (ΔCt) between the threshold cycle (Ct) of
WPRE and that of the reference gene TAF7 was calculated by applying an equal threshold. The
lower the ΔCt, the higher the gene expression level.
VCN determination
For human macrophage experiments, DNA was extracted using QIAamp DNA Micro Kit
(Qiagen), following manufacturer’s instructions. For mice experiments, DNA was extracted from
whole liver or whole spleen samples using Maxwell 16 Tissue DNA Purification Kit (Promega),
DNA was extracted from fractionated/sorted liver cells using DNeasy Blood & Tissue Kit
(Qiagen) or QIAamp DNA Micro Kit (Qiagen), according to cell number. VCN was determined
in human macrophages as described above (see “LV and RV titration”). For THP1 experiments,
because cells were collected three days after LV transduction, VCN was determined using an ad
hoc qPCR (SYBR, Applied Biosystems), which selectively amplifies the reverse transcribed
vector genome (both integrated and non-integrated) discriminating it from plasmid carried over
from the transient transfection (RT-LV; ΔU3 fw: 5’-TCACTCCCAACGAAGACAAGATC-3’,
gag rv: 5’-GAGTCCTGCGTCGAGAGAG-3’) (48). The amount of endogenous DNA was
quantified by a primers/probe set designed on the human telomerase gene, as above. Human
primary macrophages were transduced with LV produced by stable LV-producer cell lines, thus
lacking plasmid contamination. VCN in murine DNA was determined by ddPCR, starting from
5-20 ng of template gDNA using a primers/probe set designed on the primer binding site region
of LV (see “LV and RV titration” above). The amount of endogenous murine DNA was
quantified by a primers/probe set designed on the murine sema3a gene (Sema3A fw: 5’-
ACCGATTCCAGATGATTGGC-3’; Sema3A rv: 5’-TCCATATTAATGCAGTGCTTGC-3’;
Sema3A probe: HEX 5’-AGAGGCCTGTCCTGCAGCTCATGG-3’ BHQ1). The PCR reaction
was performed with each primer (900 nM) and the probe (250 nM) following manufacturer’s
instructions (Biorad), read with QX200 reader and analyzed with QuantaSoft software (Biorad).
VCN in NHP DNA was determined by qPCR starting from 50 ng of template DNA using
primers and a probe designed on the primer binding site region of LV (see “LV and RV titration”
above). The amount of endogenous DNA was quantified by a primers/probe set designed on the
TATA-Box Binding Protein Associated Factor 7 (TAF7) gene, (Applied Biosystems RH
02916247_s1), amplifying both the human and NHP TAF7 genes. Transduced CEM clones
described above (see “LV and RV titration”) were used as standard and internal control. This
analytical method was validated in compliance with the Organization for Economic Co-operation
and Development (OECD) Principles of Good Laboratory Practice (GLP) in terms of accuracy
(deviation vs. nominal VCN ≤ 30%), specificity, intra-assay precision (coefficient of variation ≤
5%), inter-assay precision (coefficient of variation ≤ 11%) and linearity (R2 ≥ 0.99) within the
range of 0.02 ng/reaction – 257.11 ng/reaction for TAF7 and 0.22 ng/reaction – 1568.4
ng/reaction for HIV. The lower limit of VCN quantification was 0.006, which resulted within the
accuracy criteria (deviation vs. nominal VCN ≤ 30%). The lower limit of VCN detection was
0.0004, calculated based on the lower limit of detection of the HIV system.
Vector IS retrieval and analysis
For the retrieval of vector IS we adopted a sonication-based linker-mediated PCR method
previously described (49, 50). Briefly, genomic DNA (1 µg for spleen tissue and 1 µg for each
liver lobe) was sheared using a Covaris E220 Ultrasonicator (Covaris Inc.), generating fragments
with an average size of 1,000 bp. The fragmented DNA was then split in 3 technical replicates
and subjected to end repair and 3’ adenylation using the NEBNext® Ultra™ DNA Library Prep
Kit for Illumina® (New England Biolabs), and then ligated (DNA Technologies ligation kit) to
linker cassettes (LC) containing: i) 8 nucleotide sequence barcode, used for sample
identification, ii) 12 random nucleotide sequence, used for quantification purposes and iii) all the
sequences required for the Read 2 Illumina paired end sequencing. Ligation products were then
subjected to 35 cycles of exponential PCR with primers complementary to LV LTR and the LC,
next 10 additional PCR cycles were done to add sequences required for sequencing. Finally, the
amplification products were sequenced using the Illumina HiSeq platform (Illumina).
Sequencing reads were processed by a dedicated bioinformatics pipeline (VISPA2) as previously
described (51). Briefly, paired sequence reads are filtered for quality standards, barcodes
identified for sample demultiplexing of the sequence reads, the cellular genomic sequence
mapped on the genome of Crab-eating macaque (Macaca_fascicularis_5.0/macFas5, Jun. 2013)
and the nearest RefSeq gene assigned to each unambiguously mapped IS. The abundance of each
LV IS was estimated as described previously by (49, 50), where the number of genomes with the
same IS is calculated by counting the different number of fragments generated by sonication-
mediated shearing. Then, the relative abundance of each LV IS is calculated as the percentage of
genomes with a specific IS over the total genomes. CIS were identified by combining the results
obtained from the algorithm based on Montecarlo simulations described by Abel et al (52) and
the Grubbs test for outliers (34, 53). Human genomic intervals (GRCh37/hg19) syntenic with the
CIS regions mapped in the Crab-eating macaque assembly (Macaca_fascicularis_5.0/macFas5)
were extracted using the UCSC Genome Browser comparative genomics alignment tool.
Supplementary Figures
Fig. S1. Fractionation and sorting of liver cell subpopulations. Schematic diagram of
fractionation of PC, nPCs and sorting strategy of Hepatocytes (Hep), LSEC, KC, pDC from total
liver. The liver was perfused through the inferior vena with a collagenase IV solution. The
digested tissue was centrifuged at low speed to obtain PC-containing pellets. PC purity was
determined by flow cytometry, quantifying as 4.8%±0.5 the proportion of CD45-positive and
CD31-positive events (mean with SEM of three independent procedures). Cells contained in the
supernatant fractions were loaded on density gradient to collect nPC at the interface. Hep were
further purified from PC by FACS; LSEC, KC and pDC were separated from nPC by FACS.
FACS-sorted population highlighted in the red boxes were >98% pure.
Fig. S2. LV biodistribution within the liver cell subpopulations. (A) Stacked bar plot of
retrieved LV VCN (%) in PC or nPC at the indicated LV doses shown in Fig. 1B, based on the
formula VCNtotal = (VCNPCx0.7) + (VCNNPCx0.3). (B) Correlation of liver VCN calculated
based on the relative contributions of VCN in fractionated PC and total nPC (X axis) and liver
VCN calculated based on the relative contributions of fractionated PC, FACS-sorted LSEC and
KC (Y axis). (C,D) Correlations between VCN measured in whole liver (X axis) and liver VCN
calculated based on the relative contributions of (C) VCN in fractionated PC and nPC, or (D)
based on the relative contributions of VCN in fractionated PC and FACS-sorted LSEC and KC
(Y axis). Formula VCNtotal = (VCNPCx0.7) + (VCNNPCx0.3) or (VCNPCx0.7) + (VCNLSECx0.15)
+ (VCNKCx0.06). (E,F) Correlations between the percentage of GFP-positive PC (Y axis) and
(E) VCN in whole liver (X axis) or (F) VCN in fractionated PC (X axis). Data are shown as
mean with SEM (n=4 at each point). (G) Correlation between human FIX antigen (Y axis) and
VCN in FACS-sorted Hep (X axis) of C57BL/6 and NOD mice, 2 months after LV
administration (1.2-2x1010
TU/kg) shown in Fig. 1E,F. (H) Stacked bar plot of retrieved LV
VCN (%) in Hep, KC or LSEC in C57BL/6 or NOD mice shown in Fig. 1F, based on the
formula VCNtotal = (VCNHepx0.7) + (VCNLSECx0.15) + (VCNKCx0.06). (I) Single values and
mean with SEM of VCN measured in total liver samples of C57BL/6 (n=15, black squares) or
NOD mice (n=13, red circles), 2 months after LV administration (1x1010
TU/kg). For
comparison, the VCN calculated based on the relative contribution of the VCN of sorted
hepatocytes and KC from Fig. 1F (formula VCNtotal = (VCNHepx0.7) + (VCNKCx0.06)) are
shown. Mann-Whitney test. (J) Single values and mean with SEM of VCN in liver FACS-sorted
Hep, LSEC, KC or pDC, and whole spleen, as indicated, of NOD mice, 2 months after
administration of LV (n=6-7, black circles) or CD47-free LV (n=7-11, light blue circles) (1.2-
2x1010
TU/kg); same data sets shown in Fig. 1F (LV-treated NOD mice) and Fig. 1I (CD47-free
LV treated NOD mice) but plotted here together for direct comparison of LV and CD47-free LV
in the same mouse strain. Mann-Whitney test.
Fig. S3. Generation of CD47-negative cells. (A) Flow cytometry analysis (contour plots with
outliers) of 293T unstained, untreated, CRISPR/Cas9 treated, CD47-negative or CD47-positive
sorted, as indicated, performed 3 days after sorting. (B) Percentage of CD47-negative cells
(white bars) and of alleles bearing indels (NHEJ, black bars) in 293T cells transiently transfected
with the 3 different sgRNAs (A, B or C) with the indicated quantities of Cas9 and sgRNA
expressing plasmids, 1 week after transfection. (C-E) Mean with SEM of (C) infectious titer
(TU/mL), (D) physical particles (ng HIV Gag p24/mL) and (E) specific infectivity (TU/ng p24)
of LV produced by CD47-positive (black bars, n=3) or by CD47-negative (white bars, n=3)
293T as indicated. No significant differences by Mann-Whitney test.
Fig. S4. Generation of CD47-overexpressing cells. (A) Flow cytometry analysis (histograms)
of 293T (left panel), LV-GFP producer cell line (middle panel) and LV-FIX producer cell line
(right panel), unstained (filled black line), untreated (black line), or transduced with CD47-
expressing self-inactivating (SIN) RV (yellow line), as indicated, performed 2-4 weeks after
transduction. Note that almost all the cells show basal CD47 expression (black lines). (B) DNA
copies of SIN RV per diploid genome (SIN-RV VCN), determined in untreated (UNT) 293T,
CD47hi 293T, beta-2 microglobulin (B2M)-negative 293T, LV-GFP or LV-FIX producer cell
lines, as indicated. B2M-negative cells are used to produce MHC-free LV as reported in (24)
(C,D) SIN-RV VCN (C) or mean fluorescence intensity (MFI) of CD47, fold to basal (D),
determined in CD47hi 293T (yellow bars) or CD47hi B2M-negative 293T (blue bars), at the
indicated time (days) upon continuous culture, showing no counter-selection of SIN-RV
transduced CD47hi cells. (E) Percentage of CD47hi 293T cells (yellow bars) and untreated 293T
cells (white bars) determined at the indicated time (days) upon continuous co-culture. (F-K)
Mean with SEM of (F,I) infectious titer (TU/mL), (G,J) physical particles (ng p24/mL) and
(H,K) specific infectivity (TU/ng p24) of LV produced by untreated (black bars, n=3), CD47hi
(yellow bars, n=3) B2M-negative (orange bars, n=3), B2M-negative CD47hi (blue bars) 293T,
LV-GFP or LV-FIX producer cell lines, as indicated. No significant differences by Mann-
Whitney test. B2M-negative CD47hi cells were used to produce MHCfree/CD47hi LV batches
then used in NHP.
Fig. S5. Cytokine and chemokine response to LV, CD47hi LV, or CD47-free LV
administration in NOD mice. (A) Single values and mean with SEM of VCN measured in KC
of LV- or CD47hi-LV treated mice, as indicated, at 4-8x109 TU/Kg (circles) or 1.2-2x10
10
TU/Kg (triangles) from Fig. 2H,I, reported here for direct comparison. Mann Whitney test. (B-F)
Single values and mean with SEM of the concentration of (B) MIP-1α, C) MIP-1β, (D) MCP1,
(E) CXCL1 and (F) G-CSF in the serum of NOD mice treated with LV, CD47hi LV or CD47-
free LV as indicated, 3 hours after LV administration, or left untreated (Untr). Kruskal-Wallis
test with Dunn’s multiple comparison test.
Fig. S6. Cytokine and chemokine response to LV or CD47hi LV administration in NHPs.
(A) Quantitative analysis of large-scale LV batches compared to lab-grade LV batches (same
dataset shown in Fig. 2D reported here for comparison) produced by control (MHC-free LV or
LV, black circles) or CD47-overexpressing (MHC-free CD47hi LV or CD47hi LV, yellow
circles) immunostained with anti-CD47 antibody, or as staining control without the primary
antibody (ctrl, black triangles) and analyzed by electron microscopy (n=14-66 virions per
sample). (B-H) Mean with SEM of the concentration of (B) IL-2, (C) IL-1RA, (D) IL-18, (E)
MIP-1α, (F) MIP-1β, (G) MCP1 and (H) IL-10 in the serum of NHP, at the indicated time after
administration of vehicle, LV or CD47hi LV.
Fig. S7. LV gene therapy in NHPs. (A) Percentage of the serum concentrations of LV particles
(measured as HIV Gag p24) recovered at the indicated time (hours) after administration of LV
(black symbols) or CD47hi LV (yellow symbols) relative to the total amount of administered LV
particles. (B) Mean with SEM of the concentration of C3a in the serum of NHP treated with LV
(n=3, black squares) or CD47hi LV (n=3, yellow squares) at the indicated time after LV
administration. (C) Concentrations of anti-VSV.G IgG in the serum of NHP at the indicated time
(days) after administration of LV (black symbols) or CD47hi LV (yellow symbols). (D)
Mathematical function (lines) of the distribution of VCN between hepatocytes (Hep) and KC of
NHP treated with LV (solid black line) or CD47hi LV (dashed black line). The grey area
represents the domain of definition of LV-function, while the yellow area represents the domain
of definition of CD47hi LV-function. (E) Equations, on the left, and their solutions, on the right,
describing the mathematical functions depicted in (C). VCN in Hep and KC of LV-treated or
CD47-hi LV treated NHP are referred to as xLV, yLV, xCD47hi-LV and yCD47hi-LV respectively; the
sum of VCN in Hep (x) and in KC (y) weighted for their relative abundance in the liver (70%
and 6% respectively) results in the measured VCN in the total liver, assuming similar
contribution of the VCN of other liver cell populations to the total liver VCN in both LV and
CD47hi LV NHP groups (equations (i) and (ii)). The ratio between the VCN in Hep is
proportional to the ratio of average human FIX output among the LV and CD47hi LV groups
(equation (iii)). Domains of definition for LV and CD47-hi LV groups are derived from the
statement that VCN in Hep and KC must be >0 (equations (iv)). Estimated mean values of VCN
in Hep are 0.65 or 0.29 for CD47hi-LV or LV-treated NHP, respectively. Estimated mean values
of VCN in KC are 7.53 or 14.7 for CD47hi-LV or LV-treated NHP, respectively. Note that
Sparrow, which appears to have cleared FIX-expressing cells (i.e. transduced hepatocytes) by
cellular immune response at the end of the experiment, despite absence of LV FIX RNA
expression and ISH signal (see Fig 5 B-D), showed a residual total liver VCN of 0.5. This value
likely reflects the extent of transduction of non-parenchymal cells, which do not express the FIX
transgene and thus are spared from immune clearance, and well matches the estimated average
VCN in KC calculated using this mathematical model (equation ii). Indeed, yCD47hi-LV=12.08 if
xCD47hi-LV=0, thus residual total VCN in the total liver would be of 0.72, according to this
mathematical model (VCNtotal=12.08x0.06), very close to the measured total liver VCN.
Fig. S8. IS analysis in CD47hi LV– or LV-treated NHP spleen. (A,B) Stacked bar plots
representing the abundance of each LV IS retrieved from the spleen of LV- or CD47hi-LV
treated NHP. In (A) each LV IS is represented by a different color with the height in relative
proportion with the number of retrieved genomes (frequency) over the total. In (B) the frequency
by which individual LV integrations are found in 1 or more genomes are plotted in groups of
increasing number of genomes.
Table S1. Large-scale LV batches used in NHP study. The table shows the results of selected quality control assays performed on the large-scale
purified LV batches used in NHP study. EU: endotoxin units. TU: transducing units.
LV BATCH code 15140 15148 16106 16094 16098 16104
Particles/ml g p24/mL 23 14 28 48 28 29
Titer TU/mL 4.5x108 2.7x10
8 4.8x10
8 8.2x10
8 5.7x10
8 5.1x10
8
Infectivity TU/ng p24 1.8x104 1.7x10
4 1.4x10
4 2.5x10
4 2.3x10
4 2.4x10
4
Transgene Activity Positive Positive Positive Positive Positive Positive
Total DNA g/mL 6.4 2.4 6.7 8.4 7.0 5.9
Host cell protein ng/mL 565 507 1171 1051 710 798
Endotoxin EU/mL <0.2 <0.5 <0.2 <0.2 <0.2 <0.2
Sterility Sterile Sterile Sterile Sterile Sterile Sterile
Batch Volume Total mL 108 102 105 94 100 99
LV version MHC-free LV MHC-free CD47hi LV
Table S2. Clinical biochemistry, hematology, and hemostasis of vehicle NHP. The tables show the results of clinical biochemistry, hematology
and hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
Table S3. Clinical biochemistry, hematology, and hemostasis of LV1. The tables show the results of clinical biochemistry, hematology and
hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
LV1
Table S4. Clinical biochemistry, hematology, and hemostasis of LV2. The tables show the results of clinical biochemistry, hematology and
hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
LV2
Table S5. Clinical biochemistry, hematology, and hemostasis of LV3. The tables show the results of clinical biochemistry, hematology and
hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
LV3
Table S6. Clinical biochemistry, hematology, and hemostasis of CD47hi LV1. The tables show the results of clinical biochemistry, hematology
and hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
CD47hi-LV1
Table S7. Clinical biochemistry, hematology, and hemostasis of CD47hi LV2. The tables show the results of clinical biochemistry, hematology
and hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
CD47hi-LV2
Table S8. Clinical biochemistry, hematology, and hemostasis of CD47hi LV3. The tables show the results of clinical biochemistry, hematology
and hemostasis parameters in serum or plasma samples of the indicated NHP analyzed at the indicated time (Days) before or after LV or vehicle
administration. The normal reference values for Macaca fascicularis are shown in the column “Reference” and the mean±3 standard deviations
calculated on 14 pre-treatment samples taken from the same animals are shown in the column “Basal Range”. Values lower than basal range are
shown in blue, values higher than basal range are shown in red. RBC: red blood cells WBC: white blood cells. Gamma GT: gamma-glutamil
transpeptidase.
CD47hi-LV3
Table S9. LV IS in NHPs. The table shows the number of IS retrieved in liver and spleen
genomic DNA samples of CD47hi-LV or LV-treated NHPs. Fisher’s exact test was used to
compare the proportions of total IS retrieved in liver and spleen from the two treatment groups
(2x2 comparison indicated by the connecting line). The proportions are significantly different
even when the number of IS compared is reduced by normalizing with the amount of genomic
DNA tested (5 and 1 µg for liver and spleen respectively).
Vector NHP
N
Liver
IS
N
Spleen
IS
Fisher's
exact test
p value
Liver
IS/ng
DNA
(5 µg)
Spleen
IS/ng
DNA
(1 µg)
Fisher's
exact test
p value
LV1 2903 1732 581 1732
LV
LV2 3420 2528 P<0.0001 684 2528 P<0.0001
LV3 2574 3042 515 3042
TOT 8897 7302 1780 7302
CD47hi LV
CD47hi-
LV1 6449 1659 1290 1659
CD47hi-
LV2 4129 1656 826 1656
CD47hi-
LV3 3982 697 796 697
TOT 14560 4012 2912 4012
Table S10. LV IS in cancer genes. The table shows the number and frequency of cancer genes
targeted by LV IS in liver and spleen genomic DNA samples of CD47hi-LV or LV-treated
NHPs. The number of IS targeting cancer genes in liver and spleen tissues was calculated for IS
with an abundance ≤4 genomes and the few IS represented by >4 genomes. Annotation of the
genes was performed as described in the Methods section.
Liver
Vector NHP Tot
IS
N IS
abundance
≤4
genomes
N IS
abundance
>4
genomes
Cancer
genes
targeted by
IS≤4
genomes
Cancer
genes
targeted by
IS>4
genomes
LV
LV1 2903 2796 107 76 (2.7%) 1 (0.93%)
LV2 3420 3381 39 78 (2.30%) 1 (2.56%)
LV3 2574 2511 63 39 (1.55%) 1 (1.58%)
Mean ± SD 2.2±0.5% 1.7±0.7%
CD47hi LV
CD47hi-
LV1 6449 6029 420 109 (1.80%) 10 (2.3%)
CD47hi-
LV2 4129 3966 163 74 (1.86%) 11 (6.7%)
CD47hi-
LV3 3982 3865 117 85 (2.1%) 1 (0.85%)
Mean ± SD
1.9±1.7% 3.3±2.5%
Spleen
Vector NHP Tot
IS
N IS
abundance
≤4
genomes
N IS
abundance
>4
genomes
Cancer
genes
targeted by
IS≤4
genomes
Cancer
genes
targeted by
IS>4
genomes
LV
LV1 1732 1728 4 44 (2.5%) 0
LV2 2528 2509 19 51 (2.03%) 0
LV3 3042 3034 8 41 (1.35%) 0
Mean ± SD 2 ± 2.3% 0
CD47hi LV
CD47hi-
LV1 1659 1658 1 33 (1.99%) 0
CD47hi-
LV2 1656 1655 1 41 (2.47%) 0
CD47hi-
LV3 697 696 1 27 (3.87%) 0
Mean ± SD
2.3 ± 0.7% 0
Table S11. LV CIS. The table shows the CIS identified from liver integration datasets obtained
from each NHP treated with LV or CD47hi-LV
a) NHP ID: Identifier of the different NHPs treated by CD47hi-LV or LV.FIX
b) CIS gene: gene identified as CIS
c) Chr: Chromosome location of the CIS gene
d) N LV IS: number of LV IS targeting the indicated CIS gene
e) Span: Genomic window (in Kb) encompassing all the LV IS that constitute the CIS.
f) P value: Statistical value of the identified CIS (by the Grubbs test for outliers)
g) Synteny NHP LV CIS with human LV CIS: Region syntenic with human genomic regions
that are known to be frequently targeted by LV IS, are indicated with genes included in the
syntenic region (34-36). NHP LV CIS genes LOC102124855, KANK2 and ARSA do not have an
obvious orthologous human LV CIS region.
NHP(a)
CIS Gene(b)
NHP
Chr(c)
N
LV
IS(d)
Span
(kb)(e)
P
value (f)
Synteny NHP LV CIS
to human LV CIS (g)
LV
LV1 LOC102124855 14 6 5 0.004
KANK2 19 6 26 0.029
LV2
LOC102124855 14 10 8 0.000
LOC102137002 14 7 48 0.004
Chr11 region
(PACS1, FRMD8, SF1)
CD
47hi-
LV
CD47hi-
LV1
LOC102137002 14 7 37 0.007
Chr11 region
(PACS1, FRMD8, SF1)
SF3B2 14 6 32 0.032
Chr11 region
(PACS1, FRMD8, SF1)
LOC102140384 4 5 11 0.040
Chr6 region
(TAP1, HLA, BRD2,
BSMB9)
CD47hi-
LV2
SF3B2 14 6 32 0.033
Chr11 region
(PACS1, FRMD8, SF1)
LOC102140384 4 5 27 0.040
Chr6 region
(TAP1, HLA, BRD2,
BSMB9)
CD47hi-
LV3
LOC102124855 14 8 9 0.001
LOC102137002 14 7 14 0.005
Chr11 region
(PACS1, FRMD8, SF1)
KIAA0195 15 7 46 0.029
LOC102140384 4 5 35 0.024
Chr6 region
(TAP1, HLA, BRD2,
BSMB9)
ARSA 10 5 15 0.036
Movie S1. IV2PM of LVs, CD47hi LVs, or CD47-free LVs upon administration. IV2PM
imaging sequences of GFP-labeled LVs, CD47hi or CD47-free LVs (as indicated) uptake by
liver KCs in C57BL/6 or NOD mice. Sinusoids are labeled in white, KCs in red. LV is
administered 2 minutes after starting the recording. Bar, 30 µm; time in minutes and seconds.