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Preparation for a first-in-man lentivirus trial in cystic fibrosis patients
Eric WFW. Alton1,11, Jeffery M Beekman2, A. Christopher Boyd3,11, June Brand3,10, Marianne
S. Carlon4, Mary M. Connolly5,11, Mario Chan1,11, Sinead Conlon1,11, Heather E Davidson3,11,
Jane C. Davies1,11, Lee A. Davies5,11, Johanna F. Dekkers2, Ann Doherty3,11, Sabrina Gea-
Sorli1,11, Deborah R. Gill5,11, Uta Griesenbach1,11, Mamoru Hasegawa6, Tracy E Higgins1,11,
Takashi Hironaka6, Laura Hyndman3,11, Gerry McLachlan7,11, Makoto Inoue6, Stephen C.
Hyde5,11, J. Alastair Innes3,11, Toby M Maher8, Caroline Moran1,11, Cuixiang Meng1,11, Michael
C Paul-Smith1,11, Ian A. Pringle5,11, Kamila M Pytel1,11, Andrea Rodriguez-Martinez1,11,
Alexander C Schmidt9, Barbara J Stevenson3,11, Stephanie G. Sumner-Jones5,11, Richard
Toshner8, Shu Tsugumine6, Marguerite W. Wasowicz1,11, Jie Zhu10
1Department of Gene Therapy, National Heart and Lung Institute, Imperial College, London,
UK
2 Department of Pediatric Pulmonology, Laboratory of Translational Immunology,
Wilhelmina Children’s Hospital, University Medical Centre, Utrecht, The Netherlands
3Centre for Genomic and Experimental Medicine, IGMM, University of Edinburgh,
Edinburgh EH4 2XU, UK
4KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Department of
Pharmaceutical and Pharmacological Sciences, Belgium
5Gene Medicine Research Group, NDCLS, John Radcliffe Hospital, Oxford OX3 9DU
6ID Pharme Co. Ltd. (DNAVEC Center), Tsukuba, Japan
7Roslin Institute & R(D)SVS, University of Edinburgh, Easter Bush , Midlothian EH25 9RG,
UK
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8Fibrosis Research Group, Inflammation, Repair & Development Section, National Heart and
Lung Institute, Sir Alexander Fleming Building, Imperial College, London SW7 2AZ
9Ave Leopold Wiener, 110, 1170 Brussels, Belgium
10Lung Pathology Unit, Dept of Airway Disease Infection, NHLI, Imperial College London.
11UK Cystic Fibrosis Gene Therapy Consortium
Corresponding authors:
Professor Uta Griesenbach, Department of Gene Therapy, Imperial College London,
[email protected], Tel:+44 207 594 7927, Fax: +44 207 351 8340
Dr Chris Boyd, Centre for Genomic and Experimental Medicine, IGMM, University of
Edinburgh, [email protected]. Tel:+44 131 651 8733
Professor Deborah Gill, Gene Medicine Research Group, NDCLS, John Radcliffe Hospital,
Oxford OX3 9DU, Deborah.gil [email protected] Tel; +44 1865 221845, Fax: +44 1865
221834
Running Title: CF lentivirus gene therapy
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What is the key question?
Is a lentiviral vector, which was pseudotyped to achieve efficient gene transfer into airway
epithelial cells, suitable for progression into a first-in-man gene therapy trial in cystic fibrosis
patients?
What is the bottom line?
The data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man
CF trial in 2017 for which funding has been obtained.
Why read on?
In contrast to other viral vectors lentiviral vectors hold substantial promise for the
development of gene therapy for a range of diseases, including chronic conditions due to their
high efficacy, duration of expression and the fact that pre-existing and acquired immune
responses do not interfere with vector efficacy on repeated administration.
Twitter feed:
Preparation for a first-in-man cystic fibrosis gene therapy trial, scheduled for mid 2017, using
a lentiviral vector.
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Abstract
We have recently shown that non-viral gene therapy can stabilise the decline of lung function
in cystic fibrosis (CF) patients. However, the effect was modest, and more potent gene
transfer agents are still required. F/HN-pseudotyped lentiviral vectors are more efficient for
lung gene transfer than non-viral vectors in pre-clinical models. In preparation for a first-in-
man CF trial using the lentiviral vector we have undertaken key translational pre-clinical
studies. Regulatory-compliant vectors carrying a range of promoter/enhancer elements were
assessed in mice and human air liquid interface cultures to select the lead candidate; CFTR
expression and function were assessed in CF models using this lead candidate vector.
Toxicity was assessed and “benchmarked” against the leading non-viral formulation recently
used in a Phase IIb clinical trial. Integration site profiles were mapped and transduction
efficiency determined to inform clinical trial dose-ranging. The impact of pre-existing and
acquired immunity against the vector and vector stability in several clinically relevant
delivery devices was assessed. A hybrid promoter (hCEF) consisting of the elongation factor
1 promoter and the CMV enhancer was most efficacious in both murine lungs and human
air liquid interface cultures (both at least 2 log orders above background). The efficacy (at
least 14% of airway cells transduced), toxicity and integration site profile supports further
progression towards clinical trial and pre-existing and acquired immune responses do not
interfere with vector efficacy. The lead rSIV.F/HN candidate expresses functional CFTR and
the vector retains 90-100% transduction efficiency in clinically relevant delivery devices. The
data support progression of the F/HN pseudotyped lentiviral vector into a first-in-man CF
trial in 2017.
Keywords: Cystic fibrosis, gene therapy, gene transfer, lentivirus, viral vector
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Introduction
Our ongoing efforts to improve pulmonary gene transfer for the treatment of lung diseases
such as cystic fibrosis (CF), have led to the assessment of a lentiviral vector (simian
immunodeficiency virus [SIV]) pseudotyped with the Sendai virus (SeV) envelope proteins F
and HN (rSIV.F/HN)(1). The latter contribute significantly to the high transduction efficiency
of Sendai virus-based vectors in the airway epithelium(2).
We have previously shown that F/HN-pseudotyped SIV vector produced gene expression in
the lungs and nose of mice for the duration of their lifetime (~2 yr). Further, this expression
was at least 2-log orders higher than our lead non-viral formulation recently shown to
produce significant effects in the lungs of CF patients. Repeated daily administration led to a
cumulative dose-related increase in gene expression, whilst repeated monthly administration
to murine lower airways was feasible without loss of gene expression. There was no evidence
of chronic toxicity during a 2-year study period and F/HN-pseudotyped SIV led to persistent
gene expression in human differentiated airway cultures and human lung slices and
transduced freshly obtained primary human airway epithelial cells(3;4). In contrast to other
pseudotypes, such as VSV-G(5) or GP64(6), the F/HN pseudotype does not require co-
administration of compounds that open tight junctions or inhibit ciliary beating (3;4) , likely
making the vector more acceptable for clinical translation, in the context of CF chronic
pulmonary bacterial infection.
It has been suggested that self-inactivating (SIN) lentiviral vectors may carry less risk of
insertional mutagenesis due to inactivation of the promoter/enhancer properties in the long-
terminal repeat (LTR) which were responsible for proto-oncogene transactivation in some -
oncoretroviral vectors trials(7). In the most studied context of haematopoetic stem cells
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(HSC), differences in insertion site profiles between -oncoretroviral and lentiviral vectors
have favoured the safety profile of the latter(7;8). Further, recent studies with -
oncoretroviral(9) and lentiviral(10) vectors in Wiskott-Aldrich Syndrome patients has
allowed, for the first time, direct comparison of safety and efficacy of these vectors in man.
To date, the data support an improved safety profile of lentiviral vectors in the context of
HSC transduction. Further, clinical studies in patients with metachromatic
leukodystrophy(11) and Parkinson’s disease(12) have not raised any safety concerns for
lentiviral vectors, although longer follow-up is required.
To catalyse translation of the lentiviral vector platform into clinic we have now selected the
clinical lead candidate by generating pharmacopoeia-compliant producer plasmids and
cGMP-compliant vector production methods (Virus Production paper; in preparation), and
comparing several promoter/enhancer elements in both IC and integrase-defective (ID) (13)
vectors in mouse lung and ex vivo human models. Further, we have a) mapped integration
sites, b) characterised transduced cell types, c) assessed acute toxicity, d) determined the
effects of pre-existing immunity on transduction efficiency and toxicity, e) assessed CFTR
function of our lead vector and f) quantified vector stability in delivery devices suitable for a
first-in-man trial. We propose that this combined body of data supports progression of the
rSIV.F/HN vector into a first-in-man CF clinical trial.
Materials and Methods
See online supplement
Statistical Analysis
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All analyses were performed using GraphPad Prism6. Parametric and non-parametric data
distribution was assessed with the Kolmogorov-Smirnov normality test. Multiple group and
two groups comparisons were performed using appropriate statistical tests for a specific data
sets (see details in individual figure legends). In figure 1B+C cross sectional statistical
analysis was performed on a selected time-point and, therefore, no adjustments for
longitudinal correlations were made. The null hypothesis was rejected at p<0.05.
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Results
Selection of lead candidate vector from murine and human tissue studies
Our previously published studies were carried out with integrase-competent (IC) vectors
using the cytomegalovirus (CMV) immediate early promoter/enhancer to regulate gene
expression(3;4). Here, we compared the CMV promoter/enhancer, with the eukaryotic
elongation factor 1 (EF1) promoter and a chimeric regulatory element consisting of the
human CMV enhancer coupled to the elongation factor 1 promoter (hCEF), with the aim to
select the most efficient construct for progression into clinical trials (see online supplements
for details on vector production). All experiments were performed with the maximum
feasible volume/dose and vector titres for each configuration (see Table S1 in the online data
supplement for details). The hCEF-IC vector configurationachieved the highest and most
persistent gene expression in the murine lung and nose (p<0.001) when compared to all other
constructs 130 days after transduction (Figure 1A-C). This ~3 month time-point was
predefined for cross sectional analysis, based on previous data. The integrase-defective (ID)
vector configurations did not differ from untreated controls.
Air liquid interface (ALI) cultures were also transduced with the five vector configurations
ranging from 6-30E7 TU/ALI (n=4/group). The ID vector configuration did not differ from
untreated controls, but all three IC configurations lead to significant (p<0.005 for CMV-IC
and EF1IC, and p<0.0001 for hCEF-IC at day 5 when compared to controls) levels of gene
expression on day 5, which persisted for the hCEF-IC vector to day 14 (p<0.01). Consistent
with data from mice, the hCEF-IC configuration achieved the highest and most persistent
gene expression in ALIs (Figure 1A+D) and was consequently selected as the lead candidate
for progression into clinical trials with the designation rSIV.F/HN-hCEF from here onwards.
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Gene expression occurs in relevant airway epithelial cells
Murine lungs were transduced with rSIV.F/HN-hCEF carrying the enhanced green
fluorescent protein cDNA(EGFP) and gene expression quantified histologically.
Representative sections of airway and alveolar regions are shown (Figures 2A+B).
Approximately 15% of the target airway epithelial cells throughout the lung expressed EGFP
(Figure 2C).
To further characterise the range of cells that rSIV.F/HN-hCEF-EGFP transduced, double-
labelling using a range of cell-type specific antibodies was performed (see Table S2 in the
online data supplement). Figure 3 shows that the vector was also able to transduce, goblet and
Club cells, as well as type I and II pneumocytes and on rare occasions basal cells. We could
not detect EGFP expression in pulmonary macrophages.
rSIV.F/HN shows a similar acute toxicology profile to liposome transfection
We have previously shown that F/HN-pseudotyped lentiviral vector administration to murine
lung does not cause chronic toxicity(4). Here, we show that the survival kinetics of mice
treated with various vectors manufactured using regulator-compliant, animal-product free,
production methods did not differ from vehicle treated littermates and the animals remained
healthy on gross observation (see Figure S1 in the online data supplement).
We next assessed acute toxicity 24 hr after a single administration, as well as 24 hr after the
final of four monthly doses of rSIV-F/HN. Mild cellular infiltrates were observed in all
groups treated with lentivirus (representative image shown in Figure 4A). However, these
responses (Figure 4B) were of similar magnitude to those produced by the non-viral
formulation, which was recently used in a Phase IIb multi-dose trial and did not result in any
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significant toxicity in CF patients(14), and were not significantly different to changes seen in
control animals.
Insertion site (IS) analysis
Integration site (IS) frequency analyses calculated by GREAT using regions defined as IS ±
10 kb and IS ± 100 kb showed that 73% and 70% respectively of each were between 5 and
500 kb from TSS (see Figure S2 and results section in the online data supplement). Although
there are insufficient IS to draw definitive conclusions, an exploratory ontological survey
revealed no preference for integration near oncogenic loci (data not shown).
Effects of acquired and pre-existing immunity
In the context of viral gene transfer, acquired and pre-existing immunity to the vector is an
important consideration. We, and others, have previously shown that in contrast to other viral
vectors lentiviruses can be repeatedly administered at doses that are in a likely therapeutic
range(3;4;15).
Acquired immunity – Induction of rSIV.F/HN neutralising antibodies?
We first confirmed that the pharmacopoeia-compliant vector configuration and serum-free
production methods did not affect efficacy of repeat administration (three doses at monthly
intervals) (Virus Production paper; in preparation). As part of these experiments we also
quantified neutralising antibodies using an in vitro transduction inhibition assay and showed
that antibodies neutralising rSIV.F/HN, but not a vector pseudotyped with the vesicular
stomatitis virus G glycoprotein (r.SIV.VSV-G, negative control) neutralising antibodies were
detectable in serum of mice 28 days after a single dose of rSIV.F/HN (see Figure S3 in the
online data supplement).
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Pre-existing immunity- passive immunisation of mice with human immunoglobulin
The key F and HN proteins on rSIV.F/HN are derived from Sendai virus (SeV) which has
high sequence homology with human parainfluenza virus 1 (hPIV1)(16;17). Approximately
70-90% of people have been infected with hPIV1 and produce anti-hPIV1 antibodies. We,
therefore, assessed whether the presence of anti-hPIV1 antibodies altered transduction
efficiency and toxicity in mice. We first incubated rSIV.F/HN with either purified IgG or IgA
anti-hPIV antibodies; both inhibited transduction of rSIV./F/HN in vitro (see Figure S4 in the
online data supplement).
To assess the in vivo significance of these findings mice were then treated with human
immunoglobulins (IVIg) intra-peritoneally, or topically to the lung by nasal sniffing. The
doses achieved, and indeed exceeded, antibody titres measured in human BALF and serum
(see Figure S5 in the online data supplement). Unsurprisingly, intraperitoneal administration
of IVIg lead to higher antibody levels in serum, whereas intratracheal administration lead to
higher titres in BALF.
We next treated mice with IVIg intraperitoneally and intranasally as described above,
followed 24 hr later by rSIV.F/HN-hCEF-EGFPLux. There was no significant reduction in
gene expression in the nose or lung 7 days (Figure 5) or 28 days after transduction (data not
shown).
We also assessed potential toxicity arising from the interaction of rSIV.F/HN with the pre-
formed hPIV1 antibodies. Gross observation showed that transduced mice pre-treated with
IVIg were indistinguishable from non-IVIG treated mice. In addition, we monitored
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temperature in the acute phase immediately post virus administration and did not observe
differences in any of the groups (see Figure S6A in the online data supplement). Further,
animal and lung weight did not differ in any of the groups (see Figures S6B+C in the online
data supplement).
Pre-existing immunity- SeV induced anti-F and HN antibodies
To further assess the effect of pre-existing immunity to the key F and HN epitopes, mice
were challenged with SeV prior to transduction with rSIV.F/HN. Mice were transduced with
two doses (1 month apart) of transmission-incompetent SeV(18;19) (1E6 or 1E7
TU/mouse/dose) followed by one dose of rSIV.F/HN-hCEF-EGFPLux. We confirmed that
high levels (p<0.001) of anti-SeV antibodies were generated in BALF and serum after SeV
transduction (Figure S7E in the online data supplement); transduction with rSIV.F/HN also
increased (p<0.001) anti-SeV antibodies in serum. There was no difference in any of the
groups (Figure S7A-D in the online data supplement) with respect to weight, food and water
consumption, or body temperature over time.
Pre-existing immunity- neutralising activity of anti-hPIAV1 in human serum
We next quantified endogenous anti-hPIV1 IgG levels in serum from adults and children to
enable IgG positive and negative samples to be selected for the in vitro transduction
inhibition assay (Figure S8A in the online data supplement). rSIV.F/HN transduction was
inhibited by anti-hPIAV1 IgG positive and negative samples (Figure S8B-E in the online data
supplement). In contrast, and similar to the in vitro data obtained using murine, rSIV.VSVG-
mediated transduction was significantly (p<0.01) less affected confirming epitope specificity
(Figure S8F in the online data supplement).
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Pre-existing immunity- neutralising activity of anti-hPIAV1 in epithelial lining fluid
To address whether these endogenous anti-hPIV1 antibodies in epithelial lining fluid may
inhibit rSIV.F/HN transduction in vivo, we first quantified anti-hPIV1 IgG and IgA
antibodies in BALF from children and adults’. Approximately 5% of children and 35% of
adult samples were positive for anti-hPIV1 IgG whilst 47% of children’s and 44% of adults’
samples were positive for anti-hPIV1 IgA (Figure E9A in the online data supplement). We
next grouped subjects into: IgG+/IgA+ (n=4), IgG+/IgA- (n=5), IgG-/IgA+ (n=15) and
IgG-/IgA- (n=17) and undertook in vitro transduction inhibition assays on 31 out of the 41
samples. The average % inhibition was 36±17%, 22±13%, 10±4% and 12±4% for the four
groups respectively. Subsequently, antibody positive samples (irrespective of type) were
pooled and compared to antibody negative samples. There was no significant difference
between the groups, suggesting that the low levels of inhibition seen were unlikely to be anti-
hPIV1 antibody specific (Figure S9B in the online data supplement). As a control we also
assessed transduction inhibition of a VSVG-pseudotyped control virus in a subset of samples,
which was not different from rSIV.F/HN, suggesting that the modest inhibition is unrelated to
anti-hPIV1 antibodies.
CFTR expression and function after rSIV.F/HN-hCEF-CFTR transduction
We have previously shown that F/HN-SIV expressing CFTR under the CMV promoter
generates CFTR chloride channels as assessed by the iodide efflux assay in vitro(3). Here, we
first confirmed that the lead candidate rSIV.F/HN-hCEF, carrying a codon-optimised and CG
nucleotide-depleted CFTR cDNA (soCFTR2) also generated cAMP-dependent CFTR
chloride channels in this assay (Figure 6A).
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We next transduced the nasal epithelium of CF knockout mice with rSIV.F/HN-hCEF-CFTR
and detected significant levels of vector-specific mRNA at both 7 (p<0.005) and 28 (p<0.01)
days (Figure 6B). We also transduced CF knockout mice with rSIV.F/HN-hCEF-CFTR (5E7
TU/mouse, n=10) and assessed nasal potential difference at time-points ranging from 7 to 90
days post transduction, but were unable to document correction of the chloride transport
defect at this titre (data not shown, see supplement for further discussion).
Forskolin-induced swelling of human intestinal organoids has recently been shown to be an
accurate readout for CFTR channel activity(20). We first assessed whether rSIV.F/HN-hCEF
carrying a secreted GLux reporter gene could transduce non-CF organoids. High levels of
GLux expression were detected in all treated samples (Figure S10 in the online data
supplement). We next transduced CF organoids with rSIV.F/HN-hCEF-CFTR. We observed
that organoid swelling increased significantly (p<0.0001) in rSIV.F/HN-hCEF-CFTR treated
cultures compared to negative controls (Figure 7A-D). These functional data were supported
by Western blot detection of CFTR protein in CF organoids (Figure 7E). In conclusion, the
data indicate that lentiviral delivery of CFTR in CF intestinal organoids partially restores
their CF characteristics.
Vector stability in clinically relevant delivery devices
Vector stability was assessed in a range of delivery devices suitable for administration of
lentivirus to the nose or a restricted region in the lungs of CF patients. Ultimately, we
anticipate the vector to be delivered via an aerosol-generating nebuliser to the whole lung.
However, first-in-man safety and proof-of-concept studies may focus on local and directed
delivery of the vector to the nasal and airway epithelium. We therefore assessed vector
stability in two catheters and in a nasal spray bottle and showed that passage through these
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devices did not reduce transduction efficiency (Figure S11 in the online data supplement).
Thus, clinically relevant delivery devices suitable for administration to both the nose, as well
as for regional lung delivery have been identified.
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Discussion
In addition to the use of SIN vectors, the regulation of gene expression by internal
promoter/enhancer elements, rather than by the viral long terminal repeats (LTR) appears to
have significantly improved biosafety of the vectors(21) and have highlighted the importance
of optimisation of the expression cassettes in improving efficacy. We compared the strong
CMV promoter/enhancer (recently used in Parkinson’s disease trials(22)), the human EF1
short promoter (a commonly used ubiquitous eukaryotic promoter capable of persistent
transgene expression in the lung following non-viral gene transfer(23) and a CpG-free hybrid
promoter (hCEF) consisting of the EF1 promoter and the human CMV enhancer, which we
initially developed for, and have recently used in, our Phase IIb non-viral CF gene therapy
trial(14;24). The hCEF regulatory element led to the highest levels of gene expression in the
murine lung and nose in vivo as well as in human air-liquid interface cultures. However, we
noted good consistency between murine lung and nose as well as the human ALI models and
these data may help inform future strategies for screening promoter/enhancer elements for
lung delivery. As part of our screening strategy we also compared standard integrase-
competent (IC) and integrase-defective (ID) vectors. In our models the profile of low
transient expression from the ID vector configurations did not support further assessment.
It is likely that the hCEF regulatory element leads to higher than physiological expression of
CFTR in transduced cells. Although it has been shown that CFTR overexpression can affect
cell proliferation rates in vitro, transgenic mice overexpressing CFTR showed no adverse
effects (25). In addition, we have not seen any evidence of CFTR-related toxicity in our
recent multi-dose non-viral gene therapy trial, which also used the hCEF regulatory element
(14).
Using our lead candidate configuration, approximately 15% of relevant target epithelial were
transduced at the titres we were able to generate for these studies. This compares favourably
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with a broadly held view that between 5 and 25% of cells may require correction to provide a
meaningful level of clinical correction. The titre of 8E8 TU of rSIV-F/HN-hCEF-EGFP used
to generate these data will form the basis for estimating suitable dose ranges for the first-in-
man clinical trial.
In addition to ciliated airway epithelial cells a range of other cell types including goblet and
Club cells as well as type I and II pneumocytes were transduced. Consistent with our
previous data in the nasal epithelium(3), we confirmed that basal cells, likely progenitor cells
located in the sub-epithelial layer, were only infrequently transduced. We did not see any
evidence for transduction of pulmonary macrophages, the preferential cell type transduced by
VSV-G-pseudotyped lentivirus when applied to the mouse lung by bolus administration and
without the addition of a tight junction opener(26). The broad transduction range of
rSIV.F/HN is not surprising considering that recombinant SeV similarly transduces a wide
range of cell types(27). In the human lung the range of transduced cell types will likely
depend on the delivery method. For example, aerosols with droplet sizes of 3 to 5 m, which
are most suitable for airway delivery, will, for example, not lead to efficient vector deposition
in the alveoli.
Consistent with our previous findings, transduction with pharmacopoeia-compliant vectors
did not cause chronic or excessive acute toxicity. The mild neutrophilic infiltrates observed in
mouse lung were of similar magnitude to those produced by our non-viral formulation
pGM169/GL67A, which has already been assessed in a multi-dose phase IIb trial(14).
Oncogenesis through viral genome integration is intrinsically less likely in terminally
differentiated cells of the airway epithelium than it is in rapidly dividing cells of the
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haematopoetic lineage. We infer from this preliminary study that there is no obvious bias in
the distribution of IS at the chromosomal level; the integration pattern in relation to
transcription start sites (TSS) is similar to that reported for an SIV-based vector in primary
haematopoietic stem cells by Nienhuis et al(28). Given the terminally differentiated nature of
the airway epithelium, it is notable that the findings are broadly consistent with lentiviral IS
distributions in the eye and brain(29;30), suggesting that the genotoxicity risk of rSIV.F/HN
vectors in the airways is comparably low.
Acquired and pre-existing immune responses have affected the use of adenoviral- and adeno-
associated virus vectors for airway gene transfer(31;32). We and others have previously
shown(4;15) that lentiviral vectors can be repeatedly administered. We also show that
repeated administration is feasible despite detection of anti-rSIV.F/HN neutralising
antibodies in serum. It is difficult to make quantitative comparisons between antibody levels
obtained in this and other studies due to the variability of the in vitro transduction inhibition
assays used. However, Sinn et al reported that induction of humoral immune responses after
lentivirus pulmonary gene transfer was significantly lower than after adenovirus-mediated
gene transfer to the lung(15). It is currently unclear whether repeated administration of
lentiviral vectors is feasible because of (a) low immunogenicity, (b) rapid cell entry thereby
avoiding contact with neutralising antibodies or (c) due to other reasons. It is also unclear
how well animal models will predict responses in man. In the context of AAV-mediated liver
transduction it has become clear that murine models did not predict immune responses in
man(33).
To assess the effects of pre-existing human antibodies that may cross-react with the F and
HN protein and affect efficacy and toxicity, we pre-treated mice with human
immunoglobulins (IVIg) which contain anti-hPIAV1 IgG. IVIg was administered either
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intraperitoneally or intranasally and the dosing strategy achieved hPIAV1 antibody titres in
ranges representative of titres in human serum and BALF. The presence of pre-existing hPIV
antibodies did not alter transduction efficiency or safety in mice. In contrast, it has previously
been shown that IVIg pre-administration (12 mg/mouse) drastically reduces AAV-mediated
gene transfer to the liver(34), thereby validating the use of human antibodies in murine
models.
The cause of death of a participant in an early adenovirus clinical trial in 1999 has been
widely debated(35). One suggestion is that the presence of high level, pre-existing, anti-
adenovirus antibodies may have led to complement activation after vector administration
initiating a severe immune reaction(36). To assess whether pre-existing antibodies to F and
HN proteins alter rSIV.F/HN toxicity we pre-treated mice with two doses of SeV prior to
lentivirus transduction and did not observe enhanced toxicity compared with controls.
The majority of humoral response against hPIV is mediated by IgG and IgA antibodies(37).
IgA is the predominant immunoglobulin in the upper respiratory tract where it is locally
synthesised by plasma cells in the lamina propria. IgG is the main immunoglobulin isotype in
blood and in the lower respiratory tract. We, therefore, next assessed transduction inhibition
of rSIV.F/HN in human serum samples that were either positive or negative for anti-hPIV1
IgG and showed that transduction inhibition occurred in both hPIV1 ELISA positive and
negative samples, whereas transduction with rSIV.VSV-G was not inhibited. The reasons for
the inhibition in hPIV1 negative samples may relate to sensitivity of the ELISA assay or
cross reactivity with antibodies directed against other hPIV serotypes, which the hPIV-
specific ELISA would not have detected, but could affect transduction efficiency. We then
assessed transduction inhibition in BALF which contains IgG (derived from blood) and IgA
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(produced locally in the lung). We analysed anti-hPIAV1 IgG/IgA positive and negative
samples and did not detect evidence for rSIV.F/HN-specific inhibition. We have previously
determined that lavage fluid represents an approximately 40-fold dilution of the epithelial
lining fluid (ELF) using a standard urea assay (data not shown) and this may affect
interpretation of the results. However, Moss et al detected anti-AAV2 neutralising antibodies
in BALF of CF patients treated with AAV2(38) and Bastian et al showed that anti-adenovirus
neutralising antibodies can be detected in human BALF(39), thus supporting the notion that
vector-neutralising antibodies can be detected in BALF despite the ELF dilution factor.
We have previously shown that F/HN-SIV vector expressing CFTR under the control of the
CMV promoter generated cAMP-dependent ion transport in an in vitro iodide efflux assay(3)
and here confirmed these data for the pharmacopoeia-compliant rSIV.F/HN vector carrying
the soCFTR2 cDNA. In addition we demonstrated rSIV.F/HN transduction and CFTR
function in a recently developed intestinal organoid model(20).
We also showed that rSIV.F/HN-hCEF-CFTR transduces the nasal epithelium of CF
knockout mice efficiently (~100% vector specific mRNA compared to endogenous murine
Cftr mRNA).
CF mice do not acquire spontaneous airway infections or develop CF lung disease, but the
nasal epithelium shows the characteristic CF chloride and sodium transport defects(40). To
further assess CFTR function, we attempted to correct ion transport in the CF mouse nasal
epithelium, but were unable to do so. For these experiments we used a dose of 5E7 TU/mouse
(maximum feasible dose based on vector availability). We cannot exclude the possibility that
this titre may have been sub-therapeutic. However, the relevance of measurement of CFTR
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function in the murine nose (via in vivo potential difference) has been called into question by
Ostrowski et al who showed that expression of human CFTR under the transcriptional control
of a cilia-specific promoter did not correct ion transport in CF knockout mice(41). In addition
Grubb et al have suggested that the olfactory, rather than the respiratory, nasal epithelium
mainly contributes to the ion transport defect in CF mice(42). Considering these data we do
not expect an increase in vector dose to alter chloride secretion because we have previously
shown that our vector does not efficiently transduce olfactory epithelial cells(3). We have
also shown that the CF mouse is of limited value as a stepping stone to human gene therapy
trials. Although GL67A-mediated CFTR gene transfer partially corrected chloride transport
in the human lung and nose, reduced bacterial adherence to epithelial cells, and decreased IL-
8 and neutrophils in CF sputum(43), we were unable to correct a panel of CFTR-specific
endpoint assays in the murine nose, including ion transport, periciliary liquid height, and ex
vivo bacterial adherence(44). Our data are also consistent with an earlier study by Jiang et
al(45), who showed that GL67A-mediated gene transfer did not lead to correction of the ion
transport defect in CF mice and our own report of successful correction of chloride transport
in the human, but not in the murine, nose after transfection with DC-Chol/DOPE(46;47).
Taken together these data suggest that the CF-knockout mouse may not be a representative
model in which to assess gene transfer efficiency to human airway epithelial cells and that
correction of ion transport in mice should not be used as a go-no-go decision point for
progression into clinical trial. We have also considered the two CF pig models that have been
developed, but these animals currently die shortly after birth due to intestinal disease and,
therefore, are (a) not available in large enough numbers to conduct meaningful studies and
(b) not compatible with the time-course of lentivirus integration and gene expression.
21 | P a g e
In preparation for a first-in-man trial, which will involve regional delivery of vector to the
airways, we assessed vector stability in a range of delivery devices suitable for focal delivery.
The virus was stable in these “single-pass” delivery devices. We will conduct a single-dose,
double-blinded, dose-escalating Phase I/IIa safety and efficacy clinical study. A total of 24
adult subjects will be recruited into four groups receiving 1E8, 5E8 and 2.5E9 TU of
rSIV.F/HN-hCEF-CFTR or placebo. Dosage levels were determined principally by
considering the titre necessary to demonstrate gene expression in the mouse nose, the target
for therapeutic expression in humans (5% cells transduced), and the inter-species scaling
factor. The trial will not be designed to detect clinical efficacy, but will focus on assessing
safety and the time-course of CFTR expression and function.
In summary, in combination with the parallel development of scalable GMP-compliant vector
production methods we suggest that the mouse and ex vivo human data presented
here support progression of rSIV.F/HN into a first-in-man clinical study for cystic fibrosis
scheduled to start in 2017. In addition, the unique feature of this vector platform also opens
opportunities for other lung and systemic diseases.
22 | P a g e
Acknowledgements
We thank Samia Soussi for help with preparing the manuscript. The research was supported
by the MRC-DPFS programme, the Cystic Fibrosis Trust, Just Gene Therapy and Medicor
Foundation and ERANDA. The research was also supported by the National Institute for
Health Research (NIHR) Respiratory Biomedical Research Unit at the Royal Brompton and
Harefield NHS Foundation Trust. MH, TH and ST are employees of ID Pharma Co. Ltd,
Tsukuba, Japan. None of the other authors has a conflict of interest to declare.
23 | P a g e
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27 | P a g e
Figure legends
Figure 1: Selection of lead candidate vector
Mice and human air liquid interface (ALI) cultures were transduced with five different
lentiviral vector configurations by nasal instillation: Integrase-competent (IC) vectors
carrying the human elongation factor 1 alpha (short) promoter (EF1(23), a ubiquitous
regulatory element which has previously been used in the context of lentivirus-mediated gene
transfer(48), an in-house, synthetic chimeric promoter/enhancer consisting of CpG-depleted
versions of the human elongation factor 1 alpha (short) promoter and the human CMV
enhancer (hCEF)(24) and the original CMV-based construct, as well as integrase-defective
(ID) vectors carrying the CMV- and hCEF- promoter/enhancers (6-30E7 TU/mouse or ALI,
n=6-10 mice/group, n=4 ALI/group). All vectors carried a luciferase reporter gene for
quantification of gene expression by bioluminescence imaging. Negative control mice and
ALIs remained untreated. Gene expression was quantified in the lungs and nose of mice and
in ALIs. Photon emission adjusted for differences in vector titre. (A) Representative images
of transduced and untreated mice and ALIs, (B) quantification of photon-emission in murine
lungs, (C) quantification of photoemission in murine nose nose, and (D) in human ALIs. (B-
D) reference untreated control values are shown as a dotted line (lung control: 182±6
p/s/cm2/sr, nose control: 200±10 p/s/cm2/sr, ALI control 598±1080 p/s/cm2/sr). For each
group the mean±SEM are shown. ****=p<0.001 in lung and nose comparing hCEF-ID to all
other vectors in mice (Anova followed by Tukey post-hoc test), ***=p<0.005 comparing
hCEF-IC to untreated ALI controls (Mann-Whitney).
Figure 2: Gene expression in relevant airway epithelial cells
28 | P a g e
Mice were transduced with rSIV.F/HN-hCEF-EGFP (8E8 TU/mouse) or remained untreated
(n=3/group). Seven days after transduction mice, were culled and the lungs processed for
quantification of airway cells expressing EGFP by immunohistochemistry. (A)
Representative image of a lentivirus transduced mouse. (B) representative image of an
untreated mouse. Scale bar =50 µm. AW=airway, P=Parenchyma. (C) Quantification of
EGFP in mouse airways. Each dot represents a randomly selected airway (n=10/mouse). For
convenience the data from the untreated control mice were pooled. The horizontal bar shows
the median. The dotted horizontal line represents the consensus therapeutic threshold of 5%
airway cells. ****=p<0.0001 comparing all treated mice to controls (Anova followed by
Dunnett’s mulitiple comparison test).
Figure 3: Characterisation of transduced cells by immunohistochemistry
Mice were transduced with rSIV.F/HN-hCEF-EGPF (8E8 TU/mouse) or remained untreated
(n=3/group). Seven days after transduction mice were culled and the lungs processed for
characterisation of EGFP expressing cells by immunohistochemistry. Tissue sections were
double-stained with anti-EGFP and cell type-specific antibodies and DAPI to visualise nuclei
(blue). The left panel shows EGFP expressing cells in green, the middle panel shows cell
type-specific staining in red and the right panel shows a merged image. Arrows highlight
double-labelled cells. The merged images do not in all cases show a yellow/orange signal
when green and red signals are overlaid because the proteins stained are localised to different
cellular compartments e.g in type 1 pneumocytes the EGFP is present in the cytoplasm
whereas podoplanin is a membrane protein. Scale bar = 10µm. (A) Anti- β tubulin antibody
identifies ciliated airway epithelial cells, (B) anti-uteroglobin antibody identifies Club cells in
the airways, (C) anti-mucin 5AC antibody identifies goblet cells in the airways, (D) anti-
29 | P a g e
cytokeratin 5 antibody identifies basal cells in the airways, (E) anti-podoplanin antibody
identifies type 1 pneumocytes and (F) anti-surfactant protein C antibody identifies type 2
pneumocytes.
Figure 4: Assessment of acute pulmonary toxicity after lentivirus transduction
Mice were transduced with one or four doses (1E8 TU/dose at monthly intervals, n=5/group)
of rSIV.F/HN-CMV vectors carrying luciferase or EGFP reporter genes and histological
analysis was performed 24 hrs after the last dose. Control groups included untreated and D-
PBS treated mice and mice treated with conventional (CpG containing) luciferase plasmid
DNA/GL67A complexes or CpG-free CFTR plasmid pGM169/GL67A (n=3-5 mice/group).
(A) Representative image of a lentivirus treated mouse. AW=airway, P=parenchyma, arrow
indicates mild cellular infiltrate, (B) semi-quantitative scoring of lung inflammation.
UT=untreated, one dose of rSIV (rSIV1x), four monthly doses of rSIV (rSIV4x). Each
symbol represents an individual mouse. The horizontal bar indicates the group median.
Figure 5: Transduction efficiency in mouse lung and nose in the presence of anti-human
hPIV1 antibodies
Mice were treated with human immunoglobulin (IVIg) intraperitoneally (IP, 400lor by
nasal instillation (IN, 100lControls did not receive IVIg (n=6/group). 24 hrs after
passive immunisation mice were transduced with rSIV.F/HN-hCEF-EGFPLux (1E8
TU/mouse). Control mice remained untreated (UT). Luciferase expression was quantified in
lung and nose using bioluminescent imaging 24 hr after virus transduction. (A)
Representative images for each cohort of mice, (B) Luciferase expression nose and (C) lung.
30 | P a g e
Each symbol represents one animal. The horizontal bar indicates the group median. Two
independent experiments were performed (n=6/group/experiment) and a representative figure
is shown.
Figure 6: Confirmation of CFTR expression and function
(A) HEK293T cells were transfected with rSIV.F/HN-hCEF-CFTR or an irrelevant control
virus (negative control) at MOIs of 10 and 100. The iodide efflux assay was performed 2
days after transduction. Data are presented as mean ± SEM. **=p<0.05 compared to negative
control (Anova followed by Dunnett’s multiple comparison test). (B) CF knockout mice were
transduced with rSIV.F/HN-hCEF-CFTR (1.6E8 TU/mouse) by nasal instillation. Negative
controls were treated with PBS (n=7-8/group). Mice were culled 7 and 28 days after vector
administration and vector-specific mRNA was quantified in the lungs. Each symbol
represents one animal. The horizontal bar shown the group median. The dotted line indicates
the detection limit of the assay. ***=p<0.005 and **=p<0.01 compared to the negative
control (Kruskal-Wallis followed by Dunn’s multiple comparison test).
Figure 7: Functional confirmation of CFTR production in CF intestinal organoids
(A-D) CF intestinal organoids, carrying two class I mutations (E60X/4015delATTT) were
transduced with rSIV.F/HN-hCEF-CFTR or an irrelevant control virus (negative control).
The doses in experiment 1 ranged from 0.45-3.6E7 TU/well and in experiment 2 from 0.06-
0.45E7 TU/well (n=4/dose/experiment). Doses greater than 1.8E7 TU/well resulted in cell
toxicity and reduced chloride transport (as measured by reduced organoid swelling upon
31 | P a g e
forskolin addition) (data not shown). Analysis of chloride transport in the CF organoids 4
days after transduction therefore focussed on samples treated with 0.23-0.9E7 TU/well
(n=15-16 wells/group in two independent experiments). Four days post-transduction organoid
swelling upon addition of forskolin was assessed (measured as area under curve (AUC) over
120 min, baseline set at t = 0). Representative organoid images are shown. Data are presented
as mean ± SEM. ****=p<0.0001 compared to negative control (non-paired student t-test).
(E) At the end of the experiment organoids were harvested for protein extraction and Western
blot analysis. Lane 1: Non-CF organoids transduced with negative control virus, Lane 2-4:
CF organoid untransduced or transduced with a negative control virus, Lane 5: CF organoids
transduced with rSIV.F/HN-hCEF-CFTR.
32 | P a g e
Online supplements
Preparation for a first-in-man lentivirus trial in cystic fibrosis patients
Eric WFW. Alton, Jeffery M Beekman, A. Christopher Boyd, June Brand, Marianne S.
Carlon, Mary M. Connolly, Mario Chan, Sinead Conlon, Heather E Davidson, Jane C.
Davies, Lee A. Davies, Johanna F. Dekkers, Ann Doherty, Sabrina Gea-Sorli, Deborah R.
Gill, Uta Griesenbach, Mamoru Hasegawa, Tracy E Higgins, Takashi Hironaka, Laura
Hyndman, Gerry McLachlan, Makoto Inoue, Stephen C. Hyde, J. Alastair Innes, Toby M
Maher, Caroline Moran, Cuixiang Meng, Michael C Paul-Smith, Ian A. Pringle, Kamila M
Pytel, Andrea Rodriguez-Martinez, Alexander C Schmidt, Barbara J Stevenson, Stephanie G.
Sumner-Jones, Richard Toshner, Shu Tsugumine, Marguerite W. Wasowicz, Jie Zhu
35 | P a g e
Materials and Methods
Generation of pharmacopoeia-compliant producer plasmid
Five producer plasmids were required to generate recombinant SIV vector. The sequences
were as described(1), except that the ampicillin antibiotic-resistance gene was replaced with
the CpG-free kanamycin antibiotic resistance gene from plasmid pGM169(2). For preparation
of lentiviral vectors pseudotyped with Vesicular Stomatitis Virus G glycoprotein (VSV-G),
the two plasmids expressing the F and HN proteins were replaced with one plasmid
expressing VSV-G; full details of the construction and sequences of the pharmacopoeia-
compliant producer plasmids will be published elsewhere (Virus Production paper; In
Preparation).
In brief, multiple vector genome plasmids were constructed containing a variety of transgenes
and transcription elements. DNA fragments encoding reporter transgenes such as Luciferase
(lux), secreted Gaussia luciferase (GLux), Enhanced Green Fluorescent Protein (EGFP), and
a fusion of EGFP and Luciferase (EGFPLux) were inserted into unique NheI and ApaI
restriction sites in the vector genome plasmid. In addition, a vector genome plasmid
expressing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein was
constructed by insertion of the CpG-free NheI-ApaI DNA fragment encoding codon-
optimised soCFTR2 from plasmid pGM169(2).
Promoter/enhancer sequences, including CMV(3), hCEF(2) and EF1α(3), were incorporated
into the vector genome plasmid via BglII and NheI restriction sites, following removal of the
intron, or synthetic intronic sequence. To generate integrase-defective (ID) vectors, a D64V
point mutation in the integrase gene(4) was incorporated into the gag/pol packaging plasmid.
Vector production and titration
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Production of recombinant SIV vector expressing a variety of transgenes was performed
using the five-plasmid transient transfection method(1), except that 25K branched
Polyethylenimine (PEI)(5) was used for transfection of producer plasmids into HEK293T
producer cells grown in suspension culture in Freestyle media (Life Technologies, Paisley,
UK). Virus supernatant was harvested at 72 hours post-transfection and purified essentially as
described by Merten et al(6) except that Mustang QXT Anion Exchange membranes (Pall,
Life Sciences, Portsmouth, UK) were used instead of DEAE columns (Virus Production
paper; In Preparation). The virus was formulated in Freestyle medium aliquoted and stored at
-800C.
The viral particle titre (VP/ml) was determined essentially as described by Mitomo et al(1),
using Real-Time Quantitative PCR (Q-PCR) with primers spanning the WPRE sequence
(Forward: TGGCGTGGTGTGCACTGT; Reverse: CCCGGAAAGGAGCTGACA; Probe:
6FAM-TTGCTGACGCAACCCCCACTGG-TAMRA). Virus RNA was prepared using
QIAamp Viral RNA kit including carrier RNA (QIAGEN, Crawley, UK), followed by in-
solution DNAse (Ambion, DNAfree) and quantified by one-step RT-qPCR using QuantiTect
(QIAGEN) against a standard curve of RNA mimics containing the WPRE sequence (Virus
Production paper; In preparation). This assay was also used to measure vector-specific RNA
expression following transduction.
Functional titre, reported as transducing units per ml (TU/ml) was calculated following
transduction of HEK293F cells with serial dilutions of viral vector and extraction of DNA
using QIAamp blood DNA kit (QIAGEN) (Virus Production paper; In preparation). Viral
DNA genomes were quantified by Q-PCR (same WPRE primers as above) against a standard
curve of plasmid DNA containing the WPRE sequence, using TaqMan Universal Mastermix
(Life Technologies), then normalised to total ng DNA using PicoGreen (LifeTechnologies) or
Nanodrop ND2000 (Thermo Scientific, MA, USA). Titre was calculated from the slope of the
37 | P a g e
best-fit line on a plot of WPRE copies per well of cells against volume of virus per well of
cells. This assay was also used to calculate Vector Copy Number (VCN) in transduced cells
and tissues.
Vector Transduction:
All animal procedures were performed in accordance with the conditions and limitation of the
UK Home Office Project and Personal licence regulations under the Animal Scientific
Procedure Act (1986). Female C57BL/6N mice (6–8 wk old, Charles River Laboratories,
UK) were used for most experiments. Male and female gut-corrected CF knockout mice (~6-
12 weeks)(7) were used for assessment of CFTR expression and function. Mouse nose and
lungs and air liquid interface (ALI) cultures were transduced with the vector as previously
described(1) (see RESULTS and FIGURES for details about vector titers used) and
transduction efficiency was quantified using bioluminescent imaging (BLI, IVIS,
PerkinElmer, USA), luciferase expression in tissue homogenates as previously described(1)
and immunohistochemistry (see below).
Integration site profiling
Genomic DNA was extracted (AllPrep, Life Technologies, Glasgow, UK) from transduced
human air liquid interface (hALI) cultures and mouse nose and lung tissue samples. Viral
copy number (VCN) was determined as described above. LAM-PCR was carried out
according to Schmidt et al(8) with minor modifications. Two cocktails of enzymes, GC
(HinPII, HpyCH4IV, HpaII, TaqI: NEB, UK) and AT (MseI, BfaI, CviQI: NEB, UK) were
used to maximise flanking gDNA amplification. 25-150 ng of template gDNA was used for
each LAM-PCR procedure. To increase the probability of IS retrieval, three separate linear
amplification reactions of 300 cycles were pooled and then divided into three identical
38 | P a g e
triplicate aliquots. Products were size-fractionated after the nested PCR stage by using low
melting point agarose gel extraction to remove DNA<100 bp. The length profile and band
diversity of amplicons were visually assessed using a bioanalyser (Agilent Technologies,
Stockport, UK) before proceeding. Purified PCR products were then prepared for Ion Torrent
sequencing as recommended by the manufacturer (Life Technologies, Glasgow, UK) and
sequenced. Sequencing reads were processed bioinformatically using a pipeline of custom
Perl scripts to validate the input and remove superfluous sequences of the primers, linkers,
remaining 32 bp of LTR and other vector sequences. The pipeline allows two levels of
quality control based on degree of homology to the reference genomes termed high (≥ 90%
match with no gaps) and medium (≥ 75% match with up to one gap occurring) stringency.
The processed reads were compared to the reference mouse (GRCm38/mm10) or human
(GRCh37/hg19) genome sequences using BLAT(9). IS in repeat sequence elements were
discarded and only those IS with three or more reads of ≥ 35 bp, or two or more reads of ≥ 50
bp per BLAT hit were further analysed. For each IS, distance to transcription start sites (TSS)
and transcription site residency were determined using GREAT(10), QuickMap(11) and the
UCSC and ENSEMBL genome browsers.
Assessment of toxicity
Mice were transduced at monthly intervals by nasal instillation with one to four doses (1E8
TU/dose) of rSIV-F/HN-CMV carrying Lux or EGFP reporter gene. Controls included
untreated and D-PBS treated mice, as well as mice treated with conventional (CpG
containing) luciferase plasmid DNA/GL67A complexes or CpG-free CFTR plasmid
pGM169/GL67A complexes which were used in our recently completed Phase IIb multi-dose
trial(12). The non-viral formulation was prepared as previously described(13). All mice were
culled 24 hr after the last dose. Lung tissue sections were stained with haematoxylin&eosin
39 | P a g e
and were scored semi-quantitatively. Scoring: 0=no inflammation; >0-0.5: =very few/few
foci of inflammation in peribronchial or perivascular walls; 1: =patchy cell infiltrates in
bronchial or vascular wall in <30% of the section; 2: = localised cell infiltrates in up to 60%
of the section; 3: =cell infiltrates in >60% of the section.
Immunohistochemistry
Lungs were processed, cut and de-paraffinised using standard histological procedures. For
antigen retrieval slides were treated with pre-heated 0.1M EDTA for 10 min at 100°C.
Details of all antibodies and dilutions used are presented in Table E2. Primary and secondary
antibodies were incubated at room temperature for 1hr, 0.1% Triton X-100 in 0.1M PBS was
used for all dilutions and washing steps. After the final washes sections were mounted with
ProLong Gold antifade medium with DAPI (Molecular Probes, Molecular Probes, Life
technology, Eugene, Or, USA). Images were generated using a Zeiss LSM-510 inverted
confocal microscope (Zeiss, Jena, Germany) using a 40x or 63x oil objective (1.4 NA). The
AlexaFluor 594 was excited with the HeNe543 laser. The emission signal was filtered by a
595 nm long pass filter. AlexaFluor 488 was excited with 488 nm and detected with a 505-
550nm band pass filter. DAPI was excited with a 405 nm laser and the detection range was
420-490 nm. EGFP expressing airway epithelial cells were quantified using a 63x objective
and an Axioskop II fluorescent microscope (Zeiss, Germany) to allow a comparatively large
number of airways and cells to be quantified. Twenty random airways and approximately
5000 airway cells were assessed per mouse. Airways were selected using DAPI staining
rather than antibody staining to avoid bias for highly transduced regions, but quantification
was performed visualising EGFP expression. At a 63x magnification individual cells were
clearly visible.
40 | P a g e
Acquired and Pre-existing immunity
a. Neutralising antibodies in mouse serum
Serum of untreated mice and mice transduced with rSIV.F/HN-Lux (1E8 TU/mouse) was
collected 28 days vector administration (n=5-6/group). Sample from each group were pooled
and an in vitro transduction inhibition assay performed as described below.
b. Anti-Human Parainfluenza virus I immunity in mice (passive immunisation)
rSIV.F/HN-CMV-EGFP was first incubated with 1:2 to 1:32 serial dilutions (duplicate
samples for each dilution) of purified anti-hPIV1-3 IgG and IgA antibodies (Abcam,
Cambridge, UK) and an in vitro transduction inhibition assay was performed as described
below.
Mice (n=5/group) received human immunoglobulins (Gamunex® 10% IVIg, (Grifols
International, S.A., Barcelona, Spain) by intraperitoneal injection (400 l, 40 mg IVIG) or
intranasal instillation (100 l, 10 mg IVIG) or remained untreated. 24 hr after passive
immunisation human IgG levels were quantified in mouse serum using an anti-hPIV1 IgG
Elisa kit according to the manufacturer’s protocol (Abcam) to confirm that antibody titres are
in a range relevant to what is detectable in human serum (see below). Separate cohorts of
mice (n=6/group) were treated with IVIg as described above and transduced with rSIV.F/HN-
hCEF-EGFPLux (1E8 TU/mice in 100 l per/animal) by nasal instillation 24 hr after passive
immunisation. Control animals received either vector but no IVIg or IVIg but no vector.
Luciferase expression in nose and lung was quantified by using BLI 7 and 28 days after
transduction. The mice were then culled and luciferase expression was also quantified in
nose and lung tissue homogenate. In addition serum was collected to quantify residual levels
of human IgG 29 days post IVIg injection. To assess vector toxicity in mice with pre-
existing immunity to hPIV1 we also monitored gross behaviour, body temperature and
41 | P a g e
weight in all groups in the acute phase after vector administration (until day 5) and
bodyweight at the end of the experiment (day 28).
c. Anti-Sendai virus immunity in mice
Mice were transduced with two doses (monthly interval) of transmission-incompetent F
protein deleted Sendai virus (F/SeV, 1E6 or 17 infectious units (IU) in 100 l per mouse,
n=8/group) by nasal instillation. The SeV virus did not carry a reporter gene (F/SeV-empty)
and was produced by DNAVEC Corporation, Tsukuba, Japan as previously described (14).
Control animals remained untransduced. Prior to transduction with rSIV.F/HN-hCEF-
EGFPLux (1E8 TU/mice in 100 l per/animal) 1 month after the second SeV transduction we
confirmed that SeV transduced mice had generated anti-SeV IgG antibodies in serum and
BALF using an anti-SeV IgG Elisa kit according to the manufacturer’s protocol (Alpha
Diagnostic International Inc., distributed by Source Bioscience Life Sciences, Nottingham,
UK). Before nasal instillation the lentivirus was nebulised through the eFlow® mesh
nebuliser (Pari Medical Ltd., West Byfleet, UK) and the aerosol was collected to mimic
clinical trial conditions as closely as possible. Control animals remained either (i)
untransduced, (ii) received two doses of F/SeV-empty and (iii) no lentivirus or (iv) received
lentivirus only.
To assess vector toxicity in mice with pre-existing immunity to SeV animals were monitored
daily and assessed using a semi-quantitative gross morphological scoring system monitoring
activity, general appearance, posture, hydration and respiration over a 3 month period. Food
and water consumption, body temperature and body weight were also monitored.
d. Pre-existing anti-Human Parainfluenza I immunity in serum and broncho-alveolar lavage
fluid (BALF)
42 | P a g e
Serum samples were obtained from the Respiratory Biomedical Research Unit (BRU)
biobank using the appropriate biobank ethical approval and consent processes. Adult BALF
was obtained as previously described(15). Briefly, all samples were collected from subjects
undergoing clinically indicated bronchoscopy. A total volume of 240 ml of warmed saline
was instilled into a segment of the right middle lobe and fluid retrieved by gentle manual
aspiration. Written informed consent was obtained from all subjects and the study was
approved by the Local Research Ethics Committee (Ref 10/H0720/12). Parents of children
undergoing a clinically indicated bronchoscopy and BAL (as previously described(16)
provided informed consent for an aliquot of BALF to be used for research purposes (Ref
10/H0504/9). Post collection aliquots of unfiltered and unprocessed BALF was immediately
placed on ice then stored at -80oC.
An in vitro transduction inhibition assay was performed as previously published (17). Briefly,
HEK 293T cells were seeded into 24-well plates (4E5 cells/well) and incubated overnight at
37ºC in 5% CO2. Heat-inactivated serum (30 min at 56ºC) and BALF were serially diluted
with D-MEM (Life Technologies, UK) in a total volume of 100 l and incubated with 100 l
rSIV.F/HN-CMV-EGFP or rSIV.VSV-G-CMV-EGFP for 1 hour at 37ºC in 5% CO2. The
samples were then added to the HEK-293T cells (n=3 wells/sample) and incubated overnight
after which 200 l D-MEM containing 20% FBS (Sigma –Aldrich, UK) were added and
incubated for a further 24 hrs. Cells were then trypsinised, resuspended in D-PBS+1% BSA
and the % of EGFP positive cells was calculated using a BDTM LRS II flow cytometer (BD,
Biosciences, Canada). A minimum of 10,000 cells per well were counted. Controls included
untransduced HEK 293T cells (negative control) and cells transduced with virus not
incubated with clinical samples (serum free and BALF free positive controls). All data were
expressed as a % of serum and BALF free positive controls, as appropriate. The neutralising
43 | P a g e
antibody titre was defined as the lowest sample dilution where transduction efficiency
reached at least 50% of the positive control sample.
Assessment of CFTR function
a. Iodide efflux measurements were carried out as previously described(1) except that A549
cells (adenocarcinoma human alveolar basal epithelial cells) were used instead of HEK293T
cells, because preliminary experiments showed that the hCEF promoter leads to higher levels
of gene expression in A549 cells (data not shown).
b. CF Organoids
The Ethics Committee of the Erasmus Medical Centre Rotterdam approved this study and
informed consent was obtained. Organoids were generated from rectal biopsies after
intestinal current measurements for standard care (subject with CF) or for diagnostic purpose
(healthy control) and cultured as described previously(18;19). For viral transduction,
organoids (passage 30–40) from a 7-day old culture were trypsinized (TrypLE, Gibco) for 5
min at 37 °C and seeded in 96-well culture plates (Nunc) in 4 l matrigel (Corning) and virus
(1:1 v/v virus:matrigel) containing 100-200 single cells and small organoid fragments as
described previously(20). These cells were incubated at 37 °C 10 min and immersed in 150
l medium. The medium was refreshed (250 l) 2 days after viral transduction. Four days
after viral transduction, organoids were incubated for 30 min with 3 M calcein-green
(Invitrogen), stimulated with forskolin (5 M) and analyzed by confocal live cell microscopy
at 37 °C for 120 min (LSM710, Zeiss). The total organoid area (xy plane) increase relative to
t = 0 of forskolin treatment per well was quantified using Volocity imaging software
(Improvision). Cell debris and unviable structures were manually excluded from image
analysis based on criteria described in detail in a standard operating procedure. The area
44 | P a g e
under the curve (AUC; t =120 min; baseline =100%) was calculated using Graphpad Prism.
After forskolin stimulation and confocal analysis, organoids were lysed in Laemmli buffer
supplemented with complete protease inhibitor tablets (Roche). Lysates were analyzed by
SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane
(Millipore). The membrane was blocked with 5% milk protein in TBST (0.3% Tween, 10
mM Tris pH8 and 150 mM NaCl in H2O) and probed 3 h at RT with mouse monoclonal E-
cadherin-specific (1:10000; DB Biosciences) or CFTR-specific antibodies (450, 570 and 596;
1:3000; Cystic Fibrosis Folding consortium), followed by incubation with HRP-conjugated
secondary antibodies and ECL development. Secreted Gaussia luciferase expression was
quantified in the medium as previously described(21).
c. Nasal potential difference measurements in CF mice
The nasal epithelium of gut-corrected cystic fibrosis knockout mice(7) was transduced with
rSIV.F/HN-hCEF-CFTR and nasal potential difference measurements were performed as
previously described(22).
Vector stability in delivery devices
Virus stability was assessed in a range of delivery devices suitable for vector administration
to the lung and nose: Polythylene endoscopic wash catheter (PEC, Olympus KeyMed, UK),
Trudell AeroProbe ® catheter (Trudell Medical International, Ontario, Canada) and nasal
spray devices (GSK parts No. AR5989 30mL bottle/AR9488 30 ml actuator). An rSIV.F/HN-
vector expressing a EGFP or Lux reporter gene was passed through the delivery device and
re-collected. HEK293T cells were transduced with the processed vector or with non-
processed control virus and EGFP or Lux. Expression was quantified 48 hrs post transduction
using routine FACS (on average 20,000 cells were counted for each well) or standard
45 | P a g e
luciferase assays, respectively. Untransduced cells served as negative control. Stability in
each delivery device was assessed in at least two independent experiments. Data are
expressed as % of non-passaged control.
Statistical Analysis
ANOVA followed by a Bonferroni post-hoc test or Kruskal-Wallis test followed by Dunns-
multiple comparison post-hoc test was performed for multiple group comparison after
assessing parametric and non-parametric data distribution with the Kolmogorov-Smirnov
normality test, respectively. An independent student t-test or a Mann-Whitney test was
performed for two group parametric and non-parametric data as appropriate. All analyses
were performed using GraphPad Prism4 and the null hypothesis was rejected at p<0.05.
Results
Generation of pharmacopoeia-compliant cGMP vector
All producer plasmids were engineered to be pharmacopoeia-compliant by removal of
unnecessary base pairs and replacing the ampicillin-resistance gene with the kanamycin-
resistance gene (Virus Production paper; in preparation). To distinguish recombinant SIV
vectors generated with the pharmacopoeia-compliant producer plasmids from vector
configurations published previously, the vectors in this study are designated rSIV.F/HN or
rSIV.VSV-G throughout.
Insertion site (IS) analysis
Samples of mouse lung transduced with rSIV.F/HN produced a total of 85 unique IS when
filtered with a high stringency filter and 107 when filtered at medium stringency; the
corresponding unique IS from murine nose samples were 14 (high stringency) and 12
46 | P a g e
(medium stringency) (see Table S3 in the online data supplement). The reason for the lower
IS retrieval rate from nose compared with lung samples, despite the higher vector copy
number (VCN) in the former is unknown; insufficient gDNA samples remained to further
investigate this observation. Only two IS (at either stringency) were obtained from human
ALI samples, an outcome likely attributable to the low DNA yield from this source. The
rejection rate of reads through filtering and removal of repeat sequences was much higher
than anticipated: this was mainly due to removal of unacceptably short (< 35 bp) sequences,
implying that the size fractionation step to exclude short DNA fragments had been
suboptimal (see Table S3 in the online data supplement).
IS from murine lung and nose samples transduced with rSIV.F/HN-hCEF-EGFPlux were
mapped onto the mouse karyogram, and the distances to transcriptional start sites (TSS)
determined (see Figure S2 in the online data supplement). Positional analysis showed that
73% of total IS were located in transcription units; six in exons and 73 in introns, of which
42% were integrated in intron 2 (data not shown). Frequency analyses calculated by GREAT
using regions defined as IS ± 10 kb and IS ± 100 kb showed that 73% and 70% respectively
of each were between 5 and 500 kb from TSS (see Figure S2 in the online data supplement).
Although there are insufficient IS to draw definitive conclusions, an exploratory ontological
survey revealed no preference for integration near oncogenic loci (data not shown).
Efficient IS retrieval was compromised because samples often produced an excess of short
sequence reads. In addition, some samples failed to produce enough DNA to perform the
LAM-PCR. Future work will concentrate on refining assay sensitivity to maximise retrieval,
and systematically analysing transduced mouse airway samples. Using the accumulated IS
data, we will also use gene ontological approaches(23) to further estimate the genotoxic risk
by investigating to what extent IS appear in proximity to known oncogenic loci.
47 | P a g e
References
(1) Mitomo K, Griesenbach U, Inoue M, et al. Toward Gene Therapy for Cystic Fibrosis Using a Lentivirus Pseudotyped With Sendai Virus Envelopes. Mol Ther 2010 March 23;18(6):1173-82.
(2) Hyde SC, Pringle IA, Abdullah S, et al. CpG-free plasmids confer reduced inflammation and sustained pulmonary gene expression. Nat Biotechnol 2008 May;26(5):549-51.
(3) Gill DR, Smyth SE, Goddard CA, et al. Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther 2001 October;8(20):1539-46.
(4) Yanez-Munoz RJ, Balaggan KS, Macneil A, et al. Effective gene therapy with nonintegrating lentiviral vectors. Nat Med 2006 March;12(3):348-53.
(5) Davies LA, Hyde SC, Nunez-Alonso G, et al. The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes. Biomaterials 2012 August;33(22):5618-27.
(6) Merten OW, Charrier S, Laroudie N, et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther 2011 March;22(3):343-56.
(7) Zhou L, Dey CR, Wert SE, et al. Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR. Science 1994 December 9;266(5191):1705-8.
(8) Schmidt M, Schwarzwaelder K, Bartholomae C, et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nat Methods 2007 December;4(12):1051-7.
(9) Kent WJ. BLAT--the BLAST-like alignment tool. Genome Res 2002 April;12(4):656-64.
(10) McLean CY, Bristor D, Hiller M, et al. GREAT improves functional interpretation of cis-regulatory regions. Nat Biotechnol 2010 May;28(5):495-501.
(11) Appelt JU, Giordano FA, Ecker M, et al. QuickMap: a public tool for large-scale gene therapy vector insertion site mapping and analysis. Gene Ther 2009 July;16(7):885-93.
(12) Alton EW, Armstrong DK, Ashby D, et al. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: a randomised, double-blind, placebo-controlled, phase 2b trial. Lancet Respir Med 2015 September;3(9):684-91.
(13) Griesenbach U, Sumner-Jones SG, Holder E, et al. Limitations of the murine nose in the development of nonviral airway gene transfer. Am J Respir Cell Mol Biol 2010 July;43(1):46-54.
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(14) Hirata T, Iida A, Shiraki-Iida T, et al. An improved method for recovery of F-defective Sendai virus expressing foreign genes from cloned cDNA. J Virol Methods 2002 July;104(2):125-33.
(15) Molyneaux PL, Cox MJ, Willis-Owen SA, et al. The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2014 October 15;190(8):906-13.
(16) Stafler P, Davies JC, Balfour-Lynn IM, et al. Bronchoscopy in cystic fibrosis infants diagnosed by newborn screening. Pediatr Pulmonol 2011 July;46(7):696-700.
(17) Calcedo R, Vandenberghe LH, Gao G, et al. Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses. J Infect Dis 2009 February 1;199(3):381-90.
(18) Dekkers JF, Wiegerinck CL, de Jonge HR, et al. A functional CFTR assay using primary cystic fibrosis intestinal organoids. Nat Med 2013 July;19(7):939-45.
(19) Sato T, Stange DE, Ferrante M, et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 2011 November;141(5):1762-72.
(20) Vidoviæ D, Carlon MS, da Cunha MF, et al. rAAV-CFTRÄR rescues the cystic fibrosis phenotype in human intestinal organoids and CF mice. Am J Respir Crit Care Med. In press 2015.
(21) Griesenbach U, Vicente CC, Roberts MJ, et al. Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer. Biomaterials 2011 April;32(10):2614-24.
(22) Griesenbach U, Smith SN, Farley R, et al. Validation of Nasal Potential Difference Measurements in Gut-corrected CF Knockout Mice. Am J Respir Cell Mol Biol 2008 May 5;39(4):490-6.
(23) Biffi A, Montini E, Lorioli L, et al. Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy. Science 2013 July 11.
(24) Griesenbach U, Inoue M, Meng C, et al. Assessment of F/HN-pseudotyped lentivirus as a clinically relevant vector for lung gene therapy. Am J Respir Crit Care Med 2012 November 1;186(9):846-56.
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Supplement Figure legends
Figure S1: Survival of lentivirus treated mice
Mice were treated with regulatory compliant (RC) lentiviral vectors (rSIV.F/HN) by nasal
sniffing (6-30E7 TU/mouse, n=70 at t=0) and followed for 8 months (n=8 at t=8 months).
The remaining mice were culled at interim time-points for other analyses. Survival was
compared to previously published data that showed no evidence of chronic toxicity during a
2-year follow-up period (24).
Figure S2. Distribution of vector integration sites (IS) in rSIV.F/HN-transduced mouse
lung and nose.
Mice were instilled with rSIV.F/HN vectors expressing either EGFPlux or soCFTR2 under
the control of the hCEF promoter (1E8 TU/mouse, n=2-4/group), and culled 5 days after
transduction. (A) IS plotted on mouse karyogram. High stringency IS are shown as red
triangles; additional medium stringency IS are shown as blue triangles. (B, C) Distance to
nearest transcription start site (TSS) of regions defined as high stringency IS ± flanking
sequence: (B) regions defined as IS ± 10kb; (C) regions defined as IS ± 100 kb.
Figure S3: Neutralising antibodies in mouse serum
Mice were treated with rSIV.F/HN-CMV-EGFP (1E8 TU/mouse) or remained untreated
(n=6/group). 28 days after transduction mice were culled and the serum pooled to perform an
in vitro transduction inhibition assay in 1:4 to 1:64 serial dilutions of serum (lower dilutions
were not feasible due to the small amounts of mouse serum available) to quantify neutralising
antibodies. (A) Inhibition of rSIV.F/HN transduction in serum of untreated mice, (B)
50 | P a g e
inhibition of rSIV.F/HN transduction in serum of transduced mice, (C) inhibition of
rSIV.VSV-G transduction in serum of untreated mice, (D) inhibition of rSIV.VSV-G
transduction in serum of transduced mice. Transduction efficiency is presented relative to
serum free (SF) controls.
Figure S4: In vitro inhibition of rSIV.F/HN by purified hPIV IgG and IgA antibodies
rSIV.F/HN-CMV-EGFP was incubated with 1:2-1:32 serial dilutions of purified IgG and
IgA anti-hPIV antibodies and an in vitro transduction inhibition assay was performed. All
data are expressed as a ratio of the no-antibody control samples (0). The assay was performed
in duplicate.
Figure S5: Anti-hPIV1 IgG in mouse serum and BALF after IVIg injections
Mice were treated with human immunoglobulin (IVIg) intraperitoneally (IP, 100 or
400lor by nasal instillation (IN, 100lControls remained untreated (n=5/group). 24
hr after passive immunisation mice were culled and hPIV1 IgG was measured in serum (A)
and broncho-alveolar lavage fluid (BALF) (B). Antibody titres (measured as OD450 nm)
were compared to titres measured in human serum (n=43) and BALF (n=47). Each symbol
represents one animal/human samples. The horizontal bar indicates the group median. The
dotted line indicates the sensitivity limit of the assay. The experiment was performed in
duplicate and a representative figure is shown.
51 | P a g e
Figure S6: Toxicity of rSIV.F/HN transduction in the presence of anti-human hPIV1
antibodies
Mice were treated with human immunoglobulin (IVIg) intraperitoneally (IP, 400lor by
nasal instillation (IN, 100lControls did not receive IVIg (n=6/group). 24 hrs after
passive immunisation mice were transduced with rSIV.F/HN-hCEF-EGFPLux (1E8
TU/mouse). Control mice remained untreated (UT). (A) Body temperature was measured in
the acute phase and is expressed as change from pre-treatment baseline. Day 1= one day after
IVIg administration, day 2= one day after SIV transduction, day 3= two days after SIV
transduction, day 4= three days after SIV transduction. Group mean±SEM are shown (B)
Body weight was measured 4 and 28 days after SIV transduction, (C) Lung weight was
determined 28 days after SIV transduction. (B+C) Each symbol represents one animal. The
horizontal bar shows the group median.
Figure S7: Toxicity of rSIV.F/HN transduction in the presence of anti-Sendai virus
antibodies
Mice were transduced with two doses of F/SeV (1E6 or 1E7 TU/dose) and one dose of
rSIV.F/HN (1E8 TU) at approximately monthly intervals by nasal instillation. Control groups
remained untransduced (UT) or received two doses of F/SeV (1E7 TU/dose) or one dose of
rSIV.F/HN (1.8E8 TU) (n=6-11/group). (A+B) Water and food consumption, (C)
Bodyweight, (D) Changes in body temperature (due to technical reasons body temperature
was only collected 30 days after the first F/SeV transduction. Circle: =untreated, square:
=SeV only, triangle: =SIV only, inverted triangle: =SeV (1E6TU/dose) + SIV, diamond:
=SeV (1E7TU/dose) + SIV, (E) Anti-SeV antibody levels were measured in serum and
BALF at post-mortem. Each symbol represents one mouse. The median is shown as a
52 | P a g e
horizontal bar. Serum: ****=p<0.001 compared to all other groups, $$$$=p<0.001 compared
to SeV treated groups, BALF: ****=p<0.001 compared to untreated mice, $$$=p<0.005
compared to SeV treated groups (Anova followed by Tukey multiple comparison test).
Figure S8: Neutralising antibodies in human serum
Anti-hPIV1 IgG antibodies were quantified in human serum (34 adults (age 21-70) and 9
children (age 2-15) (A). The horizontal line indicates the limit of detection for the assay.
Open symbols represent samples positive and negative for hPIV1 IgG antibodies that were
selected for the transduction inhibition assay. rSIV.F/HN-CMV-EGFP or rSIV.VSV-G -
CMV-EGFP were incubated with anti-hPIV1 IgG positive and negative human serum
(n=4/group) and an in vitro transduction inhibition assay with serial dilutions ranging from
1:8 to 1:1024 was performed to quantify neutralising antibodies (lower dilutions were not
feasible due to the small amounts of serum available). Transduction efficiency is presented
relative to serum free (SF) controls. Representative results are shown. (B) rSIV.F/HN in IgG
positive serum, (C) rSIV.F/HN in IgG negative serum, (D) rSIV.VSV-G in IgG positive
serum, (E) VSV-G-SIV in IgG negative serum. The same serum samples were used in B+D
and C+E. (F) Comparison of rSIV.F/HN and VSVG-SIV transduction inhibition. Data are
expressed as dilution at which 50% of transduction relative to serum controls is achieved.
Open and closed circles show IgG-positive and IgG-negative adults, open and closed squares
show IgG-positive and IgG-negative children. ***=p<0.005 (Mann-Whitney test).
53 | P a g e
Figure S9: Neutralising antibodies in human broncho-alveolar lavage fluid
Anti-hPIV1 IgG antibodies were quantified in human lavage fluid (n=19 adults (age 18-86),
n=12 children (age 2-16)) (A). The horizontal lines indicate the limit of detection for the
assay. Circles and squares represent serum from adults and children, respectively. Open and
closed symbols represent antibody negative and positive samples, respectively. rSIV.F/HN-
CMV-EGFP or rSIV.VSV-G-CMV-EGFP were incubated with anti-hPIV1 IgG- and IgA-
positive and negative human BALF and an in vitro transduction inhibition assay was
performed to quantify neutralising antibodies (B). Transduction efficiency is presented
relative to BALF free (BF) controls. Each symbol represents one sample. The horizontal line
indicates the group mean. To allow robust statistical analysis samples that were positive for
either anti-hPIV1 IgG or IgA antibodies were pooled (white symbols=IgG+/IgA+, grey
symbols= IgG+/IgA-, black symbols= IgG-/IgA+).
Figure S10: Gaussia luciferase production in intestinal organoids
CF intestinal organoids were transduced with rSIV.F/HN-hCEF-soGLux or an irrelevant
control virus (NC, n=16) or remained untransduced (UT, n=4). The doses in experiment 1
ranged from 0.45-3.6E7 TU/well and in experiment 2 from 0.06-0.45E7 TU/well (one
well/dose). Four days post-transduction Gaussia luciferase (Glux) was measured in the
culture medium. Each bar represents one sample except NC and UT where data shows
mean±SEM. ****=p<0.0001compared to NC (Unpaired student t-test).
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Figure S11: Vector stability in delivery devices
An rSIV.F/HN- vector expressing EGFP was passed through the delivery device and re-
collected. A polythylene endoscopic wash catheter, a Trudell AeroProbe ® catheter and a
metered-dose nasal spray devices were assessed. HEK293T cells were transduced with the
processed vector or with non-processed control virus and EGFP expression was quantified 48
hrs post transduction using routine FACS assays, respectively (n=6 wells/group/experiment).
Untransduced cells served as negative control. Stability in each delivery device was assessed
in at least two independent experiments. Data are expressed as % of non-passaged control.
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