Gene Therapy

https://doi.org/10.1038/s41434-019-0106-3

BRIEF COMMUNICATION

Distinct transduction of muscle tissue in mice after systemic delivery

of AAVpo1 vectors
Warut Tulalamba1,5 Jonas Weinmann2,6 Quang Hong Pham1 Jihad El Andari
● ● ●
2 ●
Thierry VandenDriessche1,3 ●

Marinee K. Chuah1,3 Dirk Grimm 2,4

●

Received: 21 June 2019 / Revised: 7 September 2019 / Accepted: 27 September 2019

© Springer Nature Limited 2019

Abstract
The human musculature is a promising and pivotal target for human gene therapy, owing to numerous diseases that affect
this tissue and that are often monogenic, making them amenable to treatment and potentially cure on the genetic level.
Particularly attractive would be the possibility to deliver clinically relevant DNA to muscle tissue from a minimally invasive,
intravenous vector delivery. To date, this aim has been approximated by the use of Adeno-associated viruses (AAV) of
different serotypes (rh.74, 8, 9) that are effective, but unfortunately not specific to the muscle and hence not ideal for use in
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patients. Here, we have thus studied the muscle tropism and activity of another AAV serotype, AAVpo1, that was previously
isolated from pigs and found to efficiently transduce muscle following direct intramuscular injection in mice. The new data
reported here substantiate the usefulness of AAVpo1 for muscle gene therapies by showing, for the first time, its ability to
robustly transduce all major muscle tissues, including heart and diaphragm, from peripheral infusion. Importantly, in stark
contrast to AAV9 that forms the basis for ongoing clinical gene therapy trials in the muscle, AAVpo1 is nearly completely
detargeted from the liver, making it a very attractive and potentially safer option.

Introduction
These authors contributed equally: Warut Tulalamba, Jonas
Weinmann, Quang Hong Pham Among the many tissues that are considered as preclinical
* Thierry VandenDriessche or clinical targets for therapeutic gene transfer, the muscu-
thierry.vandendriessche@vub.be lature including skeletal muscle, heart, and diaphragm is
* Marinee K. Chuah one of the most promising and most important, due to its
marinee.chuah@vub.be central role in numerous human diseases. The best-known
* Dirk Grimm example may be Duchenne muscular dystrophy (DMD), a
dirk.grimm@bioquant.uni-heidelberg.de devastating, rare, and hereditary muscle-wasting disorder
that is caused by mutations in the dystrophin gene and
1
Department of Gene Therapy & Regenerative Medicine, Vrije characterized by progressive skeletal muscle deterioration
Universiteit Brussel (VUB), B-1050 Brussels, Belgium
concurrent with pulmonary and cardiac failure. These
2
Department of Infectious Diseases/Virology, BioQuant Center, symptoms result from the loss of the muscle-protective
Heidelberg University Hospital, University of Heidelberg, 69120
function normally played by the dystrophin protein, which
Heidelberg, Germany
3
mechanically links the cytoskeleton and the extracellular
Department of Cardiovascular Sciences, Center for Molecular &
matrix in healthy muscle cells, thus protecting the muscle
Vascular Biology, University of Leuven, 3000 Leuven, Belgium
4
from contraction-induced damage. In the ~1 in 5000 males
German Center for Infection Research (DZIF) and German Center
who lack dystrophin, muscle fibers are damaged and can no
for Cardiovascular Research (DZHK), Partner site Heidelberg,
Heidelberg, Germany longer regenerate, causing early death in the first three
5 decades of life from cardiorespiratory failure.
Present address: Research Division, Faculty of Medicine Siriraj
Hospital, Mahidol University, 10700 Bangkok, Thailand Two other quintessential hereditary muscle disorders that
6 are also caused by single-gene defects, that likewise
Present address: Boehringer Ingelheim Pharma GmbH & Co. KG,
Drug Discovery Sciences, Birkendorfer Straße 65, 88400 provoke significant morbidity and mortality due to skeletal
Biberach an der Riß, Germany muscle, cardiac and/or diaphragm dysfunction, and for

which no effective bona fide cure is available are myo-
tubular myopathy (MTM) and glycogen storage disorder
(GSD) type II (also known as Pompe disease). MTM
patients typically exhibit hypotonia, muscle weakness, and
respiratory failure at birth due to the disease's effect on
skeletal muscle and diaphragm, and they typically quickly
succumb to the disease even under intensive support,
including gastrostomy feeding and mechanical ventilation.
In contrast to MTM, GSD II not only affects skeletal muscle
and diaphragm but also the heart. In GSD II patients,
glycogen is no longer broken down effectively into glucose,
due to a deficiency of the lysosomal enzyme acid
α-glucosidase and a resulting lysosomal storage defect.
Similar to MTM, children affected by the most severe form
of GSD II and lacking medical intervention suffer from
myopathy with progressive muscle weakness and die within
their first year of life from cardiorespiratory failure.
Fortunately, over the last years, new hope has emerged
for patients affected by these and other myopathies due to
the successful implementation and validation of gene
therapies based on the recombinant Adeno-associated virus
(AAV) vectors including through their systemic delivery
[1]. In fact, for MTM patients, muscle-directed gene therapy
using AAV is currently the only clinically relevant option.
The most notable AAV variant that has shown great pro-
mise in preclinical and clinical evaluation is serotype 9
(AAV9) due to its high efficiency in skeletal muscle, dia-
phragm, and heart, as extensively demonstrated by e.g., the
Müller lab [2, 3], the Duan lab [4, 5], and many others
[6–8]. AAV9 was also among the lead candidates in a next-
generation sequencing-based, high-throughput screen of
distinct AAV capsids variants in adult mice recently
performed in our lab (Weinmann et al. manuscript in pre-
paration). Notably, AAV9 is likewise the preferred capsid in
several clinical trials in DMD patients including those
conducted by the companies Solid Biosciences and Pfizer,
and it is the lead for Exonics Therapeutics who harnesses
AAV9 as a carrier for the CRISPR machinery, to induce
therapeutic exon skipping in mutated dystrophin genes
[9–11]. Others focus on alternative serotypes, including
Sarepta Therapeutics (AAVrh.74) or Audentes Therapeutics
(AAV8), which were shown to be effective in animal
models of MTM [12–14]. However, the high vector doses
used may be hazardous owing to possible immunotoxicity
in humans, especially after gene transfer in the liver that
also occurs with these promiscuous viral isolates [8, 15–20].
Similarly, muscle-directed gene therapy approaches using
AAV were assessed for treatment of GSD II in preclinical
models, but again required high vector doses to achieve
wide-spread muscle transduction [21–24].
In efforts to solve the problems with the current gen-
eration of AAV vectors for muscle gene therapy, others and
we have started to assess alternative serotypes or synthetic
W. Tulalamba et al.
capsids for their efficiency and specificity in skeletal mus-
cle, heart, and diaphragm. This includes a series of reports
from the Kobinger and Auricchio labs who have compre-
hensively studied a collection of capsids which they had
isolated from porcine tissues [25–27]. In the first of these
reports [25], Bello et al. centered on AAVpo1, a close
relative of AAVgo.1, and showed that intramuscular
injection into the tibialis anterior muscle resulted in rela-
tively robust transduction, comparable with AAV5 that was
used as a benchmark. Moreover, following the systemic
delivery of AAVpo1 via tail vein injection, relatively high
vector copy numbers were detected in skeletal muscle,
albeit functional transduction was not investigated. Further
noteworthy is that no neutralization of AAVpo1 was
observed, neither with antisera against a set of other wild-
type (wt) AAVs (serotypes 2–8) nor with high doses of up
to 12 mg/ml of human immunoglobulins (IVIg). In a 2014
follow-up study [26], the same labs reported similarly
encouraging data with other porcine isolates and showed
that AAVpo2.1, a hybrid created from AAVpo2 and
AAVpo6, also preferentially targets the skeletal muscle
(next to kidney and spleen) following the peripheral deliv-
ery. Again, however, this was only detected on the level of
vector DNA copy numbers, whereas functional transduction
data were absent. Moreover, AAVpo2.1 as well as three
other porcine variants (po4 to po6) robustly transduced the
skeletal muscle after direct injection, akin to AAVpo1 in
the original report [25], and all except for AAVpo6 were
largely resistant to IVIg neutralization [26].
Results and discussion
Analysis of in vivo luciferase expression reveals
a unique biodistribution pattern of systemically
delivered AAVpo1
To analyze the expression pattern of AAVpo1 in adult
CB17-SCID mice following the systemic delivery via tail
vein injection, we produced vectors encoding the Luc2
firefly luciferase reporter under the muscle-tropic, intron-
containing SPc5-12 promoter (Fig. 1a). For direct compar-
ison with the current gold standard in systemic muscle
transduction, wt AAV9, the latter was produced and
analyzed in parallel. Per vector and mouse, 4 × 1010
recombinant AAVpo1 or AAV9 particles were delivered via
intravenous injection (corresponding to roughly 1.6 × 1012
particles per kilogram), and two as well as 4 weeks later,
whole-body images of luciferase expression were acquired.
Finally, the animals were sacrificed another week later (i.e.,
5 weeks after the initial AAV injection) and all major
organs were removed for an additional ex vivo quantifica-
tion of luciferase levels (workflow in Fig. 1b).
Distinct transduction of muscle tissue in mice after systemic delivery of AAVpo1 vectors
A Quantitative ex vivo comparison of reporter gene
SPc5-12 pA
luc2 rep 2 cap 9
ITR intron ITR cap po1
B visual inspection of the whole mice (Fig. 2a) or the iso-
9 po1
2w 2w 1w
imaging: in vivo in vivo ex vivo
Fig. 1 Constructs and workflow for analysis of AAVpo1 and AAV9
vectors. a (left) AAV vector construct expressing a firefly luciferase
reporter (Luc2) from a muscle-tropic SPc5-12 promoter. ITR inverted
terminal repeat, pA synthetic polyadenylation site. (right) AAV helper
constructs co-encoding rep of AAV2 together with cap of either
AAV9 (benchmark) or AAVpo1. b Experimental workflow. Mice
were injected with one of the two vectors, followed by the whole-body
imaging of luciferase expression after 2 or 4 weeks and their sacrifice
after a total of 5 weeks for organ harvesting and ex vivo luciferase
measurements. This figure contains clipart from Servier Medical Art
(https://smart.servier.com/)
As shown in Fig. 2a, we observed an expected increase
in luciferase expression from week 2 to 4 for both vectors,
in line with the known in vivo kinetics of conventional,
single-stranded AAV vectors [28]. Intriguingly, visual
inspection of the whole-body images implied fundamental
differences in the biodistribution of the two capsid variants.
Firstly, luciferase expression was slightly reduced overall in
the AAVpo1 cohort as compared with the AAV9 standard.
Secondly, and more notably, we detected a significant
decrease of luciferase signals in the abdominal region
(in particular the area where the liver resides) in mice
treated with the AAVpo1 vector relative to the whole body
of the same mice as compared with the AAV9 group. This
indicated that, following intravenous delivery, the AAVpo1
capsid is capable to detarget vector gene expression from
the liver.
This was indeed confirmed upon comprehensive ex vivo
measurements of luciferase expression in individual organs
including various muscle types, i.e., diaphragm, quadriceps,
gastrocnemius, tibialis, biceps, triceps, heart, and liver.
Most strikingly, while all muscle tissues were robustly
transduced with both vectors (Fig. 2b), luciferase expression
was also prominently detected in the livers of the AAV9
cohort (where it was even stronger than in the diaphragm)
but at background level in the AAVpo1-transduced animals,
akin to the PBS control group.
expression confirms liver detargeting of AAVpo1
For further and more quantitative validation, we deter-
mined luciferase expression as photons per second and
square centimeter of tissue (ph/s/cm2/sr) in all samples
(Fig. 3). Indeed, this confirmed our conclusions from the
lated tissues (Fig. 2b), by showing that AAVpo1 had
robustly transduced all tissues albeit at a twofold to
threefold lower level as compared with AAV9. Impor-
tantly, congruent with the images in Fig. 2b, luciferase
expression in the liver was entirely undetectable in the
AAVpo1-treated mice, where we detected <1000 ph/s/
cm2/sr (averaged 888.6 ph/s/cm2/sr) similar to the PBS-
treated mice (averaged 642.7 ph/s/cm2/sr). This is in stark
contrast to the AAV9 cohort where we consistently
measured around 20,000 ph/s/cm2/sr.
Quantification on the DNA and RNA levels validates
the unique biodistribution of AAVpo1
The aforementioned data had revealed the ability of
AAVpo1 to functionally transduce all muscle tissues from
peripheral delivery, in the absence of detectable transgene
expression in the liver. To our knowledge, this exclusive
pattern of AAVpo1's bioactivity has not been reported
before, and it distinguishes this AAV serotype from all
other isolates that have previously been evaluated in the
muscle. To assess whether this unique biodistribution
profile was also reflected on the level of the vector DNA,
we measured AAV genome copy numbers in the same
tissues. As is evident from Fig. 4a, the results indeed
mirror the protein expression data in Figs. 2 and 3. In most
tissues, AAVpo1 copy numbers were within a twofold to
threefold range of those of AAV9, with the most notable
exception of the liver where AAVpo1 DNA was >100-
fold less abundant than that of AAV9. This striking
AAVpo1 detargeting from the liver that we consistently
observed on the protein and DNA level is even more
apparent in a direct side-by-side comparison of vector
biodistribution in all eight tissues that we studied here. As
seen in Fig. 4b, this shows a highly similar profile for both
capsids except for the liver, which is clearly the pre-
dominant target for AAV9 but inconspicuous in the case
of AAVpo1.
In addition, quantitative real-time (qRT-)PCR was per-
formed to confirm the luciferase expression profile by
measuring the levels of Luc2 mRNA in major organs of the
AAV-injected mice (Fig. 5a). The results show that the
expression level of Luc2 mRNA from the AAVpo1 was
only about twofold to threefold reduced as compared
with AAV9 in the targeted muscle tissues (diaphragm,
 W. Tulalamba et al.

A AAV9 AAVpo1 PBS

Week 2

Week 4

B AAV9 AAVpo1 PBS

Diaphragm

Quadriceps

Gastrocnemius

Tibialis

Biceps

Triceps

Heart

Liver

Kidneys

Spleen

Lungs

Brain

Fig. 2 Whole-body and tissue imaging of AAV9- or AAVpo1-injected PBS, exemplifying the lack of any luciferase signal (also at week 4,
mice. a Whole-body images of luciferase expression in mice injected not shown). b Images of luciferase expression in the shown organs
intravenously with the shown vectors, taken at 2 or 4 weeks after from the same mice in a, harvested 5 weeks after AAV injection
injection. Shown on the right are three control mice injected with (see Fig. 1b)

quadriceps, gastrocnemius, tibialis, biceps, triceps, and
heart) (Fig. 5a). On the other hand, the expression levels of
Luc2 mRNA in the livers of AAVpo1-treated mice were
substantially reduced (>100-fold reduction) in comparison
with AAV9 (Fig. 5a), confirming the liver-detargeted
AAVpo1 transduction profile shown in Fig. 4 above.
Lastly, the ratios of Luc2 mRNA and DNA copy num-
bers were calculated. Similar values were detected within all
tissues between the AAV9- or AAVpo1-treated groups.
This is consistent with a comparable expression efficiency
of the Luc2 mRNA from the same SPc5-12 promoter-driven
construct, regardless of the viral vector capsid used. Hence,
the differences in expression levels of the Luc2 reporter
gene and in the biodistribution of AAVpo1 versus AAV9
reflect differences in gene transfer per se due to the unique
properties of the AAVpo1 capsid.
Distinct transduction of muscle tissue in mice after systemic delivery of AAVpo1 vectors
Fig. 3 Quantification of luciferase protein expression as photon signals
in the organs from Fig. 2. Shown data are mean ± s.e.m. n = 5
for treated/injected group, and n = 3 for PBS/noninjected group.
Conclusion
The three previous reports [25–27] that have studied
porcine-derived AAV isolates in mice have implied their
potential for muscle-directed gene therapy of diseases, such
as DMD, MTM, or GSD II, by showing (i) robust trans-
duction of skeletal muscle after direct intramuscular
AAVpo1 injection, (ii) detection of AAVpo1 vector DNA
in skeletal muscle and heart following intravenous delivery,
and (iii) little to no neutralization of AAVpo1 vectors by
IVIg or anti-AAV sera. However, neither of these reported
studies has assessed actual transgene expression in muscle
tissues following intravenous AAVpo1 vector delivery nor
has the diaphragm been included in prior work. The data
Statistical analyses were performed using Mann–Whitney U-test. *p <
0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns nonsignificant
reported here now close these seminal gaps, by illustrating
strong transgene expression in all major muscle types,
including the diaphragm, following peripheral AAVpo1
vector infusion, to levels that are within a twofold to
threefold range of what can be achieved with AAV9 that
often serves as the gold standard for muscle-directed gene
therapies.
Most notably, in contrast to AAV9, we found that
AAVpo1 is markedly detargeted from the liver at the pro-
tein and DNA levels. This liver detargeting likely results
from the combination of the inherent tissue/cell specificity
of this AAV variant and the use of a muscle-tropic promoter
(i.e., SPc5-12 [29–31]) in our study. Conversely, significant
levels of transgene expression were detected in the liver

Fig. 4 Quantification of AAV vector DNA copy numbers in a subset
of the organs from Fig. 2. a Direct comparison of AAV9 versus
AAVpo1 vector copy numbers in eight different organs. b Compila-
tion of all copy numbers for AAV9 or AAVpo1, respectively, in the
with this promoter in the context of AAV9 but not with
AAVpo1. This highlights the unrestricted tropism of the
AAV9 serotype and its substantial activity in tissues outside
the muscle, which has important implications for its future
use in muscle-directed gene therapy trials and its develop-
ment as a therapeutic product. In particular, the significant
liver detargeting obtained with AAVpo1 may minimize the
risk of vector dose-dependent hepatotoxicity, which was a
common adverse event associated with systemic delivery of
W. Tulalamba et al.
eight studied organs. Shown data are mean ± s.e.m. n = 5 for treated/
injected group, and n = 3 for PBS/noninjected group. Statistical
analyses were performed using Mann–Whitney U-test. **p < 0.01,
****p < 0.0001. ns nonsignificant
AAV vectors of different serotypes (e.g., AAV2, AAV5,
AAV8, and AAV9) in the context of different clinical trials
[15, 32–36]. At high vector doses, this untoward liver
transduction and concomitant inflammation even resulted in
grade 4 toxicity consistent with an >30-fold increase in liver
transaminases [35], warranting the use of transient immune
suppression with corticosteroids.
Finally, we note again that other porcine AAV variants,
AAVpo2.1, po4, po5, and po6, have previously been
Distinct transduction of muscle tissue in mice after systemic delivery of AAVpo1 vectors

Fig. 5 Expression analysis of Luc2 mRNA from each organ of AAV9-
and AAVpo1-treated mice. a Luc2 mRNA expression as measured by
quantitative real-time (qRT-)PCR. Luc2 expression levels were nor-
malized to endogenous mGapdh mRNA. b Transcription efficiency of
Luc2 vectors. Levels of Luc2 mRNA were normalized to Luc2 DNA
copies within the same organs. Shown data are mean ± s.e.m. n = 5
for treated/injected group, and n = 3 for PBS/noninjected group.
Statistical analyses were performed using Mann–Whitney U-test. *p <
0.05, ****p < 0.0001. ns nonsignificant

reported to perform well in the mouse muscle following
direct intramuscular injection and that one of them,
AAVpo2.1, was also detargeted from the liver after per-
ipheral administration, at least on the vector DNA level
[26]. Combined with the new data reported here and the low
reactivity of all tested porcine AAV capsids with IVIg or
antisera [25, 26], this implies a great potential of this clade
of AAV variants and supports their further characterization
and development as vectors for muscle-directed gene
therapies, including their molecular evolution [37, 38] via
techniques, such as DNA family shuffling [39, 40] or
ancestral reconstruction [41]. More generally, it showcases
the vastly unexplored potential of AAV variants other than
the "mainstream" serotypes and motivates the further
exploration of these and other parvoviruses as vectors for
human gene therapy, including bocaviruses that we have
recently identified as another very promising vector for gene
transfer into primary skeletal muscle cells [42].
Materials and methods
AAV production
The AAV helper constructs used for the production of
AAVpo1- or AAV9-based vectors and co-expressing rep of
AAV2 together with the selected cap cDNA have recently
been reported by us [40]. Likewise, we have recently
described the SPc5-12 promoter-driven luciferase construct
that was packaged into the two capsids, as well as asso-
ciated protocols for AAV production, purification, and
titration [31].
In vivo bioluminescence analysis
Bioluminescence imaging was performed for in vivo luci-
ferase expression. Four-week-old CB17/IcrTac/Prkdcscid
male mice were intravenously injected with purified AAV9
or AAVpo1 capsids expressing Luc2 as reporter gene under
the SPc5-12 promoter (4 × 1010 vector genomes per mouse).
Mice were randomly assigned to the experimental groups,
and the number of mice in each group was estimated based
on post hoc power analysis. At 2 and 4 weeks post injection,
the mice were subjected to bioluminescence imaging ana-
lysis using an in vivo optical imaging system (Photo-
nImager, Biospace Lab, Paris, France). Prior to imaging,
mice were intravenously administered with a D-Luciferin
substrate in normal saline (30 mg/ml) at a dose of 150 mg/kg
of body weight. Quantitative image analysis of individual
organs was performed at 5 weeks post vector injection. Mice
were euthanized by cervical dislocation within 1 min post
D-Luciferin administration. In vivo bioluminescence was
expressed in ph/s/cm2/sr and displayed as a pseudo-colored
W. Tulalamba et al.
overlay onto a gray-scale animal image using a multicolor
scale. No animals were excluded from the analysis, and the
investigator was not blinded during analysis.
Transduction efficiency, biodistribution, and mRNA
levels
Transduction efficiency and biodistribution were evaluated by
quantifying Luc2 transgene copy numbers in the different
organs and tissues. Genomic DNA and total RNA were
extracted from different tissues of mice injected with the
various AAV vectors using a Qiagen AllPrep DNA/RNA
purification kit (Qiagen, Germantown, MD, USA). Subse-
quently, 100 ng of total RNA from each sample was subjected
to reverse transcription using a SuperScript IV cDNA
synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).
Next, a cDNA amount corresponding to 100 ng of total RNA
was amplified by qPCR on an ABI 7700 (Applied Biosys-
tems, Foster City, CA, USA). To quantify Luc2 mRNA levels
in different tissues, RT qPCR analysis was performed using
Luc2-specific primers F 5′-CCCACCGTCGTATTCGTGAG-
3′ and R 5′-TCAGGGCGATGGTTTTGTCCC-3′, yielding a
206 bp amplicon. Luc2 levels were normalized to mRNA
levels of the endogenous murine glyceraldehyde-3-phosphate
dehydrogenase (mGapdh) gene, using primers F 5′-TGTG
TCCGTCGTGGATCTGA-3′ and R 5′-GCCTGCTTCACC
ACCTTCTTGA-3′, yielding an 82 bp amplicon. Genomic
DNA (100 ng) from each sample was subjected to qPCR,
using the Luc2-specific primers as mentioned above. The
qPCR results were expressed as mean AAV copy number per
100 ng of genomic DNA. Known copy numbers (102–107) of
the corresponding plasmid were serially diluted and used to
generate the standard curve.
Statistics
Data were analyzed using Graphpad Prism 6.0. Values
shown in the figures are the mean + s.e.m. Statistical
significances were calculated by Mann–Whitney U-test
analysis.
Acknowledgements WT, QHP, JEA, TV, MKC, and DG are very
grateful for funding and other support from the MYOCURE project.
MYOCURE has received funding from the European Union’s Horizon
2020 research and innovation program under grant agreement no.
667751. DG is thankful for support by the German Center for Infection
Research (DZIF, BMBF; TTU-HIV 04.803 and TTU-HIV 04.815).
DG acknowledges additional funding by the German Research
Foundation (DFG) through the Cluster of Excellence CellNetworks
(EXC81) and the Collaborative Research Centers SFB1129 (Pro-
jektnummer 240245660) and TRR179 (Projektnummer 272983813).
TV and MKC obtained funding from the Fonds Wetenschappelijk
Onderzoek (FWO), VUB Industrieel Onderzoeksfonds (IOF), Koning
Boudewijn Stichting (Creemers-Opdebeek) and Association Belge
contre les Maladies Neuromusculaires (ABMM). We are grateful to
Alexander Bello and Gary Kobinger for initially supplying the
Distinct transduction of muscle tissue in mice after systemic delivery of AAVpo1 vectors
po1 sequence. The authors thank Julia Fakhiri for critical reading of
the manuscript as well as Ermira Samara for her help with AAV vector
production.
Author contributions TV, MKC, and DG conceived and designed the
experiments. WT, JW, QHP, and JEA generated constructs and per-
formed experiments. WT, JEA, TV, MKC, and DG wrote the manu-
script. All authors read the manuscript and approved the final version.
Compliance with ethical standards
Conflict of interest DG is a co-founder and shareholder of AaviGen
GmbH. JW is currently an employee of Boehringer Ingelheim Pharma
GmbH & Co. KG. All other authors declare that they have no conflict
of interest.
Ethical approval All animal procedures were approved by the insti-
tutional animal ethics committee of the Free University of Brussels
(VUB) (Brussels, Belgium). Husbandry was carried out in individually
ventilated Thoren cages that contained Hygienic Animal Bedding
(Lignocel). Temperature was maintained at ~21 °C with 50–60%
humidity. Animals were fed SsniFF laboratory animal food (ABEDD
Vertriebs GmbH, Vienna, Austria) ad libitum.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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