Understanding and tackling immune responses to AAV vectors

Abstract

As the clinical experience in Adeno-Associated Viral (AAV) vector-based gene therapies is expanding, the necessity to better understand and control the host immune responses is also increasing. Immunogenicity of AAV vectors in humans has been linked to several limitations of the platform including lack of efficacy due to antibody-mediated neutralization, tissue inflammation, loss of transgene expression, and in some cases complement activation and acute toxicities. Nevertheless, significant knowledge gaps remain in our understanding of the mechanisms of immune responses to AAV gene therapies, further hampered by the failure of preclinical animal models to recapitulate clinical findings. In this review, we focus on the current knowledge regarding immune responses, spanning from innate immunity to humoral and adaptive responses, triggered by AAV vectors and how they can be mitigated for safer, durable, and more effective gene therapies.

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Understanding and Tackling Immune Responses
to Adeno-Associated Viral Vectors
Helena Costa-Verdera,
1
Carmen Unzu,
2
Erika Valeri,
1
Sahil Adriouch,
3
Gloria Gonza
´lez Aseguinolaza,
2,4
Federico Mingozzi,
5
and Anna Kajaste-Rudnitski
1,
*
1
San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, Milan, Italy;
2
DNA and RNA Medicine Division, CIMA, Universidad de
Navarra, IdisNA, Pamplona, Spain;
3
University of Rouen, INSERM, U1234, Pathophysiology Autoimmunity and Immunotherapy (PANTHER), Normandie University,
Rouen, France;
4
Vivet Therapeutics S.L., Pamplona, Spain; and
5
Spark Therapeutics, Inc., Philadelphia, Pennsylvania, USA.
As the clinical experience in adeno-associated viral (AAV) vector-based gene therapies is expanding, the necessity to
better understand and control the host immune responses is also increasing. Immunogenicity of AAV vectors in humans
has been linked to several limitations of the platform, including lack of efficacy due to antibody-mediated neutralization,
tissue inflammation, loss of transgene expression, and in some cases, complement activation and acute toxicities.
Nevertheless, significant knowledge gaps remain in our understanding of the mechanisms of immune responses to AAV
gene therapies, further hampered by the failure of preclinical animal models to recapitulate clinical findings. In this
review, we focus on the current knowledge regarding immune responses, spanning from innate immunity to humoral and
adaptive responses, triggered by AAV vectors and how they can be mitigated for safer, durable, and more effective gene
therapies.
Keywords: gene therapy, AAV, immune responses, innate immunity, adaptive immunity, viral restriction
INTRODUCTION
FROM BACTERI A TO vertebrates, life has established sophis-
ticated mechanisms to detect and eliminate foreign mole-
cules or to restrict its function and replication. In
mammalian cells, pattern recognition receptors (PRR) have
a central role as they recognize evolutionarily conserved
structures on pathogens, termed pathogen-associated mo-
lecular patterns within the specific compartments that they
patrol. Engagement of toll-like receptors (TLRs) or of the
cytosolic nucleic acid-detecting immune receptors such as
the RIG-I family of helicases (RIG-I, MDA5, LGP2),
cGAS, IFI16, or AIM2 will ultimately elicit type-I inter-
feron (IFN)-mediated antiviral responses.
1–3
Because all
current and emerging gene transfer and editing technolo-
gies are bound to expose cells to exogenous nucleic acids
and, most often, also to viral vectors, host antiviral factors
and nucleic acid sensors may play a pivotal role in the
efficacy and safety of gene therapy.
4
These cell-intrinsic innate immune responses may also
promote both humoral and cellular adaptive immune re-
sponses against components of the viral vector and, po-
tentially, its cargo. The relevance of immune responses in
the context of other delivery platforms such as integrating
Lentiviral Vectors or nonviral delivery systems such as
Lipid Nanoparticles and their nucleic acid cargos have
been extensively reviewed elsewhere.
4–8
For adeno-
associated viral (AAV) vectors, studies indicate that the
establishment of adaptive immune responses starts by
the recognition of the AAV vector capsid and genome by
the innate immune system.
9
In particular, the stimulation of different TLRs present
on antigen-presenting cells (APCs) has been shown to be
*Correspondence: Dr. Anna Kajaste-Rudnitski, San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, Via Olgettina 58, Milan 20132,
Italy. E-mail: kajaste.anna@hsr.it
836 jHUMAN GENE THERAPY, VOLUME 34, NUMBERS 17 and 18 DOI: 10.1089/hum.2023.119
ª2023 by Mary Ann Liebert, Inc.
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involved in initial innate sensing of the vector and sub-
sequent establishment of adaptive responses, but other
PRRs and nonimmune cell types may also contribute to
this initial priming. As wild-type AAV naturally infects
humans, cross-reactive pre-existing immunity to AAV
vectors, both humoral and cell-mediated, is highly prev-
alent and can also interfere with gene transfer.
Immunogenicity of AAV vectors in humans has been
associated with several observations, including lack of
efficacy due to antibody-mediated neutralization, tissue
inflammation, and loss of transgene expression. In some
cases, acute toxicities have also been documented, trig-
gered by complement activation. Importantly, the lack of
preclinical animal models that robustly recapitulate find-
ings in patients further adds to the complexity of the un-
derstanding of the interaction between the immune system
and AAV vectors. This review will focus on the current
knowledge regarding immune responses, spanning from
innate immunity to humoral and cellular adaptive re-
sponses, triggered by AAV vectors, and how they can be
mitigated for safer, durable, and more effective gene
therapies.
INNATE IMMUNITY TO AAV
Capsid sensing
In primary human nonparenchymal liver cell cultures,
including Kupffer cells (KCs) and liver sinusoidal endo-
thelial cells, the viral capsid has been seen to contribute to
innate immunity mainly through binding to TLR2 ex-
pressed on the cell surface
10
(Fig. 1). TLR2 stimulation by
capsid proteins has been shown to signal through MyD88/
nuclear factor kappa B (NFjB) and transiently upregulate
proinflammatory cytokines such as interleukin (IL)-8, IL-
1b, tumor necrosis factor-a, or IL-6.
10
However, there is
no clear evidence so far of the role of TLR2 sensing in the
induction of adaptive immune responses.
11,12
Work by
Kuranda et al., explored innate responses stimulated by the
AAV2 capsid in the context of human peripheral blood
mononuclear cells (PBMCs), reporting that peripheral
monocyte-derived dendritic cells (moDCs) play a key role
in the establishment of humoral and cellular responses
through IL-1band IL-6 secretion.
13
Exposure of human PBMCs to either full AAV2, AAV2
empty particles, or capsid-derived peptide pools resulted
in increased IL-1band IL-6 levels in supernatants inde-
pendently of the serological status of the donors, with the
main source being moDCs compared to plasmacytoid
(pDCs) or conventional dendritic cells (cDCs). In subjects
previously exposed to wild-type AAV, these cytokines
triggered the differentiation of capsid-specific memory B
cells into antibody-secreting cells resulting in anti-capsid
antibody production, and this process could be inhibited
in vitro and in vivo by IL-6 and IL-1bblockade. On the
contrary, the AAV-seronegative individuals responded to
the AAV capsid by transient NK activation. The innate
receptors involved in the response were not directly as-
sessed in this study, and whether moDC activation results
from direct interaction with AAV peptides or via inter-
action with other APCs remains to be elucidated.
13
Vector genome sensing
As opposed to the TLR2-dependent sensing of the viral
capsid, sensing of the AAV vector genome through en-
dosomal TLR9 has been clearly linked with the subse-
quent activation of innate and adaptive responses against
the AAV capsid and the transgene product
11,14–16
(Fig. 1).
TLR9 recognizes unmethylated CpG sequences present in
viral or bacterial but not mammalian DNA. TLR9 signals
through the adapter molecule MyD88, which induces the
expression of proinflammatory cytokines through NFkB
activation, as well as type I IFNs via IRF3 and IRF7.
In 2009, Zhu et al. reported that exposure of murine
bone marrow-derived pDCs to single-stranded AAV
(ssAAV) led to the induction of type I IFN responses,
whereas no evidence of proinflammatory signaling was
detected in cDCs, macrophages, or KCs cultured in vitro.
14
Type I IFN signaling in pDCs was shown to be dependent
on the TLR9-MyD88 pathway and independent of the
nature of the transgene. Later studies have shown that
TLR9 stimulation and type I IFN responses mediated by
pDCs are important to promote cross-presentation of AAV
capsid antigens by cDCs to CD8
+
T cells, being pivotal for
anti-capsid cellular responses.
12
In vivo,Tlr9
-/-
, and
Myd88
-/-
mice injected intramuscularly with a ssAAV2
encoding the influenza virus hemagglutinin showed di-
minished T cell responses against both capsid and trans-
gene compared to wild-type controls.
14
Moreover, the authors reported that the anti-HA and
anti-capsid humoral responses were also significantly di-
minished in both models. Cytotoxicity was also absent in
Ifnr
-/-
mice together with reduced antibody responses to
HA and AAV, leading to long-term transgene expression,
highlighting a role for type I IFN in induction of adaptive
immunity in mice. Moreover, stimulation of PBMC-
derived human pDCs ex vivo with AAV2 encoding for
lacZ led to the upregulation of human IFN-a(hIFN-a) and
hIFN-bmRNA. The response was blocked by the TLR9
antagonist H154 ODN, suggesting a similar mechanism as
observed in mice.
A different study addressing innate immune responses
in the liver showed that proinflammatory responses to
ssAAV were mild in this tissue, whereas they were en-
hanced when using self-complementary AAV (scAAV)
vectors, correlating with stronger cellular and humoral
responses to the AAV capsid, but not to the transgene
product.
17
Innate responses to scAAV in the liver were
also dependent on TLR9 signaling and could be prevented
by transient inhibition of TLR9. Some inconsistent results
have been obtained regarding the role of TLR9 signaling
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on humoral responses to the transgene product upon in-
tramuscular AAV injection. While Wu et al., reported
stronger transgene-specific cellular and humoral responses
induced by scAAV compared to ssAAV,
18
Rogers and
colleagues observed enhanced cellular responses but un-
changed antibody responses.
19
Regarding the different impact of modulating TLR9
signaling on the regulation of cellular and humoral re-
sponses, different roles have been attributed to TLR9 and
MyD88 in the establishment of adaptive responses to the
capsid and transgene product.
11
In intramuscular setting,
cellular but not humoral responses to transgene product
were reported to depend both on TLR9 and MyD88. As for
anti-AAV capsid responses, only humoral immunity
seemed to partially depend on MyD88, but not on TLR9,
indicating the involvement of additional sensing mecha-
nisms in shaping adaptive responses.
11
Altogether, dif-
ferent works evidence that TLR9 is not required for
antibody formation, yet, it may have a modifying effect on
anti-AAV IgG titers and subclasses.
Another factor shown to affect the magnitude of TLR9-
mediated signaling in addition to the vector DNA structure
is the content of CpG motifs in regions such as the inverted
terminal repeats (ITRs), the enhancer and promoter re-
gions, intronic sequences, and polyA tail.
16,20–22
Im-
portantly, it has been suggested that the unexpected loss of
FIX expression observed in patients administered the in-
vestigational product BAX335 was due to an increase in
CpG content on the expression cassette resulting from
codon-optimization.
21,23
In the setting of ex vivo gene editing, while AAV effi-
ciently avoids activation of type I IFN responses in human
hematopoietic stem and progenitor cells (HSPC), it trig-
gers p53-mediated DNA damage responses (DDRs) in this
Figure 1. Immunological barriers to AAV-mediated gene therapy. (1) Pre-existing NAbs bind to AAV capsids hampering successful liver transduction. (2 and
3) TLRs recognize vital capsid (TLR2) and viral genomes (TLR9) and trigger innate immune pathways. (4) dsRNA can induce RLRs and MDA5 sensors that will
trigger an IFN type I immune response. (5) Antigenic capsid or recombinant protein peptides generated by proteasome degradation are presented by APCs via
MHC class I to CD8
+
T cells and via MHC class II to CD4
+
T cells. After presentation, antigen-specific cytotoxic CD8
+
T cells can eliminate transduced cells and
CD4
+
T cells stimulate the activation of antibody producing plasma B cells. AAV, adeno-associated viral; APCs, antigen-presenting cells; dsDNA, double-
stranded DNA; dsRNA, double-stranded RNA; IFN, interferon; MHC, major histocompatibility complex; NAbs, neutralizing antibodies; RLRs, Rig-I like receptors;
ssDNA, single-stranded DNA; TLRs, toll-like receptors.
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cell type.
24–26
AAV vector DNA genome is sensed in the
nucleus, where it triggers p53-mediated DDR upon re-
cruiting the MRE11-RAD50-NBS1 (MRN) complex on
the AAV ITRs.
24,25
AAV-mediated DDR induced apo-
ptotic responses in vitro and reduced engraftment of short-
term (ST)-HSC in vivo.
24,27
Complement activation
Recent clinical studies have shown that complement re-
sponses constitute another toxicity risk associated with high-
dose AAV gene transfer, causing variable clinical mani-
festations, including thrombotic microangiopathy (TMA),
kidney injury, thrombocytopenia, and cardiopulmonary in-
sufficiency.
28–32
Complement-related acute toxicities have
been observed only at vector doses above 1 ·10
13
vector
genomes (vg)/kg, and not consistently across trials.
Although complement factors have been shown to di-
rectly bind the AAV capsid ex vivo in the absence of anti-
capsid antibodies,
33,34
leading to increased AAV uptake
and innate responses in APCs,
33,35
the correlation between
pre-existing antibodies and the risk for complement acti-
vation is unclear. Additional studies have shown increased
complement factor binding and activation in the presence
of anti-AAV IgG,
34,35
in particular IgG1,
34
constituting a
risk in particular when dosing patients with pre-existing
humoral immunity. Nevertheless, complement responses
have been reported in clinical studies of gene therapy for
diseases, including Fabry disease, spinal muscular atrophy
(SMA), Methylmalonic acidemia,
28,31,36
and Duchenne
muscular dystrophy (DMD),
32
in which patients had been
prescreened for pre-existing anti-AAV antibody titers,
whereas no complement responses were reported in an
hemophilia B clinical trial, in which seropositive patients
were treated with AAV vectors.
37,38
Therefore, the risk for complement activation could be
influenced by the total vector dose administered and the
subclass of pre-existing or de novo formed anti-AAV an-
tibodies, the AAV serotype, or the underlying genetic
background of the host. Furthermore, while similar level
of complement activation has been observed to full and
empty AAV particles in the presence of immunoglobulins
in vitro,
35
a recent study presented at the 26th annual
meeting of American Society of Gene and Cell Therapy
(ASGCT) by Buchlis and colleagues suggests that the
vector genome could also be involved in the mechanism of
complement activation upon liver transduction in vivo.
39
Other mechanisms potentially contributing
to AAV innate immune activation
Among the potential alternative mechanisms contrib-
uting to AAV-induced innate immunity, one study re-
ported the induction of type I IFN responses by AAV
through the MDA5/MAVS axis, due to the detection of
double-stranded RNA generated from the intrinsic pro-
moter activity of the ITR regions (Fig. 1). However, the
impact of MAVS signaling on adaptive immune responses
has not been demonstrated.
40
Importantly, AAV gene transfer was also shown to in-
duce dose-dependent toxicity in sensory neurons of the
dorsal root ganglia (DRG) upon systemic and local AAV
administration in large animal models.
28,41,42
The mecha-
nism responsible for this toxicity remains still unclear since
evaluation of T cell responses and studies in the presence of
immunosuppressive drugs do not support a clear role of T
cell-mediated immunity,
41,43
but studies suggest a corre-
lation between overabundance of the transgene product and
neuron loss,
42
as reported also by Henry et al., at the 2023
ASGT meeting.
39
Ongoing works recently presented at the
ASGCT annual meeting have detected the presence of
immune cell infiltrates in spinal cord as well as different
cytokines in cerebrospinal fluid of injected nonhuman pri-
mates (NHPs), suggesting the involvement of innate im-
mune mechanisms which require further investigations.
39
Finally, one, although rare but lethal innate immune
response, was recently described as cytokine-mediated
capillary leak syndrome in a N-of-1 clinical trial for
DMD.
44
The patient experienced cardiac and pulmonary
toxicities associated to AAV9-mediated innate signaling,
which were complicated by the advanced DMD disease in
the patient, resulting in cardiorespiratory failure. Post-
injection studies in serum and pericardial fluid had re-
vealed elevation of proinflammatory cytokines and
complement factors before the cardiorespiratory arrest.
Restriction factors
The high AAV doses still required for therapeutic ef-
ficacy in some applications of gene transfer are a potential
risk factor contributing to the adverse toxicity observed in
recent clinical trials. The efficiency of viral vector-based
genetic manipulation may be limited by recognition of
exogenous components by host cell restriction factors
(RF).
4
RF are intrinsically present in different cell types
and usually part of the interferon stimulated genes, thus
further inducible upon type I IFN responses.
45
The antiviral mechanisms that interfere with AAV
transduction are not completely understood. Thus far, the
factors identified to block AAV transduction act at key
steps of the viral life cycle such as vector entry or DNA
genome conversion into dsDNA or target the AAV capsid
for proteasomal degradation (Fig. 2). A whole-genome
siRNA screen led to the identification of members of the
small ubiquitin-like modifier pathway as critical RF for
AAV transduction that acts at the level of vector entry
within the cells and are shared across different AAV se-
rotypes.
46
More recently, the VP2 capsid protein was
proposed as a direct target of SUMOylation.
47
Restriction
of AAV gene transduction can be mediated by direct
SUMOylation of the AAV capsid or by AAV-mediated
SUMOylation of cellular proteins, which then indirectly
influence vector transduction.
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More recently, the apical polarity determinant Crumbs 3
(Crb3) protein emerged as a key RF playing a role in viral
attachment and entry from a CRISPR screen in a human
hepatic cell line.
48
FKBP52, a cellular chaperone protein, is
among the factors reported to act at the level of dsDNA
conversion. Phosphorylated FKBP52 binds AAV2 ITR
49,50
inhibiting viral second-strand DNA synthesis and affecting
transduction efficiency. The dephosphorylated protein lo-
ses the ability to bind AAV genome thus allowing efficient
transgene expression. PHF5A, a U2 snRNP-associated
protein, is an additional critical host factor identified
through a siRNA screening as able to limit AAV transgene
expression in a serotype and cell type-independent manner,
influencing a step after second-strand synthesis.
51
Disruption of PHF5, a subunit of the splicing factor 3b
protein complex that forms the U2 small nuclear ribonu-
cleoproteins complex (U2 snRNP) together with other
proteins, is sufficient to increase AAV transcript levels,
suggesting a critical role for the U2 snRNP spliceosome
complex in host-mediated restriction of AAV.
51
AAV proteasome degradation can also potentially af-
fect transduction efficiency. Different AAV capsid surface
residues are targets of cellular protein kinases.
52,53
Phos-
phorylated capsids become a substrate for ubiquitin con-
jugation and proteasome-dependent degradation
53,54
with
overall decrease in transduction efficiency.
55
AAV genome silencing is another barrier to efficient
long-term transgene expression. It has been shown that the
dsDNA binding protein NP220 and the human silencing
hub (HUSH) complex mediate transcriptional silencing of
single-stranded and self-complementary rAAV genomes
by epigenetic modification of associated host histones in a
Figure 2. Innate immune restriction mechanisms to efficient AAV transduction. Host RF have been shown to limit AAV transduction efficiency acting at
different steps of the AAV life cycle. The main mechanisms are reported in the figure: (1) inhibition of viral attachment and entry (Crb3); (2) capsid ubiquitination
and proteasome degradation; (3) capsid SUMOylation and/or AAV-mediated SUMOylation of putative host AAV RF; (4) viral ITR binding and inhibition of dsDNA
conversion (FKBP52); (5) AAV genome silencing by epigenetic modification of associated histones (NP220; HUSH complex). HUSH, human silencing hub; ITR,
inverted terminal repeat; RF, restriction factors.
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serotype-dependent manner.
56
Lack of NP220 or HUSH
complex favors higher AAV transcript levels with reduced
H3K9 histone methylation marks.
56
Many other proteins involved in different cellular
processes such as cell cycle regulation, DDR, chromatin
remodeling, and transcriptional regulation, have also been
identified through cellular screenings as potential RF
limiting AAV transduction efficiency.
57,58
Silencing or
knock-out of these proteins results in improved transgene
expression from different AAV serotypes. The potential
benefits of inhibiting proteins such as the Fanconi anemia
protein FANCA, the HUSH-associated methyltransferase
SETDB1, or the nuclear matrix protein MORC3 have been
demonstrated in human primary cells, highlighting their
potential relevance in therapeutic settings.
58
CRISPR
screenings in cell lines were also used to identify factors
critical in facilitating AAV transduction. Among these
factors, knock-out of the GPR108 and TM9SF2 proteins
decreased AAV transduction using different serotypes.
59
Since GPR108 localizes to the Golgi, it has been suggested
that it may interact with AAV, playing a critical role in
viral escape or trafficking.
59
Strategies to overcome innate immune
barriers to AAV transduction
Although most strategies currently tested to prevent in-
nate immune responses to AAV are still under preclinical
research, several investigations have shown the influence of
the AAV vector genome composition on the induction of
proinflammatory signals through TLR9 and the magnitude
of cellular and humoral responses to both capsid and
transgene product, in particular, when using single-stranded
or self-complementary vectors.
11,17,18
In agreement, re-
ducing the CpG content
16,20–22
or including TLR9 inhibi-
tory sequences in cis
60
can dampen this cascade of immune
responses. CpG-depleted AAV vectors have shown re-
duced CD8
+
T cell responses to both capsid and the trans-
gene in liver and muscle gene transfer settings.
16,20,22
Yet, despite the amount of evidence regarding the link
between TLR9 stimulation and cytotoxic responses to the
capsid and transgene product, it seems that the beneficial
effect of avoiding TLR9 signaling is vector-dose depen-
dent, and responses are still induced above a certain
threshold
61,62
as reported also by Kumar et al., at the 26th
annual meeting of the ASGCT.
39
Although removing CpG
dimers from the transgene cDNA is beneficial in reducing
AAV immunogenicity, it is inherently impossible to
completely eliminate TLR9-mediated immune sensing.
As a result, even CpG depleted vectors stimulate TLR9
above a certain dose. Moreover, additional pathways have
been seen to contribute to induction of such responses,
such as the IL-1 receptor pathway reported by Kumar and
colleagues,
39,63,64
suggesting that multiple pathways may
need to be blocked at high vector doses for effective im-
mune modulation.
To prevent cell toxicity derived from DNA-damage
responses, inhibition of Ataxia-telangiectasia mutated ki-
nase, upstream of p53 activation, can be exploited to
prevent vector-induced DDRs in HSPC, rescuing delayed
engraftment.
24,27
Similarly, transient overexpression of
GSE56, a p53 dominant negative mRNA, is exploited in
AAV/Cas9 gene-editing procedure to rescue engraftment
defects and clonality of manipulated HSPC when coelec-
troporated with the genome-editing components.
25–27
The C5 inhibitor Eculizumab has been used to treat
complement-associated symptoms such as TMA in clini-
cal trials of SMA
36
and DMD,
32,65,66
however, further
studies are needed to determine the efficacy of this drug in
limiting complement responses. Other strategies have at-
tempted detargeting transgene expression from unwanted
tissues through the introduction of microRNA (miR) target
sites on the transgene sequence, such as the introduction of
targets for miR122
67
or miR183
42
to prevent transgene
overexpression and associated toxicity in liver and DRG,
respectively, as well as the use of miR142 targets to de-
target transgene expression in APCs and thus decrease
immune sensing of the transgene product.
67–69
The use of
miR targets to improve the tissue-specificity of transgene
expression is already being used in an ex vivo gene transfer
clinical trial for glioblastoma for the delivery of IFNa
specifically into the tumor microenvironment.
70
Strategies aimed at preventing AAV vector restriction
are also important to improve transduction efficiency and
decrease the vector doses needed to achieve therapeutic
benefit. For instance, targeting SUMOylation has been
reported to increase AAV transduction, constituting a
potential strategy to enhance AAV-based transgene de-
livery.
47
Proteasome inhibitors have also shown potential
to increase AAV transduction efficiency in different cell
types, both in vitro and in vivo,
71
as well as epigenetic
modulation of target cells to prevent vector silencing.
56
As the molecular understanding of the early innate
immune activation associated with AAV transduction in-
creases, it will be possible to design and test improved
vector configurations that avoid cellular sensors such as
TLRs or DNA recognizing proteins. Transduction proto-
cols adjusted to transiently prevent activation of these
pathways through targeted inhibitor delivery behold po-
tential to dampen innate immune activation and thereby
potentially diminish also subsequent humoral and adaptive
responses in vivo. While it is still unclear whether infec-
tious AAV encodes for antagonists of some of the iden-
tified RF, the field of virology offers plenty of examples of
how viruses effectively counteract these innate immune
barriers.
72,73
Further studies will help elucidate if AAV-
encoded proteins could be harnessed for such purposes or
if other viral factors or synthetic agonists could be
exploited to improve target cell permissivity to transduc-
tion, allowing the lowering of vector doses with conse-
quent lowering of the risks of adverse immune toxicity.
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ADAPTIVE IMMUNITY TO AAV
Humoral responses
Humoral immunity directed against the AAV capsid
represents one of the most important barriers to successful
gene transfer. Depending on the route of vector adminis-
tration, the impact of anti-capsids neutralizing antibodies
(NAbs) can be major (Fig. 3A), with systemic vector ad-
ministration being the setting, in which exposure to anti-
bodies and vector neutralization is the highest. The
antibody titer and the total capsid dose administered
74
may
also contribute to the outcome of gene transfer in the
presence of NAbs. Studies in animal models and in hu-
mans indicate that even low titer NAb can majorly affect
the outcome of gene transfer.
37,75,76
For example, NAb
titers of just 1:5 were shown to block AAV transduction of
the liver at vector doses of 5 ·10
12
vg/kg in NHPs.
77
Similarly, in humans, in the context of hemophilia B
trials, antibody titers of 1:17 were shown to prevent liver
transduction at a dose of 2 ·10
12
vg/kg,
37
whereas titers of
just 1:1 can reduce transgene expression by about 50%.
75
Nevertheless, an important aspect to be considered is that
NAb titers may vary from laboratory to laboratory de-
pending on the specific design of the NAb assay, due to the
lack of a standardized protocol.
Humoral immunity to AAV can be categorized in pre-
existing and postdosing. Pre-existing antibodies to AAV
originate from the exposure to the wild-type virus, which
usually occurs early in life.
78
Depending on the AAV se-
rotype, up to *70% of humans can be seropositive, with
an average of 30–40% positivity for anti-AAV NAbs
across serotypes in adults (Fig. 3B), and lower ser-
oprevalence in young children.
78,79
IgGs binding to AAV
are the antibody isotype mostly found in humans, with
positivity for IgM detected in a minority of individuals.
80
IgG-binding titers correlate with NAb titers,
81
and in the
context of natural immunity to AAV tend to be low to
moderate. Upon AAV vector administration, early pro-
duction of IgM is commonly seen, which is rapidly fol-
lowed by production of high-titer IgG binding to AAV
82
and, consequently, NAbs.
Post-treatment anti-AAV antibodies tend to persist at
high titers for several years and can cross-react with dif-
ferent AAV serotypes.
83
While pre-existing antibodies to
AAV affect eligibility to receive gene transfer, postdosing
humoral immunity prevents vector readministration, if
needed.
Anti-AAV antibodies are mainly measured with two
assays, a neutralization assay which measures inhibition of
transduction of a cell line by a reporter AAV vector, and a
binding assay measuring antibodies binding to the AAV
capsid.
84,85
Various protocols have been developed for
both the binding and neutralizing assays, and both have
been used for the prescreening of subjects ahead of AAV
vector administration. While binding and neutralizing
antibody titers correlate, some discrepancies can be found,
particularly at lower titers, with subjects scoring sero-
negative in one assay and positive in the other. The choice
of a method to screen for antibodies to AAV has important
Figure 3. Influence of pre-existing immunity on AAV delivery efficacy. (A) Potential impact of anti-AAV NAbs depending on the route of administration. Green
dots: the impact of anti-AAV antibodies is minimal when the vector is delivered intraparenchymally, subretinally, or into the cerebrospinal fluid. Yellow dot:
delivery of AAV intravitreally, appears to expose the vector to some degree of neutralization by NAbs. Red dot: systemic delivery of AAV vectors is the most
challenging when it comes to potential for vector neutralization by anti-capsid antibodies. (B) Prevalence of anti-capsid IgG in humans across several AAV
serotypes (adapted from Boutin et al.
151
).
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implications, including ease of assay setup, validation, and
throughput; ultimately the selection of the assay platform
needs to be defined in the context of both preclinical and
clinical studies and efforts to develop a companion diag-
nostic for an investigational gene therapy need to start
early in the development.
Several solutions have been proposed to address the
issue of anti-AAV antibodies. Broadly, they can be cate-
gorized as (1) methods to prevent antibody formation; (2)
methods to protect the vector from neutralization; and (3)
methods to remove or inactivate circulating NAbs. Anti-
body prevention is a potential effective strategy to allow
for vector readministration, unlikely to be useful in the
context of pre-existing immunity. In general, most strat-
egies work best in the context of low to moderate NAb
titers, like those found in the context of natural immunity
to the virus, while are less effective in the context to high
titer antibodies. Thus, redosing of AAV vectors will pos-
sibly require a combination of NAb reduction strategies.
Methods to prevent antibody formation
Prevention of antibody formation via immunomodula-
tory regimens has been proposed to allow for vector
readministration. Various combinations of T and B cell
targeting drugs have been tested to block antibody for-
mation postvector dosing. Some of these combinations
have been tested in the clinic.
86
Overall, the task has been
challenging, with preclinical data not easily scalable to
humans, possibly reflecting the higher immunogenicity of
AAV vectors in humans and the long-term persistence of
the AAV capsid antigen postvector dosing. While im-
munomodulation is almost universally used in AAV gene
transfer to block detrimental inflammatory responses, the
risk benefit of the approach in the context of vector read-
ministration requires careful evaluation.
The idea of tolerizing the host immune system against
the AAV capsid has been explored using various strate-
gies. For example, the coadministration of tolerogenic
nanoparticles containing sirolimus has shown promising
results in preclinical studies.
87
In a recent healthy volun-
teer clinical trial, empty AAV capsids were administered
together with tolerogenic nanoparticles.
88,89
The approach
was effective in reducing NAb titers in the ST, while an-
tibodies appeared to rebound at later times to levels
comparable to the control group, suggesting that repeated
antigen-nanoparticle (i.e., AAV capsid-nanoparticle) or
sirolimus-nanoparticle exposures may be required to allow
for a more persistent suppression of antibody formation.
Vector engineering and serotype switching. Several
methodologies can be used to engineer AAV capsids with
better tissue tropism or the ability to cross physical bar-
riers.
61
Similarly, the development of non-natural variants
and their selection against pooled human sera can help
identify novel AAV capsids with low seroprevalence.
90
In
the context of vector readministration, serotype switch has
been shown to be effective in preclinical models.
91
While
the effectiveness of the approach needs clinical validation,
it should be kept in mind that the use of a different capsid
for redosing purposes is highly resource intensive as in
many ways it is equivalent to developing a new investi-
gational gene therapy product.
Chemical modification of AAV vectors and the co-
purification of AAV with exosomes have been shown to
provide some resistance to Nabs.
92
Both approaches are
less effective in evading high titer antibodies, thus being
potentially useful only in the context of pre-existing hu-
moral immunity to AAV. Also physical isolation of a
target organ, like the liver, with saline flushing has been
shown to be effective against low to moderate NAb ti-
ters.
93
Ultimately the clinical feasibility of the approach
needs to be established.
As a broadly available and safe, clinically established
technology, the use of plasmapheresis/plasma exchange
has been shown to be effective in lowering antibody titers
to AAV in preclinical studies and in human samples in the
context of natural immunity to AAV.
94
While no data are
available in the context of gene therapy trials, based on the
data available, the strategy is likely to be effective in the
context of low anti-AAV NAb titers. Lowering of high-
titer NAbs would likely require several cycles of plasma
absorption/exchange, due to the rapid rebound post-
procedure linked to antibodies residing in the extravas-
cular space.
More recently, it has been shown that the application of
immune-adsorption procedure enables successful read-
ministration of AAV5 in NHPs
95
and AAV-specific col-
umns have been developed to remove AAV-specific Nabs
from circulation.
96
In addition, empty AAV capsid decoys
have been explored as a potential way to absorb pre-
existing antibodies during vector administration.
74
Nevertheless, as empty AAV capsids alone have been
suggested to trigger innate immune responses,
10
the im-
pact of such strategies on overall immune activation will
need to be carefully assessed.
IgG-cleaving endopeptidases are both in development
and approved drugs for kidney transplant and other
antibody-mediated diseases.
97
Following a single enzyme
infusion in human trials, a rapid cleavage of IgG was ob-
served. In the context of gene transfer, the approach has
been shown to be effective in allowing for the rapid in-
activation of NAbs, allowing for vector administration in
the context of pre-existing immunity to AAV and, poten-
tially, readministration.
98–100
While only preclinical data
are available to date, gene therapy trials set to start soon
will have to confirm the safety and efficacy of the
approach.
A key question is the efficacy of the approach at high
NAb titers, as F(ab’)2 fragments released upon IgG
cleavage retain some neutralization activity that can
potentially affect vector transduction. Related to that, the
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performance of current antibody detection methods needs
to be assessed carefully in the context of this technology.
Several endopeptidase variants are in development, in-
cluding a novel IgG and IgM cleaving enzyme.
101
Inhibitors of the neonatal Fc receptor are approved
drugs for antibody-mediated diseases such as myasthenia
gravis.
102
The chronic use of this class of drugs is safe, and
human studies showed lowering of antibody titers by up to
70% of baseline. Early preclinical studies show that the
approach can be useful alone or in combination with IgG-
cleaving endopeptidases to address Nabs.
103
More studies
are likely needed to define the efficacy of the approach,
along or in combination with other strategies, at different
NAb titers.
As efforts to tackle the issue of anti-AAV NAbs con-
tinue, more strategies are being developed to address the
issue. With clinical trials set to start, the issue of pre-
existing immunity to AAV, mostly characterized by low
NAb titers, is likely to be addressed effectively with some
of the approaches outlined above. Vector readministration
will likely require the combination of approaches, for
example immunomodulation at the time of the first ad-
ministration to lower antibody production followed by IgG
removal, or reduction of IgG half-life followed by IgG
removal. Of note, successful AAV administration in the
presence of humoral immunity to the capsid requires only
a temporally limited window of time to allow for the
vector to reach the target tissue.
104
Nevertheless, risk
benefit evaluation and development of suitable biomarkers
to monitor residual neutralization activity will be key to
gather much needed clinical learnings on this critical
challenge of in vivo gene transfer.
T cell immunity
The human immune system has evolved powerful cell-
mediated adaptive mechanisms to fight against and pre-
vent viral recurrent infections, which hampers the efficacy
of viral vector-based gene therapies. As nonpathogenic
viruses, and originally not associated with T cell activation
in mouse studies, AAVs were put forward as ideal viral
vectors invisible to the adaptive T cell surveillance.
However, the host cellular immune responses to AAV
have been challenging to elucidate due to several factors:
(1) AAV vector immunogenicity depends on serotype,
expression cassette, route of administration, target tissue,
and dose
105
; (2) differences in T cell responses between
animal models and humans
106
; and (3) low sensitivity of
AAV-reactive lymphocyte detection assays in peripheral
blood as well as lack of correlation with humoral
response.
81,107
In fact, most of what is known about the T cell re-
sponses to AAV come from lessons learned from gene
therapy clinical trials. As a general mechanism, upon
AAV systemic administration, transduced APC can rec-
ognize and present capsid-derived antigens to lympho-
cytes. Antigen presentation via major histocompatibility
complex (MHC) class I will activate cytotoxic CD8
+
T
cells that will eliminate transduced cells, whereas AAV
antigen presentation via MHC class II will activate CD4
+
T helper cells. Helper cells will amplify and expand both
the cellular and humoral immune response (Fig. 1) and
increase the anti-AAV antibody secretion by B cells.
108
Several assays have been adapted to detect AAV-specific
T cell responses, such as lymphocyte proliferation assays
in response to the AAV capsid or capsid-derived pep-
tides,
109
IFN-cELISPOT,
110,111
or activated lymphocyte
detection by flow cytometry.
13,110
Preclinical data and natural AAV infections
in human population
Importantly, most of the AAV immunological studies
performed in mice have shown that, upon AAV adminis-
tration, mouse CD8
+
T cells do not proliferate in vivo when
re-exposed to AAV-derived antigens (capsid or trans-
gene).
112,113
Furthermore, mouse CD8
+
cells specific for
AAV-derived epitopes fail to eliminate AAV-transduced
cells.
114
Interestingly however, ex vivo expanded capsid-
specific CD8
+
T cells can kill AAV transducer murine and
human hepatocytes in vitro (and to some degree in vivo in
mice upon adoptive transfer).
115–117
Despite the similarity
of NHP to humans in many translational aspects, it has
been also reported that AAV-specific CD8
+
cytotoxic T
cells obtained from NHP do not destroy AAV-transduced
cells
77,118
despite most of them being effector CD8
+
rather
than memory T cells.
108
AAV-specific CD4
+
T cells can
also be detected in NHP and their prevalence rates are
close to 50% in the NHP-screened population.
108
Regarding the healthy human population exposed to
natural AAV infections, Li et al., reported detection of AAV
capsid-specific CD8
+
and/or CD4
+
T cells in 50% of the
screened peripheral blood samples.
108
Half of the responsive
cells were identified as CD8
+
memory cells, 25% as CD8
+
effector cells, and the other 25% as effector memory cells, as
confirmedforAAV1inhealthydonors.
81
Of note, different
distributions were observed in NHP as humans have twice
the proportion of CD8
+
memory cells than primates. These
comprehensive studies also revealed that human CD4
+
T
cells were mainly central memory cells.
108
Clinical trials and approved AAV therapies
The development of a cellular immune response to
AAV was first described after FIX expression was lost in
one subject enrolled in the high-dose group in the first
AAV-mediated liver-directed clinical trial.
37
In this
landmark study, the vector was administered through the
hepatic artery. Unexpectedly, a loss of FIX expression in
plasma was detected around 4 weeks after AAV dosing,
overlapping with an asymptomatic rise in liver transami-
nases. For the second hemophilia B gene therapy trial, the
AAV8 genome was changed to a self-complementary
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form and administered systemically.
82,119
Four out of six
patients from the high-dose cohort (2 ·10
12
vg/kg)
showed the same transient increase of transaminases.
However, in this case, a short course of prednisolone
treatment was able to control the cytotoxic CD8
+
response
allowing for long-term FIX expression.
119
Notably, this
response was not observed at the lower vector doses tested
in the trial, which suggested that the immune response was
dose dependent. Data collected from a hemophilia A
clinical trial did not evidence a consistent increase in
capsid-specific T cells in peripheral blood,
120
although
few patients dosed with Roctavian (Valoctocogene rox-
aparvovec) showed sporadic T cell-positive responses by
IFNcELISPOT.
111
Across trials, however, increase in li-
ver enzymes has been consistently observed post-AAV
infusion, in some cases requiring the administration of
prolonged immunomodulatory regimens.
121
To date, no
transgene-specific CD8
+
responses have been reported
upon liver-directed AAV administration.
122
The route of administration is an important factor in
mounting a cell-mediated immune response. For instance,
the muscle has the potential to initiate inflammation as well
as promote infiltration of circulating immune cells, which
has been widely exploited for efficient host vaccina-
tion,
123,124
including AAV-based vaccines.
125,126
Never-
theless, data from gene therapy clinical trials with
intramuscular administration, including those for the
treatment of lipoprotein lipase (LPL) deficiency, alpha-1
antitrypsin (AAT) deficiency, and Pompe disease and Du-
chenne muscular dystrophy,
127–130
did not show a clearance
of transduced cells despite the detection of capsid-specific
T cells. This is in contrast with liver-directed clinical
studies. Furthermore, CD4
+
FoxP3 T cells were found both
in the LPL and the AAT deficiency trials, suggesting an
induction of tolerance in the administration site, preventing
the elimination of transduced muscle cells.
131,132
To treat inborn and acquired neurological diseases
AAV vectors are usually directly administered into the
central nervous system.
133–137
Because it is a local route of
administration into a site long considered im-
munopriviledged, many trials have not included T cell
response assays. Therefore, information in this regard is
limited. In some of these trials, the anti-AAV antibody
response in circulation was tested and an increase in NAbs
was detected. This suggests that vector leakage from the
injection site can trigger a peripheral B cell response and
potentially T cell responses as well. Noteworthy, in a recent
clinical trial for Tay-Sachs disease, mild T cells responses
were observed upon AAV8 delivery to the cisterna magna
and the thoracolumbar junction.
138
A different strategy was
used for the treatment of SMA. Zolgensma, an US Food and
Drug Administration (FDA) and European Medicines
Agency (EMA) approved AAV9-based vector, is adminis-
tered systemically together with prednisolone (https://www
.fda.gov/media/126109/download).
In the Clinical Review Document (https://www.fda
.gov/media/128116), it is noted that T cell responses to
AAV9 were observed in some cases, whereas the Euro-
pean Public Assessment Report includes that AAV9-
specific responses were detected in all patients by
ELISPOT (https://www.ema.europa.eu/en/documents/
assessment-report/zolgensma-epar-public-assessment-
report_en.pdf). However, none of the studies showed a
correlation between ELISPOT-positive results and drug
efficacy. Noteworthy, results from ocular genetic disease
clinical trials have shown that both intravitreal and sub-
retinal AAV administration can recruit T cells to the
eye,
139–141
in contrast to what was originally thought about
the immune privilege of the eye. Of note, however, lym-
phocyte infiltration does not always result in loss of ex-
pression.
140,141
Modulation and mitigation strategies
Thanks to these valuable clinical experiences, many
AAV-based clinical protocols include at least a corticoste-
roid regimen at the time of vector dosing, or in the event of a
rise in transaminases (https://doi.org/10.1182/blood-2019-
124091, https://www.fda.gov/media/164094/download,
https://www.fda.gov/media/126109/download). It should
be noted that disease-associated underlying liver patholo-
gies in conjunction with high AAV vector doses have been
associated with severe liver toxicities despite im-
munomodulation.
29
Additional forms of immunosuppres-
sion to control T cell-specific responses to the AAV capsid
include the use of cyclosporin, tacrolimus, or sirolimus/
rapamycin.
142
Most of these drugs are directed toward T
cell suppression, however, rapamycin has been shown to
have a beneficial effect on Treg cells and therefore capable
of inducing tolerance to the AAV capsid.
143–145
Recently, nanoparticles loaded with rapamycin have
shown a durable tolerogenic response to AAV vectors in
preclinical studies, even allowing for vector read-
ministration.
87,146
Other mitigation strategies focusing on
Treg cell modulation include the incorporation of IgG-
derived MHC class II epitopes into the therapeutic ex-
pression cassette to reduce AAV immunity
147
or the use of
AAV-specific CAR-Tregs and polyclonal Tregs to allow
for successful transgene expression in the presence of
capsid-specific T cell responses.
148
In the recent studies described above,
88,89
the specific
T cell response to AAV8 empty capsids (dose *2·10
12
capsid particles/kg) was studied in healthy volunteers, as
well as the potential of an immunotolerant treatment to
control it (ImmTOR) as reported by Gordon et al., at the
25th ASGCT meeting.
149
As in previous studies on nat-
ural AAV infections, patient-specific differences in T cell
induction were observed, together with asymptomatic
alanine transaminase increases. Notably, a major contri-
bution of CD4
+
T helper cells was reported. In addition, an
increase in IFNclevels was observed both in the group
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administered with the empty capsids only as well as in the
cohort with ImmTOR. However, the increase of IFNcwas
delayed in the latter.
These results confirmed that cell-mediated responses in
patients can be variable and suggest empty capsid removal
from AAV preparations as a mitigation strategy to reduce
T cell immunity to AAV. Finally, capsid engineering and
alternate AAV serotype readministration have been
proved as other promising strategies to evade T cell
responses.
150
CONCLUSIONS
AAV vector-mediated gene transfer has shown great
promise in the clinic in many applications of the tech-
nology, resulting in several regulatory approvals of gene
therapy drugs based on the platform. Immunogenicity of
AAV has limited the success of in vivo gene therapy af-
fecting safety, efficacy, and durability of gene transfer and
resulting in significant variability in clinical trials.
Therefore, controlling host immune responses to AAV
vectors is key to ensure long-term gene therapy efficiency
and avoid undesired related toxicities. In this regard, in-
sight from preclinical animal models has been instru-
mental for gaining understanding of the mechanisms of
AAV immunogenicity, as exemplified, although not ex-
haustively, in Table 1. Nevertheless, there are non-
negligible differences between these models and the
human immune system prompting additional efforts to be
put in gathering more data from clinical trials, including
time-course studies of AAV immune responses, possibly
also tracking of tissue infiltrating lymphocytes.
In summary, the immunology studies conducted in both
preclinical models as well as in human trials over more
than two decades have enabled the field to accumulate a
significant body of knowledge that is being applied back to
improve the AAV-based gene therapy technology. We
now know that AAV vectors can be designed to engage
less with the innate immune system that manufacturing
should aim at reducing process-related impurities such as
empty AAV capsids. We also learned how to manage
immune responses in the clinic using various immuno-
modulatory regimens, and potential solutions to the issue
of NAbs to AAV will be tested in the clinic in the near
future. As the work continues to progress, more solutions
will be added to the toolbox of gene therapy researchers,
enabling the continuing success of this promising thera-
peutic modality.
Table 1. Immune responses against transgene product documented in preclinical studies of adeno-associated viral-mediated
gene transfer
Species Pathological Model Transgene Product Target Tissues Immune Responses Observed Ref
Mouse — HSV-2 gB SM Cellular and humoral
152
Mouse — HA SM Cellular
153
Mouse — Ova SM Cellular and humoral
154–156
Mouse — b-gal SM Cellular
157
Mouse — HIV-1 gag SM Cellular and humoral
18
Mouse — Cas9 Liver Cellular
158
Mouse (F8
-/-
) Hemophilia A hFVIII Liver Humoral
159–161
Mouse (F9
-/-
) Hemophilia B hFIX Liver Humoral
162
Mouse (a-scg
-/-
) LGMD a-sg SM Cellular
163
Mouse (mdx) DMD b-gal SM Humoral
164
Mouse (ASM
-/-
) Niemann-Pick disease hASM CNS Humoral
165
Mouse (GAA
-/-
) Pompe disease GAA Heart, diaphragm, spinal cord Humoral
166
Dog Hemophilia A FVIII SM Humoral
167
Dog Hemophilia B FIX SM Humoral
168
Dog Batten disease TPP1 CNS Humoral
169
Dog DMD b-gal SM Cellular and humoral
170
Dog DMD Cas9 SM Cellular and humoral
171
Dog MPS I IDUA CNS Humoral
172
Cat MPS I IDUA CNS Humoral
173
Cat LPL deficiency LPL
S447X
SM Humoral
174
NHP — hAAT SM Humoral
175
NHP — EPO SM Humoral
176
NHP — GFP Liver Cellular
157
NHP — UGT1A1 Liver Cellular
177
NHP — Sulfamidase CNS Cellular
178
NHP — FVIII SM Cellular and humoral
179
a-scg, a-sarcoglycan; ASM, acid sphingomyelinase; b-gal, b-galactosidase; CNS, central nervous system; DMD, Duchenne muscular dystrophy; EPO,
erythropoietin; GAA, acid-a-glucosidase; GFP, green fluorescent protein; HA, hemagglutinin; hAAT, human a1-antitrypsin; hFIX, human clotting Factor IX; hFVIII,
human clotting Factor VIII; HIV1, human immunodeficiency virus 1; HSV2-gB, herpes simplex virus type 2 glycoprotein B; IDUA, a-L-iduronidase; LGMD, limb-
girdle muscular dystrophy; LPL, lipoprotein lipase; MPS I, type 1 mucopolysaccharidosis; NHP, non-human primates; Ova, ovalbumin; SM, skeletal muscle;
TPP1, tripeptidyl peptidase 1; UGT1A1, uridine diphosphate glucuronosyl transferase family 1 member A1.
846 COSTA-VERDERA ET AL.
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AUTHOR DISCLOSURE
E.V. and A.K.-R. are inventors on pending and issued
patents on lentiviral gene transfer filed by the Telethon
Foundation and the San Raffaele Scientific Institute. F.M.
is employee of Spark Therapeutics, a Roche company, and
equity holder of Roche. He is also an inventor in pending
and issued patents on the AAV vector platform and
methods to overcome immunity to AAV vectors. G.G.A. is
cofounder, shareholder, and employee of Vivet Ther-
apeutics and issued patents on the AAV vector platform.
FUNDING INFORMATION
This work was supported by grants from the European
Research Council (ERC-CoG 819815-ImmunoStem) and
the Telethon Foundation (TELE22-AK) to A.K.-R.
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Received for publication July 21, 2023;
accepted after revision September 2, 2023.
Published online: September 4, 2023.
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