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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5886_Библиотеки_им_академика_М_И_Перельмана.pdf
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K. Patel et al.
acids encoding a therapeutic gene were proposed to correct, replace, or augment a
diseased gene. However, translating these proposals into genetic medicines involved
circumventing several obstacles that had evolved over billions of years to resist
invading pathogens. These anatomical, biochemical, and immunological barriers
exist to prevent cellular entry of viruses and nucleic acids or eliminate cells that do
take them up. The eld of gene therapy then required a viral or non-viral vector that
could traverse these extracellular and intracellular barriers to deliver therapeutic
genes into cells. A key breakthrough was the discovery of latent viruses; pathogens
that infect host cells yet remain undetected by host immune systems [2]. This gave
the impetus to develop viral vectors to deliver therapeutic genes invivo to cells and
more importantly into the nucleus, a feat that is still difcult with non-viral-based
gene delivery methods [3].
In vivo viral vector-based gene therapies have now been approved to treat several
monogenic disorders [4]. Especially, gene delivery based on adeno-associated viral
(AAV) vectors has demonstrably measured stable transgene expression at therapeutic levels for several years [5]. However, initial forays in AAV invivo gene delivery
to treat patients with hemophilia B were met with loss of transgene expression stemming from host immune responses [6]. An understanding of host–vector interactions that triggered host immunity and the therapeutic interventions available to
mitigate immune activation later led to successful gene transfer [7]. Besides limiting the success of gene therapy, immune responses pose a serious safety concern
that in extreme cases can be fatal [8]. This review summarizes the host immune
response activated by AAV gene therapy vectors and the approaches currently being
pursued to mitigate immune recognition and activation.
7.1.1 AAV Gene Therapy
Risk of insertional mutagenesis [9] and immunogenicity [8] led to viral vectors
based on lentiviruses and adenoviruses being shunned for invivo gene delivery.
Instead, vectors based on adeno-associated viruses (AAV) were selected as the viral
vector of choice for invivo gene transfer. AAV is a small, single-stranded, DNA
parvovirus that demonstrated durable transgene expression with a notable absence
of immune activation in several preclinical studies [5]. However, the clinical experience with AAV gene therapy was different (Table 7.1) and early clinical trials
observed activation of host immune responses that eliminated transgene expression
[10]. These ndings were unexpected based on previous experience in animal models [11]. Strategies were soon deployed to manage host immune responses and
achieve durable transgene expression [10]. Here we review host immune responses
following AAV vector administration and discuss the approaches being pursued to
reduce immune activation by redesigning the vector as well as using immunomodulatory drugs. This review will mainly focus on the aspects of host immunity that are
activated following vector administration. Immune responses that preexist in a
7 Even aWorm Will Turn: Immunity Following AAV Vector Administration
Table 7.1 Immune responses in AAV clinical trials
Dose and
Disease Sponsor Vector
DMD Pzer [74] AAV9 2E14 vg/
Sarepta [75] AAVRh74 1.3E14
Solid [76] AAV9 0.5 and
Hemophilia ABiomarin [77] AAV5 4 to 6E13
Pzer [78] AAV6 3E13 vg/
Hemophilia BSt Jude/UCL [7] AAV8 6E11 vg/
Uniqure [79] AAV5 2E13 vg/
Pzer [80] Spark-100 5E11 vg/
Dimension
therapeutics [81]
FH REGENXBIO
[82]
OTCD Ultragenyx [83] AAV8 0.2 to
PD Asklepios [84] AAV2/8 1.6E12
SMA Novartis [85, 86] AAV9 1.1 E14
DMD Duchenne muscular dystrophy, FH Familial hypercholesterolemia, OTCD Ornithine trans-
carbamylase deciency, PD Pompe disease, UCL University College London, UPenn University
of Pennsylvania, UF University of Florida, ROA Route of administration, IV Intravenous, vg
Vector genomes
AAVrh10 1.6 to
AAV8 2.5 and
ROA Adverse events
Complement activation, persistent
kg; IV
vg/kg; IV
2E14 vg/
kg; IV
vg/kg; IV
kg; IV
kg; IV
kg; IV
kg; IV
5.0E12
vg/kg
7.5E12
vg/kg, IV
1E13 vg/
kg, IV
vg/kg; IV
vg/kg
vomiting, acute kidney injury,
thrombocytopenia
Myositis
Reduced platelets and RBCs,
complement activation, acute kidney
injury, cardiopulmonary insufciency
Transient transaminitis at 4–8weeks
after gene transfer
Transient transaminitis at 4–8weeks
after gene transfer
Transient transaminitis at 6–10weeks
after gene transfer
Transient transaminitis at 6–10weeks
after gene transfer
Transient transaminitis at 4–8weeks
after gene transfer
Transient transaminitis at 4–8weeks
after gene transfer
Transaminases 4–6weeks post-dosing,
T-cell response to the AAV capsid
None
None
Transient transaminitis between 3 and
7days after gene transfer, secondary
increase after reduction in
prednisolone
Thrombocytopenia was observed
between days 6 and 8 after gene
transfer
171
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K. Patel et al.
patient from a wild-type AAV infection and its impact on vector administration are
beyond the scope of this review.
7.1.2 Immune Response toAAV Vectors
AAV drugs comprise three components that can potentially activate host immunity;
the outer shell or capsid comprising viral proteins, the nucleic acid, and the therapeutically expressed protein (Fig.7.1). Activation of immunity to these components
has been noted in AAV clinical trials and can be classied temporally as those that
occur during (innate) or after vector uptake (adaptive). The innate immune system
is the rst arm that recognizes and responds to pathogens based on common molecular patterns that occur across microorganisms, nucleic acids, and compounds [12].
Adaptive immunity, as the name suggests, is based on selection and training a subset
of antibodies or T cells specic to a particular pathogen. Adaptive immune responses
are slower and evolve over several days but are poised to respond faster to a challenge with the same or similar pathogen [13].
Fig. 7.1 AAV drug components that activate immunity
7 Even aWorm Will Turn: Immunity Following AAV Vector Administration
173
7.2 Innate Immune Sensing ofAAV Vectors
Innate immune recognition leads to slowing down viral replication while also sending “danger” signals. The danger or inammatory signals further attract other
immune effector cells to amplify the antiviral response [14]. The pathogen sensors
of innate immunity were rst proposed [15] and later proved to involve receptors
that recognize invariant molecular patterns shared across pathogens [16]. These
receptors collectively called pattern recognition receptors (PRRs) sense pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns
(DAMPs). While PAMPs are biochemical signatures unique to pathogens, DAMPs
are molecules released by dying cells that are not commonly found in circulation [17].
The most well-studied PRRs are the toll-like receptors (TLRs), an evolutionarily
conserved set of proteins found in species as diverse as ies and man [16]. TLRs
recognize triacylated (TLR1/2) and diacylated (TLR2/6) lipopeptides, doublestranded RNA (TLR3), LPS (TLR4), agellin (TLR5), single-stranded RNA (TLR7
and 8), and CpG dinucleotides (TLR9) [18]. In general, PAMP/DAMP recognition
leads to a cascade of events coordinated to alert other arms of the immune system
via secreted factors called cytokines and chemokines [19]. For viral infections, this
establishes an early inammatory response blocking viral replication, inducing cellular destruction, and attracting other effector cells [20]. The antiviral state is maintained until the pathogen is eliminated or suppressed [14].
AAV-based vectors were initially found to be safe and rarely induced cytokines
or chemokines when administered to animals [21]. However, later studies demonstrated activation of a potent inammatory response via multiple engagement [22,
23]. Innate sensing of AAV vectors was demonstrated to involve the detection of
molecular patterns in both the capsid and the viral genome via TLR2 and TLR9
recognition, respectively. While the AAV capsid signature that leads to TLR2 activation is currently unknown, CpG dinucleotides in the genome have been implicated in activating the TLR9 receptor pathway [24]. These motifs are suppressed in
the mammalian genome but overrepresented in pathogens that increase their sensing by TLR9 [25]. Activation often leads to secretion of Type I interferons; cytokines that block viral replication while amplifying inammatory signals [26].
Knowledge of TLR9 activation by CpG dinucleotide motifs within AAV vectors has
led to efforts to reduce their numbers when designing AAV vectors [27]. The strategy has often been to reduce CpG content only in the transgene and not the regulatory elements. The benets of also reducing CpG content in the regulatory elements
are currently unknown. While CpG reduction has demonstrated some benets in
mouse models using highly immunogenic AAV vectors (AAVRh32.22), it is unclear
whether these benets extend to less immunogenic vectors. Nevertheless, CpG
reduction appears to be a strategy to reduce the overall immunogenicity of an
AAV vector.
A caveat of CpG depletion is the impact on transgene expression. Reports indicate that CpG depletion of transgene sequences reduces gene expression [28]. CpG
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K. Patel et al.
reduction in other regulatory elements may further negatively impact transgene
expression [29]. An alternate approach has relied on the inclusion of inhibitory
sequences within AAV vectors to block TLR9 activation [30]. The inclusion of such
sequences as part of a vector genome reduced TLR9 activation in the absence of
CpG depletion [31]. This approach bypasses the need to CpG deplete the transgene
and may avoid the issues of lower transgene expression when reducing CpGs from
the transgene or regulatory elements.
TLR9 activation may also occur from bacterial DNA that gets mispackaged into
AAV vectors. As mentioned earlier, bacterial DNA has higher CpG content and its
presence in AAV preparations can activate TLR9. The most common approach to
produce AAV vectors utilizes three plasmids each encoding a different function.
While packaging plasmid DNA into AAV is unintended, vector production is
prone to mispackaging plasmid DNA that can be transferred and persist in host
tissue after gene delivery [32, 33]. Even if not incorporated into the AAV genome,
bacterial DNA contamination can occur due to inefcient removal of these
sequences during production. Alternatively, fragments of plasmid or bacterial
genomic DNA may bind directly to the surface of the capsids and copurify during
vector production.
Construct design and vector production thus need to be optimized to reduce
plasmid DNA incorporation and effectively remove impurities. For instance, wildtype AAV genome is ~4.7kb; however, subgenomic size vectors can be efciently
produced and packaged. A small percentage of these undersized constructs are
likely to incorporate plasmid DNA adjacent to the ITRs in a process called “reverse
packaging” [34]. Such mispackaged constructs containing plasmid DNA are likely
to pose a risk of immune activation. Reverse packaging risk of vector constructs
<4.7kb can be minimized by including inert DNA sequences into AAV constructs
to optimize packaging length [35]. However, this requires a careful selection of
sequences to ensure inertness. Of concern is the risk of insertional mutagenesis.
While AAV vectors integrate at signicantly lower frequency [36], the risk of
activating a protooncogene close to the site of AAV integration by promoter/
enhancer activities within any non-coding sequences included to optimize vector
size is a concern that needs to be eliminated [37]. Reducing the amounts of plasmids used in AAV vector manufacturing or CpG depleting these sequences can be
alternative approaches to mitigate the risks from packed or contaminating plasmid
sequences.
Finally, pharmacological approaches can mitigate innate immune activation.
Several modulators of innate immunity are currently used to treat immune disorders
[38]. These drugs target common pathways that may be shared by AAV vectors in
activating innate immunity and hence be amenable for use with AAV administration. The dose and time course of intervention with these immunomodulatory drugs
will depend on the pharmacokinetics and pharmacodynamics of vector after
infusion.
7 Even aWorm Will Turn: Immunity Following AAV Vector Administration
Fig. 7.2 Interplay of innate and adaptive immunity upon AAV recognition
7.3 Adaptive Immune Responses toAAV
175
While innate immunity relies on the recognition of shared molecular identities
among pathogens, the adaptive immune response is customized to recognize and
respond to unique non-self peptides (Fig.7.2). Adaptive immunity results in longlasting humoral (antibody) and cell-mediated (cytotoxic T cells, CTLs) immunity
that is both rapid and more potent.
7.3.1 Humoral Immunity
Humoral immunity is activated when B cell receptors (BCRs), membrane-bound
antibodies, bind circulating capsid [39]. BCR engagement and subsequent activation result in switching antibodies from a membrane-bound form to a secreted form
[40]. In humans, AAV administration leads to an increase in circulating anticapsid
immunoglobulin M (IgM) and high-afnity immunoglobulin G (IgG) antibodies
that persist for several weeks [41].
In the course of a natural viral infection, the result of de novo antibody production most likely slows down viral replication. However, AAV is a nonreplicating
vector and the impact of these de novo anticapsid antibodies detected after vector
administrations is unclear. Nevertheless, patients administered Rituximab, a B
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K. Patel et al.
cell-depleting monoclonal antibody, along with AAV vectors resulted in higher circulating levels of vector over an extended period compared to control patients [42].
This nding suggests that de novo antibodies may have a role in reducing the
amount of circulating vector.
De novo antibodies have also been implicated in complement activation. Muruve
etal. were the rst to report AAV vectors activated complement; an ancient system
of proteins involved in binding and lysing pathogens or pathogen-infected cells
[21]. However, for many years after this initial publication, there were no reports of
complement activation in AAV preclinical studies or in the clinic. A general assumption was that while the potential always exists for complement activation, this was
an uncommon event. This changed with reports of complement activation following
high-dose systemic vector administration in AAV clinical trials for Duchenne muscular dystrophy (DMD). Two independent trials that infused high-dose AAV vector
into pediatric patients to treat DMD noted complement activation [43]. Patients
enrolled in these trials were selected to be NAb negative and therefore should not
have activated complement via preexisting anti-AAV antibodies. Emerging data
suggests that complement activation may be related to de novo antibodies [44].
Vector antibody immune complexes could also trigger a strong inammatory
response that in some instances could be fatal. Such “antibody dependent enhancement” leading to increased inammation has been observed for several viral infections [45]. Of note, a NAb-positive patient administered high-dose adenoviral vector
as part of a clinical trial for ornithine transcarbamylase deciency developed a fatal
systemic inammatory response [8]. Posterior analysis of the patient serum before
vector infusion demonstrated ADE of adenoviral infection that triggered strong
inammatory responses in APCs [46]. While ADE of AAV transduction has been
reported, its role in enhancing invivo inammatory responses is unclear. It must be
noted that these reports of ADE of viral infectivity have been related to preexisting
immunity. The risk of ADE from de novo antibodies is less clear. Since ADE is
more strongly associated with IgG and not IgM antibodies, the rapid clearance of
vector from circulation before the formation of high-afnity IgGs may reduce ADE
risk [47].
Humoral immunity to the transgene product can also occur especially when
treating patients with missense or null mutations. However, there is no evidence of
antitransgene antibodies in Hemophilia B patients treated with AAV.FIX gene therapy [48, 49]. The risk of developing antitransgene antibodies may have been reduced
by excluding patients with a history of developing anti-FIX antibodies following
factor replacement therapy [48].
Pharmacological approaches to reduce the risk of de novo antibody generation
following AAV vector administration have focused on the use of immunosuppression strategies. A combination of immunosuppressive drugs, rapamycin, and rituximab, was demonstrated to block the emergence of antibodies after AAV
administration to patients [50]. Similarly, coadministration of rapamycin nanoparticles also effectively prevented antibody generation after vector administration in
preclinical animal models and in the clinic [51]. This approach relies on tolerance
induction to the AAV capsids following the uptake of vector and rapamycin
7 Even aWorm Will Turn: Immunity Following AAV Vector Administration
177
nanoparticles by the same APC. Alternatively, coadministration of the CTLA4
mimetic, abatacept, was also demonstrated to reduce humoral immunity to AAV8
capsids in preclinical trials for hemophilia B [52]. Transient depletion of immunoglobulins can also avoid the formation of capsid–antibody complexes until antigen
is cleared [53–55]. Finally, complement activation in patients treated with AAV vectors has been managed by administering the anticomplement factor monoclonal
antibody Eculizumab [56].
7.3.2 Cell-Mediated Immunity
Cell-mediated immunity (CMI) follows the activation of cytotoxic T cells (CTLs)
that recognize and eliminate AAV transduced cells, and T helper cells (Th cells) that
promote CTL or B cell responses [57]. These distinctions are not always strict and
functional overlap can occur between cell types. CTL killing requires T cell receptor (TCR) recognition of antigen-derived peptides presented on Class I major histocompatibility complex molecules (human HLA or mouse MHC) in transduced
cells. NHP studies demonstrated that AAV transduced hepatocytes present capsid
antigen on Class I molecules that rendered them targets for CTL killing. In the
clinic, hemophilia B patients treated with an AAV2 vector expressing human FIX
demonstrated transgene expression loss after activation of capsid-specic CTLs [6].
Similarly, hemophilia B patients administered a self-complimentary AAV8 vector
expressing human FIX also demonstrated activation of AAV capsid-specic T cells
at 8weeks that coincided with a drop in FIX levels [7]. These ndings differ from a
muscle-directed AAV gene therapy for alpha-1 antitrypsin deciency that demonstrated stable transgene expression in the presence of capsid-specic T cells [58]. It
is possible that the routes of vector administration (intravenous vs intramuscular) or
capsids (AAV2 vs AAV1) that were employed in these trials differentially impact
CTL activation.
CTLs can also be activated following Class I presentation of the encoded therapeutic transgene product. However, the response to transgene has been different and
varies among animal models. In mice, the presence of transgene-specic CTL cells
was insufcient to eliminate AAV transduced cells. A second inammatory or “danger” signal via TLR activation was additionally required for efcient CTL homing
and elimination of transduced cells [59]. An exception was immunogenic AAV vectors that could bypass the need for additional inammatory signals [60]. While the
exact role of these inammatory signals is not clear, the resulting cytokines are
known to increase the expression of both Class I MHC molecules on hepatocytes
[61], and adhesion molecules on endothelial cells [59]. An increase in adhesion
molecules is expected to help with CTL extravasation; a process where leukocytes
migrate across the endothelial barrier to access inamed tissue [62]. Transgene CTL
responses in larger animals are complicated compared to genetically identical
inbred mice. For instance, while several studies have reported stable non-self GFP
transgene expression in non-human primates following AAV.GFP vector
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K. Patel et al.
administration, there is also evidence of GFP CTL responses that resulted in hepatitis and loss of transgene expression in cynomolgus macaques intraportally administered with an AAV7.GFP vector [63].
There is currently limited evidence for the loss of AAV-transduced cells in the
clinic following a transgene-specic immune response. CTL responses were
observed in three out of six DMD patients administered with an AAV vector expressing mini-dystrophin [64]. Epitope mapping conrmed that CTL responses were
directed against exons present in the therapeutic mini-dystrophin that were missing
in the patient. In some instances, transgene-specic CTL responses have been
observed to polymorphic residues following AAV vector administration to A1AT
patients [65]. This nding underscores the risk of gene transfer to patients with
polymorphism that differs from the therapeutic transgene sequence and can elicit
immunity. More recently, T-cell responses to AAV transduced dorsal root ganglions
(DRGs) have been implicated in the loss of these lower motor neurons within a few
months of vector administration in NHPs [66]. Initial ndings suggest CTL
responses to the transgene caused DRG cytotoxicity.
Besides CTLs and Th cells, a specialized type of lymphocyte, the T regulatory
cell (Treg), suppresses AAV transgene immune responses [67]. In rodents, hepatic
T regulatory cells were involved in maintaining tolerance to the transgene following
liver-directed gene transfer [68]. Similarly, muscle biopsies of patients treated with
AAV.A1AT gene therapy demonstrated Treg inltration into muscles [69].
Interestingly, these patients had sustained A1AT expression despite the presence of
capsid-specic CD8+ and CD4+ T cells. These ndings strongly suggest a role for
Tregs in mitigating anti-AAV immunity and helping maintain tolerance to capsid
and/or transgene.
Several approaches have been adopted in the clinic before and after AAV vector
administration to minimize the impact of developing capsid and/or transgenespecic CTLs (Table7.2). Reactive corticosteroid administration after an increase
in LFTs that occurred 6–7weeks after vector infusion mitigated the CTL response
and rescued transgene expression in AAV FIX clinical trials [7]. Prophylactic corticosteroid administrations starting a day before AAV administration have also been
used to suppress CTL activation [70]. A combination of immunomodulatory drugs
that suppressed both T and B cells transiently has also proved successful in keeping
CTL responses lower in Pompe patients treated with an AAV vector [50]. However,
the most effective combination of immunosuppressive drugs to transiently curtail
CTL responses and achieve stable transgene expression is still being explored. Of
concern are the different arms of the immune system that need to be suppressed and
the duration of immunosuppression. For instance, CTL responses have occurred
6–8weeks after vector administration in the clinic. Thus, one option would be to
immunosuppress patients until the risk of activating a CTL response has passed
after which immunosuppression can be withdrawn. Whether the window of immunosuppression needs to be longer or shorter has been explored and may vary based
on several factors, including the choice of capsid, route of administration, and vector dose. There is also the risk to patients from opportunistic infections that require
limiting the duration of immunosuppression [71]. Immunosuppression regimens
7 Even aWorm Will Turn: Immunity Following AAV Vector Administration
179
Daily
Cs+MMF
Rx+Rapa Daily
(diaphragm)
MP tapered at day
10 for pediatric
patients
Rx+Rapa
IV Rx+tac
5E13 vg/kg
Disease Sponsor Age Vector Dose ROA IS protocol Steroid
LPL AMT [87] Adult Glybera 1E12 vg/kg IM 12weeks
Table 7.2 Immunosuppression protocols in AAV clinical trials
Biomarin [77] AAV5 6E13 vg/kg
Hemophilia A Pzer [78] Adult AAV6 3E13 vg/kg IV – Reactive
Pzer [80] Spark100 5E11 vg/kg
Hemophilia B St.Jude/UCL [7] Adult AAV8 6E11 vg/kg IV – Reactive
Uniqure [79] AAV5 2E13 vg/kg
Pzer [74] AAV9 2E14 vg/kg Daily
DMD Solid [88] Pediatric AAV9 0.5 & 2E14 vg/kg IV Reactive
Sarepta [75] Rh74 1.3E14 vg/kg Reactive
PD UF [89] Pediatric AAV9 4.6E13 vg IM
Canavan Myrtelle [90] Adult AAV9 4.5E13+5E13 (IT) IV+IT Daily
GM1 NIH [91] Adult AAV9 1.5E13 vg/kg IV Rx+Rapa Daily
ALS Apic Bio [92] Adult AAVrh10 4.20E14 IT Rx+Rapa Daily
Danon disease Rocket [93] Pediatric RP-A501 (A)1.1E14 vg/kg (P)
LPLD Lipoprotein lipase deciency, DMD Duchenne muscular dystrophy, PD Pompe disease, ALS Amyotropic lateral sclerosis, UF University of Florida, IM
Intramuscular, IV Intravenous, IT Intratracheal, MP Methylprednisolone, MMF Mycophenolate mofetil, Rx Rituximab, Tac Tacrolimus, Rapa Rapamycin/
sirolimus