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rAAV Integration: Detection and Risk Assessment
Jing Yuan1, Irene Gil-Farina2, Raffaele Fronza2, and
Laurence O. Whiteley
1
Department of Toxicology, Kymera Therapeutics, Watertown, MA, USA
2
ProtaGene CGT GmbH, Heidelberg, Germany
3
Pfizer Inc. Drug Safety Research and Development, Cambridge MA, USA
3
13.1 Introduction
Several attributes of recombinant adeno associated virus (rAAV) vectors contribute
to the popularity of their use in invivo gene therapy (GTx). First, multiple serotypes
with different tissue tropism, second, widespread infection of wild‐type AAV
(wtAAV) in mammalian populations without evidence of pathogenicity and, third,
they are able to express a transgene without integrating into the host cell genome
(e.g. episomal). While considered to be a nonintegrating vector, data, primarily from
neonatal mice, indicates that AAV can integrate and result in genotoxic effects,
leading to the development of hepatocellular carcinoma (HCC)[1, 2]. The human
relevance of recombinant AAV vector‐induced liver tumors in neonatal mice is
controversial[3]. The uncertainty of human relevance of these findings in mice and
the increased interest in developing AAV GTx as a therapeutic modality has resulted
in intense interest in the scientific and regulatory community as evidenced by several
recent public forum that have discussed this topic: September 2021 FDA Cellular,
Tissue, and Gene Therapies Advisory Committee (CTGTAC) meeting[4], the 2021
ASGCT Policy Summit, and the November 2021 ASGCT‐FDA Liaison Meeting[5].
This chapter will: (1) review the biology of AAV integration and published literature related to AAV‐associated carcinogenesis, (2) discuss study design considerations for assessing AAV integration in nonclinical safety studies, (3) outline
317
Drug Development for Gene Therapy: Translational Biomarkers, Bioanalysis, and Companion
Diagnostics, First Edition. Edited by Yanmei Lu and Boris Gorovits.
© 2024 John Wiley & Sons, Inc. Published 2024 by John Wiley & Sons, Inc.
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318
methods for detecting and quantitating AAV integration, (4) discuss the regulatory landscape related to AAV integration, and (5) provide perspective on human
risk assessment considerations.
13.1.1 Biology of AAV Vectors as it Relates to Mechanisms of
AAV Integration
wtAAV has a single‐stranded DNA genome that is flanked by inverted terminal
repeat sequences (ITRs). The genome encodes two genes: the rep gene that is
required for replication and packaging of the DNA and cap gene that encodes the
proteins that assemble in the viral capsid. Following entry into the cell, wtAAV
can be maintained as an episome in the host genome or integrated into host
genome, AAVS1locus on chromosome 19was identified as a preferential integration region in humans. The integration is mediated by viral Rep protein. In rAAV,
the rep and cap genes are removed and replaced with the transgene expression
cassette; thus, the only remaining viral genome sequences are the ITRs that are
essential for packaging of the vector genome and second strand synthesis. rAAV
vector genome processing toward stable transduction relies on host cellular
machinery. Following AAV vector transduction, single‐stranded (ss) rAAV vector
genomes are used as template to form double‐stranded (ds) linear rAAV monomers as the intermediates which then transform to stable double‐stranded circular monomers and concatemers DNA [6]. Unlike wtAAV, rAAV DNA lacks the
Rep‐mediated active integration and primarily remains in a circular concatemeric
episomal form after transduction. However, AAV vectors passively integrate at
low frequency into the target cell genome. rAAV integration events may happen
both through nonhomologous end‐joining (NHEJ) DSB repair pathway and
homologs recombination. Unlike retroviral vectors, which induce double‐stranded
breaks to facilitate integration, AAV uses the spontaneous double‐strand breaks
that occur during the normal cell cycle to integrate into host cell DNA.
13.1.2 Literature Review of AAV Studies in Relation to
Neoplasia Development
Integration of rAAV into host cell genome has recently been reviewed and the
nonclinical studies are summarized in Tables 13.1 and 13.2 [21]. Evidence of
rAAV vector integration into the host genome with subsequent tumorigenesis first
came from rAAV‐treated neonatal mice. Mucopolysaccharidoses (MPS)VII is a
lysosomal storage disease caused by a deficiency of beta‐glucuronidase (GUSB).
When MPSVII mice were intravenously injected with an AAV2 vector containing
a cytomegalovirus early enhancer element and chicken beta‐actin promoter
(CAG) and a human GUSB cDNA at the newborn stage (postnatal day 2),
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13.1 Introduction 321
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Table13.2 List ofstudies withrAAV administration inlarge animal species.
Publication Species
Nowrouzi
NHP WT
etal.[22]
Gil Farina
NHP WT
etal.[3]
Mattar
NHP WT
etal.[23]
Spronck
NHP WT
etal.
2020[24]
Sullivan
NHP WT
etal.[25]
Niemeyer
Dog Hemophilia
etal.
2009[26]
Nguyen,
Dog Hemophilia
Everett
etal.[27]
Batty
Dog Hemophilia
etal.[28],
Batty
etal.[29]
Disease
model
18
n=
n= 6
6
n=
12
n=
n= 12
B
4
n=
A
n= 9
A
n= 8
AAV vector;
route
AAV‐RSV‐
LEA29Y
AAV1 or AAV8
IV or IM
AAV5‐hPBGD
(IV)
AAV‐hFIX
AAV8 or AAV5
(umbilical
vein)
AAV5‐hFIX
(IV)
AAV5‐hFVIII
(IV)
AAV2‐CMV‐
cFIX (IM)
AAV2‐(Apoe)4/
hAAT‐cFIX
(IV)
AAV‐TBG‐
cFVIII
AAV‐hAAT‐
cFVIII
AAV8 or AAV9
(PV or IV)
AAV‐TTR‐
cFVIII
AAV2, 6, 8
PV
AAV dose
(vg/kg)
12
5
× 10
13
× 10
1
13
5 × 10
1.4–
1.9
× 10
11
5
× 10
12
5 × 10
2 × 1013
13
9 × 10
13
× 10
2
13
6 × 10
1.1 × 10
to
3.4 × 10
13
1 × 10
13
2 × 10
13
4 × 10
12
6 × 10
to
2.7 × 10
Time of
treatment
Adult 1.2–2.8 yr
Adult 1 mo
In utero 11–71mo
13
Adult 6 mo
Adult 13 and
12
Juvenile/
Adult
12
5.5–12
mo
Juvenile/
Adult
5 mo–4 yr
Juvenile/
13
Adult
6 mo–2 yr
Duration
of F/U
26 wk
(6 mo)
8
yr
2–10 yr
8–12 yr
Source: Sabatino etal.[21]/Elsevier/Licensed under CC BY 4.0.
long‐term transgene expression, GUSB activity, and phenotypic correction were
achieved. A number of long‐lived rAAV‐treated MPSVII mice developed HCC and
angiosarcomas over a year after treatment [30]. A follow‐up study in newborn
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322
MPSVII with intravenous injection of the same AAV2 vector confirmed HCC
development in in both normal and MPSVII mice 13months after dosing. In addition, this study detected rAAV integrations in the tumor tissue. All of the integrants were rearranged such that the GUSB cDNA was deleted[1]. This set of
neonatal mice studies provided the main evidence on integration and tumorigenesis risk of AAV therapy.
Several additional studies reported liver tumorigenesis following rAAV administration in mice. However, the relevance of AAV integration as the primary event
is questionable, especially in adult mice. The confounding factors, such as
transgene expression[9, 12], genetic background in certain mouse strains[13], or
pretreatment in study mice that predispose to tumor risk maybe the primary cause
of tumor formation in many cases. The tumorigenesis findings related to AAV
integration are considered age‐specific to neonatal mice (PND1‐2)[1, 30] and, in
some cases, mouse strain specific to those sensitive to tumor formation. This argument has been supported by several studies in juvenile and adult animals where
administration of AAV either failed to induce tumorigenesis in rodents[8, 13, 31],
or the frequency of liver tumors in AAV‐treated animals was comparable with an
average frequency of spontaneous liver tumors in C57BL/6mice (0–10%). A summary of AAV integration studies in rodents is listed in Table 13.1. Neonatal‐
specific HCC in AAV‐treated mice can be explained by the fact that the neonatal
liver contains proliferating hepatocytes, which may lead to a high frequency of
rAAV integrations at the sites of DNA strand break, thus has an intrinsic susceptibility to tumor formation. This hypothesis is supported by the findings that AAV
administration in adult mice can lead to HCC in the context of chronic liver disease, a state that induces hepatocyte proliferation[19].
HCC related to AAV administration has not been identified in large animals nor
human. AAV integration and clonal expansion have been observed in hemophilia
dogs following treatment with a variety of AAV serotypes. No tumor formation
has been observed following more than 10 years observation[18, 26, 27, 32, 33]. In
a long‐term study of AAV gene therapy with hemophilia A dogs[34], two dogs
treated with AAV vectors expressing canine factor VIII (AAV‐cFVIII) and followed for up to 10 years had vector integration in host genome and clonal expansion. Integration events were enriched in or near genes involved in cell growth.
Most of the integration recovered lacked transgene sequence and showed vector
deletion and rearrangement, a phenomenon observed with many AAV integration
events. None of the dogs with clonal expansions showed evidence of tumors or
altered liver function revealed by liver enzyme levels and serum alpha‐fetoprotein
(AFP), a clinical biomarker for HCC. However, in another long‐term study with
hemophilia A dogs treated with AAV‐cFVIII, while the genome integration was
confirmed as rare events, no clonal expansion cells and tumors were detected after
8–12 years follow‐up[28]. The discrepancy may be related to different methods
13.1 Introduction 323
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and genome annotation is used for integration analysis. This highlights the
complexity of data analysis and methodological challenges in identifying the rare
integration events and clonal expansion.
Additional integration studies have been performed in large animals, including
nonhuman primates, and animals have been followed up for extended years after
various AAV treatments (Table13.2). The integration is confirmed to be rare event
and integration sites (IS) are random across the host genome with no clustering of
integration. One nonhuman primate (NHP) study delivered a self‐complementary
(sc) AAV5 and AAV8 with a LP1‐driven‐human factor IX (hFIX) transgene
(scAAV‐LP1‐hFIXco) to late‐gestation fetuses or adult animals through a single
intravenous injection[23]. Sustained clinically relevant levels of hFIX with liver‐
specific expression were observed without any clinical concerns four to six years
after AAV administration. However long‐term genotoxicity evaluation in NHP
remained to be determined.
AAV integration data in patients is generally lacking as patient liver biopsies are
not readily accessible. However, several different AAV serotypes (AAV1, AAV2,
AAV5, AAV8, and AAV9) have been or are currently being used in clinical trials
(including children) and no increase of cancer of any type has been reported
[31, 35–37]. In a study investigating the potential of AAV therapy‐induced tumorigenesis in humans, patients participating in an AAV2/5 gene therapy trial for
acute intermitted porphyria provided liver biopsies for analysis[3]. In a context of
low transduction levels, the study confirmed that AAV integration is both low in
frequency and random in nature, with no clustered IS near genes that had been
previously implicated in the mouse studies. Another study followed a small number of hemophilia B patients up to 15 years after liver‐directed AAV2‐FIX gene
transfer and found no evidence of tumor formation as assessed by liver transaminase values, serum α‐fetoprotein, and liver ultrasound[38]. Finally, FDA‐approved
AAV therapies, such as Luxturna and Zolgensma, have not reported preneoplastic
proliferative lesions after multiple years of posttreatment follow‐up.
In general, it is believed that the strong promoters, such CAG or TBG promoters, likely drive the read through after genome integration and trigger the nearby
proto‐oncogene expression that leads to HCC development[2]. This hypothesis
has been challenged by a few liver targeting AAV therapies with strong promoters,
such as Zolgensma for spinal muscular atrophy‐type 1 (SMA1), where the vectors
contain a strong CBA promoter and have been administrated at a high dose of
14
vg/kg to infants less than two years of age with rapid liver growth. Tumor
2
× 10
or neoplastic changes have not been identified in any of the hemophilia dogs that
were dosed as juveniles (1.5–10months of age) and with the vectors containing
the CBA promoter, suggesting that the combination of a strong promoter and liver
growth/cell proliferation do not necessarily contribute to enhanced neoplastic
risk in nonrodents when treated with an AAV gene therapy vector.
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Similar to AAV GTx vectors, there is little evidence that natural infection with
wtAAV in humans is associated with neoplasia. Infection of wtAAV, mainly
AAV2, is frequent in the human population. However, there is no clear evidence
of wtAAV integration leading to neoplasia in humans. Insertion analyses of the
liver tumors identified AAV host genome integration in less than 10% of the subjects with HCC[39–41]. AAV integrants were found in both tumors and non‐
tumor tissue, and in some cases, the integration rates were higher in the non‐tumor
tissues than in the tumors[39]. AAV was also detected with a similar frequency in
malignant and benign tumors. Thus, the concurrence of AAV infection and integration and HCC does not support a pathogenic role for wtAAV infection in the
HCC patients. While some of the IS were identified to be in the genes related to
cancer development, those genes are also the recurrent targets by tumorigenic
hepatitis B virus (HBV) integration[42], hypothesizing that HBV infection could
be the main driver for malignant tumor, and wtAAV integration could be bystanders in HCC development. Consistent with the notion, non‐clonal AAV2insertions
distributed throughout the genome in non‐tumor samples, and AAV integration
were only enriched in cancer genes in the malignant tumor cell.
13.2 Review of Regulatory Guidance and Discussion
Points that Are Raised on AAV Carcinogenesis
Four classes of viruses have been used as GTx vectors that are considered episomal vectors (AAV, adenovirus, poxvirus, and herpes simplex) because they do not
need to integrate into host cell genomes to produce their transgene[43]. This is in
contrast to retroviral vectors (gamma retroviruses and lentiviruses) that need to
integrate into the host cell genome to express their transgene. Amongst all these
vectors used as GTx vectors, only vectors derived from gamma retroviruses have
been definitively linked to insertional mutagenesis, leading to neoplasia in
humans[44].
Because AAV does integrate at low frequency, the risks of AAV vector genotoxicity and carcinogenicity from insertional mutagenesis remain an ongoing concern by health authorities even though FDA and EMA guidance documents
consider AAV vectors as non‐integrating[43, 45–48]. However, both agencies discuss research publications by Donsante and coworkers that described the induction of HCC, associated with AAV vector DNA integration, in mice that were
treated with AAV as neonates and imply that this should be considered in safety
evaluations[1, 7].
Based on review of regulatory document of approved AAV gene therapies
(Glybera, Luxturna, and Zolgensma), both EMA and FDA have acknowledged
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