Additional Risks of Gene Therapy
Hepatocellular Carcinoma
The Adeno-associated virus was discovered in the 1960s and for decades
was not thought to cause any human disease.29 For this
reason, it was an ideal candidate for use as a gene therapy vector.
However, in 2015, Nault et al reported wild type AAV (wAAV) insertions
near the TERT promoter in human hepatocellular carcinoma (HCC)
specimens.30 Additional wAAV insertions near 4 other
oncogenes were found, and the insertions were
clonal.30 These same oncogenes are targeted by
hepatitis B, which is known to contribute to HCC.31There remains debate as to whether the wAAV insertions are drivers of
oncogenes or benign passengers.32 As far as hemophilia
gene therapy goes, it really doesn’t matter if wAAV causes HCC or not.
wAAV is not infused into patients for gene therapy, rAAV is. The
question for hemophilia gene therapy is does rAAV cause HCC?
Unfortunately, the answer is yes, and here there is no debate.33 Pre-clinical Investigations of rAAV gene therapy
date back over 2 decades. An early investigation of rAAV involved a
mouse model of mucopolysaccharidosis VII (MPSVII). Neonatal mice with
this disorder were successfully treated with rAAV. However, a small
number developed HCC.34,35 Because MPSVII is fatal in
mice, it was unclear if the rAAV was causing the HCC or the underlying
disease was. A follow-up study included a normal control group. These
neonatal mice also developed HCC following rAAV.34Investigation over the ensuing 2 decades showed that rAAV inserts and
drives the RIAN locus, more specifically microRNA-341, in
neonatal mice. This does not occur in adult mice. Humans do not have aRIAN ortholog, but dogs do.35 The combination
of rAAV/neonatal mouse/RIAN locus are needed to induce HCC, or so
it was thought.34
Numerous trials of rAAV gene therapy for a variety of genetic disorders
have been carried out in adult mice without the development of
HCC.36 One difference in neonatal and adult mice is
that neonatal mice have more proliferating cells, notably, hepatocytes.
A recent investigation of rAAV gene therapy tested if rAAV could induce
HCC in adult mice with liver injury (proliferating
hepatocytes).36 Investigators used either partial
hepatectomy or fatty liver disease (high fat diet). Two rAAV vectors
were used, one designed to drive the RIAN locus, and another vector
control with a reporter gene. As expected, neither vector caused HCC
above baseline in control adult mice. The rAAV vector designed to drive
the RIAN locus caused HCC in both models of proliferating hepatocytes.
Surprisingly, so did the control vector in adult mice with fatty liver
disease (partial hepatectomy not tested).36 In
addition, a clinical trial of rAAV for phenylketonuria was placed on
hold due to the development of HCC in a mouse model.2Thus, rAAV can induce HCC in adult mice with proliferating hepatocytes.
What about other animal models?
To date, HCC has not been observed in dogs or non-human primate rAAV
gene therapy for hemophilia. However, clonally proliferating liver cells
have been found.37 Nguyen et al. recently reported on
a long term follow up of hemophilia A gene therapy in dogs using
rAAV.37 Nine dogs were followed for up to 10 years.
Liver samples were available in 6 of the dogs. rAAV integrations were
found in 1741 sites, and between 1 and 130 cells per integration.
Preferential integrations near oncogenes in clonally proliferating cells
were seen. The dogs were otherwise healthy. The authors did not report
on the presence or absence of fatty liver disease. A second long term
study of 8 hemophilic A dogs receiving rAAV gene therapy did not find
clonal proliferation in the liver.38 This manuscript
did not report on rAAV integrations, and additional analysis may be
forthcoming. Long term data for hemophilia B gene therapy in dogs is
also available.39,40 Many canine gene therapy
strategies for hemophilia B using rAAV targeted skeletal muscle and may
not confer the same HCC risk as strategies targeting hepatocytes. There
are no reports of HCC. However, detailed analysis for rAAV insertions
and clonal proliferation have not been forthcoming.
One subject in a human trial of rAAV for hemophilia B developed
HCC.35 This subject had risk factors for developing
HCC including hepatitis B and C. His liver specimen has been extensively
evaluated and did not reveal genetic changes related to rAAV
carcinogenesis but did have genetic changes typically found in
HCC.35 No other human subjects have been reported with
HCC following gene therapy for hemophilia. A recent report of liver
biopsy results from 5 subjects who participated in a hemophilia A gene
therapy trial did not show evidence of HCC or clonal
proliferation.41 Notably, 4 of 5 subjects had liver
steatosis, which apparently was
subclinical.
In summary, emergent data has shown that rAAV can induce HCC in adult
mice with liver disease.36 Long term studies of small
number (<15) of hemophilia A dogs have shown clonal
proliferation in the liver.37,38 If rAAV causes HCC in
a small proportion, or even 5-10% of dogs, it could easily be missed by
the current canine studies. To date, no studies have looked at the risk
of HCC development of dogs or non-human primates with fatty liver
disease following hemophilia AAV gene therapy. No humans have developed
HCC or clonal proliferation caused by rAAV. Since the latency for the
development of human HCC following rAAV may be decades, the risk of
human HCC following rAAV cannot currently be estimated.
Genome Integration
Another advantage of using rAAV as a vector is that it mostly remains
episomal following insertion into a cell.42 Until
recently, studies have suggested that 99+% of rAAV
vector was episomal, and <1% integrates. Emergent data has
shown a higher percentage of integrations. Up to 3% of rAAV may
integrate into liver cells following gene therapy.42Most hemophilia gene therapy protocols infuse 1014 –
1015 viral particles and target 1011hepatocytes. Assuming a more conservative estimate of genome
integrations of 0.1%, one could anticipate over 100 million
integrations following gene therapy. Indeed, some experimental data
confirms this notion. 42 Therefore, there are a
massive number of integrations following rAAV gene therapy. Most are not
intact vectors. With 1011 hepatocytes in a human
adult, each with a genome of 3 X 109 base pairs, most
integrations are likely to land in an inactive genetic region if they
integrate randomly. However, the above referenced dog hemophilia gene
therapy study suggests that integrations are not random, they tend to
occur near active genes, including oncogenes.37 One
driver integration near the wrong oncogene at the wrong time could start
the cell toward clonal proliferation and eventually, over years or
decades, overt cancer.
Other Cancers
In addition to the above-mentioned HCC following rAAV gene therapy for
hemophilia, 3 additional cases of cancer have been reported in humans,
and one in a dog.43-45 As with the HCC, the other
cancers (tonsillar carcinoma, salivary gland carcinoma, leukemia) have
been thoroughly investigated and shown not to be caused by
rAAV.43-45 It is unknown if immunosuppression received
during clinical trials may have contributed to oncogenesis. To date,
several hundred subjects have participated in clinical trials involving
rAAV for hemophilia with reported observation periods lasting several
years. There is easily over 1000 person years of observation. Thus 4
reports of human cancer may not be unexpected. However, a proper
epidemiological investigation seems indicated.
Unfolded Protein Response
The target cell for hemophilia gene therapy using rAAV is the
hepatocyte. Although factor VIII is made in the liver, it is not made in
the hepatocyte.46 Accordingly, hemophilia A gene
therapy targets a cell that does not typically produce factor VIII.
Factor IX is naturally made in the hepatocyte. Factor VIII is a large
protein with complex folding. Misfolded factor VIII protein can lead to
cellular toxicity via the unfolded protein response
(UPR).47 This has been shown to occur when non-native
cells are driven to express factor VIII. This occurs both in vitro
(Chinese hamster ovary cells) and in vivo (mouse hepatocytes) for factor
VIII.47,48 Factor IX expression is stable for years
following rAAV gene therapy in animal models and
humans.1,2 While factor VIII expression has been
stable following rAAV gene therapy in animals, this has not been the
case in humans when therapeutic levels are achieved. As reported above,
several clinical trials of AAV gene therapy for hemophilia A have shown
that hemostatic levels are not sustained.24,28 The
etiology for the falling levels remains unclear. An immune response has
been investigated and does not clearly seem to be the cause.35 UPR (Unfolded Protein Response) could provide
another explanation. However, as above, liver biopsies from subjects
following hemophilia A gene therapy failed to demonstrate evidence for
UPR at the time of biopsies.41 Biopsies were performed
2.6-4.1 years following infusion of rAAV. So cellular toxicity/loss from
UPR occurring prior to this would have been missed.
Although an immune response and UPR have been independently proposed as
explanations for falling factor VIII expression following AAV gene
therapy, an investigation by Butterfield et al. suggests that they may
not be mutually exclusive.49 In addition, this study
also suggested that translational shutdown (related to UPR and immune
response) rather than loss of hepatocytes could lead to falling factor
VIII expression following AAV gene therapy.
Concerns about UPR and HCC have also been raised. Kapelanski-Lamoureux
et al. have investigated UPR and HCC risk in mice.50In their study, all mice fed a high fat diet following receipt of a
B-domain deleted factor VIII gene therapy vector (non-AAV) via
hydrodynamic tail vein injection developed liver tumors. This happened
less so with a factor VIII variant vector less prone to misfolding and
not at all with a control vector. This suggests that factor VIII
misfolding in mice fed a high fight diet contributes to the development
of HCC independent of viral vector integration.
Spinal Muscular Atrophy
One of the first rAAV gene therapy treatments to achieve regulatory
approval was for the treatment of spinal muscular atrophy (SMA). This is
a degenerative neuromuscular disorder that results in early death in
those affected with severe (infantile) forms. rAAV gene therapy for this
disorder has met with widespread success. 51 While
affected infants treated with rAAV gene therapy are not normal, they are
achieving developmental milestones with an extended lifespan. Like
hemophilia rAAV trials, the principal toxicity seen during the SMA
trials was mild liver inflammation that is controlled with steroids.52 Now that over 3000 infants have received gene
therapy for this disorder, rare side effects not seen during the
clinical trials are being observed. 52 At least 9
cases of thrombotic
microangiography (TMA) have been reported in the medical literature
following rAAV gene therapy for SMA, one of which was fatal.53 Thirty (two fatalities) cases of TMA are reported
in the U.S (United States). Food and Drug Administration Adverse Event
Reporting System (FAERS). 54 It is unknown if there is
any overlap between cases reported in FAERS and the medical literature.
The manufacturer of SMA rAAV has reported two cases of hepatotoxicity
leading to fatalities. 56 FAERS reports 120 cases of
hepatobiliary disorders and 8 fatalities. 54 One
should interpret the FAERS data with caution, as the cause of death is
not listed, only “Reactions”. Duplicate reports may also be present.
Therefore, patients who died from causes unrelated to rAAV may be
included in this database. Table 1 shows the number of reported relevant
“Reactions” and deaths in the FAERS database for regulatory approved
medications for SMA.54 54-58 A comparison of clinical
trial results suggests that the event free survival rate is similar
between Onasemnogene abeparvovec and Risdiplam, and higher than
Nusinersen.58Post-marketing surveillance from
the FAERS database also shows higher mortality reporting for Nusinersen.
TMA and hepatobiliary disease following treatment for SMA seems to be
relatively unique to Onasemnogene
abeparvovec . Use of rAAV in other clinical trials has also resulted in
hepatotoxicity related fatalities. 53 Because SMA and
hemophilia are different diseases, different age groups were treated,
and different doses used, similar toxicities may not occur in hemophilia
patients following regulatory approval for rAAV gene therapy. However,
the potential for rare, serious, and potentially fatal toxicities should
be included in any risk/benefit calculation.
Dorsal Root Ganglion
Dorsal root ganglion (DRG) pathology has been commonly found in
non-human primate gene therapy using rAAV.59 This has
been found in a variety of vectors for a variety of diseases. It occurs
more commonly with central nervous system administration but is also
seen following intravenous administration of rAAV. Immunosuppression
does not seem to ameliorate the toxicity. It is not seen with
non-expressing vectors. 60 Accordingly, the toxicity
seems related to recombinant protein expression. Fortunately, the
toxicity is mild and short lived, and importantly, does not seem to
cause obvious symptoms in the non-human primates. DRG toxicity has been
reported in human trials of rAAV, but not for
hemophilia.1,2,53,61 However, it does not appear that
subjects enrolled in hemophilia rAAV gene therapy trials were carefully
evaluated for DRG toxicity, or if they were, this data is not reported
in published manuscripts.1,2,24,25