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accommodate the volume and complexity of GT product development are needed to accel-
erate the process of provi ding treatment options to patients without compromising on the de-
livery of safe and effective therapies to patients.
Considerations for dose extrapolation
Incumbent on every sponsor is a clear justification for the proposed FIH dose based on a
sound dataset that demonstrates a scientific rationale including POC for therapeutic effect,
safety, and controls over the drug manufacturing. There are no standard algorithms for
extrapolating doses of GTs to patients. Notably, because GTs must show clear prospect of
benefit prior to Phase 1 dosing for pediatric patients, there is a substantial hurdle for sponsors
in how best to translate the dose of vector from nonclinical studies to patients (Durairaj and
Bhattacharya, 2023). Adding to this complexity, many GT programs that leverage a single
species, provide only a single set of data to scale to patients. In these instances, investigators
may need to draw upon literature, current clinical trial experience, or prior submissions to
help guide and inform the dosing framework for the clinical trial. If a laboratory large animal
species has been incorporated in the nonclinical package as a second species, it offers the op-
portunity to scale distribution and gene expression more effectively in target tissues to the
intended patient population.
Traditional PK-PD models have historically not been applied to scaling GTs. In most in-
stances systemically delivered GTs have been either linearly scaled based on body weight
e.g., vector genomes per kilogram (vg/kg), or on desired target tissue levels of the transgene
product from small animals to humans. However, in some cases this may have resulted in
underdosing some patients while overdosing others, leading to toxicities emerging with
the relatively higher doses (Kishimoto and Samulski, 2022). GTs delivered to the eye or
CNS are more constrained, with prior clinical experience in delivering formulations to these
unique spaces informing limits for dosing based on surface area (vg/mm
2
) to be covered or
volume (vg/mL) of the space.
Broadly, two dose extrapolations are typically defined. One based on the MED from the
pivotal pharmacology study to set a pharmacologically active dose (PAD) and the second
from the MFD or NOAEL from the GLP toxicology study to bracket the upper limit for
FIH testing. A combination of vector titer and infusion rate limit the MFD that can be admin-
istered in both animals and humans. Initial estimates of the upper limits a priori are helpful in
assessing whether the proposed dosing plan is reasonable or if manufacturing and/or target
exposure will impede potential for success. Ideally, a clear separation in doses can be estab-
lished between what is anticipated to be PAD in patients and the maximal safe dose, e.g., a
therapeutic index (TI). This affords a safety margin to support rational incremental steps in
patient dosing cohorts. However, developers are often left with very narrow TI’s or no safety
margin at all. In these instances, biomarkers can be helpful to evaluate efficacy but also
monitor safety following dose administration. Regardless a clear rationale is needed to justify
both the methods and dose scaling in the Phase 1 trial, inclusive of any safety margins.
New allometric m odels are emerging to aid dose scaling of GTs. Recent work by Tang and
colleagues, proposed a gene efficiency factor (GEF) to describe the efficiency of AAV gene
transfer system and demonstrated an inverse linear relationship between log GEF and log
9. Preclinical development of viral vector gene therapies286
III. Specialty biologics and indications
body weight (W) using preclinical and clinical data. Using this model, the authors were a ble
to demonstrate that a previous trial using AAV2-CMV-FIX underdosed patients, explaining
the low transgene product plasma levels (Tang et al., 2021). In 2022, Zou, and separately Burr
and colleagues, expanded on Tang’s method to retrospectively compare data across species
and in vitro systems for a variety of AAV serotypes used to develop hemophilia GTs
(Zou, 2023) and confirmed the importance of body weight in scaling expression efficiency
of transgene product (Burr et al., 2022; Zhang and Zou, 2023). This work demonstrated the
hyperbolic relationship between viral dose and steady-state cell protein secretion, similar
to what is known for ligand binding relationships. Development of these models for AAV
transgene delivery are critical as it helps to guide investigators who would otherwise
consider linear dose scaling as being the most logical approach. It will be interesting to see
how this space develops and if similar principles are adopted for other routes of administra-
tion and target organs such as the eye and CNS.
Clinical trial design
Despite a temporary dip in GT study initiations due to the COVID-19 pandemic, at present
there are over 2000 active clinical trials in the space as of 2024 (Ginn et al., 2018; Arabi et al.,
2022; ASGCT, 2024; Arjmand et al., 2020). Globally, regulators have shown flexibility in mov-
ing away from the standard stepwise clinical trial development pathways, which can be
unduly burdensome. Most regulators allow combinations of Phases 1 and 2 with Phase 3 ex-
pansions with postapproval trials to confirm the long-term treatment effects and safety of the
GT under study. The compelling evidence of efficacy seen early on in many GT trials sup-
ports the development of innovative trial designs that maximize the opportunity to identify
a safe dose with therapeutic benefit. Ultimately this high initial hurdle reduces the number of
clinical trials needed for development of GTs.
Expansion cohort trial designs
Expansion cohort trial designs are intended to expedite clinical development by seamlessly
proceeding from initial determination of a tolerated dose to identifying drugs that work best
for spe cific patient populations in the context of a single trial. This replaces the need for using
separate Phase 1 trials and multiple Phase 2 trials (FDA, 2015a, 2022f). By increasing the total
number of patients treated at potentially therapeutic doses within the Phase 1 setting, expan-
sion cohorts may provide more clinically meaningful data concerning safety, tolerability, ef-
ficacy, pharmacokinetics, pha rmacodynamics, and patient enrichment.
Master clinical protocols to support multiple drugs or multiple patient
subpopulations
Published in 2022, the FDA guidance on master protocols describes the concepts and
Points to Consider in testing multiple drugs and/or multiple cancer subpopulations in par-
allel within a single protocol without a need to develop new protocols for every trial (FDA,
2022i,k). A related draft guidance issued in December 2023 provides substantial detail on
important considerations regarding the study design (controls, randomization, blinding,
etc.) and statistical analysis approaches from data coll ected from master protocols as well
as guidance for submis sion to regul atory authorities.
Translating nonclinical data to regulatory success 287
III. Specialty biologics and indications
The term “master protocol” is often used to describe the design of such trials, most often
late-stage trials, with a variety of other terms such as umbrella, basket, or platform describing
specific designs (Woodcock and LaVange, 2017). Types of master protocol design consider-
ations covered include the use of a single common control arm, the novel combination and
concomitant administration of two or more investigational drugs, and the use of multiple bio-
markers. Further, given the challenges with operational logistics and compliant execution of a
master protocol, the guidance addresses safety coverage and safety reporting. Like standard
clinical protocols and informed consent forms (ICFs), master protocols and ICF documents go
through the institutional review board (IRB)/EC review and approval process and may also
warrant a central IRB and a smaller IRB group dedicated to address emerging issues ad hoc
for expedient reviews. Further, there may be the need for multiple ICFs for the various sub-
studies within the master protocols. Approaches for the statistical analysis for nonrandom-
ized, randomized, and adaptive/Bayesian designs may also warrant discussion with
regulators.
Innovative trial designs and clinical trials for rare diseases
The use of complex innovative trial designs (CIDs) has grown to optimize the data gener-
ated from clinical trials, particularly those in rare diseases, where traditionally designed ran-
domized controlled trials (RCTs) are often infeasible due to small and frequently
heterogeneous patient populations. Trials for these smaller patient groups are frequently
impacted by the ability to recruit patients or the unwillingness of patients or trial stake-
holders to randomly assign anyone to placebo or sham procedures. Regulators have been
open to both new trial designs and to innovative approaches for statistical analyses of the
data from early phase GT studies in rare diseases to allow use of single arm trials (FDA,
2020e; Chand et al., 2021). The goal is to balance access while providing considerations for
the analysis of externally controlled trials to protect results from potential bias and allowing
the use of patient level data from other clinical trials or from rea l-world data (RWD) sources,
such as registries as well as electronic health records and medical claims (FDA, 2023f). The
CID gui dance provides points to consider and information necessary for a productive discus-
sion wi th FDA regarding approaches for trials that have never been used before or employ
the novel application of CID components.
Control subjects, placebo groups, and use of real-world data
Control subject comparisons are of particular concern in order to minimize the introduc-
tion of bias in clinical data from studies used to determine the effectiveness of a treatment.
Although various sources of data can serve as a control arm, traditional clinical trials utilize
designs with random assignment of participants either to a treatment arm or to a placebo
with specific outcomes compared within the study protocol. In an externally controlled trial,
participants receiving a treatment are compared with a different group external to the trial
who have not received the same treatment, and the randomization process is not utilized.
The external group can be an historical control, meaning that the treated or untreated patients
are either from another time or a concurrent control (treated or untreated patients) within the
same time.
A recent FDA guidance (Feb. 2023) focuses on the appropriateness and use of patient level
data from other clinical trials or from RWD gathered from other sources (FDA, 2023f). The
9. Preclinical development of viral vector gene therapies288
III. Specialty biologics and indications
guidance provides insights regarding optimizing the similarities of the compared groups
including participant baseline characteristics, demographic factors, comorbidities, disease at-
tributes (e.g., severity, symptoms, duration of illness), start of follow-up for the treatment of
interest, concomitant therapies, and the clinical observations coll ected. Factors to consider
that may adversely impact the interpretability of the clinical data obtained from external con-
trols including data integrity and missing data are detailed along with considerations for
assessing data comparability in external control populations.
Use of surrogate endpoints
Surrogate endpoints are markers that can predict clinical benefit but are not actual measure-
ments of clinical benefit as would be expected from standard clinical trials. These surrogate
markers may come from nonclinical pharmacology studies or exploratory endpoints from
the literature. The use of surrogate endpoints to support accelerated approval for GT products
in rare diseases is acknowledged as a necessary approach to ensure patients have GT treatment
options (Precision Medicine Online May 19, 2023 e FDA’ s Peter Marks Assures Flexibility in
Reviewing Gene Therapies for Accelerated Approval | Precision Medicine Online (Cohen,
2023). The challenge lies with regulatory agreement on the choice of endpoint(s) to accurately
predict clinical benefit and the supportive data needed to be convincing of a treatment effect.
Traditional Phase 1, 2, and 3 clinical trial designs and data collection approaches have been
the gold standard for evaluating the safety and efficacy of small molecules and biologics for de-
cades. GTs have introduced challenges to this paradigm through administration of a single
dose that is intended to provide a therapeutic benefit over a patient’s lifetime, usually in dis-
eases with a very small patient population. The recent trends toward increased regulatory flex-
ibility in the acceptance of innovative clinical trial designs for evaluation of GTsin the treatment
of serious and rare diseases has begun to fulfill the promise of improved efficiency and will
likely transform the future development pathways for these products.
Ethical considerations and weighing risk/benefit
The ethical considerations for GT products in development has consistently challenged
clinical investigators, ethicists, regulators, and scientists, particularly in evaluating and
communicating GT risks and potential benefits, selecting study participants fairly, and
engaging with patient communities (Lowenstein, 2008; Riva and Petrini, 2019; Iyer et al.,
2021). Currently, most GT trial participants have severe, life-threatening, progressive diseases
or rare diseases with high unmet and unaddressed medical needs. The urgency to enroll in
clinical trials for an opportunity to benefit from GT treatment continues to be weighed against
the risks that come from GT exposure. At the forefront for consideration are product-related
risks such as immunogenicity, toxicity, durability of effect, and irreversibility of treatment.
Managing the uncertainty around GT risk/benefit profiles requires a clear and thoughtful
ethical perspective. The foundational evidence in ascertaining the risk/benefit profile for a
first GT exposure in patients typically comes from nonclinical studies in mammalian cells
and in both healthy animals and animal models of disease. This places an enormous burden
on scientific, regulatory, and clinical decision-makers to predict clinical effects from the data
in the nonclinical package. For GTs, vector exposure typically produces an immune response
and informing patients or their guardians of the potential immune responses and
Translating nonclinical data to regulatory success 289
III. Specialty biologics and indications
management of the responses is only part of the ethical communications to patients. The
other and perhaps more important factor to consider is that exposure and imm une responses
to a given vector precludes study participants from subsequent dosing of the same vector. In
the case of toxicities or exaggerated pharmacology or insertional mutagenesis, drug with-
drawal is not possible; therefore, dose selection is a key decision for clinical investigators
along with communication around potential toxicities to support optimal patient selection
and informed consent. While a single GT dose is anticipated to produce therapeutic effects
to span a patient’ s lifetime, it is currently not known how long GT treatment effects will
last. The currently mandated long-term clinical follow up ranging from 5e15 years (FDA,
2020f) is the only means to satisfactorily address this question and requires commitment
from both patients, trialists and the disease stakeholder community.
In clinical trials of GT products, all stakeholders must be clear on the ethical considerations
around study enrollment. Strategies for selection of the first patient to enroll in a GT trial as
well as the process of selecting subsequent patients for enrollment are primary and should
focus on achieving the scientific goals of the study. Study designs that enhance potential ben-
efits for participants (e.g., higher starting dose, more rapid dose escalation) might be ethically
preferable although the trade-off may be an increased exposure to the risk of toxicity. The
patient population for a study of early or late-stage disease also plays a role in enrollment
decisions. Pediatric study enrollment requires unique ethical considerations relating to GT
risk-benefit in children, the impacts of their possible exclusion from future trials of more effec-
tive therapies, and the inability to withdraw from a GT treatment. Regulators frequently
request pediatric studies begin in older patients for the initial characterization of safety.
And for patients there is the potential for randomization to placebo control or sham injection
groups, but with a clear expectation that these patients will eventually receive GT treatment.
New issues on the ethics continuum will evolve as more and more clinical studies with GT
products are initiated. Ree xamination of ethical issues as GT science and technology evolve
will be critical for continued GT development to optimize benefit to the patients and under-
standing for all involved in the GT developmen t process.
Lessons learned from clinical trials to guide future development
The death of a patient receiving an AdV vector treatment in 1999, and development of leu-
kemia in four of nine children with severe combined immunodeficiency disorder (SCID)
receiving a gamma retrovirus in 2002, brought to light the risks with administering GTs to
patients (Si bbald, 2001; Howe et al., 2008). These two tragedies led to a worldwide morato-
rium on GT development, shifted the field away from AdV, and launched an effort to reen-
gineer integrating viral vectors (Milone and O’Doherty, 2018).
More recently, several other toxicities have come to light following administration of high
dose AAV vectors, including hepatotoxicity, thrombocytopenia, thrombo tic microangiop-
athy, renal injury, complement activation, cytokine storm, cardiomyocyte injury, ocular
inflammation, and neurotoxicities (Agarwal, 2020; Mullard, 2021; Rapti and Grimm, 2021;
Ertl, 2022). At least eight patients have died as a consequence of these toxicities, four from
the Audentes XL-MTM trial, two from the Novartis Zolgensma drug administration for
SMA, one from the Pfizer DMD trial, and one from Cure Rare Disease trial for DMD
9. Preclinical development of viral vector gene therapies290
III. Specialty biologics and indications
(Mullard, 2021; Ertl, 2022; Lek et al., 2023). Separately, several incidences of leukemia have
occurred in patients receiving an LV-based gene therapy to treat hematologic disorders
(Goyal et al., 2022; Kanter et al., 2023). Toxicity risks associated with AAV GT were discussed
at an FDA Advisory Committee meeting held in September 2021 and at specialty meetings
(FDA, 2021c,d; Lek et al., 2023a). So, what have we learned?
In the context of AAV-based GTs, the challenge for the field has been that to drive broad
correction of many target cells at distal sites in the body and ensure prospective clinical
benefit (as in the case of neuromuscular diseases for example), systemic viral vector dose
levels needed to increase (Kishimoto and Samulski, 2022); reaching doses in the order of
10
13
e10
4
vector genomes (vg)/kg (w10 to 100 trillion particles). This is approximately
three-times greater than the total number of cells in the adult human body, and even higher
for pediatric patients (Roy and Conroy, 2018). As a contemporaneous comparison, estimates
of total virion load at peak infection in patients with SARS-CoV2 (COVID-19) were between
10
9
and 10
11
infectious particles (Sender et al., 2021); 2 to 5-log lower than the number of in-
fectious/efficacious vector particles delivered to these patients. In this context, it is not sur-
prising that high doses of viral vectors expose patients to high levels of effective vector
genomes but also high doses of nonefficacious particles and associated elements e.g., host-
cell proteins and DNA byproducts from manufacturing. This complicates the ability to
discern whether the toxicitie s observed are driven solely by the presence of high levels of viral
particles, transgene expression, other elements, or a combination of all three. Moreover, the
viral vectors are subject to similar innate and adaptive immune responses as wild-type vi-
ruses. Innate immune receptors, or pattern recognition receptors (PRRs), detect viruses and
viral vectors through the recognition of conserved molecular motifs that trigger antiviral im-
munity and clearance in animals and humans similarly.
The challenge for the field is the ability to decrease total exposure while achieving effective
transgene expression in the target cells. So how can this be achieved? In order to reduce dose,
there are four main ways the field is advancing: (1) vector choice and optimization (e.g.,
capsid engineering), (2) viral genetic payload optimization (e.g., removal of enhancers, use
of human promoters, etc.), (3) immune modifications (e.g., TLR-9 blockade, removal of
CpG sequences), and (4) manufacturing improvements (e.g., reducing partial or empty vi-
rions). Significant advances to improve patient safety have occurred, however, there remains
important concern with the delivery of GTs. The remainder of this section focuses on what we
have learned from the known toxicities to inform nonclinical and clinical advancement.
Hepatotoxicity
There have been multiple incidences of fatal GT-related hepatotoxicity in clinical trials
including a young adult patient who received AdV fo r ornithine transcarbamylase deficiency
in 1999 (Sibbald, 2001), four pediatric patients who received high dose AAV8 for X-linked
myotubular myopathy (XL-MTM) in 2020 and 2021, and two children who died after
receiving AAV9 for spinal muscular atrophy (SMA) in 2022 (2020). Several other trials
have reported signals of hepatocellular injury for high dose AAV2 for hemophilia and
AAV9 for DMD (FDA, 2021c). Not surprisingly, these clinical events have raised significant
concern for developers and the regulatory agencies alike.
Lessons learned from clinical trials to guide future development 291
III. Specialty biologics and indications
For all viral vectors, systemic exposure results in activation of an innate immune response,
which is rapid, fixed, and dose-dependent. The goal of the innate response is neutralization
and systemic clearance of the virus (see section below on Immunogenicity). In the liver, the
cells involved in this defense are Kupffer cells, sinusoidal endothelial cells, and hepatocytes.
While the exact mechanisms involved in the initial interactions between the virus and these
cells have not been completely elucidated, and may vary between the virus types, when the
induced innate inflammatory response is robust enough, there is hepatic injury. Significant
elevations of the liver transaminases alanine transaminase (ALT) and aspartate transaminase
(AST), together with bilirubin, gamma-glutamyl transferase (GGT), and other liver specific
chemistries, are the clinical hallmarks of hepatocellular damage (Kwo et al., 2017; Delire
et al., 2021). Despite its enormous reserve capacity and ability to regenerate, the liver can
become overwhelmed when the number of virions far exceeds its capacity to process and
clear them, as might occur with high dose viral vectors. As a consequence, the liver becomes
a target organ of concern for systemically administered GTs, particularly with AdV and AAV
(Hendrickx et al., 2014; Chand et al., 2021; Servellita et al., 2023).
To date only a handful of publications have reported on the incidence, management and
mitigation of drug induced liver injury (DILI) in patients receiving GTs (Chand et al., 2021,
2022; Ruggiero et al., 2024). In the clinic, patients administered a GT now routinely receive
adjunct treatment with corticosteroids and an immunosuppressive regimen such as tacroli-
mus and mycophenylate mofetil among others. In most cases these immunosuppressives
have helped to control and reduce elevated liver enzyme levels observed in patients under-
going systemically administered GT. Altogether, these data suggest that the change in clinical
pathology endpoints exhibited by these patients stems from an acute hepatocellular and hep-
atobiliary injury of the liver. While the underlying mechanisms for this acute hepatotoxicity is
not fully understood, there is general agreement that it is triggered by activation of an innate
immune response initially that may include immunothrombosis (see discussion below on
Immunogenicity), tissue injury, followed by adaptive responses. To date, there is no clear
1:1 correlation between liver enzyme elevation and elevations in cytokines or antibody titers.
Each patient has shown differences in the chronology of liver markers and injury, sometimes
many months after initial dosing.
Nonclinically, liver injury data in rodents following GT is limited; however, it may not
mean that changes in liver function related clinical chemistries are absent. Detection may
be impacted by difficulties in repeat sampling in these small species. This is not an issue
for large laboratory animals such as NHPs, piglets, and dogs, which lend themselves to
continuous monitoring; and several reports suggest that high dose viral vectors elicit eleva-
tions in liver transaminases, which correlate with microscopic liver changes, which may be
preceded by liver platelet sequestration and sinusoidal endothelial injury (Hinderer et al.,
2018; Hordeaux et al., 2018, 2019, 2024; Batty et al., 2022; Hudry et al., 2023). There is one
report of GT-related hepatotoxicity in rodents where alterations in ALT, AST, and liver-
specific glutamate dehydrogenase (GLDH) were observed in association with hepatic injury
concurrent with transgene overexpression of Frataxin (Huichalaf et al., 2022). Preclinical
work in NHPs with empty vector capsids or promotor-less cassettes that fail to express trans-
gene show an absence of liver transaminase elevations and associated liver pathology, sug-
gesting an interplay between transgene expression and liver findings (Hudry et al., 2023).
In fact, a recent study evaluating a codon optimized hSMN1 transgene driven by a native
9. Preclinical development of viral vector gene therapies292
III. Specialty biologics and indications
hSMN1 promoter delivered in AAV9 in SMA mouse model, has shown both improved effi-
cacy and safety including significant reduction in liver transaminase levels compared with
the benc hmark vector (Xie et al., 2024). What is evolving from the nonclinical data sets is sup-
portive of an interplay between high viral vector doses, innate immune activation, vascular
damage, transgene product overexpression and liver injury.
Careful monitoring of liver function in nonclinical studies therefore is important to deter-
mine if the hepatic changes are reversible or may lead to severe tissue damage progressing to
systemic inflammation and potential acti vation of disseminated intravascular coagulopathy
(DIC) (Hinderer et al., 2018). An excellent overview of nonclinical and clinical liver toxicities
following GT administration is provided by Whiteley (2023).
Neurotoxicity
Among the viral vectors in cur rent use, most AAV serotypes (e.g., AAVs one to nine and
rh10) have demonstrated a particular tropism for sensory neurons in the peripheral nervous
system ganglia, including the trigeminal ganglia and dorsal root ganglia (DRG) of rats, rab-
bits, piglets and NHPs. In these species, there was histopathology evidence of trigeminal and
dorsal root ganglionopathy in the form of neuronal degeneration either with or without clin-
ical sensory deficits or corollary changes in sensory nerve conduction, often associated with
elevation of neurofilament light chain (Hinderer et al., 2018; Hordeaux et al., 2018, 2019; Bolt
et al., 2021; Buss et al., 2022; Tukov et al., 2022; Tien et al., 2024; Tyszkiewicz et al., 2024).
AAV-related sensory changes have been observed following direct delivery to the CNS
and follo wing systemic administration with a variety of AAV capsid types, suggesting
neither route nor serotype is essential to elicit the pathology (Bolt et al., 2021; Buss et al.,
2022). It is noteworthy that these changes were not observed with null or empty vector cap-
sids in which a transgene is not expressed (Johnson et al., 2023), nor with the addition of a
micro-RNA target sequence within the genetic payload of the vector to limit or inhibit trans-
gene expression in sensory neurons (Hordeaux et al., 2020a,b; Bolt et al., 2021; Sinnett et al.,
2021; Buss et al., 2022). Expression of the homologous animal transgene rather than the hu-
man transgene, or use of corticosteroids in animals receiving AAV GT also appears to amelio-
rate severity of findings, suggesting an immune-mediated component to the ganglionopathy
that may be similar to the underlying mechanism occurring with hepatotoxicity (Buss et al.,
2022; Tien et al., 2024). Part of the challenge in translating the observations in nonclinical spe-
cies to humans has been inconsistent implementation and reporting of changes in a neurotox-
icity biomarker or sensory nerve function that correlate with the histopathology findings
(Flotats-Bastardas et al., 2023). Finally, there is a form of high titer AAV vector dose-
dependent inflammatory neurotoxicity specific to intracerebral injections, which has been
observed in nonclinical and clinical studies by MRI, and is hypothesized to be a consequence
of blood-brain barrier disruption (Guo et al., 2023).
At least two clinical trials treating a neuromuscular disease and a neurodegenerative dis-
ease with neurological sequelae as part of their natural history showed some level of sensory
loss following AAV administration that was considered to be treatment-related (Mueller
et al., 2020; Bolt et al., 2021; Ertl, 2022; Buntz, 2022). This has raised significant concern for
regulators, because neuronal degeneration in the CNS is considered irreve rsible. The sequelae
of neuronal degeneration in the peripheral nervous system are less clear, primarily due to the
Lessons learned from clinical trials to guide future development 293
III. Specialty biologics and indications
possible resilience of peripheral nervous system neurons and the apparent regenerative ca-
pacity of Schwann cells. Direct neurotoxicity has not been documented for LV vectors; how-
ever, immune effector cell-associated neurotoxicity syndrome (ICANS) is a feature of CAR-T
administration secondary to CRS and immune effector signaling (Siegler and Kenderian,
2020; Velasco et al., 2023).
There are several challenges that arise for developers when assessing potential for neuro-
toxicity following GT administration in nonclinical studies. For one, the standard neuropa-
thology practices used during nonclinical general toxicity studies may be insufficient for
situations where the test article is designed to target a neurological disease, or it is eithe r ex-
pected or known to be neurotoxic based on prior experience (including histopathology).
Recent guidance documents recommend expanded neurohistopathology evaluation
(ENHP) in the evaluation of new GTs ( Bolon, 2023; Bangari et al., 2023). ENHP involves sam-
pling additional CNS regions and/or peripheral nervous system organs and reviewing serial
sections of selected neural tissues to identify key anatomic features or cell typeespecific bio-
markers. While these adjustments can improve the sensitivity of the neuropathology safety
assessment, the collection of additional tissues requires familiarity with neurologic tissues
that are not part of the routine toxicology study tissue list, e.g., DRG. The quality of the tissue
collection can be variable between contract vendors with the potential of negatively impact-
ing microscopic evaluation. For example, inconsistencies in DRG collection, processing, and/
or evaluation make it difficult to determine if this toxicity is widespread in nonclinical studies
of GTs. Trigeminal ganglia are more readily accessible and easier to collect and thereby
escape the collection-related damage often associated with DRG extraction; therefore, collec-
tion of trigeminal ganglia in addition to the DRG is recommended.
Another challenge is the quantification of nerve function, which is challenging to perform
and whose translation to patients is uncertain. Most toxicology species may not show out-
ward signs of sensory loss, even with careful testing. For this reason, histopathology remains
the gold standard in evaluating neuropathology. There are excellent reviews that specifically
address the collection and assessment ganglia for potential toxicities in nonclinical species
(Bangari et al., 2023; Bolon, 2023; Bennet et al., 2023). The assessment of certain neurodegen-
erative markers in cerebrospinal fluid and blood may also help monitor the underlying
microscopic changes in animals (Fader et al., 2022; Buss et al., 2022; Johnson et al., 2023).
Taken altogether, awareness for the potential for histopathologic changes in the nervous
system following GT treatment is key. Early interaction with regulatory authorities can
help address the feasibility of moving a program forward even with preclinical data with ev-
idence of findings in the nervous system. Naturally, a care ful review of the safety package is
needed that considers the severity and translatability of the findings, and if the risk:benefit
profile overwhelmingly points to patient benefit.
Cardiotoxicity
Although uncommon, adverse events associated with myocarditis have been reported in
two different AAV gene therapy trials for DMD. Two patients administered a GT using an
AAV9 vector and one patient administered a GT using an AAVrh74 vector presented with
myocarditis (Ertl, 2022; Lek et al., 2023a). In a review of real-world safety data following
9. Preclinical development of viral vector gene therapies294
III. Specialty biologics and indications
treatment with onasemnogene abeparvovec for the treatment of SMA, the emergence of signs
indicative of cardiac injury were the most frequently reported adverse events in patients (Rug-
giero et al., 2024). In each instance, the vector was delivering a transgene targeting in striated
muscle (either mini-dystrophin or SMN1), of which the cardiac muscle is a specialized form.
The heart is the first organ exposed to the full dose of the vector following systemic administra-
tion, thereby receiving a much higher vector dose than the peripheral musculature of the body.
Because both these AAV serotypes efficiently transduce muscle, expression of very high levels
of the transgene in cardiomyocytes could hypothetically trigger an inflammatory reaction.
Similar findings with different vectors and transgenes have been reported in two separate
studies evaluating GT for Pompe disease in healthy cynomolgus macaques (Egge rs et al.,
2022; Hordeaux et al., 2023). Eggers et al. administered an AAV8 vector expressing human
acid-alpha glucosidase (GAA) while Hordeaux etal. used AAVhu68 vector expressing a similar
transgene. GT-related myocardial injury was observed across both reports and necessitated
early euthanasia for the high dose animals in the Eggers et al. study. Both studies attributed
the myocardial injury to an anti-GAA immune response characterized by inflammation and
myocardial necrosis; however, Eggers et al. demonstrated in a separate study that myocardial
necrosis was not observed in macaques treated with an AAV8 vector carrying a species-specific
macaque GAA transgene and inflammation was minimal. By comparison, Hordeaux et al.
attributed the pathology to an MHC-1 haplotype that was triggered by a c-terminal peptide
in GAA. These two interpretations are not mutually exclusive. The MHC-I haplotype may
also be responding to the poorly processed hGAA that may have resulted from partially pro-
cessed human protein in the macaque as suggested in the Eggers study.
Cardiac toxicity in these nonclinical and clinical settings raises an important consideration
for GTs expressing the transgene in both on-target and off-target tissues. In the examples cited
above, the genes of interest are active in muscle, the target site for the disease. However, muscle
is a diverse tissue, having striated (skeletal and cardiac) and nonstriated (smooth surrounding
organs and blood vessels) forms that are widely distributed in the body. This makes toxicolog-
ical assessments challenging inthat safety scientists need to consider a broad set of potential off-
target regions and potential pathophysiological impacts when designing studies. It may be
difficult to translate relevance to humans particularly if some of the findings are driven in
part by species specific immune reactions to the presence of nonnative transgene sequences.
Altogether, these data suggest that while animal models are mostly helpful in identifying
safety signals, understanding the underlying mechanisms aids to inform translational rele-
vance. It is simple enough to include cardiac safety pharmacology endpoints and the mea-
surement of well-accepted biomarkers such as cardiac troponin in a toxicology study if the
therapeutic application means cardiomyocytes would be exposed to the drug. Careful consid-
eration of the limitations of animal models and how to best predict safety is needed.
Immunogenicity
Immunogenicity to viral vectors has to date broadly focused on responses mounted to the
(a) viral capsid or envelope; (b) viral genetic payload or (c) transgene product being
expressed (see below Sinnett et al., 2021). In any cas e, the body responds by first moun ting
an innate reaction followed by an adaptive reaction, much like it does to any foreign pathogen.
Lessons learned from clinical trials to guide future development 295
III. Specialty biologics and indications
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