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Over the past decade, several AAV-based gene therapies have been approved for the treat-
ment of monogenic inherited disorders, including Luxturna for RPE65-dependent retinal dis-
ease based on AAV2, Zolgensma for spi nal muscular dystrophy based on AAV9, Hemgenix
for hemophilia B and Roctavian for hemophilia A both based on AAV5, and Elevidys for
Duchenne muscular dystrophy based on AAVrh74.
Integrating viral vectors
Integrating viral vectors are developed from the retroviruses, a large and diverse family of
enveloped RNA viruses. Retroviruses are divided into seven genera but can be subdivided
either by genomic structure (simple and complex), oncogenic potential, or on their insertion
site preferences. These viruses consist of an 80e100 nm diameter lipid bilayer envelope sur-
rounding an RNA viral genome w7e12 kb in size (Coffin et al., 1997). Within the retroviral
virion are products of the viral gag and pol genes, two identical copies of positive-sense strand
genomic RNA, transcription RNA (tRNA) primer for reverse transcription, and a range of
host cell-derived protei ns (Chameettachal et al., 2023). The simple retroviruses (beta-
retrovirus, epsilon-retrovirus, and gamma-retrovirus) have four genes, gag, pro, pol and
env, which encode capsid and matrix proteins, transcr iption and integration machinery,
and transmembrane proteins and glycoproteins that determine cellular tropism, respectively
(Elsner and Bohne, 2017; Poletti and Mavilio, 2018; Chameettachal et al., 2023). The comple x
retroviruses (alpha-retrovirus, delta-retrovirus, lenti-virus, spuma-virus) have accessory
genes (tat, rev, vpu, vif, vpr, nef), which encode additional proteins involved in regulating viral
replication (Chameettachal et al., 2023). The tat and rev genes encode proteins regulating
expression of the viral genome; the vpu, vif, vpr and nef, genes encode proteins that enhance
viral replication (“virulence factors”).
Alpha-, beta-, epsilon-, delta-, and gamma-retroviruses are classified as oncogenic viruses
because they carry viral oncogenes and are capable of inducing tumors rapidly; they are often
referred to as acute-transforming retroviruses (Chameettachal et al., 2023). Many of the onco-
genic/acute transforming retroviruses are replication-defective due to substitution of a part
of their normal viral gene complement with an oncogene sequence and therefore, must be
coinfected with a related “helper virus” that provides the viral structural proteins needed
to produce infectious particles. While Lentivirus and Spumavirus lack oncogenes, their ability
to integrate into the host cell chromosome raises the possibility of insertional mutagenesis
and oncogene activation and are often referred to as nonacute retroviruses because there is
a longer latency period for tumor induction (Chameettachal et al., 2023).
In addition to being grouped by complexity and oncogenic potential, retroviruses can be
grouped by specific integration preferences, which can be retained in the derived vectors.
Integration site selection influences transcriptional regulation of the provirus and impacts
replication and latency. Integration preferences are directed by viral preintegration com-
plexes (PIC), which affect the likelihood for a provirus to interact and interfere with transcrip-
tion, splicing and polyadenylation of host cell genes, a key aspect of the safety profile of
different retroviral vectors (Poletti and Mavilio, 2018). Three groups are identified based
on integration characteristics: (1) integration around transcription start sites and
9. Preclinical development of viral vector gene therapies266
III. Specialty biologics and indications
transcriptional regulatory elements e.g., Moloney murine leukemia virus, foamy virus, (2)
integration in actively transcribed sequences e.g., human immunodeficiency virus and simian
immunodeficiency virus, and (3) random integration without preference for any genomic
feature e.g., Avian sarcoma-leukosis virus (Poletti a nd Mavilio, 2018).
The life (replication) cycle is similar among retroviruses, and attachment of the viral Env
glycoprotein (also known as the spike protein) to the cell surface receptor and fusion of the
viral envelope with host cell membranes are the first major steps in that cycle (Poletti and
Mavilio, 2018; Segev et al., 2018). The head subunit of the spike protein is a receptor binding
domain (RBD) that interacts with a specific receptor via multiple noncovalent bonds to form
an RBDereceptor pair (Chameettachal et al., 2023). A single virion contains multiple spike
proteins of one or more functional types, and multiple receptors and coreceptors can be
accommodated within the cell membrane contact area of a virion. This multivalent binding
increases the residence time on the plasma membrane, leading to optimal attachment of ret-
roviruses to the host cell. Fusion of the viral envelope and host cell plasma membrane occurs
through the action of glycoproteins on the viral surface known as fusogens (proteins, which
ensure that the fusion of unfamiliar membranes occurs in a control led and regulated manner),
and results in delivery of the viral core contents into the cytoplasm (Segev et al., 2018).
Uncoating and nuclear entry requires not only viral proteins but the circumvention of host
proteins that act to restrict viral life cycle. These restriction factors differ between host species
and even within cell types of a given host (Milone and O’Doherty, 2018; Boso and Kozak,
2020). Many of these restriction factors are encoded by interferon stimulated genes and in
part contribute to the innate immune signaling that is triggered after viral infection (Boso
and Kozak, 2020). Once in the nucleus, the viral RNA genome is reverse-transcribed into a
double-stranded DNA by viral-coded reverse transcriptase and transported to the nucleus,
where it is inserted into the host chromosome (typically in regions of open chromatin) by
the viral integrase protein to form a provirus (Poletti and Mavilio, 2018). The provirus is repli-
cated along with the host genome and passed on to descendant cel ls. The provirus is also
transcribed into mRNAs by the host cell’s protein synthesis machinery to produce the viral
proteins required for assembly of progeny virions (Poletti and Mavilio, 2018).
The ability to integrate foreign DNA into the host cell genome makes retroviral vectors
particularly attractive for genetic therapies as a stably integrated gene affords more durable
expression and can be faithfully passed on to daughter cells. Several other features also make
retroviruses attractive for GT; namely, cell specificity that can be changed by retargeting or
pseudo-typing (ability to make hybrid vectors incorporating features of various viruses),
thereby diversifying vector pools, and a larger packaging capacity allowing the potential
to deliver bigger genetic payloads (Duverge and Negroni, 2020). However, a major setback
occurred in 2002 with the emergence of oncogenic transformation in a cohort of children
with severe combined immunodeficiency (SCID) treated with a gamma-retroviral vector
(Check, 2002; Hacein-Bey-Abina et al., 2008). These leukemic events were later attributed
to the insertion of strong retroviral enhancer/promoter elements into areas near transcription
start sites of proto-oncogenes including LMO2, BMI1, and CCND2 (Hacein-Bey-Abina et al.,
2008). Today, the field has shifted to the use of modified vectors including HIV-based LVs
incorporating distinct features to enhance safety. These features include: (1) self inactivation
Background: Vectors and applications 267
III. Specialty biologics and indications
(SIN) preventing the transfer of enhancer and promoter elements in the 5
0
long terminal
repeat (LTR) of the vector to transduced cells, (2) removal of dispensable viral genes namely
env, vif, vpr, vpu , and nef genes, and (3) separation of the viral genome into multiple distinct
plasmids for manufacturing (Elsner and Bohne, 2017; Ferreira et al., 2021). Apart from the
improved safety profile, SIN vectors have been shown to exhibit improved transgene expres-
sion, particle production, and transduction efficiency compared with non-SIN platforms,
lending themselves to scalable manufacturing (Martinez-Molina et al., 2020).
Today the most prominent use of LV’s is in the genetic engineering of patient cells (ex-vivo
GT) including chimeric antigen receptor (CAR)-T cell therapies (through genetic redirection
of T-cell-mediated cytotoxicity to cancer cells in an HLA dependent manner) and the engi-
neering of hematopoietic stem cells (HSCs) to correct production of hemoglobin. This has
led to the approval of therapies for treatment of specific B-cell malignancies such as Kymriah
and Yescarta (Lu and Jiang, 2022), Zynteglo for treatment of b-Thalassemia (Asghar et al.,
2022), and most recently Casgevy and Lyfgenia for treatment of sickle cell disease (FDA,
2023a,c).
Therapeutic applications and beyond
Finding a way to safely and effectively introduce a novel gene into the body has remained
the goal of GT. Today, GT is applied in four broad areas: (1) gene replacement, (2) gene
silencing, (3) gene therapeutic, and (4) gene editing. The therapeutic applications for GT prod-
ucts are broad, and while initial in vivo GT development efforts have focused on treating
monogenic rare diseases as well as oncologic, ophthalmologic, hematologic and anti-
infective indications, the opportunity to address unmet needs in larger and more populated
therapeutic areas remains untapped. The variety of therapeutic areas targeted by GT prod-
ucts in development is shown in Fig. 9.2 (Ginn et al., 2018; Anguela and High, 2019; High,
2020; Arabi et al., 2022; Sayed et al., 2022).
Expansion into other therapeutic areas for virally delivered genetic material depends
largely on our understanding of the genetic basis of diseases. Other key development areas
that will afford expansion of GTs include: improvement of the genetic cargo design to in-
crease capability of delivering larger or multiple genes that could be regulated, and
capsid/envelope engineering to enhance or tailor delivery and potentially achieving optimal
transduction, manufacturability, and transgene expression while managing the immune re-
sponses associated with viral gene delivery (Li et al., 2023).
Beyond the delivery of a therapeutic gene to prevent, cure, or stabilize diseases, viral vec-
tors have been used in a multitude of nontherapeutic applications: production of reporter cell
lines for drug discovery in both in vitro and in vivo settings, development of models for
in vitro toxicology testing, manufacturing of biologics, vaccine development, and generation
of animal models of disease. The flexibility to interchange promoters and transgenes,
advancement of methods for stable transfection, and choice of viral constructs to fit small
and large genes, offers a diversity of tools for investigators. Some of the most promising
new developments of these vectors include the application for genomic editing and the gen-
eration of large animal models of disease. The last 10-year have seen an exponential growth in
the application and utility of viral vectors to manipulate the genome, the details of which are
beyond the scope of this chapter.
9. Preclinical development of viral vector gene therapies268
III. Specialty biologics and indications
FIGURE 9.2 Chart representing the number and percentages of active GT clinical trials (in vivo and ex vivo) by
phases and therapeutic areas. Adapted from the 2022 report from the Alliance for Regenerative Medicine (ARM, 2022).
Background: Vectors and applications 269
III. Specialty biologics and indications
General considerations regarding chemistry manufacturing and controls
(CMC): Viral vectors manufacturing, characterization, and testing
Early decisions regarding CMC
Decisions regarding chemistry manufacturing and controls (CMC) parameters are among
the most important factors in determining whether a GT will be allowed to be evaluated in
clinical studies (FDA, 2020a). CMC issues are the most frequent source of regulatory bottle-
necks during the development of GT products (Clement and Grieger, 2016; van der Loo and
Wright, 2016). The lack of standardization and characterization of manufacturing starting
materials, development of appropriate product specific-analytical testing methodology,
particularly with potency assays, and control strategy development are cited as some of
the most common challenges by GT manufacturing stakeholders. In addition, effective
CMC GT development relies on creation and implementation of best practices for consistent
characterization and comparability approaches during GT process development and scale up.
The limited number of GT approvals together with the lack of specific CMC GT regulatory
guidance means GT sponsors and industry groups are the key drivers establishing the stan-
dardization and best practices for the field. The topics below cover several of the main deci-
sion areas for consideration in CMC GT development.
Vector development
Vector design and development arecentral to translating the idea of GT appl ications that can
deliver on the promise of treatment. Any modifications of the virus or of the genetic payload
during development may require sponsors to conduct comparability bridging studies or repeat
safety studies, significantly slowing development. The earlier part of this chapter reviewed the
three most commonly usedviral vector platforms for GT products. An ideal vectoris genetically
stable, safe to handle, nontoxic for host cells, has packaging capacity for delivering the genetic
payload of interest, high transduction efficiency in the target cell, and elicits a low level of
immunogenicity. Factors affecting vector choice include: understanding the identity and repli-
cation status (dividing or nondividing) of the target cell type for delivery, ex-vivo or in vivo
application, the intended patient population and disease state, route of administration, dura-
bility desired, and manufacturability. Broadly, AAV vectors have been favored for in vivo de-
livery whereas retroviral vectors such as LV have been preferred for ex vivo applications
(Ghosh et al., 2020). Lastly, intellectual property and the landscape in which sponsors can freely
operate drives the decision for most commercial entities.
Producer cell line selection (upstream process)
The choice of producer cell line determines successful manufacturing of GTs. The producer
cell line will drive product yield, process consistency, product quality attributes and ulti-
mately, cost of goods. Selection of a producer cell line is both a time-consuming and labor-
intensive process that is limited by the number of clones that can be feasibly screened. While
the time for cell screening has been reduced significantly through the use of modalities such
as flow cytometry, fluorescent activated cell sorting (FACS) and other imaging strategies
9. Preclinical development of viral vector gene therapies270
III. Specialty biologics and indications
using robotics, cell lines must be extensively characterized. Regulatory authorities have strict
standards for confirmation of reproducibility, safety, and monoclonality in the selected cell
line that is used to produce a recombinant product. A cell line development campaign can
involve the screening of thousands of clones and may take up to 6e12 months or more,
even in the most streamlined industrial environments.
Considerations in producer cell line development focus on supporting characterization of
the clonality of the production cell bank. Factors such as master cell bank (MCB) host cell line
history, vector design and transfection method, cloning procedures used to generate the
MCB, stage at which the cells were adapted to suspension and serum free production, clonal
expansion, evaluation and selection, and risk assessment of potential cellular exposure to
adventitious agents in the presence of animal-derived reagents, are all taken into account
in the selection of the clonality of the producer cell line (Welch and Arden, 2019). To aid de-
velopers, FDA and ICH guidance documents provide critical criteria to consider in produc-
tion cell bank documentation (FDA, 2010, 2020a; ICH, 1998).
Issues in clonality range from incomplet e understanding of the causes of variation of cells
in culture to the simple fact that there is no single reliable or applicable method to model
growth characteristics of cells in culture. Mitigations to augment cell line clonality include
fluorescence in situ hybridization (FISH) to evaluate individual integration sites. For this,
an MCB vial is plated as single cells, expanded and characterized using phenotypic analyses
such as doubling time, productivity, etc., with product quality testing including adventitious
agent testing to prevent contamination of the selected clone. Additional characterization
testing of drug substance (DS) could include glycosylation patterns, peptide mapping, etc.
to support the integrity of cell line clonality.
Lastly, choice of mammalian versus nonmammalian cell lines may have potential impact
for safety. HEK293 cells, an immortal line derived from human embryonic kidney cells, are
among the most commonly used cell line for AAV vector production, whereas HEK293t cells
are more commonly used for LV production (Bae et al., 2020). HEK293t cells introduce an
additional safety concern due to the presence of the SV40 T antigen coding sequences and
the possibility that presence of residual sequences in the final drug substance might trigger
cellular transformation. By comparison, the use of insect cells such as Spodoptera frugiperda
(Sf9) for some AAV production including the approved product Glybera, while affording
high density suspension factories, raise a very different concern for increased immunoge-
nicity. For these nonmammalian cells, presence of residual host cell and extra-viral DNA
components as well as potential posttranslational modifications to the vector capsid, have
been hypothesized to increase risk of immunogenicity (Bucher et al., 2023). Irrespective of
the cell line chosen, close collaboration between the early development CMC team and safety
toxicologists is warranted to facilitate informed discussions on nonclinical and clinical conse-
quences of cell line selection.
Product format and formulation (downstream process)
Formulation development for GTs is complex and requires a thorough understanding of
vector design considerations such as size of the genetic material to be delivered, as well as
the safety, manufacturability, storage and stability of the final drug product (Wright et al.,
2003; Mer ten et al., 2016; Clement and Grieger, 2016; Valkama et al., 2020; Labbe et al.,
General considerations regarding chemistry manufacturing and controls (CMC) 271
III. Specialty biologics and indications
2021; Mohammad, 2022). The clinical route of administration (ROA) is also a factor that con-
tributes to the formulation selection process. Most virally delivered GT products are stored
frozen. Typical viral vector formulations can include buffering and tonicity agents, surfac-
tants, cryoprotectant and other stabilizers (Rodrigues et al., 2018). For a preclinical stage
GT formulation, titer and potency are generally the most important parameter s considered,
with increased focus on quality attributes such as identity, aggregation, infectivity and full
to empty particles as development progresses.
Formulation challenges for viral vectors can be divided into two categories: (1) those
related to maintaining product potency during purification and storage, and (2) those related
to efficient transduction in vivo, including immune responses. Aggregation, proteolysis,
oxidation and other postpackaging modifications, as well as nonspecific binding to materials
that contact the drug product during purification, storage and clinical delivery may diminish
vector potency, alter biodistribution or increase immune responses. These factors must be
thoroughly characterized, along with optimizing the conditions and excipients during formu-
lation development and should involve robust communication between formulation develop-
ment scientists and safety toxicologists. Further, the selection of a suitable container for the
formulated drug product to maintain stability during long-term storage and ultimately
commercialization is critical.
Development of a suitable vector formulation most often begins with identi fication of the
pH range associated with maximal stability and selection of a buffer with an adequate buff-
ering capacity in the target pH range. Changes in pH may affect capsid structure or lead to
aggregation, and chemical degradation of the drug product. Determining pH compatibility
with the vector, the product diluent and the storage container are essential. Further, since
most vect or products are stored frozen until use, characterization of pH during freezing
and thawing of the product provide insight into formulation stability.
Careful screening of formulation components and evaluation of storage conditions are
required to yield a stable GT formulation that is suitable for clinical and ultimately, commer-
cial use. An excellent review of some of these considerations is provided by Som et al. (2024).
Formulations and analytical tools to characterize them will continue to evolve as the use of
viral vectors becomes more prevalent in clinical development and as process understanding
and the number of regulatory precedents increases.
Product characterization and potency assays
Product characterization e quality attributes
The mantra in biologics drug development is “the process is the product” with close
attention paid to ensuring product identity, potency, purity, safety and stability
throughout manufacturing development. Product characterization is critical in under-
standing the impact of manufacturing process changes on GT p roducts and assuring
compliance with cGMPs. Identification of critical quality attributes (CQAs) and under-
standing the relat ionship between CQAs an d manufacturing process parameters form
the bases to establish product comparability during scale up and optimization (Simek,
2005). CQAs are physical, chemical, biological, or microbiological properties or character-
istics that should be within an appropriate limit, range, or distribution to ensure the
9. Preclinical development of viral vector gene therapies272
III. Specialty biologics and indications
desired product quality (Dev, 2009). Preliminary CQAs are defined early in Phase 1 of the
development lifecycle using risk assessments and continue to be refined during develop-
ment based on enhanced product knowledge and early clinical experience. A comprehen-
sive discussion of quality attributes is not the focus of this chapter, with excellent reviews
available (Merten et al., 2016; Tustian and Bak, 2021; Gimpel et al., 2021; Perry and Rayat,
2021; Labbe et al., 2021; Hickey et al., 2023).
Potency assays
Potency is defined as “the specific ability or capacity of a product, as indicated by appro-
priate lab tests or by adequately controlled clinical data obtained through the administration
of the product in the manner intended to effect a given result”. The potency test is an integral
part of the quality control of the GT final product intended to confirm consistency of produc-
tion, stability and comparability between batches (Torrents et al., 2023; Salmikangas et al.,
2023). The FDA and EMA issued guidance documents for potency testing of GT in 2011
and 2018, respectively, and most stakeholders achieve compliance with the regulations
through either in vitro or in vivo potency testing that is product-specific(ICH, 1998; Potency,
2011; De et al., 2018).
Nonclinical development
Principles of nonclinical development
The goal of nonclinical development is to provide a comprehensive set of data that sup-
ports a safe starting dose for the first-in-human (FIH) clinical trial. Unlike small molecules
and other biologics, GTs cannot be withdrawn or reversed; the very nature of the GT in-
troduces a new genetic element into the body that is intended to be permanent. This
means that t he Phase 1 clinical safety testing of GTs is generally in patients with a disease
and not in healthy volunteers. Of note, if the intended patient population is pediatric, the
GT must demonstrate a ‘prospect of direct benefit’ as part of the nonclinical package. This
puts a higher burden on investigators to demonstrate not only the safety of their p roduct
butalsoevidenceofefficacy in their nonclinical package. Therefore, careful attention must
be paid to the selection of appropriate in vitro and in vivo models, choice of endpoints, as
well as an understanding of reproducibility and model limitations. The nonclinical data
package must include a description of the efficacy or pharmacology, together with an un-
derstanding of the pharmacodynamic and pharmacokinetic profile (principally the bio-
distribution), as well as the safety assessment to inform the clinical plan (FDA, 2013;
Bolt et al., 2020). The current organization of the regulatory agencies including the land-
scape for regulating GTs and specific guidance documents available, are discussed in the
section titled, Translating nonclinical data to regulatory success below.
Selection of test article for nonclinical studies
In most instances, the GT viral vector expressing the human gene product is tested in an
animal model of the human disease. For these studies, research grade material is evalu-
ated in early proof-of-biology experiments. In the pivotal studies a product derived
Nonclinical development 273
III. Specialty biologics and indications
from the locked manufacturing process, usually produced under good laboratory prac-
tices (GLP), is more relevant to support identification of a therapeutic dose range in
proof-of-concept (POC) studies and safety studies. This typically means generation of ma-
terial under good manufacturing practice (GMP) and batch release testing with GMP or
GLP compliant assays. It is also imperative to demonstrate that the test article is stable
over the time of storage, shipping, test article formulation preparation, and throughout
the duration of test artic le administration in the animal studies. Stability and dose confir-
mation or dose assurance studies should be conducted and are mandatory for any GLP
studies equal or greater than 4-week in duration. T hese are often done as bench top
in vitro experiments.
The use of animal models of human disease assumes that there is sufficient homology of
the human gene product to support therapeutic correction in the animal model. Where this
is not feasible, an analogous product expressing the animal genetic homolog may be needed
to evaluate the potential for therapeutic change. In these instances, engagement with the
regulatory authorities is strongly advised to ensure acceptability of the strategy. Therefore,
choice of the GT for evaluation in the nonclinical studies needs to take into consideration
several factors, including: (1) homology between human and animal gene products, (2) abil-
ity of the animal model to appropriately transcribe and process the human gene product, (3)
presence of a therapeutic target in the model, and (4) immunoreactivity to the human gene
product.
Justification for route and method of administration for nonclinical studies
The ROA used in nonclinical in vivo studies should mimic the ROA for patients to sup-
port the clinical dosing strategy. The choice of ROA should also be justified to ensure that
it supports delivery and exposure to the intended target cells. Historically, the intrave-
nous (iv) ROA has been used for GTs because it delivers the viral vector to a broad num-
ber of highly desirable target tissues. However, there are many other ROAs available that
have a long history of drug application that are being tested for GTs e.g., intraparenchy-
mal, intrathecal, intracisternal, intracerebroventricular, or intraocular (subretinal, intravi-
treal, suprachoroidal), that support delivery to specialized tissues such as the brain, spinal
cord, or eye, respectively. When specific devices are developed for the delivery of GT
products to potentiate exposure of target cells to the vector, biodistribution of the vector
using a similar device needs to be established in the most appropriate species (anatomi-
cally close to humans).
Selection of animal model and or species for nonclinical efficacy studies
Pharmacologic POC studies supporting GT programs are typically conducted in a ge-
netic model of the human disease because the goal is to correct the function of a missing or
poorly functioning gene product, or to reduce the expression of a pathological human
gene. The genetically modified or genetically engineered mouse (GEM) model remains
the work-horse for many of these studies whereas naturally occurring mutations in
some domesticated species such as dogs, provide opportunities to evaluate efficacy in a
larger animal disease model (Hytonen and Lohi, 2016). With the advancement of gene
9. Preclinical development of viral vector gene therapies274
III. Specialty biologics and indications
editing tools such as clustered regulatory interspaced short palindromic repeats
(CRISPR)-Cas, other animal species can now be engineered to develop knockout models
of human diseases (Niu et al., 2014; Aida and Feng, 2020; Feng et al., 2018; Kim et al.,
2022). The challenge with leveraging these novel technologies is that this generates previ-
ously uncharacterized models, and investigators will need to spend time carefully docu-
menting the phenotypic similarities and limitations compared with the human disease as
part of their nonclinical package. Early interactions with the regulatory authorities are
important if novel animal models are selected to ensure costly and time-consuming
work is deemed adequate to support FIH testing. Focusing on key questions that address
the presence of the target, ROA, dose, target exposure, MOA, endpoints for demonstrating
activity, and threshold for efficacy, all help to ensure successful choice of an appropriate
nonclinical species.
Pharmacologic efficacy assessments
The goal of the nonclinical pharmacological studies supporting the development of inves-
tigational GT products is to establish a dose or range of doses to demonstrate prospective
therapeutic benefit in the intended clinical population and eventually establish a minimally
efficacious dose (MED) necessary for clinical improvement. This is particularly important
for GTs intended for pediatric populations where regulators require clear demonstration of
potential for therapeutic benefit prior to Phase 1 entry. Typically, these studies are conducted
in vivo in an animal model of the genetic disease, either in a heterozygous or homozygous
null mutant model, or in an animal model that either expresses or overexpresses a pathogenic
human transgene. In some instances, no in vivo model is available and instead sponsors are
burdened with providing a robust in vitro data package with literature that supports the
mechanism of action for the GT in the disease population. The pharmacologic studies are
often designed to gather additional information on the proportion of cells that need to be cor-
rected or on the level of transgene expression needed (in the case of a secreted transgene
product, for example) to achieve sufficient functional improvements. Distribution to on-
target and off-target sites, rate of clearance and durability of the gene product (e.g., to the
extent of the lifespan of the chosen model) are other parameters to evaluate within efficacy
studies. Inclusion of safety endpoints (hybrid safety/efficacy study) can provide important
insights regarding the tolerability of the GT in the disease context and is particularly impor-
tant for helping to scale the doses to humans and inform the potential for persistence and
long-term pharmacologic correction.
Study design, dose selection and justification of endpoints
In general, the nonclinical pharmacological assessment of GT products should be conduct-
ed in clinically relevant animal model/species to demonstrate the pharmacologic MOA of the
gene product. Ideally, study designs should include endpoints with potential translatability
to the targe t patient population. Pilot studies can be conducted in wild-type cells or animals
to initially understand transduction efficiency and transgene expression levels to guide
appropriate dose selection for the pivotal studies. The goal of a pivotal pharmacology study
Nonclinical development 275
III. Specialty biologics and indications
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