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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5703_Библиотеки_им_академика_М_И_Перельмана
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Pharmacodynamics and pharmacokinetics 276
Pharmacodynamics and minimal
efficacious dose (MED) 276
Pharmacokinetics e biodistribution,
shedding, and persistence 277
Nonclinical safety evaluation 278
General toxicity 278
Justification of dose 279
Justification of delivery method 279
Justification of the test system 279
Considerations for study designs,
safety endpoints and safety
pharmacology 280
Considerations for immunogenicity
in toxicology studies 281
Developmental and reproductive
toxicity (DART) 281
Genotoxicity: Insertional
mutagenesis, carcinogenicity, and
germline transmission 282
Translating nonclinical data to regulatory
success 283
An ever-evolving regulatory landscape 283
Considerations for dose extrapolation 286
Clinical trial design 287
Expansion cohort trial designs 287
Master clinical protocols to support
multiple drugs or multiple patient
subpopulations 287
Innovative trial designs and clinical
trials for rare diseases 288
Control subjects, placebo groups,
and use of real-world data 288
Use of surrogate endpoints 289
Ethical considerations and weighing risk/
benefit 289
Lessons learned from clinical trials to
guide future development 290
Hepatotoxicity 291
Neurotoxicity 293
Cardiotoxicity 294
Immunogenicity 295
Linking immunity and nonclinical
findings 297
Capsid/vector envelope immune
responses 297
Transgene immune responses 297
On the use of immunosuppressants
in nonclinical studies 298
Insertional mutagenesis 299
Conclusions 300
References 301
Abbreviations
AAV Adeno-associated vector
Abs Antibodies
ADME Absorption, distribution, metabolism and excretion
AdV Adenoviral vector
AML Acute myeloid leukemia
AMP Accelerating medicines partnership
ASO Antisense oligonucleotide
BGTC The Bespoke Gene Therapy Consortium
CAR Chimeric antigen receptor
CBER Center for Biologics Evaluation and Research
CID Complex innovative trial design
CMC Chemistry, manufacturing, and control
9. Preclinical development of viral vector gene therapies256
III. Specialty biologics and indications
CNS Central nervous system
CQA Critical quality attribute
CRIM Cross-reactive immunological material
CRS Cytokine release syndrome
DAMPs Damage-associated molecular patterns
DART Developmental and reproductive toxicity
DNA Deoxyribonucleic acid
DRG Dorsal root ganglia
DS Drug substance
EC European Commission
EF Effective dose
FACS Fluorescent activated cell sorting
FDA Food and Drug Administration
FIH First-in-human
FISH Fluorescence in situ hybridization
GEF Gene efficiency factor
GEM Genetically engineered mice
GLP Good laboratory process
GMP Good manufacturing process
gRV gamma retroviral vector
GT Gene therapy
HED Human equivalent dose
ICF Informed consent form
ICH International Council for Harmonization
IND Investigational new drug
IP Intellectual property
IRB Institutional Review Board
ITR Inverted terminal repeat
LTR Long terminal repeat
LV Lentiviral vector
MCB Master cell bank
MED Minimally efficacious dose
MFD Maximal feasible dose
MLD Myelodysplastic syndrome
MOA Mode of action
MTD Maximal tolerable dose
NIH National Institute of Health
NOAEL No adverse effect limit
OTAT Office of Tissues and Advanced Therapies
OTP Office of Therapeutic Products
PAD Pharmacologically active dose
PAMPs Pathogen-associated molecular patterns
PCR Polymerase chain reaction
PDUFA Prescription Drug User Fee Act
POC Proof of concept
RCT Randomized controlled trials
RMAT
Regenerative medicine advanced therapy
RNA Ribonucleic acid
RWD Real-world data
SCD Sickle cell disease
9. Preclinical development of viral vector gene therapies 257
III. Specialty biologics and indications
SIN self-inactivating
siRNA Small interfering RNA
TI Therapeutic index
vg Vector genome
Background: Vectors and applications
In vivo gene therapy (GT) products continue to capture the attention of the public, regu-
lators, patients, and prescribers as these revolutionary products edge their way toward ful-
filling the promise of long-lasting beneficial treatment effects. In vivo GTs have been
shown to restore phenotypic normality, overcome malignancies and infectious diseases by
way of introducing a healthy copy of a defective/flawed gene, reducing the expression of
a pathogenic isoform or directly correcting a gene to restore its biological function. Genetic
material (RNA or DNA) is delivered to target cells using a vehicle or carrier referred to as
a “vector” that can be of viral or nonviral origin. Viral delivery vectors are the focus of
this chapter, with recombinant adeno-a ssociated vectors (AAVs) or lentiviral vectors (LVs)
used most frequently (Bulcha et al., 2021; Paunovska et al., 2022; Zhao et al., 2022).
Enthusiasm during the first wave of GT product development led to rapid growth in the
GT pipeline, which then slowed as the complex nature of these products, their manufacturing
and testing, and adverse impacts on patient safety became evident. None theless, continuous
advances in the scientific understanding of virology, immunology, and molecular biology to
optimize vector design, along with improved understanding and management of safety con-
cerns (tissue injury, inflammatory responses, characterization of genotoxic potential) have
ignited a steady growth in GT products as these obstacles have been addressed.
Today GT products have been approved for a growing array of therapeutic targets (see
Table 9.1)(FDA, 2023e; Mendell et al., 2021). The diversity of applications has been realized
by the unwavering commitment of the scientists and clinicians to pursuing a variety of plat-
forms to deliver, replace, or modify the genes that underly human disease. The regulations
governing the development of these products have provided latitude for sponsors to pursue
novel therapeutic approaches with the recognition that a clear data driven strategy is needed
to address concerns for patient benefit and safety. The goal of this chapter is to highlight some
of the key elements for sponsors to consider in the preclinical development of in vivo GTs.
Delivering gene therapy products: Nonviral and viral vectors
Vectors are essentially vehicles designed to deliver genetic information directly into a
target cell and can be broadly subdivided into nonviral and viral. Nonviral vectors are vehi-
cles that use synthetic or natural materials to transfer genetic material while viral vectors use
viral-based carriers (capsid and/or envelo pe, genome) for delivery.
9. Preclinical development of viral vector gene therapies258
III. Specialty biologics and indications
TABLE 9.1 Gene therapy approvals up until 2024.
Gene
therapeutic Indication
In vivo or
ex vivo
Target cell
population(s) MOA
Sponsor and year
of approval
(agency)
ImlygicÒ
(talimogene
laherparepvec)
Recurrent melanoma In vivo (in situ) Melanoma cells Oncolytic
immunotherapy,
modified Herpes
Simpex 1 (HSV1)-
granulocyte-
macrophage colony-
stimulating factor (GM-
CSF) cDNA
BioVex/Amgen. 2015
(FDA, 2015c)
Strimvelis Severe combined
immunodeficiency due
to adenosine
deaminase deficiency
Ex vivo Autologous
CD34þ
hematopoietic stem
cells
Gene replacement,
gamma retroviral
vector encoding
adenosine deaminase
(ADA) cDNA
GlaxoSmithKline
Trading Services*.
(EMA, 2016) *currently
marketed by Telethon
foundation
LuxturnaÒ
(voretigene
neparvovec-rzyl)
Biallelic RPE65
mutation-associated
retinal dystrophy
In vivo RPE cells Gene replacement,
AAV2-RPE 65 protein
(RPE65) cDNA
Spark Therapeutics.
(FDA, 2017a; EMA,
2018b)
KymriahÒ
(tisagenlecleucel)
Relapsed or refractory
(r/r) follicular
lymphoma
Ex vivo
(in vitro)
Autologous T-cells Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding CD19
(CAR-T cells)
Novartis
Pharmaceuticals Corp.
(FDA, 2017c; EMA,
2018a)
YescartaÒ
(axicabtagene
ciloleucel)
Follicular cell
lymphoma and arising
B-cell lymphomas;
large B-cell lymphoma
Ex vivo
(in vitro)
Autologous T-cells Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding CD19
(CAR-T cells)
Kite Pharma, Inc., for
follicular lymphoma,
(FDA, 2017b); for non-
Hodgekins
lymphomas, (EMA,
2018c); for B-cell
lymphomas, (FDA,
2022).
ZolgensmaÒ
(onasemnogene
abeparvovec)
Spinal muscular
atrophy
In vivo Lower motor
neurons
Gene replacement,
AAV9-survival motor
neuron 1 (SMN1)
cDNA
AveXis/Novartis gene
Therapies, Inc. (FDA,
2019a;
EMA, 2020b).
TecartusÒ
(brexucabtagene
autoleucel)
Relapsed or refractory
mantle cell lymphoma
In vivo and
ex vivo
Autologous T-cells Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding CAR
T-cells with anti-B-cell
maturation antigen
(BCMA) domains.
Kite Pharma Inc.,
(EMA, 2020a)
BreyanziÒ
(lisocabtagene
maraleucel)
Relapsed or refractory
large B-cell lymphomas
In vivo and
ex vivo
Autologous T-cells Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding CD19
(CAR-T cells).
Juno Therapeutics,
Inc/Bristol-Myers
Squibb Pharma. (FDA,
2021b; EMA, 2022a)
(Continued)
Background: Vectors and applications 259
III. Specialty biologics and indications
TABLE 9.1 Gene therapy approvals up until 2024.dcont’d
Gene
therapeutic Indication
In vivo or
ex vivo
Target cell
population(s) MOA
Sponsor and year
of approval
(agency)
Abecma
(idecabtagene
vicleucel)
Relapsed or refractory
multiple myeloma
In vivo and
ex vivo
Autologous T-cells Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding CAR
T-cells with anti-B-cell
maturation antigen
(BCMA) domains.
Celgene/Bristol-Myers
Squibb Pharma. (FDA,
2021a)
ZyntegloÒ
(betibeglogene
autotemcel)
b-Thalassemia In vivo and
ex vivo
Autologous
CD34þ
hematopoietic stem
cells
Gene replacement,
CD34þ HST
transduced by BB305
lentivirus vector
encoding b
AT87Q
-
globin
Bluebird Bio, Inc. 2019
(EMA)*, (FDA, 2022e)
*later withdrawn
SkysonaÒ
(elivaldogene
autotemcel)
Cerebral
adrenoleukodystrophy
(CALD)
Ex vivo Autologous CD34
þ
hematopoietic stem
cells
Gene replacement,
CD34
þ
HST tranduced
by lentivirus vector
encoding
adrenoleukodystrophy
protein (ABCD1).
Bluebird bio, Inc. 2021
(EMA)*, (FDA, 2022d)
*Later withdrawn
CarvyktiÒ
(ciltacabtagene
autoleucel)
Relapsed or refractory
multiple myeloma
In vivo and
ex vivo
Autologous T-cell Immunotherapy,
Autologous T-cells
tranduced by lentivirus
vector encoding anti-
B-cell maturation
antigen (BCMA)
epitopes.
Janssen Biotech Inc.,
(FDA, 2022b)
VyjuvekÒ
(beremagene
geperpavec)
Wounds associated
with dystrophic
epidermolysis bullosa.
In vivo (topical
with repeat
dosing)
Type VII collagen Gene replacement,
HSV-1-collagen type
VII alpha 1 chain
(COL7A1)
Krystal Biotech, Inc.
(FDA, 2023d)
UpstazaÒ
(eladocagene
exuparvovec)
Aromatic
L-amino acid
decarboxylase (AADC)
deficiency
In vivo Putamen medium
spiny neurons
Gene replacement,
rAAV2-aromatic
L-
amino acid
decarboxylase (AADC)
cDNA
PTC Therapeutics
International Limited.
(EMA, 2022d)
AdstiladrinÒ
(nadofaragene
firadenovec)
Nonresponsive non-
muscle invasive
bladder cancer
In vivo (in situ) Urotheli al
carcinoma cells
Immunotherapy,
increased urothelial
IFNa2b expression
rAd-IFN/Syn3
Ferring
pharmaceuticals A/S.
(FDA, 2022a)
HemgenixÒ
(etranacogene
dezaparvovec-
drlb)
Hemophilia B (factor IX
deficiency)
In vivo Hepatocytes Gene replacement,
AAV5-cDNA hF IX
variant R338L (factor
IX-Padua) cDNA
CSL Behring. (FDA,
2022c);
EMA (2022b)
RoctavianÒ
(valoctocogene
roxaparvovec)
Hemophilia a (factor
VIII deficiency)
In vivo Hepatocytes Gene replacement,
AAV5-hFVIII-SQ
(factor VIII B-domain
BioMarin
Pharmaceutical Inc.
9. Preclinical development of viral vector gene therapies260
III. Specialty biologics and indications
Nonviral vectors
Nonviral vector systems include polymers, lipids, peptides, inorganic particles, and com-
binations thereof designed to encapsulate and introduce double-stranded, sense or antisense
oligonucleotides (e.g., DNA, mRNA, siRNA, miRNA, ASOs) systemically (Bulcha et al., 2021;
Paunovska et al., 2022). Advantages of their use include commercial scalability (especially
with lipid-based vectors), improved safety (less cytotoxicity, immunogenicity, insertional
mutagenesis), and large genetic material cargo capacity. Disadvantages include low tissue
specificity, poor gene transfer efficiency, and short-lived gene expression (Ramamoorth
and Narvekar, 2015; Zu and Gao, 2021). For specific applications such as in vivo gene editing,
however, the transient expression of the therapeutic gene (a Cas9 nuclease, in this case) is
considered advantageous as it will limit the risk for genotoxicity (Xu et al., 2020; Duan
et al., 2021; Gillmore et al., 2021).
TABLE 9.1 Gene therapy approvals up until 2024.dcont’d
Gene
therapeutic Indication
In vivo or
ex vivo
Target cell
population(s) MOA
Sponsor and year
of approval
(agency)
deleted SQ form)
cDNA
(EMA, 2022c; FDA,
2023d)
CasgevyÔ
(exagamglogene
autotemcel)
Sickle cell anemia and
transfusion-dependent
b-thalassemia
Ex vivo Autologous
CD34þ
hematopoietic stem
cells
Gene editing, CRISPR-
Cas9 based disruption
of GATA1 binding
leading to reduced
BCL11A expression (a
repressor of g-globin
gene expression),
resulting in increased
g-globin and fetal
hemoglobin (HbF)
protein production in
erythroid cells to treat
sickle cell disease and
transfusion-dependent
b-thalassemia patients.
Vertex
Pharmaceuticals/
CRISPR Therapeutics
AG. For sickle cell
anemia,and for b
Thalassemia, (EMA,
2023)
FDA (2023a)
ElevidysÒ
(delandistrogene
moxeparvovec-
rokl)
Duchenne muscular
dystrophy
In vivo Skeletal and
cardiac muscles
Gene replacement,
rAAVrh74.MHCK7.
micro-dystrophin
cDNA
Sarepta Therapeutics,
Inc./Catalent, Inc.
(FDA, 2023b)
LyfgeniaÔ
(lovotibeglogene
autotemcel)
Sickle cell anemia Ex vivo Autologous
CD34þ
hematopoietic stem
cells
Gene replacement,
CD34þ HST
transduced by BB305
lentivirus vector
encoding HbA
T87Q
Bluebird Bio, Inc.
(FDA, 2023c)
LenmeldyÔ
(atidarsagene
autotemcel)
Metachromatic
leukodystrophy
In vivo and
ex vivo
Autologous
CD34þ
hematopoietic stem
cells
Gene replacement,
CD34þ HST
transduced by
lentivirus vector
encoding arylsulfatase
a (ARSA)
Orchard Therapeutics,
(FDA, 2024a)
Background: Vectors and applications 261
III. Specialty biologics and indications
Most nonviral systems are cationic in nature and form strong complexes with anionic
nucleic acids via electrostatic forces. The cationic materials surround and condense the
nucleic acid core via charge neutralization, and effectively protect the genetic material
from environmental degradation. The cationic surface charges of nonviral vector complexes
bind with anionic proteoglycans on cell surfaces and allow for internalization via endocy-
tosis. The insertion of cell-specific receptors or cell-targeting ligands on the nonviral vector
surfaces drives tissue tropism, improved cellular uptake and delivery of nucleic acids to
the cyto plasm or nucleus (Bulcha et al., 2021; Paunovska et al., 2022).
Despite these challenges, nonviral methods of delivering genetic material are now
achieving success in patients, and are providing proof of concept for the delivery method,
most notably with the development of vaccines for SARS-CoV-2 (Urits et al., 2020; Hoy,
2021; Hald Albertsen et al., 2022). All the currently approved lipid nano-particle formulations
contain four lipids: (1) an ionizable cationic lipid, helper lipids, which include (2) 1,2-
distearoyl-sn-glycero-3-phosphocholine (DSPC) and (3) cholesterol, and (4) a polyethylene
glycol (PEG)-lipid conjugate (Hald Albertsen et al., 2022). Variations on these combinations
lead to vast numbers of unique formulations, each with their own distinct efficacy and safety
concerns. As most nonviral carriers are not regulated as GTs, they are beyond the scope of
this chapter.
Viral vectors
Viral vectors are gene delivery vehicles that use viral genomes to transfer genetic material
into target cells (Fig. 9.1). Viral vectors are defined by three components:
FIGURE 9.1 Schematic comparing the three major vector platforms together with typical designs of the
expression cassettes.
9. Preclinical development of viral vector gene therapies262
III. Specialty biologics and indications
1. Protein capsid and/or envelope: viral protein “box” and/or host lipid membrane with
viral glycoproteins that contains the genetic payload; both define cell tropism and
antigen recognition.
2. Transgene: encodes a specific therapeutic protein as replacement/correction therapy, or
a nontranslated small RNA to inactivate a mutated gene, or both.
3. Regulatory cassette: combines enhancer/promoter/auxiliary elements to control the
expression of a transgene.
Most viral vectors require the presence of specific receptors on the cell surface for binding
to, and internalization by target cells. After internalization by the host cell, usually by endo-
cytosis, the viral capsid is released into the cytoplasm (endosomal escape) before delivering
the genetic payload in the nucleus (Degors et al., 2019). Viral vectors are subcategorized as
nonintegrating and integrating, depending on whether the viral payload remains episomal
in the nucleus or as DNA integrated into the host genome.
Nonintegrating viral vectors
The two most commonly used nonintegrating viral vectors derive from wild type adeno-
virus (AdV) and adeno-associated virus (AAV). AdV and AAV belong to nonenveloped vi-
ruses consisting of an icosahedral viral capsid surrounding either a double or single strand of
DNA, respectively (Schnepp et al., 2003; Leopold and Crystal, 2007; Stadtfeld et al., 2008; Bul -
cha et al., 2021; Riyad and Weber, 2021 ; Lundstrom, 2023). While other nonintegrating viral
vectors are being explored, including integration-deficient LV, poxvirus, herpes virus, and
more recently Sendai virus, these will not be discussed in this chapter (Athanasopoulos
et al., 2017) as their application to the clinic remains limited.
Adenoviral vectors (AdV)
Adenoviridae is a family of 80e100 nm, nonenveloped, icosahedral capsid, double-
stranded DNA viruses comprising more than 100 serotypes that can infect vertebr ates;
more than 50 serotypes are specific to humans (Luisa and Ikner, 2017). Because broad preex-
isting immunity against AdV is common across human populations (Vannucci et al., 2013),
GT applications have focused on the rather rare serotypes 2 and 5. AdV entry into cells in-
volves attachment to a primary receptor, with the ‘spikes’ or ‘fibers’ extruding out of the
adenoviral capsid facilitating this attachment. Primary attachment is followed by interaction
with a secondary receptor, which initiates AdV internalization (Zhang and Bergelson, 2005;
Stasiak and Stehle, 2020). There is remarkable diversity with both the primar y and secondary
cell attachment/internalization receptors, which allows for AdV to have broad tissue
tropism. For the AdV2 and AdV5, the primary attachment receptors are coxsackievirus
and adenovirus receptor (CaR), as well as heparan sulfate glycosaminoglycans (HS-GAGs).
HS-GAGs are especially abundant within the extracellular matrix and the cellular glycocalyx.
Other primary receptors such as CD46 (for subgroup B AdVs), CD80 and CD86 (expressed on
antigen-presenting cells and receptors for AdV3 and other B1 subgroup members), sialic acid
(for subgroup D AdVs) and integrins ( aLb2 and aMb2) have been identified for other AdV
serotypes (Stasiak and Stehle, 2020). The secondary internalization receptors include integrins
such as vitronectin, fi bronectin, and laminin receptors (Greber and Gomez-Gonzalez, 2021).
The five main integrins utilized by AdVs as the secondary receptors are avb3, avb1, avb5,
Background: Vectors and applications 263
III. Specialty biologics and indications
a3b1, and a5b1(Hussein et al., 2015). Interestingly, while many investigators have relied on
the natural tropism of AdVs for gene transfer, pseudotyping the long Ad5 fiber shaft with
short shafts from other serotypes has been undertaken to modify or restrict transduction to
specific tissues or cell types, an approach especially relevant for cancer gene therapy (Von
Seggern et al., 2000; Uusi-Kerttula et al., 2016).
Following attachment to the plasma membrane, the AdV enters the cell via a clathrin-
dependent mechanism and is transported to endosomes, where acidificati on leads to partial
disassembly of the capsid and release of the altered virion into the cytoplasm. Capsid disas-
sembly occurs at the nuclear pore and the viral genome is then released wi thin the nucleus
(Lee et al., 2017; Greber and Suomalainen, 2022). AdV can transduce quiescent and dividing
cells, making them broadly applicable as vector platforms for a variety of target tissues. The
packaging capabilities of such vectors is also larger than 8 kb, outperforming both AAVs and
LV vectors. Most recently adenoviral vectors have caught the public eye with their application
as tools for vaccine delivery to treat a range of infectious diseases including SARS-CoV-2 (Men-
donca et al., 2021). These vaccines can be rapidly scaled up and take advantage of the ability to
package DNA or mRNA of the encoded pathogen protein in the AdV (Sakurai et al., 2022).
Further development of so called ‘gutless’ AdVs (or helper-dependent AdVs), in which the
genome is stripped from all viral coding sequences (therefore increasing their cloning capacity
up to 36 kb), has been shown to minimize cytotoxicity and circumvent anti-vector immunity,
making AdV very attractive for GT (Alba et al., 2005; Jozkowicz and Dulak, 2005).
Adeno-associated vectors (AAV)
Adeno-associated viruses (AAV) belong to the genus of Dependoparvoviruses of the Parvo-
viridae family and were initially identified as contaminants in adenoviral preparations. To
date 12 native AAV serotypes have been identified and hundreds of natural and engineered
variants have been reported (see Li and Samulski, 2020; for an excellent review). Efficient
replication and propagation of AAVs depend on the coinfection by a helper virus such as
adenovirus or herpes simplex virus. In the absence of concurrent infection, the wild-type
AAV (serotype 2) is capable of site-specific integration into the AAVS1 locus on the human
chromosome 19, a process that relies on the presence of the Rep protein and corresponds to
the latency phase for this virus (Samulski et al., 1991). Recombinant AAV (AAV) vectors,
which are designed without the rep 68/78 genes, mostly remain episomal and rarely integrate
in the host genome even though random integration through DNA chromosomal breaks re-
mains a rare possibility (Naso et al., 2017; Bijlani et al., 2021).
AAVs are nonenveloped viruses composed of a 22e26 nm proteinaceous icosahedral
capsid carrying a single-stranded molecule of DNA. The vira l genome size is relatively small
(4.7 kb) and encodes for only two genes flanked by inverted terminal repeats (ITRs): (1) the
rep gene, encoding for the Rep78, Rep58, Rep52 and Rep40 proteins, responsible for the repli-
cation and encapsidation of the genome, and (2) the cap gene, encoding for the capsid protein
subunits VP1, VP2 and VP3. AAV viruses are generally considered nonpathogenic, a very
desirable feature when leveraging viruses for in vivo gene transfer (Balakrishnan and Jayand-
haran, 2014). Viral vectors derived from AAV have been modified by removing all the viral
open reading frames apart from the ITRs, replacing the rest of the natural viral genome with
the desired therapeutic payload (Pupo et al., 2022). The ITRs are the only cis-acting elements
9. Preclinical development of viral vector gene therapies264
III. Specialty biologics and indications
required for genome replication and packaging (Wu et al., 2006). A key limitation of using
AAV for therapeutic applications is their relatively small cloning capacity (about 4.7 kb).
The understanding of the AAV transduction process is evolving (Riyad and Weber, 2021).
It is known that AAV entry into cells involves attachment via a primary receptor followed by
interaction with a secondary receptor to initiate internalization. Hepar an sulfate proteogly-
cans (HSPG) were the first attachment receptors identified for AAV and are utilized by sero-
types 2, 3, and 6. Sialic acid (N-linked or O-linked) is another primary attachment receptor,
which is utilized by AAV serotypes 1, 4, 5, and 6, while N-terminal galactose is utilized by the
AAV serotype 9 and mannose/mannosine-based receptor complex is utilized by AAV sero-
type 12 (Zengel and Carette, 2020; Issa et al., 2023). To date, the primary attachment receptor
remains unknown for AAV serotypes 7, 8, 10, 11, and 13.
The secondary internalization receptors for cell entry vary between the serotypes. AAV2
utilizes the a5b1 fibronectin receptor, vitronectin receptor aVb5, fibroblast growth factor re-
ceptor 1 (FGFR1), hepatocyte growth factor receptor (HGFR), LamR (nonintegrin laminin re-
ceptor), and AAV receptor (AAVR); AAV3 utilizes FGFR1, HGFR, LamR, and AAVR; AAV5
uses platelet derived growth factor (PDGF) a, PDGF b, and AAVR; AAV6 utilizes epidermal
growth factor receptor (EGFR) and AAVR; AAV8 and AAV9 both use LamR and AAVR;
AAV13 uses HSPG; AAV1 uses AAVR (Pillay and Carette, 2017; Zengel and Carette,
2020). The secondary receptor is unknown for AAV4, 7, 10, and 11. AAVR, a glycosylated
membrane protein that recycles from the plasma membrane to the trans-Golgi network
and back using the cellular endosomal network, is a receptor that acts as a cell entry factor
that is necessary for successful gene transduction (Pillay and Carette, 2017). GPR108, a mem-
ber of the G protein-coupled receptor superfami ly, is another AAV entry factor that is primar-
ily located in the Golgi (Pillay and Carette, 2017; Dudek et al., 2018) and appears to have a
downstream role in trafficking AAV to the peri-nuclear region or endosomal release.
The initial docking of the AAV viral particle onto the cell surface is the first step in the mul-
tiple interactions between the AAV and host cell factors, which allow for the viral capsid to
traffic through the endosomal pathway, trans Golgi network, escape and enter into the nu-
cleus where the viral genome will be uncoated (Pillay and Carette, 2017; Dudek et al.,
2018; Riyad and Weber, 2021). Over the past years, more in vitro genetic screens have
confirmed the importance of AAVR and/or GPR108 for transduction and allowed the iden-
tification of many other complex intermolecular interactions involved in this process. What is
becoming clear is that even after uncoating of the viral genome in the nucleus of target cells,
additional mechanisms are necessary for the efficient expression of a therapeutic transgene,
including overcoming host restriction factors (Coroadinha, 2023). In the case of a single-
stranded viral vector genome (corresponding to WT AAV), the ITR allows for self-priming
and complementary strand synthesis by host polymerases, a process that is bypassed
when self-complementary vectors (in which a double stranded fragment of DNA is packaged
in the capsid) are use d (McCarty, 2008). The vector genome design (promoter, regulatory se-
quences, etc.) will dictate the expression of a therapeutic gene in the native host environment
(Li and Samulski, 2020). The formation of viral genome concatemers, potential transcriptional
silencing (via epigenetic modifications), or integration into the host genomes are all depen-
dent on many intranuclear molecular factors that remain elusive (Maurer and Weitzman,
2020).
Background: Vectors and applications 265
III. Specialty biologics and indications
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