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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5423_Библиотеки_им_академика_М_И_Перельмана
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1 Introduction to AAV-based invivo Gene Therapy
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10
Table1.3 Complete and active AAV gene therapy clinical trial indications.
Indications of clinical trials Complete Active
Muscular Disorders 12 8
Vision Defects 10 4
Neurodegenerative Diseases 10 13
Retinal Diseases 9 25
Lysosomal & Glycogen Storage Disorders 7 20
Blood Coagulation Disorders 5 20
Cardiovascular Diseases 2 4
Amino Acid Metabolism Disorders 1 5
Arthritis 1 1
Infections 3
Adrenal Diseases 3
Developmental Disorders 2
Digestive System Diseases 3 1
Hearing Loss 1
Cancer 1
Despite its promise and setbacks, gene therapy’s potential across therapeutic
areas remains enormous, offering the hope of “one and done” cures for serious
diseases with significant unmet needs. AAV‐based gene therapy has received the
most attention in basic and clinical research recently, moving rapidly into the biotechnology industry, resulting in clinical trials designed to prove its efficiency,
safety, cost‐effectiveness, and range of use. Recombinant AAVs (rAAVs), engineered capsids with lower immunogenicity, the addition of synthetic promoters,
and gene editing techniques are just some of the developments in AAV vector‐
based gene therapy. Although these developments have come to fruition relatively
quickly, many challenges remain when developing and using gene therapy treatments. Several gene editing techniques have emerged over the past two decades,
which have been crucial in researching and developing disease mechanisms,
therapies, and more. Although there have been setbacks, the promise of gene
therapies and the potential of direct gene editing remains. The technological
advancements in labs, clinics, and manufacturing processes continue to make it
more and more possible for long‐lasting rare disease treatments. Thus, it is crucial
to understand how AAV‐based gene therapies are created and implemented, the
benefits and risks of these treatments, as well as the identification of their most
relevant indications.
1.1 Introduction 11
Wild-type AAV genome
rAAV particles
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1.1.3 The AAV Vector Structure
The AAV is part of the Parvoviridae family, being approximately 25 nm in diameter
with its DNA contained within an icosahedral capsid, the protein shell. Single‐
stranded DNA of approximately 4.7
kb is composed of genes for three capsid proteins[22] four rep proteins, and an assembly‐activating protein[23]. Palindromic
inverted terminal repeats (ITRs) are located at each end[24], forming T‐shaped
hairpin structures. The Rep gene is required for DNA replication and packaging,
and the Cap gene encodes the capsid assembly proteins. In studies of AAV2, it was
found that the structural proteins, VP1, 2, and 3, exist in a 1:1:10 ratio, forming
an icosahedral, symmetrical shape [25]. VP1was essential for infection while
VP2was necessary for nuclear transfer of capsid proteins[26]. VP3was found to
allow the binding of the virus to cell receptors[27, 28].
The AAV was discovered in 1965 as a contaminant of an adenovirus preparation
from rhesus monkey kidney cell cultures infected with simian adenovirus type
1[3]. AAVs rely on helper viruses in order to replicate in mammalian cells, typically adenoviruses or herpes viruses.
When using AAV as a vector, the DNA of interest replaces the genomic DNA,
the Rep and Cap genes, between the ITRs[29]. The exogenous DNA is referred to
as the transgene expression cassette. When creating a recombinant AAV, the gene
expression cassette needs to include a promoter, transgene, and a termination signal, which is specific to the goals of that particular gene therapy. Figure1.2 displays the requirements for creating a rAAV vector. Tissue and cell‐type specificity,
packaging size limits, and necessary expression level all need careful consideration when creating the gene expression cassette.
ITR
Expression vector
ITR ITR
Promotor Transgene pA
Figure1.2 Creation of recombinant AAV particles.
Packaging cells
Co-transfection
Cap
Promotor
Adenoviral helper genes
Rep
ITRRep
Helper vectors
Cap
1 Introduction to AAV-based invivo Gene Therapy
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However, the baculovirus‐infected insect (Sf9) system is increasingly used with
scale‐up production. Specifically, the OneBac system has been developed which
integrates rep and cap into one baculovirus, resulting in a decrease in the required
number of baculovirus particles for efficient rAAV production[30].
1.1.4 Cell Entry and Transduction Pathway
Wild‐type AAV viruses can infect dividing and nondividing cells. The majority of
the AAV genomes, wild‐type and recombinant, exist as circular episomes in tissues, though integration into the host cell chromosomes at AAV integration sites
can happen in low frequency[31]. AAV will not replicate without Rep proteins,
which regulate viral transcription. Rep proteins can be provided by a helper virus
and remain latent until this occurs[32]. Once the helper virus is delivered, the
Rep genes are expressed and replication can occur via the rolling hairpin mechanism, where a Rep protein binds the Rep binding element (RBE) within the hairpin, followed by duplication of the DNA strand and packaging. The cell entry and
transduction process of an rAAV is shown in Figure1.3. Cellular transduction of
an AAV vector begins with viral capsid interaction with the target cell’s receptors.
The cell uses endocytosis (clathrin‐coated pits) to internalize the virus, which
AAV binds receptor of
target cell
2
Endosome
3
Endosomal escape
4
Figure1.3 rAAV vector entry and transduction pathway. (1) AAV vectors bind to receptors
of target cells, (2) initiating endocytosis via clathrin-coated pits. (3) The endosomal
compartment contains the AAV, (5) which can escape to the nucleus through the nuclear
pore. (6) Uncoating releases single-stranded DNA (7) for a second strand to be
synthesized (de novo synthesis or base pair strand annealing). (8 and 9) This leads to the
production of the corrected/therapeutic mRNA and subsequent protein to be produced.
Endocytosis
Enters nucleus
5
Corrected/therapeutic
9
protein
Uncoating
6
7
Converts to double stranded DNA
Produce mRNA
8
then enters the endocytic/proteasomal compartment. These may be critical steps
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for transduction because injection of AAV2 directly into cells resulted in a reduced
infection rate compared to exposure[33]. Interestingly, the efficiency of transduction is largely affected by acidity of the endosomal compartment[34]. Proteasome
inhibitors also increase transduction efficiency[35].
The virus escapes the endosome and is imported through the nuclear pore complex into the nucleus, undergoes capsid uncoating, and the DNA is converted into
double‐stranded DNA via de novo synthesis or base pairing. The DNA can then be
transcribed and expressed [36]. Particles that are not able to translocate to the
nucleus are degraded by the proteasome and presented to cytotoxic T cells[37].
Thus, prior to genome integration, intracellular trafficking involves a number of
events in which breakdown at any step can result in failure of gene delivery.
De novo synthesis of the complementary DNA strand and strand annealing result
in the conversion of single‐stranded DNA to double‐stranded DNA. Strand annealing involves the base pairing of a coinfected separate AAV complementary single‐
strand genome. Due to the synthesis of the second DNA strand being considered
the rate‐limiting factor in transduction efficiency, second‐generation AAV vectors
with double‐stranded DNA have been developed, called self‐
AAV vectors, which have been shown to be safe and reliable for organ‐specific
transduction[38, 39].
complementary
131.2 Advantages and Disadvantages for AAV invivo
1.2 Advantages and Disadvantages for AAV invivo
1.2.1 Effectiveness and Advantages of AAV Vectors for invivo
Gene Therapy
AAV vector has been labeled as the safest and most effective vehicle for the delivery
of invivo gene therapies that produce long‐term expression with a single injection.
rAAV effectiveness is largely determined by the interaction of the capsid and the
cell surface receptors as well as downstream events after internalization.
Transduction efficiency can be altered due to the endosomal compartment pH and
the use of proteasome inhibitors[34, 35]. Immune reactions to AAVs significantly
impact a gene therapy’s efficiency and will be discussed further in Section 1.5.3.
Addressing Immunogenicity and Chapter5.
There are many advantages to using AAV vectors for gene therapy. The human
native AAV is not known to cause disease, has no pathogenicity[3], and has the
ability to infect numerous mammalian cell types. AAVs efficiently deliver genetic
material with low toxicity and immunogenicity, a good safety profile, and long‐
term effects[40, 41].
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1.2.2 Challenges of AAV Vectors for invivo Gene Therapy
Challenges, of course, still exist for gene therapy using AAV vectors. Large‐scale
manufacturing, particularly quality control and standardization, is difficult to
maintain for gene therapies, resulting in high costs. Although AAVs provide efficient gene delivery, they are less immunogenic than adenoviruses. Before transduction, single‐stranded DNA from AAVs must first be made into double‐stranded
DNA. AAVs have limited capacity, holding approximately 4.7kilobases (kb)[22].
Implementing a split vector approach can bypass the limitation of small capacity;
however, serogenicity and type of cell or tissue can markedly affect the expression
of split vectors. Also, replacing Rep genes with exogenous DNA lowers the transduction frequency because, without them, ITRs have little enhancer and promotor activity. Self‐complementary vector AAVs can be implemented to improve
efficiency and onset of gene therapies.
There are potential sources of toxicity that must be considered in the development
of AAV gene therapies, insertion mutagenesis, tumor induction, and immune
response, dependent upon reactions to the capsid and DNA of the AAV. Toxicity has
been reported for AAV clinical trials, causing hepatotoxicity, muscle toxicity, thrombotic microangiopathy, etc.[42]. Route of administration and number of viral particles used for gene therapy may contribute to toxicity and the immune response as
well. In two studies testing a hemophilia B gene therapy, AAV gene transfer in the
liver resulted in the development of capsid‐specific CD8+ T cells and elimination of
transduced hepatocytes. The reaction was halted by oral corticosteroids [40, 43],
although in some cases, immune‐modulating agents have been shown not able to
prevent loss of transgene expression. Reactions to natural AAV capsids may predict
the reaction and effectiveness of AAV‐based gene therapies [44]. Transient B‐cell
depletion and inducing immune tolerance with rapamycin may be good strategies to
reduce the host immune response to AAVs, though better strategies may be necessary
to prevent a decrease or, in some cases, a complete loss in transgene expression.
Immunogenicity and malignancy are other challenges that must be addressed during development and preclinical studies to ensure patient safety. Manufacturing gene
therapies can be challenging because of the quality control needed throughout and
production costs. Scalability has been a major obstacle to gene therapy production.
Due to the time it takes for clinical trials and regulatory procedures, drugs take ample
time to reach the market, even for diseases without any current treatment options.
1.3 Technology Platforms of AAV-based invivo
Gene Therapy
Genetic editing is the act of revising, removing, or replacing DNA directly, which
can be used to tailor recombinant AAVs for specific gene therapies. Gene therapy
uses gene replacement, gene addition, genome editing, and gene regulation tech-
ZFN & TALEN
DNA r
CRISPR/Cas9
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niques. Typically, ZFN, transcription activator‐like effector nucleases (TALENs),
and clustered regularly interspaced short palindromic repeats (CRISPR) are used
for various gene editing techniques. Each technology has specific uses and limitations, which are discussed in brief in this chapter. Further detail will be provided
in Chapter2.
1.3.1 cDNA Replacement
cDNA replacement is the method used in gene replacement therapies to express a
functional copy of a defective gene episomally, directed by an exogenous promoter, which results in stable gene expression. Most programs use AAV to deliver
vectors in gene replacement, although this method is limited to recessive or haploinsufficient disease targets. Artificial promoters allow for specificity but can
compromise physiological gene expression.
1.3.2 Genome Editing
Breaks and DNA repair are used in genome editing to design specific, targeted
vectors. Genome editing can be performed using various platforms, including
ZFN, TALEN, CRISPR/Cas9, base and primer editing, and RNAi gene silencing.
These technologies will be introduced next in this chapter with more detail
explained in Chapter2.
Many gene editing techniques utilize a DNA nuclease to cleave a specific site in the
genome (Figure1.4). ZFNs and TALENs have been used previously but are challenging and time‐consuming to design or have large constructs with limited transduction.
151.3 Technology Platforms of AAV-based invivo Gene Therapy
FOK1
ecognition protein motif
Figure1.4 Gene editing techniques using double-stranded breaks. ZFN and TALEN use
DNA recognition protein motifs and fused with a cleaving enzyme (FOK1). CRISPR/Cas9
uses a guide RNA that binds to a target sequence, then Cas9 binds the guide RNA to
cleave both DNA strands.
DNA recognition protein motif
FOK1
Cas9
Guide
RNA
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Genome editing platforms can be combined with viral vectors to improve a gene therapy’s efficacy and lower the risk of off‐target effects.
1.3.2.1 ZFN
Targeted gene replacement was first developed using ZFNs. Both ZFN and TALEN
technologies pair protein domains with nucleotides with a cleaving enzyme to
create a double‐stranded break (DSB). Approximately 30 amino acids make up
one zinc‐finger motif, each able to bind three nucleotides. ZFNs generally display
lower specificity than TALENs [45, 46] and relatively similar specificity to
improved CRISPR/Cas9 methods with certain guide RNAs (gRNAs) [47–49].
However, specificity can be difficult to compare and depends on the specific technological methods, including the gene editing target and nuclease architecture. It
also produces less immune reaction compared to the newer CRISPR technology.
In human studies using ZFN for gene editing was found to have a good safety
profile with evidence of successful gene editing, although sustained expression
was not detected[50].
1.3.2.2 TALENs
TALENs make use of non‐specific DNA nucleases bound to a DNA‐binding
domain that can target a specific sequence. TALENs were more quick and efficient
to use compared to earlier technologies[51]. The first use of TALEN technology in
human treatment was in 2015, treating pediatric acute B lymphoblastic leukemia
with TALEN‐engineered CAR T cells [52]. TALEN can be advantageous for
difficult‐to‐edit regions of DNA where it can be more efficient than CRISPR[53].
One disadvantage of the TALEN system is its incompatibility with AAV vectors. In
general, the targeted use of TALEN invivo is challenging because of its large size
and repetitive characteristics. Using high‐capacity adenovirus or non‐viral delivery
is best when implementing the TALEN system directly invivo[54].
1.3.2.3 CRISPR/Cas9
CRISPR/Cas9 is a robust gene editing tool that has emerged as the preferred gene‐
editing technique because of its ease of use, low cost, and high efficacy. While
ZFN and TALEN are protein‐based DNA recognition techniques, CRISPR is an
RNA‐based technique. In brief, CRISPR/Cas9 binds to DNA fragments containing
protospacer‐adjacent motif (PAM) sites and creates DSBs in the DNA. A gRNA
locates the gene of interest or location within the DNA and a Cas9nuclease
induces a DSB in the DNA. Then, DNA repair via non‐homologous end joining
(NHEJ) or homologous direct repair (HDR) is initiated[55].
As with most gene editing techniques, problems arise, particularly off‐target
effects and immunity. Off‐target effects are an inherent issue in gene editing,
being identified with use of ZFNs, TALENs, and CRISPR/Cas9[56, 57]. Cas9 from
1.3 Technology Platforms of AAV-based invivo Gene Therapy
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bacteria, used on the human genome significantly increases the chance of
off‐
target effects because the genome is much larger than that of bacteria[47].
Thus, many improvements in the CRISPR/Cas9 system have aimed at reducing
off‐
target effects and providing stable genome expression. In 2007, cells treated
with CRISPR were found to activate adaptive immunity, inspiring many improvements and modifications to the system[58].
Improving gRNA is another important area of gene therapy development
because it has significant effects on sensitivity and specificity of the system.
Truncated gRNA, or gRNA lengthened by two guanine nucleotides, has been
found to reduce off‐target effects[48, 49, 59]. Many tools have been developed to
design gRNA and detect off‐target effects invivo[60]. Integrase defective lentiviral
vectors‐capture was one of the first assays created to monitor off‐target effects
invivo following the use of ZFNs, and GUIDE‐seq is the common tool used to
identify off‐target sites with CRISPR/Cas9 treatments[61–63]. The use of nickases to create a single‐stranded rather than a double‐strand break also greatly
reduces off‐target effects[49].
1.3.3 Base Editing and Prime Editing
As mentioned, base and prime editing do not necessitate any DSB, making it safer
and more accurate than classical gene editing technologies. Base editing is a tool
for permanently correcting base pair mismatches[64]. A more recently developed
method called prime editing uses prime editing gRNA (pegRNA) and Cas‐
which can correct transition mutations, unlike base editing[65].
Base editors are classified as cytosine or adenine base editors, which need an
inactive Cas or Cas nickase coupled with deaminase to make the edit and a gRNA
to guide Cas to the targeted DNA binding site. Dual base‐editor systems have also
been created for a combinatorial editing approach[66]. Creating precise base edits
of more than four transition mutations has not been feasible with base editing,
leading the path to prime editing. An engineered reverse transcriptase bound to a
Cas9 nickase and a prime editing gRNA are necessary for prime editing[65].
PegRNA not only holds the complementary sequence but also another sequence
directing the exact sequence change. These methods will be very useful in editing
large genes and the treatment of autosomal dominant diseases[67]. Because these
technologies are quite new, much research is needed to determine their full safety
and efficacy in a variety of uses.
nickase,
17
1.3.4 RNAi Gene Silencing
Gene silencing can be performed with RNA interference (RNAi). RNAi can be
utilized for gene regulation through the knockdown of a target gene, known as
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gene silencing. This occurs through a process where double‐stranded RNA
(dsRNA) is processed into short interfering RNAs (siRNAs), resulting in transcriptional or post‐transcriptional gene silencing by degradation of complementary
mRNA[68]. dsRNA of more than 30 base pairs can result in a significant antiviral
response and apoptosis[69]. Probably the most promising area for RNAi is in
antiviral strategy for infections[70].
1.3.5 Gene Addition
Gene therapy for more complex genetic diseases, infectious diseases, and cancer
can be performed via gene addition methods to over‐express an endogenous or
synthetic gene. A herpes simplex virus (HSV) vector has been developed containing the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) gene to
help immune effector cells attack tumor cells[71]. Another example of gene addition has been done by transferring a CAR gene into T cells exvivo for treating
B‐cell malignancies[72].
1.4 AAV Serotypes and Tissue Affinity
Twelve AAV serotypes have been identified, and over 100 variants have been
found in human or nonhuman primate tissues; although, the best characterized is AAV2[29]. Optimal serotypes are specific to tissue type, including AAVs
1, 2, 4, 5, 8, and 9 for the central nervous system (CNS), AAVs 1, 8, and 9 for the
heart, AAV2 for the kidney, AAVs 7–9 for liver, AAVs 4–6 and 9 for the lung,
AAV8 for the pancreas, AAVs 2, 5, and 8 for photoreceptor cells, AAVs 1, 2, 4, 5,
and 8 for retinal pigment epithelium, and AAV1 and 6–9 for skeletal muscle.
The varying serotypes with differing tissue specificity and infection rates, make
AAV vectors a great candidate for gene therapy. AAV gene therapies commonly
target the liver, CNS, and muscle, which can become biofactories for producing
the required protein.
Other characteristics specific to an individual or a few AAV serotypes include
their transduction efficiency, homology to other serotypes, and speed of tissue
targeting. AAV1 and 6 share 99% homology, while AAV2has close homology to
most serotypes, not including AAV4, 5, 11, and 12. AAV 8 and 10 share 93% homology, and AAV11 and 12 have close homology to AAV4. AAV5 shares the least
homology with other serotypes. Zincarelli et al. found that AAV2‐5 have low
transduction efficiency, and AAV3 and 4 are slow in targeting tissues[73], but this
varies significantly depending on cell type[74].
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 19
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1.4.1 The Liver asa Biofactory
The liver is targeted in gene therapies of hemophilia for the production of factor
VIII and factor IX, missing coagulation proteins[75]. Hemophilia is an ideal target for gene therapy, especially as the genetics and pathophysiology of the disease
are well understood. The goal of gene therapy is the long‐term expression of the
missing genes in the coagulation cascade at levels high enough to be therapeutic,
a so‐called functional cure. A growing body of clinical experience supports the use
of liver‐directed rAAV as a gene therapy transporter of the replacement gene for
the treatment of hemophilia[75, 76].
1.4.2 The CNS asa Biofactory
Some neurodegenerative disease gene therapies have been successfully developed
for spinal muscular atrophy (SMA) and are being developed for other CNS diseases, including Parkinson’s and Alzheimer’s disease and ALS. The blood–brain
barrier (BBB) tends to be an obstacle for AAVs. Two AAVs are known to cross the
BBB; IV injection of AAV9 and AAVrh.10was shown to cross the BBB more effectively than AAV2 or AAV8 to transduce neurons and glial cells[77]. More research
is critical to determine how the CNS can be targeted efficiently to use as a gene
therapy biofactory for CNS diseases and disorders.
1.4.3 The Muscle asa Biofactory
Muscular dystrophies are another area of interest for gene therapy. AAV8 and
AAV9 tend to be used for targeting muscle, although AAV1, 2, 5, 6, and 7 can also
be utilized[78]. Muscle can be used as a biofactory, producing secretory factors for
infectious diseases, diabetes, atherosclerosis, hemophilia, and cancer therapeutic
agents. Muscle cells display the ability to secrete recombinant therapeutic proteins into the bloodstream after intramuscular AAV transduction, making it a useful tool for the treatment of distant organs[17].
1.5 Precision Medicine: Screening and Monitoring
Biomarkers, Companion Diagnostics
Besides standard efficacy and safety assessments using clinical outcomes, usually
primary endpoints in clinical trials, the incorporation of biomarkers can support
the monitoring of patients on gene therapies. These biomarkers include translational, structural, functional, and tumorigenesis biomarkers, which can be used
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